Response Of Rose To Downy Mildew Infection And Transcriptome Analysis

Rose（Rosa chinensis Jacq.）is one of the four major cut flowers in the world.It is widely planted all over the world and is deeply loved by people all over the world.However,in the production and cultivation of facilities,rose downy mildew is often prone to occur,causing serious economic losses to the rose cut flower industry.Evaluating the resistance of rose varieties to downy mildew and finding downy mildew resistance genes are the basis of rose molecular breeding.In this study,the pathogen of rose downy mildew was identified by morphological and molecular methods.The pathogen of rose downy mildew was cultured by’small environment culture method in greenhouse’.The resistance of 10 cut rose varieties to downy mildew was compared by natural inoculation in the field and the relationship between stomatal related indexes and disease resistance was analyzed.The related histological observation and physiological indexes of the highly susceptible variety’Floyd’were determined,and the resistance response mechanism of rose to downy mildew infection in different infection periods was analyzed by transcription sequencing.The main results are as follows:（1）The pathogens were identified by morphological and molecular methods.The cut rose variety’Hypnose’with typical Rose downy mildew was used as the material for isolation and culture.The results showed that the pathogen（DM）was firmly convinced of Peronospora sparsa,and the homology with Peronospora was 99.84%.The NJ phylogenetic tree of Peronospora was constructed,and the pathogen and Peronospora sparsa were clustered into one branch.（2）DM was cultured by’whole leaf method’,’water agar culture method’ and ’small environment culture method in greenhouse’respectively.It was found that the’small environment culture method in greenhouse’had the best culture effect,and it was easy to produce spores when the humidity in greenhouse reached about 70%.（3）The indoor artificial inoculation method and natural inoculation method were used to inoculate the rose varieties’Nightingale’,’Explorer’,’Orange Storm’and’Dark Night Phantom’,respectively.The inoculation results showed that the indoor artificial inoculation method had poor inoculation effect and could not cause symptoms.The natural inoculation method could effectively inoculate DM,and there were differences in disease resistance among the four varieties:’Nightingale’>’Explorer’>’Orange Storm’>’Dark Night Phantom’（disease resistance from large to small）.（4）The disease resistance of 10 cut rose varieties was evaluated by natural inoculation method,and the average disease index of 21 days was counted.The results showed that’Floyd’and’Peach Avalance+’were highly susceptible to downy mildew,and the disease index（DI）was respectively 63.04 and 63.57.The correlation analysis of stomatal area,density and DI of new and old leaves of 10 varieties of rose was carried out.It was found that the DI of rose downy mildew and the stomatal density of new leaves showed a significant negative correlation,the correlation coefficient was-0.684,and the stomatal area of old leaves showed a significant negative correlation,the correlation coefficient was-0.581.The stomatal density of new leaves was generally higher than that of old leaves.（5）The highly susceptible rose variety’Floyd’was used as the material.The leaves of 1d,3d,6d,9d and 12d were collected after inoculation with DM symptoms,then photographed and observed.Trypan blue was used to observe the microscopic staining of hyphae.It was found that the number of pathogens and the degree of pathogen germination in the middle of the leaves were always higher than those in other parts of the leaves,which were more susceptible to DM infection.The fresh leaves of roses on 0d,4d,8d,12d and 16d after inoculation with DM were observed by histochemical staining and the physiological indexes were determined.Trypan blue,DAB and NBT staining showed that the infection rate of DM was slow,and obvious staining began on 12d.The activity of antioxidant enzymes（POD,SOD,CAT）and the content of disease-related substances（soluble sugar,proline,chlorophyll,MDA）in the leaves of inoculated plants were measured.It was found that almost all of them showed an inverted’V’shape change that increased first and then decreased.（7）Transcriptome analysis was carried out at 4d,8d,12d and 16d after DM inoculation with 0d as control.Young and mature leaves all obtained the most differentially expressed genes at 12d after DM infection.Differential genes were mainly enriched in GO terms such as catalytic activity,binding,and metabolic processes;the main enriched metabolic pathways were photosynthesis,plant hormone signal transduction and porphyrin metabolism,and most of the differential genes showed a down-regulation pattern.In the plant-pathogen interaction pathway,the expression of related differentially expressed genes in the Ca²⁺signaling pathway was up-regulated,and the related pathogenesis proteins were induced.Analysis of all differentially expressed genes at four time points showed that 13 transcription factors were obtained in the new leaves and 10 transcription factors were obtained in the old leaves.The transcription factor family containing the most differentially expressed genes was MYB,followed by the WRKY family,and the expression pattern of the differentially expressed genes was almost up-regulated.