【作者】 姚世莉；

【导师】 冯明光；

【作者基本信息】 浙江大学 ， 微生物学， 2010， 硕士

【摘要】 球孢白僵菌（Beauveria bassiana）和绿僵菌（Metarhizium spp.）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。紫外辐射是影响生防真菌孢子制剂贮存和田间应用效果的关键因素之一,而菌剂生产所用菌种对紫外辐射的耐受能力直接关系到其制剂的田间生态适应性。本研究基于新建立的生防真菌对紫外辐射耐受力的定量评价技术体系,比较测定了不同地理和寄主来源的若干菌株的分生孢子对紫外线UV-B和UV-A的耐受力,筛选获得了较耐紫外辐射的菌株,并选取2个高毒力菌株评价了UV-B辐射对其杀蚜毒力的影响。主要内容和结果分述如下:60株生防真菌耐UV-B紫外辐射能力的定量评价基于本实验室新建立的生防真菌耐紫外辐射能力的定量评价技术体系,测定了不同寄主或地理来源的60株白僵菌和绿僵菌对UV-B辐射的耐受力。供试菌株在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。将各菌株的分生孢子粉分别悬浮于绿僵菌萌发液（0.25%蛋白胨,0.25%酵母粉加0.2%海藻糖）或白僵菌萌发液（2%蔗糖加0.5%蛋白胨）中,适量涂于凹载玻片上,在Bio-Sun⁺⁺紫外辐射箱中进行平均加权波长为312 nm的UV-B不同剂量（0～1.2 J/cm²）的辐射处理。然后,将玻片在25±1℃和12L:12D条件下保湿培养24小时,用棉蓝固定后在400×下镜检观察孢子萌发率。结果显示,各菌株的孢子萌发率（I_s）与UV-B辐射剂量（D）间的关系很好地拟合逻辑斯蒂衰变模型I_s=1/[1+exp（a+bD）]（r²≥0.97）。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0.05 J/cm²到0.65 J/cm²不等,菌株间差异显著。所测菌株中Maac 5734（一株金龟子绿僵菌蝗变种）最耐UV-B辐射,但其LD₅₀（0.65 J/cm²）仅相当于杭州夏季晴天阳光中日累积UV-B辐射量的1/4左右。24株绿僵菌耐UV-A紫外辐射能力的定量评价测定方法同上,但紫外辐射的波长和剂量不同,平均加权波长为365 nm,辐射剂量范围为1-18 J/cm²。观察孢子萌发率,结果显示,各菌株的孢子存活指数（I_s）与UV-A辐射剂量（D）间的关系拟合逻辑斯蒂衰变模型I_s=1/[1+exp（a+bD）]（r²≥0.999）。根据拟合的模型计算出UV-A对各菌株的半致死剂量LD₅₀,变化范围为2.78～10.46 J/cm²,菌株间差异显著。最耐UV-A辐射的菌株是Mfm 3606（一株黄绿僵菌小孢变种）,其LD₅₀(10.46 J cm^-2)仅为阳光中日累积UV-A辐射量的一半。不同强度紫外辐射对Bb 2860和Ma 759分生孢子杀蚜毒力的影响选取对桃蚜（Myzus persicae）具有较强毒力的球孢白僵菌Bb 2860和金龟子绿僵菌Ma 759为供试菌株。每个菌株对平皿甘蓝叶片上桃蚜的接种均以10⁸个孢子/ml的悬液在标准喷塔中喷雾接种,设不喷菌作为空白对照。喷雾接种后,各菌株的接菌处理分别接受预期可导致分生孢子失活10%、50%和90%的3个UV-B辐射剂量的处理,并设接菌但不辐射的处理作为对照。每处理3次重复,每重复（平皿叶片）30～40头无翅成蚜。辐射处理后,所有蚜虫在25℃和12L:12D条件下饲养8天,逐日观察记录死亡率,蚜尸保湿培养2～3天后镜检。结果显示,Bb 2860接种后经UV-B高剂量（0.86 J/cm²）、中剂量（0.45 J/cm²）、低剂量（0.03 J/cm²）的辐射处理,蚜虫校正死亡率分别为13.3%,58.9%和82.3%,平均致死期分别为3.69、3.84和3.94天。Ma 759在高剂量（0.65 J/cm²）、中剂量（0.30 J/cm²）、低剂量（0.13 J/cm²）的UV-B辐射下,蚜虫校正死亡率分别为18.1%,54.0%和88.7%,平均致死期分别为5.50、4.44和4.46天。由此说明,UV-B辐射显著降低Bb 2860和Ma 759对蚜虫的感染率,其辐射剂量引起的孢子失活比例与蚜虫死亡率的下降幅度基本吻合,但不影响各菌株对蚜虫的感染致死时间。以上研究结果充分揭示了生防真菌耐紫外辐射能力的种间和种内差异,筛选出了耐紫外辐射的生防菌株资源,首次定量活体测定了UV-B辐射对2个高毒力菌株杀蚜毒力的影响,将有助于推动杀虫真菌抗逆生物学和剂型生物学的研究不断深入。这些结果证明,通过筛选可以获得毒力和耐紫外性状兼顾的生防潜力菌株,同时为生防菌剂中加入紫外保护剂以及菌剂在日落后傍晚使用的应用策略提供了重要科学依据。更多还原

【Abstract】 SummaryFungal biocontrol agents,such as Beauveria bassiana and Metarhizium spp.,have been developed into a variety of formulations for use in control of insect pests worldwide. Fungal conidia usually used as active ingredients of the formulations are often exposed to environmental stresses after field application.Of those,solar UV irradiation is detrimental to the exposed conidia and thus an important factor to affect field efficacy and persistence. This study sought to assess conidial tolerances of 60 B.bassiana and Metarhizium strains to simulated UV-B and UV-A irradiations based on a novel system of quantitative stress bioassay in vitro.Two isolates of B.bassiana and M.anisopliae with high biocontrol potential were selected for further in vivo bioassays to evaluate the effects of three UV-B doses on their virulence to aphids sprayed with a concentrated spore suspension.The results are summarized below.In vitro responses of 60 fungal isolates to UV-B irradiation.All 60 isolates[24 B. bassiana（Bb）,20 M.anisopliae（Ma）,seven M.anisopliae var.acridum（Maac）,five M. anisopliae var.anisopliae（Maan）,one M.anisopliae var.majus（Mam） and three M. flavoviride var.minus（Mfm） with different host and geographic origins]were separately cultured on the slants of 1/4 SDAY（w/v:1%dextrose,0.25%peptone,0.25%yeast extract and 2%agar） for 7 days at the regime of 25℃and L:D 12:12.Conidia from the slant cultures were suspended in a species-specific germination broth in 0.02%Tween-80.The broth consisted of 0.5%peptone and 2%sucros for B.bassiana and of 0.25%peptone, 0.2%trehalose and 0.25%yeast extract for Metarhizium spp.Three 10-μl aliquots （replicates） of each suspension were dripped onto sterilized glass slides and each was smeared evenly in an area of ca.10 mm diameter.The smeared slides were placed in the sample tray（10×120×260 mm） of a UV irradiation chamber,named Bio-Sun⁺⁺（Vilber Lourmat,Marne-la-Vallee,France）,and then exposed to the irradiation of the weighted UV-B wavelength of 312 nm at the gradient doses of 0.005-1.1 J/cm²（D） After exposure, all slides were immediately transferred to moisture-saturated chambers and incubated for 24 h at 25℃and L:D 12:12.Subsequently,the smeared area of each slide was stained with cotton blue and examined for the counts of germinated and non-germinated conidia from three view fields at 400×magnification（＞100 conidia per field）.The ratio of the percent germination of the irradiated conidia over the control germination was defined as the survival index（I_s）.The I_s-D relationship observed from the stress bioassay of each isolate fit well the logistic survival equation I_s=1/[1+exp（a+bD）]（r²≥0.97）.The median lethal doses(LD₅₀) of the UV-B irradiation estimated from the fitted equations for the tested isolates fell in a range of 0.05-0.65 J/cm².The most UV-B tolerant isolate was Maac 5734, followed by Mfm 3606 and Mfm 3341.However,none of the tested isolates could survive a cumulative UV-B dose of 2.439 J/cm² measured from the sunlight of a sunny day of summer in Hangzhou.In vitro responses of 24 Metarhizium isolates to UV-A irradiation.The same stress bioassay method was adopted to assess the in vitro responses of 24 Metarhizium isolates to the irradiation of the weighted UV-A wavelength of 365 nm at the doses of 1.0-18.0 J/cm². Based on the I_s-D relationships well fitted to the survival equation（r²≥0.97）,the LD₅₀ estimates for the UV-A tolerances of the tested 24 isolates spanned from 2.78 to 10.46 J/cm². The three UV-B tolerant isolates appeared on the tope five list of the UV-A tolerance. However,the LD₅₀ for the most UV-A tolerant isolate（Mfm 3606） was far below on the UV-A dose of 20 J/cm² in accumulated in a full day of Beirut under clear-sky conditions.In vivo responses of two selected isolates to three UV-B doses.Two ARSEF isolates （Bb 2860 and Ma 759） with high biocontrol potential against aphids and spider mites were selected to assay the effects of three UV-B doses on the performance of green peach aphid Myzus persicae apterae sprayed with the spore suspension of 1×10⁸ conidia/ml in 0.02% Tween-80.Batches of 30-40 aphids on a cabbage leaf disc were separately sprayed with 1 ml spore suspension of the same concentration in an automatic Potter Spray and then exposed to the UV-B irradiations expected to cause 10%,50%and 90%viability losses, respectively.The three UV-B doses determined by the fitted I_s-D relationship for each isolate were 0.13,0.30 and 0.65 J/cm² for Ma 759,and 0.03,0.45 and 0.86 J/cm² for Bb 2860,respectively.After irradiation,all treated aphids on leaf discs were reared for 8 days in Petri dishes at 25℃and L:D 12:12 and monitored daily for mortality records.Mycosed cadavers were incubated for 2-3 days in moist chambers for fungal outgrowths.Treatments with the same fungal spray but no irradiation or with neither spray nor irradiation were included as controls for both isolates.All the assays were repeated three times.As a result, the tested UV-B doses significantly reduced the corrected mortalities of the aphids sprayed either with Bb 2860 or with Ma 759 but had no significant influence on the percentages of mycotized cadavers.Compared to the fungal treatment not irradiated,the low,medium and high UV-B doses reduced the aphid mortalities of Bb 2860 by 13.3%,58.9%and 82.3%, and of Ma 759 by 18.1%,54.0%and 88.7%,respectively.Overall mean（±SD） spray-to death periods of the aphids mycosed by Bb 2860（n=280） and Ma 759（n=257） at the low, medium and high UV-B does were estimated as 3.89±0.73 and 4.53±1.41 days.The UV-B effect on the mortality of M.persicae was proven to depend upon the irradiated dose.Conclusively,our results highlight a wide range of inter- and intra-specific variations in conidial tolerance to either UV-B or UV-A irradiation and thus the merits and feasibilities of choosing UV-tolerant candidates for fungal formulations based on their dose-survival responses in vitro,which are best represented by the absolute LD₅₀ estimates.The dose dependence of the UV-B effect on fungal infection in vivo supports photoprotection measures to reducing solar UV-B damages to fungal formulations in the field.These results would increase our knowledge on the stress biology of the fungal biocontrol agents and suggest a strategy for proper application of fungal formulations for improved field efficacy by spraying them at evening time to avoid solar UV irradiation for the first half day.更多还原