Daniel Esmenjaud

Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes.

ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for Xiphinema diversicaudatum, X. index and X. vuittenezi

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.

Inheritance of resistance to the root-knot nematode Meloidogyne arenaria in Myrobalan plum.

The inheritance of resistance of the self-incompatible Myrobalan plum Prunus cerasifera to the root-knot nematode Meloidogyne arenaria was studied using first a diallel cross between five parents of variable host suitability (including two highly resistant clones P.1079 and P.2175, a moderate host P.2032, a good host P.2646 and an excellent host P.16.5), followed by the G2 crosses P.16.5 × (P.2646 × P.1079) and P.2646 × (P.16.5 × P.1079). A total of 355 G1 and 72 G2 clones obtained from hard-wood cuttings sampled from trees in the field experimental design, then rooted in the nursery and inoculated individually in containers (5–10 replicates per clone) under greenhouse conditions, were evaluated for their host suitability based on a 0–5 gall-index rating under a high and durable inoculum pressure of the nematode. In the crosses involving the resistant P.1079 and P.2175 and the hosts P.2646 and P.16.5: (1) all of the G1 crosses of P.1079 were resistant while the G2 crosses segregated 1 resistant to 1 host, (2) the G1 crosses between P.2175 and either P.2646 or P.16.5 segregated 1 resistant to 1 host, and (3) all of the G1 progeny between P.2646 and P.16.5 were host. These results indicate that resistance is conferred by a single major dominant resistance gene (homozygous) in P.1079, and the same, or an allelic or a different, major dominant gene (heterozygous) in P.2175, and that P.2646 and P.16.5 are recessive for this (these) major resistance gene(s). As expected according to the hypothesis of a recessive genotype for P.2032, all of its hybrids with P.1079 were resistant, all of its hybrids with P.2646 and P.16.5 were host, and its hybrids with P.2175 segregated for resistance. Nevertheless, the 3∶2 segregation ratio of these latter hybrids suggests that clones bearing the P.2175 gene would have a selective advantage. Both resistance genes are completely dominant and confer a non-host behaviour that totally prevents the multiplication of the nematode. This is the first reported evidence of major nematode resistance genes towards M. arenaria in a species of the subgenus Prunophora in the genus Prunus. The symbols Ma1 for the P.2175 gene and Ma2 for the P.1079 gene are proposed.

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region.

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1–TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.

Marker-assisted selection for the wide-spectrum resistance to root-knot nematodes conferred by the Ma gene from Myrobalan plum (Prunus cerasifera) in interspecific Prunus material

Prunus species express a more or less wide spectrum of resistance to root-knot nematodes (RKN) of the genus Meloidogyne. Among them, sources from Myrobalan plum (P. cerasifera) control all major and minor RKN species tested. In this outbreeding species, the clones P.2175 and P.2980 are heterozygous for the Ma single dominant gene and carry the alleles Ma1 and Ma3, respectively. Each allele confers a high-level resistance to the predominant RKN, M. arenaria, M. incognita and M. javanica and to the Florida isolate of an unknown Meloidogyne sp. which overcomes the resistance from peach and almond sources. The polymorphism of two coupling-phase SCAR markers tightly linked to Ma, SCAL19690 and SCAFLP2202, was evaluated within diverse diploid Prunus accessions. This material belongs to the subgenera Prunophora (Myrobalan and apricot) or Amygdalus (peach, almond and almond-peach) and includes the RKN resistance sources ‘Nemared’, ‘Alnem 1’ and ‘GF.557’. The alleles SCAL19690 and SCAFLP2202 were not present in three apricot cultivars (‘Moniqui’, ‘Luizet’ and ‘Stark Early Orange’) representative of the genetic diversity of this species and they segregated in an interspecific cross between P.2980 and apricot. These results suggest that apricot, reported as resistant to M. arenaria, M. incognita and M. javanica, and the Myrobalan plum might possess two different resistance systems. SCAL19690 and SCAFLP2202 were also absent from all tested Amygdalus material, whatever its resistance to RKN. Eight Myrobalan×Amygdalus segregating progenies including bispecific (P.2175 or P.2980×peach or almond) and trispecific (P.2175 or P.2980×almond-peach) hybrids were tested with the Florida isolate to identify individuals carrying the Ma resistance alleles. Both SCARs were then evaluated for segregation in these progenies to develop marker-assisted selection of Prunus interspecific rootstocks. SCAL19690 and SCAFLP2202 could be clearly detected and their tight linkage to Ma1 and Ma3 was confirmed. Consequently these SCARs appear to be powerful tools to screen for RKN resistance conferred by the Ma gene. They should also facilitate marker-assisted pyramiding of Ma with other resistance genes from the Amygdalus subgenus or from the botanically-related Armeniaca section.

RAPD and SCAR markers linked to the Ma1 root-knot nematode resistance gene in Myrobalan plum (Prunus cerasifera Ehr.)

Abstract The Myrobalan plum (Prunus cerasifera) is a self-incompatible species in which the clones P.2175, P.1079 and P.2980 are highly resistant to all root-knot nematodes (RKN), Meloidogyne spp. Each clone bears a single major dominant gene, designated Ma1, Ma2 and Ma3 respectively, that controls a high and wide-spectrum resistance. Bulked segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) analysis were both performed to detect markers linked to the Ma1 gene using three segregating progenies from P.2175 (Ma1 ma1) crossed by three host parents (ma1 ma1). Four dominant coupling-phase markers were identified from a total of 660 10-base primers tested. The resulting linkage map spans 14.7 cM and comprises three markers located on the same side of Ma1 and one marker located on the other side. The nearest markers (OPAL19720 and OPA161400) are located at 3.7 and 6.7 cM, respectively, on each side of the gene. Among the three markers that could be successfully converted into sequence characterized amplified region (SCAR) markers, two of them (SCAL19690 and SCAN12620) were scored as dominant markers whereas the third (SCAO19770) failed to produce any polymorphism. SCAL19, and to a lesser extent SCAN12, can be used reliably in the marker-assisted selection of Prunus rootstocks. These markers are adequate to identify the Ma1 RKN resistance gene in intraspecific segregating progenies and will be suitable for the creation of interspecific rootstocks involving Myrobalan plum. Some of the RAPD and SCAR markers for Ma1 were also recovered in clones P.1079 and P.2980, but not in additional host clones, suggesting that Ma1, Ma2 and Ma3 are either allelic or at least closely linked.

Spectrum of the Ma genes for resistance to Meloidogyne spp. in Myrobalan plum

Abstract The Myrobalan plum, Prunus cerasifera, bears a complete-spectrum resistance to the root-knot nematodes (RKN) Meloidogyne spp. in comparison to the main resistance sources in Amygdalus rootstocks that have more restricted spectra, as evidenced by a differential resistance test based on the predominant species M. arenaria, M. incognita and M. javanica and the population M. sp. Floride. Resistance to M. arenaria (A) in Myrobalan plum is controlled by the Ma major resistance genes that are completely dominant and confer a non-host behaviour that totally prevents the multiplication of the nematode. The inheritance of resistance of this self-incompatible species to M. incognita (I), M. javanica (J) and the population M. sp. Floride (F), considered as belonging to a new RKN species, was studied using G1 hybrids from a diallel cross based on five parents, the two resistant P.2175 (Ma1 gene; heterozygous) and P.1079 (Ma2 gene; homozygous) and three host parents, P.2032, P.2646 and P.16.5 (recessive for both genes), completed with the G2 backcrosses P.16.5×(P.2646×P.1079), P.2646 ×(P.16.5×P.1079) and P.2175×(P.2646×P.1079). G1 and G2 clones obtained from softwood cuttings sampled from trees in the field experimental design, rooted in the nursery, and inoculated in containers (six replicates per clone) under greenhouse conditions, were simultaneously evaluated for their host suitability to two to four of the RKN species, based on a 0–5 gall index (GI) rating under a high and durable inoculum pressure of the nematode, and then classified into resistant (R; GI?0.2) or host (H; GI?1.3) classes. The resistance classification of each individual clone, evaluated to two (A/J: 319 clones), three (A/J/I: 249 clones) and four (A/J/I/F: 161 clones) RKN species, from segregating and non-segregating crosses involving either Ma1 or Ma2 or both or none, was identical whatever the species. The independence of the R/H classification from the tested RKN indicates that the Ma1 and Ma2 genes control resistance to all of them, and it is assumed that these genes also control resistance to other minor RKN species. The relationship of the Ma genes with the putative genes involved in Amygdalus sources is discussed with the objective of introducing them into new interspecific rootstocks expressing a complete-spectrum and high-level resistance.

Genetic signature of amphimixis allows for the detection and fine scale localization of sexual reproduction events in a mainly parthenogenetic nematode.

Asexuality is an important mode of reproduction in eukaryotic taxa and has a theoretical advantage over sexual reproduction because of the increased ability to propagate genes. Despite this advantage, hidden signs of cryptic sex have been discovered in the genomes of asexual organisms. This has provided an interesting way to address the evolutionary impact of sex in plant and animal populations. However, the identification of rare sexual reproduction events in mainly asexual species has remained a challenging task. We examined the reproductive history in populations of the plant parasitic nematode Xiphinema index by genotyping individuals collected from six grapevine fields using seven microsatellite markers. A high level of linkage disequilibrium and heterozygous excess suggested a clonality rate of 95–100%. However, we also detected rare sexual reproduction events within these highly clonal populations. By combining highly polymorphic markers with an appropriate hierarchical sampling, and using both Bayesian and multivariate analysis with phylogenetic reconstructions, we were able to identify a small number of sexually produced individuals at the overlapping zones between different genetic clusters. This suggested that sexual reproduction was favoured when and where two nematode patches came into contact. Among fields, a high degree of genetic differentiation indicated a low level of gene flow between populations. Rare genotypes that were shared by several populations suggested passive dispersal by human activities, possibly through the introduction of infected plants from nurseries. We conclude that our method can be used to detect and locate sexual events in various predominantly asexual species.

Characterization of the RMja gene for resistance to root-knot nematodes in almond: spectrum, location, and interest for Prunus breeding

In Prunus spp., resistance genes to root-knot nematodes (RKN), Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne floridensis, confer either a complete spectrum, e.g., the Ma and Rjap genes in Myrobalan and Japanese plums (subgenus Prunophora), respectively, or a more restricted spectrum, e.g., the RMia gene (M. arenaria + M. incognita) in peach (subgenus Amygdalus). We report here characterization data of the RMja gene from the almond Alnem1, another Amygdalus source. The study of its spectrum is hampered by the inability of almond to be propagated by cuttings; we overcame this problem by using F1 and BC1 crosses with previously genotyped Myrobalan plums that conferred their rooting ability to hybrids for simultaneous evaluation to different RKN. As expected from a homozygous dominant resistance, BC1 progenies of Alnem1 segregated for resistance to M. javanica but were uniformly susceptible to M. incognita and M. floridensis, demonstrating that RMja controlled M. javanica but not M. incognita nor M. floridensis. SSR markers covering the Prunus reference map placed RMja on LG7 in the same region as Ma and Rjap and thus showed its independence from the RMia gene (LG2) of the botanically closer peach. The spectrum of this gene allows the theoretical construction of interspecific rootstocks, Myrobalan plum × (almond × peach), which cumulate RMja with Ma and RMia and are protected from each of the predominant RKN affecting Prunus, i.e., M. arenaria, M. incognita, and M. javanica, by at least two genes. This pyramiding strategy should offer to rootstock material an unprecedented guarantee of durable RKN resistance.

Genetic dissection of resistance to root-knot nematodes Meloidogyne spp. in plum, peach, almond, and apricot from various segregating interspecific Prunus progenies

Sources of resistance in Prunus spp. exhibit different spectra to the root-knot nematodes (RKN) Meloidogyne incognita, Meloidogyne javanica and Meloidogyne floridensis. In this Prunus genus, two dominant genes, Ma with a complete spectrum from the heterozygous Myrobalan plums P.2175 and P.2980 (section Euprunus; subgenus Prunophora) and RMia with a more restricted spectrum from the peaches Nemared and Shalil (subgenus Amygdalus), have been identified. This study characterizes the resistance spectra of interspecific crosses involving (1) previous Myrobalan and peach sources, (2) two Alnem almonds (subgenus Amygdalus) resistant to M. javanica, and (3) the apricot A.3923, representing a species considered RKN-resistant (section Armeniaca; Prunophora). For both latter species, genetic data could be obtained through F1 crosses with genetically characterized Myrobalans that conferred their rooting ability for clonal multiplication of the hybrids and permitted their simultaneous evaluation to the three RKN. Crosses involving either Ma or RMia or both generated the expected resistance spectra. Nemared confirmed the species-specific resistance to M. incognita conferred by RMia. This rootstock, also previously considered resistant to M. javanica, was susceptible to the M. javanica isolate used, what illustrates an isolate-specific resistance to this species. Alnem accessions were shown homozygous resistant to M. javanica. In the progeny P.2980 × A.3923, Ma markers allowed to distinguish resistant individuals carrying that gene from resistant individuals lacking it. Distribution of non-Ma individuals in this cross suggested, in the apricot parent, (1) the absence of a major gene allelic to Ma and (2) the presence of a non RKN specific polygenic resistance.