MSc

The research work entitled “Management of Blackeye cowpea mosaic virus using natural products from botanicals and the fungal root endophyte Piriformospora indica” was conducted during the period of 2017-2019 at the Department of Plant Pathology, College of Agriculture, Vellayani to utilize natural products from potential antiviral plant extracts and the root endophyte P. indica for the management of the virus.
Blackeye cowpea mosaic virus was maintained in systemic host, vegetable cowpea (var. Sharika) and the local lesion host, C. amaranticolor by sap transmission with phosphate buffer. Symptoms produced by BlCMV in emerging trifoliate leaves include vein clearing, vein banding and blistering, later it developed mosaic, leaf size reduction, leaf distortion, flower malformation, reduced pod length and stunting. It produced yellow chlorotic local lesion progressed to necrotic lesion in inoculated C. amaranticolor leaves. Beneficial fungal root endophyte, P. indica was maintained in PDA and PDB by frequent sub-culturing as when required. The fungi covered petri plate (9 cm) with PDA in nine days and in broth, 16 days for complete mycelial mat formation.
Serological detection of the virus was done by DAC-ELISA and DIBA. Polyclonal antibodies of CABMV and PVY reacted positively with BlCMV infected leaves confirming the etiology of black-eye cowpea mosaic disease.
Standardization of co-cultivation for P. indica with cowpea and C. amaranticolor were conducted in both in vitro and in vivo. The mycelium of P. indica was observed to colonize inside cowpea roots within 5 DAC. Chlamydospores were seen on root surface at 7 DAC and chlamydospores inside the roots at 14 DAC in both in vitro and in vivo methods. P. indica took much longer time for the colonization in C. amaranticolor roots. Mycelium was observed in roots at one week after co-cultivation (WAC) whereas chlamydospores at two WAC and chlamydospores inside the roots at three WAC in both in vitro and in vivo study.
Ten plants with reported potential antiviral principles were screened against BlCMV in local lesion host, C. amaranticolor at 0.25, 0.5 and 1 per cent with pre- and post- inoculation method. P. niruri recorded more than 80 per cent inhibition of BlCMV local lesion in C. amaranticolor even at one per cent concentration by pre-inoculation method. B. diffusa inhibits 75 per cent of local lesions in C. amaranticolor at one per cent concentration in pre-treated plants.
P. indica colonized in roots of C. amaranticolor with primed leaves could significantly reduce local lesion caused by BlCMV by 68 per cent over control. Water diffusible exudate from P. indica collected at different intervals was evaluated against BlCMV in local lesion host, C. amaranticolor with pre- and post- inoculation. Pi-WDE collected after three day mycelium incubation in sterile water was found to inhibit more than 75 per cent of local lesion over control.
Bioassay in cowpea plants with best antiviral principles (P. niruri and B. diffusa), P. indica-priming and Pi-WDE were evaluated against BlCMV. P. indica-primed plants were less vulnerable to BlCMV infection followed by P. niruri pre-treated plants. BlCMV inoculated in P. indica-colonized plants enhanced root (22.7 g plant-1) and shoot (144.8 g plant-1) biomass which was significantly higher than the healthy plants. P. niruri pre-treated plants (root – 13.7 g plant-1 and shoot 113 g plant-1) were similar in biomass with healthy plants (root – 18.3 g plant-1 and shoot 124.3 g plant-1) while virus inoculated plants remarkably reduced root (4.6 g plant-1) and shoot biomass (12.4 g plant-1).
Partial characterization of antiviral principles from best leaf extracts viz., P. niruri and B. diffusa against BlCMV in C. amaranticolor was done. Leaf extracts of both the AVPs were thermostable at 121°C for 20 min and partially photo sensitive (exposed natural light at 9 h). Proteins were the active principles found in the both the AVPs and had a molecular size of more than 1 kDa.
Biochemical changes in host-pathogen interactions of pre-treated AVPs, P. indica-colonized and Pi-WDE treated cowpea plants were studied. Protein content of the virus inoculated plants increased initially up to 15 DAI, thereafter started to decline. Phenol content in healthy and virus inoculated plants increased with age of plants. P. niruri and B. diffusa treated cowpea plants induced rapid accumulation of phenol up to 10 DAI, later the induction was gradual. All the treated plants with the virus inoculation differs in PO activity, and its enhanced activity was in P. indica-primed and P. niruri treated plants at 10 DAI, later PO activity gradually declined in P. indica-primed while increased in P. niruri applied plants. Higher activity of PPO was observed in virus inoculated plants at 10 DAI, and the activity of PPO was significantly different with various treatments. Activity was enhanced during initial period of virus inoculation (upto 15 DAI), then the activity was reduced. Studies on PAL activity in cowpea plants treated with biotic agents and challenged with BlCMV inoculation resulted in the accumulation of PAL within 5 DAI, and there after decreased with age of plants except in Pi-WDE treated cowpea plants which enhanced PAL with age of plants.
Protein profile study (SDS-PAGE) of cowpea plants revealed the induction of many proteins in response to the different treatments. Induction of PR-proteins was more in P. indica-primed plants with and without virus inoculation during the initial phase of the tripartite interaction. The intensity of band having molecular weight 29.4 kDa was high in all the treatments except for the virus inoculated plants. An extra band of 24.8 kDa was observed in P. indica-colonized virus inoculated plants. P. niruri constantly maintained high level of 34 kDa protein in treated plants whereas, in BlCMV treated plants, none of the PR-proteins were induced at different intervals of the experiment.