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Angular leaf spot of Caucasian alder incited by a pathovar of Xanthomonas arboricola
Abstract
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 .
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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 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 out 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 the argue 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 due to 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. either belonging to previously described species or representing novel species which await formal description.
Background:
The species Xanthomonas arboricola comprises up to nine pathovars, two of which affect nut crops: pv. juglandis, the causal agent of walnut bacterial blight, brown apical necrosis, and the vertical oozing canker of Persian (English) walnut; and pv. corylina, the causal agent of the bacterial blight of hazelnut. Both pathovars share a complex population structure, represented by different clusters and several clades. Here we describe our current understanding of symptomatology, population dynamics, epidemiology, and disease control.
Taxonomic status:
Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Lysobacterales (earlier synonym of Xanthomonadales); Family Lysobacteraceae (earlier synonym of Xanthomonadaceae); Genus Xanthomonas; Species X. arboricola; Pathovars: pv. juglandis and pv. corylina.
Host range and symptoms:
The host range of each pathovar is not limited to a single species, but each infects mainly one plant species: Juglans regia (X. arboricola pv. juglandis) and Corylus avellana (X. arboricola. pv. corylina). Walnut bacterial blight is characterized by lesions on leaves and fruits, and cankers on twigs, branches, and trunks; brown apical necrosis symptoms consist of apical necrosis originating at the stigmatic end of the fruit. A peculiar symptom, the vertical oozing canker developing along the trunk, is elicited by a particular genetic lineage of the bacterium. Symptoms of hazelnut bacterial blight are visible on leaves and fruits as necrotic lesions, and on woody parts as cankers. A remarkable difference is that affected walnuts drop abundantly, whereas hazelnuts with symptoms do not.
Distribution:
Bacterial blight of walnut has a worldwide distribution, wherever Persian (English) walnut is cultivated; the bacterial blight of hazelnut has a more limited distribution, although disease outbreaks are currently more frequently reported. X. arboricola pv. juglandis is regulated almost nowhere, whereas X. arboricola pv. corylina is regulated in most European and Mediterranean Plant Protection Organization (EPPO) countries.
Epidemiology and control:
For both pathogens infected nursery material is the main pathway for their introduction and spread into newly cultivated areas; additionally, infected nursery material is the source of primary inoculum. X. arboricola pv. juglandis is also disseminated through pollen. Disease control is achieved through the phytosanitary certification of nursery material (hazelnut), although approved certification schemes are not currently available. Once the disease is present in walnut/hazelnut groves, copper compounds are widely used, mostly in association with dithiocarbamates; where allowed, antibiotics (preferably kasugamycin) are sprayed. The emergence of strains highly resistant to copper currently represents the major threat for effective management of the bacterial blight of walnut. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTJU, https://gd.eppo.int/taxon/XANTCY, https://www.euroxanth.eu, http://www.xanthomonas.org.
We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated
methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing
systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The
new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict
gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line.
The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command
line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput
and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
Bacterial blight (Xanthomonas arboricola pv. corylina (Xac)) of hazelnut (Corylus avellana L.) was described first in Oregon in 1915 and is now recognized as a damaging disease of young hazelnut trees worldwide. Thousands of hectares of new hazelnut cultivars that are resistant to eastern filbert blight (Anisogramma anomala) are being planted in the Willamette Valley of Oregon, where 99% of the US hazelnut crop is grown. Reports of bacterial blight on young hazelnut trees have increased but information about the causal pathogen is limited. Isolates were recovered from tissues with bacterial blight symptoms that were then characterized for their ability to grow on semi-selective media, their nutrient utilization profiles using Biolog GN2, quinate metabolism, copper tolerance, hypersensitive response on tobacco, and pathogenicity on hazelnut. Additionally, isolates were identified with a duplex PCR assay (ftsX and qumA), 16S rRNA sequence, and multi-locus sequence analysis (MLSA) using rpoD and gyrB. Pathogenic isolates were identified as Xac using morphological, biochemical, molecular, and host assays. Using MLSA, Xac isolates from Oregon separated into two clades, one clade with the type strain and a second clade previously described using isolates from Europe. The phylogenetic diversity of Xac observed in other countries also is present in Oregon.
Alnus subcordata (Caucasian Alder, Betulaceae) is known as one of the most important tree species in wood industry with high nitrogen fixation capability and high rate of biomass production. In recent years, a disease symptom with the characteristics of alder bacterial leaf spot has been reported from the forests in Mazandaran province, Iran. To characterize the leaf spot causal agent, samplings were conducted from infected trees in different regions of Mazandaran forests. Characterization of the bacteria isolated from symptomatic tissues was performed based on classical and molecular methods. Twenty colonies were purified and tested by PCR using primers X1 and X2 specific for the genus Xanthomonas and biochemical analyses as well. Based on molecular and phenotypic identifications, the isolates were identified as genus Xanthomonas. Phylogenetic analysis was carried out based on PCR amplification and sequencing of gyrB genes for 14 selected isolates. Consequently, according to the molecular data all recovered isolates were determined as X. arboricola. Furthermore, multilucus sequence analysis using gyrB, fyuA, dnaK and rpoD for various pathovars of X. arboricola showed that our isolates clustered in a single clade sister to X. arboricola pv. juglandis. The results of pathogenicity tests on alder leaves seedlings confirmed that all the isolates were pathogenic and caused leaf spot on A. subcordata. To the best of our knowledge, this is the first comprehensive study on the molecular identification of the alder bacterial leaf spot disease in Iran.
The recent advent of DNA sequencing technologies facilitates the use of genome sequencing data that provide means for more informative and precise classification and identification of Bacteria and Archaea. Because the current species definition is based on the comparison of genome sequences between type and other strains in a given species, building a genome database with correct taxonomic information is a premium need to enhance our efforts in exploring prokaryotic diversity and discovering new species as well as for routine identifications. Here we introduce an integrated database, called EzBioCloud, that holds the taxonomic hierarchy of Bacteria and Archaea that are represented by quality-controlled 16S rRNA gene and genome sequences. Whole genome assemblies in the NCBI Assembly Database were screened for low quality and subjected to a composite identification bioinformatics pipeline that employs gene-based searches followed by the calculation of average nucleotide identity. As a result, the database is made of 61,700 species/phylotypes including 13,132 with validly published names, and 62,362 whole genome assemblies that were taxonomically identified at the genus, species and subspecies levels. Genomic properties, such as genome size and GC content, and the occurrence in human microbiome data were calculated for each genus or higher taxa. This united database of taxonomy, 16S rRNA gene and genome sequences, with accompanied bioinformatics tools, should accelerate genome-based classification and identification of Bacteria and Archaea. The database and related search tools are available at http://www.ezbiocloud.net/.
A Xanthomonas species is reported as the causative agent of angular leaf spot of Persian or Asian alder in northern Iran
Xanthomonas arboricola is conventionally known as a taxon of plant-pathogenic bacteria, including seven pathovars. This study showed that X. arboricola encompasses also non-pathogenic bacteria causing no apparent disease symptoms on their hosts. The aim of this study was to assess the X. arboricola population structure associated to walnut including non-pathogenic strains, in order to gain a better understanding of the role of non-pathogenic xanthomonads in walnut microbiota. A multi-locus sequence analysis (MLSA) was performed on a collection of 100 X. arboricola strains including 27 non-pathogenic strains isolated from walnut. Non-pathogenic strains grouped outside clusters defined by pathovars and formed separate genetic lineages. A multi-locus variable-number tandem repeat analysis (MLVA) conducted on a collection of X. arboricola strains isolated from walnut, showed that non-pathogenic strains clustered separately from clonal complexes containing Xanthomonas aroboricola pv. juglandis strains. Some non-pathogenic strains of X. arboricola did not contain the canonical type III secretion system (T3SS) and harboured only one to three type III effectors (T3E) genes. In non-pathogenic strains CFBP 7640 and CFBP 7653, neither T3SS genes, nor any of the analysed T3E genes were detected. This finding raises the question about the origin of non-pathogenic strains and evolution of plant pathogenicity in X. arboricola. T3E genes that were not detected in any non-pathogenic isolates studied represent excellent candidates to be those responsible for pathogenicity in X. arboricola.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PCR-based methods offer advantages over more traditional diagnostic tests, in that organisms do not need to be cultured prior to their detection and protocols are highly sensitive and rapid. Consequently, there is a shift in research towards DNA-based techniques. Although reports already exist on a variety of PCR-based fingerprinting assays used to analyse the genetic diversity of bacterial populations and define their relationships, this review focuses on the general use of PCR in phytobacteriology for detection and diagnosis purposes. An updated and detailed list of published PCR protocols for detection and identification of plant-pathogenic bacteria is presented and discussed, aimed at facilitating access to information that could be particularly useful for diagnostic laboratories. This compilation includes and discusses 246 articles published between 1989 and 2007 addressing 23 genera, more than 50 species, 10 subspecies and more than 40 pathovars.
Deep and comprehensive knowledge of the genetic structure of pathogenic species is the cornerstone on which the design of precise molecular diagnostic tools is built. Xanthomonas arboricola is divided into pathovars, some of which are classified as quarantine organisms in many countries and which are responsible for diseases on nut and stone fruit trees that have emerged worldwide. Recent taxonomic studies of the genus Xanthomonas showed that strains isolated from other hosts should be classified in X. arboricola, extending the host range of this species. To investigate the genetic structure of X. arboricola and the genetic relationships between highly pathogenic strains and strains apparently not relevant for plant health, we conducted multilocus sequence analyses on a collection of strains representative of the known diversity of this species. Most of the pathovars were clustered in separate monophyletic groups. The pathovars pruni, corylina and juglandis, responsible for pandemics on specific hosts, were highly phylogenetically-related and clustered in three distinct clonal complexes. In contrast, strains with no or uncertain pathogenicity were represented by numerous unrelated singletons scattered in the phylogenic tree. Depending on the pathovar, intra- and interspecies recombination played a contrasted role in generating nucleotide polymorphism. This work provides a population genetics framework for molecular epidemiological surveys of emerging plant pathogens within X. arboricola. Based on our results, we propose to reclassify three former pathovars of X. campestris as X. arboricola pv. arracaciae comb. nov, X. arboricola pv. guizotiae comb. nov and X. arboricola pv. zantedeschiae comb. nov. An emended description of X. arboricola Vauterin et al. 1995 is provided.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Diversity index is the useful criteria for evaluating sustainability of forest ecosystems. Current study carried out in Alder (Alnus subcordata C.A. Meyer) stands that located in north forests of Iran. The aim of the study is express the plant diversity indices and positive role of the trees both natural and plantation forms. Data of Alder trees and associated species were recorded in sample plots which lay down in study area randomly. The abundance, density, percentage of frequency of each species was calculated by standard methods. The results of analysis revealed that, 47 species (21 trees and shrubs species and 26 herbaceous species) were abundant in 80 sample plots both in natural and plantations Alder stands. Whilst the results showed that the number of species in natural area (44 species) was more than plantation stands (37 species). Comparison of species distribution in different physiographical situation showed that some species such as Alnus subcordata, Parrotia persica, Rubus hyrcanus and Prunus sp. recorded in spread rang of physiographic variables as elevation, slopes and aspects. The biodiversity criteria as Shannon H' and Simpsons D and 1/D indexes showed that they were more in natural stands than plantation areas.
A comprehensive DNA-DNA hybridization study was performed by using 183 strains of the genus Xanthomonus. This genus was shown to comprise 20 DNA homology groups which are considered genomic species. Four groups corresponded to the previously described species Xanthomonas albilineans, Xanthomonas fragariae, Xanthomonas oryzae, and Xanthomonas populi. The previously described species Xanthomonas campestris was
heterogeneous and was divided into 16 DNA homology groups. One of these groups exhibited a high level of DNA homology with Xanthomonas axonopodis. The 62 pathovars represented in this study were. allocated to appropriate species. Our results, together with previous taxonomic data, supported a comprehensive revision of the classification of the genus Xanthomonas. The species X. albilineans, X. fragariae, X. oryzae, and X. populi
are not affected. The type species of the genus, X. campestris (Pammel 1895) Dowson 1939, is emended to include only the pathovars obtained from crucifers (i.e., X. campestris pv. aberrans, X. campestris pv. armoraciae, X. campestris pv. barbareae, X. campestris pv. campestris, X. campestris pv. incanae, and X. campestris pv. raphani). X. axonopodis Starr and Garces 1950 is emended to include 34 former X. campestris pathovars. The following
species names are proposed: Xanthomonas arboricola sp. nov., including X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. poinsettiicola (type C strains of the former X. campestris pathovar), X. arboricola pv. populi, and X. arboricola pv. pruni; Xanthomonas bromi sp. nov. for strains isolated from bromegrass; Xanthomonas cassavae (ex Wiehe and Dowson 1953) sp. nov., nom. rev.; Xanthomonas codiaei sp. nov., including
type B strains of the former taxon X. campestris pv. poinsettiicola; Xanthomonas cucurbitae (ex Bryan 1926) sp. nov., nom. rev.; Xanthomonas hortorum sp. nov., including X. hortorum pv. hederae, X. hortorum pv. pelargonii, and X. hortorum pv. vitians; Xanthomonas hyacinthi (ex Wakker 1883) sp. nov., nom. rev.; Xanthomonas melonis sp. nov.; Xanthomonas pisi (ex Goto and Okabe 1958) sp. nov., nom. rev.; Xanthomonas sacchari sp. nov. for
strains isolated from diseased sugarcane in Guadeloupe; Xanthomonas theicola sp. nov.; Xanthomonus translucent (ex Jones, Johnson, and Reddy 1917) sp. nov., nom. rev., including X. translucens pv. arrhenatheri, X. translucens pv. cerealis, X. translucens pv. graminis, X. translucens pv. hordei, X. translucens pv. phlei, X. translucens pv. phleipratensis, X. translucens pv. poae, X. translucens pv. secalis, X. translucens pv. translucens, and X. translucens pv. undulosa; Xanthomonas vasicola sp. nov., including X. vasicola pv. holcicola and X. vasicola
pv. vasculorum (type B strains of the former taxon X. campestris pv. vasculorum); and Xanthomonas vesicatoria (ex Doidge 1920) sp. nov., nom. rev., which includes the type B strains of the former taxon X. campestris pv. vesicatoria. Differentiating characteristics were determined for the new species on the basis of metabolic activity on a range of carbon substrates by using the Biolog GN microplate system.
Stone fruit, almond and walnut are among the most important woody crops worldwide. Xanthomonas arboricola causes severe damages on these plants with losses worth millions of US dollars annually. The European Union phytosanitary legislation (EU Directive 2000/29/CE) and the European and Mediterranean Plant Protection Organizations included plant-pathogenic X. arboricola pv. pruni, a cause of diseases on Prunus spp., in the A2 list of quarantine microorganisms. Because of the economic importance of the diseases it causes and the pathogen regulatory status, X. arboricola has created concern in recent years. Despite a high similarity in the biology and epidemiology of X. arboricola and the diseases it causes on stone fruit, almond and walnut, the literature on this bacterial species is fragmentary and restricted to the pathovar level. This review of the literature summarizes main aspects of X. arboricola and its pathogenesis on these species. Specific topics include: taxonomy, disease symptoms, epidemiology, pathogen diversity, the molecular basis of the pathogenesis, recent efforts on breeding for resistance and possible approaches to disease management.
This study records the presence of bacterial walnut blight, caused by Xanthomonas arboricola pv. juglandis (Xaj), on walnuts (Juglans spp.) in Lithuania and confirms that its geographic distribution has been underestimated. The pathogen caused the most serious symptoms on J. cinerea and J. mandshurica trees. DNA typing methods (rep-PCR and MLST) used for characterization of Xaj isolates disclosed a high similarity among them. Strains from Lithuania, however, showed a still not fully understood difference from Xaj strains from Poland, a neighbouring country, that needs further investigation.
Xanthomonas arboricola pv. juglandis is the causal agent of walnut blight, one of the most important and widespread diseases of Persian (English) walnut (Juglans regiaL.), causing severe damage to leaves, twigs and nuts. To investigate the genomic variability of X. arboricolapv. juglandis, 66 isolates obtained from different countries (England, France, Italy, The Netherlands, Romania, Spain, USA, and New Zealand) were analysed using the Amplified Fragment Length Polymorphism (AFLP) technique. EcoRI and MseI were used as restriction endonucleases. Primers with a core sequence including endonuclease recognition sites and a 3prime-terminal cytosine selective base for MseI primer, or no selective base for EcoRI primer, were used. Data were analysed by means of a multiple correspondence analysis. A total of 76 amplified polymorphic DNA fragments were used to compute relationships among isolates. The AFLP profiles of X. arboricola pv. juglandis isolates appeared to be reliably distinguishable from X. arboricola pv. pruni and X. arboricola pv. corylina, and from other Xanthomonas species, i.e. X. campestris pv. campestris, X. fragariae, X. hortorum,
X. axonopodis pv. vesicatoria. Though this pathogen is associated with one single host genus, a high level of genomic diversity was found. This diversity might be partly explained by the geographic origin. Nevertheless, isolates with different patterns were collected within one country, and similar molecular patterns were found in isolates collected at different sites. However, genetic diversity might be influenced by exchanging vegetative material from different countries. Mixing of X. arboricola pv. juglandis isolates might have partly concealed the influence of the geographic location from which the bacteria were isolated.
Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of approximately 470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of Neisseria meningitidis from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.
Forty-five Xanthomonas arboricola pv. juglandis (Xaj) strains originating from Juglans regia cultivation in different countries were molecularly typed by means of MultiLocus Sequence Typing (MLST), using acnB, gapA, gyrB and rpoD gene fragments. A total of 2.5 kilobases was used to infer the phylogenetic relationship among the strains and possible recombination events. Haplotype diversity, linkage disequilibrium analysis, selection tests, gene flow estimates and codon adaptation index were also assessed. The dendrograms built by maximum likelihood with concatenated nucleotide and amino acid sequences revealed two major and two minor phylotypes. The same haplotype was found in strains originating from different continents, and different haplotypes were found in strains isolated in the same year from the same location. A recombination breakpoint was detected within the rpoD gene fragment. At the pathovar level, the Xaj populations studied here are clonal and under neutral selection. However, four Xaj strains isolated from walnut fruits with apical necrosis are under diversifying selection, suggesting a possible new adaptation. Gene flow estimates do not support the hypothesis of geographic isolation of the strains, even though the genetic diversity between the strains increases as the geographic distance between them increases. A triplet deletion, causing the absence of valine, was found in the rpoD fragment of all 45 Xaj strains when compared with X. axonopodis pv. citri strain 306. The codon adaptation index was high in all four genes studied, indicating a relevant metabolic activity.
Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.
DNA primers corresponding to conserved motifs in bacterial repetitive (REP, ERIC, and BOX) elements and PCR were used to show that REP-, ERIC-, and BOX-like DNA sequences are widely distributed in phytopathogenic Xanthomonas and Pseudomonas strains. REP-, ERIC, and BOX-PCR (collectively known as rep-PCR) were used to generate genomic fingerprints of a variety of Xanthomonas and Pseudomonas isolates and to identify pathovars and strains that were previously not distinguishable by other classification methods. Analogous rep-PCR-derived genomic fingerprints were generated from purified genomic DNA, colonies on agar plates, liquid cultures, and directly from lesions on infected plants. REP, ERIC, and BOX-PCR-generated fingerprints of specific Xanthomonas and Pseudomonas strains were found to yield similar conclusions wtih regard to the identity of and relationship between these strains. This suggests that the distribution of REP-, ERIC, and BOX-like sequences in these strains is a reflection of their genomic structure. Thus, the rep-PCR technique appears to be a rapid, simple, and reproducible method to identify and classify Xanthomonas and Pseudomonas strains, and it may be a useful diagnostic tool for these important plant pathogens.
Phylogenetic relationships of 17 species of Xanthomonas were assessed based on analysis of 16S-23S rDNA intergenic spacer (ITS) sequences; a higher level of resolution was obtained than that revealed by 16S rDNA analysis. ITS sequences varied in size from 492 to 578 nt within the genus and the similarity among sequences ranged from 63 to 99%. Major differences were found for the hyacinthi group, which included Xanthomonas albilineans, Xanthomonas hyacinthi, Xanthomonas sacchari, Xanthomonas theicola and Xanthomonas translucens. A common ITS structure with tRNA(Ala) and tRNA(Ile) embedded within the sequence was found for all ITS sequences of Xanthomonas species and for Stenotrophomonas maltophilia. These tRNAs were highly conserved and divided the ITS sequence into three regions (ITS1, ITS2 and ITS3). ITS1 and ITS2 sequences of Xanthomonas species showed mean similarities of 87.1 and 86.8%, respectively, and differences consisted of substitution and addition/deletion of 1-5 nt. ITS2 showed remarkable variation in sequence length: most species had an ITS2 of 19-20 nt, whereas a long insertion of 51-56 nt was found in Xanthomonas codiaei, X. hyacinthi, Xanthomonas melonis, X. theicola and X. translucens. For ITS3 the most striking alteration was seen in X. hyacinthi, which showed a large deletion of 44 nt. The ITS phylogenetic tree grouped Xanthomonas species into six major clusters. Cluster I included Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas bromi, Xanthomonas campestris, X. campestris pv. gardneri, Xanthomonas cassavae, X. codiaei, Xanthomonas cucurbitae, Xanthomonas fragariae, Xanthomonas hortorum, X. melonis, Xanthomonas oryzae, Xanthomonas pisi, Xanthomonas vasicola and Xanthomonas vesicatoria. The species X. albilineans, X. sacchari, X. hyacinthi, X. theicola and X. translucens represented distinct clusters (II-VI). Topology of the 16S-23S rDNA ITS phylogenetic tree was very similar to that of the 16S rDNA tree reported previously, but more clusters were discriminated because of the higher level of diversity among the ITS sequences (16.2%) compared with the 16S rDNA sequences (1.8%).
There is no widely accepted concept of species for prokaryotes, and assignment of isolates to species is based on measures of phenotypic or genome similarity. The current methods for defining prokaryotic species are inadequate and incapable of keeping pace with the levels of diversity that are being uncovered in nature. Prokaryotic taxonomy is being influenced by advances in microbial population genetics, ecology and genomics, and by the ease with which sequence data can be obtained. Here, we review the classical approaches to prokaryotic species definition and discuss the current and future impact of multilocus nucleotide-sequence-based approaches to prokaryotic systematics. We also consider the potential, and difficulties, of assigning species status to biologically or ecologically meaningful sequence clusters.
Background: Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of stone fruits and almond, an important disease that may reduce the yield and vigor of the trees, as well as the marketability of affected fruits. Xap, is within the Xanthomonas genus which has been intensively studied due its strain specialization and the host range complexity. Here, we have summarized the recent advances in our understanding of the complexities of Xap, including the studies of molecular features that resulted after comparative phenotypic and genomic analyses, in order to obtain a clearer overview of the bacterial behavior and their infection mechanisms in the context of the X. arboricola species.
Taxonomic status: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species X. arboricola; Pathovar pruni.
Host range and symptoms: Xap infects most Prunus species including apricot, peach, nectarine, plum and almond, and occasionally cherry. Symptoms are found on leaves, fruits, twigs and branches or trunks. In severe infections defoliation and fruit dropping may occur.
Distribution: Bacterial spot of stone fruits and almond is worldwide in distribution with Xap being isolated in Africa, North and South America, Asia, Europe and Oceania continents. It is a common disease in geographic areas where stone fruits and almond are grown. Xap is listed as a quarantine organism in several areas of the world.
Genome: Genomes of six isolates from Xap have been publicly released. The genome consists of a single chromosome around 5,000,000 bp with a 65 mol% GC content and extrachromosomal plasmid element of around 41,000 bp with a 62 mol% GC content. Genomic comparative studies in X. arboricola allowed identification of putative virulence components associated with the infection process of bacterial spot of stone fruits and almond.
Disease control: Management of bacterial spot of stone fruits and almond is based on an integrated approach that comprises essentially measures to avoid Xap introduction in a producing zone, as well as the use of tolerant or resistant plant material and chemical treatments, mainly based in copper compounds. Management programs include also the use of proper cultivation practices when the disease is already stablished. Finally, for effective control of the disease, proper detection and characterization methods are needed to be used in symptomatic or asymptomatic samples as a first approach for pathogen exclusion. This article is protected by copyright. All rights reserved.
Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from 6 locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of Xanthomonas genus. PCR with DNA of the all isolates with X1 and X2 primers specific for Xanthomonas confirmed that they belong to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for type strain of X. arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC-, BOX-PCR, and MLSA) showed similarities of the studied isolates to the reference strain of X. a. pv. juglandis CFBP 7179 originating from France. However, reference strains I-391 from Portugal and LMG 746 from the United Kingdom were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences (from Gene Bank) of pathotype strains of other pathovars of X. arboricola showed that our X. a. pv. juglandis isolates consists of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping gene is necessary for proper identification of X. arboricola pathovars. This article is protected by copyright. All rights reserved.
Biomass analyses were conducted to evaluate the growth performance of an Alnus incana (L.) Moench stand established for the restoration of a land slide site. The study aimed to pioneer the quantification of biomass accumulation of alders which were planted for soil bioengineering and stabilisation purposes under extreme soil conditions. For above-ground phytomass estimation, allometric functions on a tree component level were created using linear regression analyses after ln-transformation. Based on D(130)-D(10)-relations D(10)-data were applied for function derivation. Best fits were computed using ln-transformed D(10) and height data. At 7,023 stems ha(-1) (all trees > 20 cm height), the total stand biomass added up to 18,000 kg ha(-1), had an LAI of 1.5 and indicated a high productivity even under unfavourable soil conditions. In the light of the results, grey alders appear to be highly valuable for growing on slide prone sites, indicating that they also exert a positive influence on the soil water regime and thus on slope stability.
Previous classification of Xanthomonas campestris has defined six pathovars (aberrans, armoraciae, barbareae, campestris, incanae, and raphani) that cause diseases on cruciferous plants. However, pathogenicity assays with a range of strains and different hosts identifies only three types of symptom: black rot, leaf spot and bacterial blight. These findings raise the question of the genetic relatedness between strains assigned to different pathovars or symptom phenotypes. Here we have addressed this issue by multilocus sequence analysis of 42 strains. The X. campestris species was polymorphic at the 8 loci analysed and had a high genetic diversity; 23 sequence types were identified of which 16 were unique. All strains that induce black rot (pathovars aberrans and campestris) were genetically close but split in two groups. Only three clonal complexes were found, all within pathovar campestris. The assignment of the genome-sequenced strain 756C to pathovar raphani suggested from disease symptoms was confirmed, although this group of strains was particularly polymorphic. Strains belonging to pathovars barbareae and incanae were closely related, but distinct from pathovar campestris. There is evidence of genetic exchanges of housekeeping genes within this species as deduced from a clear incongruence between individual gene phylogenies and from network structures from SplitsTree analysis. Overall this study showed that the high genetic diversity derived equally from recombination and point mutation accumulation. However, X. campestris remains a species with a clonal evolution driven by a differential adaptation to cruciferous hosts.
A multilocus sequence analysis (MLSA) of strains representing all validly published Xanthomonas spp. (119 strains) was conducted using four genes; dnaK, fyuA, gyrB and rpoD, a total of 440 sequences. Xanthomonas spp. were divided into two groups similar to those indicated in earlier 16S rDNA comparative analyses, and they possibly represent distinct genera. The analysis clearly differentiated most species that have been established by DNA-DNA reassociation. A similarity matrix of the data indicated clear numerical differences that could form the basis for species differentiation in the future, as an alternative to DNA-DNA reassociation. Some species, X. cynarae, X. gardneri and X. hortorum, formed a single heterogeneous group that is in need of further investigation. X. gardneri appeared to be a synonym of X. cynarae. Recently proposed new species, X. alfalfae, X. citri, X. euvesicatoria, X. fuscans and X. perforans, were not clearly differentiated as species from X. axonopodis, and X. euvesicatoria and X. perforans are very probably synonyms. MLSA offers a powerful tool for further investigation of the classification of Xanthomonas. Based on the dataset produced, the method also offers a relatively simple way of identifying strains as members of known species, or of indicating their status as members of new species.
Keywords:cladistic nested analysis;historical processes;phylogeography;population genetics;software
Examining genomic data for traces of selection provides a powerful tool for identifying genomic regions of functional importance. Many methods for identifying such regions have focused on conserved sites. However, positive selection may also be an indication of functional importance. This article provides a brief review of some of the statistical methods used to detect selection using DNA sequence data or other molecular data. Statistical tests based on allelic distributions or levels of variability often depend on strong assumptions regarding population demographics. In contrast, tests based on comparisons of the level of variability in nonsynonymous and synonymous sites can be constructed without demographic assumptions. Such tests appear to be useful for identifying specific regions or specific sites targeted by selection.
Xanthomonadins, unique yellow pigments of the genus Xanthomonas
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