Specific and sensitive detection tools for Xanthomonas arboricola pv. corylina, the causal agent of bacterial blight of hazelnut, developed with comparative genomics.

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.

Complete Genome and Plasmid Sequence Data of Three Strains of Xanthomonas arboricola pv. corylina, the Bacterium Responsible for Bacterial Blight of Hazelnut.

Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium has been listed as an A2 quarantine pathogen in Europe since 1978 and on the regulated non-quarantine pest list since 2019. Three isolates from various geographic regions and isolated at different times were sequenced using a hybrid approach with short- and long-read technologies to generate closed genome and plasmid sequences in order to better understand the biology of this pathogen.

Xanthomonas arboricola pv. juglandis and pv. corylina: Brothers or distant relatives? Genetic clues, epidemiology, and insights for disease management.

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.

Transcriptome analysis of Xanthomonas fragariae in strawberry leaves.

Xanthomonas fragariae is a quarantine bacterial pathogen that causes angular leaf spot on strawberry. The aim of our study was to analyse the mechanism of interaction of this bacterium with its host plant at the transcriptome level. For this purpose, mRNAs of X. fragariae growing in Wilbrink's medium and from infected strawberry cv. Elsanta plants were isolated and sequenced using the Illumina MiSeq platform. The expression profiles of the bacteria in Wilbrink's medium and in planta were very diverse. Of the 3939 CDSs recorded, 1995 had significantly different expression in planta (966 and 1029 genes were down- and upregulated, respectively). Among the genes showing increased expression in planta, those with eggNOG/COG (evolutionary genealogy of genes: Non-supervised Orthologous Groups/Cluster of Orthologous Groups) categories associated with bacterial cell motility, signal transduction, transport and metabolism of inorganic ions and carbohydrates and transcription were overrepresented. Among the genes with the most increased expression in planta, genes primarily associated with flagella synthesis and chemotaxis were found. It is also interesting to note that out of the 31 genes localized on a plasmid, 16 were expressed differently in planta, which may indicate their potential role in plant-pathogen interactions. Many genes with differentiated expression that were localized on chromosome and plasmid encode proteins of unknown function.

mRNA extraction of Xanthomonas fragariae in strawberry and validation of reference genes for the RT-qPCR for study of bacterial gene expression.

This is the first study which describes a unique procedure of isolating of high-quality, intact RNA from strawberry leaves of Xanthomonas fragariae, three most suitable reference genes, crucial for the normalization of RT-qPCR data for this pathogen and accurate expression analysis of target genes. In our study, various mathematic algorithms: NormFinder geNorm, BestKeeper, the delta CT method, RefFinder were adopted for validation of most stable reference genes from nine candidate genes (ffh, glyA, gyrA, gyrB, proC, pykA, recA, rpoB, rpoD). The analyses allowing to select three most suitable pioneer reference genes, gyrB, ffh, and pykA, that we recommend for the normalization of RT-qPCR data and for the study of the expression of target genes in Xf. Moreover, their combination as references allowed for an accurate expression analysis and computation of the fold change of the flhF and iroN2 genes in Xf. These two genes are important for the success of the colonization of plant tissue and pathogenicity and sequences of primers designed to study these genes, are presented.

Comparative transcriptome analysis of a lowly virulent strain of Erwinia amylovora in shoots of two apple cultivars - susceptible and resistant to fire blight.

BACKGROUND: Erwinia amylovora is generally considered to be a homogeneous species in terms of phenotypic and genetic features. However, strains show variation in their virulence, particularly on hosts with different susceptibility to fire blight. We applied the RNA-seq technique to elucidate transcriptome-level changes of the lowly virulent E. amylovora 650 strain during infection of shoots of susceptible (Idared) and resistant (Free Redstar) apple cultivars. RESULTS: The highest number of differentially expressed E. amylovora genes between the two apple genotypes was observed at 24 h after inoculation. Six days after inoculation, only a few bacterial genes were differentially expressed in the susceptible and resistant apple cultivars. The analysis of differentially expressed gene functions showed that generally, higher expression of genes related to stress response and defence against toxic compounds was observed in Free Redstar. Also in this cultivar, higher expression of flagellar genes (FlaI), which are recognized as PAMP (pathogen-associated molecular pattern) by the innate immune systems of plants, was noted. Additionally, several genes that have not yet been proven to play a role in the pathogenic abilities of E. amylovora were found to be differentially expressed in the two apple cultivars. CONCLUSIONS: This RNA-seq analysis generated a novel dataset describing the transcriptional response of the lowly virulent strain of E. amylovora in susceptible and resistant apple cultivar. Most genes were regulated in the same way in both apple cultivars, but there were also some cultivar-specific responses suggesting that the environment in Free Redstar is more stressful for bacteria what can be the reason of their inability to infect of this cultivar. Among genes with the highest fold change in expression between experimental combinations or with the highest transcript abundance, there are many genes without ascribed functions, which have never been tested for their role in pathogenicity. Overall, this study provides the first transcriptional profile by RNA-seq of E. amylovora during infection of a host plant and insights into the transcriptional response of this pathogen in the environments of susceptible and resistant apple plants.

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots.

To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.

Pseudomonas cerasi sp. nov. (non Griffin, 1911) isolated from diseased tissue of cherry.

Eight isolates of Gram-negative fluorescent bacteria (58(T), 122, 374, 791, 963, 966, 970a and 1021) were obtained from diseased tissue of cherry trees from different regions of Poland. The symptoms resembled those of bacterial canker. Based on an analysis of 16S rDNA sequences the isolates shared the highest over 99.9% similarity with Pseudomonas ficuserectae JCM 2400(T) and P. congelans DSM 14939(T). Phylogenetic analysis using housekeeping genes gyrB, rpoD and rpoB revealed that they form a separate cluster and confirmed their closest relation to P. syringae NCPPB 281(T) and P. congelans LMG 21466(T). DNA-DNA hybridization between the cherry isolate 58(T) and the type strains of these two closely related species revealed relatedness values of 58.2% and 41.9%, respectively. This was further supported by Average Nucleotide Identity (ANIb) and Genome-to-Genome Distance (GGDC) between the whole genome sequences of strain LMG 28609(T) and closely related Pseudomonas species. The major cellular fatty acids are 16:0 and summed feature 3 (16:1 ω7c/15:0 iso 2OH). Phenotypic characteristics differentiated the novel isolates from other closely related species. The G+C content of the genomic DNA of strain 58(T) was 59%. The diversity was proved by PCR MP and BOX PCR, eliminating the possibility that they constitute a clonal population. Based on the evidence of this polyphasic taxonomic study the eight strains are considered to represent a novel species of the genus Pseudomonas for which the name P. cerasi sp. nov. (non Griffin, 1911) is proposed. The type strain of this species is 58(T) (=LMG 28609(T)=CFBP 8305(T)).

Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR.

Specific primers were developed to detect the causal agent of stone fruit bacterial canker using conventional and real-time polymerase chain reaction (PCR) methods. PCR melting profile (PCR MP) used for analysis of diversity of Pseudomonas syringae strains, allowed to pinpoint the amplified fragments specific for P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2), which were sequenced. Using obtained data, specific sequence characterised amplified region (SCAR) primers were designed. Conventional and real-time PCRs, using genomic DNA isolated from different bacterial strains belonging to the Pseudomonas genus, confirmed the specificity of selected primers. Additionally, the specificity of the selected DNA regions for Psm1 and Psm2 was confirmed by dot blot hybridisation. Conventional and real-time PCR assays enabled accurate detection of Psm1 and Psm2 in pure cultures and in plant material. For conventional PCR, the detection limits were the order of magnitude ~10(0) cfu/reaction for Psm1 and 10(1) cfu/reaction for Psm2 in pure cultures, while in plant material were 10(0)-10(1) cfu/reaction using primers for Psm1 and 3 × 10(2) cfu/reaction using primers for Psm2. Real-time PCR assays with SYBR Green I showed a higher limit of detection (LOD) - 10(0) cfu/reaction in both pure culture and in plant material for each primer pairs designed, which corresponds to 30-100 and 10-50 fg of DNA of Psm1 and Psm2, respectively. To our knowledge, this is the first PCR-based method for detection of the causal agents of bacterial canker of stone fruit trees.