Boundary sample-based class-weighted semi-supervised learning for malignant tumor classification of medical imaging.

Medical image classification plays a pivotal role within the field of medicine. Existing models predominantly rely on supervised learning methods, which necessitate large volumes of labeled data for effective training. However, acquiring and annotating medical image data is both an expensive and time-consuming endeavor. In contrast, semi-supervised learning methods offer a promising approach by harnessing limited labeled data alongside abundant unlabeled data to enhance the performance of medical image classification. Nonetheless, current methods often encounter confirmation bias due to noise inherent in self-generated pseudo-labels and the presence of boundary samples from different classes. To overcome these challenges, this study introduces a novel framework known as boundary sample-based class-weighted semi-supervised learning (BSCSSL) for medical image classification. Our method aims to alleviate the impact of intra- and inter-class boundary samples derived from unlabeled data. Specifically, we address reliable confidential data and inter-class boundary samples separately through the utilization of an inter-class boundary sample mining module. Additionally, we implement an intra-class boundary sample weighting mechanism to extract class-aware features specific to intra-class boundary samples. Rather than discarding such intra-class boundary samples outright, our approach acknowledges their intrinsic value despite the difficulty associated with accurate classification, as they contribute significantly to model prediction. Experimental results on widely recognized medical image datasets demonstrate the superiority of our proposed BSCSSL method over existing semi-supervised learning approaches. By enhancing the accuracy and robustness of medical image classification, our BSCSSL approach yields considerable implications for advancing medical diagnosis and future research endeavors.

Selenite reduced cadmium uptake, interfered signal transduction of endogenous phytohormones, and stimulated secretion of tartaric acid based on a combined analysis of non-invasive micro-test technique, transcriptome and metabolome.

Selenium (Se) can reduce uptake and translocation of cadmium (Cd) in plants via plenty of ways, including regulation of root morphology. However, the underlying mechanisms on how Se will regulate root morphology under metal(loid) stresses are not fully illustrated. To fill up this knowledge gap, we investigated the effects of 0.5 mg L-1 selenite (Se(IV)) on root exudates, root morphology, root endogenous hormones, and Cd uptake efficiency of rice under the 1 mg L-1 Cd stress condition. The results showed that Se(IV) significantly reduced shoot and root Cd concentrations, and decreased Cd uptake efficiency via root hairs determined by a non-invasive micro-test (NMT) technology. When compared to the 1 mg L-1 Cd (Cd1) treatment, addition of 0.5 mg L-1 Se(IV) (1) significantly reduced root surface area and tip numbers, and non-significantly reduced root length, but significantly enhanced root diameter and root volume; (2) significantly enhanced concentrations of tartaric acid in the root exudate solution, root auxin (IAA) and root jasmonic acid (JA) via a UHPLC or a HPLC analysis; (3) significantly up-regulated metabolites correlated with synthesis of IAA, JA, gibberellin (GA), and salicylic acid, such as GA53, M-SA, (+/-)7-epi-JA, and derivatives of tryptophan and indole in the metabolome analysis. However, results of transcriptome analysis showed that (1) no upregulated differentially expressed genes (DEGs) were enriched in IAA synthesis; (2) some upregulated DEGs were found to be enriched in JA and GA53 synthesis pathways. In summary, although Se(IV) stimulated the synthesis of IAA, JA, and GA53, it significantly inhibited root growth mainly by 1) affecting signal transduction of IAA and GA; 2) altering IAA polar transport and homeostasis; and 3) regulating DEGs including SAUR32, SAUR36, SAUR76, OsSub33, OsEXPA8, OsEXPA18, and Os6bglu24.

Selenite affected photosynthesis of Oryza sativa L. exposed to antimonite: Electron transfer, carbon fixation, pigment synthesis via a combined analysis of physiology and transcriptome.

Selenium (Se) is a microelement that can counteract (a)biotic stresses in plants. Excess antimony (Sb) will inhibit plant photosynthesis, which can be alleviated by appropriate doses of Se but the associated mechanisms at the molecular levels have not been fully explored. Here, a rice variety (Yongyou 9) was exposed to selenite [Se(IV), 0.2 and 0.8 mg L-1] alone or combined with antimonite [Sb(III), 5 and 10 mg L-1]. When compared to the 10 mg L-1 Sb treatment alone, addition of Se in a dose-dependent manner 1) reduced the heat dissipation efficiency resulting from the inhibited donors, Sb concentrations in shoots and roots, leaf concentrations of fructose, H2O2 and O2•-; 2) enhanced heat dissipation efficiency resulting from the inhibited accepters value, concentrations of Chl a, sucrose and starch, and the enzyme activity of adenosine diphosphate glucose pyrophosphorylase, sucrose phosphate synthase, and sucrose synthase; but 3) did not alter gas exchange parameters, concentrations of Chl b and total Chl, enzyme activity of soluble acid invertase, and values of maximum P700 signal, photochemical efficiency of PSI and electron transport rate of PSI. Se alleviated the damage caused by Sb to the oxygen-evolving complex and promoted the transfer of electrons from QA to QB. When compared to the 10 mg L-1 Sb treatment alone, addition of Se 1) up-regulated genes correlated to synthesis pathways of Chl, carotenoid, sucrose and glucose; 2) disturbed signal transduction pathway of abscisic acid; and 3) upregulated gene expression correlated to photosynthetic complexes (OsFd1, OsFER1 and OsFER2).

Selenite reduced uptake/translocation of cadmium via regulation of assembles and interactions of pectins, hemicelluloses, lignins, callose and Casparian strips in rice roots.

Selenium (Se) can reduce cadmium (Cd) uptake/translocation via regulating pectins, hemicelluloses and lignins of plant root cell walls, but the detailed molecular mechanisms are not clear. In this study, six hydroponic experiments were set up to explore the relationships of uptake/translocation inhibition of Cd by selenite (Se(IV)) with cell wall component (CWC) synthesis and/or interactions. Cd and Se was supplied (alone or combinedly) at 1.0 mg L-1 and 0.5 mg L-1, respectively, with the treatment without Cd and Se as the control. When compared to the Cd1 treatment, the Se0.5Cd1 treatment 1) significantly increased total sugar concentrations in pectins, hemicelluloses and callose, suggesting an enhanced capacity of binding Cd or blocking Cd translocation; 2) stimulated the deposition of Casparian strips (CS) in root endodermis and exodermis to block Cd translocation; 3) stimulated the release of C-O-C (-OH- or -O-) and CO (carboxyl, carbonyl, or amide) to combine Cd; 4) regulated differential expression genes (DEGs) and metabolites (DMs) correlated with synthesis and/or interactions of CWSs to affect cell wall net structure to affect root cell division, subsequent root morphology and finally elemental uptake; and 5) stimulated de-methylesterification of pectins via reducing expression abundances of many DMs and DEGs in the Yang Cycle to reduce supply of methyls to homogalacturonan, and regulated gene expressions of pectin methylesterase to release carboxyls to combine Cd; and 6) down-regulated gene expressions associated with Cd uptake/translocation.

Toxicity of different forms of antimony to rice plants: Photosynthetic electron transfer, gas exchange, photosynthetic efficiency, and carbon assimilation combined with metabolome analysis.

Antimony (Sb) is a toxic metalloid, and excess Sb causes damage to the plant photosynthetic system. However, the underlying mechanisms of Sb toxicity in the plant photosynthetic system are not clear. Hydroponic culture experiments were conducted to illustrate the toxicity differences of antimonite [Sb(III)] and antimonate [Sb(V)] to the photosynthetic system in a rice plant (Yangdao No. 6). The results showed that Sb(III) showed a higher toxicity than Sb(V), judging from (1) lower shoot and root biomass, leaf water moisture content, water use efficiency, stomatal conductance, net photosynthetic rate, and transpiration rate; (2) higher water vapor deficit, soluble sugar content, starch content, and oligosaccharide content (sucrose, stachyose, and 1-kestose). To further analyze the direction of the photosynthetic products, we conducted a metabonomic analysis. More glycosyls were allocated to the synthesis pathways of oligosaccharides (sucrose, stachyose, and 1-kestose), anthocyanins, salicylic acid, flavones, flavonols, and lignin under Sb stress to quench excess oxygen free radicals (ROS), strengthen the cell wall structure, rebalance the cell membrane, and/or regulate cell permeability. This study provides a complete mechanism to elucidate the toxicity differences of Sb(III) and Sb(V) by exploring their effects on photosynthesis, saccharide synthesis, and the subsequent flow directions of glycosyls.

As(III) Exposure Induces a Zinc Scarcity Response and Restricts Iron Uptake in High-Level Arsenic-Resistant Paenibacillus taichungensis Strain NC1.

The Gram-positive bacterium Paenibacillus taichungensis NC1 was isolated from the Zijin gold-copper mine and shown to display high resistance to arsenic (MICs of 10 mM for arsenite in minimal medium). Genome sequencing indicated the presence of a number of potential arsenic resistance determinants in NC1. Global transcriptomic analysis under arsenic stress showed that NC1 not only directly upregulated genes in an arsenic resistance operon but also responded to arsenic toxicity by increasing the expression of genes encoding antioxidant functions, such as cat, perR, and gpx. In addition, two highly expressed genes, marR and arsV, encoding a putative flavin-dependent monooxygenase and located adjacent to the ars resistance operon, were highly induced by As(III) exposure and conferred resistance to arsenic and antimony compounds. Interestingly, the zinc scarcity response was induced under exposure to high concentrations of arsenite, and genes responsible for iron uptake were downregulated, possibly to cope with oxidative stress associated with As toxicity. IMPORTANCE Microbes have the ability to adapt and respond to a variety of conditions. To better understand these processes, we isolated the arsenic-resistant Gram-positive bacterium Paenibacillus taichungensis NC1 from a gold-copper mine. The transcriptome responding to arsenite exposure showed induction of not only genes encoding arsenic resistance determinants but also genes involved in the zinc scarcity response. In addition, many genes encoding functions involved in iron uptake were downregulated. These results help to understand how bacteria integrate specific responses to arsenite exposure with broader physiological responses.

Identification of a MarR Subfamily That Regulates Arsenic Resistance Genes.

In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. strain As-55. Genomic analyses of Achromobacter sp. As-55 showed that this marR is located adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite [MAs(III)], the organoarsenic compound roxarsone(III) [Rox(III)], and the inorganic antimonite [Sb(III)]. Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarRars. MarRars orthologs have three conserved cysteine residues, which are Cys36, Cys37, and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarRars (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV. Moreover, electrophoretic mobility shift assays (EMSAs) demonstrate that AdMarRars binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarRars is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Rox(III), and Sb(III). AdMarRars and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCE In this study, a MarR family member, AdMarRars was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarRars, regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarRars was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.

Potential of cadmium resistant Burkholderia contaminans strain ZCC in promoting growth of soy beans in the presence of cadmium.

Bioremediation of Cd contaminated environments can be assisted by plant-growth-promoting bacteria (PGPB) enabling plant growth in these sites. Here a gram-negative Burkholderia contaminans ZCC was isolated from mining soil at a copper-gold mine. When exposed to Cd(II), ZCC displayed high Cd resistance and the minimal inhibitory concentration was 7 mM in LB medium. Complete genome analysis uncovered B. contaminans ZCC contained 3 chromosomes and 2 plasmids. One of these plasmids was shown to contain a multitude of heavy metal resistance determinants including genes encoding a putative Cd-translocating PIB-type ATPase and an RND-type related to the Czc-system. These additional heavy metal resistance determinants are likely responsible for the increased resistance to Cd(II) and other heavy metals in comparison to other strains of B. contaminans. B. contaminans ZCC also displayed PGPB traits such as 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore production, organic and inorganic phosphate solubilization and indole acetic acid production. Moreover, the properties and Cd(II) binding characteristics of extracellular polymeric substances was investigated. ZCC was able to induce extracellular polymeric substances production in response to Cd and was shown to be chemically coordinated to Cd(II). It could promote the growth of soybean in the presence of elevated concentrations of Cd(II). This work will help to better understand processes important in bioremediation of Cd-contaminated environment.

Efficiency and risks of selenite combined with different water conditions in reducing uptake of arsenic and cadmium in paddy rice.

The co-contamination of arsenic (As) and cadmium (Cd) in soils is a common problem. Selenium (Se) can reduce the uptake of As and Cd in plants, and in practice, the alternate wetting and drying is a common culture mode in rice production. However, it is unknown whether Se can efficiently reduce As and Cd concentrations in crops suffering from a high-level contamination of As and Cd under different soil water conditions. In this study, we assessed the efficiency and risks of selenite [Se(IV)], in a pot experiment, to reduce the uptake of As and Cd in a rice plant (YangDao No 6) growing in a heavily contaminated soil by As and Cd (pH 7.28) under different soil water conditions. The results showed that Se(IV) failed to control the grain total As and Cd concentrations within their individual limited standard (0.2 mg kg-1) despite that Se(IV) significantly reduced the grain total As and Cd concentrations. The soil drying treatment alone could reduce the accumulation of arsenite [As(III)] in the grains, but additional Se(IV) stimulated the accumulation of As(III) in the grains under soil drying conditions. In addition, the addition of Se(IV) enhanced the As and Cd concentrations in the shoots and/or roots of rice plants under certain conditions. The above results all suggested that the utilization of Se(IV) in a high contaminated soil by As and Cd cannot well control the total concentrations of As and Cd in plants. In this study, the available concentrations of As and Cd in the rhizosphere soil, the rhizosphere soil pH, the formation of root iron/manganese plaques and the concentrations of essential elements in the grains were monitored, and the related mechanisms on the changes of these parameters were also discussed. This study will give a guideline for the safe production of rice plants in a heavily co-contaminated soil by As and Cd.

Differences in cadmium absorption by 71 leaf vegetable varieties from different families and genera and their health risk assessment.

Leaf vegetables have strong capabilities to take up cadmium (Cd) compared to other vegetable varieties. Until now, the differences in Cd uptake and accumulation by leaf vegetables from different families and genera and the related health risks were unknown. To remedy this, we studied 71 leaf vegetables (multiple genotypes within 17 categories of vegetables) in soil cultivation experiments (3 Cd treatment levels). Results showed that at 2.12 mg kg-1 Cd treatment, the dry weight of only five genotypic varieties from the families Brassicaceae and Asteraceae significantly decreased compared to the control, suggesting their weak Cd tolerances. Vegetables from the Brassicaceae, Asteraceae, Apiaceae, and Convolvulaceae families had stronger Cd absorption capabilities, whereas those from the Liliaceae and Amaranthaceae families had weaker ones. Cluster analysis found that the 17 vegetable categories could be divided into three groups: vegetables with high Cd accumulation capabilities were Lactuca sativa L.var. ramosa Hort. and Lactuca sativa var. longifoliaf. Lam. Vegetables with moderate Cd accumulation capabilities were bok choy, napa cabbage, choy sum, leaf mustard, Lactuca sativa L., Sonchus oleraceus L., celery, coriander, and water spinach. Vegetables with low Cd accumulation capabilities were cabbage, crown daisy, garlic chive, Allium ascalonicum, Gynura cusimbua, and edible amaranth. Estimated daily intake (EDI) and target hazard quotient (THQ) analysis results showed that 100% genotypes of vegetables from the Apiaceae and Convolvulaceae families had health risks; 100% genotypes of Lactuca sativa L., Sonchus oleraceus L., Lactuca sativa L. var. ramosa Hort., and Lactuca sativa var. longifoliaf. Lam from the Asteraceae family carried high risks. Of vegetables in the Brassicaceae family, 42.9% showed risks. Vegetables from the Amaranthaceae and Liliaceae families, Gynura cusimbua and crown daisy from the Asteraceae family, and cabbage from the Brassicaceae family all displayed relatively low risks (all 100%).

Safe utilization of polluted soil by arsenic, cadmium and lead through an integrated sericultural measure.

Effective remediation technologies to remediate multiple heavy metal contaminated farmlands are lacking. To make full use of farmlands and control its consequent health risks, we planted mulberry trees in arsenic (As)-cadmium (Cd)-lead (Pb) co-contaminated soils at four different sites; then reared silkworms on leaves harvested from these mulberry trees; and finally used the silkworm excrement to in situ remediate the As, Cd, and Pb polluted paddy soil. Mulberry leaves and stalks showed weak abilities to accumulate As, Cd, and Pb. As and Pb tended to accumulate in silkworm pupae and silkworm excrement, respectively, posing a potential health risk when they were used as pharmaceutical materials or foods. However, using the leaves of mulberry trees planted in Cd-contaminated soils to rear silkworms had a low health risk. Silkworm excrement significantly reduced the As, Cd and Pb concentrations in rice grains, with As and Cd concentration being lower but for Pb being higher their respective national limit standards. In conclusion, based on the rational utilization of resources such as silkworm excrement, pupae, and cocoons, the integrated measure in this study could effectively reduce the environmental health risks resulting from multiple As, Cd, and Pb contamination.

Differences in the uptake and bioconcentration of dichlorodiphenyltrichloroethane by eight vegetable cultivars and their health risk assessments.

Dichlorodiphenyltrichloroethane (DDT) is not easily degraded in soils, which will pose a threat to human health. We investigated the differences of eight vegetables' capacity to take up DDT, removing DDT from soil, and tolerating DDT (monitoring the responses of growth, root morphology and photosynthesis of vegetables to DDT). These vegetables included Chinese mustard (two genotypes, B.jf and B,jm), napa cabbage (two genotypes, B.coz and B.coc) and Bok choy (four genotypes, B.cz, B.cq, B.cs and B.chg). The results demonstrated that 5 mg kg-1 DDT did not display significant effects on the growth of most vegetables in this study. As compared to the control, 5 mg kg-1 DDT significantly increased the shoot and root biomass, the fine root numbers, and the fine root ratio for the genotype of B.chg. However, 5 mg kg-1 DDT exposure showed a negative effect on the shoot growth of two genotypes of napa cabbage. In general, 5 mg kg-1 DDT did not significantly affect the photosynthesis and root morphology of most vegetables in this study. Consuming these vegetables had a low non-cancer health risk, but showed a high cancer health risk. In addition, among the eight vegetables, B.chg accumulated less DDT in the edible parts and had low values of HRnon-cancer and HRcancer for consuming these vegetables containing DDT. Planting these vegetables might promote the degradation of DDT reducing its residual amount in soil.

Ionomic and transcriptomic analysis provides new insight into the distribution and transport of cadmium and arsenic in rice.

To identify the key barrier parts and relevant elements during Cd/As transport into brown rice, 16 elements were measured in 14 different parts of 21 rice genotypes; moreover, transcriptomic of different nodes was analyzed. Cd/As contents in root and nodes were significantly higher than those other parts. Node I had the highest Cd content among nodes, leading an increase in gene expressions involved in glycolytic and Cd detoxification. The Cu/Zn/Co distribution and transport to various parts was similar to that of Cd, and Fe/Sb distribution and transport to various parts was similar to that of As. Moreover, Cu/Zn/Co/Mg was correlated with Cd in root and nodes, as well as Fe with As. Besides, the ionomic profile showed the different parts of an organ were closely related, and the spatial distribution of different organs was consistent with the growth morphology of rice. Therefore, root and nodes are two key barriers to Cd/As transport into brown rice. Moreover, Node I has the highest Cd accumulation capacities among nodes. The ionomic profile reflects relationships among plant parts and correlations between the elements, suggesting that nodes are hubs for element distribution, as well as the correlation between Cd with Zn/Cu/Co/Mg, between Fe with As.

Root application of selenite can simultaneously reduce arsenic and cadmium accumulation and maintain grain yields, but show negative effects on the grain quality of paddy rice.

Most current technologies can hardly simultaneously reduce the accumulation of arsenic (As) and cadmium (Cd) in crops. In this study, root application of selenite [Se (IV)] and selenate [Se (VI)] was used to assess their abilities to reduce the accumulation of As and Cd, and maintain the yields and quality of rice grains. The results show that Se (IV) showed a weaker ability than Se (VI) to maintain the grain contents of many essential elements, but a stronger ability to decrease As and Cd contents in rice grains, and maintain the yields, photosynthesis rate and stomatal conductance, and increase the grain contents of several amino acids (AAs), total Se, selenomethionine (SeMet) and selenocysteine (SeCys). The best outcomes resulted at a relatively high application of 5 mg kg-1 Se (IV), reflecting in the highest total Se, SeCys and SeMet content (14.95, 118.70 and 864.73 µg kg-1, respectively) in the grains, highest grain yield, and lowest grain As and Cd content (0.36 and 0.07 mg kg-1, respectively). In addition, the application of 1-5 mg kg-1 Se (IV) seemed to facilitate the formation of SeMet in the grains, but most inorganic Se in the grains were transformed into SeCys and SeMet under Se (VI) treatments. This study provides a new idea to resolve the problems of high accumulation of As and Cd in rice grains and insufficiency of Se intake in China.

Efficiency evaluation for remediating paddy soil contaminated with cadmium and arsenic using water management, variety screening and foliage dressing technologies.

Paddy soils in many regions of China have been seriously polluted by multiple heavy metals or metalloids, such as arsenic (As), cadmium (Cd) and lead (Pb). In order to ensure the safety of food and take full advantage of the limited farmland resources of China, exploring an effective technology to repair contaminated soils is urgent and necessary. In this study, three technologies were employed, including variety screening, water management and foliage dressing, to assess their abilities to reduce the accumulation of Cd and As in the grains of different rice varieties, and meanwhile monitor the related yields. The results of variety screening under insufficient field drying condition showed that the As and Cd contents in the grains of only four varieties [Fengliangyouxiang 1 (P6), Zhongzheyou 8 (P7), Guangliangyou 1128 (P10), Y-liangyou 696 (P11)] did not exceed their individual national standard. P6 gained a relatively high grain yield but accumulated less As and Cd in the grains despite of the relatively high As and Cd concentrations in the rhizosphere soil. However, long-playing field drying in water management trial significantly increased Cd but decreased As content in the grains of all tested three varieties including P6, suggesting an important role of water supply in controlling the accumulation of grain As and Cd. Selenium (Se) showed a stronger ability than silicon (Si) to reduce As and Cd accumulation in the grains of Fengliangyou 4 (P2) and Teyou 524 (P13), and keep the yields. The results of this study suggest that combined application of water management and foliage dressing may be an efficient way to control As and Cd accumulation in the grains of paddy rice exposing to As- and Cd-contaminated soils.

Applying carbon dioxide, plant growth-promoting rhizobacterium and EDTA can enhance the phytoremediation efficiency of ryegrass in a soil polluted with zinc, arsenic, cadmium and lead.

This study was conducted to investigate the use of elevated carbon dioxide (CO2), plant growth-promoting rhizobacterium Burkholderia sp. D54 (PGPR) and ethylenediaminetetraacetic acid (EDTA) to enhance the phytoextraction efficiency of ryegrass in response to multiple heavy metal (or metalloid)-polluted soil containing zinc (Zn), arsenic (As), cadmium (Cd) and lead (Pb). All of the single or combined CO2, PGPR and EDTA treatments promoted ryegrass growth. The stimulation of ryegrass growth by CO2 and PGPR could primarily be attributed to the regulation of photosynthesis rather than decreased levels of Zn, As and Cd in the shoots. Most treatments seemed to reduce the Zn, As and Cd contents in the shoots, which might be associated with enhanced shoot biomass, thus causing a "dilution effect" regarding their levels. The combined treatments seemed to perform better than single treatments in removing Zn, As, Cd and Pb from soil, judging from the larger biomass and relatively higher total amounts (TAs) of Zn, As, Cd and Pb in both the shoots and roots. Therefore, we suggest that the CO2 plus PGPR treatment will be suitable for removing Zn, As, Cd and Pb from heavy metal (or metalloid)-polluted soils using ryegrass as a phytoremediation material.