Allelopathic Substances from a Marine Alga (Nannochloris sp.) - ACS

24 Allelopathic Substancesfroma Marine Alga (Nannochloris sp.) Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 12, 2016 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch024

R A L P H E. M O O N and D E A N F. M A R T I N Chemical and Environmental Management Services (CHEMS) Center, Department of Chemistry, University of South Florida, Tampa, F L 33620

Substances elaborated by a marine alga (Nannochloris sp.) adversely a f f e c t a red tide organism, Ptychodiscus brevis, i n two ways. One component causes c y t o l y s i s and a second causes P. brevis to shrink and enter a non-motile resting stage. The l a t t e r behavior is probably responsible f o r the mitigation of ichthyotoxic a c t i v i t y noted when P. brevis and the marine alga were mixed. Various studies indicate the substances elaborated by Nannochloris sp. are not toxic to fish and other marine organisms tested, though the tests, of course, have not been exhaustive. The c y t o l y t i c material has s i g n i f i c a n t ecological implications inasmuch as a serious red tide along the west coast of F l o r i d a can be responsible for massive m o r t a l i t i e s of marine animals and costs associated with a red tide outbreak have been estimated to exceed $17 m i l l i o n . Limited studies are available that indicate the d i s t r i b u t i o n of c y t o l y t i c agents inversely matches the locations of so-called "seed beds" of P. brevis. The release of a l l e l o p a t h i c substances i n the marine environment is of p a r t i c u l a r interest to those who are affected by red tides, i . e . periodic blooms of micro-organisms that give r i s e to discolored water and that may result i n intoxications. The background of the problem has been provided elsewhere (1-3). Red tides are found i n a variety of marine environments, but several red t i d e s , notably those i n Japan i n the Seto Inland Sea, and i n U.S. coastal waters ( c f . C a l i f o r n i a , New England, and the Gulf of Mexico) have attracted p a r t i c u l a r attent i o n , owing to the impact of red tide organisms on s h e l l f i s h (paral y t i c s h e l l f i s h poisoning) or mass mortality of marine animals associated with outbreaks of Ptychodiscus brevis (4_) . This organism, an unarmored d i n o f l a g e l l a t e , has been observed i n sporadic outbreaks i n west coast F l o r i d a waters and i n other nearshore waters of the Gulf of Mexico (1). Outbreaks have a considerable s o c i a l , public health and economic impact because of f i s h k i l l s . For example, the outbreak that lasted for three summer months during 0097-6156/ 85/0268-0371 $06.00/ 0 © 1985 American Chemical Society

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

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 12, 2016 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch024

372

THE CHEMISTRY OF ALLELOPATHY

1970 was estimated to cost about 17 m i l l i o n d o l l a r s (j>) because of l o s t t o u r i s t revenues, cleanup costs, and other economic impacts. While t h i s may be considerably smaller than the costs associated with an outbreak of Chattonella subsalsa i n the Seto Inland Sea (6) that a f f e c t s food f i s h , i t nevertheless represents a serious impact. The p o s s i b i l i t y of managing a bloom of Ptychodiscus brevis has been considered for many years, and various l i m i t a t i o n s have been noted to the use of chemical control (3) or the use of control by some organisms (7). C h i e f l y , the problems are two-fold: the volume to be treated and the l i m i t a t i o n s of the control agent. For example, a red tide may cover hundreds of square miles, i t w i l l be present i n patches, and may be unevenly distributed through the water column. One response may be that i t is possible to manage the red tide at the source of the bloom (8) or that there is no need to manage a red tide over vast areas, merely i n l o c a l i z e d ones that are of special i n terest. The point is moot u n t i l a suitable control agent is a v a i l a b l e . A considerable number of chemicals have been reviewed for possible red tide control substances, (9), and i n retrospect this type of research is subject to ultimate f a i l u r e because the f i r s t c r i t e r i o n of a successful control agent (chemical or b i o l o g i c a l ) has not been considered. A successful control agent probably should be sought i n the marine environment for three reasons. F i r s t , this is where the problem is and i f a natural control agent has developed, t h i s is where i t should be found. Second, the most potent chemical agents seem to be found i n the ocean or i n the atmosphere; potency appears to be required because of the tremendous d i l u t i o n s . Third, this is where we found what we believe to be a p o t e n t i a l l y useful control agent. An alga, isolated from a red tide outbreak area, produced chloroform-soluble material capable of causing l y s i s of P. brevis. The o r i g i n a l observation reported by Kutt and Martin (10) was subjected to a more thorough study by McCoy and Martin (11). The organism i n i t i a l l y was i d e n t i f i e d as Gomphosphaeria aponina, but based upon SEM studies was r e i d e n t i f i e d as a Nannochloris sp. (12). It has been demonstrated (11) that concentrated organisms when added to P. brevis culture led to the c y t o l y s i s of the red tide organism; that concentrated suspensions of destroyed c e l l s (destroyed by successive freeze-thawing) led to c y t o l y s i s , and that chloroform extracts of the c e l l - f r e e medium produced a residue that caused c y t o l y s i s of ]P. brevis (11) . The term aponin (apparent oceanic natural cytolin) has been used to describe the substance(s) responsible for c y t o l y s i s . Some e f f o r t has been directed toward understanding the growth requirements of the alga of interest, a l l e l o p a t h i c alga (13), the optimum s a l i n i t y (14), the d i s t r i b u t i o n of a l l e l o p a t h i c agents (15), and the temperature optimum (16). This paper reviews the approaches taken to separate the a l l e l o p a t h i c agents from the other materials and the methods used to characterize the b i o l o g i c a l a c t i v i t y of aponin from Nannochloris sp. Materials and Methods I s o l a t i o n of the Crude Extract. An i s o l a t i o n of Nannochloris sp. (previously referred to as Gomphosphaeria aponina) was cultured from



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samples of seawater from the west coast of F l o r i d a . Unialgal stock cultures were maintained i n an enriched seawater media (17) i n large scale semi-continuous cultures, grown under optimized culture conditions (18) . Nannochloris sp. cultures were harvested i n 30-35 l i t e r volumes shortly after the culture attained a stationary c e l l count (5000 7500 cells/ml) after 2 or 3 weeks. C e l l s were separated from the seawater media using a continuous centrifugation technique. The clear, c e l l - f r e e centrifugate was extracted for 24 hours (1 hour of mixing) with r e d i s t i l l e d chloroform (50 ml/1). Following a 24-hour s e t t l i n g period, the aqueous phase was decanted and the remaining chloroform extract was poured into a c y l i n d r i c a l separatory funnel. This chloroform-organic phase was c a r e f u l l y separated for residual aqueous portions, collected and reduced i n volume (to 5 to 10 mis) using a rotary evaporator (Buchi-rotovapor) and held at 4°C. This crude preparation was termed the "crude extract" and was used extensively for c y t o l y t i c and i n h i b i t o r y studies. Organismal Studies. The a l l e l o p a t h i c interaction between Nannoc h l o r i s sp. and several species of algae, bacteria and fungi were performed using the bioassay technique and paper disc method (cf. 19), The bioassay technique demonstrated a c y t o l y t i c e f f e c t (up to 100% k i l l ) when test species of Ptychodiscus brevis and Chattonella subsalsa (6) were exposed to various concentrations of the crude extract. Test results of Prymnesium parvum showed l i t t l e or no effect from the crude extract (28). The i n h i b i t o r y a c t i v i t y of the crude extract using the disc method was shown among a l l species of fungi tested including: Saccharomyces cerevisiae, Rhizopus s t o l o n i f e r a , Candida albicans, A l l i s c h e r i a boydii, and Aspergillus fumigatus. No i n h i b i t o r y e f f e c t s were noted for b a c t e r i a l species (Escherichia c o l i , Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus aureus) tested i n the same manner. Evaluation of HPLC Components from the Crude Extract. Individual peaks collected from HPLC i n j e c t i o n of the crude extract (Figure 1) showed that only one component (fraction 7) exhibited an i n h i b i t o r y effect upon Saccharomyces cerevisiae when tested using the paper disc method. A similar study using the germination test method (19) showed quite d i f f e r e n t responses. Observations of dark green Boston lettuce seeds were viewed from three perspectives including: 1) number of seeds germinated, 2) developmental stage achieved, and 3) f i n a l morphological orientation. During the six-day study period, most compounds showed l i t t l e or no s i g n i f i c a n t i n h i b i t i o n of seed germination when compared to cont r o l plants. Several fractions (Nos. 1-5, 9, 21, and 29, see FLgure 1) were notably i n h i b i t o r y with 60% (6 out of 10) not forming a hypocotyledon after the fourth day. A l l preparations of the crude extract series and the i n i t i a l i n j e c t i o n f r a c t i o n showed t o t a l i n h i b i t i o n of germination during the four day period. Following this observation, 100 y l of d i s t i l l e d water was added to each test dish to see i f i n h i b i t i o n of seed germination would remain permanent. On the f i f t h day, previously dormant seeds impregnated with compounds from fractions 105, 9, 21, and 26 and 10 y l of crude extract, showed development of a hypocotyledon. On the sixth day, almost a l l test



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ElutionTime (min)

F i g u r e 1. HPLC p r o f i l e o f m a t e r i a l s e l a b o r a t e d from c u l t u r e s of N a n n o c h l o r i s sp. F r a c t i o n 7 is c y t o l y t i c toward P^ b r e v i s . A f t e r Moon and M a r t i n ( 1 9 ) . C o p y r i g h t 1981, M i c r o b i o s L e t t .


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systems showed development of both hypocotyledon and cotyledons i n 70% of the seeds tested. Total i n h i b i t i o n was demonstrated by f r a c tions 0, 29, and crude extract series 25 y l through 100 u l .


Results and Discussion Lettuce Seed Studies. Variations i n f i n a l morphological development were noted when germinated test plants appeared d i f f e r e n t from control plants. Four orientation patterns were observed and include: a flattened appearance with dicotyledons upright but f l a t against the bottom of the weighing boat (Type 1), complete d i s o r i e n t a t i o n of the seedling where orientation appeared to be either horizontal, v e r t i c a l , and/or inverted (Type 2), oriented i n an inverted manner i . e . , hypocotyledon up and dicotyledon down (Type 3) and the normal orientation with dicotyledons raised upwards at least 1 cm by the hypocotyledons from the f i l t e r paper (Type 4). These orientations are summarized i n Table I, together with examples of fractions i n volved. Table I.

Behavior Type

Behavior of Dark Green Boston Lettuce Seeds i n the Presence of A l g a l Extracts (19) Appearance

Example

a

I

Dicotyledons upright, but flattened

5,6,8,13-19,21,24, 29

II

Seedling completely disoriented; orientation of dicotyledon could be horzontal, v e r t i c a l and/or i n verted. Considerable randomness noted.

1,4,7,9,10,12,25

III

Inverted. Hypocotyledon oriented upward, dicotyledon oriented downward.

2,3,11,20,26-28

IV

Normal orientation.

control

Fraction numbers, Figure 1 A Comparative Evaluation of the Crude Extract and an A n t i b i o t i c . Inh i b i t i o n and c y t o l y t i c studies of the polyene a n t i b i o t i c F i l i p i n (UpJohn Company of Kalamazoo, Michigan, U.S.A) assessed the c y t o l y t i c a c t i v i t y of an a n t i b i o t i c with antifungal c h a r a c t e r i s t i c s . Filipin was chosen as a test substance since i t lacked attached sugar moieties (as d i d the crude extract) and exhibited f u n g i c i d a l a c t i v i t y by binding to c e l l membrane s t e r o l s , d e s t a b i l i z i n g the c e l l membrane r e s u l t i n g i n a lysed c e l l (20). The crude extract was observed microscopically to lyse c e l l s also. Fungicidal testing demonstrated the i n h i b i t o r y effect of both F i l i p i n and the crude extract toward plated preparations of JS. cerevisiae (21). C y t o l y t i c bioassay preparations of the crude extract and F i l i p i n confirmed c y t o l y t i c a c t i v i t y i n both preparations. The c y t o l y t i c e f f e c t of F i l i p i n was


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i n i t i a t e d at 100-300 ppb with near t o t a l c y t o l y s i s at concentrations of 1 to 10 ppm. The c y t o l y t i c a c t i v i t y of the crude extract was demstrated between 800 ppb and 1 ppm. The i n h i b i t o r y e f f e c t s of i n v i t r o s t e r o l addition (21) showed large percentage k i l l s (83% and 91% r e s p e c t i v e l y ) . The i n v i t r o addition of ergosterol (10.1 mM) to P. brevis c e l l cultures with F i l i p i n (1.5 mM) showed complete i n h i b i t i o n of the c y t o l y t i c e f f e c t . Ergosterol (10.1 mM) added to P. brevis c e l l cultures with c e l l ex tract showed a 10% reduction i n c e l l mortality. The addition of ergosterol alone (control) showed no c y t o l y t i c e f f e c t at the experi mental concentration (10.1 mM). Mechanism of C y t o l y s i s . These r e s u l t s are p a r t i c u l a r l y s i g n i f i c a n t for understanding the mechanism of c y t o l y s i s . I t is known that aponin and ergosterol form an associated species (22) and that brevis contains ergosterol (23). Interaction of aponin and ergosterol (or a s t e r o l c l o s e l y related to ergosterol) should disrupt the i n t e g r i t y of a P_. brevis c e l l membrane and adversely a f f e c t the membrane osmoregulatory c a p a b i l i t i e s . The hypothesis is supported by two additional observations (24). F i r s t , mean c e l l volume for Ρ_. brevis i n the absence of aponin re mained constant for 8 hours, but, i n the presence of aponin, a notable increase was observed within an hour and continued for eight hours. Second, Trypan blue (CI 23850) tests indicated increased c e l l permeability i n the presence of aponin: v i a b l e , motile c e l l s were only s l i g h t l y stained; swollen c e l l s and c e l l debris were highly stained. Other Mechanism(s) of Red Tide Limitation. Cytolysis is not the only mechanism of a l l e l o p a t h i c a c t i v i t y of Nànnochloris sp., and, indeed, i t may not be the best a c t i v i t y to focus attention on. The release of toxins from P^. brevis i n confined volumes by aponin would hardly engender an enthusiastic response from a l l ecologists. On the other hand, we suspected that some f r a c t i o n of the crude extract was responsible for mitigating the ichthyotoxic a c t i v i t y of P. brevis i n the presence of aponin (11, 24). For example, Nànnochloris sp. c e l l s mixed with P. brevis produced two e f f e c t s , depending upon the r e l a t i v e concentrations (24). R e l a t i v e l y high concentrations of Nànnoc h l o r i s sp. resulted i n c y t o l y s i s and c e l l debris, but r e l a t i v e l y low concentrations resulted i n s e s s i l e or "resting-stage" P_. brevis c e l l s that were small, thick-walled, and non-motile. The l a t t e r behavior is s i g n i f i c a n t because the P. brevis would be rendered i n e f f e c t i v e and ichthyotoxins would presumably not be released from s e s s i l e c e l l s . A l l fractions (Fig. 1) were i n v e s t i gated by Pabon and Martin (25). After i n i t i a l screening of fractions (Figure 1) for a c t i v i t y , i t appeared that sessile-formation a c t i v i t y was associated with f i r s t ten fractions (minus c y t o l y t i c f r a c t i o n 7), and further investigation indicated that f r a c t i o n 4 was responsible for induction of c y t o l y s i s (at 500 ppb). Clearly, additional studies are i n order, but the evidence of a mechanism that causes an addit i o n a l disfunction of P. brevis c e l l s is encouraging. F i e l d Evaluation Studies for Cytolytic/Fungicidal Substances (26). The c y t o l y t i c e f f e c t of the crude extract on P. brevis prompted an



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investigation to i s o l a t e and detect c y t o l y t i c compounds i n the marine environment. Marine sediments were selected since t h i s would be most l i k e l y to contain the greatest concentration of l i p o p h i l i c substances i n contrast to the evaluation of seawater. The study (26) involved the comparison of marine sediment extracts from f i f t e e n stations along the West Coast of F l o r i d a . Comparative tests using the b i o assay technique on P. brevis showed that a l l sediment extracts were c y t o l y t i c toward P_. brevis. Sediment extracts from shallow stations showed the highest amount of c y t o l y t i c a c t i v i t y when compared to deeper station locations. In a general way, c y t o l y t i c a c t i v i t y showed an inverse relationship with location for red tide "seed beds". Those areas that are thought to be the points of o r i g i n of red tide (27) were the locations that showed the least c y t o l y t i c a c t i v i t y i n the sediments. The implications of this have been reviewed elsewhere (8). Fungicidal a c t i v i t y was determined by the disc method and zones of i n h i b i t i o n were recorded by measuring the diameter (mm) of the i n h i b i t i o n zone. Yeast cultures (S. cerevisiae) showed growth i n h i b i t i o n (clear area surrounding disc) by sediment extracts from a l l stations when compared to control discs. HPLC analysis of sediment extracts showed more than 20 components i n the migration p r o f i l e of each s t a t i o n . Of these components, a f r a c t i o n demonstrated to possess c y t o l y t i c a c t i v i t y i n the crude extract (V = 24 ml) was present i n a l l stations when compared to the migration p r o f i l e of the active f r a c t i o n (Figure 2). e

Other Organisms Tested with Aponin. I t is probably impossible to s a t i s f y a l l persons interested i n the e f f e c t of aponin or other organisms because the range of p o t e n t i a l l y affected organisms is so great. Nevertheless a number of organisms have been tested. We tested f i s h which were adapted to s a l t water (Poecilia sphenops) (11), and other f i s h have been tested at Mote Marine Laboratory (cf. 8) ; none were adversely affected by aponin or Nannochloris sp. As noted e a r l i e r , two marine algae have been tested: a Chrysomonad, Prymnesium parvum was unaffected by aponin (28), though a Japanese red tide organism Chattonella subsalsa (6) was cytolyzed by aponin. Brine shrimp, Artemia s a l i n a , were not adversely affected by Nannochloris sp. nor aponin (29). In addition, i t has been reported clams and some other organisms were not adversely affected i n studies done at Mote Marine Laboratory, Sarasota, FL (cf. .8). In summary, organisms representing a number of trophic levels have been studied and though there is always room for additional research i n t h i s area, results to date are promising. Comparative Evaluation of Related Compounds. Structural studies are incomplete, but NMR and gas chromatographic - mass spectroscopic analysis of a component (fraction 7) isolated by HPLC indicated b i phenyl c h a r a c t e r i s t i c s (30). In an attempt to evaluate the c y t o l y t i c effect of compounds with similar structure, 4-hydroxybiphenol and 2hydroxybiphenol were tested for l y t i c e f f e c t s on ]?. brevis. Bioassays of 2- and 4-hydroxybiphenol indicated that both compounds were c y t o l y t i c to 1?. brevis (31) .



378


V

e

Figure 2. HPLC chromatograms ( i s o c r a t i c mode, 60% methanol, 40% water) of sediment extracts from 15 study s i t e s i n west F l o r i d a coastal waters. Migration p r o f i l e are compared among sediment extracts and crude extract of Nànnochloris sp. c e l l - f r e e culture [See Moon and co-workers (26) for s p e c i f i c s i t e s ] .



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379 Allelopathic Substances from a Marine Alga

Summary The results presented here indicate the existence of environmentally significant allelopathic substance or substances that affect a red tide organism in laboratory studies. It would be difficult to imagine that such substances do not have some impact in the natural environment, particularly in view of the observed (26) distribution of aponin vis-a-vis the presence of "seed beds" of red tide (8) . The cytolytic agent termed aponin has been characterized in terms of biological activity, and the results to date indicate a material that does not adversely affect the organisms tested. Material isolated from cultures of Nannochloris sp. also has some phytotoxic activity as evidenced by the assays with lettuce seeds, and some antifungal activity. The materials elaborated by Nannochloris sp. thus have environmental significance, but it must be admitted that the full significance of these materials, like others, remains to be fully appreciated. Acknowledgments We gratefully acknowledge the support of the National Institute of Environmental Health Sciences (through grant number ES02810-03). Literature Cited 1. Rounsefell, G. Α.; Nelson, W. R. U.S. Fish Wildl. Serv. Spec. Sci. Rpt. Fisheries 1966, No. 535. 2. Martin, D. F.; Martin, Β. B. J. Chem. Educ. 1976, 53, 614-617. 3. Steidinger, K. A. Prog. Physiol. Res. 1983, 2, 435-442. 4. Steidinger, K. A. Proc. 2nd Internat. Conf. Toxic Dinoflagellate Blooms 1979, p. 435-442. 5. Habas, E. J.; Gilbert, C. K. Environ. Letters 1974, 6, 134-147. 6. Halvorson, M. J.; Martin, D. F. J. Environ. Sci. Health 1981, A16, 373-379. 7. Steidinger, Κ. Α.; Joyce, Jr., E. A. Fla. Dept. Nat. Resources, Educ. Ser. 1973, No. 17. 8. Martin, D. F. J. Environ. Sci. Health 1983, A18, 685-700. 9. Marvin, K. T.; Proctor, Jr., R. R. U.S. Fish Wildl. Serv. 1964, Data Rpt. 2. 10. Kutt, E. C.; Martin, D. F. Environ. Letters 1975, 9, 195-208. 11. McCoy, Jr., L. F.; Martin, D. F. Chemico-Biol. Interactions 1977, 17, 17-24. 12. Sakamoto, Y.; Krzanowski, J. J.; Lockey, R. F.; Martin, D. F. J. Environ. Sci. Health 1983, A18, 721-728. 13. Eng-Wilmot, D. L.; Martin, D. F. Microbios 1978, 19, 167-179. 14. Martin, D. F.; Gonzalez, M. G. Water Res. 1978, 12, 951-955. 15. McCoy, Jr., L. F.; Martin, D. F. J. Environ. Sci. Health 1978, A13, 517-525. 16. Eng-Wilmot, D. L.; Hitchcock, W. S.; Martin, D. F. Mar. Biol. 1977, 41, 71-77. 17. McCoy, Jr., L. F.; Eng-Wilmot, D. L.; Martin, D. F. J. Agri. Food Chem. 1979, 27, 69-74. 18. Eng-Wilmot, D. L.; Martin, D. F. Fla. Sci. 1977, 40, 193-197. 19. Moon, R. E.; Martin, D. F. Microbios Letters 1981, 18, 103110. 20. Gottlieb. D.; Carter, H. E.; Lung-Chi, W.; Sloneker, J. H. Phytopathol. 1960, 50, 594-603.



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21. Moon, R. E.; Martin, D. F. Microbios Letters 1980, 10, 115119. 22. Barltrop,J.;Martin, D. F. Microbios Letters in press, 23. Hamdy, Α.; Jahoda, S. W.; Martin, D. F. Microbios Letters in press. 24. Eng-Wilmot, D. L.; Martin, D. F. Microbios Letters 1981, 17, 109-116. 25. Pabon'de Majid, L.; Martin, D. F. Microbios Letters 1983, 22, 59-65. 26. Moon, R. E.; Krumrei, T. N.; Martin, D. F. Microbios Letters 1980, 14, 7-15. 27. Steidinger, K. A. Crit. Rev. Microbiol. 1973, 3, 4 28. Moon, R. E.; Martin, D. F. Bot. Mar. 1981, XXIV, 591-593. 29. Eng-Wilmot, D. L.; Martin, D. F. J. Pharma. Sci. 1979, 963966. 30. Moon, R. E. Doctoral Dissertation, University of South Florida, Tampa, 1980. 31. Moon, R. E.; Martin, D. F. J. Environ. Sci. Health 1981, A16, 373-379. RECEIVED July 12, 1984


Allelopathic Substances from a Marine Alga (Nannochloris sp.) - ACS

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