Project description:The worldwide increase in grapevine trunk diseases, mainly esca, represents a major threat for vineyard sustainability. Biocontrol of a pioneer fungus of esca, Phaeomoniella chlamydospora, was investigated here by deciphering the tripartite interaction between this trunk-esca pathogen, grapevine and the biocontrol-oomycete, Pythium oligandrum. When P. oligandrum colonizes grapevine roots, it was observed that the wood necroses caused by P. chlamydospora were significantly reduced. Transcriptomic analyses of plant and fungus responses were performed to determine the molecular events occurring, with the aim to relate P.chlamydospora degradation of wood to gene expression modulation. Following P. oligandrum-root colonization, major transcriptomic changes occurred both, in the grapevine-defense system and in the P. chlamydospore-virulence factors. Grapevine-defense was enhanced in response to P. chlamydospora attacks, with P. oligandrum acting as a plant-systemic resistance inducer, promoting jasmonic/ethylene signaling pathways and grapevine priming. P. chlamydospora pathogenicity genes, such as those related to secondary metabolite biosynthesis, carbohydrate-active enzymes and transcription regulators, were also affected in their expression. Shifts in grapevine responses and key-fungal functions were associated with the reduction of P. chlamydospora wood necroses. This study provides evidence of wood fungal pathogen transcriptional changes induced by a root biocontrol agent, P. oligandrum, in which there is no contact between the two microorganisms.
Project description:Grapevine trunk diseases (GTDs) are a serious problem of grapevines worldwide. The microbiota of the grapevine endosphere comprises prokaryotic and eukaryotic endophytes, which may form varied relationships with the host plant from symbiotic to pathogenic. To explore the interaction between grapevine endophytic bacteria and GTDs, the endomicrobiome associated with grapevine wood was characterized using next-generation Illumina sequencing. Wood samples were collected from grapevine trunks with and without external symptoms of GTD (cankers) from two vineyards in the Hunter Valley and Hilltops, NSW, Australia and metagenomic characterization of the endophytic community was conducted using the 16S rRNA gene (341F/806R) and ITS (1F/2R) sequences. Among the important GTD pathogens, Phaeomoniella, Phaeoacremonium, Diplodia and Cryptovalsa species were found to be abundant in both symptomatic and asymptomatic grapevines from both vineyards. Eutypa lata and Neofusicoccum parvum, two important GTD pathogens, were detected in low numbers in Hilltops and the Hunter Valley, respectively. Interestingly, Pseudomonas dominated the bacterial community in canker-free grapevine tissues in both locations, comprising 56-74% of the total bacterial population. In contrast, the Pseudomonas population in grapevines with cankers was significantly lower, representing 29 and 2% of the bacterial community in Hilltops and the Hunter Valley, respectively. The presence of Pseudomonas in healthy grapevine tissues indicates its ability to colonize and survive in the grapevine. The potential of Pseudomonas spp. as biocontrol agents against GTD pathogens was also explored. Dual culture tests with isolated fluorescent Pseudomonas against mycelial discs of nine Botryosphaeria dieback, three Eutypa dieback, and two Esca/Petri disease pathogens, revealed antagonistic activity for 10 Pseudomonas strains. These results suggest the potential of Pseudomonas species from grapevine wood to be used as biocontrol agents to manage certain GTD pathogens.
Project description:BackgroundGrapevine 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.ResultsThe 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.ConclusionsThe 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.
Project description: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.
Project description:Endophytic strains of Pseudomonas were isolated from grapevine tissues and exhibited antagonistic activity against several grapevine trunk disease pathogens. The draft genome sequences of the four strains revealed the presence of putative gene clusters that may impart biocontrol activity against plant pathogens.
Project description: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.
Project description:In California vineyards, spore dispersal of fungi that cause grapevine trunk diseases Botryosphaeria dieback and Eutypa dieback occurs with winter rains. Spores infect through pruning wounds made to the woody structure of the vine in winter. Better timing of preventative practices that minimize infection may benefit from routine spore-trapping, which could pinpoint site-specific time frames of spore dispersal. To speed pathogen detection from environmental spore samples, we identified species-specific PCR primers and protocols. Then we compared the traditional culture-based method versus our new DNA-based method.•PCR primers for Botryosphaeria-dieback pathogen Neofusicoccum parvum and Eutypa-dieback pathogen Eutypa lata were confirmed species-specific, through extensive testing of related species (in families Botryosphaeriaceae and Diatrypaceae, respectively), other trunk-disease pathogens, and saprophytic fungi that sporulate in vineyards.•Consistent detection of N. parvum was achieved from spore suspensions used fresh or stored at -20°C, whereas consistent detection of E. lata was achieved only with a new spore-lysis method, using zirconia/silica beads in a FastPrep homogenizer (MP Biomedicals; Solon, Ohio, USA), and only from spore suspensions used fresh. Freezing E. lata spores at -20°C made detection inconsistent.•From environmental samples, spores of E. lata were detected only via PCR, whereas spores of N. parvum were detected both via PCR and in culture.
Project description:Grapevine fungal trunk diseases (GTDs) have become a serious problem for grapevines worldwide. Nursery vines infected during the propagation process are considered one of the main ways of dissemination of GTD pathogens. In this study, we examined the status of GTDs in grapevine planting material, from rootstocks and scion mother cuttings to grafted rooted vines ready to plant, according to the local nursery propagation process. During 2018-2019, internal symptoms of GTDs were examined in 2400 propagation materials and fungal isolations were carried out from a subsample of 1026 selected materials. Our results revealed that nursery grapevine plants produced in Uruguay have a high incidence of GTDs, regardless of the scion/rootstock combination. Typical brown to black streaks and sectorial wood necrosis were observed in materials on all propagation stages, with a markedly increasing incidence throughout the nursery process, reaching almost 100% in grafted rooted vines ready to plant. Botryosphaeria dieback, Petri disease and black-foot disease were the main GTDs found. The results showed that Botryosphaeria dieback and Petri disease pathogens infect materials from the early stages of the process, with a marked increase towards the end of the plant production process, whereas black-foot disease pathogens were found exclusively in vines ready to plant. Diaporthe dieback pathogens were also detected in materials in all stages but in a low proportion (less than 10% of infected material). Based on single locus analysis, the 180 isolates selected were placed into eight genera and 89% identified within 22 fungal species associated with GTDs, with Phaeoacremonium oleae and Diaporthe terebinthifolii as new records on grapevine worldwide. Our results have concluded that locally produced vines are one of the main ways of dissemination of GTD pathogens and showed that a nursery sanitation programme is required to reduce the incidence of these diseases.
Project description:Esca is a Grapevine Trunk Disease (GTD) caused by a broad range of taxonomically unrelated fungal pathogens. These attack grapevine wood tissues inducing necroses even in the conductive vascular tissues, thus affecting the vine physiology and potentially leading to plant death. However, the influence of Esca on leaf and whole-plant water transport disruption remains poorly understood. In this paper, a detailed analysis of xylem-related physiological parameters in grapevines that expressed Esca-foliar symptoms was carried out. The experiments were conducted in a vineyard in the Bordeaux region (France) on cv. Cabernet-Sauvignon (Vitis vinifera L.) grapevines, which were monitored for Esca-foliar symptoms over a two-year period. Heat dissipation sap-flow sensors were installed during the summer on grapevines having expressed or not Esca-foliar symptoms. Leaf water potential, stomatal conductance and leaf transpiration were also measured. Physiological monitoring showed that sap flow density and whole-plant transpiration of Esca-infected grapevines decreased significantly a week before the first foliar symptoms appeared. When atmospheric water demand (Vapour Pressure Deficit, VPD) was the highest, both parameters tended to be about twice as low in symptomatic grapevines as in asymptomatic ones. Sap flow density data at the maximum transpiration-time, was systematically 29-30% lower in Esca-infected grapevines compared to control plants before or after the appearance of Esca-foliar symptoms. This trend was observed whatever the temperatures and VPD values measured. In Esca-diseased plants, larger amounts of necrotic wood, mainly white rot, were found in the trunk and cordon of symptomatic grapevines compared to healthy ones, suggesting necroses have an influence in reducing the whole-plant hydraulic capacity. This study reveals that the use of physiological monitoring methods, together with the visual monitoring of foliar symptoms, could prove useful in providing accurate measurements of Esca disease severity.
Project description:BackgroundDNA 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.ResultsWe 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.ConclusionsOverall, 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.