Publications

4629

Zebra Chip (ZC), an emerging disease of potato (Solanum tuberosum L.) first documented in potato fields around Saltillo in México in 1994, has been identified in the southwestern United States, México, and Central America and is causing losses of millions of dollars to the potato industry (4). Recently, this damaging potato disease was also documented in New Zealand (3). This disease is characterized by a striped pattern of necrosis in tubers produced on infected plants, and fried chips processed from these infected tubers are commercially unacceptable (4). Recent studies conducted in the United States and New Zealand have associated ZC with a new species of ‘Candidatus Liberibacter’ vectored by the potato psyllid, Bactericera cockerelli Sulc (1,3,4). A bacterium designated ‘Candidatus Liberibacter psyllaurous’ has recently been identified in potato plants with “psyllid yellows” symptoms that resemble those of ZC (2). To investigate whether liberibacter is associated with ZC in México, 11 potato (cv. Atlantic) tuber samples exhibiting strong ZC symptoms and six asymptomatic tubers were collected from a ZC-affected commercial potato field near Saltillo City, Coahuila, México in September 2008 and tested for this bacterium by PCR. Total DNA was extracted from symptomatic and asymptomatic tubers with cetyltrimethylammoniumbromide (CTAB) buffer (4). DNA samples were tested by PCR using primer pair OA2/OI2c (5′-GCGCTTATTTTTAATAGGAGCGGCA-3′ and 5′-GCCTCGCGACTTCGCAACCCAT-3′, respectively) specific for 16S rDNA and primer pair CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′ and 5′-TATATCTATCGTTGCACCAG-3′, respectively) designed from ribosomal protein genes (3). Seven of eleven (63.7%) ZC-symptomatic tubers and one of six (16.7%) asymptomatic potatoes yielded the expected 1,168-bp 16S rDNA and 669-bp CL514F/R amplicons, indicating the presence of liberibacter. Amplicons generated from symptomatic tubers were cloned into pCR2.1-Topo plasmid vectors (Invitrogen, Carlsbad, CA) and one clone of each amplicon was sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the ZC OA2/OI2c sequence (GenBank Accession No. FJ498806) showed 100% identity to liberibacter 16S rDNA sequences amplified from potato psyllids from Dalhart, TX and potato tubers from Garden City, KS (GenBank Accession Nos. EU921627 and EU921626, respectively). The ZC CL514F/R sequence (GenBank Accession No. FJ498807) was 98% identical to analogous rplJ and rplL liberibacter ribosomal protein gene sequences amplified from several solanaceous plants in New Zealand (GenBank Accession Nos. EU834131 and EU935005). The OA2/OI2c sequence was also identical to the 16S rDNA sequence (Genbank Accession No. EU812559) of ‘Ca. Liberibacter psyllaurous’ (2). To our knowledge, this is the first report of ‘Ca. Liberibacter psyllaurous’ associated with ZC-affected potatoes in México. References: (1) J. A. Abad et al. Plant Dis. 93:108, 2009. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 92:1474, 2008. (4) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007.

Summary Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite “ Candidatus Endonucleobacter bathymodioli” that invades the nuclei of deep‐sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur‐ and methane‐oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep‐sea ecosystems. We first discovered the intranuclear parasite “ Ca. E. bathymodioli” in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid‐Atlantic Ridge. Using primers and probes specific to “ Ca . E. bathymodioli” we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of “ Ca . E. bathymodioli” showed that the infection of a nucleus begins with a single rod‐shaped bacterium which grows to an unseptated filament of up to 20 μm length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by “ Ca . E. bathymodioli” were those of the gill bacteriocytes. These cells contain the symbiotic sulfur‐ and methane‐oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the “ Ca . E. bathymodioli” belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates.

ABSTRACTThe complete genomes of three strains from the phylumAcidobacteriawere compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of theProteobacteria, theCyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N2fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

In São Paulo State, Brazil, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any ‘Ca. L. americanus’–infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, ‘Ca. L. asiaticus’–infected trees had high titers at 32 and 35°C, but not at 38°C, while ‘Ca. L. americanus’–infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of ‘Ca. L. asiaticus’ is not yet affected at 35°C, while a temperature of 32°C is detrimental to ‘Ca. L. americanus’. Thus, ‘Ca. L. americanus’ is less heat tolerant than ‘Ca. L. asiaticus’. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

In Brazil ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’ cause huanglongbing (also known as greening), the most destructive citrus disease. A shift in pathogen prevalence was observed over time, with a disproportional increase in ‘Ca. L. asiaticus’ occurrence. Graft transmission experiments were used for a comparative study of both species using budsticks from symptomatic branches of field-affected trees as inoculum. The plants were inoculated with ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ alone, or simultaneously with both species. Symptom manifestation and conventional and quantitative real-time polymerase chain reaction were used for plant evaluations. ‘Ca. L. americanus’ was detected mainly in symptomatic plants and ‘Ca. L. asiaticus’ was detected in symptomatic plants as well as in infected plants prior to symptom manifestation. Transmission percentages varied from 54.7 to 88.0% for ‘Ca. L. asiaticus’ and 10.0 to 45.2% for ‘Ca. L. americanus’ in two experiments. In co-inoculated plants, 12.9% contained ‘Ca. L. americanus’ only, 40.3% contained ‘Ca. L. asiaticus’ only, and 19.3% contained both species. Average bacterial titers for ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’, in log cells per gram of leaf midrib, were 6.42 and 4.87 for the experimental plants and 6.67 and 5.74 for the field trees used as the source of inoculum. The higher bacterial populations of the ‘Ca. L. asiaticus’-infected plants provided an explanation for the disproportional increase in field prevalence of this species over time, based on the greater likelihood for pathogen transmission by the insect vector.