四种短体线虫的形态和分子生物学鉴定

 采用形态学和分子生物学方法对来自荷兰的藏橐吾和铃兰、泰国的高山榕、缅甸的香蕉等种苗(球)分别鉴定出玻利维亚短体线虫Pratylenchus bolivianus、铃兰短体线虫P. convallariae、咖啡短体线虫P. coffeae和斯佩奇短体线虫 P. speijeri。对这4种线虫的形态特征进行较为详细的描述后,认为头环、口针、侧区、生殖系统和尾形等形态特征是种类鉴定的重要依据;进一步利用引物28S-D2A/28S-D3Br扩增测序得到上述4种线虫的28S rRNA基因D2/D3区序列,序列分析发现玻利维亚短体线虫从欧洲到美洲的不同种群之间的遗传距离变异较小,仅为0~0.007;铃兰短体线虫同一种群不同个体之间的遗传变异较大,为0.006~0.029;咖啡短体线虫不同种群之间的平均遗传距离为0.013;斯佩奇短体线虫不同种群之间的平均遗传距离为0.010;咖啡短体线虫P. coffeae和斯佩奇短体线虫P. speijeri亲缘关系很近,二者种间平均遗传距离仅为0.026。本研究再次证明线虫28S rRNA基因D2/D3区基因序列可作为短体线虫种间鉴定的依据。     

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。     

山西省窖藏薯类块茎中2种短体线虫的种类鉴定

本文采用传统形态学与分子生物学相结合的方法,对采自山西省窖藏马铃薯和薯蓣块茎中的短体线虫进行了种类鉴定。结果表明,从马铃薯块茎中分离出的短体线虫形态学特征与斯克里布纳短体线虫Pratylenchus scribneri一致,其SSU序列与P. scribneri美国群体相似性达99.8%。从薯蓣块茎中分离出的短体线虫形态学特征与咖啡短体线虫P. coffeae一致,其ITS序列与P. coffeae浙江群体相似性达98.8%。斯克里布纳短体线虫首次在我国马铃薯块茎中发现,咖啡短体线虫首次在山西省薯蓣块茎中发现。     

江西省山药病原线虫种类鉴定及地理分布

为明确江西省山药病原线虫的种类及地理分布,对采自主产区的157份山药样品进行了线虫分离与形态学鉴定,并对主要病原线虫种类进行rDNA-ITS和28S的序列比对和种群系统进化分析。形态学和分子鉴定结果表明：山药病原线虫种类为咖啡短体线虫Pratylenchus coffeae和南方根结线虫Meloidogyne incognita。调查结果表明,咖啡短体线虫在江西省大部分山药产区广泛分布,是优势种群。吉安市永丰县和泰和县山药线虫危害最为严重,山药样品中线虫检出率分别为66.7%和88.9%。南方根结线虫在江西省山药产区不是优势种群,呈零星发生。此外,山药样品中存在两种线虫的单一种群侵染和复合种群侵染,分别占总样品数的87.1%和12.9%。综上,本研究明确了江西省山药主产区病原线虫的优势种是咖啡短体线虫,为江西省山药病原线虫的科学防控提供了有价值的参考依据。     

佛手山药线虫病害发生调查与病原鉴定

湖北省武穴市佛手山药是“农产品地理标志产品”, 近年佛手山药线虫病害严重, 为此在病源地采集山药土样进行了病害发生调查及病原鉴定?通过形态特征观察?形态特征测计值比较, 以及基于rDNA 28S D2/D3区序列构建短体线虫属线虫群体系统进化树并进行SCAR验证, 对引起佛手山药线虫病的病原线虫进行种类鉴定?结果表明:危害佛手山药的病原线虫的群体特征与咖啡短体线虫Pratylenchus coffeae相似?系统进化树显示:该病原群体与中国香港群体(HQ688677.1)聚类在一个分支上, 置信度达99%?因此, 将该群体线虫鉴定为咖啡短体线虫Pratylenchus coffeae?此外田间调查表明:线虫主要集中分布于10~20 cm深度的土层中, 线虫密度随土壤深度的增加而呈递减趋势, 且连作1年地较连作2年地的线虫数量少, 施用阿维菌素的地块较未施用的线虫数量少?上述结果表明, 在佛手山药生产中实行轮作和科学选择药剂防治, 避免连作, 可降低该线虫发生量?     

宁波地区蔬菜主要病原线虫的分离和种类鉴定

对宁波14个主要蔬菜基地进行土壤和植株取样,通过形态学和分子生物学鉴定明确了宁波地区蔬菜主要病原线虫为南方根结线虫［Meloidogyne incognita (Kofoid & White)］和咖啡短体线虫［Pratylenchus coffeae (Zimmer mann)］。南方根结线虫主要分布在江北费市、宁海冠庄、宁波梅墟和镇海九龙湖,咖啡短体线虫分布在江北费市。南方根结线虫的寄主为番茄、黄瓜、豇豆和白菜,咖啡短体线虫的寄主为萝卜和大豆。     

我国小麦落选短体线虫群体遗传多样性分析

 为了明确我国落选短体线虫群体的遗传多样性,利用mtCOI基因标记对落选短体线虫9个地理群体的遗传结构及遗传分化进行分析。结果显示,9个地理群体中共得到101条mtCOI序列,发现28个碱基变异位点,形成14个单倍型。其中,H1单倍型最常见,为7个地理群体的59个个体共有,推测其可能为祖先单倍型。全部地理群体在物种水平呈现中等遗传多样性(H_T = 0.706±0.131),聚类分析显示其可分为类群Ⅰ与类群Ⅱ两个类群,AMOVA分析揭示落选短体线虫整体水平的遗传分化主要来源于种群间。Mantel检验表明落选短体线虫群体的遗传距离与地理距离存在正相关性,但是不同种群之间的遗传分化程度与地理距离没有显著关系。中性检验和错配分布检验均揭示落选短体线虫在整体水平和两个类群上的群体历史动态都处于相对稳定的状态。     

进境新西兰麻中玻利维亚短体线虫的鉴定

2013年,北京口岸入境的新西兰麻中分离到一种短体线虫,通过形态学特征以及基于核糖体r RNA-ITS区及28S r RNA-D2/D3区序列比较,证实其为玻利维亚短体线虫(Pratylenchus bolivianus Corbett,1983)。该线虫的主要形态鉴别特征为:唇区有3个唇环;平均V=80%~82%;尾末端圆而光滑。该线虫至今未在我国发现,这是国际上首次从新西兰种苗中截获。     

4种短体线虫形态及rDNA-ITS的PCR-RFLP鉴定

 短体线虫隶属于垫刃目(Tylenchida Thorne, 1949),垫刃亚目(Tylenchina Chitwood, 1950),短体科(Pratylenchidae Thorne, 1949),短体亚科(Pratylenchinae Thorne, 1949),短体线虫属(Pratylenchus Filipjev, 1936),是一类重要的迁移性内寄生植物病原线虫 ^[1]。国内外已报道该属线虫百余种。其中我国已报道种类有P.alleni、 P.artemisiae、 P.coffeae、 P.crenatu、 P.cruciferus、 P.dioscoreae、 P.hexincisus、 P.loosi、 P.penetrans、 P.pratensi、 P.neglectus、 P.penetrans、 P.scribneri、 P.thornei、 P.varicaudatus、 P.vulnus及P.zeae 17种。近年来,随着世界各地贸易往来日益频繁,植物线虫的传播机会大大增加,我国口岸也先后从进境植物根围截获P.brachyurus、 P.neglectus、 P.zeae、 P.coffeae、 P.penetrans、 P.pinguicaudatus等。基于本类群线虫的重要性和国内以往的鉴定都主要单纯依据形态学特征,本研究结合形态学和rDNA的PCR-RFLP及序列分析技术对浙江和海南不同寄主上植物线虫调查时发现的4个短体线虫群体进行了鉴定。     

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。     

Distribution analyses of Meloidogyne spp. and Pratylenchus coffeae sensu lato in coffee plots in Costa Rica and Guatemala

The distribution of the plant-parasitic nematodes Pratylenchus coffeae sensu lato and Meloidogyne spp. were studied in two plots, one in Guatemala ( P. coffeae and M. paranaensis ) and the other in Costa Rica ( P. coffeae and M. exigua ). The quantity of nematodes per g fresh weight root were counted for each coffee tree sampled. The distributions were aggregated, and generally fitted well to negative binomial distributions. Population aggregation was greater when a smaller number of nematodes were involved, suggesting that initial colonization develops in foci. Analyses of the relationships between population levels of the species suggested that there was competition between Pratylenchus coffeae and Meloidogyne spp. This competition was expressed differently depending on the relative population density of the different species.     

Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of <Emphasis Type="Italic">Pratylenchus</Emphasis> from the Mediterranean region

Plant parasitic nematodes belonging to the genus Pratylenchus, also known as root lesion nematodes, cause serious economic damage to different crop plants. In order to explore genetic structures in different isolates, we investigated several P. thornei, P. neglectus and P. penetrans populations of different geographic origins. The analysis at the species level was also extended to P. penetrans, P. pinguicaudatus, P. vulnus and P. mediterraneus. Sequence analysis of a specific portion of DNA was carried out. In particular, the sequences of the D3 region of the 26S gene were obtained and compared with similar sequences available in databases. The results support the hypothesis that P. penetrans may represent a species complex, while in P. neglectus the intra-species heterogeneity observed is due to intra-individual variability. Furthermore, the specific conservation of some nucleotides in different P. thornei populations indicates their fixation in the rDNA repeats in this species. The presence of these nucleotides, the molecular signature of P. thornei, may assist in determining the nature of nematode infections.     

Detection and analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme markers

The root-lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus , P. goodeyi , P. penetrans , P. scribneri , P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis . A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.     

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。     

Molecular characterisation and diagnostics of some Longidorus species (Nematoda: Longidoridae) from Russia and other countries using rRNA genes

The needle nematodes of the genus Longidorus can cause diseases of various crops and trees, and are comprised of more than 150 valid species. Eleven valid and six unidentified species of the genus Longidorus collected in different regions of Russia, two states of USA, Germany, New Zealand and Ukraine were molecularly characterized using analysis of the partial 18S rRNA and the D2–D3 expansion segments of the 28S rRNA gene sequences. Fifty-four partial 28S rRNA and fifteen partial 18S rRNA gene sequences were obtained for the present study. Using molecular criteria, we confirmed the morphological identification and distinguished between the following species: L. aetnaeus, L. africanus, L. andalusicus, L. artemisiae, L. caespiticola, L. distinctus, L. elongatus, L. euonymus, L. intermedius, L. leptocephalus and L. lignosus. Two longidorid populations from Russia and four from California were not identified to a species level. We obtained the full length D2–D3 of 28S rRNA gene sequence from several freshly-collected L. artemisiae samples. We confirmed the identity of the D2 region of 28S rRNA gene sequence with a short D2 of 28S rRNA gene fragment sequence previously obtained from formalin-fixed nematodes embedded in the L. artemisiae paratype slides. Longidorus lignosus was molecularly characterized and L. aetnaeus was reported from Russia for the first time. PCR-D2-D3-RFLP diagnostic profiles generated by five restriction enzymes: AluI, HinfI, Bsp143I, Tru1I and RsaI are presented for sixteen Longidorus species.