Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance.

Here we report draft-quality genome sequences for pathotype strains of eight plant-pathogenic bacterial pathovars: Xanthomonas campestris pv. asclepiadis, X. campestris pv. cannae, X. campestris pv. esculenti, X. campestris pv. nigromaculans, X. campestris pv. parthenii, X. campestris pv. phormiicola, X. campestris pv. zinniae and X. dyei pv. eucalypti (= X. campestris pv. eucalypti). We also sequenced the type strain of species X. melonis and the unclassified Xanthomonas strain NCPPB 1067. These data will be useful for phylogenomic and taxonomic studies, filling some important gaps in sequence coverage of Xanthomonas phylogenetic diversity. We include representatives of previously under-sequenced pathovars and species-level clades. Furthermore, these genome sequences may be useful in elucidating the molecular basis for important phenotypes, such as biosynthesis of coronatine-related toxins and degradation of fungal toxin cercosporin.

First identification of Xanthomonas nasturtii as the cause of black rot of watercress in Hawaii.

Watercress (Nasturtium officinale) has been in continuous production in Hawaii for over a century and is part of the local diet. Black rot of watercress was first identified as caused by Xanthomonas nasturtii in Florida (Vicente et al., 2017), but symptoms of this disease have also been regularly observed in Hawaii production in all islands, mostly during the rainy season from December to April in areas with poor air circulation (McHugh & Constantinides, 2004). Initially, this disease was attributed to X. campestris due to similar symptoms to black rot of brassicas. Samples of watercress with symptoms that could be attributed to a bacterial disease including yellow spots and lesions on leaves and stunting and deformation of plants in more advanced stages, were collected from a farm in Aiea in the island of Oahu, Hawaii, in October 2017. Isolations were performed at the University of Warwick. Fluid from macerated leaves was streaked into plates of King's B (KB) medium and Yeast Dextrose Calcium Carbonate Agar (YDC). After 48-72 hrs incubation at 28°C, the plates showed a range of mixed colonies. Single cream-yellow mucoid colonies were sub-cultured several times and pure isolates including WHRI 8984 were stored at -76°C as previously described (Vicente et al., 2017). Colony morphology was observed in KB plates and, in contrast to the type strain from Florida (WHRI 8853 = NCPPB 4600), isolate WHRI 8984 did not cause browning of the medium. Pathogenicity was tested on four-week old watercress and Savoy cabbage cv. Wirosa F1 plants by inoculations on leaves as previously described (Vicente et al., 2017). WHRI 8984 did not produce symptoms when inoculated on cabbage but produced typical symptoms on watercress. A re-isolation from a leaf showing a V-shaped lesion, produced isolates with the same morphology, including isolate WHRI 10007A, that was also shown to be pathogenic to watercress therefore completing the Koch's postulates. Fatty acid profiling was performed on WHRI 8984 and 10007A and controls grown on trypticase soy broth agar (TSBA) plates at 28°C for 48 hrs as described by Weller et al. (2000). Profiles were compared with the RTSBA6 v6.21 library; as the database does not include X. nasturtii, the results were only interpreted at the genus level, and both isolates were shown to be Xanthomonas sp. For molecular analysis, DNA was extracted and the gyrB partial gene was amplified and sequenced as described by Parkinson et al. (2007). Comparisons with sequences available in the National Centre for Biotechnology Information (NCBI) databases using the Basic Local Alignment Search Tool (BLAST) showed that partial gyrB of WHRI 8984 and 10007A were identical to the type strain from Florida therefore confirming that they belong to X. nasturtii. For whole genome sequencing, genomic libraries for WHRI 8984 were prepared using Illumina's Nextera XT v2 kit and sequenced on a HiSeq Rapid Run flowcell. The sequences were processed as previously described (Vicente et al., 2017) and the whole genome assembly has been deposited in GenBank (accession QUZM00000000.1); the phylogenetic tree shows that WHRI 8984 is close, but not identical to the type strain. This is the first identification of X. nasturtii in watercress crops in Hawaii. Control of this disease generally involves the use of copper bactericides and minimizing moisture on leaves by reducing overhead irrigation and increasing air circulation (McHugh & Constantinides, 2004); seed testing might help to select batches that are disease free and, in longer term, breeding for disease resistance might produce cultivars that can be part of management strategies.

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas.

Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.

Complete Genome Sequence of Strain WHRI 3811 Race 1 of Xanthomonas campestris pv. campestris, the Causal Agent of Black Rot of Cruciferous Vegetables.

Xanthomonas campestris pv. campestris is an important bacterial pathogen that causes black rot and brings about enormous production loss for cruciferous vegetables worldwide. Currently, genome sequences for only a few X. campestris pv. campestris isolates are available, most of which are draft sequences. Based on the next-generation sequencing and single-molecule sequencing in real time technologies, we present here the complete genome sequence of strain WHRI 3811 race 1 of X. campestris pv. campestris, which is a type strain that has been extensively used. The genome data will contribute to our understanding of X. campestris pv. campestris genomic features and pave the way for research on X. campestris pv. campestris-host interactions.

Draft Genome Sequences of Pathotype Strains for Three Pathovars Belonging to Three Xanthomonas Species.

We present here the draft genome sequences of type/pathotype strains for three Xanthomonas species and pathovars with different host specificities, the Hedera helix L. pathogen Xanthomonas hortorum pv. hederae WHRI 7744 (NCPPB 939T), the rice pathogen X. oryzae pv. oryzicola WHRI 5234 (NCPPB 1585), and the cotton pathogen X. citri subsp. malvacearum WHRI 5232 (NCPPB 633).

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.

Whole-Genome Re-Alignment Facilitates Development of Specific Molecular Markers for Races 1 and 4 of Xanthomonas campestris pv. campestris, the Cause of Black Rot Disease in Brassica oleracea.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a seed borne disease of Brassicaceae. Eleven pathogenic races have been identified based on the phenotype interaction pattern of differential brassica cultivars inoculated with different strains. Race 1 and 4 are the two most frequent races found in Brassica oleracea crops. In this study, a PCR molecular diagnostic tool was developed for the identification of Xcc races 1 and 4 of this pathogen. Whole genomic sequences of races 1, 3, 4 and 9 and sequences of three other Xanthomonas pathovars/species (X. campestris pv. incanae (Xci), X. campestris pv. raphani (Xcr) and X.euvesicatoria (Xev) were aligned to identify variable regions among races. To develop specific markers for races 1 and 4, primers were developed from a region where sequences were dissimilar in other races. Sequence-characterized amplified regions (SCAR) and insertion or deletion of bases (InDel) were used to develop each specific set of primers. The specificity of the selected primers was confirmed by PCR tests using genomic DNA of seven different Xcc races, two strains of X. campestris pathovars and other species of bacteria. Bacterial samples of the races 1 and 4 isolates were collected from artificially inoculated cabbage leaves to conduct bio-PCR. Bio-PCR successfully detected the two Xcc isolates. By using our race-specific markers, a potential race 1 strain from the existing Korean Xcc collection was identified. The Xcc race 1 and 4-specific markers developed in this study are novel and can potentially be used for rapid detection of Xcc races through PCR.

Pathogenic, phenotypic and molecular characterisation of Xanthomonas nasturtii sp. nov. and Xanthomonas floridensis sp. nov., new species of Xanthomonas associated with watercress production in Florida.

We describe two new species of the genus Xanthomonas, represented by yellow mucoid bacterial strains isolated from diseased leaves of watercress (Nasturtium officinale) produced in Florida, USA. One strain was pathogenic on watercress, but not in other species including a range of brassicas; other strains were not pathogenic in any of the tested plants. Data from Biolog carbon source utilization tests and nucleotide sequence data from 16S and gyrB loci suggested that both pathogenic and non-pathogenic strains were related to, yet distinct from, previously described Xanthomonas species. Multilocus sequence analysis and whole genome-wide comparisons of the average nucleotide identity (ANI) of genomes of two strains from watercress showed that these are distinct and share less than 95 % ANI with all other known species; the non-pathogenic strain WHRI 8848 is close to Xanthomonascassavae (ANI of 93.72 %) whilst the pathogenic strain WHRI 8853 is close to a large clade of species that includes Xanthomonasvesicatoria (ANI ≤90.25 %). Based on these results, we propose that both strains represent new Xanthomonas species named Xanthomonas floridensis sp. nov. (type strain WHRI 8848=ATCC TSD-60=ICMP 21312=LMG 29665=NCPPB 4601) and Xanthomonas nasturtii sp. nov. (type strain WHRI 8853=ATCC TSD-61=ICMP 21313=LMG 29666=NCPPB 4600), respectively. The presence of non-pathogenic Xanthomonas strains in watercress and their interaction with pathogenic strains needs to be further investigated. Although the importance of the new pathogenic species is yet to be determined, the bacterial disease that it causes constitutes a threat to watercress production and its distribution should be monitored.

Genome-Wide Linkage and Association Mapping of Halo Blight Resistance in Common Bean to Race 6 of the Globally Important Bacterial Pathogen.

Pseudomonas syringae pv. phaseolicola (Psph) Race 6 is a globally prevalent and broadly virulent bacterial pathogen with devastating impact causing halo blight of common bean (Phaseolus vulgaris L.). Common bean lines PI 150414 and CAL 143 are known sources of resistance against this pathogen. We constructed high-resolution linkage maps for three recombinant inbred populations to map resistance to Psph Race 6 derived from the two common bean lines. This was complemented with a genome-wide association study (GWAS) of Race 6 resistance in an Andean Diversity Panel of common bean. Race 6 resistance from PI 150414 maps to a single major-effect quantitative trait locus (QTL; HB4.2) on chromosome Pv04 and confers broad-spectrum resistance to eight other races of the pathogen. Resistance segregating in a Rojo × CAL 143 population maps to five chromosome arms and includes HB4.2. GWAS detected one QTL (HB5.1) on chromosome Pv05 for resistance to Race 6 with significant influence on seed yield. The same HB5.1 QTL, found in both Canadian Wonder × PI 150414 and Rojo × CAL 143 populations, was effective against Race 6 but lacks broad resistance. This study provides evidence for marker-assisted breeding for more durable halo blight control in common bean by combining alleles of race-nonspecific resistance (HB4.2 from PI 150414) and race-specific resistance (HB5.1 from cv. Rojo).

R-gene variation across Arabidopsis lyrata subspecies: effects of population structure, selection and mating system.

BACKGROUND: Examining allelic variation of R-genes in closely related perennial species of Arabidopsis thaliana is critical to understanding how population structure and ecology interact with selection to shape the evolution of innate immunity in plants. We finely sampled natural populations of Arabidopsis lyrata from the Great Lakes region of North America (A. l. lyrata) and broadly sampled six European countries (A. l. petraea) to investigate allelic variation of two R-genes (RPM1 and WRR4) and neutral genetic markers (Restriction Associated DNA sequences and microsatellites) in relation to mating system, phylogeographic structure and subspecies divergence. RESULTS: Fine-scale sampling of populations revealed strong effects of mating system and population structure on patterns of polymorphism for both neutral loci and R-genes, with no strong evidence for selection. Broad geographic sampling revealed evidence of balancing selection maintaining polymorphism in R-genes, with elevated heterozygosity and diversity compared to neutral expectations and sharing of alleles among diverged subspecies. Codon-based tests detected both positive and purifying selection for both R-genes, as commonly found for animal immune genes. CONCLUSIONS: Our results highlight that combining fine and broad-scale sampling strategies can reveal the multiple factors influencing polymorphism and divergence at potentially adaptive genes such as R-genes.

Xanthomonas campestris pv. campestris (cause of black rot of crucifers) in the genomic era is still a worldwide threat to brassica crops.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson is a Gram-negative bacterium that causes black rot, the most important disease of vegetable brassica crops worldwide. Intensive molecular investigation of Xcc is gaining momentum and several whole genome sequences are available. TAXONOMY: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadacea; Genus Xanthomonas; Species X. campestris. HOST RANGE AND SYMPTOMS: Xcc can cause disease in a large number of species of Brassicaceae (ex-Cruciferae), including economically important vegetable Brassica crops and a number of other cruciferous crops, ornamentals and weeds, including the model plant Arabidopsis thaliana. Black rot is a systemic vascular disease. Typical disease symptoms include V-shaped yellow lesions starting from the leaf margins and blackening of the veins. RACE STRUCTURE, PATHOGENESIS AND EPIDEMIOLOGY: Collections of Xcc isolates have been differentiated into physiological races based on the response of several brassica species lines. Black rot is a seed-borne disease. The disease is favoured by warm, humid conditions and can spread rapidly from rain dispersal and irrigation water. DISEASE CONTROL: The control of black rot is difficult and relies on the use of pathogen-free planting material and the elimination of other potential inoculum sources (infected crop debris and cruciferous weeds). Major gene resistance is very rare in B. oleracea (brassica C genome). Resistance is more readily available in other species, including potentially useful sources of broad-spectrum resistance in B. rapa and B. carinata (A and BC genomes, respectively) and in the wild relative A. thaliana. GENOME: The reference genomes of three isolates have been released. The genome consists of a single chromosome of approximately 5 100 000 bp, with a GC content of approximately 65% and an average predicted number of coding DNA sequences (CDS) of 4308. IMPORTANT GENES IDENTIFIED: Three different secretion systems have been identified and studied in Xcc. The gene clusters xps and xcs encode a type II secretion system and xps genes have been linked to pathogenicity. The role of the type IV secretion system in pathogenicity is still uncertain. The hrp gene cluster encodes a type III secretion system that is associated with pathogenicity. An inventory of candidate effector genes has been assembled based on homology with known effectors. A range of other genes have been associated with virulence and pathogenicity, including the rpf, gum and wxc genes involved in the regulation of the synthesis of extracellular degrading enzymes, xanthan gum and lipopolysaccharides. USEFUL WEBSITE: http://www.xanthomonas.org/

Occurrence and Diversity of Xanthomonas campestris pv. campestris in Vegetable Brassica Fields in Nepal.

Black rot caused by Xanthomonas campestris pv. campestris was found in 28 sampled cabbage fields in five major cabbage-growing districts in Nepal in 2001 and in four cauliflower fields in two districts and a leaf mustard seed bed in 2003. Pathogenic X. campestris pv. campestris strains were obtained from 39 cabbage plants, 4 cauliflower plants, and 1 leaf mustard plant with typical lesions. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) using repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus, and BOX primers was used to assess the genetic diversity. Strains were also race typed using a differential series of Brassica spp. Cabbage strains belonged to five races (races 1, 4, 5, 6, and 7), with races 4, 1, and 6 the most common. All cauliflower strains were race 4 and the leaf mustard strain was race 6. A dendrogram derived from the combined rep-PCR profiles showed that the Nepalese X. campestris pv. campestris strains clustered separately from other Xanthomonas spp. and pathovars. Race 1 strains clustered together and strains of races 4, 5, and 6 were each split into at least two clusters. The presence of different races and the genetic variability of the pathogen should be considered when resistant cultivars are bred and introduced into regions in Nepal to control black rot of brassicas.

Integration of the cytogenetic and genetic linkage maps of Brassica oleracea.

We have assigned all nine linkage groups of a Brassica oleracea genetic map to each of the nine chromosomes of the karyotype derived from mitotic metaphase spreads of the B. oleracea var. alboglabra line A12DHd using FISH. The majority of probes were BACs, with A12DHd DNA inserts, which give clear, reliable FISH signals. We have added nine markers to the existing integrated linkage map, distributed over six linkage groups. BACs were definitively assigned to linkage map positions through development of locus-specific PCR assays. Integration of the cytogenetic and genetic linkage maps was achieved with 22 probes representing 19 loci. Four chromosomes (2, 4, 7, and 9) are in the same orientation as their respective linkage groups (O4, O7, O8, and O6) whereas four chromosomes (1, 3, 5, and 8) and linkage groups (O3, O9, O2, and O1) are in the opposite orientation. The remaining chromosome (6) is probably in the opposite orientation. The cytogenetic map is an important resource for locating probes with unknown genetic map positions and is also being used to analyze the relationships between genetic and cytogenetic maps.