Integrated Metabolomics and Morpho-Biochemical Analyses Reveal a Better Performance of Azospirillum brasilense over Plant-Derived Biostimulants in Counteracting Salt Stress in Tomato.

Increased soil salinity is one of the main concerns in agriculture and food production, and it negatively affects plant growth and crop productivity. In order to mitigate the adverse effects of salinity stress, plant biostimulants (PBs) have been indicated as a promising approach. Indeed, these products have a beneficial effect on plants by acting on primary and secondary metabolism and by inducing the accumulation of protective molecules against oxidative stress. In this context, the present work is aimed at comparatively investigating the effects of microbial (i.e., Azospirillum brasilense) and plant-derived biostimulants in alleviating salt stress in tomato plants by adopting a multidisciplinary approach. To do so, the morphological and biochemical effects were assessed by analyzing the biomass accumulation and root characteristics, the activity of antioxidant enzymes and osmotic stress protection. Furthermore, modifications in the metabolomic profiles of both leaves and root exudates were also investigated by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS). According to the results, biomass accumulation decreased under high salinity. However, the treatment with A. brasilense considerably improved root architecture and increased root biomass by 156% and 118% in non-saline and saline conditions, respectively. The antioxidant enzymes and proline production were enhanced in salinity stress at different levels according to the biostimulant applied. Moreover, the metabolomic analyses pointed out a wide set of processes being affected by salinity and biostimulant interactions. Crucial compounds belonging to secondary metabolism (phenylpropanoids, alkaloids and other N-containing metabolites, and membrane lipids) and phytohormones (brassinosteroids, cytokinins and methylsalicylate) showed the most pronounced modulation. Overall, our results suggest a better performance of A. brasilense in alleviating high salinity than the vegetal-derived protein hydrolysates herein evaluated.

Nutraceutical Profiles of Two Hydroponically Grown Sweet Basil Cultivars as Affected by the Composition of the Nutrient Solution and the Inoculation With Azospirillum brasilense.

Sweet basil (Ocimum basilicum L.) is one of the most produced aromatic herbs in the world, exploiting hydroponic systems. It has been widely assessed that macronutrients, like nitrogen (N) and sulfur (S), can strongly affect the organoleptic qualities of agricultural products, thus influencing their nutraceutical value. In addition, plant-growth-promoting rhizobacteria (PGPR) have been shown to affect plant growth and quality. Azospirillum brasilense is a PGPR able to colonize the root system of different crops, promoting their growth and development and influencing the acquisition of mineral nutrients. On the bases of these observations, we aimed at investigating the impact of both mineral nutrients supply and rhizobacteria inoculation on the nutraceutical value on two different sweet basil varieties, i.e., Genovese and Red Rubin. To these objectives, basil plants have been grown in hydroponics, with nutrient solutions fortified for the concentration of either S or N, supplied as SO4 2- or NO3 -, respectively. In addition, plants were either non-inoculated or inoculated with A. brasilense. At harvest, basil plants were assessed for the yield and the nutraceutical properties of the edible parts. The cultivation of basil plants in the fortified nutrient solutions showed a general increasing trend in the accumulation of the fresh biomass, albeit the inoculation with A. brasilense did not further promote the growth. The metabolomic analyses disclosed a strong effect of treatments on the differential accumulation of metabolites in basil leaves, producing the modulation of more than 400 compounds belonging to the secondary metabolism, as phenylpropanoids, isoprenoids, alkaloids, several flavonoids, and terpenoids. The primary metabolism that resulted was also influenced by the treatments showing changes in the fatty acid, carbohydrates, and amino acids metabolism. The amino acid analysis revealed that the treatments induced an increase in arginine (Arg) content in the leaves, which has been shown to have beneficial effects on human health. In conclusion, between the two cultivars studied, Red Rubin displayed the most positive effect in terms of nutritional value, which was further enhanced following A. brasilense inoculation.

Azospirillum brasilense inoculation counteracts the induction of nitrate uptake in maize plants.

Nitrogen (N) represents one of the limiting factors for crop growth and productivity and to date has been widely supplied via external application of fertilizers. However, the use of plant growth-promoting rhizobacteria (PGPR) might represent a valuable tool to further improve plant nutrition. This study examines the influence of Azospirillum brasilense strain Cd on nitrate uptake in maize (Zea mays) plants, focusing on the high-affinity transport system (HATS). Plants were induced with nitrate (500 µM) and either inoculated or not with Azospirillum. Inoculation decreased the nitrate uptake rate in induced plants, suggesting that Azospirillum may negatively affect HATS in the short term. The expression dynamics of ZmNF-YA and ZmLBD37 suggested that Azospirillum affected the N balance in the plants, most probably by supplying them with reduced N, i.e. NH4+. This was further corroborated by measurements of total N and the expression of ammonium transporter genes. Overall, our data demonstrate that Azospirillum can counteract the plant response to nitrate induction, albeit without compromising N nutrition. This suggests that the agricultural application of microbial inoculants requires fine-tuning of external fertilizer inputs.

Role of Azospirillum brasilense in triggering different Fe chelate reductase enzymes in cucumber plants subjected to both nutrient deficiency and toxicity.

Azospirillum brasilense was reported to up-regulate iron (Fe) uptake mechanisms, such as Fe reduction and rhizosphere acidification, in both Fe sufficient and deficient cucumber plants (Cucumis sativus L.). Strategy I plants take up both Fe and copper (Cu) after their reduction mediated by the ferric-chelate reductase oxidase (FRO) enzyme. Interestingly, in cucumber genome only one FRO gene is reported. Thus, in the present study we applied a bioinformatics approach to identify the member of cucumber FRO gene family and allowed the identification of at least three CsFRO genes, one of which was the already identified, i.e. CsFRO1. The expression patterns of the newly identified transcripts were investigated in hydroponically grown cucumber plants treated with different Fe and Cu nutritional regimes. Gene expression was then correlated with morphological (i.e. root architecture) and physiological (Fe(III) reducing activity) parameters to shed light on: i) the CsFRO homologue responsible of the increased reduction activity in Fe-sufficient plants inoculated with A. brasilense cucumber plants, and ii) the possible effect of A. brasilense in ameliorating the symptoms of Cu toxicity in cucumber plants. The data obtained showed that all the CsFRO genes were expressed in the root tissues of cucumber plants and responded to Cu starvation, combined Cu/Fe deficiency and Cu toxicity. Only CsFRO3 was modulated by the A. brasilense in Fe-sufficient plants suggesting for the first time a different specificity of action of the three isoenzymes depending not only on the nutritional regime (either deficiency or toxicity) but also on the presence of the PGPR. Furthermore, results suggest that the PGPR could even ameliorate the stress symptoms caused by both the double (i.e. Cu and Fe) and Cu deficiency as well as Cu toxicity modulating, on one hand, the growth of the root system and, on the other hand, the root nutrient uptake.

Growth and chemical changes in the rhizosphere of black oat (Avena strigosa) grown in soils contaminated with copper.

Copper based pesticides are used to protect vineyards from fungal infections. Plants like black oats (Avena strigosa Schreb) can promote chemical changes in the rhizosphere, reducing copper (Cu) bioavailability in contaminated soils. The objective of this study was to evaluate how copper additions would affect growth, morphology and nutrient uptake by black oats and how the plants affect the chemical composition in rhizosphere and bulk soil. The soil was collected in grassland of southern Brazil. The soil was air-dried, adjusted pH and added phosphorus and potassium amendments, and then it was incubated. Three Cu levels were established in the soil with the addition of 0, 40 and 80 mg Cu kg-1. The experimental design consisted of pots containing 8 plants with 10 kg of soil. Rhizosphere (2 kg of soil) and bulk (8 kg of soil) separated by a 30 µm nylon membrane. Black oat plants were grown for 54 days. The soil and solution were chemically characterized throughout cultivation for Cu speciation. At 54 days after emergence, the soil was sampled and proceeded chemical analysis and plants were collected to determine yield dry matter, morphological parameters and nutrient concentration. Black oat plants induce increase of pH and dissolved organic carbon in the rhizosphere. These root-induced processes increase the percentage of complexed chemical species and decrease free Cu+2 in soil solution, decreasing Cu toxicity. However, soil contamination with Cu induces morphological changes and nutritional imbalances. Black oats could thus be planted along with vineyards, for such increasing protect the soil and promote nutrient cycling, as well as reduce the free Cu available fraction due to the root-induced modifications in the rhizosphere.

Soil amendment as a strategy for the growth of young vines when replanting vineyards in soils with high copper content.

Soil contamination with copper (Cu)-based agrochemicals used in vineyards for pest control is a growing problem. In this context, the application of soil amendment to limit Cu toxicity, especially for young plants after the replanting of vineyards, has been a concern for winemakers. Therefore, the aim of this study was to evaluate how different amendments can contribute to the decrease in Cu availability in areas vocated to viticulture. Furthermore, the aim was to evaluate to the effect of Cu on the biochemical and physiological changes in the development of the young vine plants, both at the shoot and the root level. Vine plants were grown in a greenhouse using a Typic Hapludalf soil characterized by 87.5 mg of Cu kg-1 (control). Three different amendments were applied to the soil: limestone (3 Mg ha-1), calcium silicate (3 Mg ha-1) and vermicompost (30 g of C kg-1). The amendment with vermicompost and calcium silicate caused a significant alkalization of the soil solution. Moreover, specifically for the treatment with vermicompost, the levels of Cu2+ in the soil solution were consistently diminished with a clear benefit for plants (+89% biomass accumulation at the shoot level). In addition, this soil amendment led to a higher photosynthetic rate, lower superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (POD, EC 1.11.1.7) activity and a higher percentage of fine roots with diameter between 0 < L ≥ 0.2 mm (particularly active in water and nutrient acquisition). In conclusion, results showed that vermicompost effectively reduced Cu phytotoxicityin young vines grown in soils with high Cu contents. Furthermore, this amendment might be an asset in enhancing the availability of other important micronutrients such as iron.

Copper accumulation in vineyard soils: Rhizosphere processes and agronomic practices to limit its toxicity.

Viticulture represents an important agricultural practice in many countries worldwide. Yet, the continuous use of fungicides has caused copper (Cu) accumulation in soils, which represent a major environmental and toxicological concern. Despite being an important micronutrient, Cu can be a potential toxicant at high concentrations since it may cause morphological, anatomical and physiological changes in plants, decreasing both food productivity and quality. Rhizosphere processes can, however, actively control the uptake and translocation of Cu in plants. In particular, root exudates affecting the chemical, physical and biological characteristics of the rhizosphere, might reduce the availability of Cu in the soil and hence its absorption. In addition, this review will aim at discussing the advantages and disadvantages of agronomic practices, such as liming, the use of pesticides, the application of organic matter, biochar and coal fly ashes, the inoculation with bacteria and/or mycorrhizal fungi and the intercropping, in alleviating Cu toxicity symptoms.

Effects of zinc addition to a copper-contaminated vineyard soil on sorption of Zn by soil and plant physiological responses.

The occurrence of high levels of Cu in vineyard soils is often the result of intensive use of fungicides for the preventive control of foliar diseases and can cause toxicity to plants. Nowadays many grape growers in Southern Brazil have replaced Cu-based with Zn-based products. The aim of the study was to evaluate whether the increase in Zn concentration in a soil with high Cu contents can interfere with the dynamics of these elements, and if this increase in Zn may cause toxicity to maize (Zea mays L.). Soil samples were collected in two areas, one in a vineyard with more than 30 years of cultivation and high concentration of Cu and the other on a natural grassland area adjacent to the vineyard. Different doses of Cu and Zn were added to the soil, and the adsorption isotherms were built following the Langmuir's model. In a second experiment, the vineyard soil was spiked with different Zn concentrations (0, 30, 60, 90, 180, and 270mg Zn kg(-1)) in 3kg pots where maize was grown in a greenhouse for 35 days. When Cu and Zn were added together, there was a reduction in the quantities adsorbed, especially for Zn. Zn addition decreased the total plant dry matter and specific leaf mass. Furthermore, with the increase in the activity of catalase, an activation of the antioxidant system was observed. However, the system was not sufficiently effective to reverse the stress levels imposed on soil, especially in plants grown in the highest doses of Zn. At doses higher than 90Znmgkg(-1) in the Cu-contaminated vineyard soil, maize plants were no longer able to activate the protection mechanism and suffered from metal stress, resulting in suppressed dry matter yields due to impaired functioning of the photosynthetic apparatus and changes in the enzymatic activity of plants. Replacement of Cu- by Zn-based fungicides to avoid Cu toxicity has resulted in soil vineyards contaminated with these metals and damaging of plant photosynthetic apparatus and enzyme activity.