Nematode culturing
The M. incognita population used in the study was originally collected from the heavily infected tomato plants grown at Centre for Protected Cultivation Technology (CPCT), ICAR-IARI-New Delhi, India. The identification of the species was done morphologically based on the perineal pattern of mature females (Jepson 1987). The infected roots were washed, and egg masses were removed with sterile forceps and kept for hatching using the modified Baermann method. Second-stage juveniles (J2s) were collected in the Petri plate containing water after 24 h. From infested soil samples, the juveniles were extracted by Cobb’s decanting and sieving technique (Cobb 1918). Further, egg masses of uniform size were collected from the galled roots and inoculated (one egg mass/pot) into the root zones of susceptible Pusa Purple Long variety of brinjal and tomato cv. NS 4266. Pots were maintained in a greenhouse and growth chambers at 25–30 °C with a photoperiod of 12 h. For laboratory and pot experiments, egg masses from heavily galled roots were handpicked and transferred to vial containing 0.5% (v/v) sodium hypochlorite (NaOCl) and shaken for 3 min. The egg mass suspension was then passed through a series of filters with pore sizes of 74, 45, and 25 µm. Eggs that were retained on the 25-µm filter were collected with sterile distilled water (Hussey 1973) and allowed to hatch in modified Baermann setup at 28 °C to get freshly hatched second-stage juveniles (J2s), which were used for subsequent experiments (Viglierchio and Schmitt 1983).
Preparation of soil for pot experiments
Field soil from CPCT-IARI, New Delhi, was used for all the experimental purpose. The soil was mixed with sand in the ratio of 3:1. The soil sand mixture was steam-sterilized at 1.0546 kg/cm3 pressure for 4 h. and stored in polythene bags.
Raising, transplanting, and maintenance of tomato seedlings
Healthy susceptible seedlings’ of tomato cv. NS 4266 were raised using sterilized mixture of cocopeat: vermiculite: perlite (3:1:1). After attaining 21 days, seedlings were transplanted into earthen pots (6 inches size) and arranged in a completely randomized design under polyhouse condition. During the polyhouse experiments, all agronomic practices like irrigation by drip at 3–4 days interval, weeding was done thrice throughout the crop period, nutrient management (N:P:K: 19:19:19 at 3 g/L, through fertigation at 2 months interval) and training of tomato plants after attaining particular stage was done. The average temperature during the pot and field experiments under protected cultivation was 30 ± 2 °C, and the crop season was Kharif-Rabi.
Treatment details for laboratory, pots and field experiments
Ba: Bacillus amyloliquefaciens DSBA 11; Bs: Bacillus subtilis DTBS 5; Pa: Pantoea agglomerans; Ba + Bs + Pa (Consortium); NB: Nutrient broth; SDW: Sterile distilled water; VP: Velum Prime® (500 g a.i./ha) as positive control.
Laboratory experiments
Preparation of PGPR culture filtrates
All the PGPR isolates, B. amyloliquefaciens DSBA-11 (ITCC BJ-0013), B. subtilis DTBS 5 (ITCC BJ-0011) and P. agglomerans (ITCC BC-0001), used in this study were collected from the Bacteriology lab and Indian Type Culture Collection (ITCC), Division of plant pathology, ICAR-IARI, New Delhi, Delhi, India. A single colony from the pure cultures of PGPR isolates was taken from 24-h. old culture plates and inoculated into 50 mL of sterilized King’s B broth in 100-mL Erlenmeyer flasks and incubated in a shaker incubator at 150 r.p.m at 37 °C for 24 h. The bacterial growth after 24 h. was tested. Culture filtrate obtained by centrifugation at 10,000 r.p.m for 15 min at 4 °C. The supernatant culture filtrate was collected and passed through syringe filter of 0.22 µm. Consequently, collected culture filtrate was tested for the absence of any viable cell and used for bioassay (Rompalli et al. 2016).
Juvenile’s mortality bioassay
The nematode suspension of 100 J2s/10 µL was poured into each well of 24-well culture plate, and 1 mL of different concentrations of cell-free culture filtrates of each PGPR isolates at 100, 50 and 25% was added and mixed thoroughly. Nutrient broth and sterile distilled water were taken as negative control, whereas Velum Prime® (Fluopyram 400 SC) was taken as positive control and 24 well plates were incubated at 28 ± 2 °C. Observation was recorded at 24, 48, 72 and 96 h. of exposure in each treatment; all dead and alive J2s were counted with the aid of counting dish under stereoscopic binocular microscope. The ratio of dead nematodes/number of total nematodes expressed the percentage mortality. Mortality rates were calculated using Abbott’s formula (Abbott 1925).
Egg hatching inhibition bioassay
The egg suspension of M. incognita (100 eggs/10 µL) was poured into 24-well tissue culture plate, and 1 mL of different concentrations of cell-free culture filtrates of each PGPR isolates at 100, 50 and 25% was added and mixed. Nutrient broth and sterile distilled water were taken as a negative control, whereas Velum Prime® was taken as a positive control and plates were incubated at 28 ± 2 °C. Observation on egg hatching was recorded at 2, 4, 6 and 8 days of exposure in each treatment. Hatching percentage was calculated by counting the number of hatched and unhatched eggs under stereoscopic binocular microscope. The percentage suppression in hatching of juveniles (J2s) was calculated using the following formula: Percentage of hatching of eggs = [1 − (Ht/Hc)] × 100, where Ht is the number of juveniles hatched in treatment, and Hc is the number of juveniles hatched in control.
Pot experiments
Application of PGPR isolates as bare root dip treatment prior transplanting
Twenty-one days old tomato seedlings’ (cv. NS 4266) which are highly susceptible to M. incognita were raised in the nursery pro-trays. Healthy seedlings’ were uprooted carefully, and the roots were dipped for 15–20 min. in each PGPR isolates at 108 CFU/mL concentration and immediately transplanted in the earthen pots (6 inch in size) containing 1500 cc soil. After 7 days of transplanting, each pot was inoculated with freshly hatched second-stage juveniles at 2 J2s/cc soil. Plants treated with only water were taken as negative control and Velum Prime® (500 g a.i./ha) as positive control. The pots were arranged in completely randomized manner in the polyhouse. All the treatments were replicated four times. After 90 days, the plants were carefully uprooted, and observations were recorded.
Application of PGPR isolates as soil drenching
Earthen pots of 6 inches in size filled with steam-sterilized soil (1500 cc/pot) were inoculated with freshly hatched second-stage juveniles at the rate of 2 J2s/cc soil and were arranged in completely randomized design under polyhouse condition. Before transplanting, each pot containing soil and nematode inoculum (2 J2s/cc soil) was drenched with PGPR isolates at 50 mL/pot (108 CFU/mL). After 1 week, 21 days old tomato seedlings’ (cv. NS 4266) which were highly susceptible to M. incognita were transplanted into each treated soil at one seedling/pot. Soil drenched with only water was taken as negative control and Velum Prime® (500 g a.i./ha) as positive control. All the treatments were replicated four times. After 90 days, the plants were carefully uprooted, and observations were recorded.
Field experiments under protected cultivation
Two field experiments were conducted during the year 2019–2020 and 2020–2021 in field plots (28.6281° N and 77.1606° E) naturally infested with M. incognita at CPCT, ICAR-IARI, New Delhi, Delhi, India.
Application of PGPR isolates as bare root dip treatment prior transplanting
The nematode-susceptible tomato seedlings’ (21 days old) roots were dipped for about 15–20 min. in each PGPR isolates at 108 CFU/mL concentration and then transplanted in blocks (10 m2) assigned as randomized block design on the selected naturally infested polyhouse beds with an average initial soil population of 6 J2s/cc soil (2019–2020) and 4 J2s/cc soil (2020–2021) were assessed using Cobb’s decanting and sieving technique (Cobb 1918). The planting distance of (60 × 60 cm) was maintained in each block having 14 plants/block. Each block had 2 rows with 7 plants in each row separated by (0.5 m) distance. Plants treated with only water and nutrients were taken as a negative control and Velum Prime® (500 g a.i./ha) as a positive control. All the treatments were replicated five times. After 7 months at crop termination stage, observations on plant growth and nematode multiplication parameters were recorded.
Application of PGPR isolates as soil drenching
Each block (10 m2) assigned on the selected naturally infested polyhouse beds with an average initial soil population of 6 J2s/cc soil (2019–2020) and 4 J2s/cc soil (2020–2021) was drenched with each PGPR isolates at 1 L/block (108 CFU/mL). After 1 week, tomato seedlings (21 days old), susceptible to M. incognita were transplanted into each block arranged in randomized complete block design. Throughout the crop period, soil was drenched three times with each PGPR isolates and consortium at 2 months interval. Soil drenched with only water and nutrients was taken as a negative control and Velum Prime® (500 g a.i./ha) as a positive control. All the treatments were replicated five times. After 7 months at crop termination stage, observations on plant growth and nematode multiplication parameters were recorded.
Observations on plant growth parameters (shoot length, root length, fresh root weight, fresh shoot weight) and nematode multiplication parameters (No. of galls/root system, No. of egg masses/root system, No. of eggs/egg mass, and reproduction factor (RF)) were recorded in both the pot and field experiments, and fruit yield (kg/10 m2) was estimated only in field experiments. Nematode RF was calculated using the formula, RF = Pf/Pi, where Pf = final nematode population and Pi = initial nematode population in soil. Pi is determined by soil sampling from the selected nematode-infested polyhouse beds, around 25 subsamples were collected, pooled, and processed using Cobb’s decanting and sieving method (Cobb 1918), similarly Pf is calculated at the time of harvest with respect to each treatment, M. incognita infective juveniles were counted, and the RF was calculated.
Experimental designs and statistical analysis
The experiments were carried out using completely randomized and randomized block designs in pots and field beds, respectively, under polyhouse conditions. The experimental data obtained were statistically analysed using Web Agri Stat Package (WASP) version 2.0 (at 5%).
