David Correa-Galeote

Spatial distribution of N-cycling microbial communities showed complex patterns in constructed wetland sediments.

Constructed wetlands are used for biological treatment of wastewater from agricultural lands carrying pollutants such as nitrates. Nitrogen removal in wetlands occurs from direct assimilation by plants and through microbial nitrification and denitrification. We investigated the spatial distribution of N-cycling microbial communities and genes involved in nitrification and denitrification in constructed wetland sediments receiving irrigation water. We used quantitative real-time PCR (qPCR) to characterize microbial communities. Geostatistical variance analysis was used to relate them with vegetation cover and biogeochemical sediment properties. The spatial distribution of the N-cycling microbial communities of sediments was heterogeneous and complex. Total communities of bacteria and crenarchaea showed different spatial distributions. Analysis of autocorrelation patterns through semivariance indicated a tendency towards a patchy distribution over scales around 10 m for genes involved in the nitrification and denitrification processes. In contrast, biogeochemical sediment properties showed diverse spatial distributions. While almost no patchiness was found for pH and moisture, patchiness at scales between 8 and 10 m was detected for carbon, nitrate and ammonia. Denitrification variables showed spatial autocorrelation at scales comparable to genes. However, denitrifying enzyme activity and potential N(2)O production showed a common spatial pattern, different from that of the N(2)O/(N(2)O + N(2)).

Bacterial Communities in the Rhizosphere of Amilaceous Maize (Zea mays L.) as Assessed by Pyrosequencing

Maize (Zea mays L.) is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance) Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere of maize intercropped with bur clover, and Gp4, Subdivision3 genera incertae sedis of phylum Verrucomicrobia, Gp6 and Rhodoferax were the main genera in the rhizosphere of maize plants. Taken together, our results suggest that bur clover produces specific changes in rhizospheric bacterial diversity of amilaceous maize plants.

Isolation of N2-fixing rhizobacteria from Lolium perenne and evaluating their plant growth promoting traits

Twenty one dinitrogen (N2)‐fixing bacteria were isolated from the rhizosphere of Lolium perenne grown for more than 10 years without N‐fertilization. The nearly complete sequence of the 16S rRNA gene of each strain and pairwise alignments among globally aligned sequences of the 16S rRNA genes clustered them into nine different groups. Out of the 21 strains, 11 were members of genus Bacillus, 3 belonged to each one of genera Paenibacillus and Pseudoxanthomonas, and the remaining 2 strains to each one of genera Burkholderia and Staphylococcus, respectively. A representative strain from each group contained the nifH gene and fixed atmospheric N2 as determined by the acetylene‐dependent ethylene production assay (acetylene reduction activity, ARA). The nine selected strains were also examined to behave as plant growth promoting bacteria (PGPRs) including their ability to act as a biocontrol agent. The nine representative strains produced indol acetic acid (IAA) and solubilized calcium triphosphate, five of them, strains C2, C3, C12, C15, and C16, had ACC deaminase activity, and strains C2, C3, C4, C12, C16, and C17 produced siderophores. Strains C13, C16, and C17 had the capability to control growth of the pathogen Fusarium oxysporum mycelial growth in vitro. PCA analysis of determined PGPR properties showed that ARA, ACC deaminase activity, and siderophore production were the most valuable as they had the maximal contribution to the total variance.

Maize endophytic bacterial diversity as affected by soil cultivation history

The bacterial endophytic communities residing within roots of maize (Zea mays L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera Sphingomonas, Herbaspirillum, Bradyrhizobium and Methylophilus in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera Dyella and Sreptococcus were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Burkholderia, candidatus Glomeribacter, Staphylococcus, Variovorax, Bacillus and Chitinophaga were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities.

Denitrification and Biodiversity of Denitrifiers in a High-Mountain Mediterranean Lake

Wet deposition of reactive nitrogen (Nr) species is considered a main factor contributing to N inputs, of which nitrate (NO3−) is usually the major component in high-mountain lakes. The microbial group of denitrifiers are largely responsible for reduction of nitrate to molecular dinitrogen (N2) in terrestrial and aquatic ecosystems, but the role of denitrification in removal of contaminant nitrates in high-mountain lakes is not well understood. We have used the oligotrophic, high-altitude La Caldera lake in the Sierra Nevada range (Spain) as a model to study the role of denitrification in nitrate removal. Dissolved inorganic Nr concentration in the water column of la Caldera, mainly nitrate, decreased over the ice-free season which was not associated with growth of microbial plankton or variations in the ultraviolet radiation. Denitrification activity, estimated as nitrous oxide (N2O) production, was measured in the water column and in sediments of the lake, and had maximal values in the month of August. Relative abundance of denitrifying bacteria in sediments was studied by quantitative polymerase chain reaction of the 16S rRNA and the two phylogenetically distinct clades nosZI and nosZII genes encoding nitrous oxide reductases. Diversity of denitrifiers in sediments was assessed using a culture-dependent approach and after the construction of clone libraries employing the nosZI gene as a molecular marker. In addition to genera Polymorphum, Paracoccus, Azospirillum, Pseudomonas, Hyphomicrobium, Thauera, and Methylophaga, which were present in the clone libraries, Arthrobacter, Burkholderia, and Rhizobium were also detected in culture media that were not found in the clone libraries. Analysis of biological activities involved in the C, N, P, and S cycles from sediments revealed that nitrate was not a limiting nutrient in the lake, allowed N2O production and determined denitrifiers’ community structure. All these results indicate that denitrification could be a major biochemical process responsible for the N losses that occur in La Caldera lake.

Symbiotic and Agronomic Characterization of Bradyrhizobial Strains Nodulating Cowpea in Northern Peru

Eighty bacterial strains were isolated from root nodules of cowpea (Vigna unguiculata) plants agriculturally grown in Northern Peru. After isolation, fifty of them reinfected cowpea and eight showed better symbiotic performance than control plants treated with nitrate both under greenhouse and field. Repetitive extragenic palindromic polymerase chain reaction (REP-PCR) clustered the strains into 3 REP-PCR groups. The partial sequence of the 16S rRNA gene from a representative strain of each REP-PCR pattern showed that the strains were closely related to members of genus Bradyrhizobium of the Alphaproteobacteria, but affiliation at the species level was not clear. Pairwise alignments among globally aligned sequences of the 16S rRNA genes, the sequencing of the housekeeping genes atpD, glnII, and recA, and their concatenated phylogenetic analysis showed that strains Rc-391-01 and Rc-458-01 grouped with B. yuanmingense B071T and that strain Rc-352-01 clustered with Bradyrhizobium liaoningense 2281T. Examination of the three representative strains to behave as plant growth-promoting rhizobacteria (PGPRs) showed that none of them solubilized calcium triphosphate and that they all three produced siderophores and IAA-related compounds and had ACC-deaminase activity.

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as narG, napA, nirK, nirS and nosZ denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because nosZ showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the nosZ-bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof nosZ-bearing communities.