Chrysanthemum phloem necrosis: detection by epifluorescence microscopy R. J. MCGOVERN,' R. K. HORST,' AND H. W. ISRAEL Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. Received October 30, 1987 MCGOVERN, R. J., HORST, R. K., and ISRAEL, H. W. 1989. Chrysanthemum phloem necrosis: detection by epifluorescence microscopy. Can. J. Bot. 67: 1024-1031. In epifluorescence diagnostic procedures for chrysanthemum phloem necrosis, autofluorescence, aniline blue, and various nucleic acid "specific" fluorochromes such as 33258-Hoechst, 4',6-diamidino-2-phenyl-indole-dichlorid berberine sulfate, ethidium bromide, and acridine orange were utilized. Increased fluorescence in foliar phloem correlated precisely with gross symptoms of chrysanthemum phloem necrosis by all techniques and was used, in the case of berberine sulfate, to facilitate detection of mycoplasmalike organisms by transmission electron microscopy. MCGOVERN, R. J., HORST, R. K., et ISRAEL, H. W. 1989. Chrysanthemum phloem necrosis: detection by epifluorescence microscopy. Can. J. Bot. 67 : 1024- 1031. Dans des procCdCs de diagnostique en Cpifluorescence de la nCcrose du phlobme de chrysanthkme, les auteurs ont utilisC I'autofluorescence, le bleu d'aniline, et differents fluorochromes spCcifiques i des acides nuclCiques tels que le reactif 33258 de Hoechst, le dichlorure de diamidino-4',6 phCnyl-2 indole, le sulfate de berbCrine, le bromure d'kthidium, et I'acridine orange. I1 y a une corrClation Ctroite entre l'augmentation de la fluorescence dans le phlobme foliaire et les symptBmes gCnC- raux de la nCcrose du phlokme chez les chrysanthbmes, quelle que soit la technique utilisCe; I'usage du sulfate de berbCrine a de plus facilitC, en microscopie Clectronique par transmission, la dCtection d'organismes ressemblant i des mycoplasmes. [Traduit par la revue] Introduction Chrysanthemum phloem necrosis (CPN) is a newly reported disease (20) associated with mycoplasmalike organisms (MLOs), and characterized symptomatically by flecking, yellowing, and premature senescence of lower leaves of the Dendranthema grandiflora Tzveler. (syns. D. rnorifolia Ramat., Chrysanthemum morifolium Ramat.) (2) Marble culti- var group (13, 14, 18, 19, 20). As yet no detection procedures, usually difficult for MLOs, have been reported for CPN. Although CPN can be transmitted via grafts and dodder to plants used for bioassays, these procedures are time consuming and often inefficient. Fluorescence microscopy has been used on several occasions to demonstrate infection of plants by MLOs (34), as well as to describe early cytopathological effects of MLO-induced disease (5). Fluorochromes specific to deoxyribonucleic acid (DNA) have proven useful in detection of mycoplasma contamination of cell cultures and in localiza- tion of phytopathogenic MLOs (34). It was the purpose of this work to assess the use of these techniques both for routine diagnosis of CPN and for screening MLO-infected specimens prior to viewing with electron microscopy. Portions of this study have appeared in abstract form (20, 21, 22). Materials and methods Tissue sampling and preparation Petioles, midveins, and secondary veins were selected from leaves of Dendranthema grandijlora Tzveler. (syns. D. rnorifolia Ramat., Chrysanthemum morifolium Ramat.) (2) 'Pink', 'White', and 'Blue Marble' naturally affected by CPN, or from leaves of chrysanthemum 'Bonnie Jean' and 'Cornell Sparkler' ('Illini Sparkler' x 'Illini Sparkler') grafted to 'Pink Marble'. Stems and similar leaf pieces were also sampled from Madagascar periwinkle, Catharanthus roseus (L.) G. Don., infected with agents of ash decline or elm yellows, diseases of presumed MLO etiology (17, 36). Controls consisted of physiologically comparable leaves from uninfected chrysanthemum 'Present address: Frank's Nursery and Crafts, 6501 East Nevada, Detroit, MI 48234, U.S.A. 'Author to whom all correspondence should be addressed. Prinlcd in Canada 1 Imprimf. au Canada 'Bonnie Jean' and 'Cornell Sparkler', and uninfected Catharanthus roseus. An additional chrysanthemum control included leaf pieces from 'Bonnie Jean' infected with chrysanthemum stunt viroid (CSV). In some instances all leaves on entire shoots of chrysanthemums were sampled. Fixed and unfixed tissue specimens were prepared by pro- cedures outlined in a companion paper by McGovern et al. (21). Periwinkle plants and some chrysanthemums were grown in Cor- nell Peatlite (23) in the greenhouse at 18-38OC. Most chrysanthe- mums were grown in the same medium in controlled environment chambers at a constant temperature of 32°C. A photoperiod of 16 h was used for growth of chrysanthemum to prevent floral initiation. Microscopic procedures Unless otherwise indicated, all stains were dissolved in distilled water, or in 0.1 M phosphate buffer, pH 7.0; staining was done at room temperature. Following staining specimens were rinsed and mounted in distilled water or PO, buffer and viewed with eplifluores- cence optics. Epifluorescence microscopy utilized a Zeiss photo- microscope I1 with a mercury vapor lamp (Zeiss HBO 100). Epifluorescence microscopy protocols are listed in Table 1. Kodak Ektachrome 400 photographic film was used to obtain color trans- parencies from which black and white prints were made indirectly. Thin sections of epoxy-embedded material (32) were examined with a Philips EM 200 transmission electron microscope (TEM). Autojluorescence Primary fluorescence, or autofluorescence, of leaf vascular tissue was examined using a UG1 exciter, 41 banier filter, and dichroic mirror 420. Tissue ~utofluorescence was also routinely verified with the filter combinations usesd for all fluorochromes prior to staining, to differentiate secondary from primary fluorescence. Aniline blue - Sirojluor. Specimens were stained in 0.1 mg/mL aniline blue in 0.06 M K,PO, buffer, pH 8.0, after the methods of Eschrich and Cumer (9), or were mounted directly in a 0.03 mg/mL aqueous solution of Siro- fluor, 4',4-(carbonyl bis(benzine- 4,l-diyl) bis(imino)) bis-benzene- sulfonic acid (Biosupplies Australia, c/o B A Australia, 140 William St., Melbourne, Victoria 3000, Australia). Sirofluor is a synthesized aniline blue fluorochrome that has shown a high specificity in aqueous solution for 0-(1-3)-glucans such as callose (33). Nucleic acid "speci~c"jluorochrornes. Specimens were stained for 20 min in 0.1 - 1.0 g/mL solutions of 33258-Hoechst (H-stain), 2-(2-(4-hydroxypheny1)-6-benzimida-