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4721

Bell pepper (Capsicum annuum) plants exhibiting symptoms that resembled those of potato psyllid (Bactericera cockerelli Sulc) damage and “Candidatus Liberibacter solanacearum” infection (2) were observed in a pepper field in La Cruz de Elota, Sinaloa, México in March 2009, with an infection rate of 1.5%. Plants exhibited chlorotic or pale green apical growth and leaf cupping, sharp tapering of the leaf apex, shortened internodes, and an overall stunting (2). Total DNA was extracted from the top whole leaf tissue of nine symptomatic and five asymptomatic pepper plants with cetyltrimethylammoniumbromide (CTAB) buffer (3,4). Seven and eight of the nine selected symptomatic pepper plants yielded the expected 1,168-bp 16S rDNA and the expected 669-bp rplJ/rplL ribosomal protein gene amplicons with the “Ca. L. solanacearum” specific OA2/OI2c and CL514F/CL514R primer pairs, respectively, indicating the presence of liberibacter (2,4). Nucleic acid from asymptomatic pepper plants yielded no products with these primers. Three amplicons generated from symptomatic pepper plants with each primer pair were cloned into pCRII-TOPO plasmid vectors (Invitrogen, Carlsbad, CA) and three clones of each amplicon were sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the 16S rDNA consensus sequence (GenBank Accession No. FJ957896) showed 100% identity to 16S rDNA sequences of “Ca. L. solanacearum” amplified from Solanum betaceum (EU935004) and S. lycopersicum (EU834130) from New Zealand (2), and “Ca. L. psyllaurous” from potato psyllids (EU812559) (1). The ribosomal protein gene consensus sequence (GenBank Accession No. FJ957894) was 100% identical to the analogous rplJ and rplL “Ca. L. solanacearum” ribosomal protein gene sequence amplified from S. lycopersicum (EU834131) from New Zealand (2) and to ‘Ca. Liberibacter’ sp. sequence amplified from zebra chip-infected potato tubers from Lancaster, CA (FJ498803). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with bell pepper in México. “Ca. L. solanacearum” was first reported in tomato and pepper plants in 2008 in New Zealand, where it has resulted in plant decline and significant yield loss, resulting in millions of dollars in losses to the commercial glasshouse tomato and pepper industry (2). Zebra chip, a new and emerging potato disease associated with ‘Ca. Liberibacter’ sp., was first identified in México in 1994, where it has caused significant economic damage, often leading to abandonment of entire potato fields (3,4). References: (1) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) J. E. Munyaneza et al. Plant Dis. 93:552, 2009.

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.

Citrus huanglongbing is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with a low-titer, phloem-limited infection by any of three uncultured species of α-Proteobacteria, ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’. A complete circular ‘Ca. L. asiaticus’ genome has been obtained by metagenomics, using the DNA extracted from a single ‘Ca. L. asiaticus’–infected psyllid. The 1.23-Mb genome has an average 36.5% GC content. Annotation revealed a high percentage of genes involved in both cell motility (4.5%) and active transport in general (8.0%), which may contribute to its virulence. ‘Ca. L. asiaticus’ appears to have a limited ability for aerobic respiration and is likely auxotrophic for at least five amino acids. Consistent with its intracellular nature, ‘Ca. L. asiaticus’ lacks type III and type IV secretion systems as well as typical free-living or plant-colonizing extracellular degradative enzymes. ‘Ca. L. asiaticus’ appears to have all type I secretion system genes needed for both multidrug efflux and toxin effector secretion. Multi-protein phylogenetic analysis confirmed ‘Ca. L. asiaticus’ as an early-branching and highly divergent member of the family Rhizobiaceae. This is the first genome sequence of an uncultured α-proteobacteria that is both an intracellular plant pathogen and insect symbiont.

The study and revision of the unicellular cyanobacterial genus Synechocystis was based on the type species S. aquatilis Sauv. and strain PCC 6803, a reference strain for this species. Uniformity in rRNA gene sequence, morphology, and ultrastructure was observed in all available Synechocystis strains, with the exception of the strain PCC 6308, which has been considered by some to be a model strain for Synechocystis. This strain differs substantially from the typical Synechocystis cluster according to both molecular (<90% of similarity, differences in 16S–23S rRNA internal transcribed spacer [ITS] secondary structure) and phenotypic criteria (different ultrastructure of cells). This strain is herein classified into the new genus Geminocystis gen. nov., as a sister taxon to the genus Cyanobacterium. Geminocystis differs from Cyanobacterium by genetic position (<94.4% of similarity) and more importantly by its different type of cell division. Because strain PCC 6308 was designated as a reference strain of the Synechocystis cluster 1 in Bergey’s Manual, the members of this genetic cluster have to be revised and reclassified into Geminocystis gen. nov. Only the members of the Synechocystis cluster 2 allied with PCC 6803 correspond both genetically and phenotypically to the type species of the genus Synechocystis (S. aquatilis).