Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains

Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.The genus Pectobacterium (formerly Erwinia) contains both narrow- and broad-host-range bacterial plant pathogens that cause soft rot, stem rot, wilt, and blackleg in species belonging to over 35% of plant orders (20). Four Pectobacterium species have been described: Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium carotovorum, and Pectobacterium wasabiae (9). The recently described organism P. carotovorum subsp. brasiliensis is genetically distinct from previously described Pectobacterium taxa; approximately 82% of its genes are shared with P. atrosepticum, and 84% of its genes are shared with P. carotovorum subsp. carotovorum, while 13% of its genes are found in neither P. atrosepticum nor P. carotovorum subsp. carotovorum (7, 10, 20). To date, only P. carotovorum subsp. carotovorum and P. atrosepticum have been reported to occur in the same field (14, 21). P. carotovorum subsp. carotovorum is found worldwide, and P. atrosepticum is found in cool climates; while P. carotovorum subsp. brasiliensis has been found only in Brazil, Israel, and the United States, it is likely to have a wider distribution (20). Compared to the ecology and genetics of P. carotovorum subsp. carotovorum and P. atrosepticum, little is known about the ecology and genetics of P. betavasculorum, P. wasabiae, or P. carotovorum subsp. brasiliensis.Pectobacterium strains isolated from potato are diverse based on serology, genome structure, and fatty acid composition (5, 35). Previous epidemiological studies of pectolytic Enterobacteriaceae were complicated by the diversity of this group and the lack of tools capable of placing all isolates into clades. For example, Gross et al. (14) were unable to classify over 50% of Pectobacterium isolates obtained from potato, and Pitman et al. (23) were unable to type 13% of their isolates. Novel PCR-based methods potentially capable of classifying all Pectobacterium isolates have been described, but they were developed prior to the recognition of P. carotovorum subsp. brasiliensis (1, 34).The main virulence determinants of Pectobacterium are the pectolytic enzymes secreted through the type II secretion system. Although these enzymes are required for development of symptoms, many other virulence genes have been shown to contribute to Pectobacterium pathogenicity, including the type III secretion system (T3SS) genes, the cfa gene cluster, and the type IV secretion system genes (3, 15, 19). Recent genomic analysis showed that some of these gene clusters, such as the cfa and type IV secretion system cluster genes, as well as genes important for interactions with insects, are present in only some Pectobacterium species (10). Thus, Pectobacterium species appear to use different genetic tools to overcome plant host barriers and to interact with insect vectors.Many gram-negative pathogenic bacteria secrete virulence proteins, known as effectors, through the T3SS into host cells. Once inside host cells, the effectors manipulate host defenses and promote bacterial growth (13). Unlike many other gram-negative plant pathogens, Pectobacterium does not require the T3SS for pathogenicity. Rather, this secretion system makes a small, but measurable, contribution to the early stages of P. carotovorum growth in leaves of the model plant Arabidopsis thaliana (26) and contributes to the virulence of P. atrosepticum on potato (15). Recently, we isolated Pectobacterium strains that lack the T3SS from potatoes and also found P. wasabiae and P. carotovorum subsp. brasiliensis on potatoes in Wisconsin (35). The first goal of this study was to determine if P. wasabiae and P. carotovorum subsp. brasiliensis are common in agricultural fields or if soft rot disease is typically caused by P. carotovorum subsp. carotovorum and P. atrosepticum, which have been the focus of nearly all previous studies of potato soft rot, stem rot, and blackleg disease. Second, since we recently isolated a strain lacking the T3SS (35), we also aimed to determine if strains lacking the T3SS are common in infected potatoes and if these strains tend to be less virulent on potato stems and tubers than strains encoding a T3SS.     

Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Comparative Genome Analysis of Campylobacter fetus Subspecies Revealed Horizontally Acquired Genetic Elements Important for Virulence and Niche Specificity

Campylobacter fetus are important animal and human pathogens and the two major subspecies differ strikingly in pathogenicity. C. fetus subsp. venerealis is highly niche-adapted, mainly infecting the genital tract of cattle. C. fetus subsp. fetus has a wider host-range, colonizing the genital- and intestinal-tract of animals and humans. We report the complete genomic sequence of C. fetus subsp. venerealis 84-112 and comparisons to the genome of C. fetus subsp. fetus 82-40. Functional analysis of genes predicted to be involved in C. fetus virulence was performed. The two subspecies are highly syntenic with 92% sequence identity but C. fetus subsp. venerealis has a larger genome and an extra-chromosomal element. Aside from apparent gene transfer agents and hypothetical proteins, the unique genes in both subspecies comprise two known functional groups: lipopolysaccharide production, and type IV secretion machineries. Analyses of lipopolysaccharide-biosynthesis genes in C. fetus isolates showed linkage to particular pathotypes, and mutational inactivation demonstrated their roles in regulating virulence and host range. The comparative analysis presented here broadens knowledge of the genomic basis of C. fetus pathogenesis and host specificity. It further highlights the importance of surface-exposed structures to C. fetus pathogenicity and demonstrates how evolutionary forces optimize the fitness and host-adaptation of these pathogens.     

Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074

The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072+/-47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50 degrees , respectively. The Km value was 0.17 mM, with a Vmax of 1,714 mmol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.     

A New Approach Using the SYBR Green-Based Real-Time PCR Method for Detection of Soft Rot Pectobacterium odoriferum Associated with Kimchi Cabbage

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferum-specific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.     

Differential Regulation of Horizontally Acquired and Core Genome Genes by the Bacterial Modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

Siderophore-Mediated Iron Acquisition Influences Motility and Is Required for Full Virulence of the Xylem-Dwelling Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart''s wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

Biocontrol of the Potato Blackleg and Soft Rot Diseases Caused by Dickeya dianthicola

Development of protection tools targeting Dickeya species is an important issue in the potato production. Here, we present the identification and the characterization of novel biocontrol agents. Successive screenings of 10,000 bacterial isolates led us to retain 58 strains that exhibited growth inhibition properties against several Dickeya sp. and/or Pectobacterium sp. pathogens. Most of them belonged to the Pseudomonas and Bacillus genera. In vitro assays revealed a fitness decrease of the tested Dickeya sp. and Pectobacterium sp. pathogens in the presence of the biocontrol agents. In addition, four independent greenhouse assays performed to evaluate the biocontrol bacteria effect on potato plants artificially contaminated with Dickeya dianthicola revealed that a mix of three biocontrol agents, namely, Pseudomonas putida PA14H7 and Pseudomonas fluorescens PA3G8 and PA4C2, repeatedly decreased the severity of blackleg symptoms as well as the transmission of D. dianthicola to the tuber progeny. This work highlights the use of a combination of biocontrol strains as a potential strategy to limit the soft rot and blackleg diseases caused by D. dianthicola on potato plants and tubers.     

Development of a Novel Model for the Assessment of Dead-Space Management in Soft Tissue

Following extensive surgical debridement in the treatment of infection, a “dead space” can result following surgical closure that can fill with hematoma, an environment conducive to bacterial growth. The eradication of dead space is essential in order to prevent recurrent infection. This study describes a novel small animal model to investigate dead-space management in muscle tissue. Two absorbable test materials were implanted in each animal; beads of calcium sulfate alone, and beads loaded with vancomycin and tobramycin. In-life blood samples and radiographs were taken from each animal following implantation. Animals were sacrificed at 1, 7, 21, 42, and 63 days post-operatively (n = 4), and implant sites were analysed by micro-computed tomography, histology and immunohistochemistry. Complete resorption was confirmed radiographically at 3 weeks post-implantation. Histologically, the host tissue response to both materials was identical, and subsequent healing at the implant sites was observed with no dead space remaining. Vancomycin was not detected in blood serum. However, peak tobramycin levels were detected in all animals at 6 hours post-implantation with no detectable levels in any animals at 72 hours post implantation. Serological inflammatory cytokine expression for IL-6, TNF-α and IL-1β indicated no unusual inflammatory response to the implanted materials or surgical procedure. The model was found to be convenient and effective for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response.     

君子兰软腐病病原体侵染途径与致病规律的研究

本文报导了君子兰(Clivia miniata)软腐病二株病原体的分离及确定;鉴定了病原体的分类地位,一株为链球菌属的一个新种,定名为君子兰链球菌(Streptococcus miniatus Zhou,Ping et al shao sp.nov),另一株为环状芽孢杆菌(Bacillus subtilis);并对其二病原体的侵染途径与致病规律进行了研究。     

Pre-PCR Processes in the Molecular Detection of Blackleg and Soft Rot Erwiniae in Seed Potatoes

The polymerase chain reaction (PCR) based detection of blackleg and soft rot erwiniae involves pre‐PCR processing steps which may compromise the sensitivity of detection. The aim of this study was to standardize these various steps to develop reproducible diagnostic PCR protocol for the detection of the three known soft rot erwiniae as they occur in the tuber, singly or in combination. Comparison of tuber peel and stolon end tissue as a starting material for enrichment of the bacteria indicated that tuber peel samples resulted in more representative and sensitive detection of the strains than extract from stolon end tissues. Substances of potato origin in the peel extract were found to be highly inhibitory to the PCR. Addition of the antioxidant Dethiotreitol to the samples before enrichment did not have any significant effect on detection during the 24 h period incubation of the peel extract at room temperature. Bulk washing of tubers with one rotten tuber included with the working sample caused surface contamination on 67–91% of the healthy tubers. Washing tubers individually circumvents the problem. The optimum temperature for enrichment of all the three strains was 27°C. At 37°C, Pectobacterium carotovorum failed to be detected while PCR on Pectobacterium atrosepticum and isolates of Dickeya spp. always produced amplification of the specific DNA fragments. Viability test on Nutrient Agar showed that only Dickeya isolates were viable after 48 h of incubation at 37°C suggesting that the detection of P. atrosepticum at 37°C was from dead or non‐viable cells. Post cell death detection experiment further confirmed that DNA was amplified from dead cells of all the strains at 27°C and 33°C whereas at 37°C, only DNA from dead cells of isolates of Dickeya and P. atrosepticum were amplified. There was no amplification from the dead cells of all isolates of P. carotovorum following the 48 h post death incubation at 37°C. The reason for this difference in post death longevity is not clear at this stage.     

Application of Hormetic UV-C for Delayed Ripening and Reduction of Rhizopus Soft Rot in Tomatoes: the Effect of Tomatine on Storage Rot Development

The application of ultraviolet-light C (UV-C, 254 nm) hormesis on tomato fruits to stimulate beneficial responses is a new method of inducing host resistance to Rhizopus soft rot, with maximum protection at 72 h following artificial inoculation with Rhizopus stolonifer and delay fruit ripening. In the tomato-Rhizopus soft rot pathosystem, UV-C induced resistance of tomatoes to soft rot was reversed by fluorescent light, and not by storage under dark condition. The present study was aimed at finding a possible fungal toxic mechanism that was involved in the control of Rhizopus soft rot. The development of carotenoids, loss of chlorophyll and ethylene production were significantly retarded during storage after treatment with hormetic doses of UV-C. The delayed ripening of UV-C treated fruits was attributed in part to the high level of putrescine and spermine polyamines. In a time-control study, it was found that the resistance of UV-C-treated tomatoes artificially inoculated with R. stolonifer correlated with a high concentration of tomatine which accumulated up to 72 h after UV-C treatment. However, there was a higher tomatine content in UV-C-treated fruits compared to untreated fruits where it was transitory at 96 h after treatment in the time-course study.