Factors limiting short-term persistence of entomopathogenic nematodes

Abstract:  Entomopathogenic nematodes are commonly applied to soil and crops for biocontrol of insects. Typically, when nematodes are applied, recoverable numbers decline quickly. Because ultra-violet light is known to kill entomopathogenic nematodes in the laboratory, many researchers blame poor field persistence on the action of ultra-violet light. We conducted a field experiment to test this hypothesis, using Heterorhabditis bacteriophora applied to turf as a model system. We compared persistence of surface-applied nematodes with subsurface-applied nematodes and found no difference in persistence. Numbers of potential nematode antagonists (mites and collembola) were also monitored. Poor persistence was positively correlated with numbers of mites and collembola in plots where nematodes were surface-applied, but not in plots where they were subsurface-applied.     

Field evaluation of entomopathogenic nematodes for the biological control of the annual bluegrass weevil,Listronotus maculicollis (Coleoptera: Curculionidae), in golf course turfgrass

The ability of entomopathogenic nematodes to suppress larval populations of the annual bluegrass weevil, Listronotus maculicollis, was investigated under field conditions over a 3-year period (2006–2008). Combination of nematode species, application rate and timing produced strong numerical yet few statistically significant reductions. Steinernema carpocapsae Weiser, S. feltiae Filipjev, and Heterorhabditis bacteriophora Poinar applied at 2.5×10⁹ IJs/ha reduced first generation late instars between 69 and 94% in at least one field trial. Steinernema feltiae provided a high level of control (94%) to low densities (~20 larvae per 0.09 m²), but gave inadequate control for higher densities (24 and 50% suppression). No significant differences were found among treatment timings. However, applications timed to coincide with the peak of larvae entering the soil (fourth instars) generally performed better than applications made prior to (preemptive) or after the majority of the population advanced from the fourth instar. Nematode populations declined sharply between 0 and 14 days after treatment (DAT). Although nematode populations later increased (at 28 DAT), indicating an ability to recycle within hosts in the environment, they were nearly undetectable 56 DAT when the second generation host larvae were present in the soil. Applying commercially available nematode species at standard field rates cannot reliably reduce L. maculicollis immature densities on golf courses, nor will single applications suppress multiple generations. Future research will need to identify application strategies to improve biocontrol consistency.     

Entomopathogenic nematodes for control of Phyllophaga georgiana (Coleoptera: Scarabaeidae) in cranberries

A series of laboratory and greenhouse experiments evaluated the entomopathogenic nematodes Steinernema scarabaei Stock & Koppenhöfer, Heterorhabditis bacteriophora Poinar, and H. zealandica Poinar for control of second- and third-instar cranberry white grub, Phyllophaga georgiana Horn (Coleoptera: Scarabaeidae), in cranberries. Steinernema scarabaei was the most effective species with 76–100% control at a rate of 2.5×10⁹ IJ/ha in the greenhouse experiments. H. zealandica and especially H. bacteriophora were generally less effective and required rates of 5×10⁹ IJ/ha for acceptable control. Larval stage had no effect on H. zealandica and H. bacteriophora performance, whereas S. scarabaei was more effective against third instars than second instars in the laboratory but not in the greenhouse experiments. Steinernema scarabaei, should it become commercially available, could be an effective alternative to chemical insecticides for P. georgiana management.     

Toxicity of spinosad and Beauveria bassiana to the black cutworm,and the additivity of subletal doses

The main objective of this study was to assess the susceptibility of the black cutworm (Agrotis ipsilon) to the biopesticide spinosad and to a commercial formulation (GHA strain) of the entomopathogenic fungus, Beauveria bassiana. Secondly, we quantified the effects of sublethal doses of spinosad on a number of A. ipsilon fitness parameters, and interactions resulting from simultaneous applications of sub-lethal doses of spinosad and B. bassiana. Under laboratory conditions, A. ipsilon third instar larvae were highly susceptible to spinosad, with an estimated LC₅₀ of 50 ppm. The entomopathogenic fungus, B. bassiana had a lower efficacy with an estimated LC₅₀ of 7×107 spores mL^?1. Topical applications of 5, 7.5 and 10 ppm of spinosad on third instar larvae reduced larval size and increased time to pupation and to emergence. However, pupal and adult weights were not significantly different between treated and control individuals. Additivity was observed from most spinosad–B. bassiana combinations tested, thus indicating compatibility between products. We concluded that spinosad is a promising tool for controlling black cutworm larvae alone or in combination with other products.     

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

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

昆虫病原线虫与其共生细菌共生关系的研究进展

昆虫病原斯氏和异小杆线虫与共生细菌的共生关系是这类线虫作为害虫生物防治因子的基础。从线虫共生细菌的信息、营养、抗菌和病原作用,以及线虫对共生细菌的保护和媒介作用综述昆虫病原线虫与其共生细菌的共生关系;描述这一共生关系的影响因子;同时,讨论了未来的研究方向和应用前景。     

Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species

We tested the effect of host density on entomopathogenic nematode efficacy in 1-L pots with grass and soil. In four experiments, combinations ranged from somewhat resistant hosts (oriental beetle, Anomala orientalis, or northern masked chafer, Cyclocephala borealis, with Heterorhabditis bacteriophora) over more susceptible hosts (Japanese beetle, Popillia japonica, with Steinernema glaseri) to a highly susceptible host (P. japonica and S. scarabaei). In each experiment, four larval densities were exposed to two nematode rates over a 14-day period. A significant effect of host density on nematode efficacy occurred only in the A. orientalis–H. bacteriophora combination, but there was no clear trend in the data. This suggests that an exhaustion of available nematode populations to less lethal levels by high host numbers was counteracted by other factors such as increased chances for nematode-host contact and increased host susceptibility due to stress via reduced food resources and increased aggression between larvae.     

昆虫病原线虫防治烟草小地老虎技术优化研究

小地老虎Agrotis ipsilon是烟草Nicotiana tabacum移栽期主要的切根害虫,严重危害烟草生产。为研判烟草生产中符合双减（减肥、减药）要求的生物和生态防控技术,集成适合推广的虫害综合治理模式,本研究在云南玉溪华宁县以昆虫病原线虫（EPN）Steinernema carpocapsae All粉剂和化学药剂敌杀死（溴氰菊酯）处理,配合2种幼虫食诱方法（传统引诱剂、人工引诱剂）以及2种增效助剂（激健1、2）组合,调查移栽后烟草幼苗受小地老虎危害死亡率,评价生物药剂、食诱剂和助剂对烟草幼苗的保护效果。结果表明,无论是否存在食诱剂,移栽4 d各施药处理均能显著降低烟草幼苗死亡率,随时间的推移,所有处理的烟草幼苗死亡率均显著上升;无食诱剂情况下,EPN+激健1和敌杀死于移栽7 d仍具显著保苗效果;传统的糖醋酒液食诱法本身具备一定保苗效果,配合施药,初期效果最好,4 d时,烟草幼苗危害率最低达0.83%±0.008%（传统食诱剂+昆虫病原线虫）;人工食诱剂持效性最好,移栽10 d时配合EPN、EPN+激健2和敌杀死仍具明显保苗效果。施用化学药剂时,食诱剂本身对保苗效果无显著贡献;使用EPN时,传统食诱法较空白具有显著保苗效果,但与人工食诱法间差异不显著。移栽4 d时,EPN+激健1较单独施放EPN具显著保苗效果,之后调查中添加助剂小区与EPN小区无显著差异。综上所述,EPN粉剂配合人工食诱剂和激健助剂,可长期较好地抑制小地老虎对烟草幼苗危害,为集成烟草全程绿色生态治理模式提供了有效参考。     

Assessment of establishment and persistence of entomopathogenic nematodes for biological control of western corn rootworm

Abstract:  The use of entomopathogenic nematodes (EPN) is potentially one ecological approach to control the invasive alien western corn rootworm ( Diabrotica virgifera virgifera LeConte, Col., Chrysomelidae) in Europe. This study investigated the establishment and the short- and long-term persistence of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), Heterorhabditis megidis Poinar, Jackson and Klein (Rh., Heterorhabditidae) and Steinernema feltiae Filipjev (Rh., Steinernematidae) in three maize fields in southern Hungary, using the insect-baiting technique. All three EPN species equally established and persisted in maize fields. The timing of application (April or June) did not influence the establishment of EPN species. EPNs persisted for 2–5 months, i.e. they survived up to and throughout D. v. virgifera larval occurrence in the soil. Results demonstrate that D. v. virgifera larvae can potentially be controlled by EPNs during the same year of EPN application but no long-term control effect is expected under intensive maize cultivation practices.     

Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to an entomopathogenic nematode

Abstract The perennial ryegrass, Lolium perenne, forms a symbiotic relationship with Neotyphodium lolii, a fungus that produces alkaloids. This relationship provides a competitive advantage to the host plant in grassland communities by increasing drought tolerance, and disease and herbivore resistance. Black cutworm, Agrotis ipsilon, is among the few insect species that are able to feed and develop on endophytic perennial ryegrass. Some insects can use plant secondary compounds to defend themselves against predators, therefore we hypothesized that the cutworms fed on endophytic grasses would exhibit greater defense against a lethal endoparasitic nematode, Steinernema carpocapsae. Laboratory experiments involving 4–5th instars support the hypothesis that A. ipsilon feeding on grass clippings from field plots with high (> 90%) incidence of endophyte infected perennial ryegrass are less susceptible to entomopathogenic nematodes than larvae fed grass clippings from plants with little or no incidence of endophyte. Laboratory studies resulted in similar overall mortality after 48 h. Field studies, however, show decreased susceptibility to S. carpocapsae when larvae were confined to areas of endophytic grass (> 75% infected). Early instars (2–3rd) fed on endophyte free grass suffered greater overall mortality at all nematode concentrations than 4–5th instars fed similarly. Early (2–3rd) instars were equally susceptible to nematode attack regardless of food source. Our results indicate that the fungal endosymbionts of grasses can influence the biology of natural enemies of an herbivorous insect.     

Efficacy of the entomopathogenic nematodes Steinernema kraussei and Heterorhabditis megidis against the black vine weevil Otiorhynchus sulcatus in open field‐grown strawberry plants

• 1 Entomopathogenic nematodes are commercially available for inundative biological control of many insects, including the black vine weevil Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae). Currently, there is a lack of commercial application tests in field‐grown crops comparing the efficacy of different species of entomopathogenic nematodes.
• 2 Field trials were carried out under different growing conditions in Ireland and Norway to evaluate the efficacy of two commercially available nematode species on the market for control of the black vine weevil Heterorhabditis megidis and Steinernema kraussei.
• 3 Heterorhabditis megidis was evaluated not only at temperatures ideal for this species (soil temperatures above 10 °C), but also in the low temperature trials with S. kraussei as a ‘positive control’. Steinernema kraussei is sold as a cold active product and was therefore evaluated at low soil temperatures (below 10 °C).
• 4 The overall results indicated that H. megidis was effective as long as temperatures were optimum (not dropping below 10 °C). For S. kraussei, the results obtained were rather disappointing, where control barely reached 50% in the trial with the coldest temperature. Temperature and soil type appeared to be a major limiting factor for the efficacy of both nematode species.
• 5 On the basis of the results and experience obtained in these trials, the future implications for biological control of O. sulcatus with entomopathogenic nematodes in commercial field‐grown strawberry production are discussed.

     

Spatial and temporal distribution of endemic and released entomopathogenic nematode populations in turfgrass

Entomopathogenic nematodes (Rhabdita: Heterorhabditidae and Steinernematidae) have been effective as inundative biological control agents of scarab larvae (Coleoptera: Scarabaeidae) in turfgrass. Entomopathogenic nematodes also occur naturally in turfgrass and endemic or inoculated populations may be able to provide effective long-term control. Variation in Heterorhabditis bacteriophora and Steinernema carpocapsae spatial and temporal distribution along transects placed at different turfgrass sites in central New Jersey, USA, was investigated. H. bacteriophora tended to be recovered from fewer sections in a transect than S. carpocapsae, but the two species, overall, did not differ in patchiness of distribution. In one transect with a H. bacteriophora population S. feltiae was also recovered, but the two populations seldom overlapped spatially. In transects with adequate scarab larvae density for analysis, H. bacteriophora density and Popillia japonica larvae density were inversely correlated. This suggests that endemic H. bacteriophora populations may suppress P. japonica populations. In one transect, an epizootic of H. bacteriophora in an undetermined host may have occurred. Edaphic factors were relatively uniform along transects and were, at most, weakly correlated with nematode recovery. Uniform inoculative releases of H. bacteriophora tended to return to patterns of distribution typical of endemic populations.     

Post-application of anti-desiccant agents improves efficacy of entomopathogenic nematodes in formulated host cadavers or aqueous suspension against diapausing codling moth larvae (Lepidoptera: Tortricidae)

Codling moth (CM), Cydia pomonella (L.) is the most serious pest of apple and other pome fruit worldwide. In temperate climates, diapausing cocooned larvae make up 100% of the population. Control of this stage would reduce or eliminate damage by first generation CM in late spring and early summer. Entomopathogenic nematodes (EPNs) are good candidates for control of CM in the cryptic habitats where the larvae overwinter. The two predominant limiting factors for EPNs are adequate moisture and temperatures below 15°C. Formulation that maintains moisture and enables survival of EPN infective juveniles (IJs) until they can infect overwintering larvae would significantly improve their utility for protection of apple, pear and walnut. In laboratory studies conducted in moist mulch (consisting of apple and conifer wood), Galleria mellonella (L.) larvae infected with Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), or Heterorhabditis bacteriophora Poinar and coated with starch and clay, produced mean mortalities of 42, 88, and 24%, respectively in CM larvae. Mulched field plots treated with formulated S. carpocapsae- or S. feltiae-infected G. mellonella larvae, then followed by an application of wood flour foam as an anti-desiccant, resulted in 56 and 86% mortality, respectively. Comparative tests of aqueous suspensions of S. carpocapsae IJs applied to cardboard bands on apple tree trunks followed by water, fire retardant gel or foam resulted in 11, 35, and 85% respective mortalities. Identical tests with S. feltiae resulted in 20, 19, and 97% respective mortalities. Our research with cadaver formulations of EPNs in mulch and aqueous suspensions on tree trunks combined with anti-desiccant agents, demonstrated significant improvement in larvicidal activity for diapausing cocooned CM larvae.     

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

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

昆虫病原线虫对烟苗的保护效果

小地老虎Agrotis ypsilon是危害烟草的重要害虫.为了实现该害虫的绿色防控,本研究测定了小地老虎幼虫对不同食物(小白菜、蕹菜、人工饲料和麸皮)取食的偏好性,评价添加鲜叶类诱食剂时昆虫病原线虫Steinernema carpocapsae All粉剂、S.feltiae SN虫尸剂、绿僵菌Metarhizium anisopliae和化学农药敌杀死(溴氰菊酯)对小地老虎的防控而实现对移栽期烟苗的保护效果.结果表明,尽管不同时间不同食物吸引的小地老虎幼虫数量有差异,但是这些幼虫对鲜叶类蔬菜(小白菜和蕹菜)具有偏好性.当每株烟苗存在2头小地老虎4龄幼虫时,未添加幼虫诱食剂的情况下,昆虫病原线虫粉剂处理后的烟苗死亡率为56.7％,显著低于绿僵菌处理的74.2％、昆虫病原线虫虫尸剂处理70.0％和对照78.3％,但高于敌杀死(溴氰菊酯)处理的烟苗死亡率37.5％;添加诱食剂后,昆虫病原线虫粉剂处理后烟苗的死亡率为40.8％,显著低于绿僵菌62.5％和昆虫病原线虫虫尸剂67.6％处理和对照62.5％,但与敌杀死(溴氰菊酯)处理的烟苗死亡率39.2％,差异不显著.当每株烟苗存在3头小地老虎4龄幼虫时,不论是否添加小地老虎幼虫诱食剂,昆虫病原线虫粉剂处理后的烟苗死亡率显著低于绿僵菌、昆虫病原线虫虫尸剂处理和对照,但与敌杀死(溴氰菊酯)处理的烟苗死亡率差异不显著.同样,添加幼虫诱食剂后,昆虫病原线虫粉剂处理的烟苗死亡率为36.7％,显著低于未加入小地老虎幼虫诱食剂的处理71.7％.因此,烟苗周围存在2头或3头小地老虎4龄幼虫的情况下,S.carpocapsae All线虫粉剂对刚移栽烟苗具有明显保护作用,在烟苗周围添加小地老虎幼虫诱食剂能显著降低烟苗的被危害率,为烟田小地老虎幼虫防控提供了有效的方法.     

Differential susceptibility of subterranean termite castes to entomopathogenic nematodes

We examined the attachment and infectivity of two entomopathogenic nematode species, Steinernema carpocapsae and Heterorhabditis indica, on soldiers and workers in two subterranean termite species, Coptotermes formosanus and C. vastator. In attachment tests with S. carpocapsae, more nematodes attached to soldiers of C. formosanus and C. vastator in the absence of workers compared to soldiers that were in the presence of workers. In tests with soldiers alone, workers alone, and mixture of equal numbers of workers and soldiers, soldiers in the mixed groups had lower mortality than soldiers alone after 1 and 4 days for both termite species. Exposure of small groups of either termite species to S. carpocapsae resulted in higher mortality of soldiers after 1 and 4 days post exposure. Mortality in soldiers alone exposed to H. indica was not significant compared to mortality in mixed groups at 1 day exposure, but was significant after 4 days. In concentration-mortality tests, a significant two-way interaction existed between nematode concentration and termite caste for C. formosanus exposed to S. carpocapsae for 1 and 4 days. A significant effect of nematode concentration was found for C. vastator exposed to H. indica for 1 day. This termite species had lower mortality when exposed to H. indica after 1 and 4 days compared to C. formosanus. At 4 days post exposure to H. indica, the effects of nematode concentration and termite caste were significant. Steinernema carpocapsae caused higher mortality than H. indica, but mortality of workers was higher when exposed to H. indica. Soldiers of both species experienced rapid mortality when exposed to S. carpocapsae, whereas soldiers of C. vastator experienced lower mortality when exposed to H. indica. Thus, our results show that when soldiers alone or workers alone are exposed to the nematodes, there is a differential susceptibility of soldiers and workers to nematode infection with soldiers being more susceptible than workers. In a mixed group of soldiers and workers or workers alone, it appears that grooming behavior of the workers serves as a mechanism to reduce nematode infection of soldiers and workers. The reason for this differential response to nematode infection is that soldiers do not exhibit grooming behavior.     

Biological control of tobacco cutworm,Spodoptera litura Fabricius with entomopathogenic nematodes

The efficacies of several entomopathogenic nematodes ofSteinernema andHeterorhabditis spp. were examined against tobacco cutworm,Spodoptera litura Fabricius.H. bacteriophora HY showed 100% mortality after 20 h against 2nd instar of tobacco cutworm. In the case of 3–4th instar,S. carpocapsae PC.,H. bacteriophora HY andS. monticola CR showed 100% mortality after 47 h. In the case of 5–6th instar,S. carpocapsae PC proved more effective than the others. Generally, the number of nematodes harvested increased as their size decreased. Also, the highest number of nematodes was obtained in the 5–6th instar ofS. litura byH. bacteriophora HY, showing about 1.3×10⁶ nematodes per larva.In vitro culturedS. carpocapsae PG showed 100% mortality after 73 h against 5–6th instar tobacco cutworm, indicating that nematodes producedin vitro can be potentially used for the biological control ofS. litura instead of nematodesin vivo.     

昆虫病原线虫对腰果细蛾的致病力测定

室内测定了Steinernema carpocapsae All、S.carpocapsae Biosys、S.glaseri NC52、S.longicaudum X-7、Heterorhabditis bacteriophora H06和H.indica LN2共6个昆虫病原线虫品系对腰果细蛾Acrocercops syngramma Meyrick幼虫的致病力。结果表明,线虫S.carpocapsae All、Biosys品系与线虫S.glaseri NC52对腰果细蛾幼虫的致死效果显著高于其它线虫,但供试的6个线虫品系均未能在腰果细蛾幼虫体内繁殖下一代,说明供试线虫在腰果细蛾幼虫体内难以繁殖。选择小卷蛾斯氏线虫S.carpocapsae All进一步研究其对腰果细蛾的致病力,结果发现,All线虫对腰果细蛾3龄幼虫处理24 h后的致死中浓度LC50为19.88 IJs/虫。腰果细蛾各龄幼虫和蛹对小卷蛾斯氏线虫S.carpocapsae All均表现出高度的敏感性,但不同幼虫龄期和处理时间敏感程度不同。小卷蛾斯氏线虫S.carpocapsae All对不同龄期腰果细蛾幼虫和蛹均表现出较强的致病力。     

Pathogenicity of five species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) to the ixodid tick Dermacentor silvarum Olenev (Acari: Ixodidae)

We determined the mortality of adult tick Dermacentor silvarum Olenev after it was exposed to five entomopathogenic nematode species: Steinernema carpocapsae BJ (Sc BJ), S. glaseri NC32 (Sg NC32), S. ceratophorum D43 (Sc D43), S. feltiae Otio (Sf Otio) and Heterorhabditis bacteriophora E-6-7 (Hb E-6-7). All five species had lethal effects on adult ticks, and caused significant decreases in the amount of eggs laid by engorged female ticks (P < 0.05). Among them, Hb E-6-7 and Sc BJ were the most pathogenic to female adult ticks, with mortalities ranging from 70 to 90% (P < 0.05). Engorged females were more susceptible than unfed and partially fed females. The cumulative mortalities of engorged females exposed to Hb E-6-7 and Sc BJ for 14 days were 90%, with LT₅₀ at 6.3 and 7.0 days, respectively. Dose-dependent effect was observed in the oviposition of the ticks after they were exposed to nematodes at concentrations of 1000–5000 IJs/dish. When the concentration was higher than 5000 IJs/dish, dose dependent effects disappeared (P > 0.05). All five nematode species were lethal to unfed male ticks, and there was significant difference in mortality when compared to the control group (P < 0.05); however, the pathogenicity varied among them.