昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

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.     

Spatial and temporal distribution of endemic entomopathogenic nematodes in a heterogeneous vegetable production landscape

Entomopathogenic nematodes within the families Steinernematidae and Heterorhabditidae (Order: Rhabditida) are potential biological control agents for many soil-dwelling vegetable pests. However, their low persistence and efficacy after field releases have resulted in limited use in pest management programs. Understanding the factors regulating natural populations of entomopathogenic nematodes may provide insight into practices to conserve populations within production systems. A series of investigations were conducted within a vegetable production area in Willard, Ohio during 2000–2003 to gain insight into the population ecology of endemic populations of entomopathogenic nematodes. A total of 440 sites across four habitats associated with the production landscape were sampled to ascertain the natural occurrence of these beneficial nematodes. Habitats included cultivated areas, grassy banks adjacent to cultivated areas, undisturbed shrub lands and forests. Twelve sites along grassy banks were monitored over a growing season to estimate associations between abiotic and biotic factors and endemic populations. Entomopathogenic nematodes were only detected along grassy banks adjacent to the cultivated areas; nematodes were recovered from 15 to 30% of sites sampled in 2001 and 2002, respectively. Two species of nematodes were isolated, Heterorhabditis bacteriophora Poinar and Steinernema feltiae Filipjev. H. bacteriophora was the most prevalent nematode species and was recovered from 60% of positive samples. Nematode populations varied temporally and spatially along grassy banks; mean population density over the growing period was 1313 infective juveniles/m². Neither macro- nor microarthropod communities nor soil temperature differed between sites at which nematodes were detected and those at which nematodes were not detected. Soil moisture, however, was associated with the occurrence and persistence of nematodes along grassy banks; mean soil moisture at sites at which nematodes were detected and those sites at which nematodes were not detected was 37.3 and 26.8%, respectively. Water management is an important component of vegetable production and our results suggest that soil moisture manipulation would be important in the establishment and sustained presence of entomopathogenic nematode populations within cultivated areas over the growing season.     

Compatibility between Forficula auricularia and entomopathogenic nematodes to be used in pome fruit pest management

Use of predators, parasitoids and entomopathogens as biocontrol agents in pome fruit production can lead to more efficient and sustainable pest management programmes. The European earwig (Forficula auricularia Linnaeus [Dermaptera: Forficulidae]) is a major predator of key pests in pome fruit orchards, and entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae are obligate parasites of a large number of insect species. Therefore, the interaction between earwigs and EPNs can play an important role in pest management programmes. Susceptibility of the European earwig to Steinernema carpocapsae, Steinernema feltiae (Steinernematidae) and Heterorhabditis bacteriophora (Heterorhabditidae) was evaluated. S. carpocapsae was the only tested EPN capable of killing the European earwig. However, the European earwig can detect the presence of S. carpocapsae and therefore avoid nematode‐treated shelters. An earwig deterrent activity in EPN‐killed codling moth larvae that reduces the foraging of European earwig on insect cadavers containing nematodes and allows nematodes to complete their life cycle was also assessed with the three species of nematodes. These findings suggest a positive compatibility between the European earwig and EPNs.     

Overwintering behavior of the entomopathogenic nematodes Steinernema scarabaei and Heterorhabditis bacteriophora and their white grub hosts

Entomopathogenic nematodes (EPNs) are obligate pathogens known to naturally persist in many habitats. Because survival is a fundamental component of persistence, we investigated whether vertical movement and other avoidance behaviors (i.e., in‐host survival and latent infection), previously speculated as viable survival mechanisms, are exhibited during the cooler months in a temperate turfgrass habitat. The vertical distribution of populations of two EPN species, Steinernema scarabaei Stock & Koppenhöfer (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), and two important hosts of these EPN species, the white grub species Popillia japonica Newman and Anomala orientalis Waterhouse (both Coleoptera: Scarabaeidae), were regularly monitored in turf plots from October through April in two consecutive years. Entomopathogenic nematode vertical distribution showed limited changes for H. bacteriophora but none for S. scarabaei. Recovery of H. bacteriophora showed a strong and consistent decline at 0–4 cm depth in the 1st year and a weaker decline at 4–10 cm in the 1st year and at 0–4 cm in the 2nd year. Due to high variability in the data, it was not possible to determine whether the decline in the upper soil layers was due to downward migration or attrition of infective juvenile nematodes. The decline occurred mostly during the first half of the season before the soil froze to any significant extent. The vertical distribution of both white grub species changed with temperature during fall and spring, but not during winter. Overwintering infective juveniles were only recovered in the soil. There was no evidence for successful in‐host survival or latent infection by the nematodes in endemic white grub populations.     

昆虫病原线虫斯氏线虫和异小杆线虫对长角血蜱雌蜱的致病力

用昆虫病原线虫小卷蛾斯氏线虫（Sc BJ）、夜蛾斯氏线虫（Sf Otio）、拟双角斯氏线虫（Sc D43）、格氏斯氏线虫（Sg NC32）和嗜菌异小杆线虫（Hb E-6-7）对长角血蜱雌蜱进行感染试验,所用线虫剂量为4 000 Ijs/dish。结果表明,5种线虫均对长角血蜱雌蜱有致死效应。Hb E-6-7和Sc BJ两种线虫对雌蜱各发育期致病力最强,导致雌蜱的累积死亡率和半致死时间分别为饥饿雌蜱82.5%,9.0天和75.0%,8.8天;吸血雌蜱90.0%,8.0天和82.5%,8.0天;饱血雌蜱93.3%,7.3天和86.7%,7.3天。Sc D43对饱血雌蜱有较高的致死效应,为80.0%,但半致死时间较长,为11.7天。Sf Otio和Sg NC32对长角血蜱雌蜱的致死效应较低。饱血雌蜱较饥饿雌蜱和吸血雌蜱更易被线虫感染。     

Biological Control of the Shore Fly (Scatella tenuicosta) with Steinernematid Nematodes and Bacillus thuringiensis var. thuringiensis in Peat and Rockwool

Steinernema feltiae Filipjev and S. carpocapsae Weiser (Nematoda: Steinernematidae) at rates of 1, 5 and 20 million m^-2 in peat pots and at rates of 1, 2.5 and 5 million m^-2 in rockwool cubes were tested against the shore fly Scatella tenuicosta Collin (Diptera: Ephydridae) by applying the nematodes either preventatively 2 days before or curatively 9 days after, or both 2 days before and 9 days after exposing the pots and cubes to flies. Based on cumulative fly numbers that emerged from peat pots sampled weekly for 3 weeks, all application strategies with 5 or 20 million nematodes net-m^-2, irrespective of species, reduced fly numbers by 61-96% as compared to untreated controls. High temperatures in 1 week reduced control efficacy to 30-35% in some treatments. In rockwool, maximum control efficacies of 83-84% were achieved by both species in the second week in treatments that had received two applications at the rate of 5 million m^-2, but these did not differ significantly from the 69-75% efficacies achieved with 2.5 million nematodes m^-2. The cumulative control efficacy over 4 weeks was only 46% at maximum. The lower control efficacy in rockwool compared to peat was due to rapid disappearance of nematodes from rockwool.     

Parasitism of Molluscs by Nematodes: Types of Associations and Evolutionary Trends

Although there are no confirmed fossil records of mollusc parasitic nematodes, diverse associations of more than 108 described nematode species with slugs and snails provide a fertile ground for speculation of how mollusc parasitism evolved in nematodes. Current phylogenic resolution suggests that molluscs have been independently acquired as hosts on a number of occasions. However, molluscs are significant as hosts for only two major groups of nematodes: as intermediate hosts for metastrongyloids and as definitive hosts for a number of rhabditids. Of the 61 species of nematodes known to use molluscs as intermediate hosts, 49 belong to Metastrongyloidea (Order Strongylida); of the 47 species of nematodes that use molluscs as definitive hosts, 33 belong to the Order Rhabditida. Recent phylogenetic hypotheses have been unable to resolve whether metastrongyloids are sister taxa to those rhabditids that use molluscs as definitive hosts. Although most rhabditid nematodes have been reported not to kill their mollusc hosts prior to their reproduction, some species are pathogenic. In fact, infective juveniles of Phasmarhabditis hermaphrodita vector a lethal bacterium into the slug host in which they reproduce. This life cycle is remarkably similar to the entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae. Also, the discoveries of Alloionema and Pellioditis in slugs are interesting, as these species have been speculated to represent the ancestral forms of the entomopathogenic nematodes. Development of the infective stage appears to be an important step toward the acquisition of molluscs as definitive hosts, and the association with specific bacteria may have arisen in conjunction with the evolution of necromeny.     

Survival Mechanisms of Entomopathogenic Nematodes

Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) are isolated from a wide variety of ecosystems ranging from sub-Arctic to arid and tropical climates. It is expected that natural populations of these nematodes will be pre-adapted to specific ecological conditions in their environments. However, little is known about the survival strategies of entomopathogenic nematodes. This review focuses on the main survival mechanisms involved in the long-term persistence of nematodes: extreme temperature tolerance (cold and heat), desiccation tolerance, osmotic tolerance, pesticide resistance and limitations of aeration. Physiological and biochemical mechanisms for survival are discussed. The review addresses the implications of the survival strategies on the establishment of exotic and indigenous steinernematids and heterorhabditids.     

An analysis of using entomopathogenic nematodes against above-ground pests

Applications of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have traditionally been targeted against soil insects. Nonetheless, research over the last two decades highlights the potential of such agents against above-ground pests under certain circumstances. A general linear model was used to test for patterns in efficacy among 136 published trials with Steinernema carpocapsae Weiser, the most common species applied against foliar and other above-ground pests. The focus was on field and greenhouse assessments, rather than laboratory assays where relevant ecological barriers to infection are typically removed. The model showed differences in nematode treatment efficacy depending on the pests' target habitat (bore holes > cryptic foliage > exposed foliage) and trial location (greenhouse > field studies). Relative humidity and temperature during and up to 8 h post-application were also predicted to influence rates of nematode infection obtained. Conversely, spray adjuvants (both wetting agents and anti-desiccants) and nematode dosage applied (both concentration and use of consecutive applications 3-4 days apart) did not explain a significant amount of variance in nematode performance. With reference to case studies the model is used to discuss the relative importance of different factors on nematode efficacy and highlight priorities for workers considering using entomopathogenic nematodes to target pests in novel environments.     

Pathogenicity,reproduction and foraging behaviours of some entomopathogenic nematodes on a new turf pest,Dorcadion pseudopreissi (Coleoptera: Cerambycidae)

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have considerable potential as biological control agents of soil-inhabiting insect pests. In the present study, the control potential of the EPNs Steinernema carpocapsae (TUR-S4), S. feltiae (Nemaplus), S. carpocapsae (Nemastar), S. feltiae (TUR-S3) and Heterorhabditis bacteriophora (Nematop) against a new longicorn pest, Dorcadion pseudopreissi Breuning, 1962 (Coleoptera: Cerambycidae), on turf was examined in laboratory studies. Pathogenicity tests were performed at the following doses: 50, 100 and 150 Dauer Juveniles (DJs)/larva at 25°C. Highest mortalities (75–92%) of the larvae were detected at the dose of 150 DJs/larva for all nematodes used. Reproduction capabilities of the used EPNs were examined at doses of 50, 75, 100 and 150 DJs/larva at 25°C. S. carpocapsae (TUR-S4) had the most invasions (32 DJs/larva) and reproduction (28042 DJs/larva) at the dose of 100 DJs, and the highest reproduction (per invaded DJ into a larva) was observed in H. bacteriophora (Nematop) (2402.85 DJs) at a dose of 50 DJs. The foraging behaviour of the nematodes in the presence of D. pseudopreissi and Galleria mellonella L. (Lepidoptera: Galleriidae) larvae was studied using a Petri dish filled with sand at 20°C. All of the used nematodes accumulated near the larvae section of both insect species (32–53% of recovered DJs) with a higher percentage of S. carpocapsae (TUR-S4) (53%) and H. bacteriophora (48%) (Nematop) moving towards larvae of D. pseudopreissi, than the S. feltiae strains.     

Commercialisation of entomopathogenic nematodes: should import regulations be revised?

Entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae are obligate insect pathogens. Their favourable characteristics as biocontrol agents have resulted in some species of EPNs being released globally and widely used for the control of diverse insect pests. In this review, we consider the occurrence of currently described EPN species, including those that have been released globally for commercial purposes. We also discuss the contribution of regulation policies to the global distribution of these species and issues that influence import regulations. Possible non-target effects, the use of commercial versus native EPNs and the possible interaction between these species are considered. Finally, we provide a view as to whether existing policies adequately deal with the risks associated with the global movement of EPNs and we suggest future directions that should be considered for the use of EPNs as biological control agents.     

Nematodes in Dryland Field Crops in the Semiarid Pacific Northwest United States

Soils and roots of field crops in low-rainfall regions of the Pacific Northwest were surveyed for populations of plantparasitic and non-plant-parasitic nematodes. Lesion nematodes (Pratylenchus species) were recovered from 123 of 130 non-irrigated and 18 of 18 irrigated fields. Pratylenchus neglectus was more prevalent than P. thornei, but mixed populations were common. Population densities in soil were affected by crop frequency and rotation but not by tillage or soil type (P < 0.05). Many fields (25%) cropped more frequently than 2 of 4 years had potentially damaging populations of lesion nematodes. Pratylenchus neglectus density in winter wheat roots was inversely correlated with grain yield (r² = 0.64, P = 0.002), providing the first field-derived evidence that Pratylenchus is economically important in Pacific Northwest dryland field crops. Stunt nematodes (Tylenchorhynchus clarus and Geocenamus brevidens) were detected in 35% of fields and were occasionally present in high numbers. Few fields were infested with pin (Paratylenchus species) and root-knot (Meloidogyne naasi and M. chitwoodi) nematodes. Nematodes detected previously but not during this survey included cereal cyst (Heterodera avenae), dagger (Xiphinema species), and root-gall (Subanguina radicicola) nematodes.     

Compatibility of entomopathogenic nematodes with fipronil

The survival and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes, Steinernema carpocapsae Weiser, S. arenarium (Artyukhovsky) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), were determined after exposure to different concentrations (250, 500, 1000 and 2000 ppm) of fipronil, an insecticide acting on the GABA receptors to block the chloride channel. Heterorhabditis bacteriophora was very tolerant to all concentrations of fipronil, with the highest mortality of 17% being observed at 2000 ppm of fipronil after 72 h exposure. Steinernema carpocapsae showed a similar response, with the highest mortality of 11.25% of IJs being observed after 72 h exposure to 2000 ppm of fipronil. Steinernema arenarium was, however, more sensitive to fipronil, and at 2000 ppm mortality rates of 94.6% and 100% were observed after 24 and 72 h, respectively. Fipronil had negligible effects on the infectivity of the three nematode species tested. The IJs which survive exposure to all concentrations of fipronil tested can infect and reproduce in Galleria larvae. The moderate effects on entomopathogenic nematodes of a lower fipronil concentration (250 ppm) and the field rates (12-60 ppm) of fipronil used as insecticide, suggest that direct mixing of entomopathogenic nematodes and fipronil at field rates is a viable integrated pest management option.     

Distribution and molecular characterization of Wolbachia endosymbionts and filarial nematodes in Maryland populations of the lone star tick (Amblyomma americanum)

The lone star tick Amblyomma americanum is host to a wide diversity of endosymbiotic bacteria. We identified a novel Wolbachia symbiont infecting A. americanum. Multilocus sequence typing phylogenetically placed the endosymbiont in the increasingly diverse F supergroup. We assayed a total of 1031 ticks (119 females, 78 males and 834 nymphs in 89 pools) from 16 Maryland populations for infection. Infection frequencies in the natural populations were approximately 5% in females and <2% (minimum infection rate) in nymphs; infection was not detected in males. Infected populations were only observed in southern Maryland, suggesting the possibility that Wolbachia is currently invading Maryland A. americanum populations. Because F supergroup Wolbachia have been detected previously in filarial nematodes, tick samples were assayed for nematodes by PCR. Filarial nematodes were detected in 70% and 9% of Wolbachia-positive and Wolbachia-negative tick samples, respectively. While nematodes were more common in Wolbachia-positive tick samples, the lack of a strict infection concordance (Wolbachia-positive, nematode-negative and Wolbachia-negative, nematode-positive ticks) suggests that Wolbachia prevalence in ticks is not due to nematode infection. Supporting this hypothesis, phylogenetic analysis indicated that the nematodes were likely a novel species within the genus Acanthocheilonema, which has been previously shown to be Wolbachia-free.     

Ecological characterization of entomopathogenic nematodes isolated in stone fruit orchard soils of Mediterranean areas

Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples (210 sites) from Catalonia and 90 soil samples (30 sites) from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data, and cross-breeding experiments the nematode species were identified as Steinernemafeltiae and Heterorhabditis bacteriophora. Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one Steinernema carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Occurrence of Steinernema species in cabbage fields and the effect of inoculated S. feltiae on Delia radicum and its parasitoids

Abstract 1 Seven organically grown cabbage fields were surveyed for entomopathogenic nematodes in the autumn by baiting. Nematodes were obtained from three fields with bait larvae infection ranging from 1.3–4.0%. 2 Inoculation of Steinernema feltiae (Rhabditida: Steinernematidae) in the spring increased bait larvae infection to 16.0–32.0%. 3 Four different species were found (Steinernema affine, Steinernema bicornutum, S. feltiae and Steinernema C1). 4 The number of Delia radicum (L.) (Diptera: Anthomyiidae) puparia was significantly reduced at plants where S. feltiae had been inoculated. 5 Delia radicum parasitoids were also affected by S. feltiae. The results indicated that Aleochara species (Coleoptera: Staphylinidae) were more negatively affected than Trypliographa rapae (Westw.) (Hymenoptera: Cynipidae).     

Susceptibility of shore fly <Emphasis Type="Italic">Scatella stagnalis</Emphasis> to five entomopathogenic nematode strains in bioassays

The shore fly, Scatella stagnalis (Fallén) (Diptera: Ephydridae) is an important insect pest of greenhouse crops. We evaluated two different Spanish isolates of entomopathogenic nematodes, Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) and Steinernema arenarium (Artyukhovsky) (Rhabditida: Steinernematidae), and two commercially available strains, Steinernema feltiae (Nemaplus^®) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae) (Nematop^®) against shore flies. In tests conducted in 24-well plate filter paper applied at 5, 11, 22, 44 and 88 nematodes per larva, all nematodes produced significant shore fly larval mortality. The lowest concentration tested was enough to obtain high larval mortality (65.2–87.0%). The nematodes Steinernema feltiae and Steinernema arenarium, which parasitized the shore fly larvae faster, also penetrated in higher number in the shore fly larva (4.6–8.8% penetration rate). In bioassays conducted in algae, Steinernema feltiae, applied at 50 nematodes/cm², caused highest (100%) and Steinernema arenarium lowest shore fly mortality (94%). Our results suggest that entomopathogenic nematodes appear feasible for controlling shore flies but further tests are needed to determine their efficacy in the field.