Search

Summary. Grapevine yellows bois noir (BN) and the grapevine trunk disease esca complex (EC) cause serious yield losses in European vineyards and are often widespread in the same vineyard. In a Chardonnay vineyard in north-eastern Italy, evolution of the two diseases from 2007 to 2020 was compared and their possible interaction was investigated. Evolution of symptomatic grapevines over the 16 years was very different between the two diseases, with a substantial linear increase for BN and an exponential increase for EC. The BN increase from one year to another was associated with the abundance of Hyalesthes obsoletus, the BN-phytoplasma vector, whereas the exponential increase in EC was likely due to the amount of inoculum and the increased size of pruning cuts over time. The courses of the two diseases were also very different, with a much greater occurrence of dead grapevines from EC than from BN. Some grapevines showed symptoms of both diseases, but the probability was less that a grapevine symptomatic for BN or EC showed symptoms of the other disease. Examinations of the spatial distribution of the two diseases showed dissociation between them. Data indicated that mechanisms of induced defense were involved in the lower probability that a grapevine affected by one showed symptoms of the other. [ABSTRACT FROM AUTHOR]

Fungi associated with Aspergillus Vine Canker (AVC) infect vigorous new shoots and canes during plant training, entering through wounds. Recently, grapevines exhibiting symptoms resembling AVC have been observed in Mexico. Thus, this study aimed to investigate these diseased plants. Woody samples from symptomatic plants were surface sterilized with alcohol and fire and then placed onto Potato Dextrose Agar (PDA) medium. Seventeen isolates displaying morphology similar to the genus Aspergillus were characterized. Colony and microscopic characteristics were observed on Czapek Yeast Extract Agar (CYA) and Malt Extract Agar (MEA). Phylogenetic analysis using calmodulin (CaM) and β-tubulin (BenA) gene markers, revealed the presence of Aspergillus niger, A. tubingensis, and A. welwitschiae species. Notably, Aspergillus isolates have an optimal growth temperature range of 35–37 °C but some exhibited thermotolerance, growing even at 50 °C. In pathogenicity assays, A. niger A10SMX, A. niger A8SMX, and A. tubingensis A13SMX exhibited the highest virulence in cultivar 'Merlot', resulting in lesions up to 2 cm in length after 50 days of inoculation. In addition to necrotic lesions, mycelia, and black conidia were observed on the leaves. Finally, a pathogenicity assay on table grape cultivars revealed the cultivar 'Cotton Candy' as the most susceptible to Aspergillus spp., and 'Prime Seedless' as the least susceptible. This study represents the first report of Aspergillus species associated with AVC in Mexican vineyards. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases are a threat to table- and grape-wine cultivation worldwide. Phaeomoniella chlamydospora (Pch) is a vascular fungus recognized as one of the most important pathogens associated with grapevine trunk diseases. The relationships between xylem vessel features and Pch susceptibility of 10 table- and 17 wine-grape genotypes, as well as 3 rootstocks, were investigated by image analysis of 50 µm cross-sections and artificial Pch inoculation on one-year-old vine cuttings. Vessels were grouped in the diameter classes 1–30, 31–60, 61–90, 91–120, and >120 µm. Among the table-grape varieties, 'Sable', 'Timco', and 'Red Globe' showed higher densities of large vessels (>120 μm) than 'Italia', 'Sugar Crisp', and 'Sugraone'. Among the wine-grape varieties, 'Minutolo', 'Montepulciano', 'Primitivo' CDTa19, and 'Verdeca' showed higher densities of large vessels than 'Aglianico', 'Nero di Troia', 'Sangiovese', and 'Susumaniello'. In the rootstocks, the vessel diameters were 50.8, 54.0, and 60.9 μm for '34 E.M.', '140 Ruggieri', and '1103 Paulsen', in that order. For table-grape varieties, Pch was re-isolated from 13.3% for 'Sugar Crisp' and 'Sugraone' cuttings up to 93.3% for 'Timco'. For wine-grape varieties, Pch re-isolation ranged from 51.1% ('Bombino nero', 'Negroamaro' D15, and 'Sangiovese') to 81.1% ('Montepulciano'), while for the rootstocks, the values were from 33 to 51%. A principal component analysis (PCA) revealed a positive correlation between the frequencies of large vessels and Pch re-isolation. In addition, in wine grapes and rootstocks, higher Pch re-isolation frequencies in the lateral parts of cuttings were correlated (r = 0.79) to a higher frequency of large vessels. The results highlight relationships between grapevine xylem vessel sizes and susceptibility to P. chlamydospora that are worthy of further research. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Eutypa dieback of grapevine is a trunk disease that impacts vineyard productivity worldwide. Grape germplasm is typically evaluated for resistance to Eutypa dieback through controlled inoculations in the greenhouse, although the high level of replication required of this approach (40 plants per genotype) can limit the total number of genotypes evaluated. An alternative approach is to evaluate naturally infected genotypes in the field. We rated the incidence and severity of vines with the diagnostic leaf symptoms of Eutypa dieback and the incidence of mortality among such vines of 973 Vitis vinifera accessions (planted in duplicate) at the US Department of Agriculture, National Clonal Germplasm Repository in Davis, CA, USA, which is maintained as a living collection for grape research. Across 3 years and spanning a total of 5 years (2011, 2013, and 2015), 120 accessions had leaf symptoms in one or more years ("susceptible accessions"). Courbu blanc [Davis Vitis identification tag (DVIT) 2313], Frankenthal blanc (DVIT 2115), and Pinot gris (DVIT 0907) were the only accessions with leaf symptoms each year. Accessions with the most severe leaf symptoms (a rating of 5 points) were Chasselas Napoleon (DVIT 0375) and Queen of the Vineyard (DVIT 0496). We identified susceptible accessions--namely, those related to 'Chasselas' and 'Muscat'--with a shared genetic background, based on a previous single nucleotide polymorphism genotyping effort of the collection. Especially for grapevine, a long-lived perennial that is meant to produce a crop for decades, knowledge of susceptible accessions and their pedigrees can help inform breeding programs and studies on the host response to infection. [ABSTRACT FROM AUTHOR]

Esca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases. [ABSTRACT FROM AUTHOR]

A comprehensive study was conducted on the prevalence of grapevine trunk diseases in Greece, focusing specifically on contamination of grapevine propagation material by Phaeomoniella chlamydospora and black foot disease‐related species. Additionally, detection of esca pathogen Fomitiporia mediterranea, causing white rot in grapevine and other woody hosts, was assessed using a new PCR‐based assay that distinguishes F. mediterranea from F. punctata. Development of a nested PCR assay, combined with a cost‐effective DNA extraction protocol, revealed a high percentage of infection by P. chlamydospora (51%) and Ilyonectria species associated with black foot disease (28%) in different types of grapevine propagation material (dormant cuttings, field‐rooted benchgrafts and green‐growing plants). Interestingly, black foot disease‐related pathogens were more prevalent in nursery plants grown in the field for 6 months (57%) than in other types of propagation material and compared with P. chlamydospora (43%), indicating increased infection of propagation material during growth in the nursery field. The cost‐effective molecular method developed in this study could be used in mass inspections of propagation material for phytosanitary purposes. Finally, using primers specific for F. mediterranea and F. puncata developed in this study, combined with the universal ITS4 primer, a collection of Fomitiporia isolates from mature grapevine and other woody hosts from Greece and Italy (southern Europe) were characterized as F. mediterranea, whereas German and Swedish isolates from forest plants (central Europe) were classified as F. punctata. The developed primers can discriminate between the two Fomitiporia species, which are indistinguishable based on culture and morphological characteristics alone. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases (GTDs) attack the vine's wood, devastating vineyards worldwide. Chile is the world's fourth-largest wine exporter, and Cabernet Sauvignon is one of the most economically important red wine varieties. Botryosphaeria dieback is an important GTD, and Diplodia seriata is one of the main pathogenic species. Biocontrol studies of these pathogens are commonly carried out at different incubation times but at a single temperature. This study aimed to evaluate the biocontrol effect of Chilean PGPB and grapevine endophytic bacteria against D. seriata at different temperatures. We analyzed the biocontrol effect of Pseudomonas sp. GcR15a, Pseudomonas sp. AMCR2b and Rhodococcus sp. PU4, with three D. seriata isolates (PUCV 2120, PUCV 2142 and PUCV 2183) at 8, 22 and 35 °C. Two dual-culture antagonism methods (agar plug diffusion and double plate) were used to evaluate the in vitro effect, and an in vivo test was performed with Cabernet Sauvignon cuttings. In vitro, the greatest inhibitions were obtained using the agar plug diffusion method and at a temperature of 8 °C, where Rhodococcus sp. PU4 obtains a 65% control (average) and Pseudomonas sp. GcR15a a 57% average. At 22 °C, only strains of Pseudomonas sp. show control. At 35 °C, one Pseudomonas strain shows the highest control (38%), on average, similar to tebuconazole (33%), and then Rhodococcus sp. (30%). In vivo, a biocontrol effect is observed against two D. seriata isolates, while the PUCV 2142 proves to be more resistant to control. The biocontrol ability at low temperatures is promising for effective control in the field, where infections occur primarily in winter. [ABSTRACT FROM AUTHOR]

The composition, diversity and dynamics of microbial communities associated with grapevines may be influenced by various environmental factors, including terroir, vintage, and season. Among these factors, terroir stands out as a unique possible determinant of the pathobiome, the community of plant-associated pathogens. This study employed high-throughput molecular techniques, including metabarcoding and network analysis, to investigate the compositional dynamics of grapevine fungal pathobiome across three microhabitats (soil, woody tissue, and bark) using the Furmint cultivar. Samples were collected during late winter and late summer in 2020 and 2021, across three distinct terroirs in Hungary's Tokaj wine region. Of the 123 plant pathogenic genera found, Diplodia, Phaeomoniella, and Fusarium displayed the highest richness in bark, wood, and soil, respectively. Both richness and abundance exhibited significant disparities across microhabitats, with plant pathogenic fungi known to cause grapevine trunk diseases (GTDs) demonstrating highest richness and abundance in wood and bark samples, and non-GTD pathogens prevailed soil. Abundance and richness, however, followed distinct patterns Terroir accounted for a substantial portion of the variance in fungal community composition, ranging from 14.46 to 24.67%. Season and vintage also contributed to the variation, explaining 1.84 to 2.98% and 3.67 to 6.39% of the variance, respectively. Notably, significant compositional differences in fungi between healthy and diseased grapevines were only identified in wood and bark samples. Cooccurrence networks analysis, using both unweighted and weighted metrics, revealed intricate relationships among pathogenic fungal genera. This involved mostly positive associations, potentially suggesting synergism, and a few negative relationships, potentially suggesting antagonistic interactions. In essence, the observed differences among terroirs may stem from environmental filtering due to varied edaphic and mesoclimatic conditions. Temporal weather and vine management practices could explain seasonal and vintage fungal dynamics. This study provides insights into the compositional dynamics of grapevine fungal pathobiome across different microhabitats, terroirs, seasons, and health statuses. The findings emphasize the importance of considering networkbased approaches in studying microbial communities and have implications for developing improved viticultural plant health strategies. [ABSTRACT FROM AUTHOR]

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.

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.

Grapevine trunk diseases (GTDs) are caused by multiple unrelated fungal pathogens, and their management remains difficult worldwide. Biocontrol is an attractive and sustainable strategy given the current need for a cleaner viticulture. In this study, twenty commercial vineyards were sampled across California to isolate endophytic and rhizospheric bacteria from different grapevine cultivars with the presence and absence of GTD symptoms. A collection of 1344 bacterial isolates were challenged in vitro against Neofusicoccum parvum and Diplodia seriata, from which a subset of 172 isolates exerted inhibition levels of mycelial growth over 40%. Bacterial isolates were identified as Bacillus velezensis (n = 154), Pseudomonas spp. (n = 12), Serratia plymuthica (n = 2) and others that were later excluded (n = 4). Representative isolates of B. velezensis, P. chlororaphis, and S. plymuthica were challenged against six other fungal pathogens responsible for GTDs. Mycelial inhibition levels were consistent across bacterial species, being slightly higher against slow-growing fungi than against Botryosphaeriaceae. Moreover, agar-diffusible metabolites of B. velezensis strongly inhibited the growth of N. parvum and Eutypa lata, at 1, 15, and 30% v/v. The agar-diffusible metabolites of P. chlororaphis and S. plymuthica, however, caused lower inhibition levels against both pathogens, but their volatile organic compounds showed antifungal activity against both pathogens. These results suggest that B. velezensis, P. chlororaphis and S. plymuthica constitute potential biocontrol agents (BCAs) against GTDs and their application in field conditions should be further evaluated.

Grapevine trunk diseases are a threat to table- and grape-wine cultivation worldwide. Phaeomoniella chlamydospora (Pch) is a vascular fungus recognized as one of the most important pathogens associated with grapevine trunk diseases. The relationships between xylem vessel features and Pch susceptibility of 10 table- and 17 wine-grape genotypes, as well as 3 rootstocks, were investigated by image analysis of 50 µm cross-sections and artificial Pch inoculation on one-year-old vine cuttings. Vessels were grouped in the diameter classes 1–30, 31–60, 61–90, 91–120, and >120 µm. Among the table-grape varieties, ‘Sable’, ‘Timco’, and ‘Red Globe’ showed higher densities of large vessels (>120 μm) than ‘Italia’, ‘Sugar Crisp’, and ‘Sugraone’. Among the wine-grape varieties, ‘Minutolo’, ‘Montepulciano’, ‘Primitivo’ CDTa19, and ‘Verdeca’ showed higher densities of large vessels than ‘Aglianico’, ‘Nero di Troia’, ‘Sangiovese’, and ‘Susumaniello’. In the rootstocks, the vessel diameters were 50.8, 54.0, and 60.9 μm for ‘34 E.M.’, ‘140 Ruggieri’, and ‘1103 Paulsen’, in that order. For table-grape varieties, Pch was re-isolated from 13.3% for ‘Sugar Crisp’ and ‘Sugraone’ cuttings up to 93.3% for ‘Timco’. For wine-grape varieties, Pch re-isolation ranged from 51.1% (‘Bombino nero’, ‘Negroamaro’ D15, and ‘Sangiovese’) to 81.1% (‘Montepulciano’), while for the rootstocks, the values were from 33 to 51%. A principal component analysis (PCA) revealed a positive correlation between the frequencies of large vessels and Pch re-isolation. In addition, in wine grapes and rootstocks, higher Pch re-isolation frequencies in the lateral parts of cuttings were correlated (r = 0.79) to a higher frequency of large vessels. The results highlight relationships between grapevine xylem vessel sizes and susceptibility to P. chlamydospora that are worthy of further research.

Eutypa dieback of grapevine is a trunk disease that impacts vineyard productivity worldwide. Grape germplasm is typically evaluated for resistance to Eutypa dieback through controlled inoculations in the greenhouse, although the high level of replication required of this approach (40 plants per genotype) can limit the total number of genotypes evaluated. An alternative approach is to evaluate naturally infected genotypes in the field. We rated the incidence and severity of vines with the diagnostic leaf symptoms of Eutypa dieback and the incidence of mortality among such vines of 973 Vitis vinifera accessions (planted in duplicate) at the US Department of Agriculture, National Clonal Germplasm Repository in Davis, CA, USA, which is maintained as a living collection for grape research. Across 3 years and spanning a total of 5 years (2011, 2013, and 2015), 120 accessions had leaf symptoms in one or more years (“susceptible accessions”). Courbu blanc [Davis Vitis identification tag (DVIT) 2313], Frankenthal blanc (DVIT 2115), and Pinot gris (DVIT 0907) were the only accessions with leaf symptoms each year. Accessions with the most severe leaf symptoms (a rating of 5 points) were Chasselas Napoleon (DVIT 0375) and Queen of the Vineyard (DVIT 0496). We identified susceptible accessions—namely, those related to ‘Chasselas’ and ‘Muscat’—with a shared genetic background, based on a previous single nucleotide polymorphism genotyping effort of the collection. Especially for grapevine, a long-lived perennial that is meant to produce a crop for decades, knowledge of susceptible accessions and their pedigrees can help inform breeding programs and studies on the host response to infection.

The composition, diversity and dynamics of microbial communities associated with grapevines may be influenced by various environmental factors, including terroir, vintage, and season. Among these factors, terroir stands out as a unique possible determinant of the pathobiome, the community of plant-associated pathogens. This study employed high-throughput molecular techniques, including metabarcoding and network analysis, to investigate the compositional dynamics of grapevine fungal pathobiome across three microhabitats (soil, woody tissue, and bark) using the Furmint cultivar. Samples were collected during late winter and late summer in 2020 and 2021, across three distinct terroirs in Hungary’s Tokaj wine region. Of the 123 plant pathogenic genera found, Diplodia, Phaeomoniella, and Fusarium displayed the highest richness in bark, wood, and soil, respectively. Both richness and abundance exhibited significant disparities across microhabitats, with plant pathogenic fungi known to cause grapevine trunk diseases (GTDs) demonstrating highest richness and abundance in wood and bark samples, and non-GTD pathogens prevailed soil. Abundance and richness, however, followed distinct patterns Terroir accounted for a substantial portion of the variance in fungal community composition, ranging from 14.46 to 24.67%. Season and vintage also contributed to the variation, explaining 1.84 to 2.98% and 3.67 to 6.39% of the variance, respectively. Notably, significant compositional differences in fungi between healthy and diseased grapevines were only identified in wood and bark samples. Cooccurrence networks analysis, using both unweighted and weighted metrics, revealed intricate relationships among pathogenic fungal genera. This involved mostly positive associations, potentially suggesting synergism, and a few negative relationships, potentially suggesting antagonistic interactions. In essence, the observed differences among terroirs may stem from environmental filtering due to varied edaphic and mesoclimatic conditions. Temporal weather and vine management practices could explain seasonal and vintage fungal dynamics. This study provides insights into the compositional dynamics of grapevine fungal pathobiome across different microhabitats, terroirs, seasons, and health statuses. The findings emphasize the importance of considering network-based approaches in studying microbial communities and have implications for developing improved viticultural plant health strategies.

Grapevine trunk diseases (GTD) result from various fungi invading grapevine wood, leading to a decline in quality and yield. Accurate identification of fungal species is vital for effective disease management. Visual inspection through microscopy is a commonly used method, but distinguishing similar microorganisms within the same genus can be challenging. For precise identification, molecular methods are often required, despite being relatively costly and time-consuming. In this paper, we present a novel method for classifying four species of grapevine wood fungi using deep learning algorithms. We evaluate the performance of four different deep learning architectures, ResNet-50, VGG-16, MobileNet, and InceptionV3, in the classification of grapevine fungal spores from our microscope image dataset. During our tests, the proposed classification methodology achieved an accuracy of up to 97.40 %. Our approach can facilitate the development of more efficient and accurate methods for fungal species identification and has potential applications in viticulture and plant pathology research.

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. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome. [ABSTRACT FROM AUTHOR]

Abstract Background Grapevine trunk diseases (GTDs) are disease complexes that are major threats to viticulture in most grapevine growing regions. The microbiomes colonizing plant belowground components form complex associations with plants, play important roles in promoting plant productivity and health in natural environments, and may be related to GTD development. To investigate associations between belowground fungal communities and GTD symptomatic or asymptomatic grapevines, fungal communities associated with three soil–plant compartments (bulk soils, rhizospheres, and roots) were characterized by ITS high-throughput amplicon sequencing across two years. Results The fungal community diversity and composition differs according to the soil–plant compartment type (PERMANOVA, p

The Botryosphaeriaceae is a fungal family that includes many destructive vascular pathogens of woody plants (e.g., Botryosphaeria dieback of grape, Panicle blight of pistachio). Species in the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum, and Neoscytalidium attack a range of horticultural crops, but they vary in virulence and their abilities to infect their hosts via different infection courts (flowers, green shoots, woody twigs). Isolates of seventeen species, originating from symptomatic apricot, grape, pistachio, and walnut were tested for pathogenicity on grapevine wood after 4 months of incubation in potted plants in the greenhouse. Results revealed significant variation in virulence in terms of the length of the internal wood lesions caused by these seventeen species. Phylogenomic comparisons of the seventeen species of wood-colonizing fungi revealed clade-specific expansion of gene families representing putative virulence factors involved in toxin production and mobilization, wood degradation, and nutrient uptake. Statistical analyses of the evolution of the size of gene families revealed expansions of secondary metabolism and transporter gene families in Lasiodiplodia and of secreted cell wall degrading enzymes (CAZymes) in Botryosphaeria and Neofusicoccum genomes. In contrast, Diplodia, Dothiorella, and Neoscytalidium generally showed a contraction in the number of members of these gene families. Overall, species with expansions of gene families, such as secreted CAZymes, secondary metabolism, and transporters, were the most virulent (i.e., were associated with the largest lesions), based on our pathogenicity tests and published reports. This study represents the first comparative phylogenomic investigation into the evolution of possible virulence factors from diverse, cosmopolitan members of the Botryosphaeriaceae.

Grapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy-five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several fungi species, including members of the Botryosphaeriaceae family and Phaeomoniella chlamydospora, considered some of the most important causal agents for these diseases in Chile. In this study, we isolated 169 endophytic and 209 rhizospheric fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Chaetomium sp., Cladosporium sp., Clonostachys rosea, Epicoccum nigrum, Purpureocillium lilacinum, and Trichoderma sp. were evaluated for their potential of biocontrol activity against Diplodia seriata, Neofusicoccum parvum, and Pa. chlamydospora. Tests of antagonism were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. Clonostachys rosea showed 98.2% inhibition of all pathogens in the presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp. Overall, these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.

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.

Despite studies associating Fusarium spp. with grapevine decline since late 1970s, no consensus has been reached regarding the roles these fungi play in grapevine health. Recent studies in British Columbia, Canada, assessed prevalence of grapevine trunk diseases (GTDs) in young and mature vineyards, and the presence of GTD fungi in ready-to-plant nursery material sold in Canada. This study characterized the Fusarium spp. isolated from grapevines in BC by sequencing part of the translation elongation factor 1-alpha (TEF1) gene, and carried out pathogenicity studies to determine whether Fusarium plays a role in grapevine decline. Fusarium spp. were isolated from 9.8% of samples collected from young vines (≤ 8-year-old), and 7.3% from mature vines (> 8-year-old), showing decline symptoms in commercial vineyards. Fusarium was also isolated from 43.9% of ready-to-plant dormant grapevines analyzed from four nurseries. Fusarium incidence varied between plants within the same nursery and between plants from the different nurseries. DNA sequences of TEF1 allowed identification of Fusarium oxysporum, F. proliferatum, F. ramigenum, and a Fusarium sp. Pathogenicity studies were conducted in 1-year-old dormant rooted ‘Chardonnay’ plants grafted onto ‘3309C’ rootstock, and treatments included: i) whole plant, ii) trimming of roots, iii) cut at rootstock basal ends, and iv) trimming of roots plus cut at rootstock basal ends. Plants were inoculated using standardized methods, and were then planted in a greenhouse. Fusarium was compared with Dactylonectria macrodidyma, D. pauciseptata and Ilyonectria liriodendri used as positive controls. The Fusarium spp. caused necroses in rootstock roots and basal ends that were similar to those caused by blackfoot fungi. Fusarium spp. and black-foot fungi reduced root and shoot dry weights when compared with non-inoculated controls, but no statistically significant differences were recorded for most treatments. This study is the first in Canada to identify Fusarium spp. from grapevines. Though Fusarium was common in these grapevines, pathogenicity tests suggest that the identified Fusarium spp. were weakly pathogenic to ‘3309C’ rootstock grapevines. [ABSTRACT FROM AUTHOR]

Grapevine Trunk Diseases (GTDs) are major threats in Mediterranean countries, causing economic losses due to reduced grape yields and long-term vine productivity, as well as death of grapevines. A survey was conducted in Piedmont (Northern Italy) during 2021-2022 to investigate the species diversity and distribution of GTD pathogens in this important Italian wine region. Morphological and multilocus phylogenetic analyses (based on ITS, tef1, tub2, act and rpb2) identified species of Botryosphaeriaceae at high frequency, including Botryosphaeria dothidea, Diplodia mutila, Diplodia seriata and Neofusicoccum parvum. Other pathogens commonly associated with GTDs, including Eutypa lata, Fomitiporia mediterranea and Phaeomoniella chlamydospora, were also isolated. Less commonly isolated species included Neocucurbitaria juglandicola, Paraconiothyrium brasiliense, Seimatosporium vitis-viniferae and Truncatella angustata. Pathogenicity tests with two representative isolates of each species were carried out using one-year-old potted grapevine cuttings (‘Barbera’). All isolates (except N. juglandicola) caused brown wood necrotic vascular discolourations on inoculated plants and were successfully re-isolated. Effects of temperature on colony growth were also assessed. For all tested isolates there was no growth at 5°C, only four isolates (Botryosphaeriaceae) grew at 35°C, and optimum growth temperatures were between 20 and 25°C. This is the first record of Paraconiothyrium brasiliense and Neocucurbitaria juglandicola associated with symptomatic grapevines in Italy. [ABSTRACT FROM AUTHOR]

Many fungal pathogens are associated with grapevine trunk diseases (GTDs), which cause important yield and economic losses in grape production. There are no effective control methods against GTDs once plants are infected, so research is aimed at preventive measures to avoid infections in nurseries and vineyards. Inhibitory activities of the phenolic compounds eugenol, epigallocatechin–3–O–gallate (EGCG) and thymol against the GTD fungi Diplodia seriata, Eutypa lata, Fomitiporia mediterranea and Neofusicoccum parvum were assessed in vitro, and in planta as grapevine pruning wound treatments. Greatest inhibition of pathogen mycelium growth was observed with eugenol (fungistatic at 1,500 µg mL–1, fungicidal at 2,500 µg mL–1). No inhibitory activity against GTD fungi was observed with EGCG. Minimum concentrations with in vitro inhibitory effects on D. seriata and N. parvum spore germination were 360 µg mL–1 for thymol and 750 µg mL–1 for eugenol. In the grapevine wound protection tests, thymol was effective against N. parvum at 360 µg mL–1, but eugenol was not. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases cause severe damage in grapevines. Management strategies focus on protection of grapevine pruning wounds using chemical fungicides or biological control agents. Botryosphaeria dieback, caused mainly by Lasiodiplodia spp., is one of the main trunk diseases in northwest Mexico. This study obtained endophytic bacteria and fungi from the heritage grapevine Vitis vinifera cv. ‘Mission’ for potential biological control of Botryosphaeria dieback. A collection of 135 bacterial and 37 fungal isolates were obtained and initially tested for antagonistic activity against Lasiodiplodia brasiliensis. The most promising isolates belonging to Trichoderma and Bacillus spp. were selected and characterized to determine their modes of action. Bacillus isolates produced volatile organic compounds that inhibited growth of Neofusicoccum parvum, and diffusible organic compounds with antifungal effects against L. brasiliensis and N. parvum. Trichoderma isolates produced diffusible organic compounds and were mycoparasites. In greenhouse assays, plants inoculated with three Trichoderma asperellum isolates (T20BCMX, EF09BCMX, and EF11BCMX), B. amyloliquefaciens (BEVP26BCMX) or Bacillus sp. (rbES015), applied preventively in soil, gave up to 50% smaller necrotic lesions when compared with the plants inoculated only with L. brasiliensis. In the field, plants inoculated with three Bacillus isolates (BEVP02BCMX, BEVP26BCMX, BEVP31BCMX) or five Trichoderma (T11BCMX, T15BCMX, T17BCMX, T20BCMX, and EF11BCMX) had lesions up to four times smaller than control plants inoculated only with L. brasiliensis. This study has demonstrated the potential of heritage grapevines to provide biological control agents for Botryosphaeria dieback. [ABSTRACT FROM AUTHOR]

During a study of Botryosphaeriaceae species associated with grapevine trunk diseases in the Czech Republic, a collection of 22 Botryosphaeriaceae-like strains were isolated from four cultivars (Blaufränkisch, Pálava, Pinot Noir, and Welschriesling) in four distinct vineyards. Based on morphology and DNA sequence data (ITS, tub2, and tef), four species were identified: Botryosphaeria dothidea, Diplodia mutila, D. seriata, and Neofusicoccum parvum. These species are reported for the first time from grapevine in the Czech Republic. Relationships between vascular lesions and particular species were highlighted in this study. Diplodia seriata was the most frequently isolated species, present in all four sampled cultivars, while D. mutila was the least frequent, present only in 'Pálava'. The cultivar Pinot Noir was the most tolerant host for Botryosphaeriaceae fungi. [ABSTRACT FROM AUTHOR]

Background: Grapevine trunk diseases (GTDs) are a complex group of diseases that lead to major economic losses in all wine‐producing countries. The investigation of biocontrol agents (BCAs) capable of forestalling or at least minimizing the development of GTDs has, recently, become a priority. Nursery experiments were set up to (i) assess the biocontrol effect of Trichoderma atroviride (Ta) SC1 and Bacillus subtilis (Bs) PTA‐271, alone and in simultaneous application, against Botryosphaeria dieback (BOT)‐ and black‐foot (BF)‐ associated pathogens during the grapevine propagation process and (ii) evaluate the success of the BCA inoculation during the grapevine propagation process, using quantitative reverse‐transcription polymerase chain reaction techniques. RESULTS: The results demonstrated a significant reduction in the percentage of potentially infected plants and the percentage of fungal isolation from wood fragments of BOT and BF pathogens in nursery material treated with Ta SC1 and Bs PTA‐271, respectively. In one of the experiments, simultaneous treatments with Bs PTA‐271 and Ta SC1 caused a reduction in percentages of potentially infected plants and fungal isolation, from wood fragments containing BOT and BF pathogens. CONCLUSION: These biological treatments may be relevant components of an integrated approach, using complementary management strategies to limit infection by GTD pathogens, but further research is still needed to elucidate the effectiveness of Bs PTA‐271 and the benefits of simultaneous application with Ta SC1 for the control of GTD pathogens in nurseries. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Grapevine trunk diseases (GTDs) are disease complexes that are major threats to viticulture in most grapevine growing regions. The microbiomes colonizing plant belowground components form complex associations with plants, play important roles in promoting plant productivity and health in natural environments, and may be related to GTD development. To investigate associations between belowground fungal communities and GTD symptomatic or asymptomatic grapevines, fungal communities associated with three soil–plant compartments (bulk soils, rhizospheres, and roots) were characterized by ITS high-throughput amplicon sequencing across two years. Results: The fungal community diversity and composition differs according to the soil–plant compartment type (PERMANOVA, p < 0.001, 12.04% of variation explained) and sampling year (PERMANOVA, p < 0.001, 8.83%), whereas GTD symptomatology exhibited a weaker, but still significant association (PERMANOVA, p < 0.001, 1.29%). The effects of the latter were particularly prominent in root and rhizosphere community comparisons. Many GTD-associated pathogens were detected, but their relative abundances were not correlated (or were negatively correlated) to symptomatology. Fusarium spp., were enriched in symptomatic roots and rhizospheres compared to asymptomatic counterparts, suggesting that their abundances were positively correlated with symptomatic vines. Inoculation tests revealed that Fusarium isolates, similar to Dactylonectria macrodidyma, a pathogen associated with black foot disease, caused dark brown necrotic spots on stems in addition to root rot, which blackened lateral roots. Disease indices were higher with co-inoculation than single inoculation with a Fusarium isolate or D. macrodidyma, suggesting that Fusarium spp. can exacerbate disease severity when inoculated with other known GTD-associated pathogens. Conclusions: The belowground fungal microbiota of grapevines varied from soil–plant compartments, the years and whether showed GTD symptoms. The GTDs symptoms were related to the enrichment of Fusarium spp. rather than the relative abundances of GTD pathogens. These results demonstrate the effects of fungal microbiota of roots and rhizospheres on GTDs, while providing new insights into opportunistic pathogenesis of GTDs and potential control practices. [ABSTRACT FROM AUTHOR]

Fungal pathogens involved in grapevine trunk diseases (GTDs) may infect grapevines throughout their lifetime, from nursery to vineyard, via open wounds in stems, canes or roots. In vineyards, pruning wound protection products (PWPPs) offer the best means to reduce the chance of infection by GTD fungi. However, PWPPs may affect non-target microorganisms that comprise the natural endophytic mycobiome residing in treated canes, disrupting microbial homeostasis and indirectly influencing grapevine health. Using DNA metabarcoding, we characterized the endophytic mycobiome of one-year-old canes of cultivars Cabernet Sauvignon and Syrah in two vineyards in Portugal and Italy and assessed the impact of established and novel PWPPs on the fungal communities of treated canes. Our results reveal a large fungal diversity (176 taxa), and we report multiple genera never detected before in grapevine wood (e.g., Symmetrospora and Akenomyces). We found differences in mycobiome beta diversity when comparing vineyards (p = 0.01) but not cultivars (p > 0.05). When examining PWPP-treated canes, we detected cultivar- and vineyard-dependent alterations in both alpha and beta diversity. In addition, numerous fungal taxa were over- or under-represented when compared to control canes. Among them, Epicoccum sp., a beneficial genus with biological control potential, was negatively affected by selected PWPPs. This study demonstrates that PWPPs induce alterations in the fungal communities of grapevines, requiring an urgent evaluation of their direct and indirect effects on plants health with consideration of factors such as climatic conditions and yearly variations, in order to better advise viticulturists and policy makers. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome.

Grapevine trunk diseases (GTDs) attack the vine’s wood, devastating vineyards worldwide. Chile is the world’s fourth-largest wine exporter, and Cabernet Sauvignon is one of the most economically important red wine varieties. Botryosphaeria dieback is an important GTD, and Diplodia seriata is one of the main pathogenic species. Biocontrol studies of these pathogens are commonly carried out at different incubation times but at a single temperature. This study aimed to evaluate the biocontrol effect of Chilean PGPB and grapevine endophytic bacteria against D. seriata at different temperatures. We analyzed the biocontrol effect of Pseudomonas sp. GcR15a, Pseudomonas sp. AMCR2b and Rhodococcus sp. PU4, with three D. seriata isolates (PUCV 2120, PUCV 2142 and PUCV 2183) at 8, 22 and 35 °C. Two dual-culture antagonism methods (agar plug diffusion and double plate) were used to evaluate the in vitro effect, and an in vivo test was performed with Cabernet Sauvignon cuttings. In vitro, the greatest inhibitions were obtained using the agar plug diffusion method and at a temperature of 8 °C, where Rhodococcus sp. PU4 obtains a 65% control (average) and Pseudomonas sp. GcR15a a 57% average. At 22 °C, only strains of Pseudomonas sp. show control. At 35 °C, one Pseudomonas strain shows the highest control (38%), on average, similar to tebuconazole (33%), and then Rhodococcus sp. (30%). In vivo, a biocontrol effect is observed against two D. seriata isolates, while the PUCV 2142 proves to be more resistant to control. The biocontrol ability at low temperatures is promising for effective control in the field, where infections occur primarily in winter.

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.

Native strains of Trichoderma in vineyard soil represent an opportunity for reducing the incidence of grapevine trunk diseases (GTDs) in vineyards. Moreover, its relationship with the environment (physicochemical soil characteristics and farming management practices) remains unclear. In the current study, a survey was carried out on farming management used by viticulturists, and soil samples were studied to analyze their physicochemical properties and to isolate Trichoderma strains. Later, statistical analyses were performed to identify possible correlations between Trichoderma populations, soil management and soil characteristics. In addition, in vitro tests, including antibiosis and mycoparasitism, were performed to select those Trichoderma strains able to antagonize Phaeoacremonium minimum. In this study a positive correlation was found between the iron content and pH in the soil, and a lower pH increases Trichoderma populations in soils. Vineyard management also affects Trichoderma populations in the soil, negatively in the case of fertilization and tillage and positively in the case of herbicide spraying. Two Trichoderma native strains were selected as potential biocontrol agents (Trichoderma gamsii T065 and Trichoderma harzianum T087) using antibiosis and mycoparasitism as mechanisms of action. These results led to the conclusion that native Trichoderma strains hold great potential as biological control agents and as producers of secondary metabolites. [ABSTRACT FROM AUTHOR]

Grapevine trunk disease fungi infect vines through openings, primarily pruning wounds. The main objective of this study was to understand the role of sucker wounds and wounds made by the removal of green shoots from the stems of potted grapevines as potential points of infection for grapevine trunk disease pathogens. Six wine and four table grape vineyards of different ages were sampled in different production areas in the Western Cape grape region of South Africa. Isolations were made from 161 sucker wounds, and fungal pathogens were identified using morphology and DNA sequence analysis of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal RNA gene, the translation elongation factor 1alpha or the partial β-tubulin gene. The results show that 62% of the sucker wounds were infected by trunk disease pathogens, including Diaporthe ampelina, Diplodia seriata, Phaeomoniella chlamydospora, Phaeoacremonium minimum, Eutypella microtheca, Cryptovalsa ampelina and Neofusicoccum australe. Diaporthe ampelina was the most common, followed by D. seriata and P. chlamydospora, in both the wine and table grape sucker wounds. Under glasshouse conditions, wounds made by the removal of young green shoots on one-year-old potted grapevine plants were inoculated with spore suspensions of D. ampelina, E. lata, N. parvum, P. minimum and P. chlamydospora. After four months, all the inoculated pathogens could be re-isolated. This study shows that grapevine sucker and green shoot wounds are susceptible to different grapevine trunk disease pathogens and may therefore play a role in the epidemiology of trunk diseases. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases can infect nursery plants at different stages of propagation. Here, we evaluated the efficiency of aqueous ozone against two fungi associated with trunk diseases (Phaeoacremonium minimum and Phaeomoniella chlamydospora). The effect of ozone on the plant growth is also studied. Aqueous ozone completely suppressed spore germination in vitro. In planta fungal development was reduced six weeks post-inoculation. In the nursery, ozone treatment was comparable to the usual chemical treatment. Irrigation with aqueous ozone had no negative effects on plant growth. These anti-fungal properties and the absence of phytotoxicity make ozone a promising alternative for controlling microbial infection in nurseries. [ABSTRACT FROM AUTHOR]

Besides well-known grapevine trunk disease (GTD)-related pathogens, there is an increased interest in wood-colonizing fungi that infect grapevines. During 2017–2018, a survey was conducted in Cyprus and wood samples were collected from vines exhibiting typical GTD symptoms. Based on morphological and multilocus phylogenetic analyses (ITS, LSU, bt2, tef1-a), four species in the Sporocadaceae family were described and typified; two in the genus of Seimatosporium: Seim. cyprium sp. nov. and Seim. vitis-viniferae and two in Sporocadus: Spo. kurdistanicus and Spo. rosigena. The teleomorph of Seim. cyprium sp. nov. was also described. Pathogenicity trials with representative isolates of each species were performed on woody stems of two-year-old potted grapevines for 12 months under field conditions. All isolates were pathogenic, causing dark brown to black vascular discoloration, extending upward and downward from the inoculation point. Sporocadus isolates were significantly more aggressive than Seimatosporium with lesion lengths ranging from 9.24 to 6.90 and 4.13 to 4.00 cm, respectively. Successful re-isolations were also evident for all species and isolates. Seim. cyprium sp. nov. is a newly described species, while Spo. kurdistanicus and Spo. rosigena are reported for the first time in Europe on Vitis vinifera, suggesting the potential role of Sporocadaceae in the GTDs complex. [ABSTRACT FROM AUTHOR]

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 GTD 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, Botryosphaeria, 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., Competing Interests: The author(s) declare no conflict of interest.

During a study of Botryosphaeriaceae species associated with grapevine trunk diseases in the Czech Republic, a collection of 22 Botryosphaeriaceae-like strains were isolated from four cultivars (Blaufränkisch, Pálava, Pinot Noir, and Welschriesling) in four distinct vineyards. Based on morphology and DNA sequence data (ITS, tub2, and tef), four species were identified: Botryosphaeria dothidea, Diplodia mutila, D. seriata, and Neofusicoccum parvum. These species are reported for the first time from grapevine in the Czech Republic. Relationships between vascular lesions and particular species were highlighted in this study. Diplodia seriata was the most frequently isolated species, present in all four sampled cultivars, while D. mutila was the least frequent, present only in ‘Pálava’. The cultivar Pinot Noir was the most tolerant host for Botryosphaeriaceae fungi.

Grapevine is regarded as a highly profitable culture, being well spread worldwide and mostly directed to the wine-producing industry. Practices to maintain the vineyard in healthy conditions are tenuous and are exacerbated due to abiotic and biotic stresses, where fungal grapevine trunk diseases (GTDs) play a major role. The abolishment of chemical treatments and the intensification of several management practices led to an uprise in GTD outbreaks. Symptomatology of GTDs is very similar among diseases, leading to underdevelopment of the vines and death in extreme scenarios. Disease progression is widely affected by biotic and abiotic factors, and the prevalence of the pathogens varies with country and region. In this review, the state-of-the-art regarding identification and detection of GTDs is vastly analyzed. Methods and protocols used for the identification of GTDs, which are currently rather limited, are highlighted. The main conclusion is the utter need for the development of new technologies to easily and precisely detect the presence of the pathogens related to GTDs, allowing to readily take phytosanitary measures and/or proceed to plant removal in order to establish better vineyard management practices. Moreover, new practices and methods of detection, identification, and quantification of infectious material would allow imposing greater control on nurseries and plant exportation, limiting the movement of infected vines and thus avoiding the propagation of fungal inoculum throughout wine regions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Grapevine is regarded as a highly profitable culture, being well spread worldwide and mostly directed to the wine-producing industry. Practices to maintain the vineyard in healthy conditions are tenuous and are exacerbated due to abiotic and biotic stresses, where fungal grapevine trunk diseases (GTDs) play a major role. The abolishment of chemical treatments and the intensification of several management practices led to an uprise in GTD outbreaks. Symptomatology of GTDs is very similar among diseases, leading to underdevelopment of the vines and death in extreme scenarios. Disease progression is widely affected by biotic and abiotic factors, and the prevalence of the pathogens varies with country and region. In this review, the state-of-the-art regarding identification and detection of GTDs is vastly analyzed. Methods and protocols used for the identification of GTDs, which are currently rather limited, are highlighted. The main conclusion is the utter need for the development of new technologies to easily and precisely detect the presence of the pathogens related to GTDs, allowing to readily take phytosanitary measures and/or proceed to plant removal in order to establish better vineyard management practices. Moreover, new practices and methods of detection, identification, and quantification of infectious material would allow imposing greater control on nurseries and plant exportation, limiting the movement of infected vines and thus avoiding the propagation of fungal inoculum throughout wine regions.

Fungal pathogens involved in grapevine trunk diseases (GTDs) may infect grapevines throughout their lifetime, from nursery to vineyard, via open wounds in stems, canes or roots. In vineyards, pruning wound protection products (PWPPs) offer the best means to reduce the chance of infection by GTD fungi. However, PWPPs may affect non-target microorganisms that comprise the natural endophytic mycobiome residing in treated canes, disrupting microbial homeostasis and indirectly influencing grapevine health. Using DNA metabarcoding, we characterized the endophytic mycobiome of one-year-old canes of cultivars Cabernet Sauvignon and Syrah in two vineyards in Portugal and Italy and assessed the impact of established and novel PWPPs on the fungal communities of treated canes. Our results reveal a large fungal diversity (176 taxa), and we report multiple genera never detected before in grapevine wood (e.g., Symmetrospora and Akenomyces). We found differences in mycobiome beta diversity when comparing vineyards (p = 0.01) but not cultivars (p > 0.05). When examining PWPP-treated canes, we detected cultivar- and vineyard-dependent alterations in both alpha and beta diversity. In addition, numerous fungal taxa were over- or under-represented when compared to control canes. Among them, Epicoccum sp., a beneficial genus with biological control potential, was negatively affected by selected PWPPs. This study demonstrates that PWPPs induce alterations in the fungal communities of grapevines, requiring an urgent evaluation of their direct and indirect effects on plants health with consideration of factors such as climatic conditions and yearly variations, in order to better advise viticulturists and policy makers.

Given their well-known antifungal abilities, species of the genus Trichoderma are of significant interest in modern agriculture. Recent studies have shown that Trichoderma species can induce plant resistance against different phytopathogens. To further extend this line of investigation, we investigate herein the transcriptomic response of grapevine trunk to Vintec®, which is a Trichoderma atroviride SC1-based commercial formulation for biological control of grapevine trunk diseases and which reduces wood colonization. The aim of the study is to understand whether the biocontrol agent Vintec® modifies the trunk response to Phaeoacremonium minimum and Phaeomoniella chlamydospora , which are two esca-associated fungal pathogens. An analysis of transcriptional regulation identifies clusters of co-regulated genes whose transcriptomic reprogramming in response to infection depends on the absence or presence of Vintec®. On one hand, the results show that Vintec® differentially modulates the expression of putative genes involved in hormonal signaling, especially those involved in auxin signaling. On the other hand, most significant gene expression modifications occur among secondary-metabolism-related genes, especially regarding phenylpropanoid metabolism and stilbene biosynthesis. Taken together, these results suggest that the biocontrol agent Vintec® induces wood responses that counteract disease development. [ABSTRACT FROM AUTHOR]

The effective management of grapevine trunk diseases (GTDs) is an ongoing challenge. Hot water treatment (HWT) is an environmentally friendly and economically viable option; however, the short-term effects of HWT on grapevine (Vitis vinifera L.) health and production are not fully understood. The aim of this study was to compare the effects of HWT on plant growth and fungal community structure in nursery stock until plants were completely established in the field. We assessed eleven graft and three rootstock varieties from four local nurseries in a region of Catalonia (NE Spain) where GTDs are a serious threat. After treatment, the plants were left to grow under field conditions for two growing seasons. Metabarcoding of the ITS region was used to study the mycobiomes of plant graft unions and root collars. We also assessed the influence of plant physiological indicators in community composition. Hot water treatment caused lasting changes in GTD communities in both the root collar and graft union that were not always characterized as a reduction of GTD-related fungi. However, HWT reduced the relative abundance of some serious GTD-associated pathogens such as Cadophora luteo-olivacea in graft tissues, and Phaeomoniella chlamydospora and Neofusicoccum parvum in the root collar. Treatment had the greatest influence on the total and GTD-related fungal communities of Chardonnay and Xarel·lo, respectively. Total community variation was driven by treatment and nursery in rootstocks, whereas HWT most significantly affected the GTD community composition in R-110 rootstock. In conclusion, changes in fungal abundance were species-specific and mostly dependent on the plant tissue type; however, HWT did reduce plant biomass accumulation in the short-term. [ABSTRACT FROM AUTHOR]

Rootstocks are the link between the soil and scion in grapevines, can provide tolerance to abiotic and biotic stresses, and regulate yield and grape quality. The vascular system of grapevine rootstocks in nurseries is still an underexplored niche for research, despite its potential for hosting beneficial and pathogenic microorganisms. The purpose of this study was to investigate the changes in the composition of fungal communities in 110 Richter and 41 Berlandieri rootstocks at four stages of the grapevine propagation process. Taxonomic analysis revealed that the fungal community predominantly consisted of phylum Ascomycota in all stages of the propagation process. The alpha-diversity of fungal communities differed among sampling times for both rootstocks, with richness and fungal diversity in the vascular system decreasing through the propagation process. The core microbiome was composed of the genera Cadophora, Cladosporium, Penicillium and Alternaria in both rootstocks, while the pathogenic genus Neofusicoccum was identified as a persistent taxon throughout the propagation process. FUNguild analysis showed that the relative abundance of plant pathogens associated with trunk diseases increased towards the last stage in nurseries. Fungal communities in the vascular system of grapevine rootstocks differed between the different stages of the propagation process in nurseries. Numerous genera associated with potential biocontrol activity and grapevine trunk diseases were identified. Understanding the large diversity of fungi in the rootstock vascular tissue and the interactions between fungal microbiota and grapevine will help to develop sustainable strategies for grapevine protection. [ABSTRACT FROM AUTHOR]