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Central Agricultural University, College of Post Graduate Studies in Agricultural Sciences, Umiam

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Author: Reddy, Desam Sainath ×
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    Diversity of native Bacillus spp. of mid hills of Meghalaya with special reference to Bacillus thuringiensis (Berliner)
    (College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-11) Reddy, Desam Sainath; Ningthoujam, Kennedy
    Bacillus species are ubiquitous gram-positive, rod-shaped cell, aerobic or facultative anaerobic bacteria and are one of the important components of the soil microbial community and many of Bacillus bacteria are reported as beneficial microbes widely used in industry and agriculture that exists in varied ecological niches in many environments. Among the beneficial microbial Bacillus species, Bacillus thuringiensis plays a vital role as biopesticide for the control of insects and the most successfully commercialized bio-control agent worldwide for its high specificity and associated environmental safety. In an effort to understand the diversity and distribution of Bacillus spp. along with Bacillus thuringiensis and its plasmid and cry gene profile variation in a diverse range of environments, the present investigation was undertaken. The isolation protocol was standardized and Enrichment method was chosen as the best method for the isolation of maximum Bacillus species compared to Sodium acetate and Saline methods. A total of 89 putative Bacillus like isolates were isolated using enrichment method from different ecological niches, collected from 12 different locations of Meghalaya. Among them, maximum 36 isolates were obtained from East Khasi Hills followed by Ri Bhoi District (33 isolates) and West Jaintia Hills (20 isolates). Based on Paenibacillus morphological characteristics, biochemical tests and using advance bacteriological identification software (ABIS), the 89 isolates were sorted into two genera (Bacillus and Paenibacillus) with 21 different species. The highest Shannon- Weiner index was recorded in Bhoirymbong (2.043) and the lowest was observed in Jowai (1.099). The highest Margalef index (3.186) and Simsons index value (0.8642) has occurred in Bhoirymbong (3.186), while the lowest in Jowai with value of (1.82) and (0.667) respectively. The Pielou’s evenness index was seen maximum with value of 1 in five locations Nongpoh, Upper Shillong, Nartiang, Jowai, Syndai, Amlarem, while the lowest was observed in Cherrapunji (0.857). Morphological characterization of 89 Bacillus isolates showed that maximum of the isolates had irregular colonies, flat elevation and had dry rough texture with white or cream coloured colonies. Biochemical characterization revealed that majority of the isolates showed positive results for catalase, voges-proskuer, starch hydrolysis and gelatin hydrolysis tests. All the 89 isolates showed negative result for Indole test. All the 89 isolates were found positive for endospore staining and Coomassie Brilliant Blue staining for the B. thuringiensis isolates showed the occurrence of dark blue crystal or staining body in spherical shapes in most of the isolates. Detection of 8 B. thuringiensis isolates (CAU-BT1, CAU-BT2, CAU-BT3, CAU-BT4, CAU-BT5, CAU-BT6, CAU-BT7, CAU-BT8) based on 16s rRNA gene sequencing analysis with a similarity of > 98% with reference Bt. The cry gene profiling for all the eight B. thuringiensis isolates revealed that all the isolates showed the presence of Cry 1 gene. The plasmid profiling revealed that all the 8 B. thuringiensis isolates harboured a single mega plasmid DNA which was above 10000 bp size. The results of the investigation indicate that the soils of Meghalaya are rich in Bacillus-like species with high diversity, which has strong potential for exploitation in the future. There is a substantial number of B. thuringeinsis isolates in this region and the presence of lepidopteran specific cry 1 genes was confirmed which can be potentially used in the integrated pest management for sustainable agriculture.
  • Thumbnail Image
    ThesisItemEmbargo
    Diversity of native Bacillus spp. of mid hills of Meghalaya with special reference to Bacillus thuringiensis (Berliner).
    (College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-11) Reddy, Desam Sainath; Ningthoujam, Kennedy
    Bacillus species are ubiquitous gram-positive, rod-shaped cell, aerobic or facultative anaerobic bacteria and are one of the important components of the soil microbial community and many of Bacillus bacteria are reported as beneficial microbes widely used in industry and agriculture that exists in varied ecological niches in many environments. Among the beneficial microbial Bacillus species, Bacillus thuringiensis plays a vital role as biopesticide for the control of insects and the most successfully commercialized bio-control agent worldwide for its high specificity and associated environmental safety. In an effort to understand the diversity and distribution of Bacillus spp. along with Bacillus thuringiensis and its plasmid and cry gene profile variation in a diverse range of environments, the present investigation was undertaken. The isolation protocol was standardized and Enrichment method was chosen as the best method for the isolation of maximum Bacillus species compared to Sodium acetate and Saline methods. A total of 89 putative Bacillus like isolates were isolated using enrichment method from different ecological niches, collected from 12 different locations of Meghalaya. Among them, maximum 36 isolates were obtained from East Khasi Hills followed by Ri Bhoi District (33 isolates) and West Jaintia Hills (20 isolates). Based on Paenibacillus morphological characteristics, biochemical tests and using advance bacteriological identification software (ABIS), the 89 isolates were sorted into two genera (Bacillus and Paenibacillus) with 21 different species. The highest Shannon- Weiner index was recorded in Bhoirymbong (2.043) and the lowest was observed in Jowai (1.099). The highest Margalef index (3.186) and Simsons index value (0.8642) has occurred in Bhoirymbong (3.186), while the lowest in Jowai with value of (1.82) and (0.667) respectively. The Pielou’s evenness index was seen maximum with value of 1 in five locations Nongpoh, Upper Shillong, Nartiang, Jowai, Syndai, Amlarem, while the lowest was observed in Cherrapunji (0.857). Morphological characterization of 89 Bacillus isolates showed that maximum of the isolates had irregular colonies, flat elevation and had dry rough texture with white or cream coloured colonies. Biochemical characterization revealed that majority of the isolates showed positive results for catalase, voges-proskuer, starch hydrolysis and gelatin hydrolysis tests. All the 89 isolates showed negative result for Indole test. All the 89 isolates were found positive for endospore staining and Coomassie Brilliant Blue staining for the B. thuringiensis isolates showed the occurrence of dark blue crystal or staining body in spherical shapes in most of the isolates. Detection of 8 B. thuringiensis isolates (CAU-BT1, CAU-BT2, CAU-BT3, CAU-BT4, CAU-BT5, CAU-BT6, CAU-BT7, CAU-BT8) based on 16s rRNA gene sequencing analysis with a similarity of > 98% with reference Bt. The cry gene profiling for all the eight B. thuringiensis isolates revealed that all the isolates showed the presence of Cry 1 gene. The plasmid profiling revealed that all the 8 B. thuringiensis isolates harboured a single mega plasmid DNA which was above 10000 bp size. The results of the investigation indicate that the soils of Meghalaya are rich in Bacillus-like species with high diversity, which has strong potential for exploitation in the future. There is a substantial number of B. thuringeinsis isolates in this region and the presence of lepidopteran specific cry 1 genes was confirmed which can be potentially used in the integrated pest management for sustainable agriculture.