镰刀菌rDNA ITS区 RFLPs分析

本实验采用PCR-RFLP技术分析了镰刀菌菌种间的多态性。运用SDS碱裂解法抽提了采自于山西省不同地区的11株镰刀菌（Fusarium）基因组DNA，并用一对特异性引物对其rDNA ITS区段进行扩增，选用4种限制性内切酶（TaqⅠ AluⅠ HaeⅢ EcoRⅠ）酶切PCR扩增产物，共得到16条多态性片段，其中特异性条带8条，分子量大约在70-900bp之间。利用NTSYS-PC(2.1)软件包分析各菌株间的DNA限制性片段多态性，总共可以分为５大组，与传统分类一致，说明该方法可以用于镰刀菌菌株间的多态性分析。     

尖孢镰刀菌异核体及其不同核型分离子rDNA ITS区序列分析

选取从河南棉花上分离的尖孢镰刀菌（Fusarium oxysporum f.sp.vasinfectum）异核体菌株Ag149及其两个表型差异显著的同核型分离了Ag149－Ⅰ和Ag149－Ⅲ为材料，对它们的核糖体基因ITS区段进行测序分析。结果表明，供试菌株核糖体基因ITS区段碱基组成完全相同，与GenBank中镰刀菌的ITS区序列进行同源性比较发现，属内种间的同源性在34％－99％之间，而大部分尖孢镰刀菌种内不同菌株之间的同源性均在98％以上。序列分析表明，ITS区对镰刀菌属内种间的差异鉴别具有参考价值，但看来并不适合尖孢镰刀菌内或种以下水平的鉴定和分类。     

基于文献计量分析的镰刀菌枯萎病研究进展解析

镰刀菌枯萎病是一种由尖孢镰刀菌引起的、毁灭性极强的真菌作物病害,为探索镰刀菌枯萎病的相关研究进展及热点,对尖孢镰刀菌枯萎病进行了引文分析。以"Fusarium wilt"(镰刀菌枯萎病)进行主题搜索,在ISI Web of Science数据库核心集搜索1985—2019年的相关文献共4 873篇,采用CiteSpace可视化软件进行引文分析,通过合作、共现、共被引等网络分析,研究镰刀菌枯萎病的研究热点和前沿趋势。根据发文量统计分析发现:美国、中国和印度在本领域发文量排名前三位,而中介中心性指标分析则表明荷兰、美国和印度文献重要性位列三甲,我国位居前10并表现出积极上升趋势。引文分析的关键词突发性检测发现"induction(诱导)"、"geneticdiversity(遗传多样性)"、"pathogenicity(致病性)"是近十年的重要热点;文献共被引聚类分析表明解淀粉芽孢杆菌、系统抗性、非致病性尖孢镰刀菌等方面为本领域的研究前沿。随着枯萎病研究层次的不断深入,未来研究将会集中在微生物防控、抗病育种、根系-病原菌-有益菌互作机制等方向;此外,多组学技术的应用必将有利于解析镰刀菌枯萎...     

禾谷镰刀菌拮抗菌株的筛选及鉴定

禾谷镰刀菌引起的小麦赤霉病能够导致小麦产量的大幅降低和品质的严重损失。为探索赤霉病生物防治方法,本试验从扬花期被禾谷镰刀菌侵染的麦穗上分离得到28株芽孢杆菌,利用平板对峙法对其进行筛选,其中20株菌对禾谷镰刀菌表现出明显的拮抗作用。根据生理生化特性和16S rDNA序列分析,这20株拮抗菌分别被鉴定为甲基营养型芽孢杆菌(11株)、解淀粉芽孢杆菌(2株)、枯草芽孢杆菌(2株)、暹罗芽孢杆菌(3株)和特基拉芽孢杆菌(2株)。这些菌株能够有效降低小麦赤霉病的发病率、发病程度和病情指数,其中菌株JS62N、JS15E和JS29I能够将赤霉病发病率降低80%以上;菌株JS29I、JS62N、JS39C和JS39D能够将赤霉病发病程度降低80%以上。16株菌能将病情指数降低80%以上;所有菌株均能极显著增加小麦的百粒重,JS12Q效果最好(+101.4%)。本研究结果为小麦赤霉病的防治提供了生物防治材料。     

土壤环境因素对致病性尖孢镰刀菌生长的影响

为了探索土壤环境因素对致病性尖孢镰刀菌(Fusarium oxysporum)存活和生长繁殖的影响,通过设定环境温度、土壤含水量和土壤p H等,研究了这些因素对尖孢镰刀菌西瓜专化型(FON)和黄瓜专化型(FOC)生长繁殖的影响。结果发现,致病性尖孢镰刀菌在21℃~30℃范围内能快速繁殖,而在45℃条件下则无法生长;土壤含水量为20%时该菌繁殖速度最快,而含水量为5%时则受到明显抑制;p H 4~5.5的偏酸性土壤适宜尖孢镰刀菌繁殖,而p H为中性或以上土壤均不利于该菌生长。结果表明,高温、干旱和碱性是减轻土壤中致病性尖孢镰刀菌繁殖的有利环境因素,通过创造相应环境条件,可以控制枯萎病的发生或蔓延。     

植物浸提液对尖孢镰刀菌的抑菌效果

为给新型生物农药的研发提出依据,用黄连、黄芩、大蒜、金银花、柴胡和连翘6种植物的浸提液单剂及其复合剂对尖孢镰刀菌进行处理,探索浸提液单剂及复合剂对尖孢镰刀菌的影响。综合对尖孢镰刀菌菌落生长和产孢量两方面的影响,植物浸提液单剂的抑菌效果相对较好的处理分别是50%黄连、30%黄芩、20%金银花、60%柴胡、50%连翘、60%连翘以及10%大蒜、30%大蒜、60%大蒜。6种植物浸提液对尖孢镰刀菌均有抑菌作用,以黄连的抑菌效果最好,平均抑菌率为52.95%,平均抑产孢率为59.46%。     

小麦籽粒中结合态脱氧雪腐镰刀菌烯醇毒素产生规律研究

为了研究小麦籽粒中结合态脱氧雪腐镰刀菌烯醇(DON)毒素产生规律,以小麦籽粒为原料,采用人工接种禾谷镰刀菌Fg18.7产毒,三氟甲烷磺酸水解后ELISA检测结合态DON.结果表明:15℃、20℃时游离态DON含量均随着培养时间的延长逐渐增加,25℃时游离态DON含量先增加后减少;不同温度下结合态DON含量均随着培养时间的延长逐渐增加,15℃时40天后达到最高含量为129.20ng·g-t.认为禾谷镰刀菌在小麦籽粒中先产生游离态DON,之后游离态DON会与籽粒中某些成分结合产生不可溶的结合态DON.     

浙江乐清铁皮石斛rDNA ITS序列的克隆及序列比对

为判断浙江乐清铁皮石斛（Zhejiang Dendrobium officinale）与石斛属（Dendrobium）其它物种的亲缘关系,本文提取了该石斛鲜样的DNA,利用真核生物ITS序列通用引物ITS4/ITS5进行了扩增,并将扩增产物连接转化至大肠杆菌,在对阳性克隆测序。将得到的序列与GenBank上我国石斛属12个组中34个种的rDNAITS进行了比对,并在此基础上构建了系统发育树。序列比对结果表明浙江乐清铁皮石斛的rDNAITS与黄石斛（D.tosaense）一致,其次与铁皮石斛（D.officinale）最相近,序列相似性99%。系统发育树也表明浙江乐清铁皮石斛的rDNAITS序列与黄石斛（D.tosaense）的相似程度高于其与铁皮石斛（D.officinale）的相似程度。本研究将为利用ITS序列鉴别石斛物种种属关系提供参考。     

Grouping of Bradyrhizobium USDA strains by sequence analysis of 168 rDNA and 168-238 rDNA internal transcribed spacer region

In order to select appropriate Bradyrhizobium USDA reference strains for primary grouping of indigenous soybean bradyrhizobia, we systematically constructed phylogenetic trees of 20 USDA strains based on DNA sequence analysis and PCR-restriction fragment length polymorphism (RFLP) targeted to 16S rDNA and the internal transcribed spacer (ITS) region between 16S and 23S rDNAs. The phylogenetic trees of 16S rDNA showed 3 major groups, cluster USDA 110 (USDA 62, 110, 122, 125, and 129), cluster USDA 6 (USDA 4, 6^T, 38, 115, 123, 127, 135, and 3622^T) and cluster B. elkanii (USDA 31, 46, 61, 76^T, 94, and 130), as well as the phylogenetically independent strain USDA 124. The topology of the ITS trees was almost similar to that of 16S rDNA, although the positions of two extra-slow-growing strains, USDA 135 and USDA 3622^T were variable among the ITS sequences, PCR-RFLP of the ITS region and 16S rDNA. Only two strains, USDA 110 and USDA 122, harbored hup genes and they fell into the USDA 110 cluster. These results suggest that PCR-RFLP analysis of 16S rDNA and the 16S-23S rDNA ITS region may be useful for the grouping of bradyrhizobia and for the first screening of hup-positive strains. Based on the above results, we propose a minimum set of USDA strains reflecting Bradyrhizobium diversity that includes B. japonicum USDA 6^T, B. japonicum USDA 110, B. japonicum USDA 124, and B. elkanii USDA 76^T. In addition, an extra-slow-growing strain with the serotype USDA 135 might be necessary for genomic diversity analysis of bradyrhizobia, because their phylogenetic positions were variable.     

鸡生长激素基因的PCR—RLFPs分析

本实验以粤黄鸡矮脚黄系等不同蛋鸡、肉鸡品种（系）作为实验材料，研究生长激素（cGH)基因的多态性，由此计算鸡不同群体的基因型频率和基因频率，并用DT软件的类平均法（UPGMA）进行聚类分析，发现cGH内含子1有6种基因型，受3个等位基因控制；内含子4有7种基因型，受4个等位基因控制，丝毛鸡的两个个体的PCR产物则出现双带现象。还发现优质肉鸡，速生型肉鸡内含子1和4的基因型频率和基因频率比较接近，而蛋鸡与它们的差别比较明显，聚类分析有类似的结果。     

基因组ITS序列分析鉴定红曲霉菌株

为建立有效的红曲霉分类鉴定方法,进一步发掘和保护红曲霉菌株生物资源,以福建省内各类红曲霉生产菌株为主要材料,采用MEGA 5.10软件对其ITS序列进行分析,构建系统发育树,结合在麦芽汁琼脂培养基、察氏酵母提取物琼脂培养基和甘油硝酸盐琼脂培养基上的菌落形态特征以及生理生化试验对41株红曲霉进行分类鉴定。试验结果显示41个红曲霉菌株的ITS序列长度为520bp左右,嘌呤嘧啶(GC)含量比例在56.2%~58.0%,41个不同序列间的平均遗传距离为0.0052。经分子生物学手段分析,辅以菌落形态观察和生理生化试验,最终将41株红曲霉鉴定为紫色红曲霉、丛毛红曲霉、橙色红曲霉、红色红曲霉、烟灰色红曲霉和白色红曲霉6大类。     

Fungal communities in bulk soil and stone compartments of different forest and soil types as revealed by a barcoding ITS rDNA and a functional laccase encoding gene marker

The soil fungal diversity and community partitioning between the bulk soil and stone compartments was investigated using PCR based approaches targeting the barcoding internal transcribed spacer (ITS) of rDNA and the laccase encoding functional gene as genetic markers. Soil samples were collected from the B-horizon of spruce and beech forests at the Hainich Biodiversity Exploratory, central Germany. The targeted markers were amplified from the respective DNA extracts using general fungal primers and basidiomycete laccase gene specific primers, cloned and sequenced. Differences in the fungal community composition between the two forest types and the soil compartments were indicated by both markers. When the effects of ecological factors were considered, the two markers produced different patterns of results. The ITS rDNA marker revealed communities principally influenced by forest type, while those detected with the functional marker were mainly affected by soil pH. The fungal communities detected by the functional marker in particular, differed significantly between soils and stones, indicating that laccase-producing fungi are specifically adapted to degrade organic matter in soils rather than weathering of stones. The study underlines the fact that coherent and complementary results may be obtained with both genetic markers used.     

Analysis of ITS of the rDNA to infer phylogenetic relationships among Vietnamese Citrus accessions

This study focused on clarifying phylogenetic relationships among Citrus accessions from Vietnam. Our phylogenetic analysis based on nucleotide sequences from the ITS of the ribosomal DNA included 69 accessions belonging to Citrus and related (sub)genera. Maximum parsimony and Bayesian analysis confirmed a clear separation of the three ‘true’ Citrus species (C. medica, C. maxima and C. reticulata). Confirming recent taxonomic revisions, Fortunella, Poncirus trifoliata and Citrus hystrix are clustered among the accessions of subgenus Citrus. C. × sinensis accessions revealed a close evolutionary relationship to either C. maxima or C. reticulata, thereby confirming their involvement in its hybrid origin. Also, some other hybrid taxa and their proposed parental species were investigated and their origin could in some cases be confirmed using the ITS sequence data.     

沙田柚黄龙病病原16S rDNA片段的克隆与序列分析

采集田间表现斑驳症状的沙田柚叶脉,用CTAB法提取总DNA。根据柑橘黄龙病病原16S rDNA的核苷酸序列设计引物P1/P2,进行PCR扩增,获得1条大小为1 167 bp的片段。酶切分析显示,该片段可被切成大小分别约为640 bp和520 bp的2个片段。扩增产物经纯化,与pM D 18-T V ector连接,转化大肠杆菌(E scherich ia coli)DH 5α,筛选克隆重组子。对PCR产物进行测序及序列分析,结果表明,与柑橘黄龙病病原亚洲种16S rDNA的同源性为99%,与非洲种的同源性为97%,与美洲种的同源性为96%。认为,沙田柚的斑驳症状是由黄龙病病原引致的,称之为沙田柚黄龙病。该沙田柚黄龙病病原属于柑橘黄龙病病原亚洲种(L iberobacter as iaticus)中的一个成员。系统进化树分析显示,沙田柚黄龙病病原与中国柑橘黄龙病病原亲缘关系最近,推测是直接来自中国柑橘黄龙病病原。     

甘肃本地产当归、黄芪和大黄的rDNA ITS序列分析

本研究采用改良CTAB法分别提取18份甘肃本地产当归、黄芪和大黄基因组DNA,并用PCR分别扩增其ITS1-5.8S-ITS2序列、直接测序并作序列同源性比对分析。双向测序分析结果表明,甘肃6个不同产地当归rDNA的ITS1、5.8S和ITS2序列一致,片段长度分别为215bp、162bp和223bp;供试的黄芪ITS1、5.8S和ITS2序列分别为228bp、164bp和210bp;大黄ITS1、5.8S和ITS2序列分别为160bp、159bp和164bp。供试材料的ITS1-5.8S-ITS2核苷酸序列已提交GenBank。本研究为提供甘肃当归、黄芪和大黄指纹图谱鉴别的分子标记、其道地性药材的分子鉴定和品质评价提供参考。     

毛竹竹鞭内生细菌的特征及16S rDNA多样性分析

本研究利用传统的细菌分离方法,结合16S rDNA序列分析对毛竹竹鞭内生细菌的特征和多样性进行了分析。从福建省武夷山、将乐、长汀毛竹竹鞭中分离到34株内生细菌,初步归属于14属,20种。来源于不同地区的毛竹竹鞭内生细菌组成存在较大差异,其优势菌群为产碱杆菌属（Alcaligenes）和葡萄球菌属（Staphylococcus）。