Potential Role of Phytoalexins in Aflatoxin Contamination of Peanuts

Chapter 5

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch005

Potential Role of Phytoalexins in Aflatoxin Contamination of Peanuts R. J. Cole, J. W. Dorner, P. D. Blankenship, and T. H. Sanders National Peanut Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Dawson, GA 31742 Peanut phytoalexins appear to be involved in resistance to drought-induced preharvest aflatoxin contamination of immature peanuts. Mature peanuts are considerably more resistant to environmentally-induced preharvest aflatoxin contamination of peanuts than are immature peanuts. The mechanism of this latter resistance i s unknown. The identification of this resistance mechanism and other resistance may provide one approach to subsequent use of biotechnology to incorporate field resistance traits into commercially acceptable varieties. Biotechnology may also be a valuable approach to exploiting genetic resistance to preharvest aflatoxin found in wild species that have evolved in an arid environment. The concept o f p l a n t s b e i n g a b l e t o produce d e f e n s i v e s u b s t a n c e s , c a l l e d p h y t o a l e x i n s , i n response t o i n f e c t i o n was f i r s t proposed by M u l l e r and B o r g e r ( 1 ) . P h y t o a l e x i n s a r e p r e s e n t l y d e f i n e d a s l o w m o l e c u l a r weight a n t i m i c r o b i a l compounds t h a t are s y n t h e s i z e d by and accumulate i n p l a n t s a f t e r t h e i r exposure t o m i c r o o r g a n i s m s . 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 i n peanuts appeared i n 1972 when V i d h y a s e k a r a n ejt j t l . (2) r e p o r t e d t h a t they d e t e c t e d p h y t o a l e x i n p r o d u c t i o n i n response t o i n v a s i o n by s t o r a g e f u n g i . I t was observed t h a t p h y t o a l e x i n was produced t o a g r e a t e r e x t e n t i n immature pods and t o a l e s s e r e x t e n t i n mature pods. The c h e m i c a l n a t u r e o f these p h y t o a l e x i n s was not d e t e r m i n e d i n t h i s s t u d y . Keen (3) r e p o r t e d the p r o d u c t i o n o f two i n d u c i b l y formed a n t i f u n g a l c h e m i c a l s i n peanuts ( A r a c h i s hypogaea) and i n f e r r e d a p h y t o a l e x i n r o l e f o r these c h e m i c a l s . I n a f o l l o w - u p s t u d y , Keen and Ingham (4) r e p o r t e d t h a t these two p h y t o a l e x i n s from A. hypogaea were c i s - and t r a n s - i s o m e r s o f 3 , 5 , 4 ' - t r i h y d r o x y - 4 - i s o p e n t e n y l s t i l b e n e ( F i g u r e 1A) o r A r a c h i d i n I I . S i m u l t a n e o u s l y , Ingham (5) i s o l a t e d two r e l a t e d compounds from the f u n g a l - i n f e c t e d h y p o c o t y l s o f an A f r i c a n - g r o w n c u l t i v a r o f A. hypogaea. These were i d e n t i f i e d as c i s - and t r a n s - 3 , 5,4'-trihydroxystilbene or r e s v e r a t r o l (Figure IB). P r e v i o u s l y , s t i l b e n e p h y t o a l e x i n s had been a s s o c i a t e d o n l y w i t h Gymnospermae. This chapter not subject to U.S. copyright Published 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


BIOTECHNOLOGY FOR CROP PROTECTION

4-lsopentenylresveratrol

F i g u r e 1A.

Chemical s t r u c t u r e of A r a c h i d i n I I .

OH F i g u r e IB. Chemical s t r u c t u r e of R e s v e r a t r o l .



COLE ET AL.

Phytoalexins in Aflatoxin Contamination

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F i g u r e 1C.

F i g u r e 1D.

C h e m i c a l s t r u c t u r e of A r a c h i d i n I .

C h e m i c a l s t r u c t u r e of A r a c h i d i n I I I .


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Aguamah e t a l . (6) i s o l a t e d 3 a n t i f u n g a l compounds from A. hypogaea t h a t were i d e n t i f i e d as 4-(3-methyl-but-l-enyl)-3,5,3 ,4 -tetrahyd r o x y s t i l b e n e ( A r a c h i d i n I , F i g u r e 1C), S ^ ^ ' - t r i h y d r o x y ^ - i s o p e n t e n y l s t i l b e n e ( A r a c h i d i n I I , F i g u r e ΙΑ), and 4 - ( 3 - m e t h y l - b u t - l - e n y l ) 3,5,4'trihydroxystilene (Arachidin I I I , Figure ID). Narayanaswamy and Mahadevan (7) r e p o r t e d l y i s o l a t e d c i s and t r a n s 3 , 5 , 4 - t r i h y d r o x y - 4 - i s o p e n t e n y l s t i l b e n e and two o t h e r unknown p h y t o a l e x i n s from the seeds of A. hypogaea ( c u l t i v a r TMU7). These i n v e s t i g a t o r s l a t e r r e p o r t e d the i s o l a t i o n of p h y t o a l e x i n from the l e a v e s of A. hypogaea, a l t h o u g h the c h e m i c a l n a t u r e of the p h y t o a l e x i n s was not s t u d i e d . Recent s t u d i e s by Wotton and Strange (8) p r o v i d e d c i r c u m s t a n t i a l e v i d e n c e f o r p h y t o a l e x i n i n v o l v e m e n t i n the r e s i s t a n c e of peanuts t o A s p e r g i l l u s f l a v u s . T h e i r r e s u l t s i n d i c a t e d t h a t r e s i s t a n c e of peanut k e r n e l s t o i n v a s i o n by A. f l a v u s was c o r r e l a t e d w i t h t h e i r c a p a c i t y t o s y n t h e s i z e p h y t o a l e x i n s as an e a r l y r e s p o n s e t o wounding. A l s o , c o n d i t i o n s t h a t promoted i n v a s i o n of peanuts by A. f l a v u s i n h i b i t e d p h y t o a l e x i n p r o d u c t i o n . Thus, k e r n e l s o f drought s t r e s s e d p l a n t s , w h i c h a r e more s u s c e p t i b l e t o A. f l a v u s than k e r n e l s of nondrought s t r e s s e d p l a n t s , produced l e s s p h y t o a l e x i n i n response t o wounding by s l i c i n g than k e r n e l s from n o n - s t r e s s e d p l a n t s . T h i s r e v i e w summarizes a r e c e n t study t h a t e v a l u a t e d the r o l e of s t i l b e n e - b a s e d p h y t o a l e x i n r e s i s t a n c e i n peanuts u s i n g s i x e n v i r o n m e n t a l c o n t r o l p l o t s (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. P h y t o p a t h o l o g y , i n p r e s s ) . The use of b i o t e c h n o l o g y t o i n c o r p o r a t e p h y t o a l e x i n - b a s e d g e n e t i c r e s i s t a n c e from w i l d s p e c i e s t h a t have e v o l v e d i n an a r i d en vironment i n t o commercially a v a i l a b l e v a r i e t i e s i s a p o t e n t i a l l y v i a b l e approach t o d e v e l o p i n g f i e l d r e s i s t a n c e a g a i n s t A. f l a v u s i n v a s i o n and subsequent a f l a t o x i n p r o d u c t i o n i n p e a n u t s . l

f


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M a t e r i a l s and Methods C u l t u r e and Treatment of P e a n u t s . F l o r u n n e r v a r i e t y peanuts were grown i n the e n v i r o n m e n t a l c o n t r o l p l o t f a c i l i t y a t the N a t i o n a l Peanut R e s e a r c h L a b o r a t o r y , Dawson, GA ( 9 ) ( D o r n e r , J . W.; C o l e , R. J . ; Sanders, T. H.; B l a n k e n s h i p , P. D. P h y t o p a t h o l o g y , i n p r e s s ) . Con v e n t i o n a l c u l t u r a l p r a c t i c e s were used f o r a l l peanuts up t o 96 days a f t e r p l a n t i n g (DAP) a t w h i c h time drought t r e a t m e n t s were imposed. One p l o t , w h i c h s e r v e d as the c o n t r o l , r e c e i v e d o p t i m a l m o i s t u r e throughout the s t u d y . Two p l o t s were equipped w i t h t h e r m o s t a t i c a l l y c o n t r o l l e d , l e a d - s h i e l d e d h e a t i n g c a b l e s t o e l e v a t e the s o i l tempera t u r e t o a mean of c a . 29 C ( o p t i m a l f o r a f l a t o x i n c o n t a m i n a t i o n ) ( 9 ) . The o t h e r t h r e e p l o t s were equipped w i t h h e a t i n g c a b l e s ( t o e l e v a t e s o i l temperature) as w e l l as epoxy-coated copper t u b i n g t h r o u g h w h i c h c o o l water was c i r c u l a t e d ( t o reduce s o i l temperature) as n e c e s s a r y to a c h i e v e a mean s o i l t e m p e r a t u r e of c a . 25 C (not as c o n d u c i v e f o r a f l a t o x i n contamination)(10). Treatments began w i t h c o n t r o l of s o i l temperature a t 103 DAP f o r the 29°C treatment and 105 DAP f o r the 25 C treatment (Dorner, J . W.; C o l e , R. J . ; S a n d e r s , T. H.; B l a n k e n s h i p , P. D. P h y t o p a t h o l o g y , i n p r e s s ) . Sampling of P e a n u t s . Samples from the d i f f e r e n t t r e a t m e n t s were c o l l e c t e d by h a n d - d i g g i n g c a . 60 f e e t of row b e g i n n i n g a t 120 DAP 17 treatment days (TD), i n the 29°C treatment and 121 DAP (18 TD)


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the 25°C t r e a t m e n t . An i n i t i a l sample from the i r r i g a t e d treatment was taken 114 DAP and f i v e o t h e r samples were c o l l e c t e d throughout the treatment p e r i o d w i t h the f i n a l sample t a k e n 184 DAP. Samples from the two drought t r e a t m e n t s were taken a t w e e k l y i n t e r v a l s w i t h the f i n a l sample from the 29°C treatment t a k e n a t 162 DAP (59 TD) and the f i n a l 25°C sample a t 183 DAP (80 TD). A l l undamaged pods were s a n d - b l a s t e d and each pod was p l a c e d i n t o one of f i v e m a t u r i t y s t a g e s based on the method o f W i l l i a m s and D r e x l e r (11) and d e s c r i b e d by Henning ( 1 2 ) . The s t a g e s , i n o r d e r of i n c r e a s i n g m a t u r i t y , were y e l l o w 1, y e l l o w 2, orange, brown, and black. Immediately a f t e r m a t u r i t y c l a s s i f i c a t i o n , w a t e r a c t i v i t y ( a ) and m o i s t u r e were determined f o r samples a t each m a t u r i t y s t a g e . Water a c t i v i t y ( a ) i s d e f i n e d as the fundamental p r o p e r t y of aqueous s o l u t i o n s , and by d e f i n i t i o n a i s a l s o n u m e r i c a l l y e q u a l t o the c o r r e s p o n d i n g r e l a t i v e h u m i d i t y (R.H.) e x p r e s s e d as a f r a c t i o n , i . e . , R.H./100. Ten pods p e r m a t u r i t y stage were h a n d s h e l l e d , s p l i t , and p l a c e d i n s e a l e d sample d i s h e s a t 25°C f o r measurements w i t h a Beckman Model E2BFA h y g r o l i n e sensor a t t a c h e d t o a Beckman Model VFB 2 h y g r o l i n e f l a t - b e d r e c o r d e r (Beckman I n d u s t r i a l C o r p . , Cedar Grove, N J ) . F o r m o i s t u r e d e t e r m i n a t i o n s , t h r e e 50-pod samples from each m a t u r i t y stage were h a n d s h e l l e d , weighed, o v e n - d r i e d f o r s i x hours a t 130°C, and reweighed t o determine m o i s t u r e l o s s . Percent m o i s t u r e was c a l c u l a t e d as i n i t i a l w e i g h t / f i n a l w e i g h t / i n i t i a l weight. w

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E v a l u a t i o n of P h y t o a l e x i n - P r o d u c i n g P o t e n t i a l . S i x g of k e r n e l s (x3) from each m a t u r i t y stage were s l i c e d 1-2 mm t h i c k , d i s t r i b u t e d i n open 60 mm t i s s u e c u l t u r e d i s h e s , and d u s t e d w i t h s p o r e s of a nona f l a t o x i n p r o d u c i n g s t r a i n of A. p a r a s i t i c u s (CP 461; SRRC 2043) t o e l i c i t p h y t o a l e x i n p r o d u c t i o n . The open d i s h e s were i n c u b a t e d i n the dark a t 25±1.0°C f o r f o u r days i n s e a l e d d e s s i c a t o r s over u n s a t u r a t e d N a C l s o l u t i o n s of a c o r r e s p o n d i n g t o t h a t determined f o r each m a t u r i t y s t a g e . T h i s was t o m a i n t a i n a l l peanuts a t t h e i r p r e h a r v e s t a d u r i n g the i n c u b a t i o n . P h y t o a l e x i n s were e x t r a c t e d and a n a l y z e d v i a h i g h performance l i q u i d chromatography a c c o r d i n g t o Dorner e t a l . ( D o r n e r , J . W.; C o l e , R. J . ; Sanders, T. H.; B l a n k e n s h i p , P. D. P h y t o p a t h o l o g y , i n p r e s s ) . A r e a c o u n t s of peaks c o r r e s p o n d i n g t o p h y t o a l e x i n s t a n d a r d s were summed t o g i v e t o t a l p h y t o a l e x i n c o n t e n t f o r comparison of samples. w

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A f l a t o x i n A n a l y s e s . The remainder of t h e peanuts i n each m a t u r i t y stage were d r i e d , s h e l l e d , and a n a l y z e d f o r a f l a t o x i n by t h e method of Dorner and C o l e (Dorner, J . W.; C o l e , R. J . J . A s s o c . O f f . A n a l . Chem., i n p r e s s ) . Results R e l a t i o n s h i p Between Treatment and K e r n e l M o i s t u r e . Peanut k e r n e l s of a l l m a t u r i t y s t a g e s from the i r r i g a t e d c o n t r o l p l o t m a i n t a i n e d an a of 1.0 throughout the experiment p e r i o d . These peanuts c o n s i s t e n t l y produced r e l a t i v e l y h i g h q u a n t i t i e s of p h y t o a l e x i n s and no samples had s i g n i f i c a n t a f l a t o x i n c o n t a m i n a t i o n (>5 ppb) d u r i n g the p e r i o d of the experiment (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. P h y t o p a t h o l o g y , i n p r e s s ) . w


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As the s t u d y p r o g r e s s e d , the a and m o i s t u r e of d r o u g h t - s t r e s s e d peanuts d e c r e a s e d . The r a t e of m o i s t u r e l o s s was f a s t e r i n the 29 °C t r e a t m e n t t h a n i n the 25°C t r e a t m e n t , p a r t i c u l a r l y i n the more immature s t a g e s (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. P h y t o p a t h o l o g y , i n p r e s s ) . However, the m o i s t u r e l o s s was n o t u n i f o r m w i t h i n a g i v e n m a t u r i t y s t a g e i n a g i v e n t r e a t ment. F o r example, i n the 141 DAP (38 TD) samples from the 29°C t r e a t m e n t , the m o i s t u r e c o n t e n t of k e r n e l s i n the y e l l o w 2 m a t u r i t y stage (next t o most immature stage) ranged from 40.3% t o 17.0% ( a range of 1.00 t o 0.92).· The y e l l o w 1 (most immature stage) and orange stage had s i m i l a r r a n g e s , but the m o i s t u r e range i n the mature s t a g e s (brown, b l a c k ) was much more u n i f o r m . T h i s phenomenon was observed i n b o t h the 29° and 25°C t r e a t m e n t s , but as e x p e c t e d , the number o f d r i e r k e r n e l s w i t h i n a m a t u r i t y s t a g e was g r e a t e r i n the 29 C t r e a t m e n t . I n a d d i t i o n , i n the y e l l o w 2 and orange c a t e g o r i e s , pods c o n t a i n i n g v e r y low m o i s t u r e k e r n e l s had a c h a r a c t e r i s t i c " m u s t a r d - c o l o r e d " appearance a f t e r s a n d - b l a s t i n g , w h i c h proved to be a c o n s i s t e n t l y sound i n d i c a t o r of low k e r n e l m o i s t u r e . K e r n e l s i n the y e l l o w 1, y e l l o w 2, and orange m a t u r i t y s t a g e s were f u r t h e r s e g r e g a t e d based on v i s u a l assessment of k e r n e l m o i s t u r e . A l l k e r n e l s from " m u s t a r d - c o l o r e d " pods were s e p a r a t e d i n t o groups of h i g h , medium, and low m o i s t u r e b e f o r e d e t e r m i n a t i o n s of m o i s t u r e , a , p h y t o a l e x i n - p r o d u c i n g p o t e n t i a l , and a f l a t o x i n c o n t a m i n a t i o n were made. These s e g r e g a t i o n s were e s s e n t i a l t o t e s t i n g the h y p o t h e s i s t h a t k e r n e l a was the p r i m a r y f a c t o r t h a t d i r e c t l y o r i n d i r e c t l y l e d to a f l a t o x i n c o n t a m i n a t i o n . S i n c e a and m o i s t u r e of peanuts i n the brown and b l a c k m a t u r i t y s t a g e s were g e n e r a l l y q u i t e u n i f o r m , i t was not n e c e s s a r y t o f u r t h e r s e g r e g a t e k e r n e l s from those c a t e g o r i e s . w


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Comparisons of m o i s t u r e c o n t e n t o f p a r t i c u l a r m a t u r i t y s t a g e s from the 29° and 25°C t r e a t m e n t s were d i f f i c u l t , s i n c e t h e r e were up to f o u r m o i s t u r e c o n t e n t s f o r each m a t u r i t y s t a g e . F o r example, i n the y e l l o w 2 group from the 29°C treatment samples a t 148 DAP (45 treatment d a y s ) , the m o i s t u r e c o n t e n t of the d i f f e r e n t s e g r e g a t i o n s w i t h i n the group were: " m u s t a r d - c o l o r e d , " 11.7%; " n o r m a l - c o l o r e d " low m o i s t u r e , 15.0%; medium m o i s t u r e , 29.3%; h i g h m o i s t u r e , 37.3%. Each sampling from b o t h the 29° and 25°C t r e a t m e n t s y i e l d e d k e r n e l s of s i m i l a r m o i s t u r e s w i t h i n a m a t u r i t y s t a g e . However, the 29 C treatment c o n s i s t e n t l y had more k e r n e l s a t lower m o i s t u r e s t h a n the 25 C t r e a t m e n t . T h e r e f o r e , t o a r r i v e a t a s i n g l e m o i s t u r e c o n t e n t f o r a s e g r e g a t e d m a t u r i t y s t a g e , the amount of k e r n e l s a t a g i v e n m o i s t u r e was t a k e n i n t o account and a "weighted m o i s t u r e " was used f o r each m a t u r i t y stage ( D o r n e r , J . W.; C o l e , R. J . ; Sanders, T. H.; B l a n k e n s h i p , P. D. P h y t o p a t h o l o g y , i n p r e s s ) . As e x p e c t e d , the more immature peanuts l o s t m o i s t u r e sooner i n the 29°C s o i l than i n the 25°C s o i l . G e n e r a l l y , i t took about a week l o n g e r f o r k e r n e l s i n the c o o l e r t r e a t m e n t t o r e a c h m o i s t u r e s a t t a i n e d i n the warmer t r e a t m e n t . The d a t a c l e a r l y showed a d i r e c t r e l a t i o n s h i p between s o i l temperature and the r a t e of water l o s s from peanut k e r n e l s under l a t e - s e a s o n drought s t r e s s . R e l a t i o n s h i p Between K e r n e l a and P h y t o a l e x i n P r o d u c t i o n . As k e r n e l a decreased as a r e s u l t of drought s t r e s s , the c a p a c i t y of k e r n e l s t o produce p h y t o a l e x i n s a l s o decreased and e v e n t u a l l y was l o s t . T h i s t r e n d was independent of m a t u r i t y and s o i l temperature. When a w

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was h i g h (0.97-1.00), k e r n e l s o f a l l m a t u r i t i e s and from b o t h t r e a t ments produced abundant p h y t o a l e x i n s . No s i g n i f i c a n t p h y t o a l e x i n p r o d u c t i o n was observed when a^, was below 0.95. T h i s was c o n s i s t e n t t h r o u g h o u t a l l m a t u r i t i e s and i n b o t h t r e a t m e n t s . This i l l u s t r a t e d the c o r r e l a t i o n between lower a and reduced p h y t o a l e x i n p r o d u c t i o n i r r e s p e c t i v e o f m a t u r i t y and s o i l t e m p e r a t u r e .


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R e l a t i o n s h i p Between P h y t o a l e x i n P r o d u c t i o n and A f l a t o x i n Contamin a t i o n . Many samples t h a t had l o s t 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 had no d e t e c t a b l e a f l a t o x i n , w h i l e i n o t h e r s i m i l a r samples a f l a t o x i n l e v e l s ranged a s h i g h as 2400 ppb ( T a b l e I ) . S i g n i f i c a n t l y however, no sample o f peanuts t h a t had moderate t o h i g h 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 c o n t a i n e d >5 ppb a f l a t o x i n . T h e r e f o r e , i t can be s t a t e d t h a t as l o n g as peanut k e r n e l s had 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 , a f l a t o x i n was n o t formed ( T a b l e I ) . However, t h e l o s s of phytoalexin-producing c a p a c i t y r e s u l t e d i n a f l a t o x i n f o r m a t i o n b u t i n o n l y a p o r t i o n o f these s u s c e p t i b l e k e r n e l s (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. P h y t o p a t h ology, i n p r e s s ) . R e l a t i o n s h i p Between K e r n e l a and A f l a t o x i n C o n t a m i n a t i o n . I n order t o e v a l u a t e t h e r e l a t i o n s h i p between a and a f l a t o x i n c o n t a m i n a t i o n , i t was u s e f u l t o d e t e r m i n e t h e percentage o f samples 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 b o t h above and below a o f 0.95 ( t h e a below w h i c h the c a p a c i t y t o produce p h y t o a l e x i n s was l o s t ) . I n t h e 29°C t r e a t ment, when a was >0.95, o n l y 5% o f a l l samples c o n t a i n e d g r e a t e r t h a n 5 ppb a f l a t o x i n . However, 50% o f a l l samples w i t h a 0.95, b u t 67% o f y e l l o w l ' s were >5 ppb when a was >0.95, but when a was 5 ppb. P r o c e e d i n g from y e l l o w 1 (most immature) t o b l a c k (most mature) when a was 5 ppb d e c r e a s e d a c c o r d i n g l y - 67%, 50%, 24%, 19%, 17%. A h i g h e r p e r c e n t a g e o f samples from t h e 29°C treatment had c o n t a m i n a t i o n t h a n from t h e 25°C t r e a t m e n t . The p e r c e n t a g e s o f samples from each m a t u r i t y ( y e l l o w 1 - b l a c k ) c o n t a i n i n g >5 ppb a f l a t o x i n when a was

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