枯草芽孢杆菌B47菌株抗菌物质的分离纯化及其对玉米小斑病的防治作用

枯草芽孢杆菌Bacillus subtilis菌株B47为番茄内生细菌,能产生对多种植物病原菌有抑制作用的抗菌物质。通过盐酸沉淀、甲醇抽提、硅胶柱层析、凝胶过滤层析和反相高效液相色谱（RP-HPLC）法提取和纯化到该抗菌物质。理化性质测定结果表明,该物质热稳定性和抗紫外线稳定性好;在碱条件下稳定;对蛋白酶不敏感;能溶于甲醇;双缩脲反应呈阳性,茚三酮反应呈阴性,但经酸水解后茚三酮反应呈阳性,推测该物质可能为环肽类物质。该物质对测试的14种病原真菌有拮抗作用,其中,对玉米大小斑病菌和各种香蕉叶斑病菌的拮抗作用强,但对供试病原细菌无拮抗作用。该抗菌物质在0.3g.L-1浓度下对玉米小斑病的田间防治效果为53.05%。     

解淀粉芽胞杆菌HRH317抗菌蛋白的分离纯化及其抗菌作用

为探究解淀粉芽胞杆菌Bacillus amyloliquefaciens HRH317菌株所产抗菌蛋白物质对禾谷镰孢菌Fusarium graminearum的抑制机理,采用硫酸铵分级沉淀菌株发酵液,DEAE Sepharose Fast Flow柱和Sephadex G-200柱层析分离纯化抗菌蛋白,聚丙烯酰胺凝胶电泳检测抗菌蛋白分子量;并采用平板计数法和扫描电子显微镜观察病原菌孢子萌发和菌丝生长情况。结果表明,硫酸铵饱和度为50%时HRH317菌株发酵液的抑菌活性最大,抑菌圈直径可达2.02 cm,显著大于其它饱和度处理;2次柱层析可纯化出抗菌蛋白,分子量约为36.0 k D。禾谷镰孢菌经该抗菌蛋白处理后12h,病原菌孢子萌发率为55.8%,显著低于对照,且2~12 h间其孢子萌发率平均下降了57.3%;该抗菌蛋白还可造成病原菌菌丝变细、畸形与扭曲,且末端膨大等现象。表明解淀粉芽胞杆菌HRH317菌株产生的抗菌蛋白对禾谷镰孢菌孢子萌发和菌丝发育具有较强的抑制作用。     

地衣芽孢杆菌W10抗菌蛋白的分离纯化及其理化性质研究

 地衣芽孢杆菌W10是一株对多种植物病原菌有拮抗作用的生防细菌,其产生的抗菌物质主要是抗菌蛋白。细菌培养滤液用30%硫酸铵盐析,透析后再经SephadexG-75层析柱纯化得到一种具有抑菌活性的蛋白。该抗菌蛋白的分子量为46049.2Da;等电点为6.71;含有糖基,含糖量为6.83%;含有脯氨酸或羟脯氨酸;不含脂基。这种蛋白在pH6~12范围内均具有抑菌作用,pH6时活性最大;对热稳定,100℃以下水浴20min,抑菌活性与对照相比均在对照的80%以上,121℃蒸汽处理20min,活性虽有所下降,但仍达对照的65.7%;对胰蛋白酶和蛋白酶K不敏感,酶处理后蛋白的抑菌活性分别为对照的98.7%和98.3%;对氯仿和紫外线也不敏感,处理后活性仍达对照的91.0%和98.6%。     

多粘类芽孢杆菌BRF-1抗菌蛋白的分离纯化

通过硫酸铵分级沉淀、Sephadex G-50柱层析并采用抑菌活性和SDS-PAGE跟踪检测，从多粘类芽孢杆菌BRF-1菌株代谢产物中分离纯化到一种对大豆立枯丝核菌具有拮抗活性的抗菌蛋白，分子量约35．4kD。     

斜纹夜蛾抗菌物质的诱导及其理化特性

用大肠杆菌K12D31免疫斜纹夜蛾六龄幼虫，12h后收集血淋巴并通过高温处理制成粗提液。SDS-PAGE电泳结果表明：诱导后粗提液含有3条分别为43．5、15．0、4．0kD的特异性条带。诱导产生的抗菌物质在酸性、高温和常温等条件下保持稳定。抗菌谱测定结果表明，诱导产生的抗菌物质对猪霍乱沙门氏菌、枯草芽孢杆菌、铜绿假单胞菌等革兰氏阴性和阳性菌有抑制作用，而且对白色念珠球菌、酿酒酵母也有抑制作用。     

内生解淀粉芽孢杆菌LP-5抗菌蛋白的分离纯化及特性

解淀粉芽孢杆菌Bacillus amyloliquefaciens LP-5是一株对梨黑斑病菌Alternaria alternata具有抑制作用的内生细菌,其产生的抗菌物质主要是抗菌蛋白.为明确该抗菌蛋白的理化性质,采用盐酸沉淀、DEAE-52层析和Sephadex G-100层析进行纯化,测定了所得抗菌蛋白对热、蛋白酶、紫外辐射及酸、碱的稳定性.结果表明:纯化的抗菌蛋白分子量为20.3 kD,等电点为6.33.该蛋白对高温敏感,100℃处理30 min,抑菌活性仅为对照的18.4%;对蛋白酶K和胰蛋白酶稳定,酶处理后的抑菌活性分别为对照的92.5%和88.9%;对紫外辐射稳定,照射12 h后抑菌活性仍达对照的92.5%;对酸稳定、对碱敏感,pH 13处理后抑菌活性下降为原有活性的74.1%.抑菌活性测定结果显示,该蛋白对梨黑斑病菌的孢子萌发和菌丝生长有明显抑制作用,可造成孢子萌发端泡囊化、菌体破裂、原生质外渗.     

土壤拮抗放线菌的分离及其抗菌活性筛选

以常见植物病原真菌和细菌为指示菌,从分离的422株放线菌中筛选出28株可代谢抗细菌活性物质的菌株,其中N331、N393菌株发酵液对供试12种真菌有显著的抑制作用。离体条件下,N331、N393菌株发酵液对供试真菌菌丝生长的抑制中浓度(EC50)均小于15 mL/L;对玉米弯孢病菌、棉花枯萎病菌、小麦根腐病菌、黄瓜霜霉病菌孢子萌发的EC₅₀亦小于15 mL/L。盆栽试验结果表明,N331、N393菌株发酵原液对黄瓜霜霉病的保护效果分别为97.4%和85.6%,治疗效果分别为83.3%和59.3%。     

海洋放线菌BM-2菌株抗真菌活性物质的分离纯化与结构鉴定

从海洋放线菌菌株BM-1发酵液中分离抗植物病原真菌的活性物质。以对禾谷镰刀菌Fusarium graminearum的生物活性为跟踪,采用硅胶柱层析、Sephadex LH-20柱层析、薄层层析和HPLC等技术,海洋放线菌菌株BM-1发酵液中含有4种对禾谷镰刀菌具有抑菌作用的活性物质,对其进行分离纯化,得到了4种具有强抗菌作用的组分Aa、Ab、Ba、Ca。采用ESI-MS、1H NMR、13C NMR等波谱分析技术,对分离量较大、活性较强的活性组分Ca进行结构鉴定,鉴定Ca为邻苯二甲酸二丁酯(DBP)。     

油茶炭疽病拮抗细菌Y13主要抑菌物质分离纯化及作用方式

油茶内生拮抗细菌Y13是一株对油茶炭疽病菌有较强抑制作用的枯草芽胞杆菌。为了确定其抑菌物质的成分组成及其对油茶炭疽病菌的作用方式,本文通过乙醇沉淀、固相萃取、反相高效液相色谱法及LC MS对抑菌物质进行分离鉴定。一共分离纯化出16个活性组分,保留时间在10.5~26.0 min的8个组分对于油茶炭疽病菌产生明显的抑菌圈,其中保留时间为11.0 min处的色谱峰抑菌效果最强,经质谱初步鉴定确定该化合物的分子量为1 042.56 u;保留时间在27.5~39.5 min的8个组分导致油茶炭疽病病原菌菌丝颜色加深,其中以保留时间37.5 min处的色谱峰效果最为明显,质谱鉴定该化合物的分子量为1 480.85 u。显微观察发现它们通过导致菌丝断裂、畸形、原生质凝集的方式抑制菌丝生长;通过使孢子畸形、膨大、消融而抑制孢子萌发。     

枯萎菌拮抗芽孢杆菌BC98-I抗菌多肽的纯化

 枯萎菌拮抗蜡状芽孢杆菌BC98-I分泌产生的抗真菌物质经粗提、Sephadex G100和DEAE52柱层析分离纯化后,得到了抗菌馏分P₁D₄。该馏分达到了电泳纯,SDS-PAGE电泳检测其分子量约为3.5kD,等电点为4.65。又经MALDI-TOF质谱检测,该馏分为精确分子量小于或等于1531.71D的多肽。氨基酸组成分析结果表明:馏分P₁D₄由Asp、Glu、Ser等12种氨基酸组成,富含酸性和极性氨基酸。结合以往研究结果,目标产物热稳定性好,对胰蛋白酶和蛋白酶K有良好耐受性,初步认为属低分子量的环状多肽。     

Inheritance of beta-cypermethrin resistance in the housefly Musca domestica (Diptera: Muscidae)

BACKGROUND: Beta-cypermethrin, a synthetic pyrethroid insecticide, was applied frequently in the control of health pests including houseflies, Musca domestica L., in China. However, different levels of resistance to beta-cypermethrin were monitored in field strains of houseflies. A strain of M. domestica, 4420-fold resistant to beta-cypermethrin after continuous 25 generations of selection, was used in this paper to determine the mode of inheritance of pyrethroid resistance. RESULTS: The estimated realized heritability (h(2)) of beta-cypermethrin resistance was 0.30 in this resistant strain. Results of bioassays showed no significant difference in values of LD(50) and slope of log dose-probit lines between reciprocal progenies F(1) and F'(1), and yielded values of - 0.10 (F(1)) and - 0.11 (F'(1)) for the degree of dominance (D). Chi-square analysis from responses of self-bred and backcross progenies (F(2), BC(1) and BC(2) respectively) indicated that the null hypothesis, a single gene responsible for resistance, was accepted. The minimum number of independent segregation genes was 0.93 for F(1) by Lande's method. CONCLUSION: It was concluded that beta-cypermethrin resistance in the housefly was inherited as a single, major, autosomal and incompletely recessive factor. These results would provide the basic information for pest management programmes.     

Monitoring insecticide resistance in house flies (Diptera: Muscidae) from New York dairies

House flies were collected from dairies across New York state and the levels of resistance to seven insecticides were measured using standard laboratory assays with three to five diagnostic concentrations. The highest levels of resistance were found for tetrachlorvinphos, permethrin and cyfluthrin. Although levels of resistance to methomyl were somewhat lower, they were among the highest ever reported for field‐collected house flies. Resistance to pyrethrins was limited primarily to the lowest diagnostic concentration. House flies were susceptible to fipronil at all dairies, suggesting that this material would be highly effective for fly control. The levels of resistance were similar at all the dairies, irrespective of their insecticide use, suggesting substantial movement of flies between facilities. Relative to resistance levels found at New York dairies in 1987, resistance levels had increased for permethrin, were unchanged for tetrachlorvinphos and had decreased for dimethoate. To identify a single diagnostic concentration that could be used in the laboratory assays to assess accurately resistance levels in future studies, we carried out a ‘simulated’ field bioassay using formulated materials. A diagnostic concentration for each insecticide is proposed on the basis of a comparison of our bioassays. © 2001 Society of Chemical Industry     

Assessing insecticide resistance and aversion to methomyl-treated toxic baits in Musca domestica L (Diptera: Muscidae) populations in southern California

Progeny of house flies (Musca domestica L) from ten California poultry operations, three dairies and one horse-riding facility were tested for methomyl- and muscalure-treated bait resistance using up to three different assays: a topical assay, a no-choice feeding assay and a choice feeding assay. LD50 resistance factors from the topical assay, compared with a locally-derived susceptible colony, ranged between 1 and 4. LC50 resistance factors from the no-choice feeding assay ranged mostly between 2 and 5, with one value of 18. Measurable LT50 resistance ratios for female flies in the choice feeding assay ranged from 43 to 159; two populations had <10% mortality at 48h and could not be measured. LT50 resistance ratios for male flies in the choice feeding assay ranged from 26 to 96, and one population was too resistant to measure. A behavioral assay tested the feeding preference of male and female flies provided a dish of sugar and a dish of methomyl- and muscalure-treated bait. Of eight strains tested, females from seven strains and males from six strains showed significant preference for sugar over bait. Behavioral factors appear to be important in the severe resistance of house flies to baits in California.     

Susceptibility of house flies (Diptera: Muscidae) exposed to commercial insecticides on painted and unpainted plywood panels

House flies were collected from dairies in New York state and the levels of resistance to commercially available insecticide formulations were measured on painted and unpainted plywood panels. Dimethoate was ineffective on all surfaces. The wettable powder permethrin formulation was more toxic than the emulsifiable concentrate formulation. The wettable powder cyfluthrin formulation was also more toxic than the recently developed liquid formulation. In general, the best house fly control was obtained on flat latex painted plywood panels and the poorest control on gloss latex painted panels. It is highly unlikely that producers obtain adequate control with dimethoate and permethrin.     

Evaluation of semiochemical toxicity to houseflies and stable flies (Diptera: Muscidae)

BACKGROUND: The housefly, Musca domestica L., and stable fly, Stomoxys calcitrans (L.) are cosmopolitan pests of both farm and home environments. Houseflies have been shown to be resistant to a variety of insecticides, and new chemistries are slow to emerge on the market. Toxicities of selected semiochemicals with molecular structures indicative of insecticidal activity were determined against adults from an insecticide‐susceptible laboratory strain of houseflies. The three most active semiochemicals were also evaluated against recently colonized housefly and stable fly strains. RESULTS: Nineteen semiochemicals classified as aliphatic alcohols, terpenoids, ketones and carboxylic esters showed toxicity to houseflies and stable flies. Rosalva (LC₅₀ = 25.98 µg cm^?2) followed by geranyl acetone and citronellol (LC₅₀ = 49.97 and 50.02 µg cm^?2) were identified as the most toxic compounds to houseflies. Permethrin was up to 144‐fold more toxic than rosalva on the susceptible strain. However, it was only 35‐fold more toxic to the insecticide‐tolerant field strain. The compounds generated high toxicity to stable flies, with LC₅₀ values ranging from 16.30 to 40.41 µg cm^?2. CONCLUSION: Quantification of LC₅₀ values of rosalva, citronellol and geranyl acetone against susceptible housefly and field‐collected housefly and stable fly strains showed that semiochemicals could serve as potent insecticides for fly control programs. Copyright © 2010 Society of Chemical Industry     

Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies

BACKGROUND: The housefly, Musca domestica L., continues to be a major pest of confined livestock operations. Houseflies have developed resistance to most chemical classes, and new chemistries for use in animal agriculture are increasingly slow to emerge. Five adult housefly strains from four Florida dairy farms were evaluated for resistance to four insecticides (beta‐cyfluthrin, permethrin, imidacloprid and nithiazine). RESULTS: Significant levels of tolerance were found in most field strains to all insecticides, and in some cases substantial resistance was apparent (as deduced from comparison with prior published results). At the LC₉₀ level, greater than 20‐fold resistance was found in two of the fly strains for permethrin and one fly strain for imidacloprid. Beta‐cyfluthrin LC₉₀ resistance ratios exceeded tenfold resistance in three fly strains. The relatively underutilized insecticide nithiazine had the lowest resistance ratios; however, fourfold LC₉₀ resistance was observed in one southern Florida fly strain. Farm insecticide use and its impact on resistance selection in Florida housefly populations are discussed. CONCLUSION: Housefly resistance to pyrethroids is widespread in Florida. Imidacloprid resistance is emerging, and tolerance was observed to both imidacloprid and nithiazine. If these insecticides are to retain efficacy, producer use must be restrained. Copyright © 2009 Society of Chemical Industry     

芸香科三种植物精油对家蝇的熏杀活性及其混配增效作用

用水蒸汽蒸馏法从岭南花椒Zanthoxylum austrosinense果实、椿叶花椒Zanthoxylum ailanthoides果实和四数九里香Murraya hunanensis叶中提取植物精油,采用三角瓶密闭熏蒸法测试3种精油对家蝇的熏杀、击倒作用及其混配增效作用。3种植物精油对家蝇的6 h熏杀LC₅₀值分别为岭南花椒果实精油 7.55 μg/cm³、椿叶花椒果实精油11.08 μg/cm³、四数九里香叶精油11.41 μg/cm³;在熏蒸浓度均为10 μL/345 mL时精油对家蝇的熏蒸击倒效果KT₅₀分别为岭南花椒果实精油16.80 min、椿叶花椒果实精油24.69 min、四数九里香叶精油23.33 min。将岭南花椒果实精油与椿叶花椒果实精油以体积比7：3和9：1混配,其共毒系数分别为130.00和136.08;将岭南花椒果实精油与四数九里香叶精油以体积比9：1混配,其共毒系数为123.71。研究结果表明,3种植物精油对家蝇均有熏杀、击倒作用,其中岭南花椒果实精油与椿叶花椒果实精油或四数九里香叶精油以适当比例混配后具有增效作用,可利用其开发高效环保的植物源灭蝇剂。     

Characterisation of spinosad resistance in the housefly Musca domestica (Diptera: Muscidae)

BACKGROUND: Spinosad, a relatively new, effective and safe pesticide, has been widely used in pest control over the last 10 years. However, different levels of resistance to this insecticide have developed in some insects worldwide. RESULTS: After continuous selection for 27 generations, a strain (SpRR) of the housefly developed 247‐fold resistance to spinosad compared with the laboratory susceptible strain (CSS). The estimated realised heritability (h²) of spinosad resistance was 0.14. There was no significant difference in the LD₅₀ values and slopes between reciprocal progenies F₁ and F₁′, and values of 0.33 (F₁) and 0.30 (F₁′) were obtained for the degree of dominance. Chi‐square analysis from responses of self‐bred (F₂) and backcrosses (BC₁ and BC₂) were highly significant, suggesting that the resistance was probably controlled by more than one gene. Synergists piperonyl butoxide (PBO), diethyl maleate (DEM) and S,S,S‐tributyl phosphorotrithioate (DEF) affected the toxicity of spinosad at a low level, and demonstrated that metabolic‐mediated detoxification was not an important factor in conferring resistance to spinosad in the SpRR strain. CONCLUSION: It was concluded that spinosad resistance in the housefly was autosomal and incompletely dominant, and the resistance was probably controlled by more than one gene. These results provide the basic information for designing successful management programmes for the control of houseflies. Copyright © 2011 Society of Chemical Industry