Inability to culture the phloem-restricted alpha-proteobacterium "Candidatus Liberibacter asiaticus" ("Ca. L. asiaticus") or the closely related species ("Candidatus Liberibacter americanus" and "Candidatus Liberibacter africanus") that are associated with Huanglongbing (HLB) hampers the development of effective long-term control strategies for this devastating disease. Here we report successful establishment and long-term maintenance of host-free "Ca. L. asiaticus" cultures, with the bacterium growing within cultured biofilms derived from infected citrus tissue. The biofilms were grown in a newly designed growth medium under specific conditions. The initial biofilm-based culture has been successfully maintained for over two years and has undergone over a dozen subcultures. Multiple independent cultures have been established and maintained in a biofilm reactor system, opening the door to the development of pure culture of "Ca. L. asiaticus" and the use of genetics-based methods to understand and mitigate the spread of HLB.

Host-free biofilm culture of "Candidatus Liberibacter asiaticus," the bacterium associated with Huanglongbing.

Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239–401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions

/pdf/evaluation-of-disease-severity-and-global-transcriptome-413xwimn2j.pdf

Evaluation of Disease Severity and Global Transcriptome Response Induced by Citrus bark cracking viroid, Hop latent viroid, and Their Co-Infection in Hop (Humulus lupulus L.).

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

Propagation and some physiological effects of Citrus bark cracking viroid and Apple fruit crinkle viroid in multiple infected hop (Humulus lupulus L.).

Ranaviruses are pathogenic viruses for poikilothermic vertebrates worldwide. The identification of a common midwife toad virus (CMTV) associated with massive die-offs in water frogs (Pelophylax spp.) in the Netherlands has increased awareness for emerging viruses in amphibians in the country. Complete genome sequencing of 13 ranavirus isolates collected from ten different sites in the period 2011-2016 revealed three CMTV groups present in distinct geographical areas in the Netherlands. Phylogenetic analysis showed that emerging viruses from the northern part of the Netherlands belonged to CMTV-NL group I. Group II and III viruses were derived from the animals located in the center-east and south of the country, and shared a more recent common ancestor to CMTV-amphibian associated ranaviruses reported in China, Italy, Denmark, and Switzerland. Field monitoring revealed differences in water frog host abundance at sites where distinct ranavirus groups occur; with ranavirus-associated deaths, host counts decreasing progressively, and few juveniles found in the north where CMTV-NL group I occurs but not in the south with CMTV-NL group III. Investigation of tandem repeats of coding genes gave no conclusive information about phylo-geographical clustering, while genetic analysis of the genomes revealed truncations in 17 genes across CMTV-NL groups II and III compared to group I. Further studies are needed to elucidate the contribution of these genes as well as environmental variables to explain the observed differences in host abundance.

Ranavirus genotypes in the Netherlands and their potential association with virulence in water frogs (Pelophylax spp.).

Two citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), have been reported and become potential threats to the citrus industry in Taiwan. The distributions and infection rates of two viroids have not been investigated since the two diseases were presented decades ago. The genetic diversities and evolutionary relationships of two viroids also remain unclear in the mix citrus planted region. Multiplex RT-PCR was used to detect the two viroids for the first time in seven main cultivars of citrus. Multiplex real-time RT-PCR quantified the distributions of two viroids in four citrus tissues. Sequence alignment and phylogenetic analysis were performed using the ClustalW and MEGA6 (neighbor-joining with p-distance model), respectively. HSVd was found more prevalent than CEVd (32.2% vs. 30.4%). Both CEVd and HSVd were commonly found simultaneously in the different citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggested that uneven distributions of both viroids with twig bark as the most appropriate material for studies involving viroid sampling such as quarantine inspection. Sequence alignment against Taiwanese isolates, along with analysis of secondary structure, revealed the existence of 10 and 5 major mutation sites in CEVd and HSVd, respectively. The mutation sites in CEVd were located at both ends of terminal and variability domains, whereas those in HSVd were situated in left terminal and pathogenicity domains. A phylogenetic analysis incorporating worldwide viroid isolates indicated three and two clusters for the Taiwanese isolates of CEVd and HSVd, respectively. Moderately high infection and co-infection rates of two viroids in certain citrus cultivars suggest that different citrus cultivars may play important roles in viroid infection and evolution. These data also demonstrate that two multiplex molecular detection methods developed in the present study provide powerful tools to understand the genetic diversities among viroid isolates and quantify viroids in citrus host. Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.

/pdf/multiplex-detection-distribution-and-genetic-diversity-of-28maonxwny.pdf

Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are commonly found simultaneously infecting different citrus cultivars in Taiwan. A crucial question to be addressed is how accumulations of these two viroids affect each other in an infected plant. In this study, we investigated the relationship between the two viroids at macroscopic and microscopic levels. CEVd and HSVd titers were examined by real-time RT-PCR in 17 plants of two citrus cultivars (blood orange and Murcott mandarin) every 3 months (spring, summer, fall and winter) from 2011 to 2013. Three nonparametric tests (Spearman’s rank correlation coefficient, Kendall’s tau rank correlation coefficient and Hoeffding’s inequality) were performed to test the correlation between CEVd and HSVd. Cellular and subcellular localizations of the two viroids were detected by digoxigenin- and colloidal gold-labeled in situ hybridization using light and transmission electron microscopy. The two viroids were unevenly distributed in four different types of citrus tissues (rootstock bark, roots, twig bark and leaves). Compared with blood orange, Murcott mandarin was generally more susceptible to CEVd and HSVd infection. Both viroids replicated and preferentially accumulated in the underground tissues of the two citrus cultivars. Except for blood orange at high temperatures, significant positive correlations were observed between the two viroids in specific tissues of both cultivars. Relative to concentrations under single-infection conditions, the CEVd population significantly increased under double infection during half of the 12 monitored seasons; in contrast, the population of HSVd significantly increased under double infection during only one season. At cellular/subcellular levels, the two viroids showed similar localization patterns in four tissues and the cells of these tissues in the two citrus cultivars. Our findings of titer enhancement, localization similarity, and lack of symptom aggravation under CEVd and HSVd double infection suggest that the two viroids have a positive relationship in citrus. The combination of molecular and cellular techniques used in this study provided evidence of titer correlation and localization of co-infecting viroids in the host. These methods may thus be useful tools for exploring viroid–viroid and viroid–host interactions.

/pdf/a-mutual-titer-enhancing-relationship-and-similar-27npwb28b2.pdf

A mutual titer-enhancing relationship and similar localization patterns between Citrus exocortis viroid and Hop stunt viroid co-infecting two citrus cultivars

Simple Summary Controlling Asian citrus psyllid (Diaphorina citri), the insect vector of Candidatus Liberibacter asiaticus (CLas), is an important part of Huanglongbing management. Specific viruses were recently discovered in a psyllid host where they may act as biological agents for minimizing psyllid populations or CLas transmission. We analyzed the psyllid gut virome from five regions in Florida and identified four D. citri-associated viruses at different compositions. The presence of these specific viruses in the gut indicated potential interactions between these viruses and CLas. Our results provided evidence that D. citri-associated viruses located in the host gut and their potential activities may be useful for pest management. Abstract Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.

/pdf/identifying-the-gut-virome-of-diaphorina-citri-from-florida-1w4jksh8.pdf

Identifying the Gut Virome of Diaphorina citri from Florida Groves

Citrus exocortis viroid (CEVd) infects nearly one third of the citrus plants in Taiwan, thereby becoming an increasing threat to the citrus industry. Using indicator plants to detect CEVd infection remains a reliable and specific bioassay method. Tomato (Solanum lycopersicum L.) is an ideal indicator plant for CEVd because it grows easily, develops fast and distinctly visible CEVd symptoms and has a well-characterized genome. However, the commonly used indicator cultivar Rutgers is not readily available and its seeds are very expensive and can only be obtained from the Asian Vegetable Research and Development Center (AVRDC), The World Vegetable Center in Taiwan. This study focused on finding new alternative indicator tomato plants from 29 commercial cultivars in Taiwan. Our reverse transcription (RT)-PCR results showed that 24 of the 29 cultivars could be infected. We selected two ('HV-206' and 'V-191') from these 24 for in depth analyses. Both cultivars exhibited obvious stunting and severe foliage symptoms when infected by two CEVd isolates from western (Yunlin isolate) and eastern (Hualien isolate) regions under three temperature regimes. Based on symptom severity, 'HV-206' was more susceptible to the Yunlin isolate and 'V-191' was more susceptible to the Hualien isolate. RT-PCR showed that CEVd replicated at higher levels in 'V-191' than in 'HV-206' at low and high temperatures. Although temperature was not a key factor in viroid titer for a given cultivar, higher temperatures accelerated symptom development. Based on our findings, we recommend 'HV-206' and 'V- 191' for use as alternative indicator plants in CEVd detection.

Identification of two commercial tomato cultivars as new indicator plants for detecting Citrus exocortis viroid infection

Pathological categorization of the stem‐pitting and mild strains Citrus tristeza virus in Taiwan and their genomic analysis

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