黄皮酰胺类生物碱的提取及对7种水果病原真菌的抑菌活性

利用菌丝生长速率法测定黄皮果核甲醇提取物对芒果炭疽病菌(Colletotrichum gloeosporioides Penz)等7种水果病原真菌的抑制活性,结果表明该提取物10 mg/mL浓度下对芒果炭疽病菌和香蕉枯萎病菌（Fusarium oxysporum f.sp. cubense）菌丝生长抑制率分别达到87.36%和84.55%。从黄皮果核甲醇提取物中分离得到生物总碱,测定其对上述两种真菌的EC50值分别为0.78 mg/mL和0.81 mg/mL。生物总碱进一步分离纯化得到新肉桂酰胺类化合物lansiumamide B, 在0.08 mg/mL浓度下,对芒果炭疽病菌和香蕉枯萎病菌的菌丝生长抑制率分别为 83.33%和60.78%。     

中国粗榧枝叶提取物分离及其对反枝苋的除草活性

以对杂草反枝苋种子萌发及根、茎生长的抑制作用为指标,提取、分离、鉴定了中国粗榧Cephalotaxus sinensis Li.枝叶中的除草活性成分。中国粗榧枝叶乙醇提取物的酸水溶液用苯萃取,萃余相碱化并用氯仿萃取得总生物碱。以硅胶及氧化铝柱层析从中国粗榧总生物碱中分离出了3个生物碱类化合物 H1、H2和H3 ,其结构依次鉴定为桥氧三尖杉碱、三尖杉碱及11-羟基三尖杉 碱。在1.00 mg/mL的供试浓度下, H1 对反枝苋种子根、茎生长的抑制率分别为86.1％和82.4％, H3 对反枝苋种子根、茎生长的抑制率分别为84.2％和77.1％, H2 除草作用较弱。测定结果表明,萃取所得总生物碱为中国粗榧的主要活性成分。     

苦参生物碱对植物病原菌的离体抗菌活性研究

测定了苦参总碱(混和生物碱)及其单体苦参碱、氧化苦参碱、槐果碱、槐定碱、野靛碱等6个样品对11种植物病原真菌和3种细菌的离体抗菌活性。结果表明,在药剂浓度为500mg/L时,仅苦参总碱对黄瓜疫霉病和辣椒炭疽病菌、槐定碱对黄瓜疫霉病菌表现出较好的活性,菌丝生长抑制率分别为96.18%、54.90%、93.62%,苦参总碱和槐定碱对黄瓜疫霉病菌菌的EC50分别为178.21mg/L和162.13mg/L;6个样品对3种病原细菌的活性较低,在药剂浓度为100μg/mL时,生长抑制率均在20%以下。     

超高效液相色谱质谱联用检测苹果和土壤中丙撑硫脲的残留分析方法

建立了丙撑硫脲在苹果和土壤中的残留分析方法。样品经提取乙腈提取、PSA净化后质谱检测器检测。结果表明:添加质量分数为0.01~0.5mg/kg时,丙撑硫脲在苹果和土壤中平均添加回收率分别为76.5%~98.3%和84.9%~99.0%,相对标准偏差分别为3.1%~4.9%和3.2%~9.0%。丙撑硫脲在苹果中的最低检出质量分数为0.01mg/kg。该方法快速简便,准确可靠。     

气相色谱法测定20％毒死蜱·异丙威可湿性粉剂

[目的]研究20%毒死蜱·异丙威可湿性粉剂中异丙威和毒死蜱在同柱同条件下分离和测定方法,为产品的质量控制以及质检机构检测提供参考。[方法]以邻苯二甲酸丙烯酯为内标化合物,5%OV-101/Chromosorb W AW-DMCS(150-180um)为色谱柱固定相,利用FID检测器,通过一阶程序升温对20%毒死蜱·异丙威可湿性粉剂中2种组分进行分离和测定。[结果]20%毒死蜱·异丙威可湿性粉剂中2种主要组分及杂质均得到了有效分离,异丙威和毒死蜱的标准偏差分别为0.056、0.070;变异系数分别为0.53%、0.68%;回收率分别为99.08%～101.42%,99.03%～102.12%;相关系数分别为0.999 7、0.999 9。[结论]本文对20%毒死蜱·异丙威可湿性粉剂的气相色谱方法进行了反复实验,一次进样可同时测定异丙威、毒死蜱的质量分数,缩短了分析时间,达到了简捷、快速、准确的目的。     

气相色谱检测小麦中高效氯氟氰菊酯的残留分析方法

建立高效氯氟氰菊酯在小麦中的残留分析方法。样品经乙腈提取、弗罗里硅土固相萃取柱净化后,气相色谱检测。添加质量分数为0. 01～1mg/kg时,高效氯氟氰菊酯在麦粒、秸秆和青秸秆中平均添加回收率分别为98%～101%、93%～98%和87%～92%,相对标准偏差分别为3%～5%、4%～5%和2%～3%。高效氯氟氰菊酯在麦粒中的最低检出质量分数都为0. 01mg/kg。该方法快速简便,准确可靠。     

氰戊·辛硫磷35%乳油高效液相色谱分析

采用高效液相色谱法,以正己烷+无水乙醚为流动相,使用紫外检测器、SiO2不锈钢柱分离测定氰戊·辛硫磷35%乳油中有效成分氰戊菊酯和辛硫磷的质量分数。结果表明:氰戊菊酯和辛硫磷的标准偏差分别为0.027 0和0.042 8;变异系数分别为0.51%和0.14%;平均回收率为99.66%和99.90%;线性相关系数分别为0.999 9和0.999 7。     

木霉几丁质酶对烟草赤星病菌的作用

以指形管培养法分别测定几丁质酶粗酶液和纯化的几丁质酶混合液(2种几丁质酶)对烟草赤星病菌孢子萌发的抑制作用。结果表明,较高浓度(25 2U)几丁质酶粗酶液在48h内强烈抑制孢子萌发和芽管伸长,或致芽管畸形和细胞壁破裂;几丁质酶混合液对赤星病菌的孢子萌发也表现出明显的抑制作用,但在相同或相近酶活性条件下,纯化的几丁质酶混合液(9 4U)和粗酶液(12 6U)对赤星病菌孢子萌发的抑制率(处理24h时)分别为46%和84 3%,前者明显低于后者。采用孢子液悬滴法接种烟苗(K 326)叶片测定木霉几丁质酶对赤星病菌致病性的影响。结果表明,几丁质酶粗酶液浓度越高,对孢子萌发抑制时间越长,抑制率越高;其浓度为4 9、9 8、19 5U/ml的抑制率7d时分别为36 8%、56 2%和57 6%。     

几种植物源活性物质对昆虫细胞的毒力测定

本文测定几种植物源活性物质对甜菜夜蛾离体培养细胞系IOZCAS-Spex-II和草地贪夜蛾离体培养细胞系Sf 9细胞增殖活性的抑制作用.试验结果表明,喜树碱、羟基喜树碱和鱼藤酮对两种昆虫细胞具有较好的抑制作用.在测定的时间(2、6、12、24、48、72 h)和剂量(1.0×10-3、1.0×10-2、1.0×10-1、1.0×100,1.0×101、1.0×102μmol/L)范围内,喜树碱、羟基喜树碱和鱼藤酮对甜菜夜蛾IOZCAS-Spex-II和草地贪夜蛾Sf 9的细胞毒性具有较好的时间和剂量依赖性,而印楝素、乌头碱、次乌头碱、伪石榴皮碱、马钱子碱和吴茱萸碱对其细胞毒性具有一定的时间效应,无明显的剂量关系.     

Witconol NP-100 与Morwet D-425在悬浮种衣剂中的应用

以Morwet D-425和Witconol NP-100代替20%福·克和30%多·福·克 悬浮种衣剂配方中的乳化剂,结果表明:Morwet D-425和Witconol NP-100在20%福·克 种衣剂中质量分数分别为3.0%和1.0%、在30%多·福·克种衣剂中质量分数分别为4.0%和1.0%时,出料1 d后制剂粘度分别为390和510 mPa·s;经过30 d室温或14 d、(54±2)℃贮藏后,制剂的颗粒聚结数低于10%,并且无不可逆沉淀形成;热贮析水体积分别为5%和9%,较原配方种衣剂降低了20%以上,室温贮藏析水体积不足1%。用激光粒度分析仪分别检测添加Morwet D-425、Witconol NP-100或原配方乳化剂的种衣剂经过0.5、1.0、1.5、2.0 h研磨后的粒度分布,新配方种衣剂经过1.5 h研磨平均粒径分别达到1.85和1.91 μm,而原配方种衣剂经2.0 h研磨平均粒径仍大于2 μm。     

放线菌Lj20抗真菌物质的分离及其在病害防治中的作用

菌株Lj20是从健康辣椒植株分离得到的一株有杭真菌活性的植物内生放线菌,为了进一步明确其生物防治潜力,利用大孔吸附树脂、离心薄层层析以及柱层析等技术对Lj20代谢产物中的抗真菌活性物质进行了分离和提纯,并采用生长速率法测定了其代谢产物对黄瓜灰霉病菌的抑制作用,同时在温室中对Lj20发酵液粗提物的防病效果进行了试验.从Lj20代谢产物中分离得到一个抗真菌活性物质,该化合物时黄瓜灰霉病菌具有较强抑制作用,EC50值为174.94 mg/L;500mg/LLj20发酵液粗提物对黄瓜灰霉病的防治效果达62.5%;经鉴定其化合物结构为3,5-二叔丁基-4-羟基-卞基甲醚.结果表明,放线菌Lj20有进一步开发和利用的价值.     

Comparative studies of acetylcholinesterases purified from two field populations of the oriental migratory locust (Locusta migratoria manilensis): implications of insecticide resistance

Acetylcholinesterase (AChE) was purified by affinity chromatography from two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), collected from Huanghua and Pingshan Counties, Hebei Province of China. The purification factors and yields were 1661-fold and 19.3%, respectively, for the Huanghua population, and 3897-fold and 39.6% for the Pingshan population. Both the purification factor and yield were significantly lower in the Huanghua population than in the Pingshan population. AChE activity was almost completely inhibited by 10⁻⁶ M eserine and BW284C51, but ?5.8% of AChE activity was inhibited by ethopropazine at the same concentration, suggesting that purified AChE from either population was a typical insect AChE. However, AChE purified from the Huanghua population was 62-, 2.0-, and 1.6-fold less sensitive to inhibition by the three organophosphate compounds, chlorpyrifos oxon, demeton-S-methyl, and paraoxon, respectively, than that from the Pingshan population. Significantly lower purification factor and low yield associated with reduced sensitivity of AChE to inhibition by the organophosphates indicated that AChE purified from the Huanghua population was biochemically and pharmacologically different from that of the Pingshan population. Reduced sensitivity of AChE appeared to contribute to organophosphate resistance in the locust from Huanghua County, where insecticides have commonly been used to manage outbreaks of the locust.     

Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH₄)₂SO₄ precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici (Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.     

Purification and biochemical characterization of glutathione S-transferases from three strains of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae): Implication of insecticide resistance

Glutathione S-transferases (GSTs) catalyzing the conjugation of reduced glutathione (GSH) to a vast range of xenobiotics including insecticides were investigated in the psocid Liposcelis bostrychophila Badonnel. GSTs from susceptible and two resistant strains (DDVP-R for dichlorvos-resistant strain and PH₃-R for phosphine-resistant strain) of L. bostrychophila were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), in a photometric microplate assay. The specific activities of GSTs purified from two resistant strains were significantly higher than their susceptible counterpart. For the resistant strains, GSTs both showed a significantly higher affinity to the substrate GSH while a declined affinity to CDNB than those of susceptible strain. The inhibitory potential of ethacrynic acid was very effective with highest I₅₀ value (the concentration required to inhibit 50% of GSTs activity) of 1.21 μM recorded in DDVP-R. Carbosulfan also exhibited excellent inhibitory effects on purified GSTs. The N-terminus of the purified enzyme was sequenced by Edman degradation, and the alignment of first 13 amino acids of the N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.     

雷公藤总生物碱对粘虫的生物活性

为进一步评价雷公藤中主要杀虫活性成分生物碱的杀虫活性及其应用前景,采用室内生测法研究了雷公藤总生物碱对粘虫Mythimnaseparate(Walker)取食、存活及生长发育等的影响。结果表明,雷公藤总生物碱对粘虫幼虫具较强的拒食活性,3、4、5龄幼虫24、48h的拒食中浓度(AFC50)分别为37.92、50.23、119.53mg·L-1和42.39、60.47、122.91mg·L-1;具有一定的胃毒活性,对粘虫3龄幼虫4、5天的LC50值分别为157.18mg·L-1和129.92mg·L-1;对粘虫幼虫的生长发育有明显抑制作用,表现在体重、体重增加量和相对生长率均显著降低,60mg·L-1处理组幼虫第2天体重、体重增加量、相对生长率比对照分别下降26.13%、42.74%和22.26%,幼虫龄期延长,存活率、化蛹率和成虫羽化率均明显下降,成虫产卵量减少;此外,还具有一定的杀卵作用,卵孵化率和初孵幼虫存活率明显降低。     

Partial purification and characterization of a methyl-parathion resistance-associated general esterase in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Increased hydrolytic metabolism of organophosphate insecticides has been associated with resistance among Nebraska western corn rootworm populations. In this study, resistance-associated esterases were partially purified by differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor of 100-fold and recovery of approximately 10%. Kinetic analysis of the partially purified enzyme indicated that the K_m of the group II esterases was identical for the two populations, although V_max was consistently threefold higher in the resistant population. A putative esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII is a monomer with a molecular weight of approximately 66 kDa, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. Immunoassays with the Myzus persicae E4 antiserum indicated that group II esterases from D. v. virgifera were cross-reactive and expressed at much higher titers in the resistant population relative to the susceptible counterpart. These results suggest that the resistance is likely associated with overproduction of an esterase isozyme in resistant D. v. virgifera populations.     

拮抗真菌HTC的鉴定及其对辣椒疫病的生物防治潜力

为明确拮抗真菌HTC对辣椒疫霉Phytophthora capsici的拮抗机制,采用平板对峙、形态学鉴定和18S rDNA序列比对分析等方法对菌株HTC进行了鉴定,并研究其发酵液与抗生物质粗提液对辣椒疫霉不同发育阶段的影响。经鉴定,菌株HTC为金色毛壳菌Chaetomium aureum。在平皿对峙试验中,菌株HTC的红色分泌物能抑制辣椒疫霉菌丝的生长,抑制率为59.1%,后期HTC菌丝可缠绕并降解辣椒疫霉菌丝。发酵液与抗生物质粗提液对辣椒疫霉不同发育阶段均有抑制作用,发酵液对辣椒疫霉菌丝生长的抑制率高达97.58%,对辣椒疫病的防治效果均高于70%。浇灌发酵液后,可提高辣椒苗的苯丙氨酸解氨酶、过氧化物酶、多酚氧化酶的活性,并明显促进辣椒苗生长,鲜重和干重增加32.27%和18.09%。研究表明,金色毛壳菌菌株HTC是一株具有开发潜力的生防菌株。     

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol⁻¹. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml⁻¹ for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl₂, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca²⁺ and Mg²⁺ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6-8.2, 3.2 mM and 0.013-0.018 μM, respectively. The Lineweaver-Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.     

渐狭蜡蚧菌Lecanicillium attenuatum产几丁质酶的活性及对南方根结线虫卵孵化的抑制作用

为探究卵寄生真菌产生的几丁质酶对南方根结线虫卵孵化的抑制作用,采用分离自黑龙江大豆孢囊线虫孢囊上的菌株CGMCC5328,利用NAG和pNP法测定其几丁质降解酶系和外切酶的活性,分析几丁质酶粗酶液对南方根结线虫卵的孵化抑制效果.结果表明,菌株CGMCC5328经形态学和ITS序列分析鉴定为渐狭蜡蚧菌Lecanicillium attenuatum;在几丁质酶诱导培养基中培养第4天时其分泌的几丁质降解酶系达酶活高峰,为16.90 U/mL;第6天外切酶达酶活高峰,为0.59 U/mL,之后酶活趋于稳定持续至第14天;几丁质酶分子量分别为79.6、65.6、54.1、42.1和32.9 kDa;其中培养第7天的几丁质酶粗酶原液对南方根结线虫卵孵化的抑制率为83.17%;第6天菌株CGMCC5328对卵的寄生率为85.10%.表明高效产几丁质酶的卵寄生真菌渐狭蜡蚧菌菌株CGMCC5328对南方根结线虫卵孵化具有明显的抑制效果.