Strain improvement of Trichoderma spp.for management of foot rot of black pepper caused by phytophthora capsici

Abstract

The research work entitled ‘Strain improvement of Trichoderma spp. for management of foot rot of black pepper caused by Phytophthora capsici’ was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2022-2025 with an objective of enhancing the antagonistic potential and growth promotion of Trichoderma strains by protoplast fusion and mutagenesis; studying the biochemical and molecular mechanisms underlying antagonism and evaluation of its efficacy for managing P. capsici. Five promising isolates of Trichoderma spp.: TRMW-2, TRKR-2, TRPN-3, TRPN-11 and TRPN-17, previously collected from different agroclimatic zones of Kerala, along with Trichoderma asperellum, the reference culture from KAU were used as the parental isolates. Molecular characterization using species-specific primers (TEF1 and RPB2) and multi-gene phylogenetic analysis identified TRMW-2, TRKR-2, TRPN 3, TRPN-11 and TRPN-17 as Trichoderma brevicompactum [Tb], Trichoderma koningiopsis [Tk], Trichoderma pholiotae (PN3) [Tp (PN3)], T. pholiotae (PN11) [Tp(PN11)] and Trichoderma guizhouense [Tg], respectively. A total of 15 protoplast fusion combinations were performed using the six parental isolates and 30 fusants with a colony diameter exceeding 8.5 cm at 48 h were screened. Among these, the 10 most effective fusants, exhibiting a growth inhibition rate of 85.73% to 92.13% against P. capsici were selected based on their mycelial growth suppression. Simple sequence repeat (SSR) marker analysis was performed using 15 primers to determine whether the selected fusants were interspecific. Similar amplification patterns were observed between the fusants Ta × Tk-1, Tk × Tp (PN3)-1, Ta × Tp (PN3)-1 and Ta × Tp (PN3)-5 and their respective parental strains for the markers PRB ATC, TV1, KON 700, TA4, TA9 and TH5, confirming them as interspecific fusants. Strain improvement through UV irradiation (5, 10 and 15 min) exhibited an inverse correlation between exposure time and the number of regenerated colonies. Among 140 mutants screened, 30 with a colony diameter greater than 7.5 cm at 48 h were selected, which were further narrowed down to 10 with the highest inhibition rates of 80.87% - 84.47% against P. capsici. Similarly, gamma irradiation (150, 200, 250 and 300 Gy) showed a negative correlation between colony regeneration and radiation dose. Among 180 mutants screened, 10 superior mutants with a growth inhibition of 81.67% - 90.40% were selected. From the 30 improved Trichoderma spp. strains obtained through three distinct strain improvement techniques, the 10 most effective strains exhibiting significant pathogen inhibition (83.73% to 93.60%) were identified and subjected to genetic stability assessment via ten successive subculturing rounds, followed by dual culture assay against P. capsici. The five most promising Trichoderma strains: T. asperellum × T. koningiopsis-1 [Ta × Tk-1], T. koningiopsis ×T. pholiotae (PN3)-1 [Tk × Tp(PN3)-1], T. pholiotae (PN11) fifth UV mutant colony irradiated for 5 min [Tp(PN11).5m5], T. pholiotae sixth gamma mutant colony irradiated at 200 Gy [Tp(PN3).200m6] and T. asperellum first gamma mutant colony irradiated at 250 Gy [Ta.250m1] with superior mycelial growth suppression (83% to 91.27%) were selected. Culture filtrate assay at 50% concentration revealed maximum mycelial inhibition of P. capsici by Tk × Tp(PN3)-1 (98.33%) and Tp(PN11).5m5 (97.92%). Additionally, Ta × Tk-1 (76.13%) and Tp(PN3).200m6 (72.25%) exhibited significant volatile metabolite activity against P. capsici, outperforming T. asperellum (59.88%). The potential of the selected Trichoderma spp. strains to produce lytic enzymes, including β-1,3 glucanase, protease, chitinase and lipase was evaluated. Ta × Tk-1, Tk × Tp(PN3)-1, Tp(PN11).5m5 and Ta.250m1 exhibited higher enzymatic activity than T. asperellum, signifying enhanced antagonistic potential. Growth promoting attributes, including IAA (174.41 μg ml-1) and siderophore production (77.09%) were highest in the interspecific fusant Ta × Tk-1, while phosphate solubilization was most pronounced in the gamma mutant Ta.250m1(1326.90 μg ml-1). Cultural and morphological characterization of the improved strains revealed distinct variations in conidiation, pigmentation, hyphal width and conidial and phialide size compared to their parental isolates. Notably, Tp(PN3).200m6 exhibited deep yellow pigmentation, while Ta × Tk-1 and Ta.250m1 showed dense sporulation. Pot culture experiments were conducted to evaluate the biocontrol efficacy and plant growth promoting potential of five promising Trichoderma spp. along with T. asperellum in black pepper var. Panniyur - 1. Biometric characters (plant height, number of leaves, nodes and internodal length) were highest in plants treated with Ta × Tk-1, followed by Ta.250m1. Upon challenge inoculation with P. capsici, lesion size was smallest in plants treated with Tk × Tp(PN3)-1 and Tp(PN11).5m5, corresponding to a percent disease index of 20.19% and 35.44% respectively, compared to 83.57% in control plants. Elevated activity of defense related enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, catalase and superoxide dismutase) was recorded in plants treated with Tk × Tp(PN3)-1, Tp(PN11).5m5 and Ta × Tk-1. The relative expression of antagonism associated genes from different functional groups; chitinase, β-1,3-endoglucanase, subtilisin-like protease and terpene synthase were analyzed in both improved Trichoderma strains and their respective parental isolates using semi-quantitative RT-PCR. Expression levels were significantly upregulated in Tk × Tp(PN3)-1 and Tp(PN11).5m5 across all four genes compared to the parental isolates. This study identified three improved Trichoderma strains; Ta × Tk-1, Tk × Tp(PN3)-1 and Tp(PN11).5m5 that exhibited enhanced antagonistic potential, biochemical characteristics, plant growth promoting traits and defense enzyme activities, surpassing the reference strain T. asperellum. Therefore, following multi-locational field trials across different crops and comprehensive toxicological studies, these strains could be released as efficient biocontrol agents in the future.