绿僵菌Ma55抗多菌灵突变株的诱变及抑菌作用研究

在室内条件下测定了供试绿僵菌对多菌灵的抗性。结果表明, 供试菌株对多菌灵均表现为敏感, 其中, Ma55菌株对多菌灵最敏感。采用分生孢子定向筛选的方法获得了金龟子绿僵菌抗多菌灵突变株, 测定了抗性突变株对多菌灵的抗性差异。结果显示, 在获得的抗性突变株中, MC-2的EC50最大, 达到397.064 3μg/mL, 对多菌灵的抗性水平最高, 抗性水平指数达到102.35。对Ma55抗多菌灵突变株的菌丝生长速率、产孢量及对棉花枯萎病菌和棉花红腐病菌的抑制效果的研究结果表明, 抗性突变株的菌丝生长速率均比亲本菌株Ma55小, 但产孢量均比Ma55大, 其中, MC-2的产孢量最大, 为5.12×106个/mL; MC-5次之, 其产孢量为4.81×106个/mL, MC-8的产孢量在抗性突变株中最低。平板对峙培养结果显示, 金龟子绿僵菌抗多菌灵突变株对棉花枯萎病菌和棉花红腐病菌菌丝生长的抑制作用均达到显著水平, 其中, MC-2对棉花枯萎病菌、棉花红腐病菌菌丝生长的抑制作用最强, 与亲本对照菌株Ma55差异不显著。     

伯氏嗜线虫致病杆菌娩norhabdus bovienii YL002对4种抗生素抗性突变株的筛选及抑菌活性分析

为了提高伯氏嗜线虫致病杆菌Xenorhabdus bovienii YL002抑菌活性,采用紫外线照射和吖啶橙复合诱变的方法,对其进行诱变改良,并在抗生素最小抑制浓度选择压力下筛选对4种抗生素具有抗性的突变菌株.以枯草芽孢杆菌为指示菌,得到了 17 株抑菌活性有显著变化且遗传稳定的突变菌株.其中菌株L0420-3对枯草芽孢杆菌的抑制活性最强,连续转接 5 次的抑菌圈直径增大率均在 80%以上;菌株Q025-1和L0314-4对番茄灰霉病菌菌丝生长的抑制率为 75.66%和 73.41%,分别是野生菌株的 1.35 倍和 1.31 倍;接种灰霉病菌后 5 d对番茄果实灰霉病的防效为 95.74%和 93.62%,分别是野生菌株的 1.71倍和 1.67 倍.     

金龟子绿僵菌fluG基因的敲除及对产孢的影响

丝状真菌的fluG基因参与调控分生孢子的生成,然而在昆虫病原真菌金龟子绿僵菌中fluG的作用鲜有研究报道。本研究利用DNA同源重组方法,构建敲除fluG的金龟子绿僵菌突变株,分析突变株的产孢特性。以苯菌灵抗性基因ben作为筛选标记,fluG基因侧翼序列作为同源臂,构建了打靶载体pDHt/sk-fluG-Ben。利用PEG介导将打靶载体转化金龟子绿僵菌的原生质体,获得了苯菌灵抗性转化子。根据靶基因和标记基因检验,确定获得了敲除fluG的金龟子绿僵菌突变株。表型分析显示,fluG突变株继代培养5代仍保持苯菌灵抗性,菌落呈疏松毛絮状,生长相较野生型明显缓慢,不能或者仅能产生极少量分生孢子。说明fluG基因的敲除影响了菌株生长发育,并阻止了分生孢子形成,是金龟子绿僵菌产孢调节的重要基因。本研究为阐述金龟子绿僵菌产孢调控机制提供依据。     

香蕉炭疽病拮抗菌Bs6602的诱变选育及防病作用研究

为了提高拮抗菌对香蕉炭疽病的抑菌活性,本研究采用紫外线-亚硝酸钠复合诱变的方法对枯草芽胞杆菌Bs6602菌株进行诱变。结果表明,该菌株的最佳诱变条件为在距离20 W紫外灯50cm下照射15s,然后经300mmol/L NaNO2诱变处理60min。经过2次复合诱变,最终得到1株抑菌活性最强的6B8-5菌株,其抑菌活性比出发菌株提高83.80%。咪鲜胺对突变株6B8-5的最低抑菌浓度(MIC)为20.83μg/mL。连续转接7代,突变株6B8-5的抑菌活性无明显降低。6B8-5菌株的抑菌谱测试结果表明,其对13种植物病原真菌具有较强的抑菌活性,其中对多主棒孢霉的抑菌活性最强,抑菌圈直径达66.39mm。对香蕉炭疽病防治的试验结果表明,香蕉贮藏15d后,6B8-5菌株的防效明显高于出发菌株,为63.94%;6B8-5菌株与43 200倍咪鲜胺稀释液混用后,其防效最高达70.27%,与咪鲜胺1 350倍稀释液防效相当。     

微波诱变选育高效溶磷木霉菌株的研究

为了获得高效溶磷菌株,对木霉菌T6菌株进行微波诱变处理,测定并分析了微波输出功率和辐照时间等参数对诱变效果的影响。结果表明:最佳诱变条件是微波输出功率900 W,辐照时间70 s,且在此条件下得到8株具有较高溶磷能力的突变菌株T6-9、T6-33、T6-93、T6-126、T6-157、T6-188、T6-196和T6-203。经过多代转接培养最后得到一株遗传稳定的溶磷木霉菌株T6-157,该菌株在液体培养条件下溶磷量为204.46 mg·L~(-1),与出发菌株相比,溶磷量提高了107.97%,植酸酶活性提高了57.35%,且突变菌株对土传植物病原菌的拮抗能力也有所提高。     

基于CTAB的莱氏绿僵菌选择性培养基制备及分离效果研究

制备适用于分离莱氏绿僵菌(Metarhizium rileyi)的选择性培养基。以十六烷基三甲基溴化铵(cetyltrimethyl ammonium bromide,CTAB)为选择性成分,以SMAY培养基为基础培养基,分别研究CTAB对M. rileyi生长的影响、对杂菌的抑制作用,并以土壤菌群为基础对该选择性培养基进行验证。结果表明,CTAB对莱氏绿僵菌具有明显的抑制作用,当CTAB浓度高于0.4 g/L时,莱氏绿僵菌无法生长,浓度低于0.2 g/L时,能够生长、产孢。CTAB可以有效抑制杂菌的生长,浓度为0.18 g/L时,对灰霉(Botrytis cinerea)、木霉(Trichoderma longibrachiatum)的抑制作用较强,对青霉(Penicillium sp.)、大肠杆菌(Escherichia coli)则完全抑制,且莱氏绿僵菌可产孢。0.18 g/L的CTAB培养基对土壤菌群具有很强的抑制作用,可以从处理土样中分离得到莱氏绿僵菌并产孢。CTAB含量为0.18 g/L的培养基可以作为莱氏绿僵菌的专用选择性培养基。     

绿僵菌IMI330189菌株突变体库的建立及毒力相关突变株的筛选

为了研究绿僵菌毒力相关的功能基因,采用农杆菌介导的转化方法将携带苯菌灵抗性标记基因的T—DNA导入绿僵菌IMI330189中,获得了绿僵菌突变体库,采用人工饲料诱饵法测定了突变株对3龄东亚飞蝗Locustamigratoria的毒力。研究发现,突变株对东亚飞蝗的毒力都有不同程度的改变,在诱饵剂浓度为10^8孢子．g-1下,野生型菌株IMI330189对东亚飞蝗的致死中时（LT50）为4-3d;突变株189-1的毒力略有提高,其LT50值为3．5d;其余突变株的毒力均降低,且突变株189—4、189—5、189—6、189—10、189-11、189—13和189—15几乎丧失了对东亚飞蝗的毒性,校正死亡率小于50％。本研究为进一步分离毒力相关基因、探索绿僵菌的致病机制奠定了基础。     

短短芽胞杆菌Bb5911的复合诱变及其对香蕉炭疽病的防治作用

为了提高拮抗菌对香蕉炭疽病的抑菌活性并探讨其与化学药剂混用的可行性,本研究采用紫外线-亚硝酸钠复合诱变的方法对短短芽胞杆菌Bb5911进行诱变,并测试了其与咪鲜胺混配对香蕉炭疽病的防治效果。结果表明,最佳诱变条件为在距离20 W紫外灯50 cm下照射15 s,然后经300 mmol/L NaNO₂诱变处理60 min。经过2次复合诱变后得到突变株5B37-3的抑菌活性最强,比出发菌株提高了112.41%。咪鲜胺对突变株5B37-3的最低抑菌浓度（MIC）为20.83 μg/mL。连续转接7次,突变株5B37-3的抑菌活性无明显降低。抑菌谱测试结果表明,突变株5B37-3对13种植物病原真菌的抑菌活性比出发菌株均有提高,提高率18.20%~239.27%。对香蕉炭疽病防治的试验结果表明,突变株5B37-3的防效明显高于出发菌株,最高为66.55%。突变株5B37-3与10.42 μg/mL咪鲜胺溶液复配后,香蕉贮藏15 d,其防治效果为72.76%,与333.33 μg/mL咪鲜胺溶液防效相当;至香蕉贮藏17 d,其防治效果显著低于333.33 μg/mL咪鲜胺溶液。     

棉铃疫病菌(Phytophthora boeh meriae)对甲霜灵的抗性遗传研究

 本文在实验室条件下,经药物直接诱变,筛选获得棉铃疫病菌(Phytophthora boehmeriae Sawada)抗甲霜灵突变株,研究了其抗药性性状的遗传。结果表明,棉铃疫病菌易对甲霜灵产生抗性,抗性菌株对甲霜灵的抗性水平可高达其敏感亲本的5161.3倍以上;部分抗性菌株的抗药性性状在无性及有性单孢后代均可稳定遗传,另有部分抗性菌株的抗性性状在无性单孢后代出现分离甚至完全丧失。初步认为,供试棉铃疫病菌对甲霜灵的抗性遗传可能与细胞质遗传因子有关     

紫外-氯化锂复合诱变哈茨木霉产生多菌灵抗药性菌株的研究

多菌灵是植物病害防治中常用的化学杀菌剂,野生型哈茨木霉对多菌灵比较敏感。为了能够更好地将哈茨木霉菌应用于实践,本试验采用紫外线-氯化锂复合诱变的方法,实现哈茨木霉菌株对多菌灵的抗性改良。试验共获得315株正向突变菌株,其中hcb-35菌株抗性能力较强。利用毒力测定方法检测了多菌灵对hcb-35有效抑菌中浓度,及hcb-35对多菌灵的抗药遗传稳定性;并利用对峙试验及显微观察检测其抑菌能力。结果显示,与哈茨木霉出发菌株hc相比,多菌灵对哈茨木霉突变株hcb-35菌株的有效抑菌中浓度提升285%;连续转接12代后,hcb-35菌株抗药性相对稳定且抑菌效果较出发菌株无明显差异,表明应用紫外-氯化锂复合诱变哈茨木霉可以获得遗传稳定的耐多菌灵突变株。     

Cross‐resistance and possible mechanisms of chlorpyrifos resistance in Laodelphax striatellus (Fallén)

BACKGROUND: Laodelphax striatellus (Fallén) is a major pest of cultivated rice and is commonly controlled in China with the organophosphate insecticides. To develop a better resistance management strategy, a chlorpyrifos‐resistant strain of L. striatellus was selected in the laboratory, and its cross‐resistance to other insecticides and possible mechanisms of the chlorpyrifos resistance were investigated. RESULTS: After 25 generations of selection with chlorpyrifos, the selected strain of L. striatellus developed 188‐fold resistance to chlorpyrifos in comparison with the susceptible strain, and showed 14‐ and 1.6‐fold cross‐resistance to dichlorvos and thiamethoxam respectively. There was no apparent cross‐resistance to abamectin. Chlorpyrifos was synergised by the inhibitor triphenyl phosphate; the carboxylesterase synergistic ratio was 3.8 for the selected strain, but only 0.92 for the susceptible strain. The carboxylesterase activity of the selected strain was approximately 4 times that of the susceptible strain, whereas there was no significant change in the activities of alkaline phosphatase, acid phosphatase, glutathione S‐transferase and cytochrome P450 monooxygenase between the strains. The Michaelis constant of acetylcholinesterase, maximum velocity of acetylcholinesterase and median inhibitory concentration of chlorpyrifos‐oxon on acetylcholinesterase were 1.7, 2.5 and 5 times higher respectively in the selected strain. CONCLUSION: The high cross‐resistance to the organophosphate dichlorvos in the chlorpyrifos‐resistant strain suggests that other non‐organophosphate insecticides would be necessary to counter resistance, should it arise in the field. Enhanced activities of carboxylesterase and the acetylcholinesterase insensitivity appear to be important mechanisms for chlorpyrifos resistance in L. striatellus. Copyright © 2010 Society of Chemical Industry     

药剂对小菜蛾抗性及敏感品系乙酰胆碱酯酶抑制作用比较

采用浸叶法测定了云南通海、元谋和澜沧的小菜蛾plutella xylostella田间种群对常用杀虫剂的抗药性。结果表明,云南上述地区小菜蛾田间种群对各类杀虫剂均产生了不同程度的抗性。对有机磷类药剂的抗药性为1.74~31.1倍;对菊酯类药剂的抗药性为7.41~764倍;对阿维菌素类药剂则产生了 5.60~4.06×104倍的抗性。通过离体和活体试验测定了药剂对小菜蛾头部乙酰胆碱酯酶(AChE)的抑制作用。敌敌畏和灭多威对通海抗性品系AChE离体和活体内的抑制中浓度(I50)分别是敏感品系的209、26.5倍和2.21、2.16倍;敌敌畏对通海小菜蛾种群的离体和活体内抑制中时间(IT50)小于敏感品系,分别是敏感品系的0.32和0.17倍;而灭多威对通海小菜蛾种群的离体和活体内抑制中时间(IT50)则大于敏感品系,分别是敏感品系的1.37和1.74倍。     

小菜蛾对阿维菌素B_1抗药性选育及交互抗性

用阿维菌素B_1(abamectin)对小菜蛾敏感种群在室内进行抗性品系选育。经过25代连续汰选,获得抗性种群Laba-R,与选育前比较,抗性提高100倍。Laba-R种群在不接触任何药剂条件下饲养20代,抗性逐渐下降,很难恢复到选育前的敏感状态。抗性汰选前后分别测定10种药剂的剂量-死亡率毒力回归线,发现Laba-R抗性种群对乙酰甲胺磷、锐劲特、灭多威、敌敌畏不存在交互抗性;对溴氰菊酯、氯氰菊酯、杀虫双、巴丹和Bt的敏感性略有下降,但无明显交互抗性。活体增效剂试验表明,增效醚(PBO)和磷酸三苯酯(TPP)对阿维菌素B_1均有明显的增效作用,其中PBO的增效活性尤为显著,它能使对阿维菌素B_1产生100多倍抗药性的小菜蛾完全恢复其敏感性。说明多功能氧化酶解毒代谢增强可能是小菜蛾对阿维菌素B_1产生抗性的主导因素之一。     

不同种群小菜蛾对4种杀虫药剂的抗性及抗性基因频率

用 FAO推荐的点滴法测定了日本、台湾、广州、东莞等地小菜蛾 (Plutella xy-lostella L .)的抗药性。测定结果表明 :日本、广州的小菜蛾对阿维菌素显示出抗药性 ,其抗性比率分别是 4 .85倍和 10 .2 7倍。杀虫药剂抗性基因型频率测定显示 ,广州、广东东莞和北京种群的最高 ,基因型频率分别为 4 1.2 5%、4 1.2 5%和 58.2 3%。研究验证了快速测定杀虫药剂抗性基因型频率的方法 ,并提出了根据不同种群小菜蛾抗性基因型频率所应采取的综合治理策略。     

小菜蛾对溴氰菊酯抗性选育及其机理

本研究用溴氰菊酯在室内以点滴法处理小菜蛾４龄幼虫，连续继代药剂淘汰选育其抗药性，至Ｆ６５代，抗药性提高到１１６３倍，已形成高抗生品系。其抗性的形成发展趋势为前期相对缓慢，中期较快，后期迅速增长。于１９９２、１９９３、１９９４年分别以氯氰菊酯、敌敌畏、杀虫双、灭虫剂有明显 正交互抗性，对其它非菊酯类杀虫虫没有产生交互抗性。用聚丙烯酰胺凝胶电泳地（ＰＡＧＥ）测定表明，小菜蛾对溴氰菊酯抗性的产生可能与非     

Effects of time and concentration on mortality of phosphine-resistant Sitophilus oryzae (L) fumigated with phosphine

The effects of exposure period and phosphine concentration on mortality of susceptible and resistant Sitophilus oryzae (L) were investigated. Although S oryzae is one of the world's most serious pests of stored grain there are few data on the practical significance of phosphine resistance in this species. The strains investigated were an Australian susceptible strain, a homozygous resistant strain exhibiting a level of resistance common in Australia and an unselected field strain from China with a much stronger resistance. Fumigations were carried out at 25 degrees C on adults and mixed-age cultures. For adults of all three strains and mixed-age cultures of the susceptible and resistant Australian strains, the relationship between concentration and time could be described by equations of the form Cnt = k. In all cases n < 1, indicating that time was a more important variable than concentration. In all fumigations of adults the resistant strains were harder to kill than the susceptible strain. However, in fumigations of mixed-age cultures, which contained the tolerant pupal stage, the difference between susceptible and resistant strains was more pronounced at lower concentrations than higher concentrations. For example, at 0.02 mg litre-1 the estimated LT99.9 for mixed-age cultures of the Australian resistant strain (27 days) is 3.4 times that of the susceptible strain (8 days), but at 1 mg litre-1 there is no difference between the two strains (4 days). Limited data on the Chinese resistant strain supported this finding. Twenty-three days exposure at 0.02 mg litre-1 had no effect on mixed-age cultures of this strain, but there were no survivors after 5 days exposure to 1 mg litre-1.     

Efficacy of current and potential grain protectant insecticides against a fenitrothion-resistant strain of the sawtoothed grain beetle,Oryzaephilus surinamemis L

The responses of a highly fenitrothion-resistant and a susceptible strain of Oryzaephilus surinamensis L. from Australia were measured against a range of alternative grain protectants using impregnated-paper and treated-grain assays. The R-strain was also highly resistant to carbaryl, moderately resistant to malathion and pirimiphos-methyl and showed low-level resistance to methacrifos, chlorpyrifos-methyl, bioresmethrin, permethrin, cyfluthrin, cypermethrin and dichlorvos. There was marginal resistance to deltamethrin. Methoprene, to which there was no resistance, was the most effective material at suppressing progeny production of the resistant strain. At current commercial application rates chlorpyrifos-methyl was the only compound of those tested that would completely control both adults and larvae of resistant O. surinamensis for a storage period of nine months. Overall, the homogeneity of response of both strains to insecticides using the treated-grain assay was as good as the response from impregnated-paper assays. The latter also tended to exaggerate resistance factors compared to the treated-grain assays.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.