Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus(CTV),usually occurs in nature as a mixture of genotypes.Six naturally infected citrus(Citrus sinensis)trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt(Sharqia,Qalyubia and Garbia).In this study,RT-PCR,Single-Strand Conformation Polymorphism(SSCP)and nucleotide sequence analysis were used for four independent CTV genomic regions(p65,p18,p20,and p23)to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates.RTPCR products(650 bp)for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing.SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns.Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7%with T36 isolate from USA,Florida.Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate(T36 isolate group),suggesting that they may have originated from closely related ancestors.Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang,p18,p20 and p65,amplified from isolate A3,Sharqia governorate,revealed that the p18,p65,and p20 genes were related to the T3-KB isolate from South Africa with 99%–100%sequence homology.Phylogenetic relationship analysis for p65,p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group.The recombination analysis identified three of six isolates from Sharqia,and Garbia as potential recombinant for p23 gene.The isolates T36 and T3 were identified as major donors for recombination events in isolate A3.Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event.The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.

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.

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

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, Ⅲ 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.

Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.

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.

Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

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 relfect 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.

Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences

The coat protein （CP） genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus （CTV） collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations （du） to synonymous mutations （ds） was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV.

Genetic Diversity and Global Distribution of Citrus tristeza virus (CTV) Strains

Citrus tristeza virus （CTV）, the most devastating viral pathogen in citrus, causes tremendous economic losses to citrus industry worldwide. The CTV isolates exhibit variable pathogenicities on their hosts indicating a mixed population of the CTV in nature. Several fragments within the CTV genome have been used for studying the genetic diversity of the CTV, however, the best region for rapid the CTV strain differentiation is still absent at present. In present study, a systemic analysis was carried out to evaluate the best region within the CTV genome for rapid CTV strain differentiation. Results of our study showed that the major coat protein （CP） coding region was the best region for this purpose. Using pair-wise distance frequency distribution plot, a reasonable genetic distance cut-off value was set for the CTV CP gene for the CTV strain differentiation. Using this criterion, eight CTV strains, including seven well characterized and a new strain, were successfully differentiated using 537 CTV isolates reported from 38 countries. The global strain distribution pattern was then determined and discussed. Our results also provided a new insight into the evolution and spreading of the virus, as well as the information for developing proper disease management strategy.

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

Citrus tristeza virus （CTV） is one of the most economically important citrus viruses and harms the citrus industry worldwide. To develop reliable and effective serological detection assays of CTV, the major capsid protein （CP） gene of CTV was expressed in Escherichia coli BL21 （DE3） using the expression vector pET-28a and purified through Ni＊-NTA affinity chromatography. The recombinant protein was used to immunize BALB/c mice. Four hybridoma cell lines （14B10, 14Hll, 20D5, and 20G12） secreting monoclonal antibodies （MAbs） against CTV were obtained through conventional hybridoma technology. The titers of MAb-containing ascitic fluids secreted by the four hybridoma lines ranged from 10-6 to 10.7 in indirect enzyme-linked immunosorbent assay （ELISA）. Western blots showed that all four MAbs could specifically react with CTV CP. Using the prepared MAbs, dot-ELISA, Tissue print-ELISA, and triple antibody sandwich （TAS）-ELISA were developed to detect CTV in tree nurseries and epidemiological studies. The developed dot-ELISA and TAS-ELISA methods could detect CTV in crude extracts of infected citrus leaves with dilutions of 1：2560 and 1：10, 240 （w/v, g/mL）, respectively. Tissue print-ELISA was particularly useful for large-scale field sample detection, mainly owing to its simplicity and lack of sample preparation requirements. The field survey revealed that CTV is prevalent on citrus trees in the Chongqing Municipality, Jiangxi Province, and Zhejiang Province of China. The coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.

Spatial and temporal spread of Citrus tristeza virus and its aphid vectors in the North western area of Morocco

First report of Citrus tristeza virus （CTV, Closterovirus） in Morocco datesback to 1961 in collections of citrus varieties. An exhaustive survey of citrus in the north of the country in 2009 revealed that CTV was spread all over the citrus production area. We attempted to evaluate the relative contribution of different aphid species in the spread of CTV disease in a Citrus reticulata orchard at the Loukkous region during 2 years （2012 and 2013）. The overall CTV incidence estimated in the experimental site increased from 17.8% in 2012 to 31.15% in 2013. The most abundant aphid species colonising clementine trees was Aphis spiraecola and A. gossypii. Both aphid species reached their maximum peaks during the spring season. The rate of viruliferous aphids, estimated by real-time RT-PCR of single aphid, revealed that 35.4% of winged A. gossypii and 28.8% of winged A. spiraecola were viruliferous, confirming a high inoculum pressure in the area surrounding the experimental site. The aphid species Toxoptera citricida, which is able to transmit the aggressive isolates of CTV, was not found in the Loukkous region. The study of the spatial distribution of the CTV showed that in general, the disease was randomly distributed in the field. Overall, the results seem to indicate that A. spiraecola may be considered as the major aphid species contributing to CTV spread in our experimental conditions. The prevalence of mild strains in the region and the high level of aphid flight activity could explain the rapid evolution of CTV incidence in the experimental area.

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza Closterovirus

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards.The citrus species grafted on sour orange rootstock are affected by this disease.Predominantly,the sweet orange,grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline,vein clearing,pin holing,bark scaling and degeneration leading to variable symptoms.Symptomatic expression of Citrus tristeza virus(CTV)in different hosts has been attributed to virus isolates which are from severe to mild.Different serological and molecular assays have been deployed to differentiate the strains of CTV.Citrus tristeza virus is diversified towards its strains on the basis of biological,serological and molecular characterization.Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation.This review will give a brief idea about the different CTV isolates,their characterization based on nucleic acid and serological assays.Different methods along with salient features for strain characterization has also been reviewed.This review will also open the new aspects towards formulation of management strategies through different detection techniques.