Article pubs.acs.org/ac
Imprint Desorption Electrospray Ionization Mass Spectrometry Imaging for Monitoring Secondary Metabolites Production during Antagonistic Interaction of Fungi Alessandra Tata,†,‡ Consuelo Perez,† Michel L. Campos,†,§ Mark A. Bayfield,∥ Marcos N. Eberlin,‡ and Demian R. Ifa*,† †
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 § 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 ∥ Department of Biology, York University, 4700 Keele Street, M3J 1P3 Toronto, Ontario, Canada S Supporting Information *
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 DESIMS 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.
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monitor the metabolite production of live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and P. aeruginosa.6,7 As already defined elsewhere,8 DESI is a spray-based ambient technique that allows the direct sampling of surfaces in the
ass spectrometry based approaches represented a revolution in microbiology.1 Mass spectrometry imaging (MSI) is a technique used to visualize the spatial distribution of lipids, biomarker, metabolites, peptides, or proteins by their mass-to-charge ratio (m/z).2−4 MSI analysis of the spatial distribution of microbial compounds has been successfully performed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) by growing the microorganisms Pseudomonas aeruginosa and Aspergillus fumigatus directly on the MALDI targets.5 Nano-desorption electrospray ionization mass spectrometry (nano-DESI-MS) was applied to © XXXX American Chemical Society
Received: September 23, 2015 Accepted: November 23, 2015
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harzianum, several analytical tools can be applied. To correctly interpret the complex metabolic data obtained from interaction of the two microorganisms, the spectrum of secondary metabolites assigned to the coculture are compared to those produced separately by both M. roreri and T. harzianum in monoculture. Although M. roreri’s morphology,31 mitochondria genome,32 nuclear genome,33 and protein expression34 were recently characterized, a metabolic study was never carried out. On the other hand, the peptaibols released by T. harzianum have been previously identified by conventional analytical techniques such as high-pressure liquid chromatography−mass spectrometry (HPLC−MS),30 nuclear magnetic resonance (NMR),35 electrospray ionization mass spectrometry (ESIMS),36,37 fast atom bombardment mass spectrometry (FABMS),38 and MALDI-MS.39 The exact characterization of the small metabolites involved in the T. harzianum/M. roreri system could be of great interest to biotechnologists that can modulate the T. harzianum behavior improving the production process of its antibiotic compounds against M. roreri.
open air. When used with a software-controlled moving stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z is correlated with the spatial distribution of chemical species.9 Interestingly, only a few microbiology studies by DESI-MS have been reported.10−14 Because of the high spray and gas pressure, the mechanism does not allow direct imaging of soft or irregular surfaces. This limitation makes DESI-MS analysis of microorganisms grown on agar media quite challenging, especially in the case of fungi. Indeed, unlike bacteria, fungal cultures are very soft and uneven because of the simultaneous presence of aerial hyphae and spores on the agar plate. In order to overcome this restriction, the use of blotting or imprinting of fungi coupled to DESI-MS, in which the chemicals are initially transferred to flat hard surfaces, was recently proposed15 and is now tested on fungal cocultures. Therefore, in this work, we use imprint imaging desorption electrospray ionization mass spectrometry (imprint DESI-MS),16−19 confirmed by liquid chromatography−highresolution mass spectrometry (LC−HRMS), to investigate the production of secondary metabolites that could be associated with the antagonistic interaction between two fungi that cohabit in cacao plants, the phytopathogen Moniliophthora roreri and the endophyte Trichoderma harzianum. Cacao (Theobroma cacao L.) is an important tropical crop since the seeds of its fruits are the raw material for production of chocolate. However, fungal cocoa pests represent a serious threat to the commercial production of chocolate worldwide. The two most devastating fungal diseases affecting cacao production are the witches broom (WB) caused by M. perniciosa and the frosty pod rot (FPR) caused by M. roreri.20 FPR is prevalent throughout Central and South America, with the exception of Brazil,21 and can cause losses in crop yields estimated between 40% and 90% of the infected plantations, with the possibility of highly susceptible fields suffering complete yield loss resulting in cocoa farm abandonment.22 Recently, global initiatives have aimed to set up environmentally friendly disease-management strategies that include good farming practices, plantation of resistant cocoa varieties, and the rational or minimal use of chemicals as well as biological control strategies.20 One promising avenue of biological control of pests may be based on the application of endophytic microorganisms to antagonize and parasitize the phytopathogen.23−26 Indeed, coexistence of endophytic microorganisms and phytopathogens that share the same space and nutrients can affect their mutual growth, adaption pattern and development. One of the most extensively used endophytic microorganisms applied in biocontrol activities is the Trichoderma spp. Trichoderma species are soil-borne organisms associated with the roots of plants. They produce and release several secondary metabolites (such as trichoviridin, harzianic acids, gliotoxin, gliovirin, viridian, viridiol, trichorzianines) which induce local or systemic plant resistance and exhibit antagonist properties against phytopathogens.27 Although ability of T. harzianum to antagonize and parasitize M. roreri has previously been established,28,29 a precise understanding of the exchanged metabolites involved in this specific interaction is still lacking. Critically for this study, the identity and amount of small secondary metabolites produced can be altered based on the nature of the interacting phytopathogen;30 specifically, secondary metabolites produced by T. harzianum depend on the specific antagonist phytopathogen and the balance between biosynthesis and biotransformation of the metabolite. In order to study the metabolite induction caused by M. roreri in T.
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MATERIAL AND METHODS Materials. Potato dextrose broth (PDB), potato dextrose agar (PDA), yeast extract, water, acetonitrile (ACN), isopropyl alcohol, and ethyl acetate were purchased from Sigma-Aldrich (Oakville, ON, Canada). The tape used was 3M Highland, 5910. The microorganisms T. harzianum T22 strain (ATCC 20847) and M. roreri (ATCC 64239) were bought from Cedarlane (Burlington, ON, Canada). Monocultures. In order to prepare the PDA plates, 19.5 g of potato dextrose agar powder and 2.5 g of yeast extract were diluted in 500 mL of distilled water, autoclaved, poured in sterile plastic plates, and polymerized overnight. The stock pellet of T. harzianum was resuspended with 5 mL of sterile water right after delivery and rehydrated overnight. A volume of 10 μL of resuspended T. harzianum stock solution was inoculated in a PDA plate. Potato dextrose broth was prepared by solubilizing 19.5 g of potato dextrose powder in 500 mL of distilled water and autoclaved. A volume of 20 μL of ready-touse M. roreri stock was previously inoculated into PDB for 8 days at 25 °C and 200 rpm. Afterward, a volume of 20 μL of the broth was plated on PDA and kept in the incubator at 25 °C for 21 days until analysis by DESI-MSI and LC−HRMS. Cocultures. M. roreri was previously grown in PDB for 8 days at 24 °C and 200 rpm, and then a volume of 20 μL of broth was inoculated into a side of the PDA plate at a few millimeters from the edge. After 96 h, a volume of 10 μL of T. harzianum was inoculated into the opposite side of the plate. After 21 days, the cocultures were analyzed by DESI-MS, DESIMS imaging and LC−HRMS Extraction of the Metabolites. The monocultures and the cocultures were both extracted. Plugs of 6 mm diameter of the M. roreri, T .harzianum, and interaction zone of the M. roreri/T. harzianum coculture were cut and weighed. One plug of each microbial culture was put into a sterile tube, and 1 mL of ethyl acetate was added. The tube was sonicated for 30 min and centrifuged for 5 min at 20 850g. The supernatant was separated, and 1 mL of isopropyl alcohol was added to the pellet. The tube was sonicated for 30 min and then centrifuged for 5 min at 20 850g. The two supernatants were joined and dried down in a concentrator for 2 h. The samples were stored at −80 °C until LC−HRMS analysis LC−HRMS. The extracts were resuspended with 50 μL of ACN/H2O (20:80), and a volume of 10 μL (per 246 mg of B
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terized, and had its metabolites screened by imprint DESI-MS. After an initial whitish growth, M. roreri becomes salmon-cream in a few days from the inoculation (Figure 1A). At this stage of
initial plug) of this solution was diluted to 1 mL of solution in ultrapure water. All samples were filtered through 0.22 μm nylon membrane prior injection. A NanoLC-Ultra System (Eksigent, Singapore) coupled to the hybrid linear ion trap− orbitrap mass spectrometer (LTQ-Orbitrap Elite, Thermo Scientific, U.S.A.) was used for the LC−HRMS analysis. The injection was performed directly in the 10-port valve using a 2 μL loop. The separation was carried out through a Biobasic C18 column (50 mm × 0.075 mm and 5 μm particle size). The mobile phase was a gradient of water (A) and acetonitrile (B), both with 0.1% (v/v) formic acid. The gradient started at 90% A and 10% B, which was converted to 10% A and 90% B over 33 min, and this proportion remained until the end of the run at 36 min. The nanoelectrospray was obtained using a PicoTip (New Objective, U.S.A.) emitter. The spray voltage was set to 2 kV, capillary temperature to 250 °C, and S-lens rf level of 68%. Full scan mass spectra were acquired in the positive ion mode over the range of m/z 200−600. Tandem mass spectrometry (MS/MS) was performed using collision-induced dissociation with collision energy of 25−35% (manufacturer’s unit). Morphological Characterization. The morphology of the microorganisms was compared to that reported in the literature. Optical microscope pictures were taken using a Zeiss LSM 700. DESI-MS and DESI-MSI Experiments. Both monocultures and cocultures were manually imprinted for 5 s on 3M Highland, 5910 tape. The imprinted tapes were placed in a labbuilt 2D moving stage using double-sided tape and analyzed by DESI-MS in the positive ion mode. All MS experiments were performed using a Thermo Fisher Scientific LTQ mass spectrometer (San Jose, CA, U.S.A.). Data were acquired and processed using Xcalibur 2.0 software (Thermo Fisher Scientific). Typical instrumental parameters used were 4.5 kV capillary voltage and 275 °C capillary temperature. A solution of H2O/ACN (80:20) was used as spray solvent and delivered at the flow rate of 3 μL min−1. Mass spectra were acquired, in positive ion mode, over the mass range from m/z 200 to 2000. A sprayer-to-surface distance of 1.0−1.5 mm, a sprayer-to-inlet distance of 4−8 mm, an incident spray 52°, and a collection angle of 10° were used. The identification of the analyte ions was confirmed by MS/MS using collision-induced dissociation with collision energy of 25−35% (manufacturer’s unit). In order to acquire DESI-MS images from monocultures and cocultures, the imprints were scanned in horizontal rows separated from 150 to 200 μm vertical steps until the entire sample was analyzed. The mass range was generally 200−1000 Da. The lines were scanned at a constant velocity in the range of 414−714 μm, and the scan time was used in the range of 0.36−0.56 s. Under these conditions, lateral spatial resolution (pixel size) in the range of 150−200 μm could be achieved. Data Processing. The MS spectra were processed by QualBrowserXcalibur. The lab-made ImageCreator ver. 3.0 software was used to convert the Xcalibur 2.0 mass spectra files (.raw) into a format compatible with BioMap (freeware, http:// www.maldi-msi.org/), which was used to process the mass spectral data and to generate 2D spatially accurate ion images. Identification of the metabolites was performed by MS/MS, literature search, and exact mass measurement.
Figure 1. Visual comparison of M. roreri at different stages of its life cycle: (A) salmon-cream M. roreri at hyphal; (B) dark brown M. roreri with spores’s powdery mass.
growth, prior to sporulation, hyphal colonies form with diameters of a few millimeters. Afterward, the yellowishcream M. roreri started covering the surface of the agar plate. Three weeks after inoculation, massive sporulation occurred (Figure 1B), and M. roreri acquired a brown color. These observations are consistent with previous reports of M. roreri growth.40 The production of metabolites of M. roreri was monitored over 3 weeks. No significant metabolites were observed both by imprint DESI-MS and LC−HRMS analysis of the extract in M. roreri at the hyphal stage. After 2 weeks of growth on the PDA plate, direct imprint DESI-MS of early sporulating yellow cream M. roreri revealed the presence of the metabolites involved in the roquefortine/meleagrin pathway. The DESI-MS spectrum is characterized by ions of m/z 322.1, 324.1, 390.1, 392.1, 403.2, 404.2, 420.2, and 434.2 corresponding to protonated molecule of dehydrohistydiltryptophanyldiketopiperazine (DHTD), histidyltryptophanyldiketopiperazine (HTD), roquefortine C, roquefortine D, meleagrin−CH3O, glandicolineA, glandicoline B, and meleagrine, respectively. Such metabolites from the roquefortine/meleagrin pathway are closely related structurally (Supporting Information Figure S1) and are common in various filamentous fungi such as Penicillium spp.41 and Aspergillus spp.42 Starting with HTD synthesized from tryptophan and histidine as substrates, the biosynthesis of meleagrine is achieved through the intermediates DHDT, roquefortine C, roquefortine D, glandicoline A, and glandicoline B. In the high-mass range of the spectrum (Figure 2C), the ions of m/z 782.6, 804.6, and 820.6 correspond to the protonated molecules of phosphatidylcholines PC(36:1), PC(38:0), and PCe(38:0). The observed metabolites were assigned by imprint DESI-MS/MS and exact mass measurement by LC−HRMS (Table S1). The spatial assignment of metabolites in the M. roreri colony was obtained using DESIMS imaging (Supporting Information Figure S2). After 3 weeks from inoculation on PDA plates, M. roreri acquired a dark brown color due to the massive production of spores (Figure 1B). At this stage two new ions of m/z 436 and m/z 448 appeared in the imprint-DESI-MS spectrum (Supporting Information Figure S3). These two new ions were characterized as protonated molecules of epineoxaline and oxaline by DESI-MS/MS and LC−HRMS (Table S1). Recently, neoxaline was proposed as final product of the roquefortine/meleagrine pathway.41 Neoxaline appears to originate from a hydrogenation of meleagrin leading to the formation of oxaline.
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RESULTS AND DISCUSSION Before studying the metabolites exchanged during the antagonistic interaction between M. roreri and T. harzianum, each fungus was cultured separately, morphologically characC
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Figure 2. (A) Optical image of M. roreri generating spores. (B) Picture of yellow-cream M. roreri, 2 weeks old, on a PDA plate. (C) DESI-MS spectrum.
Figure 3. (A) DESI-MS spectrum of T. harzianum showing characteristic peptaibols. (B) Picture of 2 weeks old T. harzianum on a PDA plate. (C) Optical image of T. harzianum filamentous cells (40× lens).
We then moved on to metabolite screening of T. harzianum in monoculture. Imprint DESI-MS was performed on two week old T. harzianum growths (Figure 3), and its secondary metabolites were analyzed and identified by MS/MS (see Supporting Information Table S2 for assignment of the ions). T. harzianum is well-known for the large production of antibiotic peptaibols independent of the presence of another microorganism. These are bioactive linear peptides of 5−20 nonproteinogenic amino acids residues abundant in α-aminoisobutyric acid (Aib) and contain an N-acyl terminus and a Cterminal alcohol.37
Peptaibols, such as harzianins and trichotoxins, were observed in the mass range of m/z 700−1800 as protonated and sodium and potassium adducts. The spectrum is predominately characterized by doubly charged ions appearing around m/z 700−950. Many singly charged ions were observed in specific region of the spectrum such as m/z 1150−1230, 1400−1490, and 1700−1800. Peptaibolic ions observed by imprint DESI-MS as well as ultrahigh resolution mass spectrometry measurements and assignments are presented in Supporting Information Table S2. Note that, in the case of the trichotoxins (mass ranges 850−950 and 1700−1800 of the spectrum), the abundances of the C13 isotopologue are clearly D
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Figure 4. (A) DESI-MS imaging of a 3 weeks old coculture of T. harzianum (left side, labeled with the letter T) and sporulating brown M. roreri (right side, labeled with the letter M). The area where the interaction between the two fungi occurs is labeled with the letter I. In red the ions produced by M. roreri are reported ([Meleagrin+H]+ of m/z 434.2); in green are those belonging to T. harzianum ([T39Butenolide+H]+ of m/z 221.1, [Harzianolide+H]+ of m/z 223.1, [Sorbicillinol+H]+ of m/z 249.1, the N/A ion of m/z 319.1 and [Trichotoxin IV+Na+K]++ of m/z 896.5). The optical image and the blue scheme reported in the last panel aim at showing the overgrowth of the T. harzianum on top of the M. roreri colony. (B) DESI-MS spectrum of the coculture. (C) Microscopic image of the coculture using 40× lens. (D) Optical image of the coculture plate. The dashed line indicates the imaged area.
Figure 5. LC−HRMS relative intensities of the secondary metabolites of m/z 223.1, 221.1, 319.1, and 249.1 obtained by analyzing six plugs of coculture extracted along the plate. At the base of the graph a picture of the extracted coculture is shown. Increased relative intensities of the secondary metabolites can be observed in the interaction zone and where the overgrowth of the T. harzianum over M. roreri occurs. The data observed are in accordance with those obtained by simple imprinting DESI-MS imaging. In the inset: chemical structures of the identified secondary metabolites produced by T. harzianum while interacting with M. roreri.
The spatial assignment of the metabolites in the T. harzianum colony obtained using imprint-DESI-MS imaging is presented in Supporting Information Figure S4. We then used imprint-DESI-MS imaging of M. roreri and T. harzianum grown in coculture to assign and map the
higher than their theoretical values, in accordance with the ESIMS data described by Suwan et al.37 In Table S2 of the Supporting Information only the monoisotopic masses were reported. No significant relative intensities of small secondary metabolites were observed in the T. harzianum monoculture. E
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Analytical Chemistry Table 1. List of Specific Metabolites Produced by T. harzianum during Antagonistic Interaction against M. roreria m/z DESI-MS
m/z LC−HRMS
m/z theor
error (ppm)
assignment
221.1 223.1 249.1 319.1
221.1172 223.1331 249.1122 319.1182
221.11722 223.13290 249.11214 319.11816
−0.09 −0.90 −0.24 −0.13
T39 butenolide harzianolide sorbicillinol N/A
type of ion [M [M [M [M
+ + + +
H]+ H]+ H]+ H]+
MS/MS fragments 203, 205, 231, 301,
193, 159, 203, 277,
163, 177, 161, 273,
159, 133 133 120 255, 193
elemental formula C13H17O3 C13H19O23 C14H17O4 C17H19O7
a
The m/z value detected during imprint-DESI-MS analysis, the m/z values of the ions detected during the LC−HRMS experiments, the assignment, the type of ion, the fragments observed by LC−MS/MS, and elemental formula of the ion species are reported.
metabolites exchanged during phytopathogenesis. The experiment was carried out by inoculating 20 μL of M. roreri broth (grown for 8 days in PDB) on a PDA plate. After 96 h of incubation at 25 °C, a volume of 10 μL of T. harzianum stock was inoculated into the other side of the plate. In the coculture, because of their mutual competition, both fungi grow more slowly. Production of metabolites from T. harzianum grown in coculture was screened by imprint DESI-MS after 1 week, 10 days, and 3 weeks. Since the production of peptaibols is not phytopathogen-dependent, we focused our attention on the screening of low molecular weight inhibitors. Previous work has demonstrated that the production of these vary in relation to (i) the specific compound, (ii) the phytopathogen used for the elicitation, (iii) the viability of the elicitor, and (iv) the balance between elicited biosynthesis and biotransformation rates.43 In order to obtain better quality images we scanned the sample in the mass range of m/z 200−1000, limiting the analysis to the low-mass metabolites and doubly charged peptaibols. After 1 week (prior to the joining of the M. roreri and T. harzianum colonies) only the M. roreri metabolites were detected. After 10 days both meleagrin metabolites and doubly charged peptaibols could be seen in the mass range of m/z 700−950, although the two colonies were not still close enough to interact. After 3 weeks of coculture we observed the appearance of ions of m/z 221.1, 223.1, 249.1, and 319.1 in the DESI-MS spectrum, which notably we had not previously observed in the monocultures (Figure 4, parts A and B). These ions are mainly localized in the interaction and overgrowth zones (Figure 4A, last panel). The ions of m/z 221.1, 223.1, and 249.1 were assigned as protonated metabolites T39 butenolide, harzianolide, and sorbicillinol, respectively (inset of Figure 5). The data obtained by ambient mass spectrometry were confirmed by LC−HRMS and LC−HRMS/MS. Their exclusive presence in the coculture and their absence in both T. harzianum, M. roreri monocultures, and agar were also verified by analyzing the extracts by LC− HRMS (Supporting Information Figures S5−S8). The exact mass measurements, the fragmentation pattern, and molecular formula are reported in Table 1. The ion of m/z 319.1 was not identified, but its production by Trichoderma spp. has previously been reported with the same high-resolution mass measurement.44 In order to verify the DESI-MS imaging data and confirm the capability of tape imprints to extract fungal metabolites while maintaining their localization, six representative plugs of 6 mm diameter each along the entire M. roreri/T. harzianum coculture plate were removed, weighed, extracted, and analyzed by LC− HRMS. The relative abundances of the secondary metabolite ions of m/z 223.1, 221.1, 319.1, and 249.1 were then obtained by analyzing the extracts of the six plugs by LC−HRMS (Figure 5). Increased relative abundances for the four metabolites were observed in the interaction zone and in the overgrowth zone between T. harzianum over M. roreri. The overall LC−HRMS data are in good accordance with those obtained by simple tape
imprinting followed by DESI-MS imaging, validating the high efficiency of the tape imprint. The production of T39 butenolide by T. harzianum strain T22 has previously been described during coculture with the phytopathogens Botrys cinerea and Rhizoctonia solani.30,43 The production of harzianolide was stimulated in T. harzianum by the phytopathogens Phythophthora cinnamomi and B. cinerea, but it was not observed in a plate confrontation assay with R. solani. Both T39 butenolide and harzianolide have been demonstrated to harbor antifungal activity against some phythopathoges.30 Sorbicillinol is a key intermediate in the biosynthesis of bisorbicillinoids, a family of secondary metabolites produced by Trichoderma spp.40,45 To the best of our knowledge this is the first observation of sorbicillinolin T. harzianum cultures. Among varying activities, bisorbicillinoids are reported to present cytotoxicity46 as well as affect the feeding preference of insects that attack crops.47 Our data confirm the production of several secondary metabolites by T. harzianum that may rely on the phytopathogenic interaction with the antagonist microorganism M. roreri. Assay to confirm the antifungal activity of harzianolide and T39 butenolide, using isolated compounds, is ongoing in our laboratory. The validation of the antibiotic properties of these metabolites may have a significant beneficial impact on the management of FPR disease in cacao plants in the future.
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CONCLUSIONS In this study, for the first time, an ambient mass spectrometry imaging approach was applied on the study of the metabolite exchange that regulates biocontrol systems based on microbial antagonism. Specifically, the exchange of metabolites between the highly commercially relevant phytopathogen M. roreri and the biocontrol agent T. harzianum was investigated. Metabolites belonging to the roquefortine/meleagrin pathway were monitored, characterized, and mapped by imprint DESI-MS in M. roreri monoculture; this represents the first observation of these compounds in the Moniliophthora genus. Furthermore, we report the first identification of fungal peptaibols of T. harzianum by ambient mass spectrometry. Finally, four metabolites produced by T. harzianum during coculture with M. roreri were identified by imprint-DESI-MS imaging. The suitability of imprint-DESI-MS imaging to the direct screening and localization of fungal metabolites involved in the microbial networking was successfully corroborated by LC−HRMS. Our results may provide a better understanding of metabolite exchange between these two fungi, not only enhancing our knowledge of the mechanisms involved in biocontrol systems but also suggesting potential directions for improvement of the Trichoderma strains used against M. roreri. Experiments are ongoing in our laboratory to confirm the antifungal potential of harzianolide and T39 butenolide against M. roreri. NMR experiments may be beneficial to verify structural assignments. F
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03614. Observed M. roreri metabolites, DESI-MS imaging of the colony and M. roreri and T. harzianum metabolites, list of peptaibolic ions observed in T. harzianum cultures, and LC−HRMS chromatograms of secondary metabolites (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Jyotsna Vinayak for the help in taking the images by optical microscopy, Dr. Valeria Micheli and Rawaa Hussein for technical advice, the FAPESP Agency (2013/15575-9 and 2014/01724-5), and the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support.
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DOI: 10.1021/acs.analchem.5b03614 Anal. Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.analchem.5b03614 Anal. Chem. XXXX, XXX, XXX−XXX
