Zhongan Li

A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China

Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate the occurrence and distribution of citrus viroids in China.

Molecular characterization and phylogenetic analysis of Citrus viroid V isolates from Pakistan

Citrus viroid V (CVd-V) was recently characterized and belongs to the genus Apscaviroid within the family Pospiviroidae. 334 CVd-V isolates were identified from Punjab, Pakistan, where CVd-V had not been reported. A total of 68 independent cDNA clones were sequenced from 11 infected trees of different cultivars, ranging from 292 to 295 nucleotides. The nucleotide diversity estimated from the nucleotide distances of the CVd-V Pakistan population was similar to that reported from other countries. Based on genetic diversity and phylogenetic analysis, two main CVd-V groups were identified indicating that Pakistan might be one of the geographic origins of CVd-V worldwide. We demonstrated that this viroid has not emerged recently and it is more widespread than previously expected.

Detection and Characterization of Miniature Inverted-Repeat Transposable Elements in “Candidatus Liberibacter asiaticus”

Miniature inverted-repeat transposable elements (MITEs) are nonautonomous transposons (devoid of the transposase gene tps) that affect gene functions through insertion/deletion events. No transposon has yet been reported to occur in “Candidatus Liberibacter asiaticus,” an alphaproteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease). In this study, two MITEs, MCLas-A and MCLas-B, in “Ca. Liberibacter asiaticus” were detected, and the genome was characterized using 326 isolates collected in China and Florida. MCLas-A had three variants, ranging from 237 to 325 bp, and was inserted into a TTTAGG site of a prophage region. MCLas-A had a pair of 54-bp terminal inverted repeats (TIRs), which contained three tandem repeats of TGGTAACCAC. Both “filled” (with MITE) and “empty” (without MITE) states were detected, suggesting the MITE mobility. The empty sites of all bacterial isolates had TIR tandem repeat remnants (TRR). Frequencies of TRR types varied according to geographical origins. MCLas-B had four variants, ranging from 238 to 250 bp, and was inserted into a TA site of another “Ca. Liberibacter” prophage. The MITE, MCLas-B, had a pair of 23-bp TIRs containing no tandem repeats. No evidence of MCLas-B mobility was found. An identical open reading frame was found upstream of MCLas-A (229 bp) and MCLas-B (232 bp) and was predicted to be a putative tps, suggesting an in cis tps-MITE configuration. MCLas-A and MCLas-B were predominantly copresent in Florida isolates, whereas MCLas-A alone or MCLas-B alone was found in Chinese isolates.

Lack of evidence for seed transmission of Citrus yellow vein clearing virus despite its frequent detection in seed tissues.

Citrus yellow vein clearing virus (CYVCV), the putative causal agent of yellow vein clearing disease of citrus, is transmissible by grafting and is spread by aphids. However, whether CYVCV is seed-transmitted is not well documented. In this study, seed transmission of CYVCV was investigated in Daidai, Carrizo citrange and Rough lemon. CYVCV was detected in the endotesta, cotyledons and embryonic axis of seeds collected from the fruits of CYVCV-infected plants by qRT-PCR. Furthermore, the relative expression values of CYVCV were significantly higher in infected leaves than in the cotyledons or embryonic axis of seeds. Although viral RNA was found in few samples of radicles of germinating seed, none of the seedlings showed any symptoms for one year and were RT-PCR negative for CYVCV. The present study indicated that CYVCV could be localized in the seed tissues, but was not transmitted to the progeny. Absence of seed transmission may be consequent to the low CYVCV titre in embryo tissue.

Occurrence of Four Citrus Viroids in Chongqing, China

Chongqing Municipality, located in the superior citrus belt of the upper-middle Yangtze River, is one of the most important citrus-producing areas in China. A survey was performed to evaluate the occurrence and distribution of citrus viroids in this area, where Poncirus trifoliata is the main rootstock. From 2002 to 2006, 72 samples of sweet oranges (Citrus sinensis), lemons (C. jambhiri), mandarins (C. reticulata), and mandarin hybrids, which showed stunting, bark scaling, and cracking symptoms on the rootstock, were collected and graft inoculated into Arizona 861-S1 Etrog citron (C. medica) on rough lemon rootstock and maintained in a greenhouse at 28 to 32°C. Fifty-one of the seventy-two samples were cultivars imported from abroad, and the remaining samples were all local cultivars. Sixty samples induced symptoms typical of citrus viroids on the Etrog indicator plants. To identify the causal agent(s), a one-step reverse transcription (RT)-PCR protocol, using five primer pairs (1) targeting the complete genome sequences, was used to detect Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) (2). All 72 samples were infected by citrus viroids. Sixty-five and thirty-five of the seventy-two samples were positive for HSVd and CVd-III, respectively. CEVd and CBLVd were found, respectively, in 20 and 11 of 72 samples, whereas CVd-IV was not detected. Of 72 samples, 12 without typical symptoms on Etrog citrons were infected by HSVd and CBLVd. Nearly all (70 of 72) infected citrus plants harbored more than one viroid species, and two plants were both infected by CEVd, CBLVd, HSVd, and CVd-III. RT-PCR products were purified and ligated into pGEM T-Easy Vector (Promega, Madison, WI) and three clones for each of the four viroid species were sequenced and deposited in GenBank: CEVd (Accession Nos. EU382202, U382203, and EU382204); CBLVd (Accession Nos. EU382211, EU382212, and EU382213); HSVd (Accession Nos. EU382208, EU382209, and EU382210); and CVd-III (Accession Nos. EU382205, EU382206, and EU382207). BLAST analysis showed that these nucleotide sequences had greater than 94% nucleotide identity to the most similar genome sequences in GenBank. Sweet orange was more frequently infected by viroids than the other citrus cultivars. To our knowledge, this is the first report of CBLVd, HSVd, and CVd-III in Chongqing and the first report of CBLVd in China. The high incidence of citrus viroids in Chongqing necessitates rapid development of a system of propagation and testing to reduce the incidence of viroids and the associated loss of citrus production. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) K. Z. Tang et al. Acta Hortic. Sin. 32:408, 2005.

First report of citrus tristeza virus trifoliate resistance-breaking (RB) genotype in Citrus grandis in China

Citrus tristeza virus (CTV) isolates have been classified into six genotypes: T30, T36, VT, T3, T68 and RB (Harper 2013). In 2002, a CTV isolate CT9 was collected from Taibeiyou pummelo (Citrus grandis) in Santai, Sichuan Province. Biological indexing indicated that CT9 produced very mild stem pitting on Mexican lime (Citrus aurantifolia), Duncan grapefruit (C. paradisi) and Symons sweet orange (C. sinensis), and vein clearing onMexican lime (C. aurantifolia). CT9 did not produce decline on Daidai sour orange (C. aurantium). In previous studies, CT9 was used to protect Guanximiyou pummelo against a dominant severe stem-pitting CTV isolate CT3 in China. Furthermore, single strand conformation polymorphism (SSCP) analyses of the main coat protein gene of CT9 and its aphid transmission subisolates showed that CT9 contained two strains that could not be segregated by single aphid transmission using Toxoptera citricida (Zhou et al., 2005). To understand the composition of CT9 further, young leaf tissues were collected and sent to Beijing Genomics Institute for transcriptome sequencing. De novo assembly of 57.8 Mb sequenced reads was performed using CLC genomics Workbench (CLC bio v10.0, Denmark). Subsequent mapping enabled to identify two contigs similar to CTV. Sixteen primer pairs were designed according to the contigs (Table S1). RT-PCRproductswere cloned and sequenced and found to be the CTV genomic regions characterized from the contigs. The 5′ and 3′ termini were determined by rapid amplification of cDNA ends (RACE kit, Invitrogen). The two CTV full-length sequence variants, CN-RB-9 (MH558665) and CN-RB-L13 (MH558666) were respectively 19,270 nucleotides (nt) in length with a maximum identity of 99% to CTV isolate CA-RB-AT35 (KU358530) and 19,250 nt in length with a highest identity of 97% to HA18–9 (GQ454869). Phylogenetic analyses of full-genome sequence showed that CN-RB-9, CNRB-L13 and other extant RB isolates available in NCBI fell into the same clade, indicating that the two variants here characterized were RB genotype. Threemonths after CT9was graft-inoculated onto trifoliate orange (Poncirus trifoliate cv. ‘Flying dragon’) which is a true to type to Poncirus trifoliate, CT9 was detected by ELISA using the monoclonal antibody (Agdia, USA). The result indicated that CT9 could replicate inP. trifoliate, which has been extensively used as a rootstock in China over the past decades. This research provides information that may be relevant to control CTV by using cross protection.

Correction to: Molecular characterization of a novel citrivirus from citrus using next‑generation sequencing

Unfortunately, the Acknowledgement, ethical statement and the Conflict of interest statements were not included in the online publication and updated here in this Erratum.

Development of an immunochromatographic strip test for rapid detection of citrus yellow vein clearing virus

A rapid immunochromatographic strip (ICS) test for detection of citrus yellow vein clearing virus (CYVCV) was developed. The test is based on an antibody sandwich format and uses the monoclonal antibody (MAb) 1E1, which is specific for CYVCV. MAb 1E1 labeled with 30-nm colloidal gold particles was coated on a gold conjugate pad. A secondary goat anti-mouse IgG was coated on the surface of a nitrocellulose filter membrane (NC) as the control (C) line, while 1E1 was coated on the surface of the NC as the test (T) line. The ICS test was evaluated for specificity and sensitivity and then applied for virus detection in field samples. There was no cross-reaction with citrus tristeza virus (CTV), satsuma dwarf virus (SDV), citrus tatter leaf virus (CTLV), citrus exocortis viroid (CEVd), citrus mosaic virus (CiMV), citrus psorosis virus (CPV), citrus ringspot virus (RSV) or ‘Candidatus Liberibacter asiaticus’ (CLas). The ICS test was still able to detect CYVCV in tissue extracts at a dilution of 1: 320 (w/v), which is as efficient as the dot-ELISA assay. In general, the ICS assay is less expensive, faster and simpler to conduct than conventional CYVCV detection methods, so it may be useful for large-scale detection or monitoring of CYVCV.

Molecular characterization of a novel citrivirus from citrus using next-generation sequencing

A novel positive-strand RNA virus infecting citrus with the tentative name “citrus leaf blotch virus 2” (CLBV-2), was identified in the present work. The complete genome sequence of CLBV-2 comprises 8,697 nucleotides (nt) excluding a poly(A) tail and three open reading frames (ORFs), showing the highest nucleotide sequence identity with the Actinidia strain (JN983456) of citrus leaf blotch virus (CLBV). The putative movement protein (ORF2), coat protein (ORF3), and 3′ untranslated region (UTR) shared high sequence similarity with those of the extant CLBV isolates. In contrast, only low sequence similarity was observed in the 5′ UTR and putative replicase polyprotein (ORF1) regions. The distant phylogenetic relationship between CLBV-2 and CLBV was deduced based on whole-genome nucleotide and whole-ORF1 amino acid sequence comparisons. Sequence comparisons suggest that CLBV-2 acquired an ORF2-ORF3-3′ UTR region homologous to CLBV by recombination with of an unknown citrivirus. In view of the fact that this genomic recombination event appears to have occurred between members of different species in the genus Citrivirus, we propose that CLBV-2 should be considered a member of a distinct species.

Molecular characterization and phylogenetic analysis of Citrus viroid VI variants from citrus in China

Citrus viroid VI (CVd-VI) is a viroid originally found from citrus and persimmon in Japan. We report here the identification and molecular characterization of CVd-VI from four growth regions of China. A total of 90 cDNA clones from nine citrus cultivars were sequenced. The sequence homologies of the Chinese CVd-VI and the reference sequence (NC_004359) vary from 94.2% to 97%. The sequence homologies among the Chinese isolates were up to 95.2%. Phylogenetic analysis of the 23 CVd-VI variants from China and Japan showed that they were grouped into two clades, one with 20 citrus variants and another with three persimmon variants, regardless of the geographic origins. Therefore, as with Hop stunt viroid, CVd-VI could also be divided into two types, citrus and persimmon types. Sequence alignment showed that most nucleotide changes between the two clades occurred in the P, V and TL domains, and analysis indicated that these mutations influenced the predicted secondary structures under minimum energy.