Molecular Identification Of The Leptographium Qinlingensis And Its Metabolic Mechanism Of Terpenoids

Leptographium qinlingensis,as an crucial pathogenic fungi of Pinus armandi,co-infected Pinus armandi with Dendroctnus armandi,which weakened the host tree and caused economic losses to P.armandi in the Qinling mountains of China.Cytochrome P450 enzyme,as a metabolic enzyme of defense substances produced by degrading host,plays a very important role in overcoming the resistance host.Firstly,this study analyzed the taxonomic status of L.qinlingensis by means of morphology and molecular biology.Then,the P450 gene of L.qinlingensis was cloned,and the effects of different nutrients on the physiological characteristics of L.qinlingensis were compared,as well as the effects of major volatile terpenoids of P.armandi on the expression level of L.qinlingensis P450 gene,in order to reveal the metabolic mechanism of L.qinlingensis P450 enzyme on terpenoids.The following results were obtained:(1)Through comparison of morphology and gene sequence including ITS,TEF1,LSU and ?-tubulin,it was determined that L.qinlingensis belonged to Ophoostoma.(2)The day of inflection of L.qinlingensis in phloem is late than the inflexion in xylem.When the xylem and phloem were the main nutrients,the cellulase activity of L.qinlingensis did not change and the peroxidase activity of L.qinlingensis decreased.The laccase activity of L.qinlingensis was significantly increased when the xylem of Pinus armandi was used as the main nutrient.(3)According to the transcriptome sequencing results,a total of 18 P450 genes were cloned.Phylogenetic tree shows that these genes belonged to 11 families,including CYP5054,CYP5062,CYP51F1,CYP530,CYP533,CYP537,CYP539,CYP544,CYP570,CYP577 and CYP617.However,there were 6 gene that were not closely related to the known P450 gene family of blue-stain fungi.(4)The results of quantitative analysis of the P450 gene expression of 12 P450 genes in different nutrient media showed that the monoterpene mixture could inhibit the expression of 8 genes and turpentine oil could inhibit the expression of 9 genes The CYP530 and CYP5054 genes play a role in the degradetion of terpenoids.(5)The results showed that after terpenoid treatment for 36 h,CYP530,CYP539,CYP570,CYP533,CYP5054,CYP5062,CYP570 and two unknown family genes(p24,p6)showed strong tolerance to(±)?-Pinene and(+)-limonene,and the tolerance of monoterpenoid mixture and terpenoid mixture could be reduced.(6)Terpenoids could inhibit the growth of L.qinlingensis,and the effect of terpenoids mixture was the most significant,making the growth inflection point of L.qinlingensis appear at the latest.Moreover,the monoterpene mixture was more effective in reducing the concentration of spores.