Collection and Maintenance of Root-Knot Nematode Populations
Populations of root-knot nematodes (RKN) were collected from Nagpur (Cotton Gossypium hirsutum), Brinjal (Solanum melongena L.), Tomato (Solanum lycopersicum L)., Wardha (Tomato), Yavatmal (Cotton), Jalgaon (okra Abelmoschus esculentus L.), Aurangabad (Okra) and Chandrapur (Cotton) districts of Maharashtra. Individual RKN stock cultures of populations were raised from single egg masses and proliferated on tomato plants belonging to Lycopersicon esculentum cv. Pusa ruby in 30 cm diameter pots in sterilized soil.
Morphological Characterization of Root-Knot Nematode
For morphological characterization female root knot nematodes were teased from infected galled roots roots and maintained in water at 4°C until observation. For taking morphometric measurements females were placed on a glass slide with a drop of sterile tap water, covered with 0 number glass coverslip supported with glass wool rods and measured under the microscope. To observe the perineal pattern, the posterior end of each female was cut with a razor blade and mounted in lactophenol solution (Franklin and Goodey, 1949). Extraction of second-stage juveniles was done by separation of egg masses from the roots and incubation in sterile distilled water at room temperature (28-300 C) for 24h. Male specimen were obtained from egg masses as well as by washing the soil adhering to roots as per Cobb’s sieving and decanting technique followed by modified Baerrmann funnel technique (Cobb, 1918, Christie & Perry, 1951). Nematodes were killed by pouring in an equal volume of hot water (>80oC) to nematode suspension, fixed in hot 4% formalin (50-60°±2oC) and mounts were prepared in glycerine according to Seinhorst’s (1959) rapid method. The morphological features considered in the adult females were the perineal pattern, body length, body width, stylet length, neck length, and neck width. Juveniles had body length, stylet length, head to median bulb length, median bulb to excretory pore and tail length measured. Male body length, stylet length, distance to DGO, spicule length and gubernaculum length were also measured. All observations were done under Leica DBLB microscope at X100 and X400 .
DNA Extraction from female nematode
20 white gravid females were selected by random sampling for DNA extraction for molecular characterization and crushed to fine powder in liquid nitrogen. (Christoforou et al., 2017). Lysis buffer (1M Nacl, 1MTris, 0.5M EDTA, 10% SDS) was added to the pooled nematode homogenate for a total volume of 3 ml. The homogenate was incubated at 370 C for 30 minutes after addition of Proteinase K solution 20mg/ml (150 µl). DNA extraction was done with buffer saturated phenol in a 1:1 phenol:chloroform followed by chloroform:iso-amyl alcohol mixture. Quantification of DNA was done through spectrophotometry and electrophoresis on 1.5% agarose gel.
PCR amplification, Cloning and phylogenetic analysis
PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes was done using forward (5’TTTCACTCGCCGTTACTAAGG3’) and reverse (5’TTGATTACGTCCCTGCCCTTT 3’) primers (Vrain et al., 1992) with the purified gravid female DNA as a template. The master mix for the 25μl PCR reactions contained sterile distilled water (13.8 μl), 10X reaction buffer (5.0 μl), MgCl2 50mM (1.5μl), dNTPs 10mM (1.0 μl), forward primer (1.0 μl), reverse primer (1.0 μl), Taq polymerase (0.2 μl) and template (1.0 μl). Negative and positive controls were kept. Amplification reactions were performed in a Biorad thermal cycler with a PCR programme as follows: Initial denaturation - 940C for 2 minutes; 35 cycles of 940C for 30s (denaturation), 600C for 45s (primer annealing), and 720C for 45s (primer extension) then a single 5 minute cycle at 720C for final extension. Post-amplification, 5µl of the amplified product was resolved on 1% agarose gel and DNA fragments were visualized through ethidium bromide staining using the Bio-Rad Gel Documentation System. Subsequently, cloning was done in a pGEM-T vector from Promega (Madison, USA) and transformed into Escherichia coli, strain JM109 ((Cat.# L2001)procured from Promega following the manufacturer’s protocol. Transformants were identified with blue-white colony selection. LB/ampicillin plates were prepared by adding ampicillin to a final concentration of 100µg/ml using sterile filters to autoclaved and cooled LB medium. LB/ampicillin plates were stored at 4°C. 100µl of 100mM IPTG and 20µl of 50mg/ml X-Gal was spread over the surface of an LB-ampicillin plate with a sterile spreader and allowed to absorb for 30 minutes at 37°C prior to use. The plates were left to dry in laminar flow chamber with lids slightly open. 10-100 µL of transformed E. coli cells were spread onto the LB agar plates using sterile spreader and plates were incubated at 37°C for 24-48 hours. Blue and white colonies appeared after due incubation and recombinant white colonies were picked and cultured. Colony PCR was performed to confirm the presence of the insert and plasmid preparation was done using Pureyield plasmid miniprep system from Promega (New Delhi, India) after which sequencing was done through SciGenom (Kochi, India). Chromatographic quality of sequences was verified with the Applied Biosystems Sequence scanner (v1.0). The nucleotide sequences were then compared with catalogued data using NCBI BLAST to determine similarities and establish identity. The contig of partial assembled sequence were made by using CAP3: A DNA sequence assembly program (Huang and Madam,1999). These assembled sequences were submitted to NCBI through BankIt and deposited in the Nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore) .
Umarao et al. (2003) characterized races of M. incognita based on their rDNA ITS sequences and reported that variation in the sequences of the four catalogued races of M. incognita resulted in variance in their restriction sites, thus allowing for differentiation of the races based on restriction site distributions. The restriction enzymes Swal, Bsa1, BsmA1 and EcoR1 were reported for this purpose. Therefore, in our studies 10 individual colonies were tested for the restriction site unique to each enzyme with the sequences for these populations. Sequence data for M.incognita available on NCBI was also analyzed for the presence of restriction sites for EcoR1, Swal, Bsa1 and BsmA1 (Table 5). Sequences were incorporated into the BioEdit Sequence Alignment Editor v.7.2.5 and compared with catalogued sequence data for M. incognita available at the National Center for Biotechnology Information (NCBI) GenBank nucleotide database using a standard BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis was done using the MEGA X (Molecular Evolutionary Genetic Analysis) v10.1 (Kumar et al., 2018) program under maximum likelihood parameters and the resultant phylogenetic tree was generated.
Race Characterization by Differential host test.
Six differential hosts viz. Nicotiana tabacum cv. NC 95 (tobacco); Gossypium hirsutum cv. Deltapine 16 (cotton); Capsicum frutescens cv. California Wonder (pepper); Citrullus vulgaris cv. Charleston Grey (watermelon), Arachis hypogaea cv. Florunner (peanut) and Solanum lycopersicum cv. Rutgers (tomato) were used for race characterization as per Hartman & Sasser (1985; Robertson et al., 2009). Three seeds of each differential host were sown in sterilized soil in 10 cm diameter earthen pots in glass house. Plants were later thinned to retain one seedling per pot. Tomato, pepper and tobacco seedlings were transplanted. After two weeks, a prepared inoculum containing 400 second-stage juveniles in 3 ml of water was gently applied near the base of each plant using sterile pipette tips, with four replicates per differential host. After 60 days, plants were gently uprooted and number of egg masses/galls on each root system were counted and assigned a rating index number according to the following scale : 0 = no gall/egg masses, 1 = 1-2 galls/egg masses; 2 = 3-10 egg masses/galls; 3 = 11-30 egg masses/galls; 4 = 31-100 egg masses/galls and 5 = over 100 egg masses/galls. Plants with index values of 2 or less were designated as non-hosts (-) while plants with index values greater than 2 were designated as hosts (+).