Exogenous nano-silicon enhances the power of intercropped faba bean to alleviate cadmium toxicity and resist Fusarium wilt | Journal of Nanobiotechnology

Check location and take a look at supplies

A soil pot experiment was performed in a greenhouse at Yunnan Agricultural College (25°7’N, 102°44’E, altitude: 1950 m) utilizing pure day-night lighting (daylight, 11 h), with temperatures starting from 20 °C to 30 °C and relative humidity between 50% and 60%. The experiment was performed from early October 2023 to mid January 2024, utilizing synthetic paddy soil in Efeng Village, Yuxi Metropolis, Yunnan Province because the take a look at soil. The area has a convention of steady faba bean cultivation, which has led to a big accumulation of the soil-borne Fusarium pathogen within the soil, inflicting faba bean Fusarium wilt. The essential bodily and chemical properties of the pattern soil had been: natural matter 22.4 g kg^− 1, complete nitrogen 1.5 g kg^− 1, complete phosphorus 0.8 g kg^− 1, complete potassium 12.6 g kg^− 1, alkali nitrogen 102.0 mg kg^− 1, Olsen phosphorus 16.9 mg kg^− 1, out there potassium 116.5 mg kg^− 1, Cd²⁺ 0.075 mg kg^− 1, and pH 7.1. The utilized fertilizers had been urea (N 46.0%), bizarre superphosphate (P₂O₅ 16.0%), and potassium sulfate (Ok₂O 50.0%).

Cd was added utilizing CdCl₂⋅2H₂O (CAS: 10108-64-2, ≥ 99.0%), bought from Beijing Huawei Ruike Chemical Expertise Co., Ltd. (Beijing, China). Peptide-coupled fluorescent silicon nanoparticles (Si-NPs) (CAS: 7440-21-3, ≥ 99.0%) ready by microwave irradiation had been chosen as nano-silicon supplies. This materials, which has excessive stability and dispersibility and is extremely absorbable by vegetation, was obtained from Xi’an Qiyue Biotechnology Co., Ltd. (Shaanxi, China).

The examined styles of faba bean and wheat had been Vicia faba L. cv. Yuxidalidou and Triticum aestivum L. cv. Yunmai-112, bought from the Grain and Crop Analysis Institute of Yunnan Academy of Agricultural Sciences.

Experimental design and crop administration

The experiment utilized a single-factor design with three soil pot therapies: (a) a monoculture of faba bean underneath Cd stress (MFcd), with three rows per pot, three faba bean per row, plant spacing of 10 cm, and row spacing of 8 cm; (b) faba bean-wheat intercropping underneath Cd stress (IFcd); and (c) faba bean-wheat intercropping underneath Cd stress with the addition of Si-NPs (IFcd + Si). Within the intercropping therapies, the planting association consisted of 1 row of faba bean (three vegetation/row), adopted by three rows of wheat (six vegetation/row), and one other row of faba bean (three vegetation/row). Every therapy contained three organic replicates, leading to a complete of 9 pots. The plastic pots had been 22 cm in peak, 34 cm in higher diameter, and 19 cm in decrease diameter.

The topsoil (0–20 cm) collected from the sphere was air-dried in a greenhouse for one week. Afterward, particles had been eliminated, and the soil was sieved by means of a 2 mm sieve. Every pot was crammed with 9.0 kg of soil. To arrange Cd-contaminated soil (2 mg kg^− 1 Cd²⁺), 36.61 mg CdCl₂⋅2H₂O was combined with 9.0 kg of experimental soil [14]. The soil was then left to equilibrate for 4 weeks, with soil moisture maintained at 60% of the sphere capability to make sure constant Cd concentrations throughout totally different soil layers, simulating typical Cd-polluted pure soil. Si-NPs had been completely combined with distilled water after which uniformly combined with the experimental soil at a dosage ratio of 250 mg kg^− 1 [14] earlier than being transferred into the pots.

To get rid of uncertainties associated to vitamins, seed emergence charge, and lightweight publicity, fertilizers had been uniformly utilized on the time of potting, with nitrogen, phosphorus, and potassium added at equal charges (N: 100 mg kg^− 1, P₂O₅: 100 mg kg^− 1, Ok₂O: 100 mg kg^− 1) for all therapies. Earlier than beginning the pot experiment, wheat and faba bean seeds had been rigorously chosen to make sure that every seed had a uniform coloration, dimension, and plumpness. The seeds had been floor sterilized with 10% (v/v) H₂O₂ for 30 min, completely rinsed, and germinated at the hours of darkness for 3 days [7]. The greenhouse was maintained at temperatures between 20 °C and 30 °C underneath pure mild and a relative humidity of fifty%. The pots had been randomly positioned and repositioned each three days. Watering was carried out each two days with 200–250 mL of distilled water per pot, sustaining the soil moisture at 60–70% of the utmost area capability. Different day by day administration practices had been in line with typical area administration.

Plant and soil pattern assortment

After three months of co-cultivation, two vegetation had been chosen for damaging sampling. All the faba bean plant was rigorously faraway from the pot, and agronomic parameters of the aboveground elements had been rapidly recorded, together with the investigation of faba bean Fusarium wilt and biomass measurement (dried at 105℃ for 15 min after which at 65 °C to fixed weight). Rhizosphere soil was collected utilizing the “shaking soil methodology” [28]. When amassing plant roots, they had been first rinsed with distilled water, and the vegetation had been divided into totally different organs (roots and aboveground elements) utilizing scissors. Particular care was taken to take care of the integrity of root constructions. The roots had been then soaked in 20 mM Na₂-EDTA for 15 min to take away Cd and Si ions hooked up to the floor, adopted by thorough rinsing with distilled water [29]. The roots had been then ready for subsequent root scanning evaluation and willpower of protection substances and enzyme exercise parameters.

The collected rhizosphere soil samples had been sieved by means of a 2 mm aperture and divided into two elements. One half was used to find out the traditional physicochemical properties and Cd content material of the rhizosphere soil, whereas the opposite half was saved in a sterile tube at -80℃ for bacterial and metabolomic analyses. The gathering course of strictly adopted the sterile working procedures to stop contamination.

Indicator willpower and strategies

Investigation of faba bean Fusarium wilt

Wilt illness investigation was performed on all faba bean vegetation upon detachment of their aboveground elements. The classification of faba bean wilt illness adopted a 5-level classification normal [7]. After investigation, the wilt illness index was calculated utilizing the next components:undefined

{rm{index}} = {matrix{Sigma >({rm{Quantity}}>{rm{of}}>{rm{diseased}}>{rm{vegetation}}>{rm{at}} hfill cr >{rm{every}}>{rm{stage}} instances >{rm{stage}}>{rm{worth}}) hfill cr} over matrix{{rm{The}}>{rm{highest}}>{rm{stage}}> instances >{rm{complete}}> hfill cr {rm{quantity}}>{rm{of}}>{rm{vegetation}}>{rm{studied}} hfill cr} } instances >100]]>

Willpower of rhizosphere soil physicochemical properties

The collected soil samples had been sieved and analyzed utilizing the next strategies: the soil pH was measured with a pH meter in a soil/water suspension (1:2.5); the natural matter was decided utilizing the Ok₂Cr₂O₇-H₂SO₄ oxidation methodology; and ammonium nitrogen and nitrate nitrogen had been measured by way of the indigo phenol blue colorimetric methodology and ultraviolet spectrophotometry, respectively. Obtainable phosphorus was extracted with NaHCO₃ and measured utilizing the molybdenum-antimony colorimetric methodology, and complete phosphorus was decided utilizing the HClO₄-H₂SO₄ digestion methodology. The flame photometer methodology was used to find out the out there and complete potassium contents. The entire above physicochemical parameters had been decided in response to the tactic described by Bao [30]. The willpower of rhizosphere soil enzyme exercise was carried out utilizing air-dried soil (5.000 g; 1 mm sieve screening) and positioned in a 50mL centrifuge tube. The extraction resolution and buffer resolution had been then added in response to the producer’s directions for constant-temperature cultivation. The actions of catalase (CAT), impartial phosphatase (NPase), dehydrogenase, urease (URE), cellulase (cell), and sucrase (SUC) within the rhizosphere soil had been measured utilizing soil enzyme exercise assay kits bought from Beijing Solaboo Expertise Co., Ltd. (Beijing, China) in response to the detailed steps and procedures supplied within the producer’s directions [31].

The bioavailable Cd content material within the rhizosphere soil was extracted utilizing diethylenetriaminepentaacetic acid (DTPA) [4], and the entire Cd within the soil and plant leaves was analyzed utilizing a combined resolution of HNO₃-HClO₄-HF (5:1:1,v/v/v) and HNO₃ digestion [32], adopted by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500a, USA). The usual take a look at outcomes had been inside the allowable error vary with a restoration charge of 90–105%.

Scanning of root morphological traits

After washing the collected and separated roots with deionized water, they had been positioned on the foundation plate of a root scanner (EPSON Perfection 4990 Photograph, China), injected with an acceptable quantity of deionized water, and scanned utilizing preset parameters. The scanned photos had been processed utilizing picture processing software program (Adobe Photoshop CC 2019), and root morphology information, akin to complete root size, common diameter, perimeter, floor space, and root length-to-diameter ratio, had been extracted utilizing specialised root evaluation software program (WinRHIZO Professional 2007d13).

Willpower of antioxidant capability and protection enzyme exercise of faba bean

Contemporary third true leaf (0.1 g) was weighed, and 1 ml of extraction resolution (containing buffer resolution) was added to homogenize the combination in an ice bathtub. After homogenization, the supernatant was centrifuged at 4℃ (10000 ×g, 10 min), collected, and positioned on ice to detect the contents of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and superoxide anion (O₂⁻), and the actions of superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase (POD). MDA, H₂O₂, and O₂⁻ contents had been decided utilizing trichloroacetic acid-thiobarbituric acid (TCA-TBA), titanium sulfate, and hydroxylamine strategies, respectively, following the procedures outlined by Yan et al. [14]. SOD exercise was measured in response to Giannopolis and Ries, APX exercise was measured in response to Nakano and Asada [33], and POD exercise was measured in response to the guaiacol methodology described by Li et al. [34].

Willpower of rhizosphere soil micro organism

Whole DNA of the soil microbial genomes was extracted utilizing the EZ.N.A^® Soil DNA Equipment (Omega Biotek, Norcross, GA, USA) from 0.5 g of freeze-dried soil pattern. The AxyPrep DNA Gel Extraction Equipment (Axygen Biosciences, USA) was used to purify the recovered product. A 2% agarose gel electrophoresis was employed to evaluate the standard of the extracted DNA, and NanoDrop2000 (Thermo Scientific, USA) was used to measure DNA focus and purity. Genomic DNA was then despatched to GENEWIZ for relative quantitative sequencing of the extremely variable V3–V4 area of the 16S rRNA gene, which was chosen for soil bacterial sequencing. The primers used had been 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GACTACHVGGGTWTCTAAT-3’) [35]. Paired-end sequencing (2 × 250 bp) was carried out utilizing an Illumina NovaSeq 6000 sequencing platform. The PCR system and process are described within the Supporting Info. Processing, sequencing, operational taxonomic unit (OTU) clustering, and bacterial species annotation of the PCR merchandise had been performed on the cloud platform of Shanghai Meiji Biotechnology Co., Ltd. (https://cloud.majorbio.com) following the tactic described by Zhang et al. [36]. Uncooked sequence information had been first saved within the Sequence Learn Archive (SRA) of the Nationwide Heart for Biotechnology Info (NCBI) after which processed and analyzed utilizing the Quantitative Perception Microbial Ecology (QIIME) software program package deal model 1.8.0 [37]. The uncooked information supplied on this examine had been submitted to NCBI underneath the accession quantity PRJNA1190895.

Metabolomics evaluation of rhizosphere soil

Pattern preparation

First, 1000.0 ± 5.0 mg of the freeze-dried rhizosphere soil pattern was precisely weighed right into a 2 mL centrifuge tube and a 6 mm grinding bead was added. Subsequently, 1000 µL of extraction resolution (methanol: water = 4:1, v: v) containing 0.02 mg/mL of the inner normal (L-2-chloroalanine) was added for metabolite extraction. The pattern resolution was floor utilizing a frozen tissue grinder for six min (-10℃, 50 Hz), adopted by low-temperature ultrasonic extraction for 30 min (5 °C, 40 kHz). The pattern was then positioned at -20 °C for 30 min, centrifuged for 15 min (4 °C, 13,000 × g), and the supernatant was transferred to an injection vial with an internal tube for evaluation. Moreover, 20 µL of supernatant from every pattern was pooled to create a high quality management pattern. In the course of the evaluation, a QC pattern was inserted each three samples to observe the repeatability of your complete course of.

LC-MS detection

Detection was carried out utilizing a Thermo Fisher Scientific ultra-high-performance liquid chromatography-tandem Fourier remodel mass spectrometry (UHPLC-Q Exactive HF-X) system. The chromatographic column was ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 μm; Waters, Milford, USA). Cell section A consisted of 95% water and 5% acetonitrile with 0.1% formic acid, whereas cell section B contained 47.5% acetonitrile, 47.5% isopropanol, and 5% water with 0.1% formic acid. The injection quantity was 3 µL, column temperature was set to 40 °C, and stream charge was 0.4 mL/min. The gradient elution program was as follows: 0–3.5 min, 0–24.5% B; 3.5–5 min, 24.5–65% B; 5–5.5 min, 65–100% B; 5.5–7.4 min, 100% B, 0.4–0.6 mL/min; 7.4–7.6 min, 100–51.5% B; 7.6–7.8 min, 51.5–0% B, 0.6–0.5 mL/min; 7.8–9 min, 0% B, 0.5–0.4 mL/min; 9–10 min, 0% B, 0.4 mL/min. The mass spectrometry situations included a twig ion supply (ESI) temperature of 425 °C, mass spectrum voltages of 3500 V (constructive mode) and − 3500 V (destructive mode), and a mass scanning vary of 70–1050 m/z. The sheath gasoline stream charge was 50 psi, auxiliary gasoline stream charge was 13 psi, ion transport tube temperature was 325 °C, and normalized collision vitality was 20–40–60 V within the cyclic collision vitality. The decision of the first mass spectrometry was set to 60,000, and that of the secondary mass spectrometry was 7,500, with the info collected in DDA mode.

Pc information processing and differential metabolite evaluation

The uncooked information had been imported into the metabolomics processing software program Progenesis QI v2.3 (Waters Company, Milford, USA) for evaluation. The software program was used for function peak looking and library identification, matching MS and MS/MS spectrometry data with metabolic databases, with the MS error set to lower than 10 ppm. Metabolites had been recognized primarily based on secondary mass spectrometry-matching scores. Principal part evaluation (PCA) and partial least squares discriminant evaluation (PLS-DA) had been carried out on the metabolites utilizing the ropls package deal in R, and mannequin stability was evaluated utilizing a 7-cycle interplay validation. Moreover, Scholar’s t-test and fold-change evaluation had been carried out. Differential metabolites had been screened utilizing variable weight values (VIP values > 1 and p < 0.05), and the highest 30 considerably totally different metabolites in every group had been intersected. The metabolic pathway enrichment evaluation of the differential metabolites was performed utilizing the KEGG database (https://www.kegg.jp/kegg), with the P-values < 0.05, indicating the numerous pathway enrichment.

Statistics and evaluation

Microsoft Excel 2010 and GraphPad Prism 8.0 had been utilized to prepare and plot the take a look at information. Statistical evaluation was carried out utilizing one-way evaluation of variance (ANOVA) and Duncan’s a number of vary take a look at (SPSS 19.0, IBM, USA) to judge the variations in indicators, akin to Fusarium wilt incidence, root morphology attribute parameters, development parameters, soil enzyme exercise, Cd content material, and soil physicochemical elements among the many MFcd, IFcd, and IFcd + Si therapies, with statistical significance set at P < 0.05. Alpha range of bacterial communities (Chao, Shannon, and Simpson indices) was evaluated utilizing the Rogers’s [38] methodology, and inter-group variations had been assessed utilizing the Wilcoxon rank sum take a look at. Beta range evaluation primarily based on Bray-Curtis distance was used to match the microbial group construction between therapies, which was verified and visualized by means of principal coordinate evaluation (PCoA). Information within the tables and figures are offered as imply ± normal deviation (SD) of three replicates. Correlation evaluation between parameters was performed utilizing Spearman’s methodology. Metabolomics of the rhizosphere soil, microbiota, and their affiliation maps with differential metabolites had been analyzed and generated utilizing the free on-line bioinformatics cloud platform of Shanghai Meiji Biomedical Expertise Co., Ltd. (https://cloud.majorbio.com).