Unravelling the plant beneficial potential of Bacillus nakamurai BDI-IS1

Agriculture in Burundi and many other tropical developing countries experiences severe challenges such as high disease and pest incidence, and the use of highly hazardous pesticides (more than 30% banned in Europe), posing serious threats to human health (food insecurity and intoxication) and environment. Bacterial biocontrol agents are safe and eco-friendly alternatives to the use of these chemicals and some Bacillus isolates belonging to species of the B. subtilis group are particularly interesting due to their strong antagonistic activity toward microbial phytopathogens mediated by bioactive secondary metabolites (BSMs) and to their capacity to form spores facilitating formulation of stable products. These species include B. velezensis, which is among the most successful commercialized biopesticides. Unfortunately, the exploitation of these bacteria is still a myth in Burundi and many other tropical African countries. Moreover, the efficacy of these products to control diseases in the field may be inconsistent and lower than expected, notably because the bacteria are not always well adapted to the quite specific abiotic conditions prevailing in some local agro-ecosystems as observed in Burundi.
The global objective of this work was to contribute to the development of biocontrol in Burundi by implementing the use of well-adapted bacilli to local agro-ecosystems. We sampled Burundian field soils at different locations and search for Bacillus-like isolates. Out of these samples, we selected one particular isolate named BDI-IS1 displaying strong and broad-spectrum antimicrobial activity via in vitro confrontation assays. Based on phenotypic and molecular traits, BDI-IS1 was identified as a new strain of the rare species Bacillus nakamurai. By coupling comparative genomics with metabolomics, we performed the first entire characterization of the secondary metabolome for a member of this species. BDI-IS1 has the potential to secrete two cyclic lipopeptides (surfactin and iturin A), one polyketide (dihydro)bacillaene, a siderophore bacillibactin, a dipeptide bacilysin and a diversity of lanthipeptides including plantazolicin, amylocyclicin, bacinapeptin and LCI. Many of these compounds are conserved across B. nakamurai strains and may serve as a chemical fingerprint for the species with plausible taxonomical relevance. We further generated multiple BDI-IS1 mutants and showed, through reverse genetics, the specific involvement of some BSMs in the observed antagonistic activities. Additionally, we unveiled for the first time the biocontrol potential of this species B. nakamurai. Indeed, greenhouse experiments showed that BDI-IS1 provides excellent protection of maize and tomato plants (up to 65% disease reduction) against northern corn leaf blight and tomato late blight, respectively. From a mechanistic point of view, our data indicate that BDI-IS1 may act either via direct antagonism or via the stimulation of systemic resistance in the host plant or a combination of both.
We next assessed the adaptability of BDI-IS1 to the stressful conditions of low pH and low temperature, prevailing often in Burundi and many tropical agro-ecosystems. It first revealed that BDI-IS1 has a remarkable tolerance to the acidic and mild-cold stress, up to pH 4.6 and 15°C. Moreover, these stresses have contrasted effects on the production of BSMs and the associated antagonistic activities against bacterial pathogens. Except for surfactin production which is not affected by decreasing temperature and bacilysin secretion favored by low pH, the production of BSMs was negatively affected by these limiting conditions. Antibacterial activity was either preserved or suppressed at low temperature. Low pH (pH 5) led to either an increase (three-fold) or a decrease in antagonistic activity against two low pH-tolerant bacterial pathogens, P. carotovorum and C. michiganensis, respectively. This remodelling of established interspecies relationships may be explained by the abiotic stress-driven modulation of the production of known BSMs involved in inter-bacterial competition and other unknown BSMs or effectors.
In depth functional genomics showed also that BDI-IS1 is equipped with core genes related to the rhizospheric lifestyle and with additional abiotic stress resistance-related genes compared to other related Bacillus spp., suggesting an improved adaptation to environmental stress which would enable its successful root colonisation and persistence. In addition, we showed that it has the genetic ability to secrete plant growth hormones and molecules mediating plant stress tolerance, as well as the genetic potential to solubilize insoluble forms of phosphorus.
Interestingly, in many aspects, our data reveal that BDI-IS1 may be more promising than the star of the biocontrol market B. velezensis QST713 (commercialized as Serenade Aso®). It exhibited an equal or superior performance not only in terms of biocontrol potential but also considering adaptation to acidic and low temperature-related stresses.
In a more practical perspective, we also investigated cost-effective but efficient options for biomass production and formulation of BDI-IS1, according to the socio-economic context of smallholder farmers in Burundi. Solid-state fermentation using locally sourced lignocellulosic substrates derived from agricultural residues was found to be a promising way of multiplying the bacterium with a high spore content in the fermented substrate. This was further formulated as a dry mixture of residual substrate embedded with BDI-IS1 spores and secreted metabolites, and this product was found to be more effective than QST713 in protecting maize against northern leaf blight disease.
Prior to its registration and widespread dissemination to rural farmers, additional experiments are still needed to ensure BDI-IS1 overall efficacy upon application in the field at different locations in the country and to reduce disease incidence in other pathosystems. The innocuity of the bacterium towards other (beneficial) microorganisms, consistency of the efficacy of the bioproduct across different production batches and stability upon long-term storage must also be evaluated. Nevertheless, our work features this strain as a new very promising tool to foster a more productive agriculture that is respectful of human health and the environment, while also improving local organic waste management and thereby helping to achieve the UN’s sustainable development goals.