Chemical stimulation of productivity and pathogenicity of entomopathogenic nematodes

Under laboratory conditions, the treatment of Steinernema carpocapsae and Heterorhabditis bacteriophora entomopathogenic nematodes with either Mg or Mn(II) ions produced an increase in their productivity and pathogenicity against Sitona lineatus weevil, pupae, and larvae. Copyright 1999 Academic Press.     

Development of the one-on-one quality assessment assay for entomopathogenic nematodes.

The one-on-one bioassay was developed using Steinernema carpocapsae (All) nematodes against the wax moth larva, Galleria mellonella. The assay was used to develop and compare virulence profiles of both in vitro- and in vivo-produced nematodes and to provide a quality assessment 'standard' for in vitro-produced nematodes. The bioassay was subsequently used to develop virulence profiles for Steinernema carpocapsae (UK), S. feltiae (UK), S. feltiae (R1.5), S. feltiae (SN), S. glaseri (NJ-43), and S. riobrave (RGV). These profiles are unique for each species and isolate and are used as a standard of virulence in routine quality assessment of nematodes produced in liquid fermentation.     

昆虫病原线虫的耐寒性

昆虫病原线虫可开发成生物农药,广泛应用于多种地下及钻蛀害虫的安全防治。但昆虫病原线虫货架期较短,对寒冷等极端环境的耐受性较差,影响了其在生物防治方面的商业开发。本文介绍了寒区的昆虫病原线虫资源,总结了昆虫病原线虫耐寒性的测定方法及增强方法、耐寒性差异的研究进展,并对其耐寒的生理生化机制及分子机理进行了综述。研究昆虫病原线虫的耐寒性,对于解释种群动态,指导昆虫病原线虫的低温保存,以及拓展其在生物防治方面的应用具有重要意义。     

Effect of temperature on the pathogenicity of entomopathogenic nematodes (Steinernema and Heterorhabditis spp.) to Delia radicum

Susceptibility of last instarlarvae of Delia radicum to Steinernema feltiae, S. carpocapsae,S. arenarium, Heterorhabditismegidis and H. bacteriophora wasevaluated in the laboratory at 10 °C,15 °C and 20 °C. S. feltiaewas the only species that killed the larvae at10 °C; S. carpocapsae, S.arenarium and H. megidis were effectiveat 15–20 °C whereas H.bacteriophora killed the maggot only at20 °C. The temperature significantlyaffected the host searching ability of alltested species. Mobility was reduced at lowtemperatures. Significant effects of the hostpresence on nematode mobility were found forS. feltiae, S. arenarium and H. megidis but not for S. carpocapsaeand H. bacteriophora. The dynamics of theattachment to and penetration into the hostwere monitored for S. feltiae at10 °C, 15 °C or 20 °C and forS. carpocapsae at 20 °C. In theperiod of 6–30 hours after inoculation, S.carpocapsae attached in higher number at20 °C than did S. feltiae at alltemperatures. At 20 °C, S.carpocapsae penetrated the host only after 30hours while S. feltiae penetrated alreadyafter 15, 9, 6 hours at 10 °C,15 °C and 20 °C, respectively.     

Mass production of entomopathogenic nematodes for plant protection

Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are commercially used to control pest insects. They are symbiotically associated with bacteria of the genera Photorhabdus and Xenorhabdus, respectively, which are the major food source for the nematodes. The biology of the nematode-bacterium complex is described, a historical review of the development of in vitro cultivation techniques is given and the current use in agriculture is summarised. Cultures of the complex are pre-incubated with the symbiotic bacteria before the nematodes are inoculated. Whereas the inoculum preparation and preservation of bacterial stocks follow standard rules, nematodes need special treatment. Media development is mainly directed towards cost reduction, as the bacteria are able to metabolise a variety of protein sources to provide optimal conditions for nematode reproduction. The process technology is described, discussing the influence of bioreactor design and process parameters required to obtain high nematode yields. As two organisms are grown in one vessel and one of them is a multicellular organism, the population dynamics and symbiotic interactions need to be understood in order to improve process management. Major problems can originate from the delayed or slow development of the nematode inoculum and from phase variants of the symbiotic bacteria that have negative effects on nematode development and reproduction. Recent scientific progress has helped to understand the biological and technical parameters that influence the process, thus enabling transfer to an industrial scale. As a consequence, costs for nematode-based products could be significantly reduced.     

Application methods of entomopathogenic nematodes for control of Mahanarva spectabilis (Hemiptera: Cercopidae)

The pathogenicity of entomopathogenic nematodes (EPNs) against nymphs of the pasture spittlebug Mahanarva spectabilis (Distant, 1909) (Hemiptera: Cercopidae) was studied under four application methods. Nymphs of the fourth or fifth instar were placed on the roots of elephant grass plants and submitted to four EPN isolates (Steinernema carpocapsae, S. feltiae, S. riobravis and Heterorhabditis amazonensis RSC1), at two concentrations (2000 and 4000 infective juveniles/mL), with four application methods (pipetting, spraying on the nymphs after froth formation, spraying before froth formation and placement of infected host cadaver method). There was no significant difference in the nymph mortality in function of the concentration and/or the isolate used. However, the efficacy was influenced by the application method, with the most efficient being sprayed on nymphs after froth formation and infected host cadavers. Steinernema riobravis applied by spraying on nymphs with froth, at a concentration of 2000 EPNs/mL, and H. amazonensis RSC1 applied by infected host cadavers caused 71% of the nymphs to die. The use of infected host cadavers and spraying in an aqueous solution on nymphs after froth formation were found to be the most efficient methods to control Mahanarva spectabilis.     

Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes

Mutual effects between the symbiotic bacteria of entomopathogenic nematodes, Photorhabdus luminescens and Xenorhabdus poinarii, and entomopathogenic fungi were investigated in vitro. A dual culture assay on nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA) medium revealed that P. luminescens is antagonistic to Metarhizium anisopliae, Beauveria bassiana, B. brongniartii and Paecilomyces fumosoroseus by inhibiting their growth and conidial production; the fungal growth was not inhibited by X. poinarii. In a second laboratory experiment, crude extract produced by M. anisopliae was tested for its activity against P. luminescens and X. poinarii. Crude extract from M. anisopliae was antibacterial to P. luminescens and X. poinarii at 1000 g/ml and inhibited their growth on NBTA, but had no effect at 100 or 10 g/ml. The influence of the crude extract of M. anisopliae on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis and Steinernema glaseri was assayed on Sabouraud Dextrose Agar (SDA) plates. Results showed that the crude extract of M. anisopliae had no toxic effects even at highest concentration (1000 g/ml).     

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.     

Evaluation of entomopathogenic nematodes for suppression of carrot weevil

The potential of entomopathogenic nematodes as biologicalcontrol agents for carrot weevil (Listronotus oregonensis) was evaluated throughboth laboratory and field experiments. In thelaboratory, Steinernema carpocapsae, S. riobrave, S. feltiae, Heterorhabditis megidis, H. bacteriophora, and a control (water only) werecompared in sand and muck soil against adults,and in sand against larvae. All nematodespecies produced high levels of larvalmortality. S. carpocapsae producedsignificantly greater adult mortality in sandthan other species or the untreated control. H. bacteriophora caused low adultmortality in sand, but the greatest adultmortality among treatments in a similar testthat used muck soil; S. carpocapsae wasranked second on muck soil. Other speciesconsistently produced intermediate (H.megidis and S. riobrave) or low (S.feltiae) levels of mortality on bothsubstrates. In the field, we compared theeffect of early season vs. late seasonapplications of H. bacteriophora or S. carpocapsae on carrot weevil mortality andparsley survival and yield. Significantdifferences among treatments in plant survivaland yield were not found; however treatmentsinvolving H. bacteriophora had higherplant survival than other treatments. Earlierapplication of this species was associated withhigher plant survival. S. carpocapsaetreatments had similar plant survival to thecontrol. Mortality of larvae and combinedstages of carrot weevil was significantlygreater at 1 week following H.bacteriophora application than for othertreatments. H. bacteriophora also showedgreater persistence than S. carpocapsaein treated plots. We conclude that H.bacteriophora is a good candidate for furtherevaluation as a biological control agentagainst carrot weevil on muck soils in theGreat Lakes region.     

Production technology for entomopathogenic nematodes and their bacterial symbionts

Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode–bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application. Journal of Industrial Microbiology & Biotechnology (2002) 28, 137–146 DOI: 10.1038/sj/jim/7000230 Received 16 August 2001/ Accepted in revised form 10 November 2001     

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.     

Distribution of entomopathogenic nematodes in Southern Cameroon

A first survey of entomopathogenic nematodes (EPN) was conducted in three agro-ecological zones of Southern Cameroon in 2007 and 2008. Entomopathogenic nematodes were recovered from 26 of 251 soil samples (10.4%). Three species, Heterorhabditis baujardi, Steinernema sp. A and Steinernema sp. B were found. The two steinernematids were considered unidentified species. Among the positive samples, 23 samples contained only H. baujardi (88.5%), two contained Steinernema sp. A co-occurring with H. baujardi (7.7%), and one sample contained Steinernema sp. B (3.9%). H. baujardi was frequent in forest and fruit crop (cocoa and oil palm plantations). Steinernema sp. A was found in a tree plantation of teak, Steinernema sp. B in a forest habitat. Nematodes were mostly present in acidic soils with pH ranging from 3.7 to 7.0. The highest EPN presence was recorded in sandy loam, sandy clay loam, sandy clay and clay soils. EPNs were not recovered in sand, loamy sand and clay loam soils. Using principal component analysis for elucidating the major variation patterns among sampling sites, four factors explaining for 73.64% of the overall variance were extracted. Factors were a combination of geographical (latitude, longitude, altitude), soil (pH, contents of sand, silt and clay, organic carbon, texture), and moisture (wilting point, field capacity) parameters as well as climatic parameters (mean annual rainfall, mean air temperature). Logistic regression and redundancy analyses (RDA) revealed that soil pH, longitude, available water and altitude were associated with presence and absence of EPN. Both logistic regression and RDA indicated that, increasing soil pH and longitude, associated with decreasing altitude, led to higher percentages of samples containing entomopathogenic nematodes.     

Optimizing the application of entomopathogenic nematodes: experimental set-up

The complex issue concerning the spray application of Entomopathogenic Nematodes (EPNs) with a hydraulic sprayer is still not solved. This research project focuses on the effect of spray application technique on the viability and deposition of EPNs. In this paper the experimental set-up used for this evaluation is described. A modular spray application system has been developed and is currently used to evaluate the effect of different parts of a sprayer on the viability of the EPNs. Based on the results of experiments using this modular spray application system, recommendations regarding pump type, mixing system, nozzle type and filter size will be formulated. Because of the large number of experiments in this research project, an image analysis system for the determination of the viability of the nematodes is developed. This paper describes two experiments comparing the new developed image processing technique with the standard microscopic counting technique.     

昆虫病原线虫液体培养研究概况

综述昆虫病原线虫液体培养的研究概况。包括液体培养的研究历史,不同培养基组分对昆虫病原线虫产量的影响,营养源的选择,培养参数在昆虫病原线虫液体培养过程中的作用,感染期昆虫病原线虫脂肪酸含量,以及昆虫病原线虫应用前景等。     

Interactions between entomopathogenic nematodes and imidacloprid for rose sawfly control

Rose sawfly, Arge ochropus (Gmelin), is one of the most important pests of ornamental plants such as roses and wild rose bushes in Northern Iran. We investigated the interactions between the insecticides imidacloprid and the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae as control agents of fifth-instar larvae in the laboratory. The larvae were very susceptible to S. carpocapsae (LC₅₀: 21 infective juvenile per larva) and H. bacteriophora (LC₅₀: 32). Combinations of two imidacloprid rates (LC₃₀ and LC₅₀) and four rates of each nematode species (LC₂₅–LC₇₅) were tested. Combinations with the lower imidacloprid rate except for that with the highest H. bacteriophora rate caused higher mortality than both respective single-agent treatments. In combination with the higher imidacloprid rate, only one combination with H. bacteriophora and two combinations with S. carpocapsae caused higher mortality than both respective single-agent treatments. Interactions were generally stronger at the lower imidacloprid rate and were stronger for S. carpocapsae (synergistic in seven combinations, additive in one) than for H. bacteriophora (synergistic in two, additive in six). Synergistic imidacloprid-S. carpocapsae combinations could be a useful tool for the control of A. ochropus larvae that would simultaneously control other common pests susceptible to imidacloprid.     

昆虫病原线虫对黄地老虎致病力的研究

为了探索应用昆虫病原线虫防治黄地老虎Agrotis segetum的可行性,室内生测比较了9个昆虫病原线虫品系对黄地老虎4龄幼虫的致病力。生测结果表明,9个不同昆虫病原线虫品系对黄地老虎4龄幼虫的致病力差别很大,其中格氏线虫Steinernema glaseri ib(Sgib)是对黄地老虎幼虫致病力最强的昆虫病原线虫品系,侵染24、36和72 h黄地老虎幼虫的校正死亡率均高于其他线虫品系。此外,还测定了不同温度、土壤湿度以及线虫剂量对格氏线虫Sgib致病力的影响,结果表明,在21℃-27℃、20%土壤湿度以及侵染期线虫与黄地老虎幼虫数量比不少于10∶1的条件下,格氏线虫Sgib对黄地老虎幼虫的致病效果最好。田间小区实验表明,格氏线虫Sgib对黄地老虎幼虫的防效达到96.67%。     

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.     

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.     

Factors affecting the natural occurrence of entomopathogenic nematodes in turfgrass

We conducted a study to determine the relationship between turfgrass management intensity and natural occurrence of entomopathogenic nematodes (EPNs). We surveyed for EPNs on putting greens, fairways, and rough areas—three distinct surface types on golf courses that are managed with different intensities. We collected 159 soil samples from putting greens, contiguous fairways, and rough areas from 19 golf courses in Ohio, USA. Nematodes were recovered from soil samples using the insect baiting technique. We also analyzed the soil samples for texture, organic matter, pH, phosphorus, calcium, magnesium and potassium. We used principal components analysis and Pearson correlation to determine the relationship between nematode occurrence and the measured parameters. Surface type was the most important factor in predicting EPNs occurrence. Putting greens differed significantly from fairways and rough areas in the number of EPN-positive sites. No EPNs were recovered from putting greens but were recovered from 43% of the fairways and 57% of the rough areas. Putting greens also differed significantly from fairways and rough areas in organic matter, pH, calcium, and phosphorus. The fairways and rough areas did not however differ in number of EPN-positive sites and measured soil parameters. Presence of EPNs correlated significantly, although weakly, with sand, silt, phosphorus, organic matter, and magnesium content, but not with clay, pH, calcium, and potassium. Nematode isolates were identified as Heterorhabditis bacteriophora, Steinernema carpocapsae, and S. glaseri. This suggests that EPNs are more likely to occur in less intensively managed sites that receive fewer inputs and have relatively high sand, and moderate silt, organic matter, phosphorus, and magnesium content.