31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCR RFLP鉴定体系 ,鉴定了 31株Bt菌株的cry基因类型 ,并进行了SDS PAGE分析和杀虫生物活性测定。研究表明 :2 5株含cry1基因 ,表达蛋白 1 30～ 1 50kD ;其中 1 6株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因 ,其表达蛋白为 81kD ;1 5株同时含有cry1和cry2基因 ( 1 3株表达蛋白约为 60kD) ;1 0株含有未知待定基因 ;6株不含所鉴定的cry基因 (其中 2株有表达产物 )。室内生物测定表明 :cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性 ,7株对舞毒蛾和膜翅目———杨叶蜂幼虫具有较高杀虫活性 ;含有cry1Aa、cry1Ac、cry2或cry1Ab、cry1Ac、cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性 ,其中 6、1 2、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明 ,通过cry基因类型鉴定和表达产物的SDS PAGE分析可以预测菌株的杀虫活性。

生物农药苏云金芽孢杆菌的优化培养研究

本文采用光(?)度法观察苏云金芽孢杆菌发酵过程,对培养液的pH值,培养温度、合氧量和培养成分进行了系统研究。实验观察到,Bt的最佳培养的条件是:在pH值为7.4,温度为30℃的条件下,使用液体发酵培养基,以250次/分的振荡频率在恒温震荡器中培养36小时。

抗虫双价基因表达载体的构建及微束激光转化植物研究

将苏云金芽孢杆菌伴孢晶体(Bacillus thuringiensis,Bt)蛋白的基因与反枝苋菜凝集素(Amaranthus ret-roflexusagglutinin,ARA)基因构建成双价基因的表达载体,编码一种既能抗鳞翅目害虫,又能抗同翅目蚜虫的杀虫蛋白。把双价基因(Bt-BA)连接到原核表达载体pET28a和植物表达载体pBI121上,经过限制性酶切分析和PCR鉴定,结果表明:含有双价基因的原核和真核重组表达质粒均已构建成功。将该双价基因转入油菜,获得抗性小植株。

Screening on Synergist of Bacillus thuringiensis Wettable Powder

[Objective] This study aimed to screen the best synergistic material for Bt wettable powder and evaluate their synergistic effect. [Method] The synergism of six different kinds of additives for Bacillus thuringiensis wettable powder （Bt WP） on the 2^nd instar larvae of Plutella xylostella was tested by method of leaf dipping in labora- tory. [Result] The mixtures of Bt with 0.1% ZnCl2, 0.5% ZnCl2, 1.0% ZnCl2, 1.0% MgCI2, 0.5% boric acid, 1.0% boric acid, 0.5% citric acid or 1.0% citric acid all ex- hibited synergistic effect, in which the synergistic effect of mixture containing 0.5% boric acid was the highest, with 17.2 synergistic ratio; followed by the mixture containing 1.0% ZnCl2, with 15.6 synergistic ratio. Moreover, addition of 0.5% boric acid could shorten the median lethal time of Bt wettable powder by about 10 h. After the mixtures of Bt with 0.5% boracic acid or 1.0% ZnCl2 was stored for 15 d at room temperature, toxicities of the two mixtures did not change significantly. [Conclusion] Boracic acid as the synergist of Bt wettable powder could not only increase insecti- cidal effect of Bt, but also accelerate its insecticidal rate. So, boracic acid could improve the disadvantages of Bt wettable powder such as poor insecticidal effect and slow insecticidal speed in a certain degree.

抗虫杀菌融合基因表达载体的构建及微束激光转化植物研究

将苏云金芽孢杆菌伴孢晶体蛋白的基因(Bacillus thuringiesis,简称Bt)通过甘氨酸接头(Gly4Ser)3与一种人工合成的抗菌肽(antimicrobial peptides,AMP)基因与相融合,编码一种新的杀虫,并具有抗菌的蛋白。把融合基因(NAMP-Bt)连接到原核表达载体pET-28a和植物表达载体pBI-121上,经过限制性酶切分析和PCR鉴定,结果表明含有融合基因的原核和真核重组表达质粒均已构建成功,并将该融合基因转入烟草,已获得抗性小植株。

Isolation of an acid producing Bacillus sp. EEEL02: Potential for bauxite residue neutralization

Bauxite residue deposit area(BRDA)is a typical abandoned mining wasteland representing extreme hostile environment with increased alkalinity.Microbially-driven neutralization of bauxite residue,based on the microbial acid producing metabolisms,is a novel strategy for achieving rapid pH neutralization and thus improving its environmental outcomes.The hypothesis was that these extreme conditions promote microbial communities which are capable of novel ecologically relevant functions.Several alkaliphilic acid producing bacteria were isolated in this study.One strain was selected for its superior growth pattern and acid metabolism(termed EEEL02).Based on the phylogenetic analysis,this strain was identified as Bacillus thuringiensis.The optimized fermentation conditions were as follows:pH 10;NaCl concentration 5%;temperature 25℃;EEEL02 preferred glucose and peptone as carbon and nitrogen sources,respectively.Based on optimal fermentation conditions,EEEL02 induced a significant pH reduction from 10.26 to 5.62 in 5-day incubation test.Acetic acid,propionic acid and CO2(g)were the major acid metabolites of fermentation,suggesting that the pH reduction in bauxite residue may be caused by acid neutralization derived from microbial metabolism.This finding provided the basis of a novel strategy for achieving rapid pH neutralization of bauxite residue.

Ecological Consequences of Elevated CO2 and Bt Cotton on Soil Collembola

Transgenic cotton was modified to express a gene derived from the bacterium Bacillus thuringiensis （Bt） to combat agriculturally important Lepidopteran pests. Elevated CO2 is expected to further alter the chemical composition of the plant, and this change may affect the role soil fauna plays in decomposition of Bt plants. A 3 months litterbag field study, consisting of four treatments using leaves from Bt cotton and near-isolines of non-Bt cotton grown under ambient and elevated CO2 levels, was conducted to investigate the abundance and community structure of soil Collembola that developed on the decaying leaf material. A total of 4,884 collembolans, including 13 genera of five families, were extracted in the present study. These results suggest that collembolan distribution was relatively uniform among the Bt cotton, elevated concentration of CO2 and control treatments, except for a significant difference in the densities of Onychiurus and Folsomides. No significant effects were detected in the decomposition rate between the two cotton varieties and two CO2 treatments. These findings indicated that transgenic Bt cotton plants and elevated CO2 do not have any adverse effect on the soil collembolans through the decomposition way in soil ecosystem.

Properties study and phylogenetic analysis of a bacterial strain with high de-emulsification efficiency

For further practical application, the phylogenetic analysis and de-emulsification properties study of strain XH1 with high de-emulsification efficiency isolated from crude oil contaminated soil in Daqing oil field were conducted with a surfactant-stabilized water-kerosene model emulsion. The factors influencing the de-emulsification efficiency and the generation site of de-emulsification active component of the strain were also investigated. The similarity of 16SrDNA sequences between strain XH1 and Bacillus mojavensis （DQ993678）was 99%. According to the physiological biochemical test, strain XH1 was preliminarily identified as Bacillus mojavensis. The logarithmic growth, stable phase and decline phase of strain XHI were determined as 14, 18 and 28 h, respectively. The best de-emulsification activity emerged after cultivating for 18h, and the complete de-emulsification was achieved at 24 h. The most favorable incubation conditions for de-emulsification occurred with pH of 6. 0 at 30 ℃. The de-emulsification capability of strain XH1 was mainly resulted from extracellular metabolites. The above results indicate that strain XH1 has high de-emulsification efficiency and is potential as a commercial de-emulsifier.

General Evaluation of Biocide （Bt-ASF-1） Produced from Iraqi Isolate of Bacillus thuringiensis

General evaluation of isolate Bacillus thuringiensis （Bt-ASF-1） used as biocide in meddle scale application was conducted. Some morphological and confirmation tests were achieved. The sensitivity tests had been accomplished by diffusion and dilution techniques to determine the response of isolate against the antibiotics. The results of diffusion tests showed to the sensitivity of bacteria to antibiotics of cefixime, erythromycin, gentamicin and tetracycline respectively. It was resistant to trimethoprim sulfonamide （TMP）, bacitracin, penicillin and all its generations, and moderate resistance to nalidixic acid. Minimum Inhibitory Concentration （MIC） for amoxicillin was ranged between 30-40 pg/mL and these results are an approximation of the universal findings. Curing experiments showed the effective role of sodium dodecyl sulfate （SDS） （1.5%） comparing with temperature. The bacterial cells became sensitive to amoxicillin and TMP. The curing by temperature did not differ significantly from control treatment in plasmid pattern or antibiotics response. Plasmid profile referring that curing by SDS has been caused disturbance in beta -lactamase genes through the sensitivity to amoxicillin and remaining resistance to ampicillin. Curing isolate by SDS also became more sensitive to nalidixic acid, erythromycin and tetracycline respectively. It was found from the curing treatments the complexity distribution of r-genes between different plasmid size and chromosome but not effect on their insecticidal ability.

Bt生物农药的水解及水解产物酪氨酸的HPLC检测

本文对目前颇具前景的生物农药—Bt的酸水解规律进行了研究,得出酸水解的最佳时间、水解剂用量和温度,并在这一条件下水解了施安牌Bt生物农药,旋转蒸发器浓缩水解样,采用HPLC法检测了水解产物酪氨酸的含量。

A New Strain of Entomopathogenic Bacillus of the Tea Pest, Eterusia magnifica （Butl.） （Zyganiidae： Lepidoptera） from Darjeeling Foothill Region of India

An entomopathogenic strain of Bacillus sp. was isolated from diseased red slug caterpillars of the leaf-feediug pest of tea, Eterusia magnifica, from the Darjeeling foothill region. Analysis of the bacterimn based on polyphasic approach such as growth phase, biochemical tests, whole body＂ protein, crystal protein profiles along with bioassay （i.e. LC50 and LT50 values） established it as a different strain but close to Bacillus thuringiensis kurstaki （Btk）, the commercial microbial pesticides of lepidopterans. Among biochemical parameters differences were noted between the new strain and Btk in ONPG, lysine decarboxylase, omithin decarboxylase, urease, nitrate reduction, V-P and glucose utilization tests. PAGE analysis of the whole body protein for the new strain recorded a 34 kDa band which was absent in Btk （used as reference）. Crystal protein profile of the newly isolated bacterial strain showed 53 and 49 kDa bands whereas in Btk only 52 kDa band was evident. Although the LC50 values of the new strain and Btk were close, their LT50 values were much different, the new strain showing a lower value than Btk. In light of the above differences and in absence of any report of entomopathogenic bacterial strain of E. magnifica, the isolated strain of Bacillus appeared to be new to science and hence was designated as RS01. The new strain opens up the possibility of its futttre use as microbial pesticide after standardizing its formulation and checking its safety aspects.

Alkalibacillus halophilus subsp. hitensis subsp. nov.:A new subspecies of moderately halophilic bacterium isolated from a chemical plant in China

In order to develop halophilic microorganism resources to improve environment, a Gram-positive, strictly aerobic and moderately halophilic bacterial strain JSA1 was obtained from the waste water sample collected from Jinhong Chemical Plant at Weihai city, by the methods of quick isolation and screening of halophilie bacteria. Systematic studies on it were carried out. Results show that the strain JSA1 is bacillus. The temperature range most suitable for its growth is 29 - 35 ℃ and the most suitable pH is 6. 5 - 9. 0. It can grow well at the salt mass concentration of 30 - 150 g/L. The C ＋ G mole fraction of its DNA is 37.5%. The analytical result of 16S rRNA gene sequence reveals that this strain has the closest relationship with Alkalibacillus halophilus （DQ359731） of Alkalibacillts. Their similarities are as high as 99%. However, they have obvious differences in aspects of whole-cell main fatty acid components, cell size, cell morphology, motility, oxidase, gelatine liquefication, NaCl tolerance range, pH tolerance range, G ＋ C mole fraction, sole carbon source, sole nitrogen source, antibiotic sensitivity and strain source. Comparing with other species of the same genus, differences of this strain are even more obvious. In view of muhiple identification results, we believe this strain is a new subspecies ofAlkalibacillus halophilus and name it Alkalibacillus halophilus subsp, hitensis subsp, nov.

The Impact of Beauveria bassiana, Bacillus thuringiensis （Bt） Spray, Trichogramma pretiosum and Spinosad on the Lepidoptera： Crambidae, European Corn Borer （Ostrinia nubilalis）

The research cxamined the effects of microbial and novel insecticidal control strategies and also Irichogramma pretiosum on the I,epidoptera： Crambidae, the European corn borer （ECB）, （Ostrinia nubilalis）. The treatments included ~ino^d, from bacteria the entomopathogenic fungus Beauveria bassiana, the egg parasitoid Trichogramma pretiosum, and foliar applied Bt spray, and t^vo combinations Bearveria bassiana plus Bt spray and Trichogramma pretiosum and spinosad. The infestations by the European corn borer larvae on stalks of corn were very＂ severe in all the control treatments both 2006 and 2007. The microbial treatments caused various levels of European corn borer mortality. In all the parameters there was a significant difference between the control and other treatments with a P 〈 0.00 l. In this research, treatment with spinosad emerged as the most effective biological agent in the control of the European corn borer. The treatments had no significant effects on the relative abundance and composition of non-target arthropods （P 〉 0.05）. This research is relevant in boosting underutilized control strategies and increasing stakeholder adoption of integrated pest management practices and thereby＇ reducing the use of conventional insecticides especially for orgamc thrmers. This is very important especially in certain countries where the commumities are skeptical about consuming transgenic crops.

Efficacy of Trichogramma evanescens and Bacillus thuringiensis kurstaki to Control Lesser Date Moth Batrachedra amydraula Merck

Efficacy of the egg parasitoid, Trichogramma evanescens and the bio-pesticide bacteria, Bacillus thuringiensis kurstaki （Btk） in reduction of lesser date moth, Batrachedra amydraula infestations were studied during seasons of 2011 and 2012. The result of 2011 showed that the release of egg parasitoid by rate of 500 and 1,000 parasitoids/palm tree achieved 55.06% and 67.45% reduction in infestation percentage, respectively. Meanwhile high reduction in infestation was achieved by Btk （78.65%） at dose rate of 6 g Btk powder/kg talc powder. The result of 2012 showed that the highest control efficacy （58.78%） was achieved at the first time of the control process, using Btk at dose rate of 6 g/kg talc powder. This result was not different significantly from the release of egg parasitoid by rate of 1,000 parasitoids/palm tree which achieved 53% reduction in infestation percentage. At the second time （after one week from the first application）, the efficacy of Btk at dose rate of 6g/kg talc powder was increased to 78.78%, which differ significantly with the release of egg parasitoid by the same rate of 1,000 parasitoids/palm tree, having 61.79% efficacy. Reapplication of the control method achieved an increase in efficacy of the control agents, such increase was significant for both the release of the parasitoid at rate of 1,000/palm tree and for Btk at a dose of 6 g/kg talc powder.

Sex-Related Larval Susceptibility of Diamondback Moth, Plutella xylostella （Lepidoptera：Plutellidae） to some Reduced-Risk Insecticides

Five reduced-risk insecticides were compared for their toxicities to male and female third instar larvae of diamondback moth （DBM）, Plutella xylostella L. （Lepidoptera：Plutellidae） obtained from a laboratory colony. Leaf-dip bioassays were used to assess larval mortality at different insecticides rates （ranging from 0.01 mg AI/L to 100 mg A1/L） and exposure times （24, 48 and 72 h after treatment）. Toxicity of the insecticides generally increased with rate and exposure time. At 72 h after treatment, median lethal concentrations （LCs0s） of methoxyfenozide, spinosad, novaluron, indoxacarb and Bacillus thuringiensis （Bt） against the male third instar larvae of P, xylostella were 0.0524, 0.1117, 0.9149, 1.1939 and 4.4983 mg AI/L, respectively, and were 0.1008, 0.2583, 1.0649, 0.2850 and 5.3053 mg AI/L against female third instar larvae, respectively. At 0.1% of the approximate recommended field rates, methoxyfenozide （60%-75%） and spinosad （53%-57%） were the most toxic, while Bt （30%-45% mortality） was the least toxic to male and female P. xylostella larvae. Spinosad was the fastest acting and the only insecticide that caused significant larval mortality （17%） at 24 h after exposure. Significant sexual differences were recorded in the susceptibility of P. xylostella larvae to two of the insecticides： spinosad and indoxacarb. Male larvae were two-fold more susceptible to spinosad but 10-fold more tolerant to indoxacarb than female larvae.

Toxicity of Bacillus thuringiensis Crystal Toxins to Field Populations of Rice Leaf Folder, Cnaphalocrocis medinalis （Guenee） and Establishment of Baseline Susceptibility to Cry1Ab

Eight insecticidal crystal proteins of Bacillus thuringiensis, CrylAa, CrylAb, CrylAc, CrylB, Cry2Aa, CrylC, CrylDa and Cry 1Ea were assessed for toxicity against 1 st instar larvae of rice leaf folder, Cnaphalocrocis medinalis （Guenee） at 48 HAT and 72 HAT. Bioassay results depicted CrylAa was the most toxic （LCso 2.35 ppm） followed by CrylBa （LCso 8,50 ppm） and CrylAb （LCso 8.73 ppm） at 48 HAT, whereas, at 72 HAT CrylAb proved to be highly toxic （LC50 0.50 ppm） followed by CrylAa （LCso 4.07 ppm）, CrylAc （LCso 4,84 ppm） and CrylBa （LCso 6.42 ppm）. Toxins Cry2Aa, CrylCa, CrylDa and CrylEa did not resulted in any mortality at 48 HAT and 72 HAT, respectively. Baseline estimates for CrylAb against 1st instar larvae of C. medinalis sampled from seven geographical locations revealed variation in LC50＇s from 0.37 ppm to LC50 16.25 ppm at 48 HAT and LC50 0.50 ppm to LC50 6.49 ppm 72 HAT, respectively with relative resistance ratios of 44-fold and 13-fold at 48 HAT and 72 HAT over the susceptible population.

Economic and Environmental Impact Transgenically Modified Cotton Comparative with Synthetic Chemicals for Insect Control

This article combines both a review of the literature and original data from our research. The comparative efficacy of Bacillus thuringiensis （Berliner） traits Bollgard, Bollgard II, WideStrike, and non-Bt expressing cotton, Gossypium hirsutum L., for control of the noctuid complex of bollworm, Helicoverpa zea （Boddie）; fall armyworm, Spodopterafrugiperda （J.E. Smith）; beet armyworm, Spodoptera exigua （Htibner）; and cabbage looper, Trichoplusia ni （Hiibner）, were evaluated in the Lower Rio Grande Valley of Texas during 2005-2009. Noctuid larval survival and damage to leaves on non-Bt cotton were 3.6-fold greater than on Bollgard II or WideStrike cotton and 1.5-fold greater than on cotton varieties with the Bollgard trait. Transgenic cotton has reduced the need for conventional insecticides with benefits to human health and the environment. The revenue differences between Bt and conventional cotton for last four years in LRGV of Texas was 214.35/ha and profit about 94.95/ha. We also analyzed cotton noctuid losses between Bt and conventionally grown cotton in the USA summary of all states and Texas summary of all regions.

Carboxylesterase and Glutathione-S-Transferase （GST＇s） Induced Resistance to Bacillus thuringiensis Toxin CrylAb in Rice Leaf Folder, Cnaphalocrocis medinalis （Guenee） Populations

The rice leaf folder （RLF）, Cnaphalocrocis medinalis （Guenee） （lnsecta： Lepidoptera： Pyralidae）, is an important pest, widely distributed in many rice growing areas of Asia. The over-use of broad-spectrum chemical insecticides has been cited as a major cause of outbreaks of C. medinalis as excessive spraying of insecticide disrupts natural biological control insecticides still remain the major control tactics against leaf folder. Carbofuran and fenthion, bendiocarb, acephate, carbosulfan, quinolphos, monocrotophos, phosphamidon and fenvalerate are the common ones used against rice leaf folder. Genetically, modified rice lines expressing B. thuringiensis insecticidal crystal proteins produced are highly tolerant to leidopteran pests. Though economic and environmental benefits of GM crops is well established, the matter of concern is the possibility of target insect pest developing resistance to this B. thuringiensis insecticidal toxins, evident from many laboratory and field experiments against many insect pests. The involvement of GSH S-transferase, carboxylesterase, and microsomal monooxygenase in insecticide resistance has been reported in insecticide-resistant strains of many insect species. Hence, the present study was taken up to monitor for cross resistance between B. thuringiensis cry toxins and synthetic insecticides in larvae of leaf folder as it is mediated by carboxylesterase titre and other enzymes by bioassay for two selected rice leaf folder field populations at the Entomology division of Directorate of Rice Research which showed 2-fold resistance ratio. Qualitative and quantitative changes of carboxylesterase （CarE） and glutathione-s-transferase （GST＇s） were worked out with midguts extracts of the two C. medinalis populations in the presence of a-napthyl acetate and chlorodi-nitro benzene substrates.

An Advanced Catch-and-Release Trap for Controlling the Red Palm Weevil

The aim of the research was to develop a catch-and-release trap for monitoring and controlling RPW （Rhynchophorus ferrugineus, red palm weevil）. The RPW insects were attracted through the use of an aggregation pheromone located into a dispenser inside the trap. The RPW adults entered through an entrance door into a sterilization room where they were sprayed with a solution containing the chemical sterilizer product ＂lufenuron＂. The adults were then forced to move into a contamination room where it was present a wet media containing spores and hyphae of Metarhizium anisopliae and spores and crystalline insecticidal proteins of Bacillus thuringensis. The sterilized and infected RPW adults were counted and released into the environment where they reached new palms and join natural populations of RPW. Preliminary experiments showed that the catch-and-release trap was able to reduce the fertility of RPW adults and to increase their mortality after contamination with entomopathogens. Moreover, the infected RPW adults were able to infect untreated RPW larvae leading to a larvae mortality of 93% within 30 days. In conclusion, the advanced catch-and-release trap represents a safe and effective tool to monitor and control the population of RPW pest without environmental pollution and risks for human health.

Agrobacterium-Mediated Transformation of Cry8db Gene in Vietnam Sweet Potato Cultivar

Sweet potato [Ipomoea batatas （L.） Lam.] is an important food crop in the world as well as in Vietnam. It is well known as a recalcitrant crop for gene transformation and tissue culture because of its genotype dependent in vitro responses. In present study, Agrobacterium-mediated transformation of cry8Db from Bacillus thuringiensis into KB 1 sweet potato variety has been studied. The C58cv strain carrying a pBl 121 backbone which contained cry8Db delta-endotoxin gene regulated under 35 S CaMV prom oter, and the selection marker gene, neomycin phosphotransferase （npt11） gene, was subjected for plant transformation. Callus induced from shoot tips and leaf explants were inoculated and cocultured with A. tumefaciens. The selection occurred during callus producing and plant regenerating steps. A total of 201 transgenic putative plant lines were produced, and 21 transgenic lines were positively confirmed by PCR and finalized by Southern blot. Four putative transgenic lines confirming a single copy of the crySEIb gene were transferred into soil pots in greenhouse. Biological activity evaluation for the insecticidal capacity of these transgenic lines under controlled conditions showed that the level of infestation by sweet potato weevils （Cylasformicarius） in untransformed plants was higher than that of transgenic lines.