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. (1997), Burkhalid et al. (1998), species and pathovar are listed in chronological order. Sorensen et al. (1998), Kerkoud et al. (2002), Loreti and The discussion concentrates on the target sequences Gallelli (2002), Zaccardelli et al. (2005, 2007) or Cullen utilised for primer design as well as on the different and Lees (2007). DNA extraction protocols or PCR variants utilised. In other primers reported here, sequences from path- ogenicity-related genes in principle specific to a pathogen, or to a group of pathogens, have been em- DISCUSSION ployed, such as the pel gene of soft-rot diseases caused by pectolytic species or subspecies of the genus Pecto- This review presents references and details of most of bacterium (Darrase et al., 1994; Louws et al., 1999), or the available PCR protocols published between 1989 belonging to a cluster of genes involved in the virulence and 2007 for specific detection and identification of systems of different bacterial families (hrp, pth and vir plant-pathogenic bacteria. A variety of PCR-based fin- genes). The utility of PCR primers that employ specific 002_LetterEditor_249 25-06-2009 10:41 Pagina 251

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 251

sequences from known pathogenicity genes has been tion and used to design primers to detect some organ- demonstrated for a wide range of bacterial species, al- isms (Seal et al., 1992a; Manceau et al., 2005). Further- though there are also examples of the need to design more, as the field of genomics progresses, more genome new primers after the discovery of strains that lack some sequences become available and specific primers can be pathogenicity genes, previously considered universal. designed to target unique regions of the genome of a For example, the phaseolotoxin gene was considered an given pathogen. Nevertheless, only in very few cases it is excellent target for Pseudomonas savastanoi pv. phaseoli- reported that these newly available sequences have been cola detection and several sets of primers were designed employed to design specific primers (López et al., on its sequence (Prosen et al., 1993; Schaad et al., 1995, 2008). It is also necessary to check the reliability of the 2007; Audy et al., 1996; Sawada et al., 1997). However, information available in the sequence databases on the discovery of nontoxigenic strains of this pathovar which the design of specific primers is based, because showed that these primers were not as specific as ex- Arahal et al. (2004) found mistakes in primers designed pected (Rico et al., 2003). Also, the nec1 gene was previ- for Ralstonia solanacearum and Clavibacter michiganen- ously proposed for Streptomyces pathogenicity testing sis subsp. sepedonicus, when comparing their sequences (Burkhalid et al., 1998), but recent works suggest that to those of the databases. the gene seems to play a subsidiary role in pathogenicity A low copy number of initial target DNA sequences and is missing from some pathogenic strains (Wanner, makes the first amplification cycles critical and PCR in- 2004, 2006). hibitors can result in false negatives, which could have a Primers have also been designed on plasmid se- major impact, especially in quarantine settings. In this quences like those from the Ti plasmid of Agrobacteri- context, sample preparation is critical, and target DNA um species (Nesme et al., 1989; Bereswill et al., 1992; should be made as available as possible for amplifica- Dong et al., 1992; Hartung et al., 1993, 1996; Firrao and tion. Plant-derived compounds and the presence of dif- Locci, 1994; Sawada et al., 1995; Verdier et al., 1998), ferent substances, like copper products (Minsavage et although in general plasmid stability must be previously al., 1994; Hartung et al., 1996), have been reported as evaluated in order to avoid false negative results. It is as- inhibitors of PCR. To avoid this, some PCR protocols sumed that if the plasmid genes encode essential fitness reported here submit the samples to some physical or or pathogenicity traits they are stable (Eastwell et al., chemical treatments before amplification. Preparation 1995). Nevertheless, primers targeted to a plasmid re- methods listed include dilution, separation and concen- ported as universal, sometimes were not found useful tration of cells by centrifugation or washing/centrifuga- for detecting all virulent strains of a group, for example tion of plant tissue (Maes et al., 1996b; Smid et al., those based on the pEA29 plasmid of Erwinia amylovo- 1995; Pan et al., 1997), or immunomagnetic separation ra (Llop et al., 2006). to enhance sensitivity and specificity (van der Wolf et The ribosomal DNA operon has also frequently been al., 1996; Walcott and Gitaitis, 2000; Walcott et al., used to design primers that allow highly sensitive detec- 2002; Khoodoo et al., 2005), etc. tion, but due to its universal nature, the level of discrimi- Removal of PCR inhibitors from samples using sim- nation lies at the species or genus levels. The internally ple procedures is also reported, including treatment transcribed spacer region (ITS) between the 16S and 23S with cation-exchange resins (Jacobsen and Rasmussen, rRNA genes appears to be more variable than 16S and 1992) or polyvinyl-pyrrolidone (PVP), which binds to 23S rRNA genes and was used to design primers by Li phenolic compounds (Leite et al., 1995; Maes et al., and De Boer (1995), Kim and Song (1996), Maes et al. 1996a; Fegan et al., 1998; Pan et al., 1998; Robène- (1996b), Takeuchi et al. (1997), Pan et al. (1998), Whitby Soustrade et al., 2006). Besides, an increasing number et al. (1998), McDowell et al. (2001), Walcott et al. of commercial kits are available for DNA purification (2002), Song et al. (2004), Grall et al. (2005), Sayler et al. from plant material (López et al., 2006) and simple (2006) or Grisham et al. (2007), among others. However, DNA extraction protocols are advised for many targets some primers from rRNA genes, such as those designed (Llop et al., 1999). The design of internal PCR controls, for E. amylovora (Maes et al., 1996a) showed problems based on sequences from the bacteria or from the plant, due to lack of specificity because they also amplified an- has also improved sensitivity and avoided false negatives other Erwinia species (Roselló et al., 2007). The rDNA (Pastrik, 2000; Weller et al., 2000; Cubero et al., 2001, sequences from unknown bacteria associated with plant 2002; Glick et al., 2002; Pastrik et al., 2002). On the disease can be amplified by PCR, subjected to sequence other hand, simply treating the sample at high tempera- analysis and compared with strains in the RDP database tures for a few minutes has often been used as an ade- (Ribosomal Database Project) (http://rdp.cme.msu.edu) quate pre-amplification treatment for detection of the (Maidak et al., 1999), providing a phylogenetic frame- target sequence from pure bacterial cultures (Seal et al., work to identify the causal agent. 1992a, 1992b; Schulz et al., 1993; Sato et al., 1997; In other cases, DNA fragments specific to a particu- Boudazin et al., 1999; Weller et al., 2000; Weller and lar species have been cloned by subtractive hybridisa- Stead, 2002; Tan et al., 2003; Kawaguchi et al., 2005; 002_LetterEditor_249 25-06-2009 10:41 Pagina 252

252 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297

Lee et al., 2006; Milijasevic et al., 2006). tamination risks and has a level of sensitivity similar to Enrichment of the pathogen in a liquid or solid medi- nested-PCR and real-time PCR. um can increase its population prior to PCR processing Multiplex PCR protocols using specific primers have (López et al., 2003). When the sample is first plated on also been set up for simultaneous detection of two genes solid medium and micro-colonies are recovered and am- of the same bacterial pathogen, thus limiting false posi- plified the method has been named BIO-PCR (Schaad et tives, (Haas et al., 1995; Arnold et al., 1996; Kawaguchi al., 1995). In general, these enrichment methods facili- et al., 2005; Rico et al., 2006), or allowing amplification tate target detection by increasing their numbers and de- of several pathogenic bacteria in seed or plant material creasing inhibitors and have proven successful in detect- (Haas et al., 1995; Smid et al., 1995; Arnold et al., 1996; ing and identifying bacteria in seeds, soil samples and Audy et al., 1996; Mills et al., 1997; Fegan et al., 1998; symptomless plant tissues (Schaad et al., 1995, 1999, Toth et al., 1998; Catara et al., 2000; Glick et al., 2002; 2007; Ito et al., 1998; Manulis et al., 1998; Wang et al., Berg et al., 2005; Kawaguchi et al., 2005; Kabadjova- 1999; Penyalver et al., 2000; Weller et al., 2000; Sakthivel Hristova et al., 2006; Peters et al., 2007), or even detec- et al., 2001; Weller and Stead, 2002; Bertolini et al., tion of one bacterium and four viruses in olive plants 2003b). They are applicable to culturable and fast-grow- (Bertolini et al., 2003a). ing bacteria and can also detect viable but not culturable Further advances have also been made through the cells (VBNC), which are well documented in environ- use of real-time PCR, which offers advantages over con- mental samples (Roszak and Colwell, 1987) and could ventional PCR because data are available in real-time, constitute a risk as an inoculum source of plant do not require time consuming post-PCR processing pathogens (Alexander et al., 1999; Ghezzi and Steck, and can be quantitative. Moreover, it is a high-through- 1999; Grey and Steck, 2001; Ordax et al., 2006, 2009). put technique for many plant pathogens from different In this respect, nine-month-old VBNC E. amylovora sample types (Schaad et al., 2003; Alvarez, 2004; Gi- cells detected by PCR became culturable and recovered taitis and Walcott, 2007). The ability to quantify pathogenicity after brief enrichment in liquid medium pathogen populations using quantitative real-time PCR (Ordax et al., 2006). holds great potential for epidemiological studies, for de- Several variants have been developed to improve sen- termining seed-borne or plant-borne inoculum and for sitivity of conventional PCR. Among the first described, establishing and monitoring transmission thresholds as nested-PCR, with both internal and external primers to bases for improved disease management (Gitaitis and the target sequence, was reported to increase sensitivity Walcott, 2007). and reduce the effect of inhibitors (Honeycut et al., Real-time PCR and melting curve analysis (MCA) are 1995; McManus and Jones, 1995; Schaad et al., 1995; state-of-the-art techniques for quantifying nucleic acids, Hartung et al., 1996; Roberts et al., 1996; Lee et al., mutation detection, genotyping analysis as well as for 1997b; Mahuku and Goodwin, 1997; Manulis et al., detection and diagnosis purposes. Many different sys- 1998; Poussier and Luisetti, 2000; Pradhanang et al., tems have been developed, including probe-based 2000; Botha et al., 2001; Poussier et al., 2002; Kang et methods, such as TaqMan Probes, molecular beacons al., 2003; Anonymous, 2004a; Song et al., 2004; Molt- (Fanelli et al., 2007), Scorpion primers (De Bellis et al., mann and Zimmermann, 2005; Falloon et al., 2006; 2007), etc. In general, the protocols developed are Robène-Soustrade et al., 2006; Cullen and Lees, 2007). based on hybridisation of the probe to the target ampli- However, in nested-PCR the risk of cross-contamination con, thus achieving maximum sensitivity and confirm- in routine analysis of large numbers of samples is in- ing the identity of the amplified product (Schaad et al., creased by the introduction of a second round of ampli- 1999; Weller et al., 2000; Oliveira et al., 2002; Schaad et fication and the simultaneous manipulation of the previ- al., 2002; Weller and Stead, 2002; Bach et al., 2003; ously amplified products. To avoid these problems, Ozakman and Schaad, 2003; Salm and Geider, 2004; nested-PCR in a single closed tube has been developed Baumgartner and Warren, 2005; Cubero and Graham, (Llop et al., 2000; Bertolini et al., 2003b). 2005; Fatmi et al., 2005; Anonymous, 2006b; Francis et A new method named co-operational polymerase al., 2006; Koyama et al., 2006; Li et al., 2006b; Cullen chain reaction (Co-PCR) (Spanish patent 31 October and Lees, 2007; De Bellis et al., 2007; Dreo et al., 2007; 2000; P20002613) has been described for highly sensi- Fanelli et al., 2007; Li et al., 2007; Schaad et al., 2007; tive detection of plant viruses and bacteria (Olmos et Weller et al., 2007; Zhao et al., 2007). In addition, al., 2002; Caruso et al., 2003; Marco-Noales et al., Koyama et al. (2006) developed competitive quenching 2008). Co-PCR is based on the simultaneous action of probes. This new method uses a special fluorescent dye three or more primers that produce three or more am- whose fluorescence is quenched by the guanine bases in plicons by the combination of the primers and the co- DNA. The conventional real-time PCR requires real- operational action of amplicons for the production of time measurement of fluorescence intensity during the largest fragment amplified by the external primers. DNA amplification, whereas this novel method only re- As it is performed in a single reaction, it minimizes con- quires measurement of fluorescence intensity before 002_LetterEditor_249 25-06-2009 10:41 Pagina 253

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 253

and after amplification. primers. Typically, amplification is completed within 30 Real-time PCR, which can provide accurate and rap- min using a simple water bath, which is kept constantly id detection of bacterial pathogens, is becoming the at 65°C (Notomi et al., 2000). LAMP-based detection gold standard for diagnosis of plant-pathogenic bacte- could be as sensitive as a conventional PCR assay for ria, as well as of other organisms. Although only 27 practical diagnosis. The product is rapidly detected on available real-time protocols are referred to here, one nylon membranes by staining, replacing conventional should bear in mind that their number has increased electrophoresis and visualization of DNA bands under from only one in 1999 (Schaad et al., 1999) to seven in UV illumination. Thus, this method does not depend 2006 (Anonymous, 2006b; Atallah and Stevenson, 2006; upon a thermal cycler and electrophoresis apparatus Berg et al., 2006; Francis et al., 2006; Koyama et al., (Okuda et al., 2005; Li et al., 2007). 2006; Li et al., 2006b; Sayler et al., 2006) and nine in Accurate detection or diagnosis of plant pathogenic- 2007 (Cullen and Lees, 2007; De Bellis et al., 2007; bacteria often requires multiple complementary tests to Dreo et al., 2007; Fanelli et al., 2007; Grisham et al., achieve definitive identification (Alvarez, 2004; López et 2007; Li et al., 2007; Schaad et al., 2007; Weller et al., al., 2006). Besides, PCR-based approaches require thor- 2007; Zhao et al., 2007). ough studies of target pathogens to both characterize In this compilation, most of the real-time PCR proto- their diversity and identify common stable markers for cols have utilised TaqMan® probes (Applied Biosys- designing specific primers. It is necessary to indicate tems, USA), which provide greater sensitivity and speci- that, although most protocols are claimed to be specific, ficity. An alternative to probe-based methods is the use they must be validated against a large collection of of DNA intercalating dyes that bind to double-stranded strains of the target bacterium and other pathogens of DNA. Dyes have much higher fluorescence when the same host, as well as against organisms of its envi- bound to double-stranded DNA compared to the un- ronment, before they can be used as standards. The reli- bound state. SYBR Green I became the most widely ablity of the protocols will eventually be demonstrated used DNA dye for real-time PCR applications because after years of use, building confidence in their accuracy of cost efficiency, generic detection of amplified DNA, and robustness in international ring tests among labora- and its ability to differentiate PCR products by melting tories (Alvarez, 2004). Inter-laboratory evaluations of curve analysis. Several protocols in the present compila- new detection or diagnostic protocols provide essential tion utilised SYBR Green (Mavdorieva et al., 2004; information on repeatability and reproducibility, ease of Salm and Geider, 2004; Atallah and Stevenson, 2006; implementation, use and interpretation of tests, giving Sayler et al., 2006; Grisham et al., 2007). an indication of their robustness in routine analysis of In our experience, it is easy to adapt existing conven- large numbers of samples. A standard protocol must tional PCR protocols to a real-time PCR assays by using subsequently be established and optimized based on re- SYBR® Green Master Mix (Qiagen, USA) and utilising sults (López et al., 2003, 2008; Alvarez, 2004). In this them for identification of bacterial cultures. However, sense the diagnostic protocols for detection of some Eu- there are disadvantages with the use of SYBR Green I, ropean Union quarantine bacteria, as Clavibacter michi- such as inhibition of PCR amplification in a concentra- ganensis subsp. sepedonicus, Xanthomonas fragariae and tion-dependent manner, effects on DNA melting tem- E. amylovora, have been validated by ring tests in the perature and preferential binding to certain DNA se- DIAGPRO project financed by the “Standard, Mea- quences. The drawback of using SYBR Green I for surements and Testing” programme of the European melting curve analysis is that the melting temperature is Union, before being adopted by the EPPO. highly dependent on the concentration of the dye (Ririe As more PCR-based methods for detection of phy- et al., 1997) and the DNA (Xu et al., 2000). topathogenic bacteria become available, their use will Loop-mediated isothermal amplification (LAMP) is progressively increase not only for identification pur- another DNA amplification method, based on auto-cy- poses, but also for studying pathogen populations in cling strand displacement DNA synthesis by a DNA their biology, ecology, and host-pathogen interactions, polymerase, which has high strand displacement activi- thus expanding our knowledge of the hidden part of the ty, and a set of specially designed inner and outer life cycle of plant pathogenic bacteria. 002_LetterEditor_249 25-06-2009 10:41 Pagina 254

254 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 ) and Pantoea oryzicola ), syringae and pv. Pseudomonas oryzae ), Pseudomonas glumae ( (pathovars (pathovars r for the different genera of protocols Erwinia herbicola ( ted names of bacteria are indicated on the right Tumour-inducing strains. DNA extraction recommended if high level of other microflora is found after enrichment. Pseudomonas avenae Burkholderia glumae agglomerans fuscovaginae, Pseudomonas syringae Xanthomonas oryzae also amplified and differentiated by primary secondary fragments. et 2004 2003 et al., et al., et al., 2005 Walcott and Gitaitis, 2000 Nesme 1989 Song Kim and Song, 1996 Pulawska and Sobiczewski, 2005; Sobiczewski al., Acidovorax Agrobacterium Bacteria (lysed) or Bacteria (lysed) crude extract and immunocapture Bacteria (DNA extraction) Seed (washes enrichment) Bacteria (DNA extraction) Bacteria from soil (DNA extraction) Genus Genus BIO Nested Semi-nested Conventional Conventional Conventional Conventional Bacteria (boiled) Tan Variant of PCR Protocol Sample (treatment) Reference Synonyms/observations Variant of PCR Protocol Sample (treatment) Reference Synonyms/observations ´ A N A B/G , virD2E vir ´ gene e D T- /FGP vir B gene p e N T-D + 15 11+12 e p ty D2 gene region tms2B tms2 T-DNA n e n e ty i n i n tms2 ITS region ITS region RBF/RBR ITS region ocsF/ocsR l i vir Target DNA Target DNA Primer name Primer name tms2A/tms2B pTi WFB1/WFB2 R16-1/R23-2R 16S rRNA gene tms2F1/tms2R2 Intercistronic N op a O c to p to O c Aaaf5/Aaar2 (internal) Aaaf3/Aaar2 (external) FGP vir B FGP vir virD2A, virD2C FGPtmr 530/FGPtmr 701 FGPtmr avenae citrulli spp. subsp. subsp. Name of primers and target DNA, sample treatment in the original article, variant PCR protocol, reference and observations fo Species/biovars Species/subspecies A. avenae A. avenae A. avenae Agrobacterium A. tumefaciens A. tumefaciens side of the table. plant pathogenic bacteria according to ISPP nomenclature. When the nomenclature reported in articles differs, originally ci Table 1. Table 002_LetterEditor_249 25-06-2009 10:41 Pagina 255

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 255 biovar 1 biovar 2 biovar 1 Agrobacterium Agrobacterium Agrobacterium et al., et al., et al., et al., Eastwell 1995 Puopolo 2007 Schulz 1993 Szegedi and Bottka, 2002 Pulawska 2006 Weller and Stead, 2002 Bacteria (lysed) or Bacteria (lysed) plant tissue (DNA extraction) Plant roots (DNA extraction) Bacteria from soil or plant tissue (DNA extraction) Bacteria (boiled) Real-time Multiplex (TaqMan) Conventional Bacteria (boiled) Conventional Conventional Bacteria (lysed) Conventional 23S ne c e 6b/vis fi B/G i g c e e e vir as en /FGP vir B sp on s specific 23S r region n A g gene 6a 15 UF e BIR C gene ArR i AvR B2R virA gene gene gene gene 11+12 E2 gene pehA ctu rol-Pr c Probe region 6a a T-DNA a RN Primers vir r rRNA gene al vir Ri-plasmid VisF/VisR VCF/VCR rol-F/rol-R PGF/PGR ef rRNA gene virA specific 23S rRNA specific 23S specific 23S rRNA yg VirE2PF/VirE2PR tum Pol Intercistronic pTiS4 vitopine synthase A. Pectin enzyme hydrolase A. rhizogenes Universal agrobacteria 23S A. vitis A. rubi FGP vir B FGP vir Tm 4 ipt, IS866, S4 radiobacter

A. tumefaciens A. A. vitis A. vitis A. vitis A. tumefaciens A. rhizogenes 002_LetterEditor_249 25-06-2009 10:41 Pagina 256

256 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 ) ) A. vitis biovar 1 (Ti or Ri plasmid) biovar 2 (Ti or Ri plasmid) biovar 3 (Ti plasmid) biovar 3 biovar 1 biovar 3 biovar 3 A. vitis A. tumefaciens (tumorigenic A. radiobacter (nonpathogenic A. tumefaciens A. tumefaciens Agrobacterium Agrobacterium Agrobacterium et 1995 et al., et al., et al., et al., et al., et al., 2005 al., Szegedi 2005 Kawaguchi Cubero 1999 Dong 1992 Sawada 1995 Genov 2006 Haas Bacteria (boiled) or plant tissue (DNA extraction) Bacteria (DNA extraction) lysates Bacteria (cells or DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction or boiled) Multiplex Bacteria (boiled) Multiplex Conventional Bacteria (lysed) Conventional Conventional Conventional Conventional Conventional B/G ´ octopine nopaline and ´ vir /FGP vir B , E C ´ tartrate 15 C gene C gene vir C gene pTis pTis 16S rRNA gene gene 11+12 A. vitis A. vitis E2 gene D2 gene oncogene region TF/TR (NHR) T-DNA T-DNA T-DNA NF/NR A, C vir vir vir VisF/VisR VCF/VCR VCF/VCR VCF/VCR vir vir CYT/CYT ipt VCF3/VCR3 A. vitis ttuCfw/ttuCrev Ab3-F3/Ab3-R4 Agrobacterium VirE2PF/VirE2PR Narrow 1/Narrow 2 deshydrogenase gene gene of gene of Intercistronic pTiS4 vitopine synthase Wide 1/Wide 2 (WHR) FGP vir B FGP vir FGPtmr 530/FGPtmr 701 FGPtmr Rhizobium 6b 6b A. tumefaciens A. vitis A. tumefaciens A. vitis A. tumefaciens A. vitis A. vitis A. radiobacter A. tumefaciens A. rhizogenes A. vitis A. tumefaciens A. rhizogenes A. vitis A. tumefaciens A. rhizogenes 002_LetterEditor_249 25-06-2009 10:41 Pagina 257

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 257 Adviced in the EPPO . ) biovar 1 biovar 2 biovar 3 biovar 1 (Ti plasmid) biovar 2 (Ti plasmid) biovar 3 (Ti plasmid) biovar 2 biovar 1 (Ri plasmid) B. caryophilli A. tumefaciens A. rhizogenes A. tumefaciens A. tumefaciens A. radiobacter Pseudomonas andropogonis Amplify also other species (but shows a distinct profile for protocol. Agrobacterium Agrobacterium Agrobacterium et et al., et al., et al., et al., 2001 1998 Bagsic 1995 Whitby 1998; McDowell et al., Bauernfeind al., 2004 Suzaki Hauben 1998 Ponsonnet and Nesme, 1994 Brenneria Burkholderia Bacteria (DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction) Bacteria (boiled) Anon., 2006a Bacteria (cells directly added to PCR mix) Bacteria (boiled) and plant vascular fluid (DNA extraction) Bacteria (DNA extraction) Genus Genus PCR-RFLP Conventional Conventional Conventional Conventional Conventional and BOX-PCR Conventional and RFLP Variant of PCR protocol Sample (treatment) ReferenceVariant of PCR protocol Sample (treatment) Reference Synonyms/observations Synonyms/observations , ´ , ´ ´ 164 2 B /P480-5 A2275, ´ Chromosomal

vir C gene C gene ´ genes Pf/Pr G1/G2 vir vir PSL/PSR ITS region VisF/VisR Es1A/Es4B PSL1/PSR1 Target DNA Target DNA Primer name Primer name VCF5/VCR5 VCF3/VCR3 FGP FGPnos1236 FGPvir 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 23S rRNA gene Ti plasmid genes P1240-5 701, FGPnos975, CMG16-1/G-16-2 CMG-23-1/G-23-2 FGPS6, FGPS1509 FGPtmr 530, FGPtmr 530, FGPtmr Vitopine synthase gene FGPL 132 Species Species A. vitis A. tumefaciens A. rhizogenes A. vitis A. rubi A. tumefaciens A. tumefaciens A. rhizogenes A. vitis A. rhizogenes (nonpathogenic) B. salicis B. andropogonis B. caryophylli B. cepacia B. gladioli 002_LetterEditor_249 25-06-2009 10:41 Pagina 258

258 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 ) and oryzicola Pseudomonas syringae ), and pv. oryzae Erwinia herbicola ( (pathovars (pathovars Restriction enzyme analysis required for differentiation species and subspecies inside both genera. Pseudomonas glumae Pantoea agglomerans fuscovaginae, Pseudomonas syringae Xanthomonas oryzae also amplified and differentiated by primary secondary fragments. Recommended in the EPPO protocol. et et al., 1997a et al., et al., et al., et al., et al., et al., et al., et al., 1999 Samac 1998 Lee Takeuchi 1997 Takeuchi 1997 Sayler 2006 Santos 1997 Bauernfeind Kim and Song, 1996 al., Dreier 1995 Anon., 2005a; Milijasevic 2006 Hadas 2005 Clavibacter Plant tissue and seeds (DNA extraction) Bacteria (DNA extraction) Bacteria or plant tissue (boiled) Bacteria or plant tissue (boiled) Seed washes and plants (without extraction step) Bacteria, seeds (alkaline lysis and boiled) Bacteria (DNA extraction) Bacteria (DNA extraction) Plant tissue and seeds (DNA extraction) bacteria (boiled) extraction) Genus Mix) Green Master Nested ® Real-time Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Bacteria (boiled) Conventional Bacteria (DNA (SBYR Variant of PCR protocol Sample (treatment) Reference Synonyms/observations A A A ene N N N N As solate A g D 4 d D d D d D i i i RN enic i m m m as as as /CM 16Sr + l l l 3 i a i a r 16S r region i Gl-16-2 l Eub-16-1 te CM o ITS region ITS region 16S rDNA ITS region ITS region ene pene ene p ene p c Target DNA g g Primer name PSA-4/PSA-R R16-1/R23-2R PL-12f/PL-11r CIRS-1/CIRS2 adi CMM5/CMM6 GL-13f/GL-14r 1 1 b a b a CMM-5/CMM-6 CMM-5/CMM-6 -1 g-1 Insertion element Forward/Reverse R16FO/CBR16R1 u . gl at- at- at cloned pathogcloned CBR16F2/CBR16R2 E DNA fragment from a 16S-23S rDNA spacer B P P P . subsp. subsp. subsp. subsp subsp. and Species/subspecies B. gladioli B. glumae B. glumae B. glumae B. plantarii Clavibacter Rathayibacter (genus specific) C. michiganensis insidiosus C. michiganensis michiganensis C. michiganensis michiganensis C. michiganensis michiganensis C. michiganensis michiganensis 002_LetterEditor_249 25-06-2009 10:41 Pagina 259

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 259 BIO implies enrichment in solid medium. Both authors used the same primers but second protocol can be quantitative. 1996 1997 et al., 1997b 1995 et al., et al., et al., et al., et al., Slack Li and De Boer, 1995 Mills Schaad 1999 Lee Firrao and Locci, 1994 1993 Hu Schneider Pastrik, 2000 Bacteria, plant tissue (alkaline treatment) Bacteria, potato tubers (DNA extraction) Bacteria, potato tubers (DNA extraction) Bacteria (untreated) Bacteria, potato tubers (DNA extraction) Plant tissue (DNA extraction) Bacteria (untreated), potato tissue (DNA extraction) genomic BIO Nested control) Real-time standard) Multiplex Multiplex (TaqMan) Competitive Conventional Conventional Conventional Conventional Conventional Bacteria (untreated) Conventional BIO+TaqMan DNA as internal DNA as internal Arabidopsis ( (Coamplification of host (Coamplification e l ng i R 4 ex PC l /CM + p 3 ti CS1 Probe region region Nested ul CSRS-C Primers fragment fragment Spif/Sp5r CM (unknown) (unknown) m Cms 50-53T A47A/A47B PSA-1/PSA-R CMS-6/CMS-7 or NS-7-F/NS-8-R Insertion element CMSIF1/CMSIR1 CMSIF2/CMSIR2 DNA from potato, CMS50F/CMS50R CMS72F/CMS72R CMS85F/CMS85R Chromosomal DNA Chromosomal DNA eggplant and tomato 16S-23S rDNA spacer 16S-23S rDNA spacer CS1 plasmid sequence Cms 50-2F/Cms 133R DNA fragment from a CS1 plasmid sequence Inverted repeat plasmid cloned pathogenic isolate Three primer sets for s Three primer sets for s ...... subsp subsp subsp subsp subsp subsp subsp subsp C. michiganensis sepedonicus C. michiganensis sepedonicus C. michiganensis sepedonicus C. michiganensis sepedonicus C. michiganensis sepedonicus C. michiganensis sepedonicus C. michiganensis sepedonicus C. michiganensis sepedonicus 002_LetterEditor_249 25-06-2009 10:41 Pagina 260

260 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 C. nebraskensis and insidiosus sepedonicus. subsp. subsp. Restriction analysis required for differentiation of michiganensis C. michiganensis yield same band. RAPD-PCR for distinguishing subspecies. 2003 et al., 1997b et al., et al., Anon., 2006b Recommended in the EPPO protocol Lee Pastrik and Rainey, 1999 Bach Guimaraes 2001 Curtobacterium Bacteria, potato tubers (DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction) Genus Nested Real-time Real-time (TaqMan) (TaqMan) Conventional Conventional Conventional Conventional Variant of PCR protocol Sample (treatment) Reference Synonyms/observations s e 1993; 1999; ci et al., ., subsp. subsp. subsp. subsp. subsp. et al et al et al., MR16R1 MR16R2 MR16R1 C C C subspe l k, 2000 k, + l i 1/ 2/ 1/ C. m. C. m. C. m. C. m. C. m. a tr l region ( ( ( ( ( PAS-R/ Primers sa CF4/CF5 tesalarius) insidiosus) subspecies Cmi probe Cmt probe r 1996, Mills (unknown) Cms probe Pas Cmn probe Cmm probe sepedonicus) Target DNA Primer name e nebraskensis) michiganensis) v 16S rRNA gene FP Cm/RP Cm MR16F MR16F MR16F MR16F MR16F MR16F Common ITS in all Subspecies-specific PSA-2 PSA-7 Chromosomal DNA PSA-4 PSA-5 PSA-1 C C C Uni 16S-23S rDNA spacer et al., 1997, Schaad and De Boer, 1995, Slack Firrao and Locci, 1994, Li See: Schneider Subspecies specific probes . pv. subsp insidiosus, insidiosus, insidiosus, Species/pathovars C. michiganensis sepedonicus C. michiganensis subspecies: michiganensis sepedonicus, nebraskensis, tessellarius C. michiganensis subspecies: michiganensis sepedonicus, nebraskensis, tessellarius C. michiganensis subspecies: michiganensis sepedonicus, nebraskensis, tessellarius C. flaccumfaciens flaccumfaciens 002_LetterEditor_249 25-06-2009 10:41 Pagina 261

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 261 Z. restriction

and carotovorum. atroseptica carotovora atroseptica Pectobacterium also amplified. subsp. subsp. subsp. subsp.

isolates from other hosts. P. atrosepticum E. chrysanthemi E. carotovora E. carotovora Other genera amplified also. Banding patterns allow differentiation of E. chrysanthemi E. chrysanthemi Restriction analysis allows discrimination of aeothiopica E. chrysanthemi E. carotovora Erwinia chrysanthemi Restriction analysis results correlate with pathovar and biovar. Specificity of multiplex PCR is lower than single assay, whereas an undesirable band can be also obtained with P. carotovorum analysis improves discrimination. 2006 1995 2002 1999 2006 et al., et al., et al., et al., et al., et al., et al., Peters 2007 Toth Tegli Nassar 1996 Smid Dickeya Enriched tubers extracts microsphere immunoassay (MIA) Purified isolate suspension or enriched microplant tissue (untreated) Bacteria (DNA extraction) or seeds Bacteria (boiled) Lee Bacteria (DNA extraction) Bacteria (boiled), potato tubers (centrifugation and lysis buffer) Genus Multiplex Multiplex and RFLP Conventional Conventional Conventional Conventional Not indicated Chao Conventional Conventional and RFLP Variant of PCR protocol Sample (treatment) Reference Synonyms/observations V V enes ) g ) P. and RRE . ecS ecS RORE gen p H p

T p C s A + and ADE 5A/5B ADE gene idg OR+ OR/ restriction site) (unknown) (unknown) pel Target DNA . atrosepticum . Primer name atrosepticum pel SR3F/SR1cR gene (including an +ATROREV ADE1/ADE2 (specific for P 16S rRNA gene Dickeya Pca For/Pca Rev PelZ-1-F/pelZ-1-S Rev Dcd For/Dcd RWF RWF pT8-1, CffFOR2/CffREV4 Chromosomal DNA Chromosomal DNA AhdI E Metalloprotease genes E Zantedeschia aethiopica pelZ (Simultaneous detection of . pv. subsp . Species sp. sp. sp. sp. sp C. flaccumfaciens flaccumfaciens Dickeya Dickeya Dickeya Dickeya Pectobacterium atrosepticum Dickeya dianthicola Dickeya Pectobacterium carotovorum carotovorum P. atrosepticum 002_LetterEditor_249 25-06-2009 10:41 Pagina 262

262 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 isolated from Erwinia piriflorinigrans Amplifies also necrotic pear blossoms. Amplification also obtained for pathogenic strains that lack plasmid pEA29. Amplifies also pathogenic strains that lack plasmid pEA29. Amplifies also pathogenic strains that lack plasmid pEA29. et 2000 et al., et al., et al., 2004 et al., , et al., et al., 1996 Maes 1996a Salm and Geider Bereswill 1995 Taylor 2001 McManus and Jones, 1995 Guilford 1996 Llop Bereswill 1992; Brown al., Anon., 2004a Recommended in the EPPO protocol. A N uno- ) m TM m r e a s e l on) Erwinia e) Re cti a tur n e n e Bacteria (proteinase K), plant (PVP and PVPP addition) lysates Bacteria (lysed), plant (untreated) Bacteria (untreated), plant (enrichment followed by i Plant (GeneReleaser) cap Bacteria, plant (DNA extraction) Bacteria, plant (untreated) Bacteria, plant (D Bacteria (untreated), plant (DNA extraction, Ge extr Genus BIO Mix) Green Master Nested Nested Nested ® (TaqMan) Real-Time Conventional Conventional Bacteria (untreated) Conventional Conventional Conventional Conventional (SBYR Variant of PCR protocol Sample (treatment) Reference Synonyms/observations 1992; genes) EA29) EA29) EA29) EA29) EA29) EA29) 2000 ams , et al., . l A (p A (p A (p A (p A (p A (p + + N N N N N N t a A/B e Ea71

region PEA71 fD2/rP1 d D d D d D d D d D d D EaF/EaR (internal) i i i i i i op (unknown) Target DNA Primer name m m m m m m Ll A/B (external) 23S rRNA gene 16S rRNA gene P29TM (probe) as as as as as as AMSbL/AMSbR Chromosomal DNA Chromosomal DNA PEANT1/PEANT2 Pl Pl Pl Pl Pl Pl AJ75/AJ76 (internal) AJ75/AJ76 (external) See: Bereswill P29TF/P29TR (primers) Chromosomal ( Species E. amylovora E. amylovora E. amylovora E. amylovora E. amylovora E. amylovora E. amylovora E. amylovora E. amylovora 002_LetterEditor_249 25-06-2009 10:41 Pagina 263

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 263 C. xyli cynodontis. xyli subsp. subsp. C. xyli xyli and xyli subsp. Amplification also obtained for pathogenic strains that lack plasmid pEA29. Clavibacter xyli Multiplex assay allows differentiation between subsp. C. xyli Amplifies also pathogenic strains that lack plasmid pEA29. et al., 2001 1998 et al., et al., et al., et al., et al., et al., et al., et al., ger ö Falloon 2006 Kabadjova- Hristova 2006 Kim Shrestha 2007 Fegan 1998 Pan Obradovic 2007 De Bellis 2007 St 2006 Leifsonia Bacteria (DNA extraction) Bacteria (DNA extraction), plant (untreated) Bacteria (DNA extraction) Bacteria (untreated), vascular fluid (PVP) Bacteria (untreated), vascular sap (PVP and Ficoll) Bacteria (DNA extraction) Plant (DNA extraction) Genus Nested Not indicated Multiplex Multiplex Conventional Conventional Conventional Conventional Conventional Bacteria (boiled) Conventional Nested-Scorpion Real-time (duplex format of Scorpion) Variant of PCR protocol Sample (treatment) Reference Synonyms/observations region EA29) EA29) EA29) EA29) EA29) EA29) ams A (p A (p A (p A (p A (p A (p + + N N N N N N region A/B E3/E4 d D d D d D CxcREV d D d D d D i i i cps (primary) i i i ITS region ITS region ITS region ITS region (unknown) (unknown) AJ75/AJ76 Cxx1/Cxx2 (secondary) Target DNA Primer name CPS1/CPS2c m m m m m AJ245/AJ246 m RST60/RST59 EpSPF/EpSPR EP16A/EPIG2c as as as as as as RSD 33/RSD 297 pEA29A/pEA29B FER 1-F/FER 1-R CxFOR/CxxREV/ Chromosomal DNA PEANT1/PEANT2 PEANT1/PEANT2 Chromosomal DNA Pl Pl Pl Pl Pl Pl 16S rRNA/ITS region CxxITSf # 5/CxxITSr 5 Chromosomal xyli xyli xyli subsp. subsp. subsp. Species/subspecies E. amylovora E. amylovora E. amylovora E. amylovora E. pyrifoliae E. pyrifoliae L. xyli L. xyli L. xyli 002_LetterEditor_249 25-06-2009 10:41 Pagina 264

264 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 but not from was proposed in

” . Distinction of the two ” 2005) and thus is not included in Calodendrum capense L. africanus et al., Ca. Liberibacter americanus “ L. species, whereas O12c/OA1primers amplify Candidatus “ the ISPP list (updated to 2004). 2005 (Teixera PrimersOI1/OI2c and O12c/OI1/OA1 amplify both Ca. prefentially Amplification from species requires restriction analysis. citrus hosts of huanglongbing disease. et et al., et al., et al., et al., et al., et al., et al., 2005 al., Coletta-Filho Jagoueix 1996 Garnier 2000 Jagoueix 1994 Teixera 2005 Hung 1999 Grisham 2007 Okuda 2005 ” ) e r p tu p tu a Liberibacter oc n m u m Plant (DNA extraction) Plant (DNA extraction) Plant (DNA extraction) Plant (DNA extraction) Plant (DNA extraction) Plant (DNA extraction) (I Plant (DNA extraction) Plant (DNA extraction) Candidatus “ Genus Green ® Real-time Master Mix) LAMP assay Conventional Conventional Conventional Conventional Conventional Conventional Conventional (SBYR Variant of PCR protocol Sample (treatment) Reference Synonyms/observations B gene rpo gene A2/J5 A2/J5 -operon cluster Rpl-B3 operon KAJL- fD1/rP1 fD2/rD1 CAL1/J5 β OI1/OI2c OI1/OI2c OI1/OI2c GB1/GB3 ITS region O12c/OA1 Target DNA LSg2f/LSg2r Primer name rpl 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 226-primer pair O12c/OI1/OA1 Lxx82F/Lxx22R 16S rRNA gene4 G- Lxx202F/Lxx331R Universal 16S rRNA Ribosomal protein genes nus Universal 16S rRNA gene Rpl-FIP, Rpl-BIP, Rpl-F3, Ribosomal protein genes _- Specific DNA fragment (unknown) ” ” ” ” ” ” ” ” xyli ” subsp. L. asiaticus L. asiaticus L. africanus subsp. L. africanus L. americanus L. americanus L. asiaticus L. asiaticus L. africanus L. africanus Species/subspecies Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. “ “ L. xyli “ capensis “ “ “ “ “ “ 002_LetterEditor_249 25-06-2009 10:41 Pagina 265

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 265 gypsophilae pv. Direct distinction of the two species. Comparison and validation of previously published protocols. Erwinia herbicola et et al., 2006b 2007 et al., et al., 1999 et al., et al., Jagoueix 1997 Hocquellet al., Li Vorwek 2007 Li Manulis 1998 ), d ate e ntr ) (DNA Pantoea t an l Daktulosphaira Plant (DNA extraction) Plant (DNA extraction) Plant (DNA extraction) Grape phylloxera ( vitifoliae extraction) Bacteria (u p Plant (DNA extraction) Genus Single Nested Real-time (TaqMan) (TaqMan) LAMP assay Conventional Conventional Conventional Conventional Conventional Multiplex real-time Variant of PCR protocol Sample (treatment) Reference Synonyms/observations a r i xe i xe 1996; 2006b

, 1999; , .

. Te , . (common) t al t al t al t al e t al t al e

e 2005; x

i , . e Li e t e t etZI gene gene iaaH l etZII (specific to each A2/J5 Probe l region -operon COXfp e t al t al β OI2/23S1 u g ou g ou e pagF/pagR TRN1/OI4

Target DNA Primer name COXf, COXr 2005; a

16S rRNA gene 16S rRNA gene 16S rRNA gene , . Isoleucine genes/ oc q of the three species) H 16S-23S rDNA spacer Cytokinin biosynthesis Cytokinin biosynthesis t al t al See: J a a See: J Ribosomal protein genes e HLBaf, HLBam, HLBas Oku d Cytochrome oxidase gene Cytochrome oxidase gene Acetamine hydrolase gene (forward) HLBr (reverse) ” ” ” ” ” ” ” ” ” ” pv. L. asiaticus L. asiaticus L. asiaticus L. asiaticus L. africanus L. africanus L. africanus L. africanus L. americanus L. americanus Species/subspecies Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. Ca. “ “ “ “ “ “ “ “ “ “ P. agglomerans P. agglomerans gypsophilae 002_LetterEditor_249 25-06-2009 10:41 Pagina 266

266 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 ) and oryzicola Burkholderia ), P. agglomerans syringae and pv. and oryzae Pseudomonas ), atroseptica P. ananas (pathovars (pathovars Pseudomonas avenae subsp. ( Pseudomonas glumae ( Faint bands obtained for with ITS primers. Erwinia herbicola Acidovorax avenae glumae Erwinia carotovora Erwinia stewartii fuscovaginae, Pseudomonas syringae Xanthomonas oryzae also amplified and differentiated by primary secondary fragments. et et al., et al., et al., et al., 1997; Coplin and Majerczak, 2002 Walcott 2002 Anon., 2006c Rcommended in the EPPO protocol. Kim and Song, 1996 al., Hyman 1997 De Boer and Ward, 1995; van der Wolf 1996; Fraaije Wilson 1994 A N ) on) te a s cti y a l ) e on ud ti c A extr A extr a cr N Pectobacterium d n xtr Bacteria, plant (untreated) Bacteria (boiled), seed (IMS) Bacteria (boiled or alkaline lysis) Bacteria (DNA extraction) Bacteria, plant (D Tuber (immunomagnetic separation followed by alkaline lysis) Bacteria (boiled), (D enriched peel Bacteria, plant (DNA extraction a e Bacteria, potato peel (enriched) Genus separation (IMS-PCR) Conventional Conventional Conventional Conventional reaction (LCR) Immunomagnetic PCR-coupled ligase Variant of PCR protocol Sample (treatment) Reference Synonyms/observations ORF region DE region hrpS ITS region ITS region ITS region (unknown) hrpS Target DNA Primer name cps PanITS1/Gs4 ES16/ES1G2c ES16/ES1G2c ECA1f/ECA2r 16S rRNA gene 16S rRNA gene R 16-1/R 23-2R ESIG1/ESIG2c HRP1d/HRP3r HRP1d/HRP3r CPSL1/CPSR2c Chromosomal DNA 16S-P5/16S-P3 (PCR) Es1, Es2, Es3, Es4 (LCR) 16S-23S rRNA/ITS region 16S-23S rRNA/ITS region subsp. subsp. subsp. Species/subspecies P. ananatis P. stewartii stewartii P. stewartii stewartii P. stewartii stewartii Pantoea agglomerans P. atrosepticum 002_LetterEditor_249 25-06-2009 10:41 Pagina 267

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 267 from subsp. Brenneria carotovorum. ) aso amplified. ) aso Pectobacterium , as well subsp. ) also amplified. P. carotovorum also amplified. atroseptica carotovora atroseptica atroseptica also amplified. B. quercina P. carotovorum subsp. subsp. subsp. subsp. E. chrysanthemi Erwinia chrysanthemi P. betavasculorum ( (

,

and . . (distinction by restriction analysis). sp sp E. carotovora E. carotovora Dickeya Amplification obtained for other genera. Banding patterns allow differentiation of other and restriction analysis improves discrimination. Dickeya dianthicola Amplification obtained with wasabiae Lower specificity of multiplex PCR, undesirable band obtained with E. carotovora Dickeya Primers for detection of four potato tubers pathogenic fungi are also described. nigrifluens E. carotovora P. wasabiae 2003 1995 1999 2006 et al., et al., et al., et al., et al., et al., Peters 2007 Kang Toth Hyman 1997 Park Smid Atallah and Stevenson, 2006 A N ) on ti c a xtr Enriched potato tubers (microsphere immunoassay) Bacteria (untreated), plant (DNA extraction) Bacteria (untreated), microplant (enriched) Bacteria (boiled), potato peel (DNA extraction) Bacteria, potato tubers (DNA extraction) Bacteria (boiled), potato tubers (centrifugation and lysis buffer) Potato tubers (D (D Potato tubers e Nested Green ) Real-time Multiplex Multiplex Competitive Conventional Conventional Conventional (iQ Supermix SBYR- (iQ Conventional and RFLP V V ents m RRE ag RORE H C gene + ene frene genes family gene ECA4r OR/ E g mpd Nested to OR/ ATOR/ CHRREV (unknown) (unknown) (unknown) SR3F/SR1cR PEAF/PEAR ECA1f/ECA2r 16S rRNA gene Rhs AD Pca For/Pca Rev RWF el INPCCF/INPCCR RWF Dcd Forw/DcdRev Chromosomal DNA Chromosomal DNA EXPCCF/EXPCCR Chromosomal DNA MpdEc-F/MpdEc-R EXPCCF/EXPCCR p E (competitor template) E Metalloproteases coding ERWFOR+ATROREV+ Contains ECA2r sequence subsp. subsp. subsp. carotovorum P. atrosepticum P. atrosepticum P. atrosepticum P. atrosepticum P. carotovorum P. atrosepticum, P. carotovorum carotovorum P. atrosepticum, P. carotovorum carotovorum 002_LetterEditor_249 25-06-2009 10:41 Pagina 268

268 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 (1994). ) and Type et al. ). P. corrugata P. mediterranea phaseolicola glycinea pv. pv. wasabiae atroseptica carotovora betavasculorum odorifera phaseolicola phaseolicola subsp. subsp. subsp. subsp. subsp. pv. pv. C. o P. syringae Protocol slightly modified from Bereswill Differentiation between Type I ( II (proposed new species, Pseudomonas syringae P. syringae Specificity improved by annealing temperature of 80 E. carotovora PCR reaction and restriction enzyme analysis do not clearly discriminate species. E. carotovora E. carotovora E. carotovora E. carotovora Pseudomonas syringae et et al., et al., et al., et al., et al., et al., et al., et al., et al., 1998 2002 Scortichini and Marchesi, 2001; Scortichini Widmer 1998 Schaad 1995 Loreti and Gallelli, 2002 Catara 2002 Prosen 1993 Borowicz 2002 Catara 2000 Darrasse 1994; Helias al., Ullrich 1993 c on) on) eti n cti cti g a a a om n A extr A extr A extr A extr u Pseudomonas N N m m Bacteria (boiled), plant (BLOTTO) Bacteria or soil (D Seed washes (untreated) Bacteria, plant (DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction) Bacteria (boiled) after enrichment or i Bacteria, seed (D Bacteria, plant (alkaline extraction) separation) Plant, soil and water (DNA extraction) Bacteria (DNA extraction) Genus Nested Multiplex Conventional Conventional Conventional Conventional Conventional Bacteria (boiled) Conventional dependent PCR Random primer- Conventional and RFLP Conventional and RFLP Variant of PCR protocol Sample (treatment) Reference Synonyms/observations hrpW PC1/2 PC5/2 – – ) genes gene cluster Y1/Y2 PC1/2 (group I) pel WA/WC PC5/2 (group II) ( – Tn5-derived – Target DNA Primer name Pseudomonas HM6/HM13 Ps-for/Ps-rev PC1/1 PC5/1 (P. corrugata) 16S rRNA gene 16S rRNA gene PAV 1/PAV 22 HB14F/HB14R RAPD fragments RAPD fragments Phaseolotoxin gene (P. mediterranea) family of pectate lyase family of pectate P 5.1/p 3.1 (external) P 5.2/P 3.2 (internal) Harpin-encoding Y PC1/1 PC5/1 Phaseolotoxin gene cluster Phaseolotoxin gene cluster P. glycinea subsp. ( sensu ( pv. pv. pv. pv. Type II) ) Species/subspecies corrugata P. atrosepticum P. carotovorum carotovorum P. betavasculorum P. odoriferum P. wasabiae Pseudomonas P. avellanae P. avellanae P. corrugata P. corrugata P. mediterranea P. savastanoi P. savastanoi phaseolicola P. savastanoi phaseolicola P. savastanoi phaseolicola stricto 002_LetterEditor_249 25-06-2009 10:41 Pagina 269

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 269 phaseolicola pv. Toxigenic and nontaxigenic strains amplified. Toxigenic and Toxigenic and nontaxigenic strains differentiated. Toxigenic and P. syringae Bacterial identification. et al., et al., et al., et al., et al., et al., et al., et al., et al., lez á Gonz 2003 Schaad 1995; Rico 2006 Schaad 2007 Penyalver 2000 Bertolini 2003b Nou, Koh and 2002 Takahashi 1996 Marchi 2005 Cintas 2002, 2006 Bacteria (DNA extraction) Seed washes (previously plated on semiselective medium MT) Bacteria, seed washes, plant (untreated) Bacteria, plant (DNA extraction) Bacteria, pre- enriched plant (DNA extraction) Bacteria (alkaline lysis) Bacteria, plant (untreated) Bacteria (DNA extraction) Bacteria (DNA extraction) BIO Nested Real-time Multiplex (TaqMan) BOX-PCR Conventional Conventional Conventional Conventional Conventional pthE phtE gene gene gene avrPphF gene (IAA chromosomal cluster gene (levan P3004R iaal iaal ptzf/ptzr synthesis) (internal) Real-time amtA BOXA 1R lscCf/lscCr Locus Locus gene (IAA) (IAA gene (cytokinin iaaHf/iaaHr iaaMf/iaaMr biosynthesis) biosynthesis) biosynthesis) biosynthesis) P5/P8, P7/P8 Locus lscC iaaM IAALF/IAALR RAPD-fragment PHA19/PHA95 PsF-tox/PsR-tox AVR1-F/AVR1-R ptz IAALN1/IAALN2 PHTE-F/PHTE-R P5.1/P3.1+P3004L/ Probe PsF-tox-286P P1/P2, P3/P4,P1-P4, iaaH tox-argK IAALF/IAALR (external) Repetitive DNA sequences Genomic DNA (unknown) Plasmid COR1 (coronatine actinidae alisalensis pv. pv. pv. pv. pv. pv. pv. pv. pv. P. savastanoi phaseolicola P. savastanoi phaseolicola P. savastanoi phaseolicola P. savastanoi savastanoi P. savastanoi savastanoi P. savastanoi savastanoi P. syringae P. syringae P. syringae atropurpurea 002_LetterEditor_249 25-06-2009 10:41 Pagina 270

270 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 of , pv. P. syringae papulans pv. P. syringae also amplified and helianthi P. syringae pv. genomospecies 1. wehereas Pap1/Pap3 also amplify other Pap1/Pap2 amplify only Pseudomonas syringae considered as nontoxigenic form of tagetis. et et al., et al., et al., et al., et al., et al., et al., 2005 Scortichini 2005 Fanelli 2007 Kerkoud 2002 Arnold 1996 Kong 2004 Kerkoud 2002; Vanneste and Yu, 2006 Sorensen 1998 al., Zaccardelli on) cti a A A ) ) N N on on ti ti c c A extr A extr a a N xtr xtr Bacteria (boiled), plant (D e Bacteria (DNA extraction) Bacteria, plant (D Bacteria (untreated) Bacteria (DNA extraction) Bacteria (boiled), plant (D e Bacteria (DNA extraction) ERIC) Real-time Multiplex Multiplex Conventional Conventional Conventional Conventional Conventional Conventional Conventional Bacteria Conventional (molecular beacon) Rep-PCR (BOX and P. e ene en gene L g A g p . tomato RN gene hrpL gene gene gene gene gene gene gene on hr pst pst i L gene pv L1/L2 L7/L8 P0/P6 eg B1/B2 B1/B2 D1/D2 r syrB SyrB hrpL e 16S re 16S SyrD Asnb exbD HrpL Hrp hrpZ Pap1/Pap3 Pap1/Pap2 r TAGTOX9 (molecular beacon) i TAGTOX10 nal AN3/1 Type I AN3/2 Type I AN7/1 Type II AN7/2 Type II MM5F/MM5R tr RAPD fragment RAPD fragment RAPD fragment Rtimefor/RTRev RcalFor1/RTRev n syringae PapHrp1/papHrp2 nter E Full-length FP1/TAGTOX9 RP1 I FP10/TAGTOX10 RP1 16S rDNA+specific to 27F/1492R+HSP1/HSP2 Probe ) s e d ti coryli papulans papulans pisi syringae tagetis tomato tomato ep pv. pv. pv. pv. pv. pv. pv. pv. nonap i s ep od p i (strains producers of cyclic l P. syringae P. syringae P. syringae P. syringae P. syringae P. syringae P. syringae P. syringae 002_LetterEditor_249 25-06-2009 10:41 Pagina 271

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 271 and Burkholderia oryzae ), Pantoea agglomerans (pathovars (pathovars ), X. oryzae Pseudomonas avenae ( ), phaseolicola pv. ) also amplified and differentiated by primary Pseudomonas glumae ( Erwinia herbicola P. syringae Bacterial identification. Acidovorax avenae Colorimetric detection of amplicons using digoxigenin amplicons using Colorimetric detection of marked internal probes. glumae ( oryzicola and secondary fragments. et al., et al., 2002 et al., et al., et al., 2002 Sawada 1997 Vicente and Roberts, 2007 Scortichini and Marchesi, 2001; Scortichini Lydon and Patterson, 2001 Kim and Song, 1996 Bultreys and Gheysen, 1999 2000 Bertolini 2003a, b; Penyalver Bacteria (DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction) Bacteria (untreated) Lee Bacteria (DNA extraction) Bacteria (DNA extraction) Pre-enriched, plant (DNA extraction) Nested Rep-PCR Multiplex Nested and Conventional Conventional Conventional Bacteria (untreated) Conventional Conventional Conventional nested RT-PCR immunocapture-nested pv. gene gene MVi2 region SLRVi2 iaal /CLRVi2 /ArMVi2 (tabtoxin gene) SyrD BOXA1R (tabtoxin gene) resistance) PM/PtQM savastanoi tblA1/tblA2 tabA1/tabA2 Pt-1A/Pt-1D1 OCTF/OCTR R16-1/R23-2R gene (phaseolotoxin SyrD1ISyrD2 16S rRNA gene REP1R/REP2I PAV 1/PAV 22 ERIC1R/ERIC2 P. savastanoi Pt-1A/Pt-1D1+Pt- tblA tabA 16S-23S rDNA spacer argK Tolaasin biosynthesis genes CMV1/CMV2+CMVi1/C CLRV1/CLRV2+CLRVi1 SLRV1/SLRV2+SLRVi1/ ArMV1/ArMV2+ArMVi1 IAALF/IAALR (external) AALN1/IAALN2 (internal) I actinidae syringae syringae theae actinidae pv. pv. pv. (pathovars (pathovars pv. pv. pv. pv. pv. (SLRSV) P. syringae P. tolaasii P. syringae producers of toxic lipodepsipeptide) P. syringae tabtoxin) producers of P. savastanoi phaseolicola P. syringae P. savastanoi savastanoi Four viruses: Cucumber mosaic virus (CMV) Cherry leaf roll virus (CLRV) Strawberry latent ringspot virus Arabis mosaic virus (ArMV) P. syringae morsprunorum P. syringae P. fuscovaginae P. syringae P. avellanae P. syringae 002_LetterEditor_249 25-06-2009 10:41 Pagina 272

272 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 are not pathovars X. campestris ( X. gardneri phaseoli P. sryringae and phaseolicola pv. pv. a bi n a a bi vesicatoria n glycinea phaseolicola pv. c a c a . ) also amplified. pv. pv. p v p v

X. vesicatoria. a e i n g phaseoli sy r sy Pseudomonas solanacearum P. solanacearum P. solanacearum Xanthomonas axonopodis pv. Other coronatine-producing also amplified with COR primers. X. axonopodis valid names according to ISPP list. BSX primers also amplify Pseudomonas syringae P . P. syringae P. syringae 1996 1997 1992b et al., 1998 et al., et al., et al. 1998 et al., et al., et al., et al., Seal 1992a; Hartung et al., Ito Bereswill 1994 Gillings 1993 Cuppels 2006 ) on ti c a xtr e

Ralstonia A N Bacteria, plant (boiled) Bacteria, plant (D Soil suspensions plated on selective medium (DNA extraction) Bacteria (DNA extraction) Pure cultures or plant tissue (frozen- boiled method DNA extraction) Genus BIO Conventionallysis) Seeds (alkaline Audy Conventional ConventionalConventional Bacteria (boiled) Seal Conventional Conventional Bacteria (boiled) Sato Conventional and RFLP Variant of PCR protocol Sample (treatment) Reference Synonyms/observations gene ) ) ) ) cfa7 gene gene efe enzime) gene (ethylene- ing 759/760 X4c/X4e X4c+X4e T3A/T5A efe pehA (unknown) gene cluster Target DNA Primer name BSX1/BSX2 COR1/COR2 Plasmid DNA form PS96H/PS96I Pseudomonas Pseudomonas Xanthomonas Xanthomonas Partial ETH-1/ETH-2 ETH-1/ETH-3 tRNA consensus ( ( ( ( HB 14F/HB 14R HB 14F+HB 14R+ (polygalacturonase) pehA # 3/6 pehA pehA # # Chromosomal DNA Coronatine biosynthesis Entire cff primer 1/ primer 2 (simultaneous detection) Coronafacic acid Phaseolotoxin gene cluster Genomic DNA (unknown) Genomic DNA (unknown) glycinea sesami glycinea, maculicola tomato tomato pv. pv. pv. pv. pv. pv. pv. pv. pv. pv. Species/ biovars P. syringae P. syringae morsprunorum P. syringae P. cannabina P. savastanoi P. savastanoi phaseolicola P. syringae P. syringae atropurpurea P. syringae P. savastanoi phaseolicola P. syringae R. solanacearum R. solanacearum R. solanacearum R. solanacearum 002_LetterEditor_249 25-06-2009 10:41 Pagina 273

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 273 division 2 race 1. division 1 strains. R. solanacearum R. solanacearum R. solanacearum strains. OLI/Z primers identify D1/B identify et 2000 2001 Specific detection of et al., et al., et al., et al., et al., 2000 Boudazin 1999; van der Wolf Pastrik 2002 Lee and Wang, 2000 Pradhanang al., Pastrik and Maiss, 2000 Weller 2000 A N on) acti Bacteria, potato tuber (untreated) Bacteria, potato tubers (DNA extraction) Soil (DNA extraction) (D Potato tubers extr Soil suspensions (previously enriched and boiled) Bacteria, potato tubers extract (boiled) Nested Real-time (TaqMan) (TaqMan) Conventional Conventional Conventional Bacteria (untreated) Lee Conventional Conventional Conventional Multiplex real-time d ) 2A r 1405 a (primers) division I ) v division II o i (primers) (primers) (probes) + (b 1405 (probe) (probe) x gene D1/B D2/B e region/ region/ b and IS l OLI1/Z (IS RS or B2 p PS-1/PS-2 OLI-1/Y-2 PS-IS RA1 PS-IS-RB1 ti B2-P RS-P control, host) l BP4-R/BP4-L Rs-1-F/Rs-1-R Rs-1-F/Rs-3-R 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene RAPD fragment COX-P PS-IS-F/PS-IS-R 1405 Insertion sequence Flanking regions of Multiplex (internal Multiplex (generic) Mu RS-I/RS-II B2-1/B2-II IS 16S-23S rDNA spacer 16S-23S rDNA spacer OLI-1/OLI-2 + JE2/Y2 R. solanacearum COX-F/COX-R R. solanacearum Potato cytochrome oxidase R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum 002_LetterEditor_249 25-06-2009 10:41 Pagina 274

274 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 X. campestris ) and Blood (

) and Blood

pelargonii pv. Ralstonia solanacearum Ralstonia also amplified.

Pseudomonas syzygii Pseudomonas syzygii Pseudomonas syzygii and Blood Disease Bacterium also ( ( ) also amplified. pelargonii Disease Bacterium also amplified. Division I and II of differentiated. Ralstonia syzygii Xanthomonas hortorum pv. Race 3, biovar 2 strains are specifically amplified. Ralstonia syzygii amplified. P. solanacearum Ralstonia syzygii Disease Bacterium 1993, et al., 1993 et al., et al., nfeld et al., et al., ö Caruso 2003 Seal 1999 Glick 2002 Ozakman and Schaad, 2003 Sch 2003 Poussier and Luisetti, 2000 Seal A A N N on) on) cti cti a a ) ) on on ti ti c c A extr A extr A extr A extr a a N N xtr xtr Bacteria (boiled), water Bacteria, soil (D e Pure culture (D Bacteria (boiled) Bacteria or plant (D Potato tuber extract (boiled) e Bacteria (boiled), plant, water and soil (D lex p BIO Nested Multiplex Multiplex Multiplex Multi Co-operative Conventional Conventional Conventional Conventional Real-time (TaqMan)- gene

on DNA i

eg A r ene andene fliC + A g RN pelargonii (ERIC) control) OLI1/Z protein) biovar 2

RN genes cluster RS3/Rs4 RsoL OLI1/Y2 F/NS-6-R (Co-PCR) OLI-1/Y-2 DG1/DG2 OLI1/OLI2 1b (internal) OLI1/BV345 DIV2F/ITRS pv. RSC-P (probe) NS-5-F/NS-6-R 16S rRNA gene 16S rRNA gene DIV1F/DIV1R DIV2F/DIV2R

internal control) XcpM1/XcpM2 hrp 16S r OLI1/OLI2/JE-2 gene (flagellar subunit gene (flagellar OLI1+Y2+BV345 16S-23S r 18S rRNA gene (host 18S Rs-1-F/Rs-1-R+NS-5- RS30/RS31 (external) X. c. 18S rDNA (host internal 18S RSC-F/RSC-R (primers) DNA fragment specific to fliC R. solanacearum pehB RS30a/RS31a/RS30b/RS3 R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum R. solanacearum 002_LetterEditor_249 25-06-2009 10:41 Pagina 275

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 275 is not included in the ISPP

achromogenes var. . S. scabies S. scabies S.scabies list et al., et al., et al., et al., Lehtonen 2004 Burkhalid 1998 Wanner, 2006 Wanner, 2004 Stange 1996 Koyama 2006 Rhodococcus Streptomyces Bacteria, potato tubers (DNA extraction) Bacteria (DNA extraction) Bacteria (DNA extraction) Plant (DNA extraction) Potato tubers, soil (DNA extraction) Genus Genus (QCQP) Conventional Conventional Mycelium (boiled) Conventional Conventional Conventional quenching probe Variant of PCR protocol Sample (treatment) ReferenceVariant of PCR protocol Sample (treatment) Reference Synonyms/observations Synonyms/observations Quantitative competitive es S. S. abi ´ . sc and S

gene gene gene Streptomyces S. aureofaciens Nf/Nr pA/pH fic for fic Probe T Probe IS (primers) nec1 nec1 nec1 gene (cytokinin gene (thaxtomin AurI/AurII Stel3/ T2st2 Target DNA Target DNA eci Primer name Primer name biosynthesis) biosynthesis) turgidiscabies ScabI/SacbII Specific for TurgI/TurgII TxtA1/TxtA2 scab1/scab2m ASE3/scab2m europaeiscabiei Nec1F/Nec1R 16S-1F/16S-1R 16S rRNA gene S. scabies 16S rRNA gene scab1m/scab2m Sp NEC-F2/NEC-R2 Universal for for Universal fas-1 16S rDNA sequences txtA Internal standard DNA JRERIGHT/JRELEFT Species and strain-specific Specific for group spp. Species Species var. Streptomyces R. fascians S. acidiscabies S. scabiei S. turgidiscabies S. scabiei S. turgidiscabies S. aureofaciens S. acidiscabies S.scabiei S.scabies achromogenes Streptomyces S. acidiscabies S. aureofaciens S. bottropensis S. europaeiscabiei S. scabiei S. stelliscabiei S. turgidiscabies New 002_LetterEditor_249 25-06-2009 10:41 Pagina 276

276 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 Cullen and Lees, 2007 Maes, 1993 Wanner, 2007 on) cti a A extr A extr Xanthomonas N Bacteria, potato tubers and soil (D Bacteria (boiled) or seed extract Bacteria (DNA extraction) Genus Nested Real-time (TaqMan) Conventional Conventional Variant of PCR protocol Sample (treatment) Reference Synonyms/observations ) ) S. newly group) and gene gene gene gene 8/27 Nf/Nr 461/477 nec1 nec1 (internal) (primers) 16s rDNA 16S rDNA Aci1/ Aci2 Aci1/ Aci2 Stel3/ Aci2 TomA Aur1/ Aur2 TxtAB ASE3/ Aci2 ASE3/ Aci2 Target DNA Primer name Tom3/Tom4 S. acidiscabies S. stelliscabiei S. acidiscabies S. bottropensis S. aureofaciens ASE3/Scab2m S. scabies ( S. turgidiscabies europaeiscabiei 16S rRNA gene Species-specific identified group ( (Newly identified Turg1m/ Turg2m NecTqP1 (probe) NecNF1/NecNR2 Streptomyces Streptomyces 16S-1F/16S 455-435 NecTqF1/NecTqR1 NecF1/NecR1 (external) TxtAB TxtAB1/ TxtAB2 TxtAB TxtAB1/ (genus) spp. Species/pathovars Streptomyces S. acidiscabies S. aureofaciens S.europaeiscabiei S. scabiei S. stelliscabiei S. turgidiscabies Xanthomonas 002_LetterEditor_249 25-06-2009 10:41 Pagina 277

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 277 X. and pv. P. syringae phaseoli ( pv. . is not a valid name phaseoli phaseolicola fuscans fuscans pv. pv. X. arboricola var. var. Xanthomonas campestris phaseoli phaseoli phaseoli ) also amplified. phaseoli pv. pv. pv. fuscans. pv. var. phaseolicola X. campestris Xf1/Xf2 specific for X. campestris Pseudomonas savastanoi phaseoli X4c/X4e amplify both pv. campestris X. campestris Xanthomonas campestris X. campestris phaseoli according to the ISPP list. 1998 et al., et al., 1997 et al., et al., et al., et al., et al., et al., et al., ne- et al., è Audy 1996 Rob Soustrade 2006 Toth Pan Birch 1997 Honeycut 1995 Wang 1999 Pagani, 2004 Verdier 1998 Khoodoo 2005 Audy 1994 A A on) N N cti a ) on) e cti tur tur a ) p A extr A extr on N oca oca on or ti n c A extr A extr u a acti N m xtr m Seeds (alkaline treatment) Bacteria (boiled), plant (PP buffer with 5% PVP) Bacteria, plant (D Bacteria (boiled) or leaf (D Bacteria, sap, leaf (untreated) Bacteria, leaf (D Bacteria (DNA extraction) Bacteria, sap, leaf (boiled) Bacteria, plant (untreated) Plant extracts (without DNA extraction) e Bacteria, plant (D Bacteria, plant (D extr i BIO RAPD Nested Nested Previous Multiplex Multiplex Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional immunocapture r r r and 23S (internal) (external) ile /KJM12 /KJM36 /KJM73 + + + f f f rRNA Ala4/L1 OP-G11 Xf1/Xf2 X4c/X4e X4c/X4e X4c/X4e Ala4/IIe2 or tRNA (unknown) XAF1/XAR1 Plasmid DNA Plasmid DNA Plasmid DNA ala ala Random primer Region between RAPD fragment RAPD fragment RAPD fragment RAPD fragment (Xanthomonas) KJM34 KJM74 KJM11 Plasmid fragment Y17CoF/Y17CoR Inter tRNA region region Inter tRNA 16S rRNA gene and 16S 16S+IIe1 or Ala1+23S PXadU/PXadL tRNA NXadU/NXadL Genomic DNA (unknown) var. var. var. phaseoli phaseoli phaseoli phaseoli pruni pv. pv. pv. pv. pv. pv. pv. pv. X. albilineans X. albilineans X. albilineans X. arboricola X. axonopodis dieffenbachiae X. axonopodis dieffenbachiae X. axonopodis manihotis X. axonopodis fuscans X. axonopodis fuscans X. axonopodis fuscans X. axonopodis X. campestris phaseoli X. campestris phaseoli X. campestris phaseoli 002_LetterEditor_249 25-06-2009 10:41 Pagina 278

278 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 citri citri citri citri citri citri pv. pv. pv. pv. pv. pv. X. campestris X. axonopodis X. axonopodis X. axonopodis X.axonopodis X. axonopodis et et et al., et al., 2006a et al., 2006 2007 et al., al., Hartung 1993 Hartung 1996 Coletta-Filho Cubero and Graham, 2002 al., Zaccardelli Li Cubero 2001 ) e r on) on) cti cti p tu p tu a a a oc n A extr A extr A extr m u N N m Plant (i Bacteria, plant (D Bacteria, plant (D Bacteria (DNA extraction) Bacteria, plant and seeds (DNA extraction) Plant (DNA extraction) Plant (DNA extraction) Nested Competitive Conventional Conventional Conventional Conventional Conventional (Internal standard) Concurrent detection and X. ) (second citri pv. termini for a + ´ 2/3 DNA strains and C) round) (FMV) A9, A10 gene cluster X4c+X4e virulence) ITS region associated) (unknown) gene (involved in CiH2/CiH3 (first round) 4/5; 6/7; 1/5 J-pth1/J-pth2 Pseudomonas Xac01/Xac02 Plasmid DNA Plasmid DNA J-RXg/J-RXc2 gene (pathogenicity- rpf ( HB 14F/HB 14R HrcCF2/HrcCR2 HB 14F+HB 14R+ plasmid DNA of Pathotype A strains Pathotype A strains Pathotype A strains Figwort mosaic virus termini homologous to Genomic and plasmid Genomic and ´ pthA Pathotypes A, B and C Simultaneous detection axonopodis Contains 5 A5, C5, A2, D2, A3, D7, 3 94-3 bio/94-4 lac 94-3 bio/94-4 hrcC (variable for pathotypes B (variable for Phaseolotoxin gene cluster citri citri citri citri citri citri pv. subsp. subsp. subsp. subsp. subsp. subsp. (Pathotypes A, B and C) (Pathotypes X. campestris campestris X. citri X.citri X.citri X. citri X. citri X. citri 002_LetterEditor_249 25-06-2009 10:41 Pagina 279

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 279 strains Xanthomonas is not included in the ISPP is not included in the ISPP List. citri citri citrumelo pv. pv. pv. citri aurantifolii pv. pv. X. axonopodis Recommended in the EPPO protocol. X. citri X. citri list. Allelic discrimination of citrus allowed and a single nucleotide difference detected. X. axonopodis X. axonopodis et et al., et al., 2004 Hartung 1993, 1996; Cubero 2001; Cubero and Graham, 2002; Anon., 2005b Mavrodieva al., Cubero and Graham, 2005 on) on) cti cti a a A extr A extr A extr A extr N N Plant (DNA extraction) Bacteria, plant (D Bacteria, plant (D Mix) Green Master ® Real-time Real-time (TaqMan) Conventional (SBYR l a l X. a X. i i r citri citri A te ter ter s c s pv. pv. n n i citrumelo citrumelo a ba c ba ba ai citri s s tr u u st r st s tr t r r r pv. pv. pv. i 2/3 spp. Xanthomonas c gene family X. citri X. citri ci strains strains , , e nke n ke n ke virulence) J-Taqpth2 downXC2 ITS region a a J-Taq16S-1 VM1/VM2 VM3/VM4 VM5/VM6 gene (involved in n e n e c c chromosome en J-pth1/J-pth2 J-Alrpallelic1 e Plasmid DNA J-RXg/J-RXc2 J-Awlrpallelic1 g g pthA X. citri gene, gene, gene, Pathotype A strains Pathotype A strains th restricted host range th J-AdlrpU1J-AdlrpU2 J-RT pth3/J-RT pth4 Pathotype A, B and C p pthA lrp wide host range strains p Ribosomal sequence, Ribosomal sequence, lrp lrp axonopodis axonopodis J-RTRib 16Sup/J-RTRib Kingsley forward/reverse pv. citri citri citri aurantifolii pv. subsp. subsp. subsp. (Pathotypes A, B and C) (Pathotypes X. citri X. axonopodis citrumelo X. citri X .citri X. citri 002_LetterEditor_249 25-06-2009 10:41 Pagina 280

280 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 Primers pair 245A/245B and 245.5/245.267 can be used in both conventional and nested PCR. Bacterial identification. Both pairs of primers can be used in conventional or nested PCR. Recommended in the EPPO protocol. et et , et et n a ger and et al., et al., ö m et al., et al., et al., r e 2004; 1996; Anon., 1996 2004 ger and m ö Pooler 1996 2005 Roberts Roberts al., Moltmann and Zi 1996; Mahuku and Goodwin, 1997 1996; Zimmermann Zhang and Goodwing, 1997 St Ruppitsch, 2004 Opgenorth al., 2006d Pooler 1996; St Ruppitsch, 2004; Anon., 2006d al., Opgenorth al., Pooler 1996; Zimmermann on) on) on) cti cti cti a a a A extr A extr A extr A extr A extr A extr N N N Bacteria (DNA extraction) Bacteria, plant (D Plant (DNA extraction) Bacteria, plant (D Plant (DNA extraction) Plant (with or without DNA extraction) (enrichment) Bacteria, plant (D rep Bacteria (untreated) rep Nested Nested Nested Multiplex Multiplex Conventional Conventional Conventional Conventional Conventional (second (first round) (first round) (first round) (first round) gene gene gene gene + + (second round) (second round) (different primer round) JJ9/JJ12 hrp hrp hrp hrp 29 A, 295B 241A/241B 245A/245B 295A/295B XF10/XF12 245.5/245.267 (second round) RAPD fragment RAPD fragment RAPD fragment ERIC1R, ERIC2 ERIC1R, ERIC2 RAPD fragments REP1R-I, REP2-I, REP1R-I, REP2-I, 241+245, 241+295, pairs combinations) 245A-245B fragment 245A-245B fragment 245.5/245.267 245+295, 241+245+295 XF9/XF11 XF9/XF11 245A/245B 245A/245B 241A, 241B, 245A, 245B, Multiplex XF9/XF12 XF9/XF12 X. fragariae X. fragariae X. fragariae X. fragariae X. fragariae X. fragariae X. fragariae X. fragariae 002_LetterEditor_249 25-06-2009 10:41 Pagina 281

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 281 C o X. . and pv. also amplified. zinniae from pistachio and oryzae cerealis pv. incanae

results in a fluorescent signal. are amplified, whereas at 68 C both pv. o citri, X. translucens oryzae pelargonii pelargonii pelargonii carotae X. translucens pv. oryzicola pv. pv. pv. pv. pathovars and X. oryzae At an annealing of 60 campestris A fragment of the same size also obtained from Groups A and B of X.campestris X. campestris X. campestris X. campestris differentiated. only oryzicola 2007 et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Zhao Adachi and Oku, 2000 Marefat 2006 Meng 2004 Manulis 1994 Sulzinski 1996, 1997, 1998 Chittaranjan and De Boer, 1997; Manulis 1994 van Doorn 2001 Kim and Song, 1996; Adachi and Oku, 2000; Sakthivel 2001; Anon., 2007 1996; Anon., 2006d Sakthivel 2001 A N on) on) on or on cti cti cti a a a ) or BIO- ) on ti c A extr A extr A extr A extr A extr a N N N xtr Rice seeds washes (untreated) Bacteria, plant (boiled) Bacteria (DNA extraction) or plant (PVP addition) Bacteria (boiled), plant, seeds (DNA extraction) Bacteria (DNA extraction) Bacteria, plant (D Bacteria, plant (D Bacteria, plant (untreated) Bacteria, plant (D BIO-PCR from seeds without DNA extraction) Pure cultures, and plant tissue (D e PCR from seeds (without DNA extraction) BIO BIO Real-time Multiplex (TaqMan) Conventional Conventional Conventional Roberts Conventional Conventional Conventional Conventional and DNA cds gene oryzae gene PF/PR pelargonii (ERIC)

oryzicola hrp 3SF/3SR fimA ITS region ITS region ITS region ITS region insertion element insertion element XF9/XF11 JAAN/JARA TXT/TXT4R TXT/TXT4R pv. R16-1/R23-2R PAf/PBf/PABr RAPD fragment Conventional RAPD fragment Conventional RAPD fragment XcpM1/XcpM2 fimbrial-subunit) receptor gene Differentiation of XOR-F/XOR-R2 XOR-F/XOR-R2 (type IV structural c.

Putative siderophore pathovars IS1113 X. IS1113 carotae pelargonii pelargonii pelargonii oryzae oryzae oryzae oryzae oryzicola pv. pv. pv. pv. pv. pv. pv. pv. pv. X. oryzae X. hortorum X. hortorum X. hortorum X. hortorum X. hyacinthi X. oryzae X. oryzae X. oryzae X. oryzae X. translucens 002_LetterEditor_249 25-06-2009 10:41 Pagina 282

282 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 cerealis, secalis, translucens, graminis, phlei, phleipratensis,

vesicatoria pv. pathovars: X. campestris X. campestris undulosa, arrhenatheri, poae No distinction of the five cereal leaf streak pathovars from the other five pathovars. 1995 1994 2005, et al., et al., et al., et al., et al., Leite Leite Maes 1996b 2006 Berg Mitkowski 2005 A N ) (sodium ) on ti c a xtr Seed washes (D (D Seed washes e ascorbate and PVPP) Bacteria (DNA extraction) Bacteria, seeds (boiled) Bacteria, seed- washes (DNA extraction) Bacteria (DNA extraction) Mix) Green Master probes) ® Conventional Conventional Conventional Multiplex-Real-time (fluorescently labeled (fluorescently (SBYR Multiplex-Conventional Conventional and RFLP ) ) Brassica groups spp. gene gene X. campestris X. campestris + + spp. T1/T2 (hypersensitive (hypersensitive hrpF hrpF 23S rRNA ITS region gen cluster) gen cluster) Brassica RST2/RST3 RST2/RST3 RST9/RST10 RST9/RST10 RST21RST22 XAN1/XAN2 XAN3/XAN4 XAN5/XAN7 Encompassing ITS region and 16S rRNA, ITS, hrpC, hrpD DHL155/DHL156 DLH138/DLH139 DHL153/DHL154 DLH120/DLH125 hrpB hrpB 5.8S rRNA gene from (host internal control) ITS region from reaction and pathogenicity (Specific for reaction and pathogenicity (Specific for poae undulosa pv. pathovars: pathovars: and group) X. translucens X. vesicatoria X. campestris aberrans, armoriaceae, barbarae, campestris, incanae, raphani Xanthomonas Numerous pathovars (not translucens X, campestris hordei X. translucens arrhenatheri, cerealis, graminis, phlei, phleipratensis, poae secalis, translucens 002_LetterEditor_249 25-06-2009 10:41 Pagina 283

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 283 t. s and oryzae ISPP Li Li ISPP Burkholderia pathovars Xaf pep ), . to the not included in g n i pathovars pathovars assayed d Pantoea agglomerans Pseudomonas syringae cor ), and P. sryringae ac fragariae X. fragariae s and e m X. oryzae pv. also amplified. na X. vesicatoria. X. arboricola d i Pseudomonas avenae l ( vesicatoria ), pelargonii specific for pv. X. fragariae. pv. e not va also amplified and differentiated by r a

ri . Pseudomonas glumae Xf gyrB e ( rdn syringae a g Erwinia herbicola oryzicola Acidovorax avenae Differentiation between X. glumae ( X. axonopodis Pseudomonas fuscovaginae pv. primary and secondary fragments. BSX primers amplify Other coronatine-producing also amplified with COR primers. Ralstonia solanacearum primers detect other Xanthomonas arboricola the ISPP List. Primers X. campestris also, but not et al., et al., et al., Kim and Song, 1996 Cuppels 2006 Pooler and Hartung, 1995 Weller 2007 Glick 2002 d A N t ) an ) an n ) r A e d l e pl a pl N oi a s ), ) ) e l N A (b (D o n o n on a a Xylella i i Re ti ti ti r r c c c t (D t (D a a a n e n e c te c te c te c te a n l xtr xt r xt r Bacteria (DNA extraction) Bacteria, plant (freeze-boiled method DNA extraction) Ba Ba (D Bacteria, sap e e p (Ge e Genus Real-time Multiplex (TaqMan) Conventional Conventional Conventional Variant of PCR protocol Sample (treatment) Reference Synonyms/observations R gene R gene

p- B- r fragariae cfa7 strains (probe) (probe) gene pv. citrus strains / Xf gy / f Xa pe F F gene region specific p- B- (general) RS3/Rs4 (primers) (primers) r DG1/DG2 X. fragariae Target DNA Primer name BSX1/BSX2 DNA (ERIC) GyraseB COR1/COR2 R16-1/R23-2R f pe (Pseudomonas) RAPD fragment internal control) (Xanthomonas) XcpM1/XcpM2 CVC-1/272-2-int f gy f gy a propyl endopeptidase Xf gyrB-P Xf gyrB-P Xaf pep-P 272-1-int/272-2-int X. fastidiosa X X 18S rRNA gene (host 18S 16S-23S rDNA spacer X. arboricola X. arboricola X. fastidiosa pep Coronafacic acid R. solanacearum pehB Genomic DNA (unknown) pv. ) pelargonii fragariae pv. pv. / citrus oryzicola (pathovars and Species / hosts X. fragariae X. arboricola X. hortorum X. oryzae oryzae X. vesicatoria Pseudomonas syringae X. fastidiosa tomato 002_LetterEditor_249 25-06-2009 10:41 Pagina 284

284 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 C. o C. o Recommended in the EPPO protocol. Only citrus and coffe related strains amplify at 68 Strains from various hosts amplified at annealing 64 et al., et al., et al., et al., et al., et al., et al., et al., 1994; Pooler and Hartung, 1995; Anon., 2004b Minsavage Minsavage 1994 Schaad 2002 Bextine and Miller, 2004 Hernandez- Martinez 2006 Oliveira 2002 Baumgartner and Warren, 2005 Fatmi 2005 Berisha 1998 Ferreira 2000 A A A N N i ou s i ou A N e v N ate A b N ) ) ) ) ) cor s ) a on on on on on ti ti g ) g ) ti ti ti on m c c c c c ti u a a a a a i i d a ti n d xtr xtr xtr xtr xtr od Bacteria, leaf (D Bacteria, leaf (D a Bacteria, leaf (D Bacteria, leaf (D Plant and xylem fluid (PVPP and s Plant tissue (D Bacteria, xylem sap or plant (D e e Plant and xylem fluid (DNA extraction) Xylem sap (D (D Xylem sap e e e Sap and macerated chips of secondary trunks of vines xylem (untreated) Leaf and petiole r (directly or p p l Bacteria, plant tissue (DNA extraction) BIO Real-time Real-time Real-time (TaqMan) (TaqMan) (TaqMan) (TaqMan) Multiprimer Conventional Conventional Conventional Conventional Conventional Conventional (Agar absorption) Multiplex-Real-time (ITS) (16S) 6FAM- ´ grapevine citrus strains gene Probe Probe strains Probes TAMRA labeled TAMRA Primers ´ ´ JB-1/JB-2 ITS region ITS region 3 labeled ITS XfF1/XfR1 XfF2/XfR2 XfF1/XfR1 6FAM-labeled 6FAM-labeled 6FAM-labeled ´ ´ ´ RST31/RST33 RST31/RST33 RST31/RST33 RST31/RST33 16S rRNA gene RAPD fragment RAPD fragment CVC-1/CCSM-1 5 5 5 CVC-1/272-2-int XF1968-L/1968-R TAQCVC 5 TAMRA labeled TAMRA labeled X. fastidiosa ´ ´ 3 3 X. fastidiosa XF1968 methyltransferase labeled 3 Genomic DNA (unknown) Genomic DNA (unknown) Genomic DNA (unknown) Genomic DNA (unknown) Genomic DNA (unknown) / citrus and / citrus and / citrus and / grapevine, / citrus / grapevine / grapevine / grapevine / grapevine X. fastidiosa X. fastidiosa X. fastidiosa X. fastidiosa X. fastidiosa grapevine X. fastidiosa grapevine X. fastidiosa coffe X. fastidiosa and oleander X. fastidiosa almond, oleander 002_LetterEditor_249 25-06-2009 10:41 Pagina 285

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 285 2005 et al., et al., et al., et al., et al., et al., et al., et al., Pooler 1997 Francis 2006 Rodrigues 2003 Costa 2004 Manceau 2005 Botha 2001 Manceau 2000 Grall A N c eti n A g a N ) ) om on on n ti ti c c u Xylophylus a a m xtr xtr m I Plant tissue, vector insects (D e separation of insects tissue extracts Plant and insect vectors (DNA extraction) Plant (DNA extraction) extraction) Bacteria (boiled) and bleeding sap (D e Bacteria and stem sap (DNA extraction) Bacteria (DNA extraction) Genus + Nested Nested Real-time Multiplex (TaqMan) PCR-ELISA conventional Conventional Conventional Conventional Plant, sap (DNA Conventional Conventional separation (IMS) Immunomagnetic Immunocapture and Variant of PCR protocol Sample (treatment) Reference Synonyms/observations ) ) s s on er C) er , m i m B izati i r r d i p r p (external) ts A, A, ts nal gene nal e hyb gene labeled S3/S4 Probe v A1/B1 G1/L1 s (se s (se nter (exter (internal) gyrB (i TM fa HL5/HL6 ITS region ITS region ITS region . 1 acti Target DNA Primer name ALM1/ALM2 RST31/RST33 RST31/RST33 S4 S4 B tr X 16S rRNA gene RAPD fragment S- 272-1-int/272-2-int XF2542-L/XF2542 S3/ A1/ S- 272-1/272-2 Sub XATS1/XATS2-Biotin BHQ1 6FAM-labeled Xamp 1.3A/Xamp 1.3B XF2542 fimbrial protein FXYgyr499/RXYgyr907 ´ ´ Subtractive hybridization Xamp 1.27A/Xamp 1.27B Xamp 1.27A/Xamp 1.27C Xamp 1.27A/Xamp 1.27C Genomic DNA (unknown) Genomic DNA (unknown) Genomic DNA (unknown) Genomic DNA (unknown) 5 3 Species X. fastidiosa X. fastidiosa X. fastidiosa X. fastidiosa X. ampelinus X. ampelinus X. ampelinus X. ampelinus 002_LetterEditor_249 25-06-2009 10:41 Pagina 286

286 PCR and plant pathogenic bacteria Journal of Plant Pathology (2009), 91 (2), 249-297 also amplified. R. syzygii and Bacterium within group 3 of the gamma subclass Proteobacteria. Ralstonia solanacearum 1998 1998 2007 1993 et al., et al., et al., et al., et al., et al., et al., ” Botha 2001 Dreo Boudazin 1999 Zreik Davis 1998 Avila (BLOs) ” A A N N ) Seal ed l oi ) ) (b on on a i ti ti r c c a a cte xtr xtr Phlomobacter fragariae Plant (DNA extraction) Plant (DNA extraction) Phloem tissue (D Phloem tissue (D e e Bacteria-like Organisms “ Yellow vine disease (YVD) Blood Disease Bacterium (BDB) Candidatus “ Papaya bunchy top disease of Cucurbita (PBT) Papaya bunchy top disease of Nested Other bacteria Real-time (TaqMan) PCR-ELISA Conventional Bacteria (untreated) Conventional Ba Conventional Conventional Conventional Variant of PCR protocol Sample (treatment) Reference Synonyms/observations Variant of PCR protocol Sample (treatment) ReferenceVariant of PCR protocol Sample (treatment) Reference Synonyms/observations Variant of PCR protocol Sample (treatment) Reference Synonyms/bbservations Synonyms/observations Biotin 1 S3/S4 D2/B (probe) OLI1/Z OLI1/Y2 (primers) YV1/Yv3 Fra4/Fra5 YV1/YV2 YV1/YV3 YV1/YV2 (external primers) ITS region ITS region ITS region (internal primers) Target DNA Target DNA Target DNA Target DNA Primer name Primer name Primer name Primer name 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene 16S rRNA gene Xamp 14F/104 MGB XATS1/XATS2- Xamp 14F/Xamp 104R S3/S4 A1/B1 Subtractive hybridization d ) d ) e e fi fi i i s s a a l l c c Species Organism Organism Organism un un s s n n i i a a Phlomobacter e m e m X. ampelinus Ca. fragariae PBT (Gamma-3 proteobacterium associated with BLO disease) YVD (Gamma-3 proteobacterium associated with BLO disease) Blood Disease Bacterium (r Blood Disease Bacterium (r 002_LetterEditor_249 25-06-2009 10:41 Pagina 287

Journal of Plant Pathology (2009), 91 (2), 249-297 Palacio-Bielsa et al. 287

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