Speciation Features of Ferdinandcohnia quinoae sp. nov to Adapt to the Plant Host.

The bacterial strain SECRCQ15T was isolated from seeds of Chenopodium quinoa in Spain. Phylogenetic, chemotaxonomic, and phenotypic analyses, as well as genome similarity indices, support the classification of the strain into a novel species of the genus Ferdinandcohnia, for which we propose the name Ferdinandcohnia quinoae sp. nov. To dig deep into the speciation features of the strain SECRCQ15T, we performed a comparative genomic analysis of the genome of this strain and those of the type strains of species from the genus Ferdinandcohnia. We found several genes related with plant growth-promoting mechanisms within the SECRCQ15T genome. We also found that singletons of F. quinoae SECRCQ15T are mainly related to the use of carbohydrates, which is a common trait of plant-associated bacteria. To further reveal speciation events in this strain, we revealed genes undergoing diversifying selection (e.g., genes encoding ribosomal proteins) and functions likely lost due to pseudogenization. Also, we found that this novel species contains 138 plant-associated gene-cluster functions that are unique within the genus Ferdinandcohnia. These features may explain both the ecological and taxonomical differentiation of this new taxon.

High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia.

The genus Micromonospora has been found in nodules of several legumes and some new species of this genus were isolated from these plant organs. In this study we analysed the taxonomic diversity of Micromonospora strains isolated from alfalfa nodules in Spain and Australia on the basis of three phylogenetic markers, the rrs and gyrB genes and 16S-23S intergenic spacer (ITS). The genome analysis of selected strains representative of different clusters or lineages found after rrs, gyrB and ITS analyses confirmed the results obtained with these phylogenetic markers. They showed that the analysed strains belong to at least 18 Micromonospora species including previously described ones, such as Micromonospora noduli, Micromonospora ureilytica, Micromonospora taraxaci, Micromonospora zamorensis, Micromonospora aurantiaca and Micromonospora tulbaghiae. Most of these strains belong to undescribed species of Micromonospora showing the high taxonomic diversity of strains from this genus inhabiting alfalfa nodules. Although Micromonospora strains are not able to induce the formation of these nodules, and it seems that they do not contribute to fix atmospheric nitrogen, they could play a role related with the mechanisms of plant growth promotion and pathogen protection presented by Micromonospora strains isolated from legume nodules.

Heterologous Expression of Rhizobial CelC2 Cellulase Impairs Symbiotic Signaling and Nodulation in Medicago truncatula.

The infection of legume plants by rhizobia is tightly regulated to ensure accurate bacterial penetration, infection, and development of functionally efficient nitrogen-fixing root nodules. Rhizobial Nod factors (NF) have key roles in the elicitation of nodulation signaling. Infection of white clover roots also involves the tightly regulated specific breakdown of the noncrystalline apex of cell walls in growing root hairs, which is mediated by Rhizobium leguminosarum bv. trifolii cellulase CelC2. Here, we have analyzed the impact of this endoglucanase on symbiotic signaling in the model legume Medicago truncatula. Ensifer meliloti constitutively expressing celC gene exhibited delayed nodulation and elicited aberrant ineffective nodules, hampering plant growth in the absence of nitrogen. Cotreatment of roots with NF and CelC2 altered Ca2+ spiking in root hairs and induction of the early nodulin gene ENOD11. Our data suggest that CelC2 alters early signaling between partners in the rhizobia-legume interaction.

The rhizosphere microbiome of burned holm-oak: potential role of the genus Arthrobacter in the recovery of burned soils.

After a forest wildfire, the microbial communities have a transient alteration in their composition. The role of the soil microbial community in the recovery of an ecosystem following such an event remains poorly understood. Thus, it is necessary to understand the plant-microbe interactions that occur in burned soils. By high-throughput sequencing, we identified the main bacterial taxa of burnt holm-oak rhizosphere, then we obtained an isolate collection of the most abundant genus and its growth promoting activities were characterised. 16S rRNA amplicon sequencing showed that the genus Arthrobacter comprised more than 21% of the total community. 55 Arthrobacter strains were isolated and characterized using RAPDs and sequencing of the almost complete 16S rRNA gene. Our results indicate that isolated Arthrobacter strains present a very high genetic diversity, and they could play an important ecological role in interaction with the host plant by enhancing aerial growth. Most of the selected strains exhibited a great ability to degrade organic polymers in vitro as well as possibly presenting a direct mechanism for plant growth promotion. All the above data suggests that Arthrobacter can be considered as an excellent PGP rhizobacterium that may play an important role in the recovery of burned holm-oak forests.

Analysis of rhizobial endosymbionts of Vicia, Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain).

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.

Bacillus terrae sp. nov. isolated from Cistus ladanifer rhizosphere soil.

A bacterial strain designated RA9T was isolated from a root of Cistus ladanifer in Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate into the genus Bacillus with its closest relatives being Bacillus fortis R-6514T and Bacillus fordii R-7190T with 98.2 % similarity in both cases. DNA-DNA hybridization studies showed mean relatedness values of 29 and 30 %, respectively, between strain RA9T and the type strains of B. fortis and B. fordii. Cells of the isolate were Gram-stain-positive, motile, sporulating rods. Catalase and oxidase were positive. Gelatin, starch and casein were not hydrolysed. Menaquinone MK-7 was the only menaquinone detected and iso-C15 : 0 and anteiso-C15 : 0 were the major fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, one unidentifed glycolipid and one unidentified lipid. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 43.1 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RA9T should be considered as representing a novel species of the genus Bacillus, for which the name Bacillus terrae sp. nov. is proposed. The type strain is RA9T (=LMG 29736T=CECT 9170T).

Paenibacillus hispanicus sp. nov. isolated from Triticum aestivum roots.

A bacterial strain designated AMTAE16T was isolated from a root of wheat in Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate in the genus Paenibacilluswith its closest relative being Paenibacillus daejeonensis AP-20T with 99.0 % 16S rRNA gene sequence similarity. DNA-DNA hybridization studies showed a mean of 30 % DNADNA relatedness between strain AMTAE16T and the type strain of P. daejeonensis. The isolate was a Gram-stainvariable, motile and sporulating rod. Catalase and oxidase activities were positive. Gelatin and starch were hydrolysed but not casein. Growth was supported by many carbohydrates and organic acids as carbon source. MK-7 was the only menaquinone detected and anteiso-C15 : 0, C16 : 0 and iso-C16 : 0 were the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids and two unidentified lipids. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 55.4 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain AMTAE16T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus hispanicus sp. nov. is proposed. The type strain is AMTAE16T(=LMG 29501T=CECT 9124T).

Cicer canariense, an endemic legume to the Canary Islands, is nodulated in mainland Spain by fast-growing strains from symbiovar trifolii phylogenetically related to Rhizobium leguminosarum.

Cicer canariense is a threatened endemic legume from the Canary Islands where it can be nodulated by mesorhizobial strains from the symbiovar ciceri, which is the common worldwide endosymbiont of Cicer arietinum linked to the genus Mesorhizobium. However, when C. canariense was cultivated in a soil from mainland Spain, where the symbiovar ciceri is present, only fast-growing rhizobial strains were unexpectedly isolated from its nodules. These strains were classified into the genus Rhizobium by analysis of the recA and atpD genes, and they were phylogenetically related to Rhizobium leguminosarum. The analysis of the nodC gene showed that the isolated strains belonged to the symbiovar trifolii that harbored a nodC allele (ß allele) different to that harbored by other strains from this symbiovar. Nodulation experiments carried out with the lacZ-labeled strain RCCHU01, representative of the ß nodC allele, showed that it induced curling of root hairs, infected them through infection threads, and formed typical indeterminate nodules where nitrogen fixation took place. This represents a case of exceptional performance between the symbiovar trifolii and a legume from the tribe Cicereae that opens up new possibilities and provides new insights into the study of rhizobia-legume symbiosis.

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.

Revision of the taxonomic status of type strains of Mesorhizobium loti and reclassification of strain USDA 3471T as the type strain of Mesorhizobiumerdmanii sp. nov. and ATCC 33669T as the type strain of Mesorhizobiumjarvisii sp. nov.

The species Mesorhizobim loti was isolated from nodules of Lotus corniculatus and its type strain deposited in several collections. Some of these type strains, such as those deposited in the USDA and ATCC collections before 1990, are not coincident with the original strain, NZP 2213T, deposited in the NZP culture collection. The analysis of the 16S rRNA gene showed that strains USDA 3471T and ATCC 33669T formed independent branches from that occupied by Mesorhizobium loti NZP 2213T and related to those occupied by Mesorhizobium opportunistum WSM2075T and Mesorhizobium huakuii IFO 15243T, respectively, with 99.9 % similarity in both cases. However, the analysis of concatenated recA, atpD and glnII genes with similarities lower than 96, 98 and 94 %, respectively, between strains USDA 3471T and M. opportunistum WSM2075T and between strains ATCC 33669T and M. huakuii IFO 15243T, indicated that the strains USDA 3471T and ATCC 33669T represent different species of the genus Mesorhizobium. These results were confirmed by DNA-DNA hybridization experiments and phenotypic characterization. Therefore, the two strains were reclassified as representatives of the two species Mesorhizobium erdmanii sp. nov. (type strain USDA 3471T = CECT 8631T = LMG 17826t2T) and Mesorhizobium jarvisii sp. nov. (type strain ATCC 33669T = CECT 8632T = LMG 28313T).

Revision of the taxonomic status of the species Rhizobium lupini and reclassification as Bradyrhizobium lupini comb. nov.

The species Rhizobium lupini was isolated from Lupinus nodules and included in the Approved Lists of Bacterial Names in 1980. Nevertheless, on the basis of the analysis of the type strain of this species available in DSMZ, DSM 30140(T), whose 16S rRNA gene was identical to that of the type strain of Bradyrhizobium japonicum , R. lupini was considered a later synonym of this species. In this study we confirmed that the strain DSM 30140(T) belongs to the species B. japonicum , but also that it cannot be the original strain of R. lupini because this species effectively nodulated Lupinus whereas strain DSM 30140(T) was able to nodulate soybean but not Lupinus. Since the original type strain of R. lupini was deposited into the USDA collection by L. W. Erdman under the accession number USDA 3051(T) we analysed the taxonomic status of this strain showing that although it belongs to the genus Bradyrhizobium instead of genus Rhizobium , it is phylogenetically distant from B. japonicum and closely related to Bradyrhizobium canariense . The type strains R. lupini USDA 3051(T) and B. canariense BTA-1(T) share 16S rRNA, recA and glnII gene sequences with similarities of 99.8%, 96.5% and 97.1%, respectively. They presented a DNA-DNA hybridization value of 36% and also differed in phenotypic characteristics and slightly in the proportions of some fatty acids. Therefore we propose the reclassification of the species Rhizobium lupini as Bradyrhizobium lupini comb. nov. The type strain is USDA 3051(T) ( = CECT 8630(T) = LMG 28514(T)).

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.

The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense).

Genista versicolor is an endemic legume from Sierra Nevada National Park which constitutes one of the UNESCO-recognized Biosphere Reserves. In the present study, a collection of strains nodulating this legume was analysed in characteristic soils of this ecosystem. Most strains nodulating G. versicolor belonged to rrs group I within the genus Bradyrhizobium and only one strain, named GV137, belonged to rrs group II from which only a single species, B. retamae, has been described in Europe to date. Strain GV137, and some strains from rrs group I, belonged to putative new species of Bradyrhizobium, although most strains from group I belonged to B. canariense, according to the ITS fragment and atpD gene analysis. This result contrasted with those obtained in Genista tinctoria in Northeast Europe whose endosymbionts were identified as B. japonicum. The analysis of the symbiotic nodC and nifH genes carried by G. versicolor-nodulating strains showed that most of them belonged to symbiovar genistearum, as did those isolated from G. tinctoria. Nevertheless, strain GV137, belonging to rrs group II, formed a divergent lineage that constituted a novel symbiovar within the genus Bradyrhizobium for which the name sierranevadense is proposed. This finding showed that the Genisteae are not restrictive legumes only nodulated by symbiovar genistearum, since Genista is a promiscuous legume nodulated by at least two symbiovars of Bradyrhizobium, as occurs in Retama species.

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: application to the identification of Lupinus nodulating strains.

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species.

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.

The celC gene, a new phylogenetic marker useful for taxonomic studies in Rhizobium.

The celC gene codifies for a cellulase that fulfils a very significant role in the infection process of clover by Rhizobium leguminosarum. This gene is located in the celABC operon present in the chromosome of strains representing R. leguminosarum, Rhizobium etli and Rhizobium radiobacter whose genomes have been completely sequenced. Nevertheless, the existence of this gene in other species of the genus Rhizobium had not been investigated to date. In this study, the celC gene was analysed for the first time in several species of this genus isolated from legume nodules and plant tumours, in order to compare the celC phylogeny to those of other chromosomal and plasmidic genes. The results obtained showed that phylogenies of celC and chromosomal genes, such as rrs, recA and atpD, were completely congruent, whereas no relation was found with symbiotic or virulence genes. Therefore, the suitability and usefulness of the celC gene to differentiate species of the genus Rhizobium, especially those with closely related rrs genes, was highlighted. Consequently, the taxonomic status of several strains of the genus Rhizobium with completely sequenced genomes is also discussed.

Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2.

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.

Auraticoccus monumenti gen. nov., sp. nov., an actinomycete isolated from a deteriorated sandstone monument.

A Gram-type-positive, strictly aerobic actinobacterium, designated strain MON 2.2(T), was isolated from the surface of a sandstone monument. Cells with a coccoid shape, arranged in pairs or clusters, were non-motile and did not produce spores. The 10 closest 16S rRNA gene sequence matches (~95 % similarity) found in the public databases were uncultured actinobacteria, while the closest cultured members indicated a phylogenetic relationship with members of the family Propionibacteriaceae (92-95 % similarity). Subsequent phylogenetic analysis placed the new isolate within the radiation of the genera Friedmanniella and Microlunatus, but forming an independent branch. Chemotaxonomic markers were consistent with the classification of strain MON 2.2(T) in the family Propionibacteriaceae, amongst the genera containing ll-diaminopimelic acid in their peptidoglycan. Characteristic fatty acids iso-C(15 : 0) and anteiso-C(15 : 0) also supported its affiliation to this taxon; however, polar lipid and menaquinone compositions clearly differentiated strain MON 2.2(T) from other genera in the family. On the basis of these results and additional physiological data obtained in the present study, it is proposed that strain MON 2.2(T) be classified in a novel species in a new genus, for which the name Auraticoccus monumenti gen. nov., sp. nov. is proposed. The type strain of Auraticoccus monumenti is MON 2.2(T) ( = CECT 7672(T)  = DSM 23257(T)  = LMG 25551(T)).