Metabolomic and Genomic Analysis of Bioactive Compounds of Phacidium infestans Karsten DSM 5139 Cultivated on Pinus sylvestris Needles

Abstract

This study investigates how Phacidium infestans acquires nutrients on Pinus sylvestris needles, which possess antimicrobial properties. P. infestans was evaluated for its growth and enzyme production on various substrates, alongside genomic and metabolomic analysis. Direct-infusion high-resolution mass spectrometry (DI-HRMS) was performed on methanol extracts obtained from P. infestans cultivated on needles and malt extract media. DI-HRMS analysis identified 21 compounds from the malt extract and 112 from the needle samples. The resin components increased in the needle samples post-cultivation, suggesting terpenoid release from resin ducts due to fungal degradation of plant cell walls. P. infestans fully consumed sugars and antifungal compounds, including taxiresinol and salicylic acid, with control-to-sample ratios (CTR/SA) of 289.76 and 47.24, respectively. Moreover, lariciresinol and pinoresinol were reduced to undetectable levels. The genomic analysis identified 421 secreted proteins, including 128 carbohydrate-active enzymes, 3 cutinases, and 49 lipases that aid host penetration and wax degradation. Several multi-drug efflux pumps and two acyclic terpene utilisation proteins were identified as well. These proteins support the cellular integrity of P. infestans by expelling toxic compounds. Our findings provide valuable insights into the metabolic strategies of P. infestans for nutrient assimilation on pine needles.

Keywords: Pinus sylvestris; Gremmenia infestans; FT‐ICR; Phacidium infestans Karsten DSM 5139; mass spectrometry; needles; secreted carbohydrate‐active enzymes.

FIGURE 1

Growth pattern of P. infestans on agar plates that contained 1% pectin, xylan, cellulose, starch, lignin, or malt extract. The growth shown is the area of mycelial growth recorded on day 12 of cultivation.

FIGURE 2

Relative abundances of coumaric acid (p = 0.009), vanillic acid (p = 0.006), quinic acid (p = 0.14), shikimic acid (p = 0.22), sinapaldehyde (p = 0.01), and dihydrosinapyl alcohol (p < 0.001) in the needle samples generated by DI(FIA)‐ESI‐HRMS. Green is the medium control (water and needles), blue indicates the samples after P. infestans growth, and red is the solvent control (methanol). The p values for each compound represent the significance of the difference between the means of the sample (with P. infestans ) and the control (containing only needles), as determined by a two‐sample t‐test.

FIGURE 3

Relative abundances of galactosyl pinitol (p = 0.02), glactosylglycerol (p = 0.04), and glucuronic acid (p = 0.04) content of the needle samples generated by DI(FIA)‐ESI‐HRMS analysis. Green is the medium control, blue indicates the samples with P. infestans, and red is the solvent control (methanol). The p values for each compound represent the significance of the difference between the means of the sample (with P. infestans ) and the control (containing only needles), as determined by a two‐sample t‐test.

FIGURE 4

Relative abundances of some resin acid compounds identified in the needle samples: Pinifolic acid (p = 0.01), pinusolidic acid (p < 0.001), and isocupressic acid (p = 0.01). Green indicates the medium control, blue indicates the sample extracts from P. infestans cultivations, and red indicates the solvent‐control (methanol). The p values for each compound represent the significance of the difference between the means of the sample (with P. infestans ) and the control (containing only needles), as determined by a two‐sample t‐test.