Chapter 8
Insecticidal Metabolites from Fusarium avenaceum, a Fungus Associated with Foliage of Abies balsamea Infested by Spruce Budworm, Choristoneura fumiferana
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George M. Strunz and Douglas B. Strongman Canadian Forestry Service-Maritimes, P.O. Box 4000, Fredericton, New Brunswick E3B 5P7, Canada Hyphae from cultures of Fusarium avenaceum, a fungus associated with spruce budworm-infested balsam f i r foliage, were toxic to spruce budworm larvae when ingested. The toxic principle isolated from hyphal extracts was identified as enniatin complex, rich in enniatin A / A . A minor F. avenaceum product, ergosterol peroxide, caused damage to spruce budworm cell cultures, but did not display toxicity in feeding assays at the range of concentrations tested. Ecological implications of the presence of insecticidal fungi on the foliage of spruce budworm host-trees are addressed. 1
The s p r u c e budworm, C h o r i s t o n e u r a f u m i f e r a n a (Clemens), i s a phytophagous i n s e c t , whose p r i n c i p a l h o s t s a r e balsam f i r , A b i e s balsamea (L.) M i l l . , and t h r e e s p r u c e , P i c e a s p e c i e s , which t o g e t h e r dominate the c o n i f e r o u s f o r e s t s o f e a s t e r n N o r t h A m e r i c a . P o p u l a t i o n s o f t h e i n s e c t p e r i o d i c a l l y i r r u p t t o epidemic p r o p o r t i o n s , r e s u l t i n g i n widespread d e s t r u c t i o n o f s u s c e p t i b l e h o s t t r e e s , w i t h major e c o l o g i c a l and r e c e n t l y , economic consequences. The o u t b r e a k s a r e f o l l o w e d by d r a m a t i c d e c l i n e s o f the i n s e c t p o p u l a t i o n s , and t h e r e i s e v i dence i n d i c a t i n g t h a t , a t l e a s t d u r i n g t h e l a s t two c e n t u r i e s , t h e s e o s c i l l a t i o n s have o c c u r r e d q u i t e r e g u l a r l y w i t h a f r e q u e n c y o f 30-40 years (]_, 2 ) . A d e t a i l e d u n d e r s t a n d i n g o f t h e p r o c e s s e s t h a t cause the c o l l a p s e o f an epidemic spruce budworm p o p u l a t i o n i s a d e s i d e r a tum f o r both t h e e c o l o g i s t and t h e f o r e s t manager, and i n t e n s i v e r e s e a r c h on n a t u r a l spruce budworm m o r t a l i t y f a c t o r s i s being cond u c t e d i n s e v e r a l l a b o r a t o r i e s . The s t u d y d e s c r i b e d i n t h i s r e p o r t o r i g i n a t e d i n a suggestion that fungi c o l o n i z i n g the f o l i a g e of the s p r u c e budworm s h o s t t r e e s might produce t o x i c m e t a b o l i t e s , a d v e r s e l y a f f e c t i n g the i n s e c t s and p o s s i b l y c o n t r i b u t i n g t o t h e d e c l i n e of an outbreak p o p u l a t i o n (E. E v e l e i g h , T. Royama, Canadian F o r e s t r y S e r v i c e - M a r i t i m e s ; D.B. Strongman, N. Whitney, U n i v . o f New B r u n s w i c k , u n p u b l i s h e d d i s c u s s i o n s , 1984). T h i s h y p o t h e s i s r e c e i v e s some s u p p o r t from a growing l i t e r a t u r e on p l a n t - f u n g a l i n t e r a c t i o n s that r e s u l t i n p r o t e c t i o n of the plant against t h e d e p r e d a t i o n o f phytophagous i n s e c t s and o t h e r h e r b i v o r e s (3,-6). 1
0097-6156/88/0379-0110S06.00/0 Published 1988 American Chemical Society
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
8.
STRUNZ AND STRONGMAN
Insecticidal Metabolites
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E s s e n t i a l ground-work f o r the i n v e s t i g a t i o n was l a i d by t h e 1985 s t u d y o f M i l l e r e t a l . ( 7 ) , i n which f u n g i were i s o l a t e d both from f o l i a g e o f budworm-infested balsam f i r t r e e s and from budworm-free t r e e s . A l t h o u g h the presence o f s p r u c e budworm markedly a f f e c t e d t h e f o l i a g e m y c o f l o r a , more t h a n d o u b l i n g t h e number o f f u n g a l s p e c i e s i s o l a t e d , t h e m a j o r i t y o f i s o l a t e s were i d e n t i f i e d as common c o l o n i z e r s o f p l a n t s u r f a c e s . However, a p p r o x i m a t e l y 6$ o f the i s o l a t e s from the budworm-infested f o l i a g e were unusual n e e d l e p h y l l o p l a n e f u n g i t h a t were n o t found on i n s e c t - f r e e f o l i a g e . S i g n i f i c a n t l y , some of t h e f u n g i i n t h i s group a r e t o x i g e n i c , entomopathogenic or both ( 7 ) , and a d e t a i l e d e x a m i n a t i o n o f some o f the more a c t i v e i n s e c t i c i d a l i s o l a t e s i s p r e s e n t l y i n p r o g r e s s . Our o b s e r v a t i o n s t o date c o n c e r n i n g one o f the group, Fusarium avenaceum (Corda ex F r . ) S a c c . (FA 1 2 0 ) a r e d e s c r i b e d i n t h i s r e p o r t . Fusarium avenaceum has a g l o b a l d i s t r i b u t i o n as a pathogen o f c e r e a l c r o p s and many o t h e r host p l a n t s , i n c l u d i n g c o n i f e r s ( 8 , 9 ) . I t i s not however, c o n s i d e r e d t o be an i m p o r t a n t d i s e a s e organism i n the s p r u c e - f i r f o r e s t s of e a s t e r n N o r t h America (1_0) (Magasi, L.P., Canadian F o r e s t r y S e r v i c e - M a r i t i m e s , p e r s o n a l communication.) The i s o l a t i o n o f FA 120 from balsam f i r f o l i a g e appears t o be a s s o c i a t e d w i t h t h e presence o f spruce budworm l a r v a e ( 7 ) . The fungus i s known to c o l o n i z e i n s e c t s , h a v i n g been i s o l a t e d from pupae o f Oulema g a l l a e c i a n a , a c e r e a l l e a f b e e t l e (JJ_). I t h a s , f u r t h e r m o r e been observed t o grow s a p r o p h y t i c a l l y on s p r u c e budworm l a r v a e ( S t r o n g man, D.B., u n p u b l i s h e d d a t a , 1985). A f t e r i t s s e l e c t i o n i n the i n i t i a l s c r e e n i n g p r o c e s s , FA 120 was grown i n submerged c u l t u r e on a medium c o n t a i n i n g g l u c o s e , y e a s t e x t r a c t , and peptone. I n c u b a t i o n o f the fungus under s u i t a b l e c o n d i t i o n s f o r 14 days a f f o r d e d 8 g of hyphae ( d r y w e i g h t ) per l i t r e o f c u l t u r e medium. Hyphal m a t t e r was u n i f o r m l y i n c o r p o r a t e d i n t o a m e r i d i c i n s e c t d i e t (1_2) f o r b i o a s s a y , by g r i n d i n g t h e two l y o p h i l i z e d m a t e r i a l s t o g e t h e r and then r e c o n s t i t u t i n g t h e d i e t by a d d i t i o n of water (A.W. Thomas, Canadian F o r e s t r y S e r v i c e - M a r i t i m e s , p e r s o n a l communication, 1985). Spruce budworm l a r v a e , r e a r e d from the second i n s t a r f o r 14 days on d i e t c o n t a i n i n g λ% (wet w e i g h t ) ground hyphae o f FA 120 s u f f e r e d more than 50% m o r t a l i t y , and s u r v i v i n g i n s e c t s were e n e r v a t e d and r e t a r d e d i n t h e i r development r e l a t i v e t o c o n t r o l s ( o f which o n l y 7% d i e d ) . An e x t r a c t of the c u l t u r e f i l t r a t e was a l s o somewhat t o x i c t o spruce budworm l a r v a e , but t h i s was not i n v e s t i g a t e d f u r t h e r because the amount o f m a t e r i a l a v a i l a b l e was s m a l l and i t s a c t i v i t y modest. S t a n d a r d p r o c e d u r e s were f o l l o w e d f o r i s o l a t i o n o f the t o x i c p r i n c i p l e s from mycelium o f FA 120. A methylene c h l o r i d e e x t r a c t o f the f r e e z e - d r i e d hyphae was i n i t i a l l y p a r t i t i o n e d between hexane and aqueous methanol t o s e p a r a t e l i p i d s from more p o l a r m a t e r i a l . B i o a s s a y - m o n i t o r e d c h r o m a t o g r a p h i c f r a c t i o n a t i o n o f the h e x a n e - s o l u b l e m a t e r i a l l e d t o t h e i s o l a t i o n of a f r a c t i o n ( c a . 5? of t h e h y p h a l w e i g h t ) which c o u l d account f o r much o f the t o x i c i t y o f the hyphae of FA 120 t o spruce budworm l a r v a e . The s p e c t r o s c o p i c and c h e m i c a l p r o p e r t i e s of t h i s m a t e r i a l were c h a r a c t e r i s t i c o f the e n n i a t i n s , a group of c y c l i c h e x a d e p s i p e p t i d e ionophore a n t i b i o t i c s produced by s e v e r a l p l a n t p a t h o g e n i c Fusarium s p e c i e s , i n c l u d i n g F. l a t e r i t i u m
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k c u l t u r e o f FA 120 i s d e p o s i t e d i n t h e herbarium a t the B i o s y s t e m a t i c s Research I n s t i t u t e , Ottawa, O n t a r i o (DA0M #196490)
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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BIOTECHNOLOGY FOR CROP PROTECTION
and F. sambucinum (13~18) (Strongman, D.W., S t r u n z , G.M., Giguère, P., Yu, CM., and Calhoun, L., J . Chem. E c o l . I n p r e s s , 1988). Whereas s o l v e n t p a r t i t i o n l e f t most of the e n n i a t i n f r a c t i o n among the l i p i d s , the balance of t h i s m a t e r i a l was f o u n d , w i t h fewer com e t a b o l i t e s , i n the s m a l l e r aqueous methanol e x t r a c t . The e n n i a t i n s are composed of t h r e e N-methyl-L-amino a c i d r e s i dues l i n k e d i n an a l t e r n a t i n g f a s h i o n w i t h t h r e e r e s i d u e s of D-2h y d r o x y i s o v a l e r i c a c i d t o form an 18-membered c y c l i c s t r u c t u r e 1, i n which the R groups may be P r , B u , or B u . They a r e u s u a l l y i s o l a t e d as complexes which may c o n t a i n e n n i a t i n A (1; R R = R = B u ) , A (1; R ^ R ^ B u , R ^ P r ) , B (1; R ^ B u , R =R = P r ) and Β (1; R = R = R = P r ) (2£). The n a t u r a l occurrence of an isomer of e n n i a t i n A, d e s i g n a t e d e n n i a t i n C (1; R =R =R =Bu ), was i n f e r r e d from the d e t e c t i o n of N - m e t h y l l e u c i n e among the h y d r o l y s i s products of some 1
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Downloaded by AUBURN UNIV on June 21, 2016 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch008
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1 n a t u r a l e n n i a t i n m i x t u r e s ( 1j>). E n n i a t i n C, as w e l l as o t h e r e n n i a t i n s have been s y n t h e s i z e d ( 2 0 ) . C o n s i d e r i n g the d i f f i c u l t y o f sepa r a t i n g e n n i a t i n m i x t u r e s from n a t u r a l s o u r c e s i n t o t h e i r i n d i v i d u a l components, the presence of N - m e t h y l l e u c i n e i n the h y d r o l y s a t e s may suggest f u r t h e r the e x i s t e n c e of a s e r i e s of e n n i a t i n s c o n t a i n i n g t h i s amino a c i d i n p l a c e of one or more N - m e t h y l i s o l e u c i n e r e s i d u e s , (cf.1_5). E n n i a t i n Β has been o b t a i n e d i n a pure s t a t e from a s p e c i e s of Fusarium (13, 14), and a procedure was developed f o r s e p a r a t i o n of e n n i a t i n A from the complex produced by F. sambucinum (1_9). The r e s u l t i n g p u r i f i e d e n n i a t i n A was shown t o c o n t a i n both t h r e o and erythro diastereomeric N-methylisoleucine residues (19).
Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
8.
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Insecticidal Metabolites
STRUNZ AND STRONGMAN
When i t i s i m p r a c t i c a l o r unnecessary t o r e s o l v e the e n n i a t i n complex i n t o i t s c o n s t i t u e n t s , the approximate c o m p o s i t i o n of the m i x t u r e may u s u a l l y be e s t i m a t e d on the b a s i s of mass s p e c t r o m e t r y (21, 22) and h y d r o l y s i s experiments. The mass spectrum of the e n n i a t i n complex from chromatography o f the FA 120 e x t r a c t showed peaks c o r r e s p o n d i n g t o the m o l e c u l a r i o n s of e n n i a t i n s A, A and B w i t h r e l a t i v e i n t e n s i t i e s o f 3.90, 4.35, and 2.04$, r e s p e c t i v e l y . A s i n g l e c r y s t a l l i z a t i o n from aqueous e t h a n o l e f f e c t e d a s i g n i f i c a n t change i n the c o m p o s i t i o n of the complex, m a n i f e s t e d i n the new v a l u e s o f 3.85, 1.44, and
