世界上含量最高的昆虫病毒杀虫剂在中国诞生

棉铃虫病毒又称棉铃虫核型多角体病毒（NPV），是棉铃虫专一性的病原微生物。棉铃虫NPV生物农药防治效果好、药效持久，对其他生物无任何毒副作用，是世界各国竞相开发生产的昆虫杀虫剂产品，也是世界上第一个登记注册的昆虫病毒生物农药。但长期以来，棉铃虫NPV必须通过“活体”生产，多项核心技术没有得到根本突破，导致生产规模小、产品生产成本高，难以实现产业化。     

环境中棉铃虫NPV定性定量的ELISA测定

酶联免疫吸附检测法,具有较高的敏感性,特异性强、简便、容易观察结果,便于大规模检测等特点。本试验采用这个方法,对棉铃虫核型多角体病毒(Heliothis armigera NPV)杀虫剂的生产和使用环境中的水、土进行了ELIS A测定。在大量用过此杀虫剂的田里,发现NPV合量约为4—9PIB/克土,扩散区约为6—53PIB/克土,污染严重的水塘中,病毒含量约为630PIB/ml水,产品车间约为830PIB/克土以上。     

我国生物农药的发展现状

我国生物农药的研究开发始于 5 0年代 ,直到 80年代中期才开始正式列入国家科技攻关的发展计划。近十多年来 ,在生物农药的资源筛选评价、新产品开发、生产工艺、产品质量检测和新技术示范推广方面都取得了长足的进展。目前国内生物农药研究开发的热点 ,主要集中在苏云金芽胞杆菌杀虫剂、农用抗生素杀虫剂、农用抗生素杀菌剂、拮抗细菌生防制剂、昆虫病原真菌制剂和昆虫杆状病毒杀虫剂几个方面。生物农药技术产品开始逐渐走入市场。1　我国生物农药技术产品现状1.1　苏云金芽胞杆菌杀虫剂我国自 5 0年代末开始苏云金杆菌 (简称 Bt)杀虫剂的…     

棉铃虫对Bt生物农药早期抗性及与转Bt基因棉抗虫性的关系

用饲料感染法建立了棉铃虫Helicoverpa rmigera(Hubmer)敏感品系(SUS1)对Bt生物农药的敏感毒力基线和区分剂量,1995年测定了五省六县棉铃虫初孵幼虫对Bt生物农药的敏感性,结果表明：山东阳谷、河北邯郸、河南新乡、安徽萧县及江苏丰县棉铃虫已产生早期抗性,抗性个体百分率为5%～10%,与敏感品系相比,LC₅₀值稍有增加,但斜率b值明显变小;而江苏东台棉铃虫仍属敏感。这是国内外首次诊测到棉铃虫对Bt生物农药抗性。用棉叶喂饲法测定比较了转Bt基因棉花品系对不同种群棉铃虫的抗虫性效果,结果表明：用早期抗性的阳谷和新乡棉铃虫初孵幼虫接虫5d后平均死亡率较敏感品系下降16%～29%,说明棉铃虫对Bt农药与转Bt生物基因棉花品系间存在交互抗性。还讨论了Bt农药的抗性治理对策。     

棉铃虫对Bt生物农药早期抗性及与转Bt基因棉抗虫性的关系

用饲料感染法建立了棉铃虫Helicoverpa rmigera(Hubmer)敏感品系(SUS1)对Bt生物农药的敏感毒力基线和区分剂量,1995年测定了五省六县棉铃虫初孵幼虫对Bt生物农药的敏感性,结果表明：山东阳谷、河北邯郸、河南新乡、安徽萧县及江苏丰县棉铃虫已产生早期抗性,抗性个体百分率为5%～10%,与敏感品系相比,LC₅₀值稍有增加,但斜率b值明显变小;而江苏东台棉铃虫仍属敏感。这是国内外首次诊测到棉铃虫对Bt生物农药抗性。用棉叶喂饲法测定比较了转Bt基因棉花品系对不同种群棉铃虫的抗虫性效果,结果表明：用早期抗性的阳谷和新乡棉铃虫初孵幼虫接虫5d后平均死亡率较敏感品系下降16%～29%,说明棉铃虫对Bt农药与转Bt生物基因棉花品系间存在交互抗性。还讨论了Bt农药的抗性治理对策。     

荧光增白剂Tinopal LPW对蜀柏毒蛾核型多角体病毒增效作用研究

用1%TinopalLPW荧光增白剂作为蜀柏毒蛾核型多角体病毒增效剂对蜀柏毒蛾2龄幼虫进行室内毒力测定,结果表明1%TinonalLPW对ParocneriaorientaNPV有较强的增效作用。使用3.6×1011PIB/hm2 1%TinopalLPW、1.8×1011PIB/hm2 1%TinopalLPW、9.0×1010PIB/hm2 1%TinopalLPW和3.6×1011PIB/hm2、1.8×1011PIB/hm2、9.0×1010PIB/hm26种处理对林间越冬代2-3龄幼虫进行超低容量喷雾防治,结果表明除9.0×1010PIB/hm2 1%TinopalLPW表现出显著的增效作用外,其余剂量有增效作用但不显著。     

阿维菌素苯甲酸盐和苜蓿银纹夜蛾核型多角体病毒对斜纹夜蛾幼虫和卵的毒力试验

采用常规方法在室内外分别测定了0.5%甲维盐(甲胺基阿维菌素苯甲酸盐,Banlep)微乳剂、10亿PIB/mL奥绿1号(苜蓿银纹夜蛾核型多角体病毒,AcMNPV)悬浮剂对斜纹夜蛾Prodenia litura(Fabricius)幼虫、卵及其初孵幼虫的杀伤力。试验结果表明,甲维盐和奥绿1号对初孵幼虫、3龄幼虫及卵具有很强的杀虫力,其死亡率分别可达95%~100%,且较特异性农药美满(虫酰肼)见效明显要快,是当前防治斜纹夜蛾等夜蛾类害虫较理想的2种新生物农药。     

棉铃虫病毒杀虫乳悬剂的生产及其药效试验

成功研制出棉铃虫病毒（ＮＰＶ）杀虫乳悬剂,改进了生产工艺和设备,建立了棉铃虫的室内品系,提高了生产效率,建立了产品的检测方法,提高了产品质量,促使棉铃虫病毒杀虫剂商品开发获得成功。大田试验证明,病毒乳悬剂的防治效果,相当于当前推广的化学农药,优于原病毒可湿性粉剂。相同剂量（０．５３一１．０７×１０（１０）ＰＩＢ／公顷）的病毒乳悬剂可使虫口平均减退９１．７％,而病毒可湿性粉剂为８３．７％。     

荧光增白剂Tinopal LPW对蜀柏毒蛾核型多角体病毒增效作用研究

用1%TinopalLPW荧光增白剂作为蜀柏毒蛾核型多角体病毒增效剂对蜀柏毒蛾2龄幼虫进行室内毒力测定,结果表明1%Tinonal LPW对Parocneria orienta NPV有较强的增效作用.使用3.6×1011PIB/hm2+1%TinopalLPW、1.8×1011PIB/hm2+ 1%TinopalLPW、9.0×1010PIB/hm2+1%TinopalLPW和3.6×1011PIB/hm2、1.8×1011PIB/hm2、9.0×1010PIB/hm26种处理对林间越冬代2-3龄幼虫进行超低容量喷雾防治,结果表明除9.0×1010PIB /hm2+1%TinopalLPW 表现出显著的增效作用外,其余剂量有增效作用但不显著.     

灰茶尺蛾核型多角体病毒大田防治条件的探索

为了经济有效地在大田应用EgNPV防治灰茶尺蛾 ,在室内进行EgNPV的毒力测定 ,试验表明致死中量LC50 为 1.13× 10 6PIB/mL ,90 %的致死量为 1.83× 10 7PIB/mL ,在 95%置信范围内 ,上限为 1.96× 10 7PIB/mL ,下限为 1.7× 10 7PIB/mL。用 2× 10 7PIB/mL浓度对不同龄期幼虫的感染试验表明 ,1 2龄效果最好 ,达 90 %以上。不同世代感染试验表明 :第 2、3、4、7代防治效果较好 ,高达 90 %以上 ,而第 5、6代效果不好 ,因此病毒增殖与大田防治工作应尽量避免在高温 ,干旱季节进行。另外探讨了适用大田防治的病毒增殖方法。     

Interaction of HaNPVs with two novel insecticides against Helicoverpa armigera Hubner (Noctuidae: Lepidoptera)

Nucleopolyhedrosis viruses can be utilized for effective management of agriculture pests. Their efficacy can be increased if they are mixed with certain insecticides. In the current study, HaNPV was mixed with two insecticides: spinetoram and emamectin benzoate in various combinations and applied to larvae of H. armigera in laboratory conditions. There were a total of 15 combinations of HaNPV with each of the two insecticides in addition to five doses of HaNPV and three doses of insecticides alone. The synergistic and antagonistic effects of combinations were explored. The results revealed that there was synergistic effect of HaNPV @ 0.5 × 10⁹ PIB/ml × Spinetoram @ 40, 20, 10 ml/100 L of water. In case of emamectin benzoate, synergistic effects were recorded at 1 × 10⁹ PIB/ml HaNPV × emamectin benzoate @ 100 ml/100 L of water. However, 0.5 × 10⁹ PIB/ml HaNPV has synergistic effects with all three doses of emamectin benzoate. The results suggested that HaNPV can be used in combination with spinetoram and emamectin benzoate for the management of resistant population of H. armigera.     

Field evaluation of nucleopolyhedrosis virus and some biorational insecticides against Helicoverpa armigera Hubner (Noctuidae: Lepidoptera)

American bolloworm, Helicoverpa armigera Hubner (Noctuidae: Lepidoptera) is considered as a major pest of various crops all over the world. It is mainly controlled by indiscriminate use of synthetic insecticides in the world due to which this pest developed resistance to most of the available insecticides. Therefore, in the current study, the efficacy of virulent strain of HaNPV (0.5 × 10⁹ PIB/ml) alone and in combination with recommended doses of spintoram (20 g/100 L of water) and emamectin benzoate (200 ml/100 L of water) was tested in field. The combination of HaNPV with spintoram and emamectin benzoate 100% reduced the larval population as compared to emamectin benzoate and HaNPV alone. This suggested that the combination of spintoram and emamectin benzoate with HaNPV could be used in field to manage the infestation of H. armigera.     

Manifold passages in an assorted infection in a host could improve virulence of Helicoverpa armigera Nucleopolyhedrovirus (HaNPV)

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is serious pests of cotton and several other crops. Helicoverpa armigera Nucleopolyhedrovirus (HaNPV) can be important alternative to synthetic insecticides for the management of H. armigera. However, the efficacy of HaNPV can vary in horizontal and vertical transmission. In the current study, we evaluated the efficacy of HaNPV of a virulent strain (vertically transmitted up to six generations) and wild strains (used after isolation from the field infected larvae). Both strains were applied to the 2nd instar larvae of H. armigera @ 1 × 10⁹ polyhedral inclusion bodies (PIB)/ml. There were six replications of each strain (strains). The results indicated higher mortalities in larvae exposed to virulent strains (68.33 ± 6.07%) as compared to wild strain (45 ± 2.24%). Virulent strains killed the larvae quite faster than wild strain. The lethal time (LT₅₀) to kill 50% of the larvae by virulent strain was 7.15 days and for wild strain it was 19.47 days. The results showed that multiple passage of HaNPV through several generations enhances its efficacy to kill H. armigera larvae faster. The results of this study will be helpful to manage H. armigera and other related lepidopoterous pests.     

细胞松弛素D对棉铃虫核型多角体病毒复制和装配的影响

杆状病毒感染引起宿主细胞肌动蛋白骨架的构象变化 ,使之形成缆绳结构 .棉铃虫核型多角体病毒 (HaNPV)的衣壳蛋白也能使宿主昆虫的肌动蛋白发生凝聚 ,用细胞松弛素D抑制宿主肌动蛋白形成纤丝结构 ,病毒感染Hz AM1,空斑计数表明 ,0 1μg/ml细胞松弛素D可使棉铃虫核型多角体病毒的增殖下降 10 4倍 ,细胞松弛素D浓度增高到 0 5 μg/ml则测不到子代病毒粒子 .Western印迹分析表明 ,细胞松弛素D并不影响受染细胞中肌动蛋白的含量 .斑点印迹 (dotblot)也表明 ,病毒DNA的合成也没有受到影响 ,推测宿主细胞的肌动蛋白纤丝结构与病毒的复制有关 .在电子显微镜下观察超薄切片发现 ,在 0 5 μg/ml细胞松弛素D处理细胞中形成的病毒粒子形态与正常形态明显不同 ,提示细胞松弛素D抑制HaNPV的增殖是由于抑制病毒组装成完整有感染性的病毒粒子 .从而可以认为宿主昆虫细胞的丝状肌动蛋白对子代病毒的复制和组装是必需的 .     

Comparison of bioassay and enzyme-linked immunosorbent assay for quantification of Spodoptera frugiperda nuclear polyhedrosis virus in soil

Standard curves with known amounts of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in soil were established with a bioassay and with an enzyme-linked immunosorbent assay (ELISA). The bioassay detected as few as 4 × 10⁴ polyhedral inclusion bodies (PIB)/g sandy soil and <10 PIB/g soils with large amounts of silt or clay. The ELISA detected as few as 360 PIB/g in all three soil types, and absorbance values were inversely related to the amount of clay. Results of the bioassay and ELISA were significantly (P < 0.01) correlated for natural NPV from field samples of silt (R = 0.961) and sandy soil (R = 0.723). Soil samples from Louisiana pastures and corn fields contain up to 7.6 × 10⁴ PIB/g, and 2× 10⁴ PIB/g are commonly present.     

In vitro production studies with a wild-type Helicoverpa baculovirus

The potential use of a wild-type Helicoverpa baculovirus as a biopesticide, using insect cell culture for its production, has been investigated. A Helicoverpa zea cell line was adapted to grow in suspension culture using a serum-free medium, SF900II and serum supplemented SF900II. The serum supplemented cells were infected with a wild-type nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV), at different stages of growth, in conditioned and tresh medium, to determine the effect of cell density on polyhedra production. Cultures infected at low cell densities, produced similar yields of virus (20–40 PIB/cell), irrespective of medium conditions. However, in infections which occurred at high cell densities, there was a 16-fold improvement in cell specific yields, when the spent medium was renewed with fresh medium prior to infection. Results indicated that only 60–70% of the viable cells in a culture produced polyhedra as a result of infections.     

棉铃虫核型多角体病毒与化学杀虫剂和卵磷脂混用的增效作用

棉铃虫核型多角体病毒(HaNPV)分别与三氟氯氰菊酯、溴氰菊酯、氰戊菊酯、灭净菊酯、灭多威、辛硫磷、甲基对硫磷和乙酰甲胺磷等化学杀虫剂混合饲喂棉铃虫幼虫,统计致死中浓度LC₅₀,计算增效比,测定虫体内与抗性有关的三种重要酶：多功能氧化酶(MFO)、羧酸酯酶(CarE)、乙酰胆碱酯酶(AChE)的活性。研究大豆卵磷脂对HaNPV致病性的影响。结果表明：HaNPV与化学杀虫剂混合饲喂抗性棉铃虫,生测统计增效比均大于1.0,特别是病毒与甲基对硫磷混用,增效比更是达到3.53,表现出良好的增效作用。混剂感染抗性棉铃虫,虫体内MFO的活性比化学杀虫剂单用时降低3～12倍,CarE和AChE的活性也比化学杀虫剂单用时低,HaNPV明显抑制了化学杀虫剂对MFO和CarE的诱导作用。HaNPV与大豆卵磷脂混用,提高了HaNPV对棉铃虫的感染致死率,缩短了致死中时间(LT₅₀)。     

Baculovirus studies in new,indigenous lepidopteran cell lines

Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.     

混合感染后杆状病毒间的增效作用——病毒蛋白及核酸的初步分析

AsNPV+HasNPV、AsNPV+HaNPV、AsNPV+PsNPV分别感染烟青虫、棉铃虫和粘虫幼虫,对分离到的核型多角体病毒(AsNPV+HasNPV)-Helicoverpa assulta、(AsNPV+HaNPV)- H.Armigera和(AsNPV+PsNPV)-Pseudaletia separata,经电镜观察,多角体蛋白及病毒粒子蛋白SDS-PAGE电泳,病毒核酸的限制性内切酶酶解分析等研究,证明各病毒的多角体形态不规则,大小差异极大,病毒粒子为杆状,(AsNPV+HasNPV)-H.Assulta 和(AsNPV+HaNPV)-H.Armigcra病毒粒子有单粒和多粒包埋类型, (AsNPV+PsNPV)-P.Separata为多粒包埋型。各病毒的多角体蛋白基本上只有一种多肽,分子量为25 000道尔顿左右。(AsNPV+HasNPV)-H.Assulta、(AsNPV+HaNPV)-H.armigera和(AsNPV+PsNPV)-P.Separata的病毒粒子分别有10、14、5条多肽,分子量大小在13 500～98 000,13 000～88 000,18 500～52 000道尔顿之间。病毒核酸经EcoRI,HindIII,HindIII+BamHI酶解,其DNA的酶切位点,大小及DNA的总分子量与AsNPV和原寄主的Has-NPV,HaNPV和PsNPVDNA的酶切图谱存在一定差异,混合病毒侵染昆虫后新复制的病毒核酸发生一定的变化,从而导致病毒蛋白和病毒形态的变化。混合感染后AsNPV对Has-NPV、HaNPV和PsNPV的侵染有明显的增效作用,其机理有待深入研究。     

Photodynamic biofilm inactivation by SAPYR—An exclusive singlet oxygen photosensitizer

Prevention and control of biofilm-growing microorganisms are serious problems in public health due to increasing resistances of some pathogens against antimicrobial drugs and the potential of these microorganisms to cause severe infections in patients. Therefore, alternative approaches that are capable of killing pathogens are needed to supplement standard treatment modalities. One alternative is the photodynamic inactivation of bacteria (PIB). The lethal effect of PIB is based on the principle that visible light activates a photosensitizer, leading to the formation of reactive oxygen species, e.g., singlet oxygen, which induces phototoxicity immediately during illumination. SAPYR is a new generation of photosensitizers. Based on a 7-perinaphthenone structure, it shows a singlet oxygen quantum yield Φ_Δ of 99% and is water soluble and photostable. Moreover, it contains a positive charge for good adherence to cell walls of pathogens. In this study, the PIB properties of SAPYR were investigated against monospecies and polyspecies biofilms formed in vitro by oral key pathogens. SAPYR showed a dual mechanism of action against biofilms: (I) it disrupts the structure of the biofilm even without illumination; (II) when irradiated, it inactivates bacteria in a polymicrobial biofilm after one single treatment with an efficacy of ≥99.99%. These results encourage further investigation on the potential of PIB using SAPYR for the treatment of localized infectious diseases.