Screening And Identification Of Flavonoids-Producing Endophytic Fungi From Sugarcane Leaves

Using the method of tissue separation,172 endophytic fungi strains were isolated from the sugarcane leaf.From the 172 fungi strains,10 different strains were screened out according to the morphological characteristics of colony(GZ-0,GZ-1,GZ-2,GZ-4,GZ-5,GZ-7,GZ-8,GZ-10,GZ-11 and GZ-15).By using the molecular methods,the 10 fungi were identified as 10 species in 8 genera,Gibberella zeae,Glomerella tucumanensis,Phyllosticta capitalensis,Aspergillus niger,Aspergillus aculeatus,Exserohilum rostratum,Colletotrichum gloeosporioides,Leptosphaeria sp.,Colletotrichum boninense and Xylaria sp.Using the methods of chromogenic reaction,thin-layer chromatography(TLC)and UV absorption spectroscopy to assay the flavonoids in the mycelium of the 10 strains,2 flavonoids-producing strains GZ-4 and GZ-5 were screened out.It was found that the total flavonoids in the mycelium of strains GZ-4 and GZ-5 was 1.21 mg/g and 4.03 mg/g respectively.The condition for extraction of total flavonoids from the mycelium of the strain GZ-5 was optimized using the single factor tests and orthogonal tests.The optimized condition for extracting the total flavonoids was as follows:ultrasonic power 47.5 W,extraction time 25 min,extraction temperature 70?,ethanol concentration 60%and solid-liquid ratio 1:60(g/mL).Under the optimized extraction conditions,the total flavonoids extracted from the mycelium of GZ-5 was as high as 4.92 mg/g.The antioxidation in vitro for the total flavonoids from the GZ-5 were evaluated,it was found that the flavonoids exhibited remarkable antioxidant activity against the hydroxyl free radical,nitrite,DPPH and superoxide radical assays.By using the Oxford Cup method,the antimicrobial activity for the total flavonoids from the strain GZ-5 was evaluated,it was found that the flavonoids exhibited antibacterial activity against the such gram-positive bacteria as Bacillus megaterium,Bacillus subtilis,Bacillus mucilaginosus,Staphylococcus aureus,Staphylococcus albus,in which it was the strongest against Staphylococcus aureus,the antibacterial concentration was as low as 0.012 mg/mL.On the results of single factor tests and orthogonal tests,the fermentation condition to produce the flavonoids was optimized as follows:inoculum 3%,7 d,28 ?,175 r/min,80 mL in 150 mL flask.Under the condition,the output of total flavonoids was up to 7.65 mg/g mycelium,or 8.57 mg/100 mL medium.