Journal of Arboriculture 31(5): September 2005 261 PCR methods indicate that tissue sampled from the tree canopy is most reliable for routine diagnostic work. In both tests, apparently healthy samples from the canopy yielded the highest percentage of positive tests. Testing of diseased tissue and of witches’ broom shoot proliferations was superior to sampling from the root system. Comparison of DAPI and PCR Tests The DAPI staining technique was once a standard in phytoplasma detection and diagnosis (Douglas 1986; Chen et al. 1989; Sinclair et al. 1989, 1992). This test has been limited in its applications due primarily to low titers of phytoplasma particles. One of the polymerase chain reaction’s most attractive features is its ability to detect DNA from organisms even when at extremely low concentrations. Our results indicate that using PCR for routine detection and diagnosis of phytoplasma infection is indeed superior to the DAPI stain test. This is especially true for asymptom- atic individuals and in tissue where titers are low. CONCLUSIONS AND RECOMMENDATIONS Ash decline (AD) is a progressive disease of ash caused by a phytoplasma (Bricker and Stutz 2004). The causal agent of AD, as documented in this paper, has been found to infect several different types of ash species and cultivars. Interest- ingly, susceptibility to AD varies according to host type. This information can be used when selecting material for the landscape setting. Modesto ash is not recommended for the urban landscape, while a resistant species such as Fraxinus uhdei would be a more appropriate choice, especially in the Phoenix and Tucson, Arizona, metropolitan areas. Finally, the unequal distribution of phytoplasma particles should be taken into consideration when collect- ing tissue samples to diagnose AD. The results of both the DAPI and PCR methods indicate that tissue sampled from the tree canopy is most reliable for routine diagnostic work. Apparently healthy samples from the canopy yielded the highest percentage of positive tests. Testing of diseased tissue and of witches’ broom shoot proliferations was superior to sampling from the root system. LITERATURE CITED Ahrens, U., and E. Seemüller. 1992. Detection of DNA of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832. Bricker, J.S., and J.C. Stutz. 1992. Etiology of Arizona ash decline (Abstr.). Phytopathology 82:1170. ———. 2004. Phytoplasmas associated with ash decline. J. Arboric. 30(3):193–199. Castello, J.D., S.B. Silverborg, and P.D. Manion. 1985. Intensification of ash decline in New York State from 1962 through 1980. Plant Dis. 69:243–246. Cha, B., and T.A. Tattar. 1991. Symptom development of ash yellows and fluctuation of mycoplasmalike organism population in white ash (Fraxinus americana L.). Arboric. J. 15:323–343. Chen, T.A., D. Lei, and C.P. Lin. 1989. Detection and identification of plant and insect mollicutes, pp. 393– 424. In Whitcomb V. R.F. and J.G. Tully (Eds.). The Mycoplasmas. Academic Press, New York, NY. Douglas, S.M. 1986. Detection of mycoplasmalike organisms in peach and chokecherry with X-disease by fluorescence microscopy. Phytopathology 76:784–787. Han, Y., J.D. Castello, and D.J. Leopold. 1991. Ash yellows, drought, and decline in radial growth of white ash. Plant Dis. 75:18–23. Kuske, C.R., and B.C. Kirkpatrick. 1992. Distribution and multiplication of western aster yellows mycoplasmalike organisms in Catharanthus roseus as determined by DNA hybridization analysis. Phytopathology 82:457–462. Luley, C.J., M.E. Mielke, J.D. Castello, J. Cummings Carlson, J. Appleby, and R. Hatcher. 1992. Ash crown condition and the incidence of ash yellows and other insects and diseases in Illinois, Iowa, Missouri, and Wisconsin. Plant Dis. 76:1209–1212. Schneider, H. 1977. Indicator hosts for pear decline: Symptomatology, histopathology, and distribution of mycoplasmalike organisms in leaf veins. Phytopathology 67:592–601. Seemüller, E., L. Junze, and U. Schaper. 1984a. Colonization behavior of MLO and symptom expression of proliferation-diseased apple trees and decline- diseased pear trees over a period of several years. Z. Pflanzenkrankh. Pflanzenshutz 91:525–532. Seemüller, E., U. Schaper, and F. Zimbelmann. 1984b. Seasonal variation in the colonization patterns of mycoplasmalike organisms associated with apple proliferation and pear decline. Z. Pflanzenkrankh. Pflanzenshutz 91:371–382. Sheta, M.H. 1988. Focus. Plant Dis. 72:4. Silverborg, S.B., and E.W. Ross. 1968. Ash dieback development in New York state. Plant Dis. Rep. 52:105– 107. Sinclair, W.A., Griffiths, H.M., and M. Treshow. 1994. Ash yellows in velvet ash in Zion National Park, Utah: High incidence but low impact. Plant Dis. 78:486–490. Sinclair, W.A., H.M. Griffiths, R.E. Davis, and I.-M. Lee. 1992. Detection of ash yellows mycoplasmalike organisms in different tree organs and in chemically preserved specimens by a DNA probe vs. DAPI. Plant Dis. 76:154–158. Sinclair, W.A., R.J. Luli, A.T. Dyer, and A.O. Larsen. 1989. Sampling and histological procedures for diagnosis of ash yellows. Plant Dis. 73:432435. ©2005 International Society of Arboriculture
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