Abstract
Preventive measures are extremely important for control of diseases that are caused by plant pathogenic bacteria on economically important plants. For this reason, fast and reliable detection methods are required. Along with the time-consuming conventional detection methods such as isolation and culturing of bacteria on media, PCR-based methods have been introduced as supplementary tests for better diagnosis of plant pathogenic bacteria . In the case of non-culturable bacteria, such as Phytoplasma s, PCR-based tests even became indispensable. Real-time PCR has become a widely used platform in nucleic acid detection and quantification in diagnostics. Despite the general guidelines for some of the steps, there are no common guidelines or standard operating procedures for introduction of real-time PCR -based detection systems for pathogens for routine laboratory use. Four cases of detection systems for grapevine pathogenic bacteria that were developed at the National Institute of Biology are presented and discussed here, providing a practical overview of the whole process from the initial assay design to the implementation of the assay into the laboratory.
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Abbreviations
- AY:
-
Aster yellows
- BN:
-
Bois noir
- DNA:
-
Deoxyribonucleic acid
- ELISA:
-
Enzyme-linked immunosorbent assay
- EPPO:
-
European and Mediterranean Plant Protection Organisation
- FD:
-
Flavescence dorée
- GYs:
-
Grapevine yellows
- OEPP:
-
Organisation Européenne et Méditerranéenne pour la Protection des Plantes
- PCR:
-
Polymerase chain reaction
- qPCR:
-
Real-time PCR
References
Alma A, Davis RE, Vibio M, Danielli A, Bosco D, Arzone A, Bertaccini A (1996) Mixed infection of grapevines in northern Italy by Phytoplasma s including 16S rRNA RFLP subgroup 16SrI-B strains previously unreported in this host. Plant Dis 80:418–421
Angelini E, Bianchi GL, Filippin L, Morassutti C, Borgo M (2007) A new TaqMan method for the identification of Phytoplasma s associated with grapevine yellows by real-time PCR assay. J Microbiol Methods 68:613–622
Baric S, Dalla-Via J (2004). A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. J Microbiol Methods 57:135–145
Bertaccini A, 2007. Phytoplasma s: diversity, taxonomy, and epidemiology. Front Biosci 12:673–689
Bianco PA, Casati P, Marziliano N (2004) Detection of Phytoplasma s associated with grapevine flavescence dorée disease using real-time PCR . J Plant Pathol 86, 257–61
Boben J, Kramberger P, Petrovič N, Cankar K, Peterka M, Štrancar A. Ravnikar M (2007) Detection and quantification of Tomato mosaic virus in irrigation waters. Eur J Plant Pathol 118:59–71
Botha WJ, Serfontein S, Greyling MM, Berger DK (2001) Detection of Xylophilus ampelinus in grapevine cuttings using a nested polymerase chain reaction. Plant Pathol 50, 515–526
Buh Gašparič M, Cankar K, Žel J, Gruden K, 2008. Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms. BMC Biotechnol. doi:10.1186/1472-6750-8-26
Bustin SA (2005) Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences. Expert Rev Mol Diagn 5:493–498
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR-a perspective. J Mol Endocrinol 34:597–601
Bustin SA, Mueller R, 2005. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci 109:365–379
Camloh M, Dreo T, Žel J, Ravnikar M (2008) The flexible scope of accreditation in GMO testing and its applicability to plant pathogen diagnostics. EPPO Bulletin 38:178–184
Campanharo JC, Lemos MVF, Lemos E (2003). Growth optimization procedures for the phytopathogen Xylella fastidiosa. Cur Microbiol 46:99–102
Cankar K, Štebih D, Dreo T, Žel J, Gruden K (2006). Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnol. doi:10.1186/1472-6750-6-37
Carbajal D, Morano KA, Morano LD (2004). Indirect immunofluorescence microscopy for direct detection of Xylella fastidiosa in xylem sap. Cur Microbiol 49:372–375
Christensen NM, Axelsen KB, Nicolaisen M, Schulz A (2005). Phytoplasma s and their interactions with hosts. Trends Plant Sci 10:526–535
Christensen NM, Nicolaisen M, Hansen M, Schulz A (2004). Distribution of Phytoplasma s in infected plants as revealed by real-time PCR and bioimaging. Mol Plant Microbe Interact 17:1175–1184
Clair D, Larrue J, Aubert G, Gillet J, Cloquemin G, Boudon-Padieu E (2003). A multiplex nested-PCR assay for sensitive and simultaneous detection and direct identification of Phytoplasma in the Elm yellows group and Stolbur group and its use in survey of grapevine yellows in France. Vitis 42:151–57
Council Directive 2000/29/EC of 8 May 2000 on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community (L169/1,10/07/2000)
Daire X, Clair D, Reinert W, Boudon-Padieu E (1997) Detection and differentiation of grapevine yellows Phytoplasma s belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA. Eur J Plant Pathol 103:507–514
Dinnes J, Deeks J, Kirby J, Roderick P (2005) A methodological review of how heterogeneity has been examined in systematic reviews of diagnostic test accuracy. Health Technol Assess 9:1–128
Dreo T, Gruden K, Manceau C, Janse JD, Ravnikar M (2007) Development of a real-time PCR -based method for detection of Xylophilus ampelinus . Plant Pathol 56:9–16
EPPO/CABI (2003) Data sheets on quarantine pests – grapevine Flavescence dorée Phytoplasma . In: Smith IM, McNamara DG, Scott PR, Holderness M (eds) Quarantine Pests for Europe. CABI International, Wallingford, UK
EPPO Standards/Normes OEPP (2004) Diagnostic protocols for regulated pests, Xylella fastidiosa. Bulletin OEPP/EPPO: Bulletin 34:187–119
Francis M, Lin H, Rosa JC-L, Doddapaneni H, Civerolo EL (2006) Genome-based PCR primers for specific and sensitive detection and quantification of Xylella fastidiosa. Eur J Plant Pathol 115:203–213
French WJ, Stassi DL, Schaad NW (1978) The use of immunofluorescence for the identification of peach phony bacterium. Phtopathol 68:1106–1108
Galetto L, Bosco D, Marzachı C (2005) Universal and groups pecific real-time PCR diagnosis of flavescence doreé (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) Phytoplasma s from field-collected plant hosts and insect vectors. Ann Appl Biol 147:191–201
Gutiérrez-Aguirre I, Mehle N, Delić D, Gruden K, Mumford R, Ravnikar M, 2009. Real-time quantitative PCR based sensitive detection and genotype discrimination of Pepino mosaic virus. J Virol Methods 162:46–55
Hodgetts J, Boonham N, Mumford R, Dickinson M (2009) Panel of 23S rRNA gene-based real-time PCR assays for improved universal and group-specific detection of Phytoplasma s. Appl Environ Microbiol 75:2945–2950
Hogenhout SA, Loria R (2008) Virulence mechanisms of Gram-positive plant pathogenic bacteria . Cur Opin Plant Biol 11:449–456
Hopkins DL (1981) Seasonal concentration of Pierce's disease bacterium in grapevine stems, petioles and leaf veins. Phytopathol 71:415–418
Hopkins DL, Purcell AH (2002) Xylella fastidiosa: cause of Pierce's disease of grapevine and other emergent diseases. Plant Dis 86:1056–1066
Hren M, Boben J, Rotter A, Kralj P, Gruden K, Ravnikar M (2007) Real-time PCR detection systems for Flavescence dorée and Bois noir Phytoplasma s in grapevine: comparison with conventional PCR detection and application in diagnostics. Plant Pathol 56:785–796
Kogovšek P, Gow L, Pompe-Novak M, Gruden K, Foster GD, Boonham N, Ravnikar M (2008) Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates. J Virol Methods 149:1–11
Lee I–M, Gundersen–Rindal DE, Davis RE, Bartoszyk IM (1998). Revised classification scheme of Phytoplasma s based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int Journal Syst Evol Microbiol 48:1153–1169
Lelliott RA, Stead DE (1987) Diagnostic procedures for bacterial plant diseases. Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Publications, Oxford, UK
López MM, Bertolini E, Marco-Noales E, Llop P,Cambra M (2006) Update on molecular tools for detection of plant pathogenic bacteria and viruses. In: Rao JR, Fleming CC, Moore JE, (eds) Molecular diagnostics: current technology and applications. Horizon Bioscience, Norfolk, UK
Marzachi C, Palermo S, Boarino A, Veratti F, D'Aquilio M, Loria A, Boccardo G (2001) Optimization of a one–step PCR assay for the diagnosis of Flavescence dorée –related Phytoplasma s in field–grown grapevines and vector populations. Vitis 40:213–217
Margaria P, Turina M, Palmano S (2009) Detection of Flavescence doreée and Bois noir Phytoplasma s, Grapevine leafroll associated virus-1 and -3 and Grapevine virus A from the same crude extract by reverse transcription-RealTime TaqMan assays. Plant Pathol 58:838–845
Manceau C, Coutaud MG, Guyon R (2000) Assessment of subtractive hybridization to select pecies and subspecies specific DNA fragments for the identification of Xylophilus ampelinus by polymerase chain reaction (PCR ). Eur J Plant Pathol 106: 243–254
Manceau C, Grall S, Brin C, Guillaumes J (2005) Bacterial extraction from grapevine and detection of Xylophilus ampelinus by a PCR and microwell plate detection system. Bulletin OEPP 34:55–60
Minsavage GV, Thompson CM, Hopkins DL, Leite RMVBC, Stall RE (1994) Development of a polymerase chain reactionprotocol for detection of Xylella fastidiosa in plant tissue. Phytopathol 84:456–461
Pelletier C, Salar P, Gillet J, Cloquemin G, Very P, Foissac X, Malembic-Maher S (2009) Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine Phytoplasma s of the 16SrV and 16SrXII-A groups with an endogenous analytical control. Vitis 48:87–95
Pirc M, Ravnikar M, Tomlinson J, Dreo T (2009) Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA. Plant Pathol 58:872–881
Purcell AH, Hopkins DL (1996) Fastidious xylem-limited bacterial plant pathogens. Ann Rev Phytopathol 34:131–151
Schaad NW, Opgenorth D, Gaush P (2002) Real-time polymerase chain reaction for one-hour on-site diagnosis of Pierce’s disease of grape in early season asymptomatic vines. Phtopathol 92:721–728
Seddas A, Meignoz R, Daire X, Boudon-Padieu E (1996) Generation and characterization of monoclonal antibodies to Flavescence dorée Phytoplasma : serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows Phytoplasma s. European Journal of Plant Pathology 102:757–764
Smart CD, Hendson M, Guilhaert MR, Saunders S, Friebertshouser G, Purcell A, Kirkpatrick BC (1998) Seasonal detection of Xylella fastidiosa in grapevines with culture, ELISA and PCR . Phytopathol 88, S83(suppl.)
Torres E, Bertolini E, Cambra M, Monton C, Martin MP (2005) Real-time PCR for simultaneous and quantitative detection of quarantine Phytoplasma s from apple proliferation (16SrX) group. Mol Cell Probes 19:334–340
Wells JM, Raju BC, Hung HY, Weisburg WG, Mandeico-Paul L, Brenner DJ (1987) Xylella fastidiosa gen. Nov., sp. Nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int J Syst Evol Bacteriol 37:136–143
Willems A, Gillis M, Kersters K, Van Den Broecke L, de Ley J (1987) Transfer of Xanthomonas ampelina Panagopoulos 1969 to a new genus, Xylophilus gen. nov., as Xylophilus ampelinus (Panagopoulos 1969) comb. nov. Int J Syst Bacteriol 37: 422–430
Žel J, Mazzara M, Savini C, Cordeil S, Camloh M, Štebih D, Cankar K, Gruden K, Morisset D, Van den Eede G (2008) Method Validation and Quality Management in the Flexible Scope of Accreditation: An Example of Laboratories Testing for Genetically Modified Organisms. Food Anal Method 1:61–72
Acknowledgments
This work was partially supported by the Phytosanitary Administration and Phytosanitary Inspectorate of the Republic of Slovenia, Slovenian Ministry of Education, Science and Sport and Ministry of Agriculture, Forestry and Food. We thank Norman W. Schaad (USDA) and Igor Zidarič (Agricultural Institute of Slovenia) for bacterial strains, Gabrijel Seljak, Msc. for field work and Magda Tušek Žnidarič and Nataša Mehle for help with ELISA .
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Hren, M. et al. (2010). Real-Time PCR Detection Methods for Economically Important Grapevine Related Bacteria. In: Delrot, S., Medrano, H., Or, E., Bavaresco, L., Grando, S. (eds) Methodologies and Results in Grapevine Research. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9283-0_15
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DOI: https://doi.org/10.1007/978-90-481-9283-0_15
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