Phenotypic and genotypic characteristics of Iranian soft rot bacterial isolates from different hosts

Headnote

Summary. During 2005-2006, 42 soft rot bacterial strains were isolated from the infected tubers of potato, roots of carrot, sugar beet and turnip, and the leaves of lettuce and cabbage with soft rot symptoms in Iran. The isolates were rod-shaped, motile with peritrichous flagella, gram negative, facultative anaerobe, oxidase and urease negative and they rotted potato tuber slices. Of the 42 isolates, 20 were identified as Pectobacterium carotovorum subsp. carotovorum (Pcc), 6 as P. carotovorum subsp. odoriferum (Pco), 4 as P. betavasculorum (Pb) and 12 strains as Dickeya dadantii (Dda). PCR amplification of fingerprints of repetitive bacterial DNA elements using the REP, ERIC and BOX primers differentiated the soft rot bacteria to the species and subspecies level. Strains of Pcc and Dda were phenotypically and genotypically highly variable, but Pb and Pco strains had low variability. REP-PCR was found to be a promising genotypic tool for the rapid and reliable speciation and typing of soft rot bacteria..

Key words: Pectobacterium, Dickeya, Rep-PCR.

Introduction

The family of the Enterobacteriaceae groups various species of plant pathogenic bacteria that cause soft rot in a wide range of plants (Toth et al., 2003). Based on 16 ribosomal DNA (rDNA), Hauben et al. (1998), offered to re-establish the genus Pectobacterium which now comprises four species: Pectobacterium carotovorum, P. atrosepticum, P. wasabiae (Pw) and P. betavasculorum (Pb). P. carotovorum was further divided into two subspecies: P. carotovorum subsp. carotovorum (Pcc) and P. carotovorum subsp. odoriferum (Pco) (Helias et al., 1998; Garden et al., 2003). Samson et al. (2005) transferred Pectobacterium chrysanthemi to the genus Dickeya as Dickeya chrysanthemi and defined four novel species: D. dadantii (Dda), D. dianthicola, D. dieffenbachiae and D. zeae. The biochemical characteristics of 6 species of Dickeya spp. have recently been described (Palacio-Bielsa et al., 2006). Pcc, Pb and Dickeya spp. are bacterial pathogens of important crops world-wide. However, the other species and subspecies are also important (Dickey, 1979; Pérombelon and Kelman 1980). Realizing the diversity within and the relationship between pathogenic taxa is a prerequisite to identifying, detecting and studying pathogens epidemiologiy (Avrova et al., 2002). Current identification techniques are based mainly on biochemical and phenotypic characteristics, with which rapid and precise identification is not always possible (Kwon et al., 1997). Biochemical tests are currently accepted as a standard to identify and characterize pathogenic bacteria, but they are very much time consuming (Toth et al., 2001). The commonly used approaches for the diagnosis and identification of pathogens include biochemical and physiological characteristics, pathogenicity tests, as well as serological techniques and techniques based on the polymerase chain reaction (PCR). Repetitive sequence based genomic fingerprinting (REP-PCR), which uses primers matching the endogenous interspersed repetitive sequences, is one of the PCR-based techniques used to identify and classify bacteria (Versalovic et al., 1991; Louws et al., 1995 and 1999). This technique is based on the PCR-mediated amplification of DNA sequences located between specific interspersed sequences of highly conserved elements in prokaryotic genomes. These repeated sequences are named BOX, REP, and ERIC elements (Versalovic et al., 1994). The method has been applied to members of diverse bacterial genera. Iranian soft rot bacteria have been reported to be diverse in studies using phenotypic characteristics and whole-cell protein electrophoresis (Ahmadvand and Rahimian, 2002; Soltani-Nejad et al., 2005; Mahmoudi et al., 2007; Zohour Paralak et al., 2007). The aim of this study was to characterize Iranian soft rot bacteria strains on the basis of biochemical, physiological and REP-PCR genomic fingerprinting, and to determine whether REP-PCR differentiated the current species and subspecies of Pectobacterium and Dicheya isolates in Iran.

Materials and methods

Bacterial strains and isolation

Forty-two isolates from diseased plants, including potato, cabbage, carrot, onion, sugar beet, pepper and lettuce, were recovered in 2006-2007 from different counties in the Fars Province of Iran (Table 1). A small piece of tissue from the periphery of decayed lesions of these plants was homogenized in sterile water, and a loopful of the suspension was streaked on Eosin methylen blue agar (EMB) and nutrient agar (NA) (Mahmoudi et al., 2007). Colonies were further purified on NA. Reference strains of Pcc (EccSCRI 193), Dickeya sp. (EchSCRI 3739) and Pb (SCRI 479) from the Scottish Crop Research Institute (SCRI, Dundee, UK) and the type strain of Pco (CFBP 1878) from the Collection Française de Bactéries Phytopathogènes (Beaucouzé, France) were also used in this study.

Physiological and biochemical tests

The 42 isolates were compared on the basis of the following biochemical, physiological and nutritional characteristics: gram reaction, catalase production, phosphate activity, oxidase production, growth at 37oC, nitrate reduction, reducing substance from sucrose, indole production, erythromycin sensitivity, growth on 5% NaCl, urease and levan production, lecithinase, Tween 80 hydrolysis, utilization of inulin, citrate, malonate and ¿-methyl glucoside (De Boer and Kelman 2001; Schaad at al., 2001), fermentative metabolism, arginine dihydrolase, H2S production from cystein, potato soft rot, gelatin liquefaction, acetoin production (VP), and gas from glucose (Schaad et al., 2001). Carbohydrate utilization was determined using the basal medium of Ayers et al., (1919) from glucose, fructose, rhamnose, arabianose, sorbitol, raffinose, melibiose, trehalose, galactose, cellobiose, lactose, maltose, mannose, and arabitol and the results were evaluated daily for 1 month (Fahy and Persley 1983).

DNA Extraction

The SDS-boiling method was used for DNA extraction (Mahmoudi et al., 2007). Total DNA was extracted from bacterial culture in a Luria Bertani (LB) medium (0.5% yeast extract, 1% tryptone, 1% NaCl). After 24 h, 3 ml of culture was removed and centrifuged at 14000 g for 3 min. The pellet was resuspended in 500 mL extraction buffer (0.2 M tris-HCl, pH 8.0; 0.2 M EDTA; 2% SDS) and boiled at 100°C for 8 min. Then 250 ml phenol and 250 ml chloroform-isoamyl alcohol (24:1, v:v) were added to the tube and centrifuged at 13000 g for 5 min. The supernatant was transferred to a new tube. To eliminate the RNA, 3 ml of RNase-A was added to the tube, the nucleic acid solution was incubated at 37°C for 1 h and then extracted with chloroform-isoamyl alcohol (24:1, v:v). The mixture was centrifuged at 13000 g for 5 min and the upper phase was transferred to a new tube. DNA was precipitated with an equal volume of cool isopropanol, washed with 70% ethanol:water (v:v), dried, resuspended in 50 ml of TAE (4% M Tris-HCl, 20 mM sodium acetate, 2 mM EDTA) buffer, and used for PCR amplification.

Species-specific PCR for identification of Pcc

The oligonucleotide primers EXPCCR (5'-CCGTAATTGCCTACCTGCTTAAG-3') and EXPCCF (5'-AACTTCGCACCGCCGACCTTCTA-3') were used in a standard PCR assay (Kang et al., 2003). The PCR reactions were performed in a Bio-Rad I-cycler (USA) in 25 mL PCR mixture containing 10 mM tris-HCL, 1.5 mM MgCl2, 200 mm of each dNTPmix, 100 ng of primers, 1 unit of Taq polymerase (Metabion, Martinsried, Germany) and 1 mL of DNA template. The PCR reaction was carried out as follows: 94°C for 4 min, for initial denaturation; 30 cycles of 94°C for 1 min; 60°C for 1 min; and 72°C for 2 min, followed by a final elongation step of 72°C for 7 min. PCR products were analyzed on 1% agarose gel in TAE buffer and visualized by staining with ethidium bromide (Sambrook et al., 1989).

REP-PCR

Repetitive PCR (REP-PCR) was used with ERIC, BOX and REP primers as described by Louws et al. (1995). The ERIC, BOX, and REP primer sets were synthesized by Metabion. Amplification was performed in a Bio-Rad I-cycler (Hercules, CA, USA) in 25 ml volumes containing 200 mM of each dNTPmix, 2 mM MgCl^sub 2^, 1.5 pM primers, 1U of Taq polymerase and 4 mL of DNA template. Thermal cycling was carried out as described by Louws et al. (1994): an initial denaturation cycle at 95°C for 7 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C (ERIC), 53°C (BOX) and 44°C (REP) for 1 min, extension cycle at 65°C for 8 min, and a final extension cycle at 65°C for 15 min. The PCR products were separated on 1.5% gel electrophoresis agarose in TAE buffer (Sambrook et al., 1989). After staining with ethidium bromide, the gels were viewed and photographed under UV illumination.

Data analysis

Data were statistically analyzed using the numerical taxonomy and multivariate analysis system (NTSYSPC) software package version 2.1. Forty phenotypic characters were included in the analysis. Genetic Relationships within and between strains were determined by cluster analysis performed by UPGMA on distance matrices calculated with the jaccard coefficient (Rohlf, 2000).

Results

Phenotypic characteristics

The biochemical and physiological properties of the bacterial isolates are shown in Table 2. The 42 strains isolated from various hosts in Iran were identified as Pcc (20 isolates), Pco (6 isolates), Pb (4 isolates) and Dda (12 isolates).

Species-specific PCR for Pcc

The oligonucleotide primers EXPCCF and EXPCCR were used to identify the Iranian Pcc strains from the various hosts. All strains of Pcc produced the expected 550 bp product after amplification with the primers (Figure 1). No PCR product was amplified with strains from the other group, Pco, Pb, or Dda.

REP-PCR

Primers corresponding to conserved sequences of the REP, ERIC and BOX elements annealed to genomic DNA and generated unique genomic fingerprints for the strains of Pcc, Pb, Pco and Dda tested. The fingerprint of strains of Pcc, and Dda are shown in Figures 2 and 3. REP, BOX, and ERIC-PCR clearly differentiated the Iranian soft rot bacteria strains isolated from the different hosts. The PCR bands were compared based on the presence or absence of fragments at a specific position, and the similarity coefficients of pairs of isolates were calculated to determine the genetic relationship among bacterial isolates. There were differences in the intensity of some amplified fragments as well as in the presence/absence of a number of polymorphic bands. When the data were analyzed by combining each set of fingerprints, four groups were clearly separated (Figure 4). Group I comprised all Pcc strains and generated unique patterns that were characteristic of this subspecies (Figure 2, A,B,C). Group II comprised all Pb strains and generated the unique patterns characteristic of this species. Group III comprised Pco, and group IV Dda. The Eric-PCR patterns of isolates within a species and subspecies were similar. Pcc, Pco, Pb and Dda each had a unique banding pattern that clearly distinguished it from all the others (Figure 5). Complex fingerprint patterns were obtained for all the isolates studied with these primers. Reproducible genomic PCR profiles consisted of bands ranging in size from 500?-?3000 bp for BOX primers, 250-3000 bp for ERIC, and 200-2500 bp for PCR primers. The 42 strains from different hosts that had been identified as Pcc, Pb, Pco or Dda by their phenotypic characters were clearly distinguished by REP-PCR with the REP, ERIC and BOX primers.

Discussion

The main aim of this study was to investigate whether the REP-PCR technique discriminated the soft rot bacteria to the species and subspecies level in Iran. Highly conserved repetitive DNA elements, such as the repetitive extragenic palindromic (REP) elements, the enterobacterial repetitive intragenic consensus (ERIC) elements, and the BOX elements, seem to be widespread in the genomes of various bacterial groups (Versalovic et al., 1991). Amplification of the sequences between each of these repetitive elements has been used to generate DNA fingerprints of several gram-negative and gram-positive bacterial species. The soft rot bacteria Pcc, Pb, Pco and D. dadantii must be characterized if rapid diagnostic methods are to be developed. In Iran, Pcc, Pb, and Dda cause severe damage to various plants. In this study, we described 42 isolates of pectolytic bacteria that belong to Pectobacterium spp., and a Dickeya sp., obtained from diseased crops, including potato, cabbage, onion, turnip, sugar beet, pepper, lettuce and carrot. Clustering based on REP-PCR with BOX, ERIC and REP primers confirmed clustering based on phenotypic features. Pcc strains were phenotypically and genetically heterogeneous (Avrova et al., 2002; Waleron et al., 2002). Polymorphism among the REP-PCR products of the Pcc strains indicated that these strains were genetically variable. Pcc was the most diverse subspecies, but Pco and Pb were much more homogeneous. Such relatively low genetic diversity may be due to a subspecies having a more recent origin, to limited population divergence or to a limited host range. Pcc has a wider host range, which may explain the genetic diversity of this subspecies (Avrova et al., 2002).

Pectobacterium betavasculorum causes soft rot of sugar beet and was also isolated from sunflower, artichoke, and potato (Avrova et al., 2002). All the Pb isolates recovered in this study were also recovered from sugar beet. Phenotypic tests showed that the Pb strains were distinct from the other species and subspecies. Based on REP-PCR with REP, ERIC and BOX primers, there was no difference between these strains (S^sub 5^, S^sub 6^, S^sub 7^, S^sub 8^) (Figures 4 and 5). The primers EXPCCF and EXPCCR were used to detect the isolated bacteria from sugar beet. But only three sugar beet isolates (S1-S3) produced the expected 550 bp product following PCR with these primers and they were identified as Pcc. The remaining four strains of sugar beet were identified as Pb by their phenotypic characters.

Pco, previously designated as atypical P. atrosepticum (Samson et al., 1980), was isolated from witloof-chicory, leek, allium and celery (Gallois et al., 1992). Pco strains were isolated from potato, cabbage, and turnip and it seems that this subspecies has a broad host range. The Pco isolates were clearly differentiated from the isolates of other species and subspecies of soft rot bacteria based on REP-PCR (Figures 4 and 5).

Different Dickeya spp. cause disease on different hosts under different climatic condition. Dickeya spp. are recognized as important pathogens in many crops (Pérombelon, 2002). Twelve strains, isolated from various hosts, were identified as Dda by phenotypic analysis and by REP-PCR. The Dda strains isolated from potato, cabbage, onion, turnip, sugar beet, pepper, lettuce and carrot in Iran were different from the Dda strains typically isolated in the Netherlands from blacking-diseased plants (Janse and Ruissen, 1988). Polymorphisms generated with ERIC primers were mainly seen in strains Po^sub 4^, T^sub 4^, Pe^sub 3^ and S^sub 4^ of Dda, characterized by the presence of a 320 bp and 380 bp fragment (Figure 3, A).

Phenotypic tests and serological methods have been developed to detect and characterize soft rot bacteria; however, not all these tests specifically detected the species and subspecies. Biochemical tests differentiate all species and subspecies, but they are time-consuming and not sensitive enough for testing purposes (Toth et al., 2001). Serological techniques too are not sensitive enough to detect low but epidemiologically significant bacterial populations. A number of other methods have been used to identify the soft rot bacteria but all of them have their limitations. REP-PCR was considerably faster, more suitable and more accurate than the other identification methods (Toth et al., 2001). This was especially true when large numbers of isolates had to be tested. This study provides evidence for the belief that REP-PCR fingerprinting is an important tool to identify the various soft rot bacteria from different hosts and monitor them. Moreover, the differentiation capacity of this technique increased significantly if the band polymorphisms were analyzed together, and this is the first report of a combined analysis of soft rot bacteria in Iran.

References

Literature cited

Ahmadvand R. and H. Rahimian, 2002. Study on diversity of the pectolytic erwinias infecting potato in hamedan province. Proceedings of the 15th Iranian Plant Protection, 7-11 September 2002, Kermanshah, Iran, 115 (abstract).

Avrova A.O. J. Hyman, R.L. Toth and I.K. Toth, 2002. Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi. Applied and Environmental Microbiology 68, 1499-1508.

Ayers S.H., P. Rupp and W.T. Johnson, 1919. A study of the alkali-forming bacteria in milk. United States Department of Agriculture Bulletin. 787, 1-39.

De Boer S.H. and A. Kelman, 2001. Erwinia soft rot group. In: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed., (Schaad N.W., Jones J.B., Chun W. ed.), American Phytopathological Society, St. Paul, MN, USA, 56-72.

Dickey R.S., 1979. Erwinia chrysanthemi: a comparative study of phenotypic properties of strains from several hosts and other Erwinia species. Phytopathology 69, 324-329.

Fahy P.C. and G.J. Persley, 1983. Plant Bacterial Diseases: A Diagnostic Guide, 1st ed. Academic Press, Sydney, Australia, 393 pp.

Gallois A., R. Samson, E. Ageron and P.A.D. Grimont, 1992. Erwinia carotovora subsp. odorifera subsp. nov., associated with odorous soft rot of chicory (Cichorium intybus L.). International Journal of Systematic Bacteriology 42, 582-588.

Garden L., C. Gouye, R. Christen and R. Samson, 2003. Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavascularum sp. nov. and Pectobacterium wasabiae sp. nov. International Journal of Systematic Bacteriology 53, 381-391.

Hauben L., E.R.B. Moore, L. Vauterin, M. Steenackers, J. Mergaert, L. Verdonck. and J. Swing, 1998. Phylogenic position of phytopathogens within the Enterobacteriaceae. Systematic and Applied Microbiology 21, 384-397.

Helias V., L.A. Le Roux, Y. Bertheau, D. Andrivon, J. Gauthier. and B. Jouan, 1998. Characterisation of Erwinia carotovora subspecies and detection of Erwinia carotovora subsp. atroseptica in potato plants, soil and water extracts with PCR-based methods. European Journal of Plant Pathology 104, 685-699.

Janse L.D. and M.A. Ruissen, 1998. Characterization and classification of Erwinia chrysanthemi strains from several hosts in the Netherlands. Phytopathology 78, 800-807.

Kang H.W., S.W. Kown and S.J. Go, 2003. PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band. Plant Pathology 52, 127-133.

Kwon S.W., S.J. Go, H.W Kang, J.C Rya and T.K. Jo, 1997. Phylogenetic analysis of Erwinia species based on 16S rDNA gene squences. International Journal of Systematic Bacteriology 47, 1061-1067.

Louws F.J., D.W. Fulbright, C.T. Stephens and D.J. De Bruijn, 1995. Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campesteris pv. vesicatoria. Phytopathology 85, 528-536.

Louws F.J., J.L.W. Rademaker and F.J. de Bruijn, 1999. The three Ds of PCR-based genomic analysis of phytobacteria: diversity, detection, and disease diagnosis. Annual Review of Phytopathology 37, 81-125.

Mahmoudi E., M.J. Soleimani and S.M. Taghavi, 2007. Detection of bacterial soft-rot of crown imperial caused by Pectobacterium carotovorum subsp. carotovorum using specific PCR primers. Phytopathologia Mediterranea 46, 1-8.

Palacio-Bielsa A., M.A. Cambra and M.M. López, 2006. Characterization of potato isolates of Dickeya chrysanthemi in Spain by a microtitre system for biovar determination. Annals and Applied Biology 148, 157-164.

Pérombelon M.C.M., 2002. Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathology 51, 1-12.

Pérombelon M.C.M. and A. Kelman, 1980. Ecology of the soft rot Erwinias. Annual Review of Phytopathology 18, 361-387.

Rohlf F.J., 2000. NTSYS-PC, Numerical taxonomy and Multivariate analysis system, Version 2.11j. Exeter Software, Setauket, NY, USA.

Sambrook J., Fritsch E.F., Maniatis T., 1989. Molecular Cloning; A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY, USA.

Samson R., J.B. Legendre, R. Christen, W. Achuak and L. Garden, 2005. Transfer of Pectobacterium chrysanthemi (Brenner et al. 1973 ) and Brenneria paradisiaca (Hauben et al. 1998 ) to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species: Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. International Journal of Systematic and evolutionary Microbiology 55, 1415-1427.

Samson R., F. Poutier, M. Sailly, L. Hingand and B. Jouan, 1980. Bacteria associated with soft-rot of witloof-chicory. Annual Review of Phytopathology 12, 3-11.

Schaad N.W., J.B. Jones and W. Chun, 2001. Laboratory Guide for identification of Plant Pathogenic bacteria. 3rd ed. American Phytopathological Society St. Paul, MN, USA, 378 pp.

Soltani-Nejad S., S.M. Taghavi, J. Hyati and R. Mostowfizadeh Ghalamfarsa, 2005. Study of phenotypic features and pathogenicity of soft rot pectobacteria in Khuzestan Province. Iranian Journal of Plant Pathology 41, 585-612.

Toth I.K., A.O. Avrova and L.J. Hyman, 2001. Rapid identification and differentiation of the soft rot erwinias using 16S-23S intergenic transcribed spacer (ITS)- PCR and RFLP analyses. Applied and Environmental Microbiology 67, 4070-4076.

Toth I.K., K.S. Bell, C.M. Holeva and J.R. Birch, 2003. Soft rot erwiniae: from genus to genomes. Molecular Plant Pathology 4, 17-30.

Versalovic J., T. Koeuth and J.R. Lupski, 1991. Distribution of repetitive DNA-sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 19, 6823-6831.

Versalovic J., M. Schneider, F.J. de Bruijn and J.R. Lupski, 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular and Cellular Biology 5, 25-40.

Waleron M., K. Waleron and A.J. Podhajska, 2002. Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment. Microbiology 148, 583-595.

Zohour Paralak E., H. Rahimian and Z. Banihashemi , 2007. A comparative study on phenotypic erwinias isolated from potato in Fars province. Iranian Journal of Plant Pathology 43, 121-144.

Accepted for publication: June 6, 2010