Study On Endophytic Fungi Isolated From Root Of Cleisostoma Williamsonii And The Inhibition Activity

Endophytic fungi is closely related to host plant. Endophytic fungi of orchids (Orchidaceae) plants and especially the reciprocal symbiosis, endophytic fungi can provide or increase the vitamins, amino acids, sugars and other nutrients to orchid; promote seed to germination; produce the plant hormones to promote plant growth, such as gibberellin; produce the secondary metabolites such as antibacterial material, enhance host resistance to the environment. Cleisostoma williiamsonii is one plant ofgenus Cleisostoma, entertaining, germplasm resources scarce. It has important protection value, is listed as national key protected wild plants. To understand endophytic fungi diversity of wild Cleisostoma williiamsonii, giving the protection and breeding to provide theoretical basis and technical guidance. This study takes wild orchid roots of Bawangling as the materials, then proceeded with identification via traditional morphological analysis. Genetic diversity analysis were applied to isolated strains utilized by r DNA ITS technology combined with morphological observations in order to determine the basic taxonomic status. To understand part of the biological characteristics of strains, it obtained study for the standpoints of different Carbon source, N source, pH and temperature. From fungal identification of morphology; Combined the technology of using r DNA ITS genetic diversity analysis of strains, to determine the basic classification status of fungi; From C source, N source, pH and temperature test point of view, understand the strains isolated from the part of the biological characteristics; The endophytic fungi pathogens antagonist activity screening, for further research, to expect to get some bacteriostatic and antifungal activity of endophytic fungi.The main research results are as follows:The determination of optimal sterilization time of the separation of endophytic fungi. Wild Cleisostoma williiamsonii in Hainan Bawangling as test material, collecting the root samples of different habitat to separation. The sterilization time by mercury bichloride was designed for3min,5min,7min.145strains for total, and24.17strains for the average sample. Under different sterilization time, the number of endophytic fungi species exist significant differences. With5min isolated62fungus strains, accounting for42.76%of the total, isolation effect is the best; followed by3min,44strains of fungi, accounting for30.34%of the total;7min at least. Surface sterilization time for5min got the most strains.Combined with morphological identification and ITS r DNA sequence analysis, determine the classification status of the strains. A total of145endophytic fungi, were identified as15genera,17taxa according to the morphology. The total145isolates were identified into15genera, including Penicillium16strains, Aspergillus12strains, Pestalotiopsis5strains, Fusarium19strains, Neonectria2strains, Chaetomium4strains, Calonectria5strains, Xylaria5strains, Paecilomyces7strains, Alternaria2strains, Phomopsis8strains, Colletotrichum32strains, Rhizopus5strains, Mucor7strains; Albugo6strains. Acorrding to the ITS sequences, another10strains need more identification studies, which had low degree of similarity of with Botryosphaeria sp., Beltraniella sp.. It shows Colletotrichum sp., Penicillium sp., Fusarium sp. are the predominant species. Registered in the NCBI, get serial Numbers for KJ512143-KJ512165.Significant differences existed among the number of endophytic fungi in different habitats. The samples collected from lowland rain forest, tropical coniferous forest, mountain rain forest habitat.47,57,41strains of fungi were isolated and identified as15,16and14different genera. Illustrating the same species from different habitat has difference on the richness of plant endophytic fungi. Endophytic fungi from tropical coniferous forest had the highest diversity index, average of2.36, lowland rain forest and mountain rain forest followed.Carbon source:With glucose as carbon source, A101, A103, B102, B103, D104, F101, F105, F106and F107grow fastest; D101grow fastest with lactose as carbon source; A102, C104, E101, E104, E106, F102grow fastest with starch as carbon source; A104, B101, C101, C102, C103, D102, D103, E102, F102, F107had fastest growing speed with maltose as the carbon source.Nitrogen source:Isolated endophytic fungi in the yeast extract, beef extract and peptone of bacteriology, tryptone organic nitrogen source can grow well in culture medium, by contrast, the inorganic nitrogen source such as urea, NaNO3and (NH4)2SO4, fungi got poor growing. B103had adapt to a wide rangein all nitrogen source except urea can grow good.pH:Endophytic fungi were suitable for the pH range of4.0～9.0, during5.0～6.0the strains growth fastest. Strains on the culture medium pH<4.0or pH>9.0, the growth had significantly suppressed.Temperature:The optimum temperature was concentrated in25.0～28.0℃; within the range of20.0～30.0℃, fungi could grow well; growth is restrained when less than20.0℃or more than30.0℃. Most under10.0℃, the slow growth A101, B102, D103, E103, E106, F104, F106can grow; most grow slowly under35.0℃, among that A104, E102, E103, F104, F105could not grow; under40.0℃the vast majority endophytic fungi could not grow, except C104, F101.Different culture medium:Most fungi such as A101, A102, B101, D102, C103, F107, C104, E103, F103, B103had fastest growing on PDA; A103, B102, F106and E101, E104, D104, F102, E106in czapek’s medium; A104, B103, D103on SDA; C101, F101, F104, F105etc in Martin substratum.Activity screening of endophytic fungus pathogen antagonism. Most endophytic fungi had no antibacterial activity, only A101, E202against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis had a certain activity. There had18strains with activity of pathogenic fungi,12.41%of the total isolated fungus,15strains of antagonistic activity for more than two kinds of pathogenic fungi,10.34%of the total number, occupies83.33%of active fungus. Xylaria sp., Fusarium sp., Paecilomyces sp.Neonectria sp., Chaetomium sp., Calonectria sp. had wide antimicrobial activity. E102had strong inhibitory rate up to46.67%against Peronophythora litchii; E106up to47.43%against Fusarium solani, F101up to53.55%against Fusarium oxysporum f. sp. Niveum.