Fungal pathogens and culture conditions
Four soil and tuber borne pathogens of potato and one (Sheath blight) from rice were used in this study. Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Fusarium spp. were isolated from infected diseased samples collected from experimental potato fields, ICAR-Central Potato Research Institute-Regional Station, Meerut, Uttar Pradesh, India. R. solani AG1-IA was collected from sheath blight infected rice plant. S. rolfsii, Fusarium spp. and R. solani AG1-IA were cultivated on potato dextrose agar (PDA, HiMedia) in the dark at 27 ± 1 °C. R. solani AG3 and S. sclerotiorum were grown on PDA 24 ± 1 °C and 22 ± 1 °C, respectively. All pathogen cultures were stored in PDA slants at 4 °C for further use.
Soil samples collection and bacteria isolation
Rhizospheric soil samples were collected from rice, maize and potato fields in 3 major potato producing states of India, namely Uttar Pradesh, Punjab and Bihar, where rice-maize-potato/rice-potato cropping sequences were followed. A total of 22 rhizospheric soil samples were collected from different locations and stored at 4 °C for further processing. Soil bacteria were isolated using serial dilution technique. Briefly, 1 g of soil sample was suspended in 9 ml of sterile distilled water and vortexed on a vortex mixture for 5 min at room temperature. The soil mixture was diluted at 1:10 ratio with sterile distilled water up to 10–7. Aliquots of 500 μl from 10–4 to 10–7 were distributed in Nutrient Agar and Pseudomonas spp. specific media plates and spread with a sterile plastic rod spreader. The culture plates were incubated at 30 ± 1 °C for 48 h in the dark. After incubation, the culture plates were observed under UV-trans- illuminator. The morphologically distinct and fluorescent colonies were sub-cultured onto the nutrient agar medium in separate plates to isolate single colonies. The purified bacterial isolates were conserved in LB-glycerol (70:30 v/v) at − 80 °C for further use.
Morphological characterization of bacterial isolates
Cultural and morphological characters, such as colony colour, shapes, Gram reaction (Hucker and Conn 1923), spore formation, motility, and catalase activity (Whittenbury 1964) of all bacterial isolates were performed by following standard protocols. Bacterial motility was observed by hanging drop method described by Bertrand et al. (2001). Spore forming bacteria was determined by method described by Harrigan and MacCance (1976).
In vitro screening for antagonistic activity
Direct in vitro antagonism activities of bacterial isolates were assessed through dual culture technique on PDA medium in Petri dishes. For this purpose, bacterial inoculums were prepared in 25 ml Nutrient Broth (NB) medium inoculated by a single colony and incubated at 30 °C and 120 rpm for 24 h. A 5-mm agar disc covered with actively growing mycelium of each fungal pathogen was placed at the centre of Petri dishes separately. To evaluate the potential antagonistic activity against fungal pathogens, 2 streaks of 20 mm of the tested bacterial culture were made at each side of the pathogen mycelium agar disc. As control, non-inoculated NB media was streaked at each side of the pathogen mycelium agar disc. Inoculated Petri dishes were sealed with parafilm and incubated at 27 ± 2 °C in the dark until the fungal mycelia reached the edge in the control dishes. Mycelial growth inhibition towards the bacterial inoculums was indicative of antagonistic activity. The test was repeated twice with 3 replications. The mycelial radial growth of each pathogen was measured and mycelial growth inhibition (%) was calculated (Ji et al. 2013). The following formula was used:
$${\text{Mycelial}}\,{\text{growth}}\,{\text{inhibition}}\,{(}\% {)}\, = \,{\text{Mycelial}}\,{\text{growth}}\,{\text{in}}\,{\text{control}}\, - \,{\text{Mycelial}}\,{\text{growth}}\,{\text{in}}\,{\text{treatment/Mycelial}}\,{\text{growth}}\,{\text{in}}\,{\text{control}}\, \times \,100$$
Evaluation of the effect of volatile organic compounds (VOCs)
To evaluate the antifungal effect of volatile organic compounds (VOCs) produced by bacterial isolates on fungal pathogens, mouth-to-mouth sealed plate assay previously described by Raza et al. (2016) was used. Bacterial suspensions (100 ul) of 1 × 107 cfu/ml was spread on plates containing Nutrient Agar (NA) medium and incubated at 30 °C for 24 h in the dark. After incubation, the lid of each plate was replaced by a PDA plate containing an agar disc (5 mm diameter) covered with mycelium of pathogen. The pathogen containing PDA plate was placed at upper side and bacteria inoculated plate placed at lower side. Plates containing sterile distilled water without bacterial suspension served as control. All plates were sealed with a double layer of Parafilm. The sealed sets of plates were incubated at 27 ± 2 °C, and the observations were recorded at 24 h intervals for 3 days. The test was repeated twice with 3 replications. The mycelial growth inhibition (%) of each pathogen was calculated according to the Trivedi et al. (2008).
Quantitative evaluation of antagonism of the selected isolate in different media
For quantitative evaluation of antagonistic activity of the screened bacterial isolate namely Pf14, 5 different broth media like, potato dextrose, nutrient, King’s B, tryptone soya, and tryptone yeast extract broth were used. One ml (A600 = 0.2) culture broth of the selected isolate, and a 5-mm agar disc covered with actively growing mycelium of each fungal pathogen were co-cultured in 50 ml different broth mediums in separate 250 ml conical flasks. The inoculated flasks were incubated at 27 ± 2 °C and 120 rpm for 2 days on a rotary incubator shaker. Broth flasks inoculated with only fungal pathogens served as control. The test was repeated twice with 3 replications. After incubation, biomass was filtered through pre-weighed Whatman No. 1 filter paper and dried at 70 °C for 24 h. The differences in dry weights between the fungal pathogens and the bacterium or the control culture (without bacteria inoculation) were recorded. Percentage (%) reduction in fungal pathogens biomass in co-culture compared to control was calculated according to Trivedi et al. (2008).
Evaluation of the effect of cell-free culture filtrates from selected isolate
The selected isolate i.e. Pf14 was inoculated in 50 ml NB medium and grown at 30 ± 2 °C on a rotator incubator shaker at 120 rpm. Broth culture after 24 and 48 h of incubation was centrifuged at 10,000 rpm for 10 min. at 4 °C and supernatant was taken. Cell-free culture filtrate (CFCF) was obtained by passing the supernatant through 0.22 μm pore size syringe filter. A 5-mm mycelium disc of each pathogen was placed in the centre of each plate. Two wells (5 mm) were made 3 cm away from the mycelium disc and aliquoted with 100 μl of CFCF. Plates in which wells were aliquoted with non-inoculated NB served as control. The plates were incubated at 27 ± 2 °C for 5 days in the dark. The test was repeated twice with 3 replications. Percent (%) mycelial growth inhibition was calculated as described above.
Evaluation of the phosphate solubilisation activity
Bacterial isolates were evaluated for their phosphate solubilisation ability on Pikovskaya’s (PVK) agar media supplemented with insoluble tri-calcium phosphate (TCP) at final concentration of 0.5%. Each bacterial isolate was spot inoculated at the centre of PVK agar plates under aseptic condition. The plates were incubated at 28 ± 2 °C for 7 days in the dark. After incubation, colony diameter and halo zone were measured for each isolate. A clear halo zone around the growing bacterial colony indicated phosphate solubilisation. Phosphate solubilisation index (SI) and solubilisation efficiency were calculated using the formula described by Edi-Premono et al. (1996):
$$\begin{aligned} & {\text{Phosphate}}\,{\text{Solubilisation}}\,{\text{Index}}\,({\text{SI}})\, = \,\left( {{\text{Colony}}\,{\text{diameter}}\, + \,{\text{Halo}}\,{\text{zone}}\,{\text{diameter}}} \right){\text{/Colony}}\,{\text{diameter}} \\ & {\text{Phosphate}}\,{\text{Solubilisation}}\,{\text{Efficiency}}\,({\text{SE}})\, = \,\left( {{\text{Colony}}\,{\text{diameter}}\, - \,{\text{Halo}}\,{\text{zone}}\,{\text{diameter}}} \right){\text{/Colony}}\,{\text{diameter}}\, \times \,100 \\ \end{aligned}$$
Evaluation of antagonistic and growth enhancement potentials of Pf10 and Pf 14 under field conditions
Plant materials and experimental design
The experiments were laid down at Experimental Field Station, ICAR-Central Potato Research Institute, Regional station, Meerut, Uttar Pradesh, India during Rabi season (year 2020–2021) in Randomized Block Design (RBD) with 3 replicates. Fully infested seed tubers of cv. Kufri Bahar were used for planting. Spore suspension and talc based formulation of Pf10 and Pf14 were used to evaluate their efficacy against the black scurf of potato. The infested tubers were submerged in spore suspension (109 cfu/ml) and talc based formulation (0.5%), separately, for 1.5 h. The shade dried treated seed tubers were planted in second week of November in a standard plot size of 9.0 m2 size. Five rows of three meters length and 60 × 20 cm row × plant spacing were maintained. The crop was grown by the application of 180:80:100 kg N: P2O5:K2O per hectare and remaining other activities were followed as per standard recommended agronomic package and practices of the region.
Growth enhancement activities
The agronomical characteristics such as germination (%) was recorded after 30 days of planting, whereas plant height (cm), no. of stems/ plant, no. of leaves/plant and root length (cm)were recorded after 60 days of planting, following standard procedures. Marketable tuber yield was recorded at the time of harvesting.
Assessment of disease
Disease incidence of stem canker was recorded at 35 DAP (Days after Planting) and black scurf incidence was recorded at harvesting using randomly selected 75 tubers from each plot. The tubers were washed with running tap water to remove soil and visualize clear symptoms of black scurf. These tubers were categorized as a 0–4 scale as their coverage of tuber infection using the following scale:
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0: No sclerotia
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1: 1-25% of tuber surface covered
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2: 26-50% of tuber surface covered
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3: 51-75% of tuber surface covered
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4: >75% of tuber surface covered
Statistical analysis
In each experiment 3 replications was maintained for in vitro and field studies, 4 replications were maintained. Data were statistically analyzed by one-way ANOVA and two-tailed t tests using software SPSS (IBM Analytics, Armonk, NY, USA). Data were presented as mean ± standard deviations (mean ± SD) where appropriate. Percent data were analyzed after angular transformation and intergroup differences were considered to statistically significant at P ≤ 0.05.
