松茸组织分离物的rDNA-ITS序列鉴定

以采自云南丽江的松茸子实体为材料,进行组织分离后,利用一对ITS引物(ITS1-ITS4)对子实体(SR176B,SR172B)和分离物(SR176H,SR172H)进行了PCR扩增、琼脂糖凝胶电泳分析,得到了700bp左右的扩增条带,进一步对ITS序列进行同源性检索比对,结果表明SR176H与SR176B,SR172H与SR172B序列同源性均为100%,鉴定出该分离物就是松茸的纯培养物。     

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。     

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。     

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。     

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73bp和26bp的特征序列。     

苹果炭疽菌的分子鉴定与检测

测定苹果炭疽菌rDNA全序列,比对苹果炭疽菌和其它炭疽菌ITS序列以及构建系统关系树,发现苹果炭疽菌与胶孢炭疽菌的ITS序列相似性高达99．8％,并与胶孢炭疽菌聚在一起,可以明确苹果炭疽菌应属于胶孢炭疽菌。进一步的序列比对发现,苹果炭疽菌的18S rDNA3’端比其它胶孢炭疽菌多出一段379bp的序列,根据这一特有片段设计引物CgF1与通用引物ITS4配对,结果仅能从苹果炭疽菌中扩增出1232bp的特异性条带。用苹果炭疽菌接种离体苹果,以接种发病的病组织总DNA为模板,利用引物CgF1／ITS4进行PCR扩增,同样可以扩增出1232bp的特异性条带,而健康苹果组织DNA中未能扩增出任何条带,表明该方法可用于苹果炭疽菌的鉴定和快速检测。     

无绿藻PCR ITS1-ITS2基因分型法的建立及应用

目的建立无绿藻(Prototheca)PCR ITS1-ITS2基因鉴定分型方法,并通过系统发育树以研究各型别无绿藻菌株间的亲缘关系。方法抽提5株无绿藻临床分离菌株的DNA,通过特异性引物扩增ITS1及ITS2区后,根据该区域核苷酸片段的差异进行基因分型,借助MEGA6.0软件建立系统发育树,并通过SSU rDNA测序加以验证。结果 5株菌株中,1株鉴定为P.zopfii hydrocarbonea,2株鉴定为P.wickerhamii。虽然由于数据库中缺乏P.zopfii portoricensis菌株ITS序列导致1、2号菌株不能被鉴定,但ITS区系统发育树中这两株菌仍被归为P.zopfii中独立的一簇,与SSU rDNA系统发育树相近,证明此分型方法无误。结论 PCR ITS1-ITS2基因分型法可作为一种有效基因分型方法用于鉴定和监测无绿藻感染。     

可污染食品及饲料的产黄曲霉毒素真菌的多重PCR检测

根据黄曲霉毒素生化途径中的关键调控基因aflR、omt-1和ver-1的序列以及真菌共有的5.8S rDNA的ITS序列分别设计ApaF/ApaR、OmtF/OmtR、VerF/VerR及ITS1/ITS4四对引物,研究建立产黄曲霉毒素真菌及其潜在饲料或食品污染的多重PCR快速灵敏检测体系。PCR扩增的4个DNA片段中,1032bp、797bp和600bp与基因库中对应基因或DNA序列的同源性达99%以上,仅452bp片段与对应基因ver-1的同源性为98%。通过优化主要影响因子,建立了快速检测产黄曲霉毒素真菌的单管多重PCR反应体系,并用于6种曲霉和1种青霉DNA样品的检测。结果显示,上述4个片段均平行地清晰出现在2株黄曲霉Aspergillus flavus和1株寄生曲霉A.parasiticus的DNA样品中,而其余菌种只检测到ITS片段,说明检测特异性很好。灵敏性分析表明,多重PCR检测的保守灵敏度为1ng/μL样品DNA,所有目标片段的条带均很清晰;即使DNA浓度降至0.1ng/μL,除aflR之外的所有条带也可分辨。     

松口蘑与假松口蘑ITS序列测定和分析比较

对松口蘑和假松口蘑进行ITS序列测序,通过DNAStar软件比较分析,发现松口蘑与假松口蘑的5.8SrDNA序列完全一致,ITS1和ITS2呈现不同程度的多态性。松口蘑ITS序列长度为601bp,假松口蘑ITS序列长度为563bp。设计了扩增松口蘑和假松口蘑ITS1的特异性引物,能够快速地区别松口蘑与假松口蘑。     

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.     

Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and beta-tubulin gene sequences

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.     

Traceability of Asian Matsutake, specialty mushrooms produced by the ectomycorrhizal basidiomycete Tricholoma matsutake, on the basis of retroelement-based DNA markers

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies, matsutake, in forests. Here we report a PCR system targeting retroelement integration sites to differentiate among individual Asian isolates of T. matsutake based on their geographical origins, such as Japan, the area of South Korea through North Korea, the northeastern provinces of China, and the area of the southwestern provinces of China through Bhutan. The overall misjudgment rate of the analytical system was approximately 5% based on 95 samples of T. matsutake examined including those from cultures and from commodities. We also provide evidence that T. matsutake isolates grown throughout the Far East, including the northeastern provinces of China, are closely related to each other while distinct from those in the area of the southwestern provinces of China through Bhutan. The method allows us to trace back geographical origins of Asian matsutake, thus contributing to food safety, appropriate tariffs, and proper price setting.