对黄曲条跳甲高毒力Bt菌株的鉴定及田间药效评价

为了探索蔬菜害虫黄曲条跳甲的生物防治措施,从海南省土壤中分离出的一株对黄曲条跳甲高毒力Bt菌株BS128,根据形态学和分子水平鉴定,明确该菌株属于苏云金芽胞杆菌,含有cry8Ga新型杀虫编码基因。通过室内生物活性评价,苏云金芽胞杆菌BS128菌悬液对黄曲条跳甲3 d的LC50值为60.0093μg/mL,250μg/mL BS128菌悬液9000 mL/hm^(2)土壤处理对黄曲条跳甲防治效果为67.66%(15 d),250μg/mL BS128菌悬液9000 m L/hm^(2)叶面喷雾对黄曲条跳甲7 d和10 d防效分别为61.86%和62.16%。结果显示2株Bt菌株土壤处理防治跳甲效果不低于叶面喷雾防治效果。我们认为利用Bt防治黄曲条跳甲更适用土壤处理,一方面重视了土壤中黄曲条跳甲幼虫的防治,可以减缓苗期成虫的危害;另一方面Bt在土壤中更利于发挥作用,持效期长,避免了叶面喷施Bt受到高温或者强紫外线照射失活。

冻土毛霉菌株BO-1对韭菜迟眼蕈蚊的致病力及防治应用评价

冻土毛霉菌株BO-1是新报道的韭菜迟眼蕈蚊病原真菌,但是关于其致病力和田间防效的针对性研究尚未见报道。本研究分析了冻土毛霉菌株BO-1对韭菜迟眼蕈蚊幼虫的致病力,探究了温度对菌株致病力的影响,并通过防效试验评价了其应用价值。结果表明,冻土毛霉菌株BO-1对韭菜迟眼蕈蚊幼虫致病活性较高,处理3 d对2龄和4龄幼虫的致死中浓度LC50分别为3.714×10^(5)和4.680×10^(6)孢子/mL;冻土毛霉菌株BO-1发挥高致病活性的适宜温度为16~28℃,尤以20~24℃最优。盆栽和田间试验证实,菌株BO-1对韭菜迟眼蕈蚊防效突出,并具有较好的持续控害作用。当孢子浓度为1×10^(9)孢子/m L时,处理后7 d防效为95.57%,与化学杀虫剂噻虫胺(93.57%)相当。因此,将冻土毛霉菌株BO-1作为韭菜迟眼蕈蚊生防菌剂加以进一步开发可行性较强。

水环境样品体外生物测试效应触发值的研究进展

水环境样品综合毒性的体外生物测试可以直接获得水环境中众多污染物共存状态下的生物毒害信息,已成为水环境污染评估和诊断的重要手段之一。但由于目前没有用于判定水质优劣的毒性效应限值标准,导致其很难被用于水质管理。为此,越来越多的研究聚焦于推导体外生物测试的效应触发值(effect-based trigger value,EBT)。本文综述了EBT建立的背景和推导原则,并总结了以健康保护为目标和以生态保护为目标的多种EBT推导方法,其中针对饮用水水质的EBT以保护人体健康为前提,主要基于每日容许摄入量(allowable daily intake,ADI)、体内安全浓度、癌症发病率增加10%的每日剂量(TD10)和环境质量标准(environmental quality standard,EQS)中的水质指导值(guideline value,GV)推导,针对地表水的EBT以生态保护为目标,依据地表水EQS中的GV值和物种敏感度分布(SSD)曲线中的第5个百分位数的危险浓度(hazardous concentration,HC5)推导。本文系统比较了不同方法推导出的体外生物测试EBT值和特征,总结了EBT在水环境中的应用,以期为高通量体外生物测试用于水质评估提供理论依据和技术支撑。

不同遗传背景的转Cry1C基因玉米品系的室内抗虫性鉴定

以转Cry1C基因的转基因玉米C492为供体,通过回交转育的方式将目标基因分别导入不同遗传背景的玉米自交系,获得转基因玉米自交系C492/N-76、C492/N202、C492/S273、C492/DH1901和C492/DH1904,采用室内离体组织生测的方法研究各品系对玉米螟和草地贪夜蛾的抗虫性。结果表明:转基因玉米C492和5个不同遗传背景自交系均表现出高抗玉米螟,虽对草地贪夜蛾的抗性有一定差异,但接虫6 d后幼虫死亡率均达到80%以上。这说明转基因玉米C492和5个不同遗传背景自交系可用于抗虫转基因玉米新品种培育,具有较好的商业化应用前景。

Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section （influents） and 9 from post-treatment section （effluents）, were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 17β-estradiol （E2）, estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

<i>In Vitro</i>Bioassay of Allelopathy in Four Bamboo Species;<i>Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis</i>, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku;Phyllostachys bambusoides cv. Madake;P. nigra cv. Hachiku;Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

A Bioassay for the Cytotoxicity of Gemcitabine Using the Marine Ciliate Euplotes vannus

This study investigated the cytotoxicity of gemcitabine using the marine ciliate Euplotes vannus as the test organism.The median lethal concentrations(LC50 values)were determined using acute toxicity tests within an exposure time of 30 min with 0,6,12,24,and 48 mg mL^-1 gemcitabine.The median inhibition effect(IC50 value)on the growth of the ciliate cells was examined using chronic toxicity tests within 5 days(120 h)after exposure for 30 min with 0,0.7,3.5,7,and 14 mg mL^-1 gemcitabine.The 30-min LC50value was 10.66-mg mL 1.The LC50 values decreased with increasing exposure times and well fitted to the toxicity curve equation LC50=10.93+28.4e^-0.19t(R2=0.93;P<0.05,t=exposure time).The IC50 value for growth rates was 7.05 mg mL^-1,and the inhibition effect on growth rates well fitted to the model equation r%=0.8681e^-0.0782Cgem(r%means growth rate with inhibition by gemcitabine,Cgem means concentrations of gemcitabine,R^2=0.99 and P<0.05).The LC50 values of a wide range of gemcitabine concentrations could therefore be predicted for any given exposure time.These results suggest that the clinical dose of gemcitabine(20 mg mL^-1)was higher than the 30-min LC50 value,which was almost the same as the 6-min LC50 value(19.88 mg mL^-1)for E.vannus cells.The results also demonstrate that E.vannus can be used as a robust test organism for bioassays of chemotherapeutic drugs during short exposure periods.

Evaluation of Allelochemicals, Abscisic Acid and Coumarin, in Leaf-Origin Suspension Cultured Cells of <i>Prunus yedoensis</i>Using Protoplast Co-Culture Bioassay Method

Dried leaves of Prunus yedoensis and P. lannesiana (50 mg) showed strong inhibitory allelopathic activities, e.g., more than 97% growth inhibition of lettuce seedling using the sandwich method. Similarly, among suspension cultures induced from leaves and peduncles of two Prunus species, we found the strongest inhibitory allelopathic activities of protoplasts of leaf-origin suspension cells of P. yedoensis, when the protoplast co-culture method for bioassay of allelopathy was applied with lettuce as a recipient plant. Effects of two putative allelochemicals, abscisic acid and coumarin, on both protoplast cultures of lettuce and P. yedoensis were investigated. Coumarin inhibited the growth of lettuce protoplasts from low concentrations, while abscisic acid stimulated. Abscisic acid inhibited the protoplast growth of P. yedoensis from low concentrations, while coumarin did not, but inhibited only at a high concentration (1 mM). Contents of abscisic acid in protoplasts were measured using small scale purification and Enzyme Linked Immno Sorbent Assay, and contents of coumarin in leaf-origin susepension cells of P. yedoensis were measured using Gas Chromatography-Mass Spectrometry. Coumarin was more likely the allelochemical causing the strong inhibitory allelopathic activities of P. yedoensis in the protoplast co-culture bioassay. Effectiveness of the protoplast co-culture bioassay method of allelopathy was discussed.

Bioassay of Clostera anastomosis Granulosis virus

The second-instar healthy larvae of Clostera. anastomosis were collected in the artificial woodland of poplar in Shuangcheng Town, Heilongjiang Province, China. The dead larvae of C. anastomosis infected by granulosis virus （GV） of Clostera anastomosis were grinded to obtain GV. The GV viral pesticide was diluted to seven concentrations, 1.58×10^3PIB·mL^-1, 1,58×10^4PIB·mL^-1, 1.58×10^5PIB·mL^-1 1.58×10^6PIB·mL^-1, 1,58×10^7PIB·mL^-1; 1.58×10^8PIB·mL^-1 and 1.58×10^9PIB·mL^-1 and the fresh poplar leaves were dipped in the seven concentrations liquids to feed the larvae. After nine days the mortality of larvae was investigated. The minimum corrected mortality （7.32%） of larvae was observed at concentrations of 1.58×10^3PIB·mL^-1 and the maximal mortality （97,36%） was observed at concentration of 1.58×10^9PIB·mL^-1. The regression equation between the logarithm of the virus concentration and the mortality was y= 1.946＋0.558x The LC50 was 2.97×10^5PIB·mL^-1. The LT50 for the virus concentration of 1.58×10^5, 1.58×10^6, 1.58×10^7, 1,58×10^58, 1.58×10^9 PIB·mL^-1 were 8.55d, 6.89d, 5.9d, 4.65d, and 4.08d, respectively, shorting gradually with the concentration increasing, It is concluded that the toxicity of Clostera anastomosis GV is very strong and as a kind of insecticides it has big potential in practical application.

Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations

The aim of this study was to develop and validate a simple,sensitive,precise and cost-effective onelevel agar diffusion（5＋1） bioassay for estimation of potency and bioactivity of Levofioxacin in pharmaceutical preparation which has not yet been reported in any pharmacopoeia.Among 16 microbial strains.Bacillus pumilus ATCC-14884 was selected as the most significant strain against Levofioxacin.Bioassay was optimized by investigating several factors such as buffer pH,inoculums concentration and reference standard concentration.Identification of Levofioxacin in commercial sample Levoflox tablet was done by FTIR spectroscopy.Mean potency recovery value for Levofioxacin in Levoflox tablet was estimated as 100.90%.A validated bioassay method showed linearity（r^2 = 0.988）,precision（Interday RSD=1.05%,between analyst RSD=1.02%） and accuracy（101.23%,RSD=0.72%）.Bioassay was correlated with HPLC using same sample and estimated potencies were 100.90% and 99.37%.respectively.Results show that bioassay is a suitable method for estimation of potency and bioactivity of Levofioxacin pharmaceutical preparations.

Pharmacokinetic study with N-Ile^1-Thr^2-63-desulfato-r-hirudin in rabbits by means of bioassay

Aim: To study the pharmacokinetic (PK) properties in rabbits treated with N-Ile1-Thr2-63-desulfato-r-hirudin (rH) newly developed in China by means of bioassay in order to provide preclinical experiment basis for its development as a novel anticoagulant agent. Methods: rH plasma concentration was determined using bioassay based on ex vivo antithrombin activity of rH. Normal rabbits received iv rH 4.0, 2.0 and 1.0 mg/kg or sc rH 2.0 mg/kg, respectively. The rabbits with acute severe renal failure were given iv rH 2.0 mg/kg. Results: The bioassay described in this paper met requirements for study of PK in rabbits. The major PK parameters after iv dosing were as follows: t1/2β 58.4~59 min. Vd 0.09~0.12 L/kg, CL 0.0035~0.0040 L/(kgmin); AUC were proportional to the doses, t1/2 and CL did not change significantly with the doses. The sc bioavailability reached 94%. The rabbits suffering from acute severe renal failure presented 11-fold longer t1/2β and 13-fold greater AUC than normal healthy rabbits. Conclusion: rH exhibited rapid elimination, distribution was only limited to extracellular space and good absorption from sc site. The excretion of rH by kidneys played a very important role in the elimination of rH. The PK of rH could be described by the two- and one-compartment model after iv and sc dosing, respectively, and followed linear kinetics.

Evaluation of mouse bioassay results in an inter-laboratory comparison for paralytic shellfish poisoning toxins

An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.

Preparative Purification and Bioassay of Bt Toxin from Cry1Ab Transgenic Rice

A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa_2CO_3 and NaHCO_3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCl buffer+1.0 mmol/L EDTA, pH=8.0) mixed with 0.1, 0.3, 0.5 and 0.8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.

Evaluation on the Joint Action Between Chlorsulfuron and Haloxyfop-R by Bioassay

The joint action between chlorsulfuron and haloxyfop R was evaluated by bioassay with wheat and corn,respectivly.The dose response curve derived from wheat bioassay showed that the inhibition of haloxyfop R to wheat root growth wasn't affected by the increasing rate of chlorsulfuron.It indicated that chlorsulfuron had no antagonism to haloxyfop R.Meanwhile,the variation analysis of corn bioassay indicated that these two herbicides had joint action on inhibition to corn primary root growth.The joint action was evaluated as additive action by using Isobole Method.So chlorsulfuron and haloxyfop R could be used as tank mixture.

Improvement of Teaching Experimental Design of Pesticide Bioassays

For mistakes taken in pesticide bioassays, teaching experimental design is improved in the paper, so as to let students explore and analyze in teaching experiments to get a deeper understanding of theoretical knowledge, thereby effectively avoiding frequently-taken mistakes in pesticide bioassays.

Development of An ICR Mouse Bioassay for Toxicity Evaluation in Neurotoxic Poisoning Toxins-Contaminated Shellfish

Objective To develop an ICR （female） mouse bioassay （MBA） for toxicity confirmation and evaluation of neurotoxins （brevetoxins）-contaminated shellfish. Methods Brevetoxins （BTX-B） as a causative agent of neurotoxic shellfish poisoning （NSP） under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit （80 μg/100 g shellfish tissues）. The LD 50 identified was 455 g/kg for BTX-B under general shellfish matrices （excluding oyster matrices） dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Conclusion The two ELISA analyses agree favorably （correlation coefficient, r 0.96; Student＇s t-tests, P〉0.05） with the developed bioassay.

Gamma Irradiation Enhanced Leaf Bioactive Components and Bioassay Parameters in M₅Mulberry (<i>Morus</i>Sp.) Mutant

Gamma radiation is an effective tool for inducing genetic variation in plant characters. In the present experiment, M5 mulberry variety juvenile twigs were subjected to source Co60 gamma irradiation (1 kR - 10 kR) and mutants grown in triplicates in randomized block design to raise M1 and M2 generation. In M2 generation plants were subjected to phytochemical and bioassay tests. Silkworm rearing parameters and commercial characters of cocoons were recorded by feeding cross breed silkworms. Results show that M5 mutant leaves revealed significant variations in phytochemical constituents and moisture content. Bioassay tests recorded significant differences compared to control in M2 generation. Commercial characters like cocoon weight (1.41 g), shell weight (0.24 g), shell percentage (16.29 %), filament length (821.00 mts), renditta (8.2), denier (2.24) and effective rate of rearing (92.14 %) were increased. It is concluded that, gamma rays treatment enhances the mulberry plants leaf bioactive components, silkworm rearing and cocoon parameters and shows beneficial variants in M2 generation.

<i>N</i>-Acetylated Gemini Surfactants: Synthesis, Surface-Active Properties, Antifungal Activity, and Ecotoxicity Bioassays

A series of N-acetylated cationic gemini surfactants (3a-e) having dimeric structures derived from tertiary amines were synthesized. Their antifungal potency and surface properties were determined. It also studied the acute toxicity of the molecule with the best performance and the best water solubility (3e) through Chlorella vulgaris and Daphnia magna bioassays. The results were compared to those obtained for a commercially available reference compound 2-(thiocyanomethylthio) benzothiazole (TCMTB). Parameters such as surface tension (&#978CMC), critical micelle concentration (CMC), surface excess concentration (Γ), and area per molecule (A) were determined. The resulting values indicated that the five gemini surfactants are characterized by good surface-active and self-aggregation properties. All surfactants were tested to evaluate their antifungal activity. Six fungal strains were used to conduct the study. The minimum inhibitory concentration (MIC) value was measured by the fungal growth inhibition. The results of the MICs were compared with two commercially available reference compounds (Fluconazole and TCMTB). The least active molecule was 3e, but 3b and 3d were found to be the most potent compounds with a similar activity for all strains. Candida albicans was the most sensitive one. In contrast, Aspergillus niger was resistant. Ecotoxicity of gemini 3e was assessed: the commercial formulation (TCMTB) was between three and four orders of magnitude more toxic than the gemini one for the biological species tested.