Browsing by Subject "Nested PCR"

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    Detection of the Oak Wilt Pathogen in Sapwood of Northern Oak Species Using PCR Technology
    (2015-06) Yang, Anna
    Early and accurate diagnosis of oak wilt, caused by Ceratocystis fagacearum, is important when disease control action is planned. When laboratory diagnosis is needed, standard isolation protocols that are used rely on high quality samples and require up to 14 days for incubation. New methods, involving the use of polymerase chain reaction (PCR) have the potential to offer faster and more reliable plant disease diagnostics. The purpose of this thesis was to 1) compare the use of a nested PCR and real-time PCR protocol to detect C. fagacearum in sapwood drill shavings using published isolation methods and 2) develop standard sampling and processing guidelines for oak wilt diagnostics using the protocols, and 3) test and evaluate the use of the protocols in operational plant disease diagnostic clinics. All assays were performed on samples obtained from Quercus rubra/Q. ellipsoidalis (red oak), Q. macrocarpa (bur oak), and Q. alba (white oak) trees in Minnesota. Drill shavings were collected from actively wilting oak trees as well as from tissues taken the year following branch or whole tree death. Between the assays tested (nested PCR, real-time PCR, and isolation), the nested PCR assay resulted in the highest estimated probability of C. fagacearum detection for branch segments sampled from actively wilting oak trees. For bur and white oak branches that had been dead for >= 1 year, the pathogen was detected using nested PCR (55 and 87% of branch segments, respectively) and real-time PCR (19%, white oak only), while isolation was not able to detect the pathogen in any branch segments. All three detection methods were able to detect the fungus in main stem samples of red oaks with a streaking cambium, however, the molecular protocols resulted in more frequent pathogen detection. Only the molecular assays detected the pathogen in sapwood samples underlying the remnants of C. fagacearum sporulation mats on main stem samples of red oak trees. The fungus was not detected by either technique in branch segments obtained from healthy oaks (controls). Inter-laboratory testing of the nested PCR and real-time PCR protocols supported the previously described differences in assay reliability. Overall, the comparison of traditional isolation and nested PCR by the University of Minnesota Plant Disease Clinic resulted in the same number of trees testing positive for the pathogen by both detection methods. A comparison of isolation methods and real-time PCR was completed by the University of Wisconsin-Madison Plant Disease Diagnostic Clinic, which resulted in a higher rate of detection using isolation methods.