2.1 Pathogen isolation

Pathogen was isolated from the infected tomato plants on the Petri-plates containing potato dextrose agar (PDA) media (Zehra et al., 2017a). After inoculation, the plates were incubated at 25–27 °C for 5–7 days.

2.2 Isolation of Trichoderma spp.

For isolating Trichoderma spp., soil samples were collected from tomato rhizosphere located at different places of the agricultural field of Banaras Hindu University campus, Varanasi (25°20′N latitude and 83°01′E longitude), Uttar Pradesh, India and were kept at 4 °C in the laboratory. Serial dilution method was adopted for isolating different Trichoderma spp. (Zehra et al., 2017b).

2.3 Fungal DNA extraction and ITS sequencing

Genomic DNA was extracted from the 1 week old from the fungal isolate following the method of Doyle and Doyle (1987). The universal primers ITS1/ITS4 were used to amplify ITS-rDNA using polymerase chain reaction (PCR) (Meena et al., 2017b). Finally, the purified products were transferred for both end sequencing and analysis. Using, National center for biotechnology information-Basic local alignment search tool (NCBI-BLAST), the sequences were matched in GenBank (https://www.ncbi.nlm.nih.gov/).

2.4 Plant material

Tomato seeds (Lycopersicon esculentum cv. Punjab Chhuara) were collected from Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh and further surface of the seeds were sterilized in 4% sodium hypochlorite (NaOCl) solution having Tween-20 (0.02% v/v), rinsed systematically using disinfected water, and then allowed for germination (Meena et al., 2016).

2.5 Experimental design, biological treatments and growth condition

The experiment was performed in plastic pots (30 cm diameter; surface sterilized with 0.1% mercuric chloride). The treatments were arranged as (1) uninoculated control, (2) pathogen (Fol) challenged, and (3) plants primed with T. harzianum LMMT spores and challenge inoculated with pathogen (Fol). The individual seedlings were transferred to the pot filled with a sterile soil mixed with sand (autoclaved with 121 °C and 15 psi pressure for 15 min):soil (4:1) mixture along with Trichoderma inoculums 10⁶ conidia/g of soil. Experiments was performed in three biological replicates (five seedlings per replicate) for each treatment. The same amount of sand:soil mix but free from T. harzianum LMMT were added to the uninoculated control plants. All the plants were kept and grown in a green house that was maintained on a temperature 25–29 °C with 70% humidity, and 14 h light and 10 h dark cycle. After 3 weeks of seedling transplantation, plants were made infected with the Fol pathogen. For this the Fol spore suspension was inoculated in the same soil sand mixtures at a concentration of 10⁶ conidia/g in all the Fol challenged and T. harzianum LMMT supplemented plants, whereas the uninoculted control plants were kept as such. The leaves from all the three treatments were plucked at five different stages at different time intervals 0, 24, 48, 72 and 96 h post inoculation (hpi) with the pathogen and used for biochemical analysis. The expression profile changes of the defense associated genes encoding defense-related antioxidative enzymes like SOD, CAT, PO and other proteins like PR proteins, PAL and PPO were also measured. The transcript abundance and relative gene expression were measured in all the treatments during the different interval of time varying from 0 to 96 h.

2.6 Superoxide dismutase (SOD), catalase (CAT) and peroxidase (PO) activity assay

The activity of SOD (EC 1.15.1.1) was measured using a method proposed by Fridovich (1974). The estimation of CAT (EC 1.11.1.6) activity was done spectrophotometrically using the previous described method exemplified by McKersie et al. (1993). The approach given by Hammerschmidt et al. (1982) was used to calculate the activity of PO (EC 1.11.1.7).

2.7 Phenylalanine Ammonia-Lyase (PAL) and polyphenol oxidase (PPO) activity assay

PAL (EC 4.1.3.5) activity was assayed according to the procedure as described by El-Shora (2002) with minor modifications. PPO (EC 1.14.18.1) activity was performed using the method of Gauillard et al. (1993).

2.8 PR-2 protein (β-1,3-glucanase) and PR-3 protein (Chitinase) assay

The activity of β-1,3-glucanase and chitinase (EC 3.2.1.14) was measured using Pan et al. (1991) and Giri and Aggarwal's (1998) technique, respectively with some minor changes.

2.9 qRT-PCR analysis

The expression patterns of defense-related genes in RNA samples of all the treatments of tomato leaves were verified by real time-PCR. Total RNA were isolated from the control and treated tomato leaf tissues using Chromous RNA isolation Kit. First strand of cDNA was synthesized using 1 μg of RNA sample with random primers with the help of cDNA synthesis kit according to the manufacture’s instructions. Primer was designed by Primer 3 software. Primers for qRT-PCR analysis were synthesized according to the following references (Song et al., 2015; Jayanna and Umesha, 2017; Aazami et al., 2021). The RT-PCR was performed by following the method of Cheng et al. (2017). All reactions were performed in triplicates on ABI Step-one RT-PCR machine. Primers with master mix was used as a negative control in all RT-PCR runs. Real time software was used to analyze the output of RT-PCR runs for the determination of relative expression used by cycle threshold (CT) values. To normalize each reaction rubisco transcript was used. All three independent biological replicates followed the same trends of expression change, of which one such value has been represented in this study.

2.10 Statistical analysis

SPSS Version 16.0 was used to perform the statistical analysis of three independent biological replicates of all the experiments. All the data were used to analyze one-way analysis of variance (ANOVA) and Duncan’s multiple range test to determine the significance of differences at P ≤ 0.05 between the treatments.

3.1 Pathogen identification

The pathogen recovered from wilted tomato plants when grown on PDA plates showed pink-white color of the fungal hyphae with cottony surface. The hyphae when observed in the microscope showed asexual structures including microconidia and macroconidia. Koch’s postulates followed for pathogenicity test of all the isolates of Fol. A highly virulent based on percentage occurrence of disease development was further selected for experiments.

3.2 Identification of Trichoderma

The species of isolated Trichoderma was identified as Trichoderma harzianum based on the morphological and conidial characteristics using the key as suggested by Rifai (1969) and Nagamani et al. (2002). The raw data obtained from the sequencing results were submitted to NCBI after annotation. The annotated ITS sequence was perform for BLASTn analysis using the standard database nucleotide collection (nr/nt). The BLASTn results showed sequence similarity with T. harzianum.The accession number provided by NCBI Genbank for T. harzianum BHU LMMT (Th) was KX 091167.

3.3 Disease incidence and Biocontrol efficacy

The T. harzianum pre-treated induced resistance in tomato seedlings were recorded (14 days after challenging the pathogen) through the decrease in severity of wilt disease infected with Fol. Observations noted maximum disease percentage for pathogen infected plants and it was 84.16%. It was noted that Th primed tomato plants exhibited significant reduction in disease severity (10.18%) while compared with Fol challenged plants. Biocontrol efficacy of Th against Fol was found to be 87.90%.

3.4 SOD, CAT and PO activity

After inoculation both non-inoculated pathogen control and the Th inoculated plants enzyme activity enhances with time. As shown in Fig. 1A, Th primed plants showed elevation in SOD generation at 48 hpi in compare to the pathogen challenged and control plants. SOD activity level rises at 48 hpi and then decreased thereafter. Th primed plants showed 2.71- and 1.34-fold higher SOD enzyme activity in compare to pathogen challenged and control plants. Natural SOD activity was regularly present in control during the assays. Activity of enzyme in non-inoculated pathogen control and the Th inoculated plants rises with time after inoculation. CAT activity in tomato primed with Th decreased after pathogen inoculation. An increase in CAT activity was observed gradually after 24 h of pathogen challenge. Maximum activity was observed at 96 hpi in Fol + Th treated plants. Fol + Th treated showed 5.87- and 1.34-fold higher CAT enzyme activity in compare to control infected plants (Fig. 1B). Elevation in activity of PO was noted in both treatments compare to the control. At 72 h, PO activity recorded maximum in all treatment, after that it gets reduce. Th prepared plants noted 1.26- and 10.94-fold increase activity of PO in compare to pathogen-infected and without treated plants, individually (Fig. 1C). A little sum of PO action was reliably recorded in sound control plants.

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Fig. 1

Estimation of changes in the activities of (A) SOD, (B) CAT, (C) PO, (D) PAL, (E) PPO, (F) Chitinase and (G) β-1,3-Glucanase in the leaves of tomato plants pre-treated with T. harzianum, after challenge inoculation with F. oxysporum f. sp. lycopersici (Fol).

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3.5 PAL and PPO activity

PAL increases significantly in both treatments during the first 72 h, then falls. In comparison to control and pathogen-challenged plants, Th primed plants showed 1.48- and 5.70-fold higher PAL activity after 72 h (Fig. 1D). PPO activity increases in both treatments when compared to control plants. PPO activity was at its peak at 72 h in all of the treatments, however it gradually decreased. When compared to pathogen-inoculated and untreated controls, Th primed plants had 1.47- and 11.47-fold greater PPO activity, respectively (Fig. 1E). In healthy control plants, a small level of PPO activity was routinely recorded.

3.6 Chitinase and β-1,3-Glucanase activity

In comparison to control plants, chitinase activity increased after pathogen exposure. Fol + Th treated plants had a 1.62- and 5.78-fold increase in chitinase activity at 72 h as compared to pathogen challenged and control plants. Following that, all of the treatments showed a decrease in chitinase activity (Fig. 2F). In comparison to control plants, activity of β-1,3-glucanase was reported to increase after pathogen challenge. At 72 h, Fol + Th treated plants had a 1.96- and 16.08-fold increase in β-1,3-glucanase activity, respectively, when compared to pathogen challenged and control plants (Fig. 2G). Following that, all of the treatments showed a decreasing trend in β-1,3-glucanase activity.

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Fig. 2

Relative expression of different antioxidant enzyme genes (A) SOD, (B) CAT, (C) PO, (D) PAL, (E) PPO, (F) PR-3 and (G) PR-2 in the leaves of tomato plants pre-treated with T. harzianum, after challenge inoculation with F. oxysporum f. sp. lycopersici (Fol).

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3.7 Regulation of defense related genes

In control plants of 24, 48 and 72 h duration leaf samples the SOD gene expression reduces non-significantly compared to the 0 h leaf samples (Fig. 2A). Although, a non-significant rise compared to control noted in 96 h leaf sample. However, in Fol and Fol + Th treated plants the expression of SOD increases compared to the control plants. In 24 and 48 h, Fol and Fol + Th treated plants it was of 1.87, 2.33 and 2.19, 2.73-fold, respectively. Although, under similar treatment after 72 and 96 h, it was 1.67, 1.04 and 1.68, 1.09-fold, respectively (Fig. 2A).

Similar to enzyme activity catalase gene expression also recorded, in non-treated plants of 24, 48, 72 and 96 h duration a non-significant rise in catalase gene expression noted upto 48 h, then it decreases non-significantly upto 72 h, then again it increases under 96 h. Compared to control plants respective values of fold change compared to control plants in Fol treated plants was 2.33, 1.61, 3.33 and 4.94-fold, respectively under 24, 48, 72, and 96 h. Under similar condition in Fol + Th treated plants the values compared to control plants was 3.73, 2.52, 4.93 and 6.33-fold (Fig. 2B).

Similar to other enzyme gene expression the expression of peroxidase showed varied expression pattern, in control plants leaf samples of different hour sampling, it gets reduced in 24 h leaf sample while it increases significantly in 48 h leaf samples and non-significantly increases in 72 and 96 h leaf samples compared to control 0 h leaf samples. However, increased expression of same gene recorded when the leaf samples of 24, 48, 72 and 96 h is exposed to Fol and Fol + Th. In Fol treated 24, 48, 72 and 96 h leaf samples the fold change compared to 0 h leaf samples was 3.25, 4.53, 5.87 and 2.17-fold, under similar condition Fol + Th treated plant leaf sample’s gene expression fold change was 5.77, 7.33, 8.29 and 3.07-fold, respectively (Fig. 2C).

Gene expression of PAL was not uniform, in control plants compared to control in 24 h and 72 h leaves its expression increases, while it decreases in the leaf samples of 48 h and 96 h. Although in Fol and Fol + Th treated, all leaf samples it increases compared to control. It was maximum 4.97 fold compared to control in Fol treated plants after 72 h, while it was minimum 1.97-fold compared to control after 96 h under similar treatment. Similarly in Fol + Th treated plants, it was maximum 6.73-fold after 72 h and minimum 3.24-fold compared to control after 96 h (Fig. 2D). PPO gene expression was non-significantly decreased in 24, 72 and 96 h leaf samples under control condition, while it get increased non-significantly in 48 h leaf samples under control condition. However, it increases in all leaf samples compared to control plants when treated with Fol and Fol + Th. In Fol treated plants, it was maximum in 48 h leaf samples while minimum in 24 h leaf samples, however it was still higher compared to control plant. Similarly in Fol + Th treated plants, it was maximum in 48 h leaf sample and minimum in 24 h leaf sample (Fig. 2E).

Expression level of chitinase (PR-3) genes were recorded under control condition. A very slight changes noted even in case of 24, 48, 72 and 96 h of leaf samples, initially it increases upto 72 h, then decreases in 96 h leaf samples. In case of Fol and Fol + Th treatment, a significant increase noted, however, it rises sharply upto 48 h in both case and the fold change values compare to control was 2.83, 5.23 and 2.97 and 5.88-fold, respectively for Fol treatment and Fol + Th treatment. In case of Fol treatment after 72 h, a slight reduction noted, however, it was high reduction after in 96 h exposed plants. While in Fol + Th treated plants, a sharp reduction observed after 72 and 96 h of Fol + Th exposure (Fig. 2F). β-1,3 Glucanase (PR-2) gene expression under control, Fol and Fol + Th, treatment increases, however, in control plants it increases upto 48 h of compared to plants, then decreases after 72 and 96 h, however, it was still higher compared to the control plants of 0 h duration. Under Fol and Fol + Th treatment, this gene expression compared to 0 h exposed plants under 24, 48 and 72 h of Fol and Fol + Th exposure, although after 96 h of both exposure, it decreases (Fig. 2G).