Double-Stranded RNAs (dsRNAs) as a Sustainable Tool against Gray Mold (Botrytis cinerea) in Grapevine: Effectiveness of Different Application Methods in an Open-Air Environment

Abstract

Grapevine is one of the most important and globally widespread fruit species, with a high impact on the economy of many countries but with an intense environmental effect. Therefore, new environmentally friendly defense strategies against fungal pathogens are needed for more sustainable agriculture. A novel emerging approach is spray-induced gene silencing (SIGS), which concerns the exogenous application of double-stranded RNA (dsRNA) inducing enhanced plant resistance against fungal pathogens. Here, we tested the ability of SIGS to prevent and counteract infection of Botrytis cinerea, one of the most economically impacting pathogens of grapevine. In particular, we tested three independent approaches for dsRNA delivery into plants: (i) high pressure spraying of leaves; (ii) petiole adsorption of dsRNAs; (iii) postharvest spraying of bunches. We demonstrated that independently from the method of application, SIGS can reduce virulence of the fungus. Moreover, we also observed three different levels of efficacy depending on the method of application. Thus, the present data provide crucial information on the possibility to exploit SIGS as an alternative sustainable and ecofriendly strategy for grapevine pre- and postharvest protection.

RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges

“…Furthermore, the functionality of absorbed exogenous RNAi molecules offers excellent adaptability and flexibility in securing the required effects on gene expression of fungi, even without the need to genetically modify the targeted pathogen [ 11 , 32 ]. This homology-based gene silencing stimulated by transgenes (co-suppression), antisense, or dsRNAs has been demonstrated in several plant pathogenic fungi/oomycetes, including different mold fungi, such as Botrytis cinerea , Neurospora crassa , and Sclerotinia sclerotiorum [ 11 , 18 , 33 , 34 ]; blast, blight, and rust fungi, such as Fusarium asiaticum , Fusarium graminearum , Magnaporthe oryzae, and Puccinia striiformis f. sp. tritici [ 17 , 35 , 36 , 37 , 38 ]; mildew, and others, such as Blumeria graminis , Cochliobolus sativus, and Venturia inaequalis [ 39 , 40 , 41 ].…”

“…The E. coli HT115 (DE3) harbors the pro-phage λDE3 encoding the Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible T7 polymerase gene for dsRNA transcription [ 101 , 113 , 115 , 116 ]. Even though, the bacterial production systems may contain bacterial homologous DNA molecules; that may affect the RNA quality and applicability, crude extracts of dsRNA can be applied on plants to test its efficiency against plant pathogens and pests [ 18 , 117 ]. Researchers demonstrated that bacterially expressed dsRNAs can be used to induce RNAi in fungus [ 18 ], virus [ 49 ], worms [ 118 ], and in insect pests [ 56 , 119 ].…”

“…Consequently, RNAi is getting research attention also as an environmentally friendly alternative to agricultural pest and pathogen control. In fact, because of its sequence-dependent mode of action, RNAi technology has an enormous range of potential as plant protection application, including control against insects [ 13 ], mite pests [ 14 , 15 ], plant pathogens [ 11 , 16 , 17 , 18 ], nematodes, and weeds [ 10 , 19 , 20 , 21 ].…”

Biotechnological Approaches: Gene Overexpression, Gene Silencing, and Genome Editing to Control Fungal and Oomycete Diseases in Grapevine

“…Selecting fungicide sites of action, Nerva and colleagues constructed a single long dsRNA molecule that exerted protection against grey mold in vitro on grapevine detached leaves and grapes at post-harvest, applied through the high-pressure spray and petiole adsorption. Despite different levels of protection being recorded among the dsRNAs delivery methods, interestingly, all the techniques that facilitate the provision of intact dsRNA to the fungus were assumed as effective [ 128 ].…”

Epigenetic Modifications: An Unexplored Facet of Exogenous RNA Application in Plants

“…Unsurprisingly, plant researchers have lately resorted to induction of RNAi by exogenous application of RNA molecules having the potential to trigger RNAi (exogenous RNAi, exo-RNAi), as an effective and transgene-free alternative to GM crops [31][32][33][34][35]. Indeed, dsRNAs/siRNAs were applied in plants by methods such as spraying, petiole uptake, trunk injection and root absorption in order to modify plant gene expression [36][37][38][39] and to confer resistance against viruses [40][41][42][43][44], fungi [45][46][47][48][49] and insects [50][51][52][53][54]. In the near future, one may envisage that exo-RNAi could potentially replace conventional herbicides, fungicides and insecticides, and a great amount of effort is invested in this direction by most major crop industries [55].…”