刺糖多孢菌高产菌株和野生型菌株多杀菌素生物合成基因簇(spn)在发酵过程中的表达分析

多杀菌素(spinosad)作为一种高效、低毒生物杀虫剂,已在农药、兽药、卫生用药等领域得到应用。中国关于多杀菌素的研究起步较晚,进展缓慢,目前仍无法实现产业化生产。本研究旨在分析刺糖多孢菌(Saccharopolyspora spinosa)高产菌株ASAGF73与野生型菌株ATCC49460在发酵过程中多杀菌素合成基因簇(spinosyn biosynthetic gene cluster,spn)的表达变化及差异,初步判断影响多杀菌素合成的关键限速步骤。生物信息学分析表明,spn基因簇含有9个转录子,分别在各转录子中设计引物,进行RTPCR检测其表达情况。结果显示,突变株ASAGF73中多杀菌素的产量提高可能与spn基因簇中部分基因的高表达有关,其中编码聚酮合酶(polyketide synthase,PKS)的基因和参与大环内联桥形成的基因表达量远高于野生型菌株的。在ASAGF73中编码鼠李糖转移酶的spn G基因在发酵前期过量表达,而在后期大量合成多杀菌素时表达减弱,有可能成为多杀菌素合成的限速步骤。本研究初步阐明能够提高多杀菌素产量的分子机制,为构建多杀菌素高产基因工程菌提供参考。     

Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

The cross-resistance and biochemical mechanism of the beet armyworm, Spodoptera exigua (Hübner), to spinosad was studied in the laboratory. S. exigua population were collected from Shanghai suburb. After five generations of selection, the resistance of S. exigua to spinosad increased 345.4 times compared with the susceptible strain. There was no cross-resistance between spinosad and fenvalerate, phoxim, methomyl, abamectin, and cyfluthrin. When the inhibitors, PBO, TPP, DEF, and DEM were used as synergist in the susceptible strain and resistant strain, the synergistic ratio was 0.7-, 0.5-, 1.0-, and 0.6- fold for the susceptible strain, and 9.8-, 1.5-, 2.6-, and 1.5-fold for the resistant strain, respectively. The results revealed that PBO had significant synergistic effect on the resistant strain. The activity in vitro of microsomal-O-demethylase and glutathione S-transferase in the resistant strain was 5.2- and 1.0-fold of the susceptible strain, respectively. The results implied that microsomal-O-demethylase might be important in conferring spinosad resistance in the S. exigua population.     

Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections

Angiostrongylus vasorum is an increasingly reported parasite in Europe that develops in dogs after ingestion of infective third stage larvae (L3) that reside in gastropod molluscs which are needed to complete the parasite's life-cycle. Infection can produce a diversity of clinical signs, determined by involvement of the respiratory, neurological, and/or coagulation system, with a likely fatal outcome in the absence of treatment. Few drugs have been shown to reliably prevent infection, and data on treatment of infections is limited. A controlled, randomized, partially blinded laboratory study was therefore executed to evaluate the efficacy and safety of a combination tablet of spinosad/milbemycin oxime in dogs inoculated with approximately 250 A. vasorum L3. Sixteen healthy nematode free adult dogs were randomly allocated to two study groups of 8 dogs each. Thirty days post inoculation (dpi) all dogs in the fed state were treated: dogs in group B were treated with spinosad and milbemycin oxime at the dose rates of 45–60 mg/kg and 0.75–1.0 mg/kg bodyweight, respectively, approximately the lower half portion of the expected full unit dose range; dogs in group A were treated with placebo tablets. All dogs were euthanized and necropsied 56–58 dpi. The heart and lungs were examined to determine the presence of A. vasorum. All placebo group dogs were infected at necropsy with counts ranging from 22 to 98 adult worms and a geometric mean worm count of 55.2. In contrast, the geometric mean worm count in the spinosad/milbemycin oxime group was 0.7 with worm numbers ranging from 0 to 8. The results of this study demonstrate that a single treatment with the tablet combination of spinosad and milbemycin oxime administered 30 dpi provided 98.8% preventive efficacy against development of adult A. vasorum infections. Monthly treatments with spinosad and milbemycin oxime have the potential to prevent the establishment of infections with A. vasorum in dogs.     

多杀菌素对小菜蛾及其天敌的毒力研究

研究评价多杀菌素(菜喜)对小菜蛾及其主要天敌的毒力结果表明,菜喜对小菜蛾具有很高的毒力和良好的田间防治效果,菜喜对青翅蚁型隐翅虫和菜蛾绒茧蜂具有直接杀伤作用,其1000倍稀释液24h使菜蛾绒茧蜂死亡率达62.2%,36h死亡率达88.7%,药液直接处理寄生蜂茧或饲喂均导致很高的死亡率,但对拟环纹狼蛛影响较小。多杀菌素对小菜蛾有很好的控制效果,但对小菜蛾天敌,特别是寄生蜂有相当的负面影响。     

Selection,mechanism, cross resistance and stability of spinosad resistance in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Spodoptera litura is one of the most destructive pests in Pakistan and in many other regions of the world. A field collected population of S. litura was selected with spinosad for eleven generations under controlled laboratory conditions to study the cross resistance, mechanism and stability of spinosad resistance in S. litura. The resistance to spinosad in S. litura increased 3921-fold (after eleven generations of selection with spinosad) as compared to a susceptible population of S. litura. No cross resistance between spinosad and emamectin benzoate, methoxyfenozide, fipronil, indoxacarb, profenofos, lufenuron or deltamethrin was found in the spinosad-selected population of S. litura. To find the possible mechanism of spinosad resistance in S. litura two synergists, Piperonyl butoxide (PBO), S, S, S-tributyl phosphorotrithioate (DEF) were tested on the susceptible and resistant strains and on the un-selected field population. The values of the synergism ratios of PBO and DEF were 2.33 and 1.06 for the spinosad-selected strain, 1.36 and 1.06 for the un-selected field population and 1.14 and 1.00 for the susceptible strain, respectively. As high PBO ratio indicates the role of microsomal O-demethylase in causing spinosad resistance in S. litura. The spinosad-resistant and field populations of S. litura were reared without any selection pressure from the 12th to the 16th generation (G₁₂–G₁₆). The spinosad resistance decreased from 3921 to 678-fold in the spinosad-resistant population and from 31.1 to 15.1-fold in the un-selected population of S. litura as compared to the susceptible strain. Spinosad resistance in S. litura has a high reversion rate (−0.15) which indicates that spinosad resistance in S. litura is unstable and can be easily managed by switching off the selection pressure for a few generations or alternating with insecticides having different modes of action.     

Laboratory and field susceptibility of blueberry spanworm (Lepidoptera: Geometridae) to conventional and reduced-risk insecticides

Blueberry spanworm (Itame argillacearia Packard) is a key insect defoliator of wild blueberry, an important crop in north-eastern North America. We evaluated the susceptibility of blueberry spanworm to several reduced-risk insecticides in laboratory and field experiments. Two populations from two Canadian provinces were studied in the laboratory. Both were highly susceptible to flubendiamide, spinetoram and deltamethrin, although there were inter-population differences, and flubendiamide was slower acting. Flubendiamide, spinosad, spinetoram, and methoxyfenozide demonstrated good suppression of economically damaging populations of blueberry spanworm larvae in the field.     

多杀菌素对阿里山潜蝇茧蜂的亚致死效应

以对多杀菌素敏感的橘小实蝇实验种群做寄主饲养阿里山潜蝇茧蜂,毒力测定结果显示,多杀菌素对阿里山潜蝇茧蜂雌蜂和雄蜂的LC50分别为0.090 8 mg/mL和0.027 4 mg/mL,LD50分别为1.004 1 mg和0.804 3 mg。分别用1 000、2 000、3 000倍液的多杀菌素处理阿里山潜蝇茧蜂成蜂,与对照相比,不同倍液的多杀菌素亚致死剂量均使阿里山潜蝇茧蜂的寿命显著缩短;雌蜂的总产卵量、平均产卵量、寄生率、子代成蜂羽化率等均随着多杀菌素浓度的增大而减少;但子代雌雄性比之间差异不显著;阿里山潜蝇茧蜂寄生橘小实蝇卵的拟合方程符合Holling圆盘方程Ⅱ型,处理寄主的时间随着多杀菌素浓度的增大而增加、瞬时攻击率、搜寻效应等均随着多杀菌素浓度的增大而减少。结果表明,多杀菌素的亚致死剂量可通过缩短寿命、降低繁殖力、改变功能反应参数等影响阿里山潜蝇茧蜂,从而影响生物防治的效果。     

多杀菌素饵剂对红火蚁的药效试验

[目的]为红火蚁的有效防治提供依据。[方法]选择以多杀菌素为原药,制成含量不同的多杀菌素诱饵,研究其对红火蚁的室内外毒杀效果。[结果]0.02%、0.04%和0.08%的多杀菌素诱饵室内毒杀作用迅速,红火蚁全部死亡时间分别为24 h1、2 h和8 h;0.01%多杀菌素毒饵室内作用适中,药后54 h红火蚁死亡率可达到100%。0.01%多杀菌素毒饵对红火蚁野外防控效果明显,药后5 d防控效果达到71.52%,25 d后上升到92.91%。0.01%多杀菌素诱饵不会快速杀死觅食工蚁,可在红火蚁个体间传播,最后达到令其全巢覆灭的目的。[结论]多杀菌素饵剂具有药效高、防治彻底、适合于各种环境使用等优点,是防治红火蚁的理想方法。     

Resistance selection and biochemical characterization of spinosad resistance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

A Helicoverpa armigera population was collected from Shandong province, China. After 15 generations of selection in the laboratory, the H. armigera strain developed more than 20-fold resistance to spinosad. At LD₅₀ level, no significant cross-resistance was found between spinosad and chlorpyrifos, methomyl, avermectin and chlorfenapyr except for fenvalerate with a low cross-resistance of 2.4-fold. However, LD₉₉ values of fenvalerate against the parental and resistant strains were not different significantly. After inhibitors were used, spinosad resistance could be partially suppressed by piperonylbutoxide (PBO) and triphenylphosphate (TPP), but not by diethylmaleate (DEM). Activities of p-nitroanisole O-demethylase (ODM) developed to 8.26-fold compared with the parental strain, but no obvious changes were found in activities of carboxyl esterase (CarE) and glutathione-S-transferase (GST). The results indicated that resistance to spinosad in the cotton bollworm might be associated with an increase in cytochrome P450 monooxygenase.     

The Post-exposure Effects of Spinosad and Plant Allelochemicals on Biological Characteristics of Helicoverpa armigera (Lepidoptera:Noctuvidae)

In order to define the combined effects of spinosad and plant allelochemicals on Helicoverpa armigera(Lepidoptera:Noctuvidae), the research fed them on artificial diets with different doses of spinosad, gossypol, quercetin and tannicacid, and the changes of pupal and imaginal biological characteristics were observed and recorded. The results showed that spinosad at sublethal concentrations decreased pupation ratio and pupal weight, prolonged prepupal and pupal periods, and decreased emergence ratio, fecundity and longevity of adults. Combined with plant allelochemicals, spinosad significantly prolonged pupal period and shorten adult longevity. Other life-cycle parameters such as pupation ratio, pupal weights, emergence ratio, eggs laid per female and hatch ratio did not change significantly. The results clearly indicated that plant allelochemicals did not change spinosad efficiency on H. armigera, but the combination of spinosad and plant allelochemicals might affect pest population dynamics significantly by changing its development.