Characterization of the Xanthomonas translucens Complex Using Draft Genomes, Comparative Genomics, Phylogenetic Analysis, and Diagnostic LAMP Assays

Abstract

Prevalence of Xanthomonas translucens, which causes cereal leaf streak (CLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange and, thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion surrounding the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. Therefore, we sequenced genomes of 15 X. translucens strains representing six different pathovars and compared them with additional publicly available X. translucens genome sequences to obtain a genome-based phylogeny for robust classification of this species. Our results reveal three main clusters: one consisting of pv. cerealis, one consisting of pvs. undulosa and translucens, and a third consisting of pvs. arrhenatheri, graminis, phlei, and poae. Based on genomic differences, diagnostic loop-mediated isothermal amplification (LAMP) primers were developed that clearly distinguish strains that cause disease on cereals, such as pvs. undulosa, translucens, hordei, and secalis, from strains that cause disease on noncereal hosts, such as pvs. arrhenatheri, cerealis, graminis, phlei, and poae. Additional LAMP assays were developed that selectively amplify strains belonging to pvs. cerealis and poae, distinguishing them from other pathovars. These primers will be instrumental in diagnostics when implementing quarantine regulations to limit further geographic spread of X. translucens pathovars.

Complete Genome Sequencing and Targeted Mutagenesis Reveal Virulence Contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in Bacterial Leaf Streak of Wheat

“…Draft genome sequence assemblies for several X. translucens strains, including strains of Xtc, Xtt, Xtu, and others, have been published ( Wichmann et al, 2013 ; Pesce et al, 2015 ; Peng et al, 2016 ; Langlois et al, 2017 ), but, assembled from short reads, the assemblies do not include the repeat-rich, TALE-encoding sequences ( tal genes). Recently, a complete genome assembly from the U.S. Xtu strain XT4699 was generated using single molecule, real-time (SMRT) technology (Pacific Biosciences), revealing eight TALE genes, and sequences of several tal genes individually cloned from other Xtu strains were also obtained, but none was functionally characterized ( Peng et al, 2016 ).…”

The translucens group of Xanthomonas translucens: Complicated and important pathogens causing bacterial leaf streak on cereals

“…Vauterin et al (, ) proposed the re‐establishment of the species Xanthomonas translucens including strains that cause leaf streak on small grains and some grasses (the “translucens” group) and strains that cause bacterial wilt on forage grasses (the “graminis” group). This classification and nomenclature system has been supported by recent molecular and whole genome sequence data (Peng et al , ; Langlois et al , ; Hersemann et al , ). Genomic sequence data also suggest that the pathovars cerealis could be genetically separated from other translucens and graminis group pathovars.…”

“…Seed is considered an important source of primary inoculum, thus use of clean seed might be a way of reducing BLS incidence. Different methods have been developed to detect the bacterium in seeds, including dilution plating with the use of selective media, seedling infection assays, serodiagnostic assays, PCR amplification, and loop‐mediated isothermal amplification (Forster and Schaad, ; Bragard and Verhoyen, ; Maes et al , ; Langlois et al , ). Seed disinfection methods have also been developed to eliminate the bacterium by using chemical or physical methods (Atanasoff and Johnson, ; Forster et al , ).…”

“…In recent years, X. translucens taxonomy has been performed using multilocus sequence analysis with data from the housekeeping gene and/or genome sequences (Wichmann et al , ; Gardiner et al , ; Peng et al , ; Langosis et al ; Curland et al , ). The analyses with the DNA sequence data basically agreed with the previous classifications for X .…”

“…The analyses with the DNA sequence data basically agreed with the previous classifications for X. translucens at both species and pathovar levels determined by DNA-DNA hybridization and DNA marker analysis and pathogenicity tests . Evidence has also suggested that cerealis strains could be genetically separated from other translucens and graminis pathovars (Langlois et al, 2017). However, more cerealis strains need to be analysed in order to draw a solid conclusion.…”