Bio-Inspired Rhamnolipids, Cyclic Lipopeptides and a Chito-Oligosaccharide Confer Protection against Wheat Powdery Mildew and Inhibit Conidia Germination.

Plant protection is mainly based on the application of synthetic pesticides to limit yield losses resulting from diseases. However, the use of more eco-friendly strategies for sustainable plant protection has become a necessity that could contribute to controlling pathogens through a direct antimicrobial effect and/or an induction of plant resistance. Three different families of natural or bioinspired compounds originated from bacterial or fungal strains have been evaluated to protect wheat against powdery mildew, caused by the biotrophic Blumeria graminis f.sp. tritici (Bgt). Thus, three bio-inspired mono-rhamnolipids (smRLs), three cyclic lipopeptides (CLPs, mycosubtilin (M), fengycin (F), surfactin (S)) applied individually and in mixtures (M + F and M + F + S), as well as a chitosan oligosaccharide (COS) BioA187 were tested against Bgt, in planta and in vitro. Only the three smRLs (Rh-Eth-C12, Rh-Est-C12 and Rh-Succ-C12), the two CLP mixtures and the BioA187 led to a partial protection of wheat against Bgt. The higher inhibitor effects on the germination of Bgt spores in vitro were observed from smRLs Rh-Eth-C12 and Rh-Succ-C12, mycosubtilin and the two CLP mixtures. Taking together, these results revealed that such molecules could constitute promising tools for a more eco-friendly agriculture.

Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis.

Bacillus subtilis is a wealth source of lipopeptide molecules such as iturins, surfactins and fengycins or plipastatins endowed with a range of biological activities. These molecules, designated secondary metabolites, are synthesized via non-ribosomal peptides synthesis (NRPS) machinery and are most often subjected to a complex regulation with involvement of several regulatory factors. To gain novel insights on mechanism regulating fengycin production, we investigated the effect of the fascinating polynucleotide phosphorylase (PNPase), as well as the effect of lipopeptide surfactin. Compared to the wild type, the production of fengycin in the mutant strains B. subtilis BBG235 and BBG236 altered for PNPase has not only decreased to about 70 and 40%, respectively, but also hampered its antifungal activity towards the plant pathogen Botrytis cinerea. On the other hand, mutant strains BBG231 (srfAA-) and BBG232 (srfAC-) displayed different levels of fengycin production. BBG231 had registered an important decrease in fengycin production, comparable to that observed for BBG235 or BBG236. This study permitted to establish that the products of pnpA gene (PNPase), and srfAA- (surfactin synthetase) are involved in fengycin production.

Study of the correlation between fengycin promoter expression and its production by Bacillus subtilis under different culture conditions and the impact on surfactin production.

This work aimed to rely expression of the fengycin promoter to fengycin production under different culture conditions. To this end, Bacillus subtilis BBG208, derived from BBG21, which is a fengycin overproducing strain carrying the green fluorescent protein (GFP) under the control of fengycin promoter, was used to assess the effects of different carbon and nitrogen sources on surfactin and fengycin production and the fengycin promoter expression. The data showed that some carbon sources oriented synthesis of one family of lipopeptides, while most of the nitrogen sources allowed high co-production of fengycin and surfactin. High expressions of promoter Pfen and fengycin synthesis were obtained with urea or urea + ammonium mixture as nitrogen source and mannitol as carbon source. Moreover, temperature, pH and oxygenation influenced their biosynthesis based on the nutrition conditions. Optimization of the production medium increased the fengycin production to 768 mg L-1, which is the highest level reported for this strain. This study defines the suitable nutrient conditions allowing as well the highest expression of the fengycin promoter and portrays the conditions relying on the fengycin and surfactin production.

Influence of promoters on the production of fengycin in Bacillus spp.

Fengycin is a promising antifungal lipopeptide from Bacillus spp. synthesized by non-ribosomal peptide synthetases (NRPS). In this work, fengycin production of a spontaneous fengycin overproducing strain, Bacillus subtilis BBG21, was first compared to those of B. subtilis BBG111 (a 168 derivative), B. subtilis ATCC 21332 and Bacillus amyloliquefaciens FZB42 under two different experimental conditions. In both conditions, very high fengycin yields were obtained from strain BBG21 (480 mg/L) in comparison to its counterparts. The high efficiency of the fengycin promoter (Pfen) of BBG21 compared to the promoter of BBG111 and FZB42 was confirmed using a GFP reporter gene. Under all tested conditions, this promoter showed highest expression in comparison to the other strains. The highest fluorescence rate was obtained with mannitol as carbon source. In addition, when the Ppps promoter from B. subtilis BBG111 was replaced by promoter Pfen from BBG21, fengycin production increased about 10-fold, while no fengycin overproduction was observed when replacement was performed with Pfen from ATCC 21332. Comparative sequence analysis of these different promoters revealed one nucleotide modification in the UP element known for its importance in the regulation process. This point mutation is thus responsible for overproduction of fengycin in BBG21.