title: Imprint Desorption Electrospray Ionization Mass Spectrometry Imaging for Monitoring Secondary Metabolites Production during Antagonistic Interaction of Fungi.
abstract: Direct analysis of microbial cocultures grown on agar media by desorption electrospray ionization mass spectrometry (DESI-MS) is quite challenging. Due to the high gas pressure upon impact with the surface, the desorption mechanism does not allow direct imaging of soft or irregular surfaces. The divots in the agar, created by the high-pressure gas and spray, dramatically change the geometry of the system decreasing the intensity of the signal. In order to overcome this limitation, an imprinting step, in which the chemicals are initially transferred to flat hard surfaces, was coupled to DESI-MS and applied for the first time to fungal cocultures. Note that fungal cocultures are often disadvantageous in direct imaging mass spectrometry. Agar plates of fungi present a complex topography due to the simultaneous presence of dynamic mycelia and spores. One of the most devastating diseases of cocoa trees is caused by fungal phytopathogen Moniliophthora roreri. Strategies for pest management include the application of endophytic fungi, such as Trichoderma harzianum, that act as biocontrol agents by antagonizing M. roreri. However, the complex chemical communication underlying the basis for this phytopathogen-dependent biocontrol is still unknown. In this study, we investigated the metabolic exchange that takes place during the antagonistic interaction between M. roreri and T. harzianum. Using imprint-DESI-MS imaging we annotated the secondary metabolites released when T. harzianum and M. roreri were cultured in isolation and compared these to those produced after 3 weeks of coculture. We identified and localized four phytopathogen-dependent secondary metabolites, including T39 butenolide, harzianolide, and sorbicillinol. In order to verify the reliability of the imprint-DESI-MS imaging data and evaluate the capability of tape imprints to extract fungal metabolites while maintaining their localization, six representative plugs along the entire M. roreri/T. harzianum coculture plate were removed, weighed, extracted, and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Our results not only provide a better understanding of M. roreri-dependent metabolic induction in T. harzianum, but may seed novel directions for the advancement of phytopathogen-dependent biocontrol, including the generation of optimized Trichoderma strains against M. roreri, new biopesticides, and biofertilizers.
authors.affiliation: Centre for Research in Mass Spectrometry, Department of Chemistry, York University , Chemistry Building, 4700 Keele Street, M3J 1P3 Toronto, Ontario, Canada.|ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas , Rua Josué Castro, s/n Cidade Universitária, CEP 13083-970 Campinas, São Paulo, Brazil.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Tata
authors.affiliation: Centre for Research in Mass Spectrometry, Department of Chemistry, York University , Chemistry Building, 4700 Keele Street, M3J 1P3 Toronto, Ontario, Canada.
authors.forename: Consuelo
authors.initials: C
authors.lastname: Perez
authors.affiliation: Centre for Research in Mass Spectrometry, Department of Chemistry, York University , Chemistry Building, 4700 Keele Street, M3J 1P3 Toronto, Ontario, Canada.|Department of Natural Active Principles and Toxicology, School of Pharmaceutical Sciences, São Paulo State University , Rodovia Araraquara-Jaú, km 1, CEP 14 801-902 Araraquara, São Paulo, Brazil.
authors.forename: Michel L
authors.initials: ML
authors.lastname: Campos
authors.affiliation: Department of Biology, York University , 4700 Keele Street, M3J 1P3 Toronto, Ontario, Canada.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Bayfield
authors.affiliation: ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas , Rua Josué Castro, s/n Cidade Universitária, CEP 13083-970 Campinas, São Paulo, Brazil.
authors.forename: Marcos N
authors.initials: MN
authors.lastname: Eberlin
authors.affiliation: Centre for Research in Mass Spectrometry, Department of Chemistry, York University , Chemistry Building, 4700 Keele Street, M3J 1P3 Toronto, Ontario, Canada.
authors.forename: Demian R
authors.initials: DR
authors.lastname: Ifa
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000363:Agaricales / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D001688:Biological Products / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002073:Butanes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018920:Coculture Techniques; D003512:Cyclohexanones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007783:Lactones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D064210:Secondary Metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014242:Trichoderma / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D001688:Biological Products; D002073:Butanes; D003512:Cyclohexanones; D007783:Lactones; C577797:harzianolide; C453419:sorbicillinol; C004511:butenolide; D015107:4-Butyrolactone