Establishment And Application Of Field Detection For Ustilago Maydis

The smut,caused by Ustilago maydis,is one of the main diseases of corn and can occur in the whole growth period.In China,the incidence of this disease was from 5%to 10%,which can lead to economic losses of agriculture.And traditional method for the detection of Ustilago maydis was not suitable to grassroots units.Therefore,the establishment of early,rapid,sensitive and low-cost on-site techniques was important to enhance the detection and prevention of plant diseases.Based on the nature and characteristics of the three targeted Ustilago maydis effector genes,12 sets of primers,three for each gene,were designed to screen the optimization for LAMP assay.After the optimization,the specificity,sensitivity,and feasibility of application for LAMP assay were further analyzed.Based on the specificity analysis,this LAMP assay was confirmed by negative testing for other pathogens.The sensitivity analysis indicates that the detection sensitivity of the pathogen by the specific primer is as low as 44 fg·?L^-1 pEasy-Pep plasmid DNA,and the standard curve obtained from the reaction exhibited a good linearity.For soil samples of artificially inoculated with teliospores,the detection limit of LAMP was 1.6×10³ spores·g^-1.And for maize samples of artificially inoculated with teliospores,pathogens were found to increase significantly after 6day post inoculation.The Coefficient of variance of the repeatbality test indicated a good repeatability of LAMP assay which all less than 5%.At the same time,a set of fluorescent detectors for quick field detection was designed,which can meet the standard of sensitivity and precision of field detection.Specifically,the established method was used for detection of 172,and 139 samples showed Ustilago mydis positive,which were 96%coincidence rate with qPCR system.To summarize,the LAMP method established in this study has proven to provide strong specificity,high sensitivity and repeatability of the LAMP assay,which is also more efficient to monitor U.maydis in the field,as a simple,effective,and repeatable technique.