重要昆虫病原真菌粉棒束孢种群的遗传分化

粉棒束孢是一种重要的昆虫病原真菌.运用ISSR分子标记研究了安徽省6个不同地理环境的粉棒束孢种群遗传异质性.结果表明:10个多态性引物多态位点百分率高达98.5％,但种群水平的多态位点差异较大,在59.6％ - 93.2％.基于Nei遗传异质性分析得出各种群间的遗传分化系数(Gst)为0.3365,基因流(Nm)为0.4931;各种群间的遗传分化低于种群内的遗传分化,表明安徽省粉棒束孢的遗传变异主要存在于种群内.根据各菌株间的遗传相似系数进行UPGMA聚类,结果表明,西山种群是单系的同质种群,其余5个种群皆为多系的异质种群,其中鹞落坪种群的异质性最高,琅琊山种群异质性最低.各种群之间的地理距离与遗传距离之间无相关关系.根据6个种群间的遗传距离进行UPGMA聚类,可将它们分成3个类群,分类的结果与各种群的地理环境相符,反映出环境的异质性对种群异质性的影响.     

2株烟粉虱高致病力病原棒束孢菌株的毒力测定

烟粉虱(Bemisiatabaci)是世界性分布且危害严重的蔬菜花卉害虫.由于烟粉虱的抗药性及传统化学药剂施用潜在的负面影响,利用病原微生物、天敌昆虫等开展烟粉虱的生物防治一直是国内外学者们研究的重点与热点之一.本研究在室内条件下,分析了爪哇棒束孢(Isaria javanica)GZQ-1菌株和玫烟色棒束孢(Isariafumosorosea)YMS01菌株对烟粉虱(MEAM1隐种)不同虫态的致病力.结果表明,2种棒束孢昆虫病原真菌均能侵染烟粉虱各个龄期,且随着孢子浓度的升高,烟粉虱的死亡率不断上升;在1×10⁸孢子/mL时,2株棒束孢对烟粉虱2龄若虫的感染致死率分别达到92.68%和78.12%;此外,2株棒束孢菌株对2龄若虫的致病力回归方程分别为y=0.421x-1.665,y=0.33x-1.646, LC₅₀值分别为9.01×10³孢子/mL和9.74×10⁴孢子/mL, LT₅₀为3.05 d和4.56 d.综上所述,本研究所评价的2株棒束孢均对烟粉虱有较高的致病力...     

我国棒束孢一新记录种—炭角棒束孢Isaria xylariiformis

<正>棒束孢Isaria是Clements Shear于1931年以粉质棒束孢Isaria farinosa(Holmsk.)Fr作为模式种建立的。Brown Smith(1957),将原分类于棒束孢中的虫生种移入了拟青霉属Paecilomyces中。     

玫烟色棒束孢几丁质酶的转基因球孢白僵菌菌株的获得及其对马尾松毛虫的毒力增效作用

本文根据GenBank中已登录的蜡蚧蚧霉Lecanicillium lecanii,粉棒束孢Isaria farinosa,球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae的几丁质酶基因序列的同源性比较, 以它们高度保守的核苷酸序列设计一对引物,采用RT-PCR和3′/5′-RACE相结合的方法,首次从玫烟色棒束孢Isaria fumosorosea中克隆出几丁质酶Ifuchi1全长cDNA基因。其全序列长为1 542 bp,编码阅读框由1 275个核苷酸组成, 5′非翻译区（5′-UTR）与3′非翻译区（3′-UTR）分别为33个核苷酸和234个核苷酸。基因编码424个氨基酸, 信号肽长度为24个氨基酸, 成熟蛋白理论分子量为47.6 kDa,理论等电点为4.89。该蛋白可归于几丁质酶18族V类。运用制备芽生孢子转化法将Ifuchi1基因导入球孢白僵菌。最适产酶时间36 h条件下, 挑选的转基因菌株的几丁质酶活性相对于野生型菌株提高了86.2%;在对马尾松毛虫的生物测定中,在孢子浓度为1×10⁷个孢子/mL时,与野生型菌株Bb13相比,转基因菌株的致死中时间LT₅₀平均缩短了29%,同时死亡率提高了52.9%;野生型菌株和转基因菌株致死中浓度LC₅₀分别为4.71×10⁶ 个孢子/ mL和1.74×10⁶ 个孢子/ mL,降低了约1.7倍的剂量,故通过基因工程方法获得的转基因工程菌株显著地提高了球孢白僵菌的杀虫效率。     

几株类棒束孢菌株分类地位的修订

【背景】粉棒束孢是自然环境中常见的一种昆虫病原真菌,全球广泛分布且寄主多样。然而,棒束孢属许多种形态学特征相似,仅根据经典形态学鉴定容易出现错乱现象。长期以来依据形态特征鉴定为粉棒束孢的菌株,其准确的分类地位出现了越来越多的争议。最近,该属起源被确定是多系发生的。【目的】明确粉棒束孢的分类沿革并探究先前被鉴定为粉棒束孢菌株的准确分类地位。【方法】选取6株先前被鉴定为粉棒束孢的菌株,对其进行经典形态观察,并扩增5个基因位点(nrSSU、nrLSU、TEF、RPB1和RPB2)的基因序列,进行多基因分子系统发育学分析。【结果】通过查阅文献和实验研究,明确了粉棒束孢分类研究的历史沿革;供试的6株不同来源的菌株与粉棒束孢模式种亲缘关系较远,而与新成立的组合种Samsoniella hepiali完全一致。【结论】利用经典形态学特征和构建5个基因系统发育树,重新修订6株类棒束孢菌株的正确分类地位为Samsoniella hepiali。为该类昆虫病原真菌正确分类提供合理的方法,也为开发和利用该类菌株提供科学的指导。     

土壤微生态环境对冬虫夏草寄主蝙蝠蛾幼虫病原菌粉棒束孢宿存及毒力的影响研究

测定了温度、含水量、土壤微生物三个因素以探讨蝙蝠蛾幼虫生长过程中微生态环境对粉棒束孢(Isaria farinose)宿存及毒力的影响。结果表明粉棒束孢30℃保存6个月的宿存量降低至10~3个/g以下。随着保存温度降低,宿存量的下降速度变缓,粉棒束孢在-18℃保存1年后,宿存量仍基本维持在10~6个/g以上。粉棒束孢在低湿环境下(含水量35%)保存1年的宿存量维持在10~4个/g,含水量45%～65%,粉棒束孢宿存量会先增加后降低,降低的速度基本随湿度的增加而增加,1年后宿存量降低至10~3个/g以下。土壤微生物对粉棒束孢的宿存具有明显抑制作用,野外高山草甸土壤中微生物显著抑制粉棒束孢萌发,不利于粉棒束孢的宿存,而室内饲养土壤由于幼虫排泄物、饲料腐殖质可能有利于粉棒束孢的宿存。上述研究结果为进一步制定粉棒束孢防治方法,同时也为粉棒束孢作为生防菌的有效保存提供了参考。     

液体或固体培养对环链棒束孢(Isaria cateinannulata)菌丝体中挥发性成分组成的影响

[目的]测定环链棒束孢(Isaria cateinannulata)在不同培养方式下,其菌丝中挥发性代谢物组成,分析代谢产物差异与培养方式间的相互联系.[方法]分别用SDAY培养基固体平板与SDY培养基液体摇瓶的培养方式进行培养;培养温度为25℃,培养周期为8天;用同时蒸馏萃取装置提取菌丝的挥发性成分;用气质联用仪进行数据采集及分析.[结果]固体培养菌丝体含有较多种类的挥发性成分,共鉴定41种化合物,其中酯类、醌类和肟类化合物为其特有.液体培养菌丝体含有的挥发性成分相对较少,共鉴定32种化合物,其中羧酸类化合物为其特有,同时酚类含量远高于固体菌丝.固体和液体培养菌丝体的主要挥发性成分都是烃类物质,其中烯烃类物质分别占固体培养菌丝和液体培养菌丝总挥发物的57.6％和7.85％,烷烃类物质分别占固体培养菌丝和液体培养菌丝总挥发物的9.19％和22.4％.[结论]固体或液体培养条件对环链棒束孢菌丝体中挥发性成分的组成有影响.     

环链棒束孢Isaria cateinannulata发酵液的化学成分分析

环链棒束孢广泛存在于自然界,并且在植物病虫害防治中起重要作用,目前相关的研究多集中于其发酵条件、生物学特性及杀虫毒力的研究,对该菌发酵液中的化学物质研究甚少。本文依次用石油醚和乙酸乙酯对环链棒束孢发酵液进行分级萃取,借助气相色谱-质谱（GC-MS）联用技术和超高效液相色谱串联四级杆飞行时间质谱法（UPLC/Q-TOF-MS）鉴定分析石油醚萃取物、乙酸乙酯萃取物和萃余物中的化学物质组成。共鉴定69个化合物的结构,其主要化学成分为酚类、酯类、烷烃、烯烃、卤代烃和酮类非极性/弱极性的小分子化合物等,其中邻苯二甲酸二丁酯、角鲨烯、环戊烯丙菊酯物质、瓜叶菊素Ⅰ等物质具有特殊的作用。发酵液提取物中还含有多种小分子化学物质,应结合多种色谱分离手段和波普学手段对代谢产物进行分离、纯化和结构鉴定,为其次生代谢产物的广泛利用提供数据支撑。     

环链棒束孢菌株培养特征、致病性及遗传变异研究

为筛选一种对小菜蛾有高致病力的杀虫真菌,对来自不同地域的环链棒束孢菌株的培养特征尤其是孢梗束形成、对小菜蛾的致病性和基于5.8S-ITS nrDNA构建的系统发育等进行分析。结果表明,供试菌株的培养性状可分为3个类型：孢梗束浓密型、孢梗束稀疏型和不产孢梗束型。孢梗束浓密型对小菜蛾的致病性最高,平均达到88.9%,其中XS.1菌株,对小菜蛾幼虫的致死率达到98%;孢梗束稀疏型次之,为68.4%;不产孢梗束型最差,仅35%。系统发育聚类树分析表明,在环链棒束孢菌株中,致病性较高的菌株,如XS.1,XS.2和SL.7等聚在一亚分支内,致病性低的菌株8.02和468.10聚在一起;不产孢梗束的两个菌株8.02和468.10聚在一个亚支。这些结果表明环链棒束孢菌株间具有明显的种内遗传变异性。孢梗束形成与小菜蛾的致病死亡率有相关性。孢梗束的形成可作为高致病性菌株选择的一个重要指标。     

多基因联合分析棒束孢属Isaria（Ascomycota,Cordycipitaceae）系统发育关系

对棒束孢属Isaria及近缘属物种开展5基因（nrSSU、nrLSU、tef-1α、rpb1和 rpb2）测序并联合分析,结合GenBank相关类群序列,探讨棒束孢属系统发育关系,最终获得95个菌株、58个明确分类群的2-5基因序列。利用MEGA和MrBayes软件进行多基因聚类分析,结果表明棒束孢属多系起源于虫草菌科中,分3个不同分支。A支主要由Isaria cicadae、I. teniupes、I. coleopterorum、I. fumosorosea和I. cateniannulata等组成;B支包括I. poprawkii、I. locustica、I. javanica、I. amoenerosea和I. cateniobliqua;C支仅有I. farinosa。分支间被Cordyceps militaris、C. ninchukispora、C. pruinosa等隔开。棒束孢在形态上,主要以瓶梗基部膨大、尖端变细及孢子呈链状等特征与其他类群分开,但同时也发现有棒状分生孢子梗和单孢子类型。基于节点的分歧时间预测分析,推测棒束孢属首次分化于70Mya,但棒束孢属主要物种形成却在60-55Mya,且3个分支的棒束孢物种为快速同时形成,而后大多数类群表现遗传稳定。同时发现,与Isaria Clade A较近一支有粉被玛利亚霉Mariannaea pruinosa（C. pruinosa无性型）和蛹草蚧霉Lecanicillium militaris（C. militaris无性型）;与粉棒束孢距离最近一支有Akanthomyces aculeatus（C. tuberculata无性型）和L. attenuatum（C. confragosa无性型）,是两个不同的属征分类群,且相互间遗传距离较近。根据棒束孢属及其近缘种属形态特征的复杂性推测,棒束孢属在快速物种形成中,其近缘类群存在一定程度的丢失和选择性演化。     

The entomopathogenic fungi Isaria farinosa (formerly Paecilomyces farinosus) and the Isaria fumosorosea species complex (formerly Paecilomyces fumosoroseus): biology,ecology and use in biological control

The entomopathogenic fungi, Isaria farinosa and Isaria fumosorosea were known as Paecilomyces farinosus and Paecilomyces fumosoroseus, for more than 30 years. Both fungi have a worldwide distribution and a relatively wide host range. While I. farinosa currently is of minor importance in research and as biocontrol agent, I. fumosorosea is regarded as a species complex, and various strains are successfully used for biocontrol of several pest insects, mainly whiteflies. During the past 40 years, numerous scientific papers on both fungal species covering various research areas were published. This is the first monographic review on the biology, ecology and use of I. farinosa and I. fumosorosea as biocontrol agents. In the following compilation, data on the identity, natural occurrence and geographical distribution, host range, production of metabolites, effect of abiotic and biotic factors (temperature, relative humidity, solar radiation, fungus, host plant) and on the use of these species in biocontrol experiments against pest insects, plant pathogens and nematodes in the laboratory and the field are summarised. Sections on their effects on non-target organisms and safety issues are also included. The aim of this review is to increase our knowledge on these entomopathogenic fungi.     

Optimization of Fermentation Medium and Conditions for Mycelial Growth and Water-soluble Exo-polysaccharides Production by Isaria farinosa B05

Abstract

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K₂HPO₄ 0.1%, and MgSO₄ 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25°C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.     

蝙蝠蛾幼虫感染粉棒束孢后的病症、组织病理变化及侵入途径

蝙蝠蛾幼虫的饲养是冬虫夏草培植的先决条件,粉棒束孢作为蝙蝠蛾幼虫饲养过程中的一种常见和主要致病菌,具有极强的致病性和流行性。结合形态学观察和组织切片技术,本文研究了蝙蝠蛾幼虫感染病原菌粉棒束孢后的症状、主要组织病理变化及粉棒束孢侵染蝙蝠蛾幼虫的途径。结果表明,用浓度为1×10⁵个/mL的粉棒束孢分生孢子悬液处理蝙蝠蛾5龄幼虫,4d后在腹部气门处有黑斑出现,气门变黑,虫体随后瘫痪死亡、体色改变、收缩化僵、菌丝层包裹、形成众多孢梗束等症状。幼虫被粉棒束孢感染后,菌丝体首先利用血淋巴进行增殖,脂肪体、肌肉、消化道、丝腺和马氏管等主要组织器官先后出现不同程度的破坏,虫体体腔被大量菌丝充斥并最终僵化。组织切片观察结果表明,粉棒束孢可以通过气门侵入蝙蝠蛾幼虫体内并造成虫体死亡,菌丝穿透气管进入血淋巴后断裂成许多长短不一的短菌丝段,目前尚未观察到菌丝直接穿透幼虫体壁和从消化道侵入的现象。本研究为蝙蝠蛾幼虫饲养过程中的病害防治提供信息参考。     

Isolation and characterisation of Isaria farinosa and Purpureocillium lilacinum associated with emerald ash borer,Agrilus planipennis in Canada

Entomopathogenic fungi of the genera Isaria and Purpureocillium were recovered from infestation sites of emerald ash borer (EAB) in Southern Ontario, Canada. Isolates were identified using morphological characters and by sequencing the ITS1-5.8S-ITS2 ribosomal DNA gene and partial β-tubulin gene. Phylogenetic analysis and constructed trees based on the ITS and β-tubulin gene explicitly confirm isolates L66B, SY17-a and LHY46-a as Isaria farinosa and B3A, B59A and SY45B-a as Purpureocillium lilacinum. Pathogenicity was tested in the laboratory against adult EAB using a single concentration (2.0×10⁷ conidia/ml) applied topically to adults. Controls included three commercial isolates: Isaria fumosorosea LRC176, Metarhizium brunneum LRC187 and Beauveria bassiana strain GHA. The native isolates I. farinosa L66B and P. lilacinum SY45B-a killed 75 and 50% of the beetles 14 days post-inoculation. Although these indigenous entomogenous fungi were less virulent compared with the commercial isolates, yearly isolation from EAB populations suggests they are one of the natural mortality factors of EAB in Canada.     

The antitumor and antioxidative activities of polysaccharides isolated from Isaria farinosa B05

The water-soluble intra-polysaccharides WIPS1 and water-soluble extra-polysaccharides WEPS1 were isolated from Isaria farinosa B05 through ethanol precipitation and gel permeation chromatography (GPC). Their characteristics were determined by chemical analysis, gas chromatography, GPC and IR spectroscopy. The results show that WIPS1 contained 90.3% carbohydrate, 8.00% uronic acid, 7.15% protein and three kinds of monosaccharides including mannose, galactose and glucose with a molar ratio of 8.0:4.8:1.0. WEPS1 contained 93.4% carbohydrate, 8.06% uronic acid, 4.40% protein and three kinds of monosaccharides including mannose, galactose and glucose with a molar ratio of 21.6:4.7:1.0. WIPS1 and WEPS1 had a molecular weight of 42 and 208kDa, respectively. The in vivo tests in mice indicate that WIPS1 and WEPS1 had significant antitumor and antioxidative activities to some extent.     

Biochemical and molecular characteristics of indigenous strains of the entomopathogenic fungus Beauveria bassiana of Central India

Forty-eight isolates of indigenous strains of Beauveria bassiana from various insect hosts collected from Central India were characterised by employing protease zymography and RAPD analysis. Results of protease zymographic profiles were reproducible and significant enough to contribute to the biochemical diversity of this species. RAPD analysis revealed the presence of high level of genetic diversity and indicated that 0-66% significant differences has evolved between these isolates. The sets of amplified bands showing identical pattern to others were grouped at 100% similarity level. A wide range in the value (0.25-1.00) of Jaccard similarity coefficient was observed among all the isolates. The grouping of the indigenous strains, obtained from numerical analysis of these data, appears to be related to the host specificity in B. bassiana. Clear groups were seen for strains isolated from Lepidopteran and Coleopteran insect hosts.     

Conidia production by Isaria fumosorosea on solid substrates and its pathogenicity towards Bemisia tabaci

Large amounts of conidia must be recovered to use Isaria fumosorosea as a biological control agent. Aerial conidia are mass-produced in solid substrate culture. Conidia obtained from growth on different substrates must be similar in quantity and quality (viability, purity and virulence). In the present work, several solid substrates were evaluated: rice, sorghum, sugarcane, corncob, plantain and wheat (derived from the production of Trichogramma sp.) to find alternatives that are economical and available in different regions. The highest conidia production was obtained on rice, plantain and corncob substrates, which yielded 5.33 × 10⁹ conidia/g, 5.24 × 10⁹ conidia/g and 4.8 × 10⁹ conidia/g substrate, respectively. At concentrations of 1 × 10⁷ conidia/mL, the mortality rates obtained with conidia developed on rice, plantain and corncob substrates were 98%, 97% and 88%, respectively. The lethal concentration 50 (LC₅₀₎ values of conidia obtained from rice and plantain were 7.2 × 10⁴ conidia/mL and 5.1 × 10⁴ conidia/mL, respectively.     

大孢虫花生物学特性及驯化栽培

对采自辽宁省仙人洞国家级自然保护区野生大孢虫花(Isaria japonica Yasuda)的生物学特性和驯化栽培进行探究。试验结果表明:PDA培养基上适宜碳、氮源分别是蔗糖和谷氨酸,适宜p H为6;在3种供试培养料中只能在配方一(2%葡萄糖,0.1%硫酸镁,0.2%磷酸二氢钾,1.4%琼脂,96%蒸馏水,p H自然,0.3%蛋白胨)中形成子实体。