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Many filamentous fungi produce only conidia for dispersal and survival in vitro or in vivo. Here, we show that the developmental regulator WetA and the velvet protein VosA are not only required for conidial maturation but indispensable for conidiation in Beauveria bassiana, a filamentous entomopathogen. Deletion of wetA or vosA resulted in more than 90 % transcriptional depression of brlA and abaA, two activator genes in the central developmental pathway, during the critical period of conidiophore development and conidiation. Consequently, ΔwetA and ΔvosA strains lost 98 % in and 88 % of their conidiation capacities under optimal culture conditions, respectively. The conidia of ΔwetA showed more defective features than those of ΔvosA, including smaller size, lesser density, lower hydrophobicity, and impaired cell walls although intracellular trehalose content decreased more in the aging culture of ΔvosA than of ΔwetA. As a result, conidial sensitivity to cell wall perturbation was elevated in ΔwetA but unaffected in ΔvosA, which produced conidia more sensitive to the oxidant menadione and the wet-heat stress at 45 °C. Both deletion mutants showed similar defects in conidial tolerance to high osmolarity or UV-B irradiation but no change in conidial sensitivity to the other oxidant H2O2 or the fungicide carbendazim. Moreover, ΔwetA lost more virulence to Galleria mellonella larvae than ΔvosA. All these phenotypical changes were restored by either wetA or vosA complementation. Taken together, WetA and VosA are indispensable for asexual development and contribute differentially to conidial quality and hence the biological control potential of B. bassiana against insect pests.

The Hog1 mitogen-activated protein (MAP) kinase regulates environmental stress responses and virulence in the entomopathogenic fungus Beauveria bassiana. To further characterize this pathway, we constructed a subtraction library enriched for genes regulated by Hog1. One targeted gene, encoding a novel membrane protein, Ohmm (oxidative homeostasis membrane-protein-mitochondria), was uniquely identified as being downregulated in the ΔHog1 background during growth under non-stress and osmotic stress conditions, but upregulated under oxidative stress. Ohmm was an experimentally validated flavin-binding protein and targeted to the mitochondria. Deletion of Ohmm resulted in increased oxidative stress resistance, whereas overexpression caused an opposite phenotype. The ΔOhmm showed accumulation of reactive oxygen species with alterations in cell wall composition and compatible solute accumulation evident as compared with the wild type parent. Conidiation was reduced > 80%; however, conidia produced by the ΔOhmm strain germinated significantly faster than wild type cells. Insect bioassays using the greater wax moth revealed increased virulence for the ΔOhmm strain in both topical and intrahemocoel injection assays, indicating a negative effect of the presence of Ohmm with respect to pathogenesis. As predicted from our characterization, deletion of Ohmm in a ΔHog1 background rescued its oxidative sensitivity phenotype, confirming that Ohmm acts downstream of the Hog1 MAP-kinase., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

On Reunion Island successful biological control of the sugarcane white grub Hoplochelus marginalis Fairmaire (Coleoptera: Melolonthidae) has been conducted for decades with strains from the entomopathogenic fungal genus Beauveria (Ascomycota: Hypocreales). A study based on morphological characters combined with a multisequence phylogenetic analysis of genes that encode the translation elongation factor 1-alpha (TEF1), RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2) and the Bloc nuc intergenic region was carried out on Beauveria strains isolated on Reunion and Madagascar from H. marginalis. This study revealed that these strains, previously identified as Beauveria brongniartii, did not match that species and are closely related to but still distinct from B. malawiensis strains. Therefore we describe the Reunion Island fungus as the new species B. hoplocheli., (© 2015 by The Mycological Society of America.)

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.

The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Quinones are widely distributed in nature and exhibit diverse biological or pharmacological activities; however, their biosynthetic machineries are largely unknown. The bibenzoquinone oosporein was first identified from the ascomycete insect pathogen Beauveria bassiana>50 y ago. The toxin can also be produced by different plant pathogenic and endophytic fungi with an array of biological activities. Here, we report the oosporein biosynthetic machinery in fungi, a polyketide synthase (PKS) pathway including seven genes for quinone biosynthesis. The PKS oosporein synthase 1 (OpS1) produces orsellinic acid that is hydroxylated to benzenetriol by the hydroxylase OpS4. The intermediate is oxidized either nonenzymatically to 5,5'-dideoxy-oosporein or enzymatically to benzenetetrol by the putative dioxygenase OpS7. The latter is further dimerized to oosporein by the catalase OpS5. The transcription factor OpS3 regulates intrapathway gene expression. Insect bioassays revealed that oosporein is required for fungal virulence and acts by evading host immunity to facilitate fungal multiplication in insects. These results contribute to the known mechanisms of quinone biosynthesis and the understanding of small molecules deployed by fungi that interact with their hosts.

Two Ras ATPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. Here we show the significance of a novel Ras homolog (Ras3) for Beauveria bassiana. Ras3 possesses five domains and two GTP/GDP switches typical for Ras family and was proven to localize to plasma membrane despite the position change of a membrane-targeting cysteine in C-terminal CAAX motif. Deletion of ras3 altered temporal transcription pattern of ras1 instead of ras2. Compared with wild-type, Δras3 grew significantly faster in a rich medium but slower in some minimal media, and produced far fewer conidia with impaired quality, which was evident with slower germination, attenuated virulence, reduced thermotolerance and decreased UV-B resistance. Moreover, Δras3 was much more sensitive to the oxidative stress of menadione than of H2O2 and to the stress of high osmolarity than of cell wall perturbation during growth. The high sensitivity of Δras3 to menadione was concurrent with reductions in both gene transcripts and total activity of superoxide dismutases. Intriguingly, the high osmosensitivity was concurrent with not only reduced transcripts of a critical transcription factor (Msn2) and most signaling proteins in the high-osmolarity-glycerol pathway of Δras3 but nearly undetectable phosphorylation signal of Hog1 hallmarking the pathway. All the changes were restored by ras3 complementation. Taken together, Ras3 is involved in the Hog1 pathway required for osmoregulation and hence can positively regulate conidiation, germination, multi-stress tolerance and virulence linked to the biological control potential of the filamentous insect pathogen., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Transcriptional control of the cell cycle by forkhead (Fkh) transcription factors is likely associated with fungal adaptation to host and environment. Here we show that Fkh2, an ortholog of yeast Fkh1/2, orchestrates cell cycle and many cellular events of Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of Fkh2 in B. bassiana resulted in dramatic down-regulation of the cyclin-B gene cluster and hence altered cell cycle (longer G2/M and S, but shorter G0/G1, phases) in unicellular blastospores. Consequently, ΔFkh2 produced twice as many, but smaller, blastospores than wild-type under submerged conditions, and formed denser septa and shorter/broader cells in aberrantly branched hyphae. In these hyphae, clustered genes required for septation and conidiation were remarkedly up-regulated, followed by higher yield and slower germination of aerial conidia. Moreover, ΔFkh2 displayed attenuated virulence and decreased tolerance to chemical and environmental stresses, accompanied with altered transcripts and activities of phenotype-influencing proteins or enzymes. All the changes in ΔFkh2 were restored by Fkh2 complementation. All together, Fkh2-dependent transcriptional control is vital for the adaptation of B. bassiana to diverse habitats of host insects and hence contributes to its biological control potential against arthropod pests.

Conidiation promotes fungal dispersal and survival in the environment, and is a determinant for the biocontrol potential of Beauveria bassiana. The SNF1/AMPK protein kinases function as an important regulator of fungal development and energy metabolism, and play a crucial role in conidiation of the filamentous fungi. In previous study, it has been established that the B. bassiana homolog (BbSNF1) controls conidial production. This study showed that the ΔBbSNF1 mutants displayed a delayed development of mycelia and conidia, but the conidiophore morphogenesis was not significantly changed in the mutants. Ablation of BbSNF1 significantly changed the metabolic homeostasis of intracellular amino acids during conidiation, and caused a notable reduction in the contents of seven amino acids (i.e., arginine, alanine, valine, phenylalanine, lysine, leucine, and glutamic acid). All above amino acids could recover conidiation of the mutants in different extents (ranging from 43.3 to 300 %). Transcriptomic analysis revealed many putative target genes regulated by BbSNF1 and associated with conidial development, and these genes were primarily involved in metabolism, cell rescue, and transport. Particularly, four categories related to the amino acid degradation were over-represented in the up-regulated genes, and three categories related to the amino acid biosynthesis were over-represented in the down-regulated genes. Moreover, the ΔBbSNF1 mutants displayed reduced expression level of the upstream and central regulators of conidiation, as well as the other regulator and cytoskeleton genes. Our data indicate that SNF1 kinase contributes to B. bassiana conidiation by regulating the metabolism and the central regulators of conidiation.

Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The entomopathogenic fungus Beauveria bassiana is widely used in pest biocontrol strategies. We evaluated both the antioxidant response mediated by compatible solutes, trehalose or mannitol, and the expression of related genes using oxygen pulses at three oxygen concentrations in solid state culture (SSC): normal atmosphere (21% O2), low oxygen (16% O2) and enriched oxygen (26% O2). Trehalose concentration decreased 75% after atmospheric modifications in the cultures, whereas mannitol synthesis was three-fold higher under the 16% O2 pulses relative to normal atmosphere (100 and 30 μg mannitol mg(-1) biomass, respectively). Confirming this result, expression of the mpd gene, coding for mannitol-1-P dehydrogenase (MPD), increased up to 1.4 times after O2 pulses. The expression of the bbrgs1 gene, encoding a regulatory G protein related to conidiation, was analysed to explain previously reported differences in conidial production. Surprisingly, expression of bbrgs1 decreased after atmospheric modification. Finally, principal component analysis (PCA) indicated that 83.39% of the variability in the data could be explained by two components. This analysis corroborated the positive correlation between mannitol concentration and mpd gene expression, as well as the negative correlation between conidial production and bbrgs1 gene expression. This study contributes to understanding of antioxidant and molecular response of B. bassiana induced under oxidant conditions., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Fungal secondary metabolites are chemical compounds important for development, environmental adaptation and for potential biotechnological and pharmaceutical applications. Oosporein, a red-pigmented benzoquinone, produced by many fungal insect pathogenic Beauveria spp., shows remarkable functional diversity, displaying antimicrobial, antiviral and even anti-proliferative activities. A homologue of the msn2/seb1 transcription factor was identified in a Beauveria bassiana random T-DNA insertion library. Targeted gene-knockout of Bbmsn2 resulted in reduced growth and increased sensitivity to Calcofluor White, H2 O2 and Congo Red. However, when normalized to growth at 26°C, the ΔBbmsn2 mutant was more tolerant to high temperature (32°C) than the wild type parent. The ΔBbmsn2 mutant also displayed a pH-dependent growth phenotype, with little growth seen at pH < 5.0 but, better growth at alkaline conditions (pH > 8.0). Unexpectedly, a pH-dependent deregulation of a red pigment, identified as oosporein, was seen in the ΔBbmsn2 mutant. The ΔBbmsn2 strain was impaired in virulence in both topical and intrahaemocoel injection bioassays against Galleria mellonella. ΔBbmsn2 proliferation in the host haemolymph and conidiation on the host cadaver was reduced. These data indicate that Bbmsn2 acts as a negative regulator of oosporein production and contributes to virulence and growth in response to external pH in B. bassiana., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray.

Hirsutella thompsonii (Ht) is a fungal pathogen of acarines and the primary cause of epizootics among mites. The draft genomes of two isolates of Ht (MTCC 3556: Ht3, 34.6 Mb and MTCC 6686: Ht6, 34.7 Mb) are presented and compared with the genomes of Beauveria bassiana (Bb) ARSEF 2860 and Ophiocordyceps sinensis (Os) CO18. Comparative analysis of carbohydrate active enzymes, pathogen-host interaction genes, metabolism-associated genes, and genes involved in biosynthesis of secondary metabolites in the four genomes was carried out. Reduction in gene family sizes in Ht3 and Os as compared with Ht6 and Bb is observed. Analysis of the mating type genes in Ht reveals the presence of MAT idiomorphs which is suggestive of cryptic sexual traits in Ht. We further identify and classify putative chitinases that may function as virulence factors in fungal entomopathogens due to their role in degradation of arthropod cuticle., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The desire for decreased reliance on chemical pesticides continues to fuel interest in alternative means for pest control including the use of naturally occurring microbial insect pathogens. Insects, as vectors of disease causing agents or as agricultural pests, are responsible for millions of deaths and significant economic losses worldwide, placing stresses on productivity (GDP) and human health and welfare. In addition, alterations in climate change are likely to affect insect ranges, expanding their access to previously constrained geographic areas, a potentially worrisome outcome. Metarhizium anisopliae and Beauveria bassiana, two cosmopolitan fungal pathogens of insects found in almost all ecosystems, are the most commonly applied mycoinsecticides for a variety of insect control purposes. The availability of the complete genomes for both organisms coupled to robust technologies for their transformation has led to several advances in engineering these fungi for greater efficacy and/or utility in pest control applications. Here, we will provide an overview of the fungal-insect and fungal-plant interactions that occur and highlight recent advances in the genetic engineering of these fungi. The latter work has resulted in the development of strains displaying (1) increased resistance to abiotic stress, (2) increased cuticular targeting and degradation, (3) increased virulence via expression of insecticidal protein/peptide toxins, (4) the ability to block transmission of disease causing agents, and (5) the ability to target specific insect hosts, decrease host fecundity, and/or alter insect behaviors.

The fungal pathogen Beauveria bassiana causes serious economic losses in sericulture. Its origin is usually attributed to the release of B. bassiana insecticides against pine caterpillars (Dendrolimus punctuatus). In the present study, 488 B. bassiana isolates obtained from silkworm (Bombyx mori) collected from 13 Chinese provinces, and 327 B. bassiana isolates obtained from D. punctatus collected from 9 provinces, were analyzed for population genetic structure using the ISSR technique based on genetic distance. A UPGMA dendrogram clustered them into three independent clades: two B. mori clades and one D. punctatus clade. A 3-D principal component analysis further divided them into two completely independent host groups, revealing high host-specificity. This suggested that white muscardine occurring in B. mori populations throughout southern China was not caused by any B. bassiana strain either naturally prevailing in D. punctatus populations or by any strain artificially released as a fungal insecticide against D. punctatus. We further investigated the genetic differentiation coefficient Gst and gene flow between B. mori-pathogenic and D. punctatus-pathogenic B. bassiana isolates from across China and from five provinces inhabited by both B. mori and D. punctatus. The Gst value across China was computed as 0.410, while the values of the five provinces ranged from 0.508 to 0.689; all above 0.25, which is the threshold for significant genetic differentiation. This suggests that B. bassiana strains isolated from the two different hosts maintained their respective heredity without a convergent homogenization trend, and reduces the possibility that the host range of the caterpillar isolates could expand and enhance their virulence in B. mori. These findings indicate that the use of B. bassiana does not threaten the safety of sericulture., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The eukaryotic calcineurin (CN) pathway comprising catalytic A (CnA) and regulatory B subunits (CnB) is crucial for many biological processes but functionally unexplored in entomopathogenic fungi. Here, we characterise three CN subunits (CnA1, CnA2 and CnB) and a downstream CN-responsive zinc finger transcription factor (Crz1) in Beauveria bassiana. CN-mediated phosphatase activity decreased by 16-38 % in all deletion mutants compared with wild type. Growth and conidiation were most defective in ΔcnB, which showed a large proportion of abnormally branched germlings but were less defective in ΔcnA1 and ΔcnA2. Conidiation defects also occurred in Δcrz1, uniquely accompanied with slower germination. Compared with wild type, the four deletion mutants became, to varying degrees, more sensitive to Ca(2+), Mn(2+), Zn(2+), Mg(2+), two oxidants, three cell wall stressors, carbendazim, heat shock and ultraviolet (UV)-B irradiation. They were also less virulent to Spodoptera litura larvae. Only ΔcnB and Δcrz1 were less tolerant to high osmolarity. The altered phenotypes of the deletion mutants were associated with lower intracellular mannitol and trehalose levels, reduced overall activity of superoxide dismutases and catalases, altered cell wall composition and down-regulation of numerous phenotype-influencing genes. Additionally, the transcription of six cascaded genes in two stress-responsive mitogen-activated protein kinase (MAPK) pathways and the phosphorylation of hallmarking Hog1 and Slt2 were largely down-regulated in all the deletion mutants under osmotic and cell wall stresses, respectively. All the changes were restored by gene complementation. Taken together, three calcineurin subunits and Crz1 play vital, but variable, roles in B. bassiana responses to environmental stresses during development and host signals during infection.

Iron is an essential element for life. However, the iron overload can be toxic. Here, we investigated the significant increase of tenellin and iron-tenellin complex production in ferricrocin-deficient mutants of Beauveria bassiana. Our chemical analysis indicated that the ferricrocin-deficient mutants T1, T3 and T5 nearly abolished ferricrocin production. In turn, these mutants had significant accumulation of iron-tenellin complex in their mycelia at 247-289 mg g(-1) cell dry weight under iron-replete condition. Both tenellin and iron-tenellin complex were not detected in the wild-type under such condition. Mass analysis of the mutants' crude extracts demonstrated that tenellin formed a 3:1 complex with iron in the absence of ferricrocin. The unexpected link between ferricrocin and tenellin biosynthesis in ferricrocin-deficient mutants could be a survival strategy during iron-mediated oxidative stress., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The complete mitochondrial genome of the entomopathogenic fungus Beauveria pseudobassiana (Ascomycota, Cordycipitaceae) was determined and found to be 28,006 bp in length. It encodes genes for 14 proteins, 2 ribosomal RNA subunits, 25 transfer RNAs with the synteny identical to those of B. bassiana and B. brongniartii. The overall base composition is 36.7% A, 35.8% T, 15.3% C and 12.2% G with an AT content of 72.5%. Group-І introns were found in large rRNA gene (rnl) and cox2 gene that include rps3 gene and putative GIY-YIG homing endonuclease, respectively. The mitochondrial genome of B. pseudobassiana will contribute to the investigation of the phylogenetic relationship, taxonomic resolution and biogeography of Beauveria.

Multiple Vcx1 (vacuolar calcium exchanger) paralogues exist in many filamentous fungi but are functionally unexplored unlike a single Vcx1 ortholog well characterized in yeasts. Here we show that five Vcx1 paralogues (Vcx1A-E) in Beauveria bassiana are conditionally functional for intracellular Ca(2+) homeostasis and contribute differentially to multistress tolerance and virulence in the filamentous entomopathogen. Each vcx1 deletion drastically upregulated transcriptional expressions of four other partners and six P-type Ca(2+)-ATPases, resulting in elevated or lowered intracellular Ca(2+) concentration in some deletion mutants treated with Ca(2+) stress or untreated at 25 and 30 °C. When calcineurin was inactivated by cyclosporine A, Ca(2+) tolerance decreased by 11-17% in five Δvcx1 mutants, but Mn(2+) sensitivity increased only in Δvcx1A and Δvcx1D, at optimal 25 °C. These two mutants were also more sensitive to Ca(2+) stress at 30 °C when calcineurin was active, and showed minor growth defect at 25 and 30 °C when calcineurin was inactive. Moreover, all the Δvcx1 mutants were more sensitive to dithiothreitol (stress-response trigger to endoplasmic reticulum) and Congo red (cell wall stressor); three of them were consistently less tolerant to the oxidants menadione and H2O2. The fungal virulence to Galleria mellonella larvae decreased by 15-40% in four Δvcx1 mutants excluding Δvcx1E, which was uniquely defective in conidial thermotolerance. All the changes were restored by each vcx1 complementation. Our findings indicate that the five Vcx1 paralogues in B. bassiana contribute differentially to calcineurin-dependent Ca(2+)/Mn(2+) tolerance, multistress responses and virulence, and recall attention to multifunctional Vcx1 paralogues in filamentous fungi., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with <5 % of the cells remaining viable after 30 days as compared with >80 % of the WT. Reduced production of extracellular proteins was also observed for the ΔBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the ΔBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes., (© 2014 The Authors.)

Human and animal disease-transmitting hard ticks (Acari: Ixodidae) are of great concern for public health and animal farming. Alternatives to tick control by chemical acaricides are urgently needed, and one intensively evaluated biocontrol strategy is based on the use of tick-pathogenic filamentous fungi. An indispensable prerequisite for the development of tick-derived fungal isolates into registered myco-acaricides is their sound taxonomic characterisation. A set of fungal strains isolated from ixodid ticks in the Republic of Moldova was genetically characterised at the genus and species level together with further tick-derived fungal isolates from different geographic locations in Europe and North America. In a previous study, the same isolates had been assigned to the species Beauveria bassiana. Using a recent molecular taxonomic approach based on phylogenetic reconstruction from both internal transcribed spacer (ITS) and protein-encoding gene sequences, all fungi investigated were conclusively assigned to one of the two "hyphomycete" genera, Beauveria or Isaria (Ascomycota; Hypocreales; Cordycipitaceae). Within the genus Isaria, two species, Isaria farinosa and Isaria fumosorosea, were equally represented. Within the genus Beauveria, the comparatively rare species Beauveria pseudobassiana was found to strongly prevail among the isolates from Moldova, and one of the two tick-derived Beauveria strains from North America could be assigned to this species as well. In particular, the previous classification as B. bassiana could not be confirmed for any of the characterised tick pathogens from Europe and North America. The data presented here lend support to the hypothesis that within the genus Beauveria specific adaptation to ticks might have occurred within the species B. pseudobassiana. To test this hypothesis, a more extensive molecular taxonomic survey carefully reconsidering previous taxonomic assignments of tick-derived fungal isolates is needed., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

SNF1/AMPK protein kinase plays important roles in fungal development and activation of catabolite-repressed genes. In this study, we characterized the role of the Beauveria bassiana SNF1 ortholog. The vegetative growth of the ΔBbSNF1 mutant was reduced by 16 to 50 % on diverse carbon/nitrogen sources. Transcriptional analysis revealed a collection of proteases and chitinases that were not induced when the mutant was grown on complex carbon/nitrogen sources. BbSNF1 also contributes to extracellular acidification. The ΔBbSNF1 mutant had enhanced production of lactic, pyruvic, and citric acid, but oxalic acid production was partially repressed. Transcriptional analysis showed that a set of genes involved in acid biosynthesis and secretion was changed in the disruption mutant, indicating that BbSNF1 controls the production of different organic acids with different mechanisms. Deletion of BbSNF1 resulted in a significant reduction in conidiation (57-75 %) and blastospore yield (80-95 %) in the mutant. Additionally, BbSNF1 regulates the morphology of blastospore-forming structures and the in vitro blastospore size. Insect bioassays revealed that the ΔBbSNF1 strain exhibited an approximately doubled median lethal time in topical bioassays, but the decreased virulence in intrahemocoel assays (~20 % change) was not as great as in the topical bioassays. These data suggest that BbSNF1 is important in penetration through the host cuticle. Moreover, a series of genes regulated by BbSNF1 and associated with blastospore formation were primarily involved in metabolism, cell cycle, and transportation. In conclusion, the SNF1/AMPK kinase contributes to the biocontrol potential of B. bassiana by mediating cellular differentiation and utilization of carbon/nitrogen sources.

Emerald ash borer is an invasive species from Asia. Beauveria bassiana strain L49-1AA is being tested for the control of emerald ash borer in Canada, using an autocontamination trapping system. We have developed a simplified allele discrimination polymerase chain reaction (PCR) assay to screen B. bassiana strain, L49-1AA from other Beauveria species by targeting the inter-strain genetic differences in 5' end of EF1-α gene of the genus Beauveria. A single nucleotide polymorphism (SNP) site, T→C was identified only in L49-1AA and was used to develop a simplified allele discrimination polymerase chain reaction (PCR) assay based on a modified allelic inhibition of displacement activity (AIDA) approach for distinguishing B. bassiana L49-1AA from all background Beauveria isolates. The SNP site was employed to design inner primers but with a deliberate mismatch introduced at the 3' antepenultimate from the mutation site in order to maximize specificity and detection efficiency. Amplification was specific to L49-1AA without cross-reaction with DNA from other Beauveria strains. In addition, the designed primers were also tested against environmental samples in L49-1AA released plots and observed to be highly efficient in detecting and discriminating the target strain, L49-1AA from both pure and crude DNA samples. This new method can potentially allow for more discriminatory tracking and monitoring of released L49-1AA in our autocontamination and dissemination projects for managing EAB populations. Additionally, the modified-AIDA format has potential as a tool for simultaneously identifying and differentiating closely related Beauveria species, strains/isolates as well as general classification of other pathogens or organisms., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Members of α-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mnt1 in Beauveria bassiana, a filamentous enotmopathogen, and found that they were positive, but differential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on substrates with altered carbon or nitrogen sources and availability, accompanied with reduced conidial size and complexity. This contrasts to the dispensability of Ktr1 for fungal growth and conidiation. More cell wall damage occurred in Δktr4 and Δkre2 than in Δktr1, including altered contents of the cell wall components mannoproteins, α-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Δktr4 and Δkre2 became more sensitive to oxidation and cell wall perturbation than Δktr1 during colony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Δktr4 and Δkre2 but only the thermotolerance decreased in Δktr1. All the phenotypical changes were well restored to wild-type levels by the complementation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktr1 although all of them are significant contributors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The entomopathogenic fungus Beauveria bassiana (Balsamo 1835) Vuillemin is an effective alternative control agent against some agricultural pests and biological vectors of important diseases such as Chagas disease. In this work we studied an isolate of Beauveria bassiana from of the town of San Antonio Rayón, Puebla, Mexico and its entomopathogenic effects on Meccus pallidipennis (Stal 1872). Phylogenetic analysis using molecular comparison of the ITS and EF1α genes, showed that the resulting cladogram places the BUAP 04 strain with a relationship closer to the AFAO 9-6 strain, within the diversity of the B. bassiana sensu lato group. Although there was the possibility that BUAP 04 strain was a direct descendant of strains used in campaigns of biologic control, molecular study allowed us to recognize that it was a different fungus due to numerous inserts. A strain isolated from a T. dimiata was evaluated for pathogenicity against another triatoma (Meccus pallidipennis) species obtaining an LC50 of 4.16 × 10(6) spores/mL, confirming that the BUAP 04 strain is virulent for M. pallidipennis and could be a good prospect for formulations to control M. pallidipennis.

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ~3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ~24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin>laminarin>lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassianaderived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.

O-Mannosylation dependent on the protein O-mannosyltransferase (Pmt) family is an essential post-translational modification process in eukaryotes, but their connection to the biocontrol potential of a filamentous entomopathogen against arthropod pests is not understood. Here, we characterized the functions of three Pmt orthologues (Pmt1, Pmt2 and Pmt4) in the Pmt family of Beauveria bassiana and found that they were positive, but differential, regulators of the fungal growth, conidiation, multi-stress tolerance and virulence. Three Pmt2 knockdown mutants (ΔPmt2 was lethal), ΔPmt1 and ΔPmt4 grew 20-79% slower on nutrition-rich and limited media. Their conidial yields on a standard medium were reduced by 17-62%, accompanied with delayed germination. All the mutants became significantly less tolerant to most stresses of cell wall perturbation, high osmolarity, oxidation, wet heat and UV-B irradiation during colony growth and conidial germination and lost virulence by 53-62% via cuticle infection, although their virulence via hemocoel injection was not affected. Strikingly, these phenotypic defects were accompanied with remarkable cell wall damage, including thinner cell wall, lower conidial hydrophobicity and altered cell wall composition. All the changes were well restored to wild-type levels by targeted Pmt1 or Pmt4 complementation. Our results indicate for the first time that Pmt1, Pmt2 and Pmt4 are all required for the full biocontrol potential of B. bassiana despite differential contributions., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phytochromes (Phy) in filamentous fungi are Group VIII histidine kinases that share a unique N-terminal photosensory core, but their functions are largely unknown. Here we show that Beauveria bassiana Phy (Bbphy) is functionally vital for growth, conidiation and multistress tolerance of the fungal entomopathogen lacking sexual stage. Colony growth of ΔBbphy was significantly slower in a nutrition-rich medium but faster in several minimal media. Conidial yield of ΔBbphy in the rich medium increased at the fitted rate of 3.4 × 10(7) conidia h(-1) white light in the light/dark cycles of 0:24 to 16:8 h, decreased greatly in the short-, long- and full-day cycles of red/far-red light, but was unaffected under full-day blue light. Moreover, ΔBbphy showed higher osmosensitivity, increased antioxidant capability, and decreased conidial thermotolerance and UV-B resistance, accompanied with downregulation of Hog1 phosphorylation and of four Hog1-related genes under osmotic stress, and upregulation of five superoxide dismutases and four catalases under oxidative stress. All the changes were restored by the gene complementation. Taken together, Bbphy controls conidiation by responding to daylight length and red/far-red light and regulates multistress responses perhaps because of an involvement in Hog1 pathway. Our findings highlight diverse functions of Bbphy in B. bassiana., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Ecm33 is one of several glycosylphosphatidylinositol (GPI)-anchored proteins. This protein is known to be involved in fungal cell wall integrity, but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33 orthologues, i.e., Bbecm33 in Beauveria bassiana and Mrecm33 in Metarhizium robertsii. Bbecm33 and Mrecm33 were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33 and Mrecm33 caused slight growth defects, but conidial yield decreased much more in ΔBbecm33 (76 %) than in ΔMrecm33 (42 %), accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell walls in ΔBbecm33 only. Consequently, ΔBbecm33 was far more sensitive to the cell wall-perturbating agents Congo red and sodium dodecyl sulfate (SDS) than ΔMrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants (menadione and H2O2), two fungicides (carbendazim and ethirimol), osmotic salt NaCl, and Ca(2+) during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by 55 % in ΔBbecm33 but was unaffected in ΔMrecm33, unlike a similar decrease (25-28 %) of conidial thermotolerance in both. All the changes were restored to wild-type levels by gene complementation through ectopic gene integration in each fungus. However, neither ΔBbecm33 nor ΔMrecm33 showed a significant change in virulence to a susceptible insect host. Our results indicate that Bbecm33 and Mrecm33 contribute differentially to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.

Prior knowledge of the local population structure of entomopathogenic fungi is considered an important requisite when developing microbial control strategies against major pests of crops such as white grubs. An extensive survey in the estate of Guanajuato, one of the main agricultural regions of Mexico, was carried out to determine the abundance and diversity of entomopathogenic fungi in soil. Soil collected from 11 locations was baited for entomopathogenic fungi using Galleria mellonella. In addition, all isolates were morphologically identified and selected isolates of Beauveria and Metarhizium isolates identified using Bloc and ITS or Elongation Factor 1-α and ITS sequence information respectively. Genotypic diversity was then studied using microsatellite genotyping. The proportion of isolates belonging to each genus varied amongst all locations. The species Beauveria bassiana, B. pseudobassiana and Metarhizium robertsii were found, with B. bassiana being the most abundant and widely distributed. Microsatellite genotyping showed that the 36 B. bassiana isolates were grouped in 29 unique haplotypes, but with no separation according to geographical origin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ssk1-type response regulator proteins are the core elements of histidine-to-aspartate systems that mediate fungal stress tolerance, a determinant to the biocontrol potential of fungal entomopathogens. We characterized the functions of Beauveria bassiana Ssk1 (Bbssk1) by analyzing multi-phenotypic changes in ΔBbssk1 and differentially expressed genes in the digital gene expression (DGE) libraries of ΔBbssk1 and wild-type constructed under osmotic stress. The Bbssk1 disruption caused 25 % reductions in conidial yield and virulence to Spodoptera litura larvae and significant defects in tolerances to two osmotic salts (81-84 %), H2O2 oxidation (23 %), two fungicides (21-58 %), three cell wall biosynthesis inhibitors (25-36 %), and three metal ions (~8 %) during colony growth, respectively, but little changes in cell sensitivity to menadione oxidation and in conidial thermotolerance and UV-B resistance. RNA-seq analysis with the DGE libraries revealed differential expressions of 1,003 genes in the ΔBbssk1 genome. Of those, many associated with conidiation, stress response, xenobiotic transport, cell wall integrity, and protein/carbohydrate metabolism were remarkably down-regulated, including the genes involved in mitogen-activated protein kinase (MAPK) signal pathway that downstream of Bbssk1. Our results indicate that Bbssk1 regulates positively the expressions of the MAPK cascade in the pathway of B. bassiana and many more downstream genes associated with conidiation, multi-stress tolerance, and virulence.

Background: The chemical control of the Chagas disease vector Triatoma infestans is endangered by the emergence of pyrethroid resistance. An effective alternative control tool is the use of the entomopathogenic fungus Beauveria bassiana. The effect of deltamethrin on fungal growth, gene expression and enzyme activity in relation to detoxification, antioxidant response and oxidative stress levels was studied to evaluate fungal tolerance to deltamethrin., Results: The mean inhibitory concentration (IC50 ) was 50 µg deltamethrin/cm(2). Cytochrome P450 genes were differentially expressed; cyp52X1 and cyp617N1 transcripts were > 2-fold induced, followed by cyp655C1 (1.8-fold). Minor effects were observed on genes encoding for other P450s, epoxide hydrolase and glutathione S-transferase (GST). Superoxide dismutase (SOD) genes showed induction levels ≤ 2, catalase (CAT) and glutathione peroxidase genes were also induced ∼ 2-3-fold and < 2-fold, respectively. The activities of enzymes participating in the antioxidant defense system and phase II detoxification were also evaluated; SOD, CAT and GST activity showed significant differences with deltamethrin concentration. Lipid peroxidation levels and free proline content were also altered., Conclusions: Beauveria bassiana GHA can be used combined with deltamethrin without significant metabolic detrimental effects. This combination will help optimizing the benefits and increasing the efficacy of vector control tools., (© 2013 Society of Chemical Industry.)

Metarhizium robertsii is a plant root colonizing fungus that is also an insect pathogen. Its entomopathogenicity is a characteristic that was acquired during evolution from a plant endophyte ancestor. This transition provides a novel perspective on how new functional mechanisms important for host switching and virulence have evolved. From a random T-DNA insertion library, we obtained a pathogenicity defective mutant that resulted from the disruption of a sterol carrier gene (Mr-npc2a). Phylogenetic analysis revealed that Metarhizium acquired Mr-npc2a from an insect by horizontal gene transfer (HGT). Mr-NPC2a binds to cholesterol, an animal sterol, rather than the fungal sterol ergosterol, indicating it retains the specificity of insect NPC2 proteins. Mr-NPC2a is an intracellular protein and is exclusively expressed in the hemolymph of living insects. The disruption of Mr-npc2a reduced the amount of sterol in cell membranes of the yeast-like hyphal bodies that facilitate dispersal in the host body. These were consequently more susceptible to insect immune responses than the wild type. Transgenic expression of Mr-NPC2a increased the virulence of Beauveria bassiana, an endophytic insect-pathogenic fungus that lacks a Mr-NPC2a homolog.

The brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a destructive invasive pest and has become one of the most economically-important rice pests in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Clavicipitaceae) and B. brongniartii (Saccardo), have shown great potential for the management of some sucking pest species. In this study, to explore alternative strategies for sustainable control of the sucking pest population, nine isolates of Beauveria from different pests were bioassayed under the concentrated standard spray of 1000 conidia/mm(2) in laboratory. The cumulative mortalities of adults ranged from 17.2 to 79.1% 10 days after inoculation. The virulence among all tested isolates exhibited significant differences (at p = 0.05). The highest virulent isolate was Bb09, which killed 79.1% of the treated insects and had a median lethal time of 5.5 days. Its median lethal concentration values were estimated as 134 conidia/mm(2) on day 10. The chitinase activities of nine isolates were also assayed. The results showed that the chitinase activity (18.7 U/mg) of isolate Bbr09 was the highest among all tested isolates. The biological characteristics of these strains, including growth rate, sporulation, and germination rate, were further investigated. The results showed that strain Bbr09 exhibited the best biological characteristics with relatively higher hyphal growth rate, the highest spore production, and the fastest spore germination. The isolate of Bbr09 had strong pathogenicity and exhibited great potential for sustainable control of N. lugens., (This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.)

To survive, the entomopathogenic fungus Beauveria bassiana, which shows promise as a biocontrol agent for a variety of pests, including agricultural and forestry pests and vectors of human pathogens, must tailor gene expression to the particular pH of its environment. The pH response transcription factor gene BbPacC and its flanking sequence were cloned from this fungus. Quantitative reverse transcription (RT)-PCR revealed that it is highly induced by alkaline pH and salt stress, and the expression level achieved twice that of the housekeeping gene γ-actin. A microfluorometric assay indicated that the 1479 bp promoter region could activate the expression of enhanced green fluorescent protein (EGFP) under the same conditions. Truncation analysis showed that the 1479, 1274, 1040, 888 and 742 bp promoters have similar efficiencies in activating expression of β-glucuronidase (GUS). The GUS activities of corresponding transformants reached approximately 50 % that of those containing the strong constitutive promoter PtrpC. A truncation upstream at the -572 bp position (referenced to the translation start codon ATG), however, resulted in a significant loss of GUS activity. Both the upstream absences of the -502 and -387 bp positions caused almost complete loss of GUS activity. These results suggest that PPacC is an efficient, alkaline, and salt-inducible promoter, the core cis-elements are mainly located within the -742 to -502 bp region, and promoters equal to or longer than 742 bp may be feasible for regulating gene expression in response to an ambient pH or salt stress.

The possibility of using hyphomycete fungi as suitable biocontrol agents against greenhouse whitefly has led to the isolation of various insect pathogenic fungi. Among them is Beauveria bassiana, one of the most studied entomopathogenic fungi. The objective of this study was to use B. bassiana M130 as an insecticidal agent against the greenhouse whitefly. M130 isolated from infected insects is known to be a biocontrol agent against greenhouse whitefly. Phylogenetic classification of M130 was determined according to its morphological features and 18S rRNA sequence analysis. M130 was identified as B. bassiana M130 and showed chitinase (342.28 units/ml) and protease (461.70 units/ml) activities, which were involved in the invasion of the host through the outer cuticle layer, thus killing them. The insecticidal activity was 55.2% in petri-dish test, 84.6% in pot test, and 45.3% in field test. The results of this study indicate that B. bassiana has potential as a biological agent for the control of greenhouse whitefly to replace chemical pesticides.

Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of Anopheles gambiae, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negatively regulates the activity of a putative TEP1 convertase, which converts the full-length TEP1-F form into active TEP1cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including Plasmodium berghei, Escherichia coli and the entomopathogenic fungus Beauveria bassiana. We also provide further evidence for the existence of a functional link between TEP1 and activation of hemolymph prophenoloxidase during systemic infections. Interestingly, the enhanced TEP1-mediated immune response in CLIPA2 knockdown mosquitoes correlated with a significant reduction in fecundity, corroborating the existence of a trade-off between immunity and reproduction. In sum, CLIPA2 is an integral regulatory component of the mosquito complement-like pathway which functions to prevent an overwhelming response by the host in response to systemic infections.

Bioassays were designed to evaluate Beauveria bassiana (Balsamo) Vuillemin strain GHA against Listronotus maculicollis (Kirby) adults. B. bassiana and its "inert" carrier oil in the product BotaniGard and the inert carrier oil alone provided 99 and 96% mortality, respectively, in petri dish assays 1 d after treatment when applied in 1 ml water. When the same treatments were applied in 0.5 ml of carrier water, mortality was only 1.4 and 0.7%, respectively, 1 d after treatment. After 10 d in petri dishes, B. bassiana and its inert carrier oil and the inert carrier oil alone applied in 0.5 ml water showed 77 and 9% mortality, respectively. When one-tenth the label dosage of B. bassiana and inert carrier oil was combined with neonicotinoids applied in 1 ml water, there were significant increases in weevil mortality over the neonicotinoids alone 1 d after treatment. When 88.7% of one-tenth the label dosage of inert carrier oil alone was combined with neonicotinoids clothianidin, imidacloprid, and dinotefuran applied in 1 ml water, there was also a significant increases (38%) with clothianidin in weevil mortality over clothianidin alone 1 d after treatment. B. bassiana and its inert carrier oil provided 28, 50, and 78% mortality at the highest label dosage and 47, 76, and 89% mortality at 4x the highest label dosage in turf plug assays at 7, 10, and 14 d after treatment. Addition of 5 or 20% MycoMax (a nutrient source for B. bassiana) did not significantly increase mortality.

In fungi, G-protein coupled receptors (GPCRs) link ligand/nutrient sensing to growth, mating, developmental/life-stage activation and pathogenesis. A GPCR was characterized from the entomopathogenic fungus, Beauveria bassiana (BbGPCR3), which links nutrient sensing to stress response and development. ΔBbGPCR3 mutants grew slower on various carbohydrates and displayed increased sensitivity to osmotic, oxidative and cell wall stresses. Gene expression profiling revealed a set of heat-shock and antioxidant factors that failed to be induced under oxidative stress and aberrant regulation of compatible solute-forming enzymes and cell wall biosynthesis/remodelling proteins in ΔBbGPCR3 after osmotic stress. Glucose-specific developmental defects included reduced (> 90%) conidiation and reduced dimorphic transition to the production of yeast-like blastospores, effects suppressed in media containing trehalose or glycerol, but not by addition of cyclic AMP. Insect bioassays revealed reduced virulence in topical assays but no effect in intrahaemoceol injection assays, indicating that BbGPCR3 was important in sensing signals during the initial interaction with the host but dispensable for post-penetration events. Comparative gene expression profiling of ΔBbGPCR3 mutants grown in glucose media compared with wild-type/glucose and ΔBbGPCR3/trehalose grown cells revealed sets of genes misregulated and recovered, respectively. These data link BbGPCR3 to broad developmental and genetic networks that include the major MAP kinase pathways., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Fungal entomopathogens, especially Beauveria bassiana, are often studied within the context of their use in biological pest control; however, there is limited knowledge of their distributions in host plants and soil ecosystem. We examined the distribution of B. bassiana and its influence on rice plants and paddy soils. B. bassiana could only be detected on the foliar surfaces of rice plants within 15 days under Bb-4 (7.5 × 10(4) conidia/mL) and Bb-7 (7.5 × 10(7) conidia/mL) treatments. The endophytic colonization of B. bassiana could not be found in stems, roots, or seeds of rice plants under Bb-4 and Bb-7 treatments. The fungus was found only in the leaves of rice plants under Bb-4 and Bb-7 treatments at 15 days after inoculation. Moreover, B. bassiana was absent from paddy soils under Bb-4 and Bb-7 treatments at all times. Enzyme activity (urease and phosphatase) in the paddy soils of Bb-4 and Bb-7 treatments showed no significant difference from the control. It is possible that B. bassiana was not able to colonize paddy soil. Detailed understanding of distribution and ecological interactions of B. bassiana is helpful for understanding and predicting the effects of fungal entomopathogens on host populations, and the interactions among fungal entomopathogens and other organisms in the community.

Citrus thrips, Scirtothrips citri (Moulton), is a plant-feeding pest most widely recognized for causing damage to citrus and mango fruits. This insect has broadened its host range to become a significant pest of commercial blueberries grown in the San Joaquin Valley of California. We evaluated Beauveria bassiana (Balsamo) for control of citrus thrips in blueberries grown under two watering regimes (drip irrigation with and without overhead sprinklers) and using two fungal formulations (commercially available spores in suspension vs. colonized seed) over two sampling periods, that is, for two 3-d periods after treatment. We found significant differences in thrips densities as a function of water regime treatment and fungal formulation. Thrips levels were reduced significantly with both fungal treatments at 3 d after treatment, but at 6 d, only results with colonized seed differed from the control treatment. These data suggest entomopathogenic fungi might be useful for control of citrus thrips on blueberries in particular situations (in organic production or as a resistance management tool) but that traditional pesticides will likely remain the preferred management option.

The entomopathogenic fungus Beauveria bassiana is widely used as a biological control agent (BCA) for insect pest control, with fungal propagules being either incorporated into the potting media or soil or sprayed directly onto the foliage or soil. To gain a better understanding of entomopathogenic fungal ecology when applied as a BCA to the soil environment, a case study using tag-encoded 454 pyrosequencing of fungal ITS sequences was performed to assess the fate and potential effect of an artificially applied B. bassiana strain on the diversity of soil fungal communities in an agricultural field in India. Results show that the overall fungal diversity was not influenced by application of B. bassiana during the 7 weeks of investigation. Strain-specific microsatellite markers indicated both an establishment of the applied B. bassiana strain in the treated plot and its spread to the neighboring nontreated control plot. These results might be important for proper risk assessment of entomopathogenic fungi-based BCAs.

Fungal insecticides developed from filamentous pathogens of insects are notorious for their slow killing action through cuticle penetration, depressing commercial interest and practical application. Genetic engineering may accelerate their killing action but cause ecological risk. Here we show that a Beauveria bassiana formulation, HV8 (BbHV8), engineered for fast per os killing of caterpillars by an insect midgut-acting toxin (Vip3Aa1) overexpressed in conidia has both high field efficacy and safety in full-season protection of cabbage from the damage of an insect pest complex dominated by Pieris rapae larvae, followed by Plutella xylostella larvae and aphids. In two fields repeatedly sprayed during summer, BbHV8 resulted in overall mean efficacies of killing of 71% and 75%, which were similar or close to the 70% and 83% efficacies achieved by commercially recommended emamectin benzoate but much higher than the 31% and 48% efficacies achieved by the same formulation of the parental wild-type strain (WT). Both BbHV8 and WT sprays exerted no adverse effect on a nontarget spider community during the trials, and the sprays did not influence saprophytic fungi in soil samples taken from the field plots during 4 months after the last spray. Strikingly, BbHV8 and the WT showed low fitness when they were released into the environment because both were decreasingly recovered from the field lacking native B. bassiana strains (undetectable 5 months after the spray), and the recovered isolates became much less tolerant to high temperature and UV-B irradiation. Our results highlight for the first time that a rationally engineered fungal insecticide can compete with a chemical counterpart to combat insect pests at an affordable cost and with low ecological risk.

Beauveria bassiana is a known natural enemy of a number of insect pests of crop plants. In order to screen different isolates of any given entomopathogens molecular markers provide a means for constructing the molecular phylogeny, diversity and link to virulent phenotypes. Eight isolates of B. bossiona isolated from different insect hosts and from different location at Pantnagar (Uttrakhand) were characterized by PCR-based RAPD markers. Bioassays were conducted by using first, second and third instar larvae of Spilarctia oblique in order to categorize the isolates based on virulence. The isolates were arbitrarily rated as more virulent, moderately virulent and less virulent based on the speed of killing. A wide range of variation in virulence was observed and the isolates of same insect origin and location showed differences in their aggressiveness. No correlation was found between the pathogenicity of the isolates and the relatedness of the original insect host. The pathogenicity against first, second and third instar larva of Spilarctia obliqua did not reveal any relatedness with the clustering pattern.

Genetic modification of Beauveria bassiana with the scorpion neurotoxin aaIT gene can distinctly increase its insecticidal activity, whereas the effect of this exogenous gene on the metabolism of B. bassiana is unknown until now. Thus, we investigate the global metabolic profiling of mycelia and conidia of transgenic and wild-type B. bassiana by liquid chromatography-mass spectrometry (LC-MS). Principal component analysis (PCA) and orthogonal projection to latent structure discriminant analysis (OPLS-DA) reveal clear discrimination of wild-type mycelia and conidia from transgenic mycelia and conidia. The decrease of glycerophospholipids, carnitine, and fatty acids and the increase of oxylipins, glyoxylate, pyruvic acid, acetylcarnitine, fumarate, ergothioneine, and trehalose in transgenic mycelia indicate the enhanced oxidative reactions. In contrast, most metabolites related to oxidative stress are not altered significantly in conidia, which implies that there will be no significant oxidative stress reaction when the aaIT gene is quiescent in cells.

Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis.

Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Two fungal cyclooligomer depsipeptide synthetases(CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8 ± 1.4 mg/l) and bassianolide (21.7± 0.1 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding D-hydroxyisovaleric acid (D-Hiv) and the corresponding L-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of D-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins.The total titer of beauvericin and its congeners (beauvericins A-C) was increased to 61.7 ± 3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC7159. Supplement of L-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8 ± 2.1 mg/l. Using this yeast system,we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules., (Copyright © 2013 Elsevier Inc. All rights reserved.)