Application of Ligilactobacillus salivarius CECT5713 to Achieve Term Pregnancies in Women with Repetitive Abortion or Infertility of Unknown Origin by Microbiological and Immunological Modulation of the Vaginal Ecosystem

In this study, the cervicovaginal environment of women with reproductive failure (repetitive abortion, infertility of unknown origin) was assessed and compared to that of healthy fertile women. Subsequently, the ability of Ligilactobacillus salivarius CECT5713 to increase pregnancy rates in women with reproductive failure was evaluated. Vaginal pH and Nugent score were higher in women with reproductive failure than in fertile women. The opposite was observed regarding the immune factors TGF-β 1, TFG-β 2, and VEFG. Lactobacilli were detected at a higher frequency and concentration in fertile women than in women with repetitive abortion or infertility. The metataxonomic study revealed that vaginal samples from fertile women were characterized by the high abundance of Lactobacillus sequences, while DNA from this genus was practically absent in one third of samples from women with reproductive failure. Daily oral administration of L. salivarius CECT5713 (~9 log10 CFU/day) to women with reproductive failure for a maximum of 6 months resulted in an overall successful pregnancy rate of 56%. The probiotic intervention modified key microbiological, biochemical, and immunological parameters in women who got pregnant. In conclusion, L. salivarius CECT5713 has proved to be a good candidate to improve reproductive success in women with reproductive failure.Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEContract 291-2018 established between Complutense University of Madrid and Biosearch Life S. A. (Granada, Spain)Irma Castro is the recipient of a predoctoral contract (BES-2017-080713) from the Ministerio de Ciencia, Innovación y Universidades (Spain)pu

Biodegradation of Pine Processionary Caterpillar Silk Is Mediated by Elastase- and Subtilisin-like Proteases

This article belongs to the Section Biochemistry[EN] Pine processionary caterpillar nests are made from raw silk. Fibroin protein is the main component of silk which, in the case of pine processionary caterpillar, has some unusual properties such as a higher resistance to chemical hydrolysis. Isolation of microorganisms naturally present in silk nests led to identification of Bacillus licheniformis and Pseudomonas aeruginosa strains that in a defined minimal medium were able to carry out extensive silk biodegradation. A LasB elastase-like protein from P. aeruginosa was shown to be involved in silk biodegradation. A recombinant form of this protein expressed in Escherichia coli and purified by affinity chromatography was able to efficiently degrade silk in an in vitro assay. However, silk biodegradation by B. licheniformis strain was mediated by a SubC subtilisin-like protease. Homologous expression of a subtilisin Carlsberg encoding gene (subC) allowed faster degradation compared to the biodegradation kinetics of a wildtype B. licheniformis strain. This work led to the identification of new enzymes involved in biodegradation of silk materials, a finding which could lead to possible applications for controlling this pest and perhaps have importance from sanitary and biotechnological points of viewSIAlba Diez-Galán (EDU/556/2019), Ana Ibañez (EDU/529/2017) and Carla Calvo-Peña (EDU/601/2020) were supported by a predoctoral contract from the Junta de Castilla y León and the European Social Fun

Molecular Identification and Acid Stress Response of an Acidithiobacillus thiooxidans Strain Isolated from Rio Tinto (Spain)

[EN] Acidithiobacillus thiooxidans is of paramount importance in the development of biomining technologies. Being widely recognized as an extreme acidophile, extensive research has been dedicated to understanding its significant role in the extraction of several ores in recent years. However, there still exist significant molecular uncertainties surrounding this species. This study focuses on developing a taxonomic assignment method based on the sequencing of the 16S-5S rRNA cluster, along with a qPCR-based technology enabling precise growth determination. Additionally, an approach to understanding its response to acid stress is explored through RT-PCR and MALDITOF analysis. Our findings indicate that when subjected to pH levels below 1, the cell inhibits central (carbon fixation and metabolism) and energy (sulfur metabolism) metabolism, as well as chaperone synthesis, suggesting a potential cellular collapse. Nevertheless, the secretion of ammonia is enhanced to raise the environmental pH, while fatty acid synthesis is upregulated to reinforce the cell membrane.SIWe thank Instituto de Biotecnología de León (INBIOTEC) for their technical support in performing the A. thiooxidans proteome analysis. Special thanks to (i) the ESTELLA project (“DESign of bio-based Thermoset polymer with rEcycLing capabiLity by dynAmic bonds for bio-composite manufacturing”) (Project no.: 101058371) funded by the European Union through the Horizon Europe Framework Programme (call: HORIZON-CL4-2021-RESILIENCE-01-11) and (ii) the BioPAC project (Development of bioactive and lifespan-controlled bioplastics) (Ref. TED2021-131864B-C21) funded by the MCIN (Ministerio de Ciencia e Innovación)/AEI (Agencia Estatal de Investigación)/10.13039/501100011033 (DOI) and the European Union “NextGenerationEU”/PRTR (Plan de Recuperación, Transformación y Resiliencia).Ana Ibañez was supported by a “Margarita Salas” modality postdoctoral grant (Reference no.: UP2021-025) through the University of León awarded by the Spanish Ministry of Universities within the Recovery, Transformation and Resilience Plan (Modernization and digitalization of the Educational System), which funding comes from the European Recovery Instrument European Union-NextGeneration EU. Alba Diez-Galán (EDU/556/2019), and Carla Calvo-Peña (EDU/601/2020) were supported by a predoctoral contract from the Junta de Castilla y León and the European Social Fund

Advances in the control of phytopathogenic fungi that infect crops through their root system

[EN] Productivity and economic sustainability of many herbaceous and woody crops are seriously threatened by numerous phytopathogenic fungi. While symptoms associated with phytopathogenic fungal infections of aerial parts (leaves, stems and fruits) are easily observable and therefore recognizable, allowing rapid or preventive action to control this type of infection, the effects produced by soil-borne fungi that infect plants through their root system are more difficult to detect. The fact that these fungi initiate infection and damage underground implies that the first symptoms are not as easily noticeable, and therefore both crop yield and plant survival are frequently severely compromised by the time the infection is found. In this paper we will review and discuss recent insights into plant-microbiota interactions in the root system crucial to understanding the beginning of the infectious process. We will also review different methods for diminishing and controlling the infection rate by phytopathogenic fungi penetrating through the root system including both the traditional use of biocontrol agents such as antifungal compounds as well as some new strategies that could be used because of their effective application, such as nanoparticles, virus-based nanopesticides, or inoculation of plant material with selected endophytes. We will also review the possibility of modeling and influencing the composition of the microbial population in the rhizosphere environment as a strategy for nudging the plant-microbiome interactions toward enhanced beneficial outcomes for the plant, such as controlling the infectious processSIS.G.-G. was supported by a FPU fellowship (Grant number FPU15/03475) from the Ministerio de Educacio´n, Cultura y Deporte (Madrid, Spain). A.M.I. and A.D.G. were supported by a predoctoral fellowship from the Junta de Castilla y Leo´n. C.C.-P. was supported by a technician contract co-financed by the Iniciativa de Empleo Juvenil (Junta de Castilla y León) and the European Social Fun

Using Rhizosphere Phosphate Solubilizing Bacteria to Improve Barley (Hordeum vulgare) Plant Productivity

[EN] On average less than 1% of the total phosphorous present in soils is available to plants, making phosphorous one of the most limiting macronutrients for crop productivity worldwide. The aim of this work was to isolate and select phosphate solubilizing bacteria (PSB) from the barley rhizosphere, which has other growth promoting traits and can increase crop productivity. A total of 104 different bacterial isolates were extracted from the barley plant rhizosphere. In this case, 64 strains were able to solubilize phosphate in agar plates. The 24 strains exhibiting the highest solubilizing index belonged to 16 different species, of which 7 isolates were discarded since they were identified as putative phytopathogens. The remaining nine strains were tested for their ability to solubilize phosphate in liquid medium and in pot trials performed in a greenhouse. Several of the isolated strains (Advenella mimigardefordensis, Bacillus cereus, Bacillus megaterium and Burkholderia fungorum) were able to significantly improve levels of assimilated phosphate, dry weight of ears and total starch accumulated on ears compared to non-inoculated plants. Since these strains were able to increase the growth and productivity of barley crops, they could be potentially used as microbial inoculants (biofertilizers).SIAna Ibañez and Alba Diez-Gala’n were supported by a predoctoral contract from the Junta de Castilla y León and the European Social Fund. Carla Calvo-Peña was supported by a technician contract co-financed by the Iniciativa de Empleo Juvenil (Junta de Castilla y León) and the European Social Fund.We thank the Instituto de Biotecnología de León (INBIOTEC) for their technical support in performing the HPLC analysis of organic acid production by PSB

Overexpression of ZePrx in Nicotiana Tabacum Affects Lignin Biosynthesis Without Altering Redox Homeostasis

[Abstract] Class III plant peroxidases (Prxs) are involved in the oxidative polymerization of lignins. Zinnia elegans Jacq. Basic peroxidase (ZePrx) has been previously characterized as capable of catalyzing this reaction in vitro and the role in lignin biosynthesis of several of its Arabidopsis thaliana homologous has been previously confirmed. In the present work, ZePrx was overexpressed in Nicotiana tabacum to further characterize its function in planta with particular attention to its involvement in lignin biosynthesis. Since Prxs are known to alter ROS levels by using them as electron acceptor or producing them in their catalytic activity, the impact of this overexpression in redox homeostasis was studied by analyzing the metabolites and enzymes of the ascorbate-glutathione cycle. In relation to the modification induced by ZePrx overexpression in lignin composition and cellular metabolism, the carbohydrate composition of the cell wall as well as overall gene expression through RNA-Seq were analyzed. The obtained results indicate that the overexpression of ZePrx caused an increase in syringyl lignin in cell wall stems, suggesting that ZePrx is relevant for the oxidation of sinapyl alcohol during lignin biosynthesis, coherently with its S-peroxidase nature. The increase in the glucose content of the cell wall and the reduction of the expression of several genes involved in secondary cell wall biosynthesis suggests the occurrence of a possible compensatory response to maintain cell wall properties. The perturbation of cellular redox homeostasis occurring as a consequence of ZePrx overexpression was kept under control by an increase in APX activity and a reduction in ascorbate redox state. In conclusion, our results confirm the role of ZePrx in lignin biosynthesis and highlight that its activity alters cellular pathways putatively aimed at maintaining redox homeostasis.Xunta de Galicia; ED431C 2018/57AG-U held an FPU grant from MECD (Spain) (FPU13/04835). This research was possible thanks to the funding of Xunta de Galicia (Spain) (ED431C 2018/57

Use of Endophytic and Rhizosphere Actinobacteria from Grapevine Plants To Reduce Nursery Fungal Graft Infections That Lead to Young Grapevine Decline

[EN] Endophytic and rhizosphere actinobacteria isolated from the root system of 1-year-old grafted Vitis vinifera plants were evaluated for their activities against fungi that cause grapevine trunk diseases. A total of 58 endophytic and 94 rhizosphere isolates were tested. Based on an in vitro bioassay, 15.5% of the endophytic isolates and 30.8% of the rhizosphere isolates exhibited antifungal activity against the fungal pathogen Diplodia seriata, whereas 13.8% of the endophytic isolates and 16.0% of the rhizosphere isolates showed antifungal activity against Dactylonectria macrodidyma (formerly Ilyonectria macrodidyma). The strains which showed the greatest in vitro efficacy against both pathogens were further analyzed for their ability to inhibit the growth of Phaeomoniella chlamydospora and Phaeoacremonium minimum (formerly Phaeoacremonium aleophilum). Based on their antifungal activity, three rhizosphere isolates and three endophytic isolates were applied on grafts in an open-root field nursery in a 3-year trial. The field trial led to the identification of one endophytic strain, Streptomyces sp. VV/E1, and two rhizosphere isolates, Streptomyces sp. VV/R1 and Streptomyces sp. VV/R4, which significantly reduced the infection rates produced by the fungal pathogens Dactylonectria sp., Ilyonectria sp., P. chlamydospora, and P. minimum, all of which cause young grapevine decline. The VV/R1 and VV/R4 isolates also significantly reduced the mortality level of grafted plants in the nursery. This study shows that certain actinobacteria could represent a promising new tool for controlling fungal trunk pathogens that infect grapevine plants through the root system in nurseries.SIThis work was supported by Viveros Villanueva Vides S.L. (Larraga, Spain), which was financed by the Centro para el Desarrollo Tecnológico Industrial (CDTI; Madrid, Spain) and the Government of Comunidad Foral de Navarra (Spain). S. González-García was supported by a FPU fellowship of the Ministerio de Educación, Cultura y Deporte (Madrid, Spain

Necrotic and cytolytic activity on grapevine leaves produced by Nep1-like proteins of Diplodia seriata

This article is part of the Research Topic Recent Advances on Grapevine-Microbe Interactions: From Signal Perception to Resistance Response[EN] Many phytopathogenic fungi produce necrosis and ethylene inducing peptide 1 (Nep1- like proteins or NLP) that trigger leaf necrosis and the activation of defense mechanisms. These proteins have been widely studied in plant pathogens as Moniliophthora perniciosa or Botrytis cinerea between others, but little is known about their biological roles in grapevine trunk pathogens. Advances in the sequencing of genomes of several fungi involved in grapevine trunk diseases have revealed that these proteins are present in several copies in their genomes. The aim of this project was to analyze the presence of genes encoding NLP proteins in the Diplodia seriata genome and to characterize their putative role as virulence factors associated to grapevine trunk diseases. In this study, we characterized four NLPs from Diplodia seriata. All proteins showed highly similar amino acid sequences and contained the characteristic peptide motifs of NLPs. DserNEPs slightly reduced the viability of Vitis vinifera L. cell cultures. The cytolytic activity from DserNEP1 was stronger than that from DserNEP2, even at low concentrations. Purified DserNEPs also produced necrosis in leaves when they were inoculated into micropropagules of V. vinifera L. This is the first record of Nep1-like proteins from a fungus associated with grapevine trunk diseases and also from a member of the Botryosphaeriaceae familySIThis work was supported by Bodegas Vega Sicilia S.A. (Valbuena de Duero, Valladolid, Spain

Developing tools for evaluating inoculation methods of biocontrol Streptomyces sp. strains into grapevine plants

[EN] The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agentsSIThis work was supported by Viveros Villanueva Vides S.L. (Larraga, Spain) which was financed by the Centro para el Desarrollo Tecnológico Industrial–CDTI—(Madrid, Spain) through the GLOBALVITI project (CIEN Program) (http://viverosvillanueva.es/, https://www.cdti.es/). SGG was supported by an FPU fellowship (Grant number FPU15/03475) from the Ministerio de Educación, Cultura y Deporte (Madrid, Spain) (http://www.mecd.gob.es/). RC was supported by the Programa Torres Quevedo (Grant number PTQ-14-06849) from the Ministerio de Ciencia, Innovación y Universidades (Madrid, Spain), (http://www.ciencia.gob.es/

Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation