2.1 Isolation of Rhizobacterial strains and their
identification
The four bacterial isolates used in the experiments were isolated from
tomato accessions at centre for protected cultivation and technology
(CPCT), Indian Agricultural Research Institute (IARI), New Delhi
(Devindrappa et al ., 2022). The isolates were found to cause more
than 80 percent mortality in second-stage juveniles of M.
incognita in laboratory bioassays (Devindrappa et al ., 2022).For
identification of the 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 PCR
amplifications of 16S rRNA was performed using the universal primers;
27F (5-GTT TGA TCC TGG CTCAG-3) and 1494R (5-ACG GCT ACC TTG TTA CGA
CTT-3). 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.
The 16S rRNA gene sequences obtained were submitted under accession
numbers MZ675428-MZ675431 and identified as B. pumilus , B.
megaterium , B. subtilis and B. cereus .
The pure cultures were maintained on nutrient agar (NA) slants under
refrigerated conditions and in 30per cent glycerol stocks at -80C.
2.2 Identification of Meloidogyne incognita and
maintenance of its culture
The Meloidogyne incognita species was multiplied in culture pots
on tomato cv Pusa Ruby, using a single egg mass and identified based on
the perineal pattern of an adult female (Taylor et al ., 1955).
Root-knot nematode (RKN) species was confirmed by molecular method using
DNA extracted from RKN J2s. Single eggmass was picked from culture
plants and juveniles were allowed to hatch. Hatched juveniles were used
for genomic DNA extraction using soil DNA extraction kit (Quagen)
following manufactures protocol. Quality of DNA was checked and PCR
amplification carried using universal primers specific to Internal
Transcribed Region (ITS) of nematodes (Forward primer 5’
TTGATTACGTCCCTGCCCTTT 3’ and reverse primer 5’ TTTCACTCGCCGTTACTAAGG 3’)
as described by Vrain et al., 1992. PCR conditions and programme
for amplification of ITS region was followed as per the method used by
Gawade et al. , 2022. PCR product was subjected to 1.2% agarose
gel electrophoresis and visualized under gel documentation system
(G-Box, Syngene). PCR products were purified and sequenced using both
the primers to know the gene sequences. The nucleotide sequence was
analyzed and checked for sequence similarity in Genbank DNA database of
NCBI using nucleotide Basic Local Alignment Search Tool (nBLAST) of
National Center for Biotechnology Information (NCBI).
The culture of M. incognita was multiplied using J2s from a
single eggmass on tomato maintained at greenhouse, Division of
Nematology, ICAR-Indian Agricultural Research Institute (IARI), New
Delhi. The egg masses of M. incognita were hand-picked from
culture pots and kept for hatching at 210C in a BOD
incubator to collect the juveniles.
2.3 Bioefficacy of rhizobacteria against M.
incognita infecting tomato
Three week old tomato cv Pusa Ruby seedlings were transplanted in 10
inch pre-sterilized plastic pots filled with sterilized soil
individually mixed with 3% bacterial cultures (B. pumilus. B.
megaterium, B. subtilis and B. cereus ) @
~108cfu/mL. Freshly hatched second
stage juveniles (J2) of M. incognita were inoculated @ 2J2/ cc
soil after seven days of transplanting. The nematicide Velum prime
(Bayer crop science) was taken as an additional treatment for comparison
and applied @0.56µL/kg soil after mixing in 500mL sterilized water for
uniform spread. The treatments were, T1: Water + M. incognita(Mi), T2: B. pumilus + Mi, T3: B. megaterium + Mi, T4:B. subtilis + Mi, T5: B. cereus + Mi, T6: Velum Prime +Mi.
Six replications of the six treatments were maintained in a polyhouse
for 75 days. The plants were uprooted and the number of root galls per
plant, number of J2s per cc soil, number of egg masses per plant, and
nematode reproduction factor.