Î²-Glucans and Î²-Glucan Hydrolases in Plant Pathogenesis with

7 β-Glucans and β-Glucan Hydrolases in Plant Pathogenesis with Special Reference to Wilt-Inducing Toxins

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from Phytophthora Species J. R. W O O D W A R D , P. J. K E A N E , and B. A . S T O N E Departments of Biochemistry and Botany, LaTrobe University, Bundoora, Victoria, 3083, Australia

The interaction between plants and microbial pathogens can be described in molecular, physiological, morphological and ecological terms. Some of the molecular events in host-pathogen interactions are summarised in Table I. Many of these events involve carbohydrates or carbohydrate-containing molecules. Soil-borne pathogens may be attracted chemotactically to the root surface; chemotactic agents known include ethanol, amino acids and sugars. Adhesion of the invading organism to the plant surface is in some cases through a carbohydrate ligand-lectin interaction. Penetration of the host surface c e l l walls and subsequent invasion of the underlying tissues may involve hydrolytic enzymes secreted by the pathogen which can degrade wall components such as polysaccharides and cutin. Invasion of the host may be f a c i l i t a t e d by secretion of microbial toxins, some of which are polysaccharide or glycoprotein in nature. These toxins may cause symptoms such as wilting and cellular necrosis through interference with the host's metabolism. Following penetration by micro-organisms a number of resistance mechanisms may be i n i t i a t e d by the plant. These resistance mechanisms appear to be triggered by the plant's a b i l i t y to recognize the pathogen as "non-self", a characteristic also shown by plant mating systems and somatic c e l l interactions (35). Among the numerous resistance phenomena are localised metabolic responses or hypersensitive reactions which can lead to c e l l death, browning and collapse of cells around the site of infection. If the plant responds with sufficient speed, the invasion is not sustained and the interaction is said to be incompatible. One response i s modification of the c e l l wall either to provide material which may encapsulate the micro organism or to form a barrier through l i g n i f i c a t i o n or callose deposition. Another mechanism which may enable the plant to resist infection is the production of fungistatic compounds known as phytoalexins, which are e l i c i t e d by both chemical and physical stimuli. Among the chemical e l i c i t o r s are microbial poly saccharides and glycoproteins. A further defence may involve 0-8412-0555-8/80/47-126-113$07.25/0 ©

1980 A m e r i c a n C h e m i c a l Society

Sandford and Matsuda; Fungal Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

114

FUNGAL

TABLE I :

Molecular Events i n Host-pathogen I n t e r a c t i o n s

Events i n I n f e c t i o n and Symptom Induction


POLYSACCHARIDES

Agents

Chemotactic a t t r a c t i o n of s o i l organisms to r o o t surfaces.01,2)

Ethanol, amino a c i d s , sugars.

Recognition and b i n d i n g o f microorganisms to root s u r f a c e s .

Carbohydrate l i g a n d s on surfaces of micro-organisms o r roots (mucilage) , carbohydrateb i n d i n g p r o t e i n s on surfaces of micro-organisms o r r o o t s .

P e n e t r a t i o n of root s u r f a c e s , i n t e r c e l l u l a r or i n t r a c e l l u l a r i n v a s i o n . (8,9,10,11)

M i c r o b i a l polysaccharide hydrolases s p e c i f i c f o r c e l l u l o s e , xylan, pectin, 1,3-3-glucans, e t c .

Injury o r k i l l i n g o f host c e l l s . I n t e r f e r e n c e with primary metabolism, l o c a l n e c r o s i s , r e l e a s e of phytohormones, systemic e f f e c t s , e.g. w i l t i n g . Membrane leakage and d i s r u p t i o n . (12,17)

Low mol. wt. m i c r o b i a l t o x i n s , m i c r o b i a l polysaccharides and glycoproteins, microbial enzymes.

I n h i b i t i o n o f host enzymes.(18,19)

Microbial inhibitors.

Events i n Host

Resistance

Agents

Immobilisation and encapsulation of i n f e c t i n g agents. (20,21,22)

Plant c e l l wall polysaccharides and l e c t i n s .

H y p e r s e n s i t i v e r e a c t i o n s . (23,24)

Microbial polysaccharides.

Production of phytoalexins (posti n f e c t i o n , a n t i - m i c r o b i a l agents) formed de novo from remote p r e c u r s o r s . (3,23,25,26,27)

Chemical and p h y s i c a l s t i m u l i i n c l u d i n g m i c r o b i a l products, polysaccharides, glycop r o t e i n s and peptides.

Production of a n t i - m i c r o b i a l substances from p r e - e x i s t i n g s u b s t r a t e s . (17)

?

Increases i n l e v e l s o f glycan hydrolases and other enzymes. (11,28)

M i c r o b i a l metabolites e.g. 3,6-3-glucans as inducers.

Production o f ethylene. (29,109,110)

Microbial polysaccharides.

D e p o s i t i o n of c a l l o s e , gums, phenolics and polyphenolics i n and around i n f e c t e d c e l l s . Increases i n hydroxyproliner i c h glycoproteins i n c e l l w a l l s (30,31,32,33,34)

?



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115

enhancement of the l e v e l of the p l a n t ' s own complement of p o l y saccharide hydrolases which are b e l i e v e d to be i n v o l v e d i n depolymerising the c e l l w a l l s of invading micro-organisms. Recently i t has been shown by Kuc (36)that l o c a l i s e d host r e a c t i o n to the pathogen may confer r e s i s t a n c e on other p a r t s of the p l a n t . This i m p l i e s a t r a n s f e r of information through the p l a n t . The nature of the transmitted s i g n a l has not been resolved but i n the r e l a t e d phenomenon of i n s e c t - i n d u c e d r e s i s t a n c e a wound hormone i s produced at the s i t e of i n t e r a c t i o n of i n s e c t and p l a n t . This hormone i s b e l i e v e d to be carbohydrate i n nature and s t r u c t u r a l l y r e l a t e d to p l a n t c e l l w a l l s (37). I t i s the purpose of t h i s paper to summarise the present s t a t e of information concerning those aspects of p l a n t - m i c r o b i a l i n t e r a c t i o n s i n which ί^-glucans and 3-glucan hydrolases are i n v o l v e d and to make s p e c i a l reference to t h e i r r o l e i n wilt^i n d u c t i o n by Phytophthora species. I t has not been p o s s i b l e w i t h i n the framework of t h i s paper to discuss the extent to which these phenomena may be g e n e r a l l y r e p r e s e n t a t i v e of molecular events i n p l a n t pathogenesis, nor should i t be i n f e r r e d that a l l or any of the events i n v o l v i n g (3-glucans and β-glucan hydrolases are n e c e s s a r i l y encountered i n any p a r t i c u l a r plant-pathogen interaction» 2.

The Role of g-Glucans and β-Glucan Hydrolases of Pathogens i n I n f e c t i o n and Symptom Induction i n Host P l a n t s

2.1 M i c r o b i a l β-Glucan Hydrolases and L y s i s of P l a n t C e l l Wall Polymers. Many s o i l and a e r i a l pathogens gain access to host t i s s u e s by enzymic l y s i s of epidermal c e l l w a l l s of r o o t s , leaves, stems, e t c . . Phytopathogenic organisms possess an array of i n d u c i b l e polysaccharide hydrolases capable of degrading the complex polysaccharides of the p l a n t c e l l w a l l (8>, 9^, 10) . The enzymes i n c l u d e hydrolases f o r 1,4- and 1,3-3-glucans (11, 38). The hydrolases are of general importance i n the p e n e t r a t i o n and spread of the pathogen i n p l a n t t i s s u e s but are not determinants of v a r i e t a l s p e c i f i c i t y (2^3, 25) . 2.2 W i l t - i n d u c i n g β-Glucans from Micro-organisms. Some pathogens are known to produce toxins which i n t e r f e r e with the host's metabolism and growth, and may e v e n t u a l l y lead to the plant's death. The chemistry of m i c r o b i a l toxins and t h e i r e f f e c t s on the plant have been reviewed (13, 14, 15, 16, 17). W i l t i n g i s a common symptom i n many p l a n t diseases and some t o x i n s i s o l a t e d from pathogens are able to induce w i l t i n g experimentally. Among these w i l t - i n d u c i n g toxins are high molecular-weight polysaccharides and g l y c o p r o t e i n s . Table I I l i s t s those s t u d i e d and the symptoms they induce.



FUNGAL

POLYSACCHARIDES

Table I SOME HIGH MOLECULAR WEIGHT FUNGAL AND BACTERIAL PHYTOTOXINS COMPOUND

SOURCE

SYMPTOMS INDUCED

1,2-B-Glucan

Aqrobacterium tumefaciens

Wilting (39)

l,3,l,6-B-Glucan (Mycolaminarin)

Phytophthora spp.

Wilting (40)

Culture filtrate

Ρ cryptoqea

Veinal necrosis, collapse and de hydration of lamina (41)

Glycopeptide (98% galactose, 0-4% protein)

Erwinia amylovora

Wilting (42)

Glycopeptide Ceratocystis (Mainly 1,6-oculmi mannosyl substituted by 3-linked rhamnose) Glycopeptide (Highly branched, mainly mannose and glucose)

Wilting (15)

Corynebacterium Wilting (43,44) sepedonicum

Glycopeptide Corynebacterium Wilting (45) (Mostly L-fucose, insidiosum mannose, galactose, glucose) 1



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Toxins

2.2.1 W i l t - i n d u c i n g β-Glucans from Phytophthora Species. W i l t i n g i s a symptom of many diseases induced by species of Oomycetes. For example Phytophthora cinnamomi, the c a u s a l organism of "die-back" i n h o r t i c u l t u r a l p l a n t s such as f r u i t trees as w e l l as t r e e s , shrubs and w i l d f l o w e r s of A u s t r a l i a n n a t i v e f o r e s t s (46, 47) , produces w i l t i n g and s h r i v e l l i n g of cotyledons and leaves of Eucalyptus s e e d l i n g s (48, 49). We have s t u d i e d the w i l t - i n d u c i n g e f f e c t of macromolecular products of three species of Phytophthora chosen on the b a s i s of t h e i r p a t h o g e n i c i t y to the i n d i c a t o r p l a n t Eucalyptus sieberi (50). These were the extremely pathogenic P. cinnamomi, the l e s s pathogenic P. cryptogea and the non-pathogenic P. nicotianae. Fungi were grown i n batch c u l t u r e i n a l i q u i d medium (51) and the e t h a n o l - p r e c i p i t a b l e compounds c o l l e c t e d from the c u l t u r e f i l t r a t e a f t e r 5-7 days growth. The b i o l o g i c a l a c t i v i t y of t h i s m a t e r i a l on Ε. sieberi s e e d l i n g s i s shown i n Figure 1. Similar r e s u l t s were obtained with seedlings of Ε. cypellocarpa, which i s t o l e r a n t to P. cinnamomi (48), F r a c t i o n a t i o n of the crude P. cinnamomi p r e p a r a t i o n was achieved on DEAE-cellulose as shown i n Figure 2a. The unbound f r a c t i o n I was much more a c t i v e i n w i l t - i n d u c t i o n on a carbo hydrate b a s i s than the bound f r a c t i o n I I . The unbound f r a c t i o n (I) was rechromatographed on DEAE-cellulose (Figure 2b) to give a component r i c h i n carbohydrate but c o n t a i n i n g some p r o t e i n . Chromatography of t h i s component on CM-cellulose (Figure 2c) y i e l d e d an unbound carbohydrate f r a c t i o n c o n t a i n i n g 1.2% p r o t e i n . Gel f i l t r a t i o n chromatography i n d i c a t e d that the carbohydrate from the P. cinnamomi c u l t u r e s p u r i f i e d by ion-exchange chromatography was a polysaccharide which was p o l y d i s p e r s e with respect to apparent molecular weight i n the range 30,000 to 200,000 daltons. Chromatography of P. cryptogea and P. nicotianae preparations on DEAE-cellulose gave e l u t i o n p r o f i l e s s i m i l a r to that of P. cinnamomi. The unbound f r a c t i o n s were predominantly polysaccharide: the f r a c t i o n from P. nicotianae contained 0.2% p r o t e i n and that from P. cryptogea 0.4% p r o t e i n . The w i l t inducing a c t i v i t y of the p u r i f i e d polysaccharides i s shown i n Figure 3. The monosaccharide composition and s t r u c t u r e of the P. cinnamomi polysaccharide was i n v e s t i g a t e d by a c i d h y d r o l y s i s , methylation and s p e c i f i c enzymic degradation (50) Each of the polysaccharides was composed predominantly of glucose (see Table I I I ) . e



118

FUNGAL

TIME

POLYSACCHARIDES

(h)

Figure 1. E . sieberi wilting bio-assay of unfractionated ethanol-insoluble poly mers from the culture filtrates of (a)Y. cinnamomi, (b) cryptogea, and (c) P. nico tianae; polymer concentration: (%) 500 pg/mL; (O) 50 μg/mL; (A) 5 ^g/mL. The assessment of wilting was performed by introducing substances to be tested through the cut ends of the main rootlets of 1-2 month old E . sieberi seedlings immersed in solu tions of test material. Wilt assays were conducted under controlled conditions in the presence of nystatin (50 units/mL) and tetracycline (50 ^gjmL). The degree of wilting was assessed at regular intervals during 40-hr tests. An arbitrary score of wilting severity (index of wilting, IW) was used to record the seedlings' response: 1, slight wilting; 2, pronounced wilting; 3, severe wilting; and 4, death . Each compound was tested in triplicate and the IW was the mean score.



7.

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Figure

ET AL.

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2.

119

Fractionation of Phytophthora cinnamomi extracellular polymers: (O) glucose equivalents; (Φ) absorbance at 280 nm. (a) Stepwise elution of components of the ethanol-insoluble polymers from culture fil trates of P. cinnamomi on DEAE-cellulose. The column was loaded with a preparation containing 280 mg carbohydrate and 218 mg protein in 104 mL of lOmM tris-acetate (pH 8) and washed successively in 250 mL of lOmM tris-acetate (pH 8), 200 mL of same buffer 0.5m NaCI and 100 mL of the starting buffer 1.0M NaCI. 9-mL fractions were collected, (b) Gradient elution chromatography of Peak I (Figure 2a) on DEAEcellulose. Fractions 1-22 were pooled, concentrated on a rotary film evaporator under reduced pressure at 40°C, and dialyzed against lOmM tris-acetate (pH 8). The concen trated fractions (33 mL) containing 109 mg carbohydrate and 75 mg protein were loaded onto the column that was washed with 40 mL starting buffer followed by a 300-mL gradient (0-0.75M NaCI in starting buffer), (c) Gradient elution chromatography of Peak I from DEAE-cellulose. Fractions 2-11 (Figure 2b) were pooled, concentrated, and dialyzed against distilled water and then against lOmM. acetic acid-sodium acetate buffer, pH 5.0. The concentrated fraction (32 mL) containing 88 mg carbohydrate and 53 mg protein was loaded onto a column and washed with 100 mL starting buffer fol lowed by a 200-mL gradient (0-0.75M NaCI in starting buffer).


FUNGAL


120

0

25

POLYSACCHARIDES

50

TIME

(h)

Figure 3. E . seiberi wilting bio-assay of purified Phytophthora polysaccharides; polymer concentration: (%)500 ^g/mh; (O)50 μg/mL; (A) 5 \xg/mL. (a) P. cinnamomi polysaccharide from successive DEAE-cellulose and CM-cellulose chromatography, (b) P. cryptogea polysaccharide from DEAE-cellulose chromatogra phy, (c) P. nicotianae polysaccharide from DEAE-cellulose chromatography.

TABLE I I I .

Monosaccharide

Composition of the P u r i f i e d

P o l y s a c c h a r i d e s from Phytophthora Filtrates

Culture

(50)

Arabinose

Mannose

Glucose

%

%

%

P. cinnamomi

-

4

96

P. cryptogea

trace

trace

100

P. nicotianae

trace

trace

100


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M e t h y l a t i o n a n a l y s i s i n d i c a t e d t h a t there were 3 - l i n k e d g l u c o s y l residues i n the molecule, as w e l l as g l u c o s y l residues s u b s t i t u t e d at both 3- and 6 - p o s i t i o n s (see Table I V ) .


TABLE IV.

Deduced Glucosyl Component

G l u c o s i d i c Linkage Composition of the Phytophthora Polysaccharides as Determined by g.l.c./m.s. of t h e i r P a r t i a l l y Methylated A l d i t o l Acetates (50) Molar

Percentage

T* P. cinnamomi

P. cryptogea

P.

nicotianae

Terminal glucosyl

1.00

27

34

34

1,3-linked glucosyl

1.87

44

23

26

1,6-linked glucosyl

2.30

7

6

8

1,3,6-linked glucosyl

4.55

22

37

32

* Retention times r e l a t i v e to l,5-di-0-acetyl-2,3,4,6tetramethyl-D-glucitol. The p o l y s a c c h a r i d e s were β-linked-glucans as judged by t h e i r s u s c e p t i b i l i t y to h y d r o l y s i s by s p e c i f i c β-glucan hydrolases and from t h e i r i n f r a - r e d s p e c t r a . H y d r o l y s i s of the P. cinnamomi polysaccharide by a s e r i e s of β-glucan hydrolases with d e f i n e d s p e c i f i c i t y gave f u r t h e r s t r u c t u r a l i n f o r m a t i o n . The Euglena l,3-B-glucan exo-hydrolase (EC 3,2.1.58) (52) only p a r t i a l l y degraded the p o l y s a c c h a r i d e , r e l e a s i n g mainly glucose and s m a l l e r amounts o f a compound with the Rglc of g e n t i o b i o s e ; w h i l e the Rhizopus arrhizus 1,3-|3-glucan endo-hydrolase (EC 3.2.1.6) (53) produced more complete degradation, r e l e a s i n g glucose and a s e r i e s of o l i g o - g l u c o s i d e s . The B a c i l l u s s u b t i l i s 1,3:1,4-βglucan endo-hydrolase (EC 3.2.1.73) (54) d i d not degrade the p o l y s a c c h a r i d e . H y d r o l y s i s by the exo-hydrolase suggested t h a t some 1,3-linked p o r t i o n s o f the p o l y s a c c h a r i d e were l o c a t e d a t terminal non-reducing ends. In a d d i t i o n , the extensive cleavage by the Rhizopus endo-hydrolase i n d i c a t e d t h a t there are s u s c e p t i b l e 1,3-linkages throughout the polymer. The methylation and enzymic data do not d i s t i n g u i s h between a molecule having a 1,3-linked backbone and branch p o i n t s a t C-6 and one having a 1,6-linked backbone and branching at C-3.


122

FUNGAL

POLYSACCHARIDES


When the enzyme-treated preparations were t e s t e d f o r b i o l o g i c a l a c t i v i t y i n the w i l t i n d u c t i o n assay i t was found that the Rhizopus enzyme-treated p r e p a r a t i o n had l o s t i t s b i o l o g i c a l a c t i v i t y (Figure 4). The B a c i l l u s enzyme-treated preparation r e t a i n e d i t s a c t i v i t y as might be expected from the i n a b i l i t y of t h i s enzyme to depolymerise the p r e p a r a t i o n . A f t e r treatment with the Euglena exo-hydrolase, which caused only p a r t i a l degradation, the polymer r e t a i n e d most of i t s w i l t - i n d u c i n g activity. 2.2.2 R e l a t i o n s h i p of W i l t - i n d u c i n g 3,6-g-Glucans to Hyphal Wall Polysaccharides. The w i l t - i n d u c i n g 3,6-3-glucans are comparable i n s t r u c t u r e to s o l u b l e 3,6-3-glucan components of hyphal w a l l s and c u l t u r e f i l t r a t e s of other species of Oomycetes. Related polysaccharides have a l s o been found i n higher f u n g i (see Table V). T y p i c a l l y they have a l i n e a r 1,3-3-glucan backbone and are s u b s t i t u t e d by l a t e r a l s i d e chains c o n s i s t i n g of s i n g l e , g l u c o s y l residues l i n k e d through C-6 to main chain residues. The methylation and enzymic data f o r the P. cinnamomi polysaccharide are c o n s i s t e n t with such a s t r u c t u r e although other s t r u c t u r e s are a l s o compatible with the data. The hyphae of Phytophthora p a r a s i t i c a have been shown to have b i l a y e r e d c e l l w a l l s , the innermost l a y e r c o n s i s t i n g of c e l l u l o s e m i c r o f i b r i l s i n a matrix of 3,6-3-glucan and p r o t e i n and an outermost l a y e r of water-soluble 3,6-3-glucan (71). In Schizophyllum commune a filamentous m a t e r i a l c o n t a i n i n g 3,6-3glucan has been shown to cover the surface of the hyphae (72) . I t i s presumably t h i s l a y e r which i s shed i n t o the c u l t u r e medium during c u l t i v a t i o n of these fungi i n shaken f l a s k s . Phytophthora spp. and other species of Oomycetes a l s o have cytoplasmic 3,6-3-glucans (mycolaminarins) and t h e i r phosphate e s t e r s (73). These polysaccharides a l s o induce w i l t i n g i n avocado (Persea indica) and other p l a n t s (40) (see Table I I ) . 2.2.3 W i l t i n g Response Induced by Various Polysaccharides i n Ε. sieberi Seedlings. We have compared the w i l t - i n d u c i n g c a p a c i t i e s of the Phytophthora preparations and a number of polysaccharides of known s t r u c t u r e (50) . The r e s u l t s are shown i n Table VI. At comparable concentrations the degree and type of w i l t i n g response induced by n e u t r a l 3 - l i n k e d polysaccharides was i n d i s t i n g u i s h a b l e from that induced by the Phytophthora poly saccharide preparations. The charged polysaccharides, CM-pachyman and h y a l u r o n i c a c i d , had only s l i g h t a c t i v i t y i n the w i l t i n g assay. Of the two α-glucans t e s t e d , s o l u b l e s t a r c h was without e f f e c t and a dextran, 1,6-a-glucan, caused severe w i l t i n g o f the cotyledons only, a response q u i t e d i f f e r e n t from that found with the 3 - l i n k e d p o l y s a c c h a r i d e s . D i f f e r e n c e s between the wilting response given by s t a r c h , dextran and p e c t i n and that given by 3-glucans have been observed i n other s t u d i e s (40)(see Table V I ) .


Wilt-Inducing


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TIME

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Toxins

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Figure 4. E . sieberi wilting bio-assay of the purified and enzymically treated P. cinnamomi polysaccharide; (Φ) 500 μg/mL; (O) 50 fxg/mL; (A) 5 g/mL: (a) untreated control; (b) after treatment with B. subtilis 1,3;1,4-β-glucan endohydrolase; (c) after treatment with E . gracilis Ι^-β-gjLucan exohydrolase; (d) after treatment with R. arrhizus 1,3-βglucan endohydrolase.



filtrate)

SCB

Monilinia fructicola (Culture f i l t r a t e )

SCB Β

cerevisiae Term Ara 1,5 Ara

Small e x t r a cellular fraction

Β

5

16 1.1

16

15 14

43 65

10

9

12 72

18

27 18

19

32

21

10

8

24

Large e x t r a cellular fraction

Β

35

13

25

21

58

56

22

(60)

(58)

(57)

(56)

(55, 56)

References

(1,3:1,4) (61) 10

1

S t r u c t u r a l Linkages (% or r a t i o ) 1,3- 1,6- 1,3,6- 1,4-

10

Term

13

Term Man

Other Monomers or Substituants

Β

Structure

Some Water-soluble 1,3-3-Glucans from Fungi

(Hot water e x t r a c t )

ASCOMYCOTINA Saccharomyces (Autolysate)

Pythium deharyanum (Culture f i l t r a t e )

(Culture

Phytophthora (Mycelium)

megasperma

granulosum

Trichophyton

OOMYCETES

quinckeanum

Microsporum (Mycelium)

DEUTEROMYCOTINA

TABLE V:


Ο Ω

>

*

FUNGAL

POLYSACCHARIDES

Table "ΖΓ WILTING RESPONSE OF Ε SIEBERI SEEDLINGS TO VARIOUS POLYSACCHARIDES


POLYSACCHARIDE

STRUCTURE INDEX OF WILT MAIN INDUCTION LINKAGES WILTING (E SIEBERI) (PERSEA INDICA) (50 ) (40) T

P. CINNAMOMI

3-, 6- and 3,6-β3- and 3,6-β-

SCB

30

SCB

3 0

+ (1 mg/ml)

SCB

ND

+ (IO;ug/ml and 50/ig/ml)

LAMINARIN (from 3 - and Eisenia bicyclis) 6-B-

L

2-3

ND

CM-PACHYMAN

L

10

ND

L

27

ND

L

0 3

ND

L

23

ND + (lmg/ml)

LAMINARIN (from Laminaria hyperborea) MYCOLAMINARIN (phosphate)

3; 6- and 3,6-β-

3-β-

BARLEY GLUCAN 34HYALURONIC ACID 34LUTEAN

and βand 0-

6-0-

L

23

4-and STARCH (soluble) 4 , 6 - σ Ι -

Β

0

DEXTRAN

6-oc-

L

3-0"

CM-CELLULOSE

4-0-

L

ND

+ (lmg/ml)

PECTIN

4-a-GALUA

L

ND

+ (Img/mlf"

PUSTULAN

6-0-

•For explanation of index of wilting see caption to Figure I * [Polysaccharide] 500>ig/ml L Linear î Also tested on glycine max L SCB Side chain branching and Theobroma cacoa L Β Branched **0nly cotyledons affected '**0nly leaves affected ttCaused marginal necrosis in leaves ND Not determined



7.

WOODWARD ET

AL.

Wilt-Inducing

Toxins

127

2.2.4 Mechanism of W i l t - i n d u c t i o n by β-Glucans. In general, p l a n t w i l t i n g may be induced by toxins i n one of s e v e r a l ways by prevention of water uptake, by i n c r e a s e i n water l o s s through impairment of stomatal f u n c t i o n , by prevention of water t r a n s p o r t w i t h i n the p l a n t o r by a l t e r a t i o n of membrane p e r m e a b i l i t y (12, 16). L i g h t microscopy of E. s i e b e r i seedlings i n which w i l t i n g had been induced by P. cinnamomi 3,6-3-glucan f a i l e d to r e v e a l any p h y s i c a l blockage such as t y l o s e s or v a s c u l a r gels i n the root or stem v e s s e l s (50) nor were major blockages due to mycelium or t y l o s e s seen i n the v a s c u l a r t i s s u e of seedlings d i r e c t l y i n o c u l a t e d with P. cinnamomi (74) . However, i t has been suggested (12) that high molecular weight p o l y saccharides might block the p e t i o l a r v e i n l e t s and "the u l t r a f i l t e r s of the p i t membrane". U l t r a s t r u c t u r a l examination would be needed to t e s t t h i s p o s s i b i l i t y . Low molecular weight t o x i n s from Fusiococcum amygdali (75) and Helminthosporium maydis (76) are known to a f f e c t stomatal f u n c t i o n through t h e i r a c t i o n on guard c e l l s but no polymeric toxins have been shown to act at t h i s s i t e . A primary e f f e c t of w i l t - i n d u c i n g t o x i n s of low molecular weight may be through plasma membrane damage, r e s u l t i n g i n increased water p e r m e a b i l i t y (14, 16). The macromolecular w i l t inducing toxins may a l s o i n t e r a c t with membrane components and have s i m i l a r e f f e c t s on water p e r m e a b i l i t y . Thus glycopeptides from c u l t u r e f i l t r a t e s of F u l v i a fulva (syn. Cladosporium fulvum) cause leakage of e l e c t r o l y t e s from i s o l a t e d tomato mesophyll cells. I t i s suggested that the glycopeptides may b i n d r e v e r s i b l y to plasma membranes (77). Although there i s no d i r e c t evidence f o r a l t e r a t i o n of membrane p e r m e a b i l i t y by Phytophthora c e l l w a l l 3-glucans, there i s evidence that low molecular weight (

Î²-Glucans and Î²-Glucan Hydrolases in Plant Pathogenesis with

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