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

ABSTRACT

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 δ(13)C analysis using isotope ratio mass spectrometry (IRMS) to detect active parasitism or metabolic assimilation of (13)C-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 (13)C was added. The δ(13)C analysis of mycelia/cells and DNA was undertaken using IRMS at different times, to study the uptake kinetics of (13)C. The mechanisms of interaction were studied by implementing dual-culture tests and measuring the δ(13)C values of the two BCAs after 29 days of contact with the labeled pathogen. RESULTS: A. mellea absorbed (13)C more slowly (plateau at 21 days) than T. atroviride and P. fluorescens (3 and 1 day, respectively) in pure-culture. The maximum δ(13)C values were higher in A. mellea and T. atroviride mycelia (8,019.9‰ and 10,383.7‰, 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 δ(13)C value with respect to the unlabeled treatment (66.4‰ and -26.6‰, respectively), due to active interaction. Lower assimilation of (13)C 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.