Falcaria vulgaris extract: A mixture of quorum sensing inhibitors for controlling Pectobacterium carotovorum subsp. carotovorum.

A promising strategy to control bacterial diseases involves using Quorum Sensing Inhibitor (QSI) compounds. This study aimed to evaluate the potential of Falcaria vulgaris plant extract to combat the phytopathogenic Pectobacterium carotovorum subsp. carotovorum (Pcc) via its QSI activity. Using biosensors and Minimum Inhibitory Concentration (MIC) assays, the QSI and antimicrobial aspects of the extract were assessed. Furthermore, the effect of the extract on the reduction of tuber maceration in potatoes was examined. Subsequently, homology modeling based on LasR was conducted to analyze interactions between ligand 3-oxo-C8-AHL, and ExpR2 protein. Docking studies were performed on all extract compounds identified via Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The extract effectively reduced maceration at sub-MIC concentrations across various pathogenic strains. Furthermore, Cyclopentadecanone, 2-hydroxy, showed more negative docking energy than the native ligand. Z,E-2,13-Octadecadien-1-ol showed energy equivalence to the native ligand. Additionally, this plant included certain compounds or their analogs that had previously been discovered as QSI compounds. These compounds included oleic acid, n-Hexadecanoic acid, cytidine, and linoleic acid, and they had energies that were comparable to that of the native ligand. In conclusion, the remarkable QSI property showed by this plant is likely attributed to a combination of compounds possessing this characteristic.

Needle in a haystack: Antibacterial activity-guided fractionation of a potato wound tissue extract.

Erwinia carotovora is a major cause of potato tuber infection, which results in disastrous failures of this important food crop. There is currently no effective antibiotic treatment against E. carotovora. Recently we reported antibacterial assays of wound tissue extracts from four potato cultivars that exhibit a gradient of russeting character, finding the highest potency against this pathogen for a polar extract from the tissue formed immediately after wounding by an Atlantic cultivar. In the current investigation, antibacterial activity-guided fractions of this extract were analyzed by liquid chromatography-mass spectrometry (LC-MS) utilizing a quadrupole-time-of-flight (QTOF) mass spectrometer. The most active chemical compounds identified against E. carotovora were: 6-O-nonyl glucitol, Lyratol C, n-[2-(4-Hydroxyphenyl)] ethyldecanamide, α-chaconine and α-solanine. Interactions among the three compounds, ferulic acid, feruloyl putrescine, and α-chaconine, representing metabolite classes upregulated during initial stages of wound healing, were also evaluated, offering possible explanations for the burst in antibacterial activity after tuber wounding and a chemical rationale for the temporal resistance phenomenon.

Linolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria: Biophysical investigation of the mode of action.

Fatty acid hydroperoxides (HPO) are free phyto-oxylipins known for their crucial role as signalling molecules during plant defense mechanisms. They were also demonstrated to have direct biocidal activities against plant pathogens including gram negative bacteria. In the present work, the biocidal effect of one linolenic fatty acid hydroperoxide, namely 13-HPOT has been investigated on three plant pathogen gram negative bacteria: Pectobacterium carotovorum, Pseudomonas syringae and Xanthomonas translucens. We showed that 13-HPOT has a strong dose response effect on those phytopathogens. In a second part, the molecular mechanism behind the antibacterial effect of 13-HPOT was investigated at a molecular level using an integrative biophysical approach combining in vitro and in silico methods. Since other antimicrobial amphiphilic molecules have been shown to target the lipid membrane of the organisms they act on, we focused our study on the interaction of 13-HPOT with biomimetic membranes. In a first step, we hypothesized that the inner membrane of the bacteria was the main site of action of 13-HPOT and hence we used lipids representative of this membrane to form our models. Our results indicated that 13-HPOT can interact with the lipid representative of the inner bacterial plasma membrane. A strong membrane insertion is suggested but no major permeabilization of the membrane is observed. Phosphatidylethanolamine (PE) and cardiolipin (CL), present in the bacterial plasma membrane, appear to play important roles in this interaction. We suggest that the mode of action of 13-HPOT should involve either subtle changes in membrane properties, such as its lateral organization and distribution, and/or interactions with membrane proteins.

Efficacy of Argentinean propolis extracts on control of potato soft rot caused by Erwinia carotovora subsp.

BACKGROUND: Erwinia carotovora subsp. cause the potato soft rot, which is a major disease in agriculture. Antibacterial agents currently applied on potato soft rot often offer a restricted control and have several disadvantages. Propolis has shown a wide range of antimicrobial activity, although its effect has not been investigated on E. carotovora subsp. In this work, we tested extracts from propolis samples of Northwest Argentina against E. carotovora subsp. RESULTS: Ethanolic propolis extracts (EPEs) from samples of Santiago del Estero province, particularly from sample 4 (EPE4), showed the highest antibacterial activity, which was associated with the highest content of flavonoids. 2',4'-Dihydroxychalcone, 2',4'-dihydroxy-3'-methoxychalcone, galangin, and pinocembrin were identified as antibacterial constituents of EPE4. 2',4'-Dihydroxychalcone showed an antibacterial activity (minimum inhibitory concentration, MIC = 0.3-1.2 µg gallic acid equivalents (GAE) mL-1 ; minimum bactericidal concentration, MBC = 0.6-4.8 µg GAE mL-1 ) lower than that of bacterimycin (MIC = 2.4-9.6 µg mL-1 ; MBC = 19.2-38.4 µg GAE mL-1 ) and streptocycline (MIC = 19.2-38.4 µg mL-1 ; MBC = 38.4-76.8 µg mL-1 ). Preventive assays on unwounded and wounded potatoes showed that their immersion in EPE4 containing 87.5 µg GAE mL-1 or streptocycline containing 40 µg mL-1 was equally effective in controlling potato soft rot, reducing the disease incidence by 64.6-67.0% (unwounded tubers) and 88.0-86.0% (wounded tubers) and the disease severity by 49.8-49.8% (unwounded tubers) and 54.5-68.5% (wounded tubers). CONCLUSIONS: Flavonoid-rich propolis extracts from Northwest Argentina efficiently reduced in vivo the incidence and severity of potato soft rot caused by E. carotovora subsp.

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.

AhlX, an N-acylhomoserine Lactonase with Unique Properties.

N-Acylhomoserine lactonase degrades the lactone ring of N-acylhomoserine lactones (AHLs) and has been widely suggested as a promising candidate for use in bacterial disease control. While a number of AHL lactonases have been characterized, none of them has been developed as a commercially available enzymatic product for in vitro AHL quenching due to their low stability. In this study, a highly stable AHL lactonase (AhlX) was identified and isolated from the marine bacterium Salinicola salaria MCCC1A01339. AhlX is encoded by a 768-bp gene and has a predicted molecular mass of 29 kDa. The enzyme retained approximately 97% activity after incubating at 25 °C for 12 days and ~100% activity after incubating at 60 °C for 2 h. Furthermore, AhlX exhibited a high salt tolerance, retaining approximately 60% of its activity observed in the presence of 25% NaCl. In addition, an AhlX powder made by an industrial spray-drying process attenuated Erwinia carotovora infection. These results suggest that AhlX has great potential for use as an in vitro preventive and therapeutic agent for bacterial diseases.

Polyphenols, Antioxidant, Antibacterial, and Biofilm Inhibitory Activities of Peel and Pulp of Citrus medica L., Citrus bergamia, and Citrus medica cv. Salò Cultivated in Southern Italy.

The aim of this paper was to study the polyphenols of peel and pulp of three Citrus taxa-Citrus medica, Citrus bergamia, and Citrus medica cv. Salò-cultivated in the Cosenza province, Southern Italy, and to evaluate their antioxidant and antibacterial activity, performed against Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Pectobacterium carotovorum. Furthermore, we assessed the inhibitory effect of the extracts on bacterial capacity to form biofilm, and on the metabolic activity of the cells present therein. The results indicated that such extracts could find new potential applications in the field of natural antioxidant and anti-bacterial agents in pharmaceutics, agriculture, and food fields.

Aspergoterpenins A⁻D: Four New Antimicrobial Bisabolane Sesquiterpenoid Derivatives from an Endophytic Fungus Aspergillus versicolor.

Aspergoterpenins A⁻D (1⁻4), four new bisabolane sesquiterpenoid derivatives, were obtained from the endophytic fungus, Aspergillus versicolor, together with eight known compounds (5⁻12), and their structures were elucidated by a comprehensive analysis of their NMR (Nuclear Magnetic Resonance), MS (Mass Spectrum) and CD (Circular Dichroism) spectra. Aspergoterpenin A (1) was the first example with a characteristic ketal bridged-ring part in the degraded natural bisabolane-type sesquiterpene structures. The compounds 1⁻12 displayed no significant activities against four cancer cell lines (A549, Caski, HepG2 and MCF-7). Further, the antimicrobial activities to Erwinia carotovora sub sp. Carotovora were evaluated, and the results showed that compounds 1⁻12 displayed antimicrobial activities with MIC values ranging from 15.2 to 85.2 µg/mL.

Antibacterial activities of the phytochemicals-characterized extracts of Callistemon viminalis, Eucalyptus camaldulensis and Conyza dioscoridis against the growth of some phytopathogenic bacteria.

Three bacterial isolates were isolated from infected potato tubers showing soft and brown rots like symptoms as well as one isolate from infected peach tree showing crown gall symptom. The morphological, biochemical and molecular assays proved that bacterial isolates belonging to Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum, Dickeya spp. and Agrobacterium tumefaciens. The acetone (AcE) and n-butanol (ButE) extracts of Callistemon viminalis flowers and essential oil from aerial parts of Conyza dioscoridis as well as ButE of Eucalyptus camaldulensis bark are evaluated at different concentrations against the growth of the isolated bacteria. The diameter of inhibition zone (IZ) and the minimum inhibitory concentrations (MICs) are compared. Results indicated that the highest IZ values were 20.0 mm and 18.3 mm for E. camaldulensis bark ButE and C. viminalis flower ButE, respectively, against P. carotovorum; 16.3 mm and 16.0 mm for E. camaldulensis bark ButE and C. viminalis flower ButE, respectively, against R. solanacearum; 18.5 mm for C. viminalis flower AcE and C. dioscoridis aerial parts EO against Dickeya spp.; and 15.0 mm for C. viminalis flower AcE against A. tumefaciens. MICs ranged from <16 µg/mL for D. solani to >4000 µg/mL for A. tumefaciens. It was proved that C. viminalis flowers AcE contains mainly 5-hydroxymethylfurfural (20.6%), palmitic acid (18.5%), and pyrogallol (16.4%); while C. viminalis flower ButE contains palmitic acid (36.3%), 2-hydroxymyristic acid (9.4%), 5-hydroxymethylfurfural (7.2%), and shikimic acid (6.6%); whereas E. camaldulensis bark ButE contains 8-nonynoic acid methyl ester (45.6), camphor (30.9%), menthol (8.8%), and 1,8-cineole (eucalyptol) (8.2%), whilst the EO of C. dioscoridis aerial parts comprises Z-(13,14-epoxy)tetradec-11-en-1-ol acetate (11.6%), γ-elemene (10.2%), tau.-muurolol (7.1%), and cadina-3,9-diene (4.7%). It can be concluded that phytochemical extracts of C. viminalis, E. camaldulensis and C. dioscoridis demonstrated strong to moderate antibacterial effects against the studied plant bacterial pathogens.

Screening bactericidal effect of Pectobacterium carotovorum subsp. carotovorum strains against causal agent of potato soft rot.

This study focuses on the potential of Pectobacterium carotovorum subsp. carotovorum (Pcc) strains producing bacteriocin as a tool to control potato soft rot disease. Thirty out of 48 purified bacterial strains were characterized as Pcc using specific PCR and phenotypic tests. The pathogenicity and pectate degrading assays were recorded positive for 13 strains. Bacteriocin typing clustered producers into three groups according to their antimicrobial spectra. Majority of the producers except strains of group II showed antibacterial activity toward relative genus and the role of UV or mitomycin C was inductive. In addition, none of the distant genus was sensitive to Pcc bacteriocins except Rhizobium vitis. Molecular detection of four bacteriocins including carotovoricin, carosin S1, S2 and carosin D was performed. Overall, 54.5% of group I, 47.3 and 70% of groups II and III strains carried carotovoricin and four strains harbored gene corresponding to carosin S1. According to our data divers antimicrobial patterns obtained by Pcc strains and existence of new bateriocines could be possible. Moreover, our findings recommended that direct application of P29 or expression of corresponding genes of Pog22 or P21 in a nonpathogenic strain as a biocontrol agent may improve soft rot disease control.

Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses.

Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

Effect of oxygen on the growth and biofilm formation of Xylella fastidiosa in liquid media.

Xylella fastidiosa is a xylem-limited bacterial pathogen, and is the causative agent of Pierce's disease of grapevines and scorch diseases of many other plant species. The disease symptoms are putatively due to blocking of the transpiration stream by bacterial-induced biofilm formation and/or by the formation of plant-generated tylosis. Xylella fastidiosa has been classified as an obligate aerobe, which appears unusual given that dissolved O2 levels in the xylem during the growing season are often hypoxic (20-60 µmol L(-1)). We examined the growth and biofilm formation of three strains of X. fastidiosa under variable O2 conditions (21, 2.1, 0.21 and 0 % O2), in comparison to that of Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) under similar conditions. The growth of X. fastidiosa more closely resembled that of the facultative anaerobe, and not the obligate aerobe. Xanthomonas campestris, the closest genetic relative of X. fastidiosa, exhibited no growth in an N2 environment, whereas X. fastidiosa was capable of growing in an N2 environment in PW(+), CHARDS, and XDM2-PR media. The magnitude of growth and biofilm formation in the N2 (0 % O2) treatment was dependent on the specific medium. Additional studies involving the metabolism of X. fastidiosa in response to low O2 are warranted. Whether X. fastidiosa is classified as an obligate aerobe or a facultative anaerobe should be confirmed by gene activation and/or the quantification of the metabolic profiles under hypoxic conditions.

Effect of allyl-isothiocyanate on survival and antimicrobial peptide expression following oral bacterial infections in Drosophila melanogaster.

Since infections with antibiotic-resistant bacteria cause increasing problems worldwide, the identification of alternative therapies is of great importance. Plant-derived bioactives, including allyl-isothiocyanate (AITC), have received attention for their antimicrobial properties. The present study therefore investigates the impact of AITC on survival and antimicrobial peptide (AMP) levels in Drosophila melanogaster challenged with the fly pathogenic bacteria Pectobacterium carotovorum subsp. carotovorum and Leuconostoc pseudomesenteroides. AITC, a sulfur-containing compound derived from glucosinolates, exhibits antimicrobial properties and has been suggested to modulate AMP expression. By using D. melanogaster, we demonstrate that AITC treatment resulted in a concentration-dependent decrease of survival rates among female flies, particularly in the presence of the Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum, whereas AITC did not affect survival in male flies. Despite the ability of isothiocyanates to induce AMP expression in cell culture, we did not detect significant changes in AMP mRNA levels in infected flies exposed to AITC. Our findings suggest sex-specific differences in response to AITC treatment and bacterial infections, underlining the complexity of host-pathogen interactions and potential limitations of AITC as a preventive or therapeutic compound at least in D. melanogaster models of bacterial infections.

Orostachys malacophylla (pall.) fisch extracts alleviate intestinal inflammation in Drosophila.

ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys malacophylla (Pall.) Fisch (O. malacophylla) is a succulent herbaceous plant that is the Orostachys genus of Crassulaceae family. O. malacophylla has been widely used as a traditional Chinese medicine with antioxidant, anti-inflammatory, anti-febrile, antidote, anti-Toxoplasma gondii properties. However, the biological function of alleviating intestinal inflammation and key bioactive compounds were still unknown. AIM OF THE STUDY: We used a Drosophila model to study the protective effects and bioactive compounds of O. malacophylla water extract (OMWE) and butanol extract (OMBE) on intestinal inflammation. MATERIALS AND METHODS: Drosophila intestinal inflammation was induced by oral invasion of dextran sodium sulfate (DSS) or Erwinia carotovora carotovora 15 (Ecc15). We revealed the protective effects of two extracts by determining intestinal reactive oxygen species (ROS) and antimicrobial peptide (AMP) levels and intestinal integrity, and using network pharmacology analysis to identify bioactive compounds. RESULTS: We demonstrated that both OMWE and OMBE could ameliorate the detrimental effects of DSS, including a decreased survival rate, elevated ROS levels, increased cell death, excessive proliferation of ISCs, acid-base imbalance, and disruption of intestinal integrity. Moreover, the overabundance of lipid droplets (LDs) and AMPs by Ecc15 infection is mitigated by these extracts, thereby enhancing the flies' resistance to adverse stimuli. In addition, we used widely targeted metabolomics and network pharmacology analysis to identify bioactive compounds associated with IBD healing that are present in OMWE and OMBE. CONCLUSIONS: In summary, our research indicates that OMWE and OMBE significantly mitigate intestinal inflammation and have the potential to be effective therapeutic agents for IBD in humans.

Bioactivity of silverleaf nightshade (Solanum elaeagnifolium Cav.) berries parts against Galleria mellonella and Erwinia carotovora and LC-MS chemical profile of its potential extract.

Natural products received much attention as an environmentally beneficial solution for pest management. Therefore, the extracts of invasive silverleaf nightshade (Solanum elaeagnifolium Cav.) weeds using their berries parts (seeds, peels and mucilage) supported by bioassay-guided fractionation were tested against both the greater wax moth (Galleria mellonella) and Erwinia carotovora pv. carotovora causes of the blackleg of potatoes. The seeds and peels of S. elaeagnifolium were successively extracted by maceration using dichloromethane (DCM), ethyl acetate (EtOAc), and ethanol (EtOH), respectively. While, its mucilage was extracted using EtOAc. The successive EtOH extract of the plant seeds had promising inhibition efficacy and the best minimal inhibition concentration (MIC) of 50 µg/ml against E. Carotovora amongst other extracts (DCM and EtOAc of the plant berries parts). Depending on dose response activity, EtOH extract had G. mellonella larval mortality and pupal duration rates (LC50; 198.30 and LC95; 1294.73 µg/ml), respectively. Additionally, this EtOH extract of seeds was fractionated using preparative TLC to three characteristic bands. The insecticidal and bacterial activities of these isolated bands (SEA, SEB, and SEC) were evaluated at a dose of 100 µg/ml, causing mortality by 48.48, 62.63 and 92.93% (G. mellonella larvae) and inhibition by 15.22, 0.00 and 31.66 mm (E. carotovora), respectively. Moreover, the separated major three bands were tentatively identified using LC-ESI-MS analysis revealing the presence of two phenolic acids; chlorogenic acid (SEA) and dicaffeoyl quinic acid (SEB) in addition to one steroidal saponin (SEC) annotated as borassoside E or yamoscin. Finally, the plant seeds' successive EtOH extract as well as its active constituents, exhibited potential broad-spectrum activity and the ability to participate in future pest management initiatives. A field study is also recommended to validate its bio-efficacy against selected pests and to develop its formulations.

Green synthesis of metal nanoparticles and study their anti-pathogenic properties against pathogens effect on plants and animals.

According to an estimate, 30% to 40%, of global fruit are wasted, leading to post harvest losses and contributing to economic losses ranging from $10 to $100 billion worldwide. Among, all fruits the discarded portion of oranges is around 20%. A novel and value addition approach to utilize the orange peels is in nanoscience. In the present study, a synthesis approach was conducted to prepare the metallic nanoparticles (copper and silver); by utilizing food waste (Citrus plant peels) as bioactive reductants. In addition, the Citrus sinensis extracts showed the reducing activity against metallic salts copper chloride and silver nitrate to form Cu-NPs (copper nanoparticles) and Ag-NPs (Silver nanoparticles). The in vitro potential of both types of prepared nanoparticles was examined against plant pathogenic bacteria Erwinia carotovora (Pectobacterium carotovorum) and pathogens effect on human health Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Moreover, the in vivo antagonistic potential of both types of prepared nanoparticles was examined by their interaction with against plant (potato slices). Furthermore, additional antipathogenic (antiviral and antifungal) properties were also examined. The statistical analysis was done to explain the level of significance and antipathogenic effectiveness among synthesized Ag-NPs and Cu-NPs. The surface morphology, elemental description and size of particles were analyzed by scanning electron microscopy, transmission electron microscopy, energy-dispersive spectroscopy and zeta sizer (in addition polydispersity index and zeta potential). The justification for the preparation of particles was done by UV-Vis Spectroscopy (excitation peaks at 339 nm for copper and 415 nm for silver) and crystalline nature was observed by X-ray diffraction. Hence, the prepared particles are quite effective against soft rot pathogens in plants and can also be used effectively in some other multifunctional applications such as bioactive sport wear, surgical gowns, bioactive bandages and wrist or knee compression bandages, etc.

Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana.

Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 µg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the 'full virus vector strategy' with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.

Characterization of genes required for the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Pcc21 in Chinese cabbage.

Pectobacterium carotovorum subsp. carotovorum is a well-known plant pathogen that causes severe soft rot disease in various crops, resulting in considerable economic loss. To identify pathogenicity-related factors, Chinese cabbage was inoculated with 5314 transposon mutants of P. carotovorum subsp. carotovorum Pcc21 derived using Tn5 transposon mutagenesis. A total of 35 reduced-virulence or avirulent mutants were isolated, and 14 loci were identified. The 14 loci could be functionally grouped into nutrient utilization (pyrD, purH, purD, leuA and serB), production of plant cell-wall-degrading enzymes (PCWDEs) (expI, expR and PCC21_023220), motility (flgA, fliA and flhB), biofilm formation (expI, expR and qseC), susceptibility to antibacterial plant chemicals (tolC) and unknown function (ECA2640). Among the 14 genes identified, qseC, tolC and PCC21_023220 are novel pathogenicity factors of P. carotovorum subsp. carotovorum involved in biofilm formation, phytochemical resistance and PCWDE production, respectively.

[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.

Study of antibacterial activity by capillary electrophoresis using multiple UV detection points.

A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gram-negative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 µm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications.