Skip to main content

Occurrence of copper-resistant Pseudomonas syringae pv. syringae strains isolated from rain and kiwifruit orchards also infected by P. s. pv. actinidiae

Abstract

During spring 2014–2015, leaves showing extensive leaf spotting were collected from Actinidia deliciosa and A. chinensis orchards located in northern, central and southern Italy where a copper-based strategy used to control bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), was ineffective. In addition, bacterial isolates were recovered from wind-driven raindrops. Psa was isolated solely from raindrops, whereas P. s. pv. syringae was consistently isolated from both symptomatic leaves and rain. The P. s. pv. syringae isolates were molecularly typed using repetitive-sequence PCR and assessed in vitro for their resistance to copper sulphate and for the presence of the cop ABCDRS gene cluster. An ad hoc quantitative real-time PCR was performed to confirm the P. s. pv. syringae capability for Actinidia spp. leaf infection using a low dose of inoculum and co-inoculation with Psa. A copper-resistant P. s. pv. syringae strain isolated from rain effectively colonized Actinidia spp. leaves from a low dose of inoculum. When such a strain was co-inoculated with a reference Psa strain, they reached a higher density than in inoculations performed singly. Copper-resistant P. s. pv. syringae strains infecting the leaves of Actinidia spp. appear to be widespread in Italy and, in some circumstances, they could have replaced Psa in leaf colonization and disease development. Populations of this phytopathogen showing resistance to copper would appear to be dispersed in the environment by the rain. This raises questions concerning the reliability of long-term strategies to control kiwifruit bacterial canker caused by Psa solely with copper compounds.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Aiello, D., Ferrante, P., Vitale, A., Polizzi, G., Scortichini, M., & Cirvilleri, G. (2015). Characterization of Pseudomonas syringae pv. syringae isolated from mango in Sicily and occurrence of copper-resistant strains. Journal of Plant Pathology, 97, 273–282.

    Google Scholar 

  • Altimira, F., Yanez, C., Bravo, G., Gonzàlez, M., Rojas, L. A., & Seeger, M. (2012). Characterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile. BMC Microbiology, 12, 193.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Andersen, C. B., Holst-Jensen, A., Berdal, K. G., Thorstensen, T., & Tengs, T. (2006). Equal performance of TaqMan, MGB, molecular beacon, and SYBR green-based detection assays in detection and quantification of roundup ready soybean. Journal of Agriculture and Food Chemistry, 54, 9658–9663.

    CAS  Article  Google Scholar 

  • Bender, C. L., & Cooksey, C. A. (1986). Indigenous plasmids in Pseudomonas syringae pv. Tomato: Conjugative transfer and role in copper resistance. Journal of Bacteriology, 165, 534–541.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Bender, C. L., & Cooksey, C. A. (1987). Molecular cloning of copper resistance genes in Pseudomonas syringae pv. Tomato. Journal of Bacteriology, 169, 470–474.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Bowers, R. M., & Dahr, A. K. (2011). Effect of template on generating a standard curve for absolute quantification of an RNA virus by real-time reverse transcriptase-polymerase chain reaction. Molecular and Cellular Probes, 25, 60–64.

    CAS  Article  PubMed  Google Scholar 

  • Buckling, A., Harrison, F., Vos, M., Brockhurst, M. A., Gardner, A., West, S. A., & Griffin, A. (2007). Siderophore-mediated cooperation and virulence in Pseudomonas aeruginosa. FEMS Microbiology Ecology, 62, 135–141.

    CAS  Article  PubMed  Google Scholar 

  • Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611–622.

    CAS  Article  PubMed  Google Scholar 

  • Cazorla, F. M., Arrebola, E., Sesma, A., Pérez-Garcia, A., Codina, J. C., Murillo, J., et al. (2002). Copper resistance in Pseudomonas syringae strains isolated from mango is encoded mainly by plasmids. Phytopathology, 92, 909–916.

    CAS  Article  PubMed  Google Scholar 

  • Cha, J.-S., & Cooksey, D. A. (1991). Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. Proceedings of the National Academy of Sciences USA, 88, 8915–8919.

    CAS  Article  Google Scholar 

  • Ciarroni, S., Gallipoli, L., Taratufolo, M. C., Butler, M. I., Poulter, R. T. M., Pourcel, C., et al. (2015). Development of a multiple loci variable number of tandem repeats analysis (MLVA) to unravel the intra-pathovar structure of Pseudomonas syringae pv. actinidiae populations worldwide. PLoS One, 10, e0135310.

    Article  PubMed  PubMed Central  Google Scholar 

  • Colombi, E., Straub, C., Künzel, S., Templeton, M. D., Mc Cann, H. C., & Rainey, P. B. (2017). Evolution of copper resistance in the kiwifruit pathogen Pseudomonas syringae pv. actinidiae through acquisition of integrative conjugative elements and plasmids. Environmental Microbiology, 19, 819–832.

    CAS  Article  PubMed  Google Scholar 

  • Donati, I., Buriani, G., Cellini, A., Mauri, S., Costa, G., & Spinelli, F. (2014). New insights on the bacterial canker of kiwifruit (Pseudomonas syringae pv. actinidiae). Journal of Berry Research, 4, 53–67.

    Article  Google Scholar 

  • European and Mediterranean Plant Protection Organization (EPPO) (2011). First report of Pseudomonas syringae pv. actinidiae in Chile. EPPO Reporting Service n° 03.

  • Everett, K. R., Taylor, R. T., Romberg, M. K., Rees-George, J., Fullerton, R. A., Vanneste, J. L., et al. (2011). First report of Pseudomonas syringae pv. actinidiae causing kiwifruit bacterial canker in New Zealand. Australasian Plant Disease Notes, 6, 67–71.

    Article  Google Scholar 

  • Ferrante, P., & Scortichini, M. (2009). Identification of Pseudomonas syringae pv. actinidiae as causal agent of bacterial canker of yellow kiwifruit (Actinidia chinensis Planchon) in central Italy. Journal of Phytopathology, 157, 768–770.

    Article  Google Scholar 

  • Ferrante, P., & Scortichini, M. (2010). Molecular and phenotypic features of Pseudomonas syringae pv. actinidiae isolated during recent epidemics of bacterial canker on yellow kiwifruit (Actinidia chinensis) in central Italy. Plant Pathology, 59, 954–962.

    CAS  Article  Google Scholar 

  • Ferrante, P., Fiorillo, E., Marcelletti, S., Marocchi, F., Mastroleo, M., Simeoni, S., et al. (2012). The importance of the main colonization and penetration sites of Pseudomonas syringae pv. actinidiae and prevailing weather conditions in the development of epidemics in yellow kiwifruit, recently observed in central Italy. Journal of Plant Pathology, 94, 455–461.

    Google Scholar 

  • Ferrante, P., Takikawa, Y., & Scortichini, M. (2015). Pseudomonas syringae pv. actinidiae strains isolated from past and current epidemics to Actinidia spp. reveal a diverse population structure of the pathogen. European Journal of Plant Pathology, 142, 677–689.

    CAS  Article  Google Scholar 

  • Fett, W. F., & Dunn, M. F. (1989). EPSs produced by Pseudomonas syringae pathovars in infected leaves of susceptible hosts. Plant Physiology, 89, 5–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Fitt, B. D. L., Mc Cartney, H. A., & Walklate, P. J. (1989). The role of rain in dispersal of pathogen inoculums. Annual Review of Phytopathology, 27, 241–270.

    Article  Google Scholar 

  • Gallelli, A., Talocci, S., Pilotti, M., & Loreti, S. (2014). Real-time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker. Plant Pathology, 63, 264–276.

    CAS  Article  Google Scholar 

  • Gao, X., Huang, Q., Zhao, Z., Han, Q., Ke, X., Qin, H., et al. (2016). Studies on the infection, colonization, and movement of Pseudomonas syringae pv. actinidiae in kiwifruit tissue using a GFPuv-labeled strain. PLoS One, 11, e0151169.

    Article  PubMed  PubMed Central  Google Scholar 

  • Garcia-Vallvé, S., Palau, J., & Romeu, A. (1999). Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Molecular Biology and Evolution, 16, 1125–1134.

    Article  PubMed  Google Scholar 

  • Goto, M., Hikota, T., Nakajima, M., Takikawa, Y., & Tsuyumu, S. (1994). Occurrence and properties of copper-resistance in plant pathogenic bacteria. Annals of the Phytopathological Society of Japan, 60, 147–153.

    CAS  Article  Google Scholar 

  • Greer, G., & Saunders, C. (2012). The cost of Psa-V to the New Zealand kiwifruit industry and the wider community. Lincoln University Research center; agribusiness and economy Research unit, Research report n° 237.

  • Gutiérrez-Barranquero, J. A., De Vicente, A., Carrion, V. J., Sundin, G. W., & Cazorla, F. M. (2013). Recruitment and rearrangement of three different genetic determinants into a conjugative plasmid increase copper resistance in Pseudomonas syringae. Applied and Environmental Microbiology, 79, 1028–1033.

    Article  PubMed  PubMed Central  Google Scholar 

  • Huvenne, H., Debode, J., Maes, M., & Heungens, K. (2011). Real-time PCR mediated monitoring of Fusarium foetens in symptomatic and non-symptomatic hosts. European Journal of Plant Pathology, 131, 705–717.

    Article  Google Scholar 

  • Kidambi, S. P., Sundin, G. W., Palmer, D. A., Chakrabarthy, A. M., & Bender, C. L. (1995). Copper as signal for alginate synthesis in Pseudomonas syringae pv. Syringae. Applied and Environmental Microbiology, 61, 2172–2179.

    CAS  PubMed  PubMed Central  Google Scholar 

  • King, E. O., Ward, M. K., & Raney, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescein. Journal of Laboratory and Clinical Medicine, 44, 301–307.

    CAS  PubMed  Google Scholar 

  • Koh, Y. J., Lee, D. H., Shin, J. S., & Hur, J.-S. (2001). Chemical and cultural control of bacterial blossom blight of kiwifruit caused by Pseudomonas syringae in Korea. New Zealand Journal of Crop and Horticultural Science, 29, 29–34.

    Article  Google Scholar 

  • Koh, Y. J., Kim, G. H., Koh, H. S., Lee, Y. S., Kim, S. C., & Jung, J. S. (2012). Occurrence of a new type of Pseudomonas syringae pv. actinidiae strain of bacterial canker on kiwifruit in Korea. Plant Pathology Journal, 28, 423–427.

    CAS  Article  Google Scholar 

  • Kümmerli, R., Van Den Berg, P., Griffin, A. S., West, S. A., & Gardner, A. (2010). Repression of competition favours cooperation: Experimental evidence from bacteria. Journal of Evolutionary Biology, 23, 699–706.

    Article  PubMed  Google Scholar 

  • Laue, H., Schenk, A., Li, H., Lambertsen, L., Neu, T. R., Molin, S., et al. (2006). Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae. Microbiology, 152, 2909–2918.

    CAS  Article  PubMed  Google Scholar 

  • Lelliott, R.A., & Stead, D.E. (1987). Methods for the diagnosis of bacterial diseases of plants. (in T.F. Preece (ed.), Methods in Plant Pathology, 216 pp., Oxford: Blackwell scientific publications.)

  • Louws, F. J., Fullbright, D. W., Stephens, C. T., & De Brujin, F. J. (1994). Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 60, 2286–2295.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Macho, A. P., Zumaquero, A., Ortiz-Martin, I., & Beuzón, C. R. (2007). Competitive index in mixed infection: A sensitive and accurate assay for the genetic analysis of Pseudomonas syringae-plant interactions. Molecular Plant Pathology, 8, 437–450.

    Article  PubMed  Google Scholar 

  • Marcelletti, S., Ferrante, P., Petriccione, M., Firrao, G., & Scortichini, M. (2011). Pseudomonas syringae pv. actinidiae draft genomes comparison reveal strain-specific features involved in adaptation and virulence to Actinidia species. PLoS One, 6, e27297.

  • Mazzaglia, A., Studholme, D. J., Taratufolo, M. C., Cai, R., Almeida, N. F., Goodman, T., et al. (2012). Pseudomonas syringae pv. actinidiae (PSA) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage. PLoS One, 7, e36518.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Mellano, M. A., & Cooksey, D. A. (1988). Induction of the copper resistance operon from Pseudomonas syringae. Journal of Bacteriology, 170, 4399–4401.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Monteil, C. L., Bardin, M., & Morris, C. E. (2014). Features of air masses associated with the deposition of Pseudomonas syringae and Botrytis cinerea by rain and snowfall. ISME Journal, 8, 2290–2304.

    Article  PubMed  PubMed Central  Google Scholar 

  • Morris, C. E., Monteil, C. L., & Berge, O. (2013). The life history of Pseudomonas syringae: Linking agriculture to earth system processes. Annual Review of Phytopathology, 51, 85–104.

    CAS  Article  PubMed  Google Scholar 

  • Mosser, T., Talagrand-Reboul, E., Colston, S. M., Graf, J., Figueras, M. J., Jumas-Bilak, E., et al. (2015). Exposure to pairs of Aeromonas strains enhances virulence in the Caenorhabditis elegans infection model. Frontiers in Microbiology, 6, 1218.

    Article  PubMed  PubMed Central  Google Scholar 

  • Pereira, T. P., Do Amaral, F. P., Dall’Asta, P., Brod, F. C., & Arisi, A. C. (2014). Real-time PCR quantification of the plant growth promoting bacteria Herbaspirillum seropedicae strain SmR1 in maize roots. Molecular Biotechnology, 56, 660–670.

    CAS  PubMed  Google Scholar 

  • Petriccione, M., Di Cecco, I., Arena, S., Scaloni, A., & Scortichini, M. (2013). Proteomic changes in Actinidia chinensis shoot during systemic infection with a pandemic Pseudomonas syringae pv. actinidiae strain. Journal of Proteomics, 78, 461–473.

    CAS  Article  PubMed  Google Scholar 

  • Petriccione, M., Mastrobuoni, F., Zampella, L., & Scortichini, M. (2015). Reference gene selection for normalization of RT-qPCR gene expression data from Actinidia deliciosa leaves infected with Pseudomonas syringae pv. actinidiae. Scientific Reports 5, 16961.

  • Scortichini, M. (2014). Field efficacy of chitosan to control Pseudomonas syringae pv. actinidiae, the causal agent of kiwifruit bacterial canker. European Journal of Plant Pathology, 140, 887–892.

    CAS  Article  Google Scholar 

  • Scortichini, M. (2016). Field efficacy of a zinc-copper-hydracid of citric acid biocomplex compound to reduce oozing from winter cankers caused by Pseudomonas syringae pv. actinidiae to Actinidia spp. Journal of Plant Pathology, 98, 651–655.

    Google Scholar 

  • Scortichini, M., Dettori, M. T., Marchesi, U., Palombi, M. A., & Rossi, M. P. (1998). Differentiation of Pseudomonas avellanae strains from Greece and Italy by rep-PCR genomic fingerprinting. Journal of Phytopathology, 146, 417–420.

    Article  Google Scholar 

  • Scortichini, M., Marchesi, U., Dettori, M. T., & Rossi, M. P. (2003). Genetic diversity, presence of the syrB gene, host preference and virulence of Pseudomonas syringae pv. syringae strains from woody and herbaceous host plants. Plant Pathology, 52, 277–286.

    CAS  Article  Google Scholar 

  • Scortichini, M., Marcelletti, S., Ferrante, P., Petriccione, M., & Firrao, G. (2012). Pseudomonas syringae pv. actinidiae: A re-emerging, multi-faceted, pandemic pathogen. Molecular Plant Pathology, 13, 631–640.

    Article  PubMed  Google Scholar 

  • Shinozaki, T., Shimizu, S., Wiyochi, T., Kusumoto, S., Sawada, H. (2014). Occurrence of kiwifruit canker caused by virulent strain (Psa 3) of Pseudomonas syringae pv. actinidiae in Ehime prefecture. Phytopathological Society of Japan, Kansai district meeting, p. 39.

  • Sorensen, K. N., Kim, K. H., & Takemoto, J. Y. (1988). PCR detection of cyclic lipodepsinonapeptide-producing Pseudomonas syringae pv. Syringae and similarity of strains. Applied and Environmental Microbiology, 64, 226–230.

    Google Scholar 

  • Sundin, G. W., Jones, A. L., & Fulbright, D. W. (1989). Copper resistance in Pseudomonas syringae pv. syringae from cherry orchards and its associated transfer in vitro and in planta with a plasmid. Phytopathology, 79, 861–865.

    CAS  Article  Google Scholar 

  • Tyson, J. L., Curtis, C. L., Manning, M. A., Rees-George, J., Snelgar, W. P., & Blattman, P. (2014). Systemic movement of Pseudomonas syringae pv. actinidiae in kiwifruit vines in New Zealand. New Zealand Plant Protection, 67, 41–47.

    Google Scholar 

  • Vanneste, J. L., Yu, L., Cornish, D. A., Tanner, D. J., Windner, R., Chapman, J. R., et al. (2013). Identification, virulence and distribution of two biovars of Pseudomonas syringae pv. actinidiae in New Zealand. Plant Disease, 97, 708–719.

    CAS  Article  Google Scholar 

  • Yu, J., Peňaloza-Vazquez, A., Chakrabarty, A. M., & Bender, C. M. (1999). Involvement of the exopolisaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. Syringae. Molecular Microbiology, 33, 712–720.

    CAS  Article  PubMed  Google Scholar 

  • Zampella, L., Mastrobuoni, F., Petriccione, M., Ferrante, P., Marcelletti, S., Scortichini, M. (2015). First report of Pseudomons syringae pv. actinidiae on Actinidia spp. cultivated in Campania (southern Italy). Journal of Plant Pathology 97 (supplement), S77.

  • Zhang, J. H., Quigley, N. B., & Gross, D. C. (1995). Analysis of the syrB and syrC genes of Pseudomonas syringae pv. syringae indicates that syringomycin is synthesized by a thiotemplate mechanism. Journal of Bacteriology, 177, 4009–4020.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Zhao, Z., Gao, X. N., Yang, D. H., Huang, L. L., Qin, H. Q., & Kang, Z. S. (2015). Field detection of canker-causing bacteria on kiwifruit trees: Pseudomonas syringae pv. actinidiae is the major causal agent. Crop Protection, 75, 55–62.

    Article  Google Scholar 

Download references

Acknowledgments

This study has received funding from the European Union Seventh Programme (FP7/2007-2013) under the grant agreement n° 613678 (DROPSA). The authors wish to thank Y. Takikawa, Shizuoka University, Japan, for having supplied the copper-resistant Psa SUPP 765 strain.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Marco Scortichini.

Ethics declarations

Conflict of interest

The authors declare no competing interest.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Petriccione, M., Zampella, L., Mastrobuoni, F. et al. Occurrence of copper-resistant Pseudomonas syringae pv. syringae strains isolated from rain and kiwifruit orchards also infected by P. s. pv. actinidiae . Eur J Plant Pathol 149, 953–968 (2017). https://doi.org/10.1007/s10658-017-1246-1

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-017-1246-1

Keywords

  • Actinidia
  • Bacterial canker
  • Real-time PCR
  • Epidemiology
  • Plant disease management