Botryosphaeria dieback is a grapevine trunk disease with a worldwide distribution associated with Diplodia seriata and Neofusicoccum parvum, among several other Botryosphaeriaceae species. The aforementioned xylem-inhabiting fungi cause wood lesions and leaf and berry symptoms, and eventually lead to the death of the plant. The aim of this work was to develop a simple model system to reproduce the foliar symptoms caused by D. seriata and N. parvum to better characterize fungal pathogenicity and determine the mechanisms involved in symptom development. Green stems of grafted ‘Aragonez’ grapevine cuttings were inoculated with three isolates of N. parvum and two isolates of D. seriata with different degrees of virulence and the experiment was repeated four times from 2011 to 2014. Three months after inoculation, the lesions associated with N. parvum were larger than those associated with D. seriata. Similarly, 8 months after inoculation, the percentage of plants showing foliar symptoms was greater in the N. parvum treatments than in the D. seriata treatments. During the emergence of foliar symptoms, plant stress-related responses were modulated in green stems and leaves, especially a downregulation of superoxide dismutase (SOD) and fasciclin-like arabinogalactan protein (fascAGP) and an upregulation of stilbene synthase (STS) genes with an accumulation of phenolics. In conclusion, the simple model system developed allowed a good characterization of isolate pathogenicity and correlation with foliar symptoms of Botryosphaeria dieback, namely spots on leaf margin and blade.

Vitis vinifera is affected by many diseases every year, depending on causal agents, susceptibility of cultivars, and climate region. Some are caused by a single agent, such as gray mold caused by Botrytis cinerea or powdery mildew caused by Erysiphe necator. Others result from the actions of a complex of pathogens such as grapevine trunk diseases (GTDs). GTDs are presently among the most devastating diseases in viticulture worldwide because both the economic losses and the long-term sustainability of vineyards are strongly affected. The complexity of GTDs results from the diversity of associated fungi, the undetermined period of latency within the vine (asymptomatic status), the erratic foliar symptom expression from one year to the next, and, probably correlated with all of these points, the lack of efficient strategies to control them. Distinct methods can be beneficial to improve our knowledge of GTDs. In vitro bioassays with cell suspensions, calli, foliar discs, full leaves, or plantlets, and in vivo natural bioassays with cuttings, grafted plants in the greenhouse, or artificially infected ones in the vineyard, can be applied by using progressive integrative levels of in vitro and in vivo, depending on the information searched. In this review, the methods available to understand GTDs are described in terms of experimental procedures, main obtained results, and deliverable prospects. The advantages and disadvantages of each model are also discussed.

The aroma of grapes is cultivar dependent and is influenced by terroir, vineyard practices, and abiotic and biotic stresses. Trincadeira is a non-aromatic variety associated with low phenolic content and high sugar and organic acid levels. This cultivar, widely used in Portuguese wines, presents high susceptibility to Botrytis cinerea. This work aimed to characterise the volatile profile of Trincadeira grapes and how it changes under infection with B. cinerea. Thirty-six volatile organic compounds were identified, from different functional groups, namely alcohols, ester acetates, fatty acid esters, fatty acids, aldehydes, and products of the lipoxygenase pathway. Both free and glycosidic volatile organic compounds were analysed by Gas Chromatography and Gas Chromatography coupled to Mass Spectrometry for component quantification and identification, respectively. A multivariance analysis showed a clear discrimination between healthy and infected grapes with 2-trans-hexenal and isoamyl-acetate among the compounds identified as negative and positive markers of infection, respectively. Ester acetates such as 2-phenylethyl acetate, isoamyl acetate, and 2-methylbutyl acetate were present in higher contents in infected samples, whereas the contents of several fatty acid esters, such as ethyl decanoate and ethyl dodecanoate, decreased. These data were integrated with quantitative PCR data regarding genes involved in volatile metabolism and showed up-regulation of a gene coding for Hydroperoxide Lyase 2 in infected grapes. Altogether, these changes in volatile metabolism indicate an impact on the grape quality and may be related to defence against B. cinerea. The presence/absence of specific compounds might be used as infection biomarkers in the assessment of Trincadeira grapes’ quality.

Botryosphaeria dieback caused by several Botryosphaeriaceae species is one of the most important grapevine trunk diseases affecting vineyards worldwide. These fungi cause wedge-shaped perennial cankers and black streaking of the wood and have also been associated with intervein leaf chlorosis, dried or mummified berries, and eventually, the death of the plant. Early season symptoms may sometimes be disregarded by growers, being mistaken with symptoms from other diseases such as downy mildew or botrytis rot. Currently, few studies are available to determine what species may be causing these early season symptoms in grapevines. During the 2018 season, during the flowering period, grapevine samples showing necrosis on green shoots, dried inflorescences, and flowers, were collected in vineyards throughout the central regions of Portugal. Isolations were performed from symptomatic organs, and twenty-three isolates of Botryosphaeriaceae were selected. An analysis of the ITS and part of the translation elongation factor 1-α sequences was performed, revealing that the two main species apparently responsible for these symptoms were Diplodia seriata and Neofusicoccum parvum. In pathogenicity tests conducted on 1-year-old plants grown under controlled conditions in a greenhouse and on field-grown clusters, symptoms were reproduced, confirming the pathogenic behavior of the selection of isolates.

Botrytis cinerea is responsible for the gray mold disease, severely affecting Vitis vinifera grapevine and hundreds of other economically important crops. However, many mechanisms of this fruit-pathogen interaction remain unknown. The combined analysis of the transcriptome and metabolome of green fruits infected with B. cinerea from susceptible and tolerant genotypes was never performed in any fleshy fruit, mostly because green fruits are widely accepted to be resistant to this fungus. In this work, peppercorn-sized fruits were infected in the field or mock-treated, and berries were collected at green (EL32) stage from a susceptible (Trincadeira) and a tolerant (Syrah) variety. RNAseq and GC–MS data suggested that Syrah exhibited a pre-activated/basal defense relying on specific signaling pathways, hormonal regulation, namely jasmonate and ethylene metabolisms, and linked to phenylpropanoid metabolism. In addition, putative defensive metabolites such as shikimic, ursolic/ oleanolic, and trans-4-hydroxy cinnamic acids, and epigallocatechin were more abundant in Syrah than Trincadeira before infection. On the other hand, Trincadeira underwent relevant metabolic reprogramming upon infection but was unable to contain disease progression. RNA-seq analysis of the fungus in planta revealed an opposite scenario with higher gene expression activity within B. cinerea during infection of the tolerant cultivar and less activity in infected Trincadeira berries. The results suggested an activated virulence state during interaction with the tolerant cultivar without visible disease symptoms. Together, this study brings novel insights related to early infection strategies of B. cinerea and the green berry defense against necrotrophic fungi.