The Research Of Molecular Mechanism Associated With Penicillium Digitatum Resistance To Prochloraz

Citrus, as the most important economic fruit, whose production was reduced owing to the affecting by green mold (Penicillium digitatum). Citrus green mold and blue mold caused by P. digitatum and P. italicum infects the fruit during harvesting, processing and storing, and is considered as the most destructive postharvest fungal diseases, leading to serious economic losses. Sterol 14alpha-demethylase (CYP51), a key enzyme of sterol biosynthetic pathways in eukaryotes, was widely expressed in bacteria, fungi, plants and animals. It is responsible for catalyzing the reaction of sterol 14a-demethylation. Sterol biosynthesis inhibitor fungicides (SBIs) are widely used as agricultural fungicides, which can destroy the ergosterol biosynthesis pathway, through inhibit the activity of enzymes in sterol biosynthesis, and then lead to the damage of cell membrane.14a-demethylation inhibitors (DMIs) are the main group of sterol synthesis inhibitor fungicides, which contains imazalil, prochloraz, triazolone, diniconazole, tebuconazole et al. The target enzyme of DMIs is CYP51. However, the widespread usage of these DMIs compounds result in the fungus resistance to DMIs more and more frequently. The molecular mechanism associated to resistant fungus has become a hot research region in agriculture.In order to study the molecular mechanism associated to resistant fungus, wild P.digitatum isolates were selected to analysis the relationship between gene mutations and resistance by the methods of molecular biology and bioinformatics. The main results were described as following:1. A total number of 135 citrus pathogenic fungi strains were isolated from different locations of Hubei province, including 78 P.digitatum,34 P.italicum and 23 unkown pathogenic fungi. The sensitivity to prochloraz (PRC) of all strains was tested. The results showed that 25/78 strains (about32%) were found resistance. By calculating the EC50 of the 40 P.digitatum isolates (containing all PRC-R phenotype strains and 15 PRC-S phenotype strains), the tested population were divided into four different sensitivity level groups, S, LR, MR and HR.2. The genome DNA of all PRC-R phenotype strains and 15 PRC-S phenotype strains were extracted. The Pdcyp51A and Pdcyp51B upstream sequences were amplified from 40 isolate, and sequenced to detect if there are some insertions in the PRC-R strains. An extra 199-bp fragment insertion was found in the promoter regions of Pdcyp51B, while none was found in Pdcyp51A upstream sequences. The PdCYP51A/B/C genes were cloned and analysised, which showed that the three genes located in different branches in the phylogeny trees respectively, and they had only 45% similarity. Alignment analysis of PdCYP51A and PdCYP51B indicated that there was no relationship between mutations in CYP51A and P.digitatum resistance, while four substitution mutations in PdCYP51B were detected (Y136H and Q309H in HR strains, G459S and F506I in MR strains, Q309H in LR strains), corresponding to the three sensitivity levels.3. Bioinformatics methods were used to analysis the mutations in PdCYP51B associated with the resistence. The structural modeling of PdCYP51B was carried out by SWISS-MODEL. It contained twelve major helices, four antiparallel β sheets and the connecting loops. The heme was surrounded by the well conserved a-helices E, I, J, K and L. Inhibitor docking analysis of PdCYP51B was accomplished with prochloraz docked into the active site of PdCYP51B. According to the results of our experiment, Y136H and G459S in PdCYP51B were proved to be the key mutations associated with prochloraz resistance. Most of all, Y136H was proved to be involved in the interaction between the active site and prochloraz.