不同温、湿度下白僵菌对棉铃虫幼虫的致病力

测试了不同温、湿度下白僵菌Beauveria bassiana对棉铃虫Helicoverpa armigera幼虫的致病力。实验设置了10、15、20、25、30℃ 5个温度水平,用高浓度（1×10<.sup8孢子/mL）、中浓度（1×10⁷孢子/mL）、低浓度（1×10⁶孢子/mL）的白僵菌孢子液分别感染1～4龄的棉铃虫。结果表明：三个浓度处理的棉铃虫在温度为25℃时的致死中时（LT₅₀）最短,死亡速度最快,死亡率最高;高于或低于此温度时,棉铃虫的LT₅₀延长,死亡速度减慢。相对湿度发生变化时,感病棉铃虫死亡速度和死亡率明显不同。相对湿度为95%左右时,棉铃虫死亡速度最快,死亡率最高;相对湿度低于70%时,棉铃虫死亡率显著降低。     

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

球孢白僵菌不同感染方式侵染棉铃虫幼虫的毒性比较及组织病理变化

为明确球孢白僵菌在不同感染方式下对棉铃虫的侵染能力, 采用饲喂法和浸渍法测定了球孢白僵菌HFW-05对棉铃虫Helicoverpa armigera (Hübner) 2龄幼虫的致病力, 并通过组织切片显微技术、 扫描电镜技术观察了球孢白僵菌HFW-05对棉铃虫的致病方式。结果表明: 白僵菌HFW-05可通过消化道（饲喂法）成功侵染2龄棉铃虫, 接种感染6 d后的校正死亡率为75.8%。经由体表（浸渍法）接种白僵菌HFW-05的试虫, 试验中体重的变化和取食量与对照相近（6 d校正死亡率仅为17.3%, 不能通过体表达到致病效果）。组织病理学变化表明: 26±1℃条件下, HFW-05菌株对棉铃虫以消化道侵染为主, 侵染后可导致寄主中肠微绒毛脱落严重, 肠壁组织溶解并最终只剩余底膜; 马氏管变形萎缩, 边缘向外突出隆起, 管径变大; 脂肪体萎缩解体, 结构松散; 表皮下的细胞被菌丝侵染破坏。浸渍法接种的试虫, 切片观察处理6 d后试虫, 体内未发现菌丝, 肠壁组织正常完整。扫描电镜观察, 浸渍法接种的分生孢子未能穿透棉铃虫表皮, 而是贴于寄主表皮表面生长, 在湿度合适的条件下, 菌丝生长到一定时间后断裂成为芽生孢子。白僵菌HFW-05可经由消化道对棉铃虫达到较高的致病效果, 在一定程度上弥补外界环境对白僵菌侵染的不利影响, 对今后应用白僵菌进行生物防治具有重要意义。     

Effects of conidial densities and spray volume of Metarhizium anisopliae and Beauveria bassiana fungal suspensions on conidial viability,droplet size and deposition coverage in bioassay using a novel bioassay spray system

A newly developed spray tower was used to characterise droplet distribution and coverage of conidial suspensions of Metarhizium anisopliae ATCC 62176 and Beauveria bassiana NI8 with different spray volumes. ATCC 62176 and NI8 had different spray models which could be caused by the surface physicochemical characteristics of the strains and conidia.     

一株白僵菌对红火蚁的毒力及对免疫酶活性与基因表达的影响

白僵菌是最具防治害虫潜力的一类昆虫病原真菌。本研究测定了球孢白僵菌Beauveria bassiana 237菌株对红火蚁Solenopsis invicta的感染毒力,并探究了白僵菌侵染对红火蚁免疫相关酶活性及相关基因表达的影响。结果显示,该白僵菌菌株对红火蚁的毒力较强,在孢子悬浮液108孢子/mL浓度下,对红火蚁的致死中时间LT50为5.288±0.2014 d;在10^4~10^8孢子/mL的不同浓度处理下,红火蚁死亡率随孢子浓度的增加而显著增加。经计算,第4~10天致死中浓度LC50值由8.82×10^6孢子/mL降低到8.95×10^5孢子/mL。红火蚁被球孢白僵菌感染后,体内保护酶和解毒酶发生了不同程度的改变。其中保护酶类酚氧化酶（PO）的活性在白僵菌处理后的第12 h已出现抑制,而在第24 h、48 h、72 h时均显著高于对照;超氧化物歧化酶（SOD）活性在12 h时与对照无显著差异,但在24~48 h阶段酶活显著上升,之后下降;过氧化物酶（POD）在较早时段保持与对照无显著差异水平,至中后期48~72 h出现持续升高。解毒酶类混合功能氧化酶系（MFO）的活性在整个检测时间段内表现为抑制-上升-抑制-上升的波动状态;谷胱甘肽S-转移酶（GSTs）活性的变化与SOD相似,只在24~48 h阶段出现上升。白僵菌还导致红火蚁免疫信号通路Toll途径相关基因表达量变化。在处理后12 h,识别因子GNBP1、Spaetzle即被激活,维持上调-回调波动趋势;而信号传递因子Myd88、pelle在检测的12~72 h内基本处于被抑制状态,只有Myd88在48 h时表达量上升;转录因子Dorsal以及抗菌肽Defensin在12~24 h都已被显著激活,而在后续48~72 h被抑制。综上所述,球孢白僵菌237菌株通过调节红火蚁酶活以及免疫相关基因的表达量实现成功侵染和致病作用,具有很高的生防应用价值。     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the tarnished plant bug, Lygus lineolaris L., in strawberries

Beauveria bassiana has a high insecticidal potential to control the tarnished plant bug, Lygus lineolaris, a significant pest of strawberries. Screening experiments showed that L. lineolaris adults were susceptible to several B. bassiana isolates. Another screening test with Coleomegilla maculata, a natural enemy found in strawberries, was also performed in order to select the isolate having lower entomopathogenic impact on this insect. Based on data obtained from both insect species and on the ecozone origin of the B. bassiana isolates, INRS‐IP and INRS‐CFL isolates were selected for further experiments. The LC₅₀ values of these two isolates against L. lineolaris adults were 7.8 × 10⁵ and 5.3 × 10⁵ conidia/ml, and average survival time (AST) values were 4.46 and 4.37 days at a concentration of 1 × 10⁸ conidia/ml respectively. Results also indicated that L. lineolaris nymphs are susceptible to the selected isolates. During field experiments, using a randomized block design with four replicates, INRS‐IP and INRS‐CFL isolates were applied at two rates (1 × 10¹¹ and 1 × 10¹³ conidia/ha) weekly during a period of 4 weeks. These multiple applications triggered a significant reduction of L. lineolaris nymphal populations in strawberries. Twenty‐four days after the first application, a significant difference was observed between the mean population densities of surviving nymphs in all B. bassiana‐treated plots (less than one insect per five plants) compared with those in control plots (four insects per five plants). During the field experiment, persistence of insecticidal activity and viability of B. bassiana conidia were also monitored. The results showed the presence of viable and infective conidia up to 6 days after each application on strawberry foliage. Moreover, the multiple applications of B. bassiana at the rate of 1 × 10¹³ conidia/ha triggered a significant reduction in strawberry fruit injuries induced by L. lineolaris feeding behaviour compared with the control plots.     

Effect of culture substrates on virulence of Beauveria bassiana (Ascomycota: Cordycipitaceae) conidia against the browntail moth,Euproctis chrysorrhoea (Lepidoptera: Lymantriidae)

We studied the effect of different solid substrates on virulence of two Beauveria bassiana isolates against the browntail moth, Euproctis chrysorrhoea (L.) (Lep.: Lymantriidae). Conidia produced on wheat grains, wheat flour, wheat bran, rice flour, rice bran, rice paddy, corn flour, millet, and Sabouraud's dextrose agar with 1% yeast extract (SDAY) as control were compared. There were significant differences among these substrates for their effects on the virulence of produced conidia. Applying 10⁷ conidia/mL of B. bassiana EUT105, produced on rice bran caused the highest (84.9%) and on rice flour, the lowest (57.6%) mortalities. Bioassay on fifth-instar larvae using aerial conidia harvested from wheat grains, rice paddy, and SDAY indicated that conidia from wheat grains had the highest virulence while those from rice paddy, the lowest.     

Efficacy of Beauveria bassiana against the strawberry pests, Lygus lineolaris, Anthonomus signatus and Otiorhynchus ovatus

There are several insect species causing serious economic losses in strawberry, Fragaria vesca L., productions. In Quebec, Canada, the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), the strawberry bud weevil clipper, Anthonomus signatus (Say) and the strawberry root weevil, Otiorhynchus ovatus (L.) are the most important pests. We tested the susceptibility of these pests to the entomopathogenic fungus Beauveria bassiana under laboratory conditions. Sixteen isolates were evaluated for their insecticide potential against these insects. Adults of each species were infected by the immersion method. All isolates were pathogenic to adults of all three species, causing mortality rates between 23.3% and 100% at a concentration of 1 × 10⁷ conidia/ml. Based on the screening results, isolate INRS‐CFL was selected for its insecticide potential and then used for further analyses against L. lineolaris, A. signatus and O. ovatus adults. Bioassays were performed to evaluate the lethal concentration (LC₅₀) and the average survival time (AST) of this isolate against both insect species. Results of dose–response mortality bioassays using four concentrations – 1 × 10⁴, 1 × 10⁶, 1 × 10⁸ and 1 × 10⁹ conidia/ml – indicated a LC₅₀ values of 5.3 × 10⁵, 1.8 × 10⁷ and 9.9 × 10⁷ conidia/ml at 7 days after inoculation for L. lineolaris, A. signatus and O. ovatus respectively. Using a dose of 1 × 10⁸ conidia/ml, the AST values were estimated at 4.41, 7.56 and 8.29 days, respectively, at a concentration of 1 × 10⁸ conidia/ml. This study demonstrated the potential of B. bassiana for the management of L. lineolaris, A. signatus and O. ovatus. Results also suggest that the heteropteran species is more susceptible than coleopteran species to B. bassiana.     

Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil,Rhynchophorus ferrugineus (Olivier)

Nineteen different isolates of the entomopathogenic fungi Beauveria bassiana sensu lato (s.l.) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers were tested against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) at two different spore concentrations (1 × 10⁷ and 1 × 10⁸ conidia mL^?1). Three isolates of B. bassiana and two of M. anisopliae gave >88 % larval and >75 % adult mortality of R. ferrugineus on their highest dose rate respectively. More sporulating cadavers (mycosis) resulted from a high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana and M. anisopliae isolates with mortality levels usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers gave more mortality compared to the other sources. In the virulence assay two isolates of B. bassiana caused the highest percentage of both larval and adult mortality at all exposure intervals which suggest that they may be the most promising for use in sustainable management programs aimed at microbial control in date palm orchards.     

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。     

Mycelia and spent fermentation broth of Beauveria bassiana incorporated into synthetic diets affect mortality,growth and development of larval Helicoverpa zea (Lepidoptera: Noctuidae)

Beauveria bassiana endophytically colonises corn (Zea mays) reducing tunneling from European corn borer (Ostrinia nubilalis). Endophytic colonisation of other plants by B. bassiana has been reported, and potentially, may reduce insect feeding on these plants. We evaluated the effects on larval growth and development, and mortality of different rates of dried, ground mycelia and water-soluble metabolites from fermentation broth culture of different isolates of B. bassiana incorporated into a synthetic diet and fed to neonate bollworm, Helicoverpa zea larvae. Development was delayed, weights of larvae were lower, and mortality was high for larvae fed the highest rates (1.0 and 5.0%, w/v) of mycelia incorporated diet compared to control. Insects fed diets containing mycelia of B. bassiana isolate 11-98 had the greatest mortality. Mortality was 100% for larvae fed 5% (w/v) mycelia incorporated diet of isolate 11-98, and 61% for isolate 3-00. For insects fed low rates (0.1 to 0.5%, w/v) of mycelia incorporated diet, mortality was lower, approximately 5% for isolate 11-98, and 5 to 14% for isolate 3-00. At the 0.1% (w/v) rate of mycelia incorporated diet, development occurred at an accelerated rate, compared to fungus-free controls, indicating increased nutrition in the lowest rate fungal diet. Mortality was low for all larvae fed diets containing spent fermentation broth of B. bassiana; however, development was delayed. Insects fed the highest rate (0.5%, v/v) of spent fermentation broth-amended diet had lower pupal weights, and a greater number of days to pupation than insects fed the lowest (0.1%, v/v) rate. Insects fed the 5% (v/v) rate of spent fermentation broth of isolates 11-98 and 3-00 had the longest days to pupation.     

Viability of cotton and canola pollen on the proboscis of Helicoverpa armigera: implications for spread of transgenes and pollination ecology

Abstract.  1. Pollen can be transported thousands of kilometres by insects but its viability after long-distance transport is not known. Knowing the potential for this mechanism to cause outcrossing of transgenes from genetically modified (GM) plants is important for risk assessments.
2. The viability of pollen from cotton ( Gossypium hirsutum L.) and canola ( Brassica napus L.) was determined after placing it on the proboscis of Helicoverpa armigera moths for intervals of up to 32 h. Viability of both cotton and canola pollen declined at a much greater rate when in contact with the moth proboscis. Most was non-viable by 8 h compared with 16 h for control cotton pollen or 32 h for canola pollen.
3. There was no significant difference in the rate of decline of pollen viability between the five conventional cotton varieties, or between these and the one GM cotton variety used in these experiments.
4. The number of canola pollen grains remaining on the proboscis declined over time. Very few cotton pollen grains were retained on the proboscis.
5. The reduction in pollen viability during contact with the proboscis might indicate partial ingestion of the pollen via the proboscis.
6. The points above suggest that pollen is unlikely to remain attached or remain viable when carried over large distances by H. armigera . The implications for spread of pollen from transgenic plants and for pollination ecology in general are discussed.     

Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

不同温湿度下球孢白僵菌对小猿叶甲的致病力

本文在室内研究了分离自小猿叶甲的一株球孢白僵菌(SCAU-BB01D)在不同温度和湿度条件下对小猿叶甲成虫和2龄幼虫的致病力。结果显示温度和相对湿度对白僵菌的致病力有显著影响。在17℃和29℃,小猿叶甲成虫和2龄幼虫的死亡率显著低于其他温度; 在23℃下,成虫接菌后第14天的累计死亡率达到最高为95.60%; 在23℃和26℃下,2龄幼虫接菌后第10天达到最高累计死亡率96%。随着相对湿度的提高,球孢白僵菌对小猿叶甲的致病力显著增加,当相对湿度从50%增加至100%时, 成虫第14天的累计死亡率从55.70%增加到88.80%,2龄幼虫第10天的累计死亡率从62.00%增加到96.00%。机率值分析结果表明,在17、20、23、26和29℃温度下,白僵菌对小猿叶甲成虫和2龄幼虫的LT₅₀分别为 13.63、10.27、8.05、8.87、12.41天和 6.06、5.72、4.90、4.86、6.73天。随着相对湿度的升高,球孢白僵菌对小猿叶甲的致死中时(LT₅₀)呈缩短的趋势,当相对湿度为50%、60%、70%、80%、90%、100％时,成虫和2龄幼虫的LT₅₀分别为12.97、10.97、10.13、9.79、9.14、8.29天和7.68、6.92、5.76、5.57、5.48、4.82天。由实验结果可以得知,23～26℃是球孢白僵菌感染小猿叶甲最适宜的温度范围,环境湿度越高越有利于病原菌侵染,当相对湿度大于90%时,最有利于球孢白僵菌对小猿叶甲的感染。     

Potential effect of Beauveria bassiana (Hypocreales: Clavicipitaceae) on Anthonomus signatus (Coleoptera: Curculionidae) in strawberries

The strawberry bud weevil, Anthonomus signatus (Say) (Coleoptera: Curculionidae) is a serious pest of strawberry crops in Canada. The results of previous laboratory screening and bioassay experiments showed that A. signatus adults were susceptible to several isolates of Beauveria bassiana (Balsmo) Vuillemin. Based on these results, the INRS-IP and INRS-CFL isolates were selected for field trails. Using a randomized block design with four replicates, three applications of the INRS-IP and INRS-CFL isolates were applied at a rate of 1×10¹³ conidia/ha at 7-day intervals. The multiple applications of B. bassiana triggered a significant reduction of A. signatus adult populations in strawberries. Twenty-four days after the first application, a significant difference was observed between the mean numbers of surviving adults in all B. bassiana-treated plots compared to control plots. During the field experiment, the persistence of insecticidal activity and the viability of B. bassiana conidia were also monitored. Results showed the presence of viable and infective conidia on strawberry foliage up to 6 days after each application. Moreover, the multiple applications of B. bassiana at a rate of 1×10¹³ conidia/ha resulted in a significant reduction of damaged strawberry fruits compared with control plots, where fruit loss was caused by A. signatus feeding and egg laying.     

Effects of inoculation method on efficacy of wettable powder and oil dispersion formulations of Beauveria bassiana against Colorado potato beetle larvae under low-humidity conditions

The effects of inoculation method on efficacy of two formulations of Beauveria bassiana strain GHA against Colorado potato beetle larvae were investigated. Under dry greenhouse conditions, ca. 58% mortality was observed among second-instar larvae exposed directly to sprays of B. bassiana conidia, whereas mortality among larvae exposed to similarly treated foliage (either leaf dorsal or ventral surfaces) was <10%. Mortality was ca. 64% among larvae exposed to both direct sprays and foliar spray deposits. Equivalent rates of mortality were observed among larvae treated with a clay-based wettable powder versus an emulsifiable oil-based formulation of B. bassiana conidia; however, this was observed despite application of an approximately 40% greater dose of WP-formulated conidia, indicating greater efficacy of the emulsifiable oil formulation. These results suggest that, under dry conditions, potato beetle larvae do not readily acquire an effective dose of conidia from treated foliage and that development of improved application technologies to more effectively target the larvae may ultimately prove more beneficial than development of formulations with greater foliar persistence.     

不同温度下球孢白僵菌对桃蚜生殖力及生命表参数的影响

对6日龄桃蚜Myzus persicae(Sulzer)用106孢子/mL球孢白僵菌Beauveria bassiana(Balsamo)Vuillemin悬浮液接种后,饲养于4组温度(18,21,25和28℃)下,相对湿度(RH)为85%的条件下,观察其生殖力及生命表参数的变化。结果显示,与对照相比,在观察时限的10d内接种蚜日均产仔数分别减少43%,31%,53%和61%,接种蚜的净生殖力R0下降57%,73%,74%,60%,内禀增长力rm下降30%,41%,36%,22%。这表明,球孢白僵菌对桃蚜种群增长具有一定的抑制作用,其作用在21、25℃下更为明显。     

球孢白僵菌在红火蚁体表侵染的扫描电镜观察

利用扫描电镜观察了球孢白僵菌Beauveria bassiana Bb04菌株分生孢子对红火蚁Beauveria bassiana 工蚁体壁的侵染过程。结果表明: 分生孢子多分布在红火蚁工蚁节间膜、胸部的褶皱、气门、体壁的凹陷部位、刚毛窝附近, 以及着生较密刚毛的足上。萌发的分生孢子在节间膜以及体表缝隙、刚毛窝及刚毛稀少的凹陷部位、胸部褶皱和足胫节处入侵。分生孢子在附着12 h后开始萌发, 接种后18 h附着在节间膜处的孢子首先侵入成功, 接种后24 h刚毛窝附近孢子萌发入侵, 接种后60 h胸、腹和足等部位的孢子均成功穿透侵入表皮。分生孢子可以直接以芽管侵入表皮, 也可以产生附着胞再侵入。