Application of Plant Growth-Promoting Bacteria from Cape Verde to Increase Maize Tolerance to Salinity.

Salinity constitutes a major abiotic factor that negatively affects crop productivity. Inoculation with plant growth-promoting bacteria (PGPB) is proven to increase plant tolerance to abiotic stresses and enhance plant growth, development and productivity. The present study aims to increase the resilience of crops to salinity using bacteria from the microbiome of plants growing in saline environments. For that, the halotolerance of bacteria present in the roots of natural plants growing on Sal Island, which is characterized by its arid environment and maritime influence, was determined, with some strains having extreme halotolerance. Their ability to produce plant growth-promoting traits was evaluated, with most strains increasing indole acetic acid (26-418%), siderophore (>300%) and alginate (2-66%) production and phosphate solubilization (13-100%) under salt stress. The strains evidencing the best performance were inoculated in maize (Zea mays L.) plants and their influence on plant growth and biochemical status was evaluated. Results evidenced bacterial ability to especially increase proline (55-191%), whose osmotic, antioxidant and protein-protecting properties reduced protein damage in salt-stressed maize plants, evidencing the potential of PGPB to reduce the impact of salinity on crops. Enhanced nutrition, phytohormone production and osmolyte synthesis along with antioxidant response all contribute to increasing plant tolerance to salt stress.

Biochemical and Behavioural Alterations Induced by Arsenic and Temperature in Hediste diversicolor of Different Growth Stages.

Contamination with Arsenic, a toxic metalloid, is increasing in the marine environment. Additionally, global warming can alter metalloids toxicity. Polychaetes are key species in marine environments. By mobilizing sediments, they play vital roles in nutrient and element (including contaminants) cycles. Most studies with marine invertebrates focus on the effects of metalloids on either adults or larvae. Here, we bring information on the effects of temperature increase and arsenic contamination on the polychaete Hediste diversicolor in different growth stages and water temperatures. Feeding activity and biochemical responses-cholinesterase activity, indicators of cell damage, antioxidant and biotransformation enzymes and metabolic capacity-were evaluated. Temperature rise combined with As imposed alterations on feeding activity and biochemical endpoints at different growth stages. Small organisms have their antioxidant enzymes increased, avoiding lipid damage. However, larger organisms are the most affected class due to the inhibition of superoxide dismutase, which results in protein damage. Oxidative damage was observed on smaller and larger organisms exposed to As and temperature of 21 °C, demonstrating higher sensibility to the combination of temperature rise and As. The observed alterations may have ecological consequences, affecting the cycle of nutrients, sediment oxygenation and the food chain that depends on the bioturbation of this polychaete.

Do Volatiles Affect Bacteria and Plants in the Same Way? Growth and Biochemical Response of Non-Stressed and Cd-Stressed Arabidopsis thaliana and Rhizobium E20-8.

Plant roots are colonized by rhizobacteria, and these soil microorganisms can not only stimulate plant growth but also increase tolerance to stress through the production of volatile organic compounds. However, little is known about the effect that these plant beneficial volatiles may have on bacteria. In this study, the effects on growth and oxidative status of different concentrations of three volatiles already reported to have a positive influence on plant growth (2-butanone, 3-methyl-1-butanol, and 2,3-butanediol) were determined in A. thaliana and Rhizobium sp. strain E20-8 via airborne exposure in the presence and absence of Cd. It was expected to ascertain if the plant and the bacterium are influenced in the same way by the volatiles, and if exposure to stress (Cd) shifts the effects of volatiles on plants and bacteria. Results showed the antioxidant activity of the volatiles protecting the plant cell metabolism from Cd toxicity and increasing plant tolerance to Cd. Effects on bacteria were less positive. The two alcohols (3-methyl-1-butanol and 2,3-butanediol) increased Cd toxicity, and the ketone (2-butanone) was able to protect Rhizobium from Cd stress, constituting an alternative way to protect soil bacterial communities from stress. The application of 2-butanone thus emerges as an alternative way to increase crop production and crop resilience to stress in a more sustainable way, either directly or through the enhancement of PGPR activity.

Interplay of Seasonality, Major and Trace Elements: Impacts on the Polychaete Diopatra neapolitana.

Polychaetes are known to be good bioindicators of marine pollution, such as inorganic contamination. Major and trace elements are commonly present in sediment and may be accumulated by polychaetes such as the tubiculous Diopatra neapolitana. In this study, D. neapolitana individuals were collected in the autumn, winter, spring, and summer of 2018/2019 from the Ria de Aveiro lagoon (western Portugal) to understand how seasonality influences element accumulation. The impact of the interaction of seasonality and elements on oxidative status, energy metabolism, and oxidative damage was also assessed. The obtained results showed that the activity of the antioxidant enzymes catalase, glutathione S-transferases, and non-protein thiol levels were higher in summer and that superoxide dismutase, lipid peroxidation, and electron transport system activity increased in winter. The lowest glycogen levels were observed during spring, and protein carbonylation was the highest during autumn. These results could mainly be related to high temperatures and the bioaccumulation of Al, As, Mn, and Zn. Energy-related parameters increased during spring and autumn, mainly due to the bioaccumulation of the same elements during spring and summer. Lipid damage was higher during winter, which was mainly due to salinity and temperature decreases. Overall, this study demonstrates that seasonality plays a role in element accumulation by polychaetes and that both impact the oxidative status of D. neapolitana.

Effects of graphene oxide nanosheets in the polychaete Hediste diversicolor: Behavioural, physiological and biochemical responses.

Numerous applications exist for graphene-based materials, such as graphene oxide (GO) nanosheets. Increased concentrations of GO nanosheets in the environment have the potential to have a large negative effect on the aquatic environment, with consequences for benthic organisms, such as polychaetes. The polychaete Hediste diversicolor mobilises the sediments, hence altering the availability of contaminants and the nutrients biogeochemical cycle. As such, this study proposes to assess the effects of different GO nanosheet concentrations on the behaviour, feeding activity, mucus production, regenerative capacity, antioxidant status, biochemical damage and metabolism of H. diversicolor. This study evidenced that H. diversicolor exposed to GO nanosheets had a significantly lower ability to regenerate their bodies, took longer to feed and burrow into the sediment and produced more mucus. Membrane oxidative damage (lipid peroxidation) increased in exposed specimens. The increased metabolic rate (ETS) evidenced a higher energy expenditure in exposed organisms (high use of ready energy sources - soluble sugars) to fight the toxicity induced by GO nanosheets, such as SOD activity. The increase in SOD activity was enough to reduce reactive oxygen species (ROS) induced by GO on cytosol at the lowest concentrations, avoiding the damage on proteins (lower PC levels), but not on membranes (LPO increase). This study revealed that the presence of GO nanosheets, even at the lower levels tested, impaired behavioural, physiological, and biochemical traits in polychaetes, suggesting that the increase of this engineered nanomaterial in the environment can disturb these benthic organisms, affecting the H. diversicolor population. Moreover, given the important role of this group of organisms in coastal and estuarine food webs, the biogeochemical cycle of nutrients, and sediment oxygenation, there is a real possibility for repercussions into the estuarine community.

Effects of volatile sulfur compounds on growth and oxidative stress of Rhizobium leguminosarum E20-8 exposed to cadmium.

Volatile sulfur compounds (VSCs) have been reported to be produced by many bacterial species. Depending on the compound, they can negatively influence some organisms (fungi, nematodes and insects) or promote plant growth. Some of these compounds have also been hypothesized to play a role in bacterial response to cadmium (Cd) induced stress. This study aimed to assess the potential effects of four VSCs (dimethyl sulfide - DMS, dimethyl disulfide - DMDS, dimethyl trisulfide - DMTS and methyl thioacetate - MTA) on the growth and oxidative status of Rhizobium sp. strain E20-8 via airborne exposure, in order to test the hypothesis that these volatile compounds can influence growth and tolerance to cadmium. Our results show that, overall, the tested compounds triggered similar antioxidant mechanisms in Rhizobium in the presence of Cd. The protective effect at the membrane level by DMDS and DMTS particularly demonstrates the antioxidant effect of these volatiles, with reductions of up to 50% (DMS) and 80% (DMTS) in lipid peroxidation levels. Due to the volatile nature of these compounds, the low concentrations tested (1 nM to 100 mM), and considering that they are released by bacteria and other organisms such as plants, it is possible that these effects also occur in the soil ecosystem.

Rhizobium sensing of airborne saturated aldehydes of different sizes modulates the response to Cd exposure.

α,ß-unsaturated aldehydes are generally reported as being toxic, however for saturated aldehydes information is scarce. Here we report the effects on growth and biochemical endpoints related to oxidative stress of Rhizobium colonies under airborne exposure to C6 to C13 saturated aliphatic aldehydes and exposed or not to Cd. Smaller aldehydes (C6 to C10) and larger aldehydes (C11 to C13) had distinct effects on cell biochemistry. Smaller aldehydes reduced and larger ones increased lipid peroxidation. The activity of superoxide dismutase was also decreased by smaller aldehydes and increased by the larger ones. Thus, even an exposure at a distance to saturated aldehydes is able to influence the biochemical status of bacterial cells, and the effects appear to be dependent on the size and thus on distinct properties (e.g. volatility and liposolubility). Moreover, some aldehydes (the smaller saturated ones) may even have a beneficial effect, that switches when cells are in oxidative stress (exposed to Cd). This influence can be used in different contexts, by increasing the resilience of bacterial communities to environmental contaminants with oxidizing effect or by sensitizing bacteria to antimicrobial agents.

Rhizobium response to sole and combined exposure to cadmium and the phytocompounds alpha-pinene and quercetin.

Soils can be contaminated with substances arising from anthropogenic sources, but also with natural bioactive compounds produced by plants, such as terpenes and flavonoids. While terpenes and flavonoids have received much less attention from research studies than metals, the effects that phytocompounds can have on soil organisms such as beneficial microorganisms should not be neglected. Herein we report the sole and combined exposure of Rhizobium to cadmium, to the monoterpene alpha-pinene and to the flavanol quercetin. A range of environmentally relevant concentrations of the phytocompounds was tested. Physiological (growth, protein content and intracellular Cd concentration), oxidative damage (lipid peroxidation, protein carbonylation) and antioxidant mechanisms (superoxide dismutase, catalase, glutathione, glutathione-S-transferases, protein electrophoretic profiles) were assessed. Results suggest that exposure to both phytocompounds do not influence Rhizobium growth, but for combined exposure to phytocompounds and Cd, different responses are observed. At low concentrations, phytocompounds seem to relieve the stress imposed by Cd by increasing antioxidant responses, but at high concentrations this advantage is lost and membrane damage may even be exacerbated. Thus, the presence of bioactive phytocompounds in soil may influence the tolerance of microorganisms to persistent toxicants, and may change their impact on the environment.

Airborne exposure of Rhizobium leguminosarum strain E20-8 to volatile monoterpenes: Effects on cells challenged by cadmium.

Volatile organic compounds (VOCs) are produced by plants, fungi, bacteria and animals. These compounds are metabolites originated mainly in catabolic reactions and can be involved in biological processes. In this study, the airborne effects of five monoterpenes (α-pinene, limonene, eucalyptol, linalool, and menthol) on the growth and oxidative status of the rhizobial strain Rhizobium leguminosarum E20-8 were studied, testing the hypothesis that these VOCs could influence Rhizobium growth and tolerance to cadmium. The tested monoterpenes were reported to have diverse effects, such as antibacterial activity (linalool, limonene, α-pinene, eucalyptol), modulation of antioxidant response or antioxidant properties (α-pinene and menthol). Our results showed that non-stressed cells of Rhizobium E20-8 have different responses (growth, cell damage and biochemistry) to monoterpenes, with α-pinene and eucalyptol increasing colonies growth. In stressed cells the majority of monoterpenes failed to minimize the detrimental effects of Cd and increased damage, decreased growth and altered cell biochemistry were observed. However, limonene (1 and 100 mM) and eucalyptol (100 nM) were able to increase the growth of Cd-stressed cells. Our study evidences the influence at-a-distance that organisms able to produce monoterpenes may have on the growth and tolerance of bacterial cells challenged by different environmental conditions.

The role of volatiles in Rhizobium tolerance to cadmium: Effects of aldehydes and alcohols on growth and biochemical endpoints.

Rhizobia have a significant agronomic and environmental role and are eminent contributors to soil fertility. However, this group of microorganisms are affected by various environmental stresses, such as Cd contamination. High Cd concentrations change bacterial metabolism. During this metabolic shift, bacteria alter their volatilome (the set of volatile metabolites synthesized by an organism). In the presence of Cd, peak areas of saturated aldehydes and alcohols were previously reported to increase, and the consequences of this increase to cells are poorly known. In this study, Rhizobium sp. strain E20-8 cells were exposed to Cd and aldehydes or their conjugated alcohols. Exposure to Cd (100 µM) inhibited cell growth and induced several biomarkers of oxidative stress. The present study also evidenced the higher toxicity of most aldehydes relatively to the corresponding alcohol in the presence of Cd, suggesting that reduction of aldehydes into alcohols may be an effective mechanism to restrain aldehydes toxicity in Rhizobium cells under Cd toxicity. Nonetheless, the protective effect was dependent on the pair aldehyde-respective alcohol considered and it differed between Cd stressed and non-stressed cells. Differences in the ability to convert aldehydes to alcohols may emerge as a new feature helping explain the oxidative tolerance variability among bacteria.

Alginate as a feature of osmotolerance differentiation among soil bacteria isolated from wild legumes growing in Portugal.

Plants are naturally colonized by bacteria that can exert beneficial effects on growth and stress tolerance. These bacteria can be used as inoculants to boost crop productivity and plants resilience, and can be especially interesting if they are able to survive to abiotic stresses, such as drought. Herein we report the mechanisms that soil bacteria resort to tolerate drought and we also explore the influence of each mechanism to the level of drought tolerance exhibited, in order to test the hypothesis that different levels of tolerance displayed by bacteria are linked to differential efficiency of osmotolerance mechanisms. For this, the biochemical and physiological responses of bacterial strains of different genera and displaying different levels of tolerance to osmotic stress (sensitive, moderately tolerant and tolerant) induced by polyethylene glycol-6000 (PEG) were studied. Betaine, trehalose and alginate content increased in the majority of the strains exposed to PEG. Betaine was the osmolyte with higher increases, evidencing the important role of this compound in the tolerance of bacteria to drought. However, betaine and trehalose levels were not significantly different among bacteria with different osmotolerance levels. Several biochemical endpoints (protein content, superoxide dismutase, catalase, glutathione-S-transferases) related to oxidative stress were assessed, since oxidative damage has been reported in drought conditions, but little information is available. The oxidative stress parameters were not sufficient to explain differences in the osmotolerance observed for the tested strains. In contrast, alginate showed significant differences among the three levels of osmotolerance, linking the level of osmotolerance with the ability of soil bacteria to synthesize and accumulate alginate intracellularly for the first time. Moreover, our results show that this ability is present in different bacteria genera. Thus, evaluating the ability to synthesize alginate might be an important cue when considering bacterial inoculants for osmotically stressful conditions.

Bacteria from nodules of wild legume species: Phylogenetic diversity, plant growth promotion abilities and osmotolerance.

The demand for food with high nutritional value that can sustain the growth of human population while safeguarding sustainability deserves urgent attention. A possible strategy is the inoculation of crops with plant growth promoting (PGP) bacteria. Plants are naturally colonized by bacteria that can exert beneficial effects on growth and stress tolerance. N2 fixation by rhizobia in the root nodules of legumes is a well-known PGP effect. These bacteria can be used as inoculants to boost legumes productivity and can be especially interesting if they are able to survive to abiotic stresses, such as drought. Herein we report the phylogenetic diversity of bacteria colonizing the root nodules of several wild legume species, from four geographic locations in Portugal with different bioclimates. Interestingly, the vast majority of strains belonged to Flavobacterium, Pseudomonas and other genera apart from rhizobia. PGP abilities other than N2 fixation (production of indol acetic acid, siderophores and volatile organic compounds) and osmotolerance were screened. Location and host plant species did not influence PGP abilities and osmotolerance. Taken together, results evidenced that bacterial strains from wild legumes displaying PGP abilities and osmotolerance can be regarded as good candidates for inoculants of a broad range of hosts, including non-legumes.