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。     

The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region

A survey of entomopathogenic nematodes in Lebanon was conducted for the first time during 2008-2009. Samples were collected on the coastal strip and in nine vegetation types extending from the coastal line to 3088 m above sea level. Wooded and herbaceous ecosystems were considered for sampling purposes. A total of 570 samples were taken, out of which 1% were positive for entomopathogenic nematodes. Approximately, 15.8% out of the 19 sites sampled revealed entomopathogenic nematodes presence (representing three samples). Two entomopathogenic nematodes species Heterorhabditis bacteriophora and Steinernema feltiae were recovered, and identification of their symbiotic bacteria revealed the presence of a Xenorhabdus bovienii, Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii.     

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。     

Nutritive significance of crystalline inclusion proteins of Photorhabdus luminescens in Steinernema nematodes

Phase I cells of Photorhabdus luminescens produce two types of intracellular crystalline inclusion proteins designated CipA and CipB. The genes encoding CipA and CipB proteins from P. luminescens H06 were expressed respectively in Escherichia coli and these cells were used to feed the axenic first juveniles (J1) of three Steinernema nematode isolates in liquid cultures and on agar plates. In liquid cultures, the axenic J1 juveniles of all three test Steinernema nematode isolates were able to produce next dauer juveniles (DJs) in the E. coli cultures with at least one of the expressed Cip proteins, but unable to develop beyond the next J1 stage without expressed Cip proteins. For each target nematode isolate, addition of the supernatant of the bacterial culture of its Xenorhabdus symbiont to the tested liquid cultures did not induce the formation of DJs. However, on LB agar plates with different test E. coli cultures, all J1 juveniles of the three Steinernema strains finally developed into next DJs. It seemed that the metabolite pathway of the test bacteria in both culture systems was different. The presence of the Cip proteins has a significant influence on the DJ formation of the Steinernema nematodes in liquid culture system.     

Insect Biocontrol with Non-endemic Entomopathogenic Nematodes (Steinernema and Heterorhabditis spp.): Conclusions and Recommendations of a Combined OECD and COST Workshop on Scientific and Regulatory Policy Issues

Fifteen invited experts from 10 Organisation for Economic Cooperation and Development (OECD) and European countries participating in the European Commission's Cooperation in the Field of Science and Technical Research (COST) Action 819, along with 12 other participants, met to review and debate the potential problems associated with the introduction and commercial use of non-indigenous nematodes for insect biological control. The consensus view of the participants was that entomopathogenic nematodes (EPNs) possess specific biological and ecological features, which make their use in biological control exceptionally safe. All the scientific evidence available supports the conclusion that EPNs are safe to the environment, as well as to production and application personnel, the general public and the consumers of agricultural products treated with them. Only a few potential, but very remote, risks could be identified. Therefore, it was recommended that EPNs should not be subject to any kind of registration. The introduction of non-indigenous nematode species, however, should be regulated. Species should be accurately identified, and details of the origin, known distribution, probable host range and safety to the user must be provided. In addition, an expert opinion, based on available information, of the possible impact on non-target organisms must be available.     

Photorhabdus luminescens LN2转座突变菌株的研究

发光杆菌属Photorhabdus细菌与异小杆属Heterorhabditis昆虫病原线虫的共生关系是这类生物杀虫剂产业化生产和田间应用的基础。本文采用Tn5转座方法构建了共生细菌P. luminescens LN2突变体库;从中筛选出一个对其共生线虫H. indica LN2的生长繁殖有显著促进作用的突变菌株（LN2-M2716）;测定了该突变菌株的菌落特征、对大蜡螟Galleria mellonella及非特异共生线虫H. bacteriophora H06的毒性、对线虫产量的影响。结果显示,LN2-M2716菌株在菌落形态、色素分泌、过氧化氢酶反应、荧光、食物信息作用以及对大蜡螟毒力等方面与野生型菌株差异不明显;但对非特异共生线虫H. bacteriophora H06的毒性及对特异共生线虫H. indica LN2生长繁殖的促进作用方面均明显高于野生型菌株。论文结果为构建支持线虫高产的菌株提供了关键技术。     

一种从小卷蛾斯氏线虫怀卵成虫制备感染期幼虫的简易方法

昆虫病原线虫是新型的生物杀虫剂,其感染期幼虫是昆虫病原线虫产业化生产和应用的唯一虫态,对昆虫病原线虫基因功能的研究及转基因改造有助于推进昆虫病原线虫的产业化。本研究基于昆虫病原线虫\"噬母现象\"的原理,以不同的孵育液孵育小卷蛾斯氏线虫Steinernema carpocapsae的怀卵成虫,找到可以简单快速从怀卵成虫直接获得整齐龄期的感染期幼虫的方法,为该线虫卵或性腺的RNA干扰后感染期幼虫的收集及生物测定提供基础,为昆虫病原线虫的转基因改造以提高其环境耐受力提供技术支持。     

ADVANCES IN THE RESEARCH OF ENTOMOPATHOGENIC NEMATODES STEINERNEMA AND HETERORHABDITIS IN CHINA

Abstract  The current status of Steinernema and Heterorhabditis entornopathogenic nematode research in China was reviewed in the collection and identification, the application in pest control programmes, symbiotic bacteria and mass production.     

高毒力昆虫病原线虫共生细菌菌株的筛选及其杀虫活性的检测

昆虫病原线虫共生细菌是寄生在昆虫病原线虫肠道的一种细菌,二者互惠共生。实验采用6个不同种的菌株为筛选材料。共生细菌菌株的培养液经85%饱和度的(NH₄)_2SO4盐析,浓缩冻干得到杀虫粗提物。以粗提物注射大蜡螟Galleria mellonella、饲喂玉米螟Ostrinia furnacalis和棉铃虫Helicoverpa armigera,发现Xenorhabdus nematophilus D43、X.bovienii A54、Photorhabdus luminescens HZL和CB-8等4个菌株发酵液的粗提物对昆虫有高的血腔毒性,菌株A54对昆虫又有高的胃毒效果。由此确立A54为高毒力的菌株,其杀虫活性表现为：注射大蜡螟48 h的死亡率为80%,96 h为93.3%;粗提物饲喂玉米螟,72 h死亡率为53.3%,120 h死亡率为100%;饲喂棉铃虫,72 h死亡率为80.1%,120 h死亡率为90%。杀虫粗提物经DEAE-52柱层析分离,得到一个穿透峰和三个盐的梯度洗脱峰,其中穿透峰对昆虫有很好的胃毒效果,但没有血腔毒性;三个盐峰均有很高的血腔毒性,但没有胃毒作用。穿透峰样品饲喂2龄、3龄棉铃虫也有很好的杀虫活性,96 h 2龄棉铃虫的死亡率为65%,3龄棉铃虫的死亡率为30%;处理96 h的棉铃虫同处理前相比体重下降,未死棉铃虫体重明显低于对照。     

Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize

The western corn rootworm (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) is an invasive maize (Zea mays L.) pest in Europe. Crop yield is significantly impacted by the feeding of all three larval instars on maize roots, making them prime targets for control measures. Therefore, the control efficacy of three entomopathogenic nematodes (EPNs), Steinernema feltiae (Filipjev), Heterorhabditis bacteriophora Poinar, and H. megidis Poinar, Jackson and Klein (Nematoda: Rhabditida), was studied in four field plot experiments in southern Hungary in 2005 and 2006. All EPN species significantly reduced D. v. virgifera independently, whether applied as a row spray with a solid stream into the soil at sowing or onto the soil along maize rows in June. When applied at maize sowing, H. bacteriophora was more effective at reducing D. v. virgifera (81%) than H. megidis (49%) and S. feltiae (36%). When applied in June, H. bacteriophora and H. megidis were more effective at reducing D. v. virgifera (around 70%) than S. feltiae (32%). All tested EPN species significantly reduced damage on maize roots independently, whether they were applied at sowing or in June. Damage, however, was not totally prevented. The use of H. bacteriophora for the development of a biological control product for inundative releases against D. v. virgifera larvae is suggested.     

中国昆虫病原斯氏和异小杆线虫的研究概况(英语)

昆虫病原斯氏和导小杆线虫作为生物防治因子已受到世界各国的广泛重视,并得到了商品化的发展.自50年代,特别是7O年代末,我国从原捷克斯洛伐克、澳大利亚、美国等地引进了大批昆虫病原斯氏和异小杆线虫.同时也从土壤或自然寄主中分离出许多本国线虫种或品系,并对此类线虫进行了全面的研究,在昆虫病原线虫的种类调查和鉴定、田间大面积应用、安全性、共生细菌、大量繁殖等方面取得了可喜的进展.本文将综述这些方面的研究概况.     

Dispersal Behaviour and Transmission Strategies of the Entomopathogenic Nematodes Heterorhabditis and Steinernema

Entomopathogenic nematodes of the Heterorhabditidae and Steinernematidae appear to be capable of long-distance dispersal and local migration. Their transmission strategies include both highly active seek-and-destroy behaviours and ambusher strategies, and they may be sensitive to sex-related factors in their own populations. Their host-finding abilities are poorly understood, despite the fact that these abilities are fundamental to their success as biocontrol agents in soil. Like the vast numbers of exotic hymenopterans and other natural enemies that have been released for biological control over the past century, they may be used in their ecologically competent wild-type form. On the other hand, because they are applied inundatively, they may be tailored, by breeding or transformation, to their intended purpose and to ecological incompetence, improving both their efficacy and their ecological safety.     

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.     

Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Field efficacy of entomopathogenic nematodes against the beetle Maladera matrida (Coleoptera: Scarabaeidae)

Single, double and triple releases of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, reduced the population of the beetle Maladera matrida Argaman, infesting peanuts (’Shulamit’ cv.) by 70, 75 and 93% respectively in microplot tests. Simultaneous and late (2 weeks after infestation) applications reduced beetle numbers by 63 and 79% respectively, in the microplots, while early application (2 weeks prior to infestation) did not reduce the beetle population. In a field trial, reductions in insect population and damage to the crop were achieved by early treatment with the nematode as well as by Heptachlor, leading to reductions in the insect population of 60 and 90% respectively, when recorded 4 weeks after nematode application. However, the nematode treatment did not maintain its effectiveness for a longer period and pest damage increased to the same level as the untreated control after 7 weeks. When the nematodes were applied at different concentrations (0.25–1.0 x 10⁶ infective juveniles (IJs) m^‐2) their effectiveness was not related to the concentration level. The only significant (P < 0.05) reduction in insect levels was recorded in the treatment with 0.5 X 10⁶ IJs m^‐2. In a second field trial, both H. bacteriophora and Steinernema glaseri reduced insect populations significantly (P < 0.05) by approximately 50% in comparison to the control. In the third trial, treatment with H. bacteriophora resulted in a decrease in insect population of 90% while treatment with S. carpocapsae reduced the grub numbers by 40% in comparison to the control. A differential susceptibility of various grub developmental stages was recorded in the field. The small grubs (I‐4 mm long, lst‐2nd larval stage) were not affected by the nematode treatments while the numbers of medium and large size grubs were reduced by 2‐ and 3‐fold respectively in the various tests. Nematodes were recovered by ‘nematode traps’ containing Galleria mellonella larvae from treated field plots 78 days after application. The implications of the results from the present studies on the use of entomopathogenic nematodes are discussed in relation to the development of an integrated pest management programme.     

Xenorhabdus和Photorhabdus代谢产物体外抗肿瘤活性

用8株昆虫病原线虫共生菌(Xenorhabdus属和Photorhabdus属)的发酵液进行了体外抗肿瘤活性的研究,从中筛选出活性较高的菌株一嗜线虫致病杆菌(Xenorhabdus nematophila CB6)。经有机溶剂萃取和硅胶柱层析对嗜线虫致病杆菌发酵液进行进一步分离,得到5个组分。用MTT法测定体外杀肿瘤细胞作用,发现其中2个组分具有抗肿瘤活性。组分3对HepG2、Hela、BGC-823、A-549、K562和HT-29的IC50分别等于3．1μg／ml、8．5μg／ml、2．1μg／ml、3．0μg／ml、4．4μg／ml和1．5μg／ml;组分5对HepG2、Hela、BGC-823、A-549、K562和HT-29的IC50分别等于0．1μg／ml、1．8μg／ml、0．1μg／ml、0．4μg／ml、1．2μg／ml和0．6μg／ml。同时发现抗肿瘤活性组分对正常人肺成纤维细胞MRC-5和正常人肝细胞L-02生长的影响很小。该研究在国内率先报道了嗜线虫致病杆菌发酵液具有较强抗肿瘤活性,为新药物的研制和开发提供了一条崭新的途径。