2.1 Isolation of chitinolytic bacterial strains
The bacteria used in this study (Table 1) were isolated from various soil samples (rhizospheric fertile soil, rhizospheric nematode infected soil, rhizospheric nematode non-infected soil, rhizospheric saline soil, crab shell rich soil, etc.) from diverse locations across the states of Maharashtra and Gujarat in India on colloidal chitin agar (CCA) media. Colloidal chitin was prepared as per the protocol by Hsu and Lockwood (1975), using chitin flakes (Himedia-GRM1356, India). A CCA medium was prepared as described by Shahbaz and Yu (2020) and used to isolate chitinase-positive strains using a serial dilution scheme. Plates were incubated at 30 ± 2 0C for seven days. Colonies surrounded by a clear zone on the CCA plate are considered as chitinolytic bacteria. These strains were maintained as glycerol (SDFCL-38454 L05, India) stocks and stored at -80˚C till further studies.
2.2 Characterization of the selected bacterial isolates
The pure colony of each bacterial strain was inoculated separately in a 500ml Erlenmeyer flask containing 50ml nutrient broth (Himedia- M002, India) and incubated at 37 °C, at 200 rpm, for 18h to determine the Gram nature by the Gram's staining method (Coico, 2005). The sporulation capacity of the isolates was evaluated by inoculating pure colonies in nutrient broth (Himedia- M002, India) and incubating for 48 h at 37 °C, 200 rpm by spore staining technique (Schaeffer and Fulton, 1933). A hemolysis test was conducted by spot inoculating sheep blood agar plates (Himedia-MP1301, India) with the bacterial cultures cultivated overnight and further incubating the plates for 24–72 hrs at 30 ± 2 0C. The zones of clearance around the colonies were considered as an indicator of the presence of hemolysis (Nayak and Mukherjee, 2011). Further, a tube staining assay was employed to qualitatively assess the exopolysaccharide (EPS) production as an indication for biofilm formation capacity. (Jain et al., 2013).
2.3 Qualitative extra-cellular enzyme analysis
Cultures were assessed for their ability to produce extra-cellular hydrolytic enzymes like chitinase, protease, lipase, and gelatinase. A total of twenty-five Gram-positive, sporulating, and non-hemolytic bacterial isolates were selected for enzyme analysis. Enzyme analysis was qualitatively conducted using an agar (Himedia-RM026, India) medium, with a suitable substrate for each enzyme. A protease was determined on skim milk powder (Himedia- GRM1254, India) agar media as suggested by Vazquez & Mac Cormack,(2002), lipase activity was determined on tributyrin agar (Himedia- M157, FD081, India) media as described by Veerapagu et al., (2013), gelatinase activity was determined on gelatinase agar (Merck-M9512jh70, Germany) as described by Sharma et al., (2015) and colloidal chitin agar was used to determine the chitinase activity as suggested by Jha et al., (2016). The cultures' enzyme activity was reported as mentioned by (Tennalli, 2012).
2.4 Anti-fungal activity of bacterial isolates
Anti-fungal activity of the selected twenty-five strains was analyzed against common plant pathogenic fungi, Fusarium oxysporum NFCCI 651, and Alternaria alternata NFCCI 261. These fungal strains were procured from the National fungal culture collection of India (NFCCI) and preserved on potato dextrose agar (Himedia-M096, India) slants. The activity was determined by the agar well-diffusion method suggested by El Barnossi et al. (2020), with modifications as required. Briefly, an aliquot consisting of 1 mL of the inoculum, containing approximately 104 spores of the pathogenic fungal strain, was added to 20 mL of melted potato dextrose agar (Himedia- M096) medium, maintained at 45 °C. This was then poured into 90 mm diameter size sterile petri plates and allowed to solidify at room temperature for an hour. Small wells were created in the agar plates using a cork borer (5 mm). A 25µl of overnight grown bacterial isolates, exhibiting an optical density of 1.0 (pre-adjusted with sterile distilled water) at 600 nm, was loaded into the wells. The petri plates were maintained at 4 °C for 2 hrs to allow uniform diffusion of culture inoculum into the agar. Later, plates were incubated aerobically at 30 ± 2 0C for 48 h, and the diameters of the clearance zones were recorded, as published earlier. (Oyedele & Ogunbanwo, 2014).
2.5 In vitro activity
The in vitro nematicidal activity was evaluated by the egg hatching inhibition assay and by studying the mortality of second-stage juveniles (J2) of the M. incognita.
2.5.1 Preparation of nematode inoculum
The RKN-infected roots from tomato plants were used for extracting the nematode eggs. Briefly, the infected roots were chopped into pieces having a length of 2-3 cm and were treated for 3 min with 0.05% w/v of sodium hypochlorite (SDFCL-33040 L05, India) under stirring. The released eggs were harvested by passing through a series of sieve (1mm, 0.250mm, 0.045mm and 0.025mm), followed by (36%, w/v) sucrose (Himedia-MB025, India) gradient centrifugation at 3000 RPM. The supernatant was poured over a sieve of the size 500 mesh (0.025mm), washed with sterile distilled water several times, and collected as a suspension. The contents of this suspension were examined under a stereo zoom microscope (Olympus- SZ61, Japan) to identify the presence and density of eggs. Further, J2s were obtained by pouring the egg suspension over 4-ply tissue paper mounted on a piece of wire mesh, as described by Kumar et al. (2018). This assembly was fixed in a petri plate, to which fresh water was added and maintained at 24 ± 1°C till the eggs hatched. The freshly hatched J2s were used during the experiments.
2.5.2 Identification of RKNs
The galls from tomato roots were dissected to collect the female nematodes. The perineal pattern of these female nematodes was studied under a compound microscope (Olympus-CX43 RF, Japan, 40X resolution) as described by (Taylor & Netscher, 1974).
2.5.3 Effect on egg hatching
The effect of twenty-five bacterial isolates on egg hatching was evaluated by the method reported by Kumar et al. (2018), with necessary modifications. The test was conducted in pre-sterilized 6-well tissue culture (Tarson. Cat. No.980010, India) plates, with each well having a capacity of 12 ml volume. In brief, 1.0 mL of egg suspension containing approximately 100 eggs of M. incognita was mixed with 2.5 ml of sterile distilled water and 0.5 ml of bacterial suspension, having an optical density of 1.0 (pre-adjusted with sterile distilled water) at 600 nm, and grown overnight in a nutrient broth (Himedia-M002, India). All the analyses were performed with three replications, and the plates were incubated at 28°C for five days. Nutrient broth and water were maintained as controls. The effect of bacterial isolate on egg hatching was observed microscopically (Olympus- SZ61, Japan) on 3rd, 4th and 5th day of incubation and calculated by the formula (Sikandar et al., 2020) stated in equation 1.
2.5.4 Effect on mortality of juveniles
Juvenile mortality was evaluated after slight modifications in the method stated by Kumar et al. (2018). An aliquot of 0.5 mL of the respective overnight grown in a nutrient broth (Himedia-M002, India) bacterial suspensions (at 1.0 OD600nm) was mixed with 2.5 ml sterile distilled water and 1.0 mL of nematode suspension containing approximately 100 M. incognita J2's, in 6-well sterile tissue-culture plates, to test the percent mortality of nematodes. Sterile water and nutrient broth medium served as controls. The mobility of the J2's was confirmed microscopically by probing the nematodes with a needle, while the immobile ones were considered dead. All the analyses were performed with three replications. The percentage mortality was recorded after 2-4 days and calculated with the formula (Sikandar et al., 2020) stated in equation 2.
2.6 Electron microscopic observations of bacteria-treated eggs
The virulence of the isolates against M. incognita eggs was confirmed by environmental scanning electron microscopy (E-SEM) analyses. For this, 1mL of suspension containing unhatched eggs of M. incognita was treated with 0.5 mL of actively growing ZB-HT4 bacterial isolate (OD600nm-1.0) and maintained for three days at room temperature. The treated and non-treated eggs specimen were prepared for analysis as described by Sousa et al. (2020) and observed under the scanning electron microscope. The e-SEM analysis was carried out at 20kV under a low vacuum. FEI Quanta 200 (Netherlands) was used for the SEM analysis at 1000X, 2000X, and 10000X magnification.
2.7 Molecular identification of selected strains.
The ten most effective strains were selected based on their properties and antagonism against RKN eggs and juveniles for molecular identification using the Illumina MiSeq platform. Genomic DNA was isolated using the QIAampPowerFecal Pro DNA kit, Cat: 51804 (Qiagen, Germany), as per the manufacturer's instructions. A genomic library for each strain was constructed in alignment with the recommendations for whole genome sequencing using Illumina NexteraTM DNA flex library preparation kit, cat: 20060060 (Illumina Inc., USA) (Prjibelski et al., 2020) A gyrB gene was used as a marker for phylogenetic identification (Wang et al.,2007). The phylogenetic tree was constructed based on subjecting the gyrB sequence of the selected isolates to BLASTN search against the NCBI nr/nt database, using default parameters (Tatusova et al., 2016). The sequences from the top 10 hits of alignments were downloaded and aligned against the query using clustalW. The multi-aligned sequences were imported into MEGA X and Tamura Nei model, in association with gamma distribution, to construct a phylogenetic tree using the bootstrap value of 3000. The phylogenetic tree was downloaded in Newick format and visualized in iTOL.
2.8 Statistical Analysis
The data obtained from the in vitro studies were analyzed statistically using analysis of variance. Tukey's test was applied for the one-way analysis of variance (ANOVA) using SPSS 20.0 (IBM, SPSS statistics 20) with a statistical significance level of 0.05.