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

Background: 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. Results: 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). Conclusions: 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

Native Philippine Heterorhabditis indica isolates from banana and rice fields and preliminary results of their virulence against the larvae of super worm (Zophobas morio Fabricius Coleoptera: Tenebrionidae)

Background: Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are used as biological control agents of several insect pests in lieu of synthetic pesticides. Results: Using an insect-baiting technique, naturally occurring EPN isolates were collected from banana and rice fields in the municipality of Hagonoy, the Philippines. A total of 4 isolates were recovered from all the sites namely, HBP1, HBP2, HR3 and HR4. By combining morphometrical and ITS rDNA data, all the isolates were identified as Heterorhabditis indica. To initially assess their infectivity, virulence tests were conducted on the last instar larvae of the super worm, Zophobas morio Fabricius (Coleoptera: Tenebrionidae), the model insect. HR3 isolate had the highest percentage mortality (56%), followed by HBP1 and HBP2 (33%), which were comparable to the available nematode in the market, Heterorhabditis bacteriophora (44%), whereas the lowest was obtained in HR4 isolate (11%). Conclusions: This study accounted 4 more H. indica isolates in addition to pre-existing isolates in the country, expanding its habitat range and geographic distribution. This also provides baseline information on the potential biocontrol utilization of native EPNs against soil-dwelling insect pests of agricultural crops. Further investigations are required to assess their efficacy against several other insect pests of economic importance for the integrated pest management (IPM) programmes in the country

Isolation, identification and virulence of the local entomopathogenic nematode Heterorhabditis indica in the southern Philippines against the sugarcane white grub, Leucopholis irrorata (Chevrolat) (Coleoptera: Scarabaeidae)

Entomopathogenic nematodes (EPNs) are used as biocontrol agents of diverse insect pests. EPN diversity study was conducted in the southern Philippines (Cotabato province), encompassing a total of 5 municipalities with 25 Barangays (villages) for the potential biocontrol of the sugarcane white grub, Leucopholis irrorata. Of the 25 soil samples collected, only 5 harbored nematodes by using the Galleria-baiting technique. Through preliminary assessment of morphological data and confirmation analysis of D2-D3 segments of the 28S rDNA, locally-isolated nematodes were identified as Heterorhabditis indica (1 isolate), Metarhabditis rainai (3 isolates) and Oscheius rugaonensis (1 isolate). Moreover, the virulence of the H. indica (KRR4) infective juveniles (IJs) isolated from sugarcane soils was assessed against the target local insect pests, L. irrorata grubs under laboratory conditions. The mean mortality (%) caused by H. indica isolate KRR4 on L. irrorata at different dosages (10, 50, 100, 150, 200 and 300 IJs/grub) ranged from 37.5–100% after 24 h of exposure. The LD50 required to kill 50% of the L. irrorata grub population was 5.5 ± 2 IJs/grub. The virulence indicated that local H. indica

Virulence, penetration rate and reproductive potential of entomopathogenic nematodes from eastern Australia in Queensland fruit fly, Bactrocera tryoni

Queensland fruit fly (Bactrocera tryoni) is Australia's most significant horticultural pest, yet limited options are available for its biological control. Here, we assessed the virulence and reproductive potential of 32 newly isolated Australian entomopathogenic nematode (EPN) strains of Heterorhabditis bacteriophora, Heterorhabditis indica, Heterorhabditis marelatus, Heterorhabditis zealandica and Steinernema feltiae against the late third larval instar and pupal stages of B. tryoni under laboratory conditions, and compared their performance with four commercially available strains of H. bacteriophora, H. zealandica, S. feltiae and S. carpocapsae. All EPN strains caused significant larval mortality, and, remarkably, 29 also pupal mortality. Among the EPN strains the mean LD50 value, presented as the number of infective juveniles (IJs) per B. tryoni individual, ranged from 25 to 114 IJs per larva, and from 114 to 360 per pupa; therefore, 3.2 to 4.6× more IJs were required to kill pupae. The penetration rates of IJs in B. tryoni larvae varied from 2 to 6.8%, and the number of IJs recovered per larva varied between 2,100 to 5,683. Our findings highlight the potential of Australian EPN strains for controlling larval and pupal stages of B. tryoni. Overall, based on the three tested traits, one H. zealandica strain (Hz.NAR1), three H. indica strains (Hi.ECCH, Hi.HRN and Hi.HIE2), one H. bacteriophora strain (Hb.HIE), one H. marelatus strain (Hm.ENCBF2) and one S. feltiae strain (Sf.Y13) were found the most promising candidates as potential biological control agents against the soil inhabiting stages of B. tryoni. Further laboratory testing is required to assess their persistence under a broader range of environmental conditions before their biological control efficacy can be trialled under field conditions

Isolation and molecular characterization of five entomopathogenic nematode species and their bacterial symbionts from eastern Australia

Entomopathogenic nematodes (EPNs) are used in biological control of pest insects but their potential may be limited by strain availability from different bioregions and effectiveness against specific pests. Here, we isolated and characterized EPNs and their symbiotic bacteria from Australia where their diversity is scarcely known. We collected 198 soil samples from citrus orchards, grasslands and forests across temperate, subtropical and tropical eastern Australia. EPNs were isolated by baiting with mealworm, greater wax moth and Queensland fruit fly, The Australia’s most significant horticultural pest. We obtained 36 isolates which, according to DNA sequence analyses, represented five species, Heterorhabditis bacteriophora, Heterorhabditis indica, Heterorhabditis marelatus, Heterorhabditis zealandica and Steinernema feltiae, including the first report of H. marelatus from Australia, and H. indica and H. zealandica from New South Wales. Thirty-five isolates were baited with mealworm, one with fruit fly, and none with wax moth. Heterorhabditis marelatus was recovered from forests, H. bacteriophora from citrus orchards, S. feltiae from citrus orchards and grasslands, H. indica and H. zealandica from all three habitats. According to bacterial DNA analyses, Photorhabdus heterorhabditis occurred in H. zealandica and a reference strain of H. bacteriophora, Photorhabdus laumondii in H. bacteriophora and H. marelatus, Photorhabdus tasmaniensis in H. indica and H. bacteriophora, and Photorhabdus namnaonensis in H. zealandica. Unexpectedly, Pseudomonas protegens and Delftia acidovorans were found in S. feltiae while its expected symbiont Xenorhabdus remained undetected, possibly due to our approach. The newly isolated EPNs should be tested as biological control agents against pest insects