虎尾兰叶斑病病原鉴定及其对多菌灵的敏感性测定

为明确虎尾兰叶斑病致病病原,采集典型病叶进行病原菌的分离、纯化,利用形态学与分子生物学方法进行鉴定,并测定了病原菌对多菌灵的敏感性。结果表明,引起虎尾兰叶斑病的病原菌为拟轮枝镰刀菌(Fusarium verticillioides(Sacc.)Nirenberg),属于半知菌亚门丝孢纲镰刀菌属。多菌灵对该菌的有效中质量浓度(EC50)为3.11μg/m L,说明多菌灵对虎尾兰叶斑病菌有较好的抑制效果。     

Genetic Variation of Host Populations of Liriomyza sativae Blanchard

In this study, partial sequences of the mitochondrial cytochrome oxidase subunit I （mtDNA-COI） gene and the ribosomal internal transcribed spacer 1 （rDNA-ITS 1） gene, isolated from five artificial populations of Liriomyza sativae （Diptera： Agromyzidae）, were sequenced and compared, to analyze their genetic variation. Analysis of the mtDNA-CO1 gene showed that a low genetic variation was detected among the five populations and only five variable sites were found in the nucleotide sequences. Most of the observed variations that occurred within the populations were because of nucleotide transitions, whereas, the interpopulation variation was because of the differences in haplotype frequencies occurring among the host populations. Analysis of the rDNA-ITS1 gene revealed a small diversity in the five host populations. The trend of genetic differentiation in the host populations was consistent with the preference of L. sativae to the plant hosts.     

砀山梨炭疽病病原鉴定及其抑菌药剂筛选

【目的】明确近年来严重危害砀山梨的炭疽病病原菌种类,研究常用杀菌剂对致病菌菌丝生长、分生孢子萌发的抑制作用。【方法】对6个取自不同梨品种上的病果、病叶样本分别进行发病组织培养、单孢分离纯化,根据病原菌的形态特征和致病性,并结合其rDNA-ITS序列分析,进行病原菌种类鉴定。采用常用杀菌剂分别对病原菌菌丝和分生孢子进行处理,观测其化学抑制效果。【结果】从不同病斑上分离出了6个纯化菌株,其形态特征相同,且与已报道的炭疽病菌(Colletotrichum spp.)形态特征相似;用上述纯培养菌株接种于健康果实和叶片上,又引起与田间原标本相同的病害症状;通过病原菌rDNA-ITS克隆测序、BLASTn比对分析,6个菌株为同一致病菌,且该致病菌与台湾枣(Taiwan jujube)炭疽菌株(Colletotrichum sp.EXMQ-1;登录号FJ233185)、日本超市水果(Japanese fruit)炭疽菌株(Glomerella cingulata;登录号AB219012)和凤梨草莓(Fragaria ananassa)炭疽菌株(C.gloeosporioides;登录号EU200455)的rDNA-ITS序列一致;430g·L-1戊唑醇水悬浮剂等7种杀菌剂对病原菌菌丝生长抑制率达100%;70%代森锰锌可湿性粉剂等8种药剂处理,病原菌分生孢子萌发率为0。【结论】危害砀山梨的炭疽病病原菌为半知菌亚门刺盘孢属的胶胞炭疽菌(Colletotrichum gloeosporioides),其有性阶段为围小丛壳(Glomerella cingulata)。常用药剂对该菌菌丝和分生孢子的抑制作用存在着显著的差异。     

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.     

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。     

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。     

中国腐霉属新记录种——Pythium nunn的形态学鉴定及rDNA-ITS系统发育分析

为了调查北京地区苗圃基地的腐霉属种类,采用花瓣、叶片诱捕法对采集到的土样进行诱导,以分离出腐霉菌。结果表明:有5株形态相似的菌株在形态及培养形状上较为一致,且异于中国已报道的其他腐霉种。运用改进的CTAB法提取菌株的基因组DNA,测定其rDNA-ITS序列,并与模式菌株CBS808.96进行多重序列比对,相似度为99.26%。选取与Pythium nunn同组的所有腐霉种以及其他组别的部分腐霉种,以Phytophthora polymorphica为外类群,采用NJ法构建了系统进化树。根据形态特征及分子序列分析结果,将此5株菌株鉴定为Pythium nunn。Pythium nunn是一种具有生防潜力的卵菌,可以为后续的生物防治研究提供试验基础。     

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。     

中国部分省份苹果轮纹病菌及相关类群的系统学关系

【目的】对苹果轮纹病菌（Botryosphaeria dothidea）及其相关类群的rDNA-ITS序列进行分析,探讨他们之间的系统学关系,为进一步研究其系统发育和演化提供理论依据。【方法】对来源于陕西、山西、山东、河南及四川5个省份的113株苹果轮纹病菌和2株梨轮纹病菌的rDNA-ITS区段直接测序,经GenBank同源性检索比对（BLASTn）及DnaSP 4.0软件分析后,结合GenBank下载序列构建系统发育的邻接树和最大简约树,同时计算遗传距离和序列核苷酸差异数。【结果】115株供试菌株分为9个单倍型（Hap₁～Hap₉）,其中Hap₇（B.obtusa）来源于山西省;Hap₉（苹果壳色单隔孢溃疡病菌,B.stevensii）来源于四川省;其余单倍型均为B.dothidea,其中Hap₁的出现频率最高,包含86株分离株。邻接法和最大简约法分析结果显示,用于构建发育树的序列均形成了3个大的聚类组。聚类组Ⅰ包含B.obtusa、B.rhodina（柑橘葡萄座腔菌）和B.stevensii等3个种,其中B.obtusa和B.rhodina各自独立聚为一支,而B.stevensii全部序列分为3支。聚类组Ⅱ包含2个分支:其中B.lutea单独聚为一支;B.ribis（茶麃子葡萄座腔菌）和B.parva亲缘关系较近,共同聚为一支。聚类组Ⅲ由B.dothidea和贝伦格葡萄座腔菌（B.berengeriana）共同构成,支持B.dothidea与B.berengeriana为同物异名的观点。遗传距离及序列核苷酸差异数显示,B.dothidea与其他种间的遗传距离在4.91%～7.57%,序列核苷酸差异数为31～54bp;B.dothidea不同单倍型之间的遗传距离为0.19%～1.93%,序列核苷酸差异数为1～10bp。【结论】B.stevensii显示出了复合种的特性,其分类地位还有待于进一步研究。B.dothidea与其他相似种的亲缘关系较远,且在B.dothidea种内存在核苷酸序列的分化,但分化程度较低。B.dothidea不同地理来源的菌株之间存在差异,但地理分化特征不明显。     

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。