Influence of abiotic factors on in vitro growth of indigenous Trichoderma isolates of Gujarat

Introduction

Trichoderma is a genus of filamentous fungi widely recognized for its role in agriculture and biotechnology due to its potent biocontrol properties and ability to enhance plant growth (Samuels, 2006). These fungi are known for their antagonistic activity against a wide range of plant pathogens, making them valuable in integrated pest management and sustainable agriculture. Trichoderma species achieve this through various mechanisms, including the mycoparasitism, antibiosis, production of hydrolytic enzymes, secondary metabolites and competition for nutrients and space (Benitez et al., 2004) [2] . Additionally, they form symbiotic relationships with plants, promoting growth and provide resistance to biotic and abiotic stresses (Gamalero and Glick, 2011) [3] . Native Trichoderma isolates refer to strains of the Trichoderma genus that are indigenous to specific geographical regions or local environments. These isolates have adapted to the unique climatic and soil conditions of their native habitats, often displaying superior resilience and effectiveness in controlling local plant pathogens compared to non-native or commercially available strains (Harman et al., 2004) [4] . These isolates often exhibit higher efficacy in their natural environment due to their evolutionary adaptation to local biotic and abiotic factors. Additionally, native isolates may possess unique traits such as resistance to local pathogens, tolerance to specific soil pH levels, or enhanced enzyme production, making them well-suited for use in biofertilizers and soil conditioners (Singh et al., 2014) [15] . Understanding the growth and effectiveness of Trichoderma strains under various environmental conditions is crucial for optimizing their application in agricultural practices. The growth and effectiveness of Trichoderma strains are significantly influenced by abiotic factors, which include temperature, pH, salinity and water availability (Papavizas, 1985) [8] . These factors not only affect the in vitro growth rates of Trichoderma but also determine their antagonistic potential and ability to colonize various substrates. Understanding how these environmental parameters influence Trichoderma growth is essential for optimizing their application in different agro-ecological conditions (Gamalero and Glick, 2011) [3] . In this context, exploring the influence of abiotic factors on the growth of indigenous Trichoderma strains under controlled laboratory conditions provides valuable insights. Such studies can help in selecting and developing robust Trichoderma strains that are effective under a wide range of environmental conditions. This study aims to investigate the influence of abiotic factors such as temperature, pH, salinity and water availability on the in vitro growth of indigenous Trichoderma strains. By understanding how these environmental parameters affect indigenous Trichoderma, it may be possible to enhance their application as biocontrol agents and improve agricultural productivity in Gujarat.

Materials and Methods

Collection of rhizospheric soil sample and isolation of Trichoderma isolates

Rhizospheric soil samples from pulse/legume crops i.e., chickpea, pigeonpea, blackgram, greengram, cowpea, soybean and groundnut were collected during Rabi 2021 and Kharif 2022 from the eight districts viz., Anand, Kheda, Ahmedabad, Vadodara, Panchmahal, Mahisagar, Dahod and Chhota Udepur of middle Gujarat. The rhizospheric soil samples were processed by using the serial dilution technique for the isolation of Trichoderma. The samples were prepared in sterilized distilled water and serially diluted. From the dilution blank, serial dilutions of 10 -2 to 10 -6 were prepared. One millilitre of each dilution of 10 -3 to 10 -6 was pipetted out to sterile Petri plates containing Trichoderma selective media and spread using a glass spreader. The plates were incubated at 28 °C and examined regularly for the development of colonies for five days. Based on the morphological study of the different colonies, selected colonies were sub-cultured in PDA plates by the hyphal tip method.

Effect of abiotic factors on growth and sporulation of indigenous Trichoderma isolates 1. pH

The impact of pH on the radial growth of five indigenous Trichoderma isolates was assessed by placing 5 mm discs on potato dextrose agar (PDA) with pH levels of 5, 6, 7, 8 and 9, adjusting pH with 0.1 N NaOH or HCl, following the method of Prabhavathi et al. (2014) [9] . The plates were incubated at 28±1 °C for five days, with each treatment replicated four times and mycelial growth was measured after incubation

Temperature tolerance

The optimum temperature and tolerance limits of five efficient indigenous Trichoderma isolates were investigated in vitro by incubating 5 mm discs on PDA plates at temperatures of 20, 30, 35, 40 and 45 °C. The experiment, following Shim et al. (1997) [14] , involved maintaining these temperatures in a BoD incubator with four replicates per treatment. Radial growth was measured after five days of incubation.

Salinity tolerance

The NaCl tolerance of five efficient indigenous Trichoderma isolates was assessed using the poisoned food technique (Sharvelle, 1961) [13] . Isolates were exposed to PDA amended with NaCl at concentrations of 100, 500, 750, 1000 and 1250 mM. A 5 mm disc of each isolate was placed on the respective media and plates were incubated at 28±1 °C for five days with four replicates per treatment. Radial mycelial growth was measured and analyzed statistically.

Drought tolerance

Drought tolerance of five efficient indigenous Trichoderma isolates was tested by growing them on PDA plates with increasing levels of polyethylene glycol (PEG 6000 Da) at 10, 20, 30, and 40 percent. A 5 mm disc of each isolate was placed on the PEG-amended media, and plates were incubated at 28±1 °C (Kucuk, 2017) [5] . Radial growth was recorded after five days.

Results and Discussion

Isolation of indigenous Trichoderma isolates

Trichoderma isolates were obtained from collected rhizospheric soils using a serial dilution procedure with dilution factors of 10 -4 to 10 -6 on ready-made Trichoderma selective media (TSM). A total of 130 soil samples were collected from different locations, out of which, 62 soil samples were recovered Trichoderma spp. Out of these 62 indigenous Trichoderma isolates, five potent Trichoderma isolates were selected based on their in vitro antagonistic ability against Fusarium oxysporum f. sp. ciceri. The further research was conducted using these five potent indigenous Trichoderma isolates (Table 1). [9] , Mishra et al. (2016) [6] and Zehra et al. (2017) [17] who revealed that the growth of different Trichoderma isolates was highest at pH 6. (2020) [1] , Singh et al. (2021) [16] and Ramatsitsi et al. (2023) [11] also determined the effect of temperature on the growth and sporulation of different Trichoderma isolates and concluded that the optimum temperature range for the better growth and sporulation of Trichoderma is 25 to 30 °C.

Salinity tolerance

Trichoderma species, known for their adaptability in diverse environments, are significantly affected by salinity, which influences their physiological processes such as osmotic regulation, enzyme activity and nutrient uptake. A study examined the growth of Trichoderma isolates at varying salt concentrations (100, 500, 750, 1000, and 1250 mM) (Table 4). At 100 mM, all isolates grew normally, reaching 90 mm in five days. Growth declined as salinity increased, with growth ranging from 80.60 to 89.47 mm at 500 mM and a significant drop was observed at 750 mM (45.00 to 51.31 mm). At 1000 mM, growth was reduced further to 25.06-38.40 mm and at 1250 mM, it was minimal (10.34-29.62 mm). Sporulation was profuse at 100 mM, sparse at 500 mM in treatment T 1 and absent in all isolates at concentrations of 500 mM and above. A similar result was also found by Ramanagouda et al. (2021) [10] who noticed a drastic reduction in mycelial growth and morphological variations at and above 700 mM salt concentration. [7] who stated that the growth and sporulation of Trichoderma isolates were reduced with increasing PEG concentration.

Acknowledgement

AcknowledgementThe researchers highly acknowledge the Hon. Vice Chancellor, Dr. K. B. Kathiria; Director of Research and Dean PG studies, Dr. M. K. Jhala, Principal and Dean, Dr. Y. M. Shukla and Professor and Head Department of Plant Pathology Dr. R. G. Parmar for the keen guidance and support throughout the course of study.

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