Pathovar-Specific PCR Method for Detection and Identification of Xanthomonas translucens pv. undulosa.

Bacterial leaf streak disease caused by Xanthomonas translucens pv. undulosa is an economically important disease threatening wheat and barley crops around the globe. Thus far, specific PCR-based detection and identification tests for X. translucens pathovars are not available. In this study, we used comparative genomics approach to design a pathovar-specific primer pair for detection of X. translucens pv. undulosa in naturally infected seeds and its differentiation from other pathovars of the species. For this aim, complete genome sequences of strains of different X. translucens pathovars were compared and the specific PCR primer pair XtuF/XtuR was designed. These primers were strictly specific to X. translucens pv. undulosa because the expected 229-bp DNA fragment was not amplified in the closely related pathovars or in other xanthomonads, wheat-pathogenic bacteria, and other plant-pathogenic bacteria. High sensitivity of the primer pair XtuF/XtuR allowed detection of pure DNA of the pathogen in a concentration as low as 4.5 pg/µl. The pathogen was also detected in water suspension at a concentration of 8.6 × 102 CFU/ml. The PCR test was capable of detecting the pathogen in extracts of naturally infected wheat seeds at a concentration of 3.5 × 104 CFU/g while a culture-plate method was able to detect the pathogen at a concentration of 50 × 105 CFU/g of the same seeds. The PCR test developed in this study is a step forward for precise detection and identification of X. translucens pv. undulosa to prevent outbreaks of the bacterial leaf streak disease.

Taxonomic Refinement of Xanthomonas arboricola.

Xanthomonas arboricola comprises a number of economically important fruit tree pathogens classified within different pathovars. Dozens of nonpathogenic and taxonomically unvalidated strains are also designated as X. arboricola, leading to a complicated taxonomic status in the species. In this study, we have evaluated the whole-genome resources of all available Xanthomonas spp. strains designated as X. arboricola in the public databases to refine the members of the species based on DNA similarity indexes and core genome-based phylogeny. Our results show that, of the nine validly described pathovars within X. arboricola, pathotype strains of seven pathovars are taxonomically genuine, belonging to the core clade of the species regardless of their pathogenicity on the host of isolation (thus the validity of pathovar status). However, strains of X. arboricola pv. guizotiae and X. arboricola pv. populi do not belong to X. arboricola because of the low DNA similarities between the type strain of the species and the pathotype strains of these two pathovars. Thus, we propose to elevate the two pathovars to the rank of a species as X. guizotiae sp. nov. with the type strain CFBP 7408T and X. populina sp. nov. with the type strain CFBP 3123T. In addition, other mislabeled strains of X. arboricola were scattered within Xanthomonas spp. that belong to previously described species or represent novel species that await formal description.

Complete Genome Sequencing Provides Novel Insight Into the Virulence Repertories and Phylogenetic Position of Dry Beans Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens.

Bacterial wilt of dry beans (family Fabaceae) caused by the actinobacterial agent Curtobacterium flaccumfaciens pv. flaccumfaciens is one of the most important diseases threatening edible legume production around the globe. Despite the economic losses due to the bacterial wilt disease, the pathogen has not so far been investigated for its genomic features, pathogenicity determinants, and virulence strategies. Here we present the first complete genome sequence of a highly virulent bacteriocin-producing C. flaccumfaciens pv. flaccumfaciens strain P990. The bacterium has a circular chromosome consisting of 3,736 kbp with the G+C% content of 71.0%. Furthermore, a 147-kbp circular plasmid (pCff1) with 66.1% G+C% content as well as two circular plasmid-like DNAs with sizes of 25 and 22 kbp were detected within the genomic contents of C. flaccumfaciens pv. flaccumfaciens. Phylogenetic analyses revealed that only a few number of Curtobacterium sp. strains deposited in the public databases could be classified within the species C. flaccumfaciens. Comparative genomics of C. flaccumfaciens pv. flaccumfaciens using the genome sequences of actinobacterial plant pathogens revealed the presence of a set of unique low G+C% content genomic islands in the C. flaccumfaciens pv. flaccumfaciens genome. Homologs of pathogenicity-determinant loci capable of producing 1,4-beta-xylanase (xysA), pectate lyase (pelA1 and pelA2), serine protease (chpC, chpG, and pat-1), and sortase (srtA) were detected in C. flaccumfaciens pv. flaccumfaciens genome. The genomic data presented here extend our understanding of the C. flaccumfaciens pv. flaccumfaciens genomic features and pave the ways of research on functional and interaction genetics to combat the risk of bacterial wilt disease in the 21st century's dry bean industry.

Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within the Genus Clavibacter, Including Nonpathogenic Tomato-Associated Strains.

Members of the genus Clavibacter are economically important bacterial plant pathogens infecting a set of diverse agricultural crops (e.g., alfalfa, corn, potato, tomato, and wheat). Tomato-associated Clavibacter sp. strains account for a great portion of the genetic diversity of the genus, and C. michiganensissensu stricto (formerly C. michiganensis subsp. michiganensis), causing bacterial canker disease, is considered one of the most destructive seed-borne agents for the crop worldwide. However, current taxonomic descriptions of the genus do not reflect the existing diversity of the strains, resulting in unsatisfactory results in quarantine surveys for the pathogens. In this study, we used all the available genome sequences of Clavibacter sp. strains, including the type strains of newly described subspecies, to provide precise insight into the diversity of tomato-associated members of the genus and further clarify the taxonomic status of the strains using genotypic and phenotypic features. The results of phylogenetic analyses revealed the existence of nine hypothetical new species among the investigated strains. None of the three new subspecies (i.e., C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis, and C. michiganensis subsp. phaseoli) is included within the tomato-pathogenic C. michiganensissensu stricto lineage. Although comparative genomics revealed the lack of chp and tomA pathogenicity determinant gene clusters in the nonpathogenic strains, a number of pathogenicity-related genes were noted to be present in all the strains regardless of their pathogenicity characteristics. Altogether, our results indicate a need for a formal taxonomic reconsideration of tomato-associated Clavibacter sp. strains to facilitate differentiation of the lineages in quarantine inspections.IMPORTANCEClavibacter spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops, such as alfalfa, corn, pepper, potato, tomato, and wheat. A number of plant-pathogenic members of the genus (e.g., C. michiganensissensu stricto and C. sepedonicus, infecting tomato and potato plants, respectively) are included in the A2 (high-risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Although tomato-associated members of Clavibacter spp. account for a significant portion of the genetic diversity in the genus, only the strains belonging to C. michiganensissensu stricto (formerly C. michiganensis subsp. michiganensis) cause bacterial canker disease of tomato and are subjected to the quarantine inspections. Hence, discrimination between the pathogenic and nonpathogenic Clavibacter sp. strains associated with tomato seeds and transplants plays a pivotal role in the accurate detection and cost-efficient management of the disease. On the other hand, detailed information on the genetic contents of different lineages of the genus would lead to the development of genome-informed specific detection techniques. In this study, we have provided an overview of the phylogenetic and genomic differences between the pathogenic and nonpathogenic tomato-associated Clavibacter sp. strains. We also noted that the taxonomic status of newly introduced subspecies of C. michiganensis (i.e., C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis, and C. michiganensis subsp. phaseoli) should be reconsidered.

Genome annotation and comparative genomic analysis of Bacillus subtilis MJ01, a new bio-degradation strain isolated from oil-contaminated soil.

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.

Phenotypic and Molecular-Phylogenetic Analyses Reveal Distinct Features of Crown Gall-Associated Xanthomonas Strains.

In summer 2019, widespread occurrence of crown gall disease caused by Agrobacterium spp. was observed on commercially grown ornamental plants in southern Iran. Beside agrobacteria, pale yellow-pigmented Gram-negative strains resembling the members of Xanthomonas were also associated with crown gall tissues on weeping fig (Ficus benjamina) and Amaranthus sp. plants. The purpose of the present study was to characterize the crown gall-associated Xanthomonas strains using plant inoculation assays, molecular-phylogenetic analyses, and comparative genomics approaches. Pathogenicity tests showed that the Xanthomonas strains did not induce disease symptoms on their host of isolation. However, the strains induced hypersensitive reaction on tobacco, geranium, melon, squash, and tomato leaves via leaf infiltration. Multilocus sequence analysis suggested that the strains belong to clade IA of Xanthomonas, phylogenetically close to Xanthomonas translucens, X. theicola, and X. hyacinthi. Average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of the strains isolated in this study and reference Xanthomonas strains are far below the accepted thresholds for the definition of prokaryotic species, signifying that these strains could be defined as two new species within clade IA of Xanthomonas. Comparative genomics showed that the strains isolated from crown gall tissues are genetically distinct from X. translucens, as almost all the type III secretion system genes and type III effectors are lacking in the former group. The data obtained in this study provide novel insight into the breadth of genetic diversity of crown gall-associated bacteria and pave the way for research on gall-associated Xanthomonas-plant interactions. IMPORTANCE Tumorigenic agrobacteria-members of the bacterial family Rhizobiaceae-cause crown gall and hairy root diseases on a broad range of plant species. These bacteria are responsible for economic losses in nurseries of important fruit trees and ornamental plants. The microclimate of crown gall and their accompanying microorganisms has rarely been studied for the microbial diversity and population dynamics of gall-associated bacteria. Here, we employed a series of biochemical tests, pathogenicity assays, and molecular-phylogenetic analyses, supplemented with comparative genomics, to elucidate the biological features, taxonomic position, and genomic repertories of five crown gall-associated Xanthomonas strains isolated from weeping fig and Amaranthus sp. plants in Iran. The strains investigated in this study induced hypersensitive reactions (HR) on geranium, melon, squash, tobacco, and tomato leaves, while they were nonpathogenic on their host of isolation. Phylogenetic analyses and whole-genome-sequence-based average nucleotide identity (ANI)/digital DNA-DNA hybridization (dDDH) calculations suggested that the Xanthomonas strains isolated from crown gall tissues belong to two taxonomically unique clades closely related to the clade IA species of the genus, i.e., X. translucens, X. hyacinthi, and X. theicola.