Mechanism Of Synthetic Bacterial Community From Root System Of Artemisia Annua L.against Globisporangium Ultimum Var.

Artemisia annua L.is a traditional Chinese medicine with a long history.Artemisinin contained in A.annua plants.is a new antimalarial drug with high efficiency,safety and non-toxic side effects,which can effectively inhibit the occurrence of malaria.One of the main reasons for the lack of disease is the abundance of endophytic bacteria with inhibition activity.Associations between host and microbial communities can be initiated by establishing sterile hosts and then explored by inoculating specific microbial communities.Therefore,in this thesis,by first establishing the inoculant system of A.annua,we can provide experimental materials for studying the interaction between A.annua and bacterial community.By isolating and purifying the endophytic bacteria from the root system,an 8-bacteria synthectic community was constructed,which could significantly inhibit the development of A.annua disease caused by Globisporangium ultimum var..Soil is the main source of inoculation of plant endophytic bacteria,with exogenous pathogens challenge,the community structure and diversity of plant host endophytic bacteria will change.In order to further understand how A.annua selectively chooses soil bacteria under pathogen stress to enhance its own defense system,exogenous pathogens were added and potting tie-back method was adopted for treatment.At the same time,Illumina Miseq high-throughput sequencing technology was used to analyze the changes of microbial community under the interference of exogenous pathogenic fungus,to explore how pathogenic fungus affected the host of A.annua to select beneficial bacteria in numerous soil microbial communities,and to further clarify the reason why A.annua rarely suffers diseases.1.Establishment of tissue culture regeneration system of A.annua.The stem tips of A.annua were used as explants.The explants were soaked with 0.3%streptomycin solution for 3 h before disinfection,and successively disinfected with 75%ethanol for30 s,0.1%Hg Cl₂ for 6 min.After inoculation in 20 mg·L^-1 citric acid medium,the best effect was achieved,with 25.7%contamination rate,8.6%mortality rate and 22.4%Browning rate.Using MS medium+1.0 mg·L^-16-benzylaminopurine（6-BA）+0.1 mg·L^-1α-naphthoacetic acid（NAA）+20mg·L^-1 citric acid+0.2 g·L^-1 activated carbon as the optimal medium,the healing rate reached 96.9%and the budding rate reached 93.8%.The optimal subculture medium was MS medium+0.5 mg·L^-1NAA+0.1 mg·L^-1 3-indoleacetic acid（IAA）+0.2 g·L^-1 activated carbon,and the rooting rate reached95.0%.The survival rate of seedlings in humus soil was 100.0%after 4～5 days in the room before transplanting.2.Isolation and screening of endophytic fungi and endophytic bacteria from A.annua.Three endophytic fungi were isolated and purified from the leaves of A.annua.Its pathogenicity was G.ultimum>Fusarium tricinctum>Cerrena unicolor,and G.ultimum was used as a follow-up experimental pathogen.A total of 60 strains of endophytic bacteria were isolated and purified from the roots of A.annua,belonging to 18 different genera,among which 6 strains showed inhibitory effect against G.ultimum as Rhizobium pusense（N26）>Paracoccus sp.（N21）>Flavobacterium sp.（N58）>Brevundimonas sp.（N16）>Stenotrophomonas sp.（N22）>Bacillus sp.（N31）.The 6 strains assembled together were C6 bacterial community.In addition,2 strains showed strong inhibitory effect on C.unicolor,Brevibacillus nitrificans（N49）>Cupriavidus sp.（N56）,which was assembled with the above 6 endophytic bacteria to form a C8 bacterial community.3.Assembly and disease resistance of root bacterial colonies of A annua.（1）Assembly of bacterial community.Although C6 bacterial community could delay A.annua disease caused by G.ultimum,but all the plants would still be susceptible to the disease in the end.However,after the addition of N49 and N56 on the basis of the C6 bacterial community,the infected plants were significantly reduced by73.3%compared with C6 bacterial community.These results indicated that N49 and N56 played a key role in enhancing the defense system of the C8 bacterial community.Therefore,the C8 bacterial community was superior to the C6 bacterial community.（2）Inhibitory effect of assembled bacterial communities on G.ultimum.When the soil surface of G.ultimum was inoculated only,a large number of white cotton flocculous mycelia gathered on the soil surface,and the whole A.annua seedlings all withered and yellow and died of root rot,with the mortality and morbidity rate as high as 100.0%.When the soil surface of G.ultimum and C8 bacterial community were inoculated together,the mycelia on the soil surface significantly decreased,most of the plants grew normally,and the symptoms of drying and yellows were improved.The morbidity rate and Death rate decreased to 26.7%and 11.1%,respectively.（3）Effects of assembled bacterial community on growth,physiology,biochemistry and secondary metabolism of A.annua.Compared with no microbial inoculation,the growth of A.annua inoculated only with G.ultimum was significantly inhibited,and its dry weight,fresh weight,plant height and root length were significantly decreased by 350.2%,189.3%,153.9%and 40.3%,respectively.Due to severe stress,the activity of superoxide dismutase（SOD）,peroxidase（POD）and proline content were increased by 22.9%,1019.2%and 112.9%,respectively.The contents of artemisinin,scopoletin,chrysosplenol-D and chrysosplenetin were decreased by 306.6%,216.4%,332.6%and 115.8%,respectively,all due to poor plant growth.Compared with G.ultimum alone,dry weight,fresh weight,plant height,root number and root length of G.ultimum inoculated with C8 community were increased significantly by 21.7%,28.6%,182.4%,279.4%and 172.3%,respectively.The activity of SOD,POD and contents of proline were decreased by 42.1%,68.7%and 71.8%,respectively.The improvement of growth conditions significantly were increased the contents of artemisinin,scopoletin,chrysosplenol-D and chrysosplenetin by 234.5%,82.8%,35.4%and 96.2%,respectively.4.Effects of exogenous addition of G.ultimum on root bacterial community of A.annua.Based on high-throughput sequencing technology,at the phylum level,Proteobacteria,Actinobacteria and Firmicutes were the main phyla of the root bacterial community of A.annua,rice and H.Cordata.After the exogenous addition of G.ultimum,under the natural soil community,Bacteroidetes in the root of A.annua.were increased significantly by 2.8%,Bacteroidetes in the root were increased significantly by 5.7%and Proteobacteria were decreased significantly by 9.8%of H.Cordata and Actinobacteria decreased significantly by 14.5%,Proteobacteria and Chloroflexi increased significantly by 11.9%and 1.4%in the root of rice,respectively.However,there was no significant change in the synthetic community of 101 bacteria（C101）.The Alpha diversity indices of A.annua had no significant change,while the Shannon,Simpson,Chao1 and ACE index of rice in the natural soil community were increased significantly by 11.1%,6.1%,16.4%and 13.2%,respectively.In the C101 synthetic community,Shannon and Simpson index of rice significantly were increased by 25.2%and 18.7%,respectively.In the natural soil community,Shannon and Simpson index of H.Cordata significantly were increased by 8.3%and 2.6%,and Chao1 and ACE index significantly were decreased by 18.8%and 18.2%,respectively,indicating that the number and species of microorganisms in the roots of rice and H.Cordata significantly changed,and the relative abundance of various bacterial members changed significantly.