土壤耐硒菌株筛选及其硒活化能力评价研究

  目的  从湖北省恩施市双河镇新塘乡渔塘坝硒矿床采集矿渣、淤泥、农田土壤,从中筛选分离出耐硒菌株,并对筛选出菌株的活化土壤硒的能力进行评价。  方法  采用稀释涂布平板法和平板划线法分离、筛选出耐受亚硒酸钠浓度较高的菌株,根据菌株形态学特征和用分子生物学方法对其进行分类鉴定,再将分离得到的耐硒菌株分别接种于酸性富硒赤红壤中进行培养,通过测定培养后土壤中的硒含量评价两菌株活化土壤硒的能力。  结果  筛选出了细菌B-1和真菌B-2两耐硒菌株;细菌B-1被鉴定为嗜麦芽寡养单胞菌（Stenotrophomonas maltophilia）,其可耐亚硒酸钠浓度为17000 μg mL?1,在温度20 ~ 35 ℃、pH 5 ~ 9、盐浓度0 ~ 3%条件下可正常生长;真菌B-2被鉴定为聚多曲霉（Aspergillus sydowii）,该菌株可耐亚硒酸钠浓度为6000 μg mL?1,在温度25 ~ 30 ℃、pH 4 ~ 7、盐浓度2% ~ 5%条件下可正常生长。添加两耐硒菌株培养后土壤的可溶态硒和可交换态硒含量均显著提高。  结论  筛选出具有较高的硒耐受性和较强的活化酸性富硒土壤中硒的能力的菌株,可为富硒土壤资源开发利用提供生物种质资源和技术手段,对富硒微生物肥料研发、富硒农产品生产和硒污染土壤环境修复也有重要意义。     

一株高效还原亚硒酸盐的粘质沙雷氏菌的分离鉴定及特性

从广西钦州市钦北区的富硒土壤中筛选出一株可高效还原亚硒酸盐的细菌QZB-1。16S rDNA基因序列分析确定菌株为粘质沙雷氏菌(Serratia marcescens)。研究发现菌株QZB-1可耐高达180 mmol/L的亚硒酸盐。进一步研究发现菌株QZB-1在36 h之内对1 mmol/L亚硒酸盐的还原率为95%，随着硒浓度升高还原率有所下降。正交实验表明，对菌株还原亚硒酸盐的影响程度依次是初始亚硒酸盐浓度>培养时间>接种量，最佳还原条件是初始亚硒酸盐浓度为1 mmol/L，接种量为10%，培养时间为36 h，此时菌株QZB-1对亚硒酸盐的还原率大于98.55%。研究结果表明新型亚硒酸盐还原菌粘质沙雷氏菌QZB-1可高效还原亚硒酸盐为单质纳米硒，可高效应用于亚硒酸盐污染水体和土壤的治理。     

桑肠杆菌菌株的富硒特性及其喷施对玉米籽粒的硒素强化

硒是人体必需微量元素之一,对新陈代谢过程有十分重要的促进作用,通过生物强化措施可以增加人体对硒素的补充。该研究利用从植物内生菌中筛选出的一株具有将无机硒转化成生物利用率更高的有机硒的菌株IX+2 2,测定其16S r RNA基因序列,鉴定该菌株为桑肠杆菌(Enterobacter mori)。该文研究不同的硒含量、加硒时间与不同培养收集时间对菌株IX+2 2硒素转化率的影响。结果表明,培养基中硒含量越高,菌株IX+2 2胞内有机硒含量越高,硒素转化率越高,但达到某一个峰值后又开始下降;在对数生长期加硒有利于菌株IX+2 2的生长和对无机硒的转化;菌株培养24 h时收集硒素转化效果最好,继续培养菌体生长减弱。同时针对菌株对玉米籽粒富硒的强化效应做了进一步验证。该菌株对玉米籽粒富硒的强化作用表明:同等亚硒酸钠用量条件下,亚硒酸钠喷施处理后玉米籽粒中硒含量达到228.58μg/kg,而富硒微生物菌剂处理后籽粒中硒含量达到378.89μg/kg,是前者的1.66倍,富硒IX+2 2菌剂的富硒效果优于喷施亚硒酸钠溶液。该研究结果可为玉米籽粒的硒素强化提供理论支撑。     

施硒对紫云英生长、生理和硒积累特性的影响

通过盆栽试验,系统地研究了紫云英生长过程中对不同土壤硒浓度(0,0.125,1.25,2.5和5 mg kg~(-1))处理的生理生态响应以及不同土壤硒浓度处理下紫云英各部位硒的赋存形态,以期为紫云英的进一步综合开发利用提供科学理论依据。研究表明,随着土壤施硒浓度的升高,紫云英各项生长指标(株高、茎粗、根长、各部分鲜重和干重)呈先升后降趋势,土壤施硒浓度为0.125 mg kg~(-1)时,其各项生长指标达到最大值。随着土壤施硒浓度的增加,SOD活性、POD活性、可溶性蛋白和叶绿素含量均呈现先降后升再降的变化趋势;CAT活性呈先降后升的趋势;脯氨酸含量呈上升的趋势;可溶性糖含量则呈逐渐下降的趋势。随着施硒培养时间的延长,各处理组SOD活性和脯氨酸含量主要表现为先降后升再降的趋势;POD活性、可溶性糖、可溶性蛋白以及叶绿素含量主要表现为先升后降的趋势;CAT活性主要表现为先降后升的趋势。在土壤施硒浓度为0~5 mg kg~(-1)下,紫云英根部的硒含量为0.14~150.87 mg kg~(-1),地上部分硒含量为0.04~215.17 mg kg~(-1),其中,土壤施硒浓度≥2.5 mg kg~(-1)时,地上部分含硒量高于根部。硒在紫云英中主要以有机态(Se Met、Me Se Cys和Se Cys2)存在,占提取液总硒的81.43%~93.05%。综合研究结果认为,紫云英适合被开发为高硒材料,实际生产中以土壤施硒浓度为2.5 mg kg~(-1)为宜。     

车前草根际土壤细菌的筛选鉴定与功能初探

从车前草(Plantago asiatica L.)根际土壤中筛选发现潜在的具有活硒能力、抑镉能力或二者能力兼之的细菌菌株。首先,采用稀释涂布平板法从车前草根际土壤中分离纯化出耐硒耐镉细菌;将分离得到的耐硒耐镉细菌进行16S rRNA基因鉴定;然后,利用土壤固体培养基对细菌进行培养,并用氢化物发生原子荧光光谱法和电感耦合等离子体发射光谱法分别测定不同细菌对应土壤的有效硒和有效镉,计算出菌株活硒率和抑镉率;最后,初步评价菌株对土壤硒或镉活化或抑制的能力。试验结果从车前草根际土壤中分离鉴定二级细菌8株,其中活硒率超过100%的菌株有CQC-3、CQC-8、CQC-11、CQC-12,抑镉率超过20%的菌株有CQC-7、CQC-8、CQC-12、CQC-13,活硒抑镉能力兼具的菌株有CQC-8和CQC-12,此研究结果为后续解决土壤和植物硒镉伴生问题、活硒抑镉机理研究和菌剂的开发提供基础资料。     

响应面法优化制备高富硒产朊假丝酵母

考察了发酵条件对产朊假丝酵母富硒能力的影响。通过单因素的筛选,对酵母富硒能力影响较大的3个因素:亚硒酸钠浓度、初始p H值及培养温度,以胞内总硒含量为响应值,利用响应面法对其进行优化。结果显示:在培养时间30 h、加硒时间对数生长中期、亚硒酸钠浓度35 mg·L-1,初始p H 6.6、接种量10%、培养温度27℃、装液量150 m L/500 m L的条件下,最大的菌体生物量为6.87 g·L-1;胞内总硒含量达到12 639.7μg·L-1,硒含量为1 839.8μg·g-1,其中亚硒酸钠转化率为79.1%,有机硒含量占90%以上;胞内实际总硒含量与数学模型理论值12 518.8μg·L-1相差不显著,响应面法能较好地优化产朊假丝酵母富硒工艺条件。     

施用生物炭对红壤富硒区硒生物有效性的影响

研究生物炭调理措施对红壤区富硒土壤硒形态及硒生物有效性的影响,为富硒土壤硒活化及硒资源高效利用提供理论依据。通过盆栽试验,连续开展三批玉米苗期研究,设置3 个生物炭添加水平:土壤质量0.5%(T1)、1.0%(T2)和1.5%(T3),以不添加生物炭处理为对照(CK),分析生物炭添加对土壤硒形态及玉米硒素营养的影响。结果表明:施用生物炭后,T1、T2 和T3 的可溶态硒分别平均提高0.46、0.42 和0.43 个百分点;可交换态硒分别平均提高0.61、1.66 和1.50 个百分点;降低了铁锰氧化物结合态硒的比例;有机结合态硒比例先降低后逐渐提高,残渣态硒比例则先提高后逐渐降低。玉米根系硒含量与土壤可溶态、可交换态及有效硒含量均呈显著线性正相关,玉米茎叶硒含量与土壤各硒形态之间的相关性不显著。施用生物炭能提高玉米植株体内的硒累积量,T1、T2 和T3 处理玉米植株平均硒累积量分别比CK 提高9.46%、31.00% 和21.22%。可见,在红壤上施用生物炭能有效提高土壤硒的生物有效性并促进硒在植物体内的累积,其中以添加土壤质量 1.0% 的生物炭效果最好。     

施硒对暗棕壤硒状况和森林蔬菜硒积累的影响

以培育富硒森林蔬菜为目标,在暗棕壤上进行了荚果蕨、小叶芹、小根蒜、婆婆丁等4种山野菜施硒试验。硒肥种类为亚硒酸钠,施硒量为0.10~10.00mg kg-1,直接施肥范围为0~15cm表土。经过一个完整的生长周期(1a)后测定土壤和植物硒指标。结果表明,典型暗棕壤对外源亚硒酸钠态硒具有较强吸收与保持能力,从低剂量至高剂量施入的硒有90%~70%以上仍保留在原施硒土层内;其中H2O可溶态硒占全硒的6.03%~3.78%,KH2PO4浸提的有效态硒占全硒的19.36%~37.54%,而且土壤全硒、KH2PO4浸提的有效态硒及H2O可溶态硒皆与最初的施硒量呈线性正相关。在土施强化供硒条件下,4种山野菜均能高量富集硒,其含硒量为对照的1.7~158.9倍;不同植物的富硒能力有差异,它们在递增施硒情况下高量富集硒的变化模式也各有特点。总体来看,山野菜含硒量与施硒量之间为非线性正相关关系,但在一定的较低施硒量范围内这种关系则是线性的,线性范围因植物而异;土壤的可溶态硒、KH2PO4浸提的有效硒甚至全硒等指标与山野菜含硒量之间亦均呈明显的非线性正相关,且施硒量较低时其关系亦表现为线性。初步得出,典型暗棕壤控制施硒是在近自然条件下培育富硒森林蔬菜并获取高富硒生物资源的有效途径;不过,强烈搅动的砂砾质暗棕壤却不利于硒肥保持和植物吸收,不适于富硒森林蔬菜栽培。     

基于GIS的南京市溧水区富硒土地资源开发利用研究

[目的]对南京市溧水区富硒土地资源开发利用进行研究,为发展该区特色富硒农产品产业提供科学参考。[方法]在对溧水全区土壤采样并测试土壤化学性质的基础上,采用ArcGIS对土壤硒含量进行地统计和插值分析,选取最合适的拟合模型和插值方法,根据样点数据预测整个研究区土壤含硒情况,综合文献资料和溧水实际情况将富硒土地分级,明确富硒土地空间分布,结合土壤酸碱度划分出适宜种植富硒农产品的区域及种类。[结果]溧水区约有4.25%的富硒土地资源,呈块状或点状分布于各个乡镇,可以综合土壤酸碱度等因素开发作为富硒农产品的种植地。[结论]普通克里金方法中高斯模型可以很好地拟合出溧水区土壤富硒情况,并且研究区的富硒土地资源具有极大的开发空间和利用价值,需要进行合理规划,为开发区域特色农产品市场提供依据。     

一株红壤花生根际溶磷真菌的分离、鉴定及溶磷能力的研究

为提高磷素利用率,从花生根际土壤样品中筛选到一株溶磷能力强的菌株B1-A,结合菌落形态特征和ITS rDNA序列分析将其鉴定为黑曲霉(Aspergillus niger)。菌株B1-A的不同接种量(1%,3%,5%)在两种难溶性磷酸盐(FePO4,AlPO4)培养液的溶磷量和pH随时间动态变化表明溶磷量均与pH呈显著负相关,B1-A的溶磷量与接种量、培养时间、磷矿粉浓度有关,实验结果表明菌株B1-A在接种量为3%～5%、培养时间168 h、云南磷矿粉浓度为3 g L-1、江西磷矿粉浓度为5 g L-1时溶磷效果较好。菌株B1-A对磷酸三钙、磷酸铝、磷酸铁、磷矿粉有较强的溶解能力,最高溶磷量分别为418.7、942.3、242.2、177.4 mg L-1,有望为开发高效红壤微生物磷肥提供种质资源。     

小麦硒敏感性的基因型差异分析

土壤中硒分布不均匀,富硒土壤中硒含量可达1 200 mg/kg,硒含量过高对大多数植物均有毒害作用。小麦是我国重要的主要粮食作物,筛选硒耐性强的小麦品种有利于实现小麦在富硒地区更好的推广。本研究选择99份小麦品种(系)进行盆栽试验,在拔节期进行处理设置(硒处理每盆硒酸钠用量120 mg/L,记作Se120;以硒酸钠0 mg/L处理为对照,记作CK),比较不同品种(系)小麦硒处理下农艺性状的变化。结果表明:与CK相比,过量施硒后小麦籽粒千粒重显著提高(P0.01),株高、有效叶面积、单株叶片数和功能叶片数均显著下降(P0.01),SPAD值(叶绿素相对含量)则没有显著变化。以Se120和CK之间农艺性状的差异作为小麦的耐硒性指标,通过马氏距离综合评估不同小麦品种对硒的耐受性和敏感性差异,结合聚类分析,发现X325、淮麦18、扬麦15号、淮9440、扬麦11是典型的耐硒品种,而扬麦2号和Corrine是典型的硒敏感型品种。     

Uptake and remobilization of selenium in Brassica napus L. plants supplied with selenate or selenium‐enriched plant residues

Selenium (Se) biofortification via crops is one of the best strategies to elevate the daily Se intake in areas where soil Se levels are low. However, Se fertilizer recovery (SeFR) is low and most of the Se taken up accumulates in non‐harvested plant parts and returns to the soil with plant residues. A pot experiment with soil was undertaken to study the efficiency of inorganic Se (Na₂SeO₄) and Se‐enriched plant residues for biofortification, as well as to identify the bottlenecks in Se utilization by Brassica napus L. The soil was fertilized with Na₂SeO₄ (0 and 7 µg Se kg^?1) or with Se in stem or leaf residues (0 and 7 µg Se kg^?1). A treatment with autoclaved soil was included (0 and 7 µg kg^?1 as Na₂SeO₄) to unravel the impact of microbial activity on Se uptake. The Se‐enriched plant residues produced a lower Se uptake efficiency (SeUPE) and SeFR than did inorganic Se, and soil autoclaving enhanced Se accumulation in the plants. The time required for decomposition seems to preclude crop residues as an alternative source of Se. Furthermore, B. napus had a limited capacity to accumulate Se in seeds. The study shows that the bottlenecks in Se biofortification appear to be its low bioavailability in soil and poor loading from the silique walls to seeds. Thus, improved Se translocation to seeds would be a useful breeding goal in B. napus to increase SeFR.     

黑麦草对硒的吸收、分配与累积

用营养液培养方法研究了黑麦草(Lolium.multiflorum,Lamaubada)对硒的吸收、分配和累积特征以及硒对黑麦草生物量的影响,以探讨黑麦草对硒的吸收和利用规律。结果表明,不同SeO32--Se浓度处理的黑麦草在不同生育期的硒含量大小顺序均表现为分蘗期拔节期抽穗期苗期,且其差异在低硒浓度([Se4+]0.05.mg／L)时表现不明显,在非中毒范围内,黑麦草体内含硒量随供硒水平的提高而增加。在不同硒浓度处理中,黑麦草不同器官的硒含量大小顺序均为根叶茎,黑麦草吸收的SeO32--Se大部分在根部累积,少部分转移到茎与叶中,SeR／SeL3,SeR／Ses4。当营养液中加入低浓度硒([Se4+]=0-0.1.mg／L)时促进了黑麦草的生长,其生物量随外源硒浓度的增大而增加;高浓度硒([Se4+]1.0.mg／L)时抑制黑麦草的生长甚至引起中毒死亡。抽穗期黑麦草对硒的阶段累积率最大,达40.42％。     

Selenium accumulation in a rapid‐cycling Brassica oleracea population responds to increasing sodium seienate concentrations

Because of their short life cycle, rapid‐cycling base populations (RCBP) of Brassica can act as model systems for investigating selenium (Se) metabolism in high sulfur (S) accumulating plants. To establish treatment responses for a B. oleracea RCBP, plants were grown in nutrient solutions containing 0, 3, 6, and 9 mg sodium seienate (Na₂SeO₄) L^‐1. Depletion of Se from nutrient solutions increased linearly with in‐creasing Na₂SeO₄ concentrations. Selenium accumulation ranged from 551 to 1,916 μg Se g^‐1 dry weight for leaf tissue, 267 to 1,165 μg Se g^‐1 dry weight for stem tissue, and 338 to 1,636 μg Se g^‐1 dry weight for root tissue. Selenium additions also resulted in linear increases in S accumulation in leaves and stems. Selenium supplementation has been shown to improve the health of individuals with low Se status. Because Brassica species are important vegetable and forage crops, their enrichment with Se maybe a good delivery system for mammalian diets.     

The Positive Effect of Sulfur on Selenium Detoxification Under Selenite Condition in Wheat

An experiment was performed to evaluate the regulatory effects of varied amounts of sulfur (S) on selenium (Se) uptake and distribution within wheat (Triticum aestivum L.) in response to selenite (SeO₃^2-). The results showed that an appropriate amount of SeO₃^2- (≤5 mg kg^?1) improved the agronomic traits of wheat, and the addition of S significantly alleviated wheat growth inhibition caused by excessive SeO₃^2- (≥15 mg kg^?1). The Se concentration of different fractions in wheat grain showed a tendency of alkali-soluble Se > alcohol-soluble Se > water-soluble Se > salt-soluble Se. The use of S significantly reduced Se accumulation in each wheat part when the addition of SeO₃^2- was ≤15 mg kg^?1, but the Se distribution ratio of various wheat parts was not influenced by S supply. Overall, the application of S fertilizer is an effective technical measure to promote wheat production safety in high-Se areas.     

Sorption behavior of selenium and antimony in soils as a function of phosphate ion concentration

Abstract

Both selenium (Se) and antimony (Sb) are major soil and water pollutants. Their sorption behavior in a soil–plant system was studied. Soil–soil solution distribution coefficients (K _ds) for Se and Sb were measured, using a radiotracer, as an indicator of their sorption levels. Both Se and Sb behave as oxoanions (SeO^2? ₄, H₂PO^? ₄ and SO^2? ₄) in soil; thus, the effects of concentrations of two major oxoanions (SeO^2? ₄ and SeO^2? ₃) on Se and Sb sorption were also examined. The K _d values for Se for Japanese soils significantly correlated with the K _d values for Sb (n = 141). The K _ds of both Se and Sb similarly decreased with increasing SbO^? ₃ concentration. These results indicated that the sorption of Se and Sb was similarly controlled by a ligand-exchange mechanism such as phosphate sorption in soil. However, an increase in the concentration of SeO^2? ₃ did not decrease the K _ds of Se and Sb. Furthermore, the ligand-exchangeable fractions of stable Se and Sb in major Japanese soils were determined by extraction with 0.1 mol L^?1 Na₂HPO₄ solution. For both Se and Sb, the phosphate-extractable fractions were 10-fold higher for Se and fivefold higher for Sb than their water-soluble fractions. Although the total Se and Sb amounts in soils were the same, their ligand-exchangeable fractions were different. Approximately 0.9–12% of total Se and 0.2–1.3% of total Sb were extracted by the phosphate solution. These findings suggested that Se was more likely to be mobilized by the addition of phosphate than Sb. The effect of plant-available phosphate in the soil and the phosphate sorption capacity of soil on Se and Sb availabilities for plants were also examined using a pot experiment with soybean plants. The experimental results suggested that a high content of available phosphate and/or low phosphate sorption capacity of soil increased both Se and Sb availabilities to the plant. However, the results also suggested that the soil Se availability to the plant was higher than that of Sb even though the soil total Se and Sb amounts were the same.     

Effects of pH and selenium oxidation state on the selenium accumulation and yield of alfalfa

Experiments were performed in greenhouse sand culture to determine the effect of pH and Se oxidation state on the tissue composition and yield of alfalfa (Medicago sativa L.). Alfalfa was planted and irrigated with nutrient solution containing 0, 0.25, 0.5, 1.0 or 3.0 mg Se L^‐1 as Na₂SeO₃ or Na₂SeO_l4. The solution pH was maintained at 7.0 ± 0.2 or 4.5±0.2. Three harvests were made and the shoots and roots weighed and analyzed for total Se. At the levels tested, additions of Se to the treatment solutions increased the tissue Se concentrations and depressed alfalfa shoot growth regardless of Se oxidation state or pH. Root growth was reduced in the presence of Se(VI) but was unaffected by Se(IV). Selenium was accumulated to higher concentrations in the shoot when supplied as Se(VI) compared to Se(IV) and accumulated to higher concentrations in the root when added as Se(IV) compared to Se(VI). Relative shoot yields were depressed more by Se(IV) then Se(VI) at pH 7, but there was no difference between Se(IV) and Se(VI) at pH 4.5. Relative root yields were depressed by Se(VI) but not by Se(IV).     

NUTRITIONAL BALANCE CHANGES IN LETTUCE PLANT GROWN UNDER DIFFERENT DOSES AND FORMS OF SELENIUM

The mineral composition and maintenance of mineral balance are important to growth and development of plants. The selenium (Se) has not been described as an essential element for plants, although there are studies that have demonstrated to interaction between Se with other mineral nutrients. The aim was to evaluate the influence that Se application at different rates and forms exerts on the nutritional state in lettuce plants. The plants were grown under different treatments: 5, 10, 20, 40, 60, 80, 120 μmol L^?1 as sodium selenate [Na₂SeO₄ or Na₂SeO₃]. All the plants growth under controlled conditions. The results showed changes in some of the essential nutrients inside of plants such as nitrogen (N), phosphorous (P), iron (Fe), copper (Cu), calcium (Ca). The effect of Se depended largely on the Se from was applied to the culture medium. Thus, the selenite application had a stronger effect on the nutritional state of the plant.     

Effect of soil amendments on fractionation of Selenium‐enriched soil

Abstract

This study was to determine the effect of soil amendments on the fractionation of selenium (Se) using incubation experiments under simulated upland and flooded conditions. The treatments were as follows: 1) control [soil + sodium selenite (Na₂SeO₃) (1 mg Se kg^‐1)]; 2) control + calcium carbonate (CaCO₃) (5 g kg^‐1); 3) control + alfalfa (40 g kg^‐1); and 4) control + CaCO₃ (5 g kg^‐1) + alfalfa (40 g kg^‐1). After a 90‐day incubation, soil was sampled and fractionated into five fractions: 1) potassium sulfate (K₂SO₄)‐soluble fraction (available to plants); 2) potassium dihydrogen phosphate (KH₂PO₄)‐exchangeable fraction (potentially available); 3) ammonium hydroxide (NH₃H₂O)‐soluble fraction (potentially available); 4) hydrochloric acid (HCl)‐extractable fraction (unavailable); and 5) residual fraction (unavailable). Compared with the control, CaCO₃ increased the K₂SO₄ fraction at the expense of the NH₃H₂O fraction. Alfalfa increased both the K₂SO₄ and residual fractions but reduced the KH₂PO₄ and NH₃H₂O fractions. The CaCO₃‐alfalfa treatment had a similar effect to the alfalfa treatment alone. The comparison between the upland and flooded conditions showed that the flooded condition generally increased the residual fraction and decreased the potentially‐available fractions. In general, CaCO₃ was a better amendment because it not only increased the available fraction but also maintained the potentially available fractions at a high level. The application of Na₂SeO₃ and use of appropriate soil amendments can improve Se availability in soil.