Host Plant Resistance to Pests

2 Role of Elicitors of Phytoalexin Accumulation in Plant

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Disease Resistance 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 In Host Plant Resistance to Pests; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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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

In Host Plant Resistance to Pests; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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of growth of these c e l l s

Elicitors

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Phytoalexin

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(8).

The Chemical Nature of the Phytophthora megasperma var. Elicitor.


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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



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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


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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 .


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.


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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,



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. Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 10, 2014 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch002

16. 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.


Host Plant Resistance to Pests

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