Isotope ratio mass spectrometry identifies soil microbial biocontrol agents having trophic relations with the plant pathogen Armillaria mellea

An understanding of the types of interactions that take place between plant pathogens and other microorganisms in the natural environment is crucial in order to identify new potential biocontrol agents. The use of microorganisms labelled with stable isotopes is a potentially useful method for studying direct parasitisation of a given pathogen or assimilation of the pathogen’s metabolites by microorganisms. A microorganism labelled with a stable isotope can be monitored in the environment and isotope ratio mass spectrometry can detect whether it is directly parasitised or its metabolites are used by other microorganisms. In this study, we isolated 158 different species of fungi and bacteria from soil and assayed their biocontrol potential against a plant pathogen (Armillaria mellea) by coupling a dual-culture test with mass spectrometry analysis of the 13C isotope in the microorganisms in presence of 13C-labelled A. mellea. The microorganisms affected the pathogen by means of antibiosis phenomena (total or partial inhibition of pathogen growth, alteration of its morphology) and by antagonism, probably resulting from competition for space and nutrients or from mycoparasitism. Isotope ratio mass spectrometry was used to identify direct trophic interactions between microorganisms and the pathogen as in dual cultures as in soil microcosms. Six fungi and one bacterium were found to display the best active trophic behaviour against the pathogenin dual cultures; three microorganisms were discarded due to their plant pathogen potential. Trichoderma harzianum, Pseudomonas fluorescens and Rhodosporidium babjevae were selected to carry out the experiments. T. harzianum inhibited pathogen development (rate of inhibition 80±0.19%) and its ı 13C values increased (244.03±36.70‰) in contact with 13C-labelled A. mellea. Lower levels of antagonism and correspondingly lower assimilation of 13C were detected in P. fluorescens and R. babjevae. Only T. harzianum maintained mycoparasitic activity in the soil microcosm, showing a 13C value of 1.97±2.24‰after one month in co-presence with the labelled pathogen. This study provides support for the use of isotope ratio mass spectrometry as an additional tool in screening for potential biocontrol agents