High-efficiency green management of potato late blight by a self-assembled multicomponent nano-bioprotectant

Late blight, caused by the oomycete *Phytophthora infestans*, is a major potato disease, causing billions of dollars in annual losses and impacting food security. Current reliance on synthetic pesticides raises environmental and health concerns. RNA interference (RNAi), using double-stranded RNA (dsRNA) to silence pathogen genes, offers a green alternative. However, *P. infestans* has poor dsRNA uptake, hindering RNAi efficacy. This research aimed to develop a nano-bioprotectant to overcome this limitation, providing an environmentally sustainable disease management strategy. The strategy involves using nanotechnology to deliver dsRNA targeting key pathogen genes (*PiHmp1* and *PiCut3*) and a plant elicitor (cellobiose) to enhance plant defense mechanisms, leading to increased disease resistance.

Existing literature highlights the devastating impact of potato late blight and the limitations of current chemical control measures. Studies demonstrate the potential of RNAi for disease management, but the low uptake efficiency of dsRNA by *P. infestans* has been a significant hurdle. Previous research has explored the use of nanoparticles to enhance the delivery of various molecules, including dsRNA, demonstrating increased cellular uptake and improved efficacy. This study builds upon these findings, focusing specifically on the application of nanotechnology for efficient delivery of dsRNA and a plant elicitor to combat potato late blight.

A star polycation (SPc) nanocarrier, previously shown to enhance intracellular delivery, was used to encapsulate dsRNA targeting *PiHmp1* and *PiCut3*. The dsRNA was synthesized both in vitro and using a bacterial expression system (pET28-BL21(DE3) RNase III-), which was shown to significantly increase dsRNA production. Cellobiose, a plant elicitor, was incorporated into the nano-bioprotectant to stimulate plant defense responses. The self-assembly of the multicomponent nano-bioprotectant (cellobiose, SPc, and dsRNA) was characterized using isothermal titration calorimetry (ITC) and transmission electron microscopy (TEM). The protective effect of different formulations (naked dsRNA, dsRNA/SPc, cellobiose/SPc, and the multicomponent nano-bioprotectant) was evaluated on detached potato leaves and whole potato plants under greenhouse and field conditions. Disease severity was assessed by measuring lesion area and disease index. Gene expression analysis using qRT-PCR was performed to evaluate the efficacy of RNAi and the activation of plant defense genes. The quantification of coumarin, a phytoalexin, was performed using LC-MS/MS.

The SPc nanocarrier significantly enhanced the uptake of dsRNA by *P. infestans* sporangia and hyphae, overcoming the delivery bottleneck. SPc protected dsRNA from degradation, prolonging its protective effect. The bacterial expression system increased dsRNA production threefold compared to a conventional system. The co-delivery of dsRNA targeting *PiHmp1* and *PiCut3* resulted in effective silencing of both genes and significantly reduced disease symptoms. Cellobiose/SPc complex activated endocytosis, upregulating several endocytosis-related genes and enhancing the expression of plant defense genes, such as *StWRKY1*, *StPRI*, *StPPO*, and *StPTIS*. The multicomponent nano-bioprotectant exhibited a strong protective effect against potato late blight under both greenhouse and field conditions, outperforming the commercial fungicide mancozeb. ITC analysis revealed that the self-assembly of the multicomponent nano-bioprotectant was driven by non-covalent interactions such as hydrogen bonds and hydrophobic interactions. TEM analysis showed that the multicomponent nano-bioprotectant had a particle size of approximately 88 nm.

This study successfully demonstrated the efficacy of a self-assembled multicomponent nano-bioprotectant in controlling potato late blight. The combination of dsRNA-mediated gene silencing and plant elicitor-induced systemic resistance provided a superior level of protection compared to conventional chemical controls. The use of nanotechnology addressed the major limitation of dsRNA-based plant protection strategies—poor dsRNA uptake by the target pathogen. The efficient and cost-effective bacterial dsRNA production system is crucial for scalable field applications. The findings highlight the potential of this eco-friendly strategy for sustainable disease management in agriculture.

This research presents a novel and effective approach for green management of potato late blight using a self-assembled multicomponent nano-bioprotectant. The synergistic combination of dsRNA-mediated gene silencing and plant immune response activation proved highly effective in field conditions, surpassing a commercial fungicide. Future research could explore the application of this technology to other plant diseases and pests, optimizing the components and delivery system for various pathogens and crops.

While the study demonstrated excellent results under specific field conditions, further research is needed to assess the effectiveness of the nano-bioprotectant under different environmental conditions and with various potato cultivars. Long-term studies are also required to evaluate the potential for the development of resistance in *P. infestans*. The study focused on two target genes in *P. infestans*; investigating other potential targets could further enhance the efficacy of this approach.