Biological control of Botrytis cinerea using the antagonistic and endophytic Burkholderia cepacia Cs5 for vine plantlet protection

Antifungal activity of the Burkholderia cepacia Cs5 was tested in vitro and in vivo for the control of Botrytis cinerea . Bacterial biomass was significantly improved by the amendment of ZnSO(4), Mo(7)(NH(4))(6)O(24), and mannitol to the NBY medium; consequently, the amount of the secreted fungicides was increased. The quantification of B. cinerea inhibition, in liquid and solid conditions, showed an important sensitivity of this fungus to the strain Cs5 fungicides. Microscopic monitoring impact of these fungicides on mycelium structure showed an important increase in their diameter and ramifications in the presence of 0.75% supernatant. For the in vivo application of the strain Cs5, Vitis vinifera plantlets were inoculated with a Cs5 bacterial suspension, then with B. cinerea spores. The plantlets protection was total and durable when these two inoculations were made 3 weeks apart, which is the time for the endophytic bacterium to colonize the plantlets up to the top leaves. This protection is due to Cs5 antagonism and the elicitation of the plantlets self-defense via the root overgrowth.Ministry of Higher Education and Scientific ResearchL'activité antifongique de la souche (Cs5) a été testée in vitro et in vivo en vue du contrôle de . La biomasse bactérienne a été améliorée par l'ajout de ZnSO4, Mo7(NH4)6O24 et du mannitol au milieu de culture NBY qui a, par conséquent, augmenté la quantité de fongicides secrétés. La quantification de l'inhibition de B. cinerea dans les conditions de culture liquid et solide a montré une forte sensibilité de ce champignon aux fongicides de Cs5. Le suivi de l'impact de ces fongicides sur les structures des mycéliums a montré une augmentation importante de leur diamètre et de leurs ramifications en présence de 0,75 % de surnageant. Pour l'application in vivo de la souche Cs5, les plantules de ont été inoculées par une suspension bactérienne de Cs5, puis par une suspension de spores de B. cinerea. Une protection totale et durable des plantules était atteinte lorsque les deux inoculations étaient réalisées à trois semaines d'intervalle, soit le temps nécessaire à la bactérie endophyte pour coloniser les plantules jusqu'aux feuilles supérieures. Cette protection est due à l'antagonisme de Cs5 et à l'induction de l'auto-défense des plantules via la sur-croissance des racines

Heterologous expression and secretion of an antifungal Bacillus subtilis chitosanase (CSNV26) in Escherichia coli

The aims of the study were the production improvement, the purification, the characterization and the activity investigation of chitosanase CSNV26 of Bacillus subtilis (V26). The gene csnV26 encoding for this protein was amplified and cloned in the pBAD vector then expressed in Escherichia coli (Top10). The SDS-PAGE and zymogram analysis of the recombinant protein showed that it has two active forms sized 27 and 31 kDa, corresponding to the protein with and without signal peptide. This protein has the particularity of being secreted by Top10-pBAD-csnV26 with a high yield of 6.2 g/l. The HPLC purification of CSNV26 from supernatant confirmed the presence of the two sizes. The investigation of the CSNV26 thermostability showed that the pure protein is highly stable keeping 68 % of its activity after 30-min treatment at 100 °C, contrarily to the protein present within the supernatant of E. coli and B. subtilis (V26). The molecular dynamics study of the predicted structure of protein in both forms showed that the presence of the peptide signal in the form of 31 kDa gave it a remarkable thermal stability. The antifungal activity of CSNV26 was evidenced on Rhizopus nigricans and Rhizopus oryzae. Indeed, it has provoked an alteration and embrittlement of their hyphae with onset of protoplast

Le modèle atomique entre le savoir de l'expert et le savoir de l'enseignant. Quelles conséquences curriculaires.

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Le modèle atomique est-il la réalité ? Conceptions d'étudiants et d'enseignants de sciences physiques et chimiques.

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Environmental Burkholderia cepacia strain CS5 acting by two analogous alkyl-quinolones and a didecyl-phthalate against a broad spectrum of phytopathogens fungi

An environmental Burkholderia cepacia strain named Cs5 was isolated and identified first using API biochemical identification system and then with 16S rDNA and recA sequence homology search. This bacterium exhibited a broad spectrum of fungicidal activities against Alternaria alternata, Aspergillus niger, Fusarium culmorum, F. graminearum, F. oxysporum and Rhizoctonia solani. In the liquid conditions, the MIC of A. niger and R. solani were reached with, respectively, 1.25-2% of the Cs5 liquid culture supernatant. However, in the solid conditions, the same inhibition was caused in the presence of 3% of the Cs5 supernatant. The exhibition of these two fungi at low concentrations of supernatant Cs5 caused various morphological changes of their mycelia which were observed by confocal microscopy. Three antifungal compounds, named Cs5-255, Cs5-257 and Cs5-446, were purified from the Cs5 culture. The structural analysis of these molecules showed that Cs5-255 and Cs5-257 are analogous and belonged to the alkyl-quinolone family, while Cs5-446 was a didecyl-phthalate, isolated for the first time from a bacterium.Ministere de l’Enseignement Superieur et de la Recherche Scientifiqu

Antifungal activities of an endophytic Pseudomonas fluorescens strain Pf1TZ harbouring genes from pyoluteorin and phenazine clusters

Pseudomonas fluorescens Pf1TZ was inhibitory in vitro to a number of phytopathogenic fungi and could protect vine plantlets against Botrytis cinerea. Total protection was reached after 3 weeks of bacterial inoculation. The endophytism of Pf1TZ was confirmed by confocal microscopy using its inherent fluorescence. The molecular characterization of Pf1TZ indicated the presence of genes from clusters encoding pyoluteorin and phenazine. The chromatographic purification and LC-MS(n) analysis revealed that the most active one has a molecular mass of 504 Da.Ministry of Higher Education and Scientific Researc

Correlation between synthesis variation of 2-alkylquinolones and the antifungal activity of aBurkholderia cepacia strain collection

Twenty epiphytic and rhizospheric bacterial strains harbouring strong antifungal activities were isolated from the Tunisian environment. This group of bacteria was identified as Burkholderia cepacia genomovar I using 16S rDNA and recA fragment gene sequence analyses for two selected strains and RFLP technique for the eighteen other ones. This identification did not show variability between isolates despite the significant differences in the antifungal activities of their culture supernatant and the organic crude extract against Aspergillus niger and other phytopathogenic fungi. Chromatographic and mass spectrometric analyses of these extracts allowed us to confirm the difference between strains of the group. Their metabolic production showed differences in term of contents and quantities of secreted molecules, particularly those which were identified to be involved in the antifungal activities. Two metabolites, named Bc-255 and Bc-257 secreted by the entire group at different amounts, have been purified and tested separately against A. niger. Bc-255 showed an activity twice as high as those shown by Bc-257. The structural characterization of these two compounds by mass spectrometry and nuclear magnetic resonance spectroscopy allowed their identification as two analogous 2-alkylquinolones with only one difference at the alkyl chain.Ministry of Higher Education and Scientific Researc