Characterization of the Mycoplasmalike Organism Associated with Witches'-Broom Disease of Lime and Proposition of a Candidatus Taxon for the Organism, "Candidates Phytoplasma aurantifolia"

Abstract

Witches'-broom disease of small-fruited acid lime (WBDL) is a severe disease caused by a mycoplasmalike organism (MLO) in the Sultanate of Oman and the United Arab Emirates. The WBDL MLO was characterized by studying its genome size, the sequences of its 16s ribosomal DNA and the 16s-23s ribosomal DNA spacer region, and hybridization profiles obtained by using WBDL MLO-specific probes. The size of the WBDL MLO genome is 720 kbp. Genomic similarities with the MLOs of sunhemp, sesame, and alfalfa phyllodies were demonstrated, and we found that the WBDL MLO belongs to the sunhemp phyllody phylogenetic subgroup.Witches'-broom disease of small-fruited acid lime [Citrus aurantifolia (L.) Swingle] (WBDL) is a lethal disease which is caused by a mycoplasmalike organism (MLO) and appeared in the Sultanate of Oman in the late 1970s (2, 3, 7) and in the neighboring country the United Arab Emirates in 1989 (4, 7). The WBDL MLO was experimentally transmitted by dodder (Cuscuta campestris Yunker) to periwinkle (Catharantus roseus L.) plants, in which it induces characteristic symptoms different from those reported with other MLOs (7). The WBDL MLO is thought to be naturally spread by the leafhopper Hishimonus phycitis, as many WBDL MLO-infected H. phycitis individuals were collected from lime trees with WBDL in Oman (4). However, experimental transmission of the disease with H. phycitis has not been achieved yet. Widely spread in India, I-€. phycitis was not reported from the Arabic Peninsula (5) before we found it there (4). As lime trees have been grown in Oman for centuries, it is likely that the disease started with the introduction of this new insect vector, probably from India, where it is known to transmit the MLO of eggplant little-leaf disease (1). The MLO agent, however, was probably indigenous to the Sultanate of Oman since the disease has never been described in India or elsewhere.In 1991, monoclonal antibodies and DNA probes specific for the WBDL MLO were produced (7). No serological relationships with any of the other MLOs tested, including the eggplant little-leaf disease MLO from India, were detected, but in this paper we show that DNA extracted from phyllody MLOinfected sunhemp, sesame, and alfalfa plants hybridized with the DNA probes which we tested.In order to determine the phylogenetic position of the WBDL MLO among the mollicutes, we cloned and sequenced its 16s rRNA gene. Indeed, even though MLOs cannot be cultured, their phylogenic positions have been determined from the sequences of their 16s ribosomal DNAs (rDNAs) (10,11,15,19). These organisms have been shown to be true mollicutes, and the name "phytoplasma" has been proposed for them (20). In addition, the sequence of the 16s-23s rDNA spacer region and the genome sue of the WBDL MLO were determined. MATERIALS AND METHODSPlant material. Healthy periwinkle and lime plants were obtained from seeds and were maintained at 25°C during the day and at 20°C at night.Lime and periwinkle plants were infected with the WBDL MLO by graft transmission and were g...

Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows

“…There is a consensus that each group should represent at least one separate species (Gundersen et al, 1994;Jung et al, 2002;Lee et al, 1998a;Seemüller et al, 1998). So far, of the nine 'Candidatus Phytoplasma' species proposed (Davis et al, 1997;Griffiths et al, 1999b;Jung et al, 2002Jung et al, , 2003Montano et al, 2001;Sawayanagi et al, 1999;Verdin et al, 2003;White et al, 1998;Zreik et al, 1995), two ('Candidatus Phytoplasma aurantifolia' and 'Candidatus Phytoplasma australasia') belong to subgroups in the peanut witches'-broom group (=group 16SrII) according the classification scheme proposed by Lee et al (1998a). The other six are 'Candidatus Phytoplasma fraxini' (16SrVII-A), 'Candidatus Phytoplasma australiense' (16SrXII-B), 'Candidatus Phytoplasma japonicum' (a strain closely related to members of 16SrXII), 'Candidatus Phytoplasma brasiliense' (16SrXV-A), 'Candidatus Phytoplasma castaneae' (most closely related to palm lethal yellows group 16SrIV), 'Candidatus Phytoplasma phoenicium' (16SrIV-B) and 'Candidatus Phytoplasma ziziphi' (16SrV-B).…”

'Candidatus Phytoplasma castaneae', a novel phytoplasma taxon associated with chestnut witches' broom disease.

“…Sequence analysis of 16S rDNA has also played an important role in the description of novel phytoplasma species (Zreik et al, 1995 ;Davis et al, 1997 ;White et al, 1998 ;Griffiths et al, 1999 ;Sawayanagi et al, 1999 ;Montano et al, 2001). This study used similar approaches to determine the phylogenetic relationships between the CnWB phytoplasma and other phytoplasmas.…”

‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects

“…Phytoplasma', identified as described above, and a list of 16S rRNA gene sequences of related strains. The listed taxa either have already been described individually in past issues of IJSEM or are described in accompanying papers in this issue or in forthcoming papers; the reader is referred to the original papers for their detailed descriptions (Zreik et al, 1995;Davis et al, 1997;White et al, 1998;Griffiths et al, 1999;Sawayanagi et al, 1999;Montano et al, 2001;Jung et al, 2002Jung et al, , 2003aHiruki & Wang, 2004;Lee et al, 2004a, b;Marcone et al, 2004a, b;Seemüller & Schneider, 2004 Lee, I.-M., Davis, R. E. & Hiruki, C. (1991). Genetic interrelatedness among clover proliferation mycoplasmalike organisms (MLOs) and other MLOs investigated by nucleic acid hybridization and…”

“…Since then, several distinct taxa have been described as 'Candidatus (Ca.) Phytoplasma aurantifolia' (Zreik et al, 1995), 'Ca. Phytoplasma australiense' (Davis et al, 1997), 'Ca.…”