| The herb Artemisa annua L. is a significant pharmaceutical plant, which is the producer of artemisinin. Besides anti-malaria, artemisinin and its derivatives have been shown to be effective against tumor, a number of viruses, other prokaryotic and eukaryotic agents. They also show the characteristic of allelopathy. Artemisinin is present in exceedingly small amounts, thereby hindering sufficient production for treating malaria let alone any of the other diseases against which it is effective. It is widely accepted that endophyte is a rich source of functional metabolites. The aim of this study was to investigate the composition of endophytic actinobacteria associated with A. annua collected from Kunming, get valuable and (or) novel resources, and the relationship between endophytic actinobacteria and A. annua preliminarily.Four methods were employed in actinobacterial endophytes isolation. The Mini-Bead Beater and dry-heating were used firstly in actinobacterial endophytes isolation. A total of 228 endophytic actinobacteria were isolated from samples of A. annua. According to the morphological characteristics and the results of 16S rRNA gene sequences analysis, strains were identified to the genus level. The results showed that the most abundant was Streptomyces, and then Promicromonospora. A diverse range of other rare taxa of Actinomycetales were also isolated, including isolates of the genera Pseudonocardia, Nocardia, Nonomuraea, Rhodococcus, Kribbella, Micromonospora, Actinomadura, Streptosporangium, Amycolatopsis, Dactylosporangium, Blastococcus, Glycomyces, Kocuria, Micrococcus, Gordonia, Planotetraspora and an potential novel genus.Six candidates were subjected to polyphasic taxonomy study including analysis of morphological and cultural characteristics, physiological and biochemical tests, chemotaxonomic properties, phylogenetic analysis based on 16S rRNA gene sequences and DNA-DNA hybridizations. Strain YIM 65646 was identified as a member of the novel genus of the family Micromonosporaceae, the name Plantmonospora gen. nov. was proposed. Plantmonospora endophytica sp. nov. was assigned as the type species of this novel genus. Strain YIM 63111 was a novel species of the genus Pseudonocardia, and it was named as Pseudonocardia pretiosum sp. nov. Strains YIM 65594, YIM 65638 and YIM 65642 belonged to the novel species Streptomyces endophytica sp. nov., and strain YIM 65594 was assigned as the type strain. Strain YIM 65601 was a new member of the genus Nonomuraea, and the name Nonomuraea endophytica was proposed.Results of isolation and identification work showed that A. annua harbored abundant endophytic actinobacteria including a lot of undescribed novel species. Furthermore, their antimicrobial, enzyme and herbicidal activities and the potential of produing polyketide or non-ribosomal peptide were estimated.The agar plate diffusion method was used to investigate the antimicrobial activity of endophytic isolates. There were 15.8%～34.6% of tested strains showed inhibition activity to Staphylococcus aureus, Candida albicans, Helminthosporium maydis, Fusarium avenaceum, Colletotrichum sp. and Cytospora mandshurica. Among them, there were thirty-one strains (13.6%) showed strong and broad-spectrum antimicrobial activities.The presence of PKS-I, PKS-II and NRPS sequences were investigated in total of 166 strains by specific amplification of total DNA with the corresponding primers. High frequencies of positive PCR amplification were obtained for PKS-II (75 strains, 45.2%), NRPS (54 strains, 32.5%) and PKS-I (35 strains, 21.1%) biosynthetic systems.In the enzyme plate screening experiment, amount of 199 strains were tested. Seven of them showed great amylase activity, ten strains showed great protease activity, one strain showed great lipase activity (R-r >0.5cm), and seven strains showed great cellulase activity(R-r≥1.0cm).The results of herbicidal activity screening experiment indicated that 19 out of 117 samples of fermentation broth could inhibit the germination of Echinochloa crusgalli completely.The results of bioactivity evaluation indicated that endophytic actinobacteria are valuable sources of natural products mining. Moreover, we obtained a batch of bioactive strains, which were the objects in continued studies.There were 32 isolates used for inoculating A. annua seedlings. Most of them didn't show effect on the growth of seedlings. There were six strains enhanced the growth of seedlings, and four strains inhibited the growth. It was arrestive that strain YIM 63111 showed the strongest inhibition effect.Both A. annua seedlings grown in the gnotobiotic system and green house were inoculated with strain YIM 63111. The real time RT-PCR determination revealed that the transcription levels of CYP71AV1 (cytochrome P450 monooxygenase) and CPR (cytochrome P450 oxidoreductase) genes were up-regulated after inoculated with strain YIM 63111. Compared with untreated control, the artemisinin content in the treated plants were increased by 30%～40%. This was the first report about that endophytic actinobacterium was able to induce the biosynthesis of artemisinin.In this study, strain YIM 63111 was tagged with the egfp gene. Endophytic colonization was observed in the sample, which was incubated for 14 days after inoculated with this strain.The biological and ecological properties of endophytic actinobacteria isolated from A. annua were demonstrated partly in this dissertation. These findings will guide us to explore bioactive metabolites from endophytic actinobacteria, use them to enhance the growth of A. annua and accumulation of artemisinin, and understand the relationship between endophyte and plants.
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