Twenty different strains of Pectobacterium carotovorum subsp. carotovorum ( Pcc ) were recovered utilizing works host enrichment attack from dirt of different veggies turning Fieldss of Gujarat, India, during 2006-9. These strains have been classified into five different biovars by differences in physiological and biochemical characteristics, and identified as Pcc by species-specific PCR and 16s rDNA sequences. Furthermore Pcc strains were clearly differentiated based on virulency trait, works cell wall degrading enzymes production and phyletic analysis of 16s rDNA sequence and Repetitive extragenic palindromic- PCR ( rep-PCR ) . rep-PCR typing utilizing four different primer sets revealed a high familial variableness amongst these strains independent from their infective nature, with some of them demoing indistinguishable stria form although isolated from different dirts and old ages. The variableness among stray strains indicates that factors other than works host correlative to familial variableness during host-pathogen interaction of this economic of import Pcc strains. Based on these polyphasic word pictures of Pcc strains isolated from dirt have revealed heterogeneousness among them, can be considered as utile surveies for epidemiological surveillance ( or distribution ) of soft putrefaction doing Pcc in semi waterless part of India.
India is ranked worldwide as a first and 2nd largest bring forthing state in production of fruits and veggies severally ( FAO, USA ) . The bulk of veggies and fruits are classically turning in India in field conditions in contrast to cultivation in green houses for high output in developed states. Lower output of fruits and veggies in developing states is frequently caused by inappropriate direction of diseases and plagues during turning in the field. Poor managing patterns during turning every bit good as station crop conditions makes agricultural production vulnerable to microbic diseases ( Shepherd, 2006 ) .
Bacterial soft-rot caused by Pectobacterium spp. ( officially doing Pectobacterium spp. ) has been considered as one of the most perennial diseases observed in assortment of veggies and fruits species worldwide and cause great economic loss of harvests ( Yahiaoui-Zaidi et al. , 2003, Agrios, 2006, Farrar et al. , 2000, Toth et al. , 2003 ) . The genus Pectobacterium consist figure of species within the household of Enterobacteriaceae. Harmonizing to comprehensive reappraisals the most economically of import soft putrefaction causation Pectobacteriums are P. carotovorum subsp. carotovorum ( Pcc, before known as E. carotovora ssp. carotovora ) , and P. carotovorum subsp. atrosepticum ( Pca, before known as E. carotovora ssp. atroseptica ) , Dickeya chrysanthemi ( Dc, before known as E. chrysanthemi ) , which cause diseases including soft putrefaction, scab and aerial root putrefaction in broad assortment of veggies and fruits, and other commercially of import harvests ( Toth et al. , 2003, Perombelon, 2002 ) . Three more races of P. carotovorum subsp. betavasculorum, subsp. odoriferum, and subsp. wasabiae were besides described as morbific agents to specific works hosts. These five soft putrefaction doing Pectobacterium species were by and large differentiated based on physiology and biochemical characteristics, 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 races of P. carotovorum is considered as a complex taxon consisting of strains with a scope of different phenotypic, biochemical, host scope, and familial features. Soft putrefaction doing Pectobacterium species are good suited for analyzing the ecology, speciation, and pathogenicity of enterobacterial pathogens as they are widespread in the environment and can infect legion works species ( De Boer, 2003, Smith & A ; Bartz, 1990, Yap et al. , 2004, Yishay et al. , 2008, Ma et al. , 2007 ) . Among these economically of import soft putrefaction doing Pectobacterium, Pcc strains have been described with broad host scopes and distribution in both temperate and tropical zones, whilst Pca strains are by and large restricted to potato merely in cool climes ( Perombelon & A ; Kelman, 1980, Seo et al. , 2002 ) . Phenotypic and genotypic variableness have been reported so far among Pcc strains isolated in Asiatic states such as Korea, Japan, Thailand, and China ( Seo et al. , 2002, Hu et al. , 2008 ) . There are really few studies shows being of soft putrefaction doing Pectobacterium strains in South-Asia part chiefly in India covering their variableness and pathogenesis in a broad truth of works species. To our cognition most of the instances of infections caused by soft putrefaction doing Pectobacterium in India were diagnosed as top putrefaction and chaff putrefaction ( Shekhawat et al. , 1976, Rangarajan & A ; Chakravarti, 1970 ) .
The dearth of information sing distribution of the soft putrefaction doing Pectobacterium prevalent in vegetable turning Fieldss in India has prompted the present surveies. The cardinal aim of this survey was to understand the current construction of P. carotovorum subsp. carotovorum population in India to help future control schemes before soft putrefaction disease arise from dirt.
Materials and methods
Sampling, Enrichment and Isolation
As described in Table-1 dirt samples were collected from a deepness of 10-20cm in inter row spacing country of different veggies turning Fieldss were placed in unfertile plastic bags and stored at 4A°C until processed, while diseased veggies samples were collected from local markets, nearby Vadodara, Gujarat, India, during Aug 2006 to June 2009. The sampling were attempted as wide on geographical country as possible to understand the familial diverseness of soft putrefaction doing Pectobacterium population throughout the veggies turning parts of Gujarat, India. Suspensions were prepared from 2g of dirt in 10ml normal saline solution ( 0.85 % NaCl ) was used as inoculant. The morbid works samples were washed with 0.5 % Na hypochlorite ( 2 proceedingss ) , rinsed twice with deionized H2O and dried for 10 min, little piece of 0.5cmA-0.5cm septic tissues were so excised with a sterilized scalpel placed in 5ml normal saline solution ( 0.85 % NaCl ) and assorted.
Potato pieces were prepared by socked murphy tubers in 0.5 % Na hypochlorite for 10 min, rinsed with unfertile deionized H2O, immersed in 95 % ethyl alcohol ( 1 min ) for external disinfection, followed by ternary wash in unfertile deionized H2O, air-dried under a laminar goon for 10 min. 0.5 centimeter midst pieces were cut with a unfertile murphy slicer and individual piece placed in each Petri dish incorporating filter documents soaked with unfertile distilled H2O.
As illustrated in Fig.-1, 100 I?l of dirt suspension or infected tissues suspension was inoculated on each murphy piece and so incubated at 30A°C for 24h. After incubation bugs that show maceration on murphy piece, infected tissues ( 0.5cmA-0.5cm ) were excised and used for vaccination on another fresh sterile murphy piece as above for enrichment. After a sum of three rhythms of vaccination from macerated tissues were conducted followed by consecutive dilution and plating onto differential PT agar home base ( Burr & A ; Schroth, 1977 ) . After incubation at 30A°C for 24h settlement diverseness was judged and selected based pectinase activity on PT medium.
A 0.5 centimeter midst murphy tuber piece was incorporated in each unfertile flask incorporating 5g of unfertile dirt exhaustively assorted with 20ml distilled H2O. The Pcc MTCC1428 strain was grown overnight in alimentary stock at 30A°C, was serially diluted, 1 ml aliquots from each diminishing dilutions were added to unfertile field dirt mixture with potato piece or pectate broth ( Meneley & A ; Stanghellini, 1976 ) . Enrichment on murphy piece and pectate broth was followed as described before by ( Meneley & A ; Stanghellini, 1976 ) , after incubation at 30A°C for 48 hours 1 milliliters aliquots were removed, serially diluted and plated on PT agar medium. All dilutions were replicated three times and experiment repeated 10 times.
Physiological and Biochemical word pictures
After incubation on PT agar medium, the settlement diverseness was judged by settlement morphology and pectolytic activity. For designation of soft putrefaction doing pectobacterium species conventional techniques ( Dye, 1969 ) ; ( Thomson et al. , 1981 ) ; Lelliot & A ; Dickey, 1984 ; Schaad, 1988 ) were used. A sum of 20 six trials used for designation of stray strains ( Table -2 ) . In add-on, the API 20E Kit ( BioMerieux, Marcy l’Etoile, France ) was used for farther verification harmonizing to the maker ‘s protocol. The phenotypic features were compared with earlier studies for different races 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 late ( 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 suitably diluted civilization supernatant was incubated at 30A°C for an hr. After incubation released unsaturated uronic ester derived functions were estimated as described elsewhere ( Nedjma et al. , 2001 ) . One unit of pectate lyase activity is defined as the measure of enzyme needed to increase optical density by 0.1 units per hr at 30EsC, pH-8.0. Protease ( Prt ) activity was determined utilizing 0.5 % ( w/v ) casein in 0.1M Tris-HCl buffer ( pH-7.2 ) with suitably diluted civilization supernatant, and incubated at 40EsC for 30min, followed by appraisal of amino acid released as described elsewhere ( Oceguera-Cervantes et al. , 2007 ) . Tyrosine and Tryptophan were used as aminic acerb criterion. One unit of peptidase activity was defined as the sum of enzyme required to emancipate 1Aµmole of tryptophan per minute at 40EsC. Cellulase ( Cel ) activity was determined utilizing 0.2 % Carboxy methyl cellulose ( CMC ) with 0.05M ethanoate buffer ( pH 5.0 ) and suitably diluted civilization supernatant ( Marichamy & A ; Mattiasson, 2005 ) . The cut downing sugars formed were estimated by DNS reagent ( Miller, 1959 ) . Glucose was used as cut downing sugar criterion. One unit of cellulase activity is defined as the sum of enzyme required to emancipate 1I?mole of glucose per an hr at 50A°C, pH 5.0.
Virulence check was performed utilizing different types of veggies. The external surface disinfection and slice readying of veggies was performed as describe above. Vegetable pieces in gnotobiotic status were inoculated separately with 10I?l of each Pcc strain at 2.1-3.5A-106 cfu ml-1, and infected at 26A°C at 100 % comparative humidness for 48h. Aggressiveness of each strain was analyzed by mensurating macerated tissue as per centum of tissue maceration site, and comparative virulency was calculated as per centum of macerated tissue per entire weight or necrotic country ( Yap et al. , 2004, Yishay et al. , 2008 ) .
Molecular word picture
Designation based on species specific PCR and 16s rDNA sequencing
Genomic DNA extraction from pure civilization of Pectobacterium strains adult overnight in alimentary stock at 30A°C, were performed by utilizing GenElutea„?A Bacterial Genomic DNA Kit ( Sigma-Aldrich Co. , Gillingham, UK ) harmonizing to maker ‘s instructions. All strains were subjected to species specific PCR utilizing primer sets EXPCCF/EXPCCR and INPCCF/INPCCR under conditions antecedently described ( Kang et al. , 2003 ) . Nearlly full length 16s rRNA cistrons were amplified utilizing the 27f and 1492r primers ( Frank et al. , 2008 ) . PCR merchandises were purified by GenElutea„?A 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 utilizing primers sets for REP-PCR, ERIC-PCR, BOX-PCR, and ( GTG ) 5-PCR. PCR elaborations were performed with an initial denaturation measure ( 95A°C, 5 min ) followed by 35 rhythms of denaturation ( 94A°C for 30s ) , tempering ( variable temperature, 1 min ) and extension ( 72A°C for 2 min ) , and a individual concluding extension measure ( 72A°C for 10 min ) . The annealing temperature was 40A°C for REP-PCR and ( GTG ) 5-PCR, and 50A°C for ERIC-PCR and BOX-PCR. The 10 microliters of amplified merchandises were analyzed on 1.5 % agarose gels in 1A- TBE for 70 min at 90 V and gels were stained with ethidium bromide solution ( 0.5 mg ml-1 ) and photographed utilizing familial UV visible radiation at 295 nanometer. Gene Ruler 1 kilobit DNA Ladder ( MBI, Fermentas, Hanover, MD, USA ) was used to find fragment size.
The experimental mean informations were analyzed by one way-ANOVA followed by station hoc analysis utilizing Dunnet ‘s multiple comparision trials utilizing GraphPad Prism 4 package ( San Diego, CA ) . Each physiological or biochemical feature was count as a unit character: positive ( 1 ) or negative ( 0 ) of trial consequences were scored as binary traits. The distance matrix was generated utilizing the Jaccard coefficient and Cluster analysis was done by utilizing the NTSYSpc package ( version 2.0 ; Exeter Software, USA ) utilizing simple fiting harmonizing to the unweighted brace group method with arithmetic norms ( UPGMA ) ( Sutra et al. , 2001 ) . The rep-PCR stria forms of each gel were normalized by AlphaEaseFc 4.0 ( Alpha InnotechA of San Leandro, Calif. ) . Normalized sets 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 correlativity and UPGMA utilizing NTSYSpc package ( ver 2.0 ; Exeter Software, USA ) . For phyletic analysis of 16s rRNA cistron sequences were aligned with RDP ‘s aligner. A distance matrix was generated utilizing the Jukes-Cantor corrected distance theoretical account, utilizing merely alignment theoretical account places, disregarding alliance inserts and with 200 lower limit comparable place. The tree was created utilizing Weighbor with alphabet size 4 and length size 1000 ( Bruno et al. , 2000, Cole et al. , 2007 ) , the downloaded phyletic trees were show and processed utilizing MEGA 4 package ( Tamura et al. , 2007 ) .
Enrichment and isolation of enhanced virulent soft putrefaction Pectobacterium strains
The fresh enrichment technique consisted of murphy tissues as the exclusive beginning of energy was conducted to enrich soft putrefaction doing Pectobacterium bing in low cell denseness and to heighten the virulency belongingss of hibernating cells shacking in dirt. As illustrated in Fig.-1, after each rhythm the diverseness of settlement morphology was decreased, bespeaking enrichment of peculiar phenotypes. The sensitiveness of the enrichment technique with frequence of recovery was about 85 % from dirt incorporating less than five but greater than two cells per gm dry weight dirt, and 40 % with cell population less than two cells per gm dry weight dirt of the enriched population on the macerated murphy pieces obtained was in the scope of 9.57 to 10.43 log10 [ cfu/ml ] raised from an initial entire bacterial population figure of log10 [ cfu/ml ] between 2.35 and 3.12 nowadays in inoculants determined on semi selective media. Anaerobic enrichment with pectate stocks medium showed comparatively 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 putrefaction doing Pectobacterium may be in assorted populations enriched with the pectate medium, which can be eliminated by enrichment on murphy piece which allows growing of lone virulent strains of soft putrefaction doing Pectobacterium.
Word picture and designation of stray soft putrefaction doing Pectobacterium to races degree
A sum of 20 strains of stray from different beginnings during 2006-09, showed heterogeneousness in biochemical and physiological features and categorization in to five different biovars ( Table-1, 2 ) . All strains were identified as soft putrefaction doing Pectobacterium harmonizing to selected phenotypic features, most of the strains were motile, rod shaped, pectolytic, able to decompose murphy pieces, oxidase and amylase non -producers, showed catalase activity, citrate alkalization, gelatin liquefaction, use of D-galacturonic acid, turn at 37A°C and in 5 % NaCl. Biovar 4 contained 12 strains and was most similar to Pcc MTCC1428 in respects to nitrate reductase, use of cellobiose and D-Trehalose, acerb production from glucose and lactose, inability to organize cut downing substances from saccharose. Biovar 1 corresponds to 4 strains and was non similar to any other races of P. carotovorum and Dickeya sp. In relation to Pcc, biovar 1 was non nitrate cut downing but have ability to organize cut downing substances from saccharose ; acid production from D-sorbitol, D-arabitol and I±-methyl glucoside. Whilst biovar 2 correspond to one strain unable to use cellobiose, D-Trehalose ; did non demo acerb production from glucose and lactose in relation to Pcc features. Biovar 3 consist 2 strains, similar to biovar 1 in respects to separate features than Pcc, holding nitrate reductase, use of acetic acid and D-Trehalose, and inability to bring forth acid from I±-methyl glucoside. Biovar 5 contained one strain which was indistinguishable to biovar 2, but showed use of lactic acid and acerb production from glucose ( Table-2 ) . These bacteriological trials were farther 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 ) ( informations non shown ) , besides confirmed with 16s rDNA based designation.
Pathogenecity and virulency determiner enzyme production of Pcc strains
The Pcc strains were categorized in to ten different pathogenic groups based on their ability to do soft putrefaction lesions on trial veggie pieces ( Table1, 3 ) . All 20 strains of Pcc caused tissue maceration as bacterial soft putrefaction lesions on murphy piece with important differences in aggressiveness in footings of mortification country and site per centum of lesions ( Fig.-2 ) . All infective groups showed common host specificity with Daucus carota L. ( carrot ) , Solanum melongena L. ( eggplant ) , LagenariaA sicerariaA ( Molina ) Standl. ( bottle calabash ) , Cucumis sativus L. ( Cucumis sativus ) , Solanum tuberosum L. ( murphy ) . However fluctuation with staying trial vegetable hosts was observed. Eight Pcc strains caused soft putrefaction in all trial veggies and were grouped into infective group I. Whilst infective group VII consisted of three Pcc strains which did non do infection in lone Trichosanthes cucumerinaA L. ( Snake calabash ) . Pathogenic groups II and V contains two strains each, did non do infection on Beta vulgaris L. ( Sugar Beta vulgaris ) tuber piece, but differed with infection caused on Lpomoea batatas L. ( Sweet murphy ) merely by infective group V. The type strain of Pcc was grouped into infective group II ( Table 3 ) . The staying infective groups were represented by individual strains with difference in aggressiveness ( Fig.- 2a, 2b ) .
Furthermore Pcc strains were tested for production of four different works cell wall degrading enzymes ( PG, PNL, Prt, and Cel ) . The civilization supernatants of all strains grown in the presence of pectin, CMC and casein contained all four virulency deciding enzymes activity. Significant differences relative to type strain were observed in enzymes production of stray Pcc strains ( Fig.- 3 ) . Variation in specific activity was correlated to aggressiveness and host specificity of stray Pcc strains. Most aggressive strains showed important differences with type strain in their enzyme production ( p & lt ; 0.01 ) , among them BR1 strain showed higher ( p & lt ; 0.01 ) production of all four tested major virulency determiner enzymes. While PF20 strain was higher in production of pectinolytic enzymes ( PG, at P & lt ; 0.01 ; PL, at P & lt ; 0.05 ) and lower in other works cell wall degrading enzymes ( Prt, at P & lt ; 0.01 ; Cel, non significantly difference ) .Strains CF101 ( infective group IV ) , BRF302 and PR13 ( group V ) , MF8, MF11 and TF203 ( group VIII, IX and X severally ) were showed low fluctuation in host specificity have lower PCWDE productions ( P & lt ; 0.01 ) ( Fig.-2c ) .
Strain degree distinction analysis by rep-PCR and phyletic relatedness
Four different rep-PCRs utilizing two individual oligonucleotide primers, BOX A1R ( BOX-PCR ) and ( GTG ) 5 [ ( GTG ) 5-PCR ] , and two oligonucleotide primers braces, Rep 1R-I/Rep 2-I ( REP-PCR ) and ERIC 1R/ERIC2 ( ERIC-PCR ) , were assessed for their abilities to distinguish between stray strains of the same races of P. carotovorum ( Table-1 ) . With the usage of ( GTG ) 5-PCR and REP-PCR amplified DNA fingerprints runing from 6 to 18 sets, and 14 to 24 sets were found severally. While BOX-PCR and ERIC-PCR produced less than 15 sets with an norm of five and seven sets. All four rep-PCR resulted in merchandises with sizes between 100-10,000 bp. Sensitivity and duplicability of each rep-PCR were confirmed by measuring against replicates of indiscriminately selected two or three strains for indistinguishable finger-prints. With little set strength alteration, indistinguishable stria forms were observed in all the four rep-PCR for each replicates of Pcc strains. Three bunchs were revealed by the ( GTG ) 5-PCR and REP-PCR single profiles, whilst BOX-PCR and ERIC-PCR single profiles revealed 4 and 5 bunchs severally ( informations non shown ) ( Table-1 ) . Cluster analysis of combined finger-print forms of all four rep-PCR revealed 3 bunchs with the similarity coefficient of 67 % , which were subdivided to 2-4 bunchs with similarity coefficient of 76.5 % . The rep-PCR profiles showed high heterogeneousness among stray Pcc strains ( Fig.- 4 ) . All the strains identified as untypical phenotypic group were distributed into bunch 2a correspond to typical strains BR1 and MTCC1428. Conversely, the same infective groups were non indistinguishable in rep-PCR bunchs. Whilst infective group-I correspond non merely to typical but besides to two untypical strains MF106 and CT207, to boot both strains distributed in combined rep-PCR bunch 2a. However staying infective groups were distributed heterogeneously into combined rep-PCR bunchs.
Phylogenetic analysis based on 16s rRNA cistron sequences revealed that all stray strains shared & gt ; 99 % homology with Pcc strains. As shown in Fig.-5 stray strains were grouped together with different type strains and closest hit strains of Pcc, formed separate bunch to other closely related strains of works infective Enterobacteriaceae. However stray strains formed robust subset bunch from other Pcc strains, merely PF20 strain sharing bunch together with Pcc type strains DSM 30168 and LMG17566 ( Fig.-5 ) .
An attack for enrichment of soft putrefaction doing Pectobacterium to extinguish non-virulent bacterial strains during isolation was developed. Although this attack was the alternate manner to be effectual, anaerobiotic and clip disbursement stairss for enrichment to bing enrichment civilization technique for soft putrefaction doing Pectobacterium. The sensitiveness in isolation of soft putrefaction doing Pectobacterium from the dirt with this enrichment technique has been about similar to bing enrichment and isolation techniques ( Meneley & A ; Stanghellini, 1976 ) . The murphy piece used in this survey was provided selective food beginnings for rapid growing of soft putrefaction doing Pectobacterium, which did non enrich other rivals and prevailing dirt bacteriums, which allowed rapid isolation for soft putrefaction doing Pectobacterium. The selective ( PT ) medium for isolation of soft putrefaction doing Pectobacterium after the enrichment measure eliminated the possibility of other pectolytic beings ( soft-rot Pseudomonas spp. and other Fungi ) . However preliminary surveies utilizing crystal violet pectate ( CVP ) medium, direct isolation of soft putrefaction doing Pectobacterium from field dirts or unnaturally infested dirts with low population ( 3 log CFU/g dry weight of dirt ) of soft putrefaction doing Pectobacterium was non possible, which correspond to earlier studies of soft putrefaction doing Pectobacterium isolation ( Cuppels & A ; Kelman, 1974, Meneley & A ; Stanghellini, 1976 ) . ( Liao & A ; Shollenberger, 2004 ) found that three log units fewer acid-injured E. carotovora cells grew on CVP than on non-selective medium, proposing that some formulas of CVP and other selective media may neglect to insulate dormant or stressed bacterial cells from environmental beginnings. Harmonizing to their observations PT medium was more effectual than other selective media. Therefore PT medium was selected for the present surveies.
A polyphasic attack including physiological, biochemical and molecular features, was used to place stray strains of soft putrefaction doing Pectobacterium. A comparing of strains revealed the heterogeneousness within them in phenotypic and biochemical characteristics. The strains were categorized to five different biovars depending upon cardinal belongingss differentiate at races level. Consequences were compared to old studies utilizing good established differential bacteriological trials for separating Pcc with other races 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 non fit all the features antecedently reported for soft putrefaction doing Pectobacterium races. Diversity in phenotypic characteristic among stray Pcc strains from different works hosts within the same geographical parts have been normally reported in the literatures ( Duarte et al. , 2004, Gardan et al. , 2003, Seo et al. , 2003, SA‚awiak et al. , 2009, Yahiaoui-Zaidi et al. , 2003 ) . Surprisingly D. chrysanthemi was absent from the stray strains, although its being in warm climatic conditions and similar geographical parts has been extensively reported ( Mandal & A ; Maiti, 2005, Perombelon, 1992, Thind & A ; Payak, 1985 ) . Significantly stray strains belonging to biovars 1, 2 and 5 did non use D-Trehalose which is a alone belongingss of D. chrysanthemi, whilst all strains did non bring forth indole from tryptophan, a trait considered a separating feature of races of P. carotovorum from D. chrysanthemi ( Sutra et al. , 2001, Yahiaoui-Zaidi et al. , 2003 ) . All strains were identified as Pcc utilizing species specific primers, and were classified as Pcc by 16s rDNA sequence homology with the type strain.
The infective diverseness of stray Pcc strains was analyzed utilizing a assortment of works hosts. Variability in cardinal characteristics of aggressiveness and host specificity was observed among stray Pcc strains. Based on pathogenicity ten infective groups were recognized. Pcc is considered a broad-host-range pathogen relation to other races of P. carotovorum. Infective specialisation for different works hosts and variableness in pathogenicity and host scope have been noted under experimental conditions among soft putrefactions doing Pectobacterium ( Dickey, 1979, Ma et al. , 2007, Seo et al. , 2002, El-Hendawy et al. , 2002 ) , and matching observations were obtained from stray Pcc strains in this survey. Recently, as per cataloging reappraisal by ( Ma et al. , 2007 ) of the host scope of soft putrefaction doing Pectobacterium has been produced, nevertheless similar studies of host specificity of Pcc towards S. molongena L. ( eggplant ) , L. batatas L. ( sweet murphy ) , T. cucumerinaA L. ( snake calabash ) and L.A sicerariaA ( Molina ) Standl. ( bottle calabash ) have non been described. Several efforts have been made for isolation of chaff putrefaction ( similar diagnostic disease ) doing Pectobacterium from Indian Fieldss and designation of their host scope features. However categorization of bacteria doing chaff putrefaction in corn was non clarified between P. carotovorum and Dickeya spp. ( Anilkumar & A ; Chakravarti, 1971, Thind & A ; Payak, 1985, Ragarajan & A ; Chakaravarati, 1970 ) . Unfortunately, it is impossible to obtain civilizations of strains from these surveies and hence, it has non been possible to correlate the Pcc strains from the current survey with other Pectobacterium strains of Indian beginning for host scope specificity.
Plant cell wall degrading enzymes ( PCWDE ) play a cardinal function in induction of soft putrefaction mortification symptoms in host ( Hugouvieux-Cotte-Pattat et al. , 1996 ) . Production degrees of PCWDE was analyzed for each isolate to correlate fluctuation in pathogenicity of the strains towards works hosts. In vitro status isolated Pcc strains produced PG and PL during late log stage, while Cel and Prt produced during stationary stage of the growing of bacteriums, besides shows that pectinase higher bring forthing stray Pcc strains had major virulency consequence against tested works hosts. The production of pectic enzymes ( PL, PG ) are major deadly determiners of soft putrefaction doing Pectobacterium produced in initial phase of incubation with pectin substrates which are majorly present in in-between gill, among them PG produced early than PL, which facilitate farther manner 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 surveies have already correlated pathogenicity and pectinase activities as critical function in works tissue maceration during infection caused by other micro-organisms ( Johansson, 1988, Mwenje & A ; Ride, 1997, Yong Jian et al. , 2009, Barker & A ; Walker, 1962 ) .
All 20 strains of Pcc have fluctuation in their virulency, biochemical and physiological belongingss. In this survey, utilizing rep-PCR, Pcc strains were grouped in to three big bunchs, whilst ERIC-PCR grouped them into five bunchs. The ERIC-PCR analysis has been already demonstrated for variableness with in Pcc strains earlier ( Toth et al. , 1999 ) . However several surveies showed the being of high degree of familial diverseness among Pcc strains, examined by usage of assorted molecular techniques, such as PCR-restriction fragment length polymorphism ( RFLP ) ( Darrasse et al. , 1994, Waleron et al. , 2002 ) , Random amplified polymorphous DNA ( RAPD ) analysis ( Parent et al. , 1996 ) , DNA-DNA hybridisation ( Gardan et al. , 2003 ) , Amplified Fragment Length Polymorphism ( AFLP ) fingerprinting ( Avrova et al. , 2002 ) , Pulsed-field gel cataphoresis ( PFGE ) fingerprinting ( Yap et al. , 2004 ) , insistent 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 old surveies do non bespeak functional differences attributed to familial diverseness. Although PFGE and rep-PCR have about equal deciding power for distinction at races and strain degree, the rep-PCR DNA fingerprinting technique is simple and rapid, with less labour, clip and cost, can be performed with standard equipment with high throughput in comparing to PFGE ( Olive & A ; Bean, 1999, Ishii & A ; Sadowsky, 2009 ) . ( Norman et al. , 2003 ) successfully used rep-PCR fingerprinting for strain degree distinction of Pcc population isolated from a nursery keeping pool and lake H2O. In add-on to REP-PCR, ERIC-PCR and BOX-PCR, the present surveies demonstrates the usage of ( GTG ) 5-PCR for the first clip to distinguish Pcc strains isolated from field dirt and morbid veggies and fruits. The ( GTG ) 5-PCR has the highest prejudiced power and rate of right categorization comparison to other three rep-PCR ( REP-PCR, ERIC-PCR and BOX-PCR ) , and has been successfully utilised for molecular typewriting 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 infective group were grouped in to different bunchs of combined rep-PCR analysis, reflecting high degree of familial diverseness was present among the stray Pcc strains with regard to their beginning of isolation and twelvemonth. The consequences of phyletic analysis of 16S rDNA sequences of stray Pcc strains suggest heterogeneousness to certain extent was observed. Fifteen strains formed a separate subset of bunch from the type strains of Pcc and reported strains from Asiatic states, four other strains sharing common bunch with these Asiatic strains. Interestingly PF20 strain grouped with type discolorations of Pcc with in a distinguishable bunch, which correlate with bunch 3b of combined rep-PCR analysis. The most deadly strains BR1 and PF20 were clustered in to two different phylogenetic clades, which is in contrast to the phyletic association with pathogenesis traits found. The mention strain Pcc MTCC 1428 ( ATCC 15713 ) besides was clustered in distinguishable clade even though it showed indistinguishable phenotypic and virulency features with more than half of the stray strains. Different mention strains of works infective genera of Enterobacteriaceae household were used to guarantee that hardiness of subset bunch of stray strains were so alone from mention and Asiatic beginning strains of Pcc. Conversely, usage of advanced molecular techniques has elucidated a broad diverseness within Pcc from different host workss beginnings 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 surveies strongly back up the diverseness of Pcc was found independent of host scope specificity, geographical locations or works beginnings. At last the virulency and phenotypic belongingss were non associated with genotypic word picture such as rep-PCR and 16S rDNA sequence analysis. The phenotypic, infective and familial diverseness was found within Pcc strains from India as an initial measure in understanding the spacial distribution and construction of plant-pathogen population.