Bioprospection of the bacterial ß-myrcene-biotransforming trait in the rhizosphere.

The biocatalysis of ß-myrcene into value-added compounds, with enhanced organoleptic/therapeutic properties, may be performed by resorting to specialized enzymatic machinery of ß-myrcene-biotransforming bacteria. Few ß-myrcene-biotransforming bacteria have been studied, limiting the diversity of genetic modules/catabolic pathways available for biotechnological research. In our model Pseudomonas sp. strain M1, the ß-myrcene catabolic core-code was identified in a 28-kb genomic island (GI). The lack of close homologs of this ß-myrcene-associated genetic code prompted a bioprospection of cork oak and eucalyptus rhizospheres, from 4 geographic locations in Portugal, to evaluate the environmental diversity and dissemination of the ß-myrcene-biotransforming genetic trait (Myr+). Soil microbiomes were enriched in ß-myrcene-supplemented cultures, from which ß-myrcene-biotransforming bacteria were isolated, belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia classes. From a panel of representative Myr+ isolates that included 7 bacterial genera, the production of ß-myrcene derivatives previously reported in strain M1 was detected in Pseudomonas spp., Cupriavidus sp., Sphingobacterium sp., and Variovorax sp. A comparative genomics analysis against the genome of strain M1 found the M1-GI code in 11 new Pseudomonas genomes. Full nucleotide conservation of the ß-myrcene core-code was observed throughout a 76-kb locus in strain M1 and all 11 Pseudomonas spp., resembling the structure of an integrative and conjugative element (ICE), despite being isolated from different niches. Furthermore, the characterization of isolates not harboring the Myr+-related 76-kb locus suggested that they may biotransform ß-myrcene via alternative catabolic loci, being thereby a novel source of enzymes and biomolecule catalogue for biotechnological exploitation. KEY POINTS: • The isolation of 150 Myr+ bacteria hints the ubiquity of such trait in the rhizosphere. • The Myr+ trait is spread across different bacterial taxonomic classes. • The core-code for the Myr+ trait was detected in a novel ICE, only found in Pseudomonas spp.

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.

In vitro interactions between the ectomycorrhizal Pisolithus tinctorius and the saprotroph Hypholoma fasciculare fungi: morphological aspects and volatile production.

Ectomycorrhizal fungi are crucial for forests sustainability. For Castanea sativa, ectomycorrhizal fungus Pisolithus tinctorius is an important mutualist partner. Saprotrophic fungi Hypholoma fasciculare, although used for biocontrol of Armillaria root disease, it negatively affected the interaction between the P. tinctorius and plant host roots, by compromise the formation of P. tinctorius-C. sativa mycorrhizae. In this work, fungal morphology during inhibition of H. fasciculare against P. tinctorius was elucidated. P. tinctorius growth was strongly affected by H. fasciculare, which was significantly reduced after six days of co-culture and become even more significant through time. During this period, P. tinctorius developed vesicles and calcium oxalate crystals, which were described as mechanisms to stress adaption by fungi. H. fasciculare produced different volatile organic compounds in co-cultures over time and differ between single or in dual-species. H. fasciculare highly produced sesquiterpenes (namely, α-muurolene) and nitrogen-containing compounds, which are recognised as having antimicrobial activity.

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.

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.

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.