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。     

昆虫病原线虫(斯氏属与异小杆属)应用概况

<正> 在自然界,线虫与昆虫的联系是普遍的。据目前所知,至少有27个科的线虫与16个目近3000种昆虫有联系(Nickole 1984,Poinar 1983),其分布是由寄主昆虫的栖境所决定的。这些线虫对害虫的种群起着自然调节的作用。线虫与昆虫联系的方式各异,使人最感兴趣的是那些能作为害虫生物防治的病原线虫。自从Oldhan(1933)首次提出利用线虫防治害虫以来,应用病原线虫进行害虫生物防治的工作已     

我国关于昆虫病原线虫分类的研究进展

我国关于昆虫病原线虫分类的研究进展图立红（首都师范大学生物学系１０００３７）昆虫病原线虫是目前国内外发展较快的一类生物杀虫剂,随着昆虫病原线虫学科的发展,对其资源的调查、开发和利用越来越受到重视。昆虫病原线虫,寄主范围广,寄生杀虫效果好,致死速度快,...     

昆虫病原线虫共生细菌致病机理的研究进展

昆虫病原线虫共生细菌是寄生于昆虫病原线虫肠道内的一种细菌,革兰氏染色呈阴性,属肠杆菌科(Ｅｎｔｅｒｏｂａｃｔｅｒｉａｃｅａｅ)细菌[1]。它包含两个属———嗜线虫致病杆菌属(Ｘｅｎｏｒｈａｂｄｕｓ)和发光杆菌属(Ｐｈｏｔｏｒｈａｂｄｕｓ),它们分别与斯氏线虫(Ｓｔｅｉｎｅｒｎｅｍａ)和异小杆线虫(Ｈｅｔｅｒｏｒｈａｂｄｉｔｉｓ)共生。这两种线虫由于杀虫能力强,是最有应用潜力的昆虫病原线虫。它们之间的共生关系可以概括为:共生菌存在于线虫的肠道内,线虫携带共生菌进入寄主昆虫体内,并将共生菌释放到昆虫的血腔中;共生菌在昆…     

昆虫病原线虫斯氏线虫科分类研究的进展

昆虫病原线虫斯氏线虫科分类研究的进展许再福，玉国汉（茂名林业科学研究所茂名市５２５０００）（华南农业大学植保系）关键词斯氏线虫科，斯氏线虫属，分类斯氏线虫科Ｓｔｅｉｎｅｒｎｅｍａｔｉｄａｅ（异名：新线虫科Ｎｅｏａｐｌｅｃｔｉｎｉｄａｅ），属小杆总科Ｒ...     

昆虫病原线虫与环境生物、非生物因素关系的研究进展

昆虫病原线虫(Entomopathogenic nematodes)是业已商业化的昆虫寄生性天敌,对农林和卫生等重要害虫具有安全和有效的控制作用.这类线虫与环境生物和非生物因素存在密切的联系.影响昆虫病原线虫的环境生物因素包括同类线虫、共生细菌、寄主昆虫、寄生真菌以及其它昆虫病原物等;影响昆虫病原线虫的环境非生物因素主...     

昆虫病原线虫共生菌的分类

昆虫病原线虫共生菌是存在于昆虫病原线虫肠道内的一类细菌 ,属肠杆菌科 (Enterobacteriaceae) ,兼性厌氧 ,包括致病杆菌属 (Xenorhabdus) [1] 和光杆状菌属 (Photorhabdus) [2 ] ,它们分别与斯氏线虫属 (Stein ernema)和异小杆线虫属 (Heterorhabditis)的线虫共生[3] 。在自然界 ,共生菌存在于 3龄侵染期线虫的肠道中[4 ] ,不能从土壤中分离获得 ,是昆虫病原线虫的主要致病因子。昆虫病原线虫共生菌直到 2 0世纪 5 0年代末期才被发现 ,其分类地位也经常发生变动 ,鉴定工作尽管几十年来陆陆续续时有报道 ,但并不十分深入和系统。近年来 ,…     

昆虫病原线虫的共生细菌

昆虫病原线虫与其共生细菌二者互惠共生 :共生细菌需要昆虫病原线虫作为载体以寄生寄主昆虫并做为自己的营养来源 ,而昆虫病原线则需要依靠共生细菌来杀死昆虫。综述了共生细菌的病原作用、抗菌作用与杀虫作用 ,评述了共生细菌的基因工程进展 ,讨论了昆虫共生细菌在昆虫病原线虫致病性的作用。     

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%。     

昆虫病原线虫研究概况

昆虫病原线虫 (ｅｎｔｏｍｏｐａｔｈｏｇｅｎｉｃｎｅｍａｔｏｄｅｓ)是本世纪发展起来的一种有潜能的生物防治因子[1] 。它具有寄主范围广、能主动寻找寄主、对人畜及环境安全无毒 ,并能人工大量培养等优点。因此 ,在农药污染日益严重、害虫抗药性发展迅速的今天 ,昆虫病原线虫已成为可持续发展农业的迫切需要 ,受到国际生防领域的重视。特别是斯氏科线虫和小杆科线虫 ,已成为当前国际生防领域研究热点之一。本文简要概述了线虫的生物学特性、分类及致病机理等方面的研究 ,并结合目前存在的问题探讨了昆虫病原线虫的研究方向和发展前景。1…     

Effects of Density, Age and Host Cues on the Dispersal of Heterorhabditis megidis

Agar plate assays were used to assess the effect of density, incubation time and age of nematodes and the presence of insect hosts on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis (strain NLH-E87.3). IJs dispersed faster and further at high densities than at low densities. Dispersal was also influenced by the age of the IJs. Individuals stored for a period of 1.5 and 4.5 weeks showed to be more active than those stored for 2.5 and 3.5 weeks. The presence of a host insect enhanced the dispersion of nematodes. The increasing in the incubation period showed that IJs responded positively to host cues from Galleria mellonella but poorly to cues from Otiorhynchus sulcatus larvae.     

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.     

Influences of host size and host species on theinfectivity and development of Heterorhabditismegidis (strain NLH-E87.3)

The infectivity, time to first emergence of infective juveniles (IJs), total number of IJs per insect and IJs body length of the entomopathogenic nematode Heterorhabditis megidis (strain NLH-E87.3) after development in larvae of two insect hosts, Galleria mellonella (greater wax moth) and Otiorhynchus sulcatus (vine weevil) was studied. At a dose of 30 IJs, larvae of G. mellonella show to be significantly more susceptible than O. sulcatus larvae. At a dose of one IJ, vine weevil larvae were more susceptible. The number of invading infective juveniles (IJs) increased with host size while the host mortality at a dose of one IJ decreased with the increase of host size. Time to first emergence was longer at a dose of one IJ per larva and increased with the increase of host size in both insect species. Reproduction of IJs differed between host species, host sizes and doses of nematodes. Generally, the IJs body size increased with an increasing host size. The longest infective juveniles were produced at the lowest IJ doses. Results are discussed in relation to the influence of different host species and their different sizes on the performance of H. megidis (strain NLH-E87.3) as a biological control agent.     

Application of the most probable number method to estimations of entomopathogenic nematode populations in soil

The calculation of most probable numbers (mpn) was used for the estimation of numbers of infective entomopathogenic nematodes in soil. The mpn concept was first introduced in bacteriology as a means of estimating numbers of organisms in a substrate without a direct count, in cases where direct enumeration could not be applied. In the work reported here, soil samples infested with infective juveniles (IJs) of Heterorhabditis megidis (isolate HF85) were diluted with uninfested soil and the diluted soils were baited with mealworm larvae (Tenebrio molitor). The mpn of infective units of IJs in the undiluted soil was calculated. The mpn calculation was found to be applicable to entomopathogenic nematodes in soil under particular conditions. It could be successfully applied to data for the response of soil units but not to the data based on the response of individual insects, as the latter did not confirm to a Poisson series. The calculated mpn represented between 2.9 and 7.1% of the initial inoculum of IJs. It was suggested that IJs might act as a group in infecting an insect host. Using the data for Tenebrio mortality on parasitisation, the mpn based on quantal response of the mealworms would therefore not give the true density of IJs in the soil sample but the effective density, or the quantity of infective units. Although the biological significance of the infective unit needs further clarification, mpn was found to be a useful parameter for use in comparative experiments.     

A survival-reproduction trade-off in entomopathogenic nematodes mediated by their bacterial symbionts

In this work, we investigate the investment of entomopathogenic Steinernema nematodes (Rhabditidae) in their symbiotic association with Xenorhabdus bacteria (Enterobacteriaceae). Their life cycle comprises two phases: (1) a free stage in the soil, where infective juveniles (IJs) of the nematode carry bacteria in a digestive vesicle and search for insect hosts, and (2) a parasitic stage into the insect where bacterial multiplication, nematode reproduction, and production of new IJs occur. Previous studies clearly showed benefits to the association for the nematode during the parasitic stage, but preliminary data suggest the existence of costs to the association for the nematode in free stage. IJs deprived from their bacteria indeed survive longer than symbiotic ones. Here we show that those bacteria-linked costs and benefits lead to a trade-off between fitness traits of the symbiotic nematodes. Indeed IJs mortality positively correlates with their parasitic success in the insect host for symbiotic IJs and not for aposymbiotic ones. Moreover mortality and parasitic success both positively correlate with the number of bacteria carried per IJ, indicating that the trade-off is induced by symbiosis. Finally, the trade-off intensity depends on parental effects and, more generally, is greater under restrictive environmental conditions.     

Comparison of Bioassays to Measure Virulence of Different Entomopathogenic Nematodes

Five bioassays were compared for their usefulness to determine the virulence of four nematode strains. The objective of this study was to develop standard assays for particular nematode species. In all assays, the nematodes Steinernema feltiae (strain UK), S. riobravis, S. scapterisci Argentina and Heterorhabditis bacteriophora HP88 were exposed to Galleria mellonella larvae. All bioassays except the sand column assay were conducted in multi-well plastic dishes. In the penetration rate assay, the number of individual nematodes invading the insect was determined after a 48-h exposure to 200 infective juveniles (IJs). In the one-on-one assay, each larva was exposed to an individual nematode for 72 h before insect mortality was recorded. In the exposure time assay, insect mortality was recorded after exposure to 200 IJs for variable time periods. The dose-response assay involved exposing larvae to different nematode concentrations over the range 1-200 IJs/insect and recording mortality every 24 h for a 96-h period. In the sand columns assay, insects were placed in the bottom of a plastic cylinder filled with sand. Nematodes were applied on top of the sand and insect mortality was determined after IJs had migrated through the cylinder. The highest mortality level in the sand column assay was obtained with IJs of S. feltiae followed by H. bacteriophora; treatments with S. riobravis and S. scapterisci produced low levels of insect mortality. In the other four assays, S riobravis was the most virulent, followed by S. feltiae, H. bacteriophora and S. scapterisci. In the exposure time assay, rapid mortality was achieved when the insects were exposed to S. feltiae and S. riobravis. For these nematode species, a gradual increase in the number of individuals which penetrated into cadavers was recorded. Conversely, the number of nematodes in the cadavers of insects infected by H. bacteriophora and S. scapterisci remained low during the entire exposure period. In this assay, exposing the insects to these nematodes resulted in a gradual increase in mortality. In the dose-response assay, complete separation among nematode species was obtained only after 48 h of incubation at a concentration of 15 IJs/insect. LD and LD values were calculated from 50 90 dose-response assay data. However, these values did not indicate differences among the different nematode species. The present study demonstrated the variation in entomopathogenic nematode performance in different bioassays and supports the notion that one common bioassay cannot be used as a universal measure of virulence for all species and strains because nematodes differ in their behavior. Furthermore, particular assays should be used for different purposes. To select a specific population for use against a particular insect, assays that are more laborious but which simulate natural environmental conditions (e.g. the sand column assay) or invasion by the nematode (e.g. the penetration rate assay) should be considered. In cases where commercial production batches of the same nematode strains are compared, simple and fast assays are needed (e.g. the one-on-one and exposure time assays). Further studies are needed to determine the relationships between data obtained in each assay and nematode efficacy in the field.     

Orientation of Heterorhabditis megidis to insect hosts and plant roots in a Y-tube sand olfactometer

The host-searching behaviour of Heterorhabditis megidis strain NLH-E 87.3 in the presence of insect hosts and plant roots, offered individually and in combination, was studied using a newly developed Y-tube olfactometer filled with sand. Within a period of 24 hours infective juveniles (IJs) were significantly attracted to living G. mellonella larvae and caused 100% larval mortality. Otiorhynchus sulcatus larvae, however, did not elicit host-oriented movement of IJs and no larval mortality was observed. Roots of strawberry plants induced a negative response in IJs. The combination of strawberry roots and O. sulcatus larvae, however, strongly attracted IJs leading to 37% host mortality. It was shown that this type of Y-tube choice arena is a useful tool in studying the searching behaviour of entomopathogenic nematodes in a semi-natural habitat.     

Differences between the pathogenic processes induced by Steinernema and Heterorhabditis (Nemata: Rhabditida) in Pseudaletia unipuncta (Insecta: Lepidoptera)

Larvae of Pseudaletia unipuncta are moderately susceptible to infections caused by entomopathogenic nematodes, being a desirable host to study pathogenic processes caused by Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri and their associated bacteria. The ability of the infective stage of these nematodes to invade hosts is quite different. S. carpocapsae invades the highest number of insects and presents the highest penetration rate, followed by H. bacteriophora. Regression analysis between the number of insects parasitized and the number of IJs counted per insect, over time, showed a high correlation for S. carpocapsae whereas for H. bacteriophora it was low. Dose-response was most evident at a concentration below 100 IJs per insect on H. bacteriophora, whereas on S. carpocapsae it was found for doses ranging from 100 to 2,000 IJs. Student's t test analysis of dose-response showed parallel, yet unequal, slopes for both strains of H. bacteriophora, whereas distinct regressions were obtained for S. carpocapsae and S. glaseri, thus, evidencing each species develop a distinct pathogenic process. Insects injected with Photorhabdus luminescens died within 50 h after injection, whereas those treated with X. nematophila died much later. Moreover, the mortality in insects exposed to H. bacteriophora complex and injected with P. luminescens was close, but insects injected with bacteria died faster. Insect mortality in treatments with complexes S. carpocapsae and S. glaseri was significantly higher than that which was observed in insects injected with symbiotic bacteria.