Metabolic Profile of Scytalidium parasiticum- Ganoderma boninense Co-Cultures Revealed the Alkaloids, Flavonoids and Fatty Acids that Contribute to Anti-Ganoderma Activity

Abstract

In solving the issue of basal stem rot diseases caused by Ganoderma, an investigation of Scytalidium parasiticum as a biological control agent that suppresses Ganoderma infection has gained our interest, as it is more environmentally friendly. Recently, the fungal co-cultivation has emerged as a promising method to discover novel antimicrobial metabolites. In this study, an established technique of co-culturing Scytalidium parasiticum and Ganoderma boninense was applied to produce and induce metabolites that have antifungal activity against G. boninense. The crude extract from the co-culture media was applied to a High Performance Liquid Chromatography (HPLC) preparative column to isolate the bioactive compounds, which were tested against G. boninense. The fractions that showed inhibition against G. boninense were sent for a Liquid Chromatography-Time of Flight-Mass Spectrometry (LC-TOF-MS) analysis to further identify the compounds that were responsible for the microbicidal activity. Interestingly, we found that eudistomin I, naringenin 7-O-beta-D-glucoside and penipanoid A, which were present in different abundances in all the active fractions, except in the control, could be the antimicrobial metabolites. In addition, the abundance of fatty acids, such as oleic acid and stearamide in the active fraction, also enhanced the antimicrobial activity. This comprehensive metabolomics study could be used as the basis for isolating biocontrol compounds to be applied in oil palm fields to combat a Ganoderma infection.

Keywords: Ganoderma boninense; LC-TOF-MS analysis; Scytalidium parasiticum; anti-Ganoderma; biological control; metabolomics.

Figure 1

Schematic diagram of the study. The method consists of co-culturing both fungi, Ganoderma boninense and Scytalidium Parasiticum, in the same media with different growth ages (0, 3 and 5 days); liquid extraction; fractionation into recycling preparative High Performance Liquid Chromatography (HPLC); antifungal activity, a mass spectrometry analysis, including a data analysis and compounds identification.

Figure 2

Principal Component Analysis (PCA) score plot of the metabolic changes of the control and co-culture of the S. parasiticum and G. boninense fractions in the first two principal components (PC1 and PC2). G0, G3 and G5 refer to the G. boninense growth age (0, 3 and 5 days), respectively. R3 and R5 represent fractions recycled number at number 3 and number 5, respectively.

Figure 3

PCA loading plot of control and treated samples that showed the distribution of metabolites (masses) in w*c(1) and w*c(2) planes (green dots). The most important metabolites in VIP list are highlighted in red bulleted dot.

Figure 4

Representative ion intensity for the m/z value (A) 133.048 (Retention Time (RT) 2.11 min), (B) 236.162 (RT 2.13 min), (C) 296.065 (RT 2.01 min), (D) 175.130 (RT 2.00 min), (E) 325.106 (RT 2.05 min), (F) 134.020 (RT 2.03 min), (G) 147.034 (RT 2.39 min), (H) 288.284 (RT 9.52 min), (I) 316.317 (RT 10.08 min), (J) 278.061 (RT 1.95 min), (K) 476.143 (RT 2.04 min) and (L) 290.843 (RT 1.55 min) across 16 samples.

Figure 5

Dendrogram obtained after the hierarchical classification of metabolite profiles in the control and G. boninense–S. parasiticum co-culture fractions. (A) Heat map of metabolites are shown. (B) Bright red denotes highest intensities of metabolites, and light green denotes the lowest intensities or complete absence of metabolites.

Figure 6

Base peak chromatogram (BPC) (red color) and dissect chromatograms (different colors) of the G0R3 37–41 fraction by Liquid Chromatography-Time of Flight-Mass Spectrometry (LC-MS-TOF) in a positive ionization mode. Peak labeling with different numbers represent the compounds identified that correspond to Table 3. Bruker’s dissect algorithm in Data Analysis software 4.0 allows the user to observe overlapping peaks at very similar retention times.

Figure 7

Metabolites involved in the biosynthetic pathway of G. boninense–S. parasiticum interactions. Single arrows represent one-step enzymatic conversions, while dashed arrows represent multiple reactions.