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Forest ecosystems worldwide currently face worrying episodes of forest decline, which have boosted weakening and mortality of the trees. In the Mediterranean region, especially in the southeast Iberian Peninsula, Pinus sylvestris forests are severely affected by this phenomenon, and it has been commonly attributed to drought events. Remarkably, the role of root microbiota on pine decline has been overlooked and remains unclear. We therefore used metabarcoding to identify the belowground microbial communities of decline-affected and unaffected pine trees. Taxonomic composition of bacterial and fungal rhizosphere communities, and fungal populations dwelling in root endosphere showed different profiles depending on the health status of the trees. The root endosphere of asymptomatic trees was as strongly dominated by 'Candidatus Phytoplasma pini' as the root of decline-affected pines, accounting for >99 % of the total bacterial sequences in some samples. Notwithstanding, the titer of this phytopathogen was four-fold higher in symptomatic trees than in symptomless ones. Furthermore, the microbiota inhabiting the root endosphere of decline-affected trees assembled into a less complex and more modularized network. Thus, the observed changes in the microbial communities could be a cause or a consequence of forest decline phenomenon. Moreover, 'Ca. Phytoplasma pini' is positively correlated to Pinus sylvestris decline events, either as the primary cause of pine decline or as an opportunistic pathogen exacerbating the process once the tree has been weaken by other factors.

Background: The complex and co-evolved interplay between plants and their microbiota is crucial for the health and fitness of the plant holobiont. However, the microbiota of the seeds is still relatively unexplored and no studies have been conducted with olive trees so far. In this study, we aimed to characterize the bacterial, fungal and archaeal communities present in seeds of ten olive genotypes growing in the same orchard through amplicon sequencing to test whether the olive genotype is a major driver in shaping the seed microbial community, and to identify the origin of the latter. Therefore, we have developed a methodology for obtaining samples from the olive seed’s endosphere under sterile conditions. Results: A diverse microbiota was uncovered in olive seeds, the plant genotype being an important factor influencing the structure and composition of the microbial communities. The most abundant bacterial phylum was Actinobacteria, accounting for an average relative abundance of 41%. At genus level, Streptomyces stood out because of its potential influence on community structure. Within the fungal community, Basidiomycota and Ascomycota were the most abundant phyla, including the genera Malassezia, Cladosporium, and Mycosphaerella. The shared microbiome was composed of four bacterial (Stenotrophomonas, Streptomyces, Promicromonospora and Acidipropionibacterium) and three fungal (Malassezia, Cladosporium and Mycosphaerella) genera. Furthermore, a comparison between findings obtained here and earlier results from the root endosphere of the same trees indicated that genera such as Streptomyces and Malassezia were present in both olive compartments. Conclusions: This study provides the first insights into the composition of the olive seed microbiota. The highly abundant fungal genus Malassezia and the bacterial genus Streptomyces reflect a unique signature of the olive seed microbiota. The genotype clearly shaped the composition of the seed’s microbial community, al

Increasing temperatures along with severe droughts are factors that may jeopardize the survival of the forests in the Mediterranean basin. In this region, Pinus pinaster is a common conifer species, that has been used as a model species in evolutionary studies due to its adaptive response to changing environments. Although its drought tolerance mechanisms are already known, knowledge about the dynamics of its root microbiota is still scarce. We aimed to decipher the structural (bacterial abundance), compositional, functional and associative changes of the P. pinaster rhizosphere bacterial communities in spring and summer, at DNA and RNA level (environmental DNA, live and dead cells, and those synthesizing proteins). A fundamental aspect of root microbiome-based approaches is to guarantee the correct origin of the samples. Thus, we assessed the genotype of host needles and roots from which rhizosphere samples were obtained. For more than 50% of the selected trees, genotype discrepancies were found and in three cases the plant species could not be determined. Rhizosphere bacterial communities were homogeneous with respect to diversity and structural levels regardless of the host genotype in both seasons. Nonetheless, significant changes were seen in the taxonomic profiles depending on the season. Seasonal changes were also evident in the bacterial co-occurrence patterns, both in DNA and RNA libraries. While spring communities switched to more complex networks, summer populations resulted in more compartmentalized networks, suggesting that these communities were facing a disturbance. These results may mirror the future status of bacterial communities in a context of climate change. A keystone hub was ascribed to the genus Phenylobacterium in the functional network calculated for summer. Overall, it is important to validate the origin and identity of plant samples in any plant-microbiota study so that more reliable ecological analyses are performed.

The Donana ˜ wetlands comprise an emblematic Mediterranean landscape protected as a UNESCO World Heritage Site. Some parts of these wetlands have been transformed into intensive rice cultivation areas, which are currently the most productive rice-growing areas in Europe. We examined the bacterial communities in these domesticated soils as they are key for plant health and productivity and have a strong influence on biochemical cycles. To identify the bacteria, we used metabarcoding analysis coupled with metabolic predictions and cooccurrence networks. This analysis was performed in the bulk and rhizosphere soils during different stages in the growing season. These soil compartments had a greater effect on the bacterial communities than the plant phenological stages. The diversity and richness of the bacterial population inhabiting the rhizosphere was much lower than that in the bulk soil, comprising taxa that were significantly more represented in this soil compartment, such as bacteria from the genus Hydrogenophaga, three genera from the order Rhizobiales, and unclassified genera from the families Desulfocapsaceae and Actinobacteria. Rhizosphere co-occurrence networks revealed a high number of negative connections, indicating unstable bacterial communities that may be highly influenced by biotic and abiotic factors. Rhizosphere networks mostly rely on two taxa belonging to the phyla Proteobacteria and Cyanobacteria, which are the predicted network hubs in this soil compartment. The bulk soil conserved high bacterial diversity and richness that was stable throughout the growth period of rice. Anaerobic bacteria from genera Marmoricola, the uncultured Gemmatimonadota bacteria SDR1034 terrestrial group, Anaerolinea, and the sulphur oxidizer, Thiobacillus were highly represented. This analysis provides valuable information for understanding bacterial diversity in the rhizosphere of rice cultivated in this region, which is critical for enhancing plant growth and product

Quercus pyrenaica is a woody species of high landscape value, however, its forests show an advanced state of degradation in the Iberian Peninsula. Afforestation typically has low success, thus, it is necessary to improve the fitness of oaks plantlets to be transplanted, for instance, by inoculating beneficial microorganisms. In adding microorganisms to ecosystems, there must be balanced efficacy with potential effects on native microbial communities. We addressed changes in diversity, richness, composition and co-occurrence networks of prokaryotic communities in the rhizosphere of inoculated and control trees outplanted to three different sites located in the Sierra Nevada National and Natural Park (Spain). After 18 months in wild conditions, we did not detect changes due to the inoculation in the richness, diversity and structure in none of the sites. However, we observed an increase in the complexity of the co-occurrence networks in two experimental areas. Modularization of the networks changed as a result of the inoculation, although the sense of the change depended on the site. Although it was impossible to unravel the effect of bacterial inoculation, our results highlighted that inoculation alters the association of rhizosphere bacteria without entailing other changes, so networks should be analysed prior to inoculating the plantlets.

Long-term monitoring of microbial communities in the rhizosphere of post-fire forests is currently one of the key knowledge gaps. Knowing the time scale of the effects is indispensable to aiding post-fire recovery in vulnerable woodlands, including holm oak forests, that are subjected to a Mediterranean climate, as is the case with forests that are found in protected areas such as the Sierra Nevada National and Natural Park in southeastern Spain. We took rhizosphere soil samples from burned and unburned holm oak trees approximately 3, 6, and 9 years after the 2005 fire that devastated almost 3500 ha in southeastern Spain. We observed that the prokaryotic communities are recovering but have not yet reached the conditions observed in the unburned forest. A common denominator between this fire and other fires is the long-term persistence of three ecosystem recovery biomarkers—specifically, higher proportions of the genera Arthrobacter, Blastococcus, and Massilia in soil microbial communities after a forest fire. These pyrophilous microbes possess remarkable resilience against adverse conditions, exhibiting traits such as xerotolerance, nitrogen mineralization, degradation of aromatic compounds, and copiotrophy in favorable conditions. Furthermore, these biomarkers thrive in alkaline environments, which persist over the long term following forest fires. The relative abundance of these biomarkers showed a decreasing trend over time, but they were still far from the values of the control condition. In conclusion, a decade does not seem to be enough for the complete recovery of the prokaryotic communities in this Mediterranean ecosystem.

The Doñana wetlands comprise an emblematic Mediterranean landscape protected as a UNESCO World Heritage Site. Some parts of these wetlands have been transformed into intensive rice cultivation areas, which are currently the most productive rice-growing areas in Europe. We examined the bacterial communities in these domesticated soils as they are key for plant health and productivity and have a strong influence on biochemical cycles. To identify the bacteria, we used metabarcoding analysis coupled with metabolic predictions and co-occurrence networks. This analysis was performed in the bulk and rhizosphere soils during different stages in the growing season. These soil compartments had a greater effect on the bacterial communities than the plant phenological stages. The diversity and richness of the bacterial population inhabiting the rhizosphere was much lower than that in the bulk soil, comprising taxa that were significantly more represented in this soil compartment, such as bacteria from the genus Hydrogenophaga, three genera from the order Rhizobiales, and unclassified genera from the families Desulfocapsaceae and Actinobacteria. Rhizosphere co-occurrence networks revealed a high number of negative connections, indicating unstable bacterial communities that may be highly influenced by biotic and abiotic factors. Rhizosphere networks mostly rely on two taxa belonging to the phyla Proteobacteria and Cyanobacteria, which are the predicted network hubs in this soil compartment. The bulk soil conserved high bacterial diversity and richness that was stable throughout the growth period of rice. Anaerobic bacteria from genera Marmoricola, the uncultured Gemmatimonadota bacteria SDR1034 terrestrial group, Anaerolinea, and the sulphur oxidizer, Thiobacillus were highly represented. This analysis provides valuable information for understanding bacterial diversity in the rhizosphere of rice cultivated in this region, which is critical for enhancing plant growth and producti

The Mediterranean basin is drastically affected by intense and frequent droughts, which jeopardize the diversity and survival of its forest, for example, Pinus pinaster forests. The dynamics of the bacterial communities inhabiting the rhizosphere of Pinus pinaster and other plants from a pine dominated forest under contrasting hydric conditions was monitored. The forest was located in Sierra de Oria (southeast Spain), and it was mainly composed by P. pinaster, P. halepensis, woody shrub species and herbaceous plants. 18 trees visually belonging to P. pinaster located along the perimeter and across the forest were selected for the analysis. All the trees were separated at least 50 m each other. Although all of them belonged to P. pinaster morphologically according to visual identification, the genotyping of the roots confirmed that they corresponded to P. pinaster, P. halepensis, and other plant species different from genus Pinus, although in the last case it was not possible to identify the plant species. At a distance less than 50 cm from the trunk, the litter and topsoil were removed, and the soil closely attached to non-suberified roots (rhizosphere soil) was collected (depth of 5-25 cm). Sampling was carried out in two seasons with contrasting temperature and rainfall patterns: on July 18, 2017 (summer) and April 24, 2018 (spring). After rhizosphere soil DNA and RNA extraction (and cDNA synthesis), a metabarcoding approach was followed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene and its derived transcripts by Illumina MiSeq platform. Sequencing reads were bioinformatically processed; specifically, they were filtered, trimmed, clustered into ASV (Amplicon Sequence Variants), and taxonomically identified. As a result, a total of 1,123,209 and 1,089,359 quality sequences were obtained from DNA and RNA-derived libraries, which resulted in 5,241 and 5,231 ASVs, respectively. Total communities (DNA) were mainly dominated by phyla Proteobacteria

Bacterial members that comprise the rhizosphere microbiota of Pinus pinaster trees located in a forest subjected to severe drought conditions, The project SUMHAL aims at implementing a strategy for biodiversity conservation in the Mediterranean area by setting a technologically efficient and scientifically robust system. The project combines fieldwork and virtual research environments for the recording, storing, analysis, and dissemination of the conservation status and threats of biodiversity in Andalusia (Southern Spain).

RNA-based high-throughput sequencing is a valuable tool in the discernment of the implication of metabolically active bacteria during composting. In this study, “alperujo” composting was used as microbial model for the elucidation of structure-function relationships with physicochemical transformation of the organic matter. DNA and RNA, subsequently retrotranscribed into cDNA, were isolated at the mesophilic, thermophilic and maturation phases. 16S rRNA gene was amplified by quantitative PCR (qPCR) and Illumina MiSeq platform to assess bacterial abundance and diversity, respectively. The results showed that the abundance of active bacteria assessed by qPCR was maximum at thermophilic phase, which confirm it as the most active stage of the process. Concerning diversity, Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the main phyla presented in composts. Concomitantly, three different behaviours were observed for bacterial dynamics: some genera decreased during the whole process meanwhile others proliferated only at thermophilic or maturation phase. Statistical correlation between physicochemical transformations of the organic matter and bacterial diversity revealed bacterial specialisation. This result indicated that specific groups of bacteria were only involved in the organic matter degradation during bio-oxidative phase or humification at maturation. Metabolic functions predictions confirmed that active bacteria were mainly involved in carbon (C) and nitrogen (N) cycles transformations, and pathogen reduction.

It is well-known that different plant species, and even plant varieties, promote different assemblages of the microbial communities associated with them. Here, we investigate how microbial communities (bacteria and fungi) undergo changes within the influence of woody plants (two olive cultivars, one tolerant and another susceptible to the soilborne fungal pathogen Verticillium dahliae, plus wild Holm oak) grown in the same soil but with different management (agricultural versus native). By the use of metabarcoding sequencing we determined that the native Holm oak trees rhizosphere bacterial communities were different from its bulk soil, with differences in some genera like Gp4, Gp6 and Solirubrobacter. Moreover, the agricultural management used in the olive orchard led to belowground microbiota differences with respect to the natural conditions both in bulk soils and rhizospheres. Indeed, Gemmatimonas and Fusarium were more abundant in olive orchard soils. However, agricultural management removed the differences in the microbial communities between the two olive cultivars, and these differences were minor respect to the olive bulk soil. According to our results, and at least under the agronomical conditions here examined, the composition and structure of the rhizospheric microbial communities do not seem to play a major role in olive tolerance to V. dahliae.

Melojo oak (Quercus pyrenaica Willd.) is a key tree species of Mediterranean forests; however, these forests show an advanced stage of deterioration in the Iberian Peninsula. Plant-associated microorganisms play an essential role improving their host’s fitness, hence, a better understanding of oak rhizospheric microbiome, especially of those active members, could be the first step towards microbiome-based approaches for oak-forest improvement. Here we reported, for the first time, the diversity of total (DNA-based) and potentially active (RNA-based) bacterial communities of different melojo-oak forest formations through pyrosequencing of 16S rRNA gene amplicons. We found that potentially active bacterial communities were as rich and diverse as total bacterial communities, but different in terms of relative abundance patterns in some of the studied areas. Both core microbiomes were dominated by a relatively small percentage of OTUs, most of which showed positive correlation between both libraries. However, the uncoupling between abundance (rDNA) and potential activity (rRNA) for some taxa suggests that the most abundant taxa are not always the most active, and that low-abundance OTUs may have a strong influence on oak’s rhizospheric ecology. Thus, measurement of rRNA:rDNA ratio could be helpful in identifying major players for the development of bacterial bioinoculants.

Due to the ability of soil bacteria to solubilize minerals, fix N and mobilize nutrients entrapped in the organic matter, their role in nutrient turnover and plant fitness is of high relevance in forest ecosystems. Although several authors have already studied the organic matter decomposing enzymes produced by soil and plant root-interacting bacteria, most of the works did not account for the activity of cell wall-attached enzymes. Therefore, the enzyme deployment strategy of three bacterial collections (genera Luteibacter, Pseudomonas and Arthrobacter) associated with Quercus spp. roots was investigated by exploring both cell-bound and freely-released hydrolytic enzymes. We also studied the potential of these bacterial collections to produce enzymes involved in the transformation of plant and fungal biomass. Remarkably, the cell-associated enzymes accounted for the vast majority of the total activity detected among Luteibacter strains, suggesting that they could have developed a strategy to maintain the decomposition products in their vicinity, and therefore to reduce the diffusional losses of the products. The spectrum of the enzymes synthesized and the titres of activity were diverse among the three bacterial genera. While cellulolytic and hemicellulolytic enzymes were rather common among Luteibacter and Pseudomonas strains and less detected in Arthrobacter collection, the activity of lipase was widespread among all the tested strains. Our results indicate that a large fraction of the extracellular enzymatic activity is due to cell wall-attached enzymes for some bacteria, and that Quercus spp. root bacteria could contribute at different levels to carbon (C), phosphorus (P) and nitrogen (N) cycles.

We report here the draft genome sequences of two Arthrobacter strains isolated from a holm oak forest affected by wildfire. Both strains were shown to act as plant growth promoters, with AFG20 being a member of the most abundant group found in this soil and AFG7.2 being the strain with the highest indole-3-acetic acid production level.

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.

[EN] Long-term monitoring of microbial communities in the rhizosphere of post-fire forests is currently one of the key knowledge gaps. Knowing the time scale of the effects is indispensable to aiding post-fire recovery in vulnerable woodlands, including holm oak forests, that are subjected to a Mediterranean climate, as is the case with forests that are found in protected areas such as the Sierra Nevada National and Natural Park in southeastern Spain. We took rhizosphere soil samples from burned and unburned holm oak trees approximately 3, 6, and 9 years after the 2005 fire that devastated almost 3500 ha in southeastern Spain. We observed that the prokaryotic communities are recovering but have not yet reached the conditions observed in the unburned forest. A common denominator between this fire and other fires is the long-term persistence of three ecosystem recovery biomarkers—specifically, higher proportions of the genera Arthrobacter, Blastococcus, and Massilia in soil microbial communities after a forest fire. These pyrophilous microbes possess remarkable resilience against adverse conditions, exhibiting traits such as xerotolerance, nitrogen mineralization, degradation of aromatic compounds, and copiotrophy in favorable conditions. Furthermore, these biomarkers thrive in alkaline environments, which persist over the long term following forest fires. The relative abundance of these biomarkers showed a decreasing trend over time, but they were still far from the values of the control condition. In conclusion, a decade does not seem to be enough for the complete recovery of the prokaryotic communities in this Mediterranean ecosystem.