Beet Necrotic Yellow Vein Virus Isolates Research Articles

Beet necrotic yellow vein virus (benyvirus)

<i>Beet necrotic yellow vein virus</i> (<i>benyvirus</i>)

The Continuing Saga of Rhizomania of Sugar Beets in the United States.

Historically, Beet necrotic yellow vein virus (BNYVV), which causes the disease rhizomania of sugar beet, has caused major reductions in root yield and quality wherever it occurred. In the United States, the disease was first identified in California in 1984 (12), but it now occurs in every major sugar beet production region in the country. Fortunately, strong genetic tolerance to BNYVV, conferred by the Rz1 gene, was identified soon after rhizomania was discovered in the United States, and it has been incorporated into regionally adapted cultivars that allow profitable sugar beet production in areas infested with the pathogen. Plants possessing Rz1 are susceptible to infection by P. betae and BNYVV, but virus titers remain low, and typically, infected plants exhibit normal, asymptomatic growth. However, in the Imperial Valley of California in 2002, plants in a field planted to a rhizomaniatolerant cultivar began to express symptoms of rhizomania (47). Large strips of diseased plants occurred across the field, and it was soon verified that certain isolates of BNYVV from the California Imperial Valley (CIV-BNYVV) had overcome genetic resistance. In other regions of the United States, individual plants, or clusters of plants, in fields planted to rhizomaniatolerant cultivars have also become infected by BNYVV and developed diagnostic symptoms of severe rhizomania. Although patterns of disease development have varied between California fields and those in other production regions in the United States, the breakdown of genetic resistance has caused considerable concern among those involved in sugar beet production. The purpose of this article is to provide an update on rhizomania and the activities that are currently underway to manage this continuing threat to the U.S. sugar beet industry. A review on the detection and distribution of rhizomania and soilborne viruses associated with sugar beet production in the United States (61), and a second more in-depth review on BNYVV and its vector, Polymyxa betae (Keskin) (60), have been published.

Occurrence of Resistance-Breaking Beet necrotic yellow vein virus of Sugar Beet.

Rhizomania is an important virus disease of sugar beet and is caused by Beet necrotic yellow vein virus (BNYVV). During 2002-03, several sugar beet fields with cultivars partially resistant to BNYVV grown in the Imperial Valley of California were observed with severe rhizomania symptoms, suggesting that resistance conditioned by Rz1 had been compromised. Soil testing with sugar beet baiting plants followed by enzyme-linked immunosorbent assay (ELISA) was used to diagnose virus infection. Resistant varieties grown in BNYVV-infested soil from Salinas, CA, were ELISA-negative. In contrast, when grown in BNYVV-infested soil collected from the Imperial Valley, CA, all resistant varieties became infected and tested positive by ELISA. Based on host reaction, eight distinct BNYVV isolates have been identified from Imperial Valley soil (IV-BNYVV) by single local lesion isolation. Reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the eight IV-BNYVV isolates did not contain RNA-5. Singlestrand conformation polymorphism banding patterns for the IV-BNYVV isolates were identical to A-type and different from P-type. Sequence alignments of PCR products from BNYVV RNA-1 near the 3' end of IV-BNYVV isolates revealed that both IV-BNYVV and Salinas BNYVV isolates were similar to A-type and different from B-type. Our results suggest that the resistancebreaking BNYVV isolates from Imperial Valley likely evolved from existing A-type isolates.

A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus types A and B

A multiplex reverse-transcription polymerase chain assay (mRT-PCR) was developed, based on primers designed to distinguish the A and B types of beet necrotic yellow vein virus (BNYVV). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established detection method based on single strand conformation polymorphisms (SSCPs). An area of the ‘triple gene block’ region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. The A type assay produced a single 324 base-pair RT-PCR fragment when positive samples were amplified. The B type assay produced a 178 base-pair product from positive samples. No amplification was observed from healthy Chenopodium quinoa or sugar beet plants and from plants infected by others sugar beet soil-borne viruses. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. The results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples.

Detection of beet necrotic yellow vein virus using reverse transcription and polymerase chain reaction

A diagnostic test based on reverse transcription followed by the polymerase chain reaction (RT-PCR) was developed for the detection of beet necrotic yellow vein virus (BNYVV). A specific 500-base-pair fragment was amplified from the read-through region of the coat protein gene located on RNA-2. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having 94.5% homology with published sequence data for BNYVV. The assay gave a sensitivity of 800 times that of a TAS-ELISA and 50 times that of an amplified TAS-ELISA method. A range of BNYVV isolates from the UK and worldwide could be detected by this test, either as mechanically inoculated Chenopodium quinoa leaves or infected sugar beet roots. Use of the assay in routine diagnostic tests allowed a reduction of time needed for the detection of Rhizomania in soils from 7 to 4 weeks.

Production and Pathogenicity of Isolates of Beet Necrotic Yellow Vein Virus with Different Numbers of RNA Components

SUMMARY Ten Japanese field isolates of beet necrotic yellow vein virus (BNYVV) were transmitted to Tetragonia expansa by inoculation with sap from rootlets of sugar-beet seedlings, to which the virus had been transmitted by the fungus Polymyxa betae. RNA extracted from BNYVV particles obtained from the T. expansa leaves was analysed by agarose gel electrophoresis. Some isolates contained RNA-1 (7·1 kb), RNA-2 (4·8 kb), RNA-3 (1·85 kb) and RNA-4 (1·5 kb) and the others contained, in addition, RNA-5 (1·4 kb). Further isolates, derived from single lesions produced by these isolates, had a variety of RNA compositions. Some contained only RNA-1 and RNA-2. Others contained, in addition, RNA-3, RNA-4, RNA-5 or RNA-6 (1·0 kb), or combinations of two or three of these components. Such isolates generally maintained their RNA composition on further subculture, and their particles had length distributions corresponding to their RNA components. Isolates containing RNA-1 + 2 + 3 caused yellow or strongly chlorotic local lesions in T. expansa, Beta vulgaris, B. macrocarpa and Chenopodium quinoa, and caused systemic stunting and yellow mosaic in B. macrocarpa and, occasionally, in B. vulgaris. In contrast, isolates containing RNA-1 + 2 + 4 or 1 + 2 + 5 induced chlorotic lesions, those containing RNA-1 + 2 + 6 or 1 + 2 induced faint chlorotic lesions, and none of these isolates easily infected B. macrocarpa systemically. Isolates containing different combinations of RNA-3,-4 and -5 induced more severe symptoms than those containing a single RNA. Such synergistic effects occurred between RNA-3 and RNA-4 or RNA-5, or between RNA-4 and RNA-5 or RNA-6, but not between RNA-3 and RNA-6, or between RNA-5 and RNA-6. These small RNA species therefore contain the genetic determinant(s) for lesion type and for ability to infect B. vulgaris and B. macrocarpa systemically. RNA-1 and RNA-2 are viral genome components. The other RNA components have some characteristics of viral satellite nucleic acids but they may not all be dispensable if the BNYVV isolates are to survive in nature.

Biologically Active Transcripts of Beet Necrotic Yellow Vein Virus RNA-3 and RNA-4

Summary Synthetic transcripts of beet necrotic yellow vein virus (BNYVV) RNA-3 and RNA-4 were prepared from cloned cDNA in a bacteriophage T7 in vitro run-off transcription system. The RNA-3 transcripts tested had 5′ non-viral extensions of 1, 23 or 64 nucleotides and identical 3′ non-viral extensions of 12 nucleotides. An RNA-4 transcript with a 12 nucleotide 5′ non-viral extension and a 28 nucleotide 3′ non-viral extension was also synthesized. All of the transcripts were biologically active when coinoculated to Chenopodium quinoa with BNYVV isolates deficient in RNA-3 and/or RNA-4 but the presence of a long 5′ non-viral sequence on the RNA-3 transcripts was found to diminish their specific activities considerably.

Effect of beet necrotic yellow vein virus RNA composition on transmission by Polymyxa betae

Beet necrotic yellow vein virus (BNYVV) is naturally transmitted by the soil-borne fungus Polymyxa betae and usually remains confined to the roots of infected sugarbeets. In naturally infected sugarbeets the virion RNA always consists of four components which are uniform in size in different isolates but when BNYVV is propagated by mechanical inoculation to leaves of Chenopodium quinoa the two smallest RNA components, RNA-3 and -4, may undergo deletion or disappear from the isolate, suggesting that they are only essential for the natural mode of infection. To test this hypothesis, several C. quinoa isolates of BNYVV with different RNA-3 and -4 contents have been retransmitted to sugarbeet root via P. betae. The results show that the two isolates containing no detectable full-length RNA-3 and -4 are poorly transmitted and that cases of successful infection are associated with the reappearance of full-length RNA-3 and -4.

Diversity of the RNAs in Thirteen Isolates of Beet Necrotic Yellow Vein Virus in Chenopodium quinoa Detected by Means of Cloned cDNAs

AbstractComplementary deoxyribonucleic acids (cDNAs) to the four ribonucleic acid (RNA) species of isolate YU2 of beet necrotic yellow vein virus (BNYVV) were prepared by reverse transcription. The duplexes of these cDNAs were incorporated in the plasmid pBR322 and cloned into E. coli strain 5K. The insert sizes of the cDNA clones ranged from 280 to 2100 base pairs. In Southern blot hybridizations not all cDNAs specific for a particular RNA of BNYVV cross‐reacted. This suggests that they are complementaryto different regions of the respective RNA. In dot blot hybridization experiments sixteen out of eighteen cDNA clones detected all thirteen BNYVV isolates which were obtained from sugar beets originating from Yugoslavia, Italy, Switzerland, France and various regions in Germany and which were maintained in Chenopodium quinoa. Two cDNA clones specific for RNA‐3 failed to detect one German isolate. Pronounced differences between the thirteen virus isolates were seen when the electrophoretic migration of their RNAs was compared in Northern blot hybridizations. RNA‐1 and RNA‐2 of all isolates had the same sizes of 7100 and 5200 bases, respectively. The size, number and relative concentration of the smaller RNAs, however, varied greatly with different, isolates.