Molecular characterization of promising isolates of trichoderma spp. and their field evaluation against fusarium wilt of vegetable cowpea
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Date
2023-08-07
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Department of plant pathology, college of agriculture,Vellayani
Abstract
The present study entitled “Molecular characterization of promising isolates of Trichoderma spp. and their field evaluation against Fusarium wilt of vegetable cowpea” was conducted at the Department of Plant Pathology, College of Agriculture, Vellayani during 2020-2022. The study aimed at molecular characterization of potential Trichoderma isolates and their evaluation against Fusarium wilt of vegetable cowpea under field conditions. Five potent isolates of Trichoderma viz., TRMW-2, TRKR-2, TRPN-3, TRPN-11 and TRPN-17 were obtained from the previous study. The analysis of Trichoderma isolates for cell wall degrading enzymes like chitinase, protease and lipase revealed that TRPN-17 recorded the highest chitinase activity of 88.38 U ml-1 . Protease activity was found to be higher in TRPN-11 (165.86 U ml-1 ) whereas highest lipase activity was exhibited by TRPN-3 (4.86 U ml-1 ). The compatibility test among the five isolates showed that all the combinations exhibited antibiosis, lysis and overgrowth except the isolates TRPN-3 and TRPN-11 which displayed only overgrowth. A mild level of antibiosis and overgrowth was observed in the combination of the isolates TRKR-2 and TRPN-17. Assessment of competitive saprophytic ability of the Trichoderma isolates showed that per cent colonization of TRKR-2 was found to be higher (67.50 %) than other isolates and was followed by TRPN-3 and TRPN-17 (56.25 %) with no significant difference among all five isolates. In vitro screening of Trichoderma isolates by dual culture technique revealed that the per cent inhibition of TRPN-17 (63.71 %) was found to be higher, followed by TRKR-2 (58.76 %) and TRPN-3 (58.21 %). Based on the in vitro studies the effective isolates TRKR-2, TRPN-17, TRPN-3, consortia of isolates TRKR-2 and TRPN-17 and consortia of isolates TRPN-3 and TRPN-17 were selected for the in vivo evaluation against Fusarium wilt of vegetable cowpea. Molecular characterization of the isolates were done using the primers for Internal Transcribed Spacer (ITS) regions 1 and 4. All the five isolates produced a single, intact amplicon of size 600 bp approximately. The nucleotide sequences of all the ITS PCR products obtained from the five isolates were confirmed through bidirectional sequencing using the Sangers DNA sequencing. The best match of the amplified sequences was obtained from the GenBank database using NCBI-BLAST and their phylogenetic positions were explored along with the referral sequences from BLAST analysis. The oligonucleotide set of genus-specific primer TvP was used for genus-specific PCR analysis and an amplicon of approximately 245 bp was obtained. The DNA barcoding of the amplicon confirmed the genus identity of all the isolates as Trichoderma. The species-specific identification tried with translational elongation factor- 1 (tef1) and RNA polymerase II (rpb2) gene specific primers did not yield any amplicon indicating that the isolates did not belong to the species T. virens, T. longibrachiatum, T. harzianum and T. asperellum. Application of talc-based formulations of the isolates TRKR-2, TRPN-3, KAU strain and consortium of TRPN-3 and TRPN-17 as seed treatment @ of 20 g kg-1 of seed followed by soil drenching @ 2 per cent at 20, 40 and 60 DAS effectively reduced the incidence of Fusarium wilt of vegetable cowpea in pot culture study. The application of isolate TRPN-3 produced highest pod number (19) in pot culture experiment and also yield per plant (369.57 g) on par with the yield per plant produced by consortia application of TRPN-3 and TRPN-17 (344.52 g). Under field conditions, the disease was most effectively reduced by isolates TRKR-2, TRPN-3, KAU strain and consortia of TRKR-2 and TRPN-17. Lengthy pods (48.16 cm) and highest number of seeds per pod (21.73) were recorded by the application of consortium of TRPN-3 and TRPN-17 whereas application of TRPN-3 resulted in highest yield (1172.63 g/plant), number of pods per plant (19) and reduced days of flowering. Enumeration of population of Trichoderma spp. from soil in different treatments at 90 DAS revealed that, highest population was in treatment with isolate TRPN 3 under pot culture (7 ×104 cfu g-1 ) and field conditions (4 ×104 cfu g-1 ) . In the present study, Trichoderma isolates TRKR-2, TRPN-3, and consortium of TRPN-3 and TRPN-17 were found effective against Fusarium wilt of vegetable cowpea under field conditions. The genus of the isolates under study was identified as Trichoderma. The efficacy of the isolates against other soil-borne diseases of vegetable cowpea and species level identification need to be explored