Insect culture
Greater wax moth, Galleria mellonella Linnaeus, was reared on artificial diet according to the method (Patil et al. 2020). The last larval instars of G. mellonella were used for the experiment.
Biocontrol potential of EPNs was tested against Spodoptera litura Fabricius infesting cabbage Brassica oleracea var. capitata, Spilosoma obliqua Walker on brinjal, Solanum melongena Linnaeus, and Spoladea recurvalis Fabricius on amaranthus, Amaranthus tricolor Linnaeus in this study. A continuous culture of these insect hosts was maintained in an insectary at 26 ± 2 °C and 70 ± 5% RH, a photo phase of 14 h and scoto phase of 10 h. For initial establishment of the culture, different larval stages of these insects were collected from their respective host plants grown in research farm of ICAR- Indian Institute of Vegetable Research, Varanasi (25°10′55.6′′ N, 82°52′37.2′′ E; 80.71 m above sea level), Uttar Pradesh, India. The larvae were individually reared on their natural diet viz., S. litura on cabbage, S. obliqua on brinjal, and S. recurvalis on amaranthus in Petri dish (diam. 9 cm, depth 1.8 cm). Third instar larvae of these insect species were used for the bioassays.
Second instar grubs of Myllocerus subfaciatus Gurein were collected from naturally infested eggplants grown in ICAR-NBAIR research farm, Bengaluru (13° 05′48.7′′ N, 77° 34′ 02.8′′ E; 920 m above sea level), Karnataka, India. Grubs were kept in 100 ml plastic containers (diam. 5.8 cm, height 8 cm, soil capacity 115 g, total surface area 198.61 cm2, shape; round) containing 100 g of autoclaved soil with 12% moisture for 2 days at 25 ± 1 °C. Potato (Solanum tuberosum L.) pieces were provided to feed the grub. Three days after apparently healthy grubs were used for bioassay.
Isolation of native EPNs
In the present study, a survey was conducted during 2016 and 2017 in four districts of Purvanchal Region, i.e., Varanasi, Mirzapur, Sonbhadra, Deoria, and one district at Bundelkhand Region, i.e., Jhansi of Uttar Pradesh; these areas were previously unexplored. Soil samples were collected randomly using hand shovel from agricultural, horticultural, and forest ecosystems from early August to late November. In total, 130 soil samples were collected. Each representative sample (approximately 1 kg each) consists of 5 soil samples were taken at a depth of 15–20 cm. Then, soil samples were placed in polythene bags and transferred to the laboratory for investigation (Yuksel and Canhilal 2019). To isolate EPNs from the soil samples, soils were placed in 1000 ml plastic container (diam. 10.5 cm, height 14.5 cm, soil capacity 1100 g, total surface area 731.59 cm2, shape is round) with 4 individuals of last instar G. mellonella larvae. The plastic containers were covered with a lid having a small pin holes for allowing air flow, then containers were incubated at 25 ± 1 °C for 1 week. Soil samples were checked on a daily basis for 7 days to observe the cadavers of the larvae of G. mellonella. All recovered dead larvae of Galleria were placed individually on White’s traps (White 1927). Infective juveniles (IJs) emerged from these dead larvae were collected and stored at 15 °C. Two- to 3-day-old IJs of each nematode strain was tested against 20 individuals of the last instar G. mellonella larvae to confirm Koch’s postulates for pathogenicity (Pelczar and Reid 1972 and Kaya and Stock 1997).
Morphology and morphometry of native EPN strains
To study the morphology and morphometrics of EPNs strains, last instar larvae of Galleria were inoculated at rate of 200 IJs larva–1 and incubated 25 ± 1 °C in the dark. Three days after larval death, first generation adults were collected by dissecting the cadavers in Ringer’s solution. Similarly, second generation adults were collected 5th day after larval death. Infective juveniles were harvested from cadavers of the larvae of G. mellonella with the help of White’s traps. The adults of both generations and IJs were killed by pouring hot fixative and then fixed in TAF and processed, using Seinhorst I and Seinhorst II reagents (Seinhorst, 1959). The adults of both generations and IJs were mounted in dehydrated glycerin, using appropriate sized glass support. The mounted specimens were used for detailed microscopic studies (Poinar 1990). The measurements and examination of morphology were completed, using Trinocular Research Microscope (model Axio Imager Z2 by Carl Zeiss Microscopy GmbH provided with DIC optics) with suitable photomicrographs. For each Steinernema IIVR strain, 20 specimens of each developmental stage were examined. The results of morphometric and morphological characters of EPN strains isolated in this study were compared to the original description of S. siamkayai Stock, Somsook, and Reid.
Molecular characterization of native EPN strains
The genomic DNA was extracted from a single female of each Steinernema IIVR strain, using a modified method of Joyce et al. (1994). The ITS region of the EPN strains DNA was amplified by polymerase chain reaction (PCR) mixture containing 10 μl of the DNA suspension, 2.5 μl 10× PCR buffer with MgCl2, 0.5 μl dNTP mixture (10 mM each), 0.5 μl (100 pM/μl) of each primer, 0.3U Taq polymerase, and 10.7 μl double distilled water to make volume of 25 μl. The forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC- 3′) and the reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) were used in the PCR reaction for amplification of the complete ITS. The amplified PCR products were purified using a Qiagen Gel Purification Kit. The fragments of DNA were sequenced by Sanger’s method (Eurofins Genomics India Pvt., Ltd., Bengaluru, India). Sequences of the ITS-rDNA region of the EPN were used to study the phylogenetic relationships between the Steinernema IIVR strains and other related 21 Steinernema species, and Caenorhabditis elegans was used as an out group. The DNA sequences were edited using BioEdit with sequences of related species/strains. The accession numbers are cited in the phylogenetic tree were obtained from in GenBank by means of a BLAST tool of the NCBI. The phylogenetic analysis of sequence data were performed based on their ITS-rDNA region through Neighbor Joining method using MEGA 7.0 (Kumar et al. 2016).
Source of nematodes for laboratory bioassays
Entomopathogenic nematode species/strains such as Steinernema sp. (IIVR JNC01 strain), Steinernema sp. (IIVR JNC02 strain), Steinernema sp. (IIVR EPN03 strain), and Heterorhabditis indica (NBAIIH38 strain) were cultured in last instar larvae of G. mellonella (Kaya and Stock 1997). Two to 3-day-old IJs were used for laboratory bioassays. Virulence of all three Steinernema spp. IIVR strains was tested on last instar larvae of G. mellonella. Based on the better virulence, Steinernema sp. (IIVR JNC01 strain) was selected to test the biocontrol potential against lepidopteran and coleopteran pests in comparison to commercially available H. indica (NBAIIH38 strain).
Evaluation of biocontrol potential against lepidopteran pests of vegetable crops
In this study, Steinernema sp. (IIVR JNC01 strain) was tested against S. litura, S. obliqua and S. recurvalis in comparison to H. indica (NBAIIH38 strain). The study was conducted in Petri dish (diam. 9 cm, depth 1.8 cm), was filled with 20 g of sterilized soil and moisture was adjusted to 15% by adding water. Fresh leaf discs of individual host plant were placed in a Petri dish to feed insects. Each Petri dish was inoculated by each nematode species at 0, 25, 50, 100, 200 IJs larva–1. After an hour, 4 individuals of 3rd larval instar of each insect host species were released to each dish. Larval mortality was recorded 2 days after nematode inoculation, and nematode infection was confirmed by dissecting cadavers under a stereomicroscope. The whole experiment was repeated with 5 replicates.
Evaluation of biocontrol potential against eggplant ash weevil
In this experiment, biocontrol potential of Steinernema sp. (IIVR JNC01 strain) was tested against 2nd instar grubs of M. subfasciatus in comparison to H. indica (NBAIIH38 strain). For this study, 30 ml plastic cups (diam. 3.5 cm, height 3.5 cm, soil capacity 35 g, total surface area 153.93 cm2, shape is round) were filled by 25 g of sterilized soil, and moisture was adjusted to 12%. Potato pieces were added to each plastic cup to feed the grub. A single 2nd instar grub of M. subfasciatus was placed in each cup. After 24 h, grub was inoculated with each nematode species at 0, 100, 300, and 500 IJs grub–1. Then cups were placed in incubator at 25 ± 1 °C. Each treatment had 20 replicates and grub mortality was assessed on daily basis up to 7 days. Cadavers were placed on White’s traps and confirmed the death is due to EPNs by observing the nematode emergence from cadavers. The whole experiment was repeated with 20 replicates.
Statistical analysis
Before statistical analysis, percentage mortality data were normalized, using arcsine transformation. Analysis was undertaken on the transformed data. An ANOVA was conducted using PROC ANOVA (SAS version 9.3; SAS institute 2011, Cary, NC, USA). When ANOVA was significant, relevant means were compared to Tukey’s significance test values at the 5% level of significance.