Onivaldo Randig

Identification and genetic diversity of Meloidogyne spp. (Tylenchida: Meloidogynidae) on coffee from Brazil, Central America and Hawaii.

The present study was based on 18 populations of Meloidogyne spp. originating from different coffee fields in Brazil, Central America and the USA (Hawaii). The identification of the main species and an outline of the diversity of root-knot nematodes parasitising coffee in these countries with respect to esterase phenotypes, morphology and molecular polymorphism, are provided. With the present electrophoretic procedure, esterase phenotypes were demonstrated to be species-specific and constitute a good tool for identifying root-knot species from coffee, viz., M. incognita (Est I1, I2), M. paranaensis (Est P1, P2), M. arenaria (Est A2), M. arabicida (Est AR2), M. exigua (Est E1), M. mayaguensis (Est M2) and two unknown populations that probably represent new species (Est SA2, SA4). The perineal pattern is often an unreliable character when used alone for making diagnostic conclusions but, when used as a complementary tool together with enzyme characterisation, is essential for checking the morphological consistency of the identification. Male characters are important for confirming the diagnosis of some species, such as M. paranaensis, M. konaensis and M. incognita. The results showed that the RAPD markers produced are consistent with other approaches (esterase phenotypes and morphological features) for confirming species identification and for estimating genetic relationships among species and isolates. Phylogenetic analyses showed that M. mayaguensis and M. exigua are more closely related to one another than they are to the other species. This was also true for M. javanica, M. arenaria and Meloidogyne spp. Low levels of intraspecific polymorphism were detected in M. exigua (8.6%), M. incognita (11.2%) and M. paranaensis (20.3%). Conversely, M. arenaria and the two unknown Meloidogyne spp. exhibited higher levels of intra- or interspecific variability (34.9 and 29.9%, respectively).

A species‐specific satellite DNA family in the genome of the coffee root‐knot nematode Meloidogyne exigua: application to molecular diagnostics of the parasite

SUMMARY A new BglII satellite DNA has been isolated, cloned and sequenced from the coffee root-knot nematode, Meloidogyne exigua (Nematoda: Tylenchida). It is represented as tandemly repeated sequences with a monomeric unit of 277 bp. The monomers are present at approximately 17 900 copies per haploid genome, and represent about 9.7% of the total genomic DNA. Twenty randomly chosen monomers have been sequenced. The deduced unambiguous consensus sequence is 277 bp long, and displays an A + T content of 54.2%. The monomers are very homogenous in sequence, showing on average 2.4% divergence from their consensus. Therefore, it is hypothesized that this repeated family may have recently appeared in the genome of the nematode, through some extensive amplification burst. Using a cloned monomer as a probe, dot-blot experiments demonstrated the species-specific distribution of the BglII satellite DNA. Moreover, squash-blot assays allowed us to detect single M. exigua individuals, at any developmental stage, and even within root tissues, without the need for preliminary DNA purification. From these results, it is concluded that the procedure described, using the satellite DNA as a sensitive species-specific probe, should constitute an improved and accurate diagnosis method for the detection and identification of the nematode, which would contribute to the implementation of targeted pest management strategies in all coffee growing countries of South and Central America.

RAPD Characterization of Single Females of the Root-knot Nematodes, Meloidogyne Spp.

Random amplified polymorphic DNA fingerprinting was performed on single females of root-knot nematodes, Meloidogyne spp., using a new procedure for DNA isolation. One-fourth of the total DNA amount isolated from a single female proved to be sufficient as a template in a polymerase chain reaction. Electrophoretic patterns of the amplified fragments were reproducible between replicates from a single female or sister females from the same progeny, and identical to those obtained with genomic DNA purified from a large number of nematodes. Moreover, a comparative analysis over three successive generations showed stability of the amplification patterns, thus demonstrating the utility of this procedure for epidemiological and ecological studies on root-knot nematodes.