Screening Of Plant-derived Fungicides And Dessection Of The Mechanisms Of Berberine Inhibiting Alternaria Alternata

Tomato is an economically important crop with the second-biggest cultivated area in the world.The early blight caused by the fungus Alternaria alternata is a disease which severely hampers tomato production and thus leads to enormous economic loss.At present,management of early blight relies mainly on chemical synthetic fungicides.However,there are growing concerns with this strategy such as environmental pollution,zoonosis and microbial resistance.Screening of highly efficient,safe and cost-effective natural compounds may circumvent the drawbacks of chemical fungicides.Compared with chemical synthetic fungicides,plant-derived fungicides are environmental friendly,safety to non-target organisms,unable to trigger micribial resistance.Thus,plant-derived fungicides have emerged as alternatives to chemical fungicides.The objectives of this study are to(1)screen novel fungicides that may be promising alternatives to chemical fungicides,to(2)decipher the microbial resistance mechanisms,and to(3)provide insights for field use of fungicides to control early blight.The following are the main highlights of this work.1.The objective of this study is to screen cost-effective,safe and highly efficient plant-derived fungicides towards A.alternate.In vitro experiments revealed that berberine showed strongest inhibition on A.alternata among 13 compounds.The EC50 was only 60 μg/mL and MIC was 300 μg/mL.Microscope observation of berberine-treated A.alternata showed morphological deformation in the mycelium.Lesion appeared in tomato fruit,indicating the pathogenicity of A.alternata to tomato.Notably,berberine repressed the growth of A.alternata in a concentration-dependent manner.When berberine concentration reached 300 μg/mL,no lesion was observed,indicating that berberine can effectively inhibit tomato early blight.2.Among 13 candidate plant-derived compounds,berberine was shown to be most effective in inhibiting A.alte in the previous study · The EC50 of berberine to A.alternata was only 60 μg/mL,and MIC was 300μg/mL.In contrast,the EC50 values of veratrine,resveratrol,magnolol,thymol and the water extracts of black pepper were 180 μg/mL,322 μg/mL,245 μg/mL,192 μg/mL and 0.16 g/mL,respectively.Combining two or more fungicides may present higher antifungal activity than single fungicide,which is so-called synergism effect and a common strategy to reduce dose and cost.Along this thinking,veratrine,resveratrol,magnolol,thymol and the water extract of black pepper were individually mixed with berberine.Synergism effect was observed when berberine was mixted with the water extracts of black pepper at ratio of 5:1,and the addition effect occurred when berberine was mixed with water extracts of black pepper at ratio of 2:3.Addition effect was also observed when berberine was mixed with water extracts of magnolol at ratio of 1:5,1:2 and 1:1.Other combinations demonstrated antagonisms.These experiments provide basis for future development of bio-pesticides.Piperine is the key component in black pepper.The toxicity of piperine against A.alternata was studied.Results showed that the EC50 of piperine to A.alternata was 197 μg/mL,and the mixture of berberine with piperine showed antagonism effect.The active ingredient in water extracts of black pepper remains elusive.3.To date,berberine’s antifungal mechanism remains unclear and in-depth study is required.In this study the underlying mechanism was preliminarily investigated using DARSTS(Durg Affinity Responsive Target Stability)approach.DARSTS represents a typical method to uncover the targets of small molecule compounds.In this work,A.alternata was exposed to berberine-containing medium and analyzed by SDS-PAGE.Although A.alternate was digested with different doses of proteases and different ratio with proteins,no significant band was observed in the gels of SDS-PAGE.It seems that no target soluble proteins of berberin exist in A.alternata.Scanning electron microscope observation of berberine-treated A.alternata showed morphological deformation in the mycelium and spores.Specially,when berberine concentration reached 120 μg/mL,plasmolysis occurred,indicating that berberine inhibits A.alternata by acting on cell membrane.Furthermore,electrical conductivity increased when A.alternata was exposed to berberine,suggesting that berberine caused leakage of cellsap.Also,content of ergosterol was inhibited evidently after berberine treatment,indicating that berberine blocked the biosynthesis of ergosterol.Our study has shown that berberine inhibited A.alternata by acting on cell membrane.Membrane proteins are the key component of cell membrane and play an important role in pathogenicity.To exploit membrane proteins as potential targets of fungicides,the membrane proteins of A.alternata were retrieved from GenBank and subjected to bioinformatics analysis.In this work,95 membrane proteins of A.alternata with clear functions were classified according to functions.Several important membrane proteins were analyzed by TMHMM server.2.0,including the transmembrane domains,hydrophobicity and signal peptides.In particular,the transmembrane domains were analyzed through alignment of homologous sequence compared with other plant pathogens,and specific proteins and homologous proteins were discovered.