Collection of diseased samples
Chickpea plants, both healthy and diseased, showing typical wilt symptoms, were collected from Nawabganj Research Farm, C.S. Azad University of Agriculture and Technology, Kanpur and brought to the laboratory for isolation of pathogen.
Isolation and purification of pathogen
The chickpea roots affected by wilt were initially rinsed in tap water to eliminate dust particles. The affected root pieces were then sterilized in a 0.1 per cent solution of mercuric chloride for one minute, followed by thorough washing with distilled water 3-4 times. Excess water was removed by placing the root pieces between sterilized blotting papers. The PDA (Potato Dextrose Agar) medium, used for isolating the pathogen, had the following composition: 200 g peeled potato, 20 g dextrose, 20 g agar and 1000 ml distilled water, prepared according to the method described by
Johnston and Booth (1983). These pieces were then inoculated on PDA plates and placed in an incubator at 25±C for 24 hours. Single spore isolation technique was used to purify the culture and the obtained culture was maintained on potato dextrose agar medium slants for further investigation.
Identification of pathogen
The pathogen was identified by examining its cultural and morphological traits, comparing them with the characteristics described by
Booth (1971) for
Fusarium oxysporum f. sp.
ciceri given in Fig 1.
The growth pattern, cultural attributes and morphological features were analyzed on PDA to determine its identity. Growth habit characters of the fungus on seeds in Standard blotter method and cultural and morphological characters of the colony on potato dextrose agar medium in agar plate method (
ISTA, 1991) were observed. The morphological characters include mycelium colour, branching pattern, septation and width. The size of the hyphae, micro conidia, macro conidia and chlamydospores was based on the measurement. The nomenclature of the colour was based on the colour standards given by
Ridway (1912).
Sample collection, isolation and purification of Trichoderma viride
Soil samples were collected from the rhizospheric soils of chickpea fields. These samples were carefully placed in designated polythene bags, clearly labeled with collection date and transported to the laboratory for further analysis.
Trichoderma selective medium (TSM) containing specific ingredients such as MgS0
4.7H
2o, K
2HPO
4, KCl, NH
4NO
3, Glucose, Chlorempenicol, Apron 35SD, Captan, Rose Bengal, Agar-agar and distilled water was used to isolate
Trichoderma, this method was described by
Elad et al. (1981). The soil samples were processed using the serial dilution plate technique as outlined by
Johnson and Crul (1972) and the plates were then placed in incubation at 28±1°C for 5 days. Colonies were observed and recorded between the 3
rd and 5
th days and individual colonies were selected for further investigation. Purification of
Trichoderma isolates was accomplished using the single spore isolation method, where sub-culturing was carried out from the growing end of a new colony. A small amount of spores from each isolate was aseptically streaked onto potato dextrose agar (PDA) plates by using a sterilized inoculating needle.
Morphological characterization of Trichoderma spp
Isolated
Trichoderma cultures were grown on PDA media for seven days. Colonies exhibit distinctive features such as growth pattern, rate of growth, odor and coloration, which are indicative of their classification as
Trichoderma (
Gams and Bissett, 1998), were recorded.
Microscopic features
The common methods are applied for species identification typically involves using of a light microscope to examine morphological traits. These traits include the branching pattern of conidiophores, the elongation and shape of conidiophores (whether coiled, straight, or undulate), the morphology and dimensions of phialides and the shape of conidia (
Bissett, 1991; Rifai, 1969; Samuels et al., 1996) are given in Fig 2.
In vitro screening of T. viride isolates against the chickpea wilt causing pathogen
Trichoderma viride isolates were evaluated for its effectiveness against
Fusarium oxysporum f. sp.
ciceri using the dual culture technique. Both the target fungus and the fungal antagonist were cultured on Potato Dextrose Agar (PDA) medium. Bits of 5 mm diameter were cut from the periphery of seven days old cultures of the pathogen and the antagonistic fungi (
T. viride), then placed diametrically opposite each other at a 5 mm distance from the edge of the Petri dish. Each Petri dish was inoculated with a target pathogen, with one plate serving as a control without an antagonist. They were then incubated at 28±2°C in a BOD incubator. After 7 days, the presence of inhibition zones between
T. viride and the pathogen was observed. The radial growth of the pathogens on both the dual culture and control plates was measured and the percentage inhibition of the pathogen was calculated according to the method described by
Vincent and Budge (1990).
In vivo effect of seed biopriming with different doses of Trichodrema viride
The field experiment was carried out during
rabi season 2013-14 and 2014-15 over two consecutive years, at Nawabganj Research Farm, C.S. Azad University of Agriculture and Technology, Kanpur was aiming to address chickpea wilt management through the biopriming of chickpea seeds using various doses of a bio-formulation containing
Trichoderma viride. The bioformulations were prepared from isolates of
T.viride 03 which were found superior during
in vitro condition, screening against
Fusrium oxysporum f.sp.
ciceri. Fungal biomass of
T. viride 03 isolates were mixed with talcum powder @ 1:2 ratios. This experiment had 7 treatments. Prior to sowing, chickpea seeds underwent soaking (biopriming) in four different concentrations of
T. viride namely 5, 10, 15, 20 g/Kg seeds that acted as four treatments for the experiment, fifth treatment involved application of
T. viride in the soil furrows at the rate of 2.5 kg/h,In another treatment, seed biopriming was carried out with 0.2% Bavistin per kg of seed while untreated seeds served as the control treatment. Soil was inoculated with
Fusarium oxysporum f. sp.
ciceri @ of 5.0g/m2, mixed into the soil before sowing. The experiment involved the following treatment combinations as explained here:
T1: Seed biopriming with 5g of
T. viride formulation per kg of seed.
T2: Seed biopriming with 10g of
T. viride formulation per kg of seed.
T3: Seed biopriming with 15g of
T. viride formulation per kg of seed.
T4: Seed biopriming with 20g of
T. viride formulation per kg of seed.
T5: Soil application of
T. viride formulation in furrows at a rate of 2.5 kg/h.
T6: Seed biopriming with 0.2% Bavistin per kg of seed.
T7: Control (without treatment).
Observations recorded
Following observations such as seed germination%, plants survival%
, yield/ha, test weight in g, root length of plant in cm
, shoot length of plant in cm were recorded as shown in Fig 3.
Statistical analysis
The data obtained from experiment were analyzed statistically by analysis of variance (ANOVA) according to randomized block design (RBD). The treatments means were subjected to Fisher’s least square difference (LSD) post hoc test, which compared treatment means with critical differences (CD) at 5% level of significance as computed by OPSTAT software.
