RESUMO
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.
RESUMO
The reducing clade III polyketide synthase genes, including pks15, are highly conserved among entomopathogenic fungi. To examine the function of pks15, we used targeted disruption to investigate the impact of Beauveria bassiana pks15 on insect pathogenesis. Southern analysis verified that the Δpks15 mutant was disrupted by a single integration of the transformation cassette at the pks15 locus. The Δpks15 mutant had a slight reduction in radial growth, and it produced fewer spores. Our insect bioassays indicated the Δpks15 mutant to be significantly reduced in virulence against beet armyworms compared to wild type (WT), which could be partially accounted for by its markedly decreased ability to survive phagocytosis. Total haemocyte count decreased sharply by 50-fold from days 1-3 post-inoculation in insects infected with WT, compared to a 5-fold decrease in the Δpks15 mutant. The mutant also produced fewer hemolymph hyphal bodies than WT by 3-fold. In co-culture studies with amoebae that have phagocytic ability similar to that of insect haemocytes, at 48 h the mortality rate of amoebae engulfing Δpks15 decreased by 72 %, and Δpks15 CFU decreased by 83 % compared to co-culture with WT. Thus, the Δpks15 mutant had a reduced ability to cope with phagocytosis and highly reduced virulence in an insect host. These data elucidate a mechanism of insect pathogenesis associated with polyketide biosynthesis.
