Spatial dependency of soil nutrient availability and microbial properties in a mixed forest of Tsuga heterophylla and Pseudotsuga menziesii, in coastal British Columbia, Canada

Spatial variations in nutrient concentrations and turnover may contribute to variations in productivity within forest ecosystems and be responsible for creating and maintaining diversity of plant species. The aim of this study was to relate spatial patterns in soil nutrient availability and microbial properties in the forest floor and mineral soil in order to explore the controls on variations in nutrient availability in the two horizons. Microbial C, N and P, extractable N and P, and plant-available nutrients as estimated by plant root simulator probes were measured. We then used geostatistical techniques to determine the scale of spatial autocorrelation in the forest floor and mineral soil. Several of the measured variables were spatially autocorrelated at a scale of tens to hundreds of meters. Although more variables were autocorrelated in the mineral soil than in the forest floor, spatial patterns in gravimetric moisture content and nutrient concentrations in the two horizons generally overlapped. The spatial patterns were probably caused by differences in topography, except for ammonium whose shorter range of spatial autocorrelation may reflect variations in nitrogen content of the canopy.     

鉴别非洲猪瘟病毒和猪瘟病毒野毒株二重TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用

旨在建立特异、敏感的实时荧光定量PCR（FQ-PCR）方法,用于非洲猪瘟病毒（ASFV）和猪瘟病毒（CSFV）野毒株的快速鉴别检测。针对ASFV的P72基因和CSFV野毒株的5'UTR非编码区序列的保守区域分别设计1对特异性引物和1条探针,经优化反应条件,建立一种基于TaqMan MGB探针技术的FQ-PCR方法,验证方法的敏感性、特异性和稳定性,对50份临床样品进行检测,并与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法进行比较分析。结果显示：建立的鉴别ASFV和CSFV野毒株二重FQ-PCR检测方法在10⁰~10⁶拷贝·μL^-1模板范围内有良好的线性关系;对ASFV和CSFV基因出现阳性扩增信号,但对猪瘟病毒疫苗株、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型、猪细小病毒、猪乙型脑炎病毒、副猪嗜血杆菌等病原对照未出现扩增;批内、批间试验变异系数在1.18%~2.08%,重复性良好;对ASFV和CSFV的最低检测模板浓度均为10拷贝·μL^-1;利用建立的二重FQ-PCR方法对50份临床样品进行检测,检测结果与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法结果完全一致。本研究成功建立了鉴别ASFV和CSFV野毒株二重TaqMan MGB FQ-PCR方法,为ASFV和CSFV野毒株的鉴别诊断提供了快速、敏感、特异且能满足临床检测需求的检测方法。     

反刍动物埃立克体荧光定量PCR检测技术的建立和应用

为快速、准确地检测反刍动物埃立克体,本研究以反刍动物埃立克体pCS20为靶基因设计特异性引物和探针,建立了TaqMan和Eva Green荧光定量PCR方法,对其反应的特异性、敏感性和重复性进行了分析,并与OIE推荐的套式PCR方法一起对临床样品进行检测。结果显示,本方法特异性强,与牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体无交叉反应;TaqMan和Eva Green荧光定量PCR对pCS20质粒标准品的最低检测限分别为17.4拷贝·μL^-1和1.74拷贝·μL^-1,标准曲线相关系数大于0.99,组内和组间CV均小于1.5%。对420只钝眼蜱样本的检测显示,TaqMan和Eva Green荧光定量PCR的检出率分别为25.48%和29.29%,与套式PCR检测方法相比,敏感性更高。本研究为反刍动物埃立克体的检测和流行病学调查提供了一种快速、准确的检测方法。     

Digoxigenin(DIG)标记RNA探针检测侵染花生的Potyviruses

以花生条纹病毒(PStV)和花生斑驳病毒(PMV)克隆cDNA为模板,体外转录合成Digo-xgenin(DIG)标记PStV—I_9、PStV-I_(131)、PStV—I_(57-7)和PMV—A_(15)四种RNA探针。在点杂交反应中,PStV—I_9RNA探针能检测出0.25ng提纯PStV和花生病汁液62500倍稀释液中PStV。PMV—A_(16)RNA探针能检测到0.67ng提纯PMV和菜豆病汁液5120倍稀释液中PMV。两种病毒RNA探针均有高度特异性。PStV—I_(57-7)和PStV—I_(131)两种RNA探针具有与PStV—I_9RNA探针相同敏感性,但特异性稍差。     

食源性动物组织中CSFV和PRRSV双重双色荧光定量RT-PCR检测方法的建立及应用

为建立同时检检测食源性动物组织中猪瘟病毒(CSFV)和猪蓝耳病病毒(PRRSV)的双色荧光定量RT-PCR方法。本研究根据SCFV和PRRSV基因序列设计特异性的引物和不同荧光标记的TaqMan荧光探针,通过优化反应的体系和扩增条件,建立了能够检测食源性动物组织中SCFV和PRRSV的双重双色荧光定量PCR的方法。其检测下限为1×102拷贝/μL,而且与其他一些猪病病毒无交叉反应,具有良好的特异性。该方法重复性和稳定性试验表明其组内和组间的变异系数最高分别为4.2和4.5。对比试验表明,该方法对CSFV和PRRSV检验的敏感性为常规RT-PCR方法的200倍;该方法的建立为食源性动物组织中CSFV和PRRSV提供了有效手段,该方法特异性和敏感性较好,能够应用于临床检测。     

梅迪-维斯纳病毒实时荧光定量PCR检测方法的建立

为建立检测梅迪-维斯纳病毒(MVV)的TaqMan实时荧光定量PCR方法,本研究根据MVV核苷酸保守序列设计引物和探针。以梯度稀释的含有MVV目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示:5.0×105～5.0×101拷贝范围内定量PCR均有S型扩增曲线,检测灵敏度为50拷贝。对羊的其他病毒核酸均无扩增反应。本研究建立的实时定量PCR方法,灵敏度高,特异性好,在MVV的快速检测中具有良好的应用前景。     

利用实时荧光定量PCR技术检测樱桃叶斑驳病毒

樱桃叶斑驳病毒(Cherry mottle leaf virus,CMLV)是南美洲樱桃上重要的病害之一。本研究针对Genbank上公布的该病毒的核酸序列,人工合成了CMLV(AF170028.1)6741～7322 nt的核酸序列并连接载体,构建了阳性标准质粒,进行实时荧光定量PCR(Fluorescence quantitative,FQ-PCR)检测。实验结果表明,本研究设计的利用FQ-PCR检测CMLV的引物及探针特异性良好,经灵敏度检测,最低检测浓度为23 copies/μL,比普通PCR检测灵敏度高100倍。该FQ-PCR检测方法为樱桃叶斑驳病毒的防控提供了重要的技术支持。     

猪圆环病毒2型TaqMan荧光定量PCR检测方法的建立与初步应用

参照GenBank收录的猪圆环病毒2型(PCV2)基因组全序列选择保守区域设计合成了引物及其探针,并对其进行了筛选;对荧光定量PCR的反应条件进行了优化,建立了TaqM an荧光定量PCR检测方法.用建立的检测方法对临床采集的组织病料进行了检测,并与常规PCR检测方法作比较.结果显示,所建立的方法灵敏度可达3×101拷贝.μL-1,比常规PCR检测方法灵敏度高10倍,而与猪圆环病毒1型(PCV1)、猪伪狂犬病毒(PRV)、猪瘟病毒(CSFV)、猪繁殖和呼吸综合征病毒(PRRSV)没有交叉反应,具有灵敏度高、特异性强、操作简便等优点,适合于PCV2的流行病学调查和临床诊断.     

实时荧光定量TaqMan-PCR检测鸭瘟病毒方法的建立

根据GenBank中鸭瘟病毒UL6和UL7基因的序列,设计了一对特异性引物及TaqMan探针,扩增位于UL6和UL7基因第891￣991位长度为101bp片段。以DPV标准强株DNA为模板,建立了实时荧光定量PCR检测鸭瘟病毒的方法。该方法只能在鸭瘟病毒DNA样本中检出荧光信号,最小检出量为1.57×102copies(23.7fg);线性范围达8个数量级;平行复管检测,组内变异系数为0.84%(n=20),同一组样本(n=6)间隔30d重复检测,结果无显著性差异;对试验感染鸭肝脏和脑组织中鸭瘟病毒的检出率为100%(40/40),对临床病例的检测结果与病毒分离鉴定结果一致;从核酸提取到报告检测结果仅需3h;对鸭正常组织、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、鸡源大肠杆菌、鸭源多杀性巴氏杆菌、鸭副伤寒沙门氏菌等非鸭瘟病毒DNA检测,不出现非特异性荧光信号。     

The effect of nitrogen fertilization and no‐till duration on soil nitrogen supply power and post–spring thaw greenhouse‐gas emissions

With a world population now > 7 billion, it is imperative to conserve the arable land base, which is increasingly being leveraged by global demands for producing food, feed, fiber, fuel, and facilities (i.e., infra‐structure needs). The objective of this study was to determine the effect of varying fertilizer‐N rates on soil N availability, mineralization, and CO₂ and N₂O emissions of soils collected at adjacent locations with contrasting management histories: native prairie, short‐term (10 y), and long‐term (32 y) no‐till continuous‐cropping systems receiving five fertilizer‐N rates (0, 30, 60, 90, and 120 kg N ha^–1) for the previous 9 y on the same plots. Intact soil cores were collected from each site after snowmelt, maintained at field capacity, and incubated at 20°C for 6 weeks. Weekly assessments of soil nutrient availability along with CO₂ and N₂O emissions were completed. There was no difference in cumulative soil N supply between the unfertilized long‐term no‐till and native prairie soils, while annual fertilizer‐N additions of 120 kg N ha^–1 were required to restore the N‐supplying power of the short‐term no‐till soil to that of the undisturbed native prairie soil. The estimated cumulative CO₂‐C and N₂O‐N emissions among soils ranged from 231.8–474.7 g m^–2 to 183.9–862.5 mg m^–2, respectively. Highest CO₂ fluxes from the native prairie soil are consistent with its high organic matter content, elevated microbial activity, and contributions from root respiration. Repeated applications of ≥ 60 kg N ha^–1 resulted in greater residual inorganic‐N levels in the long‐term no‐till soil, which supported larger N₂O fluxes compared to the unfertilized control. The native prairie soil N₂O emissions were equal to those from both short‐ and long‐term no‐till soils receiving repeated fertilizer‐N applications at typical agronomic rates (e.g., 90 kg N ha^–1). Eighty‐eight percent of the native soil N₂O flux was emitted during the first 2 weeks and is probably characteristic of rapid denitrification rates during the dormant vegetative period after snowmelt within temperate native grasslands. There was a strong correlation (R² 0.64; p < 0.03) between measured soil Fe‐supply rate and N₂O flux, presumably due to anoxic microsites within soil aggregates resulting from increased microbial activity. The use of modern no‐till continuous diversified cropping systems, along with application of fertilizer N, enhances the soil N‐supplying power over the long‐term through the build‐up of mineralizable N and appears to be an effective management strategy for improving degraded soils, thus enhancing the productive capacity of agricultural ecosystems. However, accounting for N₂O emissions concomitant with repeated fertilizer‐N applications is imperative for properly assessing the net global warming potential of any land‐management system.