Preliminary Studies On The Action Mechanism Of Two Antimicrobial Compounds Against Plant Pathogens

The previous studies found that the compound HAINU-19 and zeylenone have good inhibitory activity against the plant pathogens fungi in our laboratory.In order to study the preliminary action mechanism of compounds HAINU-19 and zeylenone,in this work,the preliminary mechanism of the compound HAINU-19 of Colletotrichum musae and the zeylenone of Phytophthora capsici was studied.The main results are as follows:(1)The EC₅₀of compound HAINU-19 inhibiting the mycelial growth and spore germination of C.musae were 18.320?g m L^-1and 9.315?g m L^-1.The EC₅₀of zeylenone on P.capsici mycelial growth,sporangia formation and sporangia germination were was 7.025,1.488?g m L^-1and 10.646?g m L^-1,respectively.Both compounds inhibited mycelial yield.(2)Observed under an optical microscope,it was found that the hyphae treated with the compound had thickened and deformed hyphae.It was observed in transmission electron microscopy that the compound had an effect on mycelial cells.The compound HAINU-19 destroyed the integrity of C.musae cells,a large number of vacuoles appeared,and the cell membrane also wrinkled.However,zeylenone causes the malformation of P.capsici cells,the disappearance of organelles,and the blurring of cell wall boundaries.(3)Through the determination of the soluble protein and reducing sugar content of the mycelium,it was found that the soluble protein content increased after 24 hours of treatment.The compound HAINU-19 had no effect on the reducing sugar content of C.musae,while the concentration of zeylenone at 14?g m L^-1,the reducing sugar content of P.capsici was reduced by 25.58%compared with the control.(4)The content of NAG cosamine and the activity of chitinase in the hyphae were determined.It was found that the compounds HAINU-19 had no effect on the cell wall of C.musae.However,the addition of 7?g m L^-1zeylenone for 24 h resulted in an increase of about 6.71%in N-Acetyl-GLU cosamine content and an increase in chitinase activity by about 5.67%,both of which had no significant effect.(5)The results of measuring the effect of the agent on the permeability of the mycelial cell membrane show that high concentration of zeylenone can improve the permeability of the hyphal cell membrane of P.capsici.After treatment with 7.5,15,30?g m L^-1compound HAINU-19 for 6 h,the relative permeability of C.musae were27.64%,39.60%,and 50.42%,while the relative permeability of the control was17.38%,so it is inferred that the compound HAINU-19 will damage the cell membrane.HPLC and GC detected the effect of compound HAINU-19 on the content of ergosterol,squalene and lanosterol of the C.musae.The results showed that the content of ergosterol and lanosterol was significantly higher than that of the control,and the content of squalene had no significant difference.We found that the diniconazole acting on ERG11 also has no significant effect on the content of lanosterol,therefore,it is speculated that the compound HAINU-19 may act on the C-24-methylase(ERG6)which between lanosterol and eburicol.Transcriptomics analysis found that the addition of the compound HAINU-19 resulted in the down-regulation of ERG6 gene expression in the sterol biosynthesis pathway,and the fold change was less than doubled.(6)We determined the effects of compounds to consume oxygen on mycelial mycelium,and found that both compounds inhibit the respiration of the mycelium.At the same time,the effect of the compound on the content of pyruvate in the mycelium was determined.It was found that the compound HAINU-19 caused the accumulation of pyruvate in C.musae.Transcriptomics analysis found that the citrate synthase(glt A)-related genes in the TCA cycle were significantly down-regulated,and glycosylase Fructose diphosphate aldolase class I(ALDO)genes are significantly up-regulated in the hydrolysis/gluconeogenesis pathway,D-lactate dehydrogenase(cytochrome)(LDHD)and L-lactate dehydrogenase(cytochrome)in the pyruvate metabolism pathway(Lld D)gene is significantly up-regulated,leading to increased pyruvate content.The content of P.capsici treated with concentrations of 3.5,7,and14?g m L^-1zeylenone decreased by 26.32%,67.13%,and 64.08%respectively after24 h.The above results indicate that the compound HAINU-19 may affect the ERG6gene in the process of sterol biosynthesis,and affect the sterol content.At the same time,it may also affect the production of pyruvate and the expression of the citrate synthase(glt A)gene in the TCA cycle,leading to the accumulation of pyruvate and affecting mycelial growth.Zeylenone may affect the normal physiological process of mycelial by affecting the metabolism of pyruvate or glycolysis.