José M. Igual

Climate vs. soil factors in local adaptation of two common plant species

Evolutionary theory suggests that divergent natural selection in heterogeneous environments can result in locally adapted plant genotypes. To understand local adaptation it is important to study the ecological factors responsible for divergent selection. At a continental scale, variation in climate can be important while at a local scale soil properties could also play a role. We designed an experiment aimed to disentangle the role of climate and (abiotic and biotic) soil properties in local adaptation of two common plant species. A grass (Holcus lanatus) and a legume (Lotus corniculatus), as well as their local soils, were reciprocally transplanted between three sites across an Atlantic-Continental gradient in Europe and grown in common gardens in either their home soil or foreign soils. Growth and reproductive traits were measured over two growing seasons. In both species, we found significant environmental and genetic effects on most of the growth and reproductive traits and a significant interaction between the two environmental effects of soil and climate. The grass species showed significant home site advantage in most of the fitness components, which indicated adaptation to climate. We found no indication that the grass was adapted to local soil conditions. The legume showed a significant home soil advantage for number of fruits only and thus a weak indication of adaptation to soil and no adaptation to climate. Our results show that the importance of climate and soil factors as drivers of local adaptation is species-dependent. This could be related to differences in interactions between plant species and soil biota.

Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina)

Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).

Micromonospora from nitrogen fixing nodules of alfalfa ( Medicago sativa L.). A new promising Plant Probiotic Bacteria.

Biotic interactions can improve agricultural productivity without costly and environmentally challenging inputs. Micromonospora strains have recently been reported as natural endophytes of legume nodules but their significance for plant development and productivity has not yet been established. The aim of this study was to determine the diversity and function of Micromonospora isolated from Medicago sativa root nodules. Micromonospora-like strains from field alfalfa nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene sequencing. The ecological role of the interaction of the 15 selected representative Micromonospora strains was tested in M. sativa. Nodulation, plant growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain abundant and highly diverse populations of Micromonospora, both at the intra- and at interspecific level. Selected Micromonospora isolates significantly increase the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the plant for nitrogen nutrition. Moreover, they promote aerial growth, the shoot-to-root ratio, and raise the level of essential nutrients. Our results indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition efficiency. Its ecological role, biotechnological potential and advantages as a plant probiotic bacterium (PPB) are also discussed.

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.

Diversity and community structure of culturable arsenic-resistant bacteria across a soil arsenic gradient at an abandoned tungsten-tin mining area.

We studied the bacterial diversity at a single location (the Terrubias mine; Salamanca province, Spain) with a gradient of soil As contamination to test if increasing levels of As would (1) change the preponderant groups of arsenic-resistant bacteria and (2) increase the tolerance thresholds to arsenite [As(III)] and arsenate [As(V)] of such bacteria. We studied the genetic and taxonomic diversity of culturable arsenic-resistant bacteria by PCR fingerprinting techniques and 16S rRNA gene sequencing. Then, the tolerance thresholds to As(III) and As(V) were determined for representative strains and mathematically analyzed to determine relationships between tolerances to As(III) and As(V), as well as these tolerances with the soil contamination level. The diversity of the bacterial community was, as expected, inversely related to the soil As content. The overall preponderant arsenic-resistant bacteria were Firmicutes (mainly Bacillus spp.) followed by γ-Proteobacteria (mainly Pseudomonas spp.), with increasing relative frequencies of the former as the soil arsenic concentration increased. Moreover, a strain of the species Rahnella aquatilis (γ-Proteobacteria class) exhibited strong endurance to arsenic, being described for the first time in literature such a phenotype within this bacterial species. Tolerances of the isolates to As(III) and As(V) were correlated but not with their origin (soil contamination level). Most of the strains (64%) showed relatively low tolerances to As(III) and As(V), but the second most numerous group of isolates (19%) showed increased tolerance to As(III) rather than to As(V), even though the As(V) anion is the prevalent arsenic species in soil solution at this location. To our knowledge, this is the first study to report a shift towards preponderance of Gram-positive bacteria (Firmicutes) related to high concentrations of soil arsenic. It was also shown that, under aerobic conditions, strains with relatively enhanced tolerance to As(III) predominated over the most As(V)-tolerant ones.

Analysis of LMW RNA profiles of Frankia strains by staircase electrophoresis.

An optimized technique of polyacrylamide gel electrophoresis, Staircase Electrophoresis (SCE), was applied to determine the stable Low Molecular Weight RNA (LMW RNA) profiles of 25 Frankia strains from diverse geographic origins and host specificity groups as well as species from other actinomycete genera. Application of the technique permits the rapid identification of Frankia strains and their differentiation from other actinomycetes. The isolates used in this study were grouped in eight clusters, each comprising strains with identical LMW RNA profiles. Comparison of these results with others obtained from DNA sequences or DNA hybridization methods suggest a high degree of complexity in the genus Frankia. Application of SCE to profile LMW RNA should in the future facilitate biodiversity studies of Frankia and discrimination of new species.

Paenibacillus castaneae sp. nov., isolated from the phyllosphere of Castanea sativa Miller.

A bacterial strain, designated Ch-32(T), was isolated from the phyllosphere of Castanea sativa in Spain. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate in the genus Paenibacillus within the same subgroup as Paenibacillus xinjiangensis and Paenibacillus glycanilyticus, with similarities of 96.3 and 96.8 %, respectively. DNA-DNA hybridization values for strain Ch-32(T) with these two species were lower than 20 %. The novel isolate was a Gram-variable, motile, sporulating rod. It produced catalase and oxidase and hydrolysed cellulose, gelatin and aesculin. Acetoin and urease production, nitrate reduction and starch hydrolysis were negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected and anteiso-C(15 : 0), iso-C(16 : 0) and C(16 : 0) were the major fatty acids. The DNA G+C content was 46 mol%. Phylogenetic, DNA relatedness and phenotypic analyses showed that strain Ch-32(T) should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus castaneae sp. nov. is proposed; the type strain is Ch-32(T) (=CECT 7279(T)=DSM 19417(T)).

Cellulase isoenzyme profiles in Frankia strains belonging to different cross-inoculation groups

Carboxymethyl cellulase activities were evaluated in eight strains of Frankia from diverse host specificity groups and geographical origins. Cellulase activity was detected in culture supernatants of all strains in the absence of CMC (Carboxymethylcellulose) as an inducer using both double-layer plate and reducing sugar assays, indicating a constitutive production of CM-cellulases by Frankia. CM-cellulase isoenzyme profiles were visualized using activity gel electrophoresis of concentrated culture supernatants. Different electrophoretic profiles were observed among the eight strains tested, which correlate with the host specificity and taxonomic grouping of Frankia.

Casuarina cunninghamiana tissue extracts stimulate the growth of Frankia and differentially alter the growth of other soil microorganisms.

Aqueous extracts of host plant Casuarina cunninghamiana tissue altered the in vitro growth of its diazotrophic microsymbiont Frankia and a selection of other soil microorganisms. The growth of actinomycetous Frankia strains, 55005, AvcI1, CesI5, CjI82 001, and Cj was stimulated by aqueous extracts of C. cunninghamiana tissue. Green cladodes (photosynthetic branches), unsuberized roots, and suberized roots were more stimulatory than dry cladodes and seed tissue. Aqueous extracts of green cladodes of C. cunninghamiana most stimulated the growth of Casuarina-derived Frankia strains CjI82 001 and 55005. The growth of isolates of soil bacteria Bradyrhizobium japonicum, Arthrobacter globiformis, and Bacillus subtillis and of the soil fungi Penicillium oxalicum and Arthroderma cookiellum was either inhibited or not affected by cladode extracts. Cladode extracts stimulated the growth of the actinomycete Streptomyces albus and the fungus Rhizopus homothallicus. The magnitude (as great as 100%) of the increase in growth caused by tissue extracts for the Casuarina-derived Frankia strains relative to other soil microbes suggests a host-specific enhancement of the microsymbiont.

Pseudomonas coleopterorum sp. nov., a cellulase-producing bacterium isolated from the bark beetle Hylesinus fraxini

We isolated a strain coded Esc2Am(T) during a study focused on the microbial diversity of adult specimens of the bark beetle Hylesinus fraxini. Its 16S rRNA gene sequence had 99.4% similarity with respect to its closest relative, Pseudomonas rhizosphaerae IH5(T). The analysis of partial sequences of the housekeeping genes rpoB, rpoD and gyrB confirmed that strain Esc2Am(T) formed a cluster with P. rhizosphaerae IH5(T) clearly separated from the remaining species of the genus Pseudomonas. Strain Esc2Am(T) had polar flagella and could grow at temperatures from 4 °C to 30 °C. The respiratory quinone was Q9 and the main fatty acids were C16 : 0, C18 : 1ω7c and/or C18 : 1ω6c in summed feature 8 and C16 : 1ω7c and/or C16 : 1ω6c in summed feature 3. DNA-DNA hybridization results showed 51% relatedness with respect to P. rhizosphaerae IH5(T). Oxidase, catalase and urease-positive, the arginine dihydrolase system was present but nitrate reduction and β-galactosidase production were negative. Aesculin hydrolysis was positive. Based on the results from the genotypic, phenotypic and chemotaxonomic analyses, we propose the classification of strain Esc2Am(T) as representing a novel species of the genus Pseudomonas, for which we propose the name Pseudomonas coleopterorum sp. nov. The type strain is Esc2Am(T) ( = LMG 28558(T)= CECT 8695(T)).