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Average nucleotide identity based on average nucleotide identity (ANIb; lower diagonal) and digital DNA-DNA hybridization (dDDH; upper diagonal) values generated from the DNA sequence similarity comparisons among different sets of mislabeled Xanthomonas arboricola strains and the type/reference strains of Xanthomonas spp.
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Xanthomonas arboricola comprises a number of economically important fruit tree pathogens classified within different pathovars. Dozens of non-pathogenic and taxonomically un-validated 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...
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... ANI values between the former strains and the strain used to describe the proposed species X. cannabis were consistently above the accepted threshold for prokaryotic species definition (95 to 97%), in most of the cases, dDDH values between these two groups were on the border of the cutoff for species definition (68 to 69%), leading us to label the 11 X. arboricola strains in this clade as "X. cannabis"-like strains (Supplementary Table S3). The exception was strain CFBP 8594, which was undoubtedly identified as X. cannabis, sharing 97% ANI and 79% dDDH with the type strain of the species. ...Citations
... Genome-based data led Constantin et al. [33] to reclassify Xanthomonas perforans and Xanthomonas alfalfae as pathovars of X. euvesicatoria. Using whole-genome sequence data, Zarei et al. [34] refined the taxonomy of strains of X. arboricola pv. guizotiae and X. arboricola pv. ...
... In recent years, using whole genome data, several strains and species of the genus Xanthomonas have been taxonomically revised. For example, Constantin et al. [33] genetically characterized the X. axonopodis complex; and Zarei et al. [34] refined the taxonomy of Xanthomonas arboricola leading to the descriptions of 'X. populina' sp. ...
The pathovar-based taxonomy of the Xanthomonas translucens group is very confusing due to an overlap of plant host ranges and level of host specificity. Here, whole-genome sequence-based parameters (digital DNA–DNA hybridization and blast -based average nucleotide identity), phylogenomic, biochemical and phenotypical data were used to taxonomically analyse the 11 known pathovars of the X. translucens complex. This polyphasic approach taxonomically assigned the 11 pathovars of X. translucens complex into three distinct species, two of which are new: X. translucens , X. cerealis sp. nov. and X. graminis sp. nov. X. translucens consists of three pathovars: pv. translucens (=pv. hordei ), pv. pistaciae strain A ICMP 16316 PT and pv. undulosa (=pv. secalis ). X. cerealis sp. nov. encompasses the pv. cerealis strain LMG 679 PT and pv. pistaciae strain B ICMP 16317 PT with genome similarity of 92.7% (dDDH) and 99.0% (ANIb) suggesting taxonomically similar genotypes. The other new species, X. graminis sp. nov., consists of the remaining five designated pathovars (pv. graminis , pv. arrhenatheri, pv. poae , pv. phleipratensis and pv. phlei ) with highly variable dDDH and ANIb values ranging from 74.5 to 93.0% and from 96.7 to 99.2%, respectively, an indication of a very divergent taxonomic group. Only strains of pvs. phlei and phleipratensis showed the highest genomic similarities of 93.0% (dDDH) and 99.2% (ANIb), suggesting synonymic pathovars as both infect the same plant hosts. The dDDH and ANI data were corroborated by phylogenomics clustering. The fatty acid contents were similar but the type strain of X. graminis sp. nov. exhibited 20% less C 15 : 0 iso and 40% more C 17 : 0 iso fatty acids than the other species. Based on phenotypic, biochemical and whole-genome sequence data, we propose two new species, Xanthomonas cerealis sp. nov. and Xanthomonas graminis sp. nov. with type strains LMG 679 T (=NCPPB 1944 T ) and LMG 726 T (=NCPPB 2700 T ), respectively.
... In some preliminary phenotypic and genotypic studies using rep-PCR and multilocus sequence analysis (MLSA), the causative bacterium has been found to be affiliated with Xanthomonas arboricola and the accumulated data have been indicative of a relatively high genetic diversity of the inciting strains (Bafandeh et al., 2019;Ebrahimi et al., 2011;Rahimian et al., 2004). X. arboricola comprises pathogenic and non-pathogenic strains distributed in several pathovars Zarei et al., 2022). The most economically important pathovars of X. arboricola are pruni, corylina and juglandis, which affect stone fruit and nut trees and incite a range of symptoms, including canker, leaf spot, blight and fruit rot. ...
... Almost all aerial plant parts, including leaves, stems and fruits of some of the most important fruit and nut crops such as walnut, hazelnut and several Prunus species are affected (Agrios, 2005;Lamichhane, 2014;Zarei et al., 2022). Xanthomonas species and pathovars have been identified traditionally for decades by the symptomatology, host range and in some cases by DNA-DNA hybridization tests. ...
... The sequences of the ITS region of the rRNA operon and the MLSA based on the partial sequences of the housekeeping genes, . Currently, X. arboricola encompasses nine pathovars each exhibiting characteristic disease symptoms and distinct host specificities Zarei et al., 2022). ...
Caucasian alder ( Alnus subcordata ) is among the major constituents of the Hyrcanian forests extending all along the southern coast of the Caspian Sea in northern Iran. An angular leaf spot with a yellowish chlorotic halo has attained a widespread occurrence throughout the forest and suburban areas of the northern provinces including Guilan, Mazandaran and Golestan. Strains of a Xanthomonas sp. were consistently isolated from the symptomatic leaves of A. subcordata . Colonies of the isolates were yellow circular, convex and mucoid on nutrient agar containing glucose or sucrose. In multilocus sequence analysis using the genes gyrB , rpoD , dnaK , gltA , fyuA and gapA , the representative isolates occupied a clade shared by the known pathovars of Xanthomonas arboricola . The isolates were relatively heterogeneous phenotypically, but more so in their rep‐PCR fingerprints. Pathogenicity of several isolates was confirmed on seedlings of A . subcordata and Alnus rhombifolia , whereas Alnus cordata , Alnus glutinosa and Alnus crispa appeared not to be susceptible. Based on these characteristics, the isolates causing angular leaf spots of Caucasian alder appear to represent a novel pathovar of X. arboricola .
... Some pangenomics studies on certain species of Xanthomonas have been carried out to find out what makes strains more or less virulent. For instance, pan-genome tools have been addressed to aid in taxonomy revisions of X. arboricola, and also to find relationships between different lineages of Xanthomonas species based on the core genome [35][36][37]. Some effectors, such as TALEs and T3SEs, are present in most strains of X. citri pv. ...
Xanthomonas bacteria are known phytopathogens difficult to control in the field, which cause great losses in many economically important crops. Genomic islands are fragments acquired by horizontal transference that are important for evolution and adaptation to diverse ecological niches. Virulence and pathogenicity islands (PAIs) enhance molecular mechanisms related to host adaptation. In this work, we have analyzed 81 genomes belonging to X. campestris, and a complex group of X. citri, X. axonopodis, and X. fuscans belonging to nine different pathovars and three subspecies, to analyze and compare their genomic contents. Xanthomonas pan-genome is open and has a massive accessory genome. Each genome showed between three and 15 exclusive PAIs, well conserved through strains of the same pathovar or subspecies. X. axonopodis pv. anacardii had higher general similarity to X. citri subsp. citri and X. fuscans subsp. aurantifolii, with which a few PAIs were shared. Genomic synteny was even for almost all strains, with few rearrangements found in X. axonopodis pv. anacardii. The prophage regions identified in the genomes were mostly questionable or incomplete, and PAI13 in X. campestris pv. campestris ATCC33913 matched a prophage region of 19 transposable elements. Finally, PAIs in Xanthomonas are pathovar-specific, requiring individual strategies of combat.
... To address this issue and validate the results of phylogenetic analyses, the average nucleotide identity (ANI) index between the strain E4 and the type strain of E. americana CCUG 14506 T = ATCC 33852 T (GenBank accession number: NZ_JMPJ00000000.1) was calculated via the online service OrthoANI (https://www.ezbiocloud.net/tools/ani; Yoon et al. 2017) using the procedure detailed previously (Shah et al 2021;Zarei et al. 2022). Surprisingly, the strain E4 had only 81% ANI with the type strain of E. americana CCUG 14506 T , indicating that the strain E4 is mislabeled and does not belong to this species. ...
Mycopathogenic bacteria play a pivotal role in the productivity of edible mushrooms grown under controlled conditions. In this study, we carried out a comprehensive farm survey and sampling (2018-2021) on button mushroom (Agaricus bisporus) farms in 15 provinces in Iran to monitor the status of bacterial pathogens infecting the crop. Mycopathogenic bacterial strains were isolated from pins, stems, and caps, as well as the casing layer in 38 mushroom farms. The bacterial strains incited symptoms on mushroom caps ranging from faint discoloration to dark brown and blotch of the inoculated surfaces. Among the bacterial strains inciting disease symptoms on bottom mushroom, 40 strains were identified as Ewingella americana based on biochemical assays and phylogeny of 16S rRNA and gyrB gene. Ewingella americana strains differed in their aggressiveness on mushroom caps and stipes where the corresponding symptoms ranged from deep yellow to dark brown. In the phylogenetic analyses, all E. americana strains isolated in this study were clustered in a monophyletic clade closely related to the non-pathogenic and environmental strains of the species. BOX-PCR-based fingerprinting revealed intraspecific diversity. Using the cutoff level of 73-76% similarity, the strains formed six clusters. A chronological pattern was observed where the strains isolated in 2018 were differentiated from those isolated in 2020 and 2021. Taking together, due to the multifaceted nature of the pathogen, such a widespread occurrence of E. americana in mushroom farms in Iran could be an emerging threat for mushroom industry in the country.
... bacte rio. net) [8,12,41]. The OrthoANI and dDDH values of PPL560 T and PPL568 were ≤ 93.5% and < 60% with other species of genus Xanthomonas, which is below species delineation cut-offs (Supplementary Table 1). ...
Xanthomonas is a major group of pathogenic bacteria infecting staple food crops like rice. Increasingly it is being recognized that non-pathogenic Xanthomonas (NPX) are also important members of a healthy plant microbiome. However, the vast majority of the species described in this genus are of pathogenic nature, and only a few NPX species have been reported till now. Genomic and taxonogenomic analysis of NPX is needed for the management of this important group of bacteria. In this study, two yellow-pigmented bacterial isolates were obtained from healthy rice seeds in Punjab, India. The isolates designated PPL560T and PPL568 were identified as members of the genus Xanthomonas based on biochemical tests and 16S rRNA gene sequence analysis retrieved from the whole-genome sequences. Isolates formed a distinct monophyletic lineage with Xanthomonas sontii and Xanthomonas sacchari as the closest relatives in the phylogenetic tree based on core gene content shared by the representative species of the genus Xanthomonas. Pairwise ortho Average Nucleotide Identity and digital DNA-DNA hybridization values calculated against other species of Xanthomonas were below their respective cut-offs. In planta studies revealed that PPL560T and PPL568 are non-pathogenic to rice plants upon leaf clip inoculation. The absence of type III secretion system-related genes and effectors further supported their non-pathogenic status. Herein, we propose Xanthomonas indica sp. nov. as novel species of the genus Xanthomonas with PPL560T = MTCC 13185 = CFBP 9039 = ICMP 24394 as its type strain and PPL568 as another constituent member.
... populi did not belong to X. arboricola due to the low DNA similarities between the type strain of the species and the pathotype strains of these two pathovars. Thus, Zarei et al. (2022) proposed to elevate the two pathovars to the rank of species as X. guizotiae and X. populina, respectively. ...
... Several nonpathogenic (thus with no pathovar status) strains are also designated as X. arboricola in the literature and public databases (Garita-Cambronero et al., 2018;Vauterin et al., 1995;Zarei et al., 2022). Nonpathogenic strains of X. arboricola have been isolated from host tissues with and without symptoms, that is, walnut buds, sour cherry and peach leaves Garita-Cambronero et al., 2016a, 2016b. ...
Bacterial canker and spot of stone fruits, and bacterial blight of walnut caused by different pathovars of Xanthomonas arboricola i.e. X. arboricola pv. pruni and X. arboricola pv. juglandis, respectively, are considered among quarantine or regulated (non-quarantine) agents in different countries. The two pathogens were initially reported in Iran in 2005 and 1977, respectively. However, the precise identity, pathogenicity status and geographic distribution of these pathogens have been questioned. In this study, we have employed a series of comprehensive field surveys and samplings in a course of five years (2015-2019) across 16 provinces to re-assess and clarify the occurrence and distribution of X. arboricola on fruit and nut trees in Iran. Besides dozens of non-Xanthomonas and non-pathogenic bacterial strains, 27 X. arboricola strains were isolated from stone fruits (n=13) and walnut (n=14). Except for the strains Peh35, Peh78 and Peh79, the stone fruit strains were non-pathogenic on their host of isolation as well as several other plant species, while the walnut strains were pathogenic on their host of isolation. Multilocus sequence analysis (MLSA) of four housekeeping genes i.e. atpD, dnaK, gyrB, and rpoD revealed that among the stone fruit strains only Peh78 and Peh79 could be assigned into X. arboricola pv. pruni, while the walnut strains were identified as X. arboricola pv. juglandis. Based on the results of pathogenicity assays and phylogenetic analyses, we confirm the occurrence of X. arboricola pv. juglandis in Iran, while X. arboricola pv. pruni was absent in the plant samples evaluated in this study.
Bacterial blight and leaf spot of geraniums is a destructive disease of cultivated Pelargonium species around the world. During 2020-2021, surveys were conducted in seven geranium-growing provinces of Iran to monitor the status of bacterial blight and leaf spot disease. The disease was observed in six surveyed provinces varying in the extent of occurrence and severity. Twenty-two Gram-negative pale-yellow bacterial strains resembling members of Xanthomonas were isolated from symptomatic leaves and stems. Pathogenicity and host range assays showed that the bacterial strains were pathogenic on Pelargonium grandiflorum, P. graveolens, P. peltatum, and P. zonale. All strains were positive for PCR test using the primer pair XcpM1/XcpM2 which is specific for Xanthomonas hortorum pv. pelargonii. Phylogenetic analysis using the sequences of gyrB and lepA genes showed that the 22 strains clustered in a clade among the sequences of X. hortorum pv. pelargonii strains retrieved from the GenBank, while distinct from the other pathovars of X. hortorum. BOX-PCR-based fingerprinting using BOX-A1R primer revealed that the strains isolated in this study were grouped into two clusters while no distinct correlation was observed between the host/area of isolation and BOX-PCR fingerprinting. None of the strains obtained in this study nor reference strain of the pathogen did produce bacteriocin against each other. Results obtained in this study shed light on the geographic distribution, taxonomic status and host range of the bacterial blight and leaf spot pathogen of geraniums in Iran, paving the path of further research on disease management.
The Araceae family, comprising ornamentals including Anthurium, Dieffenbachia, Philodendron, Colocasia, and Zantedeschia, is susceptible to Xanthomonas pathogens. Previous analyses have established heterogeneity in aroid strains, yet unresolved taxonomic positions and dynamics between Xanthomonas and frequently associated Stenotrophomonas in aroids necessitate in-depth genetic investigation to resolve these complex relationships. This study utilized multi-locus sequence analysis (MLSA) of housekeeping genes atpD, dnaA, dnaK, gltA, and gyrB to investigate 59 aroid strains, selected based on hosts, time, and geographical origins. After adding sequences from additional strains from NCBI GenBank, analysis of 161 concatenated sequences indicated that all aroid strains fell within Xanthomonas and Stenotrophomonas. Thirty-six strains isolated from Anthurium grouped under X. phaseoli, with outliers including one strain each in X. arboricola and X. sacchari, and two in Stenotrophomonas. Six strains from Caladium, Dieffenbachia, and Philodendron formed host-specific subgroups within X. euvesicatoria. One strain from Dieffenbachia aligned with X. campestris, while strains from Colocasia, Aglaonema, and Spathiphyllum clustered with X. sacchari. Apart from the zantedeschia strain described as X. arboricola pv. zantedeschiae, two colocasia, one epipremnum, and one anthurium strain joined the X. arboricola group. Overall, this study revealed significant heterogeneity among aroid strains, with anthurium strains clustering closely despite distant geographical origins. The analysis underscores the complexity of host-pathogen specificity within Xanthomonas and emphasizes the need for further taxonomic clarification through whole genome analysis of representative strains. The finding of this research will facilitate strain selection for inclusivity and exclusivity panels in developing diagnostic assays for X. phaseoli and xanthomonads affecting aroids.
In this Opinion paper, members of the French Network on Xanthomonads give their personal view on what they consider to be some of the groundbreaking discoveries in the field of molecular plant pathology over the past 20 years. By celebrating the 20th anniversary of the first Xanthomonas genome sequences, they explain how genomics revolutionized taxonomy, provided insight into the emergence of pathogenic bacteria, enabled new fundamental discoveries and contributed to the development of novel control measures. Collectively, such new, genomics-enabled perspective will help to ensure sustainable agriculture and conservation of our environment in the future.
Xanthomonas arboricola pv. corylina (Xac; formerly Xanthomonas campestris pv. corylina) is the causal agent of the bacterial blight of hazelnuts, a devastating disease of trees in plant nurseries and young orchards. Currently, there are no PCR assays to distinguish Xac from all other pathovars of X. arboricola. A comparative genomics approach with publicly available genomes of Xac was used to identify unique sequences, conserved across the genomes of the pathogen. We identified a 2,440 bp genomic region that was unique to Xac and designed identification and detection systems for conventional PCR, qPCR (SYBR® Green and TaqMan™), and loop-mediated isothermal amplification (LAMP). All PCR assays performed on genomic DNA isolated from eight X. arboricola pathovars and closely related bacterial species confirmed the specificity of designed primers. These new multi-platform molecular diagnostic tools may be used by plant clinics and researchers to detect and identify Xac in pure cultures and hazelnut tissues rapidly and accurately.
