Alvaro Peix

Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions

The efficacy of a strain of Mesorhizobium mediterraneum to enhance the growth and phosphorous content in chickpea and barley plants was assessed in a soil with and without the addition of phospates in a growth chamber. The results obtained show that the strain PECA21 was able to mobilize phosphorous efficiently in both plants when tricalcium phosphate was added to the soil. In barley and chickpea growing in soils treated with insoluble phosphates and inoculated with strain PECA21 the phosphorous content was significantly increased in a 100 and 125%, respectively. Also, the dry matter, nitrogen, potassium, calcium and magnesium content in both plants was significantly increased in inoculated soil added with insoluble phosphate. These results show that the inoculation of a soil with rhizobia should not be based only on the effectiveness of the strains with respect to their nitrogen fixation potential, since these microorganisms can increase the growth of plants by means of other mechanisms, for example the phosphate solubilization. q 2001 Elsevier Science Ltd. All rights reserved.

Historical evolution and current status of the taxonomy of genus Pseudomonas

The genus Pseudomonas was described in 1894 and is one of the most diverse and ubiquitous bacterial genera whose species have been isolated worldwide in all kinds of environments, from the Antarctica to the Tropics, present in sediments, clinical samples, plant, fungi and diseased animal specimens, water, soil, plant rhizosphere, sea, deserts, etc. The taxonomy of the genus has been controversial for years since a lot of bacterial taxa initially included in genus Pseudomonas have been reclassified in other genera or species from a different class of Proteobacteria over the years, as techniques for characterization and classification of microorganisms improved, aiming to set a phylogenetic classification of the species. In this review, the historical evolution of the taxonomy of Pseudomonas is described, and the currently valid criteria and future challenges for taxonomy of the genus and techniques used to achieve the necessary characterization for classifying the species are discussed. Finally, all the validly published Pseudomonas species at present are listed with an overview of their diversity and ecology.

Bacterial Associations with Legumes

Legumes form a large group of plants that constitute the third largest family of angiosperms, including near 20,000 species and 750 genera. Most of them have the ability to establish symbioses with diazotrophic bacteria, collectively known as rhizobia, which induce root nodules where biological nitrogen fixation takes place, conferring legumes a relevant ecological advantage. This group of bacteria that for many years was thought to be formed by a scarce number of genera and species within alpha proteobacteria, shows nowadays an important genetic diversity including species phylogenetically divergent both in core and symbiotic genes sequences. Together with rhizobia, other endophytic bacteria are present in legume nodules coexisting with rhizobial strains and their ecological role remains unknown in most cases, but they likely have an effect in plant health, plant growth or even in the rhizobia-legume symbiosis. In this review we present an overview of the associations of bacteria with legumes, the current available knowledge on the phylogenetic diversity of both rhizobia and endophytic bacteria inhabiting root nodules, and the symbiotic features used to define symbiovars in rhizobia.

Bradyrhizobium pachyrhizi sp. nov. and Bradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachyrhizus erosus.

Several strains isolated from the legume Pachyrhizus erosus were characterized on the basis of diverse genetic, phenotypic and symbiotic approaches. These novel strains formed two groups closely related to Bradyrhizobium elkanii according to their 16S rRNA gene sequences. Strains PAC48T and PAC68T, designated as the type strains of these two groups, presented 99.8 and 99.1% similarity, respectively, in their 16S rRNA gene sequences with respect to B. elkanii USDA 76T. In spite of these high similarity values, the analysis of additional phylogenetic markers such as atpD and glnII genes and the 16S-23S intergenic spacer (ITS) showed that strains PAC48T and PAC68T represented two separate novel species of the genus Bradyrhizobium with B. elkanii as their closest relative. Phenotypic differences among the novel strains isolated from Pachyrhizus and B. elkanii were found regarding the assimilation of carbon sources and antibiotic resistance. All these differences were congruent with DNA-DNA hybridization analysis which revealed 21% genetic relatedness between strains PAC48T and PAC68T and 46% and 25%, respectively, between these strains and B. elkanii LMG 6134T. The nodD and nifH genes of strains PAC48T and PAC68T were phylogenetically divergent from those of bradyrhizobia species that nodulate soybean. Soybean was not nodulated by the novel Pachyrhizus isolates. Based on the genotypic and phenotypic data obtained in this study, the new strains represent two novel species for which the names Bradyrhizobium pachyrhizi sp. nov. (type strain PAC48T=LMG 24246T=CECT 7396T) and Bradyrhizobium jicamae sp. nov. (type strain PAC68T=LMG 24556T=CECT 7395T) are proposed.

Biodiversity of populations of phosphate solubilizing rhizobia that nodulates chickpea in different Spanish soils

Within rhizobia, two species nodulating chickpea, Mesorhizobium ciceri and Mesorhizobium mediterraneum, are known as good phosphate solubilizers. For this reason, we have analysed the ability to solubilize phosphate of a wide number of strains isolated from Cicer arietinum growing in several soils in Spain. The aim of this work was to analyse microbial populations nodulating chickpea, that are able to solubilize phosphates, using molecular techniques. In the present work we analyzed 19 strains isolated from effective nodules of C. arietinum growing in three soils from the North of Spain. Nineteen strains showed ability to solubilize phosphate in YED-P medium. These strains were separated into 4 groups according to the results obtained by 879F-RAPD fingerprinting. The 16S rDNA sequencing of a representative strain from each group allowed the identification of strains as belonging to the genus Mesorhizobium. Strains from groups I and II showed a 99.4% and 99.2% similarity with M. mediterraneum UPM-CA142T, respectively. The strains from group III were related to M. tianshanense USDA 3592T at a 99.4% similarity level. Finally, the strain from group IV was related to M. ciceri USDA 3383T with a 99.3% similarity. The LMW RNA profiles confirmed these results. Strains from groups I and II showed an identical LMW RNA profile to that of M. mediterraneum UPM-CA142T; the profile of strains from group III was identical to that of M.␣tianshanense USDA 3592T and the profile of strains from group IV was identical to that of M. ciceri USDA 3383T. Different 879F-RAPD patterns were obtained for strains of the group I, group II and the M.␣mediterraneum type strain (UPM-CA142T). The 879-RAPD patterns obtained for group III also differed from the pattern shown by M.tianshanense USDA 3592T. Finally, the patterns between group IV and M. ciceri USDA 3383T were also different. These results suggest that groups I and II may be subspecies of M. mediterraneum, group III a subspecies of M. tianshanense and group IV a subspecies of M. ciceri. Nevertheless, more studies are needed to establish the taxonomic status of strains isolated in this study.

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.

Diversity of Potassium-Solubilizing Microorganisms and Their Interactions with Plants

Potassium is, together with nitrogen and phosphorus, an essential element for plant nutrition, being the third element in the classical chemical fertilizers NPK. Deficiencies in K results in plants with poorly developed roots, small seeds and lower yields, and therefore the availability of this element is crucial for plant growth and development. The plants take this element from soil, but high amounts of K present in soils correspond to insoluble forms from rocks and silicate minerals. The solubilization of K by microorganisms is then a reliable alternative to make available this element for plants. In this chapter, we review the microorganisms reported as K solubilizers and their phylogenetic diversity, including filamentous fungi such as Penicillium or Aspergillus, yeasts such as Torulaspora and bacteria of many different genera from phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Some of these potassium-solubilizing microorganisms (KSMs) play a positive role in the growth of different plants, which is relevant to sustainable agriculture schemes, and therefore this is a promising field of study in the ‘greening’ era.

Growth promotion of common bean (Phaseolus vulgaris L.) by a strain of Burkholderia cepacia under growth chamber conditions

We isolated a strain of Burkholderia cepacia (SAOCV2), which solubilizes inorganic phosphates and antagonizes Fusarium oxysporum f. sp. phaseoli and Fusarium solani in vitro, from soil. However, this strain does not have antibiotic activity against the bacteria tested in this study, which included bacterial plant pathogens and rhizobia. The efficacy of this strain to enhance the growth and P content in common bean was assesed in a soil traditionally cultivated with this species. In this soil, the common bean is affected by Fusarium. Our results show that the strain SAOCV2 was able to mobilize P efficiently in the common bean, so that its P content was increased by 44% with respect to uninoculated plants, whereas it was not significantly different with respect to the plants treated with fungicide. Also, the N content in plants inoculated with the strain SAOCV2 was significantly higher than in uninoculated plants. This result is correlated with a larger number of nodules in plants inoculated with SAOCV2 and in plants treated with fungicide and indicates that the inhibition of fungal growth enhances the bacterial community in the plant rhizosphere, including rhizobia. Our results suggest that the inoculation with strain SAOCV2 promotes the growth of common bean by several mechanisms, that include P mobilization, antagonism towards pathogenic species of Fusarium and, indirectly, by an increase in nodulation that may lead to an increase in N2 fixation.

Bradyrhizobium cytisi sp. nov., isolated from effective nodules of Cytisus villosus

Several strains isolated from Cytisus villosus nodules have been characterized based on their diverse genetic, phenotypic and symbiotic characteristics. According to 16S rRNA gene sequence analysis, the isolates formed a group that was closely related to Bradyrhizobium canariense BTA-1(T) with 99.4% similarity. Analysis of three housekeeping genes, recA, atpD and glnII, suggested that the C. villosus strains represent a novel Bradyrhizobium species most closely related to B. canariense BTA-1(T) with similarities of 94.2, 96.7 and 94.5%, respectively. All these differences were congruent with DNA-DNA hybridization analysis, which revealed 31% relatedness between a representative strain (CTAW11(T)) isolated from C. villosus nodules and B. canariense BTA-1(T). Phenotypic differences among the strains isolated from C. villosus and B. canariense were based on assimilation of carbon and nitrogen sources. The nodC and nifH genes of strain CTAW11(T) were phylogenetically related to those of strains belonging to bv. genistearum and divergent from those of bv. glycinearum and, accordingly, they do not nodulate soybean. Based on the genotypic and phenotypic data obtained in this study, our strains should be classified as representatives of a novel species for which the name Bradyrhizobium cytisi sp. nov. is proposed; the type strain is CTAW11(T) (=LMG 25866(T)=CECT 7749(T)).

The Coexistence of Symbiosis and Pathogenicity-Determining Genes in Rhizobium rhizogenes Strains Enables Them to Induce Nodules and Tumors or Hairy Roots in Plants

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.