Mengji Cao

Distribution and Research Advances of Citrus tristeza virus

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.CTV epidemics have caused death of millions of citrus trees in many regions all over the world,where the sour orange (Citrus aurantium) was used as rootstock.Also the production of grapefruit (C.paradisi) and sweet orange (C.sinensis) has been affected by CTV strains.CTV gives uplift to three prominent syndromes,namely quick-decline (tristeza),stempitting and seedling-yellows.The disease is graft-transmissible in nature but not seed-transmitted.However,the tristeza disease in most citrus groves was a man-made problem created by the desire of horticulturists to introduce cultivars from other 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;especially on the current status of CTV prevailing and controlling in Pakistan.It provides valuable information for CTV disease and its controlling approaches.

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.

Occurrence, Distribution, and Molecular Characterization of Citrus yellow vein clearing virus in China

In 2009, a new citrus viral disease caused by Citrus yellow vein clearing virus (CYVCV) was discovered in China. To more effectively monitor the presence of CYVCV, a survey was conducted in 166 citrus orchards from 11 major citrus-growing provinces in China from May 2014 to April 2016. In all, 458 of a total of 2,350 citrus samples tested positive for CYVCV, demonstrating that the virus is widely distributed in China. In this study, the complete genome sequences of 19 CYVCV isolates from different provinces and hosts were sequenced and characterized. Comparisons of the whole-genome sequences of these 19 CYVCV isolates as well as 4 isolates previously reported from around the world revealed that the sequence identity ranged from 97.1 to 99.8%, indicating that there is a very low level of sequence heterogeneity among CYVCV isolates of different geographic origins and hosts. Phylogenetic analysis of these 23 genomic sequences suggested that all of the isolates from China were clustered into the same clade, clearly apart from the CYVCV isolates from Turkey and Pakistan. To our knowledge, this is the first extensive survey conducted in China for CYVCV incidence.

Sequence analysis and genomic organization of a new insect iflavirus, sogatella furcifera honeydew virus 1

A novel iflavirus, tentatively named “Sogatella furcifera honeydew virus 1” (SFHV1), discovered through transcriptome sequencing analysis of white-backed planthoppers (Sogatella furcifera) collected in southern China, is described here. The full genome of SFHV1 is 10,837 nucleotides (nt) long, including the polyA tail, and shares 65.5% and 64.5% genomic identity with Laodelphax striatellus picorna-like virus 2 and Laodelphax striatella honeydew virus 1, respectively. On the basis of the phylogenetic analysis of the complete genomic sequence and the deduced RdRp amino acid sequence of SFHV1 with other iflaviruses, we suggest that it is a member of a new species in the genus Iflavirus, family Iflaviridae.

Molecular characterization of a novel luteovirus infecting apple by next-generation sequencing

A new single-stranded positive-sense RNA virus, which shares the highest nucleotide (nt) sequence identity of 53.4% with the genome sequence of cherry-associated luteovirus South Korean isolate (ChALV-SK, genus Luteovirus), was discovered in this work. It is provisionally named apple-associated luteovirus (AaLV). The complete genome sequence of AaLV comprises 5,890 nt and contains eight open reading frames (ORFs), in a very similar arrangement that is typical of members of the genus Luteovirus. When compared with other members of the family Luteoviridae, ORF1 of AaLV was found to encompass another ORF, ORF1a, which encodes a putative 32.9-kDa protein. The ORF1-ORF2 region (RNA-dependent RNA polymerase, RdRP) showed the greatest amino acid (aa) sequence identity (59.7%) to that of cherry-associated luteovirus Czech Republic isolate (ChALV-CZ, genus Luteovirus). The results of genome sequence comparisons and phylogenetic analysis, suggest that AaLV should be a member of a novel species in the genus Luteovirus. To our knowledge, it is the sixth member of the genus Luteovirus reported to naturally infect rosaceous plants.

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.

Identification and Characterization of Wheat Yellow Striate Virus, a Novel Leafhopper-Transmitted Nucleorhabdovirus Infecting Wheat

A new wheat viral disease was found in China. Bullet-shaped viral particles within the nucleus of the infected wheat leave cells, which possessed 180–210 nm length and 35–40 nm width, were observed under transmission electron microscopy. A putative wheat-infecting rhabdovirus vectored by the leafhopper Psammotettix alienus was identified and tentatively named wheat yellow striate virus (WYSV). The full-length nucleotide sequence of WYSV was determined using transcriptome sequencing and RACE analysis of both wheat samples and leafhoppers P. alienus. The negative-sense RNA genome of WYSV contains 14,486 nucleotides (nt) and seven open reading frames (ORFs) encode deduced proteins in the order N-P-P3-M-P6-G-L on the antisense strand. In addition, WYSV genome has a 76-nt 3′ leader RNA and a 258-nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. The entire genome sequence shares 58.1 and 57.7% nucleotide sequence identity with two strains of rice yellow stunt virus (RYSV-A and RYSV-B) genomes, respectively. The highest amino acid sequence identity was 63.8% between the L proteins of the WYSV and RYSV-B, but the lowest was 29.5% between the P6 proteins of these viruses. Phylogenetic analysis firmly established WYSV as a new member of the genus Nucleorhabdovirus. Collectively, this study provided evidence that WYSV is likely the first nucleorhabdovirus described infecting wheat via leafhopper P. alienus transmission.

Discovery of a novel geminivirus associated with camellia chlorotic dwarf disease

Next-generation sequencing of small RNAs and PCR amplification with two pairs of back-to-back primers, followed by cloning and sequencing, allowed identification of a novel geminivirus isolate provisionally named camellia chlorotic dwarf-associated virus (CaCDaV). The complete genome sequence of CaCDaV comprises 3,687 nucleotides (nts) and six open reading frames (ORFs). The genome and putative proteins of CaCDaV were most closely related to those of two unclassified geminiviruses: citrus chlorotic dwarf-associated virus (CCDaV, 3,640 nt) and mulberry mosaic dwarf-associated virus (MMDaV, 2,952 nt). Phylogenetic analysis revealed a distinct clade for CaCDaV, CCDaV and MMDaV, supporting their inclusion in a new genus within the family Geminiviridae. Because CaCDaV and CCDaV shared a high genome nucleotide sequence identity (54.8%), we propose that the CaCDaV should be included as a member in this new genus.

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.

Complete genome sequences of four isolates of Citrus leaf blotch virus from citrus in China

Abstract Citrus leaf blotch virus (CLBV) is a member of the genus Citrivirus, in the family Betaflexiviridae. It has been reported CLBV could infect kiwi, citrus and sweet cherry in China. Of 289 citrus samples from six regions of China, 15 were detected to be infected with CLBV in this study. The complete genome of four isolates of CLBV was obtained from Reikou in Sichuan (CLBV-LH), Yura Wase in Zhejiang (CLBV-YL), Bingtangcheng in Hunan (CLBV-BT), Fengjie 72-1 in Chongqing (CLBV-FJ), respectively. While they all represented 8 747 nucleotides in monopartite size, excluding the poly(A) tail, each of the isolates coded three open reading frames (ORFs). Identity of the four isolates ranged from 98.9 to 99.8% to each other and from 96.8 to 98.1% to the citrus references in GenBank by multiple alignment of genomes. A phylogenetic tree based on the genome sequences of available CLBV isolates indicated that the four isolates were clustered together, suggesting that CLBV isolates from citrus in China did not have obvious variation. This is the first report of the complete nucleotide sequences of CLBV isolates infecting citrus in China.