Meloidogyne exigua satellite DNA
435
Fig. 6 Squash-blot experiments. (A) Meloidogyne exigua, M. incognita and M. paranaensis materials were squashed on a nylon membrane and hybridized with a cloned satellite DNA monomer. (B) Single females of Meloidogyne exigua (M.e), M. incognita (M.i ), M. mayaguensis (M.m) and M. paranaensis (M.p) were squashed on a n
ylon membrane and hybridized with a cloned satellite DNA monomer. Population codes are given in Table 1.
that the range of nucleotidic variability they displayed was representative of the whole M. exigua pMeL family. The most striking result was the high degree of sequence homogeneity among all the analysed repetition units, which deviated on average from the consensus by only 2.4%. Although some sequence variability exits, no discriminating feature of any subfamily could be detected. The second remarkable trait of these sequences is their relatively low A+ T content (54.2%) compared to what has been described for other RKN species. For example, the A+ T content in satDNAs was 68% in M. hapla (Piotte et al., 1994), 72.3% in M. fallax (Castagnone-Sereno et al., 1998), and 77% in M. incognita (Piotte et al., 1994). Since selective A+ T enrichment has been suggested as a consequence of satDNA evolution (Rojas-Rousse et al., 1993; Ugarkovic et al., 1989), this result tends to indicate that the pMeL family is a‘young’ satDNA. In that context, its relatively short life would not yet have allowed the accumulation of mutations in the repeats, which may account for the low level of variability observed among the cloned monomeric units. However, based on cytogenetic information, it has been assumed that meiotic parthenogenetic RKN, including M. exigua, are close relatives of the ancestor of the genus (Triantaphyllou, 1985). Therefore, it can be hypothesized that the pMeL repeats belong to a family that may have recently appeared in the genome of the nematode, through some extensive ampli cation burst. Indeed, the presence of internal repetitive motives in the sequence of the
monomeric unit is a good indication that ampli cation events occurred during the evolution of this satDNA. An alternative scenario to explain such a low level of sequence divergence among the pMeL monomers is that they could have been subjected to some highly effective homogenization mechanism, such as gene conversion or unequal crossing-over, as has been proposed for a number of homogeneous satDNA families (King and Cummings, 1997; Lopez-Leon et al., 1995). The observation that nucleotide changes are shared among several repeats of the pMeL family supports this hypothesis, since identical mutations shared among monomers can usually be explained as the result of partial gene conversion in the repetitive family, in contrast to independent mutational events (Drouin and Dover, 1990). However, it should be noted that homogenization mechanisms are thought to be slow for satDNA repeats (Stephan and Cho, 1994). A survey was made of other RKN species to see whether they also contained the cloned Bgl II repeat. In dot-blot experiments, hybridization was detected with M. exigua isolates only, which strongly suggests that the satDNA sequence is speci c for this taxon. However, it would be useful to test the probe against a wider range of RKN species, including other (minor) specie
s found to be associated with coffee (e.g. M. arabicida, M. konaenesis,…), in order to de nitely demonstrate its speci city. In addition, the cloned satDNA was shown to be highly abundant in the nematode genome, which allowed the unambiguous detection of single parasites independently of their developmental stage, and even in root tissues. The repeated sequence described here therefore possesses features that make it an excellent candidate for use as a speci c and extremely sensitive probe for the detection and identi cation of M. exigua. Currently, the routine identi cation of RKN associated with coffee is performed by the microscope examination of perineal patterns of females and/or by differential host tests. However, due to the limitations inherent in these methods, Meloidogyne spp. populations have frequently been misidenti ed (Campos et al., 1990). The isoenzyme electrophoresis of nematode females provides an interesting alternative analysis (Carneiro et al., 2000; Esbenshade and Triantaphyllou, 1990), but this method only works with a speci c stage of the nematode, and its detection threshold is not at all compatible with the identi cation of single individuals (Carneiro and Randig, unpublished data). Therefore, the squash-blot assay described here, using the Bgl II satDNA as a probe, should constitute an improved and accurate diagnosis method for the detection and identi cation of M. exigua. Since the experimental procedure is very easy and not time consuming, it should be possible to introduce it into eld-work without the need for a well-equipped molecular laboratory, a step which would contribute to the implementation of targeted pest management strategies in all coffee-growing countries of South and Central America.
© 2002 BLACKWELL SCIENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3(6), 431–437