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

Abstract 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 LC 50 against PWN than compound 2 (90.3 versus 145.8 μM), whereas compounds 2 and 3 had comparable estimated LC 50 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 LC 50 , engages the same response pathway remains to be determined. © 2026 Society of Chemical Industry.