马尾松内生真菌产油脂菌株的分离、筛选和鉴定

采用组织块法从马尾松(Pinus massoniana Lamb.)根、茎和叶片中分离获得内生真菌,初步筛选出产油脂菌株且对其油脂含量进行了分析;并采用形态学及分子生物学方法对产油脂菌株进行了分类鉴定。结果显示:从马尾松植株中共获得21株内生真菌菌株,其中分离自根、茎和叶片的菌株分别为4株、6株和11株。有14株菌株的菌丝中有油滴;其中,分离自叶片的菌株ZP-1、分离自根的菌株ZP-2和分离自茎的菌株ZP-3的菌丝内油滴较多且油脂含量较高,平均油脂含量分别达到29.12%、25.03%和30.56%,差异极显著(P<0.01)。不同菌株的菌落颜色、菌丝和分生孢子形态特征明显不同,菌株ZP-1、ZP-2和ZP-3的形态分别与拟青霉属(Paecilomyces Bainier)、生赤壳菌属(Bionectria Speg.)和镰刀菌属(Fusarium Link)菌种的形态相似。与GenBank中相关真菌ITS序列的比对以及NJ系统树分析结果显示:菌株ZP-1与拟青霉属、菌株ZP-2与生赤壳菌属、菌株ZP-3与镰刀菌属间的ITS片段序列相似性均达到99%,在各自的NJ系统树上它们也分别聚在一起。初步确定菌株ZP-1、ZP-2和ZP-3分别属于拟青霉属、生赤壳菌属和镰刀菌属。     

两种栽培型广藿香内生真菌群落组成变化

为探索内生真菌与广藿香互作间对宿主活性成分形成机制的影响,该研究以成分差异较大的牌香和湛香为对象,采用传统形态学方法对所获菌株归类,通过真菌通用引物ITS1/ITS4扩增菌株rDNA-ITS序列,鉴定其分类地位并研究其多样性。结果表明：(1)用PDA和LBA培养基对苗期、分枝期和成株期广藿香茎叶组织块进行内生真菌分离,共获得3 070株菌株,其中牌香(PX)分离出1 624株,鉴定出1 319株,分属于36属;湛香(ZX)分离出1 446株,鉴定出994株,分属于33属。牌香分离出7种特有内生真菌,分别为香柱菌(Epichloe typhina)、盘长孢状刺盘孢菌(Colletotrichum gloeosporioides)、座腔孢菌(Botryosphaeria sp.)、丝核菌(Rhizoctonia sp.)及截盘多毛孢菌(Truncatella sp.),并首次分离到疫霉菌(Phytophthora sp.)和指疫霉菌(Sclerophthora sp.),这2种菌属于卵菌门内生菌。湛香分离出拟青霉菌(Paecilomyces sp.)和尾孢菌(Cercospora sp.)...     

粉拟青霉种内RAPD多态性分析

对来自不同地理来源和不同寄主的13株粉拟青霉Paecilomyces farinosus和其他3种拟青霉及1株球孢白僵菌进行RAPD分析,从RAPD扩增结果可知粉拟青霉种内具丰富的遗传多样型。RAPD扩增指纹图谱能有效地分辩不同菌株的基因型,可用于监测生防效果的菌株示踪。聚类分析结果表明种间的遗传差异要明显大于种内的差异。菌株间差异是和地理来源相关,而和寄主不相关。     

果汁饮料中耐热霉菌的分离和鉴定研究

为了解果汁饮料中耐热霉菌的污染情况,本实验参照美国相关检验方法,对受微生物污染的果汁饮料进行耐热霉菌检验,将可疑的霉菌菌株进行分离、纯化及鉴定,结果表明,共检出费氏新萨托菌(Neosartorya fischeri)、黄色篮状菌(Talaromyces flavus)以及宛氏拟青霉(Paecilomyces variotii)3株耐热霉菌。通过查找其相关的背景资料,探讨饮料中耐热霉菌检验方法的科学性,为有效控制耐热霉菌的污染提供科学依据。     

木霉植酸酶编码基因多样性及作为系统发育标记潜力研究

对现有的148株木霉菌株在含植酸钙的琼脂培养基上进行了产植酸酶能力鉴定,结果表明所有菌株均产生了水解透明图,说明所有测试的木霉菌株都具有植酸酶活性,植酸酶编码基因在木霉群体中具有广泛性.选取14个种类的21株木霉,采用植酸酶保守序列设计简并引物P8205、P500-2扩增获得其中11种17株木霉植酸酶基因片段,进行了序列测定;利用ITS4、ITS5引物扩增17个木霉菌株的ITS序列并测序.分别基于植酸酶基因片段序列以及ITS序列信息,通过邻接法(N-J法)构建系统发育树,结果表明植酸酶基因序列具有多样性的特点,而基于植酸酶基因序列与基于ITS序列的分类结果基本相同,不同的是长枝木霉(Trichoderma longibrachiatum)植酸酶基因序列与哈茨木霉(Trichoderma harzianum)被分到同一分支当中,与ITS序列的进化关系相差较大,表明有可以作为木霉分类的一种新的标记的潜力,并携带部分与ITS序列不同的系统发育相关信息.     

梵净山自然保护区的一些虫生真菌

本文报道了采自梵净山自然保护区的蛹虫草(Cordyceps militaris),下垂虫草(Co-rdyceps nutans),金龟子虫草(Cordyceps scarabaeicola),蝉拟青霉(Paecilomyces cicadae),撑拟青霉(Paecilomyces suffultus),爪哇拟青霉(Paecilomyces javanicus)和暗绿拟青霉(Paecilomyces atrovirens)等14种昆虫病原真菌。其中撑拟青霉为国内首次报道的新记录种,暗绿拟青霉为一新种。此新种能以(1)在查氏琼脂上菌落暗绿、生长缓慢;(2)瓶梗基部球顶长颈瓶形;和(3)柱形分生孢子聚集成迭瓦状长链与已知种相区别。     

具有抗菌和细胞毒活性红树林真菌的鉴定分析

此次研究从海南省文昌红树林分离到的29株具有抗白色念珠菌和/或细胞毒活性真菌,分别采用核算序列分析、生理生化特征、形态观察对其进行鉴定。研究结果显示,29株真菌中,曲霉属(Aspergillus)7株,青霉属(Penicillium)16株,正青霉属(Eupenicillium)1株、新萨托菌属(Neosartorya)1株、拟青霉属(Paecilomyces Bainier)1株,暂不能定属3株。     

冬虫夏草及其相关类群的分子系统学分析

为了探明冬虫夏草及其相关类群的亲缘关系,以冬虫夏草、中国被毛孢及中华束丝孢(=冬虫夏草头孢=蝙蝠蛾多毛孢)共6株菌种作为内群和一株蛹草拟青霉作为外群进行了DNA随机多态型(RAPD)分析。此外,基于上述供试材料又在内群中增加了一株蝙蝠蛾拟青霉,并对内群和外群样品的nrDNA间隔区(ITS)碱基序列进行了测定;对于测定的8条序列连同来自GenBank中的4条相关序列进行了分子系统学分析。结果表明:中华束丝孢和中国被毛孢均系冬虫夏草菌的无性型。按照国际植物命名法规,中国被毛孢应为冬虫夏草菌无性型的正确名称。而蝙蝠蛾拟青霉为不同于冬虫夏草菌的另一种真菌;该名称由于不合格发表而不被国际植物命名法规所承认。     

拟青霉属虫生真菌核型的脉冲电泳分析

利用钳位均匀电场脉冲电泳系统,从电泳的时间、角度、电压、转换时间间隔等方面优化电泳参数,确定了适宜拟青霉属内4种虫生真菌染色体DNA电泳的最佳条件。根据电泳及软件分析结果,估算出斜链拟青霉和环链拟青霉的染色体数目和染色体组型大小,即核型(karyotype)特征。通过进行拟青霉属内4种7株虫生真菌染色体DNA核型相关的比较和分析,表明拟青霉菌种间染色体数目差异较小,核型差异较大;相同菌种不同菌株间的染色体数、核型大小并不完全相同,存在不同程度的差异;且菌株间核型大小差异程度明显小于菌种间的差异程度。     

产黄芩苷内生真菌的筛选与鉴定

从药用植物黄芩根、茎、叶和花中分离得到17株内生真菌,其发酵液对10种指示菌进行抑菌活性测定,并通过薄层色谱(TLC)和高效液相色谱(HPLC)对所分离到的内生真菌代谢产物进行分析。结果显示,所分离到的内生真菌中12株至少对一种指示菌有抑菌活性,其中3株(G2、J4和J5)具有较广的抑菌作用。从黄芩茎和花中分离得到的2株内生真菌J1、H3可以在人工培养基中产生黄芩活性成分——黄芩苷,结合菌落形态特征、显微观察及ITS序列分析,初步鉴定这2株菌株均属于青霉菌属。     

粉拟青霉种内RAPD多态性分析*

对来自不同地理来源和不同寄主的 １３株粉拟青霉Ｐａｅｃｉｌｏｍｙｃｅｓ ｆａｎｉｎｏｓｕｓ和其他３种拟青霉及１株球孢白僵菌进行ＲＡＰＤ分析,从ＲＡＰＤ扩增结果可知粉拟青霉种内具丰富的遗传多样型。ＲＡＰＤ扩增指纹图谱能有效地分辨不同菌株的基因型,可用于监测生防效果的菌株示踪。聚类分析结果表明种间的遗传差异要明显大于种内的差异。菌株间差异是和地理来源相关,而和寄主不相关。     

粉拟青霉种内RAPD多态性分析

对来自不同地理来源和不同寄主的 １３株粉拟青霉Ｐａｅｃｉｌｏｍｙｃｅｓ ｆａｎｉｎｏｓｕｓ和其他３种拟青霉及１株球孢白僵菌进行ＲＡＰＤ分析,从ＲＡＰＤ扩增结果可知粉拟青霉种内具丰富的遗传多样型。ＲＡＰＤ扩增指纹图谱能有效地分辨不同菌株的基因型,可用于监测生防效果的菌株示踪。聚类分析结果表明种间的遗传差异要明显大于种内的差异。菌株间差异是和地理来源相关,而和寄主不相关。     

rDNA internal transcribed spacer sequence analysis of Craterellus tubaeformis from North America and Europe

The basidiomycete Craterellus tubaeformis (Fries) Quélet is an important widespread ectomycorrhizal basidiomycete found in the Northern Hemisphere. In this study, 12 samples of C. tubaeformis from North America and Europe were analyzed using internal transcribed spacer (ITS) sequences to reveal the correlation between ITS genotypes and geographic locations and to provide molecular evidence for the identification of C. tubaeformis from different habitats in North America and Europe. The analyses identified abundant sequence variations within C. tubaeformis. The length of the ITS region varied from 571 to 640 bp. The proportion of variable sites was 17.6%, and the proportion of parsimony information sites was 16.7%. Phylogenetic analysis showed some correlations between the ITS genotypes and geographic locations of C. tubaeformis; however, some discrepancies between geographical location and affinity were also found. The results indicated that C. tubaeformis from different habitats in North America and Europe underwent genetic drifting and evolved into 2 different species. nrDNA ITS could be a good markers for distinguishing among C. tubaeformis from different habitats, but rational affinity should be determined by associating the available ITS data with other information sources.     

Phylogeny of mitosporic entomopathogenic fungi: is the genus Paecilomyces polyphyletic?

We analyzed sequences of the divergent domain at the 5' end of the large subunit rRNA gene from the mitosporic entomopathogenic fungi Paecilomyces sp., Paecilomyces fumosoroseus, Paecilomyces farinosus, Paecilomyces lilacinus, Verticillium lecanii, Verticillium psalliotae, Beauveria bassiana, Aschersonia sp., Aschersonia placenta, ascomycetous Cordyceps sp., and Cordyceps militaris. Phylogenetic analysis showed P. fumosorseus as the best characterized out of the analyzed species with the B. bassiana clade as its sister group. Two of the P. farinosus isolates were invariably placed within the Verticillium cluster, which also contained C. militaris. The only analyzed P. lilacinus isolate appeared on the root of the hyphomycetous fungi and was characterized as the most distinct from all the hyphomycetous fungi tested. Polyphyly of the genus Paecilomyces was well supported by the Kishino-Hasegawa test. In all trees based on the small subunit rRNA gene sequences obtained from the GenBank, V. lecanii, V. psalliotae, P. fumosoroseus, P. tenuipes and B. bassiana form, together with that of C. militaris, the best supported cluster in the tree. The rest of Cordyceps spp. constitute a distinct clade. Phylogenetic relationships derived from both tested DNA regions show polyphyly of the genus Paecilomyces and close relationships among entomopathogenic species of the genera Verticillium, Paecilomyces, and Beauveria.     

Multiple origins and nrDNA internal transcribed spacer homeologue evolution in the Glycine tomentella (Leguminosae) allopolyploid complex

Despite the importance of polyploidy in the evolution of plants, patterns of molecular evolution and genomic interactions following polyploidy are not well understood. Nuclear ribosomal DNA is particularly complex with respect to these genomic interactions. The composition of nrDNA tandem arrays is influenced by intra- and interlocus concerted evolution and their expression is characterized by patterns such as nucleolar dominance. To understand these complex interactions it is important to study them in diverse natural polyploid systems. In this study we use direct sequencing to isolate and characterize nrDNA internal transcribed spacer (ITS) homeologues from multiple accessions of six different races in the Glycine tomentella allopolyploid complex. The results indicate that in most allopolyploid accessions both homeologous nrDNA repeats are present, but that there are significant biases in copy number toward one homeologue, possibly resulting from interlocus concerted evolution. The predominant homeologue often differs between races and between accessions within a race. A phylogenetic analysis of ITS sequences provides evidence for multiple origins in several of the polyploid races. This evidence for diverse patterns of nrDNA molecular evolution and multiple origins of polyploid races will provide a useful system for future studies of natural variation in patterns of nrDNA expression.     

Internal transcribed spacer 2 amplicon as a molecular marker for identification of Peronospora parasitica (crucifer downy mildew)

AIMS: The purpose of the study was to characterize the internal transcribed spacer (ITS) regions of Peronospora parasitica (crucifer downy mildew) in order to evaluate their potential as molecular markers for pathogen identification. METHODS AND RESULTS: PCR amplification of ribosomal RNA gene block (rDNA) spacers (ITS1 and ITS2) performed in 44 P. parasitica isolates from different Brassica oleracea cultivars and distinct geographic origins, revealed no length polymorphisms. ITS restriction analysis with three endonucleases, confirmed by sequencing, showed no fragment length polymorphisms among isolates. Furthermore, ITS amplification with DNA isolated from infected host tissues also allowed the detection of the fungus in incompatible interactions. The combination of the universal ITS4 and ITS5 primers, for amplification of full ITS, with a new specific forward internal primer for ITS2 (PpITS2F), originates a P. parasitica specific amplicon, suitable for diagnosis. CONCLUSIONS: As ITS2 regions of P. parasitica, B. oleracea, other B. oleracea fungal pathogens and other Peronospora species are clearly distinct, a fast and reliable molecular identification method based on multiplex PCR amplification of full ITS and P. parasitica ITS2 is proposed for the diagnosis of crucifer downy mildew. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be applied to diagnose the disease in the absence of fungal reproductive structures, thus being useful to detect nonsporulating interactions, early stages of infection on seedlings, and infected young leaves packed in sealed plastic bags. Screening of seed stocks in sanitary control is also a major application of this diagnostic method.     

Intraspecific groups of Umbelopsis ramanniana inferred from nucleotide sequences of nuclear rDNA internal transcribed spacer regions and sporangiospore morphology

Umbelopsis ramanniana is a well-known species in this genus. A characteristic morphological feature of this fungus is the remarkable variation in the sporangiospore shape, which implies the genetic variations occur in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) in the U. ramanniana isolates. The relationship between the variations of the sequences of the nrDNA ITS regions and those of the sporangiospore morphology was investigated for 12 isolates of U. ramanniana collected in Europe. Neighbor-joining and parsimony analyses on the sequences suggested that these isolates split into three groups. Precise examination of the morphology showed that the isolates of those respective groups were different from each other in their sporangiospore shape. The present study implies at least three intraspecific groups exist in U. ramanniana and that the variations in the nucleotide sequences of the nrDNA ITS regions correlate well with those in the sporangiospore shape in these intraspecific groups.     

Evolution of the 5.8S nrDNA gene and internal transcribed spacers in Carapichea ipecacuanha (Rubiaceae) within a phylogeographic context

Nuclear ribosomal DNA (nrDNA) constitutes a multicopy gene family that is used widely to test evolutionary hypotheses across a broad range of organisms. It is presumed that, as a result of concerted evolution, tandem nrDNA repeats are homogeneous within species and different between species. We sampled 77 specimens of a disjunct species (Carapichea ipecacuanha) from throughout its three geographic ranges and obtained 266 nrDNA sequences, of which 26 were obtained by direct sequencing and 240 by cloning of PCR products. Complementary sequence analyses, which included analyses of secondary structure stability, the pattern of base substitutions, GC content, and the presence of conserved motifs, were used to characterize the internal transcribed spacer (ITS) region (ITS1-5.8S nrDNA-ITS2). Our results showed that concerted evolution of the ITS region was incomplete in C. ipecacuanha, particularly in the Atlantic range. In the highly polymorphic populations of the Atlantic range, intraindividual variation was observed and involved 56 functional paralogs and 15 pseudogenes from two highly divergent ribogroups. The Amazonian range (with 12 functional paralogs) and the Central-American range (with five functional paralogs) were genetically depauperate and exhibited no pseudogenes. In the two latter ranges, almost complete homogenization of the ITS sequences had occurred. We argue that it is important to consider past evolutionary history when making inferences about the efficiency with which concerted evolution homogenizes tandem nrDNA repeats a single sequence.     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.