The entomopathogenic fungus Beauveria bassiana and its compatibility with triflumuron: effects on the twospotted spider mite Tetranychus urticae

Laboratory studies were conducted to determine the effects of the mycoinsecticide Naturalis-L (Beauveria bassiana conidial formulation) on the twospotted spider mite Tetranychus urticae. Compatibility of B. bassiana with triflumuron (benzoylphenyl urea typically used as an insecticide, but also with acaricide effects), was also investigated in order to incorporate both in the control of this pest. For each juvenile stage, 180–22,800 viable conidia/mL on deutonymphs, 380–12,160 viable conidia/mL on protonymphs, and 712–7480 viable conidia/mL on larvae were evaluated. For the adult stage, the concentrations ranged from 213 to 54,720 viable conidia/mL. The mortality data used in the analysis were those accumulated after 5 days of treatment for deutonymphs and protonymphs, 7 days for larvae and 9 days for adults. The lethal concentration to kill 50% (LC₅₀) for the juvenile stages was 3184 viable conidia/mL (their probit-log concentration regression lines were the same), and 1949 viable conidia/mL for adults. No significant differences in mortality were observed among egg age classes (24-, 48-, 72-, and 96-h-old eggs) at the tested concentrations (1400–22,800 viable conidia/mL). When B. bassiana was combined with 0.25 g Alsystin (25% triflumuron as a wettable powder)/L, mite egg mortality decreased significantly. Triflumuron reduced mycelial growth but not conidial germination of B. bassiana. This fungus is a possible candidate to be included in integrated pest management programs with triflumuron of T. urticae. In such programs, the possible antagonist effects of triflumuron should be considered.     

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.     

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.     

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.     

Volatile organic compounds released by the entomopathogenic fungus Beauveria bassiana

The composition of volatile organic compounds (VOC) released by the entomopathogenic fungus Beauveria bassiana (Hyphomycete: Deuteromycotina) utilizing two different carbon sources was investigated. Analyses were performed by solid-phase microextraction (SPME) coupled to capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). Major components in glucose-grown cultures were diisopropyl naphthalenes, ethanol, and sesquiterpenes. Alkane-grown fungal VOC switched to a fingerprint with prevalence of n-decane. This is the first report on the volatiles released by entomopathogenic fungi.     

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.     

Molecular studies of co-formulated strains of the entomopathogenic fungus,Beauveria bassiana

A 28S rDNA intron was used as a molecular marker to distinguish between two single spore strains of Beauveria bassiana, Bb123 and Bb151. When co-formulated and assayed against larvae of Galleria mellonella these strains exhibited no synergistic increase in virulence, rather Bb123 usually dominated. This study shows that the success of any strain to infect Galleria is dependent on the dose and method of inoculation (injection versus immersion). The result of co-formulated strains grown on solid culture also showed that usually one strain dominated, i.e., strain displacement could happen both in vivo and in vitro. The speed by which one strain was displaced following successive sub-culturing on PDA partly depended on the ratio of Bb151 and Bb123. The co-formulated inoculum could widen the window over which parent strains would be active on different water activity media. Co-infection did result in heterokaryosis within the Galleria host. Molecular studies also showed that the heterokaryon was not stable and could revert back to the parent strain.     

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.     

Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

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.     

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.     

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.     

Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.     

Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a filamentous ascomycete that is pathogenic towards a broad host range of insect targets and is increasingly serving as a model for examining fungal development and host-pathogen interactions. B. bassiana displays a prohibitive level of resistance against many current fungal and/or yeast selection markers including hygromycin, neomycin, and zeocin. A genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100–150 transformants per microgram DNA/10⁸ cells and Southern blot analysis indicated that the plasmid vector was randomly integrated into the genome of B. bassiana. In addition, a construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. Over 95% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. The described transformation method increases opportunities for the genetic manipulation of B. bassiana.     

Compatibility of the insect pathogenic fungus Beauveria bassiana with neem against sweetpotato whitefly, Bemisia tabaci, on eggplant

A study on the compatibility of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) with neem was conducted against sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), on eggplant. Initially, three concentrations of B. bassiana (10⁶, 10⁷, and 10⁸ conidia ml⁻¹) and three concentrations of neem (0.25, 0.5, and 1.0%) were used as individual treatments against B. tabaci . The highest concentration of B. bassiana yielded the highest B. tabaci egg (25.2%) and nymph mortalities (73.0%), but this was not significantly different from the mortalities caused by the 10⁷ conidia ml⁻¹ suspension. Similarly, the highest concentration of neem yielded the highest egg (27.3%) and nymph mortalities (75.5%), which was also not significantly different from the 0.5% suspension. Therefore, 0.5% neem was used along with 10⁷ B. bassiana conidia ml⁻¹ suspension as an integrated pest management program against B. tabaci . The combination of B. bassiana and neem yielded the highest B. tabaci egg (29.5%) and nymph mortalities (97.2%), and the lowest LT₅₀ (2.08 day) value. Moreover, an integrated combination of B. bassiana with neem caused 27.6 and 20.5% more nymphal mortality than individual treatments of B. bassiana and neem, respectively, 7 days post-application. Thus, a combined application of an entomopathogenic fungus and a botanical insecticide may benefit from both, and it has proven effective for the control of B. tabaci on eggplant.     

Phosphorylation modification orchestrates the functionalities of peroxin 14 in filamentous entomopathogenic fungus Beauveria bassiana

Peroxin 14 (Pex14) is a component of the receptor-docking complex at peroxisomal membrane. However, its post translation modification remains largely unknown in filamentous fungi. In this study, we characterized two phosphorylation sites (S54 and T262) in Beauveria bassiana Pex14 (BbPex14). Two phosphorylation sites are dispensable for the BbPex14 role as a peroxin. The BbPex14 roles in conidiation and blastospore formation are dependent on two phosphorylation sites, and blastospore formation is more dependent on phosphorylation modification of two sites. Two phosphorylation sites differentially contribute to pexophagy during conidiation and under stress, in which the site T262 is indispensable. Evidently, the phosphorylation modification expands the functionalities of BbPex14. This study improves our understandings of the complex regulatory mechanisms underlying organellar biology in the filamentous fungi.