Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa × J. regia hybrids.

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut (Juglans regia) production worldwide. Crown gall, caused by Agrobacterium tumefaciens, and Phytophthora root and crown rots, caused by various Phytophthora spp., are among the most devastating walnut soil-borne diseases. A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks. Here, resistance to A. tumefaciens, P. cinnamomi, and P. pini is mapped in the genome of Juglans microcarpa, a North American wild relative of cultivated walnut. Half-sib J. microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J. regia cv. Serr, producing 353 and 400 hybrids, respectively. Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens. Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J. microcarpa chromosome 4D and was associated with the same haplotype, designated as haplotype b, raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens. The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

Complete Genome Sequences of Brenneria rubrifaciens Strain 6D370 and Brenneria nigrifluens Strain ATCC 13028, Causative Agents of Bark Cankers in Walnut.

Brenneria rubrifaciens and Brenneria nigrifluens, respectively, cause deep and shallow bark canker disease in walnut. B. rubrifaciens exhibits quorum sensing-controlled virulence and rubrifacine pigment production. The complete genome sequences of these species will be useful for studying the role of genes regulated by quorum sensing, including pathways mediating pathogenesis.

Draft Genome Sequence of Agrobacterium tumefaciens Biovar 1 Strain 186, Isolated from Walnut.

Agrobacterium tumefaciens biovar 1 strain 186 was isolated from a walnut tree expressing crown gall symptoms. The draft genome sequence of this strain harbored genes for crown gall formation and will be useful for understanding its virulence on Paradox, the predominant hybrid rootstock used for the cultivation of English walnut in California.

AHL signals induce rubrifacine production in a bruI mutant of Brenneria rubrifaciens.

Several members of the bacterial genus Brenneria are pathogenic on different tree species. Cell-free extracts from the bacterial phytopathogens Brenneria rubrifaciens, B. salicis, and B. nigrifluens induced production of the red pigment rubrifacine in the B. rubrifaciens bruI insertional mutant Br-212. Analysis of the bruI locus identified an adjacent open reading frame, designated bruR, with homology to luxR. High-performance liquid chromatography and mass spectrometry analysis of ethyl acetate extracts from wild-type B. rubrifaciens and Escherichia coli expressing the bruI gene identified two acyl homoserine lactone (AHL) peaks, N-(3-oxohexanoyl)-homoserine lactone (3OC6HSL) and N-hexanoyl-homoserine lactone (C6HSL). Addition of synthetic 3OC6HSL and C6HSL at 10 µM to the bruI mutant, strain Br-212, induced rubrifacine production and the ability to elicit a hypersensitive reaction (HR) in tobacco leaves. Synthetic C6HSL was less effective at inducing pigment production than 3OC6HSL at 10 µM. The bruI mutant Br-212 did not produce detectable AHLs, indicating that C6HSL and 3OC6HSL are the major AHLs produced by this species. The AHLs N-heptanoyl-DL-homoserine lactone (C7HSL), N-octanoyl-DL-homoserine lactone (C8HSL), and N-(3-oxooctanoyl)-DL-homoserine lactone (3OC8HSL) also induced pigment production in Br-212 and restored its ability to elicit an HR in tobacco, suggesting that cross-talk with other bacterial species may be possible.

Genetic loci involved in rubrifacine production in the walnut pathogen Brenneria rubrifaciens.

Brenneria rubrifaciens produces a unique red pigment known as rubrifacine that has been hypothesized to play a role in pathogenesis on walnut. Analysis of DNA flanking the Tn5 insertion site in 20 rubrifacine minus (pig(-)) mutants identified three regions required for rubrifacine production. The first region was homologous to nonribosomal peptide synthetases (NRPS), the second was homologous to autoinducer synthase genes (expI homologs), and the third region was homologous to the slyA gene of Candidatus blochmania and Escherichia coli. Pigment production was not necessary for elicitation of the hypersensitive response (HR) in tobacco and had little impact on virulence in tissue-cultured walnut plants. The expI-interrupted mutants exhibited reduced virulence on walnut and were HR negative on tobacco. Pigment production was restored in Br-212 when grown in the presence of wild-type B. rubrifaciens, E. coli carrying the cloned expI-like gene, or introduction of the cloned wild-type copy of the expI-like gene. Two Brenneria spp., B. nigrifluens and B. salicis, also restored pigment production in Br-212. These results demonstrate that rubrifacine production and virulence of B. rubrifaciens on walnut are under the control of a quorum-sensing system and are sensitive to signal molecules from other Brenneria spp.