Research On Detection Method Of Pathogen Of Aconiti Downy Mildew

Aconitum carmichaeli Debx,which belongs to the family Ranunculaceae,is the famous-region medicinal plant as well as the original plant of monkshood cultivated in Sichuan,Extensively cultivated in Jiangyou,Sichan province.Aconiti downy mildew is a kind of leaf disease which infects easily and spreads rapidly in the cultivation of A.carmichaeli.Infection of aconiti downy mildew can cause intensive affections on the production and quality of A.carmichaeli.Aconiti downy mildew is caused by which belonging to Peronospora,Peronosporaceae.Previous studies found that Aconiti downy mildew may spread through seedlings or soil transfection.The host-specific property of pathogens of Aconiti downy mildew determind the difficulty for in vitro culture of Aconiti downy mildew fungals,so as to the difficulty for future studies of the pathogens.This study tried to investigate the transmission of Aconiti downy mildew by combining the technology of paraffin section and scanning electron microscope,and establish SYBR Green I Real-time PCR reaction for quick detection kit of aconiti downy mildew pathogens by development of specific primers.These studies lay foundations for monitor of pathogens quantity,correlation analysis between desease index and pathogens quantity,safety evaluation of soil and disease prediction.The main results are as follows:1.Paraffin section of infected A.carmichaeli plant were stained by two different methods.The result indicates that PAS performs better for observation of fungal pathogens in infected tissues.The spores of pathogens were found in root,stem and leaf tissues of infected plant,they were transmited through vessel from root to leaf,then released from stoma in the blade back.2.The optimal SYBR Green I Real-time PCR reaction is 25?L.The reaction contains:12.5?L of SYBR Premix Ex Taq II,8.5?L of ddH₂O,1?L of each primer of L2A and L2B 1?L DNA template?The PCR is conducted in following conditions:initial denaturation at 95?for 30s followed by 40 cycles of amplification;each cycle consisted of 95?for 5s,57?for 30s.Then a final cycle at 95?for 10s,65?for5s was adopted to complete the reaction.The reaction exhibited high specificity.The sensitivity shows its decection limitation is 9.8×10^-4ng/?L.The function of standard curve is y=-3.724x+32.393.The correlation coefficient?R²?is 0.999,and the amplification efficiency is 85.6%.Stability tests shows a satisfactory result for replication,the coefficient of variation of each gradient is less than 2%.Sensitivity for detecting spores in soil is 40 per gram.Relation between the number of spores of soil pathogens and Ct value is:n=10?44.411-Ct?/5.3644.3.Specific primer pair L2A/L2B were designed to develop the quick detection kit of IVconite downy mildew pathogens.Tests of the kist were done as required by Chinese Pharmacopoeia,2015.The detection limitation is 5.6pg/?L.The kit is stable within 1 year under storage of 4?,highly-specific,user-friendly and capable of practices.