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9 páginas, 3 tablas, 2 figuras, Strain CRRU44T was isolated from the stems of Rubus ulmifolius plants growing in Salamanca (Spain). The phylogenetic analysis of the 16S rRNA gene sequence places this strain within the family Rhizobiaceae showing that it is equidistant to the type species of several genera from this family with similarity values ranging from 91.0 to 96.3 %. Strain CRRU44T formed a divergent lineage which clustered with Endobacterium cereale RZME27T, Neorhizobium galegae HAMBI540T and Pseudorhizobium pelagicum R1-200B4T. The phylogenomic analysis showed that strain CRRU44T was equal to or more distant from the remaining genera of the family Rhizobiaceae than other genera among them. The calculated average nucleotide identity based on blast and average amino acid identity values with respect to the type species of all genera from the family Rhizobiaceae were lower than 78.5 and 76.5 %, respectively, which are the currently cut-off values proposed to differentiate genera within this family. All these results together with those from phenotypic and chemotaxonomic analyses support that strain CRRU44T represents a novel species of a novel genus within the family Rhizobiaceae, for which the name Ferranicluibacter rubi gen. nov., sp. nov. is proposed (type strain CRRU44T=CECT 30117T=LMG 31822T)., This research was funded by MINECO (Spanish Central Government) grant PID2019-109960RB-I00 and the Strategic Research Programs for Units of Excellence from Junta de Castilla y León (CLU-2O18-04)

Ips typographus, the most serious pest of spruce forests in Europe, is associated with microorganisms facilitating its invasion and development inside spruce tissues. Despite the importance of I. typographus, little is known about its core gut microbiome. Hereby, we describe the composition of bacterial and fungal microbiomes throughout I. typographus life cycle in spring and summer generations. We used cultivation technique and molecular identification in combination with DNA and RNA metabarcoding to achieve deep inside into the beetle’s microbiome composition and structure. As it is not known whether microbiome forms stable communities inside the beetle’s gut, we observed gut epithelium for biofilm formation with Transmission Electron Microscopy. Cultivation technique together with DNA and RNA metabarcoding indicated similar dominant taxa. The bacterial community belongs almost exclusively to the phylum Proteobacteria (newly Pseudomonadota) and the most common orders and genera are Enterobacteriales (Erwinia and Serratia), Pseudomonadales (Pseudomonas) and Xanthomonadales (Pseudoxanthomonas, Stenotrophomonas). Yeasts (Saccharomycetes) highly dominated the fungal microbiome, followed by Sordariomycetes represented mainly by Ophiostoma bicolor and Endoconidiophora polonica. The most common yeasts were Wickerhamomyces bisporus, Kuraishia molischiana, Nakazawaea ambrosiae, Yamadazyma spp. and Cyberlindnera sp. The proportions of the dominant taxa belonging to the core microbiome of I. typographus change throughout its life cycle and generations. We did not observe any biofilm formation on gut epithelium, which suggests that microbial cells pass through the beetle’s gut with chyme. We propose that species belonging to the core microbiome has similar functions and alternate in the I. typographus ecosystem depending on environmental conditions.

12 páginas, 4 figuras y 2 tablas., The pine engraver beetle, Ips acuminatus Gyll, is a bark beetle that causes important damages in Scots pine (Pinus sylvestris) forests and plantations. As almost all higher organisms, Ips acuminatus harbours a microbiome, although the role of most members of its microbiome is not well understood. As part of a work in which we analysed the bacterial diversity associated to Ips acuminatus, we isolated the strain Arthrobacter sp. IA7. In order to study its potential role within the bark beetle holobiont, we sequenced and explored its genome and performed a pan-genome analysis of the genus Arthrobacter, showing specific genes of strain IA7 that might be related with its particular role in its niche. Based on these investigations, we suggest several potential roles of the bacterium within the beetle. Analysis of genes related to secondary metabolism indicated potential antifungal capability, confirmed by the inhibition of several entomopathogenic fungal strains (Metarhizium anisopliae CCF0966, Lecanicillium muscarium CCF6041, L. muscarium CCF3297, Isaria fumosorosea CCF4401, I. farinosa CCF4808, Beauveria bassiana CCF4422 and B. brongniartii CCF1547). Phylogenetic analyses of the 16S rRNA gene, six concatenated housekeeping genes (tuf-secY-rpoB-recA-fusA-atpD) and genome sequences indicated that strain IA7 is closely related to A. globiformis NBRC 12137T but forms a new species within the genus Arthrobacter; this was confirmed by digital DNA-DNA hybridization (37.10%) and average nucleotide identity (ANIb) (88.9%). Based on phenotypic and genotypic features, we propose strain IA7T as the novel species Arthrobacter ipsi sp. nov. (type strain IA7T = CECT 30100T = LMG 31782T) and suggest its protective role for its host., The project was funded by Czech Science Foundation project GACR 19-09072S. ZSS received a grant from the Junta de Castilla y León, Spanish Regional Government, and LIGD received a grant supported by Junta de Castilla y León (Escalera de Excelencia CLU-2018-04), co-funded by the P.O. FEDER of Castilla y León 2014-2020.

12 páginas, 4 figuras, Microbes host a huge variety of biosynthetic gene clusters that produce an immeasurable array of secondary metabolites with many different biological activities such as antimicrobial, anticarcinogenic and antiviral. Despite the complex task of isolating and characterizing novel natural products, microbial genomic strategies can be useful for carrying out these types of studies. However, although genomic-based research on secondary metabolism is on the increase, there is still a lack of reports focusing specifically on the genus Pseudomonas. In this work, we aimed (i) to unveil the main biosynthetic systems related to secondary metabolism in Pseudomonas type strains, (ii) to study the evolutionary processes that drive the diversification of their coding regions and (iii) to select Pseudomonas strains showing promising results in the search for useful natural products. We performed a comparative genomic study on 194 Pseudomonas species, paying special attention to the evolution and distribution of different classes of biosynthetic gene clusters and the coding features of antimicrobial peptides. Using EvoMining, a bioinformatic approach for studying evolutionary processes related to secondary metabolism, we sought to decipher the protein expansion of enzymes related to the lipid metabolism, which may have evolved toward the biosynthesis of novel secondary metabolites in Pseudomonas. The types of metabolites encoded in Pseudomonas type strains were predominantly non-ribosomal peptide synthetases, bacteriocins, N-acetylglutaminylglutamine amides and ß-lactones. Also, the evolution of genes related to secondary metabolites was found to coincide with Pseudomonas species diversification. Interestingly, only a few Pseudomonas species encode polyketide synthases, which are related to the lipid metabolism broadly distributed among bacteria. Thus, our EvoMining-based search may help to discover new types of secondary metabolite gene clusters in which lipid-related enzymes are involved. This work provides information about uncharacterized metabolites produced by Pseudomonas type strains, whose gene clusters have evolved in a species-specific way. Our results provide novel insight into the secondary metabolism of Pseudomonas and will serve as a basis for the prioritization of the isolated strains. This article contains data hosted by Microreact., Z.S.S. and E.P.A. received grants from the Regional Government of Castile and Leon. Also, this work was supported by the Regional Government of Castile and Leon (Escalera de Excelencia CLU-2018-04) and co-funded by the Operational Program of the European Regional Development Fund for Castile and Leon 2014–2020.

19 páginas, 7 figuras, Pseudomonas is a large and diverse genus broadly distributed in nature. Its species play relevant roles in the biology of earth and living beings. Because of its ubiquity, the number of new species is continuously increasing although its taxonomic organization remains quite difficult to unravel. Nowadays the use of genomics is routinely employed for the analysis of bacterial systematics. In this work, we aimed to investigate the classification of species of the genus Pseudomonas on the basis of the analyses of the type strains whose genomes are currently available. Based on these analyses, we propose the creation of three new genera (Denitrificimonas gen nov. comb. nov., Neopseudomonas gen nov. comb. nov. and Parapseudomonas gen nov. comb. nov) to encompass several species currently included within the genus Pseudomonas and the reclassification of several species of this genus in already described taxa, This work was funded by the Regional Government of Castile and Leon, Escalera de Excelencia CLU-2018-04, co-funded by the P.O. FEDER of Castilla y León 2014–2020. ZSS and EPA received grants from the Regional Government of Castile and Leon.

A phenotype is the expression of interactions between species genotype and environment. We quantified the contributions of ecological and phylogenetic associations to phenotypic variation in Geosmithia fungi. Geosmithia are symbiotic beetle-associated saprotrophs with a range of life histories and host specificities, including obligate nutritional beetle mutualists (ambrosia fungi) and phytopathogens. We hypothesized that: (1) species phenotypes are better explained by their ecology than by their phylogenetic relationships; (2) niche specialization was accompanied by enzymatic capability losses; and (3) ambrosia Geosmithia species have higher nutritional quality and antibiotic capabilities than species with facultative symbioses. Our results confirmed that long-term co-evolved specialists have reduced metabolic breadth in comparison to generalists. Phytopathogenic G. morbida produces unique enzyme suites with affinity to ligno-cellulose. Mycelia of ambrosia fungi contain large amounts of oleic fatty acid with nutritive and possibly allelopathic function. Overall, our results indicate that Geosmithia ecology have greater effect on species phenotype than their phylogenetic relationships.

13 páginas, 5 tablas, 1 figura, Today there is an urgent need to find new ways to satisfy the current and growing food demand and to maintain crop protection and food safety. One of the most promising changes is the replacement of chemical fertilizers with biofertilizers, which include plant root-associated beneficial bacteria. This work describes and shows the use of B. halotolerans SCCPVE07 and R. laguerreae PEPV40 strains as efficient biofertilizers for escarole crops, horticultural species that are widely cultivated. An in silico genome study was performed where coding genes related to plant growth promoting (PGP) mechanisms or different enzymes implicated in the metabolism of phenolic compounds were identified. An efficient bacterial root colonization process was also analyzed through fluorescence microscopy. SCCPVE07 and PEPV40 promote plant development under normal conditions and saline stress. Moreover, inoculated escarole plants showed not only an increase in potassium, iron and magnesium content but also a significant improvement in protocatechuic acid, caffeic acid or kaempferol 3-O-glucuronide plant content. Our results show for the first time the beneficial effects in plant development and the food quality of escarole crops and highlight a potential and hopeful change in the current agricultural system even under saline stress, one of the major non-biological stresses, This work was supported by Grants AGL2015-70510-R from MINECO (Spanish Ministry of Economy, Industry and Competitiveness) and Strategic Research Programs for Units of Excellence from Junta de Castilla y León (CLU-2O18-04). AJG is the recipient of a FPU predoctoral fellowship from the Central Spanish Government. IGE thanks the Spanish MICINN (Ministry of Science, Innovation and Universities) for the Juan de la Cierva-incorporación postdoctoral contract (IJCI 2017-31499).

14 páginas, 1 figura, 1 tabla, Symbiosis between microbes and insects has been raised as a promising area for understanding biological implications of microbe–host interactions. Among them, the association between fungi and bark beetles has been generally recognized as essential for the bark beetle ecology. However, many works investigating bark beetle bacterial communities and their functions usually meet in a common finding: Pseudomonas is a broadly represented genus within this holobiont and it may provide beneficial roles to its host. Thus, we aimed to review available research on this microbe–host interaction and point out the probable relevance of Pseudomonas strains for these insects, in order to guide future research toward a deeper analysis of the importance of these bacteria for the beetle’s life cycle, This research was funded by the Czech Science Foundation (GACR), grant number GACR Senior 19-09072S. The researchers also thank the Excellence Unit of the Spanish-Portuguese Institute for Agricultural Research (CIALE) (CLU-2018-04) for the provision of research facilities. ZSS received a grant from the Junta de Castilla y León, Spanish Regional Government

Bacteria-host interactions play essential roles in the biology of animals. One of such interactions implies bacteria and bark beetles. Bark beetles are considered to be serious pests that devastate entire forests worldwide. Different studies have investigated the microbiome of bark beetles. Some of them have even demonstrated the role of microbes in the protection or nutrition of their beetle host. Particularly, bacteria belonging to the genus Pseudomonas seem to be relevant players of these holobionts. Here, we review the current knowledge of Pseudomonas-bark beetle interactions, suggesting for this bacterial genus to have key roles within the core microbiome of Dendroctonus, Ips, and several other bark beetle genera. Many studies report the ability of different Pseudomonas strains to antagonize entomopathogenic fungi, to aid in the beetle nutrition by the degradation of complex nutrients which the beetle cannot digest and the capability to break down chemical defenses produced by the tree in response to bark beetles. A better understanding of the basis of this insect-bacteria interaction will enable researchers to deep in the biology of the beetle and to better design strategies to overcome this forest pest.

Because of their plant-growth-promoting (PGP) properties, microbes play crucial roles in plant development, making them potential candidates for substituting chemical fertilizers, especially when the soil conditions are not the most appropriate to obtain maximum crop yields. This is the case of strain Pseudomonas brassicacearum CDVBN10, a bacterial endophyte isolated from rapeseed (Brassica napus) roots. Previous in vitro, in planta and in silico experiments have demonstrated that strain CDVBN10 has interesting PGP potential. This endophyte synthetizes cellulose, produces siderophores, solubilizes P, promotes plant height and root length in rapeseed seedlings, and carries genes implicated in several PGP pathways. Based on these data, we conducted greenhouse experiments using this strain as a biofertilizer under normal and salinity stress conditions, finding significant improvements in those inoculated plants, compared to the negative control. To prove its biofertilizer potential under field conditions, we carried out a field trial in which plants inoculated with CDVBN10 showed a 216%, 174.3%, and 197.8% increase in number of pods, seed weight, and aerial part weight, respectively. Along-read (SMRT; PacBio) 16S rRNA gene amplicon sequencing was performed in root samples from this field trials to study possible shifts on the rapeseed root microbiome due to the inoculation with CDVBN10. Interestingly, despite the great plant improvements, there were no significant differences in root bacterial communities of inoculated plants; thus, other potential beneficial members of the plant microbiome had not been displaced. According to the results, the strain P. brassicacearum CDVBN10 is suggested as a promising bacterial biofertilizer with great performance on B. napus crops.

Some microbes are important players in plant fitness, contributing to their nutrient acquisition and protection against diverse biotic and abiotic stresses [...]

European Bark Beetle Ips typographus is a secondary pest that affects dead and weakened spruce trees (Picea genus). Under certain environmental conditions, it has massive outbreaks, resulting in the attacks of healthy trees, becoming a forest pest. It has been proposed that the bark beetle&rsquo, s microbiome plays a key role in the insect&rsquo, s ecology, providing nutrients, inhibiting pathogens, and degrading tree defense compounds, among other probable traits yet to be discovered. During a study of bacterial associates from I. typographus, we isolated three strains identified as Pseudomonas from different beetle life stages. A polyphasic taxonomical approach showed that they belong to a new species for which the name Pseudomonas typographi sp nov. is proposed. Genome sequences show their potential to hydrolyze wood compounds and synthesize several vitamins, screening for enzymes production was verified using PNP substrates. Assays in Petri dishes confirmed cellulose and xylan hydrolysis. Moreover, the genomes harbor genes encoding chitinases and gene clusters involved in the synthesis of secondary metabolites with antimicrobial potential. In vitro tests confirmed the capability of the three P. typographi strains to inhibit several Ips beetles&rsquo, pathogenic fungi. Altogether, these results suggest that P. typographi aids I. typographi nutrition and resistance to fungal pathogens.

A previous 16S rRNA gene sequence comparison had demonstrated that the type strains of Serratia vespertilionis and Serratia ficaria shared 99.5 % sequence similarity. Despite the 56.2 % homology by DNA–DNA hybridization previously found between these strains, the results of an in silico whole-genome sequence comparison and a new DNA–DNA hybridization study have clearly demonstrated that the genomes of the type strain of S. vespertilionis deposited in different Culture Collections (52T=CECT 8595T=DSM 28727T) and the type strain of S. ficaria (culture DSM 4569T), cannot support such a species differentiation. Tests for substrate utilization redone on the deposited cultures of these strains has also shown very few differences between the type strains of both species. Based on these results, and since the name S. ficaria was validly published earlier, S. vespertilionis should be considered as a later heterotypic synonym of S. ficaria , in application of the priority rule. The type strain of the species S. ficaria is strain 4024T=DSM 4569T=NCTC 12148T=ATCC 33105T=CIP 79.23T=ICPB 4050T.

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

© 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.

8 páginas, 5 tablas, 1 figura, BACKGROUND: There is an urgent need for a new sustainable way of satisfying the increasing demand for food worldwide. One of the main challenges is replacing chemical fertilizers with biofertilizers, which include plant root-associated beneficial microorganisms. The present study reports, for the first time, the effects of SCCPVE07 bacterial strain with respect to improving not only plant development, but also the nutritional content and bioactive compounds content of Coriandrum sativum L., one of the most economically important crops, even for plant growth under salinity stress. RESULTS: Innoculated coriander plants (C. sativum L.) showed an increase in potassium, carbon, calcium and iron content. A significant improvement in phenolic compounds contents was also observed. The contents of 5-O-caffeoylquinic acid, cinnamic acid, 4-methoxy-cinnamic acid hexoside, K-3-O rutinoside, Q-3-O-rutinoside, Q-3-O-glucoside and Q-3-O-glucuronide were sig nificantly enhanced. Moreover, an efficient bacterial root colonization and a noted growth promotion were demonstrated. Bacterial genome was sequenced and analysed. Gene coding related to Plant growth promotion (PGP) mechanisms and proteins involved in plant defence from salinity or in the metabolism of phenolic compounds, such as quercetin 2,3-dioxygenase and phenolic acid decarboxylase, were identified. CONCLUSION: The results obtained in the present study show, for the first time, the beneficial effects of the inoculation of a bacterial Bacillus halotolerans biofertilizer on coriander crops with respect to increasing the content in bioactive compounds and plant development., This work was supported by Grants AGL2015-70510-R from MINECO (Spanish Ministry of Economy, Industry and Competitiveness) and Strategic Research Programs for Units of Excellence from Junta de Castilla y León (CLU-2O18-04). AJG is the recipient of a FPU predoctoral fellowship from the Central Spanish Government. IGE thanks the Spanish MICINN (Ministry of Science, Innovation and Universities) for the Juan de la Cierva-incorporación postdoctoral contract (IJCI-2017-31499). The authors also thank Victoria Bishop for editing the English language of the manuscript submitted for publication.

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.

Plants harbor a diversity of microorganisms constituting the plant microbiome. Many bioinoculants for agricultural crops have been isolated from plants. Nevertheless, plants are an underexplored niche for the isolation of microorganisms with other biotechnological applications. As a part of a collection of canola endophytes, we isolated strain CDVBN77T. Its genome sequence shows not only plant growth-promoting (PGP) mechanisms, but also genetic machinery to produce secondary metabolites, with potential applications in the pharmaceutical industry, and to synthesize hydrolytic enzymes, with potential applications in biomass degradation industries. Phylogenetic analysis of the 16S rRNA gene of strain CDVBN77T shows that it belongs to the genus Microvirga, its closest related species being M. aerophila DSM 21344T (97.64% similarity) and M. flavescens c27j1T (97.50% similarity). It contains ubiquinone 10 as the predominant quinone, C19:0 cycloω8c and summed feature 8 as the major fatty acids, and phosphatidylcholine and phosphatidylethanolamine as the most abundant polar lipids. Its genomic DNA G+C content is 62.3 (mol %). Based on phylogenetic, chemotaxonomic, and phenotypic analyses, we suggest the classification of strain CDVBN77T within a new species of the genus Microvirga and propose the name Microvirga brassicacearum sp. nov. (type strain CDVBN77T = CECT 9905T = LMG 31419T)., Authors thank the European Union’s Horizon 2020 research and innovation programme for the research funding received by P.G.-F. A.J.-G. thanks the Central Spanish Government for an FPU predoctoral grant and Z.S.-S. thanks the Junta de Castilla y Leon, Spanish Regional Government, for his predoctoral grant.

The genus Pseudogymnoascus encompasses soil psychrophilic fungi living also in caves. Some are opportunistic pathogens; nevertheless, they do not cause outbreaks. Pseudogymnoascus destructans is the causative agent of the white-nose syndrome, which is decimating cave-hibernating bats. We used comparative eco-physiology to contrast the enzymatic potential and conidial resilience of P. destructans with that of phylogenetically diverse cave fungi, including Pseudogymnoascus spp., dermatophytes and outdoor saprotrophs. Enzymatic potential was assessed by Biolog MicroArray and by growth on labelled substrates and conidial viability was detected by flow cytometry. Pseudogymnoascusdestructans was specific by extensive losses of metabolic variability and by ability of lipid degradation. We suppose that lipases are important enzymes allowing fungal hyphae to digest and invade the skin. Pseudogymnoascus destructans prefers nitrogenous substrates occurring in bat skin and lipids. Additionally, P. destructans alkalizes growth medium, which points to another possible virulence mechanism. Temperature above 30 °C substantially decreases conidial viability of cave fungi including P. destructans. Nevertheless, survival of P. destructans conidia prolongs by the temperature regime simulating beginning of the flight season, what suggests that conidia could persist on the body surface of bats and contribute to disease spreading during bats active season.

13 páginas, 3 tablas y 5 figuras, Frass from the insect Tenebrio molitorcan be applied as an organic fertilizer due to its nutritional content. In this study insects were fed on different types of foodproducing excrement with different compositions. This allowed us to compare the potential effect of the excrement on plant growth promotion and to test its ability to increase tolerance to abiotic stresses such as drought, excess water and salinity. The best results were obtained when the insects were fed a diet low in fat andstarch content. Moreover, a microbiological study using the excrement produced showed that the presence of microorganisms improved plant growth parameters, inthe absence or presence of stress conditions. The analysis of the microbiome through massive parallel sequencing, as well as the analysis of the capacity of severalmicrobial isolates to promote plant growth, showed that several microorganisms present in the excrement are plant growth promoters (PGPs). Ourfindings indicatethat the frass produced by these insects, due to its nutritional content and associated microbiota, can be potentially used as a biofertilizer in organic farming.However, this potential dramatically varies according to the insect's diet. Further analyses should be performed to set the optimal time and amount of application forthe different aimed plant, The Spanish Ministry of Economy and Competitiveness (MINECO) grant PTQ-14-07381 provided the financial support to co-fund the workcarried out by PGF, JPA was co-funded by grant DI-15-07460 and ZSSwas funded by a grant from“Junta de Castilla y Leon (JCYL)”

11 páginas, 3 tablas, 2 figuras. -- The first publication is available at https://www.nature.com, Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes (azo, thiazine and anthraquinone) with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate (Ln26, approximately 4.2 Mb in size) was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.

La cadena del frío constituye un elemento clave en seguridad alimentaria, pues la mayoría de los microorganismos detienen su crecimiento a bajas temperaturas. Se han desarrollado técnicas, basadas en la secuenciación masiva, que analizan las poblaciones bacterianas de una muestra mediante secuenciación de un fragmento del gen ribosómico 16S, permitiendo la caracterización microbiana de alimentos./nEl objetivo del presente estudio consistió en la identificación y cuantificación de bacterias sobre alimentos refrigerados y sin refrigerar utilizando secuenciación masiva del ADNr16S, para describir el rol del mantenimiento de la cadena del frío sobre la evolución de comunidades microbianas en alimentos. /nAsí, se secuenció el ADN extraído de muestras de pollo, espinacas, queso fresco y yogurt, a los 2 y 4 días. Las Unidades de Operación Taxonómica (Operational Taxonomic Units: OTUs) obtenidas se compararon con bases de datos de secuencias de organismos (NCBI). Además, se realizó el cultivo de microorganismos de las muestras en el momento de su compra. /nDe forma general se observó un aumento de la cantidad y diversidad de bacterias en las muestras sin refrigerar, confirmando que la cadena del frío retrasa el desarrollo de microorganismos en los alimentos, aumentando la vida útil de los mismos. Además, el estudio demuestra la eficacia de la secuenciación masiva del ADNr16S para estudiar el efecto de la temperatura en el desarrollo de comunidades bacterianas sobre los alimentos. The cold chain is a key element in food safety since most microorganisms stop their growth at low temperatures. Techniques that analyse the bacterial populations of a sample by sequencing a fragment of the 16S ribosomal gene, allowing the microbial characterization of foods have been developed./nThe objective of the present study was to identify and quantify bacteria on refrigerated and uncooled foods using massive sequencing of rDNA16S, to describe the role of maintenance of the cold chain on the evolution of microbial communities in food. /nThus, the DNA extracted from the samples was sequenced at 2d and 4d. The OTUs obtained were compared with those of the databases and identified. The culture of microorganisms of the samples was carried out at 0d. /nIn general, an increase in the amount and diversity of bacteria was observed in the uncooled samples, confirming that the cold chain delays the development of microorganisms in foods, increasing the shelf life of them. In addition, the study demonstrates the efficacy of the massive sequencing of rDNA16S to study the effect of temperature on the development of bacterial communities on food.

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.

Understanding the activity of bacteria in coniferous forests is highly important, due to the role of these environments as a global carbon sink. In a study of the microbial biodiversity of montane coniferous forest soil in the Bohemian Forest National Park (Czech Republic), we succeeded in isolating bacterial strain S55 T , which belongs to one of the most abundant operational taxonomic units (OTUs) in active bacterial populations, according to the analysis of RNA-derived 16S rRNA amplicons. The 16S rRNA gene sequence analysis showed that the species most closely related to strain S55 T include Bryocella elongata SN10 T (95.4% identity), Acidicapsa ligni WH120 T (95.2% identity), and Telmatobacter bradus TPB6017 T (95.0% identity), revealing that strain S55 T should be classified within the phylum Acidobacteria , subdivision 1. Strain S55 T is a rod-like bacterium that grows at acidic pH (3 to 6). Its phylogenetic, genotypic, phenotypic, and chemotaxonomic characteristics indicate that strain S55 T corresponds to a new genus within the phylum Acidobacteria ; thus, we propose the name Terracidiphilus gabretensis gen. nov., sp. nov. (strain S55 T = NBRC 111238 T = CECT 8791 T ). This strain produces extracellular enzymes implicated in the degradation of plant-derived biopolymers. Moreover, analysis of the genome sequence of strain S55 T also reveals the presence of enzymatic machinery required for organic matter decomposition. Soil metatranscriptomic analyses found 132 genes from strain S55 T being expressed in the forest soil, especially during winter. Our results suggest an important contribution of T. gabretensis S55 T in the carbon cycle in the Picea abies coniferous forest.

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.

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.

Raulí is one of the most emblematic tree species of the Chilean temperate forests. Due to the high quality wood, this tree has been used for furniture and handicrafts manufacturing, which has positioned raulí as one of the most important commercial timber species in Chile. Currently, the international market demands sustainable production system for forest production, more specifically in plantlets production. In this regard, plant growth-promoting rhizobacteria (PGPR) inoculants may enhance the growth and survival of plantlets in nurseries, which means an increase in the effectiveness of replanting operations. Therefore, the aim of the present study was to isolate, characterize and screen rhizosphere-associated bacteria with PGPR potential, isolated from raulí that growth in volcanic soils in southern Chile. A total of 1,261 bacterial strains were isolated from different volcanic soils. Out of 1,261 isolates, 100 were selected based on their high levels of indole acetic acid (IAA) production. These isolates were then subjected to screening for 1-aminocyclopropane-1-carboxylic acid deaminase activity, and their ability to fix nitrogen was determined. From the 100 selected isolates, 7 were chosen for producing the highest amount of IAA to continue with genetic characterization based on their 16S rRNA gene sequences. These 7 isolates were characterized as members of the Serratia genus and were used to develop multi-strain inoculant mixtures. Later, a nursery study followed to determine the effect of inoculation with the Serratia strains on the growth of RA88 raulí clone plantlets. The nursery experiment demonstrated that Serratia strains have the potential to increase the root collar diameter, height, relative chlorophyll content, biomass and nitrogen content of raulí plantlets. The study concluded, that Serratia strains have the potential to be used as biofertilizers to increase plant growth in nursery conditions.

Strawberries (Fragaria × ananassa Duch.) are widely demanded by the consumers because they contain several bioactive compounds, mainly vitamin C and anthocyanins, which may be increased by biofertilization with some plant growth promoting bacteria. In this work we have analysed two bacterial strains, PEPV15 and PEPV16, from genera Phyllobacterium and Rhizobium, respectively, which under microcosms conditions were able to promote the strawberry growth, increasing the content of some bioactive compounds, such as vitamin C or organic acids. Here we have analysed the effect on bioactive compounds in strawberries from plants biofertilized with the strains PEPV15 and PEPV16 in field conditions. Under these conditions, the anthocyanin content was increased when plants were biofertilized with the strain PEPV15 and the pelargonidin-3-O-rutinoside content significantly increased. Besides, citric acid, vitamin C and epicatechin contents were significantly higher when either of the two strains was used as biofertilizer. Our results showed that the inoculation with Phyllobacterium and Rhizobium strains is a good agronomical practice, which improve the content of several bioactive compounds of strawberries increasing the beneficial effects on human health.

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.