腺苷酸核糖基化因子BbarfA介导白僵菌孢子萌发和毒力

【目的】探究腺苷酸糖基化因子ARF在球孢白僵菌(Beauveria bassiana)中存在种类及生物学功能。【方法】利用BLASTp搜索球孢白僵菌非冗余蛋白数据库,鉴定ARF并进行聚类分析,结合表达分析、反义抑制、超量表达野生型基因和GTP解离位点与结合位点突变的基因,解析其中1个ARF与白僵菌发育分化、逆境胁迫反应和毒力的关系。【结果】球孢白僵菌中存在至少6个ARF或类似蛋白,分别聚类于酵母、人类ARF及其类似蛋白的不同类群。其中BBA_01574与人类的ARF3、ARF4和ARF5聚为一类,命名为BbarfA。BbarfA在成熟的分生孢子和球形膨大时期表达明显高于芽管伸长期。反义抑制BbarfA加速了孢子萌发,提高了菌株毒力,而超量表达BbarfA和点突变GTP解离区域的BbarfA则延迟了孢子萌发速度,降低了菌株毒力。尽管BbarfA转录受高盐、髙渗、氧化和高温胁迫的诱导,但遗传修饰的转化子与野生菌株对上述胁迫反应的敏感性无明显差异。【结论】BbarfA介导分生孢子萌发和毒力。     

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.     

A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial hydrophobicity when expressed in Beauveria bassiana

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

Relationship between thermotolerance and hydrophobin-like proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents

AIMS: This study was to illustrate the relationship between the thermotolerance and the contents of hydrophobin-like or formic-acid-extractable (FAE) proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus produced on rice-based substrate. METHODS AND RESULTS: Survival indices of 11 isolates were separately assessed as a ratio of the viability of conidia after 3-150 min thermal stress at 48 degrees C over that of unstressed conidia and fitted well to a survival model (r(2) >/= 0.97). For a given isolate, the fitted model generated an LT(50), the time for 50% viability loss under the stress. The LT(50)s of six B. bassiana isolates (10.1-61.9 min) and five P. fumosoroseus isolates (2.8-6.2 min) were correlated (r(2) = 0.81) with FAE protein contents (6.9-23.4 microg mg(-1)). The survival indices of a fixed B. bassiana isolate after 45-min thermal stress at 48 degrees C were also correlated to the FAE protein contents from conidia produced on glucose-, sucrose-, or starch-based substrate (0.79 相似文献   

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。     

Conidiation under illumination enhances conidial tolerance of insect-pathogenic fungi to environmental stresses

Light is an important signal for fungi in the environment and induces many genes with roles in stress and virulence responses. Conidia of the entomopathogenic fungi Aschersonia aleyrodis, Beauveria bassiana, Cordyceps fumosorosea, Lecanicillium aphanocladii, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, Tolypocladium cylindrosporum, and Tolypocladium inflatum were produced on potato dextrose agar (PDA) medium under continuous white light, on PDA medium in the dark, or under nutritional stress (= Czapek medium without sucrose = MM) in the dark. The conidial tolerance of these species produced under these different conditions were evaluated in relation to heat stress, oxidative stress (menadione), osmotic stress (KCl), UV radiation, and genotoxic stress caused by 4-nitroquinoline 1-oxide (4-NQO). Several fungal species demonstrated greater stress tolerance when conidia were produced under white light than in the dark; for instance white light induced higher tolerance of A. aleyrodis to KCl and 4-NQO; B. bassiana to KCl and 4-NQO; C. fumosorosea to UV radiation; M. anisopliae to heat and menadione; M. brunneum to menadione, KCl, UV radiation, and 4-NQO; M. robertsii to heat, menadione, KCl, and UV radiation; and T. cylindrosporum to menadione and KCl. However, conidia of L. aphanocladii, S. lanosoniveum, and T. inflatum produced under white light exhibited similar tolerance as conidia produced in the dark. When conidia were produced on MM, a much stronger stress tolerance was found for B. bassiana to menadione, KCl, UV radiation, and 4-NQO; C. fumosorosea to KCl and 4-NQO; Metarhizium species to heat, menadione, KCl, and UV radiation; T. cylindrosporum to menadione and UV radiation; and T. inflatum to heat and UV radiation. Again, conidia of L. aphanocladii and S. lanosoniveum produced on MM had similar tolerance to conidia produced on PDA medium in the dark. Therefore, white light is an important factor that induces higher stress tolerance in some insect-pathogenic fungi, but growth in nutritional stress always provides in conidia with stronger stress tolerance than conidia produced under white light.     

Enhanced conidial thermotolerance and field efficacy of Beauveria bassiana by saccharides

Insecticide efficacy of Beauveria bassiana conidia was improved by optimizing the concentrations of conidial heat-protective saccharides (glucose, sucrose, maltose, trehalose, α-lactose, and mannitol) using response surface methodology. Two field trials in tea gardens were carried out to control leafhopper (Empoasca vitis) by spraying B. bassiana conidia together with the optimized saccharides (0.26 g glucose, 0.28 g lactose, 0.24 g mannitol per ml). In the field studies, B. bassiana conidia were applied to control Empoasca vitis with and without saccharides and compared with bifenthrin, a pyrethroid insecticide. With the optimal concentrations of saccharides, the conidial germination rate reached 72 % and the control efficacy of the saccharides group (65.7 %) was equal to the bifenthrin group (69.4 %), which improved by about 55 %.     

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.     

Characterization of Lactobacillus collinoides response to heat, acid and ethanol treatments

Tolerance to stress and cross-protection in Lactobacillus collinoides were examined after exposure to ethanol, acid or heat shock. Ethanol and heat-adapted cells demonstrate induced homologous␣tolerance and cross-resistance to acid stress. No cross-protection of acid-adapted cells against ethanol and heat stresses was observed. Heat was the only pretreatment leading to cross-protection against the two other stresses. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins; the synthesis of some of these polypeptides being induced by more than one condition. The greatest overlap was observed between ethanol and heat treatments. Ten proteins were found to be common to these stresses. Received: 7 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Culture degeneration in conidia of Beauveria bassiana and virulence determinants by proteomics

The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> pathogenicity to the cherry slugworm, <Emphasis Type="Italic">Caliroa cerasi</Emphasis> (Hymenoptera: Tenthredinidae) larvae

The cherry slugworm Caliroa cerasi is a significant destructive pest of sour cherries (Prunus cerasus) in Turkey. The potential of entomopathogenic fungi for controlling C. cerasi was evaluated. The effects of exposure methods and conidial concentrations (1 × 10⁶, 1.5 × 10⁶, 1 × 10⁷ and 1.5 × 10⁷ conidia/ml) on mature larvae of C. cerasi infected by Beauveria bassiana were investigated under laboratory conditions. Larvae sprayed directly with B. bassiana conidial suspensions and larvae exposed to B. bassiana-treated leaves resulted in 100% mortality within 2.90 and 2.77 days, respectively. The median lethal time (LT₅₀) and days to mortality were highest in the 1.0 × 10⁷ concentrations of conidia for both direct spray and leaf exposure. The present study suggests that B. bassiana has good potential for control of the cherry slugworm, C. cerasi.     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

Nonspecific Factors Involved in Attachment of Entomopathogenic Deuteromycetes to Host Insect Cuticle

The attachment of the conidia of the insect-pathogenic fungi Nomuraea rileyi, Beauveria bassiana, and Metarrhizium anisopliae to insect cuticle was mediated by strong binding forces. The attachment was passive and nonspecific in that the conidia adhered readily to both host and nonhost cuticle preparations. The hydrophobicity of the conidial wall and the insect epicuticle appeared to mediate the adhesion process. Detergents, solvents, and high-molecular-weight proteins known to neutralize hydrophobicity reduced conidial binding when added to conidium-cuticle preparations. However, these chemicals did not remove the hydrophobic components from the epicuticle or from conidial preparations. The outer surface of the conidium consists of a resilient layer of well-organized fascicles of rodlets. Intact rodlets extracted from B. bassiana conidia bound to insect cuticle and exhibited the hydrophobicity expressed by intact conidia. Both electrostatic charges and various hemagglutinin activities were also present on the conidial surface. However, competitive-inhibition studies indicated that these forces played little, if any, role in the adhesion process.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Influence of humidity on the infection of western flower thrips, <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> (Thysanoptera: Thripidae), by <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

The insecticidal activity of Beauveria bassiana GHA derived from a commercial mycoinsecticide BotaniGard ES against Frankliniella occidentalis was determined in a bioassay by dipping the female adults into a conidial suspension. The 90% lethal concentration of B. bassiana GHA was estimated to be 9.7 × 10⁶ conidia/ml. The lethal times for achieving 90% mortality of thrips inoculated with a 1/500-diluted solution of BotaniGard ES and a 10^7.5 (3.16 × 10⁷) conidia/ml suspension of B. bassiana GHA were estimated to be five and six days, respectively. When the treated thrips were exposed to a high relative humidity (RH) of over 99% for various periods and then transferred to 60% RH, the requisite lengths of the high-humidity period to achieve 90% mortality of the thrips at six days after inoculation were estimated to be 46 and 47 h in BotaniGard ES and B. bassiana GHA, respectively. Fungal multiplication in the thrips was detected between 48 to 60 h after inoculation by measuring Beauveria-specific DNA in the host following inoculation with a B. bassiana GHA suspension of 10^7.5 conidia/ml using a real-time quantitative PCR. The mycelial growth in the host hemocoel was not influenced by the low-humidity condition.     

Isolation of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) from the Chagas disease vector, <Emphasis Type="Italic">Triatoma infestans</Emphasis> (Hemiptera: Reduviidae) in Argentina

A survey for natural entomopathogenic fungi of the Chagas disease vector Triatoma infestans was conducted in five provinces of Argentina since 2001. Nymphs (1.5%) and adults (3.3%) infected with a strain of the fungus Beauveria bassiana were found at Dean Funes, Córdoba province, Argentina. Field collected insects that died in the laboratory were maintained in moist chambers and incubated at 22 °C. Beauveria bassiana from infected insects was cultured on SDAY media. Pathogenicity tests were conducted with a conidial suspension (1 × 10⁷ conidia/ml) of this isolate on T. infestans adults. A mortality rate of 100% was obtained at 15 days post-infection. This is the first record of natural infection of T. infestans by B. bassiana.     

Application of hydrophilic–lipophilic balance (HLB) number to optimize a compatible non-ionic surfactant for dried aerial conidia of Beauveria bassiana

The hydrophilic–lipophilic balance (HLB) number system was used to optimize a compatible non-ionic surfactant, TDA (polyoxyethylene tridecyl ether) in formulations for two Beauveria bassiana strains, NI8 and GHA. The optimal HLB number for TDA was determined on the basis of wetting times for conidial powders. The results indicated that optimal HLB number of TDA for B. bassiana strain NI8 was 8, while the optimum HLB number for strain GHA was 10. The optimized TDA surfactants required significantly less wetting times than the commonly used laboratory surfactants, Triton X-100, Span 80, and Tween 80. These optimized TDA surfactants were further characterized on their ability to produce conidial suspensions of the two strains after 5 min of mixing, TDA HLB 8 and TDA HLB 10 produced suspensions of 1.8 × 10⁸ and 1.6 × 10⁸ conidia/ml for NI8 and GHA, respectively. These conidial levels were significantly higher than those in Triton X-100, Span 80, and Tween 80 suspensions after the same mixing time. Germination assays showed that TDA HLB 8 promoted significantly higher germination rates of strain NI8 than those observed in other commonly used laboratory surfactants. However, the germination rates of the GHA strain were unaffected by any of the surfactants tested. The efficacy of the conidial suspensions was confirmed with assays against Lygus lineolaris. Bioassay results indicated that there were no significant differences in mortalities because of surfactants. These results suggest optimization based upon HLB number will not negatively impact parameters associated with efficacy, while providing desirable physical properties.     

Allee Effect in the Infection Dynamics of the Entomopathogenic Fungus Beauveria bassiana (Bals) Vuill. on the Beetle, Mylabris pustulata

Successful infection by Beauveria bassiana as with all other entomopathogenic fungi, is accomplished only at a high conidial dose while, theoretically, a single conidium should be sufficient. Indeed, this is a major deterrent in its use as a biocontrol agent. High pathogen load for infection is required by organisms which display ‘Allee’ effect. In such organisms, a threshold exists for pathogen dose, below which no infection can be caused. B. bassiana has a semelparous life cycle and, therefore, its infection dynamics are expected to conform to the mass action principle with a linear relationship between dose and successful infection observable as mortality of the insect. Whether the need for a high conidial dose to induce insect mortality by B. bassiana is due to the operation of Allee effect was examined. A sample of 34 isolates was bioassayed on Mylabris pustulata (Coleoptera: Meloidae) at four conidial concentrations. With more than half of the isolates in the sample, the lowest dose tested (10⁴ conidia/insect) did not cause insect mortality. Thus, a threshold pathogen load is required to cause successful infection. In these isolates, the dose–mortality relationship was sigmoid. Allee effect is thus identified in the infection dynamics of B. bassiana–M. pustulata system. The isolates that induced mortality at the lowest dose tested are concluded to be highly virulent with a lower threshold dose required for successful infection. With some isolates, at high conidial dose, the infection rate decreased either due to a decrease in the proportion of insects showing mycosis, to the speed of death, or both. Such a response could result from intra scramble competition arising from overload of pathogen at very high dose.     

Substrate influence on physiology and virulence of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> acting on larvae and adults of <Emphasis Type="Italic">Tenebrio molitor</Emphasis>

Two isolates of Beauveria bassiana, wild type (wt) and its mutant type (mt) were compared in terms of growth patterns on culture plates containing media based on wheat bran, grasshopper exoskeletons, colloidal chitin or Sabouraud-dextrose agar (SDA). Germination for the mt isolate was up to 33% faster in all media. Influence of media on virulence was determined against larvae and adults of Tenebrio molitor. Mortality higher than 90% was reached for adults after 6 days using conidia from all media. For larvae, a mortality of 80% was reached after 11 days with conidia collected from SDA medium and between 15 and 35% with conidia from other media. In SDA medium, conidial yield was almost ten times higher for the mt isolate compared to the wt isolate; however, virulence traits were similar against either larvae or adults. These results may influence commercial preparations of entomopathogenic fungi based on conidia.