Characterisation of isolates and strains of citrus tristeza closterovirus using restriction analysis of the coat protein gene amplified by the polymerase chain reaction (original) (raw)
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Phytopathology, 2010
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...
Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Syria in citrus groves from two Governorates (Lattakia and Tartous) and several CTV outbreaks have been reported in Apulia (southern Italy) since 2003. To molecularly characterize the CTV populations spreading in Syria and Italy, a number of isolates from each region was selected and examined by different molecular approaches including: Multiple Molecular Markers analysis (MMM), real time RT-(q)PCR, single strand conformation polymorphism (SSCP) of the major coat protein (CP) gene (P25), and sequence analysis of the CP (P25), P18, P20 and RdRp genes. SSCP analysis of CP25 yielded two distinct simple patterns among the Syrian isolates and three different patterns in the Italian isolates. Based on MMM analysis, all Syrian CTV isolates were categorized as VT-like genotype, whereas the Italian isolates reacted only with the markers specific for the T30 genotype. These findings were also confirmed by RT-qPCR and by sequencing analysis of four genomic regions. The Italian isolates had nucleotide identities which varied: from 99.5 to 99.8 for the CP gene; from 97.4% to 98.3% for the P18 gene; from 98.6% to 99.8% for the P20 and from 97.8% to 99.1% for the partial RdRp sequenced. High sequence identity was found for all genomic regions analyzed between the Syrian isolates (from 98.9% to 99.6%). These results show that the CTV populations spreading in Apulia and Syria are associated with different genotypes, indicating different potential impacts on the citrus trees in the field. Since in both areas the introduction of the virus is relatively recent, infected plants resulted to contain a single and common genotype, suggesting that CTV is spreading from the first outbreaks by aphids or local movement of autochthonous infected plant material.
Characterization of Citrus tristeza virus Isolates by Indicators and Molecular Biology Methods
Agricultural Sciences in China, 2007
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, p2YHinf 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 p23IBD-PCR group I , 111 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.
Phytopathologia Mediterranea, 2006
A panel of Citrus tristeza virus (CTV, genus Closterovirus, family Closteroviridae) isolates of different origins and with different biological properties were compared for polymorphisms in the major coat protein (CP) gene by cleavase fragment length polymorphism (CFLP) and single stranded conformation polymorphism (SSCP) analysis. The similarity between the CFLP patterns, which consisted of 15 to 20 bands, was estimated by the Pearson coefficient. The clustering patterns from the CFLP data were very similar to those from sequence data in an experiment with 16 cloned standards of the CP gene. By SSCP analysis on the other hand, most of the clones were not clustered in the same way. To assess the ability of CFLP to analyse biological samples, which may consist of a mixture of genomic variants, the CP gene of 12 CTV isolates was obtained directly from infected plants by immunocapture/ RT-PCR and analysed. With few exceptions, the isolates were correctly clustered according to the sequ...
Application of Bi-Directional PCR to Citrus Tristeza Virus: Detection and Strain Differentiation
International Organization of Citrus Virologists Conference Proceedings (1957-2010), 1996
The monoclonal antibody MCA13 reacts predominantly with severe strains of citrus tristeza virus (CTV) occurring in many regions of the world. Its specificity is based on a single nucleotide (AIT) difference a t position 371 of the capsid protein gene (CPG). Based on this single nucleotide difference, we designed two internal primers, one specific for mild strains and the other specific for severe strains of CTV. These primers, along with two terminal primers for the ends of the CPG, were used to develop a bi-directional, reverse transcription/polymerase chain reaction (BDPCR) to differentiate mild and severe strains of CTV. Under our standard PCR conditions, a full length CPG (-700 bp) and a 400 bp DNA fragment were produced with mild strains, and a 700 and a 300 bp DNA fragment were produced with severe strains. With a mixture of mild and severe strains, the 700,400 and 300 bp DNA fragments were all produced. Reducing both the terminal primer concentrations and extension times eliminated the 700 bp fragment. Thus, this method can be manipulated for different purposes. Tests of BDPCR with samples from naturallyinfected trees indicated that BDPCR were more sensitive than ELISA, because BDPCR amplified a 300 bp fragment from some samples which were MCA13 negative by ELISA tests.
All citrus trees propagated in South Africa are inoculated with a mild isolate of citrus tristeza virus (CTV) called GFMS-12. DsRNA analysis or reaction with 11 monoclonal antibodies showed only minor differences between GFMS-12-inoculated grapefruit plants maintained in the screenhouse and those exposed to natural infection in the field for 10 years. Serological differences were not detected between GFMS-12 inoculated in Marsh or Star Ruby grapefruit, Gillemberg navel, and Owari satsuma, whereas the dsRNA pattern in Star Ruby differed from that in other hosts. By a library of cDNA probes, changes in isolate composition in these four hosts were detected. Several probes that did not react with GFMS-12 or GFMS-35-infected plants kept in the screenhouse, reacted with the severe isolate GFSS-1 and with extracts from 10-yr-old field trees inoculated with GFMS-12 or GFMS-35 prior to planting. The hybridization signal with trees bearing small-sized fruits was usually stronger than that from those trees bearing normal-sized fruits. These results suggest that field trees were naturally infected by some new CTV strain(s) and that the concentration (or type) of the new strain(s) was different in trees bearing normal or small-sized fruits.
Tropical Plant Pathology, 2012
Plants of Pera sweet orange on Rangpur lime rootstocks, from orchards of the northwest and north of Paraná state, Brazil, were evaluated for severity of symptoms and genetic diversity of Citrus tristeza virus. The severity of symptoms was evaluated by the development of tree, fruit size and stem pitting symptoms. Isolates that infect these plants were compared with known mild and severe isolates by analysis of restriction fragments length polymorphism (RFLP) of the coat protein nucleotide sequences (CPNS), amplified by the polymerase chain reaction (PCR) and undergone digestion with the restriction enzymes Hinf I and Rsa I. The severity of symptoms showed that the analyzed plants from the northwest orchards presented mild to moderate tristeza symptoms, while the plants from the north orchards exhibited moderate to severe symptoms. The RFLP analysis revealed that the CTV isolates are constituted by haplotype mixtures. Rsa I was the enzyme that best discriminated the genetic diversity among the analyzed isolates of CTV. Two main groups were generated by the UPGMA analysis. The isolates from the northwest orchards grouped with most of the mild isolates used as control, and a great part of the isolates from the north orchards, was correlated with the severe isolate Capão Bonito. Correlation between the stem pitting intensity and RFLP patterns, was demonstrated with some exceptions. The failure of protection of some isolates and the contamination of the rootstocks by the severe isolates, in field nursery conditions, before grafting with scions with mild isolates, were the hypotheses considered to explain the occurrence of severe CTV isolates in the North area of Paraná State.
Molecular characterization of citrus tristeza virus (CTV) isolates in Calabria region (South Italy)
2009
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.
2007
Within the context of a program in Cyprus for the control of Citrus tristeza virus (CTV), the coat protein (CP) genes of 12 local isolates of the virus that induced different symptoms on host trees, were compared to those of known isolates. The CP genes were reverse-transcribed (RT) and amplified by polymerase chain reaction (PCR) and the resulting amplicons were cloned and sequenced. Nucleotide sequence analysis revealed no signs of geographic speciation. All the sequences obtained clustered close to those of previously known isolates of worldwide origin that are in five distinct groups. The nucleotide diversity was high compared to that found using a worldwide database of CP gene sequences. These data support the existence of different CTV introductions into Cyprus or an introduction from a location in which CTV is relatively diverse. Some of the isolates induced stem pitting on branches of grapefruit and sweet orange. Such isolates have not been noted often in the Mediterranean basin. They were close in CP sequence to isolate B249 from Venezuela, which induces stem pitting, and are of particular concern for the whole region.
African Journal of Biotechnology
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.