Development Of A Loop-mediated Isothermal Amplification Assay For Detection Of Pythium Oligandrum And Its Application

Pythium is one of the most important microbes in the world,which is closely associated with crop production and our life.The host range of Pythium is very wide.Some of them are phytopathogen and some of them are significant biocontrol strains.As phytopathogen,Pythium can infect soybean,cucumber,wheat,peanut and other crops.Soybean is an important cash crop and oil crop in China.According to research reports,soybean frequently occurring diseases mainly are soybean root rot,soybean blight,soybean seedling damping off,soybean downy mildew,soybean sclerotinia rot,soybean anthrax,soybean frogeye leaf spot,soybean cyst nematode and other soybean diseases.In recent years,soybean root rot and other soybean root diseases are common and serious.Pythium oligandrum is a typical representative of biocontrol Pythium.With the advantages of high efficiency,environmental protection and increasing production,the biocontrol microbial inoculum with oligandrin as the active component has been developed and utilized.At present,the prevention and control of soybean root disease is mainly focused on agricultural control and chemical control,while biological control is seldom applied.As a biocontrol microbe with application prospect,we should pay attention to whether Pythium oligandrum has good control effect on soybean root disease.In the first chapter,we summarized soybean diseases caused by Pythium and their comprehensive control,the biocontrol Pythium,the related molecular detection technology and LAMP assay technology.Soybean diseases caused by Pythium mainly include soybean seed rot,soybean root rot and soybean seedling damping off.These Pythium spp.mainly include Pythium ultimum,Pythium aphanidermatum,Pythium irregulare,Pythium sylvaticum,Pythium attrantheridium,Pythium perplexum,Pythium heterothallicum,Pythium myriotyum,Pythium spinosum,Pythium helicoides,Pythium terrestris,Pythium vexans,Pythium myriotylum and so on.The comprehensive control of Pythium disease mainly includes strengthening the breeding of resistant varieties,strengthening the management of farmland,rational and safe application of agents,and increasing the development and application of biological control.In addition,some Pythium spp.are important biocontrol strains,such as Pythium oligandrum,Pythium nunn and so on.At present,the reported molecular detection techniques for Pythium included common PCR,multiplex PCR,real-time PCR,high-throughput detection,molecular labeling technology,colloidal gold detection technology,loop-mediated isothermal amplification,etc.Among them,LAMP is a fast,simple and efficient molecular detection technique.LAMP can be used for the rapid detection of plant diseases,the pathogen detection of seeds,the pathogen detection of soil and inspection and quarantine of plant diseases in plant protection field.In the second chapter,based on Loop-mediated isothermal amplification(LAMP),we selected cytochrome oxidase subunit I(COI)as a target gene and screened a set of species-specific LAMP primers for Pythium oligandrum to establish a LAMP assay to rapidly detect Pythium oligandrum.Before the reaction,hydroxynaphthol blue(HNB)was added to the assay system,completed at 63℃ for 70min.The reaction results can be judged according to the color of the end product,with purple as negative and sky blue as positive.Sensitivity of the system for detecting Pythium oligandrum DNA was 100 pg/μL.This system was used to detect Pythium oligandrum oospores carried by soil,and the results showed that oospores could be detected under the condition of≥50 oospores in 0.25 g soil.This system was used to detect the hypocotyl tissue of soybean inoculated with Pythium oligandrum.The results showed that Pythium oligandrum could be detected quickly and accurately in soybean inoculated tissues.In order to verify the LAMP results,we observed the colonization of Pythium oligandrum in the soybean hypocotyl by using fluorescent inverted microscope.The observations were consistent with the LAMP assay results,indicating that Pyo 34-1,Pyo 34-2 and Pyo 34-3 strains can invade soybean hypocotyl tissues and produce lots of mycelia and oospores.The LAMP system can be used to detect the presence of Pythium oligandrum in seeds,plant,soil,act.It provides a new technical method for study of the relationship between Pythium oligandrum and soybean.In the third chapter,based on the established Pythium oligandrum LAMP system,we studied the distribution of Pythium oligandrum in the soybean roots and the colonization of Pythium oligandrum in soybean root tissues.In order to study the distribution of Pythium oligandrum strain Pyo 34-3 in the soybean roots the roots of collected soybean plants were divided into rhizosphere soil samples,root surface samples and root samples by a series of treatment methods such as shaking off and washing,ultrasonic treatment,high-speed centrifugation treatment and surface disinfection treatment.LAMP detection and plate separation were performed for the collected samples respectively.The results showed that the rhizosphere soil samples,roots surface samples and roots samples all can be detected Pythium oligandrum,and Pythium oligandrum was isolated from the rhizosphere soil samples and the roots samples.Pythium oligandrum can survive well in soybean rhizosphere soil,roots surface and root tissues.In order to study the colonization of Pythium oligandrum strain Pyo 34-3 in soybean root tissues we use fluorescent inverted microscope to observe the colonization of Pythium oligandrum in the soybean root tissues.As a result,after inoculation for 24 h-96 h,this strain produced a large number of mycelia in the soybean root tissues,and the mycelia were gradually dissolved,leaving only a small number of mycelia residues at 216 h(9 d).After inoculation for 24 h,this strain produced oospores in the root tissues.After inoculation for 72 h,some oospores were dissolved.Subsequently,the proportion of oospores digestion increased.To 216 h(9 d),only empty oogoniums were left after digestion.At the same time,the transections of the soybean roots were observed,and it was found that Pythium oligandrum can colonize the epidermis and cortex,and cannot invade stele.The results showed that soybean was the affinity host of soybean root rot.For soybean root rot and other soybean root diseases,Pythium oligandrum may have good potential for biological control.LAMP detection,microbes separation and microscopic observation were used to study the distribution,survival and colonization of Pythium oligandrum in soybean roots.It provides a new technical method for studying relationship between Pythium oligandrum and host plants.