A Simulation of the Use of High Throughput Sequencing as Pre-Screening Assay to Enhance the Surveillance of Citrus Viruses and Viroids in the EPPO Region

Citrus are affected by many viruses and viroids, some globally widespread and some restricted to particular countries or areas. In this study, we simulated the use of high throughput sequencing (HTS) and the bioinformatic analysis of small interfering RNAs (siRNA) as a pre-screening method to guide bioindexing and molecular detection to enhance the surveillance survey of some key or emerging citrus viruses, such as non-European citrus tristeza virus isolates (non-EU CTV), citrus tatter leaf virus, citrus leprosis virus, citrus yellow mosaic virus, and citrus bark cracking viroid, present in the EPPO lists, and the citrus yellow vein clearing virus. The HTS’s ability to detect other citrus viroids was also evaluated. The results demonstrate that HTS provides a comprehensive phytosanitary status of citrus samples either in single and multiple infections of viruses and viroids. It also provides effective information on citrus tristeza virus mixed infections despite not being able to identify the non-EU variants of the virus. Bioindexing checks each single virus infection but does not differentiate viroids on the Etrog citron indicator and is time-consuming. Molecular assays are valuable as confirmation tests of viruses and viroids but many pairs of primers are needed for a full screening and new or non-target pathogens remain undetected . In addition, the genomes of two isolates of the citrus yellow vein clearing virus and the citrus tatter leaf virus, detected in a sample from China, are described.

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Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing

Viruses ◽

10.3390/v13020168 ◽

2021 ◽

Vol 13 (2) ◽

pp. 168

Author(s):

Rachelle Bester ◽

Glynnis Cook ◽

Hans J. Maree

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Citrus Tristeza Virus ◽

Real Data ◽

Virus Genotype ◽

Disease Etiology ◽

Bioinformatic Pipeline ◽

Genome Coverage ◽

Tristeza Virus ◽

Citrus Tristeza

The application of high-throughput sequencing (HTS) has successfully been used for virus discovery to resolve disease etiology in many agricultural crops. The greatest advantage of HTS is that it can provide a complete viral status of a plant, including information on mixed infections of viral species or virus variants. This provides insight into the virus population structure, ecology, or evolution and can be used to differentiate among virus variants that may contribute differently toward disease etiology. In this study, the use of HTS for citrus tristeza virus (CTV) genotype detection was evaluated. A bioinformatic pipeline for CTV genotype detection was constructed and evaluated using simulated and real data sets to determine the parameters to discriminate between false positive read mappings and true genotype-specific genome coverage. A 50% genome coverage cut-off was identified for non-target read mappings. HTS with the associated bioinformatic pipeline was validated and proposed as a CTV genotyping assay.

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First Report from Syria of Citrus tristeza virus in Citrus spp.

Plant Disease ◽

10.1094/pdis-92-10-1468c ◽

2008 ◽

Vol 92 (10) ◽

pp. 1468-1468

Author(s):

R. Abou Kubaa ◽

K. Djelouah ◽

A. M. D'Onghia ◽

R. Addante ◽

M. Jamal

Keyword(s):

Citrus Tristeza Virus ◽

Sweet Orange ◽

Citrus Limon ◽

Potential Threat ◽

First Report ◽

Mexican Lime ◽

Vein Clearing ◽

Citrus Varieties ◽

Tristeza Virus ◽

Citrus Tristeza

During the spring of 2006, the main Syrian citrus-growing areas of Lattakia (Jableh, Aledyye, Eseelya, Siano, and Hresoon provinces) and Tartous (Almintar, Aljammase, Karto, Majdaloonelbahr, Yahmour, Amreet, Althawra, and Safita provinces) were surveyed to assess the presence of Citrus tristeza virus (CTV). Eight nurseries (approximately 130 plants per nursery), two budwood source fields (approximately 230 trees per field), and 19 groves (approximately 60 trees per grove) containing the main citrus varieties were visually inspected and sampled for serological assays. The hierarchical sampling method was carried out in each selected grove (2). Infected samples were collected from two nurseries, two budwood source fields, and six groves. Stems and leaf petioles from nursery trees and flower explants from the groves were collected and analyzed for CTV by direct tissue blot immunoassay (DTBIA) with the commercial kit from Plantprint (Valencia, Spain). Of 2,653 samples tested, 89 (4%) CTV-infected plants were detected. Five citrus varieties were found to be infected and Meyer lemon (Citrus limon ‘Meyer’) had the highest incidence at 16%. Numerous sweet orange varieties (Citrus sinensis L.) were found to be highly infected in the field, but only the Washington navel sweet orange was found to be infected in the nurseries. No clear CTV symptoms were observed during the survey. Samples that were positive for CTV by DTBIA were also positive by biological indexing on Mexican lime (C. aurantifolia) and immunocapture-reverse transcription-PCR as described by Nolasco et al. (3). Coat protein gene sequences obtained from five selected clones of a Syrian CTV isolate (GenBank Accession No. EU626555) showed more than 99 and 98% nucleotide sequence identity to a Jordanian CTV isolate (GenBank Accession No. AY550252) and the VT isolate (GenBank Accession No. U56902), respectively. Almost all infected samples induced moderate vein clearing symptoms when grafted to Mexican lime. Symptoms of vein clearing, leaf cupping, stunting, and stem pitting on Mexican lime were induced by graft transmission of CTV from one Valencia sample from the Tartous area. The viral inoculum is widely and randomly distributed in commercial groves, especially in the southern Tartous area and in some nurseries. To our knowledge, this is the first report of CTV in Syria. However, CTV was reported from the neighboring citrus-growing countries of Lebanon, Turkey, and Jordan (1), and the severe seedling yellows strain is present in this area, which poses a potential threat to Syrian citriculture. References: (1) G. H. Anfoka et al. Phytopathol. Mediterr. 44:17, 2005. (2) G. Hughes and T. R. Gottwald, Phytopathology 88:715, 1998. (3) G. Nolasco et al. Eur. J. Plant Pathol. 108:293, 2002.

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First Report of Citrus tristeza virus in Greece

Plant Disease ◽

10.1094/pdis.2002.86.3.329b ◽

2002 ◽

Vol 86 (3) ◽

pp. 329-329 ◽

Cited By ~ 4

Author(s):

D. Dimou ◽

J. Drossopoulou ◽

E. Moschos ◽

C. Varveri ◽

F. Bem

Keyword(s):

Citrus Tristeza Virus ◽

Sweet Orange ◽

Enzyme Linked Immunosorbent Assay ◽

First Report ◽

Vein Clearing ◽

Carrizo Citrange ◽

Sweet Lime ◽

Tristeza Virus ◽

Propagation Material ◽

Citrus Tristeza

Large-scale surveys of Citrus spp. for the presence of Citrus tristeza virus (CTV) by the Ministry of Agriculture in Greece began in 1995. Over 26,000 trees have been tested by enzyme-linked immunosorbent assay and immunoprinting (2). In summer 2000, the first CTV-infected sweet orange cv. Lane Late tree grafted on CTV-tolerant Carrizo citrange was found in Argolis County, Peloponnese. This tree belonged to a batch of CAC propagation material (20 trees) illegally introduced from Spain in 1994, which was subsequently traced and found to be infected (45%). A follow-up search of trees grafted with the above material was undertaken in the two concerned regions (Argolis and Chania-Crete), and more than 3,500 trees have been removed. Extensive surveys continue to identify and destroy new infections. Few cases (15 of 16,800) of natural transmission to cultivars other than cv. Lane Late have been found. All of these have been close to the initially infected trees in the Argolis area. Surveys in spring 2001 were extended to certified propagation material of Clemenpons mandarin on Carrizo citrange imported from Spain, and 7 of 1,038 plants were infected (0.64%). The virus was successfully graft-transmitted to sweet orange cv. Madame Vinous and sweet lime seedlings, where it was identified by immunoprinting and reverse transcription-polymerase chain reaction (1). Mild vein clearing symptoms appeared on both indicators. Vein clearing on sweet lime was also accompanied by leaf cupping. To our knowledge, this is the first report of CTV in Greece. References: (1) A. Sambade et al. J. Virol. Methods 87:25, 2000. (2) C. Vela et al. J. Gen. Virol. 67:91, 1986.

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CITRUS TRISTEZA VIRUS IN PAKISTAN: A REVIEW

Pakistan Journal of Phytopathology ◽

10.33866/phytopathol.029.02.0355 ◽

2017 ◽

Vol 29 (2) ◽

pp. 273

Author(s):

Sagheer Atta ◽

Maroof Siddiq ◽

Sidra Ashiq ◽

Abdul Hannan

Keyword(s):

Transgenic Plants ◽

Citrus Tristeza Virus ◽

Economic Losses ◽

Mild Strain ◽

Viral Pathogen ◽

Vein Clearing ◽

The World ◽

Tristeza Virus ◽

Stem Pitting ◽

Citrus Tristeza

Citrus is one of the most widely grown fruit crop in the world. Citrus tristeza virus (CTV) is one of the most economically important viral pathogen of citrus and causes different disease syndromes. The use of infected bud wood and aphids are the main cause of spread of CTV. The virus is genetically diverse and causes various symptoms like slow and quick decline, stunting, stem pitting, vein clearing and seedling yellows. Strategies have been developed to reduce the economic losses caused by CTV. Quarantine measures, bud wood certification, mild strain cross protection and eradication programs are some of the strategies that can prevent the spread of this disease. New approaches are provided by advancement in genetic engineering and molecular biology. Transgenic plants have been developed to provide resistance against the disease.

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Ketahanan Aksesi Jeruk Seedles Terhadap Tiga Strain Virus Tristeza Jeruk

Jurnal Hortikultura ◽

10.21082/jhort.v26n2.2016.p235-244 ◽

2016 ◽

Vol 26 (2) ◽

pp. 235

Author(s):

Mutia Erti Dwiastuti ◽

Sri Widyaningsih

Keyword(s):

Citrus Tristeza Virus ◽

Vein Clearing ◽

Severe Strain ◽

Strain Virus ◽

Tristeza Virus ◽

Stem Pitting ◽

Screen House ◽

Citrus Tristeza

Citrus tristeza virus (CTV) merupakan salah satu penyakit yang merugikan secara ekonomi pada jeruk. Penyakit ini telah menyebar merata di pertanaman jeruk seluruh Indonesia. Tiap varietas jeruk mempunyai ketahanan yang berbeda-beda terhadap penyakit ini. Tujuan penelitian ini adalah mengetahui tingkat ketahanan kandidat jeruk seedless hasil mutasi dengan radiasi sinar Gamma terhadap tiga strain penyakit Citrus tristeza virus. Penelitian dilaksanakan di Laboratorium Terpadu dan screen house Balai Penelitian Tanaman Jeruk dan Buah Subtropika (Balitjestro)selama 1 tahun. Tahapan yang dilakukan adalah seleksi dan perbanyakan strain CTV, pengujian ketahanan 9 kandidat mutan dan 2 tanaman berasal dari induk MT-49, MT-50, MT-52, MT-54, MT-89, MT-92 (mutasi dari tetua K SoE), MT-68 (mutasi dari tetua K Garut), KS 001(hasil silangan), KS 002 (tetua Tai Ayam), MT P2A6 (mutasi dari tetua Pamelo Nambangan1), MT P1A4 (mutasi dari tetua Pamelo Nambangan2). Pengamatan dilakukan terhadap masa inkubasi, intensitas penyakit berdasarkan gejala visual dan uji serologi Elisa. Tingkat ketahanan didasarkan pada gejala visual dan hasil pengujian dengan Elisa. Hasil penelitian menunjukkan gejala vein clearing, vein cupping, vein crocking dan stem pitting di temukan pada areal pertanaman jeruk. Masa inkubasi CTV pada kandidat mutan dengan inokulasi masing-masing strain bervariasi antara 3-5 minggu. Intensitas penyakit yang timbul akibat inokulasi masing-masing strain bervariasi, demikian juga tingkat ketahanan tanaman. Aksesi varietas yang resisten terhadap strain CTV parah (severe strain) adalah MT P2A6 dan MT P1A4; aksesi toleran terhadap strain CTV parah adalah MT 49, MT 52, MT 54, MT 68, MT 92, KS 002 dan aksesi yang peka terhadap strain CTV parah adalah MT 50, MT 89, KS 001.

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