Biocontrol and plant growth-promoting potentiality of bacteria isolated from compost extract.

The use of compost extracts is steadily increasing, offering an attractive way for plant growth enhancement and disease management replacing chemical pesticides. In this study, potential mechanisms involved in plant growth promotion and suppressive activity against fungal diseases, of a compost extract produced from poultry manure/olive husk compost, were investigated. Results of physico-chemical and microbiological investigations showed high ability to reduce Fusarium oxysporum, Alternaria alternata, Aspergillus niger and Botrytis cinerea growth. The suppressive ability detected using confrontation test and the phytostimulatory effect tested on tomato seeds were related mainly to its microbial population content. Among 150 bacterial strains, isolated from the compost extract, 13 isolates showed antifungal activity against the four tested plant pathogenic fungi. Their identification based on 16S rRNA gene sequence revealed they belonged to different species of the genus Bacillus, Alcaligenes, Providencia and Ochrobactrum. When tested for their ability to produce cell wall degradation enzymes using specific media, the majority of the 13 isolates were shown to synthesize proteases, lipases and glucanases. Similarly, the best part of them showed positive reaction for plant growth promoting substances liberation, biosurfactant production and biofilm formation. In vivo tests were carried out using tomato seeds and fruits and proved that 92% of strains improved tomato plants vigor indexes when compared to the control and 6 among them were able to reduce decay severity caused by B. cinerea over 50%. Principal component analysis showed an important correlation between in vitro and in vivo potentialities and that Bacillus siamensis CEBZ11 strain was statistically the most effective strain in protecting tomato plants from gray mould disease. This study revealed the selected strains would be useful for plant pathogenic fungi control and plant growth promotion.

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.