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Twenty different strains of Pectobacterium carotovorum subsp. carotovorum (Pcc) were recovered using plant host enrichment approach from soil of different vegetables growing fields of Gujarat, India, during 2006-9. These strains have been classified into five different biovars by differences in physiological and biochemical features, and identified as Pcc by species-specific PCR and 16s rDNA sequences. Moreover Pcc strains were clearly differentiated based on virulence trait, plant cell wall degrading enzymes production and phylogenetic analysis of 16s rDNA sequence and Repetitive extragenic palindromic- PCR (rep-PCR). rep-PCR typing using four different primer sets revealed a high genetic variability amongst these strains independent from their pathogenic nature, with some of them showing identical banding pattern although isolated from different soils and years. The variability among isolated strains indicates that factors other than plant host correlate to genetic variability during host-pathogen interaction of this economic important Pcc strains. Based on these polyphasic characterizations of Pcc strains isolated from soil have revealed heterogeneity among them, can be considered as useful studies for epidemiological surveillance (or distribution) of soft rot causing Pcc in semi arid region of India.
India is ranked worldwide as a first and second largest producing country in production of fruits and vegetables respectively (FAO, USA). The majority of vegetables and fruits are classically growing in India in field conditions in contrast to cultivation in green houses for high yield in developed countries. Lower yield of fruits and vegetables in developing countries is often caused by inappropriate management of diseases and pests during growing in the field. Poor handling practices during growing as well as post harvest conditions makes agricultural production vulnerable to microbial diseases (Shepherd, 2006).
Bacterial soft-rot caused by Pectobacterium spp. (formally causing Pectobacterium spp.) has been considered as one of the most recurrent diseases observed in variety of vegetables and fruits species worldwide and cause great economic loss of crops (Yahiaoui-Zaidi et al., 2003, Agrios, 2006, Farrar et al., 2000, Toth et al., 2003). The genus Pectobacterium consist number of species within the family of Enterobacteriaceae. According to comprehensive reviews the most economically important soft rot causing Pectobacteriums are P. carotovorum subsp. carotovorum (Pcc, earlier known as E. carotovora ssp. carotovora), and P. carotovorum subsp. atrosepticum (Pca, earlier known as E. carotovora ssp. atroseptica), Dickeya chrysanthemi (Dc, earlier known as E. chrysanthemi), which cause diseases including soft rot, blackleg and aerial stem rot in wide variety of vegetables and fruits, and other commercially important crops (Toth et al., 2003, Pérombelon, 2002). Three more subspecies of P. carotovorum subsp. betavasculorum, subsp. odoriferum, and subsp. wasabiae were also described as infective agents to specific plant hosts. These five soft rot causing Pectobacterium species were generally differentiated based on physiology and biochemical features, serological, fatty acids and molecular profiles (Thomson et al., 1981, Gardan et al., 2003, De Boer et al., 1978, De Boer, 1979). The plant-pathogenic subspecies of P. carotovorum is considered as a complex taxon consisting of strains with a range of different phenotypic, biochemical, host range, and genetic characteristics. Soft rot causing Pectobacterium species are well suited for studying the ecology, speciation, and pathogenicity of enterobacterial pathogens as they are widespread in the environment and can infect numerous plant species (De Boer, 2003, Smith & Bartz, 1990, Yap et al., 2004, Yishay et al., 2008, Ma et al., 2007). Among these economically important soft rot causing Pectobacterium, Pcc strains have been described with wide host ranges and distribution in both temperate and tropical zones, whilst Pca strains are generally restricted to potato only in cool climates (Perombelon & Kelman, 1980, Seo et al., 2002). Phenotypic and genotypic variability have been reported so far among Pcc strains isolated in Asian countries such as Korea, Japan, Thailand, and China (Seo et al., 2002, Hu et al., 2008). There are very few reports shows existence of soft rot causing Pectobacterium strains in South-Asia region mainly in India covering their variability and pathogenesis in a wide verity of plant species . To our knowledge most of the cases of infections caused by soft rot causing Pectobacterium in India were diagnosed as top rot and stalk rot (Shekhawat et al., 1976, Rangarajan & Chakravarti, 1970).
The paucity of information regarding distribution of the soft rot causing Pectobacterium prevalent in vegetable growing fields in India has prompted the present studies. The key objective of this study was to understand the current structure of P. carotovorum subsp. carotovorum population in India to aid future control strategies before soft rot disease arise from soil.
Materials and methods
Sampling, Enrichment and Isolation
As described in Table-1 soil samples were collected from a depth of 10-20cm in inter row spacing area of different vegetables growing fields were placed in sterile plastic bags and stored at 4Â°C until processed, while diseased vegetables samples were collected from local markets, nearby Vadodara, Gujarat, India, during Aug 2006 to June 2009. The sampling were attempted as broad on geographical area as possible to understand the genetic diversity of soft rot causing Pectobacterium population throughout the vegetables growing regions of Gujarat, India. Suspensions were prepared from 2g of soil in 10ml normal saline solution (0.85% NaCl) was used as inoculum. The diseased plant samples were washed with 0.5% sodium hypochlorite (2 minutes), rinsed twice with deionized water and dried for 10 min, small piece of 0.5cmÃ-0.5cm infected tissues were then excised with a sterilized scalpel placed in 5ml normal saline solution (0.85% NaCl) and mixed.
Potato slices were prepared by socked potato tubers in 0.5% sodium hypochlorite for 10 min, rinsed with sterile deionized water, immersed in 95% ethanol (1 min) for external disinfection, followed by triple wash in sterile deionized water, air-dried under a laminar hood for 10 min. 0.5 cm thick slices were cut with a sterile potato slicer and single slice placed in each Petri dish containing filter papers soaked with sterile distilled water.
As illustrated in Fig.-1, 100 Î¼l of soil suspension or infected tissues suspension was inoculated on each potato slice and then incubated at 30Â°C for 24h. After incubation microbes that show maceration on potato slice, infected tissues (0.5cmÃ-0.5cm) were excised and used for inoculation on another fresh sterile potato slice as above for enrichment. After a total of three cycles of inoculation from macerated tissues were conducted followed by serial dilution and plating onto differential PT agar plate (Burr & Schroth, 1977). After incubation at 30Â°C for 24h colony diversity was judged and selected based pectinase activity on PT medium.
A 0.5 cm thick potato tuber slice was incorporated in each sterile flask containing 5g of sterile soil thoroughly mixed with 20ml distilled water. The Pcc MTCC1428 strain was grown overnight in nutrient broth at 30Â°C, was serially diluted, 1 ml aliquots from each decreasing dilutions were added to sterile field soil mixture with potato slice or pectate broth (Meneley & Stanghellini, 1976). Enrichment on potato slice and pectate broth was followed as described earlier by (Meneley & Stanghellini, 1976), after incubation at 30Â°C for 48 hours 1 ml aliquots were removed, serially diluted and plated on PT agar medium. All dilutions were replicated three times and experiment repeated ten times.
Physiological and Biochemical characterizations
After incubation on PT agar medium, the colony diversity was judged by colony morphology and pectolytic activity. For identification of soft rot causing pectobacterium species conventional techniques (Dye, 1969); (Thomson et al., 1981); Lelliot & Dickey, 1984; Schaad, 1988) were used. A total of twenty six tests used for identification of isolated strains (Table -2). In addition, the API 20E Kit (BioMérieux, Marcy l'Etoile, France) was used for further confirmation according to the manufacturer's protocol. The phenotypic characteristics were compared with earlier reports for different subspecies of Pectobacterium (Sutra et al., 2001; Gardan et al., 2003; Duarte et al., 2004; Yahiaoui-Zaidi et al., 2003).
Polygalacturonase (PG) activity was determined as described recently (Maisuria et al., 2010). Pectate lyase (PL) activity was determined by thiobarbituric acid method, the reaction mixture contained: 1% polygalacturonic acid in 50 mM Tris-HCl buffer (pH-8.0) with 2mM CaCl2, and appropriately diluted culture supernatant was incubated at 30Â°C for an hour. After incubation released unsaturated uronic ester derivatives were estimated as described elsewhere (Nedjma et al., 2001). One unit of pectate lyase activity is defined as the quantity of enzyme needed to increase absorbance by 0.1 units per hour at 30ËšC, pH-8.0. Protease (Prt) activity was determined using 0.5% (w/v) casein in 0.1M Tris-HCl buffer (pH-7.2) with appropriately diluted culture supernatant, and incubated at 40ËšC for 30min, followed by estimation of amino acid released as described elsewhere (Oceguera-Cervantes et al., 2007). Tyrosine and Tryptophan were used as amino acid standard. One unit of protease activity was defined as the amount of enzyme required to liberate 1Âµmole of tryptophan per minute at 40ËšC. Cellulase (Cel) activity was determined using 0.2% Carboxy methyl cellulose (CMC) with 0.05M acetate buffer (pH 5.0) and appropriately diluted culture supernatant (Marichamy & Mattiasson, 2005). The reducing sugars formed were estimated by DNS reagent(Miller, 1959). Glucose was used as reducing sugar standard. One unit of cellulase activity is defined as the amount of enzyme required to liberate 1Î¼mole of glucose per an hour at 50Â°C, pH 5.0.
Virulence assay was performed using different types of vegetables. The external surface disinfection and slice preparation of vegetables was performed as describe above. Vegetable slices in gnotobiotic condition were inoculated individually with 10Î¼l of each Pcc strain at 2.1-3.5Ã-106 cfu ml-1, and infected at 26Â°C at 100% relative humidity for 48h. Aggressiveness of each strain was analyzed by measuring macerated tissue as percentage of tissue maceration site, and relative virulence was calculated as percentage of macerated tissue per total weight or necrotic area (Yap et al., 2004, Yishay et al., 2008).
Identification based on species specific PCR and 16s rDNA sequencing
Genomic DNA extraction from pure culture of Pectobacterium strains grown overnight in nutrient broth at 30Â°C, were performed by using GenEluteâ„¢Â Bacterial Genomic DNA Kit (Sigma-Aldrich Co., Gillingham, UK) according to manufacturer's instructions. All strains were subjected to species specific PCR using primer sets EXPCCF/EXPCCR and INPCCF/INPCCR under conditions previously described (Kang et al., 2003). Nearlly full length 16s rRNA genes were amplified using the 27f and 1492r primers (Frank et al., 2008). PCR products were purified by GenEluteâ„¢Â PCR Clean-Up Kit (Sigma-Aldrich Co., Gillingham, UK) and sequenced.
Rep-PCR based DNA finger printing
Four different rep-PCR genomic fingerprinting methods (Mohapatra et al., 2007) were performed using primers sets for REP-PCR, ERIC-PCR, BOX-PCR, and (GTG)5-PCR. PCR amplifications were performed with an initial denaturation step (95Â°C, 5 min) followed by 35 cycles of denaturation (94Â°C for 30s), annealing (variable temperature, 1 min) and extension (72Â°C for 2 min), and a single final extension step (72Â°C for 10 min). The annealing temperature was 40Â°C for REP-PCR and (GTG)5-PCR, and 50Â°C for ERIC-PCR and BOX-PCR. The ten microliters of amplified products were analyzed on 1.5% agarose gels in 1Ã- TBE for 70 min at 90 V and gels were stained with ethidium bromide solution (0.5 mg ml-1) and photographed using transmitted UV light at 295 nm. Gene Ruler 1 kb DNA Ladder (MBI, Fermentas, Hanover, MD, USA) was used to determine fragment size.
The experimental mean data were analyzed by one way-ANOVA followed by post hoc analysis using Dunnet's multiple comparision tests using GraphPad Prism 4 software (San Diego, CA). Each physiological or biochemical characteristic was count as a unit character: positive (1) or negative (0) of test results were scored as binary traits. The distance matrix was generated using the Jaccard coefficient and Cluster analysis was done by using the NTSYSpc software (version 2.0; Exeter Software, USA) using simple matching according to the unweighted pair group method with arithmetic averages (UPGMA) (Sutra et al., 2001). The rep-PCR banding patterns of each gel were normalized by AlphaEaseFc 4.0 (Alpha InnotechÂ of San Leandro, Calif.). Normalized bands were scored as present (1) or absent (0) for each rep-PCR. Individual and combined finger prints of four different rep-PCR were analyzed by pearson correlation and UPGMA using NTSYSpc software (ver 2.0; Exeter Software, USA). For phylogenetic analysis of 16s rRNA gene sequences were aligned with RDP's aligner. A distance matrix was generated using the Jukes-Cantor corrected distance model, using only alignment model positions, ignoring alignment inserts and with 200 minimum comparable position. The tree was created using Weighbor with alphabet size 4 and length size 1000 (Bruno et al., 2000, Cole et al., 2007), the downloaded phylogenetic trees were display and processed using MEGA 4 software (Tamura et al., 2007).
Enrichment and isolation of enhanced virulent soft rot Pectobacterium strains
The novel enrichment technique consisted of potato tissues as the sole source of energy was conducted to enrich soft rot causing Pectobacterium existing in low cell density and to enhance the virulence properties of dormant cells residing in soil. As illustrated in Fig.-1, after each cycle the diversity of colony morphology was decreased, indicating enrichment of particular phenotypes. The sensitivity of the enrichment technique with frequency of recovery was approximately 85% from soil containing less than five but greater than two cells per gram dry weight soil, and 40% with cell population less than two cells per gram dry weight soil of the enriched population on the macerated potato slices obtained was in the range of 9.57 to 10.43 log10[cfu/ml] raised from an initial total bacterial population number of log10[cfu/ml] between 2.35 and 3.12 present in inoculums determined on semi selective media. Anaerobic enrichment with pectate broth medium showed relatively similar values of enriched bacterial population between 10.32 and 11.87 log10[cfu/ml]. Since possibility of both virulent and nonvirulent strains of soft rot causing Pectobacterium may exist in mixed populations enriched with the pectate medium, which can be eliminated by enrichment on potato slice which allows growth of only virulent strains of soft rot causing Pectobacterium.
Characterization and identification of isolated soft rot causing Pectobacterium to subspecies level
A total of twenty strains of isolated from different sources during 2006-09, showed heterogeneity in biochemical and physiological characteristics and classification in to five different biovars (Table-1, 2). All strains were identified as soft rot causing Pectobacterium according to selected phenotypic characteristics, most of the strains were motile, rod shaped, pectolytic, able to rot potato slices, oxidase and amylase non -producers, showed catalase activity, citrate alkalization, gelatin liquefaction, utilization of D-galacturonic acid, grow at 37Â°C and in 5% NaCl. Biovar 4 contained 12 strains and was most similar to Pcc MTCC1428 in regards to nitrate reductase, utilization of cellobiose and D-Trehalose, acid production from glucose and lactose, inability to form reducing substances from sucrose. Biovar 1 corresponds to 4 strains and was not similar to any other subspecies of P. carotovorum and Dickeya sp. In relation to Pcc, biovar 1 was not nitrate reducing but have ability to form reducing substances from sucrose; acid production from D-sorbitol, D-arabitol and Î±-methyl glucoside. Whilst biovar 2 correspond to one strain unable to utilize cellobiose, D-Trehalose; did not show acid production from glucose and lactose in relation to Pcc characteristics. Biovar 3 consist 2 strains, similar to biovar 1 in regards to distinguished characteristics than Pcc, having nitrate reductase, utilization of acetic acid and D-Trehalose, and inability to produce acid from Î±-methyl glucoside. Biovar 5 contained one strain which was identical to biovar 2, but showed utilization of lactic acid and acid production from glucose (Table-2). These bacteriological tests were further validated with API 20E kit. All strains were confirmed as Pcc showed 550bp DNA fragment with species specific primers (EXPCC sets), and 400bp DNA fragment with Nested-PCR (INPCC primer sets) (data not shown), also confirmed with 16s rDNA based identification.
Pathogenecity and virulence determinant enzyme production of Pcc strains
The Pcc strains were categorized in to ten different pathogenic groups based on their ability to cause soft rot lesions on test vegetable slices (Table1, 3). All twenty strains of Pcc caused tissue maceration as bacterial soft rot lesions on potato slice with significant differences in aggressiveness in terms of necrosis area and site percentage of lesions (Fig.-2). All pathogenic groups showed common host specificity with Daucus carota L. (carrot), Solanum melongena L. (brinjal), LagenariaÂ sicerariaÂ (Molina) Standl. (bottle gourd), Cucumis sativus L. (cucumber), Solanum tuberosum L. (potato). However variation with remaining test vegetable hosts was observed. Eight Pcc strains caused soft rot in all test vegetables and were grouped into pathogenic group I. Whilst pathogenic group VII consisted of three Pcc strains which did not cause infection in only Trichosanthes cucumerinaÂ L. (Snake gourd). Pathogenic groups II and V contains two strains each, did not cause infection on Beta vulgaris L. (Sugar beet) tuber slice, but differed with infection caused on Lpomoea batatas L. (Sweet potato) only by pathogenic group V. The type strain of Pcc was grouped into pathogenic group II (Table 3). The remaining pathogenic groups were represented by single strains with difference in aggressiveness (Fig.- 2a, 2b).
Furthermore Pcc strains were tested for production of four different plant cell wall degrading enzymes (PG, PNL, Prt, and Cel). The culture supernatants of all strains grown in the presence of pectin, CMC and casein contained all four virulence determinant enzymes activity. Significant differences relative to type strain were observed in enzymes production of isolated Pcc strains (Fig.- 3). Variation in specific activity was correlated to aggressiveness and host specificity of isolated Pcc strains. Most aggressive strains showed significant differences with type strain in their enzyme production (p<0.01), among them BR1 strain showed higher (p<0.01) production of all four tested major virulence determinant enzymes. While PF20 strain was higher in production of pectinolytic enzymes (PG, at p<0.01; PL, at p<0.05) and lower in other plant cell wall degrading enzymes (Prt, at p<0.01; Cel, not significantly difference).Strains CF101 (pathogenic group IV), BRF302 and PR13 (group V), MF8, MF11 and TF203 (group VIII, IX and X respectively) were showed low variation in host specificity have lower PCWDE productions (p<0.01) (Fig.-2c).
Strain level differentiation analysis by rep-PCR and phylogenetic relatedness
Four different rep-PCRs using two single oligonucleotide primers, BOX A1R (BOX-PCR) and (GTG)5 [(GTG)5-PCR], and two oligonucleotide primers pairs, Rep 1R-I/Rep 2-I (REP-PCR) and ERIC 1R/ERIC2 (ERIC-PCR), were assessed for their abilities to differentiate between isolated strains of the same subspecies of P. carotovorum (Table-1). With the use of (GTG)5-PCR and REP-PCR amplified DNA fingerprints ranging from 6 to 18 bands, and 14 to 24 bands were found respectively. While BOX-PCR and ERIC-PCR produced less than 15 bands with an average of five and seven bands. All four rep-PCR resulted in products with sizes between 100-10,000 bp. Sensitivity and reproducibility of each rep-PCR were confirmed by assessing against replicates of randomly selected two or three strains for identical finger-prints. With slight band intensity change, identical banding patterns were observed in all the four rep-PCR for each replicates of Pcc strains. Three clusters were revealed by the (GTG)5-PCR and REP-PCR individual profiles, whilst BOX-PCR and ERIC-PCR individual profiles revealed 4 and 5 clusters respectively (data not shown) (Table-1). Cluster analysis of combined finger-print patterns of all four rep-PCR revealed 3 clusters with the similarity coefficient of 67%, which were subdivided to 2-4 clusters with similarity coefficient of 76.5%. The rep-PCR profiles showed high heterogeneity among isolated Pcc strains (Fig.- 4). All the strains identified as atypical phenotypic group were distributed into cluster 2a correspond to typical strains BR1 and MTCC1428. Conversely, the same pathogenic groups were not identical in rep-PCR clusters. Whilst pathogenic group-I correspond not only to typical but also to two atypical strains MF106 and CT207, additionally both strains distributed in combined rep-PCR cluster 2a. However remaining pathogenic groups were distributed heterogeneously into combined rep-PCR clusters.
Phylogenetic analysis based on 16s rRNA gene sequences revealed that all isolated strains shared >99% homology with Pcc strains. As shown in Fig.-5 isolated strains were grouped together with different type strains and closest hit strains of Pcc, formed separate cluster to other closely related strains of plant pathogenic Enterobacteriaceae. However isolated strains formed robust subset cluster from other Pcc strains, only PF20 strain sharing cluster together with Pcc type strains DSM 30168 and LMG17566 (Fig.-5).
An approach for enrichment of soft rot causing Pectobacterium to eliminate non-virulent bacterial strains during isolation was developed. Although this approach was the alternative mode to cost effective, anaerobic and time spending steps for enrichment to existing enrichment culture technique for soft rot causing Pectobacterium. The sensitivity in isolation of soft rot causing Pectobacterium from the soil with this enrichment technique has been nearly similar to existing enrichment and isolation techniques (Meneley & Stanghellini, 1976). The potato slice used in this study was provided selective nutrient sources for rapid growth of soft rot causing Pectobacterium, which did not enrich other competitors and predominant soil bacteria, which allowed rapid isolation for soft rot causing Pectobacterium. The selective (PT) medium for isolation of soft rot causing Pectobacterium after the enrichment step eliminated the possibility of other pectolytic organisms (soft-rot Pseudomonas spp. and other fungi). However preliminary studies using crystal violet pectate (CVP) medium, direct isolation of soft rot causing Pectobacterium from field soils or artificially infested soils with low population (3 log CFU/g dry weight of soil) of soft rot causing Pectobacterium was not possible, which correspond to earlier reports of soft rot causing Pectobacterium isolation (Cuppels & Kelman, 1974, Meneley & Stanghellini, 1976). (Liao & Shollenberger, 2004) found that three log units fewer acid-injured E. carotovora cells grew on CVP than on non-selective medium, suggesting that some recipes of CVP and other selective media may fail to isolate dormant or stressed bacterial cells from environmental sources. According to their observations PT medium was more effective than other selective media. Thus PT medium was selected for the present studies.
A polyphasic approach including physiological, biochemical and molecular characteristics, was used to identify isolated strains of soft rot causing Pectobacterium. A comparison of strains revealed the heterogeneity within them in phenotypic and biochemical features. The strains were categorized to five different biovars depending upon key properties differentiate at subspecies level. Results were compared to previous reports using well established differential bacteriological tests for distinguishing Pcc with other subspecies and D. chrysanthemi (De Boer et al., 1978, Gardan et al., 2003, Seo et al., 2002, Sutra et al., 2001, Yahiaoui-Zaidi et al., 2003). Eight strains of biovar 1, 2, 3 and 5 did not match all the characteristics previously reported for soft rot causing Pectobacterium subspecies. Diversity in phenotypic characteristic among isolated Pcc strains from different plant hosts within the same geographical regions have been commonly reported in the literatures (Duarte et al., 2004, Gardan et al., 2003, Seo et al., 2003, SÅ‚awiak et al., 2009, Yahiaoui-Zaidi et al., 2003). Surprisingly D. chrysanthemi was absent from the isolated strains, although its existence in warm climatic conditions and similar geographical regions has been extensively reported (Mandal & Maiti, 2005, Pérombelon, 1992, Thind & Payak, 1985) . Significantly isolated strains belonging to biovars 1, 2 and 5 did not utilize D-Trehalose which is a unique properties of D. chrysanthemi, whilst all strains did not produce indole from tryptophan, a trait considered a distinguishing characteristic of subspecies of P. carotovorum from D. chrysanthemi (Sutra et al., 2001, Yahiaoui-Zaidi et al., 2003). All strains were identified as Pcc using species specific primers, and were classified as Pcc by 16s rDNA sequence homology with the type strain.
The pathogenic diversity of isolated Pcc strains was analyzed using a variety of plant hosts. Variability in key features of aggressiveness and host specificity was observed among isolated Pcc strains. Based on pathogenicity ten pathogenic groups were recognized. Pcc is considered a broad-host-range pathogen relative to other subspecies of P. carotovorum. Pathogenic specialization for different plant hosts and variability in pathogenicity and host range have been noted under experimental conditions among soft rot causing Pectobacterium (Dickey, 1979, Ma et al., 2007, Seo et al., 2002, El-Hendawy et al., 2002), and corresponding observations were obtained from isolated Pcc strains in this study. Recently, as per cataloging review by (Ma et al., 2007) of the host range of soft rot causing Pectobacterium has been produced, however similar reports of host specificity of Pcc towards S. molongena L. (brinjal), L. batatas L. (sweet potato), T. cucumerinaÂ L. (snake gourd) and L.Â sicerariaÂ (Molina) Standl. (bottle gourd) have not been described. Several attempts have been made for isolation of stalk rot (similar symptomatic disease) causing Pectobacterium from Indian fields and identification of their host range characteristics. However classification of bacterium causing stalk rot in maize was not clarified between P. carotovorum and Dickeya spp. (Anilkumar & Chakravarti, 1971, Thind & Payak, 1985, Ragarajan & Chakaravarati, 1970). Unfortunately, it is impossible to obtain cultures of strains from these studies and therefore, it has not been possible to correlate the Pcc strains from the current study with other Pectobacterium strains of Indian origin for host range specificity.
Plant cell wall degrading enzymes (PCWDE) play a central role in initiation of soft rot necrosis symptoms in host (Hugouvieux-Cotte-Pattat et al., 1996). Production levels of PCWDE was analyzed for each isolate to correlate variation in pathogenicity of the strains towards plant hosts. In vitro condition isolated Pcc strains produced PG and PL during late log phase, while Cel and Prt produced during stationary phase of the growth of bacteria, also shows that pectinase higher producing isolated Pcc strains had major virulence effect against tested plant hosts. The production of pectic enzymes (PL, PG) are major virulent determinants of soft rot causing Pectobacterium produced in initial stage of incubation with pectin substrates which are majorly present in middle lamella, among them PG produced early than PL, which facilitate further mode of action of Cel and Prt during infection (Hugouvieux-Cotte-Pattat et al., 1996, Le Cam et al., 1997, El-Hendawy et al., 2002). Several studies have already correlated pathogenicity and pectinase activities as vital role in plant tissue maceration during infection caused by other microorganisms (Johansson, 1988, Mwenje & Ride, 1997, Yong Jian et al., 2009, Barker & Walker, 1962).
All twenty strains of Pcc have variation in their virulence, biochemical and physiological properties. In this study, using rep-PCR, Pcc strains were grouped in to three large clusters, whilst ERIC-PCR grouped them into five clusters. The ERIC-PCR analysis has been already demonstrated for variability with in Pcc strains earlier (Toth et al., 1999). However several studies showed the existence of high level of genetic diversity among Pcc strains, examined by use of various molecular techniques, such as PCR-restriction fragment length polymorphism (RFLP) (Darrasse et al., 1994, Waleron et al., 2002), Random amplified polymorphic DNA (RAPD) analysis (Parent et al., 1996), DNA-DNA hybridization (Gardan et al., 2003), Amplified Fragment Length Polymorphism (AFLP) fingerprinting (Avrova et al., 2002), Pulsed-field gel electrophoresis (PFGE) fingerprinting (Yap et al., 2004), repetitive extragenic palindromic PCR (rep-PCR) fingerprinting (Norman et al., 2003), and 16S rDNA analysis (Gardan et al., 2003, Hu et al., 2008, Yishay et al., 2008). However these previous studies do not indicate functional differences attributed to genetic diversity. Although PFGE and rep-PCR have nearly equal resolving power for differentiation at subspecies and strain level, the rep-PCR DNA fingerprinting technique is simple and rapid, with less labor, time and cost, can be performed with standard equipment with high throughput in comparison to PFGE (Olive & Bean, 1999, Ishii & Sadowsky, 2009). (Norman et al., 2003) successfully used rep-PCR fingerprinting for strain level differentiation of Pcc population isolated from a nursery retention pond and lake water. In addition to REP-PCR, ERIC-PCR and BOX-PCR, the present studies demonstrates the use of (GTG)5-PCR for the first time to differentiate Pcc strains isolated from field soil and diseased vegetables and fruits. The (GTG)5-PCR has the highest discriminatory power and rate of correct classification compare to other three rep-PCR (REP-PCR, ERIC-PCR and BOX-PCR), and has been successfully utilized for molecular typing of strains of Escherichia coli (Mohapatra et al., 2007), Salmonella enterica (Rasschaert et al., 2005), and Enterococcus spp. (Svec et al., 2005). Isolated Pcc strains with same pathogenic group were grouped in to different clusters of combined rep-PCR analysis, reflecting high level of genetic diversity was present among the isolated Pcc strains with respect to their source of isolation and year. The results of phylogenetic analysis of 16S rDNA sequences of isolated Pcc strains suggest heterogeneity to certain extent was observed. Fifteen strains formed a separate subset of cluster from the type strains of Pcc and reported strains from Asian countries, four other strains sharing common cluster with these Asian strains. Interestingly PF20 strain grouped with type stains of Pcc with in a distinct cluster, which correlate with cluster 3b of combined rep-PCR analysis. The most virulent strains BR1 and PF20 were clustered in to two different phyletic clades, which is in contrast to the phylogenetic association with pathogenesis traits found. The reference strain Pcc MTCC 1428 (ATCC 15713) also was clustered in distinct clade even though it showed identical phenotypic and virulence characteristics with more than half of the isolated strains. Different reference strains of plant pathogenic genera of Enterobacteriaceae family were used to ensure that robustness of subset clustering of isolated strains were indeed unique from reference and Asian origin strains of Pcc. Conversely, use of advanced molecular techniques has elucidated a wide diversity within Pcc from different host plants sources and even from the same geographical and ecological niches (Ma et al., 2007, Norman et al., 2003, Seo et al., 2002, Toth et al., 2003, Yap et al., 2004, Yishay et al., 2008, Avrova et al., 2002).. Present studies strongly support the diversity of Pcc was found independent of host range specificity, geographical locations or plant sources. At last the virulence and phenotypic properties were not associated with genotypic characterization such as rep-PCR and 16S rDNA sequence analysis. The phenotypic, pathogenic and genetic diversity was found within Pcc strains from India as an initial step in understanding the spatial distribution and structure of plant-pathogen population.