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1.
The objectives of this work were to evaluate the genetic variability of Meloidogyne enterolobii by molecular markers, and develop species‐specific molecular markers for application in detection. Sixteen M. enterolobii isolates from different geographical regions (Brazil and other countries) and hosts were used in this study. The identification and purification of the populations were carried out based on isoenzyme phenotype. The DNA amplification of the intergenic region (IGS) of the rDNA and of the region between the cytochrome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) produced specific fragments of the expected size for this nematode, i.e. 780 and 705 bp, respectively. Intraspecific variability among the isolates was evaluated with three different neutral molecular markers: AFLP, ISSR and RAPD. The results showed a low level of diversity among the isolates tested, indicating that M. enterolobii is a genetically homogeneous root‐knot nematode species. The RAPD method allowed the identification of a species‐specific RAPD fragment for M. enterolobii. This fragment was cloned and sequenced, and from the sequence obtained, a set of primers was designed and tested. The amplification of a 520‐bp‐long fragment occurred only for the 16 isolates of M. enterolobii and not for the 10 other Meloidogyne species tested. In addition, positive detection was achieved in a single individual female, egg‐mass and second stage juvenile of this nematode. This SCAR species‐specific marker for M. enterolobii represents a new molecular tool to be used in the detection of this nematode from field samples and as a routine diagnostic test for quarantine devices .  相似文献   

2.
In 2017, during a survey on subsistence farms and gardens in Coimbra region, Portugal, 40 infected root samples were collected and 47 root-knot nematode (RKN) isolates identified, based on esterase phenotype. The phenotypes A2, H1, Hi2/Hi4, I1/I2/I3 and J3 associated to five Meloidogyne species (M. arenaria, M. hapla, M. hispanica, M. incognita and M. javanica) were found in 43 RKN isolates. The esterase phenotype En2/En4/En5, corresponding to M. enterolobii (=M. mayaguensis), was detected in four RKN isolates from Cereus hildmannianus (Cactaceae), Lampranthus sp. (Aizoaceae), Physalis peruviana (Solanaceae) and Callistemon sp. (Myrtaceae) infected roots. In order to validate the biochemical identification of the M. enterolobii isolates, molecular studies performed with species-specific primers yielded the expected fragment of c.520 bp, and the amplification of cytochrome oxidase subunits I and II regions of 800 bp. The DNA sequences of one of the isolates were compared with available Meloidogyne species sequences in databases. The Portuguese isolate grouped with 99–100% bootstrap support with all M. enterolobii sequences included for comparison, confirming the presence of this RKN species in Portugal. In the EPPO region, M. enterolobii has been reported in France and Switzerland and intercepted in the Netherlands, Germany and the UK associated with plant material from Asia, South America and Africa. Taking into account the pathogen aggressiveness and its distribution, there is a high probability of its spread not only in the Mediterranean region but also in Europe, and of it becoming a threat to the agricultural economy, where there are no effective strategies for its control.  相似文献   

3.
A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, Mincognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.  相似文献   

4.
南方根结线虫中国分离群体种内变异分析   总被引:1,自引:0,他引:1  
为调查我国不同地区和不同寄主上的根结线虫Meloidogyne spp.种类分布以及群体变异情况,基于酯酶和苹果酸脱氢酶同工酶图谱及SCAR分子标记技术对2017—2019年从6省19种植物根部组织分离到的40个根结线虫群体进行鉴定,针对南方根结线虫M. incognita群体分别通过寄主鉴别法进行生理小种鉴别,利用携带Mi抗性基因的番茄进行毒力测试,对2龄幼虫的口针长度和体长进行测量,并对核糖体ITS和线粒体Nad5基因序列进行比较分析。结果显示:根结线虫分离群体经鉴定包括38个南方根结线虫群体和2个象耳豆根结线虫M. erterolobii群体;38个南方根结线虫群体中有35个群体被鉴别为1号生理小种,其余3个群体被鉴别为2号生理小种;发现1个南方根结线虫群体CN19可在携带Mi抗性基因的番茄上侵染繁殖,为毒性群体,其余群体无法进行侵染和繁殖,为无毒群体。南方根结线虫群体2龄幼虫的口针长度和体长均差异较大,而不同寄主来源分离群体的ITS和Nad5基因序列也存在一定变异。基于ITS和Nad5基因序列构建的系统发育树将所有根结线虫群体归为南方根结线虫和象耳豆根结线虫组成的2个独立分支,...  相似文献   

5.
A loop‐mediated isothermal amplification (LAMP) assay for detection of Meloidogyne enterolobii (Me‐LAMP) was developed based on the sequences of the 5S ribosomal DNA (5S rDNA) and intergenic spacer 2 (IGS2) segment. The LAMP amplification was achieved at 65°C isothermal conditions within 1–1·5 h. Its amplicons were confirmed using gel electrophoresis, SacI enzyme analysis, lateral flow dipstick (LFD) assay, and visual inspection through SYBR Green I and calcein staining. The results demonstrated that the Me‐LAMP was able to specifically detect M. enterolobii populations from different geographical origins, with a detection limit of about 10 fg M. enterolobii genomic DNA, which was 10–100 times more sensitive than conventional PCR. In addition, the applicability of LAMP to field detection was confirmed following its successful performance in detecting the pest on root and soil samples. The Me‐LAMP assay possessed the characteristics of simplicity, sensitivity and specificity, and is a promising and practical molecular tool for M. enterolobii diagnosis in pest quarantine and field surveys.  相似文献   

6.
Meloidogyne enterolobii (syn. M. mayaguensis) has been reported to cause severe damage in commercial guava orchards and other plants in Central and South American countries. Considering the risk of introduction and dissemination of this pest in the European region, M. enterolobii was placed on the EPPO A2 list in 2010. The use of non-host fruit species is a recommended strategy to manage root-knot nematodes in infested guava orchards. This study screened 89 plant genotypes from 25 fruit plants of economic importance, plus two susceptible controls (guava and tomato) for its host status to M. enterolobii. Three to eight months after inoculation, nematode reproduction factor (RF) was used to characterize host suitability of fruit crops to this nematode. Ten banana genotypes, six Barbados cherries, one fig, two grape rootstocks and six melons were rated as good hosts for this nematode. Sixteen fruit plants behaved either as non-hosts or poor hosts to M. enterolobii, including assaí, atemoya, avocado, cashew nut, citrus, coconut, grape, jabuticaba, mango, mulberry, papaya, passion fruit, sapodilla, soursop, starfruit and strawberry. For the future, field experiments in areas infested by this nematode are essential to confirm the greenhouse results. These non-host fruit species can replace in the future eradicated guava trees in fields severely infested by this nematode and become an economic option for growers where M. enterolobii is considered a serious problem.  相似文献   

7.
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   

8.
Root-knot nematodes (Meloidogyne spp.) are a major problem facing crop production globally including potatoes. During the 2011/2012 potato growing season, root-knot nematode infected potato tubers were obtained from different potato growing regions in South Africa for identification of Meloidogyne spp. Using the intergenic region of the ribosomal DNA (IGS-rDNA) together with the region between the cytochrome oxidase small subunit II (COII) and the 16S rRNA gene in the mitochondrial DNA (mtDNA), five of the 78 composite samples received produced amplicon sizes of 705 bp for COII and 780 bp for IGS typical of M. enterolobii. These five samples were from the KwaZulu-Natal potato producing region. Nucleotide sequencing and phylogenetic analysis of the COII and IGS fragment showed that the five Meloidogyne populations were 100 % similar and they clustered closely with those of M. enterolobii in the GenBank database. The high damage potential of resistance-breaking populations of Meloidogyne species is a threat to profitable potato production and will require effective pest management programmes to be put in place.  相似文献   

9.
为明确云南省紫茎泽兰根结线虫病的病原种类,于2019年2月在云南省澜沧县林下三七种植区采集根部带有明显根结的紫茎泽兰根系进行根结线虫分离,通过观察所分离根结线虫的2龄幼虫、雌成虫、会阴花纹特征对其进行形态学鉴定,并利用序列比对、系统发育树分析、序列特异性扩增区段(sequence characterized amplified region,SCAR)对其进行分子生物学鉴定。结果表明,该病原线虫雌成虫会阴花纹呈圆形至卵圆形,背弓中等高或低平,侧区一侧或两侧延伸形成翼状,尾区有刻点,2龄幼虫、雌成虫形态特征及形态测量指标与北方根结线虫Meloidogyne hapla相似;该病原线虫rDNA的ITS序列和mtDNA的COI序列与NCBI数据库中已登录的北方根结线虫相应序列相似度较高,分别达99.35%和98.05%以上;该病原线虫rDNA的ITS序列、mtDNA的COI序列分别以99%、100%的支持率与北方根结线虫聚为同一分支;利用SCAR特异性引物,该病原线虫均能扩增出大小约1 500 bp的基因特异性条带。综合形态学和分子生物学鉴定结果将云南省紫茎泽兰根结线虫病病原种类鉴定为北方根结线虫。  相似文献   

10.
Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.  相似文献   

11.
PCR-RFLPs of ITS-rDNA and PCR with species-specific primers were developed for identification of cysts and juveniles of the beet cyst nematode Heterodera schachtii. Restrictions of PCR product by MvaI or ScrFI distinguish H. schachtii, H. betae, H. trifolii and H. medicaginis. RFLP profiles with eight restriction enzymes for these four nematode species are presented. Based on Internal Transcribed Spacer sequences of populations from several Schachtii group species, a specific primer for H. schachtii was designed, permitting amplification of the target sequence from juveniles and cysts of the beet cyst nematode. A duplex PCR protocol tested with a wide range of nematode samples is described.  相似文献   

12.
In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum, an unusual esterase (EST) phenotype was detected in a root‐knot nematode (RKN) from potato roots collected in Coimbra. This Portuguese isolate was purified and maintained on tomato, S. lycopersicum, and morphological, biochemical and molecular characteristics were studied. Perineal pattern morphology was highly variable, similar to Meloidogyne ethiopica and not useful for identification. The EST phenotype, from young egg‐laying females, displayed three bands similar to the Brazilian M. luci (L3) and distinct from M. ethiopica (E3). Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I and the mitochondrial DNA region between COII and 16S rRNA genes revealed that the Portuguese isolate grouped with M. luci isolates close to M. ethiopica isolates. However, considering the ITS1‐5.8S‐ITS2 region, the Portuguese isolate grouped with isolates of M. luci, M. ethiopica and M. hispanica, which limits the confidence of this region for M. luci diagnosis, and its differentiation from other species with morphological similarities. The M. luci pathogenicity to potato was also assessed in 16 commercial cultivars and compared with M. chitwoodi, considered to be a quarantine RKN species by EPPO. All potato cultivars were susceptible to both Meloidogyne species with gall indices of 5 and higher reproduction factor values ranging from 12.5 to 122.3, which suggests that M. luci may constitute a potential threat to potato production. In the present study, M. luci is reported for the first time attacking potato in Portugal.  相似文献   

13.
Nine populations of Meloidogyne spp. from Greece have been identified as M. javanica or M. incognita using either isozyme phenotypes or the sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) technique. Virulence against the Mi resistance gene was assayed by pot experiments in controlled conditions and revealed the ability of five populations of M. javanica and one population of M. incognita to reproduce on tomato cultivars containing that gene. A resistance-breaking population of M. incognita is reported for the first time in the country; the M. javanica populations constitute new records for the Greek mainland.  相似文献   

14.
The root-knot nematode Meloidogyne incognita is widely distributed and a major pathogen of cotton (Gossypium spp.) worldwide. The objectives of this study were to assess the genetic variability and aggressiveness of Brazilian populations of M. incognita in cotton. Five populations of M. incognita and one isolate of M. enterolobii (outgroup) were used in the molecular analysis. Our results showed that only 2.7 % of the RAPD and AFLP fragments were polymorphic. Despite the existence of two races (races 3 and 4) and two esterase phenotypes (I1 and I2), a low genetic variability among populations was observed, which might be due to the mitotic parthenogenetic mode of reproduction of this pathogen. The aggressiveness/virulence among populations towards different cotton genotypes was also studied. None of the populations was virulent to the resistant cotton genotypes M-315 RNR, TX-25, CIR1343, Wild Mexican Jack Jones and CIR1348 (reproduction factor <1). Two populations of M. incognita from the states of Mato Grosso do Sul and Parana (Umuarama) (races 4 and 3, respectively) were highly aggressive to the susceptible control FM966 and virulent to the accessions LA-887 and Clevewilt-6 that showed moderate resistance to other populations tested.  相似文献   

15.
Thirty-two isolates of Fusarium species were obtained from wilted Welsh onion (Allium fistulosum) grown on nine farms from six regions in Japan and identified as F. oxysporum (18 isolates), F. verticillioides (7 isolates), and F. solani (7 isolates). The pathogenicity of 32 isolates was tested on five commercial cultivars of Welsh onion and two cultivars of bulb onion in a seedling assay in a greenhouse. The Fusarium isolates varied in the degree of disease severity on the cultivars. Five F. oxysporum isolates (08, 15, 17, 22, and 30) had a higher virulence on the cultivars than the other isolates. The host range of these five isolates was limited to Allium species. Molecular characterization of Fusarium isolates was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA. The 32 isolates were grouped into eight types (four types for F. oxysporum, one for F. verticillioides, and three for F. solani). Restriction patterns of the ITS region were not related to pathogenicity. However, the haplotypes obtained with five enzymes (RsaI, HinfI, HaeIII, ScrFI, and MspI) and the phylogenetic analysis permitted the discernment of the three Fusarium species. The PCR-RFLP analysis should provide a rapid, simple method for differentiating Fusaruim species isolated from wilted Welsh onion in Japan.  相似文献   

16.
A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 103cfu ml–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.  相似文献   

17.
In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.  相似文献   

18.
Mitochondrial DNA (mtDNA) extracts from 13 isolatesof Fusarium oxysporum f. sp.niveum, including 12 from widely separated geographic regions within the United States and representing the three races, and one race 2 isolate from Israel, were examined for the presence of plasmid DNA and were also subjected to restriction endonucleases analysis. None of the mtDNA from any isolate had a copurifying plasmid. The estimated size of mtDNA fromF. o. f. sp.niveum race 0, calculated as the average of the sum of restriction fragment sizes, was 45.1 ± 2.2 kb. The restriction enzymesBamHI, EcoRI, Hpal, HindIII andMbol resolved 2, 4, 9, 21, and more than 40 fragments, respectively, but no polymorphisms were observed among the 13 isolates with any of these endonucleases. However, PstI digestion showed three distinct polymorphic patterns among the isolates. Each appeared to derive from point mutations that resulted in a change of one or more restriction sites. The most common pattern was present in nine of the isolates (three of race 0, four of race 1, and two of race 2) and included a 1.5 kb fragment. A second polymorphic pattern occurred in three USA isolates (one each of race 0, race 1 and race 2) and was characterized by an apparent replacement of the 1.5 kb fragment by 0.6 and 0.9 kb fragments. The Israeli isolate [ISL-59(73) race 2| had a unique pattern lacking the 1.5 and 2.0 kb fragments present in the common pattern and, instead, had 0.6, 0.9 and 3.0 kb fragments. The mtDNA polymorphisms observed among the USA isolates were not correlated with either pathological race or geographic region of origin.  相似文献   

19.
根结线虫种群的线粒体DNA分析   总被引:7,自引:0,他引:7  
 在同工酶和形态学鉴定的基础上,利用引物#C2F3和#1108对42个根结线虫种群线粒体DNA (mtDNA)中的COⅡLrRNA间区域进行特异性PCR扩增,35个种群的扩增产物约为1.7kb,其中29个是南方根结线虫,6个是爪哇根结线虫;3个花生根结线虫种群的扩增产物约为1.1kb;1个种群的扩增产物约为0.7kb,为根结线虫属在中国的新记录种;3个北方根结线虫种群的扩增产物约为0.5kb。用单条2龄幼虫提取物作模板得到的结果与大量提取DNA作模板的结果相同。为了区分产生相同大小片段的南方根结线虫和爪哇根结线虫,用限制性内切酶HinfⅠ对扩增产物进行酶切,结果表明:所有供试的南方根结线虫都可以被HinfⅠ酶切,且产生约1.3和0.4kb的2个限制性片段;但供试的爪哇根结线虫种群不能被酶切。由此表明,利用mtDNA PCR及酶切实验可以作为快速而准确地鉴定常见根结线虫的方法。  相似文献   

20.
为明确棉铃虫Helicoverpa armigera(Hübner)在山东省不同地理种群的遗传分化及遗传多样性,对采集得到的山东省14个棉铃虫种群共109个样本进行线粒体细胞色素氧化酶亚基(I mito‐chondrial cytochrome oxidase I,mt COI)及亚基II (mitochondrial cytochrome oxidase I,mt COII)基因片段测序,并进行遗传多样性分析,探究其在山东省的种群遗传变异情况。结果表明,棉铃虫mt COImt COII基因拼接(1 306 bp)共有18个单倍型Hap1~Hap18,其中Hap2和Hap3单倍型包含的个体数量最多,分别为37个和20个。莱阳种群的单倍型多样性最高,为1.000,莱西种群的核苷酸多态性最高,为0.004 5,14个棉铃虫种群的总体固定系数FST值为0.028,说明棉铃虫种群之间几乎没有遗传分化。GENEPOP分析表明种群间遗传距离和地理距离无显著相关性。表明山东省不同地区棉铃虫遗传分化较低,各地区种群间没有显著的遗传差异。  相似文献   

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