Tissue-Print and Squash Capture Real-Time RT-PCR Method for Direct Detection of Citrus tristeza virus (CTV) in Plant or Vector Tissues

Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis.

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Discrimination Between Mild and Severe Citrus tristeza virus Isolates with a Rapid and Highly Specific Real-Time Reverse Transcription-Polymerase Chain Reaction Method Using TaqMan LNA Probes

Phytopathology ◽

10.1094/phyto-99-3-0307 ◽

2009 ◽

Vol 99 (3) ◽

pp. 307-315 ◽

Cited By ~ 31

Author(s):

S. Ruiz-Ruiz ◽

P. Moreno ◽

J. Guerri ◽

S. Ambrós

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Citrus Tristeza Virus ◽

Dynamic Range ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Tristeza Virus ◽

Citrus Tristeza

Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 102 copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates.

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Comportamento de híbridos de citros em relação à infecção natural pelo Citrus tristeza virus e à presença de sintomas de descamamento eruptivo

Revista Brasileira de Fruticultura ◽

10.1590/0100-2945-179/13 ◽

2014 ◽

Vol 36 (3) ◽

pp. 735-741 ◽

Cited By ~ 2

Author(s):

Almir Santos Rodrigues ◽

Cristiane de Jesus Barbosa ◽

Walter dos Santos Soares Filho ◽

Juliana Freitas-Astúa

Keyword(s):

Citrus Tristeza Virus ◽

Rt Pcr ◽

Tristeza Virus ◽

Citrus Tristeza

O Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura vem gerando híbridos para utilização como porta-enxertos, que necessitam ser avaliados em relação ao comportamento frente à infecção natural por isolados locais de Citrus tristeza virus (CTV) e à presença de sintomas de descamamento eruptivo (BahiaBarkScaling disease - BBS). Este trabalho apresenta resultados da avaliação do comportamento de 141 híbridos (sob a forma de pés-francos ou enxertados) estabelecidos na área experimental da Embrapa Mandioca e Fruticultura, no Recôncavo Sul da Bahia. Foram avaliadas a presença e a severidade de sintomas de caneluras e descamamento por meio de escala de notas. Para detectar a presença do CTV, foi utilizado o método sorológico de ELISA indireto e RT-PCR. Os híbridos avaliados foram classificados como imunes, tolerantes e intolerantes ao CTV. A maioria dos híbridos que apresentaram sintomas de BBS tem uma tangerineira como parental.

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Identificação de isolados de Citrus tristeza virus (CTV) protetivos para Citrus sinensis (L.) Osbeck

Summa Phytopathologica ◽

10.1590/0100-5405/172270 ◽

2018 ◽

Vol 44 (1) ◽

pp. 17-22

Author(s):

Ana Paula Gonçalves ◽

Karina Silva dos Santos ◽

Camila de Cassia Silva ◽

Tanara Garcia de Novaes ◽

Rúbia de Oliveira Molina

Keyword(s):

Citrus Sinensis ◽

Citrus Tristeza Virus ◽

Rt Pcr ◽

Tristeza Virus ◽

Citrus Tristeza

RESUMO O Citrus tristeza virus (CTV) causa significativas perdas na produtividade de laranja doce [Citrus sinensis (L.) Osbeck] e seu controle tem sido realizado principalmente com a premunização. O trabalho teve como objetivo analisar a variabilidade de isolados fortes e fracos de CTV provenientes de plantas de citros inoculadas e mantidas em casa de vegetação e amostras de campo, coletadas em pomar comercial situado no município de Rolândia, PR. Para a determinação da variabilidade e diversidade genética dos isolados foi realizada avaliação dos sintomas e empregadas as técnicas de RT– PCR e RFLP, utilizando os oligonucleotídeos específicos HCP1/HCP2 e posterior sequenciamento dos fragmentos amplificados. Na avaliação de canelura, os isolados mantidos em casa de vegetação induziram sintomas leves, com exceção do isolado severo Capão Bonito. Os sintomas mais severos ocorreram em amostras situadas no campo. De acordo com as análises multivariadas os isolados de CTV tendem a se agrupar conforme a severidade dos sintomas e condições ambientais as quais foram expostas formando agrupamentos distintos entre amostras provenientes do campo e casa de vegetação. O dendrograma gerado a partir do sequenciamento dos isolados e as análises multivariadas revelaram que o isolado proveniente da amostra “Forte Arapongas” apresentou maior similaridade com o controle padrão forte proveniente de Capão Bonito. Os isolados identificados como fracos e provenientes das amostras Pêra IAC e Rolândia 5 apresentaram maior similaridade. Pode-se aferir que plantas hospedeiras mantidas em campo possuem maior variabilidade de isolados.

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Detection of severe isolates os citrus tristeza virus by bidirectional RT-PCR (BD-RT-PCR)

VIRUS Reviews & Research ◽

10.17525/vrrjournal.v5i1.176 ◽

2000 ◽

Vol 5 (1) ◽

Author(s):

M. L. P. N. Targon ◽

M. A. Machado ◽

A. A. Souza ◽

G. W. Müller

Keyword(s):

Citrus Tristeza Virus ◽

Rt Pcr ◽

Tristeza Virus ◽

Citrus Tristeza

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

Phytopathology ◽

10.1094/phyto-04-10-0102 ◽

2010 ◽

Vol 100 (10) ◽

pp. 1077-1088 ◽

Cited By ~ 25

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.

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