Plasma pre-treatment and peroxynitric acid soaking as a novel oxidative seed disinfection strategy against seed-borne fungal pathogens in maize.

Abstract

Introduction: Seed-borne fungal contamination is an important challenge in maize production and may contribute to grain deterioration and mycotoxin contamination. This study evaluated peroxynitric acid (PNA), alone and in combination with nonequilibrium gaseous plasma pretreatment, as a rapid oxidative strategy for disinfecting maize seeds contaminated with Aspergillus flavus and Fusarium fujikuroi.

Methods: Maize seeds were artificially inoculated with approximately 10⁶ spores of either A. flavus or F. fujikuroi. For plasma pretreatment, seeds were sequentially exposed to hydrogen (0.2 s) and oxygen (0.2 s) radio-frequency plasmas in a low-pressure reactor. Hydrogen plasma was sustained in H mode at 30 Pa and 800 W, whereas oxygen plasma was sustained in E mode at 50 Pa and 300 W. Seeds were subsequently exposed to 1 -400 mM PNA for 3 -300 s. Antifungal efficacy was assessed using direct plating assays and four-parameter logistic modeling to estimate the 50% and 90% inhibitory concentrations (IC50 and IC90) and effective exposure times (ET50 and ET90). Seed viability was evaluated by germination assays.

Results: PNA exhibited rapid, concentration- and time-dependent antifungal activity against both fungal species. Strong suppression of F. fujikuroi infection was observed at PNA concentrations ≥100 mM, with complete suppression of detectable infection at 400 mM after 180 s. At 50 mM PNA, A. flavus showed substantially faster inactivation kinetics than F. fujikuroi, with estimated ET50 values of 42.3 and 95.1 s, respectively. At 100 mM PNA, plasma pretreatment shifted the fitted time -response curves toward shorter exposure times and lower infection levels, particularly for A. flavus. However, planned plasma-versus-no-plasma comparisons were not statistically significant after adjustment for overdispersion. Neither PNA nor plasma treatment significantly affected seed germination.

Discussion: PNA demonstrated rapid and effective antifungal activity against seed-borne A. flavus and F. fujikuroi, with clear species-specific differences in susceptibility. Plasma pretreatment appeared to accelerate early PNA-mediated fungal suppression but did not consistently improve final disinfection efficacy at prolonged exposure times. This effect may be associated with increased seed-surface hydrophilicity following plasma exposure. The results support PNA as a promising oxidative approach for maize seed disinfection and provide further insight into species-specific responses and plasma-associated kinetic effects in seed-borne filamentous fungi.