| R. setate×R. rugosa, a natural hybrid orgined from Rosa sertata Rolfe×Chinese traditional Rosa rugosa Thunb., has been cultivating in Kushui town Yongdeng county Gansu province at least 200 years. It is one of the major spice crops in Gansu province and the production of essiental oil accounts for more than 80% in Chinese.With the rapid increasing in market demand, the economic and social value becomes increasingly prominent, which results in the plant being a special economic crop with the purpose of perfumery, pharmaceutical, brewing,etc. The special economic value prompting a sharp increase in cultivation, which leads to extensive management and intercropping with other crops that cause the spread of pests and diseases, ultimately, severely reduced the production. In recent years, there is a high incidence of leaf spot on the plant in the field and displaying a more serious condition. In order to ascertain the impact of leaf spot on quality of R. setate×R. rugosa, reveal the mechnasim of leaf spot pathogen infecting the plant, the experiment was conducted on the impact of leaf spot on bioactive substances accumulation and physiological and biochemical changes, pathogen separation and identification, and pathogen prevention, etc. The results are as follows:1. The bioactive substances in R. setate×R. rugosa at flowering season were separated and detected by the methods of GC-MS, spectrophotometry, DPPH and FRAP, etc, and the results presented that 20 components were significantly affected by the leaf spot, 8 of them increased to some extent, while 8 components decreased. For example, the relative content of citronellol,linalool and 3-methyl-1-butanol decreased by 62.83%, 63.08% and 60.18%, respectively,compared to the control. With the increased levels of leaf spot, the content of soluble sugar,total flavonoids and phenolic compounds were significantly reduced, with the level of Ⅱ andⅢ grades compared to the control decreased by 17.64% and 28.72%, 49.15% and 76.10%,46.60% and 67.90%, respectively. The antioxidant capacity also significantly reduced with I%,FRAP values decreased by 57.24% and 73.45%, 53.64% and 74.80%, respectively.2. The changes in physiology and biochemistry of R. setate×R. rugosa at flowering season were detected by the methods of HPLC, q PCR, etc, and the results displayed that there was a significant reduction in net photosynthesis, stomatal conductance, intercellular CO2 concentration, and chlorophyll content with the increased levels of leaf spot.In contrast,transpiration rate, antioxidant enzymes SOD and PPO significantly increased.In addtion, there was a a significant increase in the key enzyme activity of PAL involved in secondary metabolism, and in over-expression of key genes PAL and CHS, wherein the expression of PAL,CHS in Ⅱ and Ⅲ grades compared to control were increased by 1.8- and 4.41-fold, 2.67-and4.23-fold, respectively.3. After isolating and identifying the leaf spot pathogen and screening the Fungicides, the finding revealed that the leaf spot pathogen belonged to Alternaria sp with the premary identifaction by Morphology and Pathogenicity Assay.The mycelium DNA was sequently amplifed by the primer of ITS1F-ITS4, sequence analysis, homology alignments and Phylogenetic analysis, and confirmed that the leaf spot pathogen belonged to the variant of Pathogens Alternaria, Alternaria arborescens(JQ676197). The use of difenoconazole and tebuconazole could effectly inhibit the leaf spot pathogen with EC50 concentrations 0.0017μg/m L and 0.0041 μg/m L, respectively.The above studied results demonstrated that leaf spot stimulated the key enzyme activity and over-expression of key genes related to secondary metabolism, reduced the accumulation of the bioactive substances, and seriously affected the quality of R. Setate×R. Rugosa. The leaf spot pathogen belonged to the variant of Alternaria arborescens(JQ676197), which could be effectly inhibited by using of difenoconazole and tebuconazole. Those research results would provide a theory foundation and technological support for large-scale cultivating, pathogen controlling of R. setate×R. rugosa. |
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