The various isolates of the pathogen tomato wilt were collected from infected tomato plants in different places of Madurai district. The isolate FO (Maa)-5 was found highly virulent. This isolate was identified as Fusarium oxysporum f.sp. lycopersici based on sequencing of ITS region (Accession number: MZ043720). The pathogen was maintained on PDA slants and used for further studies.

Soil samples from rhizosphere region (3 cm) of healthy tomato plants were collected from 15 different locations of Madurai district, Tamil Nadu. The collected samples were dried and subjected to serial dilution (up to 10-4). The biocontrol agent was isolated using the selective medium of Trichoderma (TSM) and incubated for 7 days at 25±3oC (Awad et al., 2018). Later the putative colonies were purified by single hyphal tip method. General biochemical tests were done to confirm the biocontrol agent. Later these cultures were preserved in PDA slants for further studies.

Antifungal assay was carried out to evaluate the antimicrobial efficacy of the potential isolates of Trichoderma spp. against the pathogen.

• Dual culture assay

  The dual culture described by Yassin et al. (2021) was followed to test the antagonistic ability of Trichoderma species against the pathogen. Small block (5 mm disc) of Fusarium cut from the periphery was placed at one cm away from the periphery of the Petri dish previously poured with PDA. Similarly, the Trichoderma isolate was placed one cm away from the edge of the same Petri plate aseptically on the opposite end and plates were incubated at room temperature for 5 days. The experiment was replicated thrice and per cent growth inhibition was calculated by using the following formula,

  $I=\frac{(A-B)}{A}\times 100$

  Where A is mycelial growth of pathogen in control plate, B is mycelial growth of pathogen in treatment plate and I is the percent inhibition of mycelial growth.

• Effect of culture filtrates on inhibition of pathogen

  Mycelial plugs were taken from the freshly grown Trichoderma cultures and inoculated into conical flask containing fresh 100 ml potato dextrose broth and incubated for 7 days at 150 rpm at 28oC (You et al., 2016). Supernatant of the cultures were collected and centrifuged at 9000 rpm for 10 min. Then the cell free filtrates were sterilized through a 0.22 µm millipore filters and mixed with unsolidified PDA medium at 10% (v/v) concentration. Uninoculated PDB was added to PDA with same ratio for control. Mycelial disc of the pathogen was placed in all PDA plates and kept for incubation at 28oC for 5 days. Reduction in mycelial growth of the pathogen was measured and per cent inhibiton over control was arrived by the formula of Sreedevi et al. (2011),

  $I=\frac{(C-R)}{C}\times 100$

  Where,

  C - Mean linear growth of pathogen in control , R - Mean linear growth of pathogen in treatments

The potent cultures were inoculated in conical flask containing 100 ml of potato dextrose broth and incubated in shaker 150 rpm for 7 days. The mycelial mat was sieved and pierced into powder using liquid nitrogen (Liu et al., 2020). DNA extraction of virulent isolates was done by using the procedure of Zhang et al. (2010). Genomic DNA was isolated by using CTAB method. PCR amplification was carried out using universal primers – internal transcribed spacer ITS1 (5’TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). Polymerase chain reaction was performed in a reaction mixture of 50 µl with 35 cycles including 63 ng of genomic DNA, 50 pmol of each primer, 500 µM concentrations of dNTPs and 1.25 units of Taq DNA polymerase in an Eppendorf thermal Cycler. The PCR programme was performed with initial denaturation (95oC for 2 mins), followed by the repeated cycles of denaturation (94oC 1min), annealing (56oC for 30 sec) and extension (72oC for 1 min), and final extension of 72oC for 10 min. Amplicons were detected by 2% (w/v) agarose gel electrophoresis. Sequencing of purified PCR product was done at Eurofins Genomics India Pvt. Ltd. Bangalore.

ITS region of potential isolate was sequenced and BLAST searched with sequences in the NCBI, GenBank. Phylogenetic dendrogram was constructed by the neighbor-joining method in MEGA 10.0 software depending over multiple sequence alignment with an evolutionary distance of 0.05. The tree topologies were evaluated by performing analysis of 1000 data sets. The sequence was submitted to GenBank for obtaining accession number.

Mycelial disc from an actively growing colony of Trichoderma isolate was inoculated into fresh potato dextrose broth and incubated for seven days. The culture filtrates were collected by filtering using Whatmann no.1 filter paper followed by centrifugation at 9000 rpm for 15 min and finally the metabolites were extracted using ethyl acetate as solvent (Jantarach and Thanaboripat, 2010). Further concentration of extracts and elimination of solvents were done using rotary evaporator (Sharma et al., 2016).

The extract possessing high antimicrobial property has been subjected to GCMS analysis. The antibiotics, volatiles and secondary metabolites present in the sample were detected by injecting one microlitre of sample in Capillary Standard Non – Polar Column of GC - MS in which Helium was used as carrier gas. The analytical conditions were adjusted by following the procedures given by Yassin et al., (2020). The m/ z peaks representing mass to charge ratio, characteristic of the antimicrobial fractions were compared with those of the corresponding organic compounds in the NIST library (Manigundan et al., 2020).

Thin Layer Chromatography was performed to identify the presence of antifungal compounds in crude extract of Trichoderma isolate. TLC tank was filled with acetone and chloroform solvents in the ratio of 3:1 and sealed the tank immediately (Vivek et al.,2013). Desired size of TLC plate (60 F254, Merck, India) was taken and marked 0.5 cm above the bottom corner of plate. Samples were spotted at 1 cm distance and labelled. Spotted TLC plate was allowed to run in TLC tank. Then the plate was removed and visualized in laminar under UV fluorescence light (254 nm) and marked the dark purple fluorescence with pencil. The Rf value was calculated based on the distance covered (Fried and Sherma, 1982), Rf= Distance travelled by substance / Distance travelled by solvent

Statistical analysis were performed using analysis of variance (ANOVA) by SPSS software version 16 (SPSS.Chicago). The data were tabulated as mean of triplicates ± standard error and will be considered significant when the P < 0.05 and the means were compared by Duncan’s Multiple Range Test (DMRT).

The results of antifungal assay revealed that all the Trichoderma isolates possessed certain amount of antifungal activity both in dual and culture filtrate assays.

• Dual culture assay

  A total of fifteen isolates of Trichoderma spp. were isolated from the rhizosphere soil of healthy tomato plants. Among the isolates, Trichoderma isolate TA 12 was found superior against Fusarium oxysporum with 71% mycelial inhibition over control (Fig 1). The next best isolate was TA 2 with mycelial inhibition of 68.75%. Isolate TA 5 recorded minimum inhibition percentage of 46.22 (Table 1)

• Trichoderma culture filtrate assay against Fol

  The experimental results revealed that all the isolates inhibited the mycelial growth of pathogen at significant level.. Among the isolates tested, TA12 showed the maximum mycelial inhibition of 77.77% (Fig 2).This was followed by the isolate TA 2 (75.65%). The least

Table 1
Antifungal assay of Trichoderma isolates against Fusarium oxysporum fsp lycopersici

* Mean of three replications

Values with different superscripts are significantly differ from each other at p<0.05 Values in the parenthesis are arc sine transformed values

Fig. 1
In vitro dual confrontation assay of antagonistic Trichoderma isolates against Fusarium oxysporum f.sp. lycopersici

Fig. 2
Anti-mycotic potency of culture filtrate of Trichoderma spp (TA12) against Fusarium oxysporum f.sp. lycopersici

mycelial growth was observed in TA 5 at the rate of 52.20% (Table 2).

PCR of Trichoderma isolate with ITS-1 and ITS-4 primer pairs resulted in amplification of a fragment of size 636 bp (Fig 3).

Table 2
Antifungal assay of culture filtreates of Trichoderma isolates against Fusarium oxysporum f.sp. lycopersici

* Mean of three replications

Values with different superscripts are significantly differ from each other at p<0.05 Values in the parenthesis are arc sine transformed values

Fig. 3
The results of ITS amplification in potent

Fig. 4
Phylogenetic analysis of Trichoderma atroviride using neighbor joining method. The numbers over branches indicates bootstrap coefficient.

Phylogenetic analysis of the sequence (TA 12) with existing sequences in the NCBI database showed 99% sequence similarity with Trichoderma atroviride (Fig 4). The sequence was deposited in Genbank and obtained accession number (MW984524; Fig 5).

Fig. 5
GCMS chromatogram for detection of secondary metabolites in Trichoderma atroviride using ethyl acetate as solvent.

The extracts of T. atroviride were analyzed to determine its active chemical constituents. Active constituents of T. atroviride extract were demonstrated in Fig 5 and Table 3. The results showed that a numerous compounds produced by Trichoderma atroviride, possessing high antimycotic property.

The TLC plate with the sample was observed under UV laminar fluoroscence. The spot was resolved without any smear or streak pattern in TLC plate. In case of chitinase (developed in acetone:chloroform (3:1)) distinct spots were visualized under UV light (254 nm) with Rf value of 0.84 The distance travelled by the substance was 5.1 cm.

Table 3
Identification of secondary metabolites from Trichoderma atroviride through GCMS analysis