scholarly journals First Report of Citrus tristeza virus in Citrus Orchards in Bosnia and Herzegovina

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1665-1665
Author(s):  
D. Delić ◽  
M. Afechtal ◽  
K. Djelouah ◽  
B. Lolić ◽  
A. Karačić
Keyword(s):  
Coat Protein ◽  
Bosnia And Herzegovina ◽  
Coat Protein Gene ◽  
Citrus Tristeza Virus ◽  
Protein Gene ◽  
Rt Pcr ◽  
First Report ◽  
Citrus Orchards ◽  
Tristeza Virus ◽  
Das Elisa

The citrus growing area in Bosnia and Herzegovina (B&H) is limited to the confluence of the river Neretva, which is close to the Adriatic coastal region. Approximately 6 ha are grown in the country. Mandarins (Citrus reticulata Blanco) and lemons (Citrus limon L.) grafted on trifoliate orange (Poncirus trifoliata (L.) Raf.) are the most cultivated species. In June 2012, 25 samples were collected from individual trees from three locations in Herzegovina district of B&H (Mostar, Čapljina, and Ljubuški). Samples of different Citrus spp. (C. reticulata Blanco, C. aurantium L., C. limon L., C. sinensis (L.) Osbeck, P. trifoliata (L.) Raf., and Fortunella margarita Lour) and varieties were collected from infield plants, commercial citrus orchards, and a nursery. Out of 25, 10 citrus trees exhibited leaves chlorosis, whereas as all others were apparently symptomless. Double antibody sandwich (DAS)-ELISA test, using commercial kit from the DSMZ, Germany (product code AS-0988), was carried out to confirm the presence of Citrus tristeza virus (CTV). In addition, further analyses were performed using reverse transcription (RT)-PCR targeting the coat protein gene (2). CTV was detected in 8 out of the 25 tested samples with DAS-ELISA, whereas CTV was detected in 14 samples by RT-PCR. Being grafted on P. trifoliata rootstock, no typical CTV symptoms in the field were observed on the CTV-infected trees; interestingly, the lab analyses evidenced the CTV presence in all inspected locations of the Herzegovina district. To our knowledge, this is the first report of CTV in Bosnia and Herzegovina; nevertheless, the virus presence is also reported from neighboring countries Croatia (1) and Montenegro (3). The PCR products of four samples were additionally analyzed by sequencing. The preliminary results by sequencing of the coat protein gene of four selected CTV isolates (Accessions HF947341, HF947342, HF947343, and HF947347) showed 99% nucleotide identity with the CTV resistance breaking isolates from Montenegro (FR871866) and Croatia (EU579422). Although a very small number of samples were tested in this study, CTV appears to be widely distributed in the citrus orchards of the country. This could be related to the traditional use of tolerant P. trifoliata rootstock that prevents the development of the tristeza decline as well as to the virus isolates present in the region, which appear not to cause another economically devastating CTV disease such as stem pitting. Further research will be dedicated to the biological properties of the genetic variability of these identified CTV isolates and the assessment of potential aphid vectors. References: (1) S. Černi et al. Plant Dis. 89:342, 2005. (2) M. E. Hilf et al. Options Méditerranéennes B 65:89, 2009. (3) T. Papic et al. Plant Dis. 89:434, 2005.

scholarly journals Cloning and Sequencing of the Coat Protein Gene of a New Isolate of Citrus Tristeza Virus from Mexico

Plant Disease ◽  
1997 ◽  
Vol 81 (6) ◽  
pp. 693-693
Author(s):  
A. C. Cepeda-Nieto ◽  
H. A. Barrera-Saldaña
Keyword(s):  
Coat Protein ◽  
Coat Protein Gene ◽  
Citrus Tristeza Virus ◽  
Protein Gene ◽  
Immunological Methods ◽  
Rt Pcr ◽  
Indicator Plants ◽  
Orange Trees ◽  
Tristeza Virus ◽  
Citrus Tristeza

Citrus tristeza virus (CTV) causes one of the most important citrus diseases. CTV strains cause a wide range of symptoms in infected citrus worldwide. Although it has not yet affected Mexico's citrus industry, CTV constitutes a threat since one of its most efficient vectors, Toxoptera citricida, is migrating north from South America and is now in the Caribbean region. Efforts have been made to prevent spread of the virus, through early detection with serological methods (3). Use of the polymerase chain reaction (PCR), not yet a competitor of immunological methods in field diagnosis of CTV, offers a quick detection alternative. As a first step toward using PCR in early diagnosis and characterization of CTV, we searched for the CTV coat protein gene in experimentally infected leaves of Citrus aurantifolia grown at a government research station (INIFAP) at General Terán, NL. Researchers at General Terán had collected the isolates during a random sampling of orange orchards in the Gulf state of Veracruz. The original orange trees had no apparent symptoms and had been examined as a preventive measure. Several of the original orange trees were CTV positive in their initial tests with polyclonal antibody and in subsequent tests with CTV-specific monoclonal antibody MCA-13. Characteristic CTV-like particles had also been observed by electron microscopy. The only symptom induced in indicator plants was yellowing of the leaf veins. In our laboratory, reverse transcription of RNA from one of the indicator plants, coupled with PCR (RT-PCR) (1), was used to clone and sequence one of the isolates to confirm the CTV identification and establish PCR methods. Oligonucleotide primers were derived from published sequences, but unique restriction enzyme sites (EcoRI and XbaI) were added to their 5′ ends to facilitate cloning. To exclude artifacts, nucleotide sequences were obtained from both strands after cloning in M13 vectors. Although the expected RT-PCR product of 700 bp was obtained, an unexpected EcoRI site was found at position 678 of the coat protein gene. A phenylalanine residue was found at position 124, as in severe strains of CTV from various regions of the world (2). Similarities between our CT sequence (U32116) and those of other GenBank accessions are as follows: 91.17% for M76485; 90.14% for L12175; and 89.32% for S67800. References: (1) M. D. Jones and N. S. Foulkes. Nucleic Acids Res. 17:8387, 1989. (2) H. Pappu et al. Proc. Natl. Acad. Sci. USA. 90:3641, 1993. (3) C. Vela et al. J. Gen. Virol. 67:91, 1986.

Molecular detection and coat protein gene based characterization of Citrus tristeza virus prevalent in Sikkim state of India

2019 ◽  
Vol 73 (1) ◽  
pp. 135-143 ◽  
Author(s):  
Ashish Warghane ◽  
Amol Kokane ◽  
Sunil Kokane ◽  
Manali Motghare ◽  
Datta Surwase ◽  
...  
Keyword(s):  
Coat Protein ◽  
Coat Protein Gene ◽  
Molecular Detection ◽  
Citrus Tristeza Virus ◽  
Protein Gene ◽  
Tristeza Virus ◽  
Citrus Tristeza

scholarly journals Reverse-Transcription Polymerase Chain Reaction Detection of Citrus Tristeza Virus in Aphids

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1066-1069 ◽  
Author(s):  
Prem Mehta ◽  
R. H. Brlansky ◽  
S. Gowda ◽  
R. K. Yokomi
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Citrus Tristeza Virus ◽  
Protein Gene ◽  
Aphid Species ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus

A rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection of citrus tristeza virus (CTV) in three aphid species. Seven CTV isolates from a worldwide isolate collection were used for aphid acquisition feeding by three aphid species. These included the most efficient CTV vector, the brown citrus aphid, Toxoptera citricida; the melon aphid, Aphis gossypii; and the green peach aphid, Myzus persicae, a non-vector for CTV. A short procedure for nucleic acid extraction from single or groups of aphids was developed. Nucleic acid extracts from 1, 3, 5, and 10 aphids with acquisition-access periods of 24 and 48 h were reverse transcribed and amplified using primers for the coat protein gene of the Florida B3 (T-36) isolate of CTV. PCR-amplified fragments of approximately 670 bp were obtained from all the isolates tested and the amplified product from the aphids fed on citrus infected with isolate B3 was confirmed as the CTV coat protein gene by digesting with various restriction enzymes. This technique will be useful in investigations of CTV-vector-plant interactions and CTV epidemiology.

scholarly journals First Report of Cherry virus A and Little cherry virus-1 in Poland

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 909-909 ◽  
Author(s):  
B. Komorowska ◽  
M. Cieślińska
Keyword(s):  
Nucleotide Sequence ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Germplasm Collection ◽  
Mottle Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Primer Sets

Cherry virus A (CVA), a member of the genus Capillovirus, has been reported in sweet cherry in Germany, Canada, and Great Britain. No data are available on the effects of CVA on fruit quality and yield of infected trees. Little cherry disease (LChD) occurs in most cherry growing areas of the world. Symptoms on sensitive cultivars include discolored fruit that remain small, pointed in shape, and tasteless. Three Closterovirus spp. associated with LChD have been described (Little cherry virus-1 [LChV-1], LChV-2, and LChV-3). Diseased local and commercial cultivars of sour cherry trees were found in a Prunus sp. germplasm collection and orchards in Poland during the 2003 growing season. The foliar symptoms included irregular, chlorotic mottling, distortion, and premature falling of leaves. Some of the diseased trees developed rosette as a result of decreased growth and shortened internodes. Severely infected branches exhibited dieback symptoms. Because the symptoms were suggestive of a possible virus infection, leaf samples were collected from 38 trees and assayed for Prune dwarf virus and Prunus necrotic ringspot virus using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). RNA extracted from leaves was used in a reverse transcription-polymerase chain reaction (RT-PCR) with the One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies) and primer sets specific for CVA (1), LChV-1 (3), and LChV-2 (3). The RNA samples were also tested using RT-PCR for detection of Cherry mottle leaf virus (CMLV), Cherry necrotic rusty mottle virus (CNRMV), and Cherry green ring mottle virus (CGRMV) with specific primer sets (2). Amplification of a 397-bp coat protein gene product confirmed infection of 15 trees with CVA. A 419-bp fragment corresponding to the coat protein gene of LChV-1 was amplified from cv. Gisela rootstock and local cv. WVIII/1. To confirm RT-PCR results, CVA amplification products from local cv. WX/5 and LChV-1 from cvs. Gisela and WVIII/1 were cloned in bacterial vector pCR 2.1-TOPO and then sequenced. The sequences were analyzed with the Lasergene (DNASTAR, Madison, WI) computer program. The alignment indicated that the nucleotide sequence of cv. WX/5 was closely related to the published sequences of CVA (Genbank Accession No. NC_003689) and had an 89% homology to the corresponding region. The nucleotide sequence similarity between the 419-bp fragment obtained from cvs. Gisela and WVIII/1 was 87% and 91%, respectively, compared with the reference isolate of LChV-1 (Genbank Accession No. NC_001836). The sampled trees tested negative for LChV-2, CGRMV, CMLV, and CNRMV using RT-PCR. Some trees tested positive for PNRSV and PDV. To our knowledge, this is the first report of CVA and LChV-1 in Poland. References: (1) D. James and W. Jelkmann. Acta Hortic. 472:299, 1998. (2) M. E. Rott and W. Jelkmann. Eur. J. Plant Pathol. 107:411,2001. (3) M. E. Rott and W. Jelkmann. Phytopathology. 91:61, 2001.

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