Inhibitory Effects And Mode Of Action Of Nanochitin Against Fusarium Pseudograminearum

Fusarium pseudograminearum is an important pathogenic fungus caused Wheat Crown Rot disease in wheat production.Nanochitin is a polycationic rod-like nanowhisker prepared by acidic hydrolysis from shrimp origin.The advantages of nanochitin include small size effect,high specific surface area,good biocompatibility,high bioactivity,non-toxic,and biodegradable.In this study,nanochitin was prepared by acid hydrolysis using shrimp shell as original material and characterized by dynamic light scattering method,conductometric titration and transmission electron microscopy(TEM)respectively for its physicochemical properties.F.pseudograminearum was used as the target organism,the antifungal activity and the mode of action as well as the biological mechanism of nanochitin against the fungal pathogen were investigated by means of techniques of microbiology,cell biology,and nanotechnology.The main findings are as follows:1.Physicochemical properties of nanochitin:nanochitin obtained by acidolysis exhibits an effective particle size of about 120-170 nm,zeta potential in stable nanochitin aqueous suspensions was+25-50 mV detected by dynamic light scattering method.TEM image showed that nanochitin was rod-like whisker.The net charge density of nanochitin suspension was 2.7×10-5 mol/g,which determined by conductometric titration.2.Inhibitory effects of nanochitin on F.pseudograminearum:(1)The effect of nanochitin on F.pseudograminearum growth in vitro depending on dose and application methods.The results showed that 300,1000 μg/mL nanochitin treatments could significantly reduce the growth of F.pseudograminearum among the treatment methods.However,the traditional measurement in vitro may have limited its application in nanobiomaterials due to the difference in biomass.(2)Compared with the control,after 4 h treatment,the germination rate of F.pseudograminearum treated with 30,300 μg/mL of nanochitin were decrease by 33%and 23%,respectively.(3)Electron microscopy showed that the cells of F.pseudograminearum were obviously shriveled,the cell wall was damaged or the cell interior was disordered after treatment with nanochitin,morphology,productions and germination of conidia were all significantly affected by nanochitin.Quantum dots labeled with nanochitin tracer observation pointed that nanochitin could be attached to the surface of F.pseudograminearum.3.Preliminary study on the mode of action of nanochitin against F.pseudograminearum:(1)Nanochitin could significantly increase the extracellular electrical conductivity of pathogenic fungi and affected the permeability of cell membranes.(2)Combined UV-vis spectrum,Infrared spectrum and PI-DAPI staining results showed that the ergosterol content of F.pseudograminearum cell membrane increased significantly and the structural stability of the cell wall dextran was decreased after 300 μg/mL nanochitin treatment,the integrity of cell wall and membrane was destroyed,which may cause the cell viability decline.(3)The activities of SDH and NADH oxidase decreased significantly and the energy supply of cell physiological activities was inhibited when treated with 1000 μg/mL nanochitin.(4)The content of virulent factor,deoxynivalenol(DON)was decreased significantly when treated with nanochitin,indicating the decrease of pathogen infectivity and/or pathogenicity.In short,the possible mode of action of nanochitin against fungal pathogen we hypothesized is that the positive charged nanochitin could be adsorbed and bound to the negatively charged substance of F.pseudograminearum cell membrane resulted in an increase of permeability of cell membrane.The stability of cell wall structure and the integrity of the cell membrane function were influenced by nanochitin,the infection ability and the pathogenicity of pathogen were consequently weakened.