scholarly journals Prereaction of Citrus tristeza virus (CTV) Specific Antibodies and Labeled Secondary Antibodies Increases Speed of Direct Tissue Blot Immunoassay for CTV

Plant Disease ◽  
2006 ◽  
Vol 90 (5) ◽  
pp. 675-679 ◽  
Author(s):  
Youjian Lin ◽  
Phyllis A. Rundell ◽  
Lianhui Xie ◽  
Charles A. Powell
Keyword(s):  
Citrus Tristeza Virus ◽  
Plant Viruses ◽  
Specific Antibodies ◽  
Rabbit Igg ◽  
Tissue Blot Immunoassay ◽  
Polymerase Chain ◽  
Bacteria And Fungi ◽  
Secondary Antibodies ◽  
Tristeza Virus ◽  
Citrus Tristeza

An improved direct tissue blot immunoassay (DTBIA) procedure for detection of Citrus tristeza virus (CTV) within 1 h is described. Prints of fresh young stems of citrus plants that were infected or not infected with CTV were made by gently and evenly pressing the fresh-cut surface of the stems onto a nitrocellulose membrane. The tissue blots were air-dried for 5 min, incubated with prereaction solutions of CTV-specific antibodies and labeled secondary antibodies, goat anti-mouse Ig (H+L)-alkaline phosphatase conjugate or goat anti-rabbit IgG alkaline phos-phatase conjugate, for up to 20 min, rinsed with PBST buffer for 5 min, and immersed into an NBT-BCIP substrate solution for 15 to 20 min. Then the blots were rinsed in water for a few seconds to stop the reactions, and the results were observed and recorded under a light microscope. All samples from greenhouse plants that were infected with CTV decline inducing isolate T-36 were positive to CTV-specific polyclonal antibody 1212 (PCA 1212) and monoclonal antibodies 17G11 (MAb 17G11) and MCA13 (MAb MCA13), whereas samples from greenhouse plants infected with non-decline-inducing isolate T-30 were positive to PCA 1212 and MAb 17G11, but not to MAb MCA13. The noninfected greenhouse plants were negative to all of the antibodies. The improved DTBIA was at least as reliable as other immunological procedures and almost as reliable as polymerase chain reaction for detecting CTV in field trees. The improved DTBIA enables the detection of CTV within 1 h by having a prereaction of CTV-specific antibodies and labeled secondary antibodies in solutions before they are applied to the tissue blots. This DTBIA procedure may be useful in detecting other plant viruses and other pathogens such as bacteria and fungi.

scholarly journals Walking Together: Cross-Protection, Genome Conservation, and the Replication Machinery of Citrus tristeza virus

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1353
Author(s):  
Svetlana Y. Folimonova ◽  
Diann Achor ◽  
Moshe Bar-Joseph
Keyword(s):  
Citrus Tristeza Virus ◽  
Fluorescent Protein ◽  
Field Tests ◽  
Fruit Trees ◽  
Plant Viruses ◽  
Cross Protection ◽  
Virus Variant ◽  
Tristeza Virus ◽  
Direct Confirmation ◽  
Citrus Tristeza

“Cross-protection”, a nearly 100 years-old virological term, is suggested to be changed to “close protection”. Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that “pre-immunization” of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.

scholarly journals Citrus tristeza virus P33 Protein Is Required for Efficient Transmission by the Aphid Aphis (Toxoptera) citricidus (Kirkaldy)

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1131
Author(s):  
Turksen Shilts ◽  
Choaa El-Mohtar ◽  
William O. Dawson ◽  
Nabil Killiny
Keyword(s):  
Citrus Tristeza Virus ◽  
Transmission Rate ◽  
Control Strategies ◽  
Virus Transmission ◽  
Plant Viruses ◽  
Transmission Mechanisms ◽  
Infectious Clones ◽  
Underlying Mechanisms ◽  
Tristeza Virus ◽  
Citrus Tristeza

Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered one of the most economically important plant viruses. CTV has destroyed millions of citrus trees in many regions of the world. Consequently, understanding of the transmission mechanism of CTV by its main vector, the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), may lead to better control strategies for CTV. The objective of this study was to understand the CTV–vector relationship by exploring the influence of viral genetic diversity on virus transmission. We built several infectious clones with different 5′-proximal ends from different CTV strains and assessed their transmission by the brown citrus aphid. Replacement of the 5′- end of the T36 isolate with that of the T30 strain (poorly transmitted) did not increase the transmission rate of T36, whereas replacement with that of the T68-1 isolate (highly transmitted) increased the transmission rate of T36 from 1.5 to 23%. Finally, substitution of p33 gene of the T36 strain with that of T68 increased the transmission rate from 1.5% to 17.8%. Although the underlying mechanisms that regulate the CTV transmission process by aphids have been explored in many ways, the roles of specific viral proteins are still not explicit. Our findings will improve our understanding of the transmission mechanisms of CTV by its aphid vector and may lead to the development of control strategies that interfere with its transmission by vector.

scholarly journals Development and Application of a Multiplex Reverse-Transcription Polymerase Chain Reaction Assay for Screening a Global Collection of Citrus tristeza virus Isolates

2010 ◽  
Vol 100 (10) ◽  
pp. 1077-1088 ◽  
Author(s):  
Avijit Roy ◽  
G. Ananthakrishnan ◽  
John S. Hartung ◽  
R. H. Brlansky
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Phylogenetic Analyses ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Variable Regions ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

scholarly journals First field records of the sexuales (males and oviparae) of Toxoptera aurantii (Hemiptera: Aphididae)

Zootaxa ◽  
2011 ◽  
Vol 2836 (1) ◽  
pp. 62
Author(s):  
NICOLÁS PÉREZ HIDALGO ◽  
DAVID MIFSUD
Keyword(s):  
Citrus Tristeza Virus ◽  
Plant Viruses ◽  
Toxoptera Aurantii ◽  
Tristeza Virus ◽  
Citrus Tristeza

Toxoptera aurantii (Boyer de Fonscolombe) is an aphid pest of great agricultural importance, not only due to its polyphagy but mostly due to its ability to transmit various plant viruses e.g. Citrus tristeza virus on Citrus (Blackman & Eastop, 2000). Blackman & Eastop (2000) considered this aphid to be entirely anholocyclic, because no sexual morphs have ever been observed in the field.

scholarly journals Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza virus by Real-Time Reverse Transcription-Polymerase Chain Reaction Assays

2010 ◽  
Vol 100 (4) ◽  
pp. 319-327 ◽  
Author(s):  
R. K. Yokomi ◽  
M. Saponari ◽  
P. J. Sieburth
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Citrus Tristeza Virus ◽  
Sodium Dodecyl ◽  
Potassium Acetate ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus ◽  
Citrus Tristeza

A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

scholarly journals Visualization of the Distribution Pattern of Citrus Tristeza Virus in Leaves of Mexican Lime

HortScience ◽  
2006 ◽  
Vol 41 (3) ◽  
pp. 725-728
Author(s):  
Youjian Lin ◽  
Charles A. Powell
Keyword(s):  
Distribution Pattern ◽  
Citrus Tristeza Virus ◽  
Imaging Technique ◽  
Plant Viruses ◽  
Test Results ◽  
Mexican Lime ◽  
Small Primary ◽  
Scanning Imaging ◽  
Tristeza Virus ◽  
Citrus Tristeza

The distribution pattern of citrus tristeza virus (CTV) T-36 isolate in leaves of infected mexican lime [Citrus aurantifolia (Christm.) Swingle] plants was visualized using a whole-leaf-blot immunoassay (WLBIA) procedure in combination with a computer scanning imaging technique and CTV-specific monoclonal antibody 17G11 (CTV MAb 17G11). The distribution pattern of CTV T-36 in leaves varied with the age of the leaves and shoots of infected plants. In the young leaves, especially the about 5-day-old leaves and the completed expanded leaves, CTV T-36 was easily detected in most of the leaf veins, the main veins and the large and small primary veins. In the old leaves, CTV T-36 only was detected in the main veins, sometimes in a few of the large primary veins with weak signals, and seldom in the small primary veins. The distribution density and immunoassay reaction signals of CTV T-36 reacted to CTV MAb 17G11 in leaves from new shoots were much higher than that in leaves from old shoots. ELISA test results using leaves with different ages from different shoots of the same mexican lime plants infected with CTV T-36 supported the visualized-test results obtained by the WLBIA in combination with computer scanning imaging technique. This is the first reported visual analysis of the distribution pattern of CTV in leaves of infected citrus plants. The results indicate that the WLBIA in combination with computer scanning imaging technique is a useful tool for studying the distribution of plant viruses in leaves of virus-infected plants.

scholarly journals Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus

2009 ◽  
Vol 84 (3) ◽  
pp. 1314-1325 ◽  
Author(s):  
Svetlana Y. Folimonova ◽  
Cecile J. Robertson ◽  
Turksen Shilts ◽  
Alexey S. Folimonov ◽  
Mark E. Hilf ◽  
...  
Keyword(s):  
Citrus Tristeza Virus ◽  
Secondary Infection ◽  
Severe Disease ◽  
Plant Viruses ◽  
Protective Measure ◽  
Challenge Virus ◽  
Superinfection Exclusion ◽  
Tristeza Virus ◽  
Virus Isolates ◽  
Citrus Tristeza

ABSTRACT Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.

scholarly journals Role of Defective RNAs in Citrus Tristeza Virus Diseases

2000 ◽  
Author(s):  
Moshe Bar-Joseph ◽  
William O. Dawson ◽  
Munir Mawassi
Keyword(s):  
Citrus Tristeza Virus ◽  
Genetic System ◽  
Helper Virus ◽  
Plant Viruses ◽  
Expression Vectors ◽  
The Us ◽  
Infected Cells ◽  
Tristeza Virus ◽  
Citrus Tristeza

This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.

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