Microbial Community Characteristics In Rhizosphere Soil Of Panax Notoginseng

Panax notoginseng (Burk.) F. H. Chen (Araliaceae), a well-known traditional Chinese medicinal plant which belong to Panax genus, is widely used for the treatment of cardiovascular diseases, inflammation, trauma, and internal and external bleeding due to injury. The long-term patronage, damp environment induced microecological imbalance, and root rot soil borne disease is increasingly prominent. It has not been effectively relief.The aim of this study is to investigate the dynamic processes in the composition and abundance of rhizosphere bacterial and fungal communities, as well as which the main factor lead to shifts in these soil microbial taxa, and reveal the difference in the composition of rhizosphere bacterial, fungal communities between root rot and healty notoginseng and the factors associatived with disease. To determine abundance of fungal, bacterial communities, we used fluorescent quantative PCR, and to study the diversity and structure of fungal and bacterial communities, we used a MiSeq sequencing technique. In addition, we studied the diversity and composition of cultured fungi and bacteria, and screened the active strains which can control root rot.Sample Info:Four rhizospherer soil samples of healthy P. notoginseng were collected from Yanshan in April and October from2012to2013. For each sample, a small part of each sample was used for high-throughput sequencing and fluorescent quantative PCR to study the changes of fungal and bacterial community structure and abundance in the process of notoginseng cultivation, another small part was used for the separation of fungi and actinomycetes, the rest was used to analyze the physical and chemical properties. The yellow-rot and sudden wilt-rot rhizosphere soil samples collected from Yanshan and Jinchuan were used to analyze microbial community structure and explore microbial factors causing root rot of P.notoginseng.Fluorescent quantitative PCR analyses showed that the numbers of fungi and bacteria in rhizosphere soil of P. notoginseng significantly increased in six months after transplanting, and then gradualyl decreased, and double increased in one year and a half later. Pearson correlation analyses displayed a significant positive correlation between fungal amount and soil water content (R=0.912, p<0.05). Shannon diversity index analyses indicated that the fungal diversity decreased with the increase of fungal amount, half a year later increased with the decrease of fungal amount, but the fungal diversity significantly increased after one year and a half. The bacterial diversity was consistent with the change of bacterial amount. High-throughput data analyses showed there was an obviously dynamic change in the rhizosphere soil microbial community of P. notoginseng. A large number of Mortierella existed in fungal community, which abundance significantly increased within half a year after transplanting, and then gradually decreased, stabilized after one year and a half. The abundance of Fusarium slightly changed. At the Phylum level, Ascomycota and Zygomycota were two dominant groups. The abundance change of Zygomycota was consistent with Mortierella. The abundance of Ascomycota plummeted within six months before transplanting, and then gradually increased. Proteobacteria and Bacteroidetes were two dominant groups of the bacterial community. The abundance of Proteobacteria increased gradually over time, but the abundances of Bacteroidetes and Proteobacteria appeared opposite. With the increase of planting cycle, pathogens increased and benefical microbes decreased, which indicated that the growth environment of P. notoginseng was becoming increasingly worse. Pearson correlation analyses and redundancy analyses showed that the major fungal groups were affected by the total nitrogen (TN) of soil, which did not reach significant levels. The total phosphorus (TP), available phosphorus (AP), and hydrolyzed nitrogen (AHN) were important factors affecting the composition of bacterial community.The microbial communities of rhizosphere soil from root rot in Yanshan and Matang were characterized. The results of qPCR. indicated that the quantities of bacteria and fungi from rhizosphere soils of root rot were higher than those from healthy soil. There were great differences between the microbial community of root rot and those of healthy ones, that is, the abundance of Mortierella was sharply decreased, but other pathogens increased to be the dominant groups. The dominant fungal pathogens included Fusarium、Ilyonectria、Cylindrocarpon、Volutella、 Mycocentrospora、Botryotinia、Monographella and its teleomorph Microdochium, and the dominant bacterial pathogens included Pseudomonas、Flavobacterium. The occurrence of root rots was closely related to the accumulation of those pathogens, and the coexistence of diverse pathogens was common.Two hundred and twenty-five individuals of fungi were isolated from rhizosphere soil of P. notoginseng, they were classified into86species distributed in5classes (Sordariomycetes、Dothideomycetes、Eurotiomycetes、Leotiomycetes and Zygomycetes),13orders,25families and52genera, and3unclassified species at genus level,7out of them might be new species,55.11%of the total individuals belong to Sordariomycetes.In addition,292individuals of actinomycetes were also isolated, the60representative isolates belong to7suborders(Streptomycineae、 Corynebacterineae、Micromonosporineae、Micrococcineae、Streptosporangineae、 Propionibacterineae、Pseudonocardineae)、11families and17genera.8out of them might be new species,70.34%of the total individuals belong to Streptomycineae. Species which were not detected by high-throughput sequencing were isolated by the traditional method, thus great microbial diversity including many new species in rhizosphere soil of P. notoginseng is indicated.With targeted by Fusarium solani, Phoma herbarum, Alternaria panax Alternaria panax and Plectosphaerella cucumerina, thirty-three of the149fungal and actinobacterial isolates displayed antimicrobial activity against at least one pathogenic fungus, Active strains containing13fungi and21actinobacteria accounted for22.1%. Actinobacteria showed stronger activity than fungi. It indicated that there were plenty of available resources in the rhizosphere of P.notoginseng.Based on the culture-independent and culture-dependent methods, the changing characteristics of microbial community in the rhizosphere of P.notoginseng and the occurrence of soil borne disease were illuminated for the frist time which could provide some important experimental theory for resolving continuous cropping obstacles of p.notoginseng. A number of high activity strains against root rot disease were obtained which were valuable resources in effective prevention and control of soil borne disease and development of new biological agents.