Cellular alterations in Pectobacterium carotovorum treated with nanostructured formulations during the incubation time.

Pectobacterium carotovorum was incubated in formulations of chitosan nanoparticles or thyme essential oil-loaded chitosan nanoparticles for a maximum period of 48 h time. The cellular changes and viability were evaluated by transmission electron microscopy (TEM), and two colorimetric assays 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) and alamar blue (AMB), respectively. The incubation time and the addition of the secondary metabolite to the formulation were key factors to the cell damage and cell inhibitory effects on P. carotovorum, TEM observations overall demonstrated on the treated bacterium, cell surface alterations such as deforming and disappearance of the cell wall and the plasmatic membrane, with agglomeration of nanoparticles outside and inside of the cells, loss of cell content and lysis. Cell viability was reduced about 80% (MTT) and 100% (AMB) in the applied treatment of chitosan-loaded thyme essential oil nanoparticles after 48 h incubation, in addition, the total cell inhibition was shown from 6 h incubation onwards with the AMB assay. The TEM micrographs and the cell viability assays provided enough evidence of the antimicrobial activity of the nanostructured formulations compared with the control where no damage was observed.

Structural Elucidation of Three Novel Kaempferol O-tri-Glycosides that Are Involved in the Defense Response of Hybrid Ornithogalum to Pectobacterium carotovorum.

Ornithogalum is an ornamental flowering species that grows from a bulb and is highly susceptible to soft-rot disease caused by Pectobacterium carotovorum (Pc). Interspecific hybridization between O. thyrsoides and O. dubium yielded hybrids with enhanced resistance to that pathogen. The hybrids displayed distinct phenolic-compound profiles with several peaks that were specifically heightened following Pc infection. Three of these compounds were isolated and identified as novel kaempferol O-tri-glycosides. The structures of these compounds were elucidated using reversed phase high-performance liquid chromatography (RP-LC), RP-LC coupled to high-resolution mass spectrometry (RP-LC-MS), and nuclear magnetic resonance (NMR) (1D 1H and 13C, DEPT, HMQC, HMBC, COSY, and NOE), in order to achieve pure and defined compounds data. The new compounds were finally identified as kaempferol 3-O-[4-O-α-l-(3-O-acetic)-rhamnopyranosyl-6-O-ß-d-xylopyranosyl]-ß-d-glucopyranoside, kaempferol 3-O-[4-O-α-l-(2-O-acetic)-rhamnopyranosyl-6-O-ß-d-xylopyranosyl]-ß-d-glucopyranoside and kaempferol 3-O-[4-O-α-l-(2,3-O-diacetic)-rhamnopyranosyl-6-O-ß-d-xylopyranosyl]-ß-d-glucopyranoside.

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns.

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP-PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.

Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection.

BACKGROUND: Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored. RESULTS: In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes. CONCLUSIONS: Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens.

Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots.

During their colonization of plants, human enteric pathogens, such as Salmonella enterica, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by Salmonella from the association with a soft rot caused by Pectobacterium carotovorum were shown to be dependent on Salmonella KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A Salmonella kdgR mutant was more fit in soft rots but not in the lesions caused by Xanthomonas spp. and Pseudomonas spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the kdgR phenotype.

[BACILLUS STRAINS'S SCREENING--ACTIVE ANTAGONISTS OF BACTERIAL AND FUNGAL PHYTOPATHOGENS].

Antagonistic activity 100 strains of Bacillus bacteria towards to museum and actual strains of phytopathogenic bacteria and fungy was defined. Relation between level of antagonistic activity to phytopathogenic bacteria and genus accessory of the last was shown. The medium level of antagonism to fungal phytopathogens at 30% of the studied strains of Bacillus bacteria was shown. 5 strains of Bacillus sp. with high and medium levels of antagonism to phytopathogens bacterial and fungy nature was selected and considered as perspective for creation of biological preparations for plant protection.

Characterization of the small RNA transcriptome in plant-microbe (Brassica/Erwinia) interactions by high-throughput sequencing.

Non-coding, small RNAs (sRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans; however, the role and mechanisms of these sRNA in plant immunity is largely unknown. To determine possible roles of sRNA in plant-pathogen interaction, we carried out a high-throughput sRNA sequencing of Brassica campestris using non-infected plants and plants infected with Erwinia carotovora. Consistent with our hypothesis that distinct classes of host sRNAs alerts their expression levels in response to infection, we found that: (1) host 28-nt sRNAs were strongly increased under pathogen infection; and (2) a group of host sRNAs homologous to the pathogen genome also accumulated at significantly higher level. Our data thus suggest several distinct classes of the host sRNAs may enhance their function by up-regulation of their expression/stability in response to bacterial pathogen challenges.

Colonization patterns of an mCherry-tagged Pectobacterium carotovorum subsp. brasiliense strain in potato plants.

Pectobacterium carotovorum subsp. brasiliense is a newly identified member of the potato soft rot enterobacteriaceae. The pathogenesis of this pathogen is still poorly understood. In this study, an mCherry-P. carotovorum subsp. brasiliense-tagged strain was generated to study P. carotovorum subsp. brasiliense-potato plant interactions. Prior to use, the tagged strain was evaluated for in vitro growth, plasmid stability, and virulence on potato tubers and shown to be similar to the wild type. Four potato cultivars were evaluated for stem-based resistance against P. carotovorum subsp. brasiliense. Confocal laser-scanning microscopy and in vitro viable cell counts showed that P. carotovorum subsp. brasiliense is able to penetrate roots of a susceptible potato cultivar as early as 12 h postinoculation and migrate upward into aerial stem parts. Due to the phenotypic differences observed between tolerant and susceptible cultivars, a comparison of P. carotovorum subsp. brasiliense colonization patterns in these cultivars was undertaken. In the susceptible cultivar, P. carotovorum subsp. brasiliense cells colonized the xylem tissue, forming "biofilm-like" aggregates that led to occlusion of some of the vessels. In contrast, in the tolerant cultivar, P. carotovorum subsp. brasiliense appeared as free-swimming planktonic cells with no specific tissue localization. This suggests that there are resistance mechanisms in the tolerant cultivar that limit aggregation of P. carotovorum subsp. brasiliense in planta and, hence, the lack of symptom development in this cultivar.

Consequences of disrupting Salmonella AI-2 signaling on interactions within soft rots.

Within soft rots, Salmonella spp. reach population densities 10- to 100-fold higher than within intact plants. The hypothesis that Salmonella spp. exchange AI-2 signals with Pectobacterium carotovorum to increase its competitive fitness was tested using mutants involved in AI-2 production (luxS) or perception (lsrACDBF or lsrG). Co-infections of a wild-type Salmonella sp. and its AI-2 mutants (at ≈3 to 10(4)) were established in green or red tomato ('FL 47' or 'Campari' for 3 or 5 days) as well as tomato co-infected with Pectobacterium (at 10(9)) or its luxS mutant. There were no significant differences in the competitive fitness of Salmonella, indicating that AI-2 signaling is not a major input in the interactions between these organisms under the tested conditions. A Salmonella lsrG::tnpR-lacZ resolvase in vivo expression technology (RIVET) reporter, constructed to monitor AI-2-related gene expression, responded strongly to the luxS deletion but only weakly to external sources of AI-2. Growth in soft rots generally decreased RIVET resolution; however, the effect was not correlated to the luxS genotype of the Pectobacterium sp. The results of this study show that AI-2 signaling offers no significant benefit to Salmonella spp. in this model of colonization of tomato or soft rots.

[Expression of cecropin P1 gene increases resistance of Camelina sativa (L.) plants to microbial phytopathogenes].

Transgenic plants of camelina (Camelina sativa (L.) Crantz) with the synthetic gene of antimicrobial peptide cecropin P1 (cecP1) were obtained. Agrobacterium-mediated transformation is performed using the binary vector pGA482::cecP1 by vacuum infiltration of flower buds. The presence of the cecP1 gene in the genome of plants was confirmed by PCR. cecP1 gene expression in transgenic plants was shown by Western blot analysis and by antimicrobial activity of plant extracts against the bacterial phytopathogene Erwinia carotovora. The plants of F0 and F1 generations had the normal phenotype and retained the ability to form viable seeds in self-pollination. cecP1 plants exhibit enhanced resistance to bacterial and fungal phytopathogens: Erwinia carotovora and Fusarium sporotrichioides. The increased sustainability of cecropin P1-expressing plants against salt stress is shown. The possibility of the integration of the cecP1 gene into the overall protective system of plants against biotic and abiotic stresses is discussed.

[Effect of surface-active substances of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria].

The effect of surface-active substances (SAS's) of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria has been studied. It was shown that the survival of cells (10(5)-10(7) in a milliliter) of the Pseudomonas and Xanthomonas phytopathogenic bacteria was found to be 0-33% after treatment with SAS preparations of the IMV Ac-5017 and IMV B-7241 strains for 2 h (0.15-0.4 mg/mL). In the presence of N. vaccinii K-8 SAS preparations (0.085-0.85 mg/mL), the number of cells of the majority of the studied phytopathogenic bacteria decreased by 95-100%. These data show prospects for using microbial SAS's for the construction of ecologically friendly drugs for regulating the number of phytopathogenic bacteria.

[Properties of pectolitic phytopathogenic bacteria isolates obtained in Ukraine].

Bacteria obtained from potato tubers having symptoms of soft rot and grown in different regions of Ukraine are identified as Pectobacterium carotovorum subsp. carotovorum. These bacteria strains are able to produce bacteriocines. Their killer activity in respect of P. carotovorum and Esherichia coli has been studied. The sensitivity to bactericines has been shown. Purified fractions of bacteriocines having high molecular weight (MCTV) have been obtained. The difference in composition of proteins from phage tails as compared to the ones in P. carotovorum J2 has been studied by the method of electrophoresis. It was found that the composition of MCTV major proteins of studied isolates mostly corresponds to P. carotovorum J2. The set of enzyme minor fractions has some different compositions as compared to P. carotovorum J2. It has been hypothesized that this difference is responsible for killer specificity.

Pectin methylesterase is induced in Arabidopsis upon infection and is necessary for a successful colonization by necrotrophic pathogens.

The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading pectin is often related to a successful initiation of the infective process. Pectin is synthesized in a highly methylesterified form and is subsequently de-esterified in muro by pectin methylesterase. De-esterification makes pectin more susceptible to the degradation by pectic enzymes such as endopolygalacturonases (endoPG) and pectate lyases secreted by necrotrophic pathogens during the first stages of infection. We show that, upon infection, Pectobacterium carotovorum and Botrytis cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as a susceptibility factor and is required for the initial colonization of the host tissue.

The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits.

The antibacterial potential of four strains of Bacillus subtilis, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, previously selected on the basis of their antifungal activity and efficacy against cucurbit powdery mildew, was examined. Among these strains, UMAF6614 and UMAF6639 showed the highest antibacterial activity in vitro, especially against Xanthomonas campestris pv. cucurbitae and Pectobacterium carotovorum subsp. carotovorum. These strains produced the three families of lipopeptide antibiotics known in Bacillus spp.: surfactins, iturins, and fengycins. Using thin-layer chromatography analysis and direct bioautography, the antibacterial activity could be associated with iturin lipopeptides. This result was confirmed by mutagenesis analysis using lipopeptide-defective mutants. The antibacterial activity was practically abolished in iturin-deficient mutants, whereas the fengycin mutants retained certain inhibitory capabilities. Analyses by fluorescence and transmission electron microscopy revealed the cytotoxic effect of these compounds at the bacterial plasma membrane level. Finally, biological control assays on detached melon leaves demonstrated the ability of UMAF6614 and UMAF6639 to suppress bacterial leaf spot and soft rot; accordingly, the biocontrol activity was practically abolished in mutants deficient in iturin biosynthesis. Taken together, our results highlight the potential of these B. subtilis strains as biocontrol agents against fungal and bacterial diseases of cucurbits and the versatility of iturins as antifungal and antibacterial compounds.

Application of natural-based nanocoatings for extending the shelf life of green bell pepper fruit.

Pectobacterium carotovorum is a phytopathogenic bacteria that causes significant economic loses in food crops, such as bell pepper, which is of special significance in the value of production and trade in Mexico. Therefore, a solution for fruit conservation must be sought. Due to environmental concerns, it is necessary the use of environmentally-friendly active packaging. In this article, chitosan and chitosan-thyme essential oil nanocoatings were used for the preservation of green bell pepper. Different formulations based on chitosan nanoparticles (CSNPs) and chitosan-thyme essential oil nanoparticles (15, 30, and 45%) were prepared. For uncoated and coated bell peppers, the quality and physiological variables of inoculated and uninoculated fruit with P. carotovorum during 12-day storage period were assessed. According to the results, the weight loss of the fruit remained almost constant over the storage days for the different formulations. A decrease in fruit firmness and an increase in the respiration rate and ascorbic acid content until day 8 with a decrease at the end of the storage period were observed. Of all the evaluated nanocoatings, the fruit treated with the formulation containing 15% CSNPs showed the lowest colony-forming units and disease incidence. Also, the coated bell peppers with this formulation had lower CO2 production compared to the remaining treatments, and the weight loss and firmness were maintained. Therefore, the use of CSNP coatings could represent a good alternative for the protection of bell pepper against the pathogenic bacteria P. carotovorum. PRACTICAL APPLICATION: The results of the application of nanocoatings based on chitosan and chitosan-thyme essential oil as an antibacterial agent against P. carotovorum on green bell pepper during 12-day storage period suggest that nanoparticle-based coatings can be a natural option for the preservation of fruit quality during ripening.

Proteome analysis of fungal and bacterial involvement in leaf litter decomposition.

Fungi and bacteria are key players in the decomposition of leaf litter, but their individual contributions to the process and their interactions are still poorly known. We combined semi-quantitative proteome analyses (1-D PAGE-LC-MS/MS) with qualitative and quantitative analyses of extracellular degradative enzyme activities to unravel the respective roles of a fungus and a bacterium during litter decomposition. Two model organisms, a mesophilic Gram-negative bacterium (Pectobacterium carotovorum) and an ascomycete (Aspergillus nidulans), were grown in both, pure culture and co-culture on minimal medium containing either glucose or beech leaf litter as sole carbon source. P. carotovorum grew best in co-culture with the fungus, whereas growth of A. nidulans was significantly reduced when the bacterium was present. This observation suggests that P. carotovorum has only limited capabilities to degrade leaf litter and profits from the degradation products of A. nidulans at the expense of fungal growth. In accordance with this interpretation, our proteome analysis revealed that most of the extracellular biodegradative enzymes (i.e. proteases, pectinases, and cellulases) in the cultures with beech litter were expressed by the fungus, the bacterium producing only low levels of pectinases.

GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis.

Systemic resistance is induced by necrotizing pathogenic microbes and non-pathogenic rhizobacteria and confers protection against a broad range of pathogens. Here we show that Arabidopsis GDSL LIPASE-LIKE 1 (GLIP1) plays an important role in plant immunity, eliciting both local and systemic resistance in plants. GLIP1 functions independently of salicylic acid but requires ethylene signaling. Enhancement of GLIP1 expression in plants increases resistance to pathogens including Alternaria brassicicola, Erwinia carotovora and Pseudomonas syringae, and limits their growth at the infection site. Furthermore, local treatment with GLIP1 proteins is sufficient for the activation of systemic resistance, inducing both resistance gene expression and pathogen resistance in systemic leaves. The PDF1.2-inducing activity accumulates in petiole exudates in a GLIP1-dependent manner and is fractionated in the size range of less than 10 kDa as determined by size exclusion chromatography. Our results demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 may mediate the production of a systemic signaling molecule(s).

Effect of medium composition and kinetic studies on extracellular and intracellular production of L-asparaginase from Pectobacterium carotovorum.

Microbial L-asparaginase occupies a prominent place among biocatalysts owing to their ability to catalyze the reaction that hydrolyze the asparagine molecule. Effect of various medium components on the production of L-asparaginase in submerged fermentation by Pectobacterium carotovorum was studied for optimal nutrient requirements. Six different media compositions were tested for the L-asparaginase production keeping fermentation conditions constant at temperature 30 °C, initial pH 7.0 and agitation speed of 120 rpm. Maximum intracellular and extracellular L-asparaginase activity was obtained in the medium containing tryptone, yeast extract, monosodium glutamate, K2HPO4 and L-asparagine. These medium components were further optimized by central composite experimental design using response surface methodology. Maximum intracellular and extracellular L-asparaginase activity of 2.282 U/mL and 0.587 U/mL were obtained respectively at the late logarithmic phase in optimized media. Unstructured kinetic models were used to describe the cell growth and product formation kinetics. The unstructured models predicted the cell growth and product formation profile accurately with high coefficient of determination.

[Polypeptide composition and killer specificity as indices of multiplicity of carotovoricins].

A method of separation and purification of macromolecular bacteriocins of phytopathogenic bacterium Erwinia carotovora is proposed. It is shown on the basis of polypeptide composition of the particles and their killer specificity, that E. carotovora ESP86 is a complex defective-polylysogenic system which includes no less than three different types of biologically active tails of incomplete temperate bacteriophages.

Development of medium for enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428.

Glutaminase-free L-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of L-asparaginase using the Plackett-Burman experimental design. The medium components, viz., glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O, were screened based on their high confidence levels (P<0.04). The optimum levels of glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O were found to be 2.076, 5.202, 1.773, and 0.373 g L(-1), respectively, using the central composite experimental design. The maximum specific activity of L-asparaginase in the optimized medium was 27.88 U mg(-1) of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.