Detection of Nepovirus Vector and Nonvector Xiphinema Species in Grapevine.

Fanleaf degeneration is considered the most damaging viral disease of grapevine. The two major nepoviruses involved are Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) which are respectively and specifically transmitted by the dagger nematodes Xiphinema index and X. diversicaudatum. The methods described below are aimed at detecting four prevalent grapevine Xiphinema species: the vector species previously mentioned and two nonvector species X. vuittenezi and X. italiae.

Development of nine polymorphic microsatellite markers for the phytoparasitic nematode Xiphinema index, the vector of the grapevine fanleaf virus.

We report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. index.

Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard.

The nematode Xiphinema index is, economically, the major virus vector in viticulture, transmitting specifically the Grapevine fanleaf virus (GFLV), the most severe grapevine virus disease worldwide. Increased knowledge of the spatial distribution of this nematode, both horizontally and vertically, and of correlative GFLV plant infections, is essential to efficiently control the disease. In two infested blocks of the Bordeaux vineyard, vertical distribution data showed that the highest numbers of individuals occurred at 40 to 110 cm depth, corresponding to the two layers where the highest densities of fine roots were observed. Horizontal distribution based on a 10 x 15 m grid sampling procedure revealed a significant aggregative pattern but no significant neighborhood structure of nematode densities. At a finer scale ( approximately 2 x 2 m), nematode sampling performed in a third block confirmed a significant aggregative pattern, with patches of 6 to 8 m diameter, together with a significant neighborhood structure of nematode densities, thus identifying the relevant sampling scale to describe the nematode distribution. Nematode patches correlate significantly with those of GFLV-infected grapevine plants. Finally, nematode and virus spread were shown to extend preferentially parallel to vine rows, probably due to tillage during mechanical weeding.

High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.

Microsatellite genetic linkage maps of myrobalan plum and an almond-peach hybrid--location of root-knot nematode resistance genes.

Inheritance and linkage studies were carried out with microsatellite [or simple sequence repeat (SSR)] markers in a F(1) progeny including 101 individuals of a cross between Myrobalan plum ( Prunus cerasifera Ehrh) clone P.2175 and the almond (Prunus dulcis Mill.)-peach ( Prunus persica L. Batsch) hybrid clone GN22 ["Garfi" (G) almond x "Nemared" (N) peach]. This three-way interspecific Prunus progeny was produced in order to associate high root-knot nematode (RKN) resistances from Myrobalan and peach with other favorable traits for Prunus rootstocks from plum, peach and almond. The RKN resistance genes, Ma from the Myrobalan plum clone P.2175 and R(MiaNem) from the 'N' peach, are each heterozygous in the parents P.2175 and GN22, respectively. Two hundred and seventy seven Prunus SSRs were tested for their polymorphism. One genetic map was constructed for each parent according to the "double pseudo-testcross" analysis model. The Ma gene and 93 markers [two sequence characterized amplified regions (SCARs), 91 SSRs] were placed on the P.2175 Myrobalan map covering 524.8 cM. The R(MiaNem) gene, the Gr gene controlling the color of peach leaves, and 166 markers (one SCAR, 165 SSRs) were mapped to seven linkage groups instead of the expected eight in Prunus. Markers belonging to groups 6 and 8 in previous maps formed a single group in the GN22 map. A reciprocal translocation, already reported in a G x N F(2), was detected near the Gr gene. By separating markers from linkage groups 6 and 8 from the GN22 map, it was possible to compare the eight homologous linkage groups between the two maps using the 68 SSR markers heterozygous in both parents (anchor loci). All but one of these 68 anchor markers are in the same order in the Myrobalan plum map and in the almond-peach map, as expected from the high level of synteny within Prunus. The Ma and R(MiaNem)genes confirmed their previous location in the Myrobalan linkage group 7 and in the GN22 linkage group 2, respectively. Using a GN22 F(2) progeny of 78 individuals, a microsatellite map of linkage group 2 was also constructed and provided additional evidence for the telomeric position of R(MiaNem) in group 2 of the Prunus genome.

Morphological, Molecular, and Differential-Host Characterization of Meloidogyne floridensis n. sp. (Nematoda: Meloidogynidae), a Root-Knot Nematode Parasitizing Peach in Florida.

A root-knot nematode, Meloidogyne floridensis n. sp., is described and illustrated from peach originally collected from Gainesville, Florida. This new species resembles M. incognita, M. christiei, M. graminicola, and M. hispanica, but with LM and SEM observations it differs from these species either by the body length, shape of head, tail and tail terminus of second-stage juveniles, body length and shape of spicules in males, and its distinctive female perineal pattern. This pattern has a high to narrowly rounded arch with coarsely broken and network-like striae in and around anal area, faint lateral lines interrupting transverse striae, a sunken vulva and anus, and large distinct phasmids. Molecular data from ribosomal IGS illustrate that M. floridensis n. sp. is different from the mitotic species M. arenaria, M. incognita, and M. javanica. Data from RAPDs confirm it and suggest that this new species lies in an intermediate phylogenetic position between the previous species and the meiotic species M. hapla, M. fallax, and M. chitwoodi. Differential host tests based on annual crops and on Prunus accessions are reported.

Location of independent root-knot nematode resistance genes in plum and peach.

Prunus species express different ranges and levels of resistance to the root-knot nematodes (RKN) Meloidogyne spp. In Myrobalan plum ( Prunus cerasifera), the dominant Ma gene confers a high-level and wide-spectrum resistance to the predominant RKN, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica and the isolate Meloidogyne sp. Florida which overcomes the resistance of the Amygdalus sources. In Japanese plum ( Prunus salicina), a similar wide-spectrum dominant resistance gene, termed R(jap), has been hypothesized from an intraspecific segregating cross. In peach, two crosses segregating for resistance to both M. incognita and M. arenaria were used to identify single genes that each control both RKN species in the Shalil ( R(Mia557)) and Nemared ( R(MiaNem)) sources. Localisation of these genes was made possible using the RFLP and SSR- saturated reference Prunus map TxE, combined with a BSA approach applied to some of the genes. The Ma1 allele carried by the Myrobalan plum accession P.2175 was localised on the linkage group 7 at an approximate distance of 2 cM from the SSR marker pchgms6. In the Japanese plum accession J.222, the gene R(jap) was mapped at the same position in co-segregation with the SSR markers pchgms6 and CPPCT022. The peach genes R(Mia557) and R(MiaNem), carried by two a priori unrelated resistance sources, were co-localized in a subtelomeric position on linkage group 2. This location was different from the more centromeric position previously proposed by Lu et al. (1999) for the resistance gene Mij to M. incognita and M. javanica in Nemared, near the SSR pchgms1 and the STS EAA/MCAT10. By contrast, R(Mia557) and R(MiaNem) were flanked by STS markers obtained by Yamamoto and Hayashi (2002) for the resistance gene Mia to M. incognita in the Japanese peach source Juseitou. Concordant results for the three independent sources, Shalil, Nemared and Juseitou, suggest that these peach RKN sources share at least one major gene resistance to M. incognita located in this subtelomeric position. We showed that plum and peach genes are independent and, thus, can be pyramided into interspecific hybrid rootstocks based on the plum and peach species.

Differential response to root-knot nematodes in prunus species and correlative genetic implications.

Responses of 17 Prunus rootstocks or accessions (11 from the subgenus Amygdalus and 6 from the subgenus Prunophora) were evaluated against 11 isolates of Meloidogyne spp. including one M. arenaria, four M. incognita, four M. javanica, one M. hispanica, and an unclassified population from Florida. Characterization of plant response to root-knot nematodes was based on a gall index rating. Numbers of females and juveniles plus eggs in the roots were determined for 10 of the rootstocks evaluated against one M. arenaria, one M. incognita, one M. javanica, and the Florida isolate. These 10 rootstocks plus Nemaguard and Nemared were retested by growing three different rootstock genotypes together in containers of soil infested individually with each of the above four isolates. Garfi and Garrigues almonds, GF.305 and Rutgers Red Leaf peaches, and the peach-almond GF.677 were susceptible to all isolates. Differences in resistance were detected among the other rootstocks of the subgenus Amygdalus. The peach-almond GF.557 and Summergrand peach were resistant to M. arenaria and M. incognita but susceptible to M. javanica and the Florida isolate. Nemaguard, Nemared, and its two hybrids G x N no. 15 and G x N no. 22 were resistant to all but the Florida isolate. In the subgenus Prunophora, Myrobalan plums P.1079, P.2175, P.2980, and P.2984; Marianna plum 29C; and P. insititia plum AD.101 were resistant to all isolates. Thus, two different genetic systems of RKN resistance were found in the subgenus Amygdalus: one system acting against M. arenaria and M. incognita, and another system also acting against M. javanica. Prunophora rootstocks bear a complete genetic system for resistance also acting against the Florida isolate. The hypotheses on the relationships between these systems and the corresponding putative genes of resistance are presented.

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.

Effect of Cutting Age on the Resistance of Prunus cerasifera (Myrobalan Plum) to Meloidogyne arenaria.

The response of softwood cuttings of Myrobalan plum infested after 50 and 105 days with 3,000 second-stage juveniles (J2) of Meloidogyne arenaria was compared to 15-month-old hardwood cuttings in 13 genotypes ranging from highly resistant to susceptible. Gall index and number of galls were recorded 30 days after infestation. Fifty-day-old cuttings rooted in perlite developed many rootlets, but had only incipient galls after infestation. In sand, rooting of 50-day-old cuttings not treated with indolebutyric acid (IBA) hormone was so variable that their resistance could not be assessed. Similar cuttings rooted with IBA developed more galls, but neither number of galls per plant nor gall index was a reliable criterion for determination of host suitability. Because of the better rooting results with IBA treatment, 105-day-old cuttings were first rooted with IBA in perlite and then transferred into sand for nematode inoculation. Known highly resistant genotypes of Myrobalan plum were gall-free and the responses of other genotypes paralleled that of the reference hardwood cuttings, although the test was less discriminating. Expression of M. arenaria host suitability in Myrobalan plum depends on root tissue maturation and cannot be reliably evaluated with 50-day-old cuttings.

Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index.

The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.

Effects of Six Pratylenchus vulnus Isolates on the Growth of Peach-Almond Hybrid and Apple Rootstocks.

The effects of six geographic isolates of Pratylenchus vulnus on the growth of GF-677 peach-almond hybrid and M-26 apple rootstocks were determined under greenhouse conditions. Plantlets were obtained from micropropagated plant material, and nematode isolates were reared in monoxenic cultures. All isolates suppressed growth on GF-677 compared with the uninfected controls. Isolate PvRO-S from Spain affected top weight of GF-677 more adversely than PvAT-F from France. Final population densities (Pf) of all P. vulnus on GF-677 were greater than 14.7 times the initial densities (Pi). They increased 61.5-fold on plants infected by PvWA-U from the United States. PvWA-U-, PvAT-F, and geographic undetermined PvU-UK isolates did not affect the growth of M-26 apple rootstock compared with PvAP-S, PvRO-S (both from Spain), and PvWA-A from Argentina isolates, which severely suppressed shoot growth of this rootstock. On M-26, Pf of the more parasitically fit isolates PvWA-A, PvAP-S, and PvRO-S were greater than those of nondamaging PvWA-U, PvAT-F, and PvU-UK isolates (>41.4 vs. <14.7 times the Pi). PvWA-U and PvAT-F reproduced more on GF-677 than on M-26 (>28.6 vs. <6.5 times the Pi). Isolate PvRO-S reproduced well and was quite destructive on both rootstocks. Results confirm the existence of strains with different damage potentials among geographically separated populations of P. vulnus.