【作者】 李忠；

【导师】 金道超；

【作者基本信息】 贵州大学 ， 动物学， 2008， 博士

【摘要】 蝉拟青霉（Paecilomyces cicadidae（Miquel）Samson）是我国分布广泛,极具生防应用潜力的虫生真菌。本文以蚜虫为试虫,从不同来源的蝉拟青霉菌株中筛选出对中药材板蓝根蚜虫（Lipaphis erysimi）具有较高致病性的LB菌株,并系统研究其对蚜虫的入侵和致病过程、对蚜虫的致病性及其影响因子、生物学特性、液体及固体培养条件优化、粗毒素杀蚜活性及常用化学农药与该菌的相容性等内容。本研究旨在探索适合蝉拟青霉生物学特性的基本培养方法与技术,揭示其对蚜虫的致病机理及控制潜能,明确蝉拟青霉对蚜虫的相互作用关系,为蚜虫综合治理中有效利用蝉拟青霉提供理论依据,同时也为寄主与病原物互作关系的研究提供新的例证。结果如下:1、菌株筛选。以不同昆虫寄主采集的拟青霉（Paecilomyces）和白僵菌（Beauveria）菌株对白术蚜虫（Macrosiphum sp.）进行生物测定。结果表明,不同属种的真菌菌株的致病性存在差异,其中蝉拟青霉的几个菌株表现了较强的致病力。进一步对12株蝉拟青霉菌株培养特性及其对蚜虫的致病性进行测定,结果表明不同菌株的培养特性和致病力存在差异,其中以蝉拟青霉LB菌株的致病力强、培养特性优良,对蚜虫的校正死亡率为80.97%,致死中时LT₅₀为4.44d,致死中浓度LC₅₀为0.87×10⁶孢子/mL。2、致病机理研究。通过扫描电镜观察,蝉拟青霉分生孢子在蚜虫体表萌发、形成芽管后可产生附着胞或直接侵入表皮,昆虫节间膜等薄弱处也是其侵染的主要部分,随后菌丝在昆虫体内形成,如湿度足够高,菌丝穿出体表产孢而再侵染新害虫。用明胶-琼脂平板法研究了蝉拟青霉12不同来源菌株的胞外蛋白酶产酶水平与其毒力的关系。结果表明,不同菌株的产酶水平存在差异,且产酶水平的高低与其对蚜虫的毒力存在显著性关系。因此可以认为该菌的胞外蛋白酶活力是构成毒力的主要因素之一。3、生物学特性及其与致病性的关系。温度、pH、湿度对蝉拟青霉生长、产孢及孢子萌发具有明显的影响。蝉拟青霉LB菌株生长、产孢及孢子萌发的最适温度是25℃。pH6时菌落生长最快,分生孢子产生及萌发的最适值为pH6～7。光照对菌落生长、孢子萌发没有明显影响,对产孢量有促进作用。孢子萌发的相对湿度范围为RH90%～100%,最适湿度是RH100%。在20～30℃范围内,蝉拟青霉LB菌株对蚜虫有致病性,在25℃下毒力最强;不同温度和不同pH值下培养的蝉拟青霉产孢量有差别,但所获得的分生孢子在相同条件下对蚜虫的毒力没有差异。在RH90～100%范围内,蝉拟青霉LB菌株对蚜虫有致病性,在RH100%时毒力最强。温度、湿度对致病性有重要的影响,在温度、湿度的互作中,湿度更具有重要作用。4、液体培养条件优化。通过对不同碳源、氮源、无机盐等对蝉拟青霉菌丝生长的影响研究,结果表明:葡糖糖为蝉拟青霉液体培养的最适碳源,蛋白胨为该菌生长的最适氮源,K₂HPO₄和MgSO₄作为无机盐有利于菌丝生长。经正交试验筛选出的培养基为:葡萄糖3%,蛋白胨1.5%,磷酸氢二钾0.1%,硫酸镁0.2%组合为优良的液体培养基。摇瓶发酵的适宜条件为:培养温度25～27℃,初始pH6～7,接种量6%,摇床转速为150r/min,500 mL三角瓶最适装液量为100 mL,发酵周期为7d。5、液体发酵代谢特性。随培养时间的延长,培养液中的还原糖含量、氨基氮含量、酸碱度和菌丝生物量均发生明显的变化。接种培养48h内,菌丝产量增长较慢;培养48～144h,菌丝生物量增长幅度最大,接种168h,菌丝生物量达22.45 g/L。培养液的pH值、糖含量、氮含量随培养时间的延长逐渐降低,菌丝快速生长的时期是糖、氮消耗最大的时期;192h后培养终止时,发酵液中的还原糖还有25%尚未消耗,而氨基氮的含量约为60%;接种72h后营养液的pH值降为4.56,因此接种72小时后,可适当增加pH值,以利蝉拟青霉菌丝生长。6、固体培养条件优化。经单因素、一般组合和正交试验筛选,得到其适宜的固态发酵培养基为玉米粉+麦麸+黄豆粉+米糠（6∶4∶1∶1）。培养条件筛选结果表明,该菌株固态发酵培养的优化条件为:接种量6%、料水比1∶1.2、初始pH值6～7、培养温度24℃、发酵时间22d、培养基厚度在2cm左右、光照24h/d,添加ZnSO₄或BaCl₂有利于产孢。在此优化条件下固态发酵培养,其孢子产量稳定在45×10⁸个/g以上,最高产孢量达到54.72×10⁸个/g。7、粗毒素杀蚜活性。发酵液对蚜虫的致死率达到81.11%,初步表明蝉拟青霉LB菌株液体培养能产生毒杀蚜虫的物质,而且杀虫活性较高。蚜虫对胞外粗毒素敏感,处理2d后的死亡率高,处理后蚜虫的死亡率随处理时间和浓度的提高而增加。胞内粗毒素提取物具有一定的杀蚜虫活性,但不及胞外粗毒素的效果明显。8、化学农药与蝉拟青霉的相容性及增效性。不同的化学农药对蝉拟青霉菌丝生长、分生孢子萌发均有不同程度抑制作用。杀菌剂对菌丝生长、产孢、孢子萌发抑制作用大,杀虫剂相对较小。结果表明吡虫啉对菌丝生长抑制作用小,对孢子萌发无明显的影响,因此可以作为蝉拟青霉混用的首选药剂。该药剂与菌混用后具有明显的增效作用,菌孢子液与药（1:15000或1:30000）混用,致死中时LT₅₀提前,蚜虫的校正死亡率提高。更多还原

【Abstract】 Paecilomyces cicadidae（Miquel） Samson.is an entomogenous fungus species widely distributed in China and a potential agent for biocontrol of insect pest.In the studies,a high virulent strain of Paecilomyces cicadae,LB strain,to aphid was screened out from the isolates collected in various areas of China.The studies of the strain with target aphid Lipaphis erysimi,a main pest of Chinese herbe Atractylocles macrocephala Koiolz,focus on the infection process, pathogenicity and factors related the biological characteristics,optimum fermentation conditions for liquid and solid culture medium,insecticidal activity of crude toxin,reciprocity of the fungal products mixed with pesticides.The studies aimed at establishing technical approach for producing of P.cicadae and revealing pathogenesis and potential of the fungus against the aphids. It may provide the fundamentals for the application of the fungus in the integrated control of the aphids and set a new exemplum for the research on interaction between insect host and pathogens.1.The screening of superior strains.The pathogenicity of strains of Paecilomyces and Beauveria from different host insects,were tested with pahid.The result showed that there were obvious differences in pathogenicity among the strains,of which some strains of P.cicadae showed higher pathogenicity than others.By comprehensive comparing of 12 P.cicadae,LB strains was.recognized as the most effective because of its higher pathogenicity,higher sporulation yield and higher tolerance.The corrected mortality was 80.97%,LT₅₀ was 4.44days,and LC₅₀ was 0.87×10⁶/mL in the aphids.2.The pathogenic mechanisms to aphid.Scanning electron microscopy was employed to investigate the infection process of P. cicadae on aphid.It was discovered that conidia of LB strain could germinate on the cuticle of aphid,the germ tube or by means of an appressorium of conidia might penetrate into the cuticle. Intersegmental membranes were preference sites of infection.Mycelia,which developed from the tube after penetration,might grow out of the integument and then produce new conidia for reinfecting if it was under heavy humid condition The relationship between extracellular protease and virulence in I2 strains of P.cicadae were studied by gelatin-agar plate methods.The result showed that productions of extracellar protease were significantly different among the strains,and there was a liner relationship between the protease activity level and the virulence of P.cicadae to the aphid.This indicated that activity of extracellular protease could be regarded as a main element consisting of the virulence of P. cicadae.3.Biological characteristics with emphasis on relationship with pathogenity.Biological characteristics were observed with cultures under series conditions of temperature and humidity.The results suggested that the temperature,pH values,RH have significantly effects on the mycelial growth,sporulation and conidial germination.Temperature of 25℃is optimum for the mycelial growth,sporulation and conidial germination.The pH 6.0 is optimum values for the mycelial growth and pH 6-7 for the sporulation and germination.Illumination had no effect on the growth of mycelium and the germination of conidia,but could increase spore yield.The RH for the conidial germination was 90～100%and the optimum of RH 100%.The fungus could cause disease of aphid within temperature 20-30℃and RH 90-100%, with the optimum of 25℃and RH 100%.Although temperature and pH had markable effect on the conidial yield,the spore cultured under different temperatures or pH values,had no differences in pathogenic capability to the aphids at same conditions.Temperature and humidity were the principle factors influencing pathogenicity,but the later played a key role in primary infection of aphid.4.Optimization of submerged fermentation conditions.Submerged culture ofp.cicadae was made With different sources of carbon and nitrogen,and inorganic compounds in order to selecting favorite cultural conditions.The results showed that the optimum carbon source was glucose,the optimum nitrogen source peptone and the optimum inorganic salt K₂HPO₄,MgSO₄ for liquid culture medium.The orthogonal test showed that the optimum compositions of a fermentative medium are as follows:3%glucose,1.5%peptone,0.1% K₂HPO₄,0.2%MgSO₄·7H₂O,pH 6～7.The highest yield of mycelia was obtained after fermenting for 7days under the conditions with the shaker rotation speed at 150 r/min,the temperature at 25～27℃,the inoculum amount 6%in volum,the medium 100mL in 500mL flask.5.Metabolism characters in liquid culture. The contents of reducing sugar,amino nitrogen,pH values and mycelia biomass in nutritive medium changed obviously with the time increasing during process of inoculation.The mycelial biomass increased rapidly in early 48-144h and reacbed 22.45mg/ml after 168h.The pH value,the contents of reducing sugar and amino nitrogen decreased gradually in the course of submerged culture ofp.cicadae.After 72h inoculated,pH value of the medium dropped from 5.63,the initial value,to 4.42.The acid degree should therefore be regulated reasonably for well development of the mycelia after 72h inoculated.6.Optimization of solid culture condition.The fermentation components of solid medium optimized by mono-factor,Multi-factor combination and orthogonal experiments are corn flour+wheat bran+soybean powder+chaff （6:4:1:1）.The optimal fermentation conditions were suggested as follows:the amount of inoculation 6%,initial content of water 120%,natural pH6～7,culture temperature 24℃, fermentation time 22 days,the thickness of medium about 2cm,and illumination 24h/d.ZnSO₄ or BaCl₂ can improve sporulation of the fungus.The conidia production could be a constant yield about 45×10⁸ spores per gram and the maximum yield reached to 52.32×10⁸ spores per gram in optimized medium under the optimal conditions.7.Insecticidal activity of crude toxins to aphid.Fermented broth caused higher correction mortality（81.11%） of aphids.Crude extracts of P. cicadae endotoxins and exotoxins were obtained from submerged culture with organic solution and then used in an insecticidal assay against aphid.Investigation of insecticidal characteristics of the fungal toxins revealed that the aphid was susceptible to the exotoxins and had higher mortality rate after 2 days treatment.Moreover,mortality rate of the aphid increased with prolongation of treatment duration or increasing concentration of the toxins.It showed that the aphid was also susceptible to the endotoxins,but of which insecticidal activity was not as strong as that of the exotoxins.It is suggested that the exotoxins are the active secondary metabolites which represent a new source of toxins ofp.cicadae.8.The compatibility and synergism of pesticide with P.cicadaeTwenty widely used pesticides were assessed for compatibility with P.cicadae.The affects of these pesticides on mycelial growth,sporulation yield and spore germination were quantified.The results showed that Imidacloprid has the lowest restraining rate to the myceli.al growth and insignificantly affect on the spore germination.Imidacloprid was thereby thought as the best pesticide to be blend with P.cicadae.The results of bioassay showed that there was significant synergism against the aphids using the mixture of P.cicadae（concentration of 5×10⁷ spore/mL） with Imidacloprid（1:15000 or 1:30000）,which made LT₅₀（0.37days,and 0.3 days respectively） shorter and corrected mortality rate higher than those using P.cicadae（5×10⁷ spore/mL） alone.更多还原