Integrated Metabolomics and Morphogenesis Reveal Volatile Signaling of the Nematode-Trapping Fungus Arthrobotrys oligospora

Abstract

The adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species Arthrobotrys oligospora Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms.

Keywords: Arthrobotrys oligospora; VOCs; metabolic adaptation; nematode-trapping fungi; pathogenicity; volatile organic compounds.

FIG 1

Morphological responses of A. oligospora grown on two different media, CMA and PDA, to living nematodes (L) or dead nematodes (D) under two different modes of contact, direct contact (DC) (A) and nondirect contact (NDC) (B), over 144 h. The results for 3D traps and hyphal fusions are shown. The corrected values are the differences between the data obtained for fungal strains treated with nematodes and those obtained for fungal strains treated without nematodes.

FIG 2

(A) PCA score plots between fungal samples during the saprophytic and pathogenic phases of growth on two different media, CMA and PDA (principal component 1 versus principal component 2; component 1, 0.85; component 2, 0.05); (B) PCA score plots between fungal samples during the saprophytic and pathogenic phases of growth on two different media, CMA and PDA (principal component 1 versus principal component 3; component 1, 0.85; component 2, 0.05; component 3, 0.03); (C) PCA score plot between the saprophytic and pathogenic fungal samples grown on CMA (principal component 2 versus principal component 3; component 1, 0.60; component 2, 0.12; component 3, 0.07); (D) PCA score plot between the saprophytic and pathogenic fungal samples grown on PDA (principal component 2 versus principal component 3; component 1, 0.65; component 2, 0.11; component 3, 0.08). C, A. oligospora growing without nematodes over 144 h as the control group; DC, A. oligospora growing under direct contact with living nematodes over 144 h; NDC-L, A. oligospora growing under nondirect contact with live nematodes over 144 h; NDC-D, A. oligospora growing under nondirect contact with dead nematodes over 144 h.

FIG 3

Unsupervised hierarchical clustering of the logarithmically transformed (log₂) relative concentrations of metabolites from the methanol extracts of A. oligospora YMF1.01883 at different growth phases cultivated in CMA and PDA media. (A) Unsupervised hierarchical clustering of the logarithmically transformed (log₂) relative concentrations of 33 metabolites from the methanol extracts of wild-type A. oligospora YMF1.01883 cultivated in CMA medium; (B) unsupervised hierarchical clustering of the logarithmically transformed (log₂) relative concentrations of 16 metabolites from the methanol extracts of wild-type A. oligospora cultivated in PDA medium. C, A. oligospora growing without nematodes over 144 h as the control group; DC, A. oligospora growing under direct contact with nematodes over 144 h; NDC-L, A. oligospora growing under nondirect contact with living nematodes over 144 h; NDC-D, A. oligospora growing under nondirect contact with dead nematodes over 144 h. CON, control.

FIG 4

Structures of 14 metabolites (6 metabolites in the CMA group, including metabolites 1 to 6, and 8 metabolites in the PDA group, including metabolites 7 to 14) (A), and their abundances over the time course from the saprophytic to the pathogenic lifestyle of the fungus (B). Green, control group, consisting of A. oligospora fungi growing without nematodes over 144 h; blue, direct contact group, consisting of A. oligospora fungi growing under direct contact with living nematodes over 144 h; rose, non-direct-contact live group, consisting of A. oligospora fungi growing under nondirect contact with living nematodes over 144 h; red, non-direct-contact dead group, consisting of A. oligospora fungi growing under nondirect contact with dead nematodes over 144 h (n = 4).

FIG 5

(A) Attracting activities of three metabolites, 2(5H)-furanone (compound 5), furan-2-ylmethanol (compound 6), and furan-2-carbaldehyde (compound 7), for the nematode C. elegans. (B) Effect of 5-methylfuran-2-carbaldehyde (compound 8) on the mortality of C. elegans at 12 h, with 1 μg/ml ivermectin (IVM) used as a positive control. (C) Effect of maltol (compound 10; M) at a concentration of 2.5 μg/ml on the trap formation of A. oligospora treated without maltol as a control (C). **, P < 0.01; *, P < 0.05 (n = 5).

FIG 6

HPLC analysis of the methanol extracts of the PDA culture broths from the wild-type strain (black line) and the ΔAOL_s00079g496 (Δ79g496) mutant (red line). Blue lines, the peak of 2(5H)-furanone (compound 5) at about 4 min. mAU, milli-absorbance units.

FIG 7

(A) Comparison of attracting activities of wild-type (WT) A. oligospora and the ΔAOL_s00079g496 (Δ496) mutant; (B) comparison of spore formations of wild-type A. oligospora and the ΔAOL_s00079g496 mutant; (C) comparison of spore germination rates of wild-type A. oligospora and the ΔAOL_s00079g496 mutant; (D) comparison of trap formation of wild-type A. oligospora and the ΔAOL_s00079g496 mutant; (E) comparison of nematode-capturing abilities of wild-type A. oligospora and the ΔAOL_s00079g496 mutant. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (n = 4).

FIG 8

Comparison of the mycelial morphology of the wild-type strain and the ΔAOL_s00079g496 mutant (Δ79g496-KS) on PDA plates (15 days).

FIG 9

GC-MS analysis of the methanol extracts of the wild-type strain (black line) and the ΔAOL_s00079g496 (Δ79g496) mutant (red line) on CMA. Red arrows, compounds detected in the mutant strain.

FIG 10

Morphological and metabolic adaptation of wild-type A. oligospora on two media, CMA and PDA. In direct contact with nematodes, the fungus grown on CMA develops more traps than that grown on PDA; in nondirect contact with nematodes, the fungus grown on PDA develops more hyphal fusion than that grown on CMA. The fungus grown on CMA produced an attractant molecule, 2(5H)-furanone, while the fungus grown on PDA produced a nematicide metabolite, 5-methylfuran 2-carbaldehyde, as well as a stimulator, maltol, that increased trap numbers.