Isolation and identification of a symbiotic bacterium fromSteinernema carpocapsae

Xenorhabdus nematophilus sp., an insect-pathogenic bacterium, was newly isolated from Korean entomopathogenic nematode ofSteinernema carpocapsae, which can be used as a useful bioinsecticide. Primary and secondary form variants ofXenorhabdus nematophilus were observed when culturedin vitro. Primary form variants adsorbed bromothymol blue, while secondary form did not. However, many other characters of two variants were very similar. The variants were all rod-shaped and cell size was highly variable ranging from 0.5 by 2.0 μm to 1.0 by 5.0 μm. Both produced highly toxic substances and killed the insect larva within 20–38 hr, indicating that insect pathogenicity ofXenorhabdus is not directly associated with its phase variation. In addition, cell-free culture supernatant ofXenorhabdus was sufficient to kill the insect larva by injecting it into insect hemolymph; however, cell-harboring culture broth was more effective for killing the insect. The use ofXenorhabdus nematophilus may provide a potential alternative toBacillus thuringiensis (Bt) toxins.     

Extracellular protease production fromXenorhabdus nematophilus, a symbiotic bacterium of entomopathogenic nematodes

Effects of nutrients and growth temperature on the production of an extracellular protease fromXenorhabdus nematophilus, a symbiotic bacterium of entomopathogenic nematodes were studied for batch culture. Tryptone and fructose were most effective nitrogen and carbon sources to produce high level of the protease within 24–28 hr of incubation. The stability of protease was poor during the incubation of the bacteria. The protease activity increased in parallel with cell growth then decreased significantly for extended culture periods of the bacteria over 48 hr at 22–30°C. Autolysis of the protease was not a major cause for the decreased protease activity since no decrease in the protease activity was observed for the whole culture broth of the bacteria which was stored up to 80 hr at 4°C.     

Growth and Virulence of Steinernema glaseri Influenced by Different Subspecies of Xenorhabdus nematophilus

Three Xenorhabdus nematophilus subspecies influenced Steinernema glaseri growth profiles and growth rates, but this was not necessarily because of different bacterial growth rates. Virulence of dauer nematodes in larval Galleria mellonella varied with the number of dauers retaining bacteria and the bacterial subspecies. Virulence was least for dauers grown on X. nematophilus subsp. bovienii because of the lack of retained bacteria. Virulence was subsequently restored by culturing these nematodes on X. nematophilus subsp. poinari.     

昆虫病原线虫共生菌Tc毒素研究进展

来自昆虫病原线虫共生菌的Tc毒素是一类多亚基组成的高分子蛋白复合物,对多种农业害虫具有广谱的胃毒活性,这类毒素与目前已知的其它杀虫蛋白同源性非常低,有可能成为新的杀虫资源。本文对来自昆虫病原线虫共生菌的Tc毒素的特性、作用模式等方面的国内外研究进展进行了综述。     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Infection of the Entomopathogenic Nematode,Steinernema carpocapsae,as Affected by the Presence of Steinernema glaseri

The infection behavior of Steinernema carpocapsae infective juveniles (IJ) was investigated in the presence and absence of S. glaseri. Mixed inoculation of S. carpocapsae with S. glaseri IJ significantly raised the nictation rates of S. carpocapsae IJ. Significantly more S. carpocapsae IJ migrated to the host insect in the mixed inoculation with S. glaseri IJ on agar plates. More S. carpocapsae IJ penetrated into the host insect placed 2 cm below the surface in the mixed inoculation with S. glaseri IJ. More S. glaseri than S. carpocapsae IJ penetrated into hosts placed 7 cm deep. Irrespective of host location, the male ratio of S. carpocapsae IJ established in the host body was always higher in the mixed inoculation with S. glaseri IJ.     

Location of the Phasmids on Infective Juveniles of Steinernema glaseri

Scanning electron microscopy revealed the location of the phasmids on infective juveniles of Steinernema glaseri. The phasmids are located about 40% of the tail length posterior to the anus and are at or near the same level. Instead of being in the center of the lateral fields, they are located just ventral to the lateral fields, or interrupting the ventral-most lateral ridge. The phasmids were covered often by an exudate.     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

Effects of Paenibacillus nematophilus on the entomopathogenic nematode Heterorhabditis megidis

The insect parasitic nematodes Heterorhabditis spp. are mutualistically associated with entomopathogenic bacteria, Photorhabdus spp. A novel association has been detected between H. megidis isolate EU17 and the endospore-forming bacterium Paenibacillus nematophilus. P. nematophilus sporangia adhere to infective juveniles (IJs) of H. megidis and develop in insect hosts along with the nematodes and their symbiont. We tested the effects of P. nematophilus on H. megidis. The yield and quality (size, energy reserves, and storage survival) of IJs were not affected by co-culture in insects with P. nematophilus. Dispersal of IJs in sand and on agar was inhibited by adhering P. nematophilus sporangia: fewer than 2% of IJs with P. nematophilus sporangia reached the bottom of a sand column, compared to 30% of the control treatment. Sporangia significantly reduced infectivity of H. megidis for wax moth larvae in sand, but not in a close contact (filter paper) assay. The results suggest that P. nematophilus may reduce the transmission potential of H. megidis through impeding the motility of IJs.     

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.     

Role of symbiotic and non-symbiotic bacteria in carbon dioxide production from hosts infected with Steinernema riobrave

Entomopathogenic nematodes of the family Steinernematidae and their mutualistic bacteria (Xenorhabdus spp.) are lethal endoparasites of insects. We hypothesized that growth of the nematode’s mutualistic bacteria in the insect host may contribute to the production of cues used by the infective juveniles (IJs) in responding to potential hosts for infection. Specifically, we tested if patterns of bacterial growth could explain differences in CO₂ production over the course of host infection. Growth of Xenorhabdus cabanillasii isolated from Steinernema riobrave exhibited the characteristic exponential and stationary growth phases. Other non-nematode symbiotic bacteria were also found in infected hosts and exhibited similar growth patterns to X. cabanillasii. Galleria mellonella larvae infected with S. riobrave produced two distinct peaks of CO₂ occurring at 25.6–36 h and 105–161 h post-infection, whereas larvae injected with X. cabanillasii alone showed only one peak of CO₂, occurring at 22.8–36.2 h post-injection. Tenebrio molitor larvae infected with S. riobrave or injected with bacteria alone exhibited only one peak of CO₂ production, which occurred later during S. riobrave infection (41.4–64.4 h post-infection compared to 20.4–35.9 h post-injection). These results indicate a relationship between bacterial growth and the first peak of CO₂ in both host species, but not for the second peak exhibited in G. mellonella.     

Factors influencing parasitism of adult Japanese beetles,Polillia japonica (Col.: Scarabaeidae) by entomopathogenic nematodes

Several factors that influence the activity of steinernematid and heterorhabditid nematodes against adult Japanese beetles were examined in the laboratory. The effect of nematode concentration on mortality of adult beetles was evaluated using a Petri plate bioassay. The adults were exposed to 1,000 to 10,000 infective stage juveniles (J3) ofSteinernema glaseri per 10 beetles with or without food for 24 hr after which they were held with food for an additional 6 days. The LC50s for males with and without food during exposure were 3,435 and 2,854 J3s/10 adults, respectively. The LC50s for mixtures of males and females with and without food were 5,228 and 1,762 J3s/10 adults respectively. Although mortality occurred during and shortly after exposure, significant additional mortality was observed 1–4 days following exposure. Exposure of males and females with food to 10,000 J3s/10 adults for 6, 12, 18 or 24 hr resulted in 47, 58, 72 and 77% mortality, respectively. Comparative activity ofS. glaseri, S. carpocapsae (All strain),S. feltiae (Biosys experimental cold adapted strain=bibionis),S. feltiae (Biosys experimental strain 27),Heterorhabditis bacteriophora, andHeterorhabditis sp. (Terceiran isolate) was evaluated against adult Japanese beetles using a 24 hr exposure to 8,000 J3s/10 adults. The most virulent species wereS. glaseri, S. feltiae (=bibionis), the Terceiran isolate ofHeterorhabditis andS. carpocapsae producing 55, 44, 36 and 34% mortality respectively. Our results indicate that adult Japanese beetles infected with entomopathogenic nematodes could serve as a mechanism for nematode dispersal.     

Influence of Soil pH and Oxygen on Persistence of Steinernema spp.

Survival of infective juveniles of Steinernema carpocapsae and Steinernema glaseri gradually declined during 16 weeks of observation as the tested soil pH decreased from pH 8 to pH 4. Survival of both species of Steinernema dropped sharply after 1 week at pH 10. Survival or S. carpocapsae and S. glaseri was similar at pH 4, 6, and 8 during the first 4 weeks, but S. carpocapsae survival was significantly greater than S. glaseri at pH 10 through 16 weeks. Steinernema carpocapsae and S. glaseri that had been stored at pH 4, 6, and 8 for 16 weeks, and at pH 10 for 1 or more weeks were not infective to Galleria mellonella larvae. Steinernema carpocapsae survival was significantly greater than that of S. glaseri at oxygen:nitrogen ratios of 1:99, 5:95, and 10:90 during the first 2 weeks, and survival of both nematode species declined sharply to less than 20% after 4 weeks. Survival of both nematode species significantly decreased after 8 weeks as the tested oxygen concentrations decreased from 20 to 1%, and no nematode survival was recorded after 16 weeks. Steinernema carpocapsae pathogenicity was significantly greater than that of S. glaseri during the first 2 weeks. No nematode pathogenicity was recorded at oxygen concentrations of 1, 5, and 10% after 2 weeks and at 20% after 16 weeks.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

4种昆虫病原线虫对草地贪夜蛾的致死作用

【目的】探讨昆虫病原线虫对草地贪夜蛾幼虫的侵染效果。【方法】在室内条件下采用生测试验法,测定了小卷蛾斯氏线虫AⅡ、长尾斯氏线虫X-7、夜蛾斯氏线虫SN和嗜菌异小杆线虫H06等4种昆虫病原线虫对草地贪夜蛾2龄和5龄幼虫的致死作用。【结果】小卷蛾斯氏线虫AⅡ与草地贪夜蛾数量比为50∶1时,36 h后草地贪夜蛾2龄和5龄幼虫的死亡率分别为92%和100%。长尾斯氏线虫X-7与草地贪夜蛾数量比为50∶1时,36 h后草地贪夜蛾2龄幼虫的死亡率为80%;在数量比30∶1时,36 h后草地贪夜蛾5龄幼虫死亡率为100%。夜蛾斯氏线虫SN与草地贪夜蛾数量比为400∶1时,36 h后草地贪夜蛾2龄和5龄幼虫的死亡率分别为88%和85%。嗜菌异小杆线虫H06与草地贪夜蛾在400∶1时,36 h后草地贪夜蛾2龄和5龄幼虫的死亡率分别为32%和67.5%。【结论】小卷蛾斯氏线虫AⅡ具有较好的草地贪夜蛾生物防治潜质,其次是长尾斯氏线虫X-7。     

Effect of soil type on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora

We tested the effect of soil type on the performance of the entomopathogenic pathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Soil types used were loamy sand, sandy loam, loam, silt loam, clay loam, acidic sand, and a highly organic potting mix. Infectivity was tested by exposing third-instar Anomala orientalis or Popillia japonica to nematodes in laboratory and greenhouse experiments and determining nematode establishment in the larvae and larval mortality. Infectivity of H. bacteriophora and H. zealandica was the highest in potting mix, did not differ among loamy sand and the loams, and was the lowest in acidic sand. Infectivity of S. glaseri was significantly lower in acidic sand than in loamy sand in a laboratory experiment but not in a greenhouse experiment, and did not differ among the other soils. Infectivity of S. scarabaei was lower in silt loam and clay loam than in loamy sand in a greenhouse experiment but not in a laboratory experiment, but was the lowest in acidic sand and potting mix. Persistence was determined in laboratory experiments by baiting nematode-inoculated soil with Galleria mellonella larvae. Persistence of both Heterorhabditis spp. and S. glaseri was the shortest in potting mix and showed no clear differences among the other substrates. Persistence of S. scarabaei was high in all substrates and its recovery declined significantly over time only in clay loam. In conclusion, generalizations on nematode performance in different soil types have to be done carefully as the effect of soil parameters including soil texture, pH, and organic matter may vary with nematode species.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

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

Population growth kinetics of the nematode, Steinernema feltiae, in submerged monoxenic culture

Monoxenic cultures of the nematode, Steinernema feltiae, were carried out on two complex liquid media: P1, mainly soybean flour/egg yolk/yeast extract, and P2, mainly egg yolk/yeast extract. Up to 140 000–200 000 nematodes ml^–1 were produced within 7 days, and more than 95% of the final population was in the infective juvenile stage. The total nematode concentration growth curve had a sigmoidal shape. Nematode population growth kinetics were modelled using a re-parameterised Gompertz model. Yeast extract concentration appeared to be a key factor for obtaining high nematode concentrations.     

Interaction between Endemic and Introduced Entomopathogenic Nematodes in Conventional-Till and No-Till Corn

We used entomopathogenic nematodes as a model to address the issue of environmental impact of introduced biological control agents in the soil. The study was conducted during three field seasons (1997, 1998, and 1999) in no-till and conventional-till corn near Goldsboro, North Carolina. The main objective was to evaluate the interaction of two endemic nematodes, Steinernema carpocapsae and Heterorhabditis bacteriophora, and an introduced exotic nematode, Steinernema riobrave (Texas). Two baiting methods with Galleria mellonella were used to evaluate the nematodes with regard to infected insects and nematode persistence when alone or in cohabitation in the field. We also examined the effects of soil depth on the nematodes' interactions, infectivity, and persistence. The results of the two baiting methods generally agreed with each other. The detection of H. bacteriophora was significantly suppressed in the presence of S. riobrave and slightly more so in conventional-till than in no-till. However, this endemic nematode was not completely displaced 1 and 2 years after the introduction of S. riobrave. Detection of S. carpocapsae and S. riobrave was not affected by the presence of each other, and detection of S. riobrave was not affected by the presence of H. bacteriophora. H. bacteriophora had the strongest tendency to be detected deeper in the soil profile, followed by S. riobrave and then S. carpocapsae. The nematodes' differences in environmental tolerances, differences in tendencies to disperse deeper in the soil profile, and patchy distributions may help explain their coexistence.     

The compatibility of the entomopathogenic nematode,Steinernema feltiae, and chemical insecticidesfor the control of the South American leafminer, Liriomyza huidobrensis

The compatibility of infectivejuveniles (IJs) of entomopathogenic nematodes, Steinernema feltiae, and chemical insecticides tocontrol larval stages of the South American leafminer,Liriomyza huidobrensis, was investigated.Initially the effect of direct IJ exposure to 5insecticides (abamectin, deltamethrin, dimethoate,heptenophos and trichlorfon) for 24 hours was testedagainst Galleria mellonella in a standard sandtube bioassay. Trichlorfon and dimethoate did notreduce the nematodes ability to locate and infect G. mellonella larvae to an unacceptable level. However,nematode infectivity was significantly reducedfollowing exposure to abamectin, deltamethrin andheptenophos. Secondly, IJ infectivity for L.huidobrensis in the presence of dry pesticideresidues on foliage was tested. No significantdetrimental effects on the level of control of L. huidobrensis was recorded when compared with theeffect of nematodes applied to residue free foliage.The integration of these agents into a pest managementprogramme is discussed.