Isolation, identification of entomopathogenic nematodes with insights into their distribution in the Syrian coast regions and virulence against Tuta absoluta

A total of 821 soil samples were collected randomly from 375 sites in Syrian coastal regions (Fig. 1). The survey was primarily conducted during the spring and autumn months, with 375 and 319 samples collected during those months, respectively, and also during the summer and winter seasons, with 71 and 56 samples, respectively, collected during those months between the years 2017 and 2020.

Figure 1:

In order to survey various ecological habitats, soil samples were collected from both natural and cultivated ecosystems at altitudes ranging from 0 to 1,200 m above sea level and from different habitats identified based on ecological similarities, namely coniferous forests (Pinus spp., Cedrus spp., Chamaecyparis spp., Juniperus spp., and several kinds of trees); (n = 57); broadleaf forests (Quercus spp., Acer spp., Arbutus spp., Ceratonia spp., Pistacia spp., and several kinds of trees) (n = 62); orchards (Citrus spp. (n=33); Olea spp. (n = 37); Juglans spp. (n = 25); Vitis spp. (n=25); and Pyrus spp. (n=23). They were also collected from several kinds of fruit orchards (e.g., Malus pumila, Prunus avium) (n=55); vegetable fields (Solanum melongen, Solanum tuberosum, Cucurbita pepo, Abelmoschus esculentus, and several other kinds of vegetables) (n = 40); oil crops (Zea mays, Arachis spp., Helianthus spp.) (n=39); leguminous crops (Cicer spp., Pisum spp., Phaseolus spp.( (n = 48); leafy vegetable fields (Spinacia oleracea., Brassica oleracea., Petroselinum crispum spp., Allium cepa., Brassica oleracea var. acephala, and several other kinds of plants) (n = 43); tobacco fields (American tobacco, aromatic tobacco, and semi-aromatic tobacco) (n = 24); greenhouses (Solanum lycopersicum, Capsicum annuum, Cucumis sativus, and ornamental plant (n = 86); fodder crops (Medicago sativa, Sorghum bicolor, Hordeum spp, Vicia spp) (n = 48); grassland (n=45); riverbanks (n=31); medicinal and aromatic plants (Melissa spp., Salvia officinalis, Salvia rosmarinus, Thymus spp.) (n = 39); and environments including sea coast (Palmae spp., Hibiscus spp., sand dunes) (n = 34) and public parks (Jacaranda spp., Azadirachta spp., Dodonaea spp., Cercis spp.) (n = 27).

At each site, the soil sample (approximately 2000 g) was composed of five random sub-samples taken with a hand shovel to a depth of 30 cm over an area of 10 m². The composite samples were placed in polyethylene bags to prevent water loss, and these were kept in coolers as recommended by Kaya and Stock (1997). The soil samples were transported to the Plant Protection Research Laboratory in the Faculty of Agriculture at Tishreen University for the isolation of EPNs. Soil physico-chemical analyses, including soil type, organic matter content (%), and pH, were carried out for the EPN-positive samples only in the laboratories of the Scientific Agricultural Research Center in Latakia. These characteristics, as well as other data from the positive sampling sites, are indicated in Table 1.

EPNs were isolated from soil samples using the “Galleria-bait method” (Bedding and Akhurst, 1975). Two 500-g sub-samples from each composite sample were placed in separate clean plastic containers with ten larvae of Galleria mellonella (Lepidoptera: Pyralidae). The containers were then covered with lids, turned upside down, and kept in the dark at room temperature for 14 days. This duration was chosen due to the presence of other entomopathogens within the tested soil samples. During this period, the isolation process was repeated two to three times. In each round, new G. mellonella larvae were added to the containers, and the soil moisture was adjusted as needed. The larvae in the containers were checked every two days (Kaya and Stock, 1997). Dead larvae that exhibited marks of infection with EPN were collected, washed twice in distilled water, and placed in a modified White trap (White, 1927). The emerging IJ were cleaned and used to confirm Koch's postulate, then stored in tissue culture flasks at 12 ºC.

Ten last-instar larvae of G. mellonella (L.) were exposed to ca. 300 IJ in a 9-cm-diameter petri dish (lined with two moist filter papers (Whatman No. 1, Maidstone, UK) (Nguyen, 2007). The cadavers were dissected in Ringer's solution (9.0 g Sodium chloride, 0.4 g Potassium chloride, 0.4 g Calcium chloride, 0.2 g Sodium carbonate, and 1000 ml Sterile distilled water) after four to five days post-infection to obtain both males and amphimictic females. In this research, we specifically studied the characteristics of the males, as they play a significant role in species identification, in addition to third-stage IJ that were collected within the initial two days following their emergence from the cadavers. For the light microscope observations and assessment, 20 living specimens were examined from each life stage of males and IJ. Additionally, specimens were killed and fixed in TAF (7ml Formalin, 2ml Triethanolamine, 91 ml water) (Kaya and Stock, 1997). Morphological traits and morphometric measurements were observed under a light microscope equipped with software (Optika Proview, Optika, Ponteranica, Italy) using 4×, 10×, 40×, and 60× objectives.

The following characteristic were studied and measured, as mentioned by Nguyen and Hunt (2007): total body length (L), maximum body diam (MBD), distance from anterior end to excretory pore (EP), distance from anterior end to nerve ring (NR), distance from anterior end to base of the esophagus (ES), tail length (T), anal body diam (ABD), with the ratios a = L/MBD, b = L/ES, c = L/T, D = EP/ES, and E = EP/T. Additionally for males, length of the spicules (SL), length of the gubernaculum (GL), and Testis reflexion (TR), SW% = SL/ABD×100, and GS% = GL/SL×100, were measured. To examine the bursa of males, living males were transferred at random into a small drop of lactophenol on a hot plate at 65 °C. After 30 min, one of the males was transferred to a big drop of lactophenol on a glass slide. The anterior part of the body was removed, and then the cover glass was placed on the posterior part of the male, which was moved slowly by means of a needle in order to rotate the nematode posterior region so as to present a ventral view. Thirty bursae were observed, and the distribution of bursal rays was studied (Nguyen, 2007).

Total genomic DNA was extracted from a single hermaphroditic using a DNeasy Blood and Tissues Kit (QIAGEN, Venlo, Netherlands) following the manufacturer's protocol. The entire internal transcribed spacer region (ITS) of rDNA was amplified using the primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-’3) for ward and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-’3) reverse (Torrini et al., 2020). The thermocycle protocol for the PCR was one cycle at 94 ºC for 2 min and 35 cycles at 94 ºC for 1 min, followed by 55 ºC for 45 s and 72 ºC for 2 min, then 72 ºC for 10 min. 10μL 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 in isolation under voltage 80 v for two hours. A 100-bp DNA ladder was used for approximate quantification of the DNA product. The gel was visualized under ultraviolet light and photographed using a BioDocAnalyze gel documentation system (Analytik Jena, Jena, Germany). The target bands were purified using gel purification kit NucleoSpin Extract II Kit (Thermo Fisher Scientific, Waltham, MA, USA). The purified PCR products were sequenced by Macrogen Inc. (Amsterdam, Netherlands). The sequences were further analyzed using BioEdit and Finch TV to trim the low-quality bases, then submitted to GenBank to get the accession number. Sequence verification was performed a BLAST (Basic Local Alignment Search Tool) to verify the identity of our sequences by comparing them with the reported ones from NCBI database. For phylogenetic analyses, corresponding ITS sequences of respective EPN were retrieved from the NCBI database and aligned using ClustalW. The evolutionary tree was deduced with maximum likelihood (ML) methods. The phylogenetic tree was constructed using MEGA-X, and cladograms were boot-strapped 1,000 times for statistical validation. The branches representing bootstrap support values lower than 50% were not shown. Caenorhabditis elegans (FJ58900.1) was used as an out-group (Kumar et al., 2018; Tamura et al., 2004).

Colonies of T. absoluta were originally collected from infested tomato greenhouses in Tartus governorate. The insects were reared on potted tomato plants (Solanum lycopersicum L, cv. Mandloun, 12 cm in diameter, 6–8 weeks old) within a large wooden rearing cage (210 × 120 × 240 cm) maintained at 25 °C, with a 16:8 h (L:D) photoperiod and 45–88% relative humidity (RH) (Ben Husin and Port, 2021). In this research, the choice of target instar larvae and pupae of T. absoluta for the assays was made based on their developmental stage, which was determined using developmental time data and morphological descriptions from a previous study (currently unpublished).

The virulence of native isolates of EPNs was evaluated against the third and fourth instar larvae (both outside leaves and inside mines), as well as pupae of T. absoluta. For that, three native isolates of EPNs (S. affine M.313, H. indica F., and H. bacteriophora H.) were used. The virulence experiments were carried out using 24-well tissue culture plates (3.14 cm² surface area/well) lined with circular filter paper and tomato leaf discs, as described by Türköz and Kaşkavalci (2016). A total of six larvae per culture plate were considered as one replicate. Each treatment had four replicates, each containing 24 larvae. The plates were then sealed with Parafilm and placed into an incubator at 18 °C in the dark. The insect mortality was observed and recorded 48 hours after infection in all the conducted assays. Four replicates (n = 24) for each treatment, treated with sterile water, served as control.

For the virulence assay in outside leaves, each larva from the target instar was transferred individually into one well. Then nematodes of each species were applied at dose levels of 1, 5, 10, 15, 20, 25, and 50 IJ per target tomato leaf miner larva.

For the virulence assay inside the mines, the LD₅₀ inoculation rates for the most efficient species from the previous assay (S. affine. M 313 and H. indica. F) were used. The third-instar larvae were transferred to a culture plate and given ca. 10 minutes to establish their mines. Then, the LD₅₀ of third-instar larvae from the outside-leaves assay for S.affine and H. indica were separately applied to each plate. A similar experimental design was followed for the assay of the fourth-instar larvae inside mines, with the applied inoculation rate of nematodes corresponding to the LD₅₀ for the fourth-instar larvae from outside leaves.

In the pupal experiment, LD₅₀ of third-instar larvae from outside leaves of S. affine and H. indica were used. Each pupa was individually placed into a well containing 10% moistened sterilized sand, the inoculation rates were then separately added to each well for each species in the plate (replicates = 4, n = 24).

To explore the ecology of native EPN isolates, the occurrence was assessed as recovery frequency (no. positive samples/no. total samples) and abundance (no. positive sites/no. total sites) (Campos-Herrera et al., 2007), expressed as a percentage. The distribution of the positive samples in each site was adjusted to a negative binomial distribution (Ludwig and Reynolds, 1988). The occurrence of EPN was related to vegetation habitat, altitude, and sampling seasons. To study the influence of those variables, Crosstabs and Pearson's v2 tests at P-value = 0.05 were carried out to assess the significant differences in EPN occurrence.

To assess the impact of vegetation, habitat and some abiotic variables on the occurrence and distribution of EPN within the Syrian coastal ecosystems, various types of variables were defined (Table 2), including (i) habitat; (ii) soil physico-chemical characteristics such as type, pH, and organic matter content (%); (iii) altitude; and (iv) sampling season. A co-inertia analysis (COIA) was then applied between EPN data in correspondence to habitat and abiotic variable data (Dray et al., 2003). The multivariate analyses and graphical representations were conducted using the ade4 library (Dray and Dufour, 2007).

Hierarchical cluster analysis (HCA) was performed using various morphometric measurements of males to illustrate the hierarchical relationships among the Heterorhabditis species and group them into homogeneous clusters based on the distance matrix. The variables used for the HCA included: the mean values of body length, EP, NR, ES, ABW, T, SL, and GL, as well as the values of (a), (b), (c), and the ratios of D%, E%, SW%, and GS%.

To assess the virulence of some native EPN isolates against the third- and fourth-instar larvae and the pupae of T. absoluta, insect mortality was calculated and expressed as a percentage. Mean values of mortality (%) were analyzed by one way ANOVA. Tukey's test was then used for pairwise multiple comparisons between the EPNs isolates according to ANOVA results. Means mortalities of 3^rd and 4^th instar were compared outside leaves at different IJ concentrations of EPN, and inside mines (for the 3^rd, 4^th instar and the pupae). Differences obtained at P < 0.01 were considered significant. Probit analyses of significantly different treatments were performed using Tukey's honestly significant difference (HSD) test. Means ± SD are presented.

All analyses were done using Genstat software and R version 3.3.2 (R Core Team, 2016).

Entomopathogenic nematodes were isolated from 27 out of 821 soil samples (3.28%) collected from 24 of the 375 sampling sites (6.4%). Among the 27 isolates, 25 were heterorhabditids (3.04%), and two were steinernematids (0.24%) found in the Syrian coastal regions (Fig. 1). The analysis of the frequency of positive samples showed that most of the sites were negative for EPN occurrence. Three out of 24 positive sites had two EPN species, and in all three sites, the various species were recovered from multiple habitats. For instance, in the Fneitiq site, H. indica and S. affine were recovered from walnut orchard and grassland, respectively. In the Zeghreen site, H. bacteriophora and H. indica were found in citrus orchards and olive fields, respectively. Finally, in the Al-Basa site, H.bacteriophora and H. indica were isolated from citrus orchard and vegetable field, respectively.

Table 3 shows the recovery frequency and the abundance of EPN in each habitat with altitude, and sampling season variables. We observed significant differences between vegetation habitats for each species’ recovery frequency and abundance (x² = 28.708. df = 9, P = 0.01491) and (x² = 30.08, df = 10, P = 0.01892), respectively. The highest recovery frequency was recorded within citrus orchards (27.27%), followed by grasslands (11.11%) and olive orchards (10.81%). EPN were not found in coniferous or broadleaf forests, leafy vegetables, medicinal or aromatic plants, in fodder Leguminous and oil crop, or in greenhouses, sea coast, riverbanks and public parks. Although EPN abundance did not show significant differences among altitude ranges, the recovery frequency was significantly different (x² = 24.154, df = 7, P = 0.0138). The highest EPN recovery frequency was detected at 0–200 m above sea level (8.37%). The lowest occurrence of EPN was recorded at 200–400, 400–600, 600–800 m, at 1.79, 1.93, and 1.39 %, respectively. EPNs were not detected at altitudes at > 800 m. EPN abundance was not significantly different among sampling seasons; although their recovery frequency varied significantly (x² = 26.73, df = 7, P = 0.0474), the highest EPN recovery frequency was recorded in autumn (5.01%), followed by spring (2.4%) and then winter (1.78%). The lowest EPN recovery frequency was recorded in summer (1.4%).

Based on morphological characteristics and morphometric measures of male and infective juveniles, in addition to internal transcribed spacer (ITs) analysis, 14 out of the 27 isolates were identified as H. indica (Fig. 2), nine isolates as H. bacteriophora (Fig. 3), and two isolates as H. pakistanense (Fig. 4). Additionally, two isolates were identified as S. affine, the morphological, morphometric, and molecular characterizations of which have been documented in a separate scientific publication (Ali et al., 2022).

Figure 2:

Figure 3:

Figure 4:

The morphological and morphometric characteristics of the Syrian heterorhabditid isolates were similar to the original descriptions provided by Poinar et al. (1992) for H. indica (Table 4 and Table 5), Poinar (1976) for H. bacteriophora (Table 6 and Table 7), and Shahina et al. (2016) for H. pakistanense (Table 8). According to the HCA analysis of selected males’ morphometric characteristics (Fig. 6), the Syrian heterorhabditid isolates displayed two main groups with bootstrap values ( > 94%). The first group included all H. indica isolates and the two isolates of H. pakistanense. All isolates of H. bacteriophora grouped together in a separate clade. On the other hand, the results of the phylogenetic tree analysis of the isolates, shown in Fig. 5, present the evolutionary descent among the different species, including the Syrian isolates in this study and those reported in the database. The analysis included four isolates from H. indica, seven isolates from H. bacteriophora, four isolates from H. pakistanense, and ten from different species.

Figure 5:

Figure 6:

The results of the bursa study revealed variations and irregular arrangements of the terminal bursal ribs in H. indica (Fig. 2) and H. pakistanense (Fig. 4), while H. bacteriophora's bursa appear normal and similar to the reference description (Fig. 3). In general, the numbers of genital papillae 1–6 remain unchanged and typical for Heterorhabditis: pair 1 is straight, reaching the bursal rim, and is located anterior to the spicule heads. Pairs 2 and 3 form a group located near the middle of the spicules that are straight and reach the bursal rim. Pairs 4, 5, and 6 pairs also form a group, located near the cloacal aperture. Laterally, the fourth pair turns outward and doesn’t reach the bursal rim, while the fifth and sixth pairs turn inward ventrally, and do reach the bursal rim.

In the 30 observed specimens, H. indica exhibited two forms of variations of the terminal group. The first form (63.33%) had three pairs (7, 8, and 9) on one side and two pairs (7 and 8) on the other side, while the second form (36.66%) had two pairs (7 and 8) on both sides. In both forms of the terminal bursal ribs, we observed that the papillae were sometimes modified to be short, narrow, and swollen at the base. We observed more variation in the terminal group of the H. pakistanense bursa. The bursa exhibited four distinct forms; the first form (50%), bursa had only one pair (7) on each side; in the second form (20%), bursa had three pairs (7, 8, and 9) on one side and one pair (7) on the other side; in the third form (16.66%), bursa had two pairs (7 and 8) on one side and one pair (7) on the other side; and in the fourth form (13.33%), bursa had three pairs (7, 8, and 9) on one side and two pairs (7 and 8) on the other side.

Four species of EPNs were found in this investigation, as indicated in Table 1. H. indica was the most abundant species, representing 51.58% of the identified positive samples. It was isolated from citrus, olive, and walnut orchards, grassland, and vegetable fields during the autumn and spring seasons. H. indica was recovered from eleven sites in the Latakia governorate and from three sites in the Tartus governorate, across altitudes that ranged from 36 to 200 m, 200 to 400 m, and from 400 to 600 m. The soil textures included silty loam, sandy loam, sandy, and clay. Organic matter content (%) ranged from 1.4 to 5.72%, while pH levels varied from 5.2 to 8.1.

H. bacteriophora was detected in 33.33% of positive samples, primarily in sites located within Latakia governorate, at altitudes ranging from 4 to 200 m. A single occurrence was recorded at an elevation of 486 m in the Tartus governorate. H. bacteriophora was isolated from citrus orchards, grassland, tobacco fields, and vegetable fields during autumn and spring seasons, with soil texture including clay loam, silty loam, and sandy loam. Soil samples showed ranges from pH 4.8 to 7.8 and organic matter percentage between 1.25 and 6.2%.

H. pakistanense was isolated from 2 soil samples, representing 7.2% of positive samples. It was found to be associated with pear orchard and vineyard in the Latakia governorate during the autumn and summer seasons. The pear orchard and vineyard were at altitudes of 179 and 213 m, respectively, with a soil texture of sandy loam. The pH values were 6.1 and 7.1, while organic matter content was low, between 1.3 and 1.89%.

S. affine also was isolated from two soil samples, representing 7.2% of the positive samples. The samples were recovered from altitudes of 604 and 709 m in the Tartus governorate. S. affine was isolated from grassland and walnut orchards during the winter and spring seasons, respectively. The respective soil textures were silty and silty loam, with pH of 6.4 and 6.1, and organic matter content percentages of 4.1% and 6.1%.

The distribution of our EPN species was investigated according to habitat, soil characteristics, altitude, and sampling seasons. According to the co-inertia analysis (COIA), the fraction of variance accounted for by the two first COIA axes are 47.65 and 34.64% respectively (eigenvalues) (Fig. 7). The loading plot indicated an important contribution of Heterorhabditis indica (positive values, located on the right side of the plot) in opposition to Steinernema affine (negative values, located on the left of the plot), while, the axis COIA₂ was essentially correlated with H. pakistanense (positive values), as opposed to H. bacteriophora (negative values). In correspondence with habitat and abiotic variables, the analysis also revealed distinct groupings among EPN species and the analyzed variables.

Figure 7:

Both H. indica and H. bacteriophora showed close association with citrus orchards (Hab₁), in altitudes from 0 to 200 m (Alt₁), moderate organic matter levels of 2 to 4% (OM₂), and acidic soil with a pH less than 6.5 (pH₁).

More specifically, H. indica isolates appear to be associated with olive orchards (Hab₂) and vegetables (Hab₄), as well as clay, sandy and sandy loam soil types (C, Sa, and SaL), and the Autumn season (S₁), while H. bacteriophora isolates were essentially correlated with tobacco fields (Hab₆) and grassland (Hab₃), clay loam and silty loam soils (CL, SiL), to alkaline soil higher than 7.5 (pH₃), and to spring season (S₂).

On the other hand, Heterorhabditis pakistanense isolates showed association with pear orchard and vineyard habitats (Hab₇, Hab₈), an altitude range of 200 to 400 m (Alt₂), a moderate pH range of 6.5–7.5 (pH₂), and a low organic matter content of 1 to 2% (OM1). Steinernema affine isolates were associated with walnut orchards (Hb₅), silty soil type (Si), altitude of 600 to 800 m (Alt₄) and winter season (S₄).

The results obtained from experiments conducted on outside leaves showed that all three native EPN species tested had the capability to infect and kill the third- and fourth-instar larvae of T. absoluta, as presented in Table 9. However, the virulence varied among isolates, according to the applied dose of IJ/ larva. The mortality rates of the two tested instar larvae exhibited significant differences between the isolates on one hand and the control on the other, even at the low doses (1 IJ /larva). It is also noted from Table 9 that the mortality rate (%) gradually and significantly increases with the IJ dose increase. The isolate of S. affine M.313 showed the highest virulence for the third- and fourth-instar larvae (Fig. 8), followed by H. indica F. (Fig. 9), and then H. bacteriophora H. The mortality rates of the third-instar larvae of S. affine M.313 ranged from 25% at 1 IJ/larva to 79.17% at 50 IJ/larva, and from 29.17% to 91.67% for the fourth-instar larvae at 1 IJ/larva and 50 IJ/larva, respectively. H. bacteriophora H. exhibited the lowest mortality rates, even at the highest dose (50 IJ/ larva), with rates not exceeding 46% for the third-instar larvae, and 54% for the fourth-instar larvae.

Figure 8:

Figure 9:

According to the data analysis from the outside leaf assays, the LD₅₀ for 3^rd instar larvae of T. absoluta was 15.85 IJ/larva for S. affine M.313, 24.19 IJ/larva for H. indica F, and 48.6 IJ/larva for H. bacteriophora H. For the fourth-instar larvae, LD₅₀ was 11.38 IJ/larva for S. affine M.313, 15.71 IJ/larva for H. indica F., and 37.1 IJ/larva for H. bacteriophora H.

The results of inside mines assays of the third and fourth-instar larvae, as well as pupae, are presented in Fig. 10. Both S. affine M.313 and H. indica F showed the ability to effectively kill larvae within the mines; S. affine M313 caused mortality ranging from 33% for third-instar larvae to 42% for fourth-instar larvae. The mortality rate of H. indica F was slightly lower; it achieved 25% for third-instar larvae and 42% for fourth-instar larvae. No significant difference was observed between the EPN isolates studied, but the mortality rates caused were significantly greater compared to the control (P = 0.000932 and 0.000349, respectively). Considering the pupae assays, both EPN isolates demonstrated low mortality rates, at 12.83% for S. affine and 9.5% for H. indica. No significant difference was observed between the two isolates, or compared with the control (P = 0.05).

Figure 10: