Partial Biological Characteristics And Metabolic Variation Of Cladosporium Cladosporioides Control And Mutant From Giant Panda

Objective:Giant Panda(Ailuropoda Melanoleuca)is an endangered species that is endemic to China,fungal infection is one of the main causes of giant panda skin diseases.Cladosporium cladosporioides Z20 is the causative agent of a case of giant panda phaeohyphomycosis skin disease.Strain Zt is the mutant strain of Z20 with mutant by microwave-NTG mutagennesis.This study will further explore the biological characteristics of Cladosporium cladosporioides Z20 and Zt,research the differences between the metabolites of Cladaosporium Z20 and Zt by the gas chromatography-mass spectrometric analysis.Lay the foundation for the study of the diagnosis,treatment and pathogenic mechanism of cladosporium cladosporioides.Method:Continuous cultivation of cladosporium cladosporioides Z20 and Zt on PDA observation and records colony morphology and growing every day.The ultrastructure of strain Z20 and Zt were observed under the transmission electron microscope.6 kinds of fermentation media(PDB,SDB,M-SDB,YM,DTM,MA)were used to determine the mycelial biomass and metabolites of strain Z20 and Zt under the same culture conditions.Determination of polysaccharide content in Z20 and Zt strains by sulfuric acid-phenol method.By means of micro-volume of broth dilution to determine the minimal inhibitory concentration(MIC)of Z20 and Zt to 5 common antifungal drugs.The metabolites of Z20 and Zt were measured by gas chromatography-mass spectrometry(GC-MS).The data were analyzed by principal component analysis(PCA),cluster analysis,KEGG Pathway.Result:1.Z20 and Zt of Cladosporium spp.continuous culture in PDA,The growth rate of strain Zt was higher than that of Z20,the color of colony was shallow,and the number of spores decreased.Under the transmission electron microscope,the mycelial wall thickness of strain Z20 was about 0.3 ?m,and the width of the strain was about 3.25 ?m,and the mycelial wall thickness of strain Zt was about 0.15 ?m,and the mycelial width was about 2.5 ?m.2.Strains Z20 and Zt under the same conditions in the YM medium fermentation culture achieved the biggest mycelium biomass and metabolites.the maximum biomass of Z20 was 2.3015+0.0292 g,the maximum amount of metabolites was 3.6646+0.4141 mg;the maximum biomass of Zt was 2.1509+0.0894 g,the maximum amount of metabolites was 2.4916+0.4630 mg.3.Determination of polysaccharide content in Z20 and Zt strains by sulfuric acid-phenol method,Z20 was 38.21 ?g/mg,Zt was 34.32 ?g/mg.4.For the strain Z20,the MIC value of Griseofulvin and Fluconazole was 32?g/mL,the Itraconazole,Ketoconazole and Ciclopirox were 0.5 ?g/mL,4 ?g/mL,8 ?g/mL,respectively.For the strain Zt,the MIC value of Griseofulvin,Fluconazole,Itraconazole,Ketoconazole and Ciclopirox were 64 ?g/mL,0.25 ?g/mL,2 ?g/mL,4?g/mL and 8 ?g/mL,respectively.5.Through GC-MS 930 peaks were detected,identified 370 substances,283 different metabolites were obtained,obtained the 181 differential metabolites KEGG ID,31 metabolic pathways involved were found.Conclusions:1.Cladosporium Z20 and Zt can grow in a variety of media,and there are significant differences in the biological characteristics under the same culture conditions.For the strain Z20,drug sensitivity in turns were Itraconazole>Ketoconazole>Ciclopirox>Fluconazole=Griseofulvin;For the strain Zt,drug sensitivity in turns were Itraconazole>Ketoconazole>Ciclopirox>Fluconazole>Griseofulvin.The sensitivity of Cladosporium cladosporioides Zt of 5 antibiotics were higher than Z20 except for Griseofulvin.2.The differential metabolites of strain Z20 and Zt were successfully obtained by GC-MS,and the metabolites were enriched into 31 metabolic pathways.