Emergence of Rsv-resistance breaking Soybean mosaic virus isolates from Korean soybean cultivars

Abstract

Twelve Rsv resistance-breaking (RB) isolates of Soybean mosaic virus (SMV) were obtained from field-grown soybean plants showing mosaic symptoms and subsequently examined biologically and molecularly. All of these RB isolates were identified as SMV based on serological and infectivity assays, and the amplification of P1 gene products by reverse transcription-polymerase chain reaction (RT-PCR). Differential soybean cultivars, lines or accessions Lee 68 (rsv), PI 96983, York, Marshall, Ogden, Kwanggyo, Suweon 97 (Rsv1 alleles), L29 (Rsv3), and V94-5152 (Rsv4), following inoculation with each RB isolate, showed similar systemic symptoms suggesting that these RB isolates can overcome Rsv resistance at three loci. To differentiate the 12 RB isolates molecularly, the P1 coding region for each isolate was amplified, cloned, sequenced and compared to known SMV strains. The P1 region from the RB isolates shared 86–90% and 90–99% similarities in amino acid (aa) and nucleotide sequence, respectively, with known SMV strains. Comparison of aa sequences indicated that these RB isolates are newly emerging isolates capable of breaking Rsv resistance. Phylogenetic analysis further suggested that the RB isolates can be classified as three major types. However, recombination was not observed within the coding region of P1 protein among the types. This is the first report on the emergence of SMV isolates capable of overcoming all of the known resistance alleles at the Rsv1 locus, as well as distinct resistance genes at Rsv3 and Rsv4.

Introduction

Soybean mosaic virus (SMV), a member of the genus Potyvirus, is one of the most prevalent viral pathogens of soybean [Glycine max (L.) Merr.] worldwide. The virus has a positive-sense single-stranded RNA genome of 9588 nucleotides with VPg at the 5′end and poly(A) tail at the 3′end. The genome of potyviruses encodes a single large polyprotein that is subsequently cleaved by virus-encoded proteases (Dougherty and Semler, 1993). The first protein in the N-terminal region of polyprotein, P1 protease, catalyzes the cleavage at a Tyr-Ser dipeptide between itself and helper component (HC-Pro, protease) protein (Mavankal and Rhoads, 1991, Verchot et al., 1992), and is essential for genome amplification (Verchot and Carrington, 1995). The diversity of potyviral P1 protein was proposed as an important parameter to distinguish different strains of a virus and to investigate the evolutionary relationships between different isolates (Lin et al., 2001, Domier et al., 2003). The P1 protein may also affect symptom development, host range, and geographical distribution (Lee and Wong, 1998).

Genetic resistance of soybean cultivars has been the most effective means of managing the disease caused by SMV. A single dominant gene for SMV resistance was first identified in soybean PI 96983 and designated as Rsv1 (Kiihl and Hartwig, 1979). Additional alleles at the same locus are carried by cultivars York, Marshall, Kwanggyo, Ogden, and Suweon 97 (Chen et al., 1991, Chen et al., 2002a). The Rsv1 allele-carrying genotypes exhibit different reaction combinations in response to SMV strain groups G1 through G7 described in the USA (Cho and Goodman, 1979, Cho and Goodman, 1982), Japan (Takahashi et al., 1980), China (Pu et al., 1982), and Korea (Cho and Chung, 1986). The system used to assign SMV isolate to pathotype is thus based on Rsv1-containing differential cultivars, with more virulent pathotypes overcoming a greater number of Rsv1 alleles (Chen et al., 1991, Chen et al., 2002a). In addition, the Rsv3 resistance gene has been identified in the Williams isoline L29 (Buss et al., 1999), and found to confer susceptibility to pathotypes of lesser virulence (G1–G3) but resistance to more virulent strains (G5–G7) (Gunduz et al., 2002). The Rsv4 resistance gene conferring resistance to SMV G1–G7 has been found in V94-5152 soybean derived from PI 486355 (Buss et al., 1997, Ma et al., 1995), as well as in PI 88788 and Peking (Gunduz et al., 2004).

In Korea, SMV was initially recognized as a mosaic-causing pathogen in soybean in the early 1970s (Chung et al., 1974). Soybean cultivars resistant to SMV were developed and within a short time, a severe necrotic strain, SMV-N, was found in some of the resistant cultivars, causing serious yield losses in a large production area (Cho et al., 1977). Since then, strains in soybean in Korea have been monitored by pathotype. More than seven SMV strains existed during 1980s, among which G5H was the most prevalent (Cho et al., 1983). However the relative incidence of G5H decreased gradually due to the utilization of resistant soybean cultivars such as Ilpumgeomjeongkong, Jangmikong, Jangsukong, Jinpumkong, Muhankong, Myeongjunamulkong, Pungsannamulkong, Sodamkong, and Soyangkong (Lee et al., 1992, Cho, 1995). In 1999 and 2000, SMV-G5H was estimated to represent only 4% of total SMV population in the Suweon area (Kim, 2000). In the 1990s, another severe isolate emerged, causing necrosis on cultivars Kwanggyo and Suweon 97, similar to the biological properties of SMV-G7 (Cho, 1995, Kim, 2000, Kim et al., 2003). Designated as SMV-G7H, this isolate was found to share 94% nucleotide (nt) sequence homology with G7 (Lim et al., 2003). In 1999–2000, SMV-G7H was reported as a predominant strain in Suweon, Korea, as it was isolated from more than 50% of SMV-infected plants, including those resistant to SMV-G5H (Kim, 2000).

The susceptibility of resistant cultivars in Korea is attributed to the emergence of resistance-breaking SMV strains, most likely from selection imposed on this seed-borne virus by the widespread use of Rsv resistance (Harrison, 2002). It is unclear, however, what evolutionary or genetic process changed SMV to enable the selected virus to overcome Rsv-mediated resistance. Therefore, this work was undertaken to systematically isolate and document the occurrence of new RB SMV strains in Korea and to examine their biological diversity based on reactions of several Rsv-containing differential soybean lines. Among the RB isolates are the first reported SMV strains that overcome resistance to SMV in soybean at all three of the known loci, Rsv1, Rsv3, and Rsv4. The genetic diversity of the new RB isolates was assessed at the molecular level by comparison of sequences of the P1 region with those of thirteen previous reported SMV strains.