Microbial Biological Control of Fungi Associated with Grapevine Trunk Diseases: A Review of Strain Diversity, Modes of Action, and Advantages and Limits of Current Strategies.

Grapevine trunk diseases (GTDs) are currently among the most important health challenges for viticulture in the world. Esca, Botryosphaeria dieback, and Eutypa dieback are the most current GTDs caused by fungi in mature vineyards. Their incidence has increased over the last two decades, mainly after the ban of sodium arsenate, carbendazim, and benomyl in the early 2000s. Since then, considerable efforts have been made to find alternative approaches to manage these diseases and limit their propagation. Biocontrol is a sustainable approach to fight against GTD-associated fungi and several microbiological control agents have been tested against at least one of the pathogens involved in these diseases. In this review, we provide an overview of the pathogens responsible, the various potential biocontrol microorganisms selected and used, and their origins, mechanisms of action, and efficiency in various experiments carried out in vitro, in greenhouses, and/or in vineyards. Lastly, we discuss the advantages and limitations of these approaches to protect grapevines against GTDs, as well as the future perspectives for their improvement.

Deciphering Plant-Induced Responses toward Botrytis cinerea and Plasmopara viticola Attacks in Two Grapevine Cultivars Colonized by the Root Biocontrol Oomycete, Pythium oligandrum.

Two major diseases that affect grapevine leaves and berries are controlled by the oomycete Pythium oligandrum. As the efficacy of biocontrol agents strongly depends on factors such as the trophic behaviors of pathogens and cultivar susceptibility, a two-disease approach was implemented to evaluate the activity of P. oligandrum against Botrytis cinerea (the necrotrophic fungus of gray mold) and Plasmopara viticola (the biotrophic oomycete of downy mildew) on two grapevine cultivars with different susceptibilities to these two pathogens. The results show that grapevine root inoculation with P. oligandrum significantly reduced P. viticola and B. cinerea infection on the leaves of the two cultivars, but with differences. This was observed when the relative expression of 10 genes was measured in response to each pathogen, and could be attributed to their lifestyles, i.e., biotrophic or necrotrophic, which are related to the activation of specific metabolic pathways of the plant. In response to P. viticola infection, genes from the jasmonate and ethylene pathways were mainly induced, whereas for B. cinerea, the genes induced were those of the ethylene-jasmonate pathway. The different levels of defense against B. cinerea and P. viticola could also explain the difference in cultivar susceptibility to these pathogens.

Hundred million years of landscape dynamics from catchment to global scale.

Our capability to reconstruct past landscapes and the processes that shape them underpins our understanding of paleo-Earth. We take advantage of a global-scale landscape evolution model assimilating paleoelevation and paleoclimate reconstructions over the past 100 million years. This model provides continuous quantifications of metrics critical to the understanding of the Earth system, from global physiography to sediment flux and stratigraphic architectures. We reappraise the role played by surface processes in controlling sediment delivery to the oceans and find stable sedimentation rates throughout the Cenozoic with distinct phases of sediment transfer from terrestrial to marine basins. Our simulation provides a tool for identifying inconsistencies in previous interpretations of the geological record as preserved in sedimentary strata, and in available paleoelevation and paleoclimatic reconstructions.

Exploring the Temporal Dynamics of the Fungal Microbiome in Rootstocks, the Lesser-Known Half of the Grapevine Crop.

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.

Sap Flow Disruption in Grapevine Is the Early Signal Predicting the Structural, Functional, and Genetic Responses to Esca Disease.

Fungal species involved in Esca cause the formation of grapevine wood necroses. It results in the deterioration of vascular network transport capacity and the disturbance of the physiological processes, leading to gradual or sudden grapevine death. Herein, for two consecutive growing seasons, a detailed analysis of the structural (wood necrosis and leaf discoloration) and physiological parameters related to the water use of healthy and esca-symptomatic grapevines was conducted. Measurements were carried out on 17-year-old grapevines that expressed, or not, Esca-leaf symptoms in a vineyard of the Bordeaux region (France). Whole-plant transpiration was recorded continuously from pre-veraison to harvest, using noninvasive sap flow sensors. Whole-plant transpiration was systematically about 40-50% lower in Esca-diseased grapevines compared with controls, and this difference can be observed around 2 weeks before the first Esca-foliar symptoms appeared in the vineyard. Unlike grapevine sap flow disruption, structural (e.g., leaf discolorations), functional (e.g., stomatal conductance, photosynthetic activity, phenolic compounds), and genetic (e.g., expression of leaf-targeted genes) plant responses were only significantly impacted by Esca at the onset and during leaf symptoms development. We conclude that sap flow dynamic, which was related to a high level of a white-rot necrosis, provides a useful tool to predict plant disorders due to Esca-grapevine disease.

Bacteria associated with wood tissues of Esca-diseased grapevines: functional diversity and synergy with Fomitiporia mediterranea to degrade wood components.

Fungi are considered to cause grapevine trunk diseases such as esca that result in wood degradation. For instance, the basidiomycete Fomitiporia mediterranea (Fmed) is overabundant in white rot, a key type of wood-necrosis associated with esca. However, many bacteria colonize the grapevine wood too, including the white rot. In this study, we hypothesized that bacteria colonizing grapevine wood interact, possibly synergistically, with Fmed and enhance the fungal ability to degrade wood. We isolated 237 bacterial strains from esca-affected grapevine wood. Most of them belonged to the families Xanthomonadaceae and Pseudomonadaceae. Some bacterial strains that degrade grapevine-wood components such as cellulose and hemicellulose did not inhibit Fmed growth in vitro. We proved that the fungal ability to degrade wood can be strongly influenced by bacteria inhabiting the wood. This was shown with a cellulolytic and xylanolytic strain of the Paenibacillus genus, which displays synergistic interaction with Fmed by enhancing the degradation of wood structures. Genome analysis of this Paenibacillus strain revealed several gene clusters such as those involved in the expression of carbohydrate-active enzymes, xylose utilization and vitamin metabolism. In addition, certain other genetic characteristics of the strain allow it to thrive as an endophyte in grapevine and influence the wood degradation by Fmed. This suggests that there might exist a synergistic interaction between the fungus Fmed and the bacterial strain mentioned above, enhancing grapevine wood degradation. Further step would be to point out its occurrence in mature grapevines to promote esca disease development.

Impacts of Sodium Arsenite on Wood Microbiota of Esca-Diseased Grapevines.

Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions (Alsace, Champagne, Languedoc) were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was Fomitiporia mediterranea (63-94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (-90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.

Field evaluation of biocontrol agents against black-foot and Petri diseases of grapevine.

BACKGROUND: Black-foot and Petri diseases are the main fungal diseases associated with young grapevine decline. Two field experiments were established to evaluate the preventive effect of two potential biocontrol agents (BCAs), that is Streptomyces sp. E1 + R4 and Pythium oligandrum Po37, and three BCA-commercial products containing Trichoderma atroviride SC1, Trichoderma koningii TK7 and Pseudomonas fluorescens + Bacillus atrophaeus on fungal infection in grafted plants and plant growth parameters. RESULTS: The effectiveness of some BCA in reducing the incidence and severity of both diseases was dependent on the plant part analyzed and the plant age. No single BCA application was able to control both diseases. Streptomyces sp. E1 + R4 were able to reduce significantly the infection of the most prevalent black-foot disease fungi while P. oligandrum Po37 and Trichoderma spp. were able to reduce significantly Phaeomoniella chlamydospora and Phaeoacremonium minimum (Petri disease) infection. BCA treatments had no effect on the shoot weight, and root weight was significantly lower in all BCA treatments with respect to the control. CONCLUSIONS: The combination of the disease-suppressive activity of two or more beneficial microbes in a biocontrol preparation is required to prevent infection by black-foot and Petri disease fungi in vineyards.

South China Sea documents the transition from wide continental rift to continental break up.

During extension, the continental lithosphere thins and breaks up, forming either wide or narrow rifts depending on the thermo-mechanical state of the extending lithosphere. Wide continental rifts, which can reach 1,000 km across, have been extensively studied in the North American Cordillera and in the Aegean domain. Yet, the evolutionary process from wide continental rift to continental breakup remains enigmatic due to the lack of seismically resolvable data on the distal passive margin and an absence of onshore natural exposures. Here, we show that Eocene extension across the northern margin of the South China Sea records the transition between a wide continental rift and highly extended (<15 km) continental margin. On the basis of high-resolution seismic data, we document the presence of dome structures, a corrugated and grooved detachment fault, and subdetachment deformation involving crustal-scale nappe folds and magmatic intrusions, which are coeval with supradetachment basins. The thermal and mechanical weakening of this broad continental domain allowed for the formation of metamorphic core complexes, boudinage of the upper crust and exhumation of middle/lower crust through detachment faulting. The structural architecture of the northern South China Sea continental margin is strikingly similar to the broad continental rifts in the North American Cordillera and in the Aegean domain, and reflects the transition from wide rift to continental breakup.

The Biocontrol Root-Oomycete, Pythium Oligandrum, Triggers Grapevine Resistance and Shifts in the Transcriptome of the Trunk Pathogenic Fungus, Phaeomoniella Chlamydospora.

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.

Major changes in grapevine wood microbiota are associated with the onset of esca, a devastating trunk disease.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.

Actinobacteria Associated with Vineyard Soils of Algeria: Classification, Antifungal Potential Against Grapevine Trunk Pathogens and Plant Growth-Promoting Features.

Grapevine trunk diseases (GTDs) are among the most destructive diseases of vineyards worldwide, including Algeria. In the fungal complex involved in GTD symptoms, referred as grapevine trunk-pathogens, Paeomoniella chlamydospora and Phaeoacremonium minimum have a determining infecting role as pioneer fungi. Due to the lack of efficiency of conventional disease management practices, a search for alternative strategies, such as biocontrol, is needed. Taking the approach of looking for biocontrol candidates in the environment surrounding the plant, the present study explored actinobacteria diversity within vineyard soils of six grape-producing regions in Algeria. Based on their 16S rRNA gene sequence, identification and phylogenic analysis were performed on the 40 isolates of actinobacteria obtained. Forty percent of strains were attached to Streptomyces, including two evidenced new species, and 32.5% were affiliated to Saccharothrix. The other less represented genera were Actinoplanes, Nocardia, Nocardiopsis, Lentzea, Nonomuraea, Promicromonospora, Saccharopolyspora and Streptosporangium. Screening based on antagonistic and plant growth promotion (PGP) abilities of the strains showed that 47.5% of the isolates exhibited appreciable antagonistic activities against both Pa. chlamydospora and Pm. minimum, with the two best strains being Streptomyces sp. Ms18 and Streptomyces sp. Sb11. Screening for plant growth promoting properties demonstrated that majority of the strains were able to produce indole acetic acid, siderophores, ammonia, ACC deaminase, cellulase and amylase, and fix N2. Through a PGP-traits-based cluster analysis, the most interesting strains were highlighted. Taking into account both antagonistic and PGP properties, Streptomyces sp Sb11 was selected as the most promising candidate for further evaluations of its efficiency in a GTDs context.

Ecophysiological impacts of Esca, a devastating grapevine trunk disease, on Vitis vinifera L.

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.

Bacterial Shifts in Nutrient Solutions Flowing Through Biofilters Used in Tomato Soilless Culture.

In soilless culture, slow filtration is used to eliminate plant pathogenic microorganisms from nutrient solutions. The present study focused on the characterization and the potential functions of microbial communities colonizing the nutrient solutions recycled on slow filters during a whole cultivation season of 7 months in a tomato growing system. Bacterial microflora colonizing the solutions before and after they flew through the columns were studied. Two filters were amended with Pseudomonas putida (P-filter) or Bacillus cereus strains (B-filter), and a third filter was a control (C-filter). Biological activation of filter unit through bacterial amendment enhanced very significantly filter efficacy against plant potential pathogens Pythium spp. and Fusarium oxysporum. However, numerous bacteria (103-104 CFU/mL) were detected in the effluent solutions. The community-level physiological profiling indicated a temporal shift of bacterial microflora, and the metabolism of nutrient solutions originally oriented towards carbohydrates progressively shifted towards degradation of amino acids and carboxylic acids over the 7-month period of experiment. Single-strand conformation polymorphism fingerprinting profiles showed that a shift between bacterial communities colonizing influent and effluent solutions of slow filters occurred. In comparison with influent, 16S rDNA sequencing revealed that phylotype diversity was low in the effluent of P- and C-filters, but no reduction was observed in the effluent of the B-filter. Suppressive potential of solutions filtered on a natural filter (C-filter), where the proportion of Proteobacteria (α- and ß-) increased, whereas the proportion of uncultured candidate phyla rose in P- and B-filters, is discussed.

Draft Genome Sequence of Diplodia seriata F98.1, a Fungal Species Involved in Grapevine Trunk Diseases.

The ascomycete Diplodia seriata is a causal agent of grapevine trunk diseases. Here, we present the draft genome sequence of D. seriata isolate F98.1 (37.27 Mb, 512 contigs, 112 scaffolds, and 8,087 predicted protein-coding genes).

Draft Genome Sequence of Biocontrol Agent Pythium oligandrum Strain Po37, an Oomycota.

The oomycotaPythium oligandrumPo37 is used as a biocontrol agent of plant diseases. Here, we present the first draft of theP. oligandrumPo37 genome sequence, which comprises 725 scaffolds with a total length of 35.9 Mb and 11,695 predicted protein-coding genes.

Bacteria in a wood fungal disease: characterization of bacterial communities in wood tissues of esca-foliar symptomatic and asymptomatic grapevines.

Esca is a grapevine trunk disease (GTD) associated with different pathogenic fungi inhabiting the woody tissues. Bacteria can also be found in such tissues and they may interact with these fungal colonizers. Although such types of microbial interactions have been observed for wood diseases in many trees, this has never been studied for grapevine. In this study, the bacterial microflora of different vine status (esca-symptomatic and asymptomatic), different anatomical part (trunk and cordon) and different type of tissues (necrotic or not) have been studied. Based on Single Strand Conformation Polymorphism (SSCP) analyses, data showed that (i) specific complexes of bacterial microflora colonize the wood of both necrotic and non-necrotic tissues of esca-foliar symptomatic and asymptomatic vines, and also that (ii) depending on the anatomical part of the plant, cordon or trunk, differences could be observed between the bacterial communities. Such differences were also revealed through the community-level physiological profiling (CLPP) with Biolog Ecoplates(TM). Two hundred seventeen bacterial strains were also isolated from plant samples and then assigned to bacterial species based on the 16S rRNA genes. Although Bacillus sp. and Pantoea agglomerans were the two most commonly isolated species from all kinds of tissues, various other taxa were also isolated. Inoculation of vine cuttings with 14 different bacterial species, and one GTD fungus, Neofusicoccum parvum, showed no impact of these bacteria on the size of the wood necroses caused by N. parvum. This study showed, therefore, that bacterial communities differ according to the anatomical part (trunk or cordon) and/or the type of tissue (necrotic or non-necrotic) of wood of grapevine plants showing external symptoms of esca disease. However, research into bacteria having a role in GTD development needs further studies.

Spreading continents kick-started plate tectonics.

Stresses acting on cold, thick and negatively buoyant oceanic lithosphere are thought to be crucial to the initiation of subduction and the operation of plate tectonics, which characterizes the present-day geodynamics of the Earth. Because the Earth's interior was hotter in the Archaean eon, the oceanic crust may have been thicker, thereby making the oceanic lithosphere more buoyant than at present, and whether subduction and plate tectonics occurred during this time is ambiguous, both in the geological record and in geodynamic models. Here we show that because the oceanic crust was thick and buoyant, early continents may have produced intra-lithospheric gravitational stresses large enough to drive their gravitational spreading, to initiate subduction at their margins and to trigger episodes of subduction. Our model predicts the co-occurrence of deep to progressively shallower mafic volcanics and arc magmatism within continents in a self-consistent geodynamic framework, explaining the enigmatic multimodal volcanism and tectonic record of Archaean cratons. Moreover, our model predicts a petrological stratification and tectonic structure of the sub-continental lithospheric mantle, two predictions that are consistent with xenolith and seismic studies, respectively, and consistent with the existence of a mid-lithospheric seismic discontinuity. The slow gravitational collapse of early continents could have kick-started transient episodes of plate tectonics until, as the Earth's interior cooled and oceanic lithosphere became heavier, plate tectonics became self-sustaining.

Characterization of Pythium oligandrum populations that colonize the rhizosphere of vines from the Bordeaux region.

This study focused on one oomycete, Pythium oligandrum, well-known for its plant protection abilities, which thrives in microbial environment where bacteria and fungal communities are also present. The genetic structures and dynamics of fungal and bacterial communities were studied in three Bordeaux subregions with various types of soil, using single-strand conformation polymorphism. The structure of the fungal communities colonizing the rhizosphere of vines planted in sandy-stony soils was markedly different from that those planted in silty and sandy soils; such differences were not observed for bacteria. In our 2-year experiment, the roots of all the vine samples were also colonized by echinulated oospore Pythium species, with P. oligandrum predominating. Cytochrome oxidase I and tubulin gene sequencings showed that P. oligandrum strains clustered into three groups. Based on elicitin-like genes coding for proteins able to induce plant resistance, six populations were identified. However, none of these groups was assigned to a particular subregion of Bordeaux vineyards, suggesting that these factors do not shape the genetic structure of P. oligandrum populations. Results showed that different types of rootstock and weeding management both influence root colonization by P. oligandrum. These results should prove particularly useful in improving the management of potentially plant-protective microorganisms.

Analyses of the temporal dynamics of fungal communities colonizing the healthy wood tissues of esca leaf-symptomatic and asymptomatic vines.

Esca, a Grapevine Trunk Disease (GTD), is of major concern for viticulture worldwide. Our study compares the fungal communities that inhabit the wood tissues of vines that expressed or not foliar esca-symptoms. The trunk and rootstock tissues were apparently healthy, whether the 10 year-old plants were symptomatic or not. The only difference was in the cordon, which contained white rot, a typical form of esca, in 79% of symptomatic plants. Observations over a period of one year using a fingerprint method, Single Strand Conformation Polymorphism (SSCP), and the ITS-DNA sequencing of cultivable fungi, showed that shifts occurred in the fungal communities colonizing the healthy wood tissues. However, whatever the sampling time, spring, summer, autumn or winter, the fungi colonizing the healthy tissues of asymptomatic or symptomatic plants were not significantly different. Forty-eight genera were isolated, with species of Hypocreaceae and Botryosphaeriaceae being the most abundant species. Diverse fungal assemblages, made up of potentially plant-pathogenic and -protective fungi, colonized these non-necrotic tissues. Some fungi, possibly involved in GTD, inhabited the non-necrotic wood of young plants, but no increase in necrosis areas was observed over the one-year period.