| Aconitum carmichaeli Debx is the famous-region medicinal plant, as well as the original plant of monkshood cultivated in Sichuan, and it has a long history of cultivation in Jiangyou. In the process of planting, A. carmichaeli Debx is easy to suffer from a variety of diseases and result in the reduction in production of medical materials, 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 cultivation of A. carmichaeli Debx, and possesses features of high morbidity, wide infection rate and great perniciousness. In this study, disease symptoms of A. carmichaeli Debx. infected Downy mildew in different growth stages from seedling place and cultivated land were dynamic tracking studied, and the situation of Downy mildew of A. carmichaeli Debx. was field surveyed, as well as the morbidity and disease index of downy mildew in Jiangyou were studied. The classification pathogenic bacteria was identified by the means of morphological features and rDNA-ITS sequences. At the same time, the spread mechanism of downy mildew preliminarily was studied, and the molecular method to rapid detect the pathogenic bacterium was established. The main results were as follows:1. The incidence of Downy mildew from A. carmichaeli Debx. in district of Jiangyou, Sichuan Province was investigated:According to the field survey of Aconitum downy mildew in Jiangyou, Sichuan Province. The morbidity and disease index of Aconitum downy mildew both have been counted. Disease symptoms of A. carmichaeli Debx. infected Downy mildew in different growth stages from seedling place and cultivated land were tracking studied. Aconitum downy mildew mainly hazards blades of Aconitum, showing diverse disease symptoms in different growth periods of the plant. During the period of seedling, blades began to become celadon and curly, leaves withered and curled inward at the stage of growth, in the stage of harvesting monkshood, brittle yellow leaves appeared, seriously, the plant died. A combination analysis of the weather conditions in the region, morbidity and disease, the results showed that there was a certain correlation between the morbidity of downy mildew and the rainfall and temperature.2. The types of Downy mildew from A.carmichaeli Debx. were identified: According to the microscopic examination of Pathogenic bacteria colony of blade which suffer from Aconitum downy mildew, the type of bacteria determined as Peronospora aconiti Yu by morphologies of sporangium, sporangiophore and oospore. OMEGA kit has been used to extract the DNA of pathogenic bacteria,the concentration and the quality were detected by conducting electrophoresis. ITS1/ ITS4 and NL1/NL4 primers, both were fungus general primers,which were used to PCR for regions of pathogen rDNA ITS and 28 S D1/D2, and the length of the sequences were 892 bp and 741 bp respectively. Then submitting the sequence to NCBI database we have got the login number KU161589 and KU161590 respectively. The results showed that SM11 samples all got together with Peronospora after the sequences were constructed phylogeny trees separately base on the method of Neighbor-joining tree.3. A kind of method of fast detection for Downy mildew from A. carmichaeli Debx. was preliminarily established:According to rDNA-ITS sequences of the pathogen, a specific amplification primer SMF1/SMR1 was designed. The primer can amplify a 640 bp fragment from frost mildew pathogen DNA. Through research about specific trials, the results indicated that the primer cannot amplify other pathogens and Aconitum blade, and it was regarded as a specificity primer. The primer sensitivity tests showed that the primer can amplify the aimed fragment at the lowest concentration of 4.38 x 10-4 ng/?L pathogen DNA. The method to rapidly detect the Aconitum downy mildew was preliminarily established, and it has provided a basis for the detection of Aconitum downy mildew.4. Nosogenesis of Downy mildew from A.carmichaeli Debx. was initially studied: Conducting the paraffin section of Aconitum seedlings from Jiangyou. Oospores of Peronospora Aconiti was observed in Aconitum seedlings, which showed that Aconitum downy mildew probably spread by oospores of Peronospora Aconiti overwintering in seedlings, until the weather getting warmer in spring, A. carmichaeli Debx. was infected. Ultrastructural observation of Aconitum downy mildew disease leaf, which discovered that pathogenic bacteria intruded into the blades through the germinal germ tubes from the leaf stomata or wounds. Pathogenic bacterium produced haustoria after infected the blades, which can draw nourishment from the host, then germinating sporocyst terrier, reproducing sporangium and spores, reinfecting Aconitum plants circularly. It is significant to make sure that mechanism of transmission of Downy mildew and can prevent and cure Aconitum downy mildew from fountainhead. |
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