【作者】 黄宝福；

【导师】 冯明光；

【作者基本信息】 浙江大学 ， 微生物学， 2008， 博士

【摘要】 球孢白僵菌（Beauveria bassiana）等丝孢类生防真菌已被开发成为多种制剂广泛用于农林害虫防治。然而,这些制剂的贮存和田间应用效果总是受到环境温度、湿度和紫外辐射等因素的影响。生防真菌耐受环境胁迫的能力直接关系到其制剂的田间生态适应性。本研究从研究建立球孢白僵菌对紫外辐射耐受力和氧化胁迫耐受性的定量评价技术体系入手,比较测定了若干菌株对紫外线UV-B和氧化剂亚甲基兰胁迫的耐受力,筛选获得了较耐紫外辐射和氧化胁迫的菌株;研究了不同培养条件或培养基成分对分生孢子氧化胁迫耐力的影响;测定了不同菌株胞内超氧化物歧化酶的酶活水平,分析了酶型的差异。主要内容和结果分述如下:球孢白僵菌耐紫外线辐射能力的定量评价研究建立了球孢白僵菌耐紫外线辐射能力的定量评价技术体系,并用于测定不同寄主或地理来源的20株白僵菌对紫外线UV-B辐射的耐受力。所有菌株在25±1℃和12L:12D条件下培养7天后收集孢子,所用培养基为1/4萨氏培养基（SDAY）斜面。将各菌株分生孢子粉悬浮于萌发液（2%蔗糖加0.5%蛋白胨）中,适量涂于凹载玻片上,曝露于UV-B（Bio-Sun,280～320 nm）的不同剂量（0～1.6 J/cm²）下进行辐射处理。然后,将玻片在25±1℃和12L:12D条件下保湿培养24小时,棉蓝固定,400×下镜检观察孢子萌发率。结果显示,各菌株的孢子萌发率（G_p）与UV-B辐射剂量（D）间的关系拟合逻辑斯蒂衰变模型G_p=1/(1+exp（a+bD）（r²≥0.93）。根据拟合的模型计算出UV-B对各菌株的半致死剂量LD₅₀,变化范围从0.14J/cm²到0.92 J/cm²不等,菌株间差异显著。耐紫外辐射最好的菌株Bb2861的LD₅₀与杭州夏季晴天（2007年8月6日）日累积紫外线辐射量（0.63 J/cm²）相接近。球孢白僵菌耐氧化胁迫能力的评价及影响因素研究建立了球孢白僵菌分生孢子耐氧化胁迫能力的定量评价技术体系,用于比较不同菌株及培养条件所产分生孢子的耐氧化胁迫能力。首先在25±1℃和12L:12D条件下培养和生产17株球孢白僵菌的分生孢子。继而在含不同浓度（50、100、150、200、250和300μg/ml）金属离子(Fe³⁺、Mn²⁺、Cu²⁺或Zn²⁺)、不同pH（4.0、5.0、6.0、7.0及8.0）、不同碳源及水活度的SDAY培养基上以及不同温度（15、20、25和30℃）下培养生产Bb2861菌株的分生孢子。所获分生孢子悬浮于0.02%吐温80,稀释至1000个孢子/mL的浓度,取50μL孢子悬浮液均匀涂到含10μg/ml亚甲基兰的SDAY平板（直径9 cm）上,于25±1℃和12L:12D条件下培养4天后测量菌落直径。将胁迫下菌落直径与对照组菌落直径之比定义为相对生长率（relative growth rate,RGR）,作为耐氧化胁迫的指标。结果表明,17株球孢白僵菌的分生孢子耐氧化胁迫能力存在显著性差异(F_16,64=28.61,P＜0.01)。所测菌株的菌落平均RGR值为53.74%,其中以Bb2881耐氧化胁迫能力最差,RGR仅为14.1±5.8%,而Bb2988耐氧化胁迫能力最强,RGR达到77.1±8.5%,相当于Bb2881的5.5倍。在培养基中添加金属离子的实验中,含300μg/ml Cu²⁺或50μg/ml Zn²⁺的培养基所产孢子的耐氧化胁迫能力最强,RGR值分别为71.9±7.4%和74.4±8.1%。偏高温度30℃下所产孢子的RGR值高于较低温度下生产的孢子(F_3,12=28,12,P＜0.01),但无氧化胁迫的30℃下所产孢子的菌落生长缓慢,菌落直径增长2.1±0.1 mm/d,而20℃下所产孢子的菌落直径增长3.5±0.1 mm/d。用可溶性淀粉作碳源生产分生孢子,其RGR值30.3%～42.5%,而以葡萄糖为碳源生产的孢子表现较强的耐氧化胁迫能力,RGR值达到73.9%～89.4%。其中,含葡萄糖31.7g/L、水活度a_w为0.988的培养基所产孢子的耐氧化胁迫能力最强（RGR:89.4±9.8%）。由此得出生产最耐氧化胁迫分生孢子的培养条件,即以4%葡萄糖为碳源,添加200μg/mL Mn²⁺,pH略偏酸性,培养温度为25℃。球孢白僵菌胞内超氧化物歧化酶的酶活水平及酶型类别比较为了测定球孢白僵菌的SOD酶活水平并区别酶型,将17个不同地理和寄主来源的菌株的分生孢子悬液（0.02%吐温80）分别涂于9 cm SDAY平板上,在25±1℃和12L:12D条件培养4天。取菌丝液氮冷冻研磨后,加入2 mL PBS（0.02 M,pH 8.0）溶解,4℃离心（16,600×g）15min,取上清以邻苯三酚法测定SOD酶活和总蛋白浓度。SOD粗酶液经Native-PAGE活性电泳后以四氮唑蓝（NBT法）活性染色进行观察,并根据酶液对KCN和H₂O₂的敏感性判断SOD酶型。结果表明,不同菌株菌丝中提取的SOD酶活存在显著差异(F_16,32=22.58,P＜0.01),酶活水平介于0.13～1.46 U/mg蛋白,平均为0.57 U/mg蛋白。菌株Bb2861的SOD酶活最高,而菌株Bb5965的SOD酶活最低。根据四氮唑蓝活性染色结果,17株菌的细胞提取液中存在两类SOD酶,其中3株（Bb2879、Bb2881和Bb5965）只含有一个SOD条带,而其余菌株有两类SOD酶型条带。一类可能为MnSOD,而另一类为FeSOD,后者在真菌中未曾有过报道。本研究的主要成绩和创新点,一是研究建立了的球孢白僵菌耐紫外线辐射能力的定量评价技术体系,揭示了耐紫外线辐射能力在不同菌株间的差异。二是建立了白僵菌抗氧化胁迫能力的评价技术体系,揭示了不同菌株间的差异,分析了影响分生孢子耐氧化胁迫能力的因素,获得了有利于增强耐氧化胁迫孢子粉生产的优化组合。三是比较分析了不同球孢白僵菌菌株的SOD酶活水平和酶型差异,首次在生防真菌中发现FeSOD。这些结果将有助于增进对球孢白僵菌抗逆生物学基础的认识,有助于推进害虫真菌的侵染生物学、流行学和剂型研究的不断深入,有助于提升真菌杀虫剂的田间稳定性和应用效果。更多还原

【Abstract】 The entomopathogenic fungus,Beauveria bassiana,has been developed into numerous formulations for wide application to biological control of insect pests in agriculture and forestry.It is well known that the shelf life and field persistence of a fungal formulation are affected by environmental factors,such as temperature,humidity and solar radiation.For formulation improvement,therefore,it is necessary to understand the tolerance of B. bassian towards main stressful factors so that exploring fungal biocontrol candidates and technical means to improving fungal fitness to variable environments is more effective.This study sought to evaluate quantitatively the conidial tolerances of different B.bassiana strains to ultraviolet（UV-B）radiation and oxidative stress for better candidates.The effects of different medium components and cultural conditions on the tolerance of conidia to oxidative stress were investigated.The activities and types of superoxide dismutases（SOD） in B.bassiana strains were also demonstrated.The results are summarized as follows.UV-B tolerance of B.bassiana strains.Twenty strains of B.bassiana（Bb）with different host and geographic origins were assayed for their tolerance to UV-B（290-320 nm） radiation.For each of the strains,conidia produced on 1/4-SDAY slants at 25℃were suspended in a germination broth（0.5%peptone and 2%sucrose plus 0.02%Tween-80）. Each of three 10-μl aliquots（replicates）of the suspension(1×10⁶ conidia ml^-1)was evenly smeared onto the concave round area（10 mm diameter）of sterilized glass slides.The smeared slides were then exposed to the UV-B（312 nm）irradiations of 0-1.6 J cm^-2in a UV-B generator ’Bio-Sun’.After exposure,the slides were incubated for 24 h at the regime of 25℃and L:D 12:12 with saturated humidity.Subsequently,the concave area of each slide was stained with methyl blue and then observed for the counts of germinated and ungerminated conidia under a microscope（400×magnification）.As a result,the proportions of germinated conidia（G_p）at the UV-B doses fit well the logistic survival model G_p =1/(1+exp（a+bD）for all the tested strains（r²≥0.93）.The median lethal doses(LD₅₀)of the UV-B irradiation estimated from the fitted equations of the tested strains fell in a range of 0.10-0.92 J cm^-2.The LD₅₀of the most UV-b tolerant strain Bb2861 was about the cumulative UV（200-320 nm）radiation(0.63 J cm^-2)on a fine summer day（6 August 2007） in Hangzhou.Oxidative stress tolerance or B.bassiana strains and influential factors in cultures. The conidia of 17 B.bassiana strains produced on SDAY under normal condition and those of the strain Bb2861 under different culture conditions were assayed for their tolerances to oxidative stress.For conidial production of Bb2861,SDAY plates were adjusted with ion concentrations(50,100,150,200,250 and 300μg ml^-1Fe³⁺,Mn²⁺,Cu²⁺or Zn²⁺),pH（4.0, 5.0,6.0,7.0 and 8.0）,carbon sources or water activity（a_w）or incubated at different temperatures（15,20,25 and 30℃）.For each of the strains or the concerned factors,three 50-μl aliquots（replicates）of conidial suspension(1000 conidia ml^-10.02%Tween 80)were evenly smeared on the plates（9 cm diameter）of SDAY containing 10μg ml^-1methylene blue（as a standard oxidative stress）and then incubated at the regime of 25℃and L:D 12:12 for 4 days.The ratio of the resultant colony diameter of（cross-measured）over that of blank control（not stressed）was defined as relative growth rate（RGR）to depict the tolerance of each strain or factor.The resultant RGR values differed significantly among the 17 strains tested(F_16,64= 28.61,P＜0.01).On average,the RGR ranged from 14.1±5.8%（Bb2881）to 77.11±8.51% （Bb2988）,yielding an overall mean of 53.74%for all tested strains.Inclusion of 300μg ml^-1 Cu²⁺or of 50μg ml^-1Zn²⁺in SDAY led to production of Bb2861 conidia most tolerant to the oxidative tolerance（RGR:71.90±7.43%and 74.38±8.14%）.Conidia produced at 30℃showed better oxidative tolerance than those produced at lower temperatures(F_3,12= 28.12, P＜0.01),colony growth with no stress at 30℃was slower（2.06±0.09 mm per day）than that at 20℃（3.49±0.09 mm per day）.The conidia produced on plates with starch as a mere carbon source had the RGR values of 30.3-42.5%.In contrast,the RGR values were 73.9-89.4%when glucose was used as carbon source for conidial production.The optimized conditions for producing the most stress-tolerant conidia were a medium containing 4% glucose as carbon source and 200μg ml^-1Mn²⁺as additive（with adjusted pH 4.0-6.0）,and 25℃for incubation.The activities and types of superoxide dismutases（SOD）in B.bassiana strains.To determine the activities and types of superoxide dismutases（SOD）in B.basstana,cellular extracts were produced from 4-day cellophane-plate colonies of 17 strains grown on 1/4 SDAY at 25℃.The SOD activities of the extracts were assayed based on their inhibitory effect on spontaneous autoxidation of pyrogallol.As a result,total SOD activities fell in a range of 0.13-1.46 U mg^-1proteins for the tested strains.Two SOD types were identified from all the cellular extracts in terms of their sensitivity to KCN or H₂O₂ in native polyacrylamide gel electrophoresis.Fourteen strains produced both MnSOD and FeSOD while the rest yielded only MnSOD or FeSOD,which was found for the first time in B. bassiana.Three strains showing distinctly low,medium and high SOD activities at 25℃differed significantly in relative SOD activity after their cultures were exposed to the thermal stress of 35℃for 3-18 h but had no change in SOD type.The variation of the SOD activities expressed either in normal cultures or in response to the thermal stress could be exploited for selection of stress-tolerant fungal candidates against insect pestsIn summary,quantitative assay methods were developed for successful evaluation of the tolerances of B.bassiana strains to UV-B radiation and oxidative stress.Factors including metal ions,pH,temperature,carbon source and water activity in conidial production were found affecting the tolerance of the resultant conidia to the oxidative stress. The overall activities of two SOD types in B.bassiana were generally at low levels despite significant variation among the tested strains.FeSOD was found for the first time in fungi. The results would increase our knowledge on the stress biology of B.bassiana and suggest a promising approach to improving fungal formulations for better field persistence and performance.更多还原