Significance: Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide and has caused billions of dollars in annual production losses, threatening the entire citrus industry. Despite extensive research efforts, there are still no effective management tools to treat HLB-positive trees or to prevent new infections. Current HLB management strategies include chemical application of insecticides and traditional heat-sensitive antibiotics, which pose threats to humans, animal health, and the environment, and likely generate drug resistant insects and microbes. Here, we identified a novel class of stable antimicrobial peptides (SAMPs) from Australian finger lime and other HLB-tolerant citrus close relatives, which has dual functions of inhibiting C Las growth in HLB-positive trees and activating host immunity to prevent new infections.

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.

RNA interference (RNAi)‐based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray‐induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double‐stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray‐induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.