Ph.D

The present study entitled “Endophyte mediated bio-intensive management of pests of cowpea Vigna unguiculata (L.) Walp.” was conducted in the Department of Agricultural Entomology, College of Agriculture, Vellayani, during the period 2020 2024. The primary objective of this research was the characterization and utilization of native endospore-forming endophytic bacteria (EEB) for the bio-intensive management of pests in cowpea. This approach aimed to provide an eco-friendly alternative to conventional pest control methods, contributing to sustainable agricultural practices. A total of 54 bacterial endophytes were isolated from vegetable and bush-type cowpea plants, of which 49 were confirmed as endospore-formers using the Schaeffer Fulton method. These endospore-forming bacteria underwent a series of bioassays to assess their pathogenicity and feeding deterrence against the chewing insect Spodopteralitura. The evaluation included direct exposure methods and exposure through primed cowpea seedlings in both choice and no-choice experimental conditions. Among the isolates tested, ten isolates viz., CPB 1a, CPB 2a, CPB 5a, CPB 8b, CPV 06, CPV 08, CPV 14, CPV 18, CPV 21, and CPV 32 were found effective in the in planta choice assessment. These isolates significantly reduced damage with lower damage intensity scores of 1.73 to 2.07 compared to unprimed controls (4.87) and other isolate-primed plants (2.47 - 4.93). The selected ten isolates were further subjected to in planta no-choice conditions, where the fitness of feeding insects was tested using relative growth rates and antifeedant activity of second and fourth instar S. litura. Three isolates, namely CPB 1a, CPV 18, and CPV 14, primed cowpea plants substantially reduced larval relative growth rates for both 2nd instar (0.35 mg/day, 0.46 mg/day, and 0.58 mg/day, respectively) and 4th instar (0.42 mg/day, 0.49 mg/day, and 0.59 mg/day, respectively). Similarly, the antifeedant activity, in terms of percentage weight gain reduction compared to larvae on control plants, was 80.53%, 73.58%, and 65.94% for the 2nd instar and 71%, 65.77%, and 54.44% for the 4th instar in these three isolates. The priming effects of endophytes were prominently observed in cowpea plants treated with CPB 1a and CPV 18 when exposed to S. litura. These plants showedenhanced and sustained production of antioxidants (SOD, CAT, POD, APX, GR) and defense enzymes (PAL, PPO, TP) compared to infested control plants. GC-MS analysis of plant volatiles revealed the release of 1-octen-3-ol and 9-octadecanoic acid, precursors of the plant defense signalling molecule jasmonic acid, in CPB 1a and CPV 18-primed plants, confirming their role in strengthening plant defense against S. litura. The efficacy of these bacterial isolates was evaluated against the sap-sucking insect Aphis craccivora through in-planta choice assays using the Aphid Quantity Ratio (AQR). Isolates CPB 1a, CPV 8, CPV 14, and CPV 18-primed plants demonstrated moderate resistance, with AQR values of 0.5, 0.42, 0.52, and 0.49, respectively, compared to unprimed control plants, which recorded an AQR value of 1.58, classifying them as highly susceptible. Furthermore, in-planta no-choice assays analyzing life table parameters revealed significant impacts of CPV 08- and CPV 18-primed plants on A. craccivora's biological and demographic traits. Relatively shorter oviposition periods of 6.27 and 7.25 days were observed in CPV 8 and CPV 18 treatments, respectively, whereas prolonged oviposition of 11.37 days was noticed in control plants. Consequently, the fecundity of aphids was reduced to 15.42 progeny per aphid for CPV 08 and 19.77 progeny per aphid for CPV 18, compared to 39.8 progeny per aphid in the control. Additionally, the intrinsic rate of population increase was lower for CPV 08 (1.24) and CPV 18 (1.26) than for the control (1.38). The higher production of antioxidants and defense enzymes, along with the release of volatile compounds such as methyl salicylate, betulinaldehyde, and trans-farnesol, confirmed the induction of plant defense responses by these isolates in cowpea plants challenged with A. craccivora. Promising bacterial isolates were identified through 16S rRNA gene sequencing. CPB 1a was identified as Bacillus velezensis, CPV 08 as Bacillus zanthoxyli, and CPV 18 as Bacillus subtilis, with sequence similarity percentages of 100%, 99.13%, and 99.57%, respectively. Comparative studies of potential endophytes such as B. velezensis CPB 1a and B. subtilis CPV 18 for S. litura along with other entomopathogens revealed that, upon seed priming, B. thuringiensis BTG4 and B. velezensis CPB 1a significantly reduced leaf damage to 17.29% and 20.56%, respectively. This was followed by B. subtilis CPV 18-treated plants, which recorded 30.18% leaf damage compared to77.24% in control plants exposed to feeding by S. litura. Additionally, plants primed with these bacteria exhibited enhanced resilience, maintaining better plant biometric characteristics such as increased fresh and dry weight under S. litura feeding conditions. Similarly, the comparative effectiveness of identified potential isolates against A. craccivora, such as B. zanthoxyli CPV 8 and B. subtilis CPV 18, alongside other entomopathogens, demonstrated that the priming effects of these microbes were most pronounced in B. zanthoxyli CPV 8 and B. subtilis CPV 18. These isolates effectively reduced the population build-up of A. craccivora to 29.66 and 32.66 aphids per plant, respectively, compared to 81.66 aphids per plant in the control. Furthermore, plants treated with these isolates showed significant improvements in plant biometric traits, such as improved fresh and dry weight under A. craccivora-infested conditions. All the effective isolates B. velezensis CPB 1a, B. zanthoxyli CPV 8, and B. subtilis CPV 18, including B. thuringiensis BTG4, were successfully reisolated from the primed plants during the observation period of two to four weeks after sowing ensure their effective colonisation as endophytes in cowpea. An in-vitro compatibility study was conducted to assess the potential of integrating these effective endophytic bacteria with entomopathogens. The results indicated that Beauveriabassiana NBAIR 05, Lecanicilliumlecanii NBAIR VL8, and B. subtilis CPV 18 were compatible with one another, enabling the creation of a microbial consortium. Pest incidence in cowpea plants treated with a microbial consortium, along with other promising endophytes and entomopathogens applied singly or in various combinations using different application techniques, was analyzed to assess their pest management potential under natural conditions in a pot culture experiment. Soil drenching with the microbial consortium provided early protection against A. craccivora, reducing populations by 29.45% to 31.28% up to five weeks after sowing (WAS). Foliar applications combining B. bassiana or L. lecanii with the consortium (treatments T18 and T19) yielded results comparable to chemical insecticides by 6 WAS after the first foliar application, with effects sustained until the final observation. Similarly, treatment T18 significantly reduced the incidence of S. litura after the first foliar application by 95%, matching the efficacy of chemical insecticides. T18(Consortium + B. bassiana) also demonstrated enhanced plant biometric characteristics, including increased plant height, number of leaves, number of pods, pod length, and pod yield, while effectively reducing the incidence of Maruca sp. and Riptortus sp. The microbial consortium consisting of Beauveriabassiana NBAIR-05, Lecanicilliumlecanii NBAIR VL-8, and B. subtilis CPV 18 developed in the current study along with foliar application of B. bassiana was proved to be effective in managing major insect pest of cowpea across different feeding guilds and have the potential to be integrated as one of the tactics in integrated pest management in cowpea.