Study On Antifungal Activity Of 4-Coumaric Acid Against Colletotrichum Gloeosporioides In Walnut

Walnut is an important woody oil species in China.The disastrous disease in walnut production is anthrax of walnut caused by anthrax of Colletotrichum gloeosporioides in China.The 4-coumaric acid of phenolic compound is an effective antifungal agent.Therefore,it is of great practical significance to investigate the antifungal activity of 4-coumaric acid and its synthesis structural gene C4H（cinnamate-4-hydroxylase）on walnut anthracnose.In this study,the minimum inhibitory concentration（MIC）of coumaric acid was determined,and different proportions of MIC were set to spray walnut fruits inoculated with C.gloeosporioides to observe the susceptibility and identify its antibacterial effect.Three C4H genes were identified from the transcriptome data in disease-resistant and disease-susceptible walnut cultivars,the JrC4Hs were cloned in walnut,construct target gene expression vector,transformation of walnut fruit and apple callus,and inoculated with C.gloeosporioides,explore the antifungal activity and action mechanism of JrC4Hs on walnut anthracnose.The main results are as follows:（1）The MIC of 4-coumaric acid and p-hydroxybenzoic acid are 1 g·L^-1 and 2 g·L^-1.The binary bacteriostatic agent with 4-coumaric acid and p-hydroxybenzoic acid MIC ratio of 1:2can significantly inhibit effect of C.gloeosporioides in walnut fruit.（2）Bioinformatic analysis of JrC4Hs Showed that JrC4Hs were hydrophobic and transmembrane proteins withα-helical secondary structure and obvious cytochrome P450conserved domain,relationship is closest Quercus suber.（3）The JrC4Hs were cloned and tissue expression analysis showed that the relative expression level of JrC4H1 and JrC4H3 were the highest in leaves,while the expression level of JrC4H2 was not high in all tissues tested,with little difference in expression,and the relative expression level of JrC4H2 was slightly higher in buds.The results of abiotic stress and hormone treatment showed that JrC4H1 and JrC4H3 expressions were significantly up-regulated,while JrC4H2 expression showed no significant change trend.It is speculated that JrC4H1 and JrC4H3 may play more important roles than JrC4H2 in abiotic stress and hormone regulation.（4）The walnut fruits with transient expression of JrC4Hs were inoculated with C.gloeosporioides,and it was found that compared with the control group,the area of C.gloeosporioides disease spots decreased significantly,and the area of C.gloeosporioides disease spots with transient expression of JrC4H3 was the smallest,indicating that the transient expression of JrC4H3 had the best inhibition effect on C.gloeosporioides.According to measurement of physiological indicators of walnut husk,the results showed that compared with the control group,the MDA content,relative electrical conductivity,H₂O₂content and superoxide anion generation rate were significantly decreased with transient expression of JrC4Hs;the antioxidant enzyme activity,total phenol content and DPPH free radical scavenging ability were significantly increased.These results show that transient expression of JrC4Hs in walnut husk may inhibit the activity of C.gloeosporioides by reducing cell membrane damage and ROS accumulation and enhancing antioxidant capacity.（5）Compared with wild-type apple callus,overexpression of JrC4Hs were inoculated with C.gloeosporioides the area of disease spots decreased in apple callus,and the area of C.gloeosporioides disease spots with overexpression of JrC4H3 was the smallest.By NBT staining analysis,the color of overexpressed JrC4H3 apple callus was the lightest,followed by overexpressed JrC4H1 apple callus,and the color of overexpressed JrC4H2 was not much different from that of wild-type apple callus.The results showed that the overexpression of JrC4H3 in apple callus had less superoxide anion accumulation,higher enzyme activity and the best inhibition effect.According to measurement of physiological indicators of apple callus,the results showed that the H₂O₂ content and superoxide anion generation rate of apple callus overexpressed with JrC4Hs were lower than the wild type;the antioxidant enzyme activity,total phenol content and DPPH free radical scavenging ability were higher than the wild type.These results indicated that overexpression of JrC4Hs in apple callus could inhibit the activity of C.gloeosporioides.（6）Subcellular localization showed that JrC4H3 protein was localized in the nucleus.