Fluctuating extreme weather events, coupled with rising average temperatures, can severely impact grapevine physiology and yield. While biostimulants have been gaining acceptance as a short-terms tools to enhance grapevine resilience, their adoption is hindered by inconsistent efficacy, partly driven by unpredictable plant stress levels. Over two contrasting seasons, we integrated physiological, transcriptomic, and metabolomic analyses to investigate how a plant-based biostimulant modulates the sensibility of Vitis vinifera under varying intensities of heat, drought, and their combination. This panel of water status, ranging from -0.02 to -1.6 MPa, revealed that the physiological response induced by the biostimulant treatment alleviates water stress within a field-relevant hydraulic window located between -0.4 and -1.2 MPa. Moreover, moderate but constitutive reduction of growth parameters in biostimulant plants, suggests a trade-off between vegetative development and abiotic stress responses. Accordingly, gene expression analysis revealed an interaction between water availability and the plant response to the biostimulant, which suggest an activation of priming mechanisms. Metabolic profiling supported these findings, highlighting the central role of phenylpropanoid pathway modulation, together with adjustments in ROS dynamics and stress-related hormone responses, particularly abscisic acid. Overall, this work emphasizes the need for integrating detailed plant water status and leaf gas exchange to accurately evaluate biostimulant performances under abiotic stress.

Grapevine Trunk Diseases (GTDs) have become a major challenge for viticulture. Since the ban of sodium arsenate (the only approved pesticide) in 2001, the need for alternative methods, such as biocontrol, has become increasingly urgent. One promising microorganism is the oomycete Pythium oligandrum, which is known to improve plant health by increasing natural defences and reducing disease incidence by up to 40 %. In order to use this microorganism in vineyards, it need first to be formulated and tested to ensure its safety. An experiment was therefore carried out in a greenhouse under semi-controlled conditions to assess the efficacy of the P. oligandrum biopesticide produced and its impact on microbial communities using a high-throughput sequencing approach. Vines were treated with the biopesticide and were inoculated with two fungi involved in wood diseases: Neofusicoccum parvum (involved in Botryosphaeria dieback) and Phaeomoniella chlamydospora (involved in Esca). During a three-month experiment, samples of the rhizosphere environment were collected to assess possible changes in microbial communities, either as part of GTDs or as a result of the action of P. oligandrum. The results indicated a minimal effect of the P. oligandrum biopesticide on the total microbial community of the vine rhizosphere. However, the treatment appeared to enhance several beneficial taxa that play a role as plant growth promoting rhizobacteria (PGPR) or biocontrol agents (BCA). This improvement, together with the direct effect of P. oligandrum, may explain the reduction in necroses caused by N. parvum and P. chlamydospora following the biopesticide application.

European grapevine is a complex holobiont composed of two plant genomes, that of the scion (Vitis vinifera L.) and the rootstock (Vitis spp.), and a multitude of microbial genomes that collectively form the microbiome. The grapevine microbiome has been extensively described over the last decade, primarily using metabarcoding approaches. Unfortunately, metabarcoding data alone provide little information on microbial functions and outcomes of plant-microbe interactions. Here we review knowledge about the microorganisms that have a demonstrated influence, positive or negative, on the performance of the grapevine holobiont. Our review encompasses bacteria, filamentous fungi, yeasts, oomycetes and viruses. It covers aboveground and belowground micro-organisms, including arbuscular mycorrhizal and ectomycorrhizal fungi. We focus on taxa and functions that protect the plant against pathogens and pests, promote growth, increase tolerance to abiotic stresses and highlight those involved in disease and decline. As the outcomes of plant-microbe interactions are labile, we examine the dynamics and functions of grapevine-microbiome interactions over both the plant lifetime and the plant evolutionary history, beginning with plant domestication. Based on the knowledge and gaps we identify, we suggest field sampling designs, culture-based experiments, molecular tools and theoretical analysis methods, including shotgun metagenomics and network models, that could be used in future research to uncover and leverage the full functional potential of the grapevine microbiome.

The most sustainable management of resistant varieties involves understanding which defence mechanisms the plant uses according to its resistance genes and stage of development and how effective these mechanisms are. Three grapevine varieties were compared for their susceptibility versus resistance to powdery mildew: Vitis vinifera 'Cabernet Sauvignon' (CS) and two hybrids, 'Artaban' and 'Prior'. Four different leaf ages were collected from 2-year-old grafted grapevines planted in pot or in vineyard, and several variables were assessed for each leaf: (a) morphological and physiological indicators, (b) pathogenicity traits: spore germination, infection ef?ciency, mycelial growth and sporulation, and (c) gene expression using a high-throughput quantitative reverse transcription PCR method to inform about plant defence status and functioning of primary metabolism. A significant decrease in germination and sporulation with increasing leaf age were observed for the three varieties: Prior showed a susceptibility similar to that of CS, whereas Artaban was fully resistant with no sporulation. Genes highlighted as markers of the variety were involved in defence (VvPR3, VvPR4 overexpressed in Artaban, VvPR4bis, VvAlli2 in Prior) or in primary metabolism (repression of VvCitS in Artaban, VvCHI in Prior, overexpression of VvCAD2, VvGST3 in Prior). After inoculation, more defence genes, especially PR proteins, were overexpressed in the oldest leaves as potential components of ontogenic resistance. PR proteins accounted for 25% of total disease variation. Phenylpropanoid and ethylene pathways were leaf age-dependent. The statistical method used highlighted the contributions of the genotypic expression involved in genetic (cultivar) and ontogenic (leaf age) resistances and of the metabolic pathways to the disease.

Grapevine trunk diseases (GTDs) are increasing in vineyards in many grape production regions. Among the pathogens causing these diseases, Neofusicoccum parvum, is one of the most frequent and virulent. To control GTDs, biocontrol is being developed using plant beneficial microorganisms. Strains of the oomycete Pythium oligandrum have been shown to naturally colonize grapevine roots in vineyards in several countries in Europe. This study examined the ability of the root-coloniser P. oligandrum to induce grapevine resistance against N. parvum, by deciphering the gene expression changes in a set of 62 genes involved in different grapevine defence pathways. Two greenhouse assays showed that the wood necrosis of vine cuttings caused by N. parvum was reduced by 65% when P. oligandrum colonized root systems of the plants. The relative expression levels of selected genes in the host trunks were studied by real-time PCR. Plant responses were assessed after inoculation by P. oligandrum and/or N. parvum, at three different sampling time points (0, 14 and 150 d after N. parvum inoculation). Sampling time influenced gene expressions for the different inoculation treatments. At each sampling time, specific host responses to the different treatments were also detected, for controls, and for inoculations with P. oligandrum, N. parvum or P oligandrum + N. parvum. When P. oligandrum colonized grapevine root systems, inoculation with the pathogen was associated with increased up-regulation and over-expression of particular genes, including those regulating Pathogen-Related proteins, cell wall reinforcement proteins and hormone signalling pathways. A priming effect of the grapevine defence system was induced in roots colonized by P. oligandrum.

The increasing use of plant defense stimulators (PDS) and biostimulants (BS) to make agriculture more sustainable has led to questions about their action on plants. A new PhysBioGen approach is proposed with complementary tools: PHYSiological (root weight); BIOchemical and BIOlogical (secondary metabolite quantification and Plasmopara viticola development) and expressions of 161 GENes involved in metabolic plant functions. The proposed approach investigated the effects of three phytostimulants on Vitis vinifera: one PDS (ASM) and one BS chelated (CH) and another enriched with seaweed (SW). Distinct responses were obtained between the PDS and the two BS. In particular, we observed the persistence of anti-mildew efficacy over time, correlated with differentiated expressions of defense genes (VvROMT, VvSAMT, VvPR8). As expected, the two BS displayed more similarities to each other than to the PDS (flavonols, anthocyanins, free salicylic acid). However, the two BS revealed differences in the modulation of genes involved in defense and primary metabolism and some genes were identified as potential markers of their action (VvWRKY1, VvLOX9, VvPOD, VvPDV1, VvXIP1, VVDnaJ). Our results highlight the common and the specific effects of the two BS and the PDS. These new tools could help in understanding the mode of action of phytostimulants in order to achieve better quality and production yield and/or as a way to limit chemical inputs in the vineyard.

The NeoViGen96 chip, a pioneering tool based on high-throughput reverse-transcription quantitative PCR of 85 defense-related genes, has been developed to assess the defense status of grapevine genotypes and correlate this with protection level (Dufour et al., 2016). It is useful for setting up alternative or complementary pest management methods using plant defense stimulators, associated or not with other pest management methods such as biological control or plant breeding. It can be used to monitor expression levels of a set of selected defense genes widely covering the various defense pathways (salicylic acid, jasmonate/ethylene-dependent signal transduction, pathogenesis-related proteins, phytoalexin production and cell wall reinforcement). It has been used under controlled conditions to assess the defenses of susceptible cultivars and/or grapevine hybrids partially or totally resistant to downy and powdery mildew. It can be used to visualize defense mechanisms in leaves of resistant genotypes having different resistance quantitative trait loci, and/or cultivars and hybrids such as 'Solaris', 'Regent', Rv4 and Rv5. It can help in understanding which metabolic pathways are important for protection against powdery and downy mildews, or other diseases, and can be used to select resistant genotypes.

Grapevine is subject to diseases that affect yield and wine quality caused by various pathogens including Botrytis cinerea. To limit the use of fungicides, an alternative is to use plant elicitors such as benzothiadiazole (BTH). We investigated the effect of a fungicide (Pyrimethanil) and an elicitor (benzothiadiazole) on plant defenses. Applications for two consecutive years in the vineyard significantly reduced gray mold. Two and seven days after treatments, the expressions of 48 genes involved in defenses showed differential modulation (up- or down-regulation) depending on treatment. Some genes were identified as potential markers of protection and were linked to an increase in total polyphenols (TP) in leaves. Surprisingly, the fungicide also induced the expression of defense genes and increased the polyphenol content. This suggests that BTH acts as an efficient elicitor in the vineyard and that Pyrimethanil may act, in part, as a defense-inducing agent on the vine.

Pine knot extract from Pinus pinaster byproducts was characterized by UHPLC-DAD-MS and NMR. Fourteen polyphenols divided into four classes were identified as follows: lignans (nortrachelogenin, pinoresinol, matairesinol, isolariciresinol, secoisolariciresinol), flavonoids (pinocembrin, pinobanksin, dihydrokaempferol, taxifolin), stilbenes (pinosylvin, pinosylvin monomethyl ether, pterostilbene), and phenolic acids (caffeic acid, ferulic acid). The antifungal potential of pine knot extract, as well as the main compounds, was tested in vitro against Plasmopara viticola. The ethanolic extract showed a strong antimildew activity. In addition, pinosylvins and pinocembrin demonstrated significant inhibition of zoospore mobility and mildew development. These findings strongly suggest that pine knot is a potential biomass that could be used as a natural antifungal product.

Botryosphaeriaceae is a fungal family comprising many species involved in botryosphaeria dieback, a worldwide grapevine trunk disease. Currently, the interactions between Botryosphaeriaceae species and various grapevine cultivars are poorly understood and little data is available. This study investigated various life traits of five isolates belonging to four species of Botryosphaeriaceae found in French vineyards (Diplodia mutila, Diplodia seriata, Lasiodiplodia viticola and Neofusicoccum parvum). The two species N. parvum and L. viticola exhibited the highest optimal growth temperature and the best growth rates. They were also responsible for the most extensive necrosis and cankers in three Vitis vinifera cultivars (Cabernet Sauvignon, Merlot and Ugni‐Blanc) that differed in susceptibility to botryosphaeria dieback, and in two genotypes resistant to downy and powdery mildew (RV4 and RV5). Identification of the extracellular toxins produced by isolates in culture media showed that the N. parvum isolate had a different metabolite profile from the others, producing terremutin and salicylic acid derivatives, which are known to be compounds associated with virulence. In a second step, life traits were associated with nondestructive monitoring of gene expression involved in the defence mechanisms of five grapevine cultivars and genotypes after inoculation of wood cuttings with Botryosphaeriaceae. The transcript analyses were carried out at different times and were associated with principal component analysis (PCA). Each cultivar presented a specific transcript signature and several transcripts were correlated either with the size of necrosis/cankers or with symptom reduction, thus offering useful markers for breeding or estimating the defence status of plants.

The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment.

The antagonistic activity of 46 bacterial strains isolated from Bordeaux vineyards were evaluated against Phaeomoniella chlamydospora, a major grapevine pathogen involved in Esca. The reduction of the necrosis length of stem cuttings ranged between 31.4% and 38.7% for the 8 most efficient strains. Two in planta trials allowed the selection of the two best strains, Bacillus pumilus (S32) and Paenibacillus sp. (S19). Their efficacy was not dependent on application method; co-inoculation, prevention in the wood and soil inoculation were tested. The involvement of antibiosis by the secretion of diffusible and/or volatile compounds in the antagonistic capacity of these two strains was assessed in vitro. Volatile compounds secreted by B. pumilus (S32) and Paenibacillus sp. (S19) were identified by gas chromatography/mass spectroscopy (GC/MS). The volatile compounds 1-octen-3-ol and 2,5-dimethyl pyrazine were obtained commercially and tested, and they showed strong antifungal activity against P. chlamydospora, which suggested that these compounds may play an important role in the bacterial antagonistic activity in planta. Furthermore, the expression of 10 major grapevine defense genes was quantified by real-time polymerase chain reaction, which demonstrated that the two strains significantly affected the grapevine transcripts four days after their application on the plants. High expression levels of different genes associated with P. chlamydospora infection in B. pumilus pre-treated plants suggests that this strain induces systemic resistance in grapevine. For the first time, we demonstrated the ability of two bacterial strains, B. pumilus and Paenibacillus sp., isolated from grapevine wood, to control P. chlamydospora via direct and/or indirect mechanisms.

Biological control of Phaeomoniella chlamydospora, a pathogen involved in Esca, a grapevine trunk wood disease, was performed using the oomycete, Pythium oligandrum. Three 4-month greenhouse assays showed that necrosis of Vitis vinifera L. cv. Cabernet Sauvignon cuttings caused by P. chlamydospora was significantly reduced (40-50%) when P. oligandrum colonized the plant root systems. The expression of a set of 22 grapevine defense genes was then quantified by real-time polymerase chain reaction to determine plant responses in the interaction between P. oligandrum/V. vinifera L./P. chlamydospora. In the trunk, specific grapevine responses to the different treatments (control, P. oligandrum, P. chlamydospora and P. oligandrum + P. chlamydospora treatments) were significantly differentiated. Expression levels of 6 genes associated with P. chlamydospora infection showed higher induction than when plants were pre-treated with P. oligandrum. These genes are involved in various pathways (PR proteins, phenylpropanoid pathways, oxylipin and oxydo-reduction systems). (C) 2015 Elsevier Inc. All rights reserved.

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.