昆虫病原线虫的脱水休眠

昆虫病原线虫在长期储存方面的劣势影响其广泛应用。脱水休眠是昆虫病原线虫感染期幼虫在缺水条件下进入的一种生命暂停现象,是昆虫病原线虫对抗恶劣环境的一种策略。本文介绍了昆虫病原线虫脱水休眠的研究现状,对其诱导方法、生理生化和分子机理等方面的研究进展进行了综述。     

Comparative efficacy of indigenous heterorhabditid nematodes from north western Himalaya and Heterorhabditis indica (Poinar,Karunakar & David) against the larvae of Helicoverpa armigera (Hubner)

The efficacy of three Heterorhabditis spp of entomopathogenic nematodes (EPNs) from north western Himalaya, India was studied against the larvae of pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), under the laboratory conditions. The larvae were exposed to 10, 20, 30 and 40 infective juveniles (IJs) of each nematode species for different time periods and they were found to be susceptible to all the EPNs tested. However, the susceptibility of larvae to nematode infection varied according to the concentrations of IJs and their exposure periods. The efficacy of these indigenous entomopathogenic nematodes was also compared against commercially available entomopathogenic nematode, H. indica. Appreciably good performance was achieved by H. bacteriophora (HRJ), which showed 73.3% mortality of insect larvae in 96 h exposure time against third instar larvae, while H. indica produced 80.0% mortality. However it was noticed that with the advancement of larval stage its mortality rate reduced and vice versa with the exposure period. All tested EPNs were also found to reproduce within the host and maximum production of IJs was recorded in H. bacteriophora (26.0 ± 3.76 × 10³ IJs/larva) at the concentration of 40 IJs/larva.     

Development of a new application apparatus for entomopathogenic nematodes

This paper describes the assessment of nematode spray application methods and details the redesign of a spinning disc applicator for the improved application of the infective juveniles of entomopathogenic nematodes to foliage. The assessment highlighted fundamental design features that prevent efficient and accurate nematode application and these are dealt with in turn specifically to enable improved nematode application. The redesigned or prototype spinning disc was shown to improve nematode carriage in droplets, leading to greater deposition of nematodes per unit area. There is a trade-off between spray droplet coverage of the target surface and the production of a suitable droplet spectrum for nematode carriage.     

Progeny production of Steinernema abbasi in lepidopterous larvae

Progeny production of a new entomopathogenic nematode Steinernema abbasi in various lepidopterous prepupae was investigated. Most dauer juveniles (DJs) developed in the greater wax moth, Galleria mellonella (234 000) but the bollworms Helicoverpa virescens (220 000) and Spodoptera exigua (166 000) were also good hosts. Progeny production was independent of the number of nematodes invading the host between the doses of 40 and 230 DJs. The infectivity of DJs declined when they were stored for 3 months but at 30°C approximately 11% of the DJs were still infective. S. abbasi is a nematode of the semi-arid tropics and it would be possible to use the nematode to target pests in these areas if it can be produced satisfactorily locally.     

昆虫病原线虫的贮存和剂型研究进展

阐述了昆虫病原线虫的贮存原理,介绍业已建立的昆虫病原线虫贮存方法及其优缺点,影响昆虫病原线虫贮存效果的主要因素,并讨论了昆虫病原线虫剂型制备方法。     

格氏线虫表皮蛋白和分泌蛋白抑制昆虫免疫活性的比较

格氏线虫表皮蛋白和分泌蛋白都具有抑制昆虫免疫反应的能力,能够帮助侵染期线虫侵染寄主,但两者所含蛋白组分并不相同,从而导致蛋白活性也有所差异。本研究采用乙醇萃取和昆虫匀浆诱导,分别从侵染期的格氏线虫体表及分泌物中得到了表皮蛋白和分泌蛋白,并比较了这两者之间的异同。结果表明,无论是在昆虫体内还是体外,侵染期线虫表皮蛋白和分泌蛋白都具有抑制昆虫免疫反应的生物活性,并且分泌蛋白的活性更强。本研究为进一步研究线虫侵染与昆虫免疫间的相互关系和线虫抑制昆虫免疫反应的作用机理提供了科学佐证。     

Effect of rapid and gradual increase of osmotic stress on survival of entomopathogenic nematodes

The effect of rapid and gradual exposure of entomopathogenic nematodes to osmotic stresses on the induction of a dormant state was determined with the nematodeSteinernema feltiae IS-6 infective juveniles (IJs). Rapid exposure of nematodes to glycerol at concentrations of 24% and 28% (w/w) caused the nematodes to enter a dormant state which was characterized by shrinking and impeded motility of all nematodes within 8 h. However, pre-exposure to gradually increasing glycerol concentrations of 5%, 10% and 18% at 4-h intervals resulted in dormancy after 4 h exposure to 24% glycerol. The total time of exposure to glycerol solution was 16 h in gradual osmotic stress. For nematodes exposed to 24% glycerol solution either rapidly or gradually, recovery occurred after 40 min in distilled water. Infectivity of osmotically stressedS. feltiae IJs was evaluated by two criteria, insect mortality and invasion rate. The assays indicated that infectivity of nematodes desiccated by rapid and gradual osmotic stresses was similar to that of fresh nematodes. Rapid exposure ofS. carpocapsae ‘All’,S. riobravis ‘Texas’,S. glaseri ‘NI’ andHeterorhabditis bacteriophora HP88 IJs to the 24% glycerol solution resulted in dormancy within 8 h. These treatments caused mortality of 48.4% and 11.7% amongS. glaseri Nl andH. bacteriophora HP88 IJs, respectively. Similar effects were observed when these nematode species were exposed to increasing osmotic stress of 5%, 10% and 18% at 6-h intervals. Under these same conditions, mortality ofH. bacteriophora HP88 andS. glaseri Nl IJs was 27.5% and 61.8%, respectively. http://www.phytoparasitica.org posting Sept. 29, 2004.     

Genetic improvement of the desiccation tolerance and host-seeking ability of the entomopathogenic nematode <Emphasis Type="Italic">Steinernema feltiae</Emphasis>

Entomopathogenic nematodes (EPNs) from the genus Steinernema (Steinernematidae) are used for biological control of insect pests. The infective stages of these nematodes are intolerant of extreme environmental conditions. Genetic improvement has been suggested as an approach for improving their ability to overcome these limitations. In this study, we bred a heterogeneous population of the EPN Steinernema feltiae Filipjev for desiccation tolerance (both rapid and slow) and enhanced host-seeking ability. We selected for tolerance of rapid desiccation by exposing infective juveniles (IJs) to ambient conditions (22–25°C; 50–65% r.h.) for 100 min. A survival rate of 80–90% was reached after ten selection cycles. To select for tolerance of slow desiccation, we exposed IJs to 97% r.h. for 72 h, followed by further exposure to 85% r.h. for an additional 72 h. A high survival rate (>85%) was obtained after 20 selection cycles. We selected for enhanced downward dispersal by forcing IJs to move through a sand column to reach larvae of last-instar Galleria mellonella placed at the bottom of the column. After 25 selection cycles, the majority (>75%) of these nematodes were found at the layer close to the insects. No reduction in fitness was detected in the selected populations. Nevertheless, the nematode population selected for enhanced downward dispersal displayed significantly higher infectivity than the foundation population. The population selected for slow desiccation was more tolerant of heat stress than the foundation population. These findings establish the basis for improvement of this nematode for use as a biological control agent under field conditions.     

Improved attachment and parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> in vitro

Monoclonal and polyclonal antibodies that bind to eggs and/or second-stage juveniles of the nematode Meloidogyne javanica were tested for their effects on the parasitic interactions between this nematode and the fungus Trichoderma. Parasitism of Trichoderma asperellum-203 and Trichoderma atroviride on nematode egg masses, eggs and juveniles was enhanced when antibodies were incorporated into in vitro parasitism bioassays. Parasitism on separated eggs (without gelatinous matrix) and their hatched juveniles was also improved, compared to controls without antibodies that did not attach fungal conidia. Improved parasitism could be due to bilateral binding of the antibodies to the nematodes and conidia, enabling better conidial attachment to the nematodes. Enhanced germination of antibody-bound conidia further improved parasitism. Differences were observed among antibodies in their effects on fungal parasitism and their interaction with Trichoderma species. We focused mainly on the egg- and juvenile-binding monoclonal antibody MISC that exhibited a stronger reaction with T. asperellum-203 than with T. atroviride. Pretreatment of this antibody with fucose inhibited its binding to nematodes and conidial attachment to nematodes, as well as conidial agglutination in the presence of the antibody. Antibody binding to juveniles affected their movement and viability, especially gelatinous matrix-originated juveniles. The fucose-specific lectin Ulex europaeus-I enhanced conidial attachment to nematode life-stages, and conidial agglutination occurred in its presence. These phenomena were inhibited by preincubating lectin with fucose. Our results suggest that carbohydrate residues, such as fucose, on the surface of the nematode and fungal conidia are involved in the antibody- and lectin-mediated improved parasitism.     

一种对桃小食心虫高毒力的斯氏线虫Steinernema sp.(CB-2Y)

本研究在室内用“沙埋法”比较了两种斯氏属昆虫病原线虫Steinernema sp.(CB-2Y)和S.feltiae Agriotos对桃小食心虫的侵染力,结果表明CB-2Y线虫对桃小食心虫幼虫和冬茧的侵染力都高于Agriotos线虫。接线虫笫7天桃小食心虫LD50,CB-2Y组为49条线虫,而Agriotos组为158条;冬茧的LD50分别为1330条和24046条。在桃小冬茧开始活动期(6月15日)分别施用两种线虫,一个月后检查桃小食心虫成虫羽化率,CB-2Y线虫的控制效果明显优于Agriotos,在1024条线虫/桃小食心虫冬茧时(75条/cm~2),越冬代桃小食心虫的羽化率分别为8.2％和29.2％。     

Potential of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae) for the control of codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in semi-field trials under South African conditions

Infection of codling moth, Cydia pomonella (L.), larvae by the entomopathogenic nematode, Heterorhabditis zealandica, was studied under semi-field conditions. Late-instar diapausing codling moth larvae, coccooned in perforated cardboard strips, were used in all trials to evaluate the nematode treatments involved. A morning application of 0.5, 1.0 and 1.5 MM infective juveniles (IJs) per tree resulted in 85%, 95% and 100% codling moth larval infection, respectively. Contrasting results were obtained for an evening application, with low levels of codling moth infection (<50%). Nematode treatment strips placed in direct sunlight resulted in almost no infection (<10%), opposed to strips attaining 67% infection placed in the shade.     

Effect of the spray application technique on the deposition of entomopathogenic nematodes in vegetables

BACKGROUND: The present study compared entomopathogenic nematode delivery at the base of savoy cabbage and cauliflower, at the lower side of savoy cabbage and cauliflower leaves and in leek stems and the ground deposition using a five‐nozzle spray boom equipped with an ISO 08 flat fan, an air induction flat fan and Twinjet spray nozzles. Additionally, an air support system and a row application system were evaluated. RESULTS: Approximately 40% of the applied nematodes did not reach the foot of the cabbage plants. The use of an air support system or a row application system improved nematode deposition at the savoy cabbage base. Relative nematode deposition on the lower side of savoy cabbage leaves was 27.20%, while only 2.64% of the applied nematodes reached the lower side of cauliflower leaves. After spraying leek with a standard boom, a low relative nematode deposition (26.64%) was measured in the leek stem. Nozzle type affected the distribution of nematodes in droplet spots. CONCLUSION: Nozzle type has a minor effect on the number of entomopathogenic nematodes delivered on difficult‐to‐reach targets. The use of modified spray application techniques directing the spray to the target site are necessary to increase the chances of contact of entomopathogenic nematodes with their target. Copyright © 2011 Society of Chemical Industry     

Evaluation of the entomopathogenic nematode Steinernema feltiae as a biological control agent of the whitefly,Bemisia tabaci

We evaluated the potential for using infective juveniles of the entomopathogenic nematode Steinernema feltiae to control the sweetpotato whitefly Bemisia tabaci B biotype on cucumber, hibiscus, and collard, under greenhouse conditions. The effects of four adjuvants (PVA, glycerol, Triton X-100, and horticultural spraying oil) on the efficiency of Steinernema feltiae in B. tabaci control were also assessed because they might increase nematode survival by reducing desiccation. The mortality of B. tabaci nymphs increased by 18, 18, and 20% on collard, hibiscus, and cucumber plants when S. feltiae concentration increased from 5000 to 15,000 infective juveniles/mL. When treated with S. feltiae (10000 infective juveniles/mL), second instar nymphs suffered greater mortality than the other instars of B. tabaci, regardless of host plant species. Mortality of B. tabaci nymphs was higher on hibiscus than on collard and cucumber plants. The use of adjuvants greatly increased infection of B. tabaci by S. feltiae. The combination of S. feltiae (10,000 infective juveniles/mL) and 1% horticultural spraying oil increased the mortality of second instar B. tabaci nymphs to 86% on collard and 90% on hibiscus, while the combination of S. feltiae and 0.1% Triton X-100 caused 83% mortality on collard and 89% on hibiscus. The potential for incorporating S. feltiae and the adjuvants in B. tabaci management is discussed.     

Association between entomopathogenic nematodes and non-synthetic insecticides for improved control of Nasutitermes spp. (Isoptera: Termitidae) in sugarcane plantations

The aim of this study was to evaluate the in vitro efficacy of the combination of entomopathogenic nematodes and non-synthetic insecticides in the control of Nasutitermes spp. (Isoptera: Termitidae). Bioassays were performed upon insect collection from areas with the high incidence of this pest. The experimental arena for insecticide assays consisted of 80-mL plastic containers with a screened cover. We tested six insecticides and the nematode species Heterorhabditidis bacteriophora RS58, Steinernema glaseri RS38, and Heterorhabditis sp. isolates AL39, AL40, AL41, AL42, AL43, AL44, AL46, and AL47, all of which were grown in last-instar larvae of Galleria mellonella L. (Lepidoptera: Pyralidae). The insecticides Codipirol®, Pure Neem Oil®, and Pyroligneous Acid Extract® caused high mortality in adults of Nasutitermes. The nematode Heterorhabditis sp. AL40 presented a median lethal concentration of 7976 infectious juveniles per adult of Nasutitermes sp. In addition, the mixture of these three non-synthetic products at a concentration of 3% caused complete mortality in Heterorhabditis sp. AL40 and S. glaseri RS38. Future trials should be encouraged in order to evaluate the field efficiency of non-synthetic insecticides mixed or not with entomopathogenic nematodes.     

Designing the ideal habitat for entomopathogen use in nursery production

BACKGROUND: Greenhouse and nursery producers use entomopathogens (nematodes and fungi) to control soil pests. Although it is known that the physical and chemical properties of mineral soil significantly impact upon soil pathogens, the influence of soilless media used for plant production on entomopathogen performance is poorly understood. RESULTS: Survival and foraging distance were differently affected by sand:peat, bark and sawdust media for entomopathogenic nematodes, but not for the immobile fungus Metarhizium anisopliae. Redwood sawdust medium consistently had a negative impact upon entomopathogenic nematodes. Dividing media into individual components supported the hypothesis that redwood sawdust reduced foraging and infection abilities of S. riobrave and H. bacteriophora. Physically altering the components by adding sand significantly improved foraging and infection success for S. riobrave in media not optimum for foraging. CONCLUSION: This study is the first to highlight the importance of selecting the appropriate soilless media and pathogen species combinations to increase efficacy of biological control. H. bacteriophora was able to find hosts in a wider diversity of medium components than S. riobrave, although both nematode species performed well in peat moss and recycled plant material. These results suggest that peat moss, recycled plant material and hardwood bark are components amenable to EPN biological control programs. Copyright © 2012 Society of Chemical Industry     

Steinernematid and heterorhabditid nematodes for biological control of the giant looper,Boarmia selenaria

The pathogenicity of steinernematid and heterorhabditid nematodes to caterpillars of the giant looper,Boarmia selenaria (Schiffermüller) (Lepidoptera: Geometridae), was determined under laboratory conditions. High mortality (over 80%) was achieved, in a petri dish bioassay, with a minimum of 20,000 infective juveniles (IJ) ofSteinernema carpocapsae (Filipjev) (=Neoaplectana carpocapsae Weiser), strain ‘All’. The LD₅₀ was 4250 IJ per caterpillar. Similar results were obtained withS. carpocapsae, strain ‘Mexican’,Steinernema sp., strain ‘CR’;Heterorhabditis bacteriophora, strain ‘HP88’; andHeterorhabditis sp., strain ‘IS’. The first and second instars were the most susceptible stages to nematode infection. A laboratory model assay indicated that 80–90% of the caterpillars were killed when they migrated to the soil to pupate; this occurred when IJ were applied to sandy and heavy soils at a concentration of 400 IJ/cm³ soil. A greenhouse test reconfirmed the effectiveness of utilizing the entomopathogenic nematodes in an “ambush” system against caterpillars migrating to the soil.     

The influence of herbicides on the viability of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPNs) are agents that can be used for the biological control of pests associated with pesticides in a tank mix. Compatibility studies need to be conducted to analyse which products are compatible with EPNs. The survival of infective juveniles (IJs) of four species of EPNs (Steinernema, Heterorhabditis) was determined after exposure to eight chemical herbicides. The effect of direct IJs exposure to herbicides for 1, 4 and 24 h was tested in a Petri dish at 15, 20 and 25 °C. The study showed that Steinernema kraussei was the most tolerant among the tested EPN species, while S. carpocapsae was the most sensitive to all tested herbicides. The lowest mortality of IJs was at 15 °C (19%). Our investigation showed, overall, the herbicides negatively affected EPN survival. The results confirmed that the compatibility is a species-specific characteristic, influenced by the temperature and time of exposure. Application of two different control ingredients (insecticide and herbicide) at the same time would reduce cost and time consumption in pest/weed control.     

70%增效型吡虫啉水分散粒剂的制备及其性能评价

采用滚动式挤压造粒法,通过配方筛选成功制备了70%增效型吡虫啉水分散粒剂(WG),并考察了增效剂胡椒基丁醚(PB)对制剂的热贮稳定性、表面张力及杀虫活性等性能的影响。优化配方(以质量分数计)为:吡虫啉原药(71.9%),增效剂PB(1.0%),分散剂MORWET-EFW(2.5%)、MORWET D-425或Tesperse 2700(5.0%),崩解剂硫酸铵(3.0%),粘结剂聚乙二醇(2.0%)和载体[m(白炭黑):m(高岭土)=1:4] 14.6%。所制备的70%增效型吡虫啉WG热贮(54℃±2 ℃)14 d后有效成分降解率为1.27%(≤1.5%);其表面张力无明显改变;润湿分散性良好,悬浮率≥90%。对蚜虫的室内毒力测定结果显示,含有PB的70%吡虫啉WG的毒力是不含PB成品的6～7倍,表明PB具有极显著的增效作用。     

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

Nematicidal activity of the leaf powder and extracts of Myrtus communis against the root‐knot nematode Meloidogyne javanica

Nematicidal activity of the leaf powder, leaf extracts and formulated leaf extracts of Myrtus communis, an evergreen shrub that is widely distributed in Israel and other Mediterranean countries, was evaluated using the root‐knot nematode Meloidogyne javanica in in vitro and pot experiments. Leaf powder added to sand at 0·1% (w/w) reduced the number of juveniles recovered from the sand by more than 50%. Reduction in galling index and number of nematode eggs on tomato roots was also observed by incorporating the leaf powder at 0·1–0·4% (w/w) in the soil in pot experiments. Leaf powder extracts with methanol or ethanol showed the highest nematicidal activity among all extracts tested. Emulsifiable concentrates of leaf‐paste extract at a concentration as low as 0·005% (a.i., w/w) reduced the number of juveniles recovered from treated sand and the gall index of cucumber seedlings. The extract paste at 26 g m^?2 was also effective in reducing the gall index of tomato plants in field‐plot experiments. The leaf powder at 0·2% and the formulated leaf‐paste extract at 0·02% were also nematicidal to Tylenchulus semipenetrans and Ditylenchus dipsaci, but not to Pratylenchus penetrans or Steinernema feltiae. At least three nematicidal compounds were found in the leaf extract upon fractionation by thin‐layer chromatography. The results suggest that the leaf powder and paste extract of M. communis are potential nematicides against root‐knot nematodes.