Journal of Chemical Ecology, Vol. 18, No. 10, 1992
INHIBITORY E F F E C T S OF T E R P E N E A L C O H O L S A N D A L D E H Y D E S ON G R O W T H OF G R E E N A L G A
Chlorella pyrenoidosa
M I Y O S H I I K A W A , * S T E P H E N P. M O S L E Y , and L I N D A J. B A R B E R O
Department of Biochemistry and Molecular Biology University of New Hampshire Durham, New Hampshire 03824 (Received February 27, 1992; accepted May 29, 1992) Abstract--The growth of the green alga Chlorellapyrenoidosa was inhibited by terpene alcohols and the terpene aldehyde citral. The strongest activity was shown by citrah Nerol, geraniol, and citronellol also showed pronounced activity. Strong inhibition was linked to acyclic terpenes containing a primary alcohol or aldehyde function. Inhibition appeared to be taking place through the vapor phase rather than by diffusion through the agar medium from the terpene-treated paper disks used in the system. Inhibition through agar diffusion was shown by certain aged samples of terpene hydrocarbons but not by recently purchased samples. Key Words--ChloreUa pyrenoidosa, alga, terpenes, growth inhibition. INTRODUCTION Terpene hydrocarbons, alcohols, aldehydes, and ketones have been reported to have antibacterial and antifungal activity (Morris, 1972; Smimoff, 1972; Uzdennikov, 1972; Andrews et al., 1980; Mathela, 1981), and it has been suggested that terpenes in the diet may be responsible for the gut o f the larvae of the Douglas fir tussock moth Orgyia pseudotsugata being relatively free of microflora (Andrews et al., 1980). Therefore, terpenes may have an ecological significance in certain situations involving microorganisms. Studies involving the effects o f terpenes on microalgae are more limited. The use of terpene alcohols as anti-blue-green algal agents has been described in the European patent literature (Juettner, 1983). *To whom correspondence should be addressed. 1755 0098-0331/92/1000 1755506.50/0 9 1992 Plenum Publishing Corporation
1756
IKAWAET AL.
In previous studies we have described the pronounced inhibitory effects of fungi-produced toxins (mycotoxins) on the growth of the green alga Chlorella pyrenoidosa (Ikawa et al., 1969, 1979, 1985; Sullivan and Ikawa, 1972). Many of the more active of these mycotoxins are terpenoids, and terpene intermediates are involved in their biosynthesis. This paper reports on an extension of this work to the simple terpenes themselves. METHODS AND MATERIALS
Paper disks (6 mm, Difco Laboratories, Detroit, Michigan) were dipped into test solutions in ethanol, excess solution removed by touching the disks to the edge of the container (ca. 20 #I of solution was retained on the disk), and the disks placed on the surface of light yellow-green Chlorella-seeded agar plates (100-mm-diameter Petri plates, three disks per plate) prepared as previously described (Ikawa et al., 1985). The plates were placed under a fluorescent light at room temperature and zones of inhibition read after two days under constant illumination. RESULTS AND DISCUSSION
The terpene hydrocarbons showed little or no inhibition at a disk concentration of 10 mg/ml (Table 1). However, it was found that samples of some of the terpenes that had been collected from various laboratories on campus and that presumably had been on the shelf for some time did show zones of growth inhibition (Table 1, footnote d), indicating that the older samples had probably undergone chemical changes to form inhibitory substances. These results recall those that had been observed with chlorellin, the antibacterial and autotoxic substance produced by Chlorella, where it was shown that chlorellin activity was due to photooxidation products of unsaturated fatty acids present in Chlorella (Spoehr et al., 1949). Most of the terpene alcohols and aldehydes tested showed inhibition that was evident as a lightening or total wipe out of color in the light yellow-green Chlorella lawn (Table 1). That inhibition was taking place through the vapor phase rather than by diffusion through the agar was shown by observing the same results when the paper disks were placed on slightly larger Teflon disks and thus were not in direct physical contact with the agar surface. The greatest inhibition was shown by nerol, gemniol, citronellol, and citral, all of which possess the acyclic terpene structure with a primary alcohol or aldehyde function (Figure 1). Citral appeared to be the most active, showing complete plate color wipe out with three disks at 1 mg/ml. The cyclic terpene alcohols bomeol and isobomeol and terpenes carrying the alcohol function at a nonterminal position
GROWTH INHIBITIONOF Chlorella BY TERPENES
1757
TABLE 1. INHIBITION OF Chlorella GROWTH BY TERPENES
(Net diameter of inhibition zone) (mm)b Compound Terpene hydrocarbons ( +)-Limonene a Myrcene ( +)-c~-Pinene d (-)-c~-Pinene (-)-/3-Pinene a a-Terpinene 7-Terpinene Terpene alcohols (-)-Bomeol Isobomeol Cineole Citronellol Geraniol Linalool Nerol Terpinen-4-ol et-Terpineol a Famesol (isomers) Hexenols cis-2-Hexen- 1-ol trans-2-Hexen- 1-ol cis-3-Hexen-l-ol (leaf alcohol) trans-3-Hexen- 1-ol trans-4-Hexen- 1-ol 5-Hexen- 1-ol Terpene aldehydes and ketones (+)-Camphor Citral
Source a
10 mg/mlc
1 mg/mF
Aldrich Aldrich Aldrich Aldrich Aldrich Aldrich Aldrich
2 Tr 0 0 0 0 0
0
K &K K &K Aldrich Aldrich Aldrich Aldrich K & K Aldrich Aldrich Aldrich
,e * 0 **~ ** * ** * 0 6
0 0
Aldrich Aldrich Aldrich
0 0 0
Aldrich Aldrich Aldrich
0 0 0
Aldrich
0 **
0.1 mg/mlC
0 * *
Tr.
**
~ Aldrich Chemical Co., Milwaukee, Wisconsin; K & K Laboratories, Plainview, New York. bDisk diameter (6 mm) subtracted from the total diameter of the inhibition zone. Figures represent the mean of all disks (three per plate) mn on two or more separate occasions. Standard deviations generally ran < 10% of the mean for means > 5 mm. CConcentration of test solution in ethanol. dOlder shelf samples of these compounds that were collected from various laboratories on campus showed inhibition at 10 mg/ml. ~* indicates platewise lightening of the lawn color as compared to control plates. ** indicates complete wipe out of the yellow-green lawn color with three disks per plate. Similar results were obtained when disks were prevented from direct contact with the agar surface by interposing larger diameter Teflon disks between the paper disks and the agar surface.
1758
IKAWA ET AL.
Myrcene
Limonene
~-Terpinene
C~-Pinene
~-Pinene
Camphene
0~-Terpineol
Geraniol
Terpinen-4-ol
Nerol
Cineole
Geranial (Citral a)
~-Terpinene
p-Cymene
Borneol (endo) Isoborneol (exo)
Camphor
Linalool
Citronellol
Neral (Citral b)
Farnesol
FIG. 1. Structures of terpenes tested against Chlorella. (a-terpineol, terpinen-4-ol, linalool) showed less activity than the more active alcohols. The sesquiterpene alcohol farnesol showed some activity at 10 mg/ml through agar diffusion. The leaf alcohol (cis-3-hexen-l-ol) and a series of related hexenols were also tested. As shown in Table 1, none of the compounds showed any activity at a concentration of 10 mg/rnl. In contrast to the activity of terpene hydrocarbons against bacteria and fungi, our results indicate that the hydrocarbons are not particularly toxic to the green alga Chlorella. Terpene alcohols showed activity, the highest activity being shown by acyclic compounds that carried the functional group on the terminal carbon atom. Acyclic terpene alcohols have been described as antiblue-green algal agents, with famesol being particularly active against Synechococcus (Juettner, 1983).
GROWTH INHIBITION OF
Chlorella BY TERPENES
1759
Lorber and Muller (1976) showed that when root tip cells o f Cucumis sativus were exposed to the vapors o f the volatile monoterpenes from the leaves o f Salvia leucophylla, disruption of the membranes surrounding the nuclei, mitochondria, and dictyosomes took place. Disruption o f the cytoplasmic membranes o f Bacillus thuringiensis and S a c c h a r o m y c e s cerevisiae were also observed when these microorganisms were treated with ct-pinene (Andrews et al., 1980). Lipophilicity appeared to be an important contributing factor in the inhibition o f lettuce fruit germination by terpenes (Reynolds, 1987). In this case, the terpene alcohols, aldehydes, and ketones appeared to be active, while the more lipophilic hydrocarbons did not show inhibitory activity. In our case also, the inhibitory activity of the alcohols in contrast to the inactivity o f the hydrocarbons indicated the need for a hydrophilic head and a lipophilic tail in a molecule for optimum activity. Although membrane effects may be the first actions of these compounds, these effects could, in turn, influence other cellular processes, such as respiration, photosynthesis, and protein synthesis. The trichothecenes, which are sesquiterpene alcohols, are strong inhibitors of protein synthesis (Sato and Ueno, 1977). Our previous results showed that there was good correlation between inhibition o f protein synthesis and inhibition o f Chlorella growth among the various trichothecenes (Ikawa et al., 1985). This suggests a possible link to processes beyond a primary disruption of membrane function. Acknowledgments--This project was supported by Hatch Project 205. Scientific Contribution No. 1612 from the New Hampshire Agricultural Experiment Station.
REFERENCES ANDREWS,R.E., PgRK, L.W., and SPENCE,K.D. 1980. Some effects of Douglas fir terpenes on certain microorganisms. Appl. Environ. Microbiol. 40:301-304. IKAWA,M., MA, D.S., MEEKER,G.B., and DAVlS,R.P. 1969. Use of Chlorella in mycotoxin and phycotoxin research. J. Agric. Food Chem. 17:425-429.
][KAWA,M., HARTSHORNE,T., BARBERO,L., IANNITELLI,R., and TERRANOVA, C. 1979. Use of Chlorella and other microorganisms for the detection of mycotoxins. Mycotoxin: Proceedings, Japanese Association of Mycotoxicology No. 9, pp. 26-29. IKAWA,M., CARR, C., and TATSUNO,T. 1985. Trichothecene structure and toxicity to the green alga Chlorella pyrenoidosa. Toxicon 23:535-537. JUETTNER, F. 1983. Composition and method to combat blue-green algae. Eur. Pat. Appl. EP 65,725 (Chem. Abst. 98:121355). LORBER,P., and MULLER,W.H. 1976. Volatile growth inhibitors produced by Salvia leucophylla: Effects on seedling root tip ultrastructure. Am. J. Bot. 63:196-200. MATHELA,C.S. 1981. In vitro antifungal examination of some terpenoids. Proc. Natl. Acad. Sci. India, Sect. A 51:513-516. MORRIS,O.N. 1972. Inhibitory effects of foliage extracts of some forest trees on commercial Bacillus thuringiensis. Can. Entomol. 104:1357-1361. REYNOLDS, T. 1987. Comparative effects of alicyclic compounds and quinones on inhibition of lettuce fruit germination. Ann. Bot. 60:215-223.
1760
IKAWA ET AL.
SATO, N., and UENO,Y. 1977. Comparative toxicities of the trichothecenes, pp. 295-307, in J.V. Rodricks, C.W. Hesseltine, and M.A. Mehlman (eds.). Mycotoxins in Human and Animal Health. Pathotex Publishers, Park Forest South, Illinois. SMIRNOFF, W.A. 1972. Effects of volatile substances released by foliage of Abies balsamea. J. Invert. Pathol. 19:32-35. SPOEHR,H.A., SMITH,J.H.C., STRAIN,H.H., MILNER,H.W., and HARDIN, G.J. 1949. Fatty acid antibacterials from plants. Publ 586. Carnegie Institution of Washington, Washington, D.C. SULLIVAN, J.D., JR., and IKAWA, M. 1972. Variations in inhibition of growth of five Chlorella strains by mycotoxins and other toxic substances. J. Agrie. Food Chem. 20:921-922. UZDENNIKOV, B.E. 1972. Effect of some terpenes on bacteria, fungi, and protozoa, pp. 100-101, in B.E. Aizenman (ed.). Fitontsidy: Resul'taty, Perspektivy i Zadachi Issledovanii, Materialy Soveshchaniya po Probleme Fitontsidov, 6th, Kiev, Je 10-13, 1969. Naukova Dumka, Kiev, USSR. (Chem. Abstr. 78:53205).
INHIBITORY E F F E C T S OF T E R P E N E A L C O H O L S A N D A L D E H Y D E S ON G R O W T H OF G R E E N A L G A
Chlorella pyrenoidosa
M I Y O S H I I K A W A , * S T E P H E N P. M O S L E Y , and L I N D A J. B A R B E R O
Department of Biochemistry and Molecular Biology University of New Hampshire Durham, New Hampshire 03824 (Received February 27, 1992; accepted May 29, 1992) Abstract--The growth of the green alga Chlorellapyrenoidosa was inhibited by terpene alcohols and the terpene aldehyde citral. The strongest activity was shown by citrah Nerol, geraniol, and citronellol also showed pronounced activity. Strong inhibition was linked to acyclic terpenes containing a primary alcohol or aldehyde function. Inhibition appeared to be taking place through the vapor phase rather than by diffusion through the agar medium from the terpene-treated paper disks used in the system. Inhibition through agar diffusion was shown by certain aged samples of terpene hydrocarbons but not by recently purchased samples. Key Words--ChloreUa pyrenoidosa, alga, terpenes, growth inhibition. INTRODUCTION Terpene hydrocarbons, alcohols, aldehydes, and ketones have been reported to have antibacterial and antifungal activity (Morris, 1972; Smimoff, 1972; Uzdennikov, 1972; Andrews et al., 1980; Mathela, 1981), and it has been suggested that terpenes in the diet may be responsible for the gut o f the larvae of the Douglas fir tussock moth Orgyia pseudotsugata being relatively free of microflora (Andrews et al., 1980). Therefore, terpenes may have an ecological significance in certain situations involving microorganisms. Studies involving the effects o f terpenes on microalgae are more limited. The use of terpene alcohols as anti-blue-green algal agents has been described in the European patent literature (Juettner, 1983). *To whom correspondence should be addressed. 1755 0098-0331/92/1000 1755506.50/0 9 1992 Plenum Publishing Corporation
1756
IKAWAET AL.
In previous studies we have described the pronounced inhibitory effects of fungi-produced toxins (mycotoxins) on the growth of the green alga Chlorella pyrenoidosa (Ikawa et al., 1969, 1979, 1985; Sullivan and Ikawa, 1972). Many of the more active of these mycotoxins are terpenoids, and terpene intermediates are involved in their biosynthesis. This paper reports on an extension of this work to the simple terpenes themselves. METHODS AND MATERIALS
Paper disks (6 mm, Difco Laboratories, Detroit, Michigan) were dipped into test solutions in ethanol, excess solution removed by touching the disks to the edge of the container (ca. 20 #I of solution was retained on the disk), and the disks placed on the surface of light yellow-green Chlorella-seeded agar plates (100-mm-diameter Petri plates, three disks per plate) prepared as previously described (Ikawa et al., 1985). The plates were placed under a fluorescent light at room temperature and zones of inhibition read after two days under constant illumination. RESULTS AND DISCUSSION
The terpene hydrocarbons showed little or no inhibition at a disk concentration of 10 mg/ml (Table 1). However, it was found that samples of some of the terpenes that had been collected from various laboratories on campus and that presumably had been on the shelf for some time did show zones of growth inhibition (Table 1, footnote d), indicating that the older samples had probably undergone chemical changes to form inhibitory substances. These results recall those that had been observed with chlorellin, the antibacterial and autotoxic substance produced by Chlorella, where it was shown that chlorellin activity was due to photooxidation products of unsaturated fatty acids present in Chlorella (Spoehr et al., 1949). Most of the terpene alcohols and aldehydes tested showed inhibition that was evident as a lightening or total wipe out of color in the light yellow-green Chlorella lawn (Table 1). That inhibition was taking place through the vapor phase rather than by diffusion through the agar was shown by observing the same results when the paper disks were placed on slightly larger Teflon disks and thus were not in direct physical contact with the agar surface. The greatest inhibition was shown by nerol, gemniol, citronellol, and citral, all of which possess the acyclic terpene structure with a primary alcohol or aldehyde function (Figure 1). Citral appeared to be the most active, showing complete plate color wipe out with three disks at 1 mg/ml. The cyclic terpene alcohols bomeol and isobomeol and terpenes carrying the alcohol function at a nonterminal position
GROWTH INHIBITIONOF Chlorella BY TERPENES
1757
TABLE 1. INHIBITION OF Chlorella GROWTH BY TERPENES
(Net diameter of inhibition zone) (mm)b Compound Terpene hydrocarbons ( +)-Limonene a Myrcene ( +)-c~-Pinene d (-)-c~-Pinene (-)-/3-Pinene a a-Terpinene 7-Terpinene Terpene alcohols (-)-Bomeol Isobomeol Cineole Citronellol Geraniol Linalool Nerol Terpinen-4-ol et-Terpineol a Famesol (isomers) Hexenols cis-2-Hexen- 1-ol trans-2-Hexen- 1-ol cis-3-Hexen-l-ol (leaf alcohol) trans-3-Hexen- 1-ol trans-4-Hexen- 1-ol 5-Hexen- 1-ol Terpene aldehydes and ketones (+)-Camphor Citral
Source a
10 mg/mlc
1 mg/mF
Aldrich Aldrich Aldrich Aldrich Aldrich Aldrich Aldrich
2 Tr 0 0 0 0 0
0
K &K K &K Aldrich Aldrich Aldrich Aldrich K & K Aldrich Aldrich Aldrich
,e * 0 **~ ** * ** * 0 6
0 0
Aldrich Aldrich Aldrich
0 0 0
Aldrich Aldrich Aldrich
0 0 0
Aldrich
0 **
0.1 mg/mlC
0 * *
Tr.
**
~ Aldrich Chemical Co., Milwaukee, Wisconsin; K & K Laboratories, Plainview, New York. bDisk diameter (6 mm) subtracted from the total diameter of the inhibition zone. Figures represent the mean of all disks (three per plate) mn on two or more separate occasions. Standard deviations generally ran < 10% of the mean for means > 5 mm. CConcentration of test solution in ethanol. dOlder shelf samples of these compounds that were collected from various laboratories on campus showed inhibition at 10 mg/ml. ~* indicates platewise lightening of the lawn color as compared to control plates. ** indicates complete wipe out of the yellow-green lawn color with three disks per plate. Similar results were obtained when disks were prevented from direct contact with the agar surface by interposing larger diameter Teflon disks between the paper disks and the agar surface.
1758
IKAWA ET AL.
Myrcene
Limonene
~-Terpinene
C~-Pinene
~-Pinene
Camphene
0~-Terpineol
Geraniol
Terpinen-4-ol
Nerol
Cineole
Geranial (Citral a)
~-Terpinene
p-Cymene
Borneol (endo) Isoborneol (exo)
Camphor
Linalool
Citronellol
Neral (Citral b)
Farnesol
FIG. 1. Structures of terpenes tested against Chlorella. (a-terpineol, terpinen-4-ol, linalool) showed less activity than the more active alcohols. The sesquiterpene alcohol farnesol showed some activity at 10 mg/ml through agar diffusion. The leaf alcohol (cis-3-hexen-l-ol) and a series of related hexenols were also tested. As shown in Table 1, none of the compounds showed any activity at a concentration of 10 mg/rnl. In contrast to the activity of terpene hydrocarbons against bacteria and fungi, our results indicate that the hydrocarbons are not particularly toxic to the green alga Chlorella. Terpene alcohols showed activity, the highest activity being shown by acyclic compounds that carried the functional group on the terminal carbon atom. Acyclic terpene alcohols have been described as antiblue-green algal agents, with famesol being particularly active against Synechococcus (Juettner, 1983).
GROWTH INHIBITION OF
Chlorella BY TERPENES
1759
Lorber and Muller (1976) showed that when root tip cells o f Cucumis sativus were exposed to the vapors o f the volatile monoterpenes from the leaves o f Salvia leucophylla, disruption of the membranes surrounding the nuclei, mitochondria, and dictyosomes took place. Disruption o f the cytoplasmic membranes o f Bacillus thuringiensis and S a c c h a r o m y c e s cerevisiae were also observed when these microorganisms were treated with ct-pinene (Andrews et al., 1980). Lipophilicity appeared to be an important contributing factor in the inhibition o f lettuce fruit germination by terpenes (Reynolds, 1987). In this case, the terpene alcohols, aldehydes, and ketones appeared to be active, while the more lipophilic hydrocarbons did not show inhibitory activity. In our case also, the inhibitory activity of the alcohols in contrast to the inactivity o f the hydrocarbons indicated the need for a hydrophilic head and a lipophilic tail in a molecule for optimum activity. Although membrane effects may be the first actions of these compounds, these effects could, in turn, influence other cellular processes, such as respiration, photosynthesis, and protein synthesis. The trichothecenes, which are sesquiterpene alcohols, are strong inhibitors of protein synthesis (Sato and Ueno, 1977). Our previous results showed that there was good correlation between inhibition o f protein synthesis and inhibition o f Chlorella growth among the various trichothecenes (Ikawa et al., 1985). This suggests a possible link to processes beyond a primary disruption of membrane function. Acknowledgments--This project was supported by Hatch Project 205. Scientific Contribution No. 1612 from the New Hampshire Agricultural Experiment Station.
REFERENCES ANDREWS,R.E., PgRK, L.W., and SPENCE,K.D. 1980. Some effects of Douglas fir terpenes on certain microorganisms. Appl. Environ. Microbiol. 40:301-304. IKAWA,M., MA, D.S., MEEKER,G.B., and DAVlS,R.P. 1969. Use of Chlorella in mycotoxin and phycotoxin research. J. Agric. Food Chem. 17:425-429.
][KAWA,M., HARTSHORNE,T., BARBERO,L., IANNITELLI,R., and TERRANOVA, C. 1979. Use of Chlorella and other microorganisms for the detection of mycotoxins. Mycotoxin: Proceedings, Japanese Association of Mycotoxicology No. 9, pp. 26-29. IKAWA,M., CARR, C., and TATSUNO,T. 1985. Trichothecene structure and toxicity to the green alga Chlorella pyrenoidosa. Toxicon 23:535-537. JUETTNER, F. 1983. Composition and method to combat blue-green algae. Eur. Pat. Appl. EP 65,725 (Chem. Abst. 98:121355). LORBER,P., and MULLER,W.H. 1976. Volatile growth inhibitors produced by Salvia leucophylla: Effects on seedling root tip ultrastructure. Am. J. Bot. 63:196-200. MATHELA,C.S. 1981. In vitro antifungal examination of some terpenoids. Proc. Natl. Acad. Sci. India, Sect. A 51:513-516. MORRIS,O.N. 1972. Inhibitory effects of foliage extracts of some forest trees on commercial Bacillus thuringiensis. Can. Entomol. 104:1357-1361. REYNOLDS, T. 1987. Comparative effects of alicyclic compounds and quinones on inhibition of lettuce fruit germination. Ann. Bot. 60:215-223.
1760
IKAWA ET AL.
SATO, N., and UENO,Y. 1977. Comparative toxicities of the trichothecenes, pp. 295-307, in J.V. Rodricks, C.W. Hesseltine, and M.A. Mehlman (eds.). Mycotoxins in Human and Animal Health. Pathotex Publishers, Park Forest South, Illinois. SMIRNOFF, W.A. 1972. Effects of volatile substances released by foliage of Abies balsamea. J. Invert. Pathol. 19:32-35. SPOEHR,H.A., SMITH,J.H.C., STRAIN,H.H., MILNER,H.W., and HARDIN, G.J. 1949. Fatty acid antibacterials from plants. Publ 586. Carnegie Institution of Washington, Washington, D.C. SULLIVAN, J.D., JR., and IKAWA, M. 1972. Variations in inhibition of growth of five Chlorella strains by mycotoxins and other toxic substances. J. Agrie. Food Chem. 20:921-922. UZDENNIKOV, B.E. 1972. Effect of some terpenes on bacteria, fungi, and protozoa, pp. 100-101, in B.E. Aizenman (ed.). Fitontsidy: Resul'taty, Perspektivy i Zadachi Issledovanii, Materialy Soveshchaniya po Probleme Fitontsidov, 6th, Kiev, Je 10-13, 1969. Naukova Dumka, Kiev, USSR. (Chem. Abstr. 78:53205).
