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Abstract: Entomopathogenic fungi, such as Beauveria bassiana, are key environmental pathogens of insects that have been exploited for biological control of insect pests. Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, mediating responses to the environment. Bbslt2, encoding for an Slt2 family MAPK, was isolated and characterized from B. bassiana. Gene disruption of Bbslt2 affected growth, caused a significant reduction in conidial production and viability, and increased sensitivity to Congo Red and fungal cell wall degrading enzymes. ΔBbslt2 mutants were altered in cell wall structure and composition, which included temperature dependent chitin accumulation, reductions in conidial and hyphal hydrophobicity, and alterations in cell surface carbohydrate epitopes. The ΔBbslt2 strain also showed hypersensitivity to heat shock and altered trehalose accumulation, which could only be partially attributed to changes in the expression of trehalase (ntl1). Insect bioassays revealed decreased virulence in the ΔBbslt2 strain using both topical and intrahemoceol injection assays. These results indicate that Bbslt2 plays an important role in conidiation, viability, cell wall integrity and virulence in B. bassiana. Our findings are discussed within the context of the two previous MAP kinases characterized from B. bassiana. [Copyright &y& Elsevier]

Highlights: [•] Catalases are involved in stress tolerance and infection in entomopathogenic fungi. [•] CatE7 gene was identified in B. bassiana via suppression subtractive hybridization. [•] The catalase gene (catE7) was homologous to monofunctional fungal catalases. [•] A transgenic strain over-expressing BbcatE7 displayed enhanced virulence. [Copyright &y& Elsevier]

Nymph and adult ticks from Ambylomma americanum and Ambylomma maculatum were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (90517) and Metarhizium anisopliae (20500). Fungal suspensions of conidia harvested from potato dextrose plates containing 108conidia/ml caused greater than 90% mortality in adult A. maculatum but less than 10% mortality in adult A. americanum over a 28 day time course. Similarly, infection with M. anisopliae (108conidia/ml) resulted in 60 and 15% mortality in A. maculatum and A. americanum, respectively. Nymphs of both tick species were more susceptible to fungal infection reaching mortality rates of almost 100% for A. maculatum and over 35% for A. americanum. Scanning electron microscopy of infected ticks showed rapid attachment, germination, and proliferation of fungal spores on A. maculatum cuticles, and to a much lesser extent on A. americanum cuticles. Pentane extracts of A. americanum cuticle hydrocarbons inhibited germination and hyphal growth of B. bassiana conidia, whereas no inhibition was observed using A. maculatum extracts. Significant mortality towards A. americanum was observed (>60%, 28 days) only when the ticks were treated with B. bassiana directly from the growth medium (107blastospores/ml, grown for 3–4 days in Sabouraud dextrose+0.5% yeast extract liquid media). These results indicate tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliae, and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infection and virulence. [Copyright &y& Elsevier]

The study of biological diversification of oomycetes has been a difficult task for more than a century. Pioneer researchers used morphological characters to describe this heterogeneous group, and physiological and genetic tools expanded knowledge of these microorganisms. However, research on oomycete diversification is limited by conflicting phylogenies. Using whole genomic data from 17 oomycete taxa, we obtained a dataset of 277 core orthologous genes shared among these genomes. Analyses of this dataset resulted in highly congruent and strongly supported estimates of oomycete phylogeny when we used concatenated maximum likelihood and coalescent-based methods; the one important exception was the position of Albugo . Our results supported the position of Phytopythium vexans (formerly in Pythium clade K) as a sister clade to the Phytophthora - Hyaloperonospora clade. The remaining clades comprising Pythium sensu lato formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, in agreement with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I was placed as sister to the Phytophthora-Hyaloperonospora-Phytopythium clade, thus confirming the lack of monophyly of Pythium sensu lato . Multispecies coalescent methods revealed that the white blister rust, Albugo laibachii , could not be placed with a high degree of confidence. Our analyses show that genomic data can resolve the oomycete phylogeny and provide a phylogenetic framework to study the evolution of oomycete lifestyles. [ABSTRACT FROM AUTHOR]

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 Δ cna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δ slt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δ slt2 and Δ cna1 strains during growth under cell wall stress as compared to the wild type. Both Δ slt2 and Δ cna1 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 Δ slt2 and Δ cna1 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 . [ABSTRACT FROM AUTHOR]

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