Grapevine trunk diseases (GTDs) are one of the most devastating pathologies that threaten the survival and profitability of vineyards around the world. Progressive banning of chemical pesticides and their withdrawal from the market has increased interest in the development of effective biocontrol agents (BCAs) for GTD treatment. In recent years, considerable progress has been made regarding the characterization of the grapevine microbiome, including the aerial part microbiome (flowers, berries and leaves), the wood microbiome, the root environment and vineyard soil microbiomes. In this work, we review these advances especially in relation to the etiology and the understanding of the composition of microbial populations in plants affected by GTDs. We also discuss how the grapevine microbiome is becoming a source for the isolation and characterization of new, more promising BCAs that, in the near future, could become effective tools for controlling these pathologies.

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).

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 agents.

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 family.

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 MALDI-TOF 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.

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 view.