| Pulsatilla spp. is one of the most important Chinese traditional medical plant in Liaoning Province with upper economic and medical value., With the adjustment of Agricultural Structure in Liaoning province, the wild windflower of artificial cultivation caused by more attention by people. We found that disease of windflower became more and more serious, windflower leaf spot is one of the most serious diseases. The disease caused by Ascochyta anemones Kab.et Bub. and the system research has not been reported at home and abroad. For this reason, pathogen identification, the genetic differentiation, pathogenic mechanism, infection characteristics and epidemic regulation of Ascochyta anemones were systematically studied. The results were as follows:1. Identification of pathology of windflower leaf spot. The samples which were collected from planting areas of windflower in Shenyang Agriculture University herbs garden, Yingemen Town of Qingyuan County, Xiajiapu Town of Qingyuan County from2010to2011were isolated. Based on Koch’s Rule and ITS sequence analysis, it is determined that the windflower leaf spot is caused by the fungi of Ascochyta anemones.2. The teleomorph of Ascochyta anemones has been discovered for the first time on overwintering windflower. Large numbers of documents indicated that the teleomorph is an undescribed species of Didymella.3. Genetic diversity of Ascochyta anemones of windflower leaf spot was analyzed from DNA molecular level for the first time, which showed rich polymorphic loci. RAPD and ISSR were used to explore genetic diversity of Ascochyta anemones from different localities in Liaoning,11RAPD primers and8ISSR primers were screened obtained108and95polymorphic DNA bands respectively. The result of NTS YS cluster analysis showed that the genetic similarity coefficient of RAPD among the30Ascochyta isolates was from0.56-0.98and these strains were divided into4genetic groups when the similarity coefficient was0.622. The result also showed that the genetic similarity coefficient of ISSR among these isolates was from0.58-1and these strains were divided into4genetic groups when the similarity coefficient was0.635. The analysis results of RAPD and ISSR suggested that the pathogens of windflower leaf spot in Liaoning province had rich genetic diversity. There existed definite correations between the clustering groups and their geographic distribution, and an obvious genetic difference among the isolates from different host sources.4. The study defined the pathogenic mechanism of Ascochyta anemones preliminary. A list of C WDEs as PG, PMG, PGTE, PMTE, Cx and β-glucosidase were found in A anemones, but the producing enzyme conditions of them were different significantly. Stationary culture made more for the production of cell-wall degradation compared to the shake culture. The activities of PG and PMG reached a peak when cultured6d, while PGTE, PMTE, Cx and β-glucosidase reached a peak when cultured12d.èPG and PMG showed maximum activities at25℃. PGTE, PMTE,(3-glucosidase and Cx showed maximum activities at30℃. PG and PMG showed maximum activities at culture pH5. Acidity alkali sedimentation method was used to extract the melanin from A. anemones. The basic characteristic of melanin and its effect of pathogenicity were studied, suggesting that the melnin was a DHN melanin. A novel polyketide synthase gene, named AaPKS (GenBank accession number KC190500), was cloned from A.anemones using RACE method. The full length cDNA of AaPKS consists of6278bp, with a5853bp ORF encoding1950amino acid residues. The predicted secondary structure composition for the protein contained about35.38%alpha helix,14.21%extended strand,4.15%beta turn and46.26%random coil. Phylogenetic analysis indicated that AaPKS of A.anemones shares95%identity to Ascochyta rabiei.5. Study on the infection characteristics of Ascochyta anemones of windflower leaf spot and the primary source of infection of windflower were fist define. Histological studies of the artificially inoculated leaf tissues with Ascochyta anemones conidia were observed by light and electron microscopy. Conidial germination and germ tube foramtion began after4hours of inoculation. Each conidium produced one to germ tubes to peneatrate the host surface. The fungus gain entry of the host by direct penetration of the leaf cuticle, following the formation of appressorium after12-24h of inoculation. Most appressoria were formed in the grooves between adjacent epidermal cells. Once the fungus was fully established, it destroyed internal tissues, resulting in diseased lesions on the leaves after3-6days of inoculation. The test result of the host range of Ascochyta anemones demonstrated that the pathogen could infect strawberry, apple, tomatoes and peppers. In addition, strawberry had the hightest disease rate80%.6. The temporal dynamic models of windflower leaf spot were first established. According to systematic investigation during2010-2011, making regression analysis via SPSS software, logistic model was comfirmed as the best model. The model deduced that exponential phase of windflower leaf spot was from late May to mid June, the logistic phase was from mid June to mid August, the decline phase was mid August to the end of windflower grow stage in Shenyang.7. Studies on primary management of windflower leaf spot. The fungistatic action of seven fungicides to A.anemones was determined. Fungicide tests proved that azoxystrobin25%SC and flusilazole40%EC exhibited the best inhibitory effect on the colony growth which its EC50values reached3.1741mg/L and9.3088mg/L respectively. Field trials showed that the disease could be effectively controlled by using the procymidone50%WP which control effect reached80%, pyrimethanil40%SC and azoxystrobin25%SC also have better control effect. |
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