Potential use and mode of action of the new strain<i>Bacillus thuringiensis</i>UM96 for the biological control of the grey mould phytopathogen<i>Botrytis cinerea</i>

Abstract

The potential use of <i>Bacillus thuringiensis</i> UM96 as a biocontrol agent for the grey mould phytopathogen <i>Botrytis cinerea</i> was evaluated. In order to dissect the mode of action of this UM96 strain, we also examined the role of lytic activities in the antagonism. First, <i>B. thuringiensis</i> UM96 was characterised based on 16S rRNA and <i>gyrA</i> gene sequencing and phenotypic traits. Petri dish biocontrol assays demonstrated that when strain UM96 was inoculated 24 h previous to <i>B. cinerea,</i> the mycelial growth was inhibited by up to 70%. Test for lytic enzymes activities of cellulase and glucanase was negative. Chitinase was the only positive enzyme activity in two different culture media. PCR detection of the <i>chiB</i> gene was also positive. Chitinolytic supernatants, obtained from rich and minimal media supplemented with colloidal chitin as the sole carbon source, from <i>B. thuringiensi</i>s UM96 showed a strong inhibitory effect of <i>B. cinerea</i> that was not observed with heat-treated supernatant. Interestingly, when the supernatant was supplemented with 100 µM allosamidin, a chitinase specific inhibitor, the antagonistic activity was suppressed significantly. A lack of chitinase activity was also observed in allosamidin-treated supernatants. Our pathogenic <i>B. cinerea</i> strain also exhibited susceptibility to pure <i>Streptomyces griseus</i> chitinase. Finally, the chitinolytic strain <i>B. thuringiensis</i> UM96 was able to protect <i>Medicago truncatula</i> plants in vitro from <i>B. cinerea</i> infection and significantly reduced the necrotic zones and root browning of the plants. Together, these results suggest a potential use of <i>B. thuringiensis</i> UM96 for the biological control of <i>B. cinerea</i> and a role for chitinases during the antagonism displayed.