Soil sampling and isolation of EPNs

The survey was conducted in the Syrian coastal region (4200 km2), which extends from the coastal line to 1000 m above the sea line level, during the spring and autumn months between 2017 and 2020. A total of 821 soil samples were collected randomly from different agricultural and ecological systems at the studied sites (Fig. 1). Each soil sample (approximate 2 kg) was composited of five random subsamples taken with a hand shovel to a depth of 30 cm over an area of 10 m2. The mixed samples were placed in separate polyethylene bags to prevent water loss and kept in coolers, as recommended by Kaya and Stock (1997), to be transported to the research laboratory, Department of Plant Protection, Tishreen University. EPNs were isolated from mixed soil samples using the “Galleria-bait method” (Bedding and Akhurst 1975). Ten larvae of G. mellonella were placed in each container, covered, turned upside down, and incubated in the dark at the temperature of a laboratory for 14 days. The larvae were checked every 48 h (Kaya and Stock 1997). Dead larvae, which exhibited marks of infection with EPNs, were collected and placed in a modified White trap after washing twice in distilled water (White 1929). The emerged IJs were cleaned and used to confirm Koch’s postulate of pathogenicity, then they were stored in tissue culture flasks at 10 °C. The soil characteristics (type, organic matter, PH) and habitats of the positive sampling sites are indicated in (Table 1).

Fig. 1
figure 1

Map of the Syrian coastal region showing the sites sampled for entomopathogenic nematodes. Sampling sites marked with red denote the absence of Steinernema sp., while sampling sites marked with green were positive for Steinernema

Table 1 Locality, habitat, sampling date, and soil characteristics of positive samples

Morphological and Morphometric characterization

Ten G. mellonella larvae were exposed to ca 2000 IJs in a Petri dish (9 cm diameter) lined with two moist filter papers (Whatman No. 1) (Nguyen 2007a). G. mellonella cadavers were dissected in Ringer’s solution (Sodium chloride 9.0 g, Potassium chloride 0.4 g, Calcium chloride 0.4 g, Sodium carbonate 0.2 g, Sterile distilled water to 1000 ml), to obtain the males and females of the first and second generations after 5 and 7 days post-infection, respectively. The infective juveniles (IJs) were collected during the first two days after emerging from the cadavers. For the light microscope observation and assessments, 20 specimens were examined from each life stage (males, females, and infective juveniles) alive. Additionally, specimens were killed and fixed in TAF (7 ml formalin, 2 ml Triethanolamine, 91 ml water) (Kaya and Stock 1997). Morphological traits and morphometric measurements were observed under a light microscope equipped with software (OPTIKA PROVIEW, Italy) using 4X, 10X, and 40X objectives.

The following morphometric characteristics were studied and measured, as mentioned by Nguyen (2007b). Total body length (L), maximum body diameter (MBD), distance from anterior end to excretory pore (EP), distance from anterior end to nerve ring (NR), distance from anterior end to the base of the esophagus (ES), tail length (T), anal body diameter (ABD), ratios: a = L/MBD, b = L/ES, c = L/T, D% = EP/ES × 100, E% = EP/T × 100. Additionally, for males the testis reflexion (TR), the length of the spicules (SL), the spicule width, the length of the gubernaculum (GL), the gubernaculum width, SW% = SL/ABD × 100, and GS% = GL/SL × 100 were measured. The number and arrangement of genital papillae of males were studied.

Molecular characterization

In the Laboratory of Molecular Biology, total genomic DNA was extracted from a single female of first generation using a DNeasy® Blood and Tissues Kit (Qiagen.Inc), following the manufacturer protocol. The entire internal transcribed spacer region (ITS) of rDNA was amplified using the primer TW81 (5´-GTTTCCGTAGGTGAACCTGC-´3) forward and AB28 (5´- ATATGCTTAAGTTCAGCGGGT-´3) reverse (Hominick et al. 1997). Protocol thermo cycler for the PCR was as: one cycle at 94 °C for 2 min, 35 cycles at 94 °C for 1 min, follow 55 °C for 45 s and 72 °C for 2 min then 72 °C for 10 min. Ten μl of the PCR product was loaded on a 1.2% agarose gel stained with 0.5 μg/ml ethidium bromide, with 1X TBE buffer running solation and under voltage 80 v for two hrs. 100-bp DNA ladder was used for approximant quantification of the DNA product. The gel was visualized under ultraviolet light and photographed using a gel documentation system (Bio. Doc. Analyze). The target bands were purified using the gel purification kit NucleoSpin®Extract II Kit. The purified PCR products were sequenced by Macrogen Inc. (Netherland).

Phylogenetic analysis

Evolutionary analyses were studied and the phylogenetic tree was conducted in MEGA X. for the 27 sequences of Steinernema spp. which were obtained from the NCBI database. Multiple sequence alignments of ITS gene among the extracted isolates and the existing sequences in the library were aligned using Clustal W. Caenorhabditis elegans (Maupas 1900) (accession number JN636099) was used as an out-group sequence. Ancestral states were inferred using the maximum likelihood method and Hasegawa–Kishino–Yano model (1000 bootstrap replicates) (Kumar et al. 2018).

Bioassays

Three bioassay tests were carried out to evaluate the virulence of both isolates M.22 and M.313 using the last instar larvae of G. mellonella (Glazer and Lewis 2000). All bioassays were performed using 24-well plates (1.5-cm diameter) as test arena, lined with filter paper discs (Whatman No. 1) at the bottom of each well. One-week-old IJs suspensions were used in the experiments. After washing the suspensions with distilled water several times, the concentrations of the IJs were adjusted using the formula:

$${text{Va}} = {text{Vx }}[1 – , left( {left( {i/c} right)} right]$$

where Va = the amount of water (ml) to be added (if positive) or to be removed (if negative) from the suspension. V = volume of the suspension (ml). i = initial concentration / 50 μl. c = final concentration/50 μl (Glazer and Lewis 2000). The last instar larvae of G. mellonella were reared on artificial diets (wheat flour 350 g, corn flour 200 g, milk powder 130 g,backing yeast powder 70 g, honey 100 ml, and glycerin 150 ml) (Metwally et al. 2012). The larvae were used with an average weight of (0.2–0.3) g. Each concentration was separately applied to the wells; there were 24 replicates for each treatment. Four-well plates were used. Each experiment was repeated twice. The controls were identical to the treatment except that no IJs.

Exposure time assay

The nematode suspension was adjusted at 8000 IJs/ml. After shaking the suspension, 50 µl (had around 400 IJs) was transferred to each of the filter paper-lined wells using a micropipette. Single G. mellonella larva was added to each well and the plates were sealed with their lids. All plates were kept at 18 °C. After 15, 30, 45, and 60 min, six insect larvae were removed from each plate and rinsed in water to remove nematodes from the surface. Then, they were placed in a Petri dish (5-cm diameter) lined with the moist filter paper and incubated at 22 °C. The insect mortality was recorded after 48 h.

Concentration–response assay

Different concentrations of IJs (5, 15, 25 and 45) /Larva were used. Each one was applied separately, to the wells. Single G. mellonella larva was added to each well and the plates were sealed. All plates were kept at 18 ºC. The insect mortality was recorded after 72 h of exposure.

One-on-One assay

The nematode suspension was adjusted at 100 IJs/ml. An individual infective juvenile was transferred to each of the wells in 25 µl water. It was added another 25 µl of distilled water to wash the contents of the pipette tip into each well. Single G. mellonella larva was added to each well and the plates were sealed properly. The plates were kept at 18 °C. The insect mortality was recorded after 72 h. Two plates were used in this experiment (n. 48). Half lethal concentration (LC50) and half lethal time (LT50) were estimated.

Statistical analysis

Data of experiments were analyzed in a completely randomized design. Statistical analysis was done using a statistical package for SPSS software (SPSS 16.0; IBM Corp., USA) to study the significant differences among both EPNs isolates, the means were compared using LSD test at (P < 0.01). LC50 and LT50 values were calculated using the Probit analysis. Means were compared by analysis of variance (ANOVA) and Duncan’s test of multiple comparisons at a 1% level of significance.

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