Genomic and Experimental Analysis of the Insecticidal Factors Secreted by the Entomopathogenic Fungus Beauveria pseudobassiana RGM 2184

Abstract

The entomopathogenic fungus Beauveria pseudobassiana strain RGM 2184 can reach a maximum efficacy of 80% against the quarantine pest Lobesia botrana in field assays. In this study, the RGM 2184 genome was sequenced, and genome mining analyses were performed to predict the factors involved in its insecticidal activity. Additionally, the metabolic profiling of the RMG 2184 culture’s supernatants was analyzed by mass spectrometry, and the insecticidal activity from one of these extracts was evaluated in Galleria mellonella larvae. The genome analysis resulted in 114 genes encoding for extracellular enzymes, four biosynthetic gene clusters reported as producers of insecticidal and bactericidal factors (oosporein, beauvericin, desmethylbassianin, and beauveriolide), 20 toxins, and at least 40 undescribed potential biocontrol factors (polyketides and nonribosomal peptides). Comparative genomic analysis revealed that 65–95% of these genes are Beauveria genus-specific. Metabolic profiling of supernatant extracts from RGM 2184 cultures exhibited secondary metabolites such as beauveriolide, oosporein, inflatin C, and bassiatin. However, a number of detected metabolites still remain undescribed. The metabolite extract caused 79% mortality of Galleria mellonella larvae at 28 days. The results of this research lay the groundwork for the study of new insecticidal molecules.

Identification of Exoenzymes Secreted by Entomopathogenic Fungus Beauveria pseudobassiana RGM 2184 and Their Effect on the Degradation of Cocoons and Pupae of Quarantine Pest Lobesia botrana

“…The culture media used in this study were as follows: M1 (4.9 g/L dextrose, 2.30 g/L yeast extract); M2 (M1 supplemented with mineral salts [ 7 ]); PDB (potato dextrose broth; BD, USA); G55 (glucose-based medium, 0.3 g/L dextrose, 0.1 g/L bacteriological peptone, 0.02 g/L tryptone, 0.02 g/L yeast extract, 0.06 g/L sodium chloride, 0.005 g/L magnesium chloride, and 0.007 g/L L-methionine [ 14 ] adjusted to pH 5.5); G8 (glucose-based medium, 0.3 g/L dextrose, 0.1 g/L bacteriological peptone, 0.02 g/L tryptone, 0.02 g/L yeast extract, 0.06 g/L sodium chloride, 0.005 g/l magnesium chloride, and 0.007 g/L L-methionine adjusted to pH 8.0)); A55 (starch-based medium,10 g/L starch, 10 g/L yeast extract [ 14 ] adjusted to pH 5.5); A8 (starch-based medium,10 g/L starch, 10 g/L yeast extract adjusted to pH 8.0); CN55 (intermediate C/N ratio, 40 g/L dextrose, 10 g/L bacteriological tryptone [ 15 ] adjusted to pH 5.5) and CN8 (intermediate C/N ratio, 40 g/L dextrose, 10 g/L bacteriological tryptone adjusted to pH 8); and YSM (20 g/L sucrose, 5 g/L yeast extract, 1.5 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 ∙7H 2 O, 0.01 g/L CaCl 2 , 0.00003 g/L H 3 BO 3 , 0.00004 g/L MnSO 4 ∙4H 2 O, 0.000025 g/L Na 2 MoO 4 ∙2H 2 O, 0.00008 g/L CuSO 4 ∙5H2O, 0.0004 g/L ZnSO 4 ∙7H 2 O, 0.0005 g/L FeCl 3 ∙6H 2 O, 0.0004 g/L CoCl 2 ∙6H 2 O [ 16 ]). All media were supplemented with 1.2% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA.).…”

“…The results were collected using timsControl 2.0 software (Bruker Daltonics) with 10 PASEF cycles and analyzed using PEAKS Studio X+ software (Bioinformatics Solutions, Columbia, Waterloo, CA, USA). The database used for identification was proteins predicted from the Beauveria pseudobassiana RGM 2184 genome (JAKJXD000000000.1) [ 7 ]. This mass spectrometry analysis allowed the identification of lipases, proteases, and chitinases secreted by strain RGM 2184 in the A8 and M2 culture media.…”

“…There are more than 750 species of EPF, belonging to 85 genera and infecting 20 of the 30 insect orders described to date [ 3 , 4 ]. The genera Metarhizium , Beauveria , Cordyceps , and Akanthomyces , which belong to the order Hypocreales, are the most commonly used for pest control because they are relatively easy to grow en masse, have a wide range of hosts, and exhibit similar efficacy to commercial insecticides [ 5 , 6 , 7 ].…”

“…A wettable powder formulation of the B. pseudobassiana RGM 2184 strain was shown to achieve a maximum efficacy level of 80% against pupae of L . botrana in field trials performed in two regions of Chile over autumn and winter [ 7 ]. In order to obtain an approximation of the enzymatic tools used by strain RGM 2184 to penetrate the cocoons and pupae of L. botrana at low temperatures, the objectives in this study were as follows: (1) to identify possible genes and/or gene clusters in the genome of B. pseudobassiana RGM 2184 encoding enzymes involved in its biocontrol activity; (2) to identify the enzymes secreted by the fungus RGM 2184; (3) to evaluate homologues of these genes in other EPF through comparative genomic analysis; and (4) to evaluate the insecticidal activity of exoenzymes secreted by strain RGM 2184 at low temperatures.…”

The Entomopathogenic Fungus Beauveria bassiana Shows Its Toxic Side within Insects: Expression of Genes Encoding Secondary Metabolites during Pathogenesis

“…At this stage, the insect host has very little chance of surviving the fungal infection despite the activation of the immune response (humoral and cellular) as a last-ditch attempt to overcome the fungus [ 4 ]. A successful fungal infection will then depend on the concerted combination of several events, one of the main ones being the production of a plethora of toxic secondary metabolites [ 5 ] that can either facilitate the fungal invasion [ 6 ] or act as immunosuppressive compounds, fighting against host defenses [ 7 ]. These secondary metabolites can have many different chemical natures, and include nonribosomal peptides and polyketides.…”