Study On The Cell Response Of Penicillium Thomii PT95 And Q1 Strains To Oxidative Stress

Sclerotium is an compact hyphal structures produced in fungi. Some Penicillium can produce sclerotia. Sclerotiogenic fungi has some unique biological characteristics which show a good application prospect. Two strains of Penicillium sp. PT95 and Q1 were separately isolated from soil collected close to Fenyang and Wutai Mountain, Shanxi Province. The strains can form abundant sclerotia in which carotenoid is accumulated. Sclerotia differentiation and carotenoid metabolism of two strains were preliminary studied. On the basis of preliminary work in laboratory, Penicillium sp. PT95 and Q1 strains were identified through ITS method. In order to select the optimum medium for mass production of sclerotia of two stains, traditional solid-plate culture and wet-plate culture were used to culture two strains. Penicillium sp. PT95 and Q1 strains of the cell in response to oxidative stress was studied from the effect of oxidative stress on sclerotia differentiation, lipid peroxidation, antioxidant metabolism and AsA-GSH cycle of these aspects.Solid-plate culture and new means of wet-plate culture were used. To determine the potential availability of the method for mass production of sclerotia, nine kinds of media were used to culture the PT95 and Q1 strains. Wet-plate method can be used to culture PT95 and Q1 strains, and can form sclerotia on the plate surface. The results of the solid-plate culture and wet-plate culture showed that on 25% glycerol nitrate agar medium and 25% glycerol nitrate broth medium, the growth of both strains was relatively slow, and no exudate and sclerotia were found. The time of sclerotia maturation was shortest on the potato dextrose agar (PDA) and the time of sclerotia maturation was longest on the Czapek’s agar (CA) of PT95 strain. The time of sclerotia initiation and the time of sclerotia maturation was the shortest on the PDA and malt extract agar (MEA) of Q1 strain. In the wet-plate culture, the time of sclerotia maturation was the shortest on the Czapek’s yeast extract broth (CYB), Czapek’s broth (CB) and potato dextrose broth (PDB) of PT95 strain. The time of sclerotia initiation and the time of sclerotia maturation was the shortest on the PDB of Q1 strain. The shape of PT95 strain’s scleotia were round in main, the shape of Ql strain’s sclerotia were irregular in main. The optimum medium for mass production of sclerotia was MEA medium and CYB medium for strain PT95 and MEA and PDA media and MEB for strain Q1, respectively.Using rDNA ITS method combined morphological observation, Penicillium sp. PT95 and Q1 strains were accurated species positioning. PT95 and Q1 strains’ITS gene sequences with Penicillium thomii (accession number NR077159) similarity reached 100%. PT95 and Q1 strains can be identified as Penicillium thomii.The PDA and MEA media were used as basic media. The effect of oxidative stress induced respectively by CuSO4, H2O2 and paraquat on the sclerotial differentiation, colony morphology, sclerotial morphology and sclerotial biomass and in sclerotium of PT95 and Q1 strains were studied. The two strains were no growth on the medium with paraquat. Under the condition of oxidative stress induced by CuSO4 and H2O2, the time of exudates and sclerotium initiation, the time of the sclerotium maturation of the two strains was advanced 1-3 day compared with the control. And the time of sclerotium maturation of Q1 strain was delayed 1-3 days than that of PT95 strain. Meanwhile, under different oxidative stress conditions, the colonies of two strains consisted of sclerotium with greater diameter. The sclerotial biomass of PT95 and Q1 strains had a positive correlation with the oxidative stress. The stronger the oxidative stress was, the higher the sclerotial biomass. The results showed that in unfavorable conditions, carotenoid may be produced to counter ROS formation, strains differentiate forming more sclerotia or increase the size of sclerotia for long-term survival to remedy deficiency of antioxidation.In PDA and MEA media as basic media, the influence of CuSO4 and H2O2 induced oxidative stress on lipid peroxidation and antioxidant metabolism were studied. The results showed that the value of lipid peroxidation had a positive correlation with the oxidative stress. Under the low oxidative stress conditions, the activities of SOD, CAT, POD and GPX of two strains were increased and total phenolic content, DPPH radical scavenging, ferrous ion chelating ability and reducing power of two strains were increased. And GPX could be considered as one of the main antioxidant enzymes. The enzymatic and non-enzymatic antioxidants will have antioxidant effect to a certain extent under the low oxidative stress condition, as an adaptation to the environment. When resulting in high oxidative stress conditions of free radicals than the enzymatic and non-enzymatic antioxidants’scope of protection, it neededs differentiation the formation of more sclerotia to help strains through adverse environment, in order to protect microbial organism function.By determing the contents of ascorbate and glutathione in AsA-GSH cycle and activities of APX, GR, MDHAR and DHAR, the effect of oxidative stress on the AsA-GSH cycle of two strains was studied. Results indicated that oxidative stress can improve the contents of AsA and DHA and the ratio of AsA/DHA in sclerotia of Penicillium thomii PT95 and Q1 strains. At the same oxidative stress condition, DHA content was higher than AsA content. Oxidative stress may increase the contents of GSH and GSSG in sclerotia of Penicillium thomii PT95 and Q1 strains. The value of the ratio of GSH/GSSG had a negative correlation with the oxidative stress. At the same oxidative stress condition, GSH content was higher than GSSG content. Under the low oxidative stress, the APX and GR activities were increased. The values of APX and GR activities had negative correlation with the oxidative stress. The values of MDHAR and DHAR activities had positive correlation with the oxidative stress. It contributed to the reduction of MDHA and DHA for AsA. It can catalyze the regeneration of AsA and make AsA remained higher reduced state in the body. It is very important to adapt to the stress environment. Results showed that under the low oxidative stress condition, PT95 and Q1 strains by AsA-GSH cycle in APX and GR these two kinds of antioxidant enzymes to survive adverse environment; while under the high oxidative stress condition, PT95 and Q1 strains by AsA-GSH cycle in AsA and DHA, GSH and GSSG of non enzymatic antioxidants and antioxidant enzymes MDHAR, DHAR to help strains through adverse environment.