Japanese Contributions to the Development of Allelochemicals

Chapter 3

Japanese Contributions to the Development of Allelochemicals Horace G. Cutler

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 3, 2018 | https://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch003

Richard B. Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30613

The Japanese people l i v e i n a delicate microecosystem that can be easily polluted by industrial chemical accidents and the use of persistent agricultural chemicals. An intensive s c i e n t i f i c effort has led to the isolation and identification of biodegradable natural products for potential use in agriculture. These include microbial metabolites that have activity against plants, microorganisms, nematodes, and insects. The chemical structures range from complex to simple and represent diverse classes of compounds. In addition, compounds isolated by non-Japanese researchers have been assigned specific uses by Japanese scientists for potential agricultural use. I f a s c i e n t i s t who knew n o t h i n g about t h e geography o r demography o f Japan v i s i t e d t h a t c o u n t r y he would b e g i n t o a r r i v e a t c e r t a i n c o n c l u s i o n s about t h e n a t u r e o f the c o u n t r y q u i t e q u i c k l y . Visits to r e s t a u r a n t s i n t h e c i t i e s a n d s u r r o u n d i n g c o u n t r y s i d e w o u l d indicate that vegetables a n d f i s h a r e q u i t e p l e n t i f u l and r e l a t i v e l y c h e a p ; t h a t t h e m a i n g r a i n f o r f o o d and b e v e r a g e i s r i c e ; t h a t r e d meat i s b o t h e x p e n s i v e a n d d i f f i c u l t t o o b t a i n . V i s i t s to shops and o p e n - a i r m a r k e t s t a l l s would c o n f i r m t h e s e observations. S t a c k s upon s t a c k s o f f r e s h vegetables i n c l u d i n g some t h a t a r e o n l y j u s t f i n d i n g t h e i r way i n t o w e s t e r n m a r k e t s , such as d a i k o n (Raphanus s a t i v u s l o n g i p i n n a t u s ) » greet the e y e . P l a s t i c pans o f a s s o r t e d f i s h i n a l l s h a p e s a n d s i z e s , a n d an abundance o f s h e l l f i s h , a r e c o m m o n p l a c e . While t r a v e l l i n g along t h e h i g h w a y s o u r s c i e n t i s t would n o t e t h a t a r a b l e l a n d i s used t o i t s maximum, even t o t h e edge of t h e r o a d , and t o some e x t e n t o n e i s reminded o f the a l l o t m e n t gardens o f World War II E n g l a n d , where a l l a v a i l a b l e l a n d was used t o produce v e g e t a b l e s t o supplement t h e f o o d r a t i o n i n g program. Even r i c e p a d d i e s extend to t h e r o a d edge and one becomes s u d d e n l y q u i t e aware of the v u l n e r a b i l i t y o f t h i s s t a p l e c r o p d u r i n g t h e a q u a c u l t u r a l s t a g e o f i t s l i f e c y c l e . One c h e m i c a l a c c i d e n t , o r one m a l i c i o u s a c t , can s p e l l d i s a s t e r f o r t h e T h i s chapter not subject to U.S. copyright. P u b l i s h e d 1987 A m e r i c a n C h e m i c a l Society

Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

r i c e c r o p b e c a u s e o f t h e r a p i d d i f f u s i o n o f agents i n w a t e r . In a d d i t i o n , our s c i e n t i s t would see t h e b a c k b o n e o f m o u n t a i n s , t h e o m n i p r e s e n t s e a t h a t c a n be r e a c h e d m o d e r a t e l y e a s i l y , and one m a j o r c o n c l u s i o n w o u l d be t h a t J a p a n i s a c o u n t r y t h a t c a n n o t a f f o r d to h a v e c h e m i c a l p o l l u t i o n problems from e i t h e r i n d u s t r y or agriculture. F u r t h e r m o r e , our o b s e r v e r w o u l d have b e e n s t r u c k b y t h e p o p u l a t i o n d e n s i t y and the s m a l l , compact houses t h a t are b u i l t q u i t e c l o s e l y t o g e t h e r and have s m a l l , i f any, g a r d e n s . Any e f f e c t on the food c h a i n i s immediately f e l t by the l a r g e p o p u l a t i o n . Our s c i e n t i s t would not be s u r p r i s e d i f t h e map o f J a p a n was shown t o h i m . The c o u n t r y i s b o w - s h a p e d , l o n g , and c o n s i s t s o f f o u r main i s l a n d s , H o k k a i d o , H o n s h u , K y u s h u , and S h i k o k u , p l u s several smaller islands. The l a n d mass i s 377,748 s q u a r e k i l o m e t e r s , o r about 4% the s i z e of the U n i t e d S t a t e s o f A m e r i c a , and s u p p o r t s a p o p u l a t i o n o f 119.6 m i l l i o n p e o p l e (_1 ). Mountains c o m p r i s e 71% o f t h e l a n d w i t h 29% p l a i n s and b a s i n s o f w h i c h a p p r o x i m a t e l y 15% i s s u i t a b l e f o r crop p r o d u c t i o n . The m o u n t a i n s s e r v e as a watershed and i n f l u e n c i n g f a c t o r on the e n v i r o n m e n t , and as a s o u r c e of t i m b e r . Compared w i t h many o t h e r c o u n t r i e s , J a p a n d o e s h a v e a d e n s e p o p u l a t i o n , e s p e c i a l l y when those f i g u r e s are compared on an a r a b l e l a n d r a t i o , but i t i s i n t e r e s t i n g t o s e e t h e demographic f i g u r e s for other countries. For example, the p o p u l a t i o n per square k i l o m e t e r i n 1983 was 616 f o r B a n g l a d e s h , 388 f o r S o u t h K o r e a , 346 f o r the N e t h e r l a n d s , 323 f o r B e l g i u m , 317 f o r J a p a n , 100 f o r C h i n a , and n o t s u r p r i s i n g l y , 24 f o r the USA (_1 ). A l l t h e d e m o g r a p h i c f i g u r e s and g e n e r a l o b s e r v a t i o n s l e a d one to the i n e v i t a b l e c o n c l u s i o n t h a t J a p a n has a v e r y delicate m i c r o e c o s y s tern t h a t c a n be e a s i l y damaged by i n d u s t r i a l o r pesticide s p i l l s . And J a p a n i s a h i g h l y c o m p e t i t i v e i n d u s t r i a l n a t i o n w h i l e , a t the same t i m e , an i n t e n s e l y a g r i c u l t u r a l o n e . No one i s more aware of the e f f e c t s o f p o l l u t i o n t h a n t h e J a p a n e s e . In 1 9 8 4 , t h e E n v i r o n m e n t A g e n c y o f the Government o f Japan p u b l i s h e d t h e " I l l u s t r a t e d W h i t e P a p e r on t h e E n v i r o n m e n t i n J a p a n " , i n w h i c h t h e a t t i t u d e s and law c o n c e r n i n g p o l l u t i o n and waste c o n t r o l are d i s c u s s e d . The word " i m p e r a t i v e " occurs throughout t h e t e x t and i t i s q u i t e o b v i o u s t h a t t h e c o u n t r y i n t e n d s to implement a r i g o r o u s s e t of s t a n d a r d s f o r i n d u s t r y and the p r i v a t e c i t i z e n . The v u l n e r a b i l i t y o f the environment and the p o p u l a t i o n i s v i v i d l y e x p r e s s e d i n the examples of i n d u s t r i a l spills. T h e f i r s t i n v o l v e d c a d m i u m , w h i c h a f f e c t s b o n e s and k i d n e y , t h a t s p i l l e d i n t o the J i n t s u r i v e r i n the Toyama P r e f e c t u r e to produce the i t a i - i t a i d i s e a s e . The s e c o n d , w h i c h i s b e t t e r known because of a p o i g n a n t s e t o f p i c t u r e s t h a t were p u b l i s h e d o f a mother b a t h i n g her s o n , was a methylmercury d i s c h a r g e t h a t caused b r a i n d i s o r d e r s and damage to t h e n e r v o u s s y s t e m . This occurred a l o n g t h e Y a t s u s h i r o c o a s t , Kumamoto P r e f e c t u r e , and i n the Agano R i v e r B a s i n i n the N i i g a t a P r e f e c t u r e ; the symptoms were d e s c r i b e d as t h e M i n a m a t a d i s e a s e . A g a i n , one n o t e s the r a i n washing down the m o u n t a i n s , sweeping through the i n d u s t r i a l and agrarian p i e d m o n t , c a r r y i n g p o l l u t a n t s out i n t o the bays and s e a . In s p i t e of the p o p u l a t i o n p r è s s u r e s , t h e s h o r t a g e o f a r a b l e l a n d , t h e c o n c e n t r a t i o n of i n d u s t r y , and the r e l a t i v e i s o l a t i o n o f the c o u n t r y , the J a p a n e s e have done a r e m a r k a b l e j o b i n k e e p i n g t h e i r c o u n t r y i n an o r d e r l y f a s h i o n . Parks abound and t h e r e i s a r e s p e c t f o r n a t u r e and a r t t h a t i s q u i t e e x t r a o r d i n a r y . B u t aware



3.

CUTLER

Japanese Contributions

to

Allelochemicals

25

o f what has happened i n the p a s t , and aware o f what might happen i n the f u t u r e , Japanese s c i e n t i s t s have asked themselves two i m p o r t a n t questions: "What s o r t o f c h e m i c a l s c a n we u s e on o u r c r o p s as p e s t i c i d e s t h a t have h i g h s p e c i f i c a c t i v i t y a g a i n s t target o r g a n i s m s ? " and "How b i o d e g r a d a b l e a r e t h e s e c h e m i c a l s ? " The a n s w e r t o t h e f i r s t q u e s t i o n may be m i c r o b i a l metabolites (allelochemicals) w h i c h a r e o r g a n i c n a t u r a l p r o d u c t s and to the second, n a t u r a l products are i n t r i n s i c a l l y b i o d e g r a d a b l e . In a d d i t i o n , a t h i r d q u e s t i o n i n v o l v e s the n a t u r e o f p h y t o p a t h o g e n i c m i c r o o r g a n i s m s and the p h y t o t o x i n s t h a t t h e y p r o d u c e . F o r i f one i s t o c o n t r o l t h e s e i n v a d e r s , t h e b i o c h e m i c a l pathways by which p h y t o t o x i n s a r e p r o d u c e d by t h e p a t h o g e n s must be e l u c i d a t e d . B e s i d e s , p h y t o p a t h o g e n s may p r o d u c e t o x i n s t h a t c a n be used to c o n t r o l crop p e s t s . We s h a l l s e e , i n t h i s b r i e f r e v i e w , t h a t Japanese work has been i n t e n s e i n the i s o l a t i o n and i d e n t i f i c a t i o n o f b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t s from m i c r o o r g a n i s m s . T h i s i n t e n s i t y has been marked by the f o l l o w i n g s t e p s : E x t e n s i v e s e a r c h f o r h i g h - y i e l d i n g s t r a i n s of m i c r o o r g a n i s m s , i s o l a t i o n and i d e n t i f i c a t i o n or p r o o f of s t r u c t u r e of a m e t a b o l i t e , and thorough t e s t i n g on p e s t s and c r o p s t h a t a r e o f economic importance to Japan when s u f f i c i e n t q u a n t i t i e s o f t h e n a t u r a l p r o d u c t a r e a v a i l a b l e , and s y n t h e s i s ( w h e n e v e r p o s s i b l e ) o f t h e m e t a b o l i t e so t h a t f u r t h e r s c r e e n i n g may be carried out. In some i n s t a n c e s m e t a b o l i t e s have been r e d i s c o v e r e d and new a p p l i c a t i o n s f o u n d f o r them t h a t were o v e r l o o k e d by the primary d i s c o v e r e r . W h i l e the Japanese c o n t r i b u t i o n s t o t h e a r e a o f b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t s have been q u i t e e x t e n s i v e , I have chosen a few examples to i l l u s t r a t e t h e w i d e d i v e r s i t y and a c t i v i t y o f m i c r o b i a l m e t a b o l i t e s against p l a n t s , microorganisms ( i n c l u d i n g a s e l f - i n h i b i t o r ) , nematodes, i n s e c t s , and o t h e r zoological species. C y l i n d r o c l a d i u m s c o p a r i u m i s an u b i q u i t o u s phytopathogenic fungus t h a t causes d i s e a s e s i n a wide v a r i e t y o f p l a n t s , e s p e c i a l l y o r n a m e n t a l s , a n d , more i m p o r t a n t l y , r i c e (Oryza s a t i v a L . ) , where i t induces sheath n e t - b l o t c h . H i r o t a and coworkers f i r s t p u b l i s h e d on the n a t u r e of the t o x i n s produced by t h i s o r g a n i s m , i n c u l t u r e , i n 1973 ( 2 . 3 ) and d u r i n g the course of the next e l e v e n y e a r s they c a r e f u l l y a n a l y z e d t h e s t r u c t u r e o f two m e t a b o l i t e s possessing b i o l o g i c a l a c t i v i t y w h i c h t h e y d e s i g n a t e d as c y l - 1 and c y l - 2 . F i n a l l y , i n 1984, the proposed s t r u c t u r e s f o r these compounds were p u b l i s h e d (4.) and a l l t h e e v i d e n c e p o i n t e d to two o l i g o p e p t i d e s , specifically t e t r a ρ e ρ t i d e s arranged i n the sequence of D - . O - m e t h y l t y r o s i n e , L - i s o l e u c i n e , L - p i p e c o l i c a c i d ( i n c y l - 2 ) or p r o l i n e i n ( c y l - 1 ) , and 2 - a m i n o - 8 - o x o - 9 , 1 0 - e p o x y d e c a n o i c a c i d (Figure 1). Both m e t a b o l i t e s were a c t i v e a g a i n s t p l a n t s p e c i e s b u t t h e m o r e a c t i v e o f t h e two m e t a b o l i t e s was c y l - 1 , w h i c h was i s o l a t e d i n r a t h e r s m a l l amounts. C y l - 1 , f o r example, i n h i b i t e d l e t t u c e r o o t e l o n g a t i o n 50% a t 0 . 5 ppm w h i l e c y l - 2 i n h i b i t e d e x t e n s i o n 50% o n l y at 1.0 ppm (2.). I n o t h e r t e s t s , u s i n g Avena s a t i v a L . c v . R u s s e l l c o l e o p t i l e s , c y l - 2 d i d not i n h i b i t the growth o f c o l e o p t i l e s b u t when i n d o l e - 3 - a c e t i c a c i d was a d d e d t o t h e i n c u b a t i o n medium at 1.0 ppm the e x t e n s i o n n o r m a l l y i n d u c e d by t h a t s u b s t a n c e d i d not o c c u r . That i s , c y l - 2 at 10-100 ppm a c t e d as an a n t a g o n i s t i c agent to i n d o l e - 3 - a c e t i c a c i d and w h i l e t h e m e c h a n i s m o f a c t i o n was n e i t h e r r e p o r t e d n o r suggested the o b s e r v a t i o n i s , n e v e r t h e l e s s , an i n t e r e s t i n g o n e .



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The c y c l i c t e t r a p e p t i d e s a r e a f a s c i n a t i n g group o f compounds and the f i r s t o b s e r v a t i o n t h a t one makes on v i e w i n g the s t r u c t u r e s o f t h e c y l g r o u p i s t h e s e q u e n c i n g o f t h e D and L amino a c i d s . T h i s l e a d s to some t h o u g h t s a b o u t t h e s y n t h e t i c p e r m u t a t i o n s by s u b s t i t u t i n g D and L s p e c i e s a n d , o f c o u r s e , t h e c o n c o m i t a n t b i o l o g i c a l a c t i v i t y . But an even g r e a t e r s u r p r i s e i s t h a t w h i l e t h e c y l s t r u c t u r e s a r e unique they are p a r t o f a g r e a t e r c l a s s of t o x i n s , a l l o f w h i c h p o s s e s s t h e c h a r a c t e r i s t i c s of h a v i n g p i p e c o l i c a c i d (or p r o l i n e ) and the 2 - a m i n o - 8 - o x o - 9 , 10-epoxydecanoic acid residue. Among t h e s e a r e c h l a m y d o c i n (Diheterospora c h l a m y d o s p o r i a ) (5.) , HC t o x i n ( H e l m i n t h o s p o r i u m carbonum) ( 6 , 7 ) , and WF-3161 ( P e t r i e l l a g u t t u l a t a ) (8.), t h e l a t t e r b e i n g a n o t h e r Japanese c o n t r i b u t i o n . A l l t h e s e i s o l a t i o n s and i d e n t i f i c a t i o n s are m i l e s t o n e s i n c a r e f u l l y c o n s t r u c t e d and m e t i c u l o u s work. It i s e s t i m a t e d t h a t a p p r o x i m a t e l y f i f t e e n y e a r s were spent p u t t i n g t o g e t h e r the c y l - 1 and 2 d a t a . O t h e r c y c l i c t e t r a p e p t i d e s have a l s o been i s o l a t e d by Japanese w o r k e r s and AM t o x i n s I , I I , a n d I I I , i s o l a t e d from A l t e r n a r i a m a l i « a r e e x t r e m e l y t o x i c to c e r t a i n p l a n t s p e c i e s ( 9 , 1 0 ) . These are c o n s t r u c t e d of L - A - h y d r o x y i s o v a l e r i c a c i d , L - a l a n i n e , * - a m i n o a c r y l i c a c i d a n d , i n AM t o x i n I , L-o-methoxyphenyl)v a l e r i c a c i d . The p h e n y l r e s i d u e i n AM t o x i n I I is L - 0 C - a m i n o - i - p h e n y l v a l e r i c a c i d , w h i l e i n AM t o x i n I I I , it is L-


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- a n i s y l o x y p r o p i o n a t e was s y n t h e s i z e d a n d shown t o h a v e t h e same n e m a t i c i d a l p r o p e r t i e s as the n a t u r a l product L - 3 . Another m e t a b o l i t e from A s p e r g i l l u s n i g e r i s n i g r a g i l l i n , a p i p e r a z i n e c l o s e l y r e s e m b l i n g n i g e r a z i n e Β (from A . n i g e r 1 - 6 3 9 ) , t h e s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two m o l e c u l e s b e i n g a terminal p h e n y l group in nigerazine B. Nigragillin, N - m e t h v l - t r a n s - 2 . 5 - d i m e t h y l - j T - s o r b y l p i p e r a z i n e , was o r i g i n a l l y i s o l a t e d b y C a e s a r e t a l . i n 1969 from A s p e r g i l l u s u s t u s but t h e compound was n o t t e s t e d i n b i o l o g i c a l s y s t e m s (22.) ( F i g u r e 8 ) . When s i l k w o r m , Bombyx m o r i L . , were o r a l l y dosed w i t h n i g r a g i l l i n i n c o r p o r a t e d i n t o t h e i r d i e t , 40 ppm proved t o be t o x i c w i t h i n 48 h and 40% o f t h e l a r v a e , w h i c h had been t r e a t e d a f t e r t h e t h i r d m o l t , died. A t 72 h , 70% h a d d i e d . Treatment w i t h 80 ppm proved t o be more l e t h a l and 100% of t h e l a r v a e d i e d w i t h i n 48 h . H o w e v e r , i t s h o u l d be p o i n t e d o u t t h a t w h i l e t h e m e t a b o l i t e was i n c o r p o r a t e d i n t o t h e media a t these r a t e s i t i s most p r o b a b l e t h a t o n l y s m a l l amounts w e r e i n g e s t e d b y t h e l a r v a e . A further d i l u t i o n effect o c c u r r e d because t e n l a r v a e were i n c l u d e d i n each d i e t assay. O t h e r symptoms n o t e d d u r i n g t h e course o f t h e experiments i n c l u d e d v o m i t i n g , c o n v u l s i o n s , and swooning ( 2 3 ) . The e f f e c t s o f t o p i c a l a p p l i c a t i o n o f n i g r a g i l l i n i n e t h y l a c e t a t e t o s i l k w o r m were n o t i c e a b l e almost i n s t a n t a n e o u s l y . D o s e s o f 5 yg/g caused i m m e d i a t e k n o c k d o w n a n d p o i s o n i n g , t h o u g h d e a t h d i d n o t always follow. S y n t h e t i c d _ l - n i g r a g i l 1 i n was made a f t e r t h e m e t h o d o u t l i n e d b y Caesar (22) and i t had t h e same b i o l o g i c a l a c t i v i t y as the n a t u r a l p r o d u c t . A n o t h e r compound w i t h marked i n s e c t i c i d a l p r o p e r t i e s i s



3.

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Japanese

Contribunons

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Allelochemicals

31

L - a l a n o s i n e ( F i g u r e 9 ) , i s o l a t e d from a Streptomyces s p e c i e s , which i n h i b i t e d l a r v a l e c d y s i s when a d m i n i s t e r e d to f o u r t h - i n s t a r l a r v a e o f the common armyworm, L e u c a n i a s e p a r a t a , i n a r t i f i c i a l d i e t (24). The m o l e c u l e i s q u i t e s i m p l e , L-2-amino-3-(hydroxynitrosamino)p r o p i o n i c a c i d , and the r e a d e r q u i c k l y c a l l s to mind t h e number o f ' s i m p l e ' amino compounds t h a t h a v e been d i s c o v e r e d d u r i n g r e c e n t y e a r s , t h a t range from a r t i f i c i a l s w e e t e n e r s ( a s p a r t a m e ) t o t h e herbicide glyphosate. Doubtless, w i t h a l l the possible p e r m u t a t i o n s f o r amino a c i d d e r i v a t i v e s , many more w i l l f i n d t h e i r way t o t h e m a r k e t p l a c e . The a c t i o n o f L - a l a n o s i n e appears t o be q u i t e s p e c i f i c on e c d y s i s and r a t e s as low as 5 ppm i n d i e t s c a u s e d i n h i b i t i o n o f head c a p s u l e removal i n 50% of l a r v a e . W i t h 40 ppm, not o n l y was head c a p s u l e removal t o t a l l y i n h i b i t e d , b u t c u t i c l e shed d i d n o t t a k e p l a c e . I f the m e t a b o l i t e was f e d to the i n s e c t i m m e d i a t e l y f o l l o w i n g e c d y s i s , t h e n l a r v a l g r o w t h was slightly delayed. T h e same e f f e c t s w e r e a l s o o b s e r v e d i n t h e c a b b a g e armyworm, Mamestra b r a s s i c a e . L - A l a n o s i n e was o r i g i n a l l y i s o l a t e d i n 1966 by M u r t h y and c o w o r k e r s as an a n t i v i r a l and a n t i t u m o r m e t a b o l i t e from Streptomyces alanosinicus (25.26). It also i n h i b i t e d r e p r o d u c t i o n i n the h o u s e f l y , Musca d o m e s t i c a ( 2 7 ) . Its m e c h a n i s m o f a c t i o n may i n v o l v e b l o c k i n g RNA adenine s y n t h e s i s , thereby i n h i b i t i n g p r o d u c t i o n o f t h e c u t i c u l a r p r o t e i n t h a t i s e s s e n t i a l f o r s c l e r o t i z a t i o n i n the m o l t i n g p r o c e s s ( 2 4 ) . Again, L - a l a n o s i n e i s an e x c e l l e n t example o f how p r e v i o u s l y d i s c o v e r e d n a t u r a l p r o d u c t s have been r e - i s o l a t e d by Japanese r e s e a r c h e r s and t e s t e d i n systems t h a t have p r a c t i c a l , n a t i o n a l a p p l i c a t i o n . In a d d i t i o n to c o n t r o l l i n g p l a n t g r o w t h and d e v e l o p m e n t and c e r t a i n i n s e c t p e s t s there i s great i n t e r e s t i n e l i m i n a t i n g plant pathogens. One o f t h e s e i s V a l s a c e r a t o s p e r m a , a f u n g u s that p r o d u c e s a p p l e c a n k e r and ranks among the most s e v e r e problems i n a p p l e c u l t i v a t i o n . M e c h a n i c a l l y i n j u r e d p a r t s of t r e e s are r e a d i l y a t t a c k e d by t h e f u n g u s , as a r e n e c r o t i c a r e a s , and i n v a s i o n proceeds m e t h o d i c a l l y u n t i l the t r u n k i s damaged. Infected parts become cankered by the fungus and e v e n t u a l l y d i e . In an attempt to c o n t r o l V . c e r a t o s p e r m a s e v e r a l a n t i b i o t i c s w e r e e v a l u a t e d and m i c r o b i a l products t e s t e d . Micromonospora c h a l c e a produced a n o v e l a n t i b i o t i c , d e s i g n a t e d p r o p a n o s i n e ( K - 7 6 ) , w h i c h had s p e c i f i c a c t i v i t y a g a i n s t V . c e r a t o s p e r m a (28) ( F i g u r e 1 0 ) . The compound w h i c h was l i k e L - a l a n o s i n e i n UV s p e c t r a l p r o p e r t i e s , was o f relatively simple s t r u c t u r e . D i s k a s s a y s i n p e t r i d i s h e s were conducted a g a i n s t s e v e r a l microorganisms. G r e a t e r t h a n 800 jigfmL were n e c e s s a r y to c o n t r o l C o l l e t o t r i c h u m l a g e n a r i u m . F u s a r i u m oxvsporum. f. l y c o p e r s i c i . G i b b e r e l l a f u i i k u r o i , P e l l i c u l a r i a f i l a m e n t o s a , and S a p r o l e g n i a p a r a s i t i c a . But 200 jig/mL c o n t r o l l e d A . k i k u c h i a n a , B o t r y t i s c i n e r e a . C o c h l i o b o l u s miyabeanus, D i a p o r t h e c i t r i , G r o m e r e l l a c i n g u l a t a , P y r i c u l a r i a o r v z a e . and R h i z o c t o n i a solani. In excess o f 100 jig/mL were needed to c o n t r o l J i . s u b t i l i s , A T C C 6 6 3 3 , J3. s t e a r o t h e r m o p h i l u s , M y c o b a c t e r i u m p h l e i 607 , S t a p h y l o c o c c u s a u r e u s 2 0 9 P , E . c o l i NIHJ, Pseudomonas a e r u g i n o s a M8152, S e r r a t i a m a r c e s c e n s , V i b r i o p e r c o l e n s ATCC 8461, and C a n d i d a a l b i c a n s M9001. O n l y 25 yg/mL were n e c e s s a r y to i n h i b i t S a c c h a r o m v c e s c e r e v i s i a e Y 2 1 - 1 , b u t most i m p o r t a n t l y , o n l y 1.0 μg/mL o f t h e a n t i b i o t i c was needed to c o n t r o l V a l s a c e r a t o s p e r m a . S i n c e o n l y 45 mg of the Na s a l t were i s o l a t e d i n i t i a l l y , t h e r e was insufficient material for f i e l d t r i a l s . S y n t h e t i c m a t e r i a l was


32


H Ο L-1 Η

Η

Η Ο


L-2

H CO

OCH CH COOCH

3

2

2

3

L-3 ( Irpex lacteus ) F i g u r e 7.

L-l L-2 L-3

: 5-pentylfuraldehyde, : 5 - ( 4 - p e n t y l ) - 2 - f u r a l d e h y d e , and : methyl 3 - j > - a n i s y l o x y p r o p i o n a t e .

Ο

NIGRAGILLIN ( Aspergillus niger ) Figure 8.

Nigragillin.

0 = N-N-CH -CH-COOH ι ι OH NH 2

2

L-ALANOSINE (Streptomyces sp.) Figure 9.

L-Alanosine.

CH -CH-CH OH 3 3

"Ο

2

N-OH

PROPANOSINE ( K - 7 6 ) ( Micromonospora chalcea ) F i g u r e 10.

Propanosine

(K-76).



3.

CUTLER

Japanese

Contributions

to

Allelochemicals

o b t a i n e d (.28) b u t i t was a r a c e m i c m i x t u r e and had o n l y h a l f the a c t i v i t y o f the n a t u r a l p r o d u c t . The c a t a l o g o f n a t u r a l p r o d u c t s t h a t c o n t r o l m i c r o o r g a n i s m s i s extensive. That i s , a f t e r a l l , one of the e a r l y a r e a s o f m e d i c i n a l r e s e a r c h and r e a l l y marks the b e g i n n i n g of a n t i b i o t i c s d e r i v e d from microbes. But l e s s common are n a t u r a l p r o d u c t s from m i c r o o r g a n i s m s t h a t a r e s e l f - i n h i b i t o r s a t low l e v e l s o f a p p l i c a t i o n . While h i g h e r p l a n t s c o n t a i n a p p a r e n t s e l f - i n h i b i t o r s and p l a n t growth regulators, s p e c i f i c a l l y a b s c i s i c a c i d , t h e r o l e of these m e t a b o l i t e s i n the p r o d u c e r o r g a n i s m i s n o t c l e a r l y u n d e r s t o o d . Aspermutarubrol, bis(5-methy1-2,3-dihydroxypheny1) e t h e r , from A s p e r g i l l u s svdowi i s such a compound ( F i g u r e 1 1 ) . The p r e s e n c e o f t h e o r g a n i s m on t h e s u r f a c e o f o l d shoe p o l i s h i s something o f a m y s t e r y b u t t h e c u r i o s i t y o f S a t o m u r a i s t o be commended I The f i r s t r e a c t i o n on d i s c o v e r i n g a fungus on any household i t e m i s to discard i t with a l a c r i t y . D u r i n g the c u l t u r i n g of the o r g a n i s m i t was o b s e r v e d t h a t p i g m e n t p r o d u c t i o n c o u l d be c o r r e l a t e d w i t h m y c e l i a l i n h i b i t i o n and t h a t a d d i t i o n o f s o d i u m a c e t a t e increased both responses. E v e n t u a l l y , a s p e r m u t a r u b r o l was i s o l a t e d as c o l o r l e s s c r y s t a l s f r o m c h l o r o f o r m (1 mg/L) (29) but i n aqueous s o l u t i o n the m e t a b o l i t e o x i d i z e d q u i t e r e a d i l y to give red products. T h e m e t a b o l i t e p r o v e d t o be i n e f f e c t i v e against S a c c h a r o m y c e 8 c e r e v i s i a e . P é n i c i l l i u m no t a turn, A s p e r g i l l u s n i g e r , A . o r v z a e . M u c o r m u ç e d o , R h i z o p u s i a p o n i c u s . and v e r y s l i g h t l y a c t i v e a g a i n s t JS. c o l i . but i t was v e r y a c t i v e a t 50, 100, and 200 ppm a g a i n s t t h e g r a m - p o s i t i v e jB. s u b t i l i s . S t a p h y l o c o c c u s a u r e u s , and M i c r o c o c c u s l y s o d e i k t i c u s . A t 1 2 . 5 , 2 5 , 50, and 100 ppm i t m o d e r a t e l y i n h i b i t e d A . svdowi and c o m p l e t e l y i n h i b i t e d at 200 ppm (.29.) · The a u t h o r s o f t h i s w o r k were q u i c k to p o i n t out t h a t the s e c r e t i o n o f a n t i b i o t i c s u b s t a n c e s as a c o m p e t i t i v e mechanism f o r the s u r v i v a l and b e n e f i t of the p r o d u c e r o r g a n i s m i s well e s t a b l i s h e d , but the p u z z l e posed by a s p e r m u t a r u b r o l remains to be explained. D u r i n g t h e c o u r s e o f t r y i n g t o i s o l a t e a s e l f - i n h i b i t o r from b e n o m y l - r e s i s t a n t s t r a i n s o f the c h e r r y brown r o t f u n g u s , M o n i l i n i a f r u e t i c o l a . two new m e t a b o l i t e s t h a t h a d a n t i m i c r o b i a l and p h y t o t o x i c p r o p e r t i e s , though not s e l f - i n h i b i t o r y c h a r a c t e r i s t i c s , were i s o l a t e d . These were m o n i l i d i o l and d e c h l o r o m o n i l i d i o l ( 3 0 ) , " e a l i c y l a l d e h y d e t y p e o c t a k e t i d e s (30.)", which are s t r u c t u r a l l y r e l a t e d t o t h e p h y t o t o x i n s p y r i c u l o l (31.) and p y r i c u l a r i o l (32) from P y r i c u l a r i a o r y z a e (Figure 12). M o n i l i d i o l , when a p p l i e d to c h e r r y l e a v e s a t 2 - 5 ^ g , f o l l o w e d by p i n p r i c k s , i n d u c e d d a r k necrotic spots. It a l s o i n h i b i t e d the growth of r i c e seedlings though h a r d d a t a a r e not a v a i l a b l e (30). D e c h l o r o m o n i l i d i o l was n o t as a c t i v e as t h e c h l o r i n a t e d compound i n e i t h e r c h e r r y or r i c e plants. O t h e r t e s t s were c a r r i e d o u t w i t h m o n i l i d i o l a g a i n s t s e l e c t e d o r g a n i s m s b u t g r e a t e r t h a n 100 ppm w e r e n e c e s s a r y t o i n h i b i t p l a n t p a t h o g e n s ( u n n a m e d ) , i n c l u d i n g M. f r u c t i c o l a . The compounds w e r e s y n t h e s i z e d a n d t h e p r o c e d u r e s were p u b l i s h e d i n 1983 ( 3 3 ) . No s y s t e m i s p e r f e c t and sometimes events become q u i t e t a n g l e d in research. What a p p e a r s t o be a p l a c i d a r e a o f e n d e a v o r is s u d d e n l y e n t e r e d b y s e v e r a l w o r k e r s who a r e , unknowingly, i n d e p e n d e n t l y p u r s u i n g common g o a l s . Such was t h e case w i t h the j > - t e r p h e n y l s , a c u r i o u s group o f compounds t h a t a r e expected to be


33


34

A L L E L O C H E M I C A L S : ROLE IN AGRICULTURE A N D FORESTRY

p r o d u c t s of a chemical r e a c t i o n i n v i t r o r a t h e r than n a t u r a l products. I n 1 9 7 5 , T a k a h a s h i was i n the p r o c e s s o f i s o l a t i n g and i d e n t i f y i n g t e r p h e n y l l i n ( F i g u r e 13) from A . c a n d i d u s when, as he states, " A t t h i s s t a g e o f t h e work M a r c h e l l i and V i n i n g ( 3 4 ) r e p o r t e d t h e i s o l a t i o n o f t h e t e r p h e n y l assumed to be i d e n t i c a l w i t h compound A . . . " ( 3 5 ) . The compounds were i d e n t i c a l . Takahashi h a d n o t e d t h e e f f e c t o f t e r p h e n y l l i n a g a i n s t HeLa c e l l s , where 3 . 2 ppm p r o d u c e d s l i g h t c e l l u l a r damage and h i g h e r c o n c e n t r a t i o n s i n d u c e d g r e a t e r c h a n g e s so t h a t a t 100 ppm t h e r e was c o m p l e t e cytolysis. C e l l s a l s o had v a r y i n g d e g r e e s o f R t y p e changes, s l i g h t l y e n l a r g e d c e l l s , e v e n l y d i s t r i b u t e d c h r o m a t i n , and s m a l l n u c l e o l i (.35). In 1 9 7 8 , C u t l e r , et a l . , d i s c o v e r e d h y d r o x y t e r p h e n y l l i n , a l s o f r o m A . c a n d i d u s . u s i n g t h e e t i o l a t e d wheat c o l e o p t i l e b i o a s s a y and showed t h a t i t had p l a n t growth r e g u l a t o r y a c t i v i t y (36) (Figure 1 4 ) . Furthermore, t e r p h e n y l l i n s i g n i f i c a n t l y i n h i b i t e d c o l e o p t i l e s 35% a t 1 0 " 3 , w h i l e h y d r o x y t e r p h e n y 1 1 i n i n h i b i t e d 1 0 0 , 4 2 , and 8%, a t 1 0 - 3 , i o ~ 4 IQ-5 . Peracetylt i o n of the m o l e c u l e r e n d e r e d i t b i o l o g i c a l l y i n a c t i v e and so t h e a c t i v i t y a p p e a r e d t o be r e l a t e d t o t h e number o f the OH g r o u p s . Four years l a t e r , Kobayashi et a l . , p u b l i s h e d t h e s t r u c t u r e s for c a n d i d u e i n s A a n d Β f r o m A . c a n d i d u s ( F i g u r e s 15 and 1 6 ) . Both were a n a l o g s o f t e r p h e n y l l i n and h y d r o x y t e r p h e n y 1 1 i n , r e s p e c t i v e l y . Each b l o c k e d i n i t i a l c l e a v a g e i n s e a u r c h i n e m b r y o s when a d d e d 5 m i n a f t e r f e r t i l i z a t i o n a t 1 χ 1 0 " ^ and 5 χ 10""* M, and i n h i b i t e d ! · 8 u b t i l i s a t 50 yg/mL (37). F u r t h e r experiments were conducted w i t h c a n d i d u s i n Β t o determine i t s e f f e c t s on DNA, RNA and p r o t e i n s y n t h e s i s i n t h e e a s t r u l a s t a g e o f sea u r c h i n embryos, uptake of [^H]-thymidine, [ ^ H ] - u r i d i n e , and [ 3 R ] - L - l e u c i n e was o b s e r v e d and percent i n h i b i t i o n noted. A t 0 pg/mL o f c a n d i d u s i n Β t h e r e was 0 i n h i b i t i o n o f u p t a k e o f these r a d i o i s o t o p e s , but a t 1 Mg/mL t h e r e was 8 3 , 5 8 , and 0% i n h i b i t i o n o f uptake o f r a d i o l a b e l e d t h y m i d i n e , u r i d i n e and L - l e u c i n e , r e s p e c t i v e l y . A t 10 jug/mL these f i g u r e s were 9 6 , 86 and 0%, r e s p e c t i v e l y . T h e r e f o r e , c a n d i d u s i n Β was n o t c o n s i d e r e d t o be a r e s p i r a t o r y i n h i b i t o r b e c a u s e i t h a d b e e n determined, i n sea u r c h i n embryos, t h a t r e s p i r a t o r y i n h i b i t o r s s u p p r e s s p r o t e i n s y n t h e s i s , and a l s o DNA and RNA s y n t h e s i s ( 3 7 ) . I n 1 9 8 5 , d i h y d r o x y t e r p h e n y l l i n was i s o l a t e d from A . c a n d i d u s and was f o u n d t o be a p p r o x i m a t e l y t w i c e a s a c t i v e as h y d r o x y t e r p h e n y l l i n i n b l o c k i n g f i r s t c l e a v a g e and i n d u c i n g i r r e g u l a r l y s h a p e d o r o d d - n u m b e r e d c e l l s i n s e a u r c h i n e m b r y o s (.38) . The laboratory synthesis of these terphenyl d e r i v a t i v e s is e x c e p t i o n a l l y d i f f i c u l t and has n o t y e t been a c c o m p l i s h e d , but m i c r o o r g a n i s m s appear t o make them w i t h r e l a t i v e e a s e . J

A

N

D

M

These few examples o f r e s e a r c h i n d i c a t e , at l e a s t i n p a r t , the i n t e n s i t y and p e r s e v e r a n c e w i t h w h i c h Japanese s c i e n t i s t s approach t h e i r work w i t h m i c r o b i a l m e t a b o l i t e s . We have seen t h a t , i n many i n s t a n c e s , e f f o r t s a r e made t o d u p l i c a t e , by s y n t h e s i s , these s u b s t a n c e s a n d i t may o n l y b e a q u e s t i o n o f t i m e b e f o r e a m a r k e t a b l e product i s developed. D o u b t l e s s , what we are s e e i n g i n the l i t e r a t u r e i s o n l y a s m a l l p o r t i o n of the energy b e i n g expended to s u c c e s s f u l l y produce biodegradable a g r o c h e m i c a l s b a s e d on n a t u r a l product templates. Japan i s c o m m i t t e d to p r o d u c i n g abundant crops on s m a l l p a r c e l s o f l a n d and to h a v i n g an u n p o l l u t e d environment.



3.

CUTLER

Japanese Contributions

to

35

Allelochemicals

ASPERMUTARUBROL ( Aspergillus sydowi ) Figure

11.

Aspermutarubrol.

HQ,

R MONILIDIOL

CI

DECHLOROMONILIDIOL

H

( Monilinia f r u c t i c o l a ) Figure

12.

M o n i l i d i o l and d e c h l o r o m o n i l i d i o l .

OCH

3

OH

TERPHENYLLIN ( Aspergillus candidus ) Figure

13.

Terphenyllin.




OCH

3

OH

HYDROXYTERPHENYLLIN ( Aspergillus candidus ) Figure

14,

Hydroxyterphenyllin.

CANDIDUSIN A ( Aspergillus candidus ) Figure

15·

Candidusin A.

CANDIDUSIN Β ( Aspergillus candidus ) Figure

16.

Candidusin B.


3. CUTLER Japanese Contributions to Allelochemicals

37

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. 24. 25.

Demographic and statistical data were kindly supplied by the Japanese Consulate, Atlanta, Georgia. Figures are given for 1983. The author thanks Masami Mera for these services. Hirota, Α.; Suzuki, Α.; Suzuki, H.; Tamura, S. Agric. Biol. Chem. 1973, 37, 643. Hirota, Α.; Suzuki, Α.; Aizawa, K.; Tamura, S. Agric. Biol. Chem. 1973, 37, 955. Takayama, S.; Isogai, Α.; Nakata, M.; Suzuki, H.; Suzuki, A. Agric. Biol. Chem. 1984, 48, 839. Closse, Α.; Huguenin, R. Helv. Chim. Acta 1974, 57, 533. Walton, J.D.; Earle, E.D.; Gibson, B.W. Biochem. Biophys. Res. Commun. 1982, 107, 785. Gross, M.L.; McCrery, D.; Crow, F.; Tomer, K.B.; Pope, M.R.; C i u f f e t t i , L.M.; Knoche, H.W.; Daly, J.M.; Dunkle, L.D. Tetrahedron Lett. 1982, 23, 5381. Umehara, K.; Nakahara, K.; Kiyoto, S.; Iwami, M.; Okamoto, M.; Tanaka, H . ; Kohsaka, M.; Aoki, H.; Imanaka, H. J . Antibiot. 1983, 36, 478. Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H. Agric Biol. Chem. 1975, 39, 1115. Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H. Agric Biol. Chem. 1975, 39, 2081. Iwamoto, T.; Shima, S.; Hirota, Α.; Isogai, Α.; Sakai, H. Agric. Biol. Chem. 1983, 47, 739. Hamasaki, T.; Nakajima, H.; Yokota, T.; Kimura, Y. Agric. Biol. Chem. 1983, 47, 891. El-Rayyes, N.R.; Al-Hajjar, F.H. J. Prakt. Chem. 1977, 319, 927. Isogai, Α.; Washizu, M.; Kondo, K.; Murakoshi, S.; Suzuki, A. Agric. Biol. Chem. 1984, 48, 2607. McCorkindale, N . J . ; Blackstone, W.P.; Johnstone, G.A.; Ray, T.R.; Troke, R.A. 11th IUPAC Int. Symp. Chem. Nat. Prod. Vol. I, 1978, p. 151. McCorkindale, N . J . ; Wright, J . L . C . ; Brian, P.W.; Clarke, S.M.; Hutchinson, S.A. Tetrahedron Lett. 1968, 727. Sassa, T.; Tomizuka, K.; Ikeda, M.; Miura, Y. Agric. Biol. Chem. 1973, 37, 1221. Sassa, T.; Tomizuka, K.; Ikeda, M.; Miura, Y. Tetrahedron Lett. 1973, 2333. Suzuki, Α.; Gohbara, M.; Kosuge, Y.; Tamura, S.; Ohashi, Y.; Sasada, Y. Agric. Biol. Chem. 1976, 40, 2505. Gohbara, M.; Kosuge, Y.; Yamasaki, S.; Kimura, Y.; Suzuki, Α.; Tamura, S. Agric. Biol. Chem. 1978, 42, 1037. Hayashi, M.; Wada, K.; Munakata, K. Agric. Biol. Chem. 1981, 45, 1527. Caesar, F . ; Jansson, K.; Mutschler, E. Pharm. Acta Helv. 1969, 44, 676. Isogai, Α.; Horii, T.; Suzuki, Α.; Murakoshi, S.; Ikeda, K.; Sato, S.; Tamura, S. Agric. Biol. Chem. 1975, 39, 739. Matsumoto, S.; Sakuda, S.; Isogai, Α.; Suzuki, A. Agric. Biol. Chem. 1984, 48, 827. Murthy, Y.K.S.; Thiemann, J . E . ; Coronelli, C.; Sensi, P. Nature 1966, 211, 1198.


38

26. 27. 28. 29. 30.


31. 32. 33. 34. 35. 36. 37. 38.

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Coronelli, C.; Pasqualucci, C.R.; Tamoni, G.; Gallo, G.G. II Farmaco. Ed. Sci. 1966, 21, 269. Kenaja, E.E. J. Econ. Entomol. 1969, 62, 1006. Abe, Y . ; Kadokura, J.; Shimazu, Α.; Seto, H.; Otake, N. Agric. Biol. Chem. 1983, 47, 2703. Taniguchi, M.; Kaneda, N. Shibata, K.; Kamikawa, T. Agric. Biol. Chem. 1978, 42, 1629. Sassa, T.; Nukina, M.; Sugiyama, T.; Yamashita, K. Agric. Biol. Chem. 1983, 47, 449. Iwasaki, S.; Nozoe, S.; Okuda, S.; Sato, Z . ; Kozaka, T. Tetrahedron Lett. 1969, 3977. Nukina, M.; Sassa, T.; Ikeda, M.; Umezama, T.; Tasaki, H. Agric. Biol. Chem. 1981, 45, 2161. Sugiyama, T.; Watanabe, M.; Sassa, T.; Yamashita, K. Agric. Biol. Chem. 1983, 47, 2411. Marchelli, R.; Vining, L.C. J. Chem. Soc. Chem. Commun. 1973, 555. Takahashi, C.; Yorhihira, K.; Natari, S.; Umeda, M. Chem. Pharm. Bull. 1976. 24, 613. Cutler, H.G.; Lefiles, J . H . ; Crumley, F . G . ; Cox, R.H. J. Agric. Food Chem. 1978, 26, 632. Kobayashi, Α.; Takemura, Α.; Koshimizu, K.; Nagano, H.; Kawazu, K. Agric. Biol. Chem. 1982, 46, 585. Kobayashi, Α.; Takemoto, Α.; Koshimizu, K.; Kawazu, K. Agric. Biol. Chem. 1985. 49, 867.

RECEIVED February 4, 1986


Japanese Contributions to the Development of Allelochemicals

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