Publications

4477

Abstract Background Huanglongbing (HLB) is a highly destructive citrus disease which threatens citrus production worldwide and ‘Candidatus Liberibacter asiaticus’ (Las), a non-culturable phloem-limited bacterium, is an associated causal agent of the disease. To better understand the physiological and molecular processes involved in host responses to Las, 2-DE and mass spectrometry analyses, as well as ICP spectroscopy analysis were employed to elucidate the global protein expression profiles and nutrient concentrations in leaves of Las-infected grapefruit plants at pre-symptomatic or symptomatic stages for HLB. Results This study identified 123 protein spots out of 191 spots that showed significant changes in the leaves of grapefruit plants in response to Las infection and all identified spots matched to 69 unique proteins/peptides. A down-regulation of 56 proteins including those associated with photosynthesis, protein synthesis, and metabolism was correlated with significant reductions in the concentrations of Ca, Mg, Fe, Zn, Mn, and Cu in leaves of grapefruit plants in response to Las infection, particularly in symptomatic plants. Oxygen-evolving enhancer (OEE) proteins, a PSI 9 kDa protein, and a Btf3-like protein were among a small group of proteins that were down-regulated in both pre-symptomatic and symptomatic plants in response to Las infection. Furthermore, a Las-mediated up-regulation of 13 grapefruit proteins was detected, which included Cu/Zn superoxide dismutase, chitinases, lectin-related proteins, miraculin-like proteins, peroxiredoxins and a CAP 160 protein. Interestingly, a Las-mediated up-regulation of granule-bound starch synthase was correlated with an increase in the K concentrations of pre-symptomatic and symptomatic plants. Conclusions This study constitutes the first attempt to characterize the interrelationships between protein expression and nutritional status of Las-infected pre-symptomatic or symptomatic grapefruit plants and sheds light on the physiological and molecular mechanisms associated with HLB disease development.

AbstractBackgroundHuanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. Previously, we reported a combination of Penicillin G and Streptomycin was effective in eliminating or suppressing the associated bacterium, ‘CandidatusLiberibacter asiaticus’ (Las).ResultsHere we report the bacterial composition and community structure in HLB-affected citrus plants during a growing season and while being treated with antibiotic combinations PS (Penicillin G and Streptomycin) and KO (Kasugamycin and Oxytetracycline) using the Phylochip™ G3 array. Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChip™ G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, of which five contained 100 or more OTUs,Proteobacteria(44.1%), Firmicutes(23.5%),Actinobacteria(12.4%),Bacteroidetes(6.6%) andCyanobacteria(3.2%). In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599. The over-all bacterial diversity decreased with the antibiotic treatments, as did the abundance of 11 OTUs withinProteobacteria,Firmicutes,BacteroidetesandPlanctomycetes. Within theProteobacteria, ten OTUs representing the classγ-proteobacteria increased in abundance after four months of treatment, when the Las bacterium was at its lowest level in the HLB-affected citrus field plants.ConclusionsOur data revealed thatProteobacteriawas constantly the dominant bacterial phylum recovered from citrus leaf midribs, with the α-proteobacterial and the γ-proteobacterial classes vying for prevalence. In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described. Bacterial cells in close proximity may be able to modify their microenvironment, making the composition of the microbial community an important factor in the ability of Las to cause HLB progression. A low Las level was seen as an annual fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.

Abstract Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.

During a survey carried out in Jordanian vineyards in August and October 2012, grapevine (Vitis vinifera L.) plants showing typical grapevine yellows (GY) disease symptoms, including leaf discoloration and curling, berry shriveling, and irregular maturation of wood, were observed. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing stunting and leaf chromatic alteration were found, suggesting the involvement of phytoplasmas in the disease etiology. Using a CTAB method, total DNA was extracted from leaf veins of 25 symptomatic and two asymptomatic grapevines, and from five symptomatic and two asymptomatic bindweeds for PCR analysis. DNAs from periwinkle (Catharanthus roseus (L.) G. Don) plants infected by ‘Ca. Phytoplasma asteris’ strain SAY (group 16SrI), ‘Ca. Phytoplasma solani’ strain STOL (group 16SrXII), and ‘Ca. Phytoplasma ulmi’ strain EY1 (group 16SrV), were used as positive controls. DNAs from healthy periwinkle and reactions without template DNA were employed as negative controls. 16S rDNA nested PCRs, carried out using the primer pairs P1/P7, followed by R16F2n/R16R2 (1), yielded an amplicon of the expected size (1,250-bp) in three grapevine and in five bindweed samples, and in positive controls. Amplicons were not produced with DNA from 22 symptomatic grapevines (probably because samples were collected late in the growing season and phytoplasma distribution in plants was non-uniform [2]); nor from asymptomatic plants and negative controls. PCR products were sequenced by commercial services in Italy (Primm, Milan) and Korea (Macrogen Inc., Soul). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank with accessions KC835139 (from grapevine) and KC835140 (from bindweed). The 16S rDNA nucleotide sequences of phytoplasmas identified in grapevine and bindweed in Jordan shared >99.5% sequence identity with ‘Ca. Phytoplasma solani’ reference strain STOL (AF248959), and carried identical STOL-unique signature sequences and distinguishing sequence blocks (3). Phylogenetic and in silico RFLP analyses confirmed the affiliation of phytoplasma strains identified in grapevine and bindweed in Jordan to the species ‘Ca. Phytoplasma solani’ (subgroup 16SrXII-A), opening an avenue to future studies on the dissemination and impact of Bois noir (BN) in Jordan. These studies may add new information about BN, previously reported in neighboring countries (4). Further studies will investigate the role of Hyalesthes obsoletus Signoret, a polyphagous Cixiidae responsible for the BN phytoplasma transmission in Europe, and other possible insect vector(s) in the BN spread in Jordan. References: (1) I.-M. Lee et al. Int. J. Syst. Bact. 48:1153, 1998. (2) F. E. Constable et al. Plant Pathol. 52:267, 2003. (3) F. Quaglino et al. Int. J. Syst. Evol. Microb. 63:2879. (4) E. Choueiri et al. Plant Dis. 86:697, 2002.

Three strictly anaerobic, methanogenic strains JH1T, JH4 and JH8 were isolated from rumen of the Korean native cattle (HanWoo; Bos taurus coreanae) in South Korea. The colonies were circular, opaque, and slightly yellowish. Phylogenetic analyses of 16S rRNA gene and mcrA (encoding α subunit of methyl-coenzyme M reductase) sequences confirmed the affiliation of the novel strains with the Methanobacteriales , and Methanobrevibacter wolinii SHT was the most closely related species. The 16S rRNA gene and mcrA sequence similarities between strains JH1T, JH4 and JH8 and M. wolinii SHT were 96.2 and 89.0 % respectively, and DNA–DNA hybridization of the isolates and M. wolinii DSM 11976T showed a 20 % reassociation. Strain JH1T exhibited 92 % DNA–DNA relatedness with strains JH4 and JH8, and their 16S rRNA gene and mcrA sequences were identical. Cells stained Gram-positive and were non-motile rods, 1.5–1.8 µm long and 0.6 µm wide. The strains were able to use H2/CO2 and formate. The optimum temperature and pH ranges for growth were 37–40 °C and pH 6.5–7.0. The DNA G+C content of strain JH1T was 28 mol%. Based on data from this study using a polyphasic approach, the three strains represent a novel species of genus Methanobrevibacter , for which the name Methanobrevibacter boviskoreani sp. nov. is proposed. The type strain is JH1T ( = KCTC 4102T = JCM 18376T).