India is ranked worldwide as a first and 2nd largest bring forthing state in production of fruits and veggies severally. The bulk of veggies and fruits are classically grown 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 cultivation in the field. Poor managing patterns during cultivation every bit good as station crop conditions makes agricultural production vulnerable to microbic diseases.
Bacterial soft-rot caused by Erwinia spp. ( reclassified as Pectobacterium spp. ) has been considered as one of the most perennial diseases observed in assortment of veggies and fruits worldwide and do great economic loss of harvests. The genus Pectobacterium consist figure of species within the household of Enterobacteriaceae. Harmonizing to comprehensive reappraisals the most economically of import soft putrefaction erwinia 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 like soft putrefaction, scab and aerial root putrefaction in broad assortment of veggies and fruits, and other commercially of import harvests. 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. 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. They are genetically diverse as is apparent from polymorphisms in the pixel and recA cistrons every bit good as in rrn, the ribosomal cistron bunch. 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. 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. Phenotypic and genotypic variableness have been reported so far among Pcc strains isolated in Asiatic states such as Korea, Japan, Thailand, and China. There are really few studies demoing being of soft putrefaction doing Pectobacterium strains in South-Asian part chiefly in India covering their variableness and pathogenesis in a broad assortment of works species. Most of the instances of infections caused by soft putrefaction doing erwinia in India were diagnosed as top putrefaction and chaff putrefaction boulder clay day of the month.
The primary virulency factors of soft putrefaction erwinias are extracellular works cell wall-degrading enzymes that comprises pectic enzymes viz. pectate lyase ( PL ) , pectin lyase ( PNL ) , and polygalaturonase ( PG ) , are straight involved in works tissue maceration by digesting the pectin that cement works cells together, while peptidases ( Prt ) and cellulases ( Cel ) that contribute to virulence by augmenting the activity of the pectic enzymes. Pathogenicity besides involves evading works defence mechanism and invasion of works tissue. Previous word picture of the soft putrefaction erwinia as timeserving pathogens is being replaced by a realisation that this group of bacteriums exhibits a sophisticated repertory of pathogenicity and virulency cistrons and regulators. Exoenzymes production, motility and other infective factors are under tight familial transcriptional and postranscriptional control by regulative mechanisms which includes cell denseness dependent quorum feeling and response to works signals, attest to a extremely specialised pathogen. The fact that production of works cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent mode may explicate the happening of pectolytic erwinia in symptomless works tissues. Transgenic workss showing bacterial quorum feeling signal molecules modulate this centripetal system and exhibit opposition to soft putrefaction infection. The pectolytic erwinias, being important works pathogens that are neither of quarantine concern nor a human wellness jeopardy while readily isolated from field beginnings, make an ideal theoretical account for look intoing the familial footing of works pathogenesis and environmental fittingness. The dearth of information sing distribution of the soft putrefaction doing erwinia prevalent in vegetable turning Fieldss in India, functional belongingss of virulency determiner enzymes, and endurance during control steps has prompted the present surveies to help future control schemes before soft putrefaction disease arise from dirt.
The thesis trades with the isolation and word picture of soft putrefaction Erwinia sp. , word picture of virulency determiner enzymes, and its survival behaviour against control steps followed by its biocontrol.
A fresh enrichment technique was developed dwelling of murphy tissues as the exclusive beginning of energy was conducted to enrich soft putrefaction doing erwinia bing in low cell denseness and to heighten the virulency belongingss of hibernating cells shacking in dirt. The sensitiveness of the enrichment technique with frequence of recovery from dirt was higher. Anaerobic enrichment with pectate stocks medium showed similar values as aerophilic enrichment of enriched bacterial population. Since possibility of both virulent and nonvirulent strains of soft putrefaction doing erwinias may be in assorted populations enriched with the pectate medium in anaerobiotic status, which can be eliminated by enrichment on murphy piece was done which allows growing of lone virulent strains of soft putrefaction doing erwinias. A sum of 20 strains isolated from different agricultural dirts and diseased works samples during the period 2006-09, showed heterogeneousness in biochemical and physiological features that lid to classification into five different biovars. These bacteriological trials were farther validated with API 20E kit. All strains were identified as P. carotovorum subsp. carotovorum showed 550bp DNA fragment with species specific primers ( EXPCC sets ) , and 400bp DNA fragment with Nested-PCR ( INPCC primer sets ) and were besides confirmed with 16s rDNA based designation. The sequences were submitted to NCBI-Genebank with the accession no. : FJ187821, HM150649-HM150665, HM179990, HM179991. Phylogenetic analysis based on 16s rRNA cistron sequences revealed that all stray strains shared & amp ; gt ; 99 % homology with Pcc type strains were grouped together with different type strains and closest hit strains of Pcc and formed separate bunch to other closely related strains of works infective Enterobacteriaceae. The Pcc strains were categorized into 10 different pathogenic groups based on their ability to do soft putrefaction lesions on 10 different type of veggies pieces. All 20 strains of Pcc showed important differences in aggressiveness in footings of mortification country and site per centum of lesions. Furthermore Pcc strains were tested for production of four different works cell wall degrading enzymes ( PG, PL, Prt, and Cel ) . The civilization supernatants of all strains grown in the presence of pectin, CMC and casein showed all four virulency determiner enzymes activities. Significant differences relative to type strain of Pcc were observed in exoenzymes production by stray Pcc strains. 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 & A ; lt ; 0.01 ) , among them Pcc BR1 strain showed higher ( p & A ; lt ; 0.01 ) production of all four tested major virulency determiner enzymes. Appraisal of races flat distinction among stray Pcc strains was performed by using 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 ) . Three bunchs were revealed by the ( GTG ) 5-PCR and REP-PCR single profiles, whilst BOX-PCR and ERIC-PCR single profiles revealed four and five bunchs severally. Cluster analysis of combined fingerprint forms of all four rep-PCR revealed three bunchs with the similarity coefficient of 67 % , which were subdivided to two-four bunchs with similarity coefficient of 76.5 % . The rep-PCR profiles showed high heterogeneousness amongst isolated Pcc strains.
Virulent deciding pectolytic enzymes of soft putrefaction erwinia plays important function during pathogenesis. The temperature-dependent production of these pectolytic enzymes and development of soft putrefaction disease are good known in erwinia. Purification of polygalacturonase ( PG ) and pectate lyase ( PL ) from selected stray strain of P. carotovorum subsp. carotovorum ( Pcc ) BR1 to their homogeneousness degree were done utilizing ion-exchange chromatography. With an accent on the thermic behaviour with different United States Public Health Service and salts, the kinetic and thermodynamic parametric quantities of the purified PG and PL from PccBR1 were studied. These enzymes are of import determiners of pathogenesis and besides important in the context of burgeoning biotechnological applications. The kinetic and thermodynamic parametric quantities for polygalacturonic acid hydrolysis by the purified PG and PL indicate both have high affinity for substrate and are catalytically efficient. The low heat content ( ?H* ) and low information ( ?S* ) values of the purified PG and PL showed that the formation of the passage province or activated complex with less upset between the enzyme-substrate was really efficient. Futhermore, the free energy for the activation of the substrate binding ( ?G*E-S ) and free energy for the formation of the activation composite ( ?G*E-T ) reconfirmed both enzymes had a high affinity towards substrates for hydrolysis and its self-generated transition into merchandises. The purified PG was stable within a temperature scope of 20-50 & A ; deg ; C and was deactivated at 60 & A ; deg ; C and 70 & A ; deg ; C, while PL was stable within a temperature scope of 20-60 & A ; deg ; C and was deactivated at 70 & A ; deg ; C and 80 & A ; deg ; C. The thermodynamic parametric quantities ( ?H* , ?G* , ?S* ) for the irreversible inactivation of the PG and PL at different temperatures ( 30-70 & A ; deg ; C ) were determined, where the effectivity of assorted salts and different United States Public Health Service for the thermic stableness of the PG and PL were besides characterized. Based on thermodynamic parametric quantities the impersonal pH status and MgCl2 favours thermic stableness of PG, while alkalic pH status and CaCl2 favours thermic stableness of PL.
To cut down the infection caused by soft putrefaction erwinia chemical control intervention is normally applied during pre-planting, cultivation and postharvest conditions, nevertheless the chemical may non ever penetrate outer bed of septic works parts and accordingly are uneffective. Antibiosis attack showed effectual intervention for soft putrefaction erwinia. But development of opposition to streptomycin has been reported for other species of Erwinia and the potency for tolerance or opposition in Pcc can non be ignored here. Failure of control steps due to survival schemes of infective bug is good known. Presently, no efficient chemical intervention is available for control of this bacterial soft putrefaction disease. The major restricting factors of commanding agent ‘s efficaciousness include authority and speed of microbic fond regard and growing during infection, low incursion in infection sites, internalisation of microbic contaminations within works tissues, biofilm formation etc. Exhibition of hurt and fix in soft putrefaction erwinia, may give a direct attack to measure safety steps to forestall disease. During control treatments the lasting population of infective bacteriums may hold reduced cultivability either due to cell emphasis, cell hurt or entry into altered physiological cell province.
With accent on effectivity of control steps for soft putrefaction erwinia and to cut down virulency determiner enzymes production with major cell harm, survival behaviour with regard to cell physiology were studied. Minimal repressive concentrations ( MIC ) of four short concatenation fatty acid ( SCFA ) ( formic acid, acetic acid, propionic acid and aspartic acid ) , two medium concatenation fatty acid ( MCFA ) ( caproic and caprylic acid ) , three organic salts ( K sorbate, Na benzoate and Na octanoate ) and two metal compounds ( Cu sulfate and alluminium chloride ) were determined utilizing twenty stray strains of P. carotovorum subsp. carotovorum. MIC values were increased at increasing pH-values and were two to six times lower for MCFA than for SCFA. The virulency determiner enzymes, chiefly pectin lyase ( PnlH ) destined for the outer membrane are synthesized with a signal sequence that is cleaved, either by the signal protease LepB for built-in outer membrane proteins or by LspA for lipoproteins ( lpp ) , when they cross the cytoplasmatic membrane. Besides the Tat ( duplicate arginine translocation ) like tract is required for aiming procedure. By utilizing transcriptional mergers between the strong boosters of the outer membrane protein aiming cistrons of P. carotovorum subsp. carotovorum, tatA and lpp was fused individually with luxCDABE cistrons, it was shown that MCFA decreased the look of tatA, involved in translocation tract required to aim virulency deciding enzymes to the outer membrane when they expressed. Expression of lpp, a strong booster of the cistron for the outer membrane lipoprotein, was significantly lower when cells were grown in alimentary stock supplemented with sub-MIC concentrations of caproic or caprylic acid ( 2.5 millimeter ) , potassium sorbate ( 20mM ) and acetic acid ( 5mM ) . While the look of tatA were significantly lower as compared to lpp, when cells were grown in alimentary stock incorporating sub-MIC concentrations of acetic acid ( 5 millimeter ) . Antimicrobial actions of weak acids on Pcc vary with concentration and type of weak acids and metal sanitizers used. The cell hurt was higher with all organic salts and metal compounds. At deadly concentration of K sorbate showed significantly higher cell endurance and log hurt, while metal compounds showed higher cell hurt without cell endurance. Obviously P. carotovorum subsp. carotovorum entered into altered cell physiological province ‘viable-but-non culturable ( VBNC ) ‘ as a endurance during low sub-lethal dosage of K sorbate at pH-4.
Quorum detection ( QS ) signaling molecule N-Acyl-homoserine lactone ( AHL ) plays a cardinal function in look and ordinance of virulency cistrons, production exoenzymes, secondary metabolites and carbapenem antibiotic productions in soft putrefaction erwinia. Targeting the QS regulative elements to develop biocontrol schemes for soft putrefaction doing P. carotovorum subsp. carotovorum is hence a possible attack. In present survey, bacteriums were isolated from sod and agricultural dirts, and screened for ability to use AHLs produced by P. carotovorum subsp. carotovorum BR1 ( PccBR1 ) as a exclusive energy beginning for growing. Twenty bacterial isolates which were able to degrade assorted type of man-made and natural AHLs were selected for farther surveies. Harmonizing to biochemical features and 16s rDNA sequences, isolates belong to the genera Ochrobacterum, Delftia, Sphingobacterium, Klebsiella, Stenotrophomonas, Brevundimonas, and Pseudomonas. Three isolates demoing strong AHL-inactivating enzyme activity were selected for farther competition analysis with PccBR1. The selected three isolates did non look to interfere with the normal growing of PccBR1 ; instead, potentially abolished accretion of AHL molecules and production of virulency determiner enzymes. In vitro, selected three isolates significantly decreased the incidence of PccBR1 infection and mortification symptom on murphy slices.. The biocontrol efficiency is correlated with the ability of bacterial isolates to bring forth AHL-inactiavting activity. Analysis of AHL debasement form to uncover the type of AHL- demobilizing enzyme is being studied.
In drumhead, utilizing fresh enrichment technique isolated 20 strains of soft putrefaction doing P. carotovorum subsp. carotovorum, showed familial variableness among themselves. However this was non correlated with host scope and aggressiveness. Kinetic belongingss and thermic behaviour with different pH and assorted salts of virulent determiners pectolytic enzymes of P. carotovorum subsp. carotovorum BR1 were determined proposing option for specific industrial application. Initiation of viable-but-non culturable province in P. carotovorum subsp. carotovorum was observed as survival scheme against K sorbate at low pH. AHLs degrading isolates can potentially function to command P. carotovorum subsp. carotovorum doing soft putrefaction disease in assorted works hosts.
Vadodara ________________________ ________________________
December 30, 2010 ( Maisuria Vimal B. ) ( Dr. Anuradha S. Nerurkar )