Nematicidal azaphilones from the mesophotic sponge-associated fungus Eutypella sp. NBUF234.

BACKGROUND: Plant-parasitic nematodes pose severe and growing threats to global agriculture and forestry. However, the development of effective control strategies is limited by the lack of small-molecule nematicides with well-defined molecular targets. RESULTS: Three undescribed azaphilones, eutylones A-C (1-3), were isolated from Eutypella sp. NBUF234, a fungus associated with a Cinachyrella sponge collected from the marine mesophotic zone. Their structures were established by spectroscopic analysis, X-ray crystallography, and quantum chemical calculations. Compounds 2 and 3 represent the first deflectin-type azaphilones bearing a C-5 methyl group and a C8 tetraketone side chain. Compounds 1-3 showed moderate, dose-dependent activity against Bursaphelenchus xylophilus (PWN) and Heterodera glycines (SCN). In the 72 h analysis, compound 3 had a lower estimated LC50 against PWN than compound 2 (90.3 versus 145.8 μM), whereas compounds 2 and 3 had comparable estimated LC50 values against SCN (159.1 and 159.6 μM). Compound 2 was prioritized for mechanistic profiling based on the preliminary screening results available when those experiments were initiated. Comparative RNA-seq, proteome-wide docking, and RNA interference were used to characterize the response to compound 2. These analyses prioritized CAG9107488.1 as a PWN candidate with preliminary functional support, although direct target engagement was not established. CONCLUSION: Mesophotic sponge-associated fungi provide structurally novel starting scaffolds for nematicide discovery. The activity ranking was species dependent, and the mechanistic analyses define candidate responses to compound 2 rather than a shared mechanism for all three compounds. Whether compound 3, which had the lower estimated PWN LC50, engages the same response pathway remains to be determined. © 2026 Society of Chemical Industry.