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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: Teide National Park (Canary Islands) is an alpine volcanic ecosystem with shrub vegetation in which legume Spartocytisus supranubius is the most characteristic key species for nitrogen input to the ecosystem. Aims and methods: Bacterial and fungal communities in bulk and rhizosphere soils were analysed through high-throughput sequencing in undisturbed and wildfire-impacted areas to identify key microorganisms in burned and unburned soils. Results: Microbial communities in undisturbed areas exhibited comparable diversity in bulk and rhizosphere soils, but differed in structure and composition. An unusual abundance of non-photosynthetic Chloroflexi from the oligotrotrophic class Ktedonobacteria dominated the bulk soils, surpassing Proteobacteria, Acidobacteria and Actinobacteria. The rhizosphere effect resulted in a microbiome with a more balanced abundance of these four phyla and enriched in potentially plant growth-promoting microorganisms. The impact of a wildfire on the shrub vegetation resulted in a microbial community, especially the fungal community, reduced in diversity and changed in structure and composition, with many of the most characteristic rhizosphere genera becoming vanished, while others took advantage of the postfire conditions and became predominant. Conclusion: The microbial communities of Teide National Park in fire-affected soils, particularly in the rhizosphere environment of the legume shrubland are significantly altered two years after a wildfire, remaining far from unburned scenarios, suggesting a slow recovery in alpine ecosystem with dry volcanic soils. Pseudarthrobacter (Actinobacteria) and Coprinellus (Basidiomycota), the two most fire-favoured genera, are good indicators of fire severity and are proposed as bioindicators to monitor the recovery of the soil ecosystem.

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

The plantation of exotic species has been a common practice in (semi-) arid areas worldwide aiming to restore highly degraded habitats. The effects of these plantations on plant cover or soil erosion have been widely studied, while little attention has been paid to the consequences on soil quality and belowground biological communities. This study evaluates the long-term (>60 years) effects of the exotic species Acacia cyclops and Pinus halepensis revegetation on soil properties, including microbiome, in an arid island. Soils under exotic plantation were compared to both degraded soils with a very low cover of native species and soils with well-preserved native plant communities. Seven scenarios were selected in a small area (~25 ha) with similar soil type but differing in the plant cover. Topsoils (0-15 cm) were analyzed for physical, chemical and biochemical properties, and amplicon sequencing of bacterial and fungal communities. Microbial diversity was similar among soils with exotic plants and native vegetation (Shannon's index = 5.26 and 5.34, respectively), while the most eroded soils exhibited significantly lower diversity levels (Shannon's index = 4.72). Bacterial and fungal communities' composition in degraded soils greatly differed from those in vegetated soils (Canberra index = 0.85 and 0.92, respectively) likely due to high soil sodicity, fine textures and compaction. Microbial communities' composition also differed in soils covered with exotic and native species, to a greater extent for fungi than for bacteria (Canberra index = 0.94 and 0.89, respectively), due to higher levels of nutrients, microbial biomass and activity in soils with native species. Results suggest that reforestation succeeded in avoiding further soil degradation but still leading to relevant changes in soil microbial community that may have negative effects on ecosystem stability. Information gained in this research could be useful for environmental agencies and decision makers about

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.

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 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

Soil health and root-associated microbiome are interconnected factors involved in plant health. The use of manure amendment on agricultural fields exerts a direct benefit on soil nutrient content and water retention, among others. However, little is known about the impact of manure amendment on the root-associated microbiome, particularly in woody species. In this study, we aimed to evaluate the effects of ovine manure on the microbial communities of the olive rhizosphere and root endosphere. Two adjacent orchards subjected to conventional (CM) and organic (OM) management were selected. We used metabarcoding sequencing to assess the bacterial and fungal communities. Our results point out a clear effect of manure amendment on the microbial community. Fungal richness and diversity were increased in the rhizosphere. The fungal biomass in the rhizosphere was more than doubled, ranging from 1.72 × 10 ± 1.62 × 10 (CM) to 4.54 × 10 ± 8.07 × 10 (OM) copies of the 18 S rRNA gene g soil. Soil nutrient content was also enhanced in the OM orchard. Specifically, oxidable organic matter, total nitrogen, nitrate, phosphorous, potassium and sulfate concentrations were significantly increased in the OM orchard. Moreover, we predicted a higher abundance of bacteria in OM with metabolic functions involved in pollutant degradation and defence against pathogens. Lastly, microbial co-occurrence network showed more positive interactions, complexity and shorter geodesic distance in the OM orchard. According to our results, manure amendment on olive orchards represents a promising tool for positively modulating the microbial community in direct contact with the plant.

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).

The aim of this study was to figure out the influence of manure amendment on the radicular (rhizosphere and root endosphere) microbial communities from olive trees. For this purpose, two adjacent olive orchards with different soil managements were surveyed: i) traditional management (TM) and ii) with manure amendment (MA). TM orchard was subjected to common agriculture procedures (including herbicides, mechanical plough and pesticides treatments), while MA cultivar did not receive any herbicides treatment, but a manure amendment was applied to the soil. For each orchard, 12 replicates (trees) were considered for analysis, all of them belonging to Picual variety. From each replicate and microhabitat, both bacterial and fungal communities were studied with the use of amplicon sequencing (pairedend 2x300-bp [PE 300] with the Illumina MiSeq platform). Raw reads were processed following our own tutorial, recently published in GitHub (https://nuriamw.github.io/micro4all/) and also available as an R package (micro4all). This tutorial includes analysis for ASV inferring with DADA2, ¿ and ß-diversity (index calculation, graphical representation and statistical analysis), differential abundance analysis with ANCOM-BC and taxonomical profiling (plots and tables). Moreover, microbial ecological networks were also studied with MENAP online tool. Our results showed that a higher fungal diversity was found in MA rhizosphere. However, no statistically significant differences were found for bacterial community at rhizosphere level, neither for fungal or bacterial endosphere community. Interestingly, community structure (ß-diversity) seemed to differ for both microhabitats (rhizosphere and root endosphere) and kingdoms (bacteria and fungi). Furthermore, network analysis showed that in both compartments TM microbial communities had closer interactions (i.e., a shorter geodesic distance) as well as a greater percentage of positive links. Regarding taxonomical profiles, it should be hig

Due to their widespread use in industrial applications in recent decades, Polychlorobiphenyls (PCBs) and heavy metals (HMs) are the most common soil contaminants worldwide, posing a risk for both ecosystems and human health. In this study, a poplar-assisted bioremediation strategy has been applied for more than 4 years to a historically contaminated area (PCBs and HMs) in Southern Italy using the Monviso poplar clone. This clone was effective in promoting a decrease in all contaminants and an increase in soil quality in terms of organic carbon and microbial abundance. Moreover, a significant shift in the structure and predicted function of the belowground microbial community was also observed when analyzing both DNA and cDNA sequencing data. In fact, an increase in bacterial genera belonging to Proteobacteria able to degrade PCBs and resist HMs was observed. Moreover, the functional profiling of the microbial community predicted by PICRUSt2 made it possible to identify several genes associated with PCB transformation (e.g., bphAa, bphAb, bphB, bphC), response to HM oxidative stress (e.g., catalase, superoxide reductase, peroxidase) and HM uptake and expulsion (e.g., ABC transporters). This work demonstrated the effectiveness of the poplar clone Monviso in stimulating the natural belowground microbial community to remove contaminants and improve the overall soil quality. It is a practical example of a nature based solution involving synergic interactions between plants and the belowground microbial community.

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.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic pollutants that contaminate large areas. They are mainly released to environment by anthropogenic activities principally due to the petrochemical industry. The low biodegradation rate characteristic of PAHs in aged contaminated soils could be overcome trough the chemical oxidation. In this study, composting with the soil and stimulation with mature compost were the strategies applied in soil microcosms after chemical oxidation with ammonium persulfate in a PAHs chronically contaminated soil. A 29% of PAHs elimination and an increase of their bioavailability were found after chemical oxidation with ammonium persulfate. Due to the oxidative treatment the total bacterial and the gram-positive population PAH dioxygenase genes were significantly reduced and no gram-negative PAHs degraders were detected. The following application of organic amendments produced a higher increase in total bacteria and recovery of the degrading population of GP PAH after one year of treatment, in comparison with the pre-oxidized soil bioremediation, only promoted by irrigation and aeration. Also a significant increase in the content of bioavailable PAHs was observed. However, from both composting strategies only the stimulation with mature compost led to a net PAHs removal. Taking into account the residual dissolved total carbon and humification degree (E/E ratio), it was attributed to the preferential consumption of more easily degradable compounds than hydrocarbons the low removal efficiency observed after one year of treatment. Due to the high bioavailable content of PAH and the residual sulfate, long-term treatments will require careful monitoring to reduce environmental risks.

[Background] Verticillium wilt of olive (VWO) is caused by the soilborne fungal pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant/resistant olive cultivars. Knowledge on the olive-associated microbiome and its potential relationship with tolerance to biotic constraints is almost null. The aims of this work are (1) to describe the structure, functionality, and co-occurrence interactions of the belowground (root endosphere and rhizosphere) microbial communities of two olive cultivars qualified as tolerant (Frantoio) and susceptible (Picual) to VWO, and (2) to assess whether these communities contribute to their differential disease susceptibility level., [Results] Minor differences in alpha and beta diversities of root-associated microbiota were detected between olive cultivars regardless of whether they were inoculated or not with the defoliating pathotype of V. dahliae. Nevertheless, significant differences were found in taxonomic composition of non-inoculated plants’ communities, “Frantoio” showing a higher abundance of beneficial genera in contrast to “Picual” that exhibited major abundance of potential deleterious genera. Upon inoculation with V. dahliae, significant changes at taxonomic level were found mostly in Picual plants. Relevant topological alterations were observed in microbial communities’ co-occurrence interactions after inoculation, both at structural and functional level, and in the positive/negative edges ratio. In the root endosphere, Frantoio communities switched to highly connected and low modularized networks, while Picual communities showed a sharply different behavior. In the rhizosphere, V. dahliae only irrupted in the microbial networks of Picual plants., [Conclusions] The belowground microbial communities of the two olive cultivars are very similar and pathogen introduction did not provoke significant alterations in their structure and functionality. However, notable differences were found in their networks in response to the inoculation. This phenomenon was more evident in the root endosphere communities. Thus, a correlation between modifications in the microbial networks of this microhabitat and susceptibility/tolerance to a soilborne pathogen was found. Moreover, V. dahliae irruption in the Picual microbial networks suggests a stronger impact on the belowground microbial communities of this cultivar upon inoculation. Our results suggest that changes in the co-occurrence interactions may explain, at least partially, the differential VWO susceptibility of the tested olive cultivars.