The influence of bioclimate on soil microbial communities of cork oak.

BACKGROUND: Soil microbiomes are important to maintain soil processes in forests and confer protection to plants against abiotic and biotic stresses. These microbiomes can be affected by environmental changes. In this work, soil microbial communities from different cork oak Portuguese forests under different edaphoclimatic conditions were described by using a metabarcoding strategy targeting ITS2 and 16S barcodes. RESULTS: A total of 11,974 fungal and 12,010 bacterial amplicon sequence variants (ASVs) were obtained, revealing rich and diverse microbial communities associated with different cork oak forests. Bioclimate was described as the major factor influencing variability in these communities (or bioclimates/cork oak forest for fungal community), followed by boron and granulometry. Also, pH explained variation of fungal communities, while C:N ratio contributed to bacterial variation. Fungal and bacterial biomarker genera for specific bioclimates were described. Their co-occurrence network revealed the existence of a complex and delicate balance among microbial communities. CONCLUSIONS: The findings revealed that bacterial communities are more likely to be affected by different edaphoclimatic conditions than fungal communities, also predicting a higher impact of climate change on bacterial communities. The integration of cork oak fungal and bacterial microbiota under different bioclimates could be further explored to provide information about useful interactions for increasing cork oak forest sustainability in a world subject to climate changes.

Olive Fungal Epiphytic Communities Are Affected by Their Maturation Stage.

The phyllosphere comprises the aerial parts of plants and is colonized by a great diversity of microorganisms, either growing inside (as endophytes) or on the surface (as epiphytes) of plant tissues. The factors that structure the diversity of epiphytes and the importance of these microorganisms for host plant protection have been less studied when compared to the case of endophytes. In this work, the epiphytic fungal communities from fruits of the olive tree (olives) in different maturation stages (green and semi-ripened), obtained from different olive orchard managements (integrated and organic production) and from distinct cultivars displaying different susceptibilities to olive anthracnose (Cobrançosa and Madural), are compared by using a metabarcoding approach. We discuss whether such differences in host resistance against anthracnose depend on both the fungal taxa or fungal community composition. A total of 1565 amplicon sequence variants (ASVs) were obtained, mainly belonging to the Ascomycota phylum and Saccharomycetes class. Although significant differences on epiphytic fungal richness were observed among olives obtained in different production systems and maturation stages, these factors in addition to host cultivar did not influence the composition of the epiphytes. Despite these results, a co-inertia analysis showed that Aureobasidium spp. and Sporocadaceae spp. were positively associated with the green olives of the cv. Madural produced under integrated production, while Saccharomycetales spp. (Kluyveromyces, Candida, Kazachstania and Saccharomyces) were positively associated with the semi-ripened olives of the cv. Cobrançosa obtained from organic production. The discriminant power of these fungi, some of them recognized as biocontrol agents, suggest that they might be important in conferring differences on host plant susceptibility to anthracnose.

Phylogenetic analysis and genetic diversity of the xylariaceous ascomycete Biscogniauxia mediterranea from cork oak forests in different bioclimates.

Cork oak is a tree species with ecological importance that contributes to economic and social development in the Mediterranean region. Cork oak decline is a major concern for forest sustainability and has negative impacts on cork oak growth and production. This event has been increasingly reported in the last decades and seems to be related with climate changes. Biscogniauxia mediterranea is an endophytic fungus of healthy cork oak trees that turns into a pathogen in trees weaken by environmental stress. Understanding the drivers of B. mediterranea populations diversity and differentiation is expected to allow a better control of cork oak decline and preserve forest sustainability. Endophyte isolates from different cork oak forests were identified as B. mediterranea and their genetic diversity was evaluated using phylogenetic and microsatellite-primed PCR analyses. Genetic diversity and variability of this fungus was correlated with environmental/phytosanitary conditions present in forests/trees from which isolates were collected. High genetic diversity and variability was found in B. mediterranea populations obtained from different forests, suggesting some degree of isolation by distance. Bioclimate was the most significant effect that explained the genetic variability of B. mediterranea, rather than precipitation or temperature intensities alone or disease symptoms. These findings bring new implications for the changing climate to cork oak forests sustainability, cork production and quality.

Cork Oak Forests Soil Bacteria: Potential for Sustainable Agroforest Production.

Plant growth promoting rhizobacteria (PGPR) are in increasing demand due to their role in promoting sustainable practices, not only in agriculture but also in forestry. Keeping in mind the future application of PGPR for increasing cork oak sustainability, the aim of this study was to find cork oak PGPR isolates with increased nutrient solubilisation traits, able to promote root morphological changes and/or antagonize cork oak bark phytopathogens. Soils from three cork oak forests with distinct bioclimates (humid, semi-humid and semi-arid) were used for isolating bacteria. From the 7634 colony-forming units, 323 bacterial isolates were biochemically assayed for PGPR traits (siderophores production, phosphate solubilizing and organic acids production), and 51 were found to display all these traits. These PGPR were able to induce root morphological changes on Arabidopsis thaliana, like suppression of primary root growth, increase of lateral roots or root hairs formation. However, the most proficient PGPR displayed specific ability in changing a single root morphological trait. This ability was related not only to bacterial genotype, but also with the environment where bacteria thrived and isolation temperature. Bacteria from semi-arid environments (mainly Bacillus megaterium isolates) could hold a promising tool to enhance plant development. Other isolates (Serratia quinivorens or B. cereus) could be further explored for biocontrol purposes.

Bacteria could help ectomycorrhizae establishment under climate variations.

Rhizosphere microbiome is one of the main sources of plant protection against drought. Beneficial symbiotic microorganisms, such as ectomycorrhizal fungi (ECMF) and mycorrhiza helper bacteria (MHB), interact with each other for increasing or maintaining host plant fitness. This mutual support benefits all three partners and comprises a natural system for drought acclimation in plants. Cork oak (Quercus suber L.) tolerance to drought scenarios is widely known, but adaptation to climate changes has been a challenge for forest sustainability protection. In this work, ECMF and MHB communities from cork oak forests were cross-linked and correlated with climates. Cenococcum, Russula and Tuber were the most abundant ECMF capable of interacting with MHB (ECMF~MHB) genera in cork oak stands, while Bacillus, Burkholderia and Streptomyces were the most conspicuous MHB. Integrating all microbial data, two consortia Lactarius/Bacillaceae and Russula/Burkholderaceae have singled out but revealed a negative interaction with each other. Russula/Burkholderaceae might have an important role for cork oak forest sustainability in arid environments, which will be complemented by the lower drought adaptation of competitive Lactarius/Bacillaceae. These microbial consortia could play an essential role on cork oak forest resilience to upcoming climatic changes.

Illuminating Olea europaea L. endophyte fungal community.

A wide array of fungal endophytes is known to inhabit plant tissues and were recently recognized as essential for plant health. A better description of the scarcely known endophyte microbiota in olive tree phyllosphere is the first step for elucidating the microbial interactions that lead to olive disease establishment. In this work, the fungal endophytic community of the phyllosphere of different olive tree cultivars (Cobrançosa, Galega vulgar, Madural, Picual, Verdeal Transmontana) is revealed by using a metabarcoding strategy targeting ITS1 barcode. A total of 460 OTUs were obtained, increasing the broad view of fungal endophytes inhabiting the olive tree phyllosphere, in particular yeast endophytes. New endophytes were persistently found in all cultivar tissues. Different olive tree cultivars depicted distinct endophyte communities. Olive cultivars exhibited dissimilar amounts of fungi with distinct ecological functions, which could explain at least in part their differential susceptibility/tolerance to olive diseases.

Cork Oak Endophytic Fungi as Potential Biocontrol Agents against Biscogniauxia mediterranea and Diplodia corticola.

An increase in cork oak diseases caused by Biscogniauxia mediterranea and Diplodia corticola has been reported in the last decade. Due to the high socio-economic and ecologic importance of this plant species in the Mediterranean Basin, the search for preventive or treatment measures to control these diseases is an urgent need. Fungal endophytes were recovered from cork oak trees with different disease severity levels, using culture-dependent methods. The results showed a higher number of potential pathogens than beneficial fungi such as cork oak endophytes, even in healthy plants. The antagonist potential of a selection of eight cork oak fungal endophytes was tested against B. mediterranea and D. corticola by dual-plate assays. The tested endophytes were more efficient in inhibiting D. corticola than B. mediterranea growth, but Simplicillium aogashimaense, Fimetariella rabenhorstii, Chaetomium sp. and Alternaria alternata revealed a high potential to inhibit the growth of both. Simplicillium aogashimaense caused macroscopic and microscopic mycelial/hyphal deformations and presented promising results in controlling both phytopathogens' growth in vitro. The evaluation of the antagonistic potential of non-volatile and volatile compounds also revealed that A. alternata compounds could be further explored for inhibiting both pathogens. These findings provide valuable knowledge that can be further explored in in vivo assays to find a suitable biocontrol agent for these cork oak diseases.

Ectomycorrhizal fungal diversity and community structure associated with cork oak in different landscapes.

Cork oak (Quercus suber L.) forests play an important ecological and economic role. Ectomycorrhizal fungi (ECMF) are key components for the sustainability and functioning of these ecosystems. The community structure and composition of ECMF associated with Q. suber in different landscapes of distinct Mediterranean bioclimate regions have not previously been compared. In this work, soil samples from cork oak forests residing in different bioclimates (arid, semi-arid, sub-humid, and humid) were collected and surveyed for ectomycorrhizal (ECM) root tips. A global analysis performed on 3565 ECM root tips revealed that the ECMF community is highly enriched in Russula, Tomentella, and Cenoccocum, which correspond to the ECMF genera that mainly contribute to community differences. The ECMF communities from the rainiest and the driest cork oak forests were distinct, with soils from the rainiest climates being more heterogeneous than those from the driest climates. The analyses of several abiotic factors on the ECMF communities revealed that bioclimate, precipitation, soil texture, and forest management strongly influenced ECMF structure. Shifts in ECMF with different hyphal exploration types were also detected among forests, with precipitation, forest system, and soil texture being the main drivers controlling their composition. Understanding the effects of environmental factors on the structuring of ECM communities could be the first step for promoting the sustainability of this threatened ecosystem.

Soil DNA pyrosequencing and fruitbody surveys reveal contrasting diversity for various fungal ecological guilds in chestnut orchards.

Fungal diversity in Mediterranean forest soils is poorly documented, particularly when considering saprobic and pathogenic organisms. Next-generation sequencing (NGS) methods applied to soil fungi provide the opportunity to unveil the most inconspicuous functional guilds (e.g. saprobes) and life forms (e.g. Corticiaceae) of this tremendous diversity. We used fruitbody surveys over 2 years and soil 454 metabarcoding in Castanea sativa orchards to evaluate respectively the reproductive (fruitbodies) and vegetative (mycelia) parts of fungal communities in three 100-year-old stands. Analysis of 839 fruitbodies and 210 291 ITS1 reads revealed high fungal diversity, mainly shown by belowground analysis, with high (dominant) abundance of mycorrhizal fruitbodies and reads. Both methods displayed contrasted composition and structure of fungal communities, with Basidio- and Ascomycetes dominating above- and belowground, respectively. For the two dominant fungal guilds (i.e. ectomycorrhizal and saprobic), diversity above- and belowground overlapped weakly. This study is the first assessment of the complementarity of fruitbody surveys and NGS for analysing fungal diversity in Mediterranean ecosystems and shows that belowground methods still need to be completed by fruiting diversity to provide a comprehensive overview of the different fungal guilds. The results shed light on chestnut soil biodiversity and question the spatial distribution and synergies among fungal guilds.

RNA-Seq and Gene Network Analysis Uncover Activation of an ABA-Dependent Signalosome During the Cork Oak Root Response to Drought.

Quercus suber (cork oak) is a West Mediterranean species of key economic interest, being extensively explored for its ability to generate cork. Like other Mediterranean plants, Q. suber is significantly threatened by climatic changes, imposing the need to quickly understand its physiological and molecular adaptability to drought stress imposition. In the present report, we uncovered the differential transcriptome of Q. suber roots exposed to long-term drought, using an RNA-Seq approach. 454-sequencing reads were used to de novo assemble a reference transcriptome, and mapping of reads allowed the identification of 546 differentially expressed unigenes. These were enriched in both effector genes (e.g., LEA, chaperones, transporters) as well as regulatory genes, including transcription factors (TFs) belonging to various different classes, and genes associated with protein turnover. To further extend functional characterization, we identified the orthologs of differentially expressed unigenes in the model species Arabidopsis thaliana, which then allowed us to perform in silico functional inference, including gene network analysis for protein function, protein subcellular localization and gene co-expression, and in silico enrichment analysis for TFs and cis-elements. Results indicated the existence of extensive transcriptional regulatory events, including activation of ABA-responsive genes and ABF-dependent signaling. We were then able to establish that a core ABA-signaling pathway involving PP2C-SnRK2-ABF components was induced in stressed Q. suber roots, identifying a key mechanism in this species' response to drought.

Phenotypic analysis of the Arabidopsis heat stress response during germination and early seedling development.

BACKGROUND: Phenotypic characterization of Arabidopsis thaliana gain- and loss-of-function mutants is a delicate and meticulous task that often involves the analysis of multiple parameters. Arabidopsis heat tolerance has been evaluated based on direct assessments that include seed germination, seedling survival, hypocotyl and root elongation, or indirect measurements such as chlorophyll content or ion leakage. RESULTS: In an attempt to simplify the detection of heat stress-associated phenotypes, a collection of protocols for analysis of seed germination and seedling survival to heat treatment is proposed. Temperatures and lengths of heat treatments were combined into several heat tolerance assays, to be used as a primary approach for the search and characterization of basal and acquired heat tolerance-associated phenotypes at early developmental stages. The usefulness of this methodology was illustrated through the characterization of heat-related phenotypes in different Arabidopsis ecotypes as well as in gain- and loss-of-function mutants. CONCLUSIONS: The use of standardized experimental protocols designed to detect temperature-related phenotypes is proposed. The suggested plate-based assays provide an appropriate framework of experimental conditions for detection of variability amongst natural accessions or mutants lines. Functional studies could be facilitated by using this inexpensive and undemanding approach.

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

A new effective assay to detect antimicrobial activity of filamentous fungi.

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.

A strategy for the identification of new abiotic stress determinants in Arabidopsis using web-based data mining and reverse genetics.

Since the sequencing of the Arabidopsis thaliana genome in 2000, plant researchers have faced the complex challenge of assigning function to thousands of genes. Functional discovery by in silico prediction or homology search resolved a significant number of genes, but only a minor part has been experimentally validated. Arabidopsis entry into the post-genomic era signified a massive increase in high-throughput approaches to functional discovery, which have since become available through publicly-available web-based resources. The present work focuses on an easy and straightforward strategy that couples data-mining to reverse genetics principles, to allow for the identification of new abiotic stress determinant genes. The strategy explores systematic microarray-based transcriptomics experiments, involving Arabidopsis abiotic stress responses. An overview of the most significant resources and databases for functional discovery in Arabidopsis is presented. The successful application of the outlined strategy is illustrated by the identification of a new abiotic stress determinant gene, HRR, which displays a heat-stress-related phenotype after a loss-of-function reverse genetics approach.

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells.

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius.

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H(2)O(2) and O(2) (.-) during the early stages of fungal contact. Three peaks of H(2)O(2) production were detected, the first two coinciding with O(2) (.-) bursts. The first H(2)O(2) production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H(2)O(2) scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H(2)O(2) production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H(2)O(2) results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.

Induction of phenolic compounds in Hypericum perforatum L. cells by Colletotrichum gloeosporioides elicitation.

Changes in phenolic metabolism after elicitation with Colletotrichum gloeosporioides (CG) has been studied in Hypericum perforatum L. (HP) cell suspension cultures. Soluble phenolics were analysed by HPLC-DAD and HPLC-DAD-MS/MS. HP cultures elicited with the CG elicitor showed a significant increase in xanthone accumulation. Xanthone accumulation increased twelve fold when the cells were primed with methyl-jasmonate (MeJ) or salicylic acid (SA), before elicitation. HP cultures exposed only to MeJ produced a set of flavonoids, the flavones which represent a substantial part (approx. 40%) of the total flavonoids accumulated in these cells. The possible importance of xanthones as a component of defence mechanism of HP against biotic stress is discussed.

Utilization and transport of glucose in Olea Europaea cell suspensions.

Cell suspensions of Olea europaea var. Galega Vulgar grown in batch culture with 0.5% (w/v) glucose were able to transport D-[(14)C]glucose according to Michaelis-Menten kinetics associated with a first-order kinetics. The monosaccharide carrier exhibited high affinity (K(m) approximately 50 micro M) and was able to transport D-glucose, D-fructose, D-galactose, D-xylose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not D-arabinose, D-mannitol or L-glucose. D-[(14)C]glucose uptake was associated with proton uptake, which also followed Michaelis-Menten kinetics. The transport of 3-O-methyl-D-glucose was accumulative (40-fold, at pH 5.0) and the protonophore carbonyl cyanide m-chlorophenylhydrazone strongly inhibited sugar accumulation. The results were consistent with the involvement of a monosaccharide: proton symporter with a stoichiometry of 1 : 1. When cells were grown with 3% (w/v) glucose, the uptake of D-[(14)C]glucose followed first-order kinetics and monosaccharide:proton symporter activity was not detected. The value obtained for the permeability coefficient of hexoses in O. europaea cells supported the hypothesis that the first-order kinetics observed in 0.5% and 3% sugar-grown cells was produced exclusively by passive diffusion of the sugar. The results indicate that in O. europaea cells sugar levels have a regulatory effect on sugar transport, because the activity for monosaccharide transport was repressed by high sugar concentrations.