Untargeted MS-Based Metabolomic Analysis of Termite Gut-Associated Streptomycetes with Antifungal Activity against Pyrrhoderma noxium

Abstract

Pyrrhoderma noxium is a plant fungal pathogen that induces the disease of brown root rot in a large variety of tree species. It is currently infecting many of the amenity trees within Brisbane City of Queensland, Australia. Steering away from harmful chemical fungicides, biological control agents offer environmentally friendly alternatives. Streptomycetes are known for their production of novel bioactive secondary metabolites with biocontrol potential, particularly, streptomycete symbionts isolated from unique ecological niches. In this study, 37 termite gut-associated actinomycete isolates were identified using molecular methods and screened against P. noxium. A majority of the isolates belonged to the genus Streptomyces, and 15 isolates exhibited strong antifungal activity with up to 98.5% mycelial inhibition of the fungal pathogen. MS/MS molecular networking analysis of the isolates' fermentation extracts revealed several chemical classes with polyketides being among the most abundant. Most of the metabolites, however, did not have matches to the GNPS database, indicating potential novel antifungal compounds in the active extracts obtained from the isolates. Pathway enrichment and overrepresentation analyses revealed pathways relating to polyketide antibiotic production, among other antibiotic pathways, further confirming the biosynthetic potential of the termite gut-associated streptomycetes with biocontrol potential against P. noxium.

Keywords: Pyrrhoderma noxium; Streptomyces; antifungal compounds; biological control; mass spectrometry; metabolomics; molecular networking; streptomycetes; termite gut symbiosis; termites.

Figure 1

(A) Co-culture assays of the selected 15 termite gut-associated Streptomyces isolates and the P. noxium pathogen. (B) Percent inhibition of P. noxium mycelia when grown in co-culture with Streptomyces isolates. (C) Disk-diffusion assays of the Streptomyces isolate extracts against P. noxium (the left disk contains the active extracts, and the right disk is the control and contains EtOAc).

Figure 2

(A) Two-dimensional PCA score plot demonstrating the differential production of metabolites between the fifteen Streptomyces isolates across the positive and negative ionisation modes (PC1 × PC2) and (B) a score plot comparing the active and non-active fermentation extracts.

Figure 3

Termite gut-associated streptomycetes molecular network coloured by 11 chemical class terms as indicated by the legend. Chemical class annotation was performed using MolNetEnhancer within the GNPS framework (A) class annotated MN in a positive ionisation mode and (B) class annotated MN in a negative ionisation mode.

Figure 4

Isoflavonoid metabolite cluster identified in the termite gut-associated streptomycetes molecular network. Nodes represent an individual metabolite; each node displays its parent mass and is coloured based on its retention time.

Figure 5

Macrotetrolide cluster identified in the termite gut-associated streptomycetes molecular network. Nodes represent an individual metabolite; each node displays its parent mass and is coloured based on its retention time. (A) macrotetrolides (B) macrocyclic lactones (C) polycyclic tetramate macrolactums.

Figure 6

Coumarin cluster identified in the termite gut-associated streptomycetes molecular network. Nodes represent an individual metabolite; each node displays its parent mass and is coloured based on its retention time.

Figure 7

Peptide cluster identified in the termite gut-associated streptomycetes molecular network. Nodes represent an individual metabolite; each node displays its parent mass and is coloured based on its retention time.

Figure 8

(A) Pathway enrichment and (B) overrepresentation analyses generated in Metaboanalyst 5.0 of all 15 of the Streptomyces isolates from the MS data. The size of the circle is indicative of the pathway impact score, while the colours are based on the p-value (the darker the colour, the higher the significance of the metabolites within the relevant pathway).