Isolation And Identification Of Hydroxybenzene-producing Endophytic Fungi In Magnolia Officinalis From Qinba Mountain

Magnolol and Honokiol are the main components of Magnolia officinalis.They have many pharmacological effects,such as anti-virus,anti-tumor,anti-bacterial,anti-ulcer,anti-inflammatory and so on.Magnolol and Honokiol are derived from the root and branch of Magnolia officinalis.With the expansion of its application,Magnolia officinalis resources were destroyed badly.In this study,we analyzed the diversity of endophytic fungi in order to established the germplasm resource bank of endophytic fungi from magnolia officinalis in Qinba Mountain area.Screening of endophytic fungi producing Magnolol and Honokiol by HPLC,then through the orthogonal experiment?L₉?3⁴??to optimize culture medium and culture conditions of endophytic fungi producing Magnolol and Honokiol.1.Isolation and preliminary identification of endophytic fungi from magnolia officinalis in Qinba Mountain Area.151 strains fungi were isolated from Magnolia officinalis in Qinba Mountain area by tissue culture,with the colonies and microscopic morphological characteristics,we identified the 144 strains as 6 orders,7 families and 16 generas.Among them,Penicillium?21 strains?,Fusarium?18 strains?and Mucorus?15 strains?were dominant groups,accounting for 13.91%,11.92%and 9.93%of the total.The results showed that the endophytic fungi of Magnolia officinalis in this area had certain species diversity,and different tissue sites had certain effects on the abundance of endophytic fungi.The quantity and species of endophytic fungi isolated from stems were the most,followed by the roots and leaves.In addition,there were some similarities and differences between quantity and species of endophytic fungi in Magnolia officinalis from different sampling sites.2.Screening and identification of hydroxybenzene-producing fungiAll of strains were fermented by liquid flask culture.Screening the endophytic fungi producing Magnolol and Honokiol by HPLC and UPLC-MS/MS.As the result,we found 14strains can produce Magnolol and Honokiol,account for 9.93%of the total.Among them,Penicillium?6 strains?account for 42.86%,Aspergillus?2 strains?account for 14.29%,the remaining 6 strains were Mucor,Trichoderma,Fusarium,Alternaria,Clonostachys and Lecanicillium,each one account for 7.14%of the total.Among the above strains,the yield of strain Y-18 and Y-88 was the highest.The yield of Honokiol in Y-18 can reach to 532.443ug/L,and the yield of Magnolol and Honokiol in Y-88 can reach to 579.140 ug/L and 471.627ug/L.With the microscopic morphological and ITS sequences of the strains,we identified the strain Y-18 as Clonostachys rosea and the strain Y-88 as Lecanicillium saksenaeae.3.Optimization of culture medium and fermentation conditions of strain Y-88The optimum liquid fermentation medium of strain Y-88 were optimized by orthogonal design?L₉?3⁴??based on single factor test.The best fermentation medium of Magnolol were as the follows?g/L?:glucose 20.00,peptone 6.00,KH₂PO₄ 4.00,MgSO₄·7H₂O 3.00,VB₁0.10,1 L of water.On these conditions,the yield of Magnolol can be raised to702.261±32.016 ug/L,improved 21.26%before optimization.The best fermentation medium of Honokiol were as the follows?g/L?:glucose 20.00,peptone 6.00,KH₂PO₄ 5.00,MgSO4·7H2O 5.00,VB1 0.10,1 L of water.On these conditions,the yield of Honokiol can be raised to 559.309±19.395 ug/L,improved 18.59%before optimization.The best fermentation conditions of strain Y-88 were as the follows:fermenting time 12 d,temperature 27?,pH 7.5,inoculation amount 15%,rotational speed 120 r/min.On these conditions,The yield of Magnolol and Honokiol can be raised to 1153.816±21.643 ug/L and 812.527±41.349 ug/L,improved 64.30%and 45.27%before optimization.