High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance.

Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

Phylogenetic analysis of Xanthomonas based on partial rpoB gene sequences and species differentiation by PCR-RFLP.

The rpoB gene was evaluated as an alternative molecular marker for the differentiation of Xanthomonas species and in order to understand better the phylogenetic relationships within the genus. PCR-RFLP experiments using HaeIII allowed differentiation of Xanthomonas species, particularly those that affect the same plant host such as Xanthomonas albilineans and X. sacchari, pathogenic to sugar cane, Xanthomonas cucurbitae and X. melonis, which cause disease in melon, and Xanthomonas gardneri, X. vesicatoria and X. euvesicatoria/X. perforans, pathogenic to tomato. Phylogenetic relationships within the genus Xanthomonas were also examined by comparing partial rpoB gene sequences (612 nt) and the Xanthomonas species were separated into two main groups. Group I, well supported by bootstrap values of 99 %, comprised X. euvesicatoria, X. perforans, X. alfalfae, X. citri, X. dyei, X. axonopodis, X. oryzae, X. hortorum, X. bromi, X. vasicola, X. cynarae, X. gardneri, X. campestris, X. fragariae, X. arboricola, X. cassavae, X. cucurbitae, X. pisi, X. vesicatoria, X. codiaei and X. melonis. Group II, again well supported by bootstrap values of 99 %, comprised X. albilineans, X. sacchari, X. theicola, X. translucens and X. hyacinthi. The rpoB gene sequence similarity observed among the species in this study ranged from 87.8 to 99.7 %. The results of PCR-RFLP of the rpoB gene indicated that this technique can be used for diagnosis and identification of most Xanthomonas strains, including closely related species within the genus. However, species that showed identical profiles could be differentiated clearly only by sequence analysis. The results obtained in our phylogenetic analysis suggested that the rpoB gene can be used as an alternative molecular marker for genetic relatedness in the genus Xanthomonas. The results of PCR-RFLP of the rpoB gene indicate that this technique can be used for diagnosis and identification of closely related species within the genus, representing a rapid and inexpensive tool that can be easily standardized between laboratories.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii.

BACKGROUND: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. RESULTS: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. CONCLUSION: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.