Preliminary Studies On The Isolation And Identification Of Endophytic Fungi And Their Secondary Metabolites In Tulipa Sp.

Tulipa gesneriana L.is an herbaceous plant of the genus Tulipa in the lily family（Liliaceae）.According to literature,there are about 150 species of tulips in the genus Tulipa worldwide,and about 14 species of tulips in the genus are mainly distributed in China,Eleven species of tulips,including the Junggar tulip,the Ili tulip,the Tianshan tulip,and the heterophyllous tulip,are distributed in Xinjiang.At present,research on tulips by domestic and abroad scholars has mainly focused on germplasm resources,introduction and cultivation,expansion and breeding,and chemical composition analysis.In contrast,less research has been conducted on the species and community structure and biological activities of endophytic fungi of Tulipa sp.Therefore,this experiment will be shown to investigate it accordingly.The details of the study and the experimental results are as follows:1.Isolation and purification of endophytic fungi:Tulipa thianschanica Regel,Tulipa suaveolens Roth,Tulipa heterophylla Baker,and Tulipa iliensis Regel were isolated and purified by tissue block culture and mycelial tip purification methods.(A total of 33 endophytic fungi were isolated and purified from different tissues of Tulipa suaveolens Roth,Tulipa heterophylla Baker and Tulipa iliensis Regel.13 endophytic fungi were obtained from Tulipa thianschanica Regel,five from Tulipa suaveolens Roth,seven from Tulipa heterophylla and eight from Tulipa iliensis.2.Active strain identification:The endophytic fungi with high activity were identified by morphological and molecular biology identification analysis methods.The results showed that the active strains of endophytic fungi in tulips are more diverse,with Alternaria sp.and Fusarium sp.being the dominant genera.3.Illumina high-throughput sequencing analysis:The structural composition and diversity of the endophytic fungal communities of four tulip species were determined and analyzed by Illumina Novaseq sequencing technology using rDNA of the ITS region as the target sequence.By comparing the fungal taxa detected from the four samples by high-throughput sequencing with the traditional isolation and identification methods,the comparison showed that at the genus level,the fungal species isolated and identified by the conventional techniques were fewer than those obtained by high-throughput sequencing,which might be due to the smaller number of fungi in these genera,and might also be since that the traditional culture media and culture methods were not suitable for the growth of endophytic fungi and did not satisfy the demanding growth conditions of some species of Microorganisms are not able to meet the challenging growth conditions of some species.Therefore,the use of this tool could help to discover new strains of tulip plants as well as endophytic metabolites with specific functions.4.Strain activity screening:The secondary metabolites of 33 endophytic fungal strains were treated separately according to the activity assay needs,and the antibacterial activity,antioxidant activity and cellular activity of their secondary metabolic crude products were measured to screen out the strains with high activity.The results showed that the activity status of the endophytic fungi of Tulipus sp.as a whole was up-and-coning,which laid the foundation for further development of the tulip plant.5.Chemical correlation between fungal secondary metabolites and hosts:the fungal strains capable of producing flavonoids were screened by chemical color development and UV-Vis spectrophotometry,and their total flavonoid content was determined.The experimental results showed that the strains TF17,TLBR18 and TB24 were rich in chemical composition.The preliminary screening of 11 flavonoid-producing strains from 33 endophytic fungi of the genus Tulipus using the NaNO₂-Al（NO₃）₃ colorimetric method indicated,to a certain extent,the existence of a close relationship between the secondary metabolites of endophytic fungi and their host plants,which provided a specific theoretical basis for further studies on endophytic fungi of the genus Tulipus.To summarize,33 strains of endophytic fungi of Tulipa sp.were isolated and purified in this thesis,and the morphological and molecular biological identification indicated that the strains of endophytic fungi of Tulipa sp.were diverse and had a large number of populations.The results of high-throughput sequencing analysis showed that the endophytic fungi of Tulipa sp.have a rich population structure and are potentially valuable for in-depth research and development.The endophytic fungi of Tulipus sp.were evaluated for antibacterial activity,antioxidant activity and cellular activity,and two strains with all three activities were obtained.Although there were few strains with all three activities simultaneously,the remaining different strains showed perfect antibacterial and antioxidant activities.In addition,in the experiment of chemical correlation between fungal secondary metabolites and host,11 flavonoid-producing strains were initially screened from 33 endophytic fungi of the genus Tulipus,which confirmed,to a certain extent,that endophytic fungi can produce the same or similar chemical components as their hosts,providing a certain theoretical basis for further studies of endophytic fungi of the genus Tulipus.Its comprehensive experimental results indicate that,in addition to being an ornamental crop,the endophytic fungi present inside the wild tulip genus in Ili can convert resource advantages into economic benefits,thus promoting the economic development of the Ili region.