1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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    Development of resistance against banana bract mosaic virus in musa spp. var. grand naine using small interfering RNA (siRNA)
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Jadhav Pritam Ramesh; Soni, K B
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    Exploitation of abiotic stress tolerant strains of trichoderma spp. for the management of soil borne fungal pathogens
    (Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Stella Doncy, P P; Reshmy Vijayaraghavan
    Soil borne phytopathogenic fungi are known to cause severe yield loss of several crops. To mitigate these crop ailments, farmers mostly rely on chemical methods of disease control such as fungicides and other pesticides which are deleterious to the environment. To date, biological control is an ecofriendly approach for the effective management of crop diseases. The fungi belonging to the genus Trichoderma are one among the most exploited biocontrol agents in the field of agriculture. However, the performance of Trichoderma spp. gets reduced when it is exposed to abiotic stressed conditions such as drought, high temperature, salinity, acidity and fungicides. Hence, this study was proposed to identify and exploit stress tolerant isolates of Trichoderma spp. with antagonistic potential in Kerala. Intensive soil sampling surveys were conducted across different stressed ecosystems of Kerala viz., Palakkad, Alappuzha, Vytilla, Kumarakom, Wayanad and Thrissur for the isolation and enumeration of native Trichoderma spp. A total of 24 isolates were obtained from 52 soil samples collected from different locations. Based on the number of Trichoderma spp. obtained from each district, they were serially numbered and abbreviated according to the name of the location. Accordingly, PAT 1 to PAT 6 represents number of isolates of Trichoderma spp. from Palakkad district, ALT 1 to ALT 3 from Alappuzha, VYT 1 and VYT 2 from Ernakulam district, KUT 1 from Kottayam district, WAT 1 to WAT 8 from Wayanad and THT 1 to THT 4 from Thrissur district. Cultural and morphological identification of these isolates were carried out under in vitro conditions. Isolates of Trichoderma spp. were subjected to in vitro screening for abiotic stress tolerance such as high temperature, drought, acidity, salinity and also to test their sensitivity towards copper fungicides. The isolates PAT6 and WAT2 were found as thermotolerant, VYT2 and ALT 1 as drought tolerant, ALT 3 and ALT 1 as acid tolerant and saline tolerant and the isolates ALT1, ALT3 and PAT 1 as copper fungicide tolerant. The selected six isolates were further subjected to biochemical tests and the study showed that the isolates VYT 2, ALT 3 and ALT 1 showed highest cellulase, β- 1, 3 glucanase and protease activity. Likewise, isolates PAT 1, ALT 1 and ALT 3 were found as best producers of ACC deaminase and PAT 1 and ALT 1 as the best cytokinin producer. The best performing isolates (ALT 1, ALT 3, PAT 1, PAT 6 and VYT 2) after enzyme study were subjected to dual culture experiment with five major soil borne pathogens to test their antagonistic potential. The isolates ALT 3, ALT 1, PAT 6 and PAT 1 showed more than 70 per cent inhibition of R. solani whereas, isolates ALT1 and VYT 2 showed only 57.78 and 55 per cent inhibition of S. rolfsii respectively. However, no significant difference was noticed among the isolates when grown against F. solani. Cent per cent inhibition of P. aphanidermatum was noticed with Trichoderma isolates PAT 1 and ALT 1. All five isolates showed 100 per cent inhibition on the growth of pathogen, P. capsici. Among the five, four isolates viz., ALT 1, ALT 3, PAT 1 and PAT 6 with best antagonistic potential were subjected to molecular characterization at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram and the isolates showed homology with the nucleotide sequence of Trichoderma asperellum. A pot culture experiment was laid with the selected isolates viz., ALT 1, ALT 3, PAT 1 and PAT 6 to test the growth promotion of cowpea and biocontrol efficacy against Rhizoctonia solani. It was observed that the isolate PAT 6 coated seeds showed 100 per cent germination and also recorded better biometric characters and yield. Moreover, the lowest per cent disease incidence of 11.11 per cent was only recorded with both the isolates ALT 3 and PAT 6. Thus, the study has enlightened our knowledge on the existence of abiotic stress tolerant isolates of T. asperellum which can be employed in future for the biocontrol of soil borne pathogens in such conditions.
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    Impact of pre-storage seed invigoration in ash gourd (benincasa hispida (thunb.) cogn.)
    (Department of Seed Science and Technology, College of Horticulture, Vellanikkara, 2018) Athmaja, S; Rose Mary Francies
    A study to elucidate the effect of seed invigoration on viability and quality of seeds in ash gourd variety KAU Local was conducted at College of Horticulture, Vellanikkara, Thrissur, during 2016-2018. The impact of seed invigoration on seed viability and seed quality parameters under ambient (S1) and refrigerated storage (S2) was assessed following a completely randomized design with seven invigoration treatments (I1 to I7) and three replications. Seeds were separately invigorated using CaCl2 (50 m M) for 12h (I1), CaCl2 (50 mM) for 24h (I2), kinetin (10 ppm) for 12h (I3), kinetin (10 ppm) for 24h (I4), KH2PO4 (10-1 M) for 24h (I5), Pseudomonas fluorescens (1x106 cfu.ml-1) for 12h (I6). Untreated seeds (I7) served as control. Both treated and untreated seeds were dried to < 8 per cent moisture content and packed in polythene bags (700 gauge). The seed quality parameters were recorded immediately after treatment and subsequently at monthly intervals for a period of 10 months, while, germination of stored seeds was assessed up to 14 months after storage (MAS). At bimonthly intervals, quantification of lipid peroxidation, sugar and amino acids leached out from the seeds and the seed micro flora infection was also done. Seed quality during storage and seed longevity were found to be significantly influenced by storage environment, invigoration treatment and their interaction throughout the storage period. The results revealed that germination and other seed quality parameters such as germination index, coefficient of velocity of germination, energy of germination, vigour indices I and II, in both treated and untreated seeds decreased progressively over the storage period. However, there was an increase in mean time to germination, time taken for 50 per cent germination, allometric index, electrical conductivity of seed leachate, seed infection per cent, leachate of sugar, amino acid and lipid peroxidation, towards the end of storage period. Germination of seeds stored under the refrigerated storage was lower than that under ambient storage in the initial storage period (upto 3 MAS). Henceforth, refrigerated seeds exhibited significant superior germination than that under ambient storage till the end of storage period (14 MAS). Germination of seeds under refrigeration was retained above 60 per cent (the minimum seed certification standards required for ash gourd) for 13 MAS compared to 5 MAS in ambient stored seeds. The study thus revealed that irrespective of seed invigoration treatments, to prolong seed longevity and maintain seed quality, storing seeds under refrigeration is advantageous over ambient storage. Irrespective of storage environment, priming induced early germination. The seed quality parameters of the invigorated seeds before storage were found to be superior to untreated seeds. The invigorated seeds had also exhibited a germination per cent above 80 at 1 MAS, while, the germination in untreated control (I7) during the corresponding period was below the MSCS. Seeds invigoration with calcium chloride for 12h (I1) and 24h (I2) recorded significantly high germination and other seed quality parameters during the storage period of ten months. Owing to the significant superiority of seeds invigorated with I1 (CaCl2 50mM 12h) and I2 (CaCl2 50mM 24h) with respect to germination in the initial period of storage (up to 4 MAS), superior seed qualities during storage as well as retention of germination above MSCS for 8 MAS, seed invigoration with CaCl2 50mM before storage can be advocated to help retain seed qualities and prolonging seed longevity during storage. The interaction between storage condition and invigoration treatment on germination and other seed indices pointed out that it was most advantageous to treat seeds with CaCl2 50mM for 12h (I1) before storing under ambient conditions. If provision for refrigerated storage is available, bio-priming with Pf 1x10-6 cfu.ml-1 for 12h (S2I6) or priming with CaCl2 50mM for 24h (I2), kinetin 10 ppm for 12h (I3) or kinetin 10 ppm for 24h (I4) or KH2PO4 10-1 M for 24h (I5) would be most advantageous. Analysis of the impact of pre-storage seed invigoration treatment on seed longevity subsequent to retrieval of seeds from refrigerated storage revealed that, irrespective of the storage period under refrigeration, the seeds were found to retain viability above MSCS for a minimum period of one month after retrieval from refrigerated storage. Viability retention of invigorated and untreated seeds during further periods of thawing was unpredictable. It was also evident that none of the treatments could help retain seed viability above MSCS for five months after retrieval from refrigeration. Results also revealed that seed invigoration with CaCl2 50mM 12h (I1) is advantageous, if one or two months of ambient storage after retrieval from cold storage is unavoidable. Hence, considering the impact of storage environment, invigoration treatment and their interaction on seed longevity and quality, as well as their influence on seed longevity during thawing, it can be summarised that seed invigoration with CaCl2 50mM for 12h (I1) or 24h (I2) would be beneficial.
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    Tagging of phytophthor pod rot disease resistance gene in cocoa (Theobroma cacao L.) using ISSR markers
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Jeughale Kishor, Pundlik; Minimol, J S
    Cocoa (Theobroma cacao L.) known as ‘Chocolate tree’, is a major cash crop in tropical countries. Cocoa production is seriously affected by pod rot diseases caused by many Phytophthora species. Among these, the pod rot caused by Phytophthora palmivora has been reported in India. Yearly losses to the cocoa growers around the world from Phytophthora diseases were assessed at 30 per cent of the total yield loss. Disease resistance can be scored using a number of morphological and physiological characters. However, the morpho-physiological characters greatly depend upon the environment which ultimately affect the experimental data. Hence, confirmation of transfer of genes by tagging with the help of a strong tool is of utmost importance in crop breeding. Molecular markers such as Inter simple sequence repeats (ISSRs) have already proven to be a good tool to detect and tag the genes of interest and will help to reduce the breeding cycle. In this context, the present study was taken up with an objective to develop a strategy to tag gene(s) for Phytophthora pod rot (PPR) resistance in cocoa using ISSR markers. Morphological characterization of 28 hybrid progenies of SVI 1.26 × PII 12.11 was carried out by recording five pod and bean characters. High variability was observed for characters viz., pod weight, pod length and breadth, wet bean weight per pod and single dry bean weight among the progeny of the same cross. Detached pod inoculation technique was adopted to classify the hybrids into resistant and susceptible ones. The wide variability was also recorded for disease reaction among the progenies. Based on the resistance score, three resistant and three susceptible hybrids were selected from the segregating progeny. The eight accessions were screened with fifty ISSR and 15 SSR primers to observe polymorphism between resistance and susceptible genotypes. Polymorphism was observed in 11 ISSR primers and from these, six primers viz., UBC 810, UBC 826, UBC 827, UBC 857, Oligo ISSR 04 and Oligo ISSR 08 were eluted and cloned. Plasmid DNA was isolated from clones and sequenced. Though various SSR primer sets screened were found to yield polymorphism, none of them was successful to give a clear distinction among the resistant and susceptible hybrids. This may be due to the fact that, Quantitative trait loci (QTLs) associated with these reported SSR primers may be absent in the genotypes considered for the study. BLASTn analysis specific to plants was done for all six sequences. Upon analysis, Oligo ISSR 04561 had shown 98 per cent identity with Predicted: T. cacao histidine-containing phosphotransfer protein 1 (HPt). HPts play an important role in propagating cytokinin signal transduction. Cytokinins are instrumental in mediating disease resistance by generating a green island around the infection zones, exhibiting delayed leaf senescence and upregulating the expression of the pathogenesis related (PR) gene/s. In addition to this, the auxin-cytokinin antagonism that occurs as part of a complex hormonal interplay, exerts a critical influence on the core SA-JA/ET plant immunity pathways. The BLASTn analysis of marker UBC 810877 resulted in 99 per cent sequence identity with Predicted: T. cacao phospholipid: diacylglycerol acyltransferase (PDAT) 1 mRNA. This protein regulates the synthesis of triacylglycerol, which is a building component of oils in the plant. Accumulation of oil content in plant cells could impart resistance against the pathogen. UBC 827571 had shown 73 per cent sequence identity with T. cacao clone TCC_BA049P20 complete sequence and it is reported to be QTL rich region associated with different traits of T. cacao. Moreover, ISSR markers UBC 810877, UBC 826535 and UBC 857839 are located on chromosome nine, six and four respectively as inferred from NCBI Genome Data Viewer tool through BLASTn annotations. These markers are found to be located in PPR resistance regions rich in defense associated genes. Further validation and exploitation of polymorphic amplicons or markers in response to PPR would be required. The linkage of Oligo ISSR 04561 and UBC 810877 with HPts and PDAT correspondingly have to be validated to elucidate the association and role of cytokinin and triacylglycerol with PPR disease resistance. If validated, UBC 810877, UBC 826535 and UBC 857839 and Oligo ISSR 04561 could be employed as a marker in PPR resistance breeding programmes in cocoa.
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    Refinement of macro-propagation technique for mass-multiplication of aloe (Aloe vera Burm.f.)
    (Department of Plantation Crops and Spices, College of Agriculture, Vellayani, 2017) Saranya, K S; Jessykutty, P C