Grapevine Fungal Pathogens Research Articles

Characterization of the RNA Mycovirome Associated with Grapevine Fungal Pathogens: Analysis of Mycovirus Distribution and Their Genetic Variability within a Collection of Botryosphaeriaceae Isolates.

Botryosphaeriaceae are fungi involved in the decay of various woody species, including the grapevine, leading to significant production losses. This fungal family is largely ubiquitous, and seven species of Botryosphaeriaceae have been identified in French vineyards, with variable levels of aggressiveness, both in vitro and in planta. Mycoviruses can impact the life traits of their fungal hosts, including aggressiveness, and are one of the factors influencing fungal pathogenicity. In this study, the RNA mycovirome of fifteen Botryosphaeriaceae isolates was characterized through the high-throughput sequencing of double-stranded RNA preparations from the respective samples. Eight mycoviruses were detected, including three potential novel species in the Narnaviridae family, as well as in the proposed Mycobunyaviridae and Fusagraviridae families. A large collection of Botryosphaeriaceae isolates was screened using RT-PCR assays specific for 20 Botryosphaeriaceae-infecting mycoviruses. Among the mycoviruses detected, some appeared to be specialists within a single host species, while others infected isolates belonging to multiple Botryosphaeriaceae species. This screening allowed us to conclude that one-third of the Botryosphaeriaceae isolates were infected by at least one mycovirus, and a significant proportion of isolates (43.5%) were found to be coinfected by several viruses, with very complex RNA mycoviromes for some N. parvum isolates.

The potency and effectiveness of six essential oils in controlling grapevine trunk diseases in Morocco

The objective of this study was to evaluate the antifungal activity (AA) of essential oils (EOs) extracted from Eucalyptus globulus Labill. (Eucalyptus), Citrus limonum (L.) Burm. (Lemon), Cinnamomum zeylanicum Blume. (Cinnamon), Lavandula latifolia aspic (Lavender), Rosmarinus officinalis L. (Rosemary), and Mentha spicata L. (Peppermint) against four pathogenic fungi responsible for grapevine trunk diseases in Moroccan vineyards. The EOs with the highest in vitro efficacy were selected for further testing on autoclaved grape wood assays against Diplodia mutila, Neoscytalidium novaehollandiae, Trichothecium roseum, and Neopestalotiopsis vitis. The major chemical constituents of the tested EOs were determined through GasChromatography–Mass Spectrometry (GC-MS) analysis, and their relative percentages were recorded. All tested EOs demonstrated significant antifungal activity against the four pathogens, as evidenced by in vitro results. Redundancy analysis (RDA) confirmed the efficacy of the tested EOs against grapevine fungal pathogens. The efficacy of each EO varied depending on the pathogen being targeted. For example, lavender had the highest efficacy across EOs (81.37%) against T. roseum, while peppermint was the most effective in inhibiting D. mutila (47.45%). Cinnamon was the most effective EO in controlling N. vitis (73.33%), while rosemary had the best effect in controlling N. novaehollandiae (56.79%), with the most effective EC50 value (8.66 µL/ml) being attributed to the rosemary EO. The in vitro results were further confirmed through autoclaved grape wood assays, with inhibition of proliferation ranging between 93.14% and 66.32%. These findings represent a novel application of EOs extracted from various medicinal plants to control fungal diseases in grapevine plantations.

In Vitro Evaluation of Some Endophytic Bacillus to Potentially Inhibit Grape and Grapevine Fungal Pathogens

Romania has a long history of grapevine culturing and winemaking. However, like any agricultural sector, viticulture faces devastating biological threats. Fungi responsible for grapevine trunk diseases (GTDs) and grape spoilage lead to considerable yield losses and a decline in grapevine quality. In the actual context, many countries, including Romania, have reoriented their approaches to minimize chemical inputs, which have been proven to be toxic and to have negative impacts on the environment, and to replace them with sustainable biocontrol strategies for the wine-growing sector. Within biocontrol strategies, Bacillus spp. is a well-known plant-protective bacteria with antifungal properties. Within this paper, six endophytic bacteria from various plant sources were studied. The bacterial strains were identified as B. pumilus, B. subtilis, and B. velezensis by sequencing their 16S rDNA region. Regardless of the in vitro test methods (using living bacterial cells, bacterial-cell-free supernatant (CFS), and volatile active compounds (VOCs)), B. velezensis strains revealed strong and broad antifungal activity against grape and grapevine fungal pathogens such as Aspergillus spp., Botrytis cinerea, Penicillium expansum, Diplodia seriata, Eutypa lata, Fusarium spp., Clonostachys rosea, Neofusicoccum parvum, and Stereum hirsutum. The functional antifungal genes encoding for difficidin, fengycin, iturins, macrolactin, and mycosubtilin were molecularly detected, which could support the proven antifungal activity of the endophytic strains. Lytic enzymes involved in fungal growth inhibition, such as chitinase, cellulase, and proteases, were also revealed to be produced by some of these bacterial strains. Various other in vitro tests, such as phosphate and phytate solubilization, phytohormone synthesis, the production of enzymes involved in the polyamine biosynthetic pathway, and pH as well as temperature tolerance tests were carried out to reveal the plant-beneficial potential of these bacterial strains. These results revealed that the B. velezensis strains, especially BAHs1, are the most suitable endophytes for grapevine biologic control, which could lead to the future development of sustainable management strategies.

Identification of grape berry indigenous epiphytic yeasts with <i>in vitro</i> and <i>in vivo</i> antagonistic activity towards pathogenic fungi

During their lifespan, grapes are exposed to conditions that can endanger plant health, including fluctuating climate and infection by pests and insects. In particular, attacks by filamentous fungi represent huge annual losses for grape farmers. Present concerns about rising antimicrobial resistance, fungicide prohibitions and shifting consumer tastes are driving the search for less harmful ways of preventing damage to grapevine by pathogenic fungi. In this study, the antagonist activity of yeasts isolated from grape berry surface microbiota was assessed by in vitro and in vivo assays on an assortment of grapevine fungal pathogens, including Botrytis cinerea and Aspergillus niger. The results show that W. anomalus, A. pullulans, C. intermedia, R. glutinis, Z. meyerae and H. takashimae were able to inhibit by 75 to 100 % mycelia growth and sporulation of the filamentous fungi in vitro. Strains of M. pulcherrima and S. bacillaris also showed 50 to 75 % efficacy in reducing mould growth. The strains were further assessed on incised leaves of 2-month-old grapevines for their ability to reduce disease incidence by the filamentous fungi. W. anomalus, A. pullulans, M. pulcherrima, C. intermedia and R. glutinis significantly reduced infection symptoms in the leaves, indicating their potential as effective biocontrol yeasts in the vineyard.

Isolation and Identification of Wild Yeast from Malaysian Grapevine and Evaluation of Their Potential Antimicrobial Activity against Grapevine Fungal Pathogens.

Pathogenic fungi belonging to the genera Botrytis, Phaeomoniella, Fusarium, Alternaria and Aspergillus are responsible for vines diseases that affect the growth, grapevine yield and organoleptic quality. Among innovative strategies for in-field plant disease control, one of the most promising is represented by biocontrol agents, including wild epiphytic yeast strains of grapevine berries. Twenty wild yeast, isolated and molecularly identified from three different Malaysian regions (Perlis, Perak and Pahang), were evaluated in a preliminary screening test on agar to select isolates with inhibition against Botrytis cinerea. On the basis of the results, nine yeasts belonging to genera Hanseniaspora, Starmerella, Metschnikowia, Candida were selected and then tested against five grape berry pathogens: Aspergillus carbonarius, Aspergillus ochraceus, Fusarium oxysporum, Alternaria alternata and Phaeomoniella chlamydospora. Starmerella bacillaris FE08.05 and Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19 showed the highest effect on inhibiting mycelial growth, which ranged between 15.1 and 4.3 mm for the inhibition ring. The quantitative analysis of the volatile organic compound profiles highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. The results of this study suggest that antagonist yeasts, potentially effective for the biological control of pathogenic moulds, can be found among the epiphytic microbiota associated with grape berries.

ChemInform Abstract: Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea

Abstractisolation and structure elucidation of lasiolactols A (I) and B (II)

Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea.

A strain of Lasiodiplodia mediterranea, a fungus associated with grapevine decline in Sicily, produced several metabolites in liquid medium. Two new dimeric γ-lactols, lasiolactols A and B (1 and 2), were characterized as (2S*,3S*,4R*,5R*,2'S*,3'S*,4'R*,5'R*)- and (2R*,3S*,4R*,5R*,2'R*,3'S*,4'R*,5'R*)-(5-(4-hydroxymethyl-3,5-dimethyl-tetrahydro-furan-2-yloxy)-2,4-dimethyl-tetrahydro-furan-3-yl]-methanols by IR, 1D- and 2D-NMR, and HR-ESI-MS. Other four metabolites were identified as botryosphaeriodiplodin, (5R)-5-hydroxylasiodiplodin, (-)-(1R,2R)-jasmonic acid, and (-)-(3S,4R,5R)-4-hydroxymethyl-3,5-dimethyldihydro-2-furanone (3 - 6, resp.). The absolute configuration (R) at hydroxylated secondary C-atom C(7) was also established for compound 3. The compounds 1 - 3, 5, and 6, tested for their phytotoxic activities to grapevine cv. Inzolia leaves at different concentrations (0.125, 0.25, 0.5, and 1 mg/ml) were phytotoxic and compound 5 showed the highest toxicity. All metabolites did not show in vitro antifungal activity against four plant pathogens.

Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity

Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride, by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride. Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%–70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina, Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the mycoparasitism effect of the extracellular enzymes.

Multilocus ISSR markers reveal two major genetic groups in Spanish and South African populations of the grapevine fungal pathogen Cadophora luteo-olivacea.

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.

Antifungal compounds from Vitis vinifera canes against major grapevine fungal pathogens

Antifungal compounds from Vitis vinifera canes against major grapevine fungal pathogens

Antifungal peptides produced by Bacillus amyloliquefaciens AG1 active against grapevine fungal pathogens

Antifungal metabolites produced by Bacillus amyloliquefaciens AG1, previously isolated from wood of grapevine with “esca syndrome”, were studied. The crude protein extract (CPE) obtained from culture supernatant fluid by precipitation with ammonium sulfate was assayed against many grapevine fungal pathogens. B. amyloliquefaciens strain AG1 showed a broad spectrum of antifungal activity, inhibiting mycelial growth in vitro of all tested fungal microorganisms. The metabolites contained in CPE were heat stable and remained active over a wide pH range (2–10). Their activity was not affected by proteolytic or glycolytic enzymes. Tricine-SDS-polyacrylamide gel electrophoresis revealed a single band within the range of 2,510–3,480 Da, that showed inhibitory activity when used in the antifungal assay. Mass spectrometry analysis of this band allowed the substances involved in antibiosis to be identified as two tryptic peptides that correspond to the N-terminal sequence of subtilisin BPN’. These results suggest a potential role of B. amyloliquefaciens AG1 as a biocontrol agent.

Antifungal effects of iron sulfate on grapevine fungal pathogens

The present study aimed to determine the most efficient experimental conditions of iron sulfate use leading to optimal inhibition in the development of fungal pathogens. Assays have been focused on fungal species inducing severe grapevine diseases. FeSO 4 directly inhibited the in vitro mycelial growth of Botrytis cinerea, Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Diplodia seriata, and Neofusicoccum parvum with variable efficiency in the range of 0.5–10 mM. The development was always completely inhibited at 20 mM. This inhibitory effect was greatly increased at acidic pH values. The anionic moiety of the molecule was of importance since bromide, chloride and sulfate were highly active, whereas acetate and oxalate showed a small effect. Electron microscope observations on E. lata and B. cinerea showed that a treatment with FeSO 4 induced dramatic changes in the hyphal organization leading to cell death. No toxicity was observed on grapevine leaves following repeated FeSO 4 sprays in the antifungal concentration range. Therefore, FeSO 4 may be proposed to effectively replace the long-term pollutant use of CuSO 4 as an antifungal agent, with the additional advantage of iron being an important plant micronutrient.

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.

Genetic structure of the grapevine fungal pathogen Phaeomoniella chlamydospora in southeastern Australia and southern France

In this study, 18 microsatellite loci were used to examine the genetic structure and mode of reproduction of Phaeomoniella chlamydospora, the fungus that causes Petri disease of grapevine and is involved in another grapevine disease, esca. A total of 60 southeastern Australian isolates were tested and compared with 64 isolates from southern France. The French population possessed relatively high genotypic diversity (G = 29·2) whilst the Australian population showed low genotypic diversity (G = 11·1), consistent with a limited founder population. Haplotypes from four different grapevine cultivars were not significantly differentiated (Φpt values effectively zero). Likewise, genetic differentiation of haplotypes from different regions within each country was not significant, although small but significant genetic differentiation (9%) was identified between Australia and France. Based on bootstrapped cluster analysis, there did not appear to be any genetic groups within the overall sample of isolates. Significant linkage disequilibrium identified in both countries, together with overrepresented, widespread identical haplotypes, indicated limited genetic recombination and a largely clonal structure consistent with the absence of an observed sexual cycle in this species.

AFLP markers reveal two genetic groups in the French population of the grapevine fungal pathogen Phaeomoniella chlamydospora

Phaeomoniella chlamydospora, (Chaetothyriales, Herpotrichiellaceae) is one of the main causal agents of Petri disease and esca on grapevines. We have used AFLP markers to study the population genetic structure of 74 isolates collected at different spatial scales: 56 isolates originated from vines with esca disease sampled from four French vineyards (Poitou-Charentes, Aquitaine, Languedoc-Roussillon, Alsace); 18 isolates were collected from a single plot (Aquitaine vineyard). Significant linkage disequilibrium indicated that P. chlamydospora populations are not panmictic, whereas the level of haplotypic diversity observed, 72 single multilocus haplotypes identified in total among the 74 isolates analysed, suggest that reproduction in this species may not be strictly clonal. Clustering analyses suggests the presence of two genetically differentiated but sympatric clusters of isolates. The level of differentiation between the two clusters is high (FST = 0.23) and significant at 13 out of the 21 loci analyzed. The most plausible explanation for this pattern of admixture is the coexistence in P. chlamydospora French populations of two predominant clonal lineages. Finally, the low level of spatial genetic differentiation in this study is consistent with the spread of this fungus through the transport of infected plant material by human activities.

Differential Analysis of Mycoalexins in Confrontation Zones of Grapevine Fungal Pathogens by Ultrahigh Pressure Liquid Chromatography/Time-of-Flight Mass Spectrometry and Capillary Nuclear Magnetic Resonance

An original approach was developed for the chemical and biological investigation of zone lines formed by the confrontation of fungi growing in confined spaces. Two wood-decaying fungi involved in esca disease, Eutypa lata and Botryosphaeria obtusa, were grown in Petri dishes. Metabolic profiles of pure fungal strains and confrontation zones were differentially analyzed by ultrahigh pressure liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC/TOFMS). Selected metabolites induced by the confrontation were isolated and characterized by capillary NMR (CapNMR) at the submilligram level. Fungitoxic and phytotoxic assays were applied to the crude extracts and isolated molecules. While the extracts of pure strains were inactive, the extract from confrontation zones exhibited significant activities. A very strongly induced compound, O-methylmellein, may explain these toxic properties. The developed approach demonstrates the use of fungal confrontations as an original source of bioactive molecules and gives new insights into the study of esca disease.

Development of microsatellite markers for the grapevine fungal pathogen Phaeomoniella chlamydospora

Twenty polymorphic microsatellite markers from microsatellite-enriched genomic DNA of the grapevine fungal pathogen, Phaeomoniella chlamydospora, were developed and characterized. The markers were used to genotype isolates from Australia and from Europe/Eurasia. The number of alleles per locus ranged from two to 11. Gene diversity per locus ranged from 0.08 to 0.63 among Australian isolates, and from 0.2 to 0.77 among isolates from Europe/Eurasia, demonstrating the suitability of these markers for population genetic studies of P. chlamydospora. Eighteen of the 20 markers also amplified a product in the closely related Phaeoacremonium aleophilum.

Secondary metabolites of the grapevine pathogen Eutypa lata inhibit mitochondrial respiration, based on a model bioassay using the yeast Saccharomyces cerevisiae.

Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2- isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2- isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2Delta, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1Delta, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H(2)O(2)) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.