苏云金芽孢杆菌Cry8Ca2蛋白的纯化与活性的研究

cry8 Ca2基因是本实验室自行分离克隆的一种新型苏云金芽孢杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白基因,其表达130 kDa蛋白对铜绿异丽金龟(Anomala corpulenta)和黄褐异丽金龟(A.exoleta)幼虫具有较高的活性。本研究对Cry8Ca2蛋白原毒素及胰凝乳蛋白酶消化片断的纯化条件及活性进行研究,最适的消化条件为质量比1∶20,37℃,消化1 h。通过阴离子交换层析得到纯的活性毒素蛋白。用原毒素与纯化的毒素对铜绿丽金龟幼虫进行生测,Cry8Ca2原毒素蛋白的LC₅₀为0.47μg/g土,纯化的毒素的LC50为0.08μg/g土,毒素的杀虫活性可以达到原毒素的6倍。     

苏云金杆菌制剂毒素蛋白的SDS-PAGE-TLC定量分析

为了更合理地评价生物农药苏云金杆菌产品的质量,研究了苏云金杆菌制剂中毒素蛋白定量测定的方法。样品经溶解后,用SDS-PAGE对毒素蛋白(130KD)进行分离,用薄层扫描仪扫描区带面积定量。该方法RSD为3.01％～5.10％,准确度为96.61％～99.74％,蛋白含量与经生物测定得到的毒力效价之间有较好的相关性,可以作为评价我国苏云金杆菌产品质量的标准化方法。     

对暗黑鳃金龟成虫具有杀虫活性的Bt蛋白筛选

暗黑鳃金龟Holotrichia parallela是一种重要的农业害虫,其成虫和幼虫均能对多种作物造成严重危害。为筛选对暗黑鳃金龟成虫具有毒杀活性的Bt蛋白,本研究首先对暗黑鳃金龟成虫的生活习性进行观察,用浸泡蛋白的榆树叶饲喂成虫,建立了Bt蛋白对暗黑鳃金龟成虫的生物学活性测定方法;制备了6种Bt Cry8类杀虫晶体全长蛋白和胰蛋白酶消化蛋白,对暗黑鳃金龟成虫进行了杀虫活性测定。初筛结果发现Cry8Ga1原毒素和消化蛋白均对暗黑鳃金龟成虫具有较高毒杀活性,其在浓度1.0 mg/mL时对暗黑鳃金龟成虫的7 d校正死亡率分别为100%和55%。为更进一步确定Cry8Ga1蛋白的杀虫活性,进行了致死中浓度测定,7 d生测结果显示,Cry8Ga1原毒素的LC₅₀为0.387 mg/mL （95%置信区间为0.108~0.746 mg/mL）,Cry8Ga1蛋白经胰蛋白酶消化后的LC₅₀为0.702 mg/mL （95%置信区间为0.150~2.664 mg/mL）。取食Cry8Ga1原毒素蛋白及其消化蛋白后的暗黑鳃金龟成虫中肠细胞产生显著的病理学变化。本研究筛选到对暗黑鳃金龟成虫有毒杀作用的Bt蛋白,研究结果将为金龟子的生物防治开辟新的途径。     

苏云金杆菌Bt-59菌株Cry/Cyt毒素蛋白鉴定及杀虫活性

苏云金杆菌以色列亚种（Bacillus thuringiensis subsp.israelensis，Bti）是防治蚊幼虫的重要病原细菌，不同菌株间毒素蛋白种类和分布不尽相同。本研究利用苏云金杆菌以色列亚种Bt-59菌株的全基因组数据，对潜在的毒素基因种类、分布及菌株杀虫活性进行了分析。结果表明，Bt-59菌株除了含有Bti标准菌株主要毒素基因外，还含有Cry4-like基因，且Cry10Aa和Cyt1Ca基因在质粒4上发生了易位。质粒5上的Cry60Aa和Cry60Ba基因编码的毒素蛋白含有ETX_MTX2结构域，并与苏云金杆菌jegathesan亚种的同源蛋白一致性为100%。Cyt1Ca蛋白除了含有Bac_thur_toxin结构域外，还含有2个串联的RicinB_lectin_2结构域。菌株Bt-59对蚊幼虫具有较高的杀虫活性，对白纹伊蚊Aedes albopictus和淡色库蚊Culex pipiens pallens的LC₅₀分别为1.239 μL/L和0.341 μL/L。研究结果明确了菌株Bt-59毒素蛋白的种类和特征，为菌株遗传改良提供理论基础。     

黑曲霉xj粗提物的拮抗机制及其抗氧化活性

为探究黑曲霉Aspergillus niger xj 菌株粗提物SE对魔芋白绢病病原菌——齐整小核菌Sclerotium rolfsii R-67的拮抗机制及其抗氧化活性，采用菌丝生长速率法，研究了SE对R-67的抑制作用，进而通过测定处理后菌株的电导率、核酸蛋白泄露、三羧酸循环关键酶[琥珀酸脱氢酶 (SDH) 及苹果酸脱氢酶 (MDH)]比活力、胞内活性氧 (ROS) 水平及核酸含量，对SE的拮抗机制进行了研究，并通过1,1-二苯基-2-三硝基苯肼 (DPPH) 法及2,2-联氮基-双-(3-乙基苯并噻唑啉-6-磺酸) 二铵盐 (ABTS) 法测定了其抗氧化活性。通过气相色谱-质谱联用 (GC-MS)方法，从粗提物SE中共检测到12种化合物，其中主要成分为5-羟甲基糠醛(质量分数 97.96%)。生物试验结果表明:SE能够抑制R-67菌丝的生长，其IC₅₀值为0.46 mg/mL；SE处理可导致R-67菌液的电导率升高及核酸蛋白含量增加，抑制其三羧酸循环中关键酶SDH与MDH 的活力，增加菌体细胞内ROS含量，使细胞核4',6-二脒基-2-苯基吲哚 (DAPI) 染色的荧光强度减弱，据此初步推测其作用机制是通过影响R-67菌体细胞膜完整性、损伤细胞结构、抑制三羧酸循环及核酸合成等途径而发挥抑菌作用。研究表明，黑曲霉xj粗提物SE在魔芋白绢病的生物防治中具有良好的应用前景，此外SE对DPPH及ABTS自由基有一定的清除能力，有望应用于抗氧化功能性产品中。     

杀虫荧光假单胞工程菌对棉铃虫的离体及活体生物测定

利用转座子mini Tn5将δ 毒素基因cryIA(c)整合到生防细菌荧光假单胞菌P30 3 1的染色体上 ,构建成荧光假单胞工程菌。以棉铃虫二龄幼虫作供试昆虫的离体生测表明 :在同一剂量水平上 ,PT4 5、PT51、PT6 1、PT71等工程菌株对棉铃虫幼虫的杀虫活性均高于野生菌株HD 73;浓度最高时PT4 5、PT51、PT6 1、PT71、HD 73处理棉铃虫的死亡率分别为 86 7%、76 7%、80 0 %、86 7%、70 0 % ,死亡率与浓度呈正相关 ,体重与浓度呈负相关。用上述工程菌接种棉花活体 ,喷雾处理 3周后 ,用棉叶饲喂的棉铃虫死亡率分别达 70 0 %、6 0 0 %、6 0 0 %、6 6 7%、33 3% ;用棉顶尖饲喂的棉铃虫死亡率分别达 70 0 %、6 3 3%、6 0 0 %、73 3%、30 0 % ;用棉蕾饲喂的棉铃虫死亡率分别为 6 3 3%、53 3%、50 0 %、6 3 3%、2 6 7%。     

番茄叶霉病菌不同生理小种毒素的分离纯化与生物测定

采用凝胶过滤方法对番茄叶霉病菌6个生理小种所产生的毒素进行了分离、纯化,并利用叶片针刺法分别对其蛋白组分进行了生物活性测定,以探讨番茄叶霉病菌不同生理小种毒素组分对番茄叶片的致病力的影响.结果表明,每个生理小种毒素的纯化峰蛋白组分中只有一个蛋白组分能使番茄叶片褪绿,分别为来自生理小种1.2.3的第4个峰蛋白组分(记作:1.2.3(4),下同)、1.2.4(2)、1.2.3.4(2)、1.3(2)、1.3.4(2)和1.4(2).说明这几个峰蛋白组分对番茄叶片有一定的致病力,而其它峰蛋白组分对番茄叶片则没有明显致病力.透射电子显微镜观察结果显示,叶绿体膨胀或变形、基质片层部分消解,其中1.2.3.4(2)处理的叶绿体解体最彻底.     

水中12 种农药的固相萃取及GC-MS 测定方法研究

利用气相色谱法进行测定 ,用己烷和丙酮做洗脱溶剂 ,用 Isolute TMENV+SPE柱对水中 12种农药的固相萃取方法进行了研究 ,并用 MS选择离子模式进行了定量分析。水中添加浓度为 1、5、10μg· L-1三个水平 ,其中 11种农药的添加回收率在 6 2 .3%～ 10 5.0 % ,只有克百威的回收率较低。     

DON毒素和小麦互作对小麦幼穗细胞可溶性蛋白的影响

 本研究经小麦幼穗细胞和DON毒素混合处理,研究抗赤霉病程度不同的苏麦3号(R)、望水白(R)、延冈坊主(R)、Alondra's (S)、安徽11(S)和仪宁小麦(S)的可溶性蛋白谱带的变化。用3μg/ml的脱氧雪腐镰刀菌烯酸(DON)与小麦幼穗细胞混和处理2、8、24、30h,分别提取可溶性蛋白,进行PAGE测定。结果显示,感病品种有多条谱带消失,如仪宁小麦、安徽11、Alondra's在处理2h后Rf 0.13的条带消失,处理8h后Rf 0.02条带消失,感病品种Alondra's和安徽11在处理24h后Rf 0.05的条带消失。抗病品种的电泳谱带没有变化,如苏麦3号、望水白在毒素处理后蛋白条带没有发生变化,抗病品种延冈坊主在处理24h后RfO.OS、O.11的蛋白带仅有变浅的趋势。研究还表明抗病品种苏麦3号、望水白、延冈坊主具有感病品种没有的Rf 0.3的蛋白带,且在毒素处理前后保持一致。     

5.2%乐·齐EC防治蔬菜美洲斑潜蝇田间药效试验

1　试验材料与方法供试药剂 :5.2 %乐·齐 EC(湖北省农科院农药实验工厂 ) ;对照药剂 :1 .8%爱福丁 EC(北京农业大学试验药厂 )、40 %乐果 EC(福建三农化学股份有限公司 ) ;供试作物 :秋豇豆 (高产 5号 ) ;防治对象 :美洲斑潜蝇 (Lirionyza sativae Blanchard) ;试验处理 :a.5.2 %乐·齐 EC 1 0 0 0倍 (50 ml/667m2 ) ;b.5.2 %乐·齐 EC1 50 0倍 (33.3 ml/667m2 ) ;c.5.2 %乐·齐 EC2 0 0 0倍 (2 5ml/667m2 ) ;d.1 .8%爱福丁 EC2 0 0 0倍 (2 5ml/667m2 ) ;e.40 %乐果EC1 0 0 0倍 (50 ml/667m2 ) ;f.清水对照 CK。本试验设 6个处理 ,…     

Breeding Cassava for Resistance to Bacterial Blight

Abstract

It is noted that cassava bacterial blight (CBB) has been reported in several African countries and that heavy infection with CBB can lead to total crop failure as most local varieties are very susceptible. The best control method is through varietal resistance. IITA has made progress in breeding for resistance to both CBB and cassava mosaic disease (CMD) incorporating simultaneously other desirable agronomic qualities. The cassava breeding work at IITA since 1972 is then discussed covering tests on a total of 583 cassava families from CIAT, East Africa and IITA for sources of high resistance to CBB, further tests on 748 IITA clones for CBB resistance in a given ecological setting of high rainfall, sandy and acid soil from which the cultivar 58308 emerged highly resistant. Other aspects of the breeding work treated are conditions affecting varietal selection, namely: genetic mechanism resistance: stability of resistance to CBB over time and in different areas in Africa; high heritability; genetic correlation between resistance to CBB and CMD; correlation of CBB resistance with other characteristics such as tuber yield and leaf defoliation; and finally some factors affecting CBB field scores which are time of planting, age of plant and time of observation.

It is noted that the sources of high resistance to CBB identified have been quickly and successfully incorporated into promising cassava clones.     

Genotype × environment Effects on Severity of Cassava Bacterial Blight Disease caused by Xanthomonas axonopodis pv. manihotis

Nine cassava genotypes were grown for three years at six sites representing three agro-ecological zones in Nigeria to study their reaction to cassava bacterial blight (CBB), investigate genotype × environment (G × E) interaction patterns for their reaction to CBB, and to identify genotypes with stability to the disease, using the additive main effects and multiplicative interaction (AMMI) statistical model. Environments, genotypes and G × E interactions accounted for 71.8%, 12.0% and 16.2% of the treatment sums of squares (SS), and were highly significant (P<0.0001) for the disease, indicating that genotypes responded differentially to CBB infection across environments. Clones 30555, 91934, U/41044, and 4(2)1425 showed the least CBB disease ratings. Other clones showed erratic and fluctuating reactions to CBB from environment to environment and were thus considered unstable to the disease. CBB was most severe in 1989 (with a mean score of 2.46) and least so in 1990 (with a score of 2.06). The sites with the most disease were Ibadan, Ilorin and Ubiaja (1989), Ibadan and Ubiaja (1990) and Mokwa (1991). Because of the favourable conditions for disease development at those sites, they could be appropriate for screening cassava genotypes for CBB resistance. The AMMI model selected AMMI1 as the best predictor for CBB because it had the smallest actual root mean square prediction difference (0.37646), and explained 90.7% of the G × E interaction for CBB. The AMMI model was successful in selecting the genotypes 30555, U/41044 and 4(2)1425 and the environments Ibadan 1989, Ilorin 1989 and Onne 1990 with stability of reaction to the disease.     

印楝素和阿维菌素的液相色谱法同时测定

提出了一种可同时测定印楝素·阿维菌素乳油中印楝素和阿维菌素B1a的液相色谱法。该方法使用Hypersil ODS C18(250 mm×4 mm,5 μm)色谱柱和紫外检测器(印楝素λ 215 nm,阿维菌素B1a λ 240 nm),印楝素和阿维菌素B1a的回收率分别为97.5%~105.0% 和98.0%~107.5%,相对标准偏差分别为1.70%和1.60%,达到了简便、快速、准确的目的。     

Detection of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis>, the causal agent of cassava bacterial blight diseases in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) in Ghana by polymerase chain reaction

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight (CBB) disease. CBB is a major constraint to cassava cultivation in Ghana. In this study, a survey was conducted in eight regions of Ghana to assess the presence of CBB disease. Out of the eight regions visited, CBB, though at different prevalence, was observed in five regions. Cassava plants samples showing suspected bacterial blight symptoms were collected for analysis by Polymerase Chain Reaction (PCR). The results of the analysis showed that Ashanti region had the highest prevalence in percentage of CBB, which recorded (70%), followed by Volta region (60%); Brong Ahafo region (40%); Eastern region (40%) and Greater Accra region (20%). Morphological examination of the putative pathogen was carried out on Cefazolin trehalose agar (CTA) and Nutrient agar (NA) media. The isolates were subjected to conventional PCR using Xanthomonas genus specific primer, RST2/RST3, Xam specific Variable Number Tandem Repeat (VNTRs) loci, XaG1_67F/R and X-gumD primers, which produced 840, 446 and 402 bp, respectively. The isolates also tested positive with SYBR Green fluorescent dye, using Real-time PCR. The resulting PCR products were sequenced and analyzed using a BLASTn program, which revealed homology between 93 and 100% with several other Xam strains retrieved from GenBank nucleotide database. The pathogenicity test of the isolates on the susceptible Esam cassava variety produced symptoms typical of Xam and the pathogen was consistently re-isolated from the inoculated cassava plants and thereby satisfying the Koch’s postulates.     

基质固相分散-高效液相色谱法测定玉米中 7种苯氧羧酸类除草剂的残留

采用基质固相分散-高效液相色谱法(MSPD-HPLC)测定了7种苯氧羧酸类除草剂在玉米中的残留。玉米样品与基质固相分散介质C18化学键合硅胶(ODS)一起研匀得到基质固相分散体系,将其填入事前装有弗罗里硅土的净化柱中,以环己烷洗去油脂等杂质,再用乙酸乙酯洗脱得到分析液;在反相高效液相色谱(RP-HPLC)柱上,分析液经ODS柱以甲醇-水(体积比65∶ 35)为流动相洗脱,用紫外检测器检测,以外标法定量。在0.1和1.0 mg/kg添加水平下,7种苯氧羧酸类除草剂在玉米中的添加回收率为80% ~110%,相对标准偏差RSD为3.6% ~10.3%,检出限在7.9 ~12.6 μ g/kg之间。     

5种杀虫剂灌根施药对黄瓜烟粉虱的防治效果研究

以盆栽黄瓜为供试植物,利用灌根法,研究了5种杀虫剂对黄瓜烟粉虱的防治效果。结果表明:阿维菌素对黄瓜烟粉虱最高防效不足30%,吡虫啉、噻虫嗪、啶虫脒和烯啶虫胺灌根施药后7d,防效均在60%以上,10d防效达最高,其中吡虫啉130mg/L处理防效最高为94.3%,持效期长达20d。本研究为黄瓜烟粉虱防治药剂的合理使用提供了科学依据。     

Genetic Mapping and Chromosomal Assignment of Magnaporthe oryzae Avirulence Genes AvrPik, AvrPiz, and AvrPiz-t Controlling Cultivar Specificity on Rice

ABSTRACT A genetic map including three avirulence (Avr) genes, AvrPik, AvrPiz, and AvrPiz-t, was constructed in a genetic cross of two rice field isolates, 84R-62B and Y93-245c-2. The chromosomal locations of the Avr genes were determined by using selected markers to probe Southern blots of the parental chromosomes that had been separated by contour-clamped homogenous electric fields electrophoresis. Electrophoretic karyotyping showed that both parental isolates 84R-62B and Y93-245c-2 contained seven chromosomes greater than 3.5 megabases (Mb) in size and 84R-62B possessed a small chromosome of approximately 1.6 Mb. The linkage groups containing AvrPiz and AvrPiz-t were assigned to chromosomes 3 and 7, respectively. Some markers from the linkage group that contained AvrPik hybridized with chromosome 1 and the 1.6-Mb chromosome, yet all of the cloned RAPD markers that were closely linked to AvrPik hybridized exclusively to the 1.6-Mb chromosome in 84R-62B, the parent that possesses AvrPik. Thus, we conclude that AvrPik is located on the 1.6-Mb chromosome in 84R-62B.     

Temporal Progression of Bean Common Bacterial Blight (Xanthomonas Campestris pv. phaseoli) in Sole and Intercropping Systems

The effects of four planting patterns of bean (Phaseolus vulgaris) (bean only, maize–bean (MB), sorghum–bean (SB), and maize–bean–sorghum (MBS)) and four cropping systems (sole cropping, row, mixed, and broadcast intercropping) on the temporal epidemics of bean common bacterial blight (CBB) caused by Xanthomonas campestris pv. phaseoli were studied. The experiments were conducted during two consecutive spring and summer seasons in 1999 and 2000 in replicated field experiments. The Gompertz model described disease progress curves better than the logistic model. Intercropping delayed epidemic onset, lowered disease incidence and severity, and reduced the disease progress rate. The type of cropping system and planting pattern affected CBB incidence and severity at initial, final and overall assessments and also affected the rate of disease development. Statistical significance of treatment interactions based on disease assessments was found for incidence in all four experiments and for severity in three experiments. A slower disease progress rate and lower incidence and severity occurred on beans planted with maize or sorghum in row, mixed and broadcast intercropping than on bean planted alone. Incidence was reduced 36% and severity 20% in intercropping compared to sole cropping. The built-in disease delay and the slowing of the disease progress rate could provide protection for beans from severe CBB epidemics in intercropped systems. Variation between years appeared to be related to relative humidity (RH).     

Relationship between ineffective R-genes and expansion rate of lesions on potato leaves, caused by Phytophthora infestans

Progenies from eight tetraploid potato crosses were assessed for the association between ineffective R- genes and lesion expansion rate (LER) in mm per day on detached leaves that had been inoculated with Phytophthora infestans . In two of the progenies, a significant association between ineffective R- genes and LER was measured, which could be an indication of residual effects. No association was found in the other progenies. These results and other corroborative evidence do not support an approach to plant breeding based on the stacking of R- genes to accumulate residual resistance effects. Using the same detached-leaf test, differences in aggressiveness between two isolates of P. infestans could be measured in all eight progenies. LER values were consistent with previously published ranges and were distributed continuously and unimodally in all progenies, suggesting that differences in relative aggressiveness of isolates were probably not affected by R- genes.     

复合微生物肥对植物土传病原真菌的抑制作用

测定了两株链霉菌R 1、R 2及其分别与有机肥配制的两种复合微生物肥料对几种植物病原真菌菌丝生长和孢子萌发的影响。R 1对草莓灰霉病菌、棉花黄萎病菌、棉花枯萎病菌的抑菌圈直径分别是 2 7、1 4和 2 1cm ,对孢子萌发的抑制率分别是 4 6 %、1 9%和 1 1 % ;R 2对以上 3种病菌的抑菌圈直径分别是 3 1、1 2和 1 7cm ,对孢子萌发的抑制率分别是 4 2 %、1 2 %和 4 %。苗期人工接种试验表明 ,一号复合微生物肥料对棉花黄萎病、油菜菌核病的抑制率分别为 2 8 0 %、4 1 9% ,二号肥分别为 72 0 %、97 2 %。