利用废弃物发酵生产苏云金芽胞杆菌的研究进展

苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是目前应用最广泛和产量最大的微生物杀虫剂,它对鳞翅目、双翅目、鞘翅目等许多害虫有特异毒杀作用,而对人类和其它非靶标动物以及农作物无害。长期以来,人们一直致力于苏云金芽胞杆菌的筛选及发酵研究,以期能获得高毒力的绿色生物杀虫剂产品。由于发酵培养基成本较高,利用废弃物生产Bt杀虫剂既有利于资源循环利用和环境保护,又能降低Bt杀虫剂的多重优点,因此成为近年来国内外的研究热点。本文简要介绍了筛选苏云金芽胞杆菌的基本方法以及利用废弃物发酵生产Bt杀虫剂的研究进展。方法以及利用废弃物发酵生产Bt杀虫剂的研究进展。     

不同添加物对苏云金芽孢杆菌杀虫活性的增效作用研究进展

苏云金芽孢杆菌（Bacillius thuringiensis,Bt）制剂是当前应用最广、最有效的生物杀虫剂之一,因其对多种昆虫具有特异性杀虫活性,而被广泛用于农林业和公共卫生等领域的害虫防治,但田间施用后,其速效性差、持效期短和防效不稳定等弊端限制了其进一步的推广。将Bt制剂与增效物质（剂）、因子混合使用以提高其杀虫活性和田间防效稳定性,是最快速、有效的途径之一,因而国内外对此开展了广泛而深入的研究。主要介绍了化学添加剂、化学杀虫剂和生物杀虫剂等添加物对Bt制剂杀虫活性的增效作用研究进展,并探讨了增效物质（剂）、因子的开发和应用前景,以期为开发安全、高效的Bt制剂的增效物质（剂）、因子提供一定的参考。     

Bt棉花害虫综合治理研究前沿

自1996年以来,全球Bt(Bacillus thuringiensis)棉花应用规模迅速增长,目前已占棉花种植总面积的60%左右,主要种植国家包括美国、澳大利亚、中国、印度和巴基斯坦等。大量研究表明,Bt棉花的大面积种植有效控制了多种靶标害虫的发生危害,从而大幅度减少了化学杀虫剂的使用量;化学杀虫剂的减少使用导致一些非靶标害虫的发生危害明显加重。针对Bt棉花生产中呈现出的害虫新问题,各国分别发展了由农业防治、生物防治、化学防治等不同措施构成的防控技术体系。     

Advance in insect pest management in Bt cotton worldwide

自1996年以来,全球Bt(Bacillus thuringiensis)棉花应用规模迅速增长,目前已占棉花种植总面积的60％左右,主要种植国家包括美国、澳大利亚、中国、印度和巴基斯坦等.大量研究表明,Bt棉花的大面积种植有效控制了多种靶标害虫的发生危害,从而大幅度减少了化学杀虫剂的使用量;化学杀虫剂的减少使用导致一些非靶标害虫的发生危害明显加重.针对Bt棉花生产中呈现出的害虫新问题,各国分别发展了由农业防治、生物防治、化学防治等不同措施构成的防控技术体系.     

斜纹夜蛾抗药性及其防治对策的研究进展

斜纹夜蛾Spodopteralitura是多种作物的重要害虫 ,对有机氯、有机磷、氨基甲酸酯类、拟除虫菊酯类以及Bt制剂等杀虫剂均产生抗药性。本文对斜纹夜蛾抗药性的形成与发展、抗性机理及其防治对策等方面进行了综述     

昆虫对苏云金杆菌的抗性

在害虫防治中,昆虫对化学农药的抗性已发展成为十分严重的问题。生物杀虫剂的抗性问题直到最近几年才有所察觉,今天仍有许多人认为Bacillus thuringensis(简称Bt)等生物制剂似乎不存在这个问题,事实上,最近越来越多的数据显示,Bt杀虫剂也存在抗性发展的趋向。在实验室条件下,目前已发现印度谷螟(Plodia interpunctella)、粉斑螟(Cadra cautella)、烟青虫(Heliothis virescens)对某些Bt杀虫晶体蛋白(ICP)产生明显抗性。Tabashnik等在夏威夷的田间应用中证实,小菜蛾(Plutell     

中国10年抗虫棉大田生产:Bt抗虫棉技术采用的直接效应和间接外部效应评估

中国自1997年便开始大面积种植Bt抗虫棉.本研究试图分析中国种植抗虫棉10年后减少防治棉铃虫杀虫剂用量的可持续性.通过对1999~2007年4个省16个村农户大田Bt抗虫棉生产的跟踪调查数据分析,发现生产上控制棉铃虫的杀虫剂用量不仅未增加,反而呈下降趋势;虽然没有直接回答Bt抗虫棉能够维持并增加其生产率的机理,但本研究发现,10年来不仅抗虫棉,而且非抗虫棉防治棉铃虫的杀虫剂用量也显著减少,这与抗虫棉广泛种植后棉铃虫的种群总量下降有关.这些发现对于Bt技术效应分析具有重要意义,同时,本文所采用的同时评估抗虫转基因技术生产的直接效应和间接外部性效应的方法,对于未来该领域的同类研究也具有借鉴意义.     

国外科技简讯

第三代Bt杀虫剂问世 据ANI 1995年3月报道:美国宾夕法尼亚州Ecogen公司已获得美国环境保护局的准许,将重组苏云金芽孢杆菌(Bt)生物杀虫剂“Raven”投入市场。Raven是在美国环境保护局注册的第一种重组Bt,也是第三代Bt生物杀虫剂。该杀虫剂包含了来自不同的Bt株系的复合蛋白,其有效成分为8％马铃薯甲虫活性毒素和2％蠋毒素。由于马铃薯甲虫已对化学杀虫剂产生了抗性,因此化学防治日渐失效。1994年Raven田间防治试验表明效果良好,为此科学家建议将其与化学杀虫剂结合施用。据统计,目前Raven在世界市场上的销售金额已高达5—10百万美元。     

Ecogen公司的Bt可有效控制草坪虫害

美国Ecogen公司宣布,该公司基于苏云金芽孢杆菌(Bt)的生物杀虫剂已证明对防治草坪虫害非常有效。实验是在美国ChemLawn Servies Corporation实验室和温室进行的。结果表明,这种新型Bt植株明显抗草坪及观赏植物害虫。对Bt生物杀虫剂敏感的两种普通害虫是日本甲虫和北方圆头犀金龟子,每年,美国在用于专业草坪防治这些害虫的费用就达5亿多美元。     

转Bt基因抗虫作物对非靶标害虫的影响

转基因作物的长期大面积种植, 在为农业生产带来惠益的同时, 对农业生态系统的健康和稳定可能会产生潜在的影响。转基因作物表达的Bt蛋白对靶标害虫起到较好的控制效果, 而对Bt蛋白不敏感的非靶标害虫种群可能会迅速发展起来, 对作物造成为害。随着抗虫转基因作物的连续多年种植, 科学家们对于田间杀虫剂施用量的增减看法不尽一致。通过总结已有的研究报道, 本文以Bt玉米和Bt棉花为例, 分析了大田中非靶标害虫暴发的现状, 以及暴发的主要原因(如杀虫剂的使用、害虫天敌减少和物种替代)。在生产实践中, 抗虫作物的长期大面积释放导致广谱杀虫剂施用量减少, 田间非靶标害虫数量上升。因此今后需要继续开展更多的研究来综合评估种植转Bt基因作物产生的长期潜在影响, 优化害虫防治措施, 避免非靶标害虫暴发。     

害虫Bt抗性机制研究新方向：昆虫体液免疫系统

作为最成功的生物农药,苏云金芽孢杆菌Bacillus thuringiensis (Bt)杀虫剂已在农业生产中应用了约80年。Bt由于其特异性强、安全高效的特点而得到广泛、成功的应用,极大减少了化学农药的用量,为环境保护作出了巨大贡献。然而,由于长期使用,一些靶标害虫逐渐对Bt产生抗性。本文对昆虫体液免疫及昆虫Bt抗性机制的研究成果进行了总结,已有研究认为害虫对Bt产生抗性的主要原因是毒素激活受阻及(或)毒素受体突变或减少。然而近年越来越多的研究表明,昆虫的Bt抗性还与其免疫系统,特别是与Toll, IMD和proPO-AS等体液免疫通路有关。由此,本文对昆虫体液免疫系统参与昆虫Bt抗性形成的主要通路进行了归纳和推论。IMD免疫通路可能通过MAPK信号通路参与调节昆虫Bt抗性,或可能通过多种免疫反应对抗因中肠组织被Bt破坏而引起的败血症,并通过JNK信号通路促使中肠组织愈合,进而提高其对Bt的抗性。从体液免疫系统切入研究,可能成为深入探索昆虫Bt抗性机制的新方向。     

Field efficacy of sweet corn hybrids expressing a Bacillus thuringiensis toxin for management of Ostrinia nubilalis (Lepidoptera: Crambidae) and Helicoverpa zea (Lepidoptera: Noctuidae)

Field studies were done in 1995-1996 to assess the efficacy of three sweet corn hybrids that express the Bacillus thuringiensis (Bt) toxin, CrylAb, against two lepidopteran pests, Ostrinia nubilalis (Hubner) and Helicoverpa zea (Boddie). The Bt hybrids tested were developed by Novartis Seeds, using the event BT-11, which expresses Bt toxin in green tissue as well as reproductive tissues including the tassel, silk, and kernel. Bt hybrids were compared with a standard non-Bt control or the non-Bt isoline for each hybrid; none of the hybrids were treated with insecticides during the study. Hybrid efficacy was based on larval control of each pest, as well as plant or ear damage associated with each pest. In both years, control of O. nubilalis larvae in primary ears of all Bt hybrids was 99-100% compared with the appropriate non-Bt check. Plant damage was also significantly reduced in all Bt hybrids. In 1996, control of H. zea in Bt hybrids ranged from 85 to 88% when compared with the appropriate non-Bt control. In 1996, a University of Minnesota experimental non-Bt hybrid (MN2 x MN3) performed as well as the Bt hybrids for control of O. nubilalis. Also, in 1996, two additional University of Minnesota experimental non-Bt hybrids (A684su X MN94 and MN2 X MN3) performed as well as Bt hybrids for percent marketable ears (ears with no damage or larvae). In addition, compared with the non-Bt hybrids, percent marketable ears were significantly higher for all Bt hybrids and in most cases ranged from 98 to 100%. By comparison, percent marketable ears for the non-Bt hybrids averaged 45.5 and 37.4% in 1995 and 1996, respectively. Results from the 2-yr study strongly suggest that Bt sweet corn hybrids will provide high levels of larval control for growers in both fresh and processing markets. Specifically, Bt sweet corn hybrids, in the absence of conventional insecticide use, provided excellent control of O. nubilalis, and very good control of H. zea. However, depending on location of specific production regions, and the associated insect pests of sweet corn in each area, some insecticide applications may still be necessary.     

不同生长期转Bt基因水稻秸杆还土对淹水土壤酶活性的影响

在实验室条件下通过秸杆还土试验比较了不同生长期转Bt基因克螟稻及其亲本稻秸杆对淹水土壤酶活性的影响。研究结果表明,与同一生长期的亲本稻秸杆相比,孕穗期和成熟期克螟稻秸杆对淹水土壤磷酸酶活性的影响较小;相反,对淹水土壤脱氢酶活性的影响非常显著,并且孕穗期秸杆与成熟期秸杆的添加对淹水土壤脱氢酶活性的影响趋势也存在较大差异。推测造成淹水土壤脱氢酶活性的显著性差异的主要原因可能是由于Bt插入基因表达的多效性所致。结果认为土壤脱氢酶活性可作为转Bt基因水稻生态安全风险性评价的潜在指标。     

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.     

Microbial control and biotechnology research on Bacillus thuringiensis in China

The current status of production and application of biopesticides for pest control in China is briefly reviewed, with a focus on research advances in microbial control with Bacillus thuringiensis (Bt). These have led to improvements in Bt production, exploitation of Bt gene resources, and development of engineered Bt insecticides and transgenic Bt crops that have expanded host ranges and increased efficacy against target pests. Both conventional and biotechnology approaches need to be employed to achieve further progress in discovery, production technology, formulation processing, development of quality standards and recommended use patterns.     

Regulation of steroidogenesis in rat Leydig cells in culture: effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt2cAMP (1 mM) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt2cAMP was also observed. Both hCG and Bt2cAMP increased the rates of synthesis of cytochrome P-450scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt2cAMP-treated cells the rate of synthesis of cytochrome P-450scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450scc and its associated protein, adrenodoxin. Since Bt2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450scc and adrenodoxin are mediated by increased cyclic AMP formation.     

两种防治措施下转Bt基因棉田绿盲蝽的发生与为害

2002年在河北省南皮县对2种防治措施下转Bt基因田绿盲蝽LyguslucorumMayre的发生与为害进行的系统调查表明,采用生物农药和低毒化学农药防治4次,Bt棉田绿盲蝽的发生为害较为严重,9月上旬发生高峰期种群密度为7.2头10株,显著高于防治指标,第2代绿盲蝽为害高峰期,叶片被害率为19.4%;采用当地棉农化学防治方法施用农药7次,Bt棉田绿盲蝽发生为害较轻,发生高峰期(8月中旬)种群密度为2.0头10株,第2代绿盲蝽为害高峰期,叶片被害率为4.8%。讨论指出绿盲蝽已成为转Bt基因棉生产中的重要问题,应加快绿盲蝽在转Bt基因棉田的生态调控研究。     

Increased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP

The effects of cyclic nucleotides on elastin synthesis were studied in ligamentum nuchae fibroblasts by adding exogenous cyclic nucleotide derivatives or beta-adrenergic agents to cell culture medium. Elastin synthesis was enhanced (approximately 80%) by dibutyryl cGMP (Bt2cGMP) in concentrations ranging from 0.01 to 100 nM. Two other cGMP derivatives, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and 2'-deoxy-cGMP, were also potent stimulators of elastin synthesis. In the absence of calcium, basal elastin production was substantially decreased (40% of control) and cGMP analogs no longer stimulated elastin synthesis, suggesting a role for calcium in the cGMP response. Bt2cAMP had no demonstrable effect on elastin production except at high concentrations which produced a nonspecific decrease equivalent to the decrease in total protein synthesis. Similarly, elevation of endogenous cellular cAMP levels by beta-adrenergic stimulation produced no change in elastin production. When 8-Br-cGMP was added to cells together with Bt2cAMP, cGMP-dependent stimulation of elastin production was abolished by cAMP in a dose-dependent fashion. These results suggest a coordinated means by which elastin production is controlled in ligament cells, i.e. increased cGMP levels lead to a stimulation of elastin production that is reversed by cAMP.     

Influence of plant severing on movement of Ostrinia nubilalis larvae in Zea mays hybrid seed production fields

Genetically engineered corn hybrids that contain a cry gene from the bacterium Bacillus thuringiensis Berliner (Bt) are gaining popularity for controlling the corn pest Ostrinia nubilalis (Hübner). Continuous use of Bt corn, however, could select for O. nubilalis that are resistant to this corn. Monitoring for insect resistance is important, because it could help maintain the Bt technology. A possible monitoring method is to collect larval insects in commercial drying bins after harvest from Bt seed production fields. A drawback to this method is that these collections may be contaminated by insects that moved as later instars from severed non-Bt male rows into the adjacent Bt female rows. These larvae have little to no exposure to Bt toxin, resulting in possible "false positives." The objectives of this study were to first find which combination of planting and severing dates produces the least number of larvae that move from non-Bt male plants to Bt female plants and to assess O. nubilalis larval movement from severed non-Bt male rows to Bt female rows. Field studies in 2002 and 2003 were designed to simulate a hybrid seed production field. Results suggest that movement of O. nubilalis larvae from male corn is minimized when corn is planted early and male plants are severed by 2 wk post-anthesis. This reduces the likelihood of false positives by reducing the number of susceptible larvae moving between Bt and non-Bt plants. Also, larvae moved to all four female rows that were adjacent to the severed rows, but there were significantly more larvae found in the closest row compared with the other three. These results could be used to develop a monitoring program to find O. nubilalis larvae with resistance to Bt corn in field populations of O. nubilalis.