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With a reduction in available chemical treatments, there is an increased interest in biological control of grapevine trunk diseases. Few studies have investigated the impact of introducing beneficial microorganisms in the rhizosphere on the existing indigenous soil microbiome. In this study, we explored the effect of two biocontrol agents (BCAs), Trichoderma atroviride SC1 (Ta SC1) (Vintec; Certis Belchim) and Bacillus subtilis PTA-271 (Bs PTA-271), on the grapevine rhizosphere bacterial and fungal microbiome as well as plant defense expression using high-throughput amplicon sequencing and quantitative real-time polymerase chain reaction (PCR), respectively. Additionally, we quantified both Ta SC1 and Bs PTA-271 in the rhizosphere over time using droplet digital PCR. The fungal microbiome was more affected by factors such as soil type, BCA treatment, and sampling time compared with the bacterial microbiome. Specifically, Ta SC1 application produced negative impacts on fungal diversity, whereas application of BCAs did not affect bacterial diversity. Interestingly, the survival and establishment of both BCAs showed opposite trends depending on the soil type, indicating that the physicochemical properties of soils have a role in BCA establishment. Fungal co-occurrence networks were less complex than bacterial networks but highly impacted by Ta SC1 application. Soils treated with Ta SC1 presented more complex and stable co-occurrence networks, with a higher number of positive correlations. Induced grapevine defenses also differed according to the soil, being more affected by BCA inoculation on sandy soil. The findings of this research emphasize the complex relationships among microorganisms in the rhizosphere and highlight the significance of taking into account various factors, such as soil type, sampling time, and BCA treatment, and their influence on the structure and dynamics of microbial communities.

Botryosphaeria dieback is a grapevine trunk disease caused by fungi of the Botryosphaeriaceae family, which attacks more specifically the woody tissues. The infection leads to different symptoms including a severe form with a leaf drop as well as premature plant death. Botryosphaeria dieback causes major economic losses, since no effective treatment is yet available. A better understanding is necessary to find solutions to fight this disease. In this study, our objective was to characterize the “leaf drop” form by (1) looking for the presence of pathogens in the basal internodes of stems, (2) quantifying blocked vessels by tylosis and/or gummosis, and (3) describing the impact of the disease on vine physiology (gene expression and metabolome) and development (establishment and functioning of the cambium and phellogen) at the level of xylem and phloem of basal stem internodes. Our study has shown that Botryosphaeriaceae were present in both phloem and xylem of the basal internodes of the annual stem, with xylem vessels obturated. We have also clearly demonstrated that gene expression and metabolite profiles were strongly modified in both xylem and phloem of diseased plants. Differences in stems between healthy (control, C) and diseased (D) plants were low at flowering (vines not yet symptomatic), higher at the onset of symptom expression and still present, although less marked, at full disease expression. qRT-PCR analysis showed in both phloem and xylem an overexpression of genes involved in plant defense, and a repression of genes related to meristematic activity (i.e. vascular cambium and phellogen). Metabolomic analysis showed specific fingerprints in stems of healthy and diseased plants from the onset of symptom expression, with an increase of the level of phytoalexins and mannitol, and a decrease of 1-kestose one. At the structural level, many alterations were observed in internodes, even before the onset of symptoms: a classical obstruction of xylem vessels and, for the first time, a disorganization of the secondary phloem with an obstruction of the sieve plates by callose. The disease modifies the development of both secondary phloem (liber) and phellogen. Altogether, this study combining different approaches allowed to highlight deep vine dysfunction in the internodes at the base of stems, that may explain vine decline due to Botryosphaeria dieback.

Background: Grapevine is a woody, perennial plant of high economic importance worldwide. Like other plants, it lives in close association with large numbers of microorganisms. Bacteria, fungi and viruses are structured in communities, and each individual can be beneficial, neutral or harmful to the plant. In this sense, microorganisms can interact with each other and regulate plant functions (including immunity) and even provide new ones. Thus, the grapevine associated with its microbial communities constitutes a supra-organism, also called a holobiont, whose functioning is linked to established plant-microorganism interactions. Aim of review: The overall health of the plant may be conditioned by the diversity and structure of microbial communities. Consequently, an optimal microbial composition will consist of a microbial balance allowing the plant to be healthy. Conversely, an imbalance of microbial populations could lead to (or be generated by) a decline of the plant. The microbiome is an active component of the host also responsive to biotic and abiotic changes; in that respect, a better understanding of the most important drivers of the composition of plant microbiomes is needed. Key scientific concepts of review: This article presents the current state of the art about the grapevine microbiota and its composition according to the plant compartments and the influencing factors. We also focus on situations of imbalance, in particular during plant disease or decline. Finally, we discuss the possible interest of microbial engineering in an agrosystem such as viticulture.

Esca belongs to the group of grapevine trunk diseases - fungal diseases present worldwide in all wine-growing regions. Some aspects of the disease, like the development of external symptoms, have still not been completely discovered and are believed to be affected by several factors, including interactions within the vine microbiome. The examination of the occurrence of the yeast-like fungus Aureobasidium pullulans in the healthy wood of Esca-diseased grapevines via both isolation and qPCR measurements showed a positive correlation between its abundance and the severity of foliar symptoms, suggesting the contribution of this fungus to Esca pathogenesis via an indirect action. In vitro confrontation tests revealed antagonistic interaction between A. pullulans and the Esca pathogen Phaeomoniella chlamydospora. Mutual growth inhibition and the induction of asexual sporogenesis were observed for both fungi without cytotoxic effects. In planta confrontation tests revealed that A. pullulans in combination with P. chlamydospora can lead to severe foliar damage in a strain-dependent manner. This phenomenon could be explained by the altered metabolism of the Esca pathogen in the presence of A. pullulans, or by the cumulative/synergistic effects of the secreted polysaccharides and/or proteins of the two fungi. The present study shows the importance of microbial interactions in the development of plant diseases, highlighting that even a non-pathogenic microorganism can act as a disease-enhancer.

In the past, most grapevine trunk diseases (GTDs) have been controlled by treatments with sodium arsenite. For obvious reasons, sodium arsenite was banned in vineyards, and consequently, the management of GTDs is difficult due to the lack of methods with similar effectiveness. Sodium arsenite is known to have a fungicide effect and to affect the leaf physiology, but its effect on the woody tissues where the GTD pathogens are present is still poorly understood. This study thus focuses on the effect of sodium arsenite in woody tissues, particularly in the interaction area between asymptomatic wood and necrotic wood resulting from the GTD pathogens’ activities. Metabolomics was used to obtain a metabolite fingerprint of sodium arsenite treatment and microscopy to visualize its effects at the histo-cytological level. The main results are that sodium arsenite impacts both metabolome and structural barriers in plant wood. We reported a stimulator effect on plant secondary metabolites in the wood, which add to its fungicide effect. Moreover, the pattern of some phytotoxins is affected, suggesting the possible effect of sodium arsenite in the pathogen metabolism and/or plant detoxification process. This study brings new elements to understanding the mode of action of sodium arsenite, which is useful in developing sustainable and eco-friendly strategies to better manage GTDs.

Grapevine flowering is an important stage in the epidemiology of Botrytis cinerea, the causal agent of gray mold disease. To prevent infection and to minimize postharvest losses, the control of this necrotrophic fungus is mainly based on chemical fungicides application. However, there is a growing interest in other control alternatives. Among them, the use of beneficial microorganisms appears as an eco-friendly strategy. This study aims to investigate the effect of Paraburkholderia phytofirmans PsJN, root-inoculated or directly sprayed on fruiting cuttings inflorescences to control B. cinerea growth. For this purpose, quantification by real time PCR of Botrytis development, direct effect of PsJN on fungal spore germination and chemotaxis were assayed. Our results showed a significant protective effect of PsJN only by direct spraying on inflorescences. Moreover, we demonstrated an inhibition exerted by PsJN on Botrytis spore germination, effective when there was a direct contact between the two microorganisms. This study showed that PsJN is positively attracted by the pathogenic fungus B. cinerea and forms a biofilm around the fungal hyphae in liquid co-culture. Finally, microscopic observations on fruit cuttings revealed a co-localization of both beneficial and pathogenic microorganisms on grapevine receptacle and stigma that might be correlated with the protective effect induced by PsJN against B. cinerea via a direct antimicrobial effect. Taking together, our findings allowed us to propose PsJN as a biofungicide to control grapevine gray mold disease.

Grape production worldwide is increasingly threatened by grapevine trunk diseases (GTDs). No grapevine cultivar is known to be entirely resistant to GTDs, but susceptibility varies greatly. To quantify these differences, four Hungarian grape germplasm collections containing 305 different cultivars were surveyed to determine the ratios of GTDs based on symptom expression and the proportion of plant loss within all GTD symptoms. The cultivars of monophyletic Vitis vinifera L. origin were amongst the most sensitive ones, and their sensitivity was significantly (p < 0.01) higher than that of the interspecific (hybrid) cultivars assessed, which are defined by the presence of Vitis species other than V. vinifera (e.g., V. labrusca L., V. rupestris Scheele, and V. amurensis Rupr.) in their pedigree. We conclude that the ancestral diversity of grapes confers a higher degree of resilience against GTDs.

The production of wine and table grapes is hampered by Phaeomoniella chlamydospora, a fungus related to Petri diseases and Esca, two of the major grapevine trunk diseases causing the death of vines all over the world. This study analyses the effect of a commercial-based substance (chitosan hydrochloride 2.5%) on grapevine–P. chlamydospore interaction. Two experimental scenarios were evaluated: (i) root application in a greenhouse-plant model system and (ii) foliar application in an open-field environment (Tempranillo grape cultivar). Vascular necrosis and pathogen recovery were reduced after chitosan application. The treatment increased root development and the flavonoid index. On open-field application, the treatment did not significantly help decrease the expression of the foliar symptoms of Esca. In symptomatic leaves, the results revealed a lower overexpression of defence-related genes (Chit 1b, CHV5, STS and PR6) in plants treated with chitosan than in nontreated vines. Altogether, this study attempts to provide a first insight into the potential role of chitosan hydrochloride in the treatment of P. chlamydospora and the interaction on the control of the Esca–pathosystem complex. Differences were found between the root and foliar spraying modes of action.

Abstract Background Bacillus subtilis strains have been widely studied for their numerous benefits in agriculture, including viticulture. Providing several assets, B. subtilis spp. are described as promising plant-protectors against many pathogens and as influencers to adaptations in a changing environment. This study reports the draft genome sequence of the beneficial Bacillus subtilis PTA-271, isolated from the rhizospheric soil of healthy Vitis vinifera cv. Chardonnay at Champagne Region in France, attempting to draw outlines of its full biocontrol capacity. Results The PTA-271 genome has a size of 4,001,755 bp, with 43.78% of G + C content and 3945 protein coding genes. The draft genome of PTA-271 putatively highlights a functional swarming motility system hypothesizing a colonizing capacity and a strong interacting capacity, strong survival capacities and a set of genes encoding for bioactive substances. Predicted bioactive compounds are known to: stimulate plant growth or defenses such as hormones and elicitors, influence beneficial microbiota, and counteract pathogen aggressiveness such as effectors and many kinds of detoxifying enzymes. Conclusions Plurality of the putatively encoded biomolecules by Bacillus subtilis PTA-271 genome suggests environmentally robust biocontrol potential of PTA-271, protecting plants against a broad spectrum of pathogens.

(1) Background: Grapevine trunk diseases (GTDs) have become a global threat to vineyards worldwide. These diseases share three main common features. First, they are caused by multiple pathogenic micro-organisms. Second, these pathogens often maintain a long latent phase, which makes any research in pathology and symptomatology challenging. Third, a consensus is raising to pinpoint combined abiotic stresses as a key factor contributing to disease symptom expression. (2) Methods: We analyzed the impact of combined abiotic stresses in grapevine cuttings artificially infected by two fungi involved in Botryosphaeria dieback (one of the major GTDs), Neofusicoccum parvum and Diplodia seriata. Fungal-infected and control plants were subjected to single or combined abiotic stresses (heat stress, drought stress or both). Disease intensity was monitored thanks to the measurement of necrosis area size. (3) Results and conclusions: Overall, our results suggest that combined stresses might have a stronger impact on disease intensity upon infection by the less virulent pathogen Diplodia seriata. This conclusion is discussed through the impact on plant physiology using metabolomic and transcriptomic analyses of leaves sampled for the different conditions.

Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated.

The growing concerns on human and environment health are forcing the plant protection industry toward the formulation of more eco-sustainable plant protection products (PPP), both efficient and innovative in their approach to disease control. A large number of these innovative formulations now rely on a combination of pathogens antagonistic properties and stimulation of natural plant defense to pathogens. The formulation HA + Cu(II), in which copper is delivered to the plants by the drug-delivery molecule hydroxyapatite (HA), was found efficient against the grapevine pathogens Plasmopara viticola and Phaeoacremonium minimum and able to induce the host-plant defense system. We investigated the HA + Cu(II) impacts on grapevine physiology, both in uninfected and when infected by the Botryosphaeria dieback agents Diplodia seriata and Neofusicoccum parvum. This study of plant physiology and disease impact were addressed to evaluate both the HA + Cu(II) potential as a plant defense elicitor and its possible and future use as PPP in vineyard. Our results showed that HA + Cu(II) induced several key-defense genes without negatively affecting plant growth and photosynthetic activity. In addition, fungistatic effect on the two Botryosphaeriaceae at the in planta tested concentrations is reported. Altogether, our results obtained under controlled conditions fully support the potential of HA + Cu(II) as a promising PPP toward grapevine trunk diseases in vineyard.

Fungal species involved in Esca cause the formation of grapevine wood necroses. It results in the deterioration of vascular network transport capacity and the disturbance of the physiological processes, leading to gradual or sudden grapevine death. Herein, for two consecutive growing seasons, a detailed analysis of the structural (wood necrosis and leaf discoloration) and physiological parameters related to the water use of healthy and esca-symptomatic grapevines was conducted. Measurements were carried out on 17-year-old grapevines that expressed, or not, Esca-leaf symptoms in a vineyard of the Bordeaux region (France). Whole-plant transpiration was recorded continuously from pre-veraison to harvest, using noninvasive sap flow sensors. Whole-plant transpiration was systematically about 40–50% lower in Esca-diseased grapevines compared with controls, and this difference can be observed around 2 weeks before the first Esca-foliar symptoms appeared in the vineyard. Unlike grapevine sap flow disruption, structural (e.g., leaf discolorations), functional (e.g., stomatal conductance, photosynthetic activity, phenolic compounds), and genetic (e.g., expression of leaf-targeted genes) plant responses were only significantly impacted by Esca at the onset and during leaf symptoms development. We conclude that sap flow dynamic, which was related to a high level of a white-rot necrosis, provides a useful tool to predict plant disorders due to Esca-grapevine disease.

A decline of various grapevines (Vitis vinifera L.) in the province of Doukkala in Morocco was observed in 2021. The causal pathogen was identified as Lasiodiplodia viticola based on morphological characteristics and phylogenetic analysis of the internal transcribed region (ITS), the β-tubulin gene (TUB) and calmodulin (cmdA). Koch’s postulates were confirmed by successful re-isolation of L. viticola from plants inoculated with the pathogen under controlled conditions. The disease was shown to be prevalent in Bni Hilal (71.43%), Laamria (60%), and Boulaouane (40%) districts, but was quasi-absent in Lmechrek. To understand the dominance of L. viticola as one of the grapevine trunk pathogens, effects of temperature (10–40 °C) and pH (pH 3–pH 12) on growth and sporulation were investigated. The species were able to grow in a range of temperatures ranging from 15 to 40°C and showed a higher growth rate at 35 °C. The fungus were also characterized by a broad optimum pH ranging between 3–12. This study is the first report dealing with L. viticola associated with grapevine trunk diseases in Morocco. Additional studies are therefore required to understand the high occurrence of this disease in vineyards, which is likely due to climate changes. A good understanding of this complex disease might help to develop a reliable and sustainable preventive control strategy.

Grapevine trunk diseases (GTD) are currently one of the most devastating and challenging diseases in viticulture, leading to considerable yield losses and a remarkable decline in grapevine quality. The identification of the causal agents is the cornerstone of an efficient approach to fighting against fungal diseases in a sustainable, non-chemical manner. This review attempts to describe and expose the symptoms of each pathology related to GTD, the modes of transmission, and the harmfulness of recently reported agents. Special attention was given to new diagnostic tests and technologies, grapevine defense mechanisms, molecular mechanisms of endophytes fungal colonization, and management strategies used to control these threats. The present extended review is, therefore, an updated state-of-the-art report on the progress in the management of vineyards.

Esca disease and Botryosphaeria dieback are currently considered as serious grapevine diseases which affect vineyard health and induce economic losses. Both of these trunk diseases (GTDs) are caused by a complex of pathogens, and foliar expression is influenced by several factors, including environmental factors such as water stress. To manage water stress in some vine areas, culture practice based on irrigation systems for limiting water stress have been developed; however, little knowledge of the influence of such systems on GTD emergence is currently available. The present paper addresses the impact of irrigation systems and climatic factors (rainfall and temperature) on the expression of GTDs, specifically esca and Botryosphaeria dieback. A field experiment on Chardonnay in North East Spain, a vine growing area where drought is present and which is managed by an irrigation system, was therefore carried out during a 3-year period. The water stress impact on GTD expression was evaluated by measuring the GTD incidence and analysing different physiological parameters at different phenological stages, including principal component analysis and gene expression. The main finding of this study was the significant roles of vine transpiration and water availability, which depend on irrigation volume and rainfall amount; together, they may explain the erratic symptom expression in plants infected by GTD fungi depending on the year. All these parameters are discussed to better understand the relationship between GTD expression, irrigation system and climatic factors.

Grapevine trunk diseases (GTDs) are a serious and growing threat to vineyards worldwide. The need for innovative control tools persists since pesticides used against some GTDs have been banned and only methods to prevent infections or to reduce foliar symptoms have been developed so far. In this context, the application of imaging methods, already applied to study plant–microbe interactions, represents an interesting approach to understand the effect of experimental treatments applied to reduce fungal colonization, on GTD-related pathogens activity. To this aim, trials were carried out to evaluate the efficacy of copper-based treatments, formulated with hydroxyapatite (HA) as co-adjuvant with innovative delivery properties, loaded with two different copper(II) compounds (tribasic sulfate and sulfate pentahydrate), and applied to grapevine propagation material to inhibit fungal wood colonization. The treated rootstock (Vitis berlandieri × Vitis riparia cv. K5BB) and scion cuttings (Vitis vinifera L., cv. Chardonnay) had been inoculated with a strain of Phaeoacremonium minimum (Pmi) compared to uninoculated rootstocks. Experimental treatments were applied during the water-soaking process, comparing the copper(II) compounds pure or formulated with HA, to hydrate the cuttings. After callusing, grafted vines were grown under greenhouse conditions in a nursery and inoculated with Pmi::gfp7 or with Pmi wild-type. Fifteen weeks post-inoculation, woody tissues close to the inoculation site were sampled to evaluate the efficiency of the treatments by studying the plant–microbe interaction by confocal laser scanning microscopy (CLSM). Copper and further elements were also quantified in the same tissues immediately after the treatments and on the CLSM samples. Finally, the grapevine defense responses were studied in the leaves of cuttings treated with the same formulations. The present investigation confirmed the relevant interaction of Pmi and the related transformed strain on the vascular tissues of grafted vines. Furthermore, in vitro assay revealed (i) the fungistatic effect of HA and the reduced effect of Cu fungicide when combined with HA. In planta assays showed (ii) the reduction of Pmi infection in propagation material treated with HA-Cu formulations, (iii) the movement of HA-Cu formulations inside the plant tissues and their persistence over time, and (iv) the plant defense reaction following the treatment with pure HA or Cu, or combined.

Grapevine trunk diseases (GTDs) are a complex caused by many taxonomically unrelated fungi. Diseases in this complex are responsible for different vascular and foliar symptoms, which cause overall decline and eventual death of grapevines. Though GTDs have been known since the late 19th Century their significance and impacts on vine health have been fully recognized much more recently. Today, GTDs are considered major biotic factors reducing grapevine yields and vineyard lifespans, which cause substantial economic losses to grape and wine industries worldwide. Emergence of these diseases in the early 1990s, and the urgent need for effective disease management strategies by growers and industry, focused scientific attention. As a result, a meeting in California took place in July of 1998, organized by the viticulturist Lucie Morton, who saw the potential threats posed by fungus infections of grapevine trunks. On that occasion, participants and founding members developed the structure and objectives of the International Council on Grapevine Trunk Diseases (ICGTD). Since then, the primary goal of the ICGTD has been to promote relevant research, and encourage collaboration and knowledge exchange among scientists and industry partners, on issues pertaining to GTDs. Every two years, since 1999, the ICGTD has organized the International Workshop on Grapevine Trunk Diseases (IWGTD), where scientists and industry members from around the world present, share and discuss the latest research findings on different areas concerning this increasingly important disease complex. This 11th Special Issue of Phytopathologia Mediterranea on GTDs collects together presentations given at the 11th IWGTD, which was held on July 7-12, 2019, in Penticton, British Columbia, Canada. This conference marked the 20th Anniversary of the Workshop. The Workshop was attended by 151 participants from 21 countries. A total of 64 oral and 48 poster papers were presented in four sessions, covering Pathogen Detection and Identification, Epidemiology, Host-Pathogen Interactions, and Disease Management. In addition, a special session on GTD control provided the local grapevine industry with a practical summary and overview of the current GTD management options and the economic impacts they cause. A student competition, with awards for best oral and poster presentations, was held for the first time at IWGTDs, with 20 graduate students participating. For Best Poster presentations, first place was awarded to Pierluigi Reveglia (Charles Sturt University, Wagga, New South Wales, Australia), second to Edelweiss Rangel Montoya (Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, Mexico), and third to Daina Grinbergs (Instituto Nacional de Investigaciones Agropecuarias, Chillán, Chile). For Best Oral Presentation, first place was awarded to Jinxz Pollard-Flamand (Agriculture and Agri-Food Canada Summerland RDC, British Columbia, Canada), second to Clément Labois (Université de Haute-Alsace, Colmar, France), and third to María del Pilar Martínez-Diz (Estación de Viticultura e Enoloxía de Galicia, Ourense, Spain). The ICGTD Council Members have determined that these Workshops will now be convened at 3 year intervals. The 12th IWGTD will be held in the Czech Republic in 2022. The editorial team of this Journal is indebted to all authors of papers presented in this Special Issue, for their support and contributions.

Grapevine trunk diseases constitute one of the major problems for viticulture worldwide, with Botryosphaeria dieback considered as one of the most important of these diseases. In this work, we aimed to (i) evaluate the effect of the combination of two products, Esquive® (a Trichoderma-based product) and LC2017 (a low-copper-based product), in the control of Lasiodiplodia theobromae, by evaluating the internal lesion length caused by inoculation of this pathogen on greenhouse kept grapevines of cvs Cabernet Sauvignon and Touriga Nacional and, (ii) investigating their elicitor effect on plant defense responses, through the analysis of the expression of a set of genes. The pathogen was always re-isolated from the infected tissues and was able to cause wood discoloration. Touriga Nacional exhibited longer lesions than Cabernet Sauvignon, and the application of both products did not appear to reduce lesion length when compared to LC2017 applied alone. The elicitor effect of LC2017 on plant defense was confirmed by gene expression analysis, and no significant differences were found between plants treated with LC2017 and with both products. Moreover, a specific response related to the cultivar was verified, but this apparently unique interaction between product, cultivar and pathogen remains to be further investigated.

Two Neofusicoccumparvum isolates and a UV mutant were characterized for their phytotoxin production in vitro, their pathogenicity on grapevine, and their genome sequenced. The isolate Np-Bt67 produced high level of (-)-terremutin, but almost no (R)-mellein, and it was the most aggressive on grapevine, triggering apoplexy. Similar symptoms were not induced by purified (-)-terremutin. The isolate Bourgogne S-116 (Np-B) produced 3-fold less (-)-terremutin and high amounts of (R)-mellein, but it was less aggressive on grapevine than Np-Bt67. The UV9 mutant obtained from Np-B (NpB-UV9) no longer produced (-)-terremutin but overproduced (R)-mellein by 2.5-fold, and it was as pathogenic as its parent. NpB-UV9 differed from its parent by simple mutations in two genes (transcription factor UCR-NP2_6692, regulatory protein UCR-NP2_9007), not located neither near (R)-mellein, nor (-)-terremutin biosynthetic genes, but likely involved in the control of (-)-terremutin biosynthesis. Grapevine immunity was disturbed upon challenge with these pathogens or purified phytotoxins, leading to an upregulation of SA-dependent defenses, while (-)-terremutin interfered with host JA/ET-dependent defenses. Our results suggest that neither (-)-terremutin nor (R)-mellein alone is essential for the pathogenicity of N. parvum on grapevine, since isolate/mutant non-producing these toxins in vitro is pathogenic. However, these phytotoxins could play a quantitative role in the infection process.

Esca is one of the most widespread grapevine trunk diseases affecting vineyards. This complex disease leads to leaf alterations, wood necrosis and eventually to plant death. Esca symptoms are caused by several fungi inhabiting the xylem of host plants and degrading the wood structure. The main pathogens causing the disease are Phaeomoniella chlamydospora, Phaeoacremonium minimum, Fomitiporia mediterranea and other wood-rotting basidiomycetes. Grapevine susceptibility to esca can be predisposed by several factors, especially climate, vine age, and cultivar. An experiment was carried out (in 2015) to assess if esca expression on leaves could also be clone-dependent. Chardonnay clones 76 and 95 grown in the same plot were compared according to their developmental and physiological traits, metabolome, and foliar symptom expression. Leaves were sampled during summer on visually healthy vines as controls (C), and from asymptomatic (D-) and symptomatic (D+) shoots of esca-affected vines. Analysis of their metabolomes highlighted a clone-dependent metabolite fingerprint associated to esca expression. Opposite variations of specific metabolites were found between C and D+ leaves of both clones. The experiment was repeated (in 2018). Leaf samples could be discriminated, especially the C and D+ samples for each clone, but the differences were less marked than in the first experiment. Discriminant compounds were all different between the two experiments, and showed no opposite variations between C and D+ samples of both clones, which indicated variable metabolite responses from year to year for both clones. These results confirm that the leaf metabolite fingerprint associated to esca expression is clone-dependent, and is year-dependent in intensity and nature.

Among all causes of grapevine decline, Grapevine Trunk Diseases (GTDs) are major concerns for grape growers. This paper reviews knowledge and proposes hypotheses on two major GTDs, esca and Botryosphaeria dieback, and assembles a conceptual model. The objective was to collect information into a sequence, from grapevine nursery propagation processes, through foliar symptom expression, to plant death in mature vineyards. Pathogen infection and colonization steps in woody vine tissues, and the hypotheses that have been formulated to explain the outburst of foliar symptoms, are reported and discussed. Factors that could aggravate or repress GTD symptoms and incidence expansion are also addressed. Vine physiology and pathology together could expand understanding of these diseases. Knowledge and hypotheses that need validation are summarized, and a conceptual model is proposed to explain the occurrence of symptoms and the influencing factors. The model could be useful to cope with the complexity of GTDs, and as a starting point for research to unravel knowledge gaps and suggest new disease management strategies.

Copper-based preparations have been used for more than 100 years in viticulture to control downy mildew caused by Plasmopara viticola. LC2017, and a new low-copper-based formulation, has been developed to control grapevine trunk diseases (GTDs). Previous greenhouse studies showed the potential of LC2017 to control GTDs by both fungistatic and plant defense elicitor effects. Here, we further characterize the effects of LC2017 in the field determining its impact on: (i) incidence of Esca, (ii) the vine microbiome, (iii) the vine physiology and (iv) enological parameters of juices. We observed a progressive decrease of cumulate Esca incidence in treated vines over the years with annual fluctuation related to the known erratic emergence of GTD symptoms. Neither harmful effects of LC2017 on the vine microbiota, nor on vine physiology were observed (at both transcriptomic and metabolomic levels). Similarly, no impact of LC2017 was observed on the enological properties of berries except for sugar content in juice from esca-diseased vines. The most important result concerns the transcriptomic profiles: that of diseased and LC2017 treated vines differs from that of disease untreated ones, showing a treatment effect. Moreover, the transcriptomic profile of diseased and LC2017-treated vines is similar to that of untreated asymptomatic vines, suggesting control of the disease.

Abstract Bacillus amyloliquefaciens subsp. plantarum strain Fito_F321 is a naturally occurring strain in vineyard, with the ability to colonise grapevine and which unveils a naturally antagonistic potential against phytopathogens of grapevine, including those responsible for the Botryosphaeria dieback, a GTD disease. Herein we report the draft genome sequence of B. amyloliquefaciens subsp. plantarum Fito_F321, isolated from the leaf of Vitis vinifera cv. Merlot at Bairrada appellation (Cantanhede, Portugal). The genome size is 3,856,229 bp, with a GC content of 46.54% that contains 3697 protein-coding genes, 86 tRNA coding genes and 5 rRNA genes. The draft genome of strain Fito_F321 allowed to predict a set of bioactive compounds as bacillaene, difficidin, macrolactin, surfactin and fengycin that due to their antimicrobial activity are hypothesized to be of utmost importance for biocontrol of grapevine diseases.

Esca disease is one of the major grapevine trunk diseases in Europe and the etiology is complex, since several inhabiting fungi are identified to be associated with this disease. Among the foliar symptom expressions, the apoplectic form may be distinguished and characterized by sudden dieback of shoots, leaf drop, and shriveling of grape clusters in a few days that can ultimately induce the plant death. To further understand this drastic event, we conducted transcriptomic and metabolomic analyses to characterize responses of leaves during the period preceding symptom appearance (20 and 7 days before foliar symptom expression) and at the day of apoplexy expression. Transcriptomic and metabolomic analyses provide signatures for the apoplectic leaves and most changes concerning the metabolism of carbohydrates, amino acids, and phenylpropanoids. In deciphering glutathione-S-transferase (GST), its preferential location in phloem, correlated with the upregulation of GST genes and a decrease of the glutathione level, offers further support to the putative role of glutathione during apoplexy expression.

The genus Lasiodiplodia has been reported from several grape growing regions and is considered as one of the fastest wood colonizers, causing Botryosphaeria dieback. The aim of this study was to (i) evaluate the efficacy of Esquive®, a biocontrol agent, on vineyard pruning wound protection, applied single or, in a combined protection strategy with a new site-targeted copper-based treatment (LC2017), and (ii) compare their efficacy with chemical protection provided by the commercially available product, Tessior®. For two seasons, protectants were applied onto pruning wounds, while LC2017 was applied throughout the season according to the manufacturer’s instructions. Pruning wounds of two different cultivars were inoculated with three isolates of Lasiodiplodia spp. Efficacy of the wound protectants, varied between both years of the assay and according to the cultivar studied but were able to control the pathogen to some extent. The application of LC2017 did not show clear evidence of improving the control obtained by the sole application of the other products tested. Nevertheless, LC2017 showed a fungistatic effect against Lasiodiplodia spp., in vitro, and has previously shown an elicitor effect against grapevine trunk diseases. Therefore, this combination of two protection strategies may constitute a promising long-term approach to mitigate the impact of Botryosphaeria dieback.

Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions (Alsace, Champagne, Languedoc) were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was Fomitiporia mediterranea (63–94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (−90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.

The economic impact of grapevine trunk diseases (GTDs) is increasing worldwide, due to the lack of efficient and simple control protocols for these disease complexes. Possible and efficient GTD management strategies must consider the complexity of host physiological alterations affecting metabolism and defense responses determined by GTD pathogens, and linked to disease expression. In this complexity, the use of biocontrol agents could give advantages in GTD control methods. The effect of the potential biocontrol agent (BCA) Fusarium proliferatum was evaluated using in vitro tests and in dual inoculation with the Botryosphaeria dieback agent Neofusicoc-cum parvum in planta. Artificial inoculations were performed in greenhouse and vineyard experiments at three key vine growth stages, the onset of G (separated clusters), I (flowering) or M (veraison) stages. The biocontrol potential was assessed using pathogenicity tests and transcriptomic analyses. Results showed that the F. proliferatum has potential for phytoprotection, with disease control efficiency related to host plant growth stage. Flowering was confirmed as the growth stage when disease control was least, and efficiency of activated defense responses against pathogen infection was minimum.

A survey was carried out in 22 European and some non-European Mediterranean countries in 2015 and 2016, to gain insights into the main fungal grapevine trunk diseases (GTDs) in Europe. Information was obtained from 105 viticulture regions, representing approx. four million ha of vineyards. Vintage and table wines are the main industry products, followed by nursery plants and table grapes. Diverse scion cultivars are grown, with 13 cultivars providing 50% of survey respondent production, and Riesling Italico, Chardonnay, Cabernet Sauvignon and Merlot predominating. The five most used rootstocks are SO4, 110R, K5BB, 41B and 1103P, together representing about 50% of survey respondent production. Despite the diversity of grapevine training, Guyot and cordon remain the most widespread methods, and grapevines are mainly hand-pruned. The use of pneumatic shears and mechanical pruning is becoming more common in some countries. The survey questionnaire proposed a simplified set of symptoms or diseases occurring in mature vineyards [apoplexy, esca complex leaf symptoms (including Grapevine Leaf Stripe Disease, GLSD), dead cordon, Eutypa related dieback, Botryosphaeria or unidentified agent dieback, Phomopsis cane and leaf spot]. Apoplexy and esca/GLSD, defining the esca complex, were the most frequent and increasing syndromes in almost all countries, except Israel and the United Kingdom, where these diseases are not present or not recorded in the survey. Dead cordon and Phomopsis cane and leaf spot were mentioned as occurring in a large number of regions but not frequently. Within individual countries, the profile of GTDs varied according to the region, and to pedo-climatic and production conditions. GTDs on young vines were uncommon, but remain a subject of concern in some of European countries with large nursery production. This overview on spread and relevance of GTDs in Europe and related aspects will be a useful starting point for policy makers and for collaborative research on factors contributing to the increasing disease incidence of GTDs.

Plant pathogens have evolved various strategies to enter hosts and cause diseases. Particularly Neofusicoccum parvum, a member of Botryosphaeria dieback consortium, can secrete the phytotoxins (-)-terremutin and (R)-mellein during grapevine colonization. The contribution of phytotoxins to Botryosphaeria dieback symptoms still remains unknown. Moreover, there are currently no efficient control strategies of this disease, and agro-environmental concerns have raised increasing interest in biocontrol strategies to limit disease spread in vineyards, especially by using some promising beneficial bacteria. Here, we first examined in planta the biocontrol capacity of Bacillus subtilis PTA-271 against N. parvum Np-Bt67 strain producing both (-)-terremutin and (R)-mellein. We then focused on the direct effects of PTA-271 on pathogen growth and the fate of pure phytotoxins, and explored the capacity of PTA-271 to induce or prime grapevine immunity upon pathogen infection or phytotoxin exposure. Results provided evidence that PTA-271 significantly protects grapevine cuttings against N. parvum and significantly primes the expression of PR2 (encoding a β-1,3-glucanase) and NCED2 (9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis) genes upon pathogen challenge. Using in vitro plantlets, we also showed that PTA-271 triggers the expression of salicylic acid- and jasmonic acid-responsive genes, including GST1 (encoding a glutathione-S-transferase) involved in detoxification process. However, in PTA-271-pretreated plantlets, exogenous (-)-terremutin strongly lowered the expression of most of upregulated genes, except GST1. Data also indicated that PTA-271 can detoxify both (-)-terremutin and (R)-mellein and antagonize N. parvum under in vitro conditions. Our findings highlight (-)-terremutin and (R)-mellein as key aggressive molecules produced by N. parvum that may weaken grapevine immunity to promote Botryosphaeria dieback symptoms. However, PTA-271 can efficiently attenuate Botryosphaeria dieback by enhancing some host immune responses and detoxifying both phytotoxins produced by N. parvum.

Grapevine trutk diseases, especially Esca, are of major concern since they gradually alter vineyards worldwide and cause heavy economic losses. The expression of Esca disease symptoms depends on several factors, including the grapevine cultivar. In this context, a possible clone-dependent expression of the Esca disease was studied. Two clones of ‘Chardonnay’ grown in the same plot were compared according to their developmental and physiological traits, metabolome, and foliar symptom expression. Analysis of their leaf metabolome highlighted differences related to symptom expression. Interestingly, the content of a few specific metabolites exhibited opposite variations in leaves of symptomatic shoots of clones 76 and 95. Altogether this study showed a clone-dependent expression of Esca disease in ‘Chardonnay’ and the relevance of GC-MS and 3D fluorescence methods to analyze the impact of the disease on the leaf metabolome.

Grapevine trunk diseases (GTDs) are one of the major concern amongst grapevine diseases, responsible for the decline of vineyards and for several economical losses. Since grapevine is naturally colonized by resident microorganisms such as Aureobasidium pullulans, the present challenge is to understand their biocontrol potential and how such microorganisms can be successfully integrated in the control of GTDs. In this context, the first priority consists to exploit the plant-beneficial-phytopathogen interactions in plant model systems, to identify the most prevalent equilibrium limiting expression of GTDs. In the current study, we deep characterized the interaction of a resident and abundant microorganism from grapevine – Aureobasidium pullulans strain Fito_F278 – against D. seriata F98.1, a Botryosphaeria dieback agent, and with plant (cv Chardonnay). Results revealed that A. pullulans strain Fito_F278 was able to reduce significantly the mycelium growth of D. seriata F98.1 at 33.41 ± 0.55%, under in vitro conditions, though this reduction is possibly dependent on a direct interaction between strain Fito_F278 and pathogen. Furthermore, strain Fito_F278 was able to promote an induction of some plant defense responses in cutting plants, 1 week after the D. seriata F98.1 infection. Results evidenced that strain Fito_F278 colonized efficiently grapevine at both epiphyte and endophyte level, could persist on plant roots for long-periods (up to 2 months after its inoculation) and grow at different pH and high salinity conditions. Moreover, a significant decrease of the microbial load from soil and rhizosphere was observed in plants treated with the strain Fito_F278, suggesting its competitivity potential in a microbial ecosystem. Altogether, the present study gives the first insights about the interaction of A. pullulans strain Fito_F278, a resident microorganism, with grapevine, its potential role against a Botryosphaeria dieback agent, and highlights its importance to toward more resilient grapevine.

Since the early 1990s, grapevine trunk diseases (GTDs) have posed threats for viticulture. Esca complex, Eutypa- and Botryosphaeria- diebacks, mostly detected in adult vineyards, are currently responsible for considerable economic losses in the main vine-growing areas of the world. Other GTDs, such as Petri- (Esca complex) and Black-foot diseases, are emerging problems in grapevine nurseries (resulting in grafting failures and/or loss of saleable plants) and in young vineyards. The impacts of GTDs in modern viticulture depend on several factors, some related to their complexity, and others linked to host plant characteristics, changes in vineyard management and to the scarcity of simple tools for their control. For these reasons control of GTDs remains difficult, also depending on knowledge transfer from research to field and vice versa. This paper outlines the main preventive and curative techniques currently applied, scientifically tested or not that have resulted from the outcomes of “Winetwork”, a European Union funded project with special emphasis on the promising and innovative approaches.

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.

Aim: The present study was conducted in grapevines to evaluate the influence of source/sink disturbance on reserve restoration in the entire plant (trunk, roots) and the consequences on reproduction, yield, sugar yield, potential alcohol content and total acidity over four successive years of treatment. Methods and results: At véraison, either total defoliation or total fruit removal was performed on Chardonnay plants grown in vineyards. The impact was measured each year on reproductive parameters and after four years on both plant weight and carbohydrate content. Defoliation induced an alteration in carbohydrate distribution in the entire plant, as revealed by decrease of starch content and parallel increase of soluble sugar content. These modifications affected the number of inflorescences/clusters per plant the year after. By contrast, fruit removal resulted in a significant increase of carbohydrate reserves in the whole plant, although the efficiency of sexual reproduction was not improved the year after. Conclusion: Results confirm the close correlation between carbohydrate reserves and the efficiency of sexual reproduction. Moreover, grapevine is able to adapt the number of both inflorescences and flowers according to the amount of available carbohydrate reserves in perennial organs. Significance and impact of the study: The quality of the crop is affected by defoliation, as shown by the decrease in yield and sugar yield. However, the impact on potential alcohol content and total acidity was less pronounced.

Low temperatures damage many temperate crops, including grapevine, which, when exposed to chilling, can be affected by symptoms ranging from reduced yield up to complete infertility. We have previously demonstrated that Burkholderia phytofirmans PsJN, a plant growth-promoting rhizobacteria (PGPR) that colonizes grapevine, is able to reduce chilling-induced damage. We hypothesized that the induced tolerance may be explained at least partly by the impact of bacteria on grapevine photosynthesis or carbohydrate metabolism during cold acclimation. To investigate this hypothesis, we monitored herein the fluctuations of photosynthesis parameters (net photosynthesis [Pn], intercellular CO2 concentration, stomatal conductances, ΦPSII, and total chlorophyll concentration), starch, soluble sugars (glucose, fructose, saccharose, mannose, raffinose, and maltose), and their precursors during 5 days of chilling exposure (4°C) on grapevine plantlets. Bacterization affects photosynthesis in a non–stomatal dependent pattern and reduced long-term impact of chilling on Pn. Furthermore, all studied carbohydrates known to be involved in cold stress tolerance accumulate in non-chilled bacterized plantlets, although some of them remained more concentrated in the latter after chilling exposure. Overall, our results suggest that modification of carbohydrate metabolism in bacterized grapevine plantlets may be one of the major effects by which this PGPR reduces chilling-induced damage.

Among fungal grapevine trunk diseases, esca proper poses a significant threat for viticulture. Apoplexy, mainly occurring on grapevines affected by esca proper, is also a threat. To verify if different responses are activated in the woody tissues of apoplectic (A) and esca proper-affected (E) vines, two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine changes in the trunk wood of E and A field-grown plants. Asymptomatic and black streaked trunk (BST) wood from A and E plants were compared to asymptomatic and BST wood of control plants. Twenty-seven differentially expressed protein spots were identified. For eleven targeted proteins, expression of the relative transcripts was also monitored by qRT-PCR. Hierarchical tree clustering revealed differences in the distribution of spots containing carbohydrate metabolism proteins and heat shock proteins between asymptomatic- and BST-wood of grapevine, irrespective of the type of plant examined (control or diseased grapevines). Asymptomatic wood was mainly characterized by down-expression of proteins involved in cell growth and defense responses. The proteome of BST wood, characterized by extensive presence of grapevine trunk disease agents, revealed over-expression of proteins involved in defense. There was no evidence of strong different response in the trunk wood of apoplectic and esca proper affected plants. This could mean that, despite the different feature of these external crown symptoms, grapevine responses at trunk wood level is very similar in both cases. This plant response might therefore either simply be due to the fact that plants can react in the same way to different stresses, or that apoplexy represents a different effect provoked by the same cause or causal agent(s).

The National Grapevine Trunk Disease Survey was conducted in France from 2003 to 2008 to monitor grapevine trunk diseases (GTDs), eutypa dieback and esca/black dead arm (BDA). Data collected from seven regions, 329 vineyards and 12 cultivars were analysed. There were great variations amongst regions in the incidence of GTDs. For esca/BDA, two groups were distinguished: vineyards in Jura and Charentes had greater incidence (93–95%) than those of Bordeaux, Alsace and Bourgogne (54–82%). Incidence increased in Charentes over the 6-year survey, with the highest values being recorded during the last 2 years. For eutypa dieback, all vineyards of Charentes were affected, with 17 to 25% of vines expressing symptoms; for the other regions, 52 to 80% of vineyards were affected, with incidences below 3%. Cultivars Savagnin and Trousseau in Jura were especially affected by esca/BDA. Instead, Ugni Blanc in Charentes was most affected by eutypa dieback. One cultivar could be significantly more affected in one region than in another. The global health status of the vineyards was also investigated. (i) For four regions, 82% (Jura) to 87% (Alsace) of the grapevines were healthy, but this percentage decreased steadily (67%) in Charentes. (ii) Plants infected by GTDs were 32 and 18% in Jura and Charentes respectively, and only 2.9% in the Bourgogne region. (iii) The unproductive plants, i.e. dead, missing, replanted or restored, represented a significant part of the losses (6.6% in Charentes to 9.9% in Jura). The extension of GTDs is discussed with regard to the abiotic and biotic factors that may favour the diseases.

Botrytis bunch rot of grapes is mainly controlled by applying fungicides at three crop stages: the end of flowering (BBCH 68), bunch closure (BBCH 77) and the beginning of veraison (BBCH 81). The phenylpyrroles derivative fudioxonil is among the most effective fungicides registered to control Botrytis cinerea. Its effectiveness was investigated in relation to spray timing, fungicide resistance and defence responses of grapevine. Frequencies of B. cinerea strains which were resistant to fungicides were evaluated at harvest. The frequencies of resistant phenotypes were similar in all treatments except for a class of multidrug resistant strains (MDR 1) whose frequency increased after fudioxonil applications. None of the treatments tested induced defence responses in flowers/berries after fungicide application, suggesting that fudioxonil effectiveness was not related to a stimulation of plant defence processes. The standard program of three fungicide applications provided the best control of B. cinerea in the Champagne region in comparison with a single treatment of fudioxonil at any of the crop stages tested.