增加糖基合成酶基因拷贝数对多杀菌素发酵单位的影响

多杀菌素是由刺糖多孢菌(Saccharopolyspora spinosa SIPI—A.2090)产生的重要农用抗生素,其生物合成途径已被阐明。NDP-葡萄糖合成酶(gtt)与葡萄糖脱氢酶(gdh)是多杀菌素生物合成途径中的限速酶。从SIPI-A.2090克隆gtt及gdh基因,并构建了表达这两个基因的整合型质粒,转入产多杀菌素刺糖多孢菌,发酵并验证其基因型。结果表明,阳性突变株SIPI—M.2092的多杀菌素发酵单位比出发菌株提高了173%,增加gtt和gdh基因拷贝数可以有效提高多杀菌素的发酵单位。     

新型生物农药-多杀菌素

多杀菌素是由刺糖多孢菌(Saccharopolyspora spinosa)经有氧发酵后的次级代谢产物,是一种新颖的大环内酯类抗生素,兼有生物农药的安全性和化学农药的速效性。本文主要就多杀菌素的研究历史、结构、理化性质、生物合成、作用机制、杀虫活性和非靶标生物毒性及降解途径进行了综述。     

基于核糖体工程理论的常压室温等离子体诱变筛选多杀菌素高产菌

利用核糖体工程理论,采用常压室温等离子体(ARTP)诱变对刺糖多孢菌(Saccharopolyspora spinosa)进行诱变筛选。以刺糖多孢菌QYLZ 88912菌株为出发菌株,对其进行ARTP诱变,选取三个不同致死率的照射时间,将孢子悬液混合,以增加抗性筛选几率。然后基于核糖体工程技术理论对混合孢子悬液进行初筛,最终筛选出了1株多杀菌素高产菌Sg200Rif110St40Er90-028。该菌株同时具有磺胺胍、利福平、链霉素、红霉素的多重抗性,摇瓶发酵试验表明,发酵7d后多杀菌素浓度可达到1 516.93 mg/L,较出发菌株QYLZ 88912的产量610.75 mg/L提高了148.37%,且遗传性稳定。以得到的高产菌Sg200Rif110St40Er90-028作为出发菌株,对发酵培养基进行响应面优化实验,优化后的培养基(g/L):葡萄糖46.97、麦芽糊精35、酵母粉40.36、水浸棉籽粉32.88、碳酸钙3,多杀菌素的产量为2 361.81 mg/L,比出发菌株的产量提高了286.71%。     

多杀菌素的生物合成

多杀菌素是一种新颖大环内酯类杀虫剂,具有对害虫高效、对环境安全、对哺乳动物低毒的优异特点。介绍了多杀菌素生物合成的步骤,及参与这些合成步骤的有关酶系统和基因簇。通过对刺糖多孢菌中多杀菌素合成基因的克隆鉴定与分析,已基本了解多杀菌素生物合成的限速步骤及相关控制基因,从而可通过遗传工程的办法改造刺糖多孢菌,提高多杀菌素的产量 。     

低剂量多杀菌素继代处理对小菜蛾nAChR基因表达的影响

【目的】探索多杀菌素对小菜蛾Plutella xylostella长期胁迫下的亚致死效应。【方法】采用低剂量(LC25)多杀菌素进行继代处理,应用叶片浸渍饲喂法分别测定不同处理世代小菜蛾种群Sub对多杀菌素的敏感性水平,并运用实时定量PCR法进一步检测低剂量多杀菌素处理后小菜蛾4龄幼虫烟碱型乙酰胆碱受体(n AChR)α亚基基因表达量变化。【结果】随着处理世代的增加,小菜蛾对多杀菌素的敏感性下降,至第10代(Sub10)其抗性倍数为2.70倍;同时对小菜蛾n AChRα亚基基因表达呈现一定的抑制作用,Sub10中n AChRα亚基基因的相对表达量较敏感种群SS下降了32.04%,且差异显著;而抗性种群RR(抗性倍数17.85)n AChRα亚基基因的相对表达量仅为SS的0.37倍。【结论】小菜蛾对多杀菌素敏感性可能与n AChRα亚基基因的表达量有关。本研究可为探究多杀菌素长期低剂量处理对小菜蛾抗药性发展的潜在影响提供一定依据。     

响应面法优化多杀菌素发酵培养基的研究

采用响应面分析方法,对刺糖多孢茵（Saccharopolyspora spinosa）H-2产多杀菌素的发酵培养基进行优化研究。运用单因子试验筛选出葡萄糖和棉籽粉为最适碳源和氮源,通过Plack—ett—Burman设计试验,对影响发酵培养基的8个相关因子进行评估并筛选出具有显著效应的4个因子：葡萄糖、棉籽粉、黄豆饼粉及玉米浆。通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box-Behnken响应面分析法,确定发酵产多杀菌素最佳培养基为葡萄糖64．5g,麦芽糖20g,玉米浆2g,大豆油40g,棉籽粉25g,黄豆饼粉2．4g,蛋白胨25g,CaCO35g,定容至1L,pH7．0。培养基优化后多杀菌素产量由278．1mg／L提高到508．7mg／L,比初始多杀茵素产量提高了1．83倍。     

多杀菌素生物合成的研究进展北大核心CSCD

多杀菌素是在刺糖多胞菌发酵液中提取的一种大环内酯类生物杀虫剂,它对靶标昆虫具有独特的快速触杀和摄食毒性,兼具化学农药的速效性和生物农药的安全性,具有低残留、快速降解等优点。本文介绍了多杀菌素的生物合成途径和体外合成方法,对随机诱变、代谢工程定向改造等刺糖多孢菌工业菌株育种方法进行了介绍,对多杀菌素在异源宿主中的合成进行了讨论。     

多杀菌素生物合成途径及改造策略

多杀菌素是一类由刺糖多孢菌产生的新型大环内酯类化合物,是绿色农用抗生素的杰出代表。本文简要总结了多杀菌素生物合成过程,并对其生物合成调控的关键位点进行了分析,提出用合成生物学手段组装多杀菌素细胞工厂的策略。     

响应曲面分析方法优化发酵液中多杀菌素的提取工艺

依据中心复合旋转设计原理,在单因素试验的基础上,采用三因素五水平的响应曲面分析法,建立了发酵液中多杀菌素提取的二次多项式数学模型,并以多杀菌素质量浓度为响应值作响应面和等高线,考察了提取时间、料液比和提取频率对多杀菌素质量浓度的影响。结果表明:发酵液中多杀菌素超声提取的优化工艺条件为提取频率75 KHz,提取时间74 min,料液比1∶1.5。在此工艺条件下,从发酵液中提取多杀菌素的质量浓度可达到100.75μg/mL。     

多杀菌素生产菌种复壮方法及其效应的研究

用分离纯化复壮、淘汰法复壮及虫体复壮三种复壮方法对一株多杀菌素生产能力已经衰退的刺糖多孢菌进行复壮研究。结果显示：淘汰法复壮可显著提高菌株的杀虫毒力及多杀菌素的产量。通过红霉素抗性复壮筛选得到菌株杀虫死亡率达到100％,多杀菌素相对效价达1058．83％;通过NaCl抗性复壮筛选得到一菌株杀虫死亡率达到95％,多杀菌素相对效价达825．80％,因此淘汰法可作为有效的复壮方法。     

Insecticide,sugar, and diet effects on feeding and mortality in Rhagoletis indifferens (Dipt., Tephritidae)

The effects of spinosad bait and various insecticides, the presence of sugar in insecticides, and diet on feeding responses and mortality in western cherry fruit fly, Rhagoletis indifferens Curran (Dipt., Tephritidae), were determined. Numbers of feeding events on insecticides with sugar were greater than on insecticides alone, but there was only a small effect of diet on feeding responses to insecticides with sugar. Feeding durations on imidacloprid, thiamethoxam and acetamiprid with sugar were shorter than on sugar water and spinosad bait, as the neonicotinoids paralysed flies quickly. Flies that fed on sugar only (nitrogen‐starved) suffered higher mortalities when exposed to spinosad, thiamethoxam and azinphos‐methyl than to imidacloprid, acetamiprid and indoxacarb, and mortality in between these two groups of treatments when exposed to spinosad bait. Mortalities were greater when sugar was added to insecticides, and were higher in nitrogen‐starved than fully‐fed (yeast extract + sugar fed) flies. Flies that fed once on thiamethoxam were killed more quickly than those that fed once on spinosad bait and spinosad. Results suggest that thiamethoxam is comparable to spinosad in its effects on mortality, and that using it with sugar in bait may also have similar results as using spinosad bait or spinosad. One benefit of using thiamethoxam with sugar may be that it kills flies more quickly, before they can oviposit, than spinosad bait, although whether a fly will feed on it may depend on how much sugar or nitrogenous food it has eaten.     

Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations

The toxicity of spinosad was determined in one susceptible and five insecticide-resistant laboratory strains of house fly, Musca domestica L. Spinosad was relatively slow-acting, but highly toxic to house flies. In a feeding bioassay, spinosad LC50 at 72 h was 0.51 microg of spinosad per gram of sugar, making it 6.3- and 3.5-fold more toxic to house flies compared with azamethiphos and methomyl, respectively. In topical application bioassay, the LD50 at 48 h of spinosad in susceptible house flies was 40 ng per 20 mg of house fly, making spinosad less toxic than the pyrethroid bioresmethrin synergized by piperonyl butoxide and the organophosphate dimethoate. The insecticide-resistant laboratory strains had resistance factors to spinosad at LC50 in feeding bioassay from 1.5 to 5.5 and at LD50 in topical application bioassay from 2.5 to 4.7, indicating that in house fly cross-resistance to the major insecticide classes will not initially be of major concern for the use of spinosad for house fly control. The toxicity of spinosad was also evaluated against 31 field populations of house flies collected from livestock farms across Denmark. The field populations were 2.2- to 7.5-fold resistant to spinosad at 72 h in feeding bioassay, but based on steep slopes in the bioassay and the limited variation of spinosad toxicity against the various field populations, we consider the field populations to be spinosad-susceptible. We propose a diagnostic dose of 12 microg of spinosad per gram of sugar in feeding bioassay with impregnated sugar for determination of resistant house flies, which is 10x the LC95 of the susceptible strain WHO and approximately = 2x the LD95 of the field populations. Spinosad showed no substantial cross-resistance to the pyrethroid bioresmethrin synergized by piperonyl butoxide, the anticholinesterases dimethoate, azamethiphos, methomyl, and spinosad in house fly field populations.     

In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dα6 using the insecticide spinosad

The vinegar fly, Drosophila melanogaster, has been used to identify and manipulate insecticide resistance genes. The advancement of genome engineering technology and the increasing availability of pest genome sequences has increased the predictive and diagnostic capacity of the Drosophila model. The Drosophila model can be extended to investigate the basic biology of the interaction between insecticides and the proteins they target. Recently we have developed an in vivo system that permits the expression and study of key insecticide targets, the nicotinic acetylcholine receptors (nAChRs), in controlled genetic backgrounds.Here this system is used to study the interaction between the insecticide spinosad and a nAChR subunit, Dα6. Reciprocal chimeric subunits were created from Dα6 and Dα7, a subunit that does not respond to spinosad. Using the in vivo system, the Dα6/Dα7 chimeric subunits were tested for their capacity to respond to spinosad. Only the subunits containing the C-terminal region of Dα6 were able to respond to spinosad, thus confirming the importance this region for spinosad binding.A new incompletely dominant, spinosad resistance mechanism that may evolve in pest species is also examined. First generated using chemical mutagenesis, the Dα6^P146S mutation was recreated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, the first use of this technology to introduce a resistant mutation into a controlled genetic background. Both alleles present with the same incompletely dominant, spinosad resistance phenotype, proving the P146S replacement to be the causal mutation. The proximity of the P146S mutation to the conserved Cys-loop indicates that it may impair the gating of the receptor. The results of this study enhance the understanding of nAChR structure:function relationships.     

Lethal and sub‐lethal effects of spinosad on the life‐history traits of army worm,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and its fitness cost of resistance

Spodoptera litura is one of the most destructive polyphagous insect pests, with more than 120 host‐plant species. In our present study, a field‐collected population of S. litura when selected with spinosad for 11 consecutive generations resulted in the development of 3921‐fold resistance to spinosad as compared to the susceptible strain. The spinosad‐resistant strain of S. litura had a relatively high fitness cost (0.17) as compared to the susceptible strain. Furthermore, the lethal and sub‐lethal effects of different concentrations of spinosad were checked on the susceptible strain at different levels; i.e., LC₄₀, LC₃₀, LC₂₀ and LC₁₀, which revealed that the impact of spinosad on the life‐history traits of S. litura increased with the increase in concentration of spinosad. A significant impact of spinosad was recorded on the larval duration, pre‐pupal weight, pupal duration, pupal weight, reproductive potential and adult emergence. The outcomes of the current research clearly indicate that fitness cost of spinosad and its sub‐lethal effects have a significant impact on population dynamics of S. litura, for which it can be incorporated in integrated pest management.     

多杀菌素生物合成基因簇启动子探测和转录时序

多杀菌素是对农业虫害防治及粮食仓储安全均具有重大意义的农用抗生素.为了深入揭示刺糖多孢菌合成多杀菌素的调控特点,首先通过建立基于报告基因的启动子探测技术,探测了多杀菌素生物合成基因簇的9个启动子活性.并进一步通过荧光定量PCR,分析了这9个基因和不在基因簇内的负责糖基前体供应和鼠李糖合成的4个基因的转录时序,结果表明多杀菌素生物合成基因簇内的9个基因在菌体生长进入稳定期时有较高的转录,这和发酵液中此时开始大量积累多杀菌素一致;同时还发现,簇外的4个与糖基供应相关的基因和基因簇内基因的转录时序不同,它们在菌体生长对数期有较高的转录活性,这暗示多杀菌素聚酮链的合成速率和参与后修饰的糖基底物供应的最优化匹配有可能是提高生物合成多杀菌素的前提和关键.     

Monitoring insecticide resistance in Australian Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) detects fipronil and spinosad resistance

Abstract  Insecticide resistance monitoring using a Potter precision spray tower with discriminating concentration and log dose probability techniques underpins the Australian insecticide management strategy for Frankliniella occidentalis Pergande. Abamectin, acephate, chlorpyrifos, dichlorvos, dimethoate, endosulfan, fipronil, malathion, methamidophos methidathion, methiocarb, methomyl, pyrazophos and spinosad are recommended for use against F. occidentalis but abamectin, methiocarb and pyrazophos are the only chemicals where insecticide resistance has not been detected. Although not registered, chlorfenapyr was effective against F. occidentalis and should be pursued for that purpose. In contrast, chlorpyrifos, dichlorvos and malathion resistance were detected at low to moderate levels throughout the study period putting their sustainable use for F. occidentalis control in doubt . Although it appears that acephate, dimethoate, endosulfan, fipronil, methamidophos, methidathion and spinosad remain effective, some populations contained a small percentage of thrips that survived exposure to a concentration that killed 100% of the susceptible strain. Subsequent laboratory selection of one such population separately with fipronil and spinosad caused an increase in resistance to these insecticides. These products must now be considered at risk. This is the first report of fipronil or spinosad resistance in populations of F. occidentalis.     

Expression of Xenobiotic Metabolizing Cytochrome P450 Genes in a Spinosad-Resistant Musca domestica L. Strain

Background

Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.

Results

Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.

Conclusion

CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.     

Evaluation of SPLAT with spinosad and methyl eugenol or cue-lure for "attract-and-kill" of oriental and melon fruit flies (Diptera: Tephritidae) in Hawaii

Specialized Pheromone and Lure Application Technology (SPLAT) methyl eugenol (ME) and cue-lure (C-L) "attract-and-kill" sprayable formulations containing spinosad were compared with other formulations under Hawaiian weather conditions against oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), respectively. Field tests were conducted with three different dispensers (Min-U-Gel, Acti-Gel, and SPLAT) and two different insecticides (naled and spinosad). SPLAT ME with spinosad was equal in performance to the standard Min-U-Gel ME with naled formulation up to 12 wk. SPLAT C-L with spinosad was equal in performance to the standard Min-U-Gel C-L with naled formulation during weeks 7 to12, but not during weeks 1-6. In subsequent comparative trials, SPLAT ME + spinosad compared favorably with the current standard of Min-U-Gel ME + naled for up to 6 wk, and it was superior from weeks 7 to 12 in two separate tests conducted in a papaya (Carica papaya L.) orchard and a guava (Psidium guajava L.) orchard, respectively. In outdoor paired weathering tests (fresh versus weathered), C-L dispensers (SPLAT + spinosad, SPLAT + naled, and Min-U-Gel + naled) were effective up to 70 d. Weathered ME dispensers with SPLAT + spinosad compared favorably with SPLAT + naled and Min-U-Gel + naled, and they were equal to fresh dispensers for 21-28 d, depending on location. Our current studies indicate that SPLAT ME and SPLAT C-L sprayable attract-and-kill dispensers containing spinosad are a promising substitute for current liquid organophosphate insecticide formulations used for areawide suppression of B. dorsalis and B. cucurbitae in Hawaii.