Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods
pez1 and P. Llop1 alver1, J.M. Quesada1, M.M. Lo M. Golmohammadi1, J. Cubero2, J. Pen
quera, Valencia, Spain 1 Instituto Valenciano de Investigaciones Agrarias (IVIA). Carretera Moncada- Na n y Tecnolog a Agraria y Alimentaria (INIA). Ctra. de La Corun a, Madrid, Spain 2 Instituto Nacional de Investigacio

Keywords protocol and validation, real-time PCR, sensitivity. Correspondence P. Llop, Instituto Valenciano de Investigaciones Agrarias (IVIA). Carretera quera, km 45, Moncada, 46113. Moncada-Na Valencia, Spain. E-mail: pllop@ivia.es

Abstract Aims: To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. Methods and Results: Citrus fruits with canker-like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real-time PCR with SYBR Green or a TaqMan probe, were compared. Canker-like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplication. Canker lesions, identied by PCR, showed viable bacteria in eleven of fteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves conrmed. By real-time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 3952 lesions depending on the protocol employed. Conclusions: An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real-time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. Signicance and Impact of the Study: This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present.

2007 / 0267: received 20 February 2007, revised 8 May 2007 and accepted 8 May 2007
doi:10.1111/j.1365-2672.2007.03484.x

Introduction Citrus bacterial canker (CBC), caused by Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas axonopodis pv. aurantifolii (Xaa) (Vauterin et al. 1995) is considered one of the most important bacterial diseases of citrus trees (Stall and Civerolo 1991; Das 2003). Although a new proposal has been published to rename the CBC causing agent as Xanthomonas smithii subsp. citri (Schaad

et al. 2005), in this work, we preferred to use the standard nomenclature because the new one has not been adopted by plant protection regulatory services worldwide and is still not included in ofcial taxonomy. CBC affects most of the citrus species in the Rutaceae family (Stall and Civerolo 1991; Das 2003). This disease is characterized by eruptive lesions on leaves, stems and fruits, and decreases the fruit quality and yield (Stall and Seymour 1983). It is also important due to its socioeconomic
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impact as a quarantine organism (Gottwald et al. 2001; Pruvost et al. 2002), because its presence in a producing area hinders the exportation of plants and fruits to other countries, and forces costly eradication programs and chemical treatments, and shifts to less susceptible varieties. The disease is present in more than 30 countries of citrus producing areas worldwide (Stall and Seymour 1983; Civerolo 1984; Leite and Mohan 1990; Stall and ` re et al. 1998; Civerolo 1991; Broadbent et al. 1992; Vernie Gottwald et al. 2001; Mohammadi et al. 2001). However, CBC has not been reported in any country of the European Union (EU), and it is considered as a quarantine organism according to the EU legislation (Directive 2000 / 29 / EC). Long distance spread of CBC occurs mainly by the movement of contaminated plant material and by human activity (Roberts et al. 2005) and there is evidence that CBC was introduced into the State of Florida in USA, in South Africa and in Australia by infected citrus plants (Das 2003; Graham et al. 2004). Citrus fruit exports to EU are only allowed from areas where Xac is present, according to legislation, if they have been appropriately disinfected and are free of symptoms of the disease. More precisely, fruit with suspicious symptoms are not allowed to be exported to EU despite the treatments applied on the material if it is conrmed the symptoms are produced by the citrus canker organism. The Mediter pez 2000), produce ranean countries are free of CBC (Lo more than 56 million Tm of citrus and are considered the fourth citrus producing area in the world (Anon 2003). Among them, Spain has more than 300 000 ha of citrus, and is the world leader in fresh fruit production and exportation. According to EU legislation, the European countries, among those Spain, have to analyse samples of imported fruits with suspicious symptoms, when they are imported from areas where CBC is present, like South America (Argentina, Brazil, Mexico, Paraguay and Uruguay). Several protocols have been proposed for the detection of the causal agent of CBC (Anon 1990, 2005), but they have not been specically applied to the detection of the target in commercial fruits. Rapid development of molecular methods for characterization of bacteria over the past decade has greatly simplied and improved the detection and identication of plant patho lvarez 2004; Lo pez et al. 2005) and sevgenic bacteria (A eral sets of primers have been designed for polymerase chain reaction (PCR) detection of Xac. Among them, some are based on sequences from plasmid borne genes (Hartung et al. 1993; Mavrodieva et al. 2004; Cubero and Graham 2005), rDNA sequences (Cubero and Graham 2002) and general or pathogenicity regulatory factors (Cubero and Graham 2004, 2005; Coletta-Filho et al. 2006). Although the different authors reported some data on the sensitivity of the designed primers, specic detec2310

tion of Xac in commercial citrus fruits was not evaluated in any of these publications. To achieve an accurate, fast and reliable detection of Xac, an integrated approach that combines bacterial isolation and conventional PCR has recently been proposed (Anon 2005). To estimate its usefulness for the analysis of commercial fruits, conventional PCR protocols as well as a more recently reported real-time PCR (Cubero and Graham 2005), have been compared in fruits with canker-like symptoms, exported to Spain in the last few years, from several South American countries where Xac is present. Materials and methods Bacterial isolation Citrus fresh fruits from different areas of Argentina and Uruguay showing canker-like symptoms were taken by the Spanish inspection services at several ports of entrance and in packinghouses. All of the shipments included a certicate of a postharvest treatment with bactericide compounds like chlorine or sodium orthophenylphenate (SOPP). The samples consisted in several fruits (from 120, with one to several canker-like spots on each), and the analyses were only performed on the lesions observed on the fruits. Each lesion, and 2 mm of the peel around it, was cut in pieces with a sterilized scalpel or razor blade, comminuted in PBS buffer and left for 1020 min at room temperature. 100 ll of the PBS extract were streaked onto plates of YPGA medium (Lelliot and Stead 1987) with and without cycloheximide (Cyc; 200 mg l)1) and incubated at 25C. After 37 days, Xac-like colonies were selected and puried for further analyses. Pathogenicity tests Pathogenicity of puried Xac colonies was evaluated on detached grapefruit leaves (Citrus grandis var. Duncan). Surface of young leaves was disinfected with ethanol 70%, washed with sterile water and placed on 1% agar plates. The leaf was cut with a scalpel, the edge of the wound inoculated with 10 ll of a suspension of 109 CFU ml)1, and incubated at 2528C until symptoms appearance (12 weeks). Negative controls with sterile water and positive controls with a suspension of 109 CFU ml)1 of Xac strain CFBP 2911 were performed. The laboratory practices throughout the processes involved with the management of suspected contaminated fruits, extraction procedures, microbiological techniques (isolation, purication of suspected colonies, pathogenicity tests, etc), were performed following strict safety measures (security laboratory levels P2P3) to avoid any possibility of pathogen scape. Those

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included the use of conned laboratories, biosecurity chambers, sterilization of disposal material, etc. Molecular detection DNA extraction The procedure described by Llop et al. (1999) was used to extract total DNA from pure bacterial suspensions and lesions on citrus fruits. Briey, 1000 ll of the bacterial suspension (108 CFU ml)1), or the PBS extract obtained from the lesions were taken and centrifuged at 8000 g for 5 min. The pellet was resuspended in 500 ll extraction buffer (02 mol l)1 TrisHCl pH 75, 025 mol l)1 NaCl, 0025 mol l)1 EDTA, 05% SDS, 05% PVP, sterilized by ltration) and shaken for 60 min at room temperature. Then it was centrifuged at 1000 g for 5 min, and 450 ll of the supernatant mixed with 450 ll of isopropanol and left 30 min at room temperature. After centrifugation at 8000 g for 10 min, the supernatant was discarded, the pellet dried on air, and resuspended in 100 ll sterile water. This DNA was used for PCR or stored at )20C until further use (sometimes up to 3 years). Conventional PCR Three pairs of primers were used for amplication of DNA from Xac. Primers 2 and 3, based on Hartung et al. (1993), amplify a 222 bp DNA fragment only in type A strains. Primers J-pth1 and J-pth2 allow the amplication of a 197 bp fragment of genomic DNA in type A, B and C strains (Cubero and Graham 2002), and primers Xac01 and Xac02 amplify a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world (Coletta-Filho et al. 2006). Amplications were carried out in a nal volume of 50 ll containing 3 mmol l)1 MgCl2, 01 mmol l)1 dNTPs, 1 lmol l)1 of each primer and 1U of DNA polymerase (Biotools, Madrid, Spain) for primers designed by Cubero and Graham (2002) and Hartung et al. (1993). Primers based on Coletta-Filho et al. (2006) employed 2 mmol l)1 Mgcl2, 01 mmol l)1 dNTPs and 25 lmol l)1 of primers and 1U of DNA polymerase. When the samples analysed consisted in DNA extracted from plant material, 2 U of DNA polymerase were used. In all cases, 5 ll of the extracted DNA were added to the PCR mixture. PCR reactions were run for 40 cycles based on the cycling conditions proposed by the authors. Fifteen ll of the PCR products were run in 152% (w / v) agarose gels stained with ethidium bromide and visualized under UV transilluminator. Negative samples (healthy fruits) were analysed through the different steps of the procedure to check for possible contaminations. Water was used as negative control and Xac strain CFBP 2911 as positive control for the PCR reactions.

Real-time PCR Real-time PCR was performed using SYBRGreen or a TaqMan probe described by Cubero and Graham (2005) as uorescence reporter systems. These assays were performed in a nal volume of 25 ll of a reaction mixture consisting of PCR universal master mix (Quantimix SYBR Green or Quantimix Easy Probes; Biotools), 10 lmol l)1 of primers J-RTpth3 and J-RTpth4 and 5 lmol l)1 of TaqMan probe (J-Taqpth2b) when used. Two microlitres of the extracted DNA from fruit samples, water control and positive control were used. Amplication, detection and data analysis were performed with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The thermal prole for SYBR Green and TaqMan probe consisted of an initial activation step at 95C for 10 min followed by 40 cycles at 95C for 15 s and 60C for 1 min. Negative samples were also analysed through the different steps of the procedure. Comparative sensitivity of the PCR protocols The sensitivity of conventional and real-time PCR assays was determined with 10-fold dilution series of Xac strain CFBP 2911 (10810)1 CFU ml)1) in sterile water or in orange peel extracts. DNA was extracted as described in Materials and methods. Five microlitres of the DNA extraction were used for conventional PCR, and 2 ll for real-time PCR. Statistical analyses All statistical analyses were performed using Statgraphics Plus for Windows 4.1 (Statistical Graphics, Rockville, MD, USA). Data from the samples were subjected to analysis of variance and the means separated by Fishers least signicant difference (LSD) procedure. Results Comparison of sensitivity the PCR protocols With pure cultures, the sensitivity achieved by the realtime PCR using SYBR Green or a Taqman probe was 10)1 CFU ml)1, whereas by conventional PCR with primers 2 and 3, J-pth1 and J-pth2 and Xac01, Xac02 was 102, 104 and 104 CFU ml)1 respectively (Table 1). Using bacterial suspensions in fruit extracts as targets for PCR detection after DNA extraction, real-time PCR was the most sensitive technique and Xac could be detected at a bacterial concentration of 101 CFU ml)1. Among the different protocols for conventional PCR, the protocol with primers 2 and 3 designed by Hartung et al. 1993; showed the highest sensitivity, 102 CFU ml)1 (Table 1).

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Table 1 Sensitivity of polymerase chain reaction (PCR) amplication in pure cultures and spiked citrus extracts by conventional and realtime PCR
Bacterial concentration (CFU ml)1) in bacterial suspension (Bs*) or citrus fruits extract (Cf) Primers 2 / 3 Bs Cf Xac01 / Xac02 Bs Cf J-pth1 / J-pth2 Bs Cf Real time** Bs Cf 10)1 100 101 102 103 104 105 106 107 108 ) ) ) ) ) ) + ) ) ) ) ) ) ) + ) ) ) ) ) ) ) + + + + ) ) ) ) + + + + ) ) ) ) + + + + + ) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Table 2 Individual sweet orange lesions analysed from 15 commercial shipments of fruit
Sample reference Isolation 2900 2901 2904 2906 2909 2911 3003 3005 3011 3014 3023 3024 3026 3047 3070 Total PCR 2 / 3* J-pth Xac Real-time PCR SYBR TaqMan

*Bs, bacterial suspension diluted in sterile water. Cf, bacterial suspension added to plant material extracted in PBS buffer. Both suspensions were plated on YPGA to determine the CF Uml)1 concentration. Conventional PCR with primers designed by Hartung et al. (1993). Conventional PCR with primers designed by Coletta-Filho et al. (2006). Conventional PCR with primers designed by Cubero and Graham (2002). **Real-time PCR with SYBR green uorescent or a TaqMan probe as reporter following the protocol described by Cubero and Graham (2005).

0 / 21** 4 / 21 2 / 21 2 / 21 2 / 21 3 / 21 2/9 5/9 4/9 5/9 6/9 7 / 10 3 / 10 10 / 10 7 / 10 8 / 10 10 / 10 10 / 10 1/5 3/5 3/5 3/5 4/5 4/5 0/6 5/6 1/6 0/6 5/6 6/6 2 / 12 6 / 12 3 / 12 4 / 12 6 / 12 9 / 12 0/5 4/5 3/5 2/5 4/5 5/5 1/8 4/8 4/8 3/8 8/8 8/8 1/8 3/8 3/8 3/8 4/8 6/8 2/3 3/3 3/3 3/3 3/3 3/3 1/4 2/4 2/4 2/4 2/4 3/4 0/6 1/6 1/6 1/6 1/6 5/6 1 / 11 1 / 11 1 / 11 1 / 11 1 / 11 7 / 11 1 / 11 1 / 11 1 / 11 1 / 11 1 / 11 3 / 11 1 / 11 1/7 1/7 1/7 1/7 1/7 16 / 130 52 / 130 39 / 130 39 / 130 58 / 130 80 / 130

Efciency of different methods for Xac detection in imported samples To compare the efciency of the detection among the different methods, 15 samples of several origins were selected and each lesion analysed by isolation, conventional PCR and real-time PCR. After bacterial isolation, 16 isolates of Xac could be obtained from 130 lesions analysed from sweet orange (Citrus sinensis L.) fruits exported to Spain during the years 2004 and 2005 (Table 2). It took 3 5 days to observe typical Xac colonies, mucoid, convex and yellow on YPGA medium, and in some cases the growth was delayed until 7 days. All the isolates obtained from the fruits analysed by direct isolation were pathogenic on grapefruit leaves. After 10 days, canker symptoms were clearly shown on the edge of the inoculation sites. Conventional PCR with primers designed by Hartung et al. (1993), Cubero and Graham (2002) and Coletta-Filho et al. (2006), and real-time PCR using SYBR green or a TaqMan probe were compared. With real-time PCR, Xac was detected in 632 61% of the fruit lesions analysed whereas by conventional PCR and isolation it was detected in 413 43% and 144 48% respectively. Signicant differences were shown among the detection methods used (P < 005) (Fig 1). The efciency of the detection methods evaluated according to the primers or the uorescence reporter
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Results obtained after isolation and polymerase chain reaction (PCR) amplication with different sets of primers and uorescence reporters. *Primers 2 and 3 designed by Hartung et al. (1993). Primers J-pth1 and J-pth2 designed by Cubero and Graham (2002). Primers Xac01 and Xac02 designed by Coletta-Filho et al. (2006). Real-time PCR using SYBR green as uorescence reporter (Cubero and Graham 2005). Real-time PCR using a TaqMan probe as uorescence reporter (Cubero and Graham 2005). **Number of lesions where Xac was detected in a sample per total number of lesions analysed in the sample.

% Fruit lesions where Xac was detected

80 70 60 50 40 30 20 10 0

c b a

Isolation

Conventional PCR Real time PCR Detection method used

Figure 1 Percentage of fruit lesions where Xac was detected by isolation, conventional polymerase chain reaction (PCR) or real-time PCR. The graph shows the average of 15 samples with the standard error (error bars). Mean values with differentt letters differ signicantly according to Fishers least signicant difference (LSD) procedure (P < 005).

used for real-time PCR (SYBR Green and Taqman probe), indicated that real-time PCR using the TaqMan probe was the most efcient technique, and isolation the less efcient. However, no signicant differences (P < 005) were observed among the different primers used in conventional PCR nor among conventional PCR

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90 80 70 60 50 40 30 20 10 0

c b b a b bc

Isolation

PCR/ Hartung

PCR/ Colleta

PCR/ Cubero

RTPCR/ SYBR

RTPCR/ TaqMan

Detection method used

Figure 2 Percentage of fruit lesions where Xac was detected by isolation, conventional polymerase chain reaction (PCR) with primers designed by Hartung et al. (1993), Coletta-Filho et al. (2006), and Cubero and Graham (2002), or real-time PCR (Cubero and Graham 2005). The graph shows the average of 15 samples with the standard error (error bars). Mean values with the same letter do not differ signicantly according to the Fishers least signicant difference (LSD) procedure (P < 005).

and real-time PCR using SYBR green (Fig 2). In all the analyses, the negative controls were always negative. Discussion Xac is present in several countries of South America (Argentina, Uruguay, Paraguay, Brazil), and the EU imports fresh fruits from them. Consequently, a detection system for this quarantine bacterium needs to be optimized for the analysis of fruits with suspected lesions. The detection protocol must include the fastest and most sensitive techniques currently available, and its evaluation in a real situation is presented here. Several of the available PCR protocols were compared in two types of samples, (pure cultures as well as spiked citrus fruits) after DNA extraction, and the results conrmed that the real-time PCR provided the highest level of sensitivity. When the detection methods were applied to naturally infected samples, real-time PCR was the most efcient technique for the detection of Xac, especially, if a TaqMan probe was used. However, in many samples, the bacterium was detected even using the less efcient methods. In those samples, the bacterial concentration was probably quite high and no special requirements were needed for the detection. Despite that, the use of the most sensitive and efcient technique such as real-time PCR is necessary, because in many cases there were difculties to detect Xac, in samples where the target bacteria are in low concentration or the fruits are not in good conditions. As the bacterial concentration is usually not known before the analysis, the most sensitive technique is advised to be included as routine for the screening of the samples in a detection protocol. The sensitivity assays performed denitively settle on the real-time PCR as the

most convenient technique for such rst screening to avoid the introduction of Xac into an area. In addition, real-time PCR eliminates the source of possible contaminants (amplicons) and is the fastest technique in obtaining the results, which conrms its usefulness, demonstrated in other bacterial models (Mackay 2004). These results also agree with former studies conducted by Cubero and Graham (2005) in other types of plant material collected under eld conditions. Moreover, because more than one technique is always advised for detection of a quarantine bacterium, a protocol that includes two assays by PCR (real time with a TaqMan probe, and conventional PCR), using two sets of primers from different genes can be employed with good reliability and sensitivity for a rst screening and consistent analysis of the samples. Among conventional PCR protocols, primers designed by Hartung et al. (1993) showed the highest sensitivity but were statistically no different from the other two conventional based PCR methods. In spite of no statistically signicant differences, in our hands Hartungs et al. (1993) primers were 10% more sensitive than the other two [primers designed by Coletta-Filho et al. (2006) and Cubero and Graham (2002)]. We suggest the use of Hartungs et al. (1993) primers as an additional detection technique in a routine protocol. However, it is necessary to take into account, as described before, that DNA from some strains causing CBC cannot be amplied using these primers. If the fruit sample is suspected to contain a nontypical Xac strain, such as A* or Aw or an strain of Xaa (Sun et al. 2004), due to its geographic origin or because the citrus species, another alternative should be addressed, like the use of universal primers designed by Coletta-Filho et al. (2006) or Cubero and Graham (2002). The occurrence of saprophytes on the citrus fruits with similar morphology to that of Xac on YPGA medium was observed in our analyses, and complicates the accurate diagnosis of the samples by isolation, but such colonies did not affect the PCR analysis. On the other hand, isolation is a useful technique to conrm the positive PCR results, and in some bacterial models is required to comply with regulations. Despite our expectation that bacterial isolation attempts would fail due to the chemical treatments applied to fruits to kill bacteria, the fact that isolation was successful in 11 of 15 shipments is rather signicant, and demonstrates that the disinfection protocols are not 100% effective. In addition, in our hands some samples were only positive by the PCR protocols. This is not due to a lack of specicity of the PCR protocols assayed, but probably to the disinfection treatments applied, which would reduce bacterial population, and may induce the noncultivable state in the analysed lesions. The long time required, in some cases,
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for the appearance of Xac colonies on YPGA medium (up to 7 days), suggests that the bacterial cells could be stressed in the lesions after the fruit treatments (washing, disinfection, chemical treatments, transport, and storage at low temperatures for variable periods of time). All these types of stresses may decrease bacterial multiplication ability, and make difcult the appearance of colonies. The viable but nonculturable (VBNC) state has not been yet demonstrated in Xac, but there are examples in other plant pathogenic bacteria, and in other Xanthomonads, showing that different stress factors (copper, low temperature, etc.) can induce this state (Ghezzi and Steck 1999). In this work, the 16 positive samples obtained by isolation are striking because they demonstrate that the compounds recommended for the disinfection of the fruits in the packing houses before exportation are not always sufcient to eliminate viable bacteria (Stapleton 1986; Brown and Schubert 1987). The presence of such living bacteria, that remain pathogenic, constitutes a risk of dissemination of the disease through contaminated symptomatic fruit. The pathogenicity assays on grapefruit leaves conrmed that the Xac cells remained viable and were able to produce symptoms, even after the fruit treatments and storage at low temperatures. Previous work on Xac in citrus trash has shown a rate of bacterial survival in lesions of 70% in fresh fruit, decreasing to 9% in trash fruit (Gambley et al. 2005). These data support the idea that this material with symptoms represents a threat for the introduction of the disease. The studies performed on the survival of CBC in plant material are not coincident, but in some experiments the amount of viable bacteria found inside the lesions was 105106 CFU lesion)1, and the dose required to infect leaves can be as low as 102103 bacteria in favourable conditions (Goto et al. 1978). Such bacterial populations have been found in the lesions of the imported fruits, suggesting that they could constitute a risk for the dissemination of the disease. Through the data obtained in this study, the isolation of live bacteria demonstrates that Xac can survive in lesions of commercial fruits and retain pathogenicity. Consequently, symptomatic commercial fruits represent a risk for the spread of CBC into the citrus producing countries of the EU. In conclusion, the use of the PCR as a screening method is very reliable in the detection of Xac in commercial citrus fruits. Real-time PCR with TaqMan probe is the most sensitive, specic and rapid method for detecting Xac in this material. Acknowledgements This research was supported by the agreement between n the Ministerio de Agricultura of Spain, Subdireccio
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General de Sanidad Vegetal and IVIA, through the project INIA RTA2006-00149-00-00, and by Plan de Vigilancia Fitosanitaria of the Generalitat Valenciana. M. Golmohammadi has a PhD fellowship from the Iranian Ministry of Science and Technology, JM. Quesada has a contract a de Agricultura of Valencia from Tragsa and Conseller alver has a contract from the Government, and J. Pen Spanish Ministry of Education. We thank L. Navarro for lvarez and C. Salcritical reading of the manuscript, B. A cedo for technical assistance, J. A. Pina for supplying plant material and C. Redondo for statistical advice. References
lvarez, M.A. (2004) Integrated approaches for detection of A plant pathogenic bacteria and diagnosis of bacterial diseases. Annu Rev Phytopathol 42, 339366. Anon (1990) EPPO standards PM 3 / 27 Quarantine procedure for Xanthomonas campestris pv.citri. OEPP/EPPO Bull. 20, 263272. Anon (2003) Available at: http://www.fao.org. Anon (2005) EPPO standards PM 7 / 44(1) Quarantine procedure for Xanthomonas axonopodis pv citri. OEPP/EPPO Bull. 35, 289294. Broadbent, P., Fahy, P.C., Gillings, M.R., Bradley, J.K. and Barens, D. (1992) Asiatic citrus canker detected in a pummelo orchard in northern Australia. Plant Dis 76, 824829. Brown, G.E. and Schubert, T. (1987) Use of Xanthomonas campestris pv. vesicatoria to evaluate surface disinfectants for canker quarantine treatment of citrus fruit. Plant Dis 71, 319323. Civerolo, E.L. (1984) Bacterial canker disease of citrus. J Rio Grande Valley Hortic Soc 37, 127146. Coletta-Filho, H.D., Takita, M.A., Souza, A.A., Neto, J.R., Destefano, S.A.L., Hartung, J.S. and Machado, M.A. (2006) Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and articially infected citrus plants. J Appl Microbiol 100, 279285. Cubero, J. and Graham, J.H. (2002) Genetic relationship among worldwide strains of Xanthomonas causing canker in citrus species and design of new primers. Appl Environ Microbiol 68, 12571264. Cubero, J. and Graham, J.H. (2004) The leucine responsive regulatory protein (lrp) gene for characterization of the relationship among Xanthomonas species. Int J Syst Evol Microbiol 54, 429437. Cubero, J. and Graham, J.H. (2005) Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate Xanthomonas strains on canker. Phytopathology 95, 13331340. Das, A.K. (2003) Citrus canker a review. J Appl Hort 5, 5260. Gambley, C.F., Benham, M., Parmenter, A., Miles, A.K., Doogan, V.J., Ramsden, M. and Whittle, P.J.L. (2005) The

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M. Golmohammadi et al.

Diagnosis of Xanthomonas axonopodis pv. citri

distribution of citrus canker in Emerald, Australia and bacterial survival in citrus trash. 2nd International Citrus Canker and Huanglongbing Research Workshop, November 711, Orlando, FL, p. 70. Ghezzi, J.I. and Steck, T.R. (1999) Induction of the viable but non-culturable condition in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil. FEMS Microbiol 30, 203208. Goto, M., Toyosima, A. and Yanaka, S. (1978) Studies on saprophytic survival of Xanthomonas citri (Hasse) Dowson, inoculums density of the bacterium surviving in saprophytic forms. Ann Phytopathol Soc 77, 197201. Gottwald, T.R., Hughes, G., Graham, J.H., Sun, X. and Riely, T. (2001) The citrus canker epidemic in Florida: The scientic basis of regulatory eradication policy for an invasive species. Phytopathology 91, 3034. Graham, J.H., Gottwald, T.R., Cubero, J. and Achor, D.S. (2004) Xanthomonas axonopodis pv. citri : factors affecting successful eradication of citrus canker. Mol Plant Pathol 5, 115. Hartung, J.S., Daniel, J.F. and Pruvost, O.P. (1993) Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method. Appl Environ Microbiol 59, 1143 1148. Leite, R.P. and Mohan, S.K. (1990) Integrated management of the citrus bacterial canker disease caused by Xanthomonas campestris pv. citri in the state of Parana, Brazil. Crop Prot 9, 37. Lelliot, R.A. and Stead, D.E. (1987) Methods for the diagnosis of bacteria diseases of plants. In Methods in Plant Pathology ed. Preece, T.F. p. 216. Blackwell: Oxford, UK. pez, M.M. Llop, P., Caruso, P., Cubero, J., Morente, C. and Lo (1999) A simple extraction procedure for efcient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. J Microbiol Methods 37, 2331. pez, M.M. (2000) Enfermedades producidas por bacterias. Lo tricos eds Duran-Vila, N. and In Enfermedades de los c a. ola de Fitopatolog Moreno, P. pp. 4750. Sociedad espan Ediciones Mundi-Prensa, p..165. pez, M.M., Cubero, J., Llop, P., Pen alver, J., Quesada, J.M., Lo Morente, C. and Catara, V. (2005) Standardization of diagnosis for citrus bacterial canker: the EPPO protocol and its use for detection in citrus fruits. Second International Citrus canker and Huanglongbing Research workshop, November 711, Orlando, FL, p. 21. Mackay, I.M. (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10, 190212. Mavrodieva, V., Levy, L. and Gabriel, D.W. (2004) Improved sampling methods for real-time polymerase chain reaction diagnosis of citrus canker from eld samples. Phytopathology 94, 6168.

Mohammadi, M., Mirzaei, M.R. and Rahimian, H. (2001) Physiological and biochemical characteristics of Iranian strains of Xanthomonas axonopodis pv. citri, the causal agent of citrus canker disease. J Phytopathol 149, 6575. Pruvost, O., Boher, B., Brocherieux, C., Nicole, M. and Chiroleu, F. (2002) Survival of Xanthomonas axonopodis pv. citri in leaf lesions under tropical environmental conditions and simulated splash dispersal of inoculums. Phytopathology 92, 336346. Roberts, P.D., Chamberlain, H.L., Chung, K.R., Schubert, T.S., Graham, J.H. and Timmer, L.W. (2005) Florida citrus pest management guide: citrus canker. Available at: http://edis. ifas.u.edu. Schaad, N.W., Postnikova, E., Lacy, G.H., Sechler, A., Agarkova, I., Stromberg, P.E., Stromberg, V.K. and Vidaver, A.K. (2005) Reclassication of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 forms A, B / C / D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv malvacearum (ex smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and var. fuscans of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov. Syst Appl Microbiol 28, 494518. Stall, R. and Civerolo, E. (1991) Research relating to the recent outbreak of citrus canker in Florida. Annu Rev Phytopathol 29, 399420. Stall, R.E. and Seymour, C.P. (1983) Canker, a threat to citrus in the gulf coast states. Plant Dis 67, 581585. Stapleton, J.J. (1986) Effects of post harvest chlorine and wax treatments on surface micro ora of lime fruit in relation to citrus bacteriosis disease. Plant Dis 70, 10461048. Sun, X., Stall, R.E., Jones, J.B., Cubero, J., Graham, J.H., Dixon, W.N., Schubert, T.S., Chaloux, P.H. et al. (2004) Detection and characterization of new strain of citrus canker bacteria from key / Mexican lime and alemow in South Florida. Plant Dis 88, 11791188. Vauterin, L., Hoste, B., Kersters, K. and Swings, J. (1995) Reclassication of Xanthomonas. Int J Syst Bacteriol 45, 472489. ` re, C., Hartung, J.S., Pruvost, O.P., Civerolo, E.L., AlvaVernie rez, A.M., Maestri, P. and Luisetti, J. (1998) Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from the Southwest Asia. Eur J Plant Pathol 104, 477485.

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