The polymerase chain reaction was evaluated for its ability to amplify DNA sequences specific for the root-knot nematode Meloidogyne hapla, using oligonucleotides whose sequence was deduced from the satellite DNA previously cloned in this species as primers. As expected, ladder patterns of monomers and multimers of an approximate 150-170-bp repeat were amplified from purified genomic DNA of all the M. hapla isolates studied, while no amplification was detected with the five other Meloidogyne species tested. Moreover, the satellite DNA nature of the amplification products was confirmed through Southern-blot hybridization with the previously cloned monomeric unit. In further experiments, DNA was extracted from single females, males, juveniles, or eggs according to a simple procedure, and used as a template in PCR assays. Amplification products were obtained, whose electrophoretic patterns were always very similar to those from M. hapla genomic DNA, thus demonstrating the high sensitivity of the method. This satellite DNA-based strategy can be exploited to develop species-specific primer sets for use on a routine basis as a diagnostic tool for unambiguous nematode identification procedures.
Total protein variation as revealed by two-dimensional electrophoresis (2D-E) was studied in 18 isolates from populations of Meloidogyne arenaria (six isolates), Meloidogyne incognita (10 isolates), and Meloidogyne javanica (one isolate) plus an unclassified isolate. Gels (80 x 60 x 0.75 mm) were silverstained and digitized in order to compare their protein patterns. Optical density and position of protein patterns were measured using statistical cluster analysis and computer-assisted image analysis software. Only those protein stains or positions that were clearly defined (i.e., without background) were considered. The number of positions in gels ranged from 86 to 203. Each of these positions had 95 clearly expressed proteins that were present in at least two replicates for each isolate. Spot position was considered a taxonomical character with two different states: presence (1) and absence (0). Accordingly, genetic distance was estimated among isolates and species, and a phylogenetic tree was constructed following the cladistic approach based on maximum parsimony analysis. Isolates of M. arenaria--M. javanica--Meloidogyne sp. and of M. incognita formed two separate monophyletic groups. Both groups were clearly defined on the basis of two sets of protein positions that can be considered as diagnostic characters. An attempt to identify these proteins by mass spectrometry was made. Group diagnostic proteins for M. incognita and M. arenaria (and for other proteins common to all isolates) were distinguished by protonated mass signals in the MALDI fingerprinting spectrum.
A survey of Meloidogyne in La Vera (Spain) was carried out during October 1995 and 1997. Isozyme electrophoresis (A-EST, MDH, CAT, GPI, DIA, GOT and SOD) was performed for each nematode isolate, each of which was cultured from an egg mass. In 1995 seven isolates of M. incognita, two of M. javanica and 75 of M. arenaria were found, while in 1997 one isolate of M. javanica and 72 of M. arenaria were found. Taxonomic relationships were established among 151 isolates using enzyme phenotypes by means of Multiple Correspondence and UPGM analysis. Twenty-six Multilocus Enzyme Electrophoresis (MLEE) phenotype variants were detected in ten significant taxonomic units: M. arenaria (seven units), M. incognita (two) and M. javanica (one). A total of 13 loci were identified and allelic frequencies for MLEE phenotype variants were estimated. Genetic distances among them suggested that M. arenaria is a poly- or paraphyletic group formed by at least two or three monophyletic lines of different origins. The evolutionary and epidemiological implications are discussed.
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