Isolation Of Endophytic Fungi From Opisthopappus Taihangensis And Selection Of Fungi Producing Flavonoids

Endophytic fungi of medicinal plants can metabolize and produce similar medicinal ingredients of host plants. Antibacterial and antitumor secondary metabolites of endophytic fungi causes widespread research. Opisthopappus taihangensis is currently only limited to the researches, such as taxonomy and tissue culture. The endophytic fungi of Opisthopappus taihangensis have not yet been reported.Flavonoids of Opisthopappus taihangensis is useful for the antioxidation, antitumor and treatment of cardiovascular disease.The purpose of this study lies in the selection of Opisthopappus taihangensis endophytic fungi producing flavonoids and classification and identification of the endophytic fungi.In this paper the main results are as follows:1) in this paper, the tissue separation method was taken to the separation and screening of endophytic fungi, and the use of the top of the hyphae purification method and the zigzag method were taken to purify endophytic fungi.With cant in vitro preservation method and sterile soil conservation tabernacle method endophytic fungi were saved.The experimental results showed that the best way to surface disinfection was 75% alcohol 45 s with mercuric chloride 45 s.And we seplarated 37 strains of Opisthopappus taihangensis endophytic fungi.2) in this paper, by using 5 kinds of flavonoids developer including AlCl3 agent, aluminum nitrate, sodium nitrite, NaOH, hydrochloric acid, magnesium powder and FeCl3 respectively, the endophytic fungi of the Opisthopappus taihangensis leaf were isolated.Among them, aluminum nitrate sodium nitrite is the specificity of the flavonoids of chromogenic agent, and the necessary conditions to determine flavonoid producing strains.We Choose positive results of aluminum nitrate and sodium nitrate and other two kinds of chromogenic reaction of endophytic fungi of flavonoids.Chromogenic reaction can be divided into two aspects, including the fermented liquid and the chromogenic reaction of chromogenic reaction of mycelium.We screened with flavonoids of endophytic fungi in the fermented liquid of the color reaction of mycelium,and determined fermented liquid and mycelium contain flavonoids endophytic fungi as Opisthopappus taihangensis leaf flavonoids production strains.The results showed that the flavonoids production strains were:T1, T2, T8, T9, S9, S10, R1,R2.3) Using high performance liquid chromatography (HPLC) method for the qualitative and quantitative we analysised flavonoids of endophytic fungi.This paper used 4 flavonoids standard products:quercetin, galangal, rutin and isorhamnetin.Pure methanol dissolved, diluted with high performance liquid meter peak time, peak area and detected with mass concentration (mg/ml) as the abscissa, absorbance values as ordinate and drawing standard curve, respectively.Results showed that T1, T8, R1 only produced rutin strains, T2 only producted quercetin, R2 producted rutin and quercetin, T9, S9, S10 at the same time produced three kinds of flavonoids, they were rutin, quercetin and isorhamnetin,and the maximum yields of quercetin, rutin, isorhamnetin were 0.0116 ±0.0034 mg/ml,0.0374 ±0.0055 mg/ml,0.0460 ± 0.0047 mg/ml.These common strains produced flavonoids substances rutin, and rutin content were relatively high. The strains of the experimental did not produce phenol.4) Morphological identification of the eight endophytic fungi strains producing flavonoids inclused the colony morphology, colors, shapes, sterigmata spore morphology of observation and description.Occording to reference literatures and the imperfect fungi belong to graphic (H.L. barnett, B.B. hunt) to determine 8 species of plant endophytic fungi classification status.5) In this paper, we used molecular biology method to identify 8 endophytic fungi strains.Respectively we used fungal genomic DNA extraction kit for genomic DNA to extracte ITS strains of endophytic fungi.In this article we uesd fungi universal primers ITS1 and ITS4 amplification to clone rDNA ITS sections of fungi.After the test of the agarose gel electrophoresis and PCR products were sent to sequence in Shanghai biological engineering co., LTD.The results showed that amplification of ITS section of the eight endophytic fungi strains producing flavonoids were 500-600 bp.We BLAST the sequence of sequencing in Genebank, and obtained its homologous sequence and ranked a series of nucleic acid sequence from high to low.By recording and downloading with the original sequence alignment score values higher homologous series, the phylogenetic tree analysis were established.Thus species status of endophytic fungi producing flavonoids were determined by molecular biology methods.The results showed that the results were same with morphological results, and we determined T1, T2, T8, T9, S9, S10, Rl, R2, respectively belong to Fusarium, Fusarium, Alternaria, Aspergillus,Pestalotiopsis and Penicillium, Alternaria.