Non-target effect of fungicides on root endophytic fungus Microdochium bolleyi in barley

Abstract Fungicides are widely used in crop protection to control phytopathogenic fungi, yet their effects on non-target microorganisms inhabiting plant tissues remain poorly understood. Endophytic fungi are components of the plant microbiome, but assessing how fungicide applications influence their colonisation is challenging because these interactions are often asymptomatic. In this study, a simplified barley- Microdochium bolleyi model system was used to investigate the effects of fungicide exposure on endophytic colonisation. In vitro sensitivity assays revealed differences among fungicides, with prothioconazole showing the highest activity against sixteen M. bolleyi isolates (EC 50 = 0.11 µg mL⁻¹), followed by fluxapyroxad (0.31 µg mL⁻¹), mefentrifluconazole (0.37 µg mL⁻¹), and azoxystrobin (0.50 µg mL⁻¹). Based on these results, prothioconazole was selected for greenhouse experiments. Application of this systemic fungicide as either a seed dressing or foliar treatment significantly reduced root colonisation by M. bolleyi in barley. Greenhouse experiments were conducted using nine M. bolleyi isolates with 12 biological replicates per treatment. Reduced colonisation was confirmed by microscopic detection of chlamydospores and quantitative PCR analysis of fungal DNA. Compared with untreated inoculated plants, root colonisation was reduced by approximately 83% following seed dressing and 65% following foliar application. These results demonstrate that, under the tested conditions, prothioconazole suppressed root colonisation by the non-target endophyte M. bolleyi . The study establishes an effect on colonisation of this specific endophyte but does not determine whether reduced colonisation has beneficial, neutral, or detrimental consequences for barley or the wider root-associated fungal community.