河北省玉米小斑病菌优势生理小种鉴定及ITS序列分析

2007~2016年在河北各地采集玉米小斑病标样,对分离出的356株玉米小斑病菌菌株进行生理小种鉴定。结果表明,在鉴定的年度范围内,玉米小斑病菌T小种、C小种、S生理型、O小种的分离频率在年度间存有差异,O小种的平均分离频率94.94%,是河北省玉米小斑病菌的优势小种;O小种对主栽品种郑单958和自交系C103的致病力呈下降趋势,在C103上致病力下降幅度小于郑单958。对河北省石家庄、保定、衡水、沧州邯郸、邢台采集的45株菌株进行ITS序列分析构建UPGMA进化树,分析表明,河北省内玉米小斑病菌株的遗传进化与地域有一定关系,差异不明显。     

间作栽培对宁夏南部山区马铃薯根际土壤真菌菌群结构的影响

根据宁夏南部山区气候和作物的生长特点，设计马铃薯‖玉米（P‖M，行比分别为4∶1，3∶2， 2∶3）、马铃薯‖蚕豆（P‖F，行比同前）、马铃薯不同品种（A‖B‖C，行比1∶1∶1）间作栽培模式，以马铃薯连作为对照，研究马铃薯根际土壤真菌多样性和菌群结构的变化，探寻能够减轻宁夏南部山区马铃薯连作障碍的有效栽培模式。采用基于18S rDNA的末端标记限制性片段长度多态性 (Terminal restriction fragment length polymorphism，T-RFLP) 技术研究不同间作栽培模式下马铃薯根际土壤真菌的菌群结构和多样性变化，构建真菌ITS克隆文库，利用Genbank数据库比对各栽培模式中ITS序列的测序结果，并作群落结构组成和功能分析。结果表明，马铃薯间作栽培后真菌Shannon-Wiener指数和Simpson指数均有不同程度降低；马铃薯与玉米、蚕豆间作后根际土壤真菌的物种丰富度在门、纲和目的分类学水平上与连作相比明显下降，菌群结构发生较大变化，成熟期马铃薯‖玉米3∶2行比间作模式与连作的差异最大，属的比例下降67.74%。间作栽培后，黑孢属（Nigrospora）、地丝霉属（Geomyces）、圆盘菌属（Orbilia）、枝顶孢属（Acremonium）、四枝孢属（Tetracladium）等9个属的真菌消失，同时新增刺盘孢属（Colletotrichum）、毛壳属（Chaetomium）、巨孢囊霉属（Gigaspora）、小球腔菌属（Pleosporineae）等13个属的真菌，其中马铃薯‖玉米3∶2行比间作后巨孢囊霉属比例高达60.35%。可见，马铃薯‖玉米间作栽培能有效改善马铃薯根际土壤的真菌菌群结构，使其微环境得以改善，缓解宁夏南部山区马铃薯连作障碍。     

肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。     

The true nature of Ganoderma in Iran: Taxonomy based on ITS and mtSSU rDNA

The genus Ganoderma Karst. has broad‐spectrum usage in biotechnology, medicine and the food industry. The complexity of the morphology within species has led to uncertain identification in the past, but recent advancements in molecular identification methods have provided scientists with the opportunity to better understand the taxonomy of the species. The present study attempts for the first time to elucidate the distinctiveness of the Ganoderma species growing in Iran concerning those elsewhere in the world based on mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and internal transcribed spacer (ITS) sequence data. The results disclosed that the G. lucidum Karst. samples collected in Iran are more similar to the European Ganoderma species than to the Asian (Chinese) one used in this study.     

葡萄炭疽病菌鉴定及同源性分析

为揭示葡萄炭疽病菌种类和遗传特征,给深入研究该病提供科学理论依据。本研究对不同地区的26株葡萄炭疽病菌通过形态学和r DNA-ITS序列分析进行鉴定及同源性研究。利用真菌通用引物ITS4、ITS5对病菌基因组进行PCR扩增,将测序结果与NCBI数据库上已知序列进行比对,同时利用Clustalx 1.83和MEGA 5.0软件进行聚类分析。结果表明:病菌菌落为白色或灰白色,菌丝呈绒状或絮状,分生孢子为圆柱形或圆筒状,单孢,无色;测得病菌ITS序列为500 bp左右,鉴定病原均为胶孢炭疽菌(Colletotrichum gloeosporioides)。不同地区葡萄炭疽菌的r DNA-ITS序列同源性高,亲缘关系比较近,但是各菌株间存在着遗传差异,并且菌株之间差异与地理来源和品种无明显的相关性。     

肉牛源细极链格孢菌的分离鉴定

为鉴定肉牛皮肤病病原及其致病性并筛选其敏感药物,本研究取患皮肤病肉牛病变部位皮屑、毛发和痂皮进行病原菌的分离,并对分离菌株进行了形态学鉴定、ITS序列分析、小鼠致病性试验和药敏试验。结果显示分离到1株形态特征与细极链格孢菌极其相似的菌株(PY2-1-2);经PCR扩增、测序和BLAST序列比对,结果显示分离株(PY2-1-2)的ITS基因序列与细极链格孢菌(MG975630.1)的ITS基因序列的相似性为99%,分离株PY2-1-2的ITS基因序列长度为543 bp,该片段包括ITS1、5.8S rDNA和ITS2的全部基因序列以及18S rDNA和28S rDNA的部分基因序列,提交GenBank(MH656780.1)。根据形态学结合ITS基因序列分析结果确定该分离株为细极链格孢菌。小鼠致病性试验结果显示该菌对小鼠有致病性。药敏试验结果显示:该菌对灰黄霉素、氟康唑、伊曲康唑、酮康唑的最小抑菌浓度(MIC)值分别为0.5μg/mL、4μg/mL、1μg/mL、1μg/mL。本研究为今后进一步探究细极链格孢菌引起的皮肤病诊治提供科学有效的参考。     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

基于ITS序列的曼陀罗属植物的分类学研究

根据曼陀罗属核糖体基因转录间隔区(ITS)序列设计通用引物,得到33个不同来源的曼陀罗属植物的ITS序列,并以小天仙子(Hyoscyamus bohemicus)为外群,应用遗传距离与系统树分析法对曼陀罗属植物之间的分类进行了初步探讨。结果表明:在供试样品中,紫花曼陀罗(Datura tatula)和曼陀罗(Daturastramonium)的ITS序列完全相同;多刺曼陀罗(Datura ferox)、栎叶曼陀罗(Datura quercifolia)和曼陀罗(Datura stramonium)之间的亲缘关系很近;紫花曼陀曼(Datura tatula)、曼陀罗(Datura stramonium)、无刺曼陀罗(Datura inermis)和光滑曼陀罗(Datura laevis)4个种间不存在遗传距离;传统分类中曼陀罗属的Dutra亚属内除光曼陀罗(Datura leichhardtii)和异色曼陀罗(Datura discolor)在ITS序列表现为比较独立的2个种外,其他各种间的亲缘关系也较近;在ITS序列上湿地曼陀罗(Datura ceratocaula)与曼陀罗(Daturastramonium...     

一株高雄山虫草的分子鉴定

[目的]利用生物信息学方法初步探讨虫草属日本拟青霉的分类地位。[方法]提取日本拟青霉的基因组,并利用通用引物ITSL和ITS4采用PCR技术扩增rDNA18S-28S ITS序列。采用Clustalx1.83和MEGA5软件构建系统发育树,生物信息学比较分析确定其分类地位。[结果]PCR扩增产物为603 bp。将日本拟青霉重新命名为雪花高雄山虫草(Cordyceps takaomontana strain JLsnow)。将虫草重新分类,并对该类群及其特征进行了描述。[结论]从分子水平上说明日本拟青霉与其他虫草之间的差异,该研究为虫草属真菌的分类提供新思路。     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.