scholarly journals Multiplex Reverse Transcription-PCR for Simultaneous Detection of Beet Necrotic Yellow Vein Virus, Beet Soilborne Virus, and Beet Virus Q and Their Vector Polymyxa betae KESKIN on Sugar Beet

2003 ◽  
Vol 69 (4) ◽  
pp. 2356-2360 ◽  
Author(s):  
Alexandre Meunier ◽  
Jean-François Schmit ◽  
Arnaud Stas ◽  
Nazli Kutluk ◽  
Claude Bragard

ABSTRACT Three soilborne viruses transmitted by Polymyxa betae KESKIN in sugar beet have been described: Beet necrotic yellow vein virus (BNYVV), the agent of rhizomania, Beet soilborne virus (BSBV), and Beet virus Q (BVQ). A multiplex reverse transcription-PCR technique was developed to simultaneously detect BNYVV, BSBV, and BVQ, together with their vector, P. betae. The detection threshold of the test was up to 128 times greater than that of an enzyme-linked immunosorbent assay. Systematic association of BNYVV with one or two different pomoviruses was observed. BVQ was detected in samples from Belgium, Bulgaria, France, Germany, Hungary, Italy, Sweden, and The Netherlands but not in samples from Turkey.

Plant Disease ◽  
2001 ◽  
Vol 85 (12) ◽  
pp. 1288-1288 ◽  
Author(s):  
A. Stas ◽  
A. Meunier ◽  
J.-F. Schmit ◽  
C. Bragard

Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania disease on sugar beet, has been reported in Belgium for more than 16 years. Other soilborne viruses belonging to the genus Pomovirus, such as Beet soilborne virus (BSBV) (3) and Beet virus Q (BVQ) (1), are suspected pathogens of sugar beets grown in Belgium. During the 2000 growing season, more than 20 fields showing rhizomania-like and yellowing symptoms on sugar beet leaves were investigated for the presence of BVQ, BNYVV, and BSBV. All samples were checked by enzyme-linked immunosorbent assay (ELISA) using commercial BNYVV (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) and BSBV/BVQ (DSMZ, Braunschweig, Germany - AS-0576 polyclonal, AS-0576/2 MAb) antisera. RNA was extracted from sugar beet rootlets using an RNeasy extraction kit (Qiagen, Hilden, Germany), before performing a reverse transcription-polymerase chain reaction (RT-PCR) using primers (5′-GCTGGAGTATATCACCGATGAC-3′ and 5′-AAAATC TCGGATAGCATCCAAC-3′) designed to specifically amplify a 510-bp region of BVQ RNA-1. The presence of BSBV and BNYVV was also checked by RT-PCR using previously described primers (1,2). The BVQ-derived PCR product was sequenced and proved to be more than 99% identical to the Wierthe BVQ isolate nucleotide sequence. Soil transmission of BVQ was demonstrated through a bioassay using soil dilutions with quartz and sugar beet cv. Cadyx as bait. After 6 weeks, BVQ was detected by RT-PCR in bait plants. The putative vector, Polymyxa betae, was identified by lactophenol-cotton blue staining of the roots followed by microscopic examination. BVQ produces irregularly shaped local lesions that appear ≈5 days after mechanical inoculation and tend to spread along veins. BVQ was detected in six fields located in the Polders Region and Brabant Province of Belgium. BVQ was always found in sugar beet samples coinfected with BNYVV and BSBV. The economic significance of BVQ and its interaction with other viruses is not known. References: (1) R. Koenig et al. J. Gen. Virol. 79:2027, 1998. (2) M. Saito et al. Arch. Virol. 141:2163, 1996. (3) M. Verhoyen and M. Van den Bossche. Parasitica. 44:71, 1987.


Plant Disease ◽  
2003 ◽  
Vol 87 (2) ◽  
pp. 201-201 ◽  
Author(s):  
William M. Wintermantel ◽  
Teresa Crook ◽  
Ralph Fogg

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV) and vectored by the soilborne fungus Polymyxa betae Keskin, is one of the most economically damaging diseases affecting sugar beet (Beta vulgaris L.). The virus likely originated in Europe and was first identified in California in 1983 (1). It has since spread among American sugar beet production regions in spite of vigorous sanitation efforts, quarantine, and disease monitoring (3). In the fall of 2002, mature sugar beet plants exhibiting typical rhizomania root symptoms, including proliferation of hairy roots, vascular discoloration, and some root constriction (2) were found in several fields scattered throughout central and eastern Michigan. Symptomatic beets were from numerous cultivars, all susceptible to rhizomania. Two to five sugar beet root samples were collected from each field and sent to the USDA-ARS in Salinas, CA for analysis. Hairy root tissue from symptomatic plants was used for mechanical inoculation of indicator plants. Mechanical inoculation produced necrotic lesions on Chenopodium quinoa and systemic infection of Beta vulgaris ssp. macrocarpa, both typical of BNYVV and identical to control inoculations with BNYVV. Symptomatic sugar beet roots were washed and tested using double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the presence of BNYVV using standard procedures and antiserum specific for BNYVV (3). Sugar beet roots were tested individually, and samples were considered positive when absorbance values were at least three times those of greenhouse-grown healthy sugar beet controls. Samples were tested from 16 fields, with 10 confirmed positive for BNYVV. Positive samples had mean absorbance values ranging from 0.341 to 1.631 (A405nm) after 30 min. The mean healthy control value was 0.097. Fields were considered positive if one beet tested positive for BNYVV, but in most cases, all beets tested from a field were uniformly positive or uniformly negative. In addition, soil-baiting experiments were conducted on seven of the fields. Sugar beet seedlings were grown in soil mixed with equal parts of sand for 6 weeks and were subsequently tested using DAS-ELISA for BNYVV. Results matched those of the root sampling. Fields testing positive for BNYVV were widely dispersed within a 100 square mile (160 km2) area including portions of Gratiot, Saginaw, Tuscola, and Sanilac counties in the central and eastern portions of the Lower Peninsula of Michigan. The confirmation of rhizomania in sugar beet from the Great Lakes Region marks the last major American sugar beet production region to be diagnosed with rhizomania disease, nearly 20 years after its discovery in California (1). In 2002, there were approximately 185,000 acres (approximately 75,00 ha) of sugar beet grown in the Great Lakes Region, (Michigan, Ohio, and southern Ontario, Canada). The wide geographic distribution of infested fields within the Michigan growing area suggests the entire region should monitor for symptoms, increase rotation to nonhost crops, and consider planting rhizomania resistant sugar beet cultivars to infested fields. References:(1) J. E. Duffus et al. Plant Dis. 68:251, 1984. (2) J. E. Duffus. Rhizomania. Pages 29–30 in: Compendium of Beet Diseases and Insects, E. D. Whitney and J. E. Duffus eds. The American Phytopathological Society, St. Paul, MN, 1986. (3) G. C. Wisler et al. Plant Dis. 83:864, 1999.


Plant Disease ◽  
2006 ◽  
Vol 90 (1) ◽  
pp. 110-110 ◽  
Author(s):  
C. Rubies Autonell ◽  
C. Ratti ◽  
R. Resca ◽  
M. De Biaggi ◽  
J. Ayala García

Beet virus Q (BVQ) is a member of the genus Pomovirus that is transmitted by Polymyxa betae Keskin. Initially described as the Wierthe serotype of Beet soilborne virus (BSBV), BVQ is now considered a distinct virus species based on its genomic properties (1). BVQ is commonly found in fields where BSBV and the causal agent of rhizomania disease, Beet necrotic yellow vein virus (BNYVV), are also present. Simultaneous infection of sugar beet plants with multiple virus species could affect disease symptom expression (4). For this reason, the pathogenicity of BVQ and its role in the epidemiology of rhizomania disease remain a subject of study. During 2004, six soil samples were collected from different sites in the Castilla-La Mancha Region in Spain (Albacete and Ciudad Real provinces) where rhizomania symptoms were observed in BNYVV-tolerant sugar beet cultivars. Soil from the Hainaut Region of Belgium, infected with BNYVV, BSBV, and BVQ and supplied by Prof. C. Bragard (Unité de Phytopathologie, Université Catholique de Louvain, Belgium) was used as a positive control. Sugar beet plants (cv. Asso) were grown in the soil samples for 45 days at 24°C and then root tissue was harvested. All samples were analyzed using enzyme-linked immunosorbent assay (ELISA) with commercial BNYVV antiserum (BIOREBA AG, Reinach, Switzerland) and BSBV/BVQ antisera (IC10 and 6G2) supplied by R. Koenig (Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany). Total RNA extracted from sugar beet roots as previously described (3) was tested using reverse transcription-polymerase chain reaction (RT-PCR). Primers BVQ3F (5′-GTT TTC AAA CTT GCC ATC CT-3′) and BVQ3R2 (5′-CCA CAA TGG GCC AAT AGA-3′), which amplify a 690-bp fragment of the triple gene block region of BVQ RNA 3, were designed based on the published sequence (GenBank Accession No. AJ223598). The presence of BSBV and BNYVV was assayed using RT-PCR with previously described primers (2,3). BVQ was detected from plants grown in soil collected from La Roda (Albacete) in Spain and from Hainaut in Belgium. The fragments amplified from Spanish sample with BVQ3F and BVQ3R2 (GenBank Accession No. AY849375) showed 95.9% nucleotide sequence identity with the previously published sequence of BVQ (1). The La Roda BVQ isolate was mechanically transmitted to Chenopodium quinoa from infected sugar beet root tissue. BVQ was detected using RT-PCR in local lesions that appeared approximately 5 days after inoculation and subsequently spread along veins. To our knowledge, this is the first report of BVQ in soil from Spain, although it has been previously reported in Belgium, Bulgaria, France, Germany, Hungary, and the Netherlands (2). BSBV and BNYVV (type A) were detected in all six Spanish samples, as well as in the Belgian soil. References: (1) R. Koenig et al. J. Gen. Virol. 79:2027, 1998. (2) A. Meunier et al. Appl. Environ Microbiol. 69:2356, 2003. (3) C. Ratti et al. J. Virol. Methods 124:41, 2005. (4) C. Rush Annu. Rev Phytopathol 41:567, 2003.


Plant Disease ◽  
2006 ◽  
Vol 90 (10) ◽  
pp. 1363-1363 ◽  
Author(s):  
N. Borodynko

The objective of this work was to determine whether Beet virus Q (BVQ), a member of the genus Pomovirus, is present in Poland. BVQ, like Beet necrotic yellow vein virus (BNYVV), is transmitted by Polymyxa betae Keskin. Earlier, BVQ was described as the Wierthe serotype of Beet soilborne virus (BSBV). Now, on the basis of its genomic properties (2), BVQ is recognized as a distinct virus species. BVQ is often found in fields where BSBV and BNYVV are present (4). During the fall of 2005, five plants of a cultivar susceptible to rhizomania (cv. Alyssa) and five resistant to rhizomania (cv. Henrietta) were collected from a field in the Wielkopolska Region of Poland, where BSBV and BNYVV had been previously identified, and tested for BVQ (1). All samples were analyzed by a double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) with antiserum against BNYVV (Bio-Rad, Hercules, CA). Rhizomania was identified only in sugar beet samples of the susceptible variety. The same samples were then tested using a triple antibody sandwich (TAS)-ELISA with commercial antisera against BSBV/BVQ (As-0576.2) and BSBV (As-0576.1) (DSMZ, Braunschweig, Germany). Nine sugar beet plants gave positive reactions with antiserum against BSBV/BVQ and negative reactions with antiserum specific to BSBV. Total RNA extracted from roots of 10 beet samples was then tested using a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) and specific primers designed to amplify a fragment of the RNA2 for BNYVV and BVQ (3). The primers specifically amplified fragments of 545 bp and 291 bp of the BNYVV and BVQ, respectively. BNYVV was detected in all five samples from susceptible sugar beet plants. The presence of BVQ was confirmed in nine of the sugar beer plants, and the RT-PCR products were sequenced. Sequence analysis of the 206-nt amplicon sequence of the Polish isolate of BVQ (GenBank Accession No. DQ309444) indicated 97% nucleotide and 94% amino acid sequence identity with the previously published sequence of BVQ (GenBank Accession No. AJ223596) (2). To my knowledge, this is the first report of the natural occurrence of BVQ on sugar beet in Poland. In Europe, it has been previously reported in Belgium, Bulgaria, France, Germany, Hungary, Italy, the Netherlands, Spain, and Sweden (3,4). References: (1) N. Borodynko et al. Plant Dis. 90:112, 2006. (2) R. Koenig et al. J. Gen. Virol. 79:2027, 1998. (3) A. Meunier et al. Appl. Environ. Microbiol. 69:2356, 2003. (4) C. Rubies Autonell et al. Plant Dis. 90:110, 2006.


Plant Disease ◽  
2003 ◽  
Vol 87 (10) ◽  
pp. 1170-1175 ◽  
Author(s):  
G. C. Wisler ◽  
R. T. Lewellen ◽  
J. L. Sears ◽  
J. W. Wasson ◽  
H.-Y. Liu ◽  
...  

Soils naturally infested with cultures of aviruliferous Polymyxa betae and viruliferous P. betae carrying two sugar beet benyviruses, Beet necrotic yellow vein virus (BNYVV) and Beet soilborne mosaic virus (BSBMV), alone and in combination, were compared with noninfested soil for their effects on seedling emergence, plant fresh weight, and virus content as measured by enzyme-linked immunosorbent assay (ELISA). Studies examined sugar beet with and without resistance to the disease rhizomania, caused by BNYVV. The Rz gene, conferring resistance to BNYVV, did not confer resistance to BSBMV. BSBMV ELISA values were significantly higher in single infections than in mixed infections with BNYVV, in both the rhizomania-resistant and -susceptible cultivars. In contrast, ELISA values of BNYVV were high (8 to 14 times the healthy mean) in single and mixed infections in the rhizomania-susceptible cultivar, but were low (approximately three times the healthy mean) in the rhizomania-resistant cultivar. Results indicate BNYVV may suppress BSBMV in mixed infections, even in rhizomania-resistant cultivars in which ELISA values for BNYVV are extremely low. Soils infested with P. betae, and with one or both viruses, showed significantly reduced fresh weight of seedlings, and aviruliferous P. betae significantly decreased sugar beet growth in assays.


Plant Disease ◽  
2008 ◽  
Vol 92 (7) ◽  
pp. 1043-1047 ◽  
Author(s):  
H.-Y. Liu ◽  
R. T. Lewellen

Rhizomania, a serious disease of sugar beet (Beta vulgaris), is caused by Beet necrotic yellow vein virus (BNYVV). Resistance allele Rz1 has been widely incorporated into commercial cultivars. Recently, resistance-breaking isolates of BNYVV (RB-BNYVV) were identified and characterized. When the occurrence of RB-BNYVV was surveyed throughout the sugar-beet-growing areas in the United States, most soil samples contained Beet oak-leaf virus (BOLV) as well. BNYVV and BOLV often occurred in the same field and sometimes in the same sugar beet plant. The possibility of interactions between these two Polymyxa betae-transmitted sugar beet viruses was tested. Plants grown in soils infested with aviruliferous P. betae or carrying RB-BNYVV and BOLV, alone and in combination, were compared with plants grown in noninfested soil for differences in plant fresh weight and virus content as measured by enzyme-linked immunosorbent assay (ELISA). Rz1 and Rz2 resistance genes that condition resistance to BNYVV did not confer resistance to BOLV. BNYVV ELISA values were significantly higher in single infections than in mixed infections with BOLV in both the rhizomania-resistant and -susceptible cultivars. In contrast, ELISA values of BOLV were not significantly different between single and mixed infections in both the rhizomania-resistant and -susceptible cultivars. Results indicate that BOLV may suppress BNYVV in mixed infections. Soils infested with P. betae significantly reduced fresh weight of sugar beet seedlings regardless of whether they were with or without one or both viruses or resistance genes.


2018 ◽  
Vol 28 (2) ◽  
pp. 210-217 ◽  
Author(s):  
Shin-Young Lee ◽  
Mi-Ju Kim ◽  
Hyun-Joong Kim ◽  
KwangCheol Casey Jeong ◽  
Hae-Yeong Kim

2012 ◽  
Vol 157 (6) ◽  
pp. 1063-1069 ◽  
Author(s):  
Masaharu Fukuda ◽  
Kazufumi Kuga ◽  
Ayako Miyazaki ◽  
Tohru Suzuki ◽  
Keito Tasei ◽  
...  

2007 ◽  
Vol 14 (12) ◽  
pp. 1563-1571 ◽  
Author(s):  
Noel P. Harrington ◽  
Om P. Surujballi ◽  
W. Ray Waters ◽  
John F. Prescott

ABSTRACT Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-γ)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN-γ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-γ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-γ mRNA responses correlated well with IFN-γ protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-γ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-γ expression by using consensus sequences of closely related species or of other species for which IFN-γ sequence information is available.


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