Journal of the Science of Food and Agriculture, 2008
BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were exam... more BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were examined for aerobic, coliform, and yeast and mould counts using normalised methods. Listeria spp., Listeria monocytogenes and Salmonella spp. were detected by real time polymerase chain reaction (QPCR) after enrichment. RESULTS: Aerobic plate counts ranged from <10 to >10 9 colony-forming units (CFU) g −1 , with the lowest and highest counts recorded for fruits and sprouts respectively. The highest incidence level of coliforms was found in ready-to-eat vegetables, with up to 65.7% of samples containing from 5 to 9 log 10 CFU g −1 . Yeasts and moulds showed their highest incidence level between 5 and 6 log 10 CFU g −1 , with an overall range from <2 to 9 log 10 CFU g −1 . Salmonella spp., Listeria spp. and L. monocytogenes were detected in 0.67, 2.7 and 0.9% respectively of the total samples examined.
Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy c... more Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.) À1 , which in turn decreased progressively to 4-5 log CFU (g f.w.) À1 after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.) À1 , thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.
This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and ve... more This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. [Int Microbiol 2008; 11(4):231-236]. This article contains supporting information [SI] online, consisting of one table (T1-SI) at the Int. Microbiol. website [www.im.microbios.org].
ABSTRACT The solid-phase conjugation of the antimicrobial peptide c(KKLKKFKKLQ) (BPC194) to a lin... more ABSTRACT The solid-phase conjugation of the antimicrobial peptide c(KKLKKFKKLQ) (BPC194) to a linear or cyclic sequence through a 1,2,3-triazole ring is described. Cyclic alkynyl-peptidyl resins derived from BPC194 were treated with azidopeptides derived from the antimicrobial peptide BP100 or from the bacteriocin iturin A. The cyclic alkynyl-peptidyl resins incorporated at the 3-position a propargylglycine, a glutamic acid residue derivatized with propargylamine or a lysine bearing a propioloyl group. The reactions of the cyclic alkynyl resins with the BP100-derived azidopeptides depended on the length and the sequence of the azidopeptides. The reactions were performed by treatment of the alkynyl resin with CuI and ascorbic acid, and required the presence of piperidine/DMF or DIEA in 2,6-lutidine/DMF. The latter conditions also allowed the conjugation of the alkynyl-peptidyl resin bearing a propioloyl lysine to a linear or cyclic azidopeptide derived from the cyclic moiety of iturin A.
A collection of bacterial strains obtained from a wide-range origin was screened for ability to p... more A collection of bacterial strains obtained from a wide-range origin was screened for ability to promote growth in two types of Prunus rootstocks in a commercial nursery. Only few strains promoted growth significantly and consistently, and a strong specificity for the rootstock cultivar was observed. Irrigation of plants with Pseudomonas fluorescens EPS282 and Pantoea agglomerans EPS427 significantly increased plant height and root weight of the plum Marianna 2624 and the peach-almond hybrid GF-677, respectively. Plant height showed a higher rate of growth in early stages of development (2.6-3.5 times the non-treated controls), but the effect decreased with plant age. However, in aged plants growth promotion was more significant on root weight (1.9 times the nontreated controls) than on plant height. The efficacy of growth promotion and the persistence of strains in the root environment were dependent on the bacterial inoculum concentration applied. Increases in root development were maximum at inoculum concentrations of up to 8 log 10 CFU ml −1 (ca 10 log 10 CFU L −1 of potting mix). Population levels at the optimum inoculum concentration were around 7 log 10 CFU g f.w. −1 root material at early stages of development and decreased to 4 log 10 CFU g f.w. −1 after several months of development. The best plant growthpromoting strains were very diverse in secondary metabolite production and antagonistic ability against several plant pathogens.
This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and ve... more This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. [Int Microbiol 2008; 11(4):231-236]. This article contains supporting information [SI] online, consisting of one table (T1-SI) at the Int. Microbiol. website [www.im.microbios.org].
The solid-phase synthesis of cyclic lipopeptidotriazoles derived from the cyclic decapeptide c(Ly... more The solid-phase synthesis of cyclic lipopeptidotriazoles derived from the cyclic decapeptide c(Lys-Lys 2 -Leu-Lys-Lys 5 -Phe-Lys-Lys-Leu-Gln) (BPC194), incorporating a triazolyl ring at Lys 2 and a hexanoyl group at Lys 5 , was studied. Four different strategies that required the use of five orthogonal protecting groups (Fmoc, tBu, All, pNZ, ivDde) were explored. The influence of the side-chain protection of Lys 2 and Lys 5 with the ivDde and pNZ groups was evaluated by incorporating Lys 2 (ivDde)/Lys 5 (pNZ) or Lys 2 (pNZ)/Lys 5 (ivDde).
The tree constitutes an ecosystem in which microorganisms play an essential role in its functiona... more The tree constitutes an ecosystem in which microorganisms play an essential role in its functionality. Interactions that microorganisms establish with plants may be beneficial or detrimental and are of extreme importance in the exploitation of trees in agriculture as crop production systems. Fruit trees, especially pomefruit trees including apple, pear and several ornamentals are of great economic importance but its production is affected by several diseases. Fungal and bacterial fruit tree diseases are mainly controlled with chemical fungicides and bactericides, but health and environmental concerns about the use of chemical pesticides have result in strong regulatory actions and have stimulated the development of beneficial microorganisms as microbial pesticides. Up to now, several microorganisms have been registered in different countries and in the EU as biocontrol agents (BCA) covering mainly fire blight, soil-borne fungal diseases and postharvest fruit fungal rot. The key aspects in the success of this technology for disease control are related to biosafety and environmental impact of biocontrol agents, the traceability and fate in the environment and food chain, the improvement by physiological, genetic engineering or the use of mixtures or formulations as well as the industrial production and development of delivery systems for treatment application to trees.
The control of plant pathogens is mainly based on copper compounds and antibiotics. However, the ... more The control of plant pathogens is mainly based on copper compounds and antibiotics. However, the use of these compounds has some limitations. They have a high environmental impact and the use of antibiotics is not allowed in several countries. Moreover, resistance has been developed to these pathogens. The identification of new agents able to fight plant pathogenic bacteria and fungi will represent an alternative to currently used antibiotics or pesticides. Antimicrobial peptides are widely recognized as promising candidates, however naturally occurring sequences present drawbacks that limit their development. These include susceptibility to protease degradation and low bioavailability. To overcome these problems, research has focused on the introduction of unnatural amino acids into lead peptide sequences. In particular, we have improved the biological profile of antimicrobial peptides active against plant pathogenic bacteria and fungi by incorporating triazolyl, biaryl and D-amino acids into their sequence. These modifications and their influence on the biological activity are summarized.
Pathogen aggressiveness on the host was studied as one of the influencing factors in the variabil... more Pathogen aggressiveness on the host was studied as one of the influencing factors in the variability of the efficiency of biocontrol by Pantoea agglomerans. The effect of the relative dose of the pathogen and biocontrol agent (BCA) on efficacy of biocontrol was analyzed in six postharvest pathogens (Rhizopus stolonifer, Botrytis cinerea, Penicillium expansum, Monilinia laxa, Penicillium digitatum and Penicillium italicum), five fruit types (apple, pear, nectarine, strawberry, orange) and two strains of P. agglomerans. Median effective dose (ED 50 ) of the pathogens and of the BCAs was estimated by fitting data to a hyperbolic saturation model. The raw data required were either obtained from the literature or generated by the appropriate experiments. The ED 50 of the pathogens covered a range from 1 to 475 spores/wound and of the BCA strains ranged from 207 to 30,000 cfu/wound. The efficiency of the P. agglomerans strains was estimated as the ratio between the ED 50 for the BCA and the pathogen, and ranged from 7 to 25,000 cfu/spore. Low values indicate high efficiencies. A significant inverse relationship was observed between the efficiency of biocontrol and the ED 50 of the pathogen on the corresponding host, indicating that the higher the aggressiveness of the pathogen the lower the efficiency of the BCA. It is expected that this relationship can be extended to other postharvest biocontrol-pathogen systems.
Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high... more Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants.
Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hyd... more Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of L-Phe with D-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 mM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t R ), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a D-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t R . The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.
We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheL... more We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheLysLysIleLeuLysTyrLeu-NH 2 ) by introducing a triazole ring in the peptide backbone or onto the side chain of a selected residue. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens, and for their cytotoxic effects on eukaryotic cells and tobacco leaves. Their proteolytic susceptibility was also analyzed. The antibacterial activity and the hemolysis were influenced by the amino acid that was modified with the triazole as well as by the absence of presence of a substituent in this heterocyclic ring. We identified sequences active against the bacteria Xanthomonas axonopodis pv. vesicatoria, Erwinia amylovora, Pseudomonas syringae pv. syringae (MIC of 1.6-12.5 M), and against the fungi Fusarium oxysporum (MIC < 6.2-12.5 M) with low hemolytic activity (0-23% at 50 M), high stability to protease digestion and no phytotoxicity. These peptidotriazoles constitute good candidates to design new antimicrobial agents.
A concise solid-phase synthesis of cyclic lipopeptides derived from the antimicrobial peptide c(L... more A concise solid-phase synthesis of cyclic lipopeptides derived from the antimicrobial peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln) (BPC194) was accomplished. Three different synthetic routes were explored. Best results were obtained using a protocol that includes as key steps: (i) synthesis of the cyclic peptidyl resin incorporating the Lys residue to be acylated protected at the N ε -amino group with an ivDde group, (ii) selective removal of the ivDde group, and (iii) acylation. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens and for their cytotoxic effects on eukaryotic cells. A sequence with high antimicrobial activity and low hemolysis was identified, constituting a good candidate for the design of new antimicrobial agents. † Electronic supplementary information (ESI) available: HPLC chromatograms and mass spectrometry spectra of linear peptides and linear lipopeptides. MS(ESI) of cyclic peptides, and MS(ESI) and HRMS(ESI) of cyclic lipopeptides BPC498, BPC500, BPC502, BPC524 and BPC530. See
Journal of the Science of Food and Agriculture, 2008
BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were exam... more BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were examined for aerobic, coliform, and yeast and mould counts using normalised methods. Listeria spp., Listeria monocytogenes and Salmonella spp. were detected by real time polymerase chain reaction (QPCR) after enrichment. RESULTS: Aerobic plate counts ranged from <10 to >10 9 colony-forming units (CFU) g −1 , with the lowest and highest counts recorded for fruits and sprouts respectively. The highest incidence level of coliforms was found in ready-to-eat vegetables, with up to 65.7% of samples containing from 5 to 9 log 10 CFU g −1 . Yeasts and moulds showed their highest incidence level between 5 and 6 log 10 CFU g −1 , with an overall range from <2 to 9 log 10 CFU g −1 . Salmonella spp., Listeria spp. and L. monocytogenes were detected in 0.67, 2.7 and 0.9% respectively of the total samples examined.
Journal of the Science of Food and Agriculture, 2008
BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were exam... more BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were examined for aerobic, coliform, and yeast and mould counts using normalised methods. Listeria spp., Listeria monocytogenes and Salmonella spp. were detected by real time polymerase chain reaction (QPCR) after enrichment. RESULTS: Aerobic plate counts ranged from <10 to >10 9 colony-forming units (CFU) g −1 , with the lowest and highest counts recorded for fruits and sprouts respectively. The highest incidence level of coliforms was found in ready-to-eat vegetables, with up to 65.7% of samples containing from 5 to 9 log 10 CFU g −1 . Yeasts and moulds showed their highest incidence level between 5 and 6 log 10 CFU g −1 , with an overall range from <2 to 9 log 10 CFU g −1 . Salmonella spp., Listeria spp. and L. monocytogenes were detected in 0.67, 2.7 and 0.9% respectively of the total samples examined.
Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy c... more Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.) À1 , which in turn decreased progressively to 4-5 log CFU (g f.w.) À1 after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.) À1 , thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.
This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and ve... more This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. [Int Microbiol 2008; 11(4):231-236]. This article contains supporting information [SI] online, consisting of one table (T1-SI) at the Int. Microbiol. website [www.im.microbios.org].
ABSTRACT The solid-phase conjugation of the antimicrobial peptide c(KKLKKFKKLQ) (BPC194) to a lin... more ABSTRACT The solid-phase conjugation of the antimicrobial peptide c(KKLKKFKKLQ) (BPC194) to a linear or cyclic sequence through a 1,2,3-triazole ring is described. Cyclic alkynyl-peptidyl resins derived from BPC194 were treated with azidopeptides derived from the antimicrobial peptide BP100 or from the bacteriocin iturin A. The cyclic alkynyl-peptidyl resins incorporated at the 3-position a propargylglycine, a glutamic acid residue derivatized with propargylamine or a lysine bearing a propioloyl group. The reactions of the cyclic alkynyl resins with the BP100-derived azidopeptides depended on the length and the sequence of the azidopeptides. The reactions were performed by treatment of the alkynyl resin with CuI and ascorbic acid, and required the presence of piperidine/DMF or DIEA in 2,6-lutidine/DMF. The latter conditions also allowed the conjugation of the alkynyl-peptidyl resin bearing a propioloyl lysine to a linear or cyclic azidopeptide derived from the cyclic moiety of iturin A.
A collection of bacterial strains obtained from a wide-range origin was screened for ability to p... more A collection of bacterial strains obtained from a wide-range origin was screened for ability to promote growth in two types of Prunus rootstocks in a commercial nursery. Only few strains promoted growth significantly and consistently, and a strong specificity for the rootstock cultivar was observed. Irrigation of plants with Pseudomonas fluorescens EPS282 and Pantoea agglomerans EPS427 significantly increased plant height and root weight of the plum Marianna 2624 and the peach-almond hybrid GF-677, respectively. Plant height showed a higher rate of growth in early stages of development (2.6-3.5 times the non-treated controls), but the effect decreased with plant age. However, in aged plants growth promotion was more significant on root weight (1.9 times the nontreated controls) than on plant height. The efficacy of growth promotion and the persistence of strains in the root environment were dependent on the bacterial inoculum concentration applied. Increases in root development were maximum at inoculum concentrations of up to 8 log 10 CFU ml −1 (ca 10 log 10 CFU L −1 of potting mix). Population levels at the optimum inoculum concentration were around 7 log 10 CFU g f.w. −1 root material at early stages of development and decreased to 4 log 10 CFU g f.w. −1 after several months of development. The best plant growthpromoting strains were very diverse in secondary metabolite production and antagonistic ability against several plant pathogens.
This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and ve... more This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. [Int Microbiol 2008; 11(4):231-236]. This article contains supporting information [SI] online, consisting of one table (T1-SI) at the Int. Microbiol. website [www.im.microbios.org].
The solid-phase synthesis of cyclic lipopeptidotriazoles derived from the cyclic decapeptide c(Ly... more The solid-phase synthesis of cyclic lipopeptidotriazoles derived from the cyclic decapeptide c(Lys-Lys 2 -Leu-Lys-Lys 5 -Phe-Lys-Lys-Leu-Gln) (BPC194), incorporating a triazolyl ring at Lys 2 and a hexanoyl group at Lys 5 , was studied. Four different strategies that required the use of five orthogonal protecting groups (Fmoc, tBu, All, pNZ, ivDde) were explored. The influence of the side-chain protection of Lys 2 and Lys 5 with the ivDde and pNZ groups was evaluated by incorporating Lys 2 (ivDde)/Lys 5 (pNZ) or Lys 2 (pNZ)/Lys 5 (ivDde).
The tree constitutes an ecosystem in which microorganisms play an essential role in its functiona... more The tree constitutes an ecosystem in which microorganisms play an essential role in its functionality. Interactions that microorganisms establish with plants may be beneficial or detrimental and are of extreme importance in the exploitation of trees in agriculture as crop production systems. Fruit trees, especially pomefruit trees including apple, pear and several ornamentals are of great economic importance but its production is affected by several diseases. Fungal and bacterial fruit tree diseases are mainly controlled with chemical fungicides and bactericides, but health and environmental concerns about the use of chemical pesticides have result in strong regulatory actions and have stimulated the development of beneficial microorganisms as microbial pesticides. Up to now, several microorganisms have been registered in different countries and in the EU as biocontrol agents (BCA) covering mainly fire blight, soil-borne fungal diseases and postharvest fruit fungal rot. The key aspects in the success of this technology for disease control are related to biosafety and environmental impact of biocontrol agents, the traceability and fate in the environment and food chain, the improvement by physiological, genetic engineering or the use of mixtures or formulations as well as the industrial production and development of delivery systems for treatment application to trees.
The control of plant pathogens is mainly based on copper compounds and antibiotics. However, the ... more The control of plant pathogens is mainly based on copper compounds and antibiotics. However, the use of these compounds has some limitations. They have a high environmental impact and the use of antibiotics is not allowed in several countries. Moreover, resistance has been developed to these pathogens. The identification of new agents able to fight plant pathogenic bacteria and fungi will represent an alternative to currently used antibiotics or pesticides. Antimicrobial peptides are widely recognized as promising candidates, however naturally occurring sequences present drawbacks that limit their development. These include susceptibility to protease degradation and low bioavailability. To overcome these problems, research has focused on the introduction of unnatural amino acids into lead peptide sequences. In particular, we have improved the biological profile of antimicrobial peptides active against plant pathogenic bacteria and fungi by incorporating triazolyl, biaryl and D-amino acids into their sequence. These modifications and their influence on the biological activity are summarized.
Pathogen aggressiveness on the host was studied as one of the influencing factors in the variabil... more Pathogen aggressiveness on the host was studied as one of the influencing factors in the variability of the efficiency of biocontrol by Pantoea agglomerans. The effect of the relative dose of the pathogen and biocontrol agent (BCA) on efficacy of biocontrol was analyzed in six postharvest pathogens (Rhizopus stolonifer, Botrytis cinerea, Penicillium expansum, Monilinia laxa, Penicillium digitatum and Penicillium italicum), five fruit types (apple, pear, nectarine, strawberry, orange) and two strains of P. agglomerans. Median effective dose (ED 50 ) of the pathogens and of the BCAs was estimated by fitting data to a hyperbolic saturation model. The raw data required were either obtained from the literature or generated by the appropriate experiments. The ED 50 of the pathogens covered a range from 1 to 475 spores/wound and of the BCA strains ranged from 207 to 30,000 cfu/wound. The efficiency of the P. agglomerans strains was estimated as the ratio between the ED 50 for the BCA and the pathogen, and ranged from 7 to 25,000 cfu/spore. Low values indicate high efficiencies. A significant inverse relationship was observed between the efficiency of biocontrol and the ED 50 of the pathogen on the corresponding host, indicating that the higher the aggressiveness of the pathogen the lower the efficiency of the BCA. It is expected that this relationship can be extended to other postharvest biocontrol-pathogen systems.
Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high... more Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants.
Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hyd... more Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of L-Phe with D-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 mM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t R ), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a D-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t R . The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.
We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheL... more We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheLysLysIleLeuLysTyrLeu-NH 2 ) by introducing a triazole ring in the peptide backbone or onto the side chain of a selected residue. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens, and for their cytotoxic effects on eukaryotic cells and tobacco leaves. Their proteolytic susceptibility was also analyzed. The antibacterial activity and the hemolysis were influenced by the amino acid that was modified with the triazole as well as by the absence of presence of a substituent in this heterocyclic ring. We identified sequences active against the bacteria Xanthomonas axonopodis pv. vesicatoria, Erwinia amylovora, Pseudomonas syringae pv. syringae (MIC of 1.6-12.5 M), and against the fungi Fusarium oxysporum (MIC < 6.2-12.5 M) with low hemolytic activity (0-23% at 50 M), high stability to protease digestion and no phytotoxicity. These peptidotriazoles constitute good candidates to design new antimicrobial agents.
A concise solid-phase synthesis of cyclic lipopeptides derived from the antimicrobial peptide c(L... more A concise solid-phase synthesis of cyclic lipopeptides derived from the antimicrobial peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln) (BPC194) was accomplished. Three different synthetic routes were explored. Best results were obtained using a protocol that includes as key steps: (i) synthesis of the cyclic peptidyl resin incorporating the Lys residue to be acylated protected at the N ε -amino group with an ivDde group, (ii) selective removal of the ivDde group, and (iii) acylation. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens and for their cytotoxic effects on eukaryotic cells. A sequence with high antimicrobial activity and low hemolysis was identified, constituting a good candidate for the design of new antimicrobial agents. † Electronic supplementary information (ESI) available: HPLC chromatograms and mass spectrometry spectra of linear peptides and linear lipopeptides. MS(ESI) of cyclic peptides, and MS(ESI) and HRMS(ESI) of cyclic lipopeptides BPC498, BPC500, BPC502, BPC524 and BPC530. See
Journal of the Science of Food and Agriculture, 2008
BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were exam... more BACKGROUND: Commercially available fruits and raw and ready-to-eat vegetables (n = 445) were examined for aerobic, coliform, and yeast and mould counts using normalised methods. Listeria spp., Listeria monocytogenes and Salmonella spp. were detected by real time polymerase chain reaction (QPCR) after enrichment. RESULTS: Aerobic plate counts ranged from <10 to >10 9 colony-forming units (CFU) g −1 , with the lowest and highest counts recorded for fruits and sprouts respectively. The highest incidence level of coliforms was found in ready-to-eat vegetables, with up to 65.7% of samples containing from 5 to 9 log 10 CFU g −1 . Yeasts and moulds showed their highest incidence level between 5 and 6 log 10 CFU g −1 , with an overall range from <2 to 9 log 10 CFU g −1 . Salmonella spp., Listeria spp. and L. monocytogenes were detected in 0.67, 2.7 and 0.9% respectively of the total samples examined.
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Papers by Esther Badosa