广西南宁市柑橘木虱虫生真菌种类调查

自柑橘木虱Diaphorina citri体上分离虫生真菌,通过形态特征和ITS序列分析,鉴定虫生真菌的种类;通过致病性测定,明确昆虫病原真菌种类。结果发现,自死体柑橘木虱上分离到18个虫生真菌菌株,隶属4种真菌;自活体上分离到985个虫生真菌菌株,隶属25种真菌。致病性测定结果发现,仅4种真菌对柑橘木虱成虫有致病性,包括刀孢蜡蚧菌Lecanicillium psalliotae、球孢白僵菌Beauveria bassiana、爪哇虫草Cordyceps javanica和淡紫紫孢菌Purpureocillium lilacinum,该4种真菌的分生孢子悬浮液（浓度5×10^7个孢子/mL）接种柑橘木虱成虫后10 d的累计死亡率分别为100%、100%、98.89%和43.33%。其中爪哇虫草仅自死体柑橘木虱上分离到,刀孢蜡蚧菌仅在活体上分离到,球孢白僵菌和淡紫紫孢菌在死体和活体上均被分离到。可见,柑橘木虱活体上的虫生真菌种类丰富。生产上应加强保护和利用昆虫病原真菌,提高其对柑橘木虱的自然抑制力。     

虫生真菌分子致病机理及基因工程改造研究进展

虫生真菌侵染寄主昆虫的复杂过程可分为体表附着、体壁穿透及体内定殖和致死等不同阶段。近年来, 以金龟子绿僵菌(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)为代表的基因功能研究取得了长足的进展, 从不同角度阐明了虫生真菌的分子致病机理; 同时, 基因工程技术的应用为昆虫病原真菌的遗传改良和选育高毒力杀虫菌株开辟了新的途径。对近年来昆虫病原真菌侵染寄主的分子对策及基因工程改造的研究进展进行了综述, 并就进一步研究虫生真菌的毒力基因及功能进行了探讨。     

我国虫生真菌研究的简单回顾与展望

经过几代人的努力,我国虫生真菌学科领域已从过去对少数种的观察研究（如冬虫夏草和白僵菌等）、益虫保护（如蚕僵病防治）、药用和害虫生物防治等,扩大到了对多类虫生真菌资源的系统研究和在国民经济不同领域中的开发应用。迄今,虫生真菌已不仅是昆虫病理学的组成部分及农林害虫防治中的一种途径和手段。作为菌物中的一个特殊生态系,就其现有研究内容来看,至少已发展成了如下几个各具特色的研究领域：以白僵菌为代表的昆虫病原真菌及其对农林害虫的生物防治;以蚊幼虫为主的卫生害虫的虫生真菌研究及利用;虫草及其无性型的综合研究;…     

虫生真菌分子致病机理及基因工程改造研究进展

虫生真菌侵染寄主昆虫的复杂过程可分为体表附着、体壁穿透及体内定殖和致死等不同阶段.近年来,以金龟子绿僵茵(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)为代表的基因功能研究取得了长足的进展,从不同角度阐明了虫生真菌的分子致病机理;同时,基因工程技术的应用为昆虫病原真菌的遗传改良和选育高毒力杀虫菌株开辟了新的途径.对近年来昆虫病原真菌侵染寄主的分子对策及基因工程改造的研究进展进行了综述,并就进一步研究虫生真菌的毒力基因及功能进行了探讨.     

科技信息与服务

我国农业害虫病原真菌的调查和研究昆虫病原微生物中,真菌种类约占60%。70年代末,国外记载的虫生真菌已达530余种,其中数量最多的是虫霉目真菌,约有160种。我国自60年代以来开始进行我国虫生真菌资源的调查和研究,但主要以自僵菌、绿僵菌为主要研究和应用对象。70年代以来,贵州农学院等单位从茶园中分离到害虫病原真菌20     

利用虫生真菌生物防治柑橘木虱的研究进展

柑橘木虱是柑橘黄龙病的重要传播虫媒。目前对柑橘黄龙病的防治尚缺有效的药剂和抗病品种,加强对柑橘木虱的防治,对控制柑橘黄龙病的蔓延具有重要意义。目前防治柑橘木虱多采用化学防治,杀虫剂的频繁使用造成了农药残留、环境污染、生物多样性被破坏和害虫产生抗药性等诸多问题,生物防治以其高效、低毒、低残留、不易产生抗药性等优点逐渐受到重视。昆虫病原真菌能侵入昆虫寄主体内,导致昆虫发病死亡,具有良好的病害流行潜力及生产应用便利性,利用昆虫病原真菌防治柑橘木虱具有广阔的发展空间。本文总结了用于柑橘木虱生物防治的虫生真菌种类,重点介绍了国内外利用球孢白僵菌、玫烟色棒束孢、淡紫紫孢菌、宛氏拟青霉、蜡蚧菌等虫生真菌在防治柑橘木虱中的应用,并对虫生真菌防治柑橘木虱的发展前景进行了展望,以期为柑橘黄龙病的防控提供借鉴。     

虫生真菌抗逆的生化分子基础及利用

虫生真菌在生活史各个阶段均面临多种环境因子的危害。为了避开这些不利的因素,虫生真菌进化出多种抗逆的机制。近年来,以球孢白僵菌(Beauveria bassiana)和金龟子绿僵菌(Metarhizium anisopliae)为代表的虫生真菌抗逆的生化分子基础的研究取得了一定的进展,明确了某些生化成份和抗逆基因与菌株抗逆性能的相关性。就环境胁迫因子、虫生真菌抗逆的生化分子基础及其在改良菌株抗逆性能方面的应用进行综述,这将为环境适应性强的虫生真菌制剂的研发与应用提供理论依据。     

应用虫生真菌防治烟粉虱的现状

烟粉虱Bemisia tabaci属世界性分布的大害虫.在一定条件下虫生真菌是控制烟粉虱种群的最有效天敌.本文就几种常见虫生真菌:蜡蚧轮枝菌Verticilliumlecanii、座壳孢Aschersoniaspp.、玫烟色拟青霉Paecilomycesfumosoroseus、球孢白僵菌Beauveria bassiana等防治烟粉虱的应用现状以及它们与其它天敌的关系进行了综述,以期对烟粉虱虫生真菌的合理利用提供参考.     

一株分离自土壤的蜡蚧菌的鉴定及对烟草粉斑螟的致病性

【目的】蜡蚧菌Lecanicillium是自然界一类重要的昆虫病原真菌,为了获取更多的蜡蚧菌作为控虫真菌资源,对从森林中的根际土壤分离得到的一株蜡蚧菌进行鉴定以及与其他几种病原真菌进行烟草粉斑螟Ephestia elutella的致病性对比。【方法】采用形态特征比较和转录间隔区(ITS)序列构建分子系统树对从贵州贵定松林下土壤中分离的菌株进行综合鉴定,采用浸虫法对烟草粉斑螟3龄幼虫进行致病性筛选。【结果】根据形态特征比较和系统发育分析,该真菌为丝枝蜡蚧菌Lecanicillium aphanocladii。在测试菌株中,环链棒束孢Isaria cateinannulata、球孢白僵菌Beauveria bassiana和丝枝蜡蚧菌对烟草粉斑螟3龄幼虫7 d的校正死亡率分别为79.22%,64.92%和77.92%,表现出较好的致死效果。【结论】丝枝蜡蚧菌对烟草粉斑螟幼虫的致死效果较好,可用作仓储内烟草粉斑螟的生物防治资源。     

西花蓟马高毒力虫生真菌筛选及其生防应用潜力的研究进展

西花蓟马是一种外来入侵的世界性害虫,对农林业危害巨大。查阅国内外相关文献,综述了当前防治西花蓟马的虫生真菌的种类、高毒力菌株的筛选及防治现状。现已知西花蓟马的寄生病原真菌有5种,包括蜡蚧轮枝菌(半知菌:丝孢目)、球孢白僵菌(半知菌:丝孢目)、金龟子绿僵菌(半知菌:丝孢目)、玫烟色棒束孢(半知菌:束梗孢目)和小孢新接霉。其中,球孢白僵菌、金龟子绿僵菌在西花蓟马的生物防治中应用最广,具有良好的开发应用潜力,部分防效好的虫生真菌已申请专利及实现工厂化生产。     

Entomopathogenic fungi and insect behaviour: from unsuspecting hosts to targeted vectors

The behavioural response of an insect to a fungal pathogen will have a direct effect on the efficacy of the fungus as a biological control agent. In this paper we describe two processes that have a significant effect on the interactions between insects and entomopathogenic fungi: (a) the ability of target insects to detect and avoid fungal pathogens and (b) the transmission of fungal pathogens between host insects. The behavioural interactions between insects and entomopathogenic fungi are described for a variety of fungal pathogens ranging from commercially available bio-pesticides to non-formulated naturally occurring pathogens. The artificial manipulation of insect behaviour using dissemination devices to contaminate insects with entomopathogenic fungi is then described. The implications of insect behaviour on the use of fungal pathogens as biological control agents are discussed.     

Pathogenic fungi of insects from Argentina (Zygomycetes: Entomophthorales)

Pathogenic fungi of insects from Argentina (Zygomycetes: Entomophthorales). Three species of Entomophthorales entomopathogenic fungi (Zygomycotina: Zygomycetes) have been identified from insects in agricultural crops (Buenos Aires Province, Argentina): Zoophthora radicans Batko (Brefeld); Entomophthora planchoniana Cornu and Pandora gammae (Weiser) Humber. Fungal structure measurements are reported.     

Effect of entomopathogenic fungi on detoxification enzyme activity in greater wax moth Galleria mellonella L. (Lepidoptera, Pyralidae) and role of detoxification enzymes in development of insect resistance to entomopathogenic fungi

Fungal infection of insects increases total esterase and glutathione S-transferase activities in the hemolymph. Activities of acid and alkaline phosphatases were similar in the infected and intact insects. Fungal infection increased the resistance of greater wax moth caterpillars to organophosphorus insecticide malathion 1.46 times relative to intact caterpillars. Possible involvement of detoxification enzymes in the development of insect resistance to entomopathogenic fungi and development of complex biological products based on entomopathogenic microorganisms and inhibitors of detoxification enzymes are discussed.     

Pathogenesis-related genes of entomopathogenic fungi

All living things on Earth experience various diseases such as those caused by viruses, bacteria, and fungi. Insects are no exception to this rule, and fungi that cause disease in insects are called entomopathogenic fungi. These fungi have been developed as microbial insecticides and are used to control various pests. Generally, the mode of action of entomopathogenic fungi is divided into the attachment of conidia, germination, penetration, growth, and generation of secondary infectious conidia. In each of these steps, that entomopathogenic fungi use genes in a complex manner (specific or diverse) has been shown by gene knock-out and RNA-sequencing analysis. In this review, the information mechanism of entomopathogenic fungi was divided into six steps: (1) attachment of conidia to host, (2) germination and appressorium, (3) penetration, (4) fungal growth in hemolymph, (5) conidia production on host, and (6) transmission and dispersal. The strategy used by the fungi in each step was described at the genetic level. In addition, an approach for studying the mode of action of the fungi is presented.     

Agroforestry coffee soils increase the insect‐suppressive potential offered by entomopathogenic fungi over full‐sun soils: A case proposing a “bait survival technique”

Entomopathogenic fungi are important natural enemies of insects. However, there is little information on the insect‐suppressive potential of these fungi and possible effects of farming management on this. Meanwhile, changes in natural landscapes due to agricultural intensification have caused considerable biodiversity loss and consequent decay of ecosystem services. However, the adoption of practices such as agroforestry in agroecosystems can foster abiotic and biotic conditions that conserve biodiversity, consequently restoring the provision of ecosystems services. Here, we assessed the effect of management systems (agroforestry or full‐sun) on the pest‐suppressive potential of entomopathogenic fungi in Brazilian coffee plantations. We used the insect bait method coupled with survival analyses to assess the speed of kill by entomopathogenic fungi and their presence in soil samples from both farming systems. We found that insects exposed to agroforestry soils died more quickly than insects exposed to full‐sun soils. Of the fungi isolated from the bait insects, Metarhizium was found most frequently, followed by Beauveria. Meanwhile, Fusarium was frequently isolated as primary or secondary infections. We propose that the differential survival of insects is indicative of a greater suppressive potential by entomopathogenic fungi in agroforestry, and that this could be promoted by the diversified landscape, microclimatic stability, and reduced soil disturbance in agroforestry systems. Furthermore, our results provide a useful demonstration of the potential use of the insect bait method to investigate pest‐suppressive potential through bait insect mortality, and we term this the “bait survival technique.”     

An improved method for detection and quantification of chitinase activities

Chitinases are enzymes that serve critical roles in fungal growth and development, in resistance of plants to fungal pathogens, and in parasitism of insects by entomopathogenic fungi. The term "chitinase" is used for 3 enzymatic activities: N-acetylglucosaminidases, which sequentially release N-acetylglucosamine residues from the chitin polymer; chitobiosidases, which release disaccharides; and endochitinases, which cleave within the polymer and release oligosaccharides. We describe a technique where chitinases are separated on non-denaturing polyacrylamide gels, activities are visualized and characterized with chitinase specific substrates, and specific activities are estimated by image analysis. This technique permits a rapid determination of all of the types of chitinases present within a sample as well as their activities.     

虫生真菌研究与应用的发展与机遇 ——2012年虫生真菌专刊序言

我国从事虫生真菌基础及应用研究的人员众多,各方面均取得了重要的研究成果。2012年“虫生真菌专刊”收集了国内关于虫生真菌资源、基因克隆及活性成分代谢等方面的最新研究进展。进一步的基础研究,应加强致病基因功能和真菌-昆虫分子相互作用等研究,以提高真菌杀虫剂或药用虫生真菌的应用效率。     

Cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance

Abstract

Entomopathogenic fungi are among the most successful biocontrol agents for preventing economic loss from insects. The identification of virulent species or isolates, the development of formulation technology and the improvement of efficiency are avenues being pursuing by researchers in diverse scientific disciplines. A successful entomopathogenic fungus deploys a combination of mechanical and biochemical processes to overcome the first defensive barrier in insects, the integument. A precise understanding of the mechanisms underlying fungal pathogenicity, particularly the roles of enzymes such as proteases, is essential in order to highlight the potential of entomopathogenic fungi and increase their virulence via genetic modifications. Cuticle-degrading proteases are divided into subtilisin-like (Pr1) and trypsin-like (Pr2) proteases, which are secreted in the initial stages of penetration. The biochemical structure contains the catalytic triad Asp39, His69 and Ser224 in addition to Ca²⁺ binding sites. Studies have shown a molecular weight of almost 19–47?kDa, an optimal pH of 7–12 and an optimal temperature of 35–45?°C. Different species or isolates of entomopathogenic fungi exhibit differences in the secretion and activity of cuticle-degrading proteases, which may indicate their virulence capacity. Genetic engineering techniques have been developed to create isolates with protease overexpression. Such isolates have significantly higher virulence against the host because they not only ensure fungal penetration but also exhibit direct toxicity to insects.     

Identification of the cuticular lipid composition of the Western Flower Thrips Frankliniella occidentalis

The Western Flower Thrips Frankliniella occidentalis effectively resists many insecticides, but it can be controlled by the use of bioinsecticides such as entomopathogenic fungi. The epicuticular chemistry of these insects is therefore of great interest, and accordingly, the cuticular lipid composition of F. occidentalis was analysed. It was found that the cuticular lipids of both the adult and larval stages of F. occidentalis consist of two groups of compounds--hydrocarbons and free fatty acids. The same hydrocarbon pattern was found in both adults and larvae, with the exception of n-hentriacontane, which was detected only in adult insects. The following homologous series were identified: n-alkanes from C-25 to C-29 (31) with the marked dominance of odd numbers of carbon atoms, 3-methylalkanes with 26 and 28 carbon atoms, and branched monomethylalkanes (branched at C-9, -11, -13 and -15) with 26, 28 and 30 carbon atoms. The chemical composition of the free fatty acids consists of two homologous series: saturated (C(14:0), C(16:0), C(18:0)) and unsaturated fatty acids (C(16:1) and C(18:1)). This analysis confirmed the lack of potential inhibitors of entomopathogenic fungi in the cuticular lipids of this insect species.     

Extracellular hydrolytic enzymes produced by entomopathogenic fungi--role in an infection process

Entomopathogenic fungi have a great potential in biological control of insect pest population. Fungal pathogens are promising source of insecticides and notable alterative to chemical pesticides. These fungi possess a unique mechanism of insects paralysis. As natural enemies of insects they attack direct host cuticle via a combination of mechanical pressure and cuticle-degrading enzymes. Entomopathogenic fungi produce several proteo-, chitino- and lipolytic enzymes, which are accepted as key factors in insect mycosis. The role of extracellular enzymes in pathogenesis is still not well understood. Profound understanding the mechanisms of insect paralysis by entomopathogenic fungi will help in the production of safer for environment and more efficiency mycoinsecticides.