Ribosomal Intergenic Spacer: A Polymerase Chain Reaction Diagnostic for Meloidogyne chitwoodi, M. fallax, and M. hapla

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.     

Interspecific Hybridization of Meiotic Parthenogenetic Meloidogyne chitwoodi and M. fallax

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.     

Meloidogyne chitwoodi and Meloidogyne fallax in France: initial management experiences

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.     

Identification of Meloidogyne chitwoodi, M. fallax and M. hapla Based on SCAR-PCR: A Powerful Way of Enabling Reliable Identification of Populations or Individuals that Share Common Traits

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.     

Specific Diagnosis of Two Root-Knot Nematodes, Meloidogyne chitwoodi and M. fallax, with Satellite DNA Probes

ABSTRACT Meloidogyne chitwoodi and M. fallax are serious pests of potato, and both species have been recently designated as quarantine organisms in the European Community and in Canada. The sympatric and less damaging species M. hapla is often found associated with both of them under temperate climates. Here, we describe the use of satellite DNA (satDNA) sequences previously isolated from these three root-knot nematode species for the development of specific diagnostic procedures. In dot-blot experiments, it was unambiguously possible to separate M. chitwoodi and M. fallax from M. hapla using satDNA monomers as probes. In squash-blot experiments, satDNAs allowed discrimination between single individuals of M. chitwoodi or M. fallax from M. hapla, even within root tissues, without the need for DNA purification. The same results were obtained with radioactive or digoxigenin-labeled probes with no loss of sensitivity in detection. M. fallax and M. chitwoodi could not be distinguished. From this study, it is concluded that such cloned satDNA sequences may constitute a powerful tool for the identification and management of Meloidogyne spp. populations in the field and for the implementation of quarantine regulations against these pests.     

Expression of resistance to the root-knot nematodes,Meloidogyne hapla andM. fallax,in wildSolanum spp. under field conditions

In 1995 two fields in the Netherlands, naturally infested withMeloidogyne hapla (Wageningen) andM. fallax (Baexem), were used to evaluate resistant and susceptibleSolanum genotypes under natural conditions. In April, genotypes were planted in circular microplots. Soil samples were taken and analyzed for the occurrence of second-stage juveniles every six weeks. From August onwards, large differences between resistant and susceptible genotypes in numbers of juveniles were found in the soil. For all resistant wildSolanum genotypes the level of infection in soil at the end of the growing season in October was equal to or lower than at the beginning. Glasshouse experiments were performed with the same genotypes and nematode populations (i.e. originally derived from these fields) and the results were comparable with the observations from the field. It is concluded that resistance, as selected in glasshouse trials, corresponds well with resistant behaviour in the field and that it is worthwhile to transfer the resistance from theseSolanum sources to commercial potato cultivars for successful control of root-knot nematodes.     

Assessment of PCR-based tools for the specific identification of some temperate Meloidogyne species including M. chitwoodi,M. fallax and M. minor

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.     

Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species

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.     

A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

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.     

Selection of virulence in Meloidogyne chitwoodi to resistance in the wild potato Solanum fendleri

The possibilities of selecting virulence from a virtually avirulent root-knot nematode population towards resistance in wild potato have been investigated. Single egg masses of Meloidogyne chitwoodi, which had occasionally been produced on roots of resistant Solanum fendleri gave rise to eight lines after one generation on tomato. Five lines were able to circumvent completely the resistance of S. fendleri 93-114-12 resulting in a susceptible response similar to that of the control potato cv Nicola. Subsequently, a resistance test with other resistant genotypes of S. fendleri, S. bulbocastanum, S. stoloniferum and S. hougasii revealed that the virulent lines were also able to break through the resistance in most other species, but clear differences were noticed between the virulent lines. The results suggest a simple inheritance of virulence in M. chitwoodi towards resistance in S. fendleri. However, more virulence factors are involved to explain the differences on the other Solanum species between the virulent lines. The implications of the ease to select virulence with respect to the practical use of resistance in potato breeding and growing are discussed.     

A Molecular Marker Correlated with Selected Virulence Against the Tomato Resistance Gene Mi in Meloidogyne incognita, M. javanica, and M. arenaria

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.     

输华马铃薯上哥伦比亚根结线虫风险分析

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。     

The MeloTuber Test: a real‐time TaqMan® PCR‐based assay to detect the root‐knot nematodes Meloidogyne chitwoodi and M. fallax directly in potato tubers

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.