In vitro efficacy of BLAD against Phytophthora cinnamomi : Effects on mycelial growth, sporulation, and zoospore germination of isolates from Quercus suber and Castanea sativa

Phytophthora cinnamomi Rands is a devastating soilborne oomycete responsible for root rot in Quercus suber L., Castanea sativa Mill., Quercus ilex L. and many other species worldwide, causing widespread forest decline across Mediterranean ecosystems. The BLAD-containing oligomer (BCO), isolated from Lupinus albus L. cotyledons, is a β-conglutin derived polypeptide oligomer who´s major 20.4 kDa polypeptide BLAD has been reported to exhibit potent and broad-spectrum antifungal and anti-oomycete activity, as well as protective efficacy against P. cinnamomi under greenhouse conditions. However, whether this protection reflects direct pathogen inhibition, host biostimulation or host boosting defense effects remains unresolved, and no in vitro characterization of its anti-P. cinnamomi activity had ever been established. Here, the in vitro activity of a BCO-based commercial formulation (PROBLAD®) was evaluated against five P. cinnamomi isolates (four from Q. suber, one from C. sativa) across multiple endpoints: mycelial radial growth, dry weight, chlamydospore and sporangia production, and zoospore germination. BCO inhibited radial growth in a concentration- and time-dependent manner (EC50: 4.74–6.43 mL/L). At ≥5 mL/L, colonies showed reduced compactness confirmed by dry weight (EC50: 2.63–4.08 mL/L), demonstrating that radial growth underestimates inhibitory activity by not capturing reductions in mycelial density. A transient stimulatory effect (hormesis) was observed at 1–2 mL/L across all isolates. Chlamydospore and sporangia production were fully suppressed at 1–5 mL/L, indicating interference with the induction phase of sporulation. Zoospore germination was inhibited with an EC50 of 5.72 mL/L and completely abolished at 20 mL/L. Microscopic examination revealed progressive cytoplasmic emptying and hyphal lysis, consistent with membrane disruption and corroborated by negative net absorbance values at 15–20 mL/L. Collectively, these results establish BCO as a potent direct inhibitor of P. cinnamomi across multiple developmental stages, resolving the mechanistic uncertainty of previous studies and supporting its integration into management programmes for root rot disease in Mediterranean forest ecosystems.