Purification And Characterization Of Proteins With Activity Against Phytopathogenic Fungi

Plant fungicidal disease is one of the primary reasons that cause agricultural loss.The application of traditional chemical pesticides for plant disease control has created a lot of problems,such as environmental pollution,high toxicity,drug resistance and so on.On the contrary,antifungal proteins have many advantages,especially in safety and environmental compatibility and high activity and little drug resistance.Two kinds of antifungal proteins were purified and characterized from the seeds of Cheiranthus allionii and Ophiocordyceps sobolifera respectively in this study.The main procedure for isolating and purifying the antifungal proteins included ion exchange chromatography on SP-Sepharose and Mono S,and gel filtration on Superdex 75.The antifungal proteins of C.allionii and O.sobolifera exhibited a molecular mass around 7 kDa and 17 kDa respectively.Both proteins demonstrated strong activity against phytopathogenic fungi.C.allionii antifungal peptide inhibited mycelial growth in fourteen kinds of fungi including Valsa mali,Exserohilum monoceras,Helminthosporium maydis,Mycosphaerella arachidicola,Colletotrichum gloeosporioides,Magnaporthe oryzae,Fusarium graminearum,Botrytis cinerea,Fusarium oxysporum,Fusarium solanif.sp.glycines,Colletotrichum micotianae,Colletotrichum higginsianum,Phytophthora infestans,and Sclerotium rolfsii.The IC50 values toward Fusarium oxysporum,Mycosphaerella arachidicola,Exserohilum monoceras,and Valsa mali,were 3.15 ?g/mL,5.19 ?g/mL,7.05 ?g/mL,and 7.22 ?g/mL respectively.The antifungal protein from O.sobolifO.showed antifungal activity toward various tested fungi with the exception of P.infestans,with an IC50 of 5.39 ?g/mL,9.25 ?g/mL,9.25 ?g/mL,and 14.79 ?g/mL toward C.micotianae,M.arachidicola,F.oxysporum,and H.maydis,respectively.The mass spectra of the antifungal peptide of C.allionii and the antifungal protein of O.sobolifera were studied and the results were searched in and retrieved from the NCBInr database.It turned out that the antifungal peptide of C.alionii may be a novel antifungal peptide and the antifungal protein of O.sobolifera exhibits similarity to biopolymer transporter ExbB[Bradyrhizobium sp.ARR65].Furthermore,the interaction between the purified proteins and fungi led to changes in chitin distribution on the hyphae,morphological changes and hyphal membrane permeability.In conclusion,C.allionii antifungal peptide and O.sobolifera antifungal protein present different characteristics,but both of them show broad-spectrum resistance to phytopathogenic fungi.The results will provide a scientific basis for the development of new biological agrochemicals for the biocontrol of plant fungicidal disease.