Quantifying Trade-Offs in the Choice of Ribosomal Barcoding Markers for Fungal Amplicon Sequencing: a Case Study on the Grapevine Trunk Mycobiome.

The evolution of sequencing technology and multiplexing has rapidly expanded our ability to characterize fungal diversity in the environment. However, obtaining an unbiased assessment of the fungal community using ribosomal markers remains challenging. Longer amplicons were shown to improve taxonomic resolution and resolve ambiguities by reducing the risk of spurious operational taxonomic units. We examined the implications of barcoding strategies by amplifying and sequencing two ribosomal DNA fragments. We analyzed the performance of the full internal transcribed spacer (ITS) and a longer fragment including also a part of the 28S ribosomal subunit replicated on 60 grapevine trunk core samples. Grapevine trunks harbor highly diverse fungal communities with implications for disease development. Using identical handling, amplification, and sequencing procedures, we obtained higher sequencing depths for the shorter ITS amplicon. Despite the more limited access to polymorphism, the overall diversity in amplified sequence variants was higher for the shorter ITS amplicon. We detected no meaningful bias in the phylogenetic composition due to the amplicon choice across analyzed samples. Despite the increased resolution of the longer ITS-28S amplicon, the higher and more consistent yields of the shorter amplicons produced a clearer resolution of the fungal community of grapevine stem samples. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals. IMPORTANCE Surveying fungal communities is key to our understanding of ecological functions of diverse habitats. Fungal communities can inform about the resilience of agricultural ecosystems, risks to human health, and impacts of pathogens. Community compositions are typically analyzed using ribosomal DNA sequences. Due to technical limitations, most fungal community surveys were based on amplifying a short but highly variable fragment. Advances in sequencing technology enabled the use of longer fragments that can address some limitations of species identification. In this study, we examined the implications of choosing either a short or long ribosomal sequence fragment by replicating the analyses on 60 grapevine wood core samples. Using highly accurate long-read sequencing, we found that the shorter fragment produced substantially higher yields. The shorter fragment also revealed more sequence and species diversity. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals.

Corrigendum to: Impact of crop load on nitrogen uptake and reserve mobilisation in Vitis vinifera.

Nitrogen deficit affects both crop production and composition, particularly in crops requiring an optimal fruit N content for aroma development. The adaptation of cultural practices to improve N use efficiency (NUE) (i.e. N uptake, assimilation and partitioning) is a priority for the sustainable production of high-quality crops. A trial was set on potted grapevines (Vitis vinifera L. cv. Chasselas) to investigate the potential of crop limitation (via bunch thinning) to control plant NUE and ultimately fruit N composition at harvest. A large crop load gradient was imposed by bunch thinning (0.5-2.5 kg m-2) and N traceability in the plant was realised with an isotope-labelling method (10 atom % 15N foliar urea). The results indicate that the mobilisation of root reserves plays a major role in the balance of fruit N content. Fertiliser N uptake and assimilation appeared to be strongly stimulated by high-yielding conditions. Fertilisation largely contributed to fulfilling the high fruit N demand while limiting the mobilisation of root reserves under high yield conditions. Plants were able to modulate root N reserve mobilisation and fertiliser N uptake in function of the crop load, thus maintaining a uniform N concentration in fruits. However, the fruit free amino N profile was modified, which potentially altered the fruit aromas. These findings highlight the great capacity of plants to adapt their N metabolism to constraints, crop thinning in this case. This confirms the possibility of monitoring NUE by adapting cultural practices.

Impact of crop load on nitrogen uptake and reserve mobilisation in Vitis vinifera.

Nitrogen deficit affects both crop production and composition, particularly in crops requiring an optimal fruit N content for aroma development. The adaptation of cultural practices to improve N use efficiency (NUE) (i.e. N uptake, assimilation and partitioning) is a priority for the sustainable production of high-quality crops. A trial was set on potted grapevines (Vitis vinifera L. cv. Chasselas) to investigate the potential of crop limitation (via bunch thinning) to control plant NUE and ultimately fruit N composition at harvest. A large crop load gradient was imposed by bunch thinning (0.5-2.5 kg m-2) and N traceability in the plant was realised with an isotope-labelling method (10 atom % 15N foliar urea). The results indicate that the mobilisation of root reserves plays a major role in the balance of fruit N content. Fertiliser N uptake and assimilation appeared to be strongly stimulated by high-yielding conditions. Fertilisation largely contributed to fulfilling the high fruit N demand while limiting the mobilisation of root reserves under high yield conditions. Plants were able to modulate root N reserve mobilisation and fertiliser N uptake in function of the crop load, thus maintaining a uniform N concentration in fruits. However, the fruit free amino N profile was modified, which potentially altered the fruit aromas. These findings highlight the great capacity of plants to adapt their N metabolism to constraints, crop thinning in this case. This confirms the possibility of monitoring NUE by adapting cultural practices.

Protease inhibitors decrease the resistance of Vitaceae to Plasmopara viticola.

Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll).

Susceptibility to downy mildew in grape clusters: New microscopical and biochemical insights.

Grape clusters of different Vitis genotypes, including Vitis vinifera cvs Chasselas and Merlot, and two interspecific grape varieties, Solaris (cvs. Merzling* x (Saperavi severneyi x Muscat ottonel)) and 2091 (cvs. Gamaret x Bronner), are susceptible or resistant to downy mildew. These cultivars were inoculated with Plasmopara viticola at three developmental stages (BBCH stages 53, 69 and 75). Samples were examined by scanning electron microscopy and the synthesis of stilbenes was measured. Microscopical examinations of pedicels, rachis and calyptras showed important differences in stomatal structures within seasonal development. At BBCH 53, successful infections were observed on all tested cultivars and functional stomata were present, while no infections were observed after this stage. At BBCH 69 and 75, cracks were observed around the stomata and guard cells were unstructured or completely collapsed, leading to closed-like stomata. At BBCH 53, significant stilbene accumulation was quantified in 2091 and Solaris; pterostilbene and δ-viniferin were produced in large amounts. In the susceptible varieties, only piceid and resveratrol were induced. At the other two stages, the concentration of all measured stilbenes was undetectable. The critical roles of seasonal development and stilbenes in the resistance of grape clusters towards downy mildew are discussed.

The effectiveness of stilbenes in resistant Vitaceae: ultrastructural and biochemical events during Plasmopara viticola infection process.

Leaves of different Vitis vinifera L. cultivars, susceptible or resistant to downy mildew, Chasselas, Solaris, IRAC 2091 (cvs. Gamaret x Bronner) and Muscadinia rotundifolia were inoculated with Plasmopara viticola. Samples were then examined by scanning and transmission electron microscopy, by light microscopy and for their ability to synthesise stilbenes. These phytoalexins were strictly analysed at infection sites. In the susceptible Chasselas, P. viticola colonises, at 72h post-infection (hpi), all of the spongy mesophyll with functional haustoria and produces mainly the non toxic piceide. No necrotic zone was observed on Chasselas leaves. The ultrastructural response to downy mildew infection is different in each of the other three resistant grape cultivars. In Solaris, where leaf necrosis are rapidly induced, the infection is restricted to the upper part of the loose spongy mesophyll, and associated with a rapid cell wall disruption and the dispersion of cytoplasmic content along with the production of viniferins. In IRAC 2091, leaf necrosis are quite similar to those observed on Solaris but the infected plant cell, as well as the haustoria, show high electron dense cellular particles without any recognisable organelles, probably related to the effect of the toxic compound pterostilbene, which is synthesised in this grape cultivar. In M. rotundifolia leaf necrosis are much more scarce and smaller than in other cultivars, but pathogen and plant cells are both strongly affected, with concomitant expulsion of cytoplasmic materials through the stomata after P. viticola penetration. In this cultivar, the concentration of all identified stilbenes exceeds 1×10³ µmol mg(-1) FW. The critical role of stilbenes in the resistance of Vitis spp. is discussed.

Control strategies against grey mould (Botrytis cinerea Pers.: Fr) and corresponding fungicide residues in grapes and wines.

This study examines the most effective anti-Botrytis strategies leading to possible lower pesticides residues in wine. To provide wine growers with a number of high-quality solutions for protection against Botrytis for their vineyards while minimizing pesticide residues in the final product, various treatment approaches were tested. A total of 10 strategies with different specific fungicide treatments for controlling Botrytis cinerea were applied to grapes at different growing stages: flowering, bunch closure and colour change. The type of vine chosen was Gamay, as it is very sensitive to Botrytis cinerea. In each experimental plot, disease incidence and severity were assessed at harvest. In addition, pesticide residue analysis was carried out on grapes, musts and wines to monitor residue levels in each treatment and to follow changes at each stage of the wine-making process. A correlation was established between the efficiency of anti-Botrytis fungicide treatment and pesticide residues in wine. Several strategies using various fungicides showed good results in terms of treatment efficiency while minimizing pesticide residues in wine, thus providing interesting alternatives to limit the development of fungal resistance.

Induction of defence mechanisms in grapevine leaves by emodin- and anthraquinone-rich plant extracts and their conferred resistance to downy mildew.

The ability of two plant extracts, Rheum palmatum root extract (RPRE) and Frangula alnus bark extract (FABE), to protect Vitis vinifera leaves from Plasmopara viticola infection was evaluated. These natural products are toxic to the pathogen and induce defence reactions in a susceptible cultivar of V. vinifera (V. vinifera cv. Chasselas), including stilbenic phytoalexin accumulation, enhanced peroxidase (EC 1.11.1.7) activity, and a hypersensitive reaction. Inhibition of the first stage of biotrophic hyphal development of P. Viticola by the two plant extracts was observed. HPLC-DAD-MS analysis showed that these two natural extracts contain many phenolic compounds belonging to the anthraquinone family, such as rhein, frangulin A, emodin, aloe-emodin, chrysophanol, and physcion. Emodin alone is able to impair P. viticola development and to stimulate viniferins and the accumulation of pterostilbene.

Botrytis cinerea Infection of Grape Flowers: Light and Electron Microscopical Studies of Infection Sites.

ABSTRACT Specific floral organs including the calyptra, stigma, and receptacle area of glasshouse-grown grapevines (Vitis vinifera cv. Cabernet Sauvignon) were inoculated with aqueous suspensions of Botrytis cinerea conidia, and the initial steps involved in colonization and infection of the host tissues were studied for several days postinoculation using light microscopy as well as scanning and transmission electron microscopy. Conidia germinated on all floral organs examined and became attached to the host surface within 48 h after inoculation. However, in all cases the vast majority of conidia accumulated in a channel-like gap between the ovary and the calyx that extended in a narrowing fashion into the flower interior where the ovary joined the receptacle. Very few conidial germ tubes were detected in the style following inoculation of the stigma, and no evidence for their growth toward the ovaries could be found. In contrast, hyphae were more abundant in the receptacle area, regardless of the site of inoculation. Tips of the calyx became necrotic and mycelium formed in the gap between ovary and calyx within 72 h following inoculation, providing a major point of colonization and infection. B. cinerea colonized dehiscent calyptras within 72 h of inoculation, providing a potential source of inoculum from calyptras that remained stuck in the cluster. The results suggest that the grape flower's receptacle area is the predominant site of infection for B. cinerea, although a minor portion of infections may also occur through the stigma and style.

Delta-viniferin, a resveratrol dehydrodimer: one of the major stilbenes synthesized by stressed grapevine leaves.

Delta-viniferin is a resveratrol dehydrodimer, an isomer of epsilon-viniferin. This compound has been reported as a molecule produced in vitro by the oxidative dimerization of resveratrol by plant peroxidases or fungal laccases. It was also recently identified in wines and in grape cell cultures. We have now identified this dimer by NMR, high-performance liquid chromatography-diode array detection (HPLC-DAD), and HPLC-mass spectrometry (MS) in grapevine leaves infected by Plasmopara viticola (downy mildew) or UV-C irradiated. Its concentration was higher than that of epsilon-viniferin and constitutes one of the most important phytoalexins derived from resveratrol.

Botrytis cinerea Infection in Grape Flowers: Defense Reaction, Latency, and Disease Expression.

ABSTRACT Inflorescences of field-grown grapevines (Vitis vinifera L. cv. Gamay) were inoculated with a Botrytis cinerea conidia suspension or dried conidia at different stages during bloom in moist weather. Approximately 10% of the conidia germinated within 72 h, resulting in two to three times more latent infections than uninoculated controls in pea-size (7 mm in diameter) berries. In surface-sterilized pea-size berries, latent B. cinerea was present predominantly in the receptacle area. After veraison, latent B. cinerea also was found in the style and, in mature berries, latent colonies were distributed throughout the pulp. Inoculation at full bloom led to the highest disease severity (66%) at harvest, compared with 38% in controls. Stilbene stress metabolites in the flowers were measured by high-performance liquid chromatography. Resveratrol accumulated mainly after pre-bloom and full-bloom inoculation, but did not prevent infection. Piceid levels did not change following inoculation, while epsilon-viniferin was found in necrotic tissues only, and pterostilbene and alphaviniferin were not detected at all. B. cinerea conidia suspensions also were applied to various locations on flowers of pot-grown cvs. Pinot Noir and Chardonnay. Inoculation of the receptacle area, but not that of the stigma and ovary, resulted in latent infections. Stilbene synthesis was similar to the field results, with resveratrol accumulating mainly in the calyptra and receptacle area. Constitutive soluble phenolic compounds (mainly derivatives of quercetin and hydroxy-cinnamic acid) were present at high concentrations in the calyptra but at low levels in the receptacle area. These experiments confirmed bloom as a critical time for B. cinerea infection in grapes and suggest that the most likely site of infection is the receptacle area or cap scar exposed at anthesis. Stilbenes may have a limited role in inhibition of flower infection and latency in susceptible grape cultivars, and epsilon-viniferin may be a by-product rather than a deterrent of infection.

Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR.

Random amplified polymorphic DNA (RAPD) assays were applied on 34 fungal strains isolated from strawberry and other host plants, in order to detect polymorphism to consequently identify and isolate molecular markers specific to Botrytis cinerea. Among the 26 10-mer primers tested, one primer mainly amplified a 750-bp product present in all the B. cinerea strains and absent in the other species and genera examined. This product was cloned and sequenced in order to design a specific 20-mer primer pair, which was tested on the 34 fungal isolates by polymerase chain reaction (PCR). A single 0.7-kb band was amplified in all the 13 strains of B. cinerea isolated from different host plants. Moreover, a 0.6-kb band was amplified in both Botrytis fabae strains. No band was observed for the nine other Botrytis species and 12 fungal genera isolated from strawberry plants. A comparison between the 0.7-kb B. cinerea sequence and the 0.6-kb B. fabae sequence revealed 98% homology and one 122-bp deletion for B. fabae, including an EcoRI site. Hybridization of Southern blots with RAPD and EcoRI-digested DNA confirmed the specificity of the marker. The limit of detection of B. cinerea genomic DNA was approximately 0.2 pg. The PCR procedure was able to amplify the 0.7-kb B. cinerea fragment form mixed samples of DNA as low as 2 pg B. cinerea genomic DNA and 1 microg plant DNA. Thus this PCR-based detection procedure is a powerful tool for diagnosis of B. cinerea in symptomless strawberry plants, and should allow infection and latency sites to be localized in order to improve knowledge of the epidemiology of the pathogen under field conditions.

The role of the extracellular sheath in recognition and attachment of conidia of Discula umbrinella (Berk. & Br.) Morelet to the host surface.

The role of the conidial sheath in the recognition process and in the attachment of conidia of the beech endophyte, Discula umbrinella, to the host surface was investigated. After treatment of conidia with different lectins the adhesion of conidia to the host surface was effectively inhibited. A strong fluorescence was observed after treatment of conidia with TRITC-labelled concanavalin A. Fluorescence with TRITC-labelled wheat germ agglutinin was observed only on spores subjected to enzymatic digestion with proteases. Observations with the transmission electron microscope (TEM), after labelling sections with colloidal gold conjugated lectins, confirmed the presence of mannose and/or glucose in the extracellular sheath. Chitin was present in the conidial ceil wall but not in the extracellular sheath. Enzymatic treatment of the conidial sheath resulted in strongly reduced attachment and changes in the binding of fluorescence-labelled lectins. TEM studies of partially digested conidia revealed that snail enzyme modified only slightly the structure of the sheath, while proteases completely dissolved the fibrillar sheath leaving a comparatively smooth cell wall with no fibrillar structures on its surface. We conclude that a proteinaceous sheath is responsible for the adhesion of Discula umbrinella conidia to the host and that glycoproteins are involved in the recognition and attachment process. This attachment mechanism closely parallels the only other recognition system studied biochemically in endophytes, and is similar to the few analogous cases studied in plant-pathogenic fungi, pointing to the functional relatedness of endophytic and pathogenic interactions.