Experimental site
The experiment was conducted during summer season (2015-16) in the polyhouse of the Department of Horticulture, authors institute, Hisar, Haryana (India).
Collection, isolation and maintenance of root-knot nematode and fungus
Cucumber roots showing galls were collected from the naturally infested polyhouses during survey and brought to the laboratory. Nematode and fungus were isolated, identified by Patil et. al., (2018) and pure culture of both the organisms were maintained. Fungus was grown on sand maize meal medium for mass production and nematode culture was maintained in screen house on brinjal. Nematode juveniles were collected by sodium hypochlorite method for inoculation.
Source of bioagents
Bioagents Trichoderma viride (107 cfu/g), Pseudomonas fluorescence ((108 cfu/g)), Purpureocillium lilacinum ((106 cfu/g) and liquid formulation of bio-agents (T. viride + P. fluorescence + P. lilacinum) were procured from IIHR, Bangalore were used in this study.
Seed treatment with bioagents
Carboxy methyl cellulose (CMC) was used as an adhesive for treating cucumber seeds with fungal and bacterial bioagents (1x108 cfu/ml.). For preparing 1% (v/w) adhesive solution, adhesive was added to fungal and bacterial bioagents. Now required amount of seeds was taken in a petriplate and the fungal as well as bacterial bioagent with the adhesive was added on the seeds stirring continuously and stopped when all the seeds got smeared with suspension. After treating, the seeds were dried in shade for 6 hours and used for sowing.
Extraction of nematodes from soil
Nematode extraction from soil was done by Cobb’s sieving and decanting method (Cobb, 1918) followed by modified Baermann funnel method (Whitehead & Hemming, 1965).
Experimental setup
This experiment was conducted in polyhouse ((24± 2 º C)) to evaluate the effect of seed treatment of bio-agents in cucumber for management of root-knot nematode and fungus (Fusarium oxysporum f. sp. cucumerinum). Experiment was conducted in pots (1 kg capacity). The soil of each pot (1500 g) was a mixture of sand, sandy loam and peat moss (2:1:1), which was previously steam-sterilized (15 Psi at 121 º C) with an autoclave for 30 min. In previous in vitro test, we tested bio-agents against M. incognita. Based on the results of these tests (un-published data), two promising doses namely: T. viride, P. fluorescence, P. lilacinum 10 and 20 g/kg seed and liquid formulation of bio-agents (T. viride + P. fluorescence + P. lilacinum) 10 ml/ kg and 15 ml/ kg seed were selected for the present study. Seed treatment of bio-agents were tested against nematode alone, fungus alone and nematode and fungus simultaneously inoculated. Treatments were arranged in completely randomized design with four replications. Cucumber seeds (cv. Sania) were treated with bio-agents for 2 hours dried in shade for one hour and then sown to each pot treatment wise @ 5 seeds per pot. One plant per pot was maintained. Sterilized soil was inoculated with 1000 J2/ kg soil of root-knot nematode and fungus 50g /kg soil.
Maintenance of plants
General care and maintenance of plants were done as recommended by CCS Haryana Agricultural University, Hisar. Hoagland solution was applied at 10 days’ interval. Hoeing, watering other practices were done as per recommendation.
Observations
After crop maturity at 60 days after sowing, plants were harvested and 200 cc soil was collected for estimation of nematode population. Observations viz. plant growth parameters (shoot and root length, fresh and dry shoot and root weight), number of galls per plant, number of egg masses per plant, number of eggs/egg mass, final nematode population (Cobb, 1918), pre-emergence damping off (after 15 and 30 days) were taken.
Statistical analysis
Data were analyzed in a factorial completely randomized design with four replications. Data were subjected to analysis of variance (ANOVA), and the means were compared with a critical difference. A significance level of P ≤ 0.05 was used in all analyses. Nematode data were transformed, using square root transformation to homogenize error variances. All calculations were performed with the software available online at the website www.hau.ac.in.