Quantitative Detection of Pseudomonas Corrugata and Pseudomonas Mediterranea in Tomato Plants by Duplex Real-Time Pcr

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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 oligonucleotide 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 (real., 2009; Palacio-Bielsa et al., 2009). This technique viewed by Catara, 2007). Plants usually show general combines the sensitivity of conventional PCR with specific real-time fluorescent signals throughout the reaction, allowing faster and less labour-intensive quantification 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

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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

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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). These DNA products were amplified. fragments were end-sequenced and specific primer pairs (PC1/1-PC1/2 and PC5/1-PC5/2) were developed Real-time PCR sensitivity. To determine sensitivity to be used in multiplex PCR assays (Catara et al., 2000, of the real-time PCR assay, aqueous bacterial suspen- 2002). Full nucleotide sequence of the fragments was sions of P. corrugata strain CFBP 5454 and of P. mediter- obtained by direct sequencing (Eurofins MWG operon, ranea strain CFBP 5447 at a concentration of 1 x 108 Germany). New real-time PCR primers were: PcoFw CFU ml-1 each were prepared from a 24 h-old culture (5’-GGTGGTATCGGTTGCGTAGCG-3’) and on NDA. The two bacterial suspensions were mixed PcoRew (5’-GTGGGAACGTTTGGGCCTGG-3’) for and ten-fold serial diluted in sterile distilled water used P. corrugata detection, amplifying a 149 bp DNA frag- in duplex real-time PCR reactions. Standard curves ment, and PmeFor (5’-CTGTCCGAGACGATGGC- were generated for each bacterial species from Ct-values GAC-3’) and PmeRew (5’-CAGACGTGGCCT- of dilution plotted versus the log cells ml-1. CAAGCAGAT-3’) for P. mediterranea detection, amplifying a 85 bp DNA fragment. The corresponding Plant material. One-month-old tomato plants of cv. probes were designed within the DNA segments to al- Marmande grown in nursery flats, were stab-inoculated low fluorescent detection of amplification and labelled on the stem at the axil of the first true leaf with a sterile with different reporter molecules: Pco (5’FAM-CCGC- entomological mounting pin, coated with bacterial cells CCCATCTGCCCACCCTGC-3’BHQ-1) and Pme taken from 48 h cultures on NDA of P. corrugata CFBP (5’TXR-CCCGCTCATCCGCTGGACCGGC-3’BHQ- 5454 and P. mediterranea CFBP 5447. During the trials, 2) for P. corrugata and P. mediterranea, respectively. plants were maintained in a growth chamber with 16/8 h (day/night) photoperiod at 28°C. After inoculation, PCR conditions and fluorescence detection. Real- plants were enclosed in polyethylene bags to maintain time PCR assays were performed using lyophilised 100% relative humidity for 3 days; the bags were then beads OmniMix HS (Cepheid Europe). Each bead was removed until the end of the experiment, fourteen days used for two 25 µl total final volume reactions. Opti- after inoculation (Catara et al., 1997; Licciardello et al., mization of primer and probe concentrations to mini- 2007). Artificially inoculated tomato stems were excised mize the interference from competing reactions during from 0.5 cm above to 0.5 cm below the inoculation site duplex PCR, was performed on samples with both pure and ground with a pestle in a 1.5 ml eppendorf tube in bacterial DNAs and cell suspensions present in known 1 ml of a antioxidant buffer (Gorris et al., 1996). One concentration at a range of dilution. Optimized primer hundred microliters were used for bacterial plate count concentrations were 400 nM and 800 nM (for both the using a spiral plater (Eddy Jet, IUL Instrument, Ger- forward and reverse primers) for P. corrugata and P. many) and the pellet of the residual bacterial suspension mediterranea, respectively. Probes concentrations were was used for DNA extraction with Gentra Puregene 200 nM for P. corrugata and 400 nM for P. mediterranea. Cell Kit (Qiagen, Italy). 006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 598

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Table 2. Quantification of Pseudomonas corrugata and P. mediterranea from separately and simultaneously inoculated plants by real-time PCR and plate count method.

Real-Time PCR (Ct) Bacterial quantification Channel 1 Channel 2 Real-time PCR Plate count Species (FAM) (Texas-Red) (Log cells ml-1)a (Log cfu ml-1) 23.39 0 5.26 3.08 23.65 0 5.10 4.29 23.16 0 5.31 4.50 23.32 0 5.24 4.66 22.71 0 5.62 4.45 P. corrugata 22.34 0 5.81 4.04 23.51 0 5.15 4.48 23.42 0 5.23 4.57 23.60 0 5.20 4.20 21.60 0 6.46 5.77 0 29.41 4.37 2.70 0 27.59 4.88 3.11 0 26.31 5.29 3.48 0 27.32 5.05 3.34 0 24.61 5.63 4.37 P. mediterranea 0 3173 4.28 3.48 0 34.00 4.44 3.00 0 28.34 4.28 3.92 0 28.84 4.83 3.27 0 29.41 4.63 3.75 22.62 25.17 5.74 4.00 22.12 23.96 6.47 4.78 23.28 24.70 6.61 4.38 20.82 24.91 6.81 6.00 19.45 21.69 7.17 6.30 19.71 20.00 7.82 6.54 19.77 21.56 6.88 6.04 P. corrugata + P. mediterranea 19.95 21.43 6.85 6.11 26.75 27.76 5.34 4.80 19.60 22.18 6.35 5.00 19.93 21.34 7.05 6.86 26.63 25.43 5.58 4.61 21.48 19.87 6.75 6.08 21.89 19.77 6.25 5.15 a Absolute quantification for both bacteria was calculated by interpolation on the respective standard calibration curves after duplex real-time PCR assay; for mixed inoculation individual species concentration was determined and the data reported are the log of the sum of the deduced cells ml-1.

RESULTS AND DISCUSSION strains of the two genomic groups were actually alloca- ble either in P. corrugata or in the new species P. Primer design, specificity and optimization of the re- mediterranea (Catara et al., 2002). As a consequence, al-time PCR assay. The real-time PCR protocol devel- the two species could be identified and discriminated oped for the diagnosis of Tomato pith necrosis identi- by the two primer pairs, as observed by other authors fied and discriminated the two causal agents of the dis- (Fiori, 2002; Moura et al., 2005; Basim et al., 2005; ease, P. corrugata and P. mediterranea, from pure or Quezado-Duval et al., 2007; Passo et al., 2008; Alippi mixed cultures detecting and quantifying them in and Lopez, 2010). planta. The real-time PCR assay relies on the original Since the length of the amplicons generated by PCR protocol for P. corrugata based on detection of two primer pairs PC5/1 – PC5/2 and PC1/1 – PC1/2 de- specific characterised regions with two pairs of primers, scribed by Catara et al. (2002) were not optimal for a re- PC5/1 – PC5/2 and PC1/1 – PC1/2 (Catara et al., al-time PCR assay, two new specific primer pairs and 2000). These primers used in multiplex PCR identified two TaqMan probes were designed within the specific a collection of P. corrugata strains but clearly highlighted DNA regions. the presence of two genomic groups, each detected by a Real-time PCR primer pairs, designated PcoFw/Rew specific set of primers. Further studies revealed that and PmeFw/Rew, were first tested in conventional PCR 006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 599

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Fig. 1. Amplification plot of Pseudomonas corrugata (A) and P. mediterranea (B) strains detected by duplex real-time PCR reactions recording FAM and Texas-Red fluorescence, respectively. Each species was detected on channel either 1 or 2 of the Smart Cycler TD II system according to the wavelength of emission of the respective probes. No fluorescence was recorded from bacteria belonging to other species and water controls. Data from Smart Cycler software 2.0 were exported as excel data to build black- and-white graphs by using Microsoft Excel.

(either separately and together) using a mix of the two ate assay specificity, 43 isolates of P. corrugata, 12 of P. bacterial genomic DNAs to verify the presence of the mediterranea, 9 strains of other Pseudomonas spp. and correct amplicon as well as the absence of cross reac- other tomato associated bacteria were tested (Table 1). tions. Two specific and distinct 149 bp and 85 bp DNA As detected by the Smart CyclerTM II System, the fluo- fragments for P. corrugata and P. mediterranea were ob- rescence of either the FAM or Texas-Red reporter dyes served with end point detection after agarose gel elec- unambiguously identified all the strains of P. corrugata trophoresis, respectively (not shown). The primers did and P. mediterranea, respectively. There was no fluores- not react with other Pseudomonas spp. or other tomato cence either in the FAM or TexasRed channels when bacterial pathogen DNAs (not shown). closely related plant-associated Pseudomonas or other With real-time PCR performed both in simplex and bacteria were tested, indicating that the amplification duplex reactions, primers and probes distinguished P. reactions were specific (Fig. 1). corrugata and P. mediterranea amplicons due to labelling Accurately identifying P. corrugata and P. mediter- with different reporter dyes, FAM and Texas-Red, re- ranea is important because an increasing number of spectively (Fig. 1). Using three ten-fold serial dilutions bacteria have been described as causing symptoms of of a 1:1 mixture of the DNAs of P. corrugata and P. necrosis, soft rotting or hollowing of tomato stem, such mediterranea at a concentration of 50 ng µl-1, the opti- as the fluorescent Pseudomonas strains isolated from mal primer/probe concentrations were determined: P TPN-affected plants in different countries (Dhanvan- corrugata 400 nM primers and 200 nM probe; P. tari, 1990; Catara et al., 1997; Sutra et al., 1997), as well mediterranea 800 nM primers and 400 nM probe (not as other Pseudomonas spp. such as P. cichorii (Wilkie shown). Reaction efficiency was similar both in simplex and Dye, 1974), P. viridiflava (Alivizatos, 1986), P. fluo- (P. corrugata R2=0.981, P. mediterranea R2=0.982) and rescens (Skoudridakis, 1986), P. marginalis (Bella and duplex real-time PCR (P. corrugata R2=0.975, P. mediter- Catara, 2010) and Erwinia spp. (Victoria and Granada, ranea R2= 0.975) and the mean values for the standard 1983; Alivizatos, 1985; Dhanvantari and Duks, 1987; curve slopes were not statistically different (ca. -4.6), in- Fiori et al., 2005). dicating that the PCR assays had similar reaction kinet- The duplex assay sensitivity was measured by a 10- ics. So, amplifications were routinely performed in du- fold dilution series of a bacterial suspension containing plex. No interference was detected between optical equal amounts of P. corrugata and P. mediterranea cells. channel and fluorescence emissions and there was no For all experiments, the Smart Cycler TD II System in- cross reactivity among these two closely related mi- strument recorded fluorescent light emission in two dif- croorganisms. Agarose gel electrophoresis analysis of ferent channels, accumulating with the number of cycles the real-time PCR products showed a positive correla- and depending on the initial amount of template DNA. tion between the fluorescence signal and the amplifica- Standard curves were obtained by plotting Ct values tion of the expected DNA fragment (not shown). versus the number of cells of each 10-fold dilution series (Fig. 2). The standard deviation for each point of Real-time PCR specificity and sensitivity. To evalu- the standard curve (indicated by error bars) increased 006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 600

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imental conditions the symptoms are already developed, but the tissues are still not completely necrotised (Catara et al., 1997; Licciardello et al., 2007). P. corrugata strain CFBP 5454 and P. mediterranea strain CFBP 5447 were detected by duplex real-time PCR in artificially inoculated plants, showing characteristic TPN symptoms, both when inoculated separately and simultaneously. Ct values of the DNA isolated from plant samples were plotted against the standard curve, and the inferred cell concentrations of the two bacteria were calculated. The fluorescence signals of the two bacterial probes were ab- solutely specific and recorded by each specific channel without any cross-reaction as assessed by the real-time PCR analysis of plants inoculated with a single bacterial species. No fluorescence was detected for DNA extracted from water-inoculated control plants. Aliquots of the inoculated plant extracts were also plated on NDA. Correlation and regression analysis between CFUs obtained by plate count method and the number of cells quantified by real-time PCR were sig- nificant (P<0.01; R2=0.81) (Fig. 3). However, the bacterial population detected by real-time PCR was much higher (1-2 logs) than the colony number (Table 2). This discrepancy could depend on the fact that PCR-based methods estimate microorganism DNA whether they are alive or not. In our case, another important factor which may influence quantification, is the unknown number of target DNA copies on the genome. The results of bacterial quantification upon inoculation of the two bacterial species should also be consid- ered. After a 14 days inoculation period tomato stem pith appeared hydropic, discoloured and necrotic, and Fig. 2. Calibration curves for the absolute quantification of sometime hollowed at the inoculation site as previously Pseudomonas corrugata CFBP 5454 (A) and P. mediterranea observed (Catara et al., 1997; Licciardello et al., 2007). CFBP 5447 (B) performed in real-time duplex PCR. Standard Symptom severity (i.e. the length of the necrosis) was curves, generated from threshold cycle numbers (Ct-value) of not significantly different in tomatoes inoculated with dilution plotted versus the log cfu ml-1, illustrated a six-log linearity range for both bacteria. Error bars represent standard individual Pseudomonas species or with a mixture of deviation from three replicates for each dilution obtained in three independent runs. R2 = correlation coefficient. Data from Smart Cycler software 2.0 were exported as excel data to build black-and-white graphs by using Microsoft Excel.

with decreasing initial target number. The correlation coefficients (R2) of the resulting standard curves for P. corrugata (0.996) and P. mediterranea (0.997) demon- strated the linearity of the quantification over a six-log range from 109 to 104 cells ml-1; this means a detection limit of 10 cells per reaction for both bacteria.

In planta detection of P. corrugata and P. mediterranea. To assess the applicability of real-time PCR for the direct in planta detection and quantification of P. corrugata and P. mediterranea, artificially inoculated plants Fig. 3. Correlation between CFUs obtained by plate count were analysed. The analysis was conducted on stem por- method and the number of cells quantified by real-time PCR tions at the inoculation site 14 days after inoculation from plants inoculated with Pseudomonas corrugata and/or P. since, in previous studies, it was observed in these exper- mediterranea. Data are log-transformed values. 006_JPP641RP(Catara)_595 15-11-2011 17:42 Pagina 601

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two species (not shown). However, when inoculated as time PCR assay for detection of Xanthomonas campestris a mixture, the bacteria showed a larger population size, from brassicas. Letters in Applied Microbiology 42: 624-630. suggesting a synergic effect between the two species. As Catara V., 2007. Pseudomonas corrugata: plant pathogen the two species were shown to produce the same array and/or biological resource? Molecular Plant Pathology 8: of acyl-homoserine lactones quorum sensing signal mol- 233-244. ecules, interspecies communication cannot be ruled out Catara V., Gardan L., Lopez M.M., 1997. Phenotypic hetero- (Licciardello et al., 2007, 2009; V. Catara, unpublished geneity of Pseudomonas corrugata strains from southern Italy. Journal of Applied Microbiology 83: 576-586. information). Catara V., Arnold D., Cirvilleri G., Vivian A., 2000. Specific The real-time quantitative PCR assay described here oligonucleotide primers for the rapid detection of the casu- can detect the closely related species P. corrugata and P. al agent of tomato pith necrosis, Pseudomonas corrugata: mediterranea with high sensitivity, a wide dynamic range evidence for two genomic groups. European Journal of of quantification and high specificity, thus enabling the Plant Pathology 106: 753-762. quantification of both bacteria in infected tomato plants. Catara V., Sutra L., Morineau A., Achouak W., Christen R., This method is field-transferable as it identifies more Gardan L., 2002. Phenotypic and genomic evidence for quickly and cheaply these species in tomato plants af- the revision of Pseudomonas corrugata and proposal of fected by TPN. Its applicability to other matrices (plants Pseudomonas mediterranea sp. nov. International Journal of or soil), or to other plant pathogen bacteria which affect Systematic and Evolutionary Microbiology 52: 1749-1758. the same host with similar symptoms, could be useful for Dhanvantari B.N., 1990. Stem necrosis of greenhouse tomato specific causal agent detection. Moreover, the combina- caused by a novel Pseudomonas sp. Plant Disease 74: 124-127. tion of species-specific and strain-specific primers and Dhanvantari B.N., Duks V.A., 1987. Bacterial stem rot of probes could also be used to study the colonization abili- greenhouse tomato: etiology, spatial distribution, and effect of high humidity. Phytopathology 77: 1457-1463. ty of species living in the same ecological niche and the Fiori M., 1992. A new bacterial disease of chrysanthemum: a interactions of microbial populations. stem rot by Pseudomonas corrugata Roberts et Scarlett. Phytopathologia Mediterranea 31: 110-114. Fiori M., 2002. Gravi attacchi di Pseudomonas corrugata ACKNOWLEDGEMENTS Roberts et Scarlett su pomodoro coltivato fuori suolo. In- formatore Fitopatologico 52 (1): 47-51. We thank E. Conte for helpful advices in primers and Fiori M., Virdis S., Schiaffino A., 2005. Phenotypic and genet- probes design. This work was funded by a grant from ic characterization of Erwinia carotovora sp. carotovora the University of Catania, Italy, Progetti di Ricerca di (Jones) Bergey et al. isolates from grafted tomato in Sar- Ateneo. dinia, Italy. Phytopathologia Mediterranea 44: 50-57. Gorris M.T., Cambra M., Llop P., López M.M., Lecomte P., Chartier R., Paulin J.P., 1996. A sensitive and specific de- REFERENCES tection of Erwinia amylovora based on the ELISA-DASI enrichment method with monoclonal antibodies. Acta Hor- ticulturae 411: 41-45. Achouak W., Thièry M., Roubaud P., Heulin T., 2000. Impact of crop management on intraspecific diversity of P. corruga- King E.O., Ward M.K., Raney D.E., 1954. 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Received February 2nd, 2011 Accepted April 13, 2011