辽宁省9种兰科植物根内与根际土壤中真菌群落结构的差异

兰科植物的生存及生长高度依赖其根中的共生真菌, 其中的菌根真菌更是对兰科植物的种子萌发与后续生长有着非常重要的作用, 研究兰科植物根中的真菌, 尤其是菌根真菌, 对兰科植物的保护有重要作用。该研究利用第二代测序技术, 对中国辽宁省境内的9种属于极小种群的兰科植物的根、根际土和根围土中的真菌群落和菌根真菌组成进行了研究。结果显示, 兰科植物根中的真菌群落和根际土、根围土中的真菌群落具有显著差异。兰科植物根中的总操作分类单元(OTU)数目远小于根际土和根围土中的总OTU数目。同时, 兰科植物根中菌根真菌的种类和丰度与根际土、根围土中菌根真菌的种类与丰度没有明显联系。FunGuild分析结果显示, 丛枝菌根真菌在根际土与根围土中的丰度非常高, 但在兰科植物的根中却数量极少。这些结果表明, 兰科植物根中的真菌群落与土壤中的真菌群落在一定程度上是相互独立的。     

洪湖沉积物中真菌群落结构的垂直分布

湖泊沉积物中真菌的类群及其作用所知甚少。以长江中上游代表性湖泊洪湖为研究对象,从湖心截取了一个72cm长的沉积柱,通过rDNA内转录间隔区（ITS）的变性梯度凝胶电泳（DGGE）,对洪湖沉积物中真菌群落结构的垂直变化进行了分析。结果显示,DGGE条带以14－18cm层次为过渡,在2－14cm和14－72cm之间存在一定差异。Shannon-Weaver index（H′）在上下层之间有波动。聚类分析上层2－18cm和中下层18－72cm分别以不同的小类聚在一起。表明14－18cm以上和以下的类群存在差异,     

Basidiomycete fungal communities in Australian sclerophyll forest soil are altered by repeated prescribed burning

Soil basidiomycetes play key roles in forest nutrient and carbon cycling processes, yet the diversity and structure of below ground basidiomycete communities remain poorly understood. Prescribed burning is a commonly used forest management practice and there is evidence that single fire events can have an impact on soil fungal communities but little is known about the effects of repeated prescribed burning. We have used internal transcribed spacer (ITS) terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the impacts of repeated prescribed burning every two or four years over a period of 30 years on soil basidiomycete communities in an Australian wet sclerophyll forest. Detrended correspondence analysis of ITS T-RFLP profiles separated basidiomycete communities in unburned control plots from those in burned plots, with those burned every two years being the most different from controls. Burning had no effect on basidiomycete species richness, thus these differences appear to be due to changes in community structure. Basidiomycete communities in the unburned control plots were vertically stratified in the upper 20 cm of soil, but no evidence was found for stratification in the burned plots, suggesting that repeated prescribed burning results in more uniform basidiomycete communities. Overall, the results demonstrate that repeated prescribed burning alters soil basidiomycete communities, with the effect being greater with more frequent burning.     

Native Trichoderma strains isolated from Bangladesh with broad spectrum antifungal action against fungal phytopathogens

Nineteen Trichoderma isolates, collected from different locations in Bangladesh, were characterised through phenotypic, biochemical and molecular means. Besides, they were assessed for their antifungal action in vitro. The isolates were divided into three groups: T. asperellum, T. virens and T. harzianum. A dual culture assay and a culture filtrate assay against 6 phytopathogens revealed that 9 of the 19 isolates showed significant antifungal activities. The isolate T. harzianum TR05 showed the highest inhibition against Fusarium oxysporum, Rhizoctonia solani, Fusarium circinatum and Phomopsis vexans, followed by T. asperellum TR08 and T. virens TR06. TR08 had the highest inhibition against Sclerotium rolfsii and Pythium aphanidermatum, followed by TR05 and TR06. These findings were in agreement with their activities of extracellular hydrolytic enzymes, including chitinase, β-1,3-glucanase, and proteinase. Our results suggest that isolates TR05, TR06 and TR08 have the potential to be effective biocontrol agents against the phytopathogenic fungi.     

Molecular appraisal of Bunium and some related arid and subarid geophilic Apiaceae–Apioideae taxa of the Ancient Mediterranean

Bunium is unusual in Apiaceae in having a variable cotyledon number and broad infrageneric dysploidy. To test the monophyly of the genus, phylogenetic relationships among 39 Bunium species were investigated with DNA sequence data from nuclear (nrITS) and plastid (psbA‐trnH intergenic spacer) regions. Several other taxa with a similar ecology and geography were also included in the analyses. Our results suggest that Bunium is not monophyletic. Bunium spp. in the eastern part of the study area are more closely related to the Central Asian genera Elaeosticta, Galagania, Hyalolaena, Mogoltavia and Oedibasis than to those in the western part. Our study revealed that molecular, morphological (cotyledon number and width of fruit commissure) and karyological data reveal similar patterns in the taxa studied. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 149–170.     

Ectomycorrhizal fungal succession in mixed temperate forests

Ectomycorrhizal (ECM) fungal communities of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were studied along a chronosequence of forest development after stand-replacing disturbance. Previous studies of ECM succession did not use molecular techniques for fungal identification or lacked replication, and none examined different host species. Four age classes of mixed forests were sampled: 5-, 26-, 65-, and 100-yr-old, including wildfire-origin stands from all four classes and stands of clearcut origin from the youngest two classes. Morphotyping and DNA sequences were used to identify fungi on ECM root tips. ECM fungal diversities were lower in 5-yr-old than in older stands on Douglas-fir, but were similar among age classes on paper birch. Host-specific fungi dominated in 5-yr-old stands, but host generalists were dominant in the oldest two age classes. ECM fungal community compositions were similar in 65- and 100-yr-old stands but differed among all other pairs of age classes. Within the age range studied, site-level ECM fungal diversity reached a plateau by the 26-yr-old age class, while community composition stabilized by the 65-yr-old class. Simple categories such as 'early stage', 'multi stage', and 'late stage' were insufficient to describe fungal species' successional patterns. Rather, ECM fungal succession may be best described in the context of stand development.     

Molecular phylogeny of the genus Philodendron (Araceae): delimitation and infrageneric classification

The genus Philodendron (Araceae) is a large neotropical group whose classification remains unclear. Previous classifications are based on morphological characters, mainly from the inflorescence, flower and leaf shape. The classification by Krause, with few modifications, is still the most commonly used system. To examine phylogenetic relationships in the genus, two ribosomal DNA nuclear markers, internal transcribed spacer (ITS) and external transcribed spacer (ETS), and the chloroplast intron rpl 16, were sequenced and analysed for more than 80 species of Philodendron and its close relative Homalomena . According to the resulting phylogeny, the genus Homalomena may be paraphyletic to the genus Philodendron . The inclusion of the American Homalomena species within the genus Philodendron might resolve this taxonomic problem. All three subgenera of Philodendron were revealed as monophyletic. Below the subgeneric level, the groups obtained in our phylogeny globally correspond to sections recognized in previous classifications. Among the morphological characters used by previous taxonomists to build their classifications, and which we optimized onto one of the most parsimonious trees, most characters were found to be homoplasious. However, leaf shape, characteristics of the sterile zone on the spadix and venation patterns are useful for delimiting subgenera and sections within the genus.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 13–27.     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T.giganteum野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.     

Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests

To advance our understanding of host effects on the community structure of ectomycorrhizal fungi (EMF), EMF communities were compared among different host species, genera and families in two mixed conifer-broadleaf forests in Japan. Using molecular identification methods we examined EMF root tips of eight coexisting species belonging to six genera (three families): Abies and Tsuga (Pinaceae), Betula and Carpinus (Betulaceae) and Fagus and Quercus (Fagaceae). In total, 205 EMF species were detected, and the total richness was estimated to exceed 300 species using major estimators. Of the 55 EMF species occurring three or more times, eight showed significantly biased host preference. A Mantel test showed a significantly negative correlation between EMF community similarity and host taxonomic distance. Detrended correspondence analysis separated EMF communities mainly by host taxonomic and successional status. Thus, EMF communities are similar on hosts with similar taxonomic and successional status. A significant proportion of EMF exhibited host specificity, which may contribute to the extremely diverse EMF community in conifer-broadleaf forests.     

广东湛江湾可培养丝状真菌分布状况的初步调查

通过一次航行对广东湛江湾19个站位的表层和底层海水进行采样。经过对样品的分离纯化,共获得253株丝状真菌菌株。通过测序获得了121个rDNA-ITS序列,并已提交给GenBank。经统计分析发现,湛江湾表层和底层海水真菌数量总体上表现为由湾内向湾口逐渐减少的水平分布格局;湛江湾表层丝状真菌的数量略多于底层,在海水表层和底层中真菌种类和数量的分布规律相似。根据形态特征及rDNA-ITS的Blast同源性分析,这些菌株分属于18个属和32个分类单元,其中包括7个海洋真菌新记录属。结果表明,湛江湾丝状真菌多样性Shannon指数达2.75,物种优势度变化范围为30.90%–0.02%。枝孢属Cladosporium的优势度最高,为湛江湾优势种群,其次是青霉属Penicillium、侧齿霉属Engyodontium、曲霉属Aspergillus、枝顶孢属Acremonium等。湛江湾表层和底层海水真菌群落的Jaccard相似系数为0.42。     

三种杓兰根相关真菌多样性和生态功能

【背景】除了菌根真菌(Orchid mycorrhizal fungi,OrMF)外,兰科植物根中还有其它内生真菌,称为根相关真菌(Root-associated fungi,RAF)。【目的】采用分离培养的方法获得同一栖息地针叶林和灌木林两种不同生境西藏杓兰、黄花杓兰和无苞杓兰的RAF菌株,研究其真菌谱系、多样性和生态功能结构。【方法】从杓兰根碎屑中分离RAF,通过总DNA提取、PCR扩增及测序得到ITS(Internaltranscribedspacer)序列;进行系统发育和多样性分析,并通过NCBI数据库比对得到相似性最高序列的注释信息来分析RAF生态学特性。【结果】共分离得到278株RAF,25种OTU类型,包括23个子囊菌门OTU,2个毛霉菌门OTU。RAF物种丰富度分析发现西藏杓兰的较黄花杓兰高,不同生境没有显著差异;不同杓兰物种较不同生境的RAF群落分化程度高。生态功能分析显示25个OTU包括共生型、腐生型和致病型3种营养型,以及外生菌根菌群、植物病原菌群、内生真菌群、动物病原菌群、真菌寄生菌群、杜鹃花类菌根群、未定义的腐生菌群和不确定型8种共位群。【结论】阐明不同生境采集的不同杓兰中RAF的分布特点和生态功能,为未来研究RAF与杓兰属植物的共生关系奠定基础。     

Molecular analysis of orchid pollinaria and pollinaria-remains found on insects

Direct observations of pollinator visits to orchids are often difficult and time consuming, especially in orchids with a deceptive pollination system where seed set is typically pollinator-limited. This lack of direct observations greatly inhibits our understanding of orchid-pollinator relationships and especially the degree of pollinator-specificity. Here we describe a molecular approach to the study of orchid-pollinator relationships based on the analysis of DNA recovered from pollinaria found on insects. The insects were collected from nectar-rich plants flowering near natural orchid populations, or taken from museum collections. Sequence analysis of the nuclear ribosomal ITS region allowed the identification of the orchid species or species-group from which the pollinaria originated. Four out of eight orchid-pollinator relationships established with this approach have not been reported previously, which highlights the value of molecular tools for the study of orchid pollination biology.     

Molecular investigation of genet distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests

The size and distribution of Cortinarius rotundisporus genets at three sclerophyll forest field sites in New South Wales, Australia, were estimated by using microsatellite-primed PCR (MS-PCR) of DNA extracted from sporocarp tissue. MS-PCR fingerprints generated with the primers (GTG)₅ and (GACA)₄ indicated that two to five genets were present at each site, with each site being characterized by a single large genet (9–30 m in diameter). Analysis of internal transcribed spacer (ITS)-RFLP patterns from individual sporocarps used in the study suggested that three distinct RFLP types were present in the sampled C. rotundisporus population. ITS sequence data indicate that the three RFLP types had less than 88.4% sequence identity to each other, strongly suggesting that C. rotundisporus is a complex of three species.     

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.     

Distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes

A study of the distribution and ecology of mosquitoes (Diptera: Culicidae) in Ardebil Province of northwestern Iran, with special reference to the known dirofilariasis focus in Meshkinshahr County, was carried out in July‐August 2005 and April‐October 2006. In total, 17 533 adult mosquitoes and 3090 third‐ and fourth‐instar larvae were collected on 14 occasions and identified using morphological characters and DNA sequence data. Twenty species belonging to seven genera were found: Anopheles claviger (Meigen), An. hyrcanus (Pallas), An. maculipennis Meigen, An. pseudopictus Grassi*, An. sacharovi Favre, An. superpictus Grassi, Aedes vexans (Meigen)*, Coquillettidia richiardii (Ficalbi)*, Culex hortensis Ficalbi, Cx. modestus Ficalbi, Cx. pipiens Linnaeus, Cx. theileri Theobald, Cx. torrentium Martini*, Cx. tritaeniorhynchus Giles, Culiseta longiareolata (Macquart), Cs. annulata (Schrank)*, Cs. subochrea (Edwards), Ochlerotatus caspius (Pallas) s.l.* (= Aedes caspius sensu auctorum), Oc. geniculatus (Olivier)* (= Aedes geniculatus sensu auctorum) and Uranotaenia unguiculata Edwards (asterisks indicate new occurrence records for the province). The most prevalent species in adult catches were An. maculipennis (52%), Cx. theileri (45%) and Cx. hortensis (1%); the most prevalent species caught as larvae were Cx. theileri (27%), Cx. hortensis (21%) and An. maculipennis (19%). Anopheles maculipennis, Cx. pipiens and Cx. theileri were most widely distributed in the province. The occurrence of Cx. torrentium in Iran is verified based on differential characters of fourth‐instar larvae. Anopheles maculipennis and An. sacharovi of the Maculipennis Group were identified from their diagnostic ITS2 sequences. For the first time, cytochrome c oxidase I (COI) sequences were obtained from Iranian specimens of An. hyrcanus, An. pseudopictus, Cx. theileri and Oc. caspiuss.l. Culex theileri and An. maculipennis were found naturally infected with third‐stage (infective) larvae of Dirofilaria immitis (Leidy) and Setaria labiatopapillosa (Alessandrini) (Spirurida: Onchocercidae), respectively, for the first time in Iran.