Response Processes Of Soybean Root Microbiome Exposure To Nano Zinc Oxide And Its Sulfide Products

Metal nanoparticles have been widely used in industrial and agricultural production and human daily life due to their unique physical and chemical properties.Recently,the application of metal nanoparticles in the sustainable development of agriculture has also shown great potential,including nano-pesticides,nano-fertilizers and nano-biological regulators.The ecological effects of metal nanoparticles in agricultural environments have attracted extensive attention from scientists at home and abroad.However,the current understanding and cognition of the effects of metal nanoparticles on microbial communities and metabolic functions in root microdomains are still lacking.Considering the morphological transformation of metal nanoparticles in the environment,the physical and chemical properties of the original metal nanoparticles will change,which may affect their potential ecological effects.In this study,zinc oxide metal nanoparticles（Zn O MNPs）and its sulfide products（s-Zn O MNPs）were selected as the test materials,a control and a zinc sulfate control treatment（Zn SO₄）were set,and soybean was selected as the test plant.Through a 70-day greenhouse pot experiment,high-throughput sequencing and metabolomic methods were used to study the effects of Zn O MNPs and its sulfide products on the microbial community and metabolic function of soybean roots.The main findings are as follows:1.The preparation and characterization of nanoparticles,Zn O MNPs and s-Zn O MNPs were successfully prepared by hydrothermal synthesis,and the morphological characteristics,elemental composition,hydrated particle size and zeta potential of nanoparticles were verified.2.Soybean growth and Zn accumulation,Zn SO₄,Zn O MNPs and s-Zn O MNPs treatments had no significant effect on soybean root elongation and dry weight.However,compared with Zn treatments at low doses（100 mg·kg-¹）,the high concentration treatments(500 mg·kg^-1)significantly reduced the above-ground dry weight of soybean.For Zn accumulation in soybean tissue,in the low concentration treatments,the change trend of Zn accumulation concentration in soybean roots was Zn SO₄>Zn O MNPs>s-Zn O MNPs,and the change trend of Zn accumulation concentration in shoots was s-Zn O MNPs>Zn O MNPs>Zn SO₄.However,under the high concentration treatments,the accumulation of Zn content in soybean roots and shoots showed the same trend,both of which were Zn SO₄ and Zn O MNPs treatments with no significant difference,and both were higher than s-Zn O MNPs treatment.3.Effects of soil physicochemical properties and enzyme activities,compared with control,soil p H,soluble organic carbon content and peroxidase activity were significantly inhibited in Zn SO₄ treatment at high dose.Soil ammonium nitrogen and nitrate nitrogen contents were significantly increased in s-Zn O MNPs treatment at low dose,and soil total nitrogen content was significantly increased in s-Zn O MNPs treatment at high dose.Soil alkaline phosphatase activity was significantly inhibited in Zn O MNPs and s-Zn O MNPs treatments at high doses.Soilβ-1,4-glucosidase andβ-1,4-xylosidase activities were inhibited in three Zn treatments at high doses.4.Microbial community response of root microdomains,for root zone and rhizosphere soil,compared with control,s-Zn O MNPs treatment at low dose significantly increased bacterial community Alpha diversity,Zn SO₄ treatment at high dose had the greatest effect on Beta diversity of bacterial community.For roots and nodules,compared with control,bacterial community Alpha diversity decreased in three Zn treatments,especially in high doses.s-Zn O MNPs and Zn O MNPs treatments at high doses had the greatest effect on Beta diversity of bacterial communities in roots and nodules,respectively.In conclusion,compared with soil,three Zn treatments had greater impacts on Alpha diversity of plant endophytic bacterial community.For significantly affected bacterial taxa,in root zone soil,the relative abundance of Actinobacteria was significantly decreased and the relative abundances of Bacteroidetes,Gemmatimonadetes,Caulobacterales and Bdellovibrionales were significantly increased in Zn SO₄ treatment at high dose.For rhizosphere soil,the relative abundance of Proteobacteria was significantly up-regulated,while the relative abundance of Firmicutes was significantly down-regulated in three Zn treatments at high doses.Moreover,the relative abundances of Bacteroidetes,Gammaproteobacteria and Sphingomonadales were significantly up-regulated in Zn SO₄ treatment at high dose,the relative abundances of Burkholderiaceae and Azospirillaceae were significantly up-regulated in Zn O MNPs treatment at high dose,the relative abundances of Actinobacteria,Rokubacteria and Blastocatellia＿Subgroup＿4 were significantly up-regulated in s-Zn O MNPs treatment at high dose.For roots,Streptosporangiales and Pseudonocardia were significantly enriched in Zn SO₄ treatment at high dose,Neorhizobium was significantly increased in s-Zn O MNPs treatment at high dose.5.Metabolomic responses of root microdomains,for rhizosphere soil,overall expression patterns of metabolites in Zn O MNPs and s-Zn O MNPs treatments at high doses were significantly different from control.The relative contents of organic acids,amino acids,phenolics,sugars and flavonoids metabolites were significantly affected by Zn treatments at high doses.Metabolite pathway analysis showed that tyrosine metabolism was significantly disturbed in three Zn treatments at high doses.For roots,metabolites in three Zn treatments at high doses were significantly different from those in control.Among them,most organic acids were significantly down-regulated,while most amino acids,phenolics,and sugars were significantly up-regulated after exposure to three Zn treatments at high doses.Metabolite pathway analysis showed that compared with rhizosphere soil,three Zn treatments at high doses induced more metabolite pathway disturbances in soybean roots.Galactose metabolism and phenylalanine metabolism in roots were significantly affected in three Zn treatments at high doses.Moreover,this study also found significant correlations between changed bacterial taxa and metabolites in three Zn treatments at high doses,indicating that three Zn treatments at high doses significantly interfered with metabolic changes of these microorganisms.In conclusion,three different forms Zn treatments had no significant effect on soybean root growth,but three different forms Zn treatments at high concentration inhibited the growth of soybean shoots.Compared with soil bacterial community,three different forms Zn treatments had a greater impact on the Alpha diversity of the plant endophytic bacterial community.For different root microdomains,the bacterial taxa affected by different Zn treatments were different.Regarding the metabolite expression levels,the relative contents of organic acids,amino acids and their derivatives,phenolics,sugars and flavonoids metabolites in rhizosphere soil and roots were significantly affected by three Zn treatments at high doses.Correlation analysis of differential bacterial taxa and metabolites showed that the three Zn treatments at high doses significantly interfered with the metabolic changes of these microorganisms.According to the differential responses of bacterial communities and metabolite levels in soybean root microdomains,three different forms Zn treatments had different effect mechanisms on bacterial communities and metabolites in soybean root microdomains,indicating that the ecological effects of Zn O MNPs and s-Zn O MNPs include not only the Zn ions generated by their dissolution,but also the effects of the nanoparticles themselves.Meanwhile,s-Zn O MNPs also exhibit different ecological effects from pristine Zn O MNPs,indicating the transformation of metal nanoparticles in the environment can regulate their ecological effects.This study provides a new insight on the response of soil and plant microbial communities and metabolites to three different forms Zn exposure,providing a comprehensive understanding of the environmental risks of Zn O MNPs.