The Hypersensitive Reponse Against Pseudoperonospora Cubensis In Cucumber

Cucumber (Cucumis sativus L.) is one of the vegetables produced all over theworld. Cucumber is always damaged by P. cubensis during its production, suffering aheavy loss of16%up to34%in total yield. To date, the traditional mangements tocontrol diseases are agricultural comprehensive control, ecological control andchemical control, which limited effective useness for long therapeutic effect andpesticide residue. Therefore, resistant cultivar is a basic method in cucumberproduction. However, the resistant mechanism in cucumber against P. cubensis isunclear. In order to investigate the resistant mechanism, the resistant inbred lineHNAU-0023and the susceptible inbred line IL112were inoculation with P. cubensis,then disease development, accumulation of H2O2and callose, programmed cell death,and hypersensitive response were determined. The main results were as follows:1. The disease index survey indicated that cucumber infected by P. cubensis, thedisease spot in IL112expanded rapidly to form the dispersive disease spot, while, thedisease spot of HNAU-0023formed relatively obvious in the early stage, graduallybecome into a necrosis disease spot, suggesting that the necrosis may hinder thedevelopment of the disease further.2. The electron microscopy microstructure observation showed that there was nocallose accumulation around the cell walls of the susceptible line IL112and P.cubensis appeaered in the host cells, in contrast, a large amount of calloseaccumulation appeared around the infected cell in the resistant line HNAU-0023, tohinder P. cubensis enter cells.3. The DNA agarose gel electrophoresis showed that, the genomic DNAfragments of the susceptible line IL112were relatively complete; in comparision, thatof the resistant line HNAU-0023significantly degraded in gradient arrangement,suggesting programmed cell death occurred.4. The cell microstructure observations showed that the cucumber leaves under P.cubensis stress, the contents of H2O2(48to120hpi), callose (24to72hpi) and the incidence of H2O2, callose staining in the attacked cells showing a hypersensitiveresponse in the infected leaves by P. cubensis of HNAU-0023were higher than IL112and had rapidly growth. In a word, the hypersensitive response of cucumber generatedalong with the accumulation of H2O2and callose, confirming that the H2O2andcallose involved in the process of cucumber against P. cubensis.5. The H2O2(48to120hpi) and callose (24to72hpi) contents of the infectedleaves in the resistant line HNAU-0023increased gradually, instead, the application ofH2O2inhibitor DPI/DMTU (48~96hpi) and callose inhibitor DDG (0to72hpi) couldeffective inhibit the accumulation of H2O2and callose content in the correspondingperiods, resulting in the disease development of DPI/DMTU/DDG treatments fasterthan the controls. It further confirmed that the H2O2and callose involved in theprocess of cucumber against P. cubensis.6. EF1α and UBI-ep were as reference genes for the normalization of target geneexpression. To investigate the responses of the resistant genes associated to P.cubensis, their expression profiles were analyzed by real-time quantitative RT-PCR.As a result, compared with the susceptible line IL112, the defense genes PR-1, MT,PIP, Danj, CAT, the transcription factors TR, WRKY60, TF, GNRF, WRKY30and thesignal transduction gene MAPK1were up-regulated, suggesting that resistance genesin a certain period of time may be involved in the process of cucumber against P.cubensis.After the treatments of H2O2inhibitor DPI/DMTU and callose inhibitor DDG,the defense genes PR-1, PIP and MT, the transcription factors TR, TF, WARKY60,WRKY30, MYC and the signal transduction genes RBOH, MAPK1in the resistant lineHNAU-0023were significantly down-regulated than the controls, further confirmingthat these genes were involved in the process and maybe the key resistance-relatedgenes of cucumber against P. cubensis.