First report of clover yellow vein virus on orchid (Dendrobium sp.) in South Korea

Orchid is one of the most popular and commercially important cultivated flowers in the world. Among many orchid species, Dendrobium species are popular cut flowers and potted plants in South Korea. In March 2019, 10 Dendrobium orchid plants in a greenhouse in Daegu, South Korea showed large chlorotic blotches, mosaic and mottle symptoms. One leaf each from the 10 symptomatic orchid plants by leaf dip-preparations and transmission electron microscopy (JEM-1400; JEOL Inc., Tokyo, Japan) after leaf dip-preparations (Brenner and Horne 1959; Richert-Pöggeler et al. 2019). Typical potyvirus-like particles of flexuous and filamentous shape and ∼ 760 × 15 nm length/width were observed in all tested samples. The presence of potyvirus was confirmed by serological detection with a commercially available ImmunoStrip® for potyvirus group (Agdia, Elkhart, USA). In contrast, a negative result was obtained for a virus-free Dendrobium plant by the serological test. The two most common viruses in orchids, namely odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) in all Dendrobium samples were not detected in any samples by an ImmunoStrip® for ORSV and CymMV (Agdia, Elkhart, USA). To determine the species of the virus, total RNA was extracted from all 10 ImmunoStrip®-positive samples using the RNeasy plant mini kit (Qiagen, Hilden, Germany). Subsequently, reverse transcription-PCR (RT-PCR) products (~1,625 bp) were amplified using potyvirus- specific primer pair (Gibbs and Mackenzie, 1997) and sequenced by the Sanger method at Macrogen (Seoul, South Korea). Sequencing results showed 100% nucleotide identity among 10 samples. Thus, one sequence was chosen for identification of virus species using sequence comparison. BLASTn analysis showed that the nucleotide sequence and its deduced amino acid sequence of the amplicon shared 95.4-98.7% and 96.2-99.6% identity to multiple clover yellow vein virus (ClYVV) sequences (e.g., accession no. AB011819) in GenBank. To further confirm the presence of ClYVV and determine if other viral agents were present in the samples, total RNA from three of the 10 symptomatic plants was depleted of ribosomal RNAs and subjected to high-throughput sequencing (HTS) analysis on a HiSeq 4000 platform (Macrogen Inc., Seoul, South Korea). A total of 3,764,432, 4,203,881, and 4,139,775 of 150-bp paired-end clean reads were obtained for the three samples. After de novo assembly of the reads with Trinity (Haas et al. 2013), 5, 6 and 7 contigs were obtained and searched with BLASTn against NCBI viral refseq database. Eighteen contigs from all three samples sized at 2,176-9,432 nt exhibited 94.0-97.9% nucleotide identity with the complete genome sequences of other ClYVV isolates (e.g., accession no. AB011819) deposited in Genbank; no other viruses were identified by HTS. The complete genome sequence (9,585 nucleotides in length) of ClYVV Dendrobium isolate (ClYVV-Den) was determined using ClYVV-specific primers (Takahashi et al., 1997) and the sequence of CIYVV-Den was deposited to GenBank (Accession no. LC506604). Together, these results support that symptomatic Dendrobium orchids were infected with ClYVV-Den in this study. ClYVV has been previously reported affecting Calanthe orchids in Japan (Inouye et al., 1988; Ikegami et al., 1995). Our results suggest that ClYVV may be detrimental to the production of Dendrobium orchids or commercial ornamental crops in South Korea. To our knowledge, this is the first report of ClYVV in Dendrobium sp. in South Korea.

Precise Exchange of the Helper-Component Proteinase Cistron Between Soybean mosaic virus and Clover yellow vein virus: Impact on Virus Viability and Host Range Specificity

Soybean mosaic virus and Clover yellow vein virus are two definite species of the genus Potyvirus within the family Potyviridae. Soybean mosaic virus-N (SMV-N) is well adapted to cultivated soybean (Glycine max) genotypes and wild soybean (G. soja), whereas it remains undetectable in inoculated broad bean (Vicia faba). In contrast, clover yellow vein virus No. 30 (ClYVV-No. 30) is capable of systemic infection in broad bean and wild soybean; however, it infects cultivated soybean genotypes only locally. In this study, SMV-N was shown to also infect broad bean locally; hence, broad bean is a host for SMV-N. Based on these observations, it was hypothesized that lack of systemic infection by SMV-N in broad bean and by ClYVV-No. 30 in cultivated soybean is attributable to the incompatibility of multifunctional helper-component proteinase (HC-Pro) in these hosts. The logic of selecting the HC-Pro cistron as a target is based on its established function in systemic movement and being a relevant factor in host range specificity of potyviruses. To test this hypothesis, chimeras were constructed with precise exchanges of HC-Pro cistrons between SMV-N and ClYVV-No. 30. Upon inoculation, both chimeras were viable in infection, but host range specificity of the recombinant viruses did not differ from those of the parental viruses. These observations suggest that (i) HC-Pro cistrons from SMV-N and ClYVV-No. 30 are functionally compatible in infection despite 55.6 and 48.9% nucleotide and amino acid sequence identity, respectively, and (ii) HC-Pro cistrons from SMV-N and ClYVV-No. 30 are not the determinants of host specificity on cultivated soybean or broad beans, respectively.

Recessive Resistance Governed by a Major Quantitative Trait Locus Restricts Clover Yellow Vein Virus in Mechanically but Not Graft-Inoculated Cultivated Soybeans

Clover yellow vein virus (ClYVV) infects and causes disease in legume plants. However, here, we found that ClYVV isolate No. 30 (ClYVV-No.30) inefficiently multiplied or spread via cell-to-cell movement in mechanically inoculated leaves of a dozen soybean (Glycine max) cultivars and resulted in failure to spread systemically. Soybean plants also had a similar resistance phenotype against additional ClYVV isolates. In contrast, all but one of 24 tested accessions of wild soybeans (G. soja) were susceptible to ClYVV-No.30. Graft inoculation of cultivated soybean TK780 with ClYVV-No.30–infected wild soybean B01167 scion resulted in systemic infection of the cultivated soybean rootstock. This suggests that, upon mechanical inoculation, the cultivated soybean inhibits ClYVV-No.30, at infection steps prior to the systemic spread of the virus, via vascular systems. Systemic infection of all F1 plants from crossing between TK780 and B01167 and of 68 of 76 F2 plants with ClYVV-No.30 indicated recessive inheritance of the resistance. Further genetic analysis using 64 recombinant inbred lines between TK780 and B01167 detected one major quantitative trait locus, designated d-cv, for the resistance that was positioned in the linkage group D1b (chromosome 2). The mapped region on soybean genome suggests that d-cv is not an allele of the known resistance genes against soybean mosaic virus.

P3N-PIPO, a Frameshift Product from theP3Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas

ABSTRACTPeas carrying thecyv1recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No.30 (Cl-No.30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking ofcyv1resistance by Cl-90-1 Br2 is P3N-PIPO produced from theP3gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene,Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele ofCyn1. Cl-No.30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized byCyn1and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized byCyn1in PI 226564. The level of P3N-PIPO accumulation from theP3gene of Cl-No.30 was significantly lower than that of Cl-90-1 Br2 in aNicotiana benthamianatransient assay. Therefore,Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate brokecyv1resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carryingCyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV.IMPORTANCEControl of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred bycyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No.30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in acyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominantCyn1gene, which may impose evolutionary constraints uponP3N-PIPOfor overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.

First Report of Clover yellow vein virus on White Clover (Trifolium repens) in South Korea

White clover (Trifolium repens L.) is a herbaceous, perennial plant that has become one of the most widely distributed legumes in the world. It is extensively used in grass-legume pastures, but also has the potential to invade agricultural lands and natural ecosystems. White clover is a well-known natural host for Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV), Soybean dwarf virus (SbDV), Beet western virus (BWYV), Tomato spotted wilt virus (TSWV), Zucchini yellow mosaic virus (ZYMV), etc (1). In July 2013, during a survey to determine the presence of different viruses infecting weed plants in South Korea, three white clover leaf samples showing yellow mosaic symptoms were collected from Taean County, South Chungcheong Do Province, South Korea. In order to identify the infecting virus, total RNA from three leaf samples was extracted using the Tri-reagent (MRC Reagent, Inc., OH) as described by the manufacturer, and was applied to the large-scale oligonucleotide (LSON) chip (3), wherein probes specific to a ClYVV isolate produced a positive reaction. All three samples tested were positive for ClYVV. To confirm this result, ClYVV-specific primers were designed using the sequences of four ClYVV isolates from NCBI (GenBank Accession Nos. AF185959, AF203536, DQ333346, and NC003536). Total RNA was extracted from symptomatic white clover samples using Easy-Spin Total RNA Extraction Kit (iNtRon, Daejeon, Korea) and used as template for RT-PCR. The positive control RNA was used from ClYVV GM isolate (KF975894) and negative control RNA used symptomless white clover plants. The ClYVV coat protein (CP) gene was amplified by RT-PCR using the specific primer pairs ClYVV-CP-F / ClYVV-CP-R (5′-CAAGAGCAGCACGATGAG-3′ and 5′-CTCGCTCTATAAAGATCAGAT-3′). DNA fragments of the expected size (1,042 bp) were obtained from the white clover Korea isolate (AB930132), and the PCR product was cloned into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan) and sequenced directly in both directions. BLAST analyses of the nucleotide sequence CP gene fragments revealed the highest identity with 98% with other ClYVV isolates (AF203536). To determine the experimental host range of the ClYVV Korea isolate, we inoculated five species (Chenopodium amaranticolor, C. quinoa, Nicotiana clevelandii, N. benthamiana, and Trifolium repens) in three families using this isolate. All test plants were mechanically inoculated with 0.1 M phosphate buffered saline (Takara, Tokyo, Japan). Each test plant was inoculated nine times and grown in a greenhouse maintained at 27 to 33°C. Necrotic local lesions were produced on inoculated leaves of C. amaranticolor, C. quinoa, and N. clevelandii 4 to 6 days post-inoculation. After 10 to 14 days, C. amaranticolor and C. quinoa showed systemic chlorotic spot symptoms, and N. clevelandii, N. benthamiana, and T. repens showed chlorotic spot, mild mosaic, and mosaic in the upper leaves, respectively. Up to now, in South Korea, ClYVV has been detected in gladiolus (Gladiolus gandavensis) (3) and soybean (Glycine max) (4). ClYVV can be easily transmitted by insect, aphid, or mechanical inoculation and has a host range including tobacco, soybean, etc. The presence of ClYVV could become an important threat to crop production in South Korea. To our knowledge, this is the first report of a ClYVV infection of the white clover plant in South Korea. References: (1) B. L. Denny and P. L. Guy. Australas. Plant Pathol. 38:270, 2009. (2) M. Nam et al. Plant Pathol. J. 30:51, 2014. (3) I. S. Park et al. Korean J. Plant Pathol. 14:74, 1998. (4) J. C. Shin et al. Plant Dis. 98:1283, 2014.

First Report of Clover yellow vein virus on Glycine max in Korea

Glycine max (Soybean) is the most important edible crop in Korea. In Korea, eight viruses have been reported to infect soybean, including Alfalfa mosaic virus (AMV), Cowpea mosaic virus (CPMV), Cucumber mosaic virus (CMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean yellow common mosaic virus (SYCMV), Soybean yellow mottle virus (SYMMV), and Peanut stunt virus (PSV) (1). In 2012, Glycine max were observed in Daegu, South Korea, with mosaic and mottling symptoms on leaves. Samples with virus-like symptoms (n = 151) were collected from Daegu including legume genetic resource field. Virus particles were filamentous rod shaped, average length 760 nm, and were analyzed by RT-PCR using specific primers for several Potyviruses and previously reported viruses infecting soybean. Only two samples showing mosaic and mottling symptoms were identified as Clover yellow vein virus (ClYVV) based on RT-PCR using primers specific for ClYVV (5′-GTTGGCTTGGTTGACACTGA-3′ and 5′-CTTCGATCATGGATGCACA-3′). The sequences of amplified fragments were 97 to 98% similar with ClYVV. ClYVV is a distinct species in the genus Potyvirus and family Potyviridae. ClYVV is transmitted by several species of aphids and by mechanical inoculation (2). ClYVV was first reported on Gentiana scabra, and the disease has never been reported in soybean fields in Korea. The biological properties and full genome sequence of the selected ClYVV isolate of apparent virus symptoms between two samples were analyzed. The ClYVV isolate was inoculated to local lesion plants, re-isolated from local lesions three times, and propagated in Nicotiana benthamiana, and then named ClYVV-Gm. The ClYVV-Gm induced local lesions on inoculated leaves of N. tabacum cv. Xanthi-nc, Tetragonia expansa, and systemic symptoms on upper leaves of Chenopodium amaranticolor, C. quinoa, and N. clevelandii. The ClYVV-Gm caused mosaic and mottling symptoms on Glycine max cv. Kwangan and Phaseolus vulgaris. The genome of ClYVV-Gm was determined to be 9,584 nucleotides in length (GenBank Accession No. KF975894), and it shared 83% to 97% nucleotide identity with the sequences of 27 previously reported ClYVV isolates including Vicia fava and Pisum sativum. Despite low occurrence of ClYVV in Glycine max, ClYVV has a broad host range including tobacco, weed species, and soybean, which can lead to spreading of the virus. Our results indicate that emergence of ClYVV could become a problem to Leguminosae in Korea. To our knowledge, this is the first biological and molecular report of ClYVV infecting Glycine max in Korea. References: (1) Y. H. Lee et al. Korea Soybean Digest 29:7, 2012. (2) T. Sasaya et al. Phytopathology 87:1014, 1997.