2.1 Diseased sample collection:- During the Rabi 2020–2021 Crop Research Farm Nawabganj, C.S. Azad University of Agriculture and Technology, Kanpur, naturally infected linseed plants with symptoms of wilt disease were collected. The infected plants were taken to the lab and thoroughly tested for the existence of the causative organism.
2.2 Cleaning and sterilization of glassware:-All of the glassware used in this study was first cleaned with washing powder or detergent, then with a 0.1 percent mercuric chloride solution, and finally with tap water.
2.3 Preparation of cultural media:-
The Singh and Chaubey (1970) method was used to isolate the Fusarium wilt pathogen on potato-dextrose-agar medium. For preserving a pure culture of the wilt, a potato-dextrose-agar medium was used.
Potato-Dextrose-Agar medium (PDA medium)
For this analysis, a potato-dextrose-agar medium with the following composition was prepared using the method defined by Johnston and Booth (1983).
Ingredients
|
Gm/ml
|
Peeled potato
|
200.0 g
|
Dextrose
|
20.0 g
|
Agar agar
|
20.0 g
|
Distilled water to make volume
|
1000.0 ml
|
The potatoes were peeled and cut into 12 mm cubes. 200.00 g of potato cubes were rinsed and boiled in 500 mL water for 20 minutes. Cheese cloth was used to filter the potato broth. Agar was also melted in 500 mL of water by heating at the same time. The potato broth was then poured into the molten agar, along with dextrose.
By adding distilled water, the final volume was increased to 1000 ml. The pH level was raised to 7.0. While tubing, the stock solution was agitated to ensure that each sample had a proportion of solid matter. After that, it was sterilized in an autoclave at 15 psi for 20 minutes.
2.4 Sterilization of metallic objects:-
Before usage, metallic objects such as blades, forceps, and inoculation needles were sterilized by dipping them in spirit and heating them over a flame until red hot, and laminar flow was sterilized with an ultra violet light. Hands were disinfected with spirit as a general disinfectant.
2.5 Method of isolation:-
The diseased linseed plants were collected from the experimental plots of the C.S. Azad University of Agriculture and Technology, Kanpur's crop Research farm Nawabganj (U.P.). The pathogenic species were isolated from the roots of linseed plants that had been infected. The roots were first washed in tap water to extract dust particles before being thoroughly washed with sterilized water to remove the surface contaminants. The instruments that would be used were sterilized with 95% methylated alcohol. A sterilized blade was used to cut small sections of the diseased section as well as healthy parts into pieces.
Inside the laminar flow, the cut parts were surface sterilized with 0.1 percent mercuric chloride solution and washed thoroughly 3 to 4 times with sterilized water to eliminate any traces of mercuric chloride. Placed in With the aid of sterilized needles, these fragments were transferred to 2% Potato Dextrose Agar (PDA) medium in 90 mm Petri dishes that had been autoclaved at 15 psi for 20 minutes. The petri dishes were then moved to a B.O.D. incubator and held at 282°C for 7 days.
After 24 hours of inoculation, these incubated plates were checked for mycelial growth of the causal fungus once a day until the fungus grew. The hyphal tips from the advancing mycelium were cut and transferred into the culture tubes containing Potato-Dextrose Agar medium for further purification, identification, and microbial identification as soon as mycelial growth was clear around these parts. The pure culture of fungus was obtained by adopting single spore techniques.
2.6 Pure culture of the pathogen:-
The fungal isolates were purified using the single spore isolation technique. A thin layer of dilute spore suspension was poured on plain agar Petri dishes, and spores were allowed to settle on the agar surface. Settled spores were isolated from one another, picked under the microscope, and encircled in Petri dishes using a dummy cutter.. They were lifted along with agar blocks and transferred to Petri dishes containing sterilized 2 per cent PDA medium. After proper growth of fungus obtained by single spore culture regular sub- culturing was done to check contamination, till pure cultures were obtained. These cultures were sub cultured at monthly intervals and maintained on Potato-Dextrose-Agar slants under refrigeration at 6 to 8°C for further study.
2.7 Pathogenicity Test:- To determine the pathogenic existence of the fungus, a pathogenicity test of the isolate obtained from affected linseed roots was performed on the same host. The experiment was conducted in 30 cm diameter pots filled with approximately 5 kg sterilized soil (autoclaved at 1.20 kg/cm2 pressure for 2 hours) that had been washed with a 5.0 percent formalin solution. The inoculum was made by growing a pure culture of Fusarium oxysporum f. sp. lini in 250 mL conical flasks on sand cum meal (9:1) medium and autoclaving at 15 psi for 30 minutes. After that, each flask was inoculated with a pure culture of Fusarium oxysporum f. sp. lini and incubated for 15 days at room temperature (around 28–30°C). Controlled pots were filled with soil but no inoculum was added. Seeds of the linseed variety ‘Chambal' were disinfected with 0.1 percent mercuric chloride solution for 3 minutes, then rinsed with sterilized water, dried, and sown in the pots. In control containers, surface sterilized Chambal seeds were sown in pots with sterilized sand and cum meal medium without fungal inoculum. Up to 60 days after sowing, the pots kept in the glass house were scrutinized for seedling emergence and wilt incidence symptoms, supporting Koch's hypotheses.
2.8 Varietal Management:-
In Randomized Block Design, Eleven cultivars, namely, Jeevan, Sweta, Parvati, Surbhi, NDL-2004-05, Kiran, RLC-92, Indira Alsi, Dipika, Shekhar and susceptible culture ‘Chambal’, were seed in three replicas during the month of October 2020 under Wilt sick Field at Nawabganj, C.S. Azad University of Agriculture and Technology in Kanpur (U.P) in three replications, each with a plot size of 3m x 1.5m and a plant to plant spacing of 25cm x 10cm. To ensure a healthy harvest, recommended agronomic practices (80kg N and 40kg P/ha irrigation three) were used. Crops were kept an eye out for the appearance of disease. Maximum disease severity and seed yield per plot (kg) were reported separately for each variety.
2.9 Evaluation of sowing dates:-
The experiment was carried out on various dates ranging from 08 October to 15 December, 2020, at 07-day intervals, in the wilt sick plots of the Crop Research Farm Nawabganj of the C. S. Azad University of Agriculture and Technology, Kanpur (U.P.). RBD had three replications, the plot size was 3m x 1.5m, and Chambal was used as the variety. The initial plant population was registered. Wilt occurrence was registered for each date of sowing and observed regularly and final date was recorded as a maturity of crops in each treatment reported. The per cent disease incidence was calculated as-
$$\text{P}\text{e}\text{r} \text{c}\text{e}\text{n}\text{t} \text{w}\text{i}\text{l}\text{t} \text{i}\text{n}\text{c}\text{i}\text{d}\text{e}\text{n}\text{c}\text{e}\frac{\text{N}\text{o}. \text{o}\text{f} \text{w}\text{i}\text{l}\text{t}\text{e}\text{d} \text{p}\text{l}\text{a}\text{n}\text{t}\text{s}}{\text{T}\text{o}\text{t}\text{a}\text{l} \text{n}\text{o}. \text{o}\text{f} \text{p}\text{l}\text{a}\text{n}\text{t}\text{s}}\times 100$$
2.10 Effect of different pH level on the growth and sporulation of pathogen:-
The most appropriate Potato dextrose medium was chosen for the analysis of the influence of various pH levels on the growth of the fungus. The pH of the medium was changed to 4.5, 5.0, 5.5, 6.5, 7.0, 7.5, and 8.0. Each pH value was studied in triplicate using 50 mL medium in 150 mL conical flasks of the liquid media and sterilized at 1.1 kg/cm2 for 15 minutes. For each pH level, the pH was changed by adding N/10 HCl or N/10 NaOH. Flasks containing an equivalent volume of medium is inoculated with a culture disk of the fungus cut with a sterilized cork borer. The flasks were incubated at 252°C for 10 days before the fungus's mycelial mat was filtered, thoroughly washed with distilled water, dried in an oven at 60°C for 48 hours, and weighed.
2.11 Effect of different temperatures on the growth and sporulation of pathogen:-
The experiments were carried out on a 2% potato dextrose medium. The fungus was grown at four different temperatures: 20, 24, 28, 32 and 40 degrees Celsius. 150ml flat bottom flasks were filled with 50ml liquid media and sterilized for 15 minutes at 1.1 kg/cm2. Three replications were maintained for each temperature and incubated at above mentioned temperatures. The mycelia mat was filtered 14 days after inoculation on pre-weighed Whatman's filters paper No.42. To extract traces of salt that remained with the mycelium, the fungal mat that had been preserved on filter paper was carefully washed with sterilized distilled water. The filtered mycelia mats were first air dried, then baked with filter paper at 60°C for 48 hours before being cooled in desiccators. To determine the real weight of the mycelium, the original weight of the filter paper was subtracted from the overall weight.
The weight of the fungus was reported separately for each replication.
2.12 Evaluation of Bio-products:-
In RBD, a cannabis culture analysis of 13 treatments and 3 replications in pot under wire house conditions was carried out. Linseed cultivar Chambal was grown in earthen pots (25x20cm) with approximately 40kg of potting mixture made up of sterilized farm yard manure, sand, and soil (3:2:1). The antagonist’s mass culture was prepared on Bajra grains using Singh's method at el (1996). Separately, the sterilized soil was thoroughly mixed with 1% (w/v) pure pathogen inoculums. The seed (5g/kg seed) and soil treatment (10g/kg soil) with Trichoderma viride and T. harzianum, the seed (10g/kg seed) and soil treatment with Pseudomonas fluorescens (20g/kg soil), and botanicals (seed kernel extract with Azadirachta indica (10% w/v) and extract of Allium sativum (10% w/v) were combined separately with the pathogen infected soil. Pots containing soil pathogen inoculums but no antagonist were used as controls. These treatments were compared to seed and soil treatments with the chemical fungicide carbendazin (0.2 per cent). The seeds were sown in pot soil at a rate of 10 seeds per pot after being treated with various bio-agents, plant products, and fungicides. Water was added when needed to keep the soil moist. The emergence of disease progression was noted in all of the therapies published. The final number of wilted plants was counted. The percent disease occurrence was estimated using the experiment's date of sowing, and the percent disease control was calculated using the formula below:
$$\mathbf{P}\mathbf{e}\mathbf{r} \mathbf{c}\mathbf{e}\mathbf{n}\mathbf{t} \mathbf{d}\mathbf{i}\mathbf{s}\mathbf{e}\mathbf{a}\mathbf{s}\mathbf{e} \mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}=\frac{\mathbf{U}\mathbf{T}-\mathbf{T}}{\mathbf{T}}\times 100$$
Where,
UT- Untreated and T- Treated
2.13 Experimental details:
Details of the treatments and layout are given below:-
Variety – RLC-92
Design – R B D
Replications- 3
Treatments- 13
Location - Crop Research Farm, Nawabganj, Chandrashekhar Azad University of Agriculture and Technology, Kanpur (U.P.).
2.14 Treatment details:
T1: Seed treatment with Trichoderma viride (5g/kg seed)
T2: Seed treatment + soil treatment with Trichoderma viride (10g/kg soil).
T3: Seed treatment with Trichoderma harzianum (5g/kg seed).
T4: Seed treatment + soil treatment with Trichoderma harzianum (10g/kg soil).
T5: Seed treatment with Pseudomonas fluorescens (10g/kg seed).
T6: Seed treatment + soil treatment with Pseudomonas fluorescens (20g/kg soil).
T7: Seed treatment with seed kernel extract of Azadirachta indica (10per cent w/v).
T8: Seed treatment + soil treatment with seed kernel extract of A. indica (10 per cent w/v).
T9: Seed treatment with extract of Allium sativum (10 per cent w/v).
T10: Seed treatment + soil treatment with extract of Allium sativum (10 percent w/v).
T11: Seed treatment with carbendazin (0.2 percent).
T12: Seed treatment + soil treatment with carbendazin (0.2 percent).
T13: Control.
2.15 Statistical analysis:
Both laboratory and pot culture experiments were carried out in accordance with Goon et .al’s(1931) Fully Randomized Block Design (RBD). To reach the result, the data collected and tests were statistically analysed. The major variations between therapies were evaluated using a variance ratio test at a 5% level of chance.