New Approaches to Chemical Control of Plant Pathogens - ACS

Jul 23, 2009 - Antifungal compounds currently available for disease control may have either non-specific or specific biochemical modes of action. Near...
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NANCY N. RAGSDALE and MALCOLM R. SIEGEL 1

Cooperative State Research Service, U.S. Department of Agriculture, Washington, DC 20251 Plant Pathology Department, University of Kentucky, Lexington, ΚY 40546

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New approaches to chemical control of plant pathogens emphasize selective use of fungicides based on an understanding of the physiological, biochemical, and molecular levels of the modes of action and mechanisms of resistance. Antifungal compounds currently available for disease control may have either non-specific or specific biochemical modes of action. Nearly all systemic fungicides are of the latter type. Compounds with specific modes of action are prone to a loss of efficacy through selection pressure for resistant pathogens. Evidence suggests that chemicals with certain modes of action may be less likely to encounter resistance problems. The possibility of circumventing resistance by chemically affecting metabolic pathways or through stereoselectivity are discussed. In addition, promising methods for plant disease control with chemicals that activate plant defense mechanisms or interfere with pathogenesis are considered. Most of the pests that cause infectious plant diseases may be classified as fungi, bacteria, nematodes, or viruses. Chemicals frequently are either unavailable or impractical to use in controlling viruses and most bacteria. Efforts to achieve nematode control usually involve compounds associated with insect control. Thus, this article w i l l be devoted to the use of chemicals to control fungi that cause plant diseases, a far greater use than for other disease agents. These fungicides, or antifungal agents as they are more correctly called, are but a part of disease control strategy. Plant breeding programs and cultural practices are also part of plant disease management practices. The traditional approach in the development of fungicides has involved large scale laboratory and greenhouse screening tests followed by closer examination of structurally related chemicals to optimize activity. More recently scientists have become aware that this type of assay system has limitations. There is a need to examine vulnerabilities in both the pest and the host, and to design 0097-6156/85/0276-0035$06.00/0 © 1985 American Chemical Society

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a n t i f u n g a l m a t e r i a l s t h a t have modes o f a c t i o n based on these principals. T h i s w i l l i d e n t i f y c h e m i c a l s t h a t have b o t h d i r e c t and i n d i r e c t a c t i o n s . New approaches emphasize an u n d e r s t a n d i n g a t the p h y s i o l o g i c a l , m o l e c u l a r , and b i o c h e m i c a l l e v e l s of modes of a c t i o n as w e l l as the mechanisms f o r pathogen r e s i s t a n c e . Some o f the more r e c e n t a n t i f u n g a l compounds r e s u l t e d from t h i s type o f a p p r o a c h . Not o n l y w i l l t h e s e a p p r o a c h e s p r o v i d e knowledge f o r the r a t i o n a l development of new a n t i p a t h o g e n i c compounds, they w i l l a l s o p r o v i d e s t r a t e g i e s f o r the use of e s t a b l i s h e d c h e m i c a l s , e s p e c i a l l y i n r e l a t i o n to r e d u c i n g or p r e v e n t i n g the development o f r e s i s t a n t pathogens· T h i s c h a p t e r w i l l endeavor to condense a r a t h e r wide r a n g i n g s u b j e c t i n t o some b a s i c p r i n c i p l e s r e g a r d i n g the types of c h e m i c a l s used t o c o n t r o l p l a n t pathogens; the s t r a t e g i e s b e h i n d t h e s e ; and some thoughts on new approaches to the development and use of c h e m i c a l s t o c o n t r o l f u n g a l d i s e a s e s of p l a n t s . First

Generation

Fungicides

The f i r s t g e n e r a t i o n f u n g i c i d e s a r e p r i m a r i l y a group r e f e r r e d t o as s u r f a c e p r o t e c t a n t s . These compounds a r e not taken up to any d e g r e e by the p l a n t , have a broad a n t i f u n g a l spectrum, and demonstrate m u l t i p l e s i t e s of a c t i o n ( 1 ) . The f u n c t i o n of t h e s e p e s t i c i d e s i s based on t h e i r b e i n g p r e s e n t when the fungus a r r i v e s and b e f o r e i n f e c t i o n occurs. They e x h i b i t a d i f f e r e n t i a l t o x i c i t y ; thus a s p e c i f i c dose w i l l k i l l the pathogen and n o t i n j u r e the h o s t p l a n t . D i f f e r e n t i a l t o x i c i t y i s based p r i m a r i l y on f u n g i r a p i d l y a c c u m u l a t i n g t o x i c c o n c e n t r a t i o n s of the f u n g i c i d e w h i l e p l a n t t i s s u e s do n o t ( 2 ) . The s u r f a c e p r o t e c t a n t s i n c l u d e i n o r g a n i c c h e m i c a l s such as e l e m e n t a l s u l f u r , Bordeaux m i x t u r e and copper o x i d e as w e l l as o r g a n i c f u n g i c i d e s such as d i a l k y l d i t h i o c a r b a m a t e s ( t h i r a m , ferbam), e t h y l e n e b i s d i t h i o c a r b a m a t e s (maneb, z i n e b ) , p h t h a l i m i d e s ( c a p t a n , f o l p e t ) , and c h l o r o t h a l o n i l . Because t h e s e f i r s t g e n e r a t i o n f u n g i c i d e s a r e m u l t i s i t e i n h i b i t o r s , they a r e n o t l i k e l y t o l o s e e f f e c t i v e n e s s because of f u n g a l r e s i s t a n c e . Pathogen r e s i s t a n c e has n o t been a problem w i t h t h i s group and t h e i r c o n t i n u e d use as an i m p o r t a n t p a r t o f d i s e a s e management programs i s h i g h l y likely. Second G e n e r a t i o n

Fungicides

D u r i n g the mid I960's, the s y s t e m i c t o x i c a n t s , or second g e n e r a t i o n f u n g i c i d e s as they a r e c a l l e d , e n t e r e d the m a r k e t . A l t h o u g h most o f the o l d e r m a t e r i a l s a r e s t i l l i n use, c u r r e n t e f f o r t s have s h i f t e d toward d e v e l o p i n g s y s t e m i c compounds which move i n the symplast, a p o p l a s t , o r a r e ambimoble ( 3 ) . Almost a l l the c o m m e r c i a l l y a v a i l a b l e s y s t e m i c f u n g i c i d e s move o n l y i n the a p o p l a s t and a r e t h e r e f o r e dependent on t r a n s p i r a t i o n f o r t h e i r movement and accumulation i n plant t i s s u e . The s y s t e m i c f u n g i c i d e s have a number o f advantages o v e r s u r f a c e p r o t e c t a n t s . Foremost i s t h e i r a b i l i t y to p e n e t r a t e h o s t t i s s u e and c o n t r o l o r e r a d i c a t e an e s t a b l i s h e d infection. I n o r d e r f o r t h e s e compounds to p r o v i d e i n t e r n a l t h e r a p y , they must have c e r t a i n q u a l i t i e s . They o r t h e i r a c t i v e d e r i v a t i v e s must be r e l a t i v e l y s t a b l e w i t h i n the h o s t t i s s u e w i t h e i t h e r a

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mechanism s p e c i f i c f o r f u n g a l c e l l s or a c o n c e n t r a t i o n so as not to produce p h y t o t o x i c e f f e c t s ( 4 ) . In c o n t r a s t to f i r s t g e n e r a t i o n f u n g i c i d e s , they a r e g e n e r a l l y e f f e c t i v e a g a i n s t a narrower range o f pathogens, and s y s t e m i c s have a s p e c i f i c s i t e or a l i m i t e d number of s i t e s of a c t i o n , making them more s u b j e c t to l o s s o f e f f i c a c y due to fungal resistance. F o l l o w i n g d e t e r m i n a t i o n of t h e i r modes of a c t i o n , tremendous c o n t r i b u t i o n s have been made through the use of t h e s e c h e m i c a l s t o e l u c i d a t e v a r i o u s b i o c h e m i c a l pathways and c e l l u l a r f u n c t i o n s . We w i l l l o o k a t a number of examples of t h e s e second g e n e r a t i o n s y s t e m i c s and b r i e f l y d i s c u s s t h e i r modes of a c t i o n . Cycloheximide. C y c l o h e x i m i d e , an a n t i f u n g a l a n t i b i o t i c produced by Streptomyces g r i s e u s , i n h i b i t s p r o t e i n s y n t h e s i s not o n l y i n f u n g i , but i n e u k a r y o t i c organisms i n g e n e r a l ( 5 ) . Due to p h y t o t o x i c i t y problems, c y c l o h e x i m i d e can o n l y be used s u c c e s s f u l l y f o r d i s e a s e c o n t r o l when low c o n c e n t r a t i o n s a r e e f f e c t i v e or on p l a n t s t h a t a r e r e l a t i v e l y i n s e n s i t i v e (6)· A l t h o u g h c y c l o h e x i m i d e has been r e p o r t e d to i n h i b i t a number of c e l l u l a r p r o c e s s e s i n c l u d i n g r e s p i r a t i o n and n u c l e i c a c i d s y n t h e s i s , t h e s e a r e r e g a r d e d as s e c o n d a r y e f f e c t s t h a t r e s u l t from the p r i m a r y s i t e of a c t i o n , i . e . , r i b o s o m a l p r o t e i n s y n t h e s i z i n g systems (7)· R e s i s t a n c e to c y c l o h e x i m i d e has been c o r r e l a t e d w i t h changes i n r i b o s o m a l s e n s i t i v i t y or i n c e l l u l a r permeability ( 1). Benomyl. Benomyl i s the b e s t known of s e v e r a l b e n z i m i d a z o l e fungicides. T h i s compound i s e f f e c t i v e a g a i n s t a l a r g e number o f f u n g i and p l a y s an i m p o r t a n t r o l e i n d i s e a s e management. Benomyl i n t e r f e r e s w i t h t u b u l i n p o l y m e r i z a t i o n i n f u n g i by b i n d i n g to the beta t u b u l i n subunit ( 8 ) . As a r e s u l t of the f a i l u r e to form m i c r o t u b u l e s , m i t o s i s i s b l o c k e d (9) as w e l l as o t h e r microtubular-dependent processes (10). I t has been shown t h a t a s i n g l e gene m u t a t i o n a l t e r s the a f f i n i t y of the t u b u l i n b i n d i n g s i t e f o r the i n h i b i t o r r e s u l t i n g i n f u n g a l r e s i s t a n c e ( 8 ) . The n a t u r e of t h i s h i g h l y s p e c i f i c mode of a c t i o n and the c o n t r o l of t o x i c i t y t h r o u g h a s i n g l e gene has r e s u l t e d , as one m i g h t e x p e c t , i n the development of problems i n the f i e l d w i t h f u n g a l r e s i s t a n c e . f

Metalaxyl. S i n c e i t s i n t r o d u c t i o n the l a t e 1 9 7 0 s f o r the c o n t r o l o f pathogens such as p o t a t o l a t e b l i g h t , downy mildews and soil-borne Pythium and P h y t o p h t h o r a spp., m e t a l a x y l , an a c y l a l a n i n e f u n g i c i d e , and o t h e r r e l a t e d phenylamide f u n g i c i d e s have seen i n c r e a s i n g u s a g e . A l t h o u g h the p r e c i s e mechanism of i n h i b i t i o n by m e t a l a x y l has not been d e t e r m i n e d , RNA s y n t h e s i s appears to be the most s e n s i t i v e pathway (JUL). F i e l d r e s i s t a n c e to m e t a l a x y l has d e v e l o p e d r a p i d l y i n s i t u a t i o n s t h a t i n v o l v e d e x c l u s i v e and r e p e a t e d use of the compound (11). T h i s has prompted the development of use s t r a t e g i e s s u c h as t h a t of combining c h e m i c a l s which have d i f f e r e n t modes of a c t i o n . Carboxin. C a r b o x i n i s used p r i m a r i l y to c o n t r o l B a s i d i o m y c e t e s s u c h as r u s t s , b u n t s , and smuts. I t i s used p r i m a r i l y f o r seed t r e a t m e n t , but a l s o has s o i l and f o l i a r a p p l i c a t i o n s . The mode o f a c t i o n o f t h i s and r e l a t e d compounds i s p r o b a b l y the b e s t u n d e r s t o o d of any f u n g i t o x i c mechanism. C a r b o x i n b l o c k s the t r a n s f e r o f e l e c t r o n s from

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s u c c i n i c dehydrogenase t o coenzyme Q i n t h e complex I I r e g i o n o f t h e m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t c h a i n (12, 1 3 ) . L a b o r a t o r y s t u d i e s have i n d i c a t e d t h a t r e s i s t a n c e i s s i n g l e - g e n e based and s e v e r a l l o c i a r e i n v o l v e d (11)« Based on t h e h i g h l y s p e c i f i c mode o f a c t i o n and s t u d i e s on l a b o r a t o r y d e v e l o p e d r e s i s t a n t m u t a n t s , i t i s s u r p r i s i n g t h a t i n s p i t e o f q u i t e a few y e a r s ' u s e , r e s i s t a n c e i n t h e f i e l d has not been a p r o b l e m . One would s p e c u l a t e t h a t n a t u r a l mutants must be l e s s f i t o r do n o t have t h e p a t h o g e n i c c a p a b i l i t i e s o f t h e w i l d t y p e . A more l i k e l y e x p l a n a t i o n i s r e l a t e d t o t h e p a t t e r n o f u s e . Because most c a r b o x i n i s used f o r seed t r e a t m e n t , t h e s e l e c t i o n p r e s s u r e i s r a t h e r low. Polyoxins. The p o l y o x i n a n t i b i o t i c s a r e e f f e c t i v e a g a i n s t f u n g i t h a t contain c h i t i n i n their c e l l walls. The r e s i s t a n c e t o p o l y o x i n s i n some f u n g i o f t h i s type a p p e a r s t o be r e l a t e d t o a p e r m e a b i l i t y f a c t o r r a t h e r than t o a change i n the s i t e o f a c t i o n ( 1 4 ) . Polyoxins c o m p e t i t i v e l y i n h i b i t c h i t i n s y n t h e t a s e and thus p r e v e n t t h e i n c o r p o r a t i o n o f u r i d i n e d i p h o s p h o - N - a c e t y l g l u c o s a m i n e i n t o the c h i t i n polymer ( 1 4 ) . E d i f e n p h o s . E d i f e n p h o s , an organophosphorus compound, has a r a t h e r l i m i t e d use because o f i t s h i g h l y s e l e c t i v e a c t i o n a g a i n s t t h e r i c e b l a s t fungus. Some a m b i g u i t y e x i s t s r e g a r d i n g the mode o f a c t i o n . A l t h o u g h t h e compound has been shown t o i n h i b i t c h i t i n s y n t h e t a s e thus b l o c k i n g c e l l w a l l s y n t h e s i s , i t a l s o i n h i b i t s the s y n t h e s i s o f p h o s p h a t i d y l c h o l i n e , a p h o s p h o l i p i d and i m p o r t a n t membrane component (11). The l a t t e r i n h i b i t i o n , w h i c h has been s u g g e s t e d as the p r i m a r y mechanism o f a c t i o n , r e s u l t s from b l o c k i n g t h e a c t i v i t y o f phospholipid-N-methyltransferase, which i s necessary i n the conversion of phosphatidylethanolamine to phosphatidylcholine (11). The e f f e c t s on c h i t i n s y n t h e s i s p r o b a b l y r e s u l t from a l t e r e d membrane properties. R e s i s t a n c e a g a i n s t e d i f e n p h o s e i n the f i e l d has been slow t o d e v e l o p . P o l y e n e s . A l t h o u g h a m p h o t e r i c i n Β and n y s t a t i n , b o t h p o l y e n e a n t i b i o t i c s , a r e v e r y a c t i v e a g a i n s t a wide v a r i e t y o f f u n g i , they a r e i m p r a c t i c a l as a g r i c u l t u r a l f u n g i c i d e s due t o t h e i r r a p i d photodecomposition (14). These compounds a f f e c t membrane p e r m e a b i l i t y through t h e i r i n t e r a c t i o n s with s t e r o l s (15). These i n t e r a c t i o n s cause membrane changes t h a t r e s u l t i n leakage o f s m a l l molecular materials. R e s i s t a n c e may g e n e r a l l y be a t t r i b u t e d t o a change i n s t e r o l q u a l i t y o r growth c o n d i t i o n s . D i c a r b o x i m i d e s and A r o m a t i c H y d r o c a r b o n s . The d i c a r b o x i m i d e f u n g i c i d e s , procymidone, v i n c l o z o l i n and i p r o d i o n e , and a r o m a t i c h y d r o c a r b o n s s u c h as c h l o r o n e b , d i c h l o r a n and q u i n t o z e n e , show a g r e a t d e a l o f s i m i l a r i t y i n t h e i r modes o f t o x i c i t y ( 1 6 ) . Although the e f f e c t s o f t h e s e compounds on a number o f b i o s y n t h e t i c p r o c e s s e s have been i n v e s t i g a t e d , the e x a c t mode of a c t i o n has n o t been r e s o l v e d (4^, 1 1 ) . I n t e r f e r e n c e w i t h n u c l e a r f u n c t i o n , membrane damage, and i n t e r f e r e n c e w i t h c e l l u l a r m o t i l e f u n c t i o n s , such as f l a g e l l a r movement and c y t o p l a s m i c s t r e a m i n g , have been s u g g e s t e d as possibilities. A b e t t e r u n d e r s t a n d i n g o f the mode o f a c t i o n may e x p l a i n why r e s i s t a n c e has p r e s e n t e d few problems i n t h e p r a c t i c a l use o f t h e s e f u n g i c i d e s .

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E r g o s t e r o l Biosynthesis I n h i b i t o r s (EBI). S i n c e the determination t h a t t r i a r i m o l owed i t s a n t i f u n g a l a c t i v i t y to the i n h i b i t i o n of e r g o s t e r o l b i o s y n t h e s i s ( 1 7 ) , a l a r g e number of s t r u c t u r a l l y d i v e r s e compounds t h a t b l o c k e r g o s t e r o l b i o s y n t h e s i s have become a v a i l a b l e f o r p l a n t d i s e a s e c o n t r o l . T h i s group c o n s i s t s o f p y r i m i d i n e s , i m i d a z o l e s , t r i a z o l e s , and m i s c e l l a n e o u s compounds i n the p y r i d i n e , m o r p h o l i n e and p i p e r a z i n e c l a s s e s . These compounds c o n t r o l a wide range of p l a n t d i s e a s e s i n c l u d i n g smuts, r u s t s , and powdery mildews (4). C u r r e n t l y , t h i s i s the l a r g e s t and most i m p o r t a n t group of s y s t e m i c compounds a v a i l a b l e f o r c o n t r o l l i n g f u n g a l d i s e a s e s i n p l a n t s and a n i m a l s . W h i l e t h e s e compounds a r e c o n v e n t i o n a l l y r e f e r r e d to as EBI f u n g i c i d e s , they can b l o c k s t e r o l b i o s y n t h e s i s i n organisms t h a t s y n t h e s i z e s t e r o l s other than e r g o s t e r o l (18). A l l o f the E B I ' s i n t e r f e r e w i t h s t e p s i n the b i o s y n t h e t i c pathway f o l l o w i n g the c y c l i z a t i o n of squalene. A r e c e n t l y developed c l a s s of a n t i f u n g a l a g e n t s f o r a n i m a l use the a l l y l a m i n e s , d e v e l o p e d by m o d i f i c a t i o n o f n a f t i f i n e , b l o c k the pathway p r i o r to c y c l i z a t i o n by i n h i b i t i n g squalene epoxidase (19). Most of the EBI f u n g i c i d e s a c t p r i m a r i l y by b l o c k i n g the cytochrome P-450 dependent s t e r o l C-14 d e m e t h y l a t i o n r e a c t i o n (18). A l t h o u g h t r i d e m o r p h produces e f f e c t s i n f u n g i s i m i l a r to t h o s e produced by compounds t h a t i n h i b i t s t e r o l C-14 d e m e t h y l a t i o n , i t does n o t i n t e r f e r e w i t h t h i s r e a c t i o n but i n h i b i t s the i s o m e r i z a t i o n o f the s t e r o l C-8(9) d o u b l e bond to C-7(8) (20) or the r e d u c t i o n of the s t e r o l C-14(15) d o u b l e bond ( 2 1 ) . I t i s i n t e r e s t i n g t h a t many s t r u c t u r a l l y d i v e r s e compounds a l l i n h i b i t s t e r o l C-14 d e m e t h y l a t i o n . However, w i t h the e x c e p t i o n o f t r i f o r i n e , t h e s e E B I * s have i n common a n i t r o g e n - c o n t a i n i n g h e t e r o c y c l e s u b s t i t u t e d w i t h one l a r g e l i p o p h i l i c g r o u p . I t has been proposed t h a t a n i t r o g e n atom o f the h e t e r o c y c l e i n t e r a c t s w i t h the protohaem i r o n atom of the cytochrome P-450 enzyme(s) i n v o l v e d i n s t e r o l C-14 d e m e t h y l a t i o n and t h a t the l i p o p h i l i c s u b s t i t u e n t i n c r e a s e s b i n d i n g a f f i n i t y through an i n t e r a c t i o n w i t h a nearby r e g i o n o f the enzyme (22, 2 3 ) . E v i d e n t l y , t h e r e can be a wide v a r i a t i o n i n the l i p o p h i l i c s u b s t i t u e n t ; however, v a r i a t i o n i n the s t r u c t u r e c o n t r o l s s p e c i f i c i t y as i n d i c a t e d by the f a c t t h a t compounds which e f f e c t i v e l y i n h i b i t f u n g a l s t e r o l demethylase systems may be r e l a t i v e l y i n e f f e c t i v e a g a i n s t the demethylase system from mammalian c e l l s ( 2 3 ) . The * * * * *

1

E B I s produce the f o l l o w i n g t y p i c a l s e c o n d a r y e f f e c t s ( 1 8 ) : No i n h i b i t i o n of i n i t i a l c e l l growth Morphological abnormalities No immediate e f f e c t on r e s p i r a t i o n No Immediate e f f e c t on RNA, DNA o r p r o t e i n s y n t h e s e s S t e r o l i n t e r m e d i a t e s accumulate F r e e f a t t y a c i d s accumulate The m o r p h o l o g i c a l a b n o r m a l i t i e s r e s u l t i n g from treatment w i t h t h e s e compounds a r e q u i t e s t r i k i n g ( 1 8 ) . S p o r i d i a of U s t i l a g o become m u l t i c e l l e d and o f t e n b r a n c h e d . G e r m i n a t i n g c o n i d i a o f some f u n g i show s w e l l i n g , l e a k a g e , and membrane r u p t u r e . In a d d i t i o n , e f f e c t s on membranes a p p a r e n t l y l e a d t o a b n o r m a l i t i e s i n s y n t h e s i s o f the c e l l w a l l as w e l l as t h a t of o t h e r c e l l u l a r components. Major l i p i d f r a c t i o n s o t h e r than s t e r o l s a r e not i n i t i a l l y inhibited. However, a d e l a y e d a c c u m u l a t i o n of f r e e f a t t y a c i d s β

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characteristically results after sterol inhibition. These are thought to r e s u l t from the breakdown of membranes and t r i g l y c e r i d e s t o g e t h e r w i t h a d i s p r o p o r t i o n a t e s y n t h e s i s and u t i l i z a t i o n o f f a t t y acids (18). Growth r e t a r d a t i o n i n h o s t p l a n t s has been a s s o c i a t e d w i t h the use of a number of t h e s e E B I ' s . T h i s e f f e c t r e s u l t s from the i n h i b i t i o n o f a r e a c t i o n i n the g i b b e r e l l i n b i o s y n t h e t i c pathway t h a t i n v o l v e s cytochrome P-450 enzymes ( 2 4 ) . A l t h o u g h the i n h i b i t i o n of e r g o s t e r o l b i o s y n t h e s i s i s r e g a r d e d as the p r i m a r y mode of a c t i o n , i t i s p o s s i b l e t h a t the s u c c e s s of t h e s e compounds as a n t i f u n g a l a g e n t s i s r e l a t e d to o t h e r e f f e c t s as well. Not a g r e a t d e a l i s known about f u n g a l hormones. A r e c e n t a r t i c l e c o n c e r n i n g human p a t h o g e n i c f u n g i i n d i c a t e d t h a t f u n g a l s t e r o i d s may be a s s o c i a t e d w i t h the p a t h o g e n i c i t y of t h e s e organisms (25). S i n c e the s t e r o i d s a r e s y n t h e s i z e d from s t e r o l p r e c u r s o r s , an i n h i b i t i o n of s t e r o i d b i o s y n t h e s i s i n a d d i t i o n to the e f f e c t s on p r e c u r s o r s t e r o l s may be r e s p o n s i b l e f o r the e f f e c t i v e n e s s of E B I s i n c o n t r o l l i n g plant pathogenic f u n g i . 1

R e s i s t a n c e has n o t thus f a r been a s e r i o u s problem f o r the EBI's. A l t h o u g h numerous r e s i s t a n t mutants have been d e v e l o p e d i n l a b o r a t o r y s t u d i e s , r e s i s t a n c e problems i n the f i e l d have not occurred. However, r e c e n t i n v e s t i g a t i o n s r e l a t e d t o the use of a t r i a z o l e f u n g i c i d e on b a r l e y i n d i c a t e t h a t r e s i s t a n t s t r a i n s o f p a t h o g e n i c f u n g i may be i n c r e a s i n g ( 2 6 ) . The f a c t t h a t t h e r e have not been major problems may be a s s o c i a t e d w i t h the n a t u r e of the mode of a c t i o n and the f a c t t h a t mutants appear to have r e d u c e d f i t n e s s (11, 1 8 ) . In o t h e r words, the g e n e t i c a l t e r a t i o n s c o n d u c i v e to s u r v i v a l i n the p r e s e n c e of t h e s e compounds may a l s o reduce f i t n e s s . T h i r d Generation

Fungicides

The t h i r d g e n e r a t i o n f u n g i c i d e s c o n s i s t of c h e m i c a l s d e s i g n e d t o take advantage of p r i n c i p l e s u n d e r l y i n g host and/or pathogen vulnerabilities. These compounds t y p i c a l l y a r e h i g h l y s p e c i f i c , o f t e n t a r g e t e d f o r a s p e c i f i c d i s e a s e on a s e l e c t e d h o s t . They c h a r a c t e r i s t i c a l l y show l i t t l e or no t o x i c i t y to f u n g i i n v i t r o . They a c t as a n t i p a t h o g e n i c agents and thus a f f e c t the p r o c e s s of p a t h o g e n e s i s . They may a c t on the h o s t t h r o u g h the i n d u c t i o n o f p l a n t r e s i s t a n c e mechanisms such as s t i m u l a t i o n of l i g n i f i c a t i o n o r enhancement o f p h y t o a l e x i n p r o d u c t i o n . ( P l e a s e r e f e r to the c h a p t e r by S a l t and Kuc i n t h i s volume f o r f u r t h e r d i s c u s s i o n of t h i s t y p e o f compound.) They may a c t on the pathogen to a c c e n t u a t e e l i c i t o r r e l e a s e or to p r e v e n t i n f e c t i o n ( h o s t p e n e t r a t i o n ) , c o l o n i z a t i o n ( i n h i b i t i o n o f p h y t o t o x i n s y n t h e s i s , e x t r a c e l l u l a r enzyme p r o d u c t i o n and a c t i o n , or p h y t o a l e x i n d e g r a d a t i o n ) or r e p r o d u c t i o n . M e l a n i n B i o s y n t h e s i s I n h i b i t o r s . The m e l a n i n b i o s y n t h e s i s i n h i b i t o r s , which a r e used to c o n t r o l r i c e b l a s t d i s e a s e , a r e a good example o f compounds t h a t p r e v e n t i n f e c t i o n . I n c l u d e d i n t h i s group a r e t r i c y c l a z o l e , p y r o q u i l o n , f t h a l i d e , and chlorbenthiazone. T r i c y c l a z o l e , w h i c h was the f i r s t of t h e s e compounds i d e n t i f i e d as b e i n g e f f e c t i v e t h r o u g h the i n h i b i t i o n of m e l a n i n s y n t h e s i s ( 2 7 ) , produces no e f f e c t s on f u n g a l growth a t c o n c e n t r a t i o n s o f the chemical that are e f f e c t i v e f o r disease c o n t r o l . Treated fungi

3.

RAGSDALE AND SIEGEL

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appear brown r a t h e r than g r a y i s h - b l a c k . T r i c y c l a z o l e blocks the c o n v e r s i o n o f 1 , 3 , 8 - t r i h y d r o x y n a p h t h a l e n e t o vermelone and t h e conversion o f 1,3,6,8-tetrahydroxynaphthalene to scytalone i n the p o k l y k e t i d e pathway o f m e l a n i n b i o s y n t h e s i s ( 2 8 ) . The f a i l u r e t o s y n t h e s i z e m e l a n i n was l a t e r t i e d t o f a i l u r e o f t h e fungus t o penetrate the host epidermis (29). A p p a r e n t l y m e l a n i n o r an o x i d i z e d melanin precursor i s i n v o l v e d i n the f u n g a l c e l l w a l l a r c h i t e c t u r e i n s u c h a way as t o p r o v i d e t h e r i g i d i t y n e c e s s a r y f o r h o s t p e n e t r a t i o n (4). Compounds t h a t a f f e c t H o s t R e a c t i o n s . Examples o f compounds w h i c h enhance o r i n d u c e h o s t r e a c t i o n s t o pathogens i n c l u d e 2 , 2 - d i c h l o r o - 3 , 3 - d i m e t h y l - c y c l o p r o p a n e c a r b o x y l i c a c i d (DDCC), p r o b e n a z o l e , and f o s e t y l - A l ( 4 ) . A l t h o u g h t h e s e c h e m i c a l s do n o t s t o p t h e fungus from p e n e t r a t i n g t h e p l a n t , they a r e q u i t e e f f e c t i v e a t p r e v e n t i n g c o l o n i z a t i o n through the enhancement o f the h o s t ' s r e s i s t a n c e mechanisms. F u r t h e r s t u d i e s a r e needed t o e l u c i d a t e how t h e s e r e s i s t a n c e mechanisms a r e t r i g g e r e d . Resistance

Mechanisms

W i t h t h e use o f second g e n e r a t i o n a n t i f u n g a l c h e m i c a l s t h e r e has been an i n c r e a s i n g awareness o f t h e need t o u n d e r s t a n d n o t o n l y t h e modes o f a c t i o n b u t a l s o t h e mechanisms o f f u n g a l r e s i s t a n c e . I t has l o n g been r e c o g n i z e d t h a t p l a n t s may be bred f o r two t y p e s o f d i s e a s e r e s i s t a n c e , r a c e s p e c i f i c and g e n e r a l . Highly s p e c i f i c disease r e s i s t a n c e i s very e f f e c t i v e f o r a l i m i t e d time. U s u a l l y the s e l e c t i o n p r e s s u r e on t h e d i s e a s e - p r o d u c i n g organism i s such t h a t a new s t r a i n , which c i r c u m v e n t s the h o s t ' s r e s i s t a n c e , emerges. Plants w i t h moderate r e s i s t a n c e g e n e r a l l y r e t a i n t h i s p r o p e r t y f o r l o n g periods. A p a r a l l e l e x i s t s with fungicides (30). Non-specific t y p e s , such as t h e p r e v i o u s l y m e n t i o n e d f i r s t g e n e r a t i o n f u n g i c i d e s , have been e f f e c t i v e f o r many y e a r s whereas the s i t e s p e c i f i c second and t h i r d g e n e r a t i o n compounds a r e more l i k e l y t o e n c o u n t e r r e s i s t a n c e problems. Among t h e b i o c h e m i c a l mechanisms o f f u n g i c i d e r e s i s t a n c e a r e reduced p e r m e a b i l i t y , metabolism ( i n c r e a s e d d e t o x i f i c a t i o n or d e c r e a s e d c o n v e r s i o n t o t h e t o x i c m a t e r i a l ) , and r e d u c e d a f f i n i t y o f the t a r g e t s i t e f o r t h e t o x i n . E f f o r t s a r e now b e i n g made t o a v o i d r e s i s t a n c e problems t h r o u g h s t r a t e g i e s o f u s e i n c o n j u n c t i o n w i t h knowledge on modes o f a c t i o n and mechanisms o f r e s i s t a n c e . A p p l i c a t i o n s t r a t e g i e s i n c l u d e t i m i n g of u s e , r e d u c t i o n o f u s e , c o m b i n a t i o n s o f p r o t e c t a n t s and e r a d i c a n t s t h a t have n o n - s p e c i f i c and s p e c i f i c modes o f a c t i o n , and c o m b i n a t i o n s of e r a d i c a n t s w i t h d i f f e r e n t s p e c i f i c modes o f a c t i o n . Knowledge on modes o f a c t i o n and r e s i s t a n c e mechanisms o f f e r some approaches t h a t can be d e v e l o p e d t o c i r c u m v e n t r e s i s t a n c e p r o b l e m s . Many o f t h e s e i d e a s were d i s c u s s e d a t a r e c e n t symposium a d d r e s s i n g novel approaches f o r r e s e a r c h on a g r i c u l t u r a l c h e m i c a l s ( 3 1 ) . S e l e c t i v e A c t i o n . E v i d e n c e s u g g e s t s t h a t a t t e n t i o n s h o u l d be g i v e n to t h e development and use o f c h e m i c a l s w i t h modes o f a c t i o n t h a t s e l e c t f o r r e s i s t a n t s t r a i n s which do n o t cause d i s e a s e p r o b l e m s . I n the s e c t i o n on E B I f u n g i c i d e s , i t was p o i n t e d o u t t h a t r e s i s t a n t

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mutants have been d e v e l o p e d i n the l a b o r a t o r y , but t h a t t h e r e have been no c a s e s of d i s e a s e c o n t r o l f a i l u r e a t t r i b u t e d to r e s i s t a n c e i n the f i e l d . Perhaps t h i s p a r t i c u l a r mechanism i s s u c h t h a t m u t a t i o n s to overcome i t impart c h a r a c t e r i s t i c s i n a fungus t h a t reduce pathogenicity (18). C i r c u m v e n t i o n o f S i t e M o d i f i c a t i o n . As i n d i c a t e d e a r l i e r , c a r b o x i n i n h i b i t s s u c c i n i c dehydrogenase a c t i v i t y . In s t u d i e s u s i n g r e s i s t a n t l a b o r a t o r y mutants and a l a r g e number of a n a l o g u e s o f the p a r e n t compound, d a t a i n d i c a t e d t h a t f o r any m u t a t i o n i n the l o c i a f f e c t i n g c a r b o x i n s e n s i t i v i t y , a s p e c i f i c s t r u c t u r a l change i n the p a r e n t compound c o u l d a l l e v i a t e or r e v e r s e the f a i l u r e to c o n t r o l the fungus (32). In s c r e e n i n g s t u d i e s , a number of a n a l o g u e s were i d e n t i f i e d t h a t showed i n v i t r o b i n d i n g a f f i n i t y f o r s u c c i n i c d e h y d r o g e n a s e - m i t o c h o n d r i a l complex I I . Some a n a l o g u e s were e f f e c t i v e a g a i n s t w i l d type and some a g a i n s t r e s i s t a n t mutants. A l o g i c a l s t r a t e g y would be to use m i x t u r e s of t h e s e c h e m i c a l s o r t o use them s e p a r a t e l y i n an a l t e r n a t i n g s c h e d u l e . U n f o r t u n a t e l y , this scheme would not work i n t h i s p a r t i c u l a r case because most of the a c t i v e a n a l o g u e s were not s y s t e m i c . However, m o d i f i c a t i o n of the c h e m i c a l s t r u c t u r e i s one a p p r o a c h to c o n t r o l the development o f resistance. C o n s i d e r a t i o n of A c t i v a t i o n and S t e r e o s e l e c t i v i t y . S t u d i e s on t r i a d i m e f o n , one of the EBI compounds, p r o v i d e thoughts to c o n s i d e r i n d e v e l o p i n g new compounds as w e l l as a d d r e s s i n g r e s i s t a n c e problems. In e v a l u a t i n g the e f f e c t i v e n e s s of t r i a d i m e f o n a p p l i e d t o p l a n t s f o r p e s t c o n t r o l , the f o l l o w i n g must be t a k e n i n t o c o n s i d e r a t i o n (33): E x t e n t of c o n v e r s i o n to t r i a d i m e n o l by h o s t and pathogen I n h e r e n t a c t i v i t i e s of the c h e m i c a l p r e s e n t (stereoselectivity) * Time r e q u i r e d f o r an e f f e c t i v e dose to r e a c h the s p e c i f i c i n t r a c e l l u l a r s i t e of a c t i o n * F u r t h e r m e t a b o l i s m of the c h e m i c a l by the pathogen or h o s t p l a n t to l e s s t o x i c compounds W h i l e a l l o f t h e s e f a c t o r s must be t a k e n i n t o a c c o u n t , the l a t t e r two a r e dependent on the f i r s t two, and we w i l l f o c u s on the f i r s t two as p o i n t s of c o n s i d e r a t i o n f o r r e s i s t a n c e as w e l l as s t r a t e g i e s f o r the f u t u r e e f f e c t i v e use of t h i s compound. F u n g i s e n s i t i v e to t r i a d i m e f o n c o n v e r t the p a r e n t compound t h r o u g h b i o c h e m i c a l r e d u c t i o n to t r i a d i m e n o l a t a h i g h r a t e , whereas r e s i s t a n t s p e c i e s d e m o n s t r a t e l i t t l e or no c o n v e r s i o n ( 3 4 ) . This i n v e s t i g a t i o n a l s o demonstrated t h a t p l a n t s enhance f u n g i t o x i c i t y by this conversion. Thus an a c t i v a t i o n p r o c e s s i n b o t h pathogen and h o s t c o u l d p o s s i b l y be a l t e r e d to p o t e n t i a t e e f f e c t i v e n e s s . To add t o t h i s somewhat complex s i t u a t i o n , two d i a s t e r e o i s o m e r s of t r i a d i m e n o l a r e formed i n the r e d u c t i o n p r o c e s s , and one i s more a c t i v e than the o t h e r ( 3 5 ) . A h i g h r a t e of c o n v e r s i o n to t r i a d i m e n o l i n a d d i t i o n to a h i g h r a t i o of the more a c t i v e d i a s t e r e o m e r to the l e s s a c t i v e can be assumed t o be p r e s e n t i n t r i a d i m e f o n - s e n s i t i v e f u n g i whereas the o p p o s i t e s i t u a t i o n may be found i n r e l a t i v e l y i n s e n s i t i v e f u n g i . The c o n v e r s i o n p r o c e s s i n p l a n t s a f f o r d s the o p p o r t u n i t y to c o n t r o l i n s e n s i t i v e f u n g i which l a c k the c o n v e r s i o n mechanism· β

*

3.

R AGSDALE AND

SIEGEL

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A number of f a c t o r s must be c o n s i d e r e d i n the a r e a of s t e r e o s e l e c t i v i t y (31). D i s t i n c t d i f f e r e n c e s a r e n o t e d between s t e r e o i s o m e r s i n a b s o r p t i o n / uptake, a c t i v i t y a t the t a r g e t or r e c e p t o r s i t e , pathways and r a t e of b i o c o n v e r s i o n , and p h a r m a c o k i n e t i c s . A l l o f t h e s e f a c t o r s a r e a p p l i c a b l e i n the c a s e of t r i a d i m e f o n and a r e examples of p r o c e s s e s t h a t may be m a n i p u l a t e d to enhance d i s e a s e c o n t r o l e f f e c t i v e n e s s . I n h i b i t i o n o f a R e s i s t a n c e Mechanism. Another i n t e r e s t i n g c a s e s e r v e s as an example of the importance of u n d e r s t a n d i n g r e s i s t a n c e mechanisms. I n t h i s s i t u a t i o n r e s i s t a n c e i s not r e l a t e d to the mode of a c t i o n . O b s e r v a t i o n s i n d i c a t e t h a t i n a w i l d type s t r a i n o f A s p e r g i l l u s n i d u l a n s , u p t a k e of the EBI f u n g i c i d e , f e n a r i m o l , i s a r a p i d , p a s s i v e i n f l u x w h i c h i n d u c e s an energy dependent e f f l u x r e s u l t i n g i n an energy dependent s t a t e of e q u i l i b r i u m ( 3 6 ) . Toxic a c t i o n o c c u r s p r i o r to s u f f i c i e n t e f f l u x to r e d u c e l e v e l s below toxic concentrations. T h i s same s t u d y found t h a t the e f f l u x system i n r e s i s t a n t s t r a i n s i s c o n s t i t u t i v e r e s u l t i n g i n a lower i n i t i a l uptake of the f u n g i c i d e . Other i n v e s t i g a t i o n s show t h a t a s i m i l a r mechanism i s i n v o l v e d i n n i d u l a n s s t r a i n s r e s i s t a n t to i m a z a l i l , a n o t h e r EBI f u n g i c i d e ( 3 7 ) . E f f l u x i s b l o c k e d by compounds such as r e s p i r a t o r y i n h i b i t o r s t h a t d e c r e a s e l e v e l s of ATP. A p p a r e n t l y the i n f l u x - e f f l u x mechanism i s dependent on i n t r a c e l l u l a r ATP, which i s u t i l i z e d by the plasma membrane ATPase and thus d r i v e s the e f f l u x p r o c e s s ( 3 6 ) . The s y n e r g i s t i c e f f e c t s of r e s p i r a t o r y i n h i b i t o r s c o u l d s e r v e as the b a s i s f o r u s i n g a c o m b i n a t i o n of i n h i b i t o r s . T h i s a l s o b r i n g s up the p o s s i b i l i t y t h a t t h r o u g h the use of r e s p i r a t o r y i n h i b i t o r s , f u n g i t h a t a r e n o r m a l l y i n s e n s i t i v e or o n l y s l i g h t l y s e n s i t i v e to f e n a r i m o l o r i m a z a l i l due t o e f f l u x systems c o u l d be c o n t r o l l e d . Conclusions New approaches f o r the c h e m i c a l c o n t r o l o f p l a n t pathogens emphasize the need to u n d e r s t a n d the p h y s i o l o g i c a l , b i o c h e m i c a l , and m o l e c u l a r l e v e l s of the modes o f a c t i o n and the mechanisms o f pathogen resistance. These approaches i n v o l v e the use of c h e m i c a l s i n a way t h a t w i l l r e d u c e or p r e v e n t the development o f r e s i s t a n t p a t h o g e n s . They a l s o take advantage of compounds t h a t s e r v e as antipathogenic a g e n t s to p r e v e n t d i s e a s e development, an a p p r o a c h w h i c h r e q u i r e s an u n d e r s t a n d i n g of h o s t - p a r a s i t e p h y s i o l o g y and p a t h o g e n i c p r o c e s s e s . The a n t i p a t h o g e n i c a g e n t s are h i g h l y s p e c i f i c and u s u a l l y t a r g e t e d f o r a d e s i g n a t e d pathogen on a g i v e n p l a n t . In l o o k i n g t o the f u t u r e , one must be p r a c t i c a l and take i n t o a c c o u n t the f a c t t h a t s u c h c h e m i c a l s a r e l i k e l y to be c o m m e r c i a l l y a v a i l a b l e o n l y f o r major d i s e a s e s on major c r o p s . The c u r r e n t c o s t s of development, r e g i s t r a t i o n , and m a r k e t i n g put r e s t r i c t i o n s on w i d e s p r e a d use o f t h e s e t h i r d g e n e r a t i o n f u n g i c i d e s . Thus, one must take advantage o f f i r s t and second g e n e r a t i o n type compounds. The i n f o r m a t i o n we have p r e s e n t e d on t h e s e m a t e r i a l s i n d i c a t e s t h a t a r e a s o n a b l e l e v e l o f knowledge e x i s t s on how they work. We have seen examples of the b r o a d range o f c e l l u l a r p r o c e s s e s t h a t second g e n e r a t i o n f u n g i c i d e s affect. A d d i t i o n a l d a t a w i l l u n d o u b t e d l y enhance t h i s i n f o r m a t i o n b a s e . New approaches w i l l take advantage of e x i s t i n g knowledge and

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emphasize the Importance of additional basic information on the mechanisms of action and resistance as well as host-pathogen relationships. Literature Cited 1. 2.

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

Sisler, H. D.; Ragsdale, N.N. in "Handbook of Pest Management in Agriculture"; Pimental, D., Ed.; CRC Press: Boca Raton, Florida, 1981; pp. 17-47. Siegel, M. R. in "Pesticide Selectivity"; Street, J . C . , E d . ; Marcel Dekker: New York, 1975; pp. 21-46. Edgington, L. V . ; Martin, R. Α.; Bruin, G. C . ; Parsons, I.M. Plant D i s . 1980, 64, 19-23. Sisler, H. D.; Ragsdale, Ν. N. in "Agricultural Chemicals of the Future"; Hilton, J . L., Ed.; Rowman and Allanheld Publishers: Totown, New Jersey, in press. Sisler, H. D. Annu. Rev. Phytopathology. 1969, 7, 311-30. Dekker, J. in "Fungicides", Vol. 2; Torgeson, D. C . , E d . ; Academic Press: New York, 1969; pp. 580-635. Siegel, M. R. in "Antifungal Compounds", Vol. 2; Siegel, M. R.; Sisler, H. D. , Eds.; Marcel Dekker: New York, 1977; pp. 399-438. Davidse, L. C. in "Fungicide Resistance in Crop Protection"; Dekker, J.; Georgopoulos, S. G . , Eds.; Pudoc: Wageningen, The Netherlands, 1982; pp. 60-70. Hammerschlag, R. S.; Sisler, H. D. Pestic. Biochem. Physiol. 1973, 3, 42-54. Howard, R. J.; Aist, J. R. Protoplasma 1977, 92, 195-210. Davidse, L. C.; deWaard, M. A. Adv. Plant Pathology. 1984, 2, 191-257. Mathre, D. E . Pestic. Biochem. Physiol. 1971, 1, 216-24. White, G. A. Biochem. Biophys. Res. Commun. 1971, 44, 1210-12. Misato, T . ; Kakiki, K. in "Antifungal Compounds", Vol. 2; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker: New York, 1977; pp. 277-300. Ragsdale, Ν. N. in "Antifungal Compounds, Vol. 2; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker; New York, 1977; pp. 333-63. Kaars Sypesteijn, A. in "Fungicide Resistance in Crop Protection"; Dekker, J.; Georgopoulos, S. G . , Eds.; Pudoc: Wageningen, The Netherlands, 1982; pp. 32-45. Ragsdale, Ν. N.; Sisler, H. D. Biochem. Biophys. Res. Commun. 1972, 46, 2048-53. Sisler, H. D.; Ragsdale, Ν. N. in "Mode of Action of Antifungal Agents"; Trinci, A. P . J . ; Ryley, J . F . , Eds.; Cambridge University Press: London, 1984; pp. 257-82. Petranyi, G.; Ryder, N. S.; Stütz, A. Science 1984, 224, 1239-41. Kato, T . ; Shoami, M.; Kawase, Y. J. Pestic. Sci. 1980, 5, 69-79. Kerkenaar, Α.; Uchiyama, M.; Versluis, G. G. Pestic. Biochem. Physiol. 1981, 16, 97-104. Henry, M. J.; Sisler, H. D. Pestic. Biochem. Physiol. 1984, 22, 262-75. Gadher, P.; Mercer, Ε. I . ; Baldwin, B. C . ; Wiggins, T. E . Pestic. Biochem. Physiol. 1983, 19, 1-10.

3. RAGSDALE AND SIEGEL 24. 25. 26. 27. 28. 29. 30.

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Coolbaugh, R. C.; Hirano, S. S.; West, C. A. Plant Physiol. 1978, 62, 571-6. Kolata, G. Science 1984, 225. 913-4. Wolfe, M. S. personal communication. Chrysayi Tokousbalides, M.; Sisler, H. D. Pestic. Biochem. Physiol. 1978, 8, 26-32. Chrysayi Tokousbalides, M.; Sisler, H. D. Pestic. Biochem. Physiol. 1979, 11, 64-73. Woloshuk, C. P.; Sisler, H. D. J . Pestic. Sci. 1982, 7, 161-6. Georgopoulos, S. G. in "Antifungal Compounds", Vol. 2; Siegel, M. R.; Sisler, H. D., Eds.; Marcel Dekker: New York, 1977; pp. 439-84. Fuchs, Α.; Davidse, L . C . ; deWaard, Μ. Α.; deWit, P. J . G. M. Pestic. Sci. 1983, 14, 272-93. White, G. Α.; Thorn, G. D. Pestic. Biochem. Physiol. 1980, 14, 26-40. Deas, Α. H.B.; Clifford, D. R. Pestic. Biochem. Physiol. 1982, 17, 120-33. Gasztonyi, M.; Josepovits, G. Pestic. Sci. 1979, 10, 57-65. Buchenauer, H. in "Abstracts of Papers 1X International Congress of Plant Protection", 1979, No. 939. DeWaard, Μ. Α.; van Nistelrooy, J . G. M. Pestic. Biochem. Physiol. 1980, 13, 255-66 Siegel, M. R.; Solel, J . Pestic. Biochem. Physiol. 1981, 15, 222-33.

RECEIVED January 15, 1985