Rapid Of Main Disease Pathogens Of Panax Notoginseng And Identification Of A Pathogen Of Panax Notoginseng Leaf Disease

Panax notoginseng(Burk.).F.H.Chen is a precious Chinese herbal medicine,which has been used for a long time to promote blood circulation and treat bruises.Researchs showed that P.notoginseng have many pharmacological effects,such as antiplatelet aggregation,thrombolysis,lowering blood pressure,anti-inflammatory and so on,and its clinical effect is significant.However,the long growth period and unique growth environment of P.notoginseng are easy to induce diseases.The root rot disease of P.notoginseng is a serious soil borne disease.It can happen throughout the year.The incidence rate is always 5%?20%,and it can reach 70% in severe cases.The continuous cropping obstacles caused by root rot increased the planting cost of P.notoginseng and decreased the utilization rate of soil,which is quite unfavorable to the industrialization development of P.notoginseng.The black spot is a kind of leaf disease that commonly occurs in the plantations of P.notoginseng.The incidence rate of black spot perennial is about 10%,which accounts for more than 60% of the total incidence at June.The pathogenic fungi live through the winter on the seeds,seedlings,roots,leaves,stumps and soil of P.notoginseng with hyphae or chlamydospores,and use them as the transmission medium for diffusion.Therefore,any rapid and accurate detection method of pathogenic fungi is of great significance to prevent the occurrence and epidemic of root rot and black spot.The main results are as follows:1.In order to establish an accurate and rapid molecular detection system for the root rot of P.notoginseng,a real-time fluorescent quantitative PCR method was developed for the rapid detection of Fusarium fungi.The specific gene loci of four species of Fusarium fungi were obtained through screened,they are aminoadipate reductase Lys2 gene of F.solani,fatty acid ?-hydroxylase gene of F.oxysporum,bleomycin hydrolase gene of F.verticillioides and abscisic acid Aba A gene of F.graminearum.Based on these specific loci,real-time fluorescent quantitative PCR primers were designed.And the quantitative detection standard curve was constructed by SYBR Green I real-time fluorescence quantitative PCR technology.Finally,a real-time fluorescent quantitative PCR method for the detection of Fusarium pathogenic fungi in P.notoginseng was established.The results showed that the content of Fusarium fungi in root rot plants was significantly higher than that in healthy plants.It is proved that Fusarium is one of the most important pathogens causing root rot of Panax notoginseng.At the same time,it also proved that the rapid molecular detection technology established in this experiment has high sensitivity and good specificity,which can provide basis and technical support for the rapid molecular detection of root rot,soil with bacteria and seeds and seedlings in the field.2.In order to make the diagnosis more convenient,we designed real-time fluorescent loop mediated isothermal amplification primers based on the specific aminoadipate reductase Lys2 gene of F.solani.Seven samples of P.notoginseng plants and soil were detected by real-time fluorescence loop-mediated isothermal amplification technology.Compared with real-time PCR,fluorescent quantitative loop mediated isothermal amplification is not only fast and highly specific,but also can achieve simultaneous amplification and detection.3.In order to detect the black spot of P.notoginseng quickly,fluorescence quantitative PCR method for the detection of A.panax was established.A specific gene,glyceraldehyde 3-phosphate dehydrogenase gene of A.panax,was obtained through screening genes.The recombinant plasmid standard of the gene was prepared,the standard curve of quantitative detection was constructed,and the real-time fluorescent quantitative PCR method to detection A.panax was finally established.The results showed that the content of A.panax in black spot plants was significantly higher than that in healthy plants.In addition,the method has high sensitivity and the concentration of detection template can be as low as 0.19 pg /?L.4.Alternaria fungi widely exist in nature,and it is an important crop disease.In this study,the samples of leaf disease of P.notoginseng were collected and the pathogenic fungi were isolated.The pathogenic inoculation experiment,morphological identification,multi site sequence analysis and phylogenetic tree study were carried out in this study.The results showed that the pathogen was Alternaria alternata.In addition,a real-time PCR system for A.alternata was established.The results showed that the content of A.alternata in the diseased plants was significantly higher than that in the healthy plants and the sensitivity of this method was high.In conclusion,the rapid molecular detection method established in this study is highly specific and sensitive,and can be used to reveal the dynamic changes of pathogenic fungi concentration in the complex P.notoginseng plants and planting soil.Furthermore,it can provide technical supports for the soil treatment,early diagnosis and dynamic monitoring of root rot and black spot,and the follow-up further understanding of the pathogenic process and mechanism of pathogenic fungi,and targeted research on efficient and environmental protection methods to provide technical support.This study laid a foundation for further understanding of the pathogen and molecular detection of the disease of P.notoginseng.