Publications

4731

Over the last decade, the plant disease huanglongbing (HLB) has emerged as a primary threat to citrus production worldwide. HLB is associated with infection by phloem-limited bacteria (‘Candidatus Liberibacter’ spp.) that are transmitted by the Asian citrus psyllid, Diaphorina citri. Transmission efficiency varies with vector-related aspects (e.g., developmental stage and feeding periods) but there is no information on the effects of host–pathogen interactions. Here, acquisition efficiency of ‘Candidatus Liberibacter asiaticus’ by D. citri was evaluated in relation to temporal progression of infection and pathogen titer in citrus. We graft inoculated sweet orange trees with ‘Ca. L. asiaticus’; then, at different times after inoculation, we inspected plants for HLB symptoms, measured bacterial infection levels (i.e., titer or concentration) in plants, and measured acquisition by psyllid adults that were confined on the trees. Plant infection levels increased rapidly over time, saturating at uniformly high levels (≈108 copy number of 16S ribosomal DNA/g of plant tissue) near 200 days after inoculation—the same time at which all infected trees first showed disease symptoms. Pathogen acquisition by vectors was positively associated with plant infection level and time since inoculation, with acquisition occurring as early as the first measurement, at 60 days after inoculation. These results suggest that there is ample potential for psyllids to acquire the pathogen from trees during the asymptomatic phase of infection. If so, this could limit the effectiveness of tree rouging as a disease management tool and would likely explain the rapid spread observed for this disease in the field.

AbstractHuanglongbing (HLB) is a destructive citrus disease. The leading cause of HLB is Candidatus Liberibacter asiaticus. Fatty acid biosynthesis is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterial agents. Enoyl−acyl carrier protein reductase (also called ENR or FabI and a product of the fabI gene) is an enzyme required in a critical step of bacterial fatty acid biosynthesis and has attracted attention as a target of novel antimicrobial agents. We determined the crystal structures of FabI from Ca. L. asiaticus in its apoform as well as in complex with b‐nicotinamide adenine dinucleotide (NAD) at 1.7 and 2.7 Å resolution, respectively, to facilitate the design and screening of small molecule inhibitors of FabI. The monomeric ClFabI is highly similar to other known FabI structures as expected; however, unlike the typical tetramer, ClFabI exists as a hexamer in crystal, whereas as dimer in solution, on the other hand, the substrate binding loop which always disordered in apoform FabI structures is ordered in apo‐ClFabI. Interestingly, the structure of ClFabI undergoes remarkable conformational change in the substrate‐binding loop in the presence of NAD. We conclude that the signature sequence motif of FabI can be considered as Gly‐(Xaa)5‐Ser‐(Xaa)n‐Val‐Tyr‐(Xaa)6‐Lys‐(Xaa)n‐Thr instead of Tyr‐(Xaa)6‐Lys. We have further identified isoniazid as a competitive inhibitor with NADH.

Peony (Paeonia tenuifolia L.) is a herbaceous perennial plant known for its beautiful and showy flowers. In Serbia it is native to the Deliblato Sands and is used as an ornamental and medicinal plant in folk medicine. This plant species has become a rarity and for that reason peony was introduced into a botanical collection near Backi Petrovac (northern Serbia), where it has been maintained since 1988. Reddening of lower leaves observed on 10% of plants (5 of 50) in the collection at flowering in May 2012 gradually progressed throughout affected plants by the seed maturation stage. Five leaves from each of three reddened and three symptomless plants were sampled at the end of July 2012. Total nucleic acid was extracted separately from individual leaves (30 samples) using the CTAB (cetyltrimethylammonium bromide) method (2). A nested PCR assay using universal primer pairs P1/P7, followed by R16F2n/R16R2 (4), amplified 16S rDNA fragments of 1.8 and 1.2 kb, respectively. DNA from all three reddened plants (15 samples) yielded 1.2-kb amplicons after nested PCRs. Restriction fragment length polymorphism (RFLP) patterns obtained by digestion of nested products with endonucleases AluI, TruI, HpaII, or HhaI (Thermo Scientific, Lithuania) (4) were identical to those of the STOL reference strain included for comparative purposes, indicating that symptoms were consistently associated with plant infection by ‘Ca. Phytoplasma solani’ (Stolbur) phytoplasma. The 16S rDNA amplicons from two peony plants (1.2 kb from B15 and 1.8 from B18) were sequenced (GenBank Accession No. KC960487 and KF614623, respectively). BLAST analysis revealed a 100% identity between the sequences and GenBank sequences of Stolbur phytoplasma, subgroup 16SrXII-A phytoplasma, previously detected in maize (JQ730750) in Serbia and red clover (EU814644.1) in the Czech Republic. Phytoplasma associated diseases of other species of the genus Paeonia (P. lactiflora Pall. and P. suffruticosa Andrews) have been described elsewhere. Disease symptoms on P. lactiflora from Chile were associated with the phytoplasma that belongs to the ribosomal subgroup 16SrVII-A (‘Ca. Phytoplasma fraxini’) (1). Also, Stolbur phytoplasma from the 16SrXII group was detected on P. suffruticosa plants in China, manifesting yellowing symptoms (3). To our knowledge, this is the first report of naturally occurring Stolbur phytoplasma disease of P. tenuifolia L. in Serbia. References: (1) N. Arismendi et al. Bull. Insectol. 64:S95, 2011. (2) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (3) Y. Gao et al. J. Phytopathol. 161:197, 2013. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

ABSTRACT The tick-borne bacterium “ Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii , it is one of the most common pathogens found in the tick Ixodes ricinus . Here, we compared the epidemiologies of “ Ca . Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole ( Myodes glareolus ), using real-time PCR for detection and quantification. The prevalences of “ Ca . Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “ Ca . Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “ Ca . Neoehrlichia mikurensis” was significantly higher than that of B. afzelii . We found a high frequency of double infections; 46% of all samples infected with “ Ca . Neoehrlichia mikurensis” also had a coinfection with B. afzelii . The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.

Candidatus Liberibacter asiaticus (Las) is a phloem-limited bacterium associated with huanglongbing (HLB), one of the most destructive diseases of citrus in Florida and other citrus-producing countries. Natural transmission of Las occurs by the psyllid vector Diaphorina citri, but transmission can also occur through grafting with diseased budwood. As a result of the difficulty of maintaining Las in culture, screening of citrus germplasm for HLB resistance often relies on graft inoculation as the mode of pathogen transmission. This study evaluates transmission efficiencies and HLB progression in graft-inoculated and psyllid-inoculated citrus under greenhouse and natural conditions in the field. Frequencies of transmission in graft-inoculated greenhouse-grown plants varied between experiments and were as high as 90% in susceptible sweet orange plants 6 to 12 months after inoculation. Transmission frequency in a tolerant Citrus × Poncirus genotype (US-802) was 31% to 75%. In contrast, transmission of Las after controlled psyllid inoculation did not exceed 38% in any of four experiments in this study. Whereas the time from inoculation to detection of Las by polymerase chain reaction (PCR) was faster in psyllid-inoculated US-802 plants compared with graft-inoculated US-802 plants, it was similar in graft- and psyllid-inoculated sweet orange plants. HLB symptom expression was indistinguishable in graft- and psyllid-inoculated plants but was not always associated with the number of bacteria in affected leaves. The highest number of Las genomes per gram leaf tissue measured in sweet orange plants was one to four × 107 in graft-inoculated plants and one to two × 107 in psyllid-inoculated plants. Highest numbers measured in tolerant US-802 plants were one to three × 106 and two to six × 106, respectively. Compared with artificial inoculation in a greenhouse setting, natural inoculation of field-grown sweet orange trees occurred at a much slower pace, requiring more than 1 year for infection incidence to reach 50% and a minimum of 3 years to reach 100%.

Sweet cherry (Prunus avium L.) is a deciduous tree originating in the Black Sea/Caspian Sea region where Asia and Europe converge. Being highly valued for its timber and fruit, sweet cherry has been cultivated and naturalized on all continents. Over the past decade, the area of sweet cherry cultivation increased rapidly in China and has reached 140,000 ha. In April 2013, sweet cherry trees (cv. Summit) exhibiting floral virescence symptoms were observed in two orchards located in suburban Taian, Shandong Province, China. The diseased trees developed flowers having white petals with green veins or abnormal floral structures having cupped, green petals. The affected flowers failed to set fruit. A month following the first appearance of the virescence symptoms, the diseased trees became wilted and eventually died. Leaf and stem samples were collected from nine symptomatic and two nearby symptomless trees. Total DNA was extracted from each sample using the Plant Quick DNA Extract Kit (TianGen, Beijing, China). Nested-PCR was carried out using phytoplasma-universal primer pairs P1/P7 and R16F2n/R16R2 (1). All PCR assays with DNA templates from symptomatic samples yielded an amplicon of 1.25 kb, corresponding to the full-length F2nR2 region of phytoplasmal 16S rDNA. No amplicon was generated in PCRs containing DNA templates from symptomless plants. The amplicons were cloned into plasmid vector pMD18-T (TaKaRa, Dalian, China) and sequenced. The obtained sequences were nearly identical, and a representative sequence was deposited into GenBank (Accession No. KF268424). An analysis of the sequence through the iPhyClassifier (4) revealed that the sweet cherry virescence (SCV) disease was associated with infection by a phytoplasma closely related to the reference strain of ‘Candidatus Phytoplasma ziziphi.’ The 16S rDNA F2nR2 region of the SCV phytoplasma shared 99.8% nucleotide sequence identity with that of ‘Candidatus Phytoplasma ziziphi’ reference strain (Accession No. AB052876). A computer-simulated restriction fragment length polymorphism (RFLP) analysis of the SCV phytoplasma 16S rDNA F2nR2 sequence with a set 17 restriction enzymes (3) resulted in a collective RFLP profile identical to the reference pattern of the elm yellows phytoplasma group, subgroup B (16SrV-B). Phytoplasmal diseases of sweet cherry were reported previously in Europe and the etiological agents were phytoplasmas of other groups, including the aster yellows group (16SrI), the X-disease group (16SrIII), and the apple proliferation group (16SrX) (2). To our knowledge, this is the first report of a phytoplasmal disease of sweet cherry in China, and the SCV phytoplasma is a new member of the subgroup 16SrV-B. Presence of 16SrV-B phytoplasmas and their etiological association with various plant diseases in China have been reported previously; affected host plants included jujube, hemp fiber, paper mulberry, Chinese cherry, plum, apricot, red barberry, clover, dianthus, elm, and sunshine tree. Our identification of the SCV phytoplasma expands the known plant host range of the 16SrV-B phytoplasma lineage. The impact of the SCV phytoplasma in the regional ecosystem and in sweet cherry production is being assessed. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) S. Paltrinieri et al. Acta Hort. 550:365, 2001. (3) W. Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Abstract The Miscellaneous Crenarchaeota group (MCG) Archaea is one of the predominant archaeal groups in anoxic environments and may have significant roles in the global biogeochemical cycles. However, no isolate of MCG has been cultivated or characterized to date. In this study, we investigated the genetic organization, ecophysiological properties and evolutionary relationships of MCG archaea with other archaeal members using metagenome information and the result of gene expression experiments. A comparison of the gene organizations and similarities around the 16S rRNA genes from all available MCG fosmid and cosmid clones revealed no significant synteny among genomic fragments, demonstrating that there are large genetic variations within members of the MCG. Phylogenetic analyses of large-subunit+small-subunit rRNA, concatenated ribosomal protein genes and topoisomerases IB gene (TopoIB) all demonstrate that MCG constituted a sister lineage to the newly proposed archaeal phylum Aigarchaeota and Thaumarchaeota. Genes involved in protocatechuate degradation and chemotaxis were found in a MCG fosmid 75G8 genome fragment, suggesting that this MCG member may have a role in the degradation of aromatic compounds. Moreover, the expression of a putative 4-carboxymuconolactone decarboxylase was observed when the sediment was supplemented with protocatechuate, further supporting the hypothesis that this MCG member degrades aromatic compounds.