In 2017, a total of 23 soil samples were collected randomly by adopting the technique of Wallace (1971) from marigold fields of Noida (28° 32′ 7.8612″ N and 77° 23′ 27.7044″ E, 206 m above sea level), Uttar Pradesh, India, and brought to the Nematology Laboratory in a well labelled polythene bags for the isolation of EPNs. The characteristics of the positive soil sample were sandy loam with 6.5 pH.
Insect rearing and maintenance
In the present study, 3 lepidopteran insect pests, viz., G. mellonella, H. armigera and S. litura, were reared under laboratory conditions and utilised in experimental work. Larvae of G. mellonella were brought to the laboratory from Bio-control Lab, Sardar Vallabhbhai Patel University of Agriculture and Technology, Modipuram, and reared on semisynthetic diet as suggested by David and Kurup (1988). Eggs and larvae of H. armigera (National accession no. NBAII-MP-NOC-01) and S. litura (National accession no. NBAII-MP-NOC-02) were purchased from ICAR-National Bureau of Agriculturally Important Insects (NBAII), Bangalore. Larvae of H. armigera were reared under laboratory conditions on chickpea-based diet as suggested by Nagarkatti and Prakash (1974), modified by Kalia et al. (2001), while the larvae of S. litura were reared on fresh, properly washed and well-sterilized castor leaves. The 3rd to 4th larval instars of these insects, approximately same size and weight larvae, were used in bioassay experiments. However, the fully grown larvae have also been used for further implications such as isolation and mass production of nematodes.
Isolation and culture of nematodes
EPNs were isolated from the soil samples using the Galleria soil baiting technique (Bedding and Akhurst, 1975). Ten last instar larvae of G. mellonella were placed in a 250 ml sterilized polystyrene jar containing soil sample and kept in BOD at 27 ± 1 °C. For larval mortality, samples were checked daily up to 7 days. The cadavers of G. mellonella were collected from the jar and washed thrice with double-distilled water (DDW), disinfected with 0.1% sodium hypochlorite and transferred on White trap (White, 1927) for IJs. Of the total 23 soil samples collected, only one sample (4.35%) was found positive for EPNs. Koch’s postulate was performed for the confirmation of entomopathogenic nature of the nematodes and the isolated strain was designated as CS38. Isolated IJs were washed thrice with DDW, disinfected with 0.1% sodium hypochlorite and finally, stored in sterilized DDW into vented tissue culture flasks in BOD at 15 °C.
Fully grown larvae of G. mellonella were infected with isolated IJs (3rd stage) and adults (males and females) of first and second generations were obtained from cadavers by dissection on 3rd and 5th days after infection. In this study, 20 IJs, 15 males and 15 females of each generation were used for morphology and morphometry. Collected nematodes of different generations were killed separately with hot Ringer’s solution, fixed in TAF solution (2 ml triethanolamine, 7 ml formalin, 91 ml distilled water) (Courtney et al., 1955), dehydrated by Seinhorst method (Seinhorst, 1959) and finally kept in glycerol. Then, they were mounted onto a drop of glycerine on glass slides and coverslips were sealed with paraffin wax. Morphological observations were made using a light compound microscope (Magnus MLX) and phase contrast microscope (Nikon Eclipse 50i). Morphometric measurements were taken with the help of the inbuilt software of a phase contrast microscope (Nikon DS-L1).
For molecular studies, genomic DNA was extracted from freshly emerged IJs, using a DNeasy Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions and eluted DNA was stored at − 20 °C until further use. For PCR amplification, Internal Transcribed Spacers (ITS) and 28S ribosomal DNA (D2D3) were used. The Internal Transcribed Spacers regions (ITS1, 5.8S, ITS2) of the rDNA were amplified using primers 18S: 5′-TTGATTACGTCCCTGCCCTTT-3′ (forward) and 26S: 5′-TTTCACTCGCCGTTACTAAGG-3′ (reverse) (Vrain et al., 1992). The rDNA fragment containing D2D3 regions of 28S rDNA were amplified using primers (D2F): 5′-AGCGGAGGAAAAGAAACTAA-3′ (forward) and (D2R): 5′-TCGGAAGGAACCAGCTACTA-3′ (reverse) (Joyce et al., 1994).
The 30 μl PCR reaction mixture consisted of Dream Taq green PCR master mix (15 μl), DNA extract (3 μl), nuclease free distilled water (10 μl) and forward and reverse primers (1 μl of each). The cycling parameters were used as follows: for ITS: 1 cycle of 94 °C for 7 min, followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 10 min, and for D2D3 fragment of 28S rDNA: 1 cycle of 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 7 min.
PCR was followed by gel electrophoresis, in which the PCR products (5 μl) were analysed on 1% TAE (Tris–acetic acid–EDTA) buffered agarose gel (50 ml) stained with ethidium bromide (1 μl EtBr) at 70 volts for 30 min. The amplified PCR products were purified and then sequenced in both directions using ABI 3730 (48 capillary) electrophoresis instrument by Bioserve Pvt. Ltd. (Hyderabad, India). Obtained amplified forward and reverse sequences were aligned, edited, and assembled using BioEdit (Hall, 1999), and then, the sequencing results were submitted to GenBank with accession numbers MG198915 and MG198916 for ITS and D2D3 regions of Steinernema abbasi strain CS38 respectively.
Sequence alignment and phylogenetic analysis
The sequences were compared to other already available sequences in the GenBank using the Basic Local Alignment Search Tool (BLAST) of the National Centre for Biotechnology Information (NCBI). Based on nucleotide similarities, the related sequences were aligned using default CLUSTALW parameters in MEGA 6.0 (Tamura et al., 2013) and optimized manually in BioEdit (Hall, 1999). Pairwise distances of ITS and D2D3 regions between isolated strain CS38 and other Steinernema species from ‘bicornatum group’ were computed in MEGA 6.0. Codon positions included were 1st + 2nd + 3rd + Noncoding.
Phylogenetic trees were constructed by Maximum Parsimony (MP) using MEGA 6.0 with bootstrap analysis based on 10,000 replicates. Evolutionary distances were also computed using p-distance method in MEGA 6.0 and expressed in the units of base differences per site. All the characters were treated as equally weighted. Steinernema affine and S. scapterisci and S. nepalense were used as outgroups for ITS and D2D3 regions, respectively.
Bioassay for pathogenicity and generative potential
Freshly emerged IJs were used to perform all bioassay experiments to evaluate the pathogenicity and generative potential of S. abbasi CS38 against G. mellonella, H. armigera and S. litura as target hosts. For pathogenicity, bioassay trails were carried out in 6-well plates (well size 3.5 cm) lined with a double layer of Whatman Filter Paper No. 1. Four different concentrations of isolate, viz., 25, 50, 100 and 200 IJs, were prepared with a final volume of 400 μl, using DDW, and were poured into each well of well-plates with the help of micropipette. Ten replicates of the 3 insect larvae of the same size and weight were used for each concentration along with control (only DDW). Larvae were placed individually into each well of above prepared well plates and then, the plates were incubated at 28 ± 2 °C in BOD. Mortality rate was recorded after every 12 h of post infection period (PIP) till 100% mortality was observed. Dead larvae of target insects were transferred onto the modified White Trap (White, 1927) to observe the persistence of infection and for emergence of IJs. Each bioassay was placed separately and to reach the optimum authenticity, all experiments were repeated thrice.
For generative potential, 10 fully grown larvae of each insect (same size and weight) were infected with 100 IJs/larva and incubated at 27 ± 1 °C in BOD. Cadavers were transferred to the White Trap and emerged IJs were collected daily in tissue culture flask for progeny count, up to 20 days till the emergence stopped. Collected nematodes were quantified under stereomicroscope (Nikon SMZ 645) with the help of counting dish in 1 ml suspension.
The experimental data of larval mortality bioassay was analyzed statistically, using SPSS software (version 16.0) by applying Probit analysis. To evaluate the pathogenicity, LC50 and LT50 values were also computed at 95% confidence limit. Larval mortality was recorded in the form of percentage mortality and graphical presentations were made using excel. For generative potential, the total number of produced IJs/larva of the studied nematode was analysed by descriptive analysis and presented in number of IJ ± SE (range).