The production of biological insecticides requires the identification and selection of candidate isolates of entomopathogenic fungi (EPF). The objective of this study was to characterise by virulence factors, single-spore strains of the CHE-CNRCB 303, 305, 307 strains initially identified as Isaria fumosorosea and the 224 strain of Metarhizium anisopliae to identify those with outstanding quality parameters that can serve to improve the control programme for Diaphorina citri (Hemiptera: Liviidae) in Mexico. The size of conidia and phialides was evaluated. The internal transcribed spacers established molecular identity. The selection of single-spore strains was done using radial growth (RG), the production of conidia (PC) and germination (G) rates. Principal component analysis (PCA) was used to determine the correlation between the variables and as screening criterion. Molecular and morphometric analysis confirmed that isolates 303, 305 and 307, and their single-spore strains correspond to the Isaria javanica species. The 224 strain and its single-spore strain were identified as M. anisopliae sensu lato. The statistical analysis of the RG, PC and G variables showed significant differences (P ≤ 0.05) between single-spore strains and their multi-spore strains. PCA showed a correlation between the RG and PC. The assay with Pr1 proteases associated with the surface of the conidia showed significant differences (P ≤ 0.05) for single-spore strains of I. javanica 303/2, 305/1 and 305/5 compared to the original strains. The characterisation of genetically homogeneous populations of EPF will allow us to obtain improved isolates, which are needed for the mass production of mycoinsecticides.

Background: Xyleborus affinisEichhoff (Coleoptera: Curculionidae) is an ambrosia beetle reported to affect avocado trees (Persea americanaMill.). The use of the entomopathogenic fungus (EPF)Beauveria bassiana(Bals.-Criv.) Vuill. for ambrosia beetle control represents an alternative to insecticides.Methods: This study was designed in two stages to selectB. bassianastrains with potential to controlX. affinisfemales. In the first stage, 19B. bassianaMexican strains from EPF collection, isolated from Coleoptera (CHE-CNRCB,http://www.gob.mx/senasica/documentos/coleccion-de-hongos-entomopatogenos), were tested. Analyses included radial growth rate, conidial yield, spore germination, and germ tube length. Results were analysed by Principal Component Analysis (PCA) to identify clusters within favourable growth phenotypes. For the second stage, 10 selected strains were re-analysed for virulence-related metabolic characteristic, including cell wall-bound cuticle-degrading enzymes–Pr1-like proteases and β-N-acetyl glucosaminidases (NAGase) chitinases, conidial hydrophobicity and monopolar germination parameters. A second PCA analysis was run for those virulence parameters analysed, and upon results strains CHE-CNRCB 44, 171, 431 and 485 were selected and tested againstX. affinisfemales. Females were treated with a 1 × 10⁸conidia mL⁻¹suspension (recommended rate), using a Potter Tower.Results: All strains showed insecticidal activity, inducing up to 58% mortality; about 30% dead beetles developed aerial mycelia (CHE-CNRCB 485) and the fastest mortality rate wast₀= 1.95 (CHE-CNRCB 44).Conclusion: Since all selected strains showed virulence againstX. affinisfemales, results indicated the possibility of selectingB. bassianastrains based on multiple metabolic attributes, as a preliminary test to perform bioassays against order-related target insects.