Although it is usually latent on citrus, apple, and pear, apple stem grooving virus (ASGV) poses a great risk to many sensitive cultivars.  Since severe leaf yellow mottle mosaic (LYMM) symptoms have been observed on Huangjinmiyou (HJY) pummelos (Citrus grandis cv. Huangjinmiyou), a commercial variety that is widely cultivated in South China, high throughput sequencing (HTS) was used to find potential pathogens and only three divergent ASGV variants were identified.  The three ASGV variants shared 81.03–82.34% genome-wide pairwise identities with each other, and were separately closest to other ASGV variants from different hosts and/or geographical regions, as indicated by viral phylogenies.  However, these new variants may have developed from viral interstrain interactions, based on the results of recombination analysis.  A large-scale survey using reverse transcription-PCR (RT-PCR) protocols designed for the three ASGV variants revealed a high incidence (92.7–100%) of ASGV in symptomatic HJY trees from 11 major citrus-producing regions in China.  None of ASGV were detected in asymptomatic trees.  Temperature treatments applied to the symptomatic HJY plants showed that ASGV is sensitive to high temperatures (30–35°C), at which not only the plants recovered, but also the viruses were not detected by RT-PCR, while at low temperatures (20–24°C), both the symptoms and viruses remained detectable.  These data show that ASGV is associated with the LYMM disease prevalent on HJY in China, and this is the significant basis especially of taking appropriate measures timely to manage the disease.  

‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Firstly, stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG), and it was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×10⁴ to 1.4×10⁸ cells μg^-1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×10⁸ CLas cells μg^-1 DNA were 30% and 40% at 30 and 40 DAG, respectively. Direct tissue blot immune assay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in in vitro culture. This method will be useful for studying plant–HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’ (CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.

Citrus Huanglongbing (HLB, yellow shoot disease) is one of the most serious citrus diseases worldwide.  To better improve the detection sensitivity, a droplet digital PCR (ddPCR) assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent of HLB.  The detection of sensitivity comparison using positive plasmid indicated that ddPCR was superior to quantitative PCR (qPCR) for detecting and quantifying Las at low concentrations.  The Las detection of 40 field samples also showed that six of 13 asymptomatic samples (46.15%) with high C_t value (>35) were positive by ddPCR.  This methodology showed great potential for early HLB infection diagnosis.

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-derived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study.  Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus (CYVCV) isolate YN were obtained using the small RNA deep sequencing technology.  Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 (JX040635) and YN (KP313242), both of which belong to the genus Mandarivirus in the family Alphaflexiviridae.  Mapping analysis of viral-derived siRNA (vsiRNA) profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority.  BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon.  This is the first CYVCV isolate detected in Chongqing and the second in China.  This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region. 

In order to understand molecular characterization of Citrus tatter leaf virus (CTLV) isolated from China, full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome amplification by RT-PCR. The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6 497 nucleotides in length and shared 79.9–91.0% and 78.8–98.0% nucleotide sequence identity, respectively, with other Apple stem grooving virus (ASGV) or CTLV strains available in GenBank. Unexpectedly, CTLV-MTH showed the highest nucleotide sequence identity (91%) with an apple isolate of ASGV, followed by 86.5% with ASGV-HH and 85.7% with ASGV-CHN. Furthermore, CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree, suggesting it has a closer relationship to ASGV than to CTLV. Therefore, it can be concluded roughly that CTLV may be not a distinct strains of ASGV. We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.

The complete nucleotide sequence of an isolate of Citrus vein enation virus (CVEV-XZG) from China has been determined for the first time. The genome consisted of 5 983 nucleotides, coding for five open reading frames (ORFs), had a similar genomic organization features with Pea enation mosaic virus (PEMV). Nucleotide and deduced amino acid sequence identity of the five ORFs compared to isolate CVEV VE-1 range from 97.1 to 99.0% and 97.4 to 100.0%, these values compared to isolate PEMV-1 range from 45.2 to 51.6% and 31.1 to 45.2%. Phylogenetic analysis based on the complete genome sequence showed that the isolate CVEV-XZG had close relationship with Pea enation mosaic virus. The results supports CVEV may be a new member of genus Enamovirus. The full sequence of CVEV-XZG presented here may serve as a basis for future study of CVEV in China.

Two miniature inverted-repeat transposable elements (MITEs), MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus’ isolates from China, the United States and Brazil. Out of the 12 sequence types, three (T4, T5-2, T6) were reported for the first time. Recombination events were found in the two unique sequence types (T5-2 and T6) which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, São Paulo isolates and a few Florida isolates.

RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.

The genetic variation and phylogenetic relationships of Citrus tristeza virus (CTV) isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3´ proximal region was relatively conserved, and the 5´ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not reflect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.

Citrus tristeza virus (CTV) is one of the most important causal agents of citrus diseases and exists as numerous strains.CTV is replicated in phloem cells of plants within the family Rutaceae and is transmitted by a few of aphid species. CTVepidemics have caused death of millions of citrus trees in many regions all over the world, where the sour orange (Citrusaurantium) was used as rootstock. Also the production of grapefruit (C. paradisi) and sweet orange (C. sinensis) hasbeen affected by CTV strains. CTV gives uplift to three prominent syndromes, namely quick-decline (tristeza), stempittingand seedling-yellows. The disease is graft-transmissible in nature but not seed-transmitted. However, the tristezadisease in most citrus groves was a man-made problem created by the desire of horticulturists to introduce cultivars fromother citrus growing areas. The utmost importance of the disease called for review articles in numbers of plant protection,epidemiology books, citriculture and proceedings. This review collects the information with respects to disease history,distribution host range, virus isolates association, identification and detection, transmission and management; especiallyon the current status of CTV prevailing and controlling in Pakistan. It provides valuable information for CTV disease andits controlling approaches.