Insect Pathogenic Fungus Beauveria Bassiana Research Articles

Cell detoxification of secondary metabolites by P4-ATPase-mediated vesicle transport.

Mechanisms for cellular detoxification of drug compounds are of significant interest in human health. Cyclosporine A (CsA) and tacrolimus (FK506) are widely known antifungal and immunosuppressive microbial natural products. However, both compounds can result in significant side effects when used as immunosuppressants. The insect pathogenic fungus Beauveria bassiana shows resistance to CsA and FK506. However, the mechanisms underlying the resistance have remained unknown. Here, we identify a P4-ATPase gene, BbCRPA, from the fungus, which confers resistance via a unique vesicle mediated transport pathway that targets the compounds into detoxifying vacuoles. Interestingly, the expression of BbCRPA in plants promotes resistance to the phytopathogenic fungus Verticillium dahliae via detoxification of the mycotoxin cinnamyl acetate using a similar pathway. Our data reveal a new function for a subclass of P4-ATPases in cell detoxification. The P4-ATPases conferred cross-species resistance can be exploited for plant disease control and human health protection.

Production of dried Beauveria bassiana conidia in packed-column bioreactor using agro-industrial palm oil residues

Agro-industrial waste byproducts are commonly inexpensive organic nutritional sources for production of filamentous fungi, and selection of an optimal bioreactor is a key factor for attaining satisfactory biomass yield and quality. The insect-pathogenic fungus Beauveria bassiana (Hypocreales: Cordycipitaceae) has been extensively mass produced by solid-state fermentation using precooked cereal grains. Here, we propose low-cost palm oil residues as the main substrate using a cylindrical packed-column aerobic bioreactor prototype designed for conidial production of B. bassiana. Fermentation treatments using B. bassiana strain CG1229 assessed the impact of temperature, air flow, substrate moisture content, and ratio between palm fiber (PF) and palm kernel cake (PKC) on conidial yield and desiccation tolerance. Results showed significant enhanced productivity (2 ×1010 conidia g−1 dry matter) and reduced fermentation time (from 168 h to 120 h) by this method compared with the conventional tray bioreactor. Optimal substrate conditions were 60% initial moisture and 30% PF + 70% PKC content, fermentation at 26 °C with aeration rate ≥ 0.2 L min−1. Air drying conidia inside the column yielded > 95% germination. Maximum spore production was achieved in 120 h. Our findings demonstrate the feasibility of using a packed-column bioreactor for mass production of B. bassiana conidia using low-cost agricultural residues, which contributes to a sustainable production method of this biopesticide in Brazil.

Colonization Efficacy of the Endophytic Insect-Pathogenic Fungus, Beauveria bassiana, Across the Plant Kingdom: A Meta-Analysis

The insect-pathogenic fungus Beauveria bassiana (Bb) colonizes several plant species as an endophyte. However, the diversity of plants colonized and the extent of colonization by this fungus have not been summarized comprehensively across all plant species. To fill this knowledge gap, a meta-analysis of published studies (years 2002–2018) on the percentage of B. bassiana plant colonization across the plant kingdom was conducted. We collected 232 published papers from which 1,051 studies (individual treatments) were extracted and analyzed with Comprehensive Meta-Analysis, Version 3 (CMA) software. Factors (n = 26) influencing plant colonization by B. bassiana were identified. Across all studies, the mean endophytic colonization of B. bassiana was 29% in different plant taxa. Plant colonization by B. bassiana was highest for isolates collected from insects. Most studies applied B. bassiana at 1 × 108 conidia/ml; however, plant colonization was greatest with an application rate of 1 × 1012 conidia/ml. Among all plant hosts, colonization percentage was highest in faba bean, plants in the family Brassicaceae, angiosperms (eudicots, annuals), plants classified with a forb/herb/vine growth habit, and plants with fibrous or tap root systems. As an inoculant, B. bassiana was most frequently applied to the whole plant after true leaves had developed. The foliar spray was the most studied inoculation method, but the application of solid grain substrate to foliage gave higher plant colonization. Studies conducted in controlled environments resulted in higher endophytic colonization with B. bassiana than field studies. Endophytic B. bassiana presence was confirmed primarily with microscopy, rather than molecular methods. High heterogeneity (I 2 = 97%) across studies was identified with large variability in endophytic B. bassiana colonization across a diversity of plant species. These findings increase our understanding and knowledge of the endophytic lifestyle of B. bassiana, which will facilitate the development of novel, sustainable, and eco-friendly disease management strategies with B. bassiana.

A Perilipin Affects Lipid Droplet Homeostasis and Aerial Hyphal Growth, but Has Only Small Effects on Virulence in the Insect Pathogenic Fungus Beauveria bassiana.

Lipid assimilation, storage, and turnover impact growth, development, and virulence in many microbial pathogens including fungi. Perilipins are proteins associated with lipid droplets (LDs) that mediate their assembly and turnover. Here, we characterized the Beauveria bassiana (BbPlin1) perilipin. BbPlin1 expression was higher in minimal media than in rich media, and, using a BbPlin1::eGFP fusion protein, the protein was shown to be co–localized to LDs, with the high expression seen during infection and proliferation within the insect (Galleria mellonella) host that dramatically decreased to almost no expression during fungal outgrowth on cadavers including in conidia, but that BbPlin1 production resumed in the conidia once placed in nutrient–containing media allowing for germination and growth. Characterization of a targeted gene deletion strain (ΔBbPlin1) revealed a dramatic (>30%) reduction in cellular LD content, promotion of aerial hyphal growth, and a small decrease in virulence, with little to no effects on vegetative growth and stress responses. However, in the ΔBbPlin1 strain, expression of the complementary LD–associated caleosin gene, BbCal1, was enhanced under nutrient–poor conditions, although no changes in BbPlin1 expression were seen in a ΔBbCal1 strain and the expression of BbPlin1 in the ΔBbCal1 strain did not change LD patterns in cells. Transcriptome and RT–PCR analyses indicated increased expression of lipid metabolism–related genes, including triacylglyercol lipase 3, enoyl–CoA isomerase, and diacylglycerol–O–acetyl transferase in the BbPlin1 deletion mutant. Lipid profile analyses confirmed that the loss of BbPlin1 significantly reduced the cellular levels of contents of triacylglycerol, diacylglycerol, and phosphatidylethanolamine as compared to the wild–type strain. These results demonstrate the involvement of the B. bassiana perilipin in mediating lipid homeostasis, fungal aerial hyphal growth, and virulence, revealing critical cycling from high expression during nutrient utilization within host cadavers to low expression during growth on the surface of the cadaver during the infection process.

Peroxins in Peroxisomal Receptor Export System Contribute to Development, Stress Response, and Virulence of Insect Pathogenic Fungus Beauveria bassiana.

In filamentous fungi, recycling of receptors responsible for protein targeting to peroxisomes depends on the receptor export system (RES), which consists of peroxins Pex1, Pex6, and Pex26. This study seeks to functionally characterize these peroxins in the entomopathogenic fungus Beauveria bassiana. BbPex1, BbPex6, and BbPex26 are associated with peroxisomes and interact with each other. The loss of these peroxins did not completely abolish the peroxisome biogenesis. Three peroxins were all absolutely required for PTS1 pathway; however, only BbPex6 and BbPex26 were required for protein translocation via PTS2 pathway. Three gene disruption mutants displayed the similar phenotypic defects in assimilation of nutrients (e.g., fatty acid, protein, and chitin), stress response (e.g., oxidative and osmotic stress), and virulence. Notably, all disruptant displayed significantly enhanced sensitivity to linoleic acid, a polyunsaturated fatty acid. This study reinforces the essential roles of the peroxisome in the lifecycle of entomopathogenic fungi and highlights peroxisomal roles in combating the host defense system.

Secretion and Detection of Defensive Compounds by the Red Flour Beetle Tribolium castaneum Interacting with the Insect Pathogenic Fungus Beauveria bassiana.

Entomopathogenic fungi such as Beauveria bassiana are extensively used for the control of insect pests worldwide. They infect mostly by adhesion to the insect surface and penetration through the cuticle. However, some insects, such as the red flour beetle Tribolium castaneum (Herbst), have evolved resistance by embedding their cuticle with antifungal compounds. Thus, they avoid fungal germination on the cuticle, which result in low susceptibility to entomopathogenic fungi. In adult T. castaneum, these antifungals are the well-known defensive compounds methyl-1,4- and ethyl-1,4-benzoquinone. In this study, we added B. bassiana conidia on the diet of adult beetles to study the effect of the entomopathogen on the secretion and detection of the beetle volatile blend containing both benzoquinones. The compounds were analyzed by solid phase microextraction coupled to gas chromatography–flame ionization detection, and were detected by electroantennography. In addition, we measured the expression level of four genes encoding for two odorant-binding proteins (OBP), one chemosensory protein (CSP), and one odorant receptor (OR) in both healthy and fungus-treated insects. Significant alterations in the secretion of both benzoquinones, as well as in the perception of methyl-1,4-benzoquinone, were found in fungus-treated insects. TcOBP7D, TcOBP0A and TcCSP3A genes were down-regulated in insects fed conidia for 12 and 48 h, and the latter gene was up-regulated in 72 h samples. TcOR1 expression was not altered at the feeding times studied. We conclude that fungus-treated insects alter both secretion and perception of benzoquinones, but additional functional and genetic studies are needed to fully understand the effects of fungal infection on the insect chemical ecology.

Differential Roles of Five Fluffy Genes (flbA–flbE) in the Lifecycle In Vitro and In Vivo of the Insect–Pathogenic Fungus Beauveria bassiana

The fluffy genes flbA–flbE are well-known players in the upstream developmental activation pathway that activates the key gene brlA of central developmental pathway (CDP) to initiate conidiation in Aspergillus nidulans. Here, we report insignificant roles of their orthologs in radial growth of Beauveria bassiana under normal culture conditions and different stresses although flbA and flbD were involved in respective responses to heat shock and H2O2. Aerial conidiation level was lowered in the deletion mutants of flbB and flbE (~15%) less than of flbA and flbC (~30%), in which the key CDP genes brlA and abaA were repressed consistently during normal incubation. The CDP-controlled blastospore production in submerged cultures mimicking insect hemolymph was abolished in the flbA mutant with brlA and abaA being sharply repressed, and decreased by 55% in the flbC mutant with only abaA being downregulated. The fungal virulence against a model insect was attenuated in the absence of flbA more than of flbC irrespective of normal cuticle infection or cuticle-bypassing infection (intrahemocoel injection). These findings unravel more important role of flbA than of flbC, but null roles of flbB/D/E, in B. bassiana’s insect–pathogenic lifecycle and a scenario distinctive from that in A.nidulans.

A peroxisomal sterol carrier protein 2 (Scp2) contributes to lipid trafficking in differentiation and virulence of the insect pathogenic fungus Beauveria bassiana

Sterol carrier protein 2 (SCP2) represents a family of proteins binding a variety of lipids and plays essential roles in cellular physiology. However, its physiological roles are largely unknown in filamentous fungi. In this study, we functionally characterized an orthologous Scp2 gene in the filamentous insect pathogenic fungus Beauveria bassiana (BbScp2). BbScp2 was verified to be a peroxisomal protein and displayed different affinities to various lipids, with strong affinity to palmitic acid (PA) and ergosterol (ES). No significant binding activity was detected between protein and oleic acid (OA) or linoleic acid (LA). Ablation of BbScp2 did not cause significant effects on fungal growth on various carbon sources, but resulted in a modest reduction in conidial (49%) and blastospore yield (45%). In addition, exogenous lipids could recover the defectives in conidiation of ΔBbScp2 mutant strain. BbScp2 was required for the cytomembrane functionality in germlings, and its loss resulted in a more significant decrease in virulence indicated by cuticle infection assay than intrahemocoel injection assay. Our findings indicate that Scp2 links the lipid trafficking to the asexual differentiation and virulence of B. bassiana.

Infection of the Western Flower Thrips, Frankliniella occidentalis, by the Insect Pathogenic Fungus Beauveria bassiana

The western flower thrips, Frankliniella occidentalis, is an aggressive agricultural insect pest causing significant damage to a wide range of fruit, vegetable, and ornamental crops. Beauveria bassiana is a broad-host-range entomopathogenic fungus capable of infecting and killing F. occidentalis. Infection of thrips by B. bassiana strain BbYT12 using a concentration of 1 × 108 conidia/mL resulted in 81.48% mortality in adults in 6 d (LT50 = 90 ± 15.1 h). Scanning electron microscopy of the infection process revealed preferential adhesion and germination of fungal spores to inter-segmental folds or grooves on the insect body surface with penetrating germlings and extended hyphae visualized during the initial stages of infection (6–24 h). Histological analyses showed the appearance of in vivo hyphal bodies in sagittal sections and the fat body as early as 24 h post-infection. Within 72 h, hyphal bodies and hyphae could be found throughout the infected organism including in the midgut, Malphigian tubules, alimentary canal, ovarioles (in females), and an extended hyphal network could be seen on insect cadavers (>72 h post-infection). Real-time RT-PCR analyses of the expression of select genes implicated in virulence including the Pr1 protease, beauvericin synthase, involved in the production of the secondary metabolite beauvericin, two cytochrome P450 monooxygenases implicated in cuticular hydrocarbon degradation, two multidrug efflux proteins, a perilipin involved in lipid storage, and the Hog1 MAP kinase and protein kinase A signaling factors revealed discrete patterns of infection-time dependent expression. These data provide basic insights into the process of B. bassiana infection of F. occidentalis.

The Tudor Domain-Containing Protein BbTdp1 Contributes to Fungal Cell Development, the Cell Cycle, Virulence, and Transcriptional Regulation in the Insect Pathogenic Fungus Beauveria bassiana.

ABSTRACTBeauveria bassiana is an insect pathogenic fungus that serves as a model system for exploring the mechanisms of fungal development and host-pathogen interactions. Clinical and experimental studies have indicated that SND1 is closely correlated with the progression and invasiveness of common cancers as a potential oncogene, but this gene has rarely been studied in fungi. Here, we characterized the contributions of an SND1 ortholog (Tdp1) by constructing a BbTdp1 deletion strain and a complemented strain of B. bassiana. Compared with the wild-type (WT) strain, the ΔBbTdp1 mutant lost conidiation capacity (∼87.7%) and blastospore (∼96.3%) yields, increased sensitivity to chemical stress (4.4 to 54.3%) and heat shock (∼44.2%), and decreased virulence following topical application (∼24.7%) and hemocoel injection (∼40.0%). Flow cytometry readings showed smaller sizes of both conidia and blastospores for ΔBbTdp1 mutants. Transcriptomic data revealed 4,094 differentially expressed genes (|log2 ratio| > 2 and a q value of <0.05) between ΔBbTdp1 mutants and the WT strain, which accounted for 41.6% of the total genes, indicating that extreme fluctuation in the global gene expression pattern had occurred. Moreover, deletion of BbTdp1 led to an abnormal cell cycle with a longer S phase and shorter G2/M and G0/G1 phases of blastospores, and enzyme-linked immunosorbent assay confirmed that the level of phosphorylated cyclin-dependent kinase 1 (Cdk1) in the ΔBbTdp1 strain was ∼31.5% lower than in the WT strain. In summary, our study is the first to report that BbTdp1 plays a vital role in regulating conidia and blastospore yields, fungal morphological changes, and pathogenicity in entomopathogenic fungi.IMPORTANCE In this study, we used Beauveria bassiana as a biological model to report the role of BbTdp1 in entomopathogenic fungi. Our findings indicated that BbTdp1 contributed significantly to cell development, the cell cycle, and virulence in B. bassiana. In addition, deletion of BbTdp1 led to drastic fluctuations in the transcriptional profile. BbTdp1 can be developed as a novel target for B. bassiana development and pathogenicity, which also provides a framework for the study of Tdp1 in other fungi.

HapX, an Indispensable bZIP Transcription Factor for Iron Acquisition, Regulates Infection Initiation by Orchestrating Conidial Oleic Acid Homeostasis and Cytomembrane Functionality in Mycopathogen Beauveria bassiana.

In pathogenic filamentous fungi, conidial germination not only is fundamental for propagation in the environment but is also a critical step of infection. In the insect mycopathogen Beauveria bassiana, we genetically characterized the role of the basic leucine zipper (bZIP) transcription factor HapX (BbHapX) in conidial nutrient reserves and pathogen-host interaction. Ablation of BbHapX resulted in an almost complete loss of virulence in the topical inoculation and intrahemocoel injection assays. Comparative transcriptomic analysis revealed that BbHapX is required for fatty acid (FA)/lipid metabolism, and biochemical analyses indicated that BbHapX loss caused a significant reduction in conidial FA contents. Exogenous oleic acid could partially or completely restore the impaired phenotypes of the ΔBbHapX mutant, including germination rate, membrane integrity, vegetative growth, and virulence. BbHapX mediates fungal iron acquisition which is not required for desaturation of stearic acid. Additionally, inactivation of the Δ9-fatty acid desaturase gene (BbOle1) generated defects similar to those of the ΔBbHapX mutant; oleic acid also had significant restorative effects on the defective phenotypes of the ΔBbOle1 mutant. A gel retarding assay revealed that BbHapX directly regulated the expression of BbOle1 Lipidomic analyses indicated that both BbHapX and BbOle1 contributed to the homeostasis of phospholipids with nonpolar tails derived from oleic acid; therefore, exogenous phospholipids could significantly restore membrane integrity. These data reveal that the HapX-Ole1 pathway contributes to conidial fatty acid/lipid reserves and that there are important links between the lipid biology and membrane functionality involved in the early stages of infection caused by B. bassiana IMPORTANCE Conidial maturation and germination are highly coupled physiological processes in filamentous fungi that are critical for the pathogenicity of mycopathogens. Compared to the mechanisms involved in conidial germination, those of conidial reserves during maturation are less understood. The insect-pathogenic fungus Beauveria bassiana, as a representative species of filamentous fungi, is important for applied and fundamental research. In addition to its conserved roles in fungal adaptation to iron status, the bZIP transcription factor HapX acts as a master regulator involved in conidial virulence and regulates fatty acid/lipid metabolism. Further investigation revealed that the Δ9-fatty acid desaturase gene (Ole1) is a direct downstream target of HapX. This study reveals the HapX-Ole1 pathway involved in the fatty acid/lipid accumulation associated with conidial maturation and provides new insights into the startup mechanism of infection caused by spores from pathogenic fungi.

Synergistic control against Asian corn borer (ACB) by Trichogramma vectored Beauveria bassiana infection on survival larvae escaped from parasitism

The combined application of natural enemies and entomopathogens as an alternative pest-control may act synergistically, additively or antagonistically. The objective of this study is to determine the efficacy of the combined application by parasitic Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae) carrying insect-pathogenic fungus Beauveria bassiana (Bals.) Vuill. (TCB) against Asian corn borer (ACB), Ostrinia furnacalis (Lepidoptera: Pyralidae). 0.1% (w/v) starch solution was selected from several adjuvants due to its highest ability to carry fungal conidia, over 3.60 × 104 conidia per wasp. The fungal conidia adsorbed on the surface of factitious host, Antheraea pernyi eggs, subsequently adhered on new emerged T. dendrolimi as a vector, which carried the conidia onto eggs’ surface of ACB. The B. bassiana had no influence on T. dendrolimi’s parasitic rate, whereas resulting in fungal conidia adhering on over 60.00% of hatched larvae, which consequently caused 27.00 ± 5.70% percent of muscardine cadaver rate of pest’s larvae in a screen house test. In field trial, it showed significantly lower incidence parameters regarded as number of damaged plants, wormholes, and living pests, per 100 plants in TCB treatment compared to that of sole T. dendrolimi release (non-TCB) and non-treatment as control, respectively, especially the number of muscardine cadaver per 100 plants was 6-fold amount higher than that of non-TCB treatment. In this study, the suitable adjuvant of cost-effective and safe to T. dendrolimi emergence was selected for fungal conidia adhering for TCB, and the fungal conidia transmitting process from host eggs to pest infection was visualized by fluorescence observation, on the basis, it was proved in both screen house and field experiments that the TCB was a synergistic ACB control program with labor saving, low cost and high efficient advantage compared to sole biocontrol agent application.

Photoprotective Role of Photolyase-Interacting RAD23 and Its Pleiotropic Effect on the Insect-Pathogenic Fungus Beauveria bassiana.

RAD23 can repair yeast DNA lesions through nucleotide excision repair (NER), a mechanism that is dependent on proteasome activity and ubiquitin chains but different from photolyase-depending photorepair of UV-induced DNA damages. However, this accessory NER protein remains functionally unknown in filamentous fungi. In this study, orthologous RAD23 in Beauveria bassiana, an insect-pathogenic fungus that is a main source of fungal insecticides, was found to interact with the photolyase PHR2, enabling repair of DNA lesions by degradation of UVB-induced cytotoxic (6-4)-pyrimidine-pyrimidine photoproducts under visible light, and it hence plays an essential role in the photoreactivation of UVB-inactivated conidia but no role in reactivation of such conidia through NER in dark conditions. Fluorescence-labeled RAD23 was shown to normally localize in the cytoplasm, to migrate to vacuoles in the absence of carbon, nitrogen, or both, and to enter nuclei under various stresses, which include UVB, a harmful wavelength of sunlight. Deletion of the rad23 gene resulted in an 84% decrease in conidial UVB resistance, a 95% reduction in photoreactivation rate of UVB-inactivated conidia, and a drastic repression of phr2 A yeast two-hybrid assay revealed a positive RAD23-PHR2 interaction. Overexpression of phr2 in the Δrad23 mutant largely mitigated the severe defect of the Δrad23 mutant in photoreactivation. Also, the deletion mutant was severely compromised in radial growth, conidiation, conidial quality, virulence, multiple stress tolerance, and transcriptional expression of many phenotype-related genes. These findings unveil not only the pleiotropic effects of RAD23 in B. bassiana but also a novel RAD23-PHR2 interaction that is essential for the photoprotection of filamentous fungal cells from UVB damage.IMPORTANCE RAD23 is able to repair yeast DNA lesions through nucleotide excision in full darkness, a mechanism distinct from photolyase-dependent photorepair of UV-induced DNA damage but functionally unknown in filamentous fungi. Our study unveils that the RAD23 ortholog in a filamentous fungal insect pathogen varies in subcellular localization according to external cues, interacts with a photolyase required for photorepair of cytotoxic (6-4)-pyrimidine-pyrimidine photoproducts in UV-induced DNA lesions, and plays an essential role in conidial UVB resistance and reactivation of UVB-inactivated conidia under visible light rather than in the dark, as required for nucleotide excision repair. Loss-of-function mutations of RAD23 exert pleiotropic effects on radial growth, aerial conidiation, multiple stress responses, virulence, virulence-related cellular events, and phenotype-related gene expression. These findings highlight a novel mechanism underlying the photoreactivation of UVB-impaired fungal cells by RAD23 interacting with the photolyase, as well as its essentiality for filamentous fungal life.

The Beauveria bassiana Gas3 β-Glucanosyltransferase Contributes to Fungal Adaptation to Extreme Alkaline Conditions.

Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.

Interaction between TATA-Binding Protein (TBP) and Multiprotein Bridging Factor-1 (MBF1) from the Filamentous Insect Pathogenic Fungus Beauveria bassiana.

TATA-binding protein (TBP) is a ubiquitous component of eukaryotic transcription factors that acts to nucleate assembly and position pre-initiation complexes. Multiprotein bridging factor 1 (MBF1) is thought to interconnect TBP with gene specific transcriptional activators, modulating transcriptional networks in response to specific signal and developmental programs. The insect pathogen, Beauveria bassiana, is a cosmopolitan fungus found in most ecosystems where it acts as an important regulator of insect populations and can form intimate associations with certain plants. In order to gain a better understanding of the function of MBF1 in filamentous fungi, its interaction with TBP was demonstrated. The MBF1 and TBP homologs in B. bassiana were cloned and purified from a heterologous E. coli expression system. Whereas purified BbTBP was shown to be able to bind oligonucleotide sequences containing the TATA-motif (Kd ≈ 1.3 nM) including sequences derived from the promoters of the B. bassiana chitinase and protease genes. In contrast, BbMBF1 was unable to bind to these same target sequences. However, the formation of a ternary complex between BbMBF1, BbTBP, and a TATA-containing target DNA sequence was seen in agarose gel electrophoretic mobility shift assays (EMSA). These data indicate that BbMBF1 forms direct interactions with BbTBP, and that the complex is capable of binding to DNA sequences containing TATA-motifs, confirming that BbTBP can link BbMBF1 to target sequences as part of the RNA transcriptional machinery in fungi.

Can Insects Develop Resistance to Insect Pathogenic Fungi?

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25th generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses.

Molecular characterization and comparative virulence of Beauveria bassiana isolates (Ascomycota: Hypocreales) associated with the greenhouse shore fly, Scatella tenuicosta (Diptera: Ephydridae)

Reports of natural infections of the insect pathogenic fungus Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) in greenhouses and laboratory colonies of Scatella tenuicosta Collin (Diptera: Ephydridae), a nuisance pest and vector of plant pathogens, suggest the potential for using B. bassiana for microbial control of this pest. In this study we assessed the diversity of B. bassiana isolates found associated with a colony of shore flies and compared these isolates to two commercial B. bassiana strains. Microsatellite markers were used to assess genetic variation among fungal isolates from adults and pupae of S. tenuicosta, adults of Hexacola neoscatellae Beardsley (Hymenoptera: Figitidae), a hymenopteran parasitoid of S. tenuicosta, and algal food source in greenhouses. Twenty-seven single spore isolates were resolved into three distinct haplotypes using eight microsatellite markers. Two haplotypes were most common, comprising several strains collected from infected shore flies and parasitoids. The third haplotype was observed in only one strain from a shore fly pupa. Bioassays of a representative strain of each haplotype against third-instar larvae, pupae and adults of shore flies showed that adults were more susceptible to the fungus than the other two stages. Secondary pick up of inocula from sprayed pupae by adults resulted in 52% and 55% mortality from B. bassiana strains ST1 and ST2, respectively. Direct spray application on adults resulted in 91 and 94% mortality from these two strains, suggesting their potential as biological control agents against shore flies.

Natural occurrence of Beauveria bassiana in Hypothenemus hampei (Coleoptera: Curculionidae) populations in unsprayed coffee fields

Three unsprayed coffee farms (farm 1, 2 and 3) were studied for the natural occurrence of the insect pathogenic fungus Beauveria bassiana in Hypothenemus hampei populations throughout the rainy season of 2004 (July–November) and 2005 (July–December). B. bassiana infections were found during most sampling dates in both years, on all three farms. The B. bassiana infection levels were higher in 2005 than in 2004 with mean prevalence of 12.1% and 2.7%, respectively. No consistent significant differences in infection level between farms were found in any of the years. B. bassiana infection levels fluctuated widely throughout the season, and peaked at 13.5% on farm 3 in 2004 and at 44.0% on farm 1 in 2005. The H. hampei population was significantly higher in 2004 than in 2005, with 6.9% of the berries infested in 2004 and only 0.7% in 2005. In both years, the H. hampei infestation level was significantly higher on farm 2. No consistent significant differences in H. hampei infestation levels were found between sampling dates on any of the farms. H. hampei infestation levels fluctuated throughout both seasons, and peaked at 15.3% on farm 2 in 2004 and 2.2% on farm 2 in 2005. No consistent density dependent correlation between H. hampei infestation level and B. bassiana infection level was found. Correlations between climatic conditions and B. bassiana or H. hampei were not found.

Biosynthesis of the 2‐Pyridone Tenellin (I) in the Insect Pathogenic Fungus Beauveria bassiana.

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Molecular monitoring and evaluation of the application of the insect-pathogenic fungus Beauveria bassiana in southeast China.

To monitor the fate of inundatively applied strains of Beauveria bassiana against Masson's pine caterpillar, Dendrolimus punctatus, in Magushan Forest, southeast China, and evaluate the potential environmental risks of biocontrol introduction. Filed samplings of mycosed cadavers were carried out continuously for more than one year after the inundative applications of two exotic B. bassiana strains. By employing four complementary molecular markers, the purified cultures from collections were characterized and the two released strains Bb17 and Bb13 were recovered with respective frequencies of 9.1 and 5.2%. The released strains were isolated from nontarget hosts. The genetic diversity of the population of B. bassiana in the trial plot changed dynamically over time and no single genotype strain dominated throughout the sampling period or at different seasons. The indigenous strains were observed to be predominant in the local environment. Co-infection and/or genetic recombination might occur between strains under natural conditions. Field applications of the biocontrol agent B. bassiana appear to pose no major risk to the environment. There was no evidence of displacement of indigenous strains of B. bassiana. The results indicate that the introduction of exotic B. bassiana strains can persist in local environment and infect nontarget insect hosts, highlighting the importance of safety assessment of the large-scale application of fungal biocontrol agents.