观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

观察球孢白僵菌侵染烟粉虱若虫过程的新方法——荧光显微法

【目的】介绍一种观察白僵菌Beauveria bassiana侵染烟粉虱Bemisia tabaci(Gennadius)若虫的荧光显微方法。【方法】将被白僵菌侵染的烟粉虱若虫用荧光素二乙酸酯(FDA)染色,并于波长为450~490 nm的蓝光下显微观察。【结果】在接种白僵菌12 h和24 h的烟粉虱若虫上分别观察到昆虫表皮上分生孢子的萌发和芽管穿透表皮。结合裸眼和荧光显微观察结果证实了被真菌侵染的烟粉虱若虫体色变红和菌体在虫体内增殖是同时发生的。【结论】应用荧光显微方法能观察到白僵菌在烟粉虱若虫体表和体内的侵染。     

二化螟幼虫病原真菌的分离纯化及其对烟粉虱和褐飞虱的毒力研究

通过对罹病二化螟Chilo suppressalis (Walker)幼虫病原真菌的分离纯化,筛选出5种不同真菌,探究不同营养对不同真菌生物活性的影响,并测定其对烟粉虱Bemisia tabaci (Gennadius)和褐飞虱Nilaparvata lugens (Stal)成虫和若虫的室内毒力。选择毒力最强的菌株,通过生物型的初步鉴定,明确高毒力菌株的种类。结果表明：所筛选的5种真菌对两种害虫均具有较高的毒力,其中菌株A26和Bbr14对烟粉虱和褐飞虱具有较强的毒力,在处理后第9 d,菌株A26对烟粉虱成虫和若虫的累计校正死亡率均最高分别达84.69%、77.62%,感染率为91.17%、88.24%,LT50为4.31 d、4.87 d;处理后第10 d对褐飞虱成虫和若虫的累计校正死亡率达87.54%、73.18%,感染率为90.13%、87.13%,LT50为4.27d、5.14 d,而菌株Bbr14的效果仅次于A26。鉴定的结果显示菌株A26为曲霉,菌株Bbr14为球孢白僵菌,曲霉的安全性有待研究,球孢白僵菌的应用技术已较成熟,有望能在防治这两类重要害虫中发挥作用。     

球孢白僵菌不同碳氮源的研究及对烟粉虱的时间-剂量-死亡率模型分析

【目的】明确不同碳、氮源对球孢白僵菌Beauveria bassiana生长的影响,确定适合该菌株生长的碳、氮源;通过研究球孢白僵菌对烟粉虱Bemisia tabaci的致病力,评价球孢白僵菌防治烟粉虱的潜力。【方法】在培养基中添加不同碳、氮源,研究其对球孢白僵菌生长速率和产孢量的影响,用浸叶法研究不同孢子浓度(1×10~4、1×10~5、1×10~6、1×10~7、1×10~8个/mL)的球孢白僵菌对烟粉虱的致病力并利用时间-剂量-死亡率模型对数据进行拟合,分析该模型的时间效应和剂量效应。【结果】蔗糖作为碳源、酵母作为氮源时球孢白僵菌的生长速率快和产孢量高;利用时间-剂量-死亡率模型对测定的数据进行拟合分析得出,随着接种天数的增加,球孢白僵菌的LC_(50)、LC_(90)逐渐降低,剂量效应增强;随着孢子浓度的增加,LT50逐渐降低,时间效应增强。【结论】球孢白僵菌的合适碳源为蔗糖,最适氮源为酵母。该菌株对烟粉虱有较强致病力,1×10~8个/mL的孢子浓度可作为防治烟粉虱的理想浓度,本试验为球孢白僵菌对烟粉虱的生物防治提供有力依据。     

球孢白僵菌HFW-05对烟粉虱的致病力及组织病理影响

环境温湿度是影响白僵菌菌株致病力的关键因素,本文测定了不同温、湿度下白僵菌Beauveria bassiana HFW-05对烟粉虱Bemisia tabaci若虫及卵的致病力,同时观察了烟粉虱感病若虫的组织病理变化。结果表明,各浓度下烟粉虱若虫的校正死亡率随时间变化曲线基本一致,接种3-4 d时出现死亡高峰,白僵菌HFW-05对烟粉虱1龄、2龄、3龄若虫的LC_(50)分别为5.94×10~4、1.06×10~5、5.08×10~5孢子/mL,LT50分别为2.54 d、2.68 d、3.18 d;白僵菌HFW-05对卵的致病力很低,1×108孢子/mL接种浓度下,卵的6 d感染率仅为24.9%。在25℃、30℃下,HFW-05对烟粉虱若虫表现出最强的致病力,高于或低于此温度时,烟粉虱若虫死亡速度减慢,死亡率降低。相对湿度发生变化时,烟粉虱若虫死亡速度和死亡率明显不同,相对湿度高于90%时,烟粉虱若虫死亡速度最快,死亡率最高,相对湿度介于70%-90%时,白僵菌HFW-05对烟粉虱若虫的致病力无明显差异,但校正死亡率显著低于环境湿度大于90%的处理,相对湿度低于70%时,烟粉虱死亡率也达到60%左右。组织病理学观察显示尾部舌状突是HFW-05侵染烟粉虱若虫的关键部位。研究结果表明白僵菌HFW-05在烟粉虱的生物防治中有较大的潜力。     

Virulence of Metarhizium flavoviride 82 to different developmental stages of the brown planthopper, Nilaparvata lugens (Hemiptera:Delphacidae)

对水稻重要害虫褐飞虱Nilaparvata lugens目前市场上尚无一种理想的微生物杀虫剂.昆虫病原真菌具有从体壁侵入的能力因而对刺吸性害虫的防治具有优势.为此,本研究选用不同原寄主和来源地的3种昆虫病原真菌(金龟子绿僵菌Metarhizium anisopliae、黄绿绿僵菌Metarhizium flavoviride和球孢白僵菌Beauveria bassiana)的12个不同菌株,以1 100孢子/mm2孢子悬浮液进行室内毒力测定.结果表明:在参试的不同菌种12个菌株中,黄绿绿僵菌Mf82菌株对褐飞虱成虫致病力最高,10 d累计校正死亡率为83.5％,致死中时(LT50)为4.6d.其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力,毒力大小顺序为成虫＞高龄若虫＞低龄若虫.黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、卵粒均有侵染作用,10d侵染率分别为66.7％和51.2％,卵龄越低,侵染效果越好,卵龄为0.5d时侵染率最高.本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、若虫和卵均有较强的致病性,是一株极具应用潜力的生防真菌.     

黄绿绿僵菌Mf82菌株对不同虫态褐飞虱的毒力

对水稻重要害虫褐飞虱Nilaparvata lugens目前市场上尚无一种理想的微生物杀虫剂。昆虫病原真菌具有从体壁侵入的能力因而对刺吸性害虫的防治具有优势。为此, 本研究选用不同原寄主和来源地的3种昆虫病原真菌（金龟子绿僵菌Metarhizium anisopliae、 黄绿绿僵菌Metarhizium flavoviride和球孢白僵菌Beauveria bassiana）的12个不同菌株, 以1 100孢子/mm²孢子悬浮液进行室内毒力测定。结果表明： 在参试的不同菌种12个菌株中, 黄绿绿僵菌Mf82菌株对褐飞虱成虫致病力最高, 10 d累计校正死亡率为83.5%, 致死中时（LT₅₀）为4.6 d。其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力, 毒力大小顺序为成虫>高龄若虫>低龄若虫。黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、 卵粒均有侵染作用, 10 d侵染率分别为66.7%和51.2%, 卵龄越低, 侵染效果越好, 卵龄为0.5 d时侵染率最高。本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、 若虫和卵均有较强的致病性, 是一株极具应用潜力的生防真菌。     

锌离子响应转录激活因子ZafA对球孢白僵菌锌离子利用及生防潜能的影响

球孢白僵菌作为丝孢类昆虫病原真菌,已广泛应用于农林害虫的生物防治,但是田间多变的环境影响了真菌制剂的效能。此外,真菌侵染宿主后,宿主体内的环境也影响真菌的增殖和侵染速度。为研究球孢白僵菌对环境中酸碱度及微量元素的平衡能力,本研究初步探讨了锌离子响应转录激活因子ZafA与真菌生长繁殖、抗逆能力、毒力以及锌离子利用的关系。结果表明,敲除zafA削弱了真菌生长繁殖和孢子萌发的能力,增加了菌株对氧化、高渗、孢壁干扰剂以及紫外胁迫的敏感性,杀虫毒力显著下降,并抑制了相关性状基因的表达水平。基因敲除菌株无法在锌离子缺失的条件下生长,且zafA基因和锌离子转运蛋白编码基因zrf1–8的表达水平会受到酸碱度以及锌离子浓度的影响。由此可见,ZafA不仅直接影响球孢白僵菌利用锌离子的能力,还与球孢白僵菌抗逆能力和毒力密切相关,本研究为提高生防真菌环境适应性和发挥毒力提供新的依据。     

球孢白僵菌对四种十字花科蔬菜害虫的兼控潜力评价

研究了分离自小猿叶甲的一株球孢白僵菌(Beauveria bassiana(SCAU-BB01D))对十字花科蔬菜4种非目标害虫烟粉虱Bemisia tabaci、小菜蛾Plutella xylostella、黄曲条跳甲Phyllotreta striolata和菜缢管蚜Lipaphis erysimi的致病能力。结果表明,在实验室条件下,该菌株对以上4种害虫均有不同程度的侵染力。1×108孢子/mL的白僵菌悬浮液对1,2,3,4龄烟粉虱若虫的侵染率分别为84.88,86.81,55.94和38.78%;对小菜蛾2,3和4龄幼虫的侵染率为67,59和44%;对黄曲条跳甲幼虫和成虫分别为63%和60%;对菜缢管蚜成蚜和若蚜的侵染率分别为44%和35%。通过机率值分析,得出该菌株对烟粉虱1~4龄若虫的LT50分别为4.14,3.78,6.24和7.59 d;对小菜蛾2~4龄幼虫的LT50分别为7.29,8.16和9.82d;对黄曲条跳甲成虫和幼虫的LT50分别为11.22和8.11 d;对菜缢管蚜成蚜和若蚜的LT50分别为11.01和12.15 d。说明该球孢白僵菌菌株对烟粉虱、小菜蛾和黄曲条跳甲3种非目标害虫具有一定的兼控作用,在蔬菜害虫的生物防治中具一定的应用潜力。     

几种土壤真菌对烟粉虱的生物活性研究

B型烟粉虱Bemisia tabaci是重大入侵害虫,发展生物防治技术控制其危害具有重要意义。本研究采用浸渍法测定了9种20株土壤真菌对B型烟粉虱的室内活性,结果表明,供试菌株对B型烟粉虱都有一定致病活性,1.0±10^8 孢子/mL的分生孢子悬浮液处理10 d后,烟粉虱2龄若虫的累计校正死亡率为53%~93%,其中淡紫紫孢菌Purpureocillium lilacinum PITS01、PlMS-12和PlMS-15菌株,以及刀孢蜡蚧菌Lecanicillium psalliotae LpTS01菌株引起B型烟粉虱91%以上的死亡率。进一步测定了PITS01和LpTS01菌株对烟粉虱的毒力,结果表明PITS01和LpTS01对烟粉虱2龄若虫在处理第4天的LC50分别为2.8×10^7 孢子/mL和3.6×10^7 孢子/mL,第6天分别为1.7×10^6 孢子/mL和3.6×10^5 孢子/mL;当孢子浓度为1.0×10^7 孢子/mL时,PITS01和LpTS01菌株LT50值分别为4.4 d和4.2 d。菌株PITS01和LpTS01对烟粉虱防治具有一定的应用潜力。     

Responses of diamondback moth to diverse entomopathogenic fungi collected from non-agricultural habitats – Effects of dose,temperature and starvation

We evaluated the virulence of Beauveria bassiana and Metarhizium isolates from soil collected across different vegetation types in Queensland, against chlorantraniliprole-resistant and insecticide-susceptible diamondback moth (DBM) larvae. Host insecticide resistance status had no effect on susceptibility to the pathogens when conidia were topically applied to larvae in the laboratory, and one B. bassiana isolate was significantly more virulent to larvae than the others (seven days after inoculation). The influence of temperature (15, 20, 25 or 30 °C): (i) at the point of host inoculation with conidia and (ii) when the pathogens had already initiated infection and were proliferating in the host haemocoel, was determined experimentally for its influence on virulence, disease progression, and sporulation. Temperature at inoculation had a greater effect on host insect mortality than it did when the fungus was already proliferating in the host haemocoel. The rearing temperature of hosts prior to inoculation had a greater effect on host susceptibility to disease than starvation of the larvae at the time of inoculation. Our results also show that each fungal isolate has its own temperature relations and that these can vary considerably across isolates, and at different points in the pathogen life cycle (germination and cuticular penetration versus growth in the host haemocoel). Temperature also had an idiosyncratic effect, across isolates and across the variables typically used to assess the potential of fungal entomopathogens as biological control agents (time to death, mortality and sporulation rates). This study demonstrates that in addition to pathogenicity and virulence, the temperature relationships of each fungal isolate when infecting insects needs to be taken into account if we are to understand their ecology and use them effectively in pest management.     

Effect of temperature on virulence of Beauveria bassiana and Metarhizium anisopliae isolates to Tetranychus evansi

The virulence of three isolates of Beauveria bassiana (Bals.) Vuill. and 23 isolates of Metarhizium anisopliae (Metschnik.) Sorok. (Ascomycota: Hypocreales) against the tomato spider mite, Tetranychus evansi Baker and Pritchard (Acari: Tetranychidae), was assessed in the laboratory. The effect of temperature on germination, radial growth and virulence of selected isolates (two isolates of B. bassiana and nine of M. anisopliae) on T. evansi was also investigated in the laboratory. All the fungal isolates tested were pathogenic to the adult females of T. evansi, and there were significant differences in mortality between fungal isolates. The lethal time to 50% mortality (LT(50)) values ranged from 4.2 to 8.1 days and the LT(90) values from 5.6 to 15.1 days. Temperature had significant effects on germination, radial growth and virulence of the various isolates. The best fungal germination was observed at 25 and 30 degrees C, while for the fungal radial growth it was 30 degrees C. All the isolates germinated and grew at all temperatures, but germination and radial growth varied with isolate and temperature. The selected isolates were all virulent to T. evansi, but virulence varied also with isolate and temperature.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

球孢白僵菌不同分离株对西花蓟马的毒力测定

【背景】西花蓟马是我国重要的一种入侵性害虫,已对多种化学农药产生了抗性。【方法】采用孢子悬浮液喷雾处理生测法,研究了8个球孢白僵菌菌株BbKM030716、BbCG051229、BbJS080625、BbQJ031121、BbXW060615、BbSM090521、BbYY090613和BbMZ051230对西花蓟马成虫的毒力,并用＂时间—剂量—死亡率＂模型进行了分析。【结果】供试球孢白僵菌各菌株对西花蓟马成虫侵染致病的剂量效应参数值分别为1.43、0.87、0.93、0.98、1.23、0.92、1.07和0.86。用1.25×104～1.25×108个·mL-1孢子悬浮液接种后,连续10d内西花蓟马的校正死亡率分别为44.13%～98.49%、12.63%～78.90%、30.36%～96.92%、51.36%～98.74%、26.14%～98.59%、7.27%～78.71%、49.06%～98.74%和27.67%～87.36%。球孢白僵菌对西花蓟马成虫的致死剂量是时间的函数,各菌株对西花蓟马成虫的致死中时随孢子浓度的增大而逐渐减小。【结论与意义】球孢白僵菌菌株BbKM030716、BbJS080625、BbQJ031121和BbXW060615对西花蓟马成虫具有较强毒力,可作为西花蓟马生防制剂开发的潜力菌株。     

胞外蛋白酶和脂酶活性作为球孢白僵菌毒力指标的可靠性分析

通过测定17株不同来源球孢白僵菌(Beauveria bassiana)的胞外蛋白酶活性(PU,μmol·ml~(-1)·min~(-1))和脂酶活性(LU,μmol·ml~(-1)·h~(-1))及其对血黑蝗(Melanoplussanguinipes)的毒力,分析用酶活性作为毒力指标的可靠性.结果表明,菌株间毒力和酶活性差异较大,LT_(50)的变化范围为5.27~16.89d,PU和LU分别为0.47~3.37×10~(-2)和0.00~56.75.将PU与LU分别对接种后各日累计死亡率及LT_(50)进行回归分析,发现各菌株PU与其毒力相关显著,但LU却与毒力相关不显著.对接种后7d的死亡率,PU可表达其变异的67%,也是最大表达率.因此,PU可作为大量菌株初筛的参考性毒力指标,但应谨慎使用,不能以PU测定完全取代常规的毒力测定.脂酶活性不宜作为所试菌种的毒力参考指标.     

Comparative growth kinetics and virulence of four different isolates of entomopathogenic fungi in the house fly (Muscadomestica L.)

Virulence (speed of kill) of a fungal entomopathogen against a particular host insect depends on biological properties of the specific isolate-host combination, together with factors such as fungal dose. How these intrinsic and extrinsic factors affect the actual pattern and extent of fungal growth invivo is poorly understood. In this study we exposed adult house flies (Muscadomestica L.) to surfaces treated with high and low doses of Beauveriabassiana (isolates BbGHA and Bb5344), Metarhiziumanisopliae (strain MaF52) and M.anisopliae var. acridum (isolate Ma189) and used quantitative real-time PCR with species-specific primers to examine the relationship between fungal growth kinetics and virulence. At the highest dose, all fungal isolates killed flies significantly faster than controls, with BbGHA, Bb5344 and MaF52 roughly equivalent in virulence (median survival time (±SE) = 5.0 ± 0.10, 5.0 ± 0.08 and 5.0 ± 0.12 days, respectively) and Ma189 killing more slowly (MST = 8.0 ± 0.20 days). At the lower dose, effective virulence was reduced and only flies exposed to isolates BbGHA and Bb5344 died significantly faster than controls (MST = 12 ± 1.36, 15 ± 0.64, 18 ± 0.86 and 21.0 ± 0.0 days for BbGHA, Bb5344, MaF52 and Ma189, respectively). Real-time PCR assays revealed that flies exposed to surfaces treated with the high dose of spores had greater spore pickup than flies exposed to the low dose for each isolate. After pickup, a general pattern emerged for all isolates in which there was a significant reduction of recovered fungal DNA 48 h after exposure followed by a brief recovery phase, a stable period of little net change in fungal sequence counts, and then a dramatic increase in sequence counts of up to three orders of magnitude around the time of host death. However, while the patterns of growth were similar, there were quantitative differences such that higher final sequence counts were recovered in insects infected with the most lethal isolates and with the higher dose. These results suggest that variation in virulence between isolates, species and doses is determined more by quantitative rather than qualitative differences in fungal growth kinetics.     

Laboratory Testing of Metarhizium spp. and Beauveria bassiana on Sahelian Non-target Arthropods

Laboratory experiments were conducted to investigate the host specificity of 11 isolates of Metarhizium sp. and one isolate of Beauveria bassiana , all deuteromycete entomopathogenic fungi that are being tested as potential biocontrol agents against locusts and grasshoppers. Metarhizium sp. (isolate IMI 330 189) was applied topically to Pimelia senegalensis and Trachyderma hispida, two Sahelian tenebrionid beetles. No infection was observed at a dose of ca. 2.5 x 106 conidia per insect, much higher than dose rates likely to occur in the field. The hymenopteran parasitoids Bracon hebetor (Braconidae) and Apoanagyrus lopezi (Encyrtidae) were exposed to surfaces treated with the entomopathogens at a rate equivalent to ca. 5 x 1012 spores ha-1, the recommended field rate against locusts. All 12 isolates tested resulted in 100% mortality of the parasitoids. Average survival times ranged from 1.6 to 4.1 days. In all cases there was fungal outgrowth on the cadavers. Exposure time-response experiments further confirmed the pathogenicity of these fungi to the parasitoids. The possibility of these parasitoids being affected in the field is discussed.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

Scaling up tests on virulence of the cassava green mite fungal pathogen <Emphasis Type="Italic">Neozygites tanajoae</Emphasis> (Entomophthorales: Neozygitaceae) under controlled conditions: first observations at the population level

Virulence of entomopathogens is often measured at the individual level using a single host individual or a group of host individuals. To what extent these virulence assessments reflect the impact of an entomopathogen on their host in the field remains largely untested, however. A methodology was developed to induce epizootics of the cassava green mite fungal pathogen Neozygites tanajoae under controlled conditions to evaluate population-level virulence of two (one Beninese and one Brazilian) isolates of the entomopathogen—which had shown similar individual-level virulence but different field impacts. In unrepeated separate experiments we inoculated mite-infested potted cassava plants with either 50 or 25 live mites (high and low inoculum) previously exposed to spores of N. tanajoae and monitored the development of fungal infections for each isolate under the same conditions. Both isolates caused mite infections and an associated decline in host mite populations relative to the control (without fungus) in all experiments, but prevalence of the fungus varied with isolate and increased with inoculum density. Peak infection levels were 90% for the Beninese isolate and 36% for the Brazilian isolate at high inoculum density, and respectively 17% and 25% at low inoculum density. We also measured dispersal from inoculated plants and found that spore dispersal increased with host infection levels, independent of host densities, whereas mite dispersal varied between isolates. These results demonstrate that epizootiology of N. tanajoae can be studied under controlled conditions and suggest that virulence tests at the population level may help to better predict performance of fungal isolates than individual-level tests.