| Green mold disease caused by Penicillium digitatum is one of the major citrus diseases.The disease causes huge economic losses to the citrus industry every year.At present,the control of green mold mainly through the use of chemical fungicides to achieve.Although chemical fungicides are effective,there are many drawbacks,such as easy to induce pathogenic bacteria to develop resistance,pollution of the environment and human health hazards.Therefore,there is an urgent need to find alternatives to chemical fungicides.Predecessors and our previous research found that the essential oil component citronellal has strong antibacterial activity,and is green and safe,not easy to induce resistance to pathogenic bacteria and other characteristics,is a very promising biofungicide.Previous studies have shown that citronellal treatment affects the synthesis of ergosterol in Penicillium digitatum filaments,which may involve the regulation of membrane proteins.However,which specific membrane proteins are involved in the regulation is not yet clear.In addition,whether ergosterol is affected by citronellal during spore germination has not been reported.In view of this,this study intends to compare the effect of citronellal on the ergosterol metabolic pathway in the process of spore germination of Penicillium digitatum,reveal the changes of mycelial membrane proteins during citronellal treatment by proteomics technology,and establish the preliminary gene deletion mutation technology system of Penicillium digitatum,aiming to study the effect of citronellal on ergosterol synthesis of Penicillium digitatum and the mechanism of action,and provide theoretical basis for the control of citrus green mold.The main results are as follows:(1)Effect of citronellal on the expression of membrane proteins in Penicillium digitatum filaments.The effects of citronellal on the ergosterol metabolism of Penicillium digitatum filaments were investigated on the basis of the effects of citronellal on the metabolites and gene expression related to the ergosterol synthesis pathway during spore germination of the sensitivity of Penicillium digitatum imazalil(Pds01)and the resistant strain(Pdw03,an ergosterol metabolism mutant).The results showed that the half-inhibitory concentrations of citronellal were about 0.8 μL/m L and 3.2 μL/m L for Pds01 and Pdw03,and the ergosterol and wool sterol contents of Pds01 and Pdw03 strains were measured at the half-inhibitory concentrations of citronellal.The results showed that the spore ergosterol content of both Pds01 and Pdw03 strains showed a decreasing trend followed by an increasing trend after citronellal treatment.The wool sterol content showed a change of increasing and then decreasing,and the ergosterol content of the treated group was significantly lower than that of the control group(P<0.05).The results showed that the spore ergosterol content of both Pds01 and Pdw03 strains showed a decreasing trend followed by an increasing trend after citronellal treatment.The wool sterol content showed a change of increasing and then decreasing,and the ergosterol content of the treated group was significantly lower than that of the control group(P<0.05).Fluorescent quantitative PCR analysis of seven key genes involved in the ergosterol synthesis pathway(Erg3,Erg5,Erg6,Erg7,Erg9,Erg11 and Erg26)revealed that citronellal treatment resulted in a significant down-regulation of Erg3 expression in spores of strain Pds01,while Erg11 expression was largely unaffected.Erg5,Erg6,Erg7,Erg Erg9 and Erg26 were significantly up-regulated at 10 h of treatment,but returned to normal after 18 h with time extension.For strain Pdw03,citronellal also induced a significant down-regulation of Erg3 expression,and Erg5,Erg6,Erg7,Erg9,Erg11 and Erg26 gene expression were significantly up-regulated at 10 h of treatment,and as time was extended to 18 h,Erg5,Erg6,Erg7 and Erg26 expression were significantly down-regulated,and Erg11 expression returned to normal.Erg9,on the other hand,was consistently up-regulated.It is speculated that Erg3 gene may be one of the potential targets of citronellal affecting ergosterol synthesis in Penicillium digitatum spores.(2)Effect of citronellal on the expression of membrane proteins in Penicillium digitatum filaments.Given that ergosterol synthesis pathway genes are mostly membrane proteins.It has been reported that membrane proteins may be involved in fungal ergosterol regulation,and the effects of citronellal treatment,exogenous addition of ergosterol+citronellal treatment on Pds01 membrane protein expression were analyzed using TMT(tandem mass tags)technique.The results of substance content showed that citronellal treatment could effectively reduce the mycelial ergosterol content of Pds01 and induce a significant increase in wool sterol content.The addition of exogenous ergosterol significantly increased the ergosterol and wool sterol contents of Pds01 mycelium,and the wool sterol content was significantly lower than that of citronellal treated group,which indicated that the addition of exogenous ergosterol could alleviate the inhibition of ergosterol metabolic pathway of Pds01 mycelium by citronellal.Membrane proteomics analysis identified 372 differential membrane proteins,mainly involved in β-alanine metabolism,steroid biosynthesis,base excision repair,terpene skeleton biosynthesis and other pathways,among which Erg2,Erg3,Erg4,Erg5,Erg6,Erg11,Erg24,Erg25 and Erg26 of ergosterol metabolic pathway could be detected,and there are significant differences between Erg3,Erg4,Erg25 and Erg26,in order to more fully explore the ergosterol synthesis pathway,ERG5 was also studied.With significant seventeen differential membrane proteins(containing Erg3,Erg4,Erg25,Erg26 and Sre A,etc.)and ERG5 were selected for subsequent fluorescence quantitative PCR validation.The results showed that citronellal treatment resulted in significant down-regulation(P<0.05)of Erg3,Erg25 and Erg26 expression and up-regulation(P<0.05)of Erg5 expression in mycelium of strain Pds01,and Erg4 expression was normal at 30 min of treatment,but significantly down-regulated with time extension to 60 min.The exogenous addition of ergosterol treatment resulted in significant up-regulation(P<0.05)of Erg25 and Erg26 expression in the mycelium of Pds01 strain,Erg5 expression was basically unaffected,Erg3 and Erg4 expression was significantly down-regulated(P<0.05)at 30 min,Erg4 expression was up-regulated at 60 min,and Erg3 expression was basically unaffected.It is speculated that Erg3 may be a target site for citronellal to inhibit the ergosterol synthesis pathway in Penicillium digitatum filaments.Except for C6 transcription factor 4 and SPT8 genes,which were significantly down-regulated,and C6 transcription factor 1 and MFS multidrug transporter1 genes,which were not significantly changed,the corresponding genes of all the proteins were significantly up-regulated.It is particularly interesting that the Sre A gene,which is directly involved in regulating the fungal ergosterol metabolic pathway,was significantly up-regulated at 30 min of citronellal treatment and returned to normal at 60 min,implying that it is likely to be involved in the ergosterol compensation mechanism,thus affecting ergosterol synthesis.(3)Construction of a deletion mutation system of Penicillium digitatum gene.Based on the membrane protein results,it is likely that Sre A is involved in citronellaldehyde-induced regulation of ergosterol metabolism in Penicillium digitatum.In order to shed light on this issue,a preliminary Sre A knockout system was constructed.The main results are as follows: Using the genomic DNA of Penicillium digitatum as a template,the upstream fragment of Sre A gene of 1196 bp and the downstream fragment of 1250 bp were obtained by PCR amplification,and the sequence amplification was successfully confirmed by sequencing to match 100% of the amplified fragment with the template fragment.The Sre A knockout vector with 100%sequence match was obtained by double digestion of the plasmid p CAMBIA1300-HPH with the target fragment of the ligase cut site,which was recovered and transformed by ligation with T4 ligase,and the resulting colonies were sequenced and verified,laying a theoretical foundation for subsequent gene function verification.In summary,the results of this study suggest that citronellal affects the ergosterol metabolic pathway in Penicillium digitatum spore germination,and the membrane protein results indicate that the mycelial target may also be Erg3.In addition,citronellal can affect the expression of several membrane proteins.Among them,Sre A has been found to be involved in the regulation of fungal ergosterol.On this basis,the Penicillium digitatum gene deletion mutation system was preliminarily constructed and Sre A knockout vectors were obtained.The results of this study can provide theoretical references for the study of the fungal inhibition mechanism of plant essential oils and postharvest disease control of fruits and vegetables. |
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