Different Varieties of Rice (Oryza sativa) Affect Cadmium Accumulation by Reshaping Rhizosphere Bacterial Community

Different Varieties of Rice (Oryza sativa) Affect Cadmium Accumulation by Reshaping Rhizosphere Bacterial Community | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Different Varieties of Rice (Oryza sativa) Affect Cadmium Accumulation by Reshaping Rhizosphere Bacterial Community Shangdu Zhang, Zhengliang Luo, Ju Peng, Xiang Wu, Bangzhi Shi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5369985/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cadmium rice is a serious danger to human health due to its ability to enrich cadmium from soil to rice plants. Previously, we have identified two self-bred late-season high quality rice varieties, which are "Yuzhenxiang" and "Xiangwanxian 12". However, the mechanism on the distribution and tolerance of their significant differences in cadmium accumulation have barely been studied so far. Therefore, in this study, we comparatively analyze the relationships between the rhizosphere bacterial community and Cd accumulation in these two rice varieties under three different Cd stress conditions during the maturity period. Our results firstly showed that significant differences in physicochemical properties affect the Cd content in rice roots, which increased with increasing Cd content in the soil. Notably, the spearman correlation analysis suggested that the differed enrichment of Variibacter , Nitrospira , Galella , Mycobacterium , and Desulfobacca affected by rice variety, which play key roles in root Cd accumulation. In general, our research indicate that the different rice varieties can altered the structure soil bacterial communities to affect Cd concentration in rice. This provides theoretical support to better control the Cd pollution problem through agricultural ecology protection. Rice Heavy Metal Cadmium Rhizosphere Bacterial Microbiome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Rice ( Oryza sativa L. ) is the prime staple food source for approximately half of the global population [ 1 , 2 ] . Recently, some acreage of rice crops has been cultivated in contaminated soils due to multiple factors, such as anthropogenic activities (e.g., chemical fertilizer and agrochemical abuse, solid and liquid refuse discharge, smelting, and mining), the requirement of an ever-growing global population, and a lack of land resources, which leads to the accumulation of multitudinous potentially toxic elements, including heavy metals, in rice crops, especially in grains [ 3 ] . Heavy metal cadmium (Cd) is a common heavy metal contaminant in rice cultivation [ 4 , 5 ] . Excessive Cd uptake and accumulation can hinder rice growth and reduce rice grain quality [ 6 ] . Additionally, the consumption of rice contaminated with Cd can trigger multiple health problems, such as cancer and cardiovascular, reproductive, and nervous system diseases in humans [ 3 ] . Cd accumulation in rice plants and grains has been found to be closely linked to multiple factors, including soil physicochemical properties, rhizosphere microorganisms, and rice plant genotypes [ 7 , 8 ] . Cd contamination can also disturb soil microbial communities and alter microbial diversity and community structure [ 9 – 11 ] . For instance, Song et al. showed that, relative to nonpolluted soils, Cd-polluted soils had a notable reduction in microbial community richness and evenness. The soil microbial communities of Cd-polluted soils are different from those of nonpolluted soils [ 11 ] . Additionally, related studies have reported that bacterial communities vary with rice genotype under Cd stress. For example, Li et al. demonstrated that rhizosphere microbial communities were different between 2B transgenic rice (truncated OsO3L2 overexpression) plants with low Cd accumulation and wild-type rice plants and that wild-type and 2B transgenic rice plants had different microbial responses to Cd [ 12 ] . The root endophytic bacterial communities were diverse in rice cultivars with low (RBQ (Nipponbare), 728B, and NX1B) or high (BB and S95B) Cd-accumulating ability [ 13 ] . Water management, fertilization, soil removal and replacement, chemical remediation, organic amendments (e.g., biochar, composts, and manures), and phytoremediation via plant species with strong Cd absorptive ability are typical remediation approaches for Cd-contaminated paddy fields [ 7 , 14 , 15 ] . Recently, microbial remediation has emerged as a highly acceptable technique for restoring heavy metal-polluted soils and eliminating heavy metals due to its advantages, such as high efficiency, economy, environmental compatibility, eco-friendliness, and safety, relative to conventional techniques [ 16 , 17 ] . Moreover, accumulating microorganisms have been implicated in the bioremediation of Cd-contaminated farmland, the reduction of Cd accumulation in rice plants and grains, and the protection of rice from Cd stress [ 18 – 20 ] . Microorganisms can reduce Cd toxicity and availability via multiple mechanisms, such as biomineralization, biosorption, biotransformation, and bioaccumulation [ 21 , 22 ] . Cadmium rice, which is enriched in cadmium from soil to grains and other organs, is a serious danger to human health. Our previous work revealed that cadmium accumulation significantly differed between the representative rice varieties Yuzhenxiang and Xiangwanxian 12 [ 23 – 24 ] . Therefore, in this study, we aimed to use 16S rDNA sequencing via the Illumina platform to comparatively analyze the effect of the rhizosphere bacterial community on the accumulation of Cd in these two rice varieties at maturity under three different Cd stresses. 2 Materials and Methods 2.1 Experimental design The experiment was carried out at the Chunhua Scientific Research Base of the Hunan Academy of Agricultural Sciences (Changsha, China) in an artificial intelligence-controlled greenhouse (113°26′57.2°E, 28°29′36.9°N). We selected Yujianxiang (YZX, 1), which has high grain cadmium accumulation, and Xiangwanxian 12 (XWX 12, 2), which has low cadmium accumulation, as experimental materials based on our previous research [23-24] . Three uniform pools were planted in an area with a self-closing steel greenhouse and a set of ponds for irrigation and drainage, which were controlled by remote-control system equipment to maintain the consistency of environmental factors. The soil for planting contained natural cadmium content treatments, which were conducted in three pooled cultivation identification areas with total cadmium contents of 0.16 ± 0.1 mg/kg (Low Cd_1, A), 0.5 ± 0.1 mg/kg (Medium Cd_2, B), and 0.9 ± 0.1 mg/kg (High Cd_3, C). The rice seeds were sown on June 13th, and the plants were subsequently transplanted on July 1st. Two rows of 14 seedlings were planted in each plot (repeated), with two rice seedlings planted in each row and one protective row set around each plot. There was 20 cm spacing between plants and rows × 20 cm (6 inches × 6 inches), with one row of planting space between communities. There was a total of 84 seedlings in each pool. Fertilizer management: no base fertilizer, urea (60 kg/ha) or potassium chloride (30 kg/ha) was applied to all three ponds. Water management involves early flooding, moist irrigation after heading, and natural soil drying before harvesting. Sampling and testing during the rice maturity period. After the removal of foreign matter such as impurities, stones, and plant residues, we used a spoon to scrape the rhizosphere soil from the rice roots. Our experiments were divided into 6 groups (A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates from each pool. A total of 30 rhizosphere soil samples were collected from two rice varieties under three different Cd stress conditions. 2.2 Determination of Cd content in plants The soil physical and chemical properties after transplanting and at harvest and the cadmium content in the plant roots, stem sheaths, leaves, and grains at the rice maturity stage were tested via the microwave digestion method. Our experiments were divided into 6 groups (A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates from each pool. The plant samples were ground separately using a grinder (the machine was cleaned first to avoid cross contamination between the next material and the previous one), passed through a 100 target sieve, and stored in a dryer. Dry samples (0.5000 g) were weighed and placed in a microwave digestion tank, and nitric acid and hydrogen peroxide were added. After digestion, the acid was heated until nearly dry, the digestion tank was rinsed with nitric acid solution three times, the solution was transferred to a 25 ml volumetric flask, the solution was diluted to scale with nitric acid solution, and the solution was mixed well. Moreover, reagent blank tests were performed. Atomic absorption spectrophotometry for the determination of cadmium content was performed according to the instructions of the “National Food Safety Standard for the Determination of Cadmium in Foods (GB 5009.15-2014)”. 2.3 Soil physicochemical property determination After the removal of foreign matter such as impurities, stones, and plant residues, 10 soil samples under each treatment condition were mixed, air-dried, pulverized, and filtered through 10-mesh standard sieves (2 mm sieves). Next, the physicochemical properties were determined. ①The total cadmium content (ST-Cd) in the soil was measured by the “complete digestion method” using graphite furnace atomic absorption spectrophotometry (GB/T 17141-1997). ②The effective cadmium (SE-Cd) content in the soil samples was examined by the “leaching method” using graphite furnace atomic absorption spectrophotometry (GB/T 23739-2009). ③The total nitrogen (TN) and alkaline soluble nitrogen (AN) in the soil samples were determined using the Kjeldahl and alkaline hydrolysis diffusion methods (LY/T1228-2015), respectively. ④The perchloric acid-sulfuric acid method (GB9837-88) was used to determine the total phosphorus (TP) content. ⑤The available phosphorus (AP) content was determined by a spectrophotometer (NY/T 1121.7-2014). ⑥The total potassium (TK) content was measured using the sodium hydroxide melting method (GB 9837-88). ⑦The available potassium (AK) was measured by flame atomic absorption spectrophotometry (NY/T889-2004). ⑧The pH was determined using a redox potentiometric at a water‒soil-liquid ratio of 1:2.5 (NY/T 1121.2-2006). ⑨The soil organic matter (SOM) content was measured by redox calorimetry (NY/T 1121.6-2006). Our experiments were divided into 6 groups (A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates from each pool. 2.4 Soil DNA extraction and 16S rDNA sequencing Our experiments were divided into 6 groups (i.e., A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates. A total of 30 soil samples were collected from two rice varieties under three different Cd stress conditions. The DNA of the microorganisms in the soil samples (0.5 g) was extracted using the E.Z.N.A. A soil DNA kit (Omega Biotek, Norcross, GA, USA) was used following the manufacturer’s protocols. DNA content and purity were analyzed using a NanoDrop 2000 UV‒vis spectrophotometer (Thermo Scientific, Wilmington, USA). DNA samples were stored at -80°C°C until sequencing. The V3-V4 hypervariable regions of the bacterial 16S rRNA genes were amplified using the above DNA templates and the primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) on an ABI GeneAmp 9700 PCR thermocycler (ABI, CA, USA) under the following PCR procedures: 95°C for 3 min; 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and 72°C for 10 min. The PCR products were retrieved after 2% agarose gel electrophoresis and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturer’s protocols and quantified using a Quantus Fluorometer (Promega, Madison, WI, USA). The 16S rDNA library was constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bio Scientific, Austin, Texas, USA) according to the manufacturer’s instructions and then paired-end sequenced on the Illumina MiSeq PE300 platform based on standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw seq data were deposited into the NCBI Sequence Read Archive (SRA) database (accession no. PRJNA602547). 2.5 Sequencing data processing The raw sequencing reads were filtered and processed using fastp (version 0.20.0) and FLASH (version 1.2.7) software under the following conditions: (i) reads with an average quality score of < 20, a length shorter than 50 bp, and ambiguous characters were removed; (ii) only overlapping sequences longer than 10 bp were assembled, and reads that could not be assembled were removed. The maximum mismatch ratio of the overlap region is 0.2. Operational taxonomic units (OTUs) with a 97% similarity cutoff were clustered, and chimeric sequences were discarded using UPARSE version 7.1 software. Each representative OTU sequence was classified and annotated. The taxonomy of each representative OTU sequence was compared against the 16S rRNA database (Silva v138) under a confidence threshold of 0.7 by RDP Classifier version 2.2. 2.6 Statistical analysis Alpha diversity analysis was performed using Mothur software and the Wilcoxon rank-sum test. Venn diagrams, community bar plots, and community heatmaps were drawn using R langue (version 3.3.1). Nonmetric multidimensional scaling (NMDS) analysis was conducted using the R language (version 3.3.1) vegan software package. Spearman correlation heatmaps were drawn using R (version 3.3.1). Statistical data analysis was performed using GraphPad Prism version 7 (La Jolla, CA, USA). The results are displayed as the means ± standard deviations. 3 Results 3.1 Differences in phenotypes and Cd contents between YZX and XWX12 under different Cd stress conditions A comparison of the phenotypes of YZX and XWX12 at the mature stage is shown in Fig. 1A and 1B. YZX has an average plant height of 117 cm, a grain length of 0.95 cm, and a length-to-width ratio of 4.8. However, XWX12 had an average plant height of 96 cm, a grain length of 0.76 cm, and a length-to-width ratio of 3.6. Given the variations in the absorption, translocation, and accumulation of heavy metals among plant organs and cultivars, the contents of Cd in different organs of 2 rice strains (YZX and XWX12) were examined at the A, B, and C cultivation sites (Table S1, Fig. 1C-1D). The results showed that the Cd content in roots tended to increase with increasing soil Cd content (Fig. 1E-1F). The root cadmium content (R-Cd) in YZX was significantly greater than that in XWX12. Interestingly, the cadmium content in the grains decreased in the order XWX 12>YZX, starting from content B. In addition, 2 rice cultivars had diverse Cd contents in the same organ and cultivation base (Fig. 1G-1H). The results showed that the root Cd content was greater than that in the stems, leaves, or grains of both rice cultivars. 3.2 The dynamic changes in soil physicochemical properties under different Cd contents There was a significant difference in the soil physicochemical properties (pH, AN, AP, AK, TN, TP, and SOM) between the A/B and C groups, while there was a significant difference in the soil physicochemical properties (pH, AN, AP, AK, TN, TP, TK, and SOM) between the A/C and B groups (Fig. 2A-2C). The results showed that there were significant differences in soil pH, TN, TP, and AP among the six sample groups (Fig. 2D-2G). Notably, the differences in soil pH and AP under moderate Cd stress conditions can be attributed to disparities in Cd content in plant roots. 3.3 Altered rhizosphere bacterial community structure under increasing Cd stress conditions in YZX and XWX12 The average number of reads per sample was 56,145 (Table S2). Alpha diversity indices are frequently used to reflect community richness and diversity (Fig. 3A-3C). The rhizosphere bacterial community richness and diversity were significantly different between YZX and XWX12 under the various cultivation conditions (Fig. 3D and 3E). The Venn diagram showed that there were 2602 overlapping bacterial OTUs (Fig. 4A), 43 overlapping bacterial phyla (Fig. 4B), and 607 overlapping bacterial genera (Fig. 4C) in all groups of samples. Importantly, the abundances of OTU 7161 (P_WA-aaa01f12) and OTU 1023 (P_SR1_Absconditabacteria) decreased significantly in the B group (Fig. 4D). Community bar plot analysis indicated that the most abundant bacterial phyla were Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, Firmicutes, and Nitrospirae (Fig. 4E). To determine the significant differences in the rhizosphere bacterial communities under the three Cd stress conditions, we used Kruskal‒Wallis H test analysis to identify the top 30 rhizosphere bacterial communities at both the phylum and genus levels (Fig. 5). Under low-Cd stress conditions (A), the most abundant rhizosphere phyla in YZX were Actinobacteria, Acidobacteria, Nitrospirae, Cyanobacteria, and Bacteroidetes, while the most abundant rhizosphere phyla in XWX12 were Proteobacteria, Actinobacteria, Acidobacteria, and Nitrospirae. Under medium Cd stress conditions (B), the most abundant rhizosphere phyla in YZX were Acidobacteria, Actinobacteria, Nitrospirae, Bacteroidetes, and Cyanobacteria, while the most abundant rhizosphere phyla in XWX12 were Proteobacteria, Actinobacteria, Acidobacteria, and Nitrospirae. Under high-Cd stress conditions (C), the most abundant rhizosphere phyla of YZX were Actinobacteria, Acidobacteria, Nitrospirae, Cyanobacteria, and Bacteroidetes, while the most abundant rhizosphere phyla of XWX12 were Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes (Fig. 5A-5B). The results indicated that the abundances of the phyla Actinobacteria and Acidobacteria had similar tendencies to change under increasing Cd stress in both YZX and XWX12. Under low-Cd conditions (A), the most abundant rhizosphere genera in YZX were Nitrospira , Anaeromyxobacter , Oryzihumus , Variibacter , and Bradyrhizobium , while the most abundant rhizosphere genera in XWX12 were Nitrospira , Anaeromyxobacter , Geobacter , Oryzihumus , and Variibacter . Under medium Cd stress conditions (B), the most abundant rhizosphere genera in YZX were Nitrospira , Anaeromyxobacter , Desulfobacca , Anaeromyxobacter , and Bradyrhizobium , while the most abundant rhizosphere genera in XWX12 were Nitrospira , Variibacter , Geobacter , Anaeromyxobacter , and Bradyrhizobium . Under high-Cd stress conditions (C), the most abundant rhizosphere genera in YZX were Nitrospira , Variibacter , Acidothermus , Bradyrhizobium , and Anaeromyxobacter , while the most abundant rhizosphere genera in XWX12 were Nitrospira , Variibacter , Acidothermus , Bradyrhizobium , and Anaerolinea (Fig. 5C-5D). The results indicated that the abundance of the genus Nitrospira had a similar tendency to change under increasing Cd stress in both YZX and XWX12. Then, we used the Wilcoxon rank sum test to detect significant differences in the rhizosphere bacterial communities between the two different rice varieties (Fig. 6). At the phylum level, Cyanobacteria was significantly more abundant in YZX than in XWX12 under low-Cd stress conditions (Fig. 6A); the abundances of Proteobacteria, Chloroflexi, Actinobacteria, Nitrospirae, Firmicutes, and Bacteroidetes were significantly different between the two rice varieties under moderate-Cd stress conditions (Fig. 6B); and the abundances of Bacteroidetes, Nitrospirae, and Caldiserica were significantly different between the two rice varieties under high-Cd stress conditions (Fig. 6C). Furthermore, at the genus level, Roseiflexus , Leptolyngbya , Chloronema , Pedomicrobium , SyntropHus , Arthronema , Gemmatirosa , Gemmatirosa , Methylomonas , Leptonema , and Anacrosporobactor significantly accumulated in YZX compared with in XWX 12 under low-Cd stress conditions (Fig. 6D); Nitrospira , Desulfobacca , Mycobacterium , Variibacter , Gaiella , Phormidium , Synechocystis , and Rhodoplanes exhibited significant differences in abundance between the two rice varieties under moderate-Cd stress conditions (Fig. 6E); and Geobacter , Rhodanobacter , Arlsobacter , Nocardioides , Roseomonas , Deferrisoma , Aquicella , Paludibaculum , Desulfobulbus , and ChitinopHaga exhibited significant differences in abundance between the two rice varieties under high-Cd stress conditions (Fig. 6F). 4 Discussion 4.1 How rice varieties and different Cd stress conditions influence the Cd content in plants Numerous studies have analyzed Cd content in vegetables across different regions, unequivocally confirming interspecific disparities in Cd accumulation among crops. Roots play a pivotal role in the transfer of Cd from the soil to plants, with numerous studies consistently demonstrating greater Cd accumulation in roots than in stems and leaves [25-26] . Root exudates include a variety of inorganic ions, protons, and organic compounds. These exudates are released from different segments of the root system during plant growth and are an inherent physiological characteristic of roots [27] . Notably, our results showed that the root Cd content was greater than that in the stems, leaves, or grains of both rice cultivars, which demonstrated that rice root specificity plays a pivotal role in manipulating Cd accumulation. On the other hand, Cd accumulation in rice plants and grains has been found to be closely linked to multiple factors, including soil physicochemical properties, rhizosphere microorganisms, and rice plant genotypes [7, 8]. Our study further confirmed that rice roots are key organs influencing the Cd content in plants under different soil Cd stress levels between YZX and XWX 12 (Fig. 1). Importantly, our results showed the same trend of Cd enrichment, which increased with increasing soil Cd concentration, between these two different rice cultivars, indicating that soil Cd stress plays an essential role in influencing rice Cd accumulation. Disturbed soil physicochemical properties play key roles in affecting the structure of rhizosphere microorganisms. 4.2 Relationships between the rhizosphere bacterial community and Cd accumulation in rice Previous studies have recently demonstrated that Cd contamination can disturb soil microbial ecology and alter microbial diversity and community structure [11,14,25] . The increasing research has investigated the mechanisms influencing Cd accumulation in the rhizosphere environment. For instance, the activities of functional rhizosphere microbes play key roles in cell wall retention and the secretion of organic acids, which influence Cd accumulation by altering soil pH. In response to Cd stress, plants strategically release specific root exudates, including long-chain fatty acids, amino acids, short-chain organic acids, and sugars, to recruit specific rhizosphere microbes, such as Shewanella putrefaciens, Bacillus megatherium, and Pseudomonas aeruginosa . A multitude of studies have demonstrated that microorganisms can potentially form symbiotic relationships and enhance the stability and activity of microbial structures related to Cd absorption and transport in crops [28-29] . Interestingly, our experiment indicated that the significant changes in soil pH and AP under moderate Cd stress conditions implied that these factors play important roles in affecting the Cd content in plant roots (Fig. 2). Therefore, we used Spearman correlation to analyze the environmental factors associated with the top 10 phyla and the top 30 genera under moderate Cd stress conditions. At the phylum level, Chloroflexi and Nitrospirae were positively correlated with Cd accumulation in rice roots, while Proteobacteria, Firmicutes, and Bacteroidetes were negatively correlated (Fig. 7A). Most Proteobacteria, Firmicutes, and Bacteroidetes were reported to be beneficial bacteria for improving plant growth and could inhibit Cd accumulation in rice roots [30] . Research on the function of Chloroflexi in the evolution of photosynthesis, which involves the fixation of inorganic CO 2 and aerobic oxidation of carbon and nitrite, has been reported [31-32] . Similarly, Nitrospirae play important roles in removing nitrogen and carbon [33] . Furthermore, our results also showed that the dominant genera Variibacter [34] and Nitrospira [35] may participate in the soil denitrification process to improve Cd accumulation in rice roots (Fig. 7B). Additionally, the functional prediction analysis shown in Fig. 7C revealed that bacteria containing mobile elements and bacteria related to oxidative stress, oxygen utilization, pathogenesis, and biofilm formation might play vital roles in cultivar-specific responses to soil Cd pollution and Cd-dependent responses in specific rice varieties. Moreover, the dominant bacterial genera that contributed to the abovementioned phenotypes were identified (Fig. 7D). The dominant bacterial genera that might be involved in both Cd responses and the abovementioned phenotypes were identified and are shown in Tables S3-S4. Related studies have reported that Cd pollution can induce oxidative stress and that oxidative stress is closely linked with Cd toxicity [39, 40] . For instance, oxygen is a crucial factor in the regulation of Cd accumulation in rice [41, 42] . An increase in antioxidant bacteria and antioxidant defense responses might improve the tolerance of rice plants to Cd [40] . Additionally, mobile elements, including C, O and N, might play vital roles in the adaptation, persistence, and transmission of bacteria in soils and the regulation of heavy metal resistance [43, 44] . Thus, our results indicated that Variibacter and Nitrospira can participate in the rice root oxygen, nitrogen and carbon cycles to affect cadmium accumulation. On the other hand, the negatively correlated genera Gaiella [36] , Mycobacterium [37] , and Desulfobacca [38] could play important roles in manipulating secondary succession of soil microbes to help decrease Cd accumulation in rice roots. Therefore, our research further confirmed that the rhizosphere microbial community plays pivotal roles in regulating cadmium accumulation in rice and can be influenced by specific inherent genetic genes [45] of rice varieties. The rhizosphere microbes affect rice cadmium accumulation under different cadmium concentrations in soil by changing the physical and chemical properties of the soil. However, deeper insight into the molecular mechanisms underlying the response of roots to Cd stress in the rhizosphere microbiome will contribute to better management of Cd pollution. 5 Conclusion In conclusion, our research indicate that the different rice varieties can altered the structure soil bacterial communities to affect Cd concentration in rice. This provides theoretical support to better control the Cd pollution problem through agricultural ecology protection, such as use different Cadmium tolerant rice variety. Meanwhile, spearman correlation analysis suggested that the genera of Variibacter , Nitrospira , Galella , Mycobacterium , and Desulfobacca play essential roles in affecting rice Cd accumulation. Therefore, we focus on exploring the molecular mechanisms of how rhizosphere microbiome decreases rice root cadmium accumulation, lead to create rice germplasm with harmless of Cd content and benefit human health. Declarations Conflict of Interests The authors have no conflicts of interests to declare. Funding Declaration Author Contribution Conceptualization, S. Z.; methodology, S. Z., Z. L., F. Z and L. Z.; investigation, S. Z., Z. L., J. P., X. W., X. M., and B. S.; writing-original draft preparation, S. Z. and F. Z; writing-review and editing, S. Z. and F. Z; supervision, L. B.; funding acquisition, S. Z. and L. B. All authors have read and agreed to the published version of the manuscript. 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J Basic Microbiol 61(2):88–109 Qi Zhang J, Ma A, Gonzalez-Ollauri Y, Yang F, Chen (2023) Soil microbes-mediated enzymes promoted the secondary succession in post-mining plantations on the Loess Plateau, China. Soil Ecol Lett 5(1):79–93 Zhang CJ, Pan J, Liu Y, Duan CH, Li M (2020) Genomic and transcriptomic insights into methanogenesis potential of novel methanogens from mangrove sediments. Microbiome Jun 17(1):94 Ruiting Wang J, Liu WJ (2022) Metabolomics and Microbiomics Reveal Impacts of Rhizosphere Metabolites on Alfalfa Continuous Cropping. Front Microbiol 13:833968 Liu J, Qu W, Kadiiska MB (2009) Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol Appl Pharmacol 238(3):209–214 Rizwan M, Ali S, Adrees M, Rizvi H, Zia-Ur-Rehman M, Hannan F, Qayyum MF, Hafeez F, Ok YS (2016) Cadmium stress in rice: toxic effects, tolerance mechanisms, and management: a critical review. Environ Sci Pollut Res Int 23(18):17859–17879 Li H, Zhang H, Yang Y, Fu G, Tao L, Xiong J (2022) Effects and oxygen-regulated mechanisms of water management on cadmium (Cd) accumulation in rice (Oryza sativa). Sci Total Environ 846:157484 Wang MY, Chen AK, Wong MH, Qiu RL, Cheng H, Ye ZH (2011) Cadmium accumulation in and tolerance of rice (Oryza sativa L.) varieties with different rates of radial oxygen loss. Environ Pollut 159(6):1730–1736 Castro H, Douillard FP, Korkeala H, Lindström M (2021) Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms. mSphere 6(4):e0038321 Wassenaar TM, Cabal A (2017) The mobile dso-gene-sso element in rolling-circle plasmids of staphylococci reflects the evolutionary history of its resistance gene. Lett Appl Microbiol 65(3):192–198 Su P# K, H# P, Q#, Wicaksono WA, Berg G, Liu Z, Ma J, Zhang, D* (2024) Cernava T* & Liu Y*. Microbiome homeostasis on rice leaves is regulated by a precursor molecule of lignin biosynthesis. Nat Commun 15(1):23 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1Thecadmiumcontentindifferentorgans.xlsx Supplementary Table 1. The cadmium content in different organs SupplementaryTable2.Thesequencingsampleinformation.xlsx Supplementary Table 2. Sequencing sample information SupplementaryTable3BacterialgeneracorrelatedwithCdandcorrespondingBugBasephenotypes.xlsx Supplementary Table 3. The 25 OTUs or genera that were strongly associated with the Cd concentration among the corresponding top 100 taxa. SupplementaryTable4The25OTUsorgenerathatwerehighlyassociatedwithCdcontentamongthecorrespondingtop100taxa..xlsx Supplementary Table 4. Bacterial genera correlated with Cd and corresponding BugBase phenotypes. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5369985","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374872547,"identity":"b816560f-65ba-4bca-b49d-497ce4de25af","order_by":0,"name":"Shangdu Zhang","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shangdu","middleName":"","lastName":"Zhang","suffix":""},{"id":374872551,"identity":"8bd39dc5-0c6d-4409-a020-dc49c8c6ba0d","order_by":1,"name":"Zhengliang Luo","email":"","orcid":"","institution":"Hunan Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhengliang","middleName":"","lastName":"Luo","suffix":""},{"id":374872552,"identity":"49050a43-266b-493e-9d0b-bfa5bc138fc3","order_by":2,"name":"Ju Peng","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ju","middleName":"","lastName":"Peng","suffix":""},{"id":374872553,"identity":"4babed06-e82d-4d01-9536-2f7ddec910bf","order_by":3,"name":"Xiang Wu","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Wu","suffix":""},{"id":374872554,"identity":"e2f60296-36b1-4995-b072-66e2484e1f13","order_by":4,"name":"Bangzhi Shi","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bangzhi","middleName":"","lastName":"Shi","suffix":""},{"id":374872555,"identity":"4b037fe0-30d9-48ce-ab3c-05a8e190065e","order_by":5,"name":"Xiufei Meng","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiufei","middleName":"","lastName":"Meng","suffix":""},{"id":374872556,"identity":"0fc16137-efde-47bd-9f9c-a2a8a7b474fe","order_by":6,"name":"Yuanyi Qin","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuanyi","middleName":"","lastName":"Qin","suffix":""},{"id":374872557,"identity":"e273d424-95ce-4e54-a71c-8b82fca37bd2","order_by":7,"name":"Leliang Zhou","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Leliang","middleName":"","lastName":"Zhou","suffix":""},{"id":374872558,"identity":"8cfde5e1-8bff-4552-a7a6-6123c2298268","order_by":8,"name":"Feiying Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIie3NPQrCMByH4ZRIuqR7xKFXiAhVB+1VDAUnPYFfgUK8got4hU7qWCnYpQfQzSJkE5wEN6OIOKUdBfNO/8DvIQCYTD+YzS0OeuqgAO7LERx/COqXJe+DAuyVJHYoSL6d+E0yuJ2dbdd1OZQnLcE7QViWwvZiuGk4WVCPYtSkOuITpojYI3oYrmuOgFYEMCLaX9z8RTA9DKQiM3/FiwixnmRMFEGKJIzHRQSzsMVETGkmvepSpEGUIE9P7DQ/3sXUp2kgyUWMOqt5KLVEVVGD5OsNC/bPyRWAafHMZDKZ/rcHoG5GMzWXrDIAAAAASUVORK5CYII=","orcid":"","institution":"Hunan Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Feiying","middleName":"","lastName":"Zhu","suffix":""},{"id":374872559,"identity":"80a44f1e-20b5-4254-b366-831eb5c3ca52","order_by":9,"name":"Liangyang Bai","email":"","orcid":"","institution":"Hunan Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Liangyang","middleName":"","lastName":"Bai","suffix":""}],"badges":[],"createdAt":"2024-11-01 02:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5369985/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5369985/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69253234,"identity":"1a5ee1c0-79e1-40f1-9d1e-b5ac8eb9323f","added_by":"auto","created_at":"2024-11-18 12:05:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":320313,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes and Cd content comparison of the two tested rice varieties at the mature stage.\u003c/p\u003e\n\u003cp\u003ePhenotypic comparison of the two tested rice varieties at the mature stage (A-B). C. Comparison analysis of the Cd contents in XWX12 under different Cd stress conditions. D. Comparison of Cd contents in YZX under different Cd stress conditions. Comparative analysis of the Cd contents of roots (E), stems (F), leaves (G), and grains (H) of 2 rice cultivars under different Cd stress conditions. Note: *P\u0026lt;0.05, **P\u0026lt;0.001, ***P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/1f80e6bde199586af90dea03.jpg"},{"id":69253216,"identity":"126d3d08-66f4-4527-a8ad-eb50f4ee76c3","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156090,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of soil physicochemical properties\u003c/p\u003e\n\u003cp\u003eBar plot of significantly different soil physicochemical properties between the A/B Cd content (A), A/C Cd content (B), and B/C Cd content (C) treatments (P\u0026lt;0.0001). D. Comparison of soil pH under different Cd stress conditions. E. Comparison analysis of soil AP under different Cd stress conditions. F. Comparison analysis of soil TN under different Cd stress conditions. G. Comparison analysis of soil TP under different Cd stress conditions. Note: AP, available phosphorus, TN, total nitrogen, TP, total phosphorus. *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/79dae2ca58e7ad59cf6ac3a7.jpg"},{"id":69253218,"identity":"086f4dfa-f262-4b67-b2e8-4fddb7727344","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139644,"visible":true,"origin":"","legend":"\u003cp\u003eDiversity analysis of bacteria at the OTU level in all samples\u003c/p\u003e\n\u003cp\u003eA. Rank abundance curves. B. Alpha diversity analysis of the coverage curves. C. Alpha diversity analysis of the Simpson index. D. Distances box plot of beta diversity. E. PCA at the OTU level.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/e7519a0439b142f2a2f1504e.jpg"},{"id":69254374,"identity":"e5bbc4f1-f32a-4a43-9207-1234c958de8c","added_by":"auto","created_at":"2024-11-18 12:13:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161176,"visible":true,"origin":"","legend":"\u003cp\u003eRhizosphere bacterial community composition analysis\u003c/p\u003e\n\u003cp\u003eVenn diagram of bacterial community composition between different groups at the OTU (A),\u003cstrong\u003e \u003c/strong\u003ephylum (B), and genus (C) levels. (D) Community heatmap analysis of different groups at the OTU level. (E) Bacterial community bar plot at the phylum level.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/e1dc5db9b99aadad8d1c94e1.jpg"},{"id":69254376,"identity":"049e669e-c5ac-47ca-b0d5-bfae77534899","added_by":"auto","created_at":"2024-11-18 12:13:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285975,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical differences in rhizosphere bacterial communities under different Cd stress conditions\u003c/p\u003e\n\u003cp\u003eThe top 30 significantly different phyla (A) and genera (B) in YZX (1) were identified by the Kruskal‒Wallis H test under different Cd stress conditions. The top 30 significantly different phyla (C) and genera (D) in XWX12 (2) were identified by the Kruskal‒Wallis H test under different Cd stress conditions. Note: *P\u0026lt;0.05, **P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/eb6a874101e00c1e3350d818.jpg"},{"id":69253221,"identity":"55052fc9-e3d9-4fdd-bcdb-83c605fcd1dd","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":285087,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical differences in the rhizosphere bacterial community between the two rice varieties\u003c/p\u003e\n\u003cp\u003eSignificant differences in the rhizosphere bacterial community between YZX (1) and XWX12 (2) under low-Cd conditions at the phylum (A) and genus (B) levels. Significant differences in the rhizosphere bacterial community between YZX and XWX12 under moderate Cd stress conditions at the phylum (C) and genus (D) levels. Significant differences in the rhizosphere bacterial community between YZX and XWX12 under high Cd stress conditions at the phylum (E) and genus (F) levels.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/27f8d14221ed07d233b27a83.jpg"},{"id":69254375,"identity":"77e6fd20-001a-4834-872f-cae2f8bc8d0e","added_by":"auto","created_at":"2024-11-18 12:13:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":235889,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman correlation heatmap of environmental factors and prediction analysis of bacterial functions by PICRUSt\u003c/p\u003e\n\u003cp\u003eA. Spearman correlation heatmap for environmental factors and 8. B. Spearman correlation heatmap for environmental factors and the top 30 genera under medium Cd stress conditions. C. COG functional classification of bacteria. D. Heatmap of bacterial functional pathways. Note: The correlation coefficient R is indicated by different colors.\u003c/p\u003e\n\u003cp\u003e*p value \u0026lt; 0.05, **p value \u0026lt; 0.01, ***p value \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/80a6bd82471e5f8ace805f57.jpg"},{"id":70748464,"identity":"5890b903-de3b-42ce-bbc2-61d2f6ddd725","added_by":"auto","created_at":"2024-12-06 08:54:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2144096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/c41201eb-4825-42a9-823d-d8dccb9fb1af.pdf"},{"id":69253214,"identity":"70529b45-4ef1-4992-9d02-d91dfd89e250","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9571,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. The cadmium content in different organs\u003c/p\u003e","description":"","filename":"SupplementaryTable1Thecadmiumcontentindifferentorgans.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/db4a5de793a9f74f11078e3a.xlsx"},{"id":69253219,"identity":"5c034792-581b-430c-9d97-73311ac02908","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9767,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2. Sequencing sample information\u003c/p\u003e","description":"","filename":"SupplementaryTable2.Thesequencingsampleinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/8f0616933a36850f1d0078e4.xlsx"},{"id":69253215,"identity":"ac454213-9bec-477d-82d9-2d13c9e1d3f2","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10052,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3. The 25 OTUs or genera that were strongly associated with the Cd concentration among the corresponding top 100 taxa.\u003c/p\u003e","description":"","filename":"SupplementaryTable3BacterialgeneracorrelatedwithCdandcorrespondingBugBasephenotypes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/7c4848ea6502ff4b14798813.xlsx"},{"id":69253220,"identity":"e2aeb48d-5232-4c36-852e-aa484820797f","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9571,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 4. Bacterial genera correlated with Cd and corresponding BugBase phenotypes.\u003c/p\u003e","description":"","filename":"SupplementaryTable4The25OTUsorgenerathatwerehighlyassociatedwithCdcontentamongthecorrespondingtop100taxa..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/d227a70addab530784f9a559.xlsx"},{"id":69253224,"identity":"f3de0e21-5f09-41bb-b0dd-70fef94d3094","added_by":"auto","created_at":"2024-11-18 12:05:08","extension":"zip","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":22205608,"visible":true,"origin":"","legend":"","description":"","filename":"FIGURES.zip","url":"https://assets-eu.researchsquare.com/files/rs-5369985/v1/01522e7e5ff20e4c0ffa479b.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Different Varieties of Rice (Oryza sativa) Affect Cadmium Accumulation by Reshaping Rhizosphere Bacterial Community","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa L.\u003c/em\u003e) is the prime staple food source for approximately half of the global population \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Recently, some acreage of rice crops has been cultivated in contaminated soils due to multiple factors, such as anthropogenic activities (e.g., chemical fertilizer and agrochemical abuse, solid and liquid refuse discharge, smelting, and mining), the requirement of an ever-growing global population, and a lack of land resources, which leads to the accumulation of multitudinous potentially toxic elements, including heavy metals, in rice crops, especially in grains \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Heavy metal cadmium (Cd) is a common heavy metal contaminant in rice cultivation \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Excessive Cd uptake and accumulation can hinder rice growth and reduce rice grain quality \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Additionally, the consumption of rice contaminated with Cd can trigger multiple health problems, such as cancer and cardiovascular, reproductive, and nervous system diseases in humans \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCd accumulation in rice plants and grains has been found to be closely linked to multiple factors, including soil physicochemical properties, rhizosphere microorganisms, and rice plant genotypes \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Cd contamination can also disturb soil microbial communities and alter microbial diversity and community structure \u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. For instance, Song et al. showed that, relative to nonpolluted soils, Cd-polluted soils had a notable reduction in microbial community richness and evenness. The soil microbial communities of Cd-polluted soils are different from those of nonpolluted soils \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Additionally, related studies have reported that bacterial communities vary with rice genotype under Cd stress. For example, Li et al. demonstrated that rhizosphere microbial communities were different between 2B transgenic rice (truncated OsO3L2 overexpression) plants with low Cd accumulation and wild-type rice plants and that wild-type and 2B transgenic rice plants had different microbial responses to Cd \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The root endophytic bacterial communities were diverse in rice cultivars with low (RBQ (Nipponbare), 728B, and NX1B) or high (BB and S95B) Cd-accumulating ability \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Water management, fertilization, soil removal and replacement, chemical remediation, organic amendments (e.g., biochar, composts, and manures), and phytoremediation via plant species with strong Cd absorptive ability are typical remediation approaches for Cd-contaminated paddy fields \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Recently, microbial remediation has emerged as a highly acceptable technique for restoring heavy metal-polluted soils and eliminating heavy metals due to its advantages, such as high efficiency, economy, environmental compatibility, eco-friendliness, and safety, relative to conventional techniques \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Moreover, accumulating microorganisms have been implicated in the bioremediation of Cd-contaminated farmland, the reduction of Cd accumulation in rice plants and grains, and the protection of rice from Cd stress \u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Microorganisms can reduce Cd toxicity and availability via multiple mechanisms, such as biomineralization, biosorption, biotransformation, and bioaccumulation \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCadmium rice, which is enriched in cadmium from soil to grains and other organs, is a serious danger to human health. Our previous work revealed that cadmium accumulation significantly differed between the representative rice varieties Yuzhenxiang and Xiangwanxian 12 \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Therefore, in this study, we aimed to use 16S rDNA sequencing via the Illumina platform to comparatively analyze the effect of the rhizosphere bacterial community on the accumulation of Cd in these two rice varieties at maturity under three different Cd stresses.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003ch2\u003e2.1\u0026nbsp; \u0026nbsp; \u0026nbsp;Experimental design\u003c/h2\u003e\n\u003cp\u003eThe experiment was carried out at the Chunhua Scientific Research Base of the Hunan Academy of Agricultural Sciences (Changsha, China) in an artificial intelligence-controlled greenhouse (113\u0026deg;26\u0026prime;57.2\u0026deg;E, 28\u0026deg;29\u0026prime;36.9\u0026deg;N). We selected Yujianxiang (YZX, 1), which has high grain cadmium accumulation, and Xiangwanxian 12 (XWX 12, 2), which has low cadmium accumulation, as experimental materials based on our previous research \u003csup\u003e[23-24]\u003c/sup\u003e. Three uniform pools were planted in an area with a self-closing steel greenhouse and a set of ponds for irrigation and drainage, which were controlled by remote-control system equipment to maintain the consistency of environmental factors. The soil for planting contained natural cadmium content treatments, which were conducted in three pooled cultivation identification areas with total cadmium contents of 0.16 \u0026plusmn; 0.1 mg/kg (Low Cd_1, A), 0.5 \u0026plusmn; 0.1 mg/kg (Medium Cd_2, B), and 0.9 \u0026plusmn; 0.1 mg/kg (High Cd_3, C).\u0026nbsp;The rice seeds were sown on June 13th, and the plants were subsequently transplanted on July 1st. Two rows of 14 seedlings were planted in each plot (repeated), with two rice seedlings planted in each row and one protective row set around each plot. There was 20 cm spacing between plants and rows \u0026times; 20 cm (6 inches \u0026times; 6 inches), with one row of planting space between communities. There was a total of 84 seedlings in each pool. Fertilizer management: no base fertilizer, urea (60 kg/ha) or potassium chloride (30 kg/ha) was applied to all three ponds. Water management involves early flooding, moist irrigation after heading, and natural soil drying before harvesting. Sampling and testing during the rice maturity period. After the removal of foreign matter such as impurities, stones, and plant residues, we used a spoon to scrape the rhizosphere soil from the rice roots. Our experiments were divided into 6 groups (A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates from each pool. A total of 30 rhizosphere soil samples were collected from two rice varieties under three different Cd stress conditions.\u003c/p\u003e\n\u003ch2\u003e2.2\u0026nbsp; \u0026nbsp; \u0026nbsp;Determination of\u0026nbsp;Cd content in plants\u003c/h2\u003e\n\u003cp\u003eThe soil physical and chemical properties after transplanting and at harvest and the cadmium content in the plant roots, stem sheaths, leaves, and grains at the rice maturity stage were tested via the microwave digestion method. Our experiments were divided into 6 groups (A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates from each pool. The plant samples were ground separately using a grinder (the machine was cleaned first to avoid cross contamination between the next material and the previous one), passed through a 100 target sieve, and stored in a dryer. Dry samples (0.5000 g) were weighed and placed in a microwave digestion tank, and nitric acid and hydrogen peroxide were added. After digestion, the acid was heated until nearly dry, the digestion tank was rinsed with nitric acid solution three times, the solution was transferred to a 25 ml volumetric flask, the solution was diluted to scale with nitric acid solution, and the solution was mixed well. Moreover, reagent blank tests were performed. Atomic absorption spectrophotometry for the determination of cadmium content was performed according to the instructions of the \u0026ldquo;National Food Safety Standard for the Determination of Cadmium in Foods (GB 5009.15-2014)\u0026rdquo;.\u003c/p\u003e\n\u003ch2\u003e2.3\u0026nbsp; \u0026nbsp; \u0026nbsp;Soil physicochemical property determination\u003c/h2\u003e\n\u003cp\u003eAfter the removal of foreign matter such as impurities, stones, and plant residues, 10 soil samples under each treatment condition were mixed, air-dried, pulverized, and filtered through 10-mesh standard sieves (2 mm sieves). Next, the physicochemical properties were determined.\u0026nbsp;①The total cadmium content (ST-Cd) in the soil was measured by the \u0026ldquo;complete digestion method\u0026rdquo; using graphite furnace atomic absorption spectrophotometry (GB/T 17141-1997).\u0026nbsp;②The effective cadmium (SE-Cd) content in the soil samples was examined by the \u0026ldquo;leaching method\u0026rdquo; using graphite furnace atomic absorption spectrophotometry (GB/T 23739-2009).\u0026nbsp;③The total nitrogen (TN) and alkaline soluble nitrogen (AN) in the soil samples were determined using the Kjeldahl and alkaline hydrolysis diffusion methods (LY/T1228-2015), respectively.\u0026nbsp;④The perchloric acid-sulfuric acid method (GB9837-88) was used to determine the total phosphorus (TP) content.\u0026nbsp;⑤The available phosphorus (AP) content was determined by a spectrophotometer (NY/T 1121.7-2014).\u0026nbsp;⑥The total potassium (TK) content was measured using the sodium hydroxide melting method (GB 9837-88).\u0026nbsp;⑦The available potassium (AK) was measured by flame atomic absorption spectrophotometry (NY/T889-2004).\u0026nbsp;⑧The pH was determined using a redox potentiometric at a water‒soil-liquid ratio of 1:2.5 (NY/T 1121.2-2006).\u0026nbsp;⑨The soil organic matter (SOM) content was measured by redox calorimetry (NY/T 1121.6-2006). Our experiments were divided into 6 groups (A_1, A_2, B_1, B_2, C_1, and C_2). Each group contained 5 biological replicates from each pool.\u003c/p\u003e\n\u003ch2\u003e2.4\u0026nbsp; \u0026nbsp; \u0026nbsp;Soil DNA extraction and 16S rDNA sequencing\u003c/h2\u003e\n\u003cp\u003eOur experiments were divided into 6 groups (i.e., A_1, A_2, B_1, B_2, C_1, and C_2).\u0026nbsp;Each group contained 5 biological replicates. A total of 30 soil samples were collected from two rice varieties under three different Cd stress conditions. The DNA of the microorganisms in the soil samples (0.5 g) was extracted using the E.Z.N.A. A soil DNA kit (Omega Biotek, Norcross, GA, USA) was used following the manufacturer\u0026rsquo;s protocols. DNA content and purity were analyzed using a NanoDrop 2000 UV‒vis spectrophotometer (Thermo Scientific, Wilmington, USA). DNA samples were stored at -80\u0026deg;C\u0026deg;C until sequencing. The V3-V4 hypervariable regions of the bacterial 16S rRNA genes were amplified using the above DNA templates and the primers 338F (5\u0026rsquo;-ACTCCTACGGGAGGCAGCAG-3\u0026rsquo;) and 806R (5\u0026rsquo;-GGACTACHVGGGTWTCTAAT-3\u0026rsquo;) on an ABI GeneAmp 9700 PCR thermocycler (ABI, CA, USA) under the following PCR procedures: 95\u0026deg;C for 3 min; 27 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 45 s; and 72\u0026deg;C for 10 min. The PCR products were retrieved after 2% agarose gel electrophoresis and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturer\u0026rsquo;s protocols and quantified using a Quantus Fluorometer (Promega, Madison, WI, USA). The 16S rDNA library was constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bio Scientific, Austin, Texas, USA) according to the manufacturer\u0026rsquo;s instructions and then paired-end sequenced on the Illumina MiSeq PE300 platform based on standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw seq data were deposited into the NCBI Sequence Read Archive (SRA) database (accession no. PRJNA602547).\u003c/p\u003e\n\u003ch2\u003e2.5\u0026nbsp; \u0026nbsp; \u0026nbsp;Sequencing data processing\u003c/h2\u003e\n\u003cp\u003eThe raw sequencing reads were filtered and processed using fastp (version 0.20.0) and FLASH (version 1.2.7) software under the following conditions: (i) reads with an average quality score of \u0026lt; 20, a length shorter than 50 bp, and ambiguous characters were removed; (ii) only overlapping sequences longer than 10 bp were assembled, and reads that could not be assembled were removed. The maximum mismatch ratio of the overlap region is 0.2. Operational taxonomic units (OTUs) with a 97% similarity cutoff were clustered, and chimeric sequences were discarded using UPARSE version 7.1 software. Each representative OTU sequence was classified and annotated. The taxonomy of each representative OTU sequence was compared against the 16S rRNA database (Silva v138) under a confidence threshold of 0.7 by RDP Classifier version 2.2.\u003c/p\u003e\n\u003ch2\u003e2.6\u0026nbsp; \u0026nbsp; \u0026nbsp;Statistical analysis\u003c/h2\u003e\n\u003cp\u003eAlpha diversity analysis was performed using Mothur software and the Wilcoxon rank-sum test. Venn diagrams, community bar plots, and community heatmaps were drawn using R langue (version 3.3.1). Nonmetric multidimensional scaling (NMDS) analysis was conducted using the R language (version 3.3.1) vegan software package. Spearman correlation heatmaps were drawn using R (version 3.3.1). Statistical data analysis was performed using GraphPad Prism version 7 (La Jolla, CA, USA). The results are displayed as the means \u0026plusmn; standard deviations.\u003c/p\u003e"},{"header":"3 Results","content":"\u003ch2\u003e3.1\u0026nbsp; \u0026nbsp; \u0026nbsp;Differences in phenotypes and Cd contents between YZX and XWX12 under different Cd stress conditions\u003c/h2\u003e\n\u003cp\u003eA comparison of the phenotypes of YZX and XWX12 at the mature stage is shown in Fig. 1A and 1B. YZX has an average plant height of 117 cm, a grain length of 0.95 cm, and a length-to-width ratio of 4.8. However, XWX12 had an average plant height of 96 cm, a grain length of 0.76 cm, and a length-to-width ratio of 3.6. Given the variations in the absorption, translocation, and accumulation of heavy metals among plant organs and cultivars, the contents of Cd in different organs of 2 rice strains (YZX and XWX12) were examined at the A, B, and C cultivation sites (Table S1, Fig. 1C-1D). The results showed that the Cd content in roots tended to increase with increasing soil Cd content (Fig. 1E-1F). The root cadmium content (R-Cd) in YZX was significantly greater than that in XWX12. Interestingly, the cadmium content in the grains decreased in the order XWX 12\u0026gt;YZX, starting from content B. In addition, 2 rice cultivars had diverse Cd contents in the same organ and cultivation base (Fig. 1G-1H).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe results showed that the root Cd content was greater than that in the stems, leaves, or grains of both rice cultivars.\u003c/p\u003e\n\u003ch2\u003e3.2\u0026nbsp; \u0026nbsp; \u0026nbsp;The dynamic changes in soil physicochemical properties under different Cd contents\u003c/h2\u003e\n\u003cp\u003eThere was a significant difference in the soil physicochemical properties (pH, AN, AP, AK, TN, TP, and SOM) between the A/B and C groups, while there was a significant difference in the soil physicochemical properties (pH, AN, AP, AK, TN, TP, TK, and SOM) between the A/C and B groups (Fig. 2A-2C). The results showed that there were significant differences in soil pH, TN, TP, and AP among the six sample groups (Fig. 2D-2G). Notably, the differences in soil pH and AP under moderate Cd stress conditions can be attributed to disparities in Cd content in plant roots.\u003c/p\u003e\n\u003ch2\u003e3.3\u0026nbsp; \u0026nbsp; \u0026nbsp;Altered rhizosphere bacterial community structure under increasing Cd stress conditions in YZX and XWX12\u003c/h2\u003e\n\u003cp\u003eThe average number of reads per sample was 56,145 (Table S2). Alpha diversity indices are frequently used to reflect community richness and diversity (Fig. 3A-3C). The rhizosphere bacterial community richness and diversity were significantly different between YZX and XWX12 under the various cultivation conditions (Fig. 3D and 3E). The Venn\u0026nbsp;diagram showed that there were 2602 overlapping bacterial OTUs (Fig. 4A), 43 overlapping bacterial phyla (Fig. 4B), and 607 overlapping bacterial genera (Fig. 4C) in all groups of samples. Importantly, the abundances of OTU 7161 (P_WA-aaa01f12) and OTU 1023 (P_SR1_Absconditabacteria) decreased significantly in the B group (Fig. 4D). Community bar plot analysis indicated that the most abundant bacterial phyla were Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, Firmicutes, and Nitrospirae (Fig. 4E).\u003c/p\u003e\n\u003cp\u003eTo determine the significant differences in the rhizosphere bacterial communities under the three Cd stress conditions, we used Kruskal‒Wallis H test analysis to identify\u0026nbsp;the top 30 rhizosphere bacterial communities at both the phylum and genus levels (Fig. 5). Under low-Cd stress conditions (A), the most abundant rhizosphere phyla in YZX were Actinobacteria, Acidobacteria, Nitrospirae, Cyanobacteria, and Bacteroidetes, while the most abundant rhizosphere phyla in XWX12 were Proteobacteria, Actinobacteria, Acidobacteria, and Nitrospirae. Under medium Cd stress conditions (B), the most abundant rhizosphere phyla in YZX were Acidobacteria, Actinobacteria, Nitrospirae, Bacteroidetes, and Cyanobacteria, while the most abundant rhizosphere phyla in XWX12 were Proteobacteria, Actinobacteria, Acidobacteria, and Nitrospirae. Under high-Cd stress conditions (C), the most abundant rhizosphere phyla of YZX were Actinobacteria, Acidobacteria, Nitrospirae, Cyanobacteria, and Bacteroidetes, while the most abundant rhizosphere phyla of XWX12 were Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes (Fig. 5A-5B). The results indicated that the abundances of the phyla Actinobacteria and Acidobacteria had similar tendencies to change under increasing Cd stress in both YZX and XWX12.\u0026nbsp;Under low-Cd conditions (A), the most abundant rhizosphere genera in YZX were \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, \u003cem\u003eOryzihumus\u003c/em\u003e, \u003cem\u003eVariibacter\u003c/em\u003e, and \u003cem\u003eBradyrhizobium\u003c/em\u003e, while the most abundant rhizosphere genera in XWX12 were \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, \u003cem\u003eGeobacter\u003c/em\u003e, \u003cem\u003eOryzihumus\u003c/em\u003e, and \u003cem\u003eVariibacter\u003c/em\u003e. Under medium Cd stress conditions (B), the most abundant rhizosphere genera in YZX were \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, \u003cem\u003eDesulfobacca\u003c/em\u003e, \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Bradyrhizobium\u003c/em\u003e, while the most abundant rhizosphere genera in XWX12 were \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eVariibacter\u003c/em\u003e, \u003cem\u003eGeobacter\u003c/em\u003e, \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Bradyrhizobium\u003c/em\u003e. Under high-Cd stress conditions (C), the most abundant rhizosphere genera in YZX were \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eVariibacter\u003c/em\u003e, \u003cem\u003eAcidothermus\u003c/em\u003e, \u003cem\u003eBradyrhizobium\u003c/em\u003e, and \u003cem\u003eAnaeromyxobacter\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewhile the most abundant rhizosphere genera in XWX12 were\u003cem\u003e\u0026nbsp;Nitrospira\u003c/em\u003e, \u003cem\u003eVariibacter\u003c/em\u003e, \u003cem\u003eAcidothermus\u003c/em\u003e, \u003cem\u003eBradyrhizobium\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Anaerolinea\u003c/em\u003e (Fig. 5C-5D). The results indicated that the abundance of the genus \u003cem\u003eNitrospira\u003c/em\u003e had a similar tendency to change under increasing Cd stress in both YZX and XWX12.\u003c/p\u003e\n\u003cp\u003eThen, we used the Wilcoxon rank sum test to detect significant differences in the rhizosphere bacterial communities between the two different rice varieties (Fig. 6). At the phylum level, Cyanobacteria was significantly more abundant in YZX than in XWX12 under low-Cd stress conditions (Fig. 6A); the abundances of Proteobacteria, Chloroflexi, Actinobacteria, Nitrospirae, Firmicutes, and Bacteroidetes were significantly different between the two rice varieties under moderate-Cd stress conditions (Fig. 6B); and the abundances of Bacteroidetes, Nitrospirae, and Caldiserica were significantly different between the two rice varieties under high-Cd stress conditions (Fig. 6C). Furthermore, at the genus level, \u003cem\u003eRoseiflexus\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Leptolyngbya\u003c/em\u003e, \u003cem\u003eChloronema\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Pedomicrobium\u003c/em\u003e, \u003cem\u003eSyntropHus\u003c/em\u003e, \u003cem\u003eArthronema\u003c/em\u003e, \u003cem\u003eGemmatirosa\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Gemmatirosa\u003c/em\u003e, \u003cem\u003eMethylomonas\u003c/em\u003e, \u003cem\u003eLeptonema\u003c/em\u003e, and \u003cem\u003eAnacrosporobactor\u003c/em\u003e significantly accumulated in YZX compared with in XWX 12 under low-Cd stress conditions (Fig. 6D); \u003cem\u003eNitrospira\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Desulfobacca\u003c/em\u003e, \u003cem\u003eMycobacterium\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Variibacter\u003c/em\u003e, \u003cem\u003eGaiella\u003c/em\u003e, \u003cem\u003ePhormidium\u003c/em\u003e, \u003cem\u003eSynechocystis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eRhodoplanes\u0026nbsp;\u003c/em\u003eexhibited significant differences in abundance between the two rice varieties under moderate-Cd stress conditions (Fig. 6E); and \u003cem\u003eGeobacter\u003c/em\u003e, \u003cem\u003eRhodanobacter\u003c/em\u003e, \u003cem\u003eArlsobacter\u003c/em\u003e, \u003cem\u003eNocardioides\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Roseomonas\u003c/em\u003e, \u003cem\u003eDeferrisoma\u003c/em\u003e, \u003cem\u003eAquicella\u003c/em\u003e, \u003cem\u003ePaludibaculum\u003c/em\u003e, \u003cem\u003eDesulfobulbus\u003c/em\u003e, and \u003cem\u003eChitinopHaga\u0026nbsp;\u003c/em\u003eexhibited significant differences in abundance between the two rice varieties under high-Cd stress conditions (Fig. 6F).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003ch2\u003e4.1\u0026nbsp; \u0026nbsp; \u0026nbsp;How rice varieties and different Cd stress conditions influence the Cd content in plants\u003c/h2\u003e\n\u003cp\u003eNumerous studies have analyzed Cd content in vegetables across different regions, unequivocally confirming interspecific disparities in Cd accumulation among crops. Roots play a pivotal role in the transfer of Cd from the soil to plants, with numerous studies consistently demonstrating greater Cd accumulation in roots than in stems and leaves \u003csup\u003e[25-26]\u003c/sup\u003e. Root exudates include a variety of inorganic ions, protons, and organic compounds. These exudates are released from different segments of the root system during plant growth and are an inherent physiological characteristic of roots \u003csup\u003e[27]\u003c/sup\u003e. Notably, our results showed that the root Cd content was greater than that in the stems, leaves, or grains of both rice cultivars, which demonstrated that rice root specificity plays a pivotal role in manipulating Cd accumulation.\u003c/p\u003e\n\u003cp\u003eOn the other hand, Cd accumulation in rice plants and grains has been found to be closely linked to multiple factors, including soil physicochemical properties, rhizosphere microorganisms, and rice plant genotypes \u003csup\u003e[7, 8].\u003c/sup\u003e Our study further confirmed that rice roots are key organs influencing the Cd content in plants under different soil Cd stress levels between YZX and XWX 12 (Fig. 1). Importantly, our results showed the same trend of Cd enrichment, which increased with increasing soil Cd concentration, between these two different rice cultivars, indicating that soil Cd stress plays an essential role in influencing rice Cd accumulation. Disturbed soil physicochemical properties play key roles in affecting the structure of rhizosphere microorganisms.\u003c/p\u003e\n\u003ch2\u003e4.2\u0026nbsp; \u0026nbsp; \u0026nbsp;Relationships between the rhizosphere bacterial community and Cd accumulation in rice\u003c/h2\u003e\n\u003cp\u003ePrevious studies have recently demonstrated that Cd contamination can disturb soil microbial ecology and alter microbial diversity and community structure \u003csup\u003e[11,14,25]\u003c/sup\u003e. The\u0026nbsp;increasing research has investigated the mechanisms influencing Cd accumulation in the rhizosphere environment. For instance, the activities of functional rhizosphere microbes play key roles in cell wall retention and the secretion of organic acids, which influence Cd accumulation by altering soil pH. In response to Cd stress, plants strategically release specific root exudates, including long-chain fatty acids, amino acids, short-chain organic acids, and sugars, to recruit specific rhizosphere microbes, such as \u003cem\u003eShewanella putrefaciens, Bacillus megatherium,\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. A multitude of studies have demonstrated that microorganisms can potentially form symbiotic relationships and enhance the stability and activity of microbial structures related to Cd absorption and transport in crops\u003csup\u003e\u0026nbsp;[28-29]\u003c/sup\u003e.\u0026nbsp;Interestingly, our experiment indicated that the significant changes in soil pH and AP under moderate Cd stress conditions implied that these factors play important roles in affecting the Cd content in plant roots (Fig. 2). Therefore, we used Spearman correlation to analyze the environmental factors associated with the top 10 phyla and the top 30 genera under moderate Cd stress conditions. At the phylum level, Chloroflexi and Nitrospirae were positively correlated with Cd accumulation in rice roots, while Proteobacteria, Firmicutes, and Bacteroidetes were\u0026nbsp;negatively correlated\u0026nbsp;(Fig. 7A). Most\u0026nbsp;Proteobacteria, Firmicutes, and Bacteroidetes were reported to be beneficial bacteria for improving plant growth and could inhibit Cd accumulation in rice roots\u003cem\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e[30]\u003c/sup\u003e.\u0026nbsp;Research on the function of Chloroflexi in the evolution of photosynthesis, which involves the fixation of inorganic CO\u003csub\u003e2\u003c/sub\u003e and aerobic oxidation of carbon and nitrite, has been reported\u0026nbsp;\u003csup\u003e[31-32]\u003c/sup\u003e. Similarly, Nitrospirae play important roles in removing nitrogen and carbon\u0026nbsp;\u003csup\u003e[33]\u003c/sup\u003e.\u0026nbsp;Furthermore, our results also showed that the dominant genera\u0026nbsp;\u003cem\u003eVariibacter\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[34]\u003c/sup\u003e and \u003cem\u003eNitrospira\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e[35]\u003c/sup\u003e may participate in the soil denitrification process to improve Cd accumulation in rice roots (Fig. 7B).\u0026nbsp;Additionally, the functional prediction analysis shown in Fig. 7C revealed that bacteria containing mobile elements and bacteria related to oxidative stress, oxygen utilization, pathogenesis, and biofilm formation might play vital roles in cultivar-specific responses to soil Cd pollution and Cd-dependent responses in specific rice varieties. Moreover, the dominant bacterial genera that contributed to the abovementioned\u0026nbsp;phenotypes were identified (Fig. 7D). The dominant bacterial genera that might be involved in both Cd responses and the abovementioned phenotypes were identified and are shown in Tables S3-S4.\u003c/p\u003e\n\u003cp\u003eRelated studies have reported that Cd pollution can induce oxidative stress and that oxidative stress is closely linked with Cd toxicity\u003csup\u003e\u0026nbsp;[39, 40]\u003c/sup\u003e. For instance, oxygen is a crucial factor in the regulation of Cd accumulation in rice \u003csup\u003e[41, 42]\u003c/sup\u003e. An increase in antioxidant bacteria and antioxidant defense responses might improve the tolerance of rice plants to Cd \u003csup\u003e[40]\u003c/sup\u003e. Additionally, mobile elements, including C, O and N, might play vital roles in the adaptation, persistence, and transmission of bacteria in soils and the regulation of heavy metal resistance \u003csup\u003e[43, 44]\u003c/sup\u003e. Thus, our results indicated that \u003cem\u003eVariibacter\u003c/em\u003e and \u003cem\u003eNitrospira\u003c/em\u003e can participate in the rice root oxygen, nitrogen and carbon cycles to affect cadmium accumulation.\u0026nbsp;On the other hand,\u0026nbsp;the negatively correlated genera \u003cem\u003eGaiella\u0026nbsp;\u003c/em\u003e\u003csup\u003e[36]\u003c/sup\u003e, \u003cem\u003eMycobacterium\u0026nbsp;\u003c/em\u003e\u003csup\u003e[37]\u003c/sup\u003e, and \u003cem\u003eDesulfobacca\u0026nbsp;\u003c/em\u003e\u003csup\u003e[38]\u003c/sup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ecould\u003cem\u003e\u0026nbsp;\u003c/em\u003eplay important roles in manipulating secondary succession of soil microbes to help decrease Cd accumulation in rice roots.\u0026nbsp;Therefore, our research further confirmed that the rhizosphere microbial community plays pivotal roles in regulating cadmium accumulation in rice and can be influenced by specific inherent genetic genes\u0026nbsp;\u003csup\u003e[45]\u003c/sup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eof rice varieties. The rhizosphere microbes affect rice cadmium accumulation under different cadmium concentrations in soil by changing the physical and chemical properties of the soil. However, deeper insight into the molecular mechanisms underlying the response of roots to Cd stress in the rhizosphere microbiome will contribute to better management of Cd pollution.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn conclusion, our research indicate that the different rice varieties can altered the structure soil bacterial communities to affect Cd concentration in rice. This provides theoretical support to better control the Cd pollution problem through agricultural ecology protection, such as use different Cadmium tolerant rice variety. Meanwhile, spearman correlation analysis suggested that the genera of \u003cem\u003eVariibacter\u003c/em\u003e, \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eGalella\u003c/em\u003e, \u003cem\u003eMycobacterium\u003c/em\u003e, and \u003cem\u003eDesulfobacca\u003c/em\u003e play essential roles in affecting rice Cd accumulation. Therefore, we focus on exploring the molecular mechanisms of how rhizosphere microbiome decreases rice root cadmium accumulation, lead to create rice germplasm with harmless of Cd content and benefit human health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interests\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interests to declare.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eDeclaration\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, S. Z.; methodology, S. Z., Z. L., F. Z and L. Z.; investigation, S. Z., Z. L., J. P., X. W., X. M., and B. S.; writing-original draft preparation, S. Z. and F. Z; writing-review and editing, S. Z. and F. Z; supervision, L. B.; funding acquisition, S. Z. and L. B. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe clean reads were deposited into the NCBI Sequence Read Archive (SRA) database (accession number: SUB11813545).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang W, Li Y, Dang P, Zhao S, Lai D, Zhou L (2018) Rice Secondary Metabolites: Structures, Roles, Biosynthesis, and Metabolic Regulation. Molecules. 23(12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou S, Zhu S, Cui S, Hou H, Wu H, Hao B, Cai L, Xu Z, Liu L, Jiang L (2021) Transcriptional and post-transcriptional regulation of heading date in rice. 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Nat Commun 15(1):23\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rice, Heavy Metal, Cadmium, Rhizosphere Bacterial, Microbiome","lastPublishedDoi":"10.21203/rs.3.rs-5369985/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5369985/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCadmium rice is a serious danger to human health due to its ability to enrich cadmium from soil to rice plants. Previously, we have identified two self-bred late-season high quality rice varieties, which are \"Yuzhenxiang\" and \"Xiangwanxian 12\". However, the mechanism on the distribution and tolerance of their significant differences in cadmium accumulation have barely been studied so far. Therefore, in this study, we comparatively analyze the relationships between the rhizosphere bacterial community and Cd accumulation in these two rice varieties under three different Cd stress conditions during the maturity period. Our results firstly showed that significant differences in physicochemical properties affect the Cd content in rice roots, which increased with increasing Cd content in the soil. Notably, the spearman correlation analysis suggested that the differed enrichment of \u003cem\u003eVariibacter\u003c/em\u003e, \u003cem\u003eNitrospira\u003c/em\u003e, \u003cem\u003eGalella\u003c/em\u003e, \u003cem\u003eMycobacterium\u003c/em\u003e, and \u003cem\u003eDesulfobacca\u003c/em\u003e affected by rice variety, which play key roles in root Cd accumulation. In general, our research indicate that the different rice varieties can altered the structure soil bacterial communities to affect Cd concentration in rice. This provides theoretical support to better control the Cd pollution problem through agricultural ecology protection.\u003c/p\u003e","manuscriptTitle":"Different Varieties of Rice (Oryza sativa) Affect Cadmium Accumulation by Reshaping Rhizosphere Bacterial Community","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 12:05:03","doi":"10.21203/rs.3.rs-5369985/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4aba8f08-fad1-4c6c-90cc-ccf3d028c9f4","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-06T08:53:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-18 12:05:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5369985","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5369985","identity":"rs-5369985","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}