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India is ranked worldwide as a first and second largest producing country in production of fruits and vegetables respectively. The majority of vegetables and fruits are classically grown 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 cultivation in the field. Poor handling practices during cultivation as well as post harvest conditions makes agricultural production vulnerable to microbial diseases.
Bacterial soft-rot caused by Erwinia spp. (reclassified as Pectobacterium spp.) has been considered as one of the most recurrent diseases observed in variety of vegetables and fruits worldwide and cause great economic loss of crops . The genus Pectobacterium consist number of species within the family of Enterobacteriaceae. According to comprehensive reviews the most economically important soft rot erwinia 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 like soft rot, blackleg and aerial stem rot in wide variety of vegetables and fruits, and other commercially important crops. 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. 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. They are genetically diverse as is evident from polymorphisms in the pel and recA genes as well as in rrn, the ribosomal gene cluster. 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. 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. Phenotypic and genotypic variability have been reported so far among Pcc strains isolated in Asian countries such as Korea, Japan, Thailand, and China. There are very few reports showing existence of soft rot causing Pectobacterium strains in South-Asian region mainly in India covering their variability and pathogenesis in a wide variety of plant species. Most of the cases of infections caused by soft rot causing erwinia in India were diagnosed as top rot and stalk rot till date.
The primary virulence factors of soft rot erwinias are extracellular plant cell wall-degrading enzymes that comprises pectic enzymes viz. pectate lyase (PL), pectin lyase (PNL), and polygalaturonase (PG), are directly involved in plant tissue maceration by digesting the pectin that cement plant cells together, while proteases (Prt) and cellulases (Cel) that contribute to virulence by augmenting the activity of the pectic enzymes. Pathogenicity also involves eluding plant defense mechanism and invasion of plant tissue. Previous characterization of the soft rot erwinia as opportunistic pathogens is being replaced by a realization that this group of bacteria exhibits a sophisticated repertoire of pathogenicity and virulence genes and regulators. Exoenzymes production, motility and other pathogenic factors are under tight genetic transcriptional and postranscriptional control by regulatory mechanisms which includes cell density dependent quorum sensing and response to plant signals, attest to a highly specialized pathogen. The fact that production of plant cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent manner may explain the occurrence of pectolytic erwinia in asymptomatic plant tissues. Transgenic plants expressing bacterial quorum sensing signal molecules modulate this sensory system and exhibit resistance to soft rot infection. The pectolytic erwinias, being significant plant pathogens that are neither of quarantine concern nor a human health hazard while readily isolated from field sources, make an ideal model for investigating the genetic basis of plant pathogenesis and environmental fitness. The paucity of information regarding distribution of the soft rot causing erwinia prevalent in vegetable growing fields in India, functional properties of virulence determinant enzymes, and survival during control measures has prompted the present studies to aid future control strategies before soft rot disease arise from soil.
The thesis deals with the isolation and characterization of soft rot Erwinia sp., characterization of virulence determinant enzymes, and its survival behavior against control measures followed by its biocontrol.
A novel enrichment technique was developed consisting of potato tissues as the sole source of energy was conducted to enrich soft rot causing erwinia existing in low cell density and to enhance the virulence properties of dormant cells residing in soil. The sensitivity of the enrichment technique with frequency of recovery from soil was higher. Anaerobic enrichment with pectate broth medium showed similar values as aerobic enrichment of enriched bacterial population. Since possibility of both virulent and nonvirulent strains of soft rot causing erwinias may exist in mixed populations enriched with the pectate medium in anaerobic condition, which can be eliminated by enrichment on potato slice was done which allows growth of only virulent strains of soft rot causing erwinias. A total of twenty strains isolated from different agricultural soils and diseased plant samples during the period 2006-09, showed heterogeneity in biochemical and physiological characteristics that lid to classification into five different biovars. These bacteriological tests were further 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 also confirmed with 16s rDNA based identification. The sequences were submitted to NCBI-Genebank with the accession no.: FJ187821, HM150649-HM150665, HM179990, HM179991. Phylogenetic analysis based on 16s rRNA gene sequences revealed that all isolated strains shared >99% homology with Pcc type strains were grouped together with different type strains and closest hit strains of Pcc and formed separate cluster to other closely related strains of plant pathogenic Enterobacteriaceae. The Pcc strains were categorized into ten different pathogenic groups based on their ability to cause soft rot lesions on ten different type of vegetables slices. All twenty strains of Pcc showed significant differences in aggressiveness in terms of necrosis area and site percentage of lesions. Furthermore Pcc strains were tested for production of four different plant cell wall degrading enzymes (PG, PL, Prt, and Cel). The culture supernatants of all strains grown in the presence of pectin, CMC and casein showed all four virulence determinant enzymes activities. Significant differences relative to type strain of Pcc were observed in exoenzymes production by isolated Pcc strains. 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 Pcc BR1 strain showed higher (p<0.01) production of all four tested major virulence determinant enzymes. Assessment of subspecies level differentiation among isolated Pcc strains was performed by utilizing 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). Three clusters were revealed by the (GTG)5-PCR and REP-PCR individual profiles, whilst BOX-PCR and ERIC-PCR individual profiles revealed four and five clusters respectively. Cluster analysis of combined fingerprint patterns of all four rep-PCR revealed three clusters with the similarity coefficient of 67%, which were subdivided to two-four clusters with similarity coefficient of 76.5%. The rep-PCR profiles showed high heterogeneity amongst isolated Pcc strains.
Virulent determinant pectolytic enzymes of soft rot erwinia plays significant role during pathogenesis. The temperature-dependent production of these pectolytic enzymes and development of soft rot disease are well known in erwinia. Purification of polygalacturonase (PG) and pectate lyase (PL) from selected isolated strain of P. carotovorum subsp. carotovorum (Pcc) BR1 to their homogeneity level were done using ion-exchange chromatography. With an emphasis on the thermal behavior with different pHs and salts, the kinetic and thermodynamic parameters of the purified PG and PL from PccBR1 were studied. These enzymes are important determinants of pathogenesis and also significant in the context of burgeoning biotechnological applications. The kinetic and thermodynamic parameters for polygalacturonic acid hydrolysis by the purified PG and PL indicate both have high affinity for substrate and are catalytically efficient. The low enthalpy (ΔH*) and low entropy (ΔS*) values of the purified PG and PL showed that the formation of the transition state or activated complex with less disorder between the enzyme-substrate was very efficient. Futhermore, the free energy for the activation of the substrate binding (ΔG*E-S) and free energy for the formation of the activation complex (ΔG*E-T) reconfirmed both enzymes had a high affinity towards substrates for hydrolysis and its spontaneous conversion into products. The purified PG was stable within a temperature range of 20-50°C and was deactivated at 60°C and 70°C, while PL was stable within a temperature range of 20-60 °C and was deactivated at 70°C and 80°C. The thermodynamic parameters (ΔH*, ΔG*, ΔS*) for the irreversible inactivation of the PG and PL at different temperatures (30-70°C) were determined, where the effectiveness of various salts and different pHs for the thermal stability of the PG and PL were also characterized. Based on thermodynamic parameters the neutral pH condition and MgCl2 favors thermal stability of PG, while alkaline pH condition and CaCl2 favors thermal stability of PL.
To reduce the infection caused by soft rot erwinia chemical control treatment is usually applied during pre-planting, cultivation and postharvest conditions, however the chemical may not always penetrate outer layer of infected plant parts and consequently are ineffective. Antibiosis approach showed effective treatment for soft rot erwinia. But development of resistance to streptomycin has been reported for other species of Erwinia and the potential for tolerance or resistance in Pcc cannot be ignored here. Failure of control measures due to survival strategies of pathogenic microbes is well known. Presently, no efficient chemical treatment is available for control of this bacterial soft rot disease. The major limiting factors of controlling agent's efficacy include potency and swiftness of microbial attachment and growth during infection, low penetration in infection sites, internalization of microbial contaminants within plant tissues, biofilm formation etc. Exhibition of injury and repair in soft rot erwinia, may give a direct approach to assess safety measures to prevent disease. During control treatments the surviving population of pathogenic bacteria may have reduced cultivability either due to cell stress, cell injury or entry into altered physiological cell state.
With emphasis on effectiveness of control measures for soft rot erwinia and to reduce virulence determinant enzymes production with major cell damage, survival behavior with respect to cell physiology were studied. Minimal inhibitory concentrations (MIC) of four short chain fatty acid (SCFA) (formic acid, acetic acid, propionic acid and aspartic acid), two medium chain fatty acid (MCFA) (caproic and caprylic acid), three organic salts (potassium sorbate, sodium benzoate and sodium octanoate) and two metal compounds (copper sulphate and alluminium chloride) were determined using twenty isolated 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 virulence determinant enzymes, mainly pectin lyase (PnlH) destined for the outer membrane are synthesized with a signal sequence that is cleaved, either by the signal peptidase LepB for integral outer membrane proteins or by LspA for lipoproteins (lpp), when they cross the cytoplasmic membrane. Also the Tat (twin arginine translocation) like pathway is required for targeting process. By using transcriptional fusions between the strong promoters of the outer membrane protein targeting genes of P. carotovorum subsp. carotovorum, tatA and lpp was fused separately with luxCDABE genes, it was shown that MCFA decreased the expression of tatA, involved in translocation pathway required to target virulence determinant enzymes to the outer membrane when they expressed. Expression of lpp, a strong promoter of the gene for the outer membrane lipoprotein, was significantly lower when cells were grown in nutrient broth supplemented with sub-MIC concentrations of caproic or caprylic acid (2.5 mM), potassium sorbate (20mM) and acetic acid (5mM). While the expression of tatA were significantly lower as compared to lpp, when cells were grown in nutrient broth containing sub-MIC concentrations of acetic acid (5 mM). Antimicrobial actions of weak acids on Pcc vary with concentration and type of weak acids and metal sanitizers used. The cell injury was higher with all organic salts and metal compounds. At lethal concentration of potassium sorbate showed significantly higher cell survival and log injury, while metal compounds showed higher cell injury without cell survival. Evidently P. carotovorum subsp. carotovorum entered into altered cell physiological state 'viable-but-non culturable (VBNC)' as a survival during low sub-lethal dose of potassium sorbate at pH-4.
Quorum sensing (QS) signaling molecule N-Acyl-homoserine lactone (AHL) plays a key role in expression and regulation of virulence genes, production exoenzymes, secondary metabolites and carbapenem antibiotic productions in soft rot erwinia. Targeting the QS regulatory elements to develop biocontrol strategies for soft rot causing P. carotovorum subsp. carotovorum is therefore a potential approach. In present study, bacteria were isolated from turf and agricultural soils, and screened for ability to utilize AHLs produced by P. carotovorum subsp. carotovorum BR1 (PccBR1) as a sole energy source for growth. Twenty bacterial isolates which were able to degrade various type of synthetic and natural AHLs were selected for further studies. According to biochemical characteristics and 16s rDNA sequences, isolates belong to the genera Ochrobacterum, Delftia, Sphingobacterium, Klebsiella, Stenotrophomonas, Brevundimonas, and Pseudomonas. Three isolates showing strong AHL-inactivating enzyme activity were selected for further competition analysis with PccBR1. The selected three isolates did not seem to interfere with the normal growth of PccBR1; rather, potentially abolished accumulation of AHL molecules and production of virulence determinant enzymes. In vitro, selected three isolates significantly decreased the incidence of PccBR1 infection and necrosis symptom on potato slices.. The biocontrol efficiency is correlated with the ability of bacterial isolates to produce AHL-inactiavting activity. Analysis of AHL degradation pattern to reveal the type of AHL- inactivating enzyme is being studied.
In summary, using novel enrichment technique isolated twenty strains of soft rot causing P. carotovorum subsp. carotovorum, showed genetic variability among themselves. However this was not correlated with host range and aggressiveness. Kinetic properties and thermal behavior with different pH and various salts of virulent determinants pectolytic enzymes of P. carotovorum subsp. carotovorum BR1 were determined suggesting alternative for specific industrial application. Induction of viable-but-non culturable state in P. carotovorum subsp. carotovorum was observed as survival strategy against potassium sorbate at low pH. AHLs degrading isolates can potentially serve to control P. carotovorum subsp. carotovorum causing soft rot disease in various plant hosts.
Vadodara ________________________ ________________________
December 30, 2010 (Maisuria Vimal B.) (Dr. Anuradha S. Nerurkar)