Studying trophic interactions between a plant pathogen and two different antagonistic microorganisms using a 13C-labeled compound and isotope ratio mass spectrometry

RATIONALE: The study of the interactions among microorganisms, especially between pathogens and other microorganisms, is a very useful way to identify possible biocontrol agents (BCAs). In this study we verified the capability of delta C-13 analysis using isotope ratio mass spectrometry (IRMS) to detect active parasitism or metabolic assimilation of C-13-labeled Armillaria mellea (plant pathogen) by Trichoderma atroviride and Pseudomonas fluorescens (two BCAs). METHODS: The three microorganisms were labeled in pure-culture using a specific medium to which D-glucose C-13 was added. The delta C-13 analysis of mycelia/ cells and DNA was undertaken using IRMS at different times, to study the uptake kinetics of C-13. The mechanisms of interaction were studied by implementing dual-culture tests and measuring the delta C-13 values of the two BCAs after 29 days of contact with the labeled pathogen. RESULTS: A. mellea absorbed C-13 more slowly (plateau at 21 days) than T. atroviride and P. fluorescens (3 and 1 day, respectively) in pure-culture. The maximum delta C-13 values were higher in A. mellea and T. atroviride mycelia (8,019.9 parts per thousand and 10,383.7 parts per thousand, respectively) than in P. fluorescens (953.4% in cells). In dual-culture the mycelia of T. atroviride which remained in direct contact with labeled A. mellea showed an increased delta C-13 value with respect to the unlabeled treatment (66.4% and -26.6 parts per thousand, respectively), due to active interaction. Lower assimilation of C-13 was detected in P. fluorescens. CONCLUSIONS: This work demonstrates that IRMS can be used for the in-depth study of direct parasitism and interaction process between biocontrol agents and labeled pathogens, allowing the screening of potential new BCAs. Copyright (C) 2012 John Wiley & Sons, Ltd.