Instituto de Fitopatologı´a y Fisiologı´a Vegetal-INTA, Co ´rdoba, Argentina Molecular Characterization of 16S Ribosomal DNA and Phylogenetic Analysis of Two X-disease Group Phytoplasmas Affecting China-tree (Melia azedarach L.) and Garlic (Allium sativum L.) in Argentina E. E. Galdeano Galdeano 1 , L. E. L. E. Torres Torres 2 , N. N. Meneguzzi Meneguzzi 3 , F. F. Guzman Guzma ´n 3 , G. G. G. G. Gomez Gomez 2 4 , D. M. D. M. Docampo Docampo 3 and and L. R. L. R. Conci Conci 3 Authors’ addresses: 1 Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Corrientes, Argentina; 2 Facultad de Ciencias Agropecuarias, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina; 3 Instituto de Fitopatologı´a y Fisiologı´a Vegetal-INTA, Camino 60 cuadras Km 5 1/2 (X5020ICA), Co´ rdoba, Argentina; 4 Instituto de Biologı´a Molecular y Celular de plantas, Universidad Polite´cnica de Valencia, Espan˜a (correspondence to L. R. Conci. E-mail: lconci@correo.inta.gov.ar) With 5 figures Received September 12, 2003; accepted December 5, 2003 3 Keywords: 16S rRNA gene, phytoplasma classification, phylogeny, nucleotide sequence analysis, garlic decline, China-tree decline Abstract Two phytoplasmas closely related to the X-disease group were associated with China-tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China-tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China-tree and garlic decline phytoplasmas to the 16SrIII (X-disease group), sub- groups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witchesÕ broom phytoplas- ma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China-tree decline, Chayote witchesÕ broom and Clover yellow edge phytoplasmas into a cluster separ- ated from the other phytoplasmas of the X-disease group. Introduction China-tree (Melia azedarach L.) has been introduced in South America from Asia for ornamental and industrial purposes. Two M. azedarach varieties are grown in Argentina: var. gigantea, which is commer- cially used for furniture manufacturing and has reached a forested area of 12 000 ha; and var. umbra- culifera, which is mainly grown for gardening and landscaping. In the late 1970s, the first symptomatic trees were seen, and Va´zquez et al. (1983) described the China-tree decline (declinamiento del paraı´so) as a mycoplasma-like disease. During the first stages of the disease, a few branches show symptoms of leaf yellowing and size reduction, shoot proliferation and internodes shortening. After a few years, the symptoms spread all over the tree until it finally dies. From the time it was first detected, the growing importance of the disease has brought about a decrease in the expansion of the forested area of China-tree, and previously implanted forests have been seriously damaged (Dalton et al., 1991). The association of the China-tree decline disease with a phytoplasma was later confirmed by polymerase chain reaction (PCR) with universal primers (Go´mez et al., 1996). A polyclonal antiserum, produced from infected China-tree plants, offered a rapid and simple tool to detect affected plants and to associate this pathogen with symptoms in other important crops (Go´mez et al., 1996). The antiserum, when used at a higher concentration, detected a phytoplasma in garlic (Allium sativum L.) plants that showed symptoms of decline (Conci et al., 1998). The presence of a wall-less pathogen within phloem cells has been established also by electron microscopy (EM) observation of ultra-thin sections from symptomatic garlic plants (Conci et al., 1992). The affected garlic plants show leaf reddening or yellowing, depending on the garlic variety, followed by a drought-stress appearance, general decline and plant death. These plants hardly produce bulbs or do not bear normal cloves. Because of these symptoms, the garlic decline has been commonly known in Argentina as Ôtristeza del ajoÕ (Conci et al., 1998). U. S. Copyright Clearance Centre Code Statement: 0931–1785/2004/1523–0174 $ 15.00/0 www.blackwell-synergy.com J. Phytopathology 152, 174–181 (2004) Ó 2004 Blackwell Verlag, Berlin ISSN 0931-1785