Metabolite profiling of Bacillus species with nematicidal activity

Abstract

Root-knot nematodes continue to be a global problem in agriculture causing major economic losses. Various Bacillus spp. have the potential to inhibit the Meloidogyne root-knot nematode populations. Although many studies conclude that the secondary metabolites of relevant Bacillus spp. are responsible for nematicidal activity, the specific metabolites are not characterised. Subsequently, the efficacy and reproducibility of biocontrol products are questionable. The aim of this study was to compare the metabolic profiles of Bacillus spp. with known nematicidal activity to bacteria without nematicidal activity. For this purpose, four Bacillus spp. known to exhibit inhibitory effects towards second-stage juveniles (J2) of Meloidogyne incognita, namely B. cereus, B. firmus, B. subtilis and B. pumilus were compared to two bacteria without known nematicidal activity – Escherichia coli and B. soli. Bacterial strains were cultivated in two types of media, namely Luria-Bertani (LB) broth as a complex medium and minimal broth (MB) as a simpler medium. This was done to evaluate the effect of the medium composition on bacterial metabolism and also on metabolomics analyses performed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). The first step was to do nematicidal bioassays to confirm the nematicidal activity of cell-free filtrates obtained from the bacteria. During the bioassays the motile and paralysed J2 nematodes were quantified after exposure to different concentrations of cell-free filtrates presumably containing secondary metabolites of the bacteria. From the results obtained it was evident that all of the selected Bacillus spp., as well as E. coli, had nematicidal effects on the M. incognita J2. When cultivated in LB broth with optimised incubation times, B. firmus and B. pumilus showed the highest nematode paralysis in the bioassays. However, when cultivated in MB, B. firmus and B. cereus showed the highest nematode paralysis. Due to the nematicidal activity observed for E. coli and B. soli, the bioassays were repeated using E. coli OP50 as control. This strain caused the lowest levels of paralysis in all assays and was therefore a more appropriate control that should be used in further studies. Untargeted metabolomics distinguished between metabolite profiles from the different Bacillus spp. Moreover, there was a clear difference between profiles when bacteria were cultivated in the different media. Bacterial cultures produce extracellular metabolites in response to their surrounding environment and it is critical that specific bacterial species are matched with the optimal cultivation media to ensure reproducible production of compounds of interest before identification of metabolites is attempted.