MSc

A study on “Strain improvement of Trichoderma spp. by protoplast fusion” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during the year 2018-2020, with the objective of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of improving the screened strains of Trichoderma Trichoderma Trichoderma spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for spp. by protoplast fusion for increasing the antagonistic ability and related traits against soil borne pathogens.
A survey was conducted in five agro-climatic zones of Kerala viz., Northern Zone, Central Zone, High Range Zone, Problem Area Zone and Southern Zone for collection of soil samples especially from forest soils. The collected soil samples were assessed for the population of Trichoderma spp. A total of 31 Trichoderma spp. isolates were obtained from the soil samples collected from five agro-climatic zones. Majority of the isolates were obtained from soils with pH of 6 to 7. Isolate TRMW2, TREN1, TREZ1, TREZ2, TREZ3, TRRN1, TRRN2, TRKR1, TRPN3, TRPN7, TRPN10 and TRPN18 exhibited full growth at four days after inoculation (DAI). The isolated Trichoderma spp. differed in growth rate and colony characters like colour of mycelium, texture of colony and sporulation pattern.
Isolates of Trichoderma spp. from different zones exhibited in vitro inhibition against soil borne pathogens such as Pythium aphanidermatum and Rhizoctonia solani. Majority of the isolates displayed high inhibition per cent compared to KAU strain of Trichoderma sp. TRRN1, TRRN2, TRPN3, TRPN7, TRPN11, TRPN15 and TRKR2 isolates exhibited complete inhibition of P. aphanidermatum in dual culture experiment; whereas TREN1, TRMW2, TREZ1, TREZ2, TRKM1, TRPN7, TRPN9, TRPN14, TRPN15, TRPN17, TRPN18 and TRKR2 isolates exhibited complete inhibition of R. solani. Trichoderma isolates such as TRPN7, TRPN15 and TRKR2 exhibited complete inhibition against both the pathogens. Antagonistic properties viz., antibiosis, lysis and overgrowth of Trichoderma isolates against P. aphanidermatum and R. solani were observed. During the antagonist-pathogen interaction, isolates TRSN1, TRSN2, TRPN10, TRPN14, TRPN15, TRPN17 and TRPN18 exhibited high levels of antibiosis. Most of the isolates caused lysis of mycelium of the pathogens which resulted in formation of clear zones in dual culture. Overgrowth of the
antagonist was another prominent antagonistic property observed in the majority of the isolates.
Based on the antagonistic properties, the Trichoderma isolates viz., TRSN1, TRMW2 and TRPN14 were selected for the protoplast fusion. During the protoplast isolation, the maximum number of protoplasts was obtained after 2 h of incubation of mycelia of parental isolates with the lytic enzyme. Protoplast fusion was carried out between the selected isolates (TRSN1 x TRPN14, TRSN1 x TRMW2, and TRPN14 x TRMW2) in the presence of poly ethylene glycol (PEG 6000). Three protoplast fusants were selected using carbendazim-amended PDA medium. The protoplast fusants displayed fast growth on PDA medium and completely covered the Petri dish at 5th of growth. The colony characters of fusants varied from light to dark green mycelium with fluffy growth and scattered to circular green heavy sporulation.
In vitro screening of protoplast fusants against P. aphanidermatum and R. solani revealed that highest inhibition against P. aphanidermatum was observed with fusant 3 (84.4%) followed by fusant 2 (74.44%). Highest inhibition against R. solani was observed with fusant 2 (100%) followed by fusant 3 (70.30%). Antagonistic properties viz., antibiosis, lysis and overgrowth were observed in the protoplast fusants. Among the three protoplast fusants, fusant 1 exhibited all the antagonistic properties against both the pathogens with heavy sporulation.
Thus, the present study has thrown light in understanding the potential of protoplast fusion in evolving improved strains of Trichoderma spp. Protoplast fusion enhanced sporulation in fusants compared to the parents. Further studies need to be conducted for the biochemical and molecular characterisation of parental isolates and fusants. The parents and protoplast fusants also have to be evaluated for their in vivo efficacy against soil borne pathogens in major crops of Kerala.