This review presents an overview of eutypa dieback, esca and botryosphaeria dieback, the predominant grapevine trunk diseases worldwide. It covers their symptomatologies in the trunk, leaves and berries; the characteristics of the different fungal species associated with them; and host-pathogen interactions. Here, the host-pathogen relationship is defined at the cytological, physiological and molecular levels. Currently available experimental tools for studying these diseases, both in vitro and in the field, are discussed. Finally, a progress report on their control, which, since the ban of sodium arsenite, comprises chemical, biological and ⁄ or sanitation methods, is presented.

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one-and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, (À)-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) (À)-4-chloro-terremutin hydrate (4) and(+)-4-hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with (À)-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins. and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-(À)-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)(À)-3-hydroxymellein

No abstract

Esca is a destructive disease in grapevines (Vitis vinifera L.) caused by at least three fungi and characterised by two different external symptoms, the apoplectic and leaf stripe form. This latter form can be discerned as soon as symptoms become visible, but the preceding discrete signs during incubation are poorly or not understood. To further understand the development of the leaf stripe form, the period preceding and following the appearance of symptoms was investigated by studying physiological and molecular markers associated with photosynthetic mechanisms and stress response. No perturbation of any targeted metabolism was observed in asymptomatic leaves of asymptomatic canes from vines showing the leaf stripe form of esca. Conversely, drastic alterations of photosynthesis functions were registered in presymptomatic leaves, as revealed by the decrease of gas exchange and chlorophyll fluorescence, and the repression of photosynthesis-related genes. These alterations were amplified during symptom development. Expression of defence-related genes was affected and detected early in presymptomatic leaves and amplified during symptom expression. Our results suggest that grapevines may react precociously by reducing photosynthesis and triggering defence mechanisms in response to the leaf stripe form of esca.

25Botryosphaeria dieback is a grapevine trunk disease with a worldwide distribution and associated 26with Diplodia seriata and Neofusicoccum parvum among several other Botryosphaeriaceae species. 27The aforementioned xylem-inhabiting fungi cause wood lesions, leaf and berry symptoms and 28 eventually lead to the death of the plant. The aim of this work was to develop a simple model 29 system to reproduce the foliar symptoms caused by D. seriata and N. parvum to better characterize 30 fungal pathogenicity and determine the mechanisms involved in symptom development. This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. 3Several Botryosphaeriaceae species are associated worldwide with the grapevine trunk diseases 45 (GTDs) known as Botryosphaeria dieback (Moller and Kasimatis 1978; Larignon and Dubos 1997; 46 Graniti et al. 2000; Fischer 2006;Larignon et al. 2009;Úrbez-Torres 2011; Spagnolo et al. 2014a; 47 Larignon et al. 2015). The most common species isolated from grapevine-growing regions around 48 the world include Diplodia seriata De Not. (Cristinzio 1978; Rovesti and Montermini 1987; 49 Larignon et al. 2001; Castillo-Pando et al. 2001; Phillips et al. 2007; Savocchia et al. 2007; Úrbez-50 Torres et al. 2008) and Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. 51Phillips (Crous et al. 2006). These fungi are xylem-inhabiting and attack the framework of 52 grapevines causing perennial cankers in the wood, resulting in leaf and berry symptoms and finally 53 leading to the death of the plant. Symptoms are characterized by yellowish-orange (white cultivars) 54 or wine-red (red cultivars) spots on leaf margins and blade, and in most cases, the emergence of a 55 brown stripe on the wood under the bark (Larignon et al. 2001; Spagnolo et al. 2014a). This 56 symptom is often associated with a grey sector of rotted wood. Shrivelling and drying of 57 inflorescences or fruit clusters are frequently observed. 58The incidence of Botryosphaeria dieback, together with two other trunk diseases, esca and 59Eutypa dieback, has increased over the years. In France it was estimated that 13% of productive 60 vines were affected by GTDs in 2012 (Grosman and Doublet 2012; Bruez et al. 2013). Although 61 GTDs, including Botryosphaeria dieback, appear to be increasingly common, accurate knowledge 62 of host-pathogen interactions poses certain problems, including (i) determining the seasonal 63 influence on field-collected data due to an uncontrolled environment and (ii) distinguishing 64 pathogen effects on grapevines from effects in response to other biotic agents in the field. This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. 4Eutypa dieback symptoms, the stunting of new shoots with small cup-shaped, chlorotic and tattered 70 leaves, were reproduced on greenhouse cuttings that were infected with Eutypa ...

Botryosphaeria dieback is a fungal grapevine trunk disease that represents a threat for viticulture worldwide due to the decreased production of affected plants and their premature death. This dieback is characterized by a typical wood discoloration called brown stripe. Herein, a proteome comparison of the brown striped wood from Botryosphaeria dieback-affected standing vines cultivars Chardonnay, Gewurztraminer, and Mourvèdre was performed. The transcript analysis for 15 targeted genes and the quantification of both total phenolics and specific stilbenes were also performed. Several pathogenesis-related proteins and members of the antioxidant system were more abundant in the brown striped wood of the three cultivars, whereas other defense-related proteins were less abundant. Additionally, total phenolics and some specific stilbenes were more accumulated in the brown striped wood. Strongest differences among the cultivars concerned proteins of the primary metabolism, which looked to be particularly impaired in the brown striped wood of ‘Chardonnay’. Low abundance of some proteins involved in defense response probably contributes to make global response insufficient to avoid the symptom development. The differential susceptibility of the three grapevine cultivars could be linked to the diverse expression of various proteins involved in defense response, stress tolerance, and metabolism.

For all these reasons, the goal of this work was to investigate plants affected by E and A, 101 through analyzing physiological perturbations on both herbaceous and woody samples in a 102 same plant. We focused on phenylpropanoid pathway by analysing the total phenolic 103 compounds, the stilbene content and the expression of 9 related genes. The expression of 11 104 stress defense response genes and 2 water-stress related genes as well as the abscisic acid 105 quantification were also performed. Moreover, known fungal metabolites such as 6-106 methylsalicyclic acid, terremutin, scytalone, isosclerone, (R)-mellein and (3R,4R)-4-107 hydroxymellein were quantified to characterize the fungus-plant interaction. 108 considered as E plants. Four groups of samples were defined for green stems: C (stems from 120 control plants), A and E (symptomatic stems from apoplectic (A) and Esca proper (E)-121 affected plants) and aS (asymptomatic stems from A and E plants) (Fig. 1). In woody tissues, 122 2 types of samples were studied: asymptomatic and black streaked wood. Black streaking 123 consists of single or more xylem vessels gathered into individual blackish brown bundles 124 Other fungi associated with grapevine trunk diseases, such as Botryophaeriaceae species and 138 E. lata were also isolated. In the opposite, no fungi were detected from either non discolored 139 wood of trunk and cordons, or discolored and non-discolored woody tissues of one-year-old 140 stems, as well as from green stems of control or diseased plants (Spagnolo et al. 2012). 109 MATERIAL AND METHODS 110 Plant material 111 RNA extraction 143Total RNA was isolated from 2 × 50 mg of powdered green stem tissues and 3 × 50 mg of 144 woody tissues (cordon and trunk) using the Plant RNA Purification Reagent (Invitrogen, 145Cergy Pontoise, France). The RNA pellet was re suspended in 20 µL of RNase-free water, 146 then treated with RQ1 DNase enzyme (Promega) and quantified by measuring the absorbance 147 at 260 nm following manufacturer's instructions. 148 149 Real-time RT-PCR analysis of gene expression 150In total, 150 ng of total RNA were reverse-transcribed using the Verso SYBR 2-step QRT 151 ROX enzyme (ABgene, Surrey, UK) according to the manufacturer's protocol. PCR 152

Among grapevine trunk diseases, esca proper and apoplexy commonly represent a threat for viticulture worldwide. To retrieve further information about the mechanisms activated in apoplectic and esca proper-affected plants, a two-dimensional gel electrophoresis (2-DE) based analysis was conducted on green stems from 26-year-old standing vines. Symptomatic and asymptomatic stems from both apoplectic (A) and esca proper-affected (E) plants compared to control (without visual symptom since 10 years) stems were studied. Thirty-three differentially expressed proteins were identified by nanoLC-MS/MS and included into three groups conceptually defined as proteins involved in (i) metabolism and energy, (ii) stress tolerance, and (iii) defense response. For nine of them, expression of the relative mRNA's was also monitored by qRT-PCR. Proteome variations were specifically related to apoplexy and esca proper but were more similar in asymptomatic stems than in the symptomatic ones. Remarkable quantitative differences were noted for several proteins in symptomatic stems according to the expressed form, A and E. Results further indicate that similar responses are likely activated in asymptomatic stems but a various quantitative expression is triggered upon onset of apoplexy or esca proper symptoms while both kind of plants are infected by the same pathogenic fungi.