The LAMP Method For Rapid Detection Of Pythium Deliense And Sensitivity Of Pythium Myriotylum To Metalaxyl And Mefenoxam

Pythium spp.belongs to Chromista,Oomycota,Oomycetes,Perenosporales,Pythiaceae,and Pythium genus.They were first discovered by Pringsheim in 1858.There are more than200 species of Pythium spp have been reported worldwide so far,of which more than 80Pythium spp are important pathogens that cause plant diseases,and nearly 60 of them have been found in China.Pythium spp infect a wide range of plants and causes complex types of diseases,usually related to seed and seedling diseases,also can cause damping-off,root rot,seedling rot and seed rot in Gramineae,Leguminosae,Solanaceae and Cucurbitaceae,resulting in huge economic losses.Among them,Pythium deliense and P.myriotylum are important plant pathogens,which have a wide host range and strong pathogenicity,causing damping off,dwarfing,root rot,leaf yellowing,wilting and other phenomena,and seriously affecting crop production.The study is mainly based on the loop-mediated isothermal amplification（LAMP）assay targeting the ITS（Internal transcribed spacer）sequence for visual dection of P.deliense which caused soybean diseases in filed.The ITS-Pyd-LAMP assay efficiently amplified the target gene in 70 minutes at 62°C.And specificity was evaluated against P.aphanidermatum,Pythium spp and other fungal species.A positive yellow-green color（by the naked eyes）or intense green fluorescence（under ultraviolet light）was observed only in the presence of P.deliense after adding SYBR Green I.The detection limit of the ITS-Pyd-LAMP assay was determined via amplification of 10-fold serial dilutions(from 100 ngμL^-1 to 1 fg·μL^-1)of purified DNA of P.deliense.Based on visual evaluation after addition of SYBR Green I,the minimum concentration of P.deliense DNA detected in the LAMP assay was 1 fg·μL^-1 Thus,the sensitivity of the ITS-Pyd-LAMP assay was extremely high.Using ITS-Pyd-LAMP assay established here,we successfully rapid diagnosis the suspected diseased soybean samples collected in the filed.Our studies provide a new method for the rapid diagnosis of soybean diseases or other plant diseases in the field,which may in future facilitate control of P.deliense infection on soybeans or other crops.The study was tested the sensitivity of P.myriotylum,which causes base rot of ginger stems.The disease samples were collected from the ginger planting area in Weifang City,Shandong Province.Through the isolation and identification,P.myriotylum was identified as the main pathogen.The sensitivity of P.myriotylum to metalaxyl and mefenoxam was investigated.The results showed that both metalaxyl and mefenoxam have a strong inhibitory effect on the mycelial growth of P.myriotylum.The effective medium concentration EC50is 0.8544μg·m L^-1 and 0.1010μg·m L^-1,respectively.And the minimum inhibitory concentration MIC was 25.0000μg·m L^-1 and 9.6667μg·m L^-1,respectively.The data shows that the above two chemicals can be expected to be effective chemicals for the prevention and treatment of ginger stem base rot,and P.myriotylum is more sensitive to mefenoxam.Therefore,mefenoxam can be selected as the first choice of prevention and treatment of the disease.