Ann Microbiol (2012) 62:1411–1417 DOI 10.1007/s13213-011-0391-6
ORIGINAL ARTICLE
Molecular typing of Pectobacterium carotovorum isolated from potato tuber soft rot in Morocco Meriam Terta & Souad Azelmat & Rajaa Ait M’hand & El Hassan Achbani & Mustapha Barakate & François Bouteau & Moulay Mustapha Ennaji
Received: 25 August 2011 / Accepted: 14 November 2011 / Published online: 3 December 2011 # Springer-Verlag and the University of Milan 2011
Abstract Twenty-two isolates of Pectobacterium carotovorum were collected from soft-rotted potato tubers originating from different production areas in Morocco, and their phenotypic and genetic characteristics were investigated. All of the isolates were identified as P. carotovorum by API 20E (identification kit) system, and yielded a 434-bp DNA fragment of the pectate-lyase-encoding pel gene in PCR experiments. Collected strains were also evaluated for their susceptibility to 11 different antibiotics: Sulfamethoxazole/ trimethoprim (SXT), Ceftazidime (CAZ), Ciprofloxacin M. Terta : R. A. M’hand : M. M. Ennaji (*) Laboratoire de Virologie et Hygiène & Microbiologie, Université Hassan II-Mohammedia, Casablanca, Morocco e-mail:
[email protected] M. M. Ennaji Casablanca-Faculté des Sciences et Techniques, Mohammedia, BP 146, Mohammedia 20560, Morocco S. Azelmat Hôpital Militaire Mohamed V, Rabat, Morocco E. H. Achbani Laboratoire de Bactériologie Végétale et Protection des Plantes, INRA Meknès, Meknès, Morocco M. Barakate Laboratoire de Biologie et Biotechnologie des Microorganismes, Université Cadi Ayyad -FSSM, Marrakech, Morocco F. Bouteau Laboratoire d’Electrophysiologie des Membranes, EA3514, Université Paris Diderot, Paris, France
(CIP), Cefotaxime sodium (CTX), Imipenem (IPM), Gentamicin (CN), Amikacin (AK), Colistin sulphate (CT), Netilmicin (NET), Ampicillin (AMP) and Cephalothin (KF). Of the total P. carotovorum strains isolated, 5 were resistant to only two antibiotics: Ampicillin and Cephalothin. Evaluation of genetic diversity was performed on all isolates by molecular typing with Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Results showed the isolates to be distributed on 2 major clusters subdivided into many subclusters. The genetic diversity within the potato strains of P. carotovorum subspecies is shown. No correlation between ERIC-PCR analyses, suscebtibility patterns, geographic areas and year isolated was observed. These results may indicate that the tuber could be the main source of contamination. In conclusion, the ability to distinguish Pectobacterium carotovorum strains from diseased potato with PCR based assay will be useful for strain relatedness studies of this pathogen in Morocco. Keywords Antibiotic susceptibility . ERIC-PCR . Heterogenecity . Pectobacterium carotovorum . Potato tuber . Soft rot
Introduction The plant pathogen Pectobacterium carotovorum is a Gramnegative and rod-shaped bacterium, originally described as the causal agent of soft rot disease on carrot (Whitehead et al. 2002). P. carotovorum was later reported to be a widespread species in many economically important food crops such as potatoes (Sledi et al. 2000). The soft rot Erwinias have been classified in the resuscitated genus Pectobacterium as P. carotovorum ssp. carotovorum, P. carotovorum ssp. atrosepticum and P. chrysanthemi based on 16S rDNA
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sequence studies (Hauben et al. 1998). Secreted multiple plant cell wall-degrading enzymes represent a key virulence factor that contributes to the ability of P. carotovorum to cause diseases. These phytopathogens can cause tuber soft rot of potatoes during storage and can also result in the occurrence of various field symptoms, including reduced emergence, chlorosis, wilting, stem rot, blackleg, desiccation and plant death (Fassihiani and Nedaeinia 2008; Helias et al. 2000). Several methods have been employed to distinguish between Pectobacterium species. The most commonly used methods are biochemical and pathogenicity tests (Karnjanarat el al. 1987; Seo et al. 2002). However, phenotypic studies using biochemical and physiological methods on soft rot Pectobacterium have been and still are undertaken in parallel with other methods to identify and characterize these species (Costa et al. 2006). To date, several typing methods based on molecular approaches have been developed in soft rot Erwinias, involving the application of restriction fragment length polymorphism (RFLP) analysis (Toth et al. 2001; Darrasse et al. 1994; YahiaouiZaidi et al. 2003), ribotyping (Nasser et al. 1994) and 16S rDNA (Hauben et al. 1998), and Amplified Fragment Length Polymorphism (AFLP) (Dellagi et al. 2000). In this study, the typing of P. carotovorum strains was conducted using antimicrobial susceptibility and Enterobacterial Repetitive Intergenic Concensus polymerase chain reaction (ERIC-PCR). ERIC elements are genetically stable and differ only in their copy number and chromosomal locations, making them a good target for strain differentiation (Yousr et al. 2010; Sledi et al. 2000; Zulkifli et al. 2009). They have been discovered in non-coding and intergenic regions (Lindsay et al. 2006; Sharples and Lloyd 1990). These elements are 126 bp long, highly conserved, and include a central core inverted repeat (Versalovic et al. 1991). The position of ERIC elements in Enterobacterial genomes varies between different species and has been used as a genetic marker to characterize isolates within a bacterial species (Soler et al. 2003; Sechi et al. 1998). This method has been widely used and it has been observed that it has the highest discriminatory power (Lindsay et al. 2006). The occurrence of bacterial soft rot disease on potato caused by P. carotovorum in Morocco has been reported, but little or no information on typing of P. carotovorum isolates has been generated (Snaiki et al. 2005, 2006; Baz et al. 2011; Anajjar et al. 2007; Terta et al. 2010) . The typing is important for epidemiology purposes so as to identify the sources of infection and monitoring the spread of bacteria. To meet this need, we have applied an ERIC-PCR assay. The ERIC-PCR method is a DNA-based typing technique frequently used for such a purpose and has proven to be a powerful tool for assessing genetic variation within and between bacterial species. Thus, the goal of this study is to assess the genetic diversity of P. carotovorum isolates
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causing soft rot of potato collected from different regions of Morocco. The method described here should be useful for the rapid identification of P. carotovorum isolates. Moreover, it may be that different strains of the pathogen are present in different production regions, leading to differences in disease incidence and severity.
Materials and methods Bacterial strains The 22 collected strains were obtained from infected potato tubers in various regions of Morocco and are designated: P106F1, P107F1, P603AH1, P603TM5, P507OZ1, P507Z1, P116sK5, P507BM2, P603TM1, P206C3, P606MN1, P603AH2, P126AT5, P116sK1, P507K2, P606sd2, P606sd6, P603A1, P303K1, P303K3, P116Sk2, and P303MN2 (Table 1), The samples of infected potato tuber were mixed and serially diluted up to 10−6 then spread (0.1 mL) on the selective diagnostic medium Crystal Violet Pectate (CVP). The colonies with round pits were further purified on LPGA medium [yeast extract 5 g L−1(Difco), peptone 5 gL−1(Difco), glucose 10 g L−1(Difco), and agar (Merck) 15–17 g L−1, pH 7.2±0.2]. All strains were maintained on nutrient agar during this study. Strains and biochemical characterization Identification of each isolate was performed with the API 20E system according to the instructions of manufacturer (bioMerieux, Marcy I’Etoile, France). The inoculum was obtained by selecting an isolated 24-h growth colony from
Table 1 Isolates of Pectobacterium carotovorum ssp. carotovorum used in this study Strains
Location
Year of isolation
P507OZ1 P206C3, P116C2 P507Z1 P603AH1, P603AH2 P603TM1, P603TM5 P603A1 P303MN2, P606MN1 P303K3, P507K2, P303K1
Oued zem Casablanca Oued zem Ain halouf Tit Mellil Guigo Mnasra Kenitra
2007 2006 2007 2003 2003 2003 2003, 2006 2003, 2007
P606Sd6, P606Sd2 P116Sk1, P116Sk2, P116Sk5 P107F1, P106F1 P507BM2 P126AT5
Sidi Slimane Sidi Kacem Fes Beni mellal Elattaouia
2006 2006 2006, 2007 2007 2006
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the surface of LPGA agar medium. The strips were inoculated with saline suspension of bacterial culture and then incubated for 24 h at 37°C. Interpretation of the 20 reactions, in addition to the oxidase reaction (which is done separately), was converted to a seven-digit code. These isolates were subjected to the following biochemical tests according to the methods reported by various researchers to identified the P. carotovorum subspecies (Dye 1969; Schaad 1980; Lelliott and Dickey 1984; Dickey and Kelman 1988). Tests included: acid production from lactose, trehalose, α-methyl-glucoside, and melibiose, growth in 5% NaCl, growth on nutrient agar at 37°C, and production of reducing substances from sucrose. PCR amplification DNA extraction was carried out according to the protocol of Li and De Boer (1995). Two specific primers, Y1 and Y2, were used for amplification. They are specific to amplifying 434-bp open reading frames of pectate lyase genes belonging to the Y family of P. carotovorum (Darrasse et al. 1994). Primer sequences for Y1 and Y2 primers were as follows: 5′ TTA CCG GAC GCC GAG CTG TGG CGT3′ and 5′CAG GAA GAT GTC GTT ATC GCG AGT3′. The PCR reaction mixture was performed as previously described (Terta et al. 2010). PCR products were subjected to electrophoresis in
1.5% agarose minigels. A standard 1-Kb DNA ladder was run on each gel as well as positive and negative controls. Antibiotic susceptibility testing Susceptibility profiling was performed by the standard disk diffusion method on Muller–Hinton agar as the test medium (Difco) (Ozgumus et al. 2007). The standard P. carotovorum 1232 was used as a positive control The strains were tested for antibiotic susceptibility for the following antibiotics (Oxoid) Ampicillin (AMP) (10 μg), Ceftazidime (CAZ) (30 μg), Cefotaxime sodium (CTX) (30 μg), Imipenem (IPM) (10 μg), Cephalothin (KF) (30 μg), Ciprofloxacin (CIP) (5 μg), Sulfamethoxazole/trimethoprim (SXT) (25 μg), Gentamicin (CN) (10 μg), Amikacin (AK) (30 μg), Colistin sulphate (CT), and Netilmicin (NET) (30 μg).. The diameter of the zone inhibition for each antibiotic was measured to the nearest millimeter. The results were interpreted by referring to an interpretative table to determine whether the isolates were resistant or susceptible (Sahilah et al. 2008). ERIC-PCR Amplification reaction was performed using the total DNA extracted from each strain as template for ERIC-PCR. Two
Fig. 1 Dendrogram of P carotovorum ssp. carotovorum based on ERIC-PCR Fingerprinting constructed by UPGMA algorithm I, II, III, IV and V designed subclusters of the major clusters A and B
I
P603TM1 P303K3 P303MN2 P507K2 P603A1
II
P606sd2
Major cluster A
P606sd6 P116C2 P116sK1 P116Sk2 P303K1
III
P606MN1 P126AT5 P507OZ1
IV
P206C3 P603AH1 P106F1 P603TM5
V
P507BM2 P507Z1 P116sK5 P603AH2
0.05
Major cluster B
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Table 2 The susceptibility of some P. carotovorum strains examined to antibiotics Antibiotics
S
R
SXT AMP CAZ CIP CTX IPM KF CN AK NET CT
22/22a 17/22 22/22 22/22 22/22 22/22 17/22 22/22 22/22 22/22 22/22
0/22 5/22 0/22 0/22 0/22 0/22 5/22 0/22 0/22 0/22 0/22
S Sensitive strain, R resistant strain AMP Ampicillin, CAZ Ceftazidime, CTX Cefotaxime sodium, IPM Imipenem, KF Cephalothin, CIP Ciprofloxacin, SXT Sulfamethoxazole/trimethoprim, CN Gentamicin, AK Amikacin, CT Colistin sulphate, NET Netilmicin a xi/yi, where xi 0 number of positive strain and yi 0 total number of strains
specific primers were used to correlate to ERIC sequence (Versalovic et al. 1991), ERIC 1R 5′ATG TAA GCT CCT GGG GAT TCA-3′ and ERIC2 5′ AAG TAA GTG ACT GGG GTG AGC-.3 (Leong et al. 2009; Sahilah et al. 2008). Each 20 μL PCR reaction mixture contained 1uM each of primers, 2 ul target DNA, 1.25 mM each deoxynucleoside triphosphate (dNTP) and 1.5 U of Taq DNA polymerase (Promega, USA) in the manufacturer’s provided buffer. All PCR reactions were performed in an automated thermocycler using the following program: an initial denaturation step at 95°C for 7 min; 30 cycles each consisting of denaturation at 95°C for 30 s, annealing at 50°C for 1 min and extension at 65°C for 8 min; followed by a final extension step of 16 min at 65°C. The PCR products were resolved on 1.5% agarose gel electrophoresis. The strains were clustered by the unweighted average pair-group method (UPGMA) and a dendrogram was constructed (Fig. 1) (Sneath and Sokal 1973).
Results and discussion Twenty-two isolates from soft-rotted potato tubers were subcultured on LPGA medium and were subjected for identification using API 20E biochemical identification kit. The API 20E results expressed as a seven-digit code were as following: 1207173 (86%); 1204173 (9%), and 1204153 (4%). These codes were documented to confirm that these isolates belonged to the P. carotovurum family. All isolates were identified as P. carotovurum on the basis of
biochemical profiles obtained. The API 20E identification system was very effective to identify the P. carotovorum as reported by Buesching et al. (1979) and Lacroix et al. (1995). The identity of strains was further confirmed by PCR assay using specific primers Y1 and Y2 (Darrasse et al. 1994). The results showed that Y1 and Y2 are able to amplify a fragment of 434 pb corresponding to the pel gene from the genomic DNA of all isolates. The PCR agreed with the API system in the species identification of all isolates., All bacterial isolates tested in the present study were identified as Pectobacterium carotovorum ssp. carotovorum according to the results of subspecies identification. The antimicrobial susceptibility patterns of bacterial isolates were determined and are reported in Table 2. Of the 22 P. carotovurum strains, the majority (17) showed homogeneous sensitivity to Sulfamethoxazole/trimethoprim (SXT), Ceftazidime (CAZ), Ciprofloxacin (CIP), Cefotaxime sodium (CTX), Imipenem (IPM) Gentamicin (CN) Amikacin (AK) Colistin sulphate (CT) Netilmicin (NET) Ampicillin (AMP), and Cephalothin (KF). Ampicillin and Cephalothin resistance was recorded for the five other isolates tested. The results of this study indicate that there is a strong homogeneity in the susceptibility pattern of P. carotovurum species. Similar observation has been reported for 16 Erwinia chrysanthemi strains isolated from Josapine Pineapple in Malaysia (Sahilah et al. 2008). The authors conclude that the homogeneity response makes it difficult to perform epidemiological observation and to trace the distribution of E. chrysanthemi in the different locations in Malaysia (Sahilah et al. 2008). To meet the need for the typing and the epidemiological distribution, the ERIC-PCR method was Table 3 The results obtained by ERIC–PCR for the P carotovorum ssp. carotovorum strains in the study Strains
Fragment size(s) (kbp)
P116sK1, P507K2, P606sd2, P606sd6, P603A1, P303K1, P303K3, P116Sk2, P303MN2, P116C2 P603AH2 P126AT5 P603TM1 P206C3 P606MN1 P507BM2 P507Z1 P116sK5 P603AH1 P603TM5 P507OZ1 P106F1
1, 0.75, 0.5 ,0.3, 0.65 1.5, 0.3 0.75, 0.5, 0.3 1.2 ,0.5, 0.75 1, 0.5 2, 1.2, 0.8, 0.3 3, 1.5, 0.5 6,2,1.3,.0.5 1.5, 1 4, 2, 1.5, 0.5 3, 1.8 1, 0.75, 0.3 3, 2, 1.3
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used, as it is the one of the tools for studying the genetic diversity of P. carotovurum species. This PCR finger-printing technique was assessed amongst 22 isolates recovered from tuber soft rot potato. The isolates yielded some distinct bands, demonstrating polymorphism among the various isolates. ERIC-PCR data are summarized in Table 2. The size of the ERIC bands ranged from 0.3 to 6 kbp. P.carotovorum isolates were shown to be very heterogeneous. The 9 isolates symbolized as P116sK1, P507K2, P606sd2, P606sd6, P603A1, P303K1, P303K3, P116Sk2, and P303MN2 showed fragments of approximately 1, 0.75, 0.5, 0.3 and 0.65 kpb (Table 3). Conserved amplicons of approximately 1, 0.75, 0.5 and 0.3 kbp were present more times than the others. A common band of approximately 0.5 kpb was found in 15 strains . The band sizes 0.8, 0.65, 6, 4, 1.8 kbp were not found in more than one strain (Table 3) The ERIC-PCR gel picture was change from JPEG to TIFF format and was used for analysis. A dendrogram was then constructed and the relationship between isolates was estimated. The results for ERIC-PCR in this study showed a high diversity of polymorphism between P. carotovorum. All isolates were categorized in 2 major clusters: A and B, each comprising 50% of all isolates. Moreover, two subclusters could be identified within cluster A: subclusters I and II. Subcluster I consisted of only one P. carotovurum strain, designated P603TM1. In subcluster II, we found 10 strains designated P303K1, P116Sk1, P116Sk2 P606Sd6, P606Sd2, P603A1, P507K2, P303K3, P303MN2, and P116C2. Cluster B consisted of the remaining 11 strains and can be divided into 3 subclusters (subcluster III, IV, and V). Subcluster III has one strain, P606MN1, subcluster IV has 4 strains, P126AT5, P507OZ1, P206C3, and P603AH1, and, finally, subcluster V has 6 isolates, P106F1, P603TM5, P507BM2, P507Z1, P116sK5, and P603AH2. It is interesting to note that, among the isolates of cluster A (P303K1, P116Sk1, P116Sk2, P606Sd6, P606Sd2, P603A1, P507K2, P303K3, P303MN2, and P116C2), P303MN2 was isolated from different regions but was present in the same ERIC-PCR profile. These results could indicate the probability of circulating the homogenous group of P. carotovorum in the explored Moroccan regions in this study. On the other hand, we observed the strains P116sk2 and P116sk5, which were isolated from the same sample, and which belong to the two different major clusters A and B, respectively. It is also the case for the strains P603AH1 and P603AH2 which belong to subclusters IV and V, respectively. These results tally with those of previous studies (Gallelli et al. 2009). DNA-polymorphism was investigated by rep-PCR and M13-PCR for characterizing 24 Pectobacterium carotovorum recovered from artichokes in the Sele valley (Campania, southern Italy). Results showed the isolates to be distributed
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in two main groups, totalling 14 haplotypes. Eight different haplotypes were obtained from samples collected in the same field and three different haplotypes were found in the same plant. (Gallelli et al. 2009). ERICPCR confirmed the high heterogeneity of this bacterial population, already reported by others (Yahiaoui-Zaidi et al. 2003; Seo et al. 2002; Fiori et al. 2005; Yap et al. 2004; Pitman et al. 2008; Gallelli et al. 2009; Zhu et al. 2010). Furthermore, our studies have also shown no correlation between ERIC-PCR groups and state of origin or collection year. Finally, cluster analysis further indicated wide variability among the isolates affiliated to the same antibiotic susceptibility pattern as they are included under different clusters in the phylogenetic tree. To the best of our knowledge, this is the first report from Morocco which has used the ERIC-PCR method with regard to studying and evaluating the typing of Pectobacterium spp. isolates. Enterobacterial Repetitive Intergenic Consensus (ERIC) showed a great diversity among the isolates of this subspecies. and it can be applied successfully for the genetic distribution and epidemiological typing of P. carotovorum carotovorum isolated from the tuber potato in Moroccan regions. In the previous studies, ERIC-PCR has been successfully used for genetic fingerprinting and molecular typing for many types of bacteria, including Escherichia coli, Bacillus spp., Salmonella, Vibrio cholerae, Pseudomonas and Vibrio parahaemolyticus (Zulkifli et al. 2009). Among routine typing techniques, ERIC-PCR can serve as a simpler and more economical method and many isolates can be analyzed simultaneously (Sahilah et al. 2008). These characteristics are important for effective epidemiological surveillance, prevention and control of important plant pathogen bacterial infections (Lin et al. 2005; Sahilah et al. 2008; Stöger et al. 2008). In summary, this study is considered as an initial step and first report into the evaluation of the typing of the P. carotovorum Moroccan population. This aspect may also be important for epidemiological study, by taking into consideration isolates with genetic characteristics, but it still has to be compared with other molecular methods. Acknowledgment This project was financially supported by the Moroccan and French cooperation PRAD Program (PRAD 07 07).
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