title: A protocol for determination of conidial viability of the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae from commercial products.
abstract: Techniques for directly determining conidial viability have widespread use but also have limitations for quality control assessments of formulated mycoinsecticides, especially in emulsifiable oil. This study proposes a new method based on adaptations of already established protocols that use the direct viability method to make it more economical and accurate, thus enabling its use in the evaluation of formulated products. Appropriate parameters and conditions were defined using products based on Beauveria bassiana and Metarhizium anisopliae in the forms of pure conidia, fungus-colonized rice, ground fungus-colonized rice and oil dispersion. The established protocol, named ESALQ, consists of the transfer of 150 μL of a suspension containing about 0.7 and 1 × 10(6) conidia/mL onto Rodac® plates with 5 mL of potato dextrose agar culture medium + 5 mg/L of Pentabiotic® and 10 μL/L of Derosal® (Carbendazim) and subsequent counting of germinated and non-germinated conidia. For the ground fungus-colonized rice and oil dispersion formulations, prior to transferring the fungal suspension to the medium, rice should be decanted and the oil removed, respectively. This method was compared with another direct viability method and with the Colony-forming unit (CFU) and Fluorescence viability methods, comparing the accuracy obtained using the coefficient of variation (CV) of the analysis of each method. The results showed that in addition to the ease of application, the developed method has higher accuracy than the other methods (with a CV up to seven times lower than in the Standard method and up to 32 times lower than CFU). The CFU method underestimated the concentration of viable conidia in most of the tested fungal forms, and in the emulsifiable oil products, these values were 54% lower for B. bassiana and 84% lower for M. anisopliae. The adaptations and standardizations proposed in the ESALQ method showed effective improvements for routine quality assessment of mycoinsecticides.
authors.affiliation: Biology Department, Federal University of Technology/UTFPR, 85892-000 Santa Helena, PR, Brazil. Electronic address: daianguilherme@gmail.com.
authors.forename: Daian Guilherme Pinto
authors.initials: DG
authors.lastname: Oliveira
authors.affiliation: Entomology and Acarology Department, ESALQ/University of São Paulo, P.O. Box 9, 13418-900 Piracicaba, SP, Brazil.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Pauli
authors.affiliation: Laboratory of Entomology (R&D), Embrapa Rice and Beans, Rodovia GO-462, Km 12, Zona Rural, P.O. Box 179, 75375-000 Santo Antônio de Goiás, GO, Brazil.
authors.forename: Gabriel Moura
authors.initials: GM
authors.lastname: Mascarin
authors.affiliation: Entomology and Acarology Department, ESALQ/University of São Paulo, P.O. Box 9, 13418-900 Piracicaba, SP, Brazil.
authors.forename: Italo
authors.initials: I
authors.lastname: Delalibera
mesh_terms: D000818:Animals; D052982:Beauveria / Q000254:growth & development* / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D007313:Insecta / Q000382:microbiology; D052981:Metarhizium / Q000254:growth & development* / Q000378:metabolism; D050296:Microbial Viability; D009172:Mycology / Q000379:methods*; D012275:Oryza / Q000382:microbiology*; D013172:Spores, Fungal / Q000254:growth & development* / Q000378:metabolism
chemical_list: D003470:Culture Media
keywords: Colony-forming unit; Entomopathogenic fungi; Germination; Microbial control; Viability assessment