Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis S. Krid1*, A. Rhouma1*, J.M. Quesada2, R. Penyalver2 and A. Gargouri3 1 Unité de Recherche Protection des Plantes Cultivées et Environnement, Institut de l’Olivier, Cité Mahrajène BP208 Tunis, Tunisia 2 Centro de Protección Vegetal y Biotecnologı́a. Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, Moncada, Valencia, Spain 3 Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax BP K3038, Sfax, Tunisia Keywords genotyping, olive knot disease, Pseudomonas savastanoi, RAPD. Correspondence Ali Rhouma, Institut de l’Olivier, Cité Mahrajène BP208 Tunis, Tunisia. E-mail: roumaal@yahoo.fr *These authors contributed equally to this work and are regarded as the joint first authors. 2008 ⁄ 0375: received 3 March 2008, revised 1 September 2008 and accepted 2 September 2008 doi:10.1111/j.1365-2672.2008.04058.x Abstract Aims: To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location. Methods and Results: Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi, the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia. Conclusions: The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical. Significance and Impact of the Study: This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains. Introduction Pseudomonas savastanoi are pathogens to plants of the Oleaceae family (Gardan et al. 1992). Pseudomonas savastanoi pv. savastanoi (hereafter termed Psv) is specific to olive trees. The disease is widespread in oliveproducing Mediterranean countries and has recently been detected in Australia (Hall et al. 2004). Its symptoms are tumourous overgrowths occurring on young twigs, branches and stems but occasionally on the leaves and fruits as well (Surico 1986). Knot development is caused by the phytohormones indole-3-acetic acid (IAA) and cytokinins produced by the bacterium (Smidt and Kosuge 1978; Comai and Kosuge 1980; Surico et al. 1985). The gene iaaL is involved in IAA production (Penyalver et al. 2000). The infection can 886 reduce both olive quality and yield (Schroth et al. 1968, 1973). Several studies have used phenotypic and genotypic techniques to differentiate between Psv isolated from olive trees and those obtained from other host plants (Gardan et al. 1992, 1999; Mugnai et al. 1994; Sisto et al. 2002, 2007). The phenotypic variation of Psv strains isolated from olive trees has recently been studied with respect to virulence, colony size, morphology, mobility and genetic manipulation (Penyalver et al. 2006; Pérez-Martı́nez et al. 2007); however, few studies are available on the genetic diversity of Psv strains isolated from olive trees with respect to geographic location and cultivars. Scortichini et al. (2004) used enterobacterial repetitive intergenic consensus (ERIC), the BOXA1R subunit of the BOX element of Streptococcus pneumoniae (BOX) and repetitive ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 886–894 S. Krid et al. Characterization of P. savastanoi strains using RAPD extragenic palindromic (REP)-polymerase chain reaction (PCR) methods and were unable to discriminate between a collection of 360 isolates from different provinces and olive tree cultivars in Italy. Recently, using fluorescent amplified fragment length polymorphism (AFLP), Sisto et al. (2007) found that Psv strains isolated from olive trees in the same geographical region were, generally, of related genotypes whereas isolates from different geographic sites displayed more variation. The discriminatory potential of AFLP has been well documented in numerous studies, which demonstrate its ability to distinguish closely related plant pathogenic bacterial strains belonging to several genera like Agrobacterium (Portier et al. 2006), Bacillus (Ticknor et al. 2001), Xanthomonas (Schaad et al. 2005) and Pseudomonas (Clerc et al. 1998); however, the method has limitations for routine applications. Random amplification of polymorphic DNA (RAPD) is more suitable as it is simpler to use and does not require the use of radioactive or other labelling compounds (Williams et al. 1990). RAPD has been used to characterize some bacterial species, such as Staphylococcus xylosus (Iacumin et al. 2006), Pseudomonas aeruginosa (Campbell et al. 2000) and Pseudomonas syringae (Clerc et al. 1998) but, to our knowledge, it has not been applied to Psv differentiation. Here, we report the characterization, by RAPD, of Psv strains that were implicated in olive knot disease in eight different orchards and three cultivars in Tunisia. The observed genetic differentiation is potentially of great importance for epidemiological and ecological studies of the olive knot disease. The following biochemical tests were performed: levan, oxidase, pectinolytic activity, presence of arginine dehydrolase and tobacco hypersensitivity (LOPAT) according to Lelliott et al. (1966). A pathogenicity test was carried out on a limited number of samples, representing one isolate per locality. Wounds measuring around 1 cm were made in the bark of 1-year-old olive stems (cv. Chemali) with a scalpel dipped in a bacterial suspension (108 CFU ml)1) of pure culture grown for 48 h on King’s medium B. Each isolate was inoculated at five wound sites. Wounds were protected with parafilm for 3 days. The inoculated trees were kept in a greenhouse at 25C and inspected for knot formation after 2 months. Two strains of known pathogenicity (ITM 317 and IVIA 1628-3) were used as positive control. Negative control trees were inoculated with sterile distilled water. The iaaL gene was amplified in all Tunisian isolates by PCR according to Penyalver et al. (2000); amplicons were purified by Promega purification kit (Wizard SV CleanUpSystem, CA, USA) and sequenced using an automatic DNA sequencer (ABI 3100; Applied Biosystems). Then the sequences were compared with those available in the Blastn program (http://www.ncbi.nlm.nih.gov/). Materials and methods DNA extraction Strains included in this study A total of 58 Tunisian isolates were obtained from young olive knots taken from eight orchards located at eight geographically different places and from three cultivars (Chemlali, Chetoui and Oueslati; Table 1). Twenty-one strains obtained from various other countries were included for comparison (Table 1). Sampling procedures and isolation from knots Sampling was carried out in spring and fall of 2006 and 2007 by cutting knots from three different trees per orchard. The knots were placed in sterile plastic bags, transported to the laboratory and processed immediately. The knots were surface-disinfected with a paper moistened with ethanol (Marchi et al. 2005). Small fragments (1–2 mm) were cut aseptically with a sterile scalpel then placed in 200 ll of sterile distilled water. After 30 min, a loopful of the resulting suspension was streaked on plates containing King’s medium B (King et al. 1954) and then incubated at 26C for 3–5 days. Single colonies were collected and checked for purity. Identification by biochemical analysis, pathogenicity tests and PCR Bacterial isolates were grown for 48 h at 26C on King’s medium B. DNA was extracted from bacterial suspensions (108 CFU ml)1) using the protocol described by Llop et al. (1999). The DNA was dissolved in sterile distilled water before quantification by spectrophotometer and kept at )20C until use. Amplification and sequencing of 16S ribosomal DNA Amplification of 16S rDNA genes was performed using the primers fD1 and rD1 (Weisburg et al. 1991). PCR mix contained 50 ng of DNA, 1 · PCR buffer, 20 lmol l)1 of each primer, 10 mmol l)1 dNTP and 1 U of Taq DNA polymerase. Amplification was performed for 35 cycles at 94C for 45 s, 45C for 1 min and 72C for 2 min. An initial denaturation, at 94C for 5 min and a final extension at 72C for 5 min were used at the beginning and end of the amplification, respectively. The PCR products (almost 1500 bp) were separated by gel electrophoresis on 1% agarose in TAE buffer 0Æ5 · stained ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 886–894 887 Characterization of P. savastanoi strains using RAPD S. Krid et al. Table 1 Origin and characteristics of Pseudomonas savastanoi pv. savastanoi isolates used in this study Sample origin Isolate ⁄ straina Country Orchard locality Year collected Cultivar Source Ad2, Ad3, Ad4, Ad5, Ad6, Aw7, Aw8, Aw9 Bz1, Bz2, Bz3, Bz4, Aw1, Aw2, Aw3, Aw4 KS2, KS4, KS5, KS6, KS7, KS8, KS9 OH1, OH2, OH3, OH4, OH5, OH6 K1, K2, K3, CH1, CH2, CH3, CH4 BK1, BK2, BK5, BK8, BK9, BK10, BK11 O1, O3, O6, O7, O8, O9, O10, O11 Aj1, Aj2, Aj3, Aj4, Aj5, Aj6, Aj7 CFBP 71 IVIA 1628-3 IVIA 2558-1t IVIA 1657-8Va IVIA 2549-2t IVIA 1972-1a IVIA 1974 E3C2 IVIA 2743-3 NCPPB 1479 CFBP 1670T ITM 317 PVBa 206 PVFi1 NCPPB 1506 CFBP 2074 BPIC 346 CFBP 1020 NCPPB 3335 NCPPB 64 NCPPB 1342 NCPPB 1344 Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Spain Spain Spain Spain Spain Spain Spain Serbia Serbia Italy Italy Italy Italy Algeria Greece France France Portugal USA USA Aouedna Bouzouita Ksouda Ouled Haffouz Chbika Bekalta Tunis Ain Jloula – Valencia Jaén Alicante Tarragona Jaén Córdoba Badajoz – – Basilicata Apulia Tuscany – – – – – – – – 2006 2007 2006 2006 2007 2006 2006 2006 1960 1996 2001 1996 2001 1998 1998 2003 1955 – 1982 1968 – 1963 – – 1967 1984 1954 1962 1962 Chemlali Chemlali Chemlali Chemlali Chemlali Chemlali Chetoui Oueslati – Cornicabra Picual Morrut Arbequina Picual Manzanilla Arbequina – – – – – – – – – – – – – This study This study This study This study This study This study This study This study CFBP IVIA IVIA IVIA IVIA IVIA IVIA IVIA NCPPB CFBP ITM PVBa PVFi NCPPB CFBP BPIC CFBP NCPPB NCPPB NCPPB NCPPB a CFBP, Collection Française de Bactéries Phytopathogènes, Angers, France; IVIA, Bacterial Collection from the Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain; NCPPB, National Collection of Plant Pathogenic Bacteria, York, England; ITM, Culture Collection of Instituto Tossine e Micotossine da Parassiti vegetali, CNR, Bari, Italy; PVBa, Culture Collection of Dipartamento di Patologia Vegetale, Università degli Studi, Bari, Italy; PVFi, Collection of Dipartimento di Biotecnologie Agrarie-Patologia Vegetale, Università degli Studi, Firenze, Italy; BPIC, Benaki Phytopqthological Institute Collection, Athens, Greece. with ethidium bromide and observed under ultraviolet (UV) light. After purification with a Promega PCR purification kit, the products were sequenced in both forward and reverse directions. The 16S rDNA nucleotide sequences were compared using Blastn. RAPD analysis An initial comparison was carried out with 20 decamer primers to identify suitable pimers for RAPD. Three primers were selected because they gave readily interpretable and reproducible results: OPR-12 (5¢-ACAGGTGCGT-3¢), OPX-7 (5¢-GAGCGAGGCT-3¢) and OPX-14 (5¢-ACAGGTGCTG-3¢). The reproducibility of the RAPD method was evaluated by running seven Psv isolates in triplicate with separate cell preparations and PCR trials of the same isolate, on different gels. 888 Amplifications based on RAPD method were realized following the methodology described by Llop et al. (2003). PCR mix contained 37Æ5 ng of DNA, 1 · PCR buffer [20 mmol l)1 Tris-HCl (pH 8Æ4), 50 mmol l)1 KCl]; 1Æ5 mmol l)1 MgCl2, 60 lmol l)1 dNTPs, 5 pmol of primer and 1 U of Taq DNA polymerase (Invitrogen) in a total volume of 25 ll. The amplification cycles were: a denaturation step of 94C for 3 min followed by five cycles of 94C for 30 s, 36C for 1 min and 72C for 1 min, with a ramp time of 1 min 48 s and 30 cycles of 94C for 30 s, 45C for 1 min and 72C for 1 min, with a ramp time of 1 min. The thermocycler employed was Eppendorf MasterCycler gradient. Amplified products were separated by gel electrophoresis on 1Æ5% agarose in 0.5 · TAE buffer stained with ethidium bromide and observed under UV light. ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 886–894 S. Krid et al. Data analysis Reproducible DNA bands were scored manually and weak bands with negligible intensity were excluded from final data analysis. Bands positions of clearly visible bands were combined for analysis. Band profiles were scored in a binary mode with 1 indicating its presence and 0 its absence. The simple matching coefficient (Sneath and Sokal 1973) was calculated to construct similarity matrices. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was performed using the computer programme ntsys-pc ver. 2.10e (Rohlf 1998). The heterogeneity coefficient was calculated (ratio between the number of RAPD types and the number of isolates). The bootstrap analysis has been performed using the treecon software ver. 1Æ3b (Van de Peer and De Wachter 1993). Principal coordinate analysis (PCO) of the similarity matrix was performed using options of software MultiVariate Statistical Package (MVSP) Plus ver. 3Æ12e (Kovach Computer Services, Anglesey, UK). Results Identification by biochemical analyses, pathogenicity tests and PCR Tunisian isolates listed in Table 1 were tested for their biochemical and pathogenic characteristics. They were Characterization of P. savastanoi strains using RAPD oxidase-negative, levan-negative, pectinolytic activitynegative, arginine dehydrolase-negative and tobacco hypersensitivity-positive. When inoculated on the olive plants, the isolates tested developed typical knots, like those observed with the reference Psv strains, IVIA 1628-3 and ITM 317. All Tunisian isolates and reference strains, used as positive controls, yielded an amplicon of about 454 bp indicating that iaaL gene occurs in all isolates tested (data not shown). The iaaL gene sequences were identical (98% identity of nucleotide) to those reported in the databases (accession number EU616803.1). The similarity of the 16S rDNA sequences of Tunisian isolates was more than 98% identical to the corresponding gene sequences of Psv present in the databases (accession number AM265392.1). Based on biochemical analyses, amplification of iaaL gene and sequence analysis of 16S rDNA gene, all the isolates in the collection belonged to Psv. RAPD analysis and clustering Following preliminary experiments in which 20 different primers were compared on 7 different strains, three primers generated polymorphic patterns that were easy to interpret based on the size and number of the generated band, as illustrated in Fig. 1. The size of the amplified DNA fragments generated with the three primers ranged from 400 pb to 3 kb. Primer OPR-12 generated 19 reproducible bands but only 10 bands were polymorphic. Primer OPX-7 generated 17 reproducible bands but only 9 Figure 1 RAPD amplification of genomic DNA from 16 bacterial isolates of Pseudomonas savastanoi pv. savastanoi with three selected primers OPR-12 (a), OPX-7 (b) and OPX-14 (c). Lane M: 100 bp molecular weight marker (Promega); Lane 1: Ad5; Lane 2: Bz2; Lane 3: KS2; Lane 4: K2; Lane 5: OH4; Lane 6: Aj4; Lane 7: BK9; Lane 8: O9; Lane 9: IVIA 1628-3; Lane 10: IVIA 1657-8Va; Lane 11: IVIA 2558-1t; Lane 12: IVIA 2549-2t; Lane 13: NCPPB 1479; Lane 14: ITM 317; Lane 15: CFBP 71; Lane 16: PVBa 206. ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 886–894 889 Characterization of P. savastanoi strains using RAPD were polymorphic. However, primer OPX-14 appeared less discriminative as compared with the other two primers. With primer OPX-14 seven bands were obtained of which four were conserved and three were polymorphic. Figure 1 shows the RAPD patterns obtained with the three primers of one Tunisian Psv isolate representative of each investigated locality and seven strains from other sources, added for comparison. As can be seen, the size of the amplified DNA fragments ranged from 3 kb to below 400 pb. Results revealed that isolates from similar geographic origin produced identical RAPD patterns (data S. Krid et al. not shown). As shown in Fig. 1, 21 RAPD patterns (based on 21 polymorphic bands) could be identified among the 79 Psv isolates and the heterogeneity coefficient was 0Æ32. Cluster analysis based on RAPD polymorphism grouped the isolates into three categories (Fig. 2). RAPD group 1 contained the strains isolated from the regions of southeast Tunisia (Aouedna and Bouzouita, which had identical profiles). The European and American strains, included in this study, were affiliated to this group and were divided into five subgroups. Subgroup 1 included PVBa 206 from Italy, which was more closely related to Figure 2 UPGMA clustering of Pseudomonas savastanoi pv. savastanoi listed in Table 1 based on RAPD polymorphism. The scale corresponds to the percentage of similarity. Significant bootstraps (i.e. >80) values for 100 resampling were displayed. 890 ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 886–894 S. Krid et al. Characterization of P. savastanoi strains using RAPD Figure 3 Principal coordinate analysis of Pseudomonas savastanoi pv. savastanoi strains based on RAPD polymorphisms. Numbers ported by axe 1 and axe 2 are eigenvalues. Only some isolates were selected in order to facilitate the lecture. the Tunisian isolates. Subgroup 2 encompassed seven strains (two from Italy, two from Serbia, one from France, Greece and Algeria). Subgroup 3 contained only three Spanish strains. Subgroup 4 regrouped four European strains (two from Spain, one from Italy and one from France). Subgroup 5 contained two American strains, a strain from Portugal and two Spanish strains. The results suggest that the Spanish strains IVIA 1628-3 and IVIA 1657-8Va are very similar. The two American strains (NCPPB 1342, NCPPB 1344) and the Portugal strain (NCPPB 64) were also very similar. Group 2 consisted exclusively of strains isolated from the region of Tunis, located in the north of the country (O1, O3, O6, O7, O8, O9, O10, O11). Group 3 contained strains isolated from the regions of the Centre of Tunisia (Ksouda, Ain Jloula, Bekalta, Chbika and Ouled Haffouz). Strains isolated from Ksouda and strains OH3, OH4 and OH5 isolated from Ouled Haffouz showed identical profiles. Interestingly, the reference strain CFBP 71 isolated in Tunisia and obtained from the French strain collection was affiliated to group 3. PCO was performed to visualize the positioning of the isolates with respect to the main axes. Results reported in Fig. 3 showed that the first PCO dimensions of the RAPD data cumulatively contained 67Æ6% of the total variation. PCO analysis positions selected some strains in a way that supports the tripartite division obtained by clustering. Furthermore, the isolates from southeast Tunisia were closely related to PVBa 206 and NCPPB 1479 (Fig. 3). The Spanish strains (IVIA 1628-3, IVIA 1657-8Va, IVIA 2558-1t, IVIA 2549-2t) were closely related to the reference strain ITM 317 isolated in Italy. The isolates of the north of the country were classified in a separate group. Isolates from central Tunisia were regrouped in the same group and were affiliated with the reference strain CFBP 71. Discussion This study examined the genetic variability within a collection of Psv isolates from different geographic locations. The isolates were taken from eight geographic sites and three cultivars in Tunisia and a collection of reference strains isolated from other countries (Spain, Italy, France, Greece, Portugal, Serbia, Algeria, USA) were included for comparison. The subtyping allowed us to understand the epidemiology of the bacterium disease and to see whether these strains were distributed according to the geographical location. The UPGMA analysis of DNA fingerprinting obtained by RAPD method revealed the presence of 32 patterns among the Psv isolates. The genetic similarity achieved using the hierarchical clustering analysis showed that a simple matching coefficient varying between 56% and 100% permitted strain differentiation. Similar results were obtained by Scortichini et al. (2004) for Psv strains in Italy using REP-PCR with an overall similarity of 81%, revealing the presence of 20 patterns, although, no ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 886–894 891 Characterization of P. savastanoi strains using RAPD clustering was obtained. However, phylogenetic analysis enabled us to cluster Psv strains into three groups at 67% of similarity. Isolates from the north of Tunisia were very similar and overall their similarity exceeded 91%. However, we noticed that the majority of the isolates coming from the centre of the country were regrouped in a single group, although they were isolated from different orchards and regions (Ksouda, Ain Jloula, Ouled Haffouz, Chbika and Bekalta). Interestingly, the reference strain CFBP 71 isolated in Tunisia in 1960 by Dr Ride was affiliated to this group and was closely related to the strains isolated from Chbika. It is possible that CFBP 71 had been isolated from the central region of Tunisia but this can no longer be verified. Isolates from Sfax region (southeast Tunisia) were regrouped together and were affiliated in the same group as some European strains isolated from Italy, Spain, France and Serbia. RAPD is an interesting tool to help us understand the epidemiology of the disease. Results showed clearly the strong conservation of genotypes within a region suggesting that there is little spread of bacterial sub-populations over large distances. However, two exceptions were found in this study (strains isolated from Aouedna and Bouzouita and those from Ksouda and Ouled Haffouz) where identical genotypes were seen in distant locations. These are most likely the result of human contamination, for instance by contaminated material or pruning tools. Consequently, Tunisian strains were regrouped according to the geographical site where they were isolated; confirming the results previously described by Sisto et al. (2007), who used the AFLP method with Italian strains isolated from olive trees. In the same orchard, the subpopulations of Psv had identical genotypes. The diversity among the band patterns was not large (heterogeneity coefficient of 0Æ32). This could be explained by the fact that contaminations from one tree to another could occur by rainfall and irrigation water and also by human when using pruning or harvesting tools. Our finding showed the low genetic diversity of strains isolated in the same orchard and therefore further investigations will be of interest to apply RAPD to have a bigger picture of localized populations of Psv. Analysis by coordination confirms the clustering obtained by UPGMA analysis. Cluster analysis sorted individuals into hierarchical groups, which may result in distantly related individuals being forced into closely connected clusters. The fact that Psv strains clustered according to their geographical region of isolation, suggests that Psv could be considered as an ecotype well adapted to the environmental conditions of every orchard. The Tunis region (north of Tunisia) is characterized by a humid climate 892 S. Krid et al. with rainfall between 400 and 600 mm. This region is dominated by the cultivar Chetoui with a plantation density of 100 trees per hectare. In the centre of Tunisia, rainfall is lower (between 250 and 300 mm) and the plantation densities vary between 50 and 70 trees per hectare. However, in the Sfax region (southeast Tunisia), rainfall does not exceed 250 mm and the density of the plantation is 17–25 trees per hectare. Besides, the cultivar seems not to affect the genetic diversity of Psv strains (Scortichini et al. 2004). The genetic structure of Psv could be influenced mainly by the environmental conditions of each region, the transfer of contaminated materials and pruning tools between orchards. The last two events could lead to lower genetic diversity of this bacterium strain between the different regions. New studies using more powerful methods and more Psv strains are necessary for providing useful information on this subject. Our findings using the RAPD method did indicate a clustering of Tunisian Psv strains that were related to the geographic location where they were isolated. 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