一株临沧野生灵芝的鉴定与驯化栽培

从采集于云南临沧的野生灵芝子实体中分离纯化获得纯培养物,编号为YAASM4672,结合形态学与ITS序列分析鉴定其分类学地位,并设计了9种栽培种培养基,测定其栽培特性。结果显示,采集到的菌株YAASM4672为灵芝（Ganoderma lingzhi）,其形态特征与分子鉴定结果一致。该菌在2号栽培种配方上,菌丝生长和子实体农艺性状（覆土组优于露地组）表现最好。菌丝体白色,长势浓密,38 d满袋,平均生长速度达(5.89±1.01) mm/d;菌盖黄色,直径达66.00 mm,平均生长速度最快为(4.14±1.07) mm/d,与其他配方相比,达到显著（P<0.05）或极显著差异（P<0.01）;菌柄褐黄,其长度、直径分别达到84.00 mm、18.00 mm。驯化的野生灵芝菌株YAASM4672人工栽培最优配方为：78%玉米芯、18%米糠、2%高粱粉、1%石膏粉、1%白砂糖,覆土能促进其子实体的生长发育。     

The Isolation,Identification and Phylogenetic Analysis of Theileria

Bovine theileriosis was a kind of hemo-protozoan diseases that seriously hindered the sustainable development of cattle industry.In the present study, the infection of Theileria was detected using microscopic examination and species-specifc PCR of T.annulata, T.sergenti and T.sinensis from 18 blood samples collected from a breeding cattle farm in China.Then ITS and MPSP genes were amplified and cloned using universal primers of ITS and allele-specific primers of 4 major piroplasm surface protein (MPSP) from the positive samples.The sequences were used to make alignment, polymorphism and phylogeny analysis.The results showed that 3 samples were positive for Theileria and the 3 positive samples were all the T.sergenti infection.The amplification of allelic MPSP gene of T.sergenti from the 3 positive samples showed that two of them were single infection by Ikeda-type and another one was co-infection with both Chitose-type and Ikeda-type, while Buffeli-type and Thai-type were not detected.Moreover, on the basis of phylogenetic tree constructed with MPSP gene sequences, types 1 and 2 of MPSP were confirmed to be present in the cattle farm.The results revealed that there were T.sergenti infection in the breeding cattle farm, and these parasites at least with 2 allelic MPSP gene types were present, which indicated that the immune prevention and control of the disease became more complicated.Our research laid foundation of the further study on T.sergenti infection and disease prevention and control.     

Genetic variation and demographic history analysis of Pestalotiopsis,Pseudopestalotiopsis, and Neopestalotiopsis fungi associated with tea (Camellia sinensis) inferred from the internal transcribed spacer region of the nuclear ribosomal DNA

To elucidate the demographic history within and among the genera Pseudopestalotiopsis, Neopestalotiopsis, and Pestalotiopsis and their genetic diversity, we studied internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) with no saturation effects in 172 Pestalotiopsis-like fungal accessions isolated from Camellia sinensis planted in nine provinces in China (64 from Yunnan, 4 from Guangxi, 2 from Guizhou, 42 from Jiangxi, 3 from Sichuan, 3 from Zhejiang, 1 from Henan, 18 from Fujian, 12 from Chongqing, and 18 from other provinces) and in Japan (1), France (1), Turkey (1), and Thailand (2). Among these, 44 Pestalotiopsis-like isolates from Yunnan in this study formed a clade in phylogenetic trees with Pestalotiopsis accessions in GenBank, despite their cultural characteristics on potato dextrose agar displaying no genus specificity. Based on estimated sequence polymorphism parameters of average pairwise differences (K), haplotype diversity (H_d), nucleotide diversity (Pi), θ (per site) from η, and recombination event rate (R_m), we identified moderate genetic variation in these three genera. Molecular evolution analysis with neutrality tests and mismatch distribution demonstrated that Pseudopestalotiopsis and Pestalotiopsis ribosomal ITS sequences evolved under a neutral model. However, the recent demographic expansion of the three genera promoted geographical isolation but showed no clear geographical structure. Pairwise genetic divergences between haplotypes and a median-joining network revealed Yunnan or Jiangxi as a possible centre of origin, and that Pseudopestalotiopsis and Neopestalotiopsis may derive from Pestalotiopsis.     

BtR05的鉴定及其对朱砂叶螨的毒力测定

对山东省应用微生物重点实验室分离保存的苏云金芽孢杆菌BtR05进行了16S rDNA序列分析,结合菌落形态、生理生化特征研究,将其鉴定为苏云金芽孢杆菌属.通过与已知的苏云金芽孢杆菌29个亚种间的生理生化特征进行比较,鉴定BtR05为苏云金芽孢杆菌达姆斯特亚种(Bacillus thuringiensis subsp.d...     

一株弗氏柠檬酸杆菌的分离鉴定及致病性研究

为确定导致罗非鱼患病的原因，本试验对濒临死亡的罗非鱼进行无菌解剖，从心脏部位分离得到1株细菌，通过形态学观察、生理生化鉴定和16S rDNA分析，确定菌株为弗氏柠檬酸杆菌（Citrobacter freundii）。药敏试验结果显示，该菌株具有很强的多重耐药性，测试的20种药物中，该菌株对恩诺沙星、青霉素等13种药物具有完全耐药性，仅对头孢曲松、头孢他啶、头孢拉定、哌拉西林、舒巴坦5种药物敏感。人工感染试验结果显示，该菌对草鱼、黄颡鱼、罗非鱼、禾花鲤具有较强的致病性。本试验结果阐明了罗非鱼患病的原因，同时在养殖过程中可指导养殖户合理用药。     

青海省大通县世居牦牛犊隐孢子虫感染情况调查及虫种鉴定

旨在调查青海省大通县部分地区世居牦牛犊隐孢子虫的感染情况及其虫种类型。在大通县宝库乡和良教乡采集了200份3~5月龄世居牦牛犊粪样进行隐孢子虫检测。所有样品经蔗糖密度梯度离心法纯化处理,采用免疫荧光试验（IFT）方法进行检测,阳性样品再用套式PCR扩增18S rDNA,并进行测序和同源性分析以供虫种鉴定。结果表明：免疫荧光试验（IFT）方法镜检出3份隐孢子虫阳性样品,阳性率为1.5%（3/200）;阳性样品经套式PCR扩增18S rDNA后,有2份隐孢子虫样品呈阳性,扩增产物长度为500 bp,测序后将序列命名为QHDTC201901和QHDTC201902;系统发育分析显示,种类鉴定发现2种隐孢子虫,分别为安氏隐孢子虫（Cryptosporidium andersoni）和牛隐孢子虫（Cryptosporidium bovis）,总感染率为1.0%（2/200）。综上提示,大通县部分地区牦牛犊存在隐孢子虫感染,感染虫种为安氏隐孢子虫和牛隐孢子虫。该调查研究为该地区牛隐孢子虫病的防控提供了临床基本数据。     

基于ITS序列探讨南瓜属植物系统发育

基于ITS序列对南瓜属3个种(中国南瓜、美洲南瓜和黑籽南瓜)的系统发育进行了研究。结果表明,ITS1全长188～191bp,ITS2全长251～263bp;南瓜ITS1序列的变异位点数为33,占ITS1全序列的17.55%,ITS1序列的信息位点数为24,占ITS1全序列的12.77%;南瓜ITS2序列的变异位点数为45个,占ITS2全序列的17.93%,ITS2的信息位点数为31个,占ITS2全序列的12.35%;ITS1的G+C含量变化范围为51.60%～54.50%,ITS2的G+C含量变化范围为53.78%～56.98%;基于ITS1和ITS2分别构建的系统树可以看出,中国南瓜和美洲南瓜的亲缘关系较近,与黑籽南瓜的亲缘关系较远。     

反刍动物瘤胃内古菌的研究进展

古菌是目前生物地球化学研究的热点之一,不仅能在高温、高盐、强酸、强碱、缺氧等极端条件下存在,而且能在多种生态环境中存活。其含量巨大和代谢产物多样,在全球的地球生物化学的作用中正扮演着重要角色。该领域研究对阐明生命运动的基本规律,揭示生物与环境间的相互作用具有重要意义。为便于了解这一新型领域以及进行的相关研究,着重介绍了生活在反刍动物瘤胃内的产甲烷古菌,以及研究瘤胃内古菌多样性的分子生物学方法,即16SrDNA的分析,该方法多被应用于极端环境下海洋古菌的扩增。近年来,在拓展于瘤胃内古菌的研究中,主要是应用16SrDNA基因分别建立产甲烷古菌的克隆库,对反刍动物瘤胃内古菌的多样性进行了研究。     

东祁连山高寒草地土壤5种镰孢菌的形态鉴定和ITS rDNA分析

为确定东祁连山高寒草地土壤中镰孢菌的分类地位,运用稀释平板法对从其中分离的5种镰孢菌,采用形态学和培养特征进行观察描述,同时利用ITS rDNA序列对其系统发育学进行分析,确定分类地位,并分析比较了基于形态学和ITS rDNA序列分析的两种鉴定手段。结果表明,3个菌株为三线镰孢(Fusariumtricinctum),2个菌株为木贼镰孢(F.equiseti),与传统方法相比,ITS rDNA方法有着较高的灵敏度,但对于东祁连山高寒草地土壤镰孢菌的鉴定,只有形态学与分子生物学鉴定相互结合,才能使镰孢菌的鉴定分类更加完善。     

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.