In Vitro Suppression of Mycelial Growth of Fusarium oxysporum by Extracellular Chitosanase of Sphingobacterium multivorum and Cloning of the Chitosanase Gene csnSM1

Abstract

A chitosan-degrading bacterium, isolated from field soil that had been amended with chitin, was identified as Sphingobacterium multivorum KST-009 on the basis of its bacteriological characteristics. The extracellular chitosanase (SM1) secreted by KST-009 was a 34-kDa protein and could be purified through ammonium sulfate precipitation, gel permeation column chromatography and SDS polyacrylamide gel electrophoresis. A chitosanase gene (csnSM1) was isolated from genomic DNA of the bacteria, and the entire nucleotide sequence of the gene and the partial N-terminal amino acid sequence of the purified SM1 were determined. The csnSM1 gene was found to encode 383 amino acids, 72 N-terminal amino acid residues were processed to produce the mature enzyme during the secretion process. Germinated microconidia of four formae speciales (lycopersici, radicis-lycopersici, melonis, and fragariae ) of Fusarium oxysporum were treated with SM1. Chitosanase treatment caused morphological changes, such as swelling of hyphal cells or indistinctness of hyphal cell tips and cessation or reduction of mycelial elongation.