台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutel lahuangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明:两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutella huangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明：两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutella huangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明：两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

被毛孢属一新种——长座虫草的无性型

报道长座虫草Cordyceps longissima Kobayasi的无性型为长座被毛孢新种Hirsutella longissima sp.nov.。用子囊孢子分离得到的无性型与子座柄部粘片所见的被毛孢相同;用分离菌在米饭培养基上人工诱发出的子座与天然子座相似,这表明长座被毛孢为长座虫草的无性型。     

被毛孢属一新种—长座虫草的无性型

报道长座虫草Cordyceps longissima Kobayasi的无性型为长座被毛孢新种Hirsutella longissima sp.nov.。用子囊孢子分离得到的无性型与子座柄部粘片所见的被毛孢相同;用分离菌在米饭培养基上人工诱发出的子座与天然子座相似,这表明长座被毛孢为长座虫草的无性型。     

被毛孢属一新种──长座虫草的无性型*

报道长座虫草 Ｃｏｒｄｙｃｅｐｓ　ｌｏｎｇｉｓｓｉｍａ　 Ｋｏｂａｙａｓｉ的无性型为长座被毛孢新种Ｈｉｒｓｕｔｅｌｌａ ｌｏｎｇｉｓｓｉｍａ Ｓｐ． ｎｏｖ．。用子囊孢子分离得到的无性型与子座柄部粘片所见的被毛孢相同;用分离菌在米饭培养基上人工诱发出的子座与天然子座相似,这表明长座被毛孢为长座虫草的无性型。     

根足虫草琅琊山变种新变种及其无性型被毛孢新种

报道采自安徽省滁州市琅琊山林场的一种虫草及其无性型,经鉴定为根足虫草琅琊山变种Cordycepsheteropodavar.langyashanensis新变种。多批次收集弹射的子囊孢子分离得到其无性型,经鉴定为一被毛孢新种,命名为根足被毛孢Hirsutellaheteropoda。模式标本及其无性型菌株干培养物保存在安徽农业大学虫生菌研究中心(RCEFAAU)。     

宽阔水自然保护区的虫草及其相关真菌Ⅱ．

采自贵州省宽阔水自然保护区的另6种虫草,它们是娄山虫草新种（CordycepsloushanensisLiang&Liusp.nov.）,绿核虫草新种（CordycepsaeruginosclerotaLiang&Liusp.nov.）,拟暗绿虫草（cordycepspseudoatrovirensKob.&Shim.）,布氏虫草（CordycepsbrongniartiiShimazu）,金针虫虫草（CordycepsagriotaKawam）,和幼虫虫草［Cordycepslarvarum（Westwood）Olliff］。还报道了多颈被毛孢（HirsutellapolycollutaLiang）,云南被毛孢丝梗新变种（HirsutellayunnanensisvartenuisynnemiLiangetLiuvar.nov.）,巨人被毛孢（HirsutellgiganteaPetch）和枝多头霉［Polycephalomycesramosum（Peck）Mains］等几种束梗孢科的虫生真菌,及娄山拟青霉新种（PaecilomycesloushanensisLiang&Liusp.nov.）。     

两种虫草无性型的微循环产孢确证

从贵阳地区采得两种虫草,古厄虫草Cordycepsgunnii（Bark．）Berk．和布氏虫草CordycepsbrongniartiiShimazu。经诱发它们子囊孢子的微循环产孢再次确证了古尼虫草的无性型是古尼拟青霉PaecilomycesgunniiLiang,布氏虫草的无性型是布氏白僵菌Beauveriabrongniartii（Sacc．）Peteh。     

安徽的虫草及其相关真菌Ⅰ.

报道疣孢虫草Cordyceps phymatospora为新种;球孢虫草C.bassiana及其无性型Beauveria bassiana,长座虫草C.longissima及其无性型Hirsutella longissima,台湾虫草C.formosana及其无性型Hirsutella so.,拟细虫草C.gracilioides和丝虫草C.filiformis等19种虫草及其部分相关真菌。模式标本保存在安徽农业大学虫生菌研究中心（RCEFAAU)。     

Detection and taxonomic placement of endophytic fungi within frond tissues of Livistona chinensis based on rDNA sequences.

The 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) of the rDNA were amplified from total DNA extracted from frond tissues of Livistona chinensis with universal and fungal-specific primers. These amplified fragments were cloned and sequenced. Phylogenetic analysis based on the 5.8S gene sequences indicated that the six clone sequences obtained were of different origins. Five sequences, P1-9, P2-6, P4-4, P4-5, and P4-7, belonged to the fungi and one sequence, P3-2, belonged to the plants. P1-9 was inferred to belong to the Basidiomycota based on the phylogenetic analysis of the 5.8S gene sequences but could not be identified to lower taxonomic levels. Further identification of the other four fungal clones to lower taxonomic levels was attempted based on phylogenetic analysis and sequence comparison of both the conserved 5.8S gene and the variable ITS regions. The origin of P2-6 was identified to be Glomerella and its anamorph Colletotrichum, the origins of P4-5 and P4-7 were Mycosphaerella and its anamorph Cladosporium, and the origin of P4-4 was the Herpotrichiellaceae. The direct approach to detection and taxonomic placement of endophytic fungi within host tissue without the need for conventional in vitro culturing is discussed.     

安徽的虫草及其相关真菌I.

报道疣孢虫草Cordyceps phymatospora为新种;球孢虫草C. bassiana 及其无性型Beauveria bassiana, 长座虫草C. longissima 及其无性型 Hirsutella longissima, 台湾虫草C. formosana 及其无性型 Hirsutella sp., 拟细虫草C. gracilioides和丝虫草C. filiformis 等19种虫草及其部分相关真菌。模式标本保存在安徽农业大学虫生菌研究中心(RCEFAAU)。     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

金耳与其近似种的rDNA-ITS序列分析

对金耳(Tremella aurantialba)的担子果、酵母状分生孢子培养物和菌丝培养物的核糖体DNA内部转录间隔区(ITS)序列进行PCR扩增和测序。结果表明金耳担子果的ITS区PCR产物均为碱基数不同的两条带,片段长度和序列与酵母状分生孢子培养物、菌丝培养物一致。通过对ITS1和ITS2联合进行系统发育分析表明金耳酵母状分生孢子培养物归属于银耳属的金耳,参与组成担子果的寄主菌丝为毛韧革菌(Stereum hirsutum)。结合GenBank中登录的金黄银耳、脑状银耳、橙黄银耳等近似种构建了的系统发育树,结果支持形态学证据,表明金耳是一个独立种。     

Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5.8S rDNA

The anamorph determination of Cordyceps sinensis remains problematic due to the lack of clear links between the sexual and conidial forms of the fungus. In this study, we applied molecular approaches to analyze the genetic variation of Cordyceps sinensis and its allies to identify the anamorph-teleomorph connection. The sequences of the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal RNA gene of Cordyceps sinensis (teleomorph) collected from Qingzang plateau (altitude over 4000m), Tibet and several related asexual conidial forms were determined. The sequence comparison showed that Cordyceps sinensis was most closely related to Hirsutella sinensis, and was clearly divergent from Paecilomyces sinensis, Stachybotrys sp. or Tolypocladium sp.; distance values, estimated according to Kimura two-parameter models between Cordyceps sinensis and Hirsutella sinensis, were extremely low (<0.02), whereas distance values between Cordyceps sinensis and Paecilomyces sinensis, Stachybotrys sp. and Tolypocladium sp. were 0.34, 0.21 and 0.25, respectively. Taken together, Hirsutella sinensis and Cordyceps sinensis are the different stages of the life cycle stages of the same organism. Hirsutella sinensis is therefore the anamorph of Cordyceps sinensis, rather than Paecilomyces sinensis or other species. The possible reasons as to why different taxa can be obtained when culturing Cordyceps sinensis are also discussed.     

Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences

A survey of the endophytic fungi in fronds of Livistona chinensis was carried out in Hong Kong. The endophyte assemblages identified using morphological characters consisted of 16 named species and 19 'morphospecies', the latter grouped based on cultural morphology and growth rates. Arrangement of taxa into morphospecies does not reflect species phylogeny, and therefore selected morphospecies were further identified based on ribosomal DNA (rDNA) sequence analysis. The 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) regions of rDNA from 19 representative morphospecies were amplified by the polymerase chain reaction and sequenced. Phylogenetic analysis based on 5.8S gene sequences showed that these morphospecies were filamentous Ascomycota, belonging in the Loculoascomycetes and Pyrenomycetes. Further identification was conducted by means of sequence comparison and phylogenetic analysis of both the ITS and 5.8S regions. Results showed that MS704 belonged to the genus Diaporthe and its anamorph Phomopsis of the Valsaceae. MS594 was inferred to be Mycosphaerella and its anamorph Cladosporium of the Mycosphaerellaceae. MS339, MS366, MS370, MS395, MS1033, MS1083 and MS1092 were placed in the genus Xylaria of the Xylariaceae. MS194, MS375 and MS1028 were close to the Clypeosphaeriaceae. MS191 and MS316 were closely related to the Pleosporaceae within the Dothideales. The other 5 morphospecies, MS786, MS1043, MS1065, MS1076 and MS1095, probably belong in the Xylariales. The value of using DNA sequence analysis in the identification of endophytes is discussed.     

Ophiocordyceps barnesii and its relationship to other melolonthid pathogens with dark stromata

A hypocrealean Coleoptera pathogen with characteristic part-spores, collected from Khao Yai National Park and Kaeng Krachan National Park in Thailand, is reported. The overall morphology was similar to Cordyceps barnesii, which is known from Sri Lanka, with ascospores disarticulating into four unusually long part-spores that were 30-40 μm long. This disarticulation and part-spore size is, so far, unique within Cordyceps sensu lato. The Thai material was identified with C. barnesii and its placement in the genus Ophiocordyceps was confirmed. Multigene analyses based on the ribosomal small subunit, RPB1 and RPB2 genes revealed the close relationship of the Thai material to Ophiocordyceps konnoana as well as O. ravenelii, O. superficialis, and O. nigrella (all of which have significantly smaller part-spores). However, Ophiocordyceps barnesii and these related species were all characterised by dark-brown to purplish stromata and an affinity for melolonthid larval hosts. No anamorph was seen in the field and was not produced in the slow-growing cultures.     

Molecular phylogeny of Leptosphaeria and Phaeosphaeria

The objectives of this study were to determine the phylogenetic relationships of species of Leptosphaeria and Phaeosphaeria and evaluate the phylogenetic significance of morphological characters of the teleomorph, anamorph, and host. Sequences of the entire ITS region, including the 5.8S rDNA, of 59 isolates representing 54 species were analyzed and the phylogeny inferred using parsimony and distance analyses. Isolates grouped into three well-supported clades. The results of this study support the separation of Phaeosphaeria from Leptosphaeria sensu stricto. Leptosphaeria bicolor and the morphologically similar Leptosphaeria taiwanensis formed a separate, well-supported clade. We conclude that peridial wall morphology, anamorph characteristics, and to a lesser extent host, are phylogenetically significant at the generic level. Ascospore and conidial morphology are taxonomically useful at the species level.     

古尼虫草无性型的分子鉴别

采用PCR技术,以rDNA的ITS区为分子指标,对古尼虫草Cordycepsgunnii的有性和无性阶段进行比较分析,从分子水平上证明古尼虫草的无性阶段是古尼拟青霉Paecilomycesgunnii。     

Ophiocordyceps halabalaensis: a new species of Ophiocordyceps pathogenic to Camponotus gigas in Hala Bala Wildlife Sanctuary, Southern Thailand

Several fungal pathogens of ants have been reported as members of the family Ophiocordycipitaceae in the order Hypocreales. Surveys in the south of Thailand have shown specimens showing characteristics that are morphologically similar to Ophiocordyceps unilateralis, a very common ant pathogen, by producing a lateral pad on one side of the stroma and producing whole ascospores. Phylogenetic analyses of the partial elongation factor tef1-α and the internal transcribed spacer regions ITS1-5.8S-ITS2 rDNA have shown that this is a distinct species from O. unilateralis. The morphological characters of Ophiocordyceps halabalaensis differs from O. unilateralis in the possession of bigger perithecia and ascospores, and molecular analyses have shown that this ant-specific fungus is sufficiently different from O. unilateralis, deserving the naming of a new species. Aspects of morphology, host association/host-specificity, and taxonomic position are discussed.