Antifungal Mechanism Of Matrine To Botryosphaeria Dothidea

Canker,caused by Botryosphaeria cdothidea,is one of the most severe plant diseases of Carya cathayensis Sarg.,accounting for 90%of the infection in naturally seeded Chinese hickory,resulting in considerable economic losses for the cultivators.Hitherto,fungicides such as tebuconazole and difenoconazole were used as chemical fungicides to control B.dothidea.However,these also pose increased risk of fungicide resistance and major threats to human body,animal,and environment.Therefore,developing new safe alternative botanical fungicides on B.dothidea is imperative.Matrine,a promising botanical antifungal,is a major quinolizidine alkaloid component isolated from Chinese herbal drug,Sophorae flavescentis(Kushen).Nevertheless,the activity of matrine against plant pathogenic fungi and the underlying mechanism of action are yet to be elucidated.In this study,the antifungal activity of matrine and the underlying mechanism related to cell membrane,respiratory metabolism and fungal transcription in B.dothidea were investigated.Finally,in order to search for new preparations with higher activity and lower costs,the synergy effects of matrine mixed with chemical antifungal agents were measured.Main results of this study are as follows:1.The antifungal activities of matrine on seven forestry fungal pathogens were determined by the mycelium growth rate method.And then the inhibitory activity of matrine on mycelium biomass and spore germination of B.dothidea were studied.As a result,the antifungal effect of matrine on B.dothidea(IC50=1.512 mg/mL)is the strongest,and followed by Alternaria alternata,Colletotrichum sp.,Sphaeropsis sapinea,Blastocladia pringsheimii,Pestalotiopsis sp.and Fusarium oxysporum..Matrine exhibited high inhibitory activity against mycelial growth and spore germination of B.dothidea in a does-dependent manner,and can completely inhibit the mycelia growth of B.dothidea within 96 days at 10 mg/mL.These results showed that matrine could effectively inhibit the mycelial growth and spore germination of B.dothidea in vitro.2.For the antifungal mechanism,the effect of matrine on the mycelium morphology and ultrastructure,membrane permeability,and content of MDA,ROS and antioxidant enzymes activities of B.dothidea was investigated in vitro experiments.Results showed that a series of alterations in morphology and ultrastructure related to cell membrane were observed in matrine-treated B.dothidea by inverted microscope,scanning electron microscope and transmission electron microscope,including obviously shriveled of hyphal,plasmolysis and leakage of cytoplasm.Meanwhile,matrine caused significantly higher conductivity and absorbance(260 nm)in extracellular,and markly lower the lipid contents of B.dothidea,indicating that the membrane permeability is increased.Furthermore,lipid peroxidation showed that matrine results in a increased in MDA contents,while enhancing the generation of ROS and the activities of antioxidant enzymes(SOD,CAT,POD).Our present results suggest that matrine could inhibit the mycelial growth of B.dothidea most likely to enhance of cell membrane permeability via membrane lipid peroxidation.3.In order to investigate the effect of matrine on respiratory metabolism of B.dothidea,the respiratory inhibition experiments were used to analyze respiratory inhibition pathways of matrine on B.dothidea and the change of respiratory metabolism-related enzymes activity was measured.Subsequently,oxygen consumption of mitochondrial,the activity of mitochondrial respiratory chain complexes ?/? and the effect of ROS accumulation in matrine-treatment B.dothicdea by mitochondrial respiratory chain complex I inhibitor rotenone were measured.The results showed that matrine caused an increased in ATP content and ATPase activity.Matrine resulted in an inhibition in the glycolysis pathway and enhancing in the TCA cycle.Moreover,the mitochondrial oxygen consumption and the enzyme I activity increased compared with that of the control in matrine-treatment B.dothidea.The ROS contents were dramatically decrease in matrine-treatment B.dothidea by rotenone.These results show that matrine could improved the respiratory metabolism and bind to mitochondrial respiratory chain complexes I.leading to excess ROS accumulation in matrine-treatment B.dothidea.4.To explore the antifungal mechanism of matrine against B.dothiodea,the transcriptional profiling of the control and different concentrations matrine treated B.dothidea after 24h of exposure were analyzed by RNA-seq.After filtering the dirty raw reads,58.65 Gb Clean Data was obtained and the Q30 base was over 93.39%.Totally,133,757 Transcript and 36,806 Unigene were annotated.Among them,28,077 genes were annotated in nr protein libraries,8513 genes were annotated in Swiss-Prot protein libraries,16739 genes were annotated in GO protein libraries,39,560 genes were annotated in COG protein libraries,9954 genes were annotated in KOG protein libraries,and 5678 genes were annotated in KEGG databases.Differential gene expression analysis indicated that matrine has significant impacts on gene transcription levels,a total of 250 differential genes were obtained,128 genes' expression levels were commonly up-regulated under different concentration of matrine,and 31 genes'expression levels were commonly down-regulated under the same criteria.Belong to enrichment analysis of different expression genes revealed that the antifungal mechanism of matrine is complex,but the different expression genes enrichment in the respiratory metabolism and oxidative phosphorylation pathway is the most significant,which were the same as that found in results above.5.In order to reduce the use of pesticides and increase the control effect,the synergistic effects of matrine compound with eight different chemical fungicides against Botryosphaeria dothidea were determined by the growth rate method.The results showed that matrine and thiophanate-methyl,azoxystrobin mixtures were screened as the better combinations by co-toxicity factor method.Nevertheless,there is antagonistic effect between matrine and chlorothalonil,mancozeb while there is additive effect between the matrine and tebuconazole,copper quinoline and difenoconazole.The optimum synergistic composite of matrine and thiophanate-methyl was measured by cotoxicity coefficient,which the ratio of matrine and thiophanate-methyl is 5:5 possessed higher co-toxicity coefficient of 197.605.In a word,the synergistic effects of matrine combined with thiophanate-methyl is obviously higher than the single-dose use.This experiment aims at reducing matrine consumption and environment pollution,delaying the development of antifungal-resistance,and providing the theory basis for field application and development of preparation of matrine.