Oliveira Daian Guilherme Pinto, Pauli Giuliano, Mascarin Gabriel Moura, Delalibera Italo
Biology Department, Federal University of Technology/UTFPR, 85892-000 Santa Helena, PR, Brazil.
Entomology and Acarology Department, ESALQ/University of São Paulo, P.O. Box 9, 13418-900 Piracicaba, SP, Brazil.
J Microbiol Methods. 2015 Dec;119:44-52. doi: 10.1016/j.mimet.2015.09.021. Epub 2015 Oct 6.
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.
直接测定分生孢子活力的技术应用广泛，但在配方杀真菌剂的质量控制评估中存在局限性，尤其是在乳油剂型中。本研究基于已建立的使用直接活力法的方案进行改进，提出一种新方法，使其更经济、准确，从而能够用于评估配方产品。使用球孢白僵菌和绿僵菌的产品，以纯分生孢子、带菌水稻、磨碎的带菌水稻和油悬液的形式，确定了合适的参数和条件。所建立的方案名为ESALQ，包括将150μL含约0.7和1×10⁶个分生孢子/mL的悬浮液转移到含有5 mL马铃薯葡萄糖琼脂培养基 + 5 mg/L戊菌隆和10 μL/L多菌灵（多菌灵）的罗达克®平板上，随后对已萌发和未萌发的分生孢子进行计数。对于磨碎的带菌水稻和油悬液配方，在将真菌悬浮液转移到培养基之前，应分别倒出水稻并去除油。将该方法与另一种直接活力法以及菌落形成单位（CFU）和荧光活力法进行比较，使用每种方法分析的变异系数（CV）比较所获得的准确性。结果表明，除了易于应用外，所开发的方法比其他方法具有更高的准确性（CV比标准方法低达7倍，比CFU低达32倍）。CFU方法在大多数测试的真菌形式中低估了活分生孢子的浓度，在乳油产品中，球孢白僵菌的这些值低54%，绿僵菌低84%。ESALQ方法中提出的改进和标准化显示出对杀真菌剂常规质量评估的有效改进。
