Dickinson, M.

In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise similarity values based on alignment of nearly full-length 16S rRNA gene sequences (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100 % identity with a comparable sequence derived from a phytoplasma strain (LDN) responsible for Awka wilt disease of coconut in Nigeria, and shared 99.0–99.6 % identity with 16S rRNA gene sequences from strains associated with Cape St Paul wilt (CSPW) disease of coconut in Ghana and Côte d’Ivoire. Similarity scores further determined that the 16S rRNA gene of the LYDM phytoplasma shared <97.5 % sequence identity with all previously described members of ‘Candidatus Phytoplasma ’. The presence of unique regions in the 16S rRNA gene sequence distinguished the LYDM phytoplasma from all currently described members of ‘Candidatus Phytoplasma ’, justifying its recognition as the reference strain of a novel taxon, ‘Candidatus Phytoplasma palmicola’. Virtual RFLP profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficients delineated coconut LYDM phytoplasma strains from Mozambique as novel members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d’Ivoire. On this basis, the CSPW phytoplasma strains were designated members of a novel subgroup, 16SrXXII-B.

This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytoplasma distinguished the phytoplasma from all previously described ‘ Candidatus Phytoplasma ’ species. Pairwise sequence similarity scores, calculated through alignment of full-length 16S rRNA gene sequences, revealed that the MaPV phytoplasma 16S rRNA gene shared 96.5 % or less sequence similarity with that of previously described ‘ Ca. Phytoplasma ’ species, justifying the recognition of the MaPV phytoplasma as a reference strain of a novel taxon, ‘Candidatus Phytoplasma malaysianum’. The 16S rRNA gene F2nR2 fragment from the MaPV phytoplasma exhibited a distinct restriction fragment length polymorphism (RFLP) profile and the pattern similarity coefficient values were lower than 0.85 with representative phytoplasmas classified in any of the 31 previously delineated 16Sr groups; therefore, the MaPV phytoplasma was designated a member of a new 16Sr group, 16SrXXXII. Phytoplasmas affiliated with this novel taxon and the new group included diverse strains infecting periwinkle, coconut palm and oil palm in Malaysia. Three phytoplasmas were characterized as representatives of three distinct subgroups, 16SrXXXII-A, 16SrXXXII-B and 16SrXXXII-C, respectively.

Phytoplasmas of the 16SrVII group in ornamental Fraxinus uhdei trees (1) growing in different cities of the Colombian Andes have been reported (2). In surveys made in Bogotá during March and May 2011, symptoms suggestive of phytoplasma infection were observed in ornamental woody species: Croton spp. (Euphorbiaceae), Pittosporum undulatum (Pittosporaceae) and Populus nigra (Salicaceae) trees, growing close to infected F. uhdei (Oleaceae). Symptoms included witches' broom, yellowing, dieback, epicormic sprouts, tufted foliage, abnormal elongation or shortening of internodes, and deliquescent branching leading to dramatic changes in crown architecture. P. undulatum and F. uhdei are introduced species representing the second and third most abundant trees in the city. P. nigra is an introduced species and Croton spp. is an Andean genus. In order to screen for the presence of phytoplasmas in Croton spp., P. undulatum, and P. nigra, four individuals of each species and two F. uhdei trees were sampled. For DNA extraction, 1 g of vascular tissue from young stems was used. Samples were tested by nested PCR with primers P1A/P7A (4) followed by R16F2n/R16R2 (3). The frequency of phytoplasma detection varied among species; P. undulatum and Croton spp. had three positives each, while P. nigra had one positive. Both F. uhdei were positive. Sequences from the amplicons (three reads) were aligned. BLAST analysis of 16S rDNA sequences from the four species tested had 99.2 to 99.7% similarity to 16SrI group sequences. Phylogenetic analysis further confirmed this relationship. Virtual sequence analysis using the iPhyclassifier tool ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) showed that the sequence derived from P. undulatum (JQ730861) produced an identical RFLP pattern to group 16SrI-B (reference sequence NC_005303). RFLP similarity coefficients of the phytoplasmas from F. uhdei, Croton spp., and P. nigra (JQ730859, JQ730859 and JQ730861) were less than 0.97, suggesting the presence of a new subgroup within group 16SrI. The vectors of phytoplasmas are unknown in the region. Phytoplasma hosts previously reported in Colombia are: Solanum quitoense (16SrIII), Manihot esculenta (16SrIII), Liquidambar styraciflua (16SrI and 16SrVII), Elaeis guineensis (16SrI and 16SrIII), Coffea arabica (16SrIII), Cordia alliodora (16SrIII), Solanum tuberosum (16SrV and 16SrXII), and Zea mays (16SrI). To our knowledge, this is the first report of Croton spp. and P. undulatum as phytoplasma hosts. Phytoplasmas of group 16SrI are known to infect more than 100 species of different families worldwide. Detection of this group in several tree species and the observation of similar symptoms in other trees species raises concerns about a possible epidemic affecting plants in the Andean region. Implications are at several levels: i) epidemiological, with infected trees representing a potential inoculum source for other ornamental plants or crops growing in the agricultural surrounding areas; ii) economic, since eventually it will be necessary to replace diseased plants; and iii) environmental, because of the negative impact on the services provided by trees and green areas. References: (1) J. J. Filgueira et al. Plant Pathology 53:520, 2004. (2) L. Franco-Lara et al. Fitopatología Colombiana 29:32, 2005. (3) D. E Gundersen et al. Phytopathol. Mediterr. 35:144, 1996. (4) I-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004.