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Nested-PCR Detection and Sequence Confirmation of ‘Candidatus Liberibacter asiaticus’ from Murraya paniculata in Guangdong, China

Citation
Deng et al. (2007). Plant Disease 91 (8)
Names
Ca. Liberibacter asiaticus
Subjects
Agronomy and Crop Science Plant Science
Abstract
Murraya paniculata (orange jasmine) is a popular ornamental rutaceaous plant and is known to be a preferred host for the Asian citrus psyllid, Diaphorina citri (Kuwayana), the primary vector of ‘Candidatus Liberibacter spp.’ that causes citrus Huanglongbing (HLB). HLB is a highly destructive citrus disease worldwide. However, the presence of ‘Ca. Liberibacter spp.’ in M. paniculata remains uncertain (2). Clarification of M. paniculata as a host of ‘Ca. Liberibacter spp.’ has direct impact on HLB control programs. During June of 2006, we identified three M. paniculata trees near a mandarin orchard affected by HLB in Luoding City and two trees from Guangzhou City, Guangdong Province, People's Republic of China. All trees had leaves showing yellowing and mottling symptoms characteristic of HLB. Both symptomatic and asymptomatic leaves were collected. DNA was extracted using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (3) was used for the first round of amplification. Amplification was conducted as previously described (1), and 2 μl of PCR reaction product were used for a second round of amplification using the same procedure but with 35 PCR cycles with primer set OI1/OI2c (3,4). After agarose gel electrophoresis and staining with ethidium bromide, a 1.1-kb DNA band was unambiguously associated with symptomatic but not asymptomatic leaf samples. Nonnested-PCR using primer set OI1/OI2c alone did not yield a target DNA band or yielded a very weak DNA band. XbaI digestion of the nested-PCR DNA product yielded two fragments, 520 and 640 bp long, characteristic of ‘Ca. L. asiaticus’. PCR amplicons were sequenced and were 1,095 bp long. This sequence shared >98% similarity to sequences of ‘Ca. L. asiaticus’ in the GenBank database. We observed that nested-PCR is necessary for consistent amplification of DNA from ‘Ca. L. asiaticus’ from M. paniculata. We excluded the possible nonspecific amplification associated with nested-PCR by XbaI restriction enzyme digestion and by nucleotide sequence analysis. Our data indicate that M. paniculata is a host of ‘Ca. L. asiaticus’ but the bacterial titer might be low. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) M. Garnier and J. Bove. Huanglongbing (Greening). Page 46 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al., eds. The American Phytopathological Society, St. Paul, MN, 2000. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (4) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.

‘Candidatus Phytoplasma lycopersici’, a phytoplasma associated with ‘hoja de perejil’ disease in Bolivia

Citation
Arocha et al. (2007). International Journal of Systematic and Evolutionary Microbiology 57 (8)
Names
Ca. Phytoplasma lycopersici
Subjects
Ecology, Evolution, Behavior and Systematics General Medicine Microbiology
Abstract
New diseases known locally as ‘hoja de perejil’ of tomato (Lycopersicon esculentum Mill) and ‘brotes grandes’ of potato (Solanum tuberosum L.) were first recognized in surveys of production fields in Bolivia during 2000–2003. Alfalfa (Medicago sativa) witches' broom and little leaf diseases of native weeds Morrenia variegata and mora-mora (Serjania perulacea) were also identified near to production fields. Phytoplasma aetiology was attributed to each of these diseases following detection and initial identification of aster yellows group (16SrI) phytoplasmas in all five diseased plant species. While potato, alfalfa and mora-mora plants contained indistinguishable 16SrI-B strains, ‘hoja de perejil’ (THP) and morrenia little leaf (MVLL)-associated phytoplasma strains shared 97.5 % 16S rRNA gene sequence similarity with ‘Candidatus Phytoplasma asteris’ and related strains and <95 % similarity with all other ‘Candidatus Phytoplasma’ species. Phylogenetic analysis of 16S rRNA gene sequences indicated that the THP and MVLL phytoplasmas represent a novel lineage within the aster yellows (16SrI) group and, on the basis of unique 16S rRNA gene sequences, we propose that THP and MVLL phytoplasmas represent ‘Candidatus Phytoplasma lycopersici’, with THP as the reference strain.