Michel Bongiovanni

Phylogenetic relationships between amphimictic and parthenogenetic nematodes of the genus Meloidogyne as inferred from repetitive DNA analysis

Plant-parasitic nematodes of the genus Meloidogyne are known to reproduce either by cross-fertilization (amphimixis), facultative meiotic parthenogenesis or obligatory mitotic parthenogenesis. Among them, M. incognita, M. arenaria and M. javanica are obligatory mitotic parthenogenetic species, while M. hapla can reproduce by both cross-fertilization and meiotic parthenogenesis. Phylogenetic relationships in this genus have been investigated by hybridization of BamHI-digested genomic DNAs of 18 geographical isolates belonging to six species with three homologous repeated DNA probes cloned at random from a genomic library of one population of M. incognita. Due to the repetitive nature of the probes, the autoradiograms exhibited extensive restriction fragment length polymorphisms (RFLPs) both between and within nematode species. Genetic distance values estimated from hybridization patterns were analysed by two phylogenetic tree-building distance methods, respectively based on constant (UPGMA) and varying (FITCH) rates of nucleotide substitution, and the resulting dendrograms showed a very similar clustering of species and populations. Comparison of these results with the other sources of phylogenetic data available for this genus, i.e. cytogenetic, isoenzymatic and mitochondrial DNA (mtDNA) data, revealed consistency with all but the mtDNA phylogeny. Due to the maternal inheritance of mtDNA, and the parthenogenetic reproductive mode of these organisms, which excludes any possibility of horizontal transfer, we conclude that nuclear DNA phylogeny should represent a more likely evolutionary history of this particular genus, and that interspecific hybridizations between sexual ancestors may account for the results with mtDNA. Thus the early split off of the mitotically parthenogenetic species cluster and M. hapla confirms the amphimictic ancestral mode of reproduction of root-knot nematodes. Moreover, the existence of polymorphism within each species at the repeated DNA level is discussed in relation to the adaptative evolution of these parthenogenetic species.

Genetic variation inMeloidogyne incognita virulence against the tomatoMi resistance gene: evidence from isofemale line selection studies

Resistance to the parthenogenetic root-knot nematodeMeloidogyne incognita is controlled in tomato by the single dominant geneMi, against which virulent pathotypes are able to develop. Isofemale lines (i.e., families) were established from a natural avirulent isolate ofM. incognita in order to study the genetic variability and inheritance of the nematode virulence. From the progeny of individual females, the production of egg masses on the root system of theMi-resistant tomato ‘Piersol’ was analyzed in artificial selection experiments. A family analysis revealed, after two successive generations, a strongly significant variation between the 63 isofemale lines tested, and the results obtained for the mothers and their daughters were also significantly correlated. These results together clearly demonstrate the existence of a genetic variability and inheritance for this character. In a second experiment, a four-generation selection was performed on 31 other isofemale lines. The results revealed a significant response to selection apparently limited only to the two families able to produce, in first generation, a significant minimal egg-mass number on the resistant cultivar.

The pepper resistance genes Me1 and Me3 induce differential penetration rates and temporal sequences of root cell ultrastructural changes upon nematode infection

Abstract In pepper ( Capsicum annuum L.), the resistance genes Me1 and Me3 have been shown to control the main species of root-knot nematodes, Meloidogyne spp. Here, the reactions to inoculation with Meloidogyne incognita second-stage juveniles (J2) of two resistant doubled haploid pepper lines, HDA149 and HDA330, obtained through in vitro androgenesis and carrying Me3 and Me1 respectively, were compared in time-course experiments. Although they both suppressed nematode reproduction, the two resistance genes induced very different response patterns. First, significantly fewer J2 were able to invade roots of HDA149 compared to HDA330. Second, while ultrastructural changes typical to the hypersensitive reaction (HR) occurred in root cells of HDA149 early after nematode inoculation (i.e. necrosis of cells directly involved in nematode penetration and feeding), the resistance mechanism in HDA330 involved a delayed plant response (i.e. cell senescence and death) which took place after the induction of a number of (imperfect) giant cells by the nematode. These differential responses are discussed in relation to the nematode ability to circumvent or not the resistance gene(s), and it is suggested that such virulence could result from the overcoming by the J2 of the early HR occurring within the epidermis and cortex of the root.

Selection forMeloidogyne incognita virulence against resistance genes from tomato and pepper and specificity of the virulence/resistance determinants

Experiments were designed to analyze the relationships between the root-knot nematodeMeloidogyne incognita and resistant tomato and pepper genotypes. From a natural avirulent isolate, near-isogenic nematode lineages were selected with virulence either against the tomatoMi resistance gene or the pepperMe3 resistance gene. Despite the drastic selection pressure used, nematodes appeared unable to overcome the pepperMe1 gene, therefore suggesting some differences in the resistance conferred byMe1 andMe3 in this species. Nematodes virulent onMi-resistant tomatoes were not able to reproduce onMe1-resistant nor onMe3-resistant peppers, and nematodes virulent onMe3-resistant peppers were not able to reproduce onMi-resistant tomatoes nor onMe1-resistant peppers. These results clearly demonstrate the specificity ofM. incognita virulence against resistance genes from both tomato and pepper, and indirectly suggest that gene-for-gene relationships could occur between these two solanaceous crops and the nematode.

Virulence and molecular diversity of parthenogenetic root-knot nematodes, Meloidogyne spp.

Root-knot nematodes (RKN) are sedentary endoparasites causing severe damage to a wide variety of crops, including tomato. Among them, the parthenogenetic species Meloidogyne arenaria, M. incognita and M. javanica are of particular economic importance. The genetic diversity and relationships of 17 populations belonging to these three major species, either avirulent or virulent against the tomato Mi resistance gene, were investigated in order to determine whether (a)virulence of the nematodes could be related to their molecular fingerprints. Genomic polymorphisms between populations were assessed by using amplified fragment length polymorphism (AFLP) markers, and data were treated by means of a multiple correspondence analysis. A total of 1550 polymorphic amplified DNA fragments were identified and used to compute the relationships between the populations. As expected, the three RKN species were clearly distributed into distinct groups, but combination of data for virulence phenotypes and DNA markers showed that clustering of populations was not associated with their (a)virulence against the tomato Mi resistance gene. Such a lack of correlation indicates that most of the observed DNA polymorphism is independent of virulence, which is presumably under host selection. This result demonstrates that virulent populations do not share a common origin, and strongly suggests that they might have appeared late after the establishment of these clonal lineages, as the result of independent mutational events.

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

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.

Cloning and characterization of two satellite DNAs in the low-C-value genome of the nematode Meloidogyne spp.

Two highly reiterated StyI satellite DNAs have been cloned from two nematode species: one from Meloidogyne hapla and another from M. incognita. The monomeric units of these two satellites have a repeat length of 169 and 295 bp, respectively. These StyI repeated element families constitute 5% of the M. hapla and 2.5% of the M. incognita haploid genomes. The A + T content is elevated in both families (i.e., 68% and 77%, respectively). Nucleotide methylation and transcriptional activity are negative. No similarity was found between the two satellites, nor to other known highly repetitive elements. These StyI satellite DNAs are quite homogenous in sequence, showing on average 3% and 3.5% divergence from their respective calculated consensus sequence. An internal subrepeating unit of about 11 bp is observed in the StyI satellite monomer sequences of M. hapla, suggesting that it could have evolved from a shorter sequence. Because of the small size of the Meloidogyne genome (51 Mb) and the abundance of repeated sequences, this genus approaches a limit in terms of coding fraction.

Transmission hereditaire de la resistance aux nematodes Meloidogyne Chitwood (Tylenchida) portee par 2 lignees de Capsicum annuum L. : etude de descendances homozygotes issues d'androgenese

RÉSUMÉ La lignée PM 217 de Capsicum annuum est résistante à Meloidogyne arenaria, M. incognita, M. javanica et M. sp. (Séville). La lignée PM 687 est résistante aux 3 premières espèces. Le parent-hôte opposé à chacune d’elles, « Yolo-Wonder », présente une résistance à M. javanica. Par androgenèse in vitro, on a obtenu des plantes haploïdes à partir des hybrides FI : PM 217 x « Yolo Wonder » et PM 687 x « Yolo-Wonder ». Chaque descendance autodiploïde a été soumise séparément à l’action de 5 populations-clones de Meloidogyne appartenant aux 4 espèces. Il apparaît que PM 217 porte 2 gènes distincts et, vraisemblablement, indépendants, l’un contrôlant la résistance à M. arenaria, M. incognita et M. javanica, l’autre contrôlant la résistance à M. javanica et M. sp. (Séville). PM 687 porte également 2 gènes, l’un actif sur les 3 principales espèces, à l’exception d’une souche de M. arenaria, l’autre contrôlant cette même souche. Ces 2 gènes sont probablement liés. Ces divers gènes sont distincts par leur spectre d’action et/ou leur mode d’expression au niveau des racines. Les gènes à spectre le plus large, portés par chacun des géniteurs résistants, ne sont pas allèles, mais paraissent liés au même chromosome. « Yolo Wonder » porterait un 5e gène à effet plus limité.

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.