Morpho-taxometrical and molecular characterization of Steinernema abbasi (Nematoda: Steinernematidae) and its pathogenicity and generative potential against lepidopteran pests

An entomopathogenic nematode (EPN) was recovered by using Galleria baiting technique from the soils of marigold fields of Noida, Uttar Pradesh, India. Based on morphological, morphometrical and molecular characterizations, the isolated strain was identified as Steinernema abbasi and tagged as CS38. The isolated strain was conspecific to original description with minor deviations. Infective juveniles (IJs) of present strain were longer than original description. Molecular analysis was done using ITS1-5.8S-ITS2 and D2D3 regions. Pathogenicity and generative potential of the present strain CS38 were tested against larvae of 3 lepidopteran insect pests, namely, Galleria mellonella L., Helicoverpa armigera (Hb.) and Spodoptera litura (Fab.). Different concentrations of IJs/larva, viz. 25, 50, 100, and 200 IJs/larva, were used for bioassay trails. All experiments were repeated thrice to reach the optimum authenticity. Results of bioassays revealed that isolate CS38 was highly virulent against the 3 insect pests and caused (100%) mortality within 48 h under laboratory conditions. Generative potential of the studied S. abbasi CS38 was recorded high in G. mellonella (19 × 104 IJs/larva) at 100 IJs/larva concentration, followed by H. armigera (63.4 × 103 IJs/larva) and S. litura (60.5 × 103 IJs/larva). Isolate CS38 is an indigenous, dominant and highly virulent strain that can be utilized as a biological control agent against the three studied insects. Moreover, it can be used for commercialization of the production of EPN-based biopesticide to be added under Integrated Pest Management in Indian agriculture system.


Background
Agriculture has been facing distressing harm due to various factors worldwide and highly affected by attack of pests and diseases which decline the crop production. Insect pests are one of them causing huge yield loss (15-20%) of agricultural crops in India. The most common problem in the effective control of insect pests is that they develop resistance against chemical pesticides with time. In Integrated Pest Management (IPM), the biological control method is considered to suppress the population of insect pests in agricultural fields. Biopesticide is the effective potential measure that controls the pests by biological non-toxic means without causing environmental hazards. Entomopathogenic nematodes (EPNs) are among extraordinary biocontrol agents than others. They have been studied against various insect pests infesting agricultural crops in different biocontrol programs (Ehlers, 2001) and it has been proven that EPNs, belonging to the families Steinernematidae and Heterorhabditidae, are the most promising and highly effective biological control agents against many insect pests (Kaya and Gaugler, 1993), and they are harmless to vertebrate and plants (Burnell and Stock, 2000). They are found in various habitats from cultivated land to desert (Hominick et al., 1996). EPNs are the soil-inhabiting endoparasitoids, having a great potential to control both soil dwelling and above-ground insect pests (Kaya andGaugler, 1993 andEhlers, 2005). Therefore, to control a broad range of insect pests in cryptic and epigeal habitats, they can be used efficiently as a biocontrol agent against grubs, cutworms, crown borers, corn root worm, fungus gnats, thrips, cranefly, beetles, weevils, moths, butterflies, crickets, grasshoppers, and other insect pests. EPNs are effective as the highly recommended commercial products (e.g., Entonem), chemical insecticide (e.g., abamectin and thiamethoxam) that significantly decrease insect pest populations (Laznik et al. 2010). These nematodes are symbiotically associated with entomopathogenic bacteria belonging to the family enteriobacteriacae, which makes them lethal obligatory insect parasitoids that affect a variety of insects including their larval forms and kill them within a short period of time.
Infective juveniles (IJs), the only free-living stage of EPNs, has the ability to search the insect-hosts, enters them through natural openings (Poinar, 1990) or penetrates through thin sections of cuticle (Bedding and Molyneux, 1982) which releases its symbiotic bacteria into the haemolymph after entering the host. These symbionts are the primary agents which are responsible for killing the host within 24-72 h by septicaemia (Boemare and Akhurst, 1988). Two major genera Steinernema and Heterorhabditis are symbiotically associated with bacteria of genus Xenorhabdus and Photorhabdus, respectively. EPNs of both of these genera are used as biocontrol agents because they have the ability to find and kill even deep-seated insects (Bedding and Miller, 1981). Moreover, they are fast-acting, cost-effective and easy to handle. The EPNs are eco-friendly, safe to nontarget organisms, have a great potential of reproduction (Kaya and Gaugler, 1993), wide host range, easy to mass produce, capable of resisting under unfavourable conditions (Askary and Ahmad, 2017), easy to apply, compatible with most of the insecticides (Chen, 2003) and sensitive to some insecticides (Laznik and Trdan, 2013a, b).
The greater wax moth, Galleria mellonella L., is a ubiquitous pest of honeybees and their hives. The cotton bollworm, Helicoverpa armigera (Hb.), and tobacco cutworm, Spodoptera litura (Fab.) are highly polyphagous and widely distributed insect pests.
The aim of the present study was to validate the EPN isolate Steinernema abbasi, on the basis of morphotaxometrical and molecular characterizations. Furthermore, the pathogenicity and generative potential of the EPN isolate against 3 lepidopteran pests were studied.

Soil sampling
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 3 rd to 4 th 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 Heena et al. Egyptian Journal of Biological Pest Control (2021)  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.

Morpho-taxometrical characterization
Fully grown larvae of G. mellonella were infected with isolated IJs (3 rd stage) and adults (males and females) of first and second generations were obtained from cadavers by dissection on 3 rd and 5 th 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).
PCR was followed by gel electrophoresis, in which the PCR products (5 μl) were analysed on 1% TAE (Trisacetic 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 1 st + 2 nd + 3 rd + 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.

Statistical analysis
The experimental data of larval mortality bioassay was analyzed statistically, using SPSS software (version 16.0) by applying Probit analysis. To evaluate the pathogenicity, LC 50 and LT 50 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).

Morpho-taxometrical characterization
The morpho-taxometrical characterizations of the different life stages (IJs, 1 st and 2 nd generation males and females) of the present isolate CS38 were compared to original described species S. abbasi (Elawad et al., 1997). Due to the presence of horn like cephalic armature in 3 rd stage IJs, the present isolate CS38 was characterized as a member of "bicornatum" group and the morphological and taxometrical studies of different life stages revealed that it was conspecific to S. abbasi. Hence, it is named as the same. Light microscope images of S. abbasi CS38 are shown in Fig. 1. Body of IJs was observed larger in size when compared with IJs of already described species. Tail is attenuated, gradually tapering and dorsally curved at tip with slight ventral depression. Female body is robust, strongly curved and C-shaped. Cuticle with fine striae, oesophagus muscular with slightly swollen rounded basal bulb, gonads reflexed containing eggs, vulva with double flapped epiptygma. Tail short, conoid with pointed tip and ventral post anal swelling present in both generations. Male body is slender, ventrally curved and J-shaped upon fixation. Cuticle with fine transverse striae, oesophagus muscular with rounded basal bulb, nerve ring located above the basal bulb and excretory pore present anterior to nerve ring. Testis reflexed, spicule paired and golden dark yellow in colour. Ventrally curved gubernaculum observed boatshaped and slightly swollen in the middle. Tail short and conoid with a bluntly rounded terminus. However, 2 nd generation females and males are smaller in size than first generation. Spicules and gubernaculum of 2 nd generation male are also shorter and thinner. Detailed morphometrics of all generation of present strain CS38 and comparative morphometrics of all generations of CS38 with S. abbasi are presented in Tables 1 and 2, respectively.

Molecular characterization
For molecular studies, Internal Transcribed Spacer (ITS) and D2D3 region of rDNA were analysed. In the ITS sequence of rDNA, the present strain CS38 did not show any variation with the already described S. abbasi (AY230158). The sequence length of ITS in S. abbasi CS38 was 739 bp with ITS-1 268 bp, 5.8S 157 bp and ITS-2 314 bp same as found in original description. The ITS nucleotide composition of CS38 was A = 175 bp, C = 111 bp, G = 162, T = 291 bp (Table 3). The ITS sequence of CS38 was separated from the other related species of "bicornatum" group by 33 to 191 bp. The pairwise distance matrix is presented in Table 4. The sequence of the D2D3 region was 883 bp and its nucleotide composition was A = 225 bp, C = 146 bp, G = 266 bp, T = 246 bp ( Table 5). The D2D3 sequences of CS38 were separated from the other related species of "bicornatum" group by 138 to 208 bp (Table 6). Pairwise distances of ITS and D2D3 regions of rDNA showed 100% similarity and 0 total character difference between present strain and original description.

Phylogenetic analysis
For phylogenetic analysis of ITS and D2D3 regions, the phylogenetic trees were constructed by the Maximum Parsimony Method, using Subtree-Pruning-Regrafting (SPR) algorithm. Both of these regions were too conservative among closely related species to resolve the Heena et al. Egyptian Journal of Biological Pest Control (2021) (Figs. 2 and 3). All positions containing gaps and missing data were eliminated in both ITS and D2D3 regions.

Bioassay for pathogenicity and generative potential
To test the pathogenicity, different IJ concentrations were applied to all target host larvae and different parameters were measured. The data revealed that the present strain CS38 was highly virulent against all the studied insects. Mortality was initiated in all the target insects after 24 h of post infection period (PIP) and recorded as 80, 80, 90 and 100% mortality in G. mellonella, followed by 30, 50, 50 and 60% mortality in H. armigera, and 0, 10, 10 and 20% mortality in S. litura, with 25, 50, 100 and 200 IJs/larva concentrations. At 36  To evaluate the generative potential of present isolate, the cadavers of target hosts, infected with 100 IJs/larva concentration, were used for progeny production. The highest progeny count at 100 IJs/larva concentration was recorded as 24.9 × 10 4 , 92 × 10 3 and 90 × 10 3 IJs/larva, while the lowest progeny count was 14.8 × 10 4 , 37 × 10 3 and 31 × 10 3 IJs/larva in G. mellonella, H. armigera and S. litura, respectively. The mean IJs/larva production count was 19 × 10 4 IJs/larva in case of G. mellonella Table 1 Morphometrics of all generations of Steinernema abbasi CS38. All measurements are in μm (except n, ratio and percentage) and in ± SD (range)      Table 4 Pairwise distances of the ITS region between Steinernema species from the "bicornatum" group