PG Thesis

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    Meta-analysis of QTLs associated with pest and disease resistance genes in cassava
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Reshma Parveen, J; Sreekumar, J
    Plant disease resistance is a complex trait which is controlled by quantitative trait loci (QTLs). Several QTLs have been found for cassava disease resistance. Meta-analysis provides a simple, reliable, and economical method for integrating information from multiple QTL studies across various environmental and genetic backgrounds, detecting consistent QTLs powerfully and estimating their genetic positions precisely. The study entitled "META-ANALYSIS OF QTLs ASSOCIATED WITH PEST AND DISEASE RESISTANCE GENES IN CASSAVA" was conducted at the ICAR-CTCRI, Sreekaryam, Thiruvanathapuram during October 2018 to August 2019. The objectives of this study was to combine the results of QTL detection studies conducted on Cassava Mosaic Disease (CMD) resistance genes in cassava, to integrate this data with genomic information of cassava and to validate the results obtained using resistant and susceptible varieties. 61 QTLs from 6 different published QTL mapping studies related to Cassava Mosaic Disease (CMD) and Cassava Brown Streak Disease (CBSD) were selected. A consensus map was constructed by integrating individual linkage maps of these QTLs with a reference map (Cassava genetic linkage map). 58 QTLs from the initial 61 QTLs were then projected to this consensus map. Using Biomercator V.4 software meta-analysis of these projected QTLs were carried out. Meta-analysis of CMD and CBSD-RN resistance QTLs were done separately. 11 MQTLs for CMD resistance and 10 MQTLs for CBSD-RN resistance were obtained. For mining candidate genes and for identifying their functional information the QTLs within the MQTLs with confidence interval less than 2cM and physical length less than1Mb were selected. A total of 38 candidate genes associated with CMD resistance and 30 genes associated with CBSD-RN resistance were annotated. Defence related civ 104 protein and transcription factors were found to be associated with these genes. Validation of the identified QTLs was done by using real time PCR and the genes were found to be expressed in resistant variety of cassava. The MQTLs found in this study that have small physical and genetic intervals are useful for marker-assisted selection for developing disease resistance varieties of cassava.
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    Ecofriendly pest management in brinjal (Solanum melongena L.)
    (Department of Entomology, College of Agriculture,Vellayani, 2000) Annie Bernice, T S; Krishnakumar, R
    A survey was conducted in the important brinjal growing tracts of Thiruvananthapuram district to study the population dynamics of the pests and their natural enemies in the brinjal ecosystem. Laboratory experiment and field experiments were conducted in the College of Agriculture, Vellayani to assess the deterrency and toxicity of the different treatments on aphids, epilachna and Leucinodes which were identified as important pests of brinjal from the survey and the effective treatments as identified from the laboratory experiment were carried out in the field. =<; Malathion alone or with garlic was found to be toxic to aphids. Epilachna and Leucinodes. The treatments with neem oil and Hyptis suaveolens either alone and in combination were found to have deterrent and toxic effects on aphids, Epilachna and Leucinodes in the laboratory experiment. However, the effect was much pronounced when neem oil activated with garlic was used in combination with Hyptis suaveolens. In the field, though malathion was found to contain the pests, the effect was not persistent for a long time and hence was not effective in the long run. The combination of neem oil + garlic plus Hyptis suaveolens was found to offer protection for the pest for a long period of time. On par with it were combination of nee m oil plus Hyptis suaveolens + garlic and combination of neem oil + H. suaveolens. The botanicals were also found to be safe for the natural enemies like coccinellids, staphylinids and spiders. This treatment was also found superior to other treatments in terms of net income and benefit cost ratio and the lower unit cost of the treatment with high yield was precisely the reason for its superiority.
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    Development of infectious clones of cassava mosaic virus and their validation
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Vishnu Narayanan; Makeshkumar, T
    The study entitled “Development of infectious clones of cassava mosaic virus and their validation” was conducted at the ICAR- Central Tuber Crop Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2017- 2018. The major objectives of the study were cloning and characterisation of SLCMV/ICMV infected leaf samples, construction of infectious clones of SLCMV/ICMV and agroinoculation of Nicotiana benthaminana with the partial dimers constructed in order to check the infectiousness of the viral clones. The whole genome amplification of SLCMV/ICMV DNA samples were done and cloned in pUC19 vectors to obtain pSLCMV A7 (2746 bp), pSLCMV B2 (2738 bp) and pICMV A5 (2739 bp) full length clones. The sequence of pSLCMV A7 showed maximum similarity of 99 % with ‘SLCMV-[TVM1]’ sequence in NCBI blast. While the sequence of pSLCMV B2 showed maximum similarity of 99 % with ‘SLCMV-[Ker20]’ sequence in NCBI blast. The sequence of pICMV A5 showed maximum similarity of 95 % with ‘ICMV-[Mah]’ sequence in NCBI blast. In order to develop infectious clones, partial dimers were constructed for SLCMV DNA-A and SLCMV DNA-B and cloned in binary vector pPZP201. These infectious clones were successfully transformed into wild type A. tumefaciens, Ach5 strain by triparental method. Then agroinoculation of N. benthamiana with the constructed partial dimers was found to be successful. After 14 days post inoculation, plants infected with DNA-A + DNA-B partial dimers showed severe symptoms like leaf curling, stunting. The plants infected with partial dimer of DNA-A alone showed mild symptoms like upward leaf curling which confirmed its monopartite lineage. While those plants agroinoculated with partial dimer of DNA-B alone did not show any symptoms. These efficient infectious clones of cassava mosaic virus and their subsequent inoculation technique would provide a major advancement to the resistance development in cassava.
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    Molecular cloning and characterization of virus causing leaf curl disease of capsicum spp.
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Niranjana Menon, C; Anita Cherian, K
    Chilli is one of the most important crops cultivated across the globe, as vegetable, spice and for industrial purposes. According to the statistics of National Horticulture Board (2017), the crop covers an area of 1860 ha in the state of Kerala with an average production of 12470 tonnes. During the last decade, the threats posed by the emerging begomoviruses infecting solanaceous crops have affected the economic cultivation of chilli. Chilli leaf curl disease caused by Chilli leaf curl virus belonging to the genus Begomovirus and family Geminiviridae is a serious constraint to chilli production in India which causes upto 100 per cent yield loss especially when infected at an early stage of the crop. Considering the importance of the disease, the present study was undertaken with the objective to study the incidence and symptomatology of chilli leaf curl disease and to characterize and clone the coat protein gene of the Chilli leaf curl virus isolates. The project initiated with purposive sampling surveys conducted in eleven different locations of Thrissur district, Kerala to document the incidence and symptomatology of leaf curl disease on chilli plants. The disease incidence recorded during the survey ranged from 43.30 to 85.00 per cent under open field conditions and from 45.75 to 79.40 per cent under protected conditions while the disease severity ranged from 43.60 to 81.54 per cent and from 49.40 to 87.50 per cent, respectively under open field conditions and protected conditions. The symptomatology of chilli leaf curl disease on different parts of the plant such as leaves, internodes, fruits and the whole plant under natural conditions was documented during the survey. The symptoms observed on the leaves of infected chilli plants under natural conditions include upward curling, crinkling, puckering, vein banding, interveinal chlorosis, size reduction of leaf lamina and leaf malformation. The fruits produced by the infected plants showed size reduction and deformation. The infected plants were stunted and bushy in appearance. The transmission of the virus by insect vector, Bemisia tabaci and grafting was studied and the symptoms were documented. The newly emerged leaves after artificial inoculation expressed symptoms such as curling, puckering and crinkling along with stunting of plant growth. Molecular characterization of the four virus isolates collected from various locations of Thrissur district viz., VKA1 VKA2, KAR1 and KOD4 and two isolates viz., VLNY1 and PKD1 collected from Vellayani, Thiruvanathapuram district and from Vithinasseri, Palakkad district, respectively were undertaken. The total genomic DNA from virus infected chilli leaf samples was isolated and subjected to PCR amplification of viral coat protein gene to confirm the presence of virus infection. PCR amplification of the isolated DNA was carried out using two Begomovirus specific degenerate (universal) primers, namely, AV494 / AC1048 (Wyatt and Brown, 1996) and Deng 540 / 541 (Deng et al., 1994). The amplicons of size 550 bp were obtained and were sequenced. The partial coat protein gene of size 550bp were also cloned into the vector pTZ57R/T and transformed into DH5α strain of Escherichia coli and the true recombinants with desirable insert were confirmed by colony PCR. The sequence data obtained in the study were subjected to in silico analysis to assess the diversity of the isolates. The nucleotide BLAST (BLASTn) analysis revealed more than 90 per cent sequence identity with Chilli leaf curl Vellanad virus isolate (Accession No. NC038442.1) from Vellanad region of Thiruvanathapuram district, Kerala. The translated nucleotide - protein BLAST (BLASTx) analysis of the viral sequences revealed more than 96 per cent sequence identity with Chilli leaf curl Vellanad virus coat protein sequence (accession no. YP_009506391.1). The coat protein sequences of all the six isolates were translated into corresponding amino acid sequence by ExPASy Translate tool and were used for further analysis and interpretation. The phylogenetic analysis revealed that, the isolates VKA2, KAR1 and KOD4 had very distinct sequence alignment when compared to other Chilli leaf curl virus isolates from India. The results indicated that, the three isolates viz., VKA2, KAR1 and KOD4 could be new strains of Chilli leaf curl virus infecting chilli. Three, possibly new strains of Chilli leaf curl virus infecting chilli have been identified and hence the study highlights the need for monitoring the emergence of new strains of plant viruses especially begomoviruses infecting solanaceous crops grown in Kerala. As this disease is one of the most important challenges to chilli cultivation, the information generated from the study could also be applied for the timely detection and effective disease management
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    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 V