【作者】 宋漳；

【导师】 郭文硕；

【作者基本信息】 福建农林大学 ， 森林培育， 2008， 博士

【摘要】 本文主要针对绿僵菌对马尾松毛虫致病菌株的筛选及影响致病力的温湿度因子、马尾松毛虫对绿僵菌入侵的生理反应、绿僵菌培养与菌种稳定性、绿僵菌与化学杀虫剂、白僵菌混配及其增效作用和绿僵菌对林间节肢动物群落多样性的影响等方面进行了较为系统的研究,旨在揭示绿僵菌对马尾松毛虫的生物控制潜能,为有效利用绿僵菌防治马尾松毛虫提供理论依据。现将主要研究结果摘要如下:1.供试的9个绿僵菌菌株(M₁₀₃、M₁₀₄、M₁₃₁、M₃₃₇、M₁₈₇、M₃₃₆、M₁₁₅、M₃₃₅和M₁₈₉)中,金龟子绿僵菌M₁₀₄和M₃₃₇菌株对马尾松毛虫有致病性,进一步对这2个菌株进行生物测定表明,M₃₃₇菌株的毒力高于M₁₀₄菌株,M₃₃₇菌株对马尾松毛虫有较高毒力。通过对致病菌株M₁₀₄和M₃₃₇生物学特性的研究,明确了它们生长发育所需要的营养条件、环境条件和分生孢子萌发的营养和环境条件。2个致病菌株对环境条件有较好的适应性,25～28℃、pH6.5～7.0为适宜的产孢条件;温度、湿度和pH显著影响分生孢子萌发,25～28℃,pH6.5～7.5,RH＞92.5%是分生孢子的最适萌发条件。比较而言,M₃₃₇菌株比M₁₀₄菌株的产孢量大、孢子萌发快、萌发率高,对温度、湿度等环境条件的适应力更强,表现出更好的开发应用潜力。2.以筛选出的绿僵菌M₃₃₇和M₁₀₄菌株为研究对象,对其致病力及温湿度影响因子与白僵菌(Bb₀₁和Bb₀₂菌株)进行比较。研究结果表明,绿僵菌M₃₃₇菌株与白僵菌Bb₀₁、Bb₀₂菌株对马尾松毛虫的毒力相当,但绿僵菌M₁₀₄菌株对马尾松毛虫的毒力低于白僵菌Bb₀₁和Bb₀₂菌株。与供试的白僵菌菌株相比,绿僵菌分生孢子具有较好的耐高温和耐旱性,在高温和低湿的条件下,绿僵菌的杀虫效果优于白僵菌。林间防治结果显示,绿僵菌对第1代马尾松毛虫的防治效果显著优于白僵菌,显示了较好的应用前景。3.马尾松毛虫对绿僵菌入侵的生理反应试验表明,幼虫被绿僵菌感染后1～4d,血淋巴中血细胞总数和可溶性蛋白浓度均显著高于同期未感染的幼虫。利用聚丙烯酰胺凝胶电泳技术,分析了感染后3d、4d、6d幼虫体壁组织中可溶性蛋白和过氧化物酶,以及血淋巴中过氧化物酶的变化。结果显示,绿僵菌的侵染对寄主昆虫的蛋白质代谢产生影响,过氧化物酶活性有减弱趋势。4.通过对金龟子绿僵菌M₃₃₇菌株液固两相发酵技术的研究,优化控制发酵过程,筛选出玉米粉（2份）+麦麸（2份）+谷壳（2份）为固体发酵培养基的优化配方,液体种子发酵周期72h,初始接种量控制在25%为宜。保持培养温度25～28℃和环境湿度83～94%左右,同时选择合适的覆盖物可降低杂菌的污染。固态发酵周期11d,菌粉干燥温度控制在35℃,时间24h,可缩短生产周期。此外,研究了2种含铜化学杀菌剂（30%氧氯化铜和耐克铜）对绿僵菌生长、产孢的影响和对杂菌的抑制作用,在0.05%～0.15%浓度范围内,2种杀菌剂对金龟子绿僵菌的菌落形成没有明显影响。而耐克铜在0.05%～0.15%浓度时能显著地增加金龟子绿僵菌的产孢量,并有缩短产孢时间的作用。研究还表明2种杀菌剂对杂菌（根霉、曲霉和青霉）有较强的抑制作用,并随着浓度的增大,抑制作用迅速增强。试验结果对绿僵菌菌剂生产具有重要意义。5.对6个绿僵菌菌株(M₃₃₇、M₁₀₃、M₁₀₄、M₁₁₅、M₃₃₅和M₃₃₆)的液体深层培养研究表明,绿僵菌M₃₃₇、M₁₀₃、M₁₀₄、M₁₁₅菌株能通过微循环产孢方式形成液生分生孢子,微循环产孢现象的发生与菌株有一定的相关性。绿僵菌在固体和液体培养中产生不同形态的分生孢子,绿僵菌M₃₃₇菌株在液体培养条件下产生的液生分生孢子为单孢、卵圆形到近球形,大小3.17～4.51μm×2.51～3.34μm,与气生分生孢子有明显差异。以M₃₃₇菌株为对象研究了在液体深层培养条件下绿僵菌生长和产孢的营养和环境条件。研究结果表明,绿僵菌液生分生孢子的形成与培养基成分密切相关。不同的碳、氮源对液生分生孢子形成具有显著影响。蔗糖是液体深层培养液生分生孢子的理想碳源,而花生饼粉、豆饼粉则是较理想的氮源,且液生分生孢子的产量与培养基氮源的性质有关,复杂的氮源比简单的氮源更有利于液生分生孢子的形成。在供试的8种碳源和6种氮源相互组配的培养液中,以蔗糖+花生饼粉的组合最佳,液生分生孢子产量达14.13×10⁹个孢子·L^-1。同时,液生分生孢子的形成比率与培养液的碳氮比有关,培养液的C/N为3:1时,可使液生分生孢子的产出率最高。微量元素和维生素对促进绿僵菌液生分生孢子的形成具有重要影响。Mo为最重要的影响元素,其次是Zn,而B、Cu、Fe、Mn、Mo、Zn的协同作用将有力促进液生分生孢子的产生。在培养基中添加VB₆+VH或复合维生素B能有效促进绿僵菌液生分生孢子的形成。不同的氨基酸对绿僵菌生长及液生分生孢子形成影响很大,天门冬酰胺和L-丙氨酸有利于液生分生孢子的形成。光照对绿僵菌生长和液生分生孢子产量均无显著影响。但培养温度显著影响其生物量和液生分生孢子的产出率。25～28℃为适宜的培养温度,但28℃时液生分生孢子的产量最高。同时,培养液的pH也显著影响绿僵菌的生长和产孢,以pH6.5～6.8为宜,但pH6.8时产孢量最大。对3株绿僵菌(M₃₃₇、M₁₀₃和M₁₁₅)在不同含量的吐温80培养液中进行液体深层培养研究表明,吐温80对绿僵菌液生分生孢子的形成具有显著的影响,培养基中吐温80含量在0.6～1.0%之间时,可获得最大的产孢量。磁化水对供试的3株金龟子绿僵菌(M₃₃₇、M₁₀₃、M₁₀₄)和1株贵州绿僵菌(M₁₁₅)的液生分生孢子形成影响显著,但对生物量无显著影响。适当磁化强度的磁化水能显著提高绿僵菌M₃₃₇和M₁₀₃菌株的液生分生孢子产量,但试验也显示不同菌株对磁化水的生物效应表现出明显的差异性。对马尾松毛虫毒力的生物测定显示,绿僵菌M₃₃₇菌株的液生分生孢子对马尾松毛虫具有较高的侵染力,仅比气生分生孢子的毒力略低。因此,绿僵菌液体发酵有进一步研究的价值。6.对金龟子绿僵菌M₃₃₇菌株进行继代培养,探讨了6种常见培养基、4种碳源和氮源以及6个碳氮组合培养基对菌种稳定性、产孢量、毒力的影响。结果显示,绿僵菌的变异虽然主要受控于菌株自身的遗传特性,但同时也受培养基组成性质的影响。常见的6种培养基以营养贫乏的CMA和WBA最易发生变异,PPDA较稳定。动物性氮源较植物性氮源稳定,以麦芽糖和乳糖为碳源比较稳定。在不同碳氮比试验中,供试菌株在C/N比为2:1的培养基上比较稳定。7.对8种化学杀虫剂（4.5%高效顺反氯氰菊酯乳油、2.5%敌杀死乳油、21%灭杀毙乳油、25%灭幼脲Ⅲ悬浮剂、20%杀灭菊酯乳油、40%氧化乐果乳油、40%辛硫磷乳油和18%杀虫双水剂）与绿僵菌M₃₃₇、M₁₀₃、M₁₀₄、M₁₁₅、M₃₃₅和M₃₃₆菌株的相容性以及菌药混配的增效作用进行研究,结果表明,供试的8种化学杀虫剂皆对绿僵菌分生孢子有程度不同的抑制作用,浓度愈高,抑制作用愈强,但次亚致死剂量对分生孢子萌发抑制作用较小。对马尾松毛虫的生物测定结果显示,M₃₃₇+杀灭菊酯（80000×）、M₃₃₇+敌杀死（60000×）、M₃₃₇+辛硫磷（10000×）、M₃₃₇+灭杀毙（25000×）、M₃₃₇+灭幼脲Ⅲ（15000×）,其LT₅₀比单用绿僵菌M₃₃₇(1.9×10¹⁰个孢子·L^-1)分别缩短了9d、7d、6d、5d和3d,增效作用明显。8.绿僵菌和白僵菌混合使用经共毒系数分析,对马尾松毛虫幼虫的毒力增加了1.78倍。林间防治试验结果也表明,绿僵菌与白僵菌混合使用对马尾松毛虫的防治效果显著优于单独使用绿僵菌和白僵菌,增效作用明显。9.在施用绿僵菌菌剂前后,通过对马尾松毛虫虫口密度的T测验、节肢动物群落的垂直分布格局及马尾松林群落特征参数的分析,结果表明,绿僵菌对马尾松毛虫的种群控制作用明显,对非目标无脊椎动物不具有明显的影响,并且改善了整个马尾松林节肢动物群落的多样性和稳定性。更多还原

【Abstract】 Nine strains of the entomopathogenic fungi belonging to Metarhizium used to efficient biocontrol of Dendrolimus punctatus were studied in this thesis.The researches focused on screening of high virulent strains and effects of temperature and humidity on their pathogenicity, host pest’s physiological response,cultivation and stability,compatibility with chemical insecticides,Metarhizium anisopliae mixed with other insecticides and synergism,as well as effects of M.anisopliae on the diversity of arthropod community in Masson pine forest,and aimed to evaluate the potential of Metarhizium as a biocontrol agent for D.punctatus and to provide the theoretical basis for the applications.The main results are as follows:1.The pathogenicity of nine swains of Metarhizium spp.(including M₁₀₃,M₁₀₄,M₁₃₁,M₃₃₇, M₁₈₇,M₃₃₆,M₁₁₅,M₃₃₅and M₁₈₉)to larvae of D.punctatus were studied in the laboratory.Two strains(M₁₀₄,M₃₃₇)showed pathogenicity to the larvae.The bioassays for toxicity of strains M₁₀₄ and M₃₃₇were tested to D.punctatus respectively and showed the virulence of M₃₃₇was stronger than that of M₁₀₄,and the Strain M₃₃₇was significantly more effective to control D.punctatus. Nutrition and other conditions for the two swains’ growth,sporulation and conidia germination were determined,and the results showed that the two strains had good adaptability to the conditions such as temperature,humidity and pH value.The suitable sporulation conditions were 25—28℃,pH 6.5—7.0.The germination of the conidia was remarkably affected by temperature, humidity and pH value.The optimum condition for germination were 25—28℃,pH 6.5—7.5, RH＞92.5%,however,M₃₃₇germinated faster than strain M₁₀₄and showed more tolerance to high temperature and low humidity,and higher conidia germination and production rate.These implied that the strain M₃₃₇had a great potential of exploitation and application.2.The virulence of M₁₀₄and M₃₃₇were tested and analyzed by contrasting with the biocontrol agent Beauveria bassiana(including Bb₀₁and Bb₀₂),which showed that there were no difference of virulence to D.punctatus among M₃₃₇,Bb₀₁and Bb₀₂,however,the virulence of M₁₀₄was lower than those of Bb₀₁and Bb₀₂.The strain M₃₃₇of M.anisopliae was more tolerant to high temperature and drought than strain Bb₀₁of B.bassiana.In the conditions of high temperature and low humidity,the control effects of M.anisopliae on D.punctatus were higher also.Field test indicated that M.anisopliae was great superior to control the first generation of D.punctatus.3.The research on the host insect physiological response to the pathogen showed that the total hemocyte counts and the concentration of hemolymph soluble protein were significantly higher in infected larvae than in non-infected ones,from 1d to 4d after infection.By means of polyacrylamide gel electrophoresis technique,the physiological changes of proteins and peroxidases in hemolymph and body wall of the D.punctatus which 3d,4d and 6d post infection were analyzed.The results revealed that M anisopliae had some effect on the metabolism of the host’s proteins,and the peroxidase activities showed a tendency to decline.4.The solid-submerge fermentation technology of strain M₃₃₇of M.anisopliae was studied, and the optimal control for fermentation process were determined.The optimized medium of solid state fermentation was obtained,which would be composed of corn flour（2 portion）,wheat bran（2 portion）and rice hull（2 portion）.The fermentation period for liquid seed was 72h and the appropriate initial inoculation amount was 25%.The culture temperature and humidity were at 25—28℃and 83—94%,respectively.Meanwhile,in order to control the contamination of mould, the suitable covering material was selected necessarily.The period of solid state fermentation was adjusted to 11d,and drying temperature was manipulated at 35℃for 24h,and then the production cycle would be shortened.In addition,the influence of two chemical germicides（including 30%Cuprum oxychloride and Naiketong）on growth and sporulation of strain M₃₃₇of M.anisopliae,and inhibitory effect on competed moulds were investigated.The results showed that the two chemical germicides,viz. 30%Cuprum oxychloride and Naiketong had no obvious effect on colony recovery of M. anisopliae at the concentration of 0.05—0.15%.Naiketong at the concentration of 0.05—0.15% had increased conidial output,while it also shortened the sporulation time of M.anisopliae.The two germicides inhibited the colony growth of competed moulds（Rhizopus sp.,Penicillium sp. Aspergillus sp.）strongly,and the inhibition became stronger when concentration increased.5.Six strains belonging to Metarhizium(including M₃₃₇,M₁₀₃,M₁₀₄,M₁₁₅,M₃₃₅and M₃₃₆) were cultivated in submerged culture.The results indicated that the submerged conidia could be produced by means of microcyclic sporogenesis for strains M₃₃₇,M₁₀₃,M₁₀₄and M₁₁₅,perhaps, the occurrence of microcyclic sporogenesis depended on the nature of strains.Under solid-state and liquid-state cultivations,the entomopathogenic fungus M.anisopliae produced different types of spores.The morphologic characteristics of submerged cortidia of strain M₃₃₇are ovoid to subspherical,monocellular,with the size of 3.17—4.51μm×2.51—3.34μm,quite different from the aerial conidia.Nutrition and other conditions for the growth and sporulation of M₃₃₇were emphatically studied in submerged culture.The results showed that the submerged sporulation depended on a delicate equilibrium in the composition of the medium.Various carbon and nitrogen sources had significant effect on the production of submerged conidia.Sucrose was ideal carbon source for submerged sporulation.Peanut cake powder and bean cake powder were effective for sporulation of submerged conidia.The production of submerged conidia was correlated with the properties of nitrogen sources,which complex nitrogen source for sporulation of submerged conidia was more favorable to simple nitrogen source.Liquid medium containing sucrose and peanut cake powder produced large numbers of submerged conidia(14.13×10⁹ spores·L^-1).The experiment also showed that the output of submerged conidia was relationship with carbon-to-nitrogen（C:N） ratio of medium.Highest yield of submerged conidia was obtained in medium containing C:N ratio of 3:1.Trace elements and Vitamins had remarkable impacts on submerged sporulation. Molybdenum was the most important element.Zinc was stimulatory factor.When all 6 elements （including B,Cu,Fe,Mn,Mo and Zn）were added together,highest yield of submerged conidia could be obtained.VB₆+VH and compound vitamin B were more effective for producing submerged conidia.Various amino acids had important influence on growth and sporulation of submerged conidia.Liquid medium containing L-asparagine or L-alanine produced large numbers of submerged conidia.Illumination had no effect on mycelia growth and submerged spomlation directly.The cultural temperature and pH value had significant effect on the mycelia growth and output of submerged conidia.The proper temperatures and pH values for submerged culture were 25—28℃and pH 6.5—6.8,respectively.The maximum yield of submerged conidia was obtained in the condition of temperature at 28℃and pH 6.8.Biomass and submerged conidia yield were measured for three strains of Metarhizium spp. (including M₃₃₇,M₁₀₃and M₁₁₅)grown in liquid media containing different concentrations of Tween 80.The data was showed that concentrations of Tween 80 had effect on sporulation of Metarhizium spp.significantly.The submerged conidia yield was highest in liquid media containing 0.6—1.0%Tween 80.Three strains of M.anisopliae(M₃₃₇,M₁₀₃,M₁₀₄)and one strain of Metarhizium guizhouense (M₁₁₅)were cultivated in submerged culture by magnetized water media,and the biomagnetic effect of magnetized water on submerged sporulation of Metarhizium spp.were preliminary analyzed.The results indicated that the submerged conidia yields were affected by magnetized water,but the biomass were unaffected.The outputs of submerged conidia of strains M₃₃₇and M₁₀₃were increased remarkably in proper intensity of magnetizing water.But the experiment also showed that there existed obvious difference in different strains for biological effects of magnetizing water.This results provided an experimental basis for further research on the biological effects and applications of the magnetized water.In addition,The virulence of submerged conidia of strain M₃₃₇was tested against D. punctatus in laboratory.Results showed that submerged conidia had stronger virulence to the larvae of D.punctatus.There were no apparent differences in the toxicity between submerged conidia and aerial cortidia,but the virulence of submerged conidia to larvae of D.punctatus was slightly lower.Therefore,it is of great value for further research on submerse fermentation of Metarhizium.6.The subcultures of M₃₃₇were studied in this experiment.Effects of 6 regular media,4 carbon and nitrogen sources and 6 combinations of carbon and nitrogen on strain stability,growth characters and virulence were observed.The results indicated that the variation of M.anisopliae was mainly controlled by its genetic materials,but also influenced by the composition nature of media.Among the six regular media,strain M₃₃₇grew most stably on PPDA,but those nutrition poor ones such as CMA and WBA would cause variation very frequently.Among nitrogen sources, those from animal were more stable than those from plant.Mean while,strain M₃₃₇grew most stably on media containing maltose and lactose as carbon source.The experiment results also showed that strain M₃₃₇grew most stably on medium containing C:N ratio of 2:1.7.The compatibility of 8 chemical insecticides（including 4.5%Beta-Cypermethrin EC, 2.5%Deltamethrin EC,21%Pesticide destroying EC,25%Benzoylphenal ureaⅢSC,20% Fenvalerate EC,40%Omethoate EC,40%Phoxim EC and 18%Dimehypo WA）with Metarhizium spp.(including M₃₃₇,M₁₀₃,M₁₀₄,M₁₁₅,M₃₃₅and M₃₃₆)were studied in this experiment.The results showed that all chemical insecticides inhibited the conidial germination to a certain degree,and the inhibition became stronger as concentration increased.But low sublethal dose of chemical insecticide had less inhibitory effect on conidial germination.The results of bioassay showed that it was obvious of the synergism against Masson’s pine caterpillars（D. punctatus）,when strain M₃₃₇of M.anisopliae(1.9×10¹⁰spores·L^-1)mixed with Fenvalerate （80000×）,Deltamethrin（60000×）,Phoxim（10000×）,Pesticide destroying（25000×）and Benzoylphenal ureaⅢ（15000×）,and the LT₅₀of mixtures would be shortened about 9 days,7 days,6 days,5 days and 3 days respectively,comparing with that of using fungal insecticide of M. anisopliae(1.9×10¹⁰spores·L^-1)alone.8.According to the analysis of Co-toxicity coefficient,M.anisopliae could increase 1.78 times in its toxicity to larvae of D.punctatus when it was mixed with B.bassiana.The effect of control test in the field showed that the mixture insecticide was great superior in controlling D.punctatus to using fungal insecticide of M.anisopliae and B.bassiana alone.9.The density of Masson’s pine caterpillars and arthropod communities in various layers of pine forest were investigated before and after spraying microbial insecticide of M.anisopliae in Shanghang county in Fujian province.T test of the density of Masson’s pine caterpillars,vertical pattern of arthropod community and its parameters were discussed.The results showed that M. anisopliae had obvious biocontrol effect on Masson’s pine caterpillars,but it had no impact on the non-target invertebrates apparently.The diversity and stability of arthropod community in pine stand was improved.更多还原