Insights on the Behavior of Nano-Copper in the Agroecosystem: Mycorrhizal Associations with Spearmint (Mentha spicata)
Nanotechnology offers significant potential benefits to our society, including the agriculture sector. With the advancement of nano-enabled agrochemicals towards sustainable and efficient agricultural practices, it is essential to address environmental issues associated with the use of nanoscale materials. The same properties that give promise to applications of nanotechnology in modern agriculture could have unintended consequences on ecosystem dynamics. A point of concern for risk management is the impact of engineered nanomaterials (ENMs) to beneficial microbial communities, which support a variety of ecosystem services. Use of copper (Cu) products in agriculture are based on their abundance, role as a micronutrient, and antimicrobial activity. As nanoparticles (NPs), their size allows for cost-effective application and easy absorption by plants. Although there are many benefits associated with Cu NPs in agrochemicals, in higher concentrations they may be toxic to the environment and co-existing organisms. Uptake routes of NPs may be intentional or unintentional (in the form of pollution). Therefore, understanding the fate, exposure, and toxicity of Cu NPs are important factors to consider for their safe use. The most common mutualistic association between terrestrial plants and microbes are formed by arbuscular mycorrhizal (AM) fungi. These specialized fungi colonize the roots of the host plant, providing an array of benefits in exchange for key nutrients. Among these conferred benefits are enhanced stress tolerance, including amelioration of heavy metal toxicity. Despite this, fungi are known to be sensitive to abiotic stresses. Although there has been extensive research on plant stress responses, it often does not consider fungal symbiosis. Hence, there is a critical knowledge gap on the impact of Cu NPs to mycorrhizal symbiosis. Spearmint (Mentha spicata) is characterized by its remarkable aroma and commercial value. The leaves and extracted essential oils are primarily used as a flavoring in foods and beverages, fragrance in perfumes and cosmetics, and antioxidative ingredient in traditional medicines. In addition to being widely cultivated for commercial production on a large scale, spearmint is also often grown in home gardens as a culinary herb. Several safety issues are brought forward with spearmint regarding its human uses and the intensive production systems used to meet growing demand. This research was conducted to evaluate the impact of Cu-based NPs/compounds on spearmint plants symbiotically associated with AM fungi. Short term and long term effects of Cu(OH)2 nanowires (nCu), Cu(OH)2 pesticide (Kocide 3000)(bCu), and CuSO4 solution (iCu) on AM fungi were investigated. The research was divided into three phases: Phase I assessed the acute toxicity of nCu, bCu, and iCu to AM fungi; Phase II determined the interactive effects of nCu, bCu, and iCu on agronomical and physiological parameters in mycorrhizal spearmint; and the transfer of nutrients from spearmint plants to AM fungi under exposure to nCu, bCu, and iCu was studied in Phase III. In Phase I, toxicity testing was conducted with two separate bioassays. Endomycorrhizal spores (Glomus etunicatum) were cultivated in either agar or lysogeny broth growth medium containing 0 to 500 mg L-1 of nCu, bCu, and iCu. Fungal growth was monitored over the span of 3 days through spore count (absorbance) and fungal biomass. Visual observations of fungal cultures were also made. The incidence of dark colored spores and mycelium expansion, especially in iCu treatments, indicated that Cu is likely being allocated to fungal structures as part of a metal detoxification strategy by AM fungi. However, based on our quantitative measurements, there were no significant changes in spore count or fungal biomass from exposure to Cu-based NPs/compounds. This experiment demonstrated that AM fungi (Glomus etunicatum) can tolerate Cu concentrations of up to at least 500 mg L-1 from nCu, bCu, and iCu. In Phase II, spearmint plants inoculated with arbuscular mycorrhizal (AM) fungi were sprayed with 0.66 and 1.05 mg of nCu, bCu, and iCu. After a 50-day growth period spearmint plants were harvested for agronomic, biochemical, and elemental analysis. Accumulation of Cu was highest in the roots (149.40−427.03 mg kg−1). In general, Cu-based NPs/compounds were toxic, inhibiting plant growth and element accumulation. The root biomass and length were reduced by up to 8.38 g and 11.80 cm or 59.92 and 48.15%, respectively. Most alterations in element accumulation occurred in the leaves, with significant decreases in Mg (≤10.47 mg kg−1 or 22.59%), Mn (≤0.77 mg kg−1 or 39.77%), and Zn (≤0.20 mg kg−1 or 37.03%), from the respective controls. Mycorrhization alleviated Cu toxicity, most notably in the roots, where interactive effects were observed under application of AM fungi and Cu-based NPs/compounds for root biomass and length. An interactive effect found between the absence of AM fungi and presence of Cu-based NPs/compounds resulted in an influx of leaf Na content (0.70−1.13 mg kg−1, or 219.74−355.07%, increase from the corresponding control), which can be seen as an indicator of Cu-induced stress. Overall, bulk Cu-based compounds had the greatest impact over nano or ionic Cu-based compounds, with interactive effects in root accumulation of Cu (decrease by AM fungi + bulk Cu) and leaf accumulation of Mg and Mn (decrease by AM fungi + bulk Cu). This research confirms that Cu-based NPs/compounds can be phytotoxic to spearmint plants and that mycorrhizal symbiosis can alleviate Cu toxicity. In Phase III, roots from 50-day-old mycorrhizal spearmints plants treated with 0.66 and 1.05 mg/Cu per pot of nCu, bCu, and iCu were isolated and analyzed. Protein, sugar, and starch contents were determined to further understand the transfer and regulation of nutrients from spearmint plants to AM fungi. Mycorrhizal spearmint roots had higher amounts of protein than non-mycorrhizal spearmints roots, which is likely due to glomalin production by AM fungi. Furthermore, there was a negative dose-dependent relationship between protein concentration and Cu-based NPs/compounds. Although sugar accumulation was not impacted by Cu treatments, variations in sugar concentration suggest that sugar transport, metabolism, and storage may have been influenced by exposure to Cu-based NPs/compounds. Similar to results from Phase II, bCu elicited a stronger response than nCu or iCu and differentially affected protein and sugar contents. Starch concentration was stable among all treatments. Findings from this study provides evidence that carbon exchange between spearmint plants and AM fungi is disrupted by Cu stress. This dissertation provided valuable insight on the different interactions between Cu-based NPs/compounds, AM fungi, and spearmint plants. In summary, our results indicated that AM fungi are tolerant of high Cu concentrations and alleviated a wide spectrum of Cu-induced phytotoxicity in spearmint through heavy metal stress adaptations. However, based on our observations, exposure to Cu-based NPs/compounds could negatively influence the nutrient exchange from spearmint plants to AM fungi. Furthermore, bulk treatments elicited a stronger response in mycorrhizal spearmint than nano or ionic treatments. These findings demonstrate that plant−microbe interactions can improve crop productivity and provide environmental benefits, emphasizing the need for risk assessment of crop systems incorporating symbiotic microorganisms. Our research confirms that while Cu-based NPs/compounds do not significantly impede the benefits conferred to spearmint from AM fungi, providing evidence for their safe commercial use in complex environmental systems, further study on underlying mechanisms and bulk Cu-based compounds is also needed.
Apodaca, Suzanne Annette, "Insights on the Behavior of Nano-Copper in the Agroecosystem: Mycorrhizal Associations with Spearmint (Mentha spicata)" (2022). ETD Collection for University of Texas, El Paso. AAI29324115.