In vitro antagonism of an actinobacterial Kitasatospora isolate against the plant pathogen Phytophthora citricola as elucidated with ultrahigh resolution mass spectrometry

Many soil microorganisms antagonistic to soil borne plant pathogens are well known for their ability to control diseases in situ. A variety of substances, like lytic enzymes, siderophores and antibiotics, produced by these organisms have the potential to protect roots against pathogens. Understanding the ecology and a functional assessment of antagonistic microbial communities in soil requires in-depth knowledge of the mechanisms involved in these interactions, a challenging task in complex systems if low-resolution methods are applied. We propose an information-rich strategy of general relevance, composed of adequate preconcentration in conjunction with ultrahigh resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS) and nuclear magnetic resonance (NMR) spectroscopy to identify any bioactive substances in complex systems. This approach is demonstrated on the specific example of substance identification considered responsible for in vitro antagonism of an actinobacterial antagonist isolated from European beech (Fagus sylvatica) rhizosphere soil against the oomycetous root rot pathogen Phytophthora citricola. The isolate belonging to the genus Kitasatospora exhibited strong antibiosis against the oomycete in vitro. The bioactive substance was observed to exhibit a molar mass of 281.1699 g/mol in positive electrospray ionization mass spectra, and the high mass accuracy of the ICR-FT/MS measurements allowed a precise assignment of a molecular formula that was found identical to the macrolide polyketide cycloheximide C15H23NO4 + H+; its identity was then unequivocally confirmed by the information-rich atomic signature of proton NMR spectroscopy. In conclusion, the combination of the near orthogonal methods (pre)fractionation, ultrahigh-resolution ICR-FT mass spectrometry (yielding molecular and MSn fragment signatures) and nuclear magnetic resonance spectroscopy (providing atomic signatures) has been found capable of identifying a biocontrol active compound of Kitasatospora active against Phytophthora citricola expediently, quickly, and accurately. This straightforward approach is of general applicability to elucidate biocontrol mechanisms in any complex system with improved efficiency.