Potato dextrose agar medium pda

Antagonistic bacteria were isolated from tomato rhizosphere in Madurai district, Tamil Nadu, India. The antagonistic activity of P. fluorescens VSMKU3054 strain was tested against R. solanacearum and fungal phytopathogens viz. Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum by the dual culture plate technique. A dual culture plate assay was used to assess the antagonistic activity of P. fluorescens VSMKU3054 against R. solanacearum [29 (link)]. R. solanacearum (10⁸ CFU/mL) was seeded on nutrient agar (NA) medium (Adding bacterial culture to the medium and plating) and a loopful of P. fluorescens VSMKU3054 was center-point inoculated. After two days of incubation at 30 °C, the zone of inhibition was measured to determine antagonistic activity. Potato dextrose agar medium (PDA) (Himedia Laboratories, Mumbai, India) was used for the dual culture plate technique for testing antagonistic activity of P. fluorescens VSMKU3054 against fungal pathogens. The bacterial colony was streaked on one side of the Petri plate and the fungal disk was placed on the center of the plate. PDA plates inoculated with pathogen alone served as the control. Antagonistic activities were evaluated by measuring the inhibition zones after 2–7 days of incubation at 28 °C (when the pathogen covered the entire plate in the control treatment).

The soil sample mentioned above was subjected to insect baiting to isolate entomopathogenic fungi, which were subsequently identified using our earlier methodologies and others (Vivekanandhan et al., 2021 (link); Mathulwe et al., 2023 ). The bait method employing third-instar larvae of Tenebrio molitor is an exceptionally sensitive technique used to isolate insect pathogenic fungi from soil. Fifteen T. molitor larvae in their third instar were transferred to a plastic container with 300 g of soil, measuring 15 cm (L) × 10 cm (W) × 10 cm (H). After securing the container with a lid, it was placed in an incubator set at 26 ± 1°C and 85% relative humidity. For 15 days, plastic containers were observed twice daily. After collecting the larval cadavers and sterilizing them for 2–3 min with 70% ethanol, the sterile cadavers were transferred to Petri plates (90 mm × 15 mm) containing pre-prepared potato dextrose agar medium PDA (HiMedia, India) (Perumal et al., 2023a (link)). Plates were kept at a relative humidity of 85% and a temperature of 26 ± 2°C for 7 to 10 days. The pure fungal cultures were isolated from the dead larval cadaver according to Balumahendhiran et al. (2019) (link) and Vivekanandhan et al. (2023) (link) and preserved in a biochemical oxygen demand incubator (BOD) (Smartscience, India) at 26 ± 2°C for future experiments.

The antifungal potential of strain S3 was evaluated against plant pathogen, Rhizoctonia solani obtained from Indian Agricultural Research Institute (IARI), Pusa, New Delhi, India by performing dual culture assay. The test isolate was streaked as straight line on four edges around the mycelial disc of fungal pathogen at the centre of Potato dextrose agar medium (PDA, Hi Media Chemicals, Mumbai, India) and incubated for 5 days at 28 °C.
The hyphal area of ~ 4 mm from the bacteria-fungus interaction zone (as shown in Fig. 4) was taken and placed on the glass cover slips and overnight fixation was done in 1.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.3). Similar procedure was done with fungal mycelium taken from the petri plate grown without bacterial inoculation (control). All the fixed samples were subsequently washed three times with phosphate buffer for 10 min each to remove loosely adhered cells. Followed by dehydration of samples using increasing concentration of ethanol—30–100% ethanol (v/v) at 10 min interval. Finally, the dehydrated samples were mounted on SEM stubs using carbon tapes and coated with gold: palladium (60:40) for visualizing under ZEISS EVO scanning electron microscope and photomicrographs were recorded.