1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Diversity of begomoviruses infecting major vegetable crops(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Bincy S Basheer; Umamaheswaran, KItem Identification of graft transmissible resistant factors and development of si RNA mediated resistance in cassava against cassava mosaic geminivirus(Department of Plant Pathology, College of Agriculture, Vellayani, 2017) Asha B Nair; Umamaheswaran, KItem Characterization and management of yellow mosaic disease of black gram (vigna mungo (L.) hepper)(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Divya Jayakumar, V J; Sumiya, K VItem Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex Poir)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Sahna Hamsa, N H; Girija, DThe study entitled “Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex poir.)” was carried out in the Molecular Biology Laboratory of Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2009-2011. The objectives of the study included the molecular characterization of geminivirus causing yellow vein mosaic disease in pumpkin and developing a PCR based diagnostic kit. Yellow vein mosaic infected pumpkin leaf sample was collected from the field of Olericulture Department, College of Horticulture, Vellanikkara and total genomic DNA was extracted by CTAB method. Specific primers for coat protein and movement protein genes were designed based on the sequences of geminiviruses infecting vegetables, obtained from the NCBI database. PCR amplification was carried out using these primers. Amplicons of size ~900bp and ~700bp were obtained for coat protein and movement protein genes respectively. The purified PCR products were ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin layered with X-gal and IPTG. After confirmation of the inserts by colony PCR, the clones were sequenced. Sequences of 891bp and 702 bp were obtained for coat protein and movement protein genes respectively. Sequence analysis was carried out with standard bioinformatics tools. On blastn analysis both coat protein and movement protein sequences showed maximum similarity to Squash leaf curl China virus (SLCCV) from Coimbatore. For coat protein gene, full length ORF of 771bp was obtained and the ORF of movement protein was partial. Primers MP1F and MP1R with expected amplicon size of 1363bp were designed to get full length ORF (846bp) of movement protein gene. The technique was validated with DNA from 15 PYVM infected and 4 healthy pumpkin leaf samples collected from Palakkad, Thrissur and Malappuaram districts. The virus was detected only in the diseased samples. Hence these primers could be used in developing a molecular diagnostic tool to detect the virus. PCR amplifications were carried out in weed plants like Emilia sonchifolia, Ageratum conyzoides, Hibiscus surattensis and Synedrella nodiflora and crop plants like okra and ash gourd with yellow vein symptom to check whether these plants serve as the collateral hosts of the virus. PCR amplification was also performed in bitter gourd with distortion mosaic symptom. No amplifications were obtained in plants other than pumpkin. Using the primers PYVMV was detected in apparently healthy mature leaves of infected pumpkin. Hence, these primers could be used to detect latent infection. Sequence and phylogenetic analysis of coat protein and movement protein gene sequences showed maximum similarity to bipartite Squash leaf curl china virus (SLCCV) from Coimbatore. Hence Pumpkin yellow vein mosaic virus (PYVMV) from Kerala can be considered as a strain of SLCCV.Item Molecular analysis of phylogeography of cassava mosaic disease(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Jayakrishnan, J T; Makeshkumar, TA study on “Molecular analysis of phylogeography of cassava mosaic disease” conducted at the ICAR- Central Tuber Crop Research Institute, Sreekariyam, Thiruvananthapuram during 2015-2016. During the study, a survey was conducted in all districts of Kerala to identify the different cassava mosaic viruses present in major cassava growing areas. The survey conducted revealed that cassava mosaic disease (CMD) is widespread in Kerala having high symptom severity and increased aggressiveness as compared to earlier years. Maximum intensity of CMD was observed in Wayanad followed by Malppuram, Alappuzha and Ernakulam districts. From the 53 fields visited across all districts, 115 samples were collected and their symptomatology was recorded. Serological tests like ELISA and DIBA were done to detect the presence of cassava mosaic viruses in them and all the samples were further diagnosed using multiplex PCR to differentially detect ICMV and SLCMV. From the 115 samples, 9 samples were ICMV positive, 68 samples SLCMV positive, 18 samples had both ICMV and SLCMV (mixed infection) and 20 samples had neither ICMV nor SLCMV infection. These 20 samples were selected and PCR were employed using specific primers of ACMV, EACMV, UgV to detect any such virus presence and the results showed that 3 samples from Pathanamthitta had suspected ACMV infection which were also positive for SLCMV. From PCR analysis, it was revealed that SLCMV is widespread in all districts of Kerala while ICMV infection was restricted to 9 districts only viz., Thiruvananthapuram, Kollam, Alappuzha, Kottayam, Idukki, Ernakulam, Palakkad, Malappuram and Kasargod. To identify the variation of cassava mosaic viruses within different districts, each ICMV and SLCMV representative samples from all districts were used for PCR-RFLP analysis using 6 different restriction enzymes viz., EcoRV, HindIII, XhoI, ClaI, BamHI and KpnI. The results revealed that SLCMV samples from districts viz., Kollam, Pathanamthitta, Kottayam, Idukki, Kannur and Kasargod shown variation within the EcoRV, HindIII, XhoI, ClaI sites wheras ICMV samples from Kollam and Malappuram showed variation within HindIII and XhoI sites. To identify the molecular level variations within the conserved region of both ICMV and SLCMV, multiplex PCR amplified products of 5 representative samples (3 ICMV and 2 SLCMV) were sequenced and the result indicates that the SLCMV samples had more variability than the ICMV samples and also more number of SNPs were found within the conserved region of all these samples. Whole genome amplification of selected samples was done using RCA to identify the variation within the DNA-A genome and the sequence result disclosed that high level of variability is present in the form of SNPs in the conserved Rep region within DNA-A of selected samples. To identify the phylogenetic relationship of the sequenced samples with that of available accessions, dendrograms were made using MEGA 9.0 software and the tree showed that sequences has variability eventhough lies within the group. To identify the presence of beta satellite molecules in the samples collected during the survey, multiplex positive samples and samples showing unusual symptoms and increased aggressiveness were selected and PCR was employed using beta specific primer. Only one sample (Thiruvananthapuram mixed infection) shown positive results for suspected beta molecule and this is the first report of identification of beta satellite molecules associated with cassava mosaic disease in India making the situation more crucial.