Bacillus thuringiensis populations ranged between 4.23 x 105, 6.52 x 105 cfu/g soil and consist of 11 types of isolates with 3 polymorphic, 7 spherical and 1 bipyramidal type of crystals. Polymorphic crystal containing isolates were further characterized. B. thuringiensis isolates were circular, white, flat and undulate or entire. These were gram-positive. These tolerated 5% NaCl and failed to grow anaerobically. None of these were positive for indole production. All were sensitive to streptomycin,vancomycin, polymyxin B and norfloxacin but resistant to penicillin G and ampicillin/sulbactum. All of the isolates were crystal forming rods, width of rods was >0.9 um, catalase positive and sporangia were not swollen. All of the isolates hydrolyzed protein but only BTc 175 produced arginine dihydrolase, BTc 152 and BTc 175 produced urease

Bhattacharya, T

Chandra, G

Chatterjee, SN

Dangar, TK

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 6 (13), pp. 1587-1591, 4 July 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals    Full Length Research Paper  Ecology and diversity of Bacillus thuringiensis in soil environment  Chatterjee, S. N.1, Bhattacharya, T.2, Dangar, T. K.3 and Chandra, G.1  1Microbiology Research Unit, Parasitology Laboratory, Department of Zoology, Burdwan University, Burdwan-713104. W.B., India. 2Department of Zoology, Vidyasagar University, Midnapore-721102, WB, India. 3Microbiology Laboratory, Central Rice Research Institute, Cuttack, India.  Accepted 29 November, 2006  Bacillus thuringiensis populations ranged between 4.23 x 105, 6.52 x 105 cfu/g soil and consist of 11 types of isolates with 3 polymorphic, 7 spherical and 1 bipyramidal type of crystals. Polymorphic crystal containing isolates were further characterized. B. thuringiensis isolates were circular, white, flat and undulate or entire. These were gram-positive. These tolerated 5% NaCl and failed to grow anaerobically. None of these were positive for indole production. All were sensitive to streptomycin, vancomycin, polymyxin B and norfloxacin but resistant to penicillin G and ampicillin/sulbactum. All of the isolates were crystal forming rods, width of rods was >0.9 um, catalase positive and sporangia were not swollen. All of the isolates hydrolyzed protein but only BTc 175 produced arginine dihydrolase, BTc 152 and BTc 175 produced urease.   Key words: Bacillus thuringiensis, soil, isolates, polymorphic crystal.   INTRODUCTION   Research of almost 85 years reveals that Bacillus spp., especially B. thuringiensis and Bacillus sphaericus are the most potent biopesticides (Boucias and Pendland, 1998). Available information depict that B. thuringiensis is a versatile pathogen capable of infecting protozoa, nema-todes, flatworms, mites and insects that are either plant pests or human and animal health hazards (Feitelson, 1993). B. thuringiensis has been obtained from soil, phy-llosphere, diseased insects, stored products, dumping pits, excreta of vegetarian animals etc. and about 30-100% spore formers of phyllosphere were found to be B. thuringiensis (Martin and Travers, 1989; Boucias and Pendland, 1998). An analysis of 27,000 isolates collected from ,100 soil samples all over the world demonstrated that B. thuringiensis could be isolated every where, inclu-ding desert, beach and tundra habitats (Martin and Tra-vers 1989; Attathom et al., 1995). B. thuringiensis acco-unts for about 5-8% of Bacillus spp. population in the environment  (Hastowo  et  al.,  1992). Till date more than 130  species  of  lepidopteran,  dipteran  and  coleopteran   *Corresponding author. E-mail: soumen_microbiology@redi-ffmail.com. insects are found to be controlled by B. thuringiensis (Dean, 1984). So far, 68 serotypes (81 serovars/varieties) of B. thuringiensis having wide array of host range have been isolated and characterized and some of them have already been commercially exploited directly as native form or indirectly as transgenic microbes or plants (Kratti-ger, 1997).  Insect pests of crops and forest plants and vectors of disease of human beings and other animals are serious threat for agriculture and public health. Worldwide, about US $8000 billion is spent for insecticides and estimates reveal that US $2700 can be substituted by the biopes-ticide B. thuringiensis (Krattiger, 1997). Besides exorbi-tant cost, and resistance and resurgence of the different pests, the chemical pesticides are the single main cause of health and environmental hazard (Krattiger, 1997). The situation demands the safer pesticides and biopesticides are the most desired alternatives. Bacteria, especially B. thuringiensis and B. sphaericus are the most potent and successful group of organisms for effective control of insect pests and vectors of diseases (Krattiger, 1997). Potentiality of B. thuringiensis as larvicide of Culex in India has been demonstrated by Ghosh et al. (1988). B. thuringiensis has certain  advantages  for  exploitation  as 1588           Afr. J. Biotechnol.    Table 1. Types of Bacillus thuringiensis isolated from different soils.  Soil no. Types of     Bt Isolate number Type of crystal S1. 5 BTc 152 BTc 170, 171,172, 173 Polymorphic Spherical S2. 6 BTc 61, 175 BTc 179, 176, 177 BTc 178 Polymorphic Spherical Bipyramidal   Table 2. Colony characters of B. thuringiensis isolates and standard culture on nutrient agar plates.  Bacteria no. Form Colour Elevation Margin BTc 152. Circular White Flat Undulate BTc  61 Circular White Flat Entire BTc 175 Circular White Flat Entire    biopesticide viz. B. thuringiensis can be used directly and as transgenic microbes and plants, being a prokaryote there is no dominant or recessive allele, highly vulnerable to genetic manipulation and the toxin gene is coded by single gene (monocistronic), B. thuringiensis is fermenta-tion friendly and therefore commercially exploitable and it is host specific or has narrow host range. These advanta-ges favoured development of about 100 formulations (Federici, 1993) and commercialization of 40 B. thurin-giensis products internationally and eight products in India exclusively by the multinational organizations (Sax-ena, 2000). However, none of the formulations marketed in India is an indigenous strain. Present study was envisaged to isolate and identify the B. thuringiensis of indigenous soils of Tarakeswar, Hoo-ghly, West Bengal, India and characterize the polymer-phic crystal producing strains, which may be exploited for biological control of a wide range of insect-pests and disease vectors in the long run. The investigation was under-taken with two main objectives; assessment of diversity of B. thuringiensis population producing different types of crystals, especially the polymorphic crystal producing strains, and morphological, physiological and biochemical characterization of the polymorphic crystal producing strains.   MATERIALS AND METHOD  Soil samples were collected from the rice fields at two locations (site 1 with clay soil and site 2 with sandy soil) of Tarakeswar of India. Top layer of soil (about 1 cm) was removed. From each loca-tion, five samples each of about 10 g were collected from five spots. Samples were mixed thoroughly and put in polythene packets with proper levels. The soils were air-dried up to 20% moisture level, powdered and sieved through a fine mesh and stored in sealed polythene bags within desiccators. The soil was checked time to time to maintain a moisture level of 20%. Immediately after collection, the soil pH was checked in the labo-ratory. Detailed characters of the soil were recorded. B. thuringie-nsis were isolated from the soil. One-gram soil was suspended in 10 ml sterile distilled water, logarithmic dilutions were made up to 10-2 level, pasteurized at 60°C for 30 min and 10 l suspension was added to 100 ml nutrient agar (NA) medium (≈ 45°C) and plated on 5 plates (Holt, 1984, Lacey, 1997). The plates were incubated at 30 ± 0.1°C for 72 h. After incubation, a portion of each colony was mounted on a slide with water and observed under a phase contrast microscope. The colonies depicting spores and crystals were marked and streaked on NA plates. Isolated colonies were reconfirmed to be B. thuringiensis by checking the spore and crystal production. One single and isolated colony was sub cultured on NA slant, incubated for 72 h at 30 ± 0.1°C and after adequate growth the slants were numbered and preserved at 4 ± 0.1°C. Cultures on NA stabs were preserved at 4 ± 0.1°C for long-term storage. The slant cultures were sub cultured periodically and used for different experiments. Morphological characters of the colonies and the bac-teria were studied following the standard microbiological methods (Pelczar et al., 1957; Collee and Miles, 1989; Lacey, 1997). The isolates were streaked on NA plates, incubated for 72 h at 30 ± 0.1°C. The shape, size, colour, margin and opacity were recorded from isolated colonies. The isolates were streaked on NA slants and stab cultured with a straight needle pierced through the centre of the NA stab tubes, incubated at 30 ± 0.1°C for 72 h and growth of the organism were recorded. Morphology of the vegetative cells, spores and crystals were observed under a phase-contrast micro-scope. Staining characters of the organism were studied for vegeta-tive, reproductive and crystal structure determination. Physiological and biochemical characters of the organisms were checked follow-ing the standard methods for identification of the isolates (Pelczar et al., 1957; Collee and Miles, 1989; Lacey, 1997).   RESULTS AND DISCUSSION  B. thuringiensis population ranged between 4.23x 105 - 6.52 x 105 cfu/g soil. Different types of B. thuringiensis isolated from the soils are presented in Table 1. Soil sample from site-1 resulted in 5 types of B. thuringiensis of which one produced polymorphic and others produced spherical crystals, site 2 produced 6 types of B. thuringie-nsis of which 2 produced polymorphic, 3 spherical and one bipyramidal crystals (Table 1). Thus in total, there were 11 isolates of which 3 (B. thuringiensis c 152, B. thuringiensis c 61 and B. thuringiensis c 175) isolates were with polymorphic crystals. These were further studied. The colony characters of the B. thuringiensis iso-lates under study were circular, white, flat and undulate or entire (Table 2). The characteristics of vegetative cells, Chatterjee et al.        1589    Table 3. Characters of vegetative cells of B. thuringiensis isolates and standard culture.  Length (µm)* Breadth (µm)* Bacteria no. Shape Range Mean Range Mean Motility Gram stain BTc 152. Rods with rounded ends 4- 5.06 4.70 1-2.1 1.45 Non motile + BTc 61 Rods with rounded ends 4.1-6.8 4.30 1.5-2.5 1.80 Motile + BTc 175 Rods with rounded ends 4-6 4.2 1.0-2.5 1.7 Non motile +   * Results of 10 observations.   Table 4.  Characters of spores of B. thuringiensis isolates and standard culture.  Length (µm)* Breath (µm)* Bacteria number Shape Range Mean Range Mean Spore stain  BTc 152. Oval 1-2 1.58 0.5-1.35 0.98 + BTc  61. Oval 1-2 1.83 0.5-2.01 1.25 + BTc 175. Oval 1.25-2.0 1.85 1-1.50 1.20 +  * Results of 10 observations.   Table 5.  Characters of crystals of B.  thuringiensis isolates and standard culture.  Length (µm) Breadth (µm) Bacteria number Shape Range Mean Range Mean Crystal stain  BTc 152. Polymorphic 1.25-2.8 2.20 1.0-2.0 1.34 + BTc  61. Polymorphic 1-2.60 1.66 0.5-1.80 1.25 + BTc 175. Polymorphic 1.0-2.0 1.60 0.5-2.00 0.78 +  . * Results of 10 observations.    Table 6. Growth characteristics of B. thuringiensis isolates and standard culture.  Bacteria number Medium BTc 152 BTc 61 BTc 175 NA + + + NA + Sodium chloride (%) 1 + + + 2 + + + 3 + + + 4 + + + 5 + + + 6 - - - 7 - - - 8 - - -    spores and crystals of B. thuringiensis isolates are given in Tables 3 - 5. All the bacteria were positive for gram stain, spore and crystal staining (Tables 3 - 5).Organisms tole-rated 5% NaCl (Table 6). The organisms failed to grow anaerobically. Enzymatic activities of different B. thuringiensis isolates are shown in Table 7. The physio-logical and biochemical properties of the B. thuringiensis cultures are presented in Table 8. None of the organisms were positive for indole production. Response of the org- Table 7.  Enzymatic activities of different B.  thuringiensis isolates and standard cultures.  Bacteria number (BTc) Tests 152 61 175 Protease: Growth + + + Result + + + Gelatinase Clear zone (mm) 32 34                         35 35Growth + + + Result + + + Casein hydrolysis Clear zone (mm) 2 1 2    anisms to the recommended doses of different antibiotics (Table 9) shows that all of them were sensitive to streptomycin, vancomycin, polymyxin B and norfloxacin but resistant to penicillin G and ampicillin/sulbactum. Results of biochemical and other characters (Tables 8 - 10) of the isolates were important for the identification of the isolates. All of the isolates were gram positive, spore and crystal forming rods, width of rods was >0.9 um, catalase positive sporangium was not swollen. All the organisms  did not grow anaerobically. The crystals prod- 1590           Afr. J. Biotechnol.    Table 8. Some physiological and biochemical properties of B. thuringiensis isolates and standard culture.  Bacteria number Tests BTc 152 BTc 61 BTc 175 Catalase + + + Arginine dihydrolase production - - + Indole production - - - Vogues Proskauer test + + + Nitrate reduction test     + + + Urease production test       + - +  + = Positive result.  - = Negative result.    Table 9.  Antibiotic assay of different B.  thuringiensis isolates and standard cultures.  Bacteria number BTc 152 BTc 61 BTc 175 Antibiotics  Re C Re C Re C Vancomycin (30 ug) S 16 S 21 S 19 Penicillin G (10 units) R 0 R 0 R 0 Norfloxacin (10 ug) S 15 S 17 S 16 Bacitracin (10 units) S 11 S 12 S 11 Ampicillin/Sulbactum (10/10 ug) R 0 R 0 R 0 Erythromycin (15 ug) S 25 S 27 S 27 Streptomycin (10 ug) S 23 S 21 S 33  Re = Reaction. C = Clear zone diameter in mm. S = Sensitive. R = Resistant.   Table 10.  Identification scheme of the isolates/cultures.                Bacteria number Characters BTc 152 BTc 61 BTc 175 Shape Rod Rod Rod Length (µm) 4.70 4.30 4.20 Width (µm) 1.45 1.80 1.70 Sporangium Not swollen Not swollen Not swollen Spore Oval Oval Oval Crystal + Polymorphic + Polymorphic + Polymorphic Gram stain + + + Catalase activity + + + Anaerobic growth - - - Genus Bacillus Bacillus Bacillus Species thuringiensis thuringiensis thuringiensis     uced by all of the tested isolates were polymorphic. All of the isolates hydrolyzed protein and only B. thurin-giensisc 175 produced arginine dihydrolase, B. thurin-giensis 152 and B. thuringiensis 175 produced urease. The results conclusively prove that the organisms are Bacillus sp. (Holt, 1984; Sneath, 1986; Garrity, 2001). Besides the characters of Bacillus sp. which are men-tioned above, the organisms produced galvanized metal-lic colonies, polymorphic crystals along with the spores, non-swollen sporangia, oval spores, positive for catalase and VP tests confirmed that the organisms belong to Group I of Bacillus sp. (Sneath, 1986; Smibert and Krieg, 1994; Garrity, 2001). Production of crystals and non-swollen sporangia identified the organisms as B. thuringiensis (Sneath, 1986; Smibert and Krieg, 1994; Thiery and Frachon, 1997; Garrity, 2001). Occurrence of B. thuringiensis in soil and population size is relatively higher than earlier reports (Martin and Travers, 1989; Hastowo and Ohba, 1992; Theunis et al., 1998; Kaur and Singh, 2000). Although there are several reports that all soils may or may not harbour B. thuringiensis (Martin and Travers, 1989; Hastowo et al., 1992), higher B. thuringiensis population containing different types of crystals reveal that the soils of Tarakeswar are rich in B. thuringiensis population and diversity.   REFERENCES  Attathom T, Chongrattanameteekul W, Chanpaisang J, Siriyan R (1995). Morphological diversity and toxicity of delta-endotoxin produced by various strains of Bacillus thuringiensis. Bull. Entomol. Res. 85:167-173. Boucias, DG, Pendland JC (1998). Principles of insect pathology. Kluwer Acad. Publ., Boston, USA, p. 537. Collee JG, Miles PS (1989). Tests for identification of bacteria. In: Practical medical microbiology, Eds. Collee JG, Duguid JP, Fraser AG, Marmion BP. 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Ecology and diversity of Bacillus thuringiensis in soil environment

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Ecology and diversity of Bacillus thuringiensis in soil environment

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