Pitting the olive seed microbiome.

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, although a shared microbiome was found. We identified genera that may translocate from the roots to the seeds, as they were present in both organs of the same trees. These findings set the stage for future research into potential vertical transmission of olive endophytes and the role of specific microbial taxa in seed germination, development, and seedling survival.

Mediterranean pine forest decline: A matter of root-associated microbiota and climate change.

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.

Thriving beneath olive trees: The influence of organic farming on microbial communities.

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 × 106 ± 1.62 × 105 (CM) to 4.54 × 106 ± 8.07 × 105 (OM) copies of the 18 S rRNA gene g-1 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 inoculation of Quercus pyrenaica trees alters co-occurrence patterns but not the composition of the rhizosphere bacteriome in wild conditions.

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.

How harmful are exotic plantations for soils and its microbiome? A case study in an arid island.

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 the controversial replacement of exotic plants in insular territories.

Bacteriome dataset from the rhizosphere of trees in a Pinus pinaster and Pinus halepensis dominated forest subjected to drought conditions.

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, Acidobacteria, Actinobacteria, Verrucomicrobia and Bacteroidetes in summer and spring, while potentially active populations (RNA libraries) were rich in Proteobacteria, Acidobacteria, Candidate division WPS-1, Actinobacteria and Verrucomicrobia both in summer and spring. On the other hand, DNA libraries were mainly dominated by genera Sphingomonas and acidobacterial groups Gp4 and Gp6, while potentially active bacteria (RNA) were rich in acidobacterial Gp3, Gp4, Gp6 and Phenylobacterium, although their relative abundance depended on the considered season. This dataset can provide valuable information about bacterial candidates which could be used as bioindicators of drought conditions. In addition to shifts in the bacterial relative abundance due to seasonal changes, the ratio RNA-based cDNA:DNA could be calculated as proxy of the potential activity of bacterial taxa. Moreover, these data could aid in developing bioformulations based on microorganisms which could be resistant to desiccation and involved in the drought resistance mechanisms of the host plant.

Correlating the above- and belowground genotype of Pinus pinaster trees and rhizosphere bacterial communities under drought conditions.

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.

Comparative study of neighboring Holm oak and olive trees-belowground microbial communities subjected to different soil management.

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.

Linking belowground microbial network changes to different tolerance level towards Verticillium wilt of olive.

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. Video abstract.

Metabarcoding reveals that rhizospheric microbiota of Quercus pyrenaica is composed by a relatively small number of bacterial taxa highly abundant.

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.

Defining the root endosphere and rhizosphere microbiomes from the World Olive Germplasm Collection.

The bacterial and fungal communities from the olive (Olea europaea L.) root systems have not yet been simultaneously studied. We show in this work that microbial communities from the olive root endosphere are less diverse than those from the rhizosphere. But more relevant was to unveil that olive belowground communities are mainly shaped by the genotype of the cultivar when growing under the same environmental, pedological and agronomic conditions. Furthermore, Actinophytocola, Streptomyces and Pseudonocardia are the most abundant bacterial genera in the olive root endosphere, Actinophytocola being the most prevalent genus by far. In contrast, Gp6, Gp4, Rhizobium and Sphingomonas are the main genera in the olive rhizosphere. Canalisporium, Aspergillus, Minimelanolocus and Macrophomina are the main fungal genera present in the olive root system. Interestingly enough, a large number of as yet unclassified fungal sequences (class level) were detected in the rhizosphere. From the belowground microbial profiles here reported, it can be concluded that the genus Actinophytocola may play an important role in olive adaptation to environmental stresses. Moreover, the huge unknown fungal diversity here uncovered suggests that fungi with important ecological function and biotechnological potential are yet to be identified.

The rhizosphere microbiome of burned holm-oak: potential role of the genus Arthrobacter in the recovery of burned soils.

After a forest wildfire, the microbial communities have a transient alteration in their composition. The role of the soil microbial community in the recovery of an ecosystem following such an event remains poorly understood. Thus, it is necessary to understand the plant-microbe interactions that occur in burned soils. By high-throughput sequencing, we identified the main bacterial taxa of burnt holm-oak rhizosphere, then we obtained an isolate collection of the most abundant genus and its growth promoting activities were characterised. 16S rRNA amplicon sequencing showed that the genus Arthrobacter comprised more than 21% of the total community. 55 Arthrobacter strains were isolated and characterized using RAPDs and sequencing of the almost complete 16S rRNA gene. Our results indicate that isolated Arthrobacter strains present a very high genetic diversity, and they could play an important ecological role in interaction with the host plant by enhancing aerial growth. Most of the selected strains exhibited a great ability to degrade organic polymers in vitro as well as possibly presenting a direct mechanism for plant growth promotion. All the above data suggests that Arthrobacter can be considered as an excellent PGP rhizobacterium that may play an important role in the recovery of burned holm-oak forests.

The underlying process of early ecological and genetic differentiation in a facultative mutualistic Sinorhizobium meliloti population.

The question of how genotypic and ecological units arise and spread in natural microbial populations remains controversial in the field of evolutionary biology. Here, we investigated the early stages of ecological and genetic differentiation in a highly clonal sympatric Sinorhizobium meliloti population. Whole-genome sequencing revealed that a large DNA region of the symbiotic plasmid pSymB was replaced in some isolates with a similar synteny block carrying densely clustered SNPs and displaying gene acquisition and loss. Two different versions of this genomic island of differentiation (GID) generated by multiple genetic exchanges over time appear to have arisen recently, through recombination in a particular clade within this population. In addition, these isolates display resistance to phages from the same geographic region, probably due to the modification of surface components by the acquired genes. Our results suggest that an underlying process of early ecological and genetic differentiation in S. meliloti is primarily triggered by acquisition of genes that confer resistance to soil phages within particular large genomic DNA regions prone to recombination.

Analysis of rhizobial endosymbionts of Vicia, Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain).

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.

Metagenomic assessment of the potential microbial nitrogen pathways in the rhizosphere of a mediterranean forest after a wildfire.

Wildfires are frequent in the forests of the Mediterranean Basin and have greatly influenced this ecosystem. Changes to the physical and chemical properties of the soil, due to fire and post-fire conditions, result in alterations of both the bacterial communities and the nitrogen cycle. We explored the effects of a holm oak forest wildfire on the rhizospheric bacterial communities involved in the nitrogen cycle. Metagenomic data of the genes involved in the nitrogen cycle showed that both the undisturbed and burned rhizospheres had a conservative nitrogen cycle with a larger number of sequences related to the nitrogen incorporation pathways and a lower number for nitrogen output. However, the burned rhizosphere showed a statistically significant increase in the number of sequences for nitrogen incorporation (allantoin utilization and nitrogen fixation) and a significantly lower number of sequences for denitrification and dissimilatory nitrite reductase subsystems, possibly in order to compensate for nitrogen loss from the soil after burning. The genetic potential for nitrogen incorporation into the ecosystem was assessed through the diversity of the nitrogenase reductase enzyme, which is encoded by the nifH gene. We found that nifH gene diversity and richness were lower in burned than in undisturbed rhizospheric soils. The structure of the bacterial communities involved in the nitrogen cycle showed a statistically significant increase of Actinobacteria and Firmicutes phyla after the wildfire. Both approaches showed the important role of gram-positive bacteria in the ecosystem after a wildfire.

Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa.

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.