Characterization and Management of Citrus Tristeza Virus in Hawai'i
Characterization and Management of Citrus Tristeza Virus in Hawai'i

Author: Michael J. Melzer

Publisher:

Published: 2009

Total Pages: 408

ISBN-13: 9781109405774

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Diseases associated with Citrus tristeza virus (CTV) are a major bottleneck for Hawai'i's citrus growers. To increase citrus production, the incidence, distribution, and genetic diversity of Hawaiian CTV must first be characterized. From this data, control strategies can then be employed to manage this virus. In this study, a comprehensive survey of CTV was conducted and it was found that approximately three-quarters of Hawai'i's citrus trees were infected by CTV, and that no locations where citrus is currently grown are free of the virus. Genotyping using molecular markers and sequencing of coat protein and p23 genes of CTV revealed that the genetic diversity of Hawai'i's CTV population is also very high. Many trees appeared to be infected with multiple and/or recombinant virus strains, as well as previously unreported strains. To further characterize potentially novel strains of the virus, a dsRNA cloning strategy based on random (r)PCR and degenerate oligonucleotide primer (DOP)-PCR was developed that required sub-nanogram amounts of dsRNA template to construct cDNA libraries. Two new strains of CTV were completely sequenced using this strategy, one of which appeared to have arisen through modular recombination between two distantly-related strains of the virus. Given the robustness of Hawaii's CTV population, the development of CTV-resistant citrus via transgene silencing appears to be the most effective management option. Transgenes designed to counter this genetic diversity, as well as the three suppressors of gene silencing encoded in the CTV genome, were introduced into the Mexican lime (Citrus aurantifolia (Christm.) Swingle] genome. These transgenic plants are currently under evaluation for their ability to resist CTV infection.

Molecular and Biological Characterization of Three Citrus Tristeza Virus Candidate Cross-protection Sources
Molecular and Biological Characterization of Three Citrus Tristeza Virus Candidate Cross-protection Sources

Author: Jacoba Wilhelmina Lubbe

Publisher:

Published: 2015

Total Pages: 444

ISBN-13:

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Citrus tristeza virus (CTV) is a RNA plant virus that infects the phloem cells of members of the family Rutaceae. CTV has a very important impact on the citrus industry worldwide and in South Africa especially so on grapefruit. CTV isolates can cause differing levels of severity of Tristeza disease, which can lead to quick decline as well as stem pitting and seedling yellows. Mild strain cross-protection is commonly used in South Africa to control the negative effects of the virus. This control mechanism is based on the super-infection exclusion principle where the presence of one specific genotype of CTV prevents the secondary infection of strains of the same genotype. This necessitates the characterization of CTV sources occurring within given citrus producing areas to know which genotypes to protect against, as well as the thorough characterization of potential cross-protection sources to ensure the specific genotypes that need to be protected against are present and to ensure that there are no strains within the source that would cause severe symptoms. The aim of this study was to characterize several sources of CTV which could potentially be used for cross-protection and at the same time to use and evaluate several methods for this. By doing next generation sequencing on an overlapping amplicon template of the 3’ half of the genome it was found that the three Grape Fruit Mild Strain 12 sub isolates, GFMS 12-7, 12-8 and 12-9 mostly exists of a T68 genotype previously identified as CT-ZA3. Using immuno-captured virus particles as template, followed by the production of cDNA through the use of degenerate primers and random amplification of the DNA as well as a p33 gene amplicon for next generation sequencing, it was found that the New Venture 41/2 candidate mild source is a mixed source containing at least the VT, RB, B165 and HA16-5 genotypes. The B390/3 candidate mild source was characterized through biological indexing and was found to only produce mild symptoms on the hosts used in the trial. The virus population was also characterized through Sanger and Illumina sequencing of the p33 gene as well as using genotype specific RT-PCRs. The source is dominated by a Taiwan-Pum/SP/T1–like isolate which belongs to the RB genotype. Additionally a comprehensive phylogenetic analysis was performed on 45 published complete genomes of CTV where it was shown that 9 genotypes exist, namely VT, T36, RB, T30, B165, T68, HA16-5, T3 and A18. The best method for genotyping, as found to produce the phylograms most similar to the complete genome phylograms, was found to be by doing a Bayesian analysis on a concatenated dataset of three segments of the genome, namely ORF 1b, ORF 2 and ORF 5.

Citrus Tristeza Virus
Citrus Tristeza Virus

Author: Antonino F. Catara

Publisher: Humana

Published: 2019-06-25

Total Pages: 247

ISBN-13: 9781493995578

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This book provides methods and clear protocols for the various technologies available to detect, characterize, and study Citrus tristeza virus (CTV), a member of the genus Closterovirus, family Closteroviridae. Thanks to the highly sensitive and specific diagnostic procedures developed, knowledge of the molecular characteristics, expression strategies, genetic variability, and epidemiology of the virus has improved significantly, as this volume reflects. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Citrus Tristeza Virus: Methods and Protocols serves as an ideal guide for plant pathologists, plant virologists, molecular biologists, and graduate students interested in performing qualitative and quantitative tests as well as recently-developed diagnostic methods in order to find solutions to improve the management of this disease.

Citrus Tristeza Virus
Citrus Tristeza Virus

Author: Caroline Mary Herron

Publisher:

Published: 2003

Total Pages: 0

ISBN-13:

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Citrus tristeza virus (CTV), an economically important graft-transmissible pathogen of citrus, causes major global declines in citrus production. In the commercial citrus of the Lower Rio Grande Valley of Texas (LRGV), where red grapefruit on tristeza-decline sensitive sour orange rootstocks predominate, incidence of CTV is low. The efficient CTV vector, the brown citrus aphid (BrCA, Toxoptera citricida Kirkaldy) is now established in Mexico and Florida, thus information is needed on the severity of CTV, CTV aphid transmission and the performance of transformed citrus towards CTV before T. citricida arrives in Texas so that appropriate management strategies can be selected. Biological indexing and molecular typing were performed on fifteen Texas CTV isolates. The majority of the CTV isolates tested contained the most severe CTV types known. In Florida, T. citricida were fed on crude CTV preparations in vitro and could transmit CTV to virus-free receptor plants with two CTV isolates, whereas a more highly purified CTV preparation from one CTV isolate was not transmitted by T. citricida. There were no differences in the majority of treatments in infectivity neutralizations using three CTV-derived antibodies (p25, p27 and p20). CTV p20 antibodies significantly enhanced the occurrence of CTV transmission in one test. The CTV genome of isolate H33 was sequenced using 'shot gun' methods. The H33 major component and H33 minor components were phylogenetically compared to the six other full-length CTV sequences. An untranslatable CTV coat protein gene was genetically transformed into the genome of the Texas commercial Rio Red grapefruit variety, and fifty-two independent transgenic lines were produced. CTV challenge responses by the transgenic lines were variable. Individual plants could be identified which had low virus titers by ELISA detection, a temporal decrease in virus titer, or a delay in virus titer accumulation. Comparing all wild types to all transgenic lines over every assessment revealed significant decreases in virus titer in the transgenic lines compared to that of the wild type. An RNA entity with similarities to marafiviruses was identified in a CTV infected plant. The entity appears non-graft transmissible to citrus, and non-mechanically transmissible to a range of herbaceous species.

Studies on the Citrus Tristeza Virus and Citrus Viroids in Pakistan
Studies on the Citrus Tristeza Virus and Citrus Viroids in Pakistan

Author: Sagheer Atta

Publisher: LAP Lambert Academic Publishing

Published: 2012

Total Pages: 148

ISBN-13: 9783659209963

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Citrus tristeza virus (CTV) is distributed worldwide, and is the causal agent of one of the most economically important and destructive diseases of citrus.Direct tissue blot immuno-assay (DTBIA) and reverse transcription polymerase chain reaction (RT-PCR) were used to pre-select CTV isolates. CTV-infected samples were then preserved in the sweet orange by graft-inoculation. The target sequences of the major coat protection gene (p25 gene) were amplified from the CTV-infected samples using RT-PCR, and the p25 genes of these isolates were analyzed by RFLP and SSCP for investigating the p25/Hinf I RFLP groups. Genetic differences and genetic relationships and the sequences of 4 genomic regions p23, p20, p18, and p25 of the 21 CTV isolates were amplified and sequenced using RT-PCR and with a sequencer. Using sequence analysis, the differences were therefore estimated among the CTV isolates from cultivated citrus. Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) were detected by one-step multiplex RT-PCR assay simultaneously.

Detection and Isolation of Homogeneous Genotypes of Citrus Tristeza Virus for Use in Virus Control Through Cross Protection
Detection and Isolation of Homogeneous Genotypes of Citrus Tristeza Virus for Use in Virus Control Through Cross Protection

Author: Kirsti Snyders

Publisher:

Published: 2015

Total Pages: 492

ISBN-13:

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One of the most serious diseases of citrus worldwide is caused by the viral pathogen Citrus tristeza virus (CTV) (family: Closteroviridae, genus: Closterovrius). This RNA virus is known to cause several economically important syndromes, namely quick decline , stem pitting and seedling yellows . The natural hosts for CTV include nearly all citrus species and cultivars, various citrus relatives such as Aeglopsis, Afraegle, Fortunella and Pamburus, and some intergeneric hybrids (Yokomi, 2009). The movement of CTV between different regions is mainly due to the dispersal of infected budwood (Bertolini et al., 2008). However, natural transmission and spread of CTV can also occur with the help of several aphid species of which the most effective vector is the Brown citrus aphid (Kirkaldy) Toxoptera citricida (Tsai et al., 2009). The severity of a CTV infection may vary depending on various contributing factors, such as environmental conditions, the rootstock combination used and lastly the presence of the CTV genotype predominating within the mixed infection. Currently, there are eight known and published CTV genotypes, each with their own degree of virulence, and up to date, forty-seven CTV genomes have been fully sequenced. It has been observed in numerous CTV population diversity studies, that a host is normally infected with a mixture of genotypic strains (Read and Pietersen, 2015; Roy et al., 2010; Roy and Brlansky, 2004). This complicates biological indexing as well as diagnostic procedures in identifying homogenous genotypic sources. This pathogen is endemic to the citrus producing areas in South Africa; hence, a mild strain cross-protection scheme has been initiated during the year 1973; aiming to control the viral disease.