Identification and Characterization of an Antifungal Protein, AfAFPR9, Produced by Marine-Derived Aspergillus fumigatus R9

A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFP(R9), was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFP(R9) was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFP(R9) displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 mu g/disc, respectively. Moreover, AfAFP(R9) exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFP(R9) was indispensable for its antifungal action. The cDNA encoding for AfAFP(R9) was cloned from A. fumigatus R9 by RTPCR and heterologously expressed in E. coli. The recombinant AfAFP(R9) protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member. (AfAFP(R9)) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.