Nematicidal activity of three novel extracellular proteases of the nematophagous fungus Monacrosporium sinense

Abstract

Extracellular proteases are an important virulence factor for the nematophagous fungi Monacrosporium. The objective of this study was to optimize, purify, partially characterize, and to evaluate the nematicidal activity of the proteases produced by the nematophagous fungus Monacrosporium sinense (SF53) by solid-state fermentation. Wheat bran was used as substrate for protease production. The variables moisture, pH, incubation time, temperature, glucose, yeast extract, and the number of conidia were tested for their influences on protease production by SF53. To determine the optimal level of the selected variables the central composite design was applied. The crude extract obtained was purified in two steps, an ion exchange chromatography and a gel excision. SDS-PAGE and zymogram were performed for analysis of the purification process. Proteolytic activity was also tested at different pHs and temperatures. In the in vitro assay, the nematicidal activity of the three proteases was evaluated. pH and incubation time showed a significant effect (p < 0.05) on production of protease. The highest value of activity was 38.0 (U/ml) under the conditions of pH 5.0 and incubation time of 211 h. SF53 produced three different proteases (Ms1, Ms2, and Ms3) which were directly purified from the zymogram. Ms1, Ms2, and Ms3 showed the following percentage of reduction (p < 0.05) on the number of Panagrellus redivivus compared to control after 24 h: 76.8, 68.1, and 92.1 %. This is the first report of the use of proteases of the isolate SF53 on a phytonematode, which may be a research tool in future works.