Use of molecular techniques for reporting a viral disease in vineyards in New England (first report) and RNAi for controlling a fungal disease in melon in the Brazilian Semiarid region

Abstract

The occurrence of plant diseases is one of the main factors affecting food production worldwide. In this study, we address the application of molecular techniques in detecting viral pathogens and developing new methods for managing fungal diseases in melon. In the first chapter, we describe the initial report of the detection of an important viral pathogen in grapevines. Grapevine red blotch virus (GRBV) is an emerging pathogen that significantly impacts grape production. This pathogen causes Grapevine Red Blotch Disease (GRBD). The virus has been spreading across several states in the United States, but until now, there had been no information about its occurrence in the New England (NE) region. The objective of this study was to investigate the presence of GRBV in NE vineyards and assess the degree of genetic variability among isolates. To detect GRBV, twenty-eight samples from five vineyards in Connecticut, Rhode Island, and Massachusetts were collected between 2018 and 2020. These samples underwent DNA extraction, rolling cycle amplification (RCA), and PCR using GRBV-specific primers. Samples that produced the PCR product of the expected size were considered positive. These samples had partial and complete genomes sequenced using the Sanger method. Phylogenetic analyses revealed that the samples fell into two separate clades, predominantly with isolates in clade 1, which shows a higher degree of genetic variability, possibly explained by genetic recombination events. This is the first report of the presence of GRBV in the evaluated New England states. Correct diagnosis is the first step toward implementing control measures and reducing virus transmission. In the second chapter, we describe the exploration of an innovative method for disease control in melon. The occurrence of diseases caused by soil-dwelling fungi is one of the primary factors that reduce melon productivity (Cucumis melo L.). Rhizoctonia solani is one of the species affecting the productivity and quality of melon, causing damage at all stages of development. In this study, we aim to explore the potential of RNAi technology based on SIGS (Spray-induced gene silencing) for controlling R. solani in melon. The genes responsible for important enzymes related to the pathogenicity of R. solani, polygalacturonase (PG1) and xylanase (Xly), were used as silencing targets. The coding regions of PG1 and Xly were amplified by PCR, sequenced, and used for dsRNA synthesis. For application in detached leaf assays and in melon plants, dsRNA molecules were encapsulated in chitosan nanoparticles. The potential for R. solani control via RNAi-SIGS was evaluated in in vitro experiments (culture media) containing only dsRNA-PG1 and dsRNA-Xly solutions. In the detached leaf experiments, solutions containing dsRNA and encapsulated dsRNA (Cs-PG1 and Cs-Xly) were applied to the leaves, which were subsequently inoculated with a medium disk containing the pure culture of the pathogen. In the greenhouse experiments, the same solutions were applied to melon seedlings that were inoculated using the infested toothpick method. In the in vitro assay, a reduction in mycelial growth was observed in the treatment with dsRNA-PG1 in pectin-based medium after seven days. A decrease in lesion size was observed in detached leaves treated with dsRNA and encapsulated dsRNA after five days of inoculation. In the greenhouse experiments, a reduction in disease severity and an increase in plant growth parameters were noted in the groups treated with dsRNA, with or without the presence of chitosan nanoparticles. The control of R. solani via RNAi-SIGS shows excellent potential for use under field conditions. This study presents an innovative technology for the future management of soil-borne fungi.