Enhancing Fusarium resistance in Nigella sativa via spray-induced gene silencing (SIGS) using chitosan nanoparticles with molecular and phytochemical perspectives

Fusarium oxysporum is the causal agent of vascular wilt disease, which makes it hard to grow and use Nigella sativa as medicine. This disease significantly reduces both yield and phytochemical quality. Traditional methods of control frequently utilize chemical fungicides; however, these can leave behind harmful residues and modify the bioactive profile of the crop. In this study, double-stranded RNA (dsRNA) targeting key pathogenicity genes (CYP51 and SGE1) of Fusarium oxysporum was designed and synthesized. This technique is known as Spray-Induced Gene Silencing (SIGS). We employed ionic gelation to mix the dsRNA with chitosan nanoparticles (CSNPs) to make it more stable, easier for leaves to take in, and able to release over time. Plants were sprayed with CSNP-dsRNA mixes at particular times during their growth, and a range of disease and quality measurements were systematically evaluated. Plants that had been treated with CSNP-dsRNA exhibited 68.4% reduced disease severity than plants that had just been exposed to the pathogen, with statistically significant effects (p ≤ 0.05). The severity of the disease was scored on a scale of 0-5. Quantitative PCR demonstrated that the treated plants had 70% reduced fungal biomass. This was associated with a 3.2- to 4.7-fold increase in the expression of defense-related genes (PR1, PDF1.2, and WRKY70) within 72 h of treatment. Biochemical analysis using HPLC demonstrated that the thymoquinone level in seeds from treated plants remained at approximately 94% of that in healthy controls. A 41.3% reduction seen in thymoquinone content in the seeds due to failure to treat the infection suggests that applications of SIGS followed by nanoparticle technology could have potential applications to either reduce the incidence of disease, stimulate healthy molecular defence mechanisms or protect the medicinal value of N. sativa. Moreover, SIGS is a viable, eco-friendly and harmless alternative to chemical fungicides, and could be applied to the management of profitable commercially planted crops that deliver considerable human health benefits; thus, providing a new and sustainable method to control plant disease.