Esterase phenotypes were used to characterise 25 populations of Meloidogyne spp. collected on bananas from different banana producing areas in Brazil, using a simplified technique for routine analysis. Meloidogyne javanica, M. incognita, M. arenaria, and Meloidogyne spp. were detected at percentages of 61.7, 32.2, 4.3 and 1.8, respectively. The Meloidogyne populations represented mixed species in 80% of the samples, M. javanica and M. incognita prevailing under field conditions. Genetic analyses were conducted using RAPD markers which provided, alone or in combination, reliable polymorphisms both between and within species. Based on the presence or absence of bands, RAPD analysis of the data resulted in clustering of species and isolates congruent with esterase phenotype characterisation. The intraspecific variability in M. javanica, M. incognita and M. arenaria represented 29.1, 19.5 and 40.2% of the polymorphic amplified fragments, respectively. A general lack of correlation was observed between banana cultivar group, geographical origin and differentiation of the nematodes.

Enzyme phenotypes and genetic diversity of root-knot nematodes parasitising musa in Brazil

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.

Satellite DNA as a target for PCR-specific detection of the plant-parasitic nematode Meloidogyne hapla.

Molecular and biochemical methods for parasite differentiation within the genus Trichinella

Recent advances in molecular biology have enabled the genotyping of individual nema- todes, facilitati!}g the analysis of genetic variability within and among plant-pathogenic nematode isolates. This review first describes representative examples of how RFLP, RAPD, AFLP, and DNA sequence analysis have been employed to describe populations of several phytonematodes, including the pinewood, burrowing, root-knot, and cyst nematodes. The second portion of this paper evaluates the utility of a size-variable mitochondrial DNA locus to examine the genetic structure of Meloidogyne isolates using two alternate methodologies, variable number tandem repeat (VNTR) and repeat associated poiymorphism (RAP) analysis. VNTR analysis has revealed genetic variation among in- dividual nematodes, whereas RAP may provide useful markers for species and population differ- entiation. Why study the genetic structure of nematode populations? Although this area remains the least understood aspect of nematode population biology (8), quanti- tative description of genetic variability among nematode populations must now assume a pivotal role in contemporary phytonematology. The escalating reliance on environmentally friendly, non-nema- ticidal management strategies including plant-host resistance, crop rotation, and bi- ological control are measures that likely in- volve genotypic interactions between the plant nematode and its host. As a conse- quence, the selection and efficacy of ap- propriate control strategies will be im- pacted by the genetic variation that defines targeted nematode populations. We must learn of the genetic differentiation that underlies populations and address whether standard management regimes are controlling only a portion of the com- ponent subpopulations. Within traditionally defined taxonomic groups, the variable host ranges of closely related nematodes have generated de- scriptions of sibling species and sub-

Application of mitochondrial DNA polymorphism to meloidogyne molecular population biology.

Introduccion La triquinelosis es una nematodosis de distribucion cosmopolita producida por Trichinella spp. y transmitida por el consumo de carne infectada cruda o poco hecha. En este trabajo se revisan los brotes de triquinelosis humana declarados durante 1990-2001, analizando su distribucion geografica y la fuente de infeccion Metodos Se realizo el examen parasitologico de las carnes sospechosas mediante digestion clorhidropepsica y la caracterizacion molecular de los aislamientos de Trichinella a traves de Random Amplified Polymorphic DNA (RAPD) y Western-blot con los anticuerpos monoclonales Us5 y Us9. La deteccion de anticuerpos anti- Trichinella de los pacientes se llevo a cabo por inmunofluorescencia indirecta (IFI) Resultados De los 49 brotes declarados durante 1990-2001, un 75,5% tuvo como fuente de infeccion la carne de jabali frente al 14,3% producido por consumo de carne de cerdo. La fuente de infeccion se desconocia en el 10,2% restante. En nuestro laboratorio se siguieron 21 brotes de los 49 registrados, de los cuales solo en 13 se pudo llevar a cabo la identificacion molecular de la especie de Trichinella implicada, y se encontro que el 61,5% estaba producido por T. britovi y el 38,5% por T. spiralis . El diagnostico serologico de los afectados por IFI permitio confirmar en el laboratorio la parasitosis Conclusiones La caracterizacion de marcadores moleculares para la identificacion de T. spiralis y T. britovi revela la importancia de T. britovi en la salud publica espanola

Revisión de los brotes de triquinelosis detectados en España durante 1990-2001*

DNA from species and races of plant parasitic nematodes (Meloidogyne, Globodera and Heterodera) and a human parasitic nematode (Trichinella) were subjected to polymerase chain reaction amplification using one arbitrary primer (M-10). This technique results in relatively simple DNA profiles that include polymorphic markers known as random amplified polymorphic DNA (RAPDs). The RAPD profiles of the plant nematode species of Meloidogyne made possible the identification of M. incognita and M. hapla, but no differences were found between the patterns of M. javanica, M. arenaria and M. graminicola. Moreover, the four races of M. incognita were indistinguishable by this primer. In contrast, when races of the plant nematode Globodera rostochiensis (Ro1 and Ro2/3) were studied under the same RAPDs conditions, a race specific profile allows these two most devastating races to be differentiated. When DNAs of eight Trichinella isolates were subjected to RAPD studies, four different patterns were identified, corresponding to the four Trichinella clusters previously defined by isozyme polymorphism.

M.R. Chacon

Papers

The differentiation of parasitic nematodes using random amplified polymorphic DNA