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Subject: Agricultural Biotechnology × End date: 2018 × Start date: 2018 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    A study on the role of exopolysaccharide in conferring acid tolerance in Bacillus sp.
    (AAU, Jorhat, 2018-01) Deka, Priyadarshini; Barooah, Madhumita
    Soil bacteria have evolved various mechanisms to adapt to stress environmental conditions such as temperature, salinity, drought and low pH condition of soil. Among the several environmental stress conditions, soil acidity an important factor influencing physicochemical and biological properties of soil along with microbial diversity and crop production is an emerging issue of immense concern due to its wide spread distribution across the globe. Although low soil pH restricts the number and diversity of bacteria, it is known that some soil bacteria are able to thrive in such conditions having evolved various mechanisms including production of biofilm to circumvent acid stress. Bacterial exopolysaccharide (EPS) are high-molecular-weight complex polymers composed of sugar moieties that form the main component of the biofilm which aid the bacteria to colonize substrata. In the present study, a total of 28 isolates were identified and characterized as acid tolerant EPS producing bacteria among which, B. amyloliquefaciens p16 produce the highest EPS (219.96 μg/ml). A culture medium containing sucrose (3.5%) as carbon source with pH 5.0 and incubated for 24 hrs was optimal for maximum production of EPS. The HPLC analysis of monomeric units of EPS produced by B. amyloliquefaciens p16 revealed the abundance of galactose at pH 7.0 which however, changed to arabinose when shifted to acidic condition (pH to 5.0 and 4.5). The isolate B. amyloliquefaciens p16, significantly improved soil physical properties in terms of greater soil aggregation (80.59 mm diameter aggregates) and water holding capacity (53.90%) when inoculated into soil over the control (31 mm diameter aggregates and 18.21%, respectively). The differential expression of epsA and epsB, the first two genes of the eps operon showed a 7 and 9 fold increased expression in pH 5.0 compared to pH 7.0 respectively. Disruption of the epsB gene in B. amyloliquefaciens p16 using integration vector pMUTIN4 generated mutants that produced significantly lesser EPS (33.23 μg/ml) when compared to the WT (223.87 μg/ml). The generated mutant of B. amyloliquefaciens p16 lacking the wrinkled morphology had an extended lag phase of 24 hrs and was barely able to survive in acidic medium (pH 4.5) unlike that of the WT type. Soil inoculated with generated mutants formed smaller soil aggregates (42.41±1.70 mm) and had decreased water holding capacity (27.67±1.94%) compared to the WT (80.59± 0.22 mm and 53.90± 1.66%, respectively). This study indicates that EPS secreted by acid tolerant bacteria (B. amyloliquefaciens p16) imparts acid tolerance and also aids in improving the soil physical structure through increased soil aggregation and water holding capacity.
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    ThesisItemOpen Access
    Study and characterization of the DNA virus diseases in few important crops of Assam
    (AAU, Jorhat, 2018-01) Baldodiya, Gajendra Mohan; DebNath, Palash
    Plant viruses cause many devastating diseases and are responsible for the huge loss in crop production and quality. The Infected plants may show a range of symptoms like leaf yellowing, leaf distortion, growth abnormalities and yield reduction. Although the majority of the plant viruses are RNA viruses, in the recent years, DNA viruses have emerged as a serious problem for vegetables and fiber crops, more specifically in tropical and subtropical regions of the world. In India, Begomoviruses are known for major yield loss in a variety of crops. So far, very few reports on DNA virus diseases are available from the northeastern region, including Assam. In the present study, three important single-stranded DNA viruses, banana bunchy top virus (BBTV), squash leaf curl China virus (SLCCNV) and tobacco curly shoot virus (TbCSV) were identified from Assam infecting banana, pumpkin, and chilli crops respectively. The crop cultivating areas were surveyed and 89 plant samples showing symptoms of geminivirus and BBTV infections were collected from 16 different crops. The broad-spectrum antibody combination, tomato yellow leaf curl virus (TYLCV) in DAS-ELISA (double antibody sandwich ELISA) assay revealed the all possible Begomovirus infections. The molecular detection method by using universal and gene-specific primers confirmed the presence of viral genome in positively screen samples through DAS-ELISA. The full-length genome amplification of detected viruses was achieved by designing walking primers from both ends of the partial genome and rolling circle amplification (RCA) followed by cloning process. The in silico analysis (genomic feature analysis, multiple sequence alignment and phylogenetic grouping) revealed the molecular properties of identified viruses. The BBTV-As-JOR was found closely related with BBTV-Lucknow isolate, although it stands out for few genomic features. Each genomic segment of the BBTV-As-JOR isolate was comprised of an ORF in sense orientation, encoding specific protein like other BBTV isolates. The common region major (CR-M) of DNA-R shows 26nt deletion like BBTV Lucknow but deletion of 36nt in DNA-U3 was found different. The BBTV-As-JOR clustered with PIO (Pacific Indian Ocean) isolates in the phylogenetic analysis based on the R-segment. Similarly, the clustering of other BBTV segments was also found with previously reported BBTV isolates of PIO group. The genome of SLCCNV-As-JOR-G8 pumpkin shared high nucleotide sequences and proteins similarity with SLCCNV-pumpkin-Varanasi DNA-A. The phylogenetic clustering also conferred these findings. The sequence analysis confirmed the presence of seven ORFs viz, AC1, AC2, AC3, AC4, AC5, AV1/CP and AV2 in SLCCNV-As-JOR-G8 pumpkin. The ORFs showed the typical begomoviral genome organization; however, they displayed similarity to different begomoviruses. The TbCSV-As was identified as a close relative of TbCSV)-[SC118] sharing the similarity of two major proteins, AL2 and AL3. The occurrence of TbCSV in chilli (Capsicum frutescens) host plant and causing upward curling of leaves is the first report from India in our knowledge. This study presents the first report of identification and characterization of BBTV, SLCCNV, and TbCSV from Assam which may open up a new insight into the understanding of DNA viruses infecting important crops of the state.