Molecular Networking-Based Metabolome and Bioactivity Analyses of Marine-Adapted Fungi Co-cultivated With Phytopathogens

Abstract

Fungi represent a rich source of bioactive metabolites and some are marketed as alternatives to synthetic agrochemicals against plant pathogens. However, the culturability of fungal strains in artificial laboratory conditions is still limited and the standard mono-cultures do not reflect their full spectrum chemical diversity. Phytopathogenic fungi and bacteria have successfully been used in the activation of cryptic biosynthetic pathways to promote the production of new secondary metabolites in co-culture experiments. The aim of this study was to map the fungal diversity of Windebyer Noor, a brackish lake connected to Baltic Sea (Germany), to induce the chemical space of the isolated marine-adapted fungi by co-culturing with phytopathogens, and to assess their inhibitory potential against six commercially important phytopathogens. Out of 123 marine-adapted fungal isolates obtained, 21 were selected based on their phylogenetic and metabolite diversity. They were challenged with two phytopathogenic bacteria (Pseudomonas syringae and Ralstonia solanacearum) and two phytopathogenic fungi (Magnaporthe oryzae and Botrytis cinerea) on solid agar. An in-depth untargeted metabolomics approach incorporating UPLC–QToF–HRMS/MS-based molecular networking (MN), in silico MS/MS databases, and manual dereplication was employed for comparative analysis of the extracts belonging to nine most bioactive co-cultures and their respective mono-cultures. The phytopathogens triggered interspecies chemical communications with marine-adapted fungi, leading to the production of new compounds and enhanced expression of known metabolites in co-cultures. MN successfully generated a detailed map of the chemical inventory of both mono- and co-cultures. We annotated overall 18 molecular clusters (belonging to terpenes, alkaloids, peptides, and polyketides), 9 of which were exclusively produced in co-cultures. Several clusters contained compounds, which could not be annotated to any known compounds, suggesting that they are putatively new metabolites. Direct antagonistic effects of the marine-adapted fungi on the phytopathogens were observed and anti-phytopathogenic activity was demonstrated.The untargeted metabolomics approach combined with bioactivity testing allowed prioritization of two co-cultures for purification and characterization of marine fungal metabolites with crop-protective activity. To our knowledge, this is the first study employing plant pathogens to challenge marine-adapted fungi.

Design of Fungal Co-Cultivation Based on Comparative Metabolomics and Bioactivity for Discovery of Marine Fungal Agrochemicals

“…In a previous study [21], we isolated eight marine fungi from Baltic Sea-derived sediment (Table 1). In the current investigation, we cultivated the fungal isolates individually (in triplicate) in solid potato dextrose agar (PDA) medium for 21 days.…”

“…All eight fungi were isolated from sediment samples obtained from the Windebyer Noor, Schleswig-Holstein, Germany in November 2015 [21]. Identification of the fungal strains by Sanger sequencing of the internal transcribed spacer (ITS) regions or 18S rRNA gene was reported previously [21].…”

“…All eight fungi were isolated from sediment samples obtained from the Windebyer Noor, Schleswig-Holstein, Germany in November 2015 [21]. Identification of the fungal strains by Sanger sequencing of the internal transcribed spacer (ITS) regions or 18S rRNA gene was reported previously [21]. Isolates used in this study had GenBank accession numbers MH791233 (ITS sequence), MH791253 (ITS sequence), MH791254 (ITS sequence), MH791292 (ITS sequence), MH791174 (18S rRNA gene sequence), MH791258 (ITS sequence), MH791244 (ITS sequence), and MH791275 (ITS sequence).…”

“…Extracts were tested in triplicates against the phytopathogens. Bioactivity testing procedure has been described before [21]. Briefly, the broth dilution approach in 96-well microplate was employed.…”

“…Several antibiotics (chloramphenicol, tetracycline, nystatin, and cycloheximide) served as positive controls, whereas the growth medium (PDA) and 0.5% (v/v) DMSO were tested as negative controls. Absorbances were measured at 600 nm using an Infinite M200 reader (TECAN Deutschland GmbH, Crailsheim, Germany) before and after incubation and % inhibition values computed [21]. The IC 50 values of the pure compounds were calculated using the Microsoft Excel program.…”