Survey and identification of entomopathogenic nematodes in the province of Cotabato, Philippines, for biocontrol potential against the tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

The tobacco cutworm, Spodoptera litura [Fab.] (Lepidoptera: Noctuidae), is a devastating insect pest of several crops. Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are used as an alternative control measure in lieu of the hazardous synthetic chemical applications. A survey of naturally occurring EPNs was conducted across the province of Cotabato, Philippines, covering a total of 5 municipalities with 25 villages. Galleria-baiting technique was employed to recover nematodes from peanut and grassland soils. Out of 50 soil samples collected, only 5 samples harbored nematodes, indicating a recovery of 10%. Preliminary morphological data identified only one EPN under the genera Heterorhabditis (1 isolate), whereas 4 were facultative necromenic nematodes from the genera Metarhabditis (2 isolates) and Oscheius (2 isolates). Analysis of D2D3 segments of the 28S rDNA confirmed high sequence similarity to Heterorhabditis indica, Metarhabditis rainai, Oscheius insectivora, and Oscheius sp. This is the first record of H. indica and M. rainai in the entire region, whereas the first record for Oscheius spp. in the Philippines. Furthermore, the biocontrol potential of the local H. indica infective juvenile (IJ) populations (PIGCD1) isolated from peanut was assessed against the tobacco cutworm, S. litura, under laboratory conditions. The mean percentage mortality caused by H. indica on S. litura at 7 different concentrations ranged from 0-100% at 24 h post inoculation. The lethal concentration (LC50) required to kill 50% of the S. litura larvae population with H. indica was 7.13±1 (IJs/larva). The use of Galleria-baiting method is a convenient approach to detect EPNs including other facultative necromenic nematodes from the soils. Obtained data indicated that the local H. indica isolate can be a promising alternative measure to suppress the economically important insect pest, S. litura, and this may provide significant outlook to establish the biocontrol program in the country.


Background
The tobacco cutworm, Spodoptera litura [Fabricius] (Lepidoptera: Noctuidae), is a serious polyphagous insect pest, widely distributed throughout tropical and subtropical areas of Asia and Pacific Regions (Bragard et al. 2019). In the Philippines, it is one of the most economically important pests, reported infesting several crops. Control of S. litura is mainly dependent on synthetic insecticides, many of which have developed high insecticide resistance, leading to periodic out breaks and resurgence of pest and eventually, crop failure (Shad et al. 2012). The rampant application of harmful chemicals has caused perennial environmental pollution and deleterious effects on humans including other beneficial and non-target organisms (Jeyasankar et al. 2014). The increasing awareness of their several disadvantages have further revolutionized the exploration and utilization of alternative control measures like the biological control agents (BCAs), which are effective, eco-friendly, and safe (Grewal et al. 2005).
Entomopathogenic nematodes (EPNs) of the families Heterorhabditae and Steinernematidae are known as most effective BCAs (Kaya and Gaugler 1993). The genera with the most successful stories are those of Steinernema and Heterorhabditis associated with their respective symbiont bacteria, Xenorhabdus and Photorhabdus (Ferreria and Malan 2014). The bacterial cells are released and rapidly kill the insect within 48-72 h (Boemare 2002).
The natural occurrence of EPNs has been documented in different soils, ranging from natural and managed ecosystems of all continents with the exception of Antarctica (Laznik and Trdan 2011). Numerous surveys in agricultural (managed) areas were conducted to collect and describe native EPNs using morphological and molecular diagnostic tools for the management of local target pests (Stock et al. 2008). These native EPN populations have mainly gained an increasing attention due to its beneficial attributes like better adaptation to local biotic and abiotic conditions (Stock et al. 2008 andCampos-Herrera et al. 2012). At present, about 100 Steinernema and 16 Hetererhabditis have been described worldwide (Bhat et al. 2020). These EPNs are broadly applied as biopesticides against insect pests (Lacey et al. 2015). For instance, S. carpocapsae induced a high mortality against S. litura from tobacco and cotton (Abdel-Razek and Abdelgawad 2007). In the case of H. indica, it was reported to be highly virulent and effective for the control of S. litura larvae (Acharya et al. 2020) in corn, cotton, and vegetable crops (Caoili et al. 2018).
The province of Cotabato being located in the island of Mindanao, the Philippines is considered a predominantly agricultural area. This province is under Region 12 (SOCCSKSARGEN) which shared the highest number of farms with 126.7 thousand hectares, covering 275.5 thousand hectares of agricultural land (Philippine Statistics Authority 2004). The use of EPNs for integrated pest management (IPM) in the area has never been explored, which may offer valuable contributions in different farms. Collection of native EPNs in the area is a vital step toward the sustainable pest suppression in the agricultural areas.
This present study therefore aimed to survey EPNs from managed and unmanaged areas like the peanut and grassland fields within the province and to assess their biocontrol efficacy against the target local pest, S. litura. This study provided additional account to the EPNs and other insect-associated nematode diversity in the country. More importantly, this can serve as baseline information toward the selection of efficient native isolates for peanut pest control and other economically important crops.

Culture of insects
The greater wax moth, Galleria mellonella [L.] (Lepidoptera: Pyralidae), was used as bait to isolate EPN from soil sample and for subsequent identification of EPN. Rearing was carried out, using an artificial diet comprising of wheat bran (37.5%), honey (23.4%), bees wax (11.7%), glycerol (21.4%), nipagine (0.5%), and yeast (5.5%) at 37°C with a 70% relative humidity (Kaya and Stock 1997). Egg masses of S. litura were collected from different peanut plantations in the province of Cotabato. After egg hatching, fresh vegetables, legume pods or seeds, and tomato fruits were provided for larval feeding until pupal stage under laboratory conditions, following the descriptions of Zhang et al. (2019) with modifications. Heat-treated soil was provided for pupation, after which the developed pupae were collected from the soil and subsequently placed inside a cage for adult emergence. To increase the rate of fecundity, 10% honey solution mixed with a few drops of multi-vitamin was added for adult nourishment. Folded filter papers were provided for egg laying, then egg masses were recollected and allowed to hatch. This rearing process was repeated and only a single culture was maintained for virulence assays.

Soil sampling
Soil samples were randomly collected from peanut (managed) and grassland (unmanaged) areas in the province of Cotabato, Philippines, from December 2018 to February 2019. A total of 50 samples (2 from each site) were collected from different sampling sites, covering 5 towns with 25 barangays or villages. From each site, 5 subsamples were taken from a 20-25-cm deep, using a hand shovel and mixed together to obtain approximately 1 kg of composite samples (Orozco et al. 2014). The geographical coordinates and corresponding elevations were recorded along with some important edaphic parameters such as soil temperature, pH, moisture, and texture (Abate et al. 2017). The temperature, pH, and moisture of each soil sample were recorded in situ, using a 4-in-1 soil survey instrument (BGT-SM4, Beijing, China). Soil texture was characterized by ring method (Daddow and Warrington 1983). All soil samples were labeled, sealed, stored in a Styrofoam box, and was brought to the Department of Plant Pathology, College of Agriculture at the University of Southern Mindanao in Kabacan, Cotabato, for further processing.

Insect-baiting and nematode culture
Insect-baiting was carried out according to the original description of Bedding and Akhurst (1975). Ten last instar larvae of G. mellonella were added to each container with the soil samples and then incubated at 25±2°C. The samples were regularly sprayed with water to avoid desiccation and monitored daily to check for successful insect infection. All collected cadavers were washed with distilled water and placed in a modified White trap (White 1927) until emergence of infective juveniles (IJs) was evident. IJs were then harvested, cleaned, and stored at 10-20°C. To obtain pure culture of the nematodes recovered from the soil, re-inoculation was done 3 times to G. mellonella larvae (Hoy et al. 2008).

Morphological and molecular characterization
Infective juveniles, adult male and female nematodes, were heat-killed and processed for fixation according to De Grisse (1969). A series of transfer to anhydrous glycerin was done, using the following 3 solutions: solution 1 (containing a 50:50 ratio of 4% formalin and glycerin), solution 2 (containing a 50:50 ratio of 96% ethanol and 4% formalin), and solution 3 (containing pure glycerin).
Morphological key characters of the genera were observed and further measurement of the body lengths was carried out using an Olympus CX22 compound microscope equipped with a digital camera. IJ and male measurements were undertaken, using the ImageJ software. The EPNs isolates were classified, using taxonomic keys from Nguyen and Hunt (2007), whereas the facultative necromenic nematode isolates were diagnosed based on Sudhaus (2011) until genus level only. Molecular analysis was undertaken by amplification of the D2D3 expansion segments of the 28S rDNA for species identification. For this, DNA extraction was first performed, following the method described by Spiridonov et al. (2004) with modifications and using the DNA extraction kit (Dongsheng Biotech). Succinctly, 5 IJs were individually picked, placed on top of the glass slide with a drop of lysis buffer. IJs were cut into small pieces, using a sterile scalpel and transferred into a micro-centrifuge tube by adding the remaining 350 μl of the lysis solution. A stepwise process involving Proteinase K, different solutions, wash and TE buffers coupled with vortexing, and centrifuge process were done as indicated by the kit manufacturer. The extracted DNA was then stored at −20°C in deep freezer for further processing. Products were sent for sequencing in Macrogen, Inc. (Seoul, South Korea). The universal primers D2A (5′-CAAGTACCGT GAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAA CCAGCTACT A-3′) were used to amplify the D2D3 expansion segment of 28S rDNA (de Brida et al. 2017). PCR conditions were programmed for initial denaturation at 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 60 s, and a final extension at 72°C for 15 min (Bhat et al. 2019).

Sequence alignment and phylogenetic analysis
Sequences obtained were first edited, using BioEdit 7.2. The D2D3 expansion segment of 28S rDNA sequences of all 5 isolates were aligned with their closest BLAST search matches in the National Center for Biotechnology Information (NCBI) GenBank (Altschul 1990). ClustalW multiple alignment was used for sequence alignment. A phylogenetic tree was constructed using the MEGA-7 (7.0) software (Kumar et al. 2016) by the maximum likelihood with a Tamura-Nei method (Tamura and Nei 1993). Steinernema carpocapsae (KY914572.1) and the model organism Caenorhabditis elegans (X03680.1) were used as outgroup taxa. All isolates were trimmed, annotated, and deposited in the NCBI GenBank with their accession numbers.

Virulence tests for H. indica
Virulence test was measured on insect mortality of the 3 rd instar larvae of S. litura. Following a modified version of the methods used by Godjo et al. (2018), approximately 50 g of heat-treated sand with 20% moisture content was placed in Petri dishes (60×15 mm). The following nematode concentrations or dosages were prepared and added to each plate: 10, 50, 100, 150, 200, 250, and 300 IJs/ml with 4 replications. After an hour, 10 3 rd larval instars of S. litura were subsequently added to the set-ups where they burrowed into the sand naturally. The control set-up only received sterile distilled water (SDW). After 24 h, insects were retrieved from the Petri dishes, recording the total number of dead larvae. Insect cadavers were dissected to confirm EPN infection (Sumaya et al. 2018).

Data analyses
The distribution of nematode species in the province was determined and shown in a map using QGIS version 3.10.1 A Coruna. The concentrations required to kill 50% of S. litura insect population (LC 50 ) for each replicate was determined, using Probit analysis (Finney 1952). The mortality data showing a non-normal distribution was analyzed using a Kruskal-Wallis test and a post hoc Dunn's test for multiple comparisons was used. Data analyses were performed in R version 4.0.2 (R Core Team 2020).

Nematode survey and recovery
Soil samples were randomly collected from peanut and grassland areas of Cotabato province in the Philippines. From a total of 50 soil samples collected from 5 towns covering 25 villages or barangays, only 5 soils harbored nematodes indicating a recovery rate of 10%. Out of 5 nematode-positive areas, 2 isolates were recovered from peanut fields in Kabacan (Osias) and Pigcawayan (North Manuangan), and 3 isolates from grassland areas in Libungan (Ulamian), Aleosan (Dualing), and Midsayap (Bual Norte). The elevation ranged between 12.6 and 61.9 masl with the following soil parameter values: soil pH (6.5-7), soil temperature (33-34°C), and soil texture (clay, clay loam, silty clay loam, and sandy clay loam). All soil samples were dry during the sampling period (Table 1).

Morphological and molecular characterization
Morphological key characters for the genera were examined and measurement of the body lengths (e.g., IJs and males) was carried out, using an Olympus CX22 compound microscope equipped with a digital camera. The EPN isolate was classified, using taxonomic keys of Nguyen and Hunt (2007) until genus level only. For PIGCD1 isolate, the body length range of IJs was 530-622 μm and males were from 759-808 μm, belonging under the genus Heterorhabditis.

Sequence alignment and phylogenetic analyses
The phylogenetic tree using maximum likelihood (

Virulence of H. indica against S. litura
Virulence of the lone H. indica PIGCD1 isolate from peanut field was evaluated based on the mean mortality (%) to the 3 rd instar larvae of S. litura 24 h post inoculation (Fig. 3). The larvae were exposed to 7 different concentrations, namely, 10, 50, 100, 150, 200, 250, 300 IJs/ larva, each with 4 replications. Significant differences among treatments (χ 2 = 26.85, df = 6, p = 0.0001) were observed for the mean mortality (%) at different Dichusa et al. Egyptian Journal of Biological Pest Control (2021)   concentrations with mean mortality ranging from 0-100%. While the highest mean mortality was obtained starting from the concentrations of 200-300 IJs/larva, no mortality was recorded in the concentration of 10 IJs/ larva and the control set-up. Further on, the lethal concentration required to kill 50% of the insect population (LC 50 ) was estimated to be 7.13 ±1 (IJs/larva), implying the high virulence and biocontrol potential of the local H. indica isolate.

Discussion
Entomopathogenic nematodes are used as biocontrol agents with several success stories in suppressing insect pests of different crops (Kaya et al. 2006). In this study, occurrence and distribution of EPNs in different areas of Cotabato province, Philippines, was conducted in order to assess their biocontrol potential to economically important insect pest like the tobacco cutworm, S. litura. From a total of 50 soil samples collected from peanut and grassland areas, only 5 harbored nematodes indicating a recovery rate of 10%. This low recovery rate is congruent with several previous studies on EPNs recovery in different surveys worldwide (Caoili et al. 2018 andKour et al. 2020). Several abiotic and biotic factors may likely influence the recovery of nematodes in the soil such as soil texture, moisture, pH, and vegetation (Abate et al. 2018 andCampos-Herrera et al. 2019). Although response of EPNs to these abiotic and biotic factors may vary within each species, their prevalence and abundance in sampling areas have nevertheless optimal levels, preference, and tolerance. Interestingly, by using the morphological and molecular taxonomic data, not only EPNs (one heterorhabditid) but also facultative necromenic nematodes (4 rhabditids) in the G. mellonella infected cadavers were detected. Generally, the H. indica isolate had similar IJ and male body size range and morphology as the type strain from India (Poinar et al. 1992), Vietnam (Phan et al. 2003) and the Philippines (Pascual et al. 2017). Analysis of D2D3 expansion segments of 28S rDNA region confirmed the following nematode species: H. indica, M. rainai, O. insectivora, and Oscheius sp. Obtained lone H. indica (PIGCD1 isolate) recovered from peanut fields in Pigcawayan had a ≥99% similarity to isolates from  Notably, these rhabditid nematodes were earlier found to have a biocontrol potential (Dillman et al. 2012 andTorrini et al. 2015). Other entomophilic nematodes also known as facultative necromenic nematodes from the genera Metarhabditis and Oscheius have demonstrated their biocontrol potential against vegetable cruciferous pests (Park et al. 2012 andTorrini et al. 2015). In this study, however, biocontrol potential of these 4 facultative necromenic isolates which were one of our research outlooks was not assessed. Several previous studies have demonstrated the efficacy of H. indica against a variety of insect pests including S. litura (Acharya et al. 2020). Similar result, H. indica, was reported to be very effective against S. litura under laboratory and field conditions (Gokte-Narkhedkar et al. 2019). Caoili et al. (2018) likewise reported that H. indica (PBCB), an isolate from Luzon island, Philippines, was highly virulent against S. litura, with percentage mean mortality of 88.67%. However, they had a higher LC 50 value (8.89 IJs/larva) at 48 h post inoculation. Moreover, in Egypt, Heterorhabditis sp. ELG., H. indica, and Heterorhabditis sp. ELB were found to have the highest in activity, obtaining a 100% mortality to S. littoralis larvae in a Petri dish assay 24 h post exposure (Abdel-Razek and Abdelgawad 2007), which is in agreement with obtained present data.

Conclusions
Nematode surveys in different areas of Cotabato province, using G. mellonella baiting method, were a convenient approach to detect EPNs including other facultative necromenic nematodes from the soils. One local EPN, H. indica isolate (PIGCD1) from peanut fields was documented in the town of Pigcawayan and 4 facultative necromenic rhabditid nematodes (M. rainai, O. insectivora, and Oscheius sp.), providing additional account of EPNs and other nematode species in the country and extending their habitats' range and geographic distribution. This is the first record of H. indica and M. rainai in Region 12 or SOCCKSARGEN, whereas Oscheius spp. was the first report in the Philippines. The virulence of the local H. indica isolate on the 3 rd instar larvae of S. litura was recorded to be very high. Therefore, this lone isolate can be further used as biocontrol agent of economically important insect pests.