| Crop soilborne diseases which damage the roots and basal part of stems are caused by numerous microorganisms and parasites that fully or partially live in soil,including fungi,oomycetes,bacteria,viruses and nematodes.Crop soilbome diseases occur widespread,infect and damage many host plants including cereal,cotton,oil crops,vegetables,fruits,forests,ornamental plants,grass,and medicinal plants.Crop soilborne diseases not only cause serious reduction of agricultural production,but also threaten the ecological environment.In recent years,as a result of long-term continuous cropping and straw returning,widespread application of no-tillage technology,reductive use of organic fertilizer,crop soilborne diseases have been aggravated year by year,especially in agricultural production in China.Plant diseases induced by soilborne plant pathogens are difficult to control and lack effective control techniques,resulting in great losses and risks to agricultural cultivation Because of the similar infection regions and symptoms,it is difficult to accurately diagnose the disease only by symptoms.The traditional pathogen detection technology is based on the isolation,purification and morphological characteristics of pathogens.The whole process is time-consuming and needs technical operators who should know well enough professional isolation and culture techniques and morphological characteristics of various pathogens.It is an essential step to control crop soilborne diseases using diverse molecular biology techniques to develop different detection method of pathogens.Multiplex PCR detection method of five pathogens on cucumber.The occurrence of crop soilborne diseases is complicated in the fields for the multiple infecting of diverse pathogens,which makes them difficult to be recognized and diagnosed.It is significance to establish a method to detect different pathogens simultaneously.Fusarium oxysporum,Fusarium solani,Sclerotinia sclerotiorum,Pythium aphanidermatum and Botrytis cinerea are five important cucumber pathogens that can infect diverse tissues at different growth stages,which leads to serious economic loss.In this study,one common forward primer and species-specific reverse primers of F.oxysporum,F.solani,S.sclerotiorum and B.cinerea were designed based on comparative genomics.The reaction conditions of multiplex PCR were optimized to establish a simple and efficient simultaneous detection of the five cucumber pathogens.The specificity assay indicated that the multiplex PCR could specifically amplify these five target pathogens and nonspecific amplification products were not obtained from other 21 fungal isolates belonged to 9 species of 7 genera and 32 oomycete isolates belonged to 16 species of 2 genera.The sensitivity analysis showed that the detection limitation of genomic DNA was 1 pg for P.aphanidermatum,10 pg for S.sclerotiorum and B.cinerea and 100 pg for F.oxysporum and F.solani.Furthermore,the multiplex PCR method was applied to detect the five pathogens from soil and irrigation water samples.The established multiplex PCR assay could provide a reliable diagnosis method of cucumber diseases.Equipment-free visualable detection method of two Phytophthora species based on lateral flow strip recombinase polymerase amplification(LF-RPA).Phytophthora species are an important group of soilbome pathogens.They produce and release zoospores that can freely and rapidly diffused through irrigation water and rain water,resulting in disease outbreak rapidly.Phytophthora parasitica and Phytophthora capsici both have a wide range of plant hosts and cause devastating diseases on many crops.Effective monitoring and rapid detection of the two pathogens is a key step in disease management.We developed a molecular detection method based on lateral flow strip recombinase polymerase amplification(LF-RPA)for the visualable detection of Ph.parasitica and Ph.capsici.The specific primers and probe were designed using the Ypt1(Yeast Protein Two,a Rab family GTPase)genes from Ph parasitica and Ph.capsici,separately.After incubation at 40? for 20 minutes,the detection results could be completed by naked eyes using LF-RPA assay.The detection limitation of LF-RPA was 1 pg and 10 pg genomic DNA of Ph.parasitica and Ph.capsici.The specificity assay indicated that LF-RPA assay could distinguish Ph.parasitica and Ph.capsici from the related oomycete species.By combining with simplified DNA extraction methods,the LF-RPA assay was successfully used for detection of Ph.parasitica and Ph.capsici in less than 30 min without specialized equipment.In this study,two important pathogenic oomycetes,Ph.parasitica and Ph.capsici,were studied to established a simple detection method,which is equipment-free for the whole process and will be a promising detection method to be applied in the fields.Equipment-free visualable detection method of three Pythium species based on LF-RPA.Pythium species are a kind of worldwide spread soilborne pathogens which have a broad range of plant hosts.The oospores can keep alive in soil for a long time which lead to the damping-off and root rot of newly planted crops.Pythium ultimum is one of the most destructive Pythium species in agriculture,because it has a wide range of hosts and lead to damping-off and root rot of wheat,tomato,potato,corn,soybean and other crops.Pythium irregulare prefers cool condition and it is one of the main pathogens causing soybean root rot in the north central region of the United States.Pythium helicoides can tolerate high temperature causing significant loss in greenhouse.However,few diseases were reported in China which caused by P.irregulare and P.helicoides.In this study,we developed a molecular detection method based on lateral flow strip recombinase polymerase amplification(LF-RPA)for the rapid and visualable detection of the three Pythium species by targeting their ITS sequences.After incubation at 40? for 20 minutes,the detection results could be judged by naked eyes using LF-RPA assay.The detection limitation of LF-RPA was 100 fg genomic DNA of P.ultimum and P.irregulare,10 fg genomic DNA of P.helicoides.The specificity assay was also evaluated.By combining with a simplified equipment-free DNA extraction method,the LF-RPA assay was successfully used for detection of the three Pythium species in less than 30 min.In this study,three Pythium species were studied to established equipment-free visualable detection method based on LF-RPA,respectively.The assay is a promising detection method to be applied in the fields and further enhance the knowledge of Pythium diseases.Real-time quantitative PCR detection method of P.aphanidermatum.Soilborne pathogens can survive in the soil for a long time.When the environmental conditions are suitable,soilborne pathogens infect crops and probably lead to the outbreak of diseases.The dynamic monitoring of the soilborne pathogens is of great significance for the management of soilborne diseases.P.aphanidermatum,a typical soilborne pathogen,can survive in the soil for a long time and cause damping off of many economically important crops and vegetables.In this study,a TaqMan-based real-time PCR method was developed to detect and quantify P.aphanidermatum in plant and soil samples.The specificity assay indicated that only P.aphanidermatum appeared positive reaction,while the other 32 oomycete isolates belonged to 14 species of 2 genera and 21 fungal isolates belonged to 10 species of 9 genera showed negative reaction.The sensitivity analysis indicated that 10 fg of genomic DNA or 10 copies of standard plasmid DNA could be detected by real-time PCR.The quantitative assay of P.aphanidermatum was carried out in infected plant and soil samples.P.aphanidermatum in plant leaves can be detected after inoculation for 10 minutes while symptom appeared 3 hours post-inoculation.The detection threshold of P.aphanidermatum in soil was around 1.67 zoospores.Therefore,the TaqMan-based real-time PCR could be a useful tool for monitoring the amount of P.in the fields. |
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