Research On The Mechanism Of Resistance To Gummy Stem Blight In Melon Pi420145

Gummy stem blight is one of the major disease of melon.It often results in the reduced production of melon in varying degrees and even leads to no production while the disease is serious.At present,PI140471,PI157082,PI511890 and PI420145 are widely known in the world.In this study,the comprehensive analysis of these 4 melon germplasm resources and 8 cultivars is carried out.And PI420145,which was highly resistant to GSB,was chose to analyzed the changes of the defensive enzyme activity and ultrastructure after inculated with Didymella bryoniae at different time.The homologous sequence of NBS resistance gene in PI420145 was cloned,and its expression characteristics were analyzed at different time after inoculated with Didymella bryoniae.The main research is as follows:1.Identification and evaluation of melon germplasm resources in gummy stem blight resistanceIn order to comprehensively annalyze the value of 4 melon germplasm resources(PI 140471,PI157082,PI511890 and PI420145)resistant to gummy stem blight,seedlings of 4 melon germplasm resources and 8 cultivars cultivated in Jiangsu province and the surrounding area were subjected to artificial inoculation under greenhouse condition to screen for materials resistant to gummy stem blight.And agronomical traits were observed and analyzed as well.The results showed that PI420145(Gsb-6)had the high degree of resistance to GSB.And the leaf shape index,the length of the main vine and the roughness of the main vine were similar to the 8 cultivars.And PI420145 carried some different traits as well,such as green peel,medium single fruit weight,and fragrant and sweet flesh.And the sweetness of PI420145 was 9.7.PI157082(Gsb-2)and PI511890(Gsb-3)were medium resistance to GSB.The single fruit weight were partial light.And the flesh taste acid.These 3 germplasm resources could not only be used as experimental materials to study the mechanism of disease resistance,but also used as a germplasm with excellent traits in melon disease breeding.This study could provide the basis for theresearch of muskmelon breeding through the comprehensive evaluation of four melon resistance sources.2.Changes of different defense enzyme activities and ultrastructure of PI420145 after infected with Didymella BryoniaeIn this study,PI420145 and ’Baipicui’ were used as resistant and susceptible materials to analyze the changes of 3 defense enzymes phenylalanine ammonia-lyase(PAL),polyphenol oxidase(PPO)and peroxidase(POD)activities in different time after infected with Didymella Bryoniae.The ultrastructural changes of host cells in PI420145 in different time after inoculation were also observed by transmission electron microscope(TEM).The results showed that the relationship between PAL,PPO,POD and the melon GSB resistance was very closely associated.The activity of defense enzymes in PI420145 was significantly higher than control.And the increasing rates and peaks of enzyme activity in PI420145 were higher than ’Baipicui’.The PAL and PPO activities in PI420145 showed the trend of slowly increasing first,then rapidly increasing,slowly decreasing at final and reached the highest value at 4d.The activity of POD was rapidly increasing in 3d～5d after inoculation and remained high level in the late period.The ultrastructural changes of the host cells were not obvious at the early stage of pathogen infection(1d～3d).The ultrastructural changes of the mesophyll cells were observed in the middle and late stages(4d～9d).Such as the swelling and deformation chloroplast,the increasing amount of osmiophilic granule,the reduction of starch granules,the serious deformation of the host cells,cell wall damage,chloroplast lamellar structure deformation and chloroplast disintegration.The key point of the change of host cells is during 4d after infected with pathogen.The changes of three kinds of defense enzymes and the ultrastructural changes of host cells could provide reference for the identification of early resistance of melon.3.Cloning and expression analysis of NBS-LRR resistance analogs from PI420145Most of the plant disease resistance(R)genes encode highly conserved nucleotide binding sites and leucine-rich repeats(NBS-LRR),which can be used to isolate GSB resistance-related candidate genes in melon PI420145.In this study,degenerate primers designed based on conserved domains in resistance genes were used to isolate NBS-encoding sequences from PI420145.And 80 R gene analogs(CMRGAs)were isolated and cloned from PI420145.Cluster analysis showed that 8 of the 80 CMRGAs belong to the nonTIR-NBS-LRR subfamily(CNL),and the remaining 72 CMRGAs belong to the TIR-NBS-LRR subfamily(TNL).The TNL class could be divided into 4 groups.5 genes analogs(CMRGA6,CMRGA21,CMRGA35,CMRGA69 and CMRGA73)were selected from each group to analyze the expression characteristics induced by Didymella bryoniae.The results showed that 5 genes could be induced by the pathogen,and could be involved in the defense response infected by Didymella bryoniae in PI420145.But its expression amount and the time to response the infection were different.In the middle of infection,the expression levels of CMRGA6,CMRGA35,CMRGA69 and CMRGA73,in PI420145 were significantly higher than those in the control ’ Baipicui’.These 4 genes might be involved in the middle stages of defense response.The expression level of CMRGA21 was still maintained at a high level in 5d-9d after infected by Didymella bryoniae among PI420145.And CMRGA21 might be involved in the late stage of defense response to the pathogen.Therefore,these 5 genes were synergized to participate in the defense response after infected by Didymella bryoniae among PI420145.