球孢白僵菌羧基转运蛋白基因BbJEN1及其上游序列的克隆与分析

在分析一株球孢白僵菌TDNA插入突变体T12的Tagging序列的基础上,根据与其具有高度同源性的一条金龟子绿僵菌EST序列（编号为AJ273226）设计简并引物,用YADE法从球孢白僵菌中扩增出该EST的同源序列及其延伸序列。序列分析表明,该片段与粗糙脉孢霉的羧基转运蛋白JEN1具有高度同源性,由此确定该序列为球孢白僵菌羧基转运蛋白JEN1基因的部分序列。然后利用YADE法延伸扩增该序列的上、下游序列,获得球孢白僵菌羧基转运蛋白JEN1的全长DNA序列,命名为GBbJEN1。利用3′RACE扩增出球孢白僵菌羧基转运蛋白JEN1的cDNA序列,命名为BbJEN1。BbJEN1全长1656bp,编码514个氨基酸的蛋白。推测蛋白分子量为55975.37Da,等电点9.32。氨基酸序列与金龟子绿僵菌、粗糙脉孢霉和酿酒酵母羧基转运蛋白JEN1的同源性分别为77%、66%和30%。序列分析表明,GBbJEN1含有2个内含子。Southern杂交表明,GBbJEN1基因在球孢白僵菌基因组中为单拷贝。利用RTPCR法对BbJEN1的表达特性进行了分析,结果发现BbJEN1基因的转录受蟑螂壳、蝉蜕等昆虫体壁的诱导,受葡萄糖的抑制。进一步利用YADE法获得了长为977bp的GBbJEN1上游序列,其中含有可能的葡萄糖抑制调控序列和压力反应元件。     

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。     

桃蚜脱皮对球孢白僵菌毒力的影响

室内测定了球孢白僵菌Beauveria bassiana BBSG8702菌株对桃蚜Myzus persicae 若蚜的毒力,并分析了若蚜脱皮与白僵菌有效侵染之间的关系。在21℃、1×10⁶/mL孢子浓度下, 1～4龄若蚜感菌后的累积死亡率分别为10.1%、2.1%、3.1%和40.2%,明显低于同样条件下成蚜的感菌死亡率(98.4%)。在若蚜各龄感菌死亡的个体中,1～4龄中分别有8.1%、50.0%、44.4%和98.0%的若蚜在死亡前发育到成蚜,且都能产下正常发育的后代。若蚜的感菌死亡率与接菌后到第一次脱皮的时间密切相关,脱皮早则感菌死亡率低,反之亦然。但试验中也发现有部分若蚜特别是低龄若蚜感菌后未完成第一次脱皮便死亡。观察了桃蚜在1、4龄早期感染球孢白僵菌后的发育、存活和生殖情况,结果表明：1、4龄早期若蚜接菌后第一次脱皮的时间早于未接菌若蚜,但提前幅度少于5%,在羽化为成蚜之前被致死的概率分别只有4.1%和0,在羽化为成蚜后8天内的生殖率与未接菌蚜虫的无明显差异。这些结果表明,若蚜的脱皮,尤其当蚜虫在低龄若蚜时受感染后的多次脱皮,可有效地摆脱球孢白僵菌分生孢子的侵染,降低该菌的毒力。     

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。     

球孢白僵菌菌株间差异性的研究

本文对不同来源的4个球孢白僵菌菌株进行了培养观察。在培养基量、接种量相同;培养条件一致的情况下,比较它们之间的产孢量。并通过生物检测,计算出对菜青虫的致死中时。结果表明,菌株间在培养性状、产孢量及毒力诸方面均存在差异。     

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HFW-05对烟粉虱的致病力及组织病理影响

环境温湿度是影响白僵菌菌株致病力的关键因素,本文测定了不同温、湿度下白僵菌Beauveria bassiana HFW-05对烟粉虱Bemisia tabaci若虫及卵的致病力,同时观察了烟粉虱感病若虫的组织病理变化。结果表明,各浓度下烟粉虱若虫的校正死亡率随时间变化曲线基本一致,接种3-4 d时出现死亡高峰,白僵菌HFW-05对烟粉虱1龄、2龄、3龄若虫的LC_(50)分别为5.94×10~4、1.06×10~5、5.08×10~5孢子/mL,LT50分别为2.54 d、2.68 d、3.18 d;白僵菌HFW-05对卵的致病力很低,1×108孢子/mL接种浓度下,卵的6 d感染率仅为24.9%。在25℃、30℃下,HFW-05对烟粉虱若虫表现出最强的致病力,高于或低于此温度时,烟粉虱若虫死亡速度减慢,死亡率降低。相对湿度发生变化时,烟粉虱若虫死亡速度和死亡率明显不同,相对湿度高于90%时,烟粉虱若虫死亡速度最快,死亡率最高,相对湿度介于70%-90%时,白僵菌HFW-05对烟粉虱若虫的致病力无明显差异,但校正死亡率显著低于环境湿度大于90%的处理,相对湿度低于70%时,烟粉虱死亡率也达到60%左右。组织病理学观察显示尾部舌状突是HFW-05侵染烟粉虱若虫的关键部位。研究结果表明白僵菌HFW-05在烟粉虱的生物防治中有较大的潜力。     

球孢白僵菌对小猿叶甲的致病力测定

在室内研究了分离自小猿叶甲的一株球孢白僵菌(SCAU-BB 01D)对小猿叶甲的致病力。结果表明,该菌株能感染小猿叶甲的成虫和各龄幼虫,但对不同虫期的致病力存在差异。在105~108孢子/m l的浓度范围内,随着处理浓度的升高,各虫期小猿叶甲的感病死亡率增加,在最高浓度1×108孢子/m l,处理后成虫第14天及1~3龄幼虫第10天的累计死亡率分别为84.7%、94.0%、96.0%和81.0%。用TDM模型对成虫和各龄幼虫的致病力数据进行模拟,所建模型均顺利通过Hosm er-L em eshow拟合异质性检验,表明模型拟合良好,并由模型估计出了该菌株对小猿叶甲各虫期的致死剂量与致死时间。在处理后第10天,成虫和1~3龄幼虫的致死中浓度(LC50)分别为2.68×107、1.07×106、1.63×105孢子/m l和8.31×106孢子/m l,而第14天成虫的LC50为2.38×106孢子/m l。随着浓度的增加,各虫期所需的感病死亡时间缩短,在最高浓度1×108孢子/m l,球孢白僵菌对小猿叶甲成虫及1~3龄幼虫的致死中时(LT50)分别为9.28、4.29、4.40d和5.06 d。综合分析白僵菌对各虫期的致死剂量及致死时间可以看出,不同虫期的小猿叶甲对球孢白僵菌敏感性不同。结果表明该菌株在小猿叶甲生物防治中具有一定的潜力。     

NirA1基因对球孢白僵菌生长、抗逆及毒力的影响

[目的] 探究参与硝酸盐同化的一个特异转录因子NirA1对球孢白僵菌（Beauveria bassiana）生长、抗逆及毒力的影响。[方法] 利用RT-PCR检测球孢白僵菌NirA1基因在不同培养基中的表达特征。通过构建敲除突变株ΔNirA1、超量表达OENirA1及回复互补菌株ComNirA1,解析NirA1在球孢白僵菌生长发育、逆境胁迫反应和致病昆虫中的功能。[结果] NirA1基因在营养较贫瘠的CZB培养基中表达量高于丰富培养基PDB和SDB。敲除NirA1导致了菌株在人工培养基上的生长缓慢,对NaNO₂和Urea的利用效率降低。RT-PCR显示,ΔNirA1中硝酸盐转运蛋白基因NrtA的表达较野生型显著下调。在CZA、PDA和1/4 SDAY培养基中,其分生孢子产量分别较野生型降低了21.6%、16.2%和25.6%。逆境胁迫分析发现,在高温（32℃）,高渗（1 mol/L NaCl）和H₂O₂处理后,ΔNirA1菌落生长抑制率较野生型分别降低29.0%、25.2%及49.0%;但在添加SDS和刚果红（CR）的处理中,突变体菌落的生长抑制率分别较野生型升高34.1%和96.2%。因此,缺失NirA后,菌株对SDS和CR更加敏感,但对高温、H₂O₂和NaCl的耐受性提高。以3龄的大蜡螟为试虫的毒力测定表明,敲除NirA1基因导致菌株的毒力提高,半致死时间较野生型提前17.4%。[结论] NirA1在球孢白僵菌生长过程中参与了氮源的利用,并在球孢白僵菌的菌落生长、分生孢子生产、胁迫反应和致病宿主过程中发挥了重要作用。     

三株球孢白僵菌对栗实象甲幼虫的感染和致病力研究

为了明确球孢白僵菌Beauveria bassiana 3菌株TST05、FDB01、SYN01对栗实象甲老熟幼虫的感染、致死率、致死中时、以及胞外蛋白酶、几丁质酶、脂肪酶的活性与毒力的关系,本研究采用3个菌株的孢子悬浮液(1.0×108孢子/m L),浸虫法感染栗实象甲幼虫,统计连续8 d的校正死亡率;以栗实象甲幼虫的虫体作为菌株的唯一碳源制备液体培养基,测定培养过程中各菌株的脂肪酶、几丁质酶、类枯草杆菌蛋白酶(Pr1酶)连续8 d的酶活性变化。结果表明,球孢白僵菌3个菌株TST05、FDB01、SYN01感染栗实象甲幼虫后,1-4 d内表现出染病和死亡的症状,连续8 d累积校正死亡率分别为40%、44.4%、62.22%,菌株SYN01对栗实象甲的致死率最高,与其他两菌株的差异显著;菌株TST05、FDB01、SYN01的Pr1酶活性最大峰值分别为(175.56±0.7)U/m L、(172.74±2.32)U/m L、(195.71±5.41)U/m L。几丁质酶活性最大峰值分别为(12.58±0.58)U/m L、(13.06±1.16)U/m L、(15.12±0.32)U/m L。都是菌株SYN01的酶活性最大,而TST05和FDB01之间差异不显著;球孢白僵菌3个菌株的Pr1酶、几丁质酶、脂肪酶在8 d内的酶活性平均值与染菌幼虫8 d累积校正死亡率的线性回归方程分别为y=0.4641x+122.45(R2=0.8854)、y=0.034x+10.473(R2=0.9328)、y=0.0354x+2.4586(R2=0.1201),说明菌株的Pr1酶和几丁质酶活性与幼虫死亡率之间呈显著的线性相关,而菌株的脂肪酶活性与菌株对幼虫的致死率不具有线性关系。综合分析,菌株SYN01可以作为生物防治栗实象甲的病原菌种;Pr1酶与几丁质酶的活性可作为菌株的毒力因子。     

白僵菌固态发酵试验研究

以产孢量为指标,筛选出白僵菌固态发酵的培养基为：90％麦麸＋5％玉米粉＋5％黄豆粉,原孢粉的产孢量可达244．7亿／g。在25m^3发酵罐内进行浅盘固态发酵,培养基的最适起始含水率为60％～65％;最佳通气条件为：前12h不通气,12～48h通气量为1：2,48～144h的通气量为1：1;而温度只需在12～48＋8h内进行控制。常温干燥能最大限度地维持白僵菌的活孢率。     

球孢白僵菌对黄瓜代谢产物及西花蓟马取食行为的影响

球孢白僵菌Beauveria bassiana灌根处理黄瓜后,发现西花蓟马Frankliniella occidentalis对叶片危害面积显著低于对照组;为探究产生这一现象的原因,测定黄瓜叶片中防御酶系和次生代谢产物等含量变化,采用直流刺吸电位仪(direct current electrical penetration graph,DC-EPG)分析西花蓟马成虫取食黄瓜叶片的行为.球孢白僵菌对黄瓜灌根后叶片中过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)含量均呈上升趋势,其中CAT活性增加最高,增加率为37.26％;丙二醛MDA的含量呈下降趋势;可溶性蛋白、类黄酮的含量增加.DC-EPG显示西花蓟马在经球孢白僵菌处理的黄瓜上,取食时间和次数均低于对照组.结果表明,黄瓜植株经球孢白僵菌处理后防御酶物质增加,进而导致西花蓟马对叶片危害下降,利于对西花蓟马的防治.     

Evaluation of Beauveria bassiana (Hyphomycetes) isolates as potential agents for control of Dendroctonus valens

Abstract The red turpentine beetle (RTB), Dendroctonus valens LeConte, as a destructive invasive pest, has become one of the most economically important forest pest in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana, have shown great potential for the management of some bark beetle species. In this study, 12 isolates of B. bassiana from bark beetle were examined for biological characteristics and virulence, to assess their potential as biocontrol agents for RTB. There were significant differences (at P= 0.05) in colony growth rate, conidial yield, conidial germination, tolerance to UV light and extracellular proteases activity among the tested B. bassiana isolates. Isolates, including Bb1801, Bb1906, Bb789 and Bb773, exhibited the best characteristics, because they have faster hyphal growth rate, higher spore production and faster spore germination, higher UV tolerance and protease (Pr1) production. The results of a pathogenicity test of B. bassiana on RTB larvae showed that most isolates of B. bassiana have demonstrated high efficacy and the highest virulent isolate was Bb1801, which killed 100% of the treated insects and had a median lethal time (LT₅₀) of 4.60 days at a concentration of 1×10⁷ conidia/mL. Therefore, isolate Bb1801 has a great potential for sustainable control of RTB in the forest. The correlation between biological characteristics and virulence of the fungal isolates is discussed and the possibility of combination of entomopathogenic fungi with semiochemicals, as one of the promising strategy for RTB control, is considered.     

Relationship between thermotolerance and hydrophobin-like proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents

AIMS: This study was to illustrate the relationship between the thermotolerance and the contents of hydrophobin-like or formic-acid-extractable (FAE) proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus produced on rice-based substrate. METHODS AND RESULTS: Survival indices of 11 isolates were separately assessed as a ratio of the viability of conidia after 3-150 min thermal stress at 48 degrees C over that of unstressed conidia and fitted well to a survival model (r(2) >/= 0.97). For a given isolate, the fitted model generated an LT(50), the time for 50% viability loss under the stress. The LT(50)s of six B. bassiana isolates (10.1-61.9 min) and five P. fumosoroseus isolates (2.8-6.2 min) were correlated (r(2) = 0.81) with FAE protein contents (6.9-23.4 microg mg(-1)). The survival indices of a fixed B. bassiana isolate after 45-min thermal stress at 48 degrees C were also correlated to the FAE protein contents from conidia produced on glucose-, sucrose-, or starch-based substrate (0.79 相似文献   

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

美国白蛾高毒力球孢白僵菌菌株筛选

【目的】美国白蛾Hyphantria cunea(Drury)是世界性检疫害虫,目前已经入侵到我国长江下游地区(从辽宁丹东至安徽芜湖)。作者在前期研究中,在芜湖地区成功获得了被球孢白僵菌Beauveria bassiana侵染的美国白蛾僵虫。本研究拟在前期已获取菌株的基础上,筛选生物学性状良好且对美国白蛾幼虫具有高致病力的球孢白僵菌菌株,以期为我国长江下游地区利用昆虫病原真菌防治美国白蛾奠定基础。【方法】对采集自美国白蛾僵虫的7株球孢白僵菌菌株(Bb01-1,Bb08-5,Bb08-6,Bb08-11,Bb22-2,Bb22-3和Bb22-5)进行生物学特性研究,以候选菌株菌落形态、生长速率、产孢量、萌发率和抗紫外能力等参数确定优良菌株;并将生物学性状良好的球孢白僵菌菌株配制成5个浓度(5×10~6,1×10~7,5×10~7,1×10~8和5×10~8孢子/m L)的孢悬液,采用浸渍法处理美国白蛾4龄幼虫,以幼虫死亡率、侵染率等指标评价菌株的致病力。【结果】供试菌株Bb22-2,Bb22-3和Bb22-5为粉状菌落,其菌落直径大,产孢早且孢子层厚;同时,这3个菌株在菌落生长速率和产孢量以及孢子萌发率、紫外照射处理后的萌发率也均显著优于其他4个菌株。毒力测定结果表明,这3个菌株对美国白蛾4龄幼虫表现出很强的致病力,各菌株处理11 d后美国白蛾幼虫校正死亡率为80%~90%。这3个菌株对美国白蛾幼虫的侵染率均高(侵染率53%~87%),并且呈现随浓度升高侵染率上升的趋势。其中,菌株Bb22-2侵染率均要高于其他2个菌株。【结论】本研究成功筛选获得了生物学性状良好且对美国白蛾高毒力的球孢白僵菌菌株。鉴于中国长江下游地区气候特点与美国白蛾生物学习性,可以预期采用昆虫病原真菌防治美国白蛾在该地区将具有很好的应用前景,但其实际应用价值尚需进一步的田间试验进行确认。     

Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii

The commercial use of entomopathogenic fungi and their products as mycoinsecticides necessitates their registration. Worldwide, several registration guidelines are available, however, most of them focus on similar or even the same safety issues. With respect to the two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. and Beauveria brongniartii (Sacc.) Petch, many commercial products have been developed, and numerous papers on different biological, environmental, toxicological and other safety aspects have been published during the past 30-40 years. The aim of the present review is to summarise these data. The following safety issues are presented: (1) identity of Beauveria spp.; (2) biological properties of Beauveria spp. (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors); (3) analytical methods to determine and quantify residues; (4) fate and behaviour in the environment (mobility and persistence in air, water and soil); (5) effects on non-target organisms (non-target microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms and nontarget arthropods); (6) effects on vertebrates (fish, amphibia, reptiles and birds); and (7) effects on mammals and human health. Based on the present knowledge it is concluded that both Beauveria species are considered to be safe.     

A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence

In order to investigate virulence enhancement of entomopathogenic fungi, a Beauveria bassiana-sourced Pr1 protease (CDEP-1) was expressed by a methylotrophic yeast Pichia pastoris and then used as an additive to three gradient sprays of B. bassiana strain (Bb0062) onto apterous green peach aphid Myzus persicae adults in six bioassays. The resultant data fit well to a time–concentration–mortality model. Generally, the LC₅₀ estimates of the fungal pathogen against the aphid species decreased with increasing CDEP-1 concentrations from 0 to 100 µg mL^?1. The LC₅₀s on days 5–7 after spray were reduced by 1.5–2.5-fold at the concentrations of 20–100 µg mL^?1. However, sprays of 20–100 µg CDEP-1 mL^?1 aqueous solution alone had no significant effect on aphid mortality compared to water spray only. Neither did inclusion of inactivated CDEP-1 at a concentration of 50 µg mL^?1 affect significantly the fungal virulence to aphids. Our results confirm for the first time that the cuticle-degrading protease CDEP-1 enhanced fungal virulence due to acceleration of conidial germination and cuticle penetration. This suggests a new approach to utilising the protease in microbial control.