Publications

4513

Plantas de hibisco com superbrotamento e definhamento seguido de morte têm sido observadas nos municípios de São Paulo, Campinas e Piracicaba. Como os sintomas são sugestivos daqueles induzidos por fitoplasmas, o presente trabalho buscou identificar o possível fitoplasma associado com a doença. Assim, 14 plantas sintomáticas de hibisco foram coletadas em Piracicaba (SP) e submetidas ao PCR duplo com os primers P1/Tint-R16F2n/R2 e ao exame em microscópio eletrônico de transmissão. A identificação foi realizada por análise de RFLP com as enzimas de restrição BfaI, DraI, HaeIII, HhaI, HpaII, MboI, MseI, RsaI e TaqI. Testes de transmissão foram conduzidos com enxertia de ramos e uso de Cuscuta subinclusa. Os resultados de nested-PCR revelaram a presença consistente de fitoplasmas em todas as plantas sintomáticas e foram confirmados pela observação de corpúsculos pleomórficos no floema, através da microscopia eletrônica. A análise de RFLP mostrou que o fitoplasma encontrado em hibisco pertence ao grupo 16SrXV, o mesmo grupo do Candidatus Phytoplasma brasiliense. O fitoplasma foi transmitido de planta doente para sadia, tanto pela enxertia como pela C. subinclusa, demonstrando ser o agente do superbrotamento do hibisco.

In January of 2009, potato plants (Solanum tuberosum) from a commercial crop in the Waikato Region, New Zealand were observed to have symptoms of upward rolling and purpling of the leaves. The symptoms appeared similar to those of “zebra chip”, a disorder of potato recently found to be associated with ‘Candidatus Liberibacter solanacearum’ in New Zealand and the United States (4). Total DNA from the leaf midveins and tubers from one of the symptomatic plants was separately extracted with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). DNA extracted from leaf midveins and tubers tested negative for ‘Ca. L. solanacearum’ by nested-PCR using primer pair OA2/OI2c (4) followed by Lib16S01F/Lib16S01R (5′-TTCTACGGGATAACGCACGG-3′ and 5′-CGTCAGTATCAGGCCAGTGAG-3′), which amplifies a 580-bp region of the 16S rRNA gene. However, DNA extracted from the tuber tissue tested positive for phytoplasma by TaqMan real-time PCR (3). No phytoplasma was detected in the DNA extracted from leaf tissue. The 16S rRNA gene, 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene of the phytoplasma were amplified with primers P1/P7 (1). The PCR product was cloned into the pCR 4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. FJ943262). BLAST analysis showed 100% identity to ‘Ca. Phytoplasma australiense’ (16SrXII, Stolbur group). A fragment of approximately 850-bp of the Tuf gene was also amplified (2) and sequenced directly (GenBank Accession No. FJ943263). BLAST analysis showed 100% identity to Tuf gene variant IX of ‘Ca. P. australiense’ (2). An additional 14 plants showing similar leaf symptoms and also production of aerial tubers were collected from seven different potato fields from the Auckland and Waikato regions. Total DNA from the leaf midveins, stem, and tubers were separately extracted from each of the plants. The samples were tested for phytoplasma by nested-PCR using primer pair R16F2/R16R2, followed by NGF/NGR (1), and tested for ‘Ca. L. solanacearum’ by nested-PCR as described above. Seven plants tested positive only for phytoplasma, three tested positive for only ‘Ca. L. solanacearum’, and four plants tested positive for both pathogens. The pathogens were most commonly detected in samples extracted from the stem with 9 and 5 of the 14 samples testing positive for phytoplasma and liberibacter, respectively. Six of each of the leaf and tuber samples tested positive for phytoplasma. Liberibacter was detected in one of the leaf samples and in four of the tuber samples. ‘Ca. P. australiense’ has only been reported from New Zealand and Australia. The only other known hosts of ‘Ca. P. australiense’ in New Zealand are strawberry and native plants belonging to the genera Cordyline, Coprosma, and Phormium (2). In Australia, ‘Ca. P. australiense’ is associated with Australian grapevine yellows and Papaya dieback (2). To our knowledge, this is the first report of ‘Ca. P. australiense’ infecting potato as well as the first report of phytoplasma and ‘Ca. L. solanacearum’ mixed infections in potato. References: (1) M. T. Andersen et al. Plant Pathol. 47:188, 1998. (2) M. T. Andersen et al. Phytopathology 96:838, 2006. (3) N. M. Christensen et al. Mol. Plant Microbe Interact. 17:1175, 2004. (4) L. W. Liefting et al. Plant Dis. 93:208, 2009.