【作者】 孟艳琼；

【导师】 李增智； 樊美珍；

【作者基本信息】 安徽农业大学 ， 森林保护学， 2003， 硕士

【摘要】 蜡蚧轮枝孢[Verticillium lecanii （Zimm.） Viegas]是一种重要的丝孢类昆虫病原真菌，近年来在国外深受重视，已有多种商品制剂注册生产并用于温室白粉虱、蚜虫等刺吸式口器害虫的防治。本研究对该菌的生物学特性、生理生化因子、双相发酵技术、孢子粉干燥工艺及孢子粉在贮藏过程中内源营养物质代谢和对温室桃蚜防治应用进行了较系统的研究，旨在为利用蜡蚧轮枝孢控制刺吸式口器害虫提供依据。 用11株蜡蚧轮枝孢对桃蚜（Myzus persicae Sulzer）2～3龄若虫进行生物测定。以桃蚜的校正死亡率、感染率及致死中时(LT₅₀)为指标综合评价不同菌株毒力。结果表明，11株蜡蚧轮枝孢菌株中，VL07菌株毒力最强，校正死亡率达100％，感染率达93％，致死中时为3.30d。其次是VL18菌株，致死中时为4.02d，校正死亡率和感染率分别为93.83％和84.44％。在所测试温度中，25℃是蜡蚧轮枝孢侵染桃蚜的最适温度，致死中剂量为533孢子／蚜。 温、湿度对蜡蚧轮枝孢生长和发育的影响为：蜡蚧轮枝孢在10～30℃的温度下均可生长，25℃为菌丝生长最适温度，其中VL07、VL18和VL09菌株菌丝生长量较高，14d累计生长量分别为4.69cm、4.98cm和4.72cm；不同菌株在不同温度下产孢量差异极显著，25℃为蜡蚧轮枝孢最适产孢温度，其中VL07和VL18菌株产孢量最高，14d分别达5.83×10⁹和4.64×10⁹孢子／cm²；相对湿度对分生孢子萌发试验表明，在RH为100％时，VL07、VL10和VL18菌株分生孢子萌发快而整齐，24h萌发率均达100％，当相对湿度在75％以下时，除VL07和VL18外，其余各菌株的萌发率均降低到20％以下，而且生长缓慢。 测定4种生物源农药与蜡蚧轮枝孢的相容性结果表明：4种农药高浓度下对蜡蚧轮枝孢分生孢子的萌发均有一定的影响，而在低浓度下影响较小，如在2×10⁴倍浓度下25％灭幼脲Ⅲ号悬浮剂对高毒力菌株VL07分生孢子的校正抑制率仅为1.66％。因此，可考虑亚致死剂量的灭幼脲Ⅲ号与蜡蚧轮枝孢混合使用以提高防治效果。 对蜡蚧轮枝孢生理生化因子的测定结果表明：蜡蚧轮枝孢的毒力与菌株酯酶总量及前沿带的酯酶量之间呈显著线性相关，相关系数分别为r=0.9198和r=0.8820；蜡蚧轮枝孢毒力与胞外蛋白酶产生水平以及草酸产生水平之间均无明显相关性。 通过对不同的碳、氮源、无机盐、起始pH值、接种量以及正交试验，以菌丝生物量为指标筛选出了适合于VL07菌株工业生产最佳发酵工艺配方，液相发酵培养基配方为：黄豆粉 4％，白砂糖一％，KH。PO’ 0刀2％，MgSO厂 7HZO 0刀1％，FeSO厂 7H。O0＊5％，VB。0＊5％，VC 0＊5％，pH值 6．89，初始接种量为 10％－15％；固相发酵培养基配方为：鼓皮 42％，稻壳 28％，玉米粉 20％，黄豆粉 10％。当培养基初始含水率为50％时，蜡蚊轮枝抱的产抱量最高，产抱量可达 1．05Xwi‘抱子哈。 将抱子粉进行不同干燥处理，结果表明：低热抽干24h和 30hffi子萌发率显著高于烘干粉，萌发率分别为 88刀0％和 86．75％；含水率分别为 13．38％和 12刀0％，含抱量分别达9．53X10‘’和 l．09X10”抱子伯。烘干粉虽然含水率可降到最低8．36O，含抱量可提高到 1．15 X 101‘抱子哈，但其活抱率仅有的．33％。因此可以首选操作方便，成本较低的低热抽干的干燥工艺。 在VL07菌株抱子粉贮存过程中发现：抱子粉的活抱率与其内源蛋白和内源总糖之间均呈极显著的正相关性。高温下贮存的抱子粉较低温下贮存的内源物质代谢快，冻干16h和24h及抽干30h的抱子粉在4℃下贮存Zld的活抱率分别降到63．5O、72．50和61．5％。25C下贮存各抱子粉活抱率均降到50％以下。 蜡蚊轮枝抱 VL07菌株对温室桃蚜的防治效果表明：抱悬液门 抱子／ml)防治区和混合悬液门‘抱子／ml与 25o灭幼腺Ill号 Zxlo‘混合液)防治区的控制效果较好，喷药后第m天校正虫口下降率分别达乃斗2土1．92％和85．49土3．27％；防治效果分别为 66。39土4．83％和 80．35土3．98％。更多还原

【Abstract】 The entomopathogenic fungi, Verticillium lecanii(Zimm.) Viegas, is one of the most promising fungal species which is used to control greenhouse aphids and other sucking insect pests. In recent years, different commercial formulations have been registered in many countries. In order to develop mass production and application of this fungus in China, its biological characteristics, biochemical and physiological factors, diphasic fermentation, conidium drying techniques, nutrient metabolism during storage, and field trial against greenhouse aphids. were studied in this thesis.Biological characters of 11 strains of V. lecanii isolated from different regions were investigated. At the same time, Bioassay was carried out against the 2~3th instar Myzus persicae larvae with the 11 strains. The most virulent strain was screened out after evaluated by the indices of mortality, infection rate and the median lethal time (LT50). The result showed that VL07 was the most virulent strain which caused mortality soon after 3 days, followed by VL18. The mortality reached 100% and 93.83%, with infection rate at 93.0% and 84.44%, LT50 at 3.30d and 4.02d, respectively. The optimum temperature for V. lecanii infection on M. persicae was 25℃. LD50 of VL07 reached 533 conidia per aphid.The mycelial biomass and the production of conidia of V. lecanii were studied at different temperatures. The result showed that all strains grew at 10℃ to 30℃, but 25℃ was the optimum temperature for mycelium growth and conidium production. The mycelia biomass of VL07, VL09 and VL18 was compazatively high at 25℃ after 14d. These diameters of colonies reached 4.69cm, 4.72cm and 4.98cm, respectively, obviously higher than other strains. The differentce of conidia production was significant between different strains at different temperature. For VL07, VL18, conidia production reached 5.83 X 109/cm2 and 4.64 X 109/cm2. The result of germination test of conidia showed that the conidia germinated rapidly and uniformly. The germination rates all reached 100% at RH 100%. The germination rates of all strains except for VL07, VL18 declined to 20% as RHdecreased down to below 75%, and grew very slowly.Some non-hazard insecticides were tested for their effection on conidial germina -tion of V. lecanii. The result showed that they made some effect at high concentration, but the effect was hight at low concentration. 25% flowable colloid of diflubenzuron induced little effect on the germination of the virulent strain VL07. The inhibition rate reached only 1.66% at a concentration of 2 ×104 times.Some physiological and biochemical factors of V. lecanii were tested. The result showed that the total amount of esterases and the frontier band esterase number were significantly correlated to the virulence, with r=0.9198 and r=0.8820, respectively, and the virulence was poorly correlated to extracellular protease production level and oxalic acid production level.A series experiments were conducted including screening of carbon and nitrogen resources, inorganic salt, initial value of pH, and initial inoculum by an orthogonal test. The optimum liquid medium for industrial fermentation production of the strain VL07 was determined by comparison of biomass of dry mycelia. Its main components were as follows: soybean powder 4%, white sugar 4%, KH2PO4 0.02%, MgSO4 . 7H2O 0.01%, FeSO4 . 7H2O 0.05%, Vb2 0.05%, Vc 0.05%, with pH value of 6.89. The initial inoculum ranged from 10% to 15%. 21.25mg/ml dry mycelial biomass was acquired after 72h cultivation with inoculum at 3.69-5.50mg/ml. The solid fermentation medium was screened for industrial production of strain VL07 by a L9 (34) orthogonal experiment. The optimum medium determined included bran 42%, rice shell 28%, maize powder 20% and soybean powder 10%. As the initial ratio of water content in the medium was 50%, conidiation reached the highest at 1.05 × 1010 conidia per gram.In an attempt to develop a drying method of the conidia of V. lecanii produced in diphasic fermentation, several drying methods更多还原