MSc

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

M.SC

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