【作者】 韩燕峰；

【导师】 康冀川；

【作者基本信息】 贵州大学 ， 动物学， 2007， 博士

【摘要】 拟青霉Paecilomyces Bain.的许多种是重要的昆虫病原真菌，其中一些种是虫草(Cordyceps)的无性型(anamorph)。作为一类寄生于昆虫的真菌资源，它们不仅在有害生物的控制上发挥着重要的作用，而且在医药卫生、功能食品、环境污染和治理以及基因工程等方面也有重要作用。本论文对我国拟青霉属真菌在以下方面取得了有新意的研究结果：1．中国拟青霉资源调查及经典分类鉴定结果[1]通过对全世界已发表的125种拟青霉进行研究整理，作者认定拟青霉属现仅包含64个有效分类单元，寄生昆虫的种为34个，占了该属成员的53.1％。[2]研编了以微观特征和培养性状为主的拟青霉属已知种Access检索数据库。实践结果表明，这个数据库对待定种的初步鉴定方便、快捷和有效。[3]对我国拟青霉进行了调查和分类研究。从全国32个省市采集的975份土样和164份罹病昆虫标本中分离到大量真菌菌株，经初步鉴定，共获得298株拟青霉，加上贵州大学真菌资源研究所保存的标本和菌种，作者共观察研究了313株拟青霉。在以表型特征为主的基础上，描述我国拟青霉39种，占了全球拟青霉总数的63％，这表明我国拟青霉具有丰富的物种多样性。在这39种拟青霉中，包括已知种20个，新纪录种3个，新组合1个和新种15个。此外还分离到拟青霉相近属的4个中国新纪录种：丽玛利亚霉鲜红变种Mariannaea elegans(Corda)Samson var.punicea Samson、印度金孢Chrysosporium indicum(H.S.Randhawa& R.S.Sandhu)Garg、裂叶金孢Chrysosporium lobatum Scharapov和高山弯颈霉Tolypocladium nubicola Bissett。对分离得到的一些拟青霉和相近属的中间类型菌株如P.sp A7-1、P. sp A17-2、P. spA18-1、P. sp A19-3、P. sp A40-1、P. sp A43、P. sp A62-1、P. sp DH3-1、P. sp LN2-9、P. sp 3’29-1和P. sp D23-1结合分子生物学方法进行了分类鉴定。[4]初步统计显示，中国拟青霉的地理分布面非常广。其生存范围涉及各种土壤、植物和多种罹病昆虫。寄主昆虫范围有鳞翅目、膜翅目、同翅目、半翅目和鞘翅目等昆虫的成虫、若虫和蛹，还可寄生其他动物如线虫、蜘蛛等。但不同种的分布范围有明显差异。如淡紫拟青霉和马昆德拟青霉几乎能从各个省市土样中分离到；而念珠藻状拟青霉和一些耐热的单瓶梗拟青霉的出现频率较低，分布面较窄。以秦岭——淮河为南北方划分界线发现，南方拟青霉菌株的检出率比北方要高，尤其是罹病昆虫上拟青霉的数量更为明显。从采集的各种生境和寄主看，土样中发现拟青霉的频率和种类最多，大多数拟青霉新种(23.9％)都来自土壤。2．Delta专家分类系统在我国虫生拟青霉种间的应用研究[1]在以经典分类时建立的Access检索数据库的基础上，构建了我国24种虫生拟青霉种的DELTA数据库。该数据库是实现如下功能扩展的基础。[2]中国虫生拟青霉种检索表的自动生成；中国虫生拟青霉的自然语言描述；中国虫生拟青霉种的距离矩阵及聚类树系图(Phenetic tree)和拟青霉种间的交互式自动鉴定。3．中国拟青霉的分子系统学研究[1]部分拟青霉菌株的分子鉴定。用测得的ITS区域rDNA序列，在GenBank经Blast比对后，下载与其相似性最大的一些近缘种和表型特征相近的种共同建树进行分析。在表型特征鉴定的基础上，得到分子系统发育树支持作为新种成立的拟青霉有：纤姿拟青霉P. tenuis；紫拟青霉P. purpurseus；轮生拟青霉P. verticillatus；柱孢拟青霉P. cylindricosporus；花溪拟青霉P.huaxiensis和小孢拟青霉P.parvosporus。[2]一些单瓶梗拟青霉分类地位的确定——戴氏霉新属的建立。基于ITS1-5.8S-ITS2 rDNA和18S rRNA序列拟青霉和一些近缘种的分子系统发育树分析表明，8个单瓶梗拟青霉与粪壳菌目有密切的亲缘关系。表型特征和分子系统发育综合分析，支持这些单瓶梗种作为一个新属成立。此属包括11个成员，其中3个新种，8个新组合。[3]环链拟青霉菌株间的遗传多样性。作者对收集到的16个菌株测定了ITS1-5.8S-ITS2 rDNA序列和从Genbank得到的3株共19株环链拟青霉序列进行比对，构建了系统发育树。结果表明，19株环链拟青霉表现出了极高的遗传多样性，ITS1-5.8S-ITS2序列能较好地反应种内遗传差异。环链拟青霉菌株间遗传差异与地域有一定相关性。[4]中国拟青霉菌株间的分子系统学分析。基于61株拟青霉和相关菌的ITS1-5.8S-ITS2 rDNA序列构建的中国拟青霉的系统发育树表明，拟青霉属的种类在演化上是多源发生的，交叉于肉座菌目和散囊菌目，涉及了其中多个科。[5]中间类型种的分子鉴定。用93株拟青霉以及属于散囊菌目中青霉属Penicillium，篮状霉属Talaromyces，衣丝霉属Byssochlamys和隐囊菌属Aphanoascus中的相关菌株构建了系统发育树，结果支持了之前已报道的3个新种的成立；发现待定菌株A18.1应是拟青霉的一个新种；菌株A62.1应是青霉属中的一个成员。待定菌株LN2.9，A40.1，A19.3和H3.1与所用的4个青霉都能明显区分开，与拟青霉聚在一起，且各自为一小分支，作者暂时把它们作为拟青霉属中的新分类单元。4．部分拟青霉菌株对小菜蛾致病性的初步研究本研究采用从土壤和罹病昆虫得到的部分拟青霉菌株和白僵菌共30株14种采用浸液法进行试验，得到如下结果：[1]感染症状表现。小菜蛾幼虫受所试白僵菌JYT12感染后，皮肤逐渐失去光泽，变为黄褐色。而受拟青霉菌株感染，死亡时虫体颜色不发生明显变化。这些受感染死虫若继续在25℃下保湿培养，整个虫体被白色菌丝和孢子粉覆盖；[2]不同供试菌株对小菜蛾的致病性差异。结果表明不同菌株间的致病性有明显差异，其中所试的25个菌株(83.33％)对小菜蛾有致病性。玫烟色拟青霉、环链拟青霉和球孢白僵菌是感染致死小菜蛾效果较好的三种昆虫病原真菌。拟青霉同种不同菌株对小菜蛾的致病性也表现出很大差异。如环链拟青霉中的SL.8、SL.7、XS.1和XS.2四个菌株的效果最好，在所试条件下达到了100％的致死率；环链拟青霉8.02菌株效果最差，死亡率小于＜10％。来自土壤的菌株对小菜蛾无致病性或仅具微弱致病性。[3]不同菌株的环链拟青霉产孢量差别非常大。产孢量最高的环链拟青霉XS-1菌株(4.91×10~8／cm~2)比产孢量最低的环链拟青霉XSXY-4菌株(0.45×10~8／cm~2)高出11倍之多。产孢量与致病性之间有一定的正相关，产孢量高的致病性相对较高，而产孢量低的其对小菜蛾的致病率也较低。[4]环链拟青霉不同菌株的菌落形态大致可归纳为三大类：[1]菌落隆起；[2]菌落表面花瓣状；和[3]菌落平展。初步观察说明各菌株间培养性状差异与其地理来源无明显相关性。不同菌株的菌落形态与产孢量有一定关系，菌落花瓣状，产孢量最大；对小菜蛾致病效果最好。菌落隆起次之；菌落平展的为最少。[5]菌落特征、产孢量和对小菜蛾的致病性在基于ITS1-5.8S-ITS2 rDNA序列构建的系统发育树中能表现出一定的相关性。更多还原

【Abstract】 1. The investigation and morphological studies of the genus Paecilomyces inChina.It is significantly important to investigate the biodiversity of Paecilomyces and theirgeographical distribution in china.Based on the taxonomic references, the characters and history of the genus Paecilomyces wassummarized. The studies were supported by two National Natural Science Fundation of China. theresults are as follows:[1] Author thought that the Paecilomyces genus contained sixty-four valid units by collectingand studying the references on 125 Paecilomyces species published in the whole world. Amongthem, entomopathogenic species were up to 53.1％of all members in the genus which showed thatPaecilomyces was really important resources of entomopathogenic fungi.[2] The database of known Paecilomyces species was established by Access software basedon morphological characters. This database was an important foundation for establishing the Deltasystem of entomogenous Paecilomyces species which was convenient and fast to classify theunknown isolates.[3] Systematic study of Paecilomyces in China was carried out. From 32 provinces and citiesin China, 298 Paecilomyces isolates were isolated from 975 soil samples and 164 infected insects.Along with the specimens deposited in the Institute of Fungus Resources at Guizhou University, atotal of 313 Paecilomyces isolates were observed and studied. Based on phenetic characters, 39species in China which compose 60.9％of the all species of genus Paecilomyces in the wholeworld were described in details. A diversity of Paecilomyces has been showed in China. Amongthose, 20 known species, 3 new records, 1 new combination and 15 new species。In addition, four new reports of related genus were isolated. They were Mariannaea elegans(Corda) Samson var. punicea Samson、Chrysosporium indicum (H.S. Randhawa & R.S. Sandhu)Garg、Chrysosporium lobatum Scharapov and Tolypocladium nubicola Bissett.There were also some intercalary strains between the genus Paecilomyces and its alliesincluding P.sp A7-1、P.sp A17-2、P.sp A18-1、P.sp A19-3、P.sp A40-1、P.sp A43、P.sp A62-1、P.sp DH3-1、P.sp LN2-9、P.sp 3’29-1 and P.sp D23-1. Their classification need to rely on themolecular technique.[4] Paecilomyces spp. were widely distributed in China. The Paecilomyces speciesdescribed in China composed 60.9％of the whole genus. Paecilomyces strains were isolated fromvarious habits such as soils, plants and infected insects. As far as host was concerned, they canparasitize the imago, larvae or pupae of various insects such as Leptoptera, Coleoptera, Hemiptera,Hemenoptera and other animals such as nematodes and spiders. However, different species hadobvious differences in distribution. For example, P. lilacinus and P marquandii can be isolatedfrom almost all soils, while P.nostocoides and some monophialidic species were rare.From the results, the check rate of strains in the South was higher than that in the North.Further, the check rate from soil samples was higher than other habits, which showed that the soilis the main habit of microorganisms and new species.2. The application studies of Delta system on entomogenous Paecilomycesspecies in China.Based on the access database of phenetic characters, the Delta system for twenty-four entomogenous Paecilomyces species in China were establish as follows:[1] Establishment of Delta system for entomogenous Paecilornyces species in China. Thissystem was the foundation to expand its function.[2] Automatic realization of key of entomogenous Paecilomyces species in China.Accomplishment of natural description of entomogenous Paecilomyces species in China.Establishment of distance matrix and phenetic tree of entomogenous Paecilomyces species inChina. Realization of interactive automatic identification among entomogenous Paecilomyces spp.3. Studies on the molecular phylogenetics of Paecilomyces spp. in ChinarDNA sequences from the strains under this study and their allies in Genbank were aligned.The phylogenetic trees were inferred by using Neighour-joining method (NJ), Most parsimonymethod (MP) and unweighted pair-group method with arithmetic mean (UPGMA) in Mega3.1 andPAUP4b 10 with 1000 bootstrap replications.[1] Molecular identification for new Paecilomyces spp.Based on the ITS 1-5.8S-ITS2 rDNA sequences of our new species and their allieslownloaded by blast in Genbank, the phylogenetic tree was infered. The following new specieswere supported by both phyiogenetic tree and morphological characters:P tenuis Y.F.Han & Z.Q.Liang, P. purpurseus Z.Q.Liang & Y.F.Han, P. verticillatusZ.Q.Liang, Z.Li & Y.F.Han, P cylindricosporus Z.Q.Liang & Y. F. Hart, P huaxiensis Z.Q.Liang& Y.F.Han and P parvosporus Y.F.Han & Z.Q.Liang[2] Establishment of a new genus—Taifanglan Z.Q.Liang, Y.F.Han & H.L.ChuBased on ITS 1-5.8S-ITS2 and 18S rDNA sequeces of some Paecilomyces species and theirrelated species including eight monophialidic species isolated from soil samples, the phylogenetictree was constructed. The results showed that eight monophialidic species had close relations withSordariales. The results of morphological characters and molecular phylogenetic analysis showedthe monophialidic species were distinguished. We therefore propose to establish a new genus—Taifanglan. There were eleven members in the new genus, including three new species and eightnew combination. We also described the characters of the new genus.[3] Studies on the genetic diversity of Paecilomyces cateniannulatus.Nineteen sequences consisted of sixteen strains collected by authors and three from Genbankwere aligned and used for constructing phylogenetic trees by NJ and MP in PAUP4b10. Theresults showed that these strains of P. cateniannulatus had greatly genetic diversity based on theITS 1-5.8S-ITS2 rDNA sequences. Moreover, the genetic differences had certain relativity with thegeography.[4] Molecular phylogenetic analysis ofPaecilomyces spp. in China.Based on the ITS 1-5.8S-ITS2 rDNA sequences of sixty-one Paecilomyces strains and theirrelated species, the phylogenetic tree was constructed. The results of molecular analysis showedthat the genus Paecilomyces were polyphyletic across two subclasses, Sordariomycetidae andEurotiomycetidae and two orders, Hypotiales and Eurotiales. Within the Eurotiales, Paecilomyceswas momophyletic. Within the Hypocreales, Paecilorayces spp. were polyphyletic, acrossClavicipitaceae and Hypocreaceae. From the molecular analysis, oringin temperature of specieswas one of main characters.[5] Molecular identification of some intercalary strains.Phylogenetic tree was constructed used ninety-three Paecilomyces strains and other relatedgenus such as Penicillium, Talaromyces, Byssochlamys and Aphanoascus. The molecular analysis supported the establishment of the three new species and the strains A 18.1, LN2.9, A19.3, A40.1and H3.1 were regarded as new units of Paecilomyces. strain A62.1 had affinity for the genusPenicillium, so we thought A62.1 belonged to the genus Penicillium.4. Primary studies of some Paecilomyces on the pathogenicity Plutellaxylostella (L.)Thirty strains of Paecilomyces and Beauveria isolated from soil samples and infected insectswere studied, the results were as follows:[1] The symptom of infected Plutella xylostella with infection of the strains tested.While the surface of larva infected by Beauveria bassiana became yellow brown, the surfaceinfected by Paecilomyces didn’t have any obvious change. After incubation at 25℃, theseinfected insects were covered by white mycelium and conidia.[2] The pathogenicity comparison among different strains tested to Plutella xylostella.From the results, it was obviously defferent in the pathogenicity for different species. About83.33% of all strains had certain pathogenicity on Plutella xylostella, especially P.fumosoroseus,P.cateniannulatus and Beauveria bassiana.Different strains in the same species had large pathogenicity differences on Plutella xylostella.For example, SL.8, SL.7, XS.I and XS.2 strains of P.cateniannulatus were the best in thepathogenicity on Plutella xylostella.In addition, the results showed that the Paecilomyces strains from infected insects had betterpathogenicity than those from soil samples. And it was obviously irrelated between thepathogenicity and geographic origin.[3] Comparison of the conidiation in different strains of P.cateniannulatus.There were very large differences in conidiation among different strains of P.cateniannulatus.For example, the conidiation of XS. 1 strain was more than eleven times higher than XSXY.4 strain.And it was related between conidiation and pathogenicity. The higher in conidiation, the better inpathogenicity and vice versa.[4] Comparison of colonies in different strains of P.cateniannulatus..Colonies were observed and divided into three groups: ridgy, petaline and flat. In comparisingthe colonies and geographic origin, they were found irrelated in different strains of P.cateniannulatus.Analysing the colonies and the pathogenicities on Plutella xylostella, it was found that thestrains with petaline colonies had the best pathogenic effect, secondly ridgy, lastly the flat. Thesedata would provide the information to screen the good strains for biocontrol.[5] The relationships among colony, conidiation, pathogenicity and ITS rDNA regionsequences.It had a certain relationship among colony, conidiation, pathogenicity and ITS rDNA regionfrom the phylogenetic tree by MP, especially the pathogenicity had better rule in the tree.Our studies on classicification of the genus Paecilomyces were based on the classicalclassification, Delta system and molecular phylogenetics. The results showed that it was importantto understand both the morphology and molecular phylogenetics in order to carry out taxonomicstudy of Paecilorayces.更多还原