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ThesisItem Open Access MOLECULAR CHARACTERIZATION OF FEW PLANT GROWTH PROMOTING MICROBES, THEIR COMPATIBILITY ANALYSIS AND UTILITY IN BIOINTENSIVE MANAGEMENT OF BACTERIAL WILT OF TOMATO(AAU, Jorhat, 2015) Nath, Bharat Chandra; Bora, L. C.Biointensive management of bacterial wilt of tomato (Lycopersicon esculentum Mill) caused by Ralstonia solanacearum was attempted using various bioactive (Plant Growth Promoting) microorganisms viz., Trichoderma viride, Pseudomonas fluorescens, Bacillus subtilis and Azotobacter chroococcum. The molecular characterization of these microorganisms was made to determine their distinctiveness from their close relatives through sequencing of ITS region of ribosomal DNA in case of fungal microorganisms and 16S region of ribosomal DNA in case of bacterial microorganisms. Interactive action among the four bioactive microorganisms showed compatibility in all the treatment combinations in vitro. The antagonistic potential of these bioactive microorganisms was tested in vitro singly or in consortia and per cent inhibition of target pathogen were recorded and analyzed. The highest inhibition (57.70%) against R. solanacearum was recorded against consortia of T. viride+ P. fluorescens + B. subtilis + A. chroococcum followed by T. viride + P. fluorescens + B. subtilis (39.77%) and P. fluorescens + B. subtilis + A. chroococcum (35.65%). Talc based bioformulations were prepared using the best three bioactive microorganisms and their consortia for management of bacterial wilt in pot grown tomato plants during 2013-14 and 2014-15 crop seasons. Applications of the consortial formulations were made as seed treatment, root treatment and soil application. Significantly highest reduction of bacterial wilt incidence (95.09 %) and highest yield (1.692 kg/plant) of tomato was recorded in treatment comprising of T. viride+ P. fluorescens + B. subtilis + A. chroococcum. Correlation studies revealed negative correlation (-0.993) between bacterial wilt incidence and yield of tomato. Screening of 47 tomato genotypes was done under field condition during 2012-13 and 2013-14 crop seasons to assess their degree of tolerance against bacterial wilt pathogen R. solanacearum. Highly resistance reaction (HR) was recorded in Konbilahi (L. pimpinellifolium) followed by resistance reaction (R) in Sel-35, sel-19 and Sel-9. Highest yield was recorded in 2012/TOLCVRES-3 (278.16 q/ha) followed by H-24 (276.85 q/ha) and 2012/TOLCVRES-4 (272.04 q/ha).ThesisItem Open Access Carpogenic germination of Sclerotia of Sclerotiniasclerotiorum (Lib.) de Bary and development of an IDM modules for management of Sclerotinia rot of French bean(AAU, 2015) Lalfakawma, C.; Das, B.C.Studies conducted on morphological and pathogenic variability among 30 isolates of Sclerotiniasclerotiorum (Lib) de Bary, derived from infected French bean (Phaseolus vulgaris) plants showing typical symptoms of white mold, collected from different geographical location of NE. All the isolates showed variation in morphological characters based on their mycelial growth, colony character and sclerotial formations. Out of 30 isolates, 14 isolates showed highly virulent reaction, each of 8 isolates revealed moderately and less virulent reaction respectively on French bean. HPLC analysis of mycelial culture extracts of highly, moderately and less virulent isolates from S. sclerotiorum showed that higher number of phenolic acid and higher concentration of oxalic acid were observed in highly virulent isolates of S. sclerotiorum , while lowest number of phenolic acids and least conc. of oxalic acid detected in less virulent isolates. In in vitro study of growth of highly virulent isolates of S. sclerotiorum against fungal antagonists showed that T. harzianum caused maximum inhibition of radial growth of S. sclerotiorum among the antagonists. Maximum growth and least per cent inhibition of radial growth by different antagonists was observed in AS3 (Assam) isolate of S. sclerotiorum. Effect of burial depth, soil drenching with Carbendazim and mycoparasites on carpogenic germination of Sclerotia of S. sclerotiorumwere evaluated under pot condition. It was observed that higher number of apothecia were produced when Sclerotia placed at ‘0’cm depth, while no apothecia were found at deeper depths viz,.6, 8 and 10cm respectively. Soil application of Carbendazim were more effective when applied 10 days after burial as compared to application at ‘0’ day of burial in inhibition of carpogenic germination of Sclerotia and there was a decreased sclerotial germination with increased concentration of fungicide. Amongst the antagonists, T. harzianum was found to be the most effective agent and caused highest reduction in carpogenic germination of Sclerotia followed by G. virens and T. koningii respectively. Under field condition, maximum reduction of white mold incidence and higher growth response and yield were observed when seeds were treated with Carbendazim @ 0.2% and it was integrated with soil application of T. harzianum @ 2%(w/w) of soil and Carbendazim spray @ 0.1% at 15 and 30 DAG in FYM amended soil as compared to other treatment combinations.ThesisItem Open Access Development of RNA based vaccine against Cucumber mosaic virus infecting Bhut Jolokia (Capsicum chinense Jacq.) crop and Citrus tristeza virus infecting citrus plantations of Assam(AAU, 2016) Borah, Munmi; Nath, P. D.“Development of RNA based vaccine against Cucumber mosaic virus infecting Bhut Jolokia (Capsicum chinense Jacq.) crop and Citrus tristeza virus infecting citrus plantations of Assam” were carried out at and Department of Plant Pathology, Assam Agricultural University, Assam, India and Laboratory of Plant Breeding and Biometry, Department of Crop Science, AUA, Athens, Greece. Utilizing virus genome properties enabled the design of novel, safe, and efficacious vaccines against different viral diseases infecting plants. In this study, it was shown that, dsRNA derived from viral sequences could interfere with cognate virus infection in a sequence-specific manner by delivering dsRNA to plant cells. In dsRNA-mediated protection, a dsRNA homolog of a viral silencing suppressor gene expressed in plants, which interferes with or prevents various stages of the viral life cycle, resulting in attenuated disease symptoms or resistance. It was aimed to produce CMV specific RNA vaccine to manage CMV infecting Bhut Jolokia crop of Assam. Application of these RNA based vaccines at the seedling stage could effectively reduce CMV infection at the later stage of the crop. These virus-free seedlings of Bhut Jolokia crop could give rise to a healthy crop growth. Taking it as a model system, it was further aimed to produce CTV specific RNA vaccine and to carry out a proof-of-concept to substantiate the same concept further in management of CTV infecting citrus plantations of North East India. A protocol for the synthesis of dsRNA using T7 RNA polymerase was utilized to produce RNA based vaccine against Cucumber mosaic virus infecting Bhut Jolokia (Capsicum chinense Jacq.) crop and Citrus tristeza virus infecting citrus plantations of Assam. CMV-encoded 2b gene based dsRNA was produced and tested against CMV infecting Bhut Jolokia (Capsicum chinense Jacq.) plants of Assam. The infection of CMV in Bhut Jolokia pepper plants was successfully interfered, demonstrating the applicability of RNA-based vaccination. In this study, double-stranded RNA derived from CMV-2b silencing suppressor gene sequence in Escherichia coli, could interfere with cognate virus infection. When dsRNA CMV-2b exogenously applied, along with CMV strain, onto Bhut Jolokia plants resulted in suppressing CMV infection. DAS-ELISA was used to identify the presence of CMV in the inoculated plants. Bhut Jolokia Pepper plants infected with CMV became severely stunted, nonproductive with dull light green foliage having a leathery appearance. In contrast, plants that received dsRNA of CMV-2b were less symptomatic and remained healthy as compared to those infected by CMV. Four experiments were conducted where; disease incidence was 15%, 5%, 29.5% and 0% when dsRNA of CMV-2b molecules were co-applied with CMV, as compared to 55%, 55%, 92% and 70% when only CMV was infected. As a result of dsRNA mediated resistance crop canopy increased, which is necessary for improved yields of the crop. This study constitutes a non transgenic approach of protection of Bhut Jolokia pepper plants against CMV. With the success of CMV specific RNA vaccine, the investigation further aimed towards production of a dsRNA construct coding for the three silencing suppressors of CTV to generate RNA-based resistance and to conduct a proof-of-concept of specific protection against viral infection. It was aimed to get more insight on the role of the CP, p20 and p23 genes as silencing suppressors of CTV in pathogenesis through topical application of these dsRNA molecules. The CP, p20 and p23 gene sequences of the North East India strain of Citrus tristeza virus was folded into a double-stranded (ds) RNA structure. dsRNA of sufficient quantities (several micrograms) obtained using in vitro transcription protocols for CP, p20 and p23 genes of the virus. The proof-concept experiment on application of these dsRNA against CTV infected citrus plants revealed that, while applied topically over leaf surface against the cognate virus, all three dsRNA constructs (CTV-CP, CTV-p23 and CTV-p20), could suppress the virus replication. This results successfully interpreted the proof -of -concept about the suppression of viral titre locally up to 10 days of topical application, through RNAi based method in citrus crop infected with CTV-North East India strain. The results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of post transcriptional gene silencing in natural virus infections, triggering the viral RNA for degradation. A dsRNA construct encoding silencing suppressors could be significantly suppressed the replication of viruses and confer potential resistance against the virus.