A New Disease in Limonium Hybrids. I. Molecular Identification
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HORTSCIENCE 39(5):1056–1059. 2004. carborundum. These plants were maintained in the greenhouse for one month and observed for symptom development. A New Disease in Limonium hybrids. I. Electron microscopy. Crude plant extracts in 0.1 M phosphate buffer were negatively Molecular Identifi cation stained with 20 g·L–1 uranyl acetate and examined for virus in a electron microscope 1 A. Gera, L. Maslenin, and A. Rosner (model 100CX II; Joel Ltd., Tokyo, Japan). Department of Virology, Agricultural Research Organization, The Volcani For ultrathin sections, pieces of petioles and/or Center, Bet Dagan 52050, Israel leaf veins (1 to 2 mm2) excised from healthy and symptomatic leaves of Limonium hybrids, M. Zeidan were fi xed in 2.5% glutaraldehyde buffered The Plant Protection and Inspection Services, Ministry of Agriculture, Bet with 75 mM potassium phosphate, pH 7.0, for Dagan 50250, Israel 2 h. The samples were then rinsed in the same buffer and postfi xed in 1% OsO4 for 3 h. After S. Pivonia a buffer rinse, the samples were dehydrated Northern Arava Research and Development, Sapir, D.N. Arava 86825, Israel in a graded acetone series, embedded in an Epon-Araldite resin mixture, and polymerized P.G. Weintraub as described by Orion and Franck (1990). Department of Entomology, ARO, Gilat Research Center, D.N. Negev 85280, Sectioned material was stained with uranyl acetate followed by lead nitrate and examined Israel by electron microscopy. Additional index words. polymerase chain reaction, elm yellows, almond witches broom, Polymerase chain reaction and sequence phytoplasma, Limonium latifolium analysis. DNA was prepared from leaf midribs and petioles as described by Tanne et al. (2000). Abstract. Yellows diseases in ornamental crops have become more common in Israel, DNA fragments were amplifi ed using the uni- and phytoplasmas have been detected in several crops. Recently, symptoms typical of versal phytoplasma primers P1/P7 (Schneider a phytoplasma infection were observed on a large number of Limonium hybrids grown et al., 1993). The product was further amplifi ed in commercial fi elds in Israel. Examination of samples from diseased plants by electron by nested PCR with the r16SF2/r16SR2 and microscopy revealed the presence of pleiomorphic membrane-bound bodies in the phloem fU3-fR5 primer pairs (Lee et al., 1995). The cells. Diseased plants were additionally analyzed by PCR using universal and nested primers DNA template in all PCR assays was 20 ng in and revealed upon sequencing products of 1143, 788 and 722 bp (Li-IL, Li-b2-IL, Li-v3-IL, a 50 µL assay. Standard PCR conditions were respectively). Analysis of the PCR products, associated with infected Limonium, clustered as in Tanne et al. (2000). Amplifi ed samples within two major groups of phytoplasmas (16SrV and 16SrIX), elm yellows and almond were electrophoresed in a 1.2% agarose gel, witches broom. This is the fi rst published record of these phytoplasmas in Israel. stained with 0.5 (g·mL–1 ethidium bromide, and photographed under UV illumination (Sambrook et al., 1989). DNA extracted from Although many species of Limonium Only L. latifolium hybrids were observed to symptom-free Limonium and naturally infected (Plumbaginaceae Juss.), herbaceous peren- be infected. Symptoms included: small and/or periwinkle plants served as negative and posi- nials, are native to the Mediterranean region, deformed/discolored fl owers; small, narrow tive controls, respectively. seedlings for commercial production are usu- basal leaves, often yellow in color; excessive Amplifi ed products from several Limo- ally imported from Europe, North America or leaf growth (witches broom or ‘asparagus nium samples were cleaned using QIAquick Asia. Two species and two hybrids are grown fern’); and eventual plant death. Over the course purifi cation kit (Qiagen, Hildon, Germany) in commercial production in the Arava valley of that season up to 60% of the plants were and sequenced by the dideoxynucleotide (located between the Dead Sea and Red Sea); affected in some areas. Symptoms resembled chain termination method, using r16SF2 or these include Limonium altaica (‘Emile’), L. those caused by phytoplasma infections. fU3 primers at the Weizmann Institute of sinuatum (annual statice), L. latifolium x L. The present paper reports the outbreak of Science, Rehovot, Israel. Sequence analysis caspium (‘Beltlaard’), and L. latifolium x L. bel- phytoplasma in Limonium hybrids grown in was performed with the DNAMAN pack- lidifolium (‘Misty’, ‘Supreme’ and ‘Sunglow’). commercial fi elds in Israel and the molecular age, version 4 (Lynnon Biosoft, Canada). In any one year there are a total of ≈70 ha; identifi cation and phylogenetic relationships Phytoplasma-related sequences were initially ≈15 ha are in L. sinuatum production and the of diverse phytoplasmas associated with the identifi ed using the BLASTN search program remainder in L. altaica and Limonium hybrids. disease. of the GenBank (Altschul et al., 1990). The These plants are grown primarily for export to nucleotide sequence of the phytoplasma Europe as cut fl owers for fl oral arrangements. Materials and Methods infecting Limonium was compared to the fol- Flowers are grown for 1 to 10 years in tunnels lowing: almond witches’ broom phytoplasma (7 × 100 m) covered with plastic; both ends are Mechanical inoculation. Leaf samples of (AlmWB1-4, AF390136-9), american aster open and ventilation holes are cut about every 2 Limonium grown in commercial plastic tunnels yellows phytoplasma (AAY, X68373), austra- m. For the fi rst 15 years of commercial produc- were examined for virus particles using elec- lian grapevine yellows phytoplasma (AUSGY, tion in the Arava, the crops were not affected tron microscopy and mechanical inoculation. L76865), aster yellows phytoplasma (AY1, by yellows disease. In October 2000 diseased Symptomatic samples were homogenized in AF322645), aster yellows phytoplasma (SAY, plants were fi rst observed in the northern Arava. 1% K2HPO4, and the sap was inoculated to AJ271323), Candidatus fraxini phytoplasma Capsicum annum L., Chenopodium quinoa (AshY1, AF092209); Candidatus fraxini Received for publication 23 June 2003 Accepted Willd., C. amaranticolor, Cucumis sativus, phytoplasma (AshY, X68339); Candidatus for publication 24 Jan. 2004. Contribution from the Cucurbita pepo, Datura stramonium, Gom- fraxini phytoplasma (AshY1-NY, AF092209); Institute of Plant Protection, Agricultural Research phrena globosa, Lycopersicon esculentum, Candidatus fraxini phytoplasma (AshY3, Organization, The Volcani Center, Bet Dagan, Nicotiana benthamiana Domin, N. clevelandii AF105315); Candidatus fraxini phytoplasma Israel, No. 517/02. This research was supported in Glay, N. glutinosa, N. tabacum ‘NN’, N. taba- (AshY5, AF105316); clover yellow edge part by a grant from the Chief Scientist, Ministry cum ‘Samsun’, N. tabacum ‘Samsun NN’, N. phytoplasma (CYE-C, AF175304), clover yel- of Agriculture, State of Israel. tabacum Xanthi nc, N. rustica, N. sylvestis, low edge phytoplasma (CYE-Or, AF189288), 1 Corresponding author; e-mail abedgera@volcani. N. tabacum ‘White Burley’, Petunia hybrida Clover yellow edge phytoplasma (CYE-L, agri.gov.il. and Physalis fl oridensis Rybd., predusted with AF173558), elm yellows phytoplasma (EY- 1056 HORTSCIENCE VOL. 39(5) AUGUST 2004 organism (PPER, X68374), mol- indicated that all Limonium plants expressing licutes from V. myrtillus (VAC, symptoms were infected by phytoplasma, while X76430), mollicutes from C. symptom-less plants were phytoplasma-free roseus (STOL-Cros, X76427), (Fig. 3). The PCR fragments, 1143, 788 and mollicutes from V. vinifera (VK, 722 bp of several clones, designated Li-IL, X76428), phytoplasma sp. (LfY5, Li-b2-IL, Li-v3-IL, respectively, were directly AF500334); Paulownia witches’ sequenced using r16SF2 or fU3 primers. Nu- broom phytoplasma (PauWB, cleotide sequencing of Li-IL amplifi ed product AF279271 ), peanut witches’ have shown 99.4% identity with EY-Ar1, Li-b2- broom phytoplasma (PnWB, IL product 83% identity with BLL-Bd-Brinjal AY139868), rice yellow dwarf and Li-V3-IL 91.4% with PPWB phytoplasmas phytoplasma (RYD, AY139873), respectively. A comparison with 53 related stolbur phytoplasma (STOL-Lily, sequences from the GenBank confi rmed the AY169309), stolbur phytoplasma classifi cation of these phytoplasmas associated (STOL, AF248959), sweetpotato with Limonium disease to two major groups witches’ broom phytoplasma (16SrV and 16SrIX), elm yellows and almond (SPWB, AY139866), Stylos- witches’ broom, respectively. Although, Li-IL anthes little leaf phytoplasma and Li-b2-IL were classifi ed to the same group (SLI-AUS, AJ289192); tomato they exhibit only 82.8% identity of nucleic big bud phytoplasma (BB, acid sequence. AF222064), tsuwabuki witches’ broom phytoplasma (TWB, Discussion D12580), Ziziphus jujube witches broom phytoplasma (JWB-Ch, A review of the literature revealed that AF305240); Ziziphus jujube phytoplasma infections are known from only witches broom phytoplasma a few species within the Plumbaginaceae: L. (JWB-Ko, AB052879); loofah sinuatum in Korea (Hahm et al., 1998), Japan witches-broom phytoplasma (Shiomi et al., 1999), Poland (Kaminska et al., (LfWB1, AF353090); loofah 1999), and Lithuana (Valiunas et al., 2001), witches-broom phytoplasma and Goniolimon tataricum (syns. Limonium (LfWB2, AY139871); phyto- tataricum) in Canada (Chang et al., 1996). plasma sp. LfY5-Oaxaca (LfY5, Although L. sinuatum is grown in the same area AF500333); western X phyto- of the Arava, and even immediately adjacent to plasma