Fine Mapping and Identification of Nucleotide Binding Site/Leucine-Rich Repeat Sequences at the MER Locus in Populus deltoides 'S9-2'.

ABSTRACT Melampsora larici-populina is the most damaging leaf pathogen for poplar in Europe. Previous genetic analyses have revealed both qualitative and quantitative resistance to this fungus. As a starting point for positional cloning of the gene or genes conferring qualitative resistance to M. larici-populina races E1, E2, and E3, a local genetic map of the Melampsora resistance (MER) locus was constructed based on amplified fragment length polymorphism (AFLP) markers. Eleven AFLP markers were identified by bulked segregant analysis. These markers were used to identify 17 recombinants at the MER locus, from a total of 512 progenies derived from three interspecific crosses involving the same resistant female parent, Populus deltoides 'S9-2'. The local genetic map covered a 3.4-centimorgan interval encompassing the target locus. Sequence analysis of these AFLP markers revealed similarities to the nucleotide binding site/leucine-rich repeat class of disease resistance genes.

Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar.

Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMT promoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula x Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions.

PCR-Based detection of the causal agent of watermark disease in willows (Salix spp.)

The watermark disease, caused by Brenneria salicis (formerly Erwinia salicis), is of significant concern wherever tree-forming willows are grown or occur naturally. The movement of infected, asymptomatic cuttings is a major cause of pathogen dispersal. A reliable and sensitive diagnostic procedure is necessary for the safe movement of willow planting material. We derived primers from the nucleotide sequence of the 16S rRNA gene of B. salicis for the development of a PCR to detect this pathogen. One set of primers, Es1a-Es4b, directed the amplification of a 553-bp fragment from B. salicis genomic DNA as well as B. salicis cells. PCR products were not observed when genomic DNA was tested for 27 strains of other, related plant-associated bacteria. Genomic fingerprinting by amplification fragment length polymorphism of B. salicis strains, originating from four different countries, and related Brenneria, Pectobacterium, and Erwinia strains revealed a very high similarity among the B. salicis genomes, indicating that the spread of the pathogen is mainly due to the transportation of infected cuttings. The PCR had to be preceded by a DNA extraction in order to detect the pathogen in the vascular fluid of willows. The minimum number of cells that could be detected from vascular fluid was 20 CFU/ml. The PCR assays proved to be very sensitive and reliable in detecting B. salicis in willow plant material.

Phylogenetic position of phytopathogens within the Enterobacteriaceae.

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrifluens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus.

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance (Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene.