Mechanism Study On Microbial Ecology Of Mountain-Breeding Nodes Of Ligusticum Chuanxiong Hort.

Chuanxiong is the dry rhizoma of Ligusticum chuanxiong Hort.,witch is the famous genuine medicinal materials of Sichuan province.More than 90%of Chuanxiong produced in Sichuan.The clonal propagated mode of Chuanxiong by "Mountain-Breeding Nodes" and"Pingba cultivation" in Sichuan nearly 800?1 000 years of history,witch cultivation mode of Chuanxiong only exists in Sichuan province.In there,Without the "Mountain-Breeding Nodes",Chuanxiong is often susceptible to pests and diseases,and significant dysplasia resulted in decreased yield of rhizome.On the basis of textual researching of Chuanxiong variety,origin evolution,cultivation mode,this study use of Multi-agent-parallel analysis method,PCR-DGGE,High-throughput sequencing,microbial pure culture technique to tracked microbial the endophytic microorganisms community structure changes in the process of production and circulation of Ligusticum chuanxiong Hort by"Mountain-Breeding Nodes" and "Pingba cultivation",and the to explore the scientific connotation of "Mountain-Breeding" from the perspective of micro ecology.The main research results are as follows.1.By herbalogical and ancient classics studying,This study clear that Xiongqiong,Gaoben,Miwu are different medicinal parts of the same base source plant Ligusticum sinense Oliv before the Tang Dynasty.Miwu is seedling leaves of L.sinense;Xiongqiong is with dilated nodules of the of L.Sinense rhizome,said Mahan Xiong;Gaoben is in the of L.sinense block rhizome.After the Tang Dynasty,Xiongqiong is the block rhizome of cultivated Ligusticum,and Gaoben is the wild Ligusticum rhizome.The Song Dynasty,Sichuan began planting on Xiongqiong,witch is famous for its high quality.To the Yuan Dynasty,Sichuan medicinal herb Chuanxiong instead of the name Xiongqiong.In Chuanxiong production,its unique way of asexual reproduction at least from the "Compendium of Materia Medica" era.On the one hand,parts of the function of Chuanxiong(Ligusticum)changes from the seedling leaves(Miwu)to the soil Ling Zi(Maxian Xiong)and again to the enlargement rhizome(Bird brain Xiong)?On the other hand,there having a plurality of Chuanxiong wild areas,then development of Sichuan produced the best quality of Chuanxiong.The special cultivation techniques of Sichuan Chuanxiong have played a decisive influence.2.Chuanxiong is composed of a variety of symbiotic microorganisms and plants.The purified 38 strains of 14 culturable endophytic actinomycetes and 84 strains of 17 culturable endophytic fungi of Chuanxiong in this study.The PCR-DGGE analysis technology shows there have 10?20 species endophytic fungi or bacteria in various cultivation stages;High-throughput sequencing revealed in different growth stage of Chuanxiong,endophytic fungi with an average of up to more than 100,of which there are more unable to determine the status of taxa.In the symbiosis of chuanxiong,relative abundance of minority species were dominant,while the vast majority of species relative abundance of less than 1%.Such as the relative abundance of the sordariomycetes all about endophytic fungal taxa of 1/4 in mountain period,while in Pingba stages of cultivation accounted for about all endophytic fungal taxa of 1/2.3.Two times before and after transplanting to mountain for breeding nodes,resulting in micro ecological structure recombination in Chuanxiong symbiont.PCR-DGGE and high-throughput sequencing analysis in this study showed that in the three different stages of cultivation,namely Puxiong period(Pingba cultivation),Lingzi period(mountain cultivation),herbs harvest period(Pingba cultivation),symbiotic microorganisms OTU distribution and relative abundance occurred obvious change in Chuanxiong.On the one hand,Chuanxiong in mountain stage has more abundant endophytic fungilarger than in Pingba period,such as in a mountain stage,Chuanxiong symbiotic fungus OTU average more than more than 130,Pu Xiong stage average of 110,herbs stage an average of about more than 90.The change trend of microbial diversity index was highest in the mountain stage,and the lowest in the herbs stage.It showed that different cultivation environment had a significant effect on the species composition of the endophytic.Compared with the other two stages between mountain stages,endophytic bacteria have the biggest difference.On the other hand,DGGE analysis and high-throughput sequencing showed there always have a small part of endophytic microbes associated with the various stages of the growth of Chuanxiong in the process of cultivated.OTU VENN analysis revealed the constant presence of fungi accounted for Chuanxiong growth during the whole process of endophytic fungal taxa of 10%or so,the species number is about 50 to 60 species.Thus,those microorganisms were relatively stable in Chuanxiong.After a long-term evolutionary adaptation,its host formed a relatively stable symbiotic relationship and in the cultivation process of Chuanxiong by vertical transmission of"Lingzi" into the next generation of host plants,and its has become an important part of Chuanxiong micro ecological system.But the relative abundance of these microorganisms are due to changes in environment and cultivation of Rhizoma chuanxiong.For example,dominant groups in mountain stage,such as Gibberella,Thanatephorus,ceratobasidium,Verticillium,were significantly more than those in the Pingba stages of cultivation.On the contrary,Sordariomycetes unclassified,Septoria and Ilyonectria groups were significantly more than in stage in Pingba mountain stage.The relative abundance of dominant bacteria changed with Chuanxiong planting environment change.It is showed that different cultivation environments have important effects on the stabilization of endophytic microorganisms.4.The process of "Mountain-Breeding Nodes" induced recombinant of Chuanxiong micro ecological structure,and regulate growth and disease resistance of Chuanxiong.According to main diseases of Rhizoma Chuanxiong,root rot disease,6 kinds of endophytic fungi and actinomycetes were isolated from the experiment of plate confrontation,which could have antagonistic effect on pathogenic bacteria,which were F.avenaceum,F.moniliforrme var.Subglutinans,P.Lilacinum,S.Scopuliridis,S.Griseorubiginosus,S.Agglomeratus.At the same time,through the sequencing technology,this study found that there are more abundant Gibberella in chuanxiong at mountain stage than at Pingba stages of cultivation.In the health of the host state,these fungi may through releasing amount of gibberellin to regulating Chuanxiong growth,contributed to the formation of Lingzi,while suppressing the underground biomass over accumulation,thereby expanding the source quantity.In conclusion,through the " Mountain-Breeding Nodes ",Chuanxiong has been rich resistant strains,also increased the types and quantities of beneficial microorganisms.Through the delicate compete with other potential pathogenic microorganisms,the microbial regulating the growth of Chuanxiong,increase the output of the source,enhance the resistance ability of Lingzi.5."Mountain-Breeding Nodes" cultivation caused micro ecological restructuring,affecting the formation of Chuanxiong medicinal parts and quality.Through PCA and PCoA,3 different Lingzi origin for "Mountain-Breeding Nodes" was founded has differences endophytic fungi structure,the main factor is different cultivation environment.Therefore,different mountain environment activities different Lingzi with different endophytic community structure.PCR-DGGE analysis showed that some new endophytic strains in Lingzi witch infection in a certain environment has been maintained to after transplanting to Pingba nursery period and Chuanxiong medicinal materials and harvest period.After transplanting back to Pingba,some strain was lost or reduced witch infection in the mountains,such as Gibberella.Its' reduction may cause the dwarfing of Chuanxiong,and biomass more assigned to underground,and promote the expansion of Chuanxiong rhizome,increase the yield of the crude drugs.Summary of the above,the cultivation mode by "Mountain-Breeding Nodes" and "Pingba cultivation" of Chuanxiong in Sichuan has exist 800?1 000 years of history,which is the accumulated valuable experience in planting by herbs farmers after long-term cultivation practice,exploration,summing up,and which is quality technical support of Chuanxiong genuine form.By referring to the classic books and herbal science,this study for the first time defined the transform habitats and species of Rhizoma Chuanxiong,and cultivation techniques and medicinal function of parts of the change.To understand the formation of cultivation technology has had a significant impact on the status of Rhizoma chuanxiong.From micro ecological perspective,this study reveals the scientific essence of the traditional cultivation technology by"Mountain-Breeding Nodes" and "Pingba cultivation" of Chuanxiong in Sichuan,that is,through the recombinant of the Chuanxiong micro ecology structure,regulate the Chuanxiong rhizome biomass distribution and promotes the development of aboveground parts of plant,enhance the resistance ability of Lingzi,and improve the yield and quality of Chuanxiong in Pingba stage,so as to realize the rejuvenation of Chuanxiong provenance.This study illustrates the simple wisdom in agriculture with the principle of micro ecology,and laid a theoretical foundation for the interpretation of asexual reproduction of other medicinal plants,crops,and nursery stock;this paper provides some new ideas and methods,for example in revealing the plant symbiotic micro organism on the geolism of formation mechanism,and excellent quality of medicinal materials in built mechanism,and in the sustainable utilization of Chinese medicine resources and germplasm rejuvenation etc..This study laid the important foundation for the further development of microbial agents such as biological control agents,microbial fertilizer,and has played an exemplary role for other plant micro ecological mechanism research in the development process and microbial utilization research.