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.     

Horizontal transmission of Beauveria bassiana (Bals.) Vuill

1 Factors influencing horizontal transmission of the entomopathogen Beauveria bassiana in the Colorado potato beetle (CPB) were examined through a series of laboratory studies. 2 Cadaver density, cadaver life stage, ambient temperature and conidial density were the factors manipulated. 3 Mortality and sporulation of burrowing CPB prepupae both increased significantly with increased sporulating second‐instar cadaver density on the soil surface. 4 Mortality rates were significantly higher when prepupae were released into laboratory arenas containing third‐instar cadavers compared to second‐instar cadavers. 5 Mortality and sporulation decreased significantly as temperature increased from 15 °C to 30 °C, however, no temperature‐dependent behavioural response by prepupae could be identified as a potential cause. 6 An 86.1% decrease in conidial density per cadaver had no significant effect on mortality or sporulation of prepupae, indicating that this level of environmental degradation of cadavers may not significantly reduce the probability of horizontal transmission.     

桃蚜脱皮对球孢白僵菌毒力的影响

室内测定了球孢白僵菌Beauveria bassiana BBSG8702菌株对桃蚜Myzus persicae 若蚜的毒力,并分析了若蚜脱皮与白僵菌有效侵染之间的关系。在21℃、1×10⁶/mL孢子浓度下, 1～4龄若蚜感菌后的累积死亡率分别为10.1%、2.1%、3.1%和40.2%,明显低于同样条件下成蚜的感菌死亡率(98.4%)。在若蚜各龄感菌死亡的个体中,1～4龄中分别有8.1%、50.0%、44.4%和98.0%的若蚜在死亡前发育到成蚜,且都能产下正常发育的后代。若蚜的感菌死亡率与接菌后到第一次脱皮的时间密切相关,脱皮早则感菌死亡率低,反之亦然。但试验中也发现有部分若蚜特别是低龄若蚜感菌后未完成第一次脱皮便死亡。观察了桃蚜在1、4龄早期感染球孢白僵菌后的发育、存活和生殖情况,结果表明：1、4龄早期若蚜接菌后第一次脱皮的时间早于未接菌若蚜,但提前幅度少于5%,在羽化为成蚜之前被致死的概率分别只有4.1%和0,在羽化为成蚜后8天内的生殖率与未接菌蚜虫的无明显差异。这些结果表明,若蚜的脱皮,尤其当蚜虫在低龄若蚜时受感染后的多次脱皮,可有效地摆脱球孢白僵菌分生孢子的侵染,降低该菌的毒力。     

Light stimulates conidiation of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a commercially important entomopathogenic fungus. Like other insect fungal pathogens, B. bassiana usually produces asexual reproductive bodies, conidia, for dispersal, transmission and infection of insects. Adequate mass-production of high quality conidia is crucial to development of an efficient B. bassiana insecticide. However, little is known about details of conidiation in this fungus in response to environmental signals, which limits understanding of the mechanism of conidiation and improvement in conidia production. Here, morphologenetic changes of B. bassiana under different light conditions are reported. When cultured in total darkness, B. bassiana hyphae can grow continuously with few reproductive structures differentiated, while illumination with white light resulted in prolific formation of conidiophores bearing abundant conidia, indicating that light could stimulate conidiation of B. bassiana. Among the single colour lights tested, blue light was the most effective to stimulating sporulation. Colonies became adapted for blue light stimulus only after hyphae had grown in total darkness for at least 96 h, whereas the photoadaptation obviously declined after 144 h. For the exposure time, 3 min of blue light pulse was enough to stimulate conidiation in the photoadapted mycelia, while prolonged light exposure over 3 min resulted in a decrease in conidia yield. Our results provided useful clues for understanding the mechanism of conidiation mediated by light in B. bassiana.     

Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata

The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.     

Development of the entomopathogenic fungus Beauveria bassiana in liquid cultures

Growth and development of B. bassiana was followed in four liquid media: peptone, peptone-glucose, glucose and glucose-peptone-yeast extract. Six developmental stages were defined: (I) the unswollen conidium, (II) the swollen conidium, (III) emergence of the germ tube, (IV) elongation of the germ tube and formation of the first septum, (V) polar and bipolar elongation (growth) of the resulting mycelium and initiation of a blastospore and, (VI) seccession of that blastospore. Conidia of B. bassiana produced germ tubes in all liquid media. Blastospores were produced in all liquid media except glucose. In peptone-glucose, the yield of blastospores was four-fold higher than in glucose-peptone-yeast extract. However, biomass production was highest in peptone-glucose-yeast extract.     

Cosmopolites sordidus (Germar) susceptibility to indigenous Cameroonian Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates

Management of the banana root borer (BRB), Cosmopolites sordidus (Germar; Coleoptera: Curculionidae), remains a challenge in banana and plantain production worldwide. Synthetic pesticides remain the most widely used solution while mycoinsecticides are increasingly being recommended. In this study, we selected indigenous isolates of Beauveria bassiana and Metarhizium anisopliae collected from plantain fields in Cameroon, and tested them in the laboratory for their viability, pathogenicity and virulence against all C. sordidus life stages. Of 13 isolates initially screened for spore germination and pathogenicity to adult weevils in conidial suspension of 3.2 × 10⁸ conidia/ml, eight isolates with high to moderate germination and highest weevil mortality were selected for dose–response bioassays with four concentrations per isolate: 3.2 × 10², 3.2 × 10⁴, 3.2 × 10⁶ and 3.2 × 10⁸ conidia/ml. The virulent isolates from adult bioassays were tested with eggs, larva and pupae in conidial suspension of 3.2 × 10⁸ conidia/ml. Isolates performance depended on insect life stage with significantly high pathogenicity and virulence against larval, pupa and adult stages. The Beauveria isolate BIITAC6.2.2 caused the highest mortality rates followed by MIITAC1.1.5. Lethal times and lethal concentrations were relatively low for the three M. anisopliae isolates and three B. bassiana isolates which were the best isolates in almost all insect life stages. Apart from being effective in multiple life stages, these isolates were transmitted horizontally from one stage to another when eggs and pupae were treated. The implication of these findings for integrated management of the BRB, and potential biopesticides development and commercialization are discussed.     

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.     

Dependence of the entomopathogenic fungus,Beauveria bassiana,on high humidity for infection of Rhodnius prolixus

Mycopathologia - The impact of relative humidity (RH) on the infective potential of the isolate Bb INRA 297 of Beauveriabassiana (Bals.) Vuillemin (Deuteromycotina: Hyphomycetes) against first in...     

Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin

In non-irrigated agricultural fields in tropical zones, high temperature and water stress prevail during the main cropping season. Natural epizootics of Beauveria bassiana on lepidopteran pests occur during winter. Application of B. bassiana during hot months when pest populations are at their climax may prove an effective management strategy. Therefore, 29 isolates of B. bassiana were tested for their ability to germinate and grow in temperature and water availability conditions prevailing during the pest season in these fields. The effect of temperature cycles with 8 h duration of high temperature fluctuating with 16 h duration of lower temperature (similar to field conditions); low water availability; and a combination of these two stress conditions was studied. Germination and growth assays were done at fluctuating temperature cycles of 32, 35, 38, and 42+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and in media with water stress created by 10, 20, 30, and 40% polyethylene glycol (PEG 6000). Assays set at a continuous temperature of 25+/-1 degrees C with no PEG in the medium served as controls. Stress was assessed as percentage germination or as growth relative to control. Isolates showing 90% growth relative to the control at temperature cycles including high temperatures of 35 and 38+/-1 degrees C were identified. One isolate (ARSEF 2860) had a thermal threshold above 43 degrees C. At 25 degrees C, all but one isolate of B. bassiana showed >90% growth relative to the control in 10% PEG (-0.45 MPa). Some isolates were found with >90% growth relative to control in medium having 30% PEG with water availability (1.33 MPa), nearly equivalent to that in soils which induce permanent wilting point of plants. When isolates that showed >90% growth relative to the control at both stress conditions, were stressed simultaneously, a decrease in growth was observed. Growth was reduced by approximately 20% at 35+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG and was affected to a greater degree in combinations of harsher stress conditions. The isolate ARSEF 2860 with a thermal threshold of >43 degrees C showed approximately 80% relative growth at a combined stress of 38+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG. These findings will aid the selection of isolates for use in field trials in hot or dry agricultural climates.     

Side effects of the entomopathogenic fungus Beauveria bassiana on the predator Coccinella undecimpunctata

The effect on predators of the biopesticide based on Beauveria bassiana spores was studied. A high concentration of the pathogen showed adverse effects on the predator (Coccinella undecimpunctata) and its biological aspects: 2, 4 and 8 g/L eliminated egg deposition in treated females. Spores at 4 g/L affected significantly the longevity of adults, duration of larvae and pupal period.     

Successive subculturing alters spore-bound Pr1 activity,germination and virulence of the entomopathogenic fungus,Beauveria bassiana

One of the hurdles in the development of entomopathogenic fungi such as Beauveria bassiana is loss of virulence when successively maintained in vitro. This may result in products of inferior quality in mass production programs. Also, there are many contradicting data and unclear points in this case. Three isolates of B. bassiana were subcultured successively 15 times. Spore-bound Pr1 activity, germination rate, and virulence of conidia against mealworm (Tenebrio molitor L.) larvae were studied. Results showed that isolates normally retained their virulence during 10 subculturings. However, they clearly offered decreased virulence (elevated LT₅₀ values and lower percent mortality). The activity of Pr1 bound to conidia declined as subculturing continued; the lowest spore-bound activity and germination potential of conidia was recorded for the 15th subculture. Virulence data were in agreement with Pr1 activity and germination rate as there was a positive correlation between germination rate and spore-bound Pr1 activity with fungal virulence. This explains that at least a part of attenuation in fungal virulence can be explored in enzymatic activity, especially in the important cuticle-degrading protease, Pr1.     

Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana

Background

The mechanisms for the association between birth by cesarean section and atopy and asthma are largely unknown.

Objective

To examine whether cesarean section results in neonatal secretion of cytokines that are associated with increased risk of atopy and/or asthma in childhood. To examine whether the association between mode of delivery and neonatal immune responses is explained by exposure to the maternal gut flora (a marker of the vaginal flora).

Methods

CBMCs were isolated from 37 neonates at delivery, and secretion of IL-13, IFN-γ, and IL-10 (at baseline and after stimulation with antigens [dust mite and cat dander allergens, phytohemagglutinin, and lipopolysaccharide]) was quantified by ELISA. Total and specific microbes were quantified in maternal stool. The relation between mode of delivery and cord blood cytokines was examined by linear regression. The relation between maternal stool microbes and cord blood cytokines was examined by Spearman's correlation coefficients.

Results

Cesarean section was associated with increased levels of IL-13 and IFN-γ. In multivariate analyses, cesarean section was associated with an increment of 79.4 pg/ml in secretion of IL-13 by CBMCs after stimulation with dust mite allergen (P < 0.001). Among children born by vaginal delivery, gram-positive anaerobes and total anaerobes in maternal stool were positively correlated with levels of IL-10, and gram-negative aerobic bacteria in maternal stool were negatively correlated with levels of IL-13 and IFN-γ.

Conclusion

Cesarean section is associated with increased levels of IL-13 and IFN-γ, perhaps because of lack of labor and/or reduced exposure to specific microbes (e.g., gram-positive anaerobes) at birth.     

High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana displays a broad insect host range and serves as a model for examining host-pathogen interactions. Rapid construction and screening of random-insertion mutants of B. bassiana provides a powerful tool to dissect the molecular mechanisms of fungal virulence. LiAc/DMSO treated B. bassiana blastospores were found to be highly competent to transformation using linear DNA and a polyethylene glycol-based method. Selection on cellophane-layered Czapek-Dox agar at a lowered pH (from 7.5 to 6.3) greatly decreased background growth of non-transformed cells and improved screening of transformants. Optimization of the protocol using integration of the bar phosphinothricin resistance gene resulted in high transformation rates (200-250 transformants/μg DNA/10⁸ cells). A collection of ∼4000 insertion mutants was examined via high-throughput screens for hydrocarbon utilization. One mutant was isolated that grew poorly on both n-hexadecane and tributyrin. The random insertion site was mapped to a gene that displayed homology to vitamin H (biotin)/tartrate transporters. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the mutant. This system provides a simple and rapid method for the generation and screening of insertion mutants and should expand our ability to genetically analyze the B. bassiana lifestyle.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

Attenuation of the entomopathogenic fungus Beauveria bassiana following serial in vitro transfers

Stability of pathogenicity in continuous in vitro cultivation is desirable for the purpose of large-scale production of a mycoinsecticide. Fungal biocontrol agents may lose virulence when maintain on artificial media, resulting in products of commercially inferior quality. In this research, two isolates (DEBI007 and DEBI008) of entomopathogenic fungus Beauveria bassiana were investigated for their stability following fifteen serial in vitro transfers assaying virulence to mealworm larvae, conidiation, and hyphal development on artificial culture as some fungal virulence determinants. Moreover, role of insect cuticle on fungal virulence restoration and protease 1 (Pr1) activity was considered as the most important factor. Although radial hyphal development and colony colour on in vitro culture was not affected following serial transfers, conidiation and Pr1 activity of both fungal isolates were reduced remarkably after fifteen transfers compared with control. Similarly, mean lethal concentration (LC₅₀) values were increased as the number of serial transfers on artificial diet increased, although these increases were not statistically significant in both isolates as the confidential limits of LC₅₀ values were overlapping. Our results revealed that attenuation of entomopathogenic fungi following serial in vitro transfers is a combination of interconnected factors. Other probable components such as pathogenicity determinants in this interaction should be explored in next researches.     

Selection of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate entomopathogenic fungi isolates as microbial control agents ofthe lesser mealworm, Alphitobius diaperinus (Panzer). Larvae and adults were inoculated with conidial suspension of 99 isolates of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. (10(5) to 10(9) conidia/ml). Vegetative growth on culture media and sporulation on culture media, cooked rice and lesser mealworm cadavers were also evaluated. Isolates of B. bassiana were more effective than the M. anisopliae isolates and larvae were more susceptible than adults. The isolates UNIOESTE 04 and UNIOESTE 02 were originally obtained from adults of the lesser mealworm and were the most efficient isolates. In addition, UNIOESTE 04 showed high level of sporulation on different culture media and is considered a potential useful tool to lesser mealworm control.