BIOLOG在土壤微生物群落功能多样性研究中的应用

微生物功能多样性信息对于明确不同环境中微生物群落的作用具有重要意义 ,而微生物群落的定量描述一直是微生物学家面临的最艰巨的任务之一。目前 ,以群落水平碳源利用类型为基础的BIOLOG氧化还原技术为研究土壤微生物群落功能多样性提供了一种简单、快速的方法 ,并得以广泛应用。但它仍然是一种以培养为基础的方法 ,显示的代谢多样性类型也不一定反映整个土壤微生物群落的功能多样性。因此 ,这种方法优点明显 ,缺陷也存在 ,并且在应用过程中还有很多关键的操作要点与技巧。本文综述了BIOLOG研究土壤微生物群落功能多样性的原理、BIOLOG研究土壤微生物群落功能多样性的方法与技巧、应用过程中容易产生的问题及可能克服的办法 ,同时还提出了值得进一步研究的问题。旨在促进对BIOLOG测定土壤微生物群落功能多样性的了解 ,为正确运用这种方法开展土壤微生物群落功能多样性研究提供科学依据和理论指导。     

生物复混肥对土壤微生物功能多样性及土壤酶活性的影响

在温室盆栽条件下,采用Biolog微平板技术,研究了玉米施用等养分量的无机肥、有机无机复混肥、生物复混肥后土壤微生物群落功能多样性及土壤酶活性的动态变化。结果表明,生物复混肥处理的微生物群落平均颜色变化率（AWCD）、微生物群落Shannon指数（H）、丰富度指数（S）和Shannon均匀度指数（E）均为最高;微生物群落主成分分析表明,不同施肥处理土壤微生物群落碳源利用特征有一定差异,PC1将生物复混肥与其他处理明显区分,生物复混肥处理分布在PC1的正方向,其他处理分布在PC1的负方向;起分异作用的主要碳源有糖类、羧酸类和氨基酸类;土壤蔗糖酶、脲酶活性均以生物复混肥处理最高,分别为72.74 mg glucose·g-1·（24 h）-1和1.15 mg NH3-N·g-1·（3 h）-1。研究表明,生物复混肥的施用比等养分量的有机无机复混肥处理能显著提高土壤微生物群落碳源利用率、微生物群落的丰富度和功能多样性,增强土壤蔗糖酶和脲酶活性。     

自生固氮菌对土壤钾的活化作用

以土壤为钾源,通过液培试验研究了5株自生固氮菌(Azotobacter sp.)对土壤钾的活化作用。结果表明,自生固氮菌能分泌大量的氢离子,大幅度降低培养液p H,使氢离子浓度提高40倍以上。自生固氮菌分泌有机酸的种类与数量因菌株不同而异,这些有机酸包括甲酸、乙酸、草酸、丁二酸、柠檬酸、苹果酸和乳酸等。其中,全部供试菌株均能分泌草酸和苹果酸。在自生固氮菌的培养液中,钾浓度显著高于未接种的培养液,但土壤矿物结构钾则显著降低。由于土壤是培养液钾的唯一来源,说明自生固氮菌可促进土壤矿物钾溶解。相关分析表明,土壤矿物结构钾与自生固氮菌的有机酸分泌总量呈显著负相关(r=-0.845*,n=6),与培养液p H呈显著正相关(r=0.702*,n=6),说明自生固氮菌分泌的有机酸和氢离子可能溶解土壤矿物钾。考虑到自生固氮菌的草酸分泌量最大,络合钙、镁、铁、铝的能力最强,且与有机酸分泌总量呈极显著正相关(r=0.990**,n=6),推测草酸的分泌在活化土壤无效钾的过程中起重要作用。接种自生固氮菌显著降低土壤无效钾,活化能力因菌株不同而异,其原因可能与有机酸分泌的数量和种类有关。     

荔枝园间作平托花生对土壤理化性质、酶活性和细菌群落结构及多样性的影响

研究荔枝间作平托花生对荔枝园土壤理化性质、酶活性及细菌多样性的影响,为荔枝健康栽培提供理论依据和技术支撑。分别选取荔枝/平托花生间作和荔枝单作模式下10～20 cm土层的土壤样本,测定酸性有效磷、全磷和速效钾等理化因子的含量以及蔗糖酶、蛋白酶和脲酶等酶活性,并在此基础上,利用Illumina MiSeq测序平台对荔枝/平托花生间作模式和荔枝单作模式下土壤的细菌群落进行16S rRNA V4～V5区检测。结果表明:与荔枝单作模式相比,在理化因子方面,荔枝间作平托花生模式下的速效钾含量极显著提高138.9%,碱解氮含量显著降低19.6%,pH稍降低但无显著差异性;在土壤酶活性方面,蔗糖酶、酸性蛋白酶、脲酶、过氧化氢酶和多酚氧化酶均极显著或显著提高;在土壤细菌群落多样性方面,Chao1、ACE和Simpson指数分别显著提高5.5%、5.2%和3.7%。在土壤细菌群落结构方面,两组样本的主要优势菌门为酸杆菌门、变形菌门、绿弯菌门和放线菌门;主要优势菌属为Gp1、Gp2、Gp3、Gp4、Gp6、芽孢杆菌属和Gaiella等菌属;间作后变形菌门、放线菌门、红游动菌属、Gaiella和黄杆菌属相对丰度明显提高,酸杆菌门、芽孢杆菌属和Gp2相对丰度显著降低。冗余分析结果表明,碱解氮、pH、全磷和有效磷是影响土壤细菌菌属结构的主要理化因子;单作模式的土壤细菌群落结构差异主要是由pH的变化引起的,而间作模式细菌群落结构主要受碱解氮的影响;此外,在酸杆菌门亚群中,Gp1、Gp2、Gp3及Gp7与pH呈正相关;Gp4、Gp5、Gp6与pH呈负相关;芽孢杆菌属与碱解氮呈正相关;Gaiella、红游动菌属和黄杆菌属等绝大多数菌属均与理化因子呈负相关。因此,荔枝园间作平托花生不仅能改善土壤理化性质以及酶活性,还能提高土壤细菌丰富度和多样性,促进荔枝根系对营养元素的有效吸收,为农民有效利用荔枝园行间空地、实现荔枝健康栽培探索出一条新的路径。     

不同栽培年限人参根际土壤细菌群落与土壤理化性质和酶活性的相关性

[目的]微生物和土壤酶是土壤微环境构建的重要部分,土壤质量与微生物功能多样性及酶活性等生物学指标密切相关,人参根际土壤的理化性质、微生物群落及酶活性对人参的产量和品质均有很大影响,研究不同栽培年限人参根际土壤细菌群落与土壤理化指标及酶活性的相关性,为人参的栽培管理提供科学依据.[方法]于人参红果期分别采集种植3、4和5...     

间套作玉米对线辣椒根际土壤微生物生态特征的影响

采用常规稀释平板法、氯仿熏蒸法、BIOLOG GN微平板反应系统及种间根系分隔技术, 以线辣椒单作(SC)为试验对照, 研究了间套作处理[玉米/线辣椒套作+根部塑料膜分隔(ICP)、玉米/线辣椒套作+根部尼龙网分隔(ICM)、玉米/线辣椒套作根部无分隔(ICN)]对线辣椒根际土壤微生物生态特征的影响。结果表明: 整个线辣椒生育期内, 各套作处理线辣椒根际土壤微生物总数与细菌总数具有相同的变化趋势, ICN和ICM处理的真菌、细菌、放线菌数量和细菌/真菌(B/F)、放线菌/真菌(A/F)比值均大于ICP与SC处理。盛果期, ICN处理根际土壤微生物量碳和微生物量氮比同处理其他生育期增加14.2%~54.0%和10.6%~54.7%。各处理土壤微生物群落AWCD的变化随培养时间呈现明显的"S"型曲线。间套作玉米显著提高了线辣椒根际土壤微生物群落的Shannon-Wiener指数(P<0.05)、Simpson指数、种间相遇几率和McIntosh指数(P<0.05), 并改变了土壤微生物对单一碳源的利用能力。线辣椒根际土壤微生物的不同多样性指数分别与其生物学产量之间存在显著或极显著正相关。说明间套作改善了土壤微生态环境。     

三种不同种植模式对土壤细菌群落多样性的影响

采用变性梯度凝胶电泳(DGGE )技术,研究了不同种植模式对土壤细菌群落多样性的影响。结果表明,不同种植模式下DGGE图谱条带的数量及亮度有较大区别,且有几条特征性条带发生了明显的变化。0—15 cm土层细菌群落的丰富度、多样性指数及均匀度指数均表现为:“菜稻菜模式”（RVCs） “休闲轮作模式”（FRCs） “蔬菜连作模式”（VCCs）;15—30 cm土层细菌群落的丰富度、多样性指数及均匀度指数均表现为:FRCs RVCs VCCs。UPGMC聚类分析表明,RVCs 处理的细菌群落结构相似性较低。主成分分析表明,对各主成分起分异作用的主要是 RVCs和FRCs;与主成分因子1 正相关程度较高的种植模式为:RVCs和VCCs,与主成分因子2 正相关程度较高的是 FRCs。可见,水稻土细菌群落多样性与种植模式密切相关,这些变化对土壤细菌群落有重要的调节作用。     

磷供应对玉米根际微生物碳源利用和功能多样性的影响

磷有效性能够改变根分泌物的组成和数量,调节土壤微生物的群落结构和多样性,但磷添加如何影响土壤微生物碳源利用和功能多样性尚不清楚。本研究通过盆栽土培试验,设置2个磷处理[低磷5.7 mg(P)?kg?1和高磷200 mg(P)?kg?1],以生长35 d的玉米根际土壤为研究对象,采用Biolog微平板法,分别在培养后240 h内每隔24 h检测具有31种不同碳源的微孔溶液颜色变化,揭示磷供应对玉米根际微生物碳源利用模式和功能多样性的影响。结果表明:随着培养时间的延长,土壤微生物对土壤碳源的利用呈现增加的趋势,直至碳源消耗殆尽;高磷供应显著增加了玉米根际土壤微生物群落平均颜色变化率(average well color development,AWCD),提高了对糖类及其衍生物、氨基酸和代谢产物的利用,但没有显著提高对脂肪酸和脂类的利用;在培养前72 h内,高磷供应显著增加了玉米根际微生物多样性指数、优势度指数和均匀度指数,但培养72 h后,磷供应对其没有显著的影响。主成分分析结果表明,提取的前3个主成分解释了75.15%的碳源利用,高磷和低磷处理具有显著不同的土壤微生物碳源利用模式。总之,糖类及其衍生物、氨基酸和代谢产物是玉米根际土壤微生物利用的主要碳源,短期磷添加能够显著增加土壤微生物对碳源的利用,在一定程度上能够提高土壤微生物群落功能多样性。     

不同栽培模式对黄瓜根际土壤酶活性及细菌群落结构的影响

分别以小麦、燕麦、毛葱、芹菜、白菜与黄瓜伴生或套作,研究了不同栽培模式对黄瓜根际土壤酶活性及细菌群落结构的影响,为连作土壤环境修复提供理论依据。结果表明:小麦/黄瓜、燕麦/黄瓜伴生,毛葱/黄瓜套作显著提高了根际土壤过氧化氢酶活性（P0.05）;芹菜/黄瓜套作和小麦/黄瓜伴生显著提高了根际土壤过氧化物酶活性（P0.05）;芹菜/黄瓜套作显著提高了根际土壤脲酶活性（P0.05）;不同栽培模式均显著提高了各时期根际土壤转化酶活性（P0.05）。PCR-DGGE分析结果显示,不同栽培模式在一定程度上提高了黄瓜根际土壤细菌群落结构多样性。DGGE条带测序显示,黄瓜根际土壤细菌大多与不可培养的细菌种属具有较高的同源性,测序比对推测,主要分属于-变形菌纲（Alphaproteobacteria）、-变形菌纲（Betaproteobacteria）、鞘脂杆菌纲（Sphingobacteria）和芽单胞菌纲（Gemmatimonadetes）四个纲。本研究说明不同栽培模式对土壤酶活性和土壤细菌群落结构均产生一定影响,改变了土壤环境,其中小麦与黄瓜伴生栽培模式效果较好。     

银北盐碱区植物根际土壤酶活性及微生物群落特征

开展盐碱区耐盐植物根际微生物群落多样性研究,对于盐碱土壤的植被恢复和生态修复具有重要意义。运用Biolog微平板技术,对宁夏银北盐碱区6种耐盐植物根际土壤酶活性及微生物群落代谢功能多样性进行研究。结果表明:不同植物根际土壤理化性质和酶活性存在一定的差异。与裸地相比,6种植物能显著提高盐碱地土壤酶活性,苜蓿根际土壤三种酶活性显著高于其他植物。土壤平均颜色变化率(AWCD)随培养时间的延长而逐渐增加,大小顺序依次为苜蓿(MX)、芨芨草(JJC)、柽柳(CL)、柳枝稷(LZJ)、苦豆子(KDZ)、枸杞(GQ)和裸地土壤(CK),根际土壤与盐碱裸地土壤之间差异显著(P<0.05)。土壤微生物群落香农指数、辛普森指数和麦金塔指数均以苜蓿根际土壤最高,芨芨草次之,二者较其他土壤差异显著(P<0.05)。不同植物根际土壤微生物碳源利用能力存在差异,苜蓿根际土壤微生物的利用率显著高于其他土壤(P<0.05),碳水化合物类是根际土壤微生物的主要碳源,其次为氨基酸类和羧酸类,胺类的利用率最小。主成分分析显示,对PC1和PC2起分异作用的主要碳源为碳水化合物类和羧酸类。综合各项指标,均表现...     

运用多隔层根箱研究玉米幼苗根际微域中芘的降解

采用多隔层根箱,通过尼龙撩网插片的控制,实现根室土(S0)、离根室0～2mm(S1)、2～4mm(S2)、4～6mm(S3)及6mm(S4)各室层土壤的分离采集,分析离根系表面不同距离土壤芘的根际降解,并借助脂肪酸甲酯(fattyacidmethylester,FAME)分析土壤微生物群落结构的空间响应机制。结果表明:种植玉米处理的各室层内土壤可提取态芘含量存在显著的不同,其大小顺序为S4S3S0S2S1;各室层微生物群落结构存在显著的变化,其中微生物生物量和丛枝菌根真菌特征脂肪酸含量表现出与土壤可提取态芘含量变化相反的变化趋势。未种植玉米处理的各室层土壤可提取态芘含量和微生物群落结构没有差异。土壤可提取态芘含量与微生物生物量和丛枝菌根真菌的特征脂肪酸呈显著负相关(p0.01)。     

Growth and enzymatic activity of maize (Zea mays L.) plant: Solution culture test for copper dioxide nano particles

Copper (Cu) is an essential micronutrient for plants, which acts either as the metal component of enzymes or as a functional structural or a regulatory co-factor of a large number of enzymes. To understand the possible benefits of applying nanotechnology to agriculture, the first step should be to analyze penetration and transport of nano-particles in plants. The present study was conducted to test the hypothesis that copper nanoparticle would enter into the plant cell and govern the growth of maize plant.A solution culture experiment was conducted to investigate the effect of Cu nano-particles (<50nm) on the growth and enzymatic activity of maize (Zea mays L.) plant. Bioaccumulation of Cu nano-particles in plant was also investigated. Results showed that Cu nano-particles can enter into the plant cell through roots and leaves. Bioaccumulation increased with increasing concentration of Cu nano-particles (NPs), and agglomeration of particles was observed in the cells using transmission-electron microscopy. Application of Cu nano-particles through solution culture as well as spray enhanced the growth (51%) of maize plant in comparison to control. The different enzymatic activities like glucose-6-phosphate dehydrogenase, succinate dehydrogenase, superoxide dismutase, catalase, andguaiacol peroxidase were studied to find a possible pathway through which NPs may affect the enzymatic activity of plant. Amongst the enzymes, the activity of glucose-6-phosphate dehydrogenase was highly influenced by copper oxide (CuO)nano-particles application by spray as well as in solution. Experimental results revealed that CuOnano-particles affected the pentose phosphate pathway of maize plant. The obtained experimental results provided conclusive evidence to indicate that the nano-particles considered under this study could enter into the plant cell, easily be assimilated by plants and also enhanced its growth by regulating the different enzyme activities.     

Effects of Selected Surfactants on Nutrient Uptake in Corn (Zea mays L.)

Surfactants in herbicide formulations eventually enter soil and may disrupt various processes. Research examined effects on nutrient uptake in corn caused by surfactants, herbicides, and surfactant-herbicide combinations applied to silt loam and silty clay loam soils in the greenhouse. Surfactants evaluated were Activator 90, Agri-Dex, and Thrust; herbicides were glyphosate, atrazine, and bentazon. Corn was planted in fertilized soils with moisture content maintained for optimum growth. Foliage (V8 growth stage) was collected for elemental analyses. Nutrient uptake differed with soil texture. Nutrient uptake from silty clay loam was more affected by surfactants and/or herbicides than in silt loam. Potassium uptake was significantly (P = 0.05) decreased in silt loam only by Thrust but uptake of phosphorus (P), potassium (K) calcium (Ca), sulfur (S), copper (Cu), and zinc (Zn) decreased by ≤30% in silty clay loam treated with surfactants. Surfactants and/or herbicides may interact with soil texture to affect nutrient uptake. Long-term field studies to validate changes in nutrient uptake and grain yields after annual applications of surfactants plus herbicides are needed.     

Critical limit of boron for maize (Zea mays L.) in red and lateritic soil of Jharkhand,India

ABSTRACT

To establish a critical limit in soils and plant, an experiment was conducted in red and lateritic soil (Alfisols) of farmer’s field in tribal-dominated Panchayat Kurum, Palkot block, Gumla district, Jharkhand, India. Based on the results of the field experiment, the critical limits were determined as 0.48, 0.50, 0.47, and 0.42 mg kg^?1 in the soil, respectively, for hot water, hot calcium chloride, salicylic acid, and ammonium acetate-extractable B, while a critical limit of 12.00 mg kg^?1 was observed in maize tissue using the graphical method. In an analysis of variance method, the critical limits of B in soils were found as 0.45, 0.54, 0.49, and 0.43 mg kg^?1 using hot water, hot calcium chloride, salicylic acid, and ammonium acetate extractants, respectively. Maize plants were highly responsive to B application where soil B level was below the critical limit (0.50 mg kg^?1). In a field experiment, grain yield of maize increased with increasing levels of B application, while soil application at 1.0 kg ha^?1 + two foliar application (at the knee and pre-flowering stages) of borax at 0.2% were showed significantly higher grain yield of the maize crop. The hot water, hot calcium chloride, salicylic acid, and ammonium acetate-extractable B were significantly and positively correlated with organic carbon and negatively correlated with the electrical conductivity of soils.     

Nitrogen fertilization does not affect micronutrient uptake in grain maize (Zea mays L.)

Abstract

Due to continuous single nitrogen fertilization, we hypothesized a built-up deficiency of micronutrients in crops that would limit plant growth and crop quality. In 2-year field experiments using urea-N fertilized grain maize (Zea mays L.), hybrid KWS 2376 at 0, 120 and 240 kg N ha^?1 crop uptake of Zn, Mn, Cu and Fe was studied at DC 32, DC 61 and in the grain harvested. Micronutrient contents at DC 32 stage – 1st node (aboveground phytomass) and DC 61 – flowering (ear leaf) were all at levels indicative of adequate micronutrient supply to the crop. At both sampling occasions the Fe:Zn and Fe:Mn ratios were adequate implying that Fe did not inhibit the uptake of Zn and Mn. The application of nitrogen increased the Fe content at the 1st sampling in both years; in the second year the same was also the case for the Zn content. Nitrogen nutrition increased the contents of Mn and Fe at the 2nd sampling only in year 2; in the other treatments no changes were observed in the micronutrient contents. Micronutrient correlations in the grain were discovered between Zn and Mn contents and between Fe and Mn contents. In the second year the highest N-rate significantly increased the Fe and Zn content of the grain compared with the lower rates of nitrogen fertilization. Grain yields were not affected by the rate of nitrogen and ranged between 13.65 and 14.34 t ha^?1 (1st year) and between 13.68 and 14.18 t ha^?1 (2nd year). Nitrogen fertilization did not reduce the content of micronutrients in the plant or grain of maize. It is evident that the continuous single use of N fertilization so far has not resulted in a micronutrient deficiency of the plants limiting the nutrient density of the grain or reducing its quality.     

Kinetics of 15N-labelled nitrate uptake by maize (Zea mays L.) root segments

Abstract

Isotherms and kinetic constants of nitrate uptake by excised root segments from the apical root zone of 6-d-old maize seedlings pretreated with nitrate were investigated using ¹⁵N-labelled nitrate. The isotherms were resolved into two systems namely a multiphasic saturable system at substrate concentrations lower than 2 mol m^?-3 and a linear system at higher concentrations. The detailed analysis of the multiphasic saturable system suggested the existence of at least three phases, which followed the Michaelis-Menten kinetics. The I _max and K _m of each phase increase from the lower phase to the upper phase. The distance from the root tip and the presence of stele affected considerably the linear system but only slightly the saturable system.     

毒死蜱对棉花根际土细菌群落多样性和结构的影响

通过室内盆栽试验模拟自然环境条件,采用高效液相色谱(HPLC)和末端限制性片段长度多态性(T-RFLP)技术,研究了土壤使用推荐剂量(5 mg·kg~(-1))及推荐剂量的2倍、3倍和4倍(10 mg·kg~(-1)、15 mg·kg~(-1)、20 mg·kg~(-1))毒死蜱对棉花根际土壤细菌群落多样性和结构的影响,以不施用毒死蜱的土壤为对照。结果表明,5 mg·kg~(-1)、10 mg·kg~(-1)、15 mg·kg~(-1)和20 mg·kg~(-1)毒死蜱在土壤中的半衰期分别为10.04 d、11.36 d、11.55 d和12.16 d,60 d时基本完全降解。毒死蜱处理60 d后,棉花生物量显著降低;毒死蜱浓度越高,棉花生物量越低。无毒死蜱条件下不同取样时间根际细菌多样性无显著差异,毒死蜱处理组前30 d细菌多样性均显著降低,60 d时毒死蜱处理组细菌多样性恢复到正常水平。研究发现毒死蜱浓度越高对细菌多样性抑制作用越显著,恢复越缓慢。主成分分析结果发现,第10 d、30 d和60 d毒死蜱处理组与对照组细菌群落结构差异显著,其中60 d时20 mg·kg~(-1)毒死蜱处理组差异最显著,即使土壤中毒死蜱完全降解,根际细菌群落结构仍不会恢复到正常水平。60 d时,被毒死蜱抑制的细菌有硝化刺菌属(Nitrospina sp.)和Cellulophaga sp.等,被激活的有芽孢杆菌属(Bacillus sp.)和链霉菌属(Streptomyces sp.)等。可见,毒死蜱的引入,重新构建了土壤细菌群落结构,显著影响棉花生长,对棉花根际土壤微生态环境冲击较大,应对其生态安全性予以重视。