Fungicides and the grapevine wood mycobiome: a case study on Tracheomycotic Ascomycete Phaemoniella chlamydospora reveals potential for two novel control strategies

Original ResearchPhaeomoniella chlamydospora is a tracheomycotic fungus that colonizes the xylem of grapevines (Vitis vinifera L.), causing wood discoloration, brown wood streaking, gummosis, and wood necrosis, which negatively affect the overall health, productivity, and life span of vines. Current control strategies to prevent or cope with P. chlamydospora infections are frequently ineffective. Moreover, it is unclear how fungicides commonly applied in vineyards against downy and powdery mildew agents affect the wood mycobiome, including wood pathogens such as P. chlamydospora. In this study, we used next-generation sequencing to assess the effects of foliar spray of grapevines with inorganic (copper oxychloride and sulfur), synthetic (penconazole and fosetyl-aluminum), and natural (Blad) fungicides currently used against the downy and powdery mildews. The subjects of our investigation were (i) the resident wood mycobiome, (ii) the early colonization by a consortium of fungal wood endophytes (ACEA1), (iii) the wood colonization success of P. chlamydospora, and (iv) the in planta interaction between P. chlamydospora and ACEA1, under greenhouse conditions, in rooted grapevine cuttings of cv. Cabernet Sauvignon. The data obtained suggest that the resident mycobiome is affected by different fungicide treatments. In addition, the early colonization success of the endophytes composing ACEA1 varied in response to fungicides, with relative abundances of some taxa being overrepresented or underrepresented when compared with the control. The wood colonization by P. chlamydospora comported significant changes in the mycobiome composition, and in addition, it was greatly affected by the foliar spray with Blad, which decreased the relative abundance of this pathogen 12-fold (4.9%) when compared with the control (60.7%) and other treatments. The presence of the pathogen also decreased considerably when co-inoculated into the plant with ACEA1, reaching relative abundances between 13.9% and 2.0%, depending on the fungicide treatment applied. This study shows that fungicides sprayed to prevent infections of powdery and downy mildews have an control strategies to fight P. chlamydospora, namely, the foliar spray with Blad and the use of ACEA1. Further studies to confirm these results are requiredinfo:eu-repo/semantics/publishedVersio

DNA-based detection of grapevine trunk-disease pathogens from environmental spore samples.

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

Fungal species associated with grapevine trunk diseases in Washington wine grapes and California table grapes, with novelties in the genera Cadophora, Cytospora, and Sporocadus.

Grapevine trunk diseases cause serious economic losses to grape growers worldwide. The identification of the causal fungi is critical to implementing appropriate management strategies. Through a culture-based approach, we identified the fungal species composition associated with symptomatic grapevines from wine grapes in southeastern Washington and table grapes in the southern San Joaquin Valley of California, two regions with contrasting winter climates. Species were confirmed through molecular identification, sequencing two to six gene regions per isolate. Multilocus phylogenetic analyses were used to identify novel species. We identified 36 species from 112 isolates, with a combination of species that are new to science, are known causal fungi of grapevine trunk diseases, or are known causal fungi of diseases of other woody plants. The novel species Cadophora columbiana, Cytospora macropycnidia, Cytospora yakimana, and Sporocadus incarnatus are formally described and introduced, six species are newly reported from North America, and grape is reported as a new host for three species. Six species were shared between the two regions: Cytospora viticola, Diatrype stigma, Diplodia seriata, Kalmusia variispora, Phaeoacremonium minimum, and Phaeomoniella chlamydospora. Dominating the fungal community in Washington wine grape vineyards were species in the fungal families Diatrypaceae, Cytosporaceae and Sporocadaceae, whereas in California table grape vineyards, the dominant species were in the families Diatrypaceae, Togniniaceae, Phaeomoniellaceae and Hymenochaetaceae. Pathogenicity tests demonstrated that 10 isolates caused wood discoloration similar to symptomatic wood from which they were originally isolated. Growth rates at temperatures from 5 to 35°C of 10 isolates per region, suggest that adaptation to local climate might explain their distribution

Pest risk assessment of Diaporthe vaccinii for the EU territory

24As requested by the European Commission, the EFSA Panel on Plant Health (PLH) Panel assessed therisk ofDiaporthe vacciniiin the EU, focusing on entry, establishment, spread and impacts on cultivatedand wildVacciniumspecies, the principal hosts being American and European cranberry and blueberry.Several outbreaks occurred in the EU since 1956, but most were eradicated except in Latvia. ThePanel considered entry via fruits and plants for planting. The risk of establishment from discardedinfected berries is much lower than from infected plants for planting, of which, potted plants andcuttings pose the greatest risk, while plug plants, derived from tissue culture and grown in pest freestructures, pose a low risk. Nine per cent of the EU is highly suitable for establishment of thepathogen, mostly in the SE and NE. Following establishment, the pathogen could spread naturally overshort range, and by human assistance over long range. Calculations with an integrated model forentry, establishment and spread, indicate that with current regulations, over a period of 5 years, a fewhundred cultivatedVacciniumplants and several thousandVacciniumplants in natural ecosystemswould contract the disease. The associated loss of commercial production is small, less than one tonneof berries per year. On natural vegetation, the median impact after 5 years was estimated to benegligible affecting a negligible proportion of the naturalVacciniumpopulation (29108). However,the uncertainty of this estimate was high, due to uncertainty about the rate of spread; in a worst-casescenario (99th percentile), almost 1% of plants in natural areas would become infected. Completederegulation (scenario A1) was predicted to increase the impact substantially, especially in naturalareas, while additional measures (scenario A2) would effectively eliminate the entry of infected plantsfor planting, further reducing the impacts below the current situation.openopenJeger, Michael; Bragard, Claude; Caffier, David; Candresse, Thierry; Chatzivassiliou, Elisavet; Dehnen‐Schmutz, Katharina; Gilioli, Gianni; Grégoire, Jean‐Claude; Jaques Miret, Josep Anton; MacLeod, Alan; Navarro, Maria Navajas; Niere, Björn; Parnell, Stephen; Potting, Roel; Rafoss, Trond; Rossi, Vittorio; Urek, Gregor; Van Der Werf, Wopke; West, Jonathan; Winter, Stephan; Gardi, Ciro; Mosbach‐Schulz, Olaf; Koufakis, Ioannis; Van Bruggen, ArienaJeger, Michael; Bragard, Claude; Caffier, David; Candresse, Thierry; Chatzivassiliou, Elisavet; Dehnen‐schmutz, Katharina; Gilioli, Gianni; Grégoire, Jean‐claude; Jaques Miret, Josep Anton; Macleod, Alan; Navarro, Maria Navajas; Niere, Björn; Parnell, Stephen; Potting, Roel; Rafoss, Trond; Rossi, Vittorio; Urek, Gregor; Van Der Werf, Wopke; West, Jonathan; Winter, Stephan; Gardi, Ciro; Mosbach‐schulz, Olaf; Koufakis, Ioannis; Van Bruggen, Arien

Differential physiological responses of resistant and susceptible grape cultivars to Eutypa dieback

Eutypa lata is a fungal pathogen of grapevine that causes widespread economic damage and threatens vineyard longevity worldwide. This study was initiated to further understanding of how grapevines resist E. lata infections, using an integrated approach combining inoculation assays in the greenhouse with physiological and biochemical measurements. Resistant 'Zinfandel' and susceptible 'Syrah' grapevines were subjected to control and inoculation treatments and assessed for gas exchange, water status, photosynthetic biochemistry, hydraulic conductivity, wood chemistry, and fungal spread (lesion length). Infection reduced leaf photochemical function and gas exchange in Zinfandel and increased these variables in Syrah (p<0.05). Infection produced shorter lesions in Zinfandel (p<0.05), suggesting that downregulating gas exchange limited pathogen spread by reducing the carbon supply to the pathogen or fungal movement in the transpiration stream. Neither cultivar upregulated wood defense compounds in response to infection, but proanthocyanidin and catechin levels were constitutively higher in Zinfandel and stilbenoid and flavonoid contents were constitutively higher in Syrah (p<0.05). Altogether, this study is the first to show that, counterintuitively, downregulating physiological function in response to infection improves long-term resistance to E. lata. Screening responses in photochemical function or gas exchange could provide a high-throughput alternative to measuring lesion lengths in assessing resistance

Preventative Management of Trunk Diseases in Table Grape Vitis vinifera Autumn King

Background and goals In California’s southern San Joaquin Valley, applications of pruning-wound protectants may lower the incidence of grapevine trunk diseases. Rain is important for spore dispersal of the causal fungi, which infect pruning wounds. Despite low annual rainfall in this region, pruning wounds are at risk of infection, based on reports of symptoms and pathogen detection in vineyards, and from controlled inoculations demonstrating susceptibility of Vitis vinifera table grape cultivars. Therefore, our goal was to evaluate protectants for pruning wounds, testing two commercial fungicides, thiophanate-methyl (Topsin; UPL) and pyraclostrobin + boscalid (Pristine; BASF), for three years in an 8-yr-old Autumn King vineyard. Methods and key findings For three years, liquid formulations of fungicides were applied alone or with Pentra-bark (Quest Products Corp.), an adjuvant to enhance pesticide penetration of the bark of woody crops. After spray application, pruning wounds were inoculated with spores of the fungi that cause the trunk diseases Botryosphaeria dieback (Neofusicoccum parvum), Esca (Phaeoacremonium minimum), and Eutypa dieback (Eutypa lata). Both thiophanate-methyl and pyraclostrobin + boscalid were effective against N. parvum, with greater efficacy using Pentra-bark in two study years. Both fungicides were moderately effective against E. lata, with greater efficacy using Pentra-bark in one study year. Neither fungicide, alone or with Pentrabark, was effective against P. minimum. Conclusions and significance One application of thiophanate-methyl or pyraclostrobin + boscalid to pruning wounds (after pruning and before rain) may prevent Botryosphaeria and Eutypa dieback, but not Esca. Greater fungicide efficacy with Pentra-bark against E. lata and N. parvum in some years warrants further testing, for example, at higher concentrations

Characterization of the wood mycobiome of Vitis vinifera in a vineyard affected by Esca. Spatial distribution of fungal communities and their putative relation with leaf symptoms

Original ResearchEsca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina R next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (Debaryomyces, Trematosphaeria, Biatriospora, Lopadostoma, and Malassezia) and numerous hitherto unreported species. Esca-associated fungi Phaeomoniella chlamydospora and Fomitiporia sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., Eutypa spp., Inonotus hispidus). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting ‘tiger stripes’ symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of escainfo:eu-repo/semantics/publishedVersio

Cadophora margaritata sp. nov. and other fungi associated with the longhorn beetles Anoplophora glabripennis and Saperda carcharias in Finland

Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of insects would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn beetle (Saperda carcharias) and the recently introduced Asian longhorn beetle (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from Populus tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora-Mollisia species complex. A commonly encountered fungus was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood.Peer reviewe

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens

BackgroundTrunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.ResultsThe integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution.ConclusionsThis study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management