Light-Activated Antimicrobial Chemicals from ... - ACS Publications

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 .

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RECEIVED November 20, 1986


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