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1.
This study describes the development of species-specific pairs of PCR primers for the root-knot nematodes Meloidogyne chitwoodi, M. fallax and M. hapla that amplify species-specific RAPD fragments. After sequencing the fragments, longer primers were designed to complement the terminal sequences of the polymorphic DNA fragments. The resulting pairs of primers were used to generate the sequence-characterized amplified regions (SCARs). Using the developed pairs of SCAR primers, SCAR fragments of M. chitwoodi, M. fallax or M. hapla were easily amplified from DNA extracts from juveniles, egg masses, females of the particular nematode species investigated, either present alone, in a mixture with other nematode species or in infested plant material. A specially designed multiplex assay using three pairs of SCAR primers enabled the identification of multiple species in a mixture in a single PCR step. Single juveniles were easily identified by applying this multiplex assay followed by a subsequent multiplex PCR using three pairs of nested primers. The SCAR-PCR-based assays described have potential to be optimized for routine practical diagnostic tests. The usefulness of converting RAPD markers into SCAR markers is discussed.  相似文献   

2.
Potato is one of the many important hosts for the root‐knot nematodes Meloidogyne chitwoodi and M. fallax that can infest roots as well as tubers. In the latter they may cause surface galls and necrotic spots below the skin. In the EU these pathogens are categorized as quarantine organisms and are therefore regulated. Phytosanitary measures (PMs) are implemented and one aspect involves diagnostic procedures to detect these pathogens. To date, visual screening of external and internal symptoms is combined with the specific identification of these pests, either through microscopy, biochemical or molecular tests. A disadvantage of all these tests is the requirement of the prior extraction of nematodes from the tubers, which is not suitable for high‐throughput screening. This paper describes the MeloTuber Test developed to simultaneously detect M. chitwoodi and M. fallax by triplex real‐time TaqMan® PCR directly in secondary potato tuber peelings. DNA extraction is carried out in a 96‐well plate of pooled secondary peelings from one hundred tubers. The analytical sensitivity is such that a single female can be readily detected in such a sample size. The validation data, described here, prove the suitability of this molecular test for the detection of M. chitwoodi and M. fallax in large scale screening tests.  相似文献   

3.
Two major proteins, Mcf-A67 and Mcf-B66, were identified by mini two-dimensional polyacrylamide gel electrophoresis in order to distinguish the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from eight other species. These quarantine proteinic markers have been microsequenced after enzymatic digestion. The internal amino acid sequences exhibit similarities to members of a family of low molecular weight intracellular lipid-binding proteins. Moreover, to explore a simple, rapid, and inexpensive way to identify the two quarantine nematodes, dot blot hybridizations were performed using an antiserum (A67) produced from the longest amino-acid sequence of the protein Mcf-A67. Although several proteins stained on the M. chitwoodi and M. fallax western blot membranes, the two nematodes were easily distinguished from other root-knot nematodes, on dot blot assays with soluble proteins extracted from a single female. Because of its specificity and sensitivity, the use of the A67 antiserum to improve the diagnosis of the two European quarantine root-knot nematodes is discussed.  相似文献   

4.
BACKGROUND: An important part of the production area of common bean (Phaseolus vulgaris L.) in Belgium is located on the sandy soils of the provinces of Antwerp and Limburg where Meloidogyne chitwoodi (Golden), M. fallax (Karssen) and M. hapla (Chitwood) are present. The host plant status of ten bean cultivars for root‐knot nematodes was determined by evaluating penetration, development and egg mass formation after inoculation with second‐stage juveniles. RESULTS: The tested cultivars were poor to good hosts for M. chitwoodi, non‐hosts or bad hosts for M. fallax and excellent hosts for M. hapla. Significantly fewer M. fallax were found in the roots, and their development was delayed. Penetration of M. hapla took place over a longer period than that of M. chitwoodi and M. fallax. The number of mature females of M. chitwoodi in cv. Polder 6 weeks after inoculation was no different from that in other cultivars, although fewer egg masses were found on this cultivar in the screening test. There was no influence of M. chitwoodi on vegetative growth of cv. Polder. CONCLUSION: The differences found in host plant status of bean cultivars stress the importance of a correct diagnosis of the Meloidogyne species in agricultural fields. Cultivar Polder showed potential as a trap crop for M. chitwoodi. Copyright © 2011 Society of Chemical Industry  相似文献   

5.
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   

6.
A. Gamon  N. Lenne 《EPPO Bulletin》2012,42(1):122-126
Since 2008, the French NPPO has been controlling two outbreaks of Meloidogyne chitwoodi and Meloidogyne fallax, in Picardie (open fields) and in Bretagne (glasshouses). Intensive investigations have been undertaken to delimit these outbreaks and to help formulate the best control management strategy to adopt in these two very different situations. In open fields, eradication measures have been implemented, with bare fallow in infested fields being adopted as the main measure, despite the impact on affected growers and high financial cost. Recently, soil analyses in fields after 2 years of bare fallow showed that neither M. chitwoodi nor M. fallax was detected in 99% of cases, and measures have now been reduced: crops such as cereals are now allowed in these fields, but no tubers or root crops can be grown. Under glasshouses, eradication was not considered feasible and so a containment strategy was followed. An extensive national survey of susceptible crops has also been carried out for early detection of possible new outbreaks.  相似文献   

7.
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   

8.
Xu J  Narabu T  Mizukubo T  Hibi T 《Phytopathology》2001,91(4):377-382
ABSTRACT Root-knot nematodes of the genus Meloidogyne are economically important pathogens of a wide range of crops. The tomato resistance gene Mi typically confers resistance to the three major species, M. incognita, M. javanica, and M. arenaria. However, virulent populations completely overcoming the Mi resistance still occur. In an attempt to develop molecular markers for virulence against Mi and gain insights into the genetic relationships among virulent populations of different species and origins, random amplified polymorphic DNA (RAPD) analyses of laboratory-selected virulent, field virulent, and avirulent populations of M. incognita, M. javanica, and M. arenaria were carried out. A RAPD marker, specific for selected virulent populations, was identified, and subsequently, converted to a sequence characterized amplified region (SCAR). Sequence characterization of the SCAR locus showed that alleles from laboratory- and field-selected virulent populations were highly similar to each other and clearly different from alleles from natural virulent and avirulent populations. This result suggests that the genetic mechanism for virulence against Mi may be similar among selected virulent populations of the three Meloidogyne spp., but different between selected and natural virulent populations. Based on the nucleotide polymorphisms at the SCAR locus, codominant and dominant polymerase chain reaction-based markers were developed enabling rapid diagnosis of selected virulent genotypes in M. incognita, M. javanica, and M. arenaria.  相似文献   

9.
Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.  相似文献   

10.
In late 2009, a European Food Safety Authority (EFSA)‐funded project (Prima phacie) began work to review and test methodologies for conducting pest risk assessment by means of case studies on three phytoplasmas (Candidatus Phytoplasma mali, Ca. P. prunorum, Ca. P. pyri); two bacteria (Acidovorax avenae subsp. citrulli, Xanthomonas citri [=X. axonopodis] pv. citri); two fungi (Guignardia citricarpa, Mycosphaerella dearnessii); two nematodes (Meloidogyne chitwoodi, M. fallax); and an insect (Anoplophora glabripennis). Multiple risk assessment schemes and methods will be applied to each of the case study pests, allowing for a comparative assessment of methods. Methods to assess the effectiveness of possible risk management options for each pest will also be evaluated. The project will further develop the scientific basis for pest risk assessment within the European Community and identify methodologies most suitable for conducting risk assessments and for evaluating the effectiveness of possible risk management options by the EFSA Panel on Plant Health in order to support European decision making. The project lasts 29 months, and is being conducted by an international consortium of 11 partners consisting of phytosanitary organizations, research institutes and a university. Results will be disseminated via conventional publications and at a workshop in March 2012.  相似文献   

11.
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   

12.
Root-knot nematodes (RKNs, Meloidogyne spp.) are damaging pests that can infect thousands of plant species and cause enormous crop losses worldwide. Panax notoginseng is a common host of root-knot nematodes. In this study, we surveyed notoginseng gardens and determined the incidence of RKNs. Among the gardens surveyed, 71 % were infected with RKNs, and the RKN incidence index ranged from 8 % to 47 % in three randomly infected gardens. Meloidogyne hapla was identified as the pathogenic nematode based on 18S ribosomal RNA analysis by DNA barcoding. The results were qualitatively and quantitatively confirmed using a real-time PCR assay according to variations in the ITS1 and ITS2 regions. These results indicated that the combination of DNA barcoding and real-time PCR is a reliable and precise method for identifying parasitic nematodes from mixed-infected plant roots in the field. In addition, the abundance of ITS1 and ITS2 displayed a similar trend to the numbers of RKNs in the three gardens, which suggests that the results of real-time PCR can be used to determine the damage caused by M. hapla in the field. Our studies show that RKNs are common and can cause serious damage to notoginseng. We present an integrated method of detecting mixed nematode species in the field and confirm M. hapla as the target for parasitic nematode control in notoginseng gardens. Our results contribute to the improvement of RKN control in notoginseng and further promote the sustainable development of medicinal plants.  相似文献   

13.
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, M. incognita, 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   

14.
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15.
湖南柑橘根结线虫种类鉴定及特异性PCR检测   总被引：1，自引：0，他引：1  
根结线虫病是严重危害湖南柑橘生产的主要病害,快速准确地进行病原线虫鉴定,从而制定针对性的防治措施,对柑橘产业的稳定发展至关重要。运用形态学和分子生物学技术对湖南永州地区柑橘根结线虫进行病原种类鉴定,确定其为番禺根结线虫Meloidogyne panyuensis。通过引物设计与筛选,建立了番禺根结线虫特异性PCR检测技术。结果表明,该检测方法特异性好,灵敏度高,操作简单,能有效地从多种根结线虫中特异性检测出番禺根结线虫,为柑橘病原线虫的快速检测鉴定提供技术支撑。  相似文献   

16.
Monilinia fructicola was until very recently a regulated pest in the European Union, and EU countries were requested to monitor its presence on their territories. As accredited laboratories should use validated tests, the mycological laboratory of CRA‐PAV carried out a validation process for the multiplex based PCR test (Coté et al., 2004 ), that is one of the most widely used tests for the identification of M. fructicola, although this test is not described in the EPPO diagnostic protocol PM 7/18 (2) because the validation data were lacking. The performance characteristics of this multiplex PCR test were established according to the EPPO Standard PM 7/98 (1) and the test was compared in a collaborative study with the end point PCR test (Ioos & Frey, 2000 ), considered as the ‘standard test’. The validation data were obtained using different isolates of M. fructicola, M. laxa, M. fructigena and Monilia polystroma, as well as different fruit tissues. Four series of the DNA target at different concentration, repeated three times, were analyzed in four Italian laboratories. The results showed that the multiplex PCR detection test (Coté et al., 2004 ) was fit for diagnostic purpose, although the analytical sensitivity was significantly lower compared to the conventional PCR ‘standard test’.  相似文献   

17.
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   

18.
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   

19.
Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.  相似文献   

20.
The root-knot nematode Meloidogyne chitwoodi is a severe pest on sandy soils in Belgium and causes quality damage to economically important crops such as carrot, potato and black salsify. Pre-planting soil sampling to detect infestations has proven useful to farmers when taking decisions on the crop rotation. To develop an adequate sampling strategy, the vertical distribution of M. chitwoodi was examined under summer barley, carrot, fodder beet, bean, marigold and black fallow on two fields with a sandy soil. Soil samples were collected at monthly intervals from April 2004 to April 2006. Cores were taken to a depth of 70 cm and split into 10 cm segments. Nematodes were extracted by zonal centrifugation. Fodder beet increased the population of M. chitwoodi immensely; carrot was also a good host. Barley was a moderate host and under bean and marigolds the population decreased. The relative distribution of M. chitwoodi over the different soil layers during two successive years was consistent in each field. The different successions with good, moderate and poor hosts did not influence this distribution significantly. A logistic model was fitted to the mean cumulative percentages of nematodes at increasing soil depth. Farmers are advised to take soil samples for detection of M. chitwoodi immediately after harvest, especially after crops with a long field period. Adapting the depth of the cores taken to the vertical distribution of the population can increase the chances of detection. Our results suggest that this distribution is persistent in crop rotations and depends on field characteristics.  相似文献   

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