转录组和蛋白组联合分析揭示Ubr1介导的白僵菌萌发和极性生长

【目的】通过比较球孢白僵菌野生型和ubr1基因缺失菌株分生孢子在同一时间点转录组学和蛋白组学的差异表达基因和蛋白及其所属通路,阐明Ubr1影响球孢白僵菌极性生长的机制,为提高球孢白僵菌生物防治潜能提供理论依据。【方法】通过对转录组学和蛋白组学的KEGG分析,获得差异表达基因和蛋白所在代谢调控通路,利用显微镜拍摄菌株在各萌发培养基(germination medium,GM)衍生板中分生孢子萌发的图像验证双组学分析中显著差异的调控通路对分生孢子极性生长的影响。【结果】ubr1基因缺失使分生孢子萌发受损,形成异常弯曲或钩状的芽管。且不论是以转录组,还是以蛋白质组为核心进行双组学KEGG联合分析,二者都能富集到氮代谢、精氨酸和脯氨酸代谢和醚脂类代谢通路。进一步的验证实验表明,球孢白僵菌中ubr1基因缺失引起的精氨酸代谢异常是分生孢子极性生长紊乱的一个重要原因,而半乳糖和氮代谢异常则会导致分生孢子的萌发速率变慢。【结论】Ubr1的缺失使精氨酸代谢受阻,进而导致分生孢子萌发管极性生长异常;同时,也使半乳糖和氮代谢异常导致分生孢子萌发速率延迟。本研究的发现对于认识极性生长的机制具有一定贡献,也拓展...     

球孢白僵菌羧基转运蛋白基因RNA干扰的沉默效应

羧基转运蛋白基因是近年来分离出的新基因,在虫生真菌中可能与穿透昆虫体壁时的能量代谢有关。构建了针对该基因的双链RNA干扰载体,采用芽生孢子转化法将载体质粒转入球孢白僵菌,并通过RT-PCR检测转化前后BbJEN1的表达情况。RT-PCR检测结果显示,球孢白僵菌转化子BbJEN1基因的表达水平显著下降,表明干扰载体具有明显的沉默效应。在两种培养基上,转化子与原始菌株的生长速度和产孢量差异均不显著,而分生孢子萌发则显著滞后于原始菌株。使用马尾松毛虫幼虫对转化子和原始菌株进行生物测定,原始菌株的半致死浓度、半致死剂量和半致死时间分别为2.79×106个孢子/mL、84.12个孢子/mm2和6.49d,而转化子的半致死浓度、半致死剂量和半致死时间则分别增加到1.27×107个孢子/mL、382.92个孢子/mm2和8.09d,毒力显著下降。由此表明BbJEN1基因与球孢白僵菌分生孢子发芽以及毒力均有关。     

松褐天牛球孢白僵菌高毒力航天诱变菌株的筛选

【目的】松褐天牛Monochamus alternatus是我国松材线虫病Bursaphelenchus xylophilus的主要媒介昆虫。为了更好地开发利用松褐天牛病原微生物球孢白僵菌Beauveria bassiana, 本研究通过航天搭载诱变及室内筛选, 获得球孢白僵菌高毒力诱变菌株。【方法】将经神舟八号飞船航天搭载诱变后的孢子稀释液涂布在PDA平板上培养,获得单菌落菌株,进而筛选获得高毒力诱变菌株。观察所获9个航天诱变菌株的菌落形态、菌落生长速度、产孢量、孢子萌发率及抗高温胁迫能力等生物学特性, 在此基础上筛选出生物学性状优良的菌株B159, B252和B305, 并进一步对松褐天牛4龄幼虫进行生物测定。再通过撒菌粉和注射菌液方法, 检验B252和B305对松木段内松褐天牛幼虫的杀虫效果。【结果】球孢白僵菌航天诱变菌株的生物学特性与野生型菌株cfcc81357存在分化。9个航天诱变菌株的菌落形态发生了不同程度的改变,6个菌落生长速率出现负向变异,仅诱变菌株B159, B252和B305能产生分生孢子。航天诱变菌株B252和B305在浓度为1.0×107 cfu/mL时对松褐天牛4龄幼虫的校正死亡率均为100%, 半致死中时(LT₅₀)分别为8.08和8.56 d, 明显优于野生型菌株, 显示出对松褐天牛的极强毒力。使用撒菌粉和孢子液体注射方法, 诱变菌株B252和B305对松木段内松褐天牛幼虫死亡率比野生型菌株高。【结论】诱变菌株B252和B305可能是优良菌株, 对生物防治松褐天牛方面有潜在的实用价值。     

球孢白僵菌Bb_bm01菌株PacC基因突变体的构建及其毒力和孢子萌发率的测定

通过敲除球孢白僵菌(Beauveria bassiana)菌株bm01的PacC基因,研究了PacC基因敲除对球孢白僵菌毒力和分生孢子萌发速率的影响。利用农杆菌(Agrobacterium tumefaciens)介导的转化方法转化Bb_bm01,通过同源重组敲除Bb_bm01的PacC基因,筛选ΔPacC基因敲除菌株,获得了遗传稳定的ΔPacC基因敲除菌株。通过毒力测定,对比了野生菌株和ΔPacC基因敲除菌株对家蚕和大蜡螟毒力的差异,发现ΔPacC基因敲除菌株对家蚕和大蜡螟毒力都减弱,差异具有统计学意义(p0.01),表明在Bb_bm01菌株中的PacC基因与它的致病性相关;另外,比较ΔPacC基因敲除菌株与野生型菌株Bb_bm01在SDB液体培养基中的孢子萌发率,发现ΔPacC基因敲除菌株孢子萌发明显减慢,表明PacC基因参与调控孢子萌发。     

代谢组学方法鉴定球孢白僵菌孢子萌发和杀虫毒力相关的标记物

【目的】识别球孢白僵菌萌发和杀虫毒力相关的标记物。【方法】本研究对7个球孢白僵菌菌株的孢子进行萌发率和对茶毛虫毒力进行测定,采用基于LC-MS的代谢组学方法,识别菌丝和孢子提取物中与萌发和毒力有关的标记物。【结果】高毒力菌株的菌丝中具有高含量的carnitine、hercynine、acetylcarnitine、α,α-trehalose;Octa-Me、arg-arg-gln、phosphatidylethanolamine[PE(18∶2/0∶0)]、phosphotidylcholine[PC(18∶3/0∶0)]和PC(18∶2/0∶0)。高萌发率菌株的孢子中具有高含量的2,3-dimethylmaleate、acetylcarnitine、propionyl-carnitine和PC(18∶2/0∶0)。高毒力菌株的孢子中具有高含量的histamine、2,5-pyrrolidinedicarboxylic acid;Diamide、carnitine、acetylcarnitine、propionyl-carnitine、butyrylcarnitine、PE(18∶2/0∶0)、PC(16∶1/0∶0)和PC(18∶3/0∶0)。此外,对菌丝中杀虫相关的环肽beauverolides,beauvericins和bassianolide进行相对含量比较分析,发现单独一种肽的含量高低与菌株对茶毛虫的毒力没有直接关系,但高毒力的Bb1898菌株中的9种肽同时具有较高的含量暗示它们可能发挥协同作用。【结论】毒力和萌发的共同标记物是脂酰肉碱和磷脂,它们可能具有维持附着胞膨压和为穿透寄主提供能量的功能。其它标记物,如在高毒力菌丝中发现的hercynine和α,α-trehalose;高萌发率孢子中的2,3-dimethylmaleate,高毒力孢子中的histamine和2,5-pyrrolidinedicarboxylic acid Diamide,它们的作用原理有待进一步研究。     

球孢白僵菌在棉铃虫表皮上萌发的研究

在扫描电子显微镜下,观察了球孢白僵菌三株不同毒力菌株的分生孢子在棉铃虫幼虫表皮表面的萌发,发现高、低毒力菌株的分生孢子都能在棉铃虫幼虫表皮上萌发和生长,但高毒力菌株的萌芽管伸长至分生孢子长径的1.5～2.5倍时能发生定向生长,垂直向里穿透表皮,低毒力菌株的萌芽管呈蔓延生长,极少有穿透表皮的发生,偶见极度伸长的菌丝穿透表皮。此外,还发现球孢白僵菌穿透菌丝穿透表皮的过程不是垂直向里一步完成的。     

耐低水活度高毒力虫生真菌菌株选育

以球孢白僵菌Beauveria bassiana和玫烟色拟青霉Paecilomyces fumosoroseus为研究对象,利用紫外线诱变芽生孢子和低水活度条件胁迫筛选,选育出突变株Bb07240、Pf01120和Pf01160,它们在低水活度（或相对湿度）下的生长和萌发均明显优于其相应的初始菌株。生物测定表明,突变株对桃蚜的毒力明显高于其初始菌株。     

几种杀虫真菌对二化螟幼虫的毒力测定与高毒力菌株的分子鉴定

【目的】测定杀虫真菌对二化螟Chilo suppressalis(Walker)幼虫的室内毒力,明确高毒力菌株的种类,为应用杀虫真菌防治二化螟提供理论基础。【方法】采用喷雾法测定10株杀虫真菌在同种浓度、湿度和温度下对二化螟幼虫的室内毒力,选择毒力最强的菌株,利用扫描电镜观察杀虫真菌分生孢子对二化螟的侵染过程;根据该菌株的生物学特性,利用白僵菌Beauveria sp.18S r DNA ITS序列进行PCR扩增、琼脂糖凝胶电泳检测和序列分析,并在核酸序列数据库中进行同源性序列对比。【结果】真菌YQ147、QING、Bbr14、Bbr84在浓度为1.0×108孢子/m L下均对二化螟幼虫具有较强的致病力,其中真菌YQ147在处理后第10天对二化螟累计校正死亡率达到78.4%,LT50为5.8 d,分子鉴定的结果显示该菌为白僵菌属球孢白僵菌Beauveria bassiana。【结论】明确了球孢白僵菌对二化螟的室内毒力最强,这为利用其对二化螟进行生物防治提供了基础。     

锌离子响应转录激活因子ZafA对球孢白僵菌锌离子利用及生防潜能的影响

球孢白僵菌作为丝孢类昆虫病原真菌,已广泛应用于农林害虫的生物防治,但是田间多变的环境影响了真菌制剂的效能。此外,真菌侵染宿主后,宿主体内的环境也影响真菌的增殖和侵染速度。为研究球孢白僵菌对环境中酸碱度及微量元素的平衡能力,本研究初步探讨了锌离子响应转录激活因子ZafA与真菌生长繁殖、抗逆能力、毒力以及锌离子利用的关系。结果表明,敲除zafA削弱了真菌生长繁殖和孢子萌发的能力,增加了菌株对氧化、高渗、孢壁干扰剂以及紫外胁迫的敏感性,杀虫毒力显著下降,并抑制了相关性状基因的表达水平。基因敲除菌株无法在锌离子缺失的条件下生长,且zafA基因和锌离子转运蛋白编码基因zrf1–8的表达水平会受到酸碱度以及锌离子浓度的影响。由此可见,ZafA不仅直接影响球孢白僵菌利用锌离子的能力,还与球孢白僵菌抗逆能力和毒力密切相关,本研究为提高生防真菌环境适应性和发挥毒力提供新的依据。     

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。     

The potential of Beauveria bassiana inoculum formulated into a polymeric matrix for a microbial control of spruce bark beetle

Two local strains of Beauveria bassiana originally isolated from naturally infected spruce bark beetles in Slovakia were tested for their virulence to Ips typographus (IT) and for their compatibility with a polymeric matrix composed of low-molecular polyethylene. Conidia could be homogenously immobilized in the low-molecular polyethylene matrix with no adverse effect on their viability and infectivity. At constant temperature (25°C), viability of immobilized conidial decreased only by 1–2% after 7 or 14 days when compared with non-formulated conidia. In field conditions, viability of conidia formulated in the matrix was even significantly higher than non-formulated conidia 35 days after their application in traps. Conidia incorporated into the polymeric matrix were infective to IT adults in laboratory bioassays. Mean values of LC₅₀ for native conidia (0.72–2.05?×?10⁶ conidia?ml^?1) and conidia immobilized in the polymeric matrix (0.64–1.03?×?10⁵ conidia?mm^?2) demonstrated high virulence. The efficacy of the local strains was significantly higher than that of B. bassiana strains from mycoinsecticides (Boverol^®, Botanigard^® ES and Naturalis-L^®). Results showed potential of this polymeric material for its use in microbial control of IT when mixed with conidia of B. bassiana.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> pathogenicity to the cherry slugworm, <Emphasis Type="Italic">Caliroa cerasi</Emphasis> (Hymenoptera: Tenthredinidae) larvae

The cherry slugworm Caliroa cerasi is a significant destructive pest of sour cherries (Prunus cerasus) in Turkey. The potential of entomopathogenic fungi for controlling C. cerasi was evaluated. The effects of exposure methods and conidial concentrations (1 × 10⁶, 1.5 × 10⁶, 1 × 10⁷ and 1.5 × 10⁷ conidia/ml) on mature larvae of C. cerasi infected by Beauveria bassiana were investigated under laboratory conditions. Larvae sprayed directly with B. bassiana conidial suspensions and larvae exposed to B. bassiana-treated leaves resulted in 100% mortality within 2.90 and 2.77 days, respectively. The median lethal time (LT₅₀) and days to mortality were highest in the 1.0 × 10⁷ concentrations of conidia for both direct spray and leaf exposure. The present study suggests that B. bassiana has good potential for control of the cherry slugworm, C. cerasi.     

一株感染家蚕的高致病性白僵菌的筛选及复合诱变

【背景】白僵蚕中的生物活性物质在医疗、保健品及化妆品行业有着广泛的应用。目前,许多人工养殖僵蚕基地在实际生产中使用的菌种多为未进行纯化优选的自然感病死亡僵蚕孢子粉且无固定的施用浓度,使得蚕的僵化死亡率难以保证。提高白僵菌菌株的致病力并筛选性状优良的高毒力菌株是工厂化生产白僵蚕研发的重要方向。【目的】利用紫外-微波复合诱变技术筛选高毒力菌株,为僵蚕工厂化生产提供优良菌株。【方法】利用孢子稀释法从山西省养殖农户中自然感染白僵菌的家蚕中分离获得一株原始白僵菌,运用紫外-微波复合的方式对该菌株进行诱变,并比较诱变前后菌株的产孢量及对家蚕的致病力。【结果】分离得到的原始菌株经鉴定为球孢白僵菌(Beauveria bassiana),命名为Beauveria bassiana Bb1003。通过对致死率和正突变率的考察,确定紫外-微波复合诱变的最佳诱变条件为紫外(功率为15 W)照射30 min,微波(功率为800 W、额定微波频率2 450 MHz)辐照60 s。筛选后得到6株复合诱变菌株(UMCM1、UMCM2、UMCM3、UMCM4、UMCM5和UMCM6)。菌株UMCM2对家蚕的僵化率高达...     

响应面法优化爪哇虫草菌的培养条件及其对斜纹夜蛾的毒力和保护酶活性影响

【目的】优化爪哇虫草菌Bd01的固态发酵培养条件,测定分生孢子对斜纹夜蛾3龄幼虫的毒力,研究被爪哇虫草菌侵染后寄主体内的保护酶活性变化。【方法】采用单因素试验确定爪哇虫草菌Bd01最佳的固态培养基及培养条件,利用Box-Behnken响应面法优化发酵参数,采用浸渍法测定分生孢子对斜纹夜蛾3龄幼虫的毒力,同时利用分光光度计法测定斜纹夜蛾3龄幼虫体内酶活性变化。【结果】以产孢量为指标,通过响应曲面法优化的爪哇虫草菌Bd01最佳产孢条件为：培养基营养成分含量为30.24g/L,pH值为7.55,光照时长为12.06h,在该条件下,培养基的产孢量为2.78×10⁸孢子/mL。浓度为1×10⁸孢子/mL的爪哇虫草菌孢子液对斜纹夜蛾3龄幼虫具有一定毒力,处理7 d时致死中浓度(LT₅₀)为3.11 d,致死中时(LC₅₀)为4.68×10⁵孢子/mL,校正死亡率为88.68%。处理后未死亡的斜纹夜蛾幼虫体内保护酶活性与对照组相比发生显著变化。【结论】优化后的培养基能够显著增加爪哇虫草菌的产...     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

2018年我国19株猪链球菌4型分离株的病原学特征及分析

[背景]近年来,猪链球菌4型(Streptococcus suis serotype 4,SS4)分离率逐渐上升,但是有关SS4的系统研究报道匮乏.[目的]研究19株SS4临床分离株的病原学特征.[方法]以2株猪链球菌2型(Streptococcus suis serotype 2,SS2)强毒株为参考菌株,对19株S...     

球孢白僵菌种内比较线粒体基因组学分析

【目的】明确球孢白僵菌种内线粒体基因组的分化程度。【方法】从GenBank下载已知的球孢白僵菌6个菌株线粒体基因组序列,详细分析基因组的组成结构,比较外显子区、内含子区和基因间区的碱基变异情况,分析菌株间的系统发育关系。【结果】球孢白僵菌不同菌株的线粒体基因组大小为28.8–32.3 kb,都有14个常见的核心蛋白编码基因、2个rRNA基因和25个tRNA基因,具有很强的共线性关系。但是,不同菌株含有的线粒体内含子数目存在差异(2–5个/菌株),在cox1、cox2和nad1基因中表现出内含子插入/缺失多态性,这是导致线粒体基因组大小变化的主要因素。对外显子、内含子和基因间区的碱基变异情况进行分析,发现内含子和基因间区相对变异较大,而外显子区相对变异较小。系统发育分析发现,这些球孢白僵菌菌株以很高的支持度聚在一起,具有相同内含子分布规律的菌株也具有较近的聚类关系。【结论】本研究首次报道球孢白僵菌因内含子数目不同、插入缺失突变和单核苷酸变异等在线粒体基因组上表现出较大程度的遗传分化,为认识真菌种内线粒体基因组分化提供了新的证据。     

A new microbial consortia containing entomopathogenic fungus,Beauveria bassiana and plant growth promoting rhizobacteria,Pseudomonas fluorescens for simultaneous management of leafminers and collar rot disease in groundnut

The virulence of 20 isolates of Beauveria bassiana (Balsamo) Vuillemin to larvae of the leafminer, Aproaerema modicella, was tested in the laboratory. Leafminer larvae were sprayed with a standard concentration of 1×10⁸ condia/mL of each B. bassiana isolate. All the B. bassiana isolates tested were pathogenic to A. modicella and the mortality varied between 16.7 and 68.9%. Beauveria bassiana isolate B2 was found to be the most virulent followed by isolate B4 which resulted in 59% mortality. Beauveria isolate B2 was selected for dose–response mortality studies with four different doses (1×10², 1×10⁴, 1×10⁶ and 1×10⁸ conidia/mL). Among the various doses tested, 1×10⁸ conidia/mL produced the highest mortality (70.7%). In addition, morphogenesis of the insect pest in all stages, larval, pupal and adult was greatly affected due to fungal infection. Further, B. bassiana isolate B2 and two Pseudomonas fluorescens strains, TDK1 and Pf1 were tested alone and in combination for suppression of groundnut leafminer and collar rot disease and promotion of plant growth and yield both under glasshouse and field conditions. The mixture of B. bassiana and P. fluorescens strains significantly reduced the leafminer and collar rot disease incidences when applied as talc-based formulation through seed, soil and foliar application under glasshouse and field conditions.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.