First report of the entomopathogenic nematode, Steinernema carpocapsae, from Moghan region of Iran and its efficacy against the turnip moth, Agrotis segetum Denis and Schiffermuller (Lepidoptera: Noctuidae), larvae

During a survey of entomopathogenic nematodes (EPNs) in Moghan region of Iran in 2015, a steinernematid species was isolated, using the Galleria-baiting method. Based on its morphological and phylogenetic analysis of molecular data, the isolate was identified as Steinernema carpocapsae. The ITS rDNA sequence was deposited in NCBI (National Center for Biotechnology Information) with accession number of MF187616.1. Nucleotide row data was edited, using MEGA 6.0 software, and homologous sequences were involved in analysis, using Blast software. Sequences were aligned using Clustal W. Bootstrap analysis. The phylogenetic tree was constructed by the maximum likelihood method, using MEGA 6.0 software, and Steinernema feltiae was used as out-group. The turnip moth, Agrotis segetum Denis and Schiffermuller (Lepidoptera: Noctuidae), is one of the most important and destructive cutworm pests in Moghan. Therefore, the lethal effect of S. carpocapsae isolate Moghan (IRMoghan) was evaluated in a soil assay against the last instar larvae of A. segetum under laboratory conditions. The bioassay results showed high susceptibility of the larvae to S. carpocapsae. The LC₁₀, LC₅₀, and LC₉₀ values were 9.9, 54.13, and 246.2 IJs (infective juveniles) per larva of the pest, respectively (χ² = 7.36; df = 3, P value = 0.061). Reproduction of the EPNs within the dissected cadavers was observed. The bioassay results indicated that the new isolate is a promising biocontrol agent against A. segetum larvae with success recycling through them.

Entomopathogenic nematodes (EPNs) belonging to the genera Steinernema and Heterorhabditis are obligate parasites of insects, which kill their hosts through the septicemia caused by their symbiotic bacteria. EPNs have been used for several decades for biological control of many important insect pests worldwide (Georgis et al. 2006). EPNs possess many of the attributes as effective biological control agents and have been used as classical, conservational, and augmentative biological control agents (Kaya and Gaugler, 1993 and Koppenhöfer, 2007). S. carpocapsae is the most commonly applied species for control of foliar and other aboveground pests. This species possess a sit and wait foraging strategy (ambushers) and therefore is effective in cryptic and soil surface habitats (Laznik and Trdan, 2011 and Lacey and Georgis, 2012).

The turnip moth, Agrotis segetum Denis and Schiffermuller (Lepidoptera: Noctuidae), is an important pest in Europe, Asia, and parts of Africa. It generally lives in the ground. The pest feeds on seedlings of a wide range of the most important crops including corn, sugar beet, potato, cabbage, and many other plants (El-Salamouny et al., 2003). Due to their soil-dwelling habits, cutworms are difficult to control; however, S. carpocapsae as an ambusher species is a promising biological control agent against A. segetum. Some previous studies indicated the efficacy of this EPN against Agrotis species under laboratory and field conditions (Morris et al., 1990; Levine and Oloumi-Sadeghi, 1992 and Yan et al., 2014).

Various genera and species of the EPNs have been isolated from Iran and investigated in different aspects such as morphology and phylogeny (Kary et al., 2009 and Nikdel et al., 2010, 2011), natural incidence in insect hosts (Karimi et al., 2009), efficacy against agricultural pests (Ebrahimi et al., 2011, 2014, 2016), interaction of entomopathogenic nematodes and chemical pesticides (Sheykhnejad et al., 2014), and their interaction with insect immune system (Ebrahimi et al., 2018).

Moghan region (39° 41′ N 47° 32′ E, 40–50 m above from sea level in north-west of Iran) is one of the most important agricultural regions of Iran. Despite of specific topographic conditions of the region, survey for EPNs was not carried out there. The present study aimed to report the first record of S. carpocapsae from Moghan region of Iran and document its virulence against A. segetum under laboratory conditions.

Sampling, isolation, and culturing of the nematode

Soil sampling and trapping the samples with the last instar larvae of the greater wax moth, Galleria mellonella (L.), was carried out according to Woodring and Kaya (1988) method. The samples were collected from Moghan region, Ardabil province, Iran, during 2015 and 2016. Infective juveniles of the nematodes cultured in the last instar larvae of G. mellonella were stored in 40-ml distilled water at 5 °C.

Morphological and morphometric identification of the nematode

Males and females, collected from 5-day infected Galleria cadavers, were dissected in distilled water. Infective juveniles (IJs) were collected after emergence from Galleria cadavers in White traps (Woodring and Kaya 1988). The nematodes were killed and processed to anhydrous glycerin by a slow evaporation method (Woodring and Kaya 1988), then mounted on microscopic slides. Morphological and morphometric measurements were recorded. The morphometric measurements included the following: L = body length, W = greatest body width, EP = distance from anterior end to excretory pore, NR = distance from anterior end to nerve ring, ES = esophagus length, a = L/W, b = L/ES, c = L/T (T = tail length), D% = EP/ES × 100, E% = EP/T × 100, ABW = anal body width, SL = spicule length, GL = gubernaculum length, IJ = infective juvenile, ratio SW = spicule length/ABW, ratio GS = gubernaculum length/spicule, MUC = mucron, A = absent, P = present.

Molecular identification of the nematode: DNA extraction, amplification, and sequencing

DNA was extracted from 10 IJs, using by modified method of Hominick et al. (1997). Briefly, the IJs were crashed in 8-μl deionized distilled (dd) H₂O in a sterilized 0.5-ml microcentrifuge tube. then 12-μl lysis buffer was added, and the mixture was homogenized for 1 min. After adding 2 μl proteinase K, the tube was frozen at − 20 °C for 2 h. The tube was incubated at 67 °C for 1 h, followed by a 97 °C incubation for 10 min to digest the proteins and inactivate proteinase K. Subsequently, the tube was centrifuged at 4000 rmp for 2 min. The supernatant containing the DNA was collected and used in PCR amplification. The ITS region from of the ribosomal DNA was amplified in a sterile 0.5-ml tube. The following were used to assemble the reaction: 15 μl of Master Mix, 10 μl of dd H₂O, 1 μl of 10 pM forward primer, 1 μl of 10 pM reverse primer, and 3 μl DNA extract. The primers were TW81:5′-GTTTCCGTAGGTGAACCTGC-3′ (forward) and AB28:5′-ATATGCTTAAGTTCAGCGGGT-3′ (reverse). PCR reactions were conducted in a Thermocycler. The PCR cycles were as follows (Hominick et al., 1997): 1 cycle at 95 °C for 4 min followed by 33 cycles at 95 °C for 30 s, 57 °C for 40 s, and 72 °C for 30 s. The last step was 72 °C for 5 min. PCR product was purified and sequenced by Bioneer Corporation, Korea. The obtained sequence was deposited at the NCBI (National Center for Biotechnology Information) database with the accession number of MF187616.1. Nucleotide row data was edited using MEGA X software (Kumar et al., 2018), and homologous sequences were involved in analysis using Blast software. Sequences were aligned, using Clustal W. Bootstrap analysis. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei 1993). The phylogenetic tree was constructed by the maximum likelihood method, using MEGA 6.0 software, and Steinernema feltiae was used as out-group.

Insects

Galleria mellonella

The greater wax moth, G. mellonella, was reared in plastic boxes with ventilated lids at 26 ± 2 °C, 50 ± 5% RH, and 16:8 (L:D) photoperiod and on an artificial diet composed of 1200-g wheat flour (44.1%), 600-g honey (22.1%), 500 ml of 99% glycerol (18.4%), 300-g yeast (11%), and 120-g bee wax (4.4%).

Agrotis segetum

Last instar larvae of A. segetum were collected from tomato fields in Ardebil Agricultural Research Station in Moghan region and transferred to the laboratory of Plant Protection Research Department. The larvae were reared on corn seedlings. Prepupae were placed in the egg lying dishes described by Sherlock (1979). The eggs were collected, and the developed last instar larvae were treated based on Sherlock (1979) method, with exception of using fresh corn seedlings as feeding source for the larvae. Rearing conditions were 25 ± 1 °C and of 70 ± 10% RH and photoperiod 16:8 (L:D). In 24-h time intervals, last instar larvae were collected from rearing dishes and were used in the bioassay experiments.

Nematode

Infective juveniles of S. carpocapsae IRMoghan were cultured on the last instar larvae of G. mellonella (Woodring and Kaya, 1988). The IJs were stored in distilled water at 5 °C and used in all experiments within 30 days of emerging from the host. Before starting the experiments, nematodes were kept at 25 °C for 20–30 min.

Bioassay

Last instar larvae of A. segetum were used in the bioassay experiments. The experiments were conducted in plastic cups (9 cm height and 7.5 cm diameter), filled with 250-g autoclaved moist sandy soil (85 sand, 10 silt and 5 clay and 10% moisture (w/w)). Based on preliminary experiments, a range of 10–100 IJs per insect (i.e., 10, 25, 50, 75, and 100 IJs per larva) was used. IJs were added in certain concentrations in 0.5 ml of water to the surface of the soil, separately. Finally, one larva was placed on the soil, and the cups were covered by ventilated lids to avoid desiccation. Control cups received 0.5-ml distilled water without nematodes. A piece of stem of fresh corn seedlings was used as feeding source which was renewed daily. Fifteen cups were used for each nematode concentration and the control. The dead or alive insects were counted after 7 days. The experiment was replicated 3 times. All dead insects were collected and dissected to ensure the presence of nematodes inside the cadavers.

Statistical analysis

LC₂₀, LC₅₀, and LC₈₀ values were obtained by Probit analysis, using SAS software (SAS Institute 2004). Analysis of variance was done, and the means were evaluated by Duncan’s multiple-range test (SAS Institute 2004). Lethal experimental data was transformed into square root of (x + 1) where needed, before analysis.

Morphometric and molecular identification

Morphometric data of IJs and first-generation males of the isolate are shown in Tables 1 and 2.

Infective juvenile: Body slender, enclosed in a sheath. Pharynx long and narrow, often degenerate, tail elongate and conical.

Males: Body C-shaped when heat-relaxed. Cuticle smooth. Stoma shallow. Pharynx with cylindrical procorpus, slightly enlarged metacarpus, followed by an isthmus and a basal bulb with valve. Nerve ring surrounding isthmus. Excretory pore anterior to nerve ring. Reproductive system monorchic, testis reflexed. Spicules paired, slightly curved. Gubernaculum boat-shaped in Bursa absent.

Based on its morphological and molecular properties, the isolate was identified as Steinernema carpocapse. The analysis of the ITS rDNA sequence confirmed the species identification.

The phylogenetic tree was constructed, and the tree with the highest log likelihood (− 2815.96) was shown. The relative phylogenetic position of the new isolate, S. carpocapsae MoghanIR1, was determined according to neighbor joining based on the 28S rDNA sequence (Fig. 1). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated, using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Evolutionary relationships of new EPN isolate (IRMoghan1). The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site

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Bioassay

Last instar larvae of A. segetum were found susceptible to infection with S. carpocapsae IRMoghan. Analysis of variance revealed significant differences among varied concentrations of S. carpocapsae IRMoghan on A. segetum mortality (F = 39.52; df = 5; P < 0.0001) (Table 3). There were no dead insects in control cups during experimental period. At the highest concentration, the nematode caused greater mortality than all other concentrations. All the treated nematode concentrations caused greater mortality than the control and those of 10 IJs/larvae (Table 4).

The LC₁₀, LC₅₀, and LC₉₀ values for S. carpocapsae IRMoghan1 on A. segetum were 9.9, 54.13, and 246.2 IJs per larva of the pest, respectively (χ² = 7.36; df = 3, P value = 0.061) (Table 5). Regression analysis revealed a significant correlation between log concentration and insect mortality (Fig. 2). Because goodness-of-fit Chi-Square value was insignificant for Probit analysis; no heterogeneity factor was used in the calculation of confidence limits and LC₁₀, LC_50, and LC₉₀ values. Reproduction of the EPNs within the dissected cadavers was observed.

Mortality (Probit-transformed) of Agrotis segetum treated with different concentrations of Steinernema carpocapsae IRMoghan

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This is the first record of S. carpocapsae form Moghan region of Iran. Two surveys have been conducted in Iran and several species of EPNs including S. carpocapsae were reported (Kary et al., 2009 and Nikdel et al., 2010). Both surveys were carried out in mountainous and cold climatic region of Iran, while the Moghan Plain has a completely different climate. The Moghan Plain due to the special topographic conditions basically has a contrary climate in contrast to other parts of Azerbaijan and even its southern regions. Based on the weather data of the Pars-Abad synoptic meteorological station, Moghan region has a mild semi-desert climate with mild winters and warm and humid summers (Shiri et al., 2015).

Various studies have investigated the effect of EPNs on different species of Agrotis in various methods (Unlu et al., 2007; Gokce et al., 2013; Yan et al., 2014 and Yuksel and Canhilal 2018). Obtained results are consistent with other researches concerning the effect of S. carpocapsae on A. segetum. Unlu et al. (2007) evaluated the effect of the Turkish isolate of S. feltiae, S. weiseri, and S. carpocapsae (10 to 100 IJs per insect) against A. segetum and reported a higher efficacy of S. carpocapsae than the other species. One hundred IJs per insect concentration of S. carpocapsae Turkish isolate caused (75%) mortality of A. segetum, while the same concentration of S. carpocapsae IRMoghan isolate showed (83.83%) mortality of the insect. Despite of a high mortality due to Moghan isolate in the highest concentration of nematodes, 50 IJs of Moghan isolate caused 30% mortality of the insect, while it was 60% for the Turkish isolate. Gokce et al. (2013) reported a high susceptibility of A. segetum larvae to S. websteri, as 100 IJs g⁻¹ of dry sand caused about 84% mortality rate of the insect.

The results of the bioassay revealed a high susceptibility of A. segetum to S. carpocapsae IRMoghan isolate. Overall, most of the lepidopteran insects are susceptible to EPNs due to their defense mechanisms (Lacey and Georgis, 2012). On the other hand, S. carpocapsae has a wide host of insect range (Labaude and Griffin, 2018). However, other factors like virulence of the nematodes due to the symbiotic bacteria against A. segetum and probable compatibility of the native isolate of S. carpocapsae with the pest could be effective in susceptibility of A. segetum larvae. Native species are often expected to be adapted to local conditions, and their use is preferred (Labaude and Griffin, 2018).

The results of this study indicated that S. carpocapsae IRMoghan isolate is a promising biocontrol agent against A. segetum with success recycling through the pest. Isolating S. carpocapsae from Moghan region and its compatibility with the insect pests of the region could incite more investigations on applicable use of this nematode species in integrated pest management programs in Moghan.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

EPNs:

Entomopathogenic nematodes,

IJs:

Infective juveniles

NCBI:

National Center for Biotechnology Information

We appreciate Z. Majd Taheri, Nematology Research Department, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran, due to her valuable technical helps in molecular identification of the nematode. Also, we greatly appreciate MR Shiri, Seed and Plant Improvement Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran, for his valuable helps during implementation of the project.

Funding

This study was supported financially by Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran, which is greatly appreciated.

Affiliations

1. BioControl Research Department, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran
   • Laleh Ebrahimi
2. Nematology Research Department, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran
   • Zahra TanhaMaafi
3. Plant Protection Research Department, Ardabil Agricultural and Natural Resources Research and Education Center, AREEO, Moghan, Iran
   • Parviz Sharifi

Contributions

LE carried out nematode isolation and identification and the experiments and was the major contributor in writing the manuscript. ZT participated in the morphological and morphometric and molecular identification of the nematode. PSH participated in nematode sampling and insect rearing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Laleh Ebrahimi.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

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