PG Thesis
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Item Biopriming and foliar apllication of biocontrol agents and endophytes for the management of major foliar fungal diseases of bush cowpea(Department of Plant Pathology, College of Agriculture , Vellayani, 2023-05-26) Aswathy ,V S.; Radhakrishnan, N VThe study entitled “Biopriming and foliar application of biocontrol agents and endophytes for the management of major foliar fungal diseases of bush cowpea” was conducted at College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram during 2020-2022. The objective was to develop best ecofriendly management practice involving biopriming, foliar application of endophytes and biocontrol agents for the control of major foliar fungal diseases in bush cowpea with special emphasis on Cercospora leaf spot and anthracnose. Symptomatology and etiology of anthracnose and Cercospora leaf spot were studied under field condition from different locations in Trivandrum district viz., Vellayani, Pappanchani, Venganoor, Balaramapuram, Nedumangad and Parassala. The pathogens were isolated and studied their morphological characteristics. Bush cowpea seeds were collected from five agro-ecological zones of Kerala viz., Northern, High range, Central, Special problem and Southern zones and the seeds were assessed for both externally and internally seed borne microflora. Percentage of infection was calculated by blotter method. The lowest percentage of infection was found in seed samples collected from Wayanad (16.7%) and the highest in Thrissur (38.2%). The diseased leaf samples showing anthracnose were collected from five locations of Thiruvananthapuram district and isolated Colletotrichum gloeosporioides from each location. Pathogenecity of isolated pathogen was proved by detached leaf assay and seedling assay. Vellayani isolate (C1) was found to be the most virulent pathogen and recorded lesion size of 4.33cm and 1.82cm in detached leaf assay and seedling assay on seventh day respectively. The isolate C1 was used for further studies. In vitro evaluation of biocontrol agents like Trichoderma asperellum T6 (KAU), Trichoderma koningiopsis (TRKR2), Trichoderma harzianum (TRMW2), Piriformospora indica (No. INBA 3202001787), Bacillus amyloliquiefaciens VLY 24, Bacillus velezensis (CBRE5), Bacillus amyloliquefaciens (CBSE5) and Pseudomonas fluorescens PN026 (KAU isolate) against Colletotrichum gloeosporioides and Cercospora sp. were carried out. Dual culture method of Colletotrichum gloeosporioides against biocontrol agent recorded highest percentage inhibition of mycelia by T. asperellum (64.76) followed by Trichoderma strain TRKR2 (52.63) which was on par with bacterial strain CBRE5 and least for Psuedomonas fluorescens. In vitro pathogen suppression by spore germination assay on Cercospora sp. by 145 biocontrol agents revealed that maximum inhibition of conidia germination was by T. asperellum (36.25 %) followed by Trichoderma strain TRKR2 and Trichoderma strain TRMW2 whereas least inhibition percentage was observed with Piriformospora indica (14.96). Peroxidase and polyphenol oxidase assay on bush cowpea pods and seeds showed that fungus infected tissues had relatively higher activity of these oxidase enzymes in comparison to healthy pods. Enzyme activities were higher in pods and seeds treated with Bacillus strain CBRE5, T. asperellum and Trichoderma strain TRKR2. Standardization of priming techniques revealed that soaking of seeds for 2h was found effective for Trichoderma asperellum, Bacillus velezensis (CBRE5) and Bacillus amyloliquiefaciens VLY 24 and 4h for Trichoderma strain TRKR2. The soaking duration is followed for the treatments in in vivo studies. Based on the in vitro studies, the best three treatments viz., Trichoderma asperellum, Trichoderma strain TRKR2 and Bacillus strain CBRE5 were taken for in vivo studies. In vivo studies on the effect of seed biopriming and spraying of biocontrol agent suspension at 4 leaf, 50 per cent, flowering and pod set stages revealed lowest disease severity of anthracnose was recorded for Trichoderma asperellum treated plants with disease suppression over control 41.74 per cent followed by carbendazim, Trichoderma strain TRKR2 and Bacillus strain CBRE5. In the case of Cercospora leaf spot Trichoderma asperellum treated plants shown highest disease suppression (63.47%) over control followed by carbendazim, Trichoderma strain TRKR2 and Bacillus strain CBRE5. Bacillus strain CBRE5 recorded least disease suppression over control in anthracnose and Cercospora leaf spot while comparing other treatments. Highest number of pods per plant (34.75), seeds per pod (15.25), plant height (46.05 cm) and yield (232.47 g) were shown by bacterial strain CBRE5 treated plants. Thus, the present study indicated that the seed biopriming for 2 h along with foliar application of Trichoderma asperellum suspension at 4 leaf , 50 per cent flowering and pod set stages was most effective treatment for the management of major foliar fungal diseases like anthracnose and Cercospora leaf spot of bush cowpea whereas seed biopriming for 2 h along with foliar application of Bacillus velezensis suspension at 4 leaf stage, 50 per cent flowering stage and pod set was the best treatment in plant growth promotion in vivo which could be used as an eco-friendly measure to produce safe to eat crop.Item Identification of sources of resistance and effective non chemical methods for the management for major viral disease of chilli in Kerala(Department of Plant Pathology, College of Agriculture Vellanikkara, 2023) Sujisha, C S; Sumiya, K VThe present study was undertaken in an endeavor to gain some understanding regarding the viral diseases affecting chilli and devising suitable management strategies to contain them. A purposive sampling survey conducted involving 3 districts of Kerala, namely Palakkad, Thrissur and Malappuram covering over 15 locations and 44 samples revealed symptoms descriptive of viral infection like curling, puckering, cupping and yellowing of leaves, dwarfing of plants and low fruit set in affected plants. To identify the etiological agent(s) associated with the symptoms, transmission studies were carried out. Whitefly transmission and graft transmission gave positive results whereas none of the samples that were sap transmitted exhibited symptoms indicating the presence of a geminivirus. Detection methods like Transmission Electron Microscopy (TEM) and Enzyme Linked Immunosorbant Assay (ELISA) were also carried out. Geminate particles were observed but no mosaic causing virus was detected in EM. ELISA performed on samples with mosaic like symptoms for the presence of potyvirus and CMV did not detect the presence of suspected virus in it. Hence, it was concluded that leaf curl disease was the only major disease affecting the crop in the area. Characterization of the virus (s) responsible for the leaf curl disease was attempted. DNA was isolated from seven representative samples after which PCR (Polymerase Chain Reaction) was performed with two sets of primers, degenerate (DENG) primer and a specific primer. The PCR amplified products of four representative isolates were sequenced and analysed for homology with sequences available in NCBI using BLAST software. Three of the isolates showed maximum nucleotide identity to the Pepper leaf curl Bangladesh virus isolate India/Coimbatore/Chilli/2008 segment DNA-A, complete sequence (HM007096.1) and the other isolate showed maximum identity with Chilli leaf curl virus-India isolate Raichur segment DNA-A, complete sequence (MK161454.1). A phylogenetic tree was constructed combining different geminiviruses reported from different crops to study the sequence similarity existing amongst them. The amino acid sequence of the coat protein gene of the isolate PKD-1 was predicted using the ExPASy translation tool available online. With the aim of identifying source (s) of resistance against chilli leaf curl disease in Kerala, two sets of screening experiments were carried out. In the initial field screening of 30 accessions, Violet Mulak, a local accession remained symptomless whereas Arka Lohit (IIHR) and Phule Jyothi (MPKVV) remained highly resistant. The molecular confirmation of the incidence of Chilli leaf curl virus (ChiLCV) in the field was done by performing PCR using primers specific to the CP gene region of the concerned virus. A pot experiment was set up to once again score the disease reaction of 48 accessions including those used in field screening along with a few more accessions. In this experiment, Phule Jyothi remained symptomless and Violet mulak turned out highly resistant whereas Arka Lohit remained HR. Twentysix random infected accessions were confirmed for the presence of ChiLCV in them. The three accessions that were adjudged highly resistant following the conclusions of the abovesaid two screening experiments were subjected to graft transmission. Fifty days old field exposed plants of the resistant sources were used as rootstocks in wedge grafting with scions from known susceptible variety. At 60 days of grafting, rootstocks and scions failed to amplify the primer indicating the absence of virus in them. From all these experiments, what could be inferred is that these accessions possessed true resistance against the virus up to 50 days after transplanting in the field. A management study to evaluate the efficacy of certain plant extracts and biocontrol agents in containing chilli leaf curl disease was undertaken the results of which indicate spraying the leaf extract of Azadirachta indica (10 %) or Mirabilis jalapa (10 %) as the best treatment. The only biocontrol agent with an effect on par with the best treatments was Bacillus subtilis applied as seed treatment (10g kg-1 seed) and foliar spray (20g l-1). All the four plant extracts did have a reducing effect on the disease severity with the least mean recorded in A. indica (10 %).