利用松茸ITS特异性引物对松茸分离物进行鉴定

利用一对ITS通用引物(YIS4-ITS5)和一对松茸物ITS特异性引物(TMF-TMR)对来源于云南省不同地区的6个松茸(Tricholoma matsutake)子实体及其6株分离物,3个假松茸(Tricholoma bakamatsutake)子实体及其3株分离物、侧耳(Pleurotus astreatus)、金针菇(Flammulina velutipes)和双孢蘑菇(Agaricus bisporus)的子实体进行了PCR扩增,琼脂糖凝胶电泳分析,结果表明ITS4-ITS5能将所有的样品扩增,并得到600bp左右的DNA扩增条带,TMFTMR扩增时,只有松茸子实体及其对应分离物有扩增条带,DNA片段大小在500bp左右。进一步对松茸子实体(TG25)及其分离物(TM25112．2)进行WS序列测定表明两者的DNA同源性为100％,从而证明所分离到的6个菌株确为松茸的纯培养物。     

四川省雅江松茸菌的分离与系统发育

对外生菌根真菌-松茸的纯培养条件进行了探讨,并从采集自四川省雅江县的松茸子实体中获得了10株松茸菌的纯培养物;分别以NS1和NS6 ,NS1和NS8,ITS4和ITS5为引物,对分离获得的松茸菌进行了18S r DNA PCR- RFL P和ITS PCR-RFL P分析,结果显示,用Alu I,H ae III,H inf I和Msp I四种限制性内切酶,这些松茸菌株的18S r DNA、ITS片段的酶切图谱完全相同;代表菌E7的ITS序列分析结果表明,本研究分离的松茸菌与Tricholoma matsutake的菌株在系统发育上高度同源,在分类上应属于同一个种     

云南美味牛肝菌ITS 区域结构特点

利用ITS 的通用引物(ITS5-ITS4) 对云南的美味牛肝菌( Boletus edulis) 子实体的DNA 进行PCR 扩增, 扩增产物回收后直接测序。序列的聚类分析表明, 在ITS1-5 . 8S rDNA-ITS2 区域, 云南的美味牛肝菌与欧洲的夏牛肝菌( B. aestivalis) 和铜色牛肝菌( B . aereus) 同源性较高, 但在ITS2 区域夏牛肝菌和铜色牛肝菌分别有一段美味牛肝菌没有的大小分别为73 bp 和26 bp 的特征序列。     

禽流感病毒分离株NS基因同源性及等位基因类型分析

目的　克隆测定国内具有代表性的禽流感病毒 (AIV)的非结构 (NS)蛋白基因核苷酸序列 ,分析其同源性和等位基因类型 ,为进一步探索禽流感NS蛋白抗体监测方法奠定基础。方法　经RT PCR扩增了国内 3株H9N2、2株H5N1、2株H7N2亚型AIV分离株的NS蛋白基因 ,并把扩增的基因片段克隆到pGEM T载体中测序 ,将测序结果与GenBank中的核苷酸序列进行同源性比较 ,绘制基因进化树。结果　经测序获得了各AIV分离株NS基因的完整编码序列。同源性分析表明 ,3株H9亚型AIV的NS基因之间的同源性为 96 %～ 98% ;两株H5亚型AIVNS基因同源性为 91 6 % ;两株H7亚型AIV的NS基因同源性为 98 9%。H5和H9亚型分离株的NS基因之间的同源性均高于 90 % ;而H7N2亚型分离株与其它两种亚型分离株的NS基因同源性约为 6 0 %～ 70 %。在AIVNS基因系统发育进化树中 ,H5、H9亚型分离株都处于等位基因A群内 ;3株H9亚型分离株的进化关系较近 ,与香港、广东的部分H5N1病毒株起源相同 ,而 2株H5病毒的NS基因则处于不同分枝内 ;2株H7亚型分离株的NS基因都处于等位基因B群内 ,进化关系较近。结论　这 7株国内AIV分离株的NS基因之间的同源性差异较大 ,约为 6 0 %～ 99% ,且包括A、B两种类型的等位基因     

PCR在猴B病毒与猴病毒8型鉴别中的应用

目的为了使BV147与SA8相区别,并进一步分析BV147基因结构特点.方法用PCR方法扩增BV147DNA,并对扩增片段进行克隆测序.结果该序列与美国BVE2490株部分基因(UL27)相对应位置的核苷酸同源性为99.54%,与SA8相对应位置的核苷酸同源性为89.91%.结论进一步证明了新分离物为B病毒.     

药用植物柴胡病毒病病原物的分子鉴定

利用非序列依赖性扩增(Sequence-independent amplification,SIA)方法对所采柴胡病样进行分子鉴定。序列测定及分析发现,伴有花叶症状的柴胡受到黄瓜花叶病毒(Cucumber mosaic virus,CMV)和蚕豆萎蔫病毒2号(Broad bean wilt virus 2,BBWV2)的复合侵染。为明确柴胡CMV分离物(CMV-SXCH)和柴胡BBWV2分离物(BBWV2-CH)的分类地位,进一步克隆CMV-SXCH的CP、MP和BBWV2-CH的LCP、SCP;序列比对发现,CMV-SXCH与CMV亚组ⅠB中株系XJ2相似性最高,其核苷酸和氨基酸序列同源性分别为99.1%、99.5%;BBWV2-CH与BBWV2中的K株系相似性最高,核苷酸和氨基酸序列同源性分别为95.4%、99.2%。系统进化树分析表明,CMV-SXCH与大多数CMV中国分离物聚类为一簇,同属于CMV亚组ⅠB;BBWV2-CH与BBWV2韩国K株系聚类为一簇,亲缘关系最近。     

1株产纤维素酶木霉菌种的鉴定

对自行筛选分离的1株木霉菌进行形态学及分子生物学鉴定。采用CTAB法抽提其基因组总DNA,利用真菌通用引物ITS1和ITS4扩增菌株rDNA ITS区序列,扩增产物纯化后进行测序。测序结果在GenBank中进行同源性搜索,并下载部分具有代表性种的ITS序列,利用软件MEGA4构建分子系统发育树,通过序列分析,并结合形态学鉴定该菌属于半知菌亚门,丝孢纲,丛梗孢目,木霉属,康宁木霉(Trichoderma koningii)。     

1株产漆酶白腐真菌的筛选和鉴定

从不同的生境采集生物样品,利用Bavendamm反应反复筛选产漆酶的白腐真菌。利用真菌通用引物对ITS1/ITS4扩增菌株rDNAITS区序列,对扩增产物进行测序。测序结果在GenBank中进行同源性搜索,下载部分具有代表性种的ITS序列进行序列比对,利用软件MEGA4构建分子系统发育树,通过序列分析,并结合形态学鉴定出4220为香栓孔菌(Trametes suaveolens)。     

水稻条叶枯病毒(RStV)基因组组分4的克隆与序列分析

利用RTPCR技术合成并扩增了水稻条叶枯病毒(RStV)中国云南分离物基因组组分4的全长cDNA,将PCR产物克隆在载体pCRII上,并进行全序列测定,所得核苷酸序列及推测的氨基酸序列与日本分离物T进行比较。结果表明,在核苷酸水平,两分离物的vORF、vcORF及基因间非编码区序列的同源性分别为94.9%、94.1%、86.1%,5’端非编码区序列相同,而3’非编码区同源性为96.1%,仅有两个核苷酸不同;在氨基酸水平,vORF及vcORF编码蛋白的同源性分别为99.4%和98.3%。可见,编码区的大小及其氨基酸序列和两末端序列都是很保守的。因此,中国云南分离物Y与日本分离物T可能有很近的亲缘关系。     

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。     

Detection of the ectomycorrhizal fungusTricholoma matsutake and some related species with specific ITS primers

Oligonucleotide primers (Tm1 and Tm4) were designed to amplify a 447–448 base pair fragment, comprising sections of the rDNA internal transcribed spacers (ITS) and the entire 5.8S rDNA, ofTricholoma matsutake. PCR products of predicted size were produced for six of eight isolates ofT. matsutake from across its natural range in Asia, and for isolates of some closely related fungi includingT. bakamatsutake, T. magnivelare, andT. caligatum. The closely relatedT. robustum could be discriminated fromT. matsutake by PCR fragment size. No PCR products were produced where the primers were tested against 16 species of ectomycorrhizal fungi associated withPinus spp. in the Southern Hemisphere. The specific primers were also used successfully to produce PCR products from matsutake infected roots collected in natural forests in China and Japan, and from pure culture synthesisedPinus radiata-T. matsutake material. These primers will be useful in research directed at establishing matsutake in the Southern Hemisphere, and also have the potential to be applied to the study of matsutake within its natural range.     

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。     

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。     

Saprobic potential of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>: growth over pine bark treated with surfactants

Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

Artificial shiro formation of Tricholoma matsutake

One type of soil collected from Maoer-shan in Heilongjiang Province,China was selected to induce hyphal growth of Tricholoma matsutake by a soil screening experiment.It was confirmed that hyphal growth of all the tested T.matsutake isolates was significantly stimulated in soil by supplemented with 0.5%～2.0% olive oil.The aggregation of hyphae and soil resembled natural Shiro.The biomass of hyphae in the soil increases with increasing olive oil concentrations.Moreover,seedlings of Pinus densiflora grew well in the soil containing 0.5%～1.0% olive oil and were also successfully infected by T.matsutake isolate A in the soil containing 1.0% olive oil.This study established a culture system of artificial Shiro formation and also provided a premise for formulation of culture substratum for fruit body formation of T.matsutake.     

Molecular Identification of Fomitiporia mediterranea and Eutypa lata/Libertella blepharis in Platanus × acerifolia

The studies of woody organs‐affecting diseases of Platanus × acerifolia can be hampered by the finding of fungi whose identification is difficult with mycological techniques. In a previous study on Platanus × acerifolia affected by severe cankers, Fomitiporia mediterranea/punctata‐like fungi, not associated with fruit bodies and Libertella sp. were recovered. Due to the severity of the associated symptoms, a characterization of fungal ribosomal DNA genes was undertaken in the present study, aimed to specific identification of the pathogens. From DNA of Fomitiporia‐vegetative isolates and Fomitiporia‐fruit body isolates, included in the study for comparison (from fruit body on plane trees typical of F. mediterranea/punctata), and Libertella sp., DNA fragments were amplified in polymerase chain reaction (PCR) with the use of ITS5/ITS4 and 5.8S‐R/LR7 primer pairs. Sequence alignments showed that Fomitiporia‐vegetative/fruit body isolates had highly homologous ITS5/ITS4 sequences, with a nucleotide identity of 98–100%. All the Fomitiporia isolates from plane tree showed 97–100% and 91–94% of nucleotide identity respectively with ITS5/ITS4 sequences of known strains of F. mediterranea and F. punctata, extracted from GenBank. The strong similarity of these identity ranges with those obtained within F. mediterranea (98–100%) and between the two Fomitiporia species (90–94%) confirms the identification of the isolates from plane tree as F. mediterranea. Sequence comparison between Libertella sp. from plane tree and known strains of Eutypa lata/L. blepharis showed 94–99% of nucleotide identity. The comparison with eight additional species of Eutypa showed 90–93% of nucleotide identity. As previously reported for the different taxa within Diatrypaceae, also ITS5/ITS4 sequence of Libertella sp. from plane tree exhibited 11‐bp tandem repeats motifs. Results of sequence alignments, of phylogenetic trees, and of the putative restriction map, identify the Fomitiporia isolates of this work as F. mediterranea, and the isolates of Libertella sp. as E. lata/L. blepharis. For comparative purposes, ITS5/ITS4 sequences of Spongipellis pachyodon and Fomes fomentarius from plane tree, were also obtained in this work.