Preparation, Antifungal Activity And Preliminary Study On Mechanism Of Action Of (nitrogen, Sulfur, And Phosphor)-containing Chitosan Deirvatives Against Plant Pathogenic Fungi

The frequent and wide application of traditional fungicides has caused serious problems such as environmental pollution and threats to human health. Therefore, it is critically necessary to discover new biofungicidal alternatives. Chitosan is one kind of natural polysaccharide and it is proved to have broad-spectrum antifungal activity. Chitosan possesses good biocompatibility, biodegradability, and non-toxicity. Hence, it has attracted much attention in the field of agriculture. However, chitosan can not be used directly as a fungicide due to its relatively low antifungal activity. Hence, many strategies were proposed to overcome these limitations. A widely used approach is the chemical modification of chitosan.In this study, nine kinds of chitosan derivatives were designed via localization of import active group and strategy of graft copolymerization. Totally,30target derivatives were obtained. Their structures were confirmed by FT-IR, NMR, elemental analysis, XRD, DSC, and SEM to determine position and degree of substitution, crystal properties, and thermodynamic properties. Antifungal activity of them against R. solani, A. solani, P. piricola Nose, S. solani, P. asparagi, F. oxysporum, A. porri, G. theae-sinensis was tested. And two of the chitosan derivatives were selected to study mechanism of action. The major findings and results were listed as follows:1. Three new1,2,4-triazole chitosan derivatives were prepared via cyclization of acyl thiourea chitosan. Their solubility and antifungal activity improved after chemical modification chitosan. Antifungal results exhibited the activity was greatly influenced by electronegativity of substituents in triazole and steric effect. The inhibitory effect followed a sequence of methyl＞methyl chloride＞phenyl. Two new1,2,3-triazole chitosan derivatives were prepared via "Click Chemistry". Antifungal results showed PhTACS had a better inhibitory effect than PyTACS.2. Nine acyl halogenated phenyl-thiosemicarbazone chitosan derivativess were prepared. The inhibitory effect followed a sequence of chloroacetyl thiosemicarbazone＞acetyl thiosemicarbazone＞benzoyl thiosemicarbazone. Various substituents of aromatic ring in chitosan derivatives had distinct impacts on antifungal activity: Cl＞F＞CF3. Seven alkyl(substituted phenyl) thiosemicarbazone were obtained and the antifungal activity was studied. It was found that the chitosan derivatives with substituted phenyl had stronger inhibitory effect than those with alkyl Strong electron contributing group at phenyl was helpful to enhance the antifungal properties. Two alkyl dithiocarbamates chitosan derivatives were prepared and their antifungal activity was evaluated. The results showed methyl dithiocarbamates chitosan was more effective than ethyl dithiocarbamates chitosan. Two dithiocarbamates chitosan salts were prepared and their antifungal activity was studied. Their solubility obviously improved after chemical modification but the antifungal activity enhanced slightly. It indicated that solubility was not a decisive factor for antifungal activity. Additionally, dithiocarbamate chitosan (ADTCCS) was more effective than triethylene diamine dithiocarbamate chitosan (TEDADTCCS).3. The derivative α-amino-n-propyl-o,o-diethyl phosphonate chitosan was obtained, antifungal results showed it had good activity. Two α-amino-2-(p-tolyloxy)pyrimidine phosphonate chitosan were prepared. All the chitosan derivatives had excellent and broad-spectrum antifungal activity. At250μg/mL, the derivatives inhibited growth of P. asparagi, F. oxysporum and A. porri at100%, and they were much stronger than triadimefon and polyoxin. Two α-amino-furanyl phosphonate chitosan derivatives were prepared via one pot synthesis under microwave assisted and solvent-free conditions. Their solubility and antifungal activity greatly enhanced after modification. The structure-activity relationship showed that α-amino-furanyl ethyl phosphonate chitosan was slightly effective than α-amino-furanyl methyl phosphonate chitosan.4. It was found that solubility and antifungal activity of phosphor-containing chitosan derivatives was much better than (nitrogen and sulfur)-containing chitosan based on the antifunal screening results. Considering the preparation of α-APEPCS and α-AFEPCS was simpler and their raw material was cheaper, they were selected to study the mechanism of action.5. In order to investigate the antifungal mechanisms of chitosan and its derivatives, we examined their effects on hyphal morphology, membrane permeability, pyruvate, chitosan, and soluble protein in F. oxysporum and G. theae-sinensis. SEM obversation showed that the hyphal treated with α-APEPCS and α-AFEPCS was serious damaged, part or all of the hyphal cracked at100μg/mL While the hyphal treated with polyoxin only had a slight deformation and the hyphal treated with chitosan had no obvious change. The relative permeability rates of the cell membrane treated with samples were all higher than that of blank control. The results showed that the cell membrane of fungi incubated with the samples was destroyed. And the relative permeability rates of the cell membrane treated with polyoxin changed fastest. The mycelial soluble protein content that was treated by the samples after24h was higher than the blank control, it also indicated the cell membrane of fungi was destroyed, and the mycelia soluble protein content for α-APEPCS was higher than the others. The mycelial pyruvate content of the fungi treated with the samples reduced after24h, it might be attributed to the fact that the derivatives can inhibit the activity of lactate dehydrogenase (LDH), thereby inducing the decrease of the pyruvate content.In the study, five chitosan derivatives with greatly enhanced activity were selected via antifungal screening. Their mechanism of action was preliminary studied. The new obtained chitosan derivatives may have a good biocompatibility, biodegradability, and non-toxicity because they retained the skeleton structure of chitosan. Moreover, the derivatives had a different antifungal spectrum compared with triadimefon and polyoxin. All the above results described here lend themselves to find novel low-toxicity and no-cross-resistance marine fungicides. And it is important to explore marine biological resources technology of high-value line.