Rapid and Easy Modified Plate-based Screening Methods for Quantitative and Qualitative Detection of Protease Production by Fungi

Abstract

Proteases constitute a significant part of cell wall-degrading enzymes (CWDEs) produced by fungal biocontrol agents and particularly crucial in mycoparasitism of fungal phytopathogens. Plate-based screening methods are routinely used for screening protease-producing microorganisms including fungi. Skim milk agar (SMA) is one of the most popular media for the detection of protease producing bacteria. However, SMA is not efficient to test fast growing fungi, because it does not give an estimation of the actual amount of secreted protease produced by fungal inocula. In the current study, the efficacy of two modified plate-screening methods, including split-SMA (SSMA) and minimal medium supplemented with skim milk (MSMW) was assessed for detection of protease production by three representative fungal strains including Trichoderma longibrachiatum strain N, Beauveria bassiana strain B and Purpureocillium lilacinum strain PL. Protease production was revealed on the three tested media by the three strains. However, the halo diameter of the fungal strains (a proxy for protease production) was the smallest on SMA. Furthermore, protease production could not be detected for T. longibrachiatum strain N on SMA due to its fast growth; while it showed the highest protease activity on both modified media compared with the other strains. According to the result of this study, the SSMA medium is an easy and more accurate method compared with the two other different methods as it displays the actual amount of protease produced by fungal strains and therefore this method is recommended for quantitative and qualitative detection of protease production by slow and fast growing fungi.

Identification of Protease Enzymes Involved in Biocontrol Activity

“…Plate Assay [5] 1. The qualitative assessment of the protease production (fungi, bacteria, and actinomycetes) is carried out using skim milk agar (SMA) medium (1000 ml distilled water: 4 g skim milk, 1 g mycological peptone/bacteriological peptone, 5 g NaCl, 15 g agar) [see Notes 1-3 for detail media composition].…”

“…3.1.2 Splitted-SMA (SSMA) Method [5] 1. To perform this method, use two different media, including SMA and home-made potato dextrose agar (H-PDA, 1000 ml sterile distilled water, 200 g potatoes, 20 g dextrose).…”

“…with Skim Milk Agar (MSMW) Method [5] This method is based on the supplementation of pH indicator dye in the medium. The protease production is ascertained based on the difference in diameter of violet halo formed on media plate.…”