The bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) have broad but finite host ranges. Population genetic surveys of RSSC pathogens show that many sequevars (subspecies groups) are predominantly recovered from wilting solanaceous plants. In contrast, strains in the IIB-4 sequevar have been isolated from plants in over a dozen families. Certain IIB-4 lineages have been classified as banana-virulent or “not pathogenic to banana (NPB)”. Prior analysis suggested that the NPB lineage has diverged from the banana-virulent IIB-4 strains. To test this model, we analyzed the phenotypes and phylogeny of a diverse collection of 19 IIB-4 isolates. We used Illumina sequencing to assemble draft genomes of 12 new strains. Based on whole genome phylogenetic analysis, these IIB-4 strains clustered into five subclades. We quantified virulence of each strain on tomato, banana, melon, and impatiens plants. Overall, the virulence patterns correlated with phylogeny. Banana virulence was restricted to the 4/4 IIB-4D subclade (N=4/4 strains) and IIB-4E subclade (N=1/2 strains). Subclades IIB-4D and IIB-4E are sister sublcades and their closest relative, the IIB-4A-C subclade, lacked virulence on banana. Our data support a revised model in which banana virulence is an innovation within the IIB4D/E subclades.

Bacterial leaf streak, bacterial blight and black chaff caused by Xanthomonas translucens pathovars are major diseases affecting small grains. Xanthomonas translucens pv. translucens and X. translucens pv. undulosa are seedborne pathogens that cause similar symptoms on barley, but only X. translucens pv. undulosa causes bacterial leaf streak of wheat. Recent outbreaks of X. translucens have been a concern for wheat and barley growers in the Northern Great Plains; however, there are limited diagnostic tools for pathovar differentiation. We developed a multiplex PCR based on whole-genome differences to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. We validated the primers across different Xanthomonas and non-Xanthomonas strains. To our knowledge, these are the first multiplex PCR to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. These molecular tools will support disease management strategies enabling detection and pathovar incidence analysis of X. translucens.

Bacterial leaf streak (BLS) of barley is caused by the Gram-negative bacterial pathogen Xanthomonas translucens (Sapkota et al. 2020). In 2021, we observed multiple hill plots with BLS symptomatic plants in a barley stripe rust nursery in Vancouver, BC, Canada. We collected 29 leaf samples showing typical BLS symptoms (e.g. necrotic lesions; Fig. S1) and stored at 4 oC until bacterial isolation. Samples were surface-sterilized in 10% NaOCl for 20 sec and rinsed twice. About 1 cm2 of leaf tissue containing BLS characteristic lesions was macerated in 200 μL sterile H2O on a petri dish, incubated for 15 min, and 10 μl of the homogenates was streaked onto Wilbrink’s - Boric Acid - Cephalexin (WBC) agar medium. Plates were incubated at 28-30 oC for 48 hrs. Four single colonies were obtained: BC10-1-2a (USask BC10-2a), BC10-1-2b (USask BC10-2b), UBC026 and UBC028. Colonies were grown in WBC broth and gDNA was extracted using E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek) or DNeasy Plant Pro Kit® (Qiagen) following manufacturer protocols. Genus-level identification was achieved using 16S rRNA sequencing with 27F/1492R primers (Lane 1991) of UBC026 (1,399 bp; NCBI # OP327375) and UBC028 (1,415 bp; NCBI #OP327376). Complete 16S rRNA sequences (1,533bp) of BC10-2a and BC10-2b (1,533 bp) were extracted from the draft whole-genome sequences (WGS) generated in this study. The 16S rRNA sequence homology values of 99.0-100% were recorded between the 4 strains. BLAST analyses of the 16S rRNA sequences to GenBank entries exhibited 99.5-100% similarity values (100% coverage) with the pathotype strains of Xtt DSM 18974T (LT604072) and X. translucens pv. undulosa (Xtu) CFBP 2055 (CP074361). Whole genomes of BC10-2a (JANUQY01) and BC10-2b (JANUQZ01) were sequenced (150-bp; reads 33.1 million; mean coverage 2125x) using NovaSeq Illumina, assembled (Unicycler v0.4.8; Wick et al. 2017) and analyzed to identify the strains to the species-level (Tambong et al. 2021). WGS of strains USask BC10-2a and USask BC10-2b exhibited genome-based DNA-DNA hybridization (dDDH; Meier-Kolthoff et al. 2013) and BLAST-based average nucleotide identity (ANIb; Richter et al. 2015) of 100%. The two strains also showed dDDH and ANIb of 90.4% (species-leel cut-off of 70%) and 98.780% and 98.80% (cut-off of 96%), respectively, with Xtt DSM 18974T (LT604072). In contrast, the WGS of BC10-2a and BC10-2b exhibited only 78.2% dDDH homology values with Xtu CFBP 2055T, suggesting that the strains are genetically more similar to Xtt. The assignment of these strains to Xtt is corroborated by phylogenomic analysis (Fig. S2; Meier-Kolthoff and Göker 2019) that showed the two strains clustering together (100% bootstrap) with the type strain DSM 18974T. These data suggest that these strains are taxonomically members of Xtt. Identification was also confirmed to the genus-level by LAMP assay using published X. translucens primers (Langlois et al. 2017). Pathovar-level identification was confirmed using a cbsA and S8.pep multiplex PCR diagnostic assay (Roman-Reyna et al. 2022). Koch’s postulates were verified by greenhouse inoculation via leaf infiltration of UBC026 and UBC028 on 21-day old barley plants (line HB522) using an inoculum of 108 CFU ml-1 followed by re-isolation of the bacteria on WBC. The inoculated plants showed typical BLS symptoms similar to those observed in the field (Fig. S1). Water-inoculated plants had no symptoms. To our knowledge, this is the first published report of BLS of barley in British Columbia.

The bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) have broad but finite host ranges. Population genetic surveys of RSSC pathogens show that many sequevars (subspecies groups) are predominantly recovered from wilting solanaceous plants. In contrast, strains in the IIB-4 sequevar have been isolated from plants in over a dozen families. We characterized the natural variation in host range of nineteen IIB-4 strains to explore the molecular determinants of host range within the RSSC. We used Illumina sequencing to assemble draft genomes of 12 strains. Based on whole genome phylogenetic analysis, these IIB-4 strains cluster into five subclades. We quantified virulence of each strain on tomato cv. Moneymaker (Solanaceae), banana cv. Dwarf Cavendish (Musaceae), melon cv. Sweet Granite (Cucurbitaceae), and impatiens cv. Beacon Orange (Balsaminaceae). To enable future meta-analyses that identify genetic factors that drive host-range, the raw virulence data is included as a supplemental table. Overall, the virulence patterns correlated with phylogeny. Strains from Martinique, Dominican Republic, Brazil, as well as multiple strains imported into Florida were highly virulent on tomato, melon, and impatiens. Several strains from Peru were highly virulent on tomato and had moderate-to-low virulence on banana, melon, and impatiens. One strain from Colombia was highly virulent on all hosts. Our findings reinforce the phenotypic plasticity within the Ralstonia solanacearum species complex.

Bacterial leaf streak, blight, and black chaff caused by Xanthomonas translucens pathovars are major diseases affecting small grains. Xanthomonas translucens pv. translucens and X. translucens pv. undulosa are seedborne pathogens that cause similar symptoms on barley, but only X. translucens pv. undulosa causes bacterial leaf streak of wheat. Recent outbreaks of X. translucens have been a concern for wheat and barley growers in the Northern Great Plains and Upper Midwest; however, there are limited diagnostic tools for pathovar differentiation. We developed a multiplex PCR based on whole-genome differences to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. We validated the primers across different Xanthomonas and non-Xanthomonas strains. To our knowledge, these are the first multiplex PCR to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. These molecular tools will support disease management strategies enabling detection and pathovar incidence analysis of X. translucens.