Isolation of root associated rhizobacteria from tomato crop
Sample collection and isolation of rhizobacteria
Three polyhouses at ICAR-Centre for Protection Cultivation Technology, Indian Agriculture Research Institute (IARI), New Delhi, with root-knot infestation, were selected for sampling. Root samples were collected in December, from infected and uninfected 7 accessions of tomato. Five root samples from each row were pooled to form one composite sample, which was labeled and stored in plastic bags at 4 °C until processed. Whole plants were uprooted from the soil to examine nematode infection. Infected roots had heavy galling, as compared to uninfected roots that had very few galls or no galls. For the isolation of bacteria, 10 g of tomato roots was cut into small pieces and added to 90 ml of 0.85% sodium chloride (dilution 10–1) and shaken at 200 rpm for 1 h in an orbital shaker (New Brunswick Scientific, USA). Suspensions were serially diluted with sterile distilled water to prepare 10−4 dilutions, agitated and a 100 µl of suspension was plated on 3 nutrient media, viz. Soil Plate Count Agar (SPCA), Soil Extract Agar (SEA) and King’s B procured from Hi Media, Mumbai, India. Plates were incubated for 12–48 h. in a BOD incubator at 30 °C using 5 replicates. The numbers of bacterial colonies were counted under a stereoscopic microscope at 10x. The morphologically distinct bacterial colonies were isolated based on shape, size, elevation, opacity and margin and multiplication on nutrient broth (NB) for nematicidal evaluation. The selected bacterial isolates were sub-cultured to purify and maintained in 15% glycerol vials, at − 80 °C.
In vitro screening of rhizobacteria from infected and uninfected plants for juvenile mortality
Freshly prepared NB was inoculated with the selected colonies and incubated on a shaker at 120 rpm for 48 h, followed by centrifugation at 10,000 rpm for 10 min. The supernatants were filtered using Whatman No.1 paper, followed by nitrocellulose syringe filter (0.20 µm) to obtain cell-free filtrates (CFF) with metabolites if any. The egg masses of M. incognita were hand-picked from culture pots maintained at greenhouse, Division of Nematology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi. The Meloidogyne species was identified based on the perineal pattern structure of female nematode (Taylor et al. 1955).The egg masses were kept for hatching at 21º C in a BOD incubator. A 1 mL of CFF was pipetted in each well of pre-sterilized multiwell plates, followed by addition of 100 J2s of freshly hatched M. incognita in 100 µl water and incubated at 25 °C, with sterile medium and sterile distilled water (SDW) as control treatments. The immobile J2s were counted at 24 h under a stereoscopic zoom binocular microscope and then transferred to distilled water for 24 h to confirm their mortality. Five replicates were maintained for each isolate.
Molecular Characterization of rhizobacterial isolates
The 24-h bacterial cultures were used for genomic DNA isolation by Zymo Research Crop quick DNA ™ Fungal/Bacterial Miniprep kit (Cat. No. D6005) according to manufacturer’s instructions and analyzed by 0.8% agarose gel. The purity and quantity of the DNA were checked in Nanodrop spectrophotometer (Thermo scientific, USA). The DNA yield was quantified in ng mL−1, and the purity was assessed at 260/280 nm absorbance ratio. The PCR amplifications of 16S rRNA was performed using the universal primers were 27F (5-GTTTGATCCTGGCTCAG-3) and 1494R (5-ACGGCTACCTTGTTACGACTT-3) used for 16S rRNA gene amplification. The PCR was carried out using standardized protocol, and the amplicons were sent for Sanger sequencing to ABA Biotech Pvt. Ltd. The nucleotide sequences obtained were aligned with the existing nucleotide database of NCBI GenBank, and the reference sequences were retrieved. Phylogenetic tree was constructed using MEGA6 (Molecular Evolutionary Genetics Analysis version 6.0) software to understand the phylogenetic relationship of the selected 4 isolates. The nucleotide sequences were compared against nucleotide database using the NCBI BLASTn and EzTaxon SERVER 2.1 programs to identify the closest known taxa. The 16S rRNA gene along with the closest homology sequences was aligned using multiple sequence alignment program CLUSTAL W algorithm implemented in MEGA 6 SOFTWARE using default parameters. The phylogenetic tree was constructed by neighbor joining method, using MEGA 6 PROGRAM, and evolutionary distances were compared with the help of Kimura’s parameter models. The 16S rRNA gene sequences obtained were submitted under accession numbers MZ675428-MZ675431.
Biochemical assays for plant growth-promoting (PGP) traits
Indole acetic acid (IAA) production (Gordon and Weber 1951)
The IAA was estimated using 48-h bacterial cultures grown in NB at 30 °C. The cultures were centrifuged at 10,000 rpm for 10 min, and supernatant (4 ml) was mixed with 2 drops of ortho-phosphoric acid and 4 ml of Salkowski reagent (48 mL of 35% perchloric acid and 2 mL of 0.5 M ferric chloride solution) in 1:2 ratio (bacterial supernatant/reagent) at room temperature. The appearance of pink color indicated production of IAA. The optical density was observed at 530 nm after 25 min in 96-well microtiter plates using multimode reader (Thermo scientific, USA).
Phosphate and zinc solubilization test (Pikovaskya 1948)
The estimation of phosphate solubilization was conducted by spotting the bacterial cultures on the Pikovaskya agar plates and incubated at 30 °C for 5 days. The appearance of clear zone around the colony was considered positive for phosphate solubilization. Similarly, 24-h-old bacterial culture was inoculated on Tris mineral salts agar medium amended with 0.2% soluble zinc oxide (ZnO) and incubated for 3–5 days at 30 °C. The Zn solubilization was determined by appearance of clear zone around the bacterial colony (Gaur 1990).
Hydrogen cyanide (HCN) production (Bakker and Skipper 1987)
The 24-h-old bacterial cultures were inoculated on nutrient agar amended with glycine (0.44%) by spread plate method. Whatman No. 1 filter paper pre-saturated with 0.5% picric acid in 2% sodium carbonate solution (w/v) was placed in the lid of Petri plate, and the plates were sealed with parafilm (to prevent leakage), followed by incubation at 30 °C for 3–5 days. The HCN production was observed by change in color of filter paper from yellowish brown to deep brown.
Catalase production test (Schaad 1992)
The catalase test was performed by adding drop of 3% hydrogen peroxide to 48-h bacterial colony on a clean glass slide and mixed through sterile toothpick. The appearance of effervescence indicated catalase production.
Chitinase production test (Rodriguez-Kabana et al. 1983)
The M9 Agar media amended with 1% (w/v) colloidal chitin was prepared and plates were divided into 4 equal sectors, followed by spot inoculation with 24-h-old bacterial cultures and incubation at 30 °C for 5 days. The appearance of clearance zone around the bacterial colonies indicates chitinase production.
Ammonia (NH3) production test (Cappuccino and Sherman 1992)
The overnight bacterial culture (100 μl) was mixed, inoculated into 10 ml peptone water and incubated at 30 °C for 4 days. After incubation, an 1 mL of culture broth was added with 1 ml Nessler’s reagent. Formation of a yellow to deep orange color was considered positive for ammonia production.
Seed germination test
In the seed germination test, by paper towel method, the bacterial isolates B9, B16, B26 and B31 were used for seed treatment of tomato variety Pusa Ruby. Seeds were surface sterilized with 0.1% HgCl2 (5 min), washed 4–5 time with sterile distilled water and soaked in liquid broth culture (108cells/mL) for 1 h. Each seed was placed on wet blotting paper using sterile forceps, rolled the paper and incubated in BOD for 16 days at 30 °C. Seeds treated with sterile nutrient broth and distilled water served as control, and 5 replications for each isolate were maintained. Germination percentage and seedling vigor index were assessed as per protocols of International Seed Testing Association (1985).
Bioefficacy of rhizobacteria against M.incognita infecting tomato cv Pusa Ruby
The bioefficacy trial was conducted in 10 inch pre-sterilized plastic pots using 2-week-old seedlings of tomato cv Pusa Ruby transplanted in double sterilized field soil mixed with 3% bacterial cultures (B. pumilus. B. megaterium, B. subtilis and B. cereus) @ ~ 108 cfu/mL. Freshly hatched M. incognita was inoculated @ 2J2/cc soil after 7 days of transplanting. The nematicide velum prime (VP) from Bayer Pvt. Ltd. was taken as an additional treatment for comparison and applied (0.56µL/kg soil) after mixing in 500 mL sterilized water for uniform spread. The treatments were T1: Sterilized Water, T2: Sterilized Water + Mi, T3: B. pumilus, T4: B. pumilus + Mi, T5: B. megaterium, T6: B. megaterium + Mi, T7: B. subtilis, T8: B. subtilis + Mi, T9: B. cereus, T10: B. cereus + Mi, T11: Media (NB), T12: VP (velum prime), and T13: VP + Mi. Six replications of the 13 treatments were maintained in a polyhouse for 75 days. The plants were uprooted for recording observations on number of root galls per root, number of J2 s per cc soil, number of egg masses per root, number eggs per egg mass, nematode reproduction factor and plant growth parameters.
Statistical analysis
Data of colony forming units of bacterial isolates from infected and uninfected plants were analyzed for significant mean differences via one-way ANOVA using statistical software Statistical Tool for Agricultural Research (STAR). The multiple mean comparisons were performed using Tukey’s honestly significant difference test (Tukey’s HSD).
