肾形肾状线虫（Rotylenchulus reniformis）浙江杭州 群体形态学和分子特性及寄主范围

 肾形肾状线虫（Rotylenchulus reniformis）是一种重要的蔬菜线虫,寄主范围和分布很广。本文根据形态学特征和形态测量将来自浙江省杭州市蔬菜上的肾状线虫群体鉴定为肾形肾状线虫,为浙江省首次报道。该群体的形态学特征及形态测量值与模式标本基本吻合,个别形态测量值(比值)有变异。通过对该肾形肾状线虫群体的rDNA中ITS区和编码28S RNA基因中的D2D3区的PCR扩增和序列测定,明确二序列长度分别为808 bp和786 bp;序列分析发现该群体ITS序列与肾形肾状线虫种内一些两性生殖群体的亲缘关系较近,同源性高达99.5%～100%,而与一孤雌生殖群体同源性为92.4%;编码28S RNA基因中的D2D3区与已报道群体的同源性达到99.4%,相似度较高;本研究还发现杭州肾形肾状线虫群体的寄主涉及11个科16个属的果蔬。     

温度和时间对水稻潜根线虫回收率的影响初步研究

 水稻潜根线虫(Hirschmanniella spp.)为水稻根内寄生,影响根系对水分相养分的吸收,使水稻地上部分表现缺素症状,导致水稻产量下降。在国内,水稻潜根线虫病尚属近年发现的一种水稻新病害,在南方诸省稻区广泛发生,作者从1984年开始从事此病的研究,发现以贝曼漏斗法分离水稻潜根线虫,其回收率与分离时的温度及分离时间关系甚为密切.     

水稻潜根线虫病田间消长与产量损失研究

经对水稻多品种定田定期取定量稻根进行分离观察,结果表明:早晚稻潜根线虫(Hirschmanniella spp.)分别在水稻破口期和齐穗期各有一个高峰。采用盆栽接种试验表明:接种量越大,产量越低,呈显著负相关;分蘖盛期前接种减产显著。该线虫主要影响千粒重和有效穗数。     

松材线虫组DNA条形码筛选

本文基于Gen Bank中登录的14种松材线虫组线虫的28S、18S和ITS部分基因DNA序列,利用遗传距离法及分子生物学方法评价其各自作为松材线虫组DNA条形码序列的适用性。结果显示,28S(拟松材线虫不分亚种)、28S(拟松材线虫分亚种)、18S和ITS的基因序列种内成对遗传距离平均值分别为0.0071、0.0030、0.0007、0.0043,种间成对遗传距离平均值分别为0.0476、0.0454、0.0052、0.1556,其中28S、18S基因序列种内及种间距离存在一定程度的重叠,ITS具有一定程度的Barcoding gap。3个基因序列构建的NJ进化树显示,28S、ITS构建的系统进化树具有较高的节点支持率,可以有效将14个松材线虫组线虫分离成独立分支,而且28S可以有效鉴别出拟松材线虫的2个亚种;基于18S基因构建的进化树不能对吉拉尼伞滑刃线虫、日本冷杉伞滑刃线虫、拟松材线虫进行有效区分。因此,28S、ITS区具有一定的遗传距离间隔以及相对较高的物种识别率,可以作为松材线虫组线虫候选条形码基因。     

从荷兰番红花种球中截获的次薄滑刃线虫的检疫鉴定

宁波口岸从荷兰番红花种球中分离到大量滑刃属线虫活虫,该荷兰群体的主要形态特征为:雌虫尾呈亚圆柱形,尾端半圆形,尾端腹侧有尾尖突长1.5~3.0μm;后阴子宫囊显著,长89.3~118μmm;侧线3条。基于28S、ITS和18S基因DNA序列构建NJ系统发育进化树显示该荷兰群体与次薄滑刃线虫亲缘关系极近且在同一进化分支中。通过形态特征和DNA序列分析,鉴定该荷兰群体为次薄滑刃线虫(Aphelenchoides subtenuis Cobb,1926)。     

水稻潜根线虫病发生因素调查及防治意见

水稻潜根线虫(Hirschmanniella spp.)广泛分布于世界水稻产区。在我国八十年代已发现本病广为发生。据对三明市12个县、市(区)240多万亩稻田抽样调查表明,各类稻田均有不同程度的发生,其中约有50万亩稻田受害较重,据试验测定,产量损失在10%以上。水稻潜根线虫寄生在水稻根内,影响根系对水分和营养的吸收。一般情况下,植株     

甘肃小麦禾谷孢囊线虫rDNA-ITS和28S rDNA-D2/D3区序列特征及HS-RFLP分析

禾谷孢囊线虫是危害禾谷类作物的重要病原,严重威胁我国小麦主产区的小麦产量和品质.利用通用引物对甘肃、河南、安徽禾谷孢囊线虫群体28S rDNA-D2/D3区和rDNA-ITS区进行PCR扩增和序列测定,利用UPG-MA方法分析了甘肃省7个种群、河南1个种群、安徽1个种群的禾谷孢囊线虫群体D2/D3区和ITS区的系统发育关系;用9种限制性内切酶对7个甘肃禾谷孢囊线虫群体的rDNA-ITS区进行了RFLP分析.结果表明:中国甘肃的禾谷孢囊线虫rDNA-D2/D3区片段长度约为780 bp,rDNA-ITS片段长度约为1 040 bp.7个甘肃禾谷孢囊线虫群体、1个河南安阳群体、1个安徽蚌埠群体的D2/D3区和新西兰的H.aucklandica群体亲缘关系很近;其ITS区同澳大利亚的H.australis、北京通州的H.avenae(AY148382)的亲缘关系很接近.RFLP分析表明,9种限制性内切酶酶切禾谷孢囊线虫群体的rDNA-ITS共产生了22个酶切片段,不同酶切的RFLP分布型在7个种群间没有差异.甘肃省禾谷孢囊线虫群体的rDNA-ITS区具有高度的保守性,与中国的C型群体相近,但不同于欧洲的A型群体和印度的B型群体.这是首次报道甘肃CCN种群分子特征.     

竹子根际螺旋线虫中国新记录种—尖尾螺旋线虫Helicotylenchus cuspicaudatus

在广东省园林植物线虫调查期间,从粉单竹Bambusa chungii根部土壤中分离到一种螺旋线虫。经详细的形态学观察和测量数据比较,将其鉴定为尖尾螺旋线虫Helicotylenchus cuspicaudatus,并获得了尖尾螺旋线虫rDNA的28SD2-D3区和ITS序列,为今后螺旋线虫的种类鉴定提供了分子数据。尖尾螺旋线虫为中国新记录种。     

河南省温县铁棍山药根腐线虫种类鉴定

为明确河南省温县铁棍山药病原线虫种类,在温县铁棍山药种植区采集有明显症状的块茎进行线虫分离,观察线虫的雌、雄成虫的形态特征,并扩增rDNA的28S D2-D3序列对其进行分子生物学鉴定。结果表明,从山药块茎中分离出的根腐线虫形态学特征及测量指标与咖啡短体线虫Pratylenchus coffeae基本一致,其28S D2-D3序列与P.coffeae相似性达99.87%～100%。本研究明确了河南省温县地区铁棍山药根腐线虫为咖啡短体线虫,为山药线虫病的鉴别及防控提供了科学依据。     

莲寄生潜根线虫的鉴定

1998～1999年 ,在南昌市郊和江西省广昌县莲田 ,对寄生在莲根部的潜根线虫进行了广泛调查 ,并对其测量和鉴定 ,鉴定结果为Hirschmanniella diversa。该种为国内新记录     

河南省玉米田根腐线虫三个病样品的病原种类鉴定

 短体属线虫(Pratylenchus Flipjev,1936)又称根腐线虫,是一类重要的迁移性植物内寄生线虫,世界范围内分布广泛且危害性大,在我国多个省份皆有发生危害。本文采用改进的贝尔曼漏斗法从河南省商丘市、荥阳市和洛阳市的3个玉米土壤样品中分离出根腐线虫,利用光学显微镜进行形态学鉴定,基于rDNA的 ITS和28S D2-D3区序列以及mtDNA-COI序列对供试根腐线虫进行了分子鉴定。形态学和分子生物学鉴定结果均表明来源于商丘样品的SY-1和SY-2种群分别为咖啡短体线虫(P. coffeae)和斯克里布纳短体线虫(P. scribneri);来源于荥阳样品的XY-1种群和洛阳样品的XC-250种群,均为斯克里布纳短体线虫(P. scribneri)。基于rDNA-ITS、28S D2-D3区和mtDNA-COI序列构建的系统进化树结果与已知鉴定结果相一致,本研究鉴定的短体线虫种类,分别单独聚类在各自分支。本研究明确了河南省玉米田3个病样品的病原种类,发现采自商丘的病样品存在两种根腐线虫复合侵染的现象,且3个病样品均发现斯克里布纳短体线虫(P. scribneri)的存在,研究结果为玉米根腐线虫病害的识别与防控提供了一定的科学依据。     

鸢尾种苗中截获的草莓滑刃线虫形态和分子鉴定

草莓滑刃线虫是一种重要的叶和芽寄生线虫,可侵染多种重要花卉、经济作物,被列入我国检疫性有害生物名录。目前,草莓滑刃线虫的鉴定以形态学鉴定为主,但一线口岸常分离不到雌虫,鉴定困难。本研究采用形态特征和分子特征相结合的方法对草莓滑刃线虫进行鉴定:对鸢尾种苗中截获的滑刃属线虫群体雌虫、雄虫进行形态观察和形态测计并与已报道文献进行比较;PCR扩增该群体的rDNA-ITS区并测序,构建基于ITS序列的系统发育进化树;采用草莓滑刃线虫特异引物对该群体DNA样本进行扩增。结果表明:该群体与草莓滑刃线虫俄罗斯种群的测量值相接近;系统进化关系分析显示,该群体与NCBI公布的草莓滑刃线虫均处于同一进化支;采用草莓滑刃线虫特异引物PCR扩增可产生片段大小为169 bp的目的条带。     

Molecular variability and phylogenetic relationships among different species and populations of <Emphasis Type="Italic">Pratylenchus</Emphasis> (Nematoda: Pratylenchidae) as inferred from the analysis of the ITS rDNA

Sequence comparisons and molecular phylogenetic analyses were used to describe the nucleotide variability of the ITS containing regions of eighteen Pratylenchus species and several populations. Comparative analysis of nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among Pratylenchus species used in the present study demonstrates that ITS sequences can widely vary in primary sequence and length. Alignment of eighty-seven Pratylenchus sequences and one outgroup taxon reveals the presence of ambiguous regions that have the greatest effect on phylogeny reconstruction. Phylogenetic analyses using Bayesian Inference, Neighbour Joining-LogDet, Maximum Likelihood and Maximum Parsimony, distinguished twelve highly or moderately supported major clades within Pratylenchus. Our results support the taxonomic usefulness of the ITS region to identify root-lesion nematode species of the genus Pratylenchus but the high nucleotide variability, sometimes, can preclude its use to resolve relationships among all members of the genus. In addition, the phylogenetic groupings are not congruent with those defined by characters derived by lip patterns and numbers of lip annuli.     

昆虫病原线虫rDNA多态性分析

本文对国内外昆虫病原线虫斯氏属和异小杆属的47个品系进行rDNA—ITS PCR—RFLP分析，研究其DNA多态性，并构建了分子系统发育树状图。各品系的ITS区无明显的长度差异，PCR—RFLP分析将47个品系分为斯氏属和异小杆属两大类，两属线虫又分别分为11组和4组。所得结果丰富了ITS PCR—RFLP图谱库，为弄清我国的昆虫病原线虫与国外种类的分子系统发育关系及未定名线虫的鉴定提供重要依据，同时为筛选适合的线虫种类防治害虫奠定基础。     

从日本进境罗汉松中截获移去剑线虫

2016年11月,深圳口岸从日本进境罗汉松中截获一种剑线虫。采用贝曼漏斗法分离线虫,结合形态学、28S rRNA基因序列测定以及构建系统发育树,对分离的线虫进行鉴定。该线虫的28SrRNA基因序列和GenBank数据库中的移去剑线虫Xiphinema elongatum(登录号分别为EF140790、KF430803、KF430802和KF430801)的序列同源性高达99%;其形态特征和测计值与X.elongatum最为吻合;系统发育树表明该线虫和X.elongatum聚为一组。通过以上结果判定截获的线虫为移去剑线虫。剑线虫是一种重要的植物外寄生线虫,我国口岸曾经多次截获剑线虫,剑线虫入侵我国的风险较高。本文首次对日本罗汉松中的移去剑线虫进行了详细的描述,提供了该线虫新的地理分布信息。     

山东省危害核桃的链格孢属真菌鉴定及其系统发育

为了明确危害核桃的链格孢属Alternaria真菌种类,对34个供试菌株在PCA培养基上的培养特征、产孢表型、分生孢子特征等进行了研究,并采用ITS、OPA2-1和OPA1-3序列分析其系统发育。其中33个菌株为链格孢A.alternata,1个为细极链格孢A.tenuissima。链格孢:菌落灰色;孢子链短、分枝频繁,每条分枝含4～10个孢子;分生孢子多倒梨形,孢身平均25.6μm×7.4μm,横隔膜2～8个。细极链格孢:菌落褐色;孢子链长、少分枝,孢子链约含10个孢子;分生孢子多长椭圆形,孢身平均26.3μm×13.5μm,具柱状喙及假喙,主横隔膜1～6个,孢身中部的隔膜较粗。链格孢属真菌在遗传上有较大差异,ITS、OPA2-1和OPA1-3序列对链格孢小孢子种具有一定的区分作用,链格孢是危害核桃的主要链格孢属种。     

Evidence of Two Formae Speciales in Venturia inaequalis, Responsible for Apple and Pyracantha Scab

ABSTRACT Genetic relationships, mating crosses, and host specificity of Venturia inaequalis isolates from Malus spp. and of Spilocaea pyracanthae isolates from Pyracantha spp. were evaluated. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS1-5.8S to ITS2) revealed a total similarity between these two putative species. ITS restriction fragment length polymorphism carried out with five restriction enzymes on a collection of 28 isolates confirmed a lack of diversity in this region between and within these two populations. Additional isolates from three related species (V. pirina, V. nashicola, and S. eriobotryae) were divided into two distinct monophyletic groups in a phylogenetic tree using ITS sequence comparison. These groups were related to their anamorph (i.e., Spilocaea or Fusicladium). When inoculated on their host of origin, fields isolates caused typical symptoms of scab disease, and a host specificity was demonstrated by cross pathogenicity of isolates from Malus x domestica and Pyracantha spp. Mating on dried leaves in vitro between one isolate of each putative species led to production of numerous perithecia. Ninety-six sporulating monoascosporic progenies were isolated from this cross. Based on these genetic and pathogenic data, we proposed that pathogens responsible for scab on Malus spp. and Pyracantha spp. are considered as two formae speciales belonging to V. inaequalis.     

A new root-knot nematode, Meloidogyne silvestris n. sp. (Nematoda: Meloidogynidae), parasitizing European holly in northern Spain

High infection rates of European holly ( Ilex aquifolium ) feeder roots by an unknown root-knot nematode were found in a holly forest at Arévalo de la Sierra (Soria province) in northern Spain. Holly trees infected by the root-knot nematode showed some decline and low growth. Infected feeder roots were distorted and showed numerous root galls of large (8–10 mm) to moderate (2–3 mm) size. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes and phylogenetic trees of sequences within the ribosomal DNA (rDNA) demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected European holly plants, but did not reproduce on a number of cultivated plants, including tomato, grapevine, princess-tree and olive. The species is described here, illustrated and named as Meloidogyne silvestris n. sp. The new root-knot nematode can be morphologically distinguished from other Meloidogyne spp. by: (i) roundish perineal pattern, dorsal arch low, with fine, sinuous cuticle striae, lateral fields faintly visible; (ii) female excretory pore level with stylet knobs, or just anterior to them, EP/ST ratio about 0·8; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anterior to excretory pore and short, sub-digitate tail; and (iv) males with lateral fields composed of four incisures, with areolated outer bands. Phylogenetic trees derived from maximum parsimony analysis based on 18S, ITS1-5·8S-ITS2 and D2–D3 of 28S rDNA showed that M. silvestris n. sp. can be differentiated from all described root-knot nematode species, and it is clearly separated from other species with resemblance in morphology, such as M. ardenensis , M. dunensis and M. lusitanica .     

剪股颖粒线虫幼虫形态与分子检测方法

剪股颖粒线虫(Anguina agrostis)是牧草、草坪上的重要线虫,该线虫在进口草籽中常以幼虫形态出现,与其它粒线虫属幼虫形态相似,很难快速鉴定到种.本实验通过对单条幼虫DNA进行提取和PCR扩增,获得其rDNA的ITS区域,测序后与基因库中的剪股颖粒线虫的rDNA的ITS区域序列进行比较,其结果与基因库中编码为AF396343.1的核酸序列吻合,从而鉴定为剪股颖粒线虫.此外,还使用5种限制性内切酶对PCR产物进行了酶切,获得了剪股颖粒线虫的ITS-RFLP指纹图谱.     

Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA

ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.