Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.
We report the sequence of 41 primer pairs of microsatellites from a CT-enriched genomic library of the peach cultivar 'Merrill O'Henry'. Ten microsatellite-containing clones had sequences similar to plant coding sequences in databases and could be used as markers for known functions. For microsatellites segregating at least in one of the two Prunus F(2) progenies analyzed, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was evaluated in 27 peach and 21 sweet cherry cultivars. All primer pairs gave PCR-amplification products on peach and 33 on cherry (80.5%). Six PCR-amplifications revealed several loci (14.6%) in peach and eight (19.5%) in sweet cherry. Among the 33 single-locus microsatellites amplified in peach and sweet cherry, 13 revealed polymorphism both in peach and cherry, 19 were polymorphic only on peach and one was polymorphic only on cherry. The number of alleles per locus ranged from 1 to 9 for peach and from 1 to 6 on sweet cherry with an average of 4.2 and 2.8 in peach and sweet cherry, respectively. Cross-species amplification was tested within the Prunus species: Prunus avium L. (sweet cherry and mazzard), Prunus cerasus L. (sour cherry), Prunus domestica L. (European plum), Prunus amygdalus Batsch. (almond), Prunus armeniaca L. (apricot), Prunus cerasifera Ehrh. (Myrobalan plum). Plants from other genera of the Rosaceae were also tested: Malus (apple) and Fragaria (strawberry), as well as species not belonging to the Rosaceae: Castanea (chestnut tree), Juglans (walnut tree) and Vitis (grapevine). Six microsatellites gave amplification on all the tested species. Among them, one had an amplified region homologous to sequences encoding a MADS-box protein in Malus x domestica. Twelve microsatellites (29.3%) were amplified in all the Rosaceae species tested and 31 (75.6%) were amplified in all the six Prunus species tested. Thirty three (80.5%), 18 (43.9%) and 13 (31.7%) gave amplification on chestnut tree, grapevine and walnut tree, respectively.
An improved genetic linkage map was constructed from a peach Ferjalou Jalousia ® × Fantasia (J×F) F 2 population. Ferjalou Jalousia ® is a flat low-acidity clingstone peach, and Fantasia is a round, normally acidic freestone peach. This population is segregated for six Mendelian characters: pollen sterility, peach or nectarine fruit, flat or round fruit, clingstone, or freestone fruit. It also segregates for the D major gene controlling the fruit's low acidity. A new character is reported here for the first time that segregates as a Mendelian character: trees bearing aborting fruits. These trees have flowers, but fruits start to fall 2 months after blooming. This recessive character has been named Af. We demonstrate that it is linked to the flat shape of the fruit. The previous map obtained from this cross was constructed using 63 individuals, whereas the present map was constructed using 207 individuals. Moreover, 82 simple-sequence repeat (SSR) markers, including 10 expressed sequence tag-SSRs, and 43 amplified fragment length polymorphism (AFLP) markers were added. Molecular markers linked to the six Mendelian characters were identified, and one of them has already been used for marker-assisted selection. This map will be used for detection of quantitative trait loci controlling organoleptic and nutritional fruit quality in peach.
We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14␣-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, <5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.
Ninety isolates of grape powdery mildew (Uncinula necator) from Europe (sixty-two) and India (twenty-eight) were collected. Ten of the sixty-two European isolates originated from mycelium overwintering in dormant buds ("flagshoots"). Mating types were determined, and genetic variation was assessed by random amplified polymorphic DNA (RAPD). Forty-one European isolates, including all "flagshoot" isolates, were mating type +, and twenty-one were mating type -. All Indian isolates were mating type -. Phenetic analysis based on 414 amplicons revealed three main groups. Most European isolates (53) clustered together. Nine flagshoot isolates clustered in a second distinct group. These isolates, which coexisted with other isolates in the field, may represent a genetically isolated biotype of U. necator. Indian isolates clustered into two groups. The first group (15 isolates) was a subgroup of the group containing European nonflagshoot isolates. The second group (12 isolates) was distinct from the other groups. These two groups of Indian isolates may represent genetically isolated populations with different climatic tolerances. A polymerase chain reaction primer pair, derived from a RAPD fragment specific to the Indian isolates, proved to be suitable for field studies.
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