Single-tube Multiplex Nested PCR System for Efficient Detection of Pathogenic Microorganisms in SPF Rodents.

PCR testing is increasingly important for microbial control in SPF facilities. However, most current PCR methods are timeconsuming and require compromise between high sensitivity and high multiplexing. We developed a one-tube multiplex nested PCR strategy (MN-PCR) for simultaneous direct (that is, without culturing) detection of multiple pathogens. We first aligned sequences for the 16S rDNA genes of selected target bacteria and a panel of closely related organisms. From these data, we designed a pair of universal primers and multiple sets of species-specific PCR primers to amplify the target sequences; the universal primers were modified to include various degenerate bases and locked nucleic acids. In a single tube, 16S rDNA sequences were amplified by using the nested PCR primers under high temperature (that is, above 65°C) during the first stage of the MN-PCR procedure, when the target-species-specific PCR primers do not support amplification due to their short length. In addition, the concentration of the nested PCR primers during the first stage was adjusted to ensure that they were consumed and did not yield visible bands themselves. During the second stage, the enriched 16S rDNA sequences then served as templates for amplification of the species-specific fragments by using the multiple PCR primers at low annealing temperatures (that is, below 60°C). The results showed that our MN-PCR method detected as little as 1 fg of target bacterial DNA in a 20-µL reaction volume, whereas conventional multiplex PCR detected a minimum of 1 pg only. Compared with traditional multiplex PCR assays, our MN-PCR system is an effective and efficient culture-free process.

[Effects of Different Exogenous Selenium Species Application on Growth and Cadmium Uptake of Pak Choi in Cadmium Contaminated Soil].

This study explored the discrepancy in the detoxification effects of different exogenous selenium (Se) species in cadmium (Cd)-contaminated soil to provide a scientific basis for the control of Cd pollution in the soil and the safe production of crops. A pot experiment was conducted to compare the effects of different concentrations (0, 0.5, 1.0, and 2.5 mg·kg-1) of selenite and selenate on the growth (root length, shoot height, biomass, and photosynthetic parameters), uptake, and translocation of Cd on pak choi in Cd-contaminated soil. The results indicated that the detoxification effect of a low Se concentration (≤1.0 mg·kg-1) treatment on Cd was better than that with a high Se concentration (2.5 mg·kg-1) treatment, and the selenite treatment demonstrated a greater detoxification effect on Cd than the corresponding selenate treatment. Meanwhile, the application of low-concentration selenite and selenate both increased the SPAD value, Pn, Gs, Ci, biomass, and shoot length of the pak choi, and the 1.0 mg·kg-1 selenite treatment had the most significant (P<0.05) effect (except Ci). Nevertheless, the photosynthetic parameters of the pak choi under the high-concentration Se were significantly lower than those under the low Se concentration treatment (except Tr, P<0.05). Compared with the treatment without Se (control), the uptake of Cd in the pak choi was reduced under different Se treatments. Compared with the control, the Cd concentration in the shoots of the pak choi treated with 1.0 mg·kg-1 of selenite and selenate decreased by 40.0% and 20.5% (P<0.05), respectively. In addition, the translocation of Cd from the root to the shoot was significantly reduced under the 0.5 mg·kg-1 selenate treatment, while the high-concentration treatments of either exogenous Se promoted the translocation of Cd. Overall, applying the appropriate amount of exogenous Se could promote the photosynthesis and biomass of pak choi, and reduce the accumulation of Cd in pak choi. Therefore, the 1.0 mg·kg-1 selenite treatment is recommended for the control and safe utilization of Cd in Cd-contaminated soil.

DOM derivations determine the distribution and bioavailability of DOM-Se in selenate applied soil and mechanisms.

Straw amendment and plant root exudates modify the quality and quantities of soil dissolved organic matter (DOM) and then manipulate the fractions of soil selenium (Se) and its bioavailability. Two typical soils with distinct pH were selected to investigate the effect of different contributors on DOM-Se in soil. The mechanisms relying on the variation in DOM characteristics (quality, quantity and composition) were explored by UV-Vis, ATR-FTIR and 3D-EEM. Straw amendment significantly (p < 0.05) suppressed the selenate bioavailability. The reduction in wheat Se content was greater in krasnozems than in Lou soil, as more HA fraction appeared in krasnozems. The root exudates of wheat mainly elevated the low molecular hydrophilic compounds (Hy) in soil, which contributed to the SOL-Hy-Se fractions and thus grain Se in soils (p < 0.01). However, straw amendment promoted DOM transforming from small molecules (Hy and FA) to aromatic large molecules (HA), when accompanied with the reduction and retention of Se associated with these molecules. As a result, selenium bioavailability and toxicity reduced with DOM amendment and DOM-Se transformation.

Leaching behaviors and chemical fraction distribution of exogenous selenium in three agricultural soils through simulated rainfall.

To clarify the leaching risk of selenium (Se) in agricultural soils, a laboratory column experiment was conducted to study the characteristics of leaching and chemical fractions of Se in three different soils treated with different levels of exogenous selenate under simulated local rainfall. Results demonstrated that the Se concentration in leachates of all tested soils decreased rapidly at the beginning of leaching and slowly decreased thereafter. After leaching, Se concentrations in leachates of all tested soils at 1, 3, and 6 mg/kg exogenous Se concentrations were 0.06-0.24, 0.25-0.84, and 0.60-1.65 mg/L, respectively, which exceeded the standard limit of the Chinese Environmental Quality Standards for Groundwater (<0.01 mg/L) (GB/T 14848-2017). The cumulative leached Se amount accounted for 51.27-86.22% of the total Se. Those results indicated the high risk of Se leaching in the tested soils. The Elovich model could better describe Se leaching processes in krasnozem, while the leaching processes of Se in black soil and loess soil accorded with the power function model. Se mainly existed in soluble fraction (61.33-81.05%) before leaching and residual fraction (48.91-68.04%) after leaching. The soluble and exchangeable Se fractions were the main contributors of Se in leachates. In addition, the parameters of the Uts and IR values could well describe the distribution of Se fractions in soil during leaching. In general, more attention should be placed on the assessment of Se leaching in soil.

Effects of straw amendment on selenium aging in soils: Mechanism and influential factors.

Soil dissolved organic matter (DOM) alters heavy metal availability, but whether straw amendment can manipulate soil selenium (Se) speciation and availability through DOM mineralization remains unclear. In this study, allochthonous maize straw and selenate were incubated together in four different soils for 1 y. The transformation and availability of DOM associated Se (DOM-Se) was investigated during aging. Results indicated that soil solution and soil particle surfaces were dominated by hexavalent hydrophilic acid-bound Se (Hy-Se). The amount of fulvic acid bound Se in soil solution (SOL-FA-Se) was higher than humic acid bound Se in soil solution (SOL-HA-Se), except in krasnozems, and mainly existed as hexavalent Se (Se(VI)). Tetravalent Se (Se(IV)) was the main valence state of FA-Se adsorbed on soil particle surfaces (EX-FA-Se) after 5 w of aging. The proportion of soil-available Se (SOL + EX-Se) decreased with increasing straw rate. However, under an application rate of 7500 kg·hm-2, soluble Se fraction (SOL-Se) reduction was minimal in acidic soils (18.7%-34.7%), and the organic bound Se fraction (OM-Se) was maximally promoted in alkaline soils (18.2%-39.1%). FA and HON could enhance the availability of Se in the soil solution and on particle surfaces of acidic soil with high organic matter content. While Se incorporation with HA could accelerate the fixation of Se into the solid phase of soil. Three mechanisms were involved in DOM-Se aging: (1) Reduction, ligand adsorption, and inner/outer-sphere complexation associated with the functional groups of straw-derived DOM, including hydroxyls, carboxyl, methyl, and aromatic phenolic compounds; (2) interconnection of EX-FA-Se between non-residual and residual Se pools; and (3) promotion by soil electrical conductivity (EC), clay, OM, and straw application. The dual effect of DOM on Se aging was highly reliant on the characteristics of the materials and soil properties. In conclusion, straw amendment could return selenium in soil and reduce soluble Se loss.

[Effects of Nitrogen Application on Selenium Uptake, Translocation and Distribution in Winter Wheat].

In order to better understand the effects of nitrogen application on accumulation, translocation and distribution of selenium in winter wheat and to provide theoretical reference for reasonable nitrogen application and increasing selenium content of grains. A pot experiment was carried out under greenhouse conditions with Se1 (0.74 mg·kg-1) or Se2 (2.60 mg·kg-1) levels of selenium, and each Se treatment was supplied with N1 (100 mg·kg-1) or N2 (200 mg·kg-1) levels of nitrogen, respectively. Selenium concentrations and biomass amounts of different parts of wheat were determined at jointing and maturity stage. The results showed that grain yield increased with increasing nitrogen levels by 13.2% and 24.0% in Se1 and Se2 treatment, respectively. Regardless of N rate, Se concentration of wheat increased with raising Se amended rate (P<0.01). Increasing nitrogen application could promote Se uptake of root and thus increase the selenium concentration of wheat grains and leaves, which was greater in Se1 treatment than in Se2 treatments. Se concentrations in wheat grain increased by 22.6% and 12.1% with the increasing N application rate in low and high Se treatment, respectively. The distribution ratios of Se in each organ ranked the same as BCFs, following the order of leaf > grain > glume > root. Increasing N fertilization increased the distribution ratio of Se in grains by 11.1% and 25.9% in low and high selenate treatments, respectively. High nitrogen fertilization could promote uptake and translocation of Se in wheat under low Se conditions, and improve Se use efficiency as well in the agricultural production.

[Dynamic Differences of Uptake and Translocation of Exogenous Selenium by Different Crops and Its Mechanism].

The study aimed to find out the dynamic changes of selenium (Se) uptake with crops growing and to provide better understanding on the translocation of Se in soil-plant system. Pot experiments and laboratory analysis were carried out, in which 6 crops were planted for eight weeks in the soils treated with selenite and selenate. The results showed that there was a significant difference in the plant dynamic uptake pattern for selenite and selenate. Se concentration in shoots and roots of all tested plants continuously declined with growth since the third week after planting for selenate treatment; while Se concentration in roots of plants gradually increased with the growth, and that for shoots firstly increased and then leveled off or decreased for selenite treatment. Crops could accumulate Se throughout the whole growing period, while 80% of the Se in plants was uptaken in the first 6 weeks. Among the 6 tested plants, the Se concentration in the shoots and roots of mustard was the highest, followed by the shoots of wheat for selenate treatment. The Se concentration in shoots of carrot was the highest, while that in roots was the lowest for selenite treatment. For the same Se treatment, the Se concentrations in both roots and shoots of broccoli, purple cabbage and flowering Chinese cabbage were similar. Broccoli, purple cabbage, flowering Chinese cabbage, mustard and wheat had higher translocation ability to transport selenate than selenite from roots to shoots, whereas carrot had the same ability for translocating selenite and selenate. Biological dilution affected the dynamic changes of the Se content in crops, especially for selenate treatment, and a significant negative correlation between Se concentration of plants and their biomass further verified this. However, the changes of biomass of plants with growing period couldn't explain the total inverse dynamic uptake pattern for selenite and selenate. A significant positive correlation was observed between Se accumulations amount in six crops with available Se changes in soil during the corresponding growing period for selenate treatment, while no such correlation was found for selenite treatment. Se accumulation in crops grown on selenite-treated soil accounted for 0.5%-18.1% of soil available Se, whereas that in selenate treatment was 1.1-4.5 times of soil available Se except for carrot. In conclusion, the dynamic changes of Se uptake with crop growth were the comprehensive results of the absorption and transport capacity of crops, Se availability in soil and biological dilution. It should be considered as a whole in the selenium biofortification.

[Selenate Adsorption and Desorption in 18 Kinds of Chinese Soil with Their Physicochemical Properties].

The environmental behavior and bioavailability of selenium (Se) in soils are greatly affected by its adsorption on soil components, which are largely discrepant with the different physicochemical properties of soils. 18 kinds of farmland soils with various physicochemical properties in China were used in batch adsorption experiments in this study to explore the influencs of soil pH, amorphous iron, aluminum oxides, organic matter and mechanical composition on the adsorption of SeO42-. The results showed that the adsorption of SeO42- on 18 soils was an initially fast phase followed by a slow process, during which the adsorption equilibrium was reached at 24 h. Second-order kinetic model(R2>0.976)and Freundlich isothermal model(R2>0.842)were the fittest for most of the adsorption process. SeO42- adsorption capacity of soil was negatively correlated with soil pH value (P<0.01) and the content of carbonate (P<0.05), while positively correlated with the content of amorphous iron, aluminum oxides (P<0.01) and organic matter content (P<0.05). The partition coefficient of solid to liquid (Kd values) of SeO42- in adsorption process for all the 18 soil types were very low and without big differences(0.99 L·kg-1-18.18 L·kg-1). The desorption rates for all tested soils were above 80%, which indicated the reversibility of SeO42- adsorption in soil. It was inferred from the above that the low Kd and high desorption rate reflected that selenate was featured by easy migration and leaching in soil, which should be emphasized in the regional evaluation and regulation of Se level.

[Effect of Cr (VI) anions on the Cu (II) adsorption behavior of two kinds of clay minerals in single and binary solution].

The adsorption of Cu (II) on kaolinite and montmorillonite was investigated through batch adsorption experiment. Several adsorption models were employed to describe the adsorption of Cu (II) on the two clay minerals in single Cu (II) and Cu(II)-Cr (VI) binary solutions, and the impact of solution with various pH values on the adsorption of Cu (II) on the two target mineral clays was investigated in order to explain the environmental chemical behavior of heavy metals in soil and to provide theoretical basis in remediation of multi-element contaminated soil. The results indicated that the adsorption process of Cu (II) on kaolinite and montmorillonite in both single and binary solutions was fast at the beginning and then slowed down. Adsorption equilibrium was observed within 120 min. In both single and binary solutions, pseudo-second-order model (R2 > 0.983) showed the highest agreement with the adsorption of Cu (II) on the two mineral clays, followed by the intra-particle diffusion model and pseudo-first-order model. Both Intra-particle diffusion model and Boyd model illustrated that the film diffusion process was the rate-limiting step, which mainly occurred at the edge and surface of mineral clays. Copper adsorption on kaolinite was well fitted with the Freundlich equation (R2 > 0.971), which could be attributed to the heterogeneity of kaolinite surface with adsorption sites that have different energies of adsorption. Langmuir equation was best fitted with the isotherm for montmorillonite (R2 > 0.983), which indicated that the adsorption was on a single molecular layer or chemisorptions. In both single and binary solutions, the adsorption of Cu (II ) on the two clay minerals first increased and then decreased with the rising of pH values. The maximum adsorption amount was found at pH = 5.0, and was in the order of Qmon. > Qkao. and Q(Single-Cu) > Q(Cu-Cr binary). Cr (VI) in the solution reduced the adsorption of Cu (II), and the minimal influence of Cr (VI) on Cu (I) adsorption was observed at pH = 6.0.

[Differences of selenium uptake pattern of pakchoi and the possible mechanism when amended with selenate and selenite].

In order to produce selenium-enriched crops and provide a theoretical basis of phytoremediation to selenium contaminated soil, pot experiment and lab analysis were carried out to examine the differences of selenium uptake pattern and bioavailability to pakchoi (Brassica chinensis L.) when amended with selenate and selenite. Meanwhile, dynamic changes of soil available Se was analyzed to explore the reason. The results showed that no significant differences in pakchoi shoot biomass between selenate and selenite treatment were observed. The Se concentration in pakchoi decreased rapidly across time in the selenate treatments, while increased with the growing period in the selenite treatments. For the selenate treatments, the pakchoi shoot Se concentration was significantly higher than that in roots, but the opposite results were found in the selenite treatments. In the whole growth period of pakchoi, Se concentration in selenate treatment was 2-60 times higher of that in selenite treatments. The content of available Se in soil slightly decreased with the growth of pakchoi in the selenite treatments, but it decreased rapidly in the selenate treatments. The significant correlations between soil available Se and Se concentration in shoots and roots of pakchoi was found in selenate treatments, the correlation coefficients were 0.901 and 0.864, respectively. On the contrast, no significant correlation was observed between soil available Se and Se concentration of roots and shoots in selenite treatments. The results indicated that the differences between the Se uptake pattern of selenate and selenite on pakchoi were the comprehensive function of the ability of soil providing Se and crop uptake ability.

[Transformation and influences of copper and selenium fractions on heavy metals bioavailability in co-contaminated soil].

Pot experiments and laboratory analysis methods were used to investigate the form transformation of additional copper and selenium and their bioavailability for pakchoi (Brassica chinensis) in co-contaminated soil. The results showed that Cu mainly existed in residual bound form, while selenium was present mainly in organic bound and residual form in the uncontaminated soil. In the contaminated soil, copper was mainly bounded to hydrated oxides of iron and manganese, while Se was in exchangeable and carbonate forms. After one month of growing season, Cu tended to transfer into organic bound fractions, while Se tended to bind to hydrated oxides of iron and manganese. The I(R) value of Cu decreased with increasing copper and selenium concentrations, while the I(R) value of Se decreased with increasing Se concentration and had nothing to do with the concentration of exogenous Cu for both before planting and after harvesting of pakchoi. The parameters estimated by S curve fitting indicated that suitable amount of Se (< or = 10 mg x kg(-1)) could promote the Cu uptake by pakchoi, and certain amount of Cu (< or = 400 mg x kg(-1)) could promote Se absorption by pakchoi. Both the I(R), values for Cu and Se had similar trends as the Cu, Se concentrations in pakchoi, which meant that the I(R) value could be used to evaluate the bioavailability of heavy metals in soil. Partial correlation analysis showed that Cu and Se in exchangeable and organic bound forms in soil had better bioavailability for pakchoi. Therefore, the I(R) value of elements in soils and the change of elements before planting and after harvesting of pakchoi can be used as indicators for evaluating the bioavailability of heavy metals.

[Transformation of copper and chromium in co-contaminated soil and its influence on bioavailability for pakchoi (Brassica chinensis)].

Heavy metal fractions in soil play an important role in the bioavailability for plants. A pot experiment was carried out to study the transformation and bioavailability for pakchoi of exogenetic copper and hexavalent chromium in co-contaminated soil. The results showed that one month after tested heavy metals were added into soil, the chromium was present mainly in residual and organic bonded form while copper was mainly bound to hydrated oxides of iron and manganese as well as residual form. Lower concentrations copper (< or = 400 mg x kg(-1)) in soil could promote chromium transform from other forms to exchangeable form while higher concentrations copper (> or = 800 mg x kg(-1)) in soil would inhibit this transformation. Similarly, chromium in low concentrations (< or = 5 mg x kg(-1)) could facility copper transform to residual form while chromium in high concentrations (> or = 20 mg x kg(-1)) would reduce such transformation process. Both added chromium to soil polluted by high concentrations copper and added copper to high chromium content soil could promote the polluted soil reach quasi-equilibrium state. Additionally, adding copper in soil could inhibit chromium uptake of pakchoi by promoting soil OM-Cr transformed into RES-Cr. While adding chromium in soil could reduce copper uptake of pakchoi by stimulating the EXE-Cu transformed into the RES-Cu. Therefore, it is necessary to take into consideration the synthetic effects of copper and chromium when carrying on assessment and phytoremediation to co-contaminated soil.