Light-Activated Antimicrobial Chemicals from Plants: Their Potential

Chapter 21

Light-Activated Antimicrobial Chemicals from Plants: Their Potential Role in Resistance to Disease-Causing Organisms 1

2

Kelsey R. Downum and Stan Nemec 1

Department of Biological Sciences, Florida International University, Miami, F L 33199 Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, F L 32803

Downloaded by EMORY UNIV on February 12, 2016 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch021

2

A variety of plants produce chemical constituents which are e f f e c t i v e , light-activated antimicrobials i n v i t r o . The role of such natural products i n s i t u remains unclear, but their potent biocidal a c t i v i t y suggests that they may represent an important biochemical defense against pathogenic organisms. The first part of t h i s chapter is used to examine what is known concerning the t o x i c i t y of furanocoumarin, polyacetylene and pterocarpan phototoxins toward various plant pathogens. Results of recent studies i n which the s u s c e p t i b i l i t y of nine fungal pathogens of the genus Citrus (Family Rutaceae) to leaf extracts and to various coumarins isolated from three Citrus species (C. limettoides, C. macrophylla and C. medica) are discussed i n the l a t t e r section.

I n a r e c e n t volume on p l a n t d i s e a s e , C o w l i n g and H o r s f a l l (1_) compared p l a n t defense w i t h the defense o f a m e d i e v a l c a s t l e . They p o i n t e d out t h a t h i g h e r p l a n t s , l i k e c a s t l e s , are immobile and must be p r e p a r e d t o p r o t e c t themselves from would-be a t t a c k e r s ( i . e . , h e r b i v o r e s and p o t e n t i a l pathogens) whenever c h a l l a n g e d . The f i r s t l i n e o f defense a g a i n s t i n v a d i n g armies f o r c a s t l e d w e l l e r s was an o u t e r c a s t l e w a l l . E x t e r n a l p l a n t s u r f a c e s ( e . g . , the c u t i c l e , e p i d e r m a l c e l l s , b a r k , e t c . ) s e r v e a s i m i l a r f u n c t i o n and g e n e r a l y p r e c l u d e p a t h o g e n i c organisms from g a i n i n g e n t r a n c e t o i n t e r n a l t i s s u e s . A v a r i e t y o f measures w i t h i n c a s t l e s were a l s o i n p l a c e t o defend c a s t l e i n h a b i t a n t s s h o u l d the o u t e r w a l l s be breached. Mazes of rooms and c o r r i d o r s , t r a p doors and doors w i t h s t u r d y l o c k s , as w e l l as the o c c a s i o n a l s e c r e t passage were i n t e n d e d t o impede t h e p r o g r e s s o f i n v a d i n g armies and a l l o w the c a s t l e d e f e n d e r s time t o regroup o r escape. P l a n t s a l s o have e l a b o r a t e i n t e r n a l d e f e n s e s which e f f e c t i v e l y p r e v e n t p e n e t r a t i o n by p o t e n t i a l pathogens. Such d e f e n s e s i n c l u d e a v a r i e t y o f p h y s i c a l b a r r i e r s , e.g., c e l l u l o s i c w a l l s , l i g n i f i e d and s u b e r i z e d t i s s u e s , as w e l l as a d i v e r s e a r r a y of t o x i c b i o c h e m i c a l s . 0097-6156/87/0339-0281 $06.00/0 © 1987 American Chemical Society

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

282

LIGHT-ACTIVATED PESTICIDES

Endogenous a n t i m i c r o b i a l s a r e among t h e most a c t i v e l y s t u d i e d of t h e v a r i o u s b i o c h e m i c a l defenses e v o l v e d by p l a n t s ( 2 - 7 ) . I n t h i s c h a p t e r , we w i l l r e v i e w what i s c u r r e n t l y known about t h e involvement o f s e v e r a l c l a s s e s o f l i g h t - a c t i v a t e d o r " s o l a r - p o w e r e d " a n t i m i c r o b i a l s i n p l a n t r e s i s t a n c e t o d i s e a s e - c a u s i n g organisms. The r e s u l t s o f r e c e n t work w i t h C i t r u s " p h o t o t o x i n s " o r " p h o t o s e n s i t i z e r s " and t h e i r e f f e c t s on f u n g a l pathogens o f t h a t genus a l s o w i l l be d e s c r i b e d . F i n a l l y , p h y t o c h e m i c a l c l a s s e s w h i c h may mediate p h o t o t o x i c a n t i m i c r o b i a l a c t i v i t y , but have y e t t o be examined f o r such b i o l o g i c a l a c t i o n , w i l l be p o i n t e d o u t .


P h o t o s e n s i t i z e r s As P l a n t D e f e n s i v e Agents P l a n t m e t a b o l i t e s from a t l e a s t t e n d i f f e r e n t p h y t o c h e m i c a l c l a s s e s a r e c a p a b l e o f l i g h t - e n h a n c e d t o x i c i t y and may be s y n t h e s i z e d by s p e c i e s i n as many as t e n p e r c e n t o f a l l l i v i n g p l a n t f a m i l i e s ( 8 ) . The c h e m i s t r y , d i s t r i b u t i o n , t o x i c o l o g y and e c o l o g i c a l importance o f many o f these b i o l o g i c a l l y a c t i v e m e t a b o l i t e s a r e d i s c u s s e d i n s e v e r a l r e c e n t r e v i e w s ( 8 - 1 1 ) ; however, these r e p o r t s pay l i t t l e a t t e n t i o n t o t h e p o t e n t i a l importance o f endogenous p h o t o t o x i n s i n p l a n t defense a g a i n s t d i s e a s e - c a u s i n g organisms. The involvement o f v a r i o u s p h o t o t o x i c a n t i m i c r o b i a l f u r a n o c o u m a r i n s , p o l y a c e t y l e n e s and p t e r o c a r p a n s i n p l a n t r e s i s t a n c e a r e c o n s i d e r e d s e p a r a t e l y below. Furanocoumarins. P s o r a l e n s o r furanocoumarins o c c u r w i d e l y i n t h e p l a n t kingdom and a r e c h a r a c t e r i s t i c c o n s t i t u e n t s i n t h e A p i a c e a e ( U m b e l l i f e r e a e ) , Fabaceae (Leguminosae), Moraceae and Rutaceae among o t h e r f a m i l i e s (12.13). A p p r o x i m a t e l y 120 d i f f e r e n t d e r i v a t i v e s have been i s o l a t e d and i d e n t i f i e d ( 1 3 ) ; many a r e potent p h o t o t o x i n s c a p a b l e o f k i l l i n g o r i n h i b i t i n g t h e growth o f v i r u s e s , b a c t e r i a and f u n g i as w e l l as a f f e c t i n g a broad-spectrum o f h i g h e r organisms ( s e e r e f . 2. f o r r e v i e w ) . P h o t o a c t i v e furanoucoumarins r e q u i r e e x c i t a t i o n by near u l t r a v i o l e t o r UVA wavelengths (320-400 nm) f o r f u l l expression of t h e i r t o x i c a c t i o n , although light-independent a f f e c t s a r e a l s o known ( s e e d i s c u s s i o n by I v i e , t h i s volume). Covalent bonding t o DNA presumably accounts f o r most o f t h e i r p h o t o t o x i c consequences ( 1 4 ) ; however, p h o t o o x i d a t i v e c e l l u l a r damage ( r e s u l t i n g from e x c i t e d oxygen s p e c i e s ) and p h o t o b i n d i n g t o c e l l u l a r p r o t e i n s have a l s o been demonstrated (15-17). Furanocoumarins a r e g e n e r a l l y p r e s e n t i n h e a l t h y p l a n t t i s s u e s (13,18) where they may f u n c t i o n as preformed o r p r e i n f e c t i o n a l a n t i m i c r o b i a l s which i n h i b i t t h e e s t a b l i s h m e n t o f d i s e a s e - c a u s i n g organisms. S u p r i s i n g l y l i t t l e work has been conducted on t h e t o x i c i t y o f p s o r a l e n s toward p l a n t v i r u s e s , phytopathogenic b a c t e r i a o r f u n g i w h i c h might encounter t h e s e m e t a b o l i t e s i n _ s i t u d e s p i t e our u n d e r s t a n d i n g o f t h e i r p h o t o t o x i c i t y toward o t h e r organisms. The b a c t e r i u m A g r o b a c t e r i u m t u m e f a c i a n s and members o f s e v e r a l f u n g a l genera i n c l u d i n g B o t r y t i s , C e r a t o c y s t i s , S c l e r o t i n i a , Stereum and G l e o s p o r i u m a r e among t h e p h y t o p a t h o g e n i c organisms known t o be a f f e c t e d by v a r i o u s furanocoumarins (19-23). The a c c u m u l a t i o n o r enhanced b i o s y n t h e s i s o f furanocoumarins i n p l a n t t i s s u e s and c e l l s u s p e n s i o n c u l t u r e s exposed t o b i o t i c and a b i o t i c s t r e s s e s suggests t h e i r p o t e n t i a l f o r involvement i n p o s t i n f e c t i o n a l p l a n t responses. The c o n c e n t r a t i o n o f x a n t h o t o x i n


21.

DOWNUM AND NEMEC

Light-Activated Antimicrobial Chemicals

283

o r 8-methoxypsoralen (8-MOP; I I ) , one o f t h e most f r e q u e n t l y encountered f u r a n o c o u m a r i n p h o t o s e n s i t i z e r s , d r a m a t i c a l l y i n c r e a s e s i n d i s e a s e d c a r r o t t i s s u e (Daucus c a r o t a ) ( 2 4 ) , i n p a r s n i p (Pastinaca s a t i v a ) root d i s c s inoculated with C e r a t o c y s t i s f i m b r i a t a as w e l l as o t h e r nonpathogens o f p a r s n i p (21) and i n c e l e r y (Apium g r a v e o l a n s ) f o l l o w i n g i n f e c t i o n w i t h t h e " p i n k - r o t " fungus S c l e r o t i n i a s c l e r o t i o r u m ( 1 9 ) . The b i o s y n t h e s i s and a c c u m u l a t i o n o f o t h e r p h o t o t o x i c furanocoumarins i n c l u d i n g bergapten o r 5-methoxypsoralen (5-MOP; I ) ( i n c a r r o t and c e l e r y ) and 4 , 5 , 8 - t r i m e t h y l p s o r a l e n ( i n c e l e r y ) a r e a l s o induced f o l l o w i n g i n f e c t i o n by d i s e a s e - c a u s i n g organisms (24,25). C e l l suspension c u l t u r e s o f p a r s e l y ( P e t r o s e l i n u m h o r t e n s e ) exposed t o f u n g a l e l i c i t o r s accumulate 8-MOP i n a s i m i a r response (26,27). Downloaded by EMORY UNIV on February 12, 2016 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch021

1

P o l y a c e t y l e n e s and T h e i r D e r i v a t i v e s . P o l y a c e t y l e n e s and t h e i r d e r i v a t i v e s r e p r e s e n t one o f t h e l a r g e s t and most e x h a u s t i v e l y s t u d i e d c l a s s e s o f p l a n t m e t a b o l i t e s . More than 700 d i f f e r e n t s t r u c t u r e s r a n g i n g from s t r a i g h t - c h a i n p o l y a c e t y l e n i c m o l e c u l e s t o various r i n g s t a b i l i z e d structures are included i n t h i s c l a s s (28). These p h y t o c h e m i c a l s occur p r i m a r i l y among members o f t h e A p i a c e a e ( U m b e l l i f e r a e ) , A r a l i a c e a e , A s t e r a c e a e (Compositae), Campanulaceae, P i t t o s p o r a c e a e , Oleaceae and S a n t a l a c e a e ( 2 8 ) . A t l e a s t two o t h e r f a m i l i e s , t h e Fabaceae (Leguminosae) and t h e S o l a n a c e a e , have members t h a t produce a c e t y l e n e s ; however these m e t a b o l i t e s a r e s y n t h e s i z e d o n l y i n response t o i n f e c t i o n by p a t h o g e n i c organisms (29,30). Many p o l y a c e t y l e n e s a r e potent p h o t o s e n s i t i z e r s w h i c h can e n t e r an e x c i t e d s t a t e f o l l o w i n g a b s o r p t i o n o f l i g h t energy. I n t h i s e x c i t e d s t a t e , such m o l e c u l e s mediate a v a r i e t y o f broad-spectrum p h o t o t o x i c responses ( s e e 9-11 f o r r e v i e w s ) . Two modes o f a c t i o n have been s u g g e s t e d . S t r a i g h t - c h a i n a c e t y l e n i c m o l e c u l e s tend t o i n t e r a c t d i r e c t l y w i t h t a r g e t b i o m o l e c u l e s i n c e l l s through r a d i c a l mechanisms (31,32) w h i l e t h i o p h e n e s , s u l f u r - d e r i v a t i v e s o f v a r i o u s a c e t y l e n i c p r e c u r s o r s , mediate t h e o x i d a t i o n o f a v a r i e t y o f b i o m o l e c u l e s (membrane l i p i d s and p r o t e i n s i n p a r t i c u l a r ) presumably v i a s i n g l e t oxygen g e n e r a t i o n (32-34). These c o n t r a s t i n g mechanisms a p p a r e n t l y compete i n o t h e r r i n g s t a b i l i z e d a c e t y l e n e s ( 3 2 ) . L i g h t - i n d e p e n d e n t t o x i c i t y has been noted f o r a v a r i e t y o f a c e t y l e n i c m e t a b o l i t e s (31,35); however t h e a c t u a l mechanisms i n v o l v e d i n such i n t e r a c t i o n s have n o t been a d a q u a t e l y studied. The i n v o l v e m e n t o f p o l y a c e t y l e n i c m o l e c u l e s i n p l a n t d i s e a s e r e s i s t a n c e has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n . A t l e a s t 15 p h y t o c h e m i c a l s from t h i s c l a s s have presumed r o l e s as p r e - and/or p o s t i n f e c t i o n a l a n t i m i c r o b i a l agents ( T a b l e I ) . S e v e r a l p o l y a c e t y l e n e s and t h i o p h e n e s i s o l a t e d from members o f t h e A s t e r a c e a e may f u n c t i o n as preformed a n t i b i o t i c s . Such a r o l e i s suggested by t h e i r potent p h o t o t o x i c i t y toward phytopathogens (8,41,43) and because they can be i s o l a t e d from h e a l t h y p l a n t t i s s u e s (28,53,54). Other s t u d i e s r e p o r t t h a t t h e b i o s y n t h e s i s o f a c e t y l e n e s i n whole p l a n t s and i n t i s s u e c u l t u r e s can be s t i m u l a t e d by v a r i o u s f a c t o r s (42,43,55) w h i c h may i n d i c a t e t h e i r p o t e n t i a l i n v o l v e m e n t i n p o s t i n f e c t i o n a l d e f e n s i v e responses. Kourany (43) i n v e s t i g a t e d t h e f a t e o f v a r i o u s a c e t y l e n i c t h i o p h e n e s i n Tagetes


284


I. 5-Mtthoxypsoraltn; R , « O M t , R• H II. 8-Mtthoxyptoraltn; R-H,

R „ OMt -

III. 5,8-Dimtthoxypsoraltn; R>OMt, R-OMt


T a b l e 1.

P l a n t s which c o n t a i n a n t i f u n g a l p o l y a c e t y l e n e s i n d i c a t e d i n disease resistance

P l a n t Source

Polyacetylene

References

Aegopodium p o d a g r a r i a L. (Apiaceae) Daucus c a r o t a ( A p i a c e a e ) L y c o p e r s i c o n esculentum (Solanaceae)

Heptadeca-1,9-diene-4,6diyne-3-ol ( f a l c a r i n o l ) 29,36-38 Heptadeca-1,9-diene-4,6diyne-3,8-diol ( f a l c a r i n d i o l )

Dendropanax t r i f i d u s Makino (Araliaceae)

16-Hydroxyoctadeca-9,17-diene12,14-diynoic a c i d Octadeca-9,17-diene-12,14-diyn1,16-diol

B i d e n s p i l o s a L. (Asteraceae)

l-Phenylhepta-l,3,5-triyne

Tagetes e r e c t a L. Tagetes p a t u l a L. (Asteraceae)

2,2 :5 ,2"-Terthienyl 5-(4-Hydroxy-l-butenyl)-2, 2 -bithienyl 5-(4-Acetoxy-l-butenyl)-2, 2 -bithienyl 5-(Buten-3-ynyl)-2, 2 -bithienyl

l

39

40,41

t

f

8,42,43

,

f

Lycopersicon

esculentum

Tetradeca-6-ene-l,3-diyne5,8-diol

29,44

Carthamus t i n c t o r i s (Asteraceae)

Trideca-3,1l-diene-5,7,9triyne-l,2-diol (safynol) Trideca-1l-ene-3,5,7,9-tetrayne1,2-diol (dehydrosafynol)

Lens c u l i n a r i s (Fabaceae) Lens n i g r r i c a n s V i c i a f a b a (Fabaceae) + 31 o t h e r V i c i a s p e c i e s

Wyerone Wyerone A c i d Wyerone Epoxide


45-48

30,49-52


21.

DOWNUM AND NEMEC


285

e r e c t a L. ( t h e a f r i c a n m a r i g o l d ) i n response t o exposure t o s e v e r a l pathogens. I n o c u l a t i o n of s e e d l i n g s w i t h h i g h l y pathogenic s t r a i n s of A l t e r n a r i a t a g e t i c a and F u s a r i u m oxysporum f . s p . r a d i c i s l y c o p e r i s i c i l e d t o a g e n e r a l d e c r e a s e i n t h i o p h e n e l e v e l s compared t o c o n t r o l p l a n t s . I n f e c t i o n of p l a n t s w i t h Fusarium oxysporum v a r . c a l l i s t e p h i r a c e 2, a m o d e r a t e l y v i r u l e n t pathogen, r e s u l t e d i n a c c u m u l a t i o n of a l p h a - t e r t h i e n y l ( I V ) and two b i t h i o p h e n e d e r i v a t i v e s above l e v e l s encountered i n n o n - i n f e c t e d p l a n t s . In a r e l a t e d s p e c i e s , dwarf m a r i g o l d (Tagetes p a t u l a L.) p l a n t s and t i s s u e c u l t u r e s i n f e c t e d or t r a n s f o r m e d w i t h A g r o b a c t e r i u m t u m e f a c i e n s a l s o accumulated t h i o p h e n e s ( 4 2 ) . These s t u d i e s suggest t h a t t h i o p h e n e b i o s y n t h e s i s can be s t i m u l a t e d by m o d e r a t e l y v i r u l e n t pathogens (which may l e a d t o i n c r e a s e d p l a n t r e s i s t a n c e t o i n f e c t i o n ) , but t h a t h i g h l y pathogenic s p e c i e s may a v o i d t h i s p l a n t response by s u p p r e s s i n g the p r o d u c t i o n of these t o x i c b i o c h e m i c a l s . P h e n y l h e p t a t r i y n e (PHT, V ) , a p h o t o t o x i c p o l y a c e t y l e n e which o c c u r s i n h e a l t h y t i s s u e s of B i d e n s p i l o s a L. ( A s t e r a c e a e ) , may f u n c t i o n as a p r e i n f e c t i o n a l i n h i b i t o r s i m i l a r t o the t h i o p h e n e s of T. e r e c t a d i s c u s s e d above ( 4 1 ) . Recent work w i t h t i s s u e c u l t u r e s of JS. p i l o s a i n d i c a t e s t h a t s y n t h e s i s a l s o may be s t i m u l a t e d by a f u n g a l c u l t u r e - f i l t r a t e (55). Three d i a c e t y l e n e a l c o h o l s [ f a l c a r i n o l ( V I ) , f a l c a r i n d i o l and t e t r a d e c a - 6 - e n e - l , 3 - d i y n e - 5 , 8 - d i o l ] , two t r i a c e t y l e n e a l c o h o l s [ d e h y d r o s a f y n o l ( V I I ) and s a f y n o l ] and t h r e e f u r a n o a c e t y l e n e s [wyerone ( V I I I ) , wyerone a c i d and wyerone e p o x i d e ] are a l s o i m p o r t a n t a n t i f u n g a l m e t a b o l i t e s i m p l i c a t e d i n induced r e s i s t a n c e responses i n p l a n t s (30,46-48,50,56). F a l c a r i n o l and f a l c a r i n d i o l occur i n h e a l t h y t i s s u e of F a l c a r i a v u l g a r i s ( 2 8 ) , Daucus c a r o t a (37) and Aegopodium p o d a g r a r i a ( 5 7 ) . These m o l e c u l e s a l s o accumulate r a p i d l y i n c a r r o t r o o t t i s s u e f o l l o w i n g w o u n d - i n o c u l a t i o n w i t h B o t r y t i s c i n e r e a (38) and i n tomato i n f e c t e d w i t h C l a d o s p o r i u m fulvum (29). A t h i r d l i n e a r a c e t y l e n e , c i s - t e t r a d e c a - 6 - e n e - l , 3 - d i y n e - 5 , 8 - d i o l , c o - o c u r r s w i t h f a l c a r i n o l and f a l c a r i n d o l i n d i s e a s e d tomato ( 4 4 ) . S a f y n o l and d e h y d r o s a f y n o l , two t r i a c e t y l e n e a l c o h o l s w i t h pronounced a n t i f u n g a l a c t i v i t y , occur i n s a f f l o w e r (Carthamus t i n c t o r i s ) (45-47). These p a r t i c u l a r m e t a b o l i t e s are r a p i d l y b i o s y n t h e s i z e d by t h i s p l a n t i n reponse t o i n f e c t i o n s w i t h a v i r u l e n t s t r a i n of P h y t o p h t h o r a d r e c h s l e r i and an a v i r u l e n t s t r a i n of megasperma v a r . s o j a e . W i t h i n 48 h of i n o c u l a t i o n , the l e v e l s of s a f y n o l and d e h y d r o s a f y n o l may i n c r e a s e by as much as 40 and 1,500 t i m e s , r e s p e c t i v e l y ( 4 8 ) . The r a t e of d e h y d r o s a f y n o l accumulation i s s t a t i s t i c a l l y c o r r e l a t e d w i t h high disease r e s i s t a n c e i n one p a r t i c u l a r b r e e d i n g l i n e ( B i g g s ) of s a f f l o w e r ( 4 8 ) . Wyerone o c c u r s i n h e a l t h y t i s s u e s of the broad bean V i c i a f a b a L. (49) and a c c u m u l a t e s , u s u a l l y i n c o n j u n c t i o n w i t h wyerone e p o x i d e , i n at l e a s t 28 o t h e r s p e c i e s of V i c i a and two s p e c i e s of Lens when c h a l l e n g e d by H e l m i n t h o s p o r i u m carbonum or B o t r y t i s c i n e r e a ( 3 0 ) . Wyerone a c i d c o - o c c u r s w i t h wyerone and wyerone epoxide i n broad bean p l a n t s i n f e c t e d w i t h s p e c i e s of B o t r y t i s ( 5 2 ) . Despite t h e i r s t r u c t u r a l s i m i l a r i t y with other phototoxic a c e t y l e n e s , f a l c a r i n o l and f a l c a r i n d i o l (and c l o s e l y r e l a t e d m e t a b o l i t e s l i k e f a l c a r i n o n e and f a l c a r i n d i o n e ) , a p p a r e n t l y a r e not p h o t o t o x i c (9). Whether the a n t i m i c r o b i a l a c t i v i t y of t e t r a d e c a - 6 - e n e - l , 3 - d i y n e - 5 , 8 - d i o l , s a f y n o l , d e h y d r o s a f y n o l or the


286



wyerone d e r i v a t i v e s may r e s u l t from l i g h t - a c t i v a t e d o r l i g h t - i n d e p e n d e n t p r o c e s s e s remains a c r i t i c a l p o i n t t h a t needs t o be e s t a b l i s h e d . Pterocarpans. S e v e r a l p t e r o c a r p a n d e r i v a t i v e s , most o f t e n r e f e r r e d to i n t h e l i t e r a t u r e as 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 r e d o m i n a n t l y o c c u r i n members o f t h e Fabaceae (Leguminosae) ( 3 0 ) . Like furanocoumarins and p o l y a c e t y l e n e s , t h e s e p h y t o c h e m i c a l s a r e t o x i c toward a wide range o f b i o l o g i c a l organisms and accumulate i n p l a n t t i s s u e s i n response t o a v a r i e t y o f s t r e s s e s ( p a r t i c u l a r l y i n f e c t i o n by pathgens) (30,58). F o r t h e s e r e a s o n s , c e r t a i n p t e r o c a r p a n s a r e b e l i e v e d t o p l a y an i m p o r t a n t r o l e i n t h e defense o f p r o d u c i n g p l a n t s a g a i n s t p o t e n t i a l d i s e a s e - c a u s i n g organisms. The b i o s y n t h e s i s , e l i c i t a t i o n and b i o l o g i c a l a c t i v i t y o f i s o f l a v o n o i d p h y t o a l e x i n s has been r e v i e w e d q u i t e r e c e n t l y (58) and w i l l be discussed only b r i e f l y here. C o n s i d e r a b l e i n t e r e s t has been f o c u s e d on an a r r a y o f a n t i m i c r o b i a l p t e r o c a r p a n d e r i v a t i v e s because o f t h e i r p u t a t i v e i n v o l v e m e n t i n p l a n t d e f e n s e . A l a r g e amount o f i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g t h e c e l l u l a r t a r g e t s and modes o f a c t i o n o f t h e s e p l a n t m e t a b o l i t e s ( 5 8 ) . B a c t e r i a l and f u n g a l membranes a r e p a r t i c u l a r l y s u s c e p t i b l e t o the e f f e c t s of i s o f l a v o n o i d p h y t o a l e x i n s ; however, o t h e r c e l l u l a r s i t e s have n o t been r u l e d out as t a r g e t s o f a c t i o n ( 5 8 ) . One i n v e s t i g a t i o n has examined t h e i n v o l v e m e n t o f l i g h t as an a c t i v a t i n g f a c t o r i n p t e r o c a r p a n t o x i c i t y . Bakker et^ al, (59) found t h a t s e v e r a l p t e r o c a r p a n d e r i v a t i v e s i n c l u d i n g g l y c e o l l i n I ( I X ) , p h a s e o l l i n (X) and p i s a t i n ( X I ) as w e l l as 3 , 6 a , 9 - t r i h y d r o x y p t e r o c a r p a n and t u b e r o s i n c a n form f r e e r a d i c a l s i n t h e presence o f UV i r r a d i a t i o n ( w i t h maximum i n t e n s i t y around 305 nm) and t h a t t h e s e f r e e r a d i c a l s a r e most l i k e l y i n v o l v e d i n t h e i n a c t i v a t i o n o f glucose-6-phosphate dehydrogenase a c t i v i t y i i i v i t r o . The e x t e n t t o w h i c h f r e e r a d i c a l f o r m a t i o n may c o n t r i b u t e t o t h e p t e r o c a r p a n t o x i c i t y (and presumably p l a n t d e f e n s e ) i n o t h e r s t u d i e s where t h e e f f e c t o f l i g h t was n o t c o n s i d e r e d i s n o t c l e a r , but c e r t a i n l y w a r r a n t s f u r t h e r attention. Recent I n v e s t i g a t i o n s Despite demonstrations that v a r i o u s phytochemicals a r e potent l i g h t - a c t i v a t e d a n t i m i c r o b i a l s i n v i t r o and t h a t many a l s o accumulate i n response t o i n f e c t i o n by d i s e a s e - c a u s i n g organisms o r other s t r e s s f u l s i t u a t i o n s , there i s l i t t l e d i r e c t evidence l i n k i n g such m o l e c u l e s t o p l a n t defense in_ s i t u . We have been s t u d y i n g t h e r o l e o f endogenous p h o t o s e n s i t i z e r s t o determine t h e i r i n v o l v e m e n t i n t h e r e s i s t a n c e o f C i t r u s s p e c i e s t o d i s e a s e - c a u s i n g organisms s i n c e f i n d i n g t h a t t h e l e a v e s o f many s p e c i e s c o n t a i n p h o t o s e n s i t i z e r s ( 8 ) . Our e f f o r t s thus f a r have c o n c e n t r a t e d on: 1) e s t a b l i s h i n g t h e s u s c e p t i b i l i t y o f v a r i o u s C i t r u s pathogens t o l e a f e x t r a c t s ; 2) i d e n t i f y i n g t h e p h o t o t o x i c p h y t o c h e m i c a l s i n t h e s e l e a f e x t r a c t s ; and 3) d e t e r m i n i n g pathogen s u s c e p t i b i l i t y t o t h e phytochemicals responsible f o r t h i s b i o c i d a l a c t i o n . I n i t i a l l y , we were i n t e r e s t e d i n d e t e r m i n i n g t h e s u s c e p t i b i l i t y of f u n g a l pathogens i s o l a t e d from C i t r u s t o l e a f e x t r a c t s p r e v i o u s l y



21.

DOWNUM AND NEMEC


287

shown t o e l i c i t p h o t o t o x i c a c t i v i t y ( 8 ) . Nine d i s e a s e - c a u s i n g fungi were o b t a i n e d f o r t h e s e s t u d i e s ( T a b l e I I ) i n c l u d i n g v a r i o u s r o o t , f r u i t and/or l e a f pathogens. S u s c e p t i b i l i t y was determined by s c r e e n i n g t h e p a t h o g e n i c organisms a g a i n s t e x t r a c t s o f C i t r u s l i m e t t o i d e s Tan, (sweet l i m e ) , C. m a c r o p h y l l a Wester (alemow) and C. medica L. ( c i t r o n ) u s i n g a d i s c b i o a s s a y . P l a n t e x t r a c t s were p r e p a r e d by homogenizing f r e s h l y c o l l e c t e d l e a f m a t e r i a l (100 g) i n methanol (300 ml) f o l l o w e d by f i l t r a t i o n and c o n c e n t r a t i o n o f t h e e x t r a c t t o a f i n a l volume o f 10 m l . S t e r i l e f i l t e r paper d i s c s were loaded w i t h t h e d i f f e r e n t e x t r a c t s (20 u l ) and a l l o w e d t o d r y . The d i s c s were p l a c e d onto d u p l i c a t e p o t a t o d e x t r o s e agar (PDA) p l a t e s c o n t a i n i n g e i t h e r s p o r e s o r m y c e l i a l fragments o f t h e n i n e p h y t o p a t h o g e n i c organisms and then i n c u b a t e d i n t h e dark f o r 60 min. Ong o f t h e d u p l i c a t e p l a t e s was i r r a d i a t e d f o r 2 h w i t h UVA (2 W m ) w h i l e t h e o t h e r p l a t e was kept i n t h e dark t o m o n i t o r l i g h t - i n d e p e n d e n t a n t i m i c r o b i a l a c t i o n . A l l p l a t e s were s u b s e q u e n t l y i n c u b a t e d i n t h e dark ( a t 25°C f o r 24-48 h) and then s c o r e d f o r zones o f i n h i b i t i o n s u r r o u n d i n g t h e f i l t e r paper discs.

Table I I .

Fungal pathogens o f C i t r u s

Leaf Pathogens Alternaria c i t r i - leafspot C o l l e t o t r i c h u m gleosporides - anthracnose F r u i t Pathogens A l t e r n a r i a c i t r i - black r o t C o l l e t o t r i c h u m gleosporides - anthracnose D i p l o i d i a n a t a l e n s i s - stem-end r o t Geotrichum candidum - sour r o t P e n i c i I l i u m d i g i t a t u m - green mold P e n i c i I l i u m i t a l i c u m - b l u e mold Root Pathogens Fusarium oxysporum - r o o t r o t Fusarium s o l a n i - r o o t r o t Phytophthora p a r a s i t i c a - foot r o t

Four o f t h e f u n g i t e s t e d were q u i t e s e n s i t i v e t o t h e C i t r u s e x t r a c t s i n t h e presence o f UVA, but were u n a f f e c t e d i n i t s absence ( T a b l e I I I ) . Three o f t h e s u s c e p t i b l e pathogens p r i m a r i l y i n f e c t r o o t s ( F . oxysporum, F. s o l a n i and P h y t o p h t h o r a p a r a s i t i c a ) w h i l e the f o u r t h , C o l l e t o t r i c h u m g l e o s p o r i d i e s , i n f e c t s m a i n l y l e a v e s and f r u i t . Other pathogens o f above-ground p l a n t p a r t s , namely A. c i t r i , D. n a t a l e n s i s , G. candidum and P. d i g i t a t u m , s u c c e s s f u l l y r e s i s t e d the l i g h t - a c t i v a t e d a n t i m i c r o b i a l a c t i o n of a l l three e x t r a c t s . P. i t a l i c u m , however, was s l i g h t l y a f f e c t e d by t h e C i t r u s macrophylla e x t r a c t . F i v e coumarin d e r i v a t i v e s were i d e n t i f i e d i n l e a f e x t r a c t s o f C_. l i m e t t o i d e s , C^. m a c r o p h y l l a and C. medica i n c l u d i n g 5 - h y d r o x y p s o r a l e n , 5-methoxypsoralen ( I ) , 5,8-dimethoxypsoralen ( I I I ) , 4-hydroxycoumarin and 7-hydroxycoumarin [8-MOP a l t h o u g h



[nurjLirj Q . . . . « . ^ Kt

IV. Alpha-Terthienyl

K

V. Phenylheptatriyne

HgC-CH-CH-C5C-C= C-CHgCH-CH-CCH^CH OH

3

VI. Falcarinol

H0H C-CH-C=C-C = C - C = C - C « C - C H « C H - C H ,


o

OH VII. Dehydrosafynol

C H - C H £ CH - CH - C-C-C

CH • CH - CO, Me

VIII. Wyerone

IX. Glyceollin I

X . Phaseollin

XI. Pisatin

MeO



+

No i n h i b i t i o n . I n h i b i t i o n zones below 10 mm.

C.

44+++

UVA

limettoides Dark

UVA

Dark

UVA

I n h i b i t i o n zones between 11 - 15 mm. Inhibition zones betweem 16 - 20 mm.

Dark

C_. macrophylla

C. medica

on the growth of Citrus pathogens

Inhibition Zones

UVA-Induced i n h i b i t o r y e f f e c t of crude leaf extracts

Alternaria c i t r i Colletotrichum gleosporides D i p l o i d i a natalensis Fusarium oxysporum Fusarium solani Geotrichum candidum P e n i c i l l i u m digitatum P e n i c i l l i u m italicum Phytophthora p a r a s i t i c a

Pathogens

Table I I I .



290


common i n c l o s e l y r e l a t e d genera has n o t been r e p o r t e d i n C i t r u s ( 1 3 ) ] . The p h o t o t o x i c i t i e s o f t h e s e d e r i v a t i v e s were t e s t e d a g a i n s t the n i n e pathogens l i s t e d i n T a b l e I I . The same b i o a s s a y procedures as above were used except t h a t the i n d i v i d u a l c h e m i c a l s ( d i s s o l v e d i n methanol) were a p p l i e d t o the f i l t e r paper d i s c s i n s t e a d o f l e a f e x t r a c t s . Only 5-methoxypsoralen (5-MOP) e l i c i t e d p h o t o t o x i c responses ( T a b l e I V ) . D. n a t a l e n s i s and the two P e n i c i l l i u m s p e c i e s were r e s i s t a n t i n t h e s e In v i t r o b i o a s s a y s . I n g e n e r a l , t h e s e s t u d i e s suggest t h a t f u n g i w h i c h i n f e c t above-ground p l a n t t i s s u e s are more r e s i s t a n t t o p h o t o t o x i c a c t i o n than r o o t pathogens. Such c o n t r a s t i n g responses p r o b a b l y r e f l e c t an e v o l v e d a b i l i t y by c e r t a i n l e a f and f r u i t pathogens t o c i r c u m v e n t the t o x i c a c t i o n o f t h e s e c h e m i c a l s v i a d e t o x i f i c a t i o n o r o t h e r processes. S i n c e UVA i s r a r e l y e x p e r i e n c e d i n the r h i z o s p h e r e , e v o l u t i o n o f such p r o c e s s e s by r o o t pathogens would seem t o be u n n e c e s s a r y . C o l l e t o t r i c h u m g l e o s p o r i d e s appears t o be an exception. U n l i k e the o t h e r pathogens o f above-ground t i s s u e s , t h i s fungus was s u s c e p t i b i l e t o 5-MOP and a l l t h r e e o f the C i t r u s e x t r a c t s which s u g g e s t s t h a t e s t a b l i s h m e n t o f t h i s fungus on p l a n t t i s s u e s may be i n f l u e n c e d by the presence o f endogenous photosensitizers. We have i s o l a t e d s e v e r a l o t h e r p h o t o t o x i c coumarins from v a r i o u s C i t r u s s p e c i e s u s i n g s t a n d a r d b i o a s s a y organisms ( i . e . , E. c o l i and Saccharomyces c e r e v i s i a e ) . I n a d d i t i o n t o 5-MOP, t h e c h e m i c a l s 7-methoxycoumarin and 5 - g e r a n o x y p s o r a l e n ( b e r g a m o t t i n ) have a l s o been i d e n t i f i e d . The t o x i c i t y o f t h e s e m e t a b o l i t e s have not y e t been e s t a b l i s h e d u s i n g the C i t r u s pathogens. Once t h i s has been a c c o m p l i s h e d , we i n t e n d t o q u a n t i t a t e the l e v e l s o f endogenous p h o t o s e n s i t i z e r s i n f i e l d and i n greenhouse-grown C i t r u s p l a n t s and e s t a b l i s h whether p l a n t r e s i s t a n c e t o pathogen i n f e c t i o n c a n be correlated with i n s i t u l e v e l s of p a r t i c u l a r photosensitizers. Conclusion We have d i s c u s s e d the a n t i m i c r o b i a l a c t i v i t y o f more than 20 p h y t o c h e m i c a l s ; most are p o t e n t p h o t o t o x i n s . O t h e r s are i n c l u d e d , not because they are demonstrated p h o t o s e n s i t i z e r s , but because they s h a r e common c h e m i c a l c h a r a c t e r i s t i c s w i t h these b i o l o g i c a l l y a c t i v e p l a n t m e t a b o l i t e s , i . e . , e x t e n s i v e a r o m a t i c o r c o n j u g a t e d double and/or t r i p l e bond systems, and may f u n c t i o n s i m i l a r l y i n v i v o . I n a d d i t i o n t o the m o l e c u l e s a l r e a d y d i s c u s s e d , numerous o t h e r p l a n t - d e r i v e d p h o t o s e n s i t i z e r s are known, but t h e i r r o l e i n p l a n t - p a t h o g e n i n t e r a c t i o n s have y e t t o be e s t a b l i s h e d . Included are v a r i o u s acetophenone, extended q u i n o n e , furanochromone and l i g n a n d e r i v a t i v e s as w e l l as s e v e r a l b e t a - c a r b o l i n e , f u r a n o q u i n o l i n e and i s o q u i n o l i n e a l k a l o i d s ( 8 , 9 ) . Other m e t a b o l i t e s w h i c h are i n v o l v e d w i t h p l a n t r e s p o n s e s t o p a t h o g e n i c i n v a s i o n and have s i m i l a r s t r u c t u r a l f e a t u r e s have r e c e n t l y been i s o l a t e d , e.g., naphthofuranones (60) and d i b e n z o f u r a n s ( 6 1 - 6 3 ) , and w a r r a n t f u r t h e r i n v e s t i g a t i o n w i t h regard t o t h e i r p o t e n t i a l phototoxic a c t i o n . Important a r e a s f o r f u t u r e r e s e a r c h t h a t might a i d i n f u r t h e r e l u c i d a t i n g the s i g n i f i c a n c e o f p h o t o s e n s i t i z e r s i n p l a n t d e f e n s e a g a i n s t d i s e a s e - c a u s i n g organisms i n c l u d e among o t h e r s : 1) t o x i c o l o g i c a l s t u d i e s t o determine c e l l u l a r mechanisms o f pathogen



Inhibitory

+ ++ +++ I I 11

No i n h i b i t i o n . I n h i b i t i o n zones I n h i b i t i o n zones I n h i b i t i o n zones I n h i b i t i o n zones

_

_

Dark

_ _ _

_

UVA

_

_

UVA

7-HC

below 10 ram. between 11 - 15 mm. between 16 - 20 mm. above 21 mm.

-

-

Dark

4-HC

_

_ -

-

_

+

Dark

UVA

5-MOP

+ -H-f IHI +++ _ _ + +

_

UVA

Zones

Dark

UVA

5,8-diMOP

4-HC - 4-hydroxycoumarin 7-HC - 7-hydroxycoumarin 5-HP - 5 - h y d r o x y p s o r a l e n 5-MOP - 5-methoxypsoralen 5,8-MOP - 5 , 8 - d i m e t h o x y p s o r a l e n

-

Dark

5-KP

Inhibition

e f f e c t o f v a r i o u s coumarins and f u r a n o c o u m a r i n s on C i t r u s pathogens

Alternaria c i t r i Colletotrichum gleosporides Diploidia natalensis Fusarium oxysporum Fusarium s o l a n i Penicillium digitatum Penicillium italicum Phytophthora p a r a s i t i c a

Pathogens

T a b l e IV.


292


s u s c e p t i b i l i t y ( o r r e s i s t a n c e ) t o p h o t o t o x i c m e t a b o l i t e s ; 2) p h y t o c h e m i c a l s t u d i e s t o e v a l u a t e the q u a n t i t a t i v e v a r i a t i o n o f s p e c i f i c p h o t o t o x i n s i n p l a n t p o p u l a t i o n s coupled w i t h i n s i t u s t u d i e s t h a t attempt t o c o r r e l a t e those endogenous l e v e l s w i t h p l a n t r e s i s t a n c e ( o r s u s c e p t i b i l i t y ) t o s p e c i f i c pathogens; and 3) breeding studies t o s e l e c t f o r plant l i n e s that synthesize a g r a d i e n t o f endogenous p h o t o s e n s i t i z e r c o n c e n t r a t i o n s w h i c h can be e v a l u a t e d f o r r e s i s t a n c e t o a broad range o f v i r u l e n t organisms. I n a d d i t i o n , past s t u d i e s t h a t i n v o l v e d p l a n t p h o t o s e n s i t i z e r s , but d i d not c o n s i d e r l i g h t as an a c t i v a t i n g element i n t h e i r t o x i c i t y , need t o be r e - e v a l u a t e d .


Acknowledgments

We would l i k e t o express our g r a t i t u d e t o Dr. Stewart A. Brown ( T r e n t U n i v e r s i t y ) f o r p r o v i d i n g us w i t h s t a n d a r d s o f v a r i o u s f u r a n o c o u a r i n s and L a v i n a F a l e i r o , A d e l h e i d R e i n o s o , Johanne S c o t t and Lee Swain f o r t e c h n i c a l a s s i s t a n c e . T h i s work was supported by g r a n t s from the W h i t e h a l l and FIU F o u n d a t i o n s .

Literature Cited 1.

2.

3.

4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15.

Cowling, E.B.; H o r s f a l l , J.G. In Plant Disease An Advanced Treatise. How Plants Defend Themselves; H o r s f a l l , J.C.; Cowling, E.B., Eds.; Academic: New York, 1980, Vol. V, pp. 1-16. Overeem, J.C. In Biochemical Aspects of Plant-Parasite Relationships; Friend, J . ; T h r e l f a l l , D.R., Eds.; Academic: New York, 1976, pp. 195-206. Kuc, J . ; Shain, L. In Antifungal Compounds. Interactions in Biological and Ecological Systems; Siegel, M.R.; S i s l e r , H.D., Eds.; Marcel Dekker: New York, 1977, Vol 2, pp. 497-535. Swain, T. Ann. Rev. Plant Physiol. 1977,28,479-501. B e l l , A.A. Ann. Rev. Plant Physiol. 1981,32,21-81. Smith, D.A. In Phytoalexins; Bailey, J.A.; Mansfield, J.W., Eds.; Wiley: New York, 1982, pp. 218-52. D a r v i l l , A.G.; Albersheim, P. Ann. Rev. Plant Physiol. 1984, 35, 243-75. Downum, K.R. In Natural Resistance of Plants to Pests: Roles of Allelochemicals; Green, M.B.; Hedin, P.A., Eds.; ACS SYMPOSIUM SERIES No. 296, American Chemical Society: Washington, D.C., 1986, pp. 197-205. Towers, G.H.N. Can. J . Bot. 1984,62,2900-11. Knox, J.P.; Dodge, A.D. Phytochem. 1985,24,889-96. Downum, K.R.; Rodriguez, E. J . Chem. Ecol. 1986, 12, 823-34. Gray, A.I.; Waterman, P.G. Phytochem. 1978,17,845-64. Murray, R.D.H.; Mendez, J . ; Brown, S.A. The Natural Coumarins: Occurrence, Chemistry and Biochemistry; Wiley: New York, 1982. Song, P.-S.; Tapley, K.J., J r . Photochem. Photobiol. 1979, 29, 1177-97. Veronese, F.M.; Schiavon, O.; Bevilacqua, R.; Bordin, F; Rodighiero, G. Photochem. Photobiol. 1982, 36, 25-30.


21.

DOWNUM AND NEMEC

16. 17. 18. 19. 20. 21.


22.

23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.


293

Granger, M.; Helene, C. Photochem. Photobiol. 1983, 38, 563-68. Tuveson, R.W.; Berenbaum, M.R.; Heininger, E.E. J . Chem. Ecol. 1986, 12, 933-48. Beier, R.C.; I v i e , G.W.; O e r t l i , E.H.; Holt, D.L. Fd. Chem. Toxic. 1983, 21, 163-65. Scheel, L.D.; Perone, V.B.; Larkin, R.L.; Kupel, R.E. Biochem. 1963, 2, 1127-31. Martin, J.T.; Baker, E.A.; Byrde, R.J.W. Ann. Appl. B i o l . 1966, 57, 491-500. Johnson, C.; Brannon, D.R.; Kuc, J . Phytochem. 1973, 12, 2961-62. Martin, J.T. In Fungal Pathogenicity and the Plant's Response; Byrde, R.J.W.; Cutting, C.V., Eds.; Academic: New York, 1973, pp. 333-5. Afek, U.; Sztejnberg, A. Phytochem. 1986, 25, 1855-56. Ceska, O.; Chaudhary, S.K.; Warrington, P.J.; Ashwood-Smith, M.J. Phytochem. 1986, 25, 81-83. Wu, C.M.; Koehler, P.E.; Ayres, J.C. Appl. Microbiol. 1972, 23., 852-56. Chappel, J ; Hahlbrock, K. Nature 1984,311,76-78. Hauffe, K.D.; Hahlbrock, K.; Scheel, D. Z. Naturforsch. 1986, 41c, 228-39. Bohlmann, F; Burkhardt, T; Zdero, C. Naturally Occurring Acetylenes; Academic: London, 1973. de Wit, P.J.G.M.; Kodde, E. Physiol. Plant Path. 1981, 18, 143-48. Robeson, D.J.; Harborne, J.B. Phytochem. 1980, 19, 2359-65. McLachlan, D.; Arnason, J.T.; Lam, J . Photochem. Photobiol. 1984, 39, 177-82. McLachlan, D.; Arnason, T.; Lam, J . Biochem. System. Ecol. 1986, 14, 17-23. Arnason, T.; Chan, G.F.Q.; Wat, C.-K.; Downum, K.R.; Towers, G.H.N. Photochem. Photobiol. 1981, 33, 821-24. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photochem. Photobiol. 1982, 36, 517-23. Champagne, D.E.; Arnason, J.T.; Philogene, B.J.R.; Morand, P.; Lam, J . J . Chem. Ecol. 1986, 12, 835-58. Kemp, M.S. Phytochem. 1978,17,1002. Garrod, B.; Lewis, B.G.; Coxon, D.T. Physiol. Plant Path. 1978, 13, 241-46. Harding, V.K.; Heale, J.B. Physiol. Plant Path. 1980, 17, 277-89. Hansen, L.; B o l l , P.M. Phytochem. 1986, 25, 285-93. DiCosmo, F.; Towers, G.H.N.; Lam, J . Pestic. S c i . 1982, 13, 589-94. Bourque, G.; Arnason, J.T.; Madhosingh, C.; Orr, W. Can. J . Bot. 1985, 63, 899-902. Norton, R.A.; Finlayson, A.J.; Towers, G.H.N. Phytochem. 1985, 24, 719-22. Kourany, E. M.S. Thesis, University of Ottawa, 1986, 170 pp. Elgersma, D.M.; Overeem, J.C. Neth. J . Plant Path. 1981, 87, 69-70. Thomas, C.A.; A l l e n , E.H. Phytopath. 1970, 60, 261-63. A l l e n , E.H.; Thomas, C.A. Phytochem. 1971, 10, 1579-82.


294 47. 48. 49.

50. 51. 52. 53. 54.


55. 56. 57. 58. 59. 60. 61. 62. 63.


A l l e n , E.H.; Thomas, C.A. Phytopath. 1971, 61, 1107-09. A l l e n , E.H.; Thomas, C.A. Phytopath. 1972, 62, 471-74. Fawcett, C.H.; Spencer, D.M.; Wain, R.L.; F a l l i s , A.G.; Jones, E.R.H.; Le Quan, M.; Page, C.B.; Thaller, V.; Shubrook, D.C.; Whitham, P.M. J . Chem. Soc. 1968, 2455-62. Hargreaves, J.A.; Mansfield, J.W.; Coxon, D.T; Price, K.R. Phytochem. 1976, 15, 1119-21. Hargreaves, J.A.; Mansfield, J.W.; Rossal, S. Physiol. Plant Path. 1977, 11, 227-42. Letcher, R.M.; Widdowson, D.A.; Deverall, B.J.; Mansfield, J.W. Phytochem. 1970, 9, 249-52. Downum, K.R.; Towers, G.H.N. J . Nat. Prod. 1983, 44, 98-103. Downum, K.R.; K e i l , D.J.; Rodriguez, E. Biochem. Syst. Ecol. 1985, 13, 109-13. DiCosmo, F.; Norton, R.; Towers, G.H.N. Naturwissenschaften 1982, 69S, 550-51. Garrod, B.; Lea, E.J.A.; Lewis, B.G. New Phytologist 1979, 83, 463-71. Schulte, K.E.; Wulfhorst, G. Archiv der Pharmacie 1977, 310, 285-98. Smith, D.A.; Banks, S.W. Phytochem. 1986, 25, 979-95. Bakker, J . ; Gommers, F.J.; Smits, L.; Fuchs, A.; de Vries, F.W. Photochem. Photobiol. 1983, 38, 323-29. Sutton, D.C.; G i l l a n , F.T.; Susie, M. Phytochem. 1985, 24, 2877-79. Kemp, M.S.; Burden, R.S.; L o e f f l e r , R.S.T. J . Chem. Soc. Perkin. Trans. I 1983, 2267-72. Kemp, M.S.; Burden, R.S. J . Chem. Soc. Perkin Trans. I 1984, 1441-43. Burden, R.S.; Kemp, M.S.; W i l t s h i r e , C.W. J . Chem. Soc. Perkin Trans. I 1984, 1445-48.

RECEIVED November 20, 1986


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