Isolation and In-vitro biocontrol potential of native Trichoderma spp. against Aspergillus niger van Tieghem causing collar rot of groundnut

Introduction

Groundnut (Arachis hypogaea L.) is an important oilseed and food legume crop cultivated predominantly in arid and semi-arid regions. It is valued for its high nutritional and economic importance, particularly as a rich source of vegetable oil and protein (Hammons, 1982; Reddy, 1976) [1,2] . In India, groundnut is one of the major oilseed crops, with Gujarat being one of the leading groundnut-producing states (Anonymous, 2024; Anonymous, 2025) [3,4] . Groundnut production is affected by several fungal diseases, among which collar rot caused by Aspergillus niger van Tieghem is an important soil-and seed-borne disease. The disease primarily affects the collar and hypocotyl regions of young seedlings and may occur in both pre-and post-emergence phases. Infected seeds may rot in the soil, while emerging seedlings develop brown lesions, followed by tissue decay, wilting, collapse and death. Under favourable conditions, collar rot can cause considerable plant mortality and yield losses (Chahal et al., 1974; Ghewande et al., 2002) [5,6] . Aspergillus niger is widely distributed in soil and plant debris and can survive under conditions favourable for disease development. The incidence of collar rot is influenced by factors such as high soil temperature, low soil moisture, sandy soil conditions and deep sowing of seeds (Saran et al., 2021) [7] . The pathogen may persist in the field and cause significant damage to groundnut seedlings, making effective management of the disease essential (Gajera et al., 2011) [8] . Although fungicides such as mancozeb, tebuconazole, thiram, carbendazim and vitavax are used for the management of collar rot, their repeated and extensive use has several limitations. These include environmental pollution, development of resistant pathogen populations, non-target effects, food and feed contamination and potential health hazards. Moreover, effective management of soil-borne pathogens through chemical application is difficult because of their persistence in the soil environment (Rakholiya et al., 2012) [9] . Therefore, biological control represents a promising and sustainable alternative for the management of soil-borne diseases (Amin et al., 2010; Dhotre and Vanmare, 2017) [10,11] . Among the biological control agents, Trichoderma spp. have received considerable attention because of their ability to suppress a wide range of soil-borne plant pathogens through different mechanisms, including competition, antibiosis, hyperparasitism and production of lytic enzymes and antifungal metabolites (Cook and Baker, 1983; Singh and Jadon, 2019) [12,13] . Several Trichoderma species, including T. harzianum, T. viride, T. asperellum, T. hamatum, T. virens and T. koningii, have been investigated for their biocontrol potential, with T. harzianum being one of the most extensively studied species for biological disease management (Keswani et al., 2014) [14] . The rhizosphere represents an important source of beneficial microorganisms, where plant-microbe interactions can influence plant health and development. Native Trichoderma isolates obtained from rhizospheric soils may therefore provide promising candidates for the biological management of soil-borne pathogens. Evaluation of their antagonistic potential against the target pathogen under controlled in-vitro conditions can help identify efficient isolates for further disease-management studies (Huang et al., 2014) [15] . Identification of efficient native biocontrol agents and evaluation of their antagonistic potential are essential prerequisites for developing sustainable and environmentally friendly management strategies against collar rot of groundnut. However, information on the diversity and effectiveness of native Trichoderma isolates against Aspergillus niger causing collar rot under in-vitro conditions is limited. Therefore, the present investigation was undertaken to isolate, identify and prove the pathogenicity of A. niger associated with collar rot of groundnut, isolate native Trichoderma spp. from the rhizospheric region of higher plants, and evaluate their antagonistic potential against A. niger under in-vitro conditions.

Materials and Methods: Isolation and identification of pathogen

Aspergillus niger was isolated from infected groundnut kernels by the agar plate method. The kernels were surface sterilized with 1% sodium hypochlorite (NaOCl) for one minute, rinsed three times with sterile distilled water and placed on Potato Dextrose Agar (PDA). Plates were incubated at 25 ± 2 °C for 4-5 days. The fungal isolates were purified by single-spore isolation and maintained on PDA slants. Identification was carried out based on cultural and morphological characteristics of the mycelium and conidia following Raper and Fennell (1965) [16] .

Isolation of native Trichoderma spp.

Rhizospheric soil samples were collected from higher plants at a depth of 15 cm from the Anand region (Table 1) and Trichoderma spp. were isolated by the serial dilution technique (Hirte, 1969) [17] on Trichoderma selective medium (Elad and Chet, 1983) [18] . Representative isolates were purified through single-spore isolation and preserved at 4 °C for further studies. A total of 52 soil samples were collected, out of which 25 soil samples recovered Trichoderma spp.

In-vitro antagonism between native Trichoderma spp. and pathogen -A. niger Dual culture method

The antagonistic potential of native Trichoderma isolates against Aspergillus niger was evaluated using the dual culture technique (Dennis and Webster, 1971a) [19] . A 5 mm mycelial disc of A. niger and each Trichoderma isolate were placed opposite to each other on PDA plates and incubated at 28 ± 1 °C. Three replications were maintained, with pathogen-only plates as control. Radial growth was recorded until complete growth in the control, and per cent growth inhibition was calculated according to Vincent (1947) [20] .

Where, PGI = Per cent growth inhibition DC = Growth of test pathogen (mm) in the absence of antagonistic isolate DT = Growth of test pathogen (mm) in the presence of antagonistic isolate

Volatile compounds

The inverted plate technique was employed to evaluate the ability of native Trichoderma isolates to produce volatile antifungal compounds against Aspergillus niger (Dennis and Webster, 1971b) [21] . A 5 mm mycelial disc from a threeday-old culture of each Trichoderma isolate was placed at the centre of PDA plates and incubated at 28 ± 1 °C for two days, while A. niger was inoculated separately and incubated for 12 h. The lids were removed, and the respective plate bottoms were sealed face-to-face with Parafilm and incubated at 28 ± 1 °C. Three replications were maintained for each treatment, with pathogen-only plates as control. Per cent inhibition of mycelial growth was calculated as described earlier.

Results and Discussion

A total of 52 rhizospheric soil samples collected from different locations of Anand district were processed for isolation of Trichoderma spp. using Trichoderma selective medium. Twenty-five samples yielded Trichoderma isolates, which were confirmed based on their cultural and morphological characteristics and purified through hyphal tip isolation

In-vitro antagonism between native Trichoderma spp. and pathogen -A. niger Dual culture method

The antagonistic potential of 25 native Trichoderma isolates against A. niger was evaluated using the dual culture technique. Considerable variation was observed among the isolates in their ability to inhibit mycelial growth of the pathogen. Isolate Tr6 recorded the highest inhibition of mycelial growth (87.81%), restricting the colony diameter of A. niger to 10.96 mm, followed by Tr10 (77.44%; 20.30 mm) and Tr16 (75.22%; 22.30 mm). Isolates Tr8 and Tr18 also exhibited high inhibitory activity, with 74.84 and 72.30 per cent inhibition and corresponding pathogen colony diameters of 22.64 and 24.53 mm, respectively (Table 2). The lowest inhibition was recorded by Tr5 (31.88%), with a pathogen colony diameter of 61.30 mm. The remaining isolates exhibited inhibition ranging from 35.22 to 72.14 per cent, indicating considerable variation in their antagonistic activity against A. niger. Overall, Tr6 was the most effective isolate in suppressing the mycelial growth of A. niger under in-vitro conditions (Figure 2). The present findings are in agreement with earlier reports demonstrating the antagonistic potential of Trichoderma spp. against A. niger. Nath and Patel (2022) [22] reported maximum inhibition of 63.72 per cent by T. viride, while Gajera et al. (2011) [8] recorded 86.2 per cent inhibition by T. virens isolate T6. Similarly, Swathy et al. (2024) [23 ]reported 86.33 per cent inhibition by Trichoderma isolate TVK1 using the dual culture technique. These findings support the effectiveness of Trichoderma spp. as potential biocontrol agents against A. niger.

Volatile compounds

The antagonistic potential of 25 native Trichoderma isolates against A. niger was evaluated using the inverted plate method. All the isolates inhibited the mycelial growth of the pathogen compared with the control (90.00 mm), with considerable variation in their activity. Isolate Tr18 recorded the highest growth inhibition (78.63%), followed by Tr6 (77.03%), Tr8 (76.41%), Tr10 (76.66%) and Tr16 (76.38%). The corresponding pathogen colony diameters were 19.23, 20.66, 21.23, 21.00 and 21.25 mm, respectively. The lowest inhibition was recorded by Tr5 (19.77%), with a maximum colony diameter of 72.20 mm. Overall, Tr18 exhibited the highest antagonistic activity against A. niger through volatile metabolites (Table 2) (Fig. 3). The present findings are in agreement with earlier reports on the inhibitory effect of Trichoderma volatile metabolites against Aspergillus spp. Erfandoust et al. (2020) [24] reported 71.93 per cent inhibition of A. niger by volatile compounds of T. atroviride. Similarly, Stracquadanio et al. (2020) [25] observed 45.86 and 25.56 per cent inhibition by T. asperellum and T. atroviride, respectively. Boukaew et al. (2024) [26] and Voloshchuk et al. (2024) [27] also reported significant suppression of Aspergillus spp. by volatile metabolites of Trichoderma.

Acknowledgement

AcknowledgmentsThe authors express their sincere gratitude to the Dean, B.A. College of Agriculture, Anand Agricultural University, Anand and the Head, Department of Plant pathology, B.A College of Agriculture for providing necessary facilities and guidance during the course of investigation. The authors are also thankful to the faculty members and staff of the Department of Plant pathology for their valuable support and cooperation during the experimental work conducted.

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