Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato-root-knot nematode interactions.

Here, the interaction of Melodoigyne incognita virulent and avirulent pathotypes with susceptible and Mi-resistant tomato (Solanum lycopersicon) has been studied. Significant differences in nematode penetration occurred 2 days postinoculation (dpi) and became stable from 3 dpi onwards. The hypersensitive cell response (HR) in resistant plants prevented the installation of the avirulent pathotype. The virulent pathotype overcame the Mi (nematode) resistance and induced feeding sites in root cells without triggering HR. Reactive oxygen species (ROS), visualized by subcellular reduction of nitroblue tetrazolium, accumulated in nematode penetrated cells. Quantitative analyses with dichlorofluorescein indicated that the oxidative burst occurred very early with both pathotypes, with an enhanced rate in hyper-responsive cells. Hydrogen peroxide (H(2)O(2)), detected by cerium chloride reaction, accumulated in the cell walls and especially in cells neighbouring HR. The apoplastic location of cerium perhydroxide indicated that either the plasma membrane or the cell wall was the primary site of the superoxide/H(2)O(2) generator. The data provide evidence, for the first time, for ROS-generated signals and their spatiotemporal expression in the host and nonhost interaction of tomato with nematodes.

Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.

RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures.

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.