Resistance identification and biochemistry of resistance of different grape varieties to downy mildew

Abstract: 【Objective】Downy mildew of grape is an important disease in the grape. Shandong coastal ar-ea is the main production area in China,but the resistance of different grape varieties to downy mildew isseldom reported in detail. The study is to evaluate the resistance of grape varieties to downy mildew,andto identify the biochemistry and signal pathways related to resistance. 【Methods】The occurrence ofdowny mildew in 40 varieties was investigated in the peak period of downy mildew. Forty new shoots wereselected from each variety,and the disease index was calculated by the 6 level,and the resistance todowny mildew was analyzed by using the disease index which were divided into 5 levels: immunizationwith disease index 0;high resistance with index 0.1-5.0;resistance with index 5.1-25.0;susceptiblewith index 25.0-50.0;high susceptible with index 50.0-100.0. The fresh grape leaves were washed withwater for 24 h under the condition of 22 ℃. The fresh spore capsule was brushed in sterile distilled water,and the suspension was combined with a spore suspension. The concentration of suspension of the sporecapsule was adjusted to 5×105·m L-1under microscope. The leaf disc was used for indoor inoculation ex-periment. The suspension of the spore capsule was sprayed on the health 4-6 leaves of‘Fragrant'‘Crim-son Seedless' ‘Ruby Seedless' with a small hand-held sprayer. Selected leaves preservation in liquid ni-trogen,then transferred to preserve at-70 ℃. The extract and determination of SA referenced Liu Yu-liang's method and the determination of H2O2 content referenced the method of Hao Zaibin et al. TotalRNA was isolated with the improved SDS method. Reverse transcription-polymerase chain reaction(RT-PCR) first strand c DNA was synthesized according to Ta Ka Ra company's reverse transcription kit. The UBQ gene was used as a control to verify uniform loading and amplification. The information of genes and primers employed in the study were provided in table 1. Each gene was amplified for 28 cycles. Productswere confirmed by sequencing and analyzed using the BLAST tool. RT-PCR products were resolved byelectrophoresis on an agarose gel and visualized by staining with ethidium bromide.【Results】The dis-ease index range of 40 grape varieties was 1.7-59.0,which exhibited significant difference in the resis-tance of the downy mildew. The disease index of‘Cold honey Seedless' ‘Fujiminori'‘Honey Juice'andother 9 varieties was between 1.7-4.6,and indicated high resistance. Disease index of‘Venus seedless' ‘Crimson Seedless' ‘Kyoho'and other 17 varieties of grape was between 5.2-23.4,showing resistance.Disease index of‘Jintian rose',‘purple earth',‘red milk'and other 11 varieties was between 27.6-48.0,showing susceptible. Disease index of‘Venus rose'‘Ruby Seedless' ‘Autumn black'were 52.7,54.6,59.2 respectively,showing high susceptible. The resistance of 40 cultivars to downy mildew was al-so investigated by indoor inoculation experiment,and the results showed that the disease index rangedfrom 0.0 to 59.6,which meant significant difference in the resistance to the disease. Among these culti-vars,the disease index of‘Venus seedless' ‘Sunset red'‘Yongyouyihao'varieties was 0.0,which exhib-ited completely resistance.‘Fragrant'and other 5 varieties showed high resistance(index: 1.2-5.0).‘Pur-ple seedless 'and other 13 varieties were resistant cultivars(index: 5.2-23.7).‘Hongmanai'and other 13 varieties were susceptible(index: 30.3- 48.9).‘Bixiang seedless' ‘Autumn Black'‘Ruby Seedless' ‘White Rosario'and‘Cupid rose'were high susceptible(index: 51.1-59.6). The results on the resis-tance of most grape varieties from indoor inoculation were basically consistent with the natural incidenceof downy mildew. Only a few varieties had different performance. On normal condition,there was no sig-nificant difference in SA content among high resistance variety(‘Fragrant'),resistant cultivar(‘CrimsonSeedless') and susceptible cultivar(‘Ruby Seedless'). After the inoculation of pathogens,however,compared with control,SA content of different varieties increased significantly. SA level of‘Fragrant'was up-regulated dramatically 3 h after inoculation,and reached the highest(1 032 μg·g-1) at 12 h aftertreatment. SA content of‘Crimson Seedless' and‘Ruby Seedless' began to increase at 3 h,6 h respec-tively,but the amount was significantly lower than that of‘Fragrant'. After 6 h of inoculation,the NPR1 expression in‘Fragrant'was significantly up-regulated,reached the highest at 12 h,and its expressionincreased in the detection period. Although NPR1 expression of‘Crimson Seedless' was also increased,its expression was less than that of‘Fragrant'. Only a little up-regulation of NPR1 expression in the‘Ru-by Seedless' was detected after inoculation. PR1 expression was induced in high resistant variety‘Fra-grant'and high resistant variety‘Crimson Seedless'. The PR1 expression level in‘Fragrant'was muchhigher than in‘Crimson Seedless'. PR1 expression in‘Fragrant'was significantly up-regulated after 6 hof inoculation,while the expression in‘Crimson Seedless' needed 12 h to increase,and PR1 expressionin‘Ruby Seedless' was not obvious. Before the inoculation of downy mildew,the content of endogenousH2O2 was 20.3 μmol·L-1and showed no significant difference to the other two species. After the inocula-tion,the content of H2O2 in leaves of three grape varieties increased and there were difference among vari-eties. The content of H2O2 in high resistant variety‘Fragrant'was increased rapidly after 1 h inoculation.The maximum amount of H2O2 was 35.2 μmol·L-1,and down to its original level after 7 h. The maximumamounts of H2O2in‘Crimson Seedless' and‘Ruby Seedless' were 26.8 μmol·L-1and 22.2 μmol·L-1re-spectively. These data indicated that H2O2 was involved in grape leaf downy mildew infection process. Af-ter 1 h of downy mildew inoculation,the Rboh D of‘Fragrant'was significantly up-regulated,and the ex-pression level reached the climax at 3 h. Although Rboh D was also induced in‘Crimson Seedless',it waslower than in‘Fragrant'. The expression of Rboh D in‘Ruby Seedless' was not increased.【Conclusion】More than 50% of grape varieties tested in the experiment showed good resistance to downy mildew,SAsignaling pathways and oxygen pathways involved in the resistance of grape to downy mildew.