006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 595 Journal of Plant Pathology (2011), 93 (3), 595-602 Edizioni ETS Pisa, 2011 595 QUANTITATIVE DETECTION OF PSEUDOMONAS CORRUGATA AND PSEUDOMONAS MEDITERRANEA IN TOMATO PLANTS BY DUPLEX REAL-TIME PCR G. Licciardello1, P. Bella2 and V. Catara2 1 Parco Scientifico e Tecnologico della Sicilia s.c.p.a., Z.I Blocco Palma I, Stradale V. Lancia 57, 95121 Catania, Italy 2 Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, Università di Catania, Via S. Sofia 100, 95123 Catania, Italy SUMMARY sufferance symptoms such as top chlorosis or wilting during the hottest part of the day. The most characteris- Pseudomonas corrugata and P. mediterranea are two tic symptom of the disease is visible in stem sections closely related bacterial species both causal agents of where the pith appears hydropic and/or necrotic and tomato pith necrosis. To screen tomato planting material hollowed. Production of adventitious roots and bacteri- reliably, a quantitative real-time PCR assay was devel- al ooze at leaf scars or hydropic lesions can be visible on oped, to detect and/or discriminate both bacterial the outside of the affected stem portions. As pith necro- pathogens in a single tube. So, two species-specific sis extends, the plant loses turgor and collapses. P. primer/probe sets were designed on the sequences of two corrugata has also been isolated from pepper (Lopez et DNA fragments amplified by a previously reported spe- al., 1988), chrysanthemum (Fiori, 1992) and geranium cific PCR protocol. TaqMan real-time PCR assays were (Magyarosy and Buchanan, 1995) affected by pith developed for individual (simplex PCR) and simultane- necrosis. ous (duplex PCR) amplifications. The assays were per- P. corrugata and P. mediterranea are readily isolated formed with the SmartCycler TD II System (Cepheid) from infected plants on bacteriological agar media. Accu- and the fluorescence from both FAM and Texas Red rate identification is important because other bacteria channels were recorded at the annealing step. Specificity (mainly Pseudomonas spp. but also Pectobacterium spp.) was tested with an extended range of P. corrugata and P. have also been associated with necrosis of tomato mediterranea strains, with other Pseudomonas spp. and (Catara, 2007). Two semi-selective media have been de- with a number of tomato bacterial pathogens. The detec- scribed for isolation from soil (Scortichini, 1989) and tion limit was approximately 10 cells per reaction for seeds (Kritzman, 1991). Immunotrapping associated with both bacteria, and quantification was linear over a six-log a semiselective medium was used to isolate P. corrugata range. The duplex real-time PCR assay was validated on efficiently from soil where it was present in low concen- tomato plants artificially inoculated by pricking the stem trations (Achouak et al., 2000). Nevertheless, these meth- with a strain of each species either separately or together. ods have not subsequently used for diagnostic purposes. Attempts to find rapid methods for the identification Key words: Tomato pith necrosis, Pseudomonas, mo- of P. corrugata, have encountered problems with high lecular diagnosis, in planta detection, bacterial quantifi- infraspecific variability (Siverio et al., 1993, 1996; cation. Catara et al., 1997; Sutra et al., 1997). P. corrugata and P. mediterranea are distinguishable from each other by the ability of P. mediterranea strains to utilize meso-tartrate, INTRODUCTION 2-ketogluconate and histamine. Moreover, two oligonu- cleotide pairs, designed within two RAPD genomic re- Pseudomonas corrugata (Roberts and Scarlett) emend. gions, amplify two specific DNA fragments of 600 or Sutra et al. (1997) and P. mediterranea Catara et al. 1100 bp from P. mediterranea and P. corrugata, respec- (2002) are the causal agents of “tomato pith necrosis” tively, in multiplex PCR reactions (Catara et al., 2002). (TPN). The disease occurs worldwide on greenhouse or An increasing number of real-time PCR assays has field tomatoes being reported mainly on adult plants been developed for phytopathogenic bacteria (Lopez et with first trusses but also on some nursery plantlets (re- al., 2009; Palacio-Bielsa et al., 2009). This technique viewed by Catara, 2007). Plants usually show general combines the sensitivity of conventional PCR with spe- cific real-time fluorescent signals throughout the reac- tion, allowing faster and less labour-intensive quantifica- tion of the target than with conventional PCR as it elim- Corresponding author: V. Catara Fax: +39.095.7147287 inates post-PCR processing. E-mail: [email protected] More recently, although real-time PCR protocols 006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 596 596 Duplex real-time PCR for detecting tomato pith necrosis agents Journal of Plant Pathology (2011), 93 (3), 595-602 were already available for some bacterial species, multi- sensitive and reliable tool for simultaneously detecting plexed protocols were re-designed or adapted to detect TPN causal agents and accurately identifying either P. multiple infections or discriminate species or the patho- corrugata or P. mediterranea and to assess the presence genic variants of a species as, for example, for P. savas- of mixed infections. tanoi pathovars (Bella et al., 2008; Tegli et al., 2010), Er- winia amylovora and E. pyrifoliae (Lehman et al., 2008) and Xanthomonas campestris (Berg et al., 2006). MATERIALS AND METHODS In this study, a real-time assay based on TaqMan chemistry with two primer pairs and TaqMan probes, Bacterial strains, growth conditions and DNA ex- differentially labelled, was developed to provide a fast, traction. The assay was developed using 43 P. corrugata Table 1. Bacterial strains used in this study. Matrix of Geographic Species Strains isolation origin CFBP 5454, CFBP 5436, PVCT 2.2, CFBP 5438, PVCT 4.1, PVCT 4.2, CFBP 5456 , PVCT 7.4, CFBP 5442, PVCT 10.1, P. corrugata Tomato Italy PVCT 10.2, CFBP 5449, PVCT 10.5, PVCT 10.6, PVCT 10.7, CFBP 5451, PVCT 10.9, PVCT 10.12, PVCT 10.13, PVCT 10.14, 6FP, 59a, 60b Soil Italy PVCT 5.1t ; PVCT 6.1t, CFBP 5459 CFBP 10146, CFBP 10900, CFBP 10904, CFBP 10950, CFBP Tomato Spain 12342, CFBP 12343, CFBP 10890 Tomato France CFBP 10532 Tomato UK NCPPB 2445 Tomato New Zealand CFBP 10058 Tomato USA 421 Tomato Switzerland CFBP 10938 Tomato Hungary Par8 Alfalfa roots USA NCPPB 3316, 313, 388 Raspberry USA 717 roots PVCT 1.6, PVCT 1.7, CFBP 5444, PVCT 8.3, CFBP 5447, P. mediterranea Tomato Italy CFBP 5458, PVCT P.1, B021 Tomato Spain CFBP 10148 Tomato France CFBP 10558 Pepper Spain CFBP 10894, CFBP 10961 Other Pseudomonas strains P. cichorii Endive Germany CFBP 2101 Pre-filter P. fluorescens UK CFBP 2102 tanks P. marginalis pv. Chicory USA CFBP 1387 marginalis P. putida Soil USA CFBP 2066 P. savastanoi pv. nerii Oleander Italy PVCT 89.1.1 P. savastanoi pv. Bean Ethiopia 1448a phaseolicola Lemon, P. syringae Italy PVCT 104.1.1, PVCT 10.2, PVCT 26 pear Other tomato- associated bacteria C. michiganensis Tomato Italy PVCT 156.1.1, PVCT 163.2.1, PVCT 176.1.1 subsp. michiganensis Hungary NCPPB 2979 P. syringae pv. tomato Tomato Italy PVCT 28.3.1 P. marginalis Tomato Italy PVCT 81.1.11, PVCT 81.5.1 Xanthomonas Tomato New Zealand CFBP 2537 vesicatoria CFBP: Collection Francaise des Bacteries Phytopathogenes, Angers, France; NCPPB: National Collection of Plant Pathogenic Bacteria, York, UK; PVCT: Patologia Vegetale, University of Catania, Italy. 006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 597 Journal of Plant Pathology (2011), 93 (3), 595-602 Licciardello et al. 597 and 12 P. mediterranea strains of diverse geographical One microliter of DNA from a single bacterial species origins (Table 1). The pathogenicity and taxonomy of or from DNAs of both species mixed 1:1 was used as these strains were verified by standard procedures template. For in planta detection 2 µl of DNA were (Catara et al., 1997, 2002). Pseudomonad strains be- used. Negative control reactions contained the same longing to other species and a number of tomato bacter- mixture with sterile water replacing the DNA template. ial pathogens were also tested (Table 1). P. corrugata and All PCR reactions were performed both as single and P. mediterranea strains were cultured on nutrient broth duplex PCR reactions in 25 µl Smart Cycler reaction or agar (Difco, USA) supplemented with 1% D-glucose tubes (Cepheid Europe) in a Smart Cycler TD II System (NDA), all the other pseudomonads on King’s medium (Transportable Device TD configuration) (Cepheid Eu- B (King et al., 1954), other bacteria on nutrient agar rope). The thermal cycling conditions for P. corrugata (NA). Total genomic DNA was extracted with Gentra and P. mediterranea DNA for both single and simultane- Puregene Cell Kit (Qiagen, Italy) from 1.5 ml bacterial ous amplification were initial denaturation at 95°C for broth cultures according to the manufacturer’s instruc- 30 sec followed by 40 cycles at 95°C for 10 sec, 62°C for tions. DNA was quantified by comparison with known 30 sec with heating ramp rates (max) 10°C sec-1 and standards in SYBR Safe-stained 1% agarose gel (Invit- cooling ramp rates (max) 25°C sec-1. Smart Cycler TD II rogen, Italy). System channel 1 and 2 were switched on for detection of FAM and Texas-Red, respectively. For each sample, Oligonucleotides primers and probes for real-time detection was performed by online monitoring to iden- PCR. The primer and probes were designed for a du- tify the exact threshold cycle number (Ct-value) where plex real-time PCR assay with the primer design soft- the log-linear phase could be distinguished from the ware Beacon DesignerTM 7.5 (Premierbiosoft.com) background. Reactions were analysed by 2% agarose within two species-specific DNA fragments obtained by gel electrophoresis to confirm that only specific PCR a RAPD screening (Catara et al., 2000).