Diversity and distribution of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) in South Africa

A total of 1506 soil samples from different habitats in seven geographic regions of South Africa were evaluated for the presence of entomopathogenic nematodes (EPN). Nematodes were isolated from 5% of the samples. Among the steinernematids, four Steinernema sp. were recovered including Steinernema khoisanae and three new undescribed species. Although steinernematids were recovered from both humid subtropical and semiarid regions, this family accounted for 80% of EPN recovered from the semiarid climate zones characterised by sandy, acidic soils. Eight isolates of S. khoisanae were recovered from the Western Cape province. One of the new undescribed steinernematids (Steinernema sp. 1) was recovered only from the Free State and KwaZulu-Natal provinces where humid subtropical conditions prevail and soils are generally less acidic with higher clay content. A high level of adaptation, however, was noted with Steinernema sp. 2, which was recovered from a wide range of soil conditions and habitats ranging from semiarid (Western Cape province) to humid subtropical (KwaZulu-Natal province). A third undescribed steinernematid, Steinernema sp. 3, seemed better adapted to heavier soils with more than 80% of isolates recovered from fruit orchards in the Free State province. Heterorhabditis bacteriophora was the only heterorhabditid recovered during this survey. This species was particularly prevalent in four provinces ranging from humid subtropical to semiarid regions. Isolation of EPN directly from insect cadavers included Steinernema sp. 2 and one H. bacteriophora from an unidentified white grub (Scarabaeidae) cadaver (i.e., dual infection) and H. bacteriophora from the black vine weevil, Otiorhynchus sulcatus.     

Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France)

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the “glaseri-group”. The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.     

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont–free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock’s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.     

Diversity of Xenorhabdus and Photorhabdus spp. and Their Symbiotic Entomopathogenic Nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.     

Natural occurrence of entomopathogenic nematodes in Liaoning (Northeast China)

Entomopathogenic nematodes (EPNs) can provide effective biological control of pest. In order to contribute to knowledge on these organisms for regional biological control programs, we studied EPN distribution and ecological requirements in Liaoning Province, Northeast China. One hundred and forty-nine soil samples were taken from 36 locations. EPNs were recovered from 22 of the 36 locations (61.11%). Forty-four samples contained steinernematids (89.80%) and 5 samples contained heterorhabditids (10.20%). EPN recovery varied among the different soil and habitat type. Most EPNs were isolated from sandy loam, and most of the samples containing EPNs were collected from woodland and fruit crop habitats. The morphological characters of infective juveniles were used for preliminary species diagnosis. We preliminarily identified 15 species of Steinernematidae (Steinernema litorale, Steinernema silvaticum, Steinernema feltiae, Steinernema bicornutum, Steinernema robustispiculum, Steinernema affine, Steinernema riobrave, Steinernema yirgalemense, Steinernema kushidai, Steinernema scapterisci, Steinernema carpocapsae, Steinernema ritteri, Steinernema tami, Steinernema rarum and Steinernema sasonense) and 4 species of Heterorhabditidae (Heterorhabditis megidis, Heterorhabditis zealandica, Heterorhabditis brevicaudis and Heterorhabditis bajardi).     

Susceptibility of Queensland fruit fly,Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), to entomopathogenic nematodes

Queensland fruit fly, Bactrocera tryoni (Froggatt), is the economically most significant Australian tephritid pest species with a large invasion potential, yet relatively little work on its biological control has been undertaken. Entomopathogenic nematodes (EPNs) are of potential interest for control of this fruit fly species as it pupates in the soil. Specifically, the pre-pupal stage of B. tryoni may present a unique window for EPN application, as fully developed larvae drop from infested fruit to the soil for pupation. For the first time, we tested the capacity of three EPN species with different foraging strategies, Steinernema feltiae, Steinernema carpocapsae and Heterorhabditis bacteriophora, to cause larval and pupal mortality in B. tryoni across a range of EPN concentrations (50, 100, 200, 500 and 1000 infective juveniles IJs cm^-2), substrate moisture (10, 15, 20 and 25% w/v) and temperatures (15, 20, 25 and 30 °C). We found that all EPN species tested caused environment and density dependent mortality in the third larval instar while pupae were not affected. Steinernema feltiae caused high mortality across different IJ concentrations and over a wider moisture and temperature range than the other two EPN species. High mortality caused by S. carpocapsae and H. bacteriophora was more limited to high IJ concentrations and a narrower moisture and temperature range. Our findings highlight the potential of EPNs for the control of B. tryoni and warrant further laboratory and field experiments to evaluate their efficacy under the wide environmental conditions that B. tryoni can occur in.     

Augmenting Entomopathogenic Nematodes in Soil from a Florida CitrusOrchard: Non-Target Effects of a Trophic Cascade

Laboratory experiments were conducted to study non-target effects of augmenting entomopathogenic nematode (EPN)communities in soil. When raw soil from a citrus orchard was augmented with either 2,000 Steinernema riobrave or S. diaprepesi, fewer EPN (P ≤ 0.05) survived if the soil had also been treated with 2,000 S. riobrave 7 d earlier (i.e., two augmentation events rather than one). EPN survival was unaffected by treatment (P ≤ 0.05) in soil that was air-dried to disrupt antagonist activity prior to the experiment. When S. diaprepesi, S. riobrave, Heterorhabditis zealandica or no EPN were added to raw soil and S. diaprepesi was added 5 d later, the survival of both S. diaprepesi and of total EPN was greater (P ≤ 0.05) in soil that received no pretreatment than in soilpre treated with S. riobrave. Pretreatment of soil with H. zealandica or S. diaprepesi had less or no affect on survival of S. diaprepesi or total EPN. When nematodes were recovered from soil and placed on water agar, the number of S. diaprepesi that were killed by endoparasitic and trapping nematophagous fungi was greater (P ≤ 0.05) if soil was pretreated with steinernematid species than if the soil was not pretreated or was pretreated with H. zealandica. The adverse effects of pretreating soil on EPN survival were density dependent within a range of pretreatment dosages (20–100 IJ/cm² soil surface), and the treatment effects required more time to become evident at lower than at higher dosages. These experiments suggest that non-target effects of augmenting the EPN community in soil vary among EPN species and have the potential to temporarily reduce EPN numbers below the natural equilibrium density.     

Harmful effects of mustard bio-fumigants on entomopathogenic nematodes

Mustard (Brassica and Sinapis spp.) green manures tilled into the soil preceding potato crops act as bio-fumigants that are toxic to plant–parasitic nematodes, providing an alternative to synthetic soil fumigants. However, it is not known whether mustard green manures also kill beneficial entomopathogenic nematodes (EPNs) that contribute to the control of pest insects. We used sentinel insect prey (Galleria mellonella larvae) to measure EPN infectivity in Washington State (USA) potato fields that did or did not utilize mustard green manures. We found a trend toward lower rates of EPN infection in fields, where mustard green manures were applied, compared to those not receiving this cultural control method. In a series of bioassays we then tested whether the application of two mustard (Brassica juncea) cultivars, differing in glucosinolate levels, disrupted the abilities of a diverse group of EPN species to infect insect hosts. Mustard-exposure trials were conducted first in laboratory arenas where EPNs were exposed to mustard extracts suspended in water, and then in larger microcosms in the greenhouse where EPNs were exposed to green manure grown, chopped, and incorporated into field soil. In all trials we used G. mellonella larvae as hosts and included multiple EPN species in the genera Steinernema (Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, and Steinernema riobrave) and Heterorhabditis (Heterorhabditis bacteriophora, Heterorhabditis marelatus, and Heterorhabditis megidis). In the laboratory, EPN infection rates were lower in arenas receiving mustard extracts than the control (water), and lower still when EPNs were exposed to extracts from plants with high versus low glucosinolate levels. Results were nearly identical when mustard foliage was soil-incorporated into greenhouse microcosms, except that the negative effects of mustards on EPNs developed more slowly in soil. Significantly, in arenas of both types one EPN species, S. feltiae, appeared to be relatively unaffected by mustard exposure. Together, our results suggest that the use of mustard bio-fumigants for the control of plant–parasitic nematodes has the potential to interfere with the biocontrol of insect pests using EPNs. Thus, it may be difficult to combine these two approaches in integrated pest management programs.     

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae,Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.     

New citriculture system suppresses native and augmented entomopathogenic nematodes

Since its first detection in 2005, the bacterial disease huanglongbing (HLB or citrus greening) has emerged as a critical threat to the citrus industry in Florida. An “Advanced Production System” (APS) could mitigate the impact of HLB by bringing citrus trees into production more quickly and economically than conventional citriculture methods. However, unlike conventional practices, APS fertigates plants daily, thereby changing the soil properties in ways that might impact soil biota. We tested the hypothesis that changes to soil properties caused by APS would affect the abundance of native entomopathogenic nematodes (EPNs) and/or the survival of augmented EPNs. The densities of organisms at different trophic levels were measured by real-time qPCR in three experiments conducted in an ongoing field experiment. Target organisms included 6 entompathogenic nematodes, 5 nematophagous fungi (NF) and a phoretic bacterium, Paenibacillus sp. Soil properties, free-living nematodes and citrus fibrous roots were also measured. Compared to soil under conventional citriculture (CC), APS increased soil pH and Mg content, while reducing the electrical conductivity, and content of K, Mn and Fe. The naturally occurring EPN Steinernema diaprepesi was 5 times less abundant in APS plots where these nematodes were more heavily encumbered by the phoretic bacterium Paenibacillus sp., which limits the foraging success of EPNs. In general, when EPNs were augmented in either treatment, fewer Steinernema riobrave than Heterorhabditis indica were recovered and recovery of both species declined rapidly over time. As seen with native S. diaprepesi, fewer augmented S. riobrave were recovered from APS plots in two of the three experiments, whereas the management system did not affect the recovery of H. indica. More of some endoparasitic and trapping NF were recovered from soil augmented with S. riobrave than with H. indica. However, variation in the responses of NF to the management systems suggests that these NF species were not primarily responsible for the steinernematid responses to APS. Although APS has the potential to reduce EPN populations and exacerbate herbivory by subterranean pests such as the root weevil Diaprepes abbreviatus, additional study of the physical causes of this effect may reveal ways to avoid the problem.     

Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves

Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.     

The lethal and sub-lethal consequences of entomopathogenic nematode infestation and exposure for adult pine weevils, Hylobius abietis (Coleoptera: Curculionidae)

Entomopathogenic nematodes (EPN) frequently kill their host within 1-2 days, and interest in EPN focuses mainly on their lethality. However, insects may take longer to die, or may fail to die despite being infected, but little is known about the effects of EPN infection on insects, other than death. Here we investigate both lethal and sub-lethal effects of infection by two EPN species, Steinernema carpocapsae and Heterorhabditis downesi, on adults of the large pine weevil, Hylobius abietis. Following 12 h nematode-weevil contact in peat, S. carpocapsae killed a significantly higher proportion of weevils (87-93%) than H. downesi (43-57%) at all concentrations tested. Less than 10% of weevils were dead within 2 days, and weevils continued to die for up to 10 days after exposure (LT₅₀ of 3 days or more). In a separate experiment, live weevils dissected 6 days after a 24 h exposure to nematodes on filter paper harbored encapsulated and dead nematodes, showing that weevils could defend themselves against infection. Some live weevils also harbored live nematodes 6 days after they had been removed from the nematode infested medium. Feeding by weevils was not affected by infection with, or exposure to, either species of EPN. We discuss these results in relation to the use of EPN in biological control against H. abietis.     

Long‐term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real‐time PCR assays

Quantitative real‐time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species‐complex in the Steinernema glaseri‐ group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species‐specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8‐ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.     

Rhizobium laguerreae is the main nitrogen-fixing symbiont of cultivated lentil (Lens culinaris) in Morocco

Genetic diversity and population structure of 268 Lens culinaris symbiotic rhizobia collected from 40 cultivated fields in the main lentil production regions in Morocco were estimated. Three chromosomal housekeeping genes (recA, glnII and atpD) and one common symbiotic gene (nodC) were sequenced and analyzed in order to identify the local symbionts of lentil. The molecular phylogeny of the concatenated housekeeping genes clustered more than 95% of the isolates in one main clade together with Rhizobium laguerreae species. R. laguerreae represents the main symbiont of cultivated lentil in Morocco and, for the first time, a large sample of individuals is obtained for this species. There is a significant and high genetic differentiation of bacterial populations among the four regions for their symbiotic gene, and much lower for their housekeeping genes. The reasons why R. laguerreae is so frequently recovered in our study is discussed.     

Food Web Responses to Augmenting the Entomopathogenic Nematodes in Bare and Animal Manure-Mulched Soil

Factorial treatments of entomopathogenic nematodes (EPN) and composted, manure mulches were evaluated for two years in a central Florida citrus orchard to study the post-application biology of EPN used to manage the root weevil, Diaprepes abbreviatus. Mulch treatments were applied once each year to study the effects of altering the community of EPN competitors (free-living bactivorous nematodes) and antagonists (nematophagous fungi (NF), predaceous nematodes and some microarthro-pods). EPN were augmented once with Steinernema riobrave in 2004 and twice in 2005. Adding EPN to soil affected the prevalence of organisms at several trophic levels, but the effects were often ephemeral and sometimes inconsistent. EPN augmentation always increased the mortality of sentinel weevil larvae, the prevalence of free-living nematodes in sentinel cadavers and the prevalence of trapping NF. Subsequent to the insecticidal effects of EPN augmentation in 2004, but not 2005, EPN became temporarily less prevalent, and fewer sentinel weevil larvae died in EPN-augmented compared to non-augmented plots. Manure mulch had variable effects on endoparasitic NF, but consistently decreased the prevalence of trapping NF and increased the prevalence of EPN and the sentinel mortality. Both temporal and spatial abundance of NF were inversely related to the prevalence of Steinernema diaprepesi, whereas Heterorhabditis zealandica prevalence was positively correlated with NF over time. The number of weevil larvae killed by EPN was likely greatest in 2005, due in part to non-target effects of augmentation on the endemic EPN community in 2004 that occurred during a period of peak weevil recruitment into the soil.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.     

Diversity and phylogenetic relationships of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Sky Islands of Southern Arizona

A survey for entomopathogenic nematodes was conducted in oak-juniper woodlands of four mountain ranges (Santa Rita, Santa Catalina, Pinale?o, and Chiricahuas), in southeastern Arizona. From a total of 120 soil samples, 23.3% were EPN-positive. Of them 78.5% were positive for Steinernema spp. and 21.5% were positive for Heterorhabditis spp. An integrated approach, combining both traditional (morphological) and molecular methods, was used for examining the diversity of species of these entomopathogenic nematodes. Two named-species S. oregonense and S. riobrave are reported for the first time in Arizona, expanding their currently known geographic range. In addition to this, three undescribed Steinernema and three Heterorhabditis spp. were recovered. Insular evolution, in part, could account for the geographic distribution of entomopathogenic nematodes in Arizona.     

Steinernema feltiae (DD-136) and S. glaseri: Persistence in Soil and Bark Compost and Their Influence on Native Nematodes

Infective juveniles (J3) of the entomogenous nematodes Steinernema feltiae DD-136 (ca. 10,000 J3/100 ml) and S. glaseri (ca. 2,500 J3/100 ml) were incubated in steam-sterilized and nonsterilized sandy soil and bark compost for 8 weeks at 25 C. The nematodes were recovered by a two-step extraction procedure at 1-week intervals, and their infectivity to lepidopterous larvae (Spodoptera litura and Galleria mellonella) and their effect on the population and community of native nematodes in soil were determined. Survival of inoculated nematodes and mortality of insects were enhanced in sterilized media. Nonsterilized bark compost proved to be equally as suitable a medium as sterilized compost. In nonsterilized soil, the survival curve of S.feltiae declined more rapidly than that or S. glaseri which was less infective to insects despite its greater persistence even in nonsterilized soil. Soon after the addition of steinernematids to soil, the population of native nematodes showed a fluctuation with an increase in rhabditids and a decrease in other kinds of nematodes.     

Incidence of Endemic Entomopathogenic Nematodes Following Application of Steinernema riobrave for Control of Diaprepes abbreviatus

Control of Diaprepes abbreviatus by endemic and exotic entomopathogenic nematodes (EPN) was monitored during 2000-2001 in two citrus orchards in central Florida (Bartow and Poinciana). Caged sentinel insect larvae were buried beneath citrus trees for 7 days at 1 to 2-month intervals from April to October each year. At Bartow, the survey occurred in experimental plots that were (i) not treated with commercial EPN, (ii) treated twice annually since 1998 with commercially formulated Steinernema riobrave, or (iii) treated twice annually with S. riobrave and liquid fertilization (15 times/year) occurred in place of dry fertilizer (3 times/year) used in the other treatments. Four endemic EPN species, in addition to S. riobrave, were recovered from the sandy soil at Bartow: S. diaprepesi, Heterorhabditis zealandica, H. indica, and H. bacteriophora. Mean insect mortality in control plots was 39.4% (range = 13% to 74%), with seasonal maxima in May to July each year. Endemic EPN were recovered from 55% (range = 22% to 81%) of the cadavers each month. Total numbers of endemic EPN recovered in all plots during 2 years were directly related to the numbers of adult weevils (D. abbreviatus and Pachnaeus litus) captured in modified Tedder''s traps and inversely related to recovery of S. riobrave. Insect mortality was higher and cadavers containing endemic EPN were more numerous in untreated control plots than in S. riobrave-treated plots, except during months in which S. riobrave was applied. In treated plots, endemic EPN were recovered from cadavers at twice the rate of S. riobrave. Suppression of endemic EPN in plots treated with S. riobrave, combined with inferior persistence by the introduced species, may have attenuated the net efficacy of S. riobrave against D. abbreviatus. In contrast, H. indica was the only endemic nematode recovered from the sandy clay loam soil at Poinciana, where the average mortality of D. abbreviatus was 12% (range 3% to 20%) and incidence of H. indica did not exceed 8%. Results of these surveys suggest that the regional patterns in the abundance and damage to citrus caused by D. abbreviatus in Florida are regulated by endemic EPN and other soilborne enemies of the weevil.