Studies On The Relationship Between Phylloplane Microflora And The Aconitum Downy Mildew On The Leaf Of Aconitum Carmichaeli Debx

Aconitum carmichaeli Debx is one of the most famous-region medicinal plants cultivated in Sichuan China,which has a long history of cultivation in Jiangyou area.In the process of planting,A.carmichaeli Debx is easy to suffer from a variety of diseases and cause the reduction of medicinal plant,which affect the both development of industrial chain of Aconiti Lateralis Radix Praeparata and Aconiti Radix.Downy mildew is one of the Leafy fungal disease which is easily happened in the process of Aconitum carmichaeli Debx cultivation and has features of high morbidirty,wide infection rate and great perniciousness.A large and diverse group of microorganisms settled on the surface of plant leaves.These microorganisms have an important role in promoting plant growth,enhancing plant resistance,degradation of environmental pollutants and nitrogen fixation.Studies have shown that the relationship between microorganisms and plant diseases may exist in both qualitative and quantitative,the occurrence of diseases associated with foliar microbial population level,foliar microflora imbalance is one of the direct causes of plant diseases.Correlation between the occurrence and control of microbial diversity of phylloplane and plant disease has been studied,especially in the economic crops,such as Camellia,Tobacco,but in the study of medicinal plants,rarely reported.In this paper,Aconiti leaves were taken as to take the Aconitum leaves as the object of study,preliminarily investigates the characteristics of microbial composition in the leaves of Aconitum and the dynamic changes of the microorganism in the leaf area of Aconitum under downy mildew stress,and provide basic information for prevention and cure of Downy mildew disease by microecological control.Main results of study as follows:1.Establishment and optimization of PCR-DGGE technique on phylloplane bacteria and fungi for Aconitum carmichaeli Debx.In order to get DNA sequences of phylloplane bacteria and fungi gathered from Aconitum carmichaeli Debx,Nested-PCR experiments have been conducted to the V3 region of 16 S rDNA and ITS1 region respectively.Vertical electrophoresis has been used to ameliorate degeneration gradient of gels,time interval method has been used to figure out electrophoresis time,in addition.For the phylloplane bacteria,the results suggest that the best denaturant gradient range is from 30% to 65%(Gel 10%),electrophoresis temperature at 60℃,voltage of 120 V,the optimal electrophoresis time is 7 h.For the phylloplane fungi,the best denaturant gradient range is from 25% to 52%(Gel 10%),electrophoresis temperature at 60℃,voltage of 120 V,the optimal electrophoresis time is 8 h.2.The microbial community structure and microbial flora characteristics were identified.The PCR-DGGE method was used to analyze 5 areas,the same origin of 6 periods and different leaf age sample of Aconitum phylloplane microbial community structure.The results showed that the microbial composition of Aconitum were rich.Except for uncultured bacteria in the environment,10 genera of bacteria were detected,among them,70% came from the Gamma Proteobacteria and Alpha Proteobacteria,and the largest proportion of Pseudomonas and Sphingosine.10 genera of fungi were detected,came from the Ascomycota,Basidiomycota.The diversity of bacteria and fungi on the leaf surface of Aconitum were affected by the difference of leaf characters,geographic location and growth period.Each leaf Samples of different ages has a most similar and small differences in the dominant species.The difference of growth period had a greater influence on the bacterial community composition,but less on the fungi.3.The correlation between the variation of the flora of Aconitum leaves and Aconitum downy mildew was studied.The dynamic changes of composition and quantity of the microorganisms in the whole growth period of Aconitum phylloplane were analyzed by the method of plate count and PCR-DGGE.The results shows that:(1)There are differences in the number and species diversity of the diseased and healthy sample.The number and species diversity of bacteria and fungi were increased than compared with that of healthy sample,and bacteria increased rapidly between health and 1 grade of disease sample.With the increase of disease degree,the number of fungi showed a gradual increasing trend,and the diversity of the population showed a trend of fluctuation,and showed different performance in different periods.(2)There were differences between diseased and healthy phylloplan microbial groups of Aconitum.Some microorganisms are accompanied by the proliferation of Aconitum downy mildew pathogens on the leaf surface.The dominant strains of certain healthy leaves gradually decrease with the multiplication of pathogens.(3)The diversity of normal fungal community in Aconitum leaves is related to the occurrence of Aconitum downy mildew.The population richness of healthy leaf fungi was negatively correlated with the disease index,indicating that the occurrence of downy mildew might be related to the diversity level of Fungi Community in Aconitum leaves.Under normal physiological conditions,the decrease in fungal community diversity in Aconitum leaves may be responsible for further outbreaks of downy mildew.