Post-harvest Pathology (Plant Pathology in the 21st Century)

Postharvest Pathology

Plant Pathology in the 21st Century: Contributions to the 9th International Congress

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Dov Prusky    M. Lodovica Gullino ●

Editors

Postharvest Pathology

Editors Dov Prusky Department of Postharvest Science of Fresh Produce Agricultural Research Organization Bet-Dagan Israel [email protected]

Maria Lodovica Gullino Università di Torino Grugliasco TO Italy [email protected]

ISBN 978-1-4020-8929-9 e-ISBN 978-1-4020-8930-5 DOI 10.1007/978-1-4020-8930-5 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009932670 © Springer Science+Business Media B.V. 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Contents

1 The Role of Pre-formed Antifungal Substances in the Resistance of Fruits to Postharvest Pathogens............................. Nimal Adikaram, Chathurika Karunanayake, and Charmalie Abayasekara 2 Mechanisms of Induced Resistance Against B. cinerea.......................... Tesfaye Mengiste, Kristin Laluk, and Synan AbuQamar 3 Induced Resistance in Melons by Elicitors for the Control of Postharvest Diseases.............................................................................. Bi Yang, Li Yongcai, Ge Yonghong, and Wang Yi 4 Mechanisms Modulating Postharvest Pathogen Colonization of Decaying Fruits............................................................... Dov Prusky, Noam Alkan, Itay Miyara, Shiri Barad, Maayan Davidzon, Ilana Kobiler, Sigal Brown-Horowitz, Amnon Lichter, Amir Sherman, and Robert Fluhr 5 Global Regulation of Genes in Citrus Fruit in Response to the Postharvest Pathogen Penicillium digitatum................................. L. González-Candelas, S. Alamar, A.R. Ballester, P. Sánchez-Torres, J. Forment, J. Gadea, M.T. Lafuente, L. Zacarías, and J.F. Marcos

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6 Epidemiological Assessments and Postharvest Disease Incidence....................................................................................... Themis J. Michailides, David P. Morgan, and Yong Luo

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7 Preharvest Strategies to Control Postharvest Diseases in Fruits........................................................................................ N. Teixidó, J. Usall, C. Nunes, R. Torres, M. Abadias, and I. Viñas

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Contents

  8 New Developments in Postharvest Fungicide Registrations for Edible Horticultural Crops and Use Strategies in the United States.................................................................................. 107 J.E. Adaskaveg and H. Förster   9 New Approaches for Postharvest Disease Control in Europe.............. 119 M. Mari, F. Neri, and P. Bertolini 10 Quo Vadis of Biological Control of Postharvest Diseases..................... 137 Wojciech J. Janisiewicz 11 Improving Formulation of Biocontrol Agents Manipulating Production Process................................................................................... 149 J. Usall, N. Teixidó, M. Abadias, R. Torres, T. Cañamas, and I. Viñas 12 Host Responses to Biological Control Agents........................................ 171 Raffaello Castoria and Sandra A.I. Wright 13 Non-fungicidal Control of Botrytis Storage Rot in New Zealand Kiwifruit Through Pre- and Postharvest Crop Management.............. 183 M.A. Manning, H.A. Pak, and R.M. Beresford 14 The Peach Story....................................................................................... 197 Paloma Melgarejo, Antonieta De Cal, Inmaculada Larena, Iray Gell, and Belen Guijarro Index.................................................................................................................. 209

Recent Developments in Postharvest Pathology

This collection of papers includes some of the presentations given at the International Congress for Plant Pathology held in Turin in 2008 in the session with the above title. Fruit production for human consumption is an important part of the market economy. Any waste due to spoilage and pest infestation, in the field but mostly during the postharvest phase, results in significant economic losses which are more pronounced as the losses occur closer to the time of produce sale. Careful handling of perishable produce is needed for the prevention of postharvest diseases at different stages during harvesting, handling, transport and storage in order to preserve the produce high quality. The extent of postharvest losses varies markedly depending on the commodities and country and are estimated to range between 4% and 8% in countries where postharvest refrigeration facilities are well developed to 50% where these facilities are minimal. Microbial decay is one of the main factors that determine losses compromising the quality of the fresh produce. For the development of an integrated approach for decay management, cultural, preharvest, harvest, and postharvest practices should be regarded as essential components that influence the complex interaction between host, pathogen, and environmental conditions. Orchard practices including preharvest fungicide applications can also directly reduce the development of postharvest fruit decay. Among postharvest practices, postharvest fruit treatments with fungicides are the most effective means to reduce decay. Ideally, these fungicides protect the fruit from infections that occur before treatment, including quiescent infections, as well from infections that are initiated after treatment during postharvest handling, shipment, and marketing. However the wide consumption in human diet of high-quality fresh fruits and vegetables and the increased concerns for the possible toxicity of fungicide residues have lead to the development of new alternative approaches for disease control. One of the alternatives is the use of antagonist applications, either alone or in combination with physical treatments and substances generally regarded as safe. The implementation of these alternatives techniques often requires modifying currently used postharvest practices and development of new formulation for their applications. Three chapters in this book deal with the mechanisms of host fruit and vegetable resistance. Adikaram and co-workers referred to preformed antifungal substances affecting the resistance of unripe fruits and changes in their level during fruit ripening. Mengiste and co-workers suggested that active processes related to the regulation vii

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of cell death, plant hormone signalling and synthesis are implicated in disease resistance to necrotrophic pathogens during storage. Interestingly Yang and coworkers indicated that a variety of chemical, physical and biological elicitors may modulate inducible mechanisms of resistance. Two chapters in this book deal with fungal pathogenicity factors and their relationship with the host response. Prusky and co-workers described an interesting mechanism used by postharvest pathogens to modulate host environment (alkalinization and acidification) leading to enhanced pathogenicity, while Gonzales-Candelas and co-workers presented the first wide transcriptome analysis of citrus fruit response to Penicillium digitatum infection. Four chapters in this book deal with subjects related to disease assessments before harvest and their relation to the postharvest treatment of fruits and vegetables. Michailidis and co-workers emphasized the importance of weather and environmental conditions to pathogen infection and suggested different approaches for disease assessment which could be used to predict the incidence of postharvest diseases. Teixido and co-workers suggested the importance of preharvest applications of biocontrol treatment efficacy in combination with nutrients and conclude on the importance of preharvest treatment in postharvest disease control. The other two chapters dealt specifically on the new development of postharvest edible crop in the United States by Adaskaveg and Förster, and in Europe by Mari and co-workers. Both suggested that integrations of combined technologies such as sanitation and use of fungicides, physical and biological agents are of high importance. Three chapters in this book are dealing with biological control of postharvest diseases and host responses to the biocontrol agents. Janisiewicz, presented a summary of the biocontrol developments in his “Quo Vadis of Biological Control …” chapter and their impact on the industry, while Usall and co-workers described the different technological changes made during the development of new formulations which allow the improvement of biocontrol efficiency. Castoria and Wright referred in their chapter to the different mechanisms of perception and activation of host resistance by the biocontrol agents. The remaining chapters of the book are focused in specific study cases of crops such as kiwifruit, peaches and grapes, where the integrations of different approaches at the pre and postharvest levels are combined. These represent new types of presentations which were presented in the evening workshops of the ISPP program with excellent attendance. Manning and Beresford described how management and assessment of rot-risk-factors in the vine and storage conditions may allow prevention of botrytis problems. Melgarejo and co-workers described the importance of orchard management in combination with epidemiological assessment to predict risk and optimal handling of fruits. In summary the Postharvest Pathology sessions included excellent presentations of new and exciting progress at the leading edge of Postharvest Pathology. Dov Prusky Department of Postharvest Science of Fresh Produce Agricultural Research Organization Bet Dagan, Israel

Epidemiological Assessments and Postharvest Disease Incidence Preharvest Strategies to Control Postharvest Diseases in Fruits New Developments in Postharvest Fungicide Registrations for Edible Horticultural Crops and Use Strategies in the United States New Approaches for Postharvest Disease Control in Europe Quo Vadis of Biological Control of Postharvest Diseases Improving Formulation of Biocontrol Agents Manipulating Production Process Host Responses to Biological Control Agents Non-fungicidal Control of Botrytis Storage Rot in New Zealand Kiwifruit Through Pre- and Postharvest Crop Management The Peach Story

Global Regulation of Genes in Citrus Fruit in Response to the Postharvest Pathogen Penicillium digitatum

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Raffaello Castoria and Sandra A. I. Wright M.A. Manning, H.A. Pak and R.M. Beresford Paloma Melgarejo, Antonieta De Cal, Inmaculada Larena, Iray Gell and Belen Guijarro

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M. Mari, F., Neri and P. Bertolini Wojciech J. Janisiewicz J. Usall, N. Teixidó, M. Abadias, R. Torres, T. Cañamas and I. Viñas

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Nimal Adikaram, Chathurika Karunanayake and Charmalie Abayasekara Tesfaye Mengiste, Kristin Laluk and Synan AbuQamar Bi Yang, Li Yongcai, Ge Yonghong and Wang Yi Dov Prusky, Itay Miyara, Noam Alkan, Shiri Barad, Maayan Davidzon, Ilana Kobiler, Sigal Brown-Horowitz, Amnon Lichter, Amir Sherman, and Robert Fluhr L. González-Candelas, S. Alamar, A.R. Ballester, P. Sánchez-Torres, J. Forment, J. Gadea, M.T. Lafuente, L. Zacarías and J.F. Marcos Themis J. Michailides; David P. Morgan and Yong Luo N. Teixidó, J. Usall, C. Nunes, R. Torres, M. Abadias and I. Viñas J. E. Adaskaveg and H. Förster

Author(s)

Chapter name

The Role of Pre-formed Antifungal Substances in the Resistance of Fruits to Postharvest Pathogens Mechanisms of Induced Resistance Against B. cinerea Induced Resistance in Melons by Elicitors for the Control of Postharvest Diseases Mechanisms Modulating Postharvest Pathogen Colonization of Decayed Fruits

Recent Developments in Postharvest Pathology ix

Chapter 1

The Role of Pre-formed Antifungal Substances in the Resistance of Fruits to Postharvest Pathogens Nimal Adikaram, Chathurika Karunanayake, and Charmalie Abayasekara

Abstract  Plants contain secondary metabolites with antifungal properties. In fruits they are mostly concentrated in the peel at immature stage and decline during ripening in coincidence with fungal rot development. The information on antifungal systems in immature avocado and mango, reviewed here, suggests that they play a role in natural disease resistance. Immature mangoes have evolved a formidable antifungal system comprising several resorcinols, gallotannins and chitinases. Resorcinols and gallotannins are inhibitory to major postharvest pathogens, Colletotrichum gloeosporioides causing anthracnose and Botryodiplodia theobromae causing stem-end rot. Their levels are generally higher in resistant cultivars than in susceptible ones. Mango latex, distributed in a fine network of canals in the fruit peel, contains chitinases which have the ability to rapidly digest conidia of C. gloeosporioides. Gallotannins and resorcinols decline progressively during ripening and the latex disappears when ripe rot development begins. Retention of latex in the harvested fruit reduces anthracnose and stem-end rot development during ripening. Treatment of harvested fruit with CO2 or inoculation with certain non-pathogenic fungi increased antifungal resorcinol concentration. Immature avocado fruits possess a pre-formed antifungal system comprising at least five antifungal compounds. The quiescence of C. gloeosporioides in the immature fruit has been attributed to the pre-formed antifungal activity of the peel. Lipoxygenase activity increases during fruit ripening, while epicatechin levels decline, suggesting that these events are linked to the decrease in di-ene concentrations. Inhibition of lipoxygenase activity results in retention of antifungal di-ene during ripening increasing fruit resistance. In freshly harvested avocados, the di-ene concentration can be further enhanced by treatment with biotic and abiotic agents.

N. Adikaram (*), C. Karunanayake, and C. Abayasekara Department of Botany, University of Peradeniya, Peradeniya, Sri Lanka e-mail: [email protected] D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_1, © Springer Science + Business Media B.V. 2010

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1.1 Pre-formed Antifungal Substances Plants produce a range of chemically diverse secondary metabolites with antifungal activity. Some of these exist in healthy plants in their biologically active forms and others occur as inactive precursors and are activated in response to tissue damage or pathogen attack (Schonbeck and Schlosser 1976; Osbourn 1996). VanEtten et al. (1994) have proposed the term phytoanticipin to distinguish these preformed antimicrobial compounds from phytoalexins, which are synthesized from remote precursors in response to pathogen attack, probably as a result of de novo synthesis of enzymes. Phytoanticipins are low molecular weight, antimicrobial compounds that are present in plants before challenge by microorganisms or are produced after infection solely from preexisting constituents (VanEtten et al. 1994). Certain plants contain high molecular weight constitutive hydrolytic enzymes such as chitinases with considerable antifungal activity and these cannot be accommodated within the umbrella of phytoanticipins, although they seem to play a role in plant defence. Pathogens infecting fruits have to face challenges that the pathogens infecting vegetative organs do not normally encounter. Fruits are generally protected by mechanical and chemical barriers in the peel and their physiology changes markedly during development, particularly when ripening occurs. Pathogens, when confront unripe fruits, often cause quiescent infections or minor damage. Such quiescent infections have been observed in tropical (Muirhead and Deverall 1981), subtropical (Droby et al. 1987), and deciduous fruits (Hall 1971). The resistance of unripe fruits to fungal decay has been shown to be associated with induced (Adikaram et al. 1982) or preformed (Prusky and Keen 1993) antifungal substances in the peel. The onset of decay coincides with fruit ripening and concurrent decrease in the antifungal compounds to sub-toxic levels. Thus, quiescence may therefore represent a mechanism for avoiding toxic levels of antifungal plant compounds. There is considerable interest in determining mechanisms underlying the natural resistance of unripe fruits to fungal pathogens and extending fruit resistance to postharvest ripening phase. In certain fruits, the preformed antifungal substances appear to play a supportive role to their arsenal of inducible defences by excluding saprophytic and epiphytic microorganisms. In other fruits such as avocado and mango where the inducible defence system is weaker, the preformed antifungal compounds appear to perform a definitive protective role. This Chapter reviews the preformed antifungal systems in mango and avocado.

1.2 Preformed Antifungal Compounds in Mango (Mangifera indica) fruit Unripe mango fruit peel contains three classes of preformed antifungal substances, gallotannins and resorcinols in the peel tissue, hydrolytic enzyme, chitinase, in the latex.

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1.2.1 Resorcinols Constitutive resorcinol type antifungal compounds were first isolated from the peel of unripe mango fruit cultivars Tommy Atkins and Haden and identified as a mixture of 5-substituted resorcinols, 5-(12-cis-heptadecenyl) resorcinol and 5-pentadecyl resorcinol (Fig. 1.1) (Droby et al. 1986; Cojocaru et al. 1986). Identical resorcinols were also shown in the peel of several other cultivars (Droby et  al. 1986). High Pressure Liquid Chromatography of the dichloromethane phase of peel extracts of two Sri Lankan cultivars, Karutha Colomban and Willard, showed peeks representing 5-(12-cis-heptadecenyl) resorcinol, 5-pentadecyl resorcinol and an additional peak due probably to a new resorcinol (Karunanayake 2008). Two other resorcinols, 5(7, 12-heptadecadienyl) resorcinol from the fruit peel (Prusky et al. 1996) and 5-(9, 12-heptadecadienyl) resorcinol from the latex (Oka et  al. 2004), have been reported. The concentration of the former did not change significantly during fruit ripening, therefore it did not appear to play a role in fruit resistance. Knodler et al. (2007) in a more recent study identified 3 major and 12 minor C15, C17 and C19 substituted resorcinols and related analogues, however, their antifungal properties are not yet known. Resorcinols in mango varieties have been studied in relation to black spot development by Alternaria alternata during ripening. In unripe fruit, the fungus

Fig.  1.1  Structures of three resorcinols from mango peel, 5-(7, 12-heptadecadienyl) resorcinol (a), 5-pentadecyl resorcinol (b), and 5-(12-heptadecenyl) resorcinol (c) (Kobiler et al. 1998)

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forms quiescent infections. The concentration of resorcinols in the unripe mango fruit peel ranged between 154–232 mg g−1 fresh weight and this declined during ripening to about 74–125 mg g−1 fresh weight. At the time of Alternaria rot development, the concentration also ranged between 74–125 mg g−1 FW (Droby et  al. 1986). 5-substituted resorcinols were present at fungitoxic levels in the unripe fruit peel and decreased to non-toxic levels, with ripening, in uninoculated fruits at the same time that disease symptoms appeared in inoculated fruits (Prusky and Keen 1993). Eight mango cultivars, Maya, Erwin, Palmer, Pairi, Mabroka, Tommy Atkins, Haden and Keitt tested gave similar results. Concentration of resorcinols in the mango fruit was also studied in relation to development of anthracnose disease by Colletotrichum gloeosporioides during ripening. In the unripe fruit where the fungus forms quiescent infections, the resorcinols are present at higher concentrations and declined during fruit ripening to very low levels when anthracnose rot development commenced (Fig.  1.2). Recent studies have showed the presence of significantly higher amounts of antifungal resorcinols in mango latex (Bandyopadhyay et  al. 1985; Oka et  al. 2004) than in the fruit peel (Hassan 2006). The constitutive resorcinols identified in peel extracts could actually be those present in the latex canals of the peel. The concentration of three resorcinols varied among different cultivars. In cultivars that are resistant to anthracnose disease, there was a higher concentration of 5-(12- cis-heptadecenyl), 5-pentadecyl and AR 21 resorcinol (Table 1.1), than in susceptible ones (Karunanayake 2008). A strong positive correlation was present between the level of resorcinols and the degree of resistance to C. gloeosporioides. Mango cultivars, Kensington Pride and Keitt that are more resistant to anthracnose, had the highest concentrations of 5-n- heptadecenyl resorcinol while Nam Doc Mai and Honey gold susceptible to anthracnose had the lowest (Hassan et al. 2007). As in mango peel, the concentration of 5-substituted resorcinols in mango latex also varied significantly among cultivars (Hassan 2006). A high correlation exists

Fig. 1.2  Resorcinol concentration in the unripe and ripe fruit peel of cultivar Willard

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Table 1.1  The concentration of resorcinols in five Sri Lankan mango cultivars (average of two samples) determined by HPLC Concentration of resorcinols mg/g fresh weight 5 – (12- cis-heptadecenyl) 5 – pentadecyl Resorcinol ar 21 Cultivar resorcinol resorcinol derivative Resorcinol 27.1 532.1 43.2 Karutha Colomban (R) 59.9 Rata (R) 91.8 26.8 2,306.3 79.0 Kohu (M) 38.1 8.2 911.4 139.2 Petti (S) 20.7 8.6 47.5 15.3 34.4 58.3 14.8 25.7 Willard (S) (R) cultivars more resistant, (M) moderately susceptible, and (S) susceptible to anthracnose (Karunanayake 2008)

between the concentration of resorcinols in mango latex and the percentage (w/w) of the non-aqueous phase of mango latex (Hassan 2006). The concentration of the 5-substituted resorcinols decreased faster during ripening in cultivars like Tommy Atkins susceptible to Alternaria spot, than in less susceptible cultivars, such as Haden (Droby et  al. 1986). A similar trend was observed in two Sri Lankan cultivars where the resorcinols in the cultivar Willard susceptible to anthracnose declined faster during ripening than in the resistant Karutha Colomban (Karunanayake 2008).

1.2.2 Gallotannins Methanol extract of the mango fruit peel when bioassayed with Cladosporium cladosporioides or C. gloeosporioides produced a prominent inhibition zone at Rf. 0.00 (Fig.  1.3). The compounds responsible for inhibition were purified and identified as a mixture of three closely related gallotannins (Fig.  1.3). The three compounds vary in the number and the points of attachment of sugar molecules. Earlier 18 different gallotannins have been reported from the mango fruit peel and eight in the pulp (Berardini et  al. 2004). The phenolic compounds which were accounted for antibacterial activity, observed in mango seed (Kabuki et al. 2000), showed almost an identical elution profile to gallotannins (Berardini et  al. 2004). Young and mature leaves and florets of mango also contain gallotannins (Karunanayake 2008). The gallotannins are directly inhibitory to the anthracnose pathogen, C. gloeosporioides and the stem-end rot pathogen, Botryodiplodia theobromae. Antifungal activity due to gallotannins was higher in the peel of unripe fruit at harvesting maturity and declined gradually during ripening. By colour break stage, the antifungal activity had declined by about 20% from what it was at harvest. At the ripe stage, when anthracnose development occurred in cultivar Karutha Colomban, the antifungal activity had declined to about 50% of the initial level.

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Fig. 1.3  (a) Inhibition areas on thin layer chromatography bioassay plates produced by gallotannins in the methanol (left) and resorcinols in the dicholomethane extracts (right) of mango fruit peel. (b) Structure of the antifungal gallotannins

Mango cultivars more resistant to anthracnose such as Gira and Rata show greater gallotannin activity in their peel than more susceptible cultivars such as Kohu and Willard. The decline of gallotannin activity during ripening was greater in the more susceptible cultivars.

1.2.3 Chitinase Activity The peel of unripe mango fruit consists of a network of fine canals with latex which extends to the pedicel. At the abscission point of mango pedicel, the small canals tend to coalesce to form three or four large canals. It is from these that the latex flow spurts out when the fruit is removed from the tree. When the mango latex is removed from the fruit and allowed to settle, it separates into an aqueous and oily phase. The latex is toxic to the conidia of C. gloeosporioides, the causal agent of mango anthracnose. When exposed to the undiluted aqueous phase of mango latex, the conidia were gradually digested (Fig. 1.4). During early hours, a slight granulation was visible in the conidia and later the conidial wall was gradually dissolved. A gel diffusion assay carried out on glycol chitin-enriched agarose confirmed the presence of chitinase enzyme in the aqueous phase of the mango latex. Under UV light (365 nm), the areas hydrolyzed by chitinase appeared dark against blue fluoresced areas containing undigested glycol chitin (Fig.  1.5). Three chitinases with molecular weights 47, 87, and 97 KDa were present in the mango latex (Karunanayake 2008). The level of chitinase activity in the aqueous phase varied with the mango cultivar. Development of anthracnose (Fig. 1.6) and stem-end rot (Fig. 1.6) during ripening was significantly lesser in fruits from which latex was not drained off after harvest

B &

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Fig.  1.4  The state of conidia of C. gloeosporioides after different periods of exposure to the undiluted aqueous phase of mango latex

Fig. 1.5  Chitinase activity of the aqueous phase of mango latex, papaya latex (positive control), dilution buffer (negative control) and a commercial enzyme standard from S. marcescence

Fig. 1.6  Anthracnose (left) and natural stem-end rot (right) development in fruits of cultivar ‘Willard’ (anthracnose) and ‘Karutha Colomban’ (SER) from which latex was drained and not drained

than in fruits from which the latex was drained off. It was subsequently shown that the peel of unripe mango fruits from which latex was not removed after harvest had greater chitinase activity (Table  1.2) which could be the reason for greater fruit resistance in latex-retained fruit.

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Table 1.2  Chitinase activity in the fruit peel of cultivar ‘Willard’ when the latex was drained after harvest Chitinase activity in units/gram fresh weight tissue Treatment Day 1 after harvest Day 3 after harvest Latex retained fruits 0.48 ± 0.03 0.29 ± 0.02 Latex drained fruits 0.32 ± 0.08 0.22 ± 0.01 The values given in the table are the average of two independent trials.

1.2.4 Pre- and Postharvest Treatments Enhance Fruit Resistance and Antifungal Activity Exposure of harvested mangoes to CO2 at a flow rate of 100 mL of CO2/min for 24 h significantly reduced anthracnose development, however, the optimum effective dosage of CO2 varied according to cultivar, 20% CO2 for Keitt, 60% CO2 for Tommy Atkins. A concomitant significant increase of 5-(7,12-heptadecadienyl) resorcinol was also detected in Keitt fruits in response to the 30% CO2 treatment (Kobiler et al. 1998). Dipping fruit in hot water (55° C) for 5 min also resulted in an increase in 5-(7,12-heptadecadienyl) resorcinol. Dipping the unripe mangoes in a suspension of conidia of Colletotrichum magna, not pathogenic on mango, before inoculation with C. gloeosporioides, significantly delayed the anthracnose development. A significant increase in 5-(7,12-heptadecadienyl)resorcinol was also detected in mango fruits dipped in a conidia suspension of C. magna (Kobiler et  al. 1998). Peeling increased five substituted resorcinols in the flesh of peeled mango and the fruits became resistant to A. alternata infection (Droby et al. 1987). Inoculation of unripe fruits with C. gloeosporioides resulted in enhanced gallotannins (Sinnaih, Unpublished data) and the total soluble phenol content (Karunanayake 2008). Concurrent histo-chemical tests carried out on inoculated fruit peel at different time intervals supported the findings of chemical tests that tissue phenolics increased following infection. Free phenols can directly act as antimicrobial substances and be oxidized to form quinines which can inhibit extracellular enzymes of the pathogen (Mayer 1987). Chitinase activity increased in the peel following inoculation with C. gloeosporioides, however, whether the increased activity is due the same chitinases found in the latex could not be ascertained (Karunanayake 2008). A field trial was conducted to investigate the influence of soil potassium on postharvest rot development and natural disease resistance. Stem-end rot development was significantly less in mangoes from trees which received three times the annual recommended dose of potassium (2,055 g × 3) compared to those which received the annual recommended level of potassium (2,055 g) or no potassium (Table 1.3). The fact that the tissue potassium levels were higher in the peel tissues from fruits harvested from potassium-treated trees (16.24 and 12.38 ppm) than the control (11.68 ppm) may indicate that potassium enhances fruit resistance.

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Table 1.3  Stem-end rot in fruits harvested from trees which received different levels of potassium Lesion area (mm2) at different days after inoculation Day 1 Day 2 Day 3 Day 4 Day 5 K level (g) 3,873.7a 10,572a 0 0 0 558.7a a a 2,055 0 0 458.8 4,140.1 9,109a a b 2,055 × 3 0 0 248.7 2,394.7 6,834b a, b  Values followed by the same letter do not differ significantly at the 5% probability level (Duncan’s Multiple Range Test)

Gallotannin activity was in fact higher in peel of fruits which received greater potassium, the differences in gallotannin activity were, however, not significant.

1.3 Avocado (Persea americana) Fruit Anthracnose disease caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and the stem-end rot caused by Phoma spp., Botryodiplodia theobromae and Phomopsis spp. are recognized as major diseases in ripe avocado fruit. Young fruit are usually free from visible symptoms and characteristic decay lesions develop during fruit ripening. The anthracnose disease originates from quiescent infections in the immature fruit long before harvest (Binyamini and Schiffmann-Nadel 1972). In unripe fruit the fungus produces an appressorium, then an infection peg which ceases the growth in the cuticle (Coates et al. 1993) and becomes quiescent. The quiescence of C. gloeosporioides was attributed to the presence of substantial preformed antifungal activity in the immature fruit peel (Prusky et al. 1982; Sivanathan and Adikaram 1989). Avocado peel contains antifungal monoene, 1-acetoxy-2,4dihydroxy-n-heptadeca-16-ene (2) and diene, 1-acetoxy-2-hydroxy-4-oxo-heneicosa12,15-diene (1) (Prusky et al. 1982; Prusky et al. 1991) and three other compounds 1,2,4-trihydroxyheptadec-16-yne (3), 1,2,4-trihydroxyheptadex-16-ene (4) and 1-acetoxy-2,4-dihydroxyheptadec-16-yne (5) (Adikaram et al. 1992):

CH3(CH2)4CH=CHCH2CH=CH(CH2)7COCH2CHOHCH2OCOCH3

(1)



CH2=CH(CH2)11CHOHCH2CHOCH2OCOCH3

(2)



CH2=CH(CH2)11CHOHCH2CHOHCH2OH

(3)



CH º C(CH2)11CHOHCH2CHOHCH2OH

(4)



CH º C(CH2)11CHOHCH2CHOHCH2OCOMe

(5)

The most striking structural feature among the five antifungal compounds is the presence of a trihydroxy fragment which could be a precursor to these compounds (Adikaram et al. 1992). The levels of antifungal di-ene in peel of unripe avocados are subject to complex regulation and may be modulated by lipoxygenase, for which the di-ene is a

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substrate (Prusky et al. 1983), and also by the flavan-3-ol epicatechin, an inhibitor of lipoxygenase (Ardi et  al. 1998; Prusky et  al. 1982). Lipoxygenase activity increases during fruit ripening, while epicatechin levels decline, suggesting that these events are linked to the decrease in di-ene concentrations. In freshly harvested unripe avocado fruits, di-ene concentrations can be further enhanced by a variety of biotic and abiotic treatments including challenge with C. gloeosporioides, wounding, irradiation, exposure to ethylene (at levels that do not induce ripening) or carbon dioxide, and treatment with lipoxygenase inhibitors (Prusky et al. 1990; Prusky and Keen 1995; Prusky et al. 1985; Prusky et al. 1991; Prusky et al. 1996). Treatment with lipoxygenase inhibitors results in increased disease resistance (Prusky et al. 1985, 1991), offering potential strategies for the manipulation of fruit physiology for control of postharvest diseases. Interestingly, inoculation of freshly harvested avocado fruit with a non-pathogenic mutant strain of Colletotrichum magna also confers protection against C. gloeosporioides, possibly by the induction of epicatechin and modulation of the level of the antifungal di-ene (Prusky et al. 1994). Taken together, this evidence suggests that for the avocado-C. gloeosporioides interaction, preformed antifungal compounds may contribute to the resistance of unripe fruits to decay.

References Adikaram NKB, Brown AE, Swinburne TR (1982) Phytoalexin involvement in the latent infection of Capsicum annuum L. fruit by Glomerella cingulata (Stonem.). Physiol Plant Pathol 21:161–170 Adikaram NKB, Ewing DF, Karunaratne AM, Wijeratne EMK (1992) Antifungal compounds from immature avocado fruit peel. Phytochemistry 31(1):93–96 Ardi R, Kobiler I, Jacoby B, Keen NT, Prusky D (1998) Involvement of epicatechin biosynthesis in the activation of the mechanism of resistance of avocado fruits to Colletotrichum gloeosporioides. Physiol Mol Plant Pathol 53:269–285 Bandyopadhyay C, Gholap AS, Mamdapur VR (1985) Characterization of alkenylresorcinols in mango (Mangifera indica L.) latex. J Agr Food Chem 33:377–379 Berardini N, Carle R, Schieber A (2004) Characterization of gallotannins and benzophenone derivatives from mango (Mangifera indica L. cv. Tommy Atkins) peel, pulp and kernels by high performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Comm Mass Spectrom 18:2208–2216 Binyamini N, Schiffmann-Nadel M (1972) Latent infection in avocado fruit due to Colletotrichum gloeosporioides. Phytopathology 67:315–320 Coates LM, Muirhead IF, Irwin JAG, Gowanlock DH (1993) Initial infection process by Colletotrichum gloeosporioides. Mycol Res 97(1):1363–1370 Cojocaru M, Droby S, Glotter E, Goldman A, Gottlieb HE, Jacoby B, Prusky D (1986) 5-(12-heptadecenyl)-resorcinol, the major component of the antifungal activity in the peelof mango fruit. Phytochemistry 25(5):1093–1095 Droby S, Prusky D, Jacoby B, Goldman A (1986) Presence of antifungal compounds in the peel of mango fruits and their relation to latent infections of Alternaria alternate. Physiol Mol Plant Pathol 29:173–183 Droby S, Prusky D, Jacoby B, Goldman A (1987) Induction of antifungal resorcinols in unripe mango fruits and its relation to latent infection by Alternaria alternata. Physiol Mol Plant Pathol 30:285–292 Hall R (1971) Pathogenicity of Monilinia fructicola. Part II. Penetration of peach leaf and fruit. Phytopathologische zeitschrift 72:281–290

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Hassan KM (2006) Constitutive alk(en)ylresorcinols and resistance to postharvest diseases in mango (Mangifera indica L.). Ph.D. thesis, University of Queensland, Australia, pp 229 Hassan MK, Dann EK, Irving DE, Coates LM (2007) Concentrations of constitutive alk(en)ylresorcinols in peel of commercial mango varieties and resistance to postharvest anthracnose. Physiol Mol Plant Pathol 71:158–165 Kabuki T, Nakajima H, Arai M, Ueda S, Kawabara Y, Dosako S (2000) Characterization of navel antimicrobial compounds from mango (Mangifera indica) kernal seeds. Food Chem 71:61 Karunanayake KOLC (2008) Natural defence mechanisms in mango fruit and their potential in the management of postharvest diseases. Ph.D. Thesis, University of Peradeniya, Sri Lanka, pp 297 Knodler M, Berardini N, Kammerer RD, Carle R, Sciber A (2007) Characterization of major and minor alk(en)yl resorcinols from mango (Mangifera indica L.) peels by high-performance liquid chromatography/atomic pressure chemical ionization mass spectrometry. Rapid Comm Mass Spectrom 21:945–951 Kobiler I, Reved R, Artez L, Prusky D (1998) Antifungal compounds regulating postharvest diseases in mango. In: Johnson GI, Highley E, Joyce DC (eds) Disease resistance in fruit. ACIAR Proceedings No. 80, 109–114 Mayer AM (1987) Polyphenol oxidases in plants – recent progress. Phytochemistry 26(1):11–20 Muirhead IF, Deverall BJ (1981) Role of appressoria in latent infection of banana fruits by Colletotrichum musae. Physiol Plant Pathol 19:77–84 Oka K, Saito F, Yasuhara T, Sugimoto A (2004) A study of cross-reactions between mango contact allergens and urushiol. Contact Dermatitis 51:292–296 Osbourn AE (1996) Saponins and plant defence – a soap story. Trends Plant Sci 1:4–9 Prusky D, Keen NT (1993) Involvement of preformed antifungal compounds in the resistance of subtropical fruits to fungal decay. Plant Dis 77:114–119 Prusky D, Keen NT (1995) Inducible preformed compounds and their involvement in the resistance of plants to pathogens. In: Reuveni R (ed) Novel approaches to integrated pest management. Lewis Publishers, Boca Raton, FL, pp 139–151 Prusky D, Keen NT, Sims JJ, Midland SL (1982) Possible involvement of an antifungal diene in the latency of Colletotrichum gloeosporioides on unripe avocado fruit. Phytopathology 72:1578–1582 Prusky D, Keen NT, Eaks I (1983) Further evidence for the involvement of a pre-formed antifungal compound in the latency of Colletotrichum gloeosporioides on unripe avocado fruits. Physiol Mol Plant Pathol 22:189–198 Prusky D, Kobiler I, Jacoby B, Sims JJ, Midland SL (1985) Inhibitors of avocado lipoxygenase; their possible relationship with the latency of Colletotrichum gloeosporioides. Physiol Mol Plant Pathol 27:269–279 Prusky D, Karni L, Kobiler L, Plumbley RA (1990) Induction of the antifungal diene in unripe avocado fruits: effect of inoculation with Colletotrichum gloeosporioides. Physiol Mol Plant Pathol 37:425–435 Prusky D, Kobiler I, Fishman Y, Sims JJ, Midland SL, Keen NT (1991) Identification of an antifungal compound in unripe avocado fruits and its possible involvement in the quiescent infections of Colletotrichum gloeosporioides. J Phytopathol 132:319–327 Prusky D, Freeman S, Rodriguez R, Keen NT (1994) A nonpathogenic mutant strain of Colletotrichum magna induces resistance to Colletotrichum gloeosporioides in avocado fruits. Mol Plant Microbe Interact 7:326–333 Prusky D, Wattad C, Kobiler I (1996) Effect of ethylene on the activation of quiescent infection of Colletotrichum gloeosporioides in avocado fruits. Mol Plant Microbe Interact 9:864–868 Schonbeck F, Schlosser E (1976) Preformed substances as potential protectants. In: Heitefuss R, Williams PH (eds) Physiological Plant Pathology. Springer-Verlag, Berlin, pp 653–678 Sivanathan S, Adikaram NKB (1989) Biological activity of four antifungal compounds in immature avocado. J Phytopathol 125:97–109 VanEtten HD, Mansfield JW, Bailey JA, Farmer EE (1994) Letter to the editor. Two classes of plant antibiotics: phytoalexins versus phytoanticipins Plant Cell 6:1191–1192

Chapter 2

Mechanisms of Induced Resistance Against B. cinerea Tesfaye Mengiste, Kristin Laluk, and Synan AbuQamar

Abstract  Botrytis cinerea is a widespread pre-and postharvest pathogen of diverse crops. Current crop protection methods rely on fungicide application and on horticultural practices. Variation for genetic resistance is documented in many crop plant species but has not been utilized. Studies in model and crop plant species are revealing the biological processes that underlie plant responses to infection to B. cinerea. The genetic control of pathogen recognition and activation of defense to restrict pathogen ingress and colonization is likely to emerge from such studies. Deeper understanding of resistance mechanisms and their genetic control will aid produce cultivars with genetic resistance to B. cinerea. The genetic components of induced resistance in different plant species and future implications are discussed. Keywords  Botrytis cinerea • genetic resistance • necrotrpohic pathogens • induced resistance

2.1 Introduction B. cinerea is a ubiquitious fungal pathogen with relative host unspecificity primarily attacking dicot plants but also some monocot species. The fungus causes the gray mold disease resulting in significant crop losses under different production conditions. Gray mold occurs over a wide geographical area, in the open field, in greenhouses and even in storages at 0–10°C. B. cinerea is the principal cause of pre- and postharvest disease in grapes, berries, tomatoes and many other crops (Coley-Smith et al. 1980; Williamson et al. 1995; Elad 1997). In grapes, where B. cinerea causes “bunch rot”, the estimated loss to the vineyard can amount to

T. Mengiste (*), K. Laluk, and S. AbuQamar Department of Botany and Plant Pathology, Purdue University, 915 W. State street, West Lafayette, IN 47906, USA e-mail: [email protected] D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_2, © Springer Science + Business Media B.V. 2009

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15–40% of the harvest depending upon the season. The losses to strawberries and cut flowers have been estimated at 10–20% (Legard et al. 2000). B. cinerea is regarded as an expensive pathogen because of the qualitative and quantitative crop losses it causes and because of its demand for high fungicide treatment. B. cinerea also develops fungicide resistance, limiting chemical crop protection options. Chemical protection is also discouraged due to public safety associated with fungicide residues on fresh produce. Consequently, there is an increased effort to identify genetic resistance. Genetic resistance provides cost-effective and sustainable plant protection. However, no robust genetic resistance against B. cinerea has been identified in crop plants. There is also a very limited understanding of the biological processes underlying plant responses to necrotrophic pathogens in general and B. cinerea in particular. The biology of B. cinerea has been studied extensively and the genome of the fungus has been sequenced (Elad et  al. 2004; van Kan 2006). The critical factors in B. cinerea pathogenesis, pathogen derived effectors, the molecular events associated with infection processes, infection related morphogenesis, and factors that confer the relative host unspecificity are still not fully understood. Equally unknown are the critical factors in plant disease resistance mechanisms in different plant species. In this chapter we will highlight recent progress made towards understanding of plant induced resistance to B. cinerea.

2.2 Plant Resistance to B. cinerea Plant resistance to diseases is controlled by a multitude of environmental as well as host and pathogen genetic factors that vary depending on pathogen-host combinations. Figure 2.1 summarizes B. cinerea derived disease and/or defense elicitors and the corresponding plant defense mechanisms. Necrotrophic pathogens in general and B. cinerea in particular have evolved infection strategies to breach plant defenses (Prins et  al. 2000a). These infection strategies involve the secretion of diverse chemical compounds before and during colonization. Some necrotrophic fungi are host specific, producing toxins that promote chlorosis and host cell death only in their hosts (host specific toxins, HSTs) (Wolpert et al. 2002). Many necrotrophic fungi including B. cinerea are host unspecific and produce host non-sepecific toxins. There is no HST identified from B. cinerea consistent with the host unspecificity of the pathogen. B. cinerea produces botrydial, a non-host-specific toxin implicated in the intitation and severity of disease (Colmenares et al. 2002). In addition, B. cinerea produces cell wall degrading enzymes, other extracelluar enzymes, oxalic acid, and reactive oxygen intermediates to promote disease and macerate plant tissues (Prins et al. 2000b). These infection strategies differ from obligate pathogens that suppress plant defenses through subtle mechanisms. B. cinerea promotes or benefits from host cell death during pathogenesis. Excellent reviews have recently been published on the pathogenesis of B. cinerea (van Kan 2006; Williamson et al. 2007). Plants also have counter defense mechanisms that are built as layers of constitutive and inducible resistance strategies. Broadly, plant defense is composed of primary

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Fig. 2.1  A generalized scheme showing major factors involved in the development of the gray mold disease by B. cinerea and corresponding components of plant defense. Prior to recognition of B. cinerea derived PAMPs and the activation of defense, the cuticle and the plant cell wall fend off infection. These are also targets of B. cinerea virulence by lipases, cutinases and the plant cell wall degrading enzymes. Plant defense is depicted as layers starting from the leaf epidermal cells protected by the cuticle, cell wall, membrane localized PRRs that trigger gene expression and the accumulation of molecules that confer resistance through diverse activities. Receptor mediated recognition is not defined in the case of B. cinerea and plant interactions. OG-R, oligogalacturonide receptors; PGIP, polygalacturonase-inhibiting protein; PRRs, pattern recognition receptors; ROIs, reactive oxygen species; PAMPs, pathogen associated molecular patterns

line of defense at the cuticular membrane, plant cell wall based defenses and those activated upon recognition of pathogen derived molecules by membrane localized pattern recognition receptors. It is also possible that plants recognize pathogen infection because of the stress, toxins and other pathogenesis events. Recognition mediated activation of defense controls the production of defense compounds. Plant survival under pathogen assault is a result of the combined effect of the various layers on the pathogen making the plant environment inhospitable to the pathogen. These include diverse phytoalexins, phytoanticipins, and antimicrobial peptides and antibiotics. Resistance to necrotrophs producing HSTs can be conditioned by single genes that neutralize the toxin or encode altered proteins not to be recognized by the toxin (Wolpert et al. 2002). Thus, host resistance mechanisms to biotrophic and necrotrophic pathogens differ significantly and resistance to host unspecific necrotrophs is predicted to be complex, requiring the involvement of many genes and pathways for full resistance. Genetic studies in model and crop plant species have defined some of the major plant defense pathways and their components that regulate resistance to B. cinerea at the various layers of defense (Table 2.1). Some of these defense components are discussed throughout this chapter.

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Table 2.1  Plant genes implicated in the regulation of responses to B. cinerea based on increased or decreased diseases resistance of mutants or transgenic plants Mutant Nature of protein Reference Epidermis-specific extracellular (Kurdyukov et al. 2006) Bodyguard, bdg a/ß-hydrolase fold protein Botrytis Resistant1 (bre1) Long-chain acyl-CoA synthetase 2 (Kurdyukov et al. 2006) (LACS2) PGIP1/2 Polygalacturonase-inhibiting proteins (Ferrari et al. 2003b) Pad2 Gamma-glutamylcysteine synthetase (Ferrari et al. 2003a) ) Pad3 Cytochrome P450 monooxygenase (Ferrari et al. 2003a); Coi1/Jai1 Coronatine insesitive1, JA receptor (Thomma et al. 1999; Li et al. 2004) Jasmonic acid insensitive1, MYC2 transcription factor (NICKSTADT et al. 2004) JIN1 Jasmonic acid resistant1, Adenylation of jasmonic acid (Ferrari et al. 2003a) JAR1 JMT Jasmonic acid carboxyl (Seo et al. 2001) methyltransferase (Bonaventure et al. 2007a) Fatty acid oxygenation Ca2+-permeant non-selective cation channel upregulated2, FOU2 AOS Alene oxide synthase (Bonaventure et al. 2007a) EIN2 ET signaling, Ethylene insensitivity (Thomma et al. 1999) Ethylene-response-factor1 GCC-box-binding protein (Berrocal-Lobo et al. 2002) Ethylene receptor1 Ethylene receptor (Ferrari et al. 2003a) Weak ethylene-insensitive5, EIN3-related transcription factor (Alonso et al. 2003) wei5 Suppressor of SA Stearoyl-ACP desaturase (Kachroo et al. 2001) insensitivity2 Botyrtis induced kinase 1 Ser/Thr receptor-like kinase (Veronese et al. 2006b) WRKY33, 70 WRKY transcription factors (AbuQamar et al. 2006; Zheng et al. 2006) Botrytis R2R3MYB transcription factor (Mengiste et al. 2003) susceptible1(BOS1) Botrytis susceptible 2,3,4 BOS2-BOS4 genes not identified (Veronese et al. 2004) Defense no death, DND1 Cyclic nucleotide gated ion channel (Govrin and Levine 2000) Enhanced disease EDR3 encode dynamin-related (Tang et al. 2006) resistance protein 1E Overexpressor of cationic Homeodomain transcription factor (Coego et al. 2005) peroxidase3, OCP3 TPK1b Ser/Thr receptor-like kinase (Abuqamar et al. 2008) SPR2 Tomato fatty acid desaturase (Li et al. 2003) Acx1 JA deficient, b-oxidation (Li et al. 2005) Sitens Abscisic acid-deficient (Audenaert et al. 2002) (Flors et al. 2007) Cel1, cel2 Tomato endo b-1,4-glucanase Notes: Arabidopsis OCP3, FOU2, JIN1, LACS2 and tomato Cel1, Cel2 mutations confer increased resistance whereas the other mutations cause susceptibility. Lesion mimic mutations that result in B. cinerea susceptibility due to the precocious cell death are excluded from this table. This table includes data from mutants for which B. cinerea disease have been performed and excludes many mutants that are susceptible to other necrotrophs and may also show susceptibility to B. cinerea

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2.3 The First Line of Defense Against B. cinerea Recent studies illuminate differences that underpin host responses to pathogens depending on pathogen life style. The first line of defense against pathogen attack, regardless of the type of the invader, are the barriers provided by the plant cuticle and cell wall. Initiation of infection and pathogen ingress may be halted by such structural defenses. The susceptibility of the plant cell wall to degradation by cell wall-hydrolyzing enzymes can affect the severity of disease caused by B. cinerea. Fungal polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors for some necrotrophic fungi, whereas the plant polygalacturonase-inhibiting proteins (PGIPs) contribute to resistance by counteracting fungal PGs (Powell et al. 2000; Ferrari et al. 2003b). Furthermore, attacked plants build up papillae on the inner side of epidermal cell walls at the point of attempted pathogen entry. Papillae are largely composed of callose (a b-1,3-glucan) but also contain polysaccharides, phenolic compounds and reactive oxygen intermediates (Flors et al. 2005). Contrary to intuitive expectations, defects in plant secondary cell wall caused by a mutation in cellulose synthesis resulted in resistance to necrotrophic pathogens (Hernandez-Blanco et al. 2007). Necrotrophic fungi target the plant cell wall for degradation, and the strength of the wall to such attack directly contributes to resistance. Recent data suggest that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens (Flors et al. 2007; Cantu et al. 2008). In tomato, inhibition of expansin and polygalacturonases (PGs) involved in the disassembly of the plant cell wall during fruit ripening resulted in reduced cell wall thickness and decreased the susceptibility of the fruits to B. cinerea, supporting the cell wall being an important virulence target for B. cinerea (Cantu et al. 2008). Absence of the endo-b-1,4-glucanases Cel1 and Cel2 in tomato reduces susceptibility to B. cinerea. Thus, cell wall-based defense mechanisms can decrease or enhance pathogen resistance. Traditionally, the cuticle is considered to serve as protection against abiotic stresses and a barrier to fungal infection. The plant cuticle protects against pathogen penetration and hence prevents the pathogen from establishing infection. Unexpectedly, Arabidopsis mutants defective in components of the cuticle were resistant to B. cinerea attributed to loss of virulence in the fungus in the absence of cuticle-derived signals (Kurdyukov et al. 2006; Chassot et al. 2007). Mutation in LACS2, a gene required for cutin biosynthesis, leads to altered cuticle development in Arabidopsis and, interestingly, also leads to resistance to B. cinerea infection (Bessire et  al. 2007; Tang et  al. 2007). Cutinase expressing as well as the body guard Arabidopsis mutant plants, which is unable to form a continuous cuticular membrane, is also more resistant to infection with this fungus (Chassot et al. 2007; Bessire et  al. 2007). It was argued that increased cuticle permeability facilitates resistance to necrotrophic infection by allowing diffusion of defense signals and effectors across the plant surface leading to an altered perception of fungal elicitors of defense (Bessire et al. 2007; Chassot et al. 2007). This perception leads to accumulation of antifungal compounds on the leaf surface. The degree of permeability

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directly correlates with the amount of antifungal compounds released at the site of infection and hence the degree of resistance.

2.4 Active Defense Against Botrytis cinerea Contrasts with Resistance to Biotrophic Pathogens Pathogen invasion activates diverse defense responses that have varying efficiencies and specificities in disease resistance. Race-specific resistance is predominantly effective against biotrophic pathogens (Jones and Dangl 2006). Pathogen produced effector proteins are directly or indirectly recognized by the corresponding plant resistance proteins to trigger the hypersensitive response (HR) which restricts further pathogen ingress. There is no precedence for such recognition based resistance to B. cinerea. The fungus causes necrotic cell death in plant cells that are remarkably similar to the HR cell death (Govrin and Levine 2000). Thus, cell death is a hall mark of race specific resistance but can be a typical disease symptom in plants infected with B. cinerea. R-gene mediated HR facilitates infection by B. cinerea (Govrin and Levine 2000, 2002). Cell death resulting from resistance responses or other physiological functions facilitates B. cinerea infection (Kachroo et al. 2001; Veronese et al. 2004b). Dead cells and necrotic tissues are sources of leaked nutrients and provide saprophytic growth base from which B. cinerea further colonizes healthy tissue. Accordingly, the expression of animal antiapoptotic genes in tobacco inhibits HR cell death and enhances resistance to B. cinerea (Dickman et al. 2001). No R-gene has been implicated in resistance to B. cinerea or other necrotrophic pathogens. Interestingly, an Arabidopsis R-gene that mediates susceptibility to the necrotrophic fungal pathogen Cochliobolus victoriae has been described (Lorang et  al. 2007). Mutations in genes mediating signaling downstream of Arabidopsis R genes, eds1 or ndr1, show no altered resistance to B. cinerea (Ferrari et al. 2003a). Systemic acquired resistance (SAR) is an active defense intiated by infection with certain necrotizing pathogens and confers resistance to secondary infection. SAR is effective against a broad-spectrum of pathogens including viruses, bacteria, fungi and oomycetes (Ryals et al. 1996; Sticher et al. 1997). Inhibition of salicylic acid (SA) accumulation or biosynthesis impairs SAR (Gaffney et al. 1993; Nawrath and Metraux 1999). In Arabidopsis, the biological and chemical induction of SAR failed to inhibit B. cinerea growth (Govrin and Levine 2002). Mutants impaired in the induction of SAR are not altered in B. cinerea resistance. Similarly, the HR inhibits a secondary infection by biotrophic pathogens by causing SAR in systemic tissues but facilitates infection by B. cinerea (Govrin and Levine 2002). Arabidopsis mutants that form spontaneous lesions and constitutively express SAR show increased susceptibility to B. cinerea (Kachroo et al. 2001). SA was associated with resistance at the point of infection in Arabidopsis tissue (Ferrari et al. 2003a). In tomato, benzothiadiazole (BTH), the chemical inducer of SAR, induced restance to B. cinerea (Audenaert et  al. 2002). Multiple pre-harvest treatments of grapevine with BTH enhanced trans-resveratrol content (by about 40-fold) and induced SAR

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in grapevine berries. The percentage of infected berries per cluster was significantly reduced in grapes from BTH-treated plants suggesting that BTH treatments could be exploited in vineyards to protect grapes against gray mold (Iriti et al. 2004). In strawberry plants treated with 0.25–2 mg/mL BTH, the development of gray mold was delayed by about 2 days on the harvested strawberry fruit at 5°C. This delay was equivalent to a 15–20% increase in storage life of the fruit suggesting that chemical plant activators could control grey mold on strawberry fruit and grapes (Leon and Joyce 2000). Thus, the available data point to the potential of SAR against B. cinerea in various crop plants despite evidence from Arabidopsis that show the failure of SAR in restricting B. cinerea. Induced systemic resistance (ISR) resembles SAR but is induced by root colonization of specific strains of nonpathogenic plant growth-promoting rhizobacteria in contrast to SAR that is induced by necrotizing pathogens. Unlike SAR, ISR is dependent on jasmonate and ethylene, independent on SA and not associated with PR gene expression (Pieterse et al. 1996); (van Loon et al. 1998). Molecularly, both SAR and ISR are intertwined through the Arabidopsis NPR1 gene. In Arabidopsis, the root-colonizing bacteria Pseudomonas fluorscens establishes ISR against B. cinerea (De Meyer et al. 1998; Ton et al. 2002).

2.5 The Plant Hormones Jasmonate and Ethylene Play a Central Role in Defense Against B. cinerea Most studies implicate the plant hormones jasmonic acid (JA) and ethylene (ET) as the key regulators of defense against necrotrophic pathogens such as B. cinerea (Glazebrook 2005). In Arabidopsis, pathogen infection and exogenous application of JA and/or ET induce defense gene expression including those encoding plant defensins and thionins (Epple et al. 1995, 1997); (Penninckx et al. 1996; Penninckx et al. 1998). The plant defensin PDF1.2 and the tomato protease inhibitor 2 (PI-2) genes are induced by B. cinerea and MeJA. These inductions require intact JA/ET or JA pathways in these plants (Thomma et al. 1998, ref). In addition, in both plant species, the JA receptor mutants coi1/jai1 fail to induce PDF1.2/PI-2 gene expression and exhibit enhanced susceptibility to B. cinerea (Penninckx et al., 1998; Li et al., 2004. Arabidopsis mutations blocking JA signaling and biosynthesis including allene oxide synthase (aos), jasmonic acid resistant (jar1), jasmonate insensitive4 (jin4), fatty acid desaturase (fad3/fad7/fad8) display increased susceptibility to B. cinerea and other necrotrophs (Thomma et al. 1998; Stintzi et al. 2001; Ferrari et al. 2003a). Additionally, over-expression of a jasmonic acid carboxyl methyltransferase (JMT) in Arabidopsis results in enhanced and constitutive JA responses as well as resistance against B. cinerea (Seo et  al. 2001). JMT catalyzes the production of methyl-jasmonate (MeJA) from JA, demonstrating that elevated levels of MeJA can confer resistance to necrotrophic pathogens possibly aiding in defense signal transduction (Seo et  al. 2001). Two Arabidopsis gain of function mutants, jasmonate insensitive1 (jin1) and fatty acid oxygenation upregulated2 (fou2), exhibit enhanced

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resistance to B. cinerea (Lorenzo et  al. 2004; Bonaventure et  al. 2007b). JIN1 encodes a transcription factor involved in the regulation of JA-inducible genes that are involved in pathogen as well as wounding defense responses (Lorenzo et  al. 2004). Mutation in FOU2 alters cation fluxes involved either directly or indirectly in the feed-back regulation of JA synthesis (Bonaventure et  al. 2007b). Overall, phenotypic analysis of mutants altered in JA accumulation and signaling clearly establishes JA as an important regulator of plant defense responses to B. cinerea. Similarly, genetic data from various plant species show ET to be a regulator of defense to B. cinerea infection. In Arabidopsis, ET acts in concert with jasmonate. ET insensitive mutants, etr1 and etr2, in soybean are severely susceptible to the necrotrophic pathogens Septoria glycines and Rhizoctonia solani (Hoffman et al. 1999). Arabidopsis ethylene insensitive (ein2) mutant plants which are blocked ET signaling show increased susceptibility to B. cinerea (Thomma et al. 1998; NormanSetterblad et al. 2000; Ferrari et al. 2003a). Some evidence also suggests that ET promotes disease caused by some fungal pathogens indicating that its role varies among different pathosystems (Lund et al. 1998; Ciardi et al. 2000; van Loon et al. 2006). ET induces fruit ripening as well as plant senescence, developmental processes linked to increased colonization by B. cinerea. Thus, ET may increase plant susceptibility or resistance to B. cinerea depending on the infected plant species and the kind of infected tissue (van Loon et al. 2006; Elad 1993; Hoffman et al. 1988). Interestingly, exposure of B. cinerea to ET in  vitro causes a reduction in growth and numerous transcriptional changes (Chagué et  al. 2006). However, expression of a putative pathogenicity gene was enhanced in B. cinerea growing on ET-producing plants compared to expression during infection on ET non-producing plants (Chagué et al. 2006). Additionally, in vitro and during infection, the fungus produces ET that is required for growth and sporulation through its own ET synthesis pathway. However, the amount of ET produced by B. cinerea during plant colonization is significantly reduced compared to the levels produced in vitro suggesting a suppression of biosynthesis either by the fungus or the host. Overall, the timing of plant inoculation plays a major role in determining whether ET may increase or decrease plant resistance to necrotrophic infection. Although it has been clearly established that ET generally aids in resistance, exactly what function ET production serves on the side of the pathogen during host interaction remains unclear.

2.6 The Emerging Role of Absciscic Acid as a Regulator Plant Response to B. cinerea The plant stress hormone, absciscic acid (ABA), regulates plant responses to pathogens. ABA regulated closure of stomata leads to decreased bacterial entry (Melotto et  al. 2006). There is no evidence for a defense based on the control of stomatal opening for B. cinerea or other necrotrophic fungi. Recently, ABA has emerged as a positive or negative regulator of disease resistance depending on the nature of the host-pathogen interaction (Anderson et al. 2004; Lorenzo et al. 2004; Mauch-Mani

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and Mauch 2005). Exogenous application of ABA caused susceptibility to B. cinerea (Audenaert et  al. 2002). ABA-deficiency in tomato and impaired ABA responses in Arabidopsis result in increased resistance to B. cinerea and other necrotrophic pathogens due to reduced ABA-signaling but increased JA- or ET-responsive gene expression (Audenaert et al. 2002). The enhanced response to ABA3 (ERA3) gene is allelic to EIN2 (Ghassemian et al. 2000), which is required for resistance to B. cinerea (Thomma et  al. 1999). In addition, resistance to the necrotrophic oomycete Pythium irregulare and to the bacterial necrotroph Ralstonia solanacearum requires ABA synthesis and responses indicating a positive role for ABA in disease resistance (Adie et  al. 2007b; Hernandez-Blanco et  al. 2007). Interestingly, ABA is also implicated as a positive signal required for plant resistance to Pythium irregulare and initiates callose biosynthesis, a defense response to certain pathogens (Flors et al. 2005; Adie et al. 2007a). On the other hand, mutations in ZFAR1, encoding a putative zinc-finger protein with ankyrin-repeat domains, cause sensitivity to ABA and local susceptibility to B. cinerea (AbuQamar et  al. 2006). Additionally, B. cinerea contains a gene cluster involved in the synthesis of ABA; however the involvement and impact of fungal produced ABA on virulence remains to be elucidated (Siewers et al. 2006).

2.7 Resistance to B. cinerea and Cross-Talk with Other Pathways Arabidopsis exhibits basal resistance to B. cinerea. This basal resistance was used as the basis for forward and reverse genetic screens to identify genes regulating basal resistance to B. cinerea (Mengiste et al. 2003). The BOS1 (Botrytis Susceptible1) gene is required to restrict the growth of B. cinerea in inoculated plants. Strikingly, bos1 plants have impaired tolerance to water deficit, increased salinity, and oxidative stress. BOS1 encodes an R2R3MYB transcription factor protein, and our results suggest that it mediates responses to signals, possibly mediated by reactive oxygen intermediates, from both biotic and abiotic stress agents. In addition, three B. cinerea susceptible mutants bos2, bos3, and bos4 defining independent genetic loci required for Arabidopsis resistance to B. cinerea were described (Veronese et al. 2004a). The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B. cinerea. The bos3 and bos4 mutants also show increased susceptibility to A. brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance. PR-1, a molecular marker of the SA-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge. In bos3, the mutant most susceptible to B. cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change in response to B. cinerea. Expression of the plant defensin gene PDF1-2 was generally lower in bos2, bos3 and bos4 mutants compared to wild-type plants,

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with a particularly strong reduction in bos3. Production of the phytoalexin camalexin is another well-characterized plant defense response. The bos2 mutant accumulates reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B. cinerea. These three mutants mediate disease responses through mechanisms independent of the BOS1. Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways. Similarly, through reverse genetic searches for genes affecting resistance to B. cinerea, we isolated other Arabidopsis mutants showing increased susceptibility to B. cinerea. Among these, the B. cinerea Induced Kinase 1 (BIK1) and the WRKY33 transcription factor genes have been described recently (Veronese et  al. 2006; Zheng et al. 2006). BIK1 is a positive regulator of resistance to B. cinerea (Veronese et al. 2006). Inactivation of BIK1 caused severe susceptibility to B. cinerea and A. brassicicola. In contrast, bik1 plants display enhanced resistance to the virulent strain of the bacterial pathogen P. syringae, with a significant reduction of bacterial growth and total absence of disease symptoms. JA- and ET-regulated defense response pathways, generally associated with resistance to necrotrophic fungi, are attenuated in bik1 as measured by the expression of the plant defensin PDF1-2 gene transcript. Similarly, the Arabidopsis WRKY33 gene, encoding transcription factor, regulates the antagonistic interactions between defense pathways (Zheng et  al. 2006). Mutations of the Arabidopsis WRKY33 gene cause enhanced susceptibility to B. cinerea, concomitant with reduced expression of PDF1-2. Over-expression of WRKY33, on the other hand, increases resistance to the two necrotrophic fungal pathogens. The wrky33 mutants do not show altered responses to a virulent strain of the bacterial pathogen P. syringae, although the ectopic expression of WRKY33 results in enhanced susceptibility to this pathogen. The susceptibility of plants expressing WRKY33 to P. syringae is associated with reduced expression of the salicylate-regulated PR-1 gene. Thus, WRKY33 is an important transcription factor that regulates the antagonistic relationship between defense pathways mediating responses to P. syringae and B. cinerea.

2.8 Mechanisms of B. cinerea Resistance Are Conserved Between Tomato and Arabidopsis The predominant mechanism of B. cinerea resistance appears to be conserved between tomato and Arabidopsis. Tomato mutants impaired in ET, JA, ABA responses or synthesis pathways mirror the B. cinerea susceptibility of the Arabidopsis mutants. This is encouraging for the possible transfer of resistance between the two species. However, there is also not clear data elaborating why Arabidopsis and tomato show significant differences in the rate of disease development. The rate of B. cinerea disease development is significantly faster in tomato as compared to Arabidopsis (unpublished observations). Recently, from searches for regulatory genes that control B. cinerea resistance, we isolated the tomato protein kinase 1 (TPK1b) gene encoding a receptor-like

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cytoplasmic kinase that is localized to the plasma membrane. Pathogen infection, mechanical wounding and oxidative stress induce expression of TPK1b, indicative of a role in mediating responses to diverse signals. Reduction of TPK1b gene expression through RNA interference (RNAi) increases the susceptibility of tomato plants to colonization by the necrotrophic fungus B. cinerea. Interestingly, TPK1b RNAi plants were also susceptible to feeding by larvae of tobacco hornworm (Manduca sexta). Notably, susceptibility to B. cinerea and insect feeding is correlated with reduced expression of the proteinase inhibitor II (PI-II) gene in response to B. cinerea and 1-aminocyclopropane-carboxylic acid (ACC), the natural precursor of ET, but wild type expression in response to mechanical wounding and methyl-jasmonate (MeJA). TPK1b RNAi seedlings are impaired in ET responses suggesting that B. cinerea induces PI-II gene expression through a TPK1b-dependent ET signaling pathway. TPK1b is a functional kinase with autophosphorylation and MBP (Myelin Basis Protein) phosphorylation activities. Thus, TPK1b is a key signaling component in defense response to necrotrophic fungi and herbivorous insects as well as ET mediated defense against pathogens. TPK1b is a functional kinase that localizes to the plasma membrane suggesting that TPK1b acts early in pathogen and insect response pathway and functions in recognition and/or signaling. The impaired disease and insect resistance in TPK1b RNAi plants is accompanied by altered ET and B. cinerea induced defense gene expression, supporting the role of TPK1b in ET-mediated defenses. ET integrates plant responses to developmental and environmental signals, including responses to pathogens (Klee 2004). The function of TPK1b suggest that plants deploy multiple and overlapping mechanisms of defense against necrotrophic pathogens and chewing insects, despite the disparate strategies of these organisms in deriving nutrition from plants. Interestingly, ectopic expression of TPK1b rescues the phenotype of the Arabidopsis bik1 mutant. TPK1b and BIK1 are not orthologs but perform partially redundant functions. The susceptibility of TPK1b RNAi plants to tobacco hornworm coupled with the lack of ET responses suggest that TPK1b mediated ET responses are required for defense against pathogens and insect pests. The attenuated PI-II gene expression in TPK1b RNAi plants is also consistent with the action of ET in parallel with the octadecanoid/wounding pathway for full PI-II gene expression (O’Donnell et al. 1996). Mutations causing ET insensitivity in soybean (Glycine max L.) and Arabidopsis, and inhibition of ET perception in tomato result in increased susceptibility to necrotrophic pathogens (Hoffman et  al. 1999; Thomma et al. 1999; Diaz et al. 2002). Multiple lines of evidence suggest that responses to insects and necrotrophic pathogens are mechanistically linked in tomato (McCormick 1991). B. cinerea induces wound-like responses including the expression of the wound response gene PI-II, possibly through the actions of oligogalacturonides released from plants cell walls by the action of fungal endopolygalacturonases. The tomato JA and wound response mutants show susceptibility to B. cinereaand their impaired resistance to tobacco hornworm (M. sexta) or spider mites (Tetranychus urticae) (Schilmiller and Howe 2005) demonstrate a clear overlap in the mechanisms of resistance to necrotrophic fungi and chewing insect pests. These overlapping responses are mediated in part by JA levels and signaling. JAI1, SPR2, ACX1, and DEF1 are

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required for basal resistance of tomato to B. cinerea. The acx1 mutant is JA-deficient and lacks local and systemic expression of defensive PI genes in response to wounding due to a defect in the first step of ß-oxidation in the octadecanoid pathway (Li et al. 2005). SPR2 is a fatty acid desaturase required for the synthesis of JA and the generation of a systemic wound signal mediating defense gene expression in tomato (Li et al. 2003). The def1 and jai1 mutants are impaired in JA responses and both show susceptibility to B. cinerea (Li et al. 2002; Abuqamar et al. 2008). Thus, these genes in the JA pathway are part of defense against B. cinerea and our findings suggest that B. cinerea resistance mechanisms regulated by JA are generally conserved between Arabidopsis and tomato.

2.9 Phyotalexins in B. cinerea Resistance The role of induced chemical compounds in relation to B. cinerea resistance has been studied in various plant species (Elad 1997). The accumulation of phyotalexin has been linked to B. cinerea resistance (Prins et al. 2000b). Grape plants accumulate phyotalexins, mainly stilbenes such as trans-and cis-resveratrol, a-and e-viniferin, and pterostilbene in response to infection by B. cinerea (Thomzik et  al. 1997). Over-expression of the phyotalexin biosynthesis enzymes stilbene synthase involved in the synthesis of the phytolalexins stilbene and resveratol results in resistance to B. cinerea in tobacco and tomato (Hain et al. 1993; Thomzik et al. 1997). In Arabidopsis, the phytoalexin camalexin accumulates in response to infection by B. cinerea, and reduced or delayed induction of camalexin results in increased susceptibility to the pathogen (Tierens et al. 2002; Ferrari et al. 2003a; van Wees et al. 2003). The synthesis of camalexin, the major phytoalexin in Arabidopsis, is regulated by the MPK3/MPK6 cascade (Ren et al. 2008). Induction of camalexin by B. cinerea was preceded by MPK3/MPK6 activation, and compromised in mpk3 and mpk6 mutants. MPK3/MPK6 controls the Phytoalexin Deficient 2 (PAD2) and PAD3 genes indicating that the MPK3/MPK6 cascade regulates camalexin synthesis through transcriptional regulation of the biosynthetic genes.

2.10 Changes in Genome Wide Gene Expression During B. cinerea Infection To determine the nature of the B. cinerea induced defense transcriptome and identify genes involved in host responses against the pathogen infection, the expression profiles of B. cinerea inoculated Arabidopsis plants were studied (AbuQamar et al. 2006). Wild type Arabidopsis plants showing basal resistance were compared to coi1, ein2, and nahG plants that represent defects in various defense responses and/ or show increased susceptibility to B. cinerea. In wild type plants, the expression

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of an average of 621 genes representing roughly 0.48% of the Arabidopsis transcriptome was induced by >twofold. B. cinerea induced genes (BIGs) encode diverse regulatory and structural proteins implicated in defense, abiotic and oxidative stress responses. The coi1 mutation affected the B. cinerea induced expression of the largest set of these BIGs consistent with its enhanced B. cinerea susceptibility. The ein2 and nahG plants, impaired in ET signaling and SA accumulation, respectively, affected the expression of 63 and 80 BIGs, respectively. Forty BIGs encode putative DNA binding proteins that belong to ET response, Zinc finger, MYB, WRKY and HD-ZIP family transcription factor proteins. The transcriptional activation of genes involved in cell death, removal of reactive oxygen species, plant hormone signaling and synthesis during B. cinerea infection coupled with the susceptibility of mutants of some B. cinerea induced genes indicates the multiplicity of pathways required for resistance to B. cinerea consistent with the complex inheritance of B. cinerea resistance.

2.11 Conclusion and Perspective Genetic approaches in Arabidopsis and tomato defined some components of the basal B. cinerea resistance. Generally, processes related to the regulation of cell death, plant hormone signaling and synthesis are implicated in disease resistance to necrotrophic pathogens. Future research is likely to unravel other processes that affect plant resistance to necrotrophs and the regulatory factors involved. Future focus will be on the biochemical and molecular mechanisms underlying host resistance to necrotrophs and its relationship with plant responses to other classes of pathogens. The tissue specificity of B. cinerea resistance and the extent of environmental control of defense responses need further investigation. In addition, transfer of knowledge gained in model plant systems to crop plants under greenhouse, field crop production and postharvest situation is very important.

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Ren D, Liu Y, Yang KY, Han L, Mao G, Glazebrook J, Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 105:5638–5643 Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD (1996) Systemic acquired resistance. Plant Cell 8:1809–1819 Schilmiller AL, Howe GA (2005) Systemic signaling in the wound response. Curr Opin Plant Biol 8:369–377 Seo HS, Song JT, Cheong JJ, Lee YH, Lee YW, Hwang I, Lee JS, Choi YD (2001) Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant response. Proc Natl Acad Sci USA 98:4788–4793 Siewers V, Kokkelink L, Smedsgaard J, Tudzynski P (2006) Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea. Appl Environ Microbiol 72:4619–4626 Sticher L, Mauch-Mani B, Metraux JP (1997) Systemic acquired resistance. Annu Rev Phytopathol 35:235–270 Stintzi A, Weber H, Reymond P, Browse J, Farmer EE (2001) Plant defense in the absence of jasmonic acid: the role of cyclopentenones. Proc Natl Acad Sci USA 98:12837–12842 Tang D, Ade J, Frye CA, Innes RW (2006) A mutation in the GTP hydrolysis site of Arabidopsis dynamin-related protein 1E confers enhanced cell death in response to powdery mildew infection. Plant J 47:75–84 Tang D, Simonich MT, Innes RW (2007) Mutations in LACS2, a long-chain acyl-coenzyme A synthetase, enhance susceptibility to avirulent Pseudomonas syringae but confer resistance to Botrytis cinerea in Arabidopsis. Plant Physiol 144:1093–1103 Thomma B, Eggermont K, Penninckx I, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 95:15107–15111 Thomma BP, Eggermont K, Tierens KF, Broekaert WF (1999) Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121:1093–1102 Thomzik JE, Stenzel K, Stöcker R, Schreier P, Hain R, Stahl DJ (1997) Synthesis of a grapevine phytoalexin in transgenic tomatoes (Lycopersicon esculentumMill.) conditions resistance against Phytophthora infestans. Physiol Mol Plant Pathol 51:265–278 Tierens KF, Thomma BP, Bari RP, Garmier M, Eggermont K, Brouwer M, Penninckx IA, Broekaert WF, Cammue BP (2002) Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds. Plant J 29:131–140 Ton J, De Vos M, Robben C, Buchala A, Metraux JP, Van Loon LC, Pieterse CM (2002) Characterization of Arabidopsis enhanced disease susceptibility mutants that are affected in systemically induced resistance. Plant J 29:11–21 van Kan JA (2006) Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci 11:247–253 van Loon LC, Bakker PA, Pieterse CM (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36:453–483 van Loon L, Geraats B, Linthorst H (2006) Ethylene as a modulator of disease resistance in plants. Trends Plant Sci 11:184–191 van Wees S, Chang H-S, Zhu T, Glazebrook J (2003) Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling. Plant Physiol 132:606–617 Veronese P, Chen X, Bluhm B, Salmeron J, Dietrich R, Mengiste T (2004a) The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection. Plant J 40:558–574 Veronese P, Nakagami H, Bluhm B, Abuqamar S, Chen X, Salmeron J, Dietrich RA, Hirt H, Mengiste T (2006a) The membrane-anchored Botrytis-Induced Kinase1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell 18:257–273

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Chapter 3

Induced Resistance in Melons by Elicitors for the Control of Postharvest Diseases Bi Yang, Li Yongcai, Ge Yonghong, and Wang Yi

Abstract  Melons fruit can be induced to develop enhanced resistance to pathogen infection by pre- or postharvest treatment with a variety of chemical, physical and biological elicitors. The elicitors include acibenzolar, soluble silicon, oxalic acid, chitosan, b-aminobutyric acid, 2,6-dichloroisonicotinic acid, heat treatment and harpin. Resistance induced is broad spectrum and long lasting, but rarely provides complete control of infection. The mechanism of induced resistance is involved in the accumulation of defense enzymes, antifungal compounds, increasing of reactive oxygen species and lignification of epidermal cells. In order to maximize the efficacy of resistance elicitors, it is required to understand of the mechanism of induced resistance and the effect factors of pre- or postharvest. There also needs to evaluate quality change in induced fruit. It is concluded that control of melons postharvest disease by induced resistance would be the use of integrated approach combining chemical, physical and biological control methods, and culture practices. Keywords  postharvest diseases • induced resistance • fruit • elicitors

3.1 Introduction Melons, Cucumis melo L., are well known members of the Cucurbitaceae family, has been divided into a number of botanical subspecies (Sykes 1990). The major ones are cantaloupes (Cucumis melo var. cantalupensis), muskmelons (C. melo var. reticulatus), oriental melons (C. melo var. chinensis) and winter melons (C. melo var. inodorus). China is the biggest producer of melons in the world (Bi et al. 2007b).

Bi Y.,(), Li Y.C., Ge Y.H., and Wang Y. College of Food Science and Engineering, Gansu Agricultural University, Lanzhou, 730070, China e-mail: [email protected]

D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_3, © Springer Science + Business Media B.V. 2009

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The melons are quite perishable after harvest. A various types of pathogens are involved in fruit decay (Snowdon 1990; Barkai-Golan 2001). In China, melons are infected by Alternaria rot (Alternaria alternata), blue mold rot (Penicillium spp.), Fusarium rot (Fusarium spp.), Green mold rot (Cladosporium sp.), Mucor rot (Mucor mucedo), pink mold rot (Trichothecium roseum), Rhizopus rot (Rhizopus spp.), sour rot (Geotrichum candidum) (Bi et  al. 2003). Both Alternaria and Fusarium rots are related to latent infection (Ge et al. 2005b). Postharvest losses are estimated to be as 35–50% because of rough handling after harvest, inadequate packaging and temperature management (Bi et al. 2007b). Postharvest disease of melons is often controlled by the application of synthetic fungicides, such as benomyl and guazatine (Morris and Wade 1983), imazalil (Aharoni et al. 1992), iprodione and azoxystrobin (Ma et al. 2004). However, due to problems related to fungicide residues, development of fungicide resistance by pathogens, and potential harmful effects on the environment, as well as the necessity to reduce losses with minimal use of fungicides, new strategies for controlling postharvest diseases have been proposed (Wilson et al. 1994). Induced resistance is a natural defense response triggered by pathogen or elicitors (Kuc 2001). Induced resistance includes local induced resistance and systemic acquired resistance (SAR). The former is directly producing disease resistance at the guided position; the latter is that untreated part produced disease resistance after treated the other part (Ryals et al. 1996). Induction disease resistance in harvested horticultural crops using physical, biological and chemical elicitors has received increasing attention over recent years, it being considered a preferred strategy for disease management (Terry and Joyce 2004). Fruit and vegetables can be induced to develop enhanced resistance to pathogen infection by pre- or postharvest treatment with a variety of chemical elicitors (Bi et al. 2007c). In this chapter, we provide a description of research that indicates that induced resistance by elicitors can be part of postharvest disease control, and provide a brief knowledge on the mechanism of induced resistance in melons.

3.2 Chemically Induced Resistance 3.2.1 Acibenzolar Acibenzolar (benzo-(1, 2, 3)-thiadiazole-7-carbo-thioicacidS-methylester; ASM; BTH; Bion™; Actigard™) is perhaps the most potent synthetic SAR activator discovered to date (Terry and Joyce 2004; Kessmann et al. 1994; Friedrich et al. 1996). The chemical is not phytotoxic and has proven an effective SAR elicitor in both monocotyledons (Gorlach et al. 1996) and dicotyledons (Tally et al. 2000). In recent years, ASM has been extensively studied to control postharvest diseases in apple (Spadaro et al. 2004), pear (Cao and Jiang 2006), peach (Liu et al. 2005), strawberry (Terry and Joyce 2000), passionfruit (Willingham et al. 2002), mango (Dann and Zainunuri 2008) and potato (Bokshi et al. 2003).

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Huang et al. (2000) found a pre-flowering foliar spray of ASM at 50 mg/L combined with a fruit dip in guazatine at 500 mg/L at harvest substantially decreased disease in stored rockmelons and Hami melons. Preharvest treatment with ASM at 50 mg/L four times from anthesis and one time (3 weeks before harvest) also reduced Alternaria and Fusarium rots in harvested rockmelons (Bokshi et al. 2006). A foliar spraying with ASM at 100 mg/L 1 week or 1 day before harvest decreased the lesion area of harvested muskmelons inoculated with F. semitectum and T. roseum (Ge et al. 2008). Field spraying with ASM at 100 mg/L significantly reduced the latent infection rate of muskmelons. Four sprays from anthesis reduced latent infection rate in fruit caused by A. alternata and Fusarium spp, by 66.7 and 60% (Zhang et al. 2006b). Postharvest treatment with ASM at 200 mg/L noticeably reduced decay severity caused by A. alternata, F. semitectum and T. roseum in Hami melon (Bi et al. 2006). Postharvest ASM treatment at 100 mg/L suppressed lesion diameter in treated and untreated halves of the same fruit inoculated with T. roseum, indicating that the chemical induced local and systemic resistance (Wang et al. 2008). As a functional analogue of SA, ASM acts downstream of SA and elicits accumulation of the same SAR genes and pathogenesis-related proteins (PRs) as SA (Friedrich et al. 1996). Bi et al. (2006b) reported that activity of peroxidase (POD), chitinase (CHT) and phenylalanine ammonia lyase (PAL) was significantly enhanced in harvested Hami melons treated with ASM. Similar results were also observed in rockmelons (Bokshi et al. 2006) and muskmelons (Wang et al. 2008). ASM treatment increased production of reactive oxygen species, augmented the content of the preformed antifungal compounds, total phenolics and lignin, and accumulated lignin, suberin and callose in muskmelon (Bi et al., unpublished data).

3.2.2 Silicon Silicon (Si) is the second most abundant element in the lithosphere (27.70%) and it is as important as phosphorus and magnesium (0.03%) in the biota (Exley 1998). Si is also considered to be biologically active and to trigger a faster and more extensive deployment of plant natural defenses (Fauteux et al. 2005). Si has been shown to reduce postharvest diseases in pear (Spotts and Cervantes 1989), cherry (Qin and Tian 2005) and jujube (Tian et al. 2005). Postharvest application of silicon oxide and sodium silicate tended to suppress postharvest pink rot severity caused by T. roseum in muskmelons (Guo et al. 2007). Sodium silicate at 100 mM significantly reduced decay incidence and severity of Hami melons inoculated with A. alternata, F. semitectum, and T. roseum. Si treatments at 100 mM were also effective in reducing natural infections of Hami melons. Si treatments applied at 100 mM pre-inoculation with T. roseum had lower decay incidence and severity than treatments applied post-inoculation, indicated resistance induction of occurred in melon fruit (Bi et al. 2006a). Postharvest treatment with Si proved effective for inhibiting pathogen growth as well as inducing disease resistance in melons. Sodium silicate has been shown to have direct inhibition in vitro antifungal activity against postharvest pathogens of

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melons (Bi et  al. 2006a). Si treatment resulted in enhanced activity of POD and PAL (Guo et al. 2007). Si also caused a more progressive and significant increase in POD and CHT activities in Hami melons challenged by T. roseum (Bi et  al. 2006a). Si treatment enhanced the production of reactive oxygen species and maintained fruit firmness. The accumulation of antifungal compounds was observed in Si treated muskmelons (Bi et al., unpublished data).

3.2.3 Other Chemicals The application of oxalic acid has already been shown to induce systemic resistance against field diseases in cucumber (Mucharroman and Kuc 1991). The chemical has been confirmed to reduce postharvest diseases in mango (Zheng et  al. 2007) and longan (Whangchai et al. 2006). Postharvest oxalic acid dipping at 50 mM reduced significantly decay severity of muskmelon fruit inoculated with T. roseum. A systemic resistance was observed in untreated halves of the same fruit (Deng et al. 2008). Chitosan has been considered a promising means for enhancing disease resistance in harvested horticultural commodities (Wilson et  al. 1994; El Ghaouth 1994). A significant reduction of rots has been recorded in chitosan-treated apple (Bautista-Banos et al. 2004), pear and kiwifruit (Du et al. 1997), table grape (Meng et al. 2008), strawberries (Zhang and Quantick 1998), bell pepper (El Ghaouth et al. 1997), tomato (Liu et al. 2007), carrots (Cheah et al. 1997). Preharvest treatment with chitosan at 1 mg/mL significantly reduced the latent infection rate of muskmelons. Three and four sprays from anthesis significantly reduced latent infection rate in fruit caused by A. alternata and Fusarium spp. Four sprays before harvest also decreased the lesion area of harvested muskmelons inoculated with A. alternata, F. semitectum and T. roseum, indicated resistance induced in melon fruit (Xie et al. 2008). Although b-aminobutyric acid (BABA) is only rarely found naturally in plants, it has proved to be a potent inducer of acquired resistance and has a broad spectrum of activity against many disease-causing organisms (Cohen 2002; Jakab et  al. 2001). Earlier foliage application of BABA at 2000 mg/L reduced the total storage rots, Alternaria and Fusarium rots of fruit. The chemical caused activation of CHT and POD activities in treated leaves of rockmelons (Bokshi et al. 2006). 2,6-dichloroisonicotinic acid (INA) protect many crops against their pathogens. INA is weakly fungistatic in  vitro, but effectively elicits SAR genes in tobacco prior to TMV challenge inoculation (Ward et al. 1991). The INA-mediated resistance has been reported to be against a broad spectrum of pathogens (Uknes et al. 1992) and the induced resistance has been suggested to have a long-lasting effect (Lucas 1999). Bokshi et al. (2006) found that earlier foliage application of INA at 50 mg/L significantly reduces the postharvest diseases of rockmelons. Activity of CHT and POD in treated leaves were stimulated and maintained at a higher level three days after a first spray with INA rather than BABA, and second spray of INA increased CHT and POD activities further and the increase lasted several weeks until harvest.

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3.3 Physically Induced Resistance Heat treatment is considered a promising method for reducing postharvest disease (Lurie 1998). It may be effective either by directly inhibiting pathogen development, or by inducing natural resistance in the fruit (Klein and Lurie 1991; Schirra et  al. 2000). Induction of resistance against decay due to hot water treatment of muskmelons for 3 min at 55°C before inoculation was reported by Zhang et  al. (2005). The resistance was found to be most effective when inoculation was carried out 24 h after hot water treatment. Dipping melons in hot water not only reduced pathogens causing storage disease but also significantly improved the life and marketability of fruit (Fallik et al. 2000). Teitel et al. (1989) found that with a longer immersion time, a hot water dip may have provided effective protection for melon fruit against storage rots. They observed that a reduced temperature of 52°C and a longer dip time of 2 min controlled decay from Alternaria spp., Fusarium spp., Rhizopus spp. and Mucor spp. without causing external heat injury. Similar observation made by Mayberry and Hartz (1992), and Barkai-Golan et al. (1994), who reported that hot water treatment of ‘Galia’ melon at 52–55°C effectively prevented storage losses caused by A. alternata, Fusarium spp. and T. roseum. A mixture of fungicides and hot water could result in an effective decay control of melons. Zhang et al. (2005) observed that hot water treatment at 55°C for 1 min combined with 50 mg/ L azoxystrobin or 250 mg/L imazalil significantly controlled the rots of muskmelons caused by R. stolonifer, Fusarium spp. and T. roseum.

3.4 Biologically Induced Resistance Harpin (Messenge™) is an acidic, heat-stable, glycine-rich, 44-kDa protein, encoded by the hrpN gene of the bacterium Erwinia amylovora (Wei et al. 1992). It is the first known bacterial product able to elicit the hypersensitive response (HR) and to induce systemic acquired resistance in plants (Baker et al. 1993; Dong et al. 1999; Mullin et al. 1998). Postharvest treatment with harpin has been shown to induce resistance in apple (de Capdeville et al. 2003) and pear (Wang et al. 2006). Field spraying with harpin at 50 mg/L reduced the latent infection rate of muskmelons. Three and four sprays from anthesis significantly decreased latent infection rate and relative surface of fruit (Wang and Bi, unpublished data). Postharvest harpin treatment at 90 mg/L decreased decay severity caused by A. alternata, F. semitectum and T. roseum in Hami melons (Bi et al. 2007a). Similar results were observed by Ge et al. (2005a) in muskmelons inoculated by F. semitectum and T. roseum. A higher concentration over 90 mg/L failed to promote resistance and did not cause phytotoxicity to melons. Harpin did not demonstrate any fungicide effect in  vitro, suppressed lesion diameter of fruit inoculated with T. roseum in treated and untreated halves of the same melon, indicating that the chemical induced local and systemic resistance. Besides, harpin provided greater level of

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decay control in long-storage-life cultivars (cv. 8601) than in short-storage-life ones (cv. New Queen). The time between initial treatment with harpin and subsequent inoculation with T. roseum significantly affected efficacy of the induction (Bi et al. 2005). Harpin treatment applied at early stage of maturity was more effective than at later stage (Bi et al., unpublished data). Studies have shown that harpin triggers a variety of cellular responses, such as activation of reactive oxygen species and cell membrane depolarization (Dong et al. 1999). Harpin induced a significant and progressively increasing activity of POD and CHT in muskmelon and Hami melons (Ge et al. 2005a; Bi et al. 2005; Wang et  al. 2008). Postharvest application of harpin also increased activity of defense enzymes, and resulted in a production of activated oxygen species, an increase of the preformed antifungal compounds and total phenolics, and an accumulation of lignin, suberin and callose in epidermal cell of muskmelons (Bi, unpublished data).

3.5 Conclusions Induced resistance may be an important part of postharvest disease control strategies of the melons in the future. Because of the apparent safety and the broad-spectrum nature of the induced resistance, research is underway to identify and develop elicitors that can be used to induce resistance in melons and thus make this resistance directly applicable to postharvest disease control. However, despite some studies as mentioned above, only a few cultivars have been studied in any detail. Having more concrete information on the biological spectrum of induced resistance on a cultivarby-cultivar basis will be of use in determining which cultivar may be suitable for this type of control. Having a better understanding of induced resistance response in more than one cultivar is important in developing any generalizations about this type of resistance. Information on the relative importance of putative defense mechanisms utilized in induced resistance is also limited. We know that induction of resistance is accompanied by the accumulation of PR proteins, defense enzymes and antifungal compounds, increasing of reactive oxygen and lignification of epidermal cells. There are other likely defense reactions and compounds that are also probably involved in the observed resistance. They could be more rapidly induced than the markers, and could be more effective against the pathogen (Hammerschmidt 1999). Each of the defense-related phenomena reported above have characteristics and correlations that strongly suggest that each is associated with the resistance state. However, the contribution of each to resistance appears to be small. This strongly suggests that the expression of full resistance requires the expression of several mechanisms, many of which are known to be coordinately regulated (Ward et al. 1991). Furthermore, understanding the defenses and the signals that regulate these defenses will also provide new avenues for implementation of induced resistance by genetic engineering or manipulation of signal pathway (Hammerschmidt and Becker 1997).

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In our haste to realize the potential offered by induced resistance for postharvest disease control, we have paid too little attention to the factors that are likely to influence its effectiveness, largely using it inappropriately as simply a fungicide replacement. Therefore, there is an urgent need for understanding of the various factors (such as cultivars, maturity, timing of treatment, field environment, postharvest handling and storage condition) that will influence the expressing of induced resistance in melon fruit, in order to maximize the efficacy of resistance elicitors (Walters et al. 2005). A more realistic scenario to combat decay of harvested melons would be the use of integrated control approach combining biological and physical control strategies, with or without limited quantities of fungicides pre-harvest, and with efficient management and handling practices. As the releasing of volatile compounds could be inhibited in ASM treated muskmelons (Jiang et al. 2007), there needs to evaluate quality change in melon fruit treated with elicitors and be assurance that induced natural defense compounds effective against pathogens are not present in consumed tissues at levels toxic to mammals (Paiva 2000; Dann 2003). The implementation of induced resistance by chemicals in melons should be approached with cautious optimism. The enormous potential for reducing postharvest diseases via their natural disease resistance mechanisms has been demonstrated. However, more information is required to ensure that this type of resistance offers a safe, effective and reliable complement to the existing methods. Acknowledgements  We are grateful to Dr. Dov Prusky (Department of Postharvest Science, ARO, Volcani Center, Israel) for offering the opportunity to write this chapter. This work was financially supported by National Natural Science Foundation of China (30671465), Ministry of Science and Technology of China (2001BA501A09) and Australia Center of International Agricultural Research (ACIAR, PHT/1998/140).

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Ge YH, Bi Y, Ma LY (2005b) Study on the periods and ways of latent infection of fungus on muskmelon (Cucumis melo L. cv. Huanghemi) fruit. Watermelon Melon China 3:1–3 (in Chinese with English abstract) Ge YH, Bi Y, Li X, Li Mei (2008) Induces resistance against Fusarium and pink rots by Acibenzolar-S-Methyl in harvested muskmelon (cv. Yindi). Agric Sci China 7:58–64 Gorlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U, Kogel KH, Oostendorp M, Staub T, Ward E, Kessmann H, Ryals J (1996) Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates expression and disease resistance in wheat. Plant Cell 8:629–643 Guo YR, Liu L, Zhao J, Bi Y (2007) Use of silicon oxide and sodium silicate for controlling Trichothecium roseum postharvest rot in Chinese cantaloupe (Cucumis melo L.). Int J Food Sci Technol 42:1012–1018 Hammerschmidt R (1999) Induced disease resistance: how do induced plants stop pathogens? Physiol Mol Plant Pathol 55:77–84 Hammerschmidt R, Becker JS (1997) Acquired resistance to disease in plants. Hort Rev 18:247–289 Huang Y, Deverall BJ, Tang WH, Wang W, Wu FW (2000) Foliar application of acibenzolar-Smethyl and protection of postharvest Rock melons and Hami melons from disease. Eur J Plant Pathol 106:651–656 Jakab G, Cottier V, Toquin V, Rigoli G, Zimmerli L, Metraux JP, Mauch-Mani B (2001) b-Aminobutyric acid-induced resistance in plants. Eur J Plant Pathol 107:29–37 Jiang YM, Li X, Bi Y, Zhou XP, Zhou W, Ge YH (2007) Inhibition of postharvest volatile compounds release by preharvest acibenzolar-S-methyl (BTH) treatment on muskmelons (cv. Yindi). Transactions of the CSAE 23:243–247 (in Chinese with English abstract) Kessmann H, Staub T, Hofmann C, Maetzke T, Herzog J, Ward E, Uknes S, Ryals J (1994) Induction of systemic acquired resistance in plants by chemicals. Annu Rev Phytopathol 32:439–459 Klein JD, Lurie S (1991) Postharvest heat treatment and fruit quality. Postharvest News Inform 2:15–19 Kuc J (2001) Concepts and directions of induced systemic resistance in plants and its application. Eur J Plant Pathol 107:7–12 Liu HX, Jiang WB, Bi Y, Luo YB (2005) Postharvest BTH treatment induces resistance of peach (Prunus persica L. cv. Jiubao) fruit to infection by Penicillium expansum and enhances activity of fruit defense mechanisms. Postharvest Biol Technol 35:263–269 Liu J, Tian SP, Meng XH, Xu Y (2007) Effects of chitosan on control of postharvest diseases and physiological responses of tomato fruit. Postharvest Biol Technol 44:300–306 Lucas JA (1999) Plant immunisation: from myth to SAR. Pest Sci 55:193–196 Lurie S (1998) Postharvest heat treatments of horticultural crops. Hort Rev 22:91–121 Ma LY, Bi Y, Zhang ZK, Zhao L, An L, Ma KQ (2004) Control of pre- and postharvest main diseases on melon variety Yindi with preharvest azoxystrobin spraying. J Gansu Agric University 39:14–17 (in Chinese with English abstract) Mayberry KS, Hartz TK (1992) Extension of muskmelon storage life through the use of hot water treatment and polyethylene wraps. HortScience 27:324–326 Meng XH, Li BQ, Liu J, Tian SP (2008) Physiologic responses and quality attributes of table grape fruit to chitosan preharvest spray and postharvest coating during storage. Food Chem 106:501–508 Morris S, Wade NL (1983) Control of postharvest diseases in cantaloupes by treatment with guazatine and benomyl. Plant Dis 67:792–792 Mucharroman E, Kuc J (1991) Oxalate and phosphate induce systemic induce resistance against disease caused by fungi, bacteria and viruses in cucumber. Crop Prot 10:261–265 Mullin P, Wang JS, Qiu D, Wei ZM (1998) Disease control and growth enhancement effects of harpin on tobacco. (Abstract) Phytopathology 88:S65 Paiva NL (2000) An introduction to the biosynthesis of chemical used in plant microbe communication. J Plant Growth Regul 19:131–143

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Qin GZ, Tian SP (2005) Enhancement of biocontrol activity of Cryptococcus laurentii by silicon and the possible mechanisms involved. Phytopathology 95:69–75 Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD (1996) Systemic acquired resistance. Plant Cell 8:1809–1818 Schirra M, D’hallewin G, Ben-Yehoshua S, Fallic E (2000) Host-pathogen interactions modulated by heat treatment. Postharvest Biol Technol 21:71–85 Snowdon AL (1990) A color atlas of postharvest diseases and disorders of fruits and vegetables. Vol.1. General Introduction and fruits. Wolfe Sci. Ltd, Spain. pp 12–52 Spadaro D, Garibaldi A, Gullino ML (2004) Control of Penicillium expansum and Botrytis cinerea on apple combining a biocontrol agent with hot water dipping and acibenzolar-S-methyl, baking soda, or ethanol application. Postharvest Biol Technol 33:141–151 Spotts RA, Cervantes LA (1989) Evaluation of disinfestant-flotation salt-surfactant combinations on decay fungi of pear in a model dump tank. Phytopathology 79:121–126 Sykes S (1990) Melons: new varieties for new and existing markets. Agric Sci 3:32–35 Tally A, Oostendorp M, Lawton K, Staub T, Bassi B (2000) Commercial development of elicitors of induced resistance to pathogens. In: Agrawal AA, Tuzun S, Bent E (eds) Induced plant defenses against pathogens and herbivores. APS, St. Paul, MN, pp 357–369 Teitel DC, Aharoni Y, Barkai-Golan R (1989) The use of heat treatments to extend the shelf life of ‘Galia’ melons. J Hort Sci 64:367–372 Terry LA, Joyce DC (2000) Suppression of grey mould on strawberry fruit with the chemical plant activator acibenzolar. Pest Manage Sci 56:989–992 Terry LA, Joyce DC (2004) Elicitors of induced disease resistance in postharvest horticultural crops: a brief review. Postharvest Biol Technol 32:1–13 Tian SP, Qin GZ, Xu Y (2005) Synergistic effects of combining biocontrol agents with silicon against postharvest diseases of jujube fruit. J Food Prot 68(3):544–550 Uknes S, Mauch mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J (1992) Acquired resistance in Arabidopsis. Plant Cell 4:645–656 Walters D, Walsh D, Newton A, Lyon G (2005) Induced resistance for plant disease control: maximizing the efficacy of resistance elicitors. Phytopathology 95:1368–1373 Wang JJ, Wang Y, Ge YH, Bi Y (2006) Inhibiting effect of postharvest Harpin treatment on Alternaria rot and induction to resistance enzymes of Pyrus bretschneideri Pingguoli. J Gansu Agric Univ 41:114–117 (in Chinese with English abstract) Wang Y, Li X, Bi Y, Ge YH, Li YC, Xie F (2008) Postharvest ASM or Harpin treatment induce resistance of muskmelons against Trichothecium roseum. Agric Sci China 7:217–223 Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ah1-Goy P, Metraux JP, Ryals JA (1991) Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3:1085–1094 Wei ZM, Laby RJ, Zumoff CH, Bauer DW, He SY, Collmer A, Beer SV (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:85–88 Whangchai K, Saengnil K, Uthaibutra J (2006) Effect of ozone in combination with some organic acids on the control of postharvest decay and pericarp browning of longan fruit. Crop Prot 25:821–825 Willingham SL, Pegg KG, Langdon PWB, Cooke AW, Beasley D, Mclennan R (2002) Combination of strobilurin fungicides and acibenzolar (Bion) to reduce scab on passionfruit caused by Cladosporium oxysporum. Australas Plant Pathol 31:333–336 Wilson CL, El-Ghaouth A, Chalutz E, Droby S, Stevens C, Lu JY, Khan V, Arul J (1994) Potentatial of induced resistance to control postharvest diseases of fruits and vegetables. Plant Dis 78:837–844 Xie DF, Bi Y, Deng JJ, He SK, Lv SF (2008) Control of latent infection and post harvest main diseases with preharvest chitosan sprays in muskmelons. J Gansu Agric Univ 43:96–99 (in Chinese with English abstract)

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Zhang D, Quantick PC (1998) Antifungal effects of chitosan coating on fresh strawberries and raspberries during storage. J Hort Sci Biotechnol 73:763–767 Zhang YM, An L, Bi Y, Liu JQ, Ma KQ (2005) Effect of heat treatment on the disease of Postharvest Muskmelon. J Gansu Agric Sci Technol 4:46–49 (in Chinese with English abstract) Zhang ZK, Bi Y, Wang JJ, Wang Y, Zhang HY, Li WH (2006) Defense enzymes and disease resistance substances induction in muskmelons by preharvest BTH spraying. J Gansu Agric Univ 41:122–125 (in Chinese with English abstract) Zheng X, Tian SP, Michael JG, Yue H, Li BQ (2007) Effects of exogenous oxalic acid on ripening and decay incidence in mango fruit during storage at room temperature. Postharvest Biol Technol 45:281–284

Chapter 4

Mechanisms Modulating Postharvest Pathogen Colonization of Decaying Fruits Dov Prusky, Noam Alkan, Itay Miyara, Shiri Barad, Maayan Davidzon, Ilana Kobiler, Sigal Brown-Horowitz, Amnon Lichter, Amir Sherman, and Robert Fluhr

Abstract  As biotrophs, insidious fungal infections of postharvest pathogens remain quiescent during fruit growth while at a particular phase during fruit ripening and senescence the pathogens transform to necrotrophs causing typical decay symptoms. Exposure of unripe hosts to pathogens (hemi-biotroph or necrotrophs), initiates defensive signal-transduction cascades that limit fungal growth and development. Exposure to the same pathogens during ripening and storage activates a substantially different signalling cascade which facilitates fungal colonization. This chapter will focus on modulation of postharvest host-pathogen interactions by pH and the consequences of these changes. Host pH can be raised or lowered in response to host signals, including alkalization by ammonification of the host tissue as observed in Colletotrichum and Alternaria, or acidification by secretion of organic acids as observed in Penicillium, Botrytis and Sclerotinia. These changes sensitize the host and activate transcription and secretion of fungal hydrolases that promote maceration of the host tissue. Several particular examples of coordinated responses which follow this scheme are described.

4.1 Introduction Postharvest fungal pathogens exploit three main routes to penetrate the host tissue: (i) through wounds caused by biotic and/or abiotic agents during growth and storage; (ii) through natural openings such as lenticels, stem ends and pedicel-fruit interphase, and (iii) by direct breaching of the host cuticle, which can occur throughout D. Prusky (*), N. Alkan, I. Miyara, S. Barad, M. Davidzon, I. Kobiler, S. Brown-Horowitz, and A. Lichter Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center, Bet Dagan, 50250 e-mail: [email protected] A. Sherman Department of Genomics, Agricultural Research Organization, the Volcani Center, Bet Dagan, 50250 R. Fluhr Department of Plant Genetics, The Weizmann Institute of Science Rehovot 76100, Israel D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_4, © Springer Science + Business Media B.V. 2009

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the fruit growth period. An active pathogenic process can start immediately after spores land on the wounded tissue, whereas other pathogens can breach the unripe fruit cuticle and then remain inactive for months until the harvested fruit ripens. The penetration process may go unnoticed by the host, or it may result in rapid defense signaling that results in the induction of defense molecules that will limit fungal development (Prusky and Lichter 2007). The period from host infection to the activation of fungal development and symptom expression is designated the quiescent stage (Prusky 1996). After harvest, during ripening and storage, the mechanism that protects the fruit from fungal attack becomes insufficient. This transition from a resistant to susceptible state parallels physiological changes that occur during ripening to which the pathogen senses and responds. In the present chapter, we will focus on modulation of the host environment by the pathogen as a mean for fungal colonization. We also touch upon the signals that may activate the transition from quiescent to necrotrophic mode during fruit ripening.

4.2 The Quiescent Stage During the colonization of plant hosts, postharvest fungal pathogens exploit two main modes of nutrition: biotrophy, in which the nutrients are obtained from the living host cells, and necrotrophy, in which nutrients are obtained from dead host cells killed by the fungus (Perfect et al. 1999). Both of these nutritional modes are exhibited by postharvest pathogens. Opportunistic postharvest pathogens may also be located within the fruit in an inactive mode, awaiting fruit wounding, ripening or senescence. The length of each period may vary among pathogens, hosts, and host developmental stages. Pathogens such as Colletotrichum, Monilinia, Botrytis and Alternaria may remain quiescent for long periods in developing fruit tissues, but initiate immediate necrotrophic development on ripening and senescing fruits. Colletotrichum is one of the major postharvest pathogens in which quiescence has been studied. Colletotrichum spores adhere to and germinate on the plant surface, produce germ tubes, and the tip of the germ tube developing from the appressorium sends an infection peg through the cuticle. Following penetration, Colletotrichum initiates subcuticular intramural colonization (Perfect et al. 1999) and spreads rapidly throughout the tissue with both inter- and intracellular hyphae. After colonizing one or more host cells, the infecting hyphae, which can be described as biotrophic (Kramer-Haimovich et al. 2006), subsequently give rise to secondary necrotrophic hyphae (Bailey and Jeger 1992; Coates et  al. 1993; Latunde-Dada et  al. 1996; Mendgen and Hahn 2001; O’Connell et  al. 1985). Botrytis and Monilinia can penetrate through wounds and also breach the fruit cuticle by using an infection peg from an appressorium that then remains quiescent for long periods of time (Fourie and Holz 1995; Pezet et  al. 2003). Depending on the physiological status of the organ, these hyphae may continue the infective process or remain quiescent. Based on published reports, three major hypothetical modes for the activation of quiescent biotrophic pathogens have been suggested (Prusky 1996): (i) deficiency

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in the host nutritional resources required for pathogen development; (ii) the presence of preformed or inducible fungistatic antifungal compounds in resistant unripe fruits, and (iii) an unsuitable environment for the activation of fungal pathogenicity factors. In the present work we will concentrate in the modulation of host environment by the fungus as mechanism of fungal colonization.

4.3 pH Modulation of the Environment by Postharvest Fungal Pathogens Is a Virulence Potentiation The ability to modify pH may be expressed in either direction, and fungi that raise or reduce it are described as “alkalizing fungi” or “acidifying fungi”, respectively. The mechanisms regulating ambient pH modulation are tied to the levels of ambient pH. Fine-tuning of enzyme expression in response to the ambient pH in the host by the fungus further highlights the importance of the specific regulatory system that is activated under a changing environmental pH which can lead to the activation of quiescent infections (Prusky and Yakoby 2003).

4.3.1 Alkalizing Fungi In normal ripening and senescing fruits, pH levels change as part of the ripening process: for instance, the pH of avocado fruit increases from 5.2 to 6.0 during ripening (Yakoby et al. 2000) (Fig. 4.1). Alkalinization of the fruit host environment during

Fig. 4.1  Changes in pH values of the pericarp (◊) of cultivar Fuerte avocado fruits during postharvest ripening. Firmness (¨) is presented as a parameter of ripening. Arrow indicates the time of decay initiation (DI) and of decay symptoms (DS) of C. gloeosporioides, following inoculation of freshly harvested fruits. Bars represent the standard deviations of the mean from one representative experiment. Cultivar Fuerte fruits were harvested at midseason

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Fig.  4.2  Measurement of the local pH environment in tomato fruit at the leading edge of the decay caused by C. coccodes infection. (a): pH values as a function of distance from the leading edge of the decay in infected (¨) and uninfected () tissue. (b): Fluorescence micrograph with BCECF at different distances from the region of decay. The changes in pH were detected microscopically in 10 mm long by 1 mm thick strips of tissue stained by BCECF fluorescence dye after correlating fluorescent values with direct pH determinations obtained by a piercing pH electrode. Fluorescence was detected by Leica fluorescence binocular the average value of 50 evaluated cells is reported. A representative picture of cells at 1, 5 and 10 mm distance for the hyphal front is presented in inset (b)

colonization by postharvest pathogens such as Colletotrichum and Alternaria is associated with fungal secretion of ammonia (Eshel et al. 2002; Prusky et al. 2001a). In avocado, the fruits pericarp showed a pH of 5.2 that increased up 7.5 and 8.0, during C. gloeosporioides attack (Yakoby et al. 2000). In tomato fruits, the initial pH ranged between 4.1 and 4.5 and decay development at the infection site increased the pH to 8.0 and the ammonia accumulated to 3.6 mM compared to 0.2 mM in the healthy tissue (Alkan et al. 2008; Prusky et al. 2001) (Fig. 4.2). In the case of polyphage pathogens such as A. alternata, a threefold to tenfold increase in ammonia concentration, and a pH elevation of 0.2 to 2.4 pH units were detected in several hosts: tomato, pepper, melon, cherry and persimmon (Eshel et al. 2002). Ambient alkalization by fungi is achieved by active secretion of ammonia, which is produced as a result of protease activity and deamination of amino acids (Prusky and Yakoby 2003). Interestingly, the initial acidic pH of the fruit has been shown to be conducive to the enhanced ammonium secretion and host-tissue alkalization that facilitate fungal virulence (Alkan et  al. 2008; Kramer-Haimovich et  al. 2006) (Fig.  4.3). The isolation of C. coccodes mutants with reduced ammonium production has provided direct evidence for the necessity of ammonium accumulation during

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Fig.  4.3  Effect of pH on ammonia secretion by C. gloeosporioides. Samples of medium were quantified for ammonia accumulation at different times after transfer to secondary medium containing KNO3, buffered with phthalate buffer to a pH range of 4.0 to 7.0. Symbols: ◊ pH 7, pH 6.5, ▲pH 6, □ pH 5,  pH 4. Significance was calculated by comparing the concentrations of ammonia at the different initial pHs on each sampling day and was analyzed statistically by analysis of variance of three replications. Values for points on the same day labeled with the same letter were not statistically different (P < 0.05)

Colletotrichum pathogenicity (Alkan et  al. 2008), indicating that ammonium accumulation is a critical factor contributing to Colletotrichum necrotrophic development in ripening fruits (Prusky and Yakoby 2003). Pathogens alter the local pH at the infection site to suit the increased expression of pathogenicity factors and the enzymatic arsenal (Denison 2000; Yakoby et  al. 2000; Prusky et  al. 2001; Eshel et al. 2002; Prusky and Yakoby 2003). Expression of the endoglucanase gene AaK1 by Alternaria alternata was found to be maximal at pH levels above 6.0, values that are characteristic of decayed tissue. AaK1 was not expressed at the lower pH values in which the pathogen was quiescent (Eshel et al. 2002). In the pathogen C. gloeosporioides, the gene pelB was expressed when the pH was above 5.7 and was positively correlated with ambient alkalinization (Yakoby et al. 2000, 2001). The transcription factor pac1, which is involved in pH regulation, follows a pattern similar to that of pelB, which suggests that they are co-regulated or that pac1 regulates the expression of pelB (Drori et al. 2003) (Fig. 4.4). Dpac1 mutants of C. gloeosporioides showed 85% reduction of PELB transcript, delayed PL secretion (Fig. 4.5) and dramatically reduced virulence, as demonstrated by infection assays with avocado fruits (Miyara et al. 2008).

4.3.2 Acidifying Fungi Other postharvest pathogens, such as P. expansum, P. digitatum, P. italicum (Prusky et  al. 2003), B. cinerea (Manteau et  al. 2003) and S. sclerotiorum (Bateman and Beer 1965; Ruijter et al. 1999), utilize tissue acidification to support their attack via

Fig. 4.4  Transcriptional activation of pelB and pac1 by C. gloeosporioides as a function of pH levels. Expression as a function of pH: Northern analysis of total RNA isolated from C. gloeosporioides mycelia 16 h after transfer to fresh secondary cultures buffered with phthalate. Blots were probed with pelB (pelB panel) and then sequentially stripped and reprobed with pac1 (pac1 panel) and rDNA probes

Fig. 4.5  Relative expression of PAC1, PELB and PL secretion by the wild-type and Dpac1 mutant strains of C. gloeosporioides at pH 6.0. (a) Expression of PAC1. (b) Expression of PELB and PL secretion. Relative expression was detected by RT-PCR 7 and 15 h after transfer of the growing hyphae from the primary medium to the inductive secondary medium. The relative expression values are the averages of three replications of the treatment ± standard deviations. Western blot analyses were repeated three times, and results from a representative experiment are presented

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the secretion of organic acids. Penicillium spp. acidifies the ambient environments of apple and citrus fruit during decay development. P. expansum caused decay of apple fruits of several cultivars, in which it caused pH decreases from values ranging from 3.95 to 4.31 in the healthy mesocarp to values ranging from 3.64 to 3.88 in the decaying tissue. P. digitatum and P. italicum caused similar pH declines in citrus fruits (Prusky et al. 2004). In the necrotrophic fungus Sclerotinia sclerotiorum, during plant infection, a pH gradient was established in relation to oxalic acid secretion and the pH of the host reached as low as pH 4.0 (Rollins and Dickman 2001). This production of oxalic acid during plant infection has been implicated as a primary determinant of pathogenicity in this and other phytopathogenic fungi. The size of lesions induced by different strains of B. cinerea on grapevine and bean leaves correlated with the amount of OA that these strain in vitro (Germeier et al. 1994). However it remains unclear whether the level of OA produced in planta is sufficient to cause host cells to directly collapse. It is suggested that OA may in fact be a co factor in pathogenesis rather than the primary phytotoxic agent (Manteau et al. 2003). Several mechanisms for tissue acidification have been proposed on the basis of such studies. Penicillium spp. use two mechanisms for ambient acidification: the production of organic acids, mainly citric and gluconic, and NH4+ utilization associated with H+ efflux. In decayed fruit by P. expansum and P. digitatum, both pathogens produced significant amounts of citric and gluconic acids in the decayed tissue and reduced the host pH by 0.5 to 1.0 units (Table  1). Ammonium depletion from the growth medium or from the fruit tissue was directly related to ambient pH reduction. The organic acids that accumulated during tissue acidification by P. expansum were mainly gluconic (GA) and citric acids (Prusky et al. 2003) the same as for Aspergillus (Ruijter et al. 1999) which secrete as well, gluconic and citric acids. The contribution of gluconic acid secretion to the colonization of apple tissue by P. expansum was

Table 4.1  pH levels in healthy and Penicillium-decayed fruits pH value±SE Penicillium sp.

Host

Cultivars

P. expansum

Apple

P. digitatum

Oranges Grapefruit Oranges Grapefruit

Granny Smith Gala Red Delicious Fuji Golden Delicious Naval Oro Blanco Naval Oro Blanco

P. italicum

Healthy

Decayed

3.95±0.06 4.31±0.06 4.44±0.03 4.44±0.06 4.54±0.06 4.77±0.45 4.74±0.05 4.77±0.07 4.55±0.13

3.64±0.01 3.88±0.03 4.07±0.02 3.96±0.02 3.88±0.03 3.12±0.07 3.10±0.14 3.02±0.13 3.23±0.17

* pH changes induced by P. expansum, P. digitatum and P. italicum on different cultivars of apple and citrus fruits. pH was measured directly with a micro-combination pH electrode Model 9810BN (Orion, Beverly, MA).  Measurements were taken 7 days after inoculation.

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analyzed by modulation (increase or decrease) of gluconic acid accumulation at the infection court. P. expansum isolates that express higher gox2 transcripts, glucose oxidase (GOX) activity and that secrete the highest amount of gluconic acid, caused disease of apple at the fastest rate (Fig. 4.6). Furthermore, the detection of significantly high levels of transcripts of gox2 and GOX activity at the edge of the decaying tissue emphasize the involvement of GOX in tissue acidification of the decaying tissue. Taken together, these results emphasize the importance of GOX in the production of the gluconic acid that leads, in turn, to host tissue acidification (Hadas et al. 2007). Organic acid accumulation findings, demonstrate that there is a close relation between the accumulation of gluconic acid and virulence of P. expansum, S. sclerotiorum and

Fig.  4.6  Effect of the aggressiveness of Penicillium expansum isolates on the pH and on the production of organic acids in infected Golden Delicious apples. (a) Decayed area 5 days after inoculation with P. expansum; (b) pH of the infected tissue. Average values of 10 replicates (± SD) are presented and (c) Amounts of organic acids (dark column indicates citric acid and bright column indicates gluconic acid)

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B. cinerea (Rollins and Dickman 2001; Manteau et  al. 2003). The production of oxalic acid by S. sclerotiorum is regulated by the ambient pH environment that met the pathogen. This regulation of pH responsive genes is mediated in part by the zinc finger transcription factor encoded by pac1, an orthologue of the Aspergillus nidulans pacC gene. Accumulation of pac1 transcripts paralleled increases in ambient pH, and oxalic acid production increases with the ambient pH of the growth medium, as does oxaloacetase activity, the enzyme proposed to catalyze oxalic acid production by hydrolysis of oxaloacetate (Rollins and Dickman 2001). Oxalic acid secretion is a pH-regulated process and, in turn, its accumulation, by virtue of environment acidification, may serve as a regulator of acid pH-regulated processes. Functionally characterization of the pacC gene homolog, pac1, from S. sclerotiorum' have demonstrated that although oxalic acid production is alkaline pH-responsive, the kinetics and magnitude of oxalate accumulation are dramatically altered and virulence, in loss-of-function pac1 mutants, was dramatically reduced in infection assays with tomato. Based on these results, pac1 appears to be necessary for the appropriate regulation of physiological processes (Rollins 2003). The accumulating findings suggested that pH modulation of the environment enables the pathogen the “selection” of specific virulence factors needed for the particular host. Several studies on fungal pathogen demonstrate that the ambient pH conditions in each host induce the expression of a specific subset of fungal genes, selected from large gene families that encode cell-wall-degrading enzymes (Eshel et al. 2002; Prusky and Yakoby 2003). In the case of P. expansum, the secretion of gluconic acid and, to a lesser extent, citric acid enhanced the expression of pectolytic enzymes and the establishment of conditions for necrotrophic development of P. expansum (Hadas et al. 2007; Torres and Candelas 2003; Prusky et al. 2003). Analysis of transcripts encoding the endopolygalacturonase gene, pepgl, from P expansum accumulated under acidic culture conditions from pH 3.5 to 5.0, suggesting that the acidification process is a pathogenicity enhancing factor of Penicillium spp. This hypothesis was supported by the findings that cultivars with lower pH as well as citric acid treatments that reduced tissue pH, increased P. expansum development. However, organic acid treatment could not enhance decay development in naturally acidic apples. Conversely, local alkalinization with NaHCO3 reduced decay development (Prusky et al. 2003). The accumulated findings so far demonstrate that ambient pH is a regulatory cue for processes linked to pathogenicity, and that specific genes are expressed as a result of the modified host pH created by the pathogen.

4.4 Effectors That Activate the Transition from Quiescent to Necrotrophic Infection The transition from biotrophism to a necrotrophic-saprophytic stage appears to be related to factors that are modulated at the intracellular level, and that are affected by nutrients and by ambient pH. Each of the secreted compounds (organic acid or

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ammonia) plays a critical physiological role in the initiation of necrotrophic development. Speculation regarding the mechanisms by which secretion of organic acids enhances virulence centers on three modes of action. First, oxalate may be directly toxic to host plants and may weaken them, thereby facilitating invasion. Second, it has been hypothesized that chelation of cell-wall Ca2+ by oxalic and gluconic acids loosens the plant cell wall (Bateman and Beer 1965; Hadas et al. 2007). Finally, oxalate secretion may suppress and activate ROS generation in a pH dependent manner and the associated plant defense responses, thereby contributing to activation of the necrotrophic mode of development (Cessna et al. 2000; Kim et al. 2008). Ammonification of the tissue also has significant physiological and biochemical effects both on the host and on the pathogen. Healthy plant cells have an electrochemical proton gradient across the plasma membrane that is important for ion uptake, solute transport, and cell-wall growth. Several studies have demonstrated that transient extracellular alkalization is essential for the induction of defense responses by fungal elicitors (Schaller and Oecking 1999). Ammonium-toxicity symptoms can lead to host stress responses expressing elevated ethylene synthesis and various changes in membrane flux (Ingermarsson et al. 1987). Alkan et al. (2009) demonstrate third role for ammonium secretion in the induction of host ROS accumulation through the NADPH oxidase mechanism in a pH dependent manner. ROS activation by ammonium can act as a factor promoting cell death in tomato fruit at the leading edge of the colonizing hyphae (Alkan et  al. 2009). KramerHaimovich et al. (2006) suggested a fourth way to affect colonization. Ammonium can directly affect the fungus pathogenicity by activating the expression of genes encoding pathogenicity factors, such as pelB expression and PL secretion in C. gloeosporioides. Finally ammonium can also indirectly affect the pathogen-host interaction by elevating the local pH of the infection site to create an optimal environment for production of pathogenicity factors as PL and endoglucanase (Yakoby et  al. 2001; Eshel et  al. 2002). This suggests that ammonification by Colletotrichum at the leading edge of the infection site may activate host responses leading to cell death and activate fungal pathogenicity factors. This way ammonium may act as an effector for activation of the transition from quiescent to necrotrophic mode in the alkalizing pathogen life cycle.

4.5 Summary It has become clear in recent years that the activation of quiescent infections is not a simple process whereby a decline in host resistance results in the activation of fungal attack. Activation of quiescent biotrophic infections seems to involve a coordinated series of events. The complexity of this process can be attributed to the significant physiological and biochemical changes that occur in the host during ripening and senescence, and that lead to decreased host response and increased susceptibility. In parallel, activation of quiescent fungal infections consists of processes that compromise host defenses directly or indirectly by detoxi-

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fication of antifungal agents. The physiological changes that accompany fruit ripening and host senescence, e.g. host pH, sugar content, cell-wall components and oxidation of wounded tissue, trigger fungal host modulation. The acidification of the tissue by organic acids (oxalic and gluconic) or its alkalization by ammonia and the possible modulation of ROS, may contribute to rapid necrotization of the tissue. Further amplification of the decay can result from activation of gene expression and release of cell-wall-degrading enzymes. The transition from the biotrophic stage to the necrotrophic one could then be resolved by characterizing the temporal changes in gene expression and protein during the transition. Further clarification of the role of putative signals (pH, nitrogen and sugar) in postharvest pathogenesis during fruit ripening is clearly needed. Nevertheless, the current state of knowledge of fungal modulation of host pH has already opened new avenues to the control of postharvest pathogens thus studies of virulence as a result of pH-conditioning by the pathogen support the developing of new strategies of postharvest fungal pathogen control involve local pH changes (Prusky et al. 2006).

References Alkan N, Fluhr R, Sherman A, Prusky D (2008) Role of ammonia secretion and pH modulation on pathogenicity of Colletotrichum coccodes on tomato fruit. Mol Plant Microbe Interact 21:1058–1066 Alkan N, Fluhr R, Sagi M, Davydov O, Prusky D (2009) Ammonia secreted by Colletotrichum coccodes effects host NADPH oxidase activity enhancing cell death and pathogenicity in tomato fruits. Molecular Plant-Microbe Interactions 22:In press Bailey JA, Jeger MJ (1992) Colletotrichum: biology, pathology and control. CAB International, Wallingford Bateman DF, Beer SV (1965) Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology 58:204–211 Cessna SG, Searsa VE, Dickman MB, Low PS (2000) Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12:2191–2199 Coates LM, Muirhead IF, Irwing JAG, Gowanlock DH (1993) Initial infection processes by Colletotrichum gloeosporioides on avocado fruit. Mycol Res 97:1363–1370 Denison SH (2000) pH regulation of gene expression in fungi. Fungal Genet Biol 29:61–71 Drori N, Kramer-Haimovich H, Rollins J, Dinoor A, Okon Y, Pines O, Prusky D (2003) External pH and nitrogen source affect secretion of pectate lyase by Colletotrichum gloeosporioides. Appl Environ Microbiol 69:3258–3262 Eshel D, Miyara I, Ailinng T, Dinoor A, Prusky D (2002) pH regulates endoglucanase expression and virulence of Alternaria alternata in persimmon fruits. Mol Plant Microbe Interact 15:774–779 Fourie JF, Holz G (1995) Initial infection process by Botrytis cinerea on nectarine and plum fruits and the development of decay. Phytopathology 85:82–87 Germeier C, Hedke K, Tiedemann AV (1994) The use of pH-indicators in diagnostic media for acid-producing plant pathogens. Z Pflanzenkr Pflanzenschutz 101:498–507 Hadas Y, Goldberg I, Pines O, Prusky D (2007) The relationship between expression of glucose oxidase, gluconic acid accumulation, acidification of host tissue and the pathogenicity of Penicillium expansum. Phytopathology 97:384–390

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Ingermarsson B, Oscarsson P, Ugglas MA, Larsson CM (1987) Nitrogen utilization III. Short-term effects of ammonium on nitrate uptake and nitrate reduction. Plant Physiol 85:865–867 Kim KS, Min JY, Dickman MB (2008) Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development. Mol Plant Microbe Interact 21:605–612 Kramer-Haimovich H, Servi E, Katan T, Rollins J, Okon Y, Prusky D (2006) Effect of ammonia production by Colletotrichum gloeosporioides on pelB activation, pectate lyase secretion, and fruit pathogenicity. Appl Environ Microbiol 72:1034–1039 Latunde-Dada AO, O’Connell RJ, Nash C, Pring RJ, Lucas JA, Bailey JA (1996) Injection process and identity of the hemibiotrophic anthracnose fungus (Colletotrichum destructivum O’Gara) from cowpea (Vigna unguiculata (L.) Walp.). Mycol Res 100:1133–1141 Manteau S, Abouna S, Lambert B, Legendre L (2003) Differential regulation by ambient pH of putative virulence factors secretion by the phytopathogenic fungus Botrytis cinerea. Fems Microbiol Ecol 43:359–366 Mendgen K, Hahn M (2001) Plant infection and the establishment of fungal biotrophy. Trends Plant Sci 6:496–498 Miyara I, Shafran H, Kramere-Haimovich H, Rollins J, Sherman A, Prusky D (2008) Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits. Mol Plant Pathol 9:281–291 O’Connell RJ, Bailey JA, Richmond DV (1985) Cytology and physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Physiol Plant Pathol 27:75–98 Perfect SE, Bleddyn Hughes H, O’Connell RJ, Green JR (1999) Colletotrichum: a model genus for studies on pathology and fungal-plant interactions. Fungal Genet Biol 27:186–198 Pezet R, Viret O, Perret C, Tabacchi R (2003) Latency of Botrytis cinerea Pers.:Fr. and biochemical studies during growth and ripening of two grape berry cultivars, respectively susceptible and resistant to grey mould. J Phytopathol 151:208–214 Prusky D (1996) Pathogen quiescence in postharvest diseases. Annu Rev Phytopathol 34:413–434 Prusky D, Lichter A (2007) Activation of quiescent infections by postharvest pathogens during transition from the biotrophic to the necrotrophic stage. FEMS Microbiol Lett 268:1–8 Prusky D, Yakoby N (2003) Pathogenic fungi: leading or led by ambient pH? Mol Plant Pathol 4:509–516 Prusky D, McEvoy JL, Leverentz B, Conway WS (2001a) Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence. Mol Plant Microbe Interact 14:1105–1113 Prusky D, Eshel D, Kobiler I, Yakoby N, Beno-Moualem D, Ackerman M, Zuthji Y, Ben Arie R (2001b) Postharvest chlorine treatments for the control of the persimmon black spot disease caused by Alternaria alternata. Postharvest Biol Technol 22:271–277 Prusky D, McEvoy JL, Saftner R, Conway WS, Jones R (2004) The relationship between host acidification and virulence of Penicillium spp. on apple and citrus fruit. Phytopathology 94:44–51 Prusky D, Kobiler I, Akerman M, Miyara I (2006) Effect of acidic solutions and acid Prochloraz on the control of postharvest decays caused by Alternaria alternata in mango and persimmon fruit. Postharvest Biol Technol 42:134–141 Rollins JA (2003) The Sclerotinia sclerotirum pac1 gene is required for sclerotial development and virulence. Mol Plant Microbe Interaction 16:785–795 Rollins JA, Dickman MB (2001) pH signaling in Sclerotinia sclerotiorum: identification of pacC/ RIM1 homolog. Applied Environ Microbiol 67:75–81 Ruijter GJG, van de Vondervoort PJ, Visser J (1999) Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese. Microbiology 145:2569–2576 Schaller A, Oecking C (1999) Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11:263–272

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Torres PS, Candelas LG (2003) Isolation and characterization of genes differentially expressed during the interaction between apple fruit and Penicillium expansum. Mol Plant Pathol 4:447–457 Yakoby N, Kobiler I, Dinoor A, Prusky D (2000) pH regulation of pectate lyase secretion modulates the attack of Colletotrichum gloeosporioides on avocado fruits. Appl Environ Microbiol 66:1026–1030 Yakoby N, Beno-Moualem D, Keen NT, Dinoor A, Pines O, Prusky D (2001) Colletotrichum gloeosporioides pelB, is an important factor in avocado fruit infection. Mol Plant Microbe Interact 14:988–995

Chapter 5

Global Regulation of Genes in Citrus Fruit in Response to the Postharvest Pathogen Penicillium digitatum L. González-Candelas, S. Alamar, A.R. Ballester, P. Sánchez-Torres, J. Forment, J. Gadea, M.T. Lafuente, L. Zacarías, and J.F. Marcos

Abstract  Large-scale EST sequencing projects and microarray hybridization nowadays constitute two major approaches to analyse biological systems at a molecular level. Although the use of these methodologies is becoming commonplace in plant pathology, their application to postharvest pathology has not yet been reported. In this chapter we analyze the overall response of citrus fruit to Penicillium digitatum infection from a genomic perspective. We have constructed both subtracted and regular cDNA libraries from infected fruits. Analysis of the non-subtracted library gives us a picture of what genes are being transcribed, whereas the subtracted library provides more direct information on what citrus genes are induced upon P. digitatum infection. A cDNA macroarray generated from the subtracted library has been used to identify what genes are up-regulated in response to pathogen infection, and to determine the influence of ethylene on their expression. Under the framework of the Spanish ‘Citrus Functional Genomic Project, CFGP’ more than fifty cDNA libraries have been generated encompassing more than 90,000 ESTs. These clones have been used to develop a cDNA microarray representing roughly 7,000 unigenes. With this tool we have analyzed the differences and commonalities of the citrus fruit responses to ethylene, wounding, P. digitatum infection and induced resistance. We will present the results of such analyses emphasizing not only what genes are affected to a larger extent but also describing what are the major changes in biological processes and metabolic pathways. Citrus is the major fruit crop worldwide with an estimated annual production above 100 million metric tons in 2007 (FAO, http://faostat.fao.org). Although most L. González-Candelas (*), S. Alamar, A.R.Ballester, P. Sánchez-Torres, M.T. Lafuente, L. Zacarías, and J.F. Marcos Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), PO Box 73 Burjassot, 46100, Valencia, Spain e-mail: [email protected] J. Forment and J. Gadea Instituto de Biología Molecular y Celular de Plantas (IBMCP), UPV-CSIC, Avenida de los Naranjos s/n, 46 022, Valencia, Spain D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_5, © Springer Science + Business Media B.V. 2010

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of the produce is devoted to juice production, the primary destination in Spain is for the fresh consumption market. During postharvest handling and storage, citrus fruit are prone to fungal decay. Penicillium digitatum and Penicillium italicum are the two major pathogens of citrus fruit being the causal agents of green and blue mould diseases, respectively. Control of these pathogens has classically been conducted with chemical fungicides, but the actual problems they face from a variety of points of view makes it necessary to undertake new approaches for disease control that minimize health and environmental risks. Different alternative control methods are being developed by numerous research groups worldwide. Most of these methods, either physical, chemical or biological, do not rely on the knowledge of the pathosystem (Palou et al. 2008). However, a deeper understanding of the fruit’s defence capabilities and of the pathogen’s pathogenicity mechanisms may lead us to more rational approaches for disease control. There are few studies focusing on the defence mechanisms of citrus fruit to postharvest pathogens. Only some b-1,3-glucanases (Porat et al. 2002), chitinases (Porat et al. 2001) and some phenylpropanoid (Arcas et al. 2000; Ballester et al. 2006; Droby et al. 2002; Kim et al. 1991, Lers et al. 1998; Ortuño et al. 2006) and oxidative stress (Ballester et  al. 2006) genes/compounds have been identified as components of the fruit’ defence arsenal that are induced either in response to fungal attack or to elicitor treatment. We have also recently shown the relevance of ethylene metabolism in the response of citrus fruit to P. digitatum infection (Marcos et  al. 2005). However, all these studies are narrow-focused ones. Nowadays we have the possibility of addressing a biological problem from a wider perspective by using non-biased approaches and taking advantage of the new ‘-omics’ tools that are being implemented in other fields of plant pathology. In this context we have initiated a large scale characterization of the citrus transcriptome with the aim of deciphering at the molecular level the defence responses of citrus fruit to pathogens and to compare these responses with those elicited by other treatments, such as wounding, exogenous ethylene application or with treatments that increase the resistance of the fruit to a subsequent pathogen attack. This initiative has been possible within the frame of the Spanish Citrus Functional Genomics Project (CFGP; http://bioinfo.ibmcp.upv.es/genomics/ cfgpDB/), a joint effort devoted to the development of genomic tools for the genetic improvement of citrus. To this end CFGP groups have generated a large collection of ESTs from numerous cDNA libraries that have then been used for the construction of a citrus cDNA microarray (Forment et al. 2005). We have conducted a multifaced approach, as depicted schematically in Fig. 5.1. On one hand we have obtained a subtracted cDNA library enriched in citrus genes up-regulated at 24 h after inoculation (hai) with P. digitatum spores (RindPdigS in Fig. 5.1). This library was prepared following the Suppression Substractive Hybridization technique (Diatchenko et  al. 1996) using mRNA derived from the rind of ‘Navelina’ oranges as ‘tester’ RNA and mRNA from wounded and mockinoculated oranges as ‘driver’. By using wounded tissue as a control we expected to enrich the cDNA library in genes that are induced specifically, or at least to a higher level, in response to fungal infection. More than 1,400 cDNA inserts from

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Fig. 5.1  Scheme representing the approaches undertaken to characterize the response of citrus fruit to P. digitatum infection. Dashed lines indicate experiments conducted with ‘Navelina’ oranges, dotted lines indicate experiments conducted with ‘Clemenules’ mandarins

this library were spotted onto nylon membranes in order to conduct hybridization analyses with RNA samples derived from orange fruits subjected to different treatments: (i) control fruits stored in air at 20ºC and high relative humidity for 24 h, (ii) fruits that have been wounded and mock-inoculated with sterile water and stored at the same conditions for 24 h, (iii) fruits inoculated with P. digitatum spores at 106 conidia/mL and stored for 24 h, and (iv) fruits that have been incubated with 10 ppm of ethylene in closed jars. Triplicate hybridization experiments were conducted and the resulting images were analyzed to find genes showing differential expression, either up- or down-regulation, among treatments. A first analysis between infected and wounded orange tissues was done in order to confirm whether the library was effectively enriched in fruit genes showing a higher expression level in response to Penicillium infection. This comparison revealed that the number of genes showing

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statistically higher expression in response to infection in comparison to wounding represented more than 80% of the genes showing differential expression between both treatments. An illustrative example of the macroarray hybridization with ‘wounding’ and ‘infection’ RNA samples is shown in Fig.  5.2. The overall results from the macroarray hybridization experiments showed that ‘infection’ was the treatment that triggered the largest number of changes in gene expression, both induction and repression, in comparison to control, non-treated fruit. It is interesting to note that roughly half of the genes that were up-regulated in response to P. digitatum infection were also up-regulated either in response to exogenous ethylene treatment or in response to wounding, a fact that reflects the important role of ethylene mediating the fruit responses to pathogen attack. However, the overlap between ethylene and infection treatments is not so extensive with the repressed genes. We have sequenced more than 350 clones from the RindPdigS library encompassing representatives of all different expression patterns obtained after macroarray hybridization. On the other hand, we have constructed a regular cDNA library (neither subtracted nor normalized) from the rind of ‘Clemenules’ mandarins that had been Wounding (24 hpi)

Infection (24 hpi)

Fig.  5.2  Hybridizations of the macroarray derived from the RindPdigS cDNA library. (a) Membranes were hybridized with RNA derived from wounded (W) or P. digitatum-infected (I) orange fruits 24 h after inoculation. (b) Detailed area of the membranes showing within circles several spots that show differential expression in both treatments

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inoculated 24 h earlier with P. digitatum spores (RindPdig24 in Fig.  5.1) and have sequenced more than 1100 randomly selected clones. Although the number of sequenced clones was lower in the RindPdigS library, the redundancy, which reflects the number of repeated sequences within the library, doubles that of RindPdig24. This difference reflects the complementary nature of both cDNA libraries. The number of ESTs in the subtracted cDNA library correlates with its induction level in the citrus peel in response to P. digitatum infection with respect to the wound response, even tough its absolute expression level is low. However, a highly represented gene in the regular cDNA library corresponds to a gene with a high level of expression whether it is induced or not. As a matter of fact many of the genes with a higher representation in RindPdigS are not present in RindPdig 24. One notable exception is the CsACO gene, which codes for an ACC oxidase and is highly abundant in both cDNA libraries, confirming previous results that highlighted the important role of ethylene in the defence response of citrus fruit to P. digitatum attack (Marcos et al. 2005). A global analysis that integrates all the nucleotide sequences obtained from both libraries can uncover the biological processes that are switched on or off in the peel of citrus fruit when challenged with this important pathogen. Gene ontology (GO) categorization is one of the tools that has become more popular for this purpose. As most of the citrus cDNA sequences have a homolog counterpart in Arabidopsis thaliana we have thus categorized citrus cDNA sequences according to the assignment of their closest A. thaliana homologs. Table 5.1 summarizes the distribution of the different ‘Biological Processes’ in RindPdigS and RindPdig24 cDNA libraries. For comparison purposes the distribution of the whole CFGP dataset is also included. The processes ‘response to stimulus’, ‘amino acid and derivative metabolism’ and ‘electron transport’ are over-represented in both cDNA libraries with respect to the overall CFGP dataset, but to a much higher extent in the subtracted library. Interestingly, ‘metabolism’ and ‘cell death’ processes are only more abundant in the RindPdigS library. Although some of the categories are too broad to withdraw conclusions from them, we can further analyze to a deeper level the annotation of the members of each process. For example, the analysis of the subcategories included in ‘metabolism’ indicates that secondary metabolism, and more specifically the metabolism of phenylpropanoids, is the most represented process in RindPdigS. Another interesting observation that highlights the importance of preparing specific cDNA libraries is the high amount of genes that are only present in the infection-derived libraries that otherwise would have not been isolated. Thus, one of the two most abundant contigs in the subtracted cDNA library, with 11 ESTs and that does not have homologous sequences in databases, is not present in any other of the CFGP cDNA libraries that actually comprise more than 85,000 good quality ESTs. The last approach we have undertaken to characterize the early response of citrus fruit to P. digitatum infection is to perform a global analysis of citrus gene expression by using the citrus ‘7k’ cDNA microarray containing 12,672 probes corresponding to 6,875 unigenes that has been developed in the CFGP (Forment et  al., 2005). RNA from three biological replicates of ‘Clemenules’ mandarins subjected to the same treatments described before were used to conduct hybridizations. We employed the common reference strategy by which each sample was labelled

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Table 5.1  ‘Biological process’ gene ontology annotation of RindPdigS and RindPdig24 cDNA libraries in comparison with the overall CFGP database GO biological process classification RindPdigS RindPdig24 CFGP No biological process GO annotation 45.7 46.4 54.8 Cellular physiological process 21.7 21.3 14.0 Biological process unknown 12.0 13.2 14.5 Macromolecule metabolism 12.5 18.9 12.8 Biosynthesis 11.4 12.5 6.3 Metabolism 8.7 4.4 4.2 Nucleobase- nucleoside- nucleotide and nucleic acid 3.3 4.3 5.0 metabolism Response to stimulus 8.2 4.8 3.0 Amino acid and derivative metabolism 6.0 2.7 1.7 Physiological process 0.7 0.7 0.5 Catabolism 2.7 3.6 1.5 Transport 3.8 4.9 4.3 Regulation of biological process 2.2 1.5 2.9 Electron transport 6.5 3.3 1.8 Cell communication 0.5 1.1 1.5 Development 1.1 0.7 0.6 Cell death 1.1 0.3 0.3 Numbers indicate the percentage of sequences from each dataset belonging to each process. A single sequence can be assigned to more than one process.

with Cy5, visualized as red spots in the images, and hybridized with a reference labelled with Cy3 that was made up with equal amounts of RNA from all analyzed samples. Two technical replications were conducted for each sample. Thus, a total of six hybridizations were conducted for each treatment. Differentially expressed genes among the four conditions were identified by SAM with a FDR < 0.01 (Tusher et al. 2001). When the results were compared against control fruit, infection was the condition that induced the largest number of changes in gene expression, followed by ethylene and wounding, being the difference more pronounced in down-regulated genes. The two genes showing the largest induction in response to P. digitatum infection showed homology to the cowpea CPRD2 gene, a gene that is induced in response to dehydration and with no known function yet. This result also confirms the validity of the RindPdigS library, as one of the most abundant contigs in this library also shows homology to the same gene. CPRD2 codes for an oxidoreductase that contains both a FAD-binding domain and a berberine bridge enzyme (BBE) domain. BBE converts the N-methyl group of (S)-reticuline into the methylene bridge moiety of (S)-scoulerine, a conversion that is unique in nature (Facchini et al. 2004). BBE is a key branchpoint enzyme in the biosynthesis of certain benzylisoquinoline alkaloids (Ziegler and Facchini 2008). Besides these two genes, among the top positions of genes showing the highest induction in response to the pathogen there are other genes coding for enzymes involved in secondary metabolism, such as PAL, O-methyl transferases or a cytochrome P450, as well as enzymes involved in the synthesis of ethylene, such ACC oxidase, two processes that were also highlighted in the analysis of the subtracted cDNA library.

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Although the overall results derived from both the subtracted cDNA library and the microarray analyses are qualitatively similar, surveying thousand of genes at the same time gives us the opportunity to take a deeper look inside the biology of the fruit tissue. GO classification of those genes that showed differential expression between conditions, either induction or repression, is one of the ways we can gather more information beyond the identification of genes showing the strongest up or down-regulation. Table  5.2 shows the GO classification of the genes that are

Table 5.2  Biological processes differentially represented (p < 0.05) among treatments Infection Infection vs Infection vs Biological process vs. air wounding ethylene Resistance Level 6 Amine biosynthetic process + + Amino acid derivative biosynthetic + + process Amino acid metabolic process + + + Cellular response to water − deprivation Defense response, incompatible + interaction Phenylpropanoid metabolic process + Translation + + − Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process Nucleotide biosynthetic process − Cell wall modification − Level 7 Amino acid biosynthetic process + + Aromatic amino acid family + metabolic process Aspartate family amino acid + metabolic process Methionine methabolic process + Jasmonic acid and ethylene+ dependent systemic resistance Pyridine nucleotide metabolic + process Pyruvate metabolic process + Level 8 Methionine metabolic process + Sulfur amino acid biosynthetic + + process Aromatic amino acid family + + biosynthetic process Isopentenyl diphosphate + biosynthetic process The ‘+’ value denotes that the process is over-represented in the first condition, whereas the ‘−’ value indicates that it is under-represented. Only genes that showed statistically differential expression in each comparison where consider for the analysis.

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differentially expressed in the comparison between ‘infection’ and the other three treatments. Lower levels of GO classification reflect broader processes and higher levels show more specific ones. Several over-represented processes in the infected tissue are related to secondary metabolism, either directly, as the phenylpropanoid metabolism, or indirectly through the synthesis of the major precursors of the different pathways, such as the aromatic amino acids tyrosine, tryptophan and phenylalanine, or the terpenoid’s precursor isopentenyl diphosphate. Synthesis of ethylene is included in processes involving the sulphur amino acid methionine. Defence responses typical of incompatible interactions together with the activation of systemic resistance dependent of the hormones ethylene and jasmonic acid are also processes triggered by P. digitatum, in agreement with the observed activation of cell death that was observed in the subtracted cDNA library. Classically, induction of cell death processes in the plant has been considered a landmark of the hypersensitive response triggered in an incompatible interaction by the recognition of an avirulence (avr) gene product from the pathogen by the resistant (R) gene product in the plant. Although host cell death is effective in deterring the progress of biotrophic pathogens, its role with necrotrophic fungi might be completely different (Glazebrook 2005). Host cell death favours infection progress of the necrotroph fungus Botrytis cinerea. Moreover, B. cinerea is able of triggering the death of host cells as a means to obtain nutrients (Govrin and Levine 2000; Govrin et al. 2006). According to our results the same situation may holds true for P. digitatum infection of citrus fruit. Another important source of information derived from the microarray analysis is the possibility of studying simultaneously the regulation of the genes belonging to the same metabolic pathway. As an example, the synthesis of phenylalanine from the shikimate-chorismate pathway is presented in Fig. 5.3. In the citrus 7 k microarray there are 12 genes whose homologous A. thaliana counterparts code for enzymes involved in the different reactions. Many of them are induced in the citrus peel in response to the presence of the pathogen, mainly at entry of the pathway. In summary, the results obtained give us a global view on how the flavedo and albedo cells of the fruit rind respond to the pathogen modifying their metabolism towards secondary metabolism with ethylene being a major player in the process. At least part of the response resembles the characteristics of a hypersensitive response that characterize the incompatible interaction. However, despite this deployment of responses by the fruit, the pathogen is able to successfully invade the cortical tissue. What we do not know yet is whether this is achieved by down-regulating later on the defences triggered by the fruit or by overcoming the different set of barriers deployed by the host.

5  Global Regulation of Genes in Citrus Fruit D-eritrose-4-phosphate

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Fig.  5.3  Representation of the pathway leading to the synthesis of phenylalanine. The first number in parenthesis indicates the number of citrus genes present in the 7 k microarray that have a homologous gene in A. thaliana and the second number indicates the number genes coding for the corresponding enzyme in A. thaliana. For each gene, the E, W, and I values are the Log2 ratios of the expression level for each condition (ethylene, wounding and infection, respectively) with respect to control fruits stored in air. The ‘+’sign denotes lack of expression in air, whereas the ‘-’ sign indicates lack of expression in the particular treatment

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Acknowledgments  We acknowledge A. Izquierdo and M.J. Pascual for their excellent technical assistance. This work was supported by grants from the Spanish Ministry of Science and Education (AGL2000-1443, GEN2001-4885-C05-04, AGL2002-01727, AGL2005-04921-C02-01) and the Generalitat Valenciana (AVCiT-GRUPOS03/008).

References Arcas MC, Botia JM, Ortuño AM, Del Río JA (2000) UV irradiation alters the levels of flavonoids involved in the defence mechanism of Citrus aurantium fruits against Penicillium digitatum. Eur J Plant Pathol 106:617–622 Ballester AR, Lafuente MT, González-Candelas L (2006) Spatial study of antioxidant enzymes, peroxidase and phenylalanine ammonia-lyase in the citrus fruit-Penicillium digitatum interaction. Postharvest Biol Technol 39:115–124 Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030 Droby S, Vinokur V, Weiss B, Cohen L, Daus A, Goldschmidt EE, Porat R (2002) Induction of resistance to Penicillium digitatum in grapefruit by the yeast biocontrol agent Candida oleophila. Phytopathology 92:393–399 Facchini PJ, Bird DA, St Pierre B (2004) Can Arabidopsis make complex alkaloids? Trends Plant Sci 9:116–122 Forment J, Gadea J, Huerta L, Abizanda L, Agusti J, Alamar S, Alos E, Andres F, Arribas R, Beltran JP, Berbel A, Blazquez MA, Brumos J, Canas LA, Cercos M, Colmenero-Flores JM, Conesa A, Estables B, Gandia M, Garcia-Martinez JL, Gimeno J, Gisbert A, Gomez G, González-Candelas L, Granell A, Guerri J, Lafuente MT, Madueno F, Marcos JF, Marques MC, Martinez F, Martinez-Godoy MA, Miralles S, Moreno P, Navarro L, Pallas V, Perez-Amador MA, Perez-Valle J, Pons C, Rodrigo I, Rodriguez PL, Royo C, Serrano R, Soler G, Tadeo F, Talon M, Terol J, Trenor M, Vaello L, Vicente O, Vidal CH, Zacarías L, Conejero V (2005) Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies. Plant Mol Biol 57:375–391 Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227 Govrin EM, Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr Biol 10:751–757 Govrin EM, Rachmilevitch S, Tiwari BS, Solomon M, Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other plants and promotes the gray mold disease. Phytopathology 96:299–307 Kim JJ, Ben-Yehoshua S, Shapiro B, Henis Y, Carmeli S (1991) Accumulation of scoparone in heat-treated lemon fruit inoculated with Penicillium digitatum Sacc. Plant Physiol 97:880–885 Lers A, Burd S, Lomaniec E, Droby S, Chalutz E (1998) The expression of a grapefruit gene encoding an isoflavone reductase-like protein is induced in response to UV irradiation. Plant Mol Biol 36:847–856 Marcos JF, González-Candelas L, Zacarías L (2005) Involvement of ethylene biosynthesis and perception in the susceptibility of citrus fruits to Penicillium digitatum infection and the accumulation of defense-related mRNAs. J Exp Bot 56:2183–2193 Ortuño A, Baidez A, Gomez P, Arcas MC, Porras I, Garcia-Lidon A, Del Rio JA (2006) Citrus paradisi and Citrus sinensis flavonoids: their influence in the defence mechanism against Penicillium digitatum. Food Chem 98:351–358

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Palou L, Smilanick JL, Droby S (2008) Alternatives to conventional fungicides for the control of citrus postharvest green and blue moulds. Stewart Postharvest Rev 4:1–16 Porat R, Vinokur V, Holland D, McCollum TG, Droby S (2001) Isolation of a citrus chitinase cDNA and characterization of its expression in response to elicitation of fruit pathogen resistance. J Plant Physiol 158:1585–1590 Porat R, McCollum TG, Vinokur V, Droby S (2002) Effects of various elicitors on the transcription of a beta-1, 3-endoglucanase gene in citrus fruit. J Phytopathol 150:70–75 Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121 Ziegler J, Facchini PJ (2008) Alkaloid biosynthesis: metabolism and trafficking. Annu Rev Plant Biol 59:735–769

Chapter 6

Epidemiological Assessments and Postharvest Disease Incidence Themis J. Michailides, David P. Morgan, and Yong Luo

Abstract  Postharvest plant disease can be measured by “incidence,” by recording the presence or absence of symptoms, and “severity,” the degree to which the symptoms are expressed. Weather and other environmental conditions play a significant role by causing stress in plants and lowering natural defenses, and by creating conditions suitable for pathogens to infect the plants. Specifically for postharvest diseases of fruit, infections can start as early as fruit set and continue until harvest. Although weather conditions influence the epidemiology of a disease in the field, the incidence of postharvest disease depends on the incidence of latent infections that initiate in the field during the season, contamination with fungal propagules during harvest, the effectiveness of postharvest treatments, and storage and marketing conditions. True latent infection, defined as a parasitic relationship that eventually induces macroscopic symptoms (Verhoeff, K., Annu. Rev. Phytopathol. 12:99–107, 1974) plays a major role in both the incidence and severity of postharvest disease. If conditions are favorable, incidence and severity of latent infections will be higher and the risk for postharvest disease development will increase and vice versa. For example, in California kiwifruit there is a positive relationship between the incidence of latent infection of sepals or stem ends, and the incidence of gray mold of fruit in cold storage. We visualize kiwifruit and other kinds of fruit as recording devices that copy the environmental conditions as latent infections. And in some cases, quantification of these latent infections can predict postharvest disease (i.e. BOTMON (Botrytis monitoring in kiwifruit sepals and/or fruit stems and in stems of grape berries) and ONFIT (overnight freezing incubation technique in stone fruit, other fleshy fruit, and in nut crops)). The source of inoculum that can drive an epidemic of a disease in the field can also affect

T.J. Michailides (), D.P. Morgan, and Y. Luo Department of Plant Pathology, Kearney Agricultural Center, University of California-Davis, 9240 South Riverbend Ave, Parlier, CA 93648, USA e-mail: [email protected] D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century Vol. 2, DOI 10.1007/978-1-4020-8930-5_6, © Springer Science + Business Media B.V. 2010

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the ­incidence of postharvest decay, by affecting the incidence of latent infection. Reducing the source of inoculum (sanitation) can reduce the incidence of latent infection of fruit, with the ultimate result in reducing postharvest disease (i.e., stone and pome fruit). Environmental conditions during bloom in various crops can have detrimental effects on the incidence of postharvest disease (i.e., grapes, pomegranates, prunes, etc.) by affecting the levels of latent infection or affecting the plant host directly. Altering cultural practices that may affect environmental conditions in the field or the physiology and histology of fruit can also affect the incidence of fruit diseases both in the field and postharvest. The development of efficient, accurate, and rapid molecular techniques (including real-time PCR assays) can facilitate the detection and quantification of disease inoculum and latent infection of fruit (i.e., stone fruit) and help predict incidence of postharvest disease. In addition, development of allele-specific RT-PCR methods for rapidly detecting fungicide resistant fungal pathogens will help growers to manage fungicide resistance and make correct decisions to reduce postharvest disease. The goal of our laboratory is to develop less expensive molecular techniques that determine latent infections and assess populations of fungi resistant to fungicides and enable us to process large numbers of samples at our laboratory and to provide the protocols to private laboratories.

6.1 Definitions Before proceeding with this review, we would like to define the various kinds of infection. According to Verhoeff 1974) latent infection of plants by pathogenic fungi is often considered of the highest levels of parasitism, since the host and parasite coexist for a period of time with minimal or no damage to the host. In other words, these infections are successfully established infections some of which will perish while others will survive and resume development as the physiological characteristics of the tissues where these infections reside change. A true latent infection involves a parasitic relationship that eventually induces macroscopic symptoms (Verhoeff 1974). Quiescent infection, however, is microscopically visible although mycelial development is arrested after infection and resumes only as the host plant reaches maturity and/or senescence (Sinclair and Cerkauskas 1996). Latent contamination, which we prefer the term inoculum load for it, involves fungal spores (or other type of fungal propagules) on the host’s surface which fail to germinate until the host reaches maturity or senescence, or wounded by insects and other means. Latent infection of plants by parasitic fungi is often considered one of the highest levels of parasitism, since the host and parasite coexist for a period of time with minimal damage to the host. Latency involves an asymptomatic parasitic phase that eventually gives rise to visible symptoms (Verhoeff 1974) if conditions are favorable for disease development. For instance, if latent infections are at high levels and most develop to active disease symptoms then a disease epidemic can initiate.

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6.2 Introduction Postharvest diseases of fresh produce (fruit and vegetables) and mycotoxin contamination of certain susceptible crops contribute to major economic losses to growers, processors, marketers, and consumers. Therefore, management of postharvest disease is needed to avoid losses and increase growers’ revenues. Postharvest diseases are the result of latent infections that occur in the field during the growing season and infections from wounding during harvest and handling operations (Eckert and Summer 1967; Michailides and Manganaris 2009). Although postharvest diseases are the continuum and or the result of diseases that initiate in the field, some researchers have emphasized postharvest disease per se without looking into the diseases caused by the same pathogens in the field. A good example is the brown rot disease of stone fruit caused by Monilinia fructicola or M. laxa. In California brown rot can have four distinct phases which in essence develop as a continuum on stone fruit. At bloom, for instance, if weather is wet and relatively warm, blossoms are infected and blighted: this is the blossom blight phase. Later on, if conditions continue to be favorable for the pathogen, the fungus can invade from the blossoms to the sustaining shoots causing cankers: this is the canker phase (which is more common for M. laxa). If wet conditions prevail, green or maturing fruit get infected and rot on the tree: this is the fruit rot phase. Finally, once fruit is harvested and removed from the orchard still can decay and this is the postharvest fruit rot phase which can occur either in storage, in the market (wholesale and retailed stores), in the household refrigerator, or in customer’s fruit display basket. Epidemiological knowledge of fungal diseases of fruit trees, nut crops, and vines is essential to help predict disease risk during the growing season and/or at harvest and during postharvest storage. Predicting plant disease is very challenging and requires major research efforts over several years to understand the dynamics of the four main variables involved in disease development: the presence/absence and quantity of the pathogen’s inoculum, stage and susceptibility of the crop, environmental conditions, and growers who continuously attempt to change the dynamics of the diseases in their agricultural systems. These major factors are not steady but compoundedly change and make the development of an accurate predictive model difficult, complex, and time consuming. Although increasingly accurate weather predictions are available through the National Oceanic Atmospheric Agency (NOAA), the other aspects of the disease triangle require a large database of information that includes the pathogens’ inoculum, which can be quantified by either trapping spores or predicted based on disease incidence in previous seasons (historical disease data) or by determining the incidence of latent infections. Susceptibility of the host and the most susceptible stage of the crop can be determined experimentally with periodic inoculations with the pathogen. In many diseases of fruit trees, nut crops, and vines, latency is an important stage in disease epidemiology and prediction. Although disease prediction depends on the above mentioned factors, it is sometimes accurate, depending on the specific disease and its nature, to predict the disease incidence and severity even without

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any recording and consideration of the above mentioned parameters. This is because the success for a latent infection to occur and develop to actual disease depends on all these factors (inoculum level present, susceptibility of the host, and environmental conditions conducive to infection) and these infections then can act as recording devices that “record” and accumulate all these parameters that contribute to disease expression. In this contention, then an infective propagule that has the capability to produce a latent infection and it could be considered as an alarm that will go off as soon as the “right time” has reached based on the accumulated “latency condition of the pathogen” has reached a minimum threshold. A “latency condition” is defined as the status of the pathogen subjected to diminishing adversity by the host as it changes physiologically and biochemically and changing conditions of the environment that affect both the host and the pathogen. In this case, the pathogen itself acts as a recording device that records all these changes (of the environment and the host) and develops accordingly, with the final result the expression of disease development. If the latency condition of the pathogen leads to its death, then symptoms of disease will not develop. Diseases we investigated that have a latency phase include Botrytis gray mold in grapes and kiwifruit caused by Botrytis cinerea (Pers.:Fr.), brown rot in stone fruit caused by Monilinia fructicola (G. Wint.) Honey and/or M. laxa (Aderh.) Honey, panicle and shoot blight of pistachio caused by Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not., and Alternaria late blight of pistachio caused by three species of Alternaria. Latent infections of berries, stone fruit, and nuts remain inactive until these fruit start maturing and environmental conditions are favorable for disease development. However, if environmental conditions are not favorable, the pathogen may not survive until the stage when the crop becomes conducive to the development of latent infection to disease symptoms. The incidence of latent infections has been shown to correlate with disease incidence in the field at harvest of fruit and nut crops (Michailides et al. 2000) or with the incidence of decays that develop in storage (Michailides and Morgan 1996a,b). The degree of success in management of fruit tree diseases depends on the level of disease itself, environmental conditions, effective cultural manipulations, and proper timing and type of fungicides selected against a disease. Fungicide resistance in fungal pathogens of tree fruit and vines also affects the outcome of disease management as well as choices of postharvest treatments. If growers had access to timely information on fungal resistance to fungicides, they could make correct decisions on what fungicide to apply and what rotation schedule would be essential to help avoid a failure of disease control in the field and/or prevent a build-up of resistance in the pathogen’s population in the orchard or vineyard. To base this discussion on personal experience, two examples will be emphasized as follows: the Monilinia fructicola and M. laxa causing brown rot disease of stone fruit; and the Botrytis cinerea causing gray mold of kiwifruit. These examples include good methods for the detection of latent infection during the growing season that predict disease at harvest and postharvest.

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6.2.1 Brown Rot-Monilinia fructicola and M. laxa Brown rot of stone fruit in California is caused by Monilinia fructicola and M. laxa. The latter species was the predominant in the early nineteenth century while in late century, M. fructicola became the dominant species, at least in prune orchards (Michailides et al. 1987). The disease expresses itself in two major phases, infection of blossoms leading to blossom blight and infection of fruit leading to fruit rot (both immature, green fruit and mature fruit infection). A third phase is the latent infection that can occur from bloom to harvest. The occurrence of latent infection by Monilinia spp. is known since the early 1960s various reports provided good examples of latent infection on apricot (Wade 1956; Tate and Corbin 1978; and Wade and Cruickshank 1992). On cherry (Curtis 1928; Förster and Adaskaveg 2000), and on peach (Tate and Corbin 1978; Michailides et  al. 2000). The best examples of latent infection by Monilinia spp. are on plum (Rosenberger 1983; Northover and Cerkauskas 1994) and on dried plum (Luo and Michailides 2001, 2003; Luo et  al. 2001). Specifically, on dried plum, infections by the brown rot fungus can be divided in (1) latent infections in blossoms; (2) latent infections and quiescent infections in green fruit, and (3) quiescent infections on leaves. Quiescent infections on leaves are small brown spots that upon isolations on acidified PDA the majority of them will produce colonies of Monilinia spp. Quiescent infections on immature dried plum fruit (prunes) are raised pin-head black specks (Fig. 6.1). The majority of the latent infections do not develop to disease; however, some can overcome the inhibiting factors encountered in green fruit and form a brown decay lesion in immature, green fruit. Latent infections will also affect directly the incidence of postharvest decay (see below). Because of the direct relationships between latent infection and brown rot disease, detecting latent infections could be a useful assay to determine risk of brown rot at harvest and postharvest.

Fig. 6.1  Quiescent infections of Monilinia spp. on ‘Howard Sun’ plum

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6.2.1.1 Conventional Methods Used to Detect Latent and Quiescent Infections of Monilinia spp. There are two types of methods for the detection of quiescent and latent infections. These include conventional (direct isolation – incubation) and molecular (PCR and real time PCR) techniques. These techniques have been used in our studies in the last several years with great success. Direct Agar Plating Technique (DAPT)  This technique is commonly used to isolate plant pathogens from plant tissues. The detection and quantification of the Monilinia sp. pathogen in quiescent infections can be determined using this technique. The agar medium commonly used for the DAPT technique in our laboratory is potato dextrose agar acidified with lactic acid (2.5 mL of a 25% vol./vol. lactic acid per liter of medium), resulting in a pH of 3.5 that is inhibitory to the majority of bacteria, but it allows the Monilinia spp. to grow when incubated at 20°–25°C (68°F to 77°F) for several days. The traditional way is to cut the plant tissue in 3 × 3 × 3 mm cubic pieces, surface disinfect them in a 5–10% chlorine solution (prepared from household bleach, which is a 5.25% solution of NaOCl) for 1 to few minutes, rinse them with sterile water once or twice, blot them on clean paper towels, and place 5–10 pieces in a 55- or 90-mm in diameter Petri plate. The plates are usually incubated at 25°C (77°F) for 5 to 7 days and recorded for the presence/ absence of Monilinia spp. from the isolations. The DAPT has been used for isolating latent infections and quiescent infections from the skin of various stone fruits, fruitto-fruit contact areas, stylar and basal ends of the fruit, flower petals, and leaf blades. For instance, when the weather is unusually wet, quiescent infections show as small rusty spots on the leaf blade and 3 × 3 mm square pieces of the blade can be surface sterilized and plated as described above. Flower Incubation Technique (FIT)  A second conventional technique for the detection of latent infections of brown rot in flowers is by collecting 100–150 random flowers per field, surface sterilized them in 1% chlorine solution (prepared from household bleach containing 5.25% NaOHCl), and lay them either on wet sterile paper towels or on sterile plastic screens in clean containers. Usually, the containers used in our laboratory are made of hard plastic and measure 24.5 × 18.0 × 8.0 cm. The containers with the flowers are incubated at room temperature or at 25°C for 5 days when latent infections develop on the hypatheum showing characteristic sporulation. The incidence of flowers with latent infection by M. fructicola is then determined within 5–7 days incubation. This technique can also be performed by using short (15–20 cm) twigs bearing flowers and placing flat four to five twigs on top of the plastic screens in the plastic containers. All the other steps for this procedure, incubation and determination of latent infections are the same as those used above for individual flowers (Luo et al. 2001). Overnight Freezing – Incubation Technique (ONFIT) (Table  6.1)  This is another conventional technique which developed and frequently used in our laboratory and can detect and quantify latent infections of Monilinia fructicola and M. laxa (the fungi that cause brown rot in stone fruit), Botrytis cinerea in grapes

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Table  6.1  Protocol of overnight freezing incubation technique (ONFIT)a to reveal latent infections by Monilinia fructicola or M. laxa in stone fruit   1. Collect 100 immature fruit from the orchard and bring to the laboratory in an ice chest.   2. Disinfect cleaned plastic screen racks and plastic containers (2 per site) in 1:20 bleach (5.0% sodium hypochlorite: water) solution for 5 min.   3. Place 50 fruit in the plastic mesh-stretch bags, label as desired, and secured mesh bags with plastic clips.   4. Prepare bleach solution in large (20 L) plastic containers. Solution is prepared 1:10 with 0.5 mL of Tween 20 per liter of tap water. Typical preparation is 5 L of solution with 0.125 mL of Tween 20. Place the plastic container in the sink to minimize unwanted splashing of the bleach solution.   5. Prepare 1 L of 70% ethanol in a 2 L beaker.   6. Place the samples in the ethanol solution for 10 s, shake off quickly and place in the bleach/ Tween-20 solution for 4 min. Swirl the bags in the solution for 5–10 s during every minute the bags are in solution.   7. Remove the bags, shake off the excess liquid in the sink and quickly place the bags in the appropriately labeled containers; replace the lid.   8. Arrange fruit in the containers under a hood, pour 200 mL of tap water, and cover the containers.   9. Place all plastic containers at 3°–4°F (−16°C) freezer for about 15 h (17:00 h to 8:00 h) overnight. 10. Remove containers from the freezer and place on a laboratory counter at about 77°F (25°C). 11. Record and count fruit showing brown rot symptoms and sporulation of the pathogen after 5–7 days. ONFIT can be used for any stone fruit such as apricot, cherry, nectarine, peach, plum, and prune

a

causing bunch rot, and B. dothidea and Alternaria species causing blight diseases in pistachio. The rationale of the technique is based on the fact that killing the fruit tissues at a stage when the tissues do not favor disease development triggers the development of latent infections to active disease symptoms or accelerates the growth of hidden colonists. The herbicide paraquat (1,1´ diemthyl-4.4´ bipyridinium dichloride) has been used in the past to kill plant tissues and trigger latent fungal infections in soybean (Cerkauskas and Sinclair 1980) and modified later for the detection of latent infections of plums by M. fructicola (Northover and Cerkauskas 1994). We also used paraquat in our laboratory initially, but because it is a toxic herbicide, we looked for an innocuous alternative. In contrast, the ONFIT is a safe technique because it does not require the use of any noxious pesticides, only surface disinfectants such as dilutions of household bleach and disinfectants, such as ethyl alcohol, which are generally regarded as safe. But, it is still necessary to thaw and then incubate the frozen fruit for a number of days under laboratory conditions (75°–77°F) until latent infections develop into symptoms and signs of the pathogen can easily be recorded (Fig. 6.2a) and results become available within 5–7 days. Latent infections of brown rot can also develop to disease symptoms, but fruit needs to be incubated under high humidity for at least 2–3 weeks. Using the ONFIT, a grower would need to wait only 5–7 days until he would be able to make a decision on disease risk in the field (Luo and Michailides 2003). For instance, the incidence of Monilinia spp. determined with the ONFIT performed with immature

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Fig. 6.2  (a) ONFIT on French prunes to detect latent infections after 7 days incubation, (b) incubation of surface sterilized French prunes without freezing for 4 weeks at room temperature

prune fruit collected in May/June correlated linearly with the incidence of the brown rot developed in the field at harvest (Fig. 6.3). In other words, the incidence of Monilinia determined with the ONFIT can predict the risk for fruit brown rot at harvest. Waiting one week is still a long waiting time, therefore, more efficient and quicker techniques than the conventional ones are urgently needed for the detection of latent infections in tree fruits, nut crops, and vines in California. The timing for performing ONFIT depends on various factors. One important factor is the inoculum potential of Monilinia spp. in the orchard. For example, when the inoculum potential is high in an orchard the timing of ONFIT can range from mid May to early July,

Percent branches with fruit rot (PBFR)

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16 PBFR = −2.6 + 0.3355 ILI

14

r2 = 0.82, P = 0.002

12 10 8 6 4 2 0

0

10

20

30

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Incidence of latent infection (%) Fig.  6.3  Linear correlation between incidence of latent infections (ILI) and percentage of branches with fruit rot (PBFR) caused by Monilinia fructicola on prune, detected by the ONFIT technique. Each data point represents an average value of multiple locations and inoculations

when the inoculum potential is low, the best timing will be in mid June, and when the inoculum potential is moderate, the best timing can be at any time in the month of June. But in general, the critical period to determine latent infection for prunes is in June (Luo and Michailides 2003). Incubation of fruit after surface sterilization following the steps that are used for the ONFIT procedure without freezing requires a long time until latent infections in green fruit are triggered to develop. For instance, when immature prune fruit were collected from two different sides of a large prune orchard, surface sterilized, and incubated as above (without freezing the fruit), latent infections started developing after 8–10 days incubation, reached to 5–7% levels in 15 days and to a maximum after 30 days (Fig. 6.2b). Even this procedure if it is done in May provides sufficient time for determining the risk for disease in an orchard and making decisions for pre-harvest sprays. There is linear correlation of the incidence of latent infection and postharvest decay for at least some of the stone fruit (i.e., prunes).

6.2.2 Botrytis Monitoring (BOTMON) - Botrytis cinerea (Table 6.2) The California kiwifruit industry and other kiwifruit industries suffer tremendous losses due to postharvest gray mold caused by Botrytis cinerea. Although this disease does not show any symptoms and or signs in the field in California, postharvest gray mold is considered as the number one disease of kiwifruit in cold storage. Since no disease symptoms develop in the field, it is most likely that infections

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Table  6.2  Protocol of Botrytis monitoring (BOTMON) to reveal latent infections by Botrytis cinerea by plating symptomless sepals and stem ends of kiwifruit   1. Harvest 60 kiwifruit from each 1-ha field (avoid any wounding) 4 months after fruit set (about 1 month before harvest).   2. Remove sepals (by hand) and stem end (with a cork borer) from each fruit.   3. Surface disinfect sepals and stem ends in 0.5% chlorine household bleach plus 2 drops of Triton-X-100 surfactant (per liter water).   4. Rinse the above, surface-sterilized fruit sepals and stem ends in sterile water and dry them in a positive-flow hood for 10–15 min.   5. Plate the above in Petri plates containing acidified potato-dextrose agar (pH = 3.2–3.5).   6. Incubate the petri Plates at 7°C for 6 days and record B. cinerea colonies growing from the sepals and stem ends in each plate (first Botrytis recording).   7. Move and incubate the petri Plates at 23°C for 3 more days and record additional B. cinerea colonies in each plate (second Botrytis recording).   8. Combine data from the two recordings (= total B. cinerea colonies) and determine incidence (%) of colonization of sepals or stem ends.   9. Use prepared tables or regression lines for sepal or stem end colonization to predict Botrytis gray mold after expected to develop after 3 or 5 months storage of fruit in controlled atmosphere. 10. Make decisions: (a) yes or no preharvest fungicide spray(s); (b) which fruit to store longer; (c) yes or no resorting and re-packing; etc.

by B. cinerea of kiwifruit are primarily latent. Michailides and Morgan (1996a) found that these infections occur on the fruit sepals and receptacles during the growing season starting 1 month after bloom and continuing until harvest. Furthermore, there are major differences in the epidemiology of Botrytis gray mold of kiwifruit in New Zealand and California. For instance, because of the dry climatic conditions in California, latent infections is of primary importance for the postharvest gray mold while in New Zealand, it is the infection of stem wound that affect the levels of the postharvest decay and not the latent infections of sepals by B. cinerea. Therefore, in California to develop more efficient methods for controlling gray mold in kiwifruit, it was necessary to understand the relationship between B. cinerea latent infections initiated in the field and the incidence of gray mold in cold storage. The BOTMON technique was developed and used to detect B. cinerea, causing latent infections of kiwifruit (Actinidia deliciosa) sepals in the field. The rationale of the technique is based on the fact that the incidence of latent infection is a good predictor of gray mold in cold storage (Michailides and Morgan 1996a,b; Michailides and Elmer 2000). BOTMON involves the collection of fruit samples with stems attached from the kiwifruit vineyard, removal of sepals or stem ends (receptacles; Fig. 6.4), and plating the fruit samples in plates with APDA (Fig. 6.5). It was determined that 1 month before harvest is the best time for sampling immature fruit to perform BOTMON and gray mold prediction, since the correlation coefficients of B. cinerea colonization of sepals and stem ends and gray mold in cold storage are the highest (r = 0.92–0.98). Interpretation of the technique’s results was given in previous

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Fig. 6.4  Sepals and stem-ends of kiwifruit cut from the fruit to be used in BOTMON to monitor the incidence of colonization of Botrytis cinerea

Fig. 6.5  BOTMON technique: plates containing acidified potato-dextrose agar where sepals (left) and stem ends (right) were plated to reveal colonization by Botrytis cinerea

publications (Michailides and Morgan 1996a,b). The results of the colonization of sepals and/or stem ends by B. cinerea grown become available to the grower in 9 days. After obtaining the results, growers can use Table 6.3 to predict the levels of gray mold in cold storage for a specific lot of fruit. In California more and more kiwifruit growers have been using the BOTMON technique to make decisions on the need for pre-harvest vinclozolin spray(s) since this technique uses fruit collected one month before harvest, allowing for sufficient time to apply a fungicide. Additionally, packinghouse

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Table 6.3  Relationship of levels of sepal and stem-end colonization by Botrytis cinerea in kiwifruit samples collected 4 months after fruit set and number of vineyards with low (<1%), moderate (1–3%), and high (>3%) levels of Botrytis gray mold decay after 3 months in controlled atmosphere storage (31°F (−0.5°C) and eight parts per billion ethylene) in 2 years Number of vineyards with fruit showing different levels of postharvest gray molda Sampled Colonization Low (£1%) Moderate (1–3%) High (³3%) Plant part Level Colonization Yr 1 Yr 2 Yr 1 Yr 2 Yr 1 Yr 2 % Sepals Low 0–15 6 3 0 1 0 0 Medium 16–50 0 0 2 3 1 0 High >50 0 0 0 0 0 1 Stem ends Low 0–15 4 2 0 0 0 0 Medium 16–50 2 1 2 3 0 0 High >50 0 0 0 1 1 1 In year 1 a total of nine vineyards were sampled and in year 2 a total of eight vineyards (fruit from one vineyard in Butte County was not sampled because of prescheduled grower’s harvest). Percentage of colonization was determined by plating 300 sepals and 60 stem ends per sampling in each orchard. a

operators and shippers can use the results to decide on the need for fruit sorting and re-packing to minimize secondary spread of the disease in storage and plan on the timing for marketing these fruit. When growers spray only when it is needed and only those vineyards which have a high incidence of latent infections, they reduce costs and contamination of the environment with pesticides. The only disadvantage of the technique is that it is still time consuming (6 days pre-incubation of the plated sepals and stem ends on APDA at 45°F and 3 more days at 77° F (see protocol in Table 6.2). Therefore, there is still a need for a quick technique that will provide results within one day or even within a few hours. BOTMON has been used also to detect B. cinerea in latent infections of apples, figs, grapes, pears, pistachios, pomegranates, and various stone fruit (cherry, nectarine, peach, plum, and prune). Prusky et al. (1981) developed a pre-harvest assessment of latent infections by Alternaria alternata in mango fruit and found that there was a positive correlation between the relative surface of fruit infected by latent Alternaria at harvest and the incidence of black spots that developed on the fruit during postharvest storage. 6.2.2.1 Molecular Techniques The polymerase chain reaction (PCR) and the development of thermocyclers have revolutionized the molecular biology since they were first described in 1985. PCRbased techniques have been used in various biological studies. Specifically in plant pathology, PCR has been used in identifying pathogens and determining pathogen population structures, taxonomy, and classification. Additionally, in the last several

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years, techniques that quantify the DNA of pathogens have been developed, and these can be very useful when the relationship of quantities of pathogens’ DNA, latent infections, and disease levels are established. Such techniques could aid in estimating spore inoculum that determines disease potential and levels of latent infections that relate to disease risk at harvest or after postharvest storage. Following, we present a few examples of molecular techniques we developed in the plant pathology laboratory at the Kearney Agric. Center for the early detection of pathogens and fungicide resistant pathogen genotypes and providing answers to critical epidemiological questions that help predict disease risk in the field and pathogen resistance to fungicides. We also discuss how these techniques could be used in agribusiness as decision making tools. PCR-based assays that detect M. fructicola and M. laxa in stone fruit  Brown rot, caused by M. fructicola and or M. laxa, is a destructive disease of stone fruit (Prunus spp.) in California causing initially blossom blight and later fruit rot. When the microclimatic conditions in the orchards are unfavorable for further disease development, infected blossoms develop into young fruit with latent infections. These fruits may drop naturally or be thinned, and when humidity is high, these dropped fruit produce numerous conidia which can cause fruit infections in midseason. Later, when favorable conditions and maturation of the fruit occur, a number of the latent infections may develop into fruit rot. Inoculum potential in the orchards is an important factor affecting both blossom blight and fruit infections (Luo and Michailides 2001, 2003). Thus, determination of inoculum potential (amount of pathogen’s spores in the orchard) in early- and mid-season is critical for predicting and managing brown rot. Inoculum potential is the most difficult parameter to determine in a stone fruit orchard. Spore traps have historically been used to determine the spore density for air-borne disease agents including M. fructicola. Because samples from traps require microscopic examination, it is both very time consuming and requires special training to recognize and count spores in the spore samples. Additionally, spore counts may be an unreliable indicator of inoculum potential because of the abundance of dust and other fungal species (i.e., Botrytis cinerea) having spores similar to those of M. fructicola. Furthermore, culturing airborne spores collected on sporetrap tapes or slides is also tedious and subject to frequent contamination problems. Thus, such classical methods are impractical for recording large number of spore trap samples required for a large-scale disease management. PCR-based assays have the potential to monitor airborne inoculum levels of plant pathogens because they are highly specific and sensitive. Recently, we developed a nested PCR method for the detection of M. fructicola on spore-trap tapes (Ma et al. 2003b). Nested-PCR primer pairs (an external primer pair EMfF + EMfR and the internal primer pair IMfF + IMfR) were designed based on the sequence of a microsatellite region generated by a microsatellite primer M13 (5¢-GAG GGT GGC GGT TCT-3¢). In specificity tests, we observed that the primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571- and a 468-bp DNA fragment, respectively, from all tested M. fructicola isolates collected from different stone fruit hosts

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at different locations in different years. No fragments were amplified from any other fungus associated with stone fruit. Sensitivity tests showed that the nested PCR assay could detect the specific fragment in as little as 1 fg of M. fructicola DNA (Fig. 6.6a) or in DNA from only two spores of M. fructicola (Fig. 6.6b). This nested PCR method can detect 200 spores in a spore-trap tape sample (equivalent to two spores/PCR reaction) collected from a commercial prune orchard. Using these species-specific primers, we can also detect latent infections in fruits caused by M. fructicola within hours, while direct plating on agar media, or overnight freezing-incubation technique would require at least one week (see details below:

Fig. 6.6  PCR using specific primers to detect (a) M. fructicola DNA and (b) DNA from spores

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Table 6.4). Since the nested-PCR assay cannot quantitatively detect the number of M. fructicola spores on a spore-trap tape, we are now working on a real-time PCR technique that can quantify spores of M. fructicola. The efficient, accurate, and feasible real-time PCR method could help growers in making timely decisions for fungicide application and reduction of unnecessary sprays.

6.3 Use of Species-Specific PCR to Detect M. Fructicola in Fruit and Flowers (Comparison of Conventional with PCR Techniques) In 2001, in a plum orchard cv. Howard Sun, the presence of numerous visible quiescent infections (Fig. 6.1) suggested the presence of even more latent (invisible) infections. In order to compare the direct plating technique of visible quiescent infections, with the ONFIT of invisible latent infections and a species-specific PCR technique, in mid May fruit were observed in the field and their fruit-to-fruit contact surface was marked with a permanent pen. All these fruit were collected and brought to our laboratory at Kearney Agricultural Center, surface disinfected in 10% bleach solution for 3 min, rinsed with sterile water twice, and placed on clean paper towels. The fruit samples were split in three subsamples; one subsample was used for the DAPT, the second for the ONFIT, and the third subsample for the species-specific PCR technique. (a) For the direct plating technique, latent infections (small pieces of green tissue of the fruit surface) and visible quiescent infections (small black specks on the fruit skin) were excised with a sterile razor, and plated on APDA plates as described in DAPT. (b) For the ONFIT, fruit without any visible infections were processed following the protocol in Table 6.1, and recorded for brown rot development on the fruit after 7–9 days incubation. And (c) for the species-specific PCR method, invisible latent and visible quiescent infections were excised as in (a) above, pre-incubated at 77°F for 1 day, and then DNA was extracted and diagnosed using M. fructicola specific PCR following published protocols (Boehm et al. 2001). Results from the PCR method after isolating fungal DNA can be completed in 6 h, i.e., in 30 h since the initiation of the procedure (Table 6.4).

Table  6.4  Techniques to detect latent and quiescent infections by Monilinia fructicola in ‘Howard Sun’ plums Time required for Technique Latent infections (%) Quiescent infections (%) results (days) PCR 7.9 60.5 1.25a ONFIT 6.7 – 7–9 DAPT – 54.3 5–7 a

Time includes 1-day preincubation of sample.

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Using the PCR technique, 7.9% of the samples with invisible latent infections were positive for DNA of M. fructicola and 6.7% of the fruit processed with ONFIT developed brown rot. Similarly, as expected when visible quiescent infections were used, 60.5% were positive for M. fructicola with the PCR technique and 54.3% of those plated on APDA developed colonies of M. fructicola. Interestingly, the traditional techniques required 5–9 days to completion while the PCR technique made results known within only 1.25 days (Table 6.4). In another experiment, plum flowers (cv. Royal Diamond) were collected in March-April from a commercial orchard in Reedley, CA. Flowers were divided into three groups based on visual symptoms: (+) flowers were heavily infected with M. fructicola and showed obvious signs of fungal sporulation on the stem and calyx surface; (+/−) flowers displayed brown patches on the petals but showed no external signs of fungal sporulation; and (−) flowers were asymptomatic, without any evidence of brown discoloration or fungal infection. DNA extractions from individual plum flowers were obtained using the Fast-Prep System FP-120 biohomogenizer instrument, following the manufacturer’s instructions for plant DNA extraction (Q-BIOgene, Inc.). In planta polymerase chain reaction (PCR) detection from flowers used the species-specific primer pair 210F1 + 210R1 at the high annealing temperature for retention of species specificity. The results indicated that all (100%) of the (+) series of flowers had much stronger amplification signals than either the (+/−) (80%) or the (−) flowers (only 10% of the flowers carrying M. fructicola (Fig. 6.7). Thus in approximately 8 h, one can determine the percentage of asymptomatic flowers carrying M. fructicola. Knowing the percentage of flowers latently infected by M. fructicola should prove a useful estimate of the inoculum potential in stone fruit orchards necessary for determining disease risk, assessment and blossom blight incidence, and in developing pre-harvest and postharvest chemical control strategies against brown rot. The PCR technique can replace the flower incubation technique (FIT) that can also provide an estimate of inoculum potential in stone fruit orchards.

6.4 Techniques to Monitor Resistance of Fungal Pathogens to Fungicides Fungicides are commonly used to manage plant diseases. However, the frequent use of fungicides with single mode of action incurs a high risk of selecting resistant genotypes of plant pathogens. To determine levels of resistance to fungicides in fungal populations, the most common conventional technique used is the direct plating of single-spore isolates in media amended with the fungicide of interest. Single-spore isolates of the pathogen are grown on PDA or acidified PDA or other specialized media under conditions that favor their sporulation. Media such as PDA or water agar (WA) amended with increasing concentrations of the test fungicide are used to determine either the EC50 of inhibition of hyphal growth (after placing a 5-mm in diameter mycelial plug in the center of the Petri plate) and/or EC50 of

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Fig. 6.7  PCR using specific primers to detect DNA of Monilinia fructicola in latent and quiescent infections of plum flowers

spore germination (after spreading a 0.5 mL of a dense spore suspension of the fungus on the surface of the plate) in comparison with the respective medium that is not amended with the test fungicide. EC50 is the concentration of the fungicide that provides 50% inhibition of mycelial growth or spore germination of the pathogen to be tested. This technique was used in our laboratory for detecting fungicide resistance in plant pathogens such as M. fructicola and M. laxa, Botryosphaeria dothidea, Fusarium moniliforme, B. cinerea, Alternaria alternata, A. tenuissima, and A. arborescens. This technique is time consuming, and obtaining the results of the tested isolates is usually very critical, especially since growers would rely on such results to decide what fungicide programs to use, depending on the presence and levels of resistance in a field. Depending on the pathogen species, in order to obtain mycelial plugs or adequate amounts of spores, the entire test can take 7–10 days, if not longer, if one takes into account the time required to isolate the pathogens from infected plant tissues. In 2004, a new technique was reported using a Spiral Plate® gradient dilution method to determine EC50 values that still requires 1–5 days for mycelial growth assays and 14–20 h for the spore inhibition studies to reveal results plus 2–5 days for sporulation in culture (Förster et al. 2004). Although results with this technique are available sooner than the classical technique, both the high cost of the Spiral Plate® equipment and the long time period to obtain results when one wants to check multiple isolates are prohibitive. Therefore, more efficient and rapid techniques are needed for checking resistance of pathogens to fungicides. An example will be the resistance in Monilinia spp. Since benzimidazole resistance in M. fructicola and M. laxa has been shown to be associated with point mutations in the b-tubulin gene (Ma et al. 2003a), we developed an allele-specific real-time PCR method for rapidly detecting benzimidazole-resistant M. fructicola and M. laxa in stone fruit orchards. A similar procedure was used for the detection

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of Alternaria resistance to azoxystrobin. This technique was used with blossoms collected from peaches in spring (March) and successfully detected the level of resistance to benzimidazoles in M. fructicola. Obviously, such rapid and quantitative detections of fungicide resistance in the fungal pathogen populations will be valuable for growers to manage fungicide resistance in fruit tree orchards and vineyards.

6.5 Conclusions and Future Prospects The presented examples represent a part of the methodology used at the Kearney Agricultural Center to access latent infections to help predict diseases at harvest and in postharvest storage and guide growers to disease management decisions and packinghouse operators to proper marketing of fruit. Although the conventional techniques can be accurate and may be less expensive because of specialized reagents (enzymes and equipment are not needed), only minimal information concerning a few isolates becomes available and only after 1–2 weeks. This “waitingfor-results” time often can be a very critical time for growers who need to make a decision on disease management, fungicide timing and frequency, type of fungicide, and resistance management program selection in their fields. Because of these serious drawbacks in the last 5 years we have focused our efforts on research to develop molecular techniques, which although are more costly, they can replace the conventional ones and have the potential to finally provide very accurate and timely information for disease management decisions. As shown from the examples above, molecular technology has proven very valuable in our plant pathology research program at the Kearney Agricultural Center. One disadvantage of the molecular methodology is that it does require specialized reagents, enzymes, and costly equipment (Polymerase Chain Reaction and Real Time PCR machines) and may be more expensive than the conventional methodology. However, when affordable, portable real-time PCR instruments and simple protocols are developed, routine and efficient diagnosis of many crop diseases or fungicide resistance in pathogen populations can be made on site and within one day, thus reducing the total costs of such tests. In general, molecular technology also helps us in understanding the biology and population structures of plant pathogens and provides quick and accurate answers to epidemiological questions on plant diseases. Subsequently, these techniques help us in developing effective strategies for disease control. With many diseases, latent infections were correlated with disease levels in the field and or postharvest. Although significant progress has been made in the discovery of conventional methods that help detect latent infections, latent infection detection is based mainly upon subjecting the infected tissues to surface sterilants, and tissue damaging agents (paraquat) or conditions (freezing) followed by incubation. Additionally, there are many variations in the type, number, duration and sequence of these processes. There is a need for faster and more efficient methods

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of latent infection detection and the use of real time polymerase chain reaction (RT-PCR) can provide the basis for efficient, accurate, and more rapid detection of pathogens. There are still gaps in our understanding on the trigger that activates the pathogen’s growth in latent infections. It is hoped though molecular techniques will eventually replace conventional ones in other patho-systems, help elucidate gaps in epidemiological research, and improve our understanding of plant disease. Future goals of our research are to develop more efficient, accurate, and rapid molecular procedures using RT-PCR and replace the conventional ones. Furthermore, our goal is to reduce costs for processing samples by using such techniques in large numbers of samples, a process that will provide accurate answers to epidemiological questions and allow the expansion of molecular epidemiology in plant disease. Acknowledgments  We thank M. Doster, D. Felts, L. Boeckler, H. Reyes, R. Puckett, and J. Windh for technical assistance. We also thank Drs. Z. Ma, H. Avenot, and E. Boehm, Postdoctoral Associates, and visiting Professor M. Yoshimura, for their research contributions. Funding for this research was ­provided by the California Kiwifruit Commission, California Pistachio Commission, California Dried Plum Board, California Tree Fruit Agreement, and the California Fig Institute.

References Boehm EWA, Ma Z, Michailides TJ (2001) Species-specific detection of Monilinia fructicola from California stone fruits and flowers. Phytopathology 91:428–439 Cerkauskas RF, Sinclair JB (1980) Use of paraquat to aid detection of fungi in soybean tissues. Phytopathology 70:1036–1038 Curtis KM (1928) The morphological aspect of resistance to brown rot in stone fruit. Ann Bot 42:39–68 Eckert JW, Sommer NF (1967) Control of diseases of fruits and vegetables by postharvest treatments. Annual Rev. of Phytopathology 5:391–432 Förster H, Adaskaveg JE (2000) Early brown rot infections in sweet cherry fruit are detected by Monilinia-specific DNA primers. Phytopathology 90:171–178 Förster H, Kanetis L, Adaskaveg JE (2004) Spiral gradient dilution, a rapid method for determining growth responses and 50% effective concentration values in fungus-fungicide interactions. Phytopathology 94:163–170 Luo Y, Michailides TJ (2001) Factors affecting latent infection of prune fruit by Monilinia fructicola. Phytopathology 91:864–872 Luo Y, Michailides TJ (2003) Threshold conditions that lead latent infection to prune fruit rot caused by Monilinia fructicola. Phytopathology 93:102–111 Luo Y, Morgan DP, Michailides TJ (2001) Risk analysis of brown rot blossom blight of prune caused by Monilinia fructicola. Phytopathology 91:759–768 Ma Z, Luo Y, Michailides TJ (2003a) Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California. J Phyto­ pathology 151:312–322 Ma Z, Yoshimura MA, Michailides TJ (2003b) Identification and characterization of benzimidazole resistance in Monilinia fructicola from stone fruit orchards in California. Appl Environ Microbiol 69:7145–7152 Michailides TJ, Elmer PAG (2000) Botrytis gray mold of kiwifruit caused by Botrytis cinerea in the United States and New Zealand. Plant Dis 84:208–223

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Michailides TJ, Manganaris GA (2009) Harvesting and handling effects on postharvest decay. Steward Postharvest Rev 2:3–7 Michailides TJ, Morgan DP (1996a) New technique predicts gray mold in stored kiwifruit. California Agric 50(3):34–40 Michailides TJ, Morgan DP (1996b) Using incidence of Botrytis cinerea in kiwifruit sepals and receptacles to predict gray mold decay in storage. Plant Dis 80:248–254 Michailides TJ, Ogawa JM, Opgenorth (1987) Shift of Monilinia spp. and distribution of isolates sensitive and resistant to benomyl in California prune and apricot orchards. Plant Dis 71:893–896 Michailides TJ, Morgan DP, Felts D (2000) Detection and significance of symptomless latent infection of Monilinia fructicola in California stone fruits. (Abstr.) Phytopathology 90:S53 Northover J, Cerkauskas RF (1994) Detection and significance of symptomless latent infections of Monilinia fructicola in plums. Can J Plant Pathol 16:30–36 Prusky D, Fuchs Y, Zauberman G (1981) A method for pre-harvest assessment of latent infection in fruits. Ann Appl Biol 98:79–85 Rosenberger DA (1983) Observations on quiescent brown rot infections in Grand Prize plums. In: Burr TJ (ed) Deciduous tree fruit disease workers. American Phytopathological Society, Ithaca, New York, pp 19–22 Sinclair JB, Cerkauskas RF (1996) Latent infection vs. endophytic colonization by fungi. In: Redlin SC, Carris LM (eds) Endophytic fungi in woody plants. Systematics, ecology, and evolution. APS, St. Paul, MN, Chapter 1, pp 3–29 Tate KG, Corbin JB (1978) Qiescent fruit infections of peach, apricot, and plum in New Zealand caused by the brown rot fungus Sclerotinia fructicola. NZ J Exp Agric 6:319–325 Verhoeff K (1974) Latent infections by fungi. Ann Rev Phytopathol 12:99–107 Wade GC (1956) Investigations on brown rot of apricots caused by Sclerotinia fructicola (Wint.) Behm. I. The occurrence of latent infection in fruit. Austr J Agric Res 7:504–515 Wade GC, Cruickshank RH (1992) The establishment and structure of latent infections with Monilinia fructicola on apricots. J Phytopathol 136:95–106

Chapter 7

Preharvest Strategies to Control Postharvest Diseases in Fruits N. Teixidó, J. Usall, C. Nunes, R. Torres, M. Abadias, and I. Viñas

Abstract  Postharvest diseases on citrus and pome fruits are generally controlled by chemical treatments applied in packinghouses before fruit storage. However, there are some points that make pre-harvest strategies interesting practices to control or reduce postharvest rots. (a) Field practices and fruit manipulation in general can play an important role in fruit susceptibility in front postharvest diseases. (b) In some cases, infection of fruit occurs in the field prior to harvest and it could be advantageous starting control at this point. (c) Pre-harvest strategies also decrease fruit manipulation and subsequently potential damages and injuries, which are necessary for some important fungi infection. (d) Additional contamination by pathogenic fungi present in drenching solutions used in packinghouses would also be avoided. Twelve years of research have allowed us to study different approaches and some of them are summarized in this chapter. The aim of this research was to enhance efficacy of preharvest biocontrol treatments using different strategies: combination of biocontrol agents, combination with low-risk substances such as ammonium molybdate, and finally increase environmental stress tolerance of biocontrol agents. All these experiences have let to conclude that pre-harvest practices can play an important role in postharvest disease control.

N. Teixidó (*), J. Usall, R. Torres, and M. Abadias IRTA, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198, Lleida, Catalonia, Spain e-mail: [email protected] C. Nunes University of Algarve, Centro de Desenvolvimiento de Ciências e Técnicas de Produção Vegetal, Faro, Portugal I. Viñas University of Lleida, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198, Lleida, Catalonia, Spain D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_7, © Springer Science + Business Media B.V. 2009

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7.1 Introduction The development of resistance in fungal pathogens to fungicides (Dekker and Georgopoulos 1982; Viñas et al. 1991, 1993) and the growing public concern over the health and environmental hazards associated with high levels of pesticide use into fruit orchards (Wisniewski and Wilson 1992) have resulted in a significant interest in the development of alternative non-chemical methods of disease control. Biological control using microbial antagonists has emerged as one of the most promising alternatives, either alone or as part of an integrated control strategy to reduce pesticide inputs. Infection of fruit by postharvest pathogens often occurs in the field prior to harvest (Biggs 1995; Roberts 1994), thus it would be advantageous to apply antagonists before harvest which would reduce initial infection and then remain active and suppress the pathogens in storage (Elad and Kirshner 1993; Leibinger et  al. 1997). When biocontrol agents are applied at postharvest, they often have difficulty controlling previously established or incipient infections, which originated in the field. Indeed, the effectiveness of the antagonist decrease in proportion as increase the time since pathogen infection was originated and the antagonist was applied (Ippolito and Nigro 2000). Biocontrol of fruit decay in storage with microbial antagonists has so far been mainly studied under controlled environmental conditions at postharvest: few studies have attempted to apply biocontrol agents to fruit under field conditions with the purpose of controlling postharvest decay (Benbow and Sugar 1999; Leibinger et al. 1997). The pathogens P. digitatum and P. italicum infect fruit through wounds produced by mechanical injuries during the growing season and harvest handling operations (Roberts 1994; Biggs 1995; Obagwu and Korsten 2003). An antagonist applied in the field, prior to harvest, will have longer to interact with the pathogen than one applied after harvest (Ippolito and Nigro 2000). It would therefore be advantageous to apply antagonists before harvest, which would reduce initial infection, and then let them remain active and suppress pathogens during storage (Elad and Kirshner 1992, 1993; Teixidó et  al. 1998a). However, an important consideration for the application of biocontrol agents at preharvest is their ability to colonize the surface of fruit both in the field and during storage and to persist, for as long as possible, in sufficient numbers on the fruit surface to maintain an efficient decay control (Wisniewski and Wilson 1992). Several works have demonstrated the ability of some microorganisms to survive under field conditions and consequently provide effective control against postharvest decay (Korsten et al. 1997; Tian et al. 2004). However, under field conditions, rapid fluctuations in water availability and temperature are both characteristic of this environment and constitute the main factors limiting the development of microbial populations (Köhl and Fokkema 1998). Some different strategies have been deeply studied by our group in order to enhance biocontrol efficacy in preharvest applications of two biocontrol agents: the yeast Candida sake CPA-1 and the bacterium Pantoea agglomerans CPA-2 and

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some examples have been described in this chapter: biocontrol agents combination, combination with low risk substances and enhance environmental stress tolerance. C. sake CPA-1 has demonstrated to be an effective biocontrol agent (BCA) against major postharvest diseases on pome fruits (Viñas et al. 1998; Usall et al. 2000, 2001) and currently is commercialized in Spain as a liquid formulation named Candifruit by SIPCAM-INAGRA S.A. P. agglomerans CPA-2 is an effective antagonist to the major postharvest fungal pathogens of pome and citrus fruits (Nunes et al. 2001b, 2002b; Teixidó et al. 2001)and it is in commercialization process in Spain as a solid formulation named Pantovital by BIODURCAL S.L.

7.2 Biocontrol Agents Combination The yeast Candida sake CPA-1 (Viñas et al. 1998) and the bacterium Pseudomonas syringae CPA-5 (Nunes et  al. 2007a) were isolated from apple surface. These microorganisms have been tested, for many years, for their control activity against the major postharvest diseases of pome fruits. The objective of this work was to determine the efficacy of pre-harvest application of a combined treatment of C. sake CPA-1 and P. syringae CPA-5 to control P. expansum decay of pear and apple fruits during cold storage and study the population dynamics of each biocontrol agent. A yeast and a bacterium have been used in order to have microorganisms with different nutritional requirements. This fact could be an advantage in order to avoid competence problems. The results of pre-harvest treatments to control postharvest blue mold in pears and apples are shown in Fig. 7.1. All treatments significantly reduced incidence of P. expansum on pears and apples stored at 1°C for 4 months. On ‘Blanquilla’ pears treated only with C. sake CPA-1 or only with P. syringae CPA-5, P. expansum incidence was reduced 53%. The combined treatment in pears was significantly different from the application of each antagonist alone, and it reduced blue mold incidence in 90%, and enhanced biocontrol activity of each antagonist in 78% (Fig. 7.1a). On ‘Golden Delicious’ apples the biocontrol agent P. syringae CPA-5 reduced blue mold incidence in 40%. Although no significant differences were observed between the individual applications of C. sake CPA-1 or combined with P. syringae CPA-5, blue mold was reduced 46% and 56% respectively. Regarding to the individual application of P. syringae CPA-5 the combined treatment enhanced biocontrol activity in 26% (Fig. 7.1b). The population dynamics of C. sake CPA-1 and P. syringae CPA-5 has been recorded. At day 0 and 2 the population densities of both antagonists were higher on pears than on apples. However, at the end of the experiment (day 120) population densities reached similar levels. In pears at day 0 and 2, the population level of P. syringae alone or in combination was higher than C. sake, while in apple the opposite was observed at all sampling times.

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a

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Fig. 7.1  Incidence of blue mold on (a):‘Blanquilla’ pear and (b): ‘Golden Delicious’ apple preharvest treated with C. sake CPA-1 (107 CFU mL−1), P. syringae CPA-5 (2 × 107 CFU mL−1), and their combination in a proportion of 50:50. Fruits were wounded and treated in the field 2 days before harvest. After harvest, fruits were sprayed with P. expansum (104 spores mL−1) and stored at 1°C and 90% ± 5% RH for 120 days. Columns with the same letter are not significantly different (P > 0.05) according to the least significant difference test (LSD). (Nunes et al. 2007b)

This study demonstrated that the pre-harvest treatment with a mixture of 50:50 of C. sake CPA-1 and P. syringae CPA-5 enhanced biocontrol activity against P. expansum on apples and pears in comparison with control by antagonists applied separately. Similar results were obtained in postharvest treatments using a mixture of C. sake CPA-1 and Pantoea agglomerans CPA-2 in apples and pears (Nunes et al. 2002c). In that work similar population level of C. sake was obtained. The ability of both antagonists to colonize wounded fruits was not affected by the presence of the other antagonist, since similar level was obtained either alone or in combination. This result agrees with other authors (Janisiewicz and Bors 1995) that concluded that the carrying capacity of the wounds is greater than the population of a single antagonist indicates. It seems that the enhancing effect of the mixture of both antagonists appears to be due to the depletion of nutrients by their growth of both antagonists in wounded fruits that does not allow the development of P. expansum. In conclusion, our research showed that the pre-harvest application of a combination of C. sake CPA-1 and P. syringae CPA-5 results in an improvement of each antagonist (Nunes et al. 2007b).

7.3 Biocontrol Combined with Low Risk Substances The chemical environment can be manipulated to enhance biocontrol activity of the antagonists, and in fact, several works report additives that enhance the effectiveness of microbial antagonists (McLaughlin et al. 1990; Janisiewicz 1994; Wisniewski et al. 1995; Janisiewicz et al. 1998).

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Preliminary assays in vitro and in vivo (Nunes et al. 2001a) on the combination of some nutrients with C. sake strain CPA-1 in order to enhance biological control showed that the combination of the antagonist and ammonium molybdate reduced blue mold decay more than other chemicals. However, the ability of ammonium molybdate to control disease development has not been fully explored. Ammonium molybdate affects metabolic processes in several organisms (Wang et al. 1995; Bodart et al. 1999). The basis of its biological activity was reported to be its ability to inhibit acid phosphatase which interferes with phosphorylation and dephosphorylation (Glew et al. 1988), one of the most important processes of cell regulating (Remaley et al. 1985; Hunter 1995). The efficacy of preharvest applications of C. sake CPA-1 combined with ammonium molybdate to control blue mold during cold storage on apples and pears was evaluated. In apple assays, fruits were wounded in the field 2 days before harvest and treated with the biocontrol agent, ammonium molybdate or the combination, and in pear assays the treatments were applied 7 and 2 days before harvest on unwounded fruits and wounds were made at postharvest. In both trials P. expansum was artificially inoculated before cold storage. In the case of apples the preharvest application of C. sake 107 CFU mL−1 combined with ammonium molybdate (NH4-Mo) 1 mM did not improve postharvest biocontrol of blue mold in comparison of each treatment applied alone and the efficacy was lower than the obtained with postharvest treatments. However, about 54% rot reduction was achieved with these combined preharvest treatments (Nunes et al. 2002d). The concentration of NH4-Mo used in this study was lower than that used in pears and in postharvest trials because the application of 5 mM preharvest caused spots on the fruit surface. The preharvest application 5 mM did not affect blue mold decay development in pears (Nunes et al. 2002a) and no differences in incidence of blue mold were observed among preharvest application of NH4-Mo followed by postharvest application of C. sake, postharvest application of NH4-Mo and postharvest application of C. sake (90% rot reduction in all cases); therefore, the reduction observed with treatments of preharvest application of NH4-Mo (at 7 and 2 days before harvest) followed by postharvest application of C. sake seems to be due to the effect of the antagonist. The preharvest application of NH4-Mo was made on unwounded pears and the fact that this treatment did not reduce blue mold decay is probably because a residue of NH4-Mo must remain on the wound to inhibit the infection. Smilanick et al. (1999) found that the capacity of sodium carbonate or bicarbonate to control green mold on citrus was significantly reduced when the fruits were rinsed at high-pressure. They concluded that this reduction occurred because the high-pressure removes the residues of the compounds necessary to achieve control of green mold. Toxicology date of NH4-Mo was determined by the “Centre d’Investigació i Desenvolupament Aplicat” (Barcelona, Catalonia, Spain), calculating the rat oral median lethal dose (LD50). This work showed that the LD50 of ammonium molybdate is 1714. 3 mg kg−1 of live weight of Sprague Dawley rat and, at this concentration, no mortality or alterations in tested animals were observed.

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The C. sake enhancement achieved with the combination with ammonium molybdate is better in postharvest treatments than in preharvest.

7.4 Enhancing Biocontrol Agents to Environmental Stress Conditions To improve stress response behaviour and stimulate mechanisms of environmental survival of biocontrol agents, it is very important to optimise their efficacy and suitability for practical conditions. Survival of foodborne pathogenic microorganisms under different stress treatments (e.g. heat, osmotic or water activity, low pH, etc.) have been extensively studied and reported. Initial investigations carried out on bacteria such as Escherichia coli (Poirier et  al. 1998), Salmonella spp. (Mattick et  al. 2000) and Listeria spp. (Greenacre et al. 2003) have demonstrated that they possess an inherent ability to adapt to unfavourable environments by the induction of various general and specific stress responses. These stress responses are characterised by the transient induction of general and specific proteins and by physiological changes that generally enhance the particular organism’s ability to withstand more adverse environmental conditions (Ang et al. 1991). In the case of osmotic stress, the significant physiological changes reported in bacteria include the induction of stress proteins as well as the accumulation of compatible solutes such as K+ ions, the amino acids glutamate, glutamine, proline and alanine, the quaternary amines glycine betaine, the tetrahydropyrimidine ectoine and sugars such as sucrose and trehalose (Csonka 1989; Kets et al. 1994; Ko et al. 1994). In yeasts and filamentous fungi the accumulated compatible solutes are mainly low- (glycerol and erythritol) and high- (arabitol and mannitol) molecular weight sugar alcohols (Beever and Laracy 1986; Ellis et al. 1991; Van Eck et al. 1993). These compatible solutes allow equilibration of the cytoplasmic water activity (aw) with the surrounding environment, thereby retaining water in the cell and thus, maintaining turgor pressure, and helping to preserve protein function within cells (Yancey et al. 1982; Csonka 1989; Van Eck et al. 1993). Subjection to a mild stress can make cells resistant to a lethal challenge with the same stress condition. Preadaptation to one particular stress condition can also render cells resistant to other stress imposing conditions: this phenomenon is known as cross protection (Sanders et al. 1999). First studies conducted in this sense in biological control were conducted by Hallsworth and Magan (1994a, 1995, 1996) on entomopathogenic biocontrol fungi, demonstrating that it is possible to physiologically manipulate growth conditions, carbon sources and carbon:nitrogen ratios to channel specific polyols into mycelium and fungal propagules, resulting in improved and more rapid germination and (or) germ tube extension under water stress conditions. Conidia modified in this way may be more pathogenic to target pests at low relative humidity (Hallsworth and Magan 1994b). Physiologically manipulated inocula of the biocontrol agent

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Epicoccum nigrum, containing high concentrations of glycerol and erythritol have also been found to give better control of brown rot (Monilinia laxa) of peaches than unmodified inocula (Pascual et al. 1996).

7.4.1 C. sake CPA-1 Enhancement C. sake cells have been grown under mild or sublethal stress conditions in order to adapt this biocontrol agent and render cells resistant to lethal or more stressful conditions. Significant improvements in low aw tolerance were achieved by modifying both the aw and nutrient concentration of growth media. The best results were obtained with glucose and glycerol solutes, and the intracellular accumulation of polyols and glucose and trehalose in these aw stress-improved cells was significantly different than with unmodified control cells (Teixidó et  al. 1998b,c). In laboratory studies, the low aw-tolerant cells provided significantly better disease control as compared with the unmodified cells, and reduced the number of infected wounds and lesion size by 75% and 90%, respectively, as compared with nontreated controls (Teixidó et al. 1998a). Unmodified and low water activity (aw) tolerant cells of C. sake CPA-1 applied before harvest were compared for ability to control blue mold of apples (Golden Delicious) caused by P. expansum under commercial storage conditions (Teixidó et al. 1998a). The population dynamics of strain CPA-1 on apples was studied in the orchard and during storage following application of 3 × 106 CFU mL−1 of each treatment 2 days prior to harvest. In the field, population sizes of unmodified treatment remained relatively unchanged, while the low aw-modified CPA-1 cells increased. During cold storage the populations in both treatments increased from 10 3 CFUg−1 to 105 CFU g−1 after 30 days, and then declined to about 2.5 × 10 4 CFU g −1 apple. After 4 months in cold storage both unmodified and low aw-tolerant cells of C. sake were equally effective against P. expansum on apple (>50% reduction in size of infected wounds) Fig.  7.2. In these experiments, it was also observed that unmodified yeast cells initially adhered better to the apple surface than the two low aw-tolerant treatments. Little is known about the characteristics of the polysaccharide matrix produced by yeasts such as C. sake. However, previous studies with such polysaccharide matrices of spores of other fungi suggest that they may have a number of important ecological properties, including protection against temperature extremes, desiccation and short wave radiation (Louis and Cooke 1983, 1985). It is possible that the energy requirements for the production of high concentrations of endogenous reserves during growth at low aw, such as polyols and trehalose (Teixidó et al. 1998b,c) could result in a modification of the amount or characteristics of the matrix. Probably, if adherence of modified cells could be improved with specific additives, population of C. sake on fruit surface could be increased and also efficacy could be enhanced.

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Fig. 7.2  Suppression of blue mold of Golden Delicious apples by Candida sake CPA-1 grown in different media. Fruits were wounded in the field and suspensions of the antagonist (107 CFU mL−1) were sprayed onto the apples 2 days prior to harvest. After harvest fruits were sprayed with an aqueous suspension of P. expansum at 104 conidia mL−1 and kept in cold storage for 4 months. Columns and lines with the same letter are not significantly different according to least significant difference test (LSD) (P < 0.01). The letters apply to both, % infected wounds and lesion diameter. Treatments: C. sake grown in unmodified NYDB (NYDB), in NYDB diluted by 75% with water and amended with glycerol to modify aw to 0.96 (NYDB25 + GLY) and in NYDB diluted by 50% with water and amended with glucose to modify aw to 0.96 (NYDB50 + GLU). (Teixidó et  al. 1998a)

Other stress conditions, such as high temperature have been studied with this biocontrol agent achieving thermotolerant cells that survived better under high temperature conditions and spray-drying process (Cañamás et al. 2008b).

7.4.2 P. agglomerans CPA-2 Enhancement The improvement of tolerance to low water activity (aw) and desiccation in P. agglomerans cells subjected to mild osmotic stress during growth was studied using different solutes to change aw of growth media. It was shown that cells grown in media at low aw using NaCl exhibited osmotic adaptation in solid media at low aw obtaining high production level and maintaining biocontrol efficacy (Teixidó et al. 2006). Osmotic-adapted cells also demonstrated thermotolerance (Teixidó et  al. 2005) and desiccation tolerance after spray drying (Teixidó et al. 2006). The role of different compatible solutes in adaptation of the bacterium to osmotic stress was determined and this study suggested that glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement (Teixidó et al. 2005; Cañamás et al. 2007).

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These osmotic adapted cells were used in preharvest treatments in order to control the main postharvest diseases on citrus fruit. In the first experiment (Cañamás et al. 2008a), it was observed that P. agglomerans cells (osmotic-adapted and non-adapted) were unable to maintain a stable population on the fruit surface and consequently, preharvest applications resulted ineffective against both naturally and artificially inoculated P. digitatum. This low survival rates revealed the sensibility of P. agglomerans cells to environmental field conditions. These results also led us to conclude that it is necessary a minimal antagonist population level on fruit surfaces in order to guarantee competition with pathogens for sites and nutrients, and to subsequently obtain an efficient control. The establishment of bacterial populations on plant surfaces is a critical phase in disease control (Ippolito and Nigro 2000). Microbes can be inactivated by several environmental factors, including sunlight, temperature, humidity, leaf surface exudates and competitors. They may also be physically lost from the target location due to the action of wind, rain or leaching (Jones and Burges 1998). The effect of main environmental factors on P. agglomerans cells, such as relative humidity and solar radiation was studied and different formulation strategies were used in order to enhance survival on fruit surface under field conditions and subsequently enhance biocontrol efficacy (Cañamás et al. 2008a). Osmotic adapted P. agglomerans cells, specially when these cells grew at 0.98 aw, were more resistant than non-adapted cells when both were applied in oranges and stored in chambers at a low Relative Humidity (43%). However, the minimum values of relative humidity registered during the first field assay were between 15% and 50%. These extreme values may restrict the survival and growth of P. agglomerans cells. The relative importance of each of these factors depends on why and where a particular product is used. In applications in foliar environments (which were our case), solar radiation, and especially the ultraviolet (UV) portion of the spectrum, is probably the most important factor affecting the persistence of microbial insecticides (Rhodes 1993; Filho et al. 2001). This detrimental effect of UV radiation was also checked in a laboratory assay in which a major decrease in P. agglomerans population was observed during exposure to sunlight. In laboratory studies, different additives, such as summer oils, alginate, glycerol and food additives at different concentrations were tested mixed with P. agglomerans in order to study its compatibility. The non-toxic ones (Citroline, Summer oil, Alginate, Sunspray, Glycerol, Siapton and Fungicover) were added to the biocontrol agent, sprayed on detached oranges and left outdoors. Population dynamics of the antagonist on fruit surface was determined along the time (Fig. 7.3). Fungicover was the most effective additive for improving the adherence and persistence of P. agglomerans cells on oranges and it was also compatible with the antagonist. Firstly, adherence was improved by Fungicover since the P. agglomerans cell population just after application and drying (0 h), was greater than when cells were only sprayed with water. It was also visually observed that spreading, wetting and dispersion were also clearly improved. This could have been due to the fact that the additive Fungicover contains fatty acid derivatives in an alcohol solution. These components could have reduced the surface tension of the cell suspension and

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Fig.  7.3  Effect of additives in the population level of non-adapted P. agglomerans cells on oranges during exposure outside under springtime environmental conditions. Samples were recovered 0 h (white bars) or 24 h (black bars) after spraying treatment. Treatments consisted in aqueous suspension of P. agglomerans cells alone or in combination with a respective additive. Values plotted at each time point are averages of three replications. Columns with different letters are significantly different (P < 0.05) according to Duncan’s test. (Cañamás et al. 2008a)

thereby improved the spread and wetness of the spray over the plant surface (Burges 1998). On the other hand, the persistence of P. agglomerans cells was also improved outdoors under springtime environmental conditions in the presence of Fungicover at 5%. It has not been possible to exactly elucidate the mechanism(s) by which this additive was able to protect the antagonist population. The additive Fungicover could also have protected P. agglomerans cells from solar radiation as sunscreen, physically reflecting and scattering, or selectively absorbing radiation, converting short wavelengths to harmless longer ones (Jones and Burges 1998). Fungicover is an edible film-forming compound for fruits and vegetables to reduce weight loss, delay senescence, improve natural brightness and reduce physiological disorders. It also reduces droplet size and improves uniformity of distribution on the surface to be protected. This additive did not show any fungicidal effect on P. digitatum (Cañamás et al. 2008a). In this study it has also been demonstrated that inoculum formulation can influence the persistence of P. agglomerans cells. Bacterial treatments prepared with lyophilised P. agglomerans cells become more resistant to environmental conditions than fresh cells, as Stockwell et al. (1998) observed when the bacterial antagonists Pseudomonas fluorescens A506 and Erwinia herbicola C9-1R was applied under field conditions.

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Different strategies could be used to improve P. agglomerans cell survival on oranges under non-controlled environmental conditions and all them were applied in two representative field trials on ‘Lane late’ and ‘Valencia’ oranges, in order to evaluate the effectiveness of different bacterial formulations of P. agglomerans applied at preharvest for controlling postharvest decays caused by natural infection and also by artificial infection (Penicillium digitatum) (Cañamás et al. 2008c). Population dynamics of P. agglomerans during trials (under field conditions and at postharvest) are shown in Fig. 7.4. In both experiments greater adherence and persistence of populations was observed when the biocontrol agent cells were sprayed using the additive Fungicover, being the population level similar to that treatment applied at postharvest. The use of additives to improve the adherence and persistence of biocontrol agents has been previously shown by Guijarro et  al. (2007), who added additives at different points of the production-formulation process, and showed that they improved adhesion of Penicillium frequentans conidia to fruit surfaces, providing more effective control against brown rot in peaches. Results for the efficacy of preharvest treatments for artificial infection by P. digitatum are shown in Fig.  7.5. In experiment 1 (Fig.  7.5a) the P25-LY + FC and P25-SH + POST treatments were significantly more effective than the other preharvest treatments and non differences were found between both after 15 days. In experiment 2 (Fig. 7.5b) and after 15 days of storage, all the preharvest treatments with the additive Fungicover showed effective control against P. digitatum with decay values of below 12.5%. Treatment P25-LY also showed effective control with 12.8% decay. However, only treatments P25-LY + FC, P98-LY + FC and P97-LY + FC with decay values of between 7% and 5.3%, exhibited levels of control on P. digitatum no statistically different to the antagonist postharvest treatment P25-SH + POST. The protective effect of the additive Fungicover was again confirmed by results and this effect varied according to whether or not the P. agglomerans cells had been osmotic adapted and if the cells had been lyophilised or not. It is therefore likely that adding Fungicover to formulations could protect cells against field conditions. At the same time, it was possible to observe that populations of osmotic adapted cells showed a higher level of survival than non-adapted cells when the additive FC was used. Furthermore, the positive effect of applying lyophilised P. agglomerans cells instead of fresh cells was also evident when treatments were combined with Fungicover. The ability of antagonists to survive at sufficient population levels on fruit surfaces after application is very important for achieving effective control (Benbow and Sugar 1999). Results indicated that there was a close relationship between the population level associated with a given treatment under field conditions and the level of control achieved by this treatment during storage. In the first experiment with ‘Lane late’ oranges, the P25-LY + FC treatment, which showed a high survival level, was the most effective of the six bacterial preharvest treatments for controlling artificial infection by P. digitatum showing a level of control not statistically different to the postharvest treatment (P25-SH + POST). In the second experiment with ‘Valencia late’ oranges, a relationship between population level and effectiveness was also found.

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Fig. 7.4  Population dynamics of P. agglomerans treatments during field and storage conditions. In Experiment 1(a) the following bacterial treatments were used: P25-SH (——), P-LY (——), P25-LY (—°—), P98-LY (—∆—), P97-LY (—◊—), P25-LY + FC (---°---) and P25-SH + POST(—´—). The bacterial treatments used in Experiment 2 (b) were P25-LY (—°—), P25-SH + FC (------), P-LY + FC (------), P25-LY + FC (---°---), P98-LY + FC (---∆---), P97-LY + FC (---◊---) and P25-SH + POST (—´—). Bacterial treatments were prepared from lyophilized (LY) or fresh (SH) and from non-adapted (P) or osmotic adapted P. agglomerans inocula in presence of 25 g L−1 of NaCl (P25) or at 0.98 or 0.97 aw in the medium (P98 and P97, respectively). The additive Fungicover (+FC) was used in some treatments at a concentration of 5% in order to check its adherence and persistence effect on the populations of P. agglomerans cells. An adequate volume of non-adapted or osmotic adapted P. agglomerans inocula for each bacterial treatment was mixed into 30 L of water in a plastic recipient to obtain a final concentration of 2 ×108 CFU mL−1.

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Fig. 7.5  Effectiveness of preharvest treatments against artificial infection of the fungal pathogen P. digitatum. Fruits were stored for 15 days at 20°C and 85% RH. The incidence of decayed fruits was scored after 7 (white bars) and 15 days (grey bars) of storage and expressed as % decay produced by the P. digitatum pathogen on orange cultivars, ‘Lane late’ and ‘Valencia late’ in Experiments 1(a) and 2(b), respectively. Different letters in the bars indicate significant differences between means according to a Duncan’s Multiple Range Test (P < 0.05). (Cañamás et al. 2008c)

All preharvest treatments, which showed stable population levels on the surface of orange fruits under field conditions, therefore also demonstrated greater effectiveness than the control treatment. Moreover, only bacterial treatments, which were

Bacterial treatments were sprayed onto orange fruits cv ‘Lane late’ (EX-1) or ‘Valencia late’ (EX-2) 1 week before harvest. Treatment P25-SH + POST was applied at postharvest dipping oranges in a solution at 1 ×108 CFU mL−1 before the storage period. Results are means of four independent samples and vertical bars indicate standard deviations. (Cañamás et al. 2008c)

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prepared from lyophilised and osmotic adapted cells, showed a level of control comparable to postharvest treatments with the biological control P. agglomerans when they were applied with additive Fungicover. These preharvest treatments were also associated with population levels that were higher under field conditions. These findings were in concordance with those of Tian et al. (2004) who found that only Rhodotorula glutinis and Cryptococcus laurentii, whose populations remained at high and stable levels, significantly reduced fruit decay during storage at 25°C. Moreover, applications of Bacillus subtilis, aimed at establishing antagonistic bacteria prior to arrival of Pseudocercospora purpurea inoculum, resulted in sustained control (Korsten et al. 1997). We conclude that survival and stability of P. agglomerans populations could be maintained under field condition by integrating certain formulation strategies: adding additives, ecophysiological osmotic adaptation and lyophilisation. Thus, it has highlighted that it is very important to optimize both, distribution of the biological control agent on the host surface and survival under field conditions. The additive Fungicover reduces droplet size and improves uniformity of distribution on the surface to be protected. Furthermore, Fungicover seems to bring a protective effect to the biocontrol agent against adverse environmental conditions. Osmotic adaptation and lyophilisation also provided a better performance of cells under field conditions. All these formulation strategies have consequently demonstrated that the improved formulation of P. agglomerans provided good protection for orange fruits against both natural and artificial infections, resulting preharvest biocontrol treatments effective in the control of postharvest diseases. However, although preharvest treatments were applied with the objective to control the infections produced in the field and consequently obtain a better control respect to apply biocontrol agents in postharvest treatments, the results showed that no significant differences in the level of control were found between the applications of biocontrol agent at preharvest or postharvest. The results of this work suggest that it is possible to broaden the spectrum of use of the biocontrol agent P. agglomerans and to thereby develop practical uses for this biocontrol agent under preharvest conditions. The results presented here are an example that the induction of stress adaptation responses is a useful and practical tool, that could broaden new possibilities for improving performance of biocontrol agents to other hosts and diseases and it could improve their antagonistic activity in a wide range of conditions. In this chapter it has been tried to compile some examples to show the interest of preharvest strategies in controlling postharvest diseases and it has been demonstrated that it is worthwhile to go ahead on this kind of research. Acknowledgements  The authors are grateful to Spanish government (Ministerio de Ciencia y Tecnología) for grants AGL-2002-01137 and AGL-2005-02510 and to FEDER (Fondo Europeo de Desarrollo Regional) and COST Action 864 for their financial support. Authors are also grateful to company BIODURCAL S.L. for providing the Fungicover product.

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Hallsworth JE, Magan N (1994a) Effects of KCl concentration on accumulation of acyclic sugar alcohols and trehalose in conidia of three entomopathogenic fungi. Lett Appl Microbiol 18:8–11 Hallsworth JE, Magan N (1994b) Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi. Microbiology-UK 140:2705–2713 Hallsworth JE, Magan N (1995) Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water activity. Microbiology-UK 141:1109–1115 Hallsworth JE, Magan N (1996) Culture age, temperature and pH affect the polyol and trehalose contents of fungal propagules. Appl Environ Microbiol 62:2435–2442 Hunter T (1995) Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signaling. Cell 80:225–236 Ippolito A, Nigro F (2000) Impact of preharvest application of biological control agents on postharvest diseases of fresh fruits and vegetables. Crop Protect 19:715–723 Janisiewicz WJ (1994) Enhancement of biocontrol of blue mold with the nutrient analog 2-deoxyD-glucose on apples and pears. Appl Environ Microbiol 60:2671–2676 Janisiewicz W, Bors B (1995) Development of a microbial community of bacterial and yeast antagonists to control wound-invading postharvest pathogens of fruits. Appl Environ Microbiol 61:3261–3267 Janisiewicz WJ, Conway WS, Glen DM, Sams CE (1998) Integrating biological control and calcium treatment for controlling postharvest decay of apples. HortScience 33:105–109 Jones KA, Burges HD (1998) Technology of formulation and application. In: Burges HD (ed) Formulation of microbial biopesticides. Kluwer Academic, London, pp 7–30 Kets EPW, Galinski EA, de Bont JAM (1994) Carnitine: a novel compatible solute in L. plantarum. Arch Microbiol 162:243–248 Ko R, Smith LT, Smith GM (1994) Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes. J Bacteriol 176:426–431 Köhl J, Fokkema NJ (1998) Strategies for biological control of necrotrophic fungal foliar pathogens. In: Boland GJ, Kuykendall LD (eds) Plant microbe interactions and biological control. Marcel Dekker, New York, pp 49–88 Korsten L, De Villiers EE, Wehner FC, Kotzé JM (1997) Field sprays of Bacillus subtilis and fungicides for control of preharvest fruit diseases of avocado in South Africa. Plant Dis 81:455–459 Leibinger W, Breuker B, Hahn M, Mengden K (1997) Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field. Phytopathology 87:1103–1110 Louis IFN, Cooke RC (1983) Influence of the conidial matrix of Sphaerellopsis filum (Darluca filum) on spore germination. Trans Br Mycol Soc 81:667–670 Louis IFN, Cooke RC (1985) Conidial matrix and spore germination in some plant pathogens. Trans Br Mycol Soc 84:661–667 Mattick KL, Jorgensen F, Legan JD, Lappins-Scott HM, Humphrey TJ (2000) Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance. Appl Environ Microbiol 66:4921–4925 McLaughlin RJ, Wisniewski ME, Wilson CL, Chalutz E (1990) Effect of inoculum concentration and salt solutions on biological control of postharvest diseases of apple with Candida sp. Phytopathology 80:456–461 Nunes C, Usall J, Teixidó N, Miró M, Viñas I (2001a) Nutritional enhancement of biocontrol activity of Candida sake (CPA-1) against Penicillium expansum on apples and pears. Eur J Plant Pathol 107:543–551 Nunes C, Usall J, Teixidó N, Viñas I (2001b) Biological control of postharvest pear diseases using a bacterium Pantoea agglomerans (CPA-2). Int J Food Microbiol 70:53–61 Nunes C, Usall J, Teixidó N, Abadias M, Viñas I (2002a) Improved control of postharvest decay of pears by combination of Candida sake (CPA-1) and ammonium molybdate. Phytopathology 92:281–287

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Nunes C, Usall J, Teixidó N, Fons E, Viñas I (2002b) Postharvest biological control by Pantoea agglomerans (CPA-2) on Golden Delicious apples. J Appl Microbiol 92:247–255 Nunes C, Usall J, Teixidó N, Torres R, Viñas I (2002c) Control of Penicillium expansum and Botrytis cinerea on apples and pears with the combination of Candida sake and Pantoea agglomerans. J Food Protect 65:178–184 Nunes C, Usall J, Teixidó N, Viñas I (2002d) Improvement of Candida sake biocontrol activity against postharvest decay by the addition of ammonium molybdate. J Appl Microbiol 92:927–935 Nunes C, Usall J, Teixidó N, Abadias M, Asensio A, Viñas I (2007a) Biocontrol of postharvest decay using a new strain of Pseudomonas syringae CPA-5 in different cultivars of pome fruits. Agr Food Sci 16:56–65 Nunes C, Usall J, Teixidó N, Abadias M, Viñas I (2007b) Preharvest application of a combined treatment of Candida sake (CPA-1) and Pseudomonas syringae (CPA-5) to control postharvest decay of pome fruits. IOBC wprs Bull 30:397–400 Obagwu J, Korsten L (2003) Integrated control of citrus green and blue mold using Bacillus subtilis in combination with sodium bicarbonate or hot water. Postharvest Biol Technol 28:187–194 Pascual S, Magan N, Melgarejo P (1996) Improved biocontrol of peach twig blight by physiological manipulation of Epicoccum nigrum. Proc. Br Crop Prot Conf, Pests Dis 4D:411–412 Poirier I, Maréchal PA, Evrard C, Gervais P (1998) Escherichia coli and Lactobacillus plantarum responses to osmotic stress. Appl Microbiol Biotechnol 50:704–709 Remaley AT, Das S, Campbell PI (1985) Characterization of Leishmania donovani acid phosphatases. J Biol Chem 260:880–886 Rhodes DJ (1993) Formulation of biological control agents. In: Jones DG (ed) Exploitation of microorganisms. Chapman & Hall, London, pp 411–439 Roberts RG (1994) Integrating biological control into postharvest disease management strategies. HortScience 29:758–762 Sanders JW, Venema G, Kok J (1999) Environmental stress responses in Lactococcus lactis. FEMS Microbiol Rev 23:483–501 Smilanick JL, Margosan DA, Mlikota F, Usall J, Michael IF (1999) Control of citrus green mold by carbonate and bicarbonate salts and the influence of commercial postharvest practices on their efficacy. Plant Dis 83:139–145 Stockwell VO, Johnson KB, Loper JE (1998) Establishment of bacterial antagonists of Erwinia amylovora on pear and apple blossoms as influenced by inoculum preparation. Phytopathology 88:506–513 Teixidó N, Viñas I, Usall J, Magan N (1998a) Control of blue mold of apples by preharvest application of Candida sake grown in media with different water activity. Phytopathology 88:960–964 Teixidó N, Viñas I, Usall J, Magan N (1998b) Improving ecological fitness and environmental stress tolerance of the biocontrol yeast Candida sake (strain CPA-1) by manipulation of intracellular sugar alcohol and sugar content. Mycol Res 102:1409–1417 Teixidó N, Viñas I, Usall J, Sanchis V, Magan N (1998c) Ecophysiological responses of the biocontrol yeast Candida sake CPA-1 to water, temperature and pH stress. J Appl Microbiol 84:192–200 Teixidó N, Usall J, Palou L, Asensio A, Nunes C, Viñas I (2001) Improving control of green and blue molds on oranges by combining Pantoea agglomerans (CPA-2) and sodium bicarbonate. Eur J Plant Pathol 107:685–694 Teixidó N, Cañamás TP, Usall J, Torres R, Magan N, Viñas I (2005) Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross protection in the biocontrol agent Pantoea agglomerans CPA-2. Lett Appl Microbiol 41:248–252 Teixidó N, Cañamás TP, Abadias M, Usall J, Solsona C, Casals C, Viñas I (2006) Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments. J Appl Microbiol 101:927–937

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Tian S, Qin G, Xu Y (2004) Survival of antagonistic yeasts under field conditions and their biocontrol ability against postharvest diseases of sweet cherry. Postharvest Biol Technol 33:327–331 Usall J, Teixidó N, Fons E, Viñas I (2000) Biological control of blue mould on apple by a strain of Candida sake under several controlled atmosphere conditions. Int J Food Microbiol 58:83–92 Usall J, Teixidó N, Torres R, Ochoa de Eribe X, Viñas I (2001) Pilot test of Candida sake (CPA-1) applications to control postharvest blue mold on apple fruit. Postharvest Biol Technol 21:147–156 Van Eck JH, Prior BA, Brandt EV (1993) The water relations of growth and polyhydroxy alcohol production by ascomycetous yeasts. J Gen Microbiol 139:1047–1054 Viñas I, Usall J, Sanchis V (1991) Tolerance of Penicillium expansum to postharvest fungicide treatment in apple packinghouses in Lleida (Spain). Mycopathologia 113:15–18 Viñas I, Vallverdú N, Monllao S, Usall J, Sanchis V (1993) Imazalil resistent Penicillium isolated from Spanish apple packinghouses. Mycopathologia 123:27–33 Viñas I, Usall J, Teixidó N, Sanchis V (1998) Biological control of major postharvest pathogens on apple with Candida sake. Int J Food Microbiol 40:9–16 Wang G, Morré DJ, Shewfelt RL (1995) Isolation of plasma membrane from Capsicum annum fruit tissue: prevention of acid phosphatase contamination. Postharvest Biol Technol 6:81–90 Wisniewski ME, Wilson CL (1992) Biological control of postharvest diseases of fruits and vegetables: recent advances. Hortscience 27:94–98 Wisniewski ME, Droby S, Chalutz E, Eilam Y (1995) Effects of Ca2+ and Mg2+ on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila. Plant Pathol 44:1016–1024 Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214–1222

Chapter 8

New Developments in Postharvest Fungicide Registrations for Edible Horticultural Crops and Use Strategies in the United States J.E. Adaskaveg and H. Förster

Abstract  New developments in postharvest fungicide registrations of fresh fruit and vegetable crops and use strategies in the United States are discussed for preventing decay and crop losses while minimizing the potential of selection of resistant pathogen populations. Postharvest fungicides used on agricultural commodities are among the most rigorously tested and regulated chemicals in the world and their risk assessment analysis and residue limits are extensively reviewed by multiple regulatory agencies. Novel products and pre-mixtures increase the spectrum of fungal decays managed and the number of crops labeled allowing global marketing of crops. These product registrations are part of a continuum of integrated approaches of handling agricultural commodities designed for stewardship of products and their safe usage in the worldwide distribution of fresh produce. Optimized postharvest usage strategies of fungicides include integration with other fungicides (i.e., pre-mixtures) and sanitation treatments to optimize performance while allowing identification of methods that reduce the selection of resistant subpopulations of pathogens. The competitive global marketing of fresh fruit crops that demands decay-free fruit and often involves long-distance shipping makes postharvest decay management a challenging task. In an integrated approach of decay management, cultural, preharvest, harvest, and postharvest practices are essential components that influence the complex interaction between host, pathogen, and environment. Orchard practices mainly affect crop health and pathogen inoculum levels, however, preharvest fungicide applications can also directly reduce the development of fruit decay. Among postharvest practices, postharvest fruit treatments with fungicides are the most J.E. Adaskaveg (*) Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA e-mail: address: [email protected] H. Förster Department of Plant Pathology, University of California, Davis, CA D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_8, © Springer Science + Business Media B.V. 2009

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effective means to reduce decay (Eckert and Ogawa 1988; Adaskaveg et al. 2002). Ideally, these fungicides protect the fruit from infections that occur before treatment, including quiescent infections, as well from infections that are initiated after treatment during postharvest handling, shipment, and marketing. Problems that had been arising with the use of the ‘older’ postharvest fungicides and that led to the cancellation of their registration or reduced usage initiated our extensive research on the identification and development of alternative treatments in the 1990s (Adaskaveg et  al. 2005). These problems included high non-target toxicity as for ortho-phenylphenol and biphenyl or lack of high efficacy and need for high usage rates as for captan or sulfur. For other fungicides such as iprodione and triforine, re-registration was not pursued because of potentially exceeding the exposure limit that is set for each pesticide. Furthermore, widespread resistance in pathogen populations has arisen against other materials such as benomyl, thiabendazole, and imazalil. In the development of new fungicides, emphasis was placed on minimizing human health risks and environmental toxicity. With mammalian toxicity concentrations of generally more than 2,000 mg/kg, these new treatments are the safest fungicides ever developed for postharvest use. Fungicides that meet these standards are classified as ‘reduced risk fungicides’ by the United States Environmental Protection Agency (EPA; EPA 2003). To reduce the potential of resistance development against the new treatments, application strategies have been developed based on experiences in developing postharvest fungicide treatments of agricultural commodities made in the past.

8.1 Risk Assessment, Maximum Residue Limits, and Postharvest Fungicide Registration Fresh fruits and vegetables are considered essential for a high-quality human diet. The use of postharvest fungicides dramatically reduces crop losses and allows worldwide distribution of fresh market fruit and vegetable crops. Registration of synthetic postharvest fungicides, like any other agricultural pesticide, includes a risk assessment analysis by governmental regulatory agencies to ensure that the product is safe to users, consumers, and the environment, as well as to the crop itself. Pesticides used in agriculture are among the most rigorously tested and regulated chemicals in the world. Risk assessment analysis involves evaluating the potential hazard characteristics of the product including the active ingredient and breakdown products, as well as carrier or inert ingredients in the formulation. Toxicological, ecotoxicological, and physical properties of the active ingredient and formulated product are evaluated to safeguard the use of the product, and analytical procedures for measuring the active ingredient are established during product development. In regulatory assessments of single active ingredient products in the United States, multiple exposure tests assess acute and chronic dietary, as well as short-, intermediate-, and long-term occupational, residential, and recreational exposure. In many countries, comprehensive reviews of data packages

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are done at national and regional levels for each country where the product will be sold. Furthermore, pesticides in general are subject to a re-review after some period of time. The prospect of world-wide pesticide registration is a daunting task for any manufacturer and only recently has regulatory harmonization across regions and among regulatory groups been considered. For any pesticide, concentration limits on a given commodity are set to ensure safe usage and minimal risk for consumers. In the United States, the EPA along with the Food and Drug Administration (FDA) evaluates human risk associated with pesticide exposure and sets its own standards for pesticide tolerances using chemical hazard and exposure assessment procedures in the risk assessment process as described previously. The Codex Alimentarius Commission was created in 1963 by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) to develop food standards and guidelines in order to protect consumer health, ensure fair trade practices, and to promote coordination of all food standards. The Codex Committee on Pesticide Residues (CCPR) develops and maintains acceptable maximum residue limits (MRLs) of pesticides for food commodities. Residue limits are established by CCPR and the Joint Meeting on Pesticide Residues by FAO and WHO based on toxicological data and estimates for average daily intake (ADI) and acute reference dose (ARfD) for humans, as well as data on a pesticide’s metabolism, environmental fate, and use patterns according to Good Agricultural Practices (GAPs). Postharvest fungicide residues are generally several times lower than the MRL (Table 8.1). Furthermore, MRLs and typical use residues for postharvest fungicides are similar to residues found when the fungicides are used preharvest. Any crop that exceeds the MRL must be destroyed following strict procedures. Residue monitoring is routinely done on agricultural commodities and ensures the distribution of safe, high-quality, disease-free fresh produce around the world. Unfortunately, many countries including the United States and the European Union have not accepted Codex MRLs as an international standard and instead have relied on their own national or import MRLs. These countries still have to follow Codex MRLs when exporting agricultural commodities to destination markets that have adopted these guidelines.

Table  8.1  Maximum residue limits (MRLs) and typical use residues of common postharvest fungicides of fruit crops Typical use residues Postharvest fungicide Crop MRL (mg/kg) (mg/kg) Fludioxonil Nectarine, plum 5 0.25–0.5 Peach 5 1–1.25 Pome fruit 5 0.5–1 1 (3–5) Pyrimethanil Pome fruit 3 (15) a Tebuconazole Sweet cherry 4 0.5–1  MRL for pyrimethanil recently changed to 15 mg/kg to accommodate different application methods.

a

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8.2 Registration of New Postharvest Fungicides in the United States After the cancellation of iprodione in 1996, the sterol biosynthesis inhibitor (SBI) tebuconazole was first in an unprecedented succession of new postharvest registrations that occurred during the years since that date and this process is still ongoing (Table 8.2). Tebuconazole (Elite®) was approved for use on sweet cherry in 1997. Also in 1997, the ‘reduced risk’ phenylpyrrole fludioxonil (Scholar® or Graduate®) was approved for use on stone fruit, and later on pome fruit, pomegranate, kiwifruit, and citrus. This was followed by registrations of the ‘reduced risk’ hydroxyanilide fenhexamid (Judge®), the anilinopyrimidine pyrimethanil (Penbotec®), and the QoI fungicide azoxystrobin (Diploma®), as well as the SBI propiconazole (Mentor®) on a range of fruit crops as indicated in Table 8.2. Each of the postharvest fungicides registered or in development has a distinctive spectrum of activity as indicated in Table 8.2 and for each crop, treatments are being made available to manage all major decays. Thus, brown rot (Monilinia spp.), gray mold (Botrytis cinerea), and Rhizopus rot (Rhizopus stolonifer) of stone fruit, gray mold (B. cinerea) and Penicillium decays (Penicillium expansum and other species of Penicillium) of pome fruit, and Penicillium decays (P. digitatum, P. italicum) and sour rot (Geotrichum citri-aurantii) of citrus fruit all can be effectively managed using minimal rates that result in very low fungicide residues on the crop. For several crops, more than one fungicide, each belonging to a different chemical class, is available. This is being done to expand the spectrum of activity of postharvest treatments and to accommodate export markets with specific residue tolerance requirements. In addition, the availability of multiple active ingredients for management of the same pathogen is critical for crops where a high risk for resistance development in pathogen populations is present (e.g., citrus and pome fruit) and where the use of prudent resistance management strategies is essential (see below). As also indicated in Table 8.2, the range of crops where new postharvest fungicides are being registered is expanding. Thus, for example, planned registrations include fludioxonil on tuber crops, tomato, as well as tropical fruits such as pineapple, papaya, and mango, azoxystrobin on potato, and propiconazole on tomato where currently resistant populations limit the use of registered compounds or where no highly effective compounds for decay management are available. For example, TBZ-resistant populations of Fusarium and Helminthosporium species occur on potato tubers causing dry rot and silver scurf, respectively, whereas on tomato no other fungicide is registered after the cancellation of ortho-phenylphenate. Furthermore, additional fungicides are needed for a number of crops where numerous decay organisms are not successfully managed. For instance, difenoconazole is proposed for use on pome fruit and tuber crops for managing Bull’s eye rot and decays caused by Fusarium spp., respectively.

SBI-triazole Benzimidazole

Sweet cherry Citrus, pome fruit, potato

– –

Penicillium decays Penicillium decays, brown rot, gray mold, sour rot Penicillium decays, brown rot, gray mold, bull’s eye rot Brown rot, Rhizopus and Mucor decays Penicillium decays, gray mold, Fusarium rot

Brown rot, gray mold, Rhizopus rot, Penicillium decays

Spectrum of activity Penicillium decays Penicillium decays, bull’s eye rot, Rhizopus rot, Fusarium decay Brown rot, gray mold

* - Reduced risk classification by the United States Environmental Protection Agency (US-EPA). See http://www.epa.gov/opppmsd1/PR_Notices/pr97-3.html.

Tebuconazole Thiabendazole

Table 8.2  Current and future postharvest fungicides for selected agricultural crops in the United States Fungicide Class Crops registered Crops planned Azoxystrobin* QoI Citrus Potato Difenoconazole SBI-triazole – Pome fruit, tuber crops Fenhexamid* Hydroxyanilide Stone fruit, pome fruit, – pomegranate, kiwifruit Tuber crops, Fludioxonil* Phenylpyrrole Stone fruit, pome fruit, tomato, pomegranate, kiwifruit, tropical fruit citrus Imazalil SBI-imidazole Citrus – Propiconazole SBI-triazole Stone fruit Citrus, tomato Pyrimethanil* Anilinopyrimidine Citrus, pome fruit Stone fruit

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8.3 Risk of Fungicide Resistance Development in the Postharvest Environment Fundamental to understanding the resistance potential of a fungal species against a fungicide is the fact that resistant populations develop primarily by selection from a very small number of naturally occurring, less sensitive individuals within the population (Brent and Hollomon 1998). These variants arise by continuous random changes or mutations that occur within a species. Some of these changes may be lethal or render the individual less fit while others have no apparent effect. The naturally occurring variations are a mechanism for survival of a species in an ever changing environment that applies selection pressures (for example, the use of fungicides) on sub-populations of the species. Once the size of a less sensitive, competitive sub-population reaches a threshold, treatments with the fungicide will no longer be effective. The relative frequency of these less sensitive isolates within the population is the resistance frequency, whereas the resistance factor describes the increase in fungicide sensitivity as compared to the baseline sensitivity (Beresford 1994). The extent of potential fungicide resistance development within a population is influenced by intrinsic properties of the fungicide and the pathogen in a given environment. In addition, a range of packinghouse practices that affect fungicide efficacy also affects the risk of resistance development. Because all of the new postharvest fungicides are single-site mode of action compounds that affect a single metabolic process, the selection of less sensitive or resistant individuals is more likely than for multi-site mode of action compounds. In our studies with P. digitatum on citrus we directly demonstrated that single-site mode of action fungicides may differ in their resistance frequency and, based on laboratory and packinghouse selection studies, were determined to range from 1 × 10−4 to 7 × 10−6 and from 1 × 10−6 to 1 × 10−9 for fludioxonil and pyrimethanil, respectively. Because resistance frequencies are generally very low, resistant individuals will more likely be selected for if the pathogen population is large. Among postharvest decay pathogens, this is certainly true for species of Penicillium that have a high reproductive potential due to abundant spore production as well as a short generation time and that developed resistance to the ‘older’ postharvest fungicides registered on citrus (i.e., imazalil and thiabendazole) and pome fruit (i.e., thiabendazole). Still, most of the other major decay fungi also have large population sizes.

8.4 Fungicide Usage Strategies for Preventing Fungicide Resistance in the Postharvest Environment The goal in using postharvest fungicides is to minimize the incidence of decay (survivorship) and to limit the reproduction of any surviving pathogen individuals that cause fruit decay (anti-sporulation properties). Commodities that are being stored

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for extended periods of time before marketing and that commonly develop decay in storage, benefit from the use of fungicides that suppress sporulation on decaying fruit. For example, on citrus and pome fruit, fludioxonil is more effective in inhibiting sporulation of Penicillium species than is pyrimethanil (Adaskaveg et al. 2004, Kanetis et al. 2008a). Fludioxonil, however, has a reduced post-infection or reachback activity (inhibition of infections that occur at and after harvest) as compared to pyrimethanil and azoxystrobin due to its contact properties that limit penetration into the fruit tissue. Because of often large harvest volumes and long transportation time from orchard to packinghouse, citrus fruit may not be processed and treated with a fungicide in a packinghouse until 24–36 h after harvest. Thus, postharvest fungicides with post-infection activity are needed for fruit that arrive from the field. With excellent reach-back but reduced anti-sporulation activity, pyrimethanil applications should be combined with other fungicides that provide sporulation control. A fludioxonil-azoxystrobin pre-mixture (Graduate A+®) for use on citrus fruit combines the anti-sporulation activity of fludioxonil with the post-infection activity of azoxystrobin and thus, will provide maximum decay control while reducing spore inoculum in citrus storage facilities, similar to imazalil before widespread resistance developed against this latter compound. If fludioxonil is used alone, applications during packing of lemon fruit allow for immediate protection of fruit handling injuries as fruit are removed from storage and processed for marketing. Post-infection activity is not required at this stage because of the short time interval between removal of fruit from storage and fungicide treatment. Packinghouse practices that increase the likelihood of resistance development include all methods that lead to sub-optimal fungicide coverage (i.e., uneven distribution over the fruit surface) and residue concentrations at infection sites (e.g., mostly fruit injuries). These practices include improper application methods due to non-calibrated equipment or due to cost-saving or using improper fungicides-fruit coating mixtures. The Fungicide Resistance Action Committee (FRAC) considers that reducing the treatment rate can enhance the development of resistance and that recommended rates must be maintained (Brent 1995). Based on the use of singleor multiple-site mode of action fungicides and on the ratio between resistant and surviving wild-type sensitive isolates in the pathogen population, contrary beliefs, however, exist about whether reduced-dose treatments will increase or decrease the selection of less-sensitive sub-populations (Brent 1995). The thought is that when one single-site mode of action compound is used and disease is managed, but not to zero levels, that the surviving sensitive wild-type population will eventually replace the resistant sub-population. Experimental data are very difficult to generate to prove this hypothesis. Predictive models are usually based on the assumption that the same fungicide is being used repeatedly. Because cross-resistance between different fungicide classes is rare and several effective fungicide classes are now available for many fruit crops, fungicides can be rotated or mixed effectively to obtain a high degree of decay control while minimizing the risk for resistance development. The registration of multiple new fungicides allows stipulation of the philosophy that any fruit lot should only be treated once with a fungicide of the same class or

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+

Phenylpyrrole Fludioxonil

+

Fludioxonil

+

Phenylpyrrole Fludioxonil

Anilinopyrimidine pyrimethanil QoI Azoxystrobin

Azoxystrobin

+

=

citrus - registered Graduate A+® citrus - registered

=

SBI Propiconazole

SBI Difenoconazole

Philabuster®

=

=

Citrus – in development

Pome fruit in early development

Fig. 8.1  Registered and planned pre-mixtures of postharvest fungicides in the United States as a strategy for increasing spectrum of activity and reducing the potential of resistance development in target pathogen populations

mode of action. Ideally, rotations of mixtures should be used for fruit crops that are being treated more than once, such as some citrus and pome fruits. New and planned postharvest fungicide pre-mixture registrations accommodate this strategy. Currently two pre-mixtures, i.e., imazalil-pyrimethanil (Philabuster®) and Graduate A+® are fully registered on citrus in the United States, however, several additional ones are in development (Fig.  8.1). Thus, for citrus a triple pre-mixture between azoxystrobin, fludioxonil, and propiconazole is in preparation. In this triple premixture, all three components are effective against Penicillium decays, whereas propiconazole is also active against sour rot caused by G. citri-aurantii. For pome fruit, a pre-mixture of fludioxonil and difenoconazole is in development, with both components being very active against Penicillium decays, fludioxonil also effective against gray mold, and difenoconazole also effective against bull’s eye rot caused by several species of Neofabrea. In mixture applications, the resistance potential is much reduced as compared to applications with single active ingredients because of a lower resistance frequency. For example, assuming resistance frequencies for fungicides A and B of 10−6 and 10−9, respectively, the resistance frequency of the mixture will be 10−15. Although these numbers are extremely low, the risk for resistance development will never be zero, and the full spectrum of integrated strategies should be employed. Maintaining a high efficacy of postharvest treatments is essential to minimize the incidence of decay and the number of surviving pathogen propagules. Application equipment has to be routinely monitored for optimal performance. Low- and ultra-low-volume in-line fungicide spray applications to wet, washed fruit have been the standard treatment method of fruit industries for many years because run-off is limited and, consequently, few disposal problems arise. New application technologies are being developed for some crops where

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Table 8.3  Comparison of postharvest application methods in an experimental packingline study on inoculated Bartlett pear fruit Incidence of decay gray Incidence of decay blue molda molda Treatment and rate Application method Control – 100 a 100 a Fludioxonil 225 mg/L Drench 4.2 d 5.6 c Dip 23.6 bc 11.1 c Low-volume spray 38.9 b 36.1 b Pyrimethanil 500 mg/L Drench 5.6 d 7c Dip 15.3 cd 5.6 c Low-volume spray 13.9 cd 34.7 b Fruit were wound-inoculated with conidia of Botrytis cinerea (gray mold) or Penicillium expansum (blue mold), incubated for 13–16 h at 20°C, and treated using an in-line drencher or a low-volume spray applicator at 20 gal/200,000 lb (or 75 L/91,000 Kg), or by a 30-s dip. All treatments were applied as aqueous solutions. Fruit were then incubated at 20°C for 6 days. a For each decay, values followed by the same letter are not significantly different based on an analysis of variance and least significant mean separation procedures.

efficacious fungicide residues are not easily obtained, such as for nectarines, plums, or some pome fruit that have a smooth, waxy epicarp. We evaluated the use of postharvest in-line re-circulating drench applications on several crops (Förster et al. 2007; Kanetis et al. 2008b). As shown for a trial with Bartlett pear in Table  8.3, treatment efficacies obtained in drench applications were generally significantly higher as compared to low-volume spray applications or dip treatments. This treatment strategy has been established for the use on citrus fruit in California but is also starting to be more widely used on other crops.

8.5 Sanitation and Fungicide Resistance Management Appropriate sanitation practices are another key aspect of resistance management. The goal is to keep postharvest pathogen populations that are exposed to fungicide selection pressure at a minimum. Sanitation includes the sorting of fruit to remove any decay and the proper disposal of treated decayed fruit during packinghouse handling. Thus, treated fruit should never be disposed of in the orchard to prevent the introduction of fungicide-exposed pathogen populations into the field. Sanitation also involves removal of fungal inoculum from air or water by filtration, as well as inactivation of inoculum on fruit, equipment, and in water handling systems using heat or chemical sanitizing agents. Chemical sanitation treatments inactivate microbial propagules brought into solution from fruit and equipment surfaces and thus, reduce the amount of pathogen inoculum that subsequently is being exposed to the fungicide. Thus, based on the concept of resistance frequency

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Table 8.4  Compatibility of citrus postharvest fungicides with sanitizers Sodium Fungicide hypochlorite Peroxyacetic acid Azoxystrobin + + Fludioxonil + + Imazalil − + Pyrimethanil − + Thiabendazole + +

Sodium bicarbonate − +/− + + +

Ratings: − = incompatible or negative effects on fruit (phytotoxicity) or fungicide efficacy + = compatible or not phytotoxicity on fruit and/or decrease in fungicide efficacy. Rates of sodium hypochlorite, peroxyacetic acid, and sodium bicarbonate were 100 mg/L (HOCl), 2700 mg/L (H2O2), and 30,000 mg/L or 3%, respectively.

discussed above, the probability for selection of resistance is reduced when only a small population is exposed to a selection pressure (i.e., use of a fungicide). In addition to sanitation of fruit and equipment, re-circulating fungicide solutions for fruit treatment also have to be sanitized (Kanetis et al. 2008b). Although only washed and sanitized fruit are being treated, spore inoculum will eventually build up in solutions during prolonged usage. Depending on the agricultural commodity, associated usage patterns, and costs, fungicide solutions can be filtered to remove inoculum, heated to kill temperature-sensitive inoculum, or chemically treated, most commonly with oxidizing agents. As indicated in Table 8.4, sanitizing agents including sodium hypochlorite (chlorine), peroxyacetic acid, or sodium bicarbonate are not all compatible with all postharvest fungicides. Thus, sanitizing treatments have to be selected depending on the fungicide used.

8.6 Epilogue The development of highly effective and regulated postharvest fungicides that can be quantified and tested and that can be re-evaluated over time has been a sound approach in the prevention of postharvest crop losses by fungal decay. The registration of active compounds with different modes of action in the pathogen has followed a trend to extremely low use rates. The increased arsenal of fungicides provides an increased spectrum of activity, allows for registration on additional commodities with less dependency on any single active compound, and facilitates global marketing. Usage patterns have to be optimized for each crop to obtain maximum decay control. The near simultaneous registration of several compounds for a single crop allows for new postharvest usage strategies to minimize selection of resistant pathogen sub-populations. This scenario is distinctly different from historical events where postharvest fungicides for agricultural commodities were introduced sequentially after resistance to the previously registered fungicide had already developed. The registration of fungicide pre-mixtures especially for crops

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where resistance to the ‘older’ registered compounds has developed is a built-in resistance management strategy. Integration with other practices such as sanitation, and the usage philosophy that any fruit lot should only be treated once with a fungicide of the same class should minimize any risk losing the new treatments due to the development of resistance. In addition to these pre-registration and usage strategies of postharvest fungicides, among other pivotal components in resistance management is the routine monitoring for fungicide sensitivity in the pathogen population (Adaskaveg et al. 2004). The goal is to detect any shifts in sensitivity as compared to baseline values at an early stage before field (practical) resistance and lack of fungicide efficacy occurs and when corrective measures can still alleviate the spread of resistance. Fungicide manufacturers and packinghouse managers realize the need for judicial usage patterns because the identification and registration of new compounds that qualify for the same the high standards in safety and performance is difficult due to the paucity of appropriate materials and the regulatory safeguards. Thus, every effort should be made to extend the usage time of any registered postharvest fungicide.

References Adaskaveg JE, Förster H, Sommer NF (2002) Principles of postharvest pathology and management of decays of edible horticultural crops. In: Kader A (ed) Postharvest technology of horticultural crops, 4th edn. UC DANR Publ. 3311. Oakland, CA, pp 163–195 Adaskaveg JE, Kanetis L, Soto-Estrada A, Förster H (2004) A new era of postharvest decay control in citrus with the simultaneous introduction of three new “reduced-risk” fungicides. Proc Int Soc Citriculture Vol. III: 999–1004 Adaskaveg JE, Förster H, Gubler WD, Teviotdale BL, Thompson DF (2005) Reduced-risk fungicides help manage brown rot and other fungal disease of stone fruit. Calif Agric 59:109–114 Beresford R (1994) Understanding fungicide resistance. Orchardist 67:24 Brent KJ (1995) Fungicide resistance in crop pathogens: how can it be managed? FRAC Monograph No. 1. GIFAP, Brussels Brent KJ, Hollomon DW (1998) Fungicide resistance: the assessment of risk. FRAC Monograph No. 2. GIFAP, Brussels Eckert JW, Ogawa JM (1988) The chemical control of postharvest diseases: deciduous fruits, berries, vegetables and root/tuber crops. Ann Rev Phytopathol 26:433–469 EPA (2003) Reducing pesticide risk. www.epa.gov/pesticides/controlling/reducedrisk Förster H, Driever GF, Thompson DC, Adaskaveg JE (2007) Postharvest decay management for stone fruit crops in California using the “reduced-risk” fungicides fludioxonil and fenhexamid. Plant Dis 91:209–215 Kanetis L, Förster H, Adaskaveg JE (2008a) Comparative efficacy of the new postharvest fungicides azoxystrobin, fludioxonil, and pyrimethanil for managing citrus green mold. Plant Dis 91:1502–1511 Kanetis L, Förster H, Adaskaveg JE (2008b) Optimizing efficacy of new postharvest fungicides and evaluation of sanitizing agents for managing citrus green mold. Plant Dis 92:261–269

Chapter 9

New Approaches for Postharvest Disease Control in Europe M. Mari, F. Neri, and P. Bertolini

Abstract  Alternative methods to fungicide treatments have been studied in order to prevent fruit losses in the postharvest phase. Within these methods the applications of: (a) biological control agents (BCAs), (b) plant bioactive compounds, and (c) physico-chemical methods showed interesting results but still far from a practical application in Europe. Actually, despite the substantial progress obtained with BCAs, any biofungicide has been registered in Europe to control postharvest pathogens, moreover because of their insufficient and inconsistent performance. The use of plant bioactive compounds has shown that the treatment conditions (concentration, form of application, formulation, exposure time, time of treatment, etc.) can deeply influence their efficacy. The different responses found in many studies indicate a cultivar specificity in the product-pathogen-volatile interaction. A barrier to use the plant bioactive compounds may not be efficacy, but rather the off-odours caused in fruits and vegetables and/or the phytotoxicity. Physicochemical methods include heat, ionising and ultraviolet C irradiation, food additives inducers of resistance. Heat treatments by hot water dips, hot dry air, vapour heat or very short water rinse and brushing appear promising. To overcome the drawbacks that have arisen with the these methods, the integration of the antagonist with other treatments such as low toxic substances (GRAS), heat, etc. has been proposed; this strategy could produce an additive or synergistic effect on disease control and obtain satisfactory levels of disease reduction. Keywords  Botrytis cinerea • fruit • Monilinia spp. • Penicillium spp. • Phlyctema vagabunda

M. Mari (*), F. Neri, and P. Bertolini CRIOF – DIPROVAL, University of Bologna, Via Gandolfi, 19, 40057, Cadriano, Bologna, Italy e-mail: [email protected] D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_9, © Springer Science + Business Media B.V. 2009

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9.1 Introduction Fruit production for human consumption requires time and money and is not only a biological process but also an important part of the market economy. Any waste due to spoilage and pest infestation, in the field and during the postharvest phase, represents economic losses which are greater the closer they are to the sale of the fruit. Fruit perishes quickly, and if care is not taken in its harvesting, handling and transport, it will soon decay and become unfit for human consumption (FAO 1989). The extent of postharvest losses varies in relation to commodities and country; although few up-to-date data are available (Amorim et al. 2008), they can be estimated as ranging between 4–8% in countries where refrigeration facilities are well developed to 50% where these facilities are minimal (Eckert and Ogawa 1985). Microbial decay is one of the main factors that determine losses, also compromising the quality of the fresh produce. In the past the use of new fungicides and modern storage technologies such as controlled atmosphere, ultra low oxygen atmosphere, dynamic atmosphere, etc. have extended the shelf-life of fresh fruit, reducing postharvest losses. However, in the last 20 years, the concern about public health and the environment has considerably limited the use of fungicides after harvest and consumer demand for fungicide-free fruit has also led the multiple retailers to pursue a policy of eliminating residues, following a new ‘zero residue’ integrated pest and disease management programme implemented by their produces (Cross and Berrie 2008). The interest in finding alternative approaches to control postharvest disease fitting well with the concept of safe food for human health has thus greatly increased. There are three main research fields (biological control with microbial antagonists, natural bioactive compounds, physico-chemical methods) characterized by a great number of studies and widely reviewed (Janisiewicz and Korsten 2002; Spadaro and Gullino 2004; Mari et al. 2007a).

9.2 Biological Control with Microbial Antagonists The first studies on biocontrol of postharvest diseases appeared over 20 years ago and since then important progress has been achieved. These investigations have produced commercial products: Aspire™ (Ecogen Inc., Langhore, PA) based on the yeast Candida oleophila; BioSave™ 100 and 110 (JET Harvest Solution, Longwood, FL) based on a strain of Pseudomonas syringae; YeldPlus™ (Anchor Yeast, Cape Town) based on Cryptococcus albidus; Shemer™ (AgroGreen, Asgdod) based on Metschnikovia fructicola. These biofungicides have been registered for postharvest use in the United States (Aspire™, BioSave™), in South Africa (YeldPlus™), in Israel (Shemer™) but not in Europe. CANDIFRUIT™ (SIPCAM INAGRA, S.A. Valencia) based on Candida sake is commercially available only in Spain from the 2008 season for pome fruits against postharvest pathogens. Despite these efforts biological control agents (BCAs) are still not routinely applied because of their insufficient and inconsistent performance, the difficulty in obtaining an adequate formulation and the dif-

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ficulty in controlling previously established infections. The registration process for a biofungicide in Europe is more difficult than elsewhere. In fact to register a BCA, the EPA (Environmental Protection Agency), which is in charge of biocontrol agent regulation in the USA, needs an average of 2 years while in Europe the registration of the same products takes almost 7 years. To accelerate the registration process, the European Community has supported a policy action, called REBECA (www.rebeca-net.de), that reviews the possible risks of biocontrol agents, compares regulations in the EU and the USA and proposes alternative less bureaucratic and more efficient regulation procedures maintaining the same level of safety for human health and the environment but accelerating market access and lowering registration costs.

9.3 Natural Bioactive Compounds Plants possess a diverse array of secondary metabolites which function as fungal inhibitors. The aroma component of horticultural products or the essential oils of spices and herbs commonly used in human diet appear interesting to control decay, because of their safety at low concentrations. The high volatility and low solubility in water of these compounds make them particularly suitable for application in their vapour phase by a new process defined as ‘biofumigation’. Recently, packing systems that deliver volatile antimicrobials to the packaged product, known as ‘antimicrobial active packaging’, have been evaluated (Ayala-Zavala et al. 2008). Absorption of volatile compounds in solid substances (starch, alumina, etc.) or their micro-encapsulation on the internal cavity of b-cyclodestrine could be interesting for the stabilization and controlled release of the antifungal compounds in active packaging. However, selected film types need to be used to prevent both loss of volatile compounds from permeation and accumulation of excessive concentration of carbon dioxide and fermentative metabolites causing off-flavours in products. The latter aspect is particularly important for fruits and vegetables with a high respiration rate especially, when kept at ambient temperature. Several bioactive compounds inhibited the growth of fruit and vegetable postharvest pathogens (Plotto et al. 2003; Tsao and Zhou 2000; Neri et  al. 2006a,b,c, 2007, 2009; Mari et  al. 2002a, 2008). The most consistent fungicidal activity was found with vapour of some isothyocyanates (ITCs) such as allyl-ITC and benzyl-ITC, followed by trans-2-hexenal, carvacrol, citral and transcinnamaldehyde. Nevertheless, the fungal inhibition by these compounds was not always confirmed in  vivo, showing that the treatment conditions (concentrations, temperature, form of application, exposure time, time of application in relation to the establishment of conidia in wounds, etc.) can considerably affect fruit and vegetable response to treatment (Fan et  al. 2006; Spotts et  al. 2007; Sholberg and Randall 2007). Plant volatile compounds generally have an intense and specific odour, they can be absorbed and metabolized by the commodities, often altering their sensory properties; moreover, especially when used at high concentrations, they can also be toxic for plant tissue (Mari et  al. 2008; Neri et  al. 2008). In spite of their consistent efficacy in culture, a barrier to the use of plant bioactive compounds

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as non-conventional methods for disease control may be the development of off-odours and/or phytotoxicity in treated products (specie and cultivar). There are still very few studies on the effect of treatment on sensory quality, and more investigation is required to avoid detrimental effects of plant bioactive compounds on texture and flavour of fruits and vegetables. In addition, allyl-ITC and other ITCs are available as synthetic compounds but can be produced from Brassica defatted meal with similar results in their efficacy against pathogens (Mari et al. 2008); this is an important aspect showing that biofumigation could be used for industrial applications. The use of bio-based chemicals obtained from renewable natural resources fits well with the goals of the European Technology Platform “Plants for the Future’’, and the beneficial effect of the use of Brassica plants in pharmacology has also been documented (Mennicke et al. 1998; Stoner et al. 1999).

9.4 Physico-Chemical Methods The use of heat, ionising and ultraviolet C (UV-C) irradiation has recently been proposed to control postharvest diseases. Physical stress can have a dual effect: disinfection of fruit skin and induction of resistance against future infections. The concept of induced resistance in plants to pathogens is not new (Chester 1933) but has been ignored for a long time. However, the induced/acquired resistance has recently been considered a preferred strategy for achieving integrated pest management (Kuc 2000). A pre- or postharvest treatment with chemical, physical or biological elicitors may reduce or suppress postharvest diseases. Salicylic acid (SA), methyl jasmonate, acibenzolar, chitosan, and phosphonate are some of the chemical elicitors tested and reviewed by Terry and Joyce (2004). Their activity has sometimes proved inconsistent (Yu et  al. 2007), with only a fungistatic effect (El-Ghaouth et  al. 1992) and related to treatment timing and the plant development stage (Huang et al. 2000). In addition, SA can be incompatible with integrated pest management since phytotoxic effects on leaf margin (Reglinski et al. 1997) or on fruit skin (Mari data not published) were observed. Heat treatments by hot water dips, hot dry air, vapour heat or very short water rinse and brushing have been used with success against numerous postharvest pathogens and reviewed by Lurie (1998) and Fallik (2004); the authors pointed out the beneficial effects of a pre-storage heat treatment: (a) easy to use; (b) kills the pathogens on the surface of fruits: (c) economical (d) environmentally safe. However, the physiological responses can be different with respect to cultivar, season and growing location, and a thorough evaluation of temperature and dipping time is therefore necessary. Heat may also inhibit pathogens infecting fruit prior to harvest and reduce rot development on organic produce, maintaining fruit quality. Common food additives generally recognised as safe (GRAS) include salts such as sodium carbonate, sodium bicarbonate, potassium sorbate, sodium propionate, etc., allowed with no restrictions for many applications in European and North America regulations. They have been tested in numerous trials and appear to be interesting tools to manage postharvest decay because in addition to their ­consistent

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antimicrobial activity, they are inexpensive, readily available, and suitable for the postharvest handling practices that use water to float fruit out of field bins and to remove field heat from fruit by hydrocooling. Although these salts appear fungistatic, not very persistent and with minimal risk of injury to the fruit overall when used as a heated solution, the combination of GRAS and BCA is better, being more effective than either treatment alone as shown in numerous trials (Teixido et  al. 2001; Conway et al. 2004; Karabulut et al. 2005).

9.5  Main Postharvest Diseases in Europe The main fungal pathogens that cause important postharvest losses on fruits in European growing areas are: Penicillium expansum and Phlyctema vagabunda on pome fruits, Monilinia sp. on stone fruits, P. digitatum and P. italicum on citrus fruits, Botrytis cinerea on table grape, strawberries and kiwifruits.

9.5.1 Blue Mould Blue Mould (P. expansum Link) is one of the main postharvest diseases in pome fruit (Jones and Aldwinckle 1990). In Europe, the pathogen causes extensive decay (Amiri et  al. 2008), particularly in pears penetrating through wounds and microwounds, that frequently occur during harvesting and handling (Spotts et al. 1998). Control of blue mould is based on the use of thiabendazole and imazalil, applied as a drencher or on line-sprayer treatment prior to cold storage. However, in the last decade the development of resistance to these fungicides has reduced the effectiveness of such treatments, making them useless (Vinas et al. 1993; Baraldi et al. 2003). In this situation, the need for new and alternative means to control blue mould is more and more urgent. The results obtained with BCAs in packinghouse tests on pome fruits showed a control level of natural infections of P. expansum not commercially acceptable, even less than 50%. In addition, a different level of efficacy was observed when the same BCA was applied to fruits derived from different orchards (Torres et al. 2006). This probably depends on fruit quality, inoculum density, level of fruit susceptibility to infection and the time elapsing between inoculation and treatment (Droby et al. 2003). The formulation of BCA can also considerably influence the efficacy of the antagonist but also make its application easier and less expensive (Fravel et al. 1998). Torres et al. (2006) tested seven liquid and one wettable powder formulations of C. sake (strain CPA-1) on pome fruit against blue mould and only one, a liquid formulation, was selected for commercial trials, because it was cheap to produce and easily suspended in water; however all C. sake formulations showed the same efficacy as fresh cells. To enhance the antifungal activity of BCA, the application of yeast mixtures or GRAS (sodium bicarbonate) and antagonist mixture was tested with a decay reduction of 84–97.4% compared to

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the P. expansum drench alone (Janisiewicz et al. 2008). The efficacy of a biocontrol yeast, Cryptococcus laurentii, was increased by the combination with SA (Ting et al. 2007). P. expansum is a necrotrophic pathogen and can start the active pathogenic process immediately (within the first 12–24 h of incubation), after spores land on the wounded tissue (Prusky and Lichter 2007); this may explain the inefficacy of SA treatment in some host-pathogen interactions. But when SA was used in combination with C. laurentii, the antagonist acted as the primary defence line against the pathogen, rapidly colonizing the wounds, utilizing available nutrients and inhibiting the initial attack of fungus, while SA was able to reinforce the decay control by activation of fruit natural resistance after 48 h of incubation. Within natural bioactive compounds, AITC and trans-2-exenal showed consistent fungicidal activity against P. expansum (Mari et al. 2002a; Neri et al. 2006a). In ‘Golden Delicious’ apples, the blue mould control by trans-2-exenal was particularly interesting, treatment applied 24 after inoculation significantly reduced decay and patulin content in fruits, without causing negative effects on quality traits (Neri et al. 2006a). In contrast, treatment with trans-2-exenal concentrations, effective against blue mould, caused phytotoxic symptoms on ‘Abate Fetel’ pears and affected fruit flavour in ‘Conference’ and ‘Barlett’ pears and ‘Royal Gala’ apple (Neri et al. 2006b). Another important aspect is the accumulation of a toxin: patulin in pome fruit infected by P. expansum. Morales et  al. (2008) found that the use of two BCAs (C. sake and Pantoea agglomerans) seemed to have a positive effect on decay control and patulin accumulation after apple cold storage. On the other hand, a yeast, Rodotorula glutinis (strain LS11), appears to metabolize patulin in vitro and in a model emulating decaying apple tissue. Further, the BCA is able to decrease accumulation of this toxin in P. expansum-infected apples. If confirmed, these results could pave the way for the development of new technologies for the prevention and/ or detoxification of patulin contamination in apple-based juices (Castoria et  al. 2005). The same effect on patulin accumulation was also recently observed in ‘Golden Delicious’ and ‘Granny Smith’ apples treated with natural biocides, quercitin and umbelliferone phenolic compounds (Sanzani et al. 2008).

9.5.2 Lenticel Rot Lenticel Rot [Neofabrea alba (EJ Gutrie) Verkley, anamorph P. vagabunda Desm., syn. Gloeosporium album Ostew] is one of the most frequent and damaging diseases occurring in stored apples (more rarely in pears) in Italy, France and other European apple growing countries (Pratella 2000; Amiri et al. 2008). Fruit infection occurs in the orchard, but disease symptoms appear only several months after harvest. ‘Pinova’, ‘Topaz’ and several late maturing cvs of apples such as ‘Gold Rush’ and ‘Pink Lady’ are particularly susceptible to the disease, with an incidence that can exceed 15–30% after 120 days of cold storage, particularly in organically grown fruit (Bompeix and Cholodowski-Faivre 1998; Mari et  al. 2002b; Maxin

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et  al. 2005; Weibel et  al. 2005). Current measures to control N. alba infection include pre- and postharvest treatments with synthetic fungicides. In Italy, the use of thiabendazole fungicide is allowed in postharvest treatments only for apples and pears stored longer than 31 December. Among non-chemical means to control lenticel rot in apples, hot water treatment has shown good efficacy, and seems particularly interesting for organic production (Maxin et al. 2005; Neri et al. 2008b). Dips in water at 45°C for 10 min led to a consistent reduction of infection both on artificially inoculated cv ‘Golden Delicious’ (80% of efficacy after 90 days of storage) and naturally infected cv ‘Pink Lady’ (90% of efficacy after 135 days of storage), without causing any damage to the fruit (Neri et al. 2008b). The efficacy of the treatment is likely due to effects on the pathogen; however, effects on fruit (induced resistance) could also be involved. Shorter treatments (10–30 s) would be optimal to accelerate fruit handling in packing houses and would be better for commercial application than 45°C for 10 min. However, a temperature of at least 50°C is needed to significantly reduce N. alba mycelial growth for short exposure treatment (1 min) (Neri et  al. 2008b), and apples dipped at 50–55°C for 30–180 s showed skin damage, with sensitivity varying among cultivars (Maxin et al. 2005; Burchill 1964; Spalding et al. 1969). Fumigation with plant volatile compounds (carvacrol, trans-2-hexenal, transcinnamaldehyde and citral) showed consistent efficacy only in  vitro (Neri et  al. 2008b). The failure of these volatiles in lenticel rot control on apples may be due to their insufficient vapour pressure, making the volatiles unable to penetrate through fruit lenticels and reach the pathogen site. Other studies on lenticel rot control reported the inefficacy of several natural compounds applied in dipping treatment at concentrations much higher than those used in this study and a significant disease control (64–84%) was only found with 2,000 ppm L-carvone or eugenol treatment in hot water (Bompeix and Cholodowski-Faivre 1998). However, similar or better control of the decay was found with a single hot water treatment (Bompeix and Coureau 2008).

9.5.3 Brown Rot Brown rot (Monilinia sp.) is one of the main diseases in stone fruit occurring in the field during both pre- and postharvest. In Europe there are substantially two causal agents of brown rot, Monilinia laxa (Aderhold and Ruhland) and M. fructigena (Aderhold and Ruhland); a third M. fructicola (G. Winter) has recently been detected in Spain and France, but it is not yet widespread in Europe. Losses depend on weather conditions and are especially severe if high humidity, warm temperatures and abundant rainfall prevail prior to harvest (Bonaterra et al. 2003). In some cases, infections occurring in the field can remain quiescent until fruit ripens, provoking important losses in the postharvest phase, estimated between 5% and 10% or more (Margosan et al. 1997). In European countries, this plant pathogen is controlled by fungicide spray programs only in the field, since postharvest treatment

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is not allowed. In the last two decades numerous studies have indicated the efficacy of BCAs against M. laxa on peach and nectarine; most of these studies refer to yeasts (Karabulut and Baykal 2003), bacteria (Bonaterra et  al. 2003) and fungi (Mari et  al. 2007b) alone or integrated with food additives (Qin et  al. 2006) or physical methods (Karabulut and Baykal 2002). Pre-harvest treatments can be fundamental as a consequence of disease epidemiology and the importance of controlling latent infections (Larena et  al. 2005). For this reason, a scheduled programme of brown rot management could consider a turnover of active ingredients, including BCA in an integrated control strategy. However, applied in the postharvest, BCA does not appear able to control previously established infections (latent or quiescent) that often develop within 24–48 h after harvest and their eradication could be possible with postharvest physico-chemical treatments such as hot water (Margosan et al. 1997) or potassium sorbate (Gregori et al. 2008). Fumigants are good candidates for postharvest brown rot control since their use entails minimal handling of the food product and absence of the fruit wetting involved in vapour treatments. Several studies on antifungal activity of plant aroma compounds against M. laxa and M. fructicola have been carried out in  vitro and in  vivo trials. Trans-2-hexenal (Tsao and Zhou 2000; Neri et  al. 2007), hexanal (Song et al. 2007), acetic acid (Liu et al. 2002), and isothiocyante (Mari et al. 2008) treatments obtained satisfactory control of the pathogen. However, to achieve optimal effects, it is important to establish an effective volatile concentration and exposure duration combination. Compared with previous studies, stone fruit were found to be more sensitive to trans-2-hexenal injury than were pome fruit; a differential sensitivity was also observed among stone fruit species, increasing from plum to nectarine and peach, to apricot (Neri et  al. 2007). Others bioactive molecules derived from the development of an endophytic fungus Muscodor albus applied on peaches in bugged cartons had an interesting effect; the biofumigation treatment reduced brown rot by 72% with respect to untreated fruits (Schnabel and Mercier 2006). The fungus, growing on previously colonized desiccated rye grain, produces at least 28 volatile compounds and this mixture can be more effective than other fumigant agents, which are used as single compounds.

9.5.4 Green and Blue Moulds Green and blue moulds (P. digitatum Pers.: Fr. Sacc. and P. italicum Wehmer respectively) are the most important disease of citrus fruit in growing areas characterised by low summer rains (Eckert and Eaks 1989). Both pathogens grow at the optimal temperature of 24°C and although green mould is predominant at room temperature, blue mould is more important on cold-stored citrus fruit, since P. italicum grows faster than P. digitatum below 10°C (Brown and Eckert 2000). The fungi are wound parasites and can infect fruit in the grove, the packinghouse and during distribution and marketing. The fungal inoculum is practically always present on the surface of fruit during the season and after harvest can build up high levels unless appropriate pack-

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inghouse sanitisation measures are adopted (Kanetis et  al. 2007). Application of synthetic fungicides is generally the main method to control green and blue moulds where the frequency of fungicide-resistant isolates is low; however, the intense use of fungicides such as sodium o-phenylphenate, thiabendazole, and imazalil (the only fungicides registered for use on citrus fruits in the European community) has led to the proliferation of fungicide-resistant isolates (D’Aquino et al. 2006). The alternatives to conventional fungicides for the control of green and blue moulds in citrus fruit have been widely reviewed in a recent article by Palou et al. (2008). According to their nature, these alternatives can be physical, chemical or biological. As far as physical control methods are concerned, heat (Plaza et al. 2003), ultraviolet (UV-C) (D’hallewin et  al. 1999) and ionising (Sommer et  al. 1964) radiation and ozoned atmosphere storage (Palou et al. 2001) controlled P. digitatum and italicum; nevertheless, each of them showed drawbacks and disadvantages. Typical heat treatment by curing considers the exposure of fruit for 2–3 days to an air atmosphere heated to temperatures higher than 30°C and high relative humidity. Although this procedure showed satisfactory control of green mould, blue mould was less controlled when fruit were cold-stored for long periods after treatment (Plaza et al. 2003). In addition, fruit weight loss and heat phytotoxicity can be potential risks for heat-treated fruit with respect to cultivars, season, growing location and initial conditions while fruit quality traits may not be affected by heat treatment (Porat et al. 2000). The exposure to low doses (0.5–8 kJ/m2) of UV-C or ionising radiation has significantly reduced the incidence of green and blue moulds (D’hallewin et al. 1999) and on-line UV-C equipment for fruit treatment was developed (Wilson et  al. 1997). Despite this, numerous issues have to be addressed before a practical use of this system: the entire area of the fruit has to be rapidly treated and the system should be sufficiently flexible in relation to fruit attributes and retail destination (Palou et al. 2008). Ionising radiation is associated with some beneficial effects on fruit, such as the stimulation of synthesis of antifungal compounds that extend shelf-life by delaying ripening and senescence, although the doses required exceeding 1,000 Gy (100 krad) induce apparent rind injury (Barkai-Golan 1992). Ozone (O3) is a potent biocide and has recently been approved for many food contact applications, but at non-phytotoxic concentrations (0.3–1 mL/L) it is not able to control P. digitatum and P. italicum, obtaining only a fungistatic effect; however, inhibiting aerial mycelium growth and sporulation can reduce the proliferation of fungicides-resistant strains of these pathogens (Ariza et al. 2002). Biological control using microbial antagonists is a promising alternative to fungicides to control postharvest disease of citrus fruit. During the last 20 years, strains of yeast, bacteria and filamentous fungi have been isolated, tested, selected, identified and characterized as BCA effective against citrus green and blue moulds (Palou et al. 2008). This considerable research work has led to the production and registration of three biofungicides for use against postharvest citrus pathogens. Aspire™ (C. oleophila, limited to the USA and Israel), BioSave™ (P. syringae, limited to the USA) and Shemer™ (Metschnicowia fructicola, limited to Israel) are now available. Other products, such as Biocure, Bio-Coat (based on a strain of C. saitoana), will soon be available on the marketplace. However, the commercial use of these

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products is and remains limited and accounts for only a very small fraction of the potential market. As discussed in several reviews (Droby and Wisniewski 2003; El-Ghaouth et al. 2004; Janisiewicz and Korsten 2002), the combination of BCA with other alternative control methods can be a promising approach to overcome some drawbacks in BCA activity, enhancing their efficacy. The combination of BCAs with heat (Porat et al. 2002), GRAS (Usall et al. 2001), and UV-C (Stevens et al. 1997) produced a synergic effect and was superior to all the treatments alone in controlling green and blue moulds. A sequence of treatments proposed by Usall et al. (2008) comprises initial treatment with a heated solution of sodium bicarbonate or sodium carbonate followed by the application of BCA. Such combined treatments can be easily implemented on a commercial scale in many citrus packinghouses because they are compatible with existing facilities and postharvest handling practices.

9.5.5 Grey Mould Grey mould (B. cinerea Pers. Fr.) is an important postharvest disease because environmental conditions prevailing during storage facilities are favourable to its development. B. cinerea is a widespread pathogen on many crops but is the main problem in cold storage and subsequent shipment for table grape, strawberry and kiwifruit. On table grape, B. cinerea is responsible for severe losses in the field and after harvest and is the main threat to its long-distance transport and storage (Romanazzi et al. 2007). Control of the disease is especially important in storage because it develops at low temperatures (−0.5°C) and spreads quickly among berries, forming ‘nests’ that can spread over the entire bunch. Canopy management, pre-harvest fungicide applications and postharvest sulphur dioxide fumigation are the current practices to control grey mould (Droby and Lichter 2004); however, alternatives to sulphur dioxide are urgently needed because sulfites can be harmful to allergic people and also because sulphur dioxide has been removed from the GRAS compound list by the FDA (Anonymous 1986) and is not allowed in European countries. In an integrated pest management strategy, some physicochemical methods have been tested with interesting results. Dipping bunches after harvest in ethanol has prevented B. cinerea infection partially due to the high sensitivity of conidia to ethanol (Lichter et al. 2003), although ethanol is unsuitable for latent infection. Others treatments, such as vapour heat (Lydakis and Aked 2003), hexanal fumigation (Archbold et al. 1997), UV-C (Nigro et al. 1998), resveratrol (Gonzalez Ureña et  al. 2003), and chitosan (Romanazzi et  al. 2006) alone or in combination with ethanol (Romanazzi et  al. 2007) were found to significantly inhibit the development of B. cinerea. Several reports demonstrated the efficacy or BCAs against prevention of grey mould after harvest (Lima et al. 1997; Karabulut and Baykal 2003), although no antagonist seems to be efficient enough against natural infections (Schena et al. 2003). Strawberry is a highly perishable non-climacteric fruit and has to be harvested at full maturity to achieve maximum quality (Hernández-Muñoz et  al. 2008).

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Botrytis rot is one of the most important diseases of strawberry in Europe; infections initiated in the field during flowering remain quiescent until fruit ripens, causing heavy economic losses during shipment or storage. The use of synthetic fungicides in the field is the main method for reducing postharvest diseases and involves two applications of botrycides during peak flowering periods; however these measures, when weather conditions are favourable to the pathogen, have limited effects on postharvest disease control. Biocontrol of grey mould has been attempted for 10 years now and preliminary findings have been obtained (Lima et al. 1997; Karabulut et al. 2004; Hernández-Muñoz et al. 2008). BCAs are more efficient against infections that are established during the postharvest stage; since Botrytis rot originates mainly from latent infections of floral parts, the application of the antagonists at the flower stage seems the best strategy for effective control of grey mould (Lima et al. 1997). Antagonist treatment under field conditions may reduce pathogen sporulation on crop debris and consequently flower and fruit infection. Stem-end rot is the main symptom produced by B. cinerea on kiwifruit; the fungus colonizes fruit sepals and receptacles (Michailides and Elmer 2000) and penetrates by picking wound at, or soon after, harvest. The disease appears after 3–4 months of storage at 0°C or sooner if the fruit is kept at higher temperatures. Preharvest fungicide treatments are not always effective and easy to apply; alternative control methods suggest good orchard hygiene to remove the inoculum of B. cinerea, summer pruning to increase sunlight and air exposure ( Brigati et al. 2003a) and careful postharvest handling to avoid fruit injury. A delay between harvest and cool storage results in a significant decrease of B. cinerea infections during storage. This method, also called ‘curing’, consists of keeping fruit at ambient temperature for at least 2 days before packing or cold storage and it has been implemented over the years. In Italy, ‘curing’ is now carried out directly in cold storage rooms, decreasing the temperature from 10 to 1°C in 8 days and delaying establishment of the controlled atmosphere regimes to 30–40 days after harvesting. This method reduced the incidence of stem end rot and at same time any negative effects were observed on the fruit quality (Brigati et al. 2003b). Few data are reported on the biocontrol of B. cinerea in kiwifruit; the attempts to use some antagonists such as Bacilus subtilis and P. syringae (Michailides and Elmer 2000), Aureobasidium pullulans and C. oleophila (Lima et al. 1997) revealed a variable activity. A study by Kulakiotu et al. (2004) showed the potential for successful biological control of grey mould by volatiles of ‘Isabella’ grape. However, the antibiotic action of the ’Isabella’ volatiles against B. cinerea was tested only at two temperatures (10°C and 21°C) and was stronger at 21°C than at 10°C.

9.6 Conclusions The increasing interest in alternative methods to fungicides to control postharvest diseases has produced numerous studies over the last few decades. The results obtained show some significant progress in the reduction of pesticide use; however,

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some critical points have still to be considered. It’s unrealistic to assume that biological control agents have the same fungicidal activity as pesticides; studies to improve the formulation, compatibility with current handling and storage practices and the integration with GRAS, hot or resistance elicitors treatments are in progress and new registrations of biofungicides are expected. In the future, all of these methods will be fundamental for organically grown crops that, untreated with chemical fungicides, suffer due to a relatively high rate of decay in the postharvest phase. Research should provide appropriate tools (BCAs, natural substances, GRAS, curing) to tailor a complete integrated disease management strategy specific for each situation (species, climatic and seasonal conditions, destination market, etc.).

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Weibel F, Hahn P, Lieber, S, Häseli A, Amsler T, Zingg D (2005) Lutte contre Gloeosporium (album et perennans) des pommes biologiques Post-Recolte avec des produits oxidants et des eaux électrolytiques,et Pre-Recolte avec differents produits biologiques entre 2001–2004. Journées Techniques Fruits & Légumes et Viticulture Biologiques 120 Wilson CL, Upchurch B, El-Ghaouth A, Stevens C, Khan V, Droby S, Chalutz E (1997) Using an on line UV-C apparatus to treated harvested fruit for controlling postharvest decay. HortTechnol 7:278–282 Yu T, Chen J, Huang B, Liu D, Zheng X (2007) Biocontrol of blue and grey mould diseases of pear fruit by integration of antagonistic yeast with salicylic acid. Int J Food Microbiol 116:339–345

Chapter 10

Quo Vadis of Biological Control of Postharvest Diseases Wojciech J. Janisiewicz

Abstract  Research on Biological Control of Postharvest Diseases (BCPD) has been conducted for over two decades and successes, present and future direction are being discussed. The BCPB has been accepted by the fruit and vegetable industry as the stand alone or in combination with other commercial treatments, depending on fruit and vegetable. BioSave has been on the market since 1996, and its use is expanding to control more postharvest diseases of fruits and vegetables. World-wide efforts in developing BCPD resulted in the registration of more products recently. The number of scientific publications is also increasing steadily. As postharvest biocontrol products are coming to the market, their anticipated limitations are become apparent, and much of the current research is focused on addressing these limitations. Combining antagonists with various substances Generally Regarded as Safe (GRASS), such as sodium bicarbonate, calcium chloride, diluted ethanol, or with physical treatments such as heat, or UV irradiation are typical examples of approaches being used. A mixture of two compatible biocontrol agents often showed an additive or synergistic effect in controlling fruit decays. Biocontrol agents developed for the control of fruit decays have also been shown to inhibit growth of foodborne human pathogens. This aspect gains in importance as new outbreaks are reported with increasing frequency, and fresh cut fruit and vegetables are particularly vulnerable to colonization by foodborne pathogens. Biocontrol agents can also control decays originating from wounds made during mechanical harvesting of fruits. Currently, available biocontrol products were developed for the control of decays originating from the infection of fruit wounds, but the next greatest challenge for BCPD research is the development of the next generation of biocontrol products that control latent infections. Many important diseases of temperate, subtropical and tropical fruit, including those caused by Monillinia spp. and Colletotrichum spp.., originate from these infections in the orchard and cause decay on fruits in storage. This research requires broadening the pool of microorganisms W.J. Janisiewicz Agricultural Research Service, U.S. Department of Agriculture, Appalachian Fruit Research Station, 2217, Wiltshire Road, Kearneysville, WV, 25430, USA e-mail: [email protected] D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_10, © Springer Science + Business Media B.V. 2010

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screened for biological control activity to include, in addition to those occurring naturally on fruit, microorganisms from different plants and plant parts, as well as microorganisms from different habitats. Some programs are already focusing in this direction, and there is great hope and optimism that at the next ISPP Congress in 2013 in Beijing, we will have reports on the significant progress in this area. Keywords  Biocontrol, Biocontrol products, Emerging biocontrol, Postharvest decay

10.1 Historical Perspective This is the fourth consecutive evening session on Biological Control of Postharvest Diseases (BCPD) at the ICPP and it is axiomatic that, after more than two decades of research on BCPD, we should reflect on the direction of the current research and what we can expect in the foreseeable future. A SCOPUS survey of the last 10 years of research on BCPD indicates an increasing number of peer-review publications from 19 in 1998 to 54 in 2008 (a projected number based on 38 publications by the end of August, 2008). Most of the research has been conducted in Europe (mainly Belgium, Italy and Spain, and Sweden), United States, Israel, South Africa, and more recently in China. Many smaller programs developing in other countries also contribute significantly to progress in this field, and further indicate the growing interest in BCPD. The expansion of this research began with the report by Pusey and Wilson (1984) on the successful control of brown rot of peach and other stone fruits caused by Monilinia fructicola after harvest using soil isolated Bacillus subtilis (strain B3). Postharvest application attempts were made after the field application of this bacterium to peach trees from bloom to harvest failed to control this disease. The authors concluded that controlled environmental conditions in the postharvest system provide a more stable environment for the antagonist resulting in a high level of disease control (Wilson and Pusey 1985). Results from pilot tests on the control of brown rot of peach, conducted in commercial packinghouses, were encouraging, but this antagonist was never commercialized (Pusey et  al. 1988). The discovery of bacterial and yeast antagonists effective against various postharvest diseases of pome fruits among resident microflora of apple and pear provided a new source of antagonists. It also made us realize that potential antagonists on the surfaces of these fruits have long been consumed by humans without any apparent adverse effects (Janisiewicz 1987). The next milestone was the registration by the United States Environmental Protection agency (EPA) and commercialization of the first two biocontrol agents: a yeast Candida oleophila, used in Aspire™ (Droby et  al. 1998), and a saprophytic strain of Pseudomonas syringae, used in BioSave™ (Janisiewicz and Jeffers 1997) in 1995. In this biotechnology era, each year seems to be a millennium, and the success of new commercial biocontrol products is often measured by their life span on the market. Three years of commercial use seems to be the breaking point for many biocontrol products. In

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this context, the last milestone was achieved in 2006, the 10-year anniversary of the large scale commercial use of BioSave. There have been many important advances in BCPD during the past decade, especially in the area of mechanisms of biocontrol, manipulation of antagonist physiology for the benefit of biocontrol, development of formulations, and integration with other control methods. Only time will indicate the full impact of these advances on the increased use of BCPB.

10.2 Biocontrol Products Aspire™ and BioSave™ lead the way in commercial application of biocontrol agents to fruit. Other products such as YieldPlus™ based on Cryptococcus albidus, Avogreen™ based on Bacillus subtilis, and Shemer™ based on Metschnikowia fructicola are also on the market in various countries. Aspire™ has been registered in the United States for postharvest application to citrus and pome fruits. This product was taken off the market 3 years after its large scale commercial introduction. BioSave™ (two strains of P. syringae) was originally registered for postharvest application to pome and citrus fruits, and this was later extended to cherries, potatoes, and more recently to sweet potatoes. YieldPlus™ was developed in South Africa for postharvest application to pome fruits but the success of this product is largely unknown and there is no published literature or information available to determine extent of its use. Avogreen™ has been used for control of postharvest disease of avocado. Its use has been limited (L. Korsten, personal communication), possibly due to inconsistent results. More recently, Shemer™ was registered in Israel for both pre- and postharvest application on various fruits and vegetables including apricot, citrus, grapes, peach, pepper, strawberry and sweet potato. There are three more products coming to the market: Candifruit™ based on Candida sake, developed in Spain; Boni-Protect®, based on Aureobasidium pullulans, developed in Germany and NEXY, based on Candida oleophila, developed in Belgium. All of these products have been registered for control of postharvest diseases of pome fruits. These new products are further testimony that increasing interest in BCPD is not just a matter of scientific curiosity but have resulted in diligent efforts to implement this approach. Gradual removal of the major regulatory barriers to registration of antagonists for BCPD in different countries is also very encouraging.

10.3 Postharvest System BCPD now encompasses the use of antagonists to control postharvest diseases of fruits, vegetables and grains. Postharvest biocontrol of flower diseases is another area awaiting exploration. Early attempts to use a metabolite (pyrrolnitrin) of a bacterial antagonist to control cut rose flower infections were very encouraging (Hammer et al. 1993) but little else has been done in this field. Biocontrol of grain

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spoilage during storage in silos has made significant advances and appears to be on the way to commercialization (Peterson and Schnurer 1995; Peterson et al. 1999; Druvefors 2004). Examples from commercially used biocontrol products indicate that biocontrol agents developed for BCPD on one commodity could also be effective against the same or different pathogens on other commodities. The activity of biocontrol agents is not as universal as fungicides but is less specialized than originally anticipated. Broadening the application of biocontrol agents is a good strategy for commercial success. It not only makes the product more profitable and allows for a quicker return on the investment made in the commercialization of the product, but it also allows a buffer in the fluctuation in the market due to registration of new fungicides or other alternative products. As postharvest biocontrol products are coming to the market, their anticipated limitations are elucidated and much of the current research is focused on addressing these limitations. The most commonly used approach is combining antagonists with various substances Generally Regarded as Safe (GRASS), sodium bicarbonate (SBC), calcium chloride, or ethanol (Bertolini 2008; Karabulut et al. 2003). These combinations both reduced the fluctuation and increased the level of decay control. Combining antagonists with other alternatives to fungicide treatments also showed good results, but its implementation often requires modifying currently used postharvest practices or adds substantially to the cost of the treatment. For example, a heat treatment of apples with hot air at 38°C for 4 days or oranges at 30°C for 1 day after harvest in combination with antagonists gave superior control (including eradicative activity) of blue mold and gray mold, respectively, compared to the individual treatments (Huang et al. 1995; Leverentz et al. 2000, Leverentz et al. 2003c). However, this approach will require adding heating equipment and changing the temperature in of storage rooms. Some packinghouses use high temperatures to sanitize empty bins and storage rooms, but even this practice is still very limited. It is also well established that the proper selection of the combination of two antagonists can provide superior decay control to either antagonists applied individually (Calvo et al. 2003; Janisiewicz and Bors 1995). Although using this approach is very attractive, it doubles the cost of registration because each antagonist must be registered separately. This problem could be eliminated if biocontrol products currently on the market could be combined resulting in additive or synergistic effects. There is increasing consensus that, in the future, non-fungicidal control will rely on the combination of various treatments with biocontrol. This combination of treatments is similarly to the hurdle concept developed by Leistner in 1978 for the reduction of food contamination with foodborne pathogens, where each additional treatment further reduces the possibility of food contamination (Leistner 1978, 2000).

10.4 Challenges of Latent Infection All biocontrol agents currently registered for postharvest application control fruit decays originating from wound infections made during or after harvest (see review by Janisiewicz and Korsten 2002). Although for some fruits, such as pome or citrus

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(depending on the region) fruits, wounds are the main court of entry for postharvest decay causing fungi, many postharvest decays of stone fruits and subtropical fruits develop in storage from latent infections occurring in the orchard. These infections are difficult to control because the intimate relationship of the pathogen with the host has been already established, and melanized appressoria often formed by these fungi on fruit surface are very resistant to environmental factors and penetration by fungicides. Control of latent infection with microbial antagonists is the next big challenge to BCPD. Earlier attempts to reduce decay originating from latent infections on mango fruit indicate that microbial antagonists could be effective against this type of infection (Koomen and Jeffries 1993), however this work did not continue beyond initial reports, suggesting difficulties in making further progress. New screening and fruit testing approaches must be developed to address latent infection. One of the approaches used in our laboratory employs in vitro screening for biocontrol activity against M. fructicola and Colletotrichum accutatum on crystalline cellulose membranes impregnated with waxes isolated from apple, plum or nectarine fruit. Both fungi produce melanized appressoria on these membranes when inoculated as an aqueous suspension of the conidia, and can infect fruit from these appressoria if the membrane is placed on the fruit. Bacteria and yeast can be applied to the membrane surface with the appressoria, and those showing growth around appressoria and/or reducing fruit infection from these membranes are selected for further testing directly on fruit. Our current challenge is to make this procedure more quantitative. Unlike with wound infecting fungi, where screening for antagonists was independent of the mechanism of biocontrol, screening against latent infections could benefit from focusing on organisms exhibiting mechanisms of biocontrol which could most likely succeed against the melanized structures of the pathogen. The primary candidates are microorganisms producing volatile substances and enzymes capable of degrading the melanized structure of the fungi. Predacious yeasts may also be useful. Antifungal volatiles produced by the antagonist Muscodor albus, discovered as an endophyte of Cinnamomun zeylanicium in Honduras, have been shown recently to control major postharvest pathogens on a variety of fruits (Strobel 2006; Strobel et  al. 2001; Mercier and Jiménez 2004; Mercier and Smilanick 2005). Focusing the search on this mechanism could greatly enhance opportunities of finding antagonists with volatiles capable of penetrating melanized structures of the fungi. This example also indicates that microflora of exotic plants, especially endophytes, is an untappped resource and should be explored for their biocontrol potential. With the exception of grapes, pome, citrus and several other fruits, information about resident microflora of fruit and fruit tree plants is very limited and their potential for BCPD is largely unexplored.

10.5 Biological Control Mechanism Studies of the ecology and physiology of antagonists were the main driving forces during initial development of BCPD that lead to commercial products, but research on the mechanisms of biological control (MBC), that have been lagging behind, has

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made significant progress especially during the past decade. This subject is covered in Chapter 12 by R. Castoria and Sandra A.I. Wright. Here, I would like to point out a few factors that have contributed to this progress. More focus and resources have been put into studying mechanisms of BCPD after the first commercial products began to be marketed on a large scale in late 1990s. The limitations to BCPD have become a reality in commercial settings and the knowledge of the MBC has been look upon as one of the ways that may address some of these limitations. Advances in molecular biology have allowed us to address, in a more direct manner, questions about the role of some putative MBC e.g. lytic enzymes or reactive oxygen species (ROS) (Castoria et  al. 2005; Chan et  al. 2007; Friel et  al. 2007; Macarisin et al. 2007; Massart and Jijakli 2006; Xu and Tian 2008). In most cases more than one mechanism of biocontrol has been implicated and the significance of different mechanisms of biocontrol still needs to be established. Development of a bacterial and yeast model systems, where genes for different MBC traits can be expressed, and where the transformants can be tested on fruit for biocontrol potential, could be very helpful in many cases. A good examples of what could be accomplished in the fruit system is work with, Saccharomyces cerevisiae and Picha pastoris, non-antagonistic yeasts which were transformed with genes coding for antifungal peptides cercopin (defensin from insects) and Psd1 (defensin from pea seeds), respectively. These transformants greatly reduced postharvest decays on tomato and pome fruits (Jones and Prusky 2002; Janisiewicz et al. 2008). There are genes currently available that could be tested in such a system, e.g. genes coding for antifungal pyrolnitrin, which were isolated from Pseudomonas fluorescent, and also produced by Pseudomonas cepacia (now Burkholderia cepacia), an excellent biocontrol agent against various postharvest decays on pome, stone and citrus fruits (Janisiewicz and Roitman 1988; Hammer et al. 1997). Other good candidates are genes responsible for the production of various lytic enzymes, ROS or siderophores. Eventually, this approach may lead to improvement of existing antagonists or the development of new ones (as with cercopin and Psd1 defensins), however for this to have a practical outcome, the hurdles of safety and public acceptance will have to be overcome (Janisiewicz 1998). The discovery of a very effective antagonist producing antifungal volatiles indicates that a single MBC can be sufficient to provide adequate control of a fruit decay. This also justifies screening for a single MBC, if an effective mechanism can be identified, and makes in vitro screening more meaningful and effective because it allows the screening of vast numbers of organisms in a short period of time before resorting to more expensive and time consuming tests on fruit.

10.6 Emerging New Areas in Postharvest Biocontrol During the past decade, more emphasis has been put on the safety of fruits and vegetables as an increasing number of outbreaks with foodborne human diseases are reported each year following consumption of various fruit and vegetables. The situation is worsened by growing consumption of fresh-cut fruits and vegetables,

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which provide a conducive environment for the growth of various bacterial human pathogens. This also creates an opportunity for the use of microbial antagonists to combat these foodborne pathogens. For example, Pseudomonas syringae used in Bio-Save can prevent the growth of E. coli in apple wounds (Janisiewicz et  al. 1999), and several other biocontrol agents, e.g. Gluconobacter asaii, Discofaerina fagi, or Metschnikowia pulcherrima, developed for control of postharvest decays, not only prevented growth but even reduced populations of the foodborne pathogens Listeria monocytogenes and Salmonella enterica sv. Poona on fresh cut apples (Leverentz et al. 2006). The concept and the potential for using various microorganisms against human foodborne pathogens on minimally processed fruits and vegetables has been discussed (Leverentz et al. 2003b). Lactic acid bacteria (LAB) such as Lactobacillus plantarum, Lactococcus lactis, Leuconostoc sp., or Weissella sp., occur frequently on fruits and vegetables and many strains of these LAB prevented growth or even reduced populations of the major foodborne pathogens on apples and lettuce (Trias et al. 2008). Lytic bacteriophages can be very effective in reducing populations of foodborne pathogens (Leverentz et al. 2001). They can be combined with other bacterial or yeast antagonist or the bacteriocin, niacin, to achieve the targeted level of a 6 log unit reduction in populations of the foodborne pathogens (Leverentz et  al. 2003a, Fig.  10.1). In 2005, the United States Environmental Protection Agency approved, for the first time, the use of bacteriophages as a food additive to reduce contamination of foods with foodborne pathogens. It is not incidental that the control of foodborne pathogens has recently been the topic of various sessions, including the Plenary Session, at the APS Annual Meetings, as the growth of these pathogens can be intertwined with the growth of plant pathogens. Recent studies indicate that a diseased tissue can be more prone to colonization by foodborne human pathogens than the healthy tissue. For example, apple tissue infected with Glomerella cingulata, causing bitter rot, becomes less acidic allowing colonization by L. monocytogenes (Conway et al. 2000). Labor shortages, especially in the United States and other developed countries, have increased the demand for mechanical harvesting of fruits and vegetables. Significant progress has been made during the past two decades and some fruits are currently harvested mechanically, not only for processing, but also for the fresh market. Despite these major advances, fruit wounding is still a problem and is the major limitation for developing this harvesting technology. Since the majority of postharvest pathogens of temperate fruit crops infect fruits through wounds which results in decay in storage, this presents an ideal opportunity for the use of biological control. Considering that the most successful BCPD to date is against wound invading pathogens, this area is worth of exploring (Janisiewicz and Korsten 2002). The first attempt to biologically control decay originating from wounds made during mechanical harvesting suggests that this approach is feasible (Janisiewicz and Peterson 2004). Stem loss (stempulls) on mechanically harvested apples range from 20–57%, depending on cultivar. If a portion of apple skin is removed together with the stem, flesh tissue is exposed, creating a potential entry site for the pathogen. The P. syringae (ESC-11) strain that is used in BioSave reduced blue mold decay on mechanically harvested ‘Empire’ apples with stempulls from 41% to 3.3% and this antagonist completely eliminated decay on other, less susceptible, cultivars

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logCFU/plug

Lm Lm+Phage Lm+G.asaii Lm+Phage+G.asaii

8 7 6 5 4 3 2 1 0

2

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Treatment L LP LG LP G

0

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2.64a 1z2

4.45ay 3.01bz 4.01ax 3.07bx

2.74az 2.49ay 2.79ax

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Treatment means within Da significance level.

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5 7.06ax 5.54y 3.95cx 2.50dxy

7 7.474ax 6.38bx 3.65cx 1.61dy

y with different a, b, c, d letters are different at the 0.05 y, z letters are different at

th e 0.05

Fig. 10.1  Recovery of Listeria monocytogens from honeydew wedges treated with the pathogen (104 CFU/mL) alone (L) or in combination with phage (LP), Gluconobacter asaii (LG). or a combination of the two (LPG) and stored at 10°C over 7 days (Hong Y, Leverentz B, Coway WS, Janisiewicz WJ, Abadias M, Camp MJ, unpublished)

(Table. 10.1). A similar situation may occur with harvested sweet cherries where the first mechanical prototypes are already being used commercially and all of the fruit is harvested without stems (Peterson and Wolford 2001). Commercially mechanically harvested blueberry may also provide an opportunity for biocontrol, and the development of blackberry mechanical harvesters has made significant progress recently (Peterson et al. 1997; Peterson and Takeda 2003; Takeda et al. 2008).

10.7 Conclusions We are entering into a new era in BCPD with expanding scientific interest and new products coming to the market. Commercial use of the pioneering products indicates that the fruit and vegetable industry will accept biological control, as long as

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Table 10.1  Development of blue mold in the stem cavity area of mechanically harvested apples with and without stems (stempulls) after inoculation with 25 µl of Penicillium expansum suspension (5 × 105 conidia/mL) alone or in combination with the antagonist Pseudomonas syringae (ESC11). Fruit were stored at 1°C for 2 months (Janisiewicz WJ, Peterson DL, unpublished)

it provides adequate control. The persistent efforts in many programs focusing on development of new biocontrol agents and/or studying mechanisms of biocontrol are beginning to pay off. Regulatory agencies in an increasing number of countries are accepting biocontrol products as safe alternative to fungicide treatments. Currently, most of the biocontrol products are registered in individual countries, but expanding registration to other countries would facilitate greater use of the products and possibly promote the use of mixtures of some products if more than one product is registered in a country, as combinations of compatible antagonists often provided improved decay control compared to individual antagonists used alone. Examples presented earlier indicates that greater use of BCPD can be achieved by expanding its use to new commodities, new applications, and by integrating biocontrol with other treatments. Current successes with biocontrol of decays originating from fruit wounds gives us optimism that the next great challenge, the control of latent infections, also can be achieved. This, most likely, will require broadening the pool of microorganisms screened for biocontrol activity to include epiphytes and endophytes from different plants and plant parts, as well as microorganisms from different habitats. With research in this direction already under way, there is great hope and optimism that at the next ISPP Congress in 2013 in Beijing we will have reports that the next generation of biocontrol agents are capable of controlling latent infections of fruit.

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Pusey PL, Hotchkiss MW, Dulmage HT, Baumgardner RA, Zher EI, Reilly CC, Wilson CL (1988) Pilot test for commercial production and application of Bacillus subtilis (B3) for postharvest control of peach brown rot. Plant Dis 72:622–626 Strobel G (2006) Muscodor albus and its biological promise. J Ind Microbiol Biotechnol 33:514–522 Strobel GA, Dirkse E, Sears J, Markworth C (2001) Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiology 147:2943–2950 Takeda F, Krewer G, Andrews EL, Mullinix B, Peterson DL (2008) Assessment of the V45 blueberry harvester on rabbiteye blueberry and southern highbush blueberry pruned to v-shaped canopy. HortTechnol 18:4–19 Trias R, Baneras L, Bados E, Montesinos E (2008) Bioprotection of ‘Golden Delicious’ apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria. Inter J Food Microbiol 123:50–60 Wilson CL, Pusey PL (1985) Potential for biological control of postharvest plant diseases. Plant Dis. 69:375–378 Xu XB, Tian SP (2008) Reducing oxidative stress in sweet cherry fruit by Pichia membranefaciens: a possible mode of action against Penicillium expansum. J Appl Microbiol 105:1170–1177

Chapter 11

Improving Formulation of Biocontrol Agents Manipulating Production Process J. Usall, N. Teixidó, M. Abadias, R. Torres, T. Cañamas, and I. Viñas

Abstract  There are several reasons of the limited number of commercial available biocontrol agents, such as the difficulties in developing a shelf-stable formulated product that retains biocontrol activity. This chapter shows that it is possible to improve the formulations during the production process and describes several examples of improving liquid and dry formulations using different strategies such as grow microorganisms in aw modified media, under sublethal thermal stress conditions or preservation in isotonic solutions. Liquid formulation of C. sake was improved growing the cells in molasses medium with aw modified to 0.98 with the addition of sorbitol and preserved with an isotonic trehalose solution. After 180 days of storage at 4°C, the viability of this formulate was 100% and the efficacy against Penicillium expansum on apples was more than 95% rot reduction. Spray drying formulations were improved by modifying growth media or temperatures during growing period. The biocontrol agent Pantoea agglomerans grown during 48 h in NaCl 0.97 aw modified medium could increase their viability after spray drying formulation from 6% in unmodified medium to near 30% without affecting their biocontrol potential. In contrast Candida sake cells grown in unmodified molasses medium exposed to mild heat treatments at 30°C or 33°C during mid or late-exponential or early or midstationary growth phases showed an increase of survival when are exposed to lethal shock at 40°C, but only a very reduced improvement after spray drying formulation. Finally the combination of thermal and osmotic stress was studied in order to improve fluidized bed drying formulations of P.agglomerans. The results showed than using NaCl to adjust aw to 0.988 in the growth medium and increasing the temperature to 35°C during 1 h in the early stationary phase could get a good formulate with only 0.5 log reductions during fluidized bed drying process. J. Usall (*), N. Teixidó, M. Abadias, R. Torres, and T. Cañamas IRTA, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198, Lleida, Catalonia e-mail: [email protected] I. Viñas University of Lleida, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198, Lleida, Catalonia D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_11, © Springer Science + Business Media B.V. 2010

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Keywords  Liquid formulation • spray drying • thermal stress • fluidized bed drying • osmotic stress • isotonic solutions • candida sake • pantoea agglomerans

11.1 Introduction Biological control of postharvest diseases of fruits has advanced greatly during the past decade. Concerns regarding human health and environmental risks associated with chemical residues in foods have been the main driving force of the search for new and safer control methods (Droby et al. 1998). Among the proposed alternatives, the use of naturally occurring antagonistic microorganisms has been the most extensively studied. Several microbial antagonists, based on either yeast or bacteria were developed and commercially tested. However the success of these products remains limited and just a few microorganisms are commercial available to control postharvest decay of citrus and pome fruits such as Bio-Save 10 (Pseudomonas syringae ESC-10, Jet harvest solutions, USA), Shemer (Metschnikowia fructicola, Agrogreen, Israel), Boniprotect (Aureobasidium pullulans, Bio-protect, Germany) and Candifruit (Candida sake CPA-1, Sipcam Inagra, Spain). There are several reasons of this limited success, such as the inconsistency, variability of the efficacy under commercial conditions, the limited tolerance of fluctuating environmental conditions and the difficulties in developing a shelfstable formulated product that retains biocontrol activity similar to that of the fresh cells (Janisiewicz and Jeffers 1997). Dehydration of the product and maintenance in a dry environment is one of the best ways to formulate microbial agents so that they can be handled using the normal distribution and storage channels (Rhodes 1993). Unfortunately, not all microorganisms are amenable to drying and many tend to lose viability during both the drying process and storage. The classical dehydration processes are: freeze-drying, spray-drying and fluidised bed drying. These processes change the physical state of water by varying the temperature or pressure or by the combined action of both parameters. Cells dehydrated in this way are therefore subjected to simultaneous temperature and water activity stress. Survival of foodborne pathogenic microorganisms under different stress treatments (e.g. heat, osmotic or water activity, low pH, etc.) have been extensively studied and reported. Initial investigations carried out on bacteria such as Escherichia coli (Arnold and Kaspar 1995; Poirier et al. 1998), Salmonella spp. (O’Driscoll et  al. 1996; Mattick et  al. 2000) and Listeria spp. (Jorgensen et  al. 1995; Greenacre et  al. 2003) have demonstrated that they possess an inherent ability to adapt to unfavourable environments by the induction of various general and specific stress responses. These stress responses are characterised by the transient induction of general and specific proteins and by physiological changes that generally enhance the particular organism’s ability to withstand more adverse

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environmental conditions (Ang et  al. 1991). In the case of osmotic stress, the significant physiological changes reported in bacteria include the induction of stress proteins as well as the accumulation of compatible solutes such as K+ ions, the amino acids glutamine, proline, glycine betaine, carnitine and sugars such as sucrose and trehalose (Csonka 1989; Kets et al. 1994; Ko et al. 1994). In yeasts and filamentous fungi the accumulated compatible solutes are mainly low(glycerol and erythritol) and high- (arabitol and mannitol) molecular weight sugar alcohols (Beever and Laracy 1986; Ellis et al. 1991; Van Eck et al. 1993; Hallsworth and Magan 1994, 1996). These compatible solutes allow equilibration of the cytoplasmic water activity (aw) with the surrounding environment, thereby retaining water in the cell and thus, maintaining turgor pressure, and helping to preserve protein function within cells (Yancey et  al. 1982; Csonka 1989; Van Eck et al. 1993). Subjection to a mild stress makes cells resistant to a lethal challenge with the same stress condition. Preadaptation to one particular stress condition can also render cells resistant to other stress imposing conditions: this phenomenon is known as cross protection (Sanders et al. 1999). There is significant interest in the mechanisms used by such microorganisms to tolerate some stress. It has been shown that exposure of cells to a low level of environmental stress may induce endogenous adaptation strategies, which can facilitate resistance to exposure to elevated levels of the same stress, e.g. temperature. As a result, it has been demonstrated that thermotolerance in yeasts to extreme heat treatment can be achieved following a brief shift in the incubation temperature (Tiligada et al. 1999). Control of the resistance of microorganisms to temperature stress may have potential practical benefits in the process of formulation where enhanced thermotolerance is required (Ross et al. 2005). The heat stress response is also usually characterized by the transient induction of general and specific proteins and by the physiological changes that generally enhance an organism’s ability to withstand more adverse environmental conditions (Ang et al. 1991). Some of these stresses could be improved during production process. There is little published research concerning the impact of growth culture on the viability of a formulated biocontrol agent. Some studies have demonstrated that growth of C. sake in a commercial molasses-based medium or in the same medium with lowered aw (0.98) showed high viable counts, and increased the intracellular concentration of arabitol and glycerol and the water stress resistance of the cells (Abadias et al. 2001). Moreover, the intracellular water potential of cells decreased with lowered aw of the culture medium (Abadias et al. 2000). Candida sake CPA-1 has been demonstrated to be an effective biocontrol agent (BCA) against major postharvest diseases on pome fruits (Viñas et al. 1998; Usall et al. 2000; Usall et al. 2001). Detailed studies have shown that the strain CPA-2 of Pantoea agglomerans – previously classified as Erwinia herbicola – is an effective antagonist to the major postharvest fungal pathogens of pome and citrus fruits (Teixidó et al. 2001; Nunes et al. 2001, 2002) and it is in commercialization process in Spain as a solid formulation named Pantovital by BIODURCAL S.L. Commercial

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and technical formulations of these two biocontrol agents are been developed in the Postharvest Unit of UdL-IRTA Center, Lleida, Catalonia. This chapter describe several examples of improving liquid and dry formulations of these two biocontrol agents using several strategies during the production process such as grow microorganisms in aw modified media, in sublethal thermal stress or preservation in isotonic solutions.

11.2 Liquid Formulation In this work we will describe how to improve a liquid formulation of C. sake by growing the cells in several media and preserved in a liquid with the same water potential of the cells (isotonic solutions) using different substances to adjust aw and the efficacy of these liquid formulations against P. expansum in Golden Delicious apples (Abadias et al. 2003).

11.2.1 C. sake CPA-1 Formulation Using Isotonic Solutions Viability of the postharvest biocontrol agent Candida sake CPA-1 stored as liquid formulation was evaluated by studying the effect of growth, preservation medium, and temperature. C. sake was grown in molasses medium with unmodified water activity (aw) and in the same medium with aw modified to 0.98 with the addition of several solutes (glycerol, glucose, NaCl, proline or sorbitol). Low water potential solutions used as preservation media were prepared with arabitol, erythritol, glycerol, glycine, mannitol, proline, sorbitol and trehalose. These solutes were added to de-ionized water at the concentration to obtain the same (isotonic) water potential as the cells. The best growth media were the unmodified one and those modified to 0.98 aw with the addition of glycerol or sorbitol. For all growth media, the best preservation medium was the isotonic solution prepared with trehalose. Generally, for each growth medium, viability of C. sake cells was significantly higher when cells were preserved in the trehalose isotonic solution than when cells were maintained in the other trehalose solutions (Table 11.1). Viability of cells preserved in water decreased greatly (<3%) after 30 days of storage at 4°C. Generally, at concentrations below the isotonic one, increased concentration of trehalose in the preservation medium implied an increase of the viability of C. sake cells. The best viability result was obtained when cells were grown in the sorbitol modified medium and preserved with the isotonic trehalose solution, with 110% and 77% of cells remaining viable after 180 and 210 days of storage at 4°C, respectively.

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Table  11.1  Percentage of viable cells preserved at 4°C in relation to growth medium and trehalose concentration in the preservation solution Storage time at 4°C (days) Trehalose concentration, M (mol L−1) 30 60 90 120 150 180 210 Growth medium % Viability Unmodified

Glycerol 0.98 aw

Sorbitol 0.98 aw

0 0.03 0.14y 0.81 0.96 0 0.03 0.14 0.81y 0.96 0 0.03 0.14 0.81 0.96y

51a 55a 54a 32b 38b 42c 68a 79a 65ab 51bc 23b 90a 76a 75a 96a

17c 39a 41a 40a 33b 5b 26a 36a 37a 36a 1e 45d 65c 109b 125a

7d 32c 54a 38bc 43b 1d 35c 48b 55ab 58a 2e 36d 60c 105b 121a

3d 24c 43a 39ab 36b 1b 29a 35a 33a 31a 2d 16d 37c 92b 118a

NDx 18c 42a 37b 34b ND 22b 38a 47a 40a ND 10c 38b 118a 108a

ND 18b 32a 35a 34a ND 14c 32b 45a 32b ND 27b 40b 111a 110a

ND 13c 28a 29a 21b ND 6b 20a 20a 20a ND 2d 9c 63b 77a

Viability was not determined due to its low value in bold represent the trehalose solution that is isotonic with C. sake cells. For each growth medium and storage time, LSD test for separation of means has been made (P < 0.05). Different letters (a, b, c, d, e) indicate statistical differences among means. ND-Not determinated

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11.2.2 Viability of C. sake CPA-1 After Storage at 25°C and 4°C The viability of cells preserved in Phosphate Buffer or in the isotonic solutions of glycerol or proline was similar to that obtained with water. Therefore, only results of water and trehalose are shown (Fig. 11.1). More cells remained viable at 4°C than at 25°C. The number of viable cells decreased rapidly during the first 10 days of storage at 25°C, and cells grown in sorbitol-modified media and preserved with the isotonic trehalose solution obtained again the highest viability after 10 days of storage (25%). The viability of all treatments after 30 days of storage was <7%. After 4 months of storage at 4°C and regardless of the growth media used, viability of cells preserved with the isotonic trehalose solution was >21% while cells preserved with water shown viabilities <2%. Consequently, only viability of cells preserved with isotonic trehalose solution was evaluated until 7 months of storage. Viability of C. sake cells grown in the unmodified molasses medium (Fig. 11.1a) and preserved with the isotonic trehalose solution at 4°C decreased progressively and was 72%, 59%, 30%, 21% and 9% after 30, 60, 90, 120 and

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a

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210 days of storage, respectively. Similarly, the number of viable cells grown in the glycerol modified molasses medium and preserved with the isotonic trehalose solution was 70%, 70%, 56%, 46% and 10% after 30, 60, 90, 120 and 210 days of storage at 4°C (Fig. 11.1b). In contrast, the number of C. sake cells grown in the sorbitol modified medium and stored at 4°C with the isotonic trehalose solution (Fig. 11.1c) remained stable at about 100% during the 210 days of storage period. Generally, cells grown in the sorbitol modified medium showed higher viability than cells grown in the unmodified

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medium or in that modified with glycerol. The growth medium can influence the pattern of intracellular solutes accumulated in yeast (Teixidó et al. 1998a,b) or conidia (Hallsworth and Magan 1994). In some cases, such modifications resulted in improved tolerance to water stress with retained biocontrol activity (Teixidó et al. 1998a,b). However, no significant changes in the pattern of some polyols were obtained when C. sake grew in the sorbitol-modified medium (Abadias et al. 2000). Generally for each storage time and growth medium, the increase of trehalose concentration in the preservation medium involved an increase of C. sake viability at trehalose concentrations below the isotonic ones. This could suggest that trehalose concentration is not the only factor involved in maintaining C. sake viability, but probably it interacts with other factors in the growth medium. It is known that intracellular trehalose exerts a protective effect on yeasts under extreme environmental conditions such as dessication, freezing, osmotic stress and heat shock (Rapoport et al. 1998) and it also provides thermal stability to the cells (Attfield et  al. 1992). In recent years evidence has been accumulated that trehalose may protect cell viability during drying by replacing the water around polar residues in enzymes and other essential proteins, thus maintaining their integrity in the absence of water (Leslie et al. 1994). However, there are no studies on the role played by the external addition of this sugar in protecting yeast cells in liquid formulations. Teixidó et  al. 1998b found that when C. sake was grown in a aw modified medium with the addition of trehalose, the intracellular level of trehalose increased. It is possible that C. sake could have a trehalose-specific carrier in the cells.

11.2.3 Efficacy of Trehalose Isotonic Formulations Stored at 4°C for 7 months Efficacy of the best liquid formulations stored for different periods was tested against infection by Penicillium expansum on apples. Regardless of the growth medium, isotonic trehalose formulations of C. sake stored at 4°C for 7 months showed good biocontrol effect when they were tested against P. expansum infection of apples with incidence of decay lower than 18%. In contrast, decay incidence for untreated fruits was 98%. Isotonic trehalose formulation of cells that grew in the sorbitol-modified medium performed as well as fresh cells in controlling blue mold and only 2% of inoculated apples rotted. In this study (Abadias et al. 2003) an improved liquid formulation of the biocontrol agent C. sake has been found (grown in sorbitol-modified media and preserved with the isotonic trehalose solution) with retained viability and efficacy for 7 months, which will cover all the pome fruit season, and it is composed of safe substances. However, it should be stored and distributed under refrigerated conditions. The storage and transport of cells at 4ºC appears not to be an obstacle as other similar products are handled in this way.

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11.3 Spray Drying In this work we will describe how to improve spray drying formulations of P. agglomerans or C. sake modifying growth media or the temperatures during growth period.

11.3.1 Improving Spray Drying Formulation of Pantoea agglomerans CPA-2 by Osmotic Treatments This research demonstrated that cells of P. aggolerans grown in media at low aw using NaCl exhibited osmotic adaptation in solid media at low aw obtaining high production level and maintaining biocontrol efficacy (Teixidó et al. 2006). Osmoticadapted cells also demonstrated thermotolerance (Teixidó et al. 2005) and desiccation tolerance after spray drying (Teixidó et al. 2006). The microorganism was cultured in an unmodified liquid (control) or in awmodified media with the non-ionic solutes glycerol, glucose and polyethylene glycol (PEG 600) to 0.98, 0.97, 0.96 and 0.95 aw, and viability of these cells was evaluated on unstressed (0.995) and aw-stressed (NaCl 0 96 aw) solid media, in order to check total viability and aw-stress tolerance, respectively. Significant improvements in viability on unmodified medium were observed with cells grown for 24 h in NaCl 0.98 aw, glycerol 0.98 aw and 0.97 aw and for 48 h in NaCl 0.98 aw and 0.97 aw modified media. Both yield improvements and water stress tolerance were achieved with low aw-media. Cells grown for 24 h in NaCl 0.98 aw or for 48 h in NaCl 0.98 aw, 0.97 aw and 0.96 aw, glucose 0.97 aw and glycerol 0.97 aw showed improved aw-stress tolerance in comparison with control cells (data not shown). The best results were obtained with NaCl treatments (0.98 aw and 0.97 aw), for that reason a detailed time course study was conducted from 16 to 32 h and the viability of these cells was evaluated on unstressed (0.995) and awstressed (NaCl 0 96 aw) solid media, in order to check total viability and aw-stress tolerance, respectively. Control cells (grown in unmodified medium) achieved maximum viable counts after 18 h incubation with about 4 × 109 CFU mL−1 in unstressed solid media and 9x108 CFU mL−1 in aw-stressed solid media. Their population then progressively decreased under 5 × 108 CFU mL−1 after 32 h (Fig.  11.2). However, the highest viability from the experiment was achieved with cells grown in NaCl 0.98 medium, with 7 × 109 CFU mL−1 after 22 h incubation and 3 × 109 CFU mL−1 after 26 h in unstressed and aw-stressed solid media respectively. In this case, the stationary phase was more stable than in the control cells and – throughout the process – viabilities remained greater than 1 × 109 CFU mL−1. The maximum viability of NaCl 0.97 cells under aw-stress conditions was obtained after 32 h rising more than 1 × 109 CFU mL−1.

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Sodium chloride treatments appeared to play a significant role in improving low aw tolerance and obtaining high production levels. This result contrasted with those obtained by Abadias et al. (2000) and Teixidó et al. (1998b), who reported that C. sake suffered greater inhibition by NaCl than by glycerol, glucose or prolineamended media. Cells grown in NaCl modified media which showed the best low aw adaptation were tested in spray-drying trials to check desiccation tolerance. P. agglomerans was grown on unmodified, and NaCl 0.98, 0.97 and 0 96 aw modified basic media for 24 and 48 h at 30°C and 150 rpm agitation on a rotary shaker. Harvested cells were resuspended in MgSO4 10% suspension to obtain an initial concentration of 1  × 1010 CFU mL−1. These suspensions were then incubated for 30 min at room temperature, constantly shaken to allow cell adaptation and then spray-dried at an inlet temperature of 140°C and a delivery rate of 500 mL h−1. The powder obtained was rehydrated with reconstituted nonfat skimmed milk (10%), which acted as a rehydration medium. Used methodology was optimized by Costa et al. (2002). Significant differences between growth treatments were found with respect to the survival of spray-dried cells (Fig. 11.3). The best survival was achieved with

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cells grown for 48 h in NaCl 0.97 medium (29%), followed by cells grown for 48 h in NaCl 0.98 (23%). The survival rate of cells growth in unmodified medium was always less than 7%. In the case of NaCl, the survival of pre-stressed cultures improved when cells were incubated for 48 h instead of 24 h. The opposite tendency was observed with control cells. Our research demonstrated that the treatments that showed the best adaptation to low aw, also presented better survival during the spray-drying process than control cells. These results confirm others obtained by Prasad et al. (2003), who found that when pre-stressed with either heat (50°C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 showed a significant improvement in viability compared with a non-stressed control culture after storage at 30°C in its dried form. The drying technique applied in this case was fluidised bed drying. Although desiccation improvement of modified NaCl cells is clear, it is not sufficiently good in practical terms to consider spray-drying as an appropriate strategy for dehydrating this biocontrol agent. Regardless of the growth medium, all P. agglomerans treatments showed an excellent biocontrol efficacy of P. expansum and P. digitatum on both apples and oranges. The efficacy of unmodified and of the two sodium chloride modified (0.98 and 0.97 aw) treatments was not statistically different and reductions of rot incidence higher than 77% and 73% were respectively observed on artificially wounded and inoculated apples and oranges. This investigation revealed that it is possible to improve the low aw tolerance of P. agglomerans by manipulating growth conditions. Improved cells also showed better survival during the spray-drying process. These results also suggest that it is possible to improve the stress tolerance of the microorganism and thus its behaviour under non-controlled environmental conditions and/or during its formulation process without affecting its biocontrol potential.

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11.3.2 Impact of Mild Heat Treatments on Induction of Thermotolerance in the Biocontrol Yeast Candida sake CPA-1 and Viability After Spray-Drying Other way to improve the tolerance of biocontrol agents of desiccation at high temperature during spray drying is to expose the microorganisms to mild temperatures during growth (Cañamás et al. 2008). Studies were conducted on the biocontrol agent C. sake cells grown in molasses medium at 25°C and exposed to mild temperatures of 30°C and 33°C during mid (16 h), late-exponential(24 h), early- (30 h) and mid-stationary (36 h) growth phases. The effect on viability was determined after exposed to lethal shock at 40°C for up to 120 min and after spray-drying. The log (N N0−1) values obtained from cells first exposed to mild heat treatments at 30 or 33°C and then exposed to lethal shock at 40°C for up to 120 min is shown in Fig. 11.4. Cells heat-adapted at 30 or 33°C survived exposure to 40°C from all studied phases of growth significantly (P £ 0.05) better than the control cells. This pattern was similar for all mild heat treatments although heat-adapted cells at 33°C survive lethal shock better at 40°C than those adapted at 30°C. Mild heat treatments at 33°C on cells in mid- or late-exponential, early- and mid-stationary phases of growth survived 0.91-log, 1.17-log, 1.26-log, 1.21-log, respectively, more than control cells when they were subjected to 40°C for 120 min. In order to determinate the effect on survival of cells after spray-drying C. sake was grown as previously described and then resuspended into 50 ml in 10% w/v reconstituted nonfat-skimmed milk. Suspended yeast cells were incubated at 150 rev min−1 for 30 min at room temperature. Subsequently, each sample was spraydried at an inlet temperature of 150°C and a delivery rate of 500 mL h−1 as described Abadias et al. ( 2005). The spray-dried powder was rehydrated with 5 mL of 10% nonfat-skimmed milk. The dried cells were shaken vigorously for 1 min and then allowed to rehydrate for 9 min. Survival after spray-drying of heat-adapted and non-adapted control cells of C. sake is shown in Fig. 11.5. The results showed that only mild heat treatments at 33°C from the stationary phase of growth (early or mid) were able to increase survival significantly after spray-drying in comparison to control cells. At 30°C, there was no effect on induced resistance after spray-drying. Prasad et  al. (2003) also observed that during fluidised bed drying the viability of Lactobacillus rhamnosus HN001 cells adapted from stationary phase were superior to cells that were heatadapted from the exponential phase. In contrast to this, Teixeira et  al. (1997) showed that heat adaptation increased the survival of Lactobacillus bulgaricus cells during spray-drying only when heat stress was applied to cultures in the exponential phase of growth. The possible role of heat-shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined and cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse accumulation of sugar and sugar alcohols.

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It was found that mild heat-adapted cells at sublethal temperature of 33°C from early- or mid-stationary growth phase still showed higher viability, independently of the presence of chloramphenicol or cycloheximide. This suggests that protein synthesis is not responsible for the acquision of thermotolerance in the yeast C. sake CPA-1. Other research studies mainly with bacteria have shown evidence

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Fig. 11.5  Survival (%) after spray-drying of control (CS) and heat-adapted cells of Candida sake CPA-1 at 30°C or 33°C (mild heat treatments) from mid-exponential (mid-exp), late-exponential (late-exp), early-stationary (early-sta) or mid-stationary (mid-sta) growth phase until 40 h of incubation. Results are the mean of at least three-independent spray-drying trials. Columns with different letter are statistically different according to Duncan’s multiple range test at P £ 0.05

of the relationship between protein synthesis and acquired thermotolerance. Thus, Periago et  al. (2002) showed that for heat-adaptive response in Bacillus cereus ATCC 14579 de novo protein synthesis is required. Ananta and Knorr (2004) with Lactobacillus rhamnosus GG, De Angelis et  al. (2004) with Lactobacillus plantarum and Prasad et al. (2003) with Lactobacillus rhamnosus HN001 (DR20) all found a role of protein biosynthesis in the induction of heat tolerance. However, in yeast this link may still be tenuous. This is because some studies have reported data that confirmed that incubation of cells at up to optimum growth temperature induced thermotolerance and synthesis of stress proteins (McAlister and Finkelstein 1980; Tiligada et al. 1999), while others e. g. Hall (1983), showed that when microorganisms were incubated with inducer substances of HSPs, they did not show thermotolerance. These results suggested that the stress proteins induced by these analogous substances are not sufficient condition for the acquisition of thermotolerance. It was in concordance with other reported data which is concluded that HSPs were not the direct responsible compounds of thermotolerance acquired by cells (Watson et al. 1984; Swan and Watson 1999). In this study, the correlation between acquired thermotolerance and accumulation of intracellular components, such as arabitol and glucose, was only observed when heat stress was applied to cells in stationary phase at 36 h or in log-phase at 16 or 24 h. Hottiger et al. (1989), Piper (1993), and Swan and Watson (1999) have all showed that in yeast neither HSPs nor trehalose are closely correlated with heatstress tolerance. The present study suggests that for C. sake, there is no consistent role for intracellular components in thermotolerance of cells.

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11.4 Fluidized Bed Drying In this study, adaptation stress responses (osmotic, thermal and the combination) are studied to improve survival of cells after fluidized bed drying process.

11.4.1 Screening of Thermal-Stress Treatments In the first part of the present research, the heat stress response of biocontrol agent P. agglomerans CPA-2 was analyzed subjecting cells to different thermal stress treatments (35°C and 40°C) at mid exponential (mid-ex), late exponential (late-ex), early stationary (early-st) or mid stationary (mid-st) phases of growth until the end of incubation time (24 h). Results indicated that thermal stress treatments induced thermotolerance in P. agglomerans cells which meant that cells were more resistant to a lethal temperature of 45°C. Heat-inducible thermotolerance allows bacteria, after a non-lethal heat shock, to tolerate a second heat stress higher in intensity (Boutibonnes et al. 1992). It was demonstrated that 40°C was more effective than 35°C to induce thermotolerance on P. agglomerans cells under in vitro conditions (Fig. 11.6). Results also indicated that the highest thermotolerance was obtained when thermal stress treatments were applied in late exponential or early stationary phase of growth. Consequently, it could be concluded that the application of a mild heat treatment protects P. agglomerans cells from death during subsequent more extreme heat exposure. This fact could be interesting for our objectives because extreme heat conditions could happen during formulation process and also at field conditions. The induction of thermotolerance by thermal stress treatments in bacteria has been studied by other authors such as Teixeira et  al. (1994) and Gouesbet et  al. (2002) who reported that heat adaptation increased the thermotolerance of lactobacilli. Similar approach has also been used to improve tolerance of the biocontrol agent Candida sake (Cañamás et  al. 2008) as it has been shown above in Section 11.3.2 of this chapter.

11.4.2 Improving Fluidized Bed Drying Formulation of P. agglomerans CPA-2 by Osmotic-Stress Treatments In the second part of the present study we analyzed the effect of osmotic treatments on the viability of cells after fluidized bed drying process. Assayed treatments were the same described above in Section 11.3.1 using NaCl to adjust aw (0 g/l Na Cl (0.99 aw)- P, 35 g/L NaCl (0.98 aw)-P980 or 53 g/L NaCl (0.970 aw)-P970) plus another treatment with 25 g/NaCl (0.988 aw )- P988. For an optimal level of growth in P, P988, P980 and P970 treatments, incubation times were 20, 20, 22 and

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Thermal stress treatments T35-mid-ex T35-late-ex T35-early-st T35-mid-st

0.0

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Fig. 11.6  Thermotolerance of non-adapted (P) or thermal adapted P. agglomerans cells. During growth process cultures were subjected to thermal stress treatment at a sublethal temperature of 35°C (a) or 40°C (b) in mid exponential (mid-ex), late exponential (late-ex), early stationary (early-st) or mid stationary (mid-st) phase until cultures achieved 24 h of incubation. Subsequently, a heat lethal shock was performed at 45°C for 60 min. Initial cell count was approximately 1 × 108 CFU/mL and viabilities were measured after 60 min of exposition to heat lethal shock. Levels of thermotolerance were expressed as logarithmic value of relative survival fraction log10 (N/N0). Results are the means of three independent measures. Columns with different letter are statistically different according to Duncan’s multiple range test at P < 0.05

30 h, respectively. For P980 and P970 cultures were also obtained after 48 h of incubation, corresponding this time to an optimal level of osmoresistance of P. agglomerans cells (P980 + 48 h and P970 + 48 h treatments) as it had been demonstrated by Teixidó et al. (2006). Results pointed out those treatments which were effective to improve survival of P. agglomerans cells during drying process. The best survival values of P. agglomerans cells were achieved with osmotic treatments when cells grown at aw of 0.98 and 0.97 for 48 h using NaCl to adjust aw (Fig. 11.7). These treatments also shown the highest viabilities following spray-drying process in studies carried out by Teixidó et  al. (2006). However, P988 osmotic treatment was chosen for further experiments because it was a cheaper and optimized production treatment. Although P980 + 48h and P970 + 48h treatments gave higher viabilities than P988 osmotic treatment, they showed lower levels of biomass production. Other authors such as Prasad et  al. (2003) obtained also higher viabilities in osmotic shocked cells of Lactobacillus rhamnosus HN001 (DR20) than non-adapted after drying in fluidized bed dryer.

11.4.3 Improving Fluidized Bed Drying Formulation of P. agglomerans CPA-2 by Combination of Thermal and Osmotic-Stress Treatments To our knowledge there has been no previous study in which thermal and osmotic stresses were combined to improve survival of cells after drying process. In the present study the combination of osmotic and thermal stress treatments on P. agglomerans

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Fig. 11.7  Survival of non-osmotically (P treatment) and osmotically adapted cells of P. agglomerans after dehydration in a fluidized bed dryer for 20 min at 40°C. Levels of viability after drying process were expressed as logarithmic value of survival fraction log10 (N/N0). Results are the means of at least two independent fluidized bed drying trials with two independent replications per trial. Columns with different letter are statistically different according to Duncan’s multiple range test at P < 0.05

cells resulted in improved viabilities especially when short mild heat treatments were applied in early stationary phase of growth. Log reduction values showed that non-significant differences (P > 0.05) were found between survival fractions of osmotically adapted cells from P988 treatment compared with survival fractions of osmotically and thermal adapted cells from P988 treatment combined with thermal treatments T35-early-st or T40early-st (Data not shown). The combination of osmotic treatment P988 with thermal stress treatment (T40-late-ex) showed survival values statistically lower than osmotic treatment P988 but this survival value was significantly higher than that obtained with P treatment (non-osmotic cells). The combination of osmotic and thermal treatments also resulted in concentration of viable cells higher than non-adapted cells, ranging these values between 1.2 × 1010 and 5.6 × 1010 CFU/g of fluidized product. On the other hand, osmotic P988 treatment combined with short thermal treatments, T35-early-st-1h or T35-early-st-3h, showed survival values higher than nonadapted cells after fluidized bed drying process (Fig. 11.8). In this case, log N/N0 values obtained from P988 + T35-early-st-1h combined treatment were significantly higher than when osmotic P988 treatment was applied alone with log reduction of –0.51. T35-early-st-1h or T35-early-st-3h had concentration of viable cells between 5.5 × 1010 and 1.1 × 1011 CFU/g of fluidized product, respectively. The moisture content in fluidized-dried product from osmotic and thermal treatments

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Fig. 11.8  Viability of osmotically (P988) or thermal-osmotically-adapted cells of P. agglomerans after dehydration in fluidized bed drying. Thermal-osmotic-adapted cells were obtained applying osmotic P988 treatment and subjecting P. agglomerans cells in early stationary phase of growth to sublethal temperature of 35°C for 1 or 3 h (P988 + T35-early-st-1 or P988 + T35C-early-st-3 treatment, respectively). Levels of viability after drying process were expressed as logarithmic value of relative survival fraction log10 (N/N0). Results are the means of at least two independent fluidized bed drying trials with two independent replications per trial. Columns with different letter are statistically different according to Duncan’s multiple range test at P < 0.05

ranged between 9.2% and 11.8%. Interestingly, when osmotic treatment was combined with thermal stress treatment applied in early stationary phase of growth at sublethal temperature of 35°C for 1 h higher viabilities were obtained than when osmotic treatment P988 was applied alone. These results demonstrated that the combination of different stress treatments had an accumulative effect on the resistance of cells, resulting osmotic and thermal adapted P. agglomerans cells more resistant than cells only adapted with a simple stress treatment. Protective additives are used in drying processes to protect cells against environmental damages (Corcoran et al. 2005). These additives are often native substances that the microorganisms themselves produce, such as disaccharides, amino acids and amino acid derivates. Production of these compounds is often induced upon stress or pre-conditioning (Ross et al. 2005). The accumulation of glycine-betaine and carnitine has been shown to enhance survival of L. plantarum after drying (Kets et al. 1994). Results reported by Cañamás et al. (2007) confirmed that P. agglomerans cells accumulate different compatible solutes under osmotic and thermal stress. Osmotic adapted P. agglomerans cells using NaCl as solute mainly accumulated glycine-betaine. However, there was no increase in quantities of glycine-betaine and ectoine under heat stress (Cañamás et al. 2007).

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Overall, the present study it has been demonstrated that osmotic and thermal stress treatments are a practical tool to improve survival of P. agglomerans cells under stress conditions. Moreover, the final fluidized product obtained showed adequate viabilities to formulate P. agglomerans cells by fluidized bed drying process (between 5.5 × 1010 and 1.1 × 1011 CFU/g of fluidized product).

11.5 Conclusions The importance to develop a shelf-stable formulated product that retains biocontrol activity is clear in order to obtain satisfactory commercial biocontrol agents. The results shown in this chapter indicate that it is possible to improve liquid and solid formulations using several strategies such as growing microorganisms in aw modified media, in sublethal thermal stress or using preservation in isotonic solutions. This study also demonstrates that technologically sensitive cultures as P. agglomerans or C. sake can potentially be manipulated to become more robust for survival under harsh conditions, such spray drying or fluidized bed drying processes. The use of these new techniques could help the companies to put in the market more stable and effective biocontrol products. Acknowledgements  The authors are grateful to Spanish government (Ministerio de Ciencia y Tecnología) for grants AGL-2002–01137 and AGL-2005–02510 and to FEDER (Fondo Europeo de Desarrollo Regional) for their financial support.

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Beever RE, Laracy EP (1986) Osmotic adjustment in the filamentous fungus Aspergillus nidulans. J Bacteriol 168:1358–1365 Boutibonnes P, Tranchard C, Hartke A, Thammavongs B, Auffray Y (1992) Is thermotolerance correlated to heat shock protein synthesis in Lactococcus lactis subsp. lactis? Int J Food Microbiol 16:227–236 Cañamás TP, Viñas I, Usall J, Magan N, Morelló JR, Teixidó N (2007) Relative importante of amino acids, glycine-betaine and ectoine síntesis in the biocontrol agent Pantoea agglomerans CPA-2 in response to osmotic, acidic and heat stress. Lett Appl Microbiol 45:6–12 Cañamás TP, Viñas I, Usall J, Magan N, Solsona C, Teixidó N (2008) Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying. J Appl Microbiol 104:767–775 Corcoran BM, Stanton C, Fitzgerald GF, Ross RP (2005) Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 71:3060–3067 Costa E, Teixidó N, Usall J, Fons E, Gimeno V, Delgado J, Viñas I (2002) Survival of Pantoea agglomerans strain CPA-2 in a spray-drying process. J Food Protect 65:185–191 Csonka LN (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 53:121–147 De Angelis M, Di Cagno R, Huet C, Crecchio C, Fox PF, Gobbetti M (2004) Heat-shock response in Lactobacillus plantarum. Appl Environ Microbiol 70:1336–1346 Droby S, Cohen L, Daus A, Weiss B, Horev B, Chalutz E, Katz H, Keren-Tzur M, Shachnai A (1998) Commercial testing of Aspire: A yeast preparation for the biological control of postharvest decay of citrus. Biol Control 12:97–101 Ellis SW, Grindle M, Lewis DH (1991) Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and -resistant strains of Neurospora crassa. Mycol Res 95:457–464 Gouesbet G, Jan G, Boyaval P (2002) Two-dimensional electrophoresis study of Lactobacillus delbrueckeii subsp. bulgaricus thermotolerance. Appl Environ Microbiol 68:1055–1063 Greenacre EJ, Brocklehurst TF, Waspe CR, Wilson DR, Wilson DG (2003) Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20°C: optimization and modelling. Appl Environ Microbiol 69:3945–3951 Hall BG (1983) Yeast thermotolerance does not require protein synthesis. J Bacteriol 156:1363–1365 Hallsworth JE, Magan N (1994) Effect of carbohydrate type and concentration on polydydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi. Microbiology 140:2705–2713 Hallsworth JE, Magan N (1996) Culture age, temperature and pH affect the polyol and trehalose contents of fungal propagules. Appl Environ Microbiol 62:2435–2442 Hottiger T, Boller T, Wiemken A (1989) Correlation of trehalose content and heat resistance in yeast mutants altered in the RAS / adenylate cyclase pathway: Is trehalose a thermoprotectant? FEBS Lett 255:431–434 Janisiewicz WJ, Jeffers SN (1997) Efficacy of commercial formulation of two biofungicides for control of blue mold and gray mold of apples in cold storage. Crop Protect 16:629–633 Jorgensen F, Stephens PJ, Knochel S (1995) The effect of osmotic shock and subsequent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes. J Appl Bacteriol 79:274–281 Kets EPW, Galinski EA, de Bont JAM (1994) Carnitine: a novel compatible solute in L. plantarum. Arch Microbiol 162:243–248 Ko R, Smith LT, Smith GM (1994) Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes. J Bacteriol 176:426–431 Leslie SB, Teter SA, Crowe LM, Crowe JH (1994) Trehalose lowers membrane phase transitions in dry yeast cells. Biochim Biophys Acta 1192:7–13 Mattick KL, Jorgensen F, Legan JD, Lappins-Scott HM, Humphrey TJ (2000) Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance. Appl Environ Microbiol 66:4921–4925 McAlister L, Finkelstein DB (1980) Heat-shock proteins and thermal resistance in yeast. Biochem Biophys Res Commun 93:819–824

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Nunes C, Usall J, Teixidó N, Viñas I (2001) Biological control of postharvest pear diseases using a bacterium Pantoea agglomerans (CPA-2). Int J Food Microbiol 70:53–61 Nunes C, Usall J, Teixidó N, Fons E, Viñas I (2002) Postharvest biological control by Pantoea agglomerans (CPA-2) on Golden Delicious apples. J Appl Microbiol 92:247–255 O’driscoll, Cormac GM, Gahan M, Hill C (1996). Adaptative acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl Environ Microbiol 62:1693–1698 Periago PM, van Schaik W, Abee T, Wouters JA (2002) Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579. Appl Environ Microbiol 68:3486–3495 Piper PW (1993) Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 11:339–356 Poirier I, Maréchal PA, Evrard C, Gervais P (1998) Escherichia coli and Lactobacillus plantarum responses to osmotic stress. Appl Microbiol Biotechnol 50:704–709 Prasad J, McJarrow P, Gopal P (2003) Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying. Appl Environ Microbiol 69:917–925 Rapoport AI, Puzyrevskaya OM, Saubenova MG (1998) Polyols and resistance of yeast to dehydration. Microbiology (NY) 57:269–271 Rhodes DJ (1993) Formulation of biological control agents. In: Jones DG (ed) Exploitation of microorganisms. Chapman & Hall, London, pp 411–439 Ross RP, Desmond C, Fitzgerald GF, Stanton C (2005) Overcoming the technological hurdles in the development of probiotic food. J Appl Microbiol 98:1410–1417 Sanders JW, Venema G, Kok J (1999) Environmental stress responses in Lactococcus lactis. FEMS Microbiol Rev 23:483–501 Swan TM, Watson K (1999) Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat-shock proteins and trehalose. Can J Microbiol 45:472–479 Teixeira P, Castro H, Kirby R (1994) Inducible thermotolerance in Lactobacillus bulgaricus. Lett Appl Microbiol 18:218–221 Teixeira P, Castro H, Mohacsi-Farkas C, Kirby R (1997) Identification of sites of injury in Lactobacillus bulgaricus during heat stress. J Appl Microbiol 83:219–226 Teixidó N, Viñas I, Usall J, Magan N (1998a) Control of blue mold of apples by preharvest application of Candida sake grown in media with different water activity. Phytopathology 88:960–964 Teixidó N, Viñas I, Usall J, Magan N (1998b) Improving ecological fitness and environmental stress tolerance of the biocontrol yeast Candida sake (strain CPA-1) by manipulation of intracellular sugar alcohol and sugar content. Mycol Res 102:1409–1417 Teixidó N, Usall J, Palou L, Asensio A, Nunes C, Viñas I (2001) Improving control of green and blue molds on oranges by combining Pantoea agglomerans (CPA-2) and sodium bicarbonate. Eur J Plant Pathol 107:685–694 Teixidó N, Cañamás TP, Usall J, Torres R, Magan N, Viñas I (2005) Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross protection in the biocontrol agent Pantoea agglomerans CPA-2. Lett Appl Microbiol 41:248–252 Teixidó N, Cañamás TP, Abadias M, Usall J, Solsona C, Casals C, Viñas I (2006) Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments. J Appl Microbiol 101:927–937 Tiligada E, Miligkos V, Ypsilantis E, Papamichael K, Delitheos A (1999) Molybdate induces thermotolerance in yeast. Lett Appl Microbiol 29:77–80 Usall J, Teixidó N, Fons E, Viñas I (2000) Biological control of blue mould on apple by a strain of Candida sake under several controlled atmosphere conditions. Int J Food Microbiol 58:83–92 Usall J, Teixidó N, Torres R, Ochoa de Eribe X, Viñas I (2001) Pilot test of Candida sake (CPA-1) applications to control postharvest blue mold on apple fruit. Postharvest Biol Tec 21:147–156

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Van Eck JH, Prior BA, Brandt EV (1993) The water relations of growth and polyhydroxy alcohol production by ascomycetous yeasts. J Gen Microbiol 139:1047–1054 Viñas I, Usall J, Teixidó N, Sanchis V (1998) Biological control of major postharvest pathogens on apple with Candida sake. Int J Food Microbiol 40:9–16 Watson K, Dunlop G, Cavicchioli R (1984) Mitochondrial and cytoplasmic protein syntheses are not required for heat-shock acquisition of ethanol and thermotolerance in yeast. FEBS Lett 172:299–302 Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214–1222

Chapter 12

Host Responses to Biological Control Agents Raffaello Castoria and Sandra A.I. Wright

Abstract  Host responses in stored fruits induced by biocontrol agents (BCAs) i.e. by non-pathogenic yeasts and bacteria, share many features with the defence mechanisms that are induced in actively growing plant tissues. The perception of a microorganism is accompanied by the production and activation of reactive oxygen species (ROS), antioxidant enzymes, phytoalexins, phenylalanine ammonia lyase and enzymes that degrade fungal cell walls. The responses of harvested fruit to BCAs do not fit with the existing division of induced resistance pathways into Systemic Acquired Resistance (SAR) and rhizobacteria-mediated Induced Systemic Resistance (ISR), nor are the roles of salicylic or jasmonic acid clear. These responses seem to carry elements of both pathways. Moreover, successful BCAs need to be able to resist environments rich in toxic ROS; hydrogen peroxide being the dominant species, generated both during the induction of resistance (as in the defence of citrus fruit against Penicillium digitatum) and during the attack of some necrotrophic pathogens (as in the case of Penicillium expansum invading apples). Application of BCAs to fruits can result in increased production of antioxidant enzymes (by either organism), which protect living cells from the potential damage of ROS. Induction of resistance has usually not been considered an important mechanism in the activity of postharvest biocontrol agents. A deeper understanding of fruit responses that BCAs provoke of the infection process by necrotrophic pathogens during postharvest and of the accompanying host responses is needed. In the following chapter, we present examples from diverse plant-pathogen-BCA systems and suggest approaches for future research.

R. Castoria (*)   Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via Francesco De Sanctis, 86100, Campobasso, Italy e-mail: [email protected] S.A.I. Wright Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via Francesc De Sanctis, 86100, Campobasso, Italy, and Department of Plant and Environmental Sciences, University of Gothenburg, 461SE, 405 30, Göteborg, Sweden D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_12, © Springer Science + Business Media B.V. 2009

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Keywords  Biological control • induced resistance • pathogenicity strategies of postharvest pathogenic fungi.

12.1 Introduction Biological control of plant diseases is a three-way interaction in which pathogen, plant tissue(s) and biocontrol agent (BCA) are involved. The plant tissue cannot merely be considered as the battlefield where pathogen and biocontrol agent confront each other, since it appears to perceive the presence of both the pathogen and the BCA. Perception of the pathogen or of the BCA is followed by deployment of an active response that results in induced resistance that can be localized or systemic. The localized response is based on rapid cell death, reinforcement of cell walls and accumulation of phytoalexins. Systemic resistance also involves the synthesis and accumulation of antifungal depolymerases that attack the fungal wall. Even if induced resistance is normally considered to be a response of non-senescing, growing plant tissues and intact plants, the phenomenon has also been reported in BCA-treated and stored fruits and some of the responses have commonalities with those observed during the induced resistance reactions of intact, growing plants (see below). A handful of biocontrol products for postharvest use are at present available commercially, and they represent only a small fraction of the potential market for control of postharvest diseases (Fravel 2005). Some examples of these products are Bio-Save®10 LP and Bio-Save®11 LP, which are based on strains ESC 10 and ESC 11 of the bacterium Pseudomonas syringae, respectively, and are registered in the USA by JET Harvest Solutions (Longwood, FL, www.jetharvest.com). BioSave®10 LP is active on postharvest rots caused by Penicillium digitatum, Penicillium italicum and Geotrichum candidum of citrus fruit, rots caused by Penicillium expansum, Botrytis cinerea and Mucor piriformis of pears and apples, and rots in cherries caused by P. expansum and B. cinerea. Another biocontrol product for use in postharvest systems is Shemer® (www.agrogreen.co.il/shemer. asp), based on a strain of the yeast Metschnikowia fructicola, and is registered in Israel by AgroGreen Ltd (Ashdod, Israel).) Registrations in the USA in and Europe are currently pending. Shemer® is effective against postharvest rots of apricot, grapes, strawberry, citrus, peach, pepper and sweet potato caused by species of Aspergillus, Botrytis, Penicillium and Rhizopus. BoniProtect® and BoniProtect® Forte are based on mixtures of two strains of the yeast Aureobasidium pullulans, developed and marketed in Germany by Bio-Protect (Konstanz, www. bio-protect.de). BoniProtect® is effective against postharvest rots in apple caused by B. cinerea, P. expansum and Pezicula malicortici. BoniProtect® Forte is applied at bloom to strawberries and protects them during postharvest storage against the development of grey mould caused by B. cinerea. Candifruit® is based on a strain of the yeast Candida sake, developed by Centre UdL-IRTA (Lleida, Catalonia) and marketed in Spain by SIPCAM INAGRA SA (Valencia, www.sipcam.es).

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Candifruit® is effective against postharvest rots in apple and pears caused by P. expansum, B. cinerea and Rhizopus stolonifer. In stored fruits, the defence capability becomes weaker with time. As the physiology of the fruits changes during maturation and senescence, inhibitors of fungal growth decrease and the fruit becomes more susceptible to postharvest pathogens, especially to necrotrophic ones. A clear example is the antrachnose disease of avocado caused by Colletotrichum gloeosporioides, in which fungal growth undergoes a transition from the quiescent to the necrotrophic stage, a condition that appears to be associated with fruit ripening. During this transition, the level of a preformed antifungal diene decreases in the fruit as a consequence of the decrease of the level of the protective antioxidant epicathechin (Karni et al. 1989). Physiological changes such as fruit tissue softening and the shift of plant cells to a more oxidized state negatively affect fruit resistance to necrotrophic pathogens and the antagonistic activity of biocontrol yeasts (Castoria et al. 2003). Most if not all of the studies on fruit responses to treatment with BCAs have been carried out with biocontrol yeasts or yeast-like organisms. As a consequence, the present chapter will focus on the interaction with this kind of microorganisms. The chapter is not to be taken as a comprehensive review of all the literature in this area, but an attempt to give a succinct summary of the information at present available on fruit responses to BCAs, and to present new perspectives for research in postharvest biocontrol.

12.2 Modes of Action of Postharvest Biocontrol Agents As a general introduction to mechanisms of action, some definitions will be presented in order to envisage the dynamic context in which fruit responses to BCAs operate. The modes of action of beneficial microorganisms can be divided into direct antagonism and indirect antagonism. These mechanisms do not exclude each other since they are frequently described as co-occurring within the activity of the same BCA. Direct antagonism comprises those mechanisms that are a direct result of the action of an individual BCA: competition for space and nutrients, secretion of lytic enzymes (depolymerases) such as b-1,3 glucanases and chitinases that degrade the polymers of the pathogen cell wall and mycoparasitism, the latter often documented to require direct physical interaction between the cells of the BCA and the hyphae of the pathogenic fungi (Castoria et al. 1997; Janisiewicz and Korsten 2002; Wisniewski et  al. 1991). Indirect antagonism implies mechanisms which are not a direct result of the activity of the BCA, but are the consequence of the response of the fruit tissue to the presence of these beneficial microorganisms, ultimately resulting in induction of resistance (see Section 12.2.1). Competition for space and nutrients by the BCA mainly takes place when the BCA colonizes wounds of fruit, caused by bruising during handling and transportation. These wounds represent the main ports of penetration of pathogenic fungi. In order to successfully out-compete the pathogens, postharvest BCAs need to possess a sound wound colonization competence, i.e. the ability to rapidly colonize and

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thrive in fruit wounds (Droby and Chalutz 1994). Since wounding in apple fruits causes the production of hydrogen peroxide (H2O2) and superoxide anion (•O2−), it has been suggested that resistance to the oxidative stress caused by these reactive oxygen species (ROS) plays a role in wound competence of biocontrol yeasts and, as a consequence, in their competition with the pathogen for space (and nutrients). As with other climacteric fruits, the physiology of stored apples changes during maturation and senescence. One of these changes is represented by the increased production of ROS when the apple tissue becomes wounded during storage. The increased production of ROS in apple wounds negatively affects both wound colonization by BCAs and their antagonistic activity (Castoria et  al. 2003). Mycoparasitism is mediated by the physical attachment of the biocontrol agent to the hyphae of the pathogen, and is associated with the secretion of cell wall depolymerases in causing damage to the hyphae of the pathogenic fungi, as in the case of the interaction of Trichoderma spp. with Rhizoctonia solani (Harman et al. 2004). This mechanism has not been reported to be common for postharvest BCAs but, when present, as in the case of the attachment of cells of Pichia guilliermondii to hyphae of B. cinerea, it appears to be associated with the secretion of cell wall depolymerases that cause damage to the hyphae of the pathogenic fungi (Wisniewski et al. 1991). In addition, depolymerases have been suggested to contribute to the antagonistic activity of BCAs, even in the absence of clear mycoparasitism (Castoria et al. 1997, 2001).

12.2.1 Induced Resistance Signalling Pathways Induced resistance in plants is termed localized when this response and the mechanisms it relies on occur at the same site of pathogen inoculation or treatment with any triggering factor such as elicitors (i.e. compounds that elicit a hypersensitive response) or BCAs. Conversely, resistance is systemic when it occurs at a site that is distal from the one where perturbation of the plant homeostasis initially takes place. Systemic resistance can be the consequence of local pathogen inoculation, in which case it is referred to as systemic acquired resistance (SAR). Alternatively, another form of systemic resistance, called rhizobacteria-mediated induced systemic resistance (ISR), is not a result of a localized resistance event induced by pathogens, but is triggered by beneficial microbes such as rhizobacteria, chewing insects, wounding or by biotic elicitors. SAR and ISR are generally thought to operate through different signal transduction pathways: SAR is activated through salicylic acid (SA)-dependent pathway, ISR through the jasmonic acid (JA) and ethylene – dependent pathway, which is also involved in wound-induced resistance (WIR) (Kessler and Baldwin 2002) caused by tissue damage as a result of insect feeding. Another general idea is that resistance induced by JA (and ethylene) or by SA has its own respective pattern in defence mechanisms elicited: SA-mediated responses are characterized by the synthesis of pathogenesis-related (PR) proteins, whereas JA (and ethylene)-mediated responses are not (Vallad and Goodman

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2004). Studies on defined mutants of Arabidopsis thaliana have suggested that specific plant defence responses may be active on specific types of pathogens: SA-dependent defences are effective against biotrophs, JA (and ethylene)-dependent responses are effective against necrotrophs (Glazebrook 2005). The latter comprises a category that includes most postharvest wound pathogens. However, the actual situation seems to be more complex, since there is some cross-talk between these two major signalling pathways (De Vos et  al. 2005; Glazebrook 2005), through NPR1 (Non-expressor of PR-genes 1). They can thus be turned on simultaneously, producing an additive effect, and sometimes SAR seems to have a negative effect on the JA/ethylene pathway (reviewed in Durrant and Dong 2004). In the following paragraphs of this chapter, findings on localized and systemic resistance induced in fruits by BCAs will be reviewed, and the roles of plant resistance responses and pathogenicity determinants of major postharvest pathogens will be discussed. 12.2.1.1 Localized and Systemic Resistance Induced in Fruits by BCAs An early report on the induction of local resistance mechanisms in stored fruits (Arras 1996) showed that the phytoalexin scoparone was synthesized in oranges pre-treated with the yeast Candida famata strain FS35. Ippolito et  al. (2000) reported a transient induction of glucanase, chitinase and peroxidase (POD) in apple wounds treated with A. pullulans strain L47, which, although not demonstrated, they attributed to the apple tissue. This yeast-like fungus was used to successfully protect stored apple fruits from infection by P. expansum and B. cinerea (Ippolito et al. 2000). The results obtained by Castoria et al. (2001) when using a different strain of A. pullulans, strain LS30, further substantiated the notion that BCAs can induce apple tissues to produce glucanases. Some of the glucanase activity recorded in this study most probably derived from the apple tissue, since the activity was detected also in wounds that were only treated with a preparation of cell walls of P. expansum (i.e. in the absence of any living microorganism). Tian et al. (2007) cloned two b-1,3 glucanase genes (Glu-1 and Glu-2) from Jujube fruit (Ziziphus jujuba Mill), and showed that the expression of Glu-1 was induced by the yeast Cryptococcus laurentii, starting 12 h after application of this BCA. The expression study was complemented with a biochemical analysis by assessing b-1,3 glucanase activity (although it was not ascertained if the source of enzyme activity was the yeast, the fruit or both). b-1,3 glucanase activity was the highest following treatment with the yeast at all time points tested and correlated positively with the protection against P. expansum and Alternaria alternata achieved by the yeast. It is not clear if this is an example of localized or systemic resistance, since it seems as if the authors extracted fruit tissue as a whole, not only the wounded area. The first convincing evidence of BCA-induced systemic resistance in fruit was presented by Droby et al. (2002) and El Ghaouth et al. (2003), who studied C. oleophila applied to grapefruit and C. saitoana applied to apples, respectively. In both cases, the assessment of systemic resistance was based on the application

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of a Candida strain in freshly generated wounds, inoculation of the pathogen in wounds that were spatially separated from the wounds in which the BCAs had been applied and on the evaluation of disease incidence and/or severity. Droby et  al. (2002) showed that the biocontrol yeast C. oleophila induced systemic resistance to P. digitatum in grapefruit. Protection took place 24 h after the application of the yeast, it was BCA concentration-dependent and did not occur in wounds made more than 4 cm from the wounds in which the BCA was applied. Living yeast cells were needed to ensure protection, which correlated to the induction in the fruit tissue of defence responses such as phenylalanine ammonia lyase (PAL) activity, accumulation of b-1,3 glucanase and chitinase, biosynthesis of ethylene and the accumulation of the phytoalexins umbelliferone, scoparone and scopoletin. The authors hypothesized that the dependence of systemic protection on the BCA cells being alive when applied might be due to the involvement of recognition processes between the yeast and the fruit cells and the secretion of elicitors by the BCA. The presence of BCA-derived elicitors was also tested in another yeast-plant system: plants of A. thaliana treated with autoclaved baker’s yeast were proposed to recognize compounds deriving from the yeast that could act as PAMPs or MAMPs (pathogenassociated or microbe-associated molecular patterns). These compounds induced resistance in A. thaliana to the necrotrophic pathogen B. cinerea (Raacke et al. 2006). El Ghaouth et al. (2003) demonstrated the induction of systemic protection of apples by the yeast C. saitoana, which partially protected them from infection by B. cinerea. The protection, expressed as the reduction of diameters of lesions caused by B. cinerea, occurred when the pathogen was inoculated not earlier than 48–72 h after the application of the BCA and correlated with the timing of induction of b-1,3-glucanase and chitinase activities in the systemically (distally) protected fruit tissue, i.e. where the yeast had not been applied. The capability of inducing systemic resistance depended on the age of fruits, monitored as the efficacy of protection against infection by B. cinerea, which correlated with the induction of b-1,3-glucanase and chitinase activities appeared to occur only in fresh (young) but not in stored (old) apples. The authors hypothesized that a systemic signal was produced at the site of BCA treatment. As of now, no unequivocal evidence has been reported on the involvement of SA or JA in yeast-induced resistance in fruits. In this regard, Raacke et  al. (2006) showed that A. thaliana mutants impaired in the SA-dependent or JA-dependent pathways were not negatively affected in resistance to the necrotrophic fungus B. cinerea induced by autoclaved baker’s yeast, suggesting that this resistance is independent of the SA and JA pathways. Moreover, SA and JA have been tested as possible inducers of resistance in postharvest horticultural crops. A comprehensive review of these studies is presented in Terry and Joyce (2004). SA or a derivative of JA, methyl jasmonate (Me-JA), have also been directly applied on fruits and, in some reports, their effects have been compared with those induced by BCAs. However, it is not clearly described if the examined biochemical markers of induced resistance were measured at the sites of treatment and/or in other fruit parts. In sweet cherry fruit, moderate protection from infection by Monilinia fructicola was achieved by pre-harvest treatment with 2 mM SA or 0.2

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mM Me-JA, and was correlated with the induction of glucanase, PAL and POD activities, thus suggesting that enzymes commonly known as PR proteins are induced by both SA and Me-JA (Yao and Tian 2005). However, the methods used for measuring and/or detecting the induced enzyme activities do not allow the distinction between localized and systemic resistance. In the same fruit, pre-treatment with 0.5 mM SA or the BCA Pichia membranefaciens yielded some protection against P. expansum. Both treatments appeared to influence antioxidant enzyme activities, which either increase (superoxide dismutase, SOD, through dismutation of superoxide anion, O2− to H2O2) or decrease (catalase, CAT, which converts hydrogen peroxide to molecular oxygen and water; POD, which breaks down H2O2 when using it as a cosubstrate in the oxidation of phenolic substrates) the concentration of H2O2. Following inoculation with the pathogen, yeast treatment increased SOD activity but decreased CAT activity, suggesting a role for the steady-state level of ROS in the protection achieved. The authors suggested that resistance induced by the BCA might share some commonalities with that induced by SA, but that the situation was complex and needed to be clarified (Chan and Tian 2006). It is known that ROS, in particular H2O2, and the oxidative stress these chemical species cause is involved in the signalling cascade in the induction of resistance in plants. The oxidative burst contributes to SAR expression in Arabidopsis (Alvarez et al. 1998) and in pathogenesis of necrotrophic pathogens (van Kan 2006; Hadas et al. 2007). Therefore, the complex pattern of antioxidant enzyme activities reported in sweet cherry suggests that direct measurements of H2O2 and O2− are needed to shed more light on the processes involved in protection by SA and the BCA in this system. A role for ROS in the BCA-induced defence mechanisms and in the attack on fruit by postharvest necrotrophic pathogens is also indirectly suggested in the results obtained by Chan et  al. (2007) who showed the induction of antioxidant enzymes following treatment of peach fruit with 0.5 mM SA or P. membranefaciens. Differences in proteomes after the fruits had been treated with SA and the yeast correlated with the difference in protection against P. expansum provided by treatments with SA and the yeast. Although it is not clear whether or not extracted tissue contained the wound site and the yeast cells, the expression of seven proteins (28% of those identified), comprising antioxidant and PR proteins were regulated by both the BCA and SA. Protection with these two treatments correlated with a higher production of PR-9, PR-10, CAT (as confirmed also at a transcriptional and enzyme activity level) and glutathione peroxidase (PR-9, also confirmed at an enzyme activity level). Conversely, a more rapid increase of SOD[Mn] production was induced by the BCA as compared with that induced by to SA, although this finding was not in agreement with the level of enzyme activity detected. Castoria et al. (2003) hypothesized that biocontrol yeasts could also act as exogenous suppliers of antioxidant activity to the apple tissue, thus partially accounting for their protective ability, by providing it with additional defence tools counteracting the production or induction of ROS by B. cinerea. Although it is not yet established that induction of oxidative damage to plant tissues could be a general phenomenon of fungal necrotrophic attack, results by Xu et  al. (2008) appear to support this notion. Treatment of peach fruits with different biocontrol yeasts

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(C. guillermondii, C. laurentii, Rhodotorula glutinis and P. membranefaciens) reduced M. fructicola attack. As in the case of SA treatment that offered protection to sweet cherries from attack by P. expansum, as mentioned above (Chan and Tian 2006), protection achieved by the BCAs seemed to be accompanied by induction of CAT, POD (i.e. enzymes that lower concentration of H2O2), and b-1,3-glucanase activities as well as the expression of the respective genes. Although control treatments of peaches not inoculated with Monilinia were not performed – and thus it is not possible to know if the antioxidant enzymes, chitinase and glucanase measured were of plant or yeast origin – there might have been a decrease in protein damage caused by oxidative stress when infected peaches were treated with the BCAs, as compared to infected fruits not treated with the different yeasts. The authors concluded that the proteins were protected by an antioxidant response, and that this could constitute mechanisms of protection of peach fruits by the microbial biocontrol agents (Xu et al. 2008). However, more studies are needed to confirm these results and to clarify if the BCA-dependent decrease of oxidative damage to peach proteins is an (indirect) indication of M. fructicola-induced oxidation linked to the infection process, i.e. an interference with the pathogenicity strategy of the fungus, or just a reduction of symptom development, i.e. a mere epiphenomenon. Furthermore, as mentioned above, , there was no assessment of the possible oxidative stress caused by the BCAs, i.e. the oxidative damage in healthy peaches treated and untreated with the BCAs (e.g. as a consequence of ROS-mediated signalling) was not reported. In this regard, unpublished results by Droby et al. have recently shown that application of the biocontrol yeast M. fructicola in wounded tissue of apple and citrus induces generation of H2O2. In addition, the BCA itself produces O2 in apples, peaches and citrus.

12.3 Concluding Remarks The study of fruit responses to postharvest biocontrol agents is in its infancy. Even if application of SA and JA appears to improve fruit protection, a role for these compounds in BCA-induced systemic (or even localized) resistance is yet to be established. In stored fruits, the defence responses observed do not neatly fit with the classical division into SA-dependent and JA-dependent signal transduction pathways. In contrast to the responses noted in intact plants, stored fruits produce antifungal hydrolases (and other PR proteins) following either SA or (Me)JA treatment. Furthermore, both SA and JA seem to induce defence responses that provide resistance to necrotrophs, although the precise pathways are not known. BCAs could play a role in the oxidative phenomena that occur during the induction of resistance, but more studies are needed to confirm this hypothesis. Even so, some evidence appears to support the possibility that the induction of an antioxidant response may play a role in the BCA-mediated resistance of fruit to necrotrophic fungi. Further studies are needed also in this case. Sclerotinia sclerotiorum produces oxalic acid, a pathogenicity determinant that induces programmed cell death at pHs in the range of 5 to 6, and which is necessary for a successful infection

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of the plant tissue, via the induction of ROS. As oxalic acid accumulates in the tissues, the pH is lowered and at the lower pH the host oxidative burst is down-regulated. During this phase of the infection process, after the ROS-mediated attack, Sclerotinia is thus able to avoid an excessively H2O2-rich environment induced by the plant (Kim et al. 2008). H2O2, a toxic reactive oxygen species, can in some cases be employed by plants as a defence strategy and in other instances by the necrotrophic pathogen as a weapon mediating its invasion. In the P. expansumapple fruit system, acidification through production of gluconic acid by P. expansum positively affects transcription of fungal genes encoding lytic enzymes that attack the walls of fruit cells. During this phase, there is also an accumulation of H2O2 (since it is a by-product that is formed during gluconic acid synthesis) at the limit of the decaying area (Hadas et al. 2007), thus suggesting a direct role of H2O2 in the infection process, as in the case of necrotrophic attack by B. cinerea (van Kan 2006, Williamson et al. 2007). In contrast, Torres et al. (2003) suggested the possible involvement of fruit-derived H2O2 as a mechanism of resistance of young apples to P. expansum. Similarly, the resistance of citrus fruits to the incompatible pathogen P. expansum is associated with a large local accumulation of H2O2, suggested to be of citrus origin, which ultimately leads to lignification and HR (Macarisin et al. 2007). The compatible pathogen, P. digitatum is instead able to suppress the citrus fruit defence reactions and actively lowers the accumulation of hydrogen peroxide in wounds as compared to non-inoculated but wounded control fruit. As additional weight to the above theory, exogenously added CAT enhanced the pathogenicity of both P. expansum (it became a pathogen) and P. digitatum on citrus fruit. The elucidation of the role of hydrogen peroxide in the pathogenesis of different Penicillium species pathogenic to apples and citrus fruits will be facilitated when defined mutants of P. expansum and P. digitatum become available. Taken together, healthy and infected wounds of harvested fruits present an environment that is rich in ROS, an environment in which BCAs need to operate. It is therefore an advantage when BCAs are able to resist locally produced ROS, whatever the role(s) of ROS is – as inducers of fruit resistance through ROS production and signalling or as activators of an antioxidant response in the fruit that counteracts the oxidative attack by necrotrophic pathogens. Induction of resistance is not usually considered to be a major mechanism of postharvest biocontrol agents, most probably because it has been difficult to monitor and because the BCA and the pathogen are usually applied at the same site. As has been demonstrated, this area of research is emerging. As more information on the mechanisms of pathogenesis of postharvest (necrotrophic) pathogens becomes available, the exact dynamics of the plant-pathogen signalling events and the environment that BCAs encounter will become clearer. In addition, more information on the nature of fruit responses to the BCAs is needed, preferably by using defined plant mutants, so the responses can be organized into pathways, much like has been done for intact plants. BCAs consst of diverse microorganisms. It comprises both yeasts and bacteria. For the most part, they are considered to be nonpathogenic, and in this case, according to the established induced resistance pathways of intact and actively growing plants, SAR should not be involved. However, as was shown by Giobbe et al (2007), a yeast strain (Pichia fermentans) that is a

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BCA of Monilinia brown rot of apple is a pathogen when applied to peach fruit. It is plausible that other BCAs could be pathogens in as of yet unidentified pathosystems, and if so, they would in fact be regarded as incompatible pathogens in the plant test system used to monitor biocontrol activity. This category of BCAs would be expected to cause an oxidative burst and the onset of HR-related defence mechanisms in the fruits as a pathway for induction of resistance. Future research efforts need to deepen our knowledge in all three areas mentioned - on the nature of BCAs, the responses of fruits by using defined mutants and on the mechanisms of pathogenesis of the postharvest pathogens – in order for resistance pathways to be established and characterized also in harvested fruits. Acknowledgements  The authors are sincerely grateful to David B. Wright for critical reading of the manuscript and assistance with language editing. This work was funded by the Italian Ministry of Education, University and Scientific Research (MIUR) PRIN 2006 “Pilot study on innovative systems for the reduction of patulin contamination in pome fruits”, project number 2006072204, and through the MIUR-funded: “Incentivazione alla mobilità di studiosi stranieri e italiani residenti all’estero” (DM 1.2.2005, n.18).

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El Ghaouth A, Wilson CL, Wisniewski M (2003) Control of postharvest decay of apple fruit with Candida saitoana. Phytopatology 93:344–348 Fravel DR (2005) Commercialization and Implementation of Biocontrol. Annu Rev Phytopathol 43:337–359 Giobbe S, Marceddu S, Scherm B, Zara G, Mazzarello VL, Budroni M, Migheli Q (2007) The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot of apple but is pathogenic on peach fruit. FEMS Yeast Res 7:1389–1398 Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227 Hadas Y, Goldberg I, Pines O, Prusky D (2007) Involvement of gluconic acid and glucose oxidase in the pathogenicity of Penicillium expansum in apples. Phytopathology 97:384–390 Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species — opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56 Ippolito A, El Ghaouth A, Wilson CL, Wisnievski M (2000) Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defennse responses. Postharvest Biol Technol 19:265–272 Janisiewicz WJ, Korsten L (2002) Biological control of postharvest diseases of fruits. Annu Rev Phytopathol 40:411–441 Karni L, Prusky D, Kobiler I, Bar-Shira E, Kobiler D (1989) Involvement of epicatechin in the regulation of lipoxygenase activity during activation of quiescent Colletotrichum gloeosporioides infections of ripening avocado fruits. Physiol Mol Plant Pathol 35:367–374 Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Ann Rev Plant Biol 53:299–328 Kim KS, Min J-Y, Dickman MB (2008) Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development. Mol Plant Microbe Interact 21:605–612 Macarisin D, Cohen L, Eick A, Rafael G, Belausov E, Wisnievski M, Droby S (2007) Penicillium digitatum suppresses production of hydrogen peroxide in host tissue during infection of citrus fruit. Phytopathology 97:1491–1500 Raacke IC, von Rad U, Mueller MJ, Berger S (2006) Yeast increases resistance in Arabidopsis against Pseudomonas syringae and Botrytis cinerea by salicylic acid-dependent as well as independent mechanisms. Mol Plant Microbe Interact 19:1138–1146 Terry LA, Joyce DC (2004) Elicitors of induced disease resistance in postharvest horticultural crops: a brief review. Postharvest Biol Technol 32:1–13 Tian S, Yao H, Deng X, Xu X, Qin G, Chan Z (2007) Characterization and expression of b-1,3glucanase genes in Jujube fruit induced by the microbial biocontrol agent Cryptococcus ­laurentii. Phytopathology 97:260–268 Torres R, Valentines MC, Usall J, Vinas I, Larrigaudiere C (2003) Possible involvement of hydrogen peroxide in the development of resistance mechanisms in ‘Golden Delicious’ apple fruit. Postharvest Biol Technol 27:235–242 Vallad GE, Goodman RM (2004) Systemic acquired resistance and induced systemic resistance in conventional agriculture. Crop Sci 44:1920–1934 van Kan JAL (2006b) Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci 11:247–253 Williamson B, Tudzynski B, Tudzynski P, Van Kan JAL (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8:561–580 Wisniewski M, Biles C, Droby S, McLaughlin R, Wilson C, Chalutz E (1991) Mode of action of the postharvest biocontrol yeast. Pichia guilliermondii. I. Characterization of attachment to Botrytis cinerea. Physiol Mol Plant Pathol 39:245–258 Xu X, Qin G, Tian S (2008) Effect of microbial biocontrol agents on alleviating oxidative damage of peach fruit subjected to fungal pathogen. Int J Food Microbiol 126:153–158 Yao H, Tian S (2005) Effects of pre- and postharvest application of salicylic acid or methyl jasmonate on inducing disease resistance of sweet cherry fruit in storage. Postharvest Biol Technol 35:253–262

Chapter 13

Non-fungicidal Control of Botrytis Storage Rot in New Zealand Kiwifruit Through Pre- and Postharvest Crop Management M.A. Manning, H.A. Pak, and R.M. Beresford

Abstract  Storage rot of ‘Hayward’ kiwifruit, caused by Botrytis cinerea, became a serious problem in New Zealand during the 1980s, costing the then NZ$200 million industry about NZ$10 million per year. Fruit are healthy at harvest and become infected through the picking wound during postharvest handling. Disease symptoms develop after 4–8 weeks of storage at 0°C. Ethylene produced by a single rotting fruit in a tray can cause the whole tray to soften prematurely. Control attempts with pre-harvest fungicides led to resistance in B. cinerea to dicarboximide and benzimidazole fungicides. In the orchard, B. cinerea is visible mostly on flower petals early in the season, although it occurs on all plant surfaces, in understory weeds and in necrotic kiwifruit leaves. Research into storage rot risk factors revealed a relationship between rot incidence and the incidence of B. cinerea on necrotic leaves in the orchard around harvest time. Orchard populations of B. cinerea were quantified by assessing the incidence of necrotic leaf discs that were colonised by B. cinerea after incubation. From this, a predictive system has been developed that can identify high risk orchards. The botrytis problem has largely been solved by vine management that avoids dense leaf canopies. This prevents the build-up of the necrotic leaf tissue on which B. cinerea multiplies. It was also found that storage rot incidence can be greatly reduced by “curing” the fruit after harvest. This involves storing fruit for 48 h at ambient temperature before cooling. Storage rot incidence is also reduced by harvesting fruit when they are more mature (7–8°Bx), as riper fruit are much less susceptible to botrytis than immature fruit. The botrytis storage rot problem has thus been avoided by a combination of pre-harvest orchard management and postharvest handling practices, without the need for intervention with fungicides. M.A. Manning and R.M. Beresford (*) The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research, formely HotResearch), Mt Albert Research Centre, Private Bag Auckland, 1142, New Zealand e-mail: [email protected] H. Pak Avocado Industry Council, 13 267, Tauranga, 3141, New Zealand D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_13, © Springer Science + Business Media B.V. 2010

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13.1 Botrytis Storage Rot – Historical Perspective 13.1.1 Economic Impact Exports of New Zealand kiwifruit (Actinidia spp.) currently have an annual value of NZ$700–800 million from a production base of about 12,000 ha (HortResearch 2007). Although kiwifruit production suffers from relatively few plant diseases, there was a time from the late 1970s to the early 1990s when a storage rot disease (stem end rot), caused by Botrytis cinerea, led to important economic losses (Beever et al. 1984; Pennycook 1985). At that time New Zealand kiwifruit production consisted almost entirely of the A. deliciosa cultivar ‘Hayward’ (marketed as ZESPRI™ GREEN), with an export value of about NZ$200 million. Botrytis storage rot had a significant economic impact, particularly from the cost of inspecting and repacking affected lines of fruit, e.g. in 1991, Cheah and colleagues (Cheah et al. 1993) reported a cost to the industry of NZ$10 million per annum. Even a low incidence of botrytis rot affects the marketability of fruit because ethylene production from a single infected fruit in a tray can cause the whole tray to soften prematurely (Manning and Pak 1993). The tolerance for storage rot incidence, above which lines of fruit must be repacked, is one diseased fruit per three trays, or about 1%. From the mid-1990s until the present, the A. chinensis cultivar ‘Hort16A’ (marketed as ZESPRI™ GOLD), which is not prone to botrytis storage rot, has been increasing in importance, with 20 million tray equivalents produced in 2006/07. However, ZESPRI™ GREEN, which is susceptible to botrytis storage rot, is still the main kiwifruit product exported from New Zealand, with 60 million tray equivalents in 2006/07.

13.1.2 Etiology When botrytis storage rot was first recorded as a problem in 1978 (Beever et al. 1984) the etiology and epidemiology of the disease were not understood. Fruit that developed storage rot were symptomless at harvest and the first sign of disease appeared after about 4 weeks of storage at 0°C (Pennycook 1985; Manning et al. 1995). It was initially supposed, by analogy with the etiology of botrytis fruit rot in strawberry, that infection of the fruit occurred at or just after flowering, from flower parts colonised by B. cinerea that adhered to the fruit. Infection that supposedly occurred around flowering was believed to remain latent during the fruit development period, with symptoms appearing only during cool storage (Pennycook 1985). However, field observations and early epidemiological studies could not show consistent relationships between flower botrytis and storage rot incidence. It was subsequently determined that infection occurs through the picking wound during harvest, as a result of B. cinerea spores (conidia) deposited at the picking wound

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during orchard handling, packing and packhouse grading operations (Pennycook 1985). The conidia germinate in the uppermost ruptured cell layers of the picking wound and the germ tubes grow rapidly into the fruit’s vascular tissues (Hallett and Sharrock 1993).

13.1.3 Attempts at Control Early attempts to control botrytis storage rot used fungicides applied in the orchard before harvest. Although reductions in rot incidence could be demonstrated experimentally (Pennycook 1988), fungicides were only partly effective (Beever et  al. 1984; Pyke et al. 1994). When the etiology of the disease was understood, it was realised that field-applied fungicides only indirectly affect botrytis storage rot by reducing inoculum in the orchard. They cannot directly prevent infection of the picking wound. The use of dicarboximide and benzimidazole fungicides in the orchard led to the development of populations of B. cinerea in kiwifruit orchards that were resistant to both these groups of fungicides (Manning and Pak 1995). Although postharvest treatment of fruit with fungicides showed some ability to reduce storage rot incidence (Pennycook 1985; Pyke et al. 1994), this was never adopted as a commercial practice because the New Zealand kiwifruit industry did not wish to market fruit treated with postharvest fungicides. Postharvest hot air treatment and hot water treatment were also investigated experimentally (Cheah et  al. 1993). Hot air treatment at temperatures ranging from 38–60°C for times ranging from 2–40 min gave no reduction in botrytis storage rot. Although hot water treatment at 46–48°C for 8–15 min gave a significant reduction in storage rot without apparent detrimental effects to the fruit, this approach was not adopted by the kiwifruit industry. The postharvest systems used by the kiwifruit industry were designed for handling dry fruit and hot water treatment was not compatible with this.

13.2 Epidemiology of Botrytis cinerea in Kiwifruit Orchards 13.2.1 Introduction In order to resolve the botrytis storage rot problem, new control strategies were required that did not depend on pre- or postharvest fungicides, nor on other control methods that were incompatible with the fruit handling and marketing requirements of the kiwifruit industry. Epidemiological information about B. cinerea in orchards was required, to determine whether orchard management practices could be manipulated in any way to lessen the risk of storage rot. Although it was understood as early as 1985 that the picking wound was the infection site by which B. cinerea entered the fruit (Pennycook 1985), it was not

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known where the sources of inoculum for infection were. B. cinerea had been found on kiwifruit flower parts, necrotic leaves, wind damaged canes, cane prunings on the ground, fallen fruit and in understory weeds. However, there was no knowledge about how B. cinerea populations from these sources change during the season, particularly in relation to harvest date and the likelihood of infection of the picking wound. A series of studies to resolve the epidemiology of botrytis storage rot was commissioned in the early 1990s by the then New Zealand Kiwifruit Marketing Board, which subsequently became the kiwifruit marketing company, ZESPRI™ Group. Considerable government-funded research into botrytis storage rot also occurred during the 1980s and early 1990s, which at its peak, involved up to 11 plant pathologists. In 1992 the two government departments carrying out botrytis storage rot research, Plant Diseases Division of the Department of Scientific and Industrial Research and MAF-Technology of the Ministry of Agriculture and Fisheries, were dis-established and were replaced by the Crown Research Institute, HortResearch. From 1990 onwards government funding came from a single contestable pool administered by the Foundation for Research Science and Technology. These changes resulted in a substantial shift in research funding on botrytis storage rot from mostly government to mostly industry and they were accompanied by an increased clarity of purpose to solve the storage rot problem. The botrytis epidemiology research from the 1990s was detailed in kiwifruit industry reports and an overview of the methodologies and key outcomes from these studies is summarised below.

13.2.2 Methods Populations of B. cinerea in the leaf canopy of ‘Hayward’ kiwifruit orchards, vine canopy characteristics and factors affecting infection of fruit through picking wounds at harvest were studied in commercial orchards in South Auckland and the Bay of Plenty in 1992 and 1993. These studies were made during the late fruit development period (April–May, just prior to commercial harvest) in orchards with high or low risk of botrytis storage rot, as determined by the history of storage rot incidence over the previous 4 years. In each region the high and low risk orchard blocks were approximately 1 ha in area, were within 10 km of each other and all used pergola training systems. 13.2.2.1 Manipulating Vine Canopy Density in Orchard Plots To determine how vine canopy management influenced B. cinerea populations, two canopy density treatments were imposed, each in ten replicated single vine plots in each orchard. Canopy density was manipulated with different pruning strategies, whereby vines in the low density treatment were pruned to 2.4 canes/m in winter

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and received a standard commercial summer pruning regime, whereas vines in the high density treatment were pruned to 3.3 canes/m in winter and received no summer pruning. Canopy density was measured weekly in each plot using 0.5 × 0.5 m wire quadrats at four locations per plot. Total number of leaves, total leaf area, mean leaf area index (LAI) and percentage of leaf tissue that was necrotic were determined. Total and necrotic area was measured with a LICOR leaf area meter.

13.2.2.2 Measuring B. cinerea on Leaves in the Orchard The amount of B. cinerea colonisation of necrotic leaf tissue was determined by weekly sampling of 40 discs (12 mm diameter) from necrotic leaf lesions on otherwise healthy leaves in each plot. After collection, discs were incubated at room temperature under moist conditions for 16–24 h and the degree of B. cinerea colonisation was determined as the proportion of discs with B. cinerea conidiophores.

13.2.2.3 Effect of Fruit Handling Method at Harvest on Infection by B. cinerea To test the hypothesis that infection of the fruit resulted from the transfer of B. cinerea conidia from the fruit surface to the picking wound, and to determine whether postharvest fruit handling could influence the amount of botrytis storage rot, three different methods of handling fruit after picking were investigated in 1992 and 1993: (1) direct picking into trays, (2) picking into bags followed by transfer to trays, (3) picking into bags, then jostling the fruit in the bags followed by transfer to trays. Thirty trays of fruit (33 fruit/tray) were subjected to each treatment. Fruit were assessed for incidence of botrytis stem end rot after 10 weeks of storage at 0°C in 1992 and after 20 weeks in 1993. These fruit came from an orchard with a history of high botrytis storage rot incidence.

13.2.3 Results 13.2.3.1 Necrotic Leaf Tissue in Relation to Vine Canopy Density Quadrat sampling showed that the mean number of leaves with necrosis and the percentage of leaf area that was necrotic increased markedly during the month leading up to commercial harvest (early May) in both years of the study. Data for necrotic leaf area from the high risk block in South Auckland in 1992 are shown in Fig.  13.1. Necrotic leaf area continued to increase between harvest and leaf fall (data not shown).

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Mean necrotic leaf area (%)

12

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8 Y = 1.5428 * Ln(X) + 5.9096 R2 = 0.98

6

11/05 / 1992

6/05 / 1992

1/05 / 1992

26/04 / 1992

21/04 / 1992

16/04 / 1992

11/04 / 1992

6/04 / 1992

4

Fig. 13.1  Increase in the mean percentage of necrotic leaf area per quadrat with time in a high risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992. Values are the means of 10 single vine plots (4 quadrats per plot). The X-variate for the fitted line is days from 7 April 1992

The development of necrosis appeared to be associated with leaf senescence arising from shading and ageing of leaves within the canopy. Although a substantial proportion of the necrotic leaf tissue eventually became colonised by B. cinerea, it is believed that B. cinerea was a secondary invader and not the primary cause of the leaf necrosis. The amount of leaf necrosis was greater in plots with greater canopy density, as measured by LAI. For measurements made on 13 May 1992, there was an exponential increase in both number of dead leaves and necrotic leaf area with increasing LAI (Fig. 13.2). 13.2.3.2 B. cinerea Populations on Necrotic Leaves B. cinerea populations on necrotic leaf tissue in the orchard increased with time during the weeks leading up to harvest, as shown by the increasing incidence of necrotic leaf discs that were colonised by B. cinerea (Fig. 13.3). However, the amount of necrotic leaf tissue alone was not a good predictor of the B. cinerea population, because necrotic tissue may or may not be colonised. The total size of the B. cinerea population (percentage of leaf canopy area colonised per plot) was estimated as the product of the proportion of leaf discs colonised by B. cinerea and the percentage of leaf canopy area per quadrat that was necrotic. The total B. cinerea population increased exponentially in the period leading up to harvest in both high and low risk orchards, although it occurred at a greater rate in high

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a No. necrotic leaves

6 Y = .56 + exp(5.54 * X - 15.90) 4

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Leaf area index Fig. 13.2  Mean number of necrotic leaves (a) and mean necrotic leaf area (b) per quadrat in relation to leaf area index, for the high risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992

risk orchards (Fig. 13.4). This showed that the botrytis storage rot history from the high and low risk orchards was strongly correlated with the pre-harvest B. cinerea populations. A strong relationship was found between the B. cinerea population in the orchard, measured using necrotic leaf discs, and storage rot incidence (Fig. 13.5). This relationship was robust for leaf disc samples taken at various times, including the average B. cinerea incidence for six weekly samples prior to harvest (Fig. 13.5), a single sample at harvest (data not shown) or the average of four samples during the week after harvest (data not shown). Any of these explained >70% of the variation in storage rot incidence.

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Fig. 13.3  Increase over time in the incidence of leaf discs colonised by Botrytis cinerea in the high risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992. The X-variate for the fitted line is days from 7 April 1992

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Fig. 13.4  Increase in percentage of leaf canopy area colonised by Botrytis cinerea in high and low risk ‘Hayward’ kiwifruit orchards at South Auckland in 1993. The X-variate for the fitted lines is days from 15 March 1993

Incidence of botrytis storage rot (%)

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Fig. 13.5  Incidence of botrytis storage rot in relation to incidence of Botrytis cinerea on leaf discs in the orchard, as the average of six samples taken weekly leading up to harvest in the high risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992

13.2.3.3 Effect of Fruit Handling Method at Harvest on Infection by B. cinerea Direct picking of fruit into trays resulted in lower storage rot incidence in both 1992 and 1993 than treatments that involved further handling in picking bags (Table 13.1). Jostling of fruit resulted in the greatest disease incidence, showing that further handling increases the opportunity for transfer of inoculum to the picking wound. Although commercial grading of fruit was not included as a treatment, it is reasonable to assume that the additional handling would further increase the opportunity for transfer of inoculum to the picking wound.

Table 13.1  Incidence of botrytis rot in cool stored ‘Hayward’ kiwifruit in 1992 and 1993 in relation to three fruit handling treatments. After picking, fruit were either placed directly into trays, or into picking bags with and without jostling and then into trays. In 1992 the fruit were assessed after 10 weeks at 0°C and in 1993, after 20 weeks at 0°C Botrytis storage rot Botrytis storage rot Fruit handling treatment incidence (%) 1992 trial incidence (%) 1993 trial 1.6 a Placed into trays directly 7.0 a a Placed into picking bags 15.5 b 4.4 ab Placed into picking bags then jostled 22.1 c 7.2 b a

Means within a column followed by the same letter are not significantly different (LSD0.05).

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13.2.4 Discussion These studies showed that the ‘Hayward’ kiwifruit that are most at risk from botrytis storage rot are those within dense kiwifruit vine canopies, where necrotic leaf tissue is colonised to a high degree by B. cinerea. Fruit that are least at risk are those within open canopies that are exposed to low B. cinerea inoculum levels. The most likely source of inoculum for botrytis storage rot is from populations of B. cinerea that increase on dead leaf tissue during the period leading up to harvest. Leaf necrosis within the vine canopy increases during this period as a result of ageing and shading of leaves. The denser the canopy, the greater the rate at which necrosis increases. Although the amount of necrotic leaf tissue determines the potential B. cinerea population, this tissue may or may not become colonised by B. cinerea. Colonisation presumably depends on suitable environmental conditions for growth and spread of the fungus, with more dense leaf canopies likely to provide a more favourable environment. It appears that B. cinerea is a secondary invader of necrotic tissue and not the primary cause of the necrosis. The leaf disc method was developed to quantify the degree of colonisation of necrotic leaf tissue and it was subsequently incorporated into a prediction system for botrytis storage rot (see below).

13.3 Prediction and Management of Botrytis Storage Rot 13.3.1 Prediction Systems The understanding of botrytis storage rot resulting from epidemiological studies underpinned the development of prediction systems designed to identify which lines of fruit were at greatest risk. Several postharvest prediction methods were investigated, including protocols for withdrawing sample trays from lines of fruit and accelerating the development of rots in the sample at warm temperatures. Another method used agar plugs dabbed on to the picking wounds to assess B. cinerea presence at the infection site. For various practical reasons, these methods were not taken up by the kiwifruit industry. The method that was adopted, and is still used, by the kiwifruit industry is the assessment of leaf discs, as described above, to assess pre-harvest B. cinerea inoculum in the orchard (Manning and Pak 1993). Relationships of the sort shown in Fig. 13.5 allow prediction of storage rot risk in individual lines of fruit. This information can be used to make management decisions about the fruit inventory from a particular harvest season. For example, it is possible to identify which lines of fruit should be inspected for rots before shipping or which lines are less suitable for long term controlled atmosphere storage.

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13.3.2 Vine Canopy Management to Lessen Disease Risk The demonstration of relationships between leaf canopy density, leaf necrosis and botrytis storage rot risk led to the use of canopy density manipulation as the primary means to control storage rot. Canopy density is manipulated through winter and summer pruning strategies. However, reduction in LAI for storage rot control must be balanced against the need to retain adequate LAI to sustain economic kiwifruit yield, as discussed by Buwalda and colleagues (Buwalda et  al. 1992). The mechanism by which canopy density influences B. cinerea colonisation is presumably through restricted air movement, reduced radiation and greater humidity favouring growth and sporulation of B. cinerea.

13.3.3 Management of Fruit Susceptibility to Disease 13.3.3.1 Effect of Harvest Date on Fruit Susceptibility During the 1980s there were anecdotal indications that harvest and postharvest management practices could influence the amount of botrytis storage rot. Harvest date, was one factor that came into focus, particularly the maturity at which kiwifruit were harvested. Until the early 1990s it was preferred to harvest fruit as early as possible, to exploit market opportunities and to avoid the risk of damage from early autumn frosts. Harvesting at a fruit soluble solids concentration of 6.2°Bx was recommended although there was a trend towards earlier harvesting, at about 6.0°Bx. An analysis of field data collected at eight orchards over 4 years showed that storage rot incidence tended to be greater in fruit harvested at lower soluble solids concentrations (Fig. 13.6). It was clear that fruit harvested later, at 7–8°Bx,

Botrytis storage rot incidence (%)

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1993 1994 1995 1996

50 40 30 20 10 0 5

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Soluble solids conc. at harvest (8Brix)

Fig. 13.6  Incidence of botrytis storage rot in kiwifruit after about 13 weeks of storage at 0°C in relation to the mean concentration of soluble solids in fruit at harvest. Data were collected from eight commercial ‘Hayward’ kiwifruit orchards (two orchards per year) in South Auckland and the Bay of Plenty in New Zealand over 4 years

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were much less susceptible to botrytis than fruit harvested earlier. Thus by harvesting fruit at a higher soluble solids concentration, it has been possible to lessen the risk of botrytis storage rot greatly.

13.3.3.2 Curing of the Picking Wound

Botrytis storage rot incidence (%)

Botrytis storage rot incidence is greatly reduced when the picking wound is “cured” by storing fruit for 48 h at ambient temperature before cooling. This phenomenon was first reported anecdotally during the late 1980s and was defined experimentally by Pennycook and Manning (1992). They inoculated picking wounds of fruit with B. cinerea conidia and held fruit at c. 14°C for different times before placing in cool storage at 0°C. They demonstrated a decrease in the susceptibility of fruit to botrytis storage rot when fruit were held at 14°C over a 7-day period (Fig. 13.7). Lallu et al. (1997) subsequently showed that the majority of curing occurs within the first 48 h after picking. They also showed that excessive curing times can lead to increased incidence of fruit softening, although a further delay of up to 48 h between packing and pre-cooling was not detrimental. The efficacy of curing against botrytis storage rot appeared to be enhanced by a slower rate of cooling when the fruit were brought down to storage temperature. The mechanism by which curing works is not well understood, although it appears to be related to loss of water from fruit. Curing does not occur if there is no weight loss during the curing period, and it is more effective at low humidity and where there is air movement around fruit. It does not appear to be caused merely by suberisation of the picking wound.

30 25 20 15 10 5 0 0

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Delay before start of cool storage (days)

Fig. 13.7  Incidence of botrytis storage rot in ‘Hayward’ kiwifruit after 13 weeks of storage at 0°C in relation to delay before cooling. Fruit were kept at ambient temperature (14–18°C) for various times after picking, inoculated with Botrytis cinerea conidia, then placed into storage at 0°C

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13.4 Conclusions Botrytis storage rot was a serious problem affecting production and marketing of ‘Hayward’ kiwifruit in New Zealand from the late 1970s to the early 1990s. Pre-harvest fungicide sprays used in early attempts at control failed because they did not protect the picking wound against infection and because fungicide resistance developed in B. cinerea field populations. The kiwifruit industry rejected use of postharvest fungicides to prevent infection because it did not wish to market fungicide-treated fruit. A concerted research effort, investigating interactions between the biology of B. cinerea and the physiology of kiwifruit vines and fruit, led to an understanding of pre- and postharvest factors that cause botrytis storage rot. B. cinerea is ubiquitous in kiwifruit orchards, growing mainly on dead leaf tissue in the vine canopy. Spores accumulate on the fruit during the growing season and are transferred to the picking wound at harvest, where they infect. More B. cinerea inoculum occurs in dense vine canopies because restricted light penetration leads to increased leaf necrosis. There is also less air movement in dense canopies and this provides humid conditions favourable for production of B. cinerea inoculum. High-inoculum orchards can be identified using the leaf disc method and this has been incorporated into a prediction system to determine lines of fruit that are at risk of storage rot. However, good vine canopy management means the prediction system is not often required. The successful control of this disease without fungicides has been achieved by vine canopy management that avoids dense leaf canopies. Harvesting fruit at a higher sugar content minimises fruit susceptibility and delayed postharvest cooling to “cure” the fruit also reduces their susceptibility. These research findings have been incorporated into the botrytis management programme in the industry’s manual on integrated pest management. This research has ultimately led to the current situation where botrytis storage rot is a relatively minor problem for the New Zealand kiwifruit industry. Acknowledgments  Thanks are due to Zespri™ Group for permission to summarise and present the data contained in kiwifruit industry reports and to the Foundation for Research Science and Technology for funding research presented in this chapter.

References Beever DJ, McGrath HJW, Clarke DL, Todd M (1984) Field application and residues of fungicides for control of botrytis storage rot of kiwifruit. NZ J Exp Agric 12:339–346 Buwalda JG, Meekings JS, Curtis JP (1992) Where in the canopy is photosynthesis highest? NZ Kiwifruit 95 (November):14–15 Cheah LH, Irving DE, Hunt AW (1993) Postharvest heat treatments for botrytis control. NZ Kiwifruit 96 (February):26 Hallett I, Sharrock K (1993) Penetrating the picking scar’s defences. NZ Kiwifruit 96 (February):7–9

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HortResearch 2007 Fresh facts New Zealand Horticulture (2007) HortResearch – the Horticulture and Food Research Institute of New Zealand Ltd, Private Bag 92169 Auckland (www. hortresearch.co.nz), pp 33 Lallu N, Manning M, Pak H (1997) Best curing practices for orchard and packhouse. NZ Kiwifruit J 120 (March/April):35–36 Manning MA, Pak HA (1993) Production of botrytis rots. NZ Kiwifruit 96 (February):22–23 Manning MA, Pak HA (1995) Botrytis storage rot of kiwifruit: efficacy of pre-harvest sprays in orchards with dicarboximide-resistant botrytis populations. Proceedings of the 48th New Zealand Plant Protection Conference, pp 22–26 Manning MA, Pak HA, Pennycook SR (1995) Timing condition checking to catch Botrytis rots. NZ Kiwifruit 107 (February/March):24–25 Pennycook SR (1985) Fungal fruit rots of Actinidia deliciosa (kiwifruit). NZ J Exp Agric 13:289–299 Pennycook SR (1988) Some straight talking on botrytis control. NZ Kiwifruit 43 (February):15 Pennycook SR, Manning MA (1992) Picking wound curing to reduce botrytis storage rot of kiwifruit. NZ J Crop Hortic Sci 20:357–360 Pyke NB, Manktelow DG, Elmer PAG, Tate KG (1994) Postharvest dipping of kiwifruit in iprodione to control stem-end rot caused by Botrytis cinerea. NZ J Crop Hortic Sci 22:81–86

Chapter 14

The Peach Story Paloma Melgarejo, Antonieta De Cal, Inmaculada Larena, Iray Gell, and Belen Guijarro

Abstract  One of the most important postharvest disease of peaches is brown rot caused by different species of the fungus Monilinia. Anamorphs dominate as inoculum sources especially in the Mediterranean areas of Europe, where brown rot in peaches is caused by M. laxa and M. fructigena. A third species, M. fructicola causes brown rot in other parts of the world and is included in the A2 list of quarantine organisms for Europe (organisms present in the EPPO region, but contained, under official control) because its broad dissemination in Europe would be devastating especially for peach and nectarine. These fungi overwinter and produce mycelium in fruit mummies and infected wood. This produces conidia under favorable conditions or from stromata that produce ascospores in the case of M. fructicola. Fruit infection by conidia of Monilinia spp. can occur secondarily from any infected tissue in which the moisture content is sufficient for sporulation. When the microclimate is unfavorable, infections may remain latent until conditions favor disease expression, which finally leads to fruit rot. Correlation between the incidence of rotting and latent infection caused by Monilinia spp. has been reported. Management of orchards focused to decrease postharvest brown rot will be treated here. Detection and identification methods, inoculum sources, and epidemiological factors affecting latent fruit infections and postharvest brown rot will be described, together with the models available to predict disease risk. Different integrated control strategies are presented.

14.1 Introduction Peaches are commodities produced in template areas of the world. Production in Europe is mainly restricted to Mediterranean countries such as Italy, Spain, Greece and France. In Spain crop surface is increasing in the last years being approx. 12,000 ha in 2007, with a production of approx. 110,000.000 kg. P. Melgarejo (*), A. De Cal, I. Larena, I. Gell, and B. Guijarro Department of Plant Protection, Instituto Nacional de Investigación y Tecnología Agrariay Alimentaria, Crtra. De La Coruña km. 7, 28040, Madrid, Spain e-mail: [email protected] D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_14, © Springer Science + Business Media B.V. 2010

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Several pests and diseases may cause severe losses in peach orchards. Brown rot caused by Monilinia spp. is a serious disease in Mediterranean areas. Direct yield losses result from infection of flowers (flower and twig blight) and from fruit rot at preharvest, harvest and postharvest. Three Monilinia species may cause brown rot of peaches. M. fructigena and M. laxa have been extensively reported in Europe. Monilinia fructicola occurs in North and South America, Japan and Australia (EPPO 2007) and has been recently introduced in France (Lichou et  al. 2002), Austria (NPPO of Austria 2002) and Spain (Petroczy and Palkovics 2006). This species, however is listed by EPPO as a quarantine pest within the European Union (EPPO 2007) because a broad dissemination of this species in Europe would be devastating especially for peach, nectarine and apricot. In addition this is the first time in which the three species coexist in the same area.

14.2 Management of Brown Rot The first step to manage brown rot is the detection and identification of the species of Monilinia causing the disease. This issue is not only important for quarantine purposes, such in the case of M. fructicola to avoid new introductions in areas free of this fungus, but it is also essential for managing brown rot caused by all three Monilinia spp. Currently, identification of Monilinia spp. is based on cultural and morphological characteristics and fungal isolation from the plant material (De Cal and Melgarejo 1999), and molecular DNA-based methods. We designed a detection protocol that combined a set of universal primers [(IMon3.1 (GCTCGCCAGAGAATAAYY) and IMon3.2 (AGACTCAATACCAAGCTGT)] and the inclusion of the plasmid pGMON as an Internal Control for diagnosis of all Monilinia spp, and three sets of primers to discriminate the three species of Monilinia [ILaxaS (TGAGCACGAGTGAATGTATAG) and ILaxaAS (TGAGCACGAGGGCATATC) for M. laxa, IGenaS (TGCTCTGCCCGTACCCAG) and IGenaAS (GGATTTATTGTGATGTAGTTTCG) for M. fructigena, and IColaS (GAGACGCACACAGAGTCAG) and IColaAS (GAGACGCACATAGCATTGG) for M. fructicola] (Gell et al. 2007a). In Spain control of brown rot is managed at pre- and postharvest. Pre-harvest strategies include sanitary measures (removing of mummies and diseases twigs and branches) and fungicidal treatments (at flowering and pre-harvest), and postharvest con trol is base on a careful management of fruit, a quick coolness after harvest and store at 0°C, and, in some cases, a washing with chloride solutions. Biological control is also a new tool to be applied. We have developed two biological control agents, Epicoccum nigrum (strain 282, EPI282) and Penicillium frequentans (strain 919, PF919) from the resident mycoflora of peach twigs (Melgarejo et  al. 1985). Application of conidia and

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mycelium of these fungal antagonists to peach tress reduced twig blight caused by M. laxa (De Cal et  al. 1990; Madrigal et  al. 1994). We have also developed an efficient mass production method of conidia of these fungi by solid fermentation (De Cal et al. 2002; Larena et al. 2004). Different conidia formulations of each biocontrol agents with enhanced adherence to fruit, dispersal in water or/and stability were produced and tested for controlling brown rot (Larena et al. 2005; Guijarro et al. 2007). Some of them had also and improved efficacy in controlling the disease. In the following several examples are shown of our biological control research.

14.3 Biological Control Seven field experiments were carried out in peach orchards located in Spain, Italy and France in 2001 and 2002 to develop an effective and practical method of controlling brown rot disease caused by Monilinia spp. by pre-harvest applications of EPI282 treatments in the framework of BIOPOSTHARVEST EU-Project (Larena et  al. 2005). Fresh or formulated (106–7 conidia mL−1) of EPI282 needed to be applied twice both at bloom and pre-harvest to reduce postharvest brown rot (Table 14.1). Chemical fungicides reduced disease in French and Italian trials but not in a Spanish trial (Table 14.1). Integrated control (biological and chemical) was efficient in controlling the pathogens (Table 14.1). Postharvest treatments with EPI282 were also tested in Italy on natural and artificial infections to nectarine over 3 years. EPI282, as fresh or formulated cells, at a concentration of 108 conidia mL−1, were effective, significantly reducing the incidence of brown rot compared to control, both under artificial and natural infection, from 43 to 100% (Mari et al. 2007) (Fig. 14.1). Four wettable powder formulations of PF919 conidia with measurable viability of 1 year and an improved adherence to peach surfaces were applied to fruit either as postharvest treatments or before harvest in field experiments to peach trees (Guijarro et al. 2007). In the case of postharvest treatments to fruit, reductions of brown rot were obtained with all PF919 formulations. Treatments applied before harvest were tested in six field experiments in peach orchards in Spain. PF919 formulations significantly reduced the inoculum density of the pathogen in five trials out of the six tested, better than a chemical fungicide that only showed a reduction of the pathogen conidia in two of the six trials (Table 14.2). All these trials showed that both, EPI212 and PF919, are interesting potential biocontrol agents against brown rot of peaches, but that it is necessary to improve their efficacy. Different approaches can be used such as to getting better formulations, using integrated strategies (i.e. pre-harvest treatments and physicochemical postharvest treatments). But, a better knowledge of the disease epidemiology is undoubly important. We have begun a project to study the epidemiological basis to

a

Note: Data are the mean of four replicates ± standard error of the mean. CB = biological treatments; CB(106 × 4) = biological treatments applied four times at 106 conidia mL−1; CB(106 × 2) = biological treatments applied twice at 106 conidia mL−1; NT = untreated; CBF = formulated cells of EPI282; CB(107 × 4) = biological treatments applied four times at 107 conidia mL−1; CQ = chemical treatments; CI = Integrated treatments

Table 14.1  Percentage of decayed fruit by Monilinia spp. in trials carried out in different countries during 2001 and 2002 after different treatments Spain Italy France SP1 2001 IT1 2001 IT2 2001 IT3 2002 FR1 2001 FR2 2001 FR3 2002 Treatmenta 46 ± 10 39 ± 3 37 ± 4 74 ± 8 64 ± 2 68 ± 3 61 ± 8 T1 (CB106 × 4) T2 (CB107 × 4) – – – 72 ± 6 – – – T3 (CBF106 × 4) – – – 70 ± 3 – – – T4 (CB106 × 2) 77 ± 8 59 ± 3 54 ± 15 – – – – T5 (CB106 × 2) 72 ± 11 61 ± 4 57 ± 5 – – – – T6 (CQ) 59 ± 10 29 ± 2 28 ± 6 69 ± 2 34 ± 2 44 ± 6 36 ± 5 T7 (CI1) – 42 ± 1 40 ± 7 66 ± 3 – – 70 ± 12 T8 (CI2) – 28 ± 1 29 ± 2 57 ± 7 – – 32 ± 7 T9 (NT) 80 ± 6 64 ± 2 58 ± 11 84 ± 4 67 ± 4 62 ± 5 74 ± 8

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a

b

80

Infected fruits (%)

201

a

a

a

a

60

a* a

b

40

b

b b b

20

a

a a nd

0 2001

2002

Control

2003

Fresh cells

2001

Dry cells

2002

b

nd

b

2003

FOR4

Fig.  14.1  Effects of postharvest treatments with Epicoccum nigrum fresh cells (FC), dry cells (DC) or formulation FOR1 at different concentrations on artificially (a) and naturally (b) Monilinia laxa infected nectarine. FC (at a concentration of 106 conidia mL−1) were applied in 2001; FC (107 conidia mL−1) or DC (106 conidia mL−1) in 2002, and FC or FOR1 (108 conidia mL−1) in 2003. nd: not determined. *Different letters, within the same year, show a significant difference (P < 0.05) according LSD Test

control postharvest brown rot of peaches caused by Monilinia spp. Different aspects have been already studied.

14.4 Epidemiology of Brown Rot in Spanish Conditions The sources of primary inoculum were studied in nine commercial orchards of the Ebro Valley region of Spain during 6 years (2003–2005). Mummified fruit, twigs and pits have been identified as plant organs carry the pathogen from year to year in peach grown Spanish orchards. However, no relationships between any of these sources and the numbers of conidia on the fruit surface, or incidence of latent infection, or brown rot were found (Gell et al. 2008). To evaluate the effect of conidial density of Monilinia spp on fruit surface on the incidence of latent infection and brown rot in peaches eleven field surveys were performed in commercial orchards located in Cataluña, Spain, over four growing seasons from 2002 to 2005 (Gell et  al. 2009). There was a significantly positive relationship (r = 0.69) between the numbers of conidia of Monilinia spp. on the fruit surface and the incidence of latent infections caused by Monilinia spp. in stone fruit. The effect of environmental temperature (T), intensity of solar radiation (SR),

Note: Data are the mean of four replicates with five fruit per replicate ± standard error of the mean.

Table 14.2  Effect of PF909 formulations on population of Monilinia spp. (number of conidia per fruit) in different orchards where chemical and biological treatments were applied along crop during 2003 to 2005 2003 2004 2005 ALF03 SU03 ALF04 SU04 ALF05 SU05 Treatmenta FOR3 42,969 ± 5,750 0.0 ± 0.0 2,930 ± 1,870 4,883 ± 1,224 – – FOR4 59,570 ± 6,046 6,835 ± 3,335 – – – – FOR7 – – 4,883 ± 976 – – – FOR8 – – 0.0 ± 0.0 1,953 ± 738 4,400 ± 1,871 488 ± 488 Chemical 39,062 ± 5,289 2,930 ± 1,870 4,639 ± 2,008 2,930 ± 1,224 4,394 ± 2,497 5,859 ± 1,476 Untreated 48,828 ± 6,478 6,836 ± 4,331 24,170 ± 7,191 7,812 ± 1,808 23,926 ± 6,196 12,207 ± 3,417

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rainfall (R) and wind speed (WS) on the area under the number of conidia of Monilinia spp. curve (AUncC) on peach surfaces was analysed using a multiple regression model. The results of regression analysis revealed that T, SR, R, and WS could account for 99% of area of the AUncC on peach surfaces, following the equation: AUncC (T , WS , SR, R ) = -1.9x10 7 - 4.4x10 5 xSRxRxWS + 5.6x10 4 xTxSRxR + 2.5x10 5 xWS 2 xSR - 9.8x10 4 WS 2 xT



(1)

(2.0 × 106) (4.9 × 104) (5.6 × 104) (3.9 × 104) (3.3 × 104) (R = 0.986) The main finding from this study is that in order to reduce the incidence of latent infection and brown rot it is essential not only to remove the sources of primary inoculum but to reduce also the number of Monilinia spp. conidia on the fruit surface. Furthermore, the sources of airborne conidia of Monilinia spp. that are deposited on fruit surfaces should be taken into consideration in disease management programmes in Spain. The correlation between latent infections and postharvest brown rot was demonstrated in cherries infected with M. laxa and M. fructigena (Xu et al. 2007), and plums and nectarines infected with M. fructicola (Emery et  al. 2000; Luo and Michailides 2001). Five field experiments were performed in commercial orchards located in Cataluña (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of postharvest brown rot in Spanish conditions (Gell et al. 2008). No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. M. laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of postharvest brown rot: 2



[Arcsine(z) = - 0.39 + 3.48 Arcsine(y)]

(2)

(3.77) (0.45) The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of postharvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection (y) in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection (y) by the equation:

Arcsine (y) = 12.53 – 0.35WT + 0.002T2W2 + 0.00019T3W (4.138) (0.081) (0.0004)

(0.00006)

(3)

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24 latent 100

18

50 12

0 25

W

20 6

15 T

10 5 0

0

Fig. 14.2  Response surfaces predicting the incidence of latent infection (%) in peach and nectarine fruit at a range of durations of wetness (W) and temperatures (T). The surface was generated using Eq. (3)

The model predicts no latent infections when T < 8°C, and >22 h wetness required when T = 8°C but only 5 h at 25°C necessary for latent infection to occur (Fig. 14.2). The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions was also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 104 conidia of M. laxa per ml for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions. Present results demonstrate that latent infection should be taken into consideration in disease management programmes in Spain. Although brown rot may not be severe at harvest, it could develop later because of the high incidence of latent infection. A control strategy should be based on estimation of the risk of latent infection, assessed on the basis of the effects of temperature and duration of wetness. The quantitative relationships of latent infection with temperature and duration of wetness could be used to develop a risk assessment and disease prediction system for brown rot. Finally, a study of the genetic diversity of M. laxa populations in peach orchards in Spain was carried out using 144 RAPD markers (59 polymorphic and 85 monomorphic) on 21 isolates collected from several orchards (subpopulations), in various years and in various hosts (Gell et al. 2007b). The analysis of population structure

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Fig. 14.3  The UPGMA dendrogram showing the genetic similarity among isolates of Monilinia laxa sampled in different orchards in Spain, and analyzed by RAPD. The percentages below the branches are the frequencies with which a given branch appeared in 1,000 bootstrap replications. Bootstrap values below 50% are not displayed

revealed that genetic diversity within orchards (HS) accounted for 97% of the total genetic diversity (HT), while genetic diversity among orchards represented only 3%. The relative magnitude of gene differentiation between subpopulations (GST) and the estimate of the number of migrants per generation (Nm) averaged 0.032 and 15.1, respectively. The results obtained in dendrograms were in accordance with the gene diversity analysis (Fig.  14.3). Grouping of isolates in the dendrogram was independent of whether they came from the same or different orchards. There was no relationship between clustering among isolates from distinct years and hosts. Our analysis of M. laxa populations using RAPD markers revealed that most of the genetic diversity is present within subpopulations (orchards). The hypothesis of geographic subdivision, with orchards as subpopulation units, is based on the use

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of distinct management practices in different orchards. Continuous gene flow must occur between orchards, thereby preventing differentiation between subpopulations. Our results confirm that the spread of disease from one orchard to another occurs frequently. We estimated the number of migrants between orchards to be Nm = 15.1, indicating the importance of migration in preventing genetic differentiation. This flow of migrants has severe implications for the management of brown rot, in particular for the management of sanitation practices. Orchards having good sanitation practices which maintain them free or with low densities of inoculum can be infected by inoculum coming from other orchards. Disease control strategies should be designed at the local or regional scale because practices have implications on other orchards. Another important factor to be taken into account is the potential high risk of rapid spread of M. fructicola from one orchard to another. This species has recently been detected in Spain (Petroczy and Palkovics 2006). If M. laxa inoculum easily spread from orchard to orchard, it is assumed that the same will occur with M. fructicola. Once entry of a quarantine organism has been achieve in a new area, the pathogen will encounter favourable conditions for spread and establishment. The economic impact of establishment of M. fructicola in Spain, and hence in Europe was estimated as considerable (van Leeuwen et al. 2000). Direct fruit losses will increase and export markets will be affected. Cost of control will increase, and control measures might become less efficient because of the development of fungicide resistance. In addition, and as M. fructicola shares common hosts with M. laxa and M. fructigena, interactions between the different species may occur, even at the fruit or the flower scale. At the moment M. laxa is the prevalent species in orchards in Spain, but, after introduction and establishment of M. fructicola, this situation may change. M. fructicola grows faster and sporulates more abundantly than M. fructigena and M. laxa, having a better ability for dispersal (van Leeuwen and van Kesteren 1998; De Cal and Melgarejo 1999). Screening of fungicide resistance to benzimidazole and dicarboximide fungicides was made among populations of M. laxa in Spain. No isolate out of 114 tested was found resistant to either group of fungicides (Gell 2008). Acknowledgements  We thank Dr. Usall, Dr. Torres, N. Lamarca, and M. Mari for collaboration in experiments. This work was supported by projects AGL2002-4396-CO2, RTA2005-00077-CO2 from the Ministry of Science and Innovation (Spain), and QoL-PL-1999-01065 from the European Commission. We thank X. O. de Eribe and growers for their support and collaboration.

References De Cal A, Melgarejo P (1999) Effects of long-wave UV light on Monilinia growth and identification of species. Plant Dis 83:62–65 De Cal A, M-Sagasta E, Melgarejo P (1990) Biological control of peach twig blight (Monilinia laxa) with Penicillium frequentans. Plant Pathol 39:612–618

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De Cal A, Larena I, Guijarro B, Melgarejo P (2002) Solid state fermentation to produce conidia of Penicillium frequentans, a biocontrol agent against brown rot on stone fruits. Biocontrol Sci Tech 12:715–725 Emery KM, Michailides TJ, Scherm H (2000) Incidence of latent infection of immature peach fruit by Monilinia fructicola and relationship to brown rot in Georgia. Plant Dis 84:853–857 EPPO (2007) List of A2 pests regulated as quarantine pests in the EPPO region. OEPP/EPPO from http://www.eppo.org/QUARANTINE/listA2.htm Gell I (2008) Podredumbre parda del melocotonero (Monilinia spp.): detección, identificación de especies y contribución a la epidemiología de la enfermedad. Ph.D. Thesis, Polytechnic University of Madrid Gell I, Cubero J, Melgarejo P (2007a) Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit tress. J Appl Microbiol 103:2629–2637 Gell I, Larena I, Melgarejo P (2007b) Genetic diversity in Monilinia laxa populations in peach orchards in Spain. J Phytopathol 155:549–556 Gell I, De Cal A, Torres R, Usall J, Melgarejo P (2008) Relationship between the incidence of latent infections caused by Monilinia spp. and the incidence of brown rot of peach fruit: factors affecting latent infection. Eur J Plant Pathol 121:487–498 Gell I, De Cal A, Torres R, Usall J, Melgarejo P (2009) Conidial density of Monilinia spp. on peach surfaces in relation to the incident of latent infections and brown rot. Eur J Plant Pathol, 123:415–424 Guijarro B, Melgarejo P, Torres R, Lamarca N, Usall J, De Cal A (2007) Effects of different biological formulations of Penicillium frequentans on brown rot of peach and nectarine. Biol Control 42:86–96 Larena I, De Cal A, Melgarejo P (2004) Solid substrate production of Epicoccum nigrum conidia for biological control of brown rot on stone fruits. Int J Food Microbiol 94:161–167 Larena I, Torres R, De Cal A, Liñan M, Melgarejo P, Domenichini P, Bellini A, Mandrin JF, Lichou J, de Eribe X, Usall J (2005) Biological control of postharvest brown rot (Monilinia spp.) of peaches by field applications of Epicoccum nigrum. Biol Control 32:305–310 Lichou J, Mandrin JF, Breniaux D, Mercier V, Giauque P, Desbrus D, Blanc P, Belluau E (2002) Une nouvelle moniliose. Phytoma 547:22–25 Luo Y, Michailides TJ (2001) Factors affecting latent infection of prune fruit by Monilinia fructicola. Phytopathology 91:864–872 Madrigal C, Pascual S, Melgarejo P (1994) Biological control of peach twig blight (Monilinia laxa) with Epicoccum nigrum. Plant Pathology 43:554–561 Mari M, Torres R, Casalini L, Lamarca N, Mandrin JF, Lichou J, Larena I, De Cal A, Melgarejo P, Usall J (2007) Control of postharvest brown rot on nectarine by Epicoccum nigrum and physico-chemical treatments. J Sci Food Agric 87:1271–1277 Melgarejo P, Carrillo R, M-Sagasta E (1985) Mycoflora of peach twigs and flowers and its possible significance in biological control of Monilinia laxa. Trans Br Mycol Soc 85:313–317 NPPO of Austria (2002) Monilinia fructicola found in Austria. OEPP/EPPO 11:170 Petroczy M, Palkovics L (2006) First report of brown rot caused by Monilinia fructicola on imported peach in Hungary. Plant Dis 90:375 van Leeuwen GCM, van Kesteren HA (1998) Delineation of the three brown rot fungi of fruit crops (Monilinia spp.) on the basis of quantitative characteristics. Can J Bot 76:2041–2050 van Leeuwen G, Stein A, Holb I, Jeger M (2000) Yield loss in apple caused by Monilinia fructigena (Aderh. & Ruhl.) Honey, and spatio-temporal dynamics of disease development. Eur J Plant Pathol 196:519–528 Xu X-M, Bertone C, Berrie A (2007) Effects of wounding, fruit age and wetness duration on the development of cherry brown rot in the UK. Plant Pathol 56:114–119

Index

A Acibenzolar, 32–33, 122 Active defense, 18–19 Alternaria, 3–5, 32–35, 44, 46, 47, 72, 75, 80, 85, 86, 175 Alternaria alternata, 3, 8, 32–35, 46, 47, 80, 85, 175 Alternative methods, 58, 90, 108, 120, 121, 123, 127–129 Antagonists, 22, 90, 120, 138, 150, 173, 199 Anthracnose, 4, 5–9 Antifungal substances avocado preformed, 2, 9–10 induced, 2, 10 mango preformed, 2–9 Aspergillus, 49, 51, 172 B b-Aminobutyric acid (BABA), 34 Bioactive compounds, 120–122, 124 Biocontrol agents formulations, 99, 102, 120, 123, 129, 139, 149–166, 199, 201, 202 Biocontrol treatments, 90–94, 102, 140, 145, 158–162, 173, 178, 199, 200 Biological control, 32, 33, 35–37, 58, 90, 93, 94, 102, 120–121, 127–129, 137–145, 150, 171–180, 198–201 Biotrophic pathogens, 15, 18–19, 44–45, 52–53, 64 Botryodiplodia, 5, 9 Botryosphaeria dothidea, 72, 75, 85 Botrytis cinerea, 13–25, 47, 64, 72, 110, 123, 172, 184 C Candida sake, 90–96, 120, 123, 124, 139, 150–157, 159–162, 166, 172

Chitosan, 34, 122, 128 Cladosporium, 5, 32 Colletotrichum acutatum, 141 gloeosporioides, 4–10, 45–48, 52, 173 Control postharvest diseases, 31–37, 72, 86, 89–102, 137–145 D Decaying fruits, 32, 43–53, 90, 102, 107, 112, 140, 142 E Elicitors, 14, 17, 31–37, 52, 58, 122, 130, 174, 176 Epidemiological assessment, 69–87 Epidemiology, 69–87, 125, 184–192, 199, 201–206 Ethylene (ET), 10, 16, 19–23, 25, 52, 58–65, 80, 156, 174–176, 184 F Fruit resistance, 1–10, 31–37, 58, 86, 124, 125, 173, 175–180 Fruits avocado, 2, 9–10, 45–47, 139 citrus, 49, 57–65, 91, 110, 123, 139, 150, 172 mango, 2–9, 32, 34, 80, 110, 141 melons, 31–37, 46 peach, 32, 73, 75, 80, 86, 95, 99, 109, 125, 126, 138, 139, 172, 177, 178, 180, 197–206 pome, 70, 80, 91, 109–115, 120, 123, 124, 126, 138–142, 150, 151, 155 209

210 Fruits (cont.) stone, 70–75, 77, 80–85, 110, 111, 123, 125, 126, 138, 141, 142, 201 tomato, 13, 16–19, 21–25, 34, 46, 51, 52, 110, 111, 142 Fruit susceptibility, 123, 193–195 Fungicide residues, 14, 32, 109, 115 Fungicide resistance, 14, 32, 72, 85, 86, 112–116, 195, 206 Fusarium moniliforme, 85 Fusarium rot, 32–35, 85, 110–111 G Geotrichum, 32, 110, 172 Global regulation of genes, 57–65 I Induced resistance, 13–25, 31–37, 122, 125, 159, 172, 174–179 L Latent infection, 32, 70, 126, 140–141, 201 Low risk substances, 91–94 M Mango latex, 2–8 Mechanisms of resistance, 13–25, 32, 36, 37, 175, 179 Microbial antagonists, 90, 92, 120–121, 127, 141, 143, 150 Monilinia fructicola, 71–77, 81–86, 125, 126, 138, 141, 177, 178, 198, 203, 206 Monilinia laxa, 71–77, 81–83, 85, 95, 125–126, 198–199, 201, 203–206 Monitoring resistance, 84–86 Mucor rot, 32, 35 N Necrotrophic infection, 17, 20, 51–52 Necrotrophic pathogens, 14–15, 17–23, 25, 123, 173, 176, 177, 179 Non-fungicidal control, 140, 183–195 O Osmotic treatments, 97, 150, 156–158, 162–166

Index P Pantoea agglomerans, 90–92, 96–102, 124, 151, 156–158, 162–166 Pathogenicity, 20, 45, 47, 49, 51, 52, 175, 178–179 Penicillium Penicillium digitatum, 47, 49, 57–65, 90, 97–99, 101, 112, 123, 126, 127, 158, 172, 176, 179 Penicillium expansum, 47, 49–51, 91–93, 95, 96, 110, 115, 123–124, 145, 152, 155, 158, 172, 173, 175, 177–179 Penicillium italicum, 47, 49, 58, 90, 110, 123, 126, 127, 172 pH modulation acidifying fungi, 45, 47–51 alkalizing fungi, 45–47 Phoma, 9 Phomopsis, 9 Phyotalexins, 2, 15, 24, 172, 176 Plant hormones, 19–20, 25 Plant resistance, 14–16, 18, 20, 21, 25, 175 Polygalacturonase-inhibiting proteins (PGIP), 15–17 Polygalacturonases (PGs), 17, 23 Postharvest application, 33, 36, 93, 115, 138–140 Postharvest fungicide, 107–117, 185, 195 Postharvest pathogens, 1–10, 33, 43–53, 57–65, 90, 91, 112, 115, 120–122, 141, 151, 173, 175, 180 Postharvest treatment, 8–9, 32, 33, 35, 72, 92–94, 99, 102, 110, 114, 122, 124, 125, 185, 199, 201 Potato, 32, 74, 78, 79, 110, 111, 139, 172 Prediction systems, 192, 195, 204 Preharvest application, 90–93, 97, 199 Q Quiescent infection, 2, 4, 9, 44, 45, 52, 70, 73–77, 83–85, 108, 125, 128 Quiescent stage, 44–45 R Registration of new postharvest fungicides, 107–117, 120–121, 127 Residue limits, 108–109 Resistance, 1–10, 13–25, 31–37, 52, 58, 72, 90, 108, 122, 151, 172, 195, 206 Resistance management, 86, 110, 115–117 Rhizopus rot, 32, 35, 110, 111, 172–173 Risk assessment, 84, 108–109, 204

Index S Sanitation, 70, 115–117, 126, 140, 198, 206 Sclerotinia sclerotiorum, 47, 49–51, 178 Silicon, 33–34 Storage rot, 34, 35, 183–195 Systemic acquired resistance (SAR), 18–19, 32–35, 174, 175, 177, 179

211 T Thermal-stress treatments, 162–166 Trichothecium, 32 Tuber crops, 110, 111