SEARCH WITHIN CONTENT
Keywords : Biological control, Entomopathogenic nematode, Helicoverpa armigera, Heterorhabditis indica, Internal transcribed spacer, Ostrinia furnacalis, Ribosomal DNA region, Spodoptera litura, Steinernema abbasi
Citation Information : Journal of Nematology. Volume 50, Issue 2, Pages 99-110, DOI: https://doi.org/10.21307/jofnem-2018-024
License : (PUBLISHER)
Published Online: 03-June-2018
In search for local entomopathogenic nematode (EPN) species as a biological control agent of lepidopterous insect pests of corn, a survey for EPN in the major islands in the Philippines was conducted. Seven EPN populations from 279 soil samples were isolated using
Entomopathogenic nematodes (EPN) have long been studied for their pronounced virulence against a wide range of insects belonging to orders Lepidoptera, Coleoptera, Diptera, Thysanoptera, and Orthoptera. Their parasitic life cycle is initiated by the infective juveniles (IJ), either through actively searching (cruisers) or waiting (ambushers) for their insect host. They may enter the host through body openings or by penetrating directly the cuticle to reach the hemocoel. Once in the hemocoel, they release their bacterial endosymbionts (Xenorhabdus and Photorhabdus for Steinernema and Heterorhabditis, respectively) that multiply very rapidly in the hemolymph resulting to host death within 24 to 72 hr. The nematodes then feed on the bacteria and complete their life cycle inside the insect cadavers. They emerge from the deteriorating cadaver, carrying the bacterial symbiont in the anterior part of their intestine to begin another infection cycle (Smart, 1995; Burnell and Stock, 2000).
EPN-based technology provides a biological control option on many of the most important pests of agricultural crops. In addition, EPN can be integrated with other management strategies and reduce growers’ dependence on chemical insecticides. The efficacy of EPN has been demonstrated against several insect pests in different countries such as black vine weevil on cranberries (Shanks and Agudelo-Silva, 1990), leaf miner on ornamentals and vegetables (Hara et al., 1993), citrus root weevil (Bullock et al., 1999), mole crickets on turf grass (Barbara and Buss, 2005), and peachtree borer (Shapiro-Ilan et al., 2009) in the United States; European corn borer (Ben-Yakir et al., 1998) in Israel; fall army worm (Negrisoli et al., 2010) in Brazil; western flower thrips in Germany (Ebssa et al., 2001); and Japanese pine sawyer in Asia (Phan, 2008). As a matter of fact, EPN-based commercial products are already available in Europe, the United States, Australia, and in parts of Asia (Georgis and Hom, 1992; Kaya et al., 2006).
EPN are naturally found in both agriculturally disturbed and undisturbed soil environments with reports of occurrences from many temperate and tropical countries. To date, there are at least 90 Steinernema and 20 Heterorhabditis species reported (Shapiro-Ilan et al., 2017). A number of new additions to this growing list are newly described species from Asia, indicating a high diversity of EPN in the region. Gapasin et al. (2016), although reported the occurrence of EPN in the Philippines, the identification was only at the genus level. In addition, their collection was limited to Steinernema spp. and Heterorhabditis spp. in sweet potato growing areas in the country.
Corn is one of the most economically important commodity in the Philippines. From 2010 to 2014, the area planted to corn has increased up to 2.6 Mha with a production of ~7.8 Mt (Food and Agriculture Organization of the United Nations, 2017). Many of the Filipino farmers depend on tilling their land for corn production either for feed or food. However, corn production areas have been threatened by the prevalence of different pests and pathogens with the vast majority comprised of herbivorous lepidopterous insects including the Asian corn borer, Ostrinia furnacalis Guenée, which is considered the most serious biotic constraint in corn fields in the country (Camarao, 1983; Gerpacio et al., 2004; Litsinger et al., 2007; Afidchao et al., 2013), as well as the corn earworm, Helicoverpa armigera (Hubner), and the common cutworm, Spodoptera litura Fabricius (Gerpacio et al., 2004).
Two generations of O. furnacalis are observed in a corn cropping season in the Philippines, with the first and second generation coinciding with the vegetative and pollen shedding, respectively. The early stage larvae feed on the leaves and tassel. Camarao (1976) noted that although the late 4th instar larvae tunnel into the stalks and feed until pupation, some remained in the whorled leaves and unopened tassel and spikelets until pupation. Helicoverpa armigera and S. litura also attack the leaves during the vegetative stage. During reproductive stage however, H. armigera prefers to feed on the tassel and cobs (Ramos and Morallo-Rejesus, 1981). Both lepidopterans exhibit overlapping generations in a corn cropping season. The last instar larvae of H. armigera migrate toward the soil where they pupate (Smith-Pardo, 2014). The larvae of S. litura on the other hand, prefer moist sites and often hide in the soil during the day attacking young corn plants at night. The common cutworm also pupates in the soil (Nurhidayat, 2003).
With the concerns regarding the management and control of these pests, many of the farmers depend on the use of chemical pesticides and deployment of genetically modified corn varieties. However, key issues on the deleterious effects of heavy pesticide usage (Mohankumar and Ramasubramanian, 2014) and the apparent emergence of resistant pest populations (Gassmann et al., 2011; Tabashnik et al., 2013; Tabashnik, 2015) prompted the search for a more sustainable and environmentally-sound technology to address pest problems. As many countries have proven the efficacy of EPN as biological control agents, the development of such technology in the Philippines can be tapped to formulate a new pest management strategy.
With very limited studies on EPN in the country, the study was designed to determine the presence of indigenous EPN species virulent to lepidopterous insect pests of corn using a target insect pest as bait specifically, O. furnacalis. The use of O. furnacalis as host bait was explored basically because it has the closest phylogenetic relationship with Galleria mellonella (Linn.) (Regier et al., 2012) and it is the most damaging among the lepidopteran pests of corn. The study also aimed to investigate EPN distribution, and their biological control potential in order to develop local EPN populations into a viable technology for insect management in corn-producing areas.
Soil sampling was done by initially clearing off the soil surface from litter and vegetation and digging a pit to a depth of about 10-cm using a shovel. From that pit, a soil sample weighing ~1.5 kg was collected, placed inside a resealable plastic bag and labelled accordingly. From each site, two to three samples were taken depending on the size of the area. Sampling was conducted in agricultural and forested areas in Albay, Batangas, Camarines Sur, Bukidnon, Cavite, Cebu, Isabela, Laguna, Misamis Oriental, Pangasinan, Pampanga, Quezon, and South Cotabato. Soil samples positive for EPN were analyzed for pH, organic matter, sand, silt, and clay contents. The baiting technique for nematode extraction from soil was carried out by burying five live O. furnacalis, larvae underneath the moist soil in an aerated 280 cm3 plastic container. Dry soil samples were moistened with distilled water prior to use. Two replicates were prepared for each sample. The setups were incubated in the dark for a week with daily observations to check the insect condition. Dead insects were collected and placed in modified White traps (Kaya and Stock, 1997) or incubated for 3 to 5 days in 60-mm petri plates lined with moistened filter paper for nematode harvesting.
The EPN were mass-reared in O. furnacalis larvae. The IJ were stored at ~2,500 IJ per ml in 25 cm2 polysterene tissue culture flasks (FalconTM, Corning, Inc., New York, NY) at room temperature (28.0 ± 2.0°C). For the bioassay experiments O. furnacalis (12-day old), S. litura (9-day old), and H. armigera (9-day old) larvae were used as test insects. The insects were reared in the laboratory using an artificial diet developed by Ceballo and Morallo-Rejesus (1983).
Permanent mounts of first generation female, male, and IJs were prepared for light microscopy examination. Preliminary identification of the EPN up to the genus level was based on general morphology, as described by Nguyen and Hunt (2007) and Nguyen et al. (2007). Since the local EPN were reared in O. furnacalis instead of G. mellonella, which is the model host for EPN where morphometric data were based upon, molecular characterization using the internal transcribed spacer 1-5.8S-internal transcribed spacer 2 (ITS) region of the ribosomal DNA was carried out to identify up to species level. The total DNA was extracted from a single first-generation female using a technique slightly modified from Phan et al. (2005). Briefly, each EPN was macerated in 10 µl of worm lysis buffer and transferred into a microcentrifuge tube, to which 5 µl of sterile distilled water and 1 µl proteinase K (600 µg/ml) were added. The samples were then frozen at −20°C in an absolute ethanol bath for an hour followed by incubation at 65°C for another hour and finally at 95°C for 10 min. The tube was centrifuged at 13,000g for a minute and the DNA suspension was used for polymerase chain reaction (PCR).
The ITS region of the local EPN was amplified using the general primers TW81 and AB28 (Joyce et al., 1994). The PCR profile of Nguyen et al. (2004) was followed using a 25 µl PCR reaction mixture composed of 2.5 µl of the DNA suspension, 12.5 µl 2X Taq Polymerase Mix (Vivantis Technologies, Selangor Darul Ehsan, Malaysia), 0.75 µl of 50 mM MgCl2 (Vivantis Technologies, Selangor Darul Ehsan, Malaysia), 1.0 µl each of forward and reverse primers (10 pM) and 7.25 µl nuclease-free water. The amplicons were checked in 1% agarose gel in 0.5 TBE buffer and then stained with GelRed (www.biotium.com). The amplified DNA fragments were sent to Beijing Genomics Institute-Hongkong (www.bgi.com) for sequencing. The nucleotide sequences obtained were aligned and edited using BioEdit Version 7.1.10 (Hall, 1999). The deduced consensus sequence for each EPN isolate was compared with the nucleotide sequences in NCBI database using BLASTn (Altschul et al., 1990).
The pathogenicity tests were carried in 35-mm petri plates (BD Falcon) lined with WhatmanTM filter paper No.1. Each plate, representing a replicate, was inoculated with 250 µl of the EPN suspension in distilled water containing 1,000 IJ. Five larvae of each of the test insect species namely, O. furnacalis, H. armigera, and S. litura, were transferred into a petri plate and considered as a replicate. For each insect species tested, ten replicates were used for each EPN strain per trial. Each trial included a control group treated with sterile distilled water. Three repetitions of the trial were done. The plates were then incubated at room temperature (28°C ± 2.0°C) under dark conditions. Mortality and penetration rates were scored at 48 and 72 hr post infection (HPI), respectively. Two cadavers were dissected to confirm EPN penetration, while the remaining ones were kept until IJ emergence. The harvested nematodes from these test insects were re-inoculated to further confirm pathogenicity to the test insects.
Virulence, as indicated by mortality, and penetration rates were done for O. furnacalis, H. armigera, and S. litura in 35-mm diameter petri plates lined with filter paper. Each plate was inoculated with 200 IJ in 100 µl of distilled water. A single larva of each test insect species, initially surface-sterilized using 10% hydrogen peroxide and then rinsed thrice in sterile distilled water, was transferred into each plate. The plates were incubated at 28°C ± 2.0°C for 48 hr. Adult EPN were observed and counted. Insect mortality and penetration were expressed as percentage. Five replicates per EPN isolate, with ten larvae per replicate, were used (n = 50 larvae) for each insect species in a trial. A control group treated with distilled water was included in the trial. Three repetitions of the trials were done for each test insect. Fresh IJ were used for each trial. Two-way ANOVA analysis was carried out using Prism 6.0 (GraphPad Software, Inc., www.graphpad.com).
Bioassays for LC50 estimation were carried out in sterile 24-well cell culture plates lined with sterile Whatman No.1 filter paper. A larva was transferred into each well before IJ inoculation. The LC50 of S. abbasi and H. indica on O. furnacalis larvae was estimated using the concentrations 2, 4, 8, 16, and 32 IJ per larva, while 4, 8, 16, 32, and 64 IJ per larva were used for H. armigera, and S. litura. The concentrations used for the bioassays were based on the results of the penetration assays.
Meanwhile, LT50 bioassays were carried out in sterile 35-mm diameter petri plates lined with sterile filter paper inoculated with 150 µl nematode suspension with 200 IJ. A larva was placed per plate and sealed with parafilm. Mortality was observed at 2-hour intervals in insects inoculated with H. indica, while every 4 hr in insects inoculated with S. abbasi. Mortality was recorded up to 48 HPI. LC and LT values were calculated from three trials. Each trial used 30 larvae per EPN species. A control group treated with sterile distilled water was included in each trial. The LC50 and LT50 were estimated using Polo Plus 2.0 software (LeOra Software LLC). Differences among LC50 or LT50 values were considered significant when the 95% fiducial limits did not overlap.
Steinernema spp. were detected from soil samples from Batangas, Bukidnon, Laguna, and South Cotabato, while the lone Heterorhabditis sp. was found from Batangas. Nucleotide sequence analysis of the ITS region further confirmed the presence of these EPN in the soil samples. The ITS region of the local EPNs shared 98% to 100% nucleotide identity with the nucleotide sequences in GenBank database (www.ncbi.nlm.gov/genbank/), indicating the existence of four species from the seven positive soil samples comprised of Steinernema tami (1), Steinernema minutum (4), S. abbasi (1), and H. indica (1) (Fig. 1 and Table 1).
Preliminary bioassays studies on the seven EPN isolates revealed their pathogenicity against H. armigera, O. furnacalis, and S. litura larvae. All EPN isolates infected and completed their life cycle in all of these lepidopterous insects, however, their virulence, based on larval mortality varied among the EPN isolates and insect species. Results showed that H. indica PBCB and S. abbasi MBLB, were significantly highly virulent to all the lepidopteran species tested as compared to the S. minutum and S. tami isolates. As early as 24 HPI, H. indica PBCB and S. abbasi MBLB caused significantly higher mortality rate in all the test insects (P < 0.0001; α = 0.05), a trend that continued until 48 HPI (Fig. 2). At 48 HPI, a 99.33% and 100% mortality in O. furnacalis was observed for H. indica PBCB and S. abbasi MBLB, respectively, while a highly significantly lower mortality (P < 0.0001, α = 0.05), was observed for S. minutum ASPL3 (8.0%), S. minutum CCL4 (2.0%), S. minutum PIB2 (8.0%), S. minutum SRSTB3 (16.0%) and S. tami PKSC1 (6.67%). Similarly, H. indica PBCB and S. abbasi MBLB caused significantly higher mortality in both H. armigera (88.0% and 90.0%) and S. litura (88.67% and 92.00%). Thus, since the local S. minutum and S. tami isolates exhibited relatively very low mortality on the three corn insect pests tested, only H. indica PBCB and S. abbasi MBLB were assayed in the succeeding studies.
The percentage IJ penetration at 48 HPI of H. indica PBCB and S. abbasi MBLB in the three lepidopteran species is summarized in Table 2. The penetration rate of S. abbasi MBLB in H. armigera (28.15%) and S. litura (23.15%) was significantly higher than H. indica PBCB (14.35% and 18.18%) (P < 0.0001, α = 0.05). In O. furnacalis, however, no significant difference in the penetration rate of H. indica PBCB (14.25%) and S. abbasi (14.54%) was observed.
The median lethal concentrations (LC50) of H. indica PBCB and S. abbasi MBLB at 48 HPI on the three insect pest species of corn tested were estimated and summarized in Table 3. Interestingly, the lowest LC50 was observed in S. litura (8.89 IJ per larva), however, it was not significantly different compared with that observed in O. furnacalis (10.52 IJ per larva). Nevertheless, both were significantly lower as compared with the LC50 in H. armigera (19.98 IJ per larva). A different trend in virulence based on LC50 was observed for S. abbasi MBLB. The lowest LC50 was on O. furnacalis (10.98 IJ per larva) however, when compared with S. litura (17.08 IJ per larva), the difference was not significant. The highest LC50 for S. abbasi MBLB was on H. armigera (22.57 IJ per larva), which was significantly different from the LC50 of O. furnacalis, but not with the LC50 of S. litura. In addition, the LC50 of H. indica PBCB and S. abbasi MBLB revealed their equal level of virulence against H. armigera and O. furnacalis. In S. litura, however, H. indica PBCB was significantly more virulent than S. abbasi MBLB.
The median lethal time estimates (LT50) of the H. indica PBCB and S. abbasi MBLB for the three lepidopteran pest species are presented in Table 4. Both H. indica PBCB and S. abbasi MBLB had significantly shortest LT50 of 21.90 and 24.18 hr, respectively, in O. furnacalis. In contrast, the significantly longest LT50 estimates of 30.96 and 32.64 hr were observed in H. armigera for H. indica PBCB and S. abbasi MBLB, respectively. Meanwhile, the LT50 values in S. litura of H. indica PBCB (24.31 hr) and S. abbasi MBLB (26.59 hr) were significantly different from the estimates in H. armigera and O. furnacalis. Results also showed that H. indica PBCB was significantly more virulent than S. abbasi MBLB against both O. furnacalis and S. litura but equally virulent to H. armigera.
This study is the first most extensive research on EPN in the Philippines with molecular characterization for species identification and bioefficacy assays. The experiments demonstrated the pathogenicity of the local EPN isolates to some major lepidopteran pests in corn, and the identification of the two most virulent local strains, namely H. indica PBCB and S. abbasi MBLB, to H. armigera, O. furnacalis, and S. litura.
One of the limiting factors in EPN research in the Philippines is the unavailability of Galleria mellonella, the lepidopteran species conventionally used as host bait for EPN isolation from soil (Bedding and Akhurst, 1975), in the country. Although G. mellonella has been reported in the Philippines (Laigo and Morse, 1968), no laboratory or commercially reared colonies have been established in the country. Other than it is rarely found infesting apiaries since it commonly infests colonies of the less commonly commercially reared bee species, there is no market for this insect as fishing bait or feed for pet animals in the country. Thus, the use of O. furnacalis as host bait was also explored basically because it is the most serious problem among the lepidopteran pests of corn, are easily reared in the laboratory, and has the closest phylogenetic relationship with G. mellonella (Regier et al., 2012). Ostrinia furnacalis belongs to the family Crambidae – a former subfamily of Pyralidae to where G. mellonella is still currently classified (Solis, 2007), while H. armigera and S. litura are both under the family Noctuidae.
In this study, we demonstrated the ability of O. furnacalis to recover EPN with varying virulence levels, suggesting that this insect species may serve as an alternative host bait. In addition, the use of the target insect as bait may provide a selective screening method for an increased probability of obtaining EPN population highly virulent to insect species of interest as shown in this study. However, this protocol that offers a finer screening technique might not be as efficient as the G. mellonella baiting in studying EPN diversity due to underestimation of naturally-occurring EPN populations. Bedding and Akhurst (1975) accounted the very high susceptibility of G. mellonella larvae to EPN to their non-development of protective proteins since they are not naturally exposed to nematodes as compared with soil insects. In addition, Shapiro-Ilan et al. (2003) noted that utilization of the target insect pest as bait can be used to isolate EPN from soil that maybe host specific. Nonetheless, it will be interesting to compare the efficiency of O. furnacalis as a baiting host with G. mellonella as the former, having no developmental stage in the soil, is also not normally exposed to EPN. Moreover, a comparison of morphometric data of EPN cultured in O. furnacalis with those reared in G. mellonella is another noteworthy study to undertake.
Soil surveys covering different soil and habitat types (woodland, cropland, grassland) across Luzon, Visayas, and Mindanao areas revealed low positive detection of EPN (2.5% or 7 out of 279 soil samples). In a similar type of extensive sampling, low occurrence of EPN had also been reported in Azores, Portugal with 3.9% (46 out of 1,160 soil samples) (Rosa et al., 2000), in Indonesia with 11.7% (26 out of 223 soil samples) (Griffin et al., 2000), and in Ireland with 10.5% (58 out of 551 soil samples) detection (Griffin et al., 1991). However, a relatively higher EPN occurrence was observed in a soil sampling done in Liaoning (Northeast China), where there was 32.89% detection (49 out of 149 soil samples) from the samples (Wang et al., 2014). The higher percentage of occurrence of Steinernema species observed in this study is consistent to all these aforementioned related researches as well as studies in Europe (Hominick et al., 1995). Nyasani et al. (2008) noted that Steinernema spp. are more widespread than Heterorhabditis since the former has a greater ability to adapt to their environment than the latter.
In similar studies on the use of EPN against lepidopteran pests, Kalia et al. (2014) proved the efficacy of S. thermophilum (=abbasi) against H. armigera and S. litura. With regards to host susceptibility of the different lepidopteran species to S. abbasi, they reported that the LC50 at 36 hr after treatment for S. litura and H. armigera was 85 and 54.68 IJ per larva, respectively. In comparison with the result of our study, the LC50 at 48 HPI of the local S. abbasi MBLB isolate for both test insects was relatively lower with values of 22.57 IJ per larva for H. armigera and 17.08 IJ per larva for S. litura. The variation between the studies can be attributed to the difference on the larval instars used, last instar for the former and third instars for this study, and probably the longer incubation time. However, the possibility of a higher efficacy or phenotypic fitness for the Philippine S. abbasi MBLB can also be a substantial reason for such intraspecific variation. Earlier studies had already implicated significant differences in the efficacy of various nematode species or strains for controlling a particular insect (Bedding et al., 1983) as the biological control potential of a particular EPN is greatly influenced by the penetration rate of IJs, the time of symbiotic bacteria discharge, and the virulence of these symbionts (Glazer and Navon, 1990). In a more recent study, the intraspecific variability of local strains of S. feltiae in Spain in terms of reproduction, virulence, and the physiological and molecular profile of the endosymbiont was demonstrated (Campos-Herrera and Gutierrez, 2014).
When the virulence of the two local EPN was compared within the insect species, a significant difference in the LC50 values was only observed in S. litura, in which H. indica PBCB was more virulent than S. abbasi MBLB. H. indica PBCB and S. abbasi MBLB exhibited the same degree of virulence in both H. armigera and O. furnacalis. Among the three test insects, S. litura has the softest cuticle, which probably made its intersegmental membranes more vulnerable to abrasions caused by H. indica using its dorsal tooth.
Significant difference in the LT50 values was observed in the three lepidopteran species infected with H. indica PBCB and S. abbasi MBLB. Based on LT50 estimates for both EPN, the ranking of the susceptibility of the test insects from the most susceptible to the least susceptible is as follows: O. furnacalis > S. litura > H. armigera. Significant difference in the LT50 values was observed among the three test insects infected with H. indica PBCB. The same trend was observed with those infected with S. abbasi MBLB. The innate susceptibility of O. furnacalis to both EPN can be attributed to the long-term dependence of these isolates to the insect as food source. From the time of soil-baiting to mass production, the EPN have been conditioned to consume O. furnacalis in the absence of G. mellonella.
Consistently, H. armigera infected with both EPN species displayed the highest LC50 and LT50 values for both EPN species indicative of its relative resilience against EPN parasitism. Aside from host preference and nematode fitness stated earlier, the activation of insect’s immune response, be it cuticular, humoral, cellular, or combinations, to different invading pathogens including EPNs might have played a great role in this interaction (Castillo et al., 2011; Krautz et al., 2014).
The local S. minutum and S. tami were found to be pathogenic to the three lepidopteran species tested, however, they exhibited very low virulence. Although they were able to complete their life cycle in the test insects, their ability to efficiently kill the host was not evident. These EPN could have other suitable insect species as hosts.
Surveys coupled with proper identification of EPN, and comprehensive bioassays, product formulation, and field-efficacy assessment are fundamental requirements in the employment of a viable EPN technology-based program. As EPN study in the Philippines is still in its infancy, this baseline information on the diversity of these nematodes and their occurrence across different geographical locations; and their biological control potential against lepidopteran insect pests of corn contributes to the growing basic and applied local EPN researches and provides a promising insect pest management option that answers to both food security and safety in the country. Further, the development of an indigenous EPN-based technology could lead to a formulation of an effective inoculative and/or inundative biological control strategy, which can also be incorporated in integrated pest management of local insect pests. However, whether these local EPN are more or less virulent to the lepidopterous pests tested as compared with exotic strains or even the commercial strains needs further examination, since foraging strategy in combination with the target insect pest as well as ecological cues are among the important factors in EPN bioefficacy (Berry et al., 1997; Shapiro-Ilan and Cottrell, 2005).
The Philippine map showing the sampling sites for entomopathogenic nematodes. The occurrence and distribution of
The Philippine map showing the sampling sites for entomopathogenic nematodes. The occurrence and distribution of
Mean percentage mortality of
Mean percentage mortality of