Fungicidal Activity of Naturally Occurring Photosensitizers - ACS

Chapter 16

Fungicidal Activity of Naturally Occurring Photosensitizers G. H. Neil Towers and Donald E. Champagne Department of Botany, University of British Columbia, Vancouver,

Downloaded by HARVARD UNIV on October 17, 2015 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch016

British Columbia V6T 2B1, Canada

In recent years a d i v e r s i t y of natural photosensitizers with in v i t r o f u n g i c i d a l a c t i v i t y has been described. Many type I photosensitizers photobind to DNA, including linear and angular furanocoumarins, furanochromones, furanoquinolines, and probably the ß-carboline a l k a l o i d s . Isoflavonoids and a l i p h a t i c polyacetylenes attack membrane targets v i a a free r a d i c a l mechanism. Aromatic polyacetylenes display competing type I and type II reactions and the thiophenes are strictly photodynamic s e n s i t i z e r s . The evidence f o r these toxic mechanisms i s discussed, and evidence f o r the involvement of these phytochemicals resistance to fungal attack i n vivo i s reviewed. D a n i e l s (J_) has d e s c r i b e d a s i m p l e and economic p r o c e d u r e which p e r m i t s t h e r a p i d s c r e e n i n g o f p l a n t s and p l a n t e x t r a c t s f o r photot o x i c a c t i v i t y . T h i s t e c h n i q u e was o r i g i n a l l y used t o i d e n t i f y f u r a nocoumarins as t h e compounds r e s p o n s i b l e f o r t h e p h o t o s e n s i t i z i n g action of various umbelliferous plants. More r e c e n t l y , an ever increasing number o f p h o t o t o x i c secondary m e t a b o l i t e s , i n c l u d i n g a l k a l o i d s , p h e n o l i c s , q u i n o n e s , t e r p e n o i d s , and a c e t y l e n e s and t h e i r t h i o p h e n e d e r i v a t i v e s have been i s o l a t e d from v a s c u l a r p l a n t s , f u n g i and b a c t e r i a . As y e a s t s , ( p a r t i c u l a r l y Candida, Saccharomyces, and R h o d o t o r u l a ) , and o t h e r f u n g i , a r e used i n these a s s a y s , most o f t h e known p h o t o s e n s i t i z e r s a r e f u n g i c i d a l , a l t h o u g h i t i s n o t always c l e a r t h a t such a c t i v i t y r e f l e c t s t h e r o l e o f t h e s e compounds i n t h e plant. P h o t o s e n s i t i z e r s v a r y i n b o t h t h e i r mechanisms o f a c t i o n and t a r g e t s i t e s w i t h i n the c e l l . TJie two mechanisms r e c o g n i z e d , termed t y p e I and type I I , a r e reviewed b y C S . Foote elsewhere i n t h i s volume and w i l l be d e s c r i b e d o n l y b r i e f l y h e r e . In b o t h type I and t y p e I I r e a c t i o n s the g r o u n d - s t a t e s e n s i t i z e r i s p h o t o e x c i t e d t o t h e u n s t a b l e s i n g l e t s t a t e , f o l l o w e d by i n t e r s y s t e m c r o s s i n g t o y i e l d the l o n g e r - l i v e d t r i p l e t s e n s i t i z e r (2^_3). i n type I r e a c t i o n s t h e triplet sensitizer participates i n radical or electron transfer r e a c t i o n s w i t h s u s c e p t i b l e b i o m o l e c u l e s , thus consuming t h e s e n s i tizer. Type I I s e n s i t i z a t i o n s i n v o l v e t h e t r a n s f e r o f e x c i t a t i o n

0097-6156/87/0339-0231 $06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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energy from the triplet s e n s i t i z e r to ground-state (triplet) m o l e c u l a r oxygen, r e t u r n i n g the s e n s i t i z e r to i t s ground s t a t e and generating s i n g l e t oxygen. The s e n s i t i z e r may subsequently be r e e x c i t e d and c a t a l y z e f u r t h e r r e a c t i o n s . As t y p e I I s e n s i t i z a t i o n s r e q u i r e the p a r t i c i p a t i o n o f m o l e c u l a r oxygen, the a c t i v i t y o f such p h o t o s e n s i t i z e r s i s a b o l i s h e d under a n a e r o b i c c o n d i t i o n s and can be modified by a z i d e o r D^O, which a l t e r the l i f e t i m e of s i n g l e t oxygen. Some p h o t o s e n s i t i z e r s d i s p l a y an i n t e r m e d i a t e mechanism i n which b o t h t y p e I and type I I r e a c t i o n s o c c u r c o m p e t i t i v e l y . P o t e n t i a l l y p h o t o t o x i c s e c o n d a r y m e t a b o l i t e s a r e known t o o c c u r i n over t h i r t y f a m i l i e s o f v a s c u l a r p l a n t s ; i n some f a m i l i e s a s i n g l e s p e c i e s may elaborate s e v e r a l c l a s s e s of p h o t o s e n s i t i z e r s d e r i v e d from independant b i o s y n t h e t i c r o u t e s (3) . The f u n c t i o n o f t h e s e compounds i s not easy t o e s t a b l i s h , but t h e i r broad-spectrum b i o c i d a l a c t i v i t y a g a i n s t n o t o n l y f u n g i but a l s o b a c t e r i a and i n s e c t s , and t h e i r f r e q u e n t i n v o l v e m e n t i n p h y t o a l e x i n responses, s t r o n g l y s u g g e s t s t h a t t h e y f u n c t i o n as p a r t o f a generalized d e f e n s e a g a i n s t pathogens and h e r b i v o r e s . Of c o u r s e , t h i s does not p r e c l u d e the p o s s i b i l i t y o f s i m u l t a n e o u s f u n c t i o n s i n v o l v i n g p r i m a r y m e t a b o l i s m as w e l l . Type I

Photosensitizers

Type I p h o t o s e n s i t i z e r s may c o v a l e n t l y b i n d t o a v a r i e t y o f s u s c e p t i b l e t a r g e t m o l e c u l e s , i n c l u d i n g p r o t e i n s and tRNA ( / but the m a j o r i t y appear t o form ad d u c t s with DNA, and so may be termed photogenotoxic (9-11) . Such compounds are typically planar, t r i c y c l i c molecules. The b e s t known and f i r s t d e s c r i b e d o f the p h o t o g e n o t o x i n s a r e the furanocoumarins, c h a r a c t e r i s t i c secondary metabolites of the Rutaceae, Apiaceae ( U m b e l l i f e r a e ) , and c e r t a i n other f a m i l i e s of flowering plants. Daniels (_1_) f i r s t showed t h a t f u r a n o c o u m a r i n s cause l e t h a l damage t o y e a s t s i n l i g h t , and t h i s was subsequently c o n f i r m e d i n numerous studies with y e a s t s and other fungi (1220) . T o x i c i t y r e s u l t s m a i n l y from p h o t o b i n d i n g to the pyrimidine b a s e s o f DNA by means o f double bonds a t the 3 , 4 and 4 ' , 5 ' sites, forming monoadducts (21,22) or, in the case of some linear psoralens, b i f u n c t i o n a l adducts leading to interstrand crosslinkages (23-27). This photoactivity i s clearly ecologically relevant, as furanocoumarins are involved in the phytoalexin r e s p o n s e t o f u n g a l i n f e c t i o n i n some u m b e l l i f e r s ( 2 8 - 3 0 ) and these compounds can p h o t o s e n s i t i z e i n s e c t s and o t h e r h e r b i v o r e s (3J_) • Other compounds which d i s p l a y t h i s type o f a c t i v i t y a r e the furanochromones (32) , furanochromenes (33), furanoquinolines, and certain tryptophan-derived a l k a l o i d s i n c l u d i n g the p-carbolines (34-36)• The best u n d e r s t o o d o f t h e s e are the furanoquinoline a l k a l o i d s , p a r t i c u l a r l y d i c t a m n i n e ( I ) , which o c c u r i n a number o f r u t a c e o u s s p e c i e s i n c l u d i n g Skimmia j a p o n i c a and Dictamnus a l b u s (37) . D i c t a m n i n e , skimmianine ( I I ) , m a c u l o s i d e , and m a c u l i n e were phototoxic to the yeasts Saccharomyces c e r e v i s i a e and Candida a l b i c a n s i n UVA (36); d i c t a m n i n e was a l s o p h o t o t o x i c to filamentous p h y t o p a r a s i t i c and z o o p a r a s i t i c f u n g i i n c l u d i n g Mucor h i e m a l i s , M. rammanianus, Fusariurn g r a m i n e a r u s , and P e n i c i l l i u m i t a l i c u m (38) . Both d i c t a m n i n e and skimmianine i n h i b i t e d m i t o s i s and caused g r o s s


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chromosomal changes t o C h i n e s e hamster o v a r y (CHO) c e l l s i n UVA but n e t i n the d a r k , s u g g e s t i n g a c e l l u l a r t a r g e t i n the n u c l e u s , s i m i l a r t o the f u r a n o c o u m a r i n s (39^) • S u b s e q u e n t l y i t was shown t h a t [ H ] - d i c t a m n i n e i n t e r c a l a t e s w i t h c a l f - t h y m u s DNA i n the d a r k , and a f t e r near-UV i r r a d a t i o n and g e l f i l t r a t i o n t o s e p a r a t e the unbound a l k a l o i d , the l a b e l was found t o be bound t o the DNA (40) . H y d r o x y a p a t i t e chromatography o f h e a t - d e n a t u r e d [ H]-dictamnine-DNA complex showed o n l y s i n g l e - s t r a n d e d DNA, i n d i c a t i n g the f o r m a t i o n o f monoadducts. S t u d i e s o f the p h o t o b i n d i n g o f d i c t a m n i n e towards v a r i o u s s y n t h e t i c DNA's showed t h a t the r a t i o o f b i n d i n g t o p o l y ( d A dT) . p o l y ( d A - d T ) :poly(dG-dC) • p o l y ( dG-dC) :poly( dA-dU) . p o l y (dA-U):p o l y ( d A ) • p o l y ( d T ) , i n r e l a t i o n t o t h a t o f c a l f thymus DNA, was 18:1:0.5:0.3/ s i m i l a r t o the r a t i o o b s e r v e d f o r the f u r a n o c o u m a r i n 8-methoxypsoralen (8-MOP). In a d d i t i o n , p r i o r t r e a t m e n t o f DNA w i t h d i c t a m n i n e g r e a t l y r e d u c e d i n c o r p o r a t i o n o f 8-MOP, s u g g e s t i n g t h a t the b i n d i n g s i t e s f o r the two compounds a r e p r o b a b l y i d e n t i c a l . Template a c t i v i t y o f the photomodified poly(dA-dT)•poly(dA-dT) DNA, measured by the RNA polymerase r e a c t i o n / was strongly inhibited; c a l f thymus DNA was l e s s s e v e r e l y a f f e c t e d . 3


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When c u l t u r e s o f Mucor h i e m a l i s were i n c u b a t e d with [ H]d i c t a m n i n e i n the l i g h t , 0.2% o f the a d m i n i s t e r e d l a b e l (0.18 ug-mg DNA" ) was i n c o r p o r a t e d i n t o the f u n g a l DNA i n v i v o (38) • The c h e m i s t r y o f the c o v a l e n t adducts o f dictamnine with n u c l e i c a c i d b a s e s has n o t been d e s c r i b e d . These f u r a n o q u i n o l i n e s may have a r o l e i n p r o t e c t i n g some p l a n t s a g a i n s t f u n g a l a t t a c k , but t h i s has y e t t o be demonstrated. The furanochromones khellin ( I I I ) and v i s n a g i n , t h e active p r i n c i p a l s o f the m e d i c i n a l p l a n t Ammi v i s n a g a ( 4 1 ) , a r e p h o t o t o x i c towards g r a m - p o s i t i v e b a c t e r i a (32/42)/ v i r u s e s (43) / and Saccharomyces and Candida ( 3 2 ) . K h e l l i n i n d u c e s m e l a n i z a t i o n i n r a b b i t s k i n i n s u n l i g h t (4£) , and causes g r o s s chromosomal damage i n CHO c e l l s (32) . Longwave UV i r r a d i a t i o n o f a f r o z e n aqueous s u s p e n s i o n o f k h e l l i n and thymine r e s u l t e d i n the f o r m a t i o n o f a 2-2 adduct (IV) between the 2,3 double bond o f k h e l l i n and the 5*,6* double bond o f thymine, i n d i c a t i n g t h a t the p h o t o t o x i c i t y o f k h e l l i n i s due t o a mechanism s i m i l a r t o the f u r a n o c o u m a r i n s . The incorporation of furanochromones i n t o DNA i n v i v o has y e t t o be d e m o n s t r a t e d . Some furanochromones, i n c l u d i n g k h e l l i n / a r e a l s o known t o be insect antifeedants (45) , but as y e t we can o n l y s p e c u l a t e about the ecological significance of their phototoxic a c t i v i t y . S i m i l a r l y / t h e r o l e o f p h o t o t o x i c i t y i n the a c t i v i t y o f the p o t e n t c a r c i n o g e n s , the a f l a t o x i n s ( V a , b ) , p r o d u c e d by A s p e r g i l l u s f l a v u s and r e l a t e d s p e c i e s (46) i s n o t a p p a r e n t . A f l a t o x i n s are p h o t o t o x i c t o Paramecium but n o t t o E* c o l i (47) , and have n o t y e t been t e s t e d f o r p h o t o t o x i c i t y a g a i n s t f u n g i . In the mammalian l i v e r (and hence i n the dark) t h e y a r e c o n v e r t e d t o h e p a t o c a r c i n o g e n s when t h e double bond o f the f u r a n r i n g i s e p o x i d i z e d and the p r o d u c t s u b s e q u e n t l y forms c o v a l e n t a d d u c t s t o DNA (48) . E x c i t a t i o n by UVA (365 nm) a l s o i n d u c e s the f o r m a t i o n o f adducts i n v i t r o ( 4 9 ) . The p - c a r b o l i n e o r harmane a l k a l o i d s ( V I ) , the a l k a l o i d s 6-cant h i n o n e and 5-methoxy-6-canthinone ( a l l from v a r i o u s R i t a c e a e ) and the N-methyl s u b s t i t u t e d harmane b r e v i c o l l i n e from the sedge Car ex b r e v i c o l l i s are p h o t o t o x i c to Saccharomyces and Candida ( 3 4 ) . S t r u c t u r a l s i m i l a r i t y t o the f u r a n o c o u m a r i n s s u g g e s t s t h a t t h e y t o o may 1



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p h o t o b i n d t o DNA; t h i s work i s p r e s e n t l y underway i n our l a b o r a t o r y . The i s o f l a v o n o i d p h y t o a l e x i n s p h a s e o l l i n , 3,6a,9- t r i h y d r o x y p t e r o c a r p a n , g l y c e o l l i n , t u b e r o s i n , and p i s a t i n , p r o d u c e d by s e v e r a l s p e c i e s o f Eabaceae, p h o t o i n a c t i v a t e d g l u c o s e - 6 - p h o s p h a t e dehydro genase i n an in v i t r o a s s a y (50_) • S i n g l e t oxygen q u e n c h e r s , h y d r o x y l r a d i c a l s c a v e n g e r s , and s u p e r o x i d e dismutase d i d n o t p r o t e c t t h e enzyme a g a i n s t p h o t o i n a c t i v a t i o n , r u l i n g out a type I I mechanism. ESR measurements c o n f i r m e d t h e p r o d u c t i o n o f f r e e r a d i c a l s , which were most s t a b l e i n t h e case o f p h a s e o l l i n . In t h e d a r k , g l y c e o l l i n i n h i b i t s e l e c t r o n t r a n s p o r t a t some p o i n t beyond t h e s u c c i n a t e dehydrogenase s i t e ( 5 1 ) , and p i s a t i n appears t o u n c o u p l e o x i d a t i v e phosphorylation (52). The a l i p h a t i c polyacetylenes increase membrane p e r m e a b i l i t y and a r e h i g h l y t o x i c t o Saccharomyces and o t h e r y e a s t s ; t h e i r t o x i c i t y i s not oxygen dependent and t h e i r r a p i d p o l y m e r i z a t i o n i n UVA has been taken as e v i d e n c e o f f r e e r a d i c a l f o r m a t i o n (53) • Some chromenes and b e n z o f u r a n s , i n c l u d i n g e n c e c a l i n (VII), are phototoxic t o Saccharomyces and Candida (33) . These compounds cause h e m o l y s i s o f e r y t h r o c y t e s (54) i n d i c a t i n g a t a r g e t i n the membrane, and may o p e r a t e v i a a f r e e r a d i c a l mechanism. Intermediate

Photosensitizers

Many p h o t o s e n s i t i z e r s a r e c a p a b l e o f competing type I and type I I reactions. Even 8-MOP, l o n g c o n s i d e r e d a c l a s s i c a l type I p h o t o s e n s i t i z e r , g e n e r a t e s s i n g l e t oxygen i n t h e absence o f s u i t a b l e s i t e s for photobinding (S5). The r e l a t i v e s i g n i f i c a n c e o f t h e two mechanisms i s i l l u s t r a t e d by t h e o b s e r v a t i o n t h a t JE. c o l i mutants d e f e c t i v e i n t h e r e p a i r o f o x i d a t i v e damage a r e about 15 t i m e s l e s s s e n s i t i v e t o 8-MOP i n d u c e d damage than a r e e x c i s i o n r e p a i r d e f i c i e n t m u t a n t s , b u t a r e about 100 t i m e s more s e n s i t i v e than w i l d type s t r a i n s (56). Competing type I and type I I mechanisms a r e t y p i c a l o f t h e aromatic p o l y a c e t y l e n e s , c h a r a c t e r i s t i c secondary m e t a b o l i t e s o f the Asteraceae and about twenty o t h e r families o f vascular plants. E a r l y work on t h e p o l y a c e t y l e n e phenylheptatriyne (PHT) ( V I I I ) showed reduced t o x i c i t y t o Saccharomyces c e r e v i s i a e under a e r o b i c c o n d i t i o n s , and t o x i c i t y was n o t m o d i f i e d by a z i d e (which quenches s i n g l e t oxygen) o r E^O (which i n c r e a s e s t h e l i f e t i m e o f s i n g l e t oxygen) ( 5 7 ) . Low c o n c e n t r a t i o n s o f PHT r a p i d l y i n h i b i t e d cell r e s p i r a t i o n , and d i d n o t i n c r e a s e s i s t e r c h r o m a t i d exchanges ( 5 8 ) , i n d i c a t i n g t h a t t h e n u c l e u s was n o t a t a r g e t ; t h i s compound thus d i d not resemble e i t h e r t h e f u r a n o c o u m a r i n s o r t h e photodynamic dyes i n its mechanism of action. To c o m p l i c a t e the s t o r y , i t was subsequently found t h a t PHT and o t h e r p o l y a c e t y l e n e s a r e p h o t o dynamic towards some o r g a n i s m s , i n c l u d i n g E. c o l i , b u t a r e p a r t i a l l y non-photodynamic i n o t h e r systems, i n c l u d i n g Saccharomyces (53) • With l i p o s o m e s as a model membrane system, o n l y t h e photodynamic a c t i v i t y c o u l d be demonstrated ( 5 9 ) . The e f f e c t o f PHT on membrane p e r m e a b i l i t y was shown t o depend on t h e degree o f u n s a t u r a t i o n o f the membrane lipids. Permeability was g r e a t l y i n c r e a s e d i n l i p o s o m e s composed o f d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e (PC) and o t h e r s a t u r a t e d l i p i d s , which p r e s e n t a h i g h l y o r d e r e d environment, and was i n c r e a s e d t o a l e s s e r e x t e n t i n d i s o r d e r e d membranes composed o f u n s a t u r a t e d l i p i d s ; i n t h e l a t t e r case l i p i d p e r o x i d a t i o n was shown



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to be i n v o l v e d . The d i f f e r e n t e f f e c t s o f PHT i n d i f f e r e n t o r g a n i s m s may be r e l a t e d t o d i f f e r i n g l i p i d environments i n the membranes, but PHT also inactivates membrane-bound enzymes (60) and so the r e s p o n s e s may a l s o be r e l a t e d t o a c c e s s i b i l i t y o f t a r g e t p r o t e i n s , possibly including respiratory centers. Photochemical s t u d i e s p r o v i d e f u r t h e r i n f o r m a t i o n on competing t y p e I and type I I r e a c t i o n s w i t h PHT. L a s e r e x c i t a t i o n (308 or 337 nm) l e a d s t o the f o r m a t i o n o f a s t r o n g t r i p l e t s i g n a l , w i t h a l i f e t i m e o f 28 us i n m e t h a n o l , which was e f f i c i e n t l y quenched by t h e t r i p l e t quencher 1,3 o c t a d i e n e (61) . Quenching w i t h occurred w i t h a r a t e c o n s t a n t comparable t o the r a t e o f e l e c t r o n t r a n s f e r t o methyl v i o l o g e n . The formation of both s i n g l e t oxygen and the semioxidized PHT radical are c o n s i s t e n t with the competing mechanisms o b s e r v e d in vivo. When i r r a d i a t e d w i t h UVA, liposome b i l a y e r s composed o f d i s t e a r y l PC w i t h PHT p r o d u c e d a f r e e r a d i c a l s i g n a l d e t e c t e d by e l e c t r o n s p i n r e s o n a n c e (ESR) s p e c t r o s c o p y ( 6 2 ) • The spectrum c o n s i s t e d o f one broad peak w i t h a l i n e width o f 19G and a g v a l u e o f 2.0017. The r a t e o f f o r m a t i o n was dependent on light intensity, PHT concentration within the bilayer, and concentration of the liposome-PHT s u s p e n s i o n . The signal was enhanced i n an a n a e r o b i c environment, i n d i c a t i n g a n o n - o x i d a t i v e mechanism f o r f r e e r a d i c a l f o r m a t i o n . Cnce formed, the radical s p e c i e s was v e r y s t a b l e i n the p r e s e n c e o f oxygen, d e c a y i n g s l o w l y o v e r an 8-12 hour p e r i o d . F o r m a t i o n o f the r a d i c a l was enhanced i n an o r d e r e d l i p i d environment as i n c o r p o r a t i o n o f l y s o p h o s p h a t i d y l c h o l i n e , which p e r t u r b s l i p i d p a c k i n g , d e c r e a s e d l e v e l s o f the f r e e radical species. When PHT was p r e s e n t i n a l i p o s o m e w i t h an even more f l u i d membrane, such as egg y o l k PC, the l e v e l s o f f r e e r a d i c a l g e n e r a t i o n were even l o w e r . P o l y a c e t y l e n e involvement i n r e s i s t a n c e to fungal a t t a c k i s well d o c u m e n t e d , and i n c l u d e s the phytoalexins safynol and d e h y d r o s a f y n o l from Carthamnus t i n i c t o r i u s (63^,64), wyerone from the broad bean, V i c i a faba (65) , and falcarinol, falcarindiol, and E - t e t r a d e c a - 6 - e n e - 1 , 3 - d i y n e - 5 , 8 - d i o l from the tomato, L y c o p e r s i c o n e s c u l e n t u m (66) • These p h y t o a l e x i n s a r e t o x i c w i t h o u t p h o t o a c t i v a t i o n , but t h e i r a c t i v i t y may, i n some c a s e s , be enhanced i n UVA. Few s t u d i e s s p e c i f i c a l l y a d d r e s s the r o l e o f p o l y a c e t y l e n e p h o t o s e n s i t i z a t i o n i n defense a g a i n s t f u n g a l a t t a c k . PHT, p r e s e n t i n the c u t i c l e o f Bidens p i l o s a l e a v e s a t c o n c e n t r a t i o n s up t o 600 ppm, s t r o n g l y i n h i b i t s the g e r m i n a t i o n and growth o f F u s a r i u m culmorum i n UVA but not i n the d a r k ; PHT was f u n g i t o x i c and not s i m p l y f u n g i s t a t i c (67). In t h i s case the p o l y a c e t y l e n e c o n s t i t u t e s a p r e f o r m e d barrier against fungal attack. Nineteen species of phylloplane yeasts and yeast-like fungi, isolated from Hawaiian s p e c i e s o f B i d e n s w i t h and w i t h o u t l e a f p o l y a c e t y l e n e s , were t e s t e d f o r p h o t o s e n s i t i v i t y t o those a c e t y l e n e s (68) • A l t h o u g h a l l t h e s e o r g a n i s m s , members of the Sfcorobolmycetaceae, Cryptococcaceae, and E\ingi Imperfect!, were s e n s i t i v e t o some a c e t y l e n e s and r e s i s t a n t t o o t h e r s , t h e r e was no c o r r e l a t i o n between the p r e s e n c e o r absence o f leaf polyacetylenes and the distribution of these saprophytes amongst the B i d e n s s p e c i e s , w i t h one n o t a b l e e x c e p t i o n . The o n l y p a t h o g e n i c s p e c i e s i s o l a t e d , C o l l e t o t r i c h u m g l o e o s p o r i o d e s , d i d not c o l o n i z e B i d e n s l e a v e s c o n t a i n i n g C13 a r o m a t i c p o l y a c e t y l e n e s , t o which i t i s e x t r e m l y s e n s i t i v e i n v i t r o .



236


Type I I P h o t o s e n s i t i z e r s The c l a s s i c type I I s e n s i t i z e r s a r e the photodynamic dyes; t h e most active natural type II s e n s i t i z e r s yet d i s c o v e r e d are the thiophenes, sulfur derivatives of the polyacetylenes. These compounds a r e r e s t r i c t e d t o advanced t r i b e s o f the A s t e r a c e a e , including the Vernonieae, Inuleae, Heliantheae, Anthemideae, S e n e c i o n e a e , Cynareae, and p a r t i c u l a r l y t h e Tageteae (69) . Alphat e r t h i e n y l (a-T) (IX) i s t o x i c t o b a c t e r i a , y e a s t s , and o t h e r f u n g i ( 1 1 ) , and can p h o t o s e n s i t i z e h e r b i v o r o u s i n s e c t s ( 7 0 , 7 1 ) . A strict r e q u i r e m e n t f o r oxygen i n a-T t o x i c i t y has been demonstrated b o t h i n vivo (60,72-74) and in vitro (75-77) . Although the specific c e l l u l a r t a r g e t i n v o l v e d has n o t been i d e n t i f i e d , i t i s c e r t a i n l y i n t h e membrane as a-T i s not p h o t o g e n o t o x i c , and i t i n c r e a s e s membrane p e r m e a b i l i t y and i n a c t i v a t e s membrane-bound enzymes (60,74)• The membrane-bounded v i r u s e s murine c y t o m e g a l o v i r u s (CMV) and SLndbis v i r u s were s e n s i t i v e t o a-T a t 10 ug ml i n UVA but n o t i n the d a r k , b u t the membraneless v i r u s T4 was much more r e s i s t a n t (78) • Murine CMV which had been i n a c t i v a t e d by a-T and UVA p e n e t r a t e d mouse c e l l s e f f i c i e n t l y but the v i r a l DNA c o u l d not r e p l i c a t e and l a t e r v i r a l p r o t e i n s were n o t p r o d u c e d . As v i r a l gene e x p r e s s i o n was i n h i b i t e d i t was s u g g e s t e d t h a t a-T may i n t e r a c t w i t h v i r a l p r o t e i n s as w e l l as membrane l i p i d s ; a-T c e r t a i n l y o x i d i z e s p r o t e i n s i n IS. c o l i (74) • The t h i o p h e n e s a r e i n v o l v e d i n d e f e n s e a g a i n s t f u n g a l a t t a c k i n Tagetes a t l e a s t . I n n o c u l a t i o n o f T a g e t e s e r e c t a w i t h the pathogen Fusarium oxysporum e l i c i t s t w e l v e - f o l d g r e a t e r p r o d u c t i o n o f a-T, b i t h i o p h e n e h y d r o x y l , and b i t h i o p h e n e a c e t a t e (19) . Two s t r a i n s o f t h e pathogen a r e known; t h e f a s t - g r o w i n g v i r u l e n t s t r a i n k i l l s t h e p l a n t b e f o r e s i g n i f i c a n t e l e v a t i o n o f the t h i o p h e n e l e v e l s can be a c c o m p l i s h e d , but t h e s l o w e r - g r o w i n g s t r a i n i s e f f e c t i v e l y combatted by t h e s e f u n g i c i d a l compounds. R e c e n t l y we have d e s c r i b e d the a n t i f u n g a l a c t i v i t y o f a group o f a c e t y l e n i c d i t h i a c y c l o h e x a d i e n e s , from C h a e n a c t i s d o u g l a s i i and o t h e r members o f t h e Asteraceae (80). These r e d compounds, c h r i s t e n e d t h i a r u b r i n e s ( X ) , do n o t r e q u i r e l i g h t f o r t h e i r a n t i fungal a c t i v i t y . However, when i r r a d i a t e d w i t h UVA, t h e i r a c t i v i t y i s enhanced and i s t h e n extended t o b a c t e r i a and v i r u s e s . In l i g h t t h e s e u n s t a b l e compounds l o s e one o f the s u l f u r atoms o f t h e r i n g and the r e s u l t a n t t h i o p h e n e then d i s p l a y s the photodynamic a c t i v i t y c h a r a c t e r i s t i c of t h i s c l a s s of phytochemicals. The dark a n t i f u n g a l a c t i v i t y remains u n e x p l a i n e d . The activity o f the fungal photosensitizer cercosporin, a d i h y d r o x y p e r y l e n e quinone produced by v a r i o u s C e r c o s p o r a s p e c i e s , i s r e v i e w e d by Daub e l s e w h e r e i n t h i s volume. This apparently c o n s t i t u t e s t h e f i r s t case i n which a f u n c t i o n o t h e r than d e f e n s e can be a s c r i b e d t o a p h o t o t o x i n . As C e r c o s p o r a hyphae do n o t p e n e t r a t e the c e l l s o f the h o s t p l a n t , c e r c o s p o r i n - m e d i a t e d l i p o p e r o x i d a t i o n o f h o s t membranes i s e s s e n t i a l t o r e l e a s e n u t r i e n t s r e q u i r e d by the growing pathogen. Hypericin, a related compound found i n most s p e c i e s o f Hypericum, i s a l s o a photodynamic p h o t o s e n s i t i z e r (81, P. R i o x , t h i s v o l u m e ) . The p h o t o t o x i n o c c u r s i n g l a n d u l a r t r i c h o m e s and i s e f f e c t i v e a g a i n s t i n s e c t s , but i t s s i g n i f i c a n c e as a p o s s i b l e a n t i f u n g a l compound i s unknown.


Fungicidal Activity


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238


Conclusion


The d i v e r s i t y o f p h o t o t o x i c compounds i s o l a t e d from p l a n t s , f u n g i , and b a c t e r i a i n r e c e n t y e a r s s u g g e s t s t h a t such compounds may be q u i t e common i n n a t u r e . P r o b a b l y hundreds o f such compounds remain t o be i d e n t i f i e d . Much work remains t o be done t o i d e n t i f y t h e t o x i c mechanism and t a r g e t s i t e s o f many o f t h e p h o t o s e n s i t i z e r s which a r e c u r r e n t l y known. The q u e s t i o n o f how o r g a n i s m s t h a t p r o d u c e p h o t o t o x i n s a v o i d a u t o t o x i c i t y has s c a r c e l y been a d d r e s s e d . Finally, the ecological significance o f phototoxic secondary m e t a b o l i t e s has o n l y begun t o be s t u d i e d , b u t g i v e n t h e d i v e r s i t y o f s p e c i e s which c o n t a i n t h e s e compounds many f a s c i n a t i n g i n t e r a c t i o n s await d e s c r i p t i o n .

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Fungicidal Activity of Naturally Occurring Photosensitizers - ACS

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