DNA Markers, Pathogenicity Test, and Multilocus Sequence Analysis to Differentiate and Characterize Cereal-Specific Xanthomonas translucens Strains.

Xanthomonas translucens contains a group of bacterial pathogens that are closely related and have been divided into several pathovars based on their host range. X. translucens pv. undulosa (Xtu) and X. translucens pv. translucens (Xtt) are two important pathovars that cause bacterial leaf streak disease on wheat and barley, respectively. In this study, DNA markers were developed to differentiate Xtu and Xtt and were then used to characterize a collection of X. translucens strains with diverse origins, followed by confirmation and characterization with pathogenicity tests and multilocus sequence analysis/typing (MLSA/MLST). We first developed cleaved amplified polymorphic sequence markers based on the single-nucleotide polymorphisms within a cereal pathovar-specific DNA sequence. In addition, two Xtt-specific markers, designated Xtt-XopM and Xtt-SP1, were developed from comparative genomics among the sequenced Xtt/Xtu genomes. Using the developed markers, a collection of X. translucens strains were successfully identified as Xtu or Xtt. Pathogenicity tests on wheat and barley plants and MLSA of four housekeeping genes validated the pathovar assignation of those strains. Furthermore, MLSA revealed distinct subclades within both Xtu and Xtt groups. Seven and three sequence types were identified from MLST for Xtu and Xtt strains, respectively. The establishment of efficient Xtt/Xtu differentiation methods and characterization of those strains will be useful in studying disease epidemiology and host-pathogen interactions and breeding programs when screening for sources of resistance for these two important bacterial pathogens.

Comparative Genomics of Xanthomonas translucens pv. undulosa Strains Isolated from Weedy Grasses and Cultivated Wild Rice.

Bacterial leaf streak (BLS) of wheat (Triticum aestivum), caused by Xanthomonas translucens pv. undulosa, is a disease of major concern in the Northern Great Plains. The host range for X. translucens pv. undulosa is relatively broad, including several small grains and perennial grasses. In Minnesota, X. translucens pv. undulosa was isolated from weedy grasses in and around wheat fields that exhibited BLS symptoms and from cultivated wild rice (Zizania palustris) with symptomatic leaf tissue. Currently, no genomic resources are available for X. translucens pv. undulosa strains isolated from non-wheat hosts. In this study, we sequenced and assembled the complete genomes of five strains isolated from weedy grass hosts, foxtail barley (Hordeum jubatum), green foxtail (Setaria viridis), and wild oat (Avena fatua), and from cultivated wild rice and wheat. These five genomes were compared with the publicly available genomes of seven X. translucens pv. undulosa strains originating from wheat and one genome of an X. translucens pv. secalis strain originating from rye (Secale cereale). Global alignments of the genomes revealed little variation in genomic structures. Average nucleotide identity-based phylogeny and life identification numbers revealed that the strains share ≥99.25% identity. We noted differences in the presence of Type III secreted effectors, including transcription activator-like effectors. Despite differences between strains, we did not identify unique features distinguishing strains isolated from wheat and non-wheat hosts. This study contributes to the availability of genomic data for X. translucens pv. undulosa from non-wheat hosts, thus increasing our understanding of the diversity within the pathogen population.

Genome-Wide Association Mapping of Bacterial Leaf Streak Resistance in Two Elite Barley Breeding Panels.

Bacterial leaf streak (BLS), caused chiefly by the pathogen Xanthomonas translucens pv. translucens, is becoming an increasingly important foliar disease of barley in the Upper Midwest. The deployment of resistant cultivars is the most economical and practical method of control. To identify sources of BLS resistance, we evaluated two panels of breeding lines from the University of Minnesota (UMN) and Anheuser-Busch InBev (ABI) barley improvement programs for reaction to strain CIX95 in the field at St. Paul and Crookston, MN, in 2020 and 2021. The percentage of resistant lines in the UMN and ABI panels with mid-season maturity was 1.8% (6 of 333 lines) and 5.2% (13 of 251 lines), respectively. Both panels were genotyped with the barley 50K iSelect SNP array, and then a genome-wide association study was performed. A single, highly significant association was identified for BLS resistance on chromosome 6H in the UMN panel. This association was also identified in the ABI panel. Seven other significant associations were detected in the ABI panel: two each on chromosomes 1H, 2H, and 3H and one on chromosome 5H. Of the eight associations identified in the panels, five were novel. The discovery of resistance in elite breeding lines will hasten the time needed to develop and release a BLS-resistant cultivar.

Epidemiology, Host Resistance, and Genomics of the Small Grain Cereals Pathogen Xanthomonas translucens: New Advances and Future Prospects.

Bacterial leaf streak (BLS) primarily affects barley and wheat and is mainly caused by the pathogens Xanthomonas translucens pv. translucens and X. translucens pv. undulosa, respectively. BLS is distributed globally and poses a risk to food security and the supply of malting barley. X. translucens pv. cerealis can infect both wheat and barley but is rarely isolated from these hosts in natural infections. These pathogens have undergone a confusing taxonomic history, and the biology has been poorly understood, making it difficult to develop effective control measures. Recent advancements in the ability and accessibility to sequence bacterial genomes have shed light on phylogenetic relationships between strains and identified genes that may play a role in virulence, such as those that encode Type III effectors. In addition, sources of resistance to BLS have been identified in barley and wheat lines, and ongoing efforts are being made to map these genes and evaluate germplasm. Although there are still gaps in BLS research, progress has been made in recent years to further understand epidemiology, diagnostics, pathogen virulence, and host resistance.

Comparative Genomics of Barley-Infecting Xanthomonas translucens Shows Overall Genetic Similarity but Globally Distributed Virulence Factor Diversity.

Xanthomonas translucens pv. translucens (Xtt) is a global barley patho-gen and a concern for resistance breeding and regulation. Long-read whole genome sequences allow in-depth understanding of pathogen diversity. We have completed long-read PacBio sequencing of two Minnesotan Xtt strains and an in-depth analysis of available Xtt genomes. We found that average nucleotide identity (ANI)-based approaches organize Xtt strains different from the previous standard multilocus sequencing analysis approach. According to ANI, Xtt forms a separate clade from X. translucens pv. undulosa and consists of three main groups which are represented on multiple continents. Some virulence factors, such as 17 Type III-secreted effectors, are highly conserved and offer potential targets for the elicitation of broad resistance. However, there is a high degree of variation in virulence factors, meaning that germplasm should be screened for resistance with a diverse panel of Xtt.

Development of Genome-Driven, Lifestyle-Informed Markers for Identification of the Cereal-Infecting Pathogens Xanthomonas translucens Pathovars undulosa and translucens.

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, this is 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. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Sources of Bacterial Leaf Streak Resistance Identified in a Diverse Collection of Barley Germplasm.

Bacterial leaf streak (BLS) is a sporadic yet damaging disease of cereals that is growing in importance across the Upper Midwest production region. In barley (Hordeum vulgare ssp. vulgare), this disease is caused primarily by the bacterium Xanthomonas translucens pv. translucens. Accessions resistant to BLS have been reported in past studies, but few have been rigorously validated in the field. To identify accessions carrying diverse resistance alleles to BLS, a largescale germplasm screening study was undertaken against strain CIX95 of X. translucens pv. translucens in St. Paul and Crookston, Minnesota, in 2020 and 2021. The germplasm screened was diverse and included adapted breeding lines from two improvement programs, two landrace panels (one global and one from Ethiopia/Eritrea), introgression lines from wild barley (H. vulgare ssp. spontaneum) in the genetic background of barley cultivar 'Rasmusson', and an assemblage of accessions previously reported to carry BLS resistance. Of the 2,094 accessions evaluated in this study, 32 (1.5%) exhibited a consistently high level of resistance across locations and years and had heading dates similar to standard cultivars grown in the region. Accessions resistant to BLS were identified from all germplasm panels tested, providing genetically diverse sources for barley improvement programs focused on breeding for resistance to this important bacterial disease.

Genetic and Phenotypic Characterization of Xanthomonas Species Pathogenic in Wheat in Uruguay.

Bacterial diseases affecting wheat production in Uruguay are an issue of growing concern yet remain largely uninvestigated in the region. Surveys of 61 wheat fields carried out from 2017 to 2019 yielded a regional collection of 63 strains identified by 16S rRNA gene analysis as Xanthomonas spp. A real-time PCR protocol with species-specific primers previously reported allowed the identification of 44 strains as X. translucens, the causal agent of bacterial leaf streak (BLS) in wheat and other cereal crops. Multilocus sequence analysis of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD) revealed that these strains were most closely related to X. translucens pv. undulosa, the pathovar that is most commonly associated with BLS of wheat. Multilocus sequence typing was applied to examine the genetic diversity of X. translucens strains. Strains were assigned to four different sequence types, three of which were previously reported globally. Additionally, 17 Xanthomonas strains not belonging to X. translucens were obtained from diseased wheat leaves. Phylogenetic analysis showed that these strains are closely related to X. prunicola and clustered together with previously uncharacterized Xanthomonas strains isolated from wheat in Minnesota. In planta pathogenicity assays carried out on a BLS-susceptible wheat cultivar showed that X. translucens pv. undulosa strains caused brown necrosis symptoms typical of BLS, whereas non-translucens Xanthomonas sp. strains elicited an atypical symptom of dry necrosis. These findings suggest that local wheat fields are affected by X. translucens pv. undulosa and by a new wheat pathogen within the Xanthomonas genus.

Species of Dickeya and Pectobacterium Isolated during an Outbreak of Blackleg and Soft Rot of Potato in Northeastern and North Central United States.

An outbreak of bacterial soft rot and blackleg of potato has occurred since 2014 with the epicenter being in the northeastern region of the United States. Multiple species of Pectobacterium and Dickeya are causal agents, resulting in losses to commercial and seed potato production over the past decade in the Northeastern and North Central United States. To clarify the pathogen present at the outset of the epidemic in 2015 and 2016, a phylogenetic study was made of 121 pectolytic soft rot bacteria isolated from symptomatic potato; also included were 27 type strains of Dickeya and Pectobacterium species, and 47 historic reference strains. Phylogenetic trees constructed based on multilocus sequence alignments of concatenated dnaJ, dnaX and gyrB fragments revealed the epidemic isolates to cluster with type strains of D. chrysanthemi, D. dianthicola, D. dadantii, P. atrosepticum, P. brasiliense, P. carotovorum, P. parmentieri, P. polaris, P. punjabense, and P. versatile. Genetic diversity within D. dianthicola strains was low, with one sequence type (ST1) identified in 17 of 19 strains. Pectobacterium parmentieri was more diverse, with ten sequence types detected among 37 of the 2015-2016 strains. This study can aid in monitoring future shifts in potato soft rot pathogens within the U.S. and inform strategies for disease management.

First report of bacterial leaf streak caused by Xanthomonas translucens pv. undulosa on cultivated wild rice (Zizania palustris) in Minnesota.

Known by the indigenous peoples of the Great Lakes region of North America as Manoomin, wild rice (Zizania palustris) is a native aquatic grass that is honored and central to Anishinaabe culture. Cultivated wild rice, the domesticated form of this cereal bred primarily for resistance to shattering, is grown commercially in paddies. In this study we examined four isolates (CIX303, CIX306, Xt-8, and Xt-22) of Xanthomonas translucens, the causative agent of bacterial leaf streak (BLS) on cereals and weedy grasses, in molecular and host range studies to confirm the pathovar identity of strains associated with cultivated wild rice. Two of the strains examined (CIX303 and CIX306), were isolated from cultivated wild rice in 2016 as part of a survey of the pathogen in Minnesota (Ledman 2019). Xt-8 and Xt-22 are historical strains of X. translucens isolated from symptomatic wild rice leaves collected in Minnesota in the late 1970s that were reported at the time to be X. campestris pv. cerealis (Bowden and Percich 1982). A host range assay was repeated twice in the greenhouse, where two leaves of six seedlings each of hard red spring wheat (cv. RB07), spring barley (cv. Quest), spring rye (cv. Prolific), oat (cv. Ogle), quackgrass, smooth brome grass and cultivated wild rice (cv. Itasca Cycle-12) were inoculated via leaf infiltration (Curland et al. 2020). X. translucens pv. cerealis LMG 679PT, X. translucens pv. secalis LMG 883PT, X. translucens pv. translucens LMG 876T, and X. translucens pv. undulosa LMG 892PT were included as reference strains. Host response profiles were determined for each strain by recording character states five days post inoculation. Water-soaking and necrosis were considered pathogenic reactions, whereas chlorosis was not. Three pathotype strains, LMG 679PT, LMG 876T, and LMG 892PT, caused water-soaking in cultivated wild rice, whereas LMG 883PT caused chlorosis. All four strains from cultivated wild rice produced water-soaking on wheat, barley, quackgrass, and cultivated wild rice, chlorosis or water-soaking on rye, chlorosis on oat, and a reddish water-soaking on smooth brome. The character states generated by these four isolates were identical only to the host response profile for LMG 892PT. LMG 679PT differed, causing chlorosis on wheat, no symptoms on quackgrass, and water-soaking on smooth brome. A 2645 bp concatenation of housekeeping genes (rpoD, dnaK, fyuA, gyrB) was used to perform a Bayesian analysis (GenBank accessions MW528365-MW528384) (Curland et al. 2018, Curland et al. 2020, Young et al. 2008). Subsequent phylogenies grouped all four strains from cultivated wild rice with LMG 892PT and LMG 883PT. A pairwise comparison revealed 100% identity between Xt-22 and LMG 892PT. The percentage identity of CIX303, CIX308, and Xt-8 to LMG 892PT was 99.96, 99.96, and 99.92, respectively. In contrast, when compared to LMG 679PT, the four strains from cultivated wild rice had a percent identity between 97.43 and 97.50. Based on host range studies combined with MLSA, we identified recent and historical isolates from Z. palustris as X. translucens pv. undulosa. Pathovar identity of strains causing BLS on cultivated wild rice in Minnesota is crucial when screening breeding materials for disease resistance. Furthermore, given that X. translucens pv. undulosa has been prevalent on wheat in Minnesota (Curland et al. 2018), expanding knowledge of its host range to include cultivated wild rice may inform disease management practices for both crops. References: Bowden, R., and Percich, J. 1982. Phytopath. 73:640-645. Curland, R., et al. 2018. Phytopath. 108:443-453. Curland, R., et al. 2020. Phytopath. 110:257-266. Ledman, K. 2019. M.S. Thesis, Univ. of Minnesota, St. P. Paul, USA. Young, J., et al. 2008. Syst. Appl. Microbiol. 31:366-377.

Xanthomonas translucens pv. undulosa Identified on Common Weedy Grasses in Naturally Infected Wheat Fields in Minnesota.

Bacterial leaf streak (BLS) of wheat, caused by Xanthomonas translucens pv. undulosa, has been a notable disease in Minnesota wheat fields over the past decade. Potential sources of the pathogen include infested seed and crop debris. Perennial weeds are also considered a possible inoculum source, but no surveys have been conducted to evaluate which X. translucens pathovars are present on weedy grasses that are common in Minnesota wheat fields. Multilocus sequence analysis (MLSA) of four housekeeping genes (rpoD, dnaK, fyuA, and gyrB) was used to identify 77 strains isolated from six weedy grass species, wheat, and barley in and around naturally infected wheat fields in Minnesota. The MLSA phylogeny identified all strains originating from weedy grass species, except smooth brome, as X. translucens pv. undulosa, whereas strains isolated from smooth brome were determined to be X. translucens pv. cerealis. In planta character states corroborated these identifications on a subset of 41 strains, as all strains from weedy grasses caused water-soaking on wheat and barley in greenhouse assays. Multilocus sequence typing was used to evaluate genetic diversity and revealed that sequence types of X. translucens pv. undulosa originating from weedy grass hosts are similar to those found on wheat. This study identifies both annual and perennial poaceous weeds common in Minnesota that harbor X. translucens pv. undulosa and expands our understanding of the diversity of the pathogen population.

Localized Genetic and Phenotypic Diversity of Xanthomonas translucens Associated With Bacterial Leaf Streak on Wheat and Barley in Minnesota.

Bacterial leaf streak (BLS) of wheat and barley has been a disease of increasing concern in the Upper Midwest over the past decade. In this study, intra- and interfield genetic and pathogenic diversity of bacteria causing BLS in Minnesota was evaluated. In 2015, 89 strains were isolated from 100 leaf samples collected from two wheat and two barley fields naturally infected with BLS. Virulence assays and multilocus sequence alignments of four housekeeping genes supported pathovar identifications. All wheat strains were pathogenic on wheat and barley and belonged to the same lineage as the Xanthomonas translucens pv. undulosa-type strain. All barley strains were pathogenic on barley but not on wheat. Three lineages of barley strains were detected. The frequency and number of sequence types of each pathovar varied within and between fields. A significant population variance was detected between populations of X. translucens pv. undulosa collected from different wheat fields. Population stratification of X. translucens pv. translucens was not detected. Significant differences in virulence were detected among three dominant sequence types of X. translucens pv. undulosa but not those of X. translucens pv. translucens. Field trials with wheat and barley plants inoculated with strains of known sequence type and virulence did not detect significant race structures within either pathovar. Knowledge of virulence, sequence types, and population structures of X. translucens on wheat and barley can support studies on plant-bacterial interactions and breeding for BLS disease resistance.

Genetic Diversity and Virulence of Wheat and Barley Strains of Xanthomonas translucens from the Upper Midwestern United States.

Bacterial leaf streak (BLS) of wheat and barley, caused by Xanthomonas translucens pv. undulosa and X. translucens pv. translucens, has been of growing concern in small grains production in the Upper Midwestern United States. To optimize disease resistance breeding, a greater awareness is needed of the pathovars and genetic diversity within the pathogens causing BLS in the region. Multilocus sequencing typing (MLST) and analysis (MLSA) of four common housekeeping genes (rpoD, dnaK, fyuA, and gyrB) was used to evaluate the genetic diversity of 82 strains of X. translucens isolated between 2006 and 2013 from wheat, barley, rye, and intermediate wheatgrass. In addition, in planta disease assays were conducted on 75 strains to measure relative virulence in wheat and barley. All strains were determined by MLSA to be related to X. translucens pv. undulosa and X. translucens pv. translucens. Clustering of strains based on Bayesian, network, and minimum spanning trees correlated with relative virulence levels in inoculated wheat and barley. Thus, phylogeny based on rpoD, dnaK, fyuA, and gyrB correlated with host of isolation and was an effective means for predicting virulence of strains belonging to X. translucens pv. translucens and X. translucens pv. undulosa.