Molecular characterization of a phytoplasma of the ash yellows group (16Sr VII-B) occurring in Artemisia annua and Conyza bonariensis weeds

摘要

En Sudamérica se han detectado y caracterizado fitoplasmas de diferentes grupos 16Sr. Entre éstos, se detectaron fitoplasmas del grupo 16Sr VII (ash yellows) infec- tando diferentes especies de plantas herbáceas. En Brasil, se reportaron los aislamientos EriWB y RPWB infectando Erigeron sp. y Catharanthus roseus, los que fueron asignados al nuevo subgrupo 16Sr VII-B. En la Argentina, el subgrupo 16Sr VII-C fue creado para incluir al fitoplasma ArAWB que infecta alfalfa (Medicago sativa). Nuevos aislamientos de fitoplasmas del grupo 16Sr VII se han detectado en Artemisia annua y Conyza bonariensis en la Argentina, mostrando típicos síntomas de "escoba de bruja". El objetivo de este trabajo fue caracterizar molecularmente a estos aislamientos para clasificarlos y asociarlos a los fitoplasmas más relacionados. Se analizaron patrones de restricción (PCR-RFLP) del gen 16S de ARNr y se obtuvieron secuencias de tres aislamientos para realizar un análisis cladístico y poder determinar la similitud con otros fitoplasmas. Idénticos patrones de PCR-RFLP, la alta similitud de secuencias (99,9%) y el agrupamiento cladístico resultante asociaron a estos fitoplasmas presentes en malezas en Argentina (ArtWB), al subgrupo 16Sr VII-B. Estos aislamientos representan aislamientos geográficos del fitoplasma EriWB. Este trabajo revela la amplia distribución geográfica de los fitoplasmas del subgrupo 16Sr VII-B y su capacidad de infectar diferentes hospedantes. Palabras clave: Ash yellows fitoplasma; 16Sr VII; Distribución geográfica. Fecha de recepción: 07/12/07; fecha de aceptación: 27/05/08 INTRODUCTION Phytoplasmas are phytopathogenic wall-less prokaryotes belonging to the Class Mollicutes. They inhabit the phloem tissue of infected plants and cause hundreds of plant diseases in taxonomically unrelated hosts (Mc Coy et al., 1989). In nature, phytoplasmas are transmitted by hemipterans, mainly Cicadellidae, which feed on phloem sap (Tsai, 1979). The current classification is primarily based on the analysis of restriction patterns (PCR-RFLP) and nucleotide sequences of highly conserved genomic regions, such as the ribosomal RNA, ribosomal proteins and Tu elongation factor genes. By means of the analysis of restriction patterns of the 16S rRNA gene, fifteen phytoplasma "16Sr" groups have been established, these comprise more than 40 subgroups. This classification coincides, in general, with that resulting from the phylogenetic analysis of the gene sequences mentioned above (Lee et al., 1998; Seemüller et al., 1998; Lee et al., 2000; Lee et al., 2004). During the last years, phytoplasmas from different 16Sr groups have been detected and characterized in South America, these groups infect cultivated plant species (Montano et al., 2000; Galdeano et al., 2004; Conci et al., 2005; Jones et al., 2005; Galvis et al., 2007), ornamental species (Montano et al., 2001; Harrison et al., 2003; Torres et al., 2004) and weeds (Barros et al., 2002). Phytoplasma infections can produce severe symptoms in plants and can cause a rapid host death (Harrison et al., 1999; Padovan & Gibb, 2001) in some pathologies. Conyza bonariensis (L.) Cronquist (synonym: Erigeron bonariensis) and Artemisa annua L. are herbaceous species of the Asteraceae family which grow as weeds in many crops of Argentina. While C. bonariensis is a cosmopolitan species native of South America, A. annua comes from Asia and has naturalized throughout diverse regions of America and Europe (Zuloaga & Morrone, 1999). In recent years, phytoplasma infection was reported in several C. bonariensis and A. annua plants showing yellowing and witches'-broom symptoms (Torres et al, 2002). Infected plants have been found in peach orchards and vegetable crops in the province of Córdoba, where no phytoplasma infection was detected in the crops. C. bonariensis plants with the same severe symptomatology were also observed in alfalfa (Medicago sativa L.) plots in the province of San Juan. In this area, the alfalfa crop is affected by the ArAWB (16Sr VII-C) phytoplasma, which induces witches' broom symptoms (Conci et al., 2005). Diseased plants develop severe symptoms as crop ages and can reach a leafless stunted aspect, although up to the present there is no record of crop losses due to this pathology. Considering that infected weeds can be pathogens and vector reservoirs and, given the wide distribution of A. annua and C. bonariensis, it became necessary to study the etiological agent of the diseases observed in order to establish whether these species represent a potential risk for the crops. The objectives of the present work were to determine the causal agent of the observed symptomatology, and to identify and characterize the pathogen detected in A. annua and C. bonariensis plants collected from diverse ecological regions of Argen