Publications

4497

In the summer of 2008, symptoms of leaf curling with yellow, bronze, and purple discoloration, twisting of petioles, stunting of shoots and tap roots, and proliferation of secondary roots were observed in 18 commercial carrot (Daucus carota L.) fields (~62 ha) severely infested with psyllids (mainly Bactericera sp.) from 52 fields (297 ha) located in Alicante and Albacete provinces of Spain. Incidence of symptomatic plants was variable among fields. Similar symptoms were observed in 2009, 2010, and 2011. Symptoms resembled those associated with phytoplasma, spiroplasma, or the bacterium ‘Candidatus Liberibacter solanacearum’ infections in carrot (1–4). Aster yellows and stolbur phytoplasmas and Spiroplasma citri have previously been reported from carrot in mainland Spain but liberibacter infection has not been documented in this region (1). Studies were conducted to determine if ‘Ca. L. solanacearum’ was associated with the symptoms. Petiole samples of symptomatic carrot plants were collected in 2009 (25 from 9 fields in Alicante and Albacete provinces) and early 2010 (21 from 8 fields in Alicante, Albacete, and Valencia provinces) from symptomatic fields where incidence ranged from 50 to 90%. In addition, one sample collected in 2008 in Alicante was included in the assay. Also, samples were collected from five asymptomatic carrot plants. Total DNA was extracted from 0.5 g of petiole tissue of each sample with the CTAB extraction buffer method (3,4). DNA extractions were analyzed by PCR assay using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of ‘Ca. L. solanacearum’ (3,4). DNA samples were also tested for phytoplasmas and S. citri by nested-PCR assays using primer pairs P1/P7 followed by R16F2n/R16R2n and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2, respectively (2). A 1,168-bp fragment of 16S rDNA was detected in DNA extracted from 1, 12, and 12 symptomatic samples collected in 2008, 2009, and 2010, respectively, suggesting the presence of ‘Ca. L. solanacearum’ in the carrot samples. A 669-bp rplJ/rplL fragment also was amplified from DNA of the same samples. Liberibacter was not detected in asymptomatic plants. Eight and two samples were infected with S. citri and aster yellows phytoplasmas, respectively. Three samples were infected with S. citri and ‘Ca. L. solanacearum’ and one sample was infected with all three pathogens. Three amplicons obtained from the PCR assays with both primer pairs from carrot samples collected in 2009 and 2010 were sequenced directly. BLAST analysis of the 16S rDNA sequences (GenBank Nos. HQ454302, HQ454303, and HQ454304) showed 99% nucleotide identity to those of ‘Ca. L. solanacearum’ amplified from carrot in Finland (GU373049). The rplJ/rplL nucleotide sequences (HQ454305, HQ454306, and HQ454307) were 97% identical to sequences of the analogous rplJ/rplL ‘Ca. L. solanacearum’ ribosomal protein gene from carrot in Finland (GU373051). To our knowledge, this is the first report of ‘Ca. L. solanacearum’ in carrot in mainland Spain and also the first evidence of mixed infections of S. citri, ‘Ca. L. solanacearum’, and phytoplasmas in carrot. References: (1) M. C. Cebrián et al. Plant Dis. 94:1264, 2010. (2) I.-M. Lee et al. Plant Dis. 90:989, 2006. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) J. E. Munyaneza et al. Plant Dis. 94:639, 2010.

Symptoms of abnormal proliferation of shoots resulting in formation of witches’-broom growths were observed on diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.) in Brazil. RFLP analysis of 16S rRNA gene sequences amplified in PCRs containing template DNAs extracted from diseased plants collected in Bonito (Pernambuco) and Viçosa (Minas Gerais) Brazil, indicated that such symptoms were associated with infections by two mutually distinct phytoplasmas. One phytoplasma, PassWB-Br4 from Bonito, represents a new subgroup, 16SrIII-V, in the X-disease phytoplasma group (‘Candidatus Phytoplasma pruni’-related strains). The second phytoplasma, PassWB-Br3 from Viçosa, represents a previously undescribed subgroup in group 16SrVI. Phylogenetic analyses of 16S rRNA gene sequences were consistent with the hypothesis that strain PassWB-Br3 is distinct from previously described ‘Ca. Phytoplasma ’ species. Nucleotide sequence alignments revealed that strain PassWB-Br3 shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Ca. Phytoplasma ’ species. The unique properties of its DNA, in addition to natural host and geographical occurrence, support the recognition of strain PassWB-Br3 as a representative of a novel taxon, ‘Candidatus Phytoplasma sudamericanum’.

Summary ‘ Candidatus Methylomirabilis oxyfera’ is a denitrifying methanotroph that performs nitrite‐dependent anaerobic methane oxidation through a newly discovered intra‐aerobic pathway. In this study, we investigated the response of a M. oxyfera enrichment culture to oxygen. Addition of either 2% or 8% oxygen resulted in an instant decrease of methane and nitrite conversion rates. Oxygen exposure also led to a deviation in the nitrite to methane oxidation stoichiometry. Oxygen‐uptake and inhibition studies with cell‐free extracts displayed a change from cytochrome c to quinol as electron donor after exposure to oxygen. The change in global gene expression was monitored by deep sequencing of cDNA using Illumina technology. After 24 h of oxygen exposure, transcription levels of 1109 (out of 2303) genes changed significantly when compared with the anoxic period. Most of the genes encoding enzymes of the methane oxidation pathway were constitutively expressed. Genes from the denitrification pathway, with exception of one of the putative nitric oxide reductases, norZ2 , were severely downregulated. The majority of known genes involved in the vital cellular functions, such as nucleic acid and protein biosynthesis and cell division processes, were downregulated. The alkyl hydroperoxide reductase, ahpC , and genes involved in the synthesis/repair of the iron–sulfur clusters were among the few upregulated genes. Further, transcription of the pmoCAB genes of aerobic methanotrophs present in the non‐ M. oxyfera community were triggered by the presence of oxygen. Our results show that oxygen‐exposed cells of M. oxyfera were under oxidative stress and that in spite of its oxygenic capacity, exposure to microoxic conditions has an overall detrimental effect.

Carrot (Daucus carota) plants with symptoms resembling those associated with the carrot psyllid Trioza apicalis and the bacterium “Candidatus Liberibacter solanacearum” (1–4) were observed in 70% of commercial fields in southern Sweden in August 2011, with approximately 1 to 45% symptomatic plants per field. T. apicalis, a pest of carrot in northern and central Europe, including Sweden, can cause as much as 100% crop loss and is associated with “Ca. L. solanacearum” (1–4). Symptoms on affected plants include leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots (3). Carrot plant and psyllid samples were collected from fields in the province of Halland. Total DNA was extracted from petiole and root tissues of 33 symptomatic and 16 asymptomatic plants (cvs. Nevis and Florida), with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,3). DNA was also extracted from 155 psyllids (3). DNA samples were tested by PCR using primer pairs OA2/OI2c (5′'-GCGCTTATTTTTAATAGGAGCGGCA-3′/5′-GCCTCGCGACTTCGCAACCCAT-3′) and CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′/5′-TATATCTATCGTTGCACCAG-3′), to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of “Ca. L. solanacearum” (2,3). A 1,168-bp 16S rDNA fragment was detected in the DNA from all 33 symptomatic and two asymptomatic plants, and a 668-bp rplJ/rplL fragment was amplified from the DNA of all 33 symptomatic and four asymptomatic plants, indicating the presence of liberibacter. DNA from 23 and 49 psyllid samples yielded similar amplicons with OA2/OI2c and CL514F/R primer pairs, respectively. Amplicons from the DNA of four carrot roots and three T. apicalis with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the 14 amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequences from carrot (GenBank Accession No. JN863095) and T. apicalis (GenBank Accession No. NJ863096) showed 100% identity to those of “Ca. L. solanacearum” previously amplified from carrot (GU373048 and GU373049) and T. apicalis (GU477254 and GU477255) from Finland (2,3). The rplJ/rplL consensus sequences from carrot (GenBank Accession No. JN863093) and T. apicalis (GenBank Accession No. JN863094) were 99% identical to the sequences of rplJ/rplL “Ca. L. solanacearum” ribosomal protein gene from carrots in Finland (GU373050 and GU373051). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with carrot and T. apicalis in Sweden. The disease associated with this bacterium caused millions of dollars in losses to potato and several other solanaceous crops in North and Central America and New Zealand (1). This plant pathogen is also associated with significant economic damage to carrot crops observed in Finland (2,3). References: (1) J. E. Munyaneza. Southwest. Entomol. 35:471, 2010. (2) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.