Fangyun Yang

Characterization of Citrus tristeza virus Isolates by Indicators and Molecular Biology Methods

Abstract Citrus tristeza virus (CTV) exists in citrus as a large number of distinct strains differing in biological characters. The control strategies such as mild strains cross protection (MSCP) require a clear understanding of the characterization of CTV. For better understanding of the structure of CTV population and the relationship between molecular and biological characterization, 72 CTV samples collected from five provinces in China were studied, using biological indexing, p25/Hinf I restriction fragment length polymorphism (RFLP), multiple molecular markers, and bidirectional RT-PCR assay. The mixture of severe stem pitting isolates was found to be dominant in the field. CTV isolates with p25/Hinf I RFLP group 3 and p23/BD-PCR group I, 111 were the main cause of epidemics, and most CTV isolates were found to be the mixture of T30 and VT genotypes. More accurate identification of strain mixtures in the field and better understanding of the biological traits of the isolates may be achieved by applying the three molecular detection methods simultaneously.

Molecular characterization of Citrus tristeza virus isolates from Pakistan based on CPG/Hinf I restriction fragment length polymorphism (RFLP) groups analysis

From six different districts of Punjab, Pakistan, 85 isolates of Citrus tristeza virus (CTV) were collected and characterized based on coat protein gene (CPG) analysis. All isolates were collected from field trees showing various CTV symptoms such as decline in most citrus varieties, inverse pitting on some sour orange rootstocks below bud union, mild-to-moderate stem-pitting on the trunk of some sweet orange. The CTV CP gene of all isolates was amplified by reverse transcriptase polymerase chain reaction (RT–PCR) using CP gene-specific primers yielding 672 bp. The maximum disease incidence was found in sweet orange followed by mandarin and grapefruit. These isolates were then subjected to CPG/Hinf I restriction fragment length polymorphism (RFLP) analysis. Mixed infection of CTV isolates was found very common in the field tress in Pakistan. The most dominant CPG/Hinf I RFLP groups III, I and VI are the basic causal epidemic in Pakistan. Moreover, based on symptoms in the field trees, CPG/Hinf I RFLP groups III, I and VI are considered to be the obvious causes of decline and stem-pitting in Pakistan. Key words: Citrus tristeza virus, CPG/Hinf I restriction fragment length polymorphism (RFLP) groups, decline, stem-pitting.

Preliminary Studies on CPG/Hinf I RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus (CTV) causes economically important losses to the citrus industry worldwide. Mild strain cross protection (MSCP) against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces (Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi) were tested by direct tissue blot immuno-assay (DTBIA), and 158 of them were tested positively, which therefore were subjected to coat protein gene (CPG)/Hinf I restriction fragment length polymorphism (RFLP) analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf I RFLP groups Ⅲ and I are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf I RFLP groups Ⅲ and I caused severe stem-pittings in sweet oranges in China.

Molecular characterization and phylogenetic analysis of Citrus viroid I-LSS variants from citrus in Pakistan and China reveals their possible geographic origin

CVd-I-LSS (low sequence similarity), a variant of Citrus bent leaf viroid (CBLVd), was first discovered in Japan, and its distribution is currently limited to Japan and Iran. In the present study, seven CVd-I-LSS isolates were detected from different citrus hosts (Citrus sinensis, C. reticulata and C. limettioides) in Pakistan and China. Genetic diversity analysis of 49 cDNAs of CVd-I-LSS isolates showed that the Pakistan population was more diverse than that tested from Japan or China. Phylogenetic analysis clustered the predominant sequences examined into three main clades. Only sequences from the Pakistan isolates were found in all three clades, suggesting Pakistan may be the original source of CVd-I-LSS. Cultivar import records and the close phylogenetic relationship found between CVd-I-LSS from China and Japan suggested that the viroid isolated from China might have originated from Japan.

Transcriptome sequencing reveals novel Citrus bark cracking viroid (CBCVd) variants from citrus and their molecular characterization

Citrus bark cracking viroid (CBCVd), previously called Citrus viroid IV, belongs to the genus Cocadviroid within the family Pospiviroidae. CBCVd has been identified as an important causative agent in citrus and hops. In this study, we obtained the full-length genomes of different variants of all detected citrus viroids from Pakistan through transcriptome sequencing. Different CBCVd variants were first found in Pakistan. These newly discovered Pakistani CBCVd variants were provisionally called “CBCVd-LSS” for their low sequence similarity (80.9%–88.9%) with the CBCVd RefSeq sequence (NC_003539). The two most predominant CBCVd sequences from Pakistan had the closest identity, 90.6% and 87.9%, with two CBCVd sequences isolated from hops. Identification and molecular characterization of CBCVd from citrus in Pakistan and China were also reported. The length of CBCVd from China ranged from 282 to 286 nucleotides, while that of the one from Pakistan ranged from 273 to 277 nucleotides. Based on genetic diversity and phylogenetic analysis, two main CBCVd clades were identified. CBCVd sequences from Pakistan, China, and other countries were further divided into six sub-clades. Sequence alignment revealed some nucleotide changes between these sub-clades, and analysis indicated that several mutations could significantly affect the primary and secondary structure of the viroid. Our results indicated that the CBCVd sequences from Pakistan and China were significantly different with respect to genome and secondary structure and Pakistan might be one of the independent geographical origins of CBCVd worldwide.

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.

Biological indexing and genetic analysis of Citrus tristeza virus in Pakistan

Citrus tristeza virus (CTV) is one of the major threats to production and fruit quality of citrus worldwide. In Pakistan, more than 95% of the citrus trees are grown on sour orange rootstock, which is highly susceptible to CTV. We studied the genetic variability of four genomic regions (p18, p20, p23 and p25) of CTV isolates collected from the citrus orchards. High divergence was revealed among the isolates from Pakistan and also with reference isolates. An inter-isolate identity range of 92.1–99.4% at the nucleotide level and 92.3–98.8% at the amino acid level was found. Phylogenetic analysis of the predominant sequence variants of each isolate revealed almost similar grouping of isolates for each gene. The groups revealed by phylogenetic trees include sequences from isolates that cause severe quick decline, seedling yellows and stem pitting (SP) and also from mild isolates. The high percentage of mixed infections is alarming because of the threat of further diversification and spread of severe variants into additional citrus-growing areas of Pakistan and neighboring countries. Eleven CTV isolates from Pakistan were biologically indexed, and most induced mild or mild to moderate reactions on all biological indicators. Four genomic regions of isolate 21C from the biological indexing experiment were sequenced and phylogenetically analysed. These results provide the basis for mild strain cross protection (MSCP) in Pakistan and in neighbouring countries in the near future.

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.