Guanine and β-aminobutyric acid as novel elicitors enhance barley defence against net blotch disease through oxidative stress and enzymatic defence mechanisms

Net blotch disease, caused by Pyrenophora teres, poses a growing threat to global barley production, highlighting the need for sustainable disease management strategies. Traditional control relies heavily on chemical fungicides, which raise concerns about environmental safety and resistance development. There is growing interest in sustainable, eco-friendly alternatives to enhance plant immunity. Elicitors like guanine and β-aminobutyric acid (βABA) offer promising potential for boosting resistance in barley against fungal pathogens. This study investigated the effectiveness of guanine and βABA as elicitors in boosting systemic resistance in a susceptible barley cultivar (Hordeum vulgare cv. Giza 2000) against P. teres, compared to the conventional fungicide epoxiconazole, under both greenhouse and field conditions. Barley leaves were analysed for reactive oxygen species (ROS) using 3,3'-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining, for physiological parameters measurements, including chlorophyll, carotenoids, hydrogen peroxide (H₂O₂), and superoxide anion (O₂•⁻). Enzymatic activities of key antioxidant and defence-related enzymes were spectrophotometrically assessed. All epoxiconazole, guanine, and βABA significantly reduced disease severity and infection response with epoxiconazole being the most effective, followed by guanine and βABA. Notably, guanine and βABA induced an early oxidative burst, characterised by increased endogenous production of ROS, H₂O₂, and O₂•⁻, detected in guard and mesophyll cells within 24 h post-inoculation (hpi). This response was inversely correlated with fungal colonisation. Further analyses at 3 and 15 days post-inoculation (dpi) revealed that both elicitors enhanced antioxidant enzyme activity and promoted the accumulation of osmoprotectants and non-enzymatic antioxidants, crucial for countering oxidative stress. Additionally, foliar application of guanine and βABA mitigated oxidative damage by reducing excessive ROS, lipoxygenase activity, and malondialdehyde levels. The findings underscore the potential of guanine and βABA as key immune signalling molecules in plant–microbe interactions, playing a pivotal role in plant defence. To our knowledge, this is the first study to elucidate the mechanisms behind guanine- and βABA-mediated defence activation in barley against net blotch disease, positioning them as sustainable alternatives to synthetic fungicides for integrated disease management in barley cultivation.