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La kombucha es una bebida que ha ganado popularidad en los últimos años por sus aparentes propiedades probióticas. En este estudio se aislaron e identificaron microorganismos presentes en una muestra de cultivo starter de elaboración casera y en tres marcas comerciales de kombucha para determinar y comparar sus composiciones microbianas. Para la identificación de bacterias y levaduras se emplearon tanto la espectrometría de masas Matrix Assisted Laser Desorption/Ionization-Time of Flight como secuenciación Sanger, analizándose tanto sus espectros como las secuencias derivadas de los genes marcadores taxonómicos de hongos (ITS) y bacterias (16S rRNA). Los resultados demostraron una amplia variabilidad de géneros microbianos entre muestras y una alta presencia de posibles contaminaciones. Además, la presencia de especies con potencial probiótico fue menor a la esperada. Mediante este estudio se concluyó que la kombucha es susceptible de presentar tanto microorganismos beneficiosos como perjudiciales para la salud, poniendo de manifiesto la necesidad de endurecer las medidas higiénicas y la legislación en torno a la producción de kombucha y otras bebidas probióticas.

The genus Sinorhizobium comprises rhizobia that fix nitrogen in symbiosis with legumes. To support taxonomic studies of this genus and of rhizobia more broadly, we report complete genome sequences and annotations for the species type strains Sinorhizobium garamanticum LMG 24692 and Sinorhizobium numidicum LMG 27395 and CIP 109850.

The genusSinorhizobiumcomprises rhizobia that fix nitrogen in symbiosis with legumes. To support taxonomic studies of this genus and of rhizobia more broadly, we report complete genome sequences and annotations for the species type strainsSinorhizobium garamanticumLMG 24692TandSinorhizobium numidicumLMG 27395Tand CIP 109850T. Average nucleotide identity and core-genome phylogenetic analyses confirm thatS. garamanticumandS. numidicumrepresent distinct species.

Climate change, water scarcity, population growth, and food shortage are some of the threatening challenges being faced in today's world. Among different types of stresses, drought stress presents a persistent challenge for global food production, however, its harshness and intensity are supposed to expand in the imminent future. The most striking effects of drought stress on plants are stunted growth, severe damage to photosynthetic apparatus, reduction in photosynthesis, reduction in seed germination, and nutrient uptake. To deal with the destructive effect of drought stress on plants, it is necessary to consider its effects, mechanisms of action, the agronomic and genetic basis for sustainable management. Therefore, there is an urgent need for sustainable solutions to cope up with the negative impact of drought stress. This review focuses on the detrimental effects of drought stress on plants' morphological, physiological, and biochemical characteristics and recommends suitable drought management techniques to reduce the severity of drought stress. We summarize the effect of drought stress on physiological and biochemical parameters (such as germination, photosynthesis, biomass, water status, and nutrient uptake) and yield. Overall, in this article, we have reviewed the role of different phytohormones, osmolytes, exogenous compounds, proteins, plant growth-promoting microbes (PGPM), omics approaches, and genome editing technologies like clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) in alleviating drought effects in plants. We also proposed that developing drought-tolerant plant varieties requires the combined use of biotechnological and agronomic approaches and cutting-edge genome editing (GE) tools.

12 páginas, 7 figuras y 2 tablas, Melinis minutiflora is an invasive species that threatens the biodiversity of the endemic vegetation of the campo rupestre biome in Brazil, displacing the native vegetation and favouring fire spread. As M. minutiflora invasion has been associated with a high nitrogen (N) demand, we assessed changes in N cycle under four treatments: two treatments with contrasting invasion levels (above and below 50%) and two un-invaded control treatments with native vegetation, in the presence or absence of the leguminous species Periandra mediterranea. This latter species was considered to be the main N source in this site due to its ability to fix N2 in association with Bradyrhizobia species. Soil proteolytic activity was high in treatments with P. mediterranea and in those severely invaded, but not in the first steps of invasion. While ammonium was the N-chemical species dominant in plots with native species, including P.mediterranea, soil nitrate prevailed only in fully invaded plots due to the stimulation of the nitrifying bacterial (AOB) and archaeal (AOA) populations carrying the amoA gene. However, in the presence of P. mediterranea, either in the beginning of the invasion or in uninvaded plots, we observed an inhibition of the nitrifying microbial populations and nitrate formation, suggesting that this is a biotic resistance strategy elicited by P. mediterranea to compete with M. minutiflora. Therefore, the inhibition of proteolytic activity and the nitrification process were the strategies elicited by P.mediterranea to constrain M.munitiflora invasion.

The genus Rhizobium is well known in the context of its interaction with leguminous plants. The symbiosis Rhizobium-legume constitutes a significant source of ammonia in the biosphere. Rhizobium species have been studied and applied as biofertilizers for decades in legumes and nonlegumes, due to the potential as N-fixer and plant growth promoter. Since its discovery, conventional culture-dependent techniques were used to isolate Rhizobium members from their natural niche, the nodule, and their identification was routinely performed via 16S rRNA gene and different housekeeping genes. Biotechnological advances based on the use of omics-based technologies showed that species belonging to the genus Rhizobium are keystone taxa in several diverse environments, such as forests, agricultural land, Arctic, and Antarctic ecosystems, contaminated soils and plant-associated microbiota. In this chapter, we will summarize the advances in the study of the Rhizobium genus, from culturomics strategies to modern omics methodologies, mostly based on next-generation sequencing approaches. These cutting-edge molecular approaches are fundamental in the study of the behavior of Rhizobium species in their interaction with Non-leguminous plants, supporting their potential as an ecological alternative to chemical fertilizers in the battle against Climatic Change.

In the present work, we analyse the genomic and phenotypic characteristics of a strain named RZME27T isolated from roots of a Zea mays plant grown in Spain. The phylogenetic analyses of 16S rRNA gene and whole genome sequences showed that the strain RZME27T clustered with the type strains of Neorhizobium galegae and Pseudorhizobium pelagicum from the family Rhizobiaceae. This family encompasses several genera establishing symbiosis with legumes, but the genes involved in nodulation and nitrogen fixation are absent in its genome. Nevertheless, genes related to plant colonization, such as those involved in motility, chemotaxis, quorum sensing, exopolysaccharide biosynthesis and hydrolytic enzymes production were found. The comparative pangenomic analyses showed that 78 protein clusters present in the strain RZME27T were not found in the type strains of its closest relatives N. galegae and P. pelagicum. The calculated average nucleotide identity (ANI) values between the strain RZME27T and the type strains of N. galegae and P. pelagicum were 75.61% and 75.1%, respectively, similar or lower than those found for other genera from family Rhizobiaceae. Several phenotypic differences were also found, highlighting the absence of the fatty acid C19:0 cyclo ω8c and propionate assimilation. These results support the definition of a novel genus and species named Endobacterium cerealis gen. nov. sp. nov. whose type strain is RZME27T.

Mesorhizobium contains species widely known as nitrogen-fixing bacteria with legumes, but their ability to promote the growth of non-legumes has been poorly studied. Here, we analyzed the production of indole acetic acid (IAA), siderophores and the solubilization of phosphate and potassium in a collection of 24 strains belonging to different Mesorhizobium species. All these strains produce IAA, 46% solubilized potassium, 33% solubilize phosphate and 17% produce siderophores. The highest production of IAA was found in the strains Mesorhizobium ciceri CCANP14 and Mesorhizobium tamadayense CCANP122, which were also able to solubilize potassium. Moreover, the strain CCANP14 showed the maximum phosphate solubilization index, and the strain CCANP122 was able to produce siderophores. These two strains were able to produce cellulases and cellulose and to originate biofilms in abiotic surfaces and tomato root surface. Tomato seedlings responded positively to the inoculation with these two strains, showing significantly higher plant growth traits than uninoculated seedlings. This is the first report about the potential of different Mesorhizobium species to promote the growth of a vegetable. Considering their use as safe for humans, animals and plants, they are an environmentally friendly alternative to chemical fertilizers for non-legume crops in the framework of sustainable agriculture., This research was supported by the Ministerio de Medio Ambiente y Medio Rural y Marino, Organismo Autónomo de Parques Nacionales (Ref. 111/2010) to M.L.-B. and by the Strategic Research Programs for Units of Excellence from Junta de Castilla y León (CLU-2O18-04) to E.V. It was also funded by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia, in the framework of the project POCI-01-0145-FEDER-016810 (PTDC/AGR-PRO/2978/2014 and the Project UIDB/05183/2020) to E.M. E.M. acknowledges an FCT contract from the Individual Call to Scientific Employment Stimulus 2017 (CEECIND/00270/2017), which funds her position.

5 páginas, 1 figura, 1 tabla, The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus Agrobacterium contained the non-pathogenic species Agrobacterium radiobacter and the plant pathogenic species Agrobacterium tumefaciens and Agrobacterium rhizogenes. At the end of the past century two new pathogenic species, Agrobacterium rubi and Agrobacterium vitis, were added to the genus. Already in the present century these species plus Agrobacterium larrymoorei were reclassified into genus Rhizobium. This reclassification was controversial and for a time both genus names were used when new species were described. Few years ago, after a taxonomic revision based on genomic data, the old species A. rhizogenes was maintained in the genus Rhizobium, the old species A. vitis was transferred to the genus Allorhizobium and several Rhizobium species were transferred to the genus Agrobacterium, which currently contains 14 species including the old species A. radiobacter, A. tumefaciens, A. rubi and A. larrymoorei. Most of these species are able to produce tumours in different plants, nevertheless the genus Agrobacterium also encompasses non-pathogenic species, one species able to nodulate legumes and one human pathogenic species. Taking into account that the species affiliations to five Agrobacterium genomospecies have not been determined yet, an increase in the number of species within this genus is expected in the near future., This work was supported by MINECO (Spanish Central Gov-ernment) Grant AGL2013-48098-P to EV. EM was granted by apostdoctoral contract associated to this projec

Beneficial bacteria associated with plants have evolved for thousands of years together with their hosts to an intricate communication system that allow the recognition and penetration into plant tissues without harming them. Within the molecules involved in this communication system, the enzymes produced by the bacteria have an important role and some of them have been shown essential at first steps of plant colonization. In this chapter, we analyze the implication of some of the most well-known enzymes related to plant probiotic bacteria and their hosts, the steps at which these enzymes participate to allow the recognition by the plants and the bacterial penetration into their inner tissues. Between these enzymes we will examine the importance of (i) cellulases, produced by important plant growth promoters to penetrate plant tissues; (ii) chitinases, implicated in the defense of the plant against fungi and recognition by the plants; (iii) lectins, implicated in the attachment and first recognition steps; (iv) pectinases, which are usually expressed early during infection, in the penetration steps; and (v) xylanases, implicated in the recycling at senescence, amongst others. The upregulated expression of some of these enzymes in plant growth promoting bacteria was surprising at first, as they would be expected in pathogens, not in mutualistic microorganisms. However, as more data are available, the implication of hydrolytic enzymes in beneficial plant colonization is become clear.

© 2020 by the authors., In the present work, we analyse the genomic and phenotypic characteristics of a strain named RZME27T isolated from roots of a Zea mays plant grown in Spain. The phylogenetic analyses of 16S rRNA gene and whole genome sequences showed that the strain RZME27T clustered with the type strains of Neorhizobium galegae and Pseudorhizobium pelagicum from the family Rhizobiaceae. This family encompasses several genera establishing symbiosis with legumes, but the genes involved in nodulation and nitrogen fixation are absent in its genome. Nevertheless, genes related to plant colonization, such as those involved in motility, chemotaxis, quorum sensing, exopolysaccharide biosynthesis and hydrolytic enzymes production were found. The comparative pangenomic analyses showed that 78 protein clusters present in the strain RZME27T were not found in the type strains of its closest relatives N. galegae and P. pelagicum. The calculated average nucleotide identity (ANI) values between the strain RZME27T and the type strains of N. galegae and P. pelagicum were 75.61% and 75.1%, respectively, similar or lower than those found for other genera from family Rhizobiaceae. Several phenotypic differences were also found, highlighting the absence of the fatty acid C19:0 cyclo ω8c and propionate assimilation. These results support the definition of a novel genus and species named Endobacterium cerealis gen. nov. sp. nov. whose type strain is RZME27T., This work was supported by MINECO (Spanish Central Government) Grant AGL2013-48098-P to EV. EM was supported by a postdoctoral contract associated to this project and then, with a FCT contract from the Individual Call to Scientific Employment Stimulus 2017 (CEECIND/00270/2017). J.D.F.-F. was the recipient of a predoctoral fellowship from University of Salamanca

8 páginas, 4 tablas, 3 figuras, A bacterial strain designated as RZME10T was isolated from a Zea mays L. root collected in Spain. Results of analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Agrobacterium with Agrobacterium larrymoorei ATCC 51759T being the most closely related species with 99.9 % sequence similarity. The similarity values of the rpoB, recA, gyrB, atpD and glnII genes between strain RZME10T and A. larrymoorei ATCC 51759T were 93.5, 90.0, 88.7, 87.9 and 90.1 %, respectively. The estimated average nucleotide identity using blast and digital DNA–DNA hybridization values between these two strains were 80.4 and 30.2 %, respectively. The major fatty acids of strain RZME10T are those from summed feature 8 (C18 : 1 ω6c/C18 : 1 ω7c) and C16 : 0. Pathogenicity tests on tomato and carrot roots showed that strain RZME10T was not able to induce plant tumours. Based on the results of genomic, chemotaxonomic and phenotypic analyses, we propose that strain RZME10T represents a novel species named Agrobacterium cavarae sp. nov. (type strain RZME10T=CECT 9795T=LMG 31257T)., This work was supported by an MINECO (Spanish Central Government) grant AGL2013-48098-P to E.V. E.M. was granted a postdoctoral contract associated with this project. J.D.F.F. was recipient of a predoctoral fellowship from the University of Salamanca.

The author wishes to make an erratum to the published version of the paper [...]

A bacterial strain designated CRZM18RT was isolated from a root of Zea mays in Spain. The analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Rhizobium , with Rhizobium cellulosilyticum ALA10B2T and Rhizobium yantingense H66T being the most closely related species with 98.3 and 97.9 % sequence similarity, respectively. The analysis of the concatenated recA and atpD genes showed that strain CRZM18RT forms a cluster with these species and also with Rhizobium smilacinae PTYR-5T, but the recA and atpD genes of strain CRZM18RT were phylogenetically distant, with identities lower than 90 and 96 %, respectively. DNA–DNA hybridization analysis showed mean relatedness of 43, 22 and 38 % with respect to R. cellulosilyticum ALA10B2T, R. yantingense LMG 28229T and R. smilacinae LMG 27604T. Phenotypic characteristics also differed from those of the most closely related species of the genus Rhizobium . The major fatty acids were those from summed feature 8 (C18 : 1ω6c/C18 : 1ω7c) and C16 : 0. Based on the genotypic, chemotaxonomic and phenotypic data obtained in this study, we propose to classify strain CRZM18RT in a novel species named Rhizobium zeae sp. nov. (type strain CRZM18RT=LMG 29735T=CECT 9169T).

Polyhydroxyalkanoic acids (PHAs) are natural polyesters that can be used to produce bioplastics which are biodegradable. Numerous microorganisms accumulate PHAs as energy reserves. Combinations of different PHAs monomers lead to the production of bioplastics with very different properties. In the present work, we show the capability of strains belonging to various phylogenetic lineages within the genus Mesorhizobium, isolated from Lotus corniculatus nodules, to produce different PHA monomers. Among our strains, we found the production of 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxydodecanoate, and 3-hydroxyhexadecanoate. Most of the PHA-positive strains were phylogenetically related to the species M. jarvisii. However, our findings suggest that the ability to produce different monomers forming PHAs is strain-dependent.

Bacterial endophytes, a subset of a plant’s microbiota, can facilitate plant growth by a number of different mechanisms. The aims of this study were to assess the diversity and functionality of endophytic bacterial strains from internal root tissues of native legume species grown in two distinct sites in South of Portugal and to evaluate their ability to promote plant growth. Here, 122 endophytic bacterial isolates were obtained from 12 different native legume species. Most of these bacteria possess at least one of the plant growth-promoting features tested in vitro, with indole acetic acid production being the most common feature among the isolates followed by the production of siderophores and inorganic phosphate solubilization. The results of in planta experiments revealed that co-inoculation of chickpea plants with specific endophytic bacteria along with N2-fixing symbionts significantly improved the total biomass of chickpea plants, in particular when these plants were grown under saline conditions. Altogether, this study revealed that Mediterranean native legume species are a reservoir of plant growth-promoting bacteria, that are also tolerant to salinity and to toxic levels of Mn. Thus, these bacterial endophytes are well adapted to common constraints present in soils of this region which constitutes important factors to consider in the development of bacterial inoculants for stressful conditions in the Mediterranean region.

The aim of this study was to investigate whether the overexpression of NifA and NodD regulators contribute to the symbiotic improvement of chickpea mesorhizobia. The native strains V-15b, ST-2, and PMI-6 were transformed with extra copies of nifA or nodD genes and several plants trial were performed. Plant growth assays showed that nifA overexpression was able to improve the symbiotic effectiveness of V-15b, while nodD overexpression lead to the improvement of ST-2 and PMI-6. Hydroponic assays showed that plants inoculated with V15bnifA+ and PMI6nodD+ started developing nodules earlier than those inoculated with the corresponding control strains. In addition, the number of nodules was always higher in plants inoculated with the strains overexpressing the symbiotic genes. Analysis of histological sections of nodules formed by V15bnifA+ showed a more developed fixation zone when compared with control. On the other hand, nodules induced by PMI6nodD+ did not show a senescent zone, which was observed in nodules from plants inoculated with the control strain. Plants inoculated with PMI6nodD+ and ST2nodD+ showed a higher number of infection threads than the corresponding control inoculations. These results indicate that overexpressing nifA and nodD may be an important tool to achieve the improvement of the symbiotic performance of mesorhizobia.

The aims of this study were to isolate, identify and characterize culturable endophytic bacteria from chickpea (Cicer arietinum L.) roots grown in different soils. In addition, the effects of rhizobial inoculation, soil and stress on the functionality of those culturable endophytic bacterial communities were also investigated. Phylogenetic analysis based on partial 16S rRNA gene sequences revealed that the endophytic bacteria isolated in this work belong to the phyla Proteobacteria, Firmicutes and Actinobacteria, with Enterobacter and Pseudomonas being the most frequently observed genera. Production of indoleacetic acid and ammonia were the most widespread plant growth-promoting features, while antifungal activity was relatively rare among the isolates. Despite the fact that the majority of bacterial endophytes were salt- and Mn-tolerant, the isolates obtained from soil with Mn toxicity were generally more Mn-tolerant than those obtained from the same soil amended with dolomitic limestone. Several associations between an isolate’s genus and specific plant growth-promoting mechanisms were observed. The data suggest that soil strongly impacts the Mn tolerance of endophytic bacterial communities present in chickpea roots while rhizobial inoculation induces significant changes in terms of isolates’ plant growth-promoting abilities. In addition, this study also revealed chickpea-associated endophytic bacteria that could be exploited as sources with potential application in agriculture.

The concept of “organic farming” is based on a holistic view of farming systems, in which components are completely integrated into the system, such as the soil and its microbiota, climatic conditions, plants, and/or animals. The maintenance and the increase of fertility rely on the use of management practices and the application of different products from natural origins, which have the aim of providing high availability of nutrients for plant crops in agricultural soils. In this chapter, we will summarize the available information related to several inputs and practices allowed in the organic farming concept and vision, focusing on the benefits of their single or combined application. Moreover, we will introduce the latest novel applications of some of them, such as anaerobic digestion of manure, and present new alternatives, such as the use of biochar or the design of new biofertilization schemes.

Actinobacteria is one of the largest phyla within the domain Bacteria. This phylum comprises more than 400 genera heterogeneously distributed in up to 50 families, 20 orders and 6 classes, being composed with very diverse groups of microorganisms. Members included within this phylum were recovered from a wide range of aquatic and terrestrial environments and also from a huge number of higher organisms, including plants. Actinobacteria inhabiting soils and plants are well known as producers of bioactive molecules and as biocontrol agents, possessing antimicrobial activities mostly against pathogenic fungi and/or bacteria. Moreover, some of them have the capacity to exert beneficial effects on plant growth and development via different plant growth-promoting mechanisms, i.e., phytohormones biosynthesis, siderophore production, and phosphate solubilization, among others. The available genomic data revealed that members belonging to this phylum have a huge potential as Plant Probiotic Actinobacteria. A plethora of studies reported the isolation and identification of plant endophytic actinobacteria possessing those features and also their performance under controlled conditions. However, few studies show the effects of the inoculation of these actinobacteria on real field conditions. In this chapter, we will provide an overview of the available data on the Actinobacteria displaying plant growth-promoting features, particularly in the ones that already had applications in agriculture. Together with a correct taxonomic classification, we will present evidence that the Plant Probiotic Actinobacteria should be considered as a source of bacterial candidates that will be important for a future sustainable agriculture.

A bacterial strain designated AMTAE16 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-20 with 99.0 % 16S rRNA gene sequence similarity. DNA–DNA hybridization studies showed a mean of 30 % DNADNA relatedness between strain AMTAE16 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-C, C and iso-C 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 AMTAE16represents a novel species of the genus Paenibacillus, for which the name Paenibacillus hispanicus sp. nov. is proposed. The type strain is AMTAE16(=LMG 29501=CECT 9124)., This work was supported by MINECO (Spanish Central Government) Grants AGL2013-48098-P and AGL2013-40758-R. EM is granted by a postdoctoral contract of this project.

Rapeseed (Brassica napus L.) is an important crop worldwide, due to its multiple uses, such as a human food, animal feed and a bioenergetic crop. Traditionally, its cultivation is based on the use of chemical fertilizers, known to lead to several negative effects on human health and the environment. Plant growth-promoting bacteria may be used to reduce the need for chemical fertilizers, but efficient bacteria in controlled conditions frequently fail when applied to the fields. Bacterial endophytes, protected from the rhizospheric competitors and extreme environmental conditions, could overcome those problems and successfully promote the crops under field conditions. Here, we present a screening process among rapeseed bacterial endophytes to search for an efficient bacterial strain, which could be developed as an inoculant to biofertilize rapeseed crops. Based on in vitro, in planta, and in silico tests, we selected the strain Pseudomonas brassicacearum CDVBN10 as a promising candidate, this strain produces siderophores, solubilizes P, synthesizes cellulose and promotes plant height in 5 and 15 days-post-inoculation seedlings. The inoculation of strain CDVBN10 in a field trial with no addition of fertilizers showed significant improvements in pod numbers, pod dry weight and shoot dry weight. In addition, metagenome analysis of root endophytic bacterial communities of plants from this field trial indicated no alteration of the plant root bacterial microbiome, considering that the root microbiome plays an important role in plant fitness and development, we suggest this maintenance of the plant and its bacterial microbiome homeostasis as a positive result. Thus, Pseudomonas brassicacearum CDVBN10 seems to be a good biofertilizer to improve canola crops with no addition of chemical fertilizers, this the first study in which a plant growth-promoting (PGP) inoculant specifically designed for rapeseed crops significantly improves this crop&rsquo, s yields in field conditions.

10 páginas, 4 figuras y 2 tablas, The growing interest in a healthy lifestyle and in environmental protection is changing habits regarding food consumption and agricultural practices. Good agricultural practice is indispensable, particularly for raw vegetables, and can include the use of plant probiotic bacteria for the purpose of biofertilization. In this work we analysed the probiotic potential of the rhizobial strain PEPV40, identified as Rhizobium laguerreae through the analysis of the recA and atpD genes, on the growth of spinach plants. This strain presents several in vitro plant growth promotion mechanisms, such as phosphate solubilisation and the production of indole acetic acid and siderophores. The strain PEPV40 produces cellulose and forms biofilms on abiotic surfaces. GFP labelling of this strain showed that PEPV40 colonizes the roots of spinach plants, forming microcolonies typical of biofilm initiation. Inoculation with this strain significantly increases several vegetative parameters such as leaf number, size and weight, as well as chlorophyll and nitrogen contents. Therefore, our findings indicate, for the first time, that Rhizobium laguerreae is an excellent plant probiotic, which increases the yield and quality of spinach, a vegetable that is increasingly being consumed raw worldwide., This work was supported by Grants SA169U14 and SA058U16 from the Regional Government of Castile and Leon, and AGL2015–70510-R from MINECO (Spanish Ministry of Economy, Industry and Competitiveness). AJG is the recipient of a FPU predoctoral fellowship from the Central Spanish Government, JDFF is the recipient of a predoctoral fellowship from Salamanca University. PGF receives funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 750795. The authors also thank Emma Keck, translator at the Language Centre of the University of Salamanca.

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 Ca 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., Ministerio de Economía y Competitividad Grant/Award Number: AGL2015-70510-R

Due to the increasing demand for the use of agricultural products, along with new and more restrictive policies regarding the application of fertilizers, the search for alternative ways to increase crop production in a responsible way with respect to the environment is necessary, especially considering that the use of nitrogen-based fertilizers are both very costly and polluting. As regards this chapter focuses on the production of cereals because they represent the most important source of total food consumption, particularly in developing countries with diets based mainly on these types of crops. One possible solution is the application of microbial-based fertilizers (biofertilizers) to enhance crop production. In the literature, bacteria that not only promote plant growth but are also capable of colonizing the interior of plants, known as endophytic bacteria, have been described. Several studies have characterized the different ways of locating these bacteria inside plants, as well as the effects of their colonization. In addition, some entophytes are able to fix nitrogen for their hosts, produce phytohormones (auxins, cytokinins, gibberellins), degrade harmful compounds, decrease the effects of saline stress and improve seed germination, among others benefits. Several companies have attempted to exploit the positive effects caused by endophytic bacteria on their hosts by developing different products, used worldwide (e.g. Inogro®, QuickRoots®), that are based on these types of bacteria. The application of these products occurs despite the governing legislation of the different countries where it is used, and there are usually no specific regulations controlling the process of production, security and marketing of these biofertilizers.

The Rhizobium-legume symbiosis is a complex partnership with many factors, with initial bacterial colonization of the plant root surface and primary infection as key early stages. Two molecules are strongly involved in these processes: the structural carbohydrate cellulose and the enzyme cellulase, which breaks down the former and allows rhizobia to infect the roots. Here, we report the effect on common bean (Phaseolus vulgaris L.) after co-inoculation of the non-nodulating, cellulase-overproducing strain Rhizobium cellulosilyticum ALA10B2T and the P. vulgaris-nodulating R. leguminosarum strain TPV08. In order to elucidate the effect of combined inoculation with both strains, we designed greenhouse assays, including single inoculation with strain TPV08, co-inoculation with both strains and an uninoculated treatment in non-sterile peat. Chemical fertilizers were not added. Chlorophyll content in the leaves was measured after the flowering stage by spectrophotometry and was considered to be indicative of the nutrient status of the plants. Nodule formation was observed on roots of the inoculated plants, while no nodulation was observed on roots of the uninoculated plants. The results indicate a synergistic effect between the two Rhizobium strains. Co-inoculated plants exhibited significant increases in seed yield and nitrogen content in comparison with the uninoculated control plants and with plants inoculated with a single strain. It is suggested that co-inoculation with strain ALA10B2T greatly increased the efficiency of N fixation by strain TPV08.

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.

There is increasing interest in the use of plant growth-promoting rhizobacteria (PGPR) as environmental-friendly and healthy biofertilizers. Strawberries (Fragraria x ananassa) are mainly consumed fresh and hence any PGPRs used for biofertilization must be safe for humans, which is the case for members of the genus Rhizobium. In this study, the effects of inoculation of strawberry plants with Rhizobium sp. strain PEPV16, which belongs to the phylogenetic group of R. leguminosarum, and whose plant growth promotion ability has been reported previously for lettuce (Lactuca sativa) and carrots (Daucus carota), was examined. The results demonstrated that PEPV16 promotes strawberry growth through significant increases in the number of stolons, flowers and fruits as compared with uninoculated controls. Compared to uninoculated controls, the fruits of the inoculated plants had higher concentrations of Fe, Zn, Mn and Mo, and they also had higher concentrations of organic acids, such as citric and malic acid, and lower amounts of ascorbic acid than fruits. Although decreases in ascorbic acid have previously been described after the inoculation of strawberry with strains from different PGPR genera, this is the first study to report increases in organic acids after PGPR inoculation.

Aims: To study multiple bacterial colonization In vitro, several limitations are obvious. One of these limitations is the plant autofluorescence generally between green and red fluorescence depending on the plant sections. The most important limitation is the bacterial fluorescence labelling, compromised by different kind of variables. Here we report the use of a secure stain, Calcofluor White, in rhizobial and other kind of beneficial bacteria colonization studies. Study Design: Root colonization assays were designed to confirm the stability of Calcofluor White stain (Sigma) in root cell walls. Place and Duration of Study: Every assay developed in this method article was performed using the technical resources at the Department of Microbiology and Genetics in the University of Salamanca (Spain) in 2013. Methodology: We have labelled rhizobia with two different kinds of fluorescent protein genes (gfp Method Article Flores-Felix et al.; JABB, 2(1): 65-70, 2015; Article no.JABB.2015.009 66 and rfp). We have co-inoculated Lactuca sativa and Daucus carota seedlings with two rhizobia: GFP-tagged Rhizobium sp. PEPV16 and RFP-tagged Mesorhizobium sp. CSLC01. Colonization assays were perfomed in several days post-inoculation, staining inoculated lettuce and carrot roots with Calcofluor White stain (Sigma). Samples were monitorized for several days, using a fluorescence microscope (NIKON Eclipse 80i). Results: Bacterial attachment to plant tissues is observed by fluorescence microscopy after their labelling with fluorescent proteins. Our results show how Calcofluor White staining for plant tissues improves bacterial visualization in contrast with tissues stained with propidium iodide, a carcinogenic agent that cannot be used when bacteria are tagged with red fluorescent proteins such as RFP or mCherry. Conclusion: Calcofluor white is a non-carcinogenic and low toxic compound that has been classically used to stain fungi and plant tissues for different uses. Due to its low wavelength, calcofluor white may be used in combination with several fluorophores. In the present work we showed that this compound is a reliable alternative to propidium iodide for plant tissues staining in multiple rhizobial/bacterial colonization studies.