生物杀虫剂-昆虫病原线虫的培养技术

昆虫病原线虫是新型的生物杀虫剂,对钻蛀及土栖性害虫防效较好,具有部分替代化学农药的潜力。昆虫病原线虫的产业化培养是昆虫病原线虫商业化应用的基础。本文介绍了目前世界上常用的昆虫病原线虫的培养方法,包括活体培养、半固体培养和液体培养的技术,这些技术的应用有助于昆虫病原线虫的种质保存及培养,为拓展昆虫病原线虫的产业化生产和应用奠定了基础。     

昆虫病原线虫应用研究概况

大规模工厂化离体培养技术的发展,活跃了昆虫病原线虫的实际应用研究.近20年来在生物防治实践和产业化方面已经取得了令人鼓舞的进展,已成为当前国际生防领域研究热点之一.我国在昆虫病原线虫的实际应用方面也进行了大量研究.本文从昆虫病原线虫对土栖性、钻蛀性和叶面害虫的防治效果,以及影响线虫应用的环境因子的分析,简要概述了昆虫病原线虫的应用进展,并探讨昆虫病原线虫进一步应用的前景.     

武昌罗索线虫的研究与展望

武昌罗索线虫Romanomermis wuchangensis Bao.作为一种昆虫病原线虫,在昆虫的生物防治中起着重要作用。文章综述90年代以来我国关于武昌罗索线虫的生物学特性、田间应用、体内外培养以及生化与分子生物学研究进展,并对武昌罗索线虫今后的研究提出了建议。     

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。     

中华卵索线虫的研究与应用

中华卵索线虫Ovomermis sinensis Chen etal.作为一种昆虫病原线虫,在农业害虫的生物防治中起着重大作用。文章综述90年代以来我国有关中华卵索线虫的生物学特性、田间应用、体内外培养以及生化与分子生物学研究进展,并对中华卵索线虫今后的研究方向提出建议。     

昆虫病原线虫的耐寒性

昆虫病原线虫可开发成生物农药,广泛应用于多种地下及钻蛀害虫的安全防治。但昆虫病原线虫货架期较短,对寒冷等极端环境的耐受性较差,影响了其在生物防治方面的商业开发。本文介绍了寒区的昆虫病原线虫资源,总结了昆虫病原线虫耐寒性的测定方法及增强方法、耐寒性差异的研究进展,并对其耐寒的生理生化机制及分子机理进行了综述。研究昆虫病原线虫的耐寒性,对于解释种群动态,指导昆虫病原线虫的低温保存,以及拓展其在生物防治方面的应用具有重要意义。     

应用昆虫病原线虫防控白蚁的研究现状

昆虫病原线虫是防治白蚁极具潜力的生物杀虫剂,但其在白蚁防控中的应用效果有待进一步研究。本文综述了昆虫病原线虫及其共生细菌在白蚁防治中的应用研究进展,探讨了昆虫病原线虫防治白蚁应用存在的关键问题,并对其未来的研究方向进行展望。     

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要有土壤类型、温湿度、盐度、紫外线等.本文从昆虫病原线虫与环境生物、非生物因素的关系综述这类线虫的研究进展,为昆虫病原线虫的研发和应用提供参考.     

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…     

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。     

Parameters affecting plant defense pathway mediated recruitment of entomopathogenic nematodes

Entomopathogenic nematodes are natural enemies and effective biological control agents of subterranean insect herbivores. Interactions between herbivores, plants, and entomopathogenic nematodes are mediated by plant defense pathways. These pathways can induce release of volatiles and recruit entomopathogenic nematodes. Stimulation of these plant defense pathways for induced defense against belowground herbivory may enhance biological control in the field. Knowledge of the factors affecting entomopathogenic nematode behaviour belowground is needed to effectively implement such strategies. To that end, we explore the effect of elicitor, elicitor dose, mechanical damage, and entomopathogenic nematode release distance on recruitment of entomopathogenic nematode infective juveniles to corn seedlings. Increasing doses of methyl jasmonate and methyl salicylate elicitors recruited more entomopathogenic nematodes as did mechanical damage. Recruitment of entomopathogenic nematodes was higher at greater release distances. These results suggest entomopathogenic nematodes are highly tuned to plant status and present a strategy for enhancing biological control using elicitor-stimulated recruitment of entomopathogenic nematodes.     

不同培养基上繁殖的昆虫病原线虫格氏线虫表皮蛋白的差异

表皮蛋白(surface coat proteins, SCPs)是格氏线虫Steinernema glaseri克服寄主免疫系统的关键因素, 能够抑制昆虫免疫反应, 促进线虫的侵染。为了进一步研究表皮蛋白抑制昆虫免疫的作用机理和线虫与寄主间的相互关系, 我们比较分析了不同培养基繁殖的格氏线虫表皮蛋白的差异。我们用人工培养基和大蜡螟Galleria mellonella末龄（5龄）幼虫分别培养得到格氏线虫侵染期幼虫, 利用酒精提取表皮蛋白。SDS-PAGE和非变性PAGE分析显示, 大蜡螟来源的格氏线虫的表皮蛋白具有更多的蛋白条带。体外和体内的裂解血细胞实验表明, 大蜡螟来源的格氏线虫的表皮蛋白表现出强烈的裂解血细胞活性, 而人工培养基来源的格氏线虫的表皮蛋白活力不明显; 且具有裂解血细胞活性的表皮蛋白为诱导表达型蛋白。不同培养基来源的格氏线虫的表皮蛋白组成的差异和活性的不同, 表明格氏线虫表皮蛋白的产生受线虫培养条件影响较大。     

Heritability of the Liquid Culture Mass Production Potential of the Entomopathogenic Nematode Heterorhabditis bacteriophora

The infective stage of entomopathogenic nematodes ( Heterorhabditis spp.) is the mobile, but developmentally arrested dauer juvenile (DJ). For commercial application, nematodes are produced in liquid culture. Prior to the inoculation of the DJ, their symbiotic bacterium Photorhabdus luminescens is cultured. The DJ exit from the arrested stage (recovery) and develop to reproductive adults. Recovery is a response to bacterial food signals. In liquid culture the percentage of DJs recovering from the DJ stage is highly variable, which significantly influences the number of reproducing hermaphrodites and the final DJ yields. The liquid culture yield is defined by the number of DJ mL -1 harvested from the medium. The heritability of the disposition to recover from the DJ stage and of the final DJ yield in liquid culture has been evaluated. From a hybrid strain of H. bacteriophora 30 homozygous inbred lines were established by inbreeding over seven generations. These inbred lines were propagated in liquid culture and DJ recovery and yields were recorded. The calculated heritability for the DJ recovery was low ( h 2 = 0.38). No significant genetic variability could be detected for this trait. In contrast, a high heritability ( h 2 = 0.90) was found for the total number of DJs produced in the liquid medium.     

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.     

昆虫病原线虫对非生物胁迫的响应机制

昆虫病原线虫是农林害虫生物防治中重要的生防因子之一。它对非生物胁迫的耐受能力决定着线虫在田间的个体生存及控制害虫效果。线虫对环境胁迫的响应是一个整体性的复杂过程, 体现在群体遗传、发育阶段、生理生化和抗逆相关基因的表达调控等不同层次、不同水平上。本文综述了昆虫病原线虫抗逆相关领域的主要研究进展, 重点介绍线虫响应非生物胁迫的生理生化机制和相关抗性基因的分离鉴定, 并对该研究领域发展趋势进行了讨论和展望, 期望为我国研究线虫抗逆机理提供一些新的信息。     

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。     

Production of entomopathogenic nematodes in submerged monoxenic culture: A review

Monoxenic liquid culture is the most suitable technology for scaling up to industrial production of entomopathogenic nematodes (EPNs); however, the variability of the yield production remains a current problem in the process. The aim of this study was to analyze the parameters and criteria for EPN production in liquid culture based on scientific and technological knowledge from the last two decades. While experimental research has permitted the yield production of Heterorhabditis bacteriophora (362 × 10³ infective juveniles [IJs]/ml) and Steinernema carpocapsae (252 × 10³ IJs/ml), simultaneously, theoretical approaches have contributed to the understanding of the culture process, based on biological parameters of the bacterium–nematode complex and hydrodynamic and rheological parameters of the complex gas–liquid–solid system. Under this interdisciplinary research approach, bioprocess and biosystem engineering can contribute to design the various control strategies of the process variables, increase the productivity, and reduce the variability that until now distinguishes the in vitro production of EPNs by the liquid culture.     

Insect protein digestion improves purity of Steinernema carpocapsae in vitro culture and reduces culture period

Insect protein, used for in vitro culture media for entomopathogenic nematode, produces nematodes of high quality. However, the time-consuming culture and poor purity of nematodes hinder the commercial application of insect protein media. We show that hydrolyzed insect protein improves nematode purity in in vitro culture. The results revealed that nematode purity was increased by more than 90 %, and the culture period was reduced by 6 days. Estimated economic efficiency of using hydrolyzed insect protein medium was increased by 44.25 % over that obtained with non-hydrolyzed insect medium.     

Entomopathogenic Nematode Production and Application Technology

Production and application technology is critical for the success of entomopathogenic nematodes (EPNs) in biological control. Production approaches include in vivo, and in vitro methods (solid or liquid fermentation). For laboratory use and small scale field experiments, in vivo production of EPNs appears to be the appropriate method. In vivo production is also appropriate for niche markets and small growers where a lack of capital, scientific expertise or infrastructure cannot justify large investments into in vitro culture technology. In vitro technology is used when large scale production is needed at reasonable quality and cost. Infective juveniles of entomopathogenic nematodes are usually applied using various spray equipment and standard irrigation systems. Enhanced efficacy in EPN applications can be facilitated through improved delivery mechanisms (e.g., cadaver application) or optimization of spray equipment. Substantial progress has been made in recent years in developing EPN formulations, particularly for above ground applications, e.g., mixing EPNs with surfactants or polymers or with sprayable gels. Bait formulations and insect host cadavers can enhance EPN persistence and reduce the quantity of nematodes required per unit area. This review provides a summary and analysis of factors that affect production and application of EPNs and offers insights for their future in biological insect suppression.     

Entomopathogenic nematodes,a potential microbial biopesticide: mass production and commercialisation status – a mini review

Parasitic nematodes have several important attributes that make them excellent candidates for biological control of soil insects. These nematodes can be produced by in vivo by baiting technique on insects and commercially by in vitro solid/liquid culturing. Numerous insect pests on many different crops are being controlled by these insect parasitic nematodes, including root weevils, flea beetles, mint root borer, colorado potato beetle, white grubs, caterpillars and plant parasitic root nematode, e.g. root-knot nematodes. Utilisation of entomopathogenic nematodes (EPN) has raised intense interest and has been a growing concern globally mainly because of its potential efficiency, exemption from registration and other impressive attributes for utilising against the control of soil dwelling pests. This review highlights the mass production, commercialisation and utilisation of EPN as microbial biopesticide in bio-intensive pest management programmes.