Studies On The Antifungal Activity Of 1,6-O,O-diacety-lbritannilactone Against Phytophthora Capsici

Pepper blight is a worldwide spreadeddevastating soil disease and has become an important factor limiting the production of pepper,which is caused by Phytophthora capsici.Due to the lack of the disease resistant species of pepper,the main control depends on chemical fungicide,such as metalaxyl,M8,Cyazofamid,Pyrimorph and Fluorothiazolpyridone.But excessive and long-time using brings many severe problems,such as the resitance becoming much more serious than ever.Therefore,the search for a safe substitute for chemical fungicides is highly valued.Early research has shown that 1,6-O,O-diacetylbritannilactone(ABLOO)is a key activity compound of inula flowers,which has germicidal effect on fungi.However,there are few reports has been published,and the sterilization mechanism of the compound is not clear yet.In this study,P.capsiciwas used as the target for studying on the antifungal activity of ABLOO against P.capsici at its different life stages.Moreover,absolute quantification i TRAQ technology,was used to analyze the protein expression differences of P.capsici before and after ABLOO treatment.The major findings are as follows:1.The results showed that the EC50 value of ABLOO against the mycelial growth of P.capsici was 95.89 mg/L.The ABLOO could effectively inhibit sporangium formation and sporangium germination of P.capsici with EC50 values of 30.45 mg/L and 124.59 mg/L.As the result shown,the sporangium formation was the most sensitive to the ABLOO.But the inhibitory effect on zoospore germination was not obvious.2.Observed under light microscope and transmission microscope,the hyphae of P.capsici treated with ABLOO were significantly shortened.The branches were increased,and a large number of short hyphae were generated.The morphology of mycelia was abnormal.The cell wall of the cell became thin,and some small bumps on the surface that was clearly seen on the cell wall surface.The vacuole decreases in number but increases on the volume.The vacuolar fusion occurs,and other organelles in the cell are squeezed by the vacuole.This shown that P.capsici has undergone a significant change in morphology under the influence of ABLOO.3.The mycelia of P.capsici were treated with 200 mg/L,300 mg/L and 400 mg/L ABLOO.Within the tested 360 mins,the conductivities of mycelia were increased as time passed,and showed a significant upward trend.At each time point,such as 15,30,60,120,180,and 120 min,each treated concentrationof mycelium’s conductivity was increased with the incresase of drug concentration.And they were all significantly higher than that of control.More specifically,when processing time went to 180 min and 360 min,three treatment groups’ concentrationbecame no difference.It indicated that ABLOO affected the cell membrane permeability of P.capsici.The permeation of the cell membrane is deteriorated,the ion permeability is increased,and the electrolyte leakage occurs.4.β-1,3-glucan is the main component of the cell wall of the P.capsici,which plays an important role in the growth and development of phytophthora.Treated by ABLOO,β-1,3-glucan content of P.capsici decreased significantly,the inhibition rate of high concentration treatment group reached more than 70%,thus theβ-1,3-glucan content P.capsici were decreased.The function of the cell wall was damaged,the energy was reduced.As a result,spore germination and mycelial growth were inhibited.5.In this study,using absolute quantification i TRAQ technology,we compared analysis proteome of treated and untreated mycelia of P.capsici,and the function of identified protein spots was elucidated.The total number of identified proteins was 3808 and 794 significant differential proteins included.In the 794 significant differential proteins,387 was up-regulation and the other 407 was down-regulation.These differentially expressed proteins types mainly included enzymes in the key metabolic pathways and the general stress response proteins,such as Acetyl coenzyme A synthetase(D0MRI9),DNA helicase(H3GAJ9),NADH dehydrogenase(A0A0W8C3E6),and some main protein involved in the biosynthesis and energy metabolism.Through Gene Ontology analysis,these proteins were enriched in 44 GO terms(secondary classification).According to the results,the main molecular gunctional that the significant differential proteins involved were monovalent inorganic cation transmembrane transporter activity,hydrogen ion transmembrane transporter activity,transmembrane transporter activity and so on.The main biologicalprocessthat the significant differential proteins involved were oxidation-reduction process,hydrogen ion transmembrane transport,inorganic cation transmembrane transportand so on.The main cellularcomponent that the significant differential proteins involved were cytochrome complex,intrinsic component of membrane,respiratory chain complex III,integral component of membrane and so on.These results indicated that compared with the control,the regulatory mechanismand metabolic pathways of ABLOO treated P.capsici presented significant dynamic change,shows the P.capsici affected by ABLOOmade a lot of differences in manyphysiological biological processes and morphological.And in the Kyoto Encyclopedia of Genes and Genomes(KEGG)richment analysis,those significant differential proteins distributed in 83 pathway pathways,signigicantly enriched in the pathways of carbon metabolism pathway(map01200),ribosome pathway(map03010),purine metabolism pathway(map00230),biosynthesis of amino acidspathway(map01230),fatty acid metabolism pathway(map01212),pyruvate metabolism pathway(map00640)and protein processing in endoplasmic reticulum pathway(map04141).In particular,Several differential proteins related to carbon metabolism and amino acid synthesis were selected for analysis.It was found that ABLOO has an important influence on both of the carbon metabolism and amino acid synthesis of P.capsici.P.capsici is also adjusting its own metabolic pathways to resist the inhibitory effect of ABLOO.