Biological control potential of native entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) against Spodoptera cilium (Lepidoptera: Noctuidae) in turfgrass

In laboratory studies, we demonstrated that five native entomopathogenic nematode species/isolates caused 100% mortality of Spodoptera cilium larvae, a soil surface-feeding pest of turfgrass. At 25 infective juveniles/cm² applied to sod, two selected Turkish species, Steinernema carpocapsae and Heterorhabditis bacteriophora (Sarigerme isolate), averaged 77% and 29% larval mortality, respectively.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey

The diversity and distribution of entomopathogenic nematodes in thefamilies Steinernematidae and Heterorhabditidae were assessed throughout anextensive soil survey in Turkey during 1999 and 2000. Entomopathogenic nematodeswere recovered from six out of seven regions sampled, with 22 positive sites(2%) out of 1080 sites sampled. A single nematode isolate was recovered at eachof the positive sites, of which 15 were steinernematid isolates and seven wereheterorhabditid isolates representing a total of four species. Based onmorphometric and molecular data, the nematode species were identified asHeterorhabditis bacteriophora, Steinernemafeltiae, S. affine, andSteinernema n. sp. The most common species was S.feltiae, which was isolated from 10 sites in six regions, followed byH. bacteriophora from seven sites in five regions,S. affine from four sites in two regions, andSteinernema n. sp. from one site. Heterorhabditisbacteriophora and S. feltiae have been found inmany parts of the world, whereas S. affine, so far, hasonly been recovered in Europe until our survey. Steinernemaaffine was isolated from the European (Marmara) as well as theAsiatic region (Middle Anatolia) of Turkey. A new undescribedSteinernema sp. was isolated from the most eastern region(East Anatolia) of Turkey. Soils of the positive sites were classified as sandy,sandy loam, or loam (68.2%) and sandy–clay–loam or clay loam (31.8%) and the pHranged from 5.6 to 7.9. The habitats from which the entomopathogenic nematodeswere isolated were broadly classified as disturbed (59.1%), which includedagricultural fields and poplar planted for lumber and wind breaks, andundisturbed (40.9%), which included pine forest, grassland, marsh and reed sites.Steinernema feltiae, S. affine, andH. bacteriophora were recovered from both disturbed andundisturbed habitats. The new Steinernema sp. was recoveredfrom grassland. Our survey showed that these nematodes occur widely throughoutTurkey, but at a frequency below that reported for other parts of the world.     

Susceptibility of Mocis latipes (Lepidoptera: Noctuidae) to Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae)

The susceptibility of larvae, prepupae, and pupae of the grass looper Mocis latipes (Guenée) to the entomopathogenic nematode Heterorhabditis bacteriophora (Poinar) NC strain was evaluated under laboratory conditions. Concentrations of 0, 5, 10, 20, 40, 60, and 120 nematodes per larva, applied in 1 ml of sterile-distilled water, were bioassayed, applying them to groups of 20 individuals of each instar, prepupa or pupa. Mortality was recorded daily for 5 d. All instars and the prepupal stage were the most susceptible to H. bacteriophora. Mortality ranged from 22.5 to 100%. Prepupae had 97.5-100% mortality starting at 10 nematodes per prepupa. Pupal mortality ranged from 27.5 to 41.3% as nematode concentration was increased. H. bacteriophora presented LC50 values that ranged between 5.26 and 37.66 nematodes per larva and LT50 values that ranged between 1.5 and 4.3 d. Results of this study suggest that H. bacteriophora has potential as a biocontrol agent against M. latipes.     

Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae)

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.     

Efficacy of entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae) against codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in temperate regions

The biocontrol potential of South African isolates of Heterorhabditis zealandica, Steinernema citrae, S. khoisanae, S. yirgalemense, and Steinernema sp., was evaluated against codling moth, Cydia pomonella. Codling moth was susceptible to all six nematode isolates at a concentration of 50 infective juveniles/insect (78–100% mortality). Low temperatures (10 h at 17°C; 14 h at 12°C) negatively affected larvicidal activity (≤3%) for all isolates. All tested isolates were most effective at higher levels of water activity (a _w=1). The average a _w50-values for all isolates tested was 0.94 (0.93–0.95), except S. khoisanae 0.97 (0.97–0.98). Regarding host-seeking ability, no positive attraction to host cues could be detected amongst isolates, except for H. zealandica. Three of the isolates, H. zealandica, S. khoisanae, and the undescribed Steinernema sp., were selected for field-testing and proven to be effective (mortality >50%). Insect containment methods used during field experimentation was shown to influence larvacidal activity, as different levels of mortality were obtained using various containment methods (wooden planks vs. pear tree logs vs. mesh cages). Pear tree logs were impractical. Predictive equations were subsequently developed, enabling future trials to be conducted using either planks or cages, enabling the prediction of the expected level of control on tree logs. All tested isolates therefore showed a certain degree of biological control potential, however, none of the experiments showed clear efficacy-differences amongst isolates. The study highlighted the importance of environmental factors to ensure the successful application of these nematodes for the control of diapausing codling moth larvae in temperate regions.     

Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae), an entomopathogenic nematode from Thailand

Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae) is a new entomopathogenic nematode isolated in Lohmsak, Thailand. Morphology, hybridisation and molecular studies indicated the distinctness of S. siamkayai n. sp. from other Steinernema spp. Diagnostic characters include: the total body length (398–495 m) and tail length (31-41m) of the third-stage infective juvenile and lateral field pattern with 6-8 longitudinal ridges; the presence of a tail mucro in both first and second generation females and males; the size and shape of the spicules and gubernaculum, and the arrangement of the genital papillae of the first and second generation males; and the shape of the vulva and tail of the first generation female and second generation female     

Parasitism of subterranean termites (Isoptera: Rhinotermitidae: Termitidae) by entomopathogenic nematodes (Rhabditida: Steinernematidae; Heterorhabditidae)

In laboratory bioassays, Steinernema riobrave Cabanillas, Poinar and Raulston (355 strain), Steinernema carpocapsae (Weiser) (Mexican 33 strain), Steinernemafeltiae (Filipjev) (UK76 strain), and Heterorhabditis bacteriophora Poinar (HP88 strain) were all capable of infecting and killing three termite species, Heterotermes aureus (Snyder), Gnathamitermes perplexus (Banks), and Reticulitermes flavipes (Kollar) in laboratory sand assays. S. riobrave and S. feltiae caused low levels of Reticulitermes virginicus (Banks) mortality under the same conditions. At 22 degrees C, significant mortality (> or = 80%) of worker H. aureus and G. perplexus was caused by S. riobrave, in sand assays, indicating the need for further study. Because of the short assay time (3 d maximum), reproduction of the nematodes in the target host species was not recorded. All nematode species were observed to develop to fourth-stage juveniles, preadult stages, or adults in all termite species with the exception of R. virginicus. Only S. riobrave developed in R. virginicus. Nematode concentration and incubation time had significant effects on the mortality of worker H. aureus. S. riobrave consistently generated the highest infection levels and mortality of H. aureus in sand assays.     

Haplotype identification within Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) corn and rice strains from Colombia

The fall army worm Spodoptera frugiperda (Smith) is a migratory important pest of corn, sorghum, rice, grass and bermudagrass in North and South America. This species has diverged into two genetically differentiated but morphologically identical strains, "the rice" and "the corn". They have been analyzed by sequencing the genes cytochrome oxydase I, II and ITS1 from populations from the United States and Brazil. However, no such studies were performed in Colombia. In here, we identified 43 haplotypes by sequencing a fragment of the COI gene from 102 individuals, of which 40 had already been identified as the "corn" and "rice" strains or to their hybrids from Tolima, and the rest were collected from corn, cotton, sorghum, grass and rice fields in other regions of Colombia. The corn strain haplotype H1 was the most frequently found in this country, representing the main target for FAW monitoring programs. AMOVA analysis confirmed the population structure between Colombian and North American S. frugiperda haplotypes (F(ST) = 0.76812, P < 0.001), but not within the different Colombian regions, suggesting high gene flow within the country. The ML trees obtained for Tolima and for Colombia as a whole did not generate clustering amongst S. frugiperda sequences, neither via host-plant association nor by geographical areas. The minimum spanning network for Colombia corroborated our finding that the haplotype H1 has the highest frequency in the country. Our data suggest that haplotype frequency determination will be useful in the establishment of a monitoring system for this species.     

Parasitism of bark scorpion Centruroides exilicauda (Scorpiones: Buthidae) by entomopathogenic nematodes (Rhabditida: Steinernematidae; Heterorhabditidae)

In laboratory bioassays, Steinernema glaseri Steiner, Steinernema riobrave Cabanillas, Poinar & Raulston, Heterorhabditis bacteriophora Poinar, and Heterorhabditis marelatus Liu & Berry were capable of infecting and killing the bark scorpion, Centruroides exilicauda (Wood). Steinernema feltiae (Filipjev) and Steinernema carpocapsae (Weiser) failed to infect C. exilicauda at 22 degrees C. S. glaseri, H. marelatus, and H. bacteriophora caused significant mortality at 22 degrees C, indicating the potential role of these parasites as a biocontrol option. Efficacy of S. glaseri and H. bacteriophora was reduced in an assay conducted at 25 degrees C. Only S. glaseri was able to reproduce in the target host. Dissection of scorpions at the end of the experimental periods revealed inactive juvenile S. riobrave, H. marelatus, and H. bacteriophora nematodes. Both mermithid and oxyurid nematodes have been documented as nematode parasites of scorpions, but rhabditids have not been reported until now. Field studies are warranted to assess the usefulness of entomopathogenic nematodes as biocontrol agents of bark scorpions.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Spatial distribution of plants infested with Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) on corn crop

The importance of the fall armyworm, Spodoptera frugiperda (J.E. Smith), is not only due to the damage it can cause, but also to its difficult control. It is essential to know the pest population parameters, such as its standard scattering in the crop, because depending on the space arrangement of the insects in the area, different sampling methods are required. For the present work, three experimental fields were installed in different areas: the first one had 66 plots with seven plants each; the second one had 55 plots with four plants each; and the third field had 55 plots with six plants each. The presence or absence of the pest in the plant was evaluated in the samples. According to the data observed, either to the scattering index or to the theoretical scattering models, S. frugiperda dispersion is scattered at random and strongly depends on the infestation level.     

Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae)

Anhydrobiosis is considered to be an important means of achieving storage stability of entomopathogenic nematodes that are used in biological control. This study explored the effects of anhydrobiosis on longevity and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema riobrave at 5 and 25 degrees C. Anhydrobiosis was induced in water-dispersible granules (WG) at 0.966-0.971 water activity and 25 degrees C following a 7-day preconditioning of IJs at 5 degrees C in tap water. Survival and infectivity of the desiccated (anhydrobiotic) IJs was compared with non-desiccated IJs stored in water for different periods. Anhydrobiosis increased longevity of S. carpocapsae IJs by 3 months and of S. riobrave by 1 month in WG at 25 degrees C as compared with IJs stored in water. However, desiccation decreased S. feltiae longevity at 25 degrees C and of all three species at 5 degrees C. These results demonstrate a shelf-life of 5 months for S. carpocapsae at 25 degrees C and 9 months at 5 degrees C in WG with over 90% IJ survival. For S. feltiae, over 90% survival occurred only for 2 months at 25 degrees C and 5 months at 5 degrees C in WG. Steinernema riobrave had over 90% survival only for 1 month at 25 degrees C and the survival dropped below 85% within 1 month at 5 degrees C. Induction of anhydrobiosis in WG resulted in 85, 79 and 76% reduction in oxygen consumption by S. carpocapsae, S. feltiae, and S. riobrave IJs, respectively. Differences in IJ longevity among three species in water at 25 degrees C were related both to the initial lipid content and the rate of lipid utilisation, but not at 5 degrees C. The one-on-one infection bioassays indicated that desiccation had no negative effect on the infectivity of any of the nematode species suggesting no harmful effect on the IJs and/or their symbiotic bacteria. The species differences in IJ longevity and desiccation survival at different temperatures are discussed in relation to their foraging strategy and temperature adaptation.     

Steinernema weiseri n. sp. (Rhabditida,Steinernematidae), a new entomopathogenic nematode from Europe

Steinernema weiseri n. sp. is described from a roadside with apple trees near Ceské Budejovice, Czech Republic. The species is also widely distributed in Germany and Slovakia, from where it had previously been reported as Steinernema spec. F. The British Steinernema sp. D1 is considered conspecific with S. weiseri n. sp. Males of the new species are mainly characterised by light brown, slightly curved spicules with a long manubrium and the presence of a short tail mucron in the second generation. Third-stage infective juveniles are characterised by a `medium size' body and tail length, short hyaline tail portion (mostly around 1/3 of tail length), the excretory pore situated in the mid-pharynx region, lip region slightly offset, angular and flattened, and the lateral field having nine equally developed lines separated by eight distinct ridges. S. weiseri n. sp. is most similar to S. feltiae, with which it did not hybridise. RFLP analysis of the ITS region of the rDNA repeat shows S. weiseri n. sp. to be distinct from 50 other Steinernema species and isolates. The new species was found in a wide range of habitats and is readily maintained on Galleria mellonella larvae.     

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.     

Control of Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae) with entomopathogenic nematodes (Rhabditida: Heterorhabditidae,Steinernematidae) in peach orchards

Oriental fruit moth Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae) is considered a major pest in temperate fruit trees, such as peach and apple. Entomopathogenic nematodes (EPNs) are regarded as viable for pest management control due to their efficiency against tortricid in these trees. The objective of this study was to evaluate the effectiveness of native EPNs from Rio Grande do Sul state against pre-pupae of G. molesta under laboratory and field conditions. In the laboratory, pre-pupae of G. molesta were placed in corrugated cardboard sheets inside glass tubes and exposed to 17 different EPNs strains at concentrations of 6, 12, 24, 48 and 60 IJs/cm² and maintained at 25 °C, 70 ± 10% RH and photophase of 16 h. Insect mortality was recorded 72 h after inoculation of EPNs. Steinernema rarum RS69 and Heterorhabditis bacteriophora RS33 were the most virulent strains and selected for field application (LC₉₅ of 70.5 and 53.8 IJs/cm², respectively). Both strains were highly efficient under field conditions when applied in aqueous suspension directed to larvae on peach tree trunk, causing mortality of 94 and 97.0%, respectively.     

First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica

A survey of entomopathogenic nematodes was conducted in the north Pacific (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identified as Heterorhabditis indica and the three Steinernema isolates were identified as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing.     

Host plant association and genetic differentiation of corn and rice strains of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) in Colombia

Spodoptera frugiperda (Smith) is a polifagous insect of major economic impact in the western hemisphere and exhibits two strains (i.e., corn and rice) that are morphologically identical but differ in ecology, genetics and physiology. In this work we identified these strains and their respective hybrids by using a PCR-RFLP of the COI gene and PCR of the tandem region FR. Moreover, we performed a population structure analysis by using 253 larvae from Tolima, a region where S. frugiperda is a pest on corn, rice, sorghum and cotton. Corn strain was found on 42% in corn, 34% in cotton, 19% in sorghum and 0.04 % in rice and rice strain on 35% in corn, 0.06% in cotton, 0.06% in sorghum and 53% in rice, demonstrating that corn strain specificity is superior to rice strain. Hybrids between these strains were more abundant in corn. The distributions on their host plants reflect a population genetic differentiation in S. frugiperda with values of PhiPT (COI) = 0.31, P < 0.0001, PhiPT (FR) = 0.17, P < 0.0001 for all crops and PhiPT (COI) = 0.42, P < 0.01, PhiPT (FR) = 0.13, P < 0.01 for the sixteen sampled farms. The dendrograms showed two clusters representing both strains. The results obtained in this study suggest that the management of this insect must differ on each host plant, given the specialization that both strains present, particularly in corn and rice.