Field experiments were conducted in
rabi season of 2021- 22 at Agriculture Research Farm, while
in vivo experiments were performed in the laboratory of department of Plant Pathology, Lovely Professional University, Phagwara (Punjab).
In-vitro evaluation of fungicides against A. alternata
The efficacy of the fungicides was checked
in vitro based on the mycelial growth inhibition of
A. alternata using the poisoned food technique (Plate 1). All four fungicides
viz. Copper oxychloride 50% WP, Azoxystrobin 18.2% SC + Difenoconazole 11.4% SC, Hexaconazole 5% SC, Carbendazim 12% + mancozeb 63% WP) were tested against the pathogen at four different concentrations given 25, 50, 75 and 100 ppm. In Erlenmeyer flasks (250 ml), a stock solution of 100 ml was prepared by adding distilled water to each fungicide. Later working solution was made by adding the stock solution to a 250 ml flask containing potato dextrose agar medium (PDA). The chemicals were added only after sterilization was done in the autoclave. The potato dextrose agar having the required concentrations was poured into Petri plates that were sterilized before work in a hot air oven for about an hour. After the media solidified each plate was inoculated with the pathogen culture (min 7 days old) and a single Petri plate with no fungicide was also inoculated, it served as untreated control. Each treatment consisted of 3 replicates. Later the Petri plates were kept in an incubator at 26±1°C Inspection of Petri plates was done regularly for a week. The mycelial growth was measured when the control plate was fully covered
(Noman et al., 2018).
In vitro evaluation of bio-agents against A. alternata
By using the dual culture technique three bio-agents were tested against
Alternaria alternata
viz.
Trichoderma viride,
T. harzianum and
T. asperellum with three replications each. PDA was poured into Petri plates and sterilized before the procedure later pathogen inoculum was carefully put into a corner of the Petri plate and on the other corner a species of
Trichoderma. Later kept in an incubator at 25±1°C
(Kurhade et al., 2021).
In-vivo evaluation of fungicides and bioagents against A. alternata
Trials were conducted with eight treatments in three replications using randomized block design (RBD). The variety ‘Harit Sobha’ was sown with a plot size of 6 m×2.5 m with the row-to-row distance of 12 inches and plant to plant 5 cm. The sowing of the crop was done on 10 December 2021 at the research farm of Lovely Professional University. A spore suspension of 106 conidia/ml of 14-day-old culture of
A. alternata was sprayed on 2 week old spinach plants to confirm pathogenicity. Symptoms of the disease were visible in the field after 45 days of sowing. With the advancement of the disease, the necrotic spots get turned into blight
(Aslam et al., 2019). Treatments were given at a 15 days interval. The severity of the disease was measured one week after each spray using a 0-4 scale (Biswas and Ghosh, 2018) where 0, no disease; 1, 1-10%; 2, 11-15%; 3, 16-50%; 4, 51-100% plant showing leaf spot. Four fungicides
viz. Hexaconazole 55% WP, Carbendazim 12% + Mancozeb 63% WP, Azoxystrobin 18.4% SC + Difenoconazole 11.4% and Copper oxychloride 50%WP were selected to check their efficacy against
Alternaria spp. causing Alternaria leaf spot in spinach. Three biological control agents
viz.
Trichoderma viride,
T. harzianum and
T. asperellum at various concentrations. A total of three sprays were administered in the gap of fifteen days. Data was recorded and later converted into Percent disease index.
Statistical analysis
The experiments were designed as RBD in the field and CRD under laboratory condition with 3 replicates of each treatment. To evaluate the treatment effect, ANOVA was used to compare the means between treatments and OPSTAT software was applied for data analysis.
