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

Taxonomic and Functional Diversity of a Quercus pyrenaica Willd. Rhizospheric Microbiome in the Mediterranean Mountains

Altitude significantly affects vegetation growth and distribution, including the developmental stages of a forest. We used shotgun Illumina sequencing to analyze microbial community composition and functional potential in melojo-oak (Quercus pyrenaica Willd.) rhizospheric soil for three different development stages along an altitudinal gradient: (a) a low altitude, non-optimal site for forest maintenance; (b) an intermediate altitude, optimal site for a forest; and (c) a high altitude, expansion site with isolated trees but without a real forest canopy. We observed that, at each altitude, the same microbial taxa appear both in the taxonomic analysis of the whole metagenome and in the functional analysis of the methane, sulfur and nitrogen metabolisms. Although there were no major differences at the functional level, there were significant differences in the abundance of each taxon at the phylogenetic level between the rhizospheres of the forest (low and intermediate altitudes) and the expansion site. Proteobacteria and Actinobacteria were the most differentially abundant phyla in forest soils compared to the expansion site rhizosphere. Moreover, Verrucomicrobia, Bacteroidetes and Nitrospirae phyla were more highly represented in the non-forest rhizosphere. Our study suggests that rhizospheric microbial communities of the same tree species may be affected by development stage and forest canopy cover via changes in soil pH and the C/N ratio.This work was supported by research grants including ERDF (European Regional Development Funds): P08-CVI-03549 from Consejería de Innovación, Ciencia y Empresa from Junta de Andalucía, OAPN 021/2007 from Organismo Autónomo Parques Nacionales (Spanish Ministry of Environment) and 20134R069-RECUPERA 2020 from the Spanish Ministerio de Economía y Competitividad and CSIC. We would like to thank the authorities of the Sierra Nevada National Park for the access, facilities and soil sampling, and also Edward Kirton (JGI, California) for technical assistance with bioinformatics. JFCD was awarded a postdoctoral contract from Junta de Andalucía, and AJFG was awarded a postdoctoral contract from RECUPERA 2020. The work of the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231

Análisis de la diversidad procariótica asociada a quercíneas (Quercus ilex sp. ballota y Q. pyrenaica) para la identificación de bioindicadores asociados a la evolución post-incendio y al cambio climático en Sierra Nevada

[EN] The climate change is a global ongoing process which alters earth ecosystems. Not only the industry derived processes but also other «natural» factors, such as the forest burning, contribute to the increase of atmospheric CO 2 levels and, consequently to the global warming. Adequate forest management could prevent CO 2 emission and/or favour its incorporation into the soil organic matter, thus con- tributing to the overall decrease of CO 2 concentrations. In particular, a rapid re-vegetation or restora- tion strategies with autochthonous formations such as holm-oak wood ( Quercus ilex sp. ballota ) or oakwood ( Q. pyrenaica Wild.) are desirable practices. Soil microorganisms are key elements in carbon cycle completion and positively influence soil fertility and plant growth. In this research project it was studied the prokaryotic diversity associated to the above mentioned trees to identify potential biolog- ical markers to asses both, the forest restoration after a burn and the transition from holm-oak wood to oakwood at the Sierra Nevada Natural Park, as processes contributing to the climate change. Thus total DNA extracted from Quercus rhizosphere was analyzed by different approaches and techniques. First the genetic fingerprint of the rhizosphere from different trees was analyzed by TGGE. Later genetic li- braries, of the 16S rRNA and nif H genes, were constructed in order to determine the diversity and rich- ness of microorganisms and nitrogen-fixation at the rhizospere. Moreover a deep-sequencing analysis was performed with the GS FLX Titanium system of Roche. In this way, more than 10.000 sequences of 16S rRNA amplicons per ecosystem were analyzed which allowed us to obtain a coverage of circa 90% of the total diversity. Finally metagenomic libraries were constructed with environmental DNA in order to analyze the metabolic pathways of each rhizosphere and to conserve the microbial «germoplasm».[ES]El cambio climático es un hecho constatado e incuestionable. Además de los procesos de tipo industrial existen factores «naturales», como los incendios forestales, que también contribuyen al calentamiento global dada la alta emisión de CO 2 que se produce. Disminuir la concentración de CO 2 atmosférico, evitar su incremento o ayudar a su acumulación en la materia orgánica del suelo, se puede ver beneficiado por un correcto manejo de los bosques. Una rápida re-vegetación o recuperación de las formaciones autóctonas como son los encinares y robledales (Quercus ilex sp.ballota y Q. pyrenaica Wild.) pueden ayudar en este sentido. Los microorganismos del suelo son los responsables del cierre del ciclo biogeoquímico del carbono, contribuyendo además a la fertilidad del suelo y a la promoción del creci- miento vegetal. Por ello, en este proyecto se ha estudiado, por primera vez, la diversidad procariótica asociada a estos árboles con objeto de identificar bioindicadores que nos ayuden a evaluar la recupe- ración después de un incendio y la transición robledal-encinar, en el Parque Nacional de Sierra Ne- vada, como medida del cambio climático. Así se ha analizado el ADN extraído de la rizosfera de estas quercíneas mediante distintas técnicas y aproximaciones. En primer lugar se analizó el fingerprint ge- nético de la rizosfera de los distintos árboles muestreados mediante TGGE. Posteriormente, se realiza- ron librerías génicas, mediante la amplificación con cebadores específicos de los genes 16S rRNA y nif H, para determinar la diversidad y riqueza tanto procariótica como de fijación de nitrógeno. Estos estu- dios se complementaron con la ultrasecuenciación, mediante el sistema GS FLX Titanium de Roche, de amplicones del gen 16S rRNA lo que nos permitió obtener más de 10.000 secuencias por ecosistema re- sultando en una cobertura de la diversidad cercana al 90%. Finalmente se construyeron librerías me- tagenómicas a partir del ADN ambiental para analizar las rutas metabólicas presentes en cada rizosfera y poder conservar el «germoplasma» presente.Peer reviewe

Metagenomic assesment of the potential microbial nitrogen pathways in the rhizosphere of a Mediterranean forest after wildfire

Wildfires are frecuent in the forest 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 nitrgen cycle,. We explored the effetcs 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 undistrubed and burned rhizospheres had a conservative nitrogen cycle with a larger number of sequences related to the nitrogen incorpotation pathways and 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 ecosystems was assessed through the diversity of the nitrogenase reductase enzyme, wich 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 wilfire.Peer reviewe

The rhizosphere microbiome associated with the legume Spartocytisus supranubius in the high mountain ecosystem of Teide N.P.

Resumen del poster presentado en: I Spanish-Portuguese Congress on Beneficial Plant-Microbe Interactions (BeMiPlant) and XVIII National Meeting of the Spanish Society of Nitrogen Fixation (XVIII SEFIN). Oeiras, Portugal, 17-19 octubre (2022)Consejería de Economia, Conocimiento y Empleo, Gobierno de Canarias-Cofinanciación Programa Operativo FEDER Canarias 2014-2020 (Grant ProID2020010103)

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.This research was supported by the following grants: P08-CVI-03549 from The Department of Innovation, Science and Enterprise of the Autonomous Government of Andalusia; OAPN 021/2007 from The National Parks Autonomous Body (Ministry of the Environment) and 20134R069-RECUPERA 2020 from the Spanish Ministry of Economy and Competitiveness, and CSIC, including ERDF (European Regional Development Fund). AJFG and AVL were awarded a contract from RECUPERA 2020 and AVL was also supported with an FPU fellowship from the Spanish Ministry of Education, Culture and Sport.Peer Reviewe

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

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

Efecto del manejo agrícola sobre el microbioma de la raíz de olivo

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022