Paula García-Fraile

Characterization of xylanolytic bacteria present in the bract phyllosphere of the date palm Phoenix dactylifera

Aims:  Despite the interest of phyllosphere microbiology, no studies have addressed the bacteria present in bract phyllosphere, an ecosystem that has special characteristics in palm trees because the dry bracts remain on the plant until pruning and may contain polymer‐degrading bacteria involved in plant degradation. Therefore, the aim of this work was to characterize xylanolytic bacteria isolated from palm bract phyllosphere.

Rhizobium Promotes Non-Legumes Growth and Quality in Several Production Steps: Towards a Biofertilization of Edible Raw Vegetables Healthy for Humans

The biofertilization of crops with plant-growth-promoting microorganisms is currently considered as a healthy alternative to chemical fertilization. However, only microorganisms safe for humans can be used as biofertilizers, particularly in vegetables that are raw consumed, in order to avoid sanitary problems derived from the presence of pathogenic bacteria in the final products. In the present work we showed that Rhizobium strains colonize the roots of tomato and pepper plants promoting their growth in different production stages increasing yield and quality of seedlings and fruits. Our results confirmed those obtained in cereals and alimentary oil producing plants extending the number of non-legumes susceptible to be biofertilized with rhizobia to those whose fruits are raw consumed. This is a relevant conclusion since safety of rhizobia for human health has been demonstrated after several decades of legume inoculation ensuring that they are optimal bacteria for biofertilization.

MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae.

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies.

Cohnella phaseoli sp. nov., isolated from root nodules of Phaseolus coccineus in Spain, and emended description of the genus Cohnella

A bacterial strain designated GSPC1 T was isolated from root nodules of Phaseolus coccineus in Segovia (Spain). The 16S rRNA gene sequence of this strain showed 95.9 and 94.7 % sequence similarity, respectively, with those of the type strains of Cohnella hongkongensis and Cohnella thermotolerans. Strain GSPC1 T presented phenotypic, chemotaxonomic and molecular differences with respect to Cohnella species which indicated that it belonged to a different species. The isolate was a Gram-positive, aerobic, sporulated rod, motile by means of peritrichous flagella. The strain was catalase-positive and showed weak oxidase activity. It grew in the presence of 2 % NaCl. MK-7 was the predominant menaquinone. anteiso-C15:0, iso-C15:0, iso-C16: 0 and C16:0 were the major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G+C content was 60.3 mol%. The results of this study suggest that isolate GSPC1 T should be classified within a novel Cohnella species, for which the name Cohnella phaseoli sp. nov. is proposed, with strain GSPC1T (=LMG 24086T =DSM 19269T) as the type strain.

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.

Phylogenetic diversity based on rrs, atpD, recA genes and 16S–23S intergenic sequence analyses of rhizobial strains isolated from Vicia faba and Pisum sativum in Peru

In this study 17 isolates from effective nodules of Vicia faba and Pisum sativum var. macrocarpum growing in different soils from Peru were isolated and characterized. The isolates, presenting 11 different RAPD profiles, were distributed in three groups on the basis of their 16S-RFLP patterns. The 16S rRNA gene sequences of strains from 16S-RFLP groups I, II and III were closely related (identities higher than 99.5%) to Rhizobium leguminosarum bv. trifolii DSM 30141 (=ATCC 14480), R. leguminosarum bv. viciae DSM 30132T and Rhizobium etli CFN42T (=USDA 9032T), respectively. The analysis of the 16S–23S intergenic spacer (ITS) and two housekeeping genes, atpD and recA, confirmed the identification of strains from group I, however those from groups II and III were phylogenetically divergent to strains DSM 30132T and CFN42T. These results support the fact that the 16S rRNA gene is not adequate for identification at species level within genus Rhizobium and suggest the existence of putative new species within the phylogenetic group of R. leguminosarum. They also confirm the need of a taxonomic revision of R. leguminosarum since the reference strains of the three biovars included in this study are phylogenetically divergent according to their ITS, atpD and recA gene sequences.

Plants Probiotics as a Tool to Produce Highly Functional Fruits: The Case of Phyllobacterium and Vitamin C in Strawberries

The increasing interest in the preservation of the environment and the health of consumers is changing production methods and food consumption habits. Functional foods are increasingly demanded by consumers because they contain bioactive compounds involved in health protection. In this sense biofertilization using plant probiotics is a reliable alternative to the use of chemical fertilizers, but there are few studies about the effects of plant probiotics on the yield of functional fruits and, especially, on the content of bioactive compounds. In the present work we reported that a strain of genus Phyllobacterium able to produce biofilms and to colonize strawberry roots is able to increase the yield and quality of strawberry plants. In addition, the fruits from plants inoculated with this strain have significantly higher content in vitamin C, one of the most interesting bioactive compounds in strawberries. Therefore the use of selected plant probiotics benefits the environment and human health without agronomical losses, allowing the production of highly functional foods.

Taxonomy of Bacteria Nodulating Legumes

Over the years, the term “rhizobia” has come to be used for all the bacteria that are capable of nodulation and nitrogen fixation in association with legumes but the taxonomy of rhizobia has changed considerably over the last 30 year. Recently, several non-rhizobial species belonging to alpha and beta subgroup of Proteobacteria have been identified as nitrogen-fixing legume symbionts. Here we provide an overview of the history of the rhizobia and the widespread phylogenetic diversity of nitrogen-fixing legume symbionts.

Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with Sinorhizobium meliloti isolates from mainland Spain.

ABSTRACT The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers DQ333891 and AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.

Phyllobacterium endophyticum sp. nov., isolated from nodules of Phaseolus vulgaris

A strain, PEPV15(T), was isolated from a nodule on Phaseolus vulgaris grown in soil in northern Spain. Phylogenetic analyses of 16S rRNA and atpD genes showed that this strain belongs to the genus Phyllobacterium. The most closely related species were, in both cases, Phyllobacterium brassicacearum, Phyllobacterium bourgognense and Phyllobacterium trifolii, the type strains of which gave sequence similarities of 98.9, 98.6 and 98.4 %, respectively, in the 16S rRNA gene and 88.1, 87.5 and 88.7 %, respectively, in the atpD gene. PEPV15(T) contained Q-10 as the major quinone (88 %) and low amounts of Q-9 (12 %). It differed from its closest relatives in its growth in diverse culture conditions and in the assimilation of several carbon sources. The strain was not able to produce nodules in Phaseolus vulgaris. The results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain represents a novel species of the genus Phyllobacterium for which the name Phyllobacterium endophyticum sp. nov. is proposed; the type strain is PEPV15(T) ( = LMG 26470(T) = CECT 7949(T)). An emended description of the genus Phyllobacterium is also provided.