Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae.

Spodoptera littoralis, one of the most serious and destructive agricultural pests in the world, is very susceptible to Vip3 toxin. In order to develop a new efficient bioinsecticide and to prevent the development of resistance by the target pest, insecticidal activity of biosurfactant produced by Bacillus amyloliquefaciens AG1 was evaluated against S. littoralis. Bioassays revealed the susceptibility of the first instar larvae of this pest to AG1 biosurfactant with an LC50 of 245ng/cm2. Moreover, the histopathology examination of the larval midgut treated by AG1 biosurfactant showed vacuolization, necrosis and disintegration of the basement membrane. Binding experiments revealed that the AG1 biosurfactant recognized three putative receptors located in the brush border membrane vesicles of S. littoralis with sizes of 91, 72 and 64kDa. Competition assays using biotinylated metabolites indicated that AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same S. littoralis receptors. When combined, AG1 biosurfactant and Vip3Aa16 showed an additive effect against S. littoralis larvae. These findings suggested that B. amyloliquefaciens AG1 biosurfactant could be a promising biocontrol agent to eradicate S. littoralis and to prevent resistance development by this pest.

Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato.

The aim of this study is to evaluate the efficacy of the strain Bacillus subtilis V26, a local isolate from the Tunisian soil, to control potato black scurf caused by Rhizoctonia solani. The in vitro antifungal activity of V26 significantly inhibited R. solani growth compared to the untreated control. Microscopic observations revealed that V26 caused considerable morphological deformations of the fungal hyphae such as vacuolation, protoplast leakage and mycelia crack. The most effective control was achieved when strain V26 was applied 24h prior to inoculation (protective activity) in potato slices. The antagonistic bacterium V26 induced significant suppression of root canker and black scurf tuber colonization compared to untreated controls with a decrease in incidence disease of 63% and 81%, respectively, and promoted plant growth under greenhouse conditions on potato plants. Therefore, B. subtilis V26 has a great potential to be commercialized as a biocontrol agent against R. solani on potato crops.

Bacillus amyloliquefaciens AG1 biosurfactant: Putative receptor diversity and histopathological effects on Tuta absoluta midgut.

The use of biosurfactant in pest management has received much attention for the control of plant pathogens, but few studies reported their insecticidal activity. The present study describes the insecticidal activity of biosurfactant extracted from Bacillus amyloliquefaciens strain AG1. This strain produces a lipopeptide biosurfactant exhibiting an LC50 of about 180ng/cm(2) against Tuta absoluta larvae. Accordingly, the histopathologic effect of this biosurfactant on T. absoluta larvae showed serious damages of the midgut tissues including rupture and disintegration of epithelial layer and cellular vacuolization. By PCR, we showed that this biosurfactant could be formed by several lipopeptides and polyketides including iturin, fengycin, surfactin, bacyllomicin, bacillaene, macrolactin and difficidin. Binding experiment revealed that it recognized five putative receptors located in the BBMV of T. absoluta with sizes of 68, 63, 44, 30 and 19kDa. Therefore, biosurfactant AG1 hold potential for use as an environmentally friendly agent to control the tomato leaf miner.

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.

Correlation between synthesis variation of 2-alkylquinolones and the antifungal activity of a Burkholderia 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.

Use of two bacteria for biological control of bayoud disease caused by Fusarium oxysporum in date palm (Phoenix dactylifera L) seedlings.

The Bayoud, caused by Fusarium oxysporum f. sp. albedinis (Foa), is the most destructive disease of date palm (Phoenix dactylifera L) in Morocco and Algeria, with no effective control strategy yet available. In this work, two bacteria, Bacillus amyloliquefaciens strain Ag1 (Ag) and Burkholderia cepacia strain Cs5 (Cs), were examined for their potential to control this disease. Both bacterial strains inhibited both growth and sporulation of Foa. They released compounds into the culture medium, which resulted into cytological changes in Foa's mycelial structure. When Jihel-date palm plantlets, a susceptible cultivar, were induced with these bacteria, the size of the necrosis zone, which reflected the spreading of the pathogen, was reduced by more than 70%, as compared with uninduced controls. To further investigate the mechanisms of such disease reduction, phenolic compounds and peroxidase activity were assessed. One month after inoculation, date palm defense reactions against Foa were different depending on the bacterium used, B. cepacia led to higher accumulation of constitutive caffeoylshikimic acid isomers while B. amyloliquefaciens triggered the induction of new phenolic compounds identified as hydroxycinnamic acid derivatives. Peroxidase activity has also been stimulated significantly and varied with the bacterial strain used and with Foa inoculation. These results add to the promising field of investigation in controlling Bayoud disease.

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.

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.

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.