利用GFP表达系统检测球孢白僵菌侵染昆虫过程

绿色荧光蛋白(GFP)作为一种重要的报告基因,在病原菌和寄主之间的互作研究中具有良好的应用前景。利用草丁膦抗性基因bar为筛选标记,构建了组成性表达绿色荧光蛋白基因egfp的载体pBG,并转入球孢白僵菌获得成功表达。将转化子接种菜青虫进行生物测定表明,组成性表达egfp不影响球孢白僵菌的毒力。进一步利用冰冻切片和荧光显微观察技术监测球孢白僵菌侵染寄主行为,结果显示,通过荧光观察可清楚的检测到病原菌在昆虫体表的附着、菌丝穿透体壁以及菌丝从寄主体内长出等侵染过程。由此表明,GFP可有效用于球孢白僵菌的遗传标记,进行病原菌的鉴别、侵染致病机理及相关功能基因的表达模式研究。     

利用冷冻切片对球孢白僵菌侵染西花蓟马过程的观察

为探索球孢白僵菌对西花蓟马的侵染,研究球孢白僵菌在西花蓟马体表和虫体内的侵染过程,采用冷冻切片与HE染色技术,对被球孢白僵菌侵染的西花蓟马成虫进行组织切片及HE染色。结果表明,接菌4 h,体表粘附的孢子开始萌发,8-12 h芽管继续伸长并出现穿透体壁。18 h虫菌体出现在西花蓟马体内,球孢白僵菌成功侵入西花蓟马体内。之后,西花蓟马体内虫菌体、菌丝明显增多,各组织器官均受到感染,消化道、肌肉组织等发生病变。     

球孢白僵菌对温室蚜虫的侵染生物学

研究了球孢白僵菌在不同浓度、施菌方式、温度和相对湿度下对温室蚜虫的侵染力以及接种后对蚜虫的侵染速率。结果表明,高(109个孢子.mL-1)、中(108个孢子.mL-1)、低(107个孢子.mL-1)3个浓度剂量对蚜虫都有较强的致病力,且浓度越大,蚜虫的死亡率越高,死亡时间越提前。用浸渍法和孢子浴法接种蚜虫,6 d的累积死亡率分别为100%和31.1%。在测试的3个温度(22、26、30℃)中,26℃时蚜虫侵染力最强,第3天出现死亡高峰,第5天时累积死亡率就达到100%,明显高于22、30℃的处理。相对湿度越大,球孢白僵菌的致病力越强,蚜虫死亡速度越快。在温湿度组合中相对湿度为95%时,温度对白僵菌的侵染力几乎无影响,但影响发病速度,相对湿度低于95%时,26℃的侵染力始终高于22和30℃时的侵染力。通过接种后不同时间段用0.2%百菌清处理蚜虫测定该菌株的侵染速率,结果表明接种后24 h是该球孢白僵菌有效侵染蚜虫的关键时期。     

球孢白僵菌的侵染特性及应用研究进展

球孢白僵菌是一种重要的虫生真菌,具有致病力强、侵染范围广、安全无污染等优点,其制剂可作为一种真菌类杀虫剂应用于农林业害虫的防治,也可用于人工生产中药材僵蚕。本文综述了球孢白僵菌的培养性状、侵染特性、应用生产和存在的问题,并提出了发展对策,为球孢白僵菌的进一步开发应用提供参考。     

球孢白僵菌侵染中黑盲蝽致病过程的电镜观察

[目的]为揭示白僵菌Beauveria bassiana对中黑盲蝽Adelphocoris suturalis的致病机制,并探索盲蝽生物防治的有效方法.[方法]本文利用扫描电子显微镜和透射电子显微镜观察白僵菌菌株Bb-Ⅷ对中黑盲蝽成虫的侵染过程.[结果]接种白僵菌后,其分生孢子在中黑盲蝽的体壁褶皱、凹陷处或虫体连接部位附着;接种16 h后分生孢子萌发产生芽管,并借助机械压力和酶的作用侵入中黑盲蝽体壁;36-72 h时,菌丝体相继侵入中黑盲蝽的肌肉组织、脂肪体和肠体绒毛内,并在昆虫血腔内进行出芽或分隔生殖;接种48 h后中黑盲蝽死亡,虫体内的菌丝体伸到体外并萌发成气生菌丝和分生孢子,继而布满虫体.[结论]白僵菌的附着受中黑盲蝽体表物理结构的影响,其分生孢子在接种48 h内完成对中黑盲蝽的寄生并最终导致虫体发病死亡,表现出较强的致病力.菌丝入侵的机械破坏力和酶类的水解作用是白僵菌对中黑盲蝽的重要致病因子.     

利用RAPD-PCR检测三种白僵菌及球孢白僵菌种内变异

采用PCR-RAPD技术研究了球孢白僵菌2株野生型多孢株及其各1株单孢分离株的 RAPD扩增片段的多态性,比较了彼此间的相似率。结果显示,相似率在63.6～99.3％之间变化很大,表明各分离子间的基因组DNA已发生变化。球孢白僵菌与另一种白僵菌布氏白僵菌之间的相似率在64.9％～80.9％之间,与粘孢白僵菌之间的相似率在50.0～61.9％之间。     

球孢白僵菌在红火蚁体表侵染的扫描电镜观察

利用扫描电镜观察了球孢白僵菌Beauveria bassiana Bb04菌株分生孢子对红火蚁Beauveria bassiana 工蚁体壁的侵染过程。结果表明: 分生孢子多分布在红火蚁工蚁节间膜、胸部的褶皱、气门、体壁的凹陷部位、刚毛窝附近, 以及着生较密刚毛的足上。萌发的分生孢子在节间膜以及体表缝隙、刚毛窝及刚毛稀少的凹陷部位、胸部褶皱和足胫节处入侵。分生孢子在附着12 h后开始萌发, 接种后18 h附着在节间膜处的孢子首先侵入成功, 接种后24 h刚毛窝附近孢子萌发入侵, 接种后60 h胸、腹和足等部位的孢子均成功穿透侵入表皮。分生孢子可以直接以芽管侵入表皮, 也可以产生附着胞再侵入。     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

利用RAPD—PCR检测三种白僵菌及球孢白僵菌种内变异

采用ＰＣＲ－ＲＡＰＤ技术研究了球孢白僵菌２株野生型多孢株及其各１株单孢分离株的ＲＡＰＤ扩增片段的多态性,比较了彼此间的相似率。结果显示,相似率在６３．６－９９．３％之间变化很大,表明各分离子间的基因组ＤＮＡ已发生变化。     

Susceptibility of adult Aedes aegypti (Diptera: Culicidae) to infection by Metarhizium anisopliae and Beauveria bassiana: prospects for Dengue vector control

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases, stimulating the search for alternatives to current control methods. Screening assays using a range of Metarhizium anisopliae and Beauveria bassiana isolates were performed against adult female Ae. aegypti. Four virulent isolates were selected for detailed study. Adult female mosquitoes were exposed to supports previously inoculated with fungal suspensions. Fungal isolates were suspended in Tween 80+8% vegetable oil. The isolates caused between 70 and 89% mortality as a result of fungal infection over the 7-day test period. Mean survival times varied between 3 and 5 days for treated insects, whilst control survival exceeded 40 days. The most promising isolate, M. anisopliae LPP133, based not only on virulence but facility for mass production, was used for lethal exposure time determinations. An exposure time of only 3.5 h was necessary to cause 50% mortality. Large cage trails were also carried out and mean survival time of insects exposed to fungus impregnated black cloths was significantly reduced. These results show that entomopathogenic fungi could be promising biological control agents for use against adult Ae. aegypti, by inoculating fungi onto surfaces on which the mosquitoes tend to rest. The subsequent mortality caused by the fungi could potentially reduce the populations of this insect thus reducing the incidence of Dengue.     

Effect of Plant Type on the Persistence of Beauveria bassiana

Field evaluation of isolate MK 2001 of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, revealed conidia persistence and infectivity up to 26 days on foliage of lettuce and celery, two crops with substantial plant differences. Plants were treated at a rate of 100 ml/cage at a concentration of 1×10⁸ conidia/ml. The number of colony-forming units (CFU) recovered immediately after treatments from discs sampled on lettuce and celery leaves were not significantly different, due to similar foliar coverage of conidia by application. Significant differences were observed between both host plants during the 26 days of the field trial. The number of CFUs recovered on lettuce was significantly higher than that on celery leaves. However, for each host plant, there were no significant differences either between lettuce external leaves and internal leaves or between celery canopy and bottom leaves. An in vitro pathogenicity test carried out on Lygus lineolaris adults fed with leaf discs harvested from treated cages resulted in a high pathogenicity of B. bassiana isolate MK 2001. The mortality immediately after treatment did not differ from the death counts taken every other day for 26 days. However, efficacy was significantly different between both plants. Mortality was 91% on lettuce and 78% on celery, 7 days post-treatment. This study highlighted that plant type must be taken into account in foliar application of B. bassiana.     

The potential of Metarhizium anisopliae and Beauveria bassiana isolates for the control of Aedes aegypti (Diptera: Culicidae) larvae

Fungal isolates were screened against Aedes aegypti larvae. Exposure of larvae to conidial suspensions resulted in 6–90% mortality. An inoculum persistence assay using one of the most virulent isolates showed an approximate half-life of 10 days in water containers. Not all larvae surviving to form pupae resulted in adult emergence.     

Introduction of Beauveria bassiana as a biological control agent for Tribolium castaneum in Kerman province

Barley, Hordeum vulgare, one of the important crops in the word, is used in malting, feed and food industries. The red flour beetle, Tribolium castaneum, was found wherever grains or other dried foods are stored. Disinfestations of barley using chemical methods to kill insects, in this research, for the first time we isolated the pathogenic KB512 of entomopathogenic fungi Beauveria bassiana from soil and insects, which produced aerial and submerged conidia and blastospores in laboratory conditions. We investigated the best conditions for the production and utilisation of spore suspension to spray the larvae of T. castaneum, which is one of the important pests in Kerman province (Iran). One hundred and eighty isolates that naturally infected by T. castaneum were reared during spring and summer seasons 2010–2011. The pathogenicity test was carried out with direct spray. To bioassay the isolates, three concentrations of the spore suspension were prepared as follows: 1?×?10⁶ and 1?×?10⁸ conidia/ml. The pests were sprayed by aerial conidial suspension, which was prepared by 0.01% Tween 80 in distilled water, and the controls were sprayed by 0.01% Tween 80 in distilled water. After spraying the pests, the plates were incubated at 25?±?1?°C and 80% of relative humidity. Then, the treated pests were monitored every day for the fungal growth and mortality.     

28s rDNA group-I introns: a powerful tool for identifying strains of Beauveria brongniartii

The nuclear ribosomal DNA of the entomopathogenic fungus Beauveria brongniartii is polymorphic in terms of both restriction site and length. Insertions of 350–450 bp long, identified as group-I introns, were detected in the 28 s rDNA. A panel of 47 strains of B. brongniartii , two B. bassiana and one Metarhizium anisopliae of various geographical and biological origins were found to contain 14 variant forms of intron differing in size and restriction pattern, at four different positions. Twelve types of ribosomal large subunit were defined on the basis of variant distribution and compared with strain clustering based on internal transcribed spacers analysis. There was a correlation between the characteristic introns and isolates collected from the sugar cane pest Hoplochelus marginalis . Primers for polymerase chain reaction amplification were chosen from these variants, and used to develop a specific method for detecting strains pathogenic towards Hoplochelus .     

Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker

Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana.     

基于SSR标记的琅琊山球孢白僵菌寄主专化性

球孢白僵菌Beauveria bassiana是一类常见的昆虫病原真菌,其自然侵染的寄主昆虫众多,达15目149科750种。为了解球孢白僵菌自然种群的遗传多样性,探讨种群异质性和寄主来源之间的关系,分析其寄主专化性的强弱,本研究利用SSR分子标记技术,比较了安徽琅琊山国家森林公园的85株球孢白僵菌（寄主种类涉及7目24种）群体遗传多样性差异,通过构建聚类树分析菌株基因型和寄主关联性。结果表明琅琊山球孢白僵菌群体Nei’s基因多样性指数h=0.2906,Shannon信息指数I_s=0.4510,多态位点百分率P为100%。不同寄主目球孢白僵菌遗传多样性水平由高至低为鞘翅目>膜翅目>同翅目>双翅目>鳞翅目>直翅目>半翅目,其中菌株数量较多的鞘翅目、膜翅目和同翅目3个亚种群的遗传多样性较高且水平接近。聚类分析发现8对SSR引物将85株球孢白僵菌分成29个基因型,并在遗传相似系数0.70处分别聚为3个分支。分析寄主类型发现相同基因型的株系可侵染不同目的寄主,而同一类型寄主也可被不同基因型的菌株侵染。球孢白僵菌种群的总体遗传多样性较高,遗传谱系与寄主来源无明显相关性,菌株的寄主专化性弱。     

马尾松林中球孢白僵菌寄主转移和专化性的SSR标记分析

球孢白僵菌是一种最常见的虫生真菌,已被广泛应用于害虫生物防治。利用SSR简单序列重复(simple sequence repeat,SSR)分子标记对来自安徽省麻姑山马尾松林的102株球孢白僵菌进行基因分型、寄主转移和寄主专化性研究。9对微卫星引物将102株白僵菌分成31个微卫星基因型。在这31个微卫星基因型中,有5种基因型株系为相对优势菌株,这5种基因型株系通过侵染不同寄主昆虫即寄主转移延续自身在马尾松林生态系统中的传播和流行,从而证实寄主转移是白僵菌群体中的普遍现象。同时,这些相对优势基因型并不是在各个月份均匀分布,在大部分月份中,存在1–2种优势度高的基因型。相同基因型株系可侵染不同寄主的结果揭示出白僵菌不仅在种的水平,而且在菌株水平上寄主专化性也较弱。正是白僵菌较弱的寄主专化性特征促使部分基因型株系通过寄主转移,在马尾松林生态系统中得以宿存和延续。     

Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a filamentous ascomycete that is pathogenic towards a broad host range of insect targets and is increasingly serving as a model for examining fungal development and host-pathogen interactions. B. bassiana displays a prohibitive level of resistance against many current fungal and/or yeast selection markers including hygromycin, neomycin, and zeocin. A genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100–150 transformants per microgram DNA/10⁸ cells and Southern blot analysis indicated that the plasmid vector was randomly integrated into the genome of B. bassiana. In addition, a construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. Over 95% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. The described transformation method increases opportunities for the genetic manipulation of B. bassiana.