INTRODUCTION Bacillus thuringiensis is naturally occurring, gram positive, facultative anaerobic, motile, endospore forming bacteria with rod shaped vegetative cells. This bacterium is available everywhere in the environment. It can be used as a biological control agent against many insect pests due to its entomopathogenic potential. Being omnipresent, it can be isolated from different natural habitats such as soil, water, plant surfaces dead insects, and insect cadaver (Yammamoto et al., 2014; Padole et al., 2017). It is a member of the morphological group of Bacilli named as Bacillus cerus group along with the other bacteria such as Bacillus cereus, B. anthracis, B. mycoides and B. laterosporous. Bt can be distinguished from other members of the Bc group by its defining feature which is ability to produce proteinaceous insecticidal crystal during the sporulation phase of its lifecycle. The bacteria occasionally lose their ability to form crystals and become indistinguishable from B. cereus itself. (Yammamoto and Powell 1993; Sanahuja et al., 2011). Over the recent decades, the uncontrolled and imprudent use of chemical insecticides has produced a number of environmental risks, many of which are toxic to both humans and beneficial fauna. This has caused a variety of issues, such as chemical residues, the emergence of insect pests that are resistant to treatment, resurgence, and secondary pest outbreaks (Singh and Mandal 2013). There is need of looking for better environmentally friendly control methods as a result of these undesirable side effects. The superior alternative to synthetically produced pesticides is the use of ecologically sound and target-specific pest management techniques such as use of microbial biopesticides (Majeed et al., 2017). These microbes have the potential to reduce the use of dangerous chemical pesticides because they are specific to their target and are natural enemies of insects. Hence microbial biopesticides can be used against a wide variety of agricultural insect pests in many agroecosystems (Ruiu 2018). Among all the microbes used for the purpose of pest control, Bacillus thuringiensis (Bt) is most widely used and important entomopathogen as it produces insecticidal crystal (Cry) and cytolytic (Cyt) proteins named δ-endotoxins encoded by cry and cyt genes (Crickmore et al., 2020) along with newly identified Vip protein (Yu et al., 2010). Out of the total 10% of bio pesticides used globally, approximately 90% of the microbial insecticides are derived from Bt (Osman et al., 2015). Continuously excessive application of Bt and use of a same cry gene for insect control can causes development of resistance in insect pests after few generations (Zago et al. 2014). Thus it is of much importance to search for the highly virulent and more effective indigenous Bt isolates from different unexplored natural habitat with the possibility to find with intended insecticidal genes. MATERIAL AND METHODS Collection of samples. For the purpose of this work, samples were collected from different natural habitat of Bacillus thuringiensis, such as soil, phylloplane i.e. surface of leaves and insect cadaver. Collection of soil samples. Soil samples were collected by scraping off soil surface with sterile spatula and then 10 g sample 2-5 cm below the surface and stored in sterile aluminum foil bag at 4°C from areas with no previous Bt history, neither sown nor spread (Martin and Travers 1989). Collection of leaf samples. Leaves of some important agronomical as well as horticultural crops from different locations of the university fields, of Dr. PDKV, Akola were collected. Three to five leaves from the lower middle and upper part of the canopy were collected and stored in sterile aluminum foil bags at 4 °C until further use (Asokan and Puttaswamy 2007). Collection of insect cadaver. Regular field visits were made to the university fields, of Dr. PDKV, Akola as well as farmers’ fields to check the presence of dead/diseased/ moribund larvae of insects; such insect cadavers were collected each in separate sterile micro centrifuge tube and stored at 4°C until further use (Padole et al., 2017). Isolation of Bacillus thuringiensis from the collected samples. Isolation of Bt from these sample collected from natural habitat was done by the sodium acetate selective isolation and heat shock treatment (Travers et al., 1987). Isolation from soil samples. For the purpose of isolation, one gram soilwas added to 10 mL of LB broth buffered with 0.25 M sodium acetate in falcon tubes. This mixture was shaken for 4 hours at 250 rpm at 30°C and heat shocked at 80 °C for three min. Serial dilutions (10-1 to 10-6) were made in sterile saline solution. 100 µL of each dilution was spread on petri plate containing Luria agar and incubated at 30°C overnight. Chalky white colonies were picked up and plated on T3 medium and incubated at 30°C for 72 hours. The colonies grown on T3 medium were further purified using single colony isolation technique and maintained on Luria Agar at 4°C until further use (Asokan and Puttaswamy 2007). Isolation from Phylloplane. To remove the superficially adhering micro flora, the 3-5 gram of leaves were dipped in sterile distilled water and then placed in 100 mL of sterile double distilled water and rotated at 250 rpm 30°C for 4 hours. This suspension was then poured in polypropylene falcon tubes and then centrifuged at 10,000 rpm at 4°C for 5 min. and supernatant was discarded. Further 5 mL of Luria broth buffered with 0.25 M sodium acetate was added to the pellet. This mixture was shaken for 4 hours at 250 rpm at 30°C and heat shocked at 80°C for three minutes. Serial dilutions (10-1 to 10-6) were made in sterile distilled water and 100 µL of each dilution was spread on Luria agar and incubated at 30°C overnight. Chalky white colonies were picked up and plated on T3 medium and incubated at 30°C for 72 hours. Colonies showing typical characters were selected and further purified using single colony isolation technique and maintained on Luria agar at 4°C until further use. Isolation from insect cadaver. The dead larvae were surface sterilized using rectified sprit and individual larva was homogenized in a microfuge tube in 1 mL of LB buffered with 0.25 M sodium acetate. This mixture was shaken for 4 hours at 250 rpm at 30°C and heat shocked at 80°C for three minutes. Serial dilutions (10-1 to 10-6) were made in sterile distilled water and 100 µL of each dilution was spread on Luria agar and incubated at 30°C overnight. Three replications were maintained for each dilution and chalky white colonies were picked up, plated on T3 medium and incubated at 30°C for 72 hours. Colonies which showed typical characters were selected and further purified using single colony isolation technique and maintained on Luria agar at 4°C until further use (Asokan and Puttaswamy 2007). Characterization of local isolates Characterization based on colony morphology. Single colonies were obtained by using single colony isolation technique and observations were recorded regarding colony morphology parameters namely, colony size, colony shape, colony elevation, colony margin, colony color and opacity of the bacterial colony. Strain Btk HD-1 was used as standard for the comparison with local probable Bt strains. Microscopic characterization. Microscopic characterization was carried out by different staining techniques which include, gram staining, spore staining with Malachite green and Amido black. Crystal staining was carried out by using coomassie brilliant blue. Biochemical Characterization. Morphologically characterized colonies were further be confirmed by the biochemical characterization with the help of IMVIC test, which comprises of indole production, methyl red, Voges-Proskauer, citrate utilization test (Agrahari et al. 2008). Indole Test. A loopful culture of probable Bacillus thuringiensis isolates were inoculated in tryptone broth containing NaCl. pH of culture was maintained at 7.2 and kept for incubation at 37°C in environmental shaker for 24 hours. Kovac’s reagent was added after 24 hours of incubation to the bacterial culture. Absence of pink ring at the surface of the culture indicated negative test. Methyl Red Test. MRVP medium was inoculated with a loopful of twenty four hours old cultures of probable Bacillus thuringiensis isolates. The culture was incubated at 30°C on rotary shaker at 100 rpm for 48 hours. A drop of methyl red indicator was added to the test tube, red color resulted positive test indicating presence of acid while yellow color of media indicated negative reaction to the test. Voges- Proskauer test. MVRP medium was inoculated with a loopful of twenty four hours old cultures of probable Bacillus thuringiensis isolates. Then culture was incubated at 30°C on environmental rotary shaker at 100 rpm for 48 hours. 1-2 drops of α-naphthol reagent and 4-5 drops of 40% KOH were added in culture tube. Opened tubes then were placed in slanting position in order to increase contact with air. Change of surface color to pink in 10-15 min. indicated the positive test with acetyl methyl carbinol production. Citrate Utilization test. This test was conducted to test the ability of bacterial culture to utilize citrate as a sole source of energy for its growth. For this, Simmon’s citrate agar media was autoclaved, poured into sterile test tube kept at slanting position and allowed to solidify. Solidified slants were inoculated with loopful of bacterial culture and incubated for 24 hours. Growth of bacterial culture accompanied with change in the color of media from green to blue indicates positive test whereas, no growth with no change in media color indicated negative test. RESULTS AND DISCUSSION In order to explore the different habitats for the presence of Bt isolates, samples of native ecological niche, were collected from various locations of Vidarbha region. As Bt seems to be found in wide variety of niches, samples of soil, phylloplane, and insect cadavers were collected. In order to search for more efficient Bt isolates, the soil sample were collected from the areas where there is no previous history of Bt neither sown nor sprayed, phylloplane of various crops from university fields and insect cadavers from different locations were collected for the purpose of isolation of Bt (Asokan and Puttaswamy 2007). Total 80 samples were collected from 80 different locations with coordinates mentioned in Table 1. Out of these, 67, 7, 6 samples were collected from soil, phylloplane, insect cadaver respectively. For the purpose of Bt isolation, sodium acetate selective method was used (Travers et al., 1987). With the help of Travers selective isolation method 57 probable Bt were isolated from soil, 6 from insect and 5 probable Bt were isolated. The results represent in Table 2 indicate that, out of the total samples collected maximum number of probable Bt isolates were obtained from soil (57), followed by phylloplane (6) and insect cadaver (5). The number of probable Bt isolates presented in Table 6 it can be estimated that Bt is abundantly present in the ecology of the collected samples (Table 2). Previous Travers et al. (1987) suggested the ubiquitous nature of Bt and isolated 85 of Bt out of 1,115 soil samples collected from United States and 29 other countries. Bt can be considered as part of the common leaf microflora of many plants (Smith and Couche 1991). Three common hypothetical niches of B. thuringiensis in the environment which include insect cadaver, phylloplane inhabitant, and soil are the important habitats for isolation of Bt (Meadows, 1993). Similar studies were conducted previously by Agrahari et al. (2008); Shishir et al. (2012); Padole et al. (2017); Amha et al. (2021) further confirmed that Bt can be isolated successfully from soil, phylloplane and insect cadaver with the help of Travers’ acetate selective isolation method. The morphological characterization of all the isolates obtained were carried by considering various important characters including colony size, colony shape, colony elevation, colony margin, colony color an opacity is reported in Table 3. The strain from NCBI, Bacillus thuringiensis sub sp kurstaki HD-1 (NCIM Accession No. 5118) was used as positive standard for the purpose of morphological characterization. The colony characters such as circular shape, flat elevation, opaque colony and creamy white colony color with irregular margin and wavy surface with fried egg like appearance was observed in the standard strain of Btk HD-1. Colony characters similar to the standard strain were reported from the 51 isolates out of total 68 probable isolate and remaining 17 isolates showed slight difference in colony color (off white and dirty white colony color) and slightly elevated colony. Amongst the total 51 isolates showing exactly similar characters as standard strain 44 out of 57, 4 out of 6 and 3 out of 5 isolates from soil, phylloplane and insect cadaver respectively were classified as probable Bt isolates. The different colony morphology predominantly of flat, circular, creamy white color colony with irregular margin and wavy surface was recorded (El-kersh et al., 2016; Padole et al., 2017) whereas creamish to off white color colonies with mucoid or glistening surfaces having entire edges and density ranging between translucent to opaque was recorded from native isolates from Punjab (Kaur et al., 2006). Microscopic observations of the total 68 probable isolates were taken on the basis of various staining techniques including gram staining spore staining with malachite green and amido black and crystal staining with coomassie brilliant blue (CBB G-250). In addition to the staining methods, the shape and ends of vegetative bacterial cells were also observed. Positive gram staining test, rod shaped vegetative cells having terminal spore, positive spore staining with amino black and malachite green and positive crystal staining with coomassie brilliant blue G-250 was observed in standard strain Btk HD-1. All 68 possible isolates were stained using these methods and the results were recorded in Table 4, where 55, 6 and 5 isolates from soil, phylloplane and insect cadaver respectively resulted in positive gram staining and having rod shaped vegetative cells. Positive spore staining was observed for both malachite green and amido black staining in the 52, 6 and 5 isolates from soil, phylloplane and insect cadaver respectively. With the help of spore staining it was also observed that 43, 3 and 4 isolates showed terminal spore position and 9, 2 and 1 isolates from soil, phylloplane and insect cadaver respectively showed middle spore position in vegetative cell. However crystal protein staining with CBB G–250 was positive for 28, 2 and 3 isolates from soil, leaf, and insect cadaver, respectively. Similar results were recorded that Bt is gram positive bacteria and have terminal or median endospore (Baig et al., 2010; Padole et al., 2017). Presence of crystals were recorded with help of CBB staining (Kati et al., 2007; Shishir et al., 2012). Biochemical characterization of the total 68 isolates was carried out by using indole, methyl red, Voges-Proskauer, and citrate test (IMViC). Out of 57 isolates from the soil, 6 from phylloplane, and 5 from insect cadaver, 42, 4 and 4 isolates showed Voges-Proskauer test positive, 14, 2, and 1 showed positive reaction to methyl red test whereas 55, 6, and 4 showed negative reaction to citrate utilization test. However, all the 57, 6, and 5 isolates showed negative reactions to the indole test as indicated in Table 5. Standard strain Btk HD-1 showed positive reaction Voges-Proskauer test while negative for methyl red, indole and citrate utilization test. The results regarding biochemical studies were in accordance with Eswarapriya et al. (2010), reported native Bt isolates positive to Voges- Proskauer (VP) test and negative reaction to Methyl Red (MR) test. It was recorded that Bt produces acetylmethyl carbinol from glucose fermentation as it was positive for VP test among IMViC test (Deepak et al., 2011; Ghosh et al., 2017; Purohit, 2019). Negative reaction for citrate utilization test was recorded from B. thuringiensis subsp. kurstaki (De Barjac and Frachon 1990; Abirami et al., 2016; Padole et al., 2017). It was previously recorded that isolates do not have the ability decompose the amino acid tryptophan to indole which is in accordance with present study (Yoo et al., 1996; Deepak et al., 2011; Abirami et al., 2016). The Bt isolation index was calculated by dividing the population of crystalliferous Bt isolates by the total population of Bacillus for each sample collected from different sources. The result present in Table 6 indicates that the highest Bt isolation index was obtained from soil samples of Bhandara district (0.75) followed by soil from Gadhchiroli (0.66), Gondia (0.60), Amravati (0.52), Yavatmal (0.50) and Akola (0.36) and the lowest Bt isolation index was reported in soil sample from Nagpur district. Whereas, 0.60 and 0.33 Bt index was reported for insect cadaver and phylloplane respectively. Similarly, highest Bt index of 0.67 was from soil samples and minimum 0.40 from leaf samples (Shishir et al., 2012) whereas, higher Bt index recorded from forest soils (0.60) as compare to agricultural soils (0.33) (Lone et al., 2017). Hence this study provides the idea about abundance of and diversity of Bt isolates present in the habitat of Vidarbha region.