Fe₃O₄ nanoparticles and Rhizobium inoculation enhance nodulation, nitrogen fixation and growth of common bean plants grown in soil

Highlights

• -Fe₃O₄ NPs and Rhizobium inoculation-increase nitrogenase activity, - nodulation and fixation nitrogen-.

• Fe₃O₄ NPs acted synergistically with Rhizobium to improve plant growth.

• Fe₃O₄ NPs in treatments T1 and T3 showed a superparamagnetic behavior in the nodules. -

• Fe₃O₄ NPs may serve as nanofertilizer for enriching Fe-deficit soil with Fe.

Abstract

The effects of Fe₃O₄ nanoparticles (NPs) and Rhizobium inoculation on nodulation, nitrogen fixation and plant growth of common bean (cv. Red Guama, Phaseolus vulgaris) plants were investigated in growth chambers. Plants were exposed to: Fe₃O₄ NPs (2000 mg/L) (T1), Rhizobium inoculation (T2) and Fe₃O₄ NPs + Rhizobium inoculation (T3); non-treated plants were considered as controls. Harvested 35-day-old treated plants showed improved symbiotic performance including increased nitrogenase activity (51.2–90.7%), nodule leghaemoglobin (44.8–80.9%) and iron content (83.4–84.2%), number of active nodules per plant (58.7–122%) and nodule dry weight (40.2–70.6%). This resulted in enhanced symbiotic nitrogen fixation,and increased shoot (26.5–50.2%) and root (24.1–48.2%) total nitrogen content in treated plants in comparison with the controls. The best result was obtained using treatment T3. Furthermore, Fe₃O₄ NPs were taken up by bean plants in treatments T1 and T3, and these accumulated in their organs, including in nodules. All treatments led to an increase in root (51.9–79.8%) and shoot (27.5–52.7%) lengths, in leaf area (10.9–16.8%) and in root (10.1–17.8%), stem (9.8–12.7%) and leaf dry weight (8–17.3%) compared to control plants. Thus applied treatments have the potential to improve common bean plant growth through enhancement of nodulation and nitrogen fixation during vegetative growth.This study also provides strong evidence that the presence Fe₃O₄ NPs in nodules improves the symbiotic performance between Rhizobium (leguminosarum CF1 strain) and the common bean plant, due to enhanced nodulation and nitrogen fixation.

Introduction

Nanoparticles are defined as materials with three external nanoscale dimensions in the range of 1–100 nm (Jeevanandam, 2018). They have attracted interest due to their unique properties (Gilroy et al., 2016; Liu and Di Valentin, 2019; Huang et al., 2020), including quantum confinement, a large surface area to volume ratio, high surface energy, and several other catalytic and magnetic properties (Handy et al., 2008; Vallabani et al., 2019). In particular, the effects of engineering nanoparticles (ENPs) on plants are of great interest because of their importance in ecological systems. Plants provide a potential pathway for ENP transportation into the environment and serve as a significant route for their bioaccumulation in the food chain.

There are several reports on the effects of Fe₃O₄ NPs on germination and plant growth. For example, there is an increase in chlorophyll in soybean seedlings treated with 9 nm Fe₃O₄ NPs applied in a concentration based on the quantity of iron needed for plant growth; no trace of toxicity has been observed despite the translocation of NPs into soybean stems (Ghafariyan et al., 2013). There are also significant positive effects of Fe₃O₄ NPs on plant growth characteristics of wheat and rocket (Eruca sativa)(Iannone et al., 2016; Plaksenkova et al., 2019). Fe₃O₄ NPs significantly increases plant root length (9%–32%), chlorophyll a fluorescence (1.94–2.8-fold) and miRNA expression (0.31–0.42-fold) compared to those of the control in yellow medick (Medicagofalcata L.) plants (Kokina et al., 2020). Addition of Fe₃O₄ NPs (2000 mg/L) in each heavy metal (Pb, Zn, Cd and Cu) solution (1 mM) significantly decreases the growth inhibition (193.91%, 37.56%, 97.72%, 31.89% in root and 65.75%, 25.06% 87.35%, 60.96% in shoot) and activates protective mechanisms to reduce oxidative stress induced by heavy metals in the wheat seedlings (Konate et al., 2017). A progressive and systematic increase in the magnetization signal occurs in leaves, stems, and root samples of common bean plants grown in soil irrigated with increasing concentrations of Fe₃O₄ NP suspensions. This indicates that they can be taken up by the roots, translocated to the aerial regions, and accumulated in different plant organs (Govea-Alcaide et al., 2016). Also, Fe₃O₄ NPs have remarkable positive effects on the chemical properties of the soil rhizosphere with increases in P (18–22%), K, (25–43%), Ca (35–43%), Mn (53–115%) and Fe (207–493%) and on accumulation of nutrients with increases in P(11–14%), K(21–32%), Ca (17–22%), Mn (10–17%) and Fe (192–277%) in common bean plants grown in this medium (De Souza et al., 2019). Thus their use opens up a wide range of possibilities in plant research and agronomy (Abd-Elsalam et al., 2019).

Very little information is available on the role of NPs in inducing nodulation and the biological fixation nitrogen in legumes, which depends on the effective formation of nodules by Rhizobium. Inoculation of legume seeds with the bacterium can assure its present in the root environment in adequate quantities to colonize the legume rhizosphere, thereby improving nitrogen fixation upon nodule formation (Schwember et al., 2019; Mahmud et al., 2020). However, the efficiency of Rhizobium inoculation depends on the host genotype, Rhizobium strain inefficiency, soil conditions and climatic factors (Thilakarathna and Raizada, 2017; Irisarri et al., 2019; Han et al., 2020; Yuan et al., 2020). Inoculation with Fe₃O₄ NPs-induced Rhizobium (MK358859 strain) enhances nodulation, leghaemoglobin content (110.4%), nitrogenase activity (3.7%), and growth of chickpea plants (22.3–32.7%) at salinities of 75 and 150 mM NaCl (Abd-Allaa et al., 2019), and multi-walled carbon nanotubes (3000 mg kg⁻¹) slightly increase nitrogen fixation (8%) in red clover plants (Moll et al., 2016). However, other studies have shown adverse effects of NPs on legume-rhizobia symbiosis. For instance, CeO₂ NPs diminish nitrogen fixation in soybeans (Coman et al., 2019), but there is no effect of TiO₂ and Fe₃O₄ NPs on nodule colonization (Burke et al., 2015). Delayed nitrogen fixation occurs in peas exposed to TiO₂ and ZnO NPs in hydroponic systems (Fan et al., 2014; Huang et al., 2014; Sarabia-Castillo and Fernández-Luqueño, 2016). Nodulation and nitrogenase activity in faba beans are delayed by Ag NPs (Abd-Alla et al., 2016), whereas arbuscular mycorrhizal colonization of white clover roots is increased by Ag and FeO NPs (Feng et al., 2013). The number of nodules is decreased by Ag and ZnO NPs in alfalfa plants (Moghaddam et al., 2017). Therefore, the effect of NPs on legume-rhizobia appears to be species-and NPs-dependent. Here, we assess whether Rhizobium inoculation, symbiotic performance, nodulation and nitrogen fixation in common bean plants grown in soil are affected by Fe₃O₄ NPs.

The common bean (Phaseolus vulgaris L.) is considered to be the most important grain legume (Jiang et al., 2020), but it is generally regarded as a rather inefficient fixer of nitrogen in comparison to other grain legumes (Argaw and Akuma, 2015; Wilker et al., 2019; Allito et al., 2020; Reinprecht et al., 2020).This is perhaps partly due to the absence of appropriate rhizobial strains, host cultivars, environmental variables (Chekanai et al., 2018; Reinprecht et al., 2020) and reduction in effective nodulation by competition from high populations of competitive but ineffective native Rhizobia spp. (Argaw and Akuma, 2015).Therefore, improvement of bean nitrogen fixation requires a multidisciplinary approach to increase the host capacity to fix nitrogen (Argaw and Akuma, 2015; diCenzo et al., 2018) and a selection of effective Rhizobium strains that can accomplish productive nodulation in the presence of native populations of bacteria present in most soils.

The aim of this study is to determine for the first time the effects of magnetite nanoparticles (Fe₃O₄ NPs) and Rhizobium (leguminosarum CF1 strain) inoculation treatments on nodulation, nitrogen fixation, iron content and vegetative plant growth of common bean plants in soil under growth chamber conditions, and evaluate whether their synergistic interaction can improve nodule activity, nitrogen fixation and the productivity of this important legume.