The Involvement of Membrane-Degrading Enzymes During Infection of

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Chapter 21

The Involvement of Membrane-Degrading Enzymes During Infection of Potato Leaves by Phytophthora infestans Robert A. Moreau Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118

During the infection of potato leaves by Phytoph thora infestans there is a rapid degradation of membrane components (chlorophyll, galactolipids, and phospholipids). We have identified an inducible phospholipase Β activity which is secreted by the fungus in culture and also occurs during the infection of potato leaves. Under conditions which result in maximal in duction of phospholipase activity in culture filtrates, comparable levels of galactolipase and triacylglycerol lipase are also observed. During the infection of potato leaves there was a large (>l4-fold) increase in total phospholipase activity. Since potato leaves also contain very high levels of phospholipase and galactolipase activities, experiments were conducted to elucidate the possible involvement of the lipolytic enzymes of the pathogen and the host during infection and resistance. Phytophthora infestans i s the fungal pathogen that causes late b l i g h t of potatoes. The I r i s h "potato famine" of the 1840's was caused by P. infestans. Even today, l a t e b l i g h t i s s t i l l the single most important disease of potatoes worldwide (1). With modern farming techniques approximately 10% of the world potato crop and 4% of the U.S. potato crop are l o s t i n the f i e l d to t h i s disease each year (2,3). In addition to causing these large losses i n the f i e l d , l a t e b l i g h t and the secondary b a c t e r i a l infections which often accompany i t cause comparable losses during the storage of tubers. Despite many years of intensive breeding research, no e x i s t i n g c u l t i v a r s of European or North American potatoes allow commercial c u l t i v a t i o n i n humid regions without fungicide p r o t e c t i o n (_1). At best, farmers can choose c u l t i v a r s with a moderate l e v e l of general resistance ( i . e . , Sebago) which are protected by fewer applications This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

of fungicide than are required by other c u l t i v a r s . Our laboratory i s interested i n studying disease resistance mechanisms at the subcellular l e v e l , e s p e c i a l l y at the membrane l e v e l . For our preliminary studies we have chosen to study the changes i n membrane l i p i d composition that occur during a susceptible host-pathogen i n t e r a c t i o n . We have attempted to correlate changes i n l i p i d composition with changes i n the l e v e l s of l i p o l y t i c enzymes of the host and pathogen separately and during i n f e c t i o n . This report w i l l summarize our recent studies (4-7) of the P. infestans-potato l e a f i n t e r a c t i o n and compare them with comparable studies of other host-pathogen i n t e r a c t i o n s . Changes i n Host U l t r a s t r u c t u r e and L i p i d Composition during Infection In the f i e l d , i n f e c t i o n of potato plants by P. infestans i s usually l o c a l i z e d i n the leaves. Encysted zoospores germinate on the l e a f surface, form an appressorium, and penetrate the c u t i c u l a r layers of the leaf. The recent work of Wilson and Coffey (8) indicates that d i r e c t penetration of epidermal c e l l s adjacent to stomatal c e l l s i s the most common mode of entry. Hyphae spread both i n t r a and i n t e r c e l l u l a r l y forming haustoria when host c e l l s are penetrated (9,10). In susceptible i n t e r a c t i o n s , host c e l l s contain "organelles the structure of which i s disorganized" (10) or "very disintegrated organelles" (9). In contrast, fungal organelles remain i n t a c t long a f t e r the surrounding host t i s s u e has been disrupted (10). In a r e s i s t a n t i n t e r a c t i o n the fungus a c t i v e l y i n f e c t s the leaves f o r the f i r s t 9-12 h but i s then subsequently k i l l e d by 24 h (9). Unfortunately, no histochemical or cytochemical studies of the involvement of l i p o l y t i c enzymes during the i n f e c t i o n of potato leaves by P. infestans have yet been reported. Histochemical and biochemical observations of potato tubers infected by P. infestans revealed elevated l e v e l s of esterase (measured with a-naphthol acetate) i n u n i d e n t i f i e d host organelles during i n f e c t i o n (11). Cytochemical techniques were used to i d e n t i f y l i p o l y t i c a c t i v i t y i n fungal and host c e l l walls during the i n f e c t i o n of lettuce (Lactuca sativa L.) cotyledons by Bremia lactucae (12). Very l i t t l e i s known about the changes that occur i n the composition of membrane l i p i d s during the i n f e c t i o n of plants by fungal pathogens. Three such studies have been reported (13-15) and deal with the i n f e c t i o n of susceptible leaves by rust species. In each case there was a rapid disappearance of chloroplast glycol i p i d s and phosphatidyl g l y c e r o l , and evidence f o r the presence of fungal phospholipids during l a t t e r stages of i n f e c t i o n . During the i n f e c t i o n of bean plants (Phaseolus v u l g a r i s ) by Uromyces phaseoli, there was an increase i n the proportion of unsaturated f a t t y acids and t h i s change was a t t r i b u t e d to fungal growth (16). In our preliminary studies of the i n f e c t i o n of susceptible potato leaves by P. infestans we also observed a s i m i l a r rapid degradation of g l y c o l i p i d s and phosphatidyl g l y c e r o l . However, upon further i n v e s t i g a t i o n we r e a l i z e d that the unique composition of membrane l i p i d s i n P. infestans may provide a useful marker during i n f e c t i o n . The polar l i p i d composition of healthy potato leaves and cultured P. infestans i s shown i n Table I . The l i p i d

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Infection of Pota to Leaves by Phytophthora infestans

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Table I . Polar L i p i d Composition of Potato Leaves and P. infestans. Fungal cultures were grown i n l i q u i d French bean media (4) f o r 14 days at 14° without shaking. L i p i d s were extracted (42) separated by 2-dimensional TLC (18). I n d i v i d u a l spots were i d e n t i f i e d by comparison with standards, scraped from TLC p l a t e s , and analyzed f o r phosphorous (6) and hexose (43). mole % Lipid Class

3

Healthy Potato Leaf

P. infestans

53 20 3 14 3 6 1 0

0 0 0 46 0 39 2 13

MGDG DGDG SQD PC PG PE PI CAEP

Abbreviations: MGDG = monogalactosyldiglyceride, DGDG = d i g a l a c t o s y l d i g l y c e r i d e , SQD = sulfoquinovasyldiglyceride, PC = phosphatidylcholine, PG = phosphatidylglycerol, PE = phosphatidylethanolamine, PI = p h o s p h a t i d y l i n o s i t o l , CAEP = ceramide aminoethylphosphonate. composition of potato leaves i s comparable to that of the leaves of other species of angiosperme (17). P. infestans contains high l e v e l s of phosphatidylcholine and phosphatidylethanolamine, as i s common f o r fungi (13), but i t also contains an unusual sphingol i p i d , ceramide aminoethylphosphonate (CAEP) (Figure 1). This sphingolipid was previously i d e n t i f i e d i n two c l o s e l y related fungi Pythium prolatum and Phytophthora p a r a s i t i c a var. nicotianae (18). We have recently observed that a f t e r 6 days of i n f e c t i o n CAEP i s detectable i n t h i n layer chromatograms of l i p i d s from infected leaves. Since the fungal ceramide lacks carboxyl ester bonds i t i s r e s i s t a n t to hydrolysis by phospholipase Β and i t s presence may render c e r t a i n fungal membranes less vulnerable to degradation during i n f e c t i o n . P. infestans also contains high l e v e l s of two unusual f a t t y acids, arachidonic acid (20:4) and eicosapentaenoic acid (20:5) (19) which are absent i n the potato plant and could also be used as markers of fungal l i p i d s during i n f e c t i o n . We are currently studying the quantitative changes i n these membrane l i p i d components during i n f e c t i o n . Properties of Phospholipases

from Phytopathogens

Phospholipases are enzymes that catalyze the hydrolysis of membrane phospholipids. There are s i x types of phospholipases (A A , B, C, D, and lysophospholipase). Each hydrolyses a d i f f e r e n t part of the phospholipid molecule and results i n the formation of d i f f e r e n t lt

2

346


products (Figure 2). Although some assay techniques are capable of i d e n t i f y i n g the type of phospholipase a c t i v i t y , several of the common techniques only measure t o t a l breakdown of phospholipid. Phospholipase a c t i v i t y has been reported to occur i n eleven phytopathogens (ten fungi and one bacteria) (4,16,20-27) (Table I I ) . In s i x of these species, phospholipase Β (which hydrolyzes two f a t t y acids per phospholipid molecule) was detected. Only three of these studies (20,21,22) have reported the e f f e c t of fungal phospholi pases on plant t i s s u e . In Table I I , i t i s noted whether these enzyme a c t i v i t i e s were detected i n fungal cultures ( i n t r a c e l l u l a r or e x t r a c e l l u l a r ) , or i n infected plant t i s s u e . The pH optimum for each enzyme i s also l i s t e d . Table I I . Occurrence of Phospholipase A c t i v i t y i n Phytopathogens Type of Phospholipase

Optimum pH

B o t r y t i s cinerea Erwinia carotovora

Β C

Erysiphe p i s i

A

5.0 Assayed only at 8.0 Assayed only at 8.9 4.0

Species

Fusarium solani Phoma medicaginis Phytophthora infestans Rhizoctoni solani S c l e r o t i a sclerotiorum Sclerotium r o l f s i i Thielaviopsis basicola Uromyces phaseoli

2

?

Β Β ?

Β Β Β A or Β or C

?

9.0 7.5 - 8.5 4.0 4.5 4.5 and 8.5 4.0 - 5.0

3

Localization of Enzyme Ref M Ε

21 22

I

20

Ε M Ε,I,Ρ Ε Ε,Ρ Ε,Ρ Μ,Ρ Ρ

23 24 4 23 25 26 27 16

Abbreviations: Ε = from e x t r a c e l l u l a r fungal culture; I = from i n t r a c e l l u l a r fungal culture; M = from mixture of i n t r a c e l l u l a r and e x t r a c e l l u l a r ; Ρ = from infected plant t i s s u e . The properties of the phospholipase a c t i v i t i e s from cultures of P. infestans (4) are summarized i n Table I I I . Of the f i v e types of l i q u i d media tested, the greatest phospholipase a c t i v i t y was obtained when P. infestans was grown i n rye steep media. The phospholipase a c t i v i t y i n rye culture f i l t r a t e s was stimulated 15-fold by omitting glucose from the medium. The addition of 100 mg/liter of phosphatidylcholine (PC) to the rye medium caused an a d d i t i o n a l 35-fold stimulation i n the a c t i v i t y of phospholipase. These experiments suggest that t h i s enzyme a c t i v i t y i s inducible. The addition of other l i p i d s (sunflower o i l , wax ester, and cholesterol oleate) to rye media also caused an apparent induction of phospholipase a c t i v i t y . We also detected e x t r a c e l l u l a r phospho lipase a c t i v i t y i n lima bean agar cultures of P. infestans as shown i n the second column of Table I I I . When the fungus was collected

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Infection of Potato Leaves by Phytophthora infestans

OH

Η

Η

I

I

I

c

H

S

NH

H

C—0

I

(ÇH ), 2

CH

2

(ÇH ) 2

CH

3

0 H H I

I

I

c—o—P—c—c—Nu

c

î

347

I

I

Η

0 H H

I

I

3

(-)

1 8

3

Figure 1. Ceramide aminoethylphosphonate (CAEP). When obtained from P. infestans the most common f a t t y acid i s arachidonic acid and the most common long chain base i s sphingosine.

PHOSPHOLIPASE A, PHOSPHOLIPASE Β PHOSPHOLIPASE A ->y

\

2

^

II

0 CH -0-C-R R-C-O-CH 0 " C H - 0 - Ρ - 0 - CH CH N ( CH ) 2

1

2

PHOSPHOLIPASE C

2

2

3

3

PHOSPHOLIPASE D

Figure 2. S i t e s of a c t i o n of various types of phospholipases on phosphatidylcholine. Lysophospholipase hydrolyzes the carboxyl ester bond of a lysophospholipid ( i . e . , lysophosphatidylcholine).

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

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Table I I I . A Summary of the Properties of the Phospholipase A c t i v i t y from Cultures of P. infestans (4,7) Liquid Culture Media Regulation Localization pH optimum E f f e c t of 5 mM DTT E f f e c t of 5 mM EDTA E f f e c t of 5 mM CaCl Km f o r PC Substrate s p e c i f i c i t y 2

Rye steep Repressed by glucose induced by PC Mostly e x t r a c e l l u l a r 9.0 96% I n h i b i t i o n None 5% I n h i b i t i o n 2.86 μΜ TG - PC - MGDG

Agar Culture Lima bean Not tested Extracellular 9.0 94% I n h i b i t i o n 55% I n h i b i t i o n 17% I n h i b i t i o n Not tested PC > TG

Abbreviations: PC = phosphatidylcholine, TG = t r i a c y l g l y c e r o l , MGDG = monogalactosyldiglyceride, DTT = d i t h i o t h r e i t o l , EDTA = ethylenediaminetetraacetic acid. from l i q u i d rye cultures (with added PC to induce phospholipase a c t i v i t y ) much more phospholipase a c t i v i t y (30-fold) was found e x t r a c e l l u l a r l y than i n t r a c e l l u l a r l y . The pH optimum of the enzyme a c t i v i t y from both sources was 9.0. D i t h i o t h r e i t o l severely i n h i b i t e d both enzyme a c t i v i t i e s . EDTA (5 mM) had no e f f e c t on the enzyme from l i q u i d rye c u l t u r e , but i n h i b i t e d the enzyme from agar cultures. Both enzymes were s l i g h t l y i n h i b i t e d by 5 mM CaCl . A very low Km (2.86 μΜ) f o r phosphatidylcholine was measured using induced rye culture f i l t r a t e as a source of enzyme. When l i p i d substrates other than PC were tested the enzymes i n the induced l i q u i d culture were able to hydrolyze t r i a c y l g l y c e r o l (TG) and g a l a c t o l i p i d s (GL) at rates very s i m i l a r to that f o r PC. In contrast, the enzymes from agar culture hydrolyzed PC at a rate about s i x times higher than f o r TG. We have recently completed a study of the production of extra c e l l u l a r enzymes by germinating cysts of P. infestans (7). Although we were able to i d e n t i f y an esterase a c t i v i t y (p-nitrophenyl butyrate hydrolase) that appeared to be secreted during germination (0 to 20 h ) , phospholipase and lipase a c t i v i t i e s were apparently not secreted (Table IV). We are currently studying whether t h i s esterase can hydrolyze any p h y s i o l o g i c a l substrates. This i s an example of a case where the use of a nonphysiological substrate (PNP-butyrate) resulted i n new and i n t e r e s t i n g information. However, extreme care must be exercised when t r y i n g to draw physio l o g i c a l conclusions from studies using nonphysiological substrates ( i . e . , p-nitrophenyl esters or 4-methylumbelliferyl e s t e r s ) . We are currently i n v e s t i g a t i n g a new phospholipase assay (28), which employs a fluorescent phospholipid substrate, l-acyl-2-[6-[(7nitro-2,1,3 benzoxadiazol-4*yl)amino]-caproyl] phosphatidyl choline (C -NBD-PC) . Our preliminary studies (Table IV) indicate 2

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Table IV. Changes i n the l e v e l s of e x t r a c e l l u l a r enzyme a c t i v i t i e s during germination (0 to 20 h) of cysts of P. infestans. The l e v e l s of the f i r s t three enzymes were pre v i o u s l y reported (7). C,-NBD-PC hydrolysis was measured as described (28). Enzyme A c t i v i t y η mol/min/10 Spores 6

20 h

0 h

5 h

10 h

9.0 .127

43.2 .132

315.7 .137

407.3 .138

( C-PC assay)

.590

.599

.604

.610

Phospholipase (C -NBD-PC assay)

.738

.735

.733

.732

Enzyme p-nitrophenyl butyrate hydrolase Lipase Phospholipase 14

6

that very s i m i l a r values are obtained when^phospholipase a c t i v i t y i s measured with authentic phospholipid ( C-PC) or the fluorescent phospholipid (C,-NBD-PC). I f i t proves r e l i a b l e , t h i s new fluorometric assay w i l l be much more convenient (30 assays per hour verses 15 assays per day using C-PC). 14

Properties of the Phospholipase A c t i v i t y i n Healthy Potato Leaves Many types of plant tissue contain high l e v e l s of l i p o l y t i c acyl hydrolase (LAH) a c t i v i t y (29). These enzymes are capable of hydrolyzing phospholipids (phospholipase Β), g a l a c t o l i p i d s (galact o l i p a s e ) , and acyl glycerols. The LAH s i n potato tubers and bean leaves have been p u r i f i e d and extensively studied (29). In 1979 Matsuda and Hirayama (30) reported that the t o t a l a c t i v i t y of phos pholipase i n potato leaves was about 400-fold lower than i n potato tubers. They subsequently p u r i f i e d a l i p o l y t i c acyl hydrolase from potato leaves (31). The enzyme had a molecular weight of about 110,000 and a pH optimum of 5.0. The rate of hydrolysis of galac t o l i p i d s was 7-fold higher than f o r phospholipids. The following experiments i l l u s t r a t e that when studying the involvement of phospholipase i n the host-pathogen i n t e r a c t i o n , the t o t a l contribution of enzyme of host o r i g i n may be considerably higher than previously r e a l i z e d . Rodionov and Zakharova (32) recently reported very high rates of a u t o l y t i c hydrolysis of mem brane l i p i d s i n homogenates of potato leaves (26-37% of the phospholipids were hydrolyzed a f t e r 2 h at 0-1°). Our laboratory recently confirmed t h i s observation and proceeded to study some of the properties of the l i p o l y t i c acyl hydrolase a c t i v i t y i n potato leaves (6). L i p o l y t i c acyl hydrolase a c t i v i t y i s apparently inactivated by polyphenol oxidase or i t s t o x i c quinone products. 1

350


When polyphenol oxidase a c t i v i t y was c o n t r o l l e d , phospholipase a c t i v i t i e s ranged from 1.04 to 11.60 μ mol/min/gfw i n the leaves of 41 North American c u l t i v a r s (6). These values are much higher than those previously reported f o r potato leaves (.009 pmol/min/gfw) (30) and nearly as high as i n potato tubers (2 to 30 μπιοΐ/min/gfw) (5,30). Change i n the Levels of L i p o l y t i c Enzymes during I n f e c t i o n Hoppe and Heitefuss (16) reported a 2-3-fold increase i n phospho lipase a c t i v i t y during the i n f e c t i o n of susceptible bean leaves by U. phaseoli. During the i n f e c t i o n of potato leaves by P. infestans we observed a greater than 14-fold increase i n t o t a l phospholipase a c t i v i t y (Figure 3) (4). The increase i n phospholipase a c t i v i t y was roughly proportional to the amount of leaf surface area that was covered by the fungus. When phospholipase a c t i v i t y was measured with the fluorescent substrate (C -NBD-PC), as previously described (28), i t c l o s e l y p a r a l l e l e d the curve obtained with C-PC. However, when esterase a c t i v i t y was measured with another fluorometric substrate, 4-methylumbelliferyl laurate, i t was highest at day 0 and decreased during i n f e c t i o n . This indicates that 4-methylumbelliferyl laurate appears to be hydrolyzed by a l i p o l y t i c enzyme of the host. To determine whether the increase i n phospholipase a c t i v i t y during i n f e c t i o n (Figure 3) was due to enzymes of fungal or host o r i g i n , the following experiments were performed. We observed that the pH optima of the phospholipase a c t i v i t y i n fungal cultures was about 9.0 (Figure 4A,B) (4). Phospholipase a c t i v i t y was present i n the uninfected leaves (Figure 4C), but i t was much lower and ex h i b i t e d optimum phospholipase a c t i v i t y at pH 6.0 as reported elsewhere (6). The infected l e a f had a large peak of phospholipase a c t i v i t y at pH 8.0 to 9.0 and a smaller peak a t pH 6.0. The two peaks of phospholipase a c t i v i t y i n the infected leaves were further resolved by assaying i n the presence of 5 mM DTT (Figure 4D) which i s a potent i n h i b i t o r of fungal phospholipase. The peak of phos pholipase a c t i v i t y a t pH 9.0 was DTT-sensitive and l i k e l y to be of fungal o r i g i n . The peak of phospholipase a c t i v i t y at pH 6.0 was DTT-insensitive and i s probably of host o r i g i n although further work i s required to prove t h i s beyond doubt. 6

14

Conclusions and Perspectives These results i n d i c a t e that during the i n f e c t i o n of potato leaves by P. infestans, there i s a rapid degradation of the host*s membrane l i p i d s ( e s p e c i a l l y g a l a c t o l i p i d s ) and a gradual increase i n l i p i d s of fungal o r i g i n (ceramide aminoethylphosphonate being the most unique). An analysis of phospholipase a c t i v i t y revealed that a DTT-sensitive phospholipase with an a l k a l i n e pH optima accumulated i n infected leaves and was probably of fungal o r i g i n . Although these preliminary studies i n d i c a t e that l i p o l y t i c enzymes are produced by the fungus during i n f e c t i o n , much more work i s required to determine t h e i r actual role i n i n f e c t i o n and patho genesis. Because the symptomology and biochemistry of i n f e c t i o n are very s i m i l a r to those events that occur during normal l e a f

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4 r

0

3 DAYS

AFTER

5

7

INOCULATION

Figure 3. Time-course study of t o t a l phospholipase a c t i v i t y during i n f e c t i o n of potato leaves by P. infestans. Phospholipase values were previously reported (4). C -NBD-PC hydrolysis assays were performed at pH 9.0 as described (28). Hydrolysis of 4-methylumbelliferyl laurate was measured by a fluorometric technique (44). 6

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Figure 4. E f f e c t of pH on t o t a l phospholipase a c t i v i t y i n : A. culture f i l t r a t e s of fungus grown on rye steep media + 10 mg PC for 7 days. B. Culture wash of fungus grown on lima bean agar for 14 days. C. Potato l e a f , uninfected or infected f o r 7 days. D. Infected potato leaf (7 days) assayed i n the presence of 5 mM d i t h i o r e i t o l (DTT-insensitive a c t i v i t y ) . DDT-sensitive a c t i v i t y was calculated by subtracting the phospholipase a c t i v i t y i n the presence of DDT from that measured i n the absence of DDT. Reproduced with permission from Ref. 4. Copyright 1984, Academic Press.

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senescence ( i . e . , breakdown of c h l o r o p h y l l , g a l a c t o l i p i d s , and p r o t e i n ) , the two processes need to be very c a r e f u l l y compared (33). Both disease and senescence cause increases i n membrane permeability (34,35). Recent work has shown that s i m i l a r increases i n the levels of free r a d i c a l s , lipoxygenase a c t i v i t y , and l i p i d peroxidation occur during i n f e c t i o n and senescence (36-38). Other studies indicate that under c e r t a i n conditions phospholipid d e e s t e r i f i c a t i o n can be mediated by superoxide ( 0 ) i n the absence of phospholipases (39). The most recent studies irom our laboratory suggest that some of the l i p o l y t i c enzymes i n potato leaves are regulated by calmodulin (40). Even i f produced, free f a t t y acids may not accumulate i n healthy or infected leaves because peroxisomes are capable of metabolizing f a t t y acids v i a β-oxidation (41). These factors a l l indicate that membrane l i p i d metabolism i n healthy and diseased leaves i s a dynamic process and any change i n membrane composition needs to be interpreted very carefully. 2

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Thurston, H. D.; Schultz, O. In "Compendium of Potato Diseases"; Hooker, W. J., Ed.; American Phytopathological Society: St. Paul, 1981; p. 40. Currier W. W. Trends in Biochemical Science 1981, 6, 191. Bills, D. D. In "Host Plant Resistance to Pests"; Hedin, P. Α., Ed.; ACS SYMPOSIUM SERIES No. 62, American Chemical Society: Washington, DC, 1977, p. 47. Moreau, R. Α.; Rawa, D. Physiol. Plant Path. 1984, 24, 187. Moreau, R. A. J. Ag. Fd. Chem. 1985, 33, 36. Moreau, R. A. Phytochemistry 1985, 24, 411. Moreau, R. Α.; Seibles, T. S. Can. J. Bot. (in press). Wilson, U. E . ; Coffey, M. D. Ann. Bot. 1980, 45, 81. Shimony, C.; Friend, J. New Phytol. 1975, 74, 59. Coffey, M. D.; Wilson, U. A. Can. J. Bot. 1983, 61, 2669. Pitt, D.; Coombes, C. J. Gen. Microbiol. 1969, 56, 321. Duddridge, J . Α.; Sargent, J. A. Physiol. Plant Path. 1978, 12, 289. Hoppe, H. H.; Heitefuss, R. Physiol. Plant Path. 1974, 4, 11. Lösel, D. M. New Phytol. 1978, 80, 167. Lösel, D. Μ.,; Lewis, D. H. New Phytol. 1974, 73, 1157. Hoppe, Η. H.; Heitefuss, R. Physiol. Plant Path. 1974, 4, 25. Harwood, J. L. In "The Biochemistry of Plants: A Comprehen sive Treatise"; Stumpf, P. K.; Conn, Ε. E . , Eds.; Academic Press: New York, 1980; Vol. 4, p. 1. Wassef, M. K.; Hendrix, J. W. Biochem. Biophys. Acta 1977, 486, 172. Bostock, R. M.; Kuc, J . Α.; Laine, R. A. Science 1982, 22, 67. Faull, J. L . ; Gay, J. L. Physiol. Plant Path. 1983, 22, 55. Shepard, D. V.; Pitt, D. Phytochemistry 1976, 15, 1465. Tseng, T. C.; Mount, M. S. Phytopathology 1974, 64, 229. Tseng, T. C.; Bateman, D. F. Phytopathology 1968, 58, 1437.

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24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

39. 40. 41. 42. 43. 44.


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