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.     

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。     

Sustainability of insect resistance management strategies for transgenic Bt corn

Increasing interest in the responsible management of technology in the industrial and agricultural sectors of the economy has been met thorough the development of broadly applicable tools to assess the "sustainability" of new technologies. An arena ripe for application of such analysis is the deployment of transgenic crops. The new transgenic pesticidal or plant-incorporated protectant (PIP) crops have seen widespread application in the United States based on the features of higher yield, lower applications of insecticides, and control of mycotoxin content. However, open rejection of these new crops in Europe and in other countries has been a surprising message and has limited their worldwide acceptance. The US Environmental Protection Agency's (USEPA) Office of Pesticide Programs (OPP) has worked on the development and analysis of insect resistance management (IRM) strategies and has mandated specific IRM requirements for Bacillus thuringiensis (Bt) crops since 1995 under the Food, Fungicide, Insecticide, and Rodenticide Act. Improvement of data quality and sustainability of IRM strategies have been targeted in an ongoing partnership between the USEPA Office of Research and Development and the Office of Pesticide Programs that will further enhance the agency's ability to develop sustainable insect resistance management strategies for transgenic field corn (Bt corn) producing B. thuringiensis (Bt) insecticidal proteins.     

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program. This revised version was published online in November 2006 with corrections to the Cover Date.     

转基因抗虫作物中苏云金芽孢杆菌Cry蛋白的食品安全问题

苏云金芽孢杆菌(Bt)微生物制剂是农业、林业和饮用水等领域用来控制靶标害虫幼虫的有效工具,至今已经有50余年的使用历史。同时其在美国、欧洲和其他一些国家被广泛用于经过认证的有机农业生产之中。目前已获审批的转基因Bt作物中最常使用的是Cry蛋白。Cry蛋白的作用机制、食品安全性以及致敏性已经经过啮齿类动物、农场动物和人体内试验和生物信息学研究的严格检验。Cry蛋白的杀虫作用只在靶标害虫的碱性消化道内,与中肠上皮细胞的特异蛋白受体结合才能起到杀虫作用,而其他非靶标生物体内(人类、猕猴、小鼠、大鼠和牛等)都被证明没有这种特异蛋白质受体。美国、欧洲和其他国家的管理机构都已经证实了转基因Bt作物和Cry蛋白在农作物和饮用水中残留的安全性。食物加工过程能够最大化地减少转基因作物中功能性Cry蛋白的摄入。转基因抗虫作物有利于降低农药杀虫剂的使用的同时,也能够有效防止玉米中伏马菌毒素的污染。     

Helicoverpa zea and Bt cotton in the United States

Helicoverpa zea (Boddie), the bollworm or corn earworm, is the most important lepidopteran pest of Bt cotton in the United States. Corn is the preferred host, but the insect feeds on most flowering crops and wild host plants. As a cotton pest, bollworm has been closely linked to the insecticide-resistance prone Heliothis virescens (F.), tobacco budworm. Immature stages of the two species are difficult to separate in field environments. Tobacco budworm is very susceptible to most Bt toxins, and Bt cotton is considered to be "high dose." Bollworm is less susceptible to Bt toxins, and Bt cotton is not "high dose" for this pest. Bt cotton is routinely sprayed with traditional insecticides for bollworm control. Assays of bollworm field populations for susceptibility to Bt toxins expressed in Bt cotton have produced variable results since pre-deployment of Bt cottons in 1988 and 1992. Analyses of assay response trends have been used by others to suggest that field resistance has evolved to Bt toxins in bollworm, but disagreement exists on definitions of field resistance and confidence of variable assay results to project changes in susceptibility of field populations. Given historical variability in bollworm response to Bt toxins, erratic field control requiring supplemental insecticides since early field testing of Bt cottons, and dramatic increases in corn acreage in cotton growing areas of the Southern US, continued vigilance and concern for resistance evolution are warranted.     

Mitigating the effects of insecticides on arthropod biological control at field and landscape scales

Integrated pest management (IPM) programs emphasize the combination of tactics, such as chemical and biological control, to maintain pest populations below economic thresholds. Although combining tactics may provide better long-term sustainable pest suppression than one tactic alone, in many cases, insecticides and natural enemies are incompatible. Insecticides can disrupt natural enemies through lethal and sub-lethal means causing pest resurgence or secondary pest outbreaks. Legislative actions such as the Food Quality Protection Act (US) and the Directive on Sustainable Use of Pesticides (EU) have placed greater restrictions on insecticides used in agriculture, potentially enhancing biological control. Here we focus on the effects of insecticides on biological control, and potential mitigation measures that can operate at different scales. At the farm scale, natural enemies can be conserved through the use of selective insecticides, low doses, special formulations, creation of refugia, special application methods, and targeted applications (temporal or spatial). At the landscape scale, habitat quality and composition affect the magnitude of biological control services, and the degree of mitigation against the effects of pesticides on natural enemies. Current research is teasing apart the relative importance of local and landscape effects of pesticides on natural enemies and the ecosystem services they provide, and the further development of this area will ultimately inform the decisions of policy makers and land managers in terms of how to mitigate pesticide effects through habitat manipulation.     

Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti?

The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases.     

转基因抗虫棉花和玉米与节肢动物相关的生态安全性研究进展

转基因抗虫棉花和玉米自1996年商业化种植以来,已取得显著的经济、生态和社会效益。与其相关的生态安全性,特别是其对非靶标生物的影响及靶标害虫的抗性监测和治理已成为人们普遍关注的话题。本文在大量室内和田间评价工作的基础上,系统综述了国内外研究在该领域内取得的进展。结果表明： 由于Bt棉田和玉米田杀虫剂用量的减少,某些对Bt杀虫蛋白不敏感的非靶标植食害虫种群有上升的趋势; 现阶段生产上推广种植的Bt棉花和玉米花粉对家蚕、柞蚕和蜜蜂等经济昆虫以及帝王斑蝶是安全的。杀虫剂用量的减少,降低了对天敌的杀伤力,Bt田中捕食性天敌的种类和数量均显著高于常规施药田; 但Bt田内靶标害虫数量的减少和质量的降低,在一定程度上影响了寄生性天敌的种类和数量。Bt棉花和玉米的大面积种植对农田生态系统节肢动物群落结构无明显不利影响。靶标害虫田间抗性监测结果表明,无论在以大农场单一种植经营为主的发达国家如美国或澳大利亚,还是在以小农经营为主的多种寄主作物小规模交叉混合种植模式的发展中国家如中国或印度,田间并未出现10年前人们所关注和预测的靶标害虫种群抗性上升问题。究其原因,可能与发达国家严格执行了预防性的抗性治理对策及发展中国家独特的作物种植模式有关。尽管目前在田间尚未发现害虫对Bt作物产生抗性,但应用更多年份之后,害虫对Bt作物的抗性就很可能不是“是否”发生问题,而是“何时”发生的问题。因此,今后的研究重点应放在Bt棉花和玉米长期、大面积种植后,其对非靶标生物及靶标害虫抗性发展影响的长期生态效应上。     

Combining Pest Repellent Plants and Biopesticides for Sustainable Pest Management in Chinese Kale

The objective of this investigation was to study the use of pest repellent plant (PRP) species and biopesticides for sustainable pest management in Chinesekale(Brassica oleracea L. var. alboglabra Bailey). Ten selected treatments composed of three PRP species namely citronella grass - (CG) [Cymbopogon nardus L. (Rendle)], sweet basil - (SwB) (Ocimum basilicum L.) and sacred basil - (SaB) (Ocimum sanctum L.)] were intercropped with Chinese kale, integrated with two commonly used biopesticides, viz. Bacillus thuringiensis (Bt) and neem extract (Azadirachta indica) and a control were tested in a randomized complete block design (RCBD) with 3 replicates. The insect pest species infested in Chinese kale and their populations, % pest damage, fresh weight and quality of yield were investigated. The results showed that diamondback moth - (DBM) [Plutella xylostella (Linnaeus)] density was lowest in CG alone, CG+SaB, SwB+biopesticides and SaB+biopesticides, relatively low in SwB alone, CG+SaB+SwB, and CG+biopesticides, and moderate in biopesticides and sacred basil alone, and highest in the control. The densities of common cutworm - (CCW) [Spodoptera litura (Fabricius)], cabbage webworm - (CWW) [Hellula undalis (Fabricius)] and flea beetle - (FBT) (Phyllotreta sinuata Steph.) were not significantly different among treatments. The overall pest damage was lowest in CG+biopesticides treated plots followed by SwB+ biopesticides, SaB+biopesticides and biopesticides alone, and CG+biopesticides gave the best quality of yield of Chinese kale, and hence such combinations could be integrated for managing crucial insect pests, i.e. DBM and to minimize/avoid the use of highly toxic synthetic pesticides in Chinese kale.     

Water solutions of boric acid and sugar for management of German cockroach populations in livestock production systems

Pest management in confinement swine production relies primarily on calendar-based applications of broad-spectrum insecticides. However, regulatory restrictions imposed by the U.S. Food Quality Protection Act of 1996, the large financial obligation of pesticide registration, and development of insecticide resistance have led to a renewed search for alternative control methods. Boric acid dust has long served as an insecticide in urban pest management and has been shown an effective alternative for use in sensitive environments such as swine production. However, dust formulations are difficult to apply and require specialized equipment. The purpose of this study was to evaluate the efficacy of liquid baits containing boric acid for the control of German cockroaches in a commercial swine nursery. Bait, consisting of 1 or 2% boric acid and 0.5 M sucrose, was deployed in 21 bait delivery tubes per room. Results of a 2-yr study showed significant reductions in cockroach populations. When baits were withdrawn in the summer, the cockroach population increased significantly faster than when the baits were removed during the winter. These data indicate that liquid formulations of boric acid effectively reduce the burden of cockroach infestation in swine production. This approach should have applications in structures in other urban and agricultural environments.     

Compatibility and interaction between Bacillus thuringiensis and certain insecticides: perspective in management of Tuta absoluta (Lepidoptera: Gelechiidae)

Bacillus thuringiensis var. kurstaki (Berliner) (Bt) has been suggested as a biological control agent for Tuta absoluta (Meyrick). The objective of this study was to determine the interaction between abamectin, azadirachtin, indoxacarb, chlorantraniliprole, dichlorvos and metaflumizone with Bt. Effect of recommended doses of the chemical insecticides on colonisation of Bt was also investigated in culture medium. Except for metaflumizone, none of the chemicals tested reduced the colonisation of Bt compared with control. Interaction between Bt and the chemical insecticides on 2nd-instar larvae was also assessed. In interaction tests, Bt was applied at LC₅₀ level, 0, 12, 24 or 36 h after treating the larvae with LC₁₀ or LC₂₅ of the chemical insecticides. An antagonistic effect was observed in all treatments where Bt was applied immediately after the chemical insecticide. Also, antagonism was observed when treatment with Bt was done 12 h after azadirachtin and metaflumizone applications. Applying Bt 12 and 24 h after treatment with LC₂₅ of chlorantraniliprole, dichlorvos and abamectin resulted in synergism. But, synergism with LC₁₀ of dichlorvos and abamectin was observed only after 12 h. Additive effect was observed in the rest of the time and concentration combinations. Based on the results obtained, simultaneous use of the chemical insecticides tested and Bt is not recommended for T. absoluta control; and an appropriate time interval should be taken into consideration accordingly.     

Bt杀虫基因与Bt转基因抗虫植物研究进展

苏云金杆菌是一类非常重要的昆虫病原体,它能产生特异性的杀虫结晶蛋白,对农业上和生物医学上的许多有害的昆虫有毒杀作用,这些害虫包括鳞翅目、双翅目、鞘翅目、膜翅目、螨类和线虫。近三十年来,以苏云金杆菌为基础的生物杀虫剂已在世界范围内商业化用于防治重要经济作物的害虫。近年来有关Ｂｔ基因的遗传、分子生物学和基因工程已取得显著进展。本文对苏云金杆菌杀虫晶体蛋白基因的分类和杀虫机理及用该类基因构建的工程转基因植物研究状况作一简要综述,同时对Ｂｔ基因工程存在的潜在问题和解决途径作了简单的探讨。     

Bt杀虫基因与Bt转基因抗虫植物研究进展

苏云金杆菌是一类非常重要的昆虫病原体,它能产生特异性的杀虫结晶蛋白,对农业上和生物医学上的许多有害的昆虫有毒杀作用,这些害虫包括鳞翅目、双翅目、鞘翅目、膜翅目、螨类和线虫。近三十年来,以苏云金杆菌为基础的生物杀虫剂已在世界范围内商业化用于防治重要经济作物的害虫。近年来有关Bt基因的遗传、分子生物学和基因工程已取得显著进展。本文对苏云金杆菌杀虫晶体蛋白基因的分类和杀虫机理及用该类基因构建的工程转基因植物研究状况作一简要综述,同时对Bt基因工程存在的潜在问题和解决途径作了简单的探讨。     

The economics of acetone-butanol fermentation: theoretical and market considerations

Acetone-butanol (AB) fermentation was once run commercially in many countries until these chemicals could be made more cheaply from fossil oil sources. Research into the revitalisation of the process has shown that the process could once again be run economically in niche markets if run in a relatively small industrial scale processing low-grade agricultural products. The following analysis is intended to help identify suitable niche markets.     

The mammalian safety of Bacillus thuringiensis-based insecticides

The United States Environmental Protection Agency between the years 1961 and 1995 registered 177 products containing viable Bacillus thuringiensis (Bt). Numerous laboratory studies have demonstrated that Bt and Bt products are noninfectious and are toxic to mammals only at a dose > or =10(8) colony forming units (cfu) per mouse (a human equivalent based on the weight of >10(11) cfu). In contrast, as few as three vegetative cells of Bacillus anthracis can kill mice (a human equivalent of >10(3) cfu). There are only two literature reports of Bt infection in man between the year 1997 and the present, and all infected individuals had experienced either extensive burns or a blast injury, which predisposed them to infection. Two epidemiology studies conducted during large-scale aerial Bt serovar kurstaki spray campaigns reported no increased incidence of illness. Some recent papers have expressed concern about the production of Bacillus cereus enterotoxins by Bt isolates. Laboratory studies found no evidence of illness in rats and sheep fed Bt products, nor have epidemiology studies found increased incidence of diarrhea during Bt aerial spray campaigns. Increases in human antibody levels following exposure to Bt products have been reported but there was no increased incidence in asthma or other illness. Based on laboratory studies and field experience, Bt insecticides have an excellent safety record.     

Have biopesticides come of age?

Biopesticides based on living microbes and their bioactive compounds have been researched and promoted as replacements for synthetic pesticides for many years. However, lack of efficacy, inconsistent field performance and high cost have generally relegated them to niche products. Recently, technological advances and major changes in the external environment have positively altered the outlook for biopesticides. Significant increases in market penetration have been made, but biopesticides still only make up a small percentage of pest control products. Progress in the areas of activity spectra, delivery options, persistence of effect and implementation have contributed to the increasing use of biopesticides, but technologies that are truly transformational and result in significant uptake are still lacking.     

Field tests on managing resistance to Bt-engineered plants

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic Bt crops approved by the EPA. Bt-transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a "refuge" composed of non-Bt plants to conserve susceptible alleles. We have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a "separate refuge" will be more effective at conserving susceptible larvae than a "mixed refuge" and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.     

Bt crop effects on functional guilds of non-target arthropods: a meta-analysis

Background

Uncertainty persists over the environmental effects of genetically-engineered crops that produce the insecticidal Cry proteins of Bacillus thuringiensis (Bt). We performed meta-analyses on a modified public database to synthesize current knowledge about the effects of Bt cotton, maize and potato on the abundance and interactions of arthropod non-target functional guilds.

Methodology/Principal Findings

We compared the abundance of predators, parasitoids, omnivores, detritivores and herbivores under scenarios in which neither, only the non-Bt crops, or both Bt and non-Bt crops received insecticide treatments. Predators were less abundant in Bt cotton compared to unsprayed non-Bt controls. As expected, fewer specialist parasitoids of the target pest occurred in Bt maize fields compared to unsprayed non-Bt controls, but no significant reduction was detected for other parasitoids. Numbers of predators and herbivores were higher in Bt crops compared to sprayed non-Bt controls, and type of insecticide influenced the magnitude of the difference. Omnivores and detritivores were more abundant in insecticide-treated controls and for the latter guild this was associated with reductions of their predators in sprayed non-Bt maize. No differences in abundance were found when both Bt and non-Bt crops were sprayed. Predator-to-prey ratios were unchanged by either Bt crops or the use of insecticides; ratios were higher in Bt maize relative to the sprayed non-Bt control.

Conclusions/Significance

Overall, we find no uniform effects of Bt cotton, maize and potato on the functional guilds of non-target arthropods. Use of and type of insecticides influenced the magnitude and direction of effects; insecticde effects were much larger than those of Bt crops. These meta-analyses underscore the importance of using controls not only to isolate the effects of a Bt crop per se but also to reflect the replacement of existing agricultural practices. Results will provide researchers with information to design more robust experiments and will inform the decisions of diverse stakeholders regarding the safety of transgenic insecticidal crops.     

Evaluation of soil‐applied insecticides with Bt maize for managing corn rootworm larval injury

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is considered one of the most significant insect pests of maize in North America. Larvae of other secondary subterranean pests such as grape colaspis, Colaspis brunnea (F.), and Japanese beetle, Popillia japonica Newman, can also injure maize seedlings and cause yield loss. In the past decade, maize hybrids containing Bt proteins have been used to manage the western corn rootworm; additionally, seeds are commonly treated with a neonicotinoid and fungicide combination to control secondary pests. Recently, soil‐applied insecticides have been used in conjunction with rootworm Bt hybrids (and seed‐applied insecticides) in areas with perceived risk for increased rootworm larval or secondary pest damage. We conducted a series of trials from 2009 to 2011 that examined multiple rootworm Bt hybrids and their near‐isolines, along with two soil‐applied insecticides, to determine whether the Bt plus insecticide combination resulted in an increased level of efficacy or yield. We also sampled for Japanese beetle and grape colaspis larvae to determine their potential for reducing yield. Densities of secondary pests in our trials were low and likely had no effect on maize yield. The addition of a soil‐applied insecticide to rootworm Bt hybrids improved efficacy only once across 17 location‐years, when overall corn rootworm injury was highest; an improvement in yield was never observed. Our results suggest that the use of a soil‐applied insecticide with a rootworm Bt hybrid should only be considered in scenarios with potentially significant rootworm larval populations. However, potential negative consequences related to trait durability when soil insecticides are used with rootworm Bt maize should be considered.