强还原土壤灭菌防控作物土传病的应用研究

随着集约化种植程度的不断提高,土传病原菌侵染、土壤酸化、次生盐渍化、养分平衡失调等引起的作物连作障碍发生率不断攀升,严重威胁着集约化农业的可持续发展。强还原土壤灭菌(Reductive Soil Disinfestation,RSD)法是一种作物种植前的土壤处理方法,即:在发生土传病害的土壤上,添加大量的易分解有机物料、灌溉、薄膜覆盖或淹水阻隔与大气的气体交换,快速创造土壤强还原环境,短期内杀灭土传病原菌的方法。强还原杀灭土传病原菌的作用机理包括:1)厌气杀灭好氧病原菌;2)还原过程产生有毒有害的物质杀灭土传病原菌;3)强还原改变土壤微生物群落结构,抑制土传病原菌活性。强还原土壤灭菌法还具有提高土壤p H,减轻次生盐渍化的作用,具有广谱性和环境友好性。本文介绍了该方法的起源、作用机理、影响该方法效果的因素及其应用前景。     

Simple and rapid determination of thiol- and organic disulfide-sulfur in foliage and humic soil horizons

A new method for rapid determination of thiol‒ (R‒SH) and disulfide (R‒S‒S‒R′) sulfur in soil and foliage samples is presented. Using a silver sulfide electrode, the thiol sulfur content of a sample is determined by potentiometric titration with AgNO₃. After reduction with a mixture of NaOH and ascorbic acid also its disulfide S content can be quantified subsequent to neutralization of the reductive solution with citric acid. The method was tested with eight organic standards, six humic soil samples, and three Norway spruce needle samples. Disulfide S from standards with abstrictable H‒atoms in α‒ or β‒position to the disulfide bond could — with one exception — be detected completely. For the aromatic disulfides which allow only direct nucleophilic attack, recovery was at 75%. For the soil samples, 32 to 60% of the carbon‒bonded S consisted of disulfide S, for the spruce needle samples 26 to 33%. The method provides satisfactory results for most studied standards and is applicable to various natural substances after adequate sample preparation.     

十字花科黑腐病菌分泌蛋白表达谱分析

本文旨在分析Xcc8004菌株分泌蛋白的蛋白表达谱,建立Xcc8004分泌谱分析方法。首先将诱导的Xcc8004分泌蛋白进行液体酶解,通过HPLC阳离子交换柱分离多肽,采用EASY-nLC结合Orbitrap质谱鉴定分泌谱。经过过滤筛选后,共精确鉴定蛋白1532个,SignalP分析其中包含286个含信号肽且不含跨膜区,其中118个为假定蛋白。对286个可能的分泌蛋白进行基因本体评注分析,其中,主要为行驶水解酶类,受体蛋白,信号传导等重要的微生物-植物互作介导蛋白。本研究通过液相色谱-质谱联用能够高效鉴定Xcc8004的分泌蛋白,对这些鉴定的分泌蛋白的信息学分析有利于我们进一步研究Xcc8004的致病机制,为研究微生物植物相互作用鉴定了方法与理论基础。     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.     

The DFRC method for lignin analysis. 7. Behavior of cinnamyl end groups.

The behavior of cinnamyl end groups of lignins during the derivatization followed by reductive cleavage (DFRC) procedure has been investigated using lignin model compounds. On AcBr treatment, hydroxycinnamyl alcohols give rise mainly to 1-aryl-1, 3-dibromopropanes from which 1-aryl-3-bromopropanes and arylcyclopropanes are formed by zinc reduction. Arylpropene derivatives are also significant among DFRC products of etherified cinnamyl end-group models. Major monomers from DFRC of hydroxycinnamaldehydes are arylcyclopropyl acetates produced by reductive ring closure of 1-acetoxy-3-aryl-1,3-dibromopropanes. Although the reactions are not as clean as the ether-cleaving reactions that form the basis of the DFRC method, end groups produce diagnostic compounds that provide valuable markers for studying end groups in lignins.     

Production and specificity of polyclonal antibodies to hexanal-lysine adducts

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.     

Nitrate determination in baby food, using the nitrate ion selective electrode.

The nitrate electrode has been utilized in the determination of nitrate content in food products. The AOAC xylenol method was employed for comparative results. A reasonable correlation (r=0.91) was found between the 2 methods in the analysis of 49 samples containing 30-350 ppm nitrate. At the average nitrate content (100 ppm) of these foods, the standard error was 4.3 ppm. The electrode responds directly to the ionic activity of the nitrate ion. It has a linear concentration range of 1-6000 ppm nitrate and can be used over a wide pH range. The electrode does respond to some extent to anions other than nitrate, and some interferences do occur. These interferences are easily controlled by the use of cation exchange resins. The Corning known addition (spiking) method is used on all samples to insure correct electrode response in solutions containing variable background ionic composition. The electrode has the advantage of simplicity, speed, reproducibility, and accuracy. Work time saved using the electrode as opposed to the xylenol method is about 7 hr for the analysis of 20 samples. Error, and the need for repeating analysis, is much less frequent.     

Rapid electrochemical method for the evaluation of the antioxidant power of some lipophilic food extracts.

In this paper, a novel electrochemical method to evaluate the antioxidant power of lipophilic compounds present in vegetables, such as carotenoids, chlorophylls, tocopherols, and capsaicin, is reported. The method is based on a flow injection system with an electrochemical detector equipped with a glassy carbon working electrode operating amperometrically at a potential of + 0.5 V (vs Ag/AgCl). The proposed method is selective for lipophilic compounds having antioxidant power. When applied to pure compounds, the order of antioxidant power resulted as follows: lycopene > beta-carotene > zeaxanthin > alpha-carotene > beta-cryptoxanthin > lutein > alpha-tocopherol > capsaicin > chlorophyll a > chlorophyll b > astaxanthin > canthaxanthin. Results obtained on five vegetable and two fruit extracts were compared to those obtained by the 2,2'-azinobis(3-ethylbenz-thiazoline-6-sulfonic) acid (ABTS) radical cation decolorization assay, one of the most used methods to evaluate the total antioxidant capacity of foods. A good correlation between the two methods was found, except for spinach, because of the different antioxidant powers assigned by the two methods to chlorophylls. In conclusion, results suggest that the proposed electrochemical method can be successfully employed for the direct, rapid, and reliable monitoring of the antioxidant power of lipophilic food extracts.     

Direct determination of potassium-calcium activity ratio in soils with two ion-selective electrodes. I. Method of determination

A method for the direct determination of the potassium-calcium activity ratio in soils with a potassium-selective electrode and a calcium-selective electrode was developed. The two electrodes were inserted into the soil suspension, and the potential difference was measured with a high-impedance mV-meter. The reproducibility of measurements was 2–3 mV, corresponding to 0.03–0.05 pK-0.5pCa units. Measurements in soil paste and in supernatant clear solution gave identical results. The pK-0.5pCa value varied only slightly with the variation in water to soil ratio or in salt concentration if the salt concentration was not high. At high salt concentrations, the value decreased.     

Liquid chromatographic-electrochemical detection screening procedure for six nitro-containing drugs in chicken tissues at low ppb level

A screening procedure is described for the detection of furazolidone, nitrofurazone, aklomide, zoalene, nitromide, and sulfanitran residues in a single extract of chicken liver, breast, or thigh muscle at the low ppb level. The method includes extraction of tissue with chloroformethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with pH 6.0 phosphate buffer-methanol (1 + 1). Eluants are separated on a 25 cm, 5 microns C18 column with pH 6.0 phosphate buffer-methanol (57.5 + 42.5) as mobile phase. The drugs are detected with an electrochemical detector in the reductive mode at -0.8 V. Mean recoveries from all tissues ranged from 76.5% for nitrofurazone to 97.1% for zoalene.     

Study on Water-Soluble Organic Reducing Substances. I. Determination of Organic Reducing Substances by Differential Pulse Voltammetry

A new method was proposed for study of organic reducing substances in soils.According to the theoretical relationship between the voltammetric behaviors and reduction-oxidation reaction of reducing substances,the working conditions of differential pulse voltammetry (d.p.v.)for determining the organic reducing substances produced during the processes of the anaerobic decomposition of plant materials were established with a glass carbon electrode as working electrode,1M Ag-AgCl electrode with large area as reference electrode,0.2M NH4Ac as supporting from -0.5 to 1.2 voltage(vs.M Ag-AgCl).The peak current proportional to the concentration of reducing substances,and the characteristic peak potential of each organic reducing substance were regarded as the quantitative and qualitative base,respectively.These results obtained under the conditions mentioned above directly reflect both the reducing intensity and capacity of the organic reducing system in soils.     

Determination of nitrite in cured meats by ion-exclusion chromatography with electrochemical detection

A rapid liquid chromatographic (LC) method was developed for a sensitive determination of nitrite in cured meats, using ion-exclusion chromatographic separation and electrochemical detection (IEC-EC). The current AOAC colorimetric method requires 2 h shaking in a steam bath to eliminate interference from reducing compounds such as ascorbic acid. In the present method, nitrite was analyzed in the presence of ascorbic acid without interference, and the extraction time was reduced to 1 min. The extracted nitrite was determined by ion chromatography using anion-exclusion/HS column and amperometric detector equipped with platinum or glassy carbon electrode operating at +1.0 V vs Ag/AgCl reference electrode. The detection limit was 1 ppb as NO2-. The recoveries of 50 ppm nitrite added to frankfurter and meat stick were 103 and 99.6%, respectively, with relative standard deviations less than 4%. The high speed, sensitivity, and selectivity make the new method a useful alternative to the AOAC colorimetric method.     

食品表面静电涂敷高压电极结构的试验

在研究已有的静电喷涂装置基础上,采用栅网电极作为食品表面静电涂敷高压电极。为了加强电极电晕效果,提出了用芒刺电极代替栅网电极。开发了电极试验装置,并自制了荷质比测试装置。以水、CMC和食用海藻酸钠水溶液3种雾流分别对栅网和芒刺2种电极作电晕电流试验和荷质比试验。由电晕电流试验优选出两种电极的结构及静电场电压;荷质比试验证明芒刺电极比栅网电极电晕效果好     

Electrochemical sensing of total antioxidant capacity and polyphenol content in wine samples using amperometry online-coupled with microdialysis

This work describes the method for total antioxidant capacity (TAC) and/or total content of phenolics (TCP) analysis in wines using microdialysis online-coupled with amperometric detection using a carbon microfiber working electrode. The system was tested on 10 selected wine samples, and the results were compared with total reactive antioxidant potential (TRAP), oxygen radical absorbance capacity (ORAC), and chemiluminescent determination of total antioxidant capacity (CL-TAC) methods using Trolox and catechin as standards. Microdialysis online-coupled with amperometric detection gives similar results to the widely used cyclic voltammetry methodology and closely correlates with ORAC and TRAP. The problem of electrode fouling is overcome by the introduction of an electrochemical cleaning step (1-2 min at the potential of 0 V vs Ag/AgCl). Such a procedure is sufficient to fully regenerate the electrode response for both red and white wine samples as well as catechin/Trolox standards. The appropriate size of microdialysis probes enables easy automation of the electrochemical TAC/TCP measurement using 96-well microtitration plates.     

In vitro antioxidant properties and phenolic composition of Salvia virgata Jacq. from Turkey

Antioxidant activities and phenolic compositions of the active fractions of Salvia virgata Jacq. (Lamiaceae) from Turkey were examined. The aerial part of S. virgata was extracted with different solvents in an order of increasing polarity such as hexane, ethyl acetate, methanol, and 50% aqueous methanol using a Soxhlet apparatus. Water extract was also prepared from S. virgata by reflux. All solvent fractions were investigated for their total phenolic contents, total flavonoids, flavonols, qualitative-quantitative compositions (by HPLC-PDA analysis), iron(III) reductive activities, free radical scavenging activities (using DPPH*), and effect upon linoleic acid peroxidation activities; also, the peroxidation level was determined by the TBA method. The results of activity tests given as IC50 values were estimated from nonlinear algorithm and compared with standards, viz., butylated hydroxytoluene, ascorbic acid, and gallic acid. Polar fractions were found to be more active for free radical activity whereas nonpolar fractions protected the peroxidation of linoleic acid. Rosmarinic acid was the most abundant component in the extracts, followed by caffeic acid and lutelin-7- O-glycoside.     

Determination of phenolic compounds and ascorbic acid in different fractions of tomato by capillary electrophoresis with electrochemical detection

Tomato (Lycopersicon esculentum Mill.), one of the most important crops worldwide, contains different classes of substances with antioxidant properties such as carotenoids, vitamin C, and phenolics. A method based on capillary electrophoresis with electrochemical detection has been developed to analyze ascorbic acid and phenolics in the peel, pulp, and seeds of tomatoes. Operating in a wall-jet configuration, a 300 microm diameter carbon disk electrode was used as the working electrode, which exhibits a good response at +0.90 V (vs saturated calomel electrode) for the analytes. Under optimum conditions, the analytes were baseline separated within 20 min in a 50 mmol/L borate buffer (pH 8.7). Notably, excellent linearity was obtained over 3 orders of magnitude with detection limits (S/N=3) ranging from 1x10(-8) to 2x10(-7) g/mL for all analytes. This proposed method has been successfully applied to monitor the content of ascorbic acid and phenolics in real samples, and the assay results were satisfactory.     

NMR-based screening method for transglutaminases: rapid analysis of their substrate specificities and reaction rates

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins such as tofu, boiled fish paste, and sausage. Other transglutaminases (TGases) are expected to be used in the same way, and also to extend the scope of industrial applications to materials, drugs, and so on. The TGases have great diversity, not only in amino acid sequence and size, but also in their substrate specificities and catalytic activities, and therefore, it is quite difficult to estimate their reactivity. We have developed an NMR-based method using the enzymatic labeling technique (ELT) for simultaneous analysis of the substrate specificities and reaction rates of TGases. It is quite useful for comparing the existing TGases and for screening new TGases or TGases variants. This method has shown that MTG is superior for industrial use because of its lower substrate specificity compared with those of guinea pig liver transglutaminase (GTG) and red sea bream liver transglutaminase (FTG). We have also found that an MTG variant lacking an N-terminal aspartic acid residue has higher activity than that of the native enzyme.     

Fluorometric determination of nitrite in cured meats.

An indirect fluorometric method for determining sodium nitrite in meat products is presented. The extracted sodium nitrite is consumed in a diazotization reaction with a measured excess of sulfanilic acid. Fluorescamine, which acts selectively with primary amines such as sulfanilic acid, is a fluorogenic reagent for the excess amine. The amine consumed, calculated by difference from the total originally present, is directly related to the sodium nitrite content of the sample. Interferences from amino acids and soluble proteins in the meat extract are eliminated by judicious use of a secondary peak in the fluorescence spectra (436 nm excitation, 495 nm fluorescence) combined with measurement at low pH (3.30). The recoveries of sodium nitrite ranged from 83.2 to 99.6% with an average of 93.4 and a standard deviation of +/- 5.28% for 11 determinations.     

A screening method for protein characterization and differentiation.

A circular paper chromatographic method was developed for the separation of the amino acids in proteins into 7 subgroups. Butanol-acetic acid water (4+1+1) was used as the developing solvent. Eluted ninhydrin-stained aminograms gave rise to graphic profiles or numerical indexes based on absorbance percentages. The profiles can be used to compare protein-containing samples. Twenty different samples were studied through 190 comparisons of graphic profiles and coefficients of correlation, with only 4% misleading results. The method showed excellent reproducibility for the identification or differentiation of proteins and has the advantage of being performed with low-priced apparatus and reagents.     

Amperometric determination of phenazopyridine hydrochloride in a flowing stream at the glassy carbon electrode

A flow-injection method is described for the determination of phenazopyridine hydrochloride, based on electrochemical oxidation at the glassy carbon electrode. The suggested method is highly specific and can be used to determine phenazopyridine HCl in the presence of most drugs commonly found in pharmaceutical dosage forms or administered therapeutically. Applying a constant potential of +950 mV vs Ag/AgCl/3.5M KCl reference electrode, the calibration curve was linear in the 1-30 micrograms/mL range, with minimum detectability of 0.2 ng (signal-to-noise ratio 2). Good accuracy and precision were obtained when the method was applied to some dosage forms containing phenazopyridine HCl. Although automation was not used in this study, an automated system could be incorporated because the method uses the technique of continuous analysis in a flowing stream.