This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Universitet i Tromso.
zed and pollen grains were filtered from the freshly prepared suspension after an hour, the inhibitory effect was noticed. Inhibitory effect of pollen on the growth of a fungus or pollen of other species indicates an allelopathic mechanism in which a species competes against others by releasing growth-inhibiting chemicals [4,5]. Moreover, the inhibiting potential of pollen seems to be for reasons of defense and isolation. Chemical(s) involved in this phenomenon and the effect of pH on it are being investigated in detail. (The term sporangial germination means the release of zoospores from the sporangia and their subsequent germination.) The rate of inhibition of sporangial germination by pollen in the experiment is found to be highly significant (p < 0.001).
Received May 15 and July 18, 1975
Received June 2 and July 15, 1975
nuclear proteins from amoebae and plasmodia of the heterothallic "Wisconsin"-strain of Physarurn polycephalurn [7]. We have studied the RNAse isozymes during the life cycle of this strain too and found the same RNAse pattern in plasmodia as shown in Fig. 1a and in amoebae as shown in Fig. 1b. Our findings agree with the hypothesis that different sets of genes may be active in the amoebae and plasmodia of Physarum polycephalum [1], which is based on the observation that some temperature-sensitive mutations isolated as amoebae from the homothallic strain are not expressed in the plasmodia.
1. Jockusch, B.M. : Naturwissenschaften 62, 283 (1975) 2. Wheals, A.E. : Genetics 66, 623 (1970) 3. Cooke, D.J., Dee, J. : Genet. Res. Camb. 23, 307 (1974) 4. Dee, J., in: Physarum polycephalum- Object of Research in Cell Biology, p. 93 (Hfitterman, A., ed.). Stuttgart: Fischer 1973 5. Haglund, H. : Sci. Tools LKB Instrum. J. 14, 17 (1967) 6. Haugli, F.B., Dove, W.F. : Mol. Gen. Genetics 118, 109 (1972) 7. LeSturgeon, W.M., Goodman, E.M., Rusch, H.P. : Biochim. Biophys. Acta 317, 524(1973) 8. Hfittermann, A., Gebauer, M. : Cytobiologie 7, 383 (1973) 9. Braun, R., Behrens, K.: Biochim. Biophys. Acta 195, 87 (1969)
1. Linskens, H.F. : Encycl. Plant Physiol. 18, 368 (1967) 2. Vasil, I.K. : Naturwissenschaften 60, 247 (1973) 3. Mattisson, O., et al. : Nature 247, 298 (1974) 4. Kanchan, S.D.: Proc. Indian Sci. Congr. Abstr. Part 3, p. 72 (1975) 5. Whittaker, R.H., Feeny, P.P. : Science 171, 757 (197t)
Accumulation of Polycyclic Aromatic Hydrocarbons in Cultivated Microalgae H.D. Payer and C.J. Soeder
Antifungal Activity of Pollen
Gesellschaft ffir Strahlen- und Umweltforschung mbH, Mfinchen, Abteilung ftir Algenforschung und Algentechnologie, Dortmund
M.B.S. Char and S.S. Bhat
H. Kunte
Department of Post-Graduate Studies and Research in Botany, Manasa Gangotri, University of Mysore, India
Hygiene-!nstitut der Universit/it Mainz
Pollen activity in allergy and fertilization is well known [1 -3]. Another kind of pollen activity, namely of an antifungal nature is reported here. Pollen of an Asteracean plant, Partheniurn hysterophorus Linn., exercised antifungal activity by producing an inhibitory effect when tested against sporangial germination and zoospore motility in the downy mildew fungus, Sclerospora grarninicola (Sacc.) Schroet. Fresh intact pollen grains collected from dehiscing anthers in flowers were added with known weight to a definite volume of distilled water to procure pollen suspension. Leaves of Penniseturn typhoides S. & H., infected by S. graminicola were collected, surface-sterilized with 0.2 percent chlorine water and washed in sterilized distilled water. The leaves were cut into bits, floated on water, with their abaxial surface upwards, in a Petri dish lined with moist filter paper and kept overnight. The next morning, sporangia were scraped from the abaxial surface of the leaf pieces while zoospores were obtained by sowing the sporangia in distilled water for half an hour. Sporangia and zoospores were separately transferred to distilled water, dropped onto microglass slides and kept for control and experiment. In the experiment, they were tested against pollen suspension by adding the solutions drop by drop. After 30 min in the control, zoospores were released from the sporangia. The zoospores swam for 2 0 - 3 0 min and subsequently gave rise to germ tubes. In the experiment, zoospores were released from the sporangia, swam for 1 0 - 1 5 min but eventually disintegrated. Room temperature during these observations was between 23 and 30 ~ Results reveal that a concentration of 5 mg pollen in 2 m! of distilled water inhibits zoospore motility and sporangial germination. Even when pollen suspension was steam-sterili536
P. Karuwanna Institute of Food Research and Product Development, Kasetsart University, Bangkok R. Nonhof and W. Grfif Institut ffir Umwelthygiene und Pr~ventmedizin der Universitfit Erlangen-Niirnberg Terrestrial plants growing in industrial areas contain significantly higher amounts of polycyclic aromatic hydrocarbons than those from the more remote rural locations [1]. It is the aim of the present study to determine whether a similar relation between air pollution and polycyclics concentration can be detected in microalgae cultured extensively under openair conditions. The green alga, Scenedesmus acutus (G6ttingen, strain 276-3a) was grown either at Dortmund or at Bangkok. Dried algal substances from both locations were analyzed for polycyclic compounds. Depending on the polycyclic hydrocarbon content, 2-10 g of dry algal matter were extracted three times with purified benzene at 50 ~ under a reflux cooler. The combined extracts were concentrated to 1 ml, and passed through a column of aluminum oxide in order to remove the plastid pigments. The vacuum-dried eluate was dissolved in cyclohexane and transferred under shaking into a nitromethane phase. For the quantitative determination of polycyclic hydrocarbons on twodimensional thin-layer chromatograms and on reference chromatograms [2] a Zeiss spectrofhiorometer was used in combination with the Camag scanner. For control measurements at Erlangen a different procedure [3] was followed which led to the same statistical distribution pattern of determined concentrations. Naturwissenschaften 62 (1975)
9 by Springer-Verlag 1975
50
: F[uorcmt.hene..... , ....... /
" 5 [ 3,4-Benzopyrene
,0 .
.
.
.
~
~
.
.
,00 .
.
.
_ _
I0
160 200
1603[
_ _
1:12- . , ~.y ......... 2 4 6 10 100
lv.,3,4- Benzoftuoranthene ~| i I0 160
o
50
/
-
50
L., Indenopyrene 11,12-Be nzoftuoranthene 1' 10 160 2 3 4 10/uglkg 100 Concentration in the dry matter of microa[gae Fig. 1. Statistical distribution patterns of potycyclics concentration in cultivated microalgae displayed on a probability net. Ordinate divided according to Gauss integral; abscissa scale either logarithmic or linear. ~ values from Bangkok; , , values from Dortmund With the exception of fluoranthene, 1,12-benzoperylene and 11,12-benzofluoranthenein samples from Bangkok, the polycyclic contents of Scenedesmus followed a lognormal distribution(Fig. 1). Therefore, the median values a n d n o t the averages
of values from different stations have to be used for a comparison; the differences in polycyclic hydrocarbon content between microalgae samples from Dortmund and those from Bangkok are evident. The low pollutant concentrations in algal substances from Thailand do not agree with a recently published opinion [4]. Our investigations show that it is possible to produce in open ponds microalgae with a load of environmental pollutants no higher than that of emergent parts of terrestrial higher plants. In comparing the statistical distribution of the concentrations of the individual polycyclic compounds in our samples, one realizes at once that the carcinogenic substances are always lognormally distributed, whereas the polycyclics without carcinogenic activity show a normal distribution in the samples of Bangkok (Table 1). This raises again the question of whether polycyclic hydrocarbons are endogenously synthesized by plants. While a lognormal distribution of concentrations can be attributed to environmental factors (in our case this means mainly a variability of duration of exposure to emissions), a normal distribution may indicate the accumulation of endogenous compounds. Hence, from the lognormal distribution of carcinogenic polycyclics in microalgae we may conclude that their endogenous formation, as revealed for Chlorella vulgaris [5], occurs only to such a very limited extent that it is completely masked by the accumulation of the same substances from the environment. These invesigations were supported by funds of the Kingdom o f Thailand and of the Ministry of Economic Cooperation of the Federal Republic of Germany ( G A W I - F E 1656). Received July 16, 1975 1. Grimmer, G., Hildebrand, A.: Z. Krebsforsch. 67, 272 (1965) 2. Kunte, H. : Arch. Hyg. Bakteriol. 151, 193 (1967) 3. GrSs W., Diehl, H. : ibid. 150, 49 (1966) 4. Wagner, K.-H., Siddiqi, I. : Naturwissenschaften 60, 109 (1973) 5. Borneff, J., et al. : Environ. Res. 2, 22 (1968)
Table 1. Polycyclics content of dried microalgae in gg/kg (ppb). The scattering of values is indicated by median values and scattering factors in the case of lognormal distributions or by arithmetic means and standard deviations in the case of normal distributions. The degree of carcinogenic activity is expressed by a plus/minus scale in the last column
3,4-Benzopyrene 3,4-Benzofluoranthene Indenopyrene Fluoranthene 1,12-Benzoperylene 11,12-Benzofluoranthene
Dortmund
n
Bangkok
n
CarcinOgenic activity
39.5-2.149 -+1 85.4.1.795 • I 50.9- 1.923 -+~ 444.1.505 • I 52.1 91.932 + 1 36.3.1.863 .+1
19 19 19 18 19 19
1.39.1.605 +.1 5.94.1.639 -+l 2.36-1.516 .+1 91.2 _+38.2 3.89 _+ 2.36 2.40 _+ 1.02
15 14 8 8 8 8
+ + 44- 44(_+)
Degradation of Aflatoxin B1 by Microorganisms
culture after 4 days. Mycobacterium phlei also degrades aflatoxin B1 quickly. Staphylococcus aureus, Escherichia coli,
Renate Mann and H.J. Rehm
Bacillus mycoides, Arthrobacter variabilis, Acetobacter suboxydans and Corynebacterium betae seemed not to be able to
Institut ftir Mikrobiologie, Universit~it Mtinster When the cancerogenic effect of aflatoxins became known there began an intensive screening for microorganisms which were able to degrade these coumarine derivatives [1]. Flavobacterium aurantiacum and different Rhizopus sp. were found to be potent aflatoxin-degrading microorganisms. In our experiments we found that Corynebacterium rubrum degraded more than 99% of the added aflatoxin B1 (1.48 Ixg/ml) in a liquid Naturwissenschaften 62 (1975)
9 by Springer-Verlag 1975
degrade aflatoxin B~. Anascosporogenous yeasts (Candida sp.) degraded at least 80 % of aflatoxin B t within 20 days, whereas ascosporogenous yeasts (Saccharomyces) were inactive in degradation. A m o n g molds Cunninghamella echinulata, Stachybotrys lobulata, Penicillium islandicum, Aspergillus niger and A. ochraceus showed an intensive ability for degrading aflatoxin Bt (Table 1). Only Fusarium aquaeduczum scarcely removed aflatoxin B1 from the nutrition broth. 537
zed and pollen grains were filtered from the freshly prepared suspension after an hour, the inhibitory effect was noticed. Inhibitory effect of pollen on the growth of a fungus or pollen of other species indicates an allelopathic mechanism in which a species competes against others by releasing growth-inhibiting chemicals [4,5]. Moreover, the inhibiting potential of pollen seems to be for reasons of defense and isolation. Chemical(s) involved in this phenomenon and the effect of pH on it are being investigated in detail. (The term sporangial germination means the release of zoospores from the sporangia and their subsequent germination.) The rate of inhibition of sporangial germination by pollen in the experiment is found to be highly significant (p < 0.001).
Received May 15 and July 18, 1975
Received June 2 and July 15, 1975
nuclear proteins from amoebae and plasmodia of the heterothallic "Wisconsin"-strain of Physarurn polycephalurn [7]. We have studied the RNAse isozymes during the life cycle of this strain too and found the same RNAse pattern in plasmodia as shown in Fig. 1a and in amoebae as shown in Fig. 1b. Our findings agree with the hypothesis that different sets of genes may be active in the amoebae and plasmodia of Physarum polycephalum [1], which is based on the observation that some temperature-sensitive mutations isolated as amoebae from the homothallic strain are not expressed in the plasmodia.
1. Jockusch, B.M. : Naturwissenschaften 62, 283 (1975) 2. Wheals, A.E. : Genetics 66, 623 (1970) 3. Cooke, D.J., Dee, J. : Genet. Res. Camb. 23, 307 (1974) 4. Dee, J., in: Physarum polycephalum- Object of Research in Cell Biology, p. 93 (Hfitterman, A., ed.). Stuttgart: Fischer 1973 5. Haglund, H. : Sci. Tools LKB Instrum. J. 14, 17 (1967) 6. Haugli, F.B., Dove, W.F. : Mol. Gen. Genetics 118, 109 (1972) 7. LeSturgeon, W.M., Goodman, E.M., Rusch, H.P. : Biochim. Biophys. Acta 317, 524(1973) 8. Hfittermann, A., Gebauer, M. : Cytobiologie 7, 383 (1973) 9. Braun, R., Behrens, K.: Biochim. Biophys. Acta 195, 87 (1969)
1. Linskens, H.F. : Encycl. Plant Physiol. 18, 368 (1967) 2. Vasil, I.K. : Naturwissenschaften 60, 247 (1973) 3. Mattisson, O., et al. : Nature 247, 298 (1974) 4. Kanchan, S.D.: Proc. Indian Sci. Congr. Abstr. Part 3, p. 72 (1975) 5. Whittaker, R.H., Feeny, P.P. : Science 171, 757 (197t)
Accumulation of Polycyclic Aromatic Hydrocarbons in Cultivated Microalgae H.D. Payer and C.J. Soeder
Antifungal Activity of Pollen
Gesellschaft ffir Strahlen- und Umweltforschung mbH, Mfinchen, Abteilung ftir Algenforschung und Algentechnologie, Dortmund
M.B.S. Char and S.S. Bhat
H. Kunte
Department of Post-Graduate Studies and Research in Botany, Manasa Gangotri, University of Mysore, India
Hygiene-!nstitut der Universit/it Mainz
Pollen activity in allergy and fertilization is well known [1 -3]. Another kind of pollen activity, namely of an antifungal nature is reported here. Pollen of an Asteracean plant, Partheniurn hysterophorus Linn., exercised antifungal activity by producing an inhibitory effect when tested against sporangial germination and zoospore motility in the downy mildew fungus, Sclerospora grarninicola (Sacc.) Schroet. Fresh intact pollen grains collected from dehiscing anthers in flowers were added with known weight to a definite volume of distilled water to procure pollen suspension. Leaves of Penniseturn typhoides S. & H., infected by S. graminicola were collected, surface-sterilized with 0.2 percent chlorine water and washed in sterilized distilled water. The leaves were cut into bits, floated on water, with their abaxial surface upwards, in a Petri dish lined with moist filter paper and kept overnight. The next morning, sporangia were scraped from the abaxial surface of the leaf pieces while zoospores were obtained by sowing the sporangia in distilled water for half an hour. Sporangia and zoospores were separately transferred to distilled water, dropped onto microglass slides and kept for control and experiment. In the experiment, they were tested against pollen suspension by adding the solutions drop by drop. After 30 min in the control, zoospores were released from the sporangia. The zoospores swam for 2 0 - 3 0 min and subsequently gave rise to germ tubes. In the experiment, zoospores were released from the sporangia, swam for 1 0 - 1 5 min but eventually disintegrated. Room temperature during these observations was between 23 and 30 ~ Results reveal that a concentration of 5 mg pollen in 2 m! of distilled water inhibits zoospore motility and sporangial germination. Even when pollen suspension was steam-sterili536
P. Karuwanna Institute of Food Research and Product Development, Kasetsart University, Bangkok R. Nonhof and W. Grfif Institut ffir Umwelthygiene und Pr~ventmedizin der Universitfit Erlangen-Niirnberg Terrestrial plants growing in industrial areas contain significantly higher amounts of polycyclic aromatic hydrocarbons than those from the more remote rural locations [1]. It is the aim of the present study to determine whether a similar relation between air pollution and polycyclics concentration can be detected in microalgae cultured extensively under openair conditions. The green alga, Scenedesmus acutus (G6ttingen, strain 276-3a) was grown either at Dortmund or at Bangkok. Dried algal substances from both locations were analyzed for polycyclic compounds. Depending on the polycyclic hydrocarbon content, 2-10 g of dry algal matter were extracted three times with purified benzene at 50 ~ under a reflux cooler. The combined extracts were concentrated to 1 ml, and passed through a column of aluminum oxide in order to remove the plastid pigments. The vacuum-dried eluate was dissolved in cyclohexane and transferred under shaking into a nitromethane phase. For the quantitative determination of polycyclic hydrocarbons on twodimensional thin-layer chromatograms and on reference chromatograms [2] a Zeiss spectrofhiorometer was used in combination with the Camag scanner. For control measurements at Erlangen a different procedure [3] was followed which led to the same statistical distribution pattern of determined concentrations. Naturwissenschaften 62 (1975)
9 by Springer-Verlag 1975
50
: F[uorcmt.hene..... , ....... /
" 5 [ 3,4-Benzopyrene
,0 .
.
.
.
~
~
.
.
,00 .
.
.
_ _
I0
160 200
1603[
_ _
1:12- . , ~.y ......... 2 4 6 10 100
lv.,3,4- Benzoftuoranthene ~| i I0 160
o
50
/
-
50
L., Indenopyrene 11,12-Be nzoftuoranthene 1' 10 160 2 3 4 10/uglkg 100 Concentration in the dry matter of microa[gae Fig. 1. Statistical distribution patterns of potycyclics concentration in cultivated microalgae displayed on a probability net. Ordinate divided according to Gauss integral; abscissa scale either logarithmic or linear. ~ values from Bangkok; , , values from Dortmund With the exception of fluoranthene, 1,12-benzoperylene and 11,12-benzofluoranthenein samples from Bangkok, the polycyclic contents of Scenedesmus followed a lognormal distribution(Fig. 1). Therefore, the median values a n d n o t the averages
of values from different stations have to be used for a comparison; the differences in polycyclic hydrocarbon content between microalgae samples from Dortmund and those from Bangkok are evident. The low pollutant concentrations in algal substances from Thailand do not agree with a recently published opinion [4]. Our investigations show that it is possible to produce in open ponds microalgae with a load of environmental pollutants no higher than that of emergent parts of terrestrial higher plants. In comparing the statistical distribution of the concentrations of the individual polycyclic compounds in our samples, one realizes at once that the carcinogenic substances are always lognormally distributed, whereas the polycyclics without carcinogenic activity show a normal distribution in the samples of Bangkok (Table 1). This raises again the question of whether polycyclic hydrocarbons are endogenously synthesized by plants. While a lognormal distribution of concentrations can be attributed to environmental factors (in our case this means mainly a variability of duration of exposure to emissions), a normal distribution may indicate the accumulation of endogenous compounds. Hence, from the lognormal distribution of carcinogenic polycyclics in microalgae we may conclude that their endogenous formation, as revealed for Chlorella vulgaris [5], occurs only to such a very limited extent that it is completely masked by the accumulation of the same substances from the environment. These invesigations were supported by funds of the Kingdom o f Thailand and of the Ministry of Economic Cooperation of the Federal Republic of Germany ( G A W I - F E 1656). Received July 16, 1975 1. Grimmer, G., Hildebrand, A.: Z. Krebsforsch. 67, 272 (1965) 2. Kunte, H. : Arch. Hyg. Bakteriol. 151, 193 (1967) 3. GrSs W., Diehl, H. : ibid. 150, 49 (1966) 4. Wagner, K.-H., Siddiqi, I. : Naturwissenschaften 60, 109 (1973) 5. Borneff, J., et al. : Environ. Res. 2, 22 (1968)
Table 1. Polycyclics content of dried microalgae in gg/kg (ppb). The scattering of values is indicated by median values and scattering factors in the case of lognormal distributions or by arithmetic means and standard deviations in the case of normal distributions. The degree of carcinogenic activity is expressed by a plus/minus scale in the last column
3,4-Benzopyrene 3,4-Benzofluoranthene Indenopyrene Fluoranthene 1,12-Benzoperylene 11,12-Benzofluoranthene
Dortmund
n
Bangkok
n
CarcinOgenic activity
39.5-2.149 -+1 85.4.1.795 • I 50.9- 1.923 -+~ 444.1.505 • I 52.1 91.932 + 1 36.3.1.863 .+1
19 19 19 18 19 19
1.39.1.605 +.1 5.94.1.639 -+l 2.36-1.516 .+1 91.2 _+38.2 3.89 _+ 2.36 2.40 _+ 1.02
15 14 8 8 8 8
+ + 44- 44(_+)
Degradation of Aflatoxin B1 by Microorganisms
culture after 4 days. Mycobacterium phlei also degrades aflatoxin B1 quickly. Staphylococcus aureus, Escherichia coli,
Renate Mann and H.J. Rehm
Bacillus mycoides, Arthrobacter variabilis, Acetobacter suboxydans and Corynebacterium betae seemed not to be able to
Institut ftir Mikrobiologie, Universit~it Mtinster When the cancerogenic effect of aflatoxins became known there began an intensive screening for microorganisms which were able to degrade these coumarine derivatives [1]. Flavobacterium aurantiacum and different Rhizopus sp. were found to be potent aflatoxin-degrading microorganisms. In our experiments we found that Corynebacterium rubrum degraded more than 99% of the added aflatoxin B1 (1.48 Ixg/ml) in a liquid Naturwissenschaften 62 (1975)
9 by Springer-Verlag 1975
degrade aflatoxin B~. Anascosporogenous yeasts (Candida sp.) degraded at least 80 % of aflatoxin B t within 20 days, whereas ascosporogenous yeasts (Saccharomyces) were inactive in degradation. A m o n g molds Cunninghamella echinulata, Stachybotrys lobulata, Penicillium islandicum, Aspergillus niger and A. ochraceus showed an intensive ability for degrading aflatoxin Bt (Table 1). Only Fusarium aquaeduczum scarcely removed aflatoxin B1 from the nutrition broth. 537
