Establishment Of LAMP Detection Method For Pythium Arrhenomanes And RPA Assay For Pythium Aphanidermatum

The genus Pythium is a pathogenic genus found worldwide that causes plant diseases.At present,more than 120 Pythium species have been identified,most of which were soil-borne diseases or saprophytic species,and the host range of Pythium was relatively wide.They usually invaded the seedling roots and seeds,resulting in a large number of infected seedlings.They caused the seeds rotted,the roots rotted,and the fruit rotted,causing serious agricultural economic losses.Pythium arrhenomanes was an important pathogen of corn stalk rot,which mainly caused corn root rot and stem rot,and had great harm to corn production.Pythium aphanidermaturn was one of the most pathogenic pathogens in the genus Pythium and it was widely distributed in forests,grasslands,ponds and agro-ecosystems.It had wide host ranges,such as soybeans,wheat,corn,Potatoes,and many other cash crops.It can infect plant seeds,seedlings,roots and lower stems,resulting in tripping and root rot,and the oocysts of Pythium aphanidermatum can survive in the soil for a long time,then infecting plant roots or seeds.However,a series of molecular detection methods based on polymerase chain reaction(PCR),including ordinary PCR detection,real-time PCR detection,and multiplex PCR detection methods,which required the use of expensive equipments and were not suitable for experiments with limited resources and grassroots workers.In recent years,loop-mediated isothermal amplification(LAMP)and recombinase polymerase amplification(RPA)had been developed.These nucleic acid isothermal amplification techniques were simple,sensitive,specific and did not require complicated equipment.Currently,LAMP technology had been widely used in the detection of fungi,bacteria,and viruses.RPA technology was currently used in food safety testing and disease diagnosis.This detection technique was rarely used in fungi now.We successfully established a rapid and accurate loop-mediated isothermal amplification(LAMP)method for detecting Pythium arrhenomanes.The LAMP used?-tubulin gene as the target sequence.It was evaluated for specificity,sensitivity,and actual detection.The specificity test showed that only P.arrhenomanes isolates had positive results and negative results were obtained from closely related oomycete species and fungi.The limitation of LAMP method was 10 pg·?L-1,which was about 1000 times than that of the conventional PCR.In addition,this LAMP method was successfully applied to detect P.arrhenomanes DNA extracted from inoculated com tissues.We successfully established a rapid,accurate and visual LAMP method for detecting P.arrhenomanes in 60 minutes,which provides a new method for the rapid detection of P.arrhenomanes.We successfully established a rapid and accurate recombinase polymerase amplification(RPA)method for detecting Pythium aphanidermatum.The genomic method was used to identify the specific gene of Pythium aphanidermatum as the target gene.Then RPA primers and probes were designed according to the target gene.The whole reaction was incubated at 39? for 30 min and the reaction was detected by RPA agarose gel electrophoresis detection and RPA fluorescence detection.The technique was then evaluated for specificity,sensitivity and actual disease detection.In the specific experiment,only the amplification product of Pythium aphanidermatum strained from different sources showed the target band,and other Pythium,Phytophthora and fungal strains did not show the amplified band,which indicated that the RPA primer designed in this experiment can be specifically detected and had good specificity in Pythium aphanidermatum species.The sensitivity of RPA fluorescence detection system in sensitivity experiments can reach 3.75 fg,which was equivalent to the sensitivity of Real-time PCR,and it was 10000 times more sensitive than the sensitivity of RPA agarose gel detection system.It is also 10,000 times than the sensitivity of ordinary PCR.In the artificial simulation inoculation experiment,RPA method can detect Pythium pedunculata before appearance of obvious lesions.It achieved the purpose of early detection of plant pathogens and provided technical support for the diagnosis and early detection of plant pathogens.