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Letter to the Editor Pcr Detection and Identification of Plant-Pathogenic Bacteria: Updated Review of Protocols (1989-2007)

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Letter to the Editor Pcr Detection and Identification of Plant-Pathogenic Bacteria: Updated Review of Protocols (1989-2007) 002_LetterEditor_249 25-06-2009 10:41 Pagina 249 Journal of Plant Pathology (2009), 91 (2), 249-297 Edizioni ETS Pisa, 2009 249 LETTER TO THE EDITOR PCR DETECTION AND IDENTIFICATION OF PLANT-PATHOGENIC BACTERIA: UPDATED REVIEW OF PROTOCOLS (1989-2007) A. Palacio-Bielsa1, M.A. Cambra2 and M.M. López3* 1 Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), Avenida Montañana, 930, 50059 Zaragoza, Spain 2 Centro de Protección Vegetal (CPV), Gobierno de Aragón, Avenida Montañana 930, 50059 Zaragoza, Spain 3 Centro de Protección Vegetal y Biotecnología. Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera km 4.5, 46113 Moncada, Valencia, Spain SUMMARY occur, so highly sensitive protocols are required. Nucle- ic-acid based tests offer greater sensitivity, specificity, re- PCR-based methods offer advantages over more tra- liability and may be quicker than many conventional ditional diagnostic tests, in that organisms do not need methods used to detect plant-pathogenic bacteria in dif- to be cultured prior to their detection and protocols are ferent plant hosts and environments. With the develop- highly sensitive and rapid. Consequently, there is a shift ment of polymerase chain reaction (PCR), and especial- in research towards DNA-based techniques. Although ly real-time PCR, such high sensitivity is achieved, im- reports already exist on a variety of PCR-based finger- proving the accuracy of pathogen detection and identifi- printing assays used to analyse the genetic diversity of cation (Mullis, 1987; Holland et al., 1991; Vincelli and bacterial populations and define their relationships, this Tisserat, 2008). review focuses on the general use of PCR in phytobacte- Globalisation implies that state borders have become riology for detection and diagnosis purposes. An updat- more open due to increase in free-trade agreements, and ed and detailed list of published PCR protocols for de- this can facilitate the introduction and dissemination of tection and identification of plant-pathogenic bacteria is foreign pathogens. This, in turn, leads to emerging dis- presented and discussed, aimed at facilitating access to eases, which are a growing reality for phytopathologists information that could be particularly useful for diag- worldwide. A guiding principle for disease prevention is nostic laboratories. This compilation includes and dis- that when key inoculum sources have been identified, cusses 246 articles published between 1989 and 2007 effective measures must be taken to prevent further addressing 23 genera, more than 50 species, 10 sub- spread and subsequent disease outbreaks. Consequent- species and more than 40 pathovars. ly, detection of the causal organisms becomes essential, as most bacterial diseases are transmitted through con- Key words: co-operational PCR; multiplex PCR; nest- taminated seeds or propagative plant material. Plant ed-PCR; real-time PCR. quarantine polices and regulations have been imple- mented in many countries to avoid pathogens from spreading and/or to prevent exotic pathogens from be- INTRODUCTION ing introduced with plant material. To achieve this goal, complex control systems have been designed, which of- Control of diseases caused by plant-pathogenic bac- ten include guidelines for rapid, sensitive and specific teria usually requires accurate detection, followed by pathogen detection and diagnosis and among them, proper identification of the causal organism. Although PCR is the technique of choice for rapid screening. presumptive diagnosis of bacterial diseases can be rela- Compared to conventional diagnostic methods, PCR tively simple when typical symptomatology is evident, offers several advantages, because organisms do not symptoms in plants are not always specific and can be need to be cultured prior to detection; moreover it is confused with those caused by other biotic or abiotic highly sensitive, relatively simple and fast to perform. agents. On the other hand, detection of bacteria in There has been a shift towards DNA-based protocols symptomless plant material for preventive control is developed for diagnostic purposes as well as for etiolog- necessary but can be extremely difficult, since low pop- ical or epidemiological studies, as reported by reviews ulations with uneven distribution of the pathogen can published over the past fifteen years (Henson and French, 1993; Louws et al., 1999; López et al., 2003; Schaad et al., 2003; Alvarez, 2004; López et al., 2006; Vincelli and Tisseral, 2008; López et al., 2009). Applica- tion of PCR techniques in diagnostic laboratories for Corresponding author: M.M. Lopez Fax: + 34. 963424001 routine purposes is also increasing and will continue in E-mail: [email protected] the near future, especially for the rapid screening of 002_LetterEditor_249 25-06-2009 10:41 Pagina 250 250 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 samples. PCR is now considered a routine technique gerprinting techniques have been described for classifi- and recommended in most protocols recently developed cation and typing of plant-pathogenic bacteria (Louws by the European Union and the European and Mediter- et al., 1999), such as randomly amplified polymorphic ranean Plant Protection Organization (EPPO) (Anony- DNA (RAPD) (Wang et al., 1993), repetitive sequence- mous 2004a, 2004b; 2005a, 2005b; 2006a, 2006b, based (rep-PCR) (Versalovic et al., 1998; Louws et al., 2006c, 2006d; 2007; López et al., 2006). 1994, 1995, 1998), amplified fragment length polymor- Taxonomy of plant-pathogenic bacteria has been ex- phism (AFLP) (Janssen et al., 1996), restriction frag- tensively revised in recent years. Therefore, in the pres- ment length polymorphism (RFLP) (Darrase et al., ent compilation, the names utilised are those recorded in 1994; Manceau and Horvais, 1997; Mkandawire et al., the “List of Names of Plant Pathogenic Bacteria, 1864- 2004) and others. However, the present compilation fo- 2004” of the International Society for Plant Pathology cuses solely on the PCR protocols available for routine (ISPP) (http://isppweb.org/names_bacterial.asp) and detection, diagnosis or identification of plant-pathogen- have been used to classify the listed publications. How- ic bacteria. ever, when the original bacterial genus or species differs One can appreciate from the Table, that the number from the one in the ISPP list (due to different reasons of references to the different genera is highly variable and the fact that some of the cited articles were pub- and not only related to the number of described species lished before the latest taxonomic revisions appeared) or pathovars in every genus, but also to the economic both the originally cited name and its current nomencla- importance of the diseases they cause, their distribution, ture, according to the ISPP, are indicated. whether local or widespread, and their status as quaran- A wide range of plant-pathogenic bacteria can be tine organisms. We found more than 50 protocols for currently detected by PCR in numerous hosts or envi- species of the genus Xanthomonas, more than 40 for ronmental samples (Schaad et al., 2001). This compila- Pseudomonas spp., 20 for Ralstonia spp., 19 for Clav- tion provides an updated listing of PCR published pro- ibacter and Agrobacterium spp., 16 for Erwinia and tocols for detection and identification of phytopatho- Xylella spp., 12 for Pectobacterium spp., 11 for “Candi- genic bacteria, which could be especially useful for di- datus Liberibacter” spp., nine for Burkholderia spp., agnosis laboratories. It contains a non-exhaustive list of seven for Streptomyces and Pantoea spp., six for Dickeya 246 references related to PCR protocols published from and Xylophylus spp., four for Leifsonia spp., three for 1989 up to 2007, which refers to 23 bacterial genera in- Acidovorax spp., and only one or two protocols for cluding more than 50 species, 10 subspecies and more species of other genera. than 40 pathovars. Depending on the choice of PCR primers, both nar- This work summarizes essential data from each of the row and broad specificity can be obtained, allowing de- published protocols and, in order to facilitate searches, tection of a single pathogen or of several members of a information is presented according to each bacterial group of related pathogens. Primer design requires genus in a Table, which comprises the following infor- knowledge of the target DNA sequences and the past mation: ISPP accepted nomenclature for the target bac- two decades have witnessed reports of primers used to teria and name of the bacteria in the original article, identify many plant-pathogenic bacteria (Schaad et al., primers name and target DNA, variants utilised in the 2001), multiple strategies being developed to design PCR protocol, type of sample and treatment prior to am- primers for specific detection and disease diagnosis. plification, reference and observations about the Among them, the DNA sequences from known patho- method. Protocols for specific detection of bacterial genicity/virulence genes have been used as targets to de- species, alphabetically ordered, appear first, followed by sign specific primers, as those described by Bereswill et those designed for the simultaneous detection of two or al. (1994), Darrasse et al. (1994), Dreier et al. (1995), more species, or for other genera that could also be pres- Leite et al. (1995), Nassar et al. (1996), Stange et al. ent in a given host. References for each species, sub- (1996), Sato et al.
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