Isolation, Evaluation and Characterization of Indigenous Trichoderma spp. for the Management of Wilt of Chickpea Caused by Fusarium oxysporum f. sp. ciceris

Chickpea (Cicer arietinum L.) is an annual legume crop produced worldwide for its high quality protein (20-22%), fibre, carotene and minerals. It is one of the oldest cultivated legumes, with 7500-year-old remains discovered in the Middle East. It is grown in India, Australia, Myanmar, Pakistan and the United States. After the common bean, it is the world’s second most significant pulse crop. It is also known to fix nitrogen from the atmosphere (40 kg N ha^-1) and hence reduce the demand for nitrogen fertilizers. With an output of 101.3 million tonnes from a land area of 94.4 million hectares and a productivity of 1073 kg per hectare, India is a big producer of chickpeas. Madhya Pradesh has provided 37 per cent of the total area and 46 per cent of total production, thereby ranking first in both area and production (Ministry of Agriculture and Farmers Welfare, GOI, 2019).
       
Diseases and insect pests cause major productivity losses in chickpea cultivation, ranging from 50-100 percent in tropical areas to 5-10 percent in temperate areas (Van-Emden et al., 1988). In total, roughly 172 pathogens have been identified as infecting chickpea in various parts of the world. Approximately 67 fungi, 3 bacteria, 22 viruses and 80 nematodes have been identified among these pathogens (Nene et al., 1996), although only a handful have the ability to destroy the crop.
       
Wilt, caused by an important soil borne pathogen Fusarium oxysporum f. sp. ciceris (Padwick), is considered one of the most dangerous and widespread diseases of chickpea throughout the world’s growing areas (Haware, 1990; Jalali and Chand, 1992). Wilt is a vascular disease that causes xylem browning or blackening due to melanin pigment and disrupts water and nutrient transfer, causing the plant to wilt or die. Under extreme circumstances, the wilt can entirely destroy the crop, resulting in a 100% loss.
       
Several chemicals are used to treat wilt disease, but their use has negative consequences for the environment, such as residual effects, health risks for humans and animals, soil degradation and pathogen resistance development. Biological control, which is an environmentally benign method, plays a significant role in the management of this soil-borne illness in this area.
       
Trichoderma is a filamentous fungus that reproduces asexually and has a sexual teleomorph in the genus Hypocrea. Trichoderma spp. is a flexible bio-control agent that may be utilized to effectively manage a variety of plant diseases. Mycoparasitism, antibiosis, competition for nutrients or space, tolerance to stress through improved root and plant development, solubilization and sequestration of inorganic nutrients, induced resistance and inactivation of pathogen enzymes are only a few of the strategies used by Trichoderma spp. (Lewis and Lumsden, 2001). It generates both volatile and non-volatile compounds that inhibit the growth of many fungi (Corley et al., 1994; Horvath et al., 1995). As a result, the goal of this study was to examine the indigenous fungal bio-control agent Trichoderma spp. against the wilt pathogen.

Isolation of pathogen F. oxysporum f. sp. ciceris
 
Chickpea plants with typical wilt symptoms were gathered from the field. The adhering dirt particles and other debris from the diseased stem area were thoroughly washed away with tap water. The infected stem section was chopped into small 1 cm pieces and surface sterilised by soaking for a minute in a 1% sodium hypochlorite solution. To remove traces of sodium hypochlorite, the bits were rinsed three times in sterile distilled water. The sterilized parts were inoculated onto Potato Dextrose Agar (PDA) medium and cultured for 5 to 7 days at 28±1°C (Rangaswami, 1972).
 
Collection of rhizosphre soil
 
During the cropping season, soil samples from healthy chickpea rhizosphere were obtained (rabi 2021). Six places in the field were chosen at random for soil sampling and a sample was taken from the root of each healthy plant from each spot. The top 2-3 cm of dirt was scraped away and the loose soil around the healthy root was collected. The six samples taken were thoroughly mixed to form a typical sample of that area, then placed in a polythene bag and appropriately labelled. A total of twenty samples were obtained from various taluks in North Eastern Karnataka.
 
Isolation of Trichoderma spp. from rhizosphere soil of chickpea
 
Trichoderma spp. were isolated from the collected samples at the Bio-input Entreprenureship Centre, College of Agriculture, University of Agricultural Sciences, Raichur. To obtain a 1:10 dilution, ten grams of soil sample were suspended in 90 ml of sterilised distilled water and vigorously mixed with vertex mixture (10^-1). To produce a 1:100 (10^-2) dilution, one ml of this was transferred to a test tube holding 9 ml of sterilised distilled water. Similarly, a 1:100000 dilution of the material was prepared (10^-5). Each sterile Petri plate was pipetted with one millilitre of a final dilution of each sample, followed by 15-20 ml of sterilized and melted Trichoderma Selective Medium (TSM) (Ingredients per lit: MgSO₄. 7H₂O - 0.2 g; K₂HPO₄ - 0.9 g; KCL - 0.15 g; NH₄NO₃ - 1 g; Glucose - 3 g; Rose Bengal - 0.15 g; p-dimethylamino benzene diazo sodium sulfonate - 0.3 g; Chloramphenicol - 0.25 g; Pentachloronitrobenzene-0.2 g; Agar-15 g). The plates were incubated at 28±1°C for roughly 6-10 days after being filled and gently rotated for uniform mixing. The appearance of Trichoderma colonies on the Petri plates was monitored on a daily basis. The colonies started out white and eventually turned green. An actively developing colony of Trichoderma was picked from these plates among the various colonies and plated on PDA medium, with plates incubated at 28±1°C for four days.
 
Cultural characterization of native Trichoderma isolates
 
The isolates of Trichoderma spp. were described based on their cultural characteristics. Petri plates were inoculated with pure cultures of twenty Trichoderma spp. and incubated at 28±1°C for this experiment. After 72 h of incubation, the colonies were evaluated for colony diameter, colony growth type, colony margin and the first appearance of green conidia.
 
Morphological characterization of native Trichoderma isolates
 
The cultures were incubated at 20°C for morphological characterization of Trichoderma spp. Microscopic preparations included slides made in 3% KOH followed by lactophenol-cotton blue from pustules with white conidia within 7 days of incubation at 25±2°C. The slide was examined under the microscope for mycelium type, phialides arrangement, conidial shape and colour, production of chlamydospores and their position, after the cover slip was placed. Bisset (1991) provided morphological and taxonomic keys that were used to identify the species.
 
Antagonistic potential of Trichoderma spp. against F. oxysporum f. sp. ciceris
 
Using a dual culture approach, all 20 isolates of Trichoderma spp. were tested for antagonistic capability against the pathogen. The vigorously growing culture of F. oxysporum f. sp. ciceris and Trichoderma spp. was sliced into 5 mm dia. mycelial discs and deposited on fresh PDA media on either side of the Petri plate. Control plates were inoculated with F. oxysporum f. sp. ciceris but not with Trichoderma spp. Each treatment was tested three times. Plates were cultured for 10 days at 28±2°C, until the pathogen completely covered the control plate. The degree of antagonism was assessed by comparing the pathogen’s radial development with that of a control Trichoderma culture. The percent inhibition was calculated in comparison to the control.
 
Where;
I= Per cent inhibition. 
C= Radial growth of pathogen in control.      
T= Radial growth of pathogen in treatment.
 
Volatile compounds production by Trichoderma spp
 
The inverted plate technique was used to examine isolates of Trichoderma spp. for the generation of inhibitory volatile chemicals (Dennis and Webster, 1971). PDA medium was put into sterilised Petri plates and allowed to solidify. The pathogen F. oxysporum f. sp. ciceris and the mycelial disc (5 mm) of Trichoderma spp. were infected in the centre of Petri plates. Both Trichoderma spp. and pathogen inoculated plates had their upper lids removed. The pathogen-containing plate was flipped over the Trichoderma spp. containing plate and the two were sealed with adhesive tape (parafilm), with Trichoderma spp. on the lower side and pathogen on the upper side. The pathogen-bearing Petri plate was inverted over the Petri plate containing PDA media as a control. Each treatment was repeated three times and the plates were incubated for five days at 28±1°C. The per cent inhibition was calculated by using formula of Vincent (1947).
 
Statistical analysis
 
The data obtained in the present investigations for various parameters in the experiments were subjected ANOVA for a completely randomized design (CRD) for in vitro studies by using OPSTAT programme.

Symptoms of wilt
 
Initially, indications such as yellowing of older leaves followed by drooping and drying, were noted in adult plants. Later, partial wilting, drying and death of the plant were observed. The entire plant wilted under extreme conditions. When the stem was longitudinally split apart, there were no evidence of rotting on the outside, but brown to black discoloration of internal tissue was visible (Fig 1) (Sageera et al., 2012; Kandoliya and Vakharia, 2013).
 

 
Isolation of pathogen F. oxysporum f. sp. Ciceris
 
At seven days after incubation, a whitish colony of fungus with fuzzy profuse mycelium was seen. It eventually turned to pink. The pathogen was identified using standard mycological criteria based on mycelial and conidial features (Barnett and Hunter, 1972). The fungus generated a large number of spindle-shaped, curved macroconidia with three to five septa, whereas microconidia were fusiform with rounded apex and no septa. The chlamydospores were globose to oval in shape, had a thick wall and appeared terminally or intercalarily (Fig 2). Nelson (1981) and Di-Pietro et al., (2003) made similar results, reporting that F. oxysporum develops colourless to pale mycelium that turns pink or purple with age when produced in culture with ovoid microconidia and spindle-shaped macroconidia with septa. Chlamydospores with one or two cells might be terminal or intercalary.
 

 
Isolation of Trichoderma spp. from rhizosphere soil of chickpea
 
Following incubation, all 20 isolates developed a typical greenish colony on TSM as well as features that were identical to Trichoderma under the microscope.
 
Cultural characterization of native Trichoderma isolates
 
At 72 h after incubation, all of the isolates had different radial mycelium growth. When compared to other isolates, TR-1, TR-13, TR-14 and TR-18 grew to a maximum of 90.00 mm after 72 h of incubation. Colony development was fluffy and elevated in all isolates and colony margins ranged from smooth to wavy. Green conidia production, on the other hand, was began 36 h after incubation and measured from the colony’s edge after 72 h. The results are presented in Table 1 and Fig 3.  Sharma and Singh (2014) additionally looked at the 30 Trichoderma isolates’ cultural features and growth rates. All of the isolates grew quickly and generated abnormally compact colonies with uneven margins, as well as a shift in conidial colour from white to various hues of green. Divya et al., (2015) investigated the cultural features of 44 Trichoderma spp. isolates, finding that all of the isolates grew quickly and covered the full Petri plate within 72 hours. There was some variation among the isolates in rate of growth, margin, colour of mycelia and sporulation.
 

 

 
Morphological characterization
 
Microscopic features such as mycelium, phialides organization, conidial shape and colour and chlamydospore development and location were observed. All of the isolates had hyaline, septate and branching mycelium, as well as narrow, pin and broad phialides. With terminal, intercalary and terminal to intercalary chlamydospores, pale to light green coloured conidia with globose to subglobose and oval shaped conidia were generated (Table 2 and Fig 4). Trichoderma harzianum (AF1), T. viride (AF2) and T. virens (AF3) were isolated from coconut rhizosphere soil samples by Ranjana et al., (2013). They observed that the branching of conidiophores, the shape of phialides, the emergence of phialospores and the shape of phialospores were used to make the identification.
 

 

 
Antagonistic potential of native Trichoderma spp. against F. oxysporum f. sp. ciceris
 
The findings demonstrated that all twenty Trichoderma spp. isolates were antagonistic to F. oxysporum f. sp. ciceris, with mycelium inhibition ranging from 48.52 to 91.85 per cent. TR-14 had the highest percent inhibition of 91.85 per cent, followed by TR-10 (83.33%) and TR-9 (75.93%). TR-16 showed the least inhibition (48.52%), followed by TR-15 (48.52%). (52.96%) (Table 3). T. asperellum was used by Nayak and Pandey (2017) to fight F. oxysporum f. sp. ciceris. Thaware et al., (2017) also tested Trichoderma spp. against F. oxysporum f. sp. ciceris and found that T. viride and T. harzianum inhibited the test pathogen’s mycelial growth by 75.55 and 73.77 per cent, respectively.
 

 
Volatile compounds production by native Trichoderma spp.
 
The findings revealed that each isolate produced a significant amount of volatile compounds, which differed between isolates. TR-14 (72.22%) produced the highest concentration of volatile compounds, followed by TR-19 (66.67%), TR-9 (65.93%) and TR-18 (65.93%). TR-18 produced the lowest concentration of volatile compounds (2.59%). When compared to the control, all of the isolates showed a substantial difference in mycelial growth inhibition (Table 4). Nagamani et al., (2017) also discovered that volatile metabolites produced by T. asperellum were the most effective in suppressing F. oxysporum f. sp. ciceris mycelial growth by 86.70 per cent. According to Mohit et al., (2019), volatile chemicals produced by T. harzianum had a strong inhibitory effect on the mycelial growth of F. oxysporum f. sp. ciceris (79.25%), followed by T. viride (79.25%).
 

A total of twenty native isolates Trichoderma spp. were shown to have antagonistic activity against F. oxysporum f. sp. ciceris and produced volatile chemicals that effectively reduced F. oxysporum f. sp. ciceris growth. Within twenty isolates of Trichoderma spp., there was heterogeneity in culture parameters such as colony diameter, type of colony growth, colony margin and initial appearance of green conidia. Trichoderma spp. are effective in the treatment of wilt disease.

The work has been undertaken as part of the master’s research programme at Department of Plant Pathology, College of Agriculture, University of Agricultural Sciences, Raichur. The first author is thankful to the University for providing facilities to conduct the work.

None.