Genetically encoded libraries and spider venoms as emerging sources for crop protective peptides.

Agricultural crops are targeted by various pathogens (fungi, bacteria, and viruses) and pests (herbivorous arthropods). Antimicrobial and insecticidal peptides are increasingly recognized as eco-friendly tools for crop protection due to their low propensity for resistance development and the fact that they are fully biodegradable. However, historical challenges have hindered their development, including poor stability, limited availability, reproducibility issues, high production costs, and unwanted toxicity. Toxicity is a primary concern because crop-protective peptides interact with various organisms of environmental and economic significance. This review focuses on the potential of genetically encoded peptide libraries like the use of two-hybrid-based methods for antimicrobial peptides identification and insecticidal spider venom peptides as two main approaches for targeting plant pathogens and pests. We discuss some key findings and challenges regarding the practical application of each strategy. We conclude that genetically encoded peptide library- and spider venom-derived crop protective peptides offer a sustainable and environmentally responsible approach for addressing modern crop protection needs in the agricultural sector.

Influence of Nitrogen on Grapevine Susceptibility to Downy Mildew.

Downy mildew, caused by the obligate parasite Plasmopara viticola, is one of the most important threats to viticulture. The exploitation of resistant and susceptibility traits of grapevine is one of the most promising ways to increase the sustainability of disease management. Nitrogen (N) fertilization is known for influencing disease severity in the open field, but no information is available on its effect on plant-pathogen interaction. A previous RNAseq study showed that several genes of N metabolism are differentially regulated in grapevine upon P. viticola inoculation, and could be involved in susceptibility or resistance to the pathogen. The aim of this study was to evaluate if N fertilization influences: (i) the foliar leaf content and photosynthetic activity of the plant, (ii) P. viticola infectivity, and (iii) the expression of the candidate susceptibility/resistance genes. Results showed that N level positively correlated with P. viticola infectivity, confirming that particular attention should be taken in vineyard to the fertilization, but did not influence the expression of the candidate genes. Therefore, these genes are manipulated by the pathogen and can be exploited for developing new, environmentally friendly disease management tools, such as dsRNAs, to silence the susceptibility genes or breeding for resistance.

Disease Forecasting for the Rational Management of Grapevine Mildews in the Chianti Bio-District (Tuscany).

Downy and powdery mildews are major grapevine diseases. In organic viticulture, a few fungicides with protectant activities (copper and sulphur in particular) can be used, and their preventative application frequently leads to unneeded spraying. The adoption of an epidemiological disease forecasting model could optimise the timing of treatments and achieve a good level of disease protection. In this study, the effectiveness of the EPI (Etat Potentiel d'Infection) model in predicting infection risk for downy and powdery mildews was evaluated in nine organic vineyards located in Panzano in Chianti (FI), over a 2-year period (2020-2021). The reliability of the EPI model was investigated by comparing the disease intensities, the number of fungicide sprayings, the quantities of the fungicides (kg/ha), and the costs of the treatment achieved, with or without the use of the model, in a vineyard. The results obtained over two seasons indicated that, in most cases, the use of the EPI model accurately signalled the infection risk and allowed for a reduction in the frequency and cost of spraying, particularly for powdery mildew control (-40% sprayings, -20% costs compared to the farmer's schedule), without compromising crop protection. The use of the EPI model can, therefore, contribute to more-sustainable disease management in organic viticulture.

Pathogen Adaptation to American (Rpv3-1) and Eurasian (Rpv29) Grapevine Loci Conferring Resistance to Downy Mildew.

Durable resistance is a key objective in genetic improvement for disease resistance in grapevines, which must survive for years in the field in the presence of adaptable pathogen populations. In this study, the adaptation of 72 Northern Italian isolates of Plasmopara viticola, the downy mildew agent, has been investigated into Bianca, possessing Rpv3-1, the most frequently exploited resistance locus for genetic improvement, and Mgaloblishvili, a Vitis vinifera variety possessing the newly discovered Rpv29 locus. Infection parameters (latency period, infection frequency, and disease severity) and oospore production and viability were evaluated and compared to those of Pinot noir, the susceptible reference. The expected levels of disease control were achieved by both resistant cultivars (>90% on Bianca; >25% on Mgaloblishvili), despite the high frequency of isolates able to grow on one (28%) or both (46%) accessions. The disease incidence and severity were limited by both resistant cultivars and the strains able to grow on resistant accessions showed signatures of fitness penalties (reduced virulence, infection frequency, and oospore density). Together, these results indicate an adequate pathogen control but suitable practices must be adopted in the field to prevent the diffusion of the partially adapted P. viticola strains to protect resistance genes from erosion.

RNAi of a Putative Grapevine Susceptibility Gene as a Possible Downy Mildew Control Strategy.

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the diseases causing the most severe economic losses to grapevine (Vitis vinifera) production. To date, the application of fungicides is the most efficient method to control the pathogen and the implementation of novel and sustainable disease control methods is a major challenge. RNA interference (RNAi) represents a novel biotechnological tool with a great potential for controlling fungal pathogens. Recently, a candidate susceptibility gene (VviLBDIf7) to downy mildew has been identified in V. vinifera. In this work, the efficacy of RNAi triggered by exogenous double-stranded RNA (dsRNA) in controlling P. viticola infections has been assessed in a highly susceptible grapevine cultivar (Pinot noir) by knocking down VviLBDIf7 gene. The effects of dsRNA treatment on this target gene were assessed by evaluating gene expression, disease severity, and development of vegetative and reproductive structures of P. viticola in the leaf tissues. Furthermore, the effects of dsRNA treatment on off-target (EF1α, GAPDH, PEPC, and PEPCK) and jasmonic acid metabolism (COI1) genes have been evaluated. Exogenous application of dsRNA led to significant reductions both in VviLBDIf7 gene expression, 5 days after the treatment, and in the disease severity when artificial inoculation was carried out 7 days after dsRNA treatments. The pathogen showed clear alterations to both vegetative (hyphae and haustoria) and reproductive structures (sporangiophores) that resulted in stunted growth and reduced sporulation. Treatment with dsRNA showed signatures of systemic activity and no deleterious off-target effects. These results demonstrated the potential of RNAi for silencing susceptibility factors in grapevine as a sustainable strategy for pathogen control, underlying the possibility to adopt this promising biotechnological tool in disease management strategies.