| In recent years,with the acceleration of industrialization,Cd pollution in soil has become increasingly serious.Wheat is the second largest food crop in China.The Cd accumulation of wheat not only affects wheat yield and quality,but also can harm human health through the food chain.Immobilization technology of heavy metal by bacteria is an effective remediation strategy for metal-contaminated farmland soils due to its environmental friendliness and low cost.Plant growth-promoting bacteria can not only immobilize heavy metals,reduce heavy metal uptake by plants and improve the quality of the soil,but also promote plant growth.However,the mechanisms involved are still unclear.In this study,we screened a highly effective Cd-immobilizing and plant growth-promoting bacterial strains,and studied its effect on reducing Cd uptake of wheat and the mechanisms involved.The results not only provide experimental basis for clarifying the mechanisms involved in the bacteria-induced reduced Cd accumulation of wheat plants,but also provide a new and effective approach for Cd remediation and safe production of wheat grains in Cd-contaminated soils.In this study,one highly effective Cd-resistant and immobilizing bacterial strain WRS8 was isolated from the rhizosphere soil of wheat plants grown in Cd-contaminated soil.Strain WRS8 has a high ability to immobilize heavy metal ions.In the 1/5 LB liquid medium added with 10 mg L-1 Cd2+or Pb2+ or Cu2+,the concentrations of Cd2+,Pb2+,and Cu2+ were reduced by 88%,92%,and 73%,respectively,in the presence of strain WRS8 compared to the controls.The strain WRS8 produces indoleacetic acid(IAA),1-aminocyclopropane-1-carboxylate(ACC)deaminase.and arginine decarboxylase,and can dissolve apatite.16S rDNA sequence analysis showed that strain WRS8 belongs to Pseudomonas taiwanensis.In this study,Serratia liquefaciens CL-1.a highly effective Cd-immobilizing bacterial strain was used as a reference strain.The Cd adsorption of strains WRS8 and CL-1 and the mechanisms involved were compared in the Cd(10 mg L-1)-added solution.The water-soluble Cd concentration reduced by 76-86%for WRS8 and 16-30%for CL-1 compared to that for the control between 1 and 20 days of incubation.The cell surface-adsorbed and intracellular Cd contents increased by 1.7-2.4-fold and 1.7-8.0-fold,respectively,in the presence of WRS8 than in the presence of CL-1.Notably,Cd stress promoted the floccules production by strainWRS8 and the flocculent-adsorbed Cd content ranged from 3.65 to 8.21 mg g-1.These results showed that WRS8 had a higher ability to immobilize Cd than CL-1.WRS8 increased Cd immobilization through increasing cell surface and flocculent adsorption as well as the intracellular accumulation.SEM-EDS,FTIR and XPS analysis revealed that Cd was bound to the surfaces of the bacterial cells and that hydroxyl,carboxyl,and phosphate groups might be the main functional groups involved in the Cd biosorption process.The intensity and the number of chemical reactions of hydroxyl and carbonyl groups on the cell wall surface involved in Cd2+ adsorption were higher in strain WRS8 than in strain CL-1.The effect of strains WRS8 and CL-1 on Cd-immobilization in the soils and the mechanisms involved were compared.The results showed that the cell number of strains WRS8 and CL-1 in the unsterilized and sterilized soils were 84-132 × 104 CFU g-1 soil and 44-84 × 104 CFU g-1 soil,respectively,between 15-30 days of inoculation.The exchangeable Cd content in the unsterilized soil decreased by 10-23%and 7-10%,in the presence of strains WRS8 and CL-1,respectively,compared with the control.The carbonate-and iron-manganese oxidation-bound Cd contents increased by 12-19%and 23-50%.respectively,in the presence of strain WRS8.The carbonate-and iron-manganese oxidation-bound Cd contents increased by 10-18%and 12-13%,respectively,in the presence of strain CL-1.The results showed a higher ability of strain WRS8 to colonize Cd-contaminated soil and immobilize Cd.Compared with the control,strain WRS8 significantly increased the soil pH and the proportion of agglomerates(12-25%)of small particles(<0.5 mm),thereby enhancing the Cd adsorption capacity of soil and reducing the DTPA-extractable Cd content in the soil.Strain WRS8 also significantly increased the soil urease activity(29-40%)and ureC gene copy(79-105%),thereby increasing the soil pH and CO32-content,leading to Cd2+ immobilization in the soil.The effects of strains WRS8 and CL-1 on Cd uptake by wheat plants(Ningmai 9 and Yangmai 13)and the mechanisms under hydroponic conditions added with 0 and 0.5 mg L-1 of Cd were investigated.The results showed that strains WRS8 and CL-1 increased the biomass by 15-37%and 4-20%and reduced Cd content in the wheat seedlings by 81-94%and 65-81%,respectively,compared to the controls.Strains WRS8 and CL-1 also reduced Cd content in the roots by 78-84%and 60-73%in the presence of WRS8 and CL-1,respectively,compared to the control.Strain WRS8 had a higher ability to increase the wheat biomass and reduce the Cd accumulation of the wheat plants.Under Cd stress,strains WRS8 and CL-1 could colonize the root surface(106-107 CFU g-1)and the root interiors(104-105 CFU g-1).Compared with the control,the root surface-adsorbed Cd content increased by 99-207%and 25-67%,respectively,in the presence of strains WRS8 and CL-1.The expression of the LCT1(48-49%for WRS8 and 32-38%for CL-1)and HMA2(26-27%for WRS8 and 13-14%for CL-1)genes encoding Cd transporter were down-regulated and the expression of the genes IRT(7.4-11%for WRS8 and 17-18%for CL-1)and NRAMP1(42-70%for WRS8 and 27-36%for CL-1)encoding transporter for Fe and other metal ion were up-regulated in the roots inoculated with stains WRS8 and CL-1.Strain WRS8 also had a higher ability to reduce the expression of the genes encoding Cd transporters.The effects of the strains,biochar and their combination on the wheat biomass and Cd uptake were studied.Compared with the control,strain WRS8,combination of strain WRS8 and biochar significantly increased the wheat grain biomass by 27-44%.Cd content in the grains of Ningmai 9 is 0.05 mg kg-1 in the presence of strain WRS8,which was lower than the National Food Safety Standard(GB2762-2017).Compared with the control,strain WRS8 significantly increased the pH and the proportion of small soil aggregates in rhizosphere soil by 13-21%,reduced the DTPA-extractable Cd content by 24-34%,leading to the reduced Cd accumulation by the wheat plants.By using MiSeq high-throughput sequencing and traditional culture methods,the effect of strain WRS8 and CL-1 on the root microbiome(including bulk,rhizosphere soil,and intra-root)and the flora regulation mechanism involved were studied.The results showed that the dominant bacterial phyla in this microhabitat were Proteobacteria(71.91%),Actinobacteria(11.97%),and Bacleroidetes(3.91%).From bulk soil to the rhizosphere soil and to the root interior,the ratio of Proteobacteria significantly increased.The structure of bacterial community in the wheat roots was significantly different from those in the bulk and rhizosphere soils based on PCoA analysis.Strains WRS8 and CL-1 had more effect on the bacterial community structure at the jointing and booting stages of the wheat plant(N9).RDA analysis showed that the DTPA-extractable Cd content in the soil was significantly related to the variation of the root-associated microbiome community structure.Correlation analysis showed that the relative abundances of Sphingomonas and Phenylobacterium in rhizosphere soil were significantly negatively correlated with the DTPA-extractable Cd content(r=-0.65-0.77,p<0.05).The relative abundances of Massilia,Bacillus and Nocardioides were significantly positively correlated with the DTPA-extractable Cd content in rhizosphere soil was(r=0.645-0.742,p<0.05).In addition,the relative abundances of Pseudomonas and Ramlibacter were significantly negatively(r=-0.740,p<0.05)and positively(r=0.650,p<0.05)correlated with the Cd content in the wheat roots,respectively.The relative abundances of Caulobacter,Galbitalea,Polaromonas,Rhizobacter,Sphingomonas and Umezawaea in the root interiors were positively correlated with the Cd content(r=0.584-0.803,P<0.05).The relative abundances of Polaromonas and Rhizobacter positively correlated with the Cd content in the above-ground tissues(r=0.603-0.735,p<0.05).The results suggested that strain WRS8 or CL-1 might reduce the DTPA-extractable Cd content by increasing the relative abundance of Sphingomonas or Bacillus in the rhizosphere soils.Strain WRS8 might reduce the Cd uptake of the wheat roots by increasing the relative abundance of Pseudomonas and reducing the relative abundance of Ramlibacter.By using culture-dependent method,it was found that strain WRS8 significantly increased the proportion of biofilm-forming and urease-producing bacterial strains in the rhizosphere soil of the wheat plants,which might play important role in Cd immobilization in the soil and reduced Cd accumulation of the wheat plants in Cd-contaminated soils. |
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