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Citrus huanglongbing is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of ‘Candidatus Liberibacter’, of which ‘Ca. L. asiaticus’ is the most widely distributed. An optimized system using ‘Ca. L. asiaticus’-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 μg/ml) and indole-3-butyric acid (4 μg/ml). This system allowed a plant regeneration rate of 60.6% for ‘Ca. L. asiaticus’-infected cuttings in contrast to the <1% regeneration rate with water alone. Two chemical agents, penicillin G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA), were found to be effective at eliminating or suppressing the ‘Ca. L. asiaticus’ bacterium in this periwinkle regeneration system. When treated with penicillin G sodium at 50 μg/ml, all plants regenerated from ‘Ca. L. asiaticus’-infected cuttings were ‘Ca. L. asiaticus’ negative as determined by both nested polymerase chain reaction (PCR) and quantitative real-time PCR. In addition, DBNPA was also able to significantly reduce the percentage of ‘Ca. L. asiaticus’-positive plants and the titer of the ‘Ca. L. asiaticus’ bacterium at 200 μl/liter.

From 2006 to 2009, all commercial potato fields in Azacualpa F.M. Honduras were heavily infested with the potato psyllid Bactericera cockerelli (Sulc.). Plants exhibited interveinal chlorosis, vein-greening, downward curling, stunting, above ground tuber formation, and brownish flecks in some tubers. Disease incidence ranged from 50 to 95%. Leaf samples and psyllids were collected from seven fields in two potato-growing regions of Honduras. Total DNA was purified from the leaves of 30 symptomatic and three asymptomatic plants. DNA was extracted from 20 adult and 10 immature (4th to 5th instar) psyllids according to Frohlich et al (1). PCR primers, PSY680F 5′-GTTCGGAATAACTGGGCGTA-3′ and PSY1R 5′-CCCATAAGGGCCATGAGGACT-3′, were used to amplify a 680-bp fragment of the 16S rDNA for the recently described “Candidatus Liberibacter physallaurous” (2) and “Ca. L. solanacearum” (3). PSY1R/PSY680F primer design was based on the association of a previously undescribed liberibacter with vein-greening symptoms in greenhouse tomato plants in Arizona from 2006 to 2007 (GenBank Accession No. GQ926918) that lead to the hypothesis that a similar bacterium could be associated with symptomatic potato plants in Honduras. PCR amplification, cloning, and sequencing of the resultant 16S rDNA amplicons indicated that 17 of 30 potato plants, 8 of 20 adult and 7 of 10 third to fourth instar psyllids, respectively, were positive for liberibacter based on 99 to 100% shared nucleotide sequence (nt) identity with the analogous sequence from liberibacter (EU812558 [2]). To substantiate these results, a second molecular marker was targeted using the 1611F and 480R primers (~980 bp) that amplify the 16S-23S-ITSrDNA of liberibacter (2) for selected liberibacter-positive samples (above). Amplicons of the expected size were obtained from 12 of 17 potato and 7 of 10 immature psyllids. No PCR product of the expected size was obtained from asymptomatic potato samples or the PCR negative (water) control. The resultant PCR amplicons were cloned and 12 to 15 clones per amplicon were sequenced. The sequences were aligned and the percentage pair wise nt identity was calculated by Clustal W revealing that the 16S rDNA and 16S-23S-ITS sequences, respectively, shared 99 to 100% nt identity with each other. BLAST analysis against the NCBI database indicated that the 16S rRNA sequences from potato plants (GQ926922) and immature psyllids (GQ926923), and the 16S-ITS-23S sequence from potato plants (GQ926924) and immature psyllids (GQ926925), shared 98.5 to 100% nt identity with ‘Ca. Liberibacter’ reported from potato (EU812556; [2,4]) and tomato (EU812558, EU812559, EU935005; [2,3]). Evidence for the widespread presence of liberibacter and the potato psyllid in potato fields in Honduras, together with foliar and tuber symptoms that are reminiscent of those recently described in potato plants in the United States affected with ‘zebra chip’ disease (4), suggest that a similar or identical disease of the potato also occurs in Honduras. This emergent disease poses a serious threat to potato production in Honduras and elsewhere in Central America. References: (1) D. R. Frohlich et al., Mol. Ecol. 8:1683, 1999. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 78:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007.

In India, satkara (Citrus macroptera) is found in the northeastern regions of Shella and Dowki near Cherrapunji in Meghalaya, the Jampui Hills of Tripura and Mizoram, and the Chendel District of Manipur. Locally, it is called satkara because ‘sat’ refers to multiples of seven and the fruit generally contains fourteen segments. The fruit is used in the preparation of pickles and its oil is used in the perfume industry. In January 2007, in Behliangchhip, Jampui Hills, Tripura, India, we noticed seven satkara trees of 26 showing leaf mottling and yellowing in young shoots, symptoms typical of huanglongbing (HLB). HLB is a destructive citrus disease caused by a nonculturable, phloem-limited alpha-proteobacterium of the genus ‘Candidatus Liberibacter’. Among the three known ‘Ca. Liberibacter’ species (Ca. Liberibacter asiaticus, Ca. L. africanus, and Ca. L. americanus), only Ca. L. asiaticus has been reported in India (1) but not from satkara. To identify the pathogen, total DNA was extracted and purified from 200 mg of leaf midribs from five symptomatic and five symptomless trees with the DNeasy Plant Mini Kit (Qiagen, Gmbh, Hilden, Germany) as per the manufacturer's instructions. Samples were tested for ‘Ca. L. asiaticus’ by PCR with primer sets OI1/OI2c targeting the 16S rDNA locus (3) and A2/J5 targeting the beta-operon locus of ribosomal proteins (2). DNA samples from all symptomatic leaves were positive with both primer sets. The OI1/OI2c primers generated an amplicon of approximately 1,160 bp and digestion of the amplicon by XbaI yielded two DNA fragments of approximately 640 bp and 520 bp, suggesting the presence of ‘Ca. L. asiaticus’(3). The A2/J5 primer set generated an ~703-bp amplicon, indicating ‘Ca. L. africanus’ was not present. No DNA was amplified from the asymptomatic satkara trees. One each of the OI1/OI2c and A2/J5 amplicons was purified, cloned (pGEM-T Easy vector, Promega, Hampshire, UK) and sequenced (Bangalore Genei, Bengaluru, Karnataka, India). The derived sequences, 1167 bp for the OI1/OI2c amplicon and 703 bp for the A2/J5 amplicon, have been deposited in the GenBank database under Accession Nos. GQ369792 and GU074017, respectively. Sequence comparison revealed that GQ369792 and GU074017 shared >99% identity to the corresponding regions of ‘Ca. L. asiaticus’ in the GenBank database. No DNA was amplified by PCR with primer set GB1/GB2 (4) specific for ‘Ca. L. americanus’. To our knowledge, this is the first report of molecular identification of ‘Ca. L. asiaticus’ infecting satkara in India. References: (1) A. K. Das. Curr. Sci. 87:1183, 2004. (2) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) D. C. Teixeira et al. Mol. Cell. Probes 19:173, 2005.

ABSTRACT Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “ Candidatus Amoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to the Bacteroidetes . The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. “ Ca . Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “ Ca . Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “ Ca . Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila , Rickettsia bellii , Francisella tularensis , and Mycobacterium avium ). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.