Molecular detection and quantification of Xanthomonas albilineans in juice from symptomless sugarcane stalks using a real-time quantitative PCR assay

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A very sensitive and robust qPCR assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis (PFGE) groups. The detection thresholds were 100 copies/µL of plasmid DNA, 100 fg/µL of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) symptomless stalk samples, whereas only 28.4% (155/546) samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi province) and Zhanjiang (Guangdong province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

Orthology-Based Estimate of the Contribution of Horizontal Gene Transfer from Distantly Related Bacteria to the Intraspecific Diversity and Differentiation of Xylella fastidiosa

Xylella fastidiosa is a xylem-limited bacterium phylogenetically related to the xanthomonads, with an unusually large and diversified range of plant hosts. To ascertain the origin of its peculiarities, its pan-genome was scanned to identify the genes that are not coherent with its phylogenetic position within the order Xanthomonadales. The results of the analysis revealed that a large fraction of the genes of the Xylella pan-genome have no ortholog or close paralog in the order Xanthomonadales. For a significant part of the genes, the closest homologue was found in bacteria belonging to distantly related taxonomic groups, most frequently in the Betaproteobacteria. Other species, such as Xanthomonas vasicola and Xanthomonas albilineans which were investigated for comparison, did not show a similar genetic contribution from distant branches of the prokaryotic tree of life. This finding indicates that the process of acquisition of DNA from the environment is still a relevant component of Xylella fastidiosa evolution. Although the ability of Xylella fastidiosa strains to recombine among themselves is well known, the results of the pan-genome analyses stressed the additional relevance of environmental DNA in shaping their genomes, with potential consequences on their phytopathological features.