RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用

采用随机扩增多态性DNA（RAPD）技术和核糖体RNA基因（rDNA)转录间隔区（ITS）序列分析对22种拟茎点霉共34个菌株进行了系统发育研究,RAPD分析构建的UPGMA聚类图所反映的种间,种内关系与形态学分类结果基本一致。可以清楚地将分自7科寄主植物上的不同的种分别区分开来,但分自同科或同属寄主植物上的不同的种并不具有相近的亲缘关系,ITS序列分析结果不支持将Phomopsis mangiferae Ahmad和P.cytosporella Penz.et Sacc.合并为同一个种的观点,同时还.mangiferae与P.sidii de Camara的亲缘关系非常近,可能是异名同物;而为害木棉叶的拟茎点霉与杨梅枝枯病菌,P.myricaeY.J.Huang et P.K.Chi之间的碱基差异亦属于种下不同菌株间的正常差别范围,很可能就是同一个种,对相同的供试菌株两技术所反映的亲缘关系趋势相同,表明两技术用于拟茎点霉的亲缘关系分析和种类鉴定均是可行的。     

RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用*

采用随机扩增多态性DNA(RAPD)技术和核糖体RNA基因(rDNA)转录间隔区(ITS)序列分析对22种拟茎点霉共34个菌株进行了系统发育研究。RAPD分析构建的UPGMA聚类图所反映的种间、种内关系与形态学分类结果基本一致,可以清楚地将分自7科寄主植物上的不同的种分别区分开来,但分自同科或同属寄主植物上的不同的种并不具有相近的亲缘关系。ITS序列分析结果不支持将Phomopsis mangiferae Ahmad和P. cytosporella Penz. et Sacc. 合并为同一个种的观点,同时还显示出P. mangiferae与P. psidii de Camara的亲缘关系非常近, 可能是异名同物;而为害木棉叶的拟茎点霉与杨梅枝枯病菌P. myricae Y.J.Huang et P.K.Chi之间的碱基差异亦属于种下不同菌株间的正常差别范围,很可能就是同一个种。对相同的供试菌株两技术所反映的亲缘关系趋势相同,表明两技术用于拟茎点霉的亲缘关系分析和种类鉴定均是可行的。     

湖南省木本植物上的拟茎点霉新种

报道了采自湖南省木本植物上的10个拟茎点霉Phomopsis新种,即喜树拟茎点霉Ph. camptothecae、腊梅拟茎点霉Ph.chimonanthi、杜仲生拟茎点霉Ph.eucommiicola、紫薇拟茎点霉Ph.lagerstroemiae、红继木拟茎点霉Ph.loropetali、厚朴拟茎点霉Ph.magnolina、海桐拟茎点霉Ph.pittospori、竹柏拟茎点霉Phomopsis podocarpi、石榴拟茎点霉Ph.punicicola和酸枣拟茎点霉Ph.ziziphicola。对新种与其近似种在形态及ITS序列上的差异进行了讨论。文中附有新种的拉丁文、英文描述及显微结构图。模式标本保存于华南农业大学真菌标本室(SCHM)。     

南美蟛蜞菊霜霉病病原鉴定及系统发育分析

[目的]为从天敌病原生物方面探索外来入侵植物南美蟛蜞菊的生物防治,对新发现的南美蟛蜞菊霜霉病进行病原鉴定和系统发育分析。[方法]在广东省广州市对南美蟛蜞菊霜霉病的发生及危害情况进行调查,并通过病害症状识别、病原显微形态记录与比较、病原菌及其近似种ITS序列结构比较、LSU序列和ITS序列系统发育分析,对南美蟛蜞菊霜霉病病原进行鉴定和系统发育分析。[结果]南美蟛蜞菊霜霉病在广州零星发生,但该病害在华南农业大学校园内发生较严重,发病率达50%~70%,病情指数为30~35。经鉴定,其病原菌为南美蟛蜞菊单轴霉,是国内一新记录种。基于病原菌LSU序列和ITS序列的系统发育分析显示,侵染菊科植物的单轴霉属菌种聚在一个分枝,亲缘关系密切,与侵染其他不同科寄主植物的单轴霉亲缘关系较远。ITS序列结构比较显示,寄生于菊科向日葵族植物的单轴霉属菌种的ITS2区包含多个重复序列,不同菌种间的ITS2区重复序列相似度不同,说明侵染菊科向日葵簇植物的单轴霉属菌物可细分成多个种,而不是只有向日葵单轴霉。[结论]广州发生的南美蟛蜞菊霜霉病是该寄主上首次正式报道的病害,鉴定的病原菌也是国内一新记录种;寄生在菊科植物上的单轴霉属种类不尽相同,但亲缘关系紧密。     

木霉属2中国新记录种

[目的]明确我国南部地区土壤中分离得到的2株木霉菌的分类地位.[方法]采用PDAm培养基分离木霉菌菌株,通过形态学鉴定和内转录间隔区(ITS)序列、翻译延长因子1-alpha (Tef1-α)部分序列相似性分析对菌株进行初步鉴定;选取MEGA 4.0软件用Bootstrap最大简约法、水平3、重复1 000次来构建ITS、Tef1-α序列系统进化树并分析其亲缘关系.[结果]菌株CM01的ITS序列与GenBank基因库中Trichoderma intricatum strain GJS 02-78 ITS序列同源性高达99％,在ITS序列系统进化树中与模式菌株T.intricatum GJS 02-78、Trichoderma atroviride DAOM 179514亲缘关系最近;其Tef1-α序列与GenBank基因库中Hypocrea intricate strain GJS 02-78 Tef1-α序列同源性高达99％,在Tef1-α序列系统进化树中与模式菌株H.intricata GJS 02-78亲缘关系最近;其形态描述和模式菌株一致.菌株SCGA5003的ITS序列与GenBank基因库中Trichoderma stromaticum strain GJS 97-181 ITS序列同源性高达99％,在ITS序列系统进化树中与模式菌株T.stromaticum GJS 97-179、GJS 97-180、GJS 97-181、GJS 97-182、GJS 97-183亲缘关系最近;其Tef1-α序列与GenBank基因库中T.stromatieum strain GJS 97-183 Tef1-α序列同源性高达94％,在Tef1-α序列系统进化树中与模式菌株T.stromaticum CQSQ1032、GXNN7006亲缘关系最近;其形态描述和模式菌株一致.[结论]结合形态学特征与分子鉴定结果,判定菌株CM01为交织木霉(Trichoderma intricatum Samuels&Dodd/Hypocrea intricata Samuels et Dodd)、SCGA5003 为子座木霉(Trichoderma stromaticum Samuels & Pardo-Schulth/Hypocrea stromatica Bezerra,Costa & Bastos),即发现木霉属2个中国新记录种.     

甘肃省冬虫夏草菌的RAPD和ITS序列分析

目的对甘肃省冬虫夏草分离菌株进行鉴定和系统发育研究。方法采用随机扩增多态性DNA（RAPD）分析和核糖体（rDNA）内部转录间隔区（ITS）序列分析。结果共分离到18个菌株,菌株间的遗传相似性系数为0.623～1.000,遗传分化距离为0.000～0.018,每个菌株的序列与中国被毛孢的序列一致性均高于98%且18个菌株（G＋C）%的差别最大值是4.7%。结论 18个分离菌株均为中国被毛孢;多样性分析表明,不同区域样本间的遗传分化较大;同一地点样本间的遗传分化很小;同一材料不同部位获得的菌株在RAPD标记上差异无统计学意义,两技术对相同的供试菌株所反映的遗传分化不尽相同,表明两技术用于冬虫夏草遗传多样性和种类鉴定上各有它们的优势和具体选择。     

粉拟青霉种内nrDNA ITS分析

通过对20株粉拟青霉(Paecilomyces farinosus)ITS1-5．8S-ITS2(rDNA)区域序列测定,确定了粉拟青霉ITS序列,而韩国学者测定的粉拟青霉ITS序列应为细脚拟青霉(P．tenuipes)的序列。序列比较发现,韩国2株未定名的拟青霉(Paecilomyces spp．)菌株(KACC40219、KACC40221)应为粉拟青霉。基于本研究构建的邻接树推断,粉拟青霉的有性型可能是一种虫草。粉拟青霉的起源应为单源的。不同的粉拟青霉菌株的ITS序列具有多态性,源于同一地区的菌株的ITS变异也较大。ITS序列的证据表明,粉拟青霉菌株间的差异与地理来源及寄主均无相关性。     

基因组分析揭示拟茎点霉XP-8产松脂醇及其糖苷等多种次级代谢产物的合成途径

【目的】通过解析拟茎点霉属XP-8的基因组序列信息,揭示该菌株潜在的代谢途径,并分析松脂醇及其糖苷化合物等次级代谢产物生物合成相关的关键基因。【方法】使用Illumina Hi Seq 2500高通量测序平台对拟茎点霉XP-8菌株进行全基因组测序,并通过不同软件对测序数据进行序列拼接,基因预测与功能注释。【结果】组装后的拟茎点霉XP-8基因组大小为55.2 Mb,GC含量53.5%,含有17094个蛋白编码基因和310个非编码基因。获得了松脂醇及其糖苷化合物等次级代谢产物生物合成相关的基因。系统发育分析揭示出拟茎点霉XP-8与5种子囊菌共有12635个同源基因和5626个基因家族。【结论】拟茎点霉XP-8具有用于合成松脂醇及其糖苷化合物等多种次级代谢物的基因组基础,为下一步的代谢工程改造提供依据。     

利用RAPD技术对拟青霉属菌株进行分类鉴定*

用9个引物对来自安徽和浙江的16株12种拟青霉 (Paecilomyces Bainier) 进行RAPD分析,获得的平均相似性系数表明,种间的相似系数在21%~46%之间。RAPD指纹图谱在拟青霉属不同种间具明显的种的特异性,可区别所有形态近似的种类,是鉴定菌种的有效途径。RAPD结果也暗示分离自灰僵蚕的RCEF197可能是一新种。对比RAPD聚类树状图和基于ITS构建的分子系统发育图,表明该聚类树状图不适于分析种间亲缘关系。     

利用RAPD技术对拟青霉属菌株进行分类鉴定

用9个引物对来自安徽和浙江的16株12种拟青霉 (Paecilomyces Bainier) 进行RAPD分析,获得的平均相似性系数表明,种间的相似系数在21%~46%之间。RAPD指纹图谱在拟青霉属不同种间具明显的种的特异性,可区别所有形态近似的种类,是鉴定菌种的有效途径。RAPD结果也暗示分离自灰僵蚕的RCEF197可能是一新种。对比RAPD聚类树状图和基于ITS构建的分子系统发育图,表明该聚类树状图不适于分析种间亲缘关系。     

Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.     

Morphological and molecular characterization of Phomopsis vaccinii and additional isolates of Phomopsis from blueberry and cranberry in the eastern United States

Forty isolates of Phomopsis were obtained from twigs and berries of highbush blueberry, Vaccinium corymbosum, and cranberry, Vaccinium macrocarpon, isolated primarily from plants grown in the eastern United States. They were characterized using conidiomatal morphology, conidial dimensions, colony appearance and growth rate, and sequences of ITS rDNA. Based on morphological and molecular similarities, most isolates grouped together with an authentic culture of Phomopsis vaccinii Shear. This taxon is described and illustrated. However, some Phomopsis isolates from Vaccinium differed in colony and conidiomatal morphology from P. vaccinii and, based on ITS sequences, were related to isolates of Phomopsis from diverse hosts. These isolates were excluded from P. vaccinii.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

香菇自然群体中个体间的空间分布及其遗传联系*

应用体细胞不亲和性反应、交配型因子分析和基因组ＤＮＡ的ＲＡＰＤ分析,研究了一个分布于方圆约１ｋｍ的６根倒木上的１８个香菇野生菌株间的遗传差异。结果表明,该群体大多数菌株间配对（８０．４％）体细胞不亲和,而同一倒木菌株间配对的体细胞亲和率达 ６２．５％。不同倒木菌株间未发现体细胞亲和的配对。该群体存在 １１个特异的Ａ因子和７个特异的Ｂ因子。同一倒木的菌株有的交配型因子相同,有的则不同。不同倒木的菌株大多数交配型因子不同,未发现交配型因子完全相同的菌株。ＲＡＰＤ分析显示,体细胞亲和的菌株,交配型因子完全相同的菌株,在基于ＤＮＡ相似系数的遗传相关聚类中,首先聚为小类。总起来看,在自然群体中,香菇个体间的遗传差异与其空间分布之间存在一定的联系,随着空间距离的增大,菌株间的异质性相应增高。