Bioregulation of Preharvest Aflatoxin Contamination of Peanuts - ACS

Chapter 24

Bioregulation of Preharvest Aflatoxin Contamination of Peanuts Role of Stilbene Phytoalexins 1

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Joe W. Dorner , Richard J. Cole , Boris Yagen , and Benedikte Christiansen Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch024

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National Peanut Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1011 Forrester Drive, SE, Dawson, G A 31742 School of Pharmacy, Hebrew University, Jerusalem, Israel Department of Biotechnology, Instituttet for Bioteknologi, Lyngby, Denmark 2

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A r i c h and varied assortment of phytoalexins i s produced by legumes. Peanuts (Arachis hypogaea L . ) have been shown to produce various stilbene phytoalexins in response to fungal i n f e c t i o n . Recent studies have been conducted to elucidate the role of these stilbenes i n the bioregulation of preharvest aflatoxin contamination of peanuts by Aspergillus flavus and A. parasiticus. Stilbene phytoalexins appear to provide s o i l borne peanut seed with a degree of resistance to aflatoxin contamination by i n h i b i t i n g fungal growth. Under conditions of prolonged, late-season drought stress that lead to aflatoxin contamination, phytoalexin biosynthesis breaks down, presumably due to dehydration of the peanut seed. However, seed moisture under such conditions remains adequate to support A. flavus growth and aflatoxin production. The chemistry and b i o l o g i c a l activity of stilbene phytoalexins as related to preharvest aflatoxin contamination of peanuts is described. P h y t o a l e x i n s , a n t i m i c r o b i a l s u b s t a n c e s p r o d u c e d by p l a n t s i n r e s p o n s e t o i n f e c t i o n o r s t r e s s , a r e important i n the n a t u r a l defense of plants against disease. Phytoalexins a r e extremely diverse c h e m i c a l l y , and a l t h o u g h t h e i r u b i q u i t y t h r o u g h o u t t h e p l a n t kingdom i s open t o q u e s t i o n , c e r t a i n p l a n t f a m i l i e s have been found t o c o n t a i n many s p e c i e s c a p a b l e o f p h y t o a l e x i n p r o d u c t i o n ( 1 ) . Among t h o s e i s t h e Leguminosae, an e c o n o m i c a l l y i m p o r t a n t f a m i l y o f p l a n t s t h a t i n c l u d e s t h e peanut (Arachis hypogaea L . ) ( 2 ) . Peanuts a r e grown i n many a r e a s o f t h e w o r l d and have a h i g h nutritive value. However, peanuts are subject to aflatoxin c o n t a m i n a t i o n under c e r t a i n e n v i r o n m e n t a l c o n d i t i o n s , and when t h i s happens, i t n u l l i f i e s t h e i r u s e f u l n e s s a s f o o d o r f e e d ( 3 ) . I n 1972 i t was r e p o r t e d t h a t peanut k e r n e l s c a n s y n t h e s i z e p h y t o a l e x i n s i n r e s p o n s e t o f u n g a l c h a l l e n g e ( 4 ) , and s u b s e q u e n t l y s e v e r a l o f these phytoalexins were c h e m i c a l l y c h a r a c t e r i z e d a s s t i l b e n e s (5-8). S i n c e t h a t time, s t u d i e s have shown t h a t t h e s e compounds p o s s e s s b i o l o g i c a l activity against fungi, including Aspergillus flavus , one o f t h e s p e c i e s t h a t p r o d u c e s a f l a t o x i n ( 9 ) . Thus, i t has been s p e c u l a t e d t h a t s t i l b e n e p h y t o a l e x i n s might be important i n t h e n a t u r a l d e f e n s e o f peanuts a g a i n s t aflatoxinproducing fungi.

This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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The p u r p o s e s o f t h i s c h a p t e r a r e t o r e v i e w t h e f a c t o r s i n v o l v e d i n a f l a t o x i n c o n t a m i n a t i o n o f peanuts, review the chemistry of s t i l b e n e p h y t o a l e x i n s from peanuts, d i s c u s s e v i d e n c e s u p p o r t i n g t h e involvement of these s t i l b e n e s i n the b i o r e g u l a t i o n of a f l a t o x i n c o n t a m i n a t i o n , and e x p l o r e approaches t o e x p l o i t o r enhance s u c h a b i o r e g u l a t i v e c a p a c i t y t o reduce or e l i m i n a t e p r e h a r v e s t a f l a t o x i n contamination of peanuts.

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Aflatoxin A f l a t o x i n s a r e s e c o n d a r y f u n g a l m e t a b o l i t e s p r o d u c e d by A. flavus L i n k and A. parasiticus Speare and a r e r e c o g n i z e d f o r t h e i r p o t e n t h e p a t o t o x i c , t e r a t o g e n i c , and c a r c i n o g e n i c e f f e c t s i n some a n i m a l s (10). The f o u r n a t u r a l l y o c c u r r i n g a f l a t o x i n s a r e d e s i g n a t e d B B, G and G~. O t h e r known a f l a t o x i n s a r e c h e m i c a l o r b i o l o g i c a l p r o d u c t s or t h e f o u r n a t u r a l l y o c c u r r i n g compounds. Under c e r t a i n e n v i r o n m e n t a l c o n d i t i o n s t h e a f l a t o x i n - p r o d u c i n g f u n g i can i n v a d e v a r i o u s a g r i c u l t u r a l commodities, and subsequent p r o l i f e r a t i o n by these fungi contaminates the commodity with aflatoxin. The commodities most a f f e c t e d i n t h e U n i t e d S t a t e s a r e p e a n u t s , c o r n , and cottonseed. S t r i c t r e g u l a t o r y a c t i o n l e v e l s f o r a f l a t o x i n must be met f o r t h e s e commodities t o be used as food or feed (10). T h e r e f o r e , s e v e r e economic l o s s e s can o c c u r when t h e s e commodities become c o n t a m i n a t e d and a r e d i v e r t e d from e d i b l e markets. 1#

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Preharvest A f l a t o x i n Contamination

o f Peanuts

C o n t a m i n a t i o n o f peanuts w i t h a f l a t o x i n can o c c u r d u r i n g v a r i o u s phases of production, storage, handling, and marketing (11). C o n t a m i n a t i o n t h a t o c c u r s a f t e r peanuts a r e h a r v e s t e d i s p r e v e n t a b l e i f p r o p e r s t e p s a r e t a k e n t o e n s u r e t h a t t h e m o i s t u r e o f peanuts i s maintained at a l e v e l that i s u n f a v o r a b l e f o r growth o f the a f l a t o x i n - p r o d u c i n g f u n g i . However, c o n t a m i n a t i o n t h a t o c c u r s i n t h e f i e l d p r i o r t o h a r v e s t ( p r e h a r v e s t ) i s much more d i f f i c u l t t o c o n t r o l and u s u a l l y r e s u l t s i n t h e most s e v e r e a f l a t o x i n c o n t a m i n a t i o n o f peanuts. P r e h a r v e s t a f l a t o x i n c o n t a m i n a t i o n o f peanuts o c c u r s when peanuts a r e s u b j e c t e d t o severe, prolonged drought s t r e s s d u r i n g the l a s t f o u r t o s i x weeks o f t h e growing season (12-17) . E l e v a t e d s o i l temperatures t h a t u s u a l l y accompany such p e r i o d s o f l a t e - s e a s o n d r o u g h t e x a c e r b a t e t h e problem and produce optimum c o n d i t i o n s f o r p r e h a r v e s t c o n t a m i n a t i o n (12, 1 8 ) . When c o n t a m i n a t i o n o c c u r s , i t i s n o t homogeneous t h r o u g h o u t a p o p u l a t i o n o f p e a n u t s . Immature peanuts a r e more l i k e l y t o be c o n t a m i n a t e d t h a n mature p e a n u t s , and k e r n e l s t h a t a r e damaged, p a r t i c u l a r l y by i n s e c t s , can c o n t a i n e x t r e m e l y h i g h c o n c e n t r a t i o n s o f a f l a t o x i n (13, 15, 18-20). I n v a s i o n o f peanuts by the a f l a t o x i g e n i c f u n g i does n o t n e c e s s a r i l y r e s u l t i n t h e i r b e i n g contaminated w i t h a f l a t o x i n . In t h e absence o f d r o u g h t s t r e s s , samples o f peanuts have been shown t o be c o l o n i z e d by A. flavus/parasiticus a t p e r c e n t a g e s as h i g h as 25% of t h e k e r n e l s w i t h o u t d e t e c t a b l e l e v e l s o f a f l a t o x i n (18, 19). T h i s i n d i c a t e s t h a t i n t h e absence o f d r o u g h t c o n d i t i o n s t h e f u n g i a r e a b l e t o i n v a d e peanuts, but t h e peanuts a r e p r o t e c t e d from e x t e n s i v e f u n g a l p r o l i f e r a t i o n by some i n h e r e n t d e f e n s e mechanism(s) . However, when exposed t o t h e s t r e s s e s o f d r o u g h t and h e a t , a breakdown i n t h i s d e f e n s e mechanism(s) a l l o w s f o r f u n g a l p r o l i f e r a t i o n and subsequent a f l a t o x i n contamination. S t i l b e n e P h y t o a l e x i n s from

Peanuts

The f i r s t r e p o r t o f p h y t o a l e x i n p r o d u c t i o n by peanuts appeared i n 1972 when V i d h y a s e k a r a n et al. found t h a t s e v e r a l s p e c i e s o f f u n g i , i n c l u d i n g A. flavus, i n d u c e d p r o d u c t i o n o f an i n h i b i t o r y p r i n c i p l e by

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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peanut pods (4). A l t h o u g h t h e p r i n c i p l e was n o t c h e m i c a l l y c h a r a c t e r i z e d , i t was deemed t o be a p h y t o a l e x i n because i t was p r o d u c e d by t h e peanut o n l y a f t e r i n t e r a c t i o n between t h e f u n g i and the peanut. I n t h a t s t u d y , immature p e a n u t s p r o d u c e d a h i g h e r q u a n t i t y o f t h e p h y t o a l e x i n t h a n mature p e a n u t s , and t h e a u t h o r s s u g g e s t e d t h a t r e s i s t a n c e o f immature peanut pods t o f u n g i was b a s e d on t h e i r c a p a c i t y t o p r o d u c e p h y t o a l e x i n s i n r e s p o n s e t o i n f e c t i o n . I n 1975 Keen (21) r e p o r t e d t h a t n a t i v e m i c r o f l o r a s t i m u l a t e d p r o d u c t i o n o f two a n t i f u n g a l compounds by peanut seeds t h a t were soaked i n water, s l i c e d i n t o s e c t i o n s , and i n c u b a t e d f o r 3-5 d a y s . These compounds were judged t o be p h y t o a l e x i n s and were s u b s e q u e n t l y i d e n t i f i e d a s c i s - and t r a n s - i s o m e r s o f 4 - ( 3 - m e t h y l - b u t - 2 - e n y l ) 3 , 5 , 4 ' - t r i h y d r o x y s t i l b e n e [1](5)(Figure 1). S i m u l t a n e o u s l y , Ingham (6) r e p o r t e d t h e i s o l a t i o n o f c i s - and t r a n s - r e s v e r a t r o l ( 3 , 5 , 4 t r i h y d r o x y s t i l b e n e ^ ] ) from peanut h y p o c o t y l s . Additional stilbenes have been shown t o be p r o d u c e d by peanut seeds i n r e s p o n s e t o wounding, and t h e s e i n c l u d e 4 - ( 3 - m e t h y l - b u t - l - e n y l ) - 3 , 5 , 3 , 4 ' - t e t r a hydroxystilbene [3](7), 4-(3-methyl-but-l-enyl)-3,5,4'-trihydroxystilbene [4](7), and 3 - i s o p e n t a d i e n y l - 4 , 3 ' , 5 - t r i h y d r o x y s t i l b e n e [5](8).


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F i g u r e 1. C h e m i c a l s t r u c t u r e s o f s t i l b e n e p h y t o a l e x i n s from p e a n u t s .

To stimulate peanut seeds t o produce phytoalexins f o r l a b o r a t o r y s t u d i e s , t h e d r y seeds a r e t y p i c a l l y a l l o w e d t o imbibe water f o r a p p r o x i m a t e l y 24 h o u r s . The seeds a r e t h e n s l i c e d i n t o 1-3 mm s e c t i o n s o r g e n t l y chopped t o cause e x t e n s i v e c e l l u l a r damage, and t h e s e e d s a r e e i t h e r i n o c u l a t e d w i t h a fungus and i n c u b a t e d i n t h e d a r k f o r s e v e r a l days o r i n c u b a t e d u s i n g t h e n a t i v e peanut m i c r o f l o r a t o i n d u c e p h y t o a l e x i n p r o d u c t i o n (2, 2 1 , 2 2 ) . The s t i l b e n e s a r e t y p i c a l l y e x t r a c t e d from p e a n u t s w i t h 95% e t h a n o l , and f o l l o w i n g p a r t i a l p u r i f i c a t i o n t h e samples a r e s u b j e c t e d t o e i t h e r t h i n - l a y e r c h r o m a t o g r a p h i c (TLC) o r l i q u i d c h r o m a t o g r a p h i c (LC) a n a l y s e s . On TLC p l a t e s , t h e s t i l b e n e s f l u o r e s c e b l u e under 254 nm UV l i g h t o r t h e y c a n be d e t e c t e d i n LC a n a l y s i s by UV d e t e c t i o n r a n g i n g from 290-335 nm ( 5 £ 7 , 2 1 , 2 2 ) .


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Evidence Supporting Preharvest A f l a t o x i n

Stilbene Involvement Contamination

in

Bioregulation

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N a t u r a l O c c u r r e n c e o f S t i l b e n e s i n P e a n u t s . S t i l b e n e s a r e n o t found i n sound, undamaged p e a n u t s . However, t h e compounds a r e e a s i l y d e t e c t e d i n p e a n u t s t h a t have been s u b j e c t e d t o some t y p e o f damage i n t h e f i e l d when they were a t h i g h water a c t i v i t i e s (unpublished data). I n peanut s h e l l i n g p l a n t s , damaged p e a n u t s a r e removed r o u t i n e l y by e l e c t r o n i c c o l o r s o r t i n g machines, and t h e s e machines e f f i c i e n t l y e l i m i n a t e d i s c o l o r e d k e r n e l s d u r i n g the p r o c e s s i n g of peanuts. D i s c o l o r a t i o n i s u s e d t o i d e n t i f y p e a n u t s t h a t have been damaged ( u s u a l l y i n the f i e l d , but a l s o d u r i n g s t o r a g e ) and would severely detract from the overall quality of the peanuts, p a r t i c u l a r l y w i t h r e g a r d t o f l a v o r . In many c a s e s t h i s d i s c o l o r a t i o n i s v e r y s i m i l a r t o t h e d i s c o l o r a t i o n t h a t o c c u r s when imbibed p e a n u t s a r e s t i m u l a t e d t o produce p h y t o a l e x i n s i n t h e l a b o r a t o r y . When t h e s e d i s c o l o r e d p e a n u t s were a n a l y z e d f o r s t i l b e n e p h y t o a l e x i n s , the a n a l y s e s i n v a r i a b l y showed the p r e s e n c e o f the compounds, s u g g e s t i n g t h a t they a r e n a t u r a l l y p r o d u c e d i n t h e f i e l d i n r e s p o n s e t o damage ( u n p u b l i s h e d d a t a ) . S i n c e d i s c o l o r a t i o n and s t i l b e n e p r o d u c t i o n does n o t o c c u r when peanuts a r e d r y , i t i s a p p a r e n t t h a t some minimum water a c t i v i t y i s r e q u i r e d f o r the enzyme-mediated s y n t h e s i s o f t h e stilbenes. A c l o s e e x a m i n a t i o n o f damaged peanuts grown under adequate m o i s t u r e c o n d i t i o n s r e v e a l s l i t t l e , i f any, A. flavus proliferation. Likewise, i t i s unusual t o d e t e c t even low concentrations of a f l a t o x i n i n s u c h p e a n u t s . However, s t i l b e n e p h y t o a l e x i n s a r e e a s i l y detected i n such peanuts. When s i m i l a r p e a n u t s were s u r f a c e s t e r i l i z e d and p l a t e d out t o d e t e r m i n e c o u n t s and t y p e s o f f u n g a l c o l o n i z a t i o n , t h e p e r c e n t a g e s o f k e r n e l s c o l o n i z e d by A. flavus was as h i g h as 25% ( 1 9 ) . S i n c e c o l o n i z a t i o n had o c c u r r e d and p h y t o a l e x i n s had been p r o d u c e d w i t h an absence o f a f l a t o x i n c o n t a m i n a t i o n , p h y t o a l e x i n s presumably i n h i b i t e d A. flavus growth and aflatoxin production. C o n v e r s e l y , a c l o s e e x a m i n a t i o n o f damaged p e a n u t s t h a t were s u b j e c t e d t o l a t e - s e a s o n drought c o n d i t i o n s u s u a l l y r e v e a l s s e v e r a l k e r n e l s w i t h p r o l i f i c A. flavus growth and a f l a t o x i n c o n c e n t r a t i o n s t h a t can be e x t r e m e l y h i g h . P h y t o a l e x i n c o n c e n t r a t i o n s a r e t y p i c a l l y much lower i n t h e s e peanuts compared t o damaged, non-stressed peanuts. T h e r e f o r e , t h e f a c t t h a t peanuts produce s t i l b e n e p h y t o a l e x i n s n a t u r a l l y i n r e s p o n s e t o damage i n the f i e l d but do n o t become c o n t a m i n a t e d w i t h a f l a t o x i n ( i n d i c a t i v e o f A. flavus growth) u n t i l s u b j e c t e d t o p r o l o n g e d d r o u g h t s t r e s s p o i n t s toward a p r e s u m p t i v e r o l e f o r t h e s e compounds i n t h e n a t u r a l b i o r e g u l a t i o n of a f l a t o x i n contamination. B i o l o g i c a l A c t i v i t y o f S t i l b e n e s A g a i n s t A. flavus and O t h e r F u n g i . Further evidence supporting a r o l e f o r s t i l b e n e phytoalexins i n i n h i b i t i n g f u n g a l growth i n peanuts i n v o l v e s the b i o l o g i c a l a c t i v i t y of t h e s e compounds a g a i n s t A. flavus and o t h e r f u n g i . Wotton and S t r a n g e t e s t e d [ 1 ] , [ 3 ] , and [4] f o r i n h i b i t i o n o f A. flavus spore g e r m i n a t i o n and h y p h a l e x t e n s i o n ( 9 ) . The EDgQ v a l u e s f o r i n h i b i t i o n of s p o r e g e r m i n a t i o n were 12.7, 12.8, and 8.9 ug/ml, r e s p e c t i v e l y , i n V o g e l ' s medium. S i m i l a r l y , germ tube e x t e n s i o n was a l s o i n h i b i t e d w i t h E D ^ v a l u e s o f 6.8, 4.9, and 9.7 ug/ml, r e s p e c t i v e l y , f o r [ 1 ] , [ 3 ] , ana [ 4 ] . Cooksey e t a l . r e p o r t e d E D ^ v a l u e s o f 14.0 and 11.3 ug/ml f o r i n h i b i t i o n of s p o r e g e r m i n a t i o n and h y p h a l e x t e n s i o n o f A. flavus, r e s p e c t i v e l y , by [5] ( 8 ) . I n one l a b o r a t o r y , a t i m e - c o u r s e s t u d y was c a r r i e d out t o d e t e r m i n e t h e r e l a t i o n s h i p o f growth and a f l a t o x i n p r o d u c t i o n by A. flavus t o p h y t o a l e x i n a c c u m u l a t i o n by i n t a c t peanut k e r n e l s . Wotton and S t r a n g e (23) r e p o r t e d t h a t f o l l o w i n g i n o c u l a t i o n , A. flavus grew l o g a r i t h m i c a l l y f o r 2 days. However, by t h e t h i r d day when t h e



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p h y t o a l e x i n c o n c e n t r a t i o n had exceeded 50 ug/g o f k e r n e l s , f u n g a l growth e s s e n t i a l l y c e a s e d . This provided evidence f o r both the e l i c i t a t i o n o f p h y t o a l e x i n p r o d u c t i o n by A. flavus and t h e i n h i b i t i o n o f A. flavus growth by t h e p h y t o a l e x i n s . The i n h i b i t i o n o f A. parasiticus growth by [4] was d e t e r m i n e d i n our l a b o r a t o r y u s i n g a m o d i f i c a t i o n o f the a s s a y r e p o r t e d by A r n o l d i and coworkers ( 2 4 ) . The s t i l b e n e was d i s s o l v e d i n a c e t o n e and a p p r o p r i a t e amounts were added t o s t e r i l e p o t a t o d e x t r o s e a g a r (PDA) a t 50°C t o g i v e c o n c e n t r a t i o n s o f 10, 20, 50, and 100 ug/ml. The medium was dispersed i n 60 mm t i s s u e culture dishes and i n o c u l a t e d w i t h 4 mm PDA p l u g s o f a c t i v e l y growing A. parasiticus cultures. P l a t e s were i n c u b a t e d i n t h e d a r k a t 30°C and colony d i a m e t e r s were measured d a i l y . A f t e r t h r e e days t h e percent i n h i b i t i o n o f A. parasiticus growth a t 10, 20, 50, and 100 ug/ml was 15, 26, 37, and 33%, r e s p e c t i v e l y ( u n p u b l i s h e d d a t a ) . O t h e r s t u d i e s were c o n d u c t e d i n our l a b o r a t o r y t o d e t e r m i n e t h e i n h i b i t o r y e f f e c t s o f [4] a g a i n s t A. parasiticus and s e v e r a l s p e c i e s o f Penicillium ( C h r i s t i a n s e n , u n p u b l i s h e d d a t a ) . The g e r m i n a t i o n o f f u n g i was d e t e r m i n e d i n a p r o c e d u r e t h a t was s i m i l a r t o t h a t r e p o r t e d by Wotton and S t r a n g e ( 9 ) , and r a d i a l growth a l s o was d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d (24). R e s u l t s p r e s e n t e d i n T a b l e I show t h a t both spore germination and growth o f most t e s t e d s p e c i e s were inhibited by [4], although the degree of inhibition varied c o n s i d e r a b l y among s p e c i e s . Table

I.

E f f e c t o f [4] on s p o r e g e r m i n a t i o n several fungal species.

and

Spore g e r m i n a t i o n ED^Q (ug/ml)

Fungus Aspergillus parasiticus Penicillium commune P. crustosum P. verrucosum P. aurantiogriseum P. echinulatum var. d i s c o l o r

94 132 18000 42 70 77

radial

growth

of

R a d i a l growth % inhibition 25 ug/ml 50 ug/ml 54 36 46 33 25 13

54 55 61 44 50 63

Taken t o g e t h e r , t h e s e s t u d i e s show t h a t peanut k e r n e l s t i l b e n e s p o s s e s s b i o l o g i c a l a c t i v i t y a g a i n s t s p e c i e s o f b o t h Aspergillus and Penicillium. T h i s , c o u p l e d w i t h t h e f a c t t h a t t h e s e compounds a r e p r o d u c e d in vivo as a r e s u l t o f damage, p r o v i d e s e v i d e n c e that s t i l b e n e s p l a y a p a r t i n the n a t u r a l defense of peanuts a g a i n s t fungi. Occurrence of A f l a t o x i n Contamination A f t e r C e s s a t i o n of P h y t o a l e x i n Production. I n view o f t h e f a c t t h a t s t i l b e n e p h y t o a l e x i n s are n a t u r a l l y p r o d u c e d i n peanut k e r n e l s i n r e s p o n s e t o f u n g a l i n v a s i o n and that these s t i l b e n e s possess antifungal activity against a f l a t o x i g e n i c f u n g i , t h e q u e s t i o n o f how p e a n u t s become c o n t a m i n a t e d with a f l a t o x i n remains. Simply s t a t e d , how does A. flavus overcome t h i s a p p a r e n t n a t u r a l d e f e n s e mechanism o f p e a n u t s ? Because of the association of preharvest aflatoxin c o n t a m i n a t i o n o f p e a n u t s w i t h l a t e - s e a s o n d r o u g h t s t r e s s , a s t u d y was undertaken to determine the relationship among aflatoxin c o n t a m i n a t i o n , d r o u g h t , and p h y t o a l e x i n p r o d u c t i o n ( 2 2 ) . The s t u d y i n v o l v e d s a m p l i n g o f F l o r u n n e r p e a n u t s s u b j e c t e d t o and n o t s u b j e c t e d t o l a t e - s e a s o n d r o u g h t and d e t e r m i n i n g t h e peanut k e r n e l water activity (a ), phytoalexin-producing capacity, and aflatoxin c o n c e n t r a t i o n s i n a l l m a t u r i t y s t a g e s o f t h e sampled p e a n u t s . I t was r e p o r t e d t h a t t h e a and p h y t o a l e x i n - p r o d u c i n g c a p a c i t y o f p e a n u t s n o t exposed t o d r o u g h t s t r e s s remained h i g h t h r o u g h o u t t h e s t u d y w


24.

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period. These p e a n u t s d i d n o t become c o n t a m i n a t e d w i t h a f l a t o x i n even though t h e f i n a l s a m p l i n g took p l a c e 184 days a f t e r p l a n t i n g , a p p r o x i m a t e l y 40 days beyond t h e o p t i m a l h a r v e s t d a t e . The a o f peanuts grown under l a t e - s e a s o n drought c o n d i t i o n s d e c r e a s e d d u r i n g t h e s t u d y (22). The m o i s t u r e c o n t e n t was not u n i f o r m i n a sample, i n d i c a t i n g t h a t p e a n u t s s u b j e c t e d t o drought s t r e s s d i d n o t d r y a t a u n i f o r m r a t e . T h i s appeared t o be a s s o c i a t e d w i t h i n d i v i d u a l p l a n t s i n t h a t t h e peanuts on c e r t a i n p l a n t s l o s t m o i s t u r e more e a s i l y t h a n t h o s e on o t h e r p l a n t s . However, t h e o v e r a l l e f f e c t o f drought s t r e s s was t o r e d u c e t h e m o i s t u r e o r a o f peanut k e r n e l s . As peanuts became d e h y d r a t e d d u r i n g t h e drought p e r i o d , t h e y l o s t the c a p a c i t y t o produce s t i l b e n e p h y t o a l e x i n s . This lost c a p a c i t y was n o t d i r e c t l y due t o t h e d u r a t i o n o f t h e s t r e s s , but i t was directly a s s o c i a t e d w i t h the drop in a of the peanuts. R e g a r d l e s s o f drought t r e a t m e n t s o i l temperature o r peanut m a t u r i t y , t h e p h y t o a l e x i n - p r o d u c i n g c a p a c i t y o f peanuts d e c r e a s e d as t h e a d e c r e a s e d , w i t h e s s e n t i a l l y no p h y t o a l e x i n p r o d u c t i o n below a k e r n e l a o f 0.95 (Figure 2). The o n s e t o f a f l a t o x i n c o n t a m i n a t i o n d i d not o c c u r u n t i l peanut k e r n e l s had l o s t t h e c a p a c i t y t o produce p h y t o a l e x i n s as an a p p a r e n t r e s u l t of drought-induced moisture l o s s . However, mature p e a n u t s r e t a i n e d a h i g h e r degree o f r e s i s t a n c e t o a f l a t o x i n c o n t a m i n a t i o n t h a n immature p e a n u t s even a f t e r t h e l o s s o f p h y t o a l e x i n - p r o d u c i n g capability. T h i s i n d i c a t e d t h a t immature k e r n e l s r e l y more h e a v i l y on a phytoalexin-based r e s i s t a n c e than mature k e r n e l s , which a p p a r e n t l y have some a d d i t i o n a l r e s i s t a n c e not based on s t i l b e n e phytoalexins. The e v i d e n c e c l e a r l y s u p p o r t s t h e h y p o t h e s i s t h a t s t i l b e n e p h y t o a l e x i n s i n peanuts a r e an i m p o r t a n t n a t u r a l b i o r e g u l a t o r o f p r e h a r v e s t a f l a t o x i n c o n t a m i n a t i o n . That e v i d e n c e i n c l u d e s t h e f a c t s t h a t : (1) s t i l b e n e s a r e n a t u r a l l y produced i n f i e l d - d a m a g e d p e a n u t s ; (2) s t i l b e n e s p o s s e s s b i o l o g i c a l a c t i v i t y a g a i n s t A. flavus and A. parasiticus; and (3) a l t h o u g h i n v a s i o n o f p e a n u t s by A. flavus and A. parasiticus c a n o c c u r under any c o n d i t i o n s , a f l a t o x i n c o n t a m i n a t i o n does n o t o c c u r u n t i l peanuts l o s e t h e c a p a c i t y f o r p h y t o a l e x i n p r o d u c t i o n as a r e s u l t o f d r o u g h t - i n d u c e d k e r n e l d e h y d r a t i o n .


w

w

w

T a k i n g Advantage o f t h e N a t u r a l Defense Mechanism The g o a l o f our r e s e a r c h today i s t o g r e a t l y r e d u c e o r e l i m i n a t e p r e h a r v e s t a f l a t o x i n contamination of peanuts. As t h e demand f o r more wholesome f o o d w i t h l e s s r i s k o f e x p o s u r e t o t o x i n s and c a r c i n o g e n s i n c r e a s e s , t h e c o n t i n u e d use o f p e a n u t s and peanut p r o d u c t s as f o o d becomes more dependent on e f f e c t i v e management o f t h e a f l a t o x i n problem. Many approaches a r e b e i n g t a k e n t o s o l v e t h e a f l a t o x i n problem i n peanuts. An i m p o r t a n t q u e s t i o n i s whether t h e p h y t o a l e x i n - b a s e d n a t u r a l d e f e n s e mechanism o f peanuts a g a i n s t f u n g i c a n be e x p l o i t e d i n some way t o p r o v i d e a s o l u t i o n ; and i f so, how. A l l data i n d i c a t e t h a t growing a l l peanuts w i t h adequate l a t e - s e a s o n i r r i g a t i o n would e s s e n t i a l l y s o l v e t h e p r e h a r v e s t problem, but c u r r e n t l y t h i s i s n o t a f e a s i b l e approach. T h e r e f o r e , t a k i n g advantage o f any n a t u r a l d e f e n s e mechanism must i n v o l v e i t s e f f e c t i v e n e s s d u r i n g p e r i o d s o f l a t e - s e a s o n drought s t r e s s . Two a p p r o a c h e s t o m a i n t a i n i n g p h y t o a l e x i n - p r o d u c i n g c a p a c i t y d u r i n g drought a r e a p p a r e n t . The f i r s t would be t o i d e n t i f y peanut genotypes t h a t c o u l d c o n t i n u e p r o d u c i n g p h y t o a l e x i n s as t h e m o i s t u r e of the k e r n e l s decreased. I n F l o r u n n e r p e a n u t s i t appears t h a t t h e a p p r o x i m a t e lower a l i m i t for phytoalexin production i s 0.95. Peanuts t h a t c o u l d m a i n t a i n p r o d u c t i o n o f p h y t o a l e x i n s as t h e a approached 0.90 might p o s s e s s much g r e a t e r p r o t e c t i o n from A. flavus growth and a f l a t o x i n p r o d u c t i o n . A l t h o u g h t h e lower a limit for a f l a t o x i n p r o d u c t i o n i n peanuts i s about 0.85, a practical solution w

w


In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. w

0.96 a

w

0.94

0.92

F i g u r e 2. R e l a t i o n s h i p o f p h y t o a l e x i n p r o d u c t i o n t o peanut k e r n e l water a c t i v i t y (a ). T o t a l p h y t o a l e x i n s were d e t e r m i n e d by c o m b i n i n g a r e a s under p h y t o a l e x i n peaks from l i q u i d chromatograms.

0.98

0.90

» « • — * ~ ^|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—p

1.00

50000

60000


24.

DORNER ET AL.

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t o t h e problem might n o t r e q u i r e p h y t o a l e x i n p r o d u c t i o n down t o s u c h a low a l e v e l . T h i s i s because a s t h e a c o n t i n u e s t o d e c r e a s e , s o does t h e r a t e o f f u n g a l growth and a f l a t o x i n p r o d u c t i o n . Whether o r n o t a genotype e x i s t s t h a t c a n produce p h y t o a l e x i n s a t lower a i s unknown a t t h i s t i m e . T h e r e f o r e , a r i g o r o u s s c r e e n i n g program would have t o be u n d e r t a k e n t o i d e n t i f y such a g e n e t i c c a p a b i l i t y i n peanuts. A second approach t o m a i n t a i n i n g p h y t o a l e x i n - p r o d u c i n g c a p a c i t y d u r i n g d r o u g h t would be t o i d e n t i f y d r o u g h t - t o l e r a n t g e n o t y p e s t h a t can m a i n t a i n a h i g h k e r n e l a f o r a s i g n i f i c a n t l y l o n g e r p e r i o d during drought stress. I f e i t h e r o f these approaches were s u c c e s s f u l , b i o t e c h n o l o g y t e c h n i q u e s might be used t o i n c o r p o r a t e t h e d e s i r a b l e t r a i t ( s ) i n t o c o m m e r c i a l l y - d e s i r a b l e c u l t i v a r s , such as Florunner. I t i s u n l i k e l y t h a t any s i n g l e a p p r o a c h w i l l p r o v i d e a s o l u t i o n t o t h e problem o f p r e h a r v e s t a f l a t o x i n c o n t a m i n a t i o n o f peanuts. However, a m u l t i f a c e t e d a p p r o a c h t h a t c o u l d i n c l u d e enhancement o f t h e n a t u r a l b i o r e g u l a t i v e p r o p e r t i e s o f s t i l b e n e p h y t o a l e x i n s might u l t i m a t e l y y i e l d t h e s o l u t i o n t o a s e r i o u s and complex problem. w

w

w


w

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4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Deverall, B. J. In Phytoalexins; Bailey, J . A.; Mansfield J. W., Eds.; John Wiley and Sons, Inc.: New York, 1982; pp 1-20. Ingham, J . L. In Phytoalexins; Baily, J . A.; Mansfield, J . W., Eds.; John Wiley and Sons, Inc.: New York, 1982, p 21. Cole, R. J.; Sanders, T. H.; Blankenship, P. D.; H i l l , R. A. In Xenobiotics in Foods and Feeds; Finley, J . W.; Schwass, D. E . , Eds.; ACS Symposium Series No. 234; American Chemical Society: Washington, DC, 1983; pp 233-239. Vidhyasekaran, P.; Lalithakumari, D.; Govindaswamy, C. V. Indian Phytopathol. 1972, 25, 240-45. Kenn, N. T . ; Ingham, J . L. Phytochemistry 1976, 15, 1794-95. Ingham, J . L. Phytochemistry 1976, 15, 1971-93. Aguamah, D. G.; Langcake, P.; Leworthy, D. P.; Page, J . A.; Pryce, R. J.; Strange, R. N. Phytochemistry 1981, 20, 1381-83. Cooksey, C. J.; Gaviatt, P. J.; Richards, S. E . ; Strange, R. N. Phytochemistry 1988, 27, 1015-16. Wotton, H. R.; Strange, R. N. J. Gen. Microbiol. 1985, 131, 487-94. Diener, U. L.; Cole, R. J.; Sanders, T. H.; Payne, G. A.; Lee, L. S.; Klich, M. A. Ann. Rev. Phytopathol. 1987, 25, 249-70. Diener, U. L.; Pettit, R. E.; Cole, R. J . In Peanut Science and Technology; Pattee, H. E.; Young, C. T . , Eds.; American Peanut Research and Education Society, Inc.: Yoakum, Texas, 1982; pp 486-519. Blankenship, P. D.; Cole, R. J.; Sanders, T. H.; Hill, R. A. Mycopathologia 1984, 85, 69-74. Cole, R. J.; Hill, R. A.; Blankenship, P. D.; Sanders, T. H.; Garren, K. H. Dev. Ind. Microbiol. 1982, 23, 229-36. Dickens, J . W.; Satterwhite, J . B.; Sneed, R. E. J. Am. Peanut Res. Educ. Soc. 1973, 5, 48-58. H i l l , R. A.; Blankenship, P. D.; Cole, R. J.; Sanders, T. H. Appl. Microbiol. 1983, 45, 628-33. Pettit, R. E.; Taber, R. A.; Schroeder, H. W.; Harrison, A. L. Appl. Microbiol. 1971, 22-629-34. Wilson, D. M.; Stansell, J . R. Peanut Sci. 1983, 10, 54-56. Cole, R. J.; Sanders, T. H.; Hill, R. A.; Blankenship, P. D. Mycopathologia 1985, 91, 41-46. Sanders, T. H.; Cole, R. J.; Blankenship, P. D.; H i l l , R. A. Peanut Sci. 1985, 12, 90-93. Sanders, T. H.; Hill, R. A.; Cole, R. J.; Blankenship, P. D. J. Am. Oil Chem. Soc. 1981, 58, 966A-70A. Keen, N. T. Phytopathology 1975, 65, 91-92.


360 22. 23. 24.

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Dorner, J . W.; C o l e , R. J.; Sanders, T. H.; B l a n k e n s h i p , P. D. Mycopathologia 1989, 105, 117-28. Wotton, H. R.; S t r a n g e , R. N. Appl. Environ. Microbiol. 1987, 53, 270-73. A r n o l d i , A.; C a r u g h i , M.; F a r e i n a , G.; Merlini, L.; P a r r i n o , M. G. J. Agric. Food Chem. 1989, 37, 508-12.


RECEIVED May 16, 1990


Bioregulation of Preharvest Aflatoxin Contamination of Peanuts - ACS

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