Candidate gene makers for Candidatus Liberibacter asiaticus for detecting citrus greening disease

Citrus Huanglongbing (HLB) also known as citrus greening is one of the most devastating diseases of citrus worldwide. The disease is caused by Candidatus Liberibacter bacterium, vectored by the psyllid Diaphorina citri Kuwayama and Trioza erytreae Del Guercio. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection. The aim of this study was to develop effective gene-specific primer pairs for polymerase chain reaction based method for quick screening of HLB disease. Thirty-two different gene-specific primer pairs, across the Ca. Liberibacter asiaticus genome, were successfully developed. The possibility of these primer pairs for cross-genome amplification across ‘Ca. Liberibacter africanus’ and ‘Ca. Liberibacter americanus’ were tested. The applicability of these primer pairs for detection and differentiation of Ca Liberibacter spp. is discussed.     

The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Predictive sequence analysis of the Candidatus Liberibacter asiaticus proteome

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a parasitic gram-negative bacterium that is closely associated with Huanglongbing (HLB), a worldwide citrus disease. Given the difficulty in culturing the bacterium and thus in its experimental characterization, computational analyses of the whole Ca. L. asiaticus proteome can provide much needed insights into the mechanisms of the disease and guide the development of treatment strategies. In this study, we applied state-of-the-art sequence analysis tools to every Ca. L. asiaticus protein. Our results are available as a public website at http://prodata.swmed.edu/liberibacter_asiaticus/. In particular, we manually curated the results to predict the subcellular localization, spatial structure and function of all Ca. L. asiaticus proteins (http://prodata.swmed.edu/liberibacter_asiaticus/curated/). This extensive information should facilitate the study of Ca. L. asiaticus proteome function and its relationship to disease. Pilot studies based on the information from our website have revealed several potential virulence factors, discussed herein.     

Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves

Huanglongbing is one of the most devastating diseases of citrus (Citrus spp.). One management tactic against huanglongbing is aggressive management of the vector, the Asian citrus psyllid (Diaphorina citri Kuwayama), with insecticide applications. However, D. citri in abandoned groves are not controlled and therefore pose a risk of reinfestation for nearby commercial citrus. These abandoned groves could serve as a reservoir for the vector, as well as a source of the presumed causal agent for huanglongbing in Florida, Candidatus Liberibacter asiaticus (Las). The current study was conducted to determine the degree to which Las is present in abandoned Florida citrus groves and to compare relative inoculum levels in nearby managed and abandoned groves during times of the year when D. citri are abundant (June, July, and August). In addition, the movement of Las by dispersing D. citri adults from inner and edge rows of abandoned grove plots to the corresponding rows of managed plots was quantified during the same 3 mo. The results of the current study confirmed the presence of Las in both D. citri and plant tissue in abandoned groves at statistically equivalent levels to those in nearby managed groves. The mean number of D. citri adults dispersing from abandoned to managed grove plots ranged from 7.25 +/- 1.70 to 70.25 +/- 21.25 per 4-d intervals. Of those, the mean number of dispersing D. citri adults that were carrying the Las pathogen ranged from 1.00 +/- 0.58 to 1.50 +/- 0.50. Our results indicate that abandoned citrus groves are a significant source of Ca. Las and that dispersing D. citri move this pathogen into nearby managed groves.     

Transmission parameters for Candidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae)

The purpose of this investigation was to evaluate acquisition and inoculation (together, transmission) efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with citrus huanglongbing (HLB) by the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae). In laboratory studies, nymphs reared on Las infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60 to 100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Las-infected plants. Similar rates of pathogen acquisition by psyllids after nymphal and adult feeding were observed in the field. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4 to 10%, whereas groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Las-positive adult psyllids, determined using quantitative real-time polymerase chain reaction, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long time period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Las by D. citri did not result in Las-infected plants after >1 yr of incubation after inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared with adults that acquired the pathogen as nymphs.     

柑橘黄龙病菌内参基因的筛选与评估

【背景】柑橘黄龙病是世界柑橘生产上最具毁灭性的病害之一,主要由候选韧皮部杆菌属亚洲种("Candidatus Liberibacter asiaticus",CLas)引起。CLas全基因组测序已经完成,因而该病原菌的基因表达研究和功能验证得以进行。【目的】筛选CLas内参基因并评估其不同侵染时期和在不同品种植物寄主中的表达稳定性。【方法】基于基因功能类别,利用实时荧光定量PCR技术分析23个CLas的候选内参基因相对表达情况(16Sr RNA基因作为参照基因)。结合Ct值标准差和geNorm、NormFinder、RefFinder软件,评价内参基因的表达稳定性。【结果】在感染黄龙病不同时期和不同品种的植物寄主样本中,14对引物表现出较强的特异性和稳定性。内参基因稳定性排名:ftsZgyrArpoB1ftsAsecAgapzapEgmk2rpoDsecYrpoOftsWgmk1recA,根据geNorm配对变异值Vn/n+1选择稳定性最好的ftsZ和gyrA作为内参基因作进一步评估。以ftsZ+gyrA以及16S rRNA基因分别作为内参基因检测柑橘黄龙病菌致病基因LasΔ5313的表达水平,所得的表达模式相同。【结论】柑橘黄龙病菌中涉及DNA复制和细胞分裂功能的管家基因表达较稳定,在CLas的基因表达研究中可选择ftsZ+gyrA的基因组合作为内参。本研究为后续利用实时荧光定量PCR分析CLas基因表达及研究CLas致病机理奠定基础。     

Sexual transmission of a plant pathogenic bacterium, Candidatus Liberibacter asiaticus, between conspecific insect vectors during mating

Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 μm in length and 0.19 to 0.39 μm in width. The spherical structures measured from 0.61 to 0.80 μm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees.     

Diversity of "Candidatus Liberibacter asiaticus," based on the omp gene sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of "Ca. Liberibacter asiaticus" isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species "Ca. Liberibacter asiaticus," even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating "Ca. Liberibacter asiaticus" isolates.     

Incidence of “Candidatus Liberibacter asiaticus” in a Florida population of Asian citrus psyllid

The incidence of a bacterium “Candidatus Liberibacter asiaticus” was assessed in a Florida population of Asian citrus psyllid, Diaphorina citri. The bacterium is the presumed causal agent of Asiatic huanglongbing, a serious citrus disease. Adult D. citri were periodically collected between May 2010 and September 2012 in a block of diseased trees located in east‐central Florida. The psyllids were individually subjected to molecular analyses (quantitative polymerase chain reaction assays using HLBaspr primers) to determine whether the bacterium was present and, if so, the population level of the pathogen based on qPCR cycle threshold (CT) values. Significantly greater percentages of females tested positive for the pathogen than males, but there were no significant differences between females and males with respect to population levels of the pathogen within the psyllids. No significant differences were found among the three D. citri colour morphs with respect to percentages of adults testing positive for the pathogen. Among 47 sample dates, a mean of 17.5% of adults per sample date tested positive (CT < 36) for the pathogen with a mean CT value of 31.1. The incidence of the pathogen was generally higher during late fall or early winter and often lower during mid‐ to late summer. There was a significant negative correlation between percentages of D. citri testing positive and air temperature. Increases in the incidence of the pathogen may not necessarily correspond to increases in transmission, as a number of factors both internal and external to D. citri can influence transmission. Transmission rates may be highest during periods when D. citri infestation levels are large, a high percentage of adults carry a high population of the pathogen in their salivary glands, and citrus flush is abundant.     

Differentiation of "Candidatus Liberibacter asiaticus" isolates by variable-number tandem-repeat analysis

Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of "Candidatus Liberibacter asiaticus." The Nei's measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia.     

Molecular and physiological properties associated with zebra complex disease in potatoes and its relation with Candidatus Liberibacter contents in psyllid vectors

Zebra complex (ZC) disease on potatoes is associated with Candidatus Liberibacter solanacearum (CLs), an α-proteobacterium that resides in the plant phloem and is transmitted by the potato psyllid Bactericera cockerelli (?ulc). The name ZC originates from the brown striping in fried chips of infected tubers, but the whole plants also exhibit a variety of morphological features and symptoms for which the physiological or molecular basis are not understood. We determined that compared to healthy plants, stems of ZC-plants accumulate starch and more than three-fold total protein, including gene expression regulatory factors (e.g. cyclophilin) and tuber storage proteins (e.g., patatins), indicating that ZC-affected stems are reprogrammed to exhibit tuber-like physiological properties. Furthermore, the total phenolic content in ZC potato stems was elevated two-fold, and amounts of polyphenol oxidase enzyme were also high, both serving to explain the ZC-hallmark rapid brown discoloration of air-exposed damaged tissue. Newly developed quantitative and/or conventional PCR demonstrated that the percentage of psyllids in laboratory colonies containing detectable levels of CLs and its titer could fluctuate over time with effects on colony prolificacy, but presumed reproduction-associated primary endosymbiont levels remained stable. Potato plants exposed in the laboratory to psyllid populations with relatively low-CLs content survived while exposure of plants to high-CLs psyllids rapidly culminated in a lethal collapse. In conclusion, we identified plant physiological biomarkers associated with the presence of ZC and/or CLs in the vegetative potato plant tissue and determined that the titer of CLs in the psyllid population directly affects the rate of disease development in plants.     

An HPLC-MS Characterization of the Changes in Sweet Orange Leaf Metabolite Profile following Infection by the Bacterial Pathogen Candidatus Liberibacter asiaticus

Huanglongbing (HLB) presumably caused by Candidatus Liberibacter asiaticus (CLas) threatens the commercial U.S. citrus crop of an annual value of $3 billion. The earliest shift in metabolite profiles of leaves from greenhouse-grown sweet orange trees infected with Clas, and of healthy leaves, was characterized by HPLC-MS concurrently with PCR testing for the presence of Clas bacteria and observation of disease symptoms. Twenty, 8-month-old ‘Valencia’ and ‘Hamlin’ trees were grafted with budwood from PCR-positive HLB source trees. Five graft-inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Thirteen weeks after inoculation, Clas was detected in newly growing flushes in 33% and 55% of the inoculated ‘Hamlin’ and ‘Valencia’ trees, respectively. Inoculated trees remained asymptomatic in the first 20 weeks, but developed symptoms 30 weeks after grafting. No significant differences in the leaf metabolite profiles were detected in Clas-infected trees 23 weeks after inoculation. However, 27 weeks after inoculation, differences in metabolite profiles between control leaves and those of Clas-infected trees were evident. Affected compounds were identified with authentic standards or structurally classified by their UV and mass spectra. Included among these compounds are flavonoid glycosides, polymethoxylated flavones, and hydroxycinnamates. Four structurally related hydroxycinnamate compounds increased more than 10-fold in leaves from ‘Hamlin’ and ‘Valencia’ sweet orange trees in response to Clas infection. Possible roles of these hydroxycinnamates as plant defense compounds against the Clas infection are discussed.     

Genetic Diversity of Candidatus Liberibacter asiaticus Based on Two Hypervariable Effector Genes in Thailand

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasA _I and lasA _II, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasA _I imply extensive variation exists within the full and partial repeat sequence while the single band from lasA _II indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.     

Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design

Candidatus Liberibacter asiaticus is the causal agent of Huanglongbing (HLB) disease of citrus. Current management practices have not been able to control HLB and stop the spread of HLB. The current study is focused on screening small molecule inhibitors against SecA protein of Ca. L. asiaticus. Homology modeling, structure based virtual screening and molecular docking methods have been used to find the novel inhibitory compounds against SecA activity at ATP binding region. At 20 μm 17 compounds showed >50% inhibition and four compounds had more than 65% inhibition. The most active compound has IC₅₀ value of 2.5 μM. The differences between the activities of the compounds are explained by their inter-molecular interactions at ATP binding site.