Diversity,Difference And Antimicrobial Activities In The Endophytic Fungus Of Dendrobium Huoshanense

Objective:Based on Illumina MiSeq^TM high-throughput sequencing technology?1?to compare the differences in endophytic fungi of Dendrobium.huoshanense with different growth patterns;?2?to compare the differences in endophytic fungi of D.huoshanense stems in facility cultivation with different growth years;?3?to cultivate D.huoshanense the fungi were selected and identified as endophytic bacteria with antibacterial activity in D.huoshanense.The diversity and differences of endophytic fungi of D.huoshanense with different growth patterns and different growth years were investigated.The potential of endophytic fungi to enhance the disease resistance of plants was also investigated,and the basic data for the development of fermented bacterial fertilizer for ecological cultivation was provided.Method:?1?Fungal DNA was extracted using E.Z.N.A^TM Mag-Bind Soil DNA Kit?OMEGA?according to the manufacturer's instructions,universal primers ITS1 and ITS2 were used for two rounds of PCR amplification of the genomic DNA,and paired-end sequenced on the Illumina MiSeq^TM platform.The obtained raw image data was converted to the original sequencing by CASAVA base recognition sequence.Then pre-processing the data to remove the primers,chimera and nonspecific amplification sequences were removed according to the similarity between the sequences?97%?,the sequences are divided into different OTUs,and Alpha,Beta diversity analysis,Statistical analysis of species classification analysis,multi-dimensional analysis,and bacterial population difference analysis.?2?The endophytic fungus of roots,stems and leaves of D.huoshanense were cultured by tissue section and tissue grinding.The antibacterial activity of the sputum plate method and the Oxford Cup method on the bacterial and bacterial fermentation broth was carried out to screen the antibacterial activity,and molecular identification was carried out.Results:?1?The endophytic fungus of D.huoshanense are rich,Alpha diversity index analysis shows that there is a significant difference between understory cultivation and wild D.huoshanense;endophytic community composition analysis of Alternaria in the cultivation understory,cultivation on trees,imitation of wild cultivation,facility cultivation five growth modes of D.huoshanense endophytes significant difference?P<0.01?,LEfSe was used to analyze the significant difference of endophytic fungus between the groups.There are 8 genera in the wild D.huoshanense and other four groups.Helotiales sp?Stenella?Helotiales genus?Setophoma?Articulospora?Didymosphaeriaceae sp?Teichosporaceae sp and Devriesia;There are two distinct genera of Diaporthe and Strelitziana in the wild-cultivated D.huoshanense and the other four styles,which can be distinguished from other growth methods of D.huoshanense.?2?There are no significant differences in Alpha diversity between the three groups of IY1,IIY2,IIIY3 and IIY2,IIIY3,IIIY1.The dominant fungi community of IY1,IIY2,IIY2,IIIY3,IIIY1 five groups are different,the abundance of Fusarium in IIY2,IIIY3,IIIY1 and IIIY2 are significantly higher than IY1;the abundance of Zymoseptoria IY1,IIY2,IIIY1 and IIIY2 are significantly higher than IIIY3;Compared with other groups Cladosporiumonly has a distinct advantage in IY1;Cyphellophor and Trechispora have obvious advantages in IIIY3;only in the IIIY1 and IIIY2 Zymidium are predomination.Cyphellophor has a clear advantage in IIIY3,and the abundance of Zymoseptoria in IY1,IIY2,IIIY1,IIIY2 are significantly higher than IIIY3.?3?A total of 64 endophytic fungi were isolated,and 26 strains were obtained by tissue sectioning method.The tissue grinding method was divided into 38 strains.The tissue grinding method is divided into fungi,16 strains and 11 strains from root,stem and leaf,respectively.SH01 belongs to Aspergillus fumigatus which inhibits B.subtilis,S.aureus and E.coli;GL13 and GR4 belong to Penicillium Citrinum which inhibit B.subtilis and S.aureus,SH02 is Fusarium oxysporum and GS01 is P.rubens.Conclusions:?1?The richness and diversity of endophytic fungi in D.huoshanense were affected by the cultivation methods.There were significant differences in endophytic diversity between the cultivation understory and the wild D.huoshanense.There are differences between the cultivation understory,cultivation on trees,imitation of wild cultivation,facility cultivation four cultivated products and the wild products.?2?The richness and diversity of endophytic fungi in D.huoshanense are affected by the growth years.Different growth years have different dominant fungus.IIIY1,IIIY2and IIIY3 which were separated in the same growth period?three years old?also have their own dominant endophytic fungi.?3?The tissue grinding method for separating endophytic fungi from D.huoshanense is more advantageous than the tissue section method;the endophytic abundance and richness of different tissue parts is different,and the number of endophytic fungi in the root are larger than in the stems and leaves.