NCR13 peptide protects soybean against Cercospora sojina using multi-faceted modes of action and additive interaction with chemical fungicides.

Frogeye leaf spot (FLS) disease, caused by the fungus Cercospora sojina, is a serious disease affecting soybean production. Its control is challenged by resistance to quinone outside inhibitor (QoI) fungicides, but plant antifungal peptides with multi-faceted modes of action (MoAs) offer a promising alternative. Here, we showed that chickpea nodule-specific cysteine-rich peptide, NCR13_Peptide Folding Variant1 (NCR13_PFV1), exhibited antifungal activity against QoI-sensitive and -resistant field isolates of C. sojina at low micromolar concentrations, representing the first antifungal NCR peptide reported to be effective against C. sojina. Spray application showed no phytotoxicity and effectively protected soybean against FLS. When combined with the QoI fungicide azoxystrobin, NCR13_PFV1 provided additive control of FLS. NCR13_PFV1 disrupted the plasma membrane, induced reactive oxygen species (ROS) production, and was rapidly internalized, localizing in the nucleus. It bound to fungal ribosomal RNA and inhibited protein translation. RNA-seq studies revealed the up-regulation of several genes encoding heme-binding proteins in peptide-challenged C. sojina. Notably, iron supplementation in the growth medium reduced the peptide-induced ROS and antifungal activity, revealing the importance of iron homeostasis in protection or recovery of C. sojina from oxidative stress. Overall, NCR13_PFV1 with its multi-faceted MoA holds potential as a bio-fungicide for FLS control, complementing QoI fungicides and overcoming resistance in C. sojina.