Isolation And Identification Of One Strain Of Sclerotiogenic Aspergillus And Analysis Of Its Biological Characteristics

Sclerotium is a kind of fungal dormancy,which is formed of mycelium aggregation and adhesion.There are few reports about Aspergillus that can produce sclerotia.The aim of this work was to separate sclerotiogenic Aspergillus from the forest soil of four nature reserves in our country,and identify the purpose strains through the traditional morphology methods and modern molecular biology techniques.Meanwhile,the biological characteristics of these sclerotiogenic Aspergillus strains were also studied.Finally,the extraction conditions of pigment in sclerotia of the purpose strains and the factors those affect the stability of pigment were studied.By using dilution spread plate,81 strains of Aspergillus were isolated from the soil samples collected from Huangguoshu Nature Reserve in Guizhou,Pangquangou Nature Reserve,Li Mount Nature Reserve and Mian Mount Nature Reserve in Shanxi.One strain of Aspergillus isolated from coniferous forest soil of the Huangguoshu Nature Reserve was proved to be able to produce yellow sclerotia in CYA,PDA,JM and CA medium and named as A1 strain.A1 strain was identified as Aspergillus japonicus Saito var.Japonicus through colony and individual morphology observation and ITS sequence analysis.The effects of media,pH,carbon sources,nitrogen sources,concentration of carbon source and concentration of nitrogen source on A1 strain were studied.The results showed that A1 strain could grow fastly in JM,CA and PDA medium and its sclerotial biomass was also high.JM medium gave the highest sclerotial biomass.A1 strain could grow from 15℃ to 37℃.At 30℃,both colony growth speed and sclerotial biomass reached the highest.The pH value close to neutral favored the sclerotial development.In six different carbon sources tested,glucose was the best carbon source.In selected 8 kinds of nitrogen sources,A1 strain could grow quickly,but could not form sclerotia in three kinds of organic nitrogen source media.In five kinds of inorganic nitrogen sources media tested,A1 strain could form sclerotia,and the ammonium nitrate was the best nitrogen source.A1 strain could produce a large number of sclerotia in the JM media of 14.00～28.00 g/L carbon content and 0.24～0.48 g/L nitrogen content.FeSO4,ZnCl₂ and MnCl₂ had little impact on A1 strain.K₂HPO₄ and MgSO₄·7H₂O were essential to the sclerotial formation of A1 strain.KCl had negative effect to sclerotial development of this strain.Thin-layer chromatography and full-wave UV spectra scanning results showed that the pigment in sclerotia of A1 strain was(3-carotene.The analysis results on the effects of extraction temperature,time,frequency and solid-to-liquid ratio on the extraction efficiency of pigments showed that solid-to-liquid ratio had the significant effect on the extraction of pigments.The result of orthogonal experiment indicated that the optimum extraction condition of carotenoids in sclerotia was the following:solid-to-liquid ratio of 1:15g/mL,at 50℃for 1h and extracted twice.Under the optimum condition,the yield of carotenoids could reach 16.46mg/100g.The effects of some factors such as light,heat,acid,alkali,oxidant and mental ions on the pigment stability were studied.In conclusion,H₂O₂ had little influence on the pigment of A1 strain and the pigment was sensitive to light,high temperature and acid.Carotenoids are stable in the exsistence of Na⁺,K⁺,Zn²⁺ and Mg²⁺ ions.Fe³⁺,Fe²⁺,Cu²⁺ and Al³⁺ ions could destroy the pigment greatly.