A survey was conducted across various prominent apple-growing areas in Himachal Pradesh, in particular Bilaspur, Mandi, Kullu, Solan, and Shimla ( Figure 1 ). At each selected site, four sub-sites were selected in orchards/nurseries. Soil samples were collected from the rhizospheric area of apple at 10- to 15-cm soil depth into polythene bags (Singh et al., 2003).

CCA medium (in grams per liter): MgSo₄·7H₂0, 0.3; (NH₄)₂·SO₄, 3.0; KH₂PO₄, 2.0; citric acid, 1.0; agar powder, 15; Tween-80, 200 μl; colloidal chitin, 4.5; and bromocresol purple, 0.15. pH was set to 4.7.

PDA medium (in grams per liter): potato extract, 4; dextrose, 20; and agar, 20. The final pH was adjusted to 5.6. This was used for preservation of the fungal cultures.

Colloidal chitin was prepared from chitin powder. Of the chitin powder, 10 g was treated with 40–50 ml (37%) of HCl. In a 1,000-ml beaker, HCl was slowly added into the chitin powder, with continuous stirring until becoming a slurry. The chitin–HCl mixture was then kept in a refrigerator (4°C) for 1 h incubation. The mixture was treated with 50% (pre-chilled) ethanol and kept in the refrigerator overnight to facilitate better precipitation of colloidal chitin. This mixture was filtered through Whatman no. 1 filter and washed with 1–2 L of pre-chilled distilled water. The pH was set between 6 and 7 by adding NaOH/HCl and was kept in shade after filtration for drying. The ground powder was kept at 4°C for further use.

The modified protocol of Gomez Ramirez et al. (2004) was used to prepare the CCA mixture, which was then autoclaved at 121°C for 20 min. The sterilized medium was then poured into Petri plates. After solidification of the medium, different isolates of Trichoderma were kept at the center of the plate to examine the chitinase-producing isolates with three replications each and incubated at 30°C for 3 days.

The effects of different growth parameters, such as the incubation period, pH, substrate concentration, and incubation temperature, on chitinase activity were examined. The effect of pH was assessed between 3 and 8 at 1-unit intervals with NaOH/HCl. The effects of five temperature values ranging from 20°C to 40°C with a variation of 5°C and five concentrations of colloidal chitin, i.e., 0.5%, 1%, 1.5%, 2%, and 2.5%, were evaluated for 9 days. The enzyme activity was measured (Miller, 1959).

The enzyme activity assay was performed according to the method of Miller (1959). The reaction mixture [1 ml crude enzyme solution + 1 ml colloidal chitin (1%) + 1 ml of 0.1 M citrate buffer (pH 6.0)] was incubated in a water bath at 37°C for 30 min. The reaction was stopped by adding 2 ml of a dinitrosalicylic acid (DNS) reagent and incubated in a water bath for 5 min at 60°C for color development. The reaction mixture without crude enzyme was used as blank. The optical density (OD) value was measured at 540 nm using the standard curve of N-acetyl glucosamine unit (100–600 ppm). One unit (U) of chitinase activity was defined as the amount of enzyme that released micromoles of NAG per minute per milliliter, while specific activity (SA) represents the micromoles of NAG released per minute per milligram of protein.

The concentration of protein was estimated according to Bradford (1976) using Coomassie Brilliant Blue G-250. The OD value was measured at 595 nm using the standard curve of bovine serum albumin (BSA).

Colloidal chitin broth medium with a pH of 5 was prepared in 500-ml flasks, and four to five pieces of 5 mm size were inoculated in each flask for the different isolates of Trichoderma and were incubated at 30°C for 7 days. After 7 days, the flasks were kept on a rotary shaker for 1 day and the broth centrifuged at 10,000 rpm for 20 min. The supernatant was collected and then stored in a refrigerator for purification. The supernatant was used as a crude enzyme.

Purification of the crude enzyme was performed with the ammonium sulfate precipitation method (Xu et al., 2014). Ammonium sulfate precipitation was conducted using 0%–45% concentration of ammonium sulfate under 4°C. The precipitates were collected by centrifugation at 10,000 rpm for 20 min and the supernatant was discarded. The pellet was dissolved in citrate buffer (pH 6). Ammonium sulfate traces were removed through dialysis. Sephadex G-100 beads were dissolved in distilled water (10 g of Sephadex G-100 was added into 500 ml of distilled water), which was then poured over the top of the silica and washed with a large amount of citrate buffer for the smooth downward flow of protein. The dialyzed fraction was suspended in a Sephadex G-100 chromatographic column, and the fraction showing maximum chitinase activity was pooled (Laemmli, 1970).

The molecular weight of the purified protein was determined using SDS-PAGE. The purified fraction was submitted into 12% SDS-PAGE (Laemmli, 1970), and 40 μg of the sample was loaded and electrophoresis carried out with 150 V of current for approximately 1–2 h. The gel was carefully removed and then placed into a staining solution (Coomassie Brilliant Blue G-250) (Candiano et al., 2004). Thereafter, the staining solution was poured off and the gel was kept in a destaining solution for 3–4 h with continuous shaking for band visualization.

Statistical analysis was performed with R software (R Development Core Team, 2008). Experiments were conducted using a completely randomized design (CRD) in triplicates and analyzed using one-way ANOVA. Qualitative analysis of chitinase was performed using Duncan’s multiple range test (DMRT). A p-value <0.05 was considered statistically significant.

Different isolates of Trichoderma species were isolated and used for phylogenetic analysis ( Table 1 ). The phylogenetic tree showed that Trichoderma virens UHFTV017 (QR569071.1) shared close affinity with the T. virens isolate CTCCSJ-A-CS23059 with 81% bootstrap support, T. virens UHFTC001 (PV248974) shared close affinity with the T. virens isolate LW23 with 69% bootstrap support, Trichoderma harzianum UHFTH0013 (QR569067.1) shared close affinity with the T. harzianum isolate T22 with 99% bootstrap support, Trichoderma brevicompactum UHFTB001 (PV248971) shared close affinity with the T. brevicompactum isolate TB003 with 100% bootstrap support, Trichoderma longibrachiatum UHFTS001 (PV254825) and T. longibrachiatum UHFTK001 (PV248973) shared close affinity with the T. longibrachiatum isolate Ef72 with 100% bootstrap support, Trichoderma atroviride UHFTA005 (QQ193152.1) shared close affinity with the T. atroviride isolate LM-5-5 with 100% bootstrap support, T. atroviride UHFTA006 (QR612304.1) shared close affinity with the T. atroviride isolate CBS693.94 with 52% bootstrap support, T. atroviride UHFTA003 (QQ193150.1) shared close affinity with the T. atroviride isolate CBS693.94 with 50% bootstrap support and the T. atroviride isolate CBS693.94 with 52% bootstrap support, T. atroviride UHFTA004 (QQ193151.1) shared close affinity with the T. atroviride isolate Ta56 with 37% bootstrap support, T. atroviride UHFTA002 (QQ271334.1) shared close affinity with the T. atroviride isolate ZNR19 with 83% bootstrap support, T. atroviride UHFTA001 (QR271335.1) shared close affinity with the T. atroviride isolate ZNR19 with 98% bootstrap support, and T. atroviride UHFTA007 (QQ193153.1) shared close affinity with the T. atroviride isolate ZNR19 with 81% bootstrap support ( Table 2 ; Figures 2 , 3 ).

Garcia-Nunez et al. (2017) identified 10 isolates of Trichoderma with ITS-1 and ITS-4 of 600 bp amplicon size. Hermosa et al. (2000) identified several biocontrol isolates of Trichoderma of 500–600 bp amplicon size using ITS-1 and ITS-4 primers.

The results of the qualitative screening for chitinase production showed that all isolates of Trichoderma spp. showed chitinolytic activity at varying extents. UHFTA005, UHFTA006, UHFTV017, UHFTB002, UHFTC001, UHFTK001, and UHFTB001 showed maximum chitinase activity; isolates UHFTA002, UHFTA003, UHFTA004, and UHFTA007 showed medium chitinase activity; and isolates UHFTA001 and UHFTH0013 showed minimum chitinase activity ( Table 3 ; Figure 4 ).

Trichoderma species produce the hydrolytic enzyme chitinase that is key to the hydrolysis of chitin, which is present in the cell wall of many phytopathogenic fungi. In the present study, the production of a hydrolytic zone around the colony indicated the breakdown of chitin by chitinases. The bromocresol purple dye resulted in a change in the pH toward alkalinity by changing the yellow color of the dye to purple. Different isolates of Trichoderma species showed significant differences in zone size, which is due to the varying capacities of the isolates to produce chitinase for the hydrolysis of chitin. Agrawal and Kotasthane (2012) reported that, out of 61 isolates of Trichoderma spp., 17 showed highest, 8 medium, and 12 low chitinase activity. Gonzalez et al. (2023) observed that T. harzianum, the Trichoderma asperellum isolate 12.2, and the T. asperellum isolate BP60 produced chitinase in a CCA medium by changing the color from orange to purple.

High activity (H), 76–100; moderate activity (M), 51–75; low activity (L), 25–50. Means in each column followed by the same lowercase letter(s) did not differ at CD_(0.05) according to Duncan’s multiple range test.

CD_(0.05) , critical difference at 0.05 significance

High chitinolytic activity

Medium chitinolytic activity

Low chitinolytic activity

Different isolates of Trichoderma species were evaluated for chitinase production under in vitro conditions, and chitinase activity was monitored regularly for 9 days at 30°C. Table 4 illustrates the relationship between enzyme activity and incubation period, which revealed that the highest chitinase activity of the T. atroviride isolates UHFTA001, UHFTA002, UHFTA003, and UHFTA004; T. harzianum UHFTH0013; T. virens UHFTV017; T. brevicompactum UHFTB002; and T. longibrachiatum UHFTS001 peaked on day 7 of incubation, with values of 27.36, 24.88, 24.57, 28.13, 26.74, 26.43, 27.51, and 30.93 U/ml, respectively. However, in the case of T. atroviride UHFTA005, T. virens UHFTC001, T. longibrachiatum UHFTK001, and T. brevicompactum UHFTB001, the highest chitinase activity was achieved on day 6 of incubation, with activity values of 38.52, 36.82, 28.13, and 28.91 U/ml, respectively. In most of the isolates of Trichoderma species, the chitinase activity started increasing from the second day of incubation and continued its lag phase from the second day onward until day 7, where the chitinase activity reached its peak. With the increase in incubation period, the chitinase activity also increased, but declined beyond day 7 of incubation ( Figure 5 ).

Optimization of the incubation period is a crucial factor for increasing the production of chitinase as it directly influences the secretion of enzymes and their metabolic activity. By optimizing the depletion of nutrients, instability of the enzymes and metabolic changes occur. Below the optimal incubation period, the activity decreases due to the slow binding of the substrate to the active site of the enzyme. At optimal incubation, the activity reaches its peak due to the presence of sufficient nutrients and the more efficient substrate binding to the enzyme’s active site. Other researchers have also optimized the incubation period for chitinase production across different microorganisms. El-Katatny et al. (2000) observed the maximum production of chitinase on day 7 of incubation in T. harzianum, while Gueye et al. (2020) observed a progressive increase of the chitinase activity of T. asperellum between 3 and 5 days, with the highest activity observed on day 5 of incubation with an enzyme activity of 2.81 U/ml. Brzezinska and Jankiewicz (2012) reported maximum chitinase production of the Aspergillus niger isolate LOCK 62 on day 6 of incubation. These findings provide evidence for the role of the incubation period in maximizing the enzymatic activity, with many microorganisms showing a peak in activity around days 6 and 7 of incubation.

The effect of the concentration of colloidal chitin (0.1%–2%) on enzyme production was examined to elucidate the best substrate concentration under controlled conditions of 30°C temperature and pH 5 over a period of 7 days. Table 5 reveals that the highest chitinase activity in UHFTA001 (13.10 U/ml), UHFTA002 (13.10 U/ml), UHFTA003 (18.44 U/ml), UHFTA004 (6.27 U/ml), UHFTA005 (11.55 U/ml), UHFTA006 (6.89 U/ml), UHFTA007 (5.19 U/ml), UHFTV017 (4.10 U/ml), UHFTB002 (6.58 U/ml), UHFTS001 (6.74 U/ml), UHFTC001 (9.68 U/ml), UHFTK001 (3.33 U/ml), and UHFTB001 (9.84 U/ml) was observed at 1% concentration of colloidal chitin, indicating optimal chitinase production at this concentration, except for UHFTH0013 (3.48 U/ml) that had the highest activity at 0.5% concentration of colloidal chitin ( Figure 6 ).

Initially, more active sites of the enzymes are available for binding. Therefore, with the increase in substrate concentration, the reaction rate also increases due to more substrates binding to the active sites of the enzymes, leading to the release of more products. However, at a certain point, a further increase in the substrate concentration does not increase the reaction rate due to the saturation of all active sites of the enzymes, which prevents new substrates from binding, and due to the loss of enzyme activity. Optimal concentration of the substrate is a crucial factor for maximizing the production of chitinase and for increasing its biological efficacy against the chitin-exhibited phytopathogens. El-Katatny et al. (2000) concluded that the chitinase production of T. harzianum increased with an increase in the concentration of chitin up to 1%. These results are also in accordance with those of Elad et al. (1982), who concluded that the chitinase activity of T. harzianum increased up to a concentration of 1%.

All isolates of Trichoderma species were examined for the optimization of the pH to maximize chitinase production. The enzyme activity was estimated between pH 3 and pH 8 using colloidal chitin broth medium at 30°C, incubated for 7 days. As depicted in Table 6 , there was a clear relationship between the pH of the medium and the activity of chitinase. The maximum production of chitinase of UHFTA001, UHFTA003, UHFTA004, UHFTA006, UHFTA007, UHFTH0013, UHFTB002, UHFTC001, UHFTK001, and UHFTB001 was recorded at pH 5, with enzymatic activity of 20.38, 9.53, 12.94, 14.96, 20.07, 9.68, 15.16, 14.65, 24.88, and 17.28 U/ml, respectively. On the other hand, the maximum production of chitinase of UHFTA002, UHFTA005, UHFTV017, and UHFTS001 was recorded at pH 4, with enzymatic activity of 13.35, 11.24, 27.67, and 13.72 U/ml, respectively ( Figure 7 ).

Chitinase production was at its peak in the acidic medium, which declined under an alkaline environment, clearly suggesting the importance of the pH of the medium for maximum production of enzyme. During the exponential phase, the production of chitinase was the highest, enabling a more efficient breakdown of chitin. However, beyond this phase, the production entered a death phase, which was due to the exhaustion of all active sites of the enzyme. The denaturation of chitinase beyond the optimal pH reduced the enzyme’s efficiency. Therefore, maintaining an optimum pH is critical for its effective application in the management of targeted pathogens. Ekundayo et al. (2016) reported an optimum pH of 5 for the maximum chitinolytic activity of Trichoderma viride, while Sharma et al. (2021) reported the maximum production of chitinase by T. harzianum at pH 5.5.

To maximize the production of chitinase, an optimal temperature is a crucial factor. Different isolates of Trichoderma spp. were assessed for the optimization of the temperature between 20°C and 40°C. As shown in Table 7 , the optimum temperature for chitinase production was at 30°C for most of the examined isolates of Trichoderma spp., including UHFTA001 (9.37 U/ml), UHFTA002 (5.34 U/ml), UHFTA003 (5.34 U/ml), UHFTA004 (13.56 U/ml), UHFTA005 (15.58 U/ml), UHFTA006 (7.98 U/ml), UHFTA007 (14.65 U/ml), UHFTH0013 (9.22 U/ml), UHFTS001 (7.98 U/ml), and UHFTB001 (12.12 U/ml). However, in UHFTV017 (4.57 U/ml) and UHFTB002 (7.20 U/ml), maximum chitinase activity was achieved when incubated at 25°C and 35°C in UHFTK001 (6.89 U/ml) and UHFTC001 (6.89 U/ml) ( Figure 8 ).

The active site of an enzyme is a specific region where a substrate binds. When the temperature is below optimal for chitinase production by Trichoderma spp., the enzymatic activity is low due to the slower binding of the substrate; when the temperature is higher than optimal, denaturation of the enzyme occurs due to the distortion of its active site. Optimal temperature maximizes the enzyme activity due to the perfect shape of the active site, allowing enzyme to bind to the substrate in its maximum efficiency. Mohiddin et al. (2021) reported that T. harzianum showed the highest average chitinase activity at 30°C, except for the T. harzianum isolate BT3, which exhibited maximum production at 25°C. Mukhammadiev et al. (2020) reported that the highest activity (3.2 ± 0.08 U/ml) of T. viride was observed at 30°C compared with the other temperatures tested, and the enzyme synthesis declined at temperatures over 38°C. El-Katatny et al. (2000) reported that the optimum temperature for the production of chitinase by T. harzianum was around 30°C, with practically no growth being observed beyond 40°C.

Colloidal chitin broth medium was used for the extraction of chitinases from different isolates of Trichoderma spp. The supernatant collected from the medium was used as a crude enzyme extract, which was further purified with ammonium sulfate precipitation. The crude enzyme extract was suspended in 0%–45% ammonium sulfate, and the resulting precipitates were resuspended in citrate buffer and loaded into a Sephadex G-100 column for purification.

Different isolates of Trichoderma spp. were selected on the basis of the highest specific activity of the purified chitinase and were assessed for stability at various pH levels (pH 3–7) and incubation temperatures (from 30°C to 70°C). The results demonstrated that all of the examined isolates of Trichoderma spp., including UHFTA002, UHFTA004, UHFTA005, UHFTA006, UHFTV017, UHFTB002, UHFTC001, UHFTK001, and UHFTB001, maintained high stability between a pH range of 3–7 and temperature of 30–50°C. A drastic fall in activity was observed beyond pH 5 and beyond 50°C. The stability of the chitinases from all isolates of Trichoderma spp. declined drastically, and a significant inactivation of the enzyme was observed at 70°C ( Tables 9 , 10 and Figures 10 , 11 ).

The active site of an enzyme is highly sensitive to pH change, which affects the binding of the substrate and its catalytic function. A high pH can lead to denaturation due to the disruption of hydrogen bonding. Acidic pH can reduce the binding of the substrate to the active site of the enzyme due to the unfolding of protein. Microorganisms produce enzymes that must remain active in various environmental and soil conditions; therefore, it is important to determine the stability of the particular enzyme produced by a particular microorganism. In various bioformulations such as biopesticides and biofungicides, the activity of chitinase is crucial for maintaining its stability at a broad pH range.

The incubation temperature is an important factor that influences the stability of an enzyme, as high temperatures can disrupt the structure of the protein, leading to its denaturation. This suggests that the chitinases from the different species of Trichoderma are well adapted for functioning at medium to slightly high temperatures, i.e., 30–50°C, making them suitable for use in the biological control of various phytopathogens in the different agroclimatic zones of Himachal Pradesh. Jenifer et al. (2014) concluded that the chitinase from T. viride remained stable at pH 3–6 and up to 50°C. Ekundayo et al. (2016) also concluded that the optimal temperature for the chitinolytic activity of T. viride was 50°C and that it was stable at temperatures of 40–50°C and pH 6–7.

Different concentrations of the purified chitinases of 14 isolates of Trichoderma spp. were evaluated for their antifungal activity against D. necatrix. From Table 11 , it is evident that, with the increase in the concentration of chitinase, the inhibition of D. necatrix also increased, with the highest inhibition observed in T. atroviride UHFTA005 at all concentrations, i.e., 0.15, 0.30, 0.45, and 0.60 μl with 55.55%, 64.44%, 77.77%, and 92.22% inhibition, respectively. However, minimum inhibition at 0.15 μl concentration was recorded in T. atroviride UHFTA001 (32.00%), and at 0.30 μl concentration, it was observed in T. atroviride UHFTA001 (57.77%). At 0.45 and 0.60 μl chitinase concentrations, minimum percent inhibition was recorded in T. atroviride UHFTA001, i.e., 68.88% and 83.33%, respectively, when compared with the control. All treatments were statistically significant.

The purified chitinases from the different isolates of Trichoderma spp. were assessed for their antifungal activity against S. rolfsii. As depicted in Table 12 , the highest inhibition at 0.15 μl was recorded in T. atroviride UHFTA005 (22.22%), which was statistically at par with that in T. atroviride UHFTA006 (22.22%) and T. virens UHFTV017 (22.22%). Similarly, maximum inhibition at 0.30 μl was recorded in T. atroviride UHFTA005 (44.44%), which was statistically at par with that in T. atroviride UHFTA006 (44.44%) and T. virens UHFTV017 (44.44%). However, at 0.45 and 0.60 μl concentrations, maximum inhibition was observed in T. atroviride UHFTA005, with 68.88% and 91.11%, respectively. Minimum inhibition at 0.15 μl concentration was observed in T. atroviride UHFTA001 (3.33%) when compared with the control. All treatments were statistically significant.

In the biological control of soilborne pathogens, namely, D. necatrix and S. rolfsii, through Trichoderma spp., chitinase plays a major role by degrading the chitin present in the cell wall of these fungi, thereby hydrolyzing chitin in the fungal cell wall, leading to the weakening of the cell structure, lysis of the cell, and, ultimately, death of the fungus. The degradation of chitin by chitinase also disrupts the integrity of fungal hyphae, making it more susceptible to antagonism by other microbes. In addition to suppressing the growth of fungal pathogens, chitinase plays an important role in the growth promotion of plants. Sharma et al. (2021) highlighted the potential inhibition of D. necatrix through chitinase from Trichoderma spp., while Gonzalez et al. (2023) highlighted that chitinase significantly inhibited the growth of Fusarium oxysporum.

From Table 13 , it can be inferred that the chitinases from the different isolates of Trichoderma spp. not only reduced the incidence of white root rot but also promoted the health of plants in terms of plant height, stem girth, number of branches, and leaf area. Data pertaining to the effectiveness of Trichoderma spp. in Table 13 revealed that minimum disease incidence (13.33%) was recorded in seedlings treated with 0.60 μl of T. atroviride UHFTA005 and T. atroviride UHFTA006, with 86.67% disease control compared with the control, thereby depicting maximum plant height, i.e., 89.73 and 87.70 cm, respectively. Similarly, maximum stem girth, leaf area, and number of branches were also recorded in both these treatments, i.e., 14.03 mm stem girth, 6.00 number of branches, and 15.87 cm² leaf area in seedlings treated with T. atroviride UHFTA005, followed by T. atroviride UHFTA006 with 13.27 mm stem girth, 5.66 average number of branches, and 15.04 cm² leaf area. However, minimum plant height, stem girth, number of branches, and leaf area were recorded in seedlings treated with 0.45 μl chitinase of T. brevicompactum UHFTB002, with respective values of 55.00 cm, 9.56 mm, 3.00, and 7.62 cm². All treatments were statistically significant.

Hood et al. (2023) demonstrated that T. harzianum β-1,4-glucosidase binds to and degrades the key structural polysaccharides of M. phaseolina—including α-1,3-glucan, β-1,3-glucan, β-1,3/1,4-glucan, and chitin—suggesting its role in the reduction of pathogen incidence by weakening the integrity of the fungal cell wall. Our research validated similar results, as the Trichoderma isolates producing chitinases significantly reduced the incidence of white root rot and enhanced plant health, highlighting chitinase as a crucial enzyme in the suppression of fungal pathogens. These findings underline the critical enzymatic involvement of Trichoderma in biocontrol.

From Table 14 , it can be inferred that the chitinases from the different isolates of Trichoderma spp. not only reduced the incidence of seedling blight but also promoted the health of plants in terms of plant height, stem girth, no of branches, and leaf area. Minimum disease incidence (26.67%) was recorded in seedlings treated with 0.60 μl of chitinase from T. atroviride UHFTA005, with 73.33% disease control, followed by T. atroviride UHFTA006, T. virens UHFTV017, T. brevicompactum UHFTB002, and T. virens UHFTC001, with 66.67% disease control in each case. Maximum disease incidence was recorded (86.67%) when seedlings were treated with 0.45 μl chitinase from T. brevicompactum UHFTB002. Data also revealed that maximum plant height (75.53 cm), stem girth (12.24 mm), number of branches (5.00), and leaf area (11.77 cm²) were recorded in seedlings treated with 0.60 μl chitinase from T. atroviride UHFTA005, followed by seedlings treated with 0.60 μl chitinase from T. atroviride UHFTA006, with 73.36 cm plant height, 12.14 mm stem girth, 5.00 average number of branches, and 11.75 cm² leaf area. However, minimum plant height, stem girth, number of branches, and leaf area were recorded in seedlings treated with 0.45 μl chitinase from T. brevicompactum UHFTB002, with values of 42.26 cm, 8.26 mm, 2.00, and 5.80 cm², respectively. All treatments were statistically significant.

The findings of Ribeiro dos Santos and Lima dos Santos (2025) underscored the multifaceted role of Trichoderma species in agriculture, emphasizing their capacity to colonize the rhizosphere, promote plant growth, and produce enzymes and secondary metabolites that contribute to pathogen suppression.