Entomopathogenic nematodes thesis

Sipes, Dr.

University of Tasmania

Brent advisor Tropical Plant Pathology department. Entomopathogenic nematodes EPNs are obligate parasites of insects. EPNs have a broad host range, are easily mass reared, and kill insects within 48 hours. EPNs are safe for vertebrates, plants, and non-target organism. On the other hand, EPNs have disadvantages that make them less effective against foliar insect pest because they are sensitive to desiccation, ultraviolet UV radiation and high temperatures.

The goal of this research was to improve the efficacy of aboveground application EPNs by protecting them against desiccation and UV radiation. The first objective was to determine efficacy of the desiccation protectant Barricade gel in extending the viability of EPNs. The third objective was to demonstrate enhanced insect control with EPNs protected from UV radiation and desiccation.

Back To Abstract. 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.

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, ; Burnell and Stock, EPN-based technology provides a biological control option on many of the most important pests of agricultural crops.

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, , leaf miner on ornamentals and vegetables Hara et al. EPN are naturally found in both agriculturally disturbed and undisturbed soil environments with reports of occurrences from many temperate and tropical countries.

Cold Tolerance Mechanisms of Entomopathogenic Nematodes

To date, there are at least 90 Steinernema and 20 Heterorhabditis species reported Shapiro-Ilan et al. 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. In addition, their collection was limited to Steinernema spp. Corn is one of the most economically important commodity in the Philippines. From to , the area planted to corn has increased up to 2.

Many of the Filipino farmers depend on tilling their land for corn production either for feed or food. Two generations of O. The early stage larvae feed on the leaves and tassel. Camarao 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. During reproductive stage however, H. Both lepidopterans exhibit overlapping generations in a corn cropping season. The last instar larvae of H. The larvae of S. The common cutworm also pupates in the soil Nurhidayat, 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, and the apparent emergence of resistant pest populations Gassmann et al. 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.

The use of O.

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Regier et al. 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 cm using a shovel.

From each site, two to three samples were taken depending on the size of the area. 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. 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, or incubated for 3 to 5 days in mm petri plates lined with moistened filter paper for nematode harvesting.

The EPN were mass-reared in O. For the bioassay experiments O. The insects were reared in the laboratory using an artificial diet developed by Ceballo and Morallo-Rejesus Permanent mounts of first generation female, male, and IJs were prepared for light microscopy examination.

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Preliminary identification of the EPN up to the genus level was based on general morphology, as described by Nguyen and Hunt and Nguyen et al. Since the local EPN were reared in O.

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The total DNA was extracted from a single first-generation female using a technique slightly modified from Phan et al. The PCR profile of Nguyen et al. The nucleotide sequences obtained were aligned and edited using BioEdit Version 7. Five larvae of each of the test insect species namely, O. 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.

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. Adult EPN were observed and counted.


Insect mortality and penetration were expressed as percentage. 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. Bioassays for LC 50 estimation were carried out in sterile well cell culture plates lined with sterile Whatman No.

Biocontrol of Ticks by Entomopathogenic Nematodes: Research Update

A larva was transferred into each well before IJ inoculation. The LC 50 of S. The concentrations used for the bioassays were based on the results of the penetration assays. A larva was placed per plate and sealed with parafilm. Mortality was observed at 2-hour intervals in insects inoculated with H. Mortality was recorded up to 48 HPI.

LC and LT values were calculated from three trials.