南昌市郊梨轮纹病病原菌鉴定

摘要：为了明确南昌市郊区梨轮纹病菌的种类归属，本文从果园采集呈典型症状的梨轮纹病样本，采用组织分离法进行病菌分离和病菌纯化，获得5个梨轮纹病菌分离株，该5个分离株的培养性状相同且与已报道的葡萄座腔菌Botryosphaeria dothidea的培养性状一致。用菌饼对离体梨枝条进行接种，结果5个分离株均导致典型病斑，对该病斑进行再分离，又分离到与原接种菌一致的病原菌。任选其中的一个分离株NC-1进行rDNA-ITS序列扩增和序列测定，获得片段长为543bp的核苷酸序列，通过BLAST分析，发现该序列与GenBank中葡萄座腔菌的同源性高达100%。上述结果表明南昌市郊区梨轮纹病菌属于葡萄座腔菌     

山楂叶悬钩子枯枝病病原菌形态学及分子鉴定

在东北林业大学校园内的山楂叶悬钩子(Rubus crataegifolius Bge.)上发现了1种枯枝病,对其病原菌进行分离纯化,通过PDA菌丝体对健康的山楂叶悬钩子进行柯赫氏证病法验证,20 d后烧伤处理接种病原菌的山楂叶悬钩子枝条上出现典型症状;结合形态学鉴定以及rDNA-ITS、26S rDNA D1/D2、EF-1a序列的比对分析,确认引起山楂叶悬钩子枯枝病的病原菌为火炬树暗葡腔菌(Phaeobotryon rhois),该病害为山楂叶悬钩子的国内新病害。     

Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.     

黄瓜霜霉病和白粉病病原菌的rDNA-ITS序列分析

为了应用分子特征确定黄瓜霜霉病和白粉病的病原菌种类,扩增、测定了上海地区黄瓜霜霉病菌和白粉病菌的核糖体DNA内转录间隔区(rDNA-ITS)序列,依据rDNA-ITS序列特征分析了两种病原菌种类,以及与近缘种的差异性。结果显示,黄瓜霜霉病菌的rDNA-ITS1和rDNA-ITS2长度分别为141和406 bp,rDNA-ITS1 GC含量为41.13%,rDNA-ITS2 GC含量为46.80%(闵行区株和金山区株)或46.55%(浦东新区株),rDNA-ITS序列在种内保守性很高,种间差异性与亲缘关系呈正相关,分子特征证实研究的黄瓜霜霉病病原菌为古巴拟霜霉菌;黄瓜白粉病菌的rDNA-ITS1和rDNA-ITS2长度分别为136和89 bp,GC含量分别为59.56%和66.29%,rDNA-ITS序列在研究材料中保守,与瓜类单囊壳(Sphaerotheca cucurbitae)完全相同,但与形态鉴别的结果Sphaerotheca fuliginea差异高达4.5%,提示黄瓜白粉病病原菌的种类需进一步澄清和确定。     

Molecular analysis of the major Phytophthora species on cocoa

The internally transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene cluster of 161 isolates of Phytophthora species involved in pod rot, stem canker and leaf blight of cocoa were analysed to determine inter- and intraspecific variation in this disease complex. The species P. palmivora , P. megakarya , P. capsici , P. citrophthora and P. nicotianae could all be clearly distinguished by PCR amplification of the ITS region followed by restriction analysis with Hae III, Hinf I, Pvu II and Alu I. This method provided a relatively rapid identification procedure for these species, and was able to distinguish isolates that had previously been misidentified by morphological methods. Sequence analysis showed that the four main cocoa-associated species formed two distinct groups, one comprising P. capsici and P. citrophthora , and the other P. palmivora and P. megakarya . Detailed sequence analysis and comparison with published literature suggested that P. capsici isolates from cocoa may be closely related to P. tropicalis , a species recently described from Cyclamen and Dianthus .     

引起乙烯褪绿蜜橘果实腐烂的主要致病真菌分离及鉴定

为明确贮藏过程中乙烯褪绿蜜橘果实腐烂的主要致病真菌,以重庆市北碚产区无核蜜橘为研究材料,利用乙烯利对其果实进行褪绿处理,观察贮藏期间褪绿蜜橘果实的发病症状,确定病害种类,统计发病率;并从发病果实中分离主要致病真菌,采用形态学特征观察和ITS序列分子生物学分析对其进行鉴定。结果表明,乙烯褪绿加剧贮藏蜜橘果实病害的发生。从发病果实中共分离出5株主要致病真菌菌株,分别编号为A、B、C、D、E;结合形态学特征和5株致病真菌的ITS序列分析结果,A、B菌株均被鉴定为胶孢炭疽菌Colletotrichum gloeosporioides,但这2株菌株具有不同的菌落形态及致病特性;C菌株被鉴定为焦腐病菌Lasiodiplodia theobromae;D菌株被鉴定为交链格孢菌Alternaria alternata;E菌株被鉴定为棘孢曲霉Aspergillus aculeatus。A、B菌株引起的果实腐烂占乙烯褪绿蜜橘总腐烂果实的81.96%,是引起贮藏过程中乙烯褪绿蜜橘果实腐烂的主要致病真菌,其次为C菌株,D、E菌株为次要致病真菌。     

一种绞股蓝根结线虫的研究初报

调查陕西安康地区绞股蓝根结线虫的发生情况及危害种类,揭示绞股蓝连作与根结线虫的关系,旨在为绞股蓝根结线虫防治提供理论依据。对安康主产区6个调查点的绞股蓝根结线虫病进行取样调查,并采用形态学观察及rDNA-ITS序列分析方法进行分离鉴定。结果表明,在安康市绞股蓝主产区均发现了不同程度的根结线虫危害,严重的地块发病率高达75.4%,且发病情况随种植年限的延长和海拔的降低而逐渐加重。通过分离鉴定,确定了发生在安康地区绞股蓝上的根结线虫为南方根结线虫(Meloidogyne incognita)。目前未见绞股蓝上发现根结线虫危害的报道,这是首次报道陕西省安康地区绞股蓝上发生根结线虫,为进一步分析绞股蓝根结线虫种群及该病的防治研究奠定了基础。     

猕猴桃果实贮藏期主要真菌病害的rDNA-ITS鉴定及序列分析

【目的】对陕西省周至县猕猴桃主栽品种‘华优’、‘海沃德’及‘秦美’贮藏期霉烂果实中的病原菌进行分离与鉴定,分析比较病原菌rDNA-ITS序列的差异和种内与种间群体分化状况,确定优势菌,为病害防治提供依据。【方法】采用病原菌形态鉴定与rDNA-ITS鉴定相结合的方法,确定引起猕猴桃霉烂的病原菌种类;通过构建系统发育树,分析病原菌种之间的亲缘关系。【结果】从贮藏期霉烂的猕猴桃果实中共分离出30株病原菌,主要为青霉属（Penicillium）、木霉属（Trichoderma）、交链孢霉属(Alternaria) 、毛霉属（Mucor）、拟青霉属(Simplicillium)等5个属。其中青霉属（Penicillium）19株占63.3%,为优势菌,包括Penicillium sp. 、Penicillium chrysogenum、Penicillium paneum、Penicillium purpurogenum和Penicillium commune;木霉属（Trichoderma）8株占26.7%较次之,包括Trichoderma longibrachiatum和Trichoderma sp;交链孢霉属(Alternaria) 、毛霉属（Mucor）、拟青霉属(Simplicillium)各1株,各占3.3%;聚类分析表明,30株病原聚为五大类群。【结论】引起陕西省周至县‘华优’、‘海沃德’和 ‘秦美‘猕猴桃贮藏期果实霉烂优势病原菌为Penicillium sp.、Penicillium chrysogenum、Penicillium paneum、Penicillium purpurogenum和Penicillium commune;rDNA-ITS区序列在青霉菌属内种间差异鉴别具有较高的参考价值。     

定殖烟草根结线虫卵和雌虫机会真菌的多样性

机会真菌对根结线虫的生物防治具有巨大潜力。本研究于2010—2011年针对来源于中国7个省份的156份烟草根结线虫样本,分别分离根结线虫游离卵、卵块和雌虫上定殖的机会真菌。应用18S rDNA-ITS片段测序结果结合菌株的形态学观察和培养性状鉴定定殖游离卵、卵块和雌虫上的真菌。根据分离到菌株的培养性状、形态学和rDNA-ITS序列分析,对定殖于南方根结线虫或北方根结线虫的9 839个游离卵、408个卵块和284个雌虫的真菌菌株,明确鉴定出9属13个种,其中定殖根结线虫新记录种5个,即长梗木霉、芬芳镰刀菌、渐狭蜡蚧菌、虫草棒束孢和交枝顶孢。淡紫紫孢菌Purpureocillium lilacinum(原名:淡紫拟青霉)为优势种,分布广泛,在云南、安徽、湖北、贵州和山东省均被分离到。该菌在南方根结线虫和北方根结线虫的游离卵、卵块和雌虫平均分离率分别为0.49%、24.00%和16.90%,说明其地理和生态适应性广泛。不同地区来源的烟草根结线虫卵和雌虫上定殖的机会真菌种类存在明显差异。目前尚无渐狭蜡蚧菌和虫草棒束孢作为线虫病原物的报道,有待于进一步深入研究其对根结线虫的致病性。     

香菇L26的菌丝培养特性及系统发育学分析

对香菇L26菌丝生长所需碳源、氮源、无机盐和酸碱度等因素进行了研究,结果表明,香菇L26生长所需最佳碳源为玉米粉,菌丝生长速度为0.601 cm/d;最佳氮源为蛋白胨,菌丝生长速度为0.520 cm/d;当基础培养基中MgSO4·7 H2O浓度为0.5 g/L时,菌丝生长最佳,生长速度为0.636 cm/d;培养基初始pH =6.0时,菌丝生长速度最快为0.533cm/d.采用通用引物ITS1与ITS4对香菇L26的rDNA-ITS基因序列进行扩增与测序,获得了757bp的DNA序列(GenBank accession No.JX 205093),基于5.8 S rDNA与ITS区序列构建系统发育树,对其分类学地位进行了分析研究.