| Blue mold decay,caused by species of Penicillium expansum,is one of the most important postharvest diseases of pome fruits.Besides causing economic losses,P.expansum has also potential public health significance since it produces patulin.Chitosan,is known to be one of the natural polysaccharides,and also possess antifungal effects on various plant pathogenic bacteriaand fungi.According to research reports,chitosan has a good anti-fungi effect on a variety of pathogens of fruits and vegetables,but there are some differences in the antibacterial rules of chitosan with different molecular weights,some even appears contradictory,and the mechanism of direct antibacterial action on chitosan,particularly research at the cellular level,lacks in-depth understanding in China.Previous study in our laboratory confirmed that chitosan had a good inhibitory effect on the P.expansum-caused postharvest decay in apple fruit,and pre-treatment with chitosan increased the expression levels of proteins related to cell structure,signal transduction,carbohydrates and energy metabolism,and protein degradation in apples.In this study,we aimed to investigate the inhibitory efficiency of chitosan with different molecular weights on P.expansum,to obtain insights into the changes in cell structure of P.expansum induced by chitosan treatment and to infer the underlying molecular mechanisms related to anti-fungal activity of chitosan by ultra-structural examination by electron microscopy laser confocal microscopy and high-throughput RNA-Seq analysis.The main findings are as follows:(1)Three kinds of chitosan with different molecular weight(2,48 and 100 kDa)were used in this study and the inhibitory effect of chitosan on spore germination and mycelial growth of Penicillium expansum increased with increasing molecular weight and concentration.Chitosan with molecular weight of 100 kD and concentration of0.1% exhibit the best the inhibitory efficiency,and the spore germination rate and mycelia growth were reduced by 95.6% and 44.8%,respectively.(2)The result of light microscopy and electron microscopy showed that both spores and mycelium suffered severe damage by chitosan treatment.The fungalmycelium appeared to be bifurcated,swollen and deformed after exposure to chitosan.While the conidia subject to chitosan,intense vacuolization along the cell and membrane rupture resulting in the loss of cellular content were observed with presence of chitosan.(3)RNA-Seq method was used to detect gene expression changes in P.expansum after chitosan treatment for 2 and 6 h,respectively.A total of 4551 and3887 differentially expressed genes(DEGs)with p-value(0.05)and fold change(1.5)were identified upon exposure to chitosan treatment at 2 and 6 h,respectively.Among the differentially expressed genes,2312 gens were up-regulated and 2239 genes were down-regulated at the early stage of chitosan treatment(2 h),and 1907 genes were up-regulated and 1980 genes were down-regulated at the late stage of chitosan treatment(6 h).DEG and KEGG analysis revealed that genes involved in endocytic pathway were mainly up-regulated and this biological process was activated by chitosan treatment.The possible interaction sites of chitosan in P.expansum were further tracked with FITC-labeled chitosan,and fluorescent signal was observed both on the surface of cells and in cells,suggesting chitosan can enter into the cells.Further experiment indicated that the uptake of chitosan by P.expansum might be an energy-dependent endocytosis.Rather interestingly,gel-retardation assay evidence the interaction between chitosan and DNA,which indicated that chitosan possibly has DNA as intracellular target for antifungal action against P.expansum. |
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