258 Bricker and Stutz: Host Range and Distribution of Ash Decline Phytoplasma having chlorotic symptoms, (3) branches with leaves having leaf tip and edge necrosis, (4) branches with leaves with “olive” dieback, and (5) branches with witches’ brooms. Root cuttings were collected on 13 September 1991. Duplicates were made of each sample for DAPI and PCR testing. The results of the DAPI tests and the PCR DNA amplification procedure were then compared in order to determine how suitable each was for routine phytoplasma detection. Stem samples were processed for both PCR and DAPI testing using the methods outlined in Bricker and Stutz (2004). Root samples were processed by removing dirt and debris from the tissue under running water, and removing the bark tissue for DNA extraction. RESULTS Phytoplasma Host Range in Arizona Symptoms of AD were found to occur throughout the state of Arizona. These symptoms included leaf tip or edge necrosis, chlorosis, and witches’ brooms. The survey of all the species and cultivars of ash trees in Arizona revealed that only the Modesto ash suffers from the advanced stages of AD (Table 1). Symptoms of AD in Modesto ash were observed in the Phoenix, Tucson, Wickenburg, and Miami-Globe areas, with the highest incidence of disease symptoms occurring in the Phoenix metropolitan area. AD symptoms were also observed in other ash cultivars and species but at a much lower frequency. Wild-type F. velutina ash trees were the only other ash trees, other than the Modesto ash, included within the statewide survey in which any individuals exhibited ad- vanced stages of AD (with greater than 50% of the canopy with dieback). Advanced AD symptoms were observed in one population of wild-type F. velutina trees growing along the Verde River in Clarksdale. Overall, however, the state- wide status of wild-type F. velutina was apparently healthy (Mean severity rating [MSR] = 1.21). Only a few (6%) of the Rio Grande ash trees surveyed in the Tempe area had symptoms of AD corresponding to class 2 (a few dead branches on the tree), indicating that the cultivar may be susceptible to AD. None of the evergreen ash (F. uhdei) trees included within the AD survey exhibited symptoms of AD. Comparison of DAPI and PCR Tests Using the DAPI staining technique, phytoplasmas were detected in all of the ash species and cultivars tested (Table 2). Positive DAPI tests were obtained from native Fraxinus velutina at seven of the eleven collection sites located across the range of the species in Arizona. Some plants did exhibit symptoms that might be due to AD, and, in 28.3% of these cases, a positive DAPI test was obtained (Table 2). A number of other individuals appeared completely healthy, but a positive DAPI test was obtained in 18.2% of those trees tested, thus indicating the presence of an phytoplasma infection. The overall rate of phytoplasma infection, as indicated by positive DAPI tests, in all samples of the native F. velutina, was 27.1% (Table 1). Phytoplasmas were detected using DAPI in 66.7% (Table 2) of the samples collected from symptomatic Fraxinus velutina cv. ‘Modesto’ in Arizona outside the Phoenix metropolitan area. For Modesto ash trees that were apparently healthy, a positive DAPI test was obtained in 27.3% of the trees tested. Modesto ash trees cultivated outside of the Phoenix metropoli- tan area yielded an overall 50% rate of infection as indicated the DAPI test (Table 1). The level of phytoplasma infection in the Modesto ash was found to be highest in the Phoenix metropolitan area, with 75.6% of the symptomatic trees and 68.7% of the asymptomatic trees testing DAPI positive (Table 2). The overall rate of phytoplasma infection in Modesto ash trees sampled and tested using DAPI in the Phoenix area was 76.9% (Table 1). A positive DAPI test was obtained in 38.9% of the F. uhdei trees and in 33.3% of the F. velutina cv. ‘Rio Grande’ samples tested (Table 1). Table 1. Mean severity ratings and DAPI test results for various ash trees growing in cultivation and the wild in Arizona that were surveyed for ash decline in fall 1992. Mean Species/cultivar Fraxinus velutina, wild type F. velutina ‘Modesto’ cultivated in Tempe F. velutina ‘Rio Grande’ cultivated in Tempe F. uhdei cultivated in Tempe ©2005 International Society of Arboriculture No. of trees Percentage with severity surveyed symptoms 57 108 F. velutina ‘Modesto’ cultivated outside Phoenix metropolitan area 26 50 50 19.3 100.0 57.7 6.0 0.0 *Mean severity rating = Σ class ratings ÷ number of individuals rated. rating* 1.21 4.10 2.27 1.06 1.0 Percentage DAPI positive 27.1 The DAPI staining method was performed on the 42 ash samples tested using PCR amplification. In 61.9% of these samples, similar (either both positive or both negative) DAPI staining and PCR results were obtained. The DAPI and PCR tests were not the same in 38.1% of the samples tested. For 19% (8 of 42) of the total samples, a positive DAPI test was obtained, while the PCR yielded no product. In 19% (8 of 42) of the samples, the DAPI test was negative, while a 560 bp fragment was obtained using PCR. Amplified PCR products 76.9 50.0 33.3 38.9 were obtained from six of the eight apparently healthy trees included in the study. Phytoplasmas had previously been detected in samples from one of the two negative PCR trees by repeated testing
September 2005
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