Entomopathogenic Nematode Production and Application Technology

Production and application technology is critical for the success of entomopathogenic nematodes (EPNs) in biological control. Production approaches include in vivo, and in vitro methods (solid or liquid fermentation). For laboratory use and small scale field experiments, in vivo production of EPNs appears to be the appropriate method. In vivo production is also appropriate for niche markets and small growers where a lack of capital, scientific expertise or infrastructure cannot justify large investments into in vitro culture technology. In vitro technology is used when large scale production is needed at reasonable quality and cost. Infective juveniles of entomopathogenic nematodes are usually applied using various spray equipment and standard irrigation systems. Enhanced efficacy in EPN applications can be facilitated through improved delivery mechanisms (e.g., cadaver application) or optimization of spray equipment. Substantial progress has been made in recent years in developing EPN formulations, particularly for above ground applications, e.g., mixing EPNs with surfactants or polymers or with sprayable gels. Bait formulations and insect host cadavers can enhance EPN persistence and reduce the quantity of nematodes required per unit area. This review provides a summary and analysis of factors that affect production and application of EPNs and offers insights for their future in biological insect suppression.     

Control of the Oriental Fruit Moth, Grapholita molesta, Using Entomopathogenic Nematodes in Laboratory and Fruit Bin Assays

The oriental fruit moth (OFM), Grapholita molesta (Busck), which is among the most important insect pests of peaches and nectarines, has developed resistance to a wide range of insecticides. We investigated the ability of the entomopathogenic nematodes (EPN) Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), S. riobrave (Cabanillas et al.), and Heterorhabditis marelatus (Liu and Berry) to control OFM under laboratory and fruit bin conditions. At a dosage of 10 infective juveniles (IJ)/cm² in the laboratory, S. carpocapsae caused 63%, S. feltiae 87.8%, S. riobrave 75.6%, and H. marelatus 67.1% OFM mortality. All four nematode species caused significant OFM larval mortality in comparison to the nontreated controls. Steinernema feltiae was used for the bin assays due to the higher OFM mortality it caused than the other tested EPN species and to its ability to find OFM under cryptic environments. Diapausing cocooned OFM larvae in miniature fruit bins were susceptible to IJ of S. feltiae in infested corner supports and cardboard strips. Treatment of bins with suspensions of 10 or 25 S. feltiae IJ/ml water with wetting agent (Silwet L77) resulted in 33.3 to 59% and 77.7 to 81.6% OFM mortality in corner supports and cardboard strips, respectively. This paper presents new information on the use of EPN, specifically S. feltiae, as nonchemical means of OFM control.     

Perspectives on the Behavior of Entomopathogenic Nematodes from Dispersal to Reproduction: Traits Contributing to Nematode Fitness and Biocontrol Efficacy

The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema are widely used for the biological control of insect pests and are gaining importance as model organisms for studying parasitism and symbiosis. In this paper recent advances in the understanding of EPN behavior are reviewed. The “foraging strategy” paradigm (distinction between species with ambush and cruise strategies) as applied to EPN is being challenged and alternative paradigms proposed. Infection decisions are based on condition of the potential host, and it is becoming clear that already-infected and even long-dead hosts may be invaded, as well as healthy live hosts. The state of the infective juvenile (IJ) also influences infection, and evidence for a phased increase in infectivity of EPN species is mounting. The possibility of social behavior - adaptive interactions between IJs outside the host - is discussed. EPNs’ symbiotic bacteria (Photorhabdus and Xenorhabdus) are important for killing the host and rendering it suitable for nematode reproduction, but may reduce survival of IJs, resulting in a trade-off between survival and reproduction. The symbiont also contributes to defence of the cadaver by affecting food-choice decisions of insect and avian scavengers. I review EPN reproductive behavior (including sperm competition, copulation and evidence for attractive and organizational effects of pheromones), and consider the role of endotokia matricida as parental behavior exploited by the symbiont for transmission.     

A Realistic Appraisal of Methods to Enhance Desiccation Tolerance of Entomopathogenic Nematodes

Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.     

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus

Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g^-1 insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g^-1 insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.     

Susceptibility of the Plum Curculio,Conotrachelus nenuphar,to Entomopathogenic Nematodes

The plum curculio, Conotrachelus nenuphar, is a major pest of pome and stone fruit. Our objective was to determine virulence and reproductive potential of six commercially available nematode species in C. nenuphar larvae and adults. Nematodes tested were Heterorhabditis bacteriophora (Hb strain), H. marelatus (Point Reyes strains), H. megidis (UK211 strain), Steinernema riobrave (355 strain), S. carpocapsae (All strain), and S. feltiae (SN strain). Survival of C. nenuphar larvae treated with S. feltiae and S. riobrave, and survival of adults treated with S. carpocapsae and S. riobrave, was reduced relative to non-treated insects. Other nematode treatments were not different from the control. Conotrachelus nenuphar larvae were more susceptible to S. feltiae infection than were adults, but for other nematode species there was no significant insect-stage effect. Reproduction in C. nenuphar was greatest for H. marelatus, which produced approximately 10,000 nematodes in larvae and 5,500 in adults. Other nematodes produced approximately 1,000 to 3,700 infective juveniles per C. nenuphar with no significant differences among nematode species or insect stages. We conclude that S. carpocapsae or S. riobrave appears to have the most potential for controlling adults, whereas S. feltiae or S. riobrave appears to have the most potential for larval control.     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

From Augmentation to Conservation of Entomopathogenic Nematodes: Trophic Cascades, Habitat Manipulation and Enhanced Biological Control of Diaprepes abbreviatus Root Weevils in Florida Citrus Groves

The use of entomopathogenic nematodes (EPN) for management of the root weevil, Diaprepes abbreviatus, in Florida citrus groves is considered a biological control success story and typically involves augmentation in which EPN are applied inundatively as biopesticides to quickly kill the pest. However, recent evidence indicates that efficacy of EPN applications in Florida citrus depends on soil type. They are very effective in the well drained coarse sands of the Central Ridge but often less so in poorly drained fine-textured soils of the Flatwoods. Moreover, groves on the Central Ridge can harbor rich communities of endemic EPN that might often suppress weevil populations below economic thresholds, whereas Flatwoods groves tend to have few endemic EPN and frequent weevil problems. Current research is examining the ecological dynamics of EPN in Florida citrus groves, the potential impact of EPN augmentation on soil food webs, especially endemic EPN, and whether habitat manipulation and inoculation strategies might be effective for conserving and enhancing EPN communities to achieve long-term control in problem areas. Conservation biological control could extend the usefulness of EPN in Florida citrus and be especially appropriate for groves with persistent weevil problems.     

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.     

Entomopathogenic Nematodes as a Model System for Advancing the Frontiers of Ecology

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have a mutualistic–symbiotic association with enteric γ-Proteobacteria (Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus), which confer high virulence against insects. EPNs have been studied intensively because of their role as a natural mortality factor for soil-dwelling arthropods and their potential as biological control agents for belowground insect pests. For many decades, research on EPNs focused on the taxonomy, phylogeny, biogeography, genetics, physiology, biochemistry and ecology, as well as commercial production and application technologies. More recently, EPNs and their bacterial symbionts are being viewed as a model system for advancing research in other disciplines such as soil ecology, symbiosis and evolutionary biology. Integration of existing information, particularly the accumulating information on their biology, into increasingly detailed population models is critical to improving our ability to exploit and manage EPNs as a biological control agent and to understand ecological processes in a changing world. Here, we summarize some recent advances in phylogeny, systematics, biogeography, community ecology and population dynamics models of EPNs, and describe how this research is advancing frontiers in ecology.     

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.     

Estimating Sample Size and Persistence of Entomogenous Nematodes in Sandy Soils and Their Efficacy Against the Larvae of Diaprepes abbreviatus in Florida

In two studies to estimate sampling requirements for entomogenous nematodes in the field, highest persistence of Heterorhabditis bacteriophora after application occurred beneath the canopies of mature citrus trees. Nematode persistence declined with distance from the center-line of the tree row toward the row-middles. Immediately after nematode application to soil, 32 samples (15 cm deep, 2.5-cm diameter) beneath a single tree were required to derive 95% confidence intervals that were within 40% of mean nematode population density. The estimated probability of measuring the mean density within 40%, using 32 samples, declined to 88% at 2 days post-application and to 76% at 7 days. The persistence in soil of Steinernema carpocapsae, S. riobravis, and two formulations containing H. bacteriophora and their efficacy against the larvae of Diaprepes abbreviatus were compared in a grove of 4-year-old citrus trees. Within 6 days, the recovered population densities of all nematodes declined to <5% of levels on day 0. The recovery of H. bacteriophora during the first 2 weeks was lower than that of the other two species. Steinemema riobravis and both formulations of H. bacteriophora reduced recovery of D. abbreviatus by more than 90% and 50%, respectively. Steinernema carpocapsae did not affect population levels of the insect.     

Effects of host nutrition on virulence and fitness of entomopathogenic nematodes: Lipid- and protein-based supplements in Tenebrio molitor diets

Entomopathogenic nematodes, Heterorhabditis indica and Steinernema riobrave, were tested for virulence and reproductive yield in Tenebrio molitor that were fed wheat bran diets with varying lipid- and protein-based supplements. Lipid supplements were based on 20% canola oil, peanut, pork or salmon, or a low lipid control (5% canola). Protein treatments consisted of basic supplement ingredients plus 0, 10, or 20% egg white; a bran-only control was also included. Some diet supplements had positive effects on nematode quality, whereas others had negative or neutral effects. All supplements with 20% lipids except canola oil caused increased T. molitor susceptibility to H. indica, whereas susceptibility to S. riobrave was not affected. Protein supplements did not affect host susceptibility, and neither lipid nor protein diet supplements affected reproductive capacity of either nematode species. Subsequently, we determined the pest control efficacy of progeny of nematodes that had been reared through T. molitor from different diets against Diaprepes abbreviatus and Otiorhynchus sulcatus. All nematode treatments reduced insect survival relative to the control (water only). Nematodes originating from T. molitor diets with the 0% or 20% protein exhibited lower efficacy versus D. abbreviatus than the intermediate level of protein (10%) or bran-only treatments. Nematodes originating from T. molitor lipid or control diets did not differ in virulence. Our research indicates that nutritional content of an insect host diet can affect host susceptibility to entomopathogenic nematodes and nematode fitness; therefore, host media could conceivably be optimized to increase in vivo nematode production efficiency.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.     

Screening Spanish isolates of steinernematid nematodes for use as biological control agents through laboratory and greenhouse microcosm studies

Entomopathogenic nematodes (EPNs) are one of the best non-chemical alternatives for insect pest control, with native EPN strains that are adapted to local conditions considered to be ideal candidates for regional biological control programs. Virulence screening of 17 native Mediterranean EPN strains was performed to select the most promising strain for regional insect pest control. Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) Rioja strain produced 7%, 91% and 33% larval mortality for the insects Agriotes sordidus (Illiger) (Coleoptera: Elateridae), Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), respectively, and was selected as the most promising strain. The S. feltiae Rioja strain-S. littoralis combination was considered the most suitable to develop the Rioja strain as a biocontrol agent for soil applications. The effect of soil texture on the virulence of the Rioja strain against S. littoralis was determined through dose-response experiments. The estimated LC₉₀ to kill larvae in two days was 220, 753 and 4178 IJs/cm² for soils with a clay content of 5%, 14% and 24%, respectively, which indicates that heavy soils produced negative effects on the virulence of the Rioja strain. The nematode dose corresponding to the LC₉₀ for soils with a 5% and 14% clay content reduced insect damage to Capsicum annuum Linnaeus (Solanales: Solanaceae) plants under greenhouse microcosm conditions. The results of this research suggest that an accurate characterization of new EPN strains to select the most suitable combination of insect, nematode and soil texture might provide valuable data to obtain successful biological control under different ecological scenarios in future field applications.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Comparative Estimation of Genetic Diversity in Population Studies using Molecular Sampling and Traditional Sampling Methods

Entomopathogenic nematodes (EPN) are efficient biological pest control agents. Population genetics studies on EPN are seldom known. Therefore, it is of interest to evaluate the significance of molecular sampling method (MSM) for accuracy, time needed, and cost effectiveness over traditional sampling method (TSM). The study was conducted at the Mohican Hills golf course at the state of Ohio where the EPN H. bacteriophora has been monitored for 18 years. The nematode population occupies an area of approximately 3700 m² with density range from 0.25-2 per gram soil. Genetic diversity of EPN was studied by molecular sampling method (MSM) and traditional sampling method (TSM) using the mitochondrial gene pcox1. The MSM picked 88% in compared to TSM with only 30% of sequenced cox 1 gene. All studied genetic polymorphism measures (sequence and haplotype) showed high levels of genetic diversity of MSM over TSM. MSM minimizes the chance of mitochondrial genes amplification from non target organisms (insect or other contaminating microorganisms). Moreover, it allows the sampling of more individuals with a reliable and credible representative sample size. Thus, we show that MSM supersedes TSM in labour intensity, time consumption and requirement of no special experience and efficiency.     

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.     

A Comparison of Entomopathogenic Nematode Longevity in Soil under Laboratory Conditions

We compared the longevity of 29 strains representing 11 entomopathogenic nematode species in soil over 42 to 56 d. A series of five laboratory experiments were conducted with six to eight nematode strains in each and one or more nematode strains in common, so that qualitative comparisons could be made across experiments. Nematodes included Heterorhabditis bacteriophora (four strains), H. indica (Homl), H. marelatus (Point Reyes), H megidis (UK211), H. mexicana (MX4), Steinernema carpocapsae (eight strains), S. diaprepesi, S. feltiae (SN), S. glaseri (NJ43), S. rarum (17C&E), and S. riobrave (nine strains). Substantial within-species variation in longevity was observed in S. carpocapsae, with the Sal strain exhibiting the greatest survival. The Sal strain was used as a standard in all inter-species comparisons. In contrast, little intra-species variation was observed in S. riobrave. Overall, we estimated S. carpocapsae (Sal) and S. diaprepesi to have the highest survival capability. A second level of longevity was observed in H. bacteriophora (Lewiston), H. megidis, S. feltiae, and S. riobrave (3–3 and 355). Lower levels of survivability were observed in other H. bacteriophora strains (Hb, HP88, and Oswego), as well as S. glaseri and S. rarum. Relative to S. glaseri and S. rarum, a lower tier of longevity was observed in H. indica and H. marelatus, and in H. mexicana, respectively. Although nematode persistence can vary under differing soil biotic and abiotic conditions, baseline data on longevity such as those reported herein may be helpful when choosing the best match for a particular target pest.     

Soil Applications of Steinernematid and Heterorhabditid Nematodes for Control of Colorado Potato Beetles,Leptinotarsa decemlineata (Say)

Three strains of Steinernema feltiae Filipjev (All, Mexican, and Breton strains) and one of Heterorhabditis heliothidis (Khan, Brooks, and Hirschmann) were evaluated for their potential to control Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), larvae and pupae in the soil. In laboratory studies, H. heliothidis and S. feltiae (Mexican strain) produced the highest mortality (6 days posttreatment) of CPB when applied to the surface of a soil column containing mature CPB larvae 5 cm below. Mortality ranged from 80 to 90% at rates of 79-158 nematodes/cm². Similar results were seen in a field microplot study with all four nematodes; S. feltiae (Mexican strain) and H. heliothidis were most effective. Adult CPB emergence was reduced 86.5-100% after application of 31-93 H. heliothidis/cm² and 88.4-100% with 93-155 S. feltiae (Mexican strain)/cm². The All strain of S. feltiae was moderately effective (ca. 80% reduction at 93-155 nematodes/cm²), while the Breton strain was ineffective (< 40% reduction at 155 nematodes/cm²). In small plots of potatoes enclosed in field cages, application of H. heliothidis and S. feltiae (Mexican strain) at rates of 93-155 nematodes/cm² before larval CPB burial in the soil resulted in 66-77% reduction in adult CPB emergence. Soil applications of these nematodes show potential for biological control of CPB.