携带cip基因的酿酒酵母对自由生活线虫Panagrellus redivivus生长繁殖的影响

Photorhabdus luminescens细菌与昆虫病原异小杆属Heterorhabditis线虫专性共生。初生型共生细菌产生两种胞内晶体蛋白CipA and CipB,为共生线虫提供营养。为探索Cip蛋白是否对自由生活的全齿复活线虫Panagrellus redivivus具有类似的营养功能,建立了Cip蛋白的重组酿酒酵母表达体系,并用于饲喂无菌的P. redivivus线虫J1幼虫。重组酿酒酵母表达的Cip蛋白能为线虫所利用,表现为营养支持作用,体现为线虫生长发育速度的加快以及繁殖能力的提高,说明Cip蛋白能为此种自由生活线虫提供营养来源。     

嗜菌异小杆线虫H06品系对红棕象甲致病力的时间效应及对血淋巴的影响

【目的】阐明嗜菌异小杆线虫H06品系处理对红棕象甲血淋巴免疫系统的影响。【方法】采用室内生物测定和生理生化测定法,研究了嗜菌异小杆线虫H06品系对红棕象甲不同虫态的致病力及对血淋巴各项指标的调节作用。【结果】嗜菌异小杆线虫H06品系对红棕象甲不同虫态的致病力与处理浓度和处理时间均呈正相关性,经嗜菌异小杆线虫H06品系处理后,红棕象甲血淋巴总量和血淋巴酯酶均呈前期升高、后期降低的趋势,处理后72 h的血淋巴总量和酯酶活性与对照相比分别降低39.73%和53.36%;血淋巴蛋白含量与对照相比呈下降的趋势,虽在60 h时有一个短暂的回升,之后又迅速下降,其中在处理后72 h,血淋巴的蛋白含量与对照相比下降74.96%。【结论】嗜菌异小杆线虫H06品系的侵入破坏了红棕象的血淋巴免疫系统,本研究结果可为揭示昆虫病原线虫对红棕象甲的致病机理提供依据。     

噻虫嗪对昆虫病原线虫侵染韭菜迟眼蕈蚊能力的影响

【目的】为提高昆虫病原线虫对韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang幼虫的防治效果,将昆虫病原线虫与环境友好型化学杀虫剂混用是一条有效途径。【方法】测定了噻虫嗪与6个昆虫病原线虫品系混用对韭菜迟眼蕈蚊的作用效果,以及温度和土壤含水量对作用效果的影响,并进行了田间验证。【结果】田间推荐浓度噻虫嗪(100 mg·L~(-1))对6种供试线虫存活无显著影响。处理后3 d,低浓度噻虫嗪(15 mg·L~(-1))分别与6品系线虫混合后处理韭菜迟眼蕈蚊幼虫,其死亡率明显高于线虫和噻虫嗪单用处理。小卷蛾斯氏线虫SF-SN+噻虫嗪、印度异小杆线虫LN2+噻虫嗪和小卷蛾斯氏线虫All+噻虫嗪3种组合发挥杀虫作用的最适温度范围分别为20~25℃、25~30℃和25~30℃,显著高于其他温度;最适土壤含水量范围为10%~18%,也显著高于其他湿度。大田条件下,施用后7 d,单用噻虫嗪、线虫+噻虫嗪组合处理对韭菜迟眼蕈蚊的防治效果显著高于单线虫,且以芫菁夜蛾斯氏线虫SF-SN+噻虫嗪的防治效果最高,达到93%以上。【结论】芫菁夜蛾斯氏线虫SF-SN品系与噻虫嗪组合联合防治韭菜迟眼蕈蚊效果最好。     

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。     

昆虫病原线虫共生细菌的代谢产物

昆虫病原线虫共生细菌寄生于昆虫病原线虫肠道内 ,二者互惠共生。该菌革兰氏染色阴性 ,属肠杆菌科 (Ｅｎｔｅｒｏｂａｃｔｅｒｉａｃｅａｅ) ,包含两个属———嗜线虫致病杆菌属 (Ｘｅｎｏｒｈａｂｄｕｓ)和发光杆菌属 (Ｐｈｏｔｏｒｈａｂｄｕｓ)。其中嗜线虫致病杆菌与斯氏线虫 (Ｓｔｅｉｎｅｒｎｅｍａ)共生 ,发光杆菌与异小杆线虫 (Ｈｅｔｅｒｏｒｈａｄｉｔｉｓ)共生。近几年 ,随着对共生菌研究的逐渐深入 ,发现共生菌能产生许多有应用潜力的代谢产物 ,如杀虫蛋白、抑菌物质、抗癌物质、胞外酶、胞内晶体蛋白、色素及荧光素等。特别是…     

致病杆菌属和光杆状菌属细菌杀虫毒素蛋白

致病杆菌属和光杆状菌属细菌是一类分别与斯氏线虫属和异小杆属线虫共生的昆虫病原细菌 ,属肠杆菌科 ,此类细菌产生的杀虫毒素蛋白是近年来发现的一类高效、杀虫谱广的新型杀虫蛋白。此类毒素蛋白对多种昆虫具有注射和口服毒性 ,在同一菌株中有多个杀虫基因 ,各杀虫蛋白基因之间具有协同毒力效应 ,杀虫蛋白基因在大肠杆菌和植物中表达的毒素蛋白对多种害虫具有口服毒性。     

昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

嗜菌异小杆线虫侵染后暗黑鳃金龟和大黑鳃金龟幼虫脂肪体和中肠组织超微结构观察

【目的】探究昆虫病原线虫嗜菌异小杆线虫沧州品系 Heterorhabditis bacteriophora strain Cangzhou侵染对蛴螬脂肪体和中肠的影响,进一步明确其对蛴螬的致病机理。【方法】采用透射电镜技术,观察暗黑鳃金龟 Hololtrichia oblita (Faldermann)和大黑鳃金龟 H. parallela Motschulsky 2龄幼虫被嗜菌异小杆线虫沧州品系侵染后其脂肪体和中肠组织的病理变化。【结果】血腔注射感染期病原线虫嗜菌异小杆线虫沧州品系悬浮液24和48 h后,观察发现暗黑鳃金龟和大黑鳃金龟2龄幼虫脂肪体和中肠的组织结构均按时序逐渐发生变化,起初表现为脂肪球变形或变小,颜色变浅,脂肪体细胞和中肠细胞内质网、线粒体肿胀,中肠微绒毛变形脱落等现象,48 h后包裹脂肪球的膜结构破裂,脂肪体细胞和中肠细胞线粒体破裂,内质网数量减少,中肠微绒毛大量脱落,同时核内染色质大量解离,核膜破裂。【结论】经昆虫病原线虫嗜菌异小杆线虫沧州品系处理后,暗黑鳃金龟和大黑鳃金龟两种金龟甲2龄幼虫脂肪体和中肠细胞均出现明显的病理变化过程,这是嗜菌异小杆线虫高效致死蛴螬的原因之一。本研究可为昆虫病原线虫作为一种生物防治手段在蛴螬的综合防治中更好地发挥作用提供理论依据。     

昆虫病原线虫共生细菌胞内晶体蛋白的研究进展

初生型发光杆菌属(Photorhabdus)及嗜线虫致病杆菌属(Xenorhabdus)细菌分别与异小杆线虫属(Heterorhabditis)和斯氏线虫属(Steinernema)昆虫病原线虫互惠共生.这类昆虫病原细菌在稳定生长期分别产生两种形态各异的胞内晶体蛋白.本文回顾了这类蛋白的研究历史和最新的研究进展,特别是胞内晶体蛋白的理化性质和生物学功能,同时讨论这种晶体蛋白的研究方法与技术.     

根结线虫对斜纹夜蛾幼虫生长及营养利用随龄期变化的研究

【目的】为明确南方根结线虫Meloidogyne incognita侵染对斜纹夜蛾Spodoptera litura幼虫的影响是否因幼虫的龄期而异。【方法】采用被南方根结线虫侵染和未被侵染的乌桕叶片饲喂斜纹夜蛾幼虫,并测定不同龄期幼虫的生长（幼虫体重、发育历期、相对生长率）和营养利用（取食量、近似消化率、食物转化率）情况。【结果】随着幼虫龄期的增加,斜纹夜蛾幼虫的取食量及发育历期呈上升趋势,幼虫体重、相对生长率和食物转化率先上升后下降,而近似消化率则先下降后上升。与饲喂未侵染南方根结线虫的乌桕叶片相比,取食线虫侵染的乌桕叶片的斜纹夜蛾2龄幼虫近似消化率（0.78±0.07）%显著增加了约30%,但斜纹夜蛾的4龄幼虫体重（0.12±0.04）g、相对生长率（0.20±0.06）g·g^(-1)·d(-1)·d^(-1)和食物转化率（0.54±0.18）%分别降低了61%、36%和73%。线虫侵染处理与龄期互作对斜纹夜蛾的发育历期和取食量无显著影响。【结论】根结线虫侵染对斜纹夜蛾幼虫生长及其营养利用的影响因龄期而异,线虫侵染处理虽显著增加了斜纹夜蛾2龄幼虫的近似消化率,但却抑制了其4龄幼虫的生长及其营养利用。     

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.     

重组昆虫病原细菌Cip蛋白基因工程菌发酵条件的初步研究

昆虫病原细菌产生两种胞内晶体蛋白CipA和CipB,为新型的生物农药——昆虫病原线虫提供必需的营养。在已构建原核表达载体pET-15b-cipA和pET-15b-cipB的基础上,研究了这两种重组载体在不同宿主菌中的表达情况,重组菌的生长特性,摇瓶培养时表达重组Cip蛋白工程菌的发酵条件。结果显示:以E.coli BL21(DE3)为宿主菌,在LB培养基中培养至OD600为0.8～1.0时,1 mmol/L IPTG诱导8 h,CipA和CipB的表达量可达30.9%和32.6%,重组质粒具有良好的分离稳定性和结构稳定性。     

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.     

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.     

Directional movement of entomopathogenic nematodes in response to electrical field: effects of species, magnitude of voltage, and infective juvenile age

Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported a directional response to electrical fields for two entomopathogenic nematode species; specifically, when electrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a cruiser-type foraging strategy) moved to a higher electric potential, whereas Steinernema carpocapsae, an ambush-type forager, moved to a lower potential. Thus, we hypothesized that entomopathogenic nematode directional response to electrical fields varies among species, and may be related to foraging strategy. In this study, we tested the hypothesis by comparing directional response among seven additional nematode species: Heterorhabditis bacteriophora, Heterorhabditis georgiana, Heterorhabditis indica, Heterorhabditis megidis, Steinernema feltiae, Steinernema riobrave, and Steinernema siamkayai. S. carpocapsae and S. glaseri were also included as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. siamkayai is an ambusher and S. feltiae and S. riobrave are intermediate. Additionally, we determined the lowest voltage that would elicit a directional response (tested in S. feltiae and S. carpocapsae), and we investigated the impact of nematode age on response to electrical field in S. carpocapsae. In the experiment measuring diversity of response among species, we did not detect any response to electrical fields among the heterorhabditids except for H. georgiana, which moved to a higher electrical potential; S. glaseri and S. riobrave also moved to a higher potential, whereas S. carpocapsae, S. feltiae, and S. siamkayai moved to a lower potential. Overall our hypothesis that foraging strategy can predict directional response was supported (in the nematodes that exhibited a response). The lowest electric potential that elicited a response was 0.1 V, which is comparable to electrical potential associated with some insects and plant roots. The level of response to electrical potential diminished with nematode age. These results expand our knowledge of electrical fields as cues that may be used by entomopathogenic nematodes for host-finding or other aspects of navigation in the soil.     

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.     

The Natural Host Range of Steinernema and Heterorhabditis spp. and Their Impact on Insect Populations

The natural host range of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis can be defined as the range of insects which indigenous nematode populations use for propagation. Information on the natural host range is rare. However, based on records of insects found to be naturally infected with nematodes, some conclusions regarding the natural host range of some Steinernema spp. and Heterorhabditis spp. are presented. Reports of indigenous nematode populations impacting on insect populations can be divided between relatively balanced, long-lasting nematode-host associations and unbalanced, short-lasting epizootics. Examples of the augmentation and inoculative introduction of nematodes in agriculture and forestry ecosystems are presented. Based on current knowledge, nematode reproduction strategies are discussed and indications of the risk involved in the release of non-indigenous nematodes are given.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).