Use of Unique RNA Sequence-Specific Oligonucleotide Primers for RT-PCR to Detect and Differentiate Soybean Mosaic Virus Strains

Plant Disease ◽  
1996 ◽  
Vol 80 (10) ◽  
pp. 1170 ◽  
Author(s):  
M. E. Omunyin
Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 561-561 ◽  
Author(s):  
S. Khankhum ◽  
P. Bollich ◽  
R. A. Valverde

Kudzu is an introduced legume commonly found growing as a perennial throughout the southeastern United States. This fast-growing vine was originally planted as an ornamental for forage and to prevent erosion (2), but is now considered an invasive species. During April 2011, a kudzu plant growing near a soybean field in Amite (Tangipahoa Parish, southeastern LA) was observed with foliar ringspot and mottle symptoms. Leaf samples were collected, and sap extracts (diluted 1:5 w/v in 0.02 M phosphate buffer pH 7.2) were mechanically inoculated onto carborundum-dusted leaves of at least five plants of the following species: kudzu, common bean (Phaseolus vulgaris) cv. Black Turtle Soup, globe amaranth (Gomphrena globosa), Nicotiana benthamiana, and soybean (Glycine max) cv. Asgrow AG 4801. Two plants of each species were also mock-inoculated. Eight to fourteen days after inoculation, all virus-inoculated plants showed virus symptoms that included foliar ringspots, mosaic, and mottle. Common bean and soybean also displayed necroses and were stunted. ELISA using antisera for Bean pod mottle virus, Cucumber mosaic virus, Soybean mosaic virus, and Tobacco ringspot virus (TRSV) (Agdia Inc., Elkhart, IN) were performed on field-collected kudzu and all inoculated plants species. ELISA tests resulted positive for TRSV but were negative for the other three viruses. All virus-inoculated plant species tested positive by ELISA. To confirm that TRSV was present in the samples, total RNA was extracted from infected and healthy plants and used in RT-PCR tests. The set of primers TRS-F (5′TATCCCTATGTGCTTGAGAG3′) and TRS-R (5′CATAGACCACCAGAGTCACA3′), which amplifies a 766-bp fragment of the RdRp of TRSV, were used (3). Expected amplicons were obtained with all of the TRSV-infected plants and were cloned and sequenced. Sequence analysis confirmed that TRSV was present in kudzu. Nucleotide sequence comparisons using BLAST resulted in a 95% similarity with the bud blight strain of TRSV which infects soybeans (GenBank Accession No. U50869) (1). TRSV has been reported to infect many wild plants and crops, including soybean. In soybean, this virus can reduce yield and seed quality (4). During summer 2012, three additional kudzu plants located near soybean fields showing ringspot symptoms were also found in Morehouse, Saint Landry, and West Feliciana Parishes. These three parishes correspond to the north, central, and southeast regions, respectively. These plants also tested positive for TRSV by ELISA and RT-PCR. The results of this investigation documents that TRSV was found naturally infecting kudzu near soybean fields in different geographical locations within Louisiana. Furthermore, a TRSV strain closely related to the bud blight strain that infects soybean was identified in one location (Amite). This finding is significant because infected kudzu potentially could serve as the source of TRSV for soybean and other economically important crops. To the best of our knowledge, this is the first report of TRSV infecting kudzu. References: (1) G. L. Hartman et al. 1999. Compendium of Soybean Diseases. American Phytopathological Society, St. Paul, MN. (2) J. H. Miller and B. Edwards. S. J. Appl. Forestry 7:165, 1983. (3) S. Sabanadzovic et al. Plant Dis. 94:126, 2010. (4) P. A. Zalloua et al. Virology 219:1, 1996.


2012 ◽  
Vol 38 (2) ◽  
pp. 202-209 ◽  
Author(s):  
Da-Gang WANG ◽  
Ying MA ◽  
Ning LIU ◽  
Gui-Jie ZHENG ◽  
Zhong-Lu YANG ◽  
...  

2014 ◽  
Vol 58 (01) ◽  
pp. 81-85 ◽  
Author(s):  
A. ZARZYŃSKA ◽  
M. JEŻEWSKA ◽  
K. TRZMIEL ◽  
B. HASIÓW-JAROSZEWSKA

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1285-1285 ◽  
Author(s):  
S. Lim ◽  
Y.-H. Lee ◽  
D. Igori ◽  
F. Zhao ◽  
R. H. Yoo ◽  
...  

In July 2013, soybean (Glycine max) plants at the research field in Daegu, South Korea, showed virus-like symptoms, such as mosaic, mottle, yellowing, and stunting. Overall, there were approximately 1% of soybean plants that showed these symptoms. Sixteen soybean samples were collected based on visual symptoms and subjected to laboratory characterization. Total RNA was extracted from each sample with the Tri Reagent (Molecular Research Center, Cincinnati, OH) and cDNA was synthesized using random N25 primer with RevertAid Reverse Transcriptase (Thermo Scientific, Waltham, MA), according to the manufacturers' instructions. All samples were tested by PCR with Prime Taq Premix (2X) (GeNet Bio, Daejeon, Korea) and primer sets specific to Soybean mosaic virus (SMV; 5′-CATATCAGTTTGTTGGGCA-3′ and 5′-TGCCTATACCCTCAACAT-3′), Peanut stunt virus (PSV; 5′-TGACCGCGTGCCAGTAGGAT-3′ and 5′-AGGTDGCTTTCTWTTGRATTTA-3′), Soybean yellow mottle mosaic virus (SYMMV; 5′-CAACCCTCAGCCACATTCAACTAT-3′ and 5′-TCTAACCACCCCACCCGAAGGATT-3′), and Soybean yellow common mosaic virus (SYCMV; 5′-TTGGCTGAGAGGAGTGGCTT-3′ and 5′-TGCGGTCGTGTAGTCAGTG-3′). Among 16 samples tested, five were positive for SMV and two for SYMMV. Three samples were found infected by both SMV and SYMMV and four by both SMV and PSV. Since two of the symptomatic samples were not infected by viruses described above, a pair of primers specific to Peanut mottle virus (PeMoV; 5′-GCTGTGAATTGTTGTTGAGAA-3′ and 5′-ACAATGATGAAGTTCGTTAC-3′) was tested (1). All 16 samples were subjected to RT-PCR with primers specific to PeMoV. Four were found positive for PeMoV. Two of them were already found infected with SYMMV. In order to identify the complete nucleotide sequences of PeMoV coat protein (CP), another primer set (5′-TGAGCAGGAAAGAATTGTTTC-3′ and 5′-GGAAGCGATATACACACCAAC-3′) was used. RT-PCR product was cloned into RBC TA Cloning Vector (RBC Bioscience, Taipei, Taiwan) and the nucleotide sequence of the insert was determined by Macrogen (Seoul, Korea). CP gene of the PeMoV (GenBank Accession No. KJ664838) showed the highest nucleotide sequence identity with PeMoV isolate Habin (KF977830; 99% identity), and the highest amino acid identity with GenBank Accession No. ABI97347 (100% identity). In order to fulfill Koch's postulates, several G. max cv. Williams 82 were inoculated with the extracts of PeMoV-infected leaf tissue. At 14 days post-inoculation, plants showed systemic mottle symptoms. These symptomatic plants were subjected to RT-PCR, and the nucleotide sequences of the PCR product were found identical to that of the virus in the inoculum. To our knowledge, this is the first report of soybean-infecting PeMoV, a member of the genus Potyvirus in the family Potyviridae, in South Korea. Reference: (1) R. G. Dietzgen et al. Plant Dis. 85:989, 2001.


Crop Science ◽  
1994 ◽  
Vol 34 (2) ◽  
pp. 414-422 ◽  
Author(s):  
P. Chen ◽  
G. R. Buss ◽  
C. W. Roane ◽  
S. A. Tolin

Plant Disease ◽  
2009 ◽  
Vol 93 (7) ◽  
pp. 760-760 ◽  
Author(s):  
B. D. Nelson ◽  
L. L. Domier

Soybean, Glycine max L, is grown on 1,420,000 ha in North Dakota and is the most important oilseed crop in the state. Viruses in soybean have not previously been reported from North Dakota (2). In July and August of 2007, 64 soybean fields in Cass, Richland, and Sargent counties in southeastern North Dakota were surveyed for Soybean mosaic virus (SMV). These counties have a high concentration of soybean hectares, a long history of soybean production, and soybean aphid infestations that were observed in 2004 and 2006. Fields were sampled with a grid pattern across the area with at least 8 km (5 miles) between fields. A transect of approximately 60 m through each field was made and 20 leaves were collected at random. Sap was extracted in phosphate buffer and stored at –80°C until tested first using double antibody sandwich (DAS)-ELISA with positive controls and reagents and protocol from Agdia Inc. (Elkhart, IN). Using DAS-ELISA, SMV was detected in 19 of the 64 soybean fields sampled. To confirm the presence of SMV, 12 samples that were positive for SMV by DAS-ELISA also were tested by reverse transcription (RT)-PCR. RNA was extracted from sap by a Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScrip III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen Inc., Carlsbad, CA) and SMV-specific primers (5′-TTCAGCACAATGGGTGAGGATG-3′ and 5′-AATTCTGTGTGGCTTGATGTTGC-3′) (1). Eight of the twelve ELISA-positive samples were positive for SMV by RT-PCR, confirming the presence of SMV in the samples. To our knowledge, this is the first report of SMV infecting soybean in North Dakota. References: (1) L. L. Domier et al. (Abstr.). Phytopathology 98(suppl.):S47, 2008. (2) B. D. Nelson and G. Danielson. (Abstr.). Phytopathology 95(suppl.):S164, 2005.


2021 ◽  
Author(s):  
Qiuyan Ren ◽  
Hua Jiang ◽  
Wenyang Xiang ◽  
Yang Nie ◽  
Song Xue ◽  
...  

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