Complete Genome Sequence Data of Two Xanthomonas arboricola Strains Isolated from Blueberry Plants Displaying Bacterial Leaf Blight in Poland
2022; American Phytopathological Society; Volume: 112; Issue: 8 Linguagem: Inglês
10.1094/phyto-11-21-0484-a
ISSN1943-7684
AutoresMonika Kałużna, Joël F. Pothier,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoHomePhytopathology®Vol. 112, No. 8Complete Genome Sequence Data of Two Xanthomonas arboricola Strains Isolated from Blueberry Plants Displaying Bacterial Leaf Blight in Poland PreviousNext Resource Announcement OPENOpen Access licenseComplete Genome Sequence Data of Two Xanthomonas arboricola Strains Isolated from Blueberry Plants Displaying Bacterial Leaf Blight in PolandMonika Kałużna and Joël F. PothierMonika Kałużna†Corresponding authors: M. Kałużna; E-mail Address: monika.kaluzna@inhort.pl, and J. F. Pothier; E-mail Address: joel.pothier@zhaw.chhttps://orcid.org/0000-0002-1745-1444The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland and Joël F. Pothier†Corresponding authors: M. Kałużna; E-mail Address: monika.kaluzna@inhort.pl, and J. F. Pothier; E-mail Address: joel.pothier@zhaw.chhttps://orcid.org/0000-0002-9604-7780Environmental Genomics and Systems Biology Research Group, Institute for Natural Resource Sciences, Zurich University of Applied Sciences, Wädenswil, SwitzerlandAffiliationsAuthors and Affiliations Monika Kałużna1 † Joël F. Pothier2 † 1The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland 2Environmental Genomics and Systems Biology Research Group, Institute for Natural Resource Sciences, Zurich University of Applied Sciences, Wädenswil, Switzerland Published Online:6 May 2022https://doi.org/10.1094/PHYTO-11-21-0484-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementThe cultivation of blueberry (Vaccinium corymbosum L.) is becoming increasingly important due to the high content of beneficial nutrients in the fruit, its attractiveness, and its high profitability. Both the worldwide total area harvested and production of blueberries have increased significantly since 1961 (FAOSTAT 2021). Poland is among the top 10 producers of blueberries, with an annual production that has reached the unprecedented amount of 34,770 tons in 2019 (FAOSTAT 2021) and, in 2021, the harvest was at the level of 55,300 tons (GUS 2021). For many years, this plant species remained rarely infected by bacterial pathogens. Hitherto described, but not present at a large intensity were tumorigenic Agrobacterium spp. causing crown gall (Alippi et al. 2012), Burkholderia andropogonis causing bacterial leaf spot (Kobayashi et al. 1995), and bacterial leaf scorch caused by the new emerging pathogen Xylella fastidiosa (Chang et al. 2009). Recently, blueberry plantations have been increasingly affected by Pseudomonas spp. (Kałużna et al. 2013), the causal agent of bacterial canker. New pathogenic bacteria—the subject of this study—were discovered that were never recorded on blueberry plantations. The strains reported here constitute the first report of a Xanthomonas sp. causing symptoms on this plant which might be economically relevant. The two strains were sequenced for further analysis of evolution within the species Xanthomonas arboricola, for determining whether the strains constitute a new pathovar within the species and improving the molecular diagnostics of this new pathogen.In 2013, blueberry cultivars Toro and Duke growing in a nursery located in Central Poland presented russet-brown, irregular spots on leaves (Fig. 1A). From these leaf spots, fluorescent and yellow bacteria were isolated. Colony morphology of yellow isolates resembling that of the Xanthomonas genus were obtained on yeast extract nutrient agar (YNA) medium (Schaad et al. 2001). Two yellow isolates, 1311a and 1314c, obtained from 'Toro' and 'Duke', respectively (Table 1), were positive in a PCR assay using primers X1 and X2 specific for bacteria belonging to the genus Xanthomonas (Maes 1993). The pathogenicity tests performed on blueberry cultivar Bluecrop confirmed their pathogenic ability (Fig. 1B). Based on partial sequence analysis of gyrB, the strains were not closely related to each other; however, both were placed within X. arboricola strains (Fig. 1C), a species known to cause symptoms on several fruit trees but never reported on blueberry, like any other Xanthomonas sp. so far. A further taxonomic study relying on multilocus sequence analysis of partial sequence analysis of gyrB, fuyA, and rpoD (totaling 1,635 bp) confirmed the definitive classification of these isolates (data not shown). The isolates were stored at −80°C in a mixture of glycerol 20% (vol/vol) and phosphate-buffered saline buffer (0.27% Na2HPO4, 0.04% NaH2PO4, and 0.8% NaCl) until further use. Before extraction of DNA for genomic analysis, the strains were cultured on YNA medium and incubated at 26°C for 48 h.Fig. 1. A, Leaves of Vaccinium corymbosum 'Duke' naturally infected by Xanthomonas arboricola 1314c and obtained from a nursery in Central Poland in 2013 displaying leaf spot symptoms. B, Leaf spots symptoms developing on V. corymbosum 'Bluecrop' after syringe infiltration with X. arboricola 1314c after 2 to 3 weeks postinoculation in the greenhouse under natural daylight conditions. C, Maximum-likelihood unrooted phylogenetic tree based on the analysis of 508 bp of gyrB partial sequences of Xanthomonas spp. The dendrogram was constructed based on the Tamura-Nei model. A discrete γ distribution was used to model evolutionary rate differences among sites (five categories; +G, parameter = 0.2776). Analyses were conducted using MEGA X version 10.0.5 (Kumar et al. 2018). Bootstrap values (expressed as percentages of 500 replicates) are indicated at each node and displayed only when over 50. Accession numbers or source for gyrB sequences are indicated within parentheses next to the species name, with strains sequenced in this study marked in bold. Superscripts following strain names: T indicates the type strain of a species and PT indicates the pathotype strain for a pathovar. D, Whole-genome BLAST distance phylogeny (GBDP) inferred with the Type (Strain) Genome Server (TYGS) (Meier-Kolthoff and Göker 2019). Results were provided by the TYGS on 27 October 2021 using the two genomes sequenced in this study in addition to 17 best-matching Xanthomonas type strains as determined by the TYGS platform. The tree was inferred with FastME version 2.1.6.1 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. Numbers above branches are GBDP pseudobootstrap support values >60% from 100 replicates, with an average branch support of 97.3%. The tree was rooted at the midpoint. Genome assembly accessions or accession numbers for genome sequences are indicated within parentheses next to the species name, with strains sequenced in this study marked in bold. Superscripts following strain names: T indicates the type strain of a species and PT indicates the pathotype strain for a pathovar.Download as PowerPointTable 1. Genome metrics and accession numbers of the newly sequenced Xanthomonas arboricola genomesStrainParametersa1311a1314cOrigin (year)Poland (2013)Poland (2013)HostVaccinium corymbosum 'Toro'Vaccinium corymbosum 'Duke'Genome size (bp)4,889,0214,891,115GC content (%)65.7165.7Total number of genes4,0494,069Illumina dataTotal number of reads3,579,2903,308,462Average read length (bp)251251Average coverage (×)171155Oxford Nanopore dataTotal number of reads68,923432,617Read length N50 (bp)23,76511,399Average coverage (×)149300SRA accession number (MinION/MiSeq)ERR5260057/ERR5260084ERR5260058/ERR5260086ENA accession numberHG992336HG992337ANI (%)96.5496.54dDDH (%)70.570.5BUSCO scores (%)99.899.9aSRA = Sequence Read Archive, ENA = European Nucleotide Archive, ANI = average nucleotide identity, dDDH = digital DNA-DNA hybridization, and BUSCO = benchmarking universal single-copy ortholog. ANI using BLAST (ANIb) and dDDH using the d4 formula are relative to X. arboricola pv. juglandis CFBP 2528T (GenBank genome assembly accession GCA_001013475.1). BUSCO used the xanthomonodales_odb10 (2020-03-06) lineage dataset.Table 1. Genome metrics and accession numbers of the newly sequenced Xanthomonas arboricola genomesView as image HTML Genomic DNA for short- and long-read sequencing was isolated according to the salt-extraction method described by Aljanabi and Martinez (1997), with slight modifications (Kałużna et al. 2012). A paired-end library (with insert size of approximately 350 bp) was prepared with the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB, Ipswich, MA, U.S.A.). Libraries were sequenced on a MiSeq sequencer (Illumina, San Diego, CA, U.S.A.) with 2× 250-bp paired-end reads using a MiSeq reagent kit version 2.The library for the MinION sequencing was prepared with the ligation sequencing kit (catalog number SQK-LSK1O8; Oxford Nanopore Technologies, Oxford, United Kingdom) and run on an R9.4.1 flow cell with a MinION sequencer. The native barcoding expansion kit (catalog number XP-NBD114) was used for multiplexing. Reads were base called and demultiplexed using Guppy version 3.3.3.Short- and long-read library preparation and sequencing were outsourced at Genomed S.A. (Warsaw, Poland).De novo hybrid assemblies using the MiSeq and MinION reads were conducted with Unicycler version 0.4.8 (Wick et al. 2017) and Trycycler version 0.3.3 for comparison purpose (Wick et al. 2021). In total, three and eight nucleotide changes for 1311a and 1314c, respectively, were performed during the first short-read polishing round using Pilon version 1.22. The genomes were then annotated using Prokka version 1.14.5 (Seemann 2014). All tools were run with default parameters.A single chromosomal scaffold of 4.9 Mbp with a G+C content of 65.7% was obtained for both strains (Table 1). Similar assembly results were provided by the second hybrid assembler. Genome completeness was 99.8% and 99.9% (Table 1) when assessed using the benchmarking universal single-copy ortholog (BUSCO) version 5.2.1 (Manni et al. 2021) and the xanthomonodales_odb10 (2020-03-06) lineage dataset. Whole-genome comparison based on average nucleotide identity using BLASTN (ANIb) implemented in pyANI version 0.2.10 (Pritchard et al. 2016) confirmed that the two strains had high degree of synteny (data not shown) to other X. arboricola genomes (data not shown) but particularly to X. arboricola pv. juglandis CFBP 2528T, the type strain of the X. arboricola species (Table 1). Digital DNA-DNA hybridization and genome phylogeny inferred with the Type (Strain) Genome Server (Meier-Kolthoff and Göker 2019) also clearly assigned the two strains sequenced here to the X. arboricola species (Table 1; Fig. 1D).To assess or highlight a relevant phytosanitary feature encoded by the two X. arboricola strains studied here, we analyzed the presence of genes involved in resistance to copper (Behlau et al. 2011). Results revealed the presence of the copAB operon and the copL, cutC, and pCuAC genes encoded by both strains. This observation suggests that these strains would survive at high copper concentration, as reported for other members of the species X. arboricola (Kałużna et al. 2021; Pothier et al. 2022).To limit the risk of introduction to other countries, three of the nine known X. arboricola pathovars (Kałużna et al. 2021) were listed by the European Plant Protection Organization as A2 quarantine pathogens and as Regulated Non-Quarantine Pests (Picard et al. 2018) since the end of 2019 (European Union 2019). Molecular methods are currently available for the detection and diagnostics of two of these three regulated X. arboricola pathovars (Catara et al. 2021); namely, X. arboricola pv. pruni (Bühlmann et al. 2013; Palacio-Bielsa et al. 2011; Pothier et al. 2011) and X. arboricola pv. juglandis (Fernandes et al. 2017; Martins et al. 2019). Nevertheless, for the remaining regulated pathovar (X. arboricola pv. corylina), amplicons are also obtained with the species- and pathovar-level primer sets designed for X. arboricola pv. pruni (Kałużna et al. 2021; Pothier et al. 2011; Webber et al. 2020), thus allowing the detection of both regulated pathogens. Here, once PCR was performed with the DNA of strains 1311a and 1314c, an amplicon was only observed with the species-level-specific primer set whereas no amplicon was observed with the pathovar-level primer set (pathovars pruni and corylina) or with the loop-mediated isothermal PCR set designed for X. arboricola pv. pruni (Bühlmann et al. 2013). Because our results suggest that the 1311a and 1314c strains belong to the X. arboricola species, we conclude that they also show the actual lack of a suitable molecular diagnostic for the strains causing symptoms on blueberry. Such molecular methods are required because further inspections confirmed the presence of X. arboricola on blueberry in other geographic localizations in Poland since the isolation of these two strains (M. Kałużna, unpublished data).The sequenced genomes discussed here will be used for further analysis of evolution within the species X. arboricola, as well as for the development of improved diagnostics tools for this possibly relevant pathogen of blueberry to enable an effective disease control strategy.Data AvailabilityThe raw data and assembled or annotated genome sequences have been deposited in the European Nucleotide Archive under BioProject number PRJEB42845. Genome data and raw read accession numbers for strains 1311a and 1314c are listed in Table 1.AcknowledgmentsWe thank the HPC team of the School for Life Sciences and Facility Management at the Zurich University of Applied Sciences for computer resources and support.The author(s) declare no conflict of interest.Literature CitedAlippi, A. M., López, A. C., and Balatti, P. A. 2012. Diversity among agrobacteria isolated from diseased plants of blueberry (Vaccinium corymbosum) in Argentina. Eur. J. Plant Pathol. 134:415-430. https://doi.org/10.1007/s10658-012-0001-x Crossref, ISI, Google ScholarAljanabi, S. M., and Martinez, I. 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res. 25:4692-4693. https://doi.org/10.1093/nar/25.22.4692 Crossref, Medline, ISI, Google ScholarBehlau, F., Canteros, B. I., Minsavage, G. V., Jones, J. B., and Graham, J. H. 2011. Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. citrumelonis. Appl. Environ. Microbiol. 77:4089-4096. Crossref, Medline, ISI, Google ScholarBühlmann, A., Pothier, J. F., Tomlinson, J. A., Frey, J. E., Boonham, N., Smits, T. H. M., and Duffy, B. 2013. Genomics-informed design of loop-mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level. Plant Pathol. 62:475-484. https://doi.org/10.1111/j.1365-3059.2012.02654.x Crossref, ISI, Google ScholarCatara, V., Cubero, J., Pothier, J. F., Bosis, E., Bragard, C., Đermić, E., Holeva, M. C., Jacques, M.-A., Petter, F., Pruvost, O., Robène, I., Studholme, D. J., Tavares, F., Vicente, J. G., Koebnik, R., and Costa, J. 2021. Trends in molecular diagnosis and diversity studies for phytosanitary regulated Xanthomonas. Microorganisms 9:862. https://doi.org/10.3390/microorganisms9040862 Crossref, Medline, ISI, Google ScholarChang, C.-J., Donaldson, R., Brannen, P., Krewer, G., and Boland, R. 2009. Bacterial leaf scorch, a new blueberry disease caused by Xylella fastidiosa. HortScience 44:413-417. https://doi.org/10.21273/HORTSCI.44.2.413 Crossref, ISI, Google ScholarEuropean Union. 2019. Commission implementing directive (EU) 2019/2072 of 28 November 2019 establishing uniform conditions for the implementation of regulation (EU) 2016/2031 of the European Parliament and the Council, as regards protective measures against pests of plants, and repealing commission regulation (EC) no. 690/2008 and amending commission implementing regulation (EU) 2018/2019. Off. J. Eur. Union L319:1-278. Google ScholarFAOSTAT. 2021. Food and agriculture data. Food and Agriculture Organization of the United Nations. https://www.fao.org/faostat/en/#home Google ScholarFernandes, C., Albuquerque, P., Sousa, R., Cruz, L., and Tavares, F. 2017. Multiple DNA markers for identification of Xanthomonas arboricola pv. juglandis isolates and its direct detection in plant samples. Plant Dis. 101:858-865. https://doi.org/10.1094/PDIS-10-16-1481-RE Link, ISI, Google ScholarGUS. 2021. Statistics Poland. Total cultivation area, yields and harvest of blueberry in orchards in 2021. Source of data from the Central Statistical Office of Poland, Główny Urząd Statystyczny (GUS). https://stat.gov.pl/en/ Google ScholarKałużna, M., Fischer-Le Saux, M., Pothier, J. F., Jacques, M.-A., Obradović, A., Tavares, F., and Stefani, E. 2021. Xanthomonas arboricola pv. juglandis and pv. corylina: brothers or distant relatives? Genetic clues, epidemiology, and insights for disease management. Mol. Plant Pathol. 22:1481-1499. https://doi.org/10.1111/mpp.13073 Crossref, Medline, ISI, Google ScholarKałużna, M., Janse, J. D., and Young, J. M. 2012. Detection and identification methods and new tests as used and developed in the framework of COST 873 for bacteria pathogenic to stone fruits and nuts: Pseudomonas syringae pathovars. J. Plant Pathol. 94:S1.117-S111.126. Google ScholarKałużna, M., Puławska, J., and Meszka, B. 2013. A new bacterial disease on blueberry (Vaccinium corymbosum) caused by Pseudomonas spp. J. Plant Prot. Res. 53:32-36. https://doi.org/10.2478/jppr-2013-0004 Crossref, Google ScholarKobayashi, D. Y., Stretch, A. W., and Oudemans, P. V. 1995. A bacterial leaf spot of highbush blueberry hardwood cuttings caused by Pseudomonas andropogonis. Plant Dis. 79:839-842. https://doi.org/10.1094/PD-79-0839 Crossref, ISI, Google ScholarKumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35:1547-1549. https://doi.org/10.1093/molbev/msy096 Crossref, Medline, ISI, Google ScholarMaes, M. 1993. Fast classification of plant-associated bacteria in the Xanthomonas genus. FEMS Microbiol. Lett. 113:161-165. https://doi.org/10.1111/j.1574-6968.1993.tb06508.x Crossref, ISI, Google ScholarManni, M., Berkeley, M. R., Seppey, M., Simão, F. A., and Zdobnov, E. M. 2021. BUSCO update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 38:4647-4654. https://doi.org/10.1093/molbev/msab199 Crossref, Medline, ISI, Google ScholarMartins, L., Fernandes, C., Albuquerque, P., and Tavares, F. 2019. Assessment of Xanthomonas arboricola pv. juglandis bacterial load in infected walnut fruits by quantitative PCR. Plant Dis. 103:2577-2586. https://doi.org/10.1094/PDIS-12-18-2253-RE Link, ISI, Google ScholarMeier-Kolthoff, J. P., and Göker, M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10:2182. https://doi.org/10.1038/s41467-019-10210-3 Crossref, Medline, ISI, Google ScholarPalacio-Bielsa, A., Cubero, J., Cambra, M. A., Collados, R., Berruete, I. M., and Lopez, M. M. 2011. Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species. Appl. Environ. Microbiol. 77:89-97. https://doi.org/10.1128/AEM.01593-10 Crossref, Medline, ISI, Google ScholarPicard, C., Afonso, T., Benko-Beloglavec, A., Karadjova, O., Matthews-Berry, S., Paunovic, S. A., Pietsch, M., Reed, P., van der Gaag, D. J., and Ward, M. 2018. Recommended regulated non-quarantine pests (RNQPs), associated thresholds and risk management measures in the European and Mediterranean region. Bull. OEPP/EPPO Bull. 48:552-568. https://doi.org/10.1111/epp.12500 Crossref, Google ScholarPothier, J. F., Kałużna, M., Prokić, A., Obradovic, A., and Rezzonico, F. 2022. Complete genome and plasmid sequence data of three strains of Xanthomonas arboricola pv. corylina, the bacterium responsible for bacterial blight of hazelnut. Phytopathology 112:956-960. https://doi.org/10.1094/PHYTO-08-21-0356-A Link, ISI, Google ScholarPothier, J. F., Pagani, M. C., Pelludat, C., Ritchie, D. F., and Duffy, B. 2011. A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni. J. Microbiol. Methods 86:16-24. https://doi.org/10.1016/j.mimet.2011.03.019 Crossref, Medline, ISI, Google ScholarPritchard, L., Glover, R. H., Humphris, S., Elphinstone, J. G., and Toth, I. K. 2016. Genomics and taxonomy in diagnostics for food security: Soft-rotting enterobacterial plant pathogens. Anal. Methods 8:12-24. https://doi.org/10.1039/C5AY02550H Crossref, ISI, Google ScholarSchaad, N. W., Jones, J. B., and Chun, W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. American Phytopathological Society, St. Paul, MN. Google ScholarSeemann, T. 2014. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. https://doi.org/10.1093/bioinformatics/btu153 Crossref, Medline, ISI, Google ScholarWebber, J. B., Putnam, M., Serdani, M., Pscheidt, J. W., Wiman, N. G., and Stockwell, V. O. 2020. Characterization of isolates of Xanthomonas arboricola pv. corylina, the causal agent of bacterial blight, from Oregon hazelnut orchards. J. Plant Pathol. 102:799-812. https://doi.org/10.1007/s42161-020-00505-6 Crossref, ISI, Google ScholarWick, R. R., Judd, L. M., Cerdeira, L. T., Hawkey, J., Méric, G., Vezina, B., Wyres, K. L., and Holt, K. E. 2021. Trycycler: Consensus long-read assemblies for bacterial genomes. Genome Biol. 22:266. https://doi.org/10.1101/2021.07.04.451066 Crossref, Medline, ISI, Google ScholarWick, R. R., Judd, L. M., Gorrie, C. L., and Holt, K. E. 2017. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13:e1005595. https://doi.org/10.1371/journal.pcbi.1005595 Crossref, Medline, ISI, Google ScholarM. Kałużna and J. F. Pothier contributed equally to this work.Funding: This work and the article publication charges were financed by the National Science Centre, Poland (Narodowe Centrum Nauki), grant UMO-2017/25/B/NZ9/01565 to M. Kałużna. J. F. Pothier acknowledges support from the Department of Life Sciences and Facility Management of the Zurich University of Applied Sciences (ZHAW) in Wädenswil. This article is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 112, No. 8 August 2022SubscribeISSN:0031-949Xe-ISSN:1943-7684 DownloadCaptionRing rot lesions caused by Botryosphaeria dothidea on young 'Fuji' apple fruit inoculated with conidia for 7 days (Liu et al.). Photo credit: Bao-hua Li Metrics Article History Issue Date: 28 Jul 2022Published: 6 May 2022Accepted: 15 Feb 2022 Pages: 1814-1818 Information© 2022 The American Phytopathological SocietyFundingNarodowe Centrum NaukiGrant/Award Number: UMO-2017/25/B/NZ9/01565Keywordsbacteriablueberrycomplete genomegenomicsXanthomonas arboricolaThe author(s) declare no conflict of interest.PDF download
Referência(s)