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DIVERSITY OF SOIL ACTINOMYCETES FROM SOUTH INDIA AND SOUTH CHINA R. BALAGURUNATHAN, L. XU* and C. JIANG*
Centre of Advanced Study in Marine Biology, Annamalai University,
Parangipettai 608 502, India and
Code Number:AC96013
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ABSTRACT. Characteristics of actinomycetes isolated from soil samples collected in the south of India and of China with different types of vegetation were analysed. The number of actinomycetes increased progressively from primeval forests to secondary forests and vegetable farmland, while the kinds of organisms isolated decreased according to the same order. Fourteen and twelve genera were isolated from Indian and Chinese soils respectively. Antibiotic activity of the more rare strains stressed their potential as a source of novel antibiotics.
Actinomycetes are well known producers of antibiotics and antitumour agents in addition to enzymes of industrial interest, such as protease, chitinase and glucose isomerase (Tanaka & Omura, 1990).
Isolation of new strains producing novel antibiotics is of paramount importance and rare actinomycetes are target organisms (Williams & Vickers, 1988). This is confirmed by results obtained with Micromonospora sp. and Nocardia sp. for gentamicin C complex and rifamycin group antibiotics respectively (Nisbet, 1982). In addition the isolation of diverse strains of actinomycetes provides a theoretical guide for the exploitation and utilisation of the resources of the organisms. One of the aims of biodiversity studies is the use of effective isolation procedures to analyse the distribution of actinomycetes in different climatic and ecological environments.
In the present study the actinomycete population of two different sampling areas was investigated.
MATERIALS and METHODS
Sampling. Soil samples (5 for each location) were collected from primeval forests, secondary forests and vegetable farmland in the south of India and of China (Table 1). All soils were of the laterite type.
Table 1. Characteristics of the sampling locations
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SAMPLING SITE ALTITUDE(m.s.l.) SOIL pH VEGETATION
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SALEM (SOUTH INDIA)
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Primeval Forest 800-850 5.3-6.2 Rubiaceae
Secondary Forest 575-650 6.2-6.7 Orchideaceae, Rubiaceae
Farmland 650-700 6.2-6.7 vegetables
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MENGLUN (SOUTH CHINA)
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Primeval Forest 550-925 5.1-5.8 Bignoniaceae, Datiscaceae,
Euphorbiaceae, Rubiaceae
Secondary Forest 550-890 4.8-6.4 Fagaceae, Lauraceae,
Orchideaceae Rubiaceae
Farmland 550-580 5.4 vegetables
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In India, samples were collected in and around the hilly Salem area, Tamilnadu State (Lat. 11 degrees 37'N, Long. 78 degrees 16'E), with an average temperature of 35 C and an annual rainfall of about 450-500 mm. In China sampling was carried out in the Menglun area, south of Xishuangbanna, Yunnan province (Lat. 21 degrees 50'N; Long. 98 degrees 46'E). The area, 70 km wide, is one of the national nature protection zones. The annual average temperature is 20.9 C and rainfall is 1240-1944 mm. Locally it is known as a tropical, seasonal rainy forest.
Isolation. Mesophilic actinomycetes were isolated on glycerol asparagine and on HV agar (Nonomura & Hayakawa 1988), incubated at 28 C for 10-30 dd. Colonies were tentatively identified by spore chain morphology under a light microscope and transferred to yeast extract-malt extract agar.
Thermophiles were isolated from soil samples, air dried at room temperature and heated at 120 C for one hour, on half strength nutrient, yeast extract - malt extract and on glycerol-asparagine agar, incubated at 55 C for 5-7 dd. Colonies were transferred to yeast extract-malt extract agar. Pure cultures were maintained in 20% glycerol at -70 C.
Physiological characteristics. Activity on cellulose, chitin, fibrin and mannase was determined by observing the clearing of the medium around growth (Ruan, 1976). Other enzymatic assays were carried out according to Shirling and Gottlieb (1966).
Isolates, grown on a soybean meal glucose medium, were tested for antimicrobial activity by the inhibition zone method (Ruan, 1976).
Resistance to phenol, hydrofluoric acid and HgCl2 was tested on yeast extract - malt extract broth after 15 dd at 28 C.
RESULTS and DISCUSSION:
Mesophilic actinomycetes. Number and type of mesophilic actinomycetes isolated from the Salem and the Menglun area are summarised in Table 2.
Table 2. Genera of mesophilic actinomycetes recovered in the two sampling locations (10^3 CFU/10g dry soil; Stm: Streptomyces; Mms: Micromonospora; Acp: Actinoplanes; Dac: Dactylosporangium; Cat: Catellatospora; Mbs: Microbispora; Mts: Microtetraspora; Sts: Streptosporangium; Acm: Actinomadura; Acb: Actinobispora; Sam: Saccharomonospora; Sap: Saccharopolyspora; Noc: Nocardia; UI: unidentified)
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Sampling
Site Stm Mms Acp Dac Cat Mbs Mts Sts
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SALEM (SOUTH INDIA)
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Primeval
forest 65.5 16.25 - 0.50 2.25 0.50 2.75 4.75
Secondary
forest 315.20 111.20 0.80 0.40 1.00 - 0.20 -
Farmland 618.60 175.20 0.40 - - - 0.80 -
Mean 333.10 100.80 0.24 0.18 0.65 0.16 1.25 1.59
% 73.40 22.21 0.06 0.04 0.15 0.04 0.28 0.40
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MENGLUN (SOUTH CHINA)
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Primeval
forest 30.20 8.20 - 1.80 0.60 0.40 1.00 3.60
Secondary
forest 39.25 15.75 0.50 9.50 4.50 0.75 025 1.50
Farmland 53.00 85.40 - 1.60 0.80 - 4.60 1.40
Mean 40.82 36.45 0.17 4.30 1.97 0.38 1.95 2.20
% 38.90 34.74 0.16 4.09 1.87 0.36 1.85 2.09
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Table 2 (continued)
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Sampling
Site Acm Acb Sam Sap Noc UI Total
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SALEM (SOUTH INDIA)
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Primeval
forest 9.00 - - 0.25 0.25 2.75 104.75
Secondary
forest 20.00 - - - 3.60 - 452.40
Farmland 9.80 - - - - 804.80
Mean 12.93 - - 0.08 1.28 - 453.98
% 2.90 - - 0.02 0.30 0.20 100.00
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MENGLUN (SOUTH CHINA)
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Primeval
forest 11.00 1.40 - 1.20 7.40 0.60 67.40
Secondary
forest 7.75 6.75 - 0.75 2.50 2.25 92.00
Farmland 8.40 - 0.20 - - - 155.40
Mean 9.05 2.72 0.07 0.65 3.30 0.95 104.93
% 8.62 2.60 0.06 0.62 3.14 0.90 100.00
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Streptomyces species (73.40 and 38.90% in the two locations respectively), followed by Micromonospora species (22.21 and 34.74%), represented the most frequent actinomycetes, confirming previous results (Alexander, 1961).
In both locations actinomycete counts increased progressively according to the primeval forest - secondary forest - farmland sequence (104.75 - 452.40 -804.80x10^4 and 67.40 - 92.00 - 155.40x10^4 respectively). The higher content in organic matter of vegetable farmlands may explain these results.
On the other hand with regard to the diversity of the isolates, that is in terms of different morphological types recovered, the trend appears to progress in the opposite direction (10 - 8 - 5 different genera from the Salem area and 11 - 12 - 8 from the Menglun one).
In addition to Streptomyces and Micromonospora, representatives of the following taxa were recovered: Actinoplanes, Dactylosporangium, Catellatospora, Microbispora, Microtetraspora, Streptosporangium, Actinomadura, Actinobispora, Saccharomonospora, Saccharopolyspora and Nocardia.
Thermophilic actinomycetes. Results are summarised in Table 3. Isolation of thermophiles was more successful in the Salem area, which is characterised by poor rainfall and high temperatures throughout the year. The Menglun area is represented by typical rainy forests with low temperatures (Jiang et al., 1992).
Table 3. Number (10^3 CFU/g) of thermophilic actinomycetes isolated in the different sites
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SAMPLING SITE Streptomyces Actinomadura Thermoactinomyces Total
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SALEM (SOUTH INDIA)
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Primeval Forest 6 - 42 48
Secondary Forest - - 87 87
Farmland 8 1 57 66
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MENGLUN (SOUTH CHINA)
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Primeval Forest - - 8 8
Secondary Forest - - - -
Farmland - - 3 3
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Physiological characteristics. Enzymatic and antimicrobial properties of the isolates are shown in Table 4.
Table 4. Relative enzyme and antimicrobial activity of the isolates
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BIOACTIVITY SALEM ISOLATES MENGLUN ISOLATES
Active/Tested Active/Tested
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Enzyme
Cellulase 120/300 85/250
Chitinase 265/300 215/250
Fibrinolysis 210/1200 195/950
Mannase 80/600 55/500
Protease 85/290 75/250
Antibiosis
S.aureus 40/300 26/250
B.subtilis 60/300 28/250
V.cholerae 10/300 8/250
E.coli 7/300 6/250
A.niger 32/300 24/250
C.albicans 0/300 0/250
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Fifty-five per cent of the isolates were active against Staphylococcus aureus, 40% against Escherichia coli and Vibrio cholerae and 20% against Bacillus subtilis. As for antifungal activity 56 out of 550 isolates were active against Aspergillus niger, but none against Candida albicans.
Seventy per cent of the strains were not affected by 5 ppm phenol, 40% were resistant 500 ppm hydrofluoric acid and 55% grew in the presence of HgCl2.
Further work, intended to exploit the potential of the isolates, is in progress.
ACKNOWLEDGEMENTS. The work was financed by the International Cooperation Science Foundation of Yunnan-China. One of the authors (RB) wishes to thank the Director of the C.A.S. in Marine Biology, the authorities of Annamalai University and Prof.A.Subramanian for their support.
REFERENCES Alexander, M. (1961). Introduction to Soil Microbiology. J.Wiley and Sons, New York
Jiang, C.L., L.-H.Xu, Y.-R.Yang, J.Ma & G.Y.Guo (1992). A study on thermophilic actinomycetes from various environments in Yunnan. Bull. Jap. Fed. Cult. Coll., 8(1): 2-10 Nisbet, L,.J., (1982). Current strategies in the search for bioactive microbial metabolites. J. Chem. Tech. Biotech., 32 : 251- 270
Nonomura, H. & M.Hayakawa (1988). New methods for the selective isolation of soil actinomycetes. In: Y. Okami, T.Beppu & H.Ogawa (eds.) Biology of Actinomycetes '88. Jap. Sci. Soc. Press, Japan, pp. 288- 293
Ruan, J.S. (1976). Elementary Actinomycete Classification. Academic Press, Beijing
Shirling, E.B. & D.Gottlieb (1966). Methods for classification of Streptomyces species. Int. J. Syst. Bacteriol., 16: 313-340
Tanaka, Y. & S.Omura (1990). Metabolism and products of actinomycetes - An introduction. Actinomycetologica, 4: 13-14 Williams, S.T. & J.C.Vickers (1988). Detection of actinomycetes in the natural environment. Problems and perspectives. In: Y. Okami, T.Beppu & H.Ogawa (eds.) Biology of Actinomycetes 88. Jap. Sci. Soc. Press, Japan, pp. 265-270 Copyright 1996 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia |
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