The Chemistry of Allelopathy - ACS Publications - American Chemical

26

Oxygenated Fatty A c i d s : A Class o f Allelochemicals f r o m A q u a t i c Plants ROBERT T. VAN ALLER, GEORGE F. PESSONEY, VAN A. ROGERS, EDWARDJ.WATKINS, and HAROLD G. LEGGETT

Downloaded by CORNELL UNIV on May 28, 2017 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch026

Departments of Chemistry and Biology, University of Southern Mississippi, Hattiesburg, MS 39401 Allelochemic effects of aquatic macrophytes on algae are discussed. Bioassays of chromatographic fractions from Eleocharis microcarpa Torr. indicate that oxygenated fatty acids are the causative agents. Methods of isolation of these materials from aquatic macrophytes and from natural waters are described. Purification and structure determinations show that prominent components of the fraction are C trihydroxycyclopentyl and C hydroxycyclopentenone fatty acids. Similar components were extracted from other aquatic plants. In addition, these components were extracted from pond waters. Implications to algal autoinhibition, algal succession in eutropic waters, and control of algal diversity are discussed. 20

18

Ecologiste for many years have been fascinated by the possible causes and controls of phytoplankton diversity and seasonal succession. Even from the early days, investigators have recognized that numerous factors play a part and that pH, major nutrient ions, and physical factors are important causes. See review articles by Hutchinson (1)» Dugelate (2) and Tilman, al. (3). Allelochemic effects between algal species in eutropic systems have also been recognized as important factors (4-6). Keating (6) studied algal succession in a eutropic pond and concluded that dominant blue-green algae secreted substances that inhibited predecessor species and stimulated successor species. Extracts of cultures were studied but no attempt was made to identify chemicals. Earlier, Proctor (7) grew two member algal cultures in each of several combinations from which he established a dominance pattern. He then steam-distilled a yellowishwhite substance from one of the cultures and concluded the substance was a mixture of fatty acids. Spoehr (8) isolated "chlorellin" from Chlorella sp. and concluded that the substance was a mixture of photooxidized unsaturated fatty acids. For several years, we have been interested in the effect that 0097-6156/85/0268-0387$06.00/0 © 1985 American Chemical Society

Thompson; The Chemistry of Allelopathy ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


388

THE CHEMISTRY OF ALLELOPATHY

higher aquatic plants exhibit i n c o n t r o l l i n g plankton d i v e r s i t y i n eutropic systems. In one of the few studies of allelochemic i n t e r actions between higher plants and algae, Hasler and Jones i n 1949 (9) studied the e f f e c t s Potamogeton f o l i o s u s and Anacharis canadensis on phyto- and zooplankton and found blue-green algae to be i n h i b i t e d by these plants. In a study by Fleming i n these laboratories (JO), an inverse c o r r e l a t i o n was made between the amount of coverage of Chara vulgaris and numbers and d i v e r s i t y of blue-green algae i n f i s h hatchery ponds. Our investigations of water quality i n many commercial aquacul ture ponds (11) have shown that such systems are highly eutropic and that most experience a dense bloom of blue-green^algae during^ the summer months. These blooms range from 2 χ 10 TO 5 χ 10 c e l l s / 1 of usually one or sometimes two species of the genera Micro cystis, O s c i l l a t o r i a , Anabaena, and Lyngbya. Very few rep resentatives from the other d i v i s i o n s of algae are evident during these blooms. A few ponds are seen to have dense growths of the higher plants Najas sp. and Eleocharis sp. These pondg are also eutropic but c h a r a c t e r i s t i c a l l y contain fewer than 5 χ 10 cells/1 that consist mostly of a normal d i v e r s i t y of green algae and diatoms with blue-green algae present only i n small numbers. Macronutrients exist i n both sediment and water column i n s u f f i c i e n t quantities to promote a l g a l blooms. The nutrient concentrations i n these ponds are comparable to adjacent ponds with heavy a l g a l blooms. Zooplankton are generally present i n these ponds i n normal amounts. Blue-green algae are notorious for causing oxygen depletion, toxic f i s h k i l l s , and odors i n eutropic systems. They are also im p l i c a t e d i n the widespread occurrance of Legionella pneumophila, the causative organism of Legionaires' Disease (12). We began our i n vestigations of the allelochemic effects of aquatic higher plants on blue-green algae i n the hope of i d e n t i f y i n g selective blue-green algal inhibitors. Preliminary Investigations Agar-paper disk bioassay was used to test fractions from various chromatographic separations. Twenty-four species of algae were screened i n order to select at least one that: would show good i n h i b i t i o n by a crude extract of 15. microcarpa, could be maintained i n culture, and would produce r e s u l t s whithin a reasonable period of time. Table I l i s t s organisms used i n preliminary bioassays. Cultures were obtained from the Texas C o l l e c t i o n of Algae at the University of Texas, or i s o l a t e d from l o c a l habitats. Cultures were grown under standard laboratory conditions on a l g a l growth media s o l i d i f i e d with agar. Both axenic and u n i a l g a l cultures were used. I n h i b i t i o n zones were noted from 7 to 14 days after inoculation. It can be seen that the extract was e f f e c t i v e i n i n h i b i t i n g blue-green algae to a greater extent than other taxa. After i n i t i a l screening, Anabaena flos-aquae was selected to be used exclusively to assay for b i o l o g i c a l a c t i v i t y of chromatographic f r a c t i o n s . Separation and Structural Studies A

crude

extract of ]2. microcarpa was prepared by b o i l i n g the


fresh

VAN A L L E R ET A L .

Oxygenated Fatty Acids

Table I.

Inhibition of Selected Algae

Chlorophycophyta


Cyanochloronta

Anabaena catenula A. c y l i n d r i c a A. flos-aquae Anabaena sp. Anacystis nidulans Cylindrospermum sp. Lyngbya sp. Nostoc muscorum O s c i l l a t o r i a tenuis Phormidium sp Plectonema notaturn

++ ++ ++ ++ + ++ ++ ++ ++ ++ ++

Chlamydomonas eugametos C. r e i n h a r d t i i C h l o r e l l a sp. Chlorococcum hypnosporum Scenedesmus quadricauda Scenedesmus sp. Stichococcus sp. Ulothrix fimbriata Oedogonium sp.

_

-+ + -

+

Ch ry s ophy cophyt a Euglenophycophyta

Euglena g r a c i l i s

—

Xanthophyceae Tribonema sp. Bacillariophyceae Nivicula sp.

+ +

(++) good i n h i b i t i o n , (+) detectable zone, (-) no i n h i b i t i o n



390

T H E C H E M I S T R Y OF A L L E L O P A T H Y

plant i n water for one hour and f i l t e r i n g . After a c i d i f y i n g , active materials were extracted into chloroform and the excess chloroform removed by evaporation. The resulting crude extract was used i n c o l umn and TLC separations. Figure 1 shows the results of column chromatography on s i l i c a gel followed by two consecutive preparative TLC systems. The chloroform f r a c t i o n from column chromatography gave a s l i g h t l y p o s i t i v e bioassay and contained simple saturated and unsaturated fatty acids by comparison of RRTs with authentic samples by GLC. The chloroform/acetone f r a c t i o n contained most of the extracted material and a c t i v i t y . More polar fractions did not show a c t i v i t y and were therefore not investigated further. The o i l y chloroform/acetone f r a c t i o n gave t y p i c a l broad carboxylic IR absorpt i o n between 2850 cm and 2350 cm and at 1710 cm · It was also soluble i n d i l u t e NaOH and NaHCO^. E s t e r i f i c a t i o n eliminated the broad carboxylic absorption and produced a normal carbonyl ester absorption at 1730 cm _ j A p o s i t i v e 2,4-DNP test and a strong IR absorption at 3400 cm , which was unchanged by e s t e r i f i c a t i o n , confirmed that this mixture contained keto and hydroxy f a t t y acids. Preparative TLC (System I, 85:15:2 chloroform/methanol /water followed by System IV, 50:50:2 hexane/ethyl ether/formic acid) of the chloroform/acetone f r a c t i o n produced 43 separate bands, 33 of which had d e f i n i t e a c t i v i t y . IR spectra of these active bands were s t r i k i n g l y s i m i l a r , e x i b i t i n g the same major features as noted f o r the unseparated chloroform/acetone f r a c t i o n . UV spectra were also s i m i l a r : absorption at 275 and 220 nm, with the l a t t e r being strongest. Autoxidation of unsaturated fatty acids is well known. M o d i f i cations of the extraction and separation procedures were made to i n vestigate the p o s s i b i l i t y that these oxygenated fatty acids (OFAs) arise as a r t e f a c t s . When exposure to l i g h t and a i r were minimized, no changes were noted i n TLC and HPLC. The r e l a t i v e l y large number and small amounts of these OFAs were surprising and made further s t r u c t u r a l studies d i f f i c u l t . Since f r a c t i o n 4:6 had good a c t i v i t y and was well separated from other bands, i t was used for f u r j ^ e r studies. Ozonolysis, low and high resolution MS, H-NMR and C-NMR data obtained on 4:6 allowed structure (I) (13-15) to be assigned.

This compound has not been previously reported i n the l i t e r a ture. The compound is unusual i n that the s u b s t i t u t i o n pattern of a l k y l and hydroxyl groups on the ring is analogous to the prostaglandin F series.



SATD.

mg

4 . 3 mg Rf O.04

41

H-)

3 B A N D S 3 (—)

Τ

4 6 4 mg (+)

6 BANDS 1 5 (+)

PREP T L C S Y S T E M IV

4:8

7 BANDS 3 ( - ) 4 (+)


1 4 . 2 mg Rf O.85

2 mg (+) Rf O.15

3 mg (+) Rf O.26

Rf

O.34

Institute,

45


2 3 . 9 mg Rf O.74

(—)

F i g u r e 1. Column and p r e p a r a t i v e t h i n l a y e r chromatography. Reproduced w i t h p e r m i s s i o n from Ref. 15. C o p y r i g h t 1983, The Hormel

44

5 3 8 . 6 mg Rf O.66

8 BANDS 0 ( - ) 8 eH „ 0

Q

7

o

2

-CH.

+

C

5 5°2 H

97 * 111 • 113

-CH,

+

C

4 3°2

F i g u r e 4. Low and h i g h r e s o l u t i o n mass s p e c t r a l p a t t e r n s of Component I I .

H

83 * 97 * * 99

fragmentation


VAN ALLER ET AL.



Table I I .

Carbon Thirteen NMR C o r r e l a t i o n

Ο

Carbon No.

Chemical S h i f t s

Ha

1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18

176.5 36.9 132.0 138.2 33.8 29.4 28.8 40.8 73.1 44.2 129. 1 142.5 208.2 141.0 130.0 24.7 13.7

Attached Proton

lib

176.5 36.9 131.8 138. 1 34.0 29.2 28.7 40.4 71.7 44 .2 128.9 142.4 207.9 29.3 29.7 24 .0 13.9

Test

Ha

lib

UP UP DOWN DOWN UP UP UP UP DOWN DOWN DOWN DOWN UP DOWN DOWN UP DOWN

UP UP DOWN DOWN UP UP UP UP DOWN DOWN DOWN DOWN UP UP UP UP DOWN


398


Table I I I . OFA Occurrance by HPLC


Source

Potamogeton sp. (Pond Weed) Najas sp. Thalassla sp. (Turtle Grass) Ruppia sp. (Widgeon Grass) Chara sp. ^ Oscillatoria jp. C h l o r e l l a sp. Pond A Pond Β Pond C Pond D

I

II

+ ++ + + + +

++ + ++

++ + + ++

++ ++

Legend: Pond A - normal d i v e r s i t y of phytoplankton Pond Β - Microcystis sp. bloom Pond C - very l i t t l e phytoplankton Pond D - dense growth E. microcarpa 1 - laboratory cultures (++) major peak, (+) minor peak

When Winter senescent plant material was harvested and extracted, y i e l d s of OFAs were greatly reduced. OFAs from the four ponds were extracted e a s i l y from 20-50 l i t e r s of pond water by passing the water through a tube f i l l e d with ODS s i l i c a . These ponds produced between 1 and 1.5 ppm of OFAs after e l u t i o n with methyl formate. The o r i g i n of I, l i a and l i b i n these ponds is uncertain at t h i s time. Discussion OFAs i n E. microcarpa and other higher aquatic plants, as well as i n waters where these plants grow, appear to exert d e f i n i t e effects on the phytoplankton community i n eutropic systems by showing selective i n h i b i t i o n against blue-green algae. The large number of these met abolites is surprising, but perhaps should be expected i n view of the variety of chain-lengths and unsaturated centers found i n fatty acid constituents of aquatic plants (19). At least one enzyme system from a higher plant, f l a x , has the a b i l i t y to convert C . g , C « Q , and t r i e n o i c fatty acids to cyclopentenone fatty acids (17;. Recent work i n these laboratories indicates that small amounts of the OFA mixture from 12. microcarpa, less than 1 ppm, stimulate many species that are inhibited at higher concentrations. Also, i n d i v i d u a l components of the mixture may i n h i b i t , have no effect on, or stimulate d i f f e r e n t algae. The presence of I and smaller amounts of other OFAs i n algae (Table III) is interesting i n view of autoinhibition of algae c u l tures (7, 20, 21). Autoinhibition occurs as cultures begin to age at



26. VAN ALLER ET AL.


399

high numbers of cells/liter, presumably because of cellular metabolites being released into the medium. Pratt showed that autoinhibitors from Chlorella vulgaris also inhibited C. pyrenoidosa and other algae (22). He also found, as we did with OFAs, that low concentrations of the autoinhibitors stimulated growth (23). Spoehr studied the antibiotic chlorellin produced by £. pyrenoidosa, the same substance studied by Pratt (24), and found evidence that the material was a complex mixture of oxidized unsaturated fatty acids (8). He concluded that the mixture resulted from photooxidation, since inhibition of Staphylococcus aureus was increased by exposing the inhibitors to air and sunlight. It seems possible, however, since OFAs were detected from Chlorella sp. in these laboratories, that his inhibitors were similar to ours when isolated but became more oxidized on treatment with air and sunlight. This possibility is being investigated. As stated earlier, the effects of individual OFAs on the growth of algae are complex. Liquid chromatograms of plant extracts, including the three algae, while having the same general appearance, have components in differing amounts as well as components with different retention times. These differing patterns of constituents may relate to algal succession in eutropic systems. Boyd, in a study involving two membered cultures of blue-green and green algae, found that many green algae were inhibited by blue-green algae but that none of the blue-green algae were inhibited by green algae. He also found one green alga to be stimulated by a blue-green alga. He found inconsistent patterns, however, when filtrates of water from bloom ponds were used in growth media and attributed pattern differences to extracellular substances from genera other than the dominant bloom alga (25). It remains to be demonstrated that OFAs are extracellular metabolites of algae and that they are prominant factors among the many determinants of succession and dominance patterns of phytoplankton. It appears that these compounds are worthy of further structural study, and that the effects on individual algae show promise of providing insight into the complex interactions in aquatic ecosystems. Summary A relatively large number of OFAs have been detected in several aquatic plants in small amounts. Bioassays of the OFA mixture against various algae showed them to have good specificity against blue-green algae. Three principal components of the group were identified from IS. microcarpa and were also indicated in pond water inhabited by this plant. Bioassay of these components indicate a similar specificity against blue-green algae. Further work is necessary before the overall effects of individual OFAs on dominance and inhibition patterns can be determined with reasonable certainty. Literature Cited 1. Hutchenson, G. E. Am. Nat. 1961, 95, 137-45. 2. Dugelate, R. C. Limnol. Oceanogr. 1967, 12, 685-95. 3. Tilman, D.; Kilham, S. S.; Kilham, P. Ann. Rev. Ecol. Systems 1982, 13, 349-72.


400

4. 5. 6. 7. 8. 9. 10. 11. 12.


13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.


Whittaker, R. H.; Feeny, P. P. Science 1971, 171, 757-70. Putnam, A. R. Chem. Eng. News 1983, 61(14), 34-35. Keating, Κ. I. Science 1977, 196, 885-87. Proctor, V. W. Limnol. Oceanog. 1957, 2, 125-29. Spoehr, V. W. "Fatty Acid Antibacterials From Plants"; Carnegie Institution, Washington, D.C., Publication No. 586, 1949; p. 24. Hasler, A. D.; Jones, E. Ecology 1949, 30, 359-64. Fleming, A. N. M.S. Thesis, University of Southern Mississippi, Hattiesburg, Mississippi, 1973. "Experimental Treatment of Catfish Ponds with Algal Inhibitors," National Marine and Fisheries Service, 1983. Tison, D. L.; Pope, D. H.; Cherry, W. B.; Fliermans, C. B. Journal of Applied Environmental Microbiology 1980, 39, 456459. Clark, L. R. Ph. D. Thesis, University of Southern Mississippi, Hattiesburg, Mississippi, 1980. Rogers, V. A. M.S. Thesis, University of Southern Mississippi, Hattiesburg, Mississippi, 1983. van Aller, R. T.; Clark, L. R.; Pessoney, G. F.; Rogers, V. A. Lipids 1983, 18, 617-22. Rogers, V. A.; van Aller, R. T.; Pessoney, G. F.; Watkins, E. J.; Leggett, H. G. Lipids 1984, 19, 303-305. Zimmerman, D. C.; Feng, P. Lipids 1978, 13, 313-16. Vick, Β. Α.; Zimmerman, D. C. Lipids 1979, 14, 734-40. Shorland, F. B. In "Chemical Plant Taxonomy"; Swain, T., Ed.; Academic Press: New York, 1963; pp. 255-57. Pratt, R. Amer. J. Bot. 1940, 27, 52-56. Pratt, R. Amer. J. Bot. 1940, 27, 431-36 Pratt, R. Amer. J. Bot. 1943, 30, 404-08. Pratt, R. Amer. J. Bot. 1942, 29, 32-33. Pratt, R.; Spoehr, H. A. Science 1944, 99, 351-2. Boyd, C. E. Weed Science 1973, 21, 27-37.

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The Chemistry of Allelopathy - ACS Publications - American Chemical

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