Isolation And Identification Of Epichlo? Bromicola And The Effects Of Extracts Of Its Fermentation Broth On The Gorwth Of Arabidopsis Thaliana Seedlings

Epichloe endophytes are a group of sexual (Epichloe spp.) and asexual (Neotyphodium spp.) clavicipitaceous fungi (Clavicipitaceae, Ascomycota) that form symbiotic associations with temperate grasses. The alkaloids produced by Epichloe sp. are mainly responsible for the host resistance to feeding by insects. We report here on the identification of endophytic fungi of Coix lachryma-jobi L. var. ma-yuen Stapf, and exploring the growth promoting effects of the fermentation broth of Epichloe bromicola on the growth of Arabidopsis thaliana seedlings to provide a scientific basis for future practical use. The main results of this study were summarized as follows:1. Fungal genomic DNA was extracted and fragments of translation elongation factor 1-a gene (tefl) and β-tubulin gene (tub2) from Epichloe sp. were cloned and sequenced.759-and 934-bp partial sequence of tefl and tub2, respectively, were sequenced for the representative strains of Epichloe sp.. Most of the sequences identified by BLAST in NCBI were Epichloe strains by using these sequences. Nabour-joining phylogenetic analyses of the endophyte tefl and tub2 sequences showd that the endophytic strain we isolated was closely related to Epichloe bromicola. The results showed that the sequence similarity of tefl and tub2 with these of Epichloe bromicola were 98.67% and 98.8%, respectively. Combining the results of morphology and phylogenetic evidence, the endophytic fungi was identified as Epichloe bromicola.2.75% ethanol extract of the fermentation broth of Epichloe bromicola was further divided into the extracts of petroleum ether dichloromethane, ethyl acetate and butanol. These extracts were added into 1/2 MS medium of Arabidopsis seedlings. The statistical results showed that, Epichloe bromicola and CH2C12 extract promoted adventicious root formation, number of leaves, fresh weight and dry weight of Arabidopsis seedlings. EtOAc extract promoted number of leaves, but inhibited the elongation of primary root of Arabidopsis seedlings, as was the case in CH2Cl2 and nBuOH extracts.3. In order to looking for material basis which affect the growth of Arabidopsis seedlings, CH2C12 and EtOAc extracts were analyzed by UPLC-HRMS. The results showed that indole acetic acid (IAA), indole-3-ethanol (IEtOH) and indole-3-carboxaldehyde were found in the CH2C12 and EtOAc extracts, we speculated that Auxin substance maybe the active compound affected the growth of Arabidopsis seedlings. However, further research on the expresion of early auxin response factor need to launch and elucidate its mechanism.