Biologically Active Natural Products - ACS Publications - American

Chapter 4

Toxins of Phytopathogenic Microorganisms Structural Diversity and Physiological Activity

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S. Sakamura, A. Ichihara, and T. Yoshihara Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

Plant pathogenic fungi and bacteria produce a number of phytotoxins and there is current interest in the use of their compounds and derivatives for agrochemicals. After commenting on the significance of toxin reseach, recent progress in the area of bacterial toxins and host-specific toxins is briefly mentioned. Recently, we have found several physiologically active compounds including novel ones, that is, betaenones and aphidicolanes from Phoma betae, reduced perylenequinones and anthraquinones from Stemphylium botryosum, and cyclopentanoid sesquiterpenes from timothy stalks infected with Epichloe typhina. The chemistry of toxins produced by plant pathogens has made rapid progress in the last twenty years. At the present time, the number of toxins elucidated based on their chemical structures is more than 170 compounds and the number increases every year (1). The chemical studies of toxins have been undertaken with the purpose of (a) understanding the causal factor in plant diseases and (b) discovering of physiologically active principles, including plant regulating substances. In general, isolation of these compounds is carried out starting from culture broths or infected plant material. Toxins are structurally unique and belong to the class of secondary metabolites which almost all come from fungi except a few from certain bacteria. There has been no evidence that a toxin was confirmed from biotrophic fungi or plant material infected with these fungi. Bacterial phytotoxins Several phytotoxins have been isolated from the Pseudomonas group of plant pathogenic bacteria and their structures elucidated. Though some reviews on bacterial phytotoxins have appeared (2-4), particular points require summarization. 0097-6156/88/0380-0057$06.00/0 o 1988 American Chemical Society

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Pseudomonas s y r i n g a e pv. t a b a c i , which causes w i l d f i r e d i s e a s e o f t o b a c c o , produces t a b t o x i n C5)• R e c e n t l y the s t r u c t u r e o f t a b t o x i n was c o n f i r m e d v i a t o t a l s y n t h e s i s ( 6 ) . The s t r u c t u r e o f p h a s e o l o t o x i n ( 7 ) , produced by Pseudomonas s y r i n g a e pv. p h a s e o l i c o l a which causes h a l o b l i g h t o f bean, has been r e v i s e d because new mass s p e c t r u m d a t a were p r o v i d e d ( 8 ) . More r e c e n t l y i n t e r e s t i n g b i o s y n t h e t i c s t u d i e s o f c o r o n a t i n e (9) i s o l a t e d from Pseudomonas s y r i n g a e pv. a t r o p u r p u r e a , the c a u s a l agent o f c h o c o l a t e s p o t d i s e a s e on I t a l i a n r y e g r a s s , have shown t h a t the a c i d i c component, c o r o n a f a c i c a c i d , was d e r i v e d f r o m a b r a n c h e d p o l y k e t i d e w i t h f i v e a c e t a t e u n i t s and one p y r u v a t e ( 1 0 ) . Host-Specific

Toxins

T h i r t e e n s p e c i e s o f f u n g i which produce h o s t - s e l e c t i v e o r h o s t s p e c i f i c t o x i n s a r e known and they b e l o n g to the genus A l t e r n a r i a (6 s p e c i e s ) and H e l m i n t h o s p o r i u m (4 s p e c i e s ) and the o t h e r s ( 1 1 ) . These t o x i n s have a s p e c i f i c a l l y h i g h t o x i c i t y toward the r e s t r i c t e d h o s t s , such as c u l t i v a r s and s p e c i e s o f p l a n t . Thus t h e s e compounds a r e r e g a r d e d as a p r i m a r y d e t e r m i n a n t i n p a t h o g e n e s i s and a r e sometimes c a l l e d p a t h o t o x i n s . The p r i n c i p a l t o x i n i s s t r u c t u r a l l y d i v e r s e , u s u a l l y o c c u r s t o g e t h e r w i t h c l o s e l y r e l a t e d compounds, and sometimes i n the company o f non-host s p e c i f i c t o x i n s as w e l l . Based on the c h e m i c a l s t r u c t u r e , the t o x i n s a r e c l a s s i f i e d as c y c l o p e p t i d e s , amino a c i d e s t e r s , a l i p h a t i c a m i n o p o l y o l e s t e r s , p o l y a l c o h o l s , a l i p h a t i c p o l y o l l a c t o n e s , or sesquiterpene galactosides. The pathogens o f d i f f e r e n t s p e c i e s a l s o produce t o x i n s a n a l o g u s to each o t h e r i n t h e i r s t r u c t u r e s . An example o f t h i s can be seen w i t h HMT- and PM-toxins ; they b o t h p o s s e s s an a l i p h a t i c p o l y a l c o h o l s t r u c t u r e (11). Fungal Nonhost-Specific

Toxins

A number o f p h y t o t o x i c m e t a b o l i t e s w h i c h a r e non-host s p e c i f i c have been i s o l a t e d and i d e n t i f i e d by us from the f o l l o w i n g f u n g i , Phoma b e t a e (synonym: P h y l l o s t i c t a b e t a e ) and Stemphylium botryosum. Betaenones and a p h i d i c o l a n e s (12-16). Phoma b e t a e F r i e s , a c a u s a l agent o f r o o t r o t and l e a f s p o t d i s e a s e s o f sugar b e e t s i s grown on a p o t a t o - s u g a r medium, and the c u l t u r e d f i l t r a t e s a r e s u b j e c t e d to e x t r a c t i o n . The i n d i v i d u a l t o x i n s a r e i s o l a t e d by e x t r a c t i o n w i t h EtOAc, s i l i c a g e l chromatography and p r e p a r a t i v e TLC. A b i o a s s a y i s performed w h i c h c o n s i s t s o f measuring the growth i n h i b i t i o n o f l e t t u c e s e e d l i n g s . As a r e s u l t , the p h y s i o l o g i c a l l y a c t i v e compounds i s o l a t e d were d i v i d e d i n t o two g r o u p s . One group b e l o n g s t o the n o v e l d e c a l i n d e r i v a t i v e s and a r e named betaenones A ( l ) , B ( j y , C ( 3 ) , D ( 4 ) , E(5) and F ( 6 ^ . The o t h e r was a p h i d i c o l i n (7) and i t s newly i d e n t i f i e d a n a l o g s 3 - d e o x y a p h i d i c o l i n ( 8 ) , aphidicolin-17-monoacetate (9) and a p h i d i c o l i n - 3 , 1 8 - o r t h o a c e t a t e (10). ~" ~* In our i n v i v o e x p e r i m e n t s ( d a t a not shown), a p h i d i c o l i n and i t s a n a l o g s cause a marked i n h i b i t i o n o f DNA s y n t h e s i s and show s e l e c t i v i t y f o r DNA polymerase a ( 1 7 ) . On the o t h e r hand, betaenone C(3^ s t r o n g l y i n h i b i t e d b o t h the p r o t e i n and RNA s y n t h e s i s compared


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SAKAMURAETAL.

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15

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Toxins ojPhytopathogenic Microorganisms

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w i t h A and B. Betaenone C, however, d i d n o t s i g n i f i c a n t l y i n h i b i t DNA s y n t h e s e s . Among t h e b e t a e n o n e s , C(3) was d e t e r m i n e d t o have t h e mcst i n h i b i t o r y e f f e c t as t e s t e d on growth o f r i c e s e e d l i n g s (18) ( T a b l e 1 ) . T a b l e 1. E f f e c t o f betaenones and a p h i d i c o l a n e s on r o o t elongation of r i c e seedlings Toxin Root e l o n g a t i o n ( 1 0 ~ M) (cm) Control 5.23 Betaenone A ( l ) 1.40 4.80 Betaenone B(j2) Betaenone C(3^ 0.56 1.64 Aphidicolin(7) 2.17 3-deoxyaphidocolin(8) 1.90 Aphidicolin-17-monoacetate(9) 1.75 A p h i d i c o l i n - 3 , 18-orthoacetate(10) a) Mean l e n g t h , average o f 20 s e e d l i n g s

% of i n h i b i t i o n


4

0 73 8 89 69 59 64 67

I t s h o u l d be n o t e d t h a t we e s t a b l i s h e d betaenone B(2j) t o be b i o s y n t h e s i z e d f r o m e i g h t a c e t a t e u n i t s v i a t h e p o l y k e t i d e pathway w i t h t h e o r i g i n o f t h e f i v e b r a n c h e d m e t h y l groups f r o m m e t h i o n i n e ( 1 6 ) . V e r y r e c e n t l y f e e d i n g e x p e r i m e n t s w i t h [1--^C, -^0] a c e t a t e r e v e a l e d t h a t i n t h e l^C-NMR s p e c t r u m o f e n r i c h e d 2^ o n l y t h e i s o t o p i c s h i f t e d s i g n a l was o b s e r v e d a t C-16 [A + 0.05ppm], b u t n o t a t C - l and C-18. T h i s means t h a t t h e oxygen atom a t C - l does n o t o r i g i n a t e from t h e a c e t a t e and absence o f t h e e x p e c t e d i s o t o p i c s i g n a l a s c r i b a b l e t o C-18 would be due t o t h e r a p i d exchange o f t h e l a b e l e d oxygen o f t h e end c a r b o x y l group o f t h e p o l y k e t i d e c h a i n . On t h e o t h e r hand, a d d i t i o n o f a n c y m i d o l , a p o t e n t cytochrome P-450 i n h i b i t o r , i n t o t h e c u l t u r e has caused an i n h i b i t i o n o f betaenone f o r m a t i o n and a c o n c o m i t a n t a c c u m u l a t i o n o f a p l a u s i b l e i n t e r m e d i a t e , probetaenone I (13,) . These f e e d i n g and i n h i b i t o r e x p e r i m e n t s c l e a r l y show t h a t 11 i s an i n t e r m e d i a t e i n t h e b i o s y n t h e s i s o f 2 and p r e s e n t s a unique b i o s y n t h e t i c pathway i n v o l v i n g an i n t r a m o l e c u l a r D i e l s - A l d e r r e a c t i o n o f a t r i e n e 11a as a key s t e p (Oikawa, H.; I c h i h a r a , A.; Sakamura, S., s u b m i t t e d for publication). More r e c e n t l y c l o s e l y a n a l o g o u s m e t a b o l i t e s t o b e t a e n o n e s , s t e m p h y l o x i n s I and I I have been i d e n t i f i e d from Stemphylium botryosum f . s p . l y c o p e r s i c i , and they c o r r e s p o n d t o betaenones C (3) and A(^L) r e s p e c t i v e l y . The compounds o f b o t h groups d i f f e r from one a n o t h e r i n t h a t t h e C-^ m e t h y l o f betaenones i s r e p l a c e d w i t h the h y d r o x y m e t h y l o f s t e m p h y l o x i n s . M a n u l i s e t a l . demonstrated, u s i n g growing tomato c e l l s , t h a t s t e m p h y l o x i n s and betaenones comparably i n h i b i t p r o t e i n s y n t h e s i s (19). The d a t a i n d i c a t e t h a t lower c o n c e n t r a t i o n s o f s t e m p h y l o x i n s were r e q u i r e d f o r 50% i n h i b i t i o n than f o r b e t a e n o n e s . The r e s u l t s p a r a l l e l t h o s e from o f t h e r i c e s e e d l i n g t e s t . Reduced p e r y l e n e q u i n o n e s and a n t h r a q u i n o n e s . A s t r a i n , Stemphylium botryosum i s o l a t e d from a b e e t p l a n t w i t h t h e l e a f s p o t d i s e a s e , was examined t o o b t a i n p h y t o t o x i n s from t h e c u l t u r e d b r o t h s . Procedures



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of t h e c u l t u r e of the fungus, i s o l a t i o n o f the p h y t o t o x i c compounds, b i o a s s a y and t h e s t r u c t u r a l d e t e r m i n a t i o n were s i m i l a r t o t h o s e f o r Phoma b e t a e . S e v e r a l compounds w e r e i d e n t i f i e d as t h e known compounds n a m e l y , s c y t a l o n e ( 1 2 ) , s t e m p h y p e r y l e n o l ( 1 3 ) , d a c t y l a r i o l ( 1 4 ) , m a c r o s p o r i n ( 1 5 ) , and a l t e r p o r i o l A o r B(16) (20,21). Two new c o m p o u n d s w e r e named s t e m p h y l e n o l s A ( 1 7 ) and B(18), both b e i n g atropisomers of each o t h e r . By u s i n g t h e p r e g e r m i n a t e d l e t t u c e and b e e t s e e d l i n g s b i o a s s a y , d a c t y l a r i o l (14) was m o r e i n h i b i t o r y i n t h e l e t t e r s e e d l i n g e l o n g a t i o n t e s t t h a n t h e o t h e r s , a n d a n e f f e c t i v e d o s e f o r i n h i b i t i o n was o b s e r v e d a t a c o n c e n t r a t i o n o f 12.5 ppm f o r b e e t . S t e m p h y p e r y l e n o l (13) and s c y t a l o n e (12) showed m o d e r a t e i n h i b i t o r y a c t i v i t y , w h e r e a s t h e d i m e r i c a n t n r a q u i n o n e s a l t e r p o r r i o l ( 1 6 ) , s t e m p h y l e n o l s ( 1 7 , 18^ a n d m o n o m e r i c m a c r o s p o r i n ( 1 5 ) s h o w e d no s i g n i f i c a n t e f f e c t (Teshima, Y.; I c h i h a r a , A.; S a k a m u r a , S. i n p r e p a r a t i o n ) . F u n g i t o x i c c o m p o u n d s ( 2 2 , 2 3 ) , c h o k o l s a n d c h o k o l i c a c i d A. Choke d i s e a s e f u n g i ( E p i c h l o e t y p h i n a ) i n f e c t t i m o t h y p l a n t s and f o r m s t r o m a t a on them. The s t r o m a i s t e r m e d " c h o k e " a n d i m p e d e s t h e development of the p a n i c l e . On t h e o t h e r h a n d , i n f e c t e d t i m o t h y p l a n t s a c q u i r e an i n d u c e d r e s i s t a n c e a g a i n s t a n o t h e r i n v a d e r , t h e l e a f spot disease pathogen (Cladosporium p h l e i ) . One o f t h e r e s i s t a n c e m e c h a n i s m s i s l i k e l y t o b e t h a t some m e t a b o l i t e ( s ) o f choke d i s e a s e f u n g i have i n h i b i t o r y e f f e c t s a g a i n s t the l e a f spot disease fungi. We h a v e b e e n e x p l o r i n g t h e f u n g i t o x i c c o m p o u n d s f r o m t h e c h o k e s t o e x p l a i n t h e r e s i s t a n c e phenomenon. For monitoring the a c t i v i t y , TLC b i o a u t o g r a p h y u s i n g C l a d o s p o r i u m h e r b a r u m was employed. The 7 0 % E t O H e x t r a c t s o f t h e f r e s h c h o k e s w e r e p a r t i t i o n e d i n t o n-hexane and EtOAc s o l u b l e f r a c t i o n s . E a c h f r a c t i o n was c h r o m a t o g r a p h e d on s i l i c a g e l a n d S e p h a d e x L H - 2 0 , a n d f u r t h e r p u r i f i e d b y HPLC. The f u n g i t o x i c c o m p o u n d s i s o l a t e d w e r e d e s i g n a t e d c h o k o l A ( 1 9 ) , B ( 2 0 ) , C ( 2 1 ) , D ( 2 2 ) , E(23) and F ( 2 4 ) and c h o k o l i c a c i d A(25). Tlie s p e c t r a l d a t a , t o g e t h e r w i t h s p i n d e c o u p l i n g e x p e r i m e n t s , s u g g e s t e d t h a t t h e c h o k o l s p o s s e s s t h e same f i v e membered r i n g s y s t e m w i t h v a r i e d s i d e c h a i n s . The r e l a t i v e s t e r e o c h e m i s t r y o f t h e common f i v e membered r i n g s y s t e m was d e t e r m i n e d by n u c l e a r O v e r h a u s e r e f f e c t d i f f e r e n c e s p e c t r o s c o p y o f chokol C(21). The a b s o l u t e c o n f i g u r a t i o n o f c h o k o l E ( 2 p was c o n f i r m e d by i t s CD s p e c t r u m w i t h t h e c h e l a t i n g r e a g e n t E u ( f o d ) 3 a n d i t s c h e m i c a l c o n v e r s i o n t o e p i c y c l o n e r o d i o l o x i d e (24) , t h e a b s o l u t e c o n f i g u r a t i o n o f w h i c h was e v i d e n t . Thus t h e s t e r e o c h e m i s t r y o f c h o k o l E ( 2 3 ) was d e t e r m i n e d t o b e 2 S , 3R, 6R a n d 10R i n t h e depicted structure. N e r o l i d y l pyrophosphate i s an i n t e r m e d i a t e i n t h e b i o s y n t h e s i s (25) o f c y c l o n e r o d i o l w h i c h p o s s e s s e s a n o v e l t y p e s k e l e t o n w i t h f i v e c a r b o n members i n t h e s e s q u i t e r p e n o i d s a n d t h e same a b s o l u t e c o n f i g u r a t i o n as c h o k o l s . B i o s y n t h e s e s o f c h o k o l s and c h o k o l i c a c i d A(£5,) a r e l i k e l y t o b e v i a t h e n e r o l i d y l p y r o p h o s p h a t e pathway. U s i n g TLC b i o a u t o g r a p h y , a m i n i m u m q u a n t i t y o f t h e f u n g i t o x i c a c t i v i t y p e r s p o t was o b t a i n e d f o r e a c h c o m p o u n d ; c h o k o l A ( 2 5 y g ) , B ( 5 g ) , C ( 5 g ) , D(5yg) and E ( 5 0 y g ) . Two r e p o r t s o n t h e s y n t h e s i s o f c h o k o l A ( 1 9 ) h a v e b e e n p u b l i s h e d ; o n e c o n c e r n s t h e s y n t h e s i s o f t h e r a c e m i c compound ( 2 6 ) and t h e o t h e r c o n c e r n s e n a n t i o s e l e c t i v e s y n t h e s i s ( 2 7 ) . u

y




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Significance and application of toxins


a)

Selection of disease resistant plants (28). When resistant plants are treated with a specific toxin, the visible and physiological changes similar to those found in infected plants occur. Certain pathogen-produced metabolites, pathotoxins, could serve as valid substitutes for living pathogens to develop the disease symptom. Thus, it has been possible to identify diseaseresistant individuals by selecting from populations those plants which are resistant to the toxins. In addition to these toxins, non-specific toxins that cause disease symptoms such as necrosis, browning leaf spots, chlorosis and cytotoxicity, may be used for the selection of disease resistant plants. Instead of using whole plants, the tests have been directed towards using protoplast, cell and tissue cultures. Extensive studies have been hindered by the limited supply of purified toxins but, with synthetic toxins materials and their mimics probably available, the difficulties should be reduced. b) Utilization of toxins for agrochemicals (29). Since some of phytotoxins are known to have herbicidal activity as well as various other physiological activity, they may be potent for use as herbicides or plant growth regulating substances, though complete biological testing will be required. For example, although aphidicolin is noted as an antiviral and antimitotic compound, aphidicolin has recently been found to be very effective for inducing high synchronization of plant cells in liquid culture (30). Also those compounds are useful as lead or model compounds for the design of new agrochemicals. LITERATURE CITED 1. Sakamura, S. KAGAKU TO SEIBUTSU 1985, 25, 289-298. 2. Mitchell, R. E. In Toxins in Plant Disease; Durbin, R. D., Ed,; Academic: New York, 1981; P259. 3. Macko, V. In Toxins and Plant Pathogenesis; Daly, J. M.; Deverall, B. J. Eds.; Academic: Australia, 1983; P41. 4. Ichihara, A. J. Synth. Org. Chem. 1987, 45, 357-368. 5. Stewart, W. W. Nature 1971, 229, 174-178. 6. Baldwin, J. E.; Bailey, P. D.; Gallacher, G.; Otsuka, M.; Singleton, K. A.; Wallace, P. M.; Prout, K.; Wolf, W. M. Tetrahedron 1984, 40, 3695-3708. 7. Mitchell, R. E. Phytochemistry 1976, 15, 1941-1947. 8. Moore, R. E.; Niemczura, W. P., Kwok, O. C. H., Patil, S. S. Tetrahedron Lett. 1984, 36, 3931-3934. 9. Ichihara, A.; Shiraishi, K.; Sato, H.; Sakamura, S.; Nishiyama, K.; Sakai, R.; Fukuoka, A.; Matsumoto, T. J. Am. Chem. Soc. 1977, 99, 636. 10. Parry, R. J.; Mafoti, R. J. Am. Chem. Soc. 1986, 108, 468-469. 11. Kono, Y.; Suzuki, Y.; Takeuchi, S. J. Synth. Org. Chem. 1985, 43, 980-989. 12. Ichihara, A.; Oikawa, H.; Hayashi, K.; Sakamura, S. J. Am. Chem. Soc. 1983, 105, 2907-2908.


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