Dynamics of Metabolite Induction in Fungal Co-cultures by Metabolomics at Both Volatile and Non-volatile Levels

Abstract

Fungal co-cultivation has emerged as a promising way for activating cryptic biosynthetic pathways and discovering novel antimicrobial metabolites. For the success of such studies, a key element remains the development of standardized co-cultivation methods compatible with high-throughput analytical procedures. To efficiently highlight induction processes, it is crucial to acquire a holistic view of intermicrobial communication at the molecular level. To tackle this issue, a strategy was developed based on the miniaturization of fungal cultures that allows for a concomitant survey of induction phenomena in volatile and non-volatile metabolomes. Fungi were directly grown in vials, and each sample was profiled by head space solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS), while the corresponding solid culture medium was analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS) after solvent extraction. This strategy was implemented for the screening of volatile and non-volatile metabolite inductions in an ecologically relevant fungal co-culture of Eutypa lata (Pers.) Tul. & C. Tul. (Diatrypaceae) and Botryosphaeria obtusa (Schwein.) Shoemaker (Botryosphaeriaceae), two wooddecaying fungi interacting in the context of esca disease of grapevine. For a comprehensive evaluation of the results, a multivariate data analysis combining Analysis of Variance and Partial Least Squares approaches, namely AMOPLS, was used to explore the complex LC-HRMS and GC-MS datasets and highlight dynamically induced compounds. A time-series study was carried out over 9 days, showing characteristic metabolite induction patterns in both volatile and non-volatile dimensions. Relevant links between the dynamics of expression of specific metabolite production were observed. In addition, the antifungal activity of 2-nonanone, a metabolite incrementally produced over time in the volatile fraction, was assessed against Eutypa lata and Botryosphaeria obtusa in an adapted bioassay set for volatile compounds. This compound has shown antifungal activity on both fungi and was found to be co-expressed with a known

Antifungal potential of secondary metabolites involved in the interaction between citrus pathogens

“…To screen for new antifungal compounds with potential to protect citrus fruits and control postharvest diseases, we applied a co-culture strategy involving P. digitatum and another citrus pathogen, P. citrinum . Co-culture is a strategy inspired by nature in which the competition between the microorganisms can induce the production of new metabolites 8 . In previous work, the co-cultivation between Trichophyton rubrum and Bionectria ochroleuca induced the production of a new sulfated analogue of PS-990, suggesting that this compound is further sulfated during the fungal interaction 26 .…”

“…Microorganisms are one of the main sources for natural products with useful biological activities, i.e., potential antifungals, antibiotics, anticancer agents, surfactants 8,9 . However, besides the genetic potential and diversity of microbes, many microbial biosynthetic genes are not activated in the unnatural cultivation conditions used in laboratory and only a few part of the metabolites are accessible 10 .…”

An Integrative Multi-Omics Workflow to Address Multifactorial Toxicology Experiments

“…In this approach, the ANOVA-based partitioning of the sources of variation (experimental factors) is followed by a joint analysis of the ANOVA submatrices by means of a supervised multiblock algorithm based on the OPLS framework. Several recent publications reported the successful application of the AMOPLS strategy to the analysis of multifactorial omics experiments [23,30,31].…”

Dynamics of Chemical Diversity during Co‐Cultures: An Integrative Time‐Scale Metabolomics Study of Fungal Endophytes Cophinforma mamane and Fusarium solani

“…Various co‐culture experiments have been studied, such as the co‐cultivation of Trametes versicolor and Ganoderma applanatum , the results of which have been shown to induce two new compounds, N ‐(4‐methoxyphenyl)formamide 2‐ O ‐β‐D‐xyloside and N ‐(4‐methoxyphenyl)formamide 2‐ O ‐β‐D‐xylobioside; [2] Trichophyton rubrum and Bionectria ochroleuca induced five de novo compounds including 4‐hydroxysulfoxy‐2,2‐dimethylthielavin; [3] the co‐cultures of two endophytic fungus Alternaria tenuissima and Nigrospora sphaerica significantly increased the production of some polyketides, including antifungal stemphyperylenol; [4] metabolites in Stereum hirsutum were up‐regulated after co‐cultivation with Coprinus disseminates [5] . With the combination of metabolomics approach for new compounds identification, co‐culture is a powerful strategy for new drug discovery [6] …”

“…In vitro fungal co‐cultures have traditionally been carried out on a solid medium, as fungi grow well in these conditions and are prompt to colonize unexplored regions with nutrients [7] . This approach allowed the study of interaction patterns and metabolic changes that take place at the mycelia front [6] . Fungal co‐cultures were generally performed in 9 cm–15 cm Petri dish in a given time of incubation [4,8–10] .…”