Standard protocols for detection of phytopathogenic Pseudomonas spp. and Ralstonia solanacearum in plant material, soil, water or other sources, often still rely on the isolation of bacterial colonies on appropriate media, and/or on the use of serological techniques. However, over the last several years, molecular techniques, mainly based on PCR methods after extraction of nucleic acids from samples, have improved enough to allow a more rapid, reliable detection of these bacteria. When maximum accuracy is required the use of multiple techniques in an integrated approach is advised. Other promising technologies like flow cytometry, electronic nose or microarrays are emerging due to the need for developing more rapid high throughput detection methods.
Molecular typing methods are very useful to study intra-specific diversity, to identify sources of inoculum and to track the spread of the infections, although phenotypic methods are also used. Several reliable molecular techniques including AFLP, BOX, ERIC and rep-PCR, IS-RFLP, MLST, and macrorestriction-PFGE are available. However there is no single technique that can be recommended and polyphasic analyses have provided to be most useful. The diversity of Spanish strains of R. solanacearum and P. savastanoi pv. savastanoi analysed by several techniques is discussed in detail.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Agron, P.G., Macht, M., Radnedge, L., Skowronski, E.W., Miller, W. & Andersen, G.L. (2002). Use of subtractive hybridisation for comprehensive surveys of prokaryotic genome differences. FEMS Microbiol. Lett. 211, 175–182.
Álvarez, A.M. (2004). Integrated approaches for detection of plant pathogenic bacteria and diagnosis of bacterial diseases. Annu. Rev. Phytopathol. 42, 339–366.
Anonymous (1998). Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. Official J. Eur. Communities, No. L235/1, 21.08.98.
Anonymous (2004). EPPO standards. Diagnostic protocols for regulated pests. Ralstonia solanacearum. Bull. OEPP-EPPO Bull. 34, 173–178.
Anonymous (2005). EPPO standards. Diagnostic protocols for regulated pests. Pseudomonas syringae pv. persicae. Bull. OEPP-EPPO Bull. 35, 285–287.
Anonymous (2006). Commission Directive 2006/63/EC of 14 July 2006 amending Annexes II to VII to Council.
Arahal, D.R., Llop, P., Alonso, M. & López, M.M. (2004). In silico evaluation of molecular probes for detection and identification of Ralstonia solanacearum and Clavibacter michiganensis subsp. sepedonicus. Syst. Appl. Microbiol. 27, 581–591.
Bertolini, E., Olmos, A., López, M.M. & Cambra, M. (2003a). Multiplex nested reverse transcription-polymerase chain reaction in a single closed tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology 93, 286–292.
Bertolini, E., Peñalver, R., García, A., Olmos, A., Quesada, J.M., Cambra, M. & López, M.M. (2003b). Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube. J. Microbiol. Methods 52, 261–266.
Boudazin, G., Le Roux, A.C., Josi, K., Labarre, P. & Jouan, B. (1999). Design of division-specific primers of Ralstonia solanacearum and application to the identification of European isolates. Eur. J. Plant Pathol. 105, 373–380.
Caruso, P. (2005). Detección y caracterización serológica y molecular de Ralstonia solanacearum biovar 2, causante de la marchitez y podredumbre parda en patata. Tesis doctoral, Universidad de Valencia, Spain.
Caruso, P., Bertolini, E., Cambra, M. & López, M.M. (2003). A new and co-operational polymerase chain reaction (Co-PCR) for rapid detection of Ralstonia solanacearum in water. J. Microbiol. Methods 55, 257–272.
Catara, V., Arnold, D., Cirvilleri, G. & Vivian, A. (2000). Specific oligonucleotide primers for the rapid identification and detection of the agent of tomato pith necrosis, Pseudomonas corrugata, by PCR amplification: evidence for two distinct genomic groups. Eur. J. Plant Pathol. 106, 753–762.
Cubero, J., Martínez, M.C., Llop, P. & López, M.M. (1999). A simple and efficient PCR method for the detection of Agrobacterium tumefaciens in plant tumours. J. Appl. Microbiol. 86, 591–602.
Danial, J., Mc Hugh, R.C. & Saddler, G.S. (2006). Molecular characterization of the potato brown rot pathogen Ralstonia solanacearum, race 3, biovar 2A. Proceedings of the 4th International Bacterial Wilt Symposium. CSL, York, UK, p. 28.
Davey, H.M. & Kell, D.B. (1996). Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol. Rev. 60, 641–696.
de Lacy Costello, B.P.J., Ewen, R.J., Garner, K., Ratcliffe, N.M., Schleicher, T. & Spencer-Phillips, P.T.N. (2006). Sensors for detection of bacterial infections of potato tubers: from Erwinia to Clavibacter. Proceedings of the 11th International Conference on Plant Pathogenic Bacteria. Royal College of Physicians of Edinburgh, Scotland, pp. 27–28.
de León, L., Siverio, F. & Rodríguez, A. (2006). Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation. J. Microbiol. Methods 67, 141–149.
Fegan, M. & Prior, P. (2005). How complex is the Ralstonia solanacearum species complex? In: Bacterial Wilt Disease, and the Ralstonia solanacearum Species Complex (Eds. Prior, P., Allen, J. & Hayward, A.C.). St. Paul, MN, APS Press, pp. 449–461.
Fessehaie, A.S.H., Boer, D. & Levesque, C.A. (2003). An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato. Phytopathology 93, 262–269.
Grey, B.E. & Steck, T.R. (2001). The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl. Environ. Microbiol. 67, 3866–3872.
Hayward, A.C., El-Nashaar, H.M., Nydegger, U. & De Lindo, L. (1990). Variation in nitrate metabolism in biovars of Pseudomonas solanacearum. J. Appl. Bacteriol. 69, 269–280.
Llop, P., Caruso, P., Cubero, J., Morente, C. & López, M.M. (1999). A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. J. Microbiol. Methods 37, 23–31.
López, M.M., Bertolini, E., Caruso, P., Penyalver, R., Marco-Noales, E., Gorris, M.T., Morente, C., Salcedo, C., Cambra, M. & Llop, P. (2005). Advantages of an integrated approach for diagnosis of quarantine pathogenic bacteria in plant material. Phytopathol. Polonica 35, 49–56.
López, M.M., Bertolini, E., Marco-Noales, E., Llop, P. & Cambra, M. (2006). Update on molecular tools for detection of plant pathogenic bacteria and viruses. In: Molecular Diagnostics. Current Technology and Applications (Eds. Rao, J.R., Fleming, C.C. & Moore, J.E.). Horizon Bioscience, Norfolk, UK, pp. 1–46.
López, M.M., Bertolini, E., Olmos, A., Caruso, P., Gorris, M.T., Llop, P., Penyalver, R. & Cambra, M. (2003). Innovative tools for detection of plant pathogenic viruses and bacteria. Int. Microbiol. 6, 233–243.
López, M.M., Gorris, M.T., Llop, P., Cubero, J., Vicedo, B. & Cambra, M. (1997). Selective enrichment improves the isolation, serological and molecular detection of plant pathogenic bacteria. In: Diagnosis and Identification of Plant Pathogens (Eds. Dehne, H.W., Adam, G., Diekmann, M., Frahn, J. & Mauler, A.). Kluwer.
López, M.M., Llop, P., Cubero, J., Penyalver, R., Caruso, P., Bertolini, E., Peñalver, J., Gorris, M.T., Olmos, A. & Cambra, M. (2001). Strategies for improving serological and molecular detection of plant pathogenic bacteria. Proceedings of the 10th International Conference on Plant Pathogenic Bacteria. Charlottetown, Canada, Kluwer, pp. 83–85.
Louws, F.J., Rademaker, J.L.W. & Brujin, F.J. (1999). The three Ds of PCR-based genomic analysis of phytobacteria: diversity, detection, and disease diagnosis. Ann. Rev. Phytopathol. 37, 81–125.
Mills, D., Russell, B.W. & Hanus, J.W. (1997). Specific detection of Clavibacter michiganensis subsp. sepedonicus by amplification of three unique DNA sequences isolated by subtraction hybridization. Phytopathology 87, 853–861.
Ordax, M., Marco-Noales, E., López, M.M. & Biosca, E.G. (2006). Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state. Appl. Environ. Microbiol. 72, 3482–3488.
Ozakman, M. & Schaad, N.W. (2003). A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers. Can. J. Plant Pathol. 25, 232–239.
Penyalver, R., García, A., Ferrer, A., Bertolini, E. & López, M.M. (2000). Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR. Appl. Environ. Microbiol. 66, 2673–2677.
Prosen, D., Hatziloukas, E., Schaad, N.W. & Panopoulas, N.J. (1993). Specific detection of Pseudomonas syringae pv. phaseolicola DNA in bean seed by polymerase chain reaction-based amplification of a phaseolotoxin gene region. Phytopathology 83, 965–970.
Quesada, J.M. (2007). Epidemiología y control químico de Pseudomonas savastanoi pv. savastanoi causante de la tuberculosis del olivo. Tesis doctoral, Universidad Politécnica de Valencia, Spain.
Saettler, A.W., Schaad, N.W. & Roth, D.A. (1995). Detection of bacteria in seed and other planting material. St. Paul, MN, APS, 122 pp.
Sarkar, S.F. & Guttman, D.S. (2004) Evolution of the core genome of Pseudomonas syringae, a highly clonal, endemic plant pathogen. Appl. Environ. Microbiol. 70, 1999–2012.
Schaad, N.W. & Frederick, R.D. (2002). Real-time PCR and its application for rapid plant disease diagnostics. Canadian J. Plant Pathol. 24, 250–258.
Schaad, N.W., Berthier-Schaad, Y. & Knorr, D. (2007). A high throughput membrane BIO-PCR technique for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola. Plant Pathol. 56, 1–8.
Schaad, N.W., Frederick, R.D., Shaw, J., Schneider, W.L., Hickson, R., Petrillo, M.D. & Luster, D.G. (2003). Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues. Annu. Rev. Phytopathol. 41, 305–324.
Schaad, N.W., Jones, J.B. & Chun, W. (2001). Laboratory guide for the identification of plant pathogenic bacteria, 3rd edn. St. Paul, MN, APS Press, 370 pp.
Schaad, N.W., Tamaki, S., Hatziloukas, E. & Panopoulos, N. (1995). A combined biological amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85, 243–248.
Schoen, C., De Weerdt, M., Hillhorst, R., Boender, P., Szemes, M., & Bonants, P. (2003). Multiple detection of plant (quarantine) pathogens by micro-arrays: innovative tool for plant health management. In Abstracts of the 19th International Symposium on Virus and Virus-like Diseases of Temperate Fruit Crops, Valencia, Spain, p. 108.
Schoen, C., De Weerdt, M., Hillhorst, R., Chan, A., Boender, P., Zijlstra, C. & Bonants, P. (2002). Use of novel 3D microarray flow through system for plant pathogen multiplex detection. In Abstracts Agricultural Biomarkers for Array Technology, Management Committee Meeting, Wadenswil, Switzerland, p. 11.
Scortichini, M., Rossi, M.P. & Salerno, M. (2004). Relationship of genetic structure of Pseudomonas savastanoi pv. savastanoi populations from Italian olive trees and patterns of host genetic diversity. Plant Pathol. 53, 491–497.
Seal, S., Jackson, L., Young, J.P. & Daniels, J. (1993). Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the blood disease bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. J. Gen. Microbiol. 139, 1587–1594.
Seal, S.E., Jackson, L.A., & Daniels, M.J. (1992). Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridisation and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction. Appl. Environ. Microbiol. 58, 3751–3758.
Soby, S., Kirkpatrick, B. & Kosuge, T. (1993). Characterization of an insertion sequence IS53 located within IS51 on the iaa-containing plasmid of Pseudomonas syringae pv. savastanoi. Plasmid 29, 135–141.
Stinton, J.A., Persaud, K.C., Stead, D., Bryning, G. & Parkinson, N. (2006). Using an SPME-Enose to identify quarantine pathogens of potatoes. Proceedings of the 11th International Conference on Plant Pathogenic Bacteria. Royal College of Physicians of Edinburgh, Scotland, pp. 29–30.
Tenover, F.C., Arbiet, R.D. & Goering, R.V. (1997). How to select and interpret molecular typing strain methods for epidemiologic studies of bacterial infections: a review for health care epidemiologists. Infect. Control. Hosp. Epidemiol. 18, 426–439.
Timms-Wilson, T.M., Bryant, K. & Bailey, M.J. (2001). Strain characterization and 16S–23S probe development for differentiating geographically dispersed isolates of the phytopathogen Ralstonia solanacearum. Environ. Microbiol. 3, 785–797.
Van der Wolf, J.M., Bonants, P.J.M., Smith, J.J., Hagenaar, M., Nijhuis, E., van Beckhoven, J.R.C.M., Saddler, G.S., Trigalet, A. & Feuillade, R. (1998). Genetic diversity of Ralstonia solanacearum race 3 in Western Europe determined by AFLP, RC-PFGE and Rep-PCR. In: Bacterial Wilt Disease, Molecular and Ecological Aspects (Eds. Prior, P., Allen, J. & Elphinstone, J.). Berlin, Springer, pp. 44–49.
Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K. & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60, 407–438.
Walcott, R.R. & Gitaitis, R.D. (2000). Detection of Acidovorax avenae subsp. citrulli in watermelon seed using immunomagnetic separation and the polymerase chain reaction. Plant Dis. 84, 470–474.
Weller, S.A., Elphinstone, J.G., Smith, N.C., Boonham, N. & Stead, D.E. (2000). Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl. Environ. Microbiol. 66, 2853–2858.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Springer Science + Business Media, B.V
About this chapter
Cite this chapter
López, M.M. et al. (2008). Current Technologies for Pseudomonas spp. And Ralstonia solanacearum Detection and Molecular Typing. In: Fatmi, M., et al. Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-6901-7_1
Download citation
DOI: https://doi.org/10.1007/978-1-4020-6901-7_1
Publisher Name: Springer, Dordrecht
Print ISBN: 978-1-4020-6900-0
Online ISBN: 978-1-4020-6901-7
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)