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Beauveria bassiana degenerates after repeated subcultures, demonstrating declined conidiation and insect virulence. The target of rapamycin (TOR) kinase conserved among eukaryotes is the master regulator of cellular physiology and is likely involved in culture degeneration. Indeed, the levels of TOR-associated proteins increase over successive subcultures. Here, CRISPR/Cas9 locus engineering introduced the inducible Tet-On promoter upstream of the TOR kinase 2 gene tor2 in B. bassiana. The mutant PTet-On tor2 'T41' was verified for the Tet-On integration via PCR analyses and provided a model for evaluating the fungal phenotypes according to the tor2 expression levels, induced by doxycycline (Dox) concentrations. At 0 µg·mL−1 of Dox, T41 had 68% of the wild type's (WT) tor2 expression level, hampered radial growth and relatively lower levels of oxidative stress tolerance, conidiation and virulence against Spodoptera exigua, compared to those under the presence of Dox. A low dose of Dox at 0.1–1 µg·mL−1 induced tor2 upregulation in T41 by up to 91% compared to 0 µg·mL−1 of Dox, resulting in significant increases in radial growth by 8–10% and conidiation by 8–27%. At 20 µg·mL−1 of Dox, which is 132% higher than T41's tor2 expression level at 0 µg·mL−1 of Dox, T41 showed an increased oxidative stress tolerance and a decrease in growth inhibition under iron replete by 62%, but its conidiation significantly dropped by 47% compared to 0 µg·mL−1 of Dox. T41 at 20 µg·mL−1 of Dox had a strikingly increased virulence (1.2 day lower LT50) against S. exigua. The results reflect the crucial roles of TOR kinase in the vegetative growth, conidiation, pathogenicity and oxidative stress tolerance in B. bassiana. Since TOR upregulation is correlated with culture degeneration in multiple subcultures, our data suggest that TOR signaling at relatively low levels plays an important role in growth and development, but at moderate to high levels could contribute to some degenerated phenotypes, e.g., those found in successive subcultures. [ABSTRACT FROM AUTHOR]

Beauveria bassiana is a globally distributed entomopathogenic fungus that produces various secondary metabolites to support its pathogenesis in insects. Two polyketide synthase genes, pks14 and pks15, are highly conserved in entomopathogenic fungi and are important for insect virulence. However, understanding of their mechanisms in insect pathogenicity is still limited. Here, we overexpressed these two genes in B. bassiana and compared the metabolite profiles of pks14 and pks15 overexpression strains to those of their respective knockout strains in culture and in vivo using tandem liquid chromatography-mass spectrometry (LC-MS/MS) with Global Natural Products Social Molecular Networking (GNPS). The pks14 and pks15 clusters exhibited crosstalk with biosynthetic clusters encoding insect-virulent metabolites, including beauvericins, bassianolide, enniatin A, and the intracellular siderophore ferricrocin under certain conditions. These secondary metabolites were upregulated in the pks14-overexpressing strain in culture and the pks15-overexpressing strain in vivo. These data suggest that pks14 and pks15, their proteins or their cluster components might be directly or indirectly associated with key pathways in insect pathogenesis of B. bassiana, particularly those related to secondary metabolism. Information about interactions between the polyketide clusters and other biosynthetic clusters improves scientific understanding about crosstalk among biosynthetic pathways and mechanisms of pathogenesis. [ABSTRACT FROM AUTHOR]

Simple Summary: The spider mite is a destructive pest of various crops during warm and dry conditions in tropical countries, including Thailand. The pest is difficult to control despite synthetic acaricide use. In the field, insect populations gradually develop resistance to synthetic pesticides over long-term use. The use of entomopathogenic fungi is more human- and environmentally friendly. We searched and identified a potential fungal isolate from a culture collection, focusing on the genus Metarhizium. Metarhizium sp. BCC 4849 not only controls the spider mite but also plays a significant role as a natural regulator of other insect pests. Here, we investigated its infection process on the mite, optimized a conidial formulation for extended shelf life, and conducted toxicological assays in animals to assess its biosafety in humans. The fungal genome has been sequenced. The genomic data indicated that oxidoreduction proteins; zinc-, heme-, and iron-binding proteins; and transmembrane transporters are abundant in the genome. Five isolates of Metarhizium sp. were evaluated for their pathogenicity against the spider mite (Tetranychus truncatus Ehara) (Acari: Tetranychidae) and Metarhizium sp. BCC 4849 resulted in the highest mortality (82%) on the 5th day post-inoculation (DPI). Subsequent insect bioassay data indicated similar high virulence against five other insects: African red mites (Eutetranychus africanus Tucker) (Acari: Tetranychidae), bean aphid (Aphis craccivora Koch) (Hemiptera: Aphididae), cassava mealybug (Phenacoccus manihoti Matile-Ferrero) (Hemiptera: Pseudococcidae), sweet potato weevil (Cylas formicarius Fabricius) (Coleoptera: Brentidae), and oriental fruit fly (Bactrocera dorsalis Hendel) (Diptera: Tephritidae), at mortalities of 92–99%, on 3rd–6th DPI, and in laboratory conditions. The pathogenicity assay against E. africanus in hemp plants under greenhouse conditions indicated 85–100% insect mortality on 10th DPI using the fungus alone or in combination with synthetic acaricide. Genome sequencing of Metarhizium sp. BCC 4849 revealed the high abundance of proteins associated with zinc-, heme-, and iron-binding; oxidation-reduction; and transmembrane transport, implicating its versatile mode of interaction with the environment and adaptation to various ion homeostasis. The light and scanning electron microscopy indicated that at 24 h post inoculation (PI), adhesion and appressorial formation occurred, notably near the setae. Most infected mites had stopped moving and started dying by 48–72 h PI. Elongated hyphal bodies and oval blastospores were detected in the legs. At 96–120 h PI or longer, dense mycelia and conidial mass had colonized the interior and exterior of dead mites, primarily at the bottom than the upper part. The shelf-life study also indicated that conidial formulation combined with an oxygen-moisture absorber markedly enhanced the viability and germination after storage at 35 °C for four months. The fungus was tested as safe for humans and animals, according to our toxicological assays. [ABSTRACT FROM AUTHOR]