The saskatoon (Amelanchier Alnifolia Nutt.) is a woody shrub from the rose family. One of the main challenges to its culture is Entomosporium leaf and berry spot (ELBS) disease, which is caused by a hemibiothrophic fungus Entomosporium mespili (Sacc.). Due to the limited growth of the pathogen in vitro, we tested several culture media, and potato dextrose agar remained the best tested medium to grow the pathogen in vitro. E. mespili showed high variations in the morphology and sporulation of its cultures on PDA, which were not related to the geographic origin of the tested isolates. The amount of spores produced by the fungus in vitro was significantly high compared to its sporulation in nature. The use of liquid media such as potato broth was essential to produce enough mycelium for other experiments. The pathogen produced big spherical balls of mycelium where the nutrient and the aeration were sufficient. In absence of previous protocols to extract DNA from this pathogen, we tested several methods and determined a mixture of CTAB and SDS to be best for extraction of good quality DNA from E. mespili grown in vitro. Lyophilisation of E. mespili’s tissue and the addition of proteinase K to the extraction buffer reduced DNA degradation. To avoid the co-precipitation of polysaccharides with DNA, we used a high concentration of salt to precipitate polysaccharides, followed by a separation by phenol:chloroform:isoamyl (25:24:1). Molecular markers including Random Amplified Polymorphic DNA (RAPD) and Amplified Fragments Length Polymorphism (AFLP) revealed a high level of polymorphism in E. mespili. The artificial inoculation gave different results from natural infection, with a high level of diversity both on detached leaves and seedlings. According to our tests, growing E. mespili in vitro reduced its pathogenicity.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:MWU.1993/19449 |
| Date | 23 April 2013 |
| Creators | Naoui, Abdelwahed |
| Contributors | Daayf, Fouad (Plant Science), Piercey-Normore, Michele (Biological Science) Rashid, Khalid (Plant Science) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Detected Language | English |
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