Resistance Evaluation And Gene Mapping Of Cucumber Varieties(Lines) To Pseudoperonospora Cubensis

Downy mildew of cucumber(Cucymis sativus),caused by the obligate biotrophic parasite Pseudoperonospora cubensis, is one of the most destructive diseases on cucumber. P.cubensiswas observed on stems, leaves, leaf petioles,tendrils and peduncles of heavily infected cucumber, however,symptoms exclusively showed on leaves.There are many controversial (or results) methods on P.cubensis preservation,inoculation, as well as disease resistance and resistant genemapping In this research, westarted with sporangium germination,followed by inoculation methods, finally, we established a reliable system for.Pcubensisinoculation,resistance gene identificationand evaluation, furthermore, we also located oneresistance gene, which will service cucumber breeding and production.The addition of sucrose at five percent in thesuspension increased sporangium germination compared to no sucrose.It indicates that additionof sucrose in the suspension can boost porangium germination. Besides, low temperaturesalso induce sporangium germination ratio in our experiments.The downy mildew disease indexewas significantly lower at 5 days after treatment with the inoculatedsporangium suspensions thatstored at -70? and 26? than the onesstoring at-20?,16?,-60C,-40C,-30C,3? and blank control(at 26? for Oh)at 5 days.The results indicate P.cubensiscan be stored at low temperatures,but ultralow temperature storage significantly reduced the pathogenecity.Moreover, the study shows that the disease index reduction with 26? treated suspension was mainly due to leaves senescing and other microorganisms contamination.In vivo experiments on cucumber leaves found that when inoculatedwith sporangiumat the concentration of 104/ml which stored at 5? for 2 hours, the disease index was increased compared to other temperature treatments. The results indicate low temperature could increase the diseaseincidence. In addition, studies found that the incolation effect offolair spraying method was better than the injection and root drench methods.Moreover, The effect was also better when the sporangium concentration at 10/ml than 103/ml.In vitro inoculation on different cucumber cutivars (or varieties) with the sporangium concentration at 103/ml 7 days post inoculation found that there were different resistance-susceptible degrees on these cucumber varieties, among which, thehighest disease index reachedat sixty.P.cubensis inoculation at the abaxial leaves hadmore severe disease thanat theadaxial leaves. Treatments on detached leaves kept in different micro-enviroment shew thatthe disease incidence ingreen-keeping medium was more severe than in the filter paper.Moreover, results found that adding 5?g/mlbenzimidazole into the moisture substrateprolonged the green timeof leaves. The sporangium production was much more at detached leavesinthe green-keeping medium than in the filter paper,it indicates that green-keeping medium is better for storage and multiplication of P.cwbensis.Thediseasewas more severe when adding tween 20 into the sporangium suspensions at the concentration of 0.02 han adding tween80. Besides, in addition a little of cucumber pollen into the sporangium suspensions facilitated the downy mildew disease incidences.Different inoculation concentrations and methods comparison for P.cubensiswere conducted in the field. The results point out inoculation concentration of ten thousand per milliliter caused more severe P.cubensis infection than other treatements in the field.Spraying method is best for the field inoculation than othermethods(injection, root irrigation). It isconsistent with the best inoculation method in the growth chamber.Nevertheless, the field inoculation concentration is higher than in the growth chamber..The inoculation and identification of resistance cucumber lines were conducted in the greenhouse. Inoculation of sporangium suspensionsat the concentration of thousand per milliliter,whichtreated with five degrees for two hous,causedmore severesymptoms than other two methods.Furthermore, inoculation with the same treated sporangium suspensions on different cucumber lines found that the resistance-susceptbility level were different.The disease indexeof cucumber line 7312 and 7296 was significantly lower than the line 7307,7302,7308 and 7301.Downy mildew disease indexes was investigatedtwelve daysafter inoculation in the open field, the results found that the disease index was much higher than control when inoculated with the sporangium suspension which concentration was thousand per milliliter and treated with at 5? for two hours.The resistant levels to downy mildew of eight cucumber lines were differentwhen using this inoculation mthod, and the disease indexes ranged from 8.55 to 77.06.The resistance of different cucumber lines or varieties against the downy mildew was investigated at naturally infection condition in two experiment sites.The resistance of most of the lines was consistent in two sites,butexcept line l.This indicates identification and evaluation of cucumber resistance to downy mildew was influenced by the environmental and other factors,which need us to inoculate the cucumber in experiment or the induced susceptible cultivars.According to the screening experiment which conducted in field, the variety jinyan 4was found as a resistant control variety and erzaozi as a susceptible control variety.These two lines would be used in the resistance identification and evaluation experiments in the future.Based on the result of cluster analysis of 62 different cucumber varieties and inbred lines against to downy mildew,a standard was established for cucumber resistant identification and evaluationagainst downy mildew in the field.When the disease index of variety erzaozi reached34.06,the resistant types were judged by the disease indexes as follows:HR?5,5<R?13,13< MR?19.5,19.5< MS?25,25<S<33.5,HS>33.5.Adjusted disease indexwasalso established and its availability was verified inthe successive two years experiments. Adjusted disease index= tested variety(lines)disease index at X year/(variety erzaozi disease index at X year/34.06). Different year resistance results can be comprared using the adjusted disease index.The resistance loss or weaken for cucumber production in those resistant varieties was monitored insuccessive three years.The ratio of 87.50% reduced or lose was found over three years.Therefore, monitoringand rotating the resistantlineswas necessary.The resistance gene location on the chromosomes was studied.Cucumber line S,derived from self-pollination of the varity erzaozi, is susceptible to dowany mildew. Cucumber line R,derived from self-pollination of varity line 8,has a high level of resistance to downy mildew. The genetical population was established bycrossing line R and S as well astheir descendants F1F2.The resistance identification results showed that the resistant resource R was dominant inheritance.By usingthe SLAF-seq(Specific-Locus Amplified Fragment Sequening) technique, the resistance gene of line R was identified on chromosome 6,locating the region from 8,835,226bp to 10,455,046bp.The results lay a foundation for futurefine mapping.