Isolation And Identification Of Endophytic Microorganism From Citrons

Citrus medica is one of the three basic species of the genus Citrus L.that has many germplasm resources and excellent characters.Citron C-05 is,up to now,the unique genotype identified resistant to citrus canker disease within Citrus.In the previous study,the stem segments of citron C-05 were used for tissue culture and genetic transformation of key candidate genes,but it was observed that a variety of microorganisms were easy to grow in the incision area of stem segments after disinfected.The results of sequence analysis showed that the isolated microorganisms were endophytic,and the pH sensitivity of different endophytic microorganisms was different.Therefore,this study intends to isolate and identify the endophytic microorganisms of citron C-05 and other 12 citron germplasms.Then,the morphological characteristics and pH sensitivity of endophytic microorganisms were evaluated,which laid a foundation for exploring the optimal conditions of tissue culture and genetic transformation of citrus stem segments.The main results of isolation and identification of endophytes are as follows:1.The semi-lignified stem segments of citrons grown in the room culture were disinfected with 75%C₂H₅OH and 1%Na Cl O.The stems,epidermis and stele tissues were placed on Beef Extract medium,Gorodkowa medium,PDA medium and MT medium respectively,culture at dark for 7-30 days with 28℃.The results showed that 134microorganisms were isolated from 11 kinds of citron,46 strains were identified as endophytic microorganisms,including 18 strains of endophytic bacteria,16 strains of endophytic actinomycetes and 12 strains of endophytic fungi,while no endophytic strains were isolated from Dana Citron and Round Citron.These 46 kinds of endophytic bacteria can be divided into 14 species of phyla or genus:Ascomycota、Aspergillus、Bacillus、Curtobacterium、Janibacter、Methylobacterium、Microbacterium、Pantoea、Penicillium、Phyllosticta、Pseudomonas、Rhizobium、Sphingomonas and Streptomyces.Among them,Curtobacterium sp.（23.91%）,Bacillus sp.（13.04%）,Penicillium sp.（13.04%）and Pseudomonas sp.（10.87%）were superior to other endophytic microorganisms in quantity.2.Mixing the epidermis and cortex tissue,cultivated the column tissue after grinding.Four strains of endophytic bacteria,two strains of endophytic actinomycetes and eight strains of endophytic fungi were found in epidermis and cortex.One strain of endophytic bacteria and four strains of endophytic actinomycetes were found in the stele,and the rest were found in the stem segments.3.The pH sensitivity of endophytic microorganisms showed that the growth of endophytic bacteria and actinomycetes was significantly inhibited at pH 4,and the optimal pH was 6-8.Rhizobium yanglingense grew the slowest on LB medium with pH 9;The growth inhibition of Pantoea dispersa on LB medium at pH 4 was inhibited but not significant.Among the endophytic actinomycetes,one strain grew the slowest on LB medium with pH 9,while five strains grew slowly on LB medium with pH 4,all of them belonged to the Curtobacterium sp.