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4509

Huanglongbing (HLB) disease, associated with ‘Candidatus Liberibacter asiaticus’ (CLas), is primarily spread via infected citrus nursery trees and by infective Asian citrus psyllid, the insect vector. Recently, the Texas Department of Agriculture initiated regulations requiring commercial and retail citrus nurseries in Texas to transition from traditional open-field to enclosed facilities with insect-resistant screens to mitigate the risk of nurseries serving as sources of CLas. Although several nursery production facilities have adopted this regulation, non-enclosed nurseries persist and pose a significant threat to the citrus industry as potential sources of CLas. A systematic survey for HLB was embarked on in a semi-open nursery facility in South Texas in April 2014. Leaf tissue samples taken from 94 trees representing 5% of the total number of potted trees in the nursery were tested for CLas by quantitative and conventional PCR assays. Of 94 trees tested, 3.2% (3 trees) were positive for CLas by both assays. The presence of CLas in the PCR-positive samples was confirmed by multi-locus sequence analyses. The results represent the first report of HLB in a nursery facility in Texas, and underscore the need for more intensive surveillance for HLB in citrus nursery stock as an integral component of HLB mitigation efforts in Texas. Accepted for publication 27 August 2014. Published 15 December 2014.

In April and May of 2012, bell pepper (Capsicum annuum) plants exhibiting symptoms that resembled those of the bacterium ‘Candidatus Liberibacter solanacearum’ infection (2,4) were observed in commercial pepper fields in several departments in Honduras, including Francisco Morazán, Ocotepeque, El Paraíso, and Olancho. Many of the fields were infested with the psyllid Bactericera cockerelli, a vector of ‘Ca. L. solanacearum’ (3). The plants exhibited chlorotic or pale green apical growth and leaf cupping, sharp tapering of the leaf apex, shortened internodes, and overall stunting (2,4). All cultivars grown were affected and 20 to 75% of plants in each field were symptomatic. Pepper (var. Nataly) plant samples were collected from a total of eight affected fields (two fields per department). Total DNA was extracted from the top whole leaf tissue of a total of 19 plants, including 14 symptomatic and 5 asymptomatic pepper plants, with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1). The DNA samples were then tested by PCR using specific primer sets OA2/OI2c and OMB 1482f/2086r to amplify a portion of 16S rDNA and the outer membrane protein (OMB) genes, respectively, of ‘Ca. L. solanacearum’ (1,2). OMB gene and 16S rDNA fragments of 605 and 1,168 bp, respectively, were amplified from the DNA of 7 of 14 (50%) symptomatic plants with each primer set, indicating the presence of ‘Ca. L. solanacearum.’ No ‘Ca. L. solanacearum’ was detected in the five asymptomatic plants with either primer sets. DNA amplicons with both primer sets were cloned from the DNA of plant samples collected from each of the three departments: Francisco Morazán (in the locality of Zamorano), Ocotepeque (municipality of Plan del Rancho in Sinuapa), and El Paraíso (municipality of Danlí), and four clones of each of the six amplicons were sequenced. BLASTn analysis of the 16S rDNA resulted in a single consensus sequence for all three locations (deposited in GenBank as Accession Nos. KF188226, KF188227, and KF188228) and showed 100% identity to numerous 16S rDNA sequences of ‘Ca. L. solanacearum’ in GenBank, including accessions HM245242, JF811596, and KC768319. Similarly, identical OMB consensus sequences were observed in all three locations (deposited in GenBank as KF188230, KF188231, and KF188233) that are 100% identical to several ‘Ca. L. solanacearum’ sequences in GenBank (e.g., KC768331 and CP002371) along with a second consensus sequence (deposited in GenBank as accession KF188232) from Ocotepeque that was 99% identical to the consensus sequence from the three locations and sequences in GenBank. To our knowledge, this is the first report of ‘Ca. L. solanacearum’ associated with pepper crops in Honduras, where pepper constitutes an economically important commodity. This bacterium has also caused millions of dollars in losses to potato and several other solanaceous crops in United States, Mexico, Central America, and New Zealand (1,2,3,4). Furthermore, ‘Ca. L. solanacearum’ has been reported to severely damage carrot crops in Europe, where it is transmitted to carrot by the psyllids Trioza apicalis and Bactericera trigonica (3). Monitoring this pathogen and its vectors will prevent serious damage they cause to economically important crops. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009.

Abstract ‘Candidatus Liberibacter solanacearum’ is a pathogen of solanaceous crops (Solanales: Solanaceae) that causes zebra chip disease of potato (Solanum tuberosum L.) and plant dieback in tomato (S. lycopersicum L.) and pepper (Capsicum spp.). This pathogen is vectored by the potato/ tomato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), but little is known about the interactions between B. cockerelli and ‘Ca. Liberibacter solanacearum.’ Fluorescence in situ hybridization was used to assess the incidence of ‘Ca. Liberibacter solanacearum’ in the hemolymph, bacteriomes, alimentary canals, and salivary glands of B. cockerelli. Liberibacter was observed in 66% of alimentary canals, 39% of salivary glands, and 40% of bacteriomes dissected from adult psyllids. Compared with adults, the organs of fifth instars appeared less likely to harbor Liberibacter, which was observed in 52% of alimentary canals, 10% of salivary glands, and 6% of bacteriomes dissected from the nymphs. Results of real-time polymerase chain reaction confirmed that fewer fifth instars were infected with Liberibacter compared with adults and indicated that fifth instars were less likely to transmit the pathogen to noninfected host plants. These observations of the localization of ‘Ca. Liberibacter solanacearum’ in the organs and tissues of B. cockerelli adults and nymphs will aid the study of Liberibacter-psyllid interactions and the epidemiology of ‘Ca. Liberibacter solanacearum.’

ABSTRACTNitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium “CandidatusPelagibacter ubique,” a cultured member of the orderPelagibacterales(SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation. A survey of 266Alphaproteobacteriagenomes found these five regulatory genes nearly universally conserved, absent only in intracellular parasites and members of the orderPelagibacterales, including “Ca. Pelagibacter ubique.” Global differences in mRNA and protein expression between nitrogen-limited and nitrogen-replete cultures were measured to identify nitrogen stress responses in “Ca.Pelagibacter ubique” strain HTCC1062. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating that they devote increased resources to the assimilation of nitrogenous organic compounds. Enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) were similarly upregulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in control of the nitrogen starvation response. Comparisons of the transcriptome and proteome supported previous observations of uncoupling between transcription and translation in nutrient-deprived “Ca.Pelagibacter ubique” cells. Overall, these data reveal a streamlined, PII-independent response to nitrogen stress in “Ca.Pelagibacter ubique,” and likely otherPelagibacterales, and show that they respond to nitrogen stress by allocating more resources to the assimilation of nitrogen-rich organic compounds.IMPORTANCEPelagibacteralesare extraordinarily abundant and play a pivotal role in marine geochemical cycles, as one of the major recyclers of labile dissolved organic matter. They are also models for understanding how streamlining selection can reshape chemoheterotroph metabolism. Streamlining and its broad importance to environmental microbiology are emerging slowly from studies that reveal the complete genomes of uncultured organisms. Here, we report another remarkable example of streamlined metabolism inPelagibacterales, this time in systems that control nitrogen assimilation.Pelagibacteralesare major contributors to metatranscriptomes and metaproteomes from ocean systems, where patterns of gene expression are used to gain insight into ocean conditions and geochemical cycles. The data presented here supply background that is essential to interpreting data from field studies.