Isolation of the bacterial strain
Bacterial strain KMT-4 was isolated from the nematode-infected tomato rhizosphere at a screen house of Department of Nematology, Hisar, Haryana, India (29°08′29.7″ N, 75°42′14.0″ E), using serial dilution technique. The bacterium was selected through various biochemical and antagonistic tests against M. javanica. It was cultured in King’s B medium—peptone 20g/l, glycerol 10ml/l, K2HPO4 1.5g/l, MgSO4 1.5g/l, glucose 1g/l, NaCl 3–5g/l, pH 7 (King et al., 1954) at 30°C with shaking at 300 rpm till 48h for further studies. The bacterial cells were adjusted to obtain 108 CFU/ml with sterile distilled water.
Identification of the bacterial strain KMT-4
KMT-4 was subjected to various morphological studies and Gram staining. The 16S rRNA sequence analysis was done for the identification of the bacterial strain. Genomic DNA was extracted according to the manufacturer’s protocol of the Genomic DNA isolation kit (Sigma-Aldrich, St. Louis, MO, USA), and PCR amplification of the 16S rRNA gene was done using the universal primers 8-27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGTTACCTTGTTACGACTT-3′). Sanger’s dideoxy sequencing method was used in sequence determination of 16S rRNA gene of the bacterium. The 16S rRNA gene sequence was then identified using the GenBank database at NCBI. A phylogenetic tree was constructed using the MEGA X software.
Characterization of KMT-4 for plant growth-promoting traits
The bacterium was screened for the presence of various direct as well as indirect growth-promoting attributes like IAA production, ammonia excretion, phosphate solubilization, chitinase activity, siderophore production, and hydrogen cyanide production. Three replicates were used for each experiment. KMT-4 was tested for quantitative estimation of IAA production and ammonia excretion in the culture supernatant by the method given by Tang and Bonner (1974) and Chaney and Marbach (1962), respectively. Phosphate solubilization activity was checked by the ability to form a zone around the bacterial spots on Pikovskaya agar plates supplemented with tricalcium phosphate (Pikovskaya, 1948). Similarly, chitinase activity was determined by spot test method on colloidal chitin agar plates (Murthy and Bleakley, 2012). A hollow zone formation around the spots indicated chitinase activity. Siderophore production was detected by CAS (Chrome Azurol S) assay (modified method of Schwyn and Neilands, 1987). The presence of iron chelating compounds (siderophores) was depicted by the decolorization of the blue-colored ferric dye complex, giving yellow hollow zone around the bacterial colonies. For HCN production, the bacterium was transferred to test tubes containing freshly prepared King’s B medium amended with glycine and sterile filter paper strips saturated with picric acid solution fixed inside. Change of color from yellow to reddish brown indicated cyanogenic potential (Alstrom and Burns, 1989).
Collection of nematodes
Meloidogyne javanica was cultured on brinjal and tomato plants under greenhouse conditions in Hisar, India. The infected roots were collected and rinsed with tap water after that dipped in 0.4% sodium hypochlorite (NaClO) solution (Hussey and Barker, 1973) and again rinsed for 5 min. The eggs thus released from egg masses on roots were concentrated on a 500-mesh sieve and rinsed with tap water. Juveniles (J2) were obtained from eggs after incubating them in distilled water at 25°C and collected by using modified Baermann funnel technique (MBFT) (Viglierchio and Schmitt, 1983) after 48 h. The number of J2 was standardized by counting them in 1 ml suspension; average number was used to represent the number of J2/ml. Egg masses were picked using a hand lens.
Effect of KMT-4 on hatching of egg masses and juvenile mortality in vitro
KMT-4 culture broth was grown for 72 h and centrifuged at a speed of 10,000 rpm for 10 min. The supernatant thus obtained was used as extracellular extract, and the bacterial pellet was dissolved in a 0.1M phosphate buffer of pH 7 for preparing the intracellular extract. The bacterial cells were ruptured using freezing and thawing technique (Harrison, 2011) and centrifuged at 10,000 rpm for 10 min. The supernatant obtained was used as intracellular extract, and the pellet thus formed was then discarded. Egg masses were collected from nematode M. javanica-infected brinjal roots using forceps and hand lens. Five egg masses (with a mean no. of 225 eggs) were added to 5 ml of each—untreated distilled water (control), extracellular extract, intracellular extract, and intact bacterial suspension (72 h grown culture broth) of the bacterial isolate in a 6-well culture plate. Hatching was observed at an interval of 24 till 96 h. The number of juveniles (J2) observed was recorded and compared with control. The number of J2 in suspension was standardized by counting them in 1 ml suspension; the average number was used to represent the number of J2/ml. Nematode suspension containing 100 J2 was treated by the bacterial isolate. The number of dead J2 was recorded till 48 h if their bodies were stiff and straight by pricking them with a needle. The mortality percentage was calculated using the following equation:
[(live J2 prior to treatment−live J2 after 48 h)/live J2 prior to treatment] × 100
A stereo microscope was used to observe the nematodes. All the experiments were conducted in 3 replicates. Phosphate buffer and growth medium were also used as control.
Pot house experiment on brinjal
To evaluate the effect of KMT-4 for biological control of M. javanica and growth promotion of host plant, an experiment was conducted on brinjal (Solanum melongena L.) in the pot house of Department of Microbiology, Hisar. Five kilograms sandy loam soil was used to fill up the clay pots of 8-in. diameter, and a recommended dose of fertilizers (RDF) consisting of slurry 104 kg/ha, urea 40 kg/ha, single superphosphate (SSP) 20 kg/ha, and muriate of potash (MoP) 10 kg/ha was applied to each pot. Seedlings of nematode-susceptible brinjal var. BR112 (obtained from the Department of Vegetable Science, Hisar) at 2-leaf stage were transplanted into the pots. A total of six treatments were used for the pot experiment as T1, control (RDF); T2, RDF + Azotobacter chroococcum Mac27 (108 CFU/ml); T3, RDF + M. javanica; T4, RDF + M. javanica + A. chroococcum Mac27 (108 CFU/ml); T5, RDF + M. javanica + carbofuran at 1 kg a.i/ha; and T6, RDF + M. javanica + isolate KMT-4 (108 CFU/ml). Seedlings were treated with 50 ml KMT-4 (108 CFU/ml) and 50 ml A. chroococcum Mac27 (108 CFU/ml obtained from the collection of Department of Microbiology, Hisar, Haryana), and carbofuran @ 1kg a.i/ha was added as per treatments described above. A. chroococcum Mac27 was referred to as plant growth control and carbofuran as chemical control. Plants were irrigated by tap water regularly. After 10 days of transplantation, nematode suspension containing eggs at 3000 eggs/pot was injected in the rhizosphere of plants with the help of a pipette in four 1-cm deep holes around the roots. There were 3 replicates of each treatment. The pot experiment was organized in a completely randomized design. Crop was uprooted after 45 days, and observations on number of eggs/root system, no. of galls/root system, and final nematode population/200cc soil were recorded. No. of eggs/root system was isolated by dipping the roots in 0.4% NaClO with stirring for 5 min and counted under stereomicroscope. The number of galls/root system was counted with the help of hand lens by spreading the roots in a 15-cm wide Petri plate. For determining final nematode population, soil from each treatment was mixed thoroughly, and 200cc soil from each replicate was analyzed by Cobb’s sieving and decanting method (Cobb, 1918), followed by MBFT. Obtained J2 were counted under stereomicroscope. Plant growth parameters like plant height, root fresh weight, root dry weight, shoot fresh weight, and shoot dry weight were also recorded on the 45th day of transplanting. Total bacterial populations were determined from the rhizosphere both at 0 and after 45 days of treatment.
Field trials were conducted separately during the year 2018 and 2019 on brinjal (Solanum melongena L.) and cucumber (Cucumis sativus L.) crops, respectively to check the efficacy of the bacterial isolate KMT-4 under protected cultivation at farmer’s polyhouse in Bhuna (Fatehabad), Haryana, India (29°32′17.0412″N, 75°42′28.9368″E) with initial nematode population of 275 J2/200cc of soil. Brinjal var. Hisar Shyamal and cucumber var. Multistar were selected for the experiment. A total of 3 treatments including control (water), KMT-4, and chemical carbofuran at 1 kg a.i./ha were incorporated. Brinjal seedlings (at two leaf stage) were dipped in KMT-4 broth (108 CFU/ml) for 30 min before transplantation, and cucumber seeds were coated with 50 g jaggary suspension after that mixed with 100ml of KMT-4 broth (108 CFU/ml), partially dried then sown into the field. The field experiment was arranged as a randomized block design with 6 replicate plots of each treatment. Plot size was 6×4 m. Roots from these plants were indexed for galling and egg masses on a scale of 1–5 (Heald et al., 1989). For the estimation of final nematode population, Cobb’s sieving and decanting method (Cobb, 1918) was used, followed by MBFT. The extracted J2 were counted at ×40 magnification under a stereomicroscope. Yield (q/ha) was determined after harvest. All the data were statistically analyzed and compared with control as well as chemically treated plants.
All the given values are expressed as mean ± standard error (SE). The SAS version 9.2 software (SAS Institute, Inc.) was used for all the statistical analysis. Means were separated and compared using Duncan’s multiple range test. Differences in mean values were considered significant when P <0.05.