Photoactivated Biocides from Higher Plants - ACS Symposium Series

16 Photoactivated Biocides from Higher Plants Kelsey R. Downum Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: January 16, 1986 | doi: 10.1021/bk-1986-0296.ch016

Department of Biological Sciences, Florida International University, Miami, F L 33199

Plants synthesize chemicals which possess a wide range of biological activities. Natural products that require light for expression of their toxic biological consequences are among the most interesting of these bioactivities. Such "photosensitizers" or "phototoxins" are structurally variable, are produced by widely divergent flowering plant families and mediate broad-spectrum toxic reactions. Acetophenones, alkaloids, furocoumarins, furochromones, polyines as well as one important lignan have been added to a growing list of phototoxic metabolites isolated from plant sources. The chemistry, distribution and biocidal action of these toxic allelochemicals is discussed. Plants produce an array of chemicals that can be excited by sunlight. The best known of these natural products include chlorophylls, carotenoids and phytochrome. Chlorophylls and carotenoids participate i n photosynthesis, the process evolved by plants to convert l i g h t into the chemical energy e s s e n t i a l for blosynthetic processes. Phytochrome, on the other hand, mediates a variety of growth and developmental responses such as seed germination, growth regulation and f l o r a l i n i t i a t i o n . In addition to light-activated "pigments", many plants also produce "photosensitizers" or "phototoxins". These metabolites are unique i n that they become toxic to other organisms i n the presence of sunlight ( s p e c i f i c a l l y the UVA region of sunlight; 320-400 nm). The b i o c i d a l p o t e n t i a l of such phototoxic phytochemicals has been demonstrated against microorganisms (including important b a c t e r i a l and fungal pathogens of plants), nematodes, herbivorous and non-herbivorous insects as well as numerous other organisms (1-3). Such broad-spectrum b i o a c t i v i t y suggests that plant photosensitizers may function as "solar-powered" defensive agents which discourage insect herbivory and i n h i b i t i n f e c t i o n by pathogenic organisms i n nature.

0097-6156/86/0296-0197$06.00/0 © 1986 American Chemical Society

In Natural Resistance of Plants to Pests; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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NATURAL RESISTANCE OF PLANTS TO PESTS

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: January 16, 1986 | doi: 10.1021/bk-1986-0296.ch016

Chemistry of Photosensitizers Plant metabolites that are capable of becoming toxic i n sunlight or UVA are produced by a wide variety of biochemical pathways and thus are a s t r u c t u r a l l y diverse group of natural products. Both linear and c y c l i c photosensitizers are known from plants. Linear phototoxins are generally derived from fatty acid precursors and t y p i c a l l y possess conjugated double and t r i p l e bond systems. The majority of c y c l i c photosensitizers, on the other hand, are b i - and t r i c y c l i c aromatic molecules that may contain nitrogen, oxygen or s u l f u r as heterocyclic elements. Most phototoxic phytochemicals are c y c l i c . Various a l k a l o i d s , acetophenones, extended quinones, furochromones and furocouxnarins belong to this c l a s s . Thiophenic and r i n g - s t a b i l i z e d acetylenic polyines as w e l l as the lignan nordihydroguaiaretic acid (NDGA) belong to a second group of photosensitizers that have both linear and c y c l i c moieties while a t h i r d type consists of straight-chain molecules only. Examples of photosensitizers from these categories are given i n Figure 1. D i s t r i b u t i o n of Photosensitizers i n Plants Chemicals that are p o t e n t i a l l y capable of phototoxic action have been i s o l a t e d from more than 30 flowering plant f a m i l i e s . Their occurrence among important monocot and dicot families i s shown i n Table I. Most of the taxa represented i n this table synthesize several types of photosensitizers. Members of the Asteraceae (sunflower family) and the Rutaceae (citrus family) for example synthesize the widest range of phototoxic compounds. Other families (e.g., Hypericaceae, Li11aceae, Moraceae and Orchidaceae) either lack or f a i l to express such blosynthetic d i v e r s i t y . Plants from these l a t t e r groups contain phototoxins derived from a single metabolic pathway. Acetylenes, beta-carboline a l k a l o i d s , furocoumarins and lignans occur widely among the families l i s t e d i n Table I. Acetophenones (benzofurans and benzopyrans) and extended quinones have a much more limited d i s t r i b u t i o n while furochromones, furoquinoline alkaloids and thiophenes are apparently r e s t r i c t e d to single f a m i l i e s . It should be mentioned that although acetylenic polyines are phytochemical components of plants from numerous families (4), only those derivatives produced by members of the Asteraceae have been shown to be phototoxic (1). Lignans are included i n Table I because they represent an important class of p o t e n t i a l photosensitizers. We recently i s o l a t e d and i d e n t i f i e d the f i r s t phototoxic lignan nordihydroguaiaretic acid from the leaf r e s i n of the creosote bush Larrea tridentata (Zygophyllaceae) (5). A t o t a l of more than 200 different lignans from ca. 60 plant families (6) are known and represent a s i g n i f i c a n t pool of p o t e n t i a l l y active phytochemicals. Further i n v e s t i g a t i o n i s needed to e s t a b l i s h whether t h i s b i o l o g i c a l a c t i v i t y i s unique to NDGA or representative of the action of other lignans. The ecotypic d i s t r i b u t i o n of major phototoxin-producing families i s shown i n Table I I . Plants that synthesize these b i o c i d a l components are widely d i s t r i b u t e d i n nature (after r e f . 12),


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C = C — C = C— C = C — C H

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3


I.

I

VIII. Figure 1. Structures of representative phototoxic plant products from various phytochemical classes. (I) 1-phenylhepta-l,2,3triyne (acetylenic polyine); (II) 6-methoxyeuparin (benzofuran); (III) harmane (beta-carboline alkaloid); (IV) k h e l l i n (furochromone); (V) 8-methoxypsoralen (furocoumarin); (VI) dictamnine (furoquinoline a l k a l o i d ) ; (VII) nordihydroguaiaretic acid (lignan) and (VIII) alpha-terthienyl (thiophene).



+ +

+ +

+ + +

I

+

+

+ +

II

+ + + + +

+

+

III

+

+

IV

+

V

+ +

+ +

+

+

+

VI

+

VII

+ +

+

VIII

+ + +

+

+ +

IX

D i s t r i b u t i o n of Phototoxic Phytochemicals

+

X

4,6-7 4,6 4,6-7,9-11 8-9 4,12 4,7,9 13,14 8 7 7 6,9,13 9,12 1,4,6-9,12 4,6-7,9 6,9

References

1^. Acetylenes; I I . Acetophenones; I I I . Beta-Carboline Alkaloids; IV. Extended Quinones; V. Furochromones; VI. Furocoumarins; VII. Furoquinoline A l k a l o i d s ; VIII. Isoquinoline A l k a l o i d s ; IX. Lignans; X. Thiophenes.

Apiaceae Araliaceae Asteraceae Cyperaceae Euphorbiaceae Fabaceae Hypericaceae Liliaceae Moraceae Orchidaceae Polygonaceae Rubiaceae Rutaceae Solanaceae Zygophyllaceae

Plant Family

Table I.


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Plants

but seem to occur most often i n t r o p i c a l / s u b t r o p i c a l climates. Such f a m i l i a l d i s t r i b u t i o n s are quite i n t e r e s t i n g and suggest that plants with endogenous phototoxins may be most successful i n environments where they are (or could be) exposed to intense solar i r r a d i a t i o n throughout much of the year.

Table I I .

D i s t r i b u t i o n of Phototoxin-Containing


Plant Families Dicotyledonous Apiaceae (carrot & parsnips family) Araliaceae (ivy & ginseng family) Asteraceae (sunflower family) Euphorbiaceae (spurge family) Hypericaceae Moraceae ( f i g family) Polygonaceae (buckwheat family) Rubiaceae (coffee family) Rutaceae (citrus family) Solanaceae (potatoe family) Zygophyllaceae Monocotyledonous Cyperaceae (reeds & sedges) L i l i a c e a e ( l i l y family) Orchidaceae (orchid family)

Plant Families

Main D i s t r i b u t i o n

Temperate Uplands Tropics World-Wide Tropics Tropics to Temperate Tropics/Subtropics Temperate Tropics/Subtropics Tropics to Temperate Tropics to Temperate Tropics/Subtropics Temperate World-Wide Tropics

B i o c i d a l Action of Photosensitizers The toxic mechanisms of photosensitization have been reviewed recently (1-3) and w i l l not be dealt with here. The reader i s referred to these references for detailed discussions of the c e l l u l a r targets and molecular mechanisms of phototoxicity mediated by various natural products. The broad-spectrum b i o c i d a l action induced by phototoxic allelochemicals has been demonstrated toward a range of organisms including bacteria, fungi, nematodes, insects as well as non-phototoxin-containing plants. Table III summarizes the organisms susceptible to photosensitization by alpha-terthienyl, a phototoxin c h a r a c t e r i s t i c of many Asteraceae species (4, 10-11). The t o x i c i t y of this thiophenic polyine toward plant pathogens (e.g., Agrobacterium, A l t e r n a r i a , Cladosporium, Colietotrichum, Fusarium, Pythium, Rhizoctonia), nematodes and herbivorous insects (e.g., Euxoa, Heliothus, Manduca, Ostrinia) suggests that i t may function i n nature to discourage not only herbivory, but also i n f e c t i o n by p o t e n t i a l pathogens. Many natural products react with s i m i l a r nons p e c i f i c i t y i n UVA (34-44). The function(s) of these allelochemicals remains to be demonstrated, but deterrence of deleterious organisms seems to be a p o s s i b i l i t y worthy of further investigation. The s u s c e p t i b i l i t y of p a r t i c u l a r b a c t e r i a l or fungal pathogens


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to phototoxic phytochemicals i s based exclusively on i n v i t r o studies. E f f o r t s to evaluate the involvement of these photosensi t i z e r s i n plant resistance to disease organisms have not yet been conducted. The absence of a well defined host-pathogen system for study i s one of the p r i n c i p a l reasons for this void i n our understanding. Table I I I .

UVA-Mediated Lethal A c t i v i t y of Alpha-Terthienyl Toward Various Organisms


Biocidal Activity

Susceptible Organisms

References

Bactericidal

Agrobacterium, B a c i l l u s , E. c o l i Pseudomonas, Staphlococcus

16-21

Fungicidal

A l t e r n a r i a , A s p e r g i l l u s , Candida, Cladosporium, Colletotrichum, 17-18,22-23 Fusarium, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Saprolegnia

Nematicidal

Aphelenchus, Caenorhabditis, 24-25 Ditylenchus, Meloidogyne, Pratylenchus

Insecticidal

Aedes, Euxoa, Heliothus, Manduca, O s t r i n i a , Simullum, Spodoptera

Allelopathic

Asclepias, Chenopodium, Lactuca, Phleum, T r i f o l i u m

26-31 32-33

We have recently begun to look for new plant species that contain endogenous photosensitizers which might be useful i n disease resistance studies. Preliminary e f f o r t s have centered on screening previously untested plants for UVA-induced a n t i b i o t i c a c t i v i t y . A g r i c u l t u r a l l y important species from phototoxin-containing families were given the highest p r i o r i t y for testing as d e t a i l s concerning their pathology and phytochemistry would most l i k e l y be available. Citrus and several closely related genera i n the Rutaceae were among the most active of the plants selected for examination. Methanolic leaf extracts from eight genera were applied to s t e r i l e f i l t e r paper discs and bioassayed (in UVA and dark) on agar plates using standard microbial techniques (described i n r e f . 45). The results of these bioassays are given i n Table IV. Extracts from s i x genera were phototoxic to JE. c o l i and to the yeast Saccharomyces cerevisiae while extracts of Fortunella and Glycomis species were not. A n t i b i o t i c a c t i v i t y i n the absence of UVA was not noticeable with any of the leaf extracts. These findings are of p a r t i c u l a r note because they are the f i r s t report of phototoxic action i n the genera Afraegle, A t a l a n t i a , C i t r u s , M i c r o c i t r u s , Sever!nia and Swinglea although photosensitizers have been demonstrated i n other genera of the family (7). Various furοcoumarln derivatives which have been isolated from Citrus species (46-48) are undoubtedly responsible for much of the b i o a c t i v i t y of the species assayed, however the role that other


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photosensitizers might play i n this l e t h a l action has not been established.

Table IV.

UVA-Induced A n t i b i o t i c Action of Leaf Extracts of Citrus and Related Genera of the Rutaceae


Plant Species

Inhibitory Zones E. c o l i S. cerevisiae Dark UVA Dark UVA

Afraegle paniculata (Swing.) Engler Atalantia monophylla DC. Citrus depressa Hayata (mandarin) C. grandis (L.) Osbeck (pummelo) C. limetta Risso (limetta) C. limettoides Tan. (sweet lime) C* macrophylla Wester (alemow) C. medica L. (citron) _C. miaray Wester C. sinensis (L.) Osbeck (sweet orange)C. tiawanica JC. ujukitsu Hort. ex Tan. Glycomis pentaphylla (Retz.) Correa Fortunella sp. (kumquat) Microcitrus australasica (F. Muell.) Swing. (Australian finger-lime) Severinia b u x i f o l l a (Poir.) Tenore (Chinese box-orange) Swinglea glutinosa (Blanco) Herr. (tabog or swinglea)

ïf+ ++ ++ + ++ ++ ++ ++ + + + +

-

+++ \\\ ++ + ++ +++ ++ ++ + + + +

_ -

-H-

++

-H-

-H-

No a n t i b i o t i c a c t i v i t y (-); Inhibitory zones below 14 mm between 15-20 mm (++) and larger than 21 mm (+++).

(+),

Many Citrus pathogens are known including species of A l t e r n a r i a , Cercospora, Fusarium, Mycosphaerella, Phytophthora, Pseudomonas and Xanthomonas (citrus canker). The s u s c e p t i b i l i t y of several of these organisms to endogenous Citrus photosensitizers i s currently under investigation i n my laboratory. Susceptible pathogens, based on i n v i t r o bioassays, w i l l be used to evaluate the importance of phototoxins i n the resistance of Citrus species to i n f e c t i o n by pathogenic organisms. Conclusion The number and s t r u c t u r a l d i v e r s i t y of phototoxic phytochemicals has grown tremendously over the l a s t ten years. Our knowledge and understanding of the b i o l o g i c a l a c t i v i t y of these natural products w i l l undoubtedly continue to expand as new structures are elucidated and new plant families are examined. In addition to exploratory studies, i t Is important that we begin to turn our attention toward the s i g n i f i c a n c e or function of photosensitizers. Do plants which


204


contain these chemicals have s e l e c t i v e advantages over species which lack them? Do increased levels of a p a r t i c u l a r photosensitizer increase a plants resistance to pathogens, nematodes or insect herbivory? I f so, are t h e i r autotoxic effects expressed i n tissues containing high levels of phototoxins? Answers to these and other questions are necessary i n order to c l a r i f y the role(s) of phototoxic natural products.


Acknowledgments I would l i k e to thank Drs. Eloy Rodriguez and G.H.N. Towers f o r c r i t i c a l reading of this manuscript, Dr. C. Campbell (Tropical Research and Education Center-IFAS, University of F l o r i d a , Homestead, FL) for access to the various Citrus species and related Rutaceae genera used i n this study and J.A. Downum for f i e l d and technical assistance. The support of NSF (PCM 8209100) and NIH (AI 18398) to E. Rodriguez and the FIU Foundation (571204900) i s g r a t e f u l l y acknowledged.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19.

Towers, G.H.N. Can. J. Bot. 1984, 62, 2900-11. Downum, K.R.; Rodriguez, E. J. Chem. Ecol. In press. Knox, J.P.; Dodge, A.D. Phytochem. 1985, 24, 889-96. Bohlmann, F.; Burkhardt, T.; Zdero, C. "Naturally Occurring Acetylenes"; Academic: London, 1973. Downum, K.R.; Dole, J . ; Rodriguez, E. Phytochem. In review. MacRae, W.D.; Towers, G.H.N. Phytochem. 1984, 23, 1207-21. Murray, R.D.H.; Mendez, J . ; Brown, S.A. "The Natural Coumarins: Occurrence, Chemistry and Biochemistry"; Wiley: New York, 1982. Proksch, P.; Rodriguez, E. Phytochem. 1983, 22, 2335-48. Allen, J.R.F.; Holmstedt, B.R. Phytochem. 1980, 19, 1573-82. Downum, K.R.; Towers, G.H.N. J. Nat. Prod. 1983, 44, 98-103. Downum, K.R.; Keil, D.J.; Rodriguez, E. Biochem. Syst. Ecol. In press. Raffauf, R.F. In "A Handbook of Alkaloids and Alkaloid-Contain ing Plants"; Wiley: New York, 1970. Thompson, R.H. "Naturally Occurring Quinones"; Academic: London, 1971. Arnason, T.; Towers, G.H.N.; Philogene, B.J.R.; Lambert, J.D.H. In "Plant Resistance to Insects"; Hedin, P.Α., Ed.; ACS SYMPOSIUM SERIES No. 208, American Chemical Society: Washington, D.C., 1983, pp. 139-51. Heywood, V.H. In "Flowering Plants of the World"; Mayflower: New York, 1978. Camm, E.L.; Towers, G.H.N.; Mitchell, J.C. Phytochem. 1975, 14, 2007-11. Chan, G.F.Q.; Towers, G.H.N.; Mitchell, J.C. Phytochem. 1975. 14, 2295-6. Arnason, T.; Chan, G.F.Q.; Wat, C.-K.; Downum, K.; Towers, G.H.N. Photochem. Photobiol. 1981, 33, 821-4. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photochem. Photobiol. 1982, 36, 517-23.



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20. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photobiochem. Photobiophysics 1983, 6, 145-152. 21. Norton, R.A. Personal communication. 22. DiCosmo, F.; Towers, G.H.N.; Lam, J. Pest. Sci. 1982, 13, 589-94. 23. Kourany, E.; Arnason, J.T. Personal communication. 24. Gommers, F.J.; Geerlings, J.W. Nematologica 1973, 19, 389-93. 25. Gommers, F.J.; Bakker, J . ; Wynberg, H. Photochem. Photobiol. 1982, 35, 615-19. 26. Wat, C.-K.; Prasad, S.K.; Graham, E.A.; Partington, S.; Arnason, T.; Towers, G.H.N. Biochem. Syst. Ecol. 1981, 59-62. 27. Arnason, T.; Swain, T.; Wat, C.-K.; Graham, E.A.; Partington, S.; Towers, G.H.N. Biochem. Syst. Ecol. 1981, 9, 63-8. 28. Kagan, J . ; Chan, G. Experientia 1983, 39, 402-3. 29. Kagan, J . ; Chan, G.; Dhawan, S.N.; Arora, S.K.; Prokash, I. J. Nat. Prod. 1983, 46, 646-50. 30. Downum, K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-9. 31. Champagne, D.E.; Arnason, J.T.; Philogene, B.J.R.; Morand, P.; Lam, J. J. Chem. Ecol. In Press. 32. Campbell, G.; Lambert, J.D.H.; Arnason, T.; Towers, G.H.N. J. Chem. Ecol. 1982, 8, 961-72. 33. Downum, K.R.; Quiroz, A.M.; Rodriguez, E.; Towers, G.H.N. Unpublished results. 34. Berenbaum, M. Science 1978, 201, 532-4. 35. Berenbaum, M. Ecol. Entomol. 1981, 6, 345-51. 36. Friedman, J . ; Rushkin; Walker, G.R. J. Chem. Ecol. 1982, 8, 55-65. 37. Fujita, H.; Ishii, N.; Suzuki, K. Photochem. Photobiol. 1984, 39, 831-4. 38. McKenna, D.J.; Towers, G.H.N. Phytochem. 1981, 20, 1001-4. 39. Muckensturm, B.; DuPlay, D.; Robert, P.C.; Simonis, M.T.; Kienlen, J.-C. Biochem. Syst. Ecol. 1981, 9, 289-92. 40. Oginsky, E.L.; Green, G.S.; Griffith, D.G.; Fowlks, W.L. J. Bacteriol. 1959, 78, 821-33. 41. Shimomura, H.; Sashida, Y.; Nakata, H.; Kawasaki, J . ; Ito, Y. Phytochem. 1982, 21, 2213-5. 42. Towers, G.H.N.; Graham, E.A.; Spenser, I.D.; Abramowski, Z. Planta Medica: J. Med. Plant. Res. 1981, 41, 136-42. 43. Abeysekera, B.F.; Abramowski, Z.; Towers, G.H.N. Photochem. Photobiol. 1983, 38, 311-5. 44. Towers, G.H.N.; Abramowski, Z. J. Nat. Prod. 1983, 46, 576-81. 45. Downum, K.R.; Villegas, S.; Keil, D.J.; Rodriguez, E. Biochem. Syst. Ecol. In press. 46. Kefford, J.F.; Chandler, B.V. In "The Chemical Constituents of Citrus Fruits"; Academic: New York, 1970, pp. 106-11. 47. Dreyer, D.L.; Huey, P.F. Phytochem. 1973, 12, 3011-13. 48. Fisher, J.F.; Trama, L.A. Agric. Food Chem. 1979, 27, 1334-37. RECEIVED August

9, 1985


Photoactivated Biocides from Higher Plants - ACS Symposium Series

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