Host plant resistance to pests

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.fw001

Host Plant Resistance to Pests

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.fw001

Host Plant Resistance to Pests Paul A . H e d i n ,

EDITOR

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.fw001

USDA, Boll Weevil Research Laboratory

As y m p o s i u m sponsored by the Division of Pesticide Chemistry at the 174th Meeting of the American Chemical Society, Chicago, IL, Aug. 31-Sept. 1 , 1977.

62

ACS SYMPOSIUM SERIES

AMERICAN WASHINGTON,

CHEMICAL D.

C.

SOCIETY 1977

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.fw001

Data Library of Congress Host plant resistance to pests. (ACS symposium series; 62 ISSN 0097-6156) Includes bibliographical references and index. 1. Plants—Disease and pest resistance—Congresses. 2. Plant immunochemistry—Congresses. I. Hedin, Paul Arthur. II. American Chemical Society. Division of Pesticide Chemistry. III. Series: American Chemical Society. ACS symposium series; 62. SB750.H67 632'.94 77-13823 ISBN 0-8412-0389-X ACSMC8 62 1-286 1977

Copyright © 1977 American Chemical Society All Rights Reserved. No part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN THE UNITED STATES OF AMERICA

ACS Symposium Series

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.fw001

Robert F. G o u l d , Editor

Advisory Board Donald G. Crosby Jeremiah P. Freeman E. Desmond Goddard Robert A. Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.fw001

FOREWORD The A C S

S Y M P O S I U M

SERIES

was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the IN

CHEMISTRY

SERIES

parallels that of the continuing

SERIES

ADVANCES

except that in order to save time the

papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the A C S

S Y M P O S I U M

SERIES

are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

PREFACE ΤΠΟΓ the past several decades, chemical pesticides including insecticides and herbicides have been the foundation of agricultural pest control. However, the use of these pesticides has come under increasing regula­ tory control because of their side effects on nontarget species and on the

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.pr001

environment. Moreover, there is evidence of increasing resistance through genetic selection of these pests against chemicals that still are authorized for use. Yet the urgency to control pests becomes greater because man, largely because of his own rampant increase in population, seems to be running a collision course with insects and plant diseases in competition for foods and fibers. During the past 20 years, several new concepts of pest control have evolved.

For insects, these concepts include the agents that modify

behavior and development, such as pheromones, kairomones, and hor­ mones. Other approaches encompass the biological agents such as viruses, bacteria, and protozoa. Genetic manipulation has been investigated also. There have been efforts to combine various cultural techniques with these aforementioned approaches to develop integrated pest

manage­

ment systems. The control of plant diseases has concentrated primarily on prophy­ laxis, with a lesser emphasis on immunization. T h e three major prophy­ lactic approaches include: (1)

exclusion of the pathogen by quarantine,

inspection, and certification of seed; (2)

eradication of the pathogen by

crop rotation, sanitation of equipment, elimination of overwintering hosts, eradication of host plant parts, and eradication of alternate hosts; and (3) direct protection by regulating the environment; i.e., moisture, tem­ perature, insect vectors, spraying and dusting, and treating seed and the soil. T h e control of plant diseases by immunization, as with viruses and host disease resistance, has focused on acquired resistance.

This host re­

sistance may be attributed to the plant's morphology, undefined physio­ logical factors, and/or chemicals that are either present initially or are biosynthesized in response to attack by the disease organism. These alternative approaches

to pest control generally have not

achieved the successes predicted by their original expectations, however. In some instances, the degree of control is not adequate. Often, the pro­ cedures are too complicated for the grower or pest control agent to use correctly.

Cost is usually higher and, in some cases, excessive. ix

Also,

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.pr001

because these control programs are directed toward individual crops, there may be disincentives for industrial commercial development. The development of host plant varieties has several advantages compared with the other alternative approaches to pest control. There is no complicated technology for the grower to understand, and it is inexpensive, since any increased seed costs will be more than offset by decreased costs for pesticides and their application. Decreased use of pesticides is also desirable for environmental purposes since the spread of pollutants is decreased, and energy requirements to manufacture, distribute, and apply pesticides is lessened. The major disadvantage associated with plant protection by resistant varieties is that because new races arise, germ plasm must be available from which to select new varieties. However, this is not insurmountable because plant breeders have provided successfully a series of rust resistant wheats in response to the new races of the pathogen for at least four decades. Recently, the interest in elucidating some of the chemical aspects of resistance has increased because of: (1) greater interest in the field of host plant resistance as an alternative to our current dependence on pesticides and their role in developing effective integrated pest management programs; (2) some success in recent programs; (3) information derived from basic plant—insect and plant—plant interactions; (4) greater interest by private and public agencies in supplying the support necessary to conduct more basic chemical studies; (5) a greater awareness and interest by natural product chemists of the opportunities for research contributions; (6) better methodology and technology in microchemical techniques; (7) probably most important for the future, the potential Food and D r u g Administration regulation that will require chemical research to document that changes ( toxins, nutritional, etc. ) in the plant from development of resistance to a pest will not be health or nutrition hazards to humans or to other animals that might consume the resistant crop. A l l but two of the chapters in this volume were presented originally at the symposium on "The Chemical Basis for Host Plant Resistance to Pests" at the 174th National Meeting of the American Chemical Society. Seven chapters on plant diseases are followed by nine chapters on insects. It is hoped that this volume will initiate and stimulate work by cooperating groups of chemists, biochemists, entomologists, plant pathologists, plant breeders, and other related scientists. Defining the chemical basis for plant resistance to pests requires a multidisciplinary approach.

U.S. Department of Agriculture Mississippi State, MS August 22, 1977

PAUL A. HEDIN

X

1 Phytoalexins and Chemicals That Elicit Their Production in Plants Ν. T .

KEEN

and B. B R U E G G E R

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

Department of Plant Pathology, University of California, Riverside, CA 92521

Since the discovery of phytoalexins by Müller and Börger (1), their number has steadily grown until we now know of at least 64 different chemicals for which structures have been offered and which meet at least some of the criteria for phytoalexins. Although phytoalexins have been isolated from at least 75 species representing 20 families, it is too early to say whether their production is a general feature of higher plants. With the possible exception of Ginkgo (2), phytoalexins have not been reported from plants other than angiosperms and conifers with only a few from the latter. At present, they are most studied in vegetative plants and most often in the Leguminosae and Solanaceae. There are plants in which phytoalexins have been looked for and not found (see 3), but such negative data of course does not establish that they are not made; generally, when intensively searched, plants have been observed to make phytoalexins in response to the proper challenge and with the use of proper extraction techniques. This review cannot be comprehensive and will not recite the known list of phytoalexins since this has been done many time (4-9) ; instead it will hazard to deduce some generalizations about phytoalexins, their properties, biosynthesis, and, of paramount importance, to assess their presumed association with the resistance of plants to infectious plant pathogens. We will also discuss what is known about phytoalexin elicitors—chemicals or stimuli that initiate the biochemical steps ultimately resulting in phytoalexin accumulation. All the known phytoalexins are low molecular weight products of plant biosynthesis that have antibiotic properties to one or several groups of microorganisms. The preformed levels of phytoalexins are generally very low or non-detectable in healthy plant tissue, but they accumulate to high levels at the site of attack of the plant by an invading microorganism. Phytoalexins have at times been called "abnormal metabolites" or "stress metabolites". It is our opinion that these terms are, generally speaking, synonyms, and as such need not be used. Phytoalexins are prime examples of secondary metabolites in plants; they would appear 1

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

2

HOST P L A N T RESISTANCE T O PESTS

R=H Coumestrol R = Isopentenyl · Psoralidin :

Figure 1.

R = H Genistein R = OH 2'Hydroxy genistein Phytoalexins from Phaseolus vulgaris

to p r o v i d e one l i k e l y answer t o t h e l o n g - s t a n d i n g search f o r p h y s i o l o g i c functions of these p r o d u c t s — d e f e n s e against p a r a s i t e a t t a c k ( s e e JLO) . To summarize t h i s i n t o a s i m p l e d e f i n i t i o n o f f e r i n g c r i t e r i a f o r phytoalexins, we c o n s i d e r them t o be i n d u c i b l y formed higher plant metabolites that are a n t i b i o t i c to c e r t a i n p o t e n t i a l plant pathogens. We define here some terms appearing i n t h i s paper. Disease r e s i s t a n c e i n p l a n t s i s the r e s t r i c t i o n of development o f a p a t h o g e n i c agent or p a r a s i t e and i t may v a r y i n degree from immunity (no development) to only s l i g h t r e t a r d a t i o n r e l a t i v e to a so-called susceptible reaction. We employ the term incompatible to denote h o s t - p a r a s i t e combinations i n which the plant e x h i b i t s r e s i s t a n c e and compatible to r e f e r to s u s c e p t i b l e r e a c t i o n s of the host. S i m i l a r l y , an incompatible pathogen i s one leading to a r e s i s t a n t p l a n t r e a c t i o n ) w h i l e a c o m p a t i b l e one l e a d s t o a s u s c e p t i b l e host r e a c t i o n .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

1.

K E E N

AND

BRUEGGER

Phytoalexin

Production

in

Phnts

3

The burst i n phytoalexin research i n the l a s t 10 years has e s t a b l i s h e d that many p l a n t s simultaneously make s e v e r a l d i s t i n c t but chemically r e l a t e d p h y t o a l e x i n s . For instance, the green bean p l a n t , Phaseolus v u l g a r i s , produces at l e a s t 9 i s o f l a v o n o i d compounds ( F i g u r e 1) i n r e s p o n s e to v a r i o u s m i c r o o r g a n i s m s or e l i c i t o r s . P h a s e o l l i n i s the best known and has pronounced a c t i v i t y against a broad spectrum of f u n g i , except f o r those that metabolize i t to l e s s t o x i c products. Most of the other bean phytoalexins are a n t i f u n g a l ; however, g e n i s t e i n and hydroxygenis t e i n accumulate along with the other compounds (11), but have only weak a n t i f u n g a l or a n t i b a c t e r i a l p r o p e r t i e s . Do they accu­ mulate as a mere coincidence of the general a c t i v a t i o n of i s o f l a ­ v o n o i d b i o s y n t h e s i s or do they have a n t i b i o t i c a c t i v i t y to o r g a n i s m s (nematodes? p r o t o z o a n s ? ) t h a t we don't know about? Wyman and Van E t t e n (12) reported that k i e v i t o n e and p h a s e o l l i n i s o f l a v a n possessed pronounced a n t i b a c t e r i a l a c t i v i t y against s e v e r a l pathogenic b a c t e r i a , but coumestrol and p h a s e o l l i n were g e n e r a l l y without e f f e c t . T h i s , however, has been disputed by Lyon and Wood (13) and Pat i l and Gnanamanickam (14) who observed a n t i b a c t e r i a l a c t i v i t y f o r coumestrol and p h a s e o l l i n , r e s p e c t i v e l y . Another p l a n t producing m u l t i p l e , r e l a t e d phytoalexins i s Splanum tuberosum, where at l e a s t eight sesquiterpenoid compounds are known (see Dr. S t o e s s l ' s paper i n t h i s s e r i e s ) . I t should be noted that some of these compounds may o r i g i n a t e due to d i r e c t chemical m o d i f i c a t i o n of others by the challenge microorganism, as Ward and S t o e s s l (15) have r e c e n t l y demonstrated f o r the produc­ t i o n of 1 5 - d i h y d r o l u b i m i n from l u b i m i n by f u n g i . However, r i s h i t i n , the best known potato phytoalexin, as w e l l as phytub e r i n , lubimin and phytuberol can be e l i c i t e d i n the absence of l i v i n g microorganisms (16), thus negating the p o s s i b i l i t y that they represent s t r u c t u r e m o d i f i c a t i o n s . R i s h i t i n i s a n t i b i o t i c to s e v e r a l f u n g i (17,18), having a E D ^ Q value of about 50 μ g/ml i n v i t r o a g a i n s t P h y t o p h t h o r a i n f e s t a n s , w i t h somewhat h i g h e r concentrations required against other f u n g i . Phytuberin, on the o t h e r hand, e x h i b i t e d a c t i v i t y o n l y a g a i n s t £ . i n f e s t a n s . Solavetivone and anhydro-^-rotunol were a c t i v e against a l l f u n g i t e s t e d ( 1 7 ) , but l u b i m i n was i n a c t i v e a g a i n s t infestans. In potato, green bean, soybean, and a few other p l a n t s pro­ ducing m u l t i p l e phytoalexins, the r e l a t i v e proportions of the v a r i o u s phytoalexins are not always the same (19,20 and see 21). P r i c e et^ a l . (22) n o t i c e d t h i s f o r the p o t a t o sesquiterpenes and R i c h et. al, (23) o b s e r v e d t h a t l i m a bean r o o t s p r o d u c e d coumestrol and p s o r a l i d i n i n response to the nematode pathogen Pratylenchus s c r i b n e r i , but d i d not accumulate detectable l e v e l s of k i e v i t o n e , e a r l i e r found to be made by the same p l a n t i n response to b a c t e r i a (24). The a n t i b i o t i c p r o p e r t i e s of p h y t o a l e x i n s a r e r o u t i n e l y determined i n i n v i t r o assays, and s e v e r a l t e c h n i c a l and i n t e r p r e t a t i o n a l problems can occur i n these (25,26). Since h i g h l y p u r i f i e d p h y t o a l e x i n s have r e l a t i v e l y low s o l u b i l i t y i n p u r e

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

4

H O S T P L A N T RESISTANCE T O

PESTS

water, s o l u b i l i z e r s such as ethanol or other organic solvents are g e n e r a l l y added such that t h e i r f i n a l concentration i s s u f f i c i e n t l y low as not to a f f e c t the assay organism. Some have argued t h a t t h i s r e q u i r e m e n t d e t r a c t s from the p o s s i b l e a c t i v i t y of phytoalexins i n the p l a n t , but we b e l i e v e that the opposite i s more l i k e l y to o c c u r — c o - d i s s o l u t i o n of phytoalexins i n the p l a n t t i s s u e s by r e l a t e d compounds, f a t t y a c i d s , p h o s p h o l i p i d s , s a p o n i n s , e t c . l i k e l y i n c r e a s e s t h e i r i n v i v o s o l u b i l i t y and perhaps t h e i r a c t i v i t y as w e l l . Indeed, evidence i s a v a i l a b l e (27) i n d i c a t i n g t h a t the pea p h y t o a l e x i n p i s a t i n may be more a c t i v e against i n f e c t i n g hyphae of Erysiphe graminis i n v i v o than against the same organism jln v i t r o . As already mentioned, phytoalexins are o f t e n a c t i v e against f u n g i and at l e a s t some of them a f f e c t b a c t e r i a , but what about other p l a n t pests? We know of no case of phytoalexin involvement with r e s i s t a n c e to i n s e c t s , and t h i s i s perhaps to be expected with non-sedentary p e s t s . However, what about aphids, mites, and other more s t a t i o n a r y feeders? In recent work, R i c h et a l . (23) demonstrated that the lima bean (Phaseolus lunatus L.) p h y t o a l e x i n coumestrol e x h i b i t e d a p a r a l y t i c e f f e c t on the nematode pathogen Pratylenchus s c r i b n e r i and as such could account f o r the r e s i s tance of lima beans to t h i s pathogen. Perhaps t h i s portends other cases of phytoalexin mediated r e s i s t a n c e to nematodes, but we do not have information. The r e s i s t a n t or s o - c a l l e d " l o c a l l e s i o n " r e a c t i o n of s e v e r a l p l a n t s t o v i r u s e s r e s u l t s i n s u b s t a n t i a l p h y t o a l e x i n production (28,29), and although i t i s appealing to s p e c u l a t e c a u s a l i t y , we have no d i r e c t e v i d e n c e t h a t they d e l e t e r i o u s l y a f f e c t v i r u s r e p l i c a t i o n , p r o t e i n synthesis and/or v i r i o n assembly. P t e r o c a r p i n o i d compounds have been reported to possess antitumor a c t i v i t y i n animals (30), a f i n d i n g of p o s s i b l e pharmacological s i g n i f i c a n c e . Despite considerable information on the a n t i m i c r o b i a l of a c t i v i t y p h y t o a l e x i n s , we know r e l a t i v e l y l i t t l e c o n c e r n i n g the mechanism of t h e i r t o x i c i t y . U n l i k e some m i c r o b i a l a n t i b i o t i c s and s y n t h e t i c f u n g i c i d e s , p h y t o a l e x i n s do not appear to have d i s c r e e t "biochemical t a r g e t s , " but i n s t e a d , as Van E t t e n and Bateman f i r s t showed (31,32), they l i k e l y f u n c t i o n as n o n s p e c i f i c p l e i o t r o p i c membrane a n t a g o n i s t s . For instance, p h a s e o l l i n (32) c a u s e d i o n l e a k a g e from t r e a t e d fungus c e l l s and g l y c e o l l i n , p h a s e o l l i n , and medicarpin a l s o caused b u r s t i n g of red blood c e l l s (31). S i m i l a r l y , phytuberin and other sesquiterpene phytoalexins f r o m Solanum tuberosum caused r a p i d l y s i s of P h y t o p h t h o r a i n f e s t a n s zoospores (17), an e f f e c t that would seem again to imply membrane antagonism. There i s a l s o evidence that p h a s e o l l i n and other phytoalexins i n c i t e the same e f f e c t s on p e r m e a b i l i t y of the p l a n t c e l l s where they a c c u m u l a t e ( 3 3 , 3 4 ) . It i s therefore appealing to think that the d i s o r g a n i z a t i o n noted to occur i n the h y p e r s e n s i t i v e r e a c t i o n to be discussed could be caused by the extremely high l e v e l s of phytoalexins a c t i n g on the p l a n t c e l l s . I t was suspected that the aplanar c o n f i g u r a t i o n of the pterocarpan

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

1.

KEEN

A N D BRUEGGER

Phytoalexin

Production

in

Plants

5

phytoalexins might be e s s e n t i a l f o r t h e i r f u n g i t o x i c i t y (35 ), but Van E t t e n (_360 s u b s e q u e n t l y showed t h a t s e v e r a l 6a-hydroxy pterocarp-6a-enes, which do not show the same c o n f i g u r a t i o n as the corresponding parent 6a-hydroxy pterocarpans, had e s s e n t i a l l y unchanged a c t i v i t y . As noted, s e v e r a l i s o f l a v o n e s ( v i z . g e n i s t e i n and h y d r o x y g e n i s t e i n ) , do n o t p o s s e s s pronounced a n t i f u n g a l a c t i v i t y , but isopentenyl s u b s t i t u t i o n makes them considerably more a c t i v e (37,38). This leads to the s u s p i c i o n that l i p o p h i l i c i t y i s an important c o n t r i b u t o r to a c t i v i t y . B e l l and P r e s l e y (39), obtained data with the c o t t o n Verticillium dahliae host-parasite system that satisfactorily summarizes our view of phytoalexins and t h e i r c o n t r i b u t i o n to c o m p a t i b i l i t y and i n c o m p a t i b i l i t y of plants to c e r t a i n p o t e n t i a l pathogens. We c a l l t h i s r e l a t i o n s h i p the " e q u i l i b r i u m hypothesis," the s a l i e n t point being that any modulator of the h o s t - p a r a s i t e complex p o s i t i v e l y i n f l u e n c i n g pathogen c o l o n i z a t i o n rates but i n d e p e n d e n t o f e f f e c t s on h o s t p h y t o a l e x i n p r o d u c t i o n will promote d i s e a s e s u s c e p t i b i l i t y . S i m i l a r l y , any f a c t o r t h a t p o s i t i v e l y i n f l u e n c e s the l o c a l r a t e of p h y t o a l e x i n p r o d u c t i o n independently of e f f e c t s on pathogen growth w i l l promote disease r e s i s t a n c e . The converse of both tenets produces the corresponding e f f e c t s . To i l l u s t r a t e , B e l l and Presley (39), found that i n c r e a s i n g temperature from 15 to 28 C increased the r e l a t i v e a b i l i t y of cotton plants to produce phytoalexins, and p h y t o a l e x i n p r o d u c t i o n was c o n s t a n t from 28-35 C. However, i n c r e a s i n g t e m p e r a t u r e above 25° d e l e t e r i o u s l y a f f e c t e d the growth r a t e of the fungus pathogen V e r t i c i l l i u m d a h l i a e . This was c o n s i s t e n t with the observation that disease becomes l e s s severe above ca. 25 C i n n a t u r e . Conversely, lowering temperatures caused l e s s phytoalexin production but d i d not adversely a f f e c t the fungus and d i s e a s e became more s e v e r e . S i m i l a r to these r e s u l t s , many i n v e s t i g a t i o n s have shown that treatment of i n f e c t e d p l a n t s with metabolic i n h i b i t o r s that a f f e c t only the p l a n t block phytoalexin production and cause more s u s c e p t i b l e r e a c t i o n s . On the other hand, treatment of p l a n t s with agents that s p e c i f i c a l l y i n h i b i t development of the pathogen would be expected to r e s u l t i n a more r e s i s t a n t r e a c t i o n and t h i s too has been observed e x p e r i mentally. Phytoalexin production i s often associated with a widespread but poorly understood plant disease defense r e a c t i o n c a l l e d the " h y p e r s e n s i t i v e r e a c t i o n " (HR). C l a s s i c a l l y , the h y p e r s e n s i t i v e r e a c t i o n i s observed a f t e r a few hours or a few days f o l l o w i n g i n f e c t i o n by an incompatible race or species of p l a n t pathogen as the d e a t h o r d i s o r g a n i z a t i o n of t h e p l a n t c e l l s i m m e d i a t e l y adjacent to the i n f e c t i o n s i t e concomitant with or preceeding the r e s t r i c t i o n of pathogen development. Despite the f a c t that the HR operates i n response to non-pathogenic members of a l l the known t y p e s o f p l a n t p a t h o g e n s — f u n g i , b a c t e r i a , nematodes, and v i r u s e s — n o p l a u s i b l e explanation as to why i t worked was o f f e r e d u n t i l phytoalexins appeared on the scene. As w i l l be discussed i n

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

6

H O S T P L A N T RESISTANCE T O

PESTS

more d e t a i l l a t e r , many h y p e r s e n s i t i v e r e a c t i o n s r e s u l t i n the accumulation of l a r g e q u a n t i t i e s of phytoalexins i n the "hypersens i t i v e area," up to the phenomenal concentration of 10% of the dry weight of t i s s u e (40) ! I t would seem implausible that the c e l l s i n the immediate i n f e c t i o n s i t e could themselves account f o r the r e l a t i v e l y massive b i o s y n t h e s i s r e q u i r e d f o r such high concentrations. T h e r e a r e many n o n - i n f e c t e d h o s t c e l l s s u r r o u n d i n g h y p e r s e n s i t i v e a r e a s , however, and r e c e n t evidence suggests that these m e t a b o l i c a l l y a c t i v e neighboring c e l l s both export b i o s y n t h e t i c intermediates to c e l l s immediately adjacent to the i n f e c t i o n s i t e (41,43) and b i o s y n t h e s i z e phytoalexins and export them i n t o the host c e l l s and i n t e r c e l l u l a r spaces that are a c t u a l l y c o l o n i z e d by the pathogen (41,42,43). This "dumping" of phytoalexins i n t o the h y p e r s e n s i t i v e area may account f o r the very high l o c a l concentrations present and would presumably c o n s t i t u t e a very h o s t i l e l o c a l environment f o r the pathogen. There i s good evidence that phytoalexins are i n f a c t made i n l i v i n g , metabol i c a l l y a c t i v e p l a n t c e l l s (44,45,46) and the h y p e r s e n s i t i v e death of host c e l l s may r e s u l t from the t o x i c i t y of the accumul a t e d phytoalexins (44). The observed e x t r a c e l l u l a r export of phytoalexins i s a l s o c o n s i s t e n t with the common observation that phytoalexins can be e f f i c i e n t l y e l u t e d from i n t a c t p l a n t t i s s u e s by merely p l a c i n g them into contact with water or aqueous ethanol (4,47,48). These s o - c a l l e d d i f f u s i o n techniques are u s e f u l f o r o b t a i n i n g phytoalexins f r e e from most of the i n t r a c e l l u l a r plant metabolites, and i n one case the use of t h i s technique was shown to e l u t e the p h y t o a l e x i n from host t i s s u e at a s u f f i c i e n t r a t e t h a t the n o r m a l l y r e s i s t a n t t i s s u e became s u s c e p t i b l e to the pathogen (47)· K i r a l y , Barna, and Ersek (49,50,134) applied various metab o l i c i n h i b i t o r s to t h r e e n o r m a l l y c o m p a t i b l e h o s t - p a r a s i t e combinations and reported that the i n h i b i t o r s , presumed to a f f e c t only the p a r a s i t e , caused occurrence of the same h y p e r s e n s i t i v e defense r e a c t i o n as i n g e n e t i c a l l y incompatible p l a n t s ; i n one case—the potato-Phytophthora i n f e s t a n s system—they reported that the i n h i b i t o r treated p l a n t s accumulated the phytoalexin r i s h i t i n j u s t as d i d normally incompatible p l a n t s . The authors i n t e r p r e t e d t h e i r d a t a as i n d i c a t i n g t h a t the h y p e r s e n s i t i v e r e a c t i o n and phytoalexin production were t h e r e f o r e only consequences but not c a u s e s of r e s i s t a n c e , and t h a t some unknown mechanism a c t u a l l y accounted f o r r e s i s t a n c e expression. Despite the acceptance of t h i s reasoning by some (51), the l o g i c of K i r a l y and co-workers i s flawed. Disregarding arguments (52,53) of the necessary assumption that the i n h i b i t o r s were only a f f e c t i n g the pathogens, i t i s p l a u s i b l e that i f the pathogens were i n f a c t s p e c i f i c a l l y i n h i b i t e d by the treatments, the r e l e a s e of phytoa l e x i n e l i c i t i n g s u b s t a n c e s from them c o u l d a c c o u n t f o r the observations. By applying the anti-pathogen a n t i b i o t i c s , the authors were then q u i t e simply swinging the e q u i l i b r i u m r e l a t i o n ship of the l a s t paragraph g r o s s l y i n favor of the host. Were the

1.

KEEN

A N D BRUEGGER

Phytoalexin

Production

in

Plants

7

a u t h o r s ' i n t e r p r e t a t i o n c o r r e c t , we would s i m i l a r l y have to dismiss t h e immune s y s t e m i n h i g h e r a n i m a l s as a d e f e n s e mechanism, s i n c e a t t e n u a t e d pathogens and a n t i g e n s from them t r i g g e r i t , j u s t as K i r a l y et a l . reported i n p l a n t s (49,50,134).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

Are P h y t o a l e x i n s Plants?

Really

a Mechanism f o r D i s e a s e R e s i s t a n c e i n

Since Cruickshank's c l a s s i c review (4) brought phytoalexins to the a t t e n t i o n of the s c i e n t i f i c community and f o r c e f u l l y proposed t h e i r r o l e i n disease defense, they have i n e x p l i c a b l y been under a t t a c k by some workers g e n e r a l l y n o t i n v o l v e d i n p h y t o a l e x i n r e s e a r c h on the p r i n c i p l e p r e s u m p t i o n t h a t they a r e m e r e l y spurious products or formed c o i n c i d e n t a l l y to some other as yet undescribed defense mechanism. Enough evidence i s now at hand to attempt a reasonable assessment of the proposed r o l e of phytoa l e x i n s as a d e f e n s e mechanism i n p l a n t s but n o t t o deduce the g e n e r a l i t y of t h i s r o l e . There are two types of r e s i s t a n c e to be considered, general r e s i s t a n c e of a plant species to various organisms that are non-pathogenic on i t and s p e c i f i c r e s i s t a n c e , o c c u r r i n g i n c e r t a i n c u l t i v a r s of a p l a n t species to s p e c i a l i z e d biotypes, or races, of a s i n g l e pathogen species (gene-for-gene systems [54,55,56]). General

Resistance

As with a l l p a r a s i t i e s , those of p l a n t s must possess c e r t a i n c h a r a c t e r i s t i c s that enable them to s a t i s f a c t o r i l y reach the host, p e n e t r a t e and i n f e c t , c o l o n i z e h o s t t i s s u e , e s c a p e d d e f e n s e mechanisms, and f i n a l l y to reproduce and disseminate. F a i l u r e to do any of these p r o p e r l y r e s u l t s i n i n a b i l i t y to p a r a s i t i z e the plant and we say that the p l a n t i s r e s i s t a n t or i s incompatible with the p a r a s i t e . Many mechanisms c l e a r l y can account f o r t h i s that have nothing to do with p h y t o a l e x i n s — s t r u c t u r a l features of the p l a n t such as c e l l w a l l composition, c u t i c u l a r f e a t u r e s , l e a f h a i r s , s t o m a t a l b e h a v i o r , l e c t i n s , i n d u c e d l i g n i f i c a t i o n and s u b e r i z a t i o n , p r e f o r m e d c h e m i c a l i n h i b i t o r s and o t h e r s a r e u n d o u b t e d l y i m p o r t a n t i n e x c l u d i n g a g r e a t many p o t e n t i a l l y pathogenic microorganisms (see 3,10). But there i s s u b s t a n t i a l reason to think that phytoalexins are one of the mechanisms that confer general r e s i s t a n c e , e i t h e r s i n g l y or i n concert with other mechanisms (57)· Cruickshank (4) f i r s t n o t i c e d that s u c c e s s f u l pathogens on pea and bean p l a n t s were g e n e r a l l y l e s s s e n s i t i v e to the phytoa l e x i n s produced by those p l a n t s than were non-pathogens and t h i s has been confirmed by Van Etten (58). This i n d i c a t e d that phytoa l e x i n s were perhaps r e s p o n s i b l e f o r r e s i s t a n c e to t h e nonpathogens, and that the f a i l u r e of the host to produce them i n s u f f i c i e n t quantity or the a b i l i t y of the p a r a s i t e to degrade them c o u l d a c c o u n t f o r the s u c c e s s f u l p a r a s i t e s . We now know of

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

8

H O S T P L A N T RESISTANCE T O

PESTS

s e v e r a l examples where s u c c e s s f u l p a r a s i t e s are able to degrade the p l a n t p h y t o a l e x i n s , w h i l e t a x o n o m i c a l l y closely related microorganisms cannot. For instance van den Heuvel (59) showed that three s t r a i n s of the pathogen B o t r y t i s cinerea that were pathogenic on green beans had the a b i l i t y to degrade p h a s e o l l i n , w h i l e two s t r a i n s of the same fungus that were not pathogenic lacked a b i l i t y to s i g n i f i c a n t l y degrade i t . S i m i l a r observations were made by M a n s f i e l d and Widdowson (60), who found that B o t r y t i s fabae, a pathogen of V i c i a fabae p l a n t s , degraded the major plant p h y t o a l e x i n wyerone a c i d much more r a p i d l y than the r e l a t e d non-pathogen B. c i n e r e a . The p r i n c i p l e degradation product was s u b s e q u e n t l y i d e n t i f i e d as r e d u c e d wyerone a c i d and i t had l i t t l e or no a n t i f u n g a l a c t i v i t y . Hargreaves et a l . (61) also o b s e r v e d the d e g r a d a t i o n of a n o t h e r broad bean p h y t o a l e x i n , wyerone epoxide, to wyerol epoxide by 15. cinerea and to dihydrodihydroxy wyerol epoxide by IS. fabae. Again, the degradation products had l e s s a n t i f u n g a l a c t i v i t y than the parent compound, with the B. fabae product being l e a s t a c t i v e . Jones ^ t a l . (62) showed that the pepper pathogen Phytophthora c a p s i c i was much l e s s s e n s i t i v e In v i t r o to i n h i b i t i o n by the pepper phytoalexin c a p s i d i o l than was the non-pathogen of peppers, infestans. No e v i d e n c e was o b t a i n e d f o r d e g r a d a t i o n of c a p s i d i o l by c a p s i c i ; instead the fungus was apparently more t o l e r a n t of the compound. In a d d i t i o n , _P. i n f e s t a n s a l s o e l i c i t e d much greater amounts of c a p s i d i o l i n peppers than d i d .P. c a p s i c i . Thus, i t would seem that two f a c t o r s ( d i f f e r e n t i a l s e n s i t i v i t y and d i f f e r e n t i a l e l i c i t o r a c t i v i t y ) d i s t i n g u i s h the two f u n g i . The work by the Canadian group o f f e r s considerable support f o r the r o l e of c a p s i d i o l i n determining the general r e s i s t a n c e of peppers to _P. i n f estans and t h e i r s u s c e p t i b i l i t y to JP. c a p s i c i . A major l i n e of evidence l i n k i n g phytoalexins with general r e s i s t a n c e i s that c e r t a i n treatments break the normal r e s i s t a n c e of plant t i s s u e s to non-pathogens and concomitantly negate phytoa l e x i n b i o s y n t h e s i s a f t e r i n o c u l a t i o n (53,63). For instance, Chamberlain (64,65) found that treatment of soybean hypocotyls at 44 C f o r 1 hr produced no apparent damage to the p l a n t s , but i t destroyed r e s i s t a n c e to s e v e r a l non-pathogenic fungi along with phytoalexin production, p r o v i d i n g that the plants were i n o c u l a t e d w i t h i n about 24 hr a f t e r treatment. A f t e r 1-2 days, however, r e s i s t a n c e and phytoalexin production were concomitantly r e s t o r e d . A key p o i n t (64) was the f a c t that heat treatment broke r e s i s t a n c e t o some non-pathogens of soybean but not a l l of them, thus implying that phytoalexins are associated with general r e s i s t a n c e to some but not a l l non-pathogens. Yoshikawa (69) followed t h i s w i t h the f i n d i n g t h a t the p r o t e i n s y n t h e s i s inhibitor b l a s t i c i d i n S a l s o rendered soybean p l a n t s s u s c e p t i b l e to s e v e r a l normally non-pathogenic Phytophthora sp. and that the treated p l a n t s d i d not produce the soybean phytoalexin g l y c e o l l i n a f t e r inoculation.

1.

KEEN

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

Specific

AND

BRUEGGER

Phytoalexin

Production

in

Plants

9

Resistance

Plant p a t h o l o g i s t s and plant breeders have known f o r almost 80 years that some c u l t i v a r s w i t h i n a plant species have r e s i s t a n c e to c e r t a i n p l a n t pathogens and that t h i s r e s i s t a n c e to disease i s f r e q u e n t l y i n h e r i t e d as a dominant, s i n g l e gene c h a r a c t e r . P r e d i c t a b l y , the i n c o r p o r a t i o n of these s i n g l e genes i n t o agronom i c a l l y d e s i r a b l e c u l t i v a r s of crop p l a n t s has become a mainstay of p r a c t i c a l disease c o n t r o l . The curse of the methodology i s that the pathogens involved f r e q u e n t l y mutate to biotypes or races t h a t a r e p a t h o g e n i c on the r e s i s t a n t p l a n t g e n o t y p e . This r e a c q u i s i t i o n of pathogenicity or v i r u l e n c e has a l s o been found to be i n h e r i t e d as a s i n g l e gene character i n s e v e r a l p a r a s i t e s , and thus l e d F l o r (see 56) to f i r s t f o r m a l l y recognize the complement a r i t y of host r e s i s t a n c e genes and p a r a s i t e v i r u l e n c e genes; t h i s r e l a t i o n s h i p i s c a l l e d gene-for-gene complementarity and e x i s t s i n many plant-pest r e l a t i o n s h i p s (54,55,56). Aside from being a detriment to the permanent use of disease r e s i s t a n c e genes i n p r a c t i c a l a g r i c u l t u r e , the gene-for-gene r e l a t i o n s h i p o f f e r s a s p l e n d i d system f o r the c r i t i c a l t e s t i n g of mechanisms involved i n the b i o c h e m i c a l f u n c t i o n of r e s i s t a n c e genes i n p l a n t s and v i r u l e n c e genes i n p a r a s i t e s . Rowell et a l . (55) f i r s t presented t h i s as the "quadratic check," shown i n Figure 2 f o r comparing one r e s i s t a n c e gene i n the plant and one complementary v i r u l e n c e gene i n the p a r a s i t e . S i n c e i t i s p o s s i b l e to d e v e l o p h o s t and p a r a s i t e l i n e s that are "near-isogenic" except f o r the genes i n question, use of these systems c l e a r l y minimizes the problem of i n t e r f e r e n c e by genes other than those under study (55). Two terms o r i g i n a l l y introduced by Talboys (66) are u s e f u l i n c o n c e p t u a l i z i n g events o c c u r r i n g i n p l a n t - p a r a s i t e i n t e r a c t i o n s , e s p e c i a l l y gene-for-gene systems. The determinative phase denotes those i n i t i a l r e c o g n i t i o n a l events o c c u r r i n g between host and pathogen that determine or d i c t a t e whether the p l a n t w i l l subsequently react i n a compatible or incompatible way. The expressive phase c o n s t i t u t e s those events that are, as we w i l l see, i r r e v e r s i b l y predicated by the determinative phase, and may i n v o l v e r a p i d or slow p h y t o a l e x i n p r o d u c t i o n which i n t u r n may constitute r e s i s t a n c e or s u s c e p t i b i l i t y , r e s p e c t i v e l y . Gene-for-gene h o s t - p a r a s i t e systems have g e n e r a l l y o f f e r e d the most c r i t i c a l and convincing proof that r a p i d phytoalexin product i o n c o n s t i t u t e s a mechanism f o r r e s t r i c t i o n of pathogen growth i n r e s i s t a n t p l a n t t i s s u e — c o n v e r s e l y , phytoalexins are the only mechanism that i s c u r r e n t l y known to e x p l a i n r e s i s t a n c e i n genefor-gene systems, other proposed mechanisms having n e g l i g i b l e experimental support. The e x p e r i m e n t a l p o i n t s t h a t s u p p o r t phytoalexin involvement with r e s i s t a n c e expression i n gene-forgene systems may be summarized as f o l l o w s : ( i ) incompatible r e a c t i o n s r e s u l t i n more r a p i d and higher rates of phytoalexin production than do compatible ones (13,34,41,67,68,70) and a l l races of the r e s p e c t i v e pathogens seem equally s e n s i t i v e to the

10

HOST

PLANT

RESISTANCE T O PESTS

PLANT Rps

Race I ( A

R p s

rps

)

I

c

)

c

c

PARASITE

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

Race 3 ( a

R p s

Figure 2. The quadratic check (55); gene-for-gene complementarity as illustrated by the reactions of two alleles of the Rps resistance hcus in soybeans to Phytophthora megasperma var. sojae and two races of the fungus shown with their presumed but unproved virulence genotypes. Reactions: I = incompatible; C = compatible. Resistance in the plant and avirulence in the fungus are shown as dominant characters, a general observation (54;.

phytoalexins i n v i t r o , ( i i ) i n the r e l a t i v e l y few cases i n which l o c a l i z e d l e v e l s of phytoalexins have been assessed e i t h e r by e x t r a c t i o n from s p e c i f i c host c e l l s l a y e r s or h i s t o c h e m i s t r y , they have been shown to reach h i g h l y i n h i b i t o r y l e v e l s to the pathogen at the i n f e c t i o n s i t e s i n incompatible r e a c t i o n (34,40,41,68,70); ( i i i ) a p p l i c a t i o n of m e t a b o l i c i n h i b i t o r s or heat treatments simultaneously block both r e s i s t a n c e and phytoalexin production (53,71); ( i v ) although t e c h n i c a l l y d i f f i c u l t , l i m i t e d data suggest that a p p l i c a t i o n of p u r i f i e d phytoalexins to normally compatible h o s t - p a r a s i t e i n f e c t i o n s i t e s r e s u l t s i n incompatible r e a c t i o n s (27,72). The most e x t e n s i v e l y researched and best understood gene-forgene plant-pathogen system i s the soybean-Phytophthora megasperma v a r . sojae i n t e r a c t i o n , and the evidence s t r o n g l y supports a r o l e f o r phytoalexins i n the expression of r e s i s t a n c e . Plant breeders have found s e v e r a l d i f f e r e n t s i n g l e genes f o r r e s i s t a n c e to the root and hypocotyl i n f e c t i n g fungus, but only one, the dominant Rps gene, has been incorporated i n t o near-isogenic l i n e s . As with a l l gene-for-gene systems, s e v e r a l races of the fungus have been found (9 at l a s t count), and two of these, races 1 and 3, together with the rps and Rps soybean genotypes, f i l l out the quadratic c h e c k f o r t h e h o s t - p a r a s i t e system ( F i g u r e 2 ) . Klarman and

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

1.

KEEN

A N D BRUEGGER

Sojagol Figure 3.

Phytoalexin

Production

in

Pfonts

11

Coumestrol Structures of soybean (Glycine max) phytoalexins

Gerdemann (47,74) f i r s t showed t h e p o s s i b l e i n v o l v e m e n t of phytoalexins i n the expression of r e s i s t a n c e due to the Rps gene. They demonstrated that e l u t i o n of the i n f e c t i o n s i t e of incomp a t i b l e i n o c u l a t e d soybean h y p o c o t y l s w i t h water removed the phytoalexins and made the p l a n t s s u s c e p t i b l e . They a l s o chromatog r a p h i c a l l y showed the presence of two phytoalexins i n e x t r a c t s from incompatible r e a c t i n g plants that were not recovered from compatible p l a n t s . Paxton and a s s o c i a t e s showed that phytoalexins were a s s o c i a t e d w i t h r e s i s t a n c e c o n f e r r e d by one a d d i t i o n a l r e s i s t a n c e gene (75) and found that i n o c u l a t i o n with an incomp a t i b l e fungus r a c e l e d to " c r o s s - p r o t e c t i o n " of the p l a n t s against subsequent r e - i n o c u l a t i o n with a normally compatible race. They a l s o noted that a d m i n i s t r a t i o n of two of the soybean phytoa l e x i n s (now known to be g l y c e o l l i n and PA^) t o c o m p a t i b l e h o s t - p a r a s i t e combinations r e s u l t e d i n a r e s i s t a n t r e a c t i o n (72) . B r i d g e and Klarman (76) f o l l o w e d t h i s same l o g i c by showing that u l t r a v i o l e t l i g h t e l i c i t e d g l y c e o l l i n production i n

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

12

HOST P L A N T RESISTANCE T O

PESTS

the rps genotype, normally s u s c e p t i b l e to race 1, and that the c o r t i c a l t i s s u e of such UV treated p l a n t s became r e s i s t a n t to the fungus. Klarman and Sanford (77) f i r s t i s o l a t e d the major phytoa l e x i n now thought to be involved i n r e s i s t a n c e expression and Keen et a l . (78) showed that i t e x i s t e d as three isomers by GC-MS. Sims j i t aj.. (79) f o r m u l a t e d the s t r u c t u r e of one of t h e s e as 6a-hydroxyphaseollin, but Burden and B a i l e y (80) subsequently showed that t h i s was i n c o r r e c t and that the isoprene u n i t should be placed on the A r i n g (Figure 3). Lyne et a l . (81) confirmed t h i s and formulated the c u r r e n t l y accepted s t r u c t u r e s f o r the other two g l y c e o l l i n isomers (Figure 3). A l l three of them have s i m i l a r a n t i f u n g a l p r o p e r t i e s and s i m i l a r l y i n h i b i t growth of a l l j \ megasperma races (78). Although the mixed isomers as i s o l a t e d from the plant are a n t i b a c t e r i a l (82), the i n d i v i d u a l isomers have not been tested f o r t h e i r a c t i v i t y . Keen et a l . (83) showed that the i s o f l a v o n e d a i d z e i n and the coumestans coumestrol and s o j a g o l (Figure 3) accumulated c o o r d i n a t e l y with g l y c e o l l i n during the incompatible r e a c t i o n to _P. megasperma var. sojae, but that the f o r m e r compounds p o s e s s e d much l e s s a n t i f u n g a l a c t i v i t y than glyceollin. I n t h e same p a p e r , C-phenylalanine and C - i s o l i q u i r i t i g e n i n were found to be r e a d i l y incorporated i n t o g l y c e o l l i n , t h e r e b y i n d i c a t i n g t h a t the p h y t o a l e x i n i n f a c t r e s u l t s from jie novo s y n t h e s i s and not from d e g r a d a t i o n of a preformed compound a f t e r i n o c u l a t i o n . S i m i l a r to e a r l i e r observ a t i o n s by Rathmell and Bendall (84) i n green beans, Keen ^ t a l . (83) a l s o found that incompatible soybean hypocotyls s p e c i f i c a l l y accumulated i s o f l a v o n o i d s , but not f l a v o n o i d s or other detectable phenols. G e n i s t e i n and PA^ are a l s o regarded as soybean phytoa l e x i n s because they accumulate c o o r d i n a t e l y with g l y c e o l l i n i n r e s i s t a n t r e a c t i o n s (85) and possess at l e a s t weak a n t i f u n g a l activity. Keen (40) showed that g l y c e o l l i n was produced from 10-100 times f a s t e r i n incompatible than i n compatible r e a c t i o n s , d e m o n s t r a t e d t h a t p r o d u c t i o n was s t r i c t l y l o c a l i z e d a t the i n f e c t i o n s i t e i n incompatible hypocotyls, and found that concent r a t i o n s reached exceedingly h i g h l e v e l s (up to 10% of the t i s s u e d r y w e i g h t by 48 h r ) . In common w i t h a l l o t h e r known phytoalexins, there was a ^ a . 10 hr l a g period a f t e r i n o c u l a t i o n before g l y c e o l l i n was detected i n the p l a n t s , but then i t accumul a t e d r a p i d l y i n the incompatible r e a c t i o n s u n t i l 48 hr, at which time the fungus seems dead (86). Yoshikawa jet al. (7JL) have r e c e n t l y made a c r i t i c a l and elegant study of the l e v e l s of g l y c e o l l i n o c c u r r i n g at very e a r l y times a f t e r i n o c u l a t i o n with P. megasperma var. sojae by making freeze microtome s e c t i o n s of soybean hypocotyls and i n t h i s way q u a n t i t a t i n g l e v e l s of the phytoalexin o c c u r r i n g at various c e l l depths from the i n o c u l a t i o n s i t e on the hypocotyls. Their data c o n c l u s i v e l y e s t a b l i s h e s that incompatible r e a c t i o n s are d i s t i n g u i s h a b l e from c o m p a t i b l e r e a c t i o n s a t the e a r l i e s t time of g l y c e o l l i n d e t e c t i o n at about 8 hrs a f t e r i n o c u l a t i o n when the fungus has o n l y i n v a d e d a few h o s t c e l l l a y e r s . G l y c e o l l i n

1.

KEEN

A N D BRUEGGER

Phytoalexin

Production

Fungus growth stops

DNA transcription required until here

Inoculation Increased m-RNA synthesis

Glyceollin synthesis starts m-RNA translation required until here

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

13

in Ρ fonts

Γ

4

5

6

Glyceollin reaches 90 Concentration E D

I 8

7

10

HOURS J\

DETERMINATIVE PHASE

V

EXPRESSIVE PHASE

Figure 4. Timing of events occurring in incompatible-reacting soybean hypocotyh inoculated wtih Phytophthora megasperma var. sojae. The determinative phase is de­ fined as that time required for recognitional events between parasite and plant and for DNA transcription; the expressive phase encompasses translational events and phytoalexin biosynthesis.

production was s t r i c t l y l o c a l i z e d i n the host c e l l s immediately a d j a c e n t t o t h e fungus i n t h e i n c o m p a t i b l e r e a c t i o n s and i t accumulated to s t r o n g l y i n h i b i t o r y levels ( 9 Q concentrations f o r In v i t r o fungus growth) i m m e d i a t e l y b e f o r e t h e time when fungus growth stopped at j ç a . 9 hrs a f t e r i n o c u l a t i o n (see F i g u r e 4). In compatible r e a c t i n g p l a n t s , however, l e v e l s of g l y c e o l l i n never exceeded the E D ^ Q value f o r i n h i b i t i o n of fungus growth i n host c e l l s that were near the growing margin of the invading fungus. This data s t r o n g l y supports the i d e a that g l y c e o l l i n c o n s t i t u t e s an i n h i b i t o r y mechanism i n incompatible h o s t - p a r a s i t e combinations and that the f a i l u r e of the compatible host to make the c h e m i c a l f a s t enough a c c o u n t s f o r i t s s u s c e p t i b i l i t y t o r e s t r i c t the fungus. E D

In o t h e r i m p o r t a n t work Yoshikawa et a l . (87) found t h a t incompatible r e a c t i n g soybean hypocotyls synthesized p o l y ( A ) c o n t a i n i n g mRNA a t 6 times the r a t e of non-inoculated hypocotyls,

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

14

HOST P L A N T

RESISTANCE T O PESTS

but that i n o c u l a t e d compatible hypocotyls synthesized mRNA at only 2 times that of the c o n t r o l s . These data r a i s e d the p o s s i b i l i t y that the expression of i n c o m p a t i b i l i t y i s b i o c h e m i c a l l y the r e s u l t of derepressed t r a n s c r i p t i o n of genes coding f o r the enzymes of p h y t o a l e x i n b i o s y n t h e s i s . T h i s has been f u r t h e r s u b s t a n t i a t e d (69) with the f i n d i n g that the t r a n s c r i p t i o n i n h i b i t o r actinomycin D at 10 yg^ml blocked phytoalexin production i n normally incompatible p l a n t s and c o n c o m i t a n t l y r e s u l t e d i n a c o m p a t i b l e r e a c t i o n . However, a p p l i c a t i o n of actinomycin D to the p l a n t s at more than 4 hours a f t e r i n o c u l a t i o n was i n e f f e c t i v e i n r e v e r s i n g r e s i s t a n c e e x p r e s s i o n or g l y c e o l l i n p r o d u c t i o n . S i m i l a r l y , the p r o t e i n synthesis i n h i b i t o r b l a s t i c i d i n S blocked p r o t e i n synthesis i n r e s i s t a n t i n o c u l a t e d hypocotyls and t h i s r e s u l t e d i n only low p h y t o a l e x i n l e v e l s and s u s c e p t i b i l i t y of the p l a n t s , p r o v i d i n g that the i n h i b i t o r was a p p l i e d i n the f i r s t 6 hours a f t e r i n o c u l a t i o n (69). These observations are summarized i n Figure 4 and c o n s t i t u t e an e l e g a n t e l u c i d a t i o n of the m o l e c u l a r events a s s o c i a t e d w i t h r e s i s t a n c e e x p r e s s i o n to I?, megasperma v a r . sojae i n the soybean p l a n t . I f , as appears to be the case i n the soybean-P. megasperma var. sojae h o s t - p a r a s i t e system, i n c o m p a t i b i l i t y of the host to fungus i n f e c t i o n i s due to d e r e p r e s s e d b i o s y n t h e s i s of p h y t o a l e x i n s r e s u l t i n g from s p e c i f i c de novo DNA t r a n s c r i p t i o n , does s p e c i f i c i n d u c t i o n of b i o s y n t h e t i c enzymes i n f a c t o c c u r ? We have no evidence on t h i s except f o r the work of S i t t o n and West (73) with the a n t i f u n g a l and a n t i b a c t e r i a l phytoalexin casbene. S i t t o n and West obtained a c e l l - f r e e system from fungus-challenged c a s t o r bean t i s s u e t h a t s y n t h e s i z e d casbene 20-40 times f a s t e r from l a b e l l e d mevalonic a c i d than comparable e x t r a c t s from healthy plant t i s s u e . Of considerable i n t e r e s t , r a t e s of s y n t h e s i s of other diterpene hydrocarbons was e s s e n t i a l l y the same with both c e l l - f r e e systems. This suggests that casbene b i o s y n t h e s i s i s a consequence of s p e c i f i c i n d u c t i o n of the appropriate b i o s y n t h e t i c enzymes and does not r e s u l t from a general a c c e l e r a t i o n of t e r penoid b i o s y n t h e s i s . With the legume i s o f l a v o n o i d p h y t o a l e x i n s , e a r l y enzymes i n the presumed b i o s y n t h e t i c pathways i n c l u d i n g phenylalanine-ammonia lyase, chalcone-flavonone isomerase, peroxidase and others appear to have no s p e c i f i c r o l e (88,93), and l a t e r enzymes i n the presumed pathways have not been obtained i n c e l l - f r e e extracts. A prime remaining question i n p l a n t pathology i s e l u c i d a t i o n of the s p e c i f i c i t y mechanism(s) i n p l a n t - p a r a s i t e systems that operates between p a r a s i t e and host p l a n t and d i c t a t e s whether the plant w i l l respond i n a compatible or incompatible way. It is most p l a u s i b l e that the host i n such cases s p e c i f i c a l l y recognizes some chemical f e a t u r e ( s ) of the incompatible p a r a s i t e (but not the compatible) and that t h i s i n a yet unknown manner i n t e r a c t s with the host to u l t i m a t e l y r e s u l t i n derepressed phytoalexin product i o n and r e s i s t a n c e . T h i s s u s p i c i o n has generated a great deal of i n t e r e s t and w i l l i n v o l v e the next s e c t i o n .

1.

KEEN

A N D BRUEGGER

Phytoalexin

Production

in

Plants

15

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

Phytoalexin e l i c i t o r s Phytoalexin production may be e l i c i t e d i n p l a n t t i s s u e s i n the absence of l i v i n g microorganisms by such agents as heavy metal i o n s (102), UV l i g h t ( 7 6 ) , 3',5' c y c l i c AMP ( 9 ^ ) , v a r i o u s peptides, p r o t e i n s , a n t i b i o t i c s (95) and chemicals of pathogen origin. These chemical agents have been c a l l e d " i n d u c e r s " of phytoalexin production, but we p r e f e r to c a l l them " e l i c i t o r s , " because a l l we c u r r e n t l y know i s that they i n i t i a t e or e l i c i t the production of p h y t o a l e x i n s . The n o n - s p e c i f i c i t y of p h y t o a l e x i n response to such an array of agents has been used as a d e t r a c t i n g argument against the presumed r o l e of phytoalexins i n disease r e s i s t a n c e , d e s p i t e t h e o b v i o u s f a c t t h a t pathogens do n o t r o u t i n e l y l i b e r a t e heavy metal ions, a n t i b i o t i c s , e t c . Further, many of the e l i c i t o r s from pathogens are obtained by such harsh treatments as a u t o c l a v i n g or homogenization, l e a d i n g one to doubt whether they play any p h y s i o l o g i c r o l e i n h o s t - p a r a s i t e i n t e r actions. Few a t t e m p t s have been made to c r i t i c a l l y t e s t t h e a s s o c i a t i o n of e l i c i t o r s with the d i f f e r e n t i a l phytoalexin product i o n that occurs i n gene-for-gene h o s t - p a r a s i t e systems, but l o g i c d i c t a t e s that such s p e c i f i c i t y imparting f a c t o r s must f u n c t i o n during the determinative phase. We w i l l d i s c u s s n o n - s p e c i f i c e l i c i t o r s , those with no d i f f e r e n t i a l e f f e c t s of various genotypes of a p l a n t s p e c i e s , and s p e c i f i c e l i c i t o r s , those which have d i f f e r e n t i a l e l i c i t o r e f f i c i e n c y , depending on the disease r e s i s tance genotype. Among n o n - s p e c i f i c e l i c i t o r s , we recognize b i o t i c e l i c i t o r s as those of pathogen o r i g i n , and a b i o t i c e l i c i t o r s as those from other sources. In attempts to understand the biochemical processes by which p l a n t s i n i t i a t e phytoalexin production, many a b i o t i c chemicals have been used to e l i c i t phytoalexin production i n s e v e r a l plant systems such as pea pods, bean pods and hypocotyls, sweet potato root d i s c s , soybean cotyledons, hypocotyls, suspension c e l l s , and l e a v e s , j a c k bean c a l l u s , p o t a t o t u b e r s l i c e s and cowpea hypocotyls (92,93,94,95,96). Some of the n o n - s p e c i f i c a b i o t i c chemicals that e l i c i t phytoalexin production i n various of these p l a n t s systems are i n o r g a n i c ions as those of mercury, copper and s i l v e r , metabolic i n h i b i t o r s such as sodium azide and cyanide, DNA i n t e r c a l a t i n g a g e n t s such as a c t i n o m y c i n D, m i t o m y c i n C, and a c r i d i n e orange, b a s i c p r o t e i n s and peptides such as protamine, p o l y - L - l y s i n e , and spermine, and p r o t e i n synthesis i n h i b i t o r s such as cycloheximide and puromycin (89,94,101,102). Some of the compounds such as cycloheximide and actinomycin D e l i c i t phytoalexin production a t low concentrations but a t higher concentrations are i n h i b i t o r y (94,95). One of the problems i n f o r m a l i z i n g a general hypothesis f o r p h y t o a l e x i n i n d u c t i o n from data obtained with a b i o t i c e l i c i t o r s i s that not a l l plant species respond to the same chemicals (96); even d i f f e r e n t t i s s u e s w i t h i n the same p l a n t respond d i f f e r e n t l y — f o r example g l y c e o l l i n production can be e l i c i t e d i n soybean cotyledons with K C r 0 , iodoacetate, SDS, ?

?

7

16

H O S T P L A N T RESISTANCE T O

PESTS

CuCl or H g C l , but only K^Cr^O^ iodoacetate, or C u C l elicit phytoalexin production when i n j e c t e d i n t o soybean l e a v e s — S D S or H g C l , while causing n e c r o s i s , do not e l i c i t detectable quant i t i e s of phytoalexins. Since many a b i o t i c e l i c i t o r s a f f e c t DNA s t r u c t u r e and metabolism, s p e c i f i c gene derepression has been hypothesized by Hadwiger as the mechanism by which phytoalexin production i s e l i c i t e d (89). However, s i n c e most e l i c i t o r s have not been shown to act e x c l u s i v e l y on DNA, " s i d e a f f e c t s " could account f o r t h e i r a c t i v i t y . Another idea i s that the a b i o t i c e l i c i t o r s may put the c e l l under " s t r e s s " — t h a t i s , change the n o r m a l p h y s i o l o g i c a l s t a t e of the p l a n t c e l l environment and thereby cause phytoalexin production. Although s p e c i f i c mechanisms are as yet i n d e f i n i t e and perhaps d i v e r s e , the p r e v i o u s l y d i s c u s s e d work of Yoshikawa and a s s o c i a t e s (69,71,87) p o i n t s to i n v o l v e m e n t of s p e c i f i c DNA t r a n s c r i p t i o n with glyceollin e l i c i t a t i o n i n the Phytophthora—soybean system. Reilly (100) a l s o supported t h i s i n experiments with soybean suspension c u l t u r e c e l l s with UV l i g h t as the e l i c i t i n g agent. Exposure of c e l l s to i n c r e a s i n g doses of UV l i g h t r e s u l t e d i n concomitant increases i n thymine dimer formation and g l y c e o l l i n s y n t h e s i s . Photoreversal experiments on UV-treated c e l l s using v i s i b l e l i g h t showed that thymine dimers and g l y c e o l l i n synthesis a l s o d e c l i n e d i n p a r a l l e l . The observed simultaneous e f f e c t s of UV-light and photoreversal on phytoalexin e l i c i t a t i o n and p h y s i c a l DNA permutation point to DNA as the primary e l i c i t o r target of UV l i g h t . 2

2

2

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

2

A number of apparently n o n - s p e c i f i c b i o t i c e l i c i t o r s have now been r e p o r t e d . P o l y s a c c h a r i d e s from the c u l t u r e f l u i d of 3 s p e c i e s of C o l l e t o t r i c h u m (two of which were non-pathogens) e l i c i t e d p h y t o a l e x i n p r o d u c t i o n on c o t y l e d o n s of bean p l a n t s (98). A l s o , the c u l t u r e f l u i d of Pénicillium expansum (nonpathogen) e l i c i t e d phytoalexin production on bean hypocotyls and pea pods (84) and N e c t r i a g a l l i g e n a Bres. released proteases that e l i c i t e d benzoic a c i d production i n apples (99). Culture f l u i d s as w e l l as s o l u b l e extracts from the mycelia and c o n i d i a of compatible and incompatible s t r a i n s of C e r a t o c y s t i s f i m b r i a t a e l i c i t e d ipomeamarone, ipomeamoronol, and dehydroipomeamarone i n sweet potato root t i s s u e . The e l i c i t o r was s o l u b l e i n water or 0.02M KC1, heat s t a b l e , d i a l y z a b l e , had no a n i o n i c or c a t i o n i c p r o p e r t i e s and caused c e l l u l a r i n j u r y to root t i s s u e (103). I n c o m p a t i b l e and c o m p a t i b l e r a c e s of Colletotrichum l i n d e m u t h i a n u m r e l e a s e d an e l i c i t o r i n t o c u l t u r e f l u i d s t h a t was l a r g e l y g l u c a n i n n a t u r e (104) and e f f i c i e n t l y e l i c i t e d p h a s e o l l i n production i n green beans. A s i m i l a r e l i c i t o r was a l s o i s o l a t e d from p u r i f i e d m y c e l i a l c e l l w a l l preparations of the fungus. Incompatible and compatible races of Phytophthora megasperma v a r . sojae produce a h e a t - s t a b l e 1,3-glucan that i s an extremely potent n o n - s p e c i f i c e l i c i t o r of the soybean phytoalexin g l y c e o l l i n

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

1.

KEEN

AND

BRUEGGER

Phytoalexin

Production

in

Plants

17

(105,106,107). Valent and Albersheim d i s c u s s the work elsewhere i n t h i s volume. S t e k o l l and West (108) r e c e n t l y obtained an e l i c i t o r of casbene synthesis i n castor bean from the non-pathogenic fungus Rhizopus s t o l o n i f e r that, u n l i k e the glucan e l i c i t o r s , was heat l a b i l e . The p a r t i a l l y p u r i f i e d e l i c i t o r appeared to be a g l y c o p r o t e i n of about 23,000 m o l e c u l a r w e i g h t and was a c t i v e a t 0.15 yg/ml. Varns et a l . (96) showed that s o n i c a t e s and homogenates of Phytophthora i n f e s t a n s e l i c i t e d production of phytoalexins i n potato tuber t i s s u e s , but other chemicals that caused n e c r o s i s d i d not n e c e s s a r i l y f u n c t i o n as e l i c i t o r s . L i s k e r and Kuc (16) a l s o observed that potatoes were r e l a t i v e l y i n s e n s i t i v e to some of the a b i o t i c and b i o t i c e l i c i t o r s that were e f f e c t i v e i n leguminous p l a n t s such as p o l y - L - l y s i n e and p o l y s a c c h a r i d e s such as the JP. megasperma v a r . sojae glucan (105). Only autoclaved sonicates and h e a t - k i l l e d fungi having g l u c a n - c e l l u l o s e w a l l s were a c t i v e as e l i c i t o r s of the potato p h y t o a l e x i n s . This perhaps i n d i c a t e s that potato responds to a narrower range of e l i c i t o r s than leguminous plants· Among n o n - s p e c i f i c p h y t o a l e x i n e l i c i t o r s , the f a c t i s emerging that b i o t i c e l i c i t o r s are g e n e r a l l y much more a c t i v e than a b i o t i c e l i c i t o r s — b y s e v e r a l orders of magnitude i n some cases (105,108). Although t h i s may argue f o r a p h y s i o l o g i c r o l e f o r the former, they are produced both by compatible and incompatible pathogen races and s t r a i n s , are often only recovered from fungus mycelium by h a r s h e x t r a c t i o n t r e a t m e n t s , and show no h o s t s p e c i f i c i t y (98,105). A l l t h e s e f a c t o r s would appear t o e x c l u d e a r o l e for n o n - s p e c i f i c e l i c i t o r s i n the determination of gene-for-gene specificity (see a l s o 109) but they s h o u l d n o n e t h e l e s s be u s e f u l i n s t u d i e s of e l i c i t a t i o n mechanisms i n p l a n t s and could be involved i n the c o n f e r r a l of general r e s i s t a n c e . There i s a p a u c i t y of data on s p e c i f i c p h y t o a l e x i n e l i c i t o r s . The b e s t known i s a g e n u s s p e c i f i c e l i c i t o r i s o l a t e d by Cruickshank and colleagues (110). A p o l y p e p t i d e , m o n i l i c o l i n A, was p u r i f i e d from the c u l t u r e f l u i d of M o n i l i n i a f r u c t i c o l a (a non-pathogen o f P h a s e o l u s v u l g a r i s ) and was shown to e l i c i t p h a s e o l l i n a c c u m u l a t i o n i n g r e e n bean, but n e i t h e r e l i c i t e d p i s a t i n i n peas (Pisum sativum) nor phytoalexins i n broad bean ( V i c i a faba) (110). An a c e t o n e p r e c i p i t a t e d s u p e r n a t a n t of the homogenate of R h i z o c t o n i a v e r s i c o l a r (a non-pathogenic fungus asociated with r o o t s o f the o r c h i d L o r o g l o s s u m h i r c i n u m ) e l i c i t e d h i r c i n o l synthesis i n s t e r i l e t i s s u e of the o r c h i d bulb. A s i m i l a r homogenate from R h i z o c t o n i a s o l a n i , a fungus which destroys the bulbs of 1^. hircinum, d i d not e l i c i t the p h y t o a l e x i n — a case of f u n g a l species s p e c i f i c i t y (111). S t r a i n s p e c i f i c i t y was observed i n the Fusarium s o l a n i - p e a system (112). The c u l t u r e f l u i d of a v i r u l e n t Fusarium s o l a n i s t r a i n s e l i c i t e d l a r g e r q u a n t i t i e s of p i s t i n than d i d the c u l ture f l u i d from v i r u l e n t s t r a i n s ; however, the d i f f e r e n c e was

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

18

HOST

PLANT

RESISTANCE T O PESTS

apparently due to q u a n t i t i e s of the same rather than d i f f e r e n t types of e l i c i t o r s . Keen (113,114) detected a race s p e c i f i c phytoalexin e l i c i t o r i n c u l t u r e f l u i d s of race 1 of Phytophthora megasperma v a r . sojae but not from race 3 of the fungus (see Figure 2 ) . The p a r t i a l l y p u r i f i e d race 1 f a c t o r e l i c i t e d greater production of the soybean p h y t o a l e x i n g l y c e o l l i n i n Rps genotype soybeans than i n r p s — t h a t i s , the same phytoalexin s p e c i f i c i t y on these genotypes as by the l i v i n g fungus. Despite t h i s i n i t i a l encouragement, the race 1 s p e c i f i c e l i c i t o r has p r o v e d i n s u f f i c i e n t l y s t a b l e t o a l l o w i s o l a t i o n and i d e n t i f i c a t i o n and a genetic cross of the two fungus races designed to c r i t i c a l l y t e s t the a s s o c i a t i o n of the race 1 s p e c i f i c e l i c i t o r with the race 1 phenotype has f a i l e d on t e c h ­ n i c a l grounds (115,116). Proof of involvement of the metabolite i n pathogenesis i s t h e r e f o r e l a c k i n g . Nevertheless, the i n i t i a l success prompted the hypothesis t h a t , i n gene-for-gene systems, the determinative phase i n v o l v e s production of s p e c i f i c e l i c i t o r s by the p a r a s i t e that are unique f o r each dominant a v i r u l e n c e gene and s p e c i f i c r e c e p t o r s by t h e p l a n t t h a t a r e u n i q u e f o r each dominant r e s i s t a n c e gene ( F i g u r e 5 ) . T h i s i s , o r c o u r s e , a molecular c o n c e p t u a l i z a t i o n of the gene-for-gene concept (Figure 2) and there i s nothing b i o l o g i c a l l y unusual about i t . S i m i l a r v e r s i o n s have been proposed p r e v i o u s l y by s e v e r a l workers (104, 109,117,118), and i t should be noted that i t s involvement i n the d e t e r m i n a t i v e phase c o u l d o c c u r i r r e s p e c t i v e of whether t h e expressive phase involved d i f f e r e n t i a l phytoalexin production or some other, as y e t unrecognized mechanism(s) f o r r e s t r i c t i o n of p a t h o g e n growth. The s p e c i f i c e l i c i t o r - s p e c i f i c r e c e p t o r mechanism a l s o i s v i r t u a l l y i d e n t i c a l as a r e c o g n i t i o n system to that c u r r e n t l y proposed f o r c e r t a i n p o l l e n - s t y l e i n c o m p a t i b i l i t y systems i n higher p l a n t s governed by s i n g l e genes (119,120), f o r Rhiζobium-legume s p e c i f i c i t y (see 121), and f o r receptor systems f o r c e r t a i n h o s t - s p e c i f i c p l a n t t o x i n s (122). Also i n common with these systems, the e l i c i t o r s and receptors most l i k e l y are c o n s t i ­ t u t i v e metabolites that are e i t h e r present on the c e l l w a l l or plasmalemma s u r f a c e s o r , i n t h e case of e l i c i t o r s , might be e x t r a c e l l u l a r metabolites. T h i s i s due to the r e l a t i v e l y short t i m e s a v a i l a b l e f o r r e c o g n i t i o n ; f o r example i n t h e s o y b e a n Phytophthora system, events of the determinative phase are con­ cluded by about 4 hours a f t e r i n i t i a l contact of host and p a r a s i t e (Figure 4 ) . Important recent research by Goodman and co-workers (123), Sing and Schroth (124) and Sequeira's group (125) s t r o n g l y suggests that s p e c i f i c host receptor substances, p o s s i b l y l e c t i n s (104,120), may confer s p e c i f i c i t y i n tobacco, green bean and potatoes to incompatible s t r a i n s of Pseudomonas spp. Although these are not regarded as gene-for-gene systems and the p o s s i b l e r o l e of phyto­ a l e x i n s i n the expressive phase has not been w e l l i n v e s t i g a t e d , the r e s u l t s demonstrate the s p e c i f i c r e c o g n i t i o n and "attachment"

1.

KEEN

AND

Phytoalexin

BRUEGGER

Production

R = Resistance gene (dominant) r - recessive allele

Host f

Resistance genotype

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

[r'r'

r

2

r

Resistance genotype

r

i

NO

W R Specific receptor #1

Specific elicitors#i a#2

A A 1

1

AAH

R R 2

1

1

Τ

2

2

z

RRRR

t

cfo 1

s Resistance genotype R' R R R |

2

Specific elicitor#2

(

Roce | A' A

1

ÛÛ

A 2

r

R

ÛÛ

t

1

Specific receptor #2

Specific elicitor # I

ûûûû I

Resistance genotype

2

Specific receptors

Roce

19

in Plants

Specific # 1 8 receptors #2

c

N

.°..

enVi'.or's (

Race | ο Ό Ά 2 Α 2 |

t

Roce | o ' o ' c f o

v

Parasite

^ Avirulence gene (dominant) a = recessive virulence gene =

Figure 5. Scheme of the specific elicitor-specific receptor hypothesis for determina­ tion of the hypersensitive reaction in gene-f or-gene plant-parasite interactions, consid­ ering two resistance loci in the plant and two complementary virulence loci in the parasite. Host resistance genes R and R code either directly or indirectly for specific and unique receptor substances 1 and 2, possibly proteins, that specifically recognize and bind specific phytoalexin elicitors 1 or 2 from the pathogen. The hypothesis assumes that only the domi­ nant plant resistance alleles make functional receptors. Correspondingly, for the parasite, the virulence genes code for unique specific elicitors that are specifically recognized by the com­ plementary plant receptors. Only the dominant parasite avirulence alleles make functional gene products. Plant disease resistance (R) (rapid phytoalexin production) will be expressed in any combination that involves production of either specific elicitor and its complementary receptor. On the other hand, absence of a specific elicitor or its specific receptor will lead to repressed phytoalexin production and a susceptible plant response. Thus, as the gene-forgene relationship dictates, in the absence of functional resistance genes in the host or func­ tional avirulence genes in the parasite, susceptible reactions will be observed, regardless of the genotype of the complementary partner. 1

2

2

20

H O S T P L A N T RESISTANCE T O

PESTS

of incompatible but not compatible b a c t e r i a . With the potato system, Sequeira and Graham (126) have shown that both compatible and incompatible s t r a i n s of Pseudomonas solanacearum possess s t r u c t u r a l features i n t h e i r w a l l s that bind to a potato l e c t i n , but only the compatible s t r a i n s possess an e x t r a c e l l u l a r polysaccharide that "masks" the l e c t i n b i n d i n g elements, thereby l e a d i n g to nonr e c o g n i t i o n and e v e n t u a l l y to s u s c e p t i b i l i t y of the host. Although i t i s not c l e a r whether these f i n d i n g s w i l l be d i r e c t l y a p p l i c a b l e to gene-for-gene h o s t - p a r a s i t e systems, they encourage f u r t h e r research on the s p e c i f i c i t y f a c t o r s hypothesized i n Figure 5.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

Conclusions and Future D i r e c t i o n s We conclude that phytoalexins c o n s t i t u t e a l e g i t i m a t e defense mechanism i n c e r t a i n p l a n t s that i s important i n both general and s p e c i f i c disease r e s i s t a n c e i n c e r t a i n p l a n t s . However, because of the r e l a t i v e l y l i m i t e d r e s e a r c h done, i t i s i m p o s s i b l e to assess how wide-spread phytoalexin-mediated r e s i s t a n c e i s i n the p l a n t kingdom. In the more e x t e n s i v e l y studied examples i n the S o l a n a c e a e and Leguminosae, however, t h e r e i s now c o n c l u s i v e evidence i m p l i c a t i n g p h y t o a l e x i n production with the expression of disease r e s i s t a n c e . We think that i t i s time to take t h i s as a p r o v i s i o n a l dogma, along with the c o r o l l a r y s t a t i n g that s u s c e p t i b i l i t y i s o f t e n due to the f a i l u r e of the p l a n t to make phytoa l e x i n s f a s t enough, and consider how we can use phytoalexins as a v e h i c l e to o b t a i n p r a c t i c a l p l a n t disease c o n t r o l i n the f i e l d . Several approaches to t h i s have surfaced but none i s yet near p r a c t i c a l and most i f not a l l of those devised so f a r are not l i k e l y t o become s o . However, a s t a r t has been made. Since p h y t o a l e x i n s a r e a n t i b i o t i c , one c o u l d c h e m i c a l l y s y n t h e s i z e phytoalexins and apply these to p l a n t s as " n a t u r a l p e s t i c i d e s " . T h i s has been done e x p e r i m e n t a l l y i n one case (127), but the approach i s g e n e r a l l y fraught with p i t f a l l s . F i r s t , most phytoalexins are not r e a d i l y synthesized, many having asymmetric carbons and r e l a t i v e l y l a b i l e f u n c t i o n a l g r o u p s — t h e y would not be cheap to make. Most d i s t u r b i n g , however, i s the f a c t that phytoa l e x i n s a r e not i n n o c u o u s c h e m i c a l s , d e s p i t e b e i n g " n a t u r a l p e s t i c i d e s " . As discussed e a r l i e r , at l e a s t some phytoalexins are membrane antagonists and s e v e r a l of them have been shown to be potent t o x i c a n t s i n mammals ( f o r example, 32,128,129); c l e a r l y they are not the s o r t of thing to be spread around the e n v i r o n ment, e s p e c i a l l y onto food crop p l a n t s . Instead of spraying phytoalexins on p l a n t s , Albersheim (see 130) suggests applying phytoalexin e l i c i t o r s , a n t i c i p a t i n g that the e l i c i t e d phytoalexins would ward o f f p o s s i b l e pathogens. The a p p r o a c h may have m e r i t but would be p l a g u e d by methods f o r applying the e l i c i t o r s and by dosage requirements. The danger i s that as e l i c i t o r doses are increased to l e v e l s p o s s i b l y p r o v i d i n g disease c o n t r o l , one w i l l l i k e l y observe p h y t o t o x i c i t y as a r e s u l t of the phytoalexins produced. As discussed, phytoalexins are

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

1.

KEEN

AND

BRUEGGER

Phytoalexin

Production

in

Plants

21

normally a s s o c i a t e d with the n e c r o t i c , h y p e r s e n s i t i v e r e a c t i o n i n p l a n t s ; i n the normal HR, a plant can e a s i l y a f f o r d to l o s e a few of i t s c e l l s to contain pathogens, but what happens i f an a p p l i e d e l i c i t o r causes wholesale h y p e r s e n s i t i v e necrosis? or i f foods t u f f s contain s i g n i f i c a n t q u a n t i t i e s of the e l i c i t e d phytoalexins? A. A. B e l l (unpublished) has suggested a p o t e n t i a l l y u s e f u l approach to harnessing phytoalexins based on the observed f a c t that s e v e r a l p l a n t s make m u l t i p l e , r e l a t e d phytoalexins. Bell suggests that disease r e s i s t a n c e plant breeding programs could attempt t o s e l e c t p l a n t s t h a t p r e f e r e n t i a l l y made f a v o r a b l e p h y t o a l e x i n s toward the pathogen i n q u e s t i o n — f o r instance, against a b a c t e r i a l pathogen, breed f o r genotypes that p r e f e r e n t i a l l y produce potent a n t i b a c t e r i a l phytoalexins; against fungus diseases, s e l e c t f o r p r e f e r e n t i a l production of the most a n t i f u n g a l phytoalexins; perhaps i t would even be p o s s i b l e to s e l e c t p l a n t s making p h y t o a l e x i n s t h a t a pathogen c o u l d not degrade, thereby c o n f e r r i n g general disease r e s i s t a n c e . A f i n a l approach to harnessing phytoalexins i n disease c o n t r o l i n v o l v e s applying chemicals to s u s c e p t i b l e p l a n t s that are n e i t h e r a n t i b i o t i c nor p h y t o a l e x i n e l i c i t o r s , but i n t e r a c t w i t h the p l a n t - p a r a s i t e i n t e r a c t i o n to cause higher production of phytoa l e x i n s by the p l a n t c e l l s and thus impart r e s i s t a n c e . Because of t h i s behavior we propose to c a l l such chemicals " s e n s i t i z e r s " . There are suggestions that such chemicals already e x i s t . For i n s t a n c e , P r i n g and Richmond (131) found t h a t bean p l a n t s i n o c u l a t e d with a normally compatible race of Uromyces p h a s e o l i r e s p o n d e d to t r e a t m e n t w i t h the weakly a n t i f u n g a l f u n g i c i d e oxycarboxin i n the same way as g e n e t i c a l l y r e s i s t a n t p l a n t s , thus implying that oxycarboxin somehow caused i n v o c a t i o n of a normal d e f e n s e r e a c t i o n . Langcake and W i c k i n s (132,133) have shown that 2,2-dichloro-3,3-dimethyl cyclopropane c a r b o x y l i c a c i d c o n t r o l s the b l a s t d i s e a s e caused by P i r i c u l a r i a o r y z a e on compatible r i c e v a r i e t i e s , but acts n e i t h e r as a f u n g i c i d e nor as a phytoalexin e l i c i t o r . However, i n o c u l a t e d p l a n t s that were t r e a t e d with the cyclopropane were f u l l y r e s i s t a n t to the disease, and, u n l i k e u n t r e a t e d i n o c u l a t e d p l a n t s or t r e a t e d but noni n o c u l a t e d p l a n t s , accumulated momilactones A and B, which appear to be phytoalexins i n r i c e . There are s e v e r a l other l e s s w e l l defined but p o s s i b l e examples of s e n s i t i z e r s i n the l i t e r a t u r e , but we do not yet know i f the concept i s sound or i f the required chemicals can be found. I t i s an idea worthy of c o n s i d e r a t i o n , however, s i n c e chemicals such as Langcake's cyclopropane appear r e l a t i v e l y i n n o c u o u s e c o l o g i c a l l y , and do not appear to have d e l e t e r i o u s e f f e c t s on p l a n t s that are not challenged by pathogens. The a u t h o r s ' r e s e a r c h i s Foundation grant BMS 75-03319.

supported

by

National

Science

22

1. 2. 3. 4. 5. 6. 7. Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

HOST P L A N T RESISTANCE T O PESTS

Literature Cited Müller, Κ. O. and Börger, H. (1940). Arb. Biol. Reichsanst. Land-u. Forstwiss. 23, 189-231. Christensen, T. G. and Sproston, T. (1971). Phytopathology 62, 493-494. Deverall, B. J. "Defence mechanisms of plants." Cambridge Univ. Press. London. (1977), Cruickshank, I. A. M. (1963). Annual Rev. Phytopathol. 1, 351-374. Deverall, B. J. (1972). Proc. Roy Soc. Lond. B181, 233-246. Ingham, J. L. (1972). Botan. Rev. 38, 343-424. Kuc, J. "Phytoalexins", p. 632-652, In Physiological Plant Pathology, Edit, by Heitefuss, R. and Williams, P. H. (1976). Kuc, J. and Currier, W. (1976). Adv. Chem. Ser. 149, 356-368. Wood, R. K. S. (1973). Mitteil. Biologischen Bund. fur Land. u. Fortschaft 154, 95-105. Swain, T. (1977). Annual Rev. Pit. Physiol. 28, 479-501. Biggs, R. T. (1975). Austr. J. Chem. 28, 1389-1391. Wyman, J. and Van Etten, H. D. (1975). Proc. Amer. Phytopath. Soc. 2, 110. Lyon, F. M. and Wood, R. K. S. (1975). Physiol. Plant Pathol. 6, 117-124. Patil, S. S. and Gnanamanickam, S. S. (1976). Nature 259, 486-487. Ward, Ε. W. B. and Stoessl, A. (1977). Phytopathology 67., 468-471. Lisker, N. and Kuc, J. (1977). Phytopathology (in press). Harris, J. Ε. and Dennis, C. (1976). Physiol. Plant Pathol. 9, 155-165. Ishizaka, N., Tomiyama, K., Katsui, N., Murai, A. and Masamune, T. (1969). Pit. Cell Physiol. 10, 183-192. Elliston, J., Kuc, J., Williams, Ε. B., and Rahe, J. Ε. (1977). Phytopathol. Z. 88, 114-130. Keen, Ν. T. p. 268. In "Specificity in Plant Diseases." Ed. by Wood, R. K. S. and Graniti, A. Plenum Press, New York (1976). Stoessl, Α., Stothers, J. Β., and Ward, E. W. B. (1976). Phytochemistry 15, 855-872. Price, K. R., Howard, B. and Coxon, D. T. (1976). Physiol. Plant Pathol. 9., 189-197. Rich, J. R., Keen, Ν. T. and Thomason, I. J. (1977). Physiol. Plant Pathol. 10, 105-116. Keen, Ν. T. (1975). Phytopathology 65, 91-92. Skipp, R. A. and Bailey, J. A. (1976). Physiol. Plant Pathol. 9., 253-263. Bailey, J. Α., Carter, G. Α., and Skipp, R. A. (1976). Oku, H., Shiraishi, T., and Ouchi, S. (1976). Ann. Phytopath. Soc. Japan 42, 597-600.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

1. KEEN AND BRUEGGER

Phytoalexin Production in Phnts 23

28. Klarman, W. L. and F. Hammerschlag. (1972). Phytopathology 62, 719-721. 29. Bailey, J. A. and Ingham, J. L. (1971). Physiol. Plant Pathol. 1, 451-456. 30. Kojima, R., Fukushima, S., Neno, A. and Saiki, Y. (1970). Chem. Pharm. Bull. Japan 18, 2555. 31. Van Etten, H. D. (1972). Phytopathology 62, 795. 32. Van Etten, H. D. and Bateman, D. F. (1971). Phytopathology 51, 1363-1372. 33. Shiraishi, T., Oku, H., Isono, M. and Ouchi, S. (1975) Plt. Cell Physiol. 16, 939-942. 34. El Naghy, M. A. and Heitefuss, R. (1976). Physiol. Plant Pathol. 8, 269-277. 35. Perrin, D. R. and Cruickshank, I. A. M. (1969). Phytochemistry 8, 971-978. 36. Van Etten, H. D. (1976). Phytochemistry 15, 655-659. 37. Fukui, H., Egawa, H., Koshimizu, K. and Mitsui, T. (1973). Agr. Biol. Chem. 37, 417-421. 38. Ingham, J. L., Keen, N. T., and Hymowitz, T. (1977). Phytochemistry (in press). 39. Bell, A. A. and Presley, J. T. (1969). Phytopathology 59, 1141-1146. 40. Keen, Ν. T. (1971). Physiol. Plant Pathol. 1, 265-276. 41. Nakajima, T., Tomiyama, K. and Kinukawa, M. (1975). Ann. Phytopath. Soc. Japan 41, 49-55. 42. Tomiyama, Κ., and Fukaya, M. (1975). Ann. Phytopathol. Soc. Japan 41, 418-420. 43. Imaseki, H. and Uritani, I. (1964). Plt. Cell Physiol. 5, 133-143. 44. Shiraishi, T., Oku, H., Ouchi, S. and Isono, M. (1976). Ann. Phytopath. Soc. Japan 42, 609-612. 45. Mansfield, J. W., Hargreaves, J. Α., and Boyle, F. C. (1974). Nature 252, 316-317. 46. Paxton, J., Goodchild, D. J., and Cruickshank, I. A. M. (1974). Physiol. Plant Pathol. 4, 167-171. 47. Klarman, W. L. and Gerdemann, J. W. (1963). Phytopathology 53, 863-864. 48. Higgins, V. J. and Millar, R. L. (1968). Phytopathology 58, 1377-1383. 49. Ersek, T., Barna, Β., and Kiraly, Z. (1973). Acta Phytopath. Acad. Sci. Hung. 8, 3-12. 50. Barna, B., Ersek, T., and Mashaal, S. F. (1974). Acta Phytopathol. Acad. Sci. Hung. 9, 293-300. 51. van der Plank, J. E. "Principles of Plant Infection". Academic Press, New York. (1975). 52. Kim, W. K., Rohringer, R., Samborski, D. J. and Howes, Ν. K. (1977). Can. J. Bot 55: 568-573. 53. Doke, N., Nakae, Y., and Tomiyama, K. (1976). Phytopathol. Z. 87, 337-344.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

24

HOST PLANT RESISTANCE TO PESTS

54. Day, P. R. "Genetics of host-parasite interaction". Freeman, San Francisco (1974). 55. Rowell, J. B., Loegering, W. Q., and Powers, H. R. Jr. (1963). Phytopathology 53, 932-937. 56. Flor, H. H. (1971). Annual Rev. Phytopathol. 9, 275-296. 57. Heale, J. B. and Sharman, S. (1977). Physiol. Plant Pathol. 10, 51-61. 58. Van Etten, H. D. (1973). Phytopathology 63, 1477-1482. 59. Heuvel, J. van den (1976). Neth. J. Plant Pathol. 82, 153-160. 60. Mansfield, J. W. and Widdowson, D. A. (1973). Physiol. Plant Pathol. 3, 393-404. 61. Hargreaves, J. Α., Mansfield, J. W., Coxon, D. T., and Price K. R. (1976). Phytochemistry 15, 1119-1121. 62. Jones, D. R., Unwin, C. H. and Ward, E. W. B. (1975). Phytopathology 65, 1286-1288. 63. Kojima, M. and Uritani, I. (1976). Physiol. Plant Pathol. 8, 97-111. 64. Chamberlain, D. W. (1972). Phytopathology 62, 645-646. 65. Chamberlain, D. W. and Gerdemann, J. W. (1966). Phytopathology 56, 70-73. 66. Talboys, P. W. (1957). Trans. Brit. Mycol. Soc. 40, 415-427. 67. Bailey, J. A. and Deverall, B. J. (1971) Physiol. Plant Pathol. 1, 435-449. 68. Rahe, J. E. (1973). Can. J. Bot. 51, 2423-2430. 69. Yoshikawa, M. (1977). Plt. Physiol. (submitted). 70. Shiraishi, T., Oku, H., Ouchi, S., and Tsuji, Y. (1977). Phytopathol. Z. 88, 131-135. 71. Yoshikawa, Μ., Yamauchi, Κ., and Masago, H. (1977). Physiol. Plant Pathol. (Submitted). 72. Chamberlain, D. W. and Paxton, J. D. (1968). Phytopathology 58, 1349-1350. 73. Sitton, D. and West, C. A. (1975). Phytochemistry 14, 1921-1925. 74. Klarman, W. L. and Gerdemann, J. W. (1963). Phytopathology 53, 1317-1320. 75. Paxton, J. D. and Chamberlain, D. W. (1967). Phytopathology 57, 352-353. 76. Bridge, M. A. and Klarman, W. L. (1973). Phytopathology 63, 606-609. 77. Klarman, W. L. and Sanford, J. B. (1968). Life Sci. 7, 1095-1103. 78. Keen, N. T., Sims, J. J., Erwin, D. C., Rice, E. and Partridge, J. E. (1971). Phytopathology 61, 1084-1089. 79. Sims, J. J., Keen, N. T., and Honwad, V. K. (1972). Phytochemistry 11, 827-828. 80. Burden, R. S. and Bailey, J. A. (1975). Phytochemistry 14, 1389-1390.

1. KEEN AND BRUEGGER

Phytoalexin Production in Plants 25

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

81. Lyne, R. L., Mulheirn, L. J. and LeWorthy, D. P. (1976). JCS Chem. Comm. p. 497-498. 82. Keen, Ν. T. and Kennedy, B. W. (1974). Physiol. Plant Pathol. 4, 173-185. 83. Keen, N. T., Zaki, A. I., and Sims, J. J. (1972). Phytochemistry, 11, 1031-1039. 84. Rathmell, W. G. and Bendall, D. S. (1971) Physiol. Plant Pathol. 1, 351-362. 85. Keen, Ν. T. and Paxton, J. D. (1975). Phytopathology 65, 635-637. 86. Frank, J. A. and Paxton, J. D. (1970) Phytopathology 60, 315-318. 87. Yoshikawa, Μ., Masago, Η., and Keen, Ν. T. (1977). Physiol. Plant Pathol. 10, 125-138. 88. Partridge, J. E. and Keen, Ν. T. (1977). Phytopathology 67, 50-55. 89. Schwochau, M. E., and Hadwiger, L. A. (1969). Arch. Biochem. Biophys. 134, 34-41. 90. Biggs, D. R. (1972). Plt. Physiol. 50, 660-666. 91. Uritani, I., Uritani, M. and Yamada, H. (1960). Phytopathology 50, 30-34. 92. Gustine, D. L. (1976). Fed. Proc. 35, 1552. 93. Munn, C. B., and Drysdale, R. B. (1975). Phytochemistry 14,1303-1307. 94. Bailey, J. A. (1969). Phytochemistry. 8, 1393-1395. 95. Hadwiger, L. Α., and Schwochau, M. E. (1971). Plt. Physiol. 47, 346-351. 96. Varns, J. L., Currier, W. W. and Kuc, J. (1971). Phyto­ pathology 61, 968-971. 97. Oguni, I., Suzuki, Κ., and Uritani, I. (1976). Agr. Biol. Chem. 40, 1251-1252. 98. Anderson, A. (1976). Proc. Amer. Phytopath. Soc.3,314. 99. Swinburne, T. R. (1975). Phytopathol. Z. 82, 152-162. 100. Reilly, J. J. (1975). Diss. Abstr. B36, 2552. 101. Hadwiger, L. A. & Schwochau, M. E. (1970). Biochem. Biophys. Res. Comm. 38, 683-691. 102. Perrin, D. R., and Cruickshank, I. A. M. (1965). Austr. J. Biol. Sci.18,803-816. 103. Kim, W. Κ., and Uritani, I. (1974). Plt. Cell Physiol. 15, 1093-1098. 104. Albersheim, P., and Anderson-Prouty, A. J. (1975). Ann. Rev. Plt. Physiol. 26, 31-52. 105. Ayers, A. R., Ebel, J., Finelli, F., Berger, Ν. and Albersheim, P. (1976). Plt. Physiol. 57, 751-759. 106. Ayers, A. R., Ebel, J., Valent, Β. and Albersheim, P. (1976). Plt. Physiol. 57, 760-765. 107. Ayers, A. R., Valent, Β., Ebel, J., and Albersheim, P. (1976). Plt Physiol. 57, 766-774. 108. Stekoll, M. (1976). Ph.D. Dissertation, Univ. of California, Los Angeles.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch001

26

HOST PLANT RESISTANCE TO PESTS

109. Wood, R. K. S. p. 327, In "Specificity in Plant Diseases". Edit. by Wood, R. K. S. and Graniti, A. Plenum, New York (1976). 110. Cruickshank, I. Α. Μ., and Perrin, D. R. (1968). Life Sci. 7, part II 449-458. 111. Nuesch, J. (1963). Soc. Gen. Microbiol. Symp. 13, 335-343. 112. Daniels, D. L., and Hadwiger, L. A. (1976). Physiol. Plt. Pathol. 8, 9-19. 113. Keen, Ν. T. (1975). Science 187, 74-75. 114. Keen, N. T., Partridge, J. E., and Zaki, A. I. (1972). Phytopathology 62, 768. 115. Long, M. and Keen, Ν. T. (1977). Phytopathology 67, 670-674. 116. Long, M. and Keen, Ν. T. (1977). Phytopathology 67, 675-677. 117. Ercolani, G. L. (1970). Phytopathol. Med. 9, 151-159. 118. Hadwiger, L. A. and Schwochau, M. E. (1969). Phytopathology 59, 223-227. 119. Heslop-Harrison, J., Heslop-Harrison, Y., and Barker, J. (1975). Proc. Roy. Soc. B188, 287-297. 120. Linskens, H. F. p. 311, In "Specificity in Plant Diseases." Edit. by Wood, R. K. S. and Graniti, A. Plenum, New York. (1976). 121. Dazzo, F. B. and Brill, W. J. (1977). Appl. Environ. Microbiol. 33, 132-136. 122. Strobel, G. A. (1975). Sci. Amer. 232, 80-89. 123. Goodman, R. N., Huang, P. and White, J. A. (1976). Phytopathology 66, 754-764. 124. Sing, V. and Schroth, M. N. (1977). Science (In press). 125. Sequeira, L., Gaard, G. and De Zoeten, G. A. (1977). Physiol. Plant Pathol. 10, 43-50. 126. Sequeira, L. and Graham, T. C. (1977). Physiol. Plant Pathol. (in press). 127. Ward, E. W. B., Unwin, C. H. and Stoessl, A. (1975). Phytopathology 65, 168-169. 128. Uritani, I. (1967). J. Assoc. Agric. Chemists 50, 105-113. 129. Beradi, L. C. and Goldblatt, L. A. p. 212. In "Org. Const. Plt. Foodstuffs." Liener, I. E. (ed.). Academic Press, New York. (1969). 130. Maugh, T. H. (1976). Science 192, 874-876. 131. Pring, R. J. and Richmond, D. V. (1976). Physiol. Plant. Pathol. 8, 155-162. 132. Langcake, P. and Wickins, S. G. A. (1975). Physiol. Plant Pathol. 7, 113-126. 133. Langcake, P. (1977). Nature 267, 511. 134. Kiraly, Z., Barna, B., and T. Ersek. (1972) Nature 239, 456-458.

2 Role of Elicitors of Phytoalexin Accumulation in Plant Disease Resistance

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

BARBARA S. VALENT and PETER ALBERSHEIM Department of Chemistry, University of Colorado, Boulder, CO 80309 Plants are exposed to attack by an immense array of microorganisms, and yet plants are resistant to almost all of these potential pests. Many plant tissues have been observed to respond to an invasion by a pathogenic or nonpathogenic microorganism, whether a fungus, a bacterium or a virus, by accumulating phytoalexins, low molecular weight compounds which inhibit the growth of microorganisms. The production of phytoalexins appears to be a widespread mechanism by which plants attempt to defend themselves against pests (1, 2, 3). The molecules of microbial origin which trigger phytoalexin accumulation in plants have been called elicitors (4). Plants recognize and respond to elicitors as foreign molecules. It is highly improbable that plants have evolved separate recognition systems for every bacterial species and strain and every fungal race and every virus that plants are exposed to. Thus, elicitors are likely to be molecules common to many microbes and, in fact, the one to be described in this paper is a fungal polysaccharide, a polysaccharide which is a structural component of the mycelial walls of many fungi. Most plants produce several structurally related phytoalexins. The most studied phytoalexin of soybeans is glyceollin (5). Lyne et al. (6) have characterized two additional soybean phytoalexins which are structural isomers of glyceollin and which appear to have similar antibiotic characteristics. The synthesis of glyceollin, a phenylpropanoid derivative, is probably initiated from phenylalanine via the reaction catalyzed by phenylalanine ammonia-lyase, but, as yet, no biosynthetic pathway for the production of a phytoalexin has been completely described. Steven Thomas, in our laboratory, has been studying the effect of glyceollin on a variety of microorganisms, for the mechanism by which phytoalexins work is unknown. Glyceollin is a static agent rather than a toxic agent, a trait which seems to be common to many phytoalexins. Glyceollin inhibits the growth, in vitro, of the soybean pathogen, Phytophthora megasperma var. sojae (Pms), the causal agent of root and stem rot. In addition, 27

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

28

H O S T P L A N T RESISTANCE

T O PESTS

Steven has found that g l y c e o l l i n w i l l stop the growth of three Gram-negative b a c t e r i a , Pseudomonas g l y c i n e a , Rhizobium t r i f o l i i , and Rhizobium japonicum, of the Gram-positive bacterium, B a c i l l u s s u b t i l i s , and of baker's y e a s t , Saccharomyces c e r e v i s i a e . I n t e r e s t i n g l y , i t requires about 25 yg/ml of g l y c e o l l i n to i n h i b i t by 50% and 100 yg/ml to i n h i b i t by 100% the growth of a l l of these d i f f e r e n t organisms. Thus, i t appears that a p l a n t ' s phytoalexins can p o t e n t i a l l y p r o t e c t the plant from a broad spectrum of microorganisms. G l y c e o l l i n i s accumulated by soybean t i s s u e s i n response to i n f e c t i o n by Pms, the soybean pathogen. G l y c e o l l i n accumulates i n soybean hypocotyls, w i t h i n 9 hours of i n f e c t i o n w i t h Pms myc e l i a , to l e v e l s which are i n h i b i t o r y to the growth of Pms i n vitro. A component of Pms m y c e l i a l w a l l s has been demonstrated to s t i m u l a t e g l y c e o l l i n accumulation at the same r a t e as l i v e Pms mycelia. This observation and other data have convinced us that t h i s m y c e l i a l w a l l component i s r e s p o n s i b l e f o r t r i g g e r i n g g l y c e o l l i n accumulation during i n f e c t i o n by the l i v e fungus; and, t h e r e f o r e , we b e l i e v e that the m y c e l i a l w a l l component i s the n a t u r a l e l i c i t o r of t h i s system. Three d i f f e r e n t soybean t i s s u e s respond to the Pms e l i c i t o r by accumulating g l y c e o l l i n , and these have been used f o r b i o l o g i c a l assays of e l i c i t o r a c t i v i t y . An assay using 8-day o l d cotyledons (seed leaves) was used f o r the p u r i f i c a t i o n of the e l i c i t o r s i n c e t h i s was the l e a s t l a b o r i o u s assay developed (7). A second bioassay uses the hypocotyls (upper stems) of 5-day o l d soybean s e e d l i n g s (7) and a t h i r d assay uses suspension-cultured soybean c e l l s (8). In a l l three assays, the p r o d u c t i o n of g l y c e o l l i n i s p r o p o r t i o n a l to the amount of e l i c i t o r a p p l i e d . The time course of e l i c i t o r - s t i m u l a t e d g l y c e o l l i n accumulation and the amount of e l i c i t o r r e q u i r e d i s very s i m i l a r i n a l l three soybean t i s s u e s . Soybean t i s s u e s are s e n s i t i v e to extremely small amounts of Pms e l i c i t o r . About 10 moles of e l i c i t o r a p p l i e d to a s i n g l e hypocotyl s t i m u l a t e s q u a n t i t i e s of g l y c e o l l i n s u f f i c i e n t to p r e vent the growth of Pms and other microorganisms i n v i t r o . I t i s impressive, too, to observe the e f f e c t s on the growing suspension-cultured soybean c e l l s caused by the a d d i t i o n of submicromolar q u a n t i t i e s of the p o l y s a c c h a r i d e e l i c i t o r . These c e l l s respond to the small amount of e l i c i t o r even though the c e l l s are growing i n the presence of 50 mM sucrose. W i t h i n a few hours, the c e l l s t u r n l i g h t brown. At the same time, the a c t i v i t y i n the c e l l s of at l e a s t one of the enzyme b e l i e v e d to be i n v o l v e d i n the s y n t h e s i s of g l y c e o l l i n , phenylalanine ammonial y a s e , i s g r e a t l y increased. The increase i n a c t i v i t y of the phenylalanine ammonia-lyase precedes the accumulation of g l y c e o l l i n both i n the c e l l s and i n the c u l t u r e medium. The growth of the suspension-cultured c e l l s , as measured by f r e s h weight, stops upon a d d i t i o n of the e l i c i t o r . The c e l l s a l s o stop t a k i n g up ions from the media, which i s another i n d i c a t i o n of the l a c k

2.

VALENT

AND

ALBERSHEIM

of growth of these c e l l s

Elicitors

of

Phytoalexin

Accumulation

(8).

The Chemical Nature of the Phytophthora megasperma var. Elicitor.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

29

sojae

The component of the Pms m y c e l i a l w a l l s which stimulates g l y c e o l l i n accumulation by soybean t i s s u e s i s a s t r u c t u r a l p o l y saccharide. The e l i c i t o r was f i r s t found i n the f l u i d of o l d c u l t u r e s of Pms, probably being r e l e a s e d i n t o the c u l t u r e f l u i d by a u t o l y s i s . I t was l a t e r demonstrated that e l i c i t o r - a c t i v e molecules w i t h the same p r o p e r t i e s as the c u l t u r e f l u i d e l i c i t o r could be i s o l a t e d from the m y c e l i a l w a l l s of Pms by a heat t r e a t ment s i m i l a r to that used to s o l u b i l i z e the surface antigens from the c e l l w a l l s of JS. c e r e v i s i a e (_§ _) . The best method f o r obt a i n i n g l a r g e amounts of Pms e l i c i t o r i s p a r t i a l a c i d h y d r o l y s i s of the m y c e l i a l w a l l s . The s e r i e s of o l i g o s a c c h a r i d e s so obt a i n e d are extremely a c t i v e as e l i c i t o r s and possess c h a r a c t e r i s t i c s i d e n t i c a l to the c u l t u r e f l u i d e l i c i t o r . A l l elicitora c t i v e molecules examined have been found to be glucans. Methyla t i o n a n a l y s i s of the p u r i f i e d e l i c i t o r has demonstrated that t h i s glucan i s l a r g e l y a 3-linked polymer with g l u c o s y l branches to carbon 6 of about one out of every three of the backbone g l u c o s y l residues. The e l i c i t o r - a c t i v e glucan i s s u s c e p t i b l e to h y d r o l y s i s by an exo-3-l,3-glucanase i s o l a t e d from Euglena g r a c i l i s (10), i n d i c a t i n g that the Pms m y c e l i a l w a l l glucan i s a 3 - l i n k e d p o l y mer. O p t i c a l r o t a t i o n and NMR s t u d i e s have confirmed that the glucan i s 3 - l i n k e d . This i s not s u r p r i s i n g as other Phytophthora c e l l w a l l s have a q u a n t i t a t i v e l y dominant component which i s a 3-1,3-linked glucan with some branches to carbon 6 (11). Indeed, i t appears that as much as 60% of the m y c e l i a l w a l l of the Pms i s composed of t h i s polymer. The Pms m y c e l i a l w a l l e l i c i t o r has been w e l l c h a r a c t e r i z e d (12) and unpublished r e s u l t s of t h i s l a b o r a t o r y ) . That p o r t i o n of the e l i c i t o r , which i s r e l e a s e d from the w a l l s by aqueous ext r a c t i o n at 121 C, i s heterogeneous i n s i z e with an average mol e c u l a r weight of approximately 100,000. The E. g r a c i l i s enzyme hydrolyzes glucans from the non-reducing end and i s capable of h y d r o l y z i n g the the g l y c o s i d i c bond of 3-linked g l u c o s y l r e s i dues that have other g l u c o s y l residues attached to carbon 6. The product of the exoglucanase-degraded m y c e l i a l w a l l - r e l e a s e d e l i c i t o r i s s t i l l s i z e heterogeneous, but has an average molecular weight of approximately 10,000. This h i g h l y branched glucan f r a g ment r e t a i n s as much a c t i v i t y as the undegraded e l i c i t o r . The predominant g l y c o s i d i c linkages remaining a f t e r extensive exoglucanase treatment are 3-linked, 3,6-linked and t e r m i n a l glucos y l linkages i n a r a t i o of 1:1:1. SmaU amounts of 4-linked and 6-linked g l u c o s y l residues are a l s o present. The evidence that demonstrated that the Pms e l i c i t o r i s a polysaccharide included the f i n d i n g s that the e l i c i t o r i s s t a b l e to a u t o c l a v i n g at 121 C f o r s e v e r a l hours, l a c k s a f f i n i t y f o r

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

30

HOST P L A N T RESISTANCE T O

PESTS

both anion and c a t i o n exchange r e s i n s , i s completely s t a b l e to treatment by pronase, and i s s i z e heterogeneous. Periodate treatment of the w a l l - r e l e a s e d e l i c i t o r confirms the polysaccharide nature of the a c t i v e component and demonstrates the e s s e n t i a l r o l e of a branched o l i g o s a c c h a r i d e having terminal g l y c o s y l r e s i dues. Exposing the e l i c i t o r to periodate eliminates almost a l l of the e l i c i t o r a c t i v i t y . On the other hand, i f the p e r i o d a t e degraded polymers are reduced with sodium borohydride and then are subjected to m i l d a c i d h y d r o l y s i s , a considerable p o r t i o n of the e l i c i t o r a c t i v i t y i s regained. Since the 3- and 3,6-linked g l u c o s y l residues l a c k v i c i n y l hydroxyIs and are, t h e r e f o r e , r e s i s t a n t to periodate degradation, i t seems l i k e l y that the p e r i odate has destroyed the e l i c i t o r a c t i v i t y by modifying the t e r minal g l u c o s y l residues of the e l i c i t o r . The e l i c i t o r a c t i v i t y may be recovered a f t e r p a r t i a l a c i d h y d r o l y s i s of the p e r i o d a t e t r e a t e d e l i c i t o r because new terminal g l u c o s y l residues have been exposed and are able to provide the proper s t r u c t u r e of an active e l i c i t o r . The requirement f o r e l i c i t o r a c t i v i t y of a branched o l i g o saccharide i s supported by our observation that 3-linked glucans which l a c k branches to carbon 6 or have only a s i n g l e carbon 6branched g l u c o s y l r e s i d u e , such as l a m i n a r i n , have l i t t l e or no e l i c i t o r a c t i v i t y ( l e s s than one thousandth of the Pms e l i c i t o r ) . Indeed, a s e r i e s of commercially a v a i l a b l e p o l y s a c c h a r i d e s , o l i g o saccharides, methylglycosides, and simple sugars have been t e s t e d for e l i c i t o r a c t i v i t y , and, besides l a m i n a r i n , the only comm e r c i a l l y a v a i l a b l e product found with e l i c i t o r a c t i v i t y was nigeran, a m y c e l i a l w a l l component from the fungus, A s p e r g i l l u s niger. A major goal of our research i s the determination of the d e t a i l e d molecular s t r u c t u r e of the a c t i v e - s i t e of the Pms e l i citor. I t i s expected that t h i s goal w i l l be achieved by the i s o l a t i o n and s t r u c t u r a l c h a r a c t e r i z a t i o n of the smallest p o s s i b l e e l i c i t o r - a c t i v e o l i g o s a c c h a r i d e which can be derived from the glucan e l i c i t o r . This smallest e l i c i t o r - a c t i v e o l i g o s a c c h a r i d e has been produced by p a r t i a l a c i d h y d r o l y s i s of Pms m y c e l i a l w a l l s . The s e r i e s of o l i g o s a c c h a r i d e s obtained by t h i s p a r t i a l h y d r o l y s i s have been p a r t i a l l y r e s o l v e d by high r e s o l u t i o n B i o Gel P-2 g e l permeation chromatography. I t was found that o l i g o saccharides c o n t a i n i n g as few as 7 or 8 g l u c o s y l residues s t i l l retain e l i c i t o r activity. Glucose i s the only detected component of these o l i g o s a c c h a r i d e s . The smallest e l i c i t o r - a c t i v e o l i g o s a c c h a r i d e - c o n t a i n i n g f r a c t i o n s from the P-2 column have been f r a c t i o n a t e d by high pressure l i q u i d chromatography i n t o at l e a s t 5 o l i g o s a c c h a r i d e s . Two of the 5 o l i g o s a c c h a r i d e s obtained by high pressure l i q u i d chromatography can be e l i m i n a t e d by treatment of the mixture of o l i g o s a c c h a r i d e s with the IS. g r a c i l i s exoglucanase. There appears to be l i t t l e l o s s of e l i c i t o r a c t i v i t y by treatment with the exoglucanase. Of the three o l i g o s a c c h a r i d e s remaining (there may

2.

VALENT

A N D ALBERSHEIM

Elicitors

of Phytoalexin

Accumulation

31

s t i l l be one or more a d d i t i o n a l o l i g o s a c c h a r i d e s which have not been detected by the f r a c t i o n a t i o n procedures used), two a c t i v e l y e l i c i t soybean t i s s u e s t o accumulate g l y c e o l l i n . Sufficient q u a n t i t i e s o f these oligomers are now being produced t o t e s t f o r chemical p u r i t y and to permit s t r u c t u r a l c h a r a c t e r i z a t i o n .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

E l i c i t o r s Lack Species S p e c i f i c i t y Our experiments have shown that the e l i c i t o r s of p h y t o a l e x i n accumulation are not the s p e c i f i c i t y determining f a c t o r s i n the Pms-soybean system. Three Pms races (races 1, 2, and 3) are d i s t i n g u i s h e d by t h e i r d i f f e r i n g a b i l i t i e s to i n f e c t v a r i o u s soybean c u l t i v a r s . The e l i c i t o r obtained from each of the three Pms races p u r i f i e s i n e x a c t l y the same manner, and at l e a s t the major s t r u c t u r a l f e a t u r e s of the e l i c i t o r s from the three races a r e i d e n t i c a l (12). The a c t i v i t i e s of the e l i c i t o r s p u r i f i e d from the three Pms races were c a r e f u l l y examined u s i n g the three separ a t e bioassays: the cotyledon assay ( _7), the hypocotyl assay ( 7 ) , and the c e l l suspension c u l t u r e assay ( 8). A l l three assays gave the same r e s u l t s , that i s , the a c t i v i t i e s of the e l i c i t o r s from d i f f e r e n t Pms races are i d e n t i c a l . These f i n d i n g s i n d i c a t e that the three races of Pms a r e e q u a l l y e f f e c t i v e at s t i m u l a t i n g p h y t o a l e x i n accumulation i n t h e i r host soybean tissues. The r e s u l t s of another type of experiment support our conclus i o n that e l i c i t o r s are not r e s p o n s i b l e f o r r a c e - s p e c i f i c r e s i s t a n c e i n the Pms-soybean system. Soybean hypocotyls accumul a t e g l y c e o l l i n when i n o c u l a t e d with l i v i n g mycelia of Pms. The response which i s c h a r a c t e r i s t i c of n a t u r a l i n f e c t i o n s w i t h e i t h e r an i n f e c t i v e or a n o n - i n f e c t i v e race of Pms i s r e t a i n e d with t h i s i n o c u l a t i o n technique. We have compared the r e l a t i v e e f f e c t i v e n e s s of l i v e mycelia and p u r i f i e d e l i c i t o r i n stimulating g l y c e o l l i n accumulation. The r e s u l t i s the f o l l o w i n g : the onset and the r a t e of g l y c e o l l i n accumulation i n s e e d l i n g s i n o c u l a t e d with i n f e c t i v e mycelia was i n d i s t i n g u i s h a b l e from the onset and r a t e of g l y c e o l l i n accumulation i n seedlings i n o c u l a t e d w i t h e i t h e r n o n - i n f e c t i v e mycelia or p u r i f i e d e l i c i t o r . These r e s u l t s demonstrate that d i f f e r e n c e s i n r a t e s of g l y c e o l l i n accumulation i n response to d i f f e r e n t races of Pms do not account f o r the r e s i s t a n c e or s u s c e p t i b i l i t y of v a r i o u s soybean c u l t i v a r s to the Pms races. The a v a i l a b l e evidence does i n d i c a t e that e l i c i t o r s have a r o l e i n r e s i s t a n c e even though they are not determinants of race specificity. The e l i c i t o r i s o l a t e d from Pms i s capable of prot e c t i n g soybean hypocotyls from i n f e c t i o n by an i n f e c t i v e race of Pms i f the e l i c i t o r i s a p p l i e d to the hypotocyls 6 hours p r i o r to i n o c u l a t i o n with Pms. The e l i c i t o r cannot p r o t e c t soybean t i s s u e when a p p l i e d simultaneously with an i n f e c t i v e race of Pms.

32

HOST P L A N T

RESISTANCE T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

Phytoalexins are not Capable of P r o t e c t i n g P l a n t s from T h e i r Pathogens A microorganism which has evolved the a b i l i t y to grow succ e s s f u l l y on a p l a n t and thus become pathogenic to that p l a n t must a l s o have evolved a mechanism of a v o i d i n g the t o x i c e f f e c t s of phytoalexins. There are s e v e r a l p l a u s i b l e mechanisms f o r such avoidance by s u c c e s s f u l pathogens. One such mechanism might be simply the a b i l i t y of an i n f e c t i v e s t r a i n of a pathogen to grow away from the area i n which the p l a n t accumulates t o x i c l e v e l s of p h y t o a l e x i n . This p o s s i b i l i t y seems l i k e l y as an explanation f o r the avoidance of the e f f e c t s of g l y c e o l l i n i n soybean p l a n t s by i n f e c t i v e races of Pms. There are other mechanisms by which a pathogen might prevent a p l a n t from stopping the growth of the pathogen by accumulation of p h y t o a l e x i n s . For example, a pathogen might k i l l the p l a n t c e l l s i n the r e g i o n of the pathogen before those c e l l s are capable of s y n t h e s i z i n g the enzymes necessary f o r s y n t h e s i s of the phytoalexin. S t i l l another mechanism by which a s u c c e s s f u l pathogen might prevent a p l a n t from accumulating s u f f i c i e n t phytoalexins might be to repress s y n t h e s i s of enzymes i n v o l v e d i n p h y t o a l e x i n synthesis or e l s e to i n h i b i t the enzymes once they are synthes i z e d . A known mechanism by which pathogens overcome p h y t o a l e x i n i n h i b i t i o n i s m e t a b o l i z i n g the phytoalexins to l e s s t o x i c or uns t a b l e compounds (13, 14, 15, 16). A f i n a l p o s s i b l e mechanism might be the production by the pathogen of p r o t e i n s or other molecules which s p e c i f i c a l l y i n h i b i t the enzymes of the host which s o l u b i l i z e e l i c i t o r s from the m y c e l i a l w a l l s of the pathogen. Evidence suggestive of t h i s type of mechanism has a l s o been obtained (17). E l i c i t o r s are Widespread i n Nature Soybean p l a n t s have evolved the a b i l i t y to recognize and respond to the s t r u c t u r a l 3-glucan of Phytophthora mycelia w a l l s . S i m i l a r 3-glucans are found i n the w a l l s of a wide range of f u n g i . One fungus c o n t a i n i n g such 3-glucans i s brewer's yeast, S_. cerev i s i a e , a non-pathogen of p l a n t s . An e l i c i t o r has now been p u r i f i e d from a commercially a v a i l a b l e e x t r a c t of brewer's yeast (Difco) (M. Hahn, unpublished r e s u l t s ) . The 80% ethanol insoluble f r a c t i o n of the yeast e x t r a c t contains a very a c t i v e e l i c i t o r of g l y c e o l l i n accumulation i n soybeans. Most of the p o l y s a c c h a r i d e i n t h i s 80% ethanol i n s o l u b l e f r a c t i o n i s a mannan. However, yeast e x t r a c t does c o n t a i n small amounts of the 3-glucan. The glucan can be almost completely separated from the mannan by b i n d i n g the mannan to an a f f i n i t y column c o n s i s t i n g of Concanav a l i n A c o v a l e n t l y attached to Sepharose. The glucan can be separated from g l y c o p r o t e i n s by b i n d i n g the p r o t e i n s to s u l f o p r o p y l Sephadex. Both the p u r i f i e d mannan and p u r i f i e d glucan remain contaminated by small amounts (-2%) of arabinogalactan. Ribose,

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

2. VALENT AND ALBERSHEIM

Elicitors of Phytoalexin Accumulation

which contaminates the 80% ethanol insoluble fraction, is removed on a DEAE-cellulose column. The elicitor activity of the crude 80% ethanol yeast extract precipitate resides in the glucan component. The small amount of residual activity remaining in the mannan fraction can be attributed to the minor contamination of this fraction by glucan. The glucan is composed of the same glucosyl linkages found in the Pms elicitor. The same quantities of the yeast and Pms elicitor are required to stimulate glyceollin accumulation in soybeans. Our laboratory has obtained other evidence that the elicitorphytoalexin story is a general one. For example, the Pms elicitor stimulates suspension-cultured cells of sycamore and parsley to produce large amounts of phenylalanine ammonia lyase activity (8). In addition, we have obtained evidence that the Pms elicitor stimulates Phaseolus vulgaris, the true bean, to accumulate its phytoalexins (K. Cline, unpublished results). We have already reported (18) that a wall glucan from Colletotrichum lindemuthianum, a pathogen of P^. vulgaris, stimulates P^. vulgaris to produce its phytoalexin. The £. lindemuthianum glucan also stimulates soybeans to produce glyceollin (K. Cline, unpublished results). And, finally, we have recently demonstrated that the Pms elicitor stimulates potato tubers to accumulate their phytoalexins (M. Wade, unpublished results). In summary, elicitors appear to be general in nature, and diverse plants are able to respond to a single elicitor. Elicitors may therefore provide a new way of protecting plants against their pests, for elicitors may activate the plant's own defense mechanism and thereby eliminate some of the need for spraying agricultural crops with poisonous pesticides. Several industrial firms have recognized this potential and have initiated their own research or are supporting out-of-house research on elicitors. Literature Cited 1. 2. 3. 4.

Ingham, J.L., Botanical Rev. (1972) 38, 343. Kuć, J., Ann. Rev. Phytopathol. (1972) 10, 207. Deverall, B.J., Proc. R. Soc. Lond. B. (1972) 181, 233. Keen, N.T., Partridge, J.E. & Zaki, A.I., Phytopathology (1972) 62, 768. 5. Burden, R.S. & Bailey, J.A., Phytochemistry (1975) 14, 1389. 6. Lyne, R.L., Mulheirn, L.J. & Leworthy, D.P., J.C.S. Chem. Commun. (1976) 497. 7. Ayers, A.R., Ebel, J., Finelli, F., Berger, Ν. & Albersheim, P. Plant Physiol. (1976) 57, 751. 8. Ebel, J., Ayers, A.R. & Albersheim, P. Plant Physiol. (1976) 57, 775. 9. Ayers, A.R., Ebel, J., Valent, Β. & Albersheim, P., Plant Physiol. (1976) 57, 760. 10. Barras, D.R. & Stone, B.A., Biochim. Biophys. Acta (1969) 191, 342.

34 11. 12. 13. 14. 15. 16.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

17. 18.

HOST PLANT RESISTANCE TO PESTS Bartnicki-Garcia, S., J. Gen. Microbiol. (1966) 42, 57. Ayers, A.R., Valent, Β., Ebel, J. & Albersheim, P. Plant Physiol. (1976) 57, 766. Higgins, V.J., Physiol. Plant Pathol. (1975) 6, 5. Higgins, V.J., Stoessl, A. & Heath, M.C., Phytopathology (1974) 64, 105. Van Den Huevel, J. & VanEtten, H.D., Physiol. Plant Pathol. ( 1973) 3, 327. Van Den Huevel, J., VanEtten, H.D., Coffen, D.L. & Williams, T.H., Phytochemistry (1974) 13, 1129. Albersheim, P. & Valent, B.S. Plant Physiol. (1974) 53, 684. Anderson-Prouty, A.J. & Albersheim, P. Plant Physiol. (1975) 56, 286.

3 Biochemical Aspects of Plant Disease Resistance and Susceptibility

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

DOUG S. KENFIELD and GARY A. STROBEL Department of Plant Pathology, Montana State University, Bozeman, MT 59715

The events that either permit or preclude a pathogenic association between a host and its pathogen can manifest them­ selves at many different levels of the interaction (1). A complete knowledge of the interaction necessitates elucidation of the molecular events in connection with and pertinent to the genetics of pathogenicity and resistance. The purpose of this report is both to propose and illustrate a few of the possible molecular interactions between host and parasite and to discuss how these interactions relate to resistance or susceptibility in plant diseases. Molecular Interactions The molecular interplay that can occur between a plant and its pathogen can be divided into five categories. The first two categories arise when small molecules from either the pathogen or host interact with one or more structures in the other. The second two occur when the active constituents are macromolecules from either source. Such molecules may exert either positive or negative effects on their targets, and more than one type of interaction can and often does occur in a given disease. A fifth type of interaction may involve the decrease or elevation of the concentration of a normal constituent in either the host or the pathogen as influenced by the other. This is best exemplified in the case of pathogens that may u t i l i z e some constituent which ultimately results in an imbalance in metabolism in the host (2). Parasite-Produced Small Molecules It is conceivable that a small molecule produced by a patho­ gen could act selectively on a plant and actuate either a suscep­ tible or resistant reaction. In the latter case, Keen (3.) reported on the presence of a dialyzable compound in culture f i l ­ trates of an incompatible race of Phytophthora megasperma var. sojae that specifically elicited the increased production of the 35

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

36

HOST P L A N T RESISTANCE T O PESTS

antifungal phytoalexin hydroxy-phaseollin i n a resistant c u l t i v a r o f soybean but not i n a n e a r - i s o g e n i c , s u s c e p t i b l e c u l t i v a r . Compatible races o f t h i s fungus d i d not produce t h i s e l i c i t o r . W h i l e a t t e m p t s b y some t o r e p e a t t h e s e i n t e r e s t i n g o b s e r v a t i o n s h a v e n o t b e e n s u c c e s s f u l (k), i t seems l i k e l y t h a t s u c h a p h e ­ nomenon does e x i s t somewhere i n n a t u r e . Pathogens a r e a l s o capable o f p r o d u c i n g s m a l l molecules w h i c h a c t a s t o x i n s i n t h e p l a n t , a n d numerous i n s t a n c e s a r e known w h e r e t h e s e t o x i n s e x h i b i t h o s t s e l e c t i v i t y (5.). Charac­ t e r i z a t i o n o f such t o x i n s i s a v i t a l step t h a t can u l t i m a t e l y l e a d t o t h e d i s c o v e r y o f t h e i r mode o f a c t i o n , b i o s y n t h e s i s , p r e p a r a t i o n b y o r g a n i c t e c h n i q u e s , a n d , most i m p o r t a n t l y , t o serve as t o o l s i n d i s c o v e r i n g t h e m o l e c u l a r b a s i s o f d i s e a s e specificity. A t o x i n w h i c h e x h i b i t s h o s t s p e c i f i c i t y may a l s o be o f v a l u e i n s c r e e n i n g f o r r e s i s t a n t p l a n t s i n a g e n e t i c s o r b r e e d i n g program. Highly v i r u l e n t strains o f A l t e r n a r i a mali, the causal a g e n t o f a p p l e b l o t c h , p r o d u c e a s many a s s e v e n h o s t - s p e c i f i c toxins i n culture while less v i r u l e n t strains lack the a b i l i t y t o p r o d u c e some o f t h e t o x i n s (6_,J_) · The two m a j o r t o x i n s h a v e been c h a r a c t e r i z e d as a l t e r n a r i o l i d e ( c y c l o - [a-hydroxyisovaleryl-a-amino-p-methoxyphenylvaleryl-a-amino-acryl-alanyll a c t o n e ] ) a n d i t s demethoxy d e r i v a t i v e (Q) . R e s i s t a n c e i n a p p l e t r e e s h a s b e e n shown t o be c o n t r o l l e d b y m u l t i p l e d o m i n a n t g e n e s , thus t h i s system o f f e r s e x c e l l e n t o p p o r t u n i t i e s t o e l u c i d a t e t h e attack-counterattack hypotheses o f species e v o l v i n g i n c l o s e a s s o c i a t i o n w i t h one a n o t h e r . A l t e r n a r i a t e n u i s produces a t o x i n c a l l e d t e n t o x i n . This molecule i s also a c y c l i c tetrapeptide with the structure cyclo (L-leucyl-N-methyl-(Ζ)-dehydrophenylalanyl-glycyl-N-methyl-La l a n y l ) (£). The t o x i n e x h i b i t s some s p e c i e s s p e c i f i c i t y a s i t causes c h l o r o s i s i n seedlings o f c e r t a i n species but not o t h e r s , notably t h e Cruciferae (10). Steele, et a l . ( l l ) reported that the a c t i v e s i t e f o r t h i s t o x i n i n s e n s i t i v e plants i s t h e chloroplast coupling factor l ( C F l ) . Evidence supporting t h i s hypothe­ s i s i s t h a t , i n a s e n s i t i v e s p e c i e s , b o t h CFl-ATPase and t h e c o u p l e d e l e c t r o n t r a n s p o r t s y s t e m (ETS) i n c h l o r o p l a s t s a r e i n h i b i t e d by t e n t o x i n . A l s o , r a d i o - l a b e l l e d t e n t o x i n appears t o b i n d i n a m o l a r r a t i o t o p a r t i a l l y p u r i f i e d CF1 a s m e a s u r e d b y a time-release o f r a d i o - l a b e l l e d tentoxin u t i l i z i n g u l t r a - f i l t r a ­ t i o n techniques. An a f f i n i t y c o n s t a n t o f l O ^ M " was r e p o r t e d f o r t h e t o x i n t o CF1 f r o m a s e n s i t i v e s p e c i e s . I n an i n s e n s i t i v e s p e c i e s , a 2 3 - f o l d i n c r e a s e i n t e n t o x i n was r e q u i r e d t o i n h i b i t C F l - A T P a s e , c o u p l e d - E T S was n o t i n h i b i t e d , a n d t e n t o x i n bound t o t h e p a r t i a l l y p u r i f i e d CF1 w i t h an a p p a r e n t a f f i n i t y o f l e s s than 1 0 % - ! . More c o n v i n c i n g e v i d e n c e o f a d i r e c t i n v o l v e m e n t o f CF1 i s r e q u i r e d . Coincident m i g r a t i o n o f r a d i o a c t i v i t y from b o u n d , l a b e l l e d t e n t o x i n a n d CF1 d u r i n g g e l e l e c t r o p h o r e s i s would be s u b s t a n t i a t i v e e v i d e n c e , as would r e s o l u t i o n o f t h e d i f f e r e n c e s between i n v i v o and i n v i t r o r e s u l t s (12,13). 1

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

3.

K E N F i E L D A N D STROBEL

Disease

Resistance

and

Susceptibility

37

T e n t o x i n has b e e n s y n t h e s i z e d a n d a d e s c r i p t i o n o f t h e a c t i v e p a r t o f t h e m o l e c u l e i s b e i n g a t t e m p t e d (ik). Results o f t h i s a p p r o a c h may a i d i n a more r i g o r o u s c h a r a c t e r i z a t i o n o f t h e a c t i v e s i t e i n the p l a n t and allow a f u l l e r understanding o f the b i o l o g i c a l a c t i v i t y o f such c y c l i c t e t r a p e p t i d e s . The H e l m i n t h o s p o r i u m s p e c i e s o f f u n g i c o n s t i t u t e a n i m p o r ­ t a n t g r o u p i n t e r m s o f s p e c i f i c i t y a s many o f t h e m p r o d u c e h o s t s p e c i f i c t o x i n s . H. v i c t o r i a e a n d H. carbonum p r o d u c e p e p t i d y l t o x i n s w h i c h a c t s e l e c t i v e l y o n t h e p l a s m a membrane. The complete s t r u c t u r e s o f n e i t h e r o f these t o x i n s n o r s p e c i f i c s i t e s o f a c t i v i t y have been c h a r a c t e r i z e d , however. S o u t h e r n c o r n l e a f b l i g h t , t h e most d e v a s t a t i n g p l a n t disease epidemic i n recent h i s t o r y , i s caused by Helminthosporium maydis. D i f f e r e n t i a l s u s c e p t i b i l i t y i n corn i s r e l a t e d t o the p r e s e n c e o f T e x a s m a l e s t e r i l e (Terns) o r n o r m a l (N) c y t o p l a s m . P a t h o g e n i c i t y o f the fungus i s determined b y the a b i l i t y t o p r o d u c e a s many a s f i v e d i f f e r e n t h o s t s p e c i f i c t o x i n s ( t o x i n s I-V) w h i c h a p p e a r t o b e t e r p e n o i d s a n d t h e i r d e r i v a t i v e s ( l 5 , l 6 ) . T o x i n p r e p a r a t i o n s h a v e b e e n shown t o s t i m u l a t e t h e o x i d a t i o n o f malate and pyruvate, i n h i b i t p h o s p h o r y l a t i o n , and induce s w e l l i n g of m i t o c h o n d r i a from s u s c e p t i b l e but n o t r e s i s t a n t p l a n t s ( 1 7 ) . S u c h p r e p a r a t i o n s h a v e a l s o b e e n shown t o c a u s e i o n l e a k a g e , r e d u c e ADP c o n t e n t , i n h i b i t r o o t g r o w t h , a n d d i r e c t l y a f f e c t stomatal f u n c t i o n i n g (l8). Toxin preparations i n h i b i t oxidative p h o s p h o r y l a t i o n a n d s t i m u l a t e A T P a s e a c t i v i t y i n Terns b u t n o t Ν m i t o c h o n d r i a (19). While these d i v e r s e e f f e c t s are i n t e r e s t i n g , c r u d e t o x i n p r e p a r a t i o n s were u t i l i z e d i n t h e s e s t u d i e s w h i c h makes i t d i f f i c u l t t o e v a l u a t e t h e f i n d i n g s . Watrud, e t a l . (20) demonstrated t h a t malate dehydrogenase was i n h i b i t e d i n m i t o c h o n d r i a f r o m Terns c o r n b u t n o t i n t h o s e from Ν corn. Upon r u p t u r e o r r e m o v a l o f t h e o u t e r m i t o c h o n d r i a l membrane, h o w e v e r , m i t o c h o n d r i a f r o m Ν c y t o p l a s m became s e n s i ­ tive. T o x i n - b i n d i n g s t u d i e s showed t h a t m i t o c h o n d r i a f r o m b o t h Ν a n d Terns c y t o p l a s m b o u n d t o x i n s I I a n d I , b u t t h a t t h e g r e a t e s t b i n d i n g a c t i v i t y was a s s o c i a t e d w i t h t h e i n n e r m i t o c h o n d r i a l membrane o f Terns m i t o c h o n d r i a . Toxin-binding i n plant extracts was a l s o d e m o n s t r a t e d b y I r e l a n d a n d S t r o b e l ( 21 ) . U t i l i z i n g dextran-coated c h a r c o a l t o adsorb u n r e a c t e d t o x i n from assay m i x t u r e s , t h e y found t h a t e x t r a c t s from a s u s c e p t i b l e corn l i n e (W6ÎA. Terns) c o n t a i n e d a p r o t e i n l o c a l i z e d p r i m a r i l y i n t h e c y t o s o l which binds t o x i n s I and I I w i t h a i n t h e o r d e r o f 10~ The b i n d i n g a c t i v i t y i s r e l a t i v e l y h e a t i n s e n s i t i v e b u t i s destroyed by treatment with papain o r f i c i n . Binding a c t i v i t y was f o u n d i n e x t r a c t s o f b o t h s e n s i t i v e a n d i n s e n s i t i v e c o r n l i n e s , a s w e l l a s o t h e r p l a n t s p e c i e s , t h u s a l l o w i n g no d i r e c t c o r r e l a t i o n between b i n d i n g and r e s i s t a n c e o r s u s c e p t i b i l i t y . M e r t z , e t a l . ( 2 2 ) a r g u e t h a t t h e plasmalemma c o n t a i n s t h e s i t e o f s p e c i f i c i t y f o r t o x i n a c t i o n . They f o u n d t h a t a t o x i n p r e p a r a t i o n c o n t a i n i n g p r i m a r i l y t o x i n I I (10-6 t o lO-^M) i n h i b i t e d K u p t a k e i n l e a f d i s c s a n d a p i c a l r o o t segments o f +

38

HOST P L A N T RESISTANCE T O

PESTS

b o t h Terns and Ν c o r n ; h o w e v e r , a 3 0 - f o l d i n c r e a s e i n t o x i n c o n ­ c e n t r a t i o n was r e q u i r e d t o o b t a i n a s i m i l a r i n h i b i t i o n i n Ν c o r n . I n Terns c o r n , i n c r e a s i n g t h e c o n c e n t r a t i o n o f t o x i n o r t i m e o f t r e a t m e n t , d e c r e a s i n g pH, d e c r e a s i n g t h e e x t r a c e l l u l a r K concen­ t r a t i o n , and t r e a t i n g w i t h l i g h t a l l i n c r e a s e d t h e i n h i b i t o r y activity. F u r t h e r , s i x g e n o t y p e s o f c o r n w h i c h show d i f f e r e n t d e g r e e s o f s u s c e p t i b i l i t y t o t h e f u n g u s a l s o showed a c o r r e s p o n ­ d i n g d i f f e r e n t i a l i n h i b i t i o n o f K u p t a k e upon t r e a t m e n t w i t h toxin. A s e p a r a t e s t u d y has a l s o r e v e a l e d a d i f f e r e n t i a l s e n s i ­ t i v i t y of potassium uptake i n r o o t s t o t o x i n preparations (23). An a c c u r a t e p i c t u r e o f t h e a c t i v i t y o f t h e s e h o s t - s p e c i f i c t o x i n s i s not y e t p o s s i b l e . D e f i n i t i v e experiments await t h e i d e n t i f i c a t i o n o f t h e i n d i v i d u a l t o x i n s a n d t h e i r u s e a s homo­ geneous p r e p a r a t i o n s . I t d o e s a p p e a r t h a t m u l t i p l e r e a c t i o n s o c c u r b o t h a t t h e plasmalemma and i n t h e c y t o s o l , b u t t h e s i t e ( s ) of s p e c i f i c i t y remain obscure. H e l m i n t h o s p o r i u m s a c c h a r i c a u s e s e y e s p o t on s u g a r c a n e . This fungus produces a g l y c o s i d e c a l l e d h e l m i n t h o s p o r o s i d e w i t h t h e proposed s t r u c t u r e 2-hydroxycyclopropyl-a-D-galactoside (2k). The p a t h o g e n i c i t y o f t h i s f u n g u s d e p e n d s upon i t s a b i l i t y t o produce h e l m i n t h o s p o r o s i d e . M u l t i p l e t r a n s f e r s o f the patho­ g e n i c , t o x i n - p r o d u c i n g f u n g u s on s y n t h e t i c m e d i a r e s u l t e d i n a l o s s o f t o x i n p r o d u c t i o n and a t e m p o r a r y i n a b i l i t y o f t h e fungus t o c a u s e t y p i c a l symptoms o n s u s c e p t i b l e l e a v e s (25.). S u s c e p t i ­ b i l i t y i n t h e p l a n t i s governed by t h e presence o f a p r o t e i n i n t h e plasmalemma h a v i n g t h e a b i l i t y t o b i n d h e l m i n t h o s p o r o s i d e ( 2 6 , 2 7 , 2 8 ) , t h i s b i n d i n g b e i n g t h e f i r s t and s p e c i f i c s t e p o f t h e molecular events o f pathogenesis. One mode o f r e s i s t a n c e i n sugarcane i s c o n f e r r e d by t h e i n a b i l i t y o f t h e p l a n t t o b i n d t h e toxin. An o r i g i n a l l y s u s c e p t i b l e c a n e c l o n e was m u t a g e n i z e d v i a i r r a d i a t i o n and t h r e e r e s i s t a n t c l o n e s w e r e o b t a i n e d , e a c h o f w h i c h l a c k e d t h e a b i l i t y t o b i n d t h e t o x i n (2$). In another e x p e r i m e n t , s u s c e p t i b i l i t y t o h e l m i n t h o s p o r o s i d e was t r a n s f e r r e d i n v i t r o t o t o b a c c o and r e s i s t a n t s u g a r c a n e p r o t o p l a s t s b y i n c o r ­ p o r a t i n g t h e t o x i n - b i n d i n g p r o t e i n from s u s c e p t i b l e sugarcane into the normally r e s i s t a n t p r o t o p l a s t s (30). E l u c i d a t i o n of t h i s p a t h o g e n i c i n t e r a c t i o n has had an i m p a c t on s u g a r c a n e b r e e d i n g s i n c e g e n e t i c i s t s c u r r e n t l y spray clones of sugarcane s e e d l i n g s w i t h h e l m i n t h o s p o r o s i d e i n o r d e r t o s o r t out v a r i e t i e s s u s c e p t i b l e t o t h i s world-wide disease (31). +

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

+

Plant-Produced Small

Molecules

P h y t o a l e x i n s , a heterogeneous a r r a y o f substances having f u n g i s t a t i c o r f u n g i c i d a l p r o p e r t i e s , h a v e r e c e i v e d m a j o r empha­ s i s o f compounds i n t h i s c a t e g o r y . The p r o d u c t i o n o f p h y t o ­ a l e x i n s b y p l a n t s upon t r e a t m e n t w i t h v a r i o u s s t i m u l i undoubted­ l y p l a y s a r o l e i n t h e h o s t - p a r a s i t e i n t e r a c t i o n , and t h i s response i s d e a l t w i t h e l s e w h e r e i n t h i s volume. S p e c i f i c i t y as a t i m e r e l a t e d phenomenon a p p e a r s t o be

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

3.

KENFiELD

AND

STROBEL

Disease

Resistance

and

Susceptibility

39

e x h i b i t e d i n t h e i n t e r a c t i o n o f F u s a r i u m graminearurn a n d wheat i n t h e d i s e a s e known as h e a d b l i g h t . Wheat i s s u s c e p t i b l e t o h e a d b l i g h t a f t e r t h e a n t h e r s h a v e emerged and i s r e s i s t a n t p r i o r t o a n t h e s i s o r i f t h e a n t h e r s h a v e b e e n removed. Two r e l a t e d compounds, b e t a i n e and c h o l i n e , have b e e n i s o l a t e d f r o m t h e anthers b o t h o f which s t i m u l a t e t h e iri v i t r o growth o f t h e fungus and i n c r e a s e t h e v i r u l e n c e o f t h e m a c r o c o n i d i a (32). I t i s not known, h o w e v e r , i f r e s i s t a n c e t o h e a d b l i g h t i s a s p e c i f i c r e s p o n s e r e l a t e d t o t h e a b s e n c e o f t h e s e compounds. I n t h e c a s e o f H. s a c c h a r i on s u g a r c a n e i t a p p e a r s as i f a s e c o n d mode o f d i s e a s e r e s i s t a n c e c o u l d a r i s e b y t h e i n a b i l i t y o f t h e p l a n t t o " a c t i v a t e " t o x i n p r o d u c t i o n i n t h e fungus. P i n k e r t o n and S t r o b e l o b s e r v e d t h a t t h e f u n g u s l o s t i t s a b i l i t y t o p r o d u c e t h e t o x i n i f grown f o r an e x t e n d e d t i m e o n s y n t h e t i c media (33). The f u n g u s r e g a i n e d i t s p a t h o g e n i c i t y i f grown o n agar d e r i v e d from s u s c e p t i b l e sugarcane o r i f crude m a t e r i a l from a w a t e r wash o f s u s c e p t i b l e s u g a r c a n e l e a v e s was a d d e d t o t h e s y n t h e t i c media. They i s o l a t e d , i d e n t i f i e d , a n d s y n t h e s i z e d one o f t h e a c t i v a t o r s and i t was shown t o be s e r i n o l , 2 - a m i n o - l , 3 propanediol. E v i d e n c e i n d i c a t e d t h a t s e r i n o l was o f p l a n t o r i g i n a n d , s u b s e q u e n t l y , B a b c z i n s k i e t a l . (3U_) d e m o n s t r a t e d t h a t t h e b i o s y n t h e s i s o f s e r i n o l proceeds v i a s e r i n o l phosphate from dihydroxyacetone phosphate i n a t r a n s a m i n a t i o n r e a c t i o n i n ext r a c t s o f s u s c e p t i b l e s u g a r c a n e . R e s i s t a n t cane d i d not p o s s e s s s e r i n o l , nor t h e m e t a b o l i c pathway f o r i t s p r o d u c t i o n . A second i n d u c e r ( m o l e c u l a r w e i g h t = 700) i s c u r r e n t l y b e i n g c h a r a c t e r i z e d b y U. M a t e r n o f t h i s l a b o r a t o r y . I t i s n o t known how t h e s e compounds a c t t o s t i m u l a t e t h e s y n t h e s i s o f h e l m i n t h o s p o r o s i d e . I t w o u l d a l s o a p p e a r l i k e l y t h a t numerous o t h e r h o s t - p a r a s i t e c o m b i n a t i o n s a r e o p e r a t i n g i n a s i m i l a r mode, a s l o s s o f p a t h o g e n i c i t y d u r i n g extended c u l t u r e i s f r e q u e n t l y observed. The a n t i t h e s i s o f a h o s t - f o r m e d a c t i v a t o r w o u l d be a h o s t formed i n h i b i t o r o f t o x i n p r o d u c t i o n . To o u r k n o w l e d g e , no one h a s y e t d e m o n s t r a t e d t h e e x i s t e n c e o f s u c h a compound. Macromolecules

from

Pathogens

The p r o d u c t i o n o f a m a c r o m o l e c u l e b y t h e p a t h o g e n t h a t confers s p e c i f i c i t y i n the host-parasite i n t e r a c t i o n i s a v i a b l e h y p o t h e s i s , but i s not s u p p o r t e d by s t r o n g e v i d e n c e . Pathogens do p r o d u c e numerous d e g r a d a t i v e enzymes s u c h as c e l l u l a s e s , p e c t i n a s e s , a n d p r o t e a s e s , b u t t h e r e i s no e v i d e n c e f o r h o s t s p e c i f i c i t y b e i n g p r e s c r i b e d b y t h e s e enzymes. A model system i n w h i c h t h e pathogen a p p a r e n t l y c o u n t e r s a p o s s i b l e defense r e a c t i o n by t h e host o c c u r s between C o l l e t o t r i c h u m l i n d e m u t h i a n u m a n d P h a s e o l i s v u l g a r i s (35)· A major component o f t h e f u n g a l c e l l w a l l i s a 3 - 1 , 3 g l u c a n , and P_. v u l g a r i s p o s s e s e s a 3-1,3 g l u c a n a s e t h a t c o u l d pose a t h r e a t t o t h e p a t h o g e n s i n c e i t s w a l l m a t e r i a l c o u l d be d e s t r o y e d . A l b e r s h e i m , e t a l . , i s o l a t e d a p r o t e i n a c e o u s compound f r o m t h e f u n g u s

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

40

H O S T P L A N T RESISTANCE T O

PESTS

t h a t i n h i b i t s t h e a c t i v i t y o f t h e h o s t ' s $-1,3 glucanase. These o b s e r v a t i o n s a r e w o r t h y o f b e i n g t e s t e d i n o t h e r s y s t e m s and s h o u l d be f u r t h e r s t u d i e d t o a s c e r t a i n how i m p o r t a n t t h i s phenomenon i s t o p a t h o g e n s p e c i f i c i t y and t h e d e f e n s e r e a c t i o n o f the host. B o t h b a c t e r i a and f u n g i p r o d u c e l a r g e p h y t o t o x i c g l y c o p e p t i d e s w h i c h h a v e b e e n shown t o p o s s e s s some h o s t s p e c i f i c i t y (36^37). F o r i n s t a n c e , R i e s and S t r o b e l i s o l a t e d and c h a r a c t e r i z e d a glycopeptide from Corynebacterium i n s i d i o s u m t h a t causes w i l t i n g i n a l f a l f a (38). The t o x i n i s a g l y c o f u c a n w i t h a m o l e c u l a r w e i g h t o f 5 x 10^. At a c r i t i c a l c o n c e n t r a t i o n of 0 . 0 5 $ , t h e t o x i n was e f f e c t i v e as a s c r e e n i n g t o o l i n s e l e c t i n g a l f a l f a c l o n e s r e s i s t a n t t o C_. i n s i d i o s u m . Phoma t r a c h e i p h i l a , t h e c a u s a l a g e n t o f m a l s e c c o d i s e a s e of c i t r u s , produced a glycopeptide capable of i n d u c i n g v e i n a l n e c r o s i s and v e i n c l e a r i n g f o l l o w e d b y w i l t and l e a f s h e d d i n g in susceptible citrus. The t o x i n has a m o l e c u l a r w e i g h t o f 93,000. Amino a c i d s c o n t r i b u t e 36% o f i t s w e i g h t , w h i c h i s c o n s i d e r a b l y g r e a t e r t h a n e x h i b i t e d by o t h e r p h y t o t o x i c g l y c o p e p t i d e s (39)» The t o x i n c a u s e s d e a t h o f s u s c e p t i b l e l e m o n c a l l u s , b u t has no d e t r i m e n t a l e f f e c t on r e s i s t a n t o r a n g e c a l l u s . T h i s g l y c o p e p t i d e has enormous p o t e n t i a l f o r s c r e e n i n g c i t r u s p r o t o p l a s t s and/or c a l l u s t i s s u e f o r t o x i n r e s i s t a n c e . This t e c h n o l o g i c a l a d v a n c e seems f e a s i b l e s i n c e c i t r u s t r e e s h a v e r e c e n t l y been r e g e n e r a t e d from p r o t o p l a s t s . A h o s t - p a r a s i t e i n t e r a c t i o n t h a t may i n v o l v e a t r a n s f e r o f macromolecules between s p e c i e s i s t h e stem r u s t - w h e a t a s s o c i a tion. T h i s disease i s c h a r a c t e r i z e d by a h i g h degree o f s p e c i f i c i t y a t t h e g e n e t i c l e v e l i n t h a t c u l t i v a r s o f wheat c a r r y genes f o r r e s i s t a n c e (R) and s u s c e p t i b i l i t y ( r ) t o r a c e s o f t h e f u n g u s c o n t a i n i n g c o r r e s p o n d i n g genes f o r v i r u l e n c e ( a ) o r a v i r u l e n c e ( A ) . Of t h e f o u r p o s s i b l e c o m b i n a t i o n s R/A, R/a, r/A, and r / a , o n l y t h e R/A i n t e r a c t i o n l e a d s t o a r e s i s t a n t r e a c t i o n c h a r a c t e r i z e d by l o c a l i z e d n e c r o s i s (ho). R o h r i n g e r , e t a l . (hl k2) p u b l i s h e d e v i d e n c e t h a t a genes p e c i f i c RNA, p o s s i b l y t h e t r a n s c r i p t i o n p r o d u c t o f t h e gene f o r a v i r u l e n c e i n the fungus, i s d i r e c t l y i n v o l v e d i n the r e s i s t a n t r e a c t i o n o f wheat t o stem r u s t . More r i g o r o u s s u p p o r t o f t h i s h y p o t h e s i s i s n e e d e d , h o w e v e r , as t h e s e r e s e a r c h e r s r e c e n t l y reported t h e i r f a i l u r e t o c o n s i s t e n t l y reproduce t h e i r o r i g i n a l r e s u l t s (1+3). One o f t h e b e s t i l l u s t r a t i o n s o f t h e i n v o l v e m e n t o f a m a c r o m o l e c u l e i n p l a n t d i s e a s e s p e c i f i c i t y i s t h e phenomenon o f c r o w n g a l l , a tumorous d i s e a s e o f p l a n t s caused by A g r o b a c t e r i u m t u m e f a c i e n s . A m a c r o m o l e c u l e ( b a c t e r i a l p l a s m i d ) has b e e n i m p l i c a t e d a s t h e k e y f a c t o r i n i n f e c t i o u s A. t u m e f a c i e n s t h a t b r i n g s a b o u t t r a n s f o r m a t i o n o f p l a n t c e l l s (hh). Nevertheless, the u l t i m a t e gene p r o d u c t ( s ) t h a t b r i n g a b o u t t h e a b n o r m a l b e h a v i o r o f t h e p l a n t c e l l r e m a i n s t o be i d e n t i f i e d . I t i s apparent from t h e s e and o t h e r s t u d i e s t h a t t h e f i e l d o f m a c r o m o l e c u l a r i n t e r 9

3.

KENFiELD

AND

STROBEL

Disease Resistance

a c t i o n s b e t w e e n p a t h o g e n s and dial state.

and

41

Susceptibility

t h e i r plant hosts i s i n a

primor-

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

Macromolecules from P l a n t s P o s s i b i l i t i e s f o r s p e c i f i c i t y at the macromolecular l e v e l i n p l a n t s i n c l u d e unique or novel c u t i n or c e l l w a l l s t r u c t u r e s , isozymes t h a t d i c t a t e s p e c i f i c i t y , o r even t h e p r o d u c t i o n o f enzymes t h a t d e s t r o y o r a l t e r s p e c i f i c t o x i n s p r o d u c e d by t h e pathogen. E v i d e n c e f o r t h e d i r e c t , s p e c i f i c i n v o l v e m e n t o f any of these macromolecules i n the h o s t - p a r a s i t e i n t e r a c t i o n i s t o be forthcoming. An e x a m p l e o f s p e c i f i c i t y i n p l a n t - d i s e a s e r e s i s t a n c e e s t a b l i s h e d a t t h e m a c r o m o l e c u l a r l e v e l i s t h e H. s a c c h a r i s u g a r c a n e i n t e r a c t i o n w h e r e i n r e s i s t a n c e i s e x p r e s s e d as an a l t e r e d p r o t e i n r e c e p t o r t h a t cannot b i n d t h e h o s t - s p e c i f i c f u n g a l t o x i n (27). From a d d i t i o n a l w o r k on t h i s s y s t e m , i t has become a p p a r e n t t o us t h a t i t i s i n c o r r e c t t o b e l i e v e t h a t t h e m o l e c u l a r e v e n t t h a t c o n f e r s s p e c i f i c i t y a l s o must c o n s t i t u t e the c r u c i a l detrimental e f f e c t i n the disease process. A certain dogma i n p h y t o p a t h o l o g y i s t h a t a h o s t - s p e c i f i c t o x i n must r e p r o d u c e a l l o f t h e symptoms o f t h e d i s e a s e i n c i t e d b y t h e p a t h o gen. This concept formulates a workable b a s i s f o r i s o l a t i n g host s p e c i f i c t o x i n s , b u t i t must n o t be m i s c o n s t r u e d t o mean t h a t t h e s p e c i f i c i n t e r a c t i o n o f a t o x i n w i t h i t s a c t i v e s i t e comprises the e n t i r e molecular events o f pathogenesis. The s p e c i f i c e v e n t may s i m p l y be a p o t e n t i a t o r f o r t h e c r u c i a l e v e n t . T h e s e p o i n t s a r e i l l u s t r a t e d i n t h e H_. s a c c h a r i - s u g a r c a n e system. The b i n d i n g p r o t e i n f o r t h e t o x i n i s l o c a l i z e d on t h e p l a s m a membrane, and a l l s u s c e p t i b l e c l o n e s o f s u g a r c a n e t e s t e d t h u s f a r p o s s e s s an a c t i v e b i n d i n g p r o t e i n . Clones t h a t are r e s i s t a n t possess a p r o t e i n which l a c k s detectable t o x i n b i n d i n g activity. C e r t a i n l i n e s of evidence i n d i c a t e t h a t t o x i n b i n d i n g i s only a potentiating reaction that i s c r u c i a l to s p e c i f i c i t y b u t may be j u s t a k e y t o o t h e r e v e n t s i n t h e c e l l l e a d i n g t o i t s demise. Upon t r e a t m e n t w i t h t h e t o x i n , p r o t o p l a s t s f r o m s u s c e p t i b l e c a n e s w e l l and e v e n t u a l l y b u r s t , t i s s u e f r o m s u s c e p t i b l e c a n e u n d e r g o e s an i m m e d i a t e d r o p i n membrane p o t e n t i a l and shows l e a k i n e s s o f i o n s , t i s s u e ATP l e v e l s d r o p , and photosynthesis d e c l i n e s (k5). Pretreatment with cycloheximide, or elevated t e m p e r a t u r e c a u s e s u s c e p t i b l e l e a v e s t o be r e s i s t a n t (k6). Such t r e a t m e n t s , h o w e v e r , do n o t a f f e c t t h e t o x i n - b i n d i n g a c t i v i t y o f t h e p r o t e i n . The t o x i n a c t i v a t e s ( K , M g ) - A T P a s e a c t i v i t y i n p l a s m a membrane p r e p a r a t i o n s f r o m s u s c e p t i b l e t i s s u e s b u t n o t f r o m h e a t - t r e a t e d t i s s u e s jhj). Arrhenius p l o t s o f heat e f f e c t s on A T P a s e a c t i v i t y and symptom e x p r e s s i o n r e v e a l a s h a r p b r e a k a t 32°C f o r b o t h ( u n p u b l i s h e d ) . A l s o , common i n h i b i t o r s o f ATPase a c t i v i t y c o n f e r r e s i s t a n c e t o s u s c e p t i b l e t i s s u e . I t appears t h a t a s p e c i f i c , h e a t - i n s e n s i t i v e step o f t o x i n +

+ +

42

HOST P L A N T RESISTANCE T O

PESTS

b i n d i n g p r e c e d e s a h e a t - s e n s i t i v e e v e n t c r u c i a l f o r symptom expression. The s e c o n d e v e n t may -well be an a c t i v a t i o n o f p l a s m a membrane ( K , M g ) - A T P a s e a c t i v i t y . That s p e c i f i c i t y r e s i d e s i n the t o x i n - b i n d i n g step i s f u r t h e r s u p p o r t e d by t h e f a c t t h a t s e n s i t i v i t y t o h e l m i n t h o s p o r o ­ s i d e c a n be t r a n s f e r r e d i n v i t r o t o b o t h t o b a c c o and r e s i s t a n t sugarcane p r o t o p l a s t s v i a t r a n s f e r of the b i n d i n g p r o t e i n . The b i n d i n g a c t i v i t y a l o n e i s not s u f f i c i e n t t o promote d i s e a s e symptoms, h o w e v e r , b e c a u s e t o x i n - b i n d i n g a c t i v i t y has b e e n found i n T C A - s o l u b i l i z e d e x t r a c t s o f plasmalemma-enriched f r a c ­ t i o n s f r o m d i v e r s e p l a n t s (See T a b l e I ) . M i n t shows a n e a r l y t w o - f o l d i n c r e a s e i n s p e c i f i c b i n d i n g a c t i v i t y over t h a t from s u s c e p t i b l e sugarcane, yet t o x i n a p p l i e d to mint leaves f a i l s to educe any o b s e r v a b l e symptoms. The t o x i n a l s o f a i l s t o c a u s e an a c t i v a t i o n o f t h e plasma-membrane (ΚΓ*", M g ) - A T P a s e f r o m m i n t (unpublished). I n f a c t , t h e t o x i n does n o t c a u s e symptoms on any o f t h e p l a n t s t e s t e d except s u s c e p t i b l e sugarcane. Note a l s o that while tobacco possesses t o x i n - b i n d i n g a b i l i t y , protoplasts w e r e n o t k i l l e d b y h e l m i n t h o s p o r o s i d e u n t i l t h e y had b e e n t r e a t e d w i t h the b i n d i n g p r o t e i n from sugarcane. C l e a r l y , b i n d i n g a c t i v i t y i s e s s e n t i a l but i n s u f f i c i e n t t o cause d i s e a s e . Y e t a n o t h e r i n t e r e s t i n g o b s e r v a t i o n i s t h a t t h e TCA e x t r a c t s o f a l l t h e p l a n t s t e s t e d c a n b i n d r a f f i n o s e and g a l a c t i n o l ; y e t r e s i s t a n t s u g a r c a n e , p o t a t o , and c o r n l a c k t h e a b i l i t y t o b i n d helminthosporoside. I f , i n t r u t h , what we a r e s t u d y i n g i s a f a c e t o f α-galactoside t r a n s p o r t i n p l a n t s , i t a p p e a r s t h a t a l t e r a t i o n s can o c c u r w h i c h d r a s t i c a l l y a f f e c t l i g a n d s p e c i f i c i t y y e t do n o t i n t e r f e r e w i t h a p p a r e n t l y n o r m a l p h y s i o l o g i c a l f u n c ­ tions. C u r r e n t l y i n o u r l a b o r a t o r y , i s o l a t i o n and c h a r a c t e r i z a ­ t i o n o f t h e b i n d i n g p r o t e i n s f r o m s u g a r c a n e , m i n t , and t o b a c c o a r e i n p r o g r e s s i n an a t t e m p t t o d i s c e r n t h e u n i q u e n e s s o f t h e p r o t e i n f r o m s u s c e p t i b l e s u g a r c a n e and i t s i n v o l v e m e n t i n d i s e a s e specificity. +

+ +

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

+ +

Implications What a r e t h e i m p l i c a t i o n s o f m o l e c u l a r r e s e a r c h i n t o t h e s p e c i f i c i t y of p l a n t diseases? C e r t a i n l y a primary goal of plant p a t h o l o g i s t s i s t h e d e v e l o p m e n t o f more e f f e c t i v e c o n t r o l o f plant diseases. A v e r y i m p o r t a n t a p p l i c a t i o n l e a d i n g t o enhance­ ment o f c o n t r o l m e a s u r e s i s t h e i d e n t i f i c a t i o n o f b i o c h e m i c a l m a r k e r s f o r p l a n t b r e e d e r s t h e r e b y i n c r e a s i n g t h e e f f i c i e n c y and c e l e r i t y o f t h e i r e f f o r t s t o produce d i s e a s e - r e s i s t a n t p l a n t s . C u r r e n t l y i n t h i s l a b o r a t o r y , J . P. B e l t r a n i s c h a r a c t e r i z i n g i n barley cultivars d i f f e r e n t i a l binding a c t i v i t y for a glycosidic t o x i n produced by Rhyncosporium s e c a l i s , the c a u s a l agent o f b a r l e y s c a l d . I f s u c c e s s f u l , t h i s work w i l l p r o v i d e t h e f i r s t b i o c h e m i c a l marker of t o x i n r e s i s t a n c e i n a p l a n t , the g e n e t i c s o f w h i c h have been e x t e n s i v e l y c h a r a c t e r i z e d . A l t e r n a t i v e l y , the i d e n t i f i c a t i o n of molecules a c t i v e i n

3.

KENFiELD

Table I .

A N D STROBEL

Resistance

and

Susceptibility

Binding a c t i v i t y (m m o l e s u b s t r a t e / g p r o t e i n ) G a l a c t i n o l Helminthosporoside Raffinose

0.05

0.19

0.0k

0.16

Mint

1.07

O.Vf

k.l

Tobacco

0.08

0.06

0.2k

Potato

0.06

0.13

<0.005

Beet

0.11

0.05

1.9

Wheat

0.07

0.10

0.012

Barley

0.10

0.0k

0.020

Corn

0.08

0.03

<0.005

susceptible 51NG97 resistant H50-7209

43

α-Galactoside b i n d i n g a c t i v i t y o f t r i c h l o r o a c e t a t e s o l u b i l i z e d , plasma-membrane-enriched f r a c t i o n s .

Source

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

Disease

sugarcane

sugarcane

2.7

<0.005

Note: A p e l l e t e n r i c h e d i n p l a s m a membranes was o b t a i n e d a s i n (25). T h i s p e l l e t was h o m o g e n i z e d i n T r i s - H C l , 0 . 0 5 M, pH 7 - 0 c o n t a i n i n g 0 . 5 M t r i c h l o r o a c e t a t e (TCA) a n d i n c u b a t e d a t kC f o r k hours. A f t e r c e n t r i f u g a t i o n a t U 8 , 0 0 0 g f o r 20 m i n u t e s , 0 . 5 m l o f t h e s u p e r n a t a n t was i n c u b a t e d a t 25C f o r 30 m i n u t e s w i t h l^C-ligand (l0 M). The a s s a y m i x t u r e was t h e n chromât ο g r a p h e d o v e r B i o - g e l P - 2 ( 1 . 5 x ^5 c m ) , e l u t i n g w i t h b u f f e r . The f r a c t i o n s e l u t i n g a t t h e v o i d volume were c o l l e c t e d , p l a c e d i n 5 ml o f A q u a s o l , and counted i n a P a c k a r d T r i - C a r b L i q u i d S c i n ­ t i l l a t i o n Spectrometer. T o t a l p r o t e i n was m e a s u r e d a c c o r d i n g t o L o w r y , e t a l . (k6). S p e c i f i c r a d i o a c t i v i t i e s o f t h e l i g a n d s (DPM/ymole) w e r e : r a f f i n o s e - 3 5 1 , 5 0 0 ; g a l a c t i n o l - 3 3 , 7 0 0 ; and h e l m i n t h o s p o r o s i d e - U , 0 0 0 . B o v i n e serum a l b u m i n a n d b o i l e d TCA e x t r a c t s w e r e u s e d as c o n t r o l s . _ i +

44

HOST PLANT RESISTANCE TO PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

specificity may promote a rational approach to development and application of fungicides and herbicides, allowing design of more effective and more selective poisons. This approach is especial­ ly important in crop systems where weeds are closely related to the commercial plants (i.e. wild oats and wheat) and high degrees of selectivity are required for efficient control. Research on specificity will also increase our knowledge on the basic processes of life. As examples: tentoxin studies may well reveal important events in the phenomenon of uncoupling of oxidation-phosphorylation; aspects of protein-protein inter­ action in membranes and their effect on active transport are possible extensions of the work on the activity of helminthospo­ roside; the study of southern corn leaf blight should increase our knowledge of interactions of nuclear and cytoplasmic genes. Acknowledgement s Certain aspects of the research work presented in this report were supported in part by the National Science Foundation Grant PCM 76-19565, a grant from the Herman Frasch Foundation and the Montana Agricultural Experiment Station. Literature Cited 1. Brian, P. W., "Specificity in Plant Diseases", pp.15-26, Plenum Press, New York, 1976. 2. MacDonald, P. W. and Strobel, G. Α., Plant Physiol., (1970), 46, 126-135. 3. Keen, N. T., Science, (1975), 187, 74-75. 4. Ayers, A. R., Ebel, J . , Valent, Β., and Albersheim, P., Plant Physiol., (1976), 51, 760-766. 5. Scheffer, R. P., "Physiological Plant Pathology", pp.247269, Springer-Verlag, New York, 1976. 6. Kohmoto, K., Khan, I. D., Renbutso, Y., Taniguchi, T., and Nishimura, S., Physiol. Plant Pathol., (1976), 8, 141-153. 7. Nishimura, S., Kohmoto, K., Otani, H., Fukami, H., and Ueno, T., "Biochemistry and Cytology of Plant Parasite Inter­ action", pp.94-101, Elsevier Scientific Publishing Co., New York, 1976. 8. Okuno, T., Ishita, Y., Sawai, K., and Matsumoto, T., Chem Lett, (1974), 635-638. 9. Meyer, W. L., Templeton, G. Ε., Grable, C. I., Jones, R., Kuyper, L. F., Lewis, R. B., Sigel, C. W., and Woodhead, S. H., J. Am. Chem. Soc., (1975), 97, 3802-3809. 10. Durbin, R. D. and Uchytil, T. F., Phytopathology, (1977), 67, 602-603. 11. Steele, J. Α., Uchytil, T. F., Durbin, R. D., Bhatnagar, P., and Rich, D. H., Proc. Nat. Acad. Sci., USA, (1976), 73, 2245-2248. 12. Arntzen, C. H., Biochem. Biophys. Acta, (1972), 283,

3. KENFIELD AND STROBEL 13. 14. 15. 16. 17. 18. Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

45 Disease Resistance and Susceptibility

539-542. Fulton, N. D., Bollenbacher, K., and Templeton, G. Ε., Phytopathology, (1965), 55, 49-51. Rich, D. H., "Specificity in Plant Diseases," pp.l69-l84, Plenum Press, New York, 1976. Karr, A. L., Karr, D. B., and Strobel, G. Α., Plant Physiol., (1974), 53, 250-257. Karr, D. Β., Karr, A. L., and Strobel, G. Α., Plant Physiol., (1975), 55, 727-730. Miller, R.J . and Koeppe, D. Ε., Science, (1971), 173, 67-69. Arntzen, C. J., Koeppe, D. Ε., Miller, R. J., and Peverly, J. H., Physiol. Plant Pathol., (1973), 3, 79-90. Bednarski, Μ. Α., Izawa, S., and Scheffer, R. P., Plant Physiol., (1977), 59, 540-545. Watrud, L. S., Baldwin, J. Κ., Miller, R. J., and Koeppe, D. Ε., Plant Physiol., (1975), 56, 216-221. Ireland, C. A. and Strobel, G. Α., Plant Physiol., in press. Mertz, S. M. and Arntzen, C. J., Plant Physiol., in press. Frick, H., Bauman, L. F., Nicholson, R. L., and Hodges, Τ. Κ., Plant Physiol., (1977), 59, 103-106. Steiner, G. W. and Strobel, G. Α., J. Biol. Chem., (1971), 246, 4350-4357. Pinkerton, F., Ph.D. Thesis, Montana State University, (1976). Strobel, G. Α., J. Biol. Chem., (1973), 248, 1321-1328. Strobel, G. Α., Proc. Nat. Acad. Sci., USA, (1973), 70, 1693-1696. Strobel, G. A. and Hess, W. Μ., Proc. Nat. Acad. Sci. USA, (1974), 71, 1413-1417. Strobel, G. Α., Steiner, G. W., and Byther, R. S., Biochem. Genet., (1975), 13, 557-565. Strobel, G. A. and Hapner, K. D., Biochem. Biophys. Res. Comm., (1975), 63, 1151-1156. Byther, R. S. and Steiner, G. W., Phytopathology, (1972), 62, 466-470. Strange, R. Ν., Majer, J. R., and Smith, Η., Physiol. Plant Pathol., (1974), 4, 277-290. Pinkerton, F. and Strobel, G. Α., Proc. Nat. Acad. Sci. USA, (1976), 73, 4007-4011. Babczinski, P., Matern, U., and Strobel, G. Α., Plant Physiol., in press. Albersheim, P. and Valent, B. S., Plant Physiol., (1974), 53, 684-687. Strobel, G. Α., Ann. Rev. Plant Physiol., (1974), 25, 541-566. Strobel, G. Α., Ann. Rev. Microbiol., (1977), 31, 205-224. Ries, S. M. and Strobel, G. Α., Physiol. Plant Pathol., (1972), 2, 133-142. Nachmias, Α., Barash, I., Solel, Z., and Strobel, G. Α.,

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch003

46

HOST PLANT RESISTANCE TO PESTS

Physiol. Plant Pathol., (1977), 10, 147-157. 40. Flor, H. H., J. Agric. Res., (l946), 73, 335-357. 41. Rohringer, R., Howes, Ν. Κ., Kim, W. Κ., and Samborski, D. J., Nature, (1974), 249, 585-588. 42. Rohringer, R., "Specificity in Plant Diseases", pp.185198, Plenum Press, New York, 1976. 43. Rohringer, R., Howes, Ν. Κ., Kim, W. Κ., and Samborski, D. J., Can. J. Bot., (1977), 55, 851-852. 44. Kado, C. I., Ann. Rev. Phytopathol., (1976), 14, 265-308. 45. Strobel, G. Α., Trends in Biochem. Sci., (1976), 1, 247250. 46. Byther, R. S. and Steiner, G. W., Plant Physiol., (1975), 56, 415-419. 47. Strobel, G. Α., Proc. Nat. Acad. Sci., USA, (1974), 71, 4232-4236. 48. Lowry, O. Η., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem., (1951), 193, 265-275.

4 Interactions between Phytophthora Infestans and Potato Host

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

DONALD D. BILLS Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118

The white potato (Solanum tuberosum) was unknown outside of South America until the sixteenth century, but by the nineteenth century, it was widely cultivated in the British Isles, Europe, Russia, and the United States. Ireland, in particular, shifted its agricultural emphasis from cereal grains to the more productive potato with such success that the population of Ireland increased considerably to about 7 million by 1844. In 1845, climatic conditions and widespread growth of the potato in Ireland permitted catastrophic field infection by Phytophthora infestans and resulted in the loss of nearly the entire potato crop in that year and the subsequent year. The results for Ireland were devastating; over one million people died of starvation or diseases associated with malnutrition, and over one million more emigrated. P. infestans, the pathogen responsible for potato late blight, remains the most significant fungal parasite of the potato. The Nature of Late Blight The symptoms of late blight on the potato plant are visible first as dark brown patches on the leaflets, usually near the margin. On the leaflets of varieties that are incompatible with the infecting race of P. infestans, the necrosis is restricted to small spots or flecks that do not enlarge. In a susceptible interaction, the necrotic areas expand, and white mold growth is usually visible along their edges on the underside of the leaflet. If weather conditions remain sufficiently damp, the infection can spread and destroy the entire haulm in a period of several weeks. During the course of progressive infection, spores are spread to other plants by wind, rain, or insects and transferred to the soil and the tubers of infected plants by rain. The means by which late blight is perpetuated are shown in Fig. 1. In the vicinity of potato fields, elimination of cull piles containing blighted tubers is a well-accepted practice for 47 American Chemical Society Library 1155 16th St.N.W. Washington, D. C. 20036

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

48

HOST P L A N T RESISTANCE T O

PESTS

c o n t r o l l i n g the spread of i n f e c t i o n from t h i s source, and the s e l e c t i o n of seed tubers as f r e e from i n f e c t i o n as p o s s i b l e i s recognized as a n e c e s s i t y . One b l i g h t e d seed tuber i n 100,000 provides an adequate i n i t i a l inoculum f o r a major epidemic (1). C o n t r o l of the spread of i n f e c t i o n between potato p l a n t s i s dependent upon the use of f u n g i c i d e s such as the e t h y l e n e b i s d i t h i o carbamates and the p l a n t i n g of potato v a r i e t i e s that have some degree of r e s i s t a n c e . In the United States, the use of f u n g i c i d e s to prevent the spread of l a t e b l i g h t g e n e r a l l y i s p r a c t i c e d i n a l l areas except the western s t a t e s where the summer climate i s u s u a l l y dry and unconducive to the development of l a t e b l i g h t . Under current management p r a c t i c e s i n the United States, l a t e b l i g h t decreases the y i e l d of potatoes at harvest by about 4%. Further l o s s e s are encountered i n storage when tubers with minor b l i g h t e d areas become mixed i n a d v e r t e n t l y w i t h sound tubers. P^. i n f e s t a n s does not g e n e r a l l y spread to healthy tubers during storage, but the l o c i of i n f e c t i o n provide footholds f o r secondary b a c t e r i a l i n f e c t i o n s that spread to other tubers and cause s i g n i f i cant l o s s e s (2). Potato Resistance

to Fungal I n f e c t i o n

The potato i s immune to p a r a s i t i z a t i o n by the vast m a j o r i t y of f u n g i ; there are, i n c l u d i n g _P. i n f e s t a n s , only s i x or seven fungal p a r a s i t e s of the p l a n t and tuber that are economically s i g n i f i c a n t (2). The term immunity i s used to denote complete and apparently permanent p r o t e c t i o n against a given p a r a s i t e , whereas r e s i s t a n c e denotes e i t h e r incomplete or impermanent p r o t e c t i o n . P a r a l l e l s have been drawn between immunity and r e s i s t a n c e (1_, 3), but Robinson (_1) suggests that immunity i s outside of the conceptual bounds of p a r a s i t i s m . Perhaps the major d i f f i c u l t y i n c o n c e p t u a l i z i n g immunity i s that no one yet has formulated a research approach that might lead to a d e s c r i p t i o n of the circumstances r e s p o n s i b l e f o r the immune r e l a t i o n s h i p of p l a n t s to f u n g i . Resistance, on the other hand, provides chemically and p h y s i c a l l y observable and measurable phenomena as a b a s i s f o r research. Resistance to fungal p a r a s i t e s commonly i s d i v i d e d i n t o two general c a t e g o r i e s , h o r i z o n t a l r e s i s t a n c e and v e r t i c a l r e s i s t a n c e . H o r i z o n t a l r e s i s t a n c e i s s a i d to provide a constant l e v e l (high, moderate, low, or n e a r l y zero) of i n f e c t a b i l i t y w i t h a l l e x i s t i n g and p o t e n t i a l races of a given fungal s p e c i e s . From the constant l e v e l of i n f e c t a b i l i t y , the term and concept of h o r i z o n t a l r e s i s t ance was formulated by van der Plank (4). I t i s b e l i e v e d that h o r i z o n t a l r e s i s t a n c e u s u a l l y has a polygenic o r i g i n i n the host and that mechanisms r e s p o n s i b l e f o r h o r i z o n t a l r e s i s t a n c e are l i k e l y to be numerous and complex. I f t h i s were not the usual case, minor genetic changes i n a fungal p a r a s i t e would be more l i k e l y to r e s u l t i n new races capable of p a r a s i t i z i n g the host at higher l e v e l s .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

4.

BILLS

Phytophthora Infestans and

Potato

Host

49

H o r i z o n t a l r e s i s t a n c e can be viewed s p e c u l a t i v e l y as a b a r r i e r of p h y s i c a l or chemical defenses which slow fungal i n v a s i o n . U n a v a i l a b i l i t y i n the host of adequate e s s e n t i a l n u t r i e n t s required f o r fungal growth would c o n s t i t u t e a passive form of h o r i z o n t a l r e s i s t a n c e . The concept of h o r i z o n t a l r e s i s t a n c e can a l s o be extended to chemicals a p p l i e d e x t e r n a l l y . The e f f e c t i v e n e s s of new f u n g i c i d e s i s o f t e n temporary because the f u n g i produce new races unaffected by them. Other f u n g i c i d e s , such as Bordeaux Mixture, appear to have " h o r i z o n t a l p r o p e r t i e s " and maintain t h e i r e f f e c t i v e n e s s permanently, i n d i c a t i n g that the f u n g i are unable to produce new races able to t o l e r a t e them. The i n a b i l i t y of f u n g i to circumvent the t o x i c i t y of the " h o r i z o n t a l " d i t h i o carbamate f u n g i c i d e s , which i n h i b i t twenty or more enzymes (5), i s e a s i l y understood. The presence of a " n a t u r a l h o r i z o n t a l f u n g i c i d e " i n host t i s s u e would confer some degree of r e s i s t a n c e , and there i s evidence, c i t e d l a t e r , that the potato tuber contains indigenous compounds with f u n g i t o x i c p r o p e r t i e s . While potato v a r i e t i e s can e x h i b i t v a r y i n g degrees of h o r i ­ z o n t a l r e s i s t a n c e to P^. i n f e s t a n s , most commercial v a r i e t i e s have (and most breeding programs are aimed at producing) v e r t i c a l r e s i s t a n c e ( 1 ) . The term v e r t i c a l r e s i s t a n c e i s derived from the observation that i n some h o s t - p a r a s i t e i n t e r a c t i o n s i n f e c t i o n of a given host v a r i e t y by d i f f e r e n t fungal races can occur at e i t h e r a high l e v e l ( s u s c e p t i b l e or compatible i n t e r a c t i o n ) or a very low l e v e l ( r e s i s t a n t , incompatible, or h y p e r s e n s i t i v e i n t e r a c t i o n ) ( 4 ) . The terms compatible and incompatible are used i n the remainder of t h i s paper. V e r t i c a l r e s i s t a n c e i s o f t e n r e f e r r e d to as a gene-for-gene r e l a t i o n s h i p between host and fungal p a r a s i t e ( 1 ). Obviously, i t i s not the genes but the chemical and p h y s i c a l c h a r a c t e r i s t i c s whose i n h e r i t a n c e i s g e n e t i c a l l y c o n t r o l l e d that e i t h e r match or do not match each other. Albersheim and AndersonProuty ( 6 ) e x t e n s i v e l y reviewed the evidence f o r the existence of g e n e t i c a l l y c o n t r o l l e d r e c o g n i t i o n mechanisms that r e s u l t i n an incompatible response between fungal p a r a s i t e and p l a n t h o s t . A formal c l a s s i f i c a t i o n scheme has been e s t a b l i s h e d to designate the genetic r e l a t i o n s h i p between potato v a r i e t i e s and ]?. i n f e s t a n s races i n terms of c o m p a t i b i l i t y and i n c o m p a t i b i l i t y ( 7 ) . In t h i s widely accepted scheme, potato v a r i e t i e s are described as posses­ s i n g v e r t i c a l r e s i s t a n c e genes designated as R i , R 2 , R 3 , e t c . A s i n g l e potato v a r i e t y may possess zero, one, or m u l t i p l e r e s i s t a n c e genes. Ρ_. i n f e s t a n s races are described as possessing pathogenici­ ty genes designated as v^, V 2 , V 3 , e t c . (or as race 1 , race 2 , race 3 , e t c . ) , and a s i n g l e race may possess zero, one, or m u l t i p l e p a t h o g e n i c i t y genes. A potato v a r i e t y having the s i n g l e r e s i s t a n c e gene R 2 i n t e r a c t s i n a compatible manner w i t h P^. i n f e s t a n s races with the V 2 gene and i n an incompatible manner with a l l other races. Such a scheme has great u t i l i t y as a c l a s s i f i c a t i o n system, but i t s e m p i r i c a l nature must be kept i n mind. The designations used have no r e a l meaning apart from the observed i n t e r a c t i o n s between host and p a r a s i t e .

50

HOST P L A N T

RESISTANCE T O PESTS

Not a l l i n v e s t i g a t o r s b e l i e v e that r e s i s t a n c e can be n e a t l y categorized as e i t h e r h o r i z o n t a l or v e r t i c a l . A l s o , both types of r e s i s t a n c e may e x i s t simultaneously i n a s i n g l e c u l t i v a r , although strong v e r t i c a l r e s i s t a n c e u s u a l l y tends to mask h o r i zontal resistance.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

Native P r o p e r t i e s Related to Resistance The potato p l a n t and tuber have n a t i v e p h y s i c a l and chemical p r o p e r t i e s r e l a t e d to r e s i s t a n c e . The epidermis of the f o l i a r p o r t i o n of the p l a n t and the s k i n of the tuber present p h y s i c a l b a r r i e r s to fungal and b a c t e r i a l i n f e c t i o n . A moist environment i s r e q u i r e d to permit spore germination and f u n g a l i n v a s i o n across these primary defenses. The stomata of the l e a f l e t s and the l e n t i c e l s of the tuber represent weak p o i n t s i n the primary defenses, but wounding provides a more immediate f o o t h o l d f o r fungal i n v a s i o n . As described by Kuc and C u r r i e r (8), a number of indigenous compounds which have some degree of f u n g i t o x i c i t y are found a t higher l e v e l s i n the outer few m i l l i m e t e r s of the tuber. These compounds are c h l o r o g e n i c a c i d , c a f f e i c a c i d , g l y c o a l k a l o i d s , and s c o p o l i n . The l e v e l s of c h l o r o g e n i c a c i d (9), c a f f e i c a c i d (10), and g l y c o a l k a l o i d s (11, 12) i n c r e a s e i n mechanically wounded tuber t i s s u e , thus adding, perhaps, some degree of p r o t e c t i o n i n an otherwise v u l n e r a b l e s i t u a t i o n . Rapid s u b e r i z a t i o n followed by formation of a wound periderm i n as l i t t l e as 24 hr (2) i s probably more important as a defense. Scopolin and i t s aglycone s c o p o l e t i n are normally present i n potato tubers. The s c o p o l e t i n content of tubers v a r i e s with the p h y s i o l o g i c a l s t a t e , being highest i n newly-harvested, dormant tubers (13). Mechanical d i s r u p t i o n of tuber t i s s u e does not i n c r e a s e the c o n c e n t r a t i o n of s c o p o l i n , but i n f e c t i o n by P^. i n f e s t a n s , other f u n g i , b a c t e r i a , and v i r u s e s causes s i g n i f i c a n t increases (14, 15). Compounds Produced i n I n f e c t e d Potato T i s s u e The incompatible i n t e r a c t i o n of P^. i n f e s t a n s with potato t i s s u e leads to the production of a number of compounds that are not n a t i v e to the host. Studies have been c a r r i e d out predomin a n t l y with tuber t i s s u e d i s r u p t e d by s l i c i n g p r i o r to i n o c u l a t i o n and care must be e x e r c i s e d i n attempting to e x t r a p o l a t e such f i n d i n g s to events that may t r a n s p i r e i n i n t a c t f o l i a r t i s s u e during f i e l d i n f e c t i o n . Zacharius e t a l . (16) have shown that tuber t i s s u e may not even provide a corresponding compatible or incompatible response when compared to the l e a f l e t s of the same potato v a r i e t y . The terpenoid s t r u c t u r e s that have been i s o l a t e d and charact e r i z e d as products of an incompatible i n t e r a c t i o n between _P. i n f e s t a n s and potato tuber t i s s u e are shown i n F i g . 2. Tomiyama

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

4.

BILLS

Phytophthora Infestans and Potato

51

Host

Figure 1. Cycle of Phytophthora infestans in­ fection of Solanum tuberosum. Points at which the spread of infection may be disrupted effec­ tively are indicated at 1,2, and 3.

KATAHDINONE

LUBIMIN

DEHYDROKATAHDINONE

ISOLUBIMIN

OXYLUBIMIN

RISHITIN

Ο RISHITINOL

PHYTUBERIN

Figure 2. Compounds produced by the potato tuber host in response to infection. Most of these compounds have dem­ onstrated fungitoxic properties and are often referred to as phytoalexins.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

52

HOST

PLANT

RESISTANCE T O

PESTS

et a l . i s o l a t e d r i s h i t i n from i n f e c t e d R i s h i r i v a r i e t y potatoes (17), and K a t s u i et a l . c h a r a c t e r i z e d t h i s compound (18). K a t s u i ejt a l . (19, 20) i s o l a t e d and c h a r a c t e r i z e d r i s h i t i n o l . Varns et_ a l . (21) i s o l a t e d phytuberin, and Hughes and Coxon (22) e s t a b l i s h e d i t s s t r u c t u r e . M e t l i t s k i i et a l . (23) f i r s t i s o l a t e d l u b i m i n from a Soviet potato v a r i e t y , Lyubimets, and S t o e s s l et a l . (24) and K a t s u i et a l . (25) determined i t s s t r u c t u r e . K a t s u i et a l . (25) i s o l a t e d and i d e n t i f i e d oxylubimin; Kalan and Osman (26), i s o l u b i m i n . I n v e s t i g a t o r s i n England and i n the U.S. Department of A g r i c u l t u r e (27) simultaneously i s o l a t e d and i d e n t i f i e d katahdinone and dehydrokatahdinone. The t r i v i a l name, katahdinone, was d e r i v e d from the Katahdin potato v a r i e t y from which the compound was i s o l a t e d i n the United S t a t e s . The use of potato tuber s l i c e s i n the above s t u d i e s i s advantageous i n that many potato c e l l s are i n f e c t e d , i n o c u l a t e d s l i c e s can be incubated under c o n t r o l l e d c o n d i t i o n s , and the production of terpenoids i s high per u n i t mass of potato t i s s u e . The production of compounds i n the incompatible i n t e r a c t i o n i n i n t a c t f o l i a r t i s s u e i s more d i f f i c u l t to study because the i n f e c t i o n i s r e s t r i c t e d to very small areas. M e t l i t s k i i et a l . reported lubimin production i n potato l e a f l e t s i n o c u l a t e d with ]?. i n f e s t a n s (28). T h i s suggests, a t l e a s t , that terpenoid production may not be p e c u l i a r to tuber t i s s u e . While the above terpenoids have been i s o l a t e d from incomp a t i b l e i n t e r a c t i o n s between !P. i n f es tans and potato tuber t i s s u e , t h i s i s not the e x c l u s i v e mechanism f o r t h e i r p r o d u c t i o n . Zacharius et a l . (29) reported that r i s h i t i n was produced i n both a compatible i n t e r a c t i o n and an incompatible i n t e r a c t i o n a t a s i m i l a r r a t e during the f i r s t and second days f o l l o w i n g i n o c u l a t i o n . Other r e p o r t s document the production of r i s h i t i n , phytuberin, and lubimin i n potato tuber t i s s u e invaded by E r w i n i a carotovora v a r . a t r o s e p t i c a (30, 31, 32). The formation of terpenoids i n potato tuber t i s s u e c h e m i c a l l y t r e a t e d with NaF was reported by M e t l i t s k i i et a l . (33), but t h i s experiment could not be d u p l i c a t e d by Zacharius et a l . (34). In a d d i t i o n to the production of terpenoids, d e t e c t a b l e changes i n the s o l u b l e p r o t e i n patterns of potato t i s s u e i n t e r a c t i n g incompatibly with ]?. i n f e s t a n s have been r e p o r t e d . In i n f e c t e d tuber t i s s u e , Tomiyama and Stahman found an i n c r e a s e d number of e l e c t r o p h o r e t i c a l l y separable p r o t e i n bands (35). Yamamoto and Konno observed a new p r o t e i n i n l e a f l e t s of the potato p l a n t 6 hr a f t e r i n o c u l a t i o n w i t h an incompatible race of P_. i n f e s t a n s (36). I t i s p o s s i b l e that the incompatible i n t e r a c t i o n r e s u l t s i n the i n d u c t i o n of enzymes not normally present i n the tuber and that such enzymes are the new, observable p r o t e i n s . Further work i s needed to determine the r o l e , i f any, of such proteins i n resistance.

4.

BILLS

Phytophthora Infestans and

Potato

53

Host

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

Elicitors C e l l - f r e e fungal preparations that i n i t i a t e an incompatible response i n host t i s s u e have been r e f e r r e d to as e l i c i t o r s or i n d u c e r s . S o n i c a t i o n of P^. i n f e s t a n s mycelia y i e l d s c e l l - f r e e , autoclavable preparations that i n i t i a t e a t y p i c a l incompatible response, i n c l u d i n g terpenoid production and n e c r o s i s , when a p p l i e d to potato tuber s l i c e s . Varns et a l . (37) found that sonicates of both compatible and incompatible races of _P. i n f e s t a n s produced an incompatible response when a p p l i e d to tuber t i s s u e . Glucans i s o l a t e d from Phytophthora megasperma v a r . sojae, a fungal p a r a s i t e of the soy bean p l a n t , were reported by Ayers et a l . to i n i t i a t e an incompatible response i n host t i s s u e s (38). Albersheim and Anderson-Prouty suggested that e l i c i t o r s are carbohydrate-containing molecules f o r which r e c e p t o r s i t e s e x i s t on the plasma membrane of host c e l l s (6). They f u r t h e r conjectured that the s p e c i f i c i t y of the receptor s i t e f o r i t s "matching ' carbohydrate e l i c i t o r may be the b a s i s f o r incompatible i n t e r a c t i o n s as the phenotypic expression of v e r t i c a l r e s i s t a n c e . In other words, receptor s i t e s and e l i c i t o r s may represent the g e n e t i c a l l y determined primary r e c o g n i t i o n f a c t o r f o r a potato v a r i e t y and an incompatible race of _P. i n f e s t a n s . In a recent study, Kota and S t e l z i g demonstrated that prepa r a t i o n s which a c t as e l i c i t o r s caused membrane d e p o l a r i z a t i o n of potato p e t i o l e c e l l s i n l e s s than 1 min f o l l o w i n g exposure (39). These preparations were a s o n i c a t e , a 3-1,3-glucan, and a l i p o p o l y s a c c h a r i d e ( a l l obtained from P_. i n f e s t a n s ) and a p r e p a r a t i o n from ]P. megasperma. Among e i g h t other carbohydrates which are not e l i c i t o r s , only p e c t i n produced d e p o l a r i z a t i o n . This r e p o r t i s supportive of the e l i c i t o r theory of Albersheim and AndersonProuty (6) and the mechanism of a c t i o n of f u n g a l phytotoxins advanced by S t r o b e l (40). In c o n t r a s t to the theory of Albersheim and Anderson-Prouty (6), Ward and S t o e s s l proposed that a r e c o g n i t i o n mechanism (or i n c o m p a t i b i l i t y suppression mechanism) i s i n v o l v e d i n an i n t e r a c t i o n between molecules produced by the fungus and the compatible host (41). M e t l i t s k i i et a l . added another dimension to s p e c u l a t i o n about the incompatible i n t e r a c t i o n by proposing that the potato host produces compounds which induce the formation of compounds by P_. i n f e s t a n s which i n turn induce the incompatible i n t e r a c t i o n i n the host c e l l (42). Keen r e f e r r e d to the e l i c i t o r phenomenon as a derepression of the production of an a n t i f u n g a l compound i n the soy bean host c e l l (43). More work o b v i o u s l y i s needed to determine the nature of the primary events that d e t e r mine c o m p a t i b i l i t y or i n c o m p a t i b i l i t y between host and fungal parasite. 1

54

HOST

PLANT

RESISTANCE T O

PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

The P h y t o a l e x i n Theory Potato tuber t i s s u e i n o c u l a t e d with an incompatible race of Ζ.· i n f e s t a n s was reported to d i s p l a y an incompatible i n t e r a c t i o n when l a t e r i n o c u l a t e d with a normally compatible race (44). This observation l e d to the "phytoalexin theory" (45), which holds that f u n g i t o x i c compounds, p h y t o a l e x i n s , are produced by host c e l l s as a r e s u l t of i n v a s i o n by an incompatible fungus. Kud and C u r r i e r (8) r e c e n t l y reviewed p h y t o a l e x i n s t u d i e s , most of which were conducted with members of the f a m i l i e s Solanaceae (mainly the potato) and Leguminosae. For the potato, the compounds shown i n F i g . 2 are o f t e n r e f e r r e d to as phytoalexins and most of them have demonstrated f u n g i t o x i c p r o p e r t i e s . The f u n g i t o x i c i t y of r i s h i t i n , phytuberin, l u b i m i n , katahdinone, and dehydrokatahdinone was t e s t e d against m y c e l i a l growth of 1?. i n f e s t a n s by Beczner and Ersek (46). At l e v e l s of 25 to 100 ppm i n s o l i d media i n P e t r i d i s h e s , a l l of the compounds e x h i b i t e d moderate to strong i n h i b i t o r y e f f e c t s toward the f u r t h e r spread of 7-day-old c u l t u r e s of P_. i n f e s t a n s t r a n s ­ f e r r e d to the media. Ward et_ a l . (47) reported s i m i l a r i n h i b i t i o n of m y c e l i a l growth with somewhat lower l e v e l s of r i s h i t i n . Ger­ mination of P_. i n f e s t a n s zoospores was a l s o i n h i b i t e d by s i m i l a r l y low amounts of phytuberin (48), lubimin (49), and r i s h i t i n (50, 51). Since the l e v e l s of phytoalexins able to i n h i b i t pathogen growth i n v i t r o are a t t a i n e d i n many incompatible i n t e r a c t i o n s , i n v e s t i g a t o r s have suggested that phytoalexins p l a y an important r o l e i n i n h i b i t i n g fungal i n v a s i o n (6, 52-57). However, u l t r a s t r u c t u r a l s t u d i e s ( c i t e d l a t e r ) i n d i c a t e observable d i f f e r e n c e s between the compatible and incompatible i n t e r a c t i o n s w e l l i n advance of the production of d e t e c t a b l e l e v e l s of p h y t o a l e x i n s . T h i s does not disprove the theory, s i n c e no one has yet determined the time of appearance or c o n c e n t r a t i o n of phytoalexins i n the microenvironment of the i n d i v i d u a l host c e l l . Conversely, the proof of the theory would r e s t i n determining that phytoalexins are present i n s u f f i c i e n t q u a n t i t i e s e a r l y enough i n the i n f e c t e d , incompatible c e l l to i n h i b i t fungal i n v a s i o n and i n i d e n t i f y i n g the mechanism of i n h i b i t i o n . Hohl and S t o s s e l speculated that phytoalexins i n h i b i t fungal glucanases necessary f o r both f u n g a l growth and d i s r u p t i o n of 0-1,3-glucan b a r r i e r s i n the host c e l l (58). U l t r a s t r u c t u r a l Studies The compatible and incompatible i n t e r a c t i o n s between P^. i n f e s t a n s and potato host c e l l s have been s t u d i e d by scanning e l e c t r o n microscopy and transmission e l e c t r o n microscopy to y i e l d information complementary to biochemical s t u d i e s . Using a scanning e l e c t r o n microscope, Jones et a l . (59) noted sharp boundaries between obviously i n f e c t e d c e l l s near the s u r f a c e and u n i n f e c t e d c e l l s below the s u r f a c e i n the compatible i n t e r a c t i o n

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

4.

BILLS

Phytophthora Infestans and

Potato

Host

55

of P_. i n f e s t a n s with potato tuber t i s s u e . In t h i s i n t e r a c t i o n , hyphae penetrated i n t e r c e l l u l a r l y deep i n t o the t i s s u e where i n t a c t , l i v e potato c e l l s remained to support the colony with n u t r i e n t s . A sharp boundary between n e c r o t i c c e l l s and l i v e c e l l s was not observed i n the incompatible i n t e r a c t i o n . In e l e c t r o n microscopy s t u d i e s of the i n f e c t i o n of potato l e a f c e l l s , Shimony and F r i e n d (60) observed p e n e t r a t i o n of host c e l l s by P^. i n f e s t a n s hyphae i n both compatible and incompatible i n t e r a c t i o n s . Although the r a t e of i n i t i a l p e n e t r a t i o n d i d not d i f f e r , they reported v i s i b l e u l t r a s t r u c t u r a l d i f f e r e n c e s between the compatible and incompatible i n t e r a c t i o n s 7 hr a f t e r i n o c u l a t i o n (with p e n e t r a t i o n o c c u r r i n g as l a t e as 4-1/2 to 6-1/2 hr a f t e r i n o c u l a t i o n ) . Between 9 and 12 h r , host c e l l s surrounding the s i t e of i n f e c t i o n on the incompatible v a r i e t y appeared to be dead and fungal hyphae were contained w i t h i n the n e c r o t i c area; a t 24 hr, s e v e r e l y damaged hyphae were observed; a f t e r 48 h r , there were no d e t e c t a b l e l i v i n g c e l l s of e i t h e r fungus or host i n the small l e s i o n . An observed f e a t u r e of both the compatible and incompatible i n t e r a c t i o n s was the formation of a sheath or encaps u l a t i o n surrounding the i n t r a c e l l u l a r hyphae. In a p a r a l l e l u l t r a s t r u c t u r a l study of the incompatible i n t e r a c t i o n between l e t t u c e (Lactuca s a t i v a ) l e a f t i s s u e and a f u n g a l p a r a s i t e (Bremia l a c t u c a e ) , Maclean et_ a l . (61) a l s o observed that death of penet r a t e d host c e l l s preceded the c e s s a t i o n of growth and death of fungal c e l l s by s e v e r a l hours. Hohl and S t o s s e l (58) a l s o reported that f u n g a l hyphae penet r a t e d potato tuber host c e l l s i n both the compatible and incomp a t i b l e i n t e r a c t i o n s . The type of e n c a p s u l a t i o n d e s c r i b e d by Shimony and F r i e n d (60) was observed i n both incompatible and compatible i n t e r a c t i o n s and was r e f e r r e d to as an e x t r a h a u s t o r i a l matrix. Hohl and S t o s s e l , however, reported that the t y p i c a l haustorium found i n the incompatible tuber host c e l l d i f f e r e d from that of the compatible host c e l l by the presence of an a d d i t i o n a l e n t i t y , w a l l a p p o s i t i o n s , which surrounded and encased the haustorium and the e x t r a h a u s t o r i a l matrix. The encasement of h a u s t o r i a has a l s o been reported f o r the incompatible, but not the compatible, i n t e r a c t i o n of Uromyces p h a s e o l i v a r . vignae with host c e l l s of the cowpea (62). In two a d d i t i o n a l s t u d i e s of a Phytophthora pathogen i n compatible and incompatible i n t e r a c t i o n s , the presence of w a l l a p p o s i t i o n s was not reported (63, 64). In s t u d i e s of l e a f t i s s u e of the same two potato v a r i e t i e s employed i n tuber t i s s u e s t u d i e s by Hohl and S t o s s e l (58), Hohl and Suter (65) found that h a u s t o r i a i n both the compatible and incompatible i n t e r a c t i o n s were s i m i l a r and were always surrounded by an e x t r a h a u s t o r i a l matrix and sometimes a l s o by w a l l a p p o s i t i o n s . As a p o s s i b l e explanation f o r the marked u l t r a s t r u c t u r a l d i f ferences i n h a u s t o r i a observed between the two types of tubers but not i n the l e a f t i s s u e s , the authors pointed out that the l e a f l e t s of the two v a r i e t i e s d i f f e r l e s s i n r e s i s t a n c e than the corresponding tubers. Various types of f u n g a l i n v a s i o n of potato l e a f c e l l s

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

56

HOST P L A N T

RESISTANCE T O PESTS

ranging from hyphae which penetrated c e l l s and then emerged a t another p o i n t to f u l l y developed h a u s t o r i a were observed by Hohl and Suter. Various degrees of h a u s t o r i a l encasement were a l s o observed as shown i n F i g . 3, and the authors suggested that host c e l l r e s i s t a n c e may be r e l a t e d to the degree of encasement that takes p l a c e . They f u r t h e r noted that hyphae enter the l e a f p r i m a r i l y through stomata but are a l s o a b l e to penetrate epidermal c e l l s or h a i r c e l l s . In the incompatible i n t e r a c t i o n , the p a r a s i t e d i d not penetrate deeply i n t o the host t i s s u e and r a r e l y developed s u f f i c i e n t l y to produce sporangia. While u l t r a s t r u c t u r a l s t u d i e s of compatible and incompatible i n t e r a c t i o n s of IP. i n f e s t a n s with the potato host are not i n complete agreement, common f i n d i n g s have emerged: c e l l penetrat i o n occurs i n both circumstances; i n the incompatible i n t e r a c t i o n , p e n e t r a t i o n occurs to a depth of very few c e l l s followed by death of the host c e l l s and l a t e r death of the f u n g a l c e l l s . Conclusion C e r t a i n aspects of the incompatible i n t e r a c t i o n between P_. i n f e s t a n s and the potato host seem to be reasonably well-documented. A number of compounds w i t h demonstrated f u n g i t o x i c i t y are produced when tuber t i s s u e i s invaded, host c e l l s become n e c r o t i c and f u n g a l c e l l s l a t e r d i e . I d e n t i c a l symptoms i n tuber host c e l l s can be i n i t i a t e d by the a p p l i c a t i o n of c e l l - f r e e preparations of the fungus. However, the observed symptoms of i n c o m p a t i b i l i t y may be f a r removed from the primary event that t r i g g e r s the sequence of known events. The f a c t that f u n g i t o x i c compounds are evolved i s not complete proof that they are, indeed, the cause of incomp a t i b i l i t y i n the complex c e l l - c e l l i n t e r a c t i o n . Gene-for-gene i n t e r a c t i o n s between host and p a r a s i t e can be s y s t e m a t i c a l l y tabulated, but only i n the e m p i r i c a l terms of compatible or incomp a t i b l e . The phenotypic mechanisms i n v o l v e d i n i n t e r a c t i o n s a t the c e l l and molecular l e v e l s remain to be d e s c r i b e d . E l u c i d a t i o n of the molecular b a s i s f o r the s p e c i f i c i t y e x h i b i t e d i n gene-forgene, host-pathogen i n t e r a c t i o n s i s w i t h i n reach and i s an important o b j e c t i v e . Although v a l u a b l e i n f o r m a t i o n has been gained through s t u d i e s with tuber t i s s u e , i t appears that a d d i t i o n a l s t u d i e s w i t h f o l i a r t i s s u e should be emphasized. Phenomena observed i n i n t e r a c t i o n s of P_. i n f e s t a n s w i t h s l i c e d or otherwise d i s r u p t e d tuber t i s s u e cannot be assumed to occur i n f o l i a r t i s s u e . Since r e s i s t a n c e to _P. i n f e s t a n s must be expressed i n the f o l i a r p o r t i o n of the potato p l a n t i n order to break the c y c l e of i n f e c t i o n shown i n F i g . 1, i t i s of primary importance to d e s c r i b e and understand r e s i s t a n c e i n terms of the l e a f l e t s r a t h e r than the tubers. Great emphasis has been placed on understanding v e r t i c a l r e s i s t a n c e i n the potato, while h o r i z o n t a l r e s i s t a n c e has r e c e i v e d comparatively l i t t l e study. Since v e r t i c a l r e s i s t a n c e i s temporary and e f f e c t i v e only u n t i l a new race of P_. i n f e s t a n s capable of

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

4.

BILLS

Phytophthora Infestans and Potato

Host

Canadian Journal of Botany

Figure S. Diagram of haustorial development of Phytophthora infestans on potato leaves, (a) Early penetration of host wall with incipient extrahaustorial matrix formation between host wall and host plasmalemma (hp), (b) Haustorium ensheathed by extrahaustorial matrix and collared by wall appositions at its neck, (c) Progressive encasement of haustorium (ha) by wall appositions (wa). The host plasma membrane (hp) may double-over on itself at the interface of the extrahaustorial matrix (ema) and the wall apposition, (d) Fully encased haustorium with compressed extrahaustorial matrix. Haustorial development may terminate at (b), (c), or (d), probably reflecting rising degrees of host cell resistance (65).

57

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

58

HOST PLANT RESISTANCE TO PESTS bypassing the resistance mechanism evolves, additional studies aimed at identifying the physical and chemical features of the potato that contribute to horizontal resistance seem in order. Despite the extensive efforts that have been devoted to breeding potato varieties resistant to P_. infestans, potato growers must rely heavily on the use of fungicides to control late blight. Environmental and economic considerations discourage the continued use of conventional fungicides and encourage further research to understand the chemical basis for potato plant re­ sistance to P_. infestans. Robinson (1), critical of the unholistic approach to disease resistance, pointed out that the disciplines involved have concentrated too heavily upon their particular interests. Effective teams that include plant breeders, plant pathologists, microscopists, and chemists will probably have the greatest impact in the further conceptualization of resistance and the practical application of the concepts to the development of varieties with improved resistance. Literature Cited 1. Robinson, R. A. "Plant Pathosystems," pp 15-54, SpringerVerlag, Berlin, Heidelberg, New York, 1976. 2. Burton, W. G. "The Potato," pp 97-107, 248-253, H. Veenman and Zonen, Ν. V., Wageningen, Holland, 1966. 3. Plank, J. E., van der. "Principles of Plant Infection," p 216, Academic Press, New York-London, 1975. 4. Plank, J. E., van der. "Plant Diseases. Epidemics and Control," p 349, Academic Press, New York-London, 1963. 5. Day, P. R. "Genetics of Host-Parasite Interaction," p 238, W. H. Freeman and Co., San Francisco, 1974. 6. Albersheim, P., and Anderson-Prouty, A. J., Ann. Rev. Plant Physiol. (1975) 26, 31-52. 7. Black, W., Mastenbroek, C., Mills, W. R., and Peterson, L. C., Euphytica (1953) 2, 173-178. 8. Kuć, J., Currier, W. "Mycotoxins and Other Fungal Related Food Problems," Advances in Chemistry Series 149, pp 356-368, American Chemical Society, Washington, D.C., 1976. 9. Sakuma, T., and Tomiyama, Κ., Phytopathol. Soc., Jap. (1967) 33, 48-58. 10. Kuć, J., Henze, R. E., and Ullstrup, A. J., J. Amer. Chem. Soc. (1956) 78, 3123-3125. 11. McKee, R., Ann. Appl. Biol. (1955) 43, 147-148. 12. Locci, R., andKuć,J., Phytopathology (1967) 57, 1272-1273. 13. Korableva, N. P., Morozova, Ε. V., and Metlitskii, L. V., Dokl. Akad. Nauk SSSR (1973) 212, 1000-1002. 14. Clarke, D. D., Phytochemistry (1969) 8, 7. 15. Clarke, D. D. and Baines, P. S., Physiol. Plant Pathol. (1976) 9, 199-203. 16. Zacharius, R. M., Osman, S. F., Heisler, E. G., and Kissinger, J. C., Phytopathology (1976) 66, 964-966.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

4. BILLS

Phytophthora Infestans and Potato Host

17. Tomiyama, K., Sakuma, T., Ishizaka, Ν., Sato, Ν., Katsui, Ν., Takasugi, Μ., and Masamune, T., Phytopathology (1968) 58, 115-116. 18. Katsui, N., Murai, Α., Takasugi, M., Imaizumi, Κ., and Masamune, T., Chem. Commun. (Sect. D) (1968), 43-44. 19. Katsui, N., Matsunaga, Α., Imaizumi, Κ., and Masamune, T., Tetrahedron Lett. (1971) No. 2, 83-86. 20. Katsui, N., Matsunaga, Α., Imaizumi, K., and Masamune, T., Bull. Chem. Soc. Jap. (1973) 45, 2871-2877. 21. Varns, J., Kuć, J., and Williams, Ε. B., Phytopathology (1971) 61, 174-177. 22. Hughes, D. L., and Coxon, D. T., J. Chem. Soc., D. (1974), 822-823. 23. Metlitskii, L. V., Ozeretskovskaya, O. L., Chalova, L. I., Vasyukova, Ν. I., and Davydova, Μ. Α., Mikol. Fitopatol. (1971) 5, 263-271. 24. Stoessl, Α., Strothers, J. B., and Ward, E. W. B., J. Chem. Soc., D. (1974), 709-710. 25. Katsui, N., Matsunaga, Α., and Masamune, T., Tetrahedron Lett. (1974) No. 51-52, 4483-4486. 26. Kalan, Ε. B., and Osman, S. F., Phytochemistry (1976) 15, 775-776. 27. Coxon, D. T., Price, K. R., Howard, B., Osman, S. F., Kalan, Ε. Β., and Zacharius, R. M., Tetrahedron Lett. (1974) No. 34, 2921-2924. 28. Metlitskii, L. V., Ozeretskovskaya, O. L., Vasyukova, Ν. I., Davydova, Μ. Α., Savel'eva, O. H., and D'yakov, Yu, T., Mikol. Fitopatol. (1974) 8, 42-49. 29. Zacharius, R. M., Osman, S. F., Kalan, Ε. Β., and Thomas, S. R., Proc. Am. Phytopath. Soc. (1974) 1, 63. 30. Lyon, G. D., Physiol. Plant Pathol. (1972) 2, 411-416. 31. Lyon, G. D., Lund, B. M., Bayliss, C. E., and Wyatt, G. M., Physiol. Plant Pathol. (1975) 6, 43-50. 32. Beczner, J., and Lund, B. M., Acta Phytopathologica Academiae Scientiarum Hungaricae (1975) 10, 269-274. 33. Metlitskii, L. V., D'yakov, Yu. T., Ozeretskovskaya, O. L., Yurganova, L. Α., Chalova, L. I., and Vasyukova, Ν. I., Izvestiya Akad. Nauk SSSR, Ser. Biol. (1971), 399-407. 34. Zacharius, R. M., Kalan, Ε. B., Osman, S. F., and Herb, S. F., Physiol. Plant Pathol. (1975)6,301-305. 35. Tomiyama, Κ., and Stahmann, M. Α., Plant Physiol. (1964) 39, 483-490. 36. Yamamoto, M., and Konno, K., Plant and Cell Physiol. (1976) 17, 843-846. 37. Varns, J. L., Currier, W. W., and Kuć, J., Phytopathology (1971) 61, 968-971. 38. Ayers, A. R., Ebel, J., Valent, B., and Albersheim, P., Plant Physiol. (1976) 57, 760-765. 39. Kota, D. Α., Stelzig, D. Α., Proc. Am. Phytopathol. Soc. (1977) 4, in press.

59

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch004

60

HOST PLANT RESISTANCE TO PESTS

40. Strobel, G. Α., Scientific American (1975) 232, 81-88. 41. Ward, E. W. B., Stoessl, Α., Phytopathology (1976) 66, 940941. 42. Metlitskii, L. V., D'yakov, Yu. T., and Ozeretskovskaya, O. L., Biol. Abs. (1974) 58, No. 5, 2978. 43. Keen, N. T., Science (1975) 187, 74-75. 44. Müller, Κ., and Borger, H., Arb. Biol. Reichsanst. Land Forstwirt, Berlin (1940) 23, 189-231. 45. Müller, Κ., and Behr, L., Nature (1949) 163, 498-499. 46. Beczner, J., and Érsek, T., Acta Phytopathologica Academiae Scientiarum Hungaricae (1976) 11, 59-64. 47. Ward, E. W. B., Unwin, C. Η., and Stoessl, Α., Can. J. Botany (1974) 52, 2481-2488. 48. Varns, J. L. "Biochemical Response and its Control in the Irish Potato (Solanum tuberosum) -Phytophthora infestans Interactions," Ph.D. Thesis, Purdue University, Lafayette, Indiana, 1970. 49. Metlitskii, L. V., and Ozeretskovskaya, O. L., Fitoalexini. Akad. Nauk, SSSR, Nauka, Moskva, pp 176, 1973. 50. Tomiyama, Κ., Ishizaka, N., Sato, N., Masamune, T., and Katsui, N. "Biochemical Regulation in Diseased Plants or Injury," pp 287-292, Phytopath. Soc., Japan, Tokyo, 1968. 51. Ishizaka, N., Tomiyama, Κ., Katsui, N., Murai, Α., and Masamune, T., Plant Cell Physiol. (1969) 10, 183-192. 52. Keen, N. T., Physiol. Plant Pathol. (1971) 1, 265-275. 53. Rahe, J. E., Kuć, J., Chuang, C. M., and Williams, Ε. B., Neth. J. Plant Pathol. (1969) 75, 58-71. 54. Sato, N., Kitazawa, Κ., and Tomiyama, Κ., Physiol. Plant Pathol. (1971) 1, 289-295. 55. Skipp, R. Α., Physiol. Plant Pathol. (1972)2,357-374. 56. Bailey, J. Α., and Deverall, B. J., Physiol. Plant Pathol. (1971) 1, 435-439. 57. Frank, J. Α., and Paxton, J. D., Phytopathology (1970) 60, 315-318. 58. Hohl, H. R., and Stossel, P., Can. J. Bot. (1976) 54, 900912. 59. Jones, S. B., Carroll, R. J., and Kalan, Ε. B. "Scanning Electron Microscopy, Part 2, Proceedings of the Workshop on Scanning Electron Microscopy and the Plant Sciences," pp 397404, IIT Research Institute, Chicago, Illinois, 1974. 60. Shimony, C., and Friend, J., New Phytol. (1975) 74, 59-65. 61. Maclean, D. J., Sargent, J. Α., Tommerup, I. C., and Ingram, D. S., Nature (1974) 249, 186-187. 62. Heath, M. C., and Heath, I. B., Physiol. Plant Pathol. (1971) 1, 277-287. 63. Klarman, W. L., and Corbett, Μ. Κ., Phytopathology (1974) 64, 971-975. 64. Hanchey, P., and Wheeler, Η., Phytopathology (1971) 61, 33-39. 65. Hohl, H. R., and Suter, Elisabeth, Can. J. Bot. (1976) 54, 1956-1970.

5 Biosynthetic Relationships of Sesquiterpenoidal Stress Compounds from the Solanaceae

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

ALBERT STOESSL and E. W. B. WARD Agriculture Canada, Research Institute, University Sub Post Office, London, Ontario, Canada N6A 5B7 J. B. STOTHERS Chemistry Department, University of Western Ontario, London, Ontario, Canada N6A 3K7 Rishitin (I) was reported as a phytoalexin of potatoes (1) and tomatoes (2) in 1968 and was the first sesquiterpenoidal stress compound to be isolated and characterized from the Solanaceae. In the 9 years since, some 20 other, structurally more or less closely related sesquiterpenes have been described from potato, aubergine, thornapple, sweet pepper, and tobacco species (Tables I - V) (3 - 19). All of these compounds are produced essentially only under stress conditions, that is, after infection of the plant with fungi, bacteria, or viruses, or through wounding, exposure to UV, or treatment with deleterious substances. Not all, however, are necessarily phytoalexins because some lack the antifungal activity which is requisite for that function. In addition, several other compounds (Table VI) (20 - 22) which are structurally and biogenetically closely related to the stress metabolites, have been obtained in very low amounts from cured tobacco leaves. Because of this special circumstance, it is largely unclear whether these are stress compounds which were formed through undetected infection, or through other stress processes in the ageing or curing of the leaf, or whether they may have been present as normal constituents of the healthy tissue. In any event, their structures render them relevant to the present discussion. All bicyclic compounds in Tables (I - VI) are either eudesmanes or can be regarded as derived from eudesmanes by plausible rearrangements. Except for the single case of rishitinol (II), the rearrangements can be represented formally as proceeding through intermediates with C-4 and C-5 as cationic migration termini. Such a formal intermediate (XXVII) and its relationship with some of the metabolites are set out in Scheme 1. It is also notable that the bicyclic compounds, again with the exception of rishitinol, carry oxygen atoms on one or more of carbons 1 to 4 but none on carbons 6 to 9. Rishitinol is oxygenated at C-8, a position which corresponds to a carbon atom which is also oxygenated in the acyclic nerolidol derivatives XIV and XV from eggplant. These close structural relationships must reflect largely common biogenetic origins and pathways, and also, since they are 61

62

H O S T P L A N T RESISTANCE T O PESTS

Table I .

Splanum tuberosum s t r e s s

metabolites

15

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

rishitin (I) (1,2)

rishitinol ( I I ) (3)

phytuberin ( I I I , R=Ac) (4,5) phytuberol (IV, R=H) (6,2)

lubimin (V, R=H) (8,9)

15-dihydrolubimin (VII) (11)

15-dihydro-10-epilubimin (IX, R=CH 0H) (11)

hydroxylubimin (VI, R=OH) (10)

isolubimin (X) (12)

10-epilubimin ( V I I I , R-CHO) (11)

2

solavetivone (XI) (13)

anhydro-3 - r o t u n o l (XII) (13)

5.

STOESSL

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

Table I I .

Table I I I .

E T AL.

Sesquiterpenoidal

Stress Compounds

from

Solanaceae

Solanurn melongena s t r e s s m e t a b o l i t e s (9)

Datura stramonium s t r e s s m e t a b o l i t e s (10)

lubimin (V, R=H) hydroxylubimin (VI, R=0H)

germacrene-A-diol (XVII)

capsidiol (XVIII)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

64

H O S T P L A N T RESISTANCE T O

Table V.

Nlcotlana stress metabolites

OH

HO

capsidiol (XVIII) (17)

glutinosone (XX) (18)

*possibly of fungal o r i g i n ; biogenetic d e r i v a t i o n uncertain

quiesone* (XXI) (19)

PESTS

5.

STOESSL

Table VI.

Sesquiterpenoidal

E T AL.

Stress Compounds

from Solanaceae

65

Nlcotiana sesquiterpenes not proven as stress compounds

HO

1-keto-a-cyperone (XXI) (20)

solavetivone (XI, R^R^H) (21)

11,13-dihydroxysolavetivone* (XXVI)

3-ot-hydroxysolavetivone* (XXIII, R^ctH, R =H

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

2

3-3-hydroxys ο1ave t ivone * (XXIV, Rj=BH , R =H 2

13-hydroxysolavetivone* (XXV, R =H, R =0H)

OPP XXIX

XXVIII

τ

OH XIV, XV

T

XIII, XXII Scheme 1.

*occur and i s o l a t e d as glucosides (22)

I, XX

XVIII, xix

Biogenetic interrelations of sesquiterpenes from the Solanaceae

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

66

HOST P L A N T RESISTANCE T O

PESTS

unique to the Solanaceae, a considerable degree of genetic specialization. Several more or l e s s e q u i v a l e n t schemes which set out p o s s i b l e b i o g e n e t i c routes i n greater though s p e c u l a t i v e d e t a i l were discussed p r e v i o u s l y (23). The examples shown i n Scheme 2 i l l u s t r a t e the stereochemical features of such p r o p o s a l s . A key element i s the germacrenediol (XVII, R=OH) from Datura stramonium and i t s h y p o t h e t i c a l monohydroxy analogue (XVII, R=H). As judged by molecular models, these compounds are f l e x i b l e and can r e a d i l y adopt conformations which would allow them to be converted to the b i c y c l i c metabolites, both of Datura and other Solanaceae, by the pathways i n d i c a t e d . The Scheme i s only i l l u s t r a t i v e and the i n termediacy of other, d i f f e r e n t l y s u b s t i t u t e d germacrenes i s not excluded and i s probable f o r at l e a s t some of the Solanaceae metab o l i t e s . In general, however, a f u n c t i o n of germacrenes as b i o genetic precursors of b i c y c l i c sesquiterpenes has long been p o s t u l a t e d and i s widely accepted (24) . Many of the features of these suggested pathways should be q u i t e r e a d i l y s u s c e p t i b l e to experimental t e s t and some progress i n t h i s d i r e c t i o n has been made. Thus, Brooks and a s s o c i a t e s have u t i l i z e d acetate, d o u b l y - l a b e l l e d with ^ C , to demonstrate that c a p s i d i o l i s formed from a c e t a t e , v i a mevalonate, with the p o s t u l a t e d methyl migration from the C-10 to the C-5 p o s i t i o n i n a presumably eudesmanoid precursor (28). An a l t e r n a t i v e proposal invoking a two-fold rearrangement v i a a s p i r o - i n t e r m e d i a t e (29) was thereby e l i m i n a t e d . In an analogous experiment with Datura stramonium, we could show (30) that the germacrenediol (XVII) and the v e t i s p i r a n e s , lubimin (V) and hydroxylubimin ( V I ) , i n c o r p o r a t e d l,2-13c -acetate P a t t e r n which conformed p r e c i s e l y to the p o s t u l a t e d b i o g e n e t i c pathways. Our more recent work has a l s o r e l i e d almost e x c l u s i v e l y on the use of l,2-^^C2-acetate, and a b r i e f o u t l i n e of the e s s e n t i a l s of t h i s powerful and r e l a t i v e l y new methodology (31 - 34) may be i n order. Unlike carbon-12, carbon-13 has a magnetic moment and can undergo energy t r a n s i t i o n s which can be observed i n an NMR s p e c t r o meter. However, because the magnetic moment of carbon-13 i s s m a l l e r and a l s o because of the low n a t u r a l abundance (1%) of the i s o t o p e , the s e n s i t i v i t y f o r i t s d e t e c t i o n i s only about 1/6000 of that f o r protons. This problem has been l a r g e l y overcome, through the advent of F o u r i e r transform spectroscopy around 1970, and l^C-NMR s p e c t r a can now be obtained r o u t i n e l y on 10-20 mg samples, but smaller amounts can s u f f i c e i f necessary. The low n a t u r a l abundance of C has emerged as a b l e s s i n g because i t i s the necessary c o n d i t i o n f o r the use of the isotope as a t r a c e r . In b i o s y n t h e t i c s t u d i e s , t h i s can be e x p l o i t e d with great advantage because any incorporated l a b e l can be observed d i r e c t l y i n the NMR spectrum which w i l l define the molecular s i t e s as w e l l as the extent of i n c o r p o r a t i o n . Low l e v e l s of i m p u r i t i e s i n the product u s u a l l y w i l l n e i t h e r mislead nor i n t e r f e r e i n the determination. Thus, the !3c-NMR methodology obviates both the i n

2

1

a

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

5.

STOESSL E T AL.

τ

XI

Scheme 2.

Sesquiterpenoidal

Stress Compounds

τ

VII, IX

Stereochemistry of possible routes to bicyclic Solanaceae

from

Solanaceae

τ

XX

sesquiterpenes

of

68

H O S T P L A N T RESISTANCE T O

r i g o r o u s p u r i f i c a t i o n requirements of r a d i o a c t i v e t r a c e r methods and p a r t i c u l a r l y , a l s o the o f t e n d i f f i c u l t , m a t e r i a l - and timeconsuming degradative procedures which are needed to l o c a t e the i n c o r p o r a t i o n s i t e s of radio-atoms. ^ Both s i n g l y - and d o u b l y - l a b e l l e d C-acetate are much used because of t h e i r ready a v a i l a b i l i t y commercially i n 90% i s o t o p i c p u r i t y . I n c o r p o r a t i o n from s i n g l y - l a b e l l e d acetate i s detected by intensity-enhanced s i g n a l s from the i n c o r p o r a t i o n s i t e s . The s i g n a l s of a proton-noise decoupled spectrum are s i n g l e t s and an enhancement of ca 20% U a 0.2 percent enrichment) can be detected. The use of doubly l a b e l l e d , C - a c e t a t e o f f e r s the f u r t h e r advantages of somewhat g r e a t e r s e n s i t i v i t y and of p r o v i d i n g more s t r u c t u r a l i n f o r m a t i o n . Carbon-13 has a spin-quantum number I=h and t h e r e f o r e continguous -^C atoms w i l l be spin-coupled much l i k e protons. In unenriched molecules, such coupling i s normally not detected because of the very low i n c i d e n c e (ca 1 χ 10"^) of ad­ j a c e n t 13c atoms. S i m i l a r l y , except when enrichment i s of the order of s e v e r a l atom percent, i n c o r p o r a t i o n of s i n g l y - l a b e l l e d acetate a l s o does not l e a d to e a s i l y observed s p i n - s p i n m u l t i p l e t s of d i r e c t l y bonded carbons. However, d o u b l y - ^ c - l a b e l l e d acetate, i f i t i s i n c o r p o r a t e d i n t a c t and without subsequent s c i s s i o n , must n e c e s s a r i l y give r i s e to a s p i n - s p i n system which, i n a proton-noise decoupled 13ς;_^^ spectrum, w i l l appear as a p a i r of doublets. In p r a c t i c e , the s p e c t r a of b i o s y n t h e t i c a l l y enriched molecules w i l l e x h i b i t t r i p l e t s because the doublets from i n c o r ­ porated acetate w i l l be superimposed on the s i n g l e t s from n a t u r a l abundance l ^ C atoms . The outer components of t r i p l e t s are r e ­ f e r r e d to as s a t e l l i t e s . T h e i r s e p a r a t i o n i s the coupling con­ stant J and, as i n the case of protons, i t s magnitude i s char­ a c t e r i s t i c f o r the p a i r of coupled atoms. The i n t e n s i t y of the s a t e l l i t e s r e l a t i v e to the c e n t r a l peak of the t r i p l e t i s a f u n c t i o n of the enrichment. A r e l a t i v e i n t e n s i t y of about 5% f o r each s a t e l l i t e w i l l r e a d i l y s u f f i c e f o r d e t e c t i o n and w i l l cor­ respond to roughly 0.14% enrichment at each s i t e . The same s p e c t r a may a l s o contain enhanced s i n g l e t s unaccompanied by s a t e l l i t e s . These i n d i c a t e carbon atoms derived from acetate u n i t s that were cleaved by e i t h e r degradation or rearrangement during the b i o s y n t h e t i c process. Some of these p o i n t s are n i c e l y i l l u s t r a t e d by c o n s i d e r i n g the b i o s y n t h e s i s of f a r n e s y l pyrophosphate (XXVIII), the accepted common precursor of sesquiterpenes i n g e n e r a l . The pathway, which was e l u c i d a t e d i n remarkable d e t a i l Ç35, _36) and before the CNMR methodology had become a v a i l a b l e , i s represented i n Scheme 3. A noteworthy feature i s the high degree of s t e r e o s p e c i f i c i t y which i s c h a r a c t e r i s t i c of each of the i n d i v i d u a l steps shown and which i s maintained a l s o i n subsequent transformations. Another feature, p a r t i c u l a r l y r e l e v a n t i n the present context, i s that the t r a n s formation of mevalonate i n t o isopentenylpyrophosphate i n v o l v e s the l o s s of carbon dioxide from one of i t s 3 c o n s t i t u t i v e acetate u n i t s . F a r n e s o l , being b u i l t up from 3 isoprene r e s i d u e s , w i l l 1 3

2

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

PESTS

5.

STOESSL

ET

AL.

Sesquiterpenoidal

from Solanaceae

69

t h e r e f o r e comprise 6 i n t a c t acetate r e s i d u e s , shown i n the scheme by heavy bonds, and 3 lone carbon atoms derived from C-2 of mevalonate and i n d i c a t e d by heavy dots. In the l^C-NMR spectrum of f a r n e s o l b i o s y n t h e s i z e d from d o u b l y - l a b e l l e d acetate, the atoms l i n k e d by heavy bonds t h e r e f o r e would e x h i b i t t r i p l e t while the lone carbons w i l l show s i n g l e t absorption. This p a t t e r n w i l l be maintained i f the f a r n e s o l undergoes subsequent s u b s t i t u t i o n and c y c l i z a t i o n r e a c t i o n s without rearrangement or carbon l o s s , that i s , i f i t i s converted i n t o sesquiterpenes which obey Ruzicka's isoprene r u l e . T h i s i s e x a c t l y what i s observed, f o r instance i n the b i o s y n t h e t i c study (30) of the germacrenediol (XVII) from D. stramonium. The l^C-NMR P enriched compound i s shown i n F i g . 1 and e x h i b i t s a l l the expected f e a t u r e s . D o u b l y - l a b e l l e d acetate i s p a r t i c u l a r l y w e l l s u i t e d f o r studying the b i o s y n t h e s i s of sesquiterpenes which do not obey the isoprene r u l e . This i s i l l u s t r a t e d by the already c i t e d study of c a p s i d i o l by Brooks and coworkers (28) and by the recent work with the potato sesquiterpenes (37) which i s now to be des c r i b e d i n some d e t a i l . To induce the b i o s y n t h e s i s of the sesquiterpenes, f r e s h l y cut potato s l i c e s were i n o c u l a t e d with spore suspensions of M o n i l i n i a f r u c t i c o l a i n one experiment, and of Glomerella c i n g u l a t a i n another. The i n o c u l a t i o n s i t e s c o n s i s t e d of hemis p h e r i c a l w e l l s of ca 1 cm. radius cut i n t o the s l i c e s w i t h a melon b a i l e r . Each r e c e i v e d 2 ml of spore suspension and ^Csodium acetate, of about 90% i s o t o p i c p u r i t y at both C - l and C-2, was added a day l a t e r , at the r a t e of 0.5 mg i n 0.2 ml water per w e l l . The l i q u i d contents of the w e l l s were c o l l e c t e d a f t e r another day and e x t r a c t e d with ether. The product so obtained was chromatographed over a l a r g e column (1 g/mg e x t r a c t ) of s i l i c a as used f o r TLC (but not a c t i v a t e d by h e a t i n g ) . E t h e r - l i g h t p e t r o l 1:1 or methanol-chloroform 5:95 are s u i t a b l e s o l v e n t s . The m a t e r i a l s obtained i n t h i s b a s i c f r a c t i o n a t i o n were then f u r t h e r p u r i f i e d as necessary by one or more column or p r e p a r a t i v e TLC s e p a r a t i o n s . In t h i s manner, the i n t e r a c t i o n of the potatoes with M. f r u c t i c o l a y i e l d e d the compounds l i s t e d i n Table VTI, i n the amounts i n d i c a t e d . In t h e i r ^C-NMR P » isolates e x h i b i t e d the expected l a b e l l i n g p a t t e r n s . For example, r i s h i t i n ( I ) , the main component of the product, gave r i s e to 5 p a i r s of t r i p l e t s and 4 s i n g l e t s , e n t i r e l y c o n s i s t e n t with the o x i d a t i v e l o s s of C-15 i n i t s formation ( F i g . 2). In t h i s spectrum, two other features can be observed which were seen a l s o i n the s p e c t r a of a l l the metabolites which we examined. F i r s t l y , i n t e n s i t i e s of the s a t e l l i t e s r e l a t i v e to each c e n t r a l peak are a l l of the same order of magnitude, implying that the enrichment l e v e l i s roughly the same at a l l s i t e s . This i s c o n s i s t e n t with r a p i d de novo s y n t h e s i s without recourse to already present metabolic pools. Secondly, although an a l l y l i c s h i f t e q u i l i b r a t i o n of the s i d e chain double bond between the 11, 12 and 11, 13 p o s i t i o n s can be envisaged as e a s i l y o c c u r r i n g throughout the b i o s y n t h e t i c sequence, s

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

Stress Compounds

e

c

t

r

u

m

o f

s

t

n

e

e c t r a

a

1

1

t

n

e

70

H O S T P L A N T RESISTANCE T O PESTS

OH CI^-COS Co A

H0 C

C0 H

2

2

H

R

H

H

s

H

R

s

3-hydroxy-3-methylglutarate

a n t l

..

U

+

«η"

H«

/*·

PPO

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

Hp

H

R

Η

1 R

H

Hp

s

H

H

H R

IPP

H

3

. S

H

H

H

H

R

H

H

S R

H

H

R . S

S

R

\

•

PPO

syn ; inversion

H

R

s

R

Hp H

8

H

R

Hp

geranylpyrophosphate

Scheme 3.

Biosynthesis of farnesol

\ — /

Figure 1.

C NMR spectrum of 2,3-dihydroxygermacrene (XVII) biosynthesized from 1,2- C-acetate

13

13

5.

STOESSL E T A L .

Sesquiterpenoidal

Stress Compounds

from Solanaceae

Table VII ^ C-Labelled Compounds from the Potato - M o n i l i n i a fructicola interaction. Compound

y i e l d (mg)*

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

r i s h i t i n (I) lubimin (V) e p i l u b i m i n (VIII) hydroxylubimin (VI) dihydroepilubimin (IX) phytuberin ( I I I )

69 15 3 8 4 <1

* a f t e r f i n a l p u r i f i c a t i o n ; from 120 l b s potatoes

<SkOH T

Ji

CDCI

Figure 2.

3

C NMR spectrum of rishitin biosynthesized from 1,2- C-acetate

13

13

71

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

72

HOST

PLANT

RESISTANCE T O PESTS

the s p e c t r a show that such scrambling does not, i n f a c t , occur. Thus, the only carbon detectable as coupled t o C - l l i s o l e f i n i c and, s i m i l a r l y , the s i g n a l from the i s o p r o p e n y l methyl group i s of s i n g l e t character only. The spectrum o f lubimin i n F i g . 3 i l l u s t r a t e s the case o f a metabolite which e x h i b i t s the normal t r i p l e t s and 3 s i n g l e t s a l though a molecular arrangement d i d intervene i n i t s b i o s y n t h e s i s . Again, t h i s i s the expected r e s u l t because the bond broken i n the rearrangement was between two acetate residues and not w i t h i n one. E n t i r e l y analogous s p e c t r a were obtained a l s o from hydroxylubimin (VT) and from 10-epi- and 15-dihydro-10-epilubimin (VII and I X ) . The l a s t two compounds have not been p r e v i o u s l y Reported. T h e i r s t r u c t u r e s followed r e a d i l y from t h e i r % - and C-NMR s p e c t r a and i n passing, i t may be noted that the -^C- coupling patterns were themselves u s e f u l i n the s t r u c t u r e e l u c i d a t i o n . The b i o g e n e t i c o r i g i n o f the two compounds has not been c l a r i f i e d but p o s s i b l y , e p i l u b i m i n (VIII) i s formed v i a the e n o l i s a t i o n o f the aldehyde group o f lubimin (V). I t may then give r i s e to the d i hydro compound IX by r e d u c t i o n . However, the a l t e r n a t i v e pathway, from a germacrene to dihydroepilubimin (IX) and hence to lubimin (V) v i a e p i l u b i m i n ( V I I I ) , appears e q u a l l y a t t r a c t i v e . The mother l i q u o r s from which d i h y d r o e p i l u b i m i n (IX) had separated a l s o contained a l i t t l e 15-dihydrolubimin ( V I I ) . The amount was too s m a l l to allow i t s i s o l a t i o n but i t s presence was i n d i c a t e d by chromatography and e s t a b l i s h e d by the ^H-NMR spectrum of the mother l i q u o r s which was the composite o f the s p e c t r a o f pure VII and IX. Again, i t i s not c l e a r whether dihydrolubimin (VII) precedes lubimin (V) b i o g e n e t i c a l l y o r i s formed from i t by r e d u c t i o n . Formally, a t l e a s t one o f the two dihydro-compounds must be a p r e c u r s o r o f l u b i m i n but p o s s i b l y only i n an enzymebound form. I t i s , indeed, conceivable that the four compounds p a r t i c i p a t e i n a dynamic e q u i l i b r i u m . This problem i s r e c e i v i n g further attention. The l a s t compound from the potato - M. f r u c t i c o l a i n t e r a c t i o n which needs to be discussed i s phytuberin. Its predicated b i o s y n t h e s i s i s shown i n Scheme 4. I n the M. f r u c t i c o l a experiment, only a very s m a l l amount, l e s s than 1 mg, was produced but t h i s s u f f i c e d to give a - ^ c - N ^ r spectrum i n which the 5 p r e d i c t e d , enr i c h e d s i n g l e t absorptions were v i s i b l e . However, the t r i p l e t s r e s u l t i n g from l a b e l l e d carbon p a i r s and n a t u r a l abundance were too weak to be observed. Because o f the unusual nature o f the b i o s y n t h e t i c route to phytuberin, a more e x p l i c i t l y informat i v e spectrum was d e s i r a b l e and i t i s f o r t h i s reason that we turned to an i n c o r p o r a t i o n experiment w i t h the potato - Glomerella c i n g u l a t a system. Other experiments had shown that t h i s cons i s t e n t l y f u r n i s h e d phytuberin i n good y i e l d s . The metabolites which were i s o l a t e d are l i s t e d i n Table V I I I . Phytuberin ( I I I ) was obtained i n g r a t i f y i n g amount and gave the spectrum shown i n F i g . 4. The spectrum i s e n t i r e l y c o n s i s t e n t with the p o s t u l a t e d rearrangement of the carbon skeleton o f a

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

5.

STOESSL E T AL.

Sesquiterpenoidal

Stress Compounds

CD2CI 147

from Sofonaceae

2

109

Figure 3.

C NMR spectrum of lubimin biosynthesized from

13

l 2- C-acetate y

13

H I I I o r IV

Scheme 4.

Postulated biosynthesis of phytuberin

73

74

HOST P L A N T RESISTANCE T O PESTS

l 3

Table VIII C - L a b e l l e d Compounds from the Potato - Glomerella cingulata interaction.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

Compound r i s h i t i n (I) lubimin (V) e p i l u b i m i n (VIII) dihydrolubimin (VII) s o l a v e t i v o n e (XI) phytuberin ( I I I ) phytuberol (IV)

y i e l d (mg)* 53 27 79 19 39 11

* a f t e r f i n a l p u r i f i c a t i o n ; from 64 l b s potatoes

J Figure 4.

C NMR spectrum of enriched phytuberin

13

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

5. STOESSL ET AL.

Sesquiterpenoidal Stress Compounds from S

eudesmane precursor, and in particular, with the scission of a C-l - C-2 bond and reattachment of C-2 to C-5 via oxygen. En­ tirely analogous results were obtained from phytuberol (desacetylphytuberin; IV) which was isolated in the same experiment. The spectra do not provide evidence other than that pertaining to the making and breaking of skeletal bonds and many of the details of this intriguing biosynthesis remain to be studied. Experiments to this end are in hand. Finally, a few words about the other metabolites isolated from the cingulata interaction. The spectrum obtained from rishitin (I) did not differ in any essential respect from that discussed earlier. Lubimin (V) was isolated only in admixture with small amounts of epilubimin (VIII) and of a third, as yet unidentified component, but the spectrum of the mixture incorpo­ rated all the features observed earlier in the enriched pure samples of V and VIII. The spectrum of solavetivone (XE) , a com­ pound which was not detected in the M^ fructicola interaction, showed, as expected, the same coupling pattern as that shown earlier for lubimin and this pattern could also be seen in the spectrum of dihydrοlubimin (VII). The high yield in which VII was isolated from the potato Gj_ cingulata interaction contrasts strongly with its trace occurence in the fructicola experiment and is, without doubt, to a large extent a consequence of its formation from V by the fungus. The reduction of V to VII is carried out readily by G>_ cingulata and several other fungi in pure culture (38). However, this conversion does not appear to take place in M. fructicola cultures. For this reason, the occurrence of traces of VII in the potato fructicola inter­ action suggests that VII is formed by potato enzymes as discussed earlier. This problem, too, should be amenable to solution by further tracer studies. Footnotes An example of the conformational mobility of germacrenes was seen by Yamamura and coworkers in the cyclization of acoragermacrone by chemical means to both cis- and trans-decalins (25;. However, in laboratory cyclizations, 1(10):4-germacrenes usually exhibit strong conformational preferences which lead to transdecalins (26) and, as was pointed out (26) , Yamamuras results are not unambiguous. Nevertheless, when enzyme-bound in bio­ synthetic reactions, the compounds may well adopt conformations that are not favoured in the laboratory. Certainly cis-decalins, inclduing cis-eudesmanes, occur in nature. The problem is briefly but lucidly discussed in a recent monograph (27). Commercially available l,2- C2~acetate always contains an appreciable amount of singly labelled (both 1- and 2-) acetate, which will be incorporated at the same rate and will also con­ tribute to the singlet absorption. Literature Cited ( 1) Tomiyama, K., Sakuma, T., Ishizaka, Ν., Sato, Ν., Katsui, N., Takasugi, Μ., and Masamune, T. Phytopathology (1968) (58), 1

13

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

76

HOST PLANT RESISTANCE TO PESTS

115. ( 2) Sato, Ν., Tomiyama, Κ., Katsui, Ν., and Masamune, T. Ann. Phytopathol. Soc. Japan (1968) 34, 344. ( 3) Katsui, Ν., Matsunaga, Α., Imaizumi, Κ., Masamune, T., and Tomiyama, K. Tetrahedron Letters (1971) 83. ( 4) Varns, J. L., Kuć, J., and Williams, Ε. B. Phytopathology (1971) 61, 174. ( 5) Coxon, D. T., Price, K. R., Howard, Β., and Curtis, R. F. J. Chem. Soc. Perkin I (1977) 53. ( 6) Currier, W. W. Diss. Abstr. Intern. B. (1975) 36, 685. ( 7) Price, K. R., Howard, Β., and Coxon, D. T. Physiol. Plant Path. (1976)9,189. ( 8) Ozeretskovskaya, O. L., Vasyukova, Ν. I., and Metlitskii, L. V. Dokl. Bot. Sci. (1969) 187-189, 158. ( 9) Stoessl, Α., Stothers, J. B., and Ward, E. W. B. Can. J. Chem. (1975) 53, 3351. (10) Stoessl, Α., Stothers, J. B., and Ward, E. W. B. J. Chem. Soc. Chem. Commun. (1975) 431. (11) Stoessl, Α., Ward, E. W. B., and Stothers, J. B. in pre­ paration. (12) Kalan, Ε. B. and Osman, S. F. Phytochemistry (1976) 15, 775. (13) Coxon, D. T., Price, K. R., Howard, B., Osman, S. F., Kalan, Ε. Β., and Zacharius, R. M. Tetrahedron Letters (1974) 2921. (14) Stoessl, Α., Unwin, C. Η., and Ward, E. W. B. Phytopathol. Z. (1972) 74, 141. (15) Birnbaum, G. I., Stoessl, Α., Grover, S. Η., and Stothers, J. B. Can. J. Chem. (1974) 52, 993. (16) Ward, E. W. Β., Stoessl, Α., and Stothers, J. B. Phyto­ chemistry, in press, 1977. (17) Bailey, J. Α., Burden, R. S., and Vincent, G. G. Phyto­ chemistry (1975) 14, 597. (18) Burden, R. S., Bailey, J. Α., and Vincent, G. G. Phyto­ chemistry (1975) 14, 221. (19) Leppik, R. Α., Hollomon, D. W., and Bottomley, W. Phyto­ chemistry (1972) 11, 2055. (20) Roberts, D. L. Phytochemistry (1972) 11, 2077. (21) Fujimori, T., Kasuga, R., Kaneko, Η., and Noguchi, M. Phytochemistry (1977) 16, 392. (22) Anderson, R. C., Gunn, D. Μ., Murray-Rust, J., Murray-Rust, P., and Roberts, J. S. J. Chem. Soc. Chem. Commun. (1977) 27. (23) Stoessl, Α., Stothers, J. B., and Ward, E. W. B. Phyto­ chemistry (1976) 15, 855. (24) Parker, W., Roberts, J. S., and Ramage, R. Quart. Rev. (1967) 21, 331. (25) Iguchi, Μ., Niwa, Μ., and Yamamura, S. Tetrahedron Lett. (1973) 1687, 4367, and references there cited. (26) Sutherland, J. K. Tetrahedron (1974) 30, 1651. (27) Coates, R. M. Fortschr. Chem. Org. Naturstoffe (1976) 33,

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch005

5. STOESSL ET AL.

Sesquiterpenoidal Stress Compounds from

73. (28) Baker, F. C., Brooks, C. J. W., and Hutchinson, S. A. J. Chem. Soc. Chem. Commun. (1975) 293. (29) Dunham, D. J. and Lawton, R. G. J. Amer. Chem. Soc. (1971) 93, 2075. (30) Birnbaum, G. I., Huber, C. P., Post, M. L., Stothers, J. B., Robinson, J. R., Stoessl, Α., and Ward, E. W. B. J. Chem. Soc. Chem. Commun. (1976) 330. (31) Stothers, J. B. "Carbon-13 NMR Spectroscopy" Academic Press, New York, 1972. (32) Grutzner, J. B. Lloydia (1972) 35, 375. (33) Séquin, V. and Scott, Α. I. Science (1974) 186, 101. (34) McInnes, A. G. and Wright, J. L. C. Accts. Chem. Res. (1975) 8, 313. (35) Cornforth, J. W. Angew. Chem. Internat. Edn. (1968)7,903. (36) Cordell, J. A. Chem. Rev. (1976) 76, 425. (37) Stoessl, Α., Ward, E. W. B., and Stothers, J. B. Tetra­ hedron Lett. (1976) 3271. (38) Ward, E. W. B. and Stoessl, A. Phytopathology (1977) 67, 468.

6 Activated Coordinated Chemical Defense against Disease in Plants

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

JOSEPH KUĆand FRANK L. CARUSO Department of Plant Pathology, University of Kentucky, Lexington, KY 40506 It has been repeatedly demonstrated that plants are protected against disease by using procedures that are essentially identical to those used to immunize animals (1-9). Plants inoculated or treated with cultivar nonpathogenic races of pathogens, avirulent forms of pathogens, non-pathogens, heat-attenuated pathogens and high molecular weight products of infectious agents are protected against disease caused by subsequent infection by pathogens (1, 2, 4-6, 10-23). Recovery from disease or limited disease caused by a pathogen also systemically protect plants against disease caused by subsequent infection by the same pathogen or occasionally unrelated pathogens (4, 5, 17, 24-26). It is also evident that infectious agents elicit the accumulation of antibiotic chemicals (phytoalexins or stress metabolites) around sites of infection, and in resistant interactions these substances often rapidly attain concentrations that inhibit development of many infectious agents (1, 18, 27-32). Clearly, the elicitation of disease resistance in a animals and plants is not baseduponthe introduction of new genetic information for resistance into all cells of the host. The basic metabolic mechanisms are present and the success and versatility of the immune response in animals is based upon its rapid expression. It is not clear, however, that the immune response in plants depends upon the activation of a mechanism in the host which is unique for its role in defense against disease, and, which in turn, is elicited by structural components or metabolites unique to the infectious agents. Though the literature reporting induced resistance in plants dates back at least 100 years (33, 34), the reports of the successful control of plant disease in the field using the technique are very recent. This is puzzling since iimunization rapidly became and still frcms the basis of preventative medicine against infectious disease in animals. The review by Chester (34), though critical of experimental procedures in many reports he cited, did include reports that complete or partial immunity to disease occurred in seme plants after recovery frcm seme diseases. Hypovirulent strains of tobacco mo78

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

6.

K U C A N D CARUSO

Chemical

Defense

against

Disease

79

saic virus have been employed i n the Netherlands (35), Japan (36) and the U.S.S.R. (personal communication) t o control the disease caused by virulent strains of the virus on tcmato. A mechanism to explain this protection has not been established. Chestnut tree blight, incited by Endothia parasitica, has v i r t u a l l y made the American Chestnut extinct. Recently, limited biological control of the disease has been attained by inoculating plants with a hypovirulent strain of the fungus (37, 38). The mechanism f o r the protection i s not understood. I t i s possible that hypovirulence i s determined by a transmissable factor, possibly a virus. Thus, virulent strains of the fungus may become hypovirulent. I t i s uncertain, however, whether the suppression of virulence i t s e l f i s solely responsible for the protection or whether the suppressed hypovirulent strains of the pathogen also e l i c i t a defense reaction i n the chestnut soon enough and with s u f f i c i e n t magnitude to control development of the fungus. Plants susceptible t o flgrobacterium tumefaciens, the incitant of crown g a l l , are protected frcm the disease by prior inoculation with the nonpathogen A^ radiobacter (39, 40). This has proven a practical means for protection against the disease. The mechanism for protection i s uncertain. Evidence exists for a chemical antagonism due to a bacteriocin produced by A^ radiobacter as well as compétitive blocking of i n f e c t i b l e binding sites by A^ radiobacter (39, 41). Perhaps more than one mechanism explains the protection. Two plant-pathogen interactions (green bean - Colletotrichum 1 i ndemuthianum and cucurbits - Colletotrichun lagenarium) have received considerable attention i n our laboratory. In both the activation of chemical defense appears part of the plant's r e s i s tance mechanism to disease, and, i n both, protection against d i sease can be e l i c i t e d by methods which resentole immunization i n animals. Protection of green bean (Phaseolus vulgaris L.) against Colletotrichum lindpmuthianum Protection of green bean against bean anthracnose, caused by C. lindemuthianxam, was f i r s t demonstrated by infecting the hypocot y l s of bean cultivars, resistant to seme but not a l l races of the fungus (Figure 1) with cultivar nonpathogenic races of the organism. The inoculated plants were then l o c a l l y (inducer inoculum and challenge inoculum applied to the same site) and systenically (inducer inoculum and challenge inoculum applied to different sites) protected from disease caused by subsequent infection with cultivar pathogenic races of the fungus (12, 42, 43). Symptoms on protected plants were microscopically and macroscopically i n d i s tinguishable from the normal resistance reactions produced i n response to inoculation of a cultivar with a cultivar nonpathogenic race (12). I t i s apparent, therefore, that i f a cultivar has resistance to a race of a pathogen, i t could be made resistant t o a l l races of the pathogen.

80

H O S T P L A N T RESISTANCE T O PESTS

A large number of bean cultivars are, however, susceptible t o a l l known races o f lindentuthianum (44, 45), and the question remained whether these were susceptible because of an absolute lack of resistance mechanisms, or the i n a b i l i t y t o e l i c i t resistance soon enough and with sufficient magnitude to contain an infectious agent. Evidence existed t o support the l a t t e r p o s s i b i l i t y (46).

SUSChPl'JBTXITY

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

Infection with cultivar pathogenic race of pathogen (Challenge) I

RESISTANCE Infection witri cultivar nonpathogenic race of pathogen (Inducer A) or nonpathogen of bean Colletotrichum l a genarium (Inducer B)

BEAN CULTIVAR SUSCEPTIBLE TO SOME BUT NOT ALL RACES OF THE PATHOGEN œLLETOTRICTUM IJNDEMOTîIANUM Infection with inducer A^or Β followed i n 18 hr^by challenge Inducer A or Β and challenge a t same s i t e (local protection) I

RESISTANCE Figure 1.

Inducer A or Β and challenge on separate sites (systemic protection)

RESISTANCE

Elicitation of resistance in a cultivar of bean susceptible to some but not all races of Colletotrichum lindemuthianum

Hypocotyl tissue of even completely susceptible cultivars i s re­ sistant close t o and within the root zone (nonexpanding mature t i s ­ sue) . An effective resistance mechanism i s functional i n this t i s ­ sue and the c l a s s i f i c a t i o n of a c u l t i v a r as susceptible t o C^ l i n ­ demuthianum i s based only on the reaction of hypocotyl tissue d i s ­ tant from the root zone. Further experiments i n our laboratory demonstrated that cultivars of bean susceptible t o a l l races o f C. lindemuthianum (Figure 2) were locally and systemically protected against the disease by infection with C^ lagenarium, a pathogen of cucurbits, prior t o infection with the pathogen of bean (13). C. lagenarium was equally effective i n inducing systemic protection i n cultivars susceptible t o a l l races of C^ linriprnuthianum as i n cultivars resistant to one or more races o f the pathogen (Figure 1). Protection induced by races of C\_ lagenarium was generally

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

6.

K U C A N D CARUSO

Chemical

Defense

against

81

Disease

not e l i c i t e d as rapidly nor as extensively as protection e l i c i t e d by cultivar nonpathogenic races of lindenuthianum. However, l i k e protection e l i c i t e d by lindemuthianun, systemic protection e l i c i t e d by lagenarium became effective e a r l i e s t i n the most mature region of the hypocotyl. The challenge fungus developed i n protected tissue t o the same extent as i n tissue protected by c u l t i v a r nonpathogenic races of lindemuthianun. Protection e l i cited by lagenarium protected against a l l races of lindemuthianum tested as did protection e l i c i t e d by cultivar nonpathogeni c races of lindemuthianum. These observations suggest that the same mechanisms may be involved i n protection e l i c i t e d by both fungi. Additional evidence for the a b i l i t y to activate a r e s i s tance mechanism i n cultivars susceptible to a l l races of lindemuthianum was obtained by heat-attenuating cultivar pathogenic races of the pathogen (Figure 2) i n host tissue prior to the expression of symptoms (47). Such plants were protected from d i sease caused by infection with the same or other cultivar pathogenic races of the fungus. This experiment also suggested that fungal components or metabolites, even i n cultivar pathogenic races, can e l i c i t resistance. I t i s evident, therefore, that resistance mechanisms exist and can be activated i n bean cultivars resistant to seme and susceptible to other races of lindemuthianum as well as i n cultivars susceptible to a l l races of the pathogen. I t i s also evident that even cultivar pathogenic races have the chemical potential to e l i c i t resistance.

SUSCEPTIBILITY Infection wAh any race of the pathogeji (challenge) BEAN CULTIVAR SUSCEPTIBLE TO ALL RACES OF THE PATHOGEN œLIETOTRICHUM LINDEMUTHIANUM Challenge followed by heatattenuation i n host and rechallenge with the same race or other race of pathogen

t

RESISTANCE

Figure 2.

Infection^with nonpathogen of bean Colletotrichum lagenarium (inducer) followed i n 18 hr by ctellenge |

Inducer and challenge a t same s i t e (local protection)

Inducer and challenge on separate sites (systemic protection)

RESISTANCE

:JIANCE RESI!

Elicitation of resistance in a cultivar of bean susceptible to all races of Colletotrichum lindemuthianum

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

82

H O S T P L A N T RESISTANCE T O

PESTS

Systemic protection e l i c i t e d by c u l t i v a r nonpathogenic races of lindemuthianum and by C\_ lagenariim may be mechanistically similar to the resistance of mature tissue (14, 15). The nature and extent of fungal development i n these tissues i s similar and heat treatment p r i o r to infection with the inducer fungus markedly reduced systemic protection and mature tissue resistance. Heat treatment d i d not reduce the effectiveness of race-specific r e s i s tance to 1 i ndemuthianxjm^ resistance to lagenarium or l o c a l protection e l i c i t e d by these fungi. Though phytoalexin accumulation may explain race-specific resistance of bean cultivars to C\_ lindemuthianum as well as induced l o c a l protection by C\_ lindemuthianum or C. lagenarium, i t does not i t s e l f explain systemic protection (16]". Phaseollin and other phytoalexins were not detected i n systemically protected unchallenged tissue, and became apparent i n systemically protected challenged tissue only when restricted collapse and browning of host c e l l s became evident. I t i s possible, therefore, to separate two facets of the induced resistance phenomenon: (1) the chemical agents including phaseollin and other isof lavcnoid phytoalexins which accumulate around the s i t e of infection and which contribute to the inhibition of fungal development; (2) the signal which commits c e l l s removed from the s i t e of an inducing inoculation (systemic protection) to resistance. Further evidence indicates resistance i n bean cannot be attributed solely to the accumulation of phytoalexins (16). qossypii, C. phcmoides and trifolii, ronpathogens of bean, did not cause hypersensiteve browning of host c e l l s and did not e l i c i t the accumulation of phaseollin, phaseollidin, phaseollinisoflavan, kievitone or other phytoalexins detectible by the bioassays employed. Conidia of these fungi germinated and formed appressoria on the host surface, but development i n the host was rapidly restricted. Though C\_ t r i f o l i i did not e l i c i t phytoalexin accunulation or c e l l collapse, i t was extremely effective i n e l i c i t i n g l o c a l but not systemic protection against a l l races of lindemuthianiin (13). Though bean tissue infected with lindemuthianum accumulated high levels of phaseollin, high levels of phaseollinisoflavan and l i t t l e or no phaseollin accumulated i n tissue infected with lagenarium. I t appears that chemical defense against anthracnose i n bean includes the following considerations: (1) production or release of a signal which catmits c e l l s of even susceptible cultivars to resistance; therefore, susceptible plants have the potential for resistance i f i t i s expressed soon enough and with s u f f i c i e n t magnitude. (2) c u l t i v a r nonpathogenic races of a pathogen, as well as some nonpathogens of bean, can produce or e l i c i t production of t h i s signal. (3) systemically protected unchallenged tissue does not contain isof lavcnoid phytoalexins. (4) phytoalexins are not detected i n a l l reactions between nonpathogens and bean.

6.

K U C A N D CARUSO

Chemical

Defense

against

Disease

83

(5) the fungus influences which phytoalexins accumilate. (6) induced protection i s not evident before spore germination, appressorium formation and penetration into the host.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

Protection of cucurbits against Colletotrichum lagenarium - aspects of the phenomenon Recent work i n our laboratory using the interaction of cucurb i t s with the pathogen C\_ lagenarium has supported the work with the interaction of bean and the pathogen C^ lindemuthianum. Infection of a cotyledon or f i r s t true leaf (leaf one) of cwcumber with Cj_ lagenarium systemically protected tissue above (developed or not yet developed) against disease caused by the pathogen (£). Physical damage or chemical injury did not e l i c i t protection. Susceptibility i n this interaction i s characterized by the formation of large but defined lesions. Since many such lesions may form on leaves, stems and f r u i t of cucurbits, the growth of plants and their productivity, as well as the quality of f r u i t , are adversely affected. Nevertheless, the eventual r e s t r i c t i o n of lesion development suggests the presence of a mechanism f o r resistance i n susceptible plants. Whether the mechanism which r e s t r i c t s the development of lesions i s identical to that which i s systemically induced by the pathogen i s unknown, but s i m i l a r i t i e s are evident and w i l l be discussed later. Systemically induced protection against the pathogen by the pathogen i s evident as a delay i n symptom expression and a reduction i n the number and size of lesions (5). Infection of leaf one vtfien the second true leaf was one fourth to one t h i r d expanded, systemically protected plants for 4-5 wk. A second or booster inoculation 3 wk after the f i r s t inoculation extended the time of protection into the f r u i t i n g period. Protection was e l i c i t e d by and effective against six races of the fungus and was evident not only with more than 20 susceptible cultivars but also with two cultivars which express some reisitance t o the pathogen. Resistance i n these two cultivars i s expressed, as i s systemic induced resistance, by a delay i n symptom appearance and a reduction i n the number and size of lesions. A single lesion on leaf one produced significant protection (Figure 3). Protection was evident on the second leaf 72-96 hr after inoculating leaf one. Excising leaf one 72-96 hr. after inoculating leaf one did not reduce protection of leaf two. Leaf two was protected i f excised 96-120 hr. after leaf one was inoculated. I t i s evident that i t i s not necessary for the inducer to be present once protection has been i n i tiated and protection continues to be expressed i n a l l developing leaves for 4-5 wk. I t i s also evident that the protected leaf need not be attached to the plant t o maintain protection. The pattern of protection i n watermelon and muskmelon resembles that i n cucumber (48, 49). Infection of the cotyledons or f i r s t true leaf of four cultivars o f watermelon and four c u l t i v a r s of muskmelon with C^ lagenarium systemically protected the plants

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

84

HOST P L A N T RESISTANCE T O

PESTS

from disease caused by subsequent infection with the pathogen. Protection was noted as a reduction i n the number and size of l e sions, and plants remained protected 4 wk. after the protecting inoculation. Race 1, 2 and 3 of the fungus e l i c i t e d protection and a single lesion on leaf one e l i c i t e d significant protection. In three separate f i e l d t r i a l s , cucuirber plants were protected against a challenge inoculation with lagenarium by a p r i o r inoculation with the pathogen (50). Protection of watermelon was evident i n two t r i a l s and indications were that muskmelon could also be protected. Lesions on protected plants were reduced i n number and size as compared to lesions on unprotected plants (Figure 4). Though chemical or physical injury without the presence of an infectious agent did not e l i c i t protection, protection against C. lagenarium was also e l i c i t e d by the infection of cotyledons with tobacco necrosis virus (TNV) (17) and the infection of leaf one with the bacterial incitant of angular leaf spot, Pseudcmonas lachrymans (51). Infection with lagenarium also protected cucumber against disease caused by P^ lachrymans, and plant breeders have observed that cultivars are either resistant to both or susceptible to both pathogens (personal communication). Infection with lagenarium did not protect plants against TW and TOV did not protect against TOV. The above data support the hypothesis that even susceptible plants have effective mechanisms for disease resistance i f they can be expressed soon enough and with sufficient magnitude. Induced resistance can be systemic and of quite long duration. The phenomenon of systemic induced protection i n plants and immunization i n animals are similar at least i n outward appearance, and the phenomenon suggests a new approach for the practical control of disease i n plants. Mechanisms for induced systemic protection Investigators i n the f i e l d may argue about which i s the i n i t i a l or most important mechanism f o r disease resistance (or susceptibility) i n plants. Such arguments may be f r u i t l e s s i n that too or more d i s t i n c t mechanisms may be operative and the presence of both mechanisms and their coordination may determine the specif i c i t y of the interaction. Interactions i n which phytoalexins accumulate but the plant i s judged susceptible may be due to a lack of or inadequacy i n a second mechanism. The second mechanism may be the presence or induced production of agglutinins. An extremely stimulating and thought-provoking paper by U r i tani and colleagues (52) presents data which support this contention. They report that resistance of sweet potato root to nonpathogenic isolates of fimbriata i s based on at least two mechanisms. In the i n i t i a l stage of infection, agglutinating factors i n host c e l l s may agglutinate spores and germinating hyphae thereby localizing the fungus i n the host. In the second stage, both

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

6.

K U C A N D CARUSO

Chemical

Defense

against Disease

Figure 3. The effect of the number of lesions on leaf one on the protection of leaf two of cucumber against Colletotrichum lagenarium

Figure 4. Protection of cucumber against Colletotrichum lagenarium by Colletotrichum lagenarium in the field. The area of lesions per leaf eight days after the inoculation of leaf two is given in parentheses.

85

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

86

HOST P L A N T

RESISTANCE T O PESTS

pathogenic and nonpathogenic strains of the fungus e l i c i t the accumulation of furanoterpenoids i n host tissue. The duplication i s that the speed of development o f the pathogenic strain exceeds the acxwmulation of fungitoxic levels of furanoterpenoids due to a lack of agglutination, whereas spread of the nonpathogenic strain i s limited by agglutination which allows furanoterpenoids to accumulate to fungitoxic levels i n and around the s i t e of interaction between host and fungus. This concept i s consistant with our investigations of the systemically induced resistance of craaimber to lagenarium by lagenarium. There i s no evidence that the systemic aœimilation of c l a s s i cal phytoalexins i s alone responsible for the systemic protection of cucurbits. A chloroform-soluble inhibitor of the growth of C. lagenarium has been obtained from tissue surrounding lesions on protected and unprotected plants. The inhibitor may be important i n restricting lesion development and account for the normally res t r i c t e d lesions characteristic of cucurbit anthracnose. Spore germination and appressorium formation are not inhibited on protected plants, but development from the appressoria appears reduced i n protected plants. In a series of tests, penetration from appressoria was 20 to 40% and from less than 1 to 5% i n unprotected and protected plants, respectively. The presence of preformed agglutinins appears closely associated with resistance of cucumber to scab incited by Cladosporium cucumerinum (53). Studies also indicate an agglutinating factor accumulates i n unchallenged plants systemically protected by lagenariun. I t i s possible, therefore, that at least three mechanisms contribute to acquired r e s i s tance of cucurbits to C\_ lagenarium. One mechanism r e s t r i c t s penetration into the host, a second agglutinates developing hyphae i n penetrated tissue to minimize spread, and the t h i r d i s the production of c l a s s i c a l phytoalexins around the s i t e of infection. I t i s entirely possible that s t i l l a fourth mechanism may be operative. The agglutinins may also react with extracellular hydrolyt i c enzymes or toxins produced by infectious agents thereby neut r a l i z i n g their "synptom-causing" a c t i v i t y . The systemic nature of the protection of lagenarium by lagenarium resembles the systemic e l i c i t a t i o n of protease inhibitors i n wounded plants (5457), though injury does not i t s e l f e l i c i t protection. Injury of leaves of plants representing several plant families or the introduction of extracts from injured tissues into plants i s s u f f i c i e n t to e l i c i t the systemic accumulation of protease inhibitors. The character of the protein i n responding tissues markedly changes and as much as 12% of the protein i n affected tissues i s protease inhibitor with a high content of d i s u l f i d e cross linkages. We are investigating the relationship between the production of extrac e l l u l a r proteases by the pathogen and the induction of protease and other enzymatic inhibitors i n systemically protected plants. The systemic e l i c i t o r of protease aœunulation reported by Ryan and his colleagues i s s t i l l uncharacterized but the implications of this "long distance" ccnmunicaticn are profound i n r e l a -

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

6.

K U C A N D CARUSO

Chemical

Defense

against

Disease

87

tien to disease resistance and p r a c t i c a l control of disease. Seve r a l possible ingredients of a defense system become apparent: (1) a rapid "long distance" system of ccitmunicaticn (2) presence and/or e l i c i t a t i o n of protein or glycoprotein enzyme inhibitors and agglutinins (3) presence and/or e l i c i t a t i o n of c l a s s i c a l lew molecular weight phytoalexins. Hew far apart i n basic metabolic mechanisms are the immune systems i n plants and animals? Genes for resistance and susceptibility can control the presence or absecce of any one of the mechanisms and the rates of the mechanisms. In a coordinated defense against disease, any one or two mechanisms for resistance may be active, but i f a third i s inactive, i t may become the limiting factor for resistance and the plant i s susceptible. Plants with mechanisms for resistance, therefore, can i n fact be susceptible. Induced resistance may be based on the marked activation of a mechanism, which i s not limiting to defense, to a level that overcomes the limitation of a deficient factor, e . g . , phytoalexins accumulate rapidly enough and with suff i c i e n t magnitude to eliminate the need of an agglutinin. In other situations, induced resistance may activate a deficient or limiting factor i n the coordinated mechanisms. The key to disease resistance i n plants i s the functioning of multiple mechanisms for resistance and the key concept i n understanding their interaction i s one of "coordinated defense". Of course the infectious agent also influences the effectiveness or coordination of host defense. The active contribution of the infectious agent makes the interaction extremely complex but at the same time increases many fold the p o s s i b i l i t i e s for s p e c i f i c i t y . This i s paper 77-10-124 of the Kentucky Agricultural Experiment Station, Lexington, Kentucky 40506. The authors' work reported i n this manuscript was supported i n part by a grant from the Herman Frasch Foundation and grant 316-15-51 of the Cooperative State Research Service of the United States Department of Agriculture.

Literature Cited 1. Kuć, J. Annu. Rev. Phytopathology (1972) 10, 207-232. 2. Kuć, J. World Rev. of Pest Control (1968) 7, 42-55. 3. Hammerschmidt, R., Acres, S., Kuć, J. Phytopathology (1976) 66, 790-794. 4. Kuć , J . , Shockley, G., Kearney, K. Physiol. Plant Pathology (1975) 7, 195-199. 5. Kuć, J . , Richmond, S. Phytopathology (1977) 67, 533-536. 6. Matta, A. Annu. Rev. Phytopathology (1971) 9, 387-410. 7. Ross, A. Proceed. Internatl. Conf. Plant Viruses (1966) July, 127-150. 8. Yarwood, C. Proceed. Natl. Acad. Sci. U.S.A. (1954) 40, 374374-377. 9. Yarwood, C. Phytopathology (1956) 46, 540-544. 10. Bell, Α., Presley, J. Phytopathology (1969) 59, 1147-1151.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

88

HOST PLANT RESISTANCE TO PESTS

11. Deverall, B. "Defense Mechanisms in Plants" 110 p., Cambridge Univ. Press, London, 1977. 12. Elliston, J., Kuć, J . , Williams, E. Phytopathology (1971) 61, 1110-1112. 13. Elliston, J., Kuć, J . , Williams, E. Phytopathol. Z. (1976) 86, 117-126. 14. Elliston, J . , Kuć, J . , Williams, E. Phytopathol. Z. (1976) 87, 289-303. 15. Eliston, J . , Kuć, J . , Williams, E. Phytopathol. Z. (1977) 88, 43-52. 16. Elliston, J . , Kuć, J . , Williams, Ε., Rahe, J. Phytopathol. Z. (1977) 88, 114-130. 17. Jenns, Α., Kuć, J. Physiol. Plant Pathol. (1977) In press. 18. Kuć, J. "Specificity in Plant Disease" pp. 253-271, Plenum Press, N.Y. 1976. 19. Kuć, J . , Currier, W., Elliston, J . , McIntyre, J. "Biochemistry and Cytology of Plant-Parasite Interaction" pp. 168-180, Elsevier, N.Y. 1976. 20. Lovrekovich, L., Farkas, G. Nature (1965) 205, 823-824. 21. Main, C. Phytopathology (1968) 58, 1058-1059. 22. McIntyre, J . , Kuć, J . , Williams, E. Physiol. Plant Pathol. (1975) 7, 153-170. 23. Heale, J. B., Sharman, S. Physiol. Plant Pathol. (1977) 10, 51-61. 24. Braun, J . , Helton, A. Phytopathology (1971) 61, 685-687. 25. Randall, R., Helton, A. Phytopathology (1976) 66, 206-207. 26. Cruickshank, I., Mandryk, M. J. Australian Inst. Agric. Sci. (1960) 26, 369-372. 27. Kuć, J. "Encyclopedia of Plant Physiology" Vol. 4, pp. 632652, Springer-Verlag, Berlin, 1976. 28. Kuć, J . , Currier, W., Shih, M. "Biochemical Aspects of Plant­ -Parasite Relationships" pp. 225-257, Academic Press, London 1976. 29. Kosuge, T. Annu. Rev. Phytopathology (1969) 7, 195-222. 30. Stoessel, Α., Stothers, J . , Ward, E. Phytochem. (1976) 15, 855-872. 31. Ingham, J. Botan, Rev. (1972) 38, 343-424. 32. Cruickshank, I., Biggs, D., Perrin, D. J. Indian Botan. Soc. (1971) 50A, 1-11. 33. Bernard, N. Ann. Sci. Natl. Bot. (1909) 1, 1-196. 34. Chester, K. S. Quart. Rev. Biol. (1933) 8, 129-154, 275-324. 35. Rast, A. Agricultural Research Report of the Inst. of Phyto­ pathol. Res., Wageningen, The Netherlands (1975) 834, 1-76. 36. Komochi, S., Goto, T., Oshima, N. J. Hort. Assn. Japan (1966) 35, 269-276. 37. Anagnostakis, S., Jaynes, R. Plant Dis. Reptr. (1973) 57, 225-226. 38. Van Alfen, Ν., Jaynes, R., Anagnostakis, S. Science (1975) 189, 890-891. 39. New, P., Kerr, A. J. Applied Bacteriol. (1972) 35, 279-287.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch006

6. KUC AND CARUSO

Chemical Defense against Disease 89

40. Kerr, A. J. Applied Bacteriol. (1972) 35, 493-497. 41. Lippincott, Β., Lippincott, J. J. Bacteriol. (1969) 97, 620-628. 42. Rahe, J., Kuć, J., Chuang, C., Williams, E. Phytopathology (1969) 59, 1641-1645. 43. Skipp, R., Deverall, B. Physiol. Plant Pathol. (1973) 3, 299-313. 44. Bannerot, H. Ann. Amel. Plantes (1965) 15, 201-222. 45. Goth, R., Zaumeyer, W. Plant Dis. Reptr. (1965) 49, 815-818. 46. Leach, J. Minn. Agric. Expt. Sta. Techn. Bull. (1923) 14, 1-41. 47. Rahe, J. Phytopathology (1973) 63, 572-577. 48. Caruso, F., Kuć, J. Phytopathology In Press. 49. Caruso, F., Elliston, J., Kuć, J. Proc. Amer. Phytopathol. Soc. (1976) 3, 259. 50. Caruso, F., Kuć, J. Phytopathology In Press. 51. Caruso, F. Proc. Amer. Phytopathol. Soc. (1977) In Press. 52. Uritani, I., Oba, K., Kojima Μ., Kim, W., Ohuni, I., Suzuki, H. "Biochemistry andCytologyof Plant-Parasite Interaction" pp. 239-252, Elsevier, N.Y. 1976. 53. Hammerschmidt, R., Kuc , J. Proc. Amer. Phytopathol. Soc. (1977) In Press. 54. Peng, J., Black, L. Phytopathology (1976) 66, 958-963. 55. Ryan, C. Annu. Rev. Plant Physiol. (1973) 24, 173-196. 56. Gustafson, G., Ryan, C. J. Biol. Chem. (1976) 251, 7004-7010. 57. Walker-Simmons, M., Ryan, C. Plant Physiol. (1977) 59, 437439.

7 Biochemical and Ultrastructural Aspects of Southern Corn Leaf Blight Disease

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

PETER GREGORY, ELIZABETH D. EARLE, and VERNON E. GRACEN Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853

The study of southern corn leaf blight disease is of major economic and scientific value. The economic importance of this fungal disease was dramatically emphasized in 1970 when it reached nearly epidemic proportions in the U.S.A., resulting in severe losses for corn growers. Scientifically, research on the disease may result in novel and major gains in plant pathology, plant breeding and higher plant genetics. The main aims of this paper are to critically describe the present state of biochemical and ultrastructural knowledge about the mechanism of the disease, to highlight the most important gaps in our knowledge and to discuss some of the ways in which these gaps can be filled. General Characteristics of Southern Corn Leaf Blight Disease The causal organism of southern corn leaf blight disease is the fungus Helminthosporium maydis Race Τ (HmT). This is one of several members of the genus Helminthosporium to produce host­ -specific toxins. HmT and the toxin(s) from HmT (HmT toxin) only affect corn which contains the Texas (T) source of male sterile cytoplasm. Plants containing the non-male sterile (N) or the C or S types of male sterile cytoplasms are resistant to the fungus and insensitive to HmT toxin (1,2). Nuclear genes which restore Τ cytoplasm to male fertility (Rf genes) may slightly modify the disease reaction and toxin response of corn varieties containing the Τ cytoplasm (3,4). Nuclear genes other than Rf genes can affect the HmT disease reaction (5,6,7) with some inbred lines exhibiting a higher level of resistance in Τ cytoplasm than others. In all cases, however, each inbred is more susceptible in Τ than in Ν cytoplasm. Toxin sensitivity of different inbred lines in Τ cytoplasm also varies but there is no apparent correlation between nuclear control of toxin sensitivity and the degree of disease resistance (8). It is of considerable interest that another race of H. maydis, Race 0, produces a toxin but shows no specificity for Τ cytoplasm ( 9. ) . Η. maydis Race Τ might essentially represent a Race 0 90

7.

GREGORY

ET

AL.

Southern

Corn

Leaf Blight

i s o l a t e that has accumulated a d d i t i o n a l s p e c i f i c t o x i n production ( 9 ,10). The s p e c i f i c a l l y damage Τ cytoplasm i n the gives southern corn l e a f b l i g h t disease model system i n p l a n t pathology.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

E f f e c t s of HmT

Disease

91

genes f o r cytoplasmf a c t that HmT t o x i n can absence of the fungus (11) great p o t e n t i a l as a

Toxin on Τ Cytoplasm

Treatment with HmT t o x i n produces morphological, p h y s i o l o g i ­ c a l , u l t r a s t r u c t u r a l and biochemical changes i n Τ cytoplasm m a t e r i a l . Table I l i s t s these e f f e c t s , seen i n systems ranging i n complexity from whole p l a n t s to s u b c e l l u l a r f r a c t i o n s . The e f f e c t s l i s t e d are s p e c i f i c f o r Τ cytoplasm; Ν cytoplasm m a t e r i a l i s e i t h e r u n a l t e r e d by any t o x i n c o n c e n t r a t i o n t e s t e d or i s a f f e c t e d l e s s than Τ cytoplasm at a given c o n c e n t r a t i o n . Non­ s p e c i f i c e f f e c t s (e.g. carbohydrate leakage (12)) are not i n c l u d e d i n Table I. I t can be seen i n Table I that the timing, s e n s i ­ t i v i t y and degree o f s p e c i f i c i t y of the d i f f e r e n t e f f e c t s vary considerably. The r e l a t i v e importance o f the r e p o r t e d e f f e c t s i s d i f f i c u l t to evaluate. Some e f f e c t s , such as i n h i b i t i o n of r o o t growth and changes i n i s o l a t e d mitochondria, have been observed by many workers; others have been documented only by work from a s i n g l e l a b o r a t o r y or even by a s i n g l e p u b l i c a t i o n . Comparison of work i n d i f f e r e n t l a b o r a t o r i e s i s d i f f i c u l t because no standard pro­ cedures f o r p r e p a r a t i o n and assay of HmT t o x i n e x i s t . Many d i f ­ f e r e n t schemes f o r t o x i n production and p u r i f i c a t i o n are i n use (Table I I ) . Toxin a c t i v i t y i n the i n i t i a l t o x i n source i s i n f l u e n c e d by c u l t u r e c o n d i t i o n s and the f u n g a l i s o l a t e used, but the i n i t i a l t o x i n a c t i v i t y i s r a r e l y r e p o r t e d . The e f f e c t i v e n e s s of the p u r i f i c a t i o n procedures, the l e v e l of p u r i t y a t t a i n e d and the amount of n o n s p e c i f i c m a t e r i a l remaining are u s u a l l y d i f f i c u l t to assess. The use of d i f f e r e n t corn l i n e s i n d i f f e r e n t s t u d i e s or even as the source of Ν and Τ cytoplasm i n a g i v e n experiment (17) causes f u r t h e r c o m p l i c a t i o n s . In s p i t e o f problems i n i n t e r p r e t a t i o n and comparison, s t u d i e s of t o x i n e f f e c t s on Τ cytoplasm serve s e v e r a l purposes. 1) They provide convenient c r i t e r i a f o r i d e n t i f y i n g Τ cytoplasm and Τ cytoplasm components i n many d i f f e r e n t systems, a t the f i e l d l e v e l , i n seed or p o l l e n populations or i n c e l l c u l t u r e s . Usually, only Τ cytoplasm m a t e r i a l i s s e n s i t i v e to t o x i n . Other male s t e r i l e and non-male s t e r i l e cytoplasms are r e s i s t a n t . Pro­ cedures that a l t e r the usual response of Ν and Τ cytoplasm to t o x i n may y i e l d m a t e r i a l which w i l l f u r t h e r understanding of the d i f f e r e n c e between the two cytoplasms. Because c a l l u s from Τ cytoplasm seeds u s u a l l y f a i l s to grow on n u t r i e n t medium c o n t a i n ­ ing t o x i n , the t o x i n r e s i s t a n t c a l l u s o c c a s i o n a l l y seen i n Τ cytoplasm c u l t u r e s i s easy to i s o l a t e f o r f u r t h e r a n a l y s i s (25, 29). Biochemical and genetic a n a l y s i s o f such t o x i n - r e s i s t a n t T c a l l u s i s now i n progress i n our l a b o r a t o r y and i n s e v e r a l other laboratories. S i m i l a r l y f a i l u r e of Τ cytoplasm p r o t o p l a s t s to !

T

92

HOST P L A N T RESISTANCE T O PESTS

Table I .

E f f e c t s of H. maydis Race Τ t o x i n on Τ cytoplasm material

Toxin a p p l i c a ­ t i o n ( i f not i n ambient s o l u t i o n

Effect

Timing

References

P L A N T S sprayed

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

injected stem

into

p i p e t t e d onto l e a f who r 1 , imma ture t i s s u e , punctured w i t h needle

l e s i o n formation

3-6 days

(13)

chlorosis, necrosis of leaves

2-4 days

c h l o r o t i c streaks

2-5 days

(10,14,15)

24-48hours

(1,15,16, 17,18)

(13,4(0

S E E D L I N G S i n h i b i t i o n of r o o t growth

30 min

(il)

i n h i b i t i o n of u r a n y l uptake i n t o vacuoles of r o o t c e l l s

21-28 hours

(19)

s t i m u l a t i o n of e l e c ­ t r o l y t e leakage

1-4 hours

(iZ)

u l t r a s t r u c t u r a l damage to mitochondria i n root c e l l s

15-120 min

(20)

90 min f a i l u r e of root mito­ chondria to respond to deoxyglucose treatment d e p o l a r i z a t i o n of mem­ brane p o t e n t i a l d i f ­ ference of root c e l l s D E T A C H E D l e a f f l o a t e d on toxin

2-5 min

L E A V E S - L E A F

water soaking

(21)

(22)

D I S C S 3-4

days

(1)

i n j e c t e d i n t o l e a f / l e s i o n formation

3 days

(23)

a p p l i e d to p u n c t u r e / l e s i o n formation

12 hours 48 hours

(24)

3 days

(18)

cut end i n t o x i n

chlorophyll retention i n dark

(11)

7.

Southern

GREGORY E T A L .

Table I .

Corn

Leaf Blight

93

Disease

(cont.)

Toxin a p p l i c a ­ t i o n ( i f not i n ambient s o l u t i o n )

Timing

Effect

a

3 days

References

leaf piece floated on t o x i n

chlorophyll i n dark

leaf

i n h i b i t i o n o f dark C0 fixation

9-10 hours

i n h i b i t i o n of l i g h t C0 fixation

9 hours

(18)

i n h i b i t i o n of l i g h t CO f i x a t i o n

15-60 min

(26)

stimulation of elec­ t r o l y t e leakage

7-23 hours

(27)

discs

retention

(25) (15,18)

2

leaf

discs

2

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

cut end i n t o x i n

2

cut end i n t o x i n

3 hours

(19)

vacuum i n f i l t r a s t i m u l a t i o n o f e l e c t r o - 2 hours t i o n of l e a f pieces l y t e leakage

(12)

epidermal peels f l o a t e d on t o x i n

inhibition ο f l i g h t stimulated Κ uptake by guard c e l l s

3 hours

(26)

cut end i n t o x i n

i n h i b i t i o n of trans­ piration

3 hours

(19)

5-60 min

(26)

I S 0 L A T E D

R O O T S

s t i m u l a t i o n of e l e c ­ t r o l y t e leakage i n h i b i t i o n of uptake t o x i n present during a e r a t i o n of roots

8 6

Rb

i n h i b i t i o n of develop­ ment o f augmented R b uptake

2 hours (12) (10 min i n 1 expt) 90 min

(Z)

2 hours

(28,47)

28 days

(25,29)

86

C A L L U S i n agar medium

i n h i b i t i o n o f growth

P O L L E N (from Τ cytoplasm p l a n t s w i t h TRf genes) i n agar medium

i n h i b i t i o n of germin­ a t i o n and p o l l e n tube growth

2 hours

(30)

HOST

94

Table I .

PLANT

RESISTANCE T O PESTS

(cont.) Timing

References

i n h i b i t i o n of volume increase

1 day

(31)

collapse

1-3 days

(31.32)

u l t r a s t r u c t u r a l damage to mitochondria

5-60 min

(20.33)

Effect

Toxin a p p l i c a ­ t i o n ( i f not i n ambient s o l u t i o n )

P R O T O P L A S T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

I S O L A T E D

M I T O C H O N D R I A

uncoupling o f o x i d a ­ t i v e phosphorylation s t i m u l a t i o n o f NADH oxidation

(34-39) (15,23,34 35,37,38 40,41)

i n h i b i t i o n of malatepyruvate o x i d a t i o n

(15,34,35 37,38,40 41)

i n h i b i t i o n o f a-ketoglutarate oxidation

(37,41)

p a r t i a l i n h i b i t i o n of succinate oxidation ( i n sucrose o r KC1 assay media)

(15_,34,35 38)

stimulation of succinate o x i d a t i o n ( i n mannitol or sucrose assay media)

(41,42)

s t i m u l a t i o n of ATPase activity

(38,41)

a c t i v a t i o n o f cytochrome oxidase

(41)

a c t i v a t i o n of s u c c i n a t e cytochrome C reductase 32 Ρ acinhibition of cumulation

(41)

increase i n l i g h t trans­ mit tance ( s w e l l i n g )

(17,38) (17,23,34 43)

7.

GREGORY E T A L .

Table I .

Southern

Corn Leaf

Blight

95

Disease

(cont.)

Toxin a p p l i c a t i o n ( i f not i n ambient s o l u t i o n )

Effect

Timing

3

References

(33,43)

u l t r a s t r u c t u r a l changes (swelling, disruption of inner membranes) M I C R O S O M E S +

i n h i b i t i o n of K stimulated ATPase

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

a

30-60 min

(44,45)

are noted approximate time from exposure to t o x i n u n t i l e f f e c t s and/or measured e f f e c t s seen w i t h i n seconds o r a few minutes

HOST P L A N T RESISTANCE T O PESTS

96 Table I I .

Toxin Preparations Used i n P h y s i o l o g i c a l , Biochemical and U l t r a s t r u c t u r a l Studies Processing

i n f e c t e d leaves

b o i l i n g water e x t r a c t i o n

(30)

methanol e x t r a c t i o n , d i a l y s i s

(11)

methanol e x t r a c t i o n

(14)

methanol, e t h y l acetate ex­ traction

(12)

methanol, e t h y l acetate ex­ t r a c t i o n , chromatography (chloroform-methanol), e t h y l acetate e l u t i o n

(37,39,40, 41,44,45)

H. maydis race Τ mycelium + agar c u l ­ ture medium

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

References

I n i t i a l Toxin Source

(abridged)

H. maydis race Τ mycelium + c u l t u r e filtrate

chloroform e x t r a c t i o n o f ground mycelium and c u l ­ ture f i l t r a t e

H. maydis race Τ c u l ­ ture f i l t r a t e

none

(18)

(.3,11,13,16, 46)

dialysis desalting,

(1,34) ultrafiltration

(17,23,26, 43,70)

desalting, u l t r a f i l t r a t i o n , p a r t i a l Sephadex p u r i f i c a t i o n

(17)

f r e e z e d r y i n g , methanol ex­ traction

(19)

e t h y l acetate e x t r a c t i o n

(29)

methanol-water, chloroform extraction methanol-water, butanol extraction chloroform

extraction

(8,15,38)

(28,47)

(7,20,31, 32,33)

7.

GREGORY E T A L .

Southern

Corn

Leaf Blight

97

Disease

1

s u r v i v e t o x i n treatment may permit d e t e c t i o n of 'T p r o t o p l a s t s whose response to t o x i n has been a l t e r e d by genetic manipulations l i k e p r o t o p l a s t f u s i o n or o r g a n e l l e t r a n s f e r . 2) They provide a v a r i e t y o f assays f o r HmT t o x i n a c t i v i t y . S e n s i t i v e bioassays are e s s e n t i a l both f o r rigorous s t u d i e s o f t o x i n a c t i o n and f o r t o x i n p u r i f i c a t i o n procedures. E v a l u a t i o n of a s i n g l e t o x i n p r e ­ p a r a t i o n i n assays i n v o l v i n g succinate, NADH or m a l a t e - d i c h l o r o phenol indophenol (DCPIP) r e s p i r a t i o n by i s o l a t e d mitochondria and dark C 0 f i x a t i o n by l e a f d i s c s showed a q u a n t i t a t i v e i n h i b i t i o n by the t o x i n (15). Moreover, these assays are 5-10X as s e n s i t i v e to t o x i n as the s e e d l i n g root growth assay and 20-100X as s e n s i t i v e as l e a f l e s i o n assays (15). I n h i b i t i o n of p r o t o p l a s t s u r v i v a l i s a l s o 5-10X as s e n s i t i v e to t o x i n as s e e d l i n g root growth (32). E l e c t r o l y t e leakage i s one o f the l e a s t s e n s i t i v e bioassays (18, 19). 3) They provide information about the mode of t o x i n a c t i o n i n Τ cytoplasm c e l l s . The slower e f f e c t s l i s t e d i n Table I are probably secondary e f f e c t s , f a r removed from the i n i t i a l i n t e r ­ a c t i o n of t o x i n with Τ cytoplasm. Primary e f f e c t s should be r a p i d , h i g h l y s p e c i f i c f o r Τ cytoplasm and a t l e a s t as s e n s i t i v e to t o x i n as l a t e r e f f e c t s . I t should a l s o be p o s s i b l e to r e l a t e the primary e f f e c t s to the observed cytoplasmic i n h e r i t a n c e o f t o x i n s e n s i t i v i t y . Both mitochondria and plasma membranes have been i m p l i c a t e d as primary s i t e s o f e a r l y t o x i n a c t i o n .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

2

Evidence f o r the Plasma Membrane as a S i t e of Toxin A c t i o n Some p h y s i o l o g i c a l s t u d i e s suggest that plasma membranes and mi­ tochondrial membranes are a f f e c t e d by HmT t o x i n . Evidence f o r plasma membrane e f f e c t s i s based on observations t h a t a f t e r t o x i n treatment e l e c t r o l y t e leakage occurs (12,17,19,27), Κ or R b uptake i s i n h i b i t e d (7,28,47), l i g h t - s t i m u l a t e d K+ movement i n t o guard c e l l s i s i n h i b i t e d (26), p a r t i a l d e p o l a r i z a t i o n o f the plasma membrane p o t e n t i a l d i f f e r e n c e occurs (22), and K*" stimu­ l a t e d ATPase from corn root microsomes i s i n h i b i t e d (44,45). This l a t t e r p o i n t i s s i g n i f i c a n t because microsomal p r e p a r a t i o n s from oat roots contain fragments o f plasma membranes ( 4 8 ) . Because some o f these e f f e c t s occur w i t h i n minutes a f t e r t o x i n treatment (12,22) and because of the t o x i n e f f e c t on micro­ somal K stimulated ATPase, i t has been suggested that HmT t o x i n acts d i r e c t l y on plasma membranes. However the importance o f some of the observations i m p l i c a t i n g plasma membranes i s ques­ t i o n a b l e because of the weak s p e c i f i c i t y f o r Τ cytoplasm (28,47), the p r e l i m i n a r y nature o f the r e p o r t (22), the i n a b i l i t y o f other workers to repeat the microsomal ATPase r e s u l t s (49), and the high l e v e l s o f crude t o x i n o f t e n needed. There i s the f u r t h e r problem o f i n t e r p r e t i n g cytoplasmic i n h e r i t a n c e o f d i f f e r e n t i a l membrane s e n s i t i v i t y . When a h i g h l y a c t i v e , Τ c y t o p l a s m - s p e c i f i c chloroform e x t r a c t a b l e t o x i n i s used, no u l t r a s t r u c t u r a l damage to plasma membranes of Τ p r o t o p l a s t s i s seen a t times when m i t o c h o n d r i a l d i s r u p t i o n i s +

86

+

98

HOST P L A N T

RESISTANCE T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

apparent (20,33). Although crude HmT c u l t u r e f i l t r a t e caused plasma membrane damage i n Ν and Τ corn (50), the l a t t e r u l t r a s t r u c t u r a l e f f e c t s could be due to the presence o f contaminants i n HmT crude f i l t r a t e . I t seems p o s s i b l e that some o f the observed plasma membrane e f f e c t s may r e s u l t from p r i o r m i t o c h o n d r i a l i n a c t i v a t i o n . We cannot e l i m i n a t e the p o s s i b i l i t y that r a p i d and s p e c i f i c e f f e c t s on plasma membranes occur but are below the l e v e l o f u l t r a s t r u c t u r a l r e s o l u t i o n , although we a l s o have p r e ­ l i m i n a r y f r e e z e - e t c h data that f a i l s to d e t e c t plasma membrane damage with chloroform-extracted t o x i n (50). At present, i t appears that s p e c i f i c i t y of HmT t o x i n f o r Τ cytoplasm r e s i d e s i n i t s i n t e r a c t i o n with mitochondria. I d e n t i f i c a t i o n of the d i f f e r e n c e s between Ν and Τ mitochondria which l i m i t the e f f e c t of t o x i n to Τ mitochondria and the mechanism o f a c t i o n o f HmT t o x i n on Τ mitochondria i s o f c o n s i d e r a b l e i n t e r e s t . Evidence f o r Mitochondria as a S i t e o f Toxin A c t i o n The cytoplasmic i n h e r i t a n c e o f t o x i n s e n s i t i v i t y suggests that an a l t e r e d m i t o c h o n d r i a l o r c h l o r o p l a s t genome i s i n v o l v e d . A c h l o r o p l a s t s i t e seems u n l i k e l y both because no e f f e c t of t o x i n on enzyme a c t i v i t i e s i n i s o l a t e d c h l o r o p l a s t l a m e l l a e has been observed (26) and because non-green t i s s u e s l i k e roots and c a l l u s are very s e n s i t i v e to t o x i n . Several l i n e s o f evidence p o i n t to the mitochondria as an important and p o s s i b l y primary s i t e of HmT t o x i n a c t i o n . E f f e c t s of HmT Toxin on I s o l a t e d Mitochondria. Evidence f o r an e f f e c t o f HmT t o x i n on m i t o c h o n d r i a l physiology comes from s e v e r a l r e p o r t s o f toxin-induced a l t e r a t i o n i n r e s p i r a t i o n r a t e s with s e v e r a l s u b s t r a t e s and uncoupling o f o x i d a t i v e phosphoryla­ t i o n i n mitochondria i s o l a t e d from corn c o n t a i n i n g Τ cytoplasm (Table I, Table I I I ) . Evidence f o r HmT toxin-induced s t r u c t u r a l changes comes from l i g h t transmittance and u l t r a s t r u c t u r a l s t u d i e s which have shown that i s o l a t e d Τ mitochondria s w e l l a f t e r t o x i n treatment (Table I ) . Toxin treatment o f i s o l a t e d mitochondria a l s o r e s u l t s i n damage to c r i s t a e and l o s s of matrix d e n s i t y (Table I ) . Outer m i t o c h o n d r i a l membranes appear to be u l t r a s t r u c t u r a l l y u n a f f e c t e d by HmT t o x i n . These m u l t i p l e e f f e c t s of HmT t o x i n are very r a p i d (Table I ) . Mitochondria i s o l a t e d from Τ corn w i t h nuclear genes f o r f e r t i l i t y r e s t o r a t i o n (TRf) a r e a l s o s e n s i t i v e to t o x i n (23,37), but a longer time i s r e q u i r e d f o r complete i n h i b i t i o n o f malate o x i d a t i o n than when Τ mitochondria are used. The HmT t o x i n e f f e c t s are s p e c i f i c f o r Τ (and TRf) mitochondria. Even very high concentrations of t o x i n have no e f f e c t s on mitochondria i s o l a t e d from p l a n t s with Ν cytoplasm (Table I , Table I I I ) . However, the removal o f the outer membrane of Ν mitochondria r e s u l t s i n sen­ s i t i v i t y o f the inner membranes to t o x i n as shown by i n h i b i t i o n of malate-DCPIP r e s p i r a t i o n (70).

7.

GREGORY E T A L .

Table I I I .

Southern

Corn

Leaf Blight

99

Disease

E f f e c t of HmT Toxin on R e s p i r a t o r y A c t i v i t i e s of Root Mitochondria from T-cytoplasm and N-cytoplasm Corn Rate of r e s p i r a t i o n (nmol 02/min/mg p r o t e i n )

Substrate

W64A(T) NADH

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

Malate + pyruvate

State 3

243

273

State 4

79

88

+ Toxin

407*

88

State 3

80

98

State 4

20

21

+ Toxin Succinate

W64A(N)

21

0*

State 3

175

193

State 4

86

69

+ Toxin

150*

69

* A d d i t i o n of ADP o r the uncoupler 2,4-dinitrophenol (40 μΜ) d i d not s t i m u l a t e the r a t e o f oxygen uptake by Τ mitochondria a f t e r treatment with HmT t o x i n . Ν mitochondria remained well-coupled i n the presence of HmT t o x i n .

Oxygen uptake was measured with a Clark-type oxygen e l e c ­ trode (Yellow Springs Instrument Company) i n 3 ml of a medium c o n t a i n i n g 0.4 M sucrose, 10 mM KC1, 2.5 mM M g C l , 4 mM Κ Η Ρ 0 , 20 mM HEPES, pH 7.4, χ mg/ml bovine serum albumin and 0.4 mg of mitochondria i s o l a t e d from roots of 3-day-old W64A(T) or W64A(N) s e e d l i n g s . Other a d d i t i o n s , as i n d i c a t e d , were 1.5 ymol NADH, 30 ymol malate plus 30 ymol pyruvate, 30 ymol s u c c i n a t e , and 33 yg HmT t o x i n (from a p r e p a r a t i o n which caused 50% i n h i b i t i o n of r o o t growth o f W64A(T) seedlings a t a c o n c e n t r a t i o n o f 0.65 yg/ ml). State 3 rates were measured a f t e r a d d i t i o n of 150 to 300 nmol of ADP, and State 4 r a t e s a f t e r subsequent exhaustion of the added ADP. HmT t o x i n was added during State 4 r e s p i r a t i o n . 2

2

4

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

100

H O S T P L A N T RESISTANCE T O

PESTS

E f f e c t s of HmT Toxin on Mitochondria i n s i t u . Although the evidence f o r r a p i d , s e n s i t i v e and s p e c i f i c e f f e c t s of HmT t o x i n on i s o l a t e d Τ mitochondria i s c l e a r , the argument f o r mitochondria as the primary s i t e of t o x i n a c t i o n i n i n t a c t c e l l s has been weakened by a f r e q u e n t l y c i t e d r e p o r t that r e s p i r a t i o n and ATP content of s e e d l i n g t i s s u e are not s p e c i f i c a l l y a f f e c t e d by t o x i n i n time periods during which some i n h i b i t i o n of root growth can be detec­ ted (17). Unfortunately the method f o r measuring r e s p i r a t i o n was not given and the t i s s u e s assayed were s e e d l i n g shoots, f o r which no r a p i d growth i n h i b i t i o n by t o x i n was described. Toxin penetra­ t i o n i n t o the i n t a c t 2.5 cm detached shoots used may have been a l i m i t i n g f a c t o r i n the experiment. Recent u l t r a s t r u c t u r a l s t u d i e s (20,33) by our group should r e s o l v e the apparent c o n t r a d i c t i o n between t o x i n e f f e c t s on i s o ­ l a t e d mitochondria and mitochondria i n s i t u . A f t e r t o x i n t r e a t ­ ment of roots and mesophyll p r o t o p l a s t s of Τ corn, the f i r s t u l t r a s t r u c t u r a l e f f e c t s observed were changes i n the mitochondria s i m i l a r to those seen i n t o x i n - t r e a t e d i s o l a t e d mitochondria. F i f t e e n minutes exposure of roots to t o x i n r e s u l t e d i n some damage to the mitochondria of c e l l s i n the root cap and elongation zone. By two hours, many root mitochondria showed s w e l l i n g , r e d u c t i o n i n c r i s t a e and l o s s of matrix d e n s i t y (Figure 1 ) . HmT t o x i n had no e f f e c t on mitochondria i n Ν roots (Figure 1). Mitochondria w i t h i n mesophyll p r o t o p l a s t s from Τ corn were a f f e c t e d even more r a p i d l y and at lower t o x i n concentrations probably because pene­ t r a t i o n problems were minimized (33). Some p r o t o p l a s t mitochon­ d r i a were damaged w i t h i n 5 minutes and by 30 minutes, almost a l l of the p r o t o p l a s t s were a f f e c t e d . P r o t o p l a s t s t r e a t e d with t o x i n f o r 30 minutes and then washed thoroughly and c u l t u r e d a l l c o l l a p s e d w i t h i n a few days, l i k e p r o t o p l a s t s continuously exposed to t o x i n . The r a p i d t o x i n damage to the p r o t o p l a s t s was apparent­ l y i r r e v e r s i b l e (32). Toxin p a r t i a l l y p u r i f i e d by chloroform e x t r a c t i o n had no e f f e c t on mitochondria i n Ν p r o t o p l a s t s , even when high concentrations were used (33). Plasma membranes and other components of Ν and Τ p r o t o p l a s t s looked normal a f t e r 60 minutes exposure to chloroform e x t r a c t a b l e t o x i n . Physiological and biochemical studies of p r o t o p l a s t s c o n t a i n i n g mitochondria damaged by b r i e f t o x i n treatments have not yet been done; however, i t seems l i k e l y that r a p i d t o x i n e f f e c t s on mitochondrial func­ t i o n s i n v i v o w i l l be detected, perhaps even before u l t r a s t r u c ­ t u r a l damage i s apparent. R e v e r s i b i l i t y o f Toxin A c t i o n There i s some evidence which suggests that HmT t o x i n a c t i o n i s r e v e r s i b l e . Arntzen e t a l . (17) i n a study on Τ corn seedlings showed that roots t r e a t e d with t o x i n f o r one hour and then r e t u r n ­ ed to a t o x i n - f r e e medium grew more slowly than the c o n t r o l f o r approximately 6 hours, then s t a r t e d to grow f a s t e r , and by 10 hours were growing as v i g o r o u s l y as the c o n t r o l s . The degree to

GREGORY

E T A L .

Southern

Corn

Leaf

Blight

Disease

101

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

7.

Figure 1. Ultrastructural effects of chhrοform-extractable HmT toxin on mitochondria in the region of elongation of susceptible (W64A(T)) and resistant (W64A(N)) corn roots. Experimental conditions are exactly as described in Ref. 20. Treatments were (A) W64A(T), treated for 2 hr with chloroform-extractable toxin; (B) W64A(T), no toxin added; (C) W64A(N), treated ior 2 hr with chloroform-extractable toxin; (D) W64A(N), no toxin added. The calibration bar represents 0.1 μ in each case.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

102

H O S T P L A N T RESISTANCE T O PESTS

w h i c h t h e r o o t s grew a f t e r t h e t o x i n t r e a t m e n t was d e p e n d e n t o n t o x i n c o n c e n t r a t i o n ( 1 7 ) . H a l l o i n e£ a l (12) t r e a t e d l e a v e s o f Τ c o r n w i t h t o x i n f o r 3 h o u r s a n d t h e n washed t h e l e a v e s f o r 1 h o u r i n t o x i n - f r e e medium. The t o x i n - t r e a t e d l e a v e s showed much h i g h e r r a t e s o f e l e c t r o l y t e l e a k a g e t h a n t h e u n t r e a t e d controls. I n t e r p r e t a t i o n of t h i s l a t t e r data i s d i f f i c u l t be­ c a u s e i t was n o t s t a t e d w h e t h e r t h e i n c r e a s e d r a t e was d i f f e r e n t f r o m t h e r a t e s o b t a i n e d f o r l e a v e s c o n t i n u o u s l y grown i n t o x i n . R e c e n t e x p e r i m e n t s i n o u r l a b o r a t o r y (32) showed t h a t w a s h i n g o f Τ c o r n p r o t o p l a s t s , f o l l o w i n g 30 m i n u t e t r e a t m e n t s w i t h chloroform-extractable t o x i n , d i d not prevent p r o t o p l a s t c o l ­ lapse. I n t h e same s e t o f e x p e r i m e n t s T - c o r n p r o t o p l a s t c o l ­ l a p s e was p r e v e n t e d b y w a s h i n g t h e p r o t o p l a s t s a f t e r 15 m i n u t e t o x i n t r e a t m e n t s . Two l i n e s o f e v i d e n c e s u g g e s t t h a t t h e e f f e c t o f HmT t o x i n o n o x i d a t i v e p h o s p h o r y l a t i o n i n i s o l a t e d Τ m i t o ­ c h o n d r i a i s r e v e r s i b l e . F i r s t l y , w i t h NADH a s t h e s u b s t r a t e , t h e d e g r e e o f t o x i n - i n d u c e d i n h i b i t i o n o f m i t o c h o n d r i a l ATP formation increases with increasing toxin concentration; the h y p e r b o l i c nature o f the curves suggests r e v e r s i b i l i t y o f t o x i n binding (38). Secondly, t o x i n - t r e a t e d Τ mitochondria recovered most o f t h e i r p h o s p h o r y l a t i n g c a p a c i t y when t o x i n was removed by w a s h i n g t h e m i t o c h o n d r i a w i t h t o x i n - f r e e medium ( 3 8 ) . The d a t a o n r o o t s , p r o t o p l a s t s , a n d i s o l a t e d m i t o c h o n d r i a s u g g e s t t h a t HmT t o x i n does n o t b i n d f i r m l y t o t o x i n - s e n s i t i v e s i t e s i n Τ c e l l s , but r a t h e r t h a t an e q u i l i b r i u m e x i s t s between bound a n d unbound t o x i n . Speculations Mitochondria

o n t h e M e c h a n i s m o f T o x i n - I n d u c e d Damage t o Τ

The mechanism b y w h i c h HmT t o x i n damages Τ m i t o c h o n d r i a i s unknown. A l t h o u g h HmT t o x i n i n d u c e s m u l t i p l e e f f e c t s i n Τ m i t o c h o n d r i a (Table I , Table I I I ) i t i s t h e o r e t i c a l l y p o s s i b l e that a l l of the observed e f f e c t s r e s u l t from a s i n g l e , p r i m a r y e f f e c t caused by a s i n g l e type of t o x i n molecule. A l i k e l y primary e f f e c t i s a t o x i n - i n d u c e d i n c r e a s e i n t h e i o n p e r m e a b i l i t y o f t h e i n n e r mem­ branes o f Τ mitochondria. Toxin-induced i o n leakage might occur i n one o f t h r e e ways. F i r s t l y , t h e t o x i n c o u l d a c t a s a n i n n e r m i t o c h o n d r i a l mem­ b r a n e d i s r u p t a n t a s s u g g e s t e d b y Gengenbach elt a l (35) o n t h e basis of u l t r a s t r u c t u r a l data. Such a c t i o n w o u l d r e s u l t i n e x ­ t e n s i v e s t r u c t u r a l damage, i n a d d i t i o n t o c a u s i n g i n c r e a s e d i o n p e r m e a b i l i t y , a n d s h o u l d l e a d t o l e a k a g e o f i n n e r membrane m a t r i x components s u c h a s m a l a t e d e h y d r o g e n a s e . However, r e c e n t e x p e r i ­ ments i n our l a b o r a t o r y (42) h a v e shown t h a t t o x i n t r e a t m e n t does not induce leakage o f malate dehydrogenase from Τ m i t o c h o n d r i a ( T a b l e I V ) . One c r i t i c i s m o f the l a t t e r e x p e r i m e n t i s t h a t o n

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

7.

GREGORY

E T A L .

Southern

Corn

Leaf

Blight

Disease

103

a d d i t i o n of t o x i n , malate dehydrogenase could have leaked from the Τ m i t o c h o n d r i a l inner membrane but may have been h e l d w i t h ­ i n the mitochondria by the outer membrane. Secondly, the t o x i n may a c t as a c l a s s i c a l uncoupler and promote the passage of protons through the Τ m i t o c h o n d r i a l inner membrane by a c t i n g as a l i p o p h i l i c weak a c i d . A t h i r d p o s s i b i l i t y i s that the t o x i n i s an ionophore and induces increased p e r m e a b i l i t y of Τ m i t o c h o n d r i a l inner membranes to one or more types of c a t i o n by complexing w i t h the appropriate c a t i o n t o form a l i p i d s o l u b l e complex which can pass through the inner membrane. Whichever mode of a c t i o n i s c o r r e c t , we w i l l be faced with e x p l a i n i n g why Ν mitochondria are completely r e s i s t a n t to HmT t o x i n . At present we do not know whether the inner or the outer m i t o c h o n d r i a l membrane mediates r e s i s t a n c e . A p o s s i b l e r o l e of the outer m i t o c h o n d r i a l membrane i n r e s i s t a n c e was suggested by Watrud £t a l (70) because removal of the outer membrane from Ν mitochondria y i e l d e d a t o x i n - s e n s i t i v e inner membrane p r e p a r a t i o n as judged by t o x i n i n h i b i t i o n of the malate-DCPIP r e a c t i o n . I n t e r p r e t a t i o n of t h i s l a t t e r experiment should be cautious be­ cause the technique used to prepare the inner membranes i n v o l v e d osmotic s w e l l i n g and s h r i n k i n g of mitochondria i n combination with h i g h speed discontinuous sucrose gradient c e n t r i f u g a t i o n . The l a t t e r procedure could w e l l have damaged the i n n e r membranes and no p h y s i o l o g i c a l data were presented to the contrary (70). Consequently, i t i s p o s s i b l e that t o x i n s e n s i t i v i t y of the inner membranes of Ν mitochondria (70) was due to a b n o r m a l i t i e s brought about by the inner membrane i s o l a t i o n technique r a t h e r than the removal of the outer m i t o c h o n d r i a l membrane per se. Much c a r e f u l work on whole mitochondria and on subm i t o c h o n d r i a l f r a c t i o n s i s needed before we can d i s c o v e r the nature of HmT t o x i n a c t i o n on Τ mitochondria and the l a c k of t o x i n a c t i o n on Ν mitochondria. T h i s work i s a f o c a l p o i n t of our present experiments because i t i s l i k e l y to r e v e a l the biochemical b a s i s of southern corn l e a f b l i g h t d i s e a s e .

Comparative Biochemistry of Mitochondria from Ν and Τ Cytoplasms The A c t i o n of Chemicals (Other than HmT Toxin) on Ν and Τ Mitochondria. S e v e r a l chemicals known to induce one or more responses s i m i l a r to those of HmT t o x i n have been a p p l i e d to Ν and Τ mitochondria. Q u a n t i t a t i v e d i s t i n c t i o n s between Ν and Τ mitochondria have a r i s e n from these experiments. The f o l l o w i n g chemicals were used: Valinomycin. Valinomycin (VAL) s t i m u l a t e s NADH o x i d a t i o n and s w e l l i n g i n mitochondria under c e r t a i n r e a c t i o n c o n d i t i o n s by a c t -

104

HOST

Table IV.

RESISTANCE T O PESTS

E f f e c t o f HmT Toxin on the Release o f Malate Dehydro­ genase (MDH) from Τ Mitochondria

Treatment

MDH a c t i v i t y (nmol NADH oxidized/min) Supernatant

None

+ HmT Toxin

+ T r i t o n X-100 Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

PLANT

MDH a c t i v i t y r e l e a s e d i n t o supernatant % of t o t a l a c t i v i t y Mitochondria

190

3,200

6%

150

2,400

6%

150

3,600

4%

190

3,600

5%

3,600

50

99%

3,500

20

99%

Mitochondria from W64A(T) r o o t s (about 0.5 mg p r o t e i n ) were suspended i n 3 ml o f 0.4 M sucrose, 10 mM KC1, 2.5 mM M g C l , 4 mM KH P0 , 20 mM HEPES, pH 7.4 (Medium A) c o n t a i n i n g 1.5 ymol NADH and 300 nmole ADP. As i n d i c a t e d , HmT t o x i n (33 yg) o r the d e t e r ­ gent T r i t o n X-100 (to a c o n c e n t r a t i o n o f 0.15%) were added. A f t e r i n c u b a t i o n f o r 5 min a t room temperature the samples were cent r i f u g e d a t 18,000 xg f o r 18 min a t 25°. Each p e l l e t was sus­ pended i n 0.5 ml o f M e t ï u m A, and s o l u b i l i z e d with T r i t o n X-100 (0.15%) to r e l e a s e the t o t a l enzyme a c t i v i t y . Appropriate a l i q u o t s of the supernatants and p e l l e t s were assayed f o r MDH a c t i v i t y i n 3 ml o f 0.4 M sucrose, 20 mM HEPES, 0.25 mM NADH, 1 mM oxaloacetate, 1 mM KCN, pH 7.4. The o x i d a t i o n of NADH was monitored by the change i n absorbance a t 340 nm. 2

2

4

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

7.

GREGORY

ET AL.

Southern

Corn

Leaf Blight

Disease

105

ing as a Κ - s p e c i f i c ionophore (51). VAL induced s i m i l a r respons­ es i n Ν and Τ mitochondria with respect to o x i d a t i o n o f exogenous NADH and m i t o c h o n d r i a l s w e l l i n g , although the l a t t e r was more marked i n Ν mitochondria a t low VAL concentrations (38,43). Gramicidin D. Gramicidin (GRAM) causes s w e l l i n g i n KC1 o r NaCl media, uncouples o x i d a t i v e phosphorylation, and stimulates NADH o x i d a t i o n i n KC1 o r sucrose media (52). GRAM caused more s t i m u l a t i o n o f NADH o x i d a t i o n by Τ mitochondria than by Ν mito­ chondria, although GRAM-induced s w e l l i n g of Ν mitochondria was always s l i g h t l y greater than that i n Τ mitochondria (43). 2,4-Dinitrophenol (DNP) uncouples o x i d a t i v e phosphoryla­ t i o n . DNP caused dramatic s t i m u l a t i o n o f NADH r e s p i r a t i o n i n both Ν and Τ mitochondria. However, a t high l e v e l s o f DNP, the s t i m u l a t i o n was s i g n i f i c a n t l y higher f o r Τ mitochondria than f o r Ν mitochondria (43). Bednarski e t a l . (38) could not detect s i g ­ n i f i c a n t d i f f e r e n c e s between Τ and Ν mitochondria i n s e n s i t i v i t y to DNP. Sodium azide i s a terminal oxidase i n h i b i t o r . The degree of i n h i b i t i o n of NADH o x i d a t i o n by sodium azide was the same f o r Ν and Τ mitochondria (43). N i g e r i c i n (NIG) plus Κ causes uncoupling i n mitochondria. Τ mitochondria were m o r e s e n s i t i v e than were Ν mitochondria to uncoupling by NIG plus Κ (38). D e c e n y l s u c c i n i c A c i d . D e c e n y l s u c c i n i c a c i d (DSA) causes m i t o c h o n d r i a l s w e l l i n g by mediating membrane d i s r u p t i o n . DSA t r e a t ­ ments a l s o s t i m u l a t e exogenous NADH o x i d a t i o n and r e s u l t i n l o s s of o x i d a t i v e phosphorylation (53). DSA s t i m u l a t e d NADH o x i d a t i o n a t low concentrations but caused i n h i b i t i o n a t higher concentra­ t i o n s . The Ν and Τ mitochondria responded s i m i l a r l y to DSA with respect to NADH o x i d a t i o n . However, DSA caused c o n s i d e r a b l y more s w e l l i n g i n Ν mitochondria than i n Τ mitochondria (43). Calcium ( i n the presence and absence o f phosphate). Calcium i n the absence o f i n o r g a n i c phosphate stimulated NADH o x i d a t i o n i n Ν mitochondria somewhat more than i n Τ mitochondria. Calcium plus phosphate induced s i m i l a r s t i m u l a t i o n s o f NADH o x i d a t i o n i n the Ν and Τ mitochondria (43). D i g i t o n i n . D i g i t o n i n i s a saponin which can d i s r u p t b i o l o g i ­ c a l membranes. A t low concentrations d i g i t o n i n i s s e l e c t i v e f o r s t e r o i d - r i c h membranes. Evidence f o r t h i s s e l e c t i v i t y comes from the f i n d i n g o f Schnaitman et^ al^. (54) that the outer membrane o f r a t l i v e r mitochondria i s more d i g i t o n i n - s e n s i t i v e than the inner membrane. These authors suggested that as d i g i t o n i n i s known to combine with c h o l e s t e r o l (a major membrane s t e r o i d i n animals) on a 1:1 b a s i s , i t was p o s s i b l e that the outer m i t o c h o n d r i a l mem­ branes were higher i n c h o l e s t e r o l than the inner membrane. Sub­ sequent s t u d i e s with guinea p i g l i v e r mitochondria showed that the s t e r o i d content o f the outer m i t o c h o n d r i a l membrane was s i x times more concentrated, on a p r o t e i n b a s i s , than i n the inner membrane (55) . Gregory e_t aJU (56) have r e c e n t l y s t u d i e d the r e s p i r a t o r y e f f e c t s o f low concentrations o f d i g i t o n i n on N, C, +

HOST P L A N T RESISTANCE TO

106

PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

and Τ mitochondria (Figure 2 ) . The Τ mitochondria were dramati­ c a l l y more d i g i t o n i n - s e n s i t i v e than the Ν o r C mitochondria, as i n d i c a t e d by i n h i b i t i o n o f malate and s u c c i n a t e r e s p i r a t i o n and uncoupling o f o x i d a t i v e phosphorylation (Figure 2 ) . There were a l s o d i f f e r e n c e s between the Ν and C mitochondria with r e s p e c t to d i g i t o n i n s e n s i t i v i t y (Figure 2 ) . These data were thought (56) to r e f l e c t d i f f e r e n c e s i n membrane s t e r o i d composition between the N, Τ and C mitochondria. I t was noted (56) that the d i g i t o n i n e f ­ f e c t s were somewhat s i m i l a r to those o f HmT t o x i n on Τ mitochon­ dria. The data o u t l i n e d above showed that compounds other than HmT toxin can produce d i f f e r e n t i a l e f f e c t s on v a r i o u s inner mem­ brane f u n c t i o n s i n Ν and Τ mitochondria and thus s t r o n g l y sup­ ports the concept that those mitochondria d i f f e r with r e s p e c t to membrane s t r u c t u r e .

0

2

4

6

.8

1.0

1.2

14

16

18

20

Digitonin Concentration (mg digitonin/mg mitochondrial protein) Figure 2. Effects of digitonin on respira­ tion and oxidative phosphorylation in mito­ chondria isolated from W64A(N), W64A(T), and W64A(C) roots. Experimental condi­ tions are found in Ref. 56.

7.

GREGORY

ET

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

Chemical

AL.

Southern

Corn

Leaf Blight

Disease

107

Composition o f Ν and Τ Mitochondria.

N u c l e i c A c i d s . Levings and P r i n g (57) have shown that Ν and Τ cytoplasms d i f f e r with respect to m i t o c h o n d r i a l DNA (mt DNA). In these experiments, mt DNA was i s o l a t e d from the Ν and Τ cytoplasms of s e v e r a l corn l i n e s . The mt DNA's were subjected to r e s t r i c t i o n enzyme fragment a n a l y s i s using each of the r e s t r i c t i o n endonucleases Eco RI (52,58) , H i n D I I I (57.,5j0 , Bam I (57), and Sal I (57). Each of these enzymes cut DNA a t d i f f e r e n t sequence s p e c i f i c s i t e s and each y i e l d e d d i s t i n c t i o n s between the mt DNA from Ν and Τ cytoplasms (57). In a d d i t i o n , i t was shown (57) that the male s t e r i l e cytoplasms other than T, namely S, C and EP, d i f f e r e d with r e s p e c t to mt DNA. As these d i f f e r e n c e s may have been due to contaminating DNA from n u c l e a r , b a c t e r i a l or v i r a l sources, Levings and P r i n g (58) were c a r e f u l to confirm the i d e n t i t y of t h e i r mt DNA preparations by using buoyant d e n s i t y determinations i n n e u t r a l cesium c h l o r i d e and showing that t h e i r mt DNA p r e p a r a t i o n s appeared as a s i n g l e band with a buoyant d e n s i t y of 1.706 g/cm which i s c h a r a c t e r i s t i c of higher p l a n t mt DNA (59). In a d d i t i o n , the upper and lower DNA bands obtained from cesium c h l o r i d e - e t h i d i u m bromide g r a d i e n t s were i n agreement with the expected value f o r mt DNA. There was no i n d i c a t i o n of n u c l e a r DNA contamination because the upper and lower DNA bands on cesium c h l o r i d e - e t h i d i u m bromide gradients were i n d i s t i n g u i s h a b l e from each other and, i n any case, r e s t r i c t i o n enzyme fragment a n a l y s i s of the complex nuclear DNA does not y i e l d d i s t i n c t bands which c o u l d be confused with mt DNA bands (58). The p o s s i b i l i t y of contamination o f the mt DNA prepara­ t i o n s by v i r a l DNA was not r u l e d out (58). Previous work i n ­ v o l v i n g buoyant d e n s i t y a n a l y s i s o f mt DNA's on n e u t r a l cesium c h l o r i d e had f a i l e d to demonstrate d i f f e r e n c e s between Ν and Τ cytoplasms (60). D i f f e r e n c e s between the mt DNA o f suscep­ t i b l e and r e s i s t a n t c e l l s are a p r e r e q u i s i t e f o r t h e o r i e s i n ­ v o l v i n g Τ mitochondria as primary f a c t o r s i n c y t o p l a s m i c a l l y i n h e r i t e d s u s c e p t i b i l i t y to southern corn l e a f b l i g h t d i s e a s e . Whether or not the observed mt DNA d i f f e r e n c e s are a s s o c i a t e d with d i f f e r e n c e s i n disease s u s c e p t i b i l i t y remains to be seen. 3

Proteins 1. Subunit f i n g e r p r i n t s o f t o t a l m i t o c h o n d r i a l p r o t e i n . The use of high r e s o l u t i o n two-dimensional e l e c t r o p h o r e s i s i n our l a b o r a t o r y has f a i l e d to show q u a l i t a t i v e d i f f e r e n c e s between N, C and Τ mitochondria with r e s p e c t to t o t a l p r o t e i n subunits (61). The a n a l y t i c a l technique, developed by O ' F a r r e l l (62), i n v o l v e s s e p a r a t i o n of m i t o c h o n d r i a l p r o t e i n according to i s o ­ e l e c t r i c p o i n t by i s o e l e c t r i c f o c u s i n g i n the f i r s t dimension, followed by s e p a r a t i o n according to molecular weight by sodium dodecyl s u l f a t e e l e c t r o p h o r e s i s i n the second dimension. The

108

HOST P L A N T RESISTANCE T O

r e s u l t i s a " f i n g e r p r i n t " of the mitochondrial p r o t e i n subunits. The technique i s h i g h l y r e p r o d u c i b l e so that each spot on one separation can be matched with a corresponding spot on a d i f ­ ferent separation. In a d d i t i o n the technique can r e s o l v e p r o t e i n s d i f f e r i n g i n a s i n g l e charge (62). In our experiments, v i s u a l i z a t i o n of the p r o t e i n f i n g e r p r i n t s was by means of s t a i n ­ ing r a t h e r than by autoradiography which i s more s e n s i t i v e . I t i s therefore very p o s s i b l e that one or more p r o t e i n s were un­ detected i n our experiments and that q u a n t i t a t i v e d i f f e r e n c e s e x i s t between the p r o t e i n s of N, C and Τ mitochondria. Future experiments i n our laboratory i n v o l v i n g a combination of auto­ radiography and two-dimensional e l e c t r o p h o r e s i s of mitochondrial p r o t e i n s which are l a b e l l e d with e i t h e r C or S , w i l l f a c i l ­ i t a t e b e t t e r s e n s i t i v i t y and q u a n t i f i c a t i o n of the system. 2. Cytochromes. P r i n g (63) has reported that Τ mitochon­ d r i a contain a f u l l complement of cytochromes a + a$ b and c as detected by d i f f e r e n c e spectra at 25°C. I t has a l s o been shown that Τ cytoplasm contains 7-12% more cytochrome b than does Ν (63) and that TRf mitochondria contain s l i g h t l y more c y t o ­ chrome a + a 3 than do Ν mitochondria (64) i n each of f i v e inbred lines. Small d i f f e r e n c e s i n cytochrome b and c content of Ν and TRf mitochondria were noted, but these d i f f e r e n c e s were not ap­ parent f o r a l l of the inbred l i n e s t e s t e d (64). S t e r o i d s . Q u a n t i t a t i v e and q u a l i t a t i v e determinations of m i t o c h o n d r i a l s t e r o i d s i n our l a b o r a t o r y have y i e l d e d no r e p r o ­ d u c i b l e d i f f e r e n c e s between Ν and Τ cytoplasms (65). Lack of r e p r o d u c i b i l i t y may be a f u n c t i o n of v a r y i n g p u r i t y among the d i f f e r e n t mitochondrial preparations. The experiments i n v o l v e d e x t r a c t i o n of a crude s t e r o i d p r e p a r a t i o n from the i s o l a t e d mito­ chondria, p u r i f i c a t i o n of the s t e r o i d s by s i l i c i c a c i d column chromatography, followed by GLC of the f r e e s t e r o i d s . Three major s t e r o i d s were found i n the Ν and the Τ mito­ chondria: campesterol, s t i g m a s t e r o l and s i t o s t e r o l . The i d e n t i t y of these compounds was confirmed by GLC-mass spectroscopy i n c o l l a b o r a t i o n with Dr. S. F. Osman, ARS-USDA, Eastern Regional Research Center, Wyndmoor, Pa. A minor s t e r o i d was a l s o found, but the i d e n t i t y of t h i s compound i s not known at present. The f a c t that Ν and Τ mitochondria are d i f f e r e n t i a l l y s e n s i t i v e to d i g i t o n i n (56) as explained p r e v i o u s l y , suggests that f u r t h e r experiments i n v o l v i n g f r a c t i o n a t i o n , p u r i f i c a t i o n , and s t e r o i d analyses of inner and outer m i t o c h o n d r i a l membranes may y i e l d d i f f e r e n c e s between these mitochondria. I t i s of i n t e r e s t that s t e r o l d i f f e r e n c e s have been found between Ν and Τ t a s s e l s during development (66). llf

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

PESTS

3 5

9

Studies on the Molecular S p e c i f i c Toxin

Structure of H. maydis Race Τ Host

The exact molecular s t r u c t u r e of HmT

t o x i n i s not yet known

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

7.

GREGORY

E T

AL.

Southern

Corn

Leaf

Blight

Disease

109

and there i s disagreement as to which c l a s s of compounds HmT t o x i n belongs. Our current information on HmT t o x i n s t r u c t u r e c o n s i s t s of p u b l i s h e d data by Karr e_t a l . ( 2 4 ) and a recent personal com­ munication from J . M. Daly of the U n i v e r s i t y o f Nebraska (67). Karr e t a l . ( 2 4 ) i s o l a t e d f i v e t o x i c compounds from crude HmT culture f i l t r a t e . I t was p o s t u l a t e d that the t o x i c compounds were terpenoids, e x i s t i n g f r e e or as g l y c o s i d e s , ranging i n molecular weight from 350 to 600 and a chemical assay f o r HmT t o x i n was developed (68). This work i n v o l v e d f r a c t i o n a t i o n and p u r i f i c a t i o n of the t o x i c components i n combination with a l e a f puncture b i o assay f o r t o x i n a c t i v i t y i n each f r a c t i o n . The f r a c t i o n s which e x h i b i t e d a c t i v i t y i n the bioassay were analysed f o r chemical s t r u c t u r e by chemical t e s t s , chromatography, mass spectroscopy and nuclear magnetic resonance a n a l y s i s . The chemical assay f o r HmT t o x i n was a g e n e r a l i z e d t e s t f o r terpenoids using s u l f u r i c a c i d - a c e t i c anhydride reagent. The involvement of the f i v e t e r ­ penoid compounds i n HmT t o x i n s p e c i f i c i t y i s open to q u e s t i o n . A subsequent comparison (69) of the chemical assay d e s c r i b e d by Karr ejt a l . (68) to measure the f i v e terpenoid t o x i n components i n p a r t i a l l y p u r i f i e d preparations r e v e a l e d that both HmT and HmO (which does not produce a h o s t - s p e c i f i c toxin) produce compounds s i m i l a r to the toxins i d e n t i f i e d by Karr e t a l . ( 2 4 ) . Consequently i t i s p o s s i b l e that the f i v e terpenoid compounds c h a r a c t e r i z e d by Karr ejt S L L . ( 2 4 ) are not a s s o c i a t e d w i t h the h o s t - s p e c i f i c a c t i ­ v i t y of HmT t o x i n . I t i s i n t e r e s t i n g , however, that when Watrud et a l . (70) t r e a t e d Τ mitochondria w i t h a mixture of toxins I and I I (see r e f e r e n c e 2JL f ° terminology), the c h a r a c t e r i s t i c r e ­ sponses were e l i c i t e d . Another problem i n the work of Karr e t a l . ( 2 4 ) d e r i v e s from the use o f the i n s e n s i t i v e l e a f puncture b i o ­ assay i n the assessment of t o x i c a c t i v i t y of the v a r i o u s f r a c t i o n s (15,69). I t i s p o s s i b l e that one or more of the discarded f r a c ­ tions contained non-terpenoid h o s t - s p e c i f i c t o x i n a c t i v i t y . r

A non-terpenoid s t r u c t u r e f o r HmT t o x i n has been proposed r e c e n t l y by Kono and Daly (67). Some d e t a i l s of the s t r u c t u r e have not been f i n a l i z e d , but a great deal of u s e f u l i n f o r m a t i o n has been gained. The molecule has an e m p i r i c a l formula of 2 9 5 0 ° 9 (542 daltons) and i s extremely a c t i v e a g a i n s t Τ corn, causing complete n e c r o s i s of the f i r s t true l e a f a t l e v e l s as low as 5 to 10 ng. The t o x i c i t y of the molecule was s p e c i f i c f o r Τ corn as no t o x i c e f f e c t was observed on Ν corn or on other p l a n t s p e c i e s . The use of a very s e n s i t i v e t o x i n bioassay, i n which dark C 0 f i x a t i o n by corn l e a f d i s c s was measured (18) insured that no b i o l o g i c a l l y a c t i v e t o x i n f r a c t i o n s were discarded p r i o r to s t r u c t u r a l a n a l y s i s . In aqueous s o l v e n t s the m a t e r i a l chromatographed as a s i n g l e d i f f u s e spot, but i n mixtures of methanol and non-polar organic compounds a major and two minor components with host s p e c i f i c i t y were observed. These a d d i t i o n a l compounds were probably isomeric o r homologous because a c e t y l a t i o n of t o x i n r e ­ s u l t e d i n the formation of a t e t r a a c e t a t e of e m p i r i c a l formula C37H58O13 (710 daltons) which a l s o r e s o l v e d i n t o a major and two C

H

2

HOST P L A N T

110

RESISTANCE T O PESTS

minor components. The IR, NMR and UV s p e c t r a of two of the a c e t y l a t e d d e r i v a t i v e s could be d i s t i n g u i s h e d only by the e x t i n c ­ t i o n c o e f f i c i e n t i n the UV. There were i n s u f f i c i e n t amounts o f the t h i r d component to make comparisons. C and proton NMR of the major a c e t y l a t e d d e r i v a t i v e support the e x i s t e n c e of four hydroxyl groups and f i v e carbonyl groups i n the o r i g i n a l t o x i n . On the b a s i s of IR, C and proton decoupling NMR and mass s p e c t r a of the component i s o l a t e d i n the l a r g e s t y i e l d , two p o s s i b l e s t r u c t u r e s have been proposed f o r HmT t o x i n (Figure 3). Work to determine the exact s t r u c t u r e of the molecule i s s t i l l i n pro­ gress. Whether the s i n g l e p u r i f i e d t o x i n molecule of Kono and Daly (67) mimics the m u l t i p l e m i t o c h o n d r i a l e f f e c t s induced by crude t o x i c f i l t r a t e and c h l o r o f o r m - t o x i n i s a c r i t i c a l and ex­ c i t i n g question. 1 3

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

1 3

Concluding Remarks Mechanism o f Southern Corn Leaf B l i g h t Disease. In our o p i n i o n present data suggests that a major component (and perhaps the primary component) of southern corn l e a f b l i g h t disease i s the HmT toxin-induced damage of mitochondria i n s u s c e p t i b l e (T c y t o ­ plasm) corn. This o p i n i o n i s based on two major pieces of exper­ imental evidence. F i r s t l y , s e v e r a l groups have shown that i s o ­ l a t e d s u s c e p t i b l e Τ mitochondria (but not the r e s i s t a n t mito­ chondria) are s e v e r e l y damaged by HmT t o x i n . The p h y s i o l o g i c a l m a n i f e s t a t i o n of t h i s damage, should the damage a l s o occur i n s i t u , would l e a d to the death of corn c e l l s which c o n t a i n Τ cytoplasm. Secondly, u l t r a s t r u c t u r a l s t u d i e s by our group have shown that HmT t o x i n can damage Τ mitochondria i n s i t u and that t h i s damage i s s i m i l a r to that observed i n t o x i n - t r e a t e d , i s o l a t e d Τ mito­ chondria. However, i t i s of extreme importance to e l u c i d a t e whether or not HmT t o x i n a f f e c t s Τ m i t o c h o n d r i a l physiology i n v i v o , because a t present there i s no c o n c l u s i v e evidence i n t h i s area. S e n s i t i v e measurements o f i n v i v o r e s p i r a t i o n and phos­ p h o r y l a t i o n i n t o x i n - t r e a t e d corn p r o t o p l a s t s are i n progress i n our l a b o r a t o r y and may have great relevance to the nature of southern corn l e a f b l i g h t d i s e a s e . The cytoplasmic i n h e r i t a n c e of s u s c e p t i b i l i t y to the disease may a l s o be c o n s i s t e n t w i t h the mitochondria (which c o n t a i n DNA) being of primary importance. Should the t r a n s f e r of r e s i s t a n t (N) mitochondria to s u s c e p t i b l e (T) p r o t o p l a s t s r e s u l t i n a c o n f e r r i n g of r e s i s t a n c e to these p r o t o p l a s t s , there w i l l be l i t t l e doubt that mitochondria are a major f a c t o r i n the r e s i s t a n c e of corn to southern corn l e a f b l i g h t d i s e a s e . This experiment i s i n progress i n our l a b o r a t o r y . The Use of the Southern Corn Leaf B l i g h t System i n Studies on M i t o c h o n d r i a l Genetics i n Higher P l a n t s . L i t t l e i s known about the m i t o c h o n d r i a l genetics of higher p l a n t s . Although asexual genetic manipulation such as p r o t o p l a s t f u s i o n and o r g a n e l l e t r a n s f e r can b r i n g together cytoplasmic components from d i v e r s e c e l l types, few m i t o c h o n d r i a l markers are a v a i l a b l e i n higher

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

7.

GREGORY

E TA L .

OH

α.

O H

Corn Leaf

Ο

Ο

Ο

Ο

Blight

O

H

Ο

111

Disease

O H

Ο

Ο

/WWWWWWA OH

Figure 3.

Southern

O H

O H

Ο

Ο

O H

Ο

Two possible structures for HmT toxin recently proposed by Kono and Daly

(67)

112

HOST PLANT RESISTANCE TO PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

plants. Because HmT toxin rapidly and specifically destroys both Τ cytoplasm protoplasts and Τ mitochondria within protoplasts (20,31,32,33) the toxin will be a useful probe for following mitochondrial transfers. The response of protoplasts and mito­ chondria to HmT toxin after fusion of Ν and Τ cytoplasm proto­ plasts, or after transfer of isolated Ν mitochondria to Τ proto­ plasts, may answer several important questions: can "foreign" mitochondria be transferred to a different cytoplasm?; can such mitochondria survive, function, replicate, and alter the survival of host protoplasts?; do mitochondria carry all the genetic material that determines cytoplasmic response to toxin? Tech­ niques developed using HmT toxin as a probe for successful genetic manipulations with mitochondria may facilitate a new experimental approach to the study of mitochondrial genetics in higher plants. Literature Cited 1. Hooker, A.L., Smith, D.R., Lim, S.M., Beckett, J.B., Plant Dis. Rep. (1970) 54, 708. 2. Smith, D.R., Hooker, A.L., Lim, S.M., Beckett, J.B., Crop Sci. (1971) 11, 772. 3. Gracen, V.E., Grogan, C.O., Plant Dis. Rep. (1972) 56, 432. 4. Berquist, R.R., Peverly, G., Plant Dis. Rep. (1972) 56, 112. 5. Hallauer, A.R., Martinson, C.A., Agronomy J. (1975) 67, 497. 6. Lim, S.M., Plant Dis. Rep. (1974) 58, 811. 7. Caunter, I.G., Ph.D. thesis, 1977, Cornell University, "A Genetic Study of Nuclear Controlled Resistance to Southern and Yellow Leaf Blights in Maize" (Zea mays L.). 8. Payne, G.A., Yoder, O.C., Phytopathology (1977)67,in press. 9. Lim, S.M., Hooker, A.L., Genetics (1971) 69, 115. 10. Yoder, O.C., Gracen, V.E., Phytopathology (1975) 65, 273. 11. Lim, S.M., Hooker, A.L., Phytopathology (1972) 62, 968. 12. Halloin, J.M., Comstock, J.C., Martinson, C.A., Tipton, C.L., Phytopathology (1973)63,640. 13. Gracen, V.E., Forster, M.J., Sayre, K.D., Grogan, C.O., Plant Dis. Rep. (1971) 55, 469. 14. Turner, M.T., Martinson, C.A., Plant Dis. Rep. (1972) 56, 29. 15. Yoder, O.C., Payne, G.A. Gregory, P., Gracen, V.E., Physiol. Plant Path. (1977) 10, 237. 16. Wheeler, H., Williams, A.S., Young, L.D., Plant Dis. Rep. (1971) 55, 667. 17. Arntzen, C.J., Koeppe, D.E., Miller, R.J., Peverly, J.H., Physiol. Plant Path. (1973) 3, 79. 18. Bhullar, B.S., Daly, J.M., Rehfeld, D.W., Plant Physiol. (1975) 56, 1. 19. Wheeler, H., Ammon, V.D., Phytopathology (1977)67,325. 20. Aldrich, H.C., Gracen, V.E., York, D., Earle, E.D., Yoder, O.C., Tissue and Cell (1977) 9, 167. 21. Koeppe, D.E., Malone, C.P., Miller, R.J., Plant Physiol. (supp.) (1973) 51, 10.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

113 7. GREGORY ET AL. Southern Corn Leaf Blight Disease 22. Mertz, S.M. Jr., Arntzen, C.J., Plant Physiol. (supp.) (1973) 51, 16. 23. Watrud, L.S., Hooker, A.L., Koeppe, D.E., Phytopathology (1975) 65, 178. 24. Karr, A.L. Jr., Karr, D.B., Strobel, G.A., Plant Physiol. (1974) 53, 250. 25. Earle, E.D., unpublished. 26. Arntzen, C.J., Haugh, M.F., Bobick, S., Plant Physiol. (1973) 52, 569. 27. Gracen, V.E., Grogan, C.O., Forster, M.J., Can. J. Bot. (1972) 50, 2167. 28. Frick, H., Bauman, L.F., Nicholson, R.L., Hodges, T.K., Plant Physiol. (1977) 59, 103. 29. Gengenbach, B.G., Green, C.E., Crop Sci. (1975) 15, 645. 30. Laughnan, J.R., Gabay, S.J., Crop Sci. (1973) 13, 681. 31. Pelcher, L.E., Kao, K.N., Gamborg, O.L., Yoder, O.C., Gracen, V.E., Can. J. Bot. (1975) 53, 427. 32. Earle, E.D., Gracen, V.E., Yoder, O.C., Gemmill, K.P., submitted to Plant Physiol. 33. York, D.W., unpublished. 34. Miller, R.J., Koeppe, D.E., Science (1971) 173, 67. 35. Gengenbach, B.G., Miller, R.J., Koeppe, D.E., Arntzen, C.J., Can. J. Bot. (1973) 51, 2119. 36. Krueger, W.A., Josephson, L.M., Hilty, J.W., Phytopathology (1974) 64, 735. 37. Barratt, D.H.P., Flavell, R.B., Theoretical and Applied Genetics (1975) 45, 315. 38. Bednarski, M.A., Izawa, S., Scheffer, R.P., Plant Physiol. (1977) 59, 540. 39. Peterson, P.Α., Flavell, R.B., Barratt, D.H.P., Theoreti­ cal and Applied Genetics (1975) 45, 309. 40. Flavell, R., Physiol. Plant Path. (1975)6,107. 41. Peterson, P.Α., Flavell, R.B., Barratt, D.H.P., Plant Dis. Rep. (1974) 58, 777. 42. Matthews, D.E., unpublished. 43. Gengenbach, B., Koeppe, D.E., Miller, R.J., Physiol. Plant. (1973) 29, 103. 44. Tipton, C.L., Mondal, M.H., Uhlig, J., Biochem. Biophys. Res. Comm. (1973) 51, 725. 45. Tipton, C.L., Mondal, M.H., Benson, M.J., Physiol. Plant Path. (1975)7,277. 46. Gracen, V.E., Forster, M.J., Grogan, C.O., Plant Dis. Rep. (1971) 55, 938. 47. Frick, H., Nicholson, R.L., Hodges, T.K., Bauman, L.F., Plant Physiol. (1976) 57, 171. 48. Hodges, T.K., Leonard, R.T., Bracker, C.E., Keenan, T.W., Proc. Nat. Acad. Sci. U.S.A. (1972) 69, 3307. 49. Scheffer, R.P. IN Biochemistry & Cytology of Plant-Parasite Interaction [K. Tomiyama, J.M. Daly, I. Uritani, H. Oku and S. Ouchi, eds], p. 118, Elsevier Scientific Publishing Company, 1976.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch007

114

HOST PLANT RESISTANCE TO PESTS

50. Gracen, V.E., unpublished. 51. Hanson, J.B., Bertognolli, B.L., Shepherd, W.D., Plant Physiol. (1972) 50, 347. 52. Miller, R.J., Dumford, W.S., Koeppe, D.E., Plant Physiol. (1970) 46, 471. 53. Koeppe, D.E., Miller, R.J., Plant Physiol. (1971) 48, 659. 54. Schnaitman, C., Erwin, V.G., Greenawalt, J.W., J. Cell Biol. (1967) 32, 719. 55. Parsons, D.F., Yano, Y., Biochem. Biophys. Acta (1967) 135, 362. 56. Gregory, P., Gracen, V.E., Yoder, O.C., Steinkraus, N.A., Plant Sci. Lett. (1977) 9, 17. 57. Levings, C.S. III, Pring, D.R., Proc. 31st Annu. Corn and Sorghum Res. Conf. (1976) 110. 58. Levings, C.S. III, Pring, D.R., Science (1976) 193, 158. 59. Wells, R., Ingle, J., Plant Physiol. (1970) 46, 178. 60. Shah, D.M., Levings, C.S. III, Crop Sci. (1974) 14, 852. 61. Gemmill, R., Gregory, P., unpublished. 62. O'Farrell, P.H., J. Biol. Chem. (1975) 250, 4007. 63. Pring, D.R., Plant Physiol. (1975) 55, 203. 64. Watrud, L.S., Laughnan, J.R., Gabay, S.J., Koeppe, D.E., Can. J. Bot. (1976) 54, 2718. 65. Gregory, P., unpublished. 66. Comita, J.J., Klosterman, H.J., Phytochem. (1976) 15, 917. 67. Kono, Y., Daly, J.M., personal communication. 68. Karr, D.B., Karr, A.L., Strobel, G.A., Plant Physiol. (1975) 55, 727. 69. Yoder, O.C., Gracen, V.E., Plant Physiol. (1977) 59, 792. 70. Watrud, L.S., Baldwin, J.K., Miller, R.J., Koeppe, D.E., Plant Physiol. (1975) 56, 216.

Acknowledgements The experiments performed in our laboratory were supported in part by grant 75002 from the Rockefeller Foundation. We gratefully acknowledge the help of Dr. D.E. Matthews, H. Pham, and D.W. York in the preparation of this paper.

8 Host Plant Resistance to Insects

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

A. C. WAISS, JR., B. G. CHAN, and C. A. ELLIGER Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, CA 94710

In order to use chemical insecticides more advantageously, systems for integrated pest management were recommended in 1975 by the National Academy of Sciences (1,2), U. S. Department of Agriculture (3) and the Entomological Soc. of America (4). Among the various alternatives to chemical insecticides, the use of insect resistant plants, in combination with good cultural practice, is perhaps the most effective, convenient, economical and environmentally acceptable method of insect control. In addition, it is a method that is completely compatible with both chemical and other biological control measures. Host Plant Resistance (HPR) to Insects Since many excellent reviews (5, 6, 7, 8, 9) have been written on the subject of plant-insect interactions, no attempt will be made in this paper to fully review past literature. Rather, we will use a few selected examples stressing the problems that may be encountered by chemists working in this relatively unexplored area of research, as well as future research needs of this very important field. Host plant resistance (HPR) has been recognized since the early 1800's. Because plants, which are generally immobile, lack a circulatory system, the existence of the classical antigen-antibody immune system would not be possible. Through evolutionary processes plants have elaborated an array of "secondary metabolic products" of which many function in HPR. From the standpoint of natural selection in evolution HPR is a preadaptive characteristic of plants. Before being cultivated by man, plants, co-evolving with insects, either intrinsically possessed or have developed means of surviving attack by arthropods. Although the first significant economic contribution by HPR to agriculture occurred in 1890, this was the successful grafting of European grape vines onto resistant root stock from North America (10) to save the French wine industry from Phylloxera vitifolia (Fitch), the potential of 115

HOST P L A N T RESISTANCE TO

116

H P R as an insect

control method,

however,

PESTS

w a s not fully-

a p p r e c i a t e d u n t i l t h e m i d I960's p a r t l y d u e to t h e a v a i l a b i l i t y of e c o n o m i c a l ,

effective and persistent

insecticides which had

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

b e e n i n t r o d u c e d s o m e two d e c a d e s e a r l i e r . W h i l e o v e r a m i l l i o n s p e c i e s of i n s e c t s e x i s t , o n l y a f e w t h o u s a n d m a y be c l a s s i f i e d a s p e s t s ; a n d , of t h e s e , o n l y a b o u t 500 s p e c i e s p r o d u c e a p p r e c i a b l e e c o n o m i c d a m a g e . The g e n e r a l a s y n c h r o n y i n s p a c e a n d t i m e b e t w e e n the g r o w t h a n d d e v e l o p m e n t of p l a n t s a n d i n s e c t s , c a u s e d b y b o t h g e o g r a p h i c a l a n d o t h e r e n v i r o n m e n t a l f a c t o r s i s the m a j o r e s c a p e m e c h a n i s m of the h o s t . T h e " e m p h e r m e r a l i t y " (9) o r l a c k o f " a p p a r e n c y " (8) o f m a n y d e c i d u o u s a n d a n n u a l p l a n t s c a u s e s f u r t h e r s e l e c ­ tive p r e s s u r e against s p e c i a l i z e d h e r b i v o r e s . In n a t u r e , H P R p r o v i d e s s t i l l a n o t h e r d e f e n s e f o r p l a n t s to m a i n t a i n e c o l o g i c a l balance with their predators. T h r e e m e c h a n i s m s to a c c o u n t f o r t h e insect damage were p r o p o s e d by P a i n t e r 1. N o n p r e f e r e n c e - In c o - e v o l u t i o n , to t h e i r h o s t s t h r o u g h s o u r c e a t t r a c t a n t s position stimulants. N o n p r e f e r e n c e is a t h e s e s t i m u l a n t s o r , o f t e n , the p r e s e n c e physical deterrents.

p l a n t ' s r e s i s t a n c e to ( Π , 12): insects have adapted and feeding and o v i r e s u l t of the l a c k of of c h e m i c a l a n d

2. T o l e r a n c e - T h i s i s t h e a b i l i t y o f c e r t a i n p l a n t s t o w i t h ­ stand insect attack without a p p r e c i a b l e l o s s in v i g o r or c r o p yield. 3. A n t i b i o s i s - T h i s c o m p r i s e s t h e d e f e n s i v e m e c h a n i s m of p l a n t s a g a i n s t t h e i r p e s t s t h r o u g h a d v e r s e i n f l u e n c e o n g r o w t h , s u r v i v a l o r r e p r o d u c t i o n of the i n s e c t s b y m e a n s of chemical or morphological factors. H P R in Non-Agricultural

Plants

F o r c e n t u r i e s p l a n t s h a v e b e e n k n o w n to p o s s e s s i n s e c t i cidal activities. Nicotine and other tobacco alkaloids were e x p o r t e d f r o m A m e r i c a a s e a r l y a s 1690 f o r i n s e c t c o n t r o l (13). O t h e r n a t u r a l i n s e c t i c i d e s s u c h a s t h e p y r e t h o i d s a n d r o t e n o i d s h a v e b e e n u s e d i n the M i d d l e E a s t a n d f a r e a s t e r n t r o p i c s s i n c e a n c i e n t t i m e (14, 15). A r e c e n t r e v i e w (16) c i t e d n e a r l y 1500 s p e c i e s o f p l a n t s c o n t a i n i n g t o x i c a n t a n d pest control agents. D e t a i l e d s t u d i e s of h o s t p l a n t r e s i s t a n c e h a v e b e e n c o n ­ f i n e d a l m o s t e x c l u s i v e l y to n o n - a g r i c u l t u r a l p l a n t s . The i n t r i c a t e r e l a t i o n s h i p b e t w e e n the o a k t r e e a n d i t s p r i m a r y p r e d a t o r , the w i n t e r m o t h , h a s b e e n e x p l a i n e d b o t h i n t e r m s of e s c a p e i n s p a c e a n d t i m e a s w e l l a s b y c h e m i c a l a n t i b i o s i s (8, 17). A p p a r e n t l y t h e q u a n t i t y o f p o l y p h e n o l i c t a n n i n a n d iFs p r e c u r s o r s , c h a n g i n g f r o m 0. 5% o f t h e d r y l e a f w e i g h t i n A p r i l , to 5% i n S e p t e m b e r , c o r r e s p o n d to r e l a t i v e i n f e s t a t i o n a n d g r o w t h of the a d a p t e d p e s t . S i m i l a r i l y , the

8.

wAiss E T A L .

Host Plant Resistance

117

to Insects

distribution of polyphenols helped to explain host plant resistance of creosote bush, L a r r e a tridentata (9), bracken fern, P t e r i d i u m a g u i l i n u m (18) and several other plants ( 3 7 ) . T h e subject of antifeedants, repellents and deterrents, and the role of alkaloids and terpenoids in insect control will be presented by other speakers in this symposium and so will not be treated here.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

HPR

in A g r i c u l t u r a l

Crops

T h e main reason for the recent surge of interest in H P R to insects for crops of economic importance is due to the fact that over 50% of the approximately 5 0 0 million lbs of pesticides applied to U n i t e d S t a t e s cropland are insecticides (2). W h i l e they are essential to agricultural production at the present t i m e , these c h e m i c a l s present serious questions in regard to environmental quality, public health, and the existence of many species of animals. T h e severe restriction on the use of D D T , aldrin, dieldrin, chlordane and heptachlor increases the need for alternatives. A l t h o u g h , at present, over 1 0 0 insect resistant cultivars are grown in the U S A ( 13, 1 4 ) , little has been reported on the nature of resistance at the chemical or molecular level. H P R is an inheritable characteristic of plants; knowledge about its chemical basis would play an immense role in plant breeding and genetic engineering in the future. T h e following examples represent the challenge, and frustrations for chemists working in a biological field and illustrate the tremendous need for a multidisciplinary approach to solving this problem. R e s i s t a n c e of C o t t o n to T o b a c c o B u d w o r m ,

Heliothis

virescens ( F . ) I n the early 1 9 0 0 ' s it was suggested that the presence of dark internal glands in G o s s y p i u m plants was a s s o c i a t e d w i t h resistance to insects

22). G o s s y p o l ,

I,

isolated f r o m these glands, was considered to be the main c o n t r i b u t i n g f a c t o r against tobacco budworm, v i r e s c e n s (F) (23,

24,

25,

26).

Heliothis

C e r t a i n morpKôTôgTcâT c h a r a c -

teristics as observed in glabrous and nectariless strains, also appeared to add to cotton's defense against t h i s insect ( 2 7 ) . A glandless variety of cotton was developed in 1 9 5 9 (.28) that c o n t a i n s no gossypol ( 2 9 ) . A l t h o u g h glanded cottons, in general, s h o w h i g h e r r e s i s t a n c e to H e l i o t h i s ( 3 0 ) , recent studies indicate

that a similar level of resistance could be found in certain glandless varieties as well ( 3 1 , 3 2 ) . It is l o g i c a l to assume therefore that if gossypol-free cotton w e r e indeed resistant to H e l i o t h i s attack there may exist yet other resistance factors. S u c c e s s i v e solvent extraction of cotton flower buds (squares) in our laboratory (33) has indicated that while hexane extractables (containing gossypol) were toxic ( T a b l e 1 ) , the subsequent

HOST P L A N T RESISTANCE T O PESTS

118

methanol extract was even m o r e active. A n appreciable level of t o x i c i t y a l s o a p p e a r s i n t h e e x h a u s t i v e l y e x t r a c t e d r e s i d u e . E t h e r p r e c i p i t a t i o n of the m e t h a n o l e x t r a c t , f o l l o w e d b y c h r o m a t o g r a p h i c separation on Sephadex (G-25) gave a p u r i f i e d t o x i c f r a c t i o n , i d e n t i f i e d a s c o n d e n s e d t a n n i n , II.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

CHO OH

OH CHO

Table

1

B i o a s s a y of s u c c e s s i v e s o l v e n t e x t r a c t s f r o m cotton b u d s ( T e x a s -254) w i t h H e l i o t h i s v i r e s c e n s (F).

Extract

L a r v a l weight

3 ,

Hexane Acetone Methanol Water Residue Control

(mg)

48 280 1 260 1 267

Solvent extract f r o m 6 g. synthetic

flower

o f d r i e d f l o w e r b u d a d d e d t o 30

g of

diet.

A v e r a g e w e i g h t o f 10 l a r v a e

after

14 d a y s a t

28 ° C .

W h e n c o t t o n t a n n i n w a s a d d e d a t 0. 1 to 0 . 3% t o a n a d e q u a t e s y n t h e t i c d i e t , the l a r v a l w e i g h t of t o b a c c o b u d w o r m w a s r e d u c e d c o r r e s p o n d i n g l y ( T a b l e II). T h e r a t e of p u p a t i o n , p e r c e n t of l a r v a e p u p a t i n g a n d p e r c e n t of m o t h e m e r g e n c e f r o m p u p a e w e r e r e d u c e d e v e n t h o u g h the l a r v a e w e r e f e d w i t h t a n n i n d u r i n g o n l y p a r t o f t h e g r o w t h p e r i o d (7 d a y s ) . In n a t u r e r e d u c t i o n o f l a r v a l w e i g h t a n d v i g o r u n d o u b t e d l y e x p o s e s the i n s e c t s to f u r t h e r p r é d a t i o n , diseases, and other mortality factors and, thereby, d i m i n i s h e s d a m a g e to the h o s t p l a n t .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

Table

II

T h e r e s i d u a l e f f e c t of c o t t o n t a n n i n a n d t a n n i c ment

on l a r v a l weight,

days

to p u p a t i o n ,

p e r c e n t e m e r g e n c e of H e l i o t h i s v i r e s c e n s

Treatment

Mean larval weight

(%

i n diet)

Cotton 0. 0. 0. 0.

0. 0. 0. 0.

0 1 2 3

after 7 days

treat-

(F)

Days

to

%

reach pupating

3 ,

pupal stage " 3

Emergence"

tannin

0 1 2 3

Tannic

% Larval

in m g

acid

percent pupation and

299 160 84 37

81 69 50 63

14. 9 15. 2 17.4 18. 1

100 91 75 80

290 15 14 15

75 69 38 31

14.9 19. 1 19. 8 19. 8

100 82 83 40

acid

L a r v a e w e r e t r a n s f e r e d to a d a q u a t e s y n t h e t i c d i e t a f t e r d a y s on t a n n i n o r t a n n i c a c i d c o n t a i n i n g d i e t .

7

120

HOST P L A N T RESISTANCE T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

W h i l e t h e e x a c t m o d e of t a n n i n ' s t o x i c a c t i o n t o H . v i r e s c e n s h a s not b e e n e l u c i d a t e d , t h e r e i s l i t t l e d o u b t that the phenolic function is involved. T a n n i c acid, a polygallic acid e s t e r of g l u c o s e h a v i n g o r t h o - p h e n o l i c g r o u p s s i m i l a r to t h o s e of c o t t o n t a n n i n p r o d u c e s s i m i l a r a n t i b i o t i c a c t i v i t y . Methylat i o n of b o t h t a n n i n a n d t a n n i c a c i d w i t h d i a z o m e t h a n e d r a s t i c a l l y r e d u c e s t h e i r t o x i c i t y to t h e i n s e c t ( 2 3 ) . T h e a n t i b i o t i c a c t i o n of c o n d e n s e d t a n n i n f o u n d i n c o t t o n a g a i n s t H . v i r e s c e n s is not s u r p r i s i n g s i n c e tannin i s w e l l r e c o g n i z e d a s a r e s i s t a n c e f a c t o r i n the o a k t r e e a g a i n s t the o a k m o t h , O p e r o p h t e r a b r u m a t a , (17) a n d i n a l f a l f a a g a i n s t t h e alfalfa w e e v i l , H y p e r a postica, ( 34)» In t h e s e i n s t a n c e s t a n n i n w a s s u s p e c t e d to h a v e r e d u c e d t h e a v a i l a b i l i t y o f n u t r i e n t to t h e i n s e c t b y i n t e r a c t i n g w i t h p r o t e i n a n d d e a c t i v a t i o n o f d i g e s t i v e a n d o t h e r e n z y m e s ( 8 , 9, 35). O u r w o r k w o u l d h a v e b e e n m u c h s i m p l i f i e d at this p o i n t if the t a n n i n c o n t e n t s i n c o t t o n f l o w e r b u d s ( s q u a r e s w e r e i n d i r e c t r e l a t i o n to t h e d e g r e e o f s u s c e p t i b i l i t y o r "resistance". In f a c t , f r e e z e d r i e d s q u a r e p o w d e r s f r o m b o t h " r e s i s t a n t " a n d " s u s c e p t i b l e " v a r i e t i e s s h o w e d a p p r e c i a b l e t o x i c i t y at l e s s than 10% e q u i v a l e n t w e i g h t o f f r e s h p l a n t m a t e r i a l i n t h e s y n t h e t i c d i e t (29, 31, 36). P r e l i m i n a r y e x p e r i m e n t s i n o u r l a b o r a t o r y i n d i c a t e that one o r m o r e o f t h e f o l l o w i n g f a c t o r s m a y h e l p to e x p l a i n t h e a p p a r e n t l y h i g h t o x i c i t y i n c o t t o n a n d d i f f e r e n t i a t e the l e v e l of resistance among them: 1. T h e d e g r e e of p o l y m e r i z a t i o n i n t a n n i n a n d t o x i c i t y i n s y n t h e t i c d i e t m a y b e i n c r e a s e d b y e x p o s u r e to a i r o x i d a t i o n , t h e r e b y o v e r s h a d o w i n g the d i f f e r e n c e b e t w e e n " r e s i s t a n t " a n d "susceptible" varieties. 2. T h e p r e s e n t m e t h o d f o r t a n n i n a n a l y s i s (37) i s i n n â c u r a t e a n d a s it d o e s n o t d i f f e r e n t i a t e d i f f e r e n t m o l e c u l a r s i z e s of t a n n i n . 3. P r e s e n t k n o w l e d g e of the f e e d i n g b e h a v i o r of H . virescens is inadequate. It i s s u s p e c t e d , a t t h e p r e s e n t t i m e , that f i r s t i n s t a r l a r v a e f e e d on the y o u n g t e r m i n a l l e a v e s f o r s e v e r a l d a y s b e f o r e m i g r a t i n g to t h e f l o w e r b u d ( 3 8 ) . As y o u n g l a r v a e a r e m o r e s u s c e p t i b l e to t o x i c a n t s ( 3 9 ) , a more c a r e f u l o b s e r v a t i o n of f e e d i n g b e h a v i o r a n d a c c u r a t e a n a l y s e s of g o s s y p o l , t a n n i n , o r o t h e r t o x i c a n t s a n d s y n e r g i s t s a s w e l l a s a n t a g o n i s t s i n the t e r m i n a l l e a v e s s h o u l d be m a d e . Prelimi n a r y e x p e r i m e n t s i n d i c a t e that y o u n g c o t t o n l e a v e s c o n t a i n l i t t l e o r n o g o s s y p o l but s u b s t a n t i a l a m o u n t s of c o n d e n s e d t a n n i n . 4. E s t i m a t i o n s o f t a n n i n c o n t e n t s (40) i n f l o r a l p a r t s s h o w a s m u c h a s 4 - f o l d d i f f e r e n c e b e t w e e n the a n t h e r w h e r e l a r v a e of the s e c o n d o r o l d e r i n s t a r f e e d (38), a n d the total f l o w e r b u d T h i s o b s e r v a t i o n m a y h e l p to e x p l a i n t h e e x c e s s i v e t o x i c i t y o f the t o t a l s q u a r e i n the s y n t h e t i c d i e t a n d the l a c k of c o r r e l a t i o n b e t w e e n g o s s y p o l (35) a n d t a n n i n c o n t e n t w i t h r e s i s t a n c e of cotton v a r i e t i e s .

8.

WAiss E T A L .

Host

Plant Resistance

to Insects

121

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

5. T h e p o s s i b i l i t y of i n d u c e d r e s i s t a n c e c a n n o t be ignored. T h e i n c r e a s e d c o n c e n t r a t i o n of p h e n o l i c s a n d o t h e r phytoalexins in plants after fungal attack has been well d o c u m e n t e d (42, 43). T h e p r o f i l e a n d b i o s y n t h e s i s of p o l y p h e n o l s i n P i n u s r a d i a t a a r e r e p o r t e d to b e a f f e c t e d b y t h e a t t a c k o f t h e w o o d w a s p , S i r e x n o c t i l i o (44). 6. Significant v a r i a n c e in insect feeding behavior on d i f f e r e n t c u l t i v a r s m a y o c c u r d u e to d i f f e r e n c e s i n c h e m i c a l attractants or morphological factors. R e s i s t a n c e o f C o r n to E u r o p e a n c o r n b o r e r , O s t r i n i a n u b i l a l i s ( H ) . - T h e m o s t c e l e b r a t e d c l a s s i c a l e x a m p l e of H P R i n a n e c o n o m i c c r o p i s c o r n ' s c h e m i c a l d e f e n s e a g a i n s t the f i r s t g e n e r a t i o n of c o r n b o r e r . It w a s e s t a b l i s h e d t h a t 2, 4dihydroxy-7-methoxy-l, 4-benzoxazin-3-one (DIMBOA), IV, w a s r e s p o n s i b l e f o r the i n h i b i t o r y e f f e c t o n c o r n b o r e r (45, 46. 4:7). D I M B O A i s the e n z y m a t i c h y d r o l y s i s p r o d u c t of i t s n a t u r a l g l y c o s i d e , III, a n d i s l i b e r a t e d w h i l e t h e i n s e c t f e e d s o n t h e w h o l e t i s s u e of c o r n . D i r e c t c o r r e l a t i o n b e t w e e n the a g e of p l a n t (48), a n d c o n c e n t r a t i o n of 6 - m e t h o x y - 2 - b e n z o x a z o l i n o n e , ( M B O A ) , V , a m o r e s t a b l e d e g r a d a t i o n p r o d u c t of D I M B O A , a n d r e s i s t a n c e to f e e d i n g , h a s b e e n f i r m l y e s t a b l i s h e d (49).

IDE

R = GI

1Z

R= H

-j-

M u c h plant damage by c o r n b o r e r , h o w e v e r , is inflicted by the s e c o n d b r o o d l a r v a e f e e d i n g o n the p o l l e n , s i l k , c o l l a r , a n d sheath. E v i d e n c e i n d i c a t e s that s o m e f a c t o r s , o t h e r than D I M B O A , a r e r e s p o n s i b l e f o r r e s i s t a n c e to 2 n d - g e n e r a t i o n b o r e r s (50, 51). In a d d i t i o n , it h a s b e e n e s t a b l i s h e d that r e s i s t a n c e to 1st a n d 2 n d g e n e r a t i o n s of the E u r o p e a n c o r n b o r e r i n m a i z e i s not c o n f e r r e d b y the s a m e g e n e s . P r e l i m i n a r y e x p e r i m e n t s i n o u r l a b o r a t o r y i n d i c a t e that the c a u s a t i v e agent f o r 2 n d - b r o o d r e s i s t a n c e m a y be m u c h m o r e p o l a r than D I M B O A . R e s i s t a n c e of c o r n t o c o r n e a r w o r m , H e l i o t h i s z e a ( B ) . It i s g e n e r a l l y a c c e p t e d that the a d u l t m o t h s of c o r n e a r w o r m l a y s t h e i r e g g s o n the c o r n s i l k . A s the l a r v a e h a t c h a n d g r o w , they f e e d a n d t r a v e l t o w a r d the h u s k , a n d e v e n t u a l l y i n f l i c t i n g d a m a g e

122

HOST P L A N T RESISTANCE T O PESTS

to t h e k e r n e l s . It w a s o b s e r v e d i n t h e e a r l y p a r t o f t h i s c e n t u r y t h a t t h e r e s e e m e d to b e a p o s i t i v e c o r r e l a t i o n b e t w e e n l o n g , tight h u s k s a n d e a r w o r m r e s i s t a n c e i n c o r n (52), perhaps b e c a u s e of i n c r e a s e d p r o b a b i l i t y of c a n n i b a l i s m (L2), o r p r o l o n g e d e x p o s u r e to l e t h a l toxicants(_53). Chemical antibiosis w a s d e m o n s t r a t e d i n l y o p h i l i z e d c o r n s i l k a n d p l a n t e x t r a c t s (54, 55). Table

III

Bioassay

of s u c c e s s i v e

Zopalote

Chico) with Heliothis Z e a

solvent extracts f r o m c o r n silk (B.)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

Sequential extracts

(var.

a

L a v a l wt

Hexane

515

Acetone Methanol Hot water Residue Control

468 98 65 45 567

(mg)

a Ave. corn

of 10 l a r v a e f e d f o r s i l k / 3 0 g. diet.

11 d a y s w i t h e x t r a c t s f r o m 6 g .

Table The

effect

IV

of m e t h y l a t i o n o n t h e t o x i c i t y

fraction f r o m corn silk (var.

Z.

of e x t r a c t a n d

Methanol Methanol, Water Control

before methylation

Chrom.

on L H - 2 0

purified

C h i c o ) to H e l i o t h i s Z e a ( B .

L a r v a l wt Extract

of

)

a

(mg) after

methylation

88 50

243 562

20 581

579 581

A v e r a g e o f 10 l a r v a e f e d f o r 12 d a y s w i t h e x t r a c t s o r f i e d e x t r a c t s . f r o m 5 g o f l y p h o l i z e d c o r n s i l k / 3 0 g of synthetic diet.

puri-

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

8.

wAiss E T A L .

Host

Plant Resistance

123

to Insects

S u c c e s s i v e solvent: e x t r a c t i o n of s i l k s f r o m s e v e r a l c o r n v a r i e t i e s showed toxic m a t e r i a l s in both methanol and water e x t r a c t s , a s w e l l a s i n the r e s i d u e a f t e r w a t e r e x t r a c t i o n ( T a b l e III). T h e t o x i c f a c t o r i n the m e t h a n o l e x t r a c t w a s p u r i f i e d b y chromatographic separation. T h e t o x i c i t y of b o t h the m e t h a n o l and water f r a c t i o n s was d r a s t i c a l l y reduced by m e t h y l a t i o n with diazomethane (Table IV). P r e s e n t e v i d e n c e i n d i c a t e s that t h e s e toxic compounds a r e p o l y p h e n o l s . H P R to i n s e c t s i n o t h e r a g r i c u l t u r a l c r o p s . - R e s i s t a n c e of S u n f l o w e r s to s u n f l o w e r m o t h , H o m e o s o m a e l e c t e l l u m , ( Η ) , h a s b e e n r e p o r t e d to b e a s s o c i a t e d w i t h t h e i n a b i l i t y o f t h e i n s e c t t o p e n e t r a t e the s e e d c o a t of the r e s i s t a n t v a r i e t i e s (56). Recently two d i t e r p e n e a c i d s , t r a c h y l o b a n - 1 9 - o i c a c i d (VI) a n d ( - - ) - l 6 k a u r e n - 1 9 - o i c a c i d (VII), i s o l a t e d f r o m s u n f l o w e r f l o r e t s w e r e s h o w n to p o s s e s s l a r v a c i d a l a c t i v i t y to s u n f l o w e r m o t h a n d s e v e r a l o t h e r L e p i d o p t e r a s p e c i e s (57, 58). W h i l e the c o n c e n ­ t r a t i o n of t h e s e a c i d s v a r i e d w i t h a g e a n d v a r i e t y of s u n f l o w e r , t h e i r c o n t r i b u t i o n to t h e h o s t d e f e n s e m e c h a n i s m h a s n o t y e t been fully elucidated.

3ΖΓ

3ΠΕ

In a d d i t i o n to t h e p r e v i o u s e x a m p l e s , g e r m p l a s m o f m a n y o t h e r c r o p s w h i c h a r e r e s i s t a n t to i n s e c t s h a s b e e n i d e n t i f i e d ( 19, 20, 59). S o m e of the m o r e p r o m i n e n t e x a m p l e s a r e l i s t e d i n Table V . While morphological or physical characteristics may b e i n v o l v e d i n the h o s t - i n s e c t r e l a t i o n s h i p s i n s o m e o f t h e s e c a s e s , c h e m i c a l a n t i b i o s i s h a s b e e n s u g g e s t e d to h a v e t h e d o m i ­ n a n t r o l e f o r t h e p l a n t s ' r e s i s t a n c e to t h e i r p r e d a t o r s . It t h u s a p p e a r s that t h e r e i s f u n d a m e n t a l n e e d f o r the p a r t i c i p a t i o n of the c h e m i s t i n t h i s i m p o r t a n t a r e a of r e s e a r c h .

124

HOST P L A N T RESISTANCE T O PESTS

Table Some c o m m e r c i a l

crops

that e x i b i t r e s i s t a n c e

Crop

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

Alfalfa B a r l e y and other Corn Cotton Potato Rice Soy Wheat

V to i n s e c t

attack.

Insect

cereal

grains

Alfalfa aphids and weavil Greenbugs Western rootworm L y g u s bug Potato beetle and leafhopper R i c e borer and hoppers M e x i c a n bean beetle and potworm H e s s i a n fly and greenbug

Concluding Remarks R e l a t i v e l y l i t t l e e f f o r t h a s b e e n i n v e s t e d i n e s t a b l i s h i n g the u n d e r s t a n d i n g o f c r o p p l a n t s ' r e s i s t a n c e to i n s e c t s a t t h e chemical and cellular l e v e l . T h e f e e d i n g b e h a v i o r of i n s e c t s a n d o t h e r h o s t - p r e d a t o r i n t e r a c t i o n s i n m o s t of the e c o n o m i c a l l y i m p o r t a n t c r o p s n e e d to b e t h o r o u g h l y e s t a b l i s h e d . T h e k n o w l e d g e of H P R a t t h e c h e m i c a l l e v e l i s i m p e r a t i v e n o t o n l y i n p r o m o t i n g m o r e r a p i d a n d e f f e c t u a l d e v e l o p m e n t of i n s e c t r e s i s t a n t c u l t i v a r s b u t , a l s o , to m o n i t o r t h e p r e s e n c e a n d d i s t r i b u t i o n of t h e s e c h e m i c a l s . T o m o s t of u s c h e m i s t s , t h e i s o l a t i o n o f a n i n s e c t g r o w t h i n h i b i t o r , antifeedant, o r other toxicant f r o m a plant is not un s u r m o u n t a b l e , p r o v i d i n g a convenient and r e l i a b l e b i o a s s a y is a v a i l a b l e to m o n i t o r t h e p u r i f i c a t i o n p r o c e s s . T o e l u c i d a t e the d e l i c a t e b a l a n c e b e t w e e n a n i n s e c t a n d i t s h o s t i n the f i e l d i s a m u c h m o r e f o r m i d a b l e task. F r o m our experience in studying the n a t u r e of H P R , w e f e e l that the f o l l o w i n g s u g g e s t i o n s m a y be w o r t h c o n s i d e r i n g b y s c i e n t i s t s i n t e r e s t e d i n t h i s a r e a of research. 1. S i n c e the c h e m i c a l b a s i s f o r H P R i n v o l v e s the c h e m i c a l i n t e r a c t i o n s b e t w e e n two b i o l o g i c a l s y s t e m s , the h o s t p l a n t a n d its p r e d a t o r , a c l o s e l y c o o r d i n a t e d m u l t i d i s c i p l i n a r y a p p r o a c h to t h e i n v e s t i g a t i o n c a n n o t b e o v e r - s t r e s s e d . It i s i m p e r a t i v e that the c h e m i s t d e v e l o p s a n a p p r e c i a t i o n f o r the a l m o s t i n f i n i t e v a r i a b i l i t y a n d c o n s t a n t c h a n g e i n the p l a n t ' s c h e m i c a l c o m p o sition d u r i n g its growth, d e v e l o p m e n t , and s t r e s s , as w e l l as t h e i n s e c t ' s a b i l i t y to d e v e l o p , e v o l v e , a n d c o e x i s t w i t h i t s h o s t , g u i d e d a n d s t i m u l a t e d b y the p l a n t ' s u n i q u e c o m b i n a t i o n of c h e m i c a l s f o r f o o d , s h e l t e r , a n d r e p r o d u c t i o n . A biological s y s t e m is a d y n a m i c one, and in each i n s e c t - h o s t plant i n t e r action there exists a special biological relationship.

8.

WAISS E T A L .

Host

Plant Resistance

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

Thus, solely chemical approaches inadequate.

125

to Insects to t h e s e i n v e s t i g a t i o n s

are

2. T h e e s t a b l i s h m e n t of a n a p p r o p r i a t e , c o n v e n i e n t a n d r e p r o ­ d u c i b l e b i o a s s a y p r o c e d u r e i s a p r e r e q u i s i t e to t h e s u c c e s s o f H P R research. A l t h o u g h , b y w a y of e x a m p l e , the b l a c k a r m y w o r m has been used extensively in bioassays for feeding deter­ r e n t s a n d o t h e r a l l e l o c h e m i c s , the a p p r o p r i a t e i n s e c t s h o u l d b e u s e d i n the s t u d y of p l a n t - i n s e c t s i n t e r a c t i o n . While gossypol i n c o t t o n , f o r e x a m p l e , i s t o x i c to s e v e r a l l e p i d o p t e r o u s i n s e c t s , it i s a f e e d i n g s t i m u l a n t f o r t h e m o n o p h a g o u s c o t t o n b o l l w e e v i l A n t h o n o m u s g r a n d i s Β . (60) A c o n v e n i e n t p r o c e d u r e to i n c o r p o ­ r a t e p l a n t e x t r a c t s a n d p u r i f i e d f r a c t i o n s i n t o s m a l l q u a n t i t i e s of d i e t i s y e t to b e f o u n d , a l t h o u g h s e v e r a l s y n t h e t i c d i e t s f o r Lepidoptera have been reported. In a d d i t i o n , t h e r e i s s t i l l n e e d for i m p r o v e m e n t in bioassay p r o c e d u r e s for other arthropods such as C o l e o p t e r a (beetle), D i p t e r a (flies and mosquitos), H e t e r o p t e r a (true bugs) and A r a c h n i d s ( s p i d e r s ) . 3. T h e d e v e l o p m e n t of g c - m a s s s p e c t r o m e t r y h a s g r e a t l y f a c i l i t a t e d the p u r i f i c a t i o n a n d i d e n t i f i c a t i o n of v o l a t i l e o r g a n i c c o m p o u n d s i n the l a s t d e c a d e . A t present, i m p r o v e d techniques a r e n e e d e d f o r s e p a r a t i o n a n d p u r i f i c a t i o n of h i g h e r m o l e c u l a r weight polar compounds. T e c h n i q u e s of p r o t e i n , a n d o t h e r h i g h p o l y m e r c h e m i s t r y m a y be m o d i f i e d a n d e m p l o y e d h e r e . 4. F r o m o u r e x p e r i e n c e w i t h c o t t o n ' s r e s i s t a n c e to b o l l w o r m s ( H e l i o t h i s s p p . ) w e f e e l that u n l e s s the a n t i b i o t i c f a c t o r ( g o s s y ­ p o l i n t h i s c a s e ) i s l o c a t e d i n the p l a n t p a r t i n w h i c h i n s e c t feeding actually o c c u r s , and is present in an effective concentra­ t i o n at the t i m e of i n s e c t d a m a g e , the p r e s e n c e of a t o x i c a n t i n "resistant" varieties (glanded lines) and absence in " s u s c e p t i b l e " c u l t i v a r s ( g l a n d l e s s l i n e s ) m a y not be s u f f i c i e n t p r o o f f o r the toxicants role in H P R . 5. It m a y b e u s e f u l t o c o n s i d e r , i n H P R r e s e a r c h , t h a t o n a s c a l e o f 0 to 10 ( F i g u r e 1), w h e r e 10 r e p r e s e n t s a p l a n t t o t a l l y i m m u n e to c e r t a i n s p e c i f i c i n s e c t a t t a c k a n d " 0 " r e p r e s e n t s a plant w h i c h supports m a x i m u m insect growth, d e v e l o p m e n t , and reproduction, resistance and susceptibility are relative phenom­ e n a r a n g i n g b e t w e e n the t w o e x t r e m e s .

"Susceptible"

<

Total

susceptibility Figure 1.

"Resistant"

>

Immunity

Relative scale of insect growth and plant*s response

126

HOST P L A N T RESISTANCE T O PESTS

B y d e f i n i t i o n , a p l a n t i s n o l o n g e r a h o s t to a n i n s e c t w h e n it i s t o t a l l y i m m u n e to i t s e n e m y ' s a t t a c k . O n e s h o u l d not e x p e c t , t h e r e f o r e , i n H P R r e s e a r c h to f i n d a t o x i c a n t o n l y i n t h e " r e s i s t a n t " v a r i e t i e s (a q u a l i t a t i v e p h e n o m e n o n ) b u t , r a t h e r , m o r e often a v a r y i n g concentration (quantitative difference) of a n t i b i o t i c f a c t o r i n " r e s i s t a n t " a n d " s u s c e p t i b l e " l i n e s , com­ plicated further by both insect b e h a v i o r and plant d e v e l o p m e n t . Literature

1.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Cited

Smith, Ε. H. "Pest Control Strategies for the Future". National Academy of Sciences, Washington, D. C. 1972. Kennedy, D., chairman, "Pest Control: An Assessment of Present and Alternative Technology" 5 vol., National Academy of Sciences, Washington, D. C. 1975. Klassen, W. in "Insect, Science and Society" (D. Pimentel, ed.) Academic Press, New York, 1975. Glass, Ε. H. "Integrated Pest Management: Rational Potential, Needs and Implementation" Entomological Soc. Amer. Baltimore, MD, 1975. Beck, S. D., Ann. Entomol. Soc. Amer. (1965) 53 207 Van Emden, H. F. and May, M. J. in "Insect-Plant Relationship" H. F. Van Emden, ed. ) Wiley, 1973. Kogan, M. In "Introduction to Insect Pest Management (W. H. Luckmann and R. H. Metcaff, ed. ) Wiley, New York 1975. Feeny, P. in "Recent Adv. in Phytochem. - Biochemical Interaction Between Plants and Insects" (J. W. Wallace and R. L. Mansell, ed.), Plenum, New York, 1976. Rhodes, D. F. and Cates, R. G., ibid. Balachowsky, A. S. "La Lutte Contre les Insects Payot", Paris, 1951. Painter, R. Η., J. Econ, Entomol. (1941) 34:358-67. Painter, R. H. "Insect Resistance in Crop Plants, MacMillan, New York, 1951. Schweltz, I. in "Naturally Ocurring Insecticides" (M. Jacobson and D. G. Crosby, ed. ) M. Dekker, Inc. 1971. Matsui, M. and Yamamoto, I. In ibid. Fukami, H. and Nakajima, M. In ibid. Jacobson, M. "Insecticides from Plants - A Review of the Literature 1954-1971". Agric. Handbook No. 461. Feeny, P. O. J. Insect. Physiol. (1968) 14, 805-17. Lawton, J. H. in "The Biology of Bracken" (F. H. Perring ed.) Academic Press, New YOrk 1976. Gallun, R. L., J. Environ. Quality (1972) 1 259-65 Schalk, J. M. and Ratcliffe, R. H. Bull. Ent. Soc. Am. (1976) 3. 7-10. Quaintance, L. L. and Brues, C. T. USDA Bur. Entom. Bull. (1905) 50:150.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

8.

wAiss E T A L .

Host Vlant Resistance

to Insects

127

22. Cook, O. F. USDA Tech Bull. (1906) 88. 23. Adams, R., Geissman, T. A. and Edwards, J. D. Chem. Rev. (1960) 60, 555-74. 24. Bottger, G. T., Sheehan, Ε. X. and Lukefahr, M. J., J. Εcon. Entomol. (1964) 57, 283-5. 25. Lukefahr, M. J. and Mattin, D. F. Ibid (1969) 59, 176-9. 26. Lukefahr, M. J. and Houghtaling, J. E. ibid. (1969). 27. Lukefahr, M. J., Houghtaling, J. E. and Cruhm, D. G. ibid. (1975) 68, 743-6. 28. McMichael, S. C. Agron. J. ( 1959) 51 630. 29. Waiss, A. C., Jr., Chan, B. G., Benson, M. and Lukefahr, M. J. J. Ass. Off. Anal. Chem., in press. 30. Lukefahr, M. J., Noble, L. W. and Houghtaling, J. E. J. Econ. Entomol. (1966) 59, 817-20. 31. Shaver, Τ. Ν., Garcaa, J. A., and Dilday, R. Η., Environ. Entomol. (1977) 6, 82-4. 32. Malm, N. R., et al., Am. Rep., Agric. Exp. Station, Las Cruces, N. Mexico (1976). 33. Chan, B. G., Waiss, A. C., Jr., and Lukefahr, M. J. Insect. Physol., in press. 34. Bennett, S. E. J. Econ. Entomol. (1966) 58, 372-3. 35. Pridham, J. Β., "Enzyme Chemistry of Phenolic Compounds". MacMillan. New York. 1963. 36. Shaver, T. N. and Lukfahr, M. J. J. Econ. Entomol. (1971) 64, 1274-7. 37. Swain, T. and Hillis, W. E. J. Sci. Food Agri. (1959) 10, 63-68. 38. Private communication from Drs. M. J. Lukefahr and W. L. Parrott. 39. Shaver, T. H. and Parrott, W. L. J. Econ. Entomol. (1970) 63, 1802-4. 40. Data to be published. 41. Lukefahr, M. J., Shaver, Τ. Ν., Cruhm, D. E. and Houghtaling, J. E. Beltwide Cotton Prod. Res. Conf. Proced. 1974. 42. Levin, D. A. Amer. Naturalist (1971) 105, 157-81. 43. Ingham, J. L. Bot. Rev. (1972) 38 343-417. 44. Hillis, W. E. and Inone, T. Phytochem. (1968) 7, 13-22. 45. Beck, S. D. J. Insect Physiol. (1957) 1, 158-77. 46. Beck, S. D. and Stauffer, J. F. Ann. Entomol. Soc. Amer. (1957) 50, 166-70. 47. Klun, J. A., Tipton, C. L. and Brindley, T. A. J. Econ. Entomol. (1967) 60, 1529-33. 48. Beck, S. D., Ann. Rev. Entom. (1965) 10, 207-32. 49. Klun, J. A., and Brindley, T. A. J. Econ. Entomol. (1966) 59, 711-8. 50. Sulivan, S. L., Gracen, V. Ε. and Ortega, A. Environ. Entomol. (1974)3,718-20. 51. Russell, W. Α., Guthrie, W. D., Klun, J. A. and Grindeland, R. J. Econ. Entomol. (1975) 68, 31-34.

128

52. 53. 54.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch008

55.

HOST P L A N T RESISTANCE T O PESTS

Colling, G. N. and Kempton, H. H. J. Agr. Res. (1917) 11, 549-72. Guthrie, W. D. and Walter, Ε. V. J. Econ. Entomol. (1961) 54, 1248-50. McMillan, W. W., Wiseman, B. R. and Sekul, A. A. Am. Entomol. Soc. Amer, (1970) 63, 371-78 Straub, R. W. and Fairchield, M. L. J. Econ. Entomol. (1970) 63 1901-3.

9 The Effects of Plant Biochemicals on Insect Growth and Nutritional Physiology J O H N C.

REESE

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

Department of Entomology, 237 Madison, W I 53706

Russell Labs, University of Wisconsin,

In terms of their effects on insects, plant biochemicals may be divided into nutrients and non-nutrients. Nutrients have received a great deal of attention over the years. They were even thought to be the basis for host plant specificity at one time. Research in recent decades has shown, however, that most species of insects do not differ greatly in their qualitative requirements for nutrients. Thus, although the host plant obviously has to satisfy the nutritional requirements of the insect, it does not seem likely that the insects' nutritional requirements play more than a minor role in host plant specificity (1). Non-nutrients, or allelochemics (non-nutritional chemicals produced by an organism of one species, and which affect the growth, health, behavior, or population biology of another species (2)), can be extremely important factors in host plant resistance. I will concentrate my discussion of the effects of these allelochemics on insect growth and nutritional physiology, how these non-nutrients may be interacting with nutrients, and on recent work I have done in Dr. Beck's laboratory at the University of Wisconsin on the effects of various allelochemics and dietary moisture levels on insect nutritional physiology. Current Areas of Active Research. Plant Apparency. Recent investigations have indicated that evolutionary strategies in plant defense mechnisms may be based on host plant specificity as well as on the population densities and successional status of the plant species (3-5). Within a particular ecosystem, some species of plants will be predictable or apparent in both time and space (i.g. an oak tree). These plants can be easily found by herbivores. Other species are less predictable (and thus less apparent) and so are less likely to be found by herbivores. The predictable species are probably subjected to greater feeding pressure by herbivores and tend to contain high concentration of dosage-dependent (quantitative) inhibitors of digestion and assimilation. These substances are 129

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

130

HOST P L A N T RESISTANCE T O

PESTS

u s u a l l y not h i g h l y t o x i c , but slow the growth of i n s e c t s . The l e s s apparent species tend t o contain lower concentrations of more h i g h l y t o x i c ( q u a l i t a t i v e ) a l l e l o c h e m i c s . Insects tend to be more l i k e l y to evolve d e t o x i f y i n g mechanisms f o r these substances, and so to become adapted or s p e c i a l i z e d f o r feeding on c e r t a i n s p e c i e s . Such p l a n t species r e l y mainly on escape as a defense a g a i n s t the adapted species of i n s e c t s . The t o x i n s and d e t e r r e n t s i n these p l a n t s are most e f f e c t i v e a g a i n s t the nonadapted g e n e r a l i z e d feeders. Unfortunately, our a g r i c u l t u r a l p r a c t i c e s have taken many p l a n t s t h a t probably evolved under the unapparent category (and so contain the chemical defenses charact e r i s t i c o f t h i s group) and have made these p l a n t s h i g h l y p r e d i c t a b l e and t h e r e f o r e apparent by p l a n t i n g them i n huge f i e l d s and by p l a n t i n g the same species year a f t e r year on the same ground. Perhaps a p r o d u c t i v e d i r e c t i o n f o r host p l a n t r e s i s t a n c e research would be to attempt to i n c r e a s e concentrations o f dosage-dependent f a c t o r s c h a r a c t e r i s t i c of apparent p l a n t s i n agronomically accept a b l e v a r i e t i e s o f our crop p l a n t s . Metabolic E f f e c t s . Such hypotheses as the p l a n t apparency hypothesis are examples of a general s h i f t i n emphasis from b e h a v i o r a l e f f e c t s toward metabolic e f f e c t s . For a number of years the token s t i m u l i (6) theory o f host s e l e c t i o n formed the b a s i s o f a g r e a t d e a l o f research. A rough i d e a o f the research emphasis i n p a s t years can be gained by comparing the approximately 400 references c i t e d by Hedin e t a l Ç 7 ) i n t h e i r compil a t i o n o f b e h a v i o r a l chemicals, to the approximately 100 r e f e rences c i t e d by Beck and Reese (1) i n t h e i r compilation of chemicals having metabolic e f f e c t s . Despite t h i s approximately four-to-one r a t i o o f work on b e h a v i o r a l aspects to work on metabolic e f f e c t s , there are r e a l l y very few good examples o f a r e s i s t a n c e v a r i e t y u t i l i z i n g a b e h a v i o r a l chemical f o r i t s r e s i s tance mechanism (8). Chronic E f f e c t s Hypothesis. For s e v e r a l years I have been t e s t i n g the hypothesis that some a l l e l o c h e m i c s may have, i n a d d i t i o n to immediate e f f e c t s on s u r v i v a l and feeding behavior, s u b t l e c h r o n i c e f f e c t s , even at low concentrations, on r a t e of growth, u t i l i z a t i o n of food and pupation (1, 9-12). In other words, I have been i n t e r e s t e d i n the things Feeny would c a l l "dosage-dependent" f a c t o r s . The i n h i b i t i o n o f growth may be due t o an i n h i b i t i o n o f i n g e s t i o n , a s s i m i l a t i o n , or e f f i c i e n c y of conversion o f a s s i m i l a t e d o r ingested food. The more b i o l o g i c a l l y a c t i v e compounds r e l i e d on i n h i b i t i o n of v a r i o u s combinat i o n s o f the above processes f o r t h e i r e f f e c t s . The r e s u l t s o f these experiments w i l l be d i s c u s s e d i n more d e t a i l i n a l a t e r section. Insect D i e t e t i c s . Over the years, each o f the major ideas concerning host p l a n t s p e c i f i c i t y ( n u t r i t i o n , token s t i m u l i , and metabolic e f f e c t s ) have played r o l e s i n broadening our understanding of the biochemical b a s i s (or b e t t e r , bases) of p l a n t -

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and

Insect

Growth

131

insect interactions. In general, each idea was considered more or l e s s separate from each of the others, and c e r t a i n l y most research p r o j e c t s have been l a r g e l y based on only one of these ideas. We are now s t a r t i n g to recognize that any given p l a n t i n s e c t i n t e r a c t i o n must s u r e l y depend upon a l l of these f a c t o r s (If 13, 14, Γ7). Under the concept of i n s e c t d i e t e t i c s (15, 16) i t i s recognized that the feeding i n s e c t must i n g e s t food "that not only meets i t s n u t r i t i o n a l requirements, but t h a t i s a l s o capable of being a s s i m i l a t e d and converted i n t o the energy and s t r u c t u r a l substances required f o r normal a c t i v i t y and develop­ ment" (1). Therefore, i n s e c t d i e t e t i c s encompasses i n s e c t n u t r i ­ t i o n i n the c l a s s i c a l sense, any a l l e l o c h e m i c e f f e c t s on feeding behavior, the e f f e c t s of any a l l e l o c h e m i c s on s u r v i v a l , and the e f f e c t s of any compounds which have chronic a f f e c t s on growth, development, or reproduction. A l l of these aspects are c r u c i a l to a given p l a n t - i n s e c t i n t e r a c t i o n . A d e l e t e r i o u s e f f e c t i n any one o f these areas could cause a p l a n t to become more r e s i s t a n t to the attack of i n s e c t s . These d i f f e r e n t areas of i n s e c t d i e t e ­ t i c s undoubtedly i n t e r a c t with each other i n ways unknown to us a t present. Some of these i n t e r a c t i o n s may prove to be very important i n host p l a n t r e s i s t a n c e research of future years. In a d d i t i o n t o p h y s i o l o g i c a l i n t e r a c t i o n s between d i f f e r e n t aspects of i n s e c t d i e t e t i c s , there may be some important b i o l o g i ­ c a l i n t e r a c t i o n s i n r e l a t i o n to the permanence of a r e s i s t a n t variety. There are some outstanding examples of r e s i s t a n t v a r i e ­ t i e s r e t a i n i n g t h e i r r e s i s t a n c e over long periods of time. Such e x c e p t i o n a l l y s t a b l e r e s i s t a n t v a r i e t i e s must s u r e l y have a number of r e s i s t a n c e mechanisms such that the l i k e l i h o o d of a r e s i s t a n t i n s e c t biotype a r i s i n g i s lessened. A r e s i s t a n t c u l t i ­ var probably has a greater p r o b a b i l i t y of r e l a t i v e permanence, i f i t contains a l l e l o c h e m i c s which a f f e c t both behavior and metabolism. Thus b e h a v i o r a l errancy i s punished by metabolic e f f e c t s (17, 18). Such e f f e c t s may come from the same or d i f f e r e n t compounds. For an i n s e c t to counteradapt to such a s i t u a t i o n would r e q u i r e at l e a s t two mutations, one b e h a v i o r a l and one metabolic. I n t e r a c t i o n s between A l l e l o c h e m i c s and N u t r i e n t s . Allelo­ chemics may i n t e r a c t with e s s e n t i a l n u t r i e n t s of i n s e c t food. Indeed, many of the d e l e t e r i o u s metabolic or chronic e f f e c t s of p l a n t a l l e l o c h e m i s may be due p r i m a r i l y to v a r i o u s i n t e r a c t i o n s between a l l e l o c h e m i c s and n u t r i e n t s . Except f o r a few examples that w i l l be discussed l a t e r , l i t t l e work has been done on the i n t e r a c t i o n s between a l l e l o c h e ­ mics and n u t r i e n t s i n i n s e c t s and the p o s s i b l e u t i l i z a t i o n of such knowledge i n host p l a n t r e s i s t a n c e . More i n v e s t i g a t i o n s have been conducted on v e r t e b r a t e s . A b r i e f review of some of t h i s work may prove to be u s e f u l i n g i v i n g us clues as to how c e r t a i n a l l e l o c h e m i c s i n h i b i t i n s e c t growth. H a t f i e l d (21) has reviewed s e v e r a l examples from the v e r t e ­ brate l i t e r a t u r e i n which there seemed to be i n t e r a c t i o n s between

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

132

HOST P L A N T RESISTANCE T O

PESTS

a l l e l o c h e m i c s and n u t r i e n t s . Chestnut tannins have been found to reduce the a v a i l a b i l i t y of l y s i n e i n various d i e t s . I n t e r a c t i o n s between gossypol, cyclopropene f a t t y a c i d s , and p r o t e i n have been reported. A l l e l o c h e m i c s may serve as antioxidants and thus p r o t e c t c e r t a i n n u t r i e n t s from o x i d a t i o n (22), so i n t e r a c t i o n s are not always negative. Various t o x i c f a c t o r s , e s p e c i a l l y enzyme i n h i b i t o r s , from soybeans have been known f o r many years (23). Some of these f a c t o r s render c e r t a i n elements such as nitrogen or s u l f u r unavailable. Since s e v e r a l of these f a c t o r s are heat l a b i l e , and so i n many cases are r e a l l y not much of a problem i n human n u t r i ­ t i o n (24), such f a c t o r s might prove e s p e c i a l l y u s e f u l i n r e s i s ­ tance programs. U n l i k e many p o t e n t i a l r e s i s t a n c e f a c t o r s , t h e i r d e l e t e r i o u s e f f e c t s might be f e l t p r i m a r i l y by the i n s e c t p e s t s , r a t h e r than by the human consumer. This i s an area that c e r t a i n ­ l y warrants f u r t h e r research. Phytate i s a compound which can be found i n r e l a t i v e l y high amounts i n cottonseed meal (23). I t may form complexes with such minerals as z i n c , thus rendering them unavailable to monogastric animals. C e r t a i n p r o t e i n s may a l s o be complexed by phytate. Experiments i n which cottonseed meal was t r e a t e d with the enzyme phytase suggest t h a t there are i n t e r a c t i o n s between phytate and such things as phosphorous and gossypol. The i n t e r a c t i o n s between dicoumarol i n sweet c l o v e r hay and vitamin Κ have been known f o r a number of years. Supplementing the d i e t with vitamin Κ can lessen the hemorrhagic e f f e c t s of the dicoumarol (23). From the preceding d i s c u s s i o n of i n t e r a c t i o n s between a l l e l o ­ chemics and n u t r i e n t s i n v e r t e b r a t e s , i t i s apparent that ento­ mologists may p r o f i t from t e s t i n g some o f the same types o f hypo­ theses t h a t have been t e s t e d with v e r t e b r a t e s . C e r t a i n l y , some o f the d e l e t e r i o u s e f f e c t s of p l a n t a l l e l o c h e m i c s may be due,to i n t e r a c t i o n s between the a l l e l o c h e m i c and some n u t r i e n t . Several such i n t e r a c t i o n s have already been demonstrated. For example, c e r t a i n a l l e l o c h e m i c s resemble c e r t a i n n u t r i e n t s so c l o s e l y t h a t they may compete m e t a b o l i c a l l y . L-Canavanie i s q u i t e s i m i l a r to L-arginine. I t s t o x i c i t y (25-29) may be due to the formation of d e f e c t i v e canavanyl p r o t e i n s . L-Canavanine and various other amino a c i d analogues may a l s o a c t as i n h i b i t o r s o f i n s e c t repro­ d u c t i o n (30), p o s s i b l y through t h i s same mechanism. C e r t a i n a l l e l o c h e m i s may i n t e r f e r e with n u t r i e n t s by b l o c k i n g their availability. This seems to be the case f o r oak l e a f tan­ n i n s . The tannins apparently form a complex with p r o t e i n s such t h a t the p r o t e i n s are l e s s a v a i l a b l e to winter moth (Operophthera brumata) l a r v a e (31, 32). S i m i l a r l y , the " d i g e s t i b i l i t y - r e d u c i n g " f a c t o r s i n creosote r e s i n s seem to somehow block d i g e s t i b i l i t y (5). Recently, i n t e r a c t i o n s between c e r t a i n diterpene acids and c h o l e s t e r o l have been demonstrated (20). The p a r t i a l r e v e r s a l of growth i n h i b i t i o n i n the presence of r e l a t i v e l y l a r g e amounts o f c h o l e s t e r o l suggested to these workers that these diterpene a c i d s (e.g. levopimaric acid) a f f e c t the i n s e c t s ' hormonal system.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and

Insect

Growth

133

As i n the vertebrate l i t e r a t u r e discussed above, protease i n h i b i t o r s have been shown to have d e l e t e r i o u s e f f e c t s on various i n s e c t s . Most have d e a l t with t r y p s i n i n h i b i t o r s . Ryan (33) and Ryan and Green (34), have reviewed t h i s subject i n d e t a i l . B i r k and Applebaum (35) s t u d i e d the e f f e c t s of soybean t r y p s i n i n h i b i t o r s on development and p r o t e i o l y t i c a c t i v i t y of T r i b o l i u m castaneum. Su e t a l (36) found that r e l a t i v e l y high doses of soybean t r y p s i n i n h i b i t o r caused i n c r e a s e d a d u l t m o r t a l i t y i n S i t o p h i l u s oryzae. A p a r t i c u l a r l y e x c i t i n g aspect of t h i s area o f research i s the p o s s i b i l i t y that the p l a n t may be able t o produce much higher l e v e l s of enzyme i n h i b i t o r s a f t e r being attacked. T h i s feature gives the p l a n t an adaptive advantage (and an agronomic advantage) i n that i t does not expend energy f o r the synthesis o f such m a t e r i a l s u n t i l they are a c t u a l l y needed f o r defense. Green and Ryan (37.) made the e x c i t i n g d i s c o v e r y t h a t the wounding of the leaves of potato o r tomato p l a n t s by a d u l t Colorado potato b e e t l e s induces a r a p i d accumulat i o n o f protease i n h i b i t o r . Further, t h i s response was not confined t o immediate area o f the attack, but spread to other p a r t s o f the p l a n t . Recently, some work has been done on the e f f e c t s of s p e c i f i c a l l e l o c h e m i c s on a s s i m i l a t i o n of food (passage across the gut w a l l ) , e f f i c i e n c y of conversion of a s s i m i l a t e d food i n t o i n s e c t t i s s u e , and e f f i c i e n c y o f conversion of ingested food. A reduct i o n i n n u t r i t i o n a l i n d i c e s , such as those mentioned above, r e s u l t i n g from the i n g e s t i o n o f d e l e t e r i o u s a l l e l o c h e m i c s may be due to a number o f f a c t o r s , most of which r e l a t e i n one way or another to i n t e r a c t i o n s between a l l e l o c h e m i c s and n u t r i e n t s . For example, some d e l e t e r i o u s a l l e l o c h e m i c s may b i n d to a s p e c i f i c n u t r i e n t . They may a l s o bind to and i n a c t i v a t e d i g e s t i v e enzymes o r membrane c a r r i e r p r o t e i n s . A l l e l o c h e m i c s with hydroxyl groups on adjacent carbon atoms may chelate c e r t a i n e s s e n t i a l minerals. The l i t e r a t u r e d e a l i n g with the e f f e c t s o f s p e c i f i c a l l e l o chemics on these n u t r i t i o n a l i n d i c e s reamins sparse, but i s i n c r e a s i n g . Shaver e t a l (19) found t h a t gossypol decreased a s s i m i l a t i o n by bollworm l a r v a e , H e l i o t h i s zea, but had no measurable e f f e c t on u t i l i z a t i o n by tobacco budworm l a r v a e , H_. v i r e s c e n s . E r i c k s o n and Feeny (18) t e s t e d the hypothesis t h a t the l a r v a l feeding niche of P a p i l i o polyxenes a s t e r i u s i s p a r t i a l l y bounded by a l l e l o c h e m i c s which are not r e q u i r e d f o r p e r c e p t i o n o f host or non-host p l a n t s . They demonstrated t h a t s i n i g r i n , or one o f i t s breakdown products, reduced a s s i m i l a t i o n but d i d not reduce the e f f i c i e n c y with which a s s i m i l a t e d food was converted i n t o t i s s u e . Dr. Beck and I have examined the e f f e c t s of a number of p l a n t a l l e l o c h e m i c s on growth and development o f black cutworm (Agrotis i p s i l o n ) l a r v a e (1, 9-12). We have shown that growth i n h i b i t i o n may be due to i n h i b i t i o n of e i t h e r a s s i m i l a t i o n , or e f f i c i e n c y of conversion of a s s i m i l a t e d food, or a combination of both. I n h i b i t i o n of e i t h e r o f these processes w i l l , o f course, i n h i b i t the e f f i c i e n c y of conversion of ingested food. The s p e c i f i c r e s u l t s o f these experiments w i l l be summarized i n a l a t e r s e c t i o n .

134

HOST P L A N T RESISTANCE T O

PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

P o t e n t i a l of N u t r i t i o n a l Index Techniques As discussed i n the previous s e c t i o n , n u t r i t i o n a l index techniques have been used i n f r e q u e n t l y to demonstrate p o s s i b l e n u t r i e n t - a l l e l o c h e m i c i n t e r a c t i o n s or mechanisms by which d e l e ­ t e r i o u s p l a n t compounds may i n h i b i t herbivore growth. P l a n t feeding i n s e c t s c e r t a i n l y do not grow e q u a l l y w e l l on a l l p l a n t s o r p l a n t t i s s u e s , even when there are no apparent b e h a v i o r a l b a r r i e r s to t h e i r feeding. The d i f f e r e n c e s are much greater than can be explained i n terms o f p o s s i b l e d i f f e r e n c e s i n n u t r i e n t content as such o f the d i f f e r e n t p l a n t t i s s u e s . I t seems l i k e l y , t h e r e f o r e , that a l l e l o c h e m i c s c h a r a c t e r i s t i c s of various p l a n t species exert strong i n f l u e n c e s on the growth and development of insects. The n u t r i t i o n a l i n d i c e s I found most u s e f u l are a s s i m i l a t i o n (AD), e f f i c i e n c y o f conversion of a s s i m i l a t e d food (ECD), and e f f i c i e n c y o f conversion o f ingested food (ECI). _ amount ingested (mg) - feces amount ingested (mg) ECD

=

ECI =

— weight gain (mg) amount ingested (mg) - feces

(mg)

χ

1 Q Q

χ

1 Q Q

χ

1 Q Q

(mg)

weight gain (mg) amount ingested (mg)

AD (approximate d i g e s t i b i l i t y ) measures the a s s i m i l a t i o n of food and i s termed "approximate" because i t does not s u b t r a c t the weight o f waste products i n the feces or such metabolic products as the p e r i t r o p h i c membrane (38) and exuviae from the t o t a l weight o f the feces. ECD measures the e f f i c i e n c y with which a s s i m i l a t e d food i s converted i n t o i n s e c t t i s s u e . This index w i l l decrease as the p r o p o r t i o n of a s s i m i l a t e d food metabolized f o r energy increases (38). ECI measures the o v e r a l l a b i l i t y of the i n s e c t to convert ingested food i n t o t i s s u e (38). N u t r i t i o n a l i n d i c e s f o r i n s e c t s on host and non-host p l a n t s vary a great d e a l . Using the southern armyworm, Prodenia e r i d a n i a , Soo Hoo and Fraenkel (39, 40) compared the feeding behavior, growth, s u r v i v a l , and n u t r i t i o n a l i n d i c e s o f the i n s e c t on p l a n t t i s s u e s r e p r e s e n t i n g 32 f a m i l i e s . Most of the p l a n t s t e s t e d were fed upon, but host s u i t a b i l i t y was q u i t e v a r i e d , ranging from normal r a p i d larvae growth to complete m o r t a l i t y . N u t r i t i o n a l i n d i c e s were determined f o r l a r v e feeding on f r e s h p l a n t t i s s u e s representing 12 d i f f e r e n t p l a n t f a m i l i e s . AD ranged from 36% on a "poor" host t o 73% on a "good" host. ECD values ranged from 16% to 57%; ECI values ranged from 10% to 38%. Waldbauer (41) determined n u t r i t i o n a l i n d i c e s o f the tobacco homworm, Manduca s ex t a , l a r v a e on both host p l a n t s and on non-

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and

Insect

Growth

135

host p l a n t s . In the l a t t e r case, the larvae were induced to feed on non-host p l a n t t i s s u e s by removing the sense organs that enable the i n s e c t to d i s t i n g u i s h host p l a n t s from non-host p l a n t s . The use o f n u t r i t i o n a l i n d i c e s as a b a s i s f o r comparing host p l a n t u t i l i z a t i o n by a phytophagous i n s e c t being reared on d i f f e r e n t host p l a n t s has some l i m i t a t i o n s (38). V a r i a t i o n s i n water and f i b e r contents may r e s u l t i n v a r i a t i o n s i n the i n d i c e s (39-42) (see l a t e r s e c t i o n o f t h i s paper). A r e l a t i v e l y low AD might be the r e s u l t of high f i b e r content of the d i e t , but might a l s o be due t o wound-induced p r o t e o l y t i c enzyme i n h i b i t o r s (34, 37) which would reduce the d i g e s t i b i l i t y of the food. A low ECD c o u l d r e s u l t from a n t i m e t a b o l i t e s present i n the d i e t a r y m a t e r i a l , but could a l s o be produced by unfavorable amino a c i d r a t i o s t h a t would prevent the synthesis of normal s t r u c t u r a l p r o t e i n s . Plant m a t e r i a l c o n t a i n i n g acute t o x i n s preclude the determination of meaningful n u t r i t i o n a l i n d i c e s because the larvae would sicken and/or d i e during the experimental p e r i o d . Accordingly, nu­ t r i t i o n a l i n d i c e s are u s e f u l i n a p r e l i m i n a r y assessment o f host p l a n t u t i l i z a t i o n , but are not s u f f i c i e n t t o i d e n t i f y the s p e c i f i c f a c t o r s i n f l u e n c i n g the e f f i c i e n c y o f that u t i l i z a t i o n . The e f f e c t s o f chemical p l a n t f a c t o r s , i n c l u d i n g both a l l e l o c h e m i c s and n u t r i t i o n a l f a c t o r s , on the e f f i c i e n c y of d i e t a r y u t i l i z a t i o n can best be i n v e s t i g a t e d by i n c o r p o r a t i n g them i n t o a standardized a r t i f i c i a l d i e t and then determining n u t r i t i o n a l i n d i c e s . In t h i s way chemical f a c t o r s can be studied with much l e s s equivoca­ t i o n . A l s o , by using the technique o f p e r f u s i o n o f known amounts o f a compound i n t o p l a n t m a t e r i a l , most o f the l i m i t a t i o n s discussed above can be avoided. E r i c k s o n and Feeny (18) were q u i t e s u c c e s s f u l i n t h e i r use o f t h i s technique. They demonstrated t h a t P a p i l i o polyxenes a s t e r i u s larvae ate j u s t as much wen fed on p l a n t t i s s u e perfused with s i n i g r i n as when fed c o n t r o l t i s s u e . The s i n i g r i n i n h i b i t e d a s s i m i l a t i o n , though. In a d d i t i o n t o the n u t r i t i o n a l index experiments d i s c u s s e d above, I a l s o s t u d i e d the e f f e c t s o f a l l e l o c h e m i c s on some other aspects o f d i e t e t i c s o f the black cutworm. The i n s e c t s were reared on an a r t i f i c i a l d i e t (43). A l l e l o c h e m i c s were added at known concentrations. A number of p l a n t a l l e l o c h e m i c s with p o s s i b l e allomone f u n c t i o n s were t e s t e d over a 10,000-fold range o f concentrations f o r e f f e c t s on l a r v a l s u r v i v a l , growth, pupation r a t e , and pupal weight. I f i n c o r p o r a t i o n of a compound i n t o the d i e t r e s u l t e d i n a s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n between i t s d i e t a r y concentration and one of the above parameters, then the n u t r i t i o n a l i n d i c e s were determined f o r that compound a t 3.75 χ 10 M. A more d e t a i l e d d e s c r i p t i o n of the methods used f o r these experiments are described elsewhere (9). The experimental r e s u l t s (Table I) (summarized from Reese and Beck (9-12) p l u s some m a t e r i a l being prepared f o r p u b l i c a t i o n ) show a number o f things. p-Benzoquinone ( F i g . 1) reduced the amount o f i n g e s t i o n and a s s i m i l a t i o n . The reduced a s s i m i l a t i o n appeared t o be compensated f o r by an increase i n ECD. The i n t e r a c t i o n of these two parameters r e s u l t e d i n an o v e r a l l ECI

HOST P L A N T RESISTANCE T O

136

Table I.

E f f e c t s o f some a l l e l o c h e m i c s on black cutworm s u r v i v a l , weight at 10 days, pupation at 28 days, pupal weight, pupation at 35 days, i n g e s t i o n , dry weight gain, AD, ECD, and ECI. + indicates s i g n i f i c a n t c o r r e l a t i o n between compound concentration and parameter measured f o r s u r v i v a l , weight, 28-day pupat i o n , and 35-day pupation. + i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e between experimental and c o n t r o l i n s e c t s f o r i n g e s t i o n , dry weight gain, AD, ECD, and ECI. Survival

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

PESTS

10Day Wt.

p-Benzoquinone Dur oqui none

+

28Day Wt.

Pupal Wt.

+

+

+

+

+

+

+

+

35Day Pup.

+

Ingestion

Dry AD ECD Wt. Gain

+

+

+

+

+

+

+

+

+

Hydroquinone Catechol L-Dopa

+

+

+

+

+

Dopamine Chlorogenic Acid Resorcinol

+

Phloroglucinol +

+

G a l l i c Acid

+

+ +

% +

+

p-Coumaric A c i d

+ +

Cinnamic A c i d

+

+

Ferulic Acid Benzyl A l c o h o l Pyrogallol Orcinol

+ +

+

+

+

+

+

ECI

Plant Biochemicals

REESE

P-BENZOQUINONE

and Insect

HYDROQUINONE

Growth

ρ - C O U M A R I C ACID OH

CINNAMIC A C I D

V Ο

DUROQUINONE

BENZYL A L C O H O L

FERULIC

ACID

Ο

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

H

3

c A c H

3

PHLOROGLUCINOL

CATECHOL OH

J\0H

V

GALLIC

CHLOROGENIC

ACID

ACID H C I ^ \ 0 H

J\PH

V

Y

C O O H

^CH-COO

y h

o

Figure 1.

\çoo \ _ / ° h

Plant allelochemics bioassay ed with black cutworm larvae (duroquinone is probably not found in phnts)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

138

HOST P L A N T

RESISTANCE T O PESTS

that was s i m i l a r t o the c o n t r o l l a r v a e . The reduced form o f pbenzoquinone, hydroquinone, showed l i t t l e b i o l o g i c a l a c t i v i t y . Catechol i n h i b i t e d ECD, r e s u l t i n g i n a low ECI, while the amount ingested was not a f f e c t e d . L-Dopa a l s o exerted no e f f e c t on i n g e s t i o n , but i n h i b i t e d both AD and ECD. I n t e r e s t i n g l y , the s t r u c t u r e s o f both c a t e c h o l and L-dopa i n c l u d e hydroxyl groups l o c a t e d on adjacent carbon atoms. No compound that we have t e s t e d to data that i n c l u d e d t h i s s t r u c t u r e has had any e f f e c t on i n g e s t i o n . A l s o , compounds having t h i s s t r u c t u r e may a c t as c h e l a t i n g agents, thus b i n d i n g some e s s e n t i a l mineral or t r a c e element. L-Dopa might a l s o be a c t i n g as some type o f competitor with t y r o s i n e o r phenylalanine. L-Dopa without the carboxyl group (dopamine) had no d e t e c t a b l e b i o l o g i c a l a c t i v i t y . Thus, having hydroxyl groups on adjacent carbon atoms c e r t a i n l y d i d not guarantee a c t i v i t y . R e s o r c i n o l had no apparent e f f e c t on the n u t r i t i o n a l i n d i c e s ; the data suggest the reduced growth was mainly due to a reduced r a t e o f i n g e s t i o n . P h l o r o g l u c i n o l (with one more hydroxyl group than r e s o r c i n o l ) ( F i g . 1) i n h i b i t e d growth mainly through a r e d u c t i o n o f ECD and thus a reduced ECI. O r c i n o l , d i f f e r i n g from p h l o r o g l u c i n o l i n having a methyl group s u b s t i t u t e d f o r a hydroxyl group ( F i g . 1), showed no b i o l o g i c a l a c t i v i t y . Although cinnamic a c i d i n h i b i t e d pupation i n the p r e l i m i n a r y t e s t s , i t had no e f f e c t on the n u t r i t i o n a l i n d i c e s . p-Coumaric a c i d , s t r u c t u r a l l y s i m i l a r t o cinnamic a c i d , a f f e c t e d only pupal weight and ECD. F e r u l i c a c i d showed no apparent a c t i v i t y . Benzyl a l c o h o l i n h i b i t e d pupation, and i n h i b i t e d ECD s l i g h t l y . P y r o g a l l o l i n h i b i t e d growth, apparently through an accumulation o f s t a t i s t i c a l l y i n s i g n i f i c a n t e f f e c t s on v a r i o u s , parameters such as i n g e s t i o n and ECI. The a d d i t i o n o f a carboxyl group t o get g a l l i c a c i d lessened the a c t i v i t y , at l e a s t i n terms o f growth. Duroquinone was i n c l u d e d i n these experiments because o f i t s s t r u c t u r a l s i m i l a r i t i e s to the p l a n t compound, p-benzoquinone, although I am not aware o f duroquinone having been reported to occur i n p l a n t t i s s u e s . Duroquinone i n c r e a s e d AD but s t r o n g l y i n h i b i t e d other parameters. Growth was e x c e p t i o n a l l y slow i n the presence of duroquinone. In r e l a t i o n to the chronic e f f e c t s hypothesis, i t i s i n t e r e s t i n g t o note t h a t o f 37 compounds t e s t e d (not a l l shown i n Table I due t o incomplete n u t r i t i o n a l index d a t a ) , only 10 compounds reduce s u r v i v a l ( t o x i c i t y ) , whereas 25 compounds reduced growth, pupation, o r pupal weight. Thus, the chronic e f f e c t s hypothesis i s supported. I t h i n k t h a t n u t r i t i o n a l index data are extremely u s e f u l i n s t a r t i n g t o i d e n t i f y p h y s i o l o g i c a l processes i n f l u e n c e d by p l a n t a l l e l o c h e m i c s , p a r t i c u l a r l y i f the chemicals are t e s t e d s i n g l y under d e f i n e d experimental c o n d i t i o n s . Unfortunately, t h i s type o f data does not e l u c i d a t e s p e c i f i c biochemical modes o f a c t i o n . A l s o , i t does not e l i m i n a t e the p o s s i b i l i t y of a d d i t i o n a l o r nonn u t r i t i o n a l e f f e c t s . However, n u t r i t i o n a l index techniques w i l l s u r e l y help us make progress i n these d i r e c t i o n s . In a d d i t i o n ,

9.

REESE

Plant Biochemicals

and

Insect

Growth

139

these techniques have already shown t h a t growth can be i n h i b i t e d by p l a n t a l l e l o c h e m i c s i n d i f f e r e n t ways. A l s o , they c l e a r l y demonstrate t h a t the n u t r i t i o n a l physiology ( " d i e t e t i c s ) of i n s e c t s can be i n f l u e n c e d by n o n - n u t r i t i o n a l substances.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

I n t e r r e l a t i o n s h i p s o f N u t r i t i o n a l Indices and D i e t a r y Moisture Levels Recently, I examined the i n t e r r e l a t i o n s h i p s between various growth-related parameters and the e f f e c t s of d i e t a r y moisture l e v e l s on these parameters. Although water i s o f obvious importance to animal n u t r i t i o n , l i t t l e q u a n t i t a t i v e work has been done on how water intake a f f e c t s the dynamics of growth. Storedproducts i n s e c t s have remarkable a b i l i t i e s to conserve water (44), while a number of phytophagous i n s e c t s s u f f e r d e l e t e r i o u s e f f e c t s i f d i e t a r y moisture i s not r e l a t i v e l y high (38, 41, 45). House (46) found t h a t as the n u t r i e n t s o f food were d i l u t e d by water, C e l e r i o euphorbiae larvae tended to eat more. House d i d not f i n d s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the e f f i c i e n c i e s o f conversion o f ingested food between the c o n t r o l and experimental i n s e c t s over the range o f moisure l e v e l s he t e s t e d . However, I c a l c u l a t e d the c o r r e l a t i o n c o e f f i c i e n t between percentage of n u t r i e n t s ( i n g r e d i e n t s other than agar, c e l l u l o s e f l o u r , water, and f o l i a g e e x t r a c t ) and the mean e f f i c i e n c y of conversion of ingested food i n House's experiment, and I found i t to be negative and h i g h l y s i g n i f i c a n t . Soo Hoo and Fraenkei (39, 40) suggested t h a t water content o f the d i e t was important to e f f i ciency of conversion i n Prodenia e r i d a n i a ; d i l u t i o n o f the d i e t caused an apparent increase i n e f f i c i e n c y . Feeny (_3) found t h a t e f f i c i e n c y of conversion o f a s s i m i l a t e d food decreased with decreasing moisture o f the r e s p e c t i v e food p l a n t s o f various lepiodopterous l a r v a e . Hoekstra and Beenakkers (47) suggested t h a t p a r t o f the d i f f e r e n c e i n e f f i c i e n c i e s of conversion f o r Locusta m i g r a t o r i a were due to d i f f e r e n c e s i n moisture content o f the d i f f e r e n t species of p l a n t s they were fed. S c r i b e r (48) r e c e n t l y i n v e s t i g a t e d the e f f e c t s of v a r i e d moisture l e v e l s i n host leaves on Hyalophora c e c r o p i a l a r v a e . He, too, found t h a t lower moisture l e v e l s decrease the e f f i c i e n c y o f conversion of food i n t o biomass, as w e l l as the e f f i c i e n c y o f conversion o f the c a l o r i c content and the e f f i c i e n c y o f conversion o f the n i t r o genous content of the food. The f o l l o w i n g abbreviations w i l l be used i n t h i s s e c t i o n : IFrWtL — DWG DWE FWE DML uWF

— — — — —

i n i t i a l f r e s h weight o f larvae (weight o f larvae at beginning o f experiment) dry weight gain dry weight eaten f r e s h weight eaten percent dry matter of larvae at end o f experiment dry weight o f feces

H O S T P L A N T RESISTANCE

140

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

DMF DMD

— —

T O PESTS

percent dry matter o f feces at end o f experiment percent dry matter o f d i e t at beginning o f e x p e r i ment

A stock c u l t u r e o f the b l a c k cutworm was maintained using the methods of Reese e t a l (43) as modified by Reese and Beck (j)) . C o r r e l a t i o n c o e f f i c i e n t s were c a l c u l a t e d between a l l p o s s i b l e combinations o f p a i r s o f growth-related parameters from the cont r o l s o f p u b l i s h e d and unpublished a l l e l o c h e m i c experiments (ji12). The mean values from each o f 18 experiments were used i n t h i s p o r t i o n o f the study. In each o f these 18 experiments, there were 20 l a r v a e i n each group and the experiments were performed from the 10th through the 20th day o f l a r v a l l i t e . In a second s e r i e s o f experiments, the d i e t a r y moisture l e v e l s were v a r i e d . There were 20 larvae i n each c o n t r o l group and 20 l a r v a e i n each experimental group. The experimental p e r i o d was again from the 10th through the 20th day o f l a r v a l l i f e . Remaining d e t a i l s o f the n u t r i t i o n a l index techniques employed have been d i s c u s s e d e a r l i e r i n t h i s paper. I n t e r r e l a t i o n s h i p s o f I n d i c e s . IFrWtL v a r i e d from e x p e r i ment t o experiment, apparently due t o s l i g h t v a r i a t i o n s i n r e a r i n g c o n d i t i o n s and v a r i a t i o n s w i t h i n the p o p u l a t i o n ; the importance o f running p r e c i s e c o n t r o l s along with experiments i s apparent. In such a s i t u a t i o n , i t would be reasonable t o expect t h a t l a r v a e t h a t s t a r t e d the experiment a t a h e a v i e r weight would have a h i g h e r DWE, DWG, and DWF during the experiment. T h i s i s c l e a r l y shown i n Table I I . Likewise, i t would be reasonable t o assume that DWG (other f a c t o r s being e q u a l ) , should be d i r e c t l y p r o p o r t i o n a l to DWE. T h i s too proved to be the case (Table I I and F i g . 2) and confirmed the work of Kogan and Cope (42) who used Pseudoplusia i n c l u d e n s feeding on soybeans. Kogan and Cope presented the l i n e a r r e g r e s s i o n c h a r a c t e r i s t i c s f o r the r e l a t i o n s h i p they found between DWE and DWG. I used these c h a r a c t e r i s t i c s to c a l c u l a t e DWG values i n our cutworm experiments ( F i g . 2 ) . The DWG v a l u e s c a l c u l a t e d from the Kogan and Cope r e g r e s s i o n were higher than those f o r the b l a c k cutworm. T h i s i n d i c a t e s t h a t P_. includens was more e f f i c i e n t a t converting soybeans i n t o i n s e c t t i s s u e than the b l a c k cutworm was a t converting the a r t i f i c i a l d i e t used i n my experiments. Since IFrWtL and DWG were p o s i t i v e l y c o r r e l a t e d , and since IFrWtL and DWF were p o s i t i v e l y c o r r e l a t e d , i t i s reasonable to suspect t h a t a r e l a t i o n s h i p might e x i s t between DWG and DWF. T h i s was the case, as shown i n Table I I . Dr. Beck and I have s t a t e d (9) that i t may not be necessary to use s p e c i f i c i n s t a r t s f o r n u t r i t i o n a l index experiments, espec a l l y when c o n t r o l s are always run. I t was t h e r e f o r e i n t e r e s t i n g t h a t there was no s i g n i f i c a n t c o r r e l a t i o n between IFrWtL and any of the three n u t r i t i o n a l i n d i c e s (AD, ECD, and E C I ) ; i n f a c t , the c o r r e l a t i o n s were e x c e p t i o n a l l y low (Table I I ) . In other words, as long as the experiments were s t a r t e d with 10 day o l d l a r v a e ,

ECD

AD

DMF

DWF

DWG

DML

DWE

IFrWtL

Table I I .

+0.866 ***

DWE

* ** ***

+0.611 **

+0.541 *

DML

+0.703

+0.912 ***

+0.795 ***

DWG

+0.892 ***

+0.652

+0.958 ***

+0.822 ***

DWF

•

+0.004 -0.291

+0.126 -0.828 ***

-0.142

+0.749 ***

+0.001

+0.166

-0.311

+0.331

+0.236

-0.072

-0.093

ECI

-0.344

+0.209

+0.206

-0.065

-0.076

ECD

-0.106

-0.151

-0.037

-0.001

AD

-0.397

+0.179

-0.453

-0.409

DMF

< 0.05 l e v e l .

(17 degrees o f freedom i n each case) between p a i r s o f n u t r i -

indicates s t a t i s t i c a l significance at Ρ s i g n i f i c a n c e a t Ρ < 0.01 l e v e l . s i g n i f i c a n c e a t Ρ < 0.0005 l e v e l .

Coefficients of correlation t i o n a l index parameters.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

^

;s-

s? 3

| ο

•—I

a

g

2

S*

g3

*α §

g w w

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

HOST P L A N T RESISTANCE

D W E (g) Figure 2. Refotionship between DWG and DWE in black cutworm larvae. Kogan and Cope 1974 line refers to line calculated from data in Ref. 42.

T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and

Insect

Growth

143

d i f f e r e n c e s i n weight (and i n i n s t a r s ) w i t h i n the range s t u d i e d , had no apparent r e l a t i o n s h i p to AD, ECD, o r ECI. A s i m i l a r l a c k of r e l a t i o n s h i p s i s suggested by Kogan and Cope (42). They found ECI t o be roughly s i m i l a r from day 6 through day 12, when c a l c u l a t e d i n terms o f f r e s h weights. I t was more e r a t i c when c a l c u l a t e d i n terms o f dry weights, but there was s t i l l l i t t l e apparent trend up o r down, as the i n s e c t grew. A l l o f these l i n e s o f evidence suggest t h a t e f f i c i e n c y changes l i t t l e d u r i n g the l o g phase o f growth. T h i s f u r t h e r strengthens our proposal (9) t h a t i t i s during t h i s l o g phase that i t i s a p p r o p r i a t e to conduct experiments on growth-reducing d i e t a r y f a c t o r s . AD i n c r e a s e d as ECD decreased (Table I I ) . In other words, as more food was a s s i m i l a t e d , a s m a l l e r p r o p o r t i o n of the a s s i m i l a t e d food was converted i n t o i n s e c t biomass. Since ECI depends d i r e c t l y upon a s s i m i l a t i o n and the e f f i c i e n c y with which a s s i m i l a t e d food i s converted, t h i s compensating mechanism had the e f f e c t o f decreasing the v a r i a b i l i t y of ECI. ECI had a range of o n l y 7.6 percentage p o i n t s , while ECD and AD had ranges o f 39.5 and 19.0 percentage p o i n t s , r e s p e c t i v e l y , over the 18 e x p e r i ments. DML and DMF I n t e r r e l a t i o n s h i p s . Larvae that s t a r t e d the experimental p e r i o d at a h e a v i e r weight tended to end the e x p e r i ment a t a higher DML (Table I I ) . I t would appear from t h i s t h a t black cutworm larvae may have some a b i l i t y to conserve moisture. T h i s i s s u b s t a n t i a t e d by the f a c t that DML f o r c o n t r o l s was u s u a l l y around 16%, whereas DMD a t the beginning o f experiments was u s u a l l y c l o s e to 21%. A l s o , i t f i t s w e l l with the f a c t t h a t many i n s e c t s i n c r e a s e i n percent dry matter as they i n c r e a s e i n age (49, 50). Given that b l a c k cutworm l a r v a e can apparently conserve moisture when feeding on a d i e t o f 21% DMD, how much a b i l i t y do they have to maintain a constant moisture l e v e l when fed d i e t s o f v a r y i n g moisture l e v e l s ? T h i s and other questions prompted us to vary the d i e t a r y moisture l e v e l . The r e s u l t s o f these experiments w i l l be d i s c u s s e d i n a subsequent s e c t i o n . Considering the r e l a t i o n s h i p between IFrWtL and DML, and c o n s i d e r i n g the r e l a t i o n s h i p s between IFrWtL and DWE, DWG, and DWF, i t was not s u r p r i s i n g to f i n d p o s i t i v e c o r r e l a t i o n s between DML and DWE, DWG, and DWF (Table II) . DMF, however, was appar e n t l y independent o f any other parameter, i n c l u d i n g DML. This was somewhat unexpected, since i f there were a r e g u l a t o r y mechanism f o r DML, one would assume t h a t i t would operate by absorbing v a r i o u s amounts o f water from the hindgut. I n t e r e s t i n g l y , DML and DMF were both very p r e c i s e parameters i n any given experiment. Standard e r r o r o f the mean values were almost always l e s s than 1% and were u s u a l l y l e s s than 0.5%. The range o f DMF values over the 18 experiments was 15.1 percentage p o i n t s , while the range o f DML values was only 3.4 percentage p o i n t s . Compared t o the wide i n d i v i d u a l v a r i a t i o n o f such things as l a r v a l weight, t h i s v a r i a t i o n i n DML seemed e x c e p t i o n a l l y

144

HOST P L A N T RESISTANCE T O

PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

small. We have a l s o noted t h a t i n c e r t a i n o f our a l l e l o c h e m i c experiments, DMF and DML seemed to be among the most s e n s i t i v e parameters. In some experiments i n which parameters ( i . g . DWG) were not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l s , DMF or DML showed s i g n f i c a n t d i f f e r e n c e s . A l s o , such small v a r i a t i o n over many experiments i m p l i e s t h a t black cutworm larvae have the a b i l i t y to regulate DML. To i n v e s t i g a t e t h i s p o s s i b i l i t y and to t r y to answer questions discussed e a r l i e r , a s e r i e s o f e x p e r i ments were performed i n which the experimental i n s e c t s were fed on d i e t s o f varying moisture content. E f f e c t s of Various D i e t a r y Moisture L e v e l s . FWE was not a f f e c t e d by d i l u t i o n with water ( F i g . 3). U n l i k e C e l e r i o euphorbiae larvae (46), black cutworm larvae seem to have l i t t l e a b i l i t y t o compensate f o r a d i l u t i o n of the d i e t . D i e t s d r i e r than the c o n t r o l s were consumed much more p o o r l y ( F i g . 3). With FWE remaining e s s e n t i a l l y constant f o r the d i l u t e d d i e t s , the a c t u a l DWE n e c e s s a r i l y decreased with i n c r e a s i n g moisture from the c o n t r o l l e v e l ( F i g . 4 ) . With d i e t s d r i e r than the c o n t r o l s , DWE a l s o decreased. Thus, the optimal moisture content i n terms o f DWE was a d i e t s l i g h t l y d r i e r than the c o n t r o l s . DWF followed a s i m i l a r p a t t e r n . As DMD increased, ECD decreased over the e n t i r e range of concentrations t e s t e d ( F i g . 5 ) . Note t h a t the optimal DMD i n terms o f ECD i s not at a l l c l o s e to the c o n t r o l (100%) l e v e l . These data confirmed what Feeny (3), S c r i b e r (48), and House (46) found with other lepidopterous l a r v a e . I f e e l t h i s i s a s i g n i f i cant f i n d i n g s i n c e Feeny (_3) and S c r i b e r (48) used d i f f e r e n t p l a n t s (among which water content happened to v a r y ) , or the water content o f a p l a n t was changed a r t i f i c i a l l y . In e i t h e r case, many other biochemical f a c t o r s may a l s o have been d i f f e r e n t . In my experiments, a standardized d i e t was used, t h e r e f o r e , I was confident when I changed the d i e t a r y moisture l e v e l , that t h i s was the only t h i n g t h a t was v a r i e d . In a d d i t i o n , I was able to study moisture l e v e l s above the optimal growth l e v e l , as w e l l as those below t h i s l e v e l . As might be expected from the r e l a t i o n s h i p between ECD and ECI, ECI was a l s o i n v e r s e l y p r o p r t i o n a l to DMD ( F i g . 6 ) . As discussed above, ECD and AD were i n v e r s e l y p r o p o r t i o n a l to each other; t h i s apparently had a s t a b i l i z i n g e f f e c t on ECI. In the experiments i n which d i e t a r y moisture was v a r i e d , ECD was i n v e r s e l y c o r r e l a t e d with DMD, so i f AD and ECD were r e l a t e d to each other, one would expect DMD and AD to be c o r r e l a t e d . This was not the case. Thus, over a winde range o f d i e t a r y moisture l e v e l s , the compensating mechanism between ECD and AD apparently does not operate a t a detectable l e v e l . A l s o , the f a c t that AD was not a f f e c t e d by d i e t a r y moisture was i n i t s e l f somewhat s u r p r i s i n g , s i n c e one would think t h a t the moisture content o f food would have some e f f e c t on how r e a d i l y i t passes across the gut w a l l .

9.

REESE

Plant Biochemicals

and Insect

145

Growth

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

120 r

40

60

80

100

120

140

160

DMD (% of Control) Figure 3.

Effect of DMD on FWE laroae

in black cutworm

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

HOST P L A N T RESISTANCE T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and

Insect

Growth

147

Growth depends upon a combination o f how much an organism eats and how e f f i c i e n t l y t h a t m a t e r i a l i s converted i n t o t i s s u e . T h i s i s c l e a r l y demonstrated and q u a n t i f i e d by our data. Figs. 4 and 6 show the r e l a t i o n s h i p between DMD and DWE, and between DMD and ECI, r e s p e c t i v e l y . The r e l a t i o n s h i p of DMD and DWG ( F i g . 7) can be viewed as a r e s u l t a n t o f F i g s . 4 and 6. On low DMD d i e t s , the l a r v a e ate l e s s , but converted i t more e f f i c i e n t l y , g i v i n g net DWG values t h a t were somewhat lower than the c o n t r o l s . Had the i n f l u e n c e o f a higher ECI and a b i l i t y o f the l a r v a e to regulate moisure l e v e l s to some extent (discussed i n a l a t e r section) not been f a c t o r s , the curve f o r the moist d i e t s i n F i g . 7 might have followed the dashed l i n e more c l o s e l y . This dashed l i n e was c a l c u l a t e d on the b a s i s of the three p o i n t s between 80% and 100% DMD. For d i e t s d r i e r than the c o n t r o l s , both ECI and DWE dropped sharply, and combined to give g r e a t l y reduced growth. Note t h a t although the optimal DWE was a p o i n t above the c o n t r o l s (higher DMD than c o n t r o l s ) , t h a t the optimal DWG was q u i t e c l o s e to the c o n t r o l s , due to the i n f l u e n c e s of ECI. The d i e t developed e m p i r i c a l l y by Reese e t a l (43) was thus shown to be very c l o s e to the optimum i n terms o f moisture l e v e l . These experiments were o r i g i n a l l y conducted f o r the purpose of i n v e s t i g a t i n g the a b i l i t y o f black cutworm larvae to deal p h y s i o l o g i c a l l y with various d i e t a r y moisture l e v e l s . As has been shown, d i e t a r y moisture has s t r i k i n g e f f e c t s on growth and e f f i c i e n c y of conversion, as w e l l as r e l a t e d parameters. I t a l s o had r a t h e r p r e c i s e e f f e c t s on DMF, as shown i n F i g . 8. Fecal percent dry matter i n terms o f percent of the c o n t r o l s , was e s s e n t i a l l y the same as d i e t a r y dry matter ( F i g . 8). The dashed l i n e i n F i g . 8 i l l u s t r a t e s the s i t u a t i o n i f DMF were e x a c t l y equal to DMD, i n terms o f percent of the c o n t r o l s . The larvae seemed t o have l i t t l e a b i l i t y to regulate dry matter compared to many stored-products i n s e c t s . Nevertheless, evidence f o r a c e r t a i n amount o f water conserving a b i l i t y i s presented i n F i g . 9. Note the p o s i t i o n o f the curve i n r e l a t i o n to the dashed l i n e , which shows the s i t u a t i o n i f DML were e x a c t l y equal to DMD, i n terms o f percent of the c o n t r o l s . The f a c t that the observed data f a l l s above the dashed l i n e f o r low dry matter d i e t s , but lags behind the dashed l i n e f o r d r i e r d i e t s , i n d i c a t e d a degree of r e g u l a t o r y a b i l i t y . A l s o , when viewed i n terms of a c t u a l DML and DMD values (rather than i n terms of percent o f c o n t r o l s ) , DML was always s l i g h t l y lower than DMD, demonstrating some a b i l i t y to conserve water. The a b i l i t y i s not very great, though, as i n d i cated by the f a c t that as DMD increased, DML a l s o increased over the range o f d i e t a r y moisture l e v e l s t e s t e d . Apparently the mechanism was not p u t t i n g excess moisture back i n t o the gut tract. I f t h i s were happening, the feces from larvae on low dry matter d i e t s should have been much lower i n dry matter. From these data the mechanism seems to be a water conservation mechanism i n the sense of maintaining DML lower than DMD, but not a r e g u l a t o r y mechanism i n the sense of maintaining a constant DML over a range o f DMD values.

American Chemicaf Society Library 1155 16th St. N. W. Washington, D. C. 20036

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

HOST P L A N T RESISTANCE

Figure 7.

Effect of DMD on DWG in black cutworm larvae. Dashed line based on three points closest to it.

160

ο

140

5

120

H

CO

Φ

I g

100

fi*

80

60

4

0

60

80

100

120

140

160

% Dry Matter of Diet (% of Control) Figure 8. Effect of DMD on DMF in black cut­ worm larvae. Dashed line represents situation if DMF equaled DMD, in terms of percent of control.

T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and Insect

Growth

Figure 9. Effect of DMD on DML in black cutworm larvae. Dashed line represents situation if DML equaled DMD, in terms of percent of control.

149

HOST P L A N T

150

RESISTANCE T O PESTS

Moisture l e v e l i s c e r t a i n l y an important f a c t o r i n p l a n t i n s e c t i n t e r a c t i o n s . Black cutworm l a r v a e , although h i g h l y polyphagous, are undoubtedly capable of s u c c e s s f u l l y l i v i n g only on p l a n t s with f a i r l y high moisture l e v e l s . Outbreaks of t h i s p e s t are a s s o c i a t e d with f l o o d s and unusually wet years. T h e i r a b i l i t y to r e g u l a t e t h e i r moisture l e v e l i s l i m i t e d . Moisture l e v e l may be a u s e f u l f a c t o r i n host p l a n t r e s i s t a n c e . I f t h i s proves to be the case, i t would c e r t a i n l y s i m p l i f y some o f the problems encountered i n biochemically-based r e s i s t a n c e , i n which we must be concerned with p o s s i b l e e f f e c t s of these biochemicals on l i v e s t o c k and man. I b e l i e v e t h i s area of research warrants f u r t h e r study.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

Concluding Remarks T h i s summary o f i n t e r a c t i o n s between p l a n t biochemicals and i n s e c t d i e t e t i c s i s c e r t a i n l y not complete or comprehensive. I t does p o i n t out some o f the c u r r e n t areas o f research. More important, I hope that i t may stimulate more work i n areas where our knowledge i s s t i l l l a c k i n g depth. We know l i t t l e about the e f f e c t s o f a l l e l o c h e m i c s on the a s s i m i l a t i o n and conversion processes i n the gut and body t i s s u e s o f the i n s e c t . Nor do we have a very broad understanding o f the e f f e c t s o f a l l e l o c h e m i c s on amino a c i d u t i l i z a t i o n i n p r o t e i n s y n t h e s i s and other synthet i c pathways i n the developmental and reproductive physiology of the i n s e c t . The n u t r i t i o n a l index techniques discussed i n t h i s paper o f f e r a beginning, but only a beginning. Nutritional i n d i c e s can serve as i n d i c a t o r s i d e n t i f y i n g some o f the aspects t h a t should be s t u d i e d i n d e t a i l with biochemical techniques. Acknowledgement T h i s research was supported by the College o f A g r i c u l t u r a l and L i f e Sciences, U n i v e r s i t y o f Wisconsin and by a research grant (PCM 74-24001) t o Dr. Stanley D. Beck from the N a t i o n a l Science Foundation. I would l i k e to thank Dr. Dale M. N o r r i s f o r h e l p f u l suggestions f o r improving t h i s manuscript. I a l s o thank H o l l y Beermann f o r t e c h n i c a l a s s i s t a n c e . Literature Cited

1. Beck, S. D., Reese, J. C., Recent Adv. Phytochem, (1976), 10, 4192. 2. Whittaker, R. H., pp. 43-70, "Chemical Ecology", Sondheimer, E., Simeone, J. B., Eds., Academic Press, New York, 1970. 3. Feeny, P. P., pp. 3-19, "Coevolution of Animals and Plants". Gilbert, L. E., Raven, P. H., Eds., University of Texas Press, Austin, 1975. 4. Feeny, P. P., Recent Adv. Phytochem., (1976), 10, 1-40. 5. Rhoades, D. F., Cates, R. G., Recent Adv. Phytochem, (1976), 10, 168-213.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

9.

REESE

Plant Biochemicals

and Insect Growth

151

6. Fraenkel, G. S., Science, (1959), 129, 1466-1470. 7. Hedin, P. Α., Maxwell, F. G., Jenkins, J. Ν., pp. 494-527, "Proceedings of the Summer Institute on Biological Control of Plant Insects and Diseases", Maxwell, F. G., Harris, F. Α., Eds., University Press of Mississippi, Jackson, 1974. 8. Kogan, Μ., pp. 103-146, "Introduction to Insect Pest Manage­ ment", Metcalf, R. L., Luckmann, W., Eds., John Wiley and Sons, New York, 1975. 9. Reese, J. C., Beck, S. D., Ann. Entomol. Soc. Am., (1976a), 69, 59-67. 10. Reese, J. C., Beck, S. D., Ann. Entomol. Soc. Am., (1976b), 69, 68-72. 11. Reese, J. C., Beck, S. D., Ann. Entomol. Soc. Am., (1976c), 69, 999-1003. 12. Reese, J. C., Beck, S. D., pp. 217-221, "The Host-Plant in Relation to Insect Behaviour and Reproduction", Jermy, T., Ed., Plenum Press, New York, 1976. 13. Kogan, M., pp. 211-227, "Proc. XV Internat. Congress Ent.", Packer, J. S., D. White, Eds., 1977. 14. Beck, S. D., Ann. Rev. Entomol., (1965), 10, 207-232. 15. Beck, S. D., pp. 1-6, "Insect and Mite Nutrition", Rodriguez, J. G., Ed., North Holland Publishing Co., Amsterdam, 1972. 16. Beck, S. D., pp. 290-311, "Proceedings of the Summer Insti­ tute on Biological Control of Plant Insects and Diseases", Maxwell, F. G., Harris, F. Α., Eds., University Press of Mississippi, Jackson, 1974. 17. Beck, S. D., Schoonhoven, L. Μ., "Breeding Plants Resistant to Insects", Maxwell, F. G., Jennings, P. R., Eds., John Wiley and Sons, New York, (In Press). 18. Erickson, J. Μ., Feeny, P. P., Ecology, (1974), 55: 103-111. 19. Shaver, T. N., Lukefahr, M. J., Garcia, J. Α., J. Econ. Entomol., (1970), 63: 1544-1546. 20. Elliger, C. Α., Zinkel, D. F., Chan, B. G., Waiss, A. C., Jr., Experientia, (1976), 32: 1364-1365. 21. Hatfield, Ε. E., pp. 171-179, "Effect of Processing on the Nutritional Value of Feeds", National Acad. Sci., Washing­ ton, D. C., 1973. 22. Cheeke, P. R., Nutrition Reports Internat., (1972), 5: 159-170. 23. Stephenson, E. L., pp. 67-71, "Effect of Processing on the Nutritional Value of Feeds", National Acad. Sci., Washington, D. C., 1973. 24. Liener, I. E., J. Food. Sci., (1976), 41: 1076-1081. 25. Rosenthal, G. Α., Janzen, D. H., Dahlman, D. L., Science (1976), 192: 256-258. 26. Vandersant, E. S., Chremos, J. H., Ann. Entomol. Soc. Am., (1971), 64: 480-485. 27. Isogai, Α., Chang, C., Murakoshi, S., Suzuki, Α., Tamura, S., J. Agr. Chem. Soc. Japan, (1973a), 47: 443-447. 28. Isogai, Α., Murakoshi, S., Suzuki, Α., Tamura, S., J. Agr. Chem. Soc. Japan, (1973b), 47: 449-453.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch009

152

HOST

PLANT

RESISTANCE

TO

PESTS

29. Dahlman, D. L., Rosenthal, G. Α., Comp. Biochem. Physiol., (1975), 51A: 33-36. 30. Hegdekar, D. Μ., J. Econ. Entomol., (1970), 63: 1950-1956. 31. Feeny, P. P., J. Insect Physiol., (1968), 14: 805-817. 32. Feeny, P. P., Ecology, (1970), 51: 565-581. 33. Ryan, C. Α., Ann. Rev. Plant Physiol., (1973), 24: 173-196. 34. Ryan, C. Α., Green, T. R., Recent, Adv. Phytochem., (1974), 8: 123-140. 35. Birk, Y., Applebaum, S. W., Enzymologia, (1960), 22: 318326. 36. Su, H. C. F., Speirs, R. D., Mahany, P. G., J. Georgia Entomol. Soc., (1974), 9: 86-87. 37. Green, T. R., Ryan, C. Α., Science, (1972), 175: 776-777. 38. Waldbauer, G. P., Adv. Insect Physiol., (1968), 5: 229288. 39. Soo Hoo, C. F., Fraenkel, G., J. Insect Physiol., (1966a), 12: 693-709. 40. Soo Hoo, C. F., Fraenkel, G., J. Insect Physiol., (1966b), 12: 711-730. 41. Waldbauer, G. P., Entomol. Exp. Appl., (1964),7: 253-269. 42. Kogan, Μ., Cope, D., Ann. Entomol. Soc. Am., (1974), 67: 66-72. 43. Reese, J. C., English, L. M., Yonke, T. R., Fairchild, M. L., J. Econ. Entomol., (1972), 65: 1047-1050. 44. Fraenkel, G., Blewett, Μ., Bull. Entomol. Res., (1944), 35: 127-139. 45. Waldbauer, G. P., Entomol. Exp. Appl., (1962), 5: 147-158. 46. House, H. L., Canad. Entomol., (1965), 97: 62-68. 47. Hoekstra, Α., Beenakkers, A. M. T., Entomol. Exp. Appl., (1976), 19: 130-138. 48. Scriber, J. Μ., Oecologia (Berl.), (1977), 28: 269-287. 49. Wigglesworth, V. B., "The Principles of Insect Physiology", 741 pp., Methuen and Co. Ltd., London, 1965. 50. Beck, S. D., Hanec, W., J. Insect Physiol., (1960), 4: 304-418.

10 Isolation and Identification of Toxic Agents from Plants MARTIN JACOBSON

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

Biologically Active Natural Products Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Methods, techniques, and instrumentation for the improved, rapid isolation and identification of physiologically active plant constituents are available today that were lacking 25, 10, or even 5 years ago. The time period between the isolation of a pure compound and its complete identification has often been frustratingly protracted, but that period has recently been narrowed. Perhaps one of the classical examples of the lengthy time span between isolation and complete structural determination is the case of the well known insect toxicant pyrethrum, whose active components—the pyrethrins, cinerins, and jasmolins—were isolated in 1910-1916 by Staudinger and Ruzicka (1), in 1944 by LaForge and Barthel (2), and in 1965 by Godin et al. (3), respectively; however, their complete structures were not obtained until years after they were isolated. No attempt has been made here to present a comprehensive review of a l l of the methods useful for isolating and identifying pesticidal toxicants from plants. Rather those methods that are most generally useful and those pesticidal compounds that are perhaps of major interest are treated. These include pyrethrum, rotenoids, nicotine and nicotinoids, other well-known alkaloids such as those from Veratrum, unsaturated isobutylamides, bitter substances (especially insect feeding deterrents), fungal peptides, gossypol, mycotoxins, and insect growth regulators. The first section treats the isolation and general methods of structure elucidation for the toxicants in each of these categories; the second section cites additional information for each of the most significant methods. For further general reference your attention is directed to the two recent volumes by Nakanishi et al. (4_,5) entitled "Natural Products Chemistry" and to "Naturally Occurring Insecticides" (6) edited by Jacobson and Crosby. Pyrethrum The term "pyrethrum" usually refers to the dried flower heads

153

154

HOST P L A N T RESISTANCE

T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

or to an e x t r a c t o f the d r i e d heads of Chrysanthemum c i n e r a r i i f o l i u m V i s , (family Compositae). The a c t i v e components are t o x i c to house f l i e s , mosquitoes, cockroaches, l i c e , and a number of stored product s p e c i e s . I s o l a t i o n . P a r t i t i o n o f a petroleum ether s o l u t i o n of p e t r o leum ether e x t r a c t from d r i e d flower heads between the hydrocarbon and nitromethane, followed by passage o f the nitromethane s o l u t i o n through a short column of a c t i v a t e d c h a r c o a l gives an a c t i v e conc e n t r a t e of 90% p u r i t y or above (7). Molecular d i s t i l l a t i o n can then be used to p u r i f y and d e c o l o r i z e the concentrate (8). The i n s e c t i c i d a l a c t i v i t y o f pyrethrum i s a t t r i b u t e d to the a c t i o n of 6 c o n s t i t u e n t s , namely p y r e t h r i n s I and I I , c i n e r i n s I and I I , and jasmolins I and I I . P y r e t h r i n I and c i n e r i n I are f a i r l y e a s i l y separated from p y r e t h r i n I I and c i n e r i n I I by column chromatography on alumina or s i l i c a g e l , and good s e p a r a t i o n may a l s o be obtained by paper and t h i n - l a y e r chromatography (TLC) (9). I d e n t i f i c a t i o n . The p y r e t h r i n s and c i n e r i n s form c r y s t a l l i n e 2,4-dinitrophenylhydrazones and are f u r t h e r i d e n t i f i e d by degradat i o n , b y d r o g e n o l y s i s , o z o n o l y s i s , u l t r a v i o l e t (UV), i n f r a r e d (IR), and n u c l e a r magnetic resonance (NMR) spectroscopy, and o p t i c a l r o t a t i o n (9). The j a s m o l i n s are i d e n t i f i e d by UV, IR, and NMR spectroscopy (10). Rotenone and Rotenoids T h i s group of compounds comprises mainly rotenone, though i t i n c l u d e s minor amounts of deguelin, tephrosin, t o x i c a r o l , sumat r o l , malaccol, and e l l i p t o n e . The m a t e r i a l s are obtained from v a r i o u s p a r t s ( u s u a l l y the r o o t s ) o f species of D e r r i s , Lonchocarpus, Tephrosia, and Mundulea (family Leguminosae). They a c t as contact o r stomach poisons f o r aphids, house f l i e s , and v a r i o u s species of chewing i n s e c t s . I s o l a t i o n . The a c t i v e compounds are obtained by e x t r a c t i n g the crushed, d r i e d root w i t h ether or methylene c h l o r i d e , followed by overnight c o o l i n g of a carbon t e t r a c h l o r i d e s o l u t i o n of the concentrate at 0°. The carbon t e t r a c h l o r i d e s o l v a t e of rotenone c r y s t a l l i z e s out and i s f i l t e r e d o f f and taken up i n t r i c h l o r o ethylene. When t h i s s o l u t i o n i s d i l u t e d w i t h methanol, n e a r l y pure rotenone separates out and can be f u r t h e r p u r i f i e d by c r y s t a l l i z a t i o n from t r i c h l o r o e t h y l e n e . I s o l a t i o n of rotenone from seeds i s s i m p l i f i e d i f the seeds are d e f a t t e d with petroleum ether p r i o r to e x t r a c t i o n w i t h ether (11). The r e s o l u t i o n of r o t e n o i d mixtures can be e f f e c t e d by column chromatography, but the use of a l k a l i n e alumina must be avoided s i n c e i t causes r a c e m i z a t i o n ; n e u t r a l or a c i d grades of adsorbents must be used. Countercurrent d i s t r i b u t i o n (CCD) i s u s e f u l f o r d e a l i n g w i t h i n t r a c t a b l e gums, and paper, TLC, and

10.

JACOBSON

Toxic Agents from

Plants

high-performance l i q u i d chromatography (11,12).

155

(HPLC) have also been used

I d e n t i f i c a t i o n . The s t r u c t u r e s are i d e n t i f i e d by chemical degradation and by the use of IR and NMR spectroscopy, and o p t i ­ c a l r o t a t o r y d i s p e r s i o n (ORD). The o p t i c a l l y a c t i v e forms of rotenone are much more t o x i c to i n s e c t s than the racemic form (13).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

N i c o t i n e and N i c o t i n o i d s N i c o t i n e and n o r n i c o t i n e occur i n the leaves o f the tobacco p l a n t , N i c o t i a n a tabacum L. Although these compounds were used to a c o n s i d e r a b l e extent as i n s e c t stomach poisons before the ad­ vent of DDT, they are seldom used as i n s e c t i c i d e s today i n the United States. Anabasine, obtained from the roots of Anabasis a p h y l l a L. and N. glauca Graham (tree tobacco), i s e x t e n s i v e l y used i n the Soviet Union to combat t h r i p s , mites, aphids, l e a f hoppers, s a w f l i e s , l i c e , and mosquitoes (14). N. tabacum and N. glauca are members of the f a m i l y Solanaceae, and A. a p h y l l a i s i n the f a m i l y Chenopodiaceae. I s o l a t i o n . The t e r t i a r y amine, n i c o t i n e , was f i r s t obtained i n pure form i n 1828 by steam d i s t i l l i n g the b a s i f i e d ether ex­ t r a c t of tobacco leaves (15). Although n i c o t i n e sometimes occurs i n the p l a n t i n the f r e e s t a t e , i t i s u s u a l l y present as the monoa c i d i c base of c i t r i c or m a l i c a c i d (16). Anabasine i s obtained by mixing ground tree tobacco w i t h ether and d i s t i l l i n g the a l k a l i n e e x t r a c t . Since t h i s compound and n o r n i c o t i n e have a secondary amino Ν group, they may be separated from n i c o t i n e by c o n v e r t i n g them to N - d e r i v a t i v e s . I d e n t i f i c a t i o n . The s t r u c t u r e s of n i c o t i n e , n o r n i c o t i n e , and anabasine were determined by degrading the molecules w i t h a c i d s , as w e l l as by TLC and ORD. Natural nicotine i s levorotatory; d e x t r o r o t a t o r y n i c o t i n e i s much l e s s t o x i c to i n s e c t s (16). Other A l k a l o i d s The major i n s e c t i c i d a l a l k a l o i d s other than those from t o ­ bacco are j e r v i n e , veratramine, cevine, genuine, zygadenine, and p r o t o v e r i n e ; they are present i n many s p e c i e s of Veratrum and Zygadenus, and i n s a b a d i l l a (Schoenocaulon o f f i c i n a l e A. Gray), members of the f a m i l y L i l i a c e a e . I s o l a t i o n . The a l k a l o i d s are e x t r a c t e d from the powdered p l a n t with a c i d i c a l c o h o l o r an o r g a n i c s o l v e n t w i t h o r without ammonia. The i n d i v i d u a l a l k a l o i d s are separated by f r a c t i o n a l c r y s t a l l i z a t i o n , chromatography on alumina, s i l i c a g e l , k i e s e l guhr, or an ion-exchange r e s i n * or by CCD (17).

156

HOST P L A N T RESISTANCE

T O PESTS

I d e n t i f i c a t i o n . The veratrum a l k a l o i d s are i d e n t i f i e d by i n f r a r e d and NMR spectroscopy, and by mass spectrometry (MS).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

Unsaturated Isobutylamides A number of i n s e c t i c i d a l isobutylamides of unsaturated, a l i p h a t i c , s t r a i g h t - c h a i n C-10 to C-18 acids have been i s o l a t e d from p l a n t s of the f a m i l i e s Compositae and Rutaceae. Although most of these compounds, which are h i g h l y t o x i c to f l i e s and mosquitoes, have been i d e n t i f i e d , and i n some cases s y n t h e s i z e d , others have been only p a r t i a l l y c h a r a c t e r i z e d . They possess two p r o p e r t i e s i n common w i t h one another and with the p y r e t h r i n s pungency, and r a p i d knockdown and k i l l of f l y i n g i n s e c t s . Examples of these compounds are a n a c y c l i n from Anacyclus pyrethrum DC, s p i l a n t h o l from S p i l a n t h e s spp., a f f i n i n from H e l i o p s i s longipes (A. Gray) Blake, s c a b r i n and h e l i o p s i n from H. scabra Dunal., e c h i n a c e i n from Echinacea a n g u s t i f o l i a DC, and h e r c u l i n from Zanthoxylum c l a v a - h e r c u l i s L. (18). I s o l a t i o n . These compounds are i s o l a t e d by the same procedures a p p l i c a b l e to the p y r e t h r i n s ; namely, by p a r t i t i o n of a petroleum ether e x t r a c t of the p l a n t between t h i s s o l v e n t and nitromethane followed by passage of the nitromethane s o l u t i o n through a c t i v a t e d charcoal and e i t h e r high-vacuum d i s t i l l a t i o n of the f i l t r a t e or column chromatography on alumina or s i l i c a g e l

(18). I d e n t i f i c a t i o n . The s t r u c t u r e s are determined by h y d r o l y s i s to the component amine and a c i d m o i e t i e s and by UV, IR, and NMR spectroscopy, and MS (18). Bitter

Substances

The limonoid b i t t e r p r i n c i p l e s are a c l a s s of C 2 6 t r i t e r p e n e s b e l i e v e d to a r i s e i n p l a n t s of the Meliaceae and Rutaceae f a m i l i e s as o x i d a t i o n products o f t e t r a c y c l i c t r i t e r p e n e s . They i n c l u d e such i n s e c t i c i d e s and feeding d e t e r r e n t s as l i m o n i n , n o m i l i n , melianone, nimbin, a z i d i r o n e , nimbalide, and s a l a n n i n ; melianone occurs i n M e l i a azedarach L. and the others occur i n neem, A z a d i r a c h t a i n d i c a (L.) Juss. Other limonoid b i t t e r p r i n c i p l e s are r a t h e r p l e n t i f u l among the Simaroubaceae. Limonoids o c c u r r i n g among the Meliaceae show, i n general, much g r e a t e r s t r u c t u r a l v a r i a t i o n and complexity than those i n the Rutaceae. The chemist r y of the b i t t e r substances was w e l l reviewed by Korte (19) i n 1959 and by Dreyer (20) i n 1968. I s o l a t i o n . Column chromatography of the f l a v o n o i d s and pept i d e s w i t h v a r i o u s types of packings has been compared (21). The most r e c e n t l y described method f o r s e p a r a t i n g f l a v o n o i d s and p i g ments from c i t r u s o i l s i n v o l v e s g e l permeation chromatography

10.

JACOBSON

Toxic

Agents from

Plants

157

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

followed by t h i n - l a y e r chromatography (22). One o f the most potent i n s e c t f e e d i n g d e t e r r e n t s , a z a d i r a c h t i n , occurs i n v a r i o u s p a r t s (mainly the seed k e r n e l s ) of A. i n d i c a . Although f i r s t i s o l a t e d from neem i n 1968 (23,24) by tedious column chromatography o f the ethanol e x t r a c t , i t was ob­ tained i n much higher y i e l d by Nakanishi's group a t Columbia U n i v e r s i t y (25) i n 1974 by means of a s i n g l e column chromatogra­ phy of the e x t r a c t on s i l i c a g e l followed by p r e p a r a t i v e t h i n l a y e r chromatography. I d e n t i f i c a t i o n . Limonin, the major limonoid i n c i t r u s seeds, has been known f o r over 100 years and, as a reasonably a c c e s s i b l e m a t e r i a l , has been the most e x t e n s i v e l y s t u d i e d member o f the limonoids. I t s s t r u c t u r e determination was reported i n 1960 and confirmed soon afterward by X-ray c r y s t a l l o g r a p h i c s t u d i e s on the iodoacetate of e p i l i m o n o l . The s t r u c t u r e determination o f many limonoids subsequently i s o l a t e d has followed the same p a t t e r n , that i s , degradation, IR, and NMR spectroscopy, and MS, as w e l l as ORD (20). The determination o f the exceedingly complex s t r u c t u r e o f a z a d i r a c h t i n would not have been p o s s i b l e without the simultane­ ous use of p a r t i a l l y r e l a x e d F o u r i e r transform and continuous wave decoupling carbon-13 NMR techniques (25). Toxic Fungal Peptides Isolation. I n s e c t i c i d a l peptides such as p h a l l o i d i n , p h a l l o i n , amanitin, and amanin were i s o l a t e d from Amanita pantherina (Fr.) Quelet (deadly a g a r i c ) and A. muscaria ( F r . ) S. F. Gray ( f l y a g a r i c ) as described by Wieland (26) and Eugster (27). The mushrooms were steeped i n methanol, the steepate was evaporated to a small volume, and the p r e c i p i t a t e d i n o r g a n i c sub­ stances were f i l t e r e d o f f . The f i l t r a t e was f r e e d o f s o l v e n t , d i s s o l v e d i n water, t r e a t e d with l e a d acetate and f i l t e r e d ; the f i l t r a t e , f r e e d o f l e a d with s u l f u r i c a c i d , was s a t u r a t e d with ammonium s u l f a t e a t pH 4 to p r e c i p i t a t e the p e p t i d e s . These toxic peptides are separable by p a r t i t i o n chromatography f o l l o w i n g t h e i r s e p a r a t i o n i n t o l i p o p h i l i c and h y d r o p h i l i c f r a c t i o n s by treatment with methyl e t h y l ketone-acetone-water (20:6:5). Paper and TLC are a l s o s a t i s f a c t o r y . Amanitin i s separable i n t o i t s α-, 3 - , and γ- forms (26). The peptides o f A. muscaria, which are h i g h l y t o x i c to house f l i e s and mosquitoes, may a l s o be i s o l a t e d by chromatography of an a l c o h o l i c e x t r a c t on a column o f Dowex 50 o r Amberlite IR-120 ( H ) , washing f i r s t with water and then with 2N formic a c i d . P u r i f i c a t i o n on Amberlite IRC-50(H ) y i e l d s i b o t e n i c a c i d and muscazone; d e c a r b o x y l a t i o n of i b o t e n i c a c i d gives muscinol. E l e c t r o p h o r e s i s may a l s o be used. An improved method g i v i n g l a r g e r y i e l d s o f the t o x i c compounds i n v o l v e s chromatography of a butanol e x t r a c t of the fungus on alumina, i n which water-saturated +

158

HOST P L A N T

RESISTANCE T O PESTS

butanol i s used as s o l v e n t (27). I d e n t i f i c a t i o n . The peptides a r e i d e n t i f i e d by degradation, by UV, IR, and NMR spectroscopy, and by MS (27). Gossypol

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

Gossypol i s a yellow c o l o r i n g matter o c c u r r i n g i n species o f the genus Gossypium that i s r e s p o n s i b l e f o r the r e s i s t a n c e o f c e r t a i n v a r i e t i e s o f cotton, G. hirsutum L. (family Malvaceae) to cotton i n s e c t s such as the b o l l w e e v i l . I t i s a l s o t o x i c to r a t s , guinea p i g s , and r a b b i t s , but not to c a t t l e f e e d i n g on cottonseed meal. I s o l a t i o n . Cottonseed k e r n e l s are e x t r a c t e d with petroleum ether to remove the o i l , and the gossypol i s then e x t r a c t e d with ether. A d d i t i o n o f a,cetic a c i d to the ether s o l u t i o n gives a c r y s t a l l i n e g o s s y p o l - a c e t i c a c i d complex (28). I d e n t i f i c a t i o n . Gossypol was i d e n t i f i e d by degradation and combination o f v a r i o u s forms o f spectroscopy, i n c l u d i n g MS (28). Mycotoxins The most important o f the mycotoxins today i s a f l a t o x i n , which i s found i n peanuts, cottonseed, and s t o r e d products contaminated w i t h the common mold A s p e r g i l l u s f l a v u s L i n k . Soon a f t e r attempts were made to i s o l a t e a f l a t o x i n i t became c l e a r that a t l e a s t 4 major a f l a t o x i n s , as determined by t h i n - l a y e r chromatography o f the b l u e - and g r e e n - f l u o r e s c i n g compounds, were involved; these were designated a f l a t o x i n s B-^, G^, and G 2 . At l e a s t 8 minor a f l a t o x i n s a r e now known. The a f l a t o x i n s a r e h i g h l y t o x i c to warm-blooded animals feeding on contaminated f o o d s t u f f s ; they are a l s o c a r c i n o g e n i c . I s o l a t i o n . A r a p i d procedure f o r s e p a r a t i n g the a f l a t o x i n s i n r o a s t e d peanuts u t i l i z e s t h i n - l a y e r p l a t e s coated w i t h Adsorbo s i l No. 1 and the s o l v e n t system benzene-ethanol-water (40:6:3) (29). E s p e c i a l l y recommended f o r the s o l v e n t e x t r a c t i o n o f a f l a toxins from mold-damaged commodities i s the use o f the azeotrope of 2-propanol-water (87.7:12.3) (30). I d e n t i f i c a t i o n . The a f l a t o x i n s are i d e n t i f i e d by chemical degradation, UV, IR, and NMR spectroscopy, and fluorometry. An e x c e l l e n t , e n t e r t a i n i n g d i s c u s s i o n o f the e n t i r e a f l a t o x i n problem, i n c l u d i n g methods f o r i s o l a t i o n and i d e n t i f i c a t i o n , appeared t h i s year i n a r e p o r t by G o l d b l a t t (31). Insect Growth Regulators ( J u v e n i l e Hormones)

10.

JACOBSON

Toxic Agents from

Plants

159

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

Many species of p l a n t s have been extracted and t e s t e d f o r the presence of j u v e n i l e hormone (JH)-mimicking a c t i v i t y (see references 32^ and 33 f o r recent reviews). These compounds have profound e f f e c t s on many species of i n s e c t s when a p p l i e d to the l a r v a l or pupal stage; i f a d u l t s do develop from such species they are almost always deformed i n one or s e v e r a l ways and may be incapable of feeding and(or) reproducing. The c l a s s i c a l example of a p l a n t - d e r i v e d i n s e c t juvenoid i s the s o - c a l l e d "paper f a c t o r " (juvabione) reported by Slama and Williams (34) i n 1965 and subsequently i s o l a t e d by Bowers e t a l . (35) from the wood of balsam f i r , Abies balsamea (L.) M i l l , (family Pinaceae). Juvabione was i d e n t i f i e d as the methyl e s t e r of todomatuic a c i d f o l l o w i n g chromatographic i s o l a t i o n . I s o l a t i o n . A more recent example of a juvenoid obtained from a p l a n t i s echinolone [(+)-(E)-10-hydroxy-4,10-dimethyl-4,lldodecadien-2-one], i s o l a t e d from the roots of the common American coneflower, Echinacea a n g u s t i f o l i a DC ( f a m i l y Compositae) (36). Echinolone was obtained i n pure form by p a r t i t i o n i n g a pentane ext r a c t of the roots between pentane and nitromethane, s e p a r a t i n g the pentane-soluble p o r t i o n i n t o n e u t r a l , a c i d i c , and b a s i c f r a c t i o n s , and chromatographing the n e u t r a l f r a c t i o n on successive columns of F l o r i s i l , s i l i c a g e l , and s i l v e r n i t r a t e - c o a t e d s i l i c a g e l p r i o r to p r e p a r a t i v e gas chromatography. Identification. Echinolone was i d e n t i f i e d by IR and NMR spectroscopy, MS, microozonolysis, and ORD measurements (36). Insect Growth Regulators

(Molting Hormones)

The best-known examples of these compounds are the p o l y hydroxy s t e r o l ecdysone and i t s analogs i s o l a t e d from i n s e c t s and p l a n t s (37). Treatment of i n s e c t pupae with these compounds causes g r e a t l y a c c e l e r a t e d growth and development that r e s u l t i n premature breaking of diapause and, i n some cases, g i a n t a d u l t s , c o n d i t i o n s that are detrimental to the normal l i f e of the i n s e c t . Isolation. Since the polyhydroxy s t e r o l s are h i g h l y s o l u b l e i n p o l a r s o l v e n t s and s p a r i n g l y s o l u b l e i n nonpolar s o l v e n t s , the p l a n t m a t e r i a l s are extracted with a l c o h o l s . P u r i f i c a t i o n of the e x t r a c t s i s obtained by a combination of p a r t i t i o n , l i q u i d chromatography, p r e p a r a t i v e TLC, and c r y s t a l l i z a t i o n . CCD i s a l s o used. A mixture of e c d y s t e r o l s that i s d i f f i c u l t to separate may be a c e t y l a t e d to g i v e a mixture of acetates whose s e p a r a t i o n may be much e a s i e r , a f f o r d i n g pure acetates from which the f r e e ecdys t e r o l s can be regenerated by h y d r o l y s i s (38). The i s o l a t i o n of e c d y s t e r o l s i s now most commonly c a r r i e d out by TLC on s i l i c a gel-coated p l a t e s o r by column chromatography on Amberlite XAD-2 with gradient e l u t i o n by using 30 to 70% ethanol while monitoring the eluates by absorption at 254 nm (38).

160

HOST P L A N T

RESISTANCE T O PESTS

A s i m p l i f i e d procedure f o r f r a c t i o n a t i n g p l a n t m a t e r i a l s by s e q u e n t i a l e x t r a c t i o n w i t h methanol-chloroform-water and phenola c e t i c acid-water mixtures gives water-soluble, low molecular weight compounds w i t h l i t t l e chemical damage (39). Identification. S t r u c t u r a l e l u c i d a t i o n of ecdysone w i t h UV, IR, and NMR spectroscopy r e s u l t e d i n a p a r t i a l s t r u c t u r e , which was completed only with the help of i t s X-ray d i f f r a c t i o n p a t t e r n and MS. A comprehensive review of ecdysone chemistry i s that by Horn (37).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

A d d i t i o n a l Information on Methods of I s o l a t i o n and

Identification

Thin-Layer Chromatography. New procedures f o r s e p a r a t i n g compounds by programmed m u l t i p l e development TLC e q u i v a l e n t to 5,000-8,000 t h e o r e t i c a l p l a t e s have r e c e n t l y been d e s c r i b e d (40). The usual s o l v e n t system i s e t h y l acetate-ethylene d i c h l o r i d e (1:10). The most recent book on TLC i s that e n t i t l e d "HPTLC. High Performance Thin Layer Chromatography, e d i t e d by Z l a t k i s and K a i s e r , which j u s t appeared (41). 11

High-Performance L i q u i d Chromatography (HPLC). Hostettmann et a l . (42) have very r e c e n t l y reported a method f o r o b t a i n i n g pure compounds d i r e c t l y from crude p l a n t e x t r a c t s by f i l t r a t i o n of a hexane e x t r a c t through a column of s i l i c a g e l with e t h e r hexane (1:1) followed by p r e p a r a t i v e HPLC on s i l i c a g e l w i t h ether-hexane (1:9) and two runs with e t h y l acetate-hexane (1:4). This procedure r e q u i r e d only 2-3 hours compared w i t h conventional column chromatography, which r e q u i r e d 2 weeks. No degradation of unstable compounds was observed. HPLC on s i l v e r n i t r a t e - c o a t e d s i l i c a g e l (43) and a reverse phase method on μ Bondapak C-18 (44) i s of value i n s e p a r a t i n g c i s and trans isomers of unsaturated compounds. I n f r a r e d Spectroscopy. The a p p l i c a t i o n of IR spectroscopy to the i d e n t i f i c a t i o n of n a t u r a l products was reported by Cole (45) i n 1956. The use o f F o u r i e r transform IR i n research has brought new and extended c a p a b i l i t i e s i n s p e c t r a l s e n s i t i v i t y and resolution. V i b r a t i o n a l frequency assignments were p u b l i s h e d i n book form by S i l v e r s t e i n e t a l . (46) i n 1974, by Bellamy (47) i n 1975, and by Pearse and Gaydon (48) i n 1976. NMR Spectroscopy. In 1939, Linus P a u l i n g (49) published a review on the c o n f i g u r a t i o n and e l e c t r o n i c s t r u c t u r e o f molecules w i t h a p p l i c a t i o n to n a t u r a l products. T h i s may be considered the forerunner of the use o f NMR f o r the s t r u c t u r a l determination of n a t u r a l products. In 1965, Jackman (50) reviewed the use of NMR spectroscopy f o r determining e m p i r i c a l formulae, the c l a s s e s of

10.

JACOBSON

Toxic

Agents from

Plants

161

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

protons i n a molecule, the sequence of groups i n a molecule, r e l a t i v e stereochemistry, and conformation as a p p l i e d to n a t u r a l products. 23 The use of C NMR spectroscopy has helped immensely i n determining n a t u r a l products s t r u c t u r e , s i n c e the -^C n u c l e i are u s u a l l y completely decoupled from a l l o f the n u c l e i by the use of double resonance. The spectrum i s thus simply a s e r i e s o f s i n g l e t s corresponding to each v a r i e t y of carbon atom present. Textbooks on NMR spectroscopy o r i e n t e d toward organic chemists are those by Stothers (51) and by Levy and Nelson (52), both publ i s h e d i n 1972, by Levy (53) i n 1974 and 1975, and by Muellen and Pregosin (54), published i n 1977. Mass Spectrometry. In 1966, Biemann (55) published a review of MS as a p p l i e d to n a t u r a l products, e s p e c i a l l y v a r i o u s types of alkaloids. S t e r o i d s have been very thoroughly i n v e s t i g a t e d by t h i s technique by D j e r a s s i ' s group, and the i n f o r m a t i o n gained has helped considerably with the complicated e l e c t r o n impact i n duced fragmentation r e a c t i o n s o f organic molecules. The p o l y c y c l i c nature o f many p h y s i o l o g i c a l l y a c t i v e n a t u r a l products p r a c t i c a l l y excludes simple fragmentation processes and r e q u i r e s cleavage of a number o f bonds f o r the production o f a fragment. However, c o n s u l t a t i o n of a v a i l a b l e t a b l e s o f f r e q u e n t l y encountered fragment ions and c o l l e c t i o n s o f mass s p e c t r a f o r the c l a s s of compound under c o n s i d e r a t i o n makes s t r u c t u r a l determination much e a s i e r . F i e l d d e s o r p t i o n and e l e c t r o n impact MS are recent innovations of considerable importance. An e x c e l l e n t book f o r a i d i n i n t e r p r e t i n g mass s p e c t r a i s that published by M c L a f f e r t y (56) i n 1973. A novel r e p r e s e n t a t i o n of data o b t a i n a b l e from doublef o c u s s i n g mass spectrometers has been developed. I t can d i s p l a y on a s i n g l e three-dimensional s u r f a c e the normal mass spectrum together with peaks due to a l l metastable t r a n s i t i o n s o c c u r r i n g i n the instrument. Developed by Drs. Macdonald and Lacey, o f the C.S.I.R.O. D i v i s i o n of Entomology i n Canberra, A u s t r a l i a , t h i s three-dimensional r e p r e s e n t a t i o n i s more s e n s i t i v e to molecular s t r u c t u r e than any o f the two-dimensional r e p r e s e n t a t i o n s commonly used by mass s p e c t r o m e t r i s t s (57). An e x c e l l e n t review o f m i c r o a n a l y t i c a l methodology u s e f u l i n i d e n t i f y i n g unsaturated compounds i s that published i n 1975 by Beroza (58). I t includes b r i e f d i s c u s s i o n s of s p e c t r a l a n a l y s i s , chemical reagents u s e f u l i n determining f u n c t i o n a l groups, ozonol y s i s to determine double bond p o s i t i o n , and carbon-skeleton chromatography. Disclaimer The use of trade or p r o p r i e t a r y names does not n e c e s s a r i l y imply the endorsement of these products by the U.S. Department of Agriculture.

162

HOST P L A N T RESISTANCE

T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

Literature Cited

1. Staudinger, Η., and Ruzicka, L. Helv. Chim. Acta (1924), 7, 177-83. 2. LaForge, F. B., and Barthel, W. F. J. Org. Chem. (1944), 9, 242-9. 3. Godin, P. J., Sleeman, R. J., Snarey, Μ., and Thain, Ε. M. J. Chem. Soc. (C) (1966), 332-4. 4. Nakanishi, Κ., Goto, T., Ito, S., Natori, S., and Nozoe, S., eds., "Natural Products Chemistry," vol. 1, 562 pp., Academ­ ic, New York (1974). 5. Nakanishi, Κ., Goto, T., Ito, S., Natori, S., and Nozoe, S., eds., "Natural Products Chemistry," vol. 2, 586 pp., Academ­ ic, New York (1975). 6. Jacobson, Μ., and Crosby, D. G., eds., "Naturally Occurring Insecticides," 585 pp., Marcel Dekker, New York (1971). 7. Barthel, W. F., and Haller, H. L. U.S. Patent 2,372,183 (1945). 8. Elliott, M., Olejniczak, J. S., and Garner, J. J. Pyrethrum Post (1959), 5(2), 8-11. 9. Crombie, L., and Elliott, M. Fortschr. Chem. Org. Natur­ stoffe (1961), 19, 120-64. 10. Matsui, M., and Yamamoto, I. In reference 6, pp. 3-70. 11. Crombie, L. Fortschr. Chem. Org. Naturstoffe (1963), 21, 275-325. 12. Freudenthal, R. I., Emmerling, D. C., and Baron, R. L. J. Chromatog. (1977), 134, 207-10. 13. Fukami, Η., and Nakajima, M. In reference 6, pp. 81-97. 14. Feinstein, L., and Jacobson, M. Fortschr. Chem. Org. Natur­ stoffe (1953), 10, 423-76. 15. Spaeth, Ε., and Kuffner, F. Fortschr. Chem. Org. Naturstoffe (1939), 2, 248-300. 16. Schmeltz, I. In reference 6, pp. 99-116. 17. Narayanan, C. R. Fortschr. Chem. Org. Naturstoffe (1962), 20, 298-371. 18. Jacobson, M. In reference 6, pp. 137-76. 19. Korte, F., Barkemeyer, Η., and Korte, I. Fortschr. Chem. Org. Naturstoffe (1959), 17, 124-82. 20. Dreyer, D. L. Fortschr. Chem. Org. Naturstoffe (1968), 26, 190-244. 21. Ward, R. S., and Pelter, A. J. Chromatog. Sci. (1974), 12, 570-4. 22. Wilson, C. W., III, and Shaw, P. E. J. Agr. Food Chem. (1977), 25, 211-4. 23. Butterworth, J. Η., and Morgan, E. D. Chem. Commun. (1968), 23-4. 24. Butterworth, J. H., Morgan, E. D., and Percy, G. R. J. Chem. Soc. Perkin Trans. (1972), 1, 2445-50. 25. Zanno, P. R., Miura, I., Nakanishi, Κ., and Elder, D. L. J. Am. Chem. Soc. (1975), 97, 1975-7.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

10.

JACOBSON

Toxic Agents

from

Pfonts

163

26. Wieland, T. Fortschr. Chem. Org. Naturstoffe (1967), 25, 214-50. 27. Eugster, C. H. Fortschr. Chem. Org. Naturstoffe (1969), 27, 261-321. 28. Adams, R., Geissman, Τ. Α., and Edwards, J. D. Chem. Rev. (1960), 555-74. 29. Waltking, A. E., Bleffert, G. W., Chick, Μ., and Fogerty, M. Oils Oilseeds J. (Bombay) (1975), 28(2), 32-3. 30. Rayner, E. T., Koltun, S. P., and Dollear, F. G. J. Am. Oil Chem. Soc. (1977), 54, 242A-4A. 31. Goldblatt, L. A. J. Am. Oil Chem. Soc. (1977), 54, 302A-9A. 32. Jacobson, M. Mitt. Schweiz. Entomol. Ges. (1971), 44, 73-7. 33. Jacobson, M., Redfern, R. E., and Mills, G. D., Jr. Lloydia (J. Nat. Prod.) (1975), 38, 455-72. 34. Slama, Κ., and Williams, C. M. Proc. Natl. Acad. Sci. U.S. (1965), 154, 411-14. 35. Bowers, W. S., Fales, Η. Μ., Thompson, M. J., and Uebel, E. C. Science (1966), 154, 1020-2. 36. Jacobson, M., Redfern, R. Ε., and Mills, G. D., Jr. Lloydia (J. Nat. Prod.) (1975), 38, 473-6. 37. Horn, D. H. S. In referency 6, pp. 333-459. 38. Hikino, H., and Hikino, Y. Fortschr. Chem. Org. Naturstoffe (1970), 28, 256-312. 39. Laird, W. M., Mbadiwe, Ε. I., and Synge, R. L. M. J. Sci. Food Agr. (1976), 27, 127-30. 40. Jupille, T. H. J. Am. Oil Chem. Soc. (1977), 54, 179-82. 41. Zlatkis, Α., and Kaiser, R. E., eds., "HPTLC. High Pressure Thin Layer Chromatography," 240 pp., Elsevier, New York (1977). 42. Hostettmann, Κ., Pettei, M. J., Kubo, I., and Nakanishi, K. Helv. Chim. Acta (1977), 60, 670-2. 43. Heath, R. R., Tumlinson, J. H., Doolittle, R. E., and Proveaux, A. T. J. Chromatog. Sci. (1975), 13, 380-2. 44. Warthen, J. D., Jr. J. Am. Oil Chem. Soc. (1975), 52, 151-3. 45. Cole, A. R. H. Fortschr. Chem. Org. Naturstoffe (1956), 13, 1-69. 46. Silverstein, R. M., Bassler, G. C., and Morrill, T. C. "Spectrometric Identification of Organic Compounds," 3rd ed., pp. 73-119, Wiley, New York (1974). 47. Bellamy, L. J. "The Infra-red Spectra of Complex Molecules," 3rd ed., 433 pp., Wiley, New York (1975). 48. Pearse, R. W. B., and Gaydon, A. G. "The Identification of Molecular Spectra," 4th ed., 408 pp., Halsted (Wiley), New York (1976). 49. Pauling, L. Fortschr. Chem. Org. Naturstoffe (1939),3,20335. 50. Jackman, L. M. Fortschr. Chem. Org. Naturstoffe (1965), 21, 275-325. 51. Stothers, J. B. "Carbon-13 NMR Spectroscopy," Academic, New York (1972).

164

HOST P L A N T RESISTANCE

T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

52. Levy, G. C., and Nelson, G. L. "Organic Carbon-13 Nuclear Magnetic Resonance for Organic Chemists," Wiley, New York (1972). 53. Levy, G. C. (ed.), "Topics in Carbon-13 NMR Spectrometry," Wiley, New York, vol. 1 (1974); vol. II (1975). 54. Muellen, Κ., and Pregosin, P. S. "Fourier Transform NMR Techniques," Academic, London (1977). 55. Biemann, K. Fortschr. Chem. Org. Naturstoffe (1966),24,198. 56. McLafferty, F. W. "Interpretation of Mass Spectra," 2nd ed., W. A. Benjamin, New York (1973). 57. Lacey, M. J. and Macdonald, C. G. "Organic Mass Spectro­ metry," 1977, in press. 58. Beroza, M. J. Chromatog. Sci. (1975), 13, 314-21.

11 Insect Antifeedants and Repellents from African Plants I. KUBO and K. NAKANISHI

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

Department of Chemistry, Columbia University, New York, NY 10027

There is little doubt t h a t the tropical flora which a r e c o n s t a n t l y exposed t o a t t a c k by v a r i o u s p a r a sites such as v i r u s e s , b a c t e r i a , p r o t o z o a n s , f u n g i , and i n s e c t s a r e c o n f r o n t e d w i t h much h a r s h e r c o n d i t i o n s f o r survival than their temperate c o u n t e r p a r t s . T h i s necessarily leads to efficient built-in defense mechanisms and it is presumably f o r this r e a s o n t h a t tropical flora o f f e r a rich and intriguing source f o r isolating natural products possessing attractive pesticidal or medicinal properties. The results o u t l i n e d in the f o l l o w i n g stem from the s t u d i e s initiated in 1974 and more s y s t e m a t i c a l l y i n mid-1975 a t the I n t e r n a t i o n a l C e n t r e o f I n s e c t P h y s i o l o g y and E c o l o g y (ICIPE), N a i r o b i , Kenya. The g e n e s i s o f this unique c e n t e r goes back t o 1968 when C a r l D j e r a s s i (1) proposed a p l a n f o r establishing an international institute i n a d e v e l o p i n g country and Thomas R. Odhiambo, P r o f e s s o r o f Entomology a t the U n i v e r s i t y o f N a i r o b i , responded enthusiastically to initiate an i n s e c t o r i e n t e d institute in Nairobi. ICIPE was i n a u g u r a t e d in e a r l y 1970 w i t h generous s u p p o r t from the U n i t e d N a t i o n s Development Program, various o t h e r f o u n d a t i o n s , and f e d e r a l governments. Of a total of about 130 p e r s o n n e l c u r r e n t l y a t the C e n t r e , 25 are R e s e a r c h S c i e n t i s t s (post P h . D . ' s ) , m o s t l y b i o l o g i s t s , from a l l over the w o r l d who come f o r a p e r i o d o f s e v e r a l y e a r s t o be engaged i n b a s i c and i n t e r d i s c i p l i n ary s t u d i e s o f a r t h r o p o d s o f a g r i c u l t u r a l and m e d i c a l importance. An u n u s u a l system adopted a t ICIPE i s t h a t of V i s i t i n g D i r e c t o r s o f R e s e a r c h . These 10-15 VDR's from v a r i o u s i n s t i t u t i o n s t h r o u g h o u t the w o r l d pay one t o s e v e r a l v i s i t s a y e a r t o the C e n t r e t o c o n s u l t and d i r e c t (?) the r e s e a r c h . The C h e m i s t r y u n i t has had two R e s e a r c h S c i e n t i s t s (ex-members, D r s . D . L . E l d e r , T . G e b r e y s u s , W . F . Wood, I . Kubo, G . D . P r e s t w i c h ; and

165

166

HOST P L A N T RESISTANCE T O

PESTS

c u r r e n t members, Drs. A. Maradufu and K. W i l s o n ) and one S e n i o r T e c h n i c i a n , A. Chapya; the two VDR's have been J . Meinwald and K. N a k a n i s h i . The s t u d i e s on p l a n t c o n s t i t u e n t s comprise one h a l f o f the work c a r r i e d o u t by the C h e m i s t r y u n i t , the o t h e r h a l f b e i n g s t u d i e s on i n s e c t pheromones and d e f e n s e s e c r e t i o n s (2) .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

Bioassay

of Phagostimulants

and

Antifeedants

Two s p e c i e s o f the A f r i c a n armyworm, Spodoptera exempta and S. l i t t o r a l i s , were m a i n l y employed f o r the b i o a s s a y s , e i t h e r by e l e c t r o p h y s i o l o g y o r by t h e l e a f d i s k method. The monophagous S. exempta has l o n g been known as a major graminaceous c r o p p e s t i n E a s t and South A f r i c a , whereas the polyphagous S. l i t t o r a l i s i s a major c o t t o n p e s t . Other s p e c i e s o f the genus Spodop­ t e r a are d i s t r i b u t e d t h r o u g h o u t the w o r l d and c o n s t i t u t e a major p e s t . However, most o f the a n t i f e e d a n t s i s o ­ l a t e d so far appear t o be s p e c i f i c t o c e r t a i n i n s e c t s . F o r i n s t a n c e , w a r b u r g a n a l (see below) (_3) r which i s one of the most p o t e n t a n t i f e e d a n t s a g a i n s t S. exempta was h a r d l y a c t i v e a g a i n s t Manduca s e x t a (tobacco hornworm) and S c h i s t o c e r c a vaga (vagrant g r a s s h o p p e r ) ( 4 ) . Hence two i n s e c t s w h i c h seem t o have complementary t a s t e senses are c u r r e n t l y used i n our l a b o r a t o r y , namely, E p i l a c h n i a v a r i v e s t i s (Mexican bean b e e t l e ) and S. e r i d a n i a ( s o u t h e r n armyworm) . The most r e v e a l i n g b i o a s s a y f o r f o l l o w i n g phago­ s t i m u l a n t s and d e t e r r e n t s i s t h a t o f e l e c t r o p h y s i o l o g y w i t h the e i g h t s e n s i l l a a t the t i p o f t h e m a x i l l a r y p a l p ( 5 ) . These c o r r e s p o n d t o t h e t a s t e buds and a r e shown Γη F i g u r e 1 ( s c a n n i n g e l e c t r o m i c r o g r a p h ) . As d e p i c t e d s c h e m a t i c a l l y i n F i g u r e 2, a m i c r o e l e c t r o d e i s i n s e r t e d i n t o the m a x i l l a r y p a l p and another i n t o one of the s e n s i l l u m . The t i p o f the s e n s i l l u m i s b r o u g h t i n t o c o n t a c t w i t h a f i l t e r paper impregnated w i t h t h e t e s t s o l u t i o n , and the s c o r i n g , w h i c h i s r e c o r d e d as i m p u l s e s p e r second, i s done w i t h an o s c i l l o s c o p e . The maximum number o f i m p u l s e s t h a t can be evoked i s o f the o r d e r o f 200. The f u n c t i o n s o f t h e e i g h t s e n s i l l a appear t o d i f f e r b u t the d e t a i l s have n o t y e t been c l a r i f i e d (5) . Most of the compounds d e s c r i b e d i n t h i s a r t i c l e were i s o l a t e d by f o l l o w i n g the f r a c t i o n a t i o n w i t h more conventional bioassays. The l e a f d i s k method i s shown i n F i g u r e 3 (see 6) . L e a f d i s k s made w i t h a 20 mm cork b o r e r a r e immersed f o r 2 seconds i n the t e s t a c e t o n e s o l u t i o n ; immersion f o r l o n g e r than 2 seconds s h o u l d be a v o i d e d because the p h a g o s t i m u l a n t s (see below) are ex­ t r a c t e d out. These a r e p l a c e d i n a P e t r i d i s h w i t h con-

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

11.

KUBO

A N D NAKANISHI

Insect Antifeedants

and

Repellents

167

Figure 1. Scanning electromicrograph (X1650) of tip region of African armyworm (Spodoptera exempta) larva. The maxillary palp of eight sensilla are seen.

Spodoptera exempta

Feeding

Stimulants

sucrose inositol adenosine

Figure 2.

Schematic of electrophycological experiment with sensilla

168

HOST P L A N T RESISTANCE T O PESTS

Spodoptera exempta S. littoralis (African army worms)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

Zea mays "~

Test

Ten Insect larvae I

Observation Acetone or Hexane

2, 4, 6, 8, 10, 12, 24, 48 hours Control disks were eaten usually within 2 hours (CHOICE Test)

Figure 3.

The leaf disk bioassay—a "choice" method

t r o l l e a f d i s k s and t e n t h i r d i n s t a r l a r v a e o f S. exempta. T h i s c o n s t i t u t e s a " c h o i c e " t e s t , whereas d e l e t i o n o f t h e c o n t r o l d i s k s c o n s t i t u t e s a "no c h o i c e " test. The s c o r i n g i s c a r r i e d o u t o v e r a p e r i o d o f up t o 2 days depending on t h e a n t i f e e d a n t p o t e n c y . A l t e r n a t i v e l y , t h e sample s o l u t i o n c a n be a p p l i e d d i r e c t l y o n t o s e v e r a l l e a v e s o f p l a n t i n a p o t and scored against c o n t r o l leaves. A f u r t h e r method o f b i o a s s a y i s t o c o a t t h e sample on s t y r o f o a m l a m e l l a e and measure t h e w e i g h t d i f f e r e n c e between u n t r e a t e d l a m e l l a e . The e l e c t r o p h y s i o l o g i c a l method has c l a r i f i e d t h e sugar r e c e p t o r s on S. exempta l a r v a l s e n s i l l a (j>) , and by f o l l o w i n g s y s t e m a t i c f r a c t i o n a t i o n s , two o f t h e p h a g o s t i m u l a n t s c o n t a i n e d i n maize l e a v e s have been i s o l a t e d and c h e m i c a l l y c h a r a c t e r i z e d as s u c r o s e and adenosine ( 7 ) . An i m p o r t a n t f i n d i n g from t h e sugar

11.

KUBO

AND

NAKANISHI

Insect Antifeedants

and

Repellents

169

r e c e p t o r e x p e r i m e n t s i s the f a c t t h a t a few c o n t a c t s o f the s e n s i l l a w i t h f i l t e r paper impregnated w i t h p o t e n t a n t i f e e d a n t s such as w a r b u r g a n a l l e a d t o i r r e v e r s i b l e l o s s o f t a s t e r e s p o n s e ; i n a d d i t i o n , the a n t i f e e d a n t a c t i v i t y o f w a r b u r g a n a l i s i n h i b i t e d by a d d i t i o n o f an e q u i m o l a r amount o f the S H - c o n t a i n i n g L - c y s t e i n e (1).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

Antifeedants F o r the sake o f s e l f - d e f e n s e , i t i s not a t a l l s u r ­ p r i s i n g t h a t p l a n t s would c o n t a i n i n s e c t a n t i f e e d a n t s , as w e l l as p e s t i c i d e s and r e p e l l e n t s (7a). S i n c e t h e l i f e cycle o f an i n s e c t i s deranged i f the up-take o f exogenous m o u l t i n g hormones (ecdysones) f a r exceeds the b i o s y n t h e t i c l e v e l , t h e wide o c c u r r e n c e i n p l a n t s o f compounds w i t h m o u l t i n g hormone a c t i v i t y (phytoecdysones) ( 8 ^ , 1 0 ) presumably i s a l s o a s e l f - d e f e n d i n g e v o l u t i o n a r y development. W i t h the u n d e r s t a n d i n g t h a t the term f e e d i n g i s not c l a r i f i e d w i t h i n s e c t s , i . e . , i t may i n v o l v e b i t i n g and/or swallowing f a c t o r s , we would l i k e t o b r o a d l y d e f i n e i n s e c t a n t i f e e d a n t s as s u b s t a n c e s which when t a s t e d can r e s u l t i n c e s s a t i o n o f f e e d i n g e i t h e r t e m p o r a r i l y o r permanently depending upon p o t e n c y (3>) · Some n a t u r a l l y o c c u r r i n g a n t i f e e d a n t s which have been p u b l i s h e d a r e : g l y c o s i d e s o f s t e r o i d a l a l k a l o i d s , d e m i s s i n e (11), s o l a c a u l i n e (12), t o m a t i n e (13), l e p t i n e s I and I I (14); r i n g A enone and/or r i n g D a r o m a t i c s t e r o i d s , the n i c a n d r e n o n e s and o t h e r s (15, 16,Γ7,19^); j u g l o n e (5-hydroxynaphthoquinone) (19); the i s o q u i n o l i n e a l k a l o i d i s o b o l d i n e (6); p h e n y l p r o p a n o i d s (20); germacrane s e s q u i t e r p e n e s s h i r o m o d i o l and s h i r o mol (21,22) ; e n t - c l e r o d a n e and c l e r o d a n e d i t e r p e n e s , c l e r o d e n d r i n (23) , c a r y o p t i n and o t h e r s (24), and the h y d r o x y l a t e d s t e r o i d m e l i a n t r i o l (25). The c o l l e c t i o n s i n E a s t A f r i c a were c a r r i e d o u t on the b a s i s o f i n f o r m a t i o n g a t h e r e d from books (26,27) and e s p e c i a l l y from l o c a l p e o p l e on i n s e c t - r e s i s t a n t plants. In f a c t , some o f the p l a n t s a r e w i d e l y used t o c o n t r o l pest i n s e c t s or f o r medicinal purposes. The e x t r a c t i o n s were f o l l o w e d by b i o a s s a y s , and s t r u c t u r e s were d e t e r m i n e d by a c o m b i n a t i o n o f v a r i o u s s p e c t r a l methods combined o c c a s i o n a l l y w i t h c h e m i c a l r e a c t i o n s . a) A z a d i r a c h t i n from Leaves and B e r r i e s o f A z a d i r achta"""Indica ( I n d i a n neem t r e e ) and tlaLia, a z e d a r a c h (28). The t r e e s o c c u r commonly i n I n d i a and E a s t and West A f r i c a and are w i d e l y used f o r chewing s t i c k s f o r c l e a n i n g the t e e t h and as a remedy a g a i n s t m a l a r i a . The S c h i s t o c e r c a g r e g a r i a ( d e s e r t l o c u s t ) a n t i f e e d a n t m e l i a n t r i o l (25) was i s o l a t e d from the l e a v e s . Azadir­ a c h t i n was a n o n c r y s t a l l i n e compound reknown f o r i t s

170

HOST P L A N T RESISTANCE T O

PESTS

very strong antifeedant p r o p e r t i e s a g a i n s t S. g r e g a r i a , i . e . , 100% i n h i b i t i o n of feeding i s caused by 40 y g / l , o r when impregnated on f i l t e r paper by 1 ng/cm AcO' (29,20). A l t h o u g h p a r MeOOC t i a l s t r u c t u r e s and the c o r r e c t molecular formula Azadirachtin 35 44°l6 forwarded (29), c o m p l e x i t y o f the s t r u c t u r e e l u d e d e l u c i d a t i o n w i t h o u t t h e usage o f CMR. The s t r u c t u r e i s based on e x t e n s i v e NMR s t u d i e s on a z a d i r a c h t i n and i t s 14-monoa c e t a t e ( p r o b a b l y formed by a c y l m i g r a t i o n from 7-OH), i n p a r t i c u l a r the t e c h n i q u e o f simultaneous p a r t i a l l y r e l a x e d F o u r i e r t r a n s f o r m (PRFT) and o f f - r e s o n a n c e d e c o u p l i n g , which was used f o r the f i r s t t i m e . It is a l i m o n o i d c o n t a i n i n g a c l e a v e d C r i n g and an u n s t a b l e d i h y d r o f u r a n moiety. I t i s one o f t h e most p o t e n t a n t i f e e d a n t s a g a i n s t S. exempta ( r o u g h l y o n e - h a l f o f the warburganal p o t e n c y ) . The s t r u c t u r e i s t o o complex t o be s y n t h e s i z e d on a p r a c t i c a l l e v e l b u t s i n c e the y i e l d i s q u i t e h i g h (we were a b l e t o o b t a i n 800 mg from 300 g o f seeds) and the t r e e i s easy t o c u l t i v a t e , p r a c t i c a l usage o f the neem t r e e may not be o u t o f the q u e s t i o n . b) X y l o m o l i n from U n r i p e F r u i t s o f X v l o c a r p u s mpluccënsls. Roem. (Meliaceae) ( 3 1 , 3 Τ Π The u n r i p e f r u i t s (weighing about 200 g) COOCH. are b i t t e r b u t the r i p e f r u i t s a r e t r e a s u r e d by ..OCH^ E a s t A f r i c a n s and are r e p u t e d t o have a p h r o d i s ­ i a c p r o p e r t i e s . Since a l l o f the s k e l e t a l p r o ­ t o n s are c o n t i g u o u s , PMR e l u c i d a t e d the e n t i r e Xylomolin p r o t o n system. The con­ f i g u r a t i o n s a t c h i r a l c e n t e r s a r e based on the f a c t s t h a t no J v a l u e s were l a r g e r than 3 Hz, and n e i t h e r NOE nor W-type c o u p l i n g s c o u l d be o b s e r v e d . Presence o f the a c e t a l i c methoxyl group was deduced from the p r e s ­ ence o f a d o u b l e t o f q u a r t e t s s i g n a l i n the undecoupled CMR spectrum; t h i s i s due t o a d d i t i o n a l c o u p l i n g o f the methoxyl carbon t o 3-H. I t i s a s e c o i r i d o i d with a s t r u c t u r e c l o s e l y r e l a t e d t o s e c o l o g a n i n , the o r i g i n o f the t e r p e n o i d d e r i v e d m o i e t i e s i n i n d o l e a l k a l o i d s . It i s possible that xylomolin i s a progenitor of alkaloids which may be c o n t a i n e d i n the r i p e f r u i t s b u t t h i s has n o t been i n v e s t i g a t e d . The a n t i f e e d a n t l e v e l i s moder2

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

C

H

w

e

r

e

11.

KUBO

A N D NAKANISHI

Insect Antifeedants

and

Repellents

171

12

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

Ar-I

Ar-H

Ar — III

a t e , i . e . , 100 ppm a g a i n s t S. exempta; i t s t r o n g l y i n h i b i t s the r e s p i r a t i o n o f r a t l i v e r mitochondria. c) A j u g a r i n s from Aiucra remota ( L a b i a t a e ) Leaves (33). As quoted above, o t h e r c l e r o d a n e a n t i f e e d a n t s have been i s o l a t e d p r e v i o u s l y (23,24) . However, i t i s i n t e r e s t i n g t o note t h a t b o t h a n t i p o d a l s k e l e t o n s a r e a c t i v e , as e x e m p l i f i e d by c l e r o d e n d r i n (23), an e n t c l e r o d a n e d i t e r p e n e , and c a r y o p t i n (24), a c l e r o d a n e diterpene. The a b s o l u t e c o n f i g u r a t i o n o f t h e a j u g a r i n s ( e n t - c l e r o d a n e ) was d e t e r m i n e d (3J3) by c o n v e r s i o n t o a 6-keto compound and comparing i t s CD w i t h a r e f e r e n c e compound d e r i v e d from c l e r o d i n (24). The a n t i f e e d a n t a c t i v i t y i s 100 ppm a g a i n s t S. exempta. d) H a r r i s o n i n from Leaves o f H a r r i s o n i a a b v s s i n i c a O i v (Simarubaceae) ("Msabubini" o r "Mpapura-doko" i n T h i s shrub i s used w i d e l y i n E a s t Swahili) (34) A f r i c a n f o l k remedies i n c l u d i n g treatment f o r bubonic p l a g u e , hemorrhoid, snake b i t e , etc. The chopped r o o t (650 g) gave 70 mg o f h a r r i s o n i n and 25 mg o f obacunone, a r e l a t e d limonoid b i t t e r p r i n c i p l e (35, 36). The s t r u c t u r e was d e t e r mined by e x t e n s i v e NMR s t u d i e s , e s p e c i a l l y CMR (PND, PRFT and 28H undecoupled) and comparisons o f peaks w i t h t h o s e o f obacuHarrisonin

172

HOST P L A N T RESISTANCE T O

TESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

none. A l t h o u g h i t was not c l e a r whether the h e m i k e t a l f u n c t i o n a t C-7 was an a r t e f a c t o r n o t , subsequent s t u d i e s have shown t h a t the n a t u r a l p r o d u c t i s a 6,7alpha-diketone. The S. exempta a n t i f e e d a n t a c t i v i t y i s 20 ppm; i t a l s o e x h i b i t s a n t i b i o t i c a c t i v i t y (5 yg/ml a g a i n s t B a c i l l u s s u b t i l i s ) (37) and c y t o t o x i c i t y (2.2 yg/ml, KB t e s t ) (WT. e) P o l y g o d i a l , U g a n d e n s i d i a l and Warburganal from the Bark o f W a r b u r a i a s t u h l m a n n i i and W. u a a n d e n s i s (39).

Polygodial

Warburganal

Ugandensidial

The b a r k s o f b o t h t r e e s are t r e a s u r e d i n f o l k m e d i c i n e and a l s o as f o o d s p i c e . W. s t u h l m a n n i gave the known p o l y g o d i a l (40,41,42) i n moderate y i e l d w h i l e W. ugand e n s i s gave the known u g a n d e n s i d i a l (43) and new warburganal. The y i e l d o f the l a t t e r two are n o t o n l y low b u t t h e y are d i f f i c u l t t o s e p a r a t e , even by h p l c , from a n o t h e r c o n s t i t u e n t and t h i s l e d t o c o n f u s i o n i n s t r u c t u r a l s t u d i e s . Of the t h r e e , w a r b u r g a n a l i s by f a r the most a c t i v e a n t i f e e d a n t (0.1 ppm a g a i n s t S. exempta), b u t more e x t e n s i v e s t u d i e s w i l l have t o depend on s y n t h e t i c m a t e r i a l because o f the l i m i t e d q u a n t i t y . Some s t r u c t u r e / a c t i v i t y s t u d i e s c a r r i e d o u t w i t h the simple p o l y g o d i a l i n d i c a t e d t h a t the a c t i v i t y was l o s t when one o r b o t h a l d e h y d e s were r e d u c e d t o h y d r o x y l groups o r o x i d i z e d t o c a r b o x y l groups. E p i m e r i z a t i o n o f the e q u a t o r i a l 9B-CHO t o the more s t a b l e a x i a l 9a-CHO a l s o r e s u l t e d i n l o s s o f a c t i v i t y (the e q u a t o r i a l epimer p r o b a b l y owes i t s i n s t a b i l i t y t o d i p o l e - d i p o l e r e p u l s i o n between the two aldehyde f u n c t i o n s ) . These r e s u l t s i n c o n j u n c t i o n w i t h the b l o c k i n g o f a n t i f e e d a n t a c t i v i t y upon a d m i n i s t r a t i o n o f an e q u i m o l a r amount o f c y s t e i n e (1) s u g g e s t t h a t a t l e a s t p a r t o f the a n t i f e e d a n t a c t i v i t y i n the case o f S. exempta i s c a u s e d by M i c h a e l type a d d i t i o n o f the e l e c t r o p h i l i c a n t i f e e d a n t t o a n u c l e o p h i l i c t e r m i n a l i n the r e c e p t o r s i t e , and t h a t , as shown by the h i g h a c t i v i t y o f war-

11.

KUBO

A N D NAKANISHI

Insect Antifeedants

and

Repellents

173

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

b u r g a n a l , t h e s t r u c t u r a l c o m p l e x i t y o f w h i c h i s i n between those o f t h e o t h e r two, t h e s t e r i c r e q u i r e m e n t of the receptor s i t e i s f a i r l y s e l e c t i v e . Interestingly, the a n t i f e e d a n t s a g a i n s t t h e armyworm t a s t e s p i c y t o the human tongue. f) I n f l e x i n from Leaves o f i s o d o n i n f l e x u s ( L a b i a teae) (44) and Isodomedin from Leaves o f I. s h i k o k i a n u s v a r intermedins. (45) . These two compounds^ were i s o l a t e d

AcO

AcO

inflexin

4

isodomedin

from Japanese s o u r c e s b u t a r e quoted here because t h e y b o t h e x h i b i t a n t i f e e d a n t a c t i v i t y as t e s t e d a g a i n s t t h e A f r i c a n armyworm. They a r e a l s o c y t o t o x i c , t h e l e v e l s o f i n f l e x i n and isodomedin b e i n g L D 5.4 yg/ml and 4.0 yg/ml, r e s p e c t i v e l y . They a r e e n t - k a u r e n o i d d i t e r p e n e s with c l o s e l y r e l a t e d structures. g) C r o t e p o x i d e from Leaves o f C r o t o n m a c r o s t a c h y s (46). The a n t i f e e d a n t p r i n c i p l e was i s o l a t e d a c c o r d i n g t o t h e b i o a s s a y w i t h S. exempta and t h e s t r u c t u r e was d e t e r m i n e d by d e t a i l e d NMR and o t h e r methods. To o u r s u r p r i s e the s t r u c t u r e was t h a t l_IQ o f c r o t e p o x i d e (47,48, 49), a compound e x h i b i t ing tumor-inhibitory, a n t i l e u k e m i c and a n t i 0 biotic activity. The c y t o t o x i c i t y and a n t i OGluc feedant a c t i v i t y are b o t h dependent, a t l e a s t p a r t i a l l y , on t h e r e a c Crotepoxide t i o n between t h e n u c l e o 5 0

'Mr

HOST P L A N T RESISTANCE

174

T O PESTS

p h i l i e r e c e p t o r and e l e c t r o p h i l i c moiety o f t h e a c t i v e m o l e c u l e ; i s o l a t i o n o f t h e same m o l e c u l e b y two t o t a l l y d i f f e r e n t b i o a s s a y s proves the s i m i l a r i t y i n c e r t a i n a s p e c t s o f t h e s e two s e e m i n g l y d i f f e r e n t a c t i v i t i e s . h) Unedoside from t h e bark o f Canthium e u r o i d e s (50). Structure determination o f the antifeedant contained i n t h i s Ο p l a n t t u r n e d o u t t o be t h e known u d e s i d e which had been CHoOC i s o l a t e d from A r b u t u s unedo (51) · ,OAc

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

OAc

Unedoside

i ) Prep LC o f Crude P l a n t E x t r a c t s ( 5 2 ) .

3

EtOAc/hexane 1=4

1 ·' mixture (19) 4 : mixture (l80mg) OCHj

5 : pure compound (40 mg)

[<3

ι CH,

2 : germacrone

3 : dihydrochelerythrine

6: N-methylflindersine Helvetica Chimica Acta

Figure 4.

Direct obtention of pure compounds from crude plant extracts by preparative liquid chromatography (52)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

11.

KUBO

AND

NAKANISHI

Insect Antifeedants

and

Repellents

175

The r e s u l t s shown i n F i g u r e 4 were o b t a i n e d w i t h a Waters Prep LC-500 system. The d r i e d bark (150 g) o f the I n d i a n and A f r i c a n m e d i c i n a l p l a n t F a g a r a c h a l y b e a ENGL. (Rutaceae), which e x h i b i t e d a n t i f e e d a n t , a n t i ­ b a c t e r i a l and a n t i f u n g a l a c t i v i t y was e x t r a c t e d w i t h hexane. The 3.5 g o f s t i c k y o i l r e m a i n i n g a f t e r evap­ o r a t i o n o f the s o l v e n t was p r e f i l t e r e d t h r o u g h 100 g o f s i l i c a g e l and the f i l t r a t e was i n j e c t e d d i r e c t l y i n t o the p r e p LC. Peaks 1 (1 g o f a m i x t u r e o f n o n - p o l a r m a t e r i a l s ) and 2 (850 mg o f pure germacrone) were e l u t e d i n 6 min. The remainder was c o l l e c t e d as one f r a c t i o n , and was rechromatographed by p r e p LC employing a more p o l a r s o l v e n t system, upon which peaks 3 (120 mg o f d i ­ h y d r o c h e l e r y t h r i n e ) , 4 (180 mg o f a m i x t u r e o f s e v e r a l components), 5 (40 mg o f pure compound, s t r u c t u r e t o be p u b l i s h e d , r e f . S3) and 6 (125 mg o f N - m e t h y l f l i n d e r ­ s i n e ) were o b t a i n e d i n a n o t h e r 25 min. The e n t i r e o p e r a t i o n was a c h i e v e d i n 2 h o u r s as compared t o 2 weeks r e q u i r e d by c o n v e n t i o n a l open column chromatography employing a v a r i e t y o f c h r o m a t o g r a p h i c c o n d i t i o n s i n c l u d ­ ing gradient elution. The more p o l a r f r a c t i o n s a r e a l s o b e i n g s u b m i t t e d t o p r e p LC. T h i s d i r e c t a p p l i c a t i o n o f p r e p LC t o crude p l a n t e x p i a n t s c l e a r l y r e s u l t s i n g r e a t s h o r t e n ­ i n g o f the t e d i o u s e x t r a c t i o n p r o c e d u r e . Furthermore, the reduced time on the column m i n i m i z e s d e t e r i o r a t i o n o f sample on the column. F o r example, the r e l a t i v e amount o f d i h y d r o c h e l e r y t h r i n e (peak 3) o b t a i n e d from a r e g u l a r open column o p e r a t i o n was much l e s s . Acknowledgment T h i s r e s e a r c h has been s u p p o r t e d by NIH G r a n t AI 10187 (to K. N . ) a n d U.N. Development Program ( t o I C I P E ) . We a r e g r e a t l y i n d e b t e d t o our c o l l e a g u e s c i t e d i n the r e f e r e n c e s , namely, Dr. V. B a l o g h - N a i r , Dr. D.L. E l d e r , Dr. K. Hostettmann, Y.-W. Lee, I . M i u r a , M. P e t t e i , Dr. F. P i l k i e w i c z , S. T a n i s , F. Yeh, Dr. P. Zanno (Columbia U n i v e r s i t y ) and A. Chapya, and Dr. W.-C. Ma (ICIPE). We a l s o acknowledge the c o l l a b o r a t i o n w i t h P r o f . T. Kubota and h i s group ( K i n k i U n i v e r s i t y and Osaka C i t y U n i v e r s i t y ) . Literature Cited

1. Djerassi, C., Bull. Atomic Sci. (1968) 24, 22. 2. Meinwald, J., Prestwich, G., Kubo, I., and Nakanishi, Κ., Science, in press. 3. Kubo, I., Lee, Y.-W., Pettei, M., Pilkiewicz, F., Nakanishi, K., J.C.S. Chem. Comm. (1976), 1013.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

176

HOST P L A N T RESISTANCE

T O PESTS

4. These bioassays were carried out by Dr. G.B. Staal and K.J. Judy, Zoecon Corporation. 5. Ma, W.-C., unpublished results. However, see ICIPE Annual Reports (1974), p. 7-11, and (1975), p. 2230 for preliminary accounts. These Annual Reports are available on request to Librarian, ICIPE, P.O. Box 30772, Nairobi, Kenya. 6. cf. Wada, K. and Munakata, K., Agr. Food Chem. (1968), 17, 471. 7. Ma, W.-C. and Kubo, I., unpublished results. cf. ICIPE Annual Reports (1975), p. 22-30 for prelimin­ ary account. 7a. Jacobson, M. and Crosby, D.G. ed., "Naturally Occur­ ring Insecticides", Marcel Dekker Inc., New York (1971). 8. Reviews: Hikino, H. and Hikino, Y. in Zechmeister, L. ed., "Fortschritte der Chemie Organischer Natur­ stoffe" Springer Verlag (1970), 28, 256. 9. Horn, D.H.S. in Ref. 7a, p. 330. 10. Nakanishi, Κ., Pure and Applied Chem. (1971), 25, 167, and XXIII rd Intern. Congress Pure and Applied Chem. (1971), Vol. III, 29. 11. Kuhn, R. and Guhe, Ζ., Z. Nat. Forsch. (1947), 26, 467. 12. Schreiber, K., Zuchter (1957), 27, 289. 13. Buhr, R., Toball, R., and Schreiber, K., Ent. Exp. Appl. (1958), 1, 209. 14. Kuhn, R. and Low, I., Chem. Ber. (1961), 94, 1096. 15. Bates, R.B. and Eckert, D.J., J. Am. Chem. Soc. (1972), 94, 8258. 16. Begley, M.J., Crombie, L., Ham, P.J., and Whiting, D.A., J.C.S. Chem. Comm. (1972), 1108. 17. Begley, M.J., Crombie, L., Ham, P.J., and Whiting, D.A., J.C.S. Chem. Comm. (1972), 1250. 18. Bates, R.B. and Morehead, S.R., J.C.S. Chem. Comm. (1974), 125. 19. Gilbert, B.L. and Norris, D.M., J. Insect Physiol. (1968), 14, 1063. 20. Isogai, Α., Murakoshi, S., Suzuki, Α., and Tamura, S., Agri. Biol. Chem(1973), 37, 889. 21. Wada, K., Enomoto, Y., and Munakata, K., Agr. Biol. Chem. (1970), 34, 941. 22. Wada, Κ., Enomoto, Υ., and Munakata, K., Agr. Biol. Chem. (1970), 34, 946. 23. Kato, N., Shibayama, S., Munakata, Κ., and Katayama, C., J.C.S. Chem. (1971), 1632. 24. Hosozawa, S., Kato, N., and Munakata, K., Tetrahed­ ron Letters (1974), 3753. 25. Lavie, D., Jain, M.K., and Shpan-Gabrielith, S.R., Chem. Comm. (1967), 910.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

11.

KUBO

A N D NAKANISHI

Insect Antifeedants

and Repellents

177

26. Watt, J.M. and Breyer-Brandwijk, M.G., "Medicinal and Poisonous Plants of Southern and Eastern Africa", E.S. Livingstone Ltd., Edinburgh and London (1962). 27. Kokwaro, J.O. "Medicinal Plants of East Africa", East African Literature Bureau, Nairobi (1976). 28. Zanno, P.R., Miura, I., Nakanishi, K., and Elder, D.L., J. Am. Chem. Soc. (1975), 2, 1975. 29. Butterworth, J.H., Morgan, E.D., and Percy, G.R., J. Chem., Soc. Perkin Trans. (1972), 1, 2445. 30. Morgan, E.D. and Thornton, M.D., Phytochem. (1973), 12, 391. 31. Kubo, I., Miura, I., and Nakanishi, K., J. Am. Chem. Soc. (1976), 98, 6704. 32. According to Trof. D.A.H. Taylor, i t should be X. granatum. However, the literature is confused and the identity is being checked. 33. Kubo, I., Lee, Y.-W., Balogh-Nair, V., Nakanishi, K., and C hapya, Α., J.C.S. Chem. Comm (1976), 949. 34. Kubo, I., Tanis, S.P., Lee, Y.-W., Miura, I., Nakanishi, K., and Chapya, Α., Heterocycles (1976), 5, 485. 35. Arigoni, D., Barton, D.H.R., Corey, E.J., Jeger, O., Caglioti, L., Dev, S., Ferrini, P.G., Glazier, E.R., Melera, Α., Pradham, S.K., Schaffner, K., Sternhell, S., Templeton, J.E., and Tobinaga, S., Experientia (1960), 16, 41. 36. Kubota, T., Matsuura, T., Tokoroyama, T., Kamikawa, T., and Matsumoto, T., Tetrahedron Letters (1961), 325. 37. Kindly measured by Dr. M. Taniguchi, Osaka City University. 38. Kindly measured by Dr. F.J. Schmitz, University of Oklahoma. 39. Kubo, I., Lee, Y.-W., Pettei, Μ., Pilkiewicz, F., and Nakanishi, K., J.C.S. Chem. Comm. (1976), 1013. 40. Barnes, C.S. and Loder, J.W., Austral. J. Chem. (1962), 15, 222. 41. cf. also Ohsuka, Α., Nippon Kagaku Zasshi (1962), 83, 757. 42. Synthesis: Kato, T., Suzuki, T., Tanemura, M., Kumanireng, A.S., Ototani, N., and Kitahara, Y., Tetrahedron Letters (1971), 1961. 43. Brook, C.J.W. and Draffan, G.H., Tetrahedron (1969), 25, 2887. 44. Kubo, I., Nakanishi, K., Kamikawa, T., Isobe, T., and Kubota, T., Chem. Letters (1977), 99. 45. Kubo, I., Miura, I., Nakanishi, K., Kamikawa, T., Isobe, T., and Kubota, T., J.C.S. Chem. Comm. (1977), in press.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch011

178

HOST PLANT RESISTANCE TO PESTS

46· Kubo, I., Miura, I., and Nakanishi, K., to be published. 47. Kupchan, S.M., Hemingway, R.J., and Smith, R.M., J. Org. Chem. (1969), 34, 3898. 48. Synthesis: Oda, K., Ichihara, Α., and Sakamura, S., Tetrahedron Letters (1975), 3187. 49. Synthesis: Demuth, M.R., Garrett, P.E., and White, J.D., J. Am. Chem. Soc. (1976), 98, 634. 50. Kubo, I., Miura, I., and Nakanishi, Κ., unpublished results. 51. Geisman, T.A., Knaack, W.F.Jr., and Knight, J.O., Tetrahedron Letters (1966), 1245. 52. Hostettmann, K., Pettei, M.J., Kubo, I., and Nakanishi, K., Helv. Chim. Acta (1977), 60, 670. 53. Yeh, F., Miura, I., Hostettmann, Κ., Kubo, I., and Nakanishi, K., to be published.

12 Antifeedant Sesquiterpene Lactones in the Compositae T O M J. MABRY and JAMES E. GILL Department of Botany, University of Texas, Austin, T X 78712 WILLIAM C. BURNETT, JR. and SAMUEL B. JONES, JR.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch012

Department of Botany, University of Georgia, Athens, GA 30602

Most of the known 700 or so sesquiterpene lactones are characteristic constituents of many members of the Compositae, occurring only infrequently in a few other higher and lower plants (1,2)· Whether or not the a , β-unsaturated γ-lactone function present in most of these terpenoids i s responsible f o r their antifeedant properties i s not known; however, this functionality has been implicated i n most of the other biological activities known f o r these substances: e.g. antitumor, cytotoxicity, contact dermatitis, and antifungal properties. Since we recently described i n detail the role of sesquiterpene lactones i n plant-animal interactions (2), the present account w i l l only summarize our specific findings concerning the way the germacranolide glaucolide-A (I) controls the level of feeding of insects and mammals on members of the genus Vernonia (3) and our general conclusions concerning the function of sesquiterpene lactones as antifeedants. In connection with establishing the antifeedant and growth inhibition properties of glaucolide-A (I), a germacranolide-type sesquiterpene lactone from Vernonia (Compositae), six species of Lepidoptera as well as two species of mammals were u t i l i z e d i n a series of laboratory and f i e l d tests with V. gigantea and V. glauca, both of which contain glaucolide-A, and with V. f l a c c i d i f o l i a , a species which does not produce any sesquiterpene lactones. Some of the major findings of these tests are summarized i n Table I and Figure 1. Insect Antifeedant Tests All of the six lepidopterous larvae used i n testing the feeding deterrent properties of glaucolide-A i n the laboratory tests are sympatric with the species of Vernonia tested and three of them, the yellowstriped armyworm, (Spodoptera ornithogalli), cabbage looper (Trichoplusia n i ) , and the yellow woollybear (Diacrisia virginica), feed i n nature on Vernonias containing glaucolide-A; a fourth species, the saddleback caterpillar

179

HOST P L A N T RESISTANCE T O PESTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch012

180

( S i b i n e s t i m u l e a ) , feeds on V. f l a c c i d i f o l i a , a species l a c k i n g glaucolide-A. Two g e n e r a l i s t feeders, the f a l l and southern armyworms (Spodoptera e r i d a n i a and j>. f r u g i p e r d a ) , were not detected on Vernonias i n the f i e l d . The l a b o r a t o r y t e s t s e s t a b l i s h e d two trends (Table I ) : firstly, when the i n s e c t s had a choice of Vernonia f l a c c i d i f o l i a l e a f m a t e r i a l prepared i n the form of a p e l l e t with and without added g l a u c o l i d e - A , a l l the larvae p r e f e r r e d the d i e t without the sesquiterpene lactone; secondly, when the l a r v a e were o f f e r e d a choice of f r e s h leaves of g l a u c o l i d e - A species versus f r e s h leaves of V. f l a c c i d i f o l i a , they tended to e x h i b i t the same feeding patterns observed i n nature (4). That i s , except f o r the y e l l o w s t r i p e d armyworm, those species observed i n the f i e l d on Vernonias containing g l a u c o l i d e - A p r e f e r r e d these same p l a n t s over V. f l a c c i d i f o l i a i n the l a b o r a t o r y ; similarly, the one species observed on V. f l a c c i d i f o l i a under n a t u r a l conditions s t i l l p r e f e r r e d t h i s species i n the l a b o r a t o r y . Two g e n e r a l i s t feeders, the southern and f a l l armyworms, always avoided the g l a u c o l i d e - A p l a n t s . In t e s t s with the f a l l armyworm l a r v a e , i t was f u r t h e r determined that l e v e l s of about 1% g l a u c o l i d e - A ( i n a p e l l e t ) gave maximum deterrence; t h i s i s roughly the quantity of l a c t o n e m a t e r i a l synthesized i n the leaves of many sesquiterpene l a c t o n e - c o n t a i n i n g Compositaes.

Q

Glaucolide-A (Vernonia) (i)

L a r v a l Growth I n h i b i t i o n Tests Newly hatched larvae of the southern, fall, and y e l l o w s t r i p e d armyworms, cabbage looper, and yellow woollybear were reared on d i e t s c o n t a i n i n g g l a u c o l i d e - A i n amounts ranging up to 0.5%. The growth of the three armyworms was g r e a t l y reduced (Figure 1) and the r e d u c t i o n i n growth f o r the f a l l and southern armyworms was considerably greater than the decrease i n feeding. Moreover, except f o r the yellow woollybear, the

12.

MABRY ET A L .

Table I*

Antifeedant

Sesquiterpene

Lactones

181

Insect Antifeedant Tests

D i e t Preference i n Laboratory

Leaf Glaucolide-A Species

P e l l e t Choice

Choice V.

flaccidifolia

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch012

Insects:

V. f l a c . With Added Glauco­ lide-A

V. f l a c . Without Glauco­ lide-A

Observed on Glaucolide-A Vernonias 1. 2. 3.

Yellow Woollybear Cabbage Looper Yellowstriped Armyworm

χ (usually)

X

X

X X

X

X

*

X

X

X

X

Not Observed On Glaucolide-A Vernonias 1. 2. 3.

Saddleback Caterpiller Fall Armyworm Southern Armyworm

*Not