Determining the Timing of Spore Release by Botryosphaeriaceae Species in Oregon Vineyards.

Botryosphaeria dieback, caused by a group of pathogens in the Botryosphaeriaceae family, is one of the most common grapevine trunk disease complexes (GTDs) found in Oregon vineyards. To understand the period of spores released by Botryosphaeria spp. in Oregon vineyards, four Burkard 7-day recording volumetric spore traps were placed in vineyard blocks in northern and southern Oregon. Each trap was placed near a younger (<10 years) and older (>30 years) block in both regions. Spore traps were deployed at the beginning of December 2019 and continued until March 2021. Between these timeframes, 475 and 477 days of samples were collected from each spore trap in northern and southern Oregon, respectively. DNA extraction was performed from individual day samples and followed by qPCR analysis of Botryosphaeria spores trapped in each tape sample. Weather data such as temperature, precipitation, relative humidity (RH), and wind speed were collected from nearby weather stations. Association between these data and number of spores detected were analyzed using Pearson correlation analysis. In northern Oregon, the detection occurred between December and February, and the first spore detection occurred when cumulative growing degree day (GDD) totaled to 4,357 and 4,351 units (TBase = 0°C, biofix date = January 1) during the first and second seasons, respectively. Similarly, in southern Oregon, the detection occurred between November and January, and the first spore detection occurred when cumulative GDD was 4,405 units during the second season. Hours of continuous RH >86% was significantly associated with number of spores released (P = 0.026; r = 0.42). During the spore detected dates, the RH was >86% for at least 19 consecutive hours. These findings provide an important implication to manage Botryosphaeria dieback by protecting pruning wounds during the most-spore-released periods. Furthermore, the knowledge of weather variables and their possible association with spore detection provides important information towards developing predictive models in future studies.

Identification and Pathogenicity of Fusarium Species Associated with Young Vine Decline in California.

Grapevine trunk diseases are caused by a broad diversity of fungal taxa that have serious impacts on the worldwide viticulture industry due to significant reductions in vineyards yield and lifespan. Field surveys carried out from 2018 to 2022 in California nurseries and young vineyards revealed a high incidence of Fusarium. Since Fusarium species are important pathogens of other perennial crops, the present study aimed to identify and determine the pathogenicity of the Fusarium species on grapevines. Morphology of the fungal colonies coupled with multilocus phylogenetic analyses using nucleotide sequences of the translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2) genes revealed the occurrence of 10 species clustering in six species complexes, namely F. fujikuroi (FFSC), F. oxysporum (FOSC), F. solani (FSSC), F. sambucinum (FSAMSC), F. incarnatum-equiseti (FIESC), and F. tricinctum (FTSC) species complexes. The species F. annulatum (FFSC) was the most prevalent in samples from both symptomatic young vineyards (73.5% incidence) and nursery propagation material (62.5% incidence). Pathogenicity of the 10 most frequent species was confirmed by fulfilling Koch's postulates on living woody tissue of 1103 Paulsen rootstocks. Our results suggest that Fusarium spp. are involved in the development of young vine decline, probably as opportunistic pathogens when grapevines are under stress conditions.

Evaluating treatments for the protection of grapevine pruning wounds from natural infection by trunk disease fungi.

Infection of grapevines by fungal pathogens causing grapevine trunk diseases (GTDs) primarily arises from annual pruning wounds made during the dormant season. While various studies have showcased the efficacy of products in shielding pruning wounds against GTDs infections, most of these investigations hinge on artificial pathogen inoculations, which may not faithfully mirror real field conditions. This study aimed to evaluate and compare the efficacy of various liquid formulation fungicides (pyraclostrobin + boscalid) and paste treatments, as well as biological control agents (BCA: Trichoderma atroviride SC1, T. atroviride I-1237, and T. asperellum ICC012 + T. gamsii ICC080), for their potential to prevent natural infection of grapevine pruning wounds by trunk disease fungi in two field trials located in Samaniego (Northern Spain) and Madiran (Southern France) over three growing seasons. Wound treatments were applied immediately after pruning in February. One year after pruning, canes were harvested from vines and brought to the laboratory for assessment of Trichoderma spp. and fungal trunk pathogens. More than 1,200 fungal isolates associated with five GTDs (esca, Botryophaeria, Diaporthe and Eutypa diebacks, and Cytospora canker) were collected from the two vineyards each growing season. Our findings reveal that none of the products under investigation exhibited complete effectiveness against all the GTDs. The efficacy of these products was particularly influenced by the specific year of study. A notable exception was observed with the biocontrol agent T. atroviride I-1237, which consistently demonstrated effectiveness against Botryosphaeria dieback infections throughout each year of the study, irrespective of the location. The remaining products exhibited efficacy in specific years or locations against particular diseases, with the physical barrier (paste) showing the least overall effectiveness. The recovery rates of Trichoderma spp. in treated plants were highly variable, ranging from 17% to 100%, with both strains of T. atroviride yielding the highest isolation rates. This study underscores the importance of customizing treatments for specific diseases, taking into account the influence of environmental factors for BCA applications.

In vitro evaluation of the efficacy of 8-hydroxyquinoline derivatives for the control of Phaeomoniella chlamydospora, the causative agent of Petri disease in grapevines.

AIM: This study evaluates the in vitro efficacy of 8-hydroxyquinoline (8HQ) derivatives in controlling the phytopathogenic fungus Phaeomoniella chlamydospora. METHODS AND RESULTS: The in vitro tests assessed the susceptibility to the minimum inhibitory concentration (MIC), checkerboard assay, mycelial growth (MG) inhibition, and EC50 determination. Among the seven agricultural fungicides tested, tebuconazole (TEB) displayed the lowest MIC, 1.01 µg mL-1, followed by captan (CAP), thiophanate methyl (TM), and mancozeb with MICs of 4.06, 5.46, and 10.62 µg mL-1, respectively. The 8HQ derivatives used in this study were clioquinol and PH 151 (PH) with MICs of 1.09 and 2.02 µg mL-1, respectively. PH associated with TEB and CAP showed synergism and inhibited 95.8% of MG at the highest dose. TEB inhibited 100% of MG at the three highest doses, while associated with PH exhibited the lowest EC50 (0.863 + 0.0381 µg mL-1). CONCLUSIONS: We concluded that the 8HQ derivatives tested controlled effectively the P. chlamydospora in vitro. PH associated with CAP and TEB exhibited a synergistic effect. The association between PH and TM was considered indifferent. IMPACT STATEMENT: This study expands the list of active ingredients tested against P. chlamydospora, with the PH 151 and clioquinol derivatives being tested for the first time. The in vitro efficacy and synergistic action with other fungicides suggest a potential use as a grapevine wound protectant. This association makes it possible to reduce doses and increase the potency of both drugs, reducing the risk of resistance development and harm to humans and the environment.

Effects of Temperature and Moisture Duration on Spore Germination of Four Fungi that Cause Grapevine Trunk Diseases.

Grapevine trunk diseases (GTDs) are serious threats worldwide and are difficult to control, in part because the environmental requirements for epidemiological processes of the causal fungi are poorly understood. Therefore, we investigated the effects of temperature and moisture duration on spore germination of four fungi associated with two GTDs (esca complex and Eutypa dieback): Phaeomoniella chlamydospora, Phaeoacremonium minimum, Cadophora luteo-olivacea, and Eutypa lata. Conidia of Phaeomoniella chlamydospora, Phaeoacremonium minimum, and C. luteo-olivacea were similar: conidia of these fungi germinated profusely (>90%) between 20 and 30°C; Phaeomoniella chlamydospora and Phaeoacremonium minimum tended to germinate at higher temperatures (up to 40°C for P. minimum), and C. luteo-olivacea at lower temperatures (as low as 5°C). E. lata ascospores germinated between 10 and 30°C. The required duration of moist periods for germination was shortest for C. luteo-olivacea (about 6 h), followed by P. minimum and E. lata (about 12 h) and Phaeomoniella chlamydospora (about 24 h). Further research on the environmental requirements of GTD fungi may increase our ability to predict infection periods and, thereby, improve disease control.

Temperature-Dependent Growth and Spore Germination of Fungi Causing Grapevine Trunk Diseases: Quantitative Analysis of Literature Data.

Grapevine trunk diseases (GTDs) are serious threats in all viticultural areas of the world, and their management is always complex and usually inadequate. Fragmented and inconsistent information on the epidemiology and environmental requirements of the causal fungi is among the reasons for poor disease control. Therefore, we conducted a quantitative analysis of literature data to determine the effects of temperature on mycelial growth and the effects of temperature and moisture duration on spore germination. Using the collected information, we then developed mathematical equations describing the response of mycelial growth to temperature, and the response of spore germination to temperature and moisture for the different species and disease syndromes. We considered 27 articles (selected from a total of 207 articles found through a systematic literature search) and 116 cases; these involved 43 fungal species belonging to three disease syndromes. The mycelial growth of the fungi causing Botryosphaeria dieback (BD) and the esca complex (EC) responded similarly to temperature, and preferred higher temperatures than those causing Eutypa dieback (ED) (with optimal temperature of 25.3, 26.5, and 23.3°C, respectively). At any temperature, the minimal duration of the moist period required for 50% spore germination was shorter for BD (3.0 h) than for EC (17.2 h) or ED (15.5 h). Mathematical equations were developed accounting for temperature-moisture relationships of GTD fungi, which showed concordance correlation coefficients ≥0.888; such equations should be useful for reducing the risk of infection.

Seasonal periodicity of the airborne spores of fungi causing grapevine trunk diseases: an analysis of 247 studies published worldwide.

Grapevine trunk diseases (GTDs) are among the most devastating grapevine diseases globally. GTDs are caused by multiple fungi from various taxa, which release spores into the vineyard and infect wood tissue, mainly through wounds caused by viticultural operations. The timing of operations to avoid infection is critical concerning the periodicity of GTD spores in vineyards, and many studies have been conducted in different grape-growing areas worldwide. However, these studies provide conflicting and fragmented information. To synthesize current knowledge, we performed a systematic literature review, extracted quantitative data from published papers, and used these data to identify trends and knowledge gaps to be addressed in future studies. Our database included 26 papers covering 247 studies and 3,529 spore sampling records concerning a total of 29 fungal taxa responsible for Botryosphaeria dieback (BD), Esca complex (EC), and Eutypa dieback (ED). We found a clear seasonality in the presence and abundance of BD spores, with a peak from fall to spring, more in the northern than in the southern hemisphere, but not for EC and ED. Spores of these fungi were present throughout the growing season in both hemispheres, possibly due to higher variability in spore types, sporulation conditions, and spore release mechanisms in EC and ED fungi compared to BD. Our analysis has limitations due to knowledge gaps and data availability for some fungi (e.g., Basidiomycetes, causing EC). These limitations are discussed to facilitate further research.

Role of rain in the spore dispersal of fungal pathogens associated with grapevine trunk diseases.

Grapevine trunk diseases are caused by a complex of fungi that belong to different taxa, which produce different spore types and have different spore dispersal mechanisms. It is commonly accepted that rainfall plays a key role in spore dispersal, but there is conflicting information in the literature on the relationship between rain and spore trapping in aerobiology studies. We conducted a systematic literature review, extracted quantitative data from published papers, and used the pooled data for Bayesian analysis of the effect of rain on spore trapping. We selected 17 papers covering 95 studies and 8,778 trapping periods, concerning a total of 26 fungal taxa causing Botryosphaeria dieback (BD), Esca complex (EC), and Eutypa dieback (ED). Results confirmed the role of rain in the spore dispersal of these fungi, but revealed differences among the different fungi. Rain was a good predictor of spore trapping for ED (AUROC = 0.820) and BD (0.766) but not for the Ascomycetes involved in EC (0.569) and not for the only Basidiomycetes, Fomitiporella viticola, studied as for spore discharge (AUROC not significant). Prediction of spore trapping was more accurate for negative than for positive prognosis; a rain cutoff of ≥0.2 mm provided an overall accuracy ≥0.61 for correct prognoses. Spores trapped in rainless periods accounted for only <10% of the total spores. Our analysis had some drawbacks, which were mainly caused by knowledge gaps and limited data availability; these drawbacks are discussed to facilitate further research.

Characterization of Grapevine Wood Microbiome Through a Metatranscriptomic Approach.

Grapevine trunk diseases threaten wine and table grape production worldwide, primarily by reducing yields and, in its advanced stages, causing plant death. Among those diseases, the complex etiology disease known as hoja de malvón (HDM) significantly concerns Argentinian and Uruguayan viticulture. At least four different fungi are associated with this disease, but their role and interactions with other wood microorganisms are understudied. In this sense, analyzing grapevine wood microbiome composition could help understand microbial interactions occurring in HDM onset. Hence, a metatranscriptomic study was performed for the microbiome characterization of mature field-grown Vitis vinifera cv. Malbec, leaf-symptomatic or leaf-asymptomatic. The microbiome was mainly represented by Dothideomycetes and Actinobacteria. In the plant with more marked symptoms, higher levels of the Basidiomycota Arambarria destruens and Phellinus laevigatus were detected. Despite this particular difference, discriminating symptomatic from asymptomatic plants based on the presence or abundance of HDM pathogens was not possible. Alpha diversity and rank-abundance curve analyses indicated that plants with foliar symptoms have lower microbial evenness than asymptomatic plants. The co-occurrence network modeled microbial interkingdom interactions. Molecular data generated in this study will help develop future targeted molecular quantification for specific taxa.

First report of Seimatosporium vitifusiforme causing trunk disease in Chilean grapevines (Vitis vinifera).

Grapevine is one of the most important fruit crops in Chile and trunk diseases reduce the productivity, quality, and longevity of the vineyards. A survey was conducted in ancient (> 50 years) vineyards of Cauquenes (35°57´14´´S 72°17´07´´W) and Itata valleys (36°38´13´´S 72°30´57´´W), located in the central area of Chile, during 2019. Trunks and cordons showing dieback and dark brown to black wood discoloration were collected from 50 to 200-year-old plants of six cultivars: País, Moscatel, Torontel Amarilla, Carignan, Aliatica and Aligote. The bark was removed and 0.5-cm sections were cut from the edges of necrotic wood lesions. Subsequently, pieces were surface disinfected using 10% v/v sodium hypochlorite bleach (4.9% chlorine), plated on acidified quarter-strength potato dextrose agar (APDA) (25% PDA, acidified with 0.1% v/v 85% lactic acid) and incubated at 25°C, for 14 to 28 days. Hyphal tips were excised and transferred to PDA to obtain pure cultures. Along with the conidiomata and conidia produced, growth rate, color and shape of the colonies on PDA, after 7 and 14 days of incubation at 25°C (n=17), were recorded. DNA was extracted from pure cultures of three isolates on PDA: HMV3, HMV64 and HMV81. The internal transcribed spacer region and partial ß-tubulin genes were amplified, using ITS1/ITS4 (White et al. 1990) and bt2A/bt2B (Glass & Donaldson 1995) primers, respectively. Sequences were subjected to NCBI BLAST search and compared to the published sequences. Isolated colonies were whitish to light-brown, cottony with a smooth margin (n=37). Their mycelium grew 1.9 cm after 7-days and 3.2 cm after 14-days of incubation on PDA, at 25°C. Colonies produced black globose pycnidia and curved, slightly-pigmentated, three-septated conidia 22.3-(29.8)-32.2 x 3.9-(4.8)-5.3 µm (n=30), with apical and basal flexuous appendages 4.3-(12.7)-21.5 µm (n=20). When compared to type sequences of Seimatosporium vitifusiforme (Lawrence et al. 2018), ITS and ßtub sequences identity of these isolates were 99 to 100% identical. To produce uniform healthy plants for pathogenicity tests, Petit Syrah canes (1-year old) were rooted in tap water amended with 500 ppm of indole-butyric acid, for 30 days. Plants were inoculated with 0.5-cm diameter mycelial plugs of actively growing colonies of the isolates HMV3, HMV64 and HMV81 (GenBank accessions no. MW026664, MW048518; MW026665, MW048519, and MW026666, MW048520, respectively). Sterile agar plugs were used for controls. Five plants per pathogen isolate were incubated at 25°C, in a humid chamber, for 25 days, and seven additional plants per isolate were incubated in aerated tap water, for 55 days. After the incubation period, the bark was removed and the lesions were measured. Dark necrotic lesions identical to the original observations were reproduced, both in the high humidity chamber (6% length) and water (10% length). There were no differences in lesion length among the isolates (P < 0.05). Control vines remained asymptomatic. To fulfill Koch´s postulates, isolations were made from symptomatic vines and compared to the ones used for inoculation, and found to be identical. Seimatosporium vitifusiforme was previously reported as a pathogen of Vitis vinifera in California, USA (Lawrence et al. 2018). Consequently, this is the second report of this fungus as a grapevine pathogen and the first one affecting Latin-American grapevines.

Hunting modulators of plant defence: the grapevine trunk disease fungus Eutypa lata secretes an amplifier for plant basal immunity.

Grapevine trunk diseases (GTDs) are progressively affecting vineyard longevity and productivity worldwide. To be able to understand and combat these diseases, we need a different concept of the signals exchanged between the grapevine and fungi than the well-studied pathogen-associated molecular pattern and effector concepts. We screened extracts from fungi associated with GTDs for their association with basal defence responses in suspension cells of grapevine. By activity-guided fractionation of the two selected extracts, O-methylmellein was identified as a candidate modulator of grapevine immunity. O-Methylmellein could not induce immune responses by itself (i.e. does not act as an elicitor), but could amplify some of the defence responses triggered by the bacterial elicitor flg22, such as the induction level of defence genes and actin remodelling. These findings show that Eutypa lata, exemplarily selected as an endophytic fungus linked with GTDs, can secrete compounds that act as amplifiers of basal immunity. Thus, in addition to elicitors that can trigger basal immunity, and effectors that down-modulate antibacterial basal immunity, once it had been activated, E. lata seems to secrete a third type of chemical signal that amplifies basal immunity and may play a role in the context of consortia of mutually competing microorganisms.

Cultivar- and Wood Area-Dependent Metabolomic Fingerprints of Grapevine Infected by Botryosphaeria Dieback.

Botryosphaeria dieback is one of the most significant grapevine trunk diseases that affects the sustainability of the vineyards and provokes economic losses. The causal agents, Botryosphaeriaceae species, live in and colonize the wood of the perennial organs causing wood necrosis. Diseased vines show foliar symptoms, chlorosis, or apoplexy, associated to a characteristic brown stripe under the bark. According to the susceptibility of the cultivars, specific proteins such as PR-proteins and other defense-related proteins are accumulated in the brown stripe compared with the healthy woody tissues. In this study, we enhanced the characterization of the brown stripe and the healthy wood by obtaining a metabolite profiling for the three cultivars Chardonnay, Gewurztraminer, and Mourvèdre to deeper understand the interaction between the Botryosphaeria dieback pathogens and grapevine. The study confirmed a specific pattern according to the cultivar and revealed significant differences between the brown stripe and the healthy wood, especially for phytochemical and lipid compounds. This is the first time that such chemical discrimination was made and that lipids were so remarkably highlighted in the interaction of Botryosphaeriaceae species and grapevine. Their role in the disease development is discussed.

Temporal Dispersal Patterns of Phaeomoniella chlamydospora, Causal Agent of Petri Disease and Esca, in Vineyards.

Although the fungus Phaeomoniella chlamydospora is the most commonly detected causal agent of Petri disease and esca, two important fungal grapevine trunk diseases, little is known about the dispersal patterns of P. chlamydospora inoculum. In this work, we studied the dispersal of P. chlamydospora airborne inoculum from 2016 to 2018 in two viticultural areas of eastern (Ontinyent) and northern (Logroño) Spain. The vineyards were monitored weekly from November to April using microscope slide traps, and P. chlamydospora was detected and quantified by a specific real-time quantitative (qPCR) method set up in this work. The method was found to be sensitive, and a good correlation was observed between numbers of P. chlamydospora conidia (counted by microscope) and DNA copy numbers (quantified by qPCR). We consistently detected DNA of P. chlamydospora at both locations and in all seasons but in different quantities. In most cases, DNA was first detected in the last half of November, and most of the DNA was detected from December to early April. When rain was used as a predictor of P. chlamydospora DNA detection in traps, false-negative detections were observed, but these involved only 4% of the total. The dispersal pattern of P. chlamydospora DNA over time was best described (R2 = 0.765 and concordance correlation coefficient = 0.870) by a Gompertz equation, with time expressed as hydrothermal time (a physiological time accounting for the effects of temperature and rain). This equation could be used to predict periods with a high risk of dispersal of P. chlamydospora.

Grapevine trunk diseases under thermal and water stresses.

MAIN CONCLUSION: Heat and water stresses, individually or combined, affect both the plant (development, physiology, and production) and the pathogens (growth, morphology, dissemination, distribution, and virulence). The grapevine response to combined abiotic and biotic stresses is complex and cannot be inferred from the response to each single stress. Several factors might impact the response and the recovery of the grapevine, such as the intensity, duration, and timing of the stresses. In the heat/water stress-GTDs-grapevine interaction, the nature of the pathogens, and the host, i.e., the nature of the rootstock, the cultivar and the clone, has a great importance. This review highlights the lack of studies investigating the response to combined stresses, in particular molecular studies, and the misreading of the relationship between rootstock and scion in the relationship GTDs/abiotic stresses. Grapevine trunk diseases (GTDs) are one of the biggest threats to vineyard sustainability in the next 30 years. Although many treatments and practices are used to manage GTDs, there has been an increase in the prevalence of these diseases due to several factors such as vineyard intensification, aging vineyards, or nursery practices. The ban of efficient treatments, i.e., sodium arsenite, carbendazim, and benomyl, in the early 2000s may be partly responsible for the fast spread of these diseases. However, GTD-associated fungi can act as endophytes for several years on, or inside the vine until the appearance of the first symptoms. This prompted several researchers to hypothesise that abiotic conditions, especially thermal and water stresses, were involved in the initiation of GTD symptoms. Unfortunately, the frequency of these abiotic conditions occurring is likely to increase according to the recent consensus scenario of climate change, especially in wine-growing areas. In this article, following a review on the impact of combined thermal and water stresses on grapevine physiology, we will examine (1) how this combination of stresses might influence the lifestyle of GTD pathogens, (2) learnings from grapevine field experiments and modelling aiming at studying biotic and abiotic stresses, and (3) what mechanistic concepts can be used to explain how these stresses might affect the grapevine plant status.

Profiling grapevine trunk pathogens in planta: a case for community-targeted DNA metabarcoding.

BACKGROUND: DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS: We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS: Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Physiological and developmental disturbances caused by Botryosphaeria dieback in the annual stems of grapevine.

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.

Trichoderma carraovejensis: a new species from vineyard ecosystem with biocontrol abilities against grapevine trunk disease pathogens and ecological adaptation.

Trichoderma strains used in vineyards for the control of grapevine trunk diseases (GTDs) present a promising alternative to chemical products. Therefore, the isolation and characterization of new indigenous Trichoderma strains for these purposes is a valuable strategy to favor the adaptation of these strains to the environment, thus improving their efficacy in the field. In this research, a new Trichoderma species, Trichoderma carraovejensis, isolated from vineyards in Ribera de Duero (Spain) area, has been identified and phylogenetically analyzed using 20 housekeeping genes isolated from the genome of 24 Trichoderma species. A morphological description and comparison of the new species has also been carried out. In order to corroborate the potential of T. carraovejensis as a biological control agent (BCA), confrontation tests against pathogenic fungi, causing various GTDs, have been performed in the laboratory. The compatibility of T. carraovejensis with different pesticides and biostimulants has also been assessed. This new Trichoderma species demonstrates the ability to control pathogens such as Diplodia seriata, as well as high compatibility with powdered sulfur-based pesticides. In conclusion, the autochthonous species T. carraovejensis can be an effective alternative to complement the currently used strategies for the control of wood diseases in its region of origin.

Arthropods as Vectors of Grapevine Trunk Disease Pathogens: Quantification of Phaeomoniella chlamydospora on Arthropods and Mycobiome Analysis of Earwig Exoskeletons.

Viticulture worldwide is challenged by grapevine trunk diseases (GTDs). Involvement of arthropods in the dissemination process of GTD pathogens, notably esca pathogens, is indicated after detection of associated pathogens on arthropod exoskeletons, and demonstration of transmission under artificial conditions. The present study is the first to quantify spore loads via qPCR of the esca-relevant pathogen Phaeomoniella chlamydospora on arthropods collected in German vineyards, i.e., European earwigs (Forficula auricularia), ants (Formicidae), and two species of jumping spiders (Marpissa muscosa and Synageles venator). Quantification of spore loads showed acquisition on exoskeletons, but most arthropods carried only low amounts. The mycobiome on earwig exoskeletons was described for the first time to reveal involvement of earwigs in the dispersal of GTDs in general. Metabarcoding data support the potential risk of earwigs as vectors for predominantly Pa. chlamydospora and possibly Eutypa lata (causative agent of Eutypa dieback), as respective operational taxonomical unit (OTU) assigned genera had relative abundances of 6.6% and 2.8% in total reads, even though with great variation between samples. Seven further GTD-related genera were present at a very low level. As various factors influence the successful transmission of GTD pathogens, we hypothesize that arthropods might irregularly act as direct vectors. Our results highlight the importance of minimizing and protecting pruning wounds in the field.

Effects of Growing Areas, Pruning Wound Protection Products, and Phenological Stage on the Stilbene Composition of Grapevine (Vitis vinifera L.) Canes.

Applying plant protection products (PPP) on grapevine pruning wounds is a viticultural practice used to mitigate the spread of grapevine tuck disease, which is posing serious economic losses in the vine-wine industry. However, the impact of PPP on woody tissues remains unclear. Our study, conducted in two European vineyards, investigated the effects of Cuprocol, Tessior, Esquive, and Bentogran on stilbenes, in canes of Cabernet sauvignon and Syrah, at three phenological stages. Main stilbenes, quantified by HPLC-UV-DAD (1260 Agilent Infinity System) and identified by HPLC-ESI/MS (Thermo Scientific LCQ FLEET system), included E-resveratrol, E-ε-viniferin, E-piceatannol, and E-polydatin. Canes exhibited varying proportions of individual stilbenes, reflecting differences based on climatic conditions and phenological phases, rather than on the application of specific PPP. Vines grown in cool-climate conditions exhibited higher levels of E-resveratrol, whereas vines from the Mediterranean climate area exhibited higher levels of E-ε-viniferin. We also observed divergences in the accumulation trend of wood stilbenes throughout the season in canes collected in the two different growing areas.