Titanium biogenic nanoparticles to help the growth of Trichoderma harzianum to be used in biological control.

BACKGROUND: The biogenic synthesis of metallic nanoparticles is a green alternative that reduces the toxicity of this nanomaterials and may enable a synergy between the metallic core and the biomolecules employed in the process enhancing biological activity. The aim of this study was to synthesize biogenic titanium nanoparticles using the filtrate of the fungus Trichoderma harzianum as a stabilizing agent, to obtain a potential biological activity against phytopathogens and mainly stimulate the growth of T. harzianum, enhancing its efficacy for biological control. RESULTS: The synthesis was successful and reproductive structures remained in the suspension, showing faster and larger mycelial growth compared to commercial T. harzianum and filtrate. The nanoparticles with residual T. harzianum growth showed inhibitory potential against Sclerotinia sclerotiorum mycelial growth and the formation of new resistant structures. A great chitinolytic activity of the nanoparticles was observed in comparison with T. harzianum. In regard to toxicity evaluation, an absence of cytotoxicity and a protective effect of the nanoparticles was observed through MTT and Trypan blue assay. No genotoxicity was observed on V79-4 and 3T3 cell lines while HaCat showed higher sensitivity. Microorganisms of agricultural importance were not affected by the exposure to the nanoparticles, however a decrease in the number of nitrogen cycling bacteria was observed. In regard to phytotoxicity, the nanoparticles did not cause morphological and biochemical changes on soybean plants. CONCLUSION: The production of biogenic nanoparticles was an essential factor in stimulating or maintaining structures that are important for biological control, showing that this may be an essential strategy to stimulate the growth of biocontrol organisms to promote more sustainable agriculture.

Encapsulated plant growth regulators and associative microorganisms: Nature-based solutions to mitigate the effects of climate change on plants.

Over the past decades, the atmospheric CO2 concentration and global average temperature have been increasing, and this trend is projected to soon become more severe. This scenario of climate change intensifies abiotic stress factors (such as drought, flooding, salinity, and ultraviolet radiation) that threaten forest and associated ecosystems as well as crop production. These factors can negatively affect plant growth and development with a consequent reduction in plant biomass accumulation and yield, in addition to increasing plant susceptibility to biotic stresses. Recently, biostimulants have become a hotspot as an effective and sustainable alternative to alleviate the negative effects of stresses on plants. However, the majority of biostimulants have poor stability under environmental conditions, which leads to premature degradation, shortening their biological activity. To solve these bottlenecks, micro- and nano-based formulations containing biostimulant molecules and/or microorganisms are gaining attention, as they demonstrate several advantages over their conventional formulations. In this review, we focus on the encapsulation of plant growth regulators and plant associative microorganisms as a strategy to boost their application for plant protection against abiotic stresses. We also address the potential limitations and challenges faced for the implementation of this technology, as well as possibilities regarding future research.

Nanoencapsulation Boosts the Copper-Induced Defense Responses of a Susceptible Coffea arabica Cultivar against Hemileia vastatrix.

Due to the environmental risks of conventional Cu-based fungicides, Cu-loaded chitosan nanoparticles have been developed as nano-pesticides, aiming to protect plants against different diseases. In this sense, the objective was to verify the effects of chitosan nanoparticles containing Cu2+ ions on leaf discs of Coffea arabica cv. IPR 100 infected with Hemileia vastatrix. The treatments were water as a control (CONT), unloaded chitosan nanoparticles (NP), chitosan nanoparticles containing Cu2+ ions (NPCu), and free Cu2+ ions (Cu). Different concentrations of NP (0.25; 0.5; 1 g L-1) and Cu2+ ions (1.25; 2.5; 5 mmol L-1) were tested. The severity of the coffee rust was 42% in the CONT treatment, 22% in NP, and 2% in NPCu and Cu. The treatments protected coffee leaves; however, NPCu stood out for initial stress reduction, decreasing Cu phytotoxicity, promoting photosynthetic activity maintenance, and increasing antioxidant responses, conferring significant protection against coffee rust. At low concentrations (1.25 mmol L-1), NPCu showed higher bioactivity than Cu. These results suggest that Cu-loaded chitosan nanoparticles can induce a more significant plant defense response to the infection of Hemileia vastatrix than conventional Cu, avoiding the toxic effects of high Cu concentrations. Thus, this nanomaterial has great potential to be used as nano-pesticides for disease management.

Soil Treatment with Nitric Oxide-Releasing Chitosan Nanoparticles Protects the Root System and Promotes the Growth of Soybean Plants under Copper Stress.

The nanoencapsulation of nitric oxide (NO) donors is an attractive technique to protect these molecules from rapid degradation, expanding, and enabling their use in agriculture. Here, we evaluated the effect of the soil application of chitosan nanoparticles containing S-nitroso-MSA (a S-nitrosothiol) on the protection of soybeans (Glycine max cv. BRS 257) against copper (Cu) stress. Soybeans were grown in a greenhouse in soil supplemented with 164 and 244 mg kg-1 Cu and treated with a free or nanoencapsulated NO donor at 1 mM, as well as with nanoparticles without NO. There were also soybean plants treated with distilled water and maintained in soil without Cu addition (control), and with Cu addition (water). The exogenous application of the nanoencapsulated and free S-nitroso-MSA improved the growth and promoted the maintenance of the photosynthetic activity in Cu-stressed plants. However, only the nanoencapsulated S-nitroso-MSA increased the bioavailability of NO in the roots, providing a more significant induction of the antioxidant activity, the attenuation of oxidative damage, and a greater capacity to mitigate the root nutritional imbalance triggered by Cu stress. The results suggest that the nanoencapsulation of the NO donors enables a more efficient delivery of NO for the protection of soybean plants under Cu stress.

Nitric oxide-releasing nanomaterials: from basic research to potential biotechnological applications in agriculture.

Nitric oxide (NO) is a multifunctional gaseous signal that modulates the growth, development and stress tolerance of higher plants. NO donors have been used to boost plant endogenous NO levels and to activate NO-related responses, but this strategy is often hindered by the relative instability of donors. Alternatively, nanoscience offers a new, promising way to enhance NO delivery to plants, as NO-releasing nanomaterials (e.g. S-nitrosothiol-containing chitosan nanoparticles) have many beneficial physicochemical and biochemical properties compared to non-encapsulated NO donors. Nano NO donors are effective in increasing tissue NO levels and enhancing NO effects both in animal and human systems. The authors believe, and would like to emphasize, that new trends and technologies are essential for advancing plant NO research and nanotechnology may represent a breakthrough in traditional agriculture and environmental science. Herein, we aim to draw the attention of the scientific community to the potential of NO-releasing nanomaterials in both basic and applied plant research as alternatives to conventional NO donors, providing a brief overview of the current knowledge and identifying future research directions. We also express our opinion about the challenges for the application of nano NO donors, such as the environmental footprint and stakeholder's acceptance of these materials.

Regression models to stratify the copper toxicity responses and tolerance mechanisms of Glycine max (L.) Merr. plants.

MAIN CONCLUSION: Root antioxidant defense, restricted root-to-shoot Cu translocation, altered nutrient partition, and leaf gas exchange adjustments occurred as tolerance mechanisms of soybean plants to increasing soil Cu levels. The intensive application of copper (Cu) fungicides has been related to the accumulation of this metal in agricultural soils. This study aimed to evaluate the effects of increasing soil Cu levels on soybean (Glycine max) plants. Soybean was cultivated under greenhouse conditions in soils containing different Cu concentrations (11.2, 52.3, 79.4, 133.5, 164.0, 205.1, or 243.8 mg kg-1), and biochemical and morphophysiological plant responses were analyzed through linear and nonlinear regression models. Although Cu concentrations around 50 mg kg-1 promoted some positive effects on the initial development of soybean plants (e.g., increased root length and dry weight), these Cu concentrations also induced root oxidative stress and activated defense mechanisms (such as the induction of antioxidant response, N and S accumulation in the roots). At higher concentrations, Cu led to growth inhibition (mainly of the root), nutritional imbalance, and damage to the photosynthetic apparatus of soybean plants, resulting in decreased CO2 assimilation and stomatal conductance. In contrast, low translocation of Cu to the leaves, conservative water use, and increased carboxylation efficiency contributed to the partial mitigation of Cu-induced stress. These responses allowed soybean plants treated with Cu levels in the soil as high as 90 mg kg-1 to maintain growth parameters higher than or similar to those of plants in the non-contaminated soil. These data provide a warning for the potentially deleterious consequences of the increasing use of Cu-based fungicides. However, it is necessary to verify how the responses to Cu contamination are affected by different types of soil and soybean cultivars.

Atrazine nanoencapsulation improves pre-emergence herbicidal activity against Bidens pilosa without enhancing long-term residual effect on Glycine max.

BACKGROUND: Poly(ϵ-caprolactone) nanocapsules (NC + ATZ) are an efficient carrier system for atrazine and were developed as an alternative to reduce the harmful environmental effects of this herbicide. Here, we analyzed the pre-emergence herbicidal activity of NC + ATZ against Bidens pilosa and evaluated its residual effect on soybean plants after different periods of soil treatment with the formulations. RESULTS: In contrast to non-nanoatrazine, NC + ATZ treatment led to very high mortality rates of B. pilosa seedlings even after a tenfold dilution, which suggests that atrazine nanoencapsulation improved its pre-emergence herbicidal activity. In a short-term assay (17 days), soil treatment with all atrazine-containing formulations resulted in intense toxicity to soybean plants. NC + ATZ at 200 g ha-1 had the same inhibitory effects on the physiological and growth parameters of soybean plants compared with non-nanoatrazine at 2000 g ha-1 , which suggests that atrazine nanoencapsulation increased the short-term residual effect of the herbicide. In a long-term assay (60 days), a gradual recovery of soybean plants from atrazine phytotoxicity was observed. When comparing the effects of nano- and non-nanoatrazine at the same concentrations, the growth and physiological parameters of soybean plants were mainly affected to the same extent. This indicates that encapsulation of atrazine into poly(ϵ-caprolactone) nanocapsules did not enhance the long-term residual effect of the herbicide on soybean. CONCLUSION: NC + ATZ could be applied for efficient weed control without additional phytotoxicity to susceptible crops compared with non-nanoatrazine, provided that a safe interval is respected from atrazine application to sowing. © 2019 Society of Chemical Industry.

Nanocapsules Containing Neem (Azadirachta Indica) Oil: Development, Characterization, And Toxicity Evaluation.

In this study, we prepared, characterized, and performed toxicity analyses of poly(ε-caprolactone) nanocapsules loaded with neem oil. Three formulations were prepared by the emulsion/solvent evaporation method. The nanocapsules showed a mean size distribution around 400 nm, with polydispersity below 0.2 and were stable for 120 days. Cytotoxicity and genotoxicity results showed an increase in toxicity of the oleic acid + neem formulations according to the amount of oleic acid used. The minimum inhibitory concentrations demonstrated that all the formulations containing neem oil were active. The nanocapsules containing neem oil did not affect the soil microbiota during 300 days of exposure compared to the control. Phytotoxicity studies indicated that NC_20 (200 mg of neem oil) did not affect the net photosynthesis and stomatal conductance of maize plants, whereas use of NC_10 (100:100 of neem:oleic acid) and NC_15 (150:50 of neem:oleic acid) led to negative effects on these physiological parameters. Hence, the use of oleic acid as a complement in the nanocapsules was not a good strategy, since the nanocapsules that only contained neem oil showed lower toxicity. These results demonstrate that evaluation of the toxicity of nanopesticides is essential for the development of environmentally friendly formulations intended for applications in agriculture.

Evaluation of the effects of polymeric chitosan/tripolyphosphate and solid lipid nanoparticles on germination of Zea mays, Brassica rapa and Pisum sativum.

Although the potential toxicity of many metallic and carbon nanoparticles to plants has been reported, few studies have evaluated the phytotoxic effects of polymeric and solid lipid nanoparticles. The present work described the preparation and characterization of chitosan/tripolyphosphate (CS/TPP) nanoparticles and solid lipid nanoparticles (SLN) and evaluated the effects of different concentrations of these nanoparticles on germination of Zea mays, Brassica rapa, and Pisum sativum. CS/TPP nanoparticles presented an average size of 233.6±12.1nm, polydispersity index (PDI) of 0.30±0.02, and zeta potential of +21.4±1.7mV. SLN showed an average size of 323.25±41.4nm, PDI of 0.23±0.103, and zeta potential of -13.25±3.2mV. Nanotracking analysis enabled determination of concentrations of 1.33×1010 (CS/TPP) and 3.64×1012 (SLN) nanoparticles per mL. At high concentrations, CS/TPP nanoparticles caused complete inhibition of germination, and thus negatively affected the initial growth of all tested species. Differently, SLN presented no phytotoxic effects. The different size and composition and the opposite charges of SLN and CS/TPP nanoparticles could be associated with the differential phytotoxicity of these nanomaterials. The present study reports the phytotoxic potential of polymeric CS/TPP nanoparticles towards plants, indicating that further investigation is needed on the effects of such formulations intended for future use in agricultural systems, in order to avoid damage to the environment.

Nitric oxide-releasing chitosan nanoparticles alleviate the effects of salt stress in maize plants.

Nitric oxide (NO) is a signaling molecule involved in plant response to various abiotic stresses. However, the application of NO donors in agriculture is hampered by the instability of these compounds. Despite the successful uses of NO-releasing nanoparticles for biomedical purposes and the variety of nanomaterials developed as carrier systems of agrochemicals, the potential applications of nanocarriers for NO delivery in plants have not yet been tested. Herein, we report the synthesis and characterization of chitosan nanoparticles (CS NPs) containing the NO donor S-nitroso-mercaptosuccinic acid (S-nitroso-MSA). The efficiency of these NO-releasing NPs in mitigating the deleterious effects of salinity on maize plants was compared to that of the non-encapsulated NO donor. The NPs were synthesized through ionotropic gelation process, and mercaptosuccinic acid (MSA), the NO donor precursor, was encapsulated into CS NPs (91.07% encapsulation efficiency). Free thiol groups of MSA-CS NPs were nitrosated, leading to S-nitroso-MSA-CS NPs (NO-releasing NPs). The incorporation of S-nitroso-MSA into CS NPs allowed a sustained NO release. Treatments of salt-stressed maize plants with S-nitroso-MSA-CS NPs resulted in a higher leaf S-nitrosothiols content compared to that of free S-nitroso-MSA. Moreover, S-nitroso-MSA-CS NPs were more efficient than was the free NO donor in the amelioration of the deleterious effects of salinity in photosystem II activity, chlorophyll content and growth of maize plants because the protective action of the nanoencapsulated S-nitroso-MSA was achieved at lower dosages. Overall, these results demonstrate the positive impact of S-nitroso-MSA nanoencapsulation in increasing NO bioactivity in maize plants under salt stress.

Evaluation of the side effects of poly(epsilon-caprolactone) nanocapsules containing atrazine toward maize plants.

Poly(epsilon-caprolactone) (PCL) nanocapsules have been used as a carrier system for the herbicide atrazine, which is commonly applied to maize. We demonstrated previously that these atrazine containing polymeric nanocapsules were 10-fold more effective in the control of mustard plants (a target species), as compared to a commercial atrazine formulation. Since atrazine can have adverse effects on non-target crops, here we analyzed the effect of encapsulated atrazine on growth, physiological and oxidative stress parameters of soil-grown maize plants (Zea mays L.). One day after the post-emergence treatment with PCL nanocapsules containing atrazine (1 mg mL(-1)), maize plants presented 15 and 21% decreases in maximum quantum yield of photosystem II (PSII) and in net CO2 assimilation rate, respectively, as compared to water-sprayed plants. The same treatment led to a 1.8-fold increase in leaf lipid peroxidation in comparison with control plants. However, all of these parameters were unaffected 4 and 8 days after the application of encapsulated atrazine. These results suggested that the negative effects of atrazine were transient, probably due to the ability of maize plants to detoxify the herbicide. When encapsulated atrazine was applied at a 10-fold lower concentration (0.1 mg mL(-1)), a dosage that is still effective for weed control, no effects were detected even shortly after application. Regardless of the herbicide concentration, neither pre- nor post-emergence treatment with the PCL nanocapsules carrying atrazine resulted in the development of any macroscopic symptoms in maize leaves, and there were no impacts on shoot growth. Additionally, no effects were observed when plants were sprayed with PCL nanocapsules without atrazine. Overall, these results suggested that the use of PCL nanocapsules containing atrazine did not lead to persistent side effects in maize plants, and that the technique could offer a safe tool for weed control without affecting crop growth.

Nitrate reductase is required for the transcriptional modulation and bactericidal activity of nitric oxide during the defense response of Arabidopsis thaliana against Pseudomonas syringae.

Nitrate reductase (NR) has emerged as a potential NO source in plants. Indeed, the Arabidopsis thaliana NR double-deficient mutant (nia1 nia2) produces low NO and develops abnormal susceptibility to bacterial infection. We have employed quantitative real-time polymerase chain reactions to analyze the effects of NO gas on the expression of defense-related genes in wild-type and nia1 nia2 A. thaliana plants that were inoculated with an avirulent strain of Pseudomonas syringae pv. tomato. The pathogenesis-related gene 1 (PR1) was up-regulated by bacterial infection, and its expression was higher in the wild type than in nia1 nia2. Fumigation with NO attenuated the expression of PR1 and other salicylic acid-related genes in plants that had been inoculated with P. syringae. Nevertheless, NO inhibited the most intense bacterial growth and disease symptoms in nia1 nia2 leaves. The NO fumigation also directly modulated lignin biosynthesis-related gene expression (CAD1) and parts of the auxin (TIR1, ILL1, GH3) and ethylene (ACCS7) pathways, among other defense-related genes, and their modulation was more intense in the NR-deficient mutant. Pathogen inoculation induced delayed but intense H2O2 production in mutant leaves in comparison with the wild type. Hydrogen peroxide potentiated the microbicidal effects of NO against bacterial cultures. These results suggest that NO has a direct microbicidal effect in combination with H2O2 to allow for the attenuation of the SA-mediated defense response, thereby reducing the energy expenditure associated with defense-related gene transcription. Overall, these results highlight the importance of NR-dependent NO production in the establishment of disease resistance.

Nitrogen metabolism and translocation in soybean plants subjected to root oxygen deficiency.

Although nitrate (NO3(-)) but not ammonium (NH4(+)) improves plant tolerance to oxygen deficiency, the mechanisms involved in this phenomenon are just beginning to be understood. By using gas chromatography-mass spectrometry, we investigated the metabolic fate of (15)NO3(-) and (15)NH4(+) in soybean plants (Glycine max L. Merril cv. IAC-23) subjected to root hypoxia. This stress reduced the uptake of (15)NO3(-) and (15)NH4(+) from the medium and decreased the overall assimilation of these nitrogen sources into amino acids in roots and leaves. Root (15)NO3(-) assimilation was more affected by hypoxia than that of (15)NH4(+), resulting in enhanced nitrite and nitric oxide release in the solution. However, (15)NO3(-) was translocated in substantial amounts by xylem sap and considerable (15)NO3(-) assimilation into amino acids also occurred in the leaves, both under hypoxia and normoxia. By contrast, (15)NH4(+) assimilation occurred predominantly in roots, resulting in accumulation of mainly (15)N-alanine in this tissue during hypoxia. Analysis of lactate levels suggested higher fermentation in roots from NH4(+)-treated plants compared to the NO3(-) treatment. Thus, foliar NO3(-) assimilation may be relevant to plant tolerance to oxygen deficiency, since it would economize energy expenditure by hypoxic roots. Additionally, the involvement of nitric oxide synthesis from nitrite in the beneficial effect of NO3(-) is discussed.

Nitrite decreases ethanol production by intact soybean roots submitted to oxygen deficiency: a role for mitochondrial nitric oxide synthesis?

Nitrate increases the tolerance of plants to hypoxia, although the mechanisms related to this beneficial effect are still unclear. Recently, we observed that cultivation of soybean plants with nitrate reduced hypoxic accumulation of fermentation end products by isolated root segments compared with the ammonium treatment. Interestingly, the same decrease in the intensity of fermentation was detected when ammonium-grown root segments were incubated with nitrite, suggesting the involvement of this anion in the nitrate-mediated modulation of fermentative metabolism. Here we extended these experiments to intact plants subjected to root hypoxia and observed similar effects of nitrate and nitrite in reducing root ethanol production, which indicates the physiological relevance of the in vitro results. In both experimental systems, nitrite stimulated nitric oxide emission by ammonium-grown roots to levels similar to that of nitrate-cultivated ones. The involvement of mitochondrial reduction of nitrite to nitric oxide in the root response to hypoxia is suggested.

Involvement of nitrite in the nitrate-mediated modulation of fermentative metabolism and nitric oxide production of soybean roots during hypoxia.

It is widely accepted that nitrate but not ammonium improves tolerance of plants to hypoxic stress, although the mechanisms related to this beneficial effect are not well understood. Recently, nitrite derived from nitrate reduction has emerged as the major substrate for the synthesis of nitric oxide (NO), an important signaling molecule in plants. Here, we analyzed the effect of different nitrogen sources (nitrate, nitrite and ammonium) on the metabolic response and NO production of soybean roots under hypoxia. Organic acid analysis showed that root segments isolated from nitrate-cultivated plants presented a lower accumulation of lactate and succinate in response to oxygen deficiency in relation to those from ammonium-cultivated plants. The more pronounced lactate accumulation by root segments of ammonium-grown plants was followed by a higher ethanol release in the medium, evidencing a more intense fermentation under oxygen deficiency than those from nitrate-grown plants. As expected, root segments from nitrate-cultivated plants produced higher amounts of nitrite and NO during hypoxia compared to ammonium cultivation. Exogenous nitrite supplied during hypoxia reduced both ethanol and lactate production and stimulated cyanide-sensitive NO emission by root segments from ammonium-cultivated plants, independent of nitrate. On the other hand, treatments with a NO donor or a NO scavenger did not affect the intensity of fermentation of soybean roots. Overall, these results indicate that nitrite participates in the nitrate-mediated modulation of the fermentative metabolism of soybean roots during oxygen deficiency. The involvement of mitochondrial reduction of nitrite to NO in this mechanism is discussed.

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development.

The tropical pathogen Moniliophthora perniciosa causes witches' broom disease in cacao. As a hemibiotrophic fungus, it initially colonizes the living host tissues (biotrophic phase), and later grows over the dead plant (necrotrophic phase). Little is known about the mechanisms that promote these distinct fungal phases or mediate the transition between them. An alternative oxidase gene (Mp-aox) was identified in the M. perniciosa genome and its expression was analyzed througout the fungal life cycle. In addition, the effects of inhibitors of the cytochrome-dependent respiratory chain (CRC) and alternative oxidase (AOX) were evaluated on the in vitro development of M. perniciosa. Larger numbers of Mp-aox transcripts were observed in the biotrophic hyphae, which accordingly showed elevated sensitivity to AOX inhibitors. More importantly, the inhibition of CRC prevented the transition from the biotrophic to the necrotrophic phase, and the combined use of a CRC and AOX inhibitor completely halted fungal growth. On the basis of these results, a novel mechanism is presented in which AOX plays a role in the biotrophic development of M. perniciosa and regulates the transition to its necrotrophic stage. Strikingly, this model correlates well with the infection strategy of animal pathogens, particularly Trypanosoma brucei, which uses AOX as a strategy for pathogenicity.