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Chapter 12

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Functional Significance of Adhesion to the Preparation of the Infection Court by Plant Pathogenic Fungi Ralph L. Nicholson Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907

Adhesion is a component of fungal survival. For plant pathogens it represents an early event in host recognition, and may be hostspecific, as with the nematophagous fungi, or non-specific, with attachment to any surface. Regardless of specificity, adhesion initiates the infection process, and in its absence infection is prevented. Adhesives are components of the fungal extracellular matrix. Adhesion often involves the binding of lectins and carbohydrate polymers on the fungus and host surfaces and may involve the morphological differentiation of specific fungal structures. Stimulation of adhesion often results from developmental changes in the fungus that are stimulated by the host. Enzymes in the fungal matrix are also determinants of attachment and penetration. Cuticular erosion may expose aspects of surface topography necessary for recognition of the infection court. Other components of the matrix ensure survival by their antidesiccant properties and others by the selective binding of toxic polyphenols by specific proteins. In a t r a d i t i o n a l s e n s e , p l a n t p a t h o l o g i s t s seek t o prevent disease i n economically important crops and t o t h i s e n d we o f t e n s t u d y w h y a n o r g a n i s m i s s u c c e s s f u l . I d e a l l y we h o p e t o learn information that w i l l be useful i n the application of disease control measures. The s u b j e c t o f t h i s volume i s t h e c o n t r o l o f weeds through the use of plant pathogens. Thus, the goal i s q u i t e d i f f e r e n t from that o f t r a d i t i o n a l pathology as we a r e a t t e m p t i n g t o e n s u r e t h e s u c c e s s o f a d i s e a s e 0097-6156V90A)439-0218$06.25A) © 1990 American Chemical Society

Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Functional Sipufkxuxx of Adhesion

r e l a t i o n s h i p to c o n t r o l an unwanted p l a n t . A phenomenon of great importance t o p a r a s i t i s m i s the i n f e c t i o n process, an area that encompasses the e a r l i e s t events i n the i n t e r a c t i o n of a pathogen with a host. One of these events i s the adhesion of the pathogen to the p l a n t . The s i g n i f i c a n c e of adhesion to p a r a s i t i s m i s evident when one r e a l i z e s that f o r most pathogens adhesion must occur i f the organism i s to penetrate and s u c c e s s f u l l y cause disease. In the present review I w i l l consider the complexity of the adhesion process. It seems appropriate that adhesion should be considered as a target of importance f o r the s u c c e s s f u l use of a pathogen as a b i o h e r b i c i d e . For some time, i t was considered that attachment of fungal pathogens was an event that occurred by chance, o f t e n through simple entrapment of propagules amongst l e a f h a i r s or i n a n t i c l i n a l grooves on the l e a f surface. This i s i n s p i t e of the f a c t that i t was recognized e a r l y that the hyphae of fungal germlings are t y p i c a l l y surrounded or encased i n an amorphous matrix or f i l m (1). We now know that attachment i s an a c t i v e process that involves the s e c r e t i o n of adhesive materials by the fungal conidium or mycelium, materials often r e f e r r e d to as components of e x t r a c e l l u l a r matrices. In some cases the s e c r e t i o n of adhesives occurs i n response to s p e c i f i c s t i m u l i from the host plant whereas i n other instances adhesion occurs i n a n o n - s p e c i f i c manner t o any substratum. Where adhesion i s t r i g g e r e d by a s p e c i f i c stimulus i t i s often accompanied by the formation of stages of morphological development that are necessary f o r the attainment of adhesive competence. Adhesion may a l s o involve another c r i t i c a l phenomenon. It i s l i k e l y to be a s s o c i a t e d with events necessary f o r host r e c o g n i t i o n and eventual penetration, a phenomenon best termed preparation of the i n f e c t i o n court. Adhesion i s most e a s i l y recognized during the formation and attachment of appressoria, the s t r u c t u r e s from which penetration occurs, to the host surface. Indeed, appressoria are often r e f e r r e d to as adhesive s t r u c t u r e s (2.) . However, adhesion may begin p r i o r to c o n i d i a l germination and occur during the process of germ tube elongation that leads up to appressorium formation ( 2 ) . F u n c t i o n a l s i g n i f i c a n c e of adhesion and

mucilages.

The s i g n i f i c a n c e of adhesion to fungal s u r v i v a l and spread, as w e l l as the chemistry of adhesives and e x t r a c e l l u l a r mucilages have been discussed e x t e n s i v e l y i n recent reviews Q, ±, ϋ ) . Perhaps the most important aspect of fungal adhesion, regardless of the mechanism by which i t occurs, i s the prevention


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MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES of displacement. Fungi encounter plant surfaces i n both dry and aqueous environments. It seems reasonable that the movement of wind and water can e a s i l y d i s p l a c e inoculum from the p o t e n t i a l i n f e c t i o n court and must be dealt with by the organism. Thus, attachment of propagules to the host i s an important p r e r e q u i s i t e to penetration. Attachment or adhesion may a l s o e s t a b l i s h the f i r s t p h y s i o l o g i c a l contact between a host and a pathogen and ensure that the s t i m u l i that account f o r d i r e c t i o n a l growth or the necessary changes i n morphogenesis (appressorium formation) are recognized (£, 2 ) . The p h y s i c a l contact between a pathogen and a host a l s o provides a s i t e at which r e c o g n i t i o n of the 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 of the host i s e s t a b l i s h e d (£). As most fungi require water as a medium f o r germination and growth on the plant surface i t i s a l s o reasonable to assume that adhesives must, to some extent, be water i n s o l u b l e . Mims and Richardson (2) have presented evidence that i n Gymnospnrangium juniperi-virgrinianae the adhesive that binds basidiospores changes from a d i f f u s e network to a compacted, e l e c t r o n dense substance with time. Thus, adhesives may at f i r s t be r e l a t i v e l y water soluble but t h e i r composition may change r a p i d l y a f t e r s e c r e t i o n from the fungus. In some fungi, such as Colletotrichum musae the appressorium functions as a dormant s t r u c t u r e that may be bound to the host surface f o r a considerable p e r i o d p r i o r t o the event of penetration (10). The need f o r a f i r m anchoring of the pathogen during the p e r i o d of latency i s obvious. Other fungi, such as Zygophiala jamaicensis the causal agent of flyspeck of various f r u i t s , grow e n t i r e l y on the plant c u t i c l e and t h e i r f i r m adhesion t o t h i s surface i s necessary f o r t h e i r saprophytic mode of growth. £. jamaicensis produces copious amounts of e x t r a c e l l u l a r mucilage, apparently as an adhesive, and degrades the host c u t i c l e (Figure 1) which i t uses as i t s sole source of carbon (11, 1 2 , 12) . f

Zoospore Encystment i n the Oomycetes. The best examples of adhesion r e s u l t i n g from the stimulus of contact are found among the Oomycetes where zoospore encystment occurs upon contact with the host. Zoospores are motile propagules that lack c e l l w a l l s . These c e l l s often require s p e c i f i c s i t e s on a host root f o r attachment and eventual penetration. For example, zoospores of Pythium aphanidermatum and Phytophthora cinnamomi adhere p r i m a r i l y i n the zone of root elongation (14., 12) where terminal f u c o s y l


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Functional Sign^cance ofAdhesion

Figure 1.

Scanning e l e c t r o n micrograph of Zygophiala growing on the surface of a plum f r u i t . Note the extensive areas free of wax c r y s t a l s (arrows) surrounding the hyphae (h) of the fungus. Bar represents 10 |Im. Reproduced with permission from Ref. 12. Copyright 1987 American Phytopathological Society. jamaicensis


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MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES residues i n the root mucilage act to bind a l e c t i n l i k e substance on the zoospore s u r f a c e . Encystment can be a r t i f i c i a l l y induced i n clnnamoml by treatment of zoospores w i t h the l e c t i n Concanavalin A (1Ê). Evidence from s t u d i e s w i t h Pythium spp. i n a s s o c i a t i o n w i t h host and non-host r o o t s suggests t h a t species pathogenic t o graminicolous hosts are s t i m u l a t e d t o encyst s p e c i f i c a l l y by r o o t s of grass species (12, IS.) i n d i c a t i n g that host r e c o g n i t i o n i s r e l a t e d t o the s p e c i f i c i t y f o r encystment and adhesion. Contact t r i g g e r s encystment of the zoospore which e v e n t u a l l y r e s u l t s i n the formation of a c e l l w a l l . Encystment a l s o r e s u l t s i n the r e l e a s e of an adhesive f i l m t h a t covers the c y s t and a l l o w s f o r the i n i t i a l b i n d i n g of the c y s t t o the host s u r f a c e . G e n e r a l l y , encystment occurs w i t h i n minutes, as i n Pythium aphanirtermatnm (12), and r e s u l t s i n the formation of a cyst w i t h an adhesive outer coat. The o r i g i n of the adhesive coat has been demonstrated t o be p e r i p h e r a l v e s i c l e s i n the e n c y s t i n g zoospores. Hardham (22) showed t h a t b i n d i n g was s p e c i f i c t o the c y s t , and not the zoospore, of Phytophthora cinnamomi and t h a t the adhesive was preformed and present i n s m a l l v e s i c l e s at the zoospore p e r i p h e r y (21) The adhesive was composed of high molecular weight g l y c o p r o t e i n s t h a t bound s p e c i f i c a l l y w i t h soybean a g g l u t i n i n suggesting t h a t the hapten was N-acetyl-Dgalactosamine. In c o n t r a s t t o the apparent involvement of l e c t i n s i n adhesion of Phytophthora i s the a d s o r p t i o n of Fusarium moniliforme and PfaialQPhpra r a d l c i o l a t o maize root mucilage. In these f u n g i adhesion appears t o occur through i o n i c i n t e r a c t i o n s between mucilage polymers on the root and f u n g a l c o n i d i a (22). As i n the case of Phytophthora, however, b i n d i n g appeared t o depend on the stage of fungal development suggesting t h a t o n l y s p e c i f i c propagules are capable of developing adhesive competence, and i t i s p r e c i s e l y those propagules that are i n v o l v e d i n attachment and p e n e t r a t i o n of the host. In Plasmodlophora b r a s s i c a e an organism c l o s e l y r e l a t e d t o the Oomycetes, A i s t and W i l l i a m s (22) have demonstrated t h a t adhesion and p e n e t r a t i o n occurs i n a succession of organized steps t h a t are h i g h l y s p e c i f i c . Zoospores r e l e a s e an adhesive coat t h a t binds the cyst t o the cabbage root h a i r . The bound c y s t then i n i t i a t e s the process of p e n e t r a t i o n which a l s o i n v o l v e s adhesion. A v e s i c u l a r s w e l l i n g c a l l e d the adhesorium forms, and through a bulbous extension binds t o the host surface. P e n e t r a t i o n occurs when r

the cyst f o r c e s a r o d - l i k e s t r u c t u r e , termed the

S t a c h e l , through the adhesorium and into the host


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Functional Stfgnfficance of Adhesion

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c e l l . These events occur i n a p e r i o d o f but 2 . 5 t o 3 . 5 hours. Preformed adhesives t o host

and adhesives

formed i n response

stimuli.

The i n f e c t i o u s propagules of some f u n g i are produced w i t h a preformed adhesive which ensures t h a t the propagule, g e n e r a l l y a conidium, w i l l b i n d upon contact. This b i n d i n g o f t e n occurs n o n - s p e c i f i c a l l y and t o any substratum. For example, i n Magnaporthe g r i s e a t h e c a u s a l agent of r i c e b l a s t , the apex o f the conidium contains a preformed adhesive m a t e r i a l l o c a t e d o u t s i d e the c o n i d i a l plasma membrane but w i t h i n t h e c o n i d i a l w a l l (24.). The w a l l a t t h e apex of the conidium b u r s t s upon h y d r a t i o n and r e l e a s e s the adhesive which allows f o r b i n d i n g t o any s u r f a c e . The nematophagous fungus Meria coniospora has a mechanism f o r attachment s i m i l a r t o t h a t o f M . g r i s e a . The apices o f c o n i d i a possess a k n o b - l i k e s t r u c t u r e t h a t i s covered w i t h a l a y e r o f adhesive mucilage t h a t allows attachment t o the nematode host (25.) . I t was l a t e r shown t h a t adhesion occurs only w i t h s p e c i f i c nematode species and only t o the t a i l o f males o r t o the c e p h a l i c region o f both males and females (2&)· Adhesion o f M . coniospora t o the nematode Panagrrellus redivivus occurred s p e c i f i c a l l y a t the sensory organs i n the region o f the nematode mouth and appeared t o be mediated by the presence o f s i a l i c a c i d i n these regions ( 2 2 ) , again suggesting t h a t a l e c t i n - h a p t e n r e l a t i o n s h i p mediates the process of adhesion. The p l a n t pathogenic fungus Dilophospora a l o p e c u r i produces c o n i d i a w i t h e x t e n s i v e l y convoluted t e r m i n a l appendages. Adhesion t o the nematode Anguina a g r o s t i s (the v e c t o r o f annual ryegrass t o x i c i t y ) e f f e c t s e n t r y o f the fungus i n t o the p l a n t and occurs by attachment o f c o n i d i a l appendages t o the nematode. B i n d i n g i s mediated through a mucilagenous f i b r i l l a r m a t e r i a l so t h a t c o n i d i a c o l l e c t i n the t r a n s v e r s e annulations o f the nematode (22) . Although there i s no apparent damage t o the nematode c u t i c l e by t h i s adhesion, attachment does a l t e r the nematode l i f e c y c l e and r e s u l t s e v e n t u a l l y i n nematode death. This apparent c o - p a r a s i t i s m has been suggested as a c o n t r o l f o r annual ryegrass t o x i c i t y as the nematode serves as the v e c t o r f o r the c a u s a l bacterium, Corynebacterium r a t h a y i . The fungus i t s e l f i s not d i r e c t l y i n v o l v e d i n ryegrass t o x i c i t y . A carbohydrate b i n d i n g l e c t i n has been demonstrated t o occur on the surface o f the nematophagous fungus A r t h r o b o t r y s o l i g o s p o r a (29) . r


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MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES

Synthesis of the p r o t e i n occurs i n the fungus when i t produces trap structures f o r nematode capture. The l e c t i n , which has s p e c i f i c i t y f o r binding t o N-acetylD-galactosamine, occurs only on the surface o f t r a p s . Adhesion only occurs at these s i t e s and penetration of the nematode by the fungus only occurs at those s i t e s (20.) . That l e c t i n s may be involved as a general mechanism f o r adhesion of nematophagous fungi i s consistent with f i n d i n g s that the surface of nematodes i s i n t e r s p e r s e d with various saccharide residues often s p e c i f i c t o a stage of nematode development ( 2 1 ) · As the nematode c u t i c l e i s a l s o r i c h i n l i p i d s , free hydroxyl groups i n these compounds may a l s o be involved i n adhesion. Lectin-hapten r e l a t i o n s h i p s are a l s o important t o the establishment of mycorrhizal r e l a t i o n s h i p s . In the e r i c o i d mycorrhizal fungus Hymenoscyphus e r i c a e there are binding s i t e s s p e c i f i c f o r Concanavalin A i n an e x t r a c e l l u l a r matrix that surrounds hyphal walls (22/ 2 2 ) . When c u l t u r e d i n the presence of host roots the matrix and associated hapten (mannose) increase i n abundance but gradually disappear a f t e r penetration and establishment of symbiosis ( 2 1 ) . Noninfective s t r a i n s o f the fungus lack the hapten even when grown i n the presence of a p o t e n t i a l host ( 2 2 ) . Preparation of the i n f e c t i o n court by fungal pathogens. The establishment of fungal pathogens, e s p e c i a l l y those that occur on a e r i a l plant p a r t s , depends on the r e c o g n i t i o n of the plant as a p o t e n t i a l host. In most cases such r e c o g n i t i o n i s subtle and not w e l l understood. An exception i s the i n t e r a c t i o n of the rust Uromyces appendiculatus with i t s bean host Phaseolus v u l g a r i s . Urediospores germinate and subsequent germ tube growth occurs i n a d i r e c t e d manner so that when a stomate i s encountered penetration can occur. Such d i r e c t e d growth (thigmotrophism) i s probably stimulated by mechanical s i g n a l s inherent t o the l e a f surface and perceived by the fungus (£, 2 ) . The formation of appressoria, a change i n morphogenesis, i s a l s o stimulated by a s i g n a l from the plant surface. This response i s termed t h i g m o d i f f e r e n t i a t i o n and i n II. appendiculatus i t r e s u l t s when the germ tube encounters a guard c e l l l i p of a s p e c i f i c height ( 2 £ ) . Epstein et a l . (22) presented evidence t o show that an e x t r a c e l l u l a r p r o t e i n from the fungus i s required f o r II. appendiculatus germling adhesion t o a substrate and that adhesion i t s e l f i s a p r e r e q u i s i t e t o d i r e c t e d growth and t h i g m o d i f f e r e n t i a t i o n . Of course, adhesion


12. NICHOLSON

Functional Ζίχηφεαηπ ofAdhesiw

i s required f o r the s u c c e s s f u l penetration of the leaf. Growth of germ tubes and attachment of germlings to the surface of a e r i a l plant parts such as leaves i s a l s o conditioned by the hydrophobic nature of the l e a f (32) . Although below ground plant parts can e a s i l y be envisioned to e f f e c t adhesion through lectin-hapten binding, above ground parts do not appear to be amenable to such mechanisms. This i s i n s p i t e of the f a c t that c o n i d i a of fungi can be shown to possess surface carbohydrates that could serve as haptens (22) . Probably the lack of l e c t i n mediated binding on f o l i a g e r e s u l t s from the hydrophobic nature and chemistry of waxes and c u t i n that cover the surfaces of leaves. The establishment of contact on the l e a f surface and growth of the germling over the surface often involves the erosion of the l e a f c u t i c l e . Two fundamental questions r e l a t i v e t o such erosion are whether fungi produce e x t r a c e l l u l a r matrices that contain cutinases or other enzymes capable of degrading c u t i c l e components, and whether r e c o g n i t i o n of the host surface r e s u l t s from erosion. F i r s t i s the question of whether fungi c o n s i s t e n t l y produce an e x t r a c e l l u l a r matrix that surrounds germ tubes. Evidence that t h i s does occur i s s u b s t a n t i a l (2) and i s best v i s u a l i z e d i n B i p o l a r i s spp. where m u l t i p l e layered hyphal sheaths have been demonstrated (42, 4JL) . Sheath deposition even occurs p r i o r to the time that the germ tube emerges from the conidium (42, 4JL) . C u t i c u l a r erosion r e s u l t s i n the formation of imprints on the l e a f surface that can e a s i l y be detected by scanning e l e c t r o n microscopy ( 4 2 ) . Comparison of Erysiphe graminis with £. cichoracearum demonstrated that £. g r a m i n i s erodes both barley and cucumber c u t i c l e s , but that £. c i c h o r a c e a r u m only eroded or l e f t imprints on cucumber c u t i c l e s (42) . These r e s u l t s suggest a l e v e l of host s p e c i f i c i t y , p o s s i b l y f o r the enzymatic erosion of the c u t i c l e , that may a l s o be involved i n the s u c c e s s f u l penetration of the l e a f . Erosion and wax degradation a l s o occurs i n the c u t i c l e of r i c e i n contact with Helminthosporium oryzae germlings (44.) and the fungus produces excessive amounts of an e x t r a c e l l u l a r matrix around germ tubes and appressoria. The e x t r a c e l l u l a r sheath or matrix a l s o was shown t o adhere t e n a c i o u s l y to the c u t i c l e and associated wax components. E x t r a c e l l u l a r sheaths have a l s o been demonstrated to be important i n the interorganismal contact that occurs during fungal hyperparasitism. Trichoderma spp. p a r a s i t i z i n g Rhizoctonia s o l a n i and Sclerotium


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MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES r o l f s i i deposit a mucilage-like m a t e r i a l on t h e i r hosts (45.) . Erosion of the surface of the host mycelium occurs i n a manner apparently s i m i l a r t o t h a t of f u n g a l - p l a n t i n t e r a c t i o n s . The l e v e l s of e x t r a c e l l u l a r c h i t i n a s e and glucanase from the pathogen were a l s o shown t o increase when the pathogen was grown i n the presence of e i t h e r fungal host or t h e i r w a l l m a t e r i a l s . Thus e r o s i o n i n t h i s instance may be a s s o c i a t e d w i t h the simultaneous production of these enzymes w i t h the apparently adhesive sheath material. The time at which r e l e a s e of e x t r a c e l l u l a r matrices by c o n i d i a or germlings occurs i s a l s o important t o understanding t h e i r involvement i n disease development. In some cases e x t r a c e l l u l a r mucilages or matrices are r e l e a s e d upon h y d r a t i o n (24), at the time of germination (41r 4fi) * as the germ tube grows along the l e a f surface (2, 42) / and i n a s s o c i a t i o n w i t h the morphogenesis of a p p r e s s o r i a (42) . For Eryslphe graminis i t i s now known t h a t the stimulus of contact, e i t h e r w i t h the l e a f or w i t h cellophane, r e s u l t s i n the r e l e a s e of a l i q u i d from the ungerminated conidium (42). The surface of the n a t i v e , unstimulated conidium i s covered by a r e t i c u l a t e network of r i d g e s i n t e r s p e r s e d w i t h s p i n e l i k e w a l l p r o j e c t i o n s . Within 10 min of contact the conidium r e l e a s e s a l i q u i d f i l m t h a t accumulates t o the extent t h a t the r e t i c u l a t e network i s covered. In l e s s than 30 min a f t e r contact, the f i l m i s c o n s i d e r a b l y thickened, and prominent globose bodies are found i n t e r m i t t e n t l y over the s u r f a c e . Components of the f i l m are deposited onto the contact surface and flow outward (15-20 μχη) forming a zone around the conidium. The i n t e r f a c e of the conidium and the l e a f surface i s covered w i t h m a t e r i a l r e l e a s e d by the conidium suggestive of the adhesive mucilages produced by other f u n g i . Whether the m a t e r i a l serves as an adhesive i s unknown. In the area of the l e a f surface i n contact w i t h the £. graminis f i l m , the c u t i c l e l o s e s i t s o r i g i n a l i n t e g r i t y as evidenced by the apparent d i s s o l u t i o n of surface wax c r y s t a l s . W i t h i n 60 min the f i l m disappears and the surface of the conidium again assumes the morphology of the unstimulated s t a t e . These events a l l occur p r i o r t o the time of c o n i d i a l germination. Concurrent w i t h the r e l e a s e of the l i q u i d f i l m (42) i t was shown t h a t there i s a r e l e a s e of esterase w i t h a c t i v i t y against p - n i t r o p h e n y l butyrate (42) . A c t i v i t y was r e l e a s e d i n two stages, the f i r s t o c c u r r i n g within 2 min of contact and the second between 10 and 15 min of contact (Figure 2 ) . f


12. NICHOLSON

0 2 5

in

Fun*io^Sig*WcaMxofAdhcfa

10

15

20

30

45

60

T I M E A F T E R I N O C U L A T I O N ( min )

Figure 2. Contact s t i m u l a t i o n of the release of esterase from c o n i d i a of Erysiphe graminis. a) c o n i d i a on b a r l e y leaves; b) c o n i d i a on a cellophane surface. Reproduced with permission from Ref. 49. Copyright 1988 Academic Press, Inc.


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MICROBES A N D MICROBIAL PRODUCTS AS

HERBICIDES

The f i r s t phase of esterase release coincides with the appearance of the f i l m on the c o n i d i a l surface and the second phase of release coincides with the time of appearance of the globose bodies on the c o n i d i a . A n a l y s i s of the l i q u i d f o r p r o t e i n composition by native PAGE and SDS-PAGE gels demonstrated the presence of several peptides ranging i n molecular weight up to 94 kDa and the presence of three esterase a c t i v e components. It was suggested that the release of l i q u i d was an a i d to the establishment of the i n f e c t i o n court by the fungus and that one r e s u l t of the release was the degradation of components of the c u t i c l e . Degradation of the c u t i c l e was subsequently shown to occur a f t e r incubation of the p a r t i a l l y p u r i f i e d esterase preparation on the b a r l e y l e a f surface suggesting that the esterase was a cutinase type enzyme. The exact r o l e that c u t i c u l a r degradation plays to ensure the success of the pathogen i s unknown; however, r e s u l t s i n d i c a t e that the a p p r e s s o r i a l germ tube only elongates to the margin of deposition of the l i q u i d f i l m that surrounds the conidium (Kunoh and Nicholson, unpublished). Thus, e i t h e r the l i q u i d f i l m i t s e l f or the enzymatic degradation of the c u t i c l e may be involved i n the r e c o g n i t i o n of a zone s u i t a b l e f o r penetration. Composition of Adhesives. There i s l i t t l e work that d e f i n i t i v e l y describes the composition of fungal adhesives. However, i t i s apparent that fungi do not share e i t h e r common adhesives or mechanisms f o r adhesion. One f a c t o r that does appear with r e g u l a r i t y i s that the surface of fungal germ tubes are t y p i c a l l y ensheathed by a glycoprotein/carbohydrate matrix (41, 2Û* 51* 22) / and i t appears that polysaccharides or glycoproteins are often involved i n the process of adhesion. One study suggests that the a p p r e s s o r i a l adhesive of Colletotrichum araminicola i s a hemicellulose (22); however, t h i s may be questioned on the b a s i s of the p u r i t y of enzymes involved i n s t r u c t u r e e l u c i d a t i o n . In BJPQlarJS sorokiniana (24) and Neurospora crassa (22) a galactosaminoglycan appears to serve as a surface-binding compound and a p r o t e i n or g l y c o p r o t e i n f u n c t i o n i n the binding of bean rust germlings ( 2 2 ) , Phytophthora cinnamomi and £. palmivora zoospores (12, 2 2 ) f Bueraenerula spartlnae hyphopodia (22) , and Candida a l b i c a n s c e l l s (22). L e c t i n binding to s p e c i f i c haptens has been reported f o r fungi that adhere t o roots, nematodes, or other f u n g i . The chemistry of adhesion i s b e t t e r understood f o r lectin-hapten r e l a t i o n s h i p s than f o r


Functkmal Significance of ΑαΊκ^

NICHOLSON

229

adhesion that occurs i n the apparent absence of l e c t i n s . The f i r m attachment of nematophagous fungi to t h e i r hosts i s required f o r p a r a s i t i s m (22) and the surface of nematodes has been shown to be i n t e r s p e r s e d with s p e c i f i c saccharides, often s p e c i f i c to stages of nematode development (21) . As discussed above, a carbohydrate-binding p r o t e i n (approximately 20 kDa) with s p e c i f i c i t y f o r N-acetyl-D-galactosamine, present on the nematode surface has been p a r t i a l l y c h a r a c t e r i z e d from t r a p structures of the fungus Arthrobotrys oligospora (22). Synthesis of the l e c t i n was shown to be regulated by fungal development as i t occurred only on the surface of trap-bearing hyphae that form i n response to the presence of the nematode. In Meria coniospora, another nematophagous species, adhesion to the nematode i s a l s o mediated through a l e c t i n . Conidia possess a knoblike structure at t h e i r apices which i s covered by a l a y e r of mucilage that attaches the conidium to the nematode (22). Adhesion of the fungus to s p e c i f i c nematode species and regions of the nematode body i s mediated by a fungal l e c t i n s p e c i f i c f o r N-acetylneuraminic a c i d ( s i a l i c acid) (22). I t has since been suggested that s t e r i c c o n f i g u r a t i o n of s i a l i c a c i d residues on the nematode surface serve as s t i m u l i f o r the processes of adhesion and i n f e c t i o n (22). It has been demonstrated that i n the mycoparasite Trichoderma l e c t i n binding probably accounts f o r the s p e c i f i c i t y of host i d e n t i f i c a t i o n (42, 22)· Two host fungi (RhizQCtonia s o l a n i and SclerQtium r o l f s i i ) possess surface l e c t i n s with binding s p e c i f i c i t y f o r galactose residues that are present on the mycoparasite surface. Glucose- and mannose-rich s i t e s f o r l e c t i n binding have been detected on the surface of the fungal w a l l and i n an e x t r a c e l l u l a r m a t e r i a l surrounding the wall of e r i c o i d mycorrhizal fungi (22/ 2 1 ) . Concanavalin A binding s i t e s were l o c a l i z e d i n an e x t r a c e l l u l a r m a t e r i a l that r a d i a t e d from the fungal w a l l of the mycorrhizal fungus Hymenoscyphus e r i c a e . The frequency of binding s i t e s was s i g n i f i c a n t l y increased by growth of the fungus i n the presence of host roots (22). In contrast, a n o n i n f e c t i v e s t r a i n of the fungus lacked the hapten regardless of the presence of the host. The presence of mannose binding s i t e s i n the e x t r a c e l l u l a r fungal matrix was necessary f o r mycorrhizal establishment. I n t e r e s t i n g l y , once adhesion and host penetration occurred, the binding s i t e s and e x t r a c e l l u l a r matrix disappeared from the fungus (24). This induction of hapten binding s i t e s p r i o r to adhesion resembles the induction of adhesion i n Candida albicans to human r


230

MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES e p i t h e l i u m , a process that i s induced by contact and mediated by l e c t i n s b i n d i n g t o glucose and mannose haptens on the fungal surface (21/ 22). The e x t r a c e l l u l a r mucilage of C o l l e t o t r i c h u m graminlcola.

N e i t h e r the composition nor the f u n c t i o n s of f u n g a l e x t r a c e l l u l a r matrices has r e c e i v e d s i g n i f i c a n t a t t e n t i o n . Numerous f u n g i , e s p e c i a l l y those w i t h i n the genus Colletotrichum,. produce t h e i r c o n i d i a embedded w i t h i n a mucilagenous m a t e r i a l . The best s t u d i e d of these organisms i s Colletotrichum graminlcola the c a u s a l agent of anthracnose of a v a r i e t y o f grasses. The mucilage possesses remarkable p r o p e r t i e s t h a t a l l o w i t t o f u n c t i o n i n the s u r v i v a l of the organism and t o ensure i t s s u c c e s s f u l secondary spread. The mucilage of £. g r a m i n l c o l a i s produced d u r i n g s p o r u l a t i o n on i n f e c t e d host t i s s u e i n a c e r v u l i (Figure 3) and i n c u l t u r e , and c o n s i s t s of a complex mixture of high molecular weight g l y c o p r o t e i n s , enzymes, and s m a l l peptides (22, £ 4 ) · S p o r u l a t i o n and mucilage p r o d u c t i o n appear t o be light-dependent as n e i t h e r event occurs i n dark grown c u l t u r e s . The carbohydrate composition of the g l y c o p r o t e i n s i n c l u d e s mannose, rhamnose, g a l a c t o s e , and glucose i n l e v e l s of 66, 22, 10, and 2 mole %, r e s p e c t i v e l y . The peptide p o r t i o n s of the g l y c o p r o t e i n s are approximately 50% hydrophobic amino a c i d s and c o n t a i n very low l e v e l s of aromatic amino a c i d s . A very h i g h l e v e l of p r o l i n e (11 mole %) i s a l s o e v i d e n t . This amino a c i d and carbohydrate composition c l o s e l y resembles t h a t of animal mucins (22/ 2 2 ) · One such g l y c o p r o t e i n of p a r t i c u l a r importance i s a p r o l i n e - r i c h p r o t e i n (GP-66sm) t h a t i s produced by the mouse submandibular gland i n response t o high l e v e l s of d i e t a r y t a n n i n (22). The p r o t e i n s e l e c t i v e l y binds t o tannins and p r e c i p i t a t e s them from s o l u t i o n . This p r o t e c t s the mouse from the harmful e f f e c t s of d i e t a r y tannins which otherwise would reduce the e f f e c t i v e uptake of necessary amino a c i d s from d i e t a r y p r o t e i n . The resemblance of c e r t a i n of the £. graminlcola g l y c o p r o t e i n s t o animal mucins suggested t h a t the f u n g a l mucilage would a l s o c o n t a i n p r o l i n e - r i c h p r o t e i n s capable of b i n d i n g t o p o t e n t i a l l y i n h i b i t o r y t o x i c phenols. Indeed, we have demonstrated the presence of 3 p r o l i n e - r i c h p r o t e i n s i n the spore mucilage t h a t b i n d s p e c i f i c a l l y t o the phenylpropanoids p-coumaric and f e r u l i c a c i d ( 2 2 ) . Both phenols are produced by the corn p l a n t i n r

response t o i n f e c t i o n during the p e r i o d of

lesion


12. NICHOLSON

Fumctiomal Significance of Adhesion

231

Figure 3. Scanning e l e c t r o n micrographs of Colletotrifihum g r a m i n i n n i a . A) F u l l y developed acervulus of the fungus on a corn l e a f showing c o n i d i a (c) i n t e r s p e r s e d with s t e r i l e setae ( s t ) . Bar represents 10 [lm. B) The cut surface of an acervular mass of c o n i d i a . The cut surfaces of c o n i d i a (c) are v i s i b l e and demonstrate that c o n i d i a are embedded within an e x t r a c e l l u l a r mucilage (em). Bar represents 10 Jim. F i g . 3A reproduced with permission from Ref. 69. Copyright 1989 Academic Press, Inc.


232


r e s t r i c t i o n and a i d i n the containment of the fungus. In the absence of the mucilage and a s s o c i a t e d p r o l i n e r i c h p r o t e i n s the fungus i s i n h i b i t e d by these phenols completely whereas i n i t s presence the fungus i s not i n h i b i t e d (£2) . Comparison of the fungal p r o t e i n s w i t h the mouse p r o t e i n showed t h a t the fungal complex of p r o t e i n s was f i v e - f o l d more e f f e c t i v e i n sequestering phenols than the p u r i f i e d mouse p r o t e i n (£2) -

The p r o l i n e - r i c h p r o t e i n s are thought t o f u n c t i o n as p r o t e c t a n t s of spores i n the secondary spread of the fungus (£2) . Secondary spread occurs from leaves when spores are c a r r i e d across n e c r o t i c t i s s u e of l e s i o n margins onto healthy, u n i n f e c t e d areas of the l e a f . I t has now been shown t h a t both p-coumaric and f e r u l i c a c i d s as w e l l as g l y c o s i d e s and e s t e r s of these compounds leach r e a d i l y from the n e c r o t i c t i s s u e (Figure 4) and i n h i b i t fungal development i n v i v o i f the mucilage i s absent 169) . Enzymes i n the mucilage i n c l u d e an i n v e r t a s e (20.) / n o n - s p e c i f i c esterase (£2), β-glucosidase (£4.) , and DNase (21). Evidence suggests t h a t the esterase a i d s i n the e r o s i o n of the p l a n t c u t i c l e ; r o l e s f o r the other enzymes have not been a s c e r t a i n e d . A f e a t u r e of the £. g r a m i n l c o l a mucilage t h a t may be common amongst numerous f u n g i i s i t s a n t i d e s i c c a n t property (£2) . Spore masses dry w i t h i n minutes at r e l a t i v e h u m i d i t i e s l e s s than 90 %. Spores i n the absence of mucilage become d e s i c c a t e d and d i e w i t h i n hours, whereas i n i t s presence spores s u r v i v e f o r s e v e r a l months, even at r e l a t i v e h u m i d i t i e s as low as 45 %. Upon d r y i n g the mucilage forms a t h i n f i l m (< 0.01 Jim) t h a t surrounds spores and p r o t e c t s them (Figure 5). The mechanism through which a n t i d e s i c c a n t a c t i v i t y i s expressed i s unknown. D i s p e r s a l of the fungal spores i s a l s o f a c i l i t a t e d by the d r y i n g of the mucilage as spores are bound together i n c l u s t e r s that may be spread by wind as dry, p a r t i c u l a t e matter ( £ 2 ) . As the theme of t h i s volume i s the c o n t r o l of weed species i t i s appropriate t o report the presence of a low molecular weight (< 5 kDa) peptide i n the mucilage of £. graminlcola that f u n c t i o n s as an e l i c i t o r of p h y t o a l e x i n synthesis i n sorghum. Grain sorghum produces 2 phytoalexins of the deoxyanthocyanidin c l a s s , a p i g e n i n i d i n and l u t e o l i n i d i n (Figure 6) (22, 2 2 ) . Importantly, s y n t h e s i s of these compounds not only i n h i b i t s fungal growth and development but a l s o k i l l s the sorghum t i s s u e i t s e l f (Snyder and Nicholson, unpublished). Both shattercane and johnsongrass are important weed species of the genus Sorghum and both have the c a p a c i t y t o produce the t o x i c deoxyanthocyanidins. We


12. NICHOLSON

233

Functional Significance ojAdhesion

0.010 -

(a) p-coumarie acid

ferulic acid

0.005

/

0.000 0.010

0.005

0.000 10

20 30 40 50 ELUTION TIME,min

Figure 4. HPLC separation of phenolic components of an aqueous leachate from the surface o f corn leaves i n f e c t e d with Colletotrichum graminicola. a) untreated leachate; b) base hydrolyzed leachate; c) a c i d hydrolyzed leachate. Compounds 1 and 2 are isomers o f p-coumaric a c i d . Compounds separated i s o c r a t i c a l l y on a reversed phase C-18 column with a 70 % t o 30 % mixture of absolute methanol and 1 % a c e t i c a c i d . Reproduced with permission from Ref. 69. Copyright 1989 Academic Press, Inc.


234


Figure 5. Scanning e l e c t r o n micrograph of the surface of a d r i e d mass of Colletotrichum graminicola c o n i d i a showing that the c o n i d i a l mucilage d r i e s t o a t h i n f i l m (arrows) that surrounds c o n i d i a ( c ) . Bar represents 10 Jim. Reproduced with permission from Ref. 69. Copyright 1989 Academic Press, Inc.

Figure 6. Structures of the 3-deoxyanthocyanidin phytoalexins from sorghum. (I) a p i g e n i n i d i n ; (II) luteolinidin.


12. NICHOLSON

Functional Significant

235

have now demonstrated that the £. graminicola e l i c i t o r a l s o stimulates deoxyanthocyanidin synthesis i n these weeds with the r e s u l t that the t i s s u e i s k i l l e d (Yamaoka and Nicholson, unpublished). To date no other p l a n t species, i n c l u d i n g corn, cotton, soybeans and a v a r i e t y of vegetables, are a f f e c t e d by the elicitor. Present i n v e s t i g a t i o n s emphasize the p o t e n t i a l of the e l i c i t o r t o serve as a b i o c o n t r o l agent f o r e i t h e r shattercane or johnsongrass. Acknowledgments. This review i s a r t i c l e number 1 2 1 1 7 of the Purdue U n i v e r s i t y A g r i c u l t u r a l Experiment Station.

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