The formation of the trapping device induced by nematodes has been assumed as an indicator for a switch from saprophytic to predacious lifestyles for nematode-trapping fungi. However, fungal nematocidal activity is not completely synonymous with fungal trap formation. We found that the predominant nematode-trapping fungus Arthrobotrys oligospora harbored a rare NRPS (Ao415) gene cluster that was mainly distributed in nematode-trapping fungi. The gene Ao415 putatively encodes a protein with a unique domain organization, distinct from other NRPSs in other fungi. Mutation of the two key biosynthetic genes Ao415 and Ao414 combined with nontarget metabolic analysis revealed that the Ao415 gene cluster was responsible for the biosynthesis of a hydroxamate siderophore, desferriferrichrome (1). Lack of desferriferrichrome (1) and its hydroxamate precursor (3) could lead to significantly increased Fe3+ content, which induced fungal trap formation without a nematode inducer. Furthermore, the addition of Fe3+ strongly improved fungal trap formation but deleteriously caused broken traps. The addition of 1 significantly attenuated trap formation but enhanced fungal nematicidal activity. Our findings indicate that iron is a key factor for trap formation and provide a new insight into the underlying mechanism of siderophores in nematode-trapping fungi.

The objective of this paper is to explore the function of the AOL-s00215g415 (Aog415) gene, which encodes for the synthesis of siderophore in the nematode trapping fungal model strain A. oligospora, in order to understand the relationship between siderophore biosynthesis and nematode trapping activity. After a through sequence analysis, it was determined that Aog415 is a siderophore-synthesizing NRPS. The product of this gene was then identified to be the hydroxamate siderophore desferriferrichrome, using mass spectrometry analysis. When compared to the WT strains, the Aog415 knockout strain exhibited a 60% decrease in siderophore content in fermentation broth. Additionally, the number of predatory rings of decreased by 23.21%, while the spore yield increased by 37.34%. The deletion of Aog415 did not affect the growth of A. oligospora in diverse nutrient medium. Lipid metabolism-related pathways were the primary targets of Aog415 disruption as revealed by the metabolomic analysis. In comparison to the WT, a significant reduction in the levels of glycerophospholipids, and glycolipids was observed in the mutation. The metabolic alteration in fatty acyls and amino acid-like molecules were significantly disrupted. The knockout of Aog415 impaired the biosynthesis of the hydroxamate siderophore desferriferrichrome, remodeled the flow of fatty acid in A. oligospora, and mainly reprogrammed the membrane lipid metabolism in cells. Desferriferrichrome, a hydroxamate siderophore affects the growth, metabolism and nematode trapping ability of A. oligospora by regulating iron intake and cell membrane homeostasis. Our study uncovered the significant contribution of siderophores to the growth and nematode trapping ability and constructed the relationship among siderophores biosynthesis, lipid metabolism and nematode trapping activity of A. oligospora, which provides a new insight for the development of nematode biocontrol agents based on nematode trapping fungi.

Nematode-trapping fungi are natural antagonists of nematodes. These predatory fungi are capable of switching their lifestyle from a saprophytic to predatory stage in the presence of nematodes by developing specialized trapping devices to capture and consume nematodes. The biochemical mechanisms of such predator-prey interaction have become increasingly studied given the potential application of nematode-trapping fungi as biocontrol agents, but the involved fungal metabolites remain underexplored. Here, we report a comprehensive liquid-chromatography mass spectrometry (LC-MS) metabolomics study on one hundred wild isolates of nematode-trapping fungi in three different species, Arthrobotrys oligospora, Arthrobotrys thaumasia, and Arthrobotrys musiformis. Molecular networking analysis revealed that the fungi were capable of producing thousands of metabolites, and such chemical diversity of metabolites was notably increased as the fungi switched lifestyle to the predatory stage. Structural annotations by tandem mass spectrometry revealed that those fungal metabolites belonged to various structural families, such as peptide, siderophore, fatty alcohol, and fatty acid amide, and their production exhibited species specificity. Several small peptides (<1.5 kDa) produced by A. musiformis in the predatory stage were found, with their partial amino acid sequences resolved by the tandem mass spectra. Four fungal metabolites (desferriferrichrome, linoleyl alcohol, nonadecanamide, and citicoline) that were significantly enriched in the predatory stage were identified and validated by chemical standards, and their bioactivities against nematode prey were assessed. The availability of the metabolomics datasets will facilitate comparative studies on the metabolites of nematode-trapping fungi in the future.