Application of biostimulant products and biological control agents in sustainable viticulture: A review.

Current and continuing climate change in the Anthropocene epoch requires sustainable agricultural practices. Additionally, due to changing consumer preferences, organic approaches to cultivation are gaining popularity. The global market for organic grapes, grape products, and wine is growing. Biostimulant and biocontrol products are often applied in organic vineyards and can reduce the synthetic fertilizer, pesticide, and fungicide requirements of a vineyard. Plant growth promotion following application is also observed under a variety of challenging conditions associated with global warming. This paper reviews different groups of biostimulants and their effects on viticulture, including microorganisms, protein hydrolysates, humic acids, pyrogenic materials, and seaweed extracts. Of special interest are biostimulants with utility in protecting plants against the effects of climate change, including drought and heat stress. While many beneficial effects have been reported following the application of these materials, most studies lack a mechanistic explanation, and important parameters are often undefined (e.g., soil characteristics and nutrient availability). We recommend an increased study of the underlying mechanisms of these products to enable the selection of proper biostimulants, application methods, and dosage in viticulture. A detailed understanding of processes dictating beneficial effects in vineyards following application may allow for biostimulants with increased efficacy, uptake, and sustainability.

Plant growth-stimulating rhizobacteria capable of producing L-amino acids.

Pseudomonas putida BIRD-1 is a microorganism that inhabits the rhizosphere and solubilizes phosphate and iron and produces indolacetic acid [Roca, A., Pizarro-Tobías, P., Udaondo, Z., Fernández, M., Matilla, M.A., Molina-Henares, M.A., et al. (2013) Analysis of plant growth-promoting properties encoded by the genome of the rhizobacterium Pseudomonas putida BIRD-1. Environ Microbiol 15: 780-794]. In this study, we generated mutant strains that are capable of producing the plant growth stimulating compounds L-tryptophan and L-phenylalanine. We prepared clones that overproduce L-tryptophan by first mutagenizing P. putida BIRD-1, then by selecting for clones in the presence of inhibitory concentrations of 5-fluoro-D,L-tryptophan. The production of this aromatic amino acid was confirmed by chemical analysis and cross-feeding experiments with auxotrophs. One of the mutants, named P. putida BIRD-1-12, was mutagenized again to isolate clones that are also able to grow in the presence of inhibitory concentrations of p-fluoro-D,L-phenylalanine. One of these resulting clones was then isolated and named BIRD-1-12F. Our analysis revealed that the strains that either overproduce L-tryptophan, or L-tryptophan and L-phenylalanine, excel at promoting the growth of a number of plant crops of agricultural interest.

Developing robust protein analysis profiles to identify bacterial acid phosphatases in genomes and metagenomic libraries.

Phylogenetic analysis of more than 4000 annotated bacterial acid phosphatases was carried out. Our analysis enabled us to sort these enzymes into the following three types: (1) class B acid phosphatases, which were distantly related to the other types, (2) class C acid phosphatases and (3) generic acid phosphatases (GAP). Although class B phosphatases are found in a limited number of bacterial families, which include known pathogens, class C acid phosphatases and GAP proteins are found in a variety of microbes that inhabit soil, fresh water and marine environments. As part of our analysis, we developed three profiles, named Pfr-B-Phos, Pfr-C-Phos and Pfr-GAP, to describe the three groups of acid phosphatases. These sequence-based profiles were then used to scan genomes and metagenomes to identify a large number of formerly unknown acid phosphatases. A number of proteins in databases annotated as hypothetical proteins were also identified by these profiles as putative acid phosphatases. To validate these in silico results, we cloned genes encoding candidate acid phosphatases from genomic DNA or recovered from metagenomic libraries or genes synthesized in vitro based on protein sequences recovered from metagenomic data. Expression of a number of these genes, followed by enzymatic analysis of the proteins, further confirmed that sequence similarity searches using our profiles could successfully identify previously unknown acid phosphatases.

Indigenous Pseudomonas spp. Strains from the Olive (Olea europaea L.) Rhizosphere as Effective Biocontrol Agents against Verticillium dahliae: From the Host Roots to the Bacterial Genomes.

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive (Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae, were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the "Pseudomonas mandelii subgroup," within the "Pseudomonas fluorescens group," Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the "Pseudomonas aeruginosa group," Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs.

Field trial on removal of petroleum-hydrocarbon pollutants using a microbial consortium for bioremediation and rhizoremediation.

Petroleum waste sludges are toxic and dangerous that is why environmental protection agencies have declared their treatment top priority. Physicochemical treatments are expensive and environmentally unfriendly, while alternative biological treatments are less costly but, in general, work at a slower pace. An in situ bioremediation and rhizoremediation field scale trial was performed in an area contaminated with oil refinery sludge under semiarid climate. The bioremediation and rhizoremediation treatments included the use of an artificial consortium made up of plant growth-promoting rhizobacteria and polycyclic aromatic hydrocarbon-degrading bacteria,and the combined use of the mentioned consortium along with pasture plants respectively. Rhizoremediation revealed that the development of vegetation favoured the evolution of indigenous microbiota with potential to remove petroleum wastes. This was inferred as the decline of total petroleum hydrocarbons 7 months after the biological treatment.

Restoration of a Mediterranean forest after a fire: bioremediation and rhizoremediation field-scale trial.

Forest fires pose a serious threat to countries in the Mediterranean basin, often razing large areas of land each year. After fires, soils are more likely to erode and resilience is inhibited in part by the toxic aromatic hydrocarbons produced during the combustion of cellulose and lignins. In this study, we explored the use of bioremediation and rhizoremediation techniques for soil restoration in a field-scale trial in a protected Mediterranean ecosystem after a controlled fire. Our bioremediation strategy combined the use of Pseudomonas putida strains, indigenous culturable microbes and annual grasses. After 8 months of monitoring soil quality parameters, including the removal of monoaromatic and polycyclic aromatic hydrocarbons as well as vegetation cover, we found that the site had returned to pre-fire status. Microbial population analysis revealed that fires induced changes in the indigenous microbiota and that rhizoremediation favours the recovery of soil microbiota in time. The results obtained in this study indicate that the rhizoremediation strategy could be presented as a viable and cost-effective alternative for the treatment of ecosystems affected by fires.

Bacterial diversity in the rhizosphere of maize and the surrounding carbonate-rich bulk soil.

Maize represents one of the main cultivar for food and energy and crop yields are influenced by soil physicochemical and climatic conditions. To study how maize plants influence soil microbes we have examined microbial communities that colonize maize plants grown in carbonate-rich soil (pH 8.5) using culture-independent, PCR-based methods. We observed a low proportion of unclassified bacteria in this soil whether it was planted or unplanted. Our results indicate that a higher complexity of the bacterial community is present in bulk soil with microbes from nine phyla, while in the rhizosphere microbes from only six phyla were found. The predominant microbes in bulk soil were bacteria of the phyla Acidobacteria, Bacteroidetes and Proteobacteria, while Gammaproteobacteria of the genera Pseudomonas and Lysobacter were the predominant in the rhizosphere. As Gammaproteobacteria respond chemotactically to exudates and are efficient in the utilization of plants exudate products, microbial communities associated to the rhizosphere seem to be plant-driven. It should be noted that Gammaproteobacteria made available inorganic nutrients to the plants favouring plant growth and then the benefit of the interaction is common.

Analysis of the plant growth-promoting properties encoded by the genome of the rhizobacterium Pseudomonas putida BIRD-1.

Pseudomonas putida BIRD-1 is a plant growth-promoting rhizobacterium whose genome size is 5.7 Mbp. It adheres to plant roots and colonizes the rhizosphere to high cell densities even in soils with low moisture. This property is linked to its ability to synthesize trehalose, since a mutant deficient in the synthesis of trehalose exhibited less tolerance to desiccation than the parental strain. The genome of BIRD-1 encodes a wide range of proteins that help it to deal with reactive oxygen stress generated in the plant rhizosphere. BIRD-1 plant growth-promoting rhizobacteria properties derive from its ability to enhance phosphorous and iron solubilization and to produce phytohormones. BIRD-1 is capable of solubilizing insoluble inorganic phosphate forms through acid production. The genome of BIRD-1 encodes at least five phosphatases related to phosphorous solubilization, one of them being a phytase that facilitates the utilization of phytic acid, the main storage form of phosphorous in plants. Pyoverdine is the siderophore produced by this strain, a mutant that in the FvpD siderophore synthase failed to grow on medium without supplementary iron, but the mutant was as competitive as the parental strain in soils because it captures the siderophores produced by other microbes. BIRD-1 overproduces indole-3-acetic acid through convergent pathways.

Laboratory research aimed at closing the gaps in microbial bioremediation.

The industrial revolution, the first agricultural 'green revolution', and the development of antibiotics and therapeutic chemicals have brought significant and undeniable benefits to the human race. However, these advances demand high levels of energy, exploit natural resources and create large amounts of waste that creates an environmental burden for our planet. The pollution rate and character of many of the pollutants results in a rapid deterioration of the environment. Bioremediation functions to isolate and select microorganisms that operate under aerobic and anoxic conditions to remove these harmful pollutants. Current 'omics' technologies allow the exploitation of the catabolic potential of microbes without the need to cultivate them. Synthetic microbiology builds new catabolic pathways to remove recalcitrant pollutants from the environment.

Complete genome of the plant growth-promoting rhizobacterium Pseudomonas putida BIRD-1.

We report the complete sequence of the 5.7-Mbp genome of Pseudomonas putida BIRD-1, a metabolically versatile plant growth-promoting rhizobacterium that is highly tolerant to desiccation and capable of solubilizing inorganic phosphate and iron and of synthesizing phytohormones that stimulate seed germination and plant growth.

Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6-trinitrotoluene.

Pseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6-trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification-related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up- and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1-oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants.