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1.
ABSTRACT Polymerase chain reaction amplification of the intergenic spacer region between the 5S and 18S genes from Meloidogyne chitwoodi, M. fallax, and M. hapla enabled these three important temperate species to be differentiated. Length polymorphism was found between M. chitwoodi and M. fallax as a result of differing numbers of short repeats located between the 5S and 18S genes. The presence of the 5S gene within the rDNA cistrons was confirmed in the Meloidogyne spp. included in this study. The region between the 28S and 5S genes for M. chitwoodi and M. fallax was short and lacked variability in repeated sequences compared with the main tropical Meloidogyne spp. and M. hapla. Differences in the number of these repeats resulted in intraspecific length polymorphism for M.hapla. 相似文献
2.
ABSTRACT The virulence of Meloidogyne hapla, M. chitwoodi, and M. fallax was studied on genotypes of Solanum spp. in a greenhouse. Juveniles of 11 M. hapla race A isolates, 3 M. hapla race B isolates, and 5 mono-female lines of a M. hapla race A isolate were inoculated on S. chacoense, S. hougasii, and S. sparsipilum. Juveniles of eight M. chitwoodi isolates, five M. fallax isolates, and six mono-female lines of a M. chitwoodi isolate were inoculated on S. bulbocastanum, S. chacoense, S. hougasii, S. stoloniferum, and S. tuberosum. Virulence was expressed as nematode reproduction 8 weeks after inoculation. Nematode reproduction was estimated by the number of egg masses and, in one experiment, by the number of hatched second-stage juveniles per inoculated juvenile. Considerable variation in virulence and resistance was observed among M. hapla isolates and plant genotypes, respectively. The M. hapla isolate-plant species interaction was highly significant. The response to M. chitwoodi ranged from susceptible (S. tuberosum and S. chacoense) to highly resistant (S. bulbocastanum and S. hougasii). S. tuberosum was susceptible to M. fallax, whereas all four wild species were resistant. In contrast to M. hapla, no significant isolate-plant genotype interaction was obtained for M. chitwoodi or M. fallax, indicating no or little intraspecific variation in virulence. M. chitwoodi juveniles in species mixtures with M. fallax isolates appeared to be able to break the resistance of S. bulbocastanum and S. hougasii. Significant differences among mono-female lines of M. hapla and M. chitwoodi were observed, indicating heterogeneity of pathogenicity within meiotic parthenogenic Meloidogyne populations. 相似文献
3.
ABSTRACT This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present. 相似文献
4.
A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp. DNA was extracted from individual second-stage juvenile (J2) nematodes of Meloidogyne spp. and amplified by PCR (polymerase chain reaction). Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA. This enabled a molecular diagnostic key for M. incognita , M. javanica , M. arenaria , M. mayaguensis , M. hapla , M. chitwoodi and M. fallax to be designed. The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically. The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species. The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described. 相似文献
5.
‘99昆明世博会参展植物寄生线虫的检疫初报 总被引：1，自引：0，他引：1
本次博览会参展植物种类多,来源广,带土普遍疫情复杂。在严处国内外的参展植物中截获矛线目和垫刃目的１３个科的５６个属,其中在参展植物中截获我国对外检疫潜在危险性线。 相似文献
6.
依据国际植物检疫措施标准(ISPM)规定的有害生物风险分析程序(PRAS),利用多指标综合评估方法,通过对哥伦比亚根结线虫(Meloidogyne chitwoodi)的生物学特性、地理分布、寄主、经济意义和危害性等方面进行分析,结合我国进口马铃薯种薯和马铃薯种植情况,确定了其在中国具有传入、定殖和扩散的可能性,对各指标值进行赋值运算,获得总指标值R为2.25,符合高风险的检疫性有害生物标准,因此建议将其列入输华马铃薯检疫性有害生物名单,实施风险管理。本文提出了风险管理措施方案。 相似文献
7.
ABSTRACT Hybridization between two meiotic parthenogenetic species of root-knot nematodes, Meloidogyne chitwoodi and M. fallax, was investigated in two different crossing experiments on tomato plants grown in sand. The first experiment was a controlled cross between the two species. The second experiment was a bulk mating in a 1:1 mixture of two isolates. The haploid chromosome number of the parental isolates was n = 18. Successful interspecific hybridization was obtained, and the resulting hybrids produced egg masses. In eggs, cell division was observed, but most of them were without clear differentiation and consequently were sterile. Hatched F(2) juveniles were small in number, not viable, and showed morphological distortions. In the progeny of the isolate mixture of the bulk mating experiment, parental-type females of the two isolates were present in equal numbers, and 10% of all females were nonviable hybrids. Similar ratios of parental-type and hybrid females were detected in roots of test plants grown in soil from a field sample that contained a mixture of M. chitwoodi and M. fallax populations. In the controlled cross experiment, isozyme electrophoresis of malate dehydrogenase was applied to distinguish the two species and their hybrids. In the bulk mating experiment, malate dehydrogenase, esterase, and glucose 6-phosphate dehydrogenase were used as markers, two by two simultaneously on the same individual females, providing conclusive evidence for the occurrence of hybrids. This is the first report on interspecific hybridization in Meloidogyne. The possible role of interspecific hybridization in species differentiation and interspecific exchange of genetic material within Meloidogyne is discussed. 相似文献
8.
南方、爪哇和花生根结线虫的快速灵敏的PCR鉴定方法 总被引：7，自引：0，他引：7
为了研制南方、爪哇和花生根结线虫快速灵敏的检测和鉴定方法,分别分离了4个南方根结线虫和3个爪哇根结线虫特异性的随机扩增多态性DNA (RAPD)片段。在这些RAPD标记DNA序列的基础上,设计了多对SCAR PCR引物,并用源于国内外的南方、爪哇、花生、北方和象耳豆根结线虫群体验证其扩增特异性和灵敏度。最终确定了3对高效扩增的SCAR引物,它们组合使用可以可靠灵敏地鉴定南方、爪哇和花生根结线虫。3对引物的扩增灵敏度达1/3条的二龄幼虫、雄虫或雌虫,这表明本研究研制的PCR鉴定法可用于生产实践中土样和根样中3种根结线虫快速灵敏的鉴定。 相似文献
9.
4种常见根结线虫基因组DNA的RAPD分析 总被引：9，自引：2，他引：7
用120个随机引物对4种常见根结线虫10个小种和类型进行了全基因组随机扩增DNA多态性(RAPD)分析,筛选出的11个适宜引物共扩增出91条RAPD谱带,86条是多态性谱带,占总谱带的94.5%;OPL12、OPK01对4种根结线虫种及其小种扩增的谱型有明显的特异性。聚类分析显示在种间水平上4种根结线虫中花生根结线虫和爪哇根结线虫亲缘关系最近,遗传距离为0.532,北方根结线虫与另外3种根结线虫的亲缘关系最远,平均遗传距离为0.786;种下水平上同种根结线虫的不同小种和类型间存在不同程度的遗传差异,南方根结线虫4个生理小种间,花生根结线虫2个生理小种间亲缘关系较近,爪哇根结线虫2个酯酶谱带类型间,北方根结线虫2个细胞生物学小种间遗传差异较大。在RAPD技术的基础上探索根结线虫分类鉴定的分子方法有着良好的前景。 相似文献
10.
我国南方地区主要根结线虫DNA变异的RAPD分析 总被引：2，自引：0，他引：2
本试验用5组随机引物对来自我国南方地区的30个根结线虫种群进行RAPD分析,并从中筛选出多态性较好的引物12个。共扩增出179条DNA多态带,各供试种群间存在着丰富的遗传多态性。扩增结果表现出种间差异大于种内差异的共同趋势,这表明上述12个引物能够较客观地反映种群间亲缘关系的远近。北方根结线虫与另外3种线虫(南方根结线虫、爪哇根结线虫、花生根结线虫)的亲缘关系最远;在3种主要根结线虫中,爪哇根结线虫与南方根结线虫的亲缘关系相对较近。基于种群间的相似系数分析和应用UPGMA法构建的聚类树状图,显示出不同的根结线虫在较低的相似性系数范围聚类,而绝大多数种内的不同种群均以较高的相似性系数聚在一起,这与形态分类基本一致,反映了形态学分类的分子遗传本质,同时也表明了应用RAPD技术进行根结线虫亲缘关系分析和种类鉴定具有合理性和可行性。本文还对RAPD方法对南方根结线虫小种鉴定的可能性进行了初步探讨。 相似文献
11.
通过形态学与分子生物学相结合的方法对引起甘肃省陇西县黄芩根结线虫病的病原种类进行了鉴定,并采用室内人工接种的方法测定其对黄芩的致病性。结果表明,从黄芩根系分离的根结线虫其形态特征及测量值与北方根结线虫Meloidogyne hapla基本一致。该线虫种群rDNA-ITS和28S rDNA D2/D3序列与NCBI数据库中的北方根结线虫序列相似性分别为99.60%和99.74% (rDNA-ITS: JX024147; 28S rDNA D2/D3: MW288147)。贝叶斯系统发育树显示,该线虫群体与其他北方根结线虫群体聚为一支,置信度在95%以上。利用北方根结线虫特异性引物Mh-F/Mh-R扩增并测序得到457 bp的特异性片段。因此,基于形态学结合rDNA-ITS、28S rDNA D2/D3序列和特异性片段长度,将黄芩根结线虫病病原鉴定为北方根结线虫M.hapla。接种该线虫后的黄芩生长缓慢,叶片黄化,40 d后须根上有明显的根结。经分离鉴定,致病群体为北方根结线虫。综上所述,甘肃省陇西县黄芩根结线虫病病原为北方根结线虫M.hapla,该线虫对黄芩具有较强的致病性,其繁殖系数为1.997。 相似文献
12.
象耳豆根结线虫的PCR鉴定和检测方法 总被引：5，自引：1，他引：5
象耳豆根结线虫是一种在中国具有潜在经济重要性的农作物病原物。为提供有助于控制象耳豆根结线虫传播扩散的方法,研制了该线虫的快速PCR鉴定和检测法。该方法PCR引物的扩增目标为rDNA-IGS2区域,其设计依据象耳豆根结线虫与南方、爪哇、花生和北方根结线虫在该区域核酸序列的差异。通过对6种近似根结线虫的不同地理群体及自然土壤线虫群体的测试,验证了设计的PCR引物针对象耳豆根结线虫的特异性和可靠性。本方法具有快速灵敏的特点,可用于象耳豆根结线虫单条线虫的直接鉴定以及混合土壤线虫群体中象耳豆根结线虫的检测。 相似文献
13.
根萤叶甲属(Diabrotica)被我国列为进境植物检疫性有害生物,该属中的玉米根萤叶甲(D.virgifera virgifera)、十一星根萤叶甲(D.undecimpunctata)、巴氏根萤叶甲(D.barberi)是北美的主要农业害虫。为了建立一种快速的分子鉴定方法以鉴定这3种根萤叶甲,本研究从田间采集的成虫样品中获得其部分mtDNA COI序列,与Gen Bank中的相关序列进行比对,设计筛选出3种根萤叶甲的特异性引物和TaqMan探针,并进行实时荧光PCR特异性和灵敏度检测。特异性检测结果表明,用目标种根萤叶甲DNA和其特异性探针和引物进行实时荧光PCR反应时,在30个循环反应内(Ct值30)有近S型扩增曲线出现,同时其他种均无荧光信号增长。此外,灵敏度结果显示,玉米根萤叶甲和巴氏根萤叶甲可检测的最小DNA模板浓度,即实时荧光PCR反应的灵敏度为0.1 ng/μL,十一星根萤叶甲为0.01 ng/μL。 相似文献
14.
Onivaldo Randig Florence Deau Marcilene F. A. dos Santos Myrian S. Tigano Regina M. D. G. Carneiro Philippe Castagnone-Sereno 《European journal of plant pathology / European Foundation for Plant Pathology》2009,125(3):485-495
The root-knot nematode (RKN) Meloidogyne mayaguensis is considered as one of the most damaging RKN species because of its extremely wide host range. Recent surveys have shown
the rapid spread of this parasite in agro-ecosytems, often making crop cultivation not viable in the heavily infested areas.
Here, we report the identification, molecular cloning, genomic organisation and sequence analysis of a new satellite DNA (satDNA)
family from M. mayaguensis (named pMmPet). It is comprised of two groups of A+T rich, tandemly repeated units of 174 and 180 bp, respectively. Using
these sequences as targets, hybridisation and PCR experiments performed on a wide collection of 44 populations belonging to
15 RKN species showed that the pMmPet family could only be detected in the 16 M. mayaguensis populations tested. In addition, because of their repetitive nature, positive detection of pMmPet sequences was achieved
in single individual nematodes. Therefore, the repeated sequence described here possesses features that make it an excellent
candidate for use as a specific and extremely sensitive tool for the accurate detection and identification of this invasive
pest on a routine basis. Clearly, monitoring the occurrence and spread of M. mayaguensis at the domestic and international levels are needed to avoid wholesale loss of agricultural resources in the infested regions. 相似文献
15.
Sebastian Kiewnick Martijn Holterman Sven van den Elsen Hanny van Megen Juerg Ernst Frey Johannes Helder 《European journal of plant pathology / European Foundation for Plant Pathology》2014,140(1):97-110
Root-knot nematodes (Meloidogyne spp.) are important pests of numerous crops worldwide. Some members of this genus have a quarantine status, and accurate species identification is required to prevent further spreading. DNA barcoding is a method for organism identification in non-complex DNA backgrounds based on informative motifs in short DNA stretches (≈600 bp). As part of the EU 7th Framework project QBOL, 15 Meloidogyne species were chosen to compare the resolutions offered by two typical DNA barcoding loci, COI and COII, with the distinguishing signals produced by two ribosomal DNA genes (small and large subunit rDNA; SSU?≈?1,700 and LSU?≈?3,400 bp). None of the four markers distinguished between the tropical species Meloidogyne incognita, M. javanica and M. arenaria. Taking P ID (Liberal) values ≥0.93 as a measure for species delimitation, the four mtDNA and rDNA markers performed well for the tropical Meloidogyne species complex, M. enterolobii, M. hapla, and M. maritima. Within cluster III A (Holterman et al. Phytopathology, 99, 227–235, 2009), SSU rDNA did not offer resolution at species level. Both mtDNA loci COI and COII did, whereas for LSU rDNA a longer fragment (≥700 bp) is required. The high level of mitochondrial heteroplasmy recently reported for M. chitwoodi (Humphreys-Pereira and Elling Nematology, 15, 315–327, 2013) was not found in the populations under investigation, suggesting this could be a regional phenomenon. For identification of RKNs, we suggest the combined use of SSU rDNA with one of three other markers presented here. 相似文献
16.
本文汇总了2008～2010年南海口岸进境林木种苗的线虫截获情况,共截获15科26属超过40种植物寄生线虫,其中检疫性线虫有7种,分别为短体线虫(非中国种)Pratylenchus Filipjev(non-Chinese species)、毛刺线虫属(传毒种类)Trichodorus Cobb(The species transmit viruses)、根结线虫(非中国种)Meloidogyne Goeldi(non-Chinese species)等;根据携带线虫状况分析了检疫性线虫的截获情况,提出了防止危险性线虫入境、扩散措施,为口岸防止线虫入境提供借鉴。 相似文献
17.
为明确甘肃省党参根结线虫病的发生、分布及病原种类,在甘肃省党参主产区调查、统计田间根结线虫病发生情况,并通过形态学和分子生物学方法进行种类鉴定。结果表明,甘肃省6个党参主产区均有根结线虫病发生,病田率、病株率分别为40.7%、13.2%,病情指数为10.4。依据根结线虫各虫态的形态测量值和雌虫会阴花纹等特征,将危害党参的根结线虫初步鉴定为北方根结线虫Meloidogyne hapla Chitwood, 1949。rDNA-ITS区段和28S rDNA D2D3区段序列比对和系统发育树显示,6个根结线虫群体无核苷酸差异,与多个北方根结线虫聚为一支且有较高的置信度。利用特异性引物Mh-F/Mh-R扩增得到北方根结线虫的特异性片段,大小为462 bp。故综合形态学和分子生物学特征,将危害甘肃省党参的根结线虫鉴定为北方根结线虫。 相似文献
18.
Cs. BUDAI 《EPPO Bulletin》1994,24(2):511-514
Under Hungarian climatic conditions, root-knot nematodes, and other phytoparasitic nematodes, generally cause damage in certain plant growing areas. These pests are present in glasshouses throughout the country. In the field the nematodes occur in sandy soil in the vegetable-growing areas of southern and central Hungary. The following species of root-knot nematodes have so far been reported to occur in Hungary: Meloidogyne incognita, M. arenaria, M. hapla, M. javanica, M. incognita acrita, M. thamesi and M. naasi. There are 10 specific nematicides registered for nematode control in the country. These chemicals are mainly applied in glasshouses where their use produces most economic return. Nematoderesistant cultivars are only available in the case of tomato. The nematodes cause the heaviest damage on glasshouse cucumber. 相似文献
19.
最近,南方根结线虫（Meloidogyne incognita）和北方根结线虫（M. hapla）的基因组测序工作已经完成,而大豆孢囊线虫（Heterodera glycines）和马铃薯白线虫(Globodera pallida)的基因组测序工作也在进行之中。截至2008年,Nematode.net数据库中已经有近500 000个表达序列标签ESTs序列和1 200 000个来自30种线虫的基因组序列。如何从众多的序列中筛选出有实际应用价值的信息是线虫学研究面临的一大挑战。对于缺乏有效转化系统的植物寄生线虫来说,RNAi无疑是研究植物寄生线虫基因功能的有力工具。本文介绍了RNAi及其在植物寄生线虫基因功能研究中的应用。 相似文献
20.
Sylvie Gamel Emilie Huchet Anne-Claire Le Roux-Nio Géraldine Anthoine 《European journal of plant pathology / European Foundation for Plant Pathology》2014,138(4):807-817
Several conventional PCR tests have been developed for the identification of the European quarantine root-knot nematodes Meloidogyne chitwoodi and M. fallax but data are lacking for the evaluation of their performance in terms of sensitivity, repeatability, reproducibility and specificity against a large range of populations. This study evaluated the performance criteria of three conventional PCR tests recommended by the consensus diagnostic protocol for Meloidogyne chitwoodi and Meloidogyne fallax published by the European and Mediterranean Plant Protection Organization (EPPO): a species-specific PCR (IGS target), a SCAR PCR, and a rDNA ITS PCR-RFLP. Evaluation was carried out with DNA extracts from juveniles, males and females according to EPPO recommendations for test validation. A minimum of 34 populations of target and non target nematode species were tested to check the specificity of these three PCR assays. The three PCR tests were ranked according to their specificity (with regard to cross reaction with other nematodes species or genus) and their sensitivity (detection of a single juvenile or mixed with other species). The species-specific PCR proved to be more sensitive but less specific than the SCAR PCR. The PCR-RFLP enables the identification of several Meloidogyne species but profile analysis can be difficult when several species are present in the mixture. Specific PCR products and RFLP profiles were also observed for M. arenaria and M. enterolobii, and described for M. minor and M. artiellia. 相似文献