Species confirmation of fungal isolates by molecular analysis

Traditional taxonomy of hyphomycetes has been based on conidial morphology and development. In order to confirm species level for the detection and identification of the entomopathogenic fungus, we analysed the species-specific fingerprints to investigate molecular characteristics within isolates of six species and to resolve morphologically atypical isolates. The extent of fingerprint profile observed by RAPD was sufficient to confirm the species level of all the isolates. The genetic similarity among morphologically identified isolates of each species was considerably higher, allowing us to conclude that all the isolates are of same species. These results establish a molecular framework for further taxonomic, phylogenetic and comparative biological investigations.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

House flies delay fungal infection by fevering: at a cost

1. Many ectothermic species have evolved the ability to invoke a ‘behavioural fever’ when infected with a pathogen. The relative costs and benefits of this response, however, have rarely been quantified. 2. The aim of this study was investigate the nature and consequences of behavioural fever in the house fly, Musca domestica L., in response to infection with a possible biocontrol agent, the fungal entomopathogen, Beauveria bassiana (Balsamo) Vuillemin. 3. It was found that infected flies preferred higher temperatures and allocated more effort to thermoregulation than uninfected flies. Flies could not overcome infection but the altered thermal behaviour allowed infected flies to extend their survival and to lay more eggs relative to infected flies maintained under constant conditions. However, flies allowed to fever had lower egg viability suggesting a possible cost. 4. Under the present experimental conditions, the putative costs and benefits fever balanced one another resulting in no net change in fitness. Fever did not, therefore, limit the control potential of the fungus. We discuss whether the costs and benefits of behavioural fever might differ in other ecological contexts.     

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。     

Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in coffee seedlings grown in vitro and inoculated with B. bassiana suspensions in the radicle. The fungus was recovered as an endophyte 30 and 60 days postinoculation, from stems, leaves, and roots, and at 60 days postinoculation one of the isolates was also recovered as an epiphyte. Fusarium sp., Rhodotorula sp., and four bacterial morpho-species were also detected, indicating these were present as endophytes in the seed.     

Impact of moisture and UV degradation on Beauveria bassiana (Balsamo) Vuillemin conidial viability in turfgrass

Beauveria bassiana conidial viability in turfgrass was evaluated using a two-component nucleic acid stain and fluorescence microscopy. Turfgrass samples along with the top 5 cm of soil were used for conidial extraction and viability evaluation on 1, 2, 3, 7, 14 and 21 days after treatment. There were no differences in conidial viability between two Orthoptera strains, 3622 and 5977, and both strains were able to persist in a sandy loam soil for up to three weeks after application. High and low irrigation levels were applied to each of the two strains and results show that higher irrigation (5.1 cm/week) maintains conidial viability better than a low irrigation level (2.5 cm/week). Mean conidial viability was approximately 8–12% greater in plots with the high irrigation regime. Rather than significantly increase soil moisture levels, it is hypothesized that the greater amount of irrigation helps to move the conidia deeper into the thatch layer and soil profile, an area that provides protection from damaging surface temperatures and UV exposure. Rainfall that occurred during the beginning of the 2005 test minimized the irrigation effect, and the irrigation treatment differences were more pronounced after 48 h. Four different UV protectants were evaluated for an impact on conidial viability of strain 3622. Two protectants, an optical brightener and magnesium silicate clay, when added to an emulsifiable oil formulation, significantly increased conidial viability on all evaluation dates. The clay particles act as a sunlight blocker while the optical brightener absorbs UV light. The combined protection from the oil and the additive increased conidial viability by approximately 10% on all evaluation dates. Results from this study provide insight into ways to increase entomopathogenic fungal viability under field conditions.     

杀菌剂对感染越冬桃小食心虫的白僵菌的抑制作用

【目的】桃小食心虫是我国果树上发生最普遍的食心类害虫,其室内人工饲养是综合防治研究的基础,但面临着田间越冬种群大量感染白僵菌死亡,给实验室种群的建立带来很大困难。因此筛选出对球孢白僵菌高效的杀菌剂用于解决桃小食心虫种群建立时感染白僵菌的问题。【方法】利用液体摇床振荡法和平皿十字交叉法测定16种常见杀菌剂对球孢白僵菌孢子萌发和菌丝生长的影响。【结果】筛选出9种杀菌剂,其中,腐霉利和中生菌素对孢子萌发的抑制效果好,常规用量抑制效果分别为97.88%±1.53%和93.22%±2.36%;抑霉唑和戊唑醇对菌丝生长的抑制效果明显,常规用量抑制效果分别达100.00%±0.00%和98.43%±0.99%;咪酰胺、丙环唑、噻菌灵、腈菌唑和吡唑醚菌酯对孢子萌发和菌丝生长的抑制效果都好。同时测定了这9种杀菌剂常规浓度及其5倍和10倍稀释液对桃小食心虫的毒力,结果表明有5种杀菌剂对桃小食心虫稍有不利影响。【结论】4种杀菌剂,中生菌素、戊唑醇、吡唑醚菌酯和噻菌灵可用于解决桃小食心虫种群建立时越冬幼虫的感染白僵菌问题。     

Laboratory and greenhouse evaluation of a new entomopathogenic strain of Beauveria bassiana for control of the onion thrips Thrips tabaci

Abstract

The onion thrips Thrips tabaci is one of the most important pests of greenhouse and open-field broccoli, onion and other crops. However, the current strategy of using synthetic pesticides for its control is inadequate and unsustainable, leading to a growing interest in novel and effective biological control alternatives such as entomopathogenic fungi. Among 20 isolates of Beauveria bassiana tested for virulence against T. tabaci in laboratory bioassays, we found strain SZ-26 as the most potent, causing 83–100% mortality in adults at 1×10⁷ mL^?1conidia after 4–7 days. Further experiments in greenhouses showed the strain SZ-26 significantly lowered the numbers of adult and larval stages.     

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.     

Detection and avoidance of an entomopathogenic fungus by a generalist insect predator

Abstract.  1. Increasing evidence suggests that insects can assess their environment based on cues related to mortality risks to themselves or their offspring. Limited knowledge is available on such abilities in relation to entomopathogenic fungi, which can cause significant mortality in insect populations. In laboratory bioassays, the ability of the generalist predator Anthocoris nemorum L. (Heteroptera: Anthocoridae) to detect the presence of its natural enemy, the fungal pathogen Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was investigated.
2. Behavioural observations were conducted on adults of A. nemorum foraging in choice and non-choice arenas treated with conidia suspensions of B. bassiana or just the carrier (control). The arenas consisted either of nettle leaves or soil. Additionally, behaviours in response to sporulating nettle aphid cadavers compared with uninfected aphids or paper balls were evaluated on nettle leaves. An oviposition experiment was also conducted in choice arenas on conidia-treated and untreated nettle leaves.
3. Males and females detected and avoided contact with leaf surfaces inoculated with B. bassiana . Females that were forced to enter fungus-treated leaf surfaces were very reluctant to do so. When females encountered cadavers sporulating with B. bassiana they rapidly withdrew compared with harmless paper ball dummies. Soil inoculated with B. bassiana did not affect A. nemorum behaviour or residence time compared with control soil. Females inserted significantly more eggs in control leaf areas compared with areas treated with B. bassiana conidia.
4. All results suggest that A. nemorum detects and avoids the pathogen B. bassiana when it forages on host plants with which it is adapted but not on soil surfaces. The adaptive significance of detection of entomopathogenic fungi is discussed.