Natural Diversity of Nodular Microsymbionts of Alnus glutinosa in the Tormes River Basin

The genetic diversity of Frankia strains nodulating Alnus glutinosa along the basin of the Tormes River was studied on DNA extracted directly from nodules. Frankia strains inhabiting root nodules at 12 different locations, ranging in altitude from 409 to 1181 m, were characterized. For that, we amplified the whole IGS region between 16S–23S rDNA and performed a restriction fragment length polymorphism (RFLP) analysis with four restriction enzymes. Two different RFLP patterns (termed A and B) were obtained with HaeIII, indicating the existence of two different groups of Frankia strains. Three different nodule extracts from each of the two RFLP groups were selected for further analyses. Sequencing of the 16S–23S rDNA IGS showed a 100% of intragroup homology and also confirmed the difference (98.4% level of similarity) between the Frankia strains in the two nodule extract groups. The phylogenetic analyses based on the two 16S–23S rDNA IGS sequences obtained in this study and other previously published sequences indicated that Frankia strains TFAg5 and TFAg23 (chosen as representative of HaeIII–RFLP group A and B, respectively) are quite similar to other strains nodulating plants of A. rhombifolia and A. viridis in California (pairwise levels of similarity including gaps ranged from 97.8% to 98.6%), together with which they form a single group. To put the Frankia strains representative of each HaeIII–RFLP group in the context of overall Frankia diversity we amplified and sequenced the 16S rDNA and glnII gene from nodular DNA. An also remarkable fact found in this study was that Frankia strains belonging to the HaeIII–RFLP group A were distributed all along the river course, from the lowest site sampled to the highest, while Frankia strains placed into RFLP group B were restricted to the upper Tormes River, being exclusively found at altitudes of 946 m or higher.     

青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定

首次从采自青藏高原、与高原牧草嵩草属Kobresia草本植物形成外生菌根的黄绿蜜环菌Armillarialuteo-virens子实体中分离获得一组织分离菌株,运用rDNA-ITS和rDNA-IGS-1测序技术对该组织分离菌株是否为黄绿蜜环菌的纯培养菌种进行分子鉴定,并基于黄绿蜜环菌的5.8S/ITS和IGS-1序列进行核酸序列数据库GenBank同源性检索比对、构建系统发育树。结果表明,本研究获得的黄绿蜜环菌子实体组织分离菌株即为其纯培养菌种。基于ITS的系统发育分析表明黄绿蜜环菌与口蘑科内其它属间物种的系统发育关系较远;基于IGS-1的系统发育分析表明黄绿蜜环菌与蜜环菌属内的其它种序列差异较大,系统发育关系较远,而与Lepiota属内的部分种具有较近的系统发育关系。本研究首次基于分子手段对我国青藏高原的黄绿蜜环菌种进行了分离培养、分子鉴定和系统发育分析,为黄绿蜜环菌的科学分类提供了分子依据。     

高黎贡山旱冬瓜Frankia的IGS PCR-RFLP分析

在云南省高黎贡山自然保护区海拔1310～2400m的范围内,采集30个旱冬瓜根瘤样品,直接从根瘤中提取Frankia DNA,对其,nifD-nifK基因间隔区(intergenic spacer,IGS)和16S-23S rDNA IGS进行PCR—RFLP分析．结果表明,nifD-nifK IGS的PCR产物长度差异很大,经HaeⅢ和Afa I双酶切后,得到15种酶切带型,检测到多种基因型的菌株同时与同一株宿主植物共生;16S-23S rDNA IGS的PCR产物长度相似,酶切后亦区分出15种酶切带型．通过对两个基因间隔区的PCR-RFLP联合分析,发现高黎贡山旱冬瓜Frankia存在20种基因型．     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Differentiation of commercial strains of <Emphasis Type="Italic">Agaricus</Emphasis> species in China with inter-simple sequence repeat marker

In recent years, Agaricus mushroom production has undergone an important increase in the world. However, there is no consensus over the taxonomy of some commercial strains. So rapid and reliable methods to identify Agaricus strains are urgently needed. In this paper, six inter-simple sequence repeat (ISSR) primers were screened from 20 primers to analyse the genetic diversities of the 12 main commercial strains of Agaricus in China. The results showed that a total of 124 bands were produced, among which 80.6% was polymorphic, the similarity coefficients ranged from 0.69 to 0.98. Twelve tested strains were divided into 2 groups on the similarity coefficient of 0.75. Three Agaricus bitorquis strains were clustered in one group, and seven Agaricus bisporus strains were clustered with Agaricus strains S1 and S2 in the other group. As comparison with ISSR, intergenic spacers combined with restriction fragment length polymorphisms (IGS-RFLP) was employed to analyse genetic diversities of the tested strains by digesting IGS amplified products with restriction enzymes Hae III, Alu I, Rsa I, respectively, and IGS-RFLP cluster results were similar with those of ISSR. However, ISSR was superior to IGS-RFLP in differentiating closely related strains. It is suggested that the ISSR marker is an alternative method to differentiate Agaricus strains.     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences

Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.     

Genotype analysis of Malassezia restricta as the major cutaneous flora in patients with atopic dermatitis and healthy subjects

Lipophilic yeasts of the genus Malassezia colonize the skin surface of humans and are an exacerbating factor in atopic dermatitis (AD). Two species, M. restricta and M. globosa are major cutaneous microflora in both AD patients and healthy subjects. We compared the DNA sequences of the intergenic spacer (IGS) region, located between the 26S and 5S rRNA genes of M. restricta colonizing the skin surfaces of 13 AD patients and 12 healthy subjects, and of three CBS stock strains as references. The IGS 1 sequences were divided into two major groups, corresponding to AD patients and healthy subjects. These findings suggest that a specific genotype of M. restricta plays a significant role in AD, although M. restricta commonly colonizes both AD patients and healthy subjects.     

Development of an in vitro clonal culture and characterization of the rRNA gene cluster of Perkinsus atlanticus,a protistan parasite of the clam Tapes decussatus

Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.