Effect of Cell Wall of Yeast on Inhibiting Postharvest Pathogenic Fungi by Inducing Resistance in Pear and Tomato Fruits and the Possible Defense Mechanisms Involved

Author:Sun Cui

Supervisor:yu ting


Degree Year:2019





Fungal disease is one of the causes of great loss of fruits and vegetables after postharvest.The use of antagonistic yeast to inhibit postharvest diseases of fruits is considered to be the most promising alternative to chemical fungicides,which has attracted more and more attention and research at home and abroad.Over the past 3decades,studies have shown that antagonistic mechanisms of biocontrol yeast mainly include competition for nutrients and space,inducing host resistance,secreting hydrolase and adsorption.Among these,inducing host resistance is one of the important factors to inhibit pathogens,which is closely related to a variety of physiological and metabolic activities of fruits.In the previous studies,we preliminarily demonstrated that the autoclaved yeast and their cell walls could induce resistance against Penicillium expansum in pear fruit.However,the molecular mechanisms which the antagonistic yeast induced fruit disease resistance are still unclear.This study aimed to investigate the substance basis in which yeast cell wall induced resistance against pathogens in pear and tomato fruits and its possible biological mechanisms were also discussed.The main findings are as follows:(1)Substance basis analysis of resistance to P.expansum in pear fruit induced by yeast cell wall.The inhibitory effects of eight antagonistic yeasts,Saccharomyces cerevisiae and Pichia pastoris on fruit diseases were compared and analyzed.The results showed that except S.cerevisiae and P.pastoris,eight antagonistic yeasts could significantly reduce the occurrence of blue mold rot in postharvest pear fruits.Meanwhile,the autoclaved yeast and their cell wall have the ability to induce pear fruit resistance against P.expansum.Among the ten selected yeasts,the autoclaved Rhodosporidium paludigenum and its cell wall were the most effective in the induction of resistance,respectively.The analysis of cell wall components of yeast showed that cell wall components of different antagonistic yeasts have the similar patterns,but the great changes were observed in different species of yeast.Moreover,except for mannoprotein,the other component of cell wall,such as chitin,β-1,3-D-glucan andβ-1,6-D-glucan,display the excellent induction resistance against P.expansum in pear fruit.(2)The chemical structure identification of cell wall chitin from S.cerevisiae and its effect on inducing resistance in tomato fruit.The structure of cell wall chitin from S.cerevisiae was characterized by Ubbelohde viscometer,high performance liquid chromatography(HPLC),Fourier transform infrared spectroscopy(FTIR)and nuclear magnetic resonance(NMR),and its control effect and mechanisms on Botrytis cinerea in tomato fruit were explored.The results showed that the chitin sample consisted of N-acetylglucosamine with a molecular weight of 4.68×10~5 Dalton and the degree of deacetylation was 45.83%.In vitro,chitin could not directly inhibit the growth of B.cinerea,but it could significantly induce the resistance of tomato fruit to gray mold,and the induction effect was closely related to the treatment concentration and induction time.Further analysis showed that chitin induced the accumulation of reactive oxygen species(ROS)and the deposition of callose in fruit tissues.Meanwhile,the activities of the defense-related enzymes(SOD,CAT,POD,PAL,GLU and CHI)were increased and the corresponding genes were up-regulated.In addition,chitin treatment also up-regulated the of key genes in salicylic acid signaling pathway(ICS,NPR1,TGA1a,TGA2,PR1b1 and PR5).(3)Chemical structure identification and biocontrol efficacy evaluation of cell wallβ-1,3-D-glucan from S.cerevisiae.The molecular weight(Mw),monosaccharide composition,characteristic functional groups and carbon/hydrocarbon signals ofβ-1,3-D-glucan were characterized by Ubbelohde viscometer,HPLC,FTIR and NMR.The results showed that the extracted samples ofβ-1,3-D-glucan consisted of glucose and the molecular weight was 165 kDa.The glucose molecules were linked byβ-1,3 glycoside bonds.As for the induction of fruit resistance,β-1,3-D-glucan could not directly affect the growth of P.expansum spores in vitro and in the fruit wounds,while the induction treatment byβ-1,3-D-glucan significantly inhibited the spore germination of P.expansum in the fruit wounds.Scanning electron microscopy(SEM)was used to observe the effect ofβ-1,3-D-glucan treatment on the ultrastructure of pear fruit wound tissue.The results indicated thatβ-1,3-D-glucan could enhance the resistance to P.expansum at the cellular and tissue level.RT-qPCR results showed thatβ-1,3-D-glucan up-regulated the gene expression of PR1,GLU,endoGLU9,CHI3,CHI4,endoCHI,PR4,PR5 and PAL around the wound of pear tissues,thereby reducing the disease incidence and the lesion diameter of pear.(4)Genetic transformation of cell wall glucan synthesis related genes Rho1 and evaluation of their biocontrol efficacy.The full-length sequences of the target gene Rho1 was obtained from genomic DNA of S.cerevisiae by specific primers and the target gene Rho1 was cloned into the vector pYES6/CT by clonexpress technology.Then the recombinant vector was introduced into S.cerevisiae by electro-transformation.After sequencing and identification,the over-expressed recombinant yeast strain SC/Rho1 was successfully obtained.RT-qPCR results showed that the gene expression of Rho1 in SC/Rho1 was up-regulated by 107.89,compared with wild-type strains.Furthermore,compared with the wild type strain,the content of glucan in the cell wall of recombinant SC/Rho1increased by 9.26%.The results of biocontrol effect showed that the cell wall of the recombinant yeast was more effective in inducing pear fruit resistance against P.expansum.(5)Transcriptionomic analysis of cell wallβ-1,3-D-glucan from S.cerevisiae on inducing resistance to blue mold in pear fruit.The tissues of pear fruit treated withβ-1,3-D-glucan plus P.expansum was analyzed by transcrptome.The results showed that 5984 genes were up-regulated and8769 genes were down-regulated between the P.expansum treatment andβ-1,3-D-glucan plus P.expansum treatment.β-1,3-D-glucan may activate the recepors of FLS2,CNGCs and calcium channel,triggering the downstream hormone signaling transduction of auxin,jasmonic acid,gibberellin and ethylene and then leading to the transcription of defense-related transcription factors(MYC2,PTI5,PTI6,WRKY29 and WRKY33),thereby generating a series of defense-related responses.These responses including the changes of defense-related genes of POD,PAL,HSP90 and PR1,the biosynthesis and metabolism of amino acids and the activation of secondary metabolic pathways.Finally,the occurrence and development of blue mold rot in pear fruit wound were inhibited.