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Reaction of soybean to single and double inoculation with different Soybean mosaic virus strains
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Crop Protection 23 (2004) 965–971
Reaction of soybean to single and double inoculation with different Soybean mosaic virus strains Pengyin Chena,*, Glenn R. Bussa, Sue A. Tolinb b
a Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Received 18 July 2003; received in revised form 12 February 2004; accepted 17 February 2004
Abstract Two soybean genotypes [Glycine max (L.) Merr.] were inoculated either singly with eight Soybean mosaic virus (SMV) strains, or doubly with combinations of two strains, to examine the interactions between strains and determine if complementation, interference, or synergism is evident. Inoculated plants were monitored for symptom development and assayed for strain identity. Virus replication and movement were also monitored by enzyme-linked immunosorbent assay and by a leaf imprint immunoassay. Plants with resistance genes inoculated with two avirulent SMV strains remained healthy and virus was not detected by immunoassays, indicating no apparent complementation between avirulent strains to break resistance. Virulent necrosis-inducing or mosaic-inducing strains, in the presence of an avirulent strain, induced necrosis or mosaic symptoms, respectively, and only the virulent strains were recoverable from the mix-inoculated plants. A necrotic strain inoculated together with a mosaic strain resulted in mosaic symptoms and only the mosaic strain was recovered. Inoculation with two mosaic strains gave rise to mosaic symptoms and only the more virulent mosaic strain was recovered. Thus, following mixed inoculation, the interaction appears to be that of interference. Mosaic strains predominate over necrotic strains, and both predominate over avirulent strains. There is no evident complementation among avirulent strains or phenotypic synergism between two mosaic strains. r 2004 Elsevier Ltd. All rights reserved. Keywords: Glycine max; Viral interaction; Symptom expression; Pathogenicity; Complementation; Interference; Synergism
1. Introduction Soybean mosaic virus (SMV; genus Potyvirus; family Potyviridae) occurs in soybean [Glycine max (L.) Merr.] worldwide and causes significant yield loss and seed quality reduction (Hill, 1999). The virus is seedtransmitted and also readily sap- and aphid-transmissible. Various SMV isolates have been found to differ in pathogenicity and symptom expression on soybean (Cho and Goodman, 1979; Conover, 1948; Ross, 1969, 1975). A variety of symptoms ranging from mild mosaic to severe necrosis have been observed in various soybean cultivars (Cho et al., 1977; Cho and Goodman, 1979; Conover, 1948; Hill, 1999; Ross, 1969, 1975). Cho and Goodman (1979) developed a classification system for SMV isolates based on their virulence on *Corresponding author. Tel.: +1-479-575-7564; fax: +1-479-5757465. E-mail address: [email protected] (P. Chen). 0261-2194/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2004.02.009
eight soybean cultivars and assigned 98 isolates to seven strain groups, designated G1–G7. The reactions of these cultivars to SMV strains were described as resistant (symptomless), necrotic (localized and/or systemic necrosis), or susceptible (mosaic). Two additional isolates, G7A (Buzzell and Tu, 1984) and C14 (Lim, 1985), were later reported and used in SMV-resistance studies. A necrotic strain, SMV-N, occurred in Korea, causing an outbreak of severe necrosis on the cultivar Kwanggyo and four other leading cultivars possessing SMV-resistance genes (Cho et al., 1977). The infected plants produced virtually no seeds. Another necrotic strain was reported in Canada causing stem-tip necrosis on cultivar Columbia (Tu and Buzzell, 1987) and its derivatives. Takahashi and coworkers (1980) reported five SMV strains (A–E) in Japan. In addition, six SMV strains have been identified and characterized in China (Gai et al., 1989). However, the pathotypic relationships among the SMV strains from different countries are unknown.
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of soybean cv. York. All eight strains were verified for their pathotype identities by inoculation to a set of differential soybean genotypes consisting of ‘Lee 68’, ‘York’, ‘Marshall’, ‘Kwanggyo’, ‘Ogden’, and ‘PI 96983’ (Table 1) prior to the inoculation experiment. Two SMV-resistant soybean genotypes, PI 96983 (Rsv1) and York (Rsv1-y) (Chen et al., 1991, 1994), were used in the experiments. Soybean seeds were planted in 15-cm pots (12–15 seeds/pot) containing composite soil (60% Metro Mix 360 and 40% top soil). Eight to 12 plants were maintained in each pot (experiment unit) under greenhouse conditions described previously (Chen et al., 1991). Each inoculation treatment was assigned to a pot with 8–12 plants and replicated twice. Inoculation was performed at the unifoliolate leaf stage (V1) (Chen et al., 1991). Uninoculated plants and mock inoculated plants served as checks. The inoculation treatments included single inoculation, separate double inoculation, and mixed double inoculation. The single inoculation was conducted by rubbing both unifoliolate leaves of each test plant with inoculum of only one SMV strain. The separate double inoculations were performed by inoculating one unifoliolate leaf of each plant with one strain, and the other unifoliolate with a second strain. Mixed double inoculations were done by inoculating both unifoliolate leaves of each plant with mixed inoculum (equal volume) from two SMV strains. The strain combinations for separateand mixed-double inoculation included all possible combinations of any two of the eight SMV strains, except for the combination of G5 and G6 that are similar in pathogenicity and cannot be differentiated by the soybean genotypes used for double inoculation. The inoculum of each SMV strain was prepared separately from leaves showing typical mosaic symptoms 2–3 weeks after inoculation of the virus-maintenance plants. Infected trifoliolate leaves were homogenized in a chilled mortar and pestle in 0.01 M sodium phosphate buffer (pH 7.0) at a ratio of 1 g infected tissue per 10 ml buffer. The inoculum prepared for each strain was used for single and separate double inoculations. For the mixed double inoculation, equal
The worldwide occurrence of multiple strains of SMV has epidemiological importance for soybean production, and has the potential to jeopardize the value of resistant cultivars and affect exchange of soybean germplasm. For example, resistance to a mild strain in a resistant cultivar can be overcome by a more virulent strain resulting in mosaic or necrotic symptoms (Cho and Goodman, 1979; Cho et al., 1977). It is not yet known whether a mosaic or necrotic stain will be predominant if both are present in a soybean production region. In addition, stains with different virulence can be spread with germplam exchange worldwide causing epidemic problems since SMV is highly seed-transmissible. The necrotic reaction, once occurring, causes stem tip necrosis and bud blight which eventually lead to total plant death. It is thus critical to determine the interactions and dominance relationships among various SMV strains in a single plant. This investigation was initiated to examine which, if any, of the possible interactions (complementation, interference, or synergism) occur upon simultaneous inoculation of two SMV strains to soybean cultivars having SMV-strain-specific resistance genes.
2. Materials and methods The SMV strains used in this study were G1–G7 and G7A. The G1 strain, designated SMV-VA previously, was originally isolated from ‘Lee’ soybean in Virginia and is analogous to Cho and Goodman’s (1979) G1 (Hunst and Tolin, 1982). Strains G2–G7 and G7A were obtained from Dr. R. M. Goodman in 1984, then at the University of Illinois. Cultures of these SMV strains have been deposited in the American Type Culture Collection (Rockville, MD) as PV-571 (SMV-G1/VA, a G1 isolate from Virginia), PV-572 (SMV-G4), PV-573 (SMV-G5), PV-612 (SMV-G6), PV-613 (SMV-G7), and PV-614 (SMV-G7A) (Chen et al., 1994). Strains G1–G4 were mechanically transmitted and individually maintained on soybean cv. Lee 68 in the greenhouse. Other strains (G5–G7 and G7A) were maintained separately by successive inoculations on greenhouse-grown plants
Table 1 Differential reactions of soybean genotypes with different resistance alleles to Soybean mosaic virus strains identified in the US Cultivar/line
Lee 68 York Marshall Kwanggyo Ogden PI 96983 a
Genotype
rsv Rsv1-y Rsv1-m Rsv1-k Rsv1-t Rsv1
Reaction to SMV strainsa G1
G2
G3
G4
G5
G6
G7
G7A
M R R R R R
M R N R R R
M R N R N R
M N R R R R
M M R N R R
M M N N R R
M M N N N N
M M M N M M
R=resistant (symptomless), N=necrotic (systemic necrosis), M=mosaic.
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volumes of inoculum from each of two SMV strains were taken and mixed well in a test tube. The inoculum was kept on ice until inoculation was completed. Inoculum was applied to the 75% expanded unifoliolate leaves using a pestle dipped in the inoculum (one dip per leaflet, approximately 100 ml inoculum per leaflet) to rub the entire leaf surface. The leaves for inoculation were pre-dusted with 600-mesh carborundum as abrasive before inoculation and rinsed with tap water after inoculation. Buffer (mock) inoculation and/or noninoculation of York and PI 96983 plants served as controls. Spot inoculation for tracking the virus replication and movement in York, Kwanggyo, and Lee 68 soybean was done by applying inoculum of G4 or G5 with a glass rod onto one or two 3–4 mm diameter sites on an unifoliolate leaf. York is necrotic to G4 and mosaic to G5, whereas Kwanggyo is resistant to G4 and necrotic to G5. Lee 68 is mosaic to both G4 and G5. These genotype and stain combinations allow the detection and tracking of virus activities and comparisons among different reactions. Inoculated plants were monitored for symptom development on a regular basis and notes were taken for 4 weeks after inoculation. Plants were classified as resistant (R) if no symptoms developed, necrotic (N) if stem tip necrosis or systemic necrotic lesions or systemic vein, stem, and petiole necrosis occurred, and mosaic (M) if systemic mosaic symptoms were observed. When mosaic or necrotic symptoms were observed on plants inoculated with two SMV strains involving G4–G6, or G7, trifoliolate leaf samples were taken from those symptomatic plants to produce inoculum for inoculation of another set of York or PI 96983 plants, which would differentiate the two strains (Table 1). ELISA was conducted according to Lister (1978) using trifoliolate leaf samples taken from PI 96983 plants inoculated with one or two SMV strains. Each sample was replicated twice and values of absorbance at 405 nm were determined 1 h after adding the substrate. Data for ELISA were analyzed using the general linear model procedure (PROC GLM) in SAS (SAS Institute In., Carry, NC). Fisher’s protected least significant difference (LSD) at P ¼ 0:05 was used to compare mean ELISA values of different inoculation treatments. Leaf imprint immunoassay (LIIA) was conducted according to methods described previously (Holt, 1992; Lin et al., 1990; Mansky, 1990; Mansky et al., 1990) with modifications to detect virus movement and replication at exact locations in the leaves. The leaf was imprinted onto a hardened filter paper (Schleicher & Schuell #8) rather than nitrocellulose membranes. The paper was first immersed in 5% Triton X-100 (Sigma Laboratories) to remove the residual green color caused by the leaf imprinting (Srinivasan and Tolin, 1992), and then rinsed in 1X KPS-T buffer (0.1 M K2HPO4, 0.75 M NaCl, and 0.05% Tween-20, pH 7.4). Blocking was with
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4% non-fat dry milk in 1X KPS-T buffer and 0.5% bovine serum albumin (BSA) (Sigma Laboratories). Following incubation with SMV-specific whole serum from rabbit (primary antibody) (Hunst and Tolin, 1982) in 1X KPS-T buffer, and then in goat anti-rabbit antibody (secondary antibody) (Hunst and Tolin, 1982) conjugated to alkaline phosphatase (Sigma Laboratories), color was developed in nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl-phosphate (BCIP) (Zymed Laboratories). The results were analyzed visually using the leaf imprints and compared with photographs of leaves before blotting.
3. Results and discussion York exhibited a resistant reaction to single inoculation of SMV strains G1–G3, but was mosaic to strains G5–G7 and G7A. PI 96983 was infected only when inoculated with G7 and G7A and showed necrosis and mosaic symptoms, respectively (Table 2). The inoculation results for strains G1–G7 are consistent with those obtained by Cho and Goodman (1979, 1982). However, in our tests York did not give 100% necrotic plants when inoculated with G4. The non-symptomatic York plants were apparently result of escapes from the virus infection. Mixed reactions of resistance and necrosis from other soybean genotype SMV strain combinations were also observed on occasion in genetic studies (Chen et al., 1991, 1994). It also appears that the necrotic reaction is somewhat affected by environmental conditions. Tu and Buzzell (1987) found that the Table 2 Reaction of York and PI 96983 soybean to single inoculation with different Soybean mosaic virus strains SMV strain
G1 G2 G3 G4 G5 G6 G7 G7A Uninoculated Mock inoculated LSD0.05
Phenotypic reactiona
A405 nmb
York
PI 96983
PI 96983
R R R N/R M M M M R R
R R R R/N R R N M R R
0.074 0.055 0.060 0.060 0.062 0.078 0.179 0.250 0.077 0.011 0.019
a R=resistant (symptomless), N=necrosis (systemic necrosis), M=mosaic, N/R=most plants (two pots of 8–12 plants for each inoculation) were necrotic and mixed with a few resistant plants (escapes), R/N=most plants were resistant and mixed with a few necrotic plants. b Mean values of absorbance at A405 nm, determined by four replications (two samples from each of the two pots), readings were taken 1 h after addition of the substrate.
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development of stem tip necrosis on cultivar Columbia was temperature dependent; inoculated plants developed necrosis at 20–24 C, but not at 28–32 C. Similar thermosensitive responses have been reported in the Tobacco mosaic virus (TMV) tobacco N gene interaction system (Kassanis, 1952). PI 96983 plants inoculated singly with strains G1–G6 remained healthy and no virus was detected in trifoliolate leaves by ELISA (Table 2). Cho and Goodman (1979, 1982) also found that symptomless plants inoculated with SMV did not contain virus, as determined by indexing to Topcrop bean. Positive results were obtained by ELISA for necrotic and mosaic PI 96983 plants inoculated with SMV-G7 and G7A, respectively. However, the ELISA values were higher for the mosaic plants than that for the necrotic plants. Apparently, virus replication and movement were limited in the necrotic response. Tu and Buzzell (1987) were able to isolate SMV from necrotic tissue of Columbia soybean, but did not estimate the virus concentration. We observed that plants exhibiting the stem–tip necrosis reaction usually showed mosaic symptoms on the first trifoliolate leaf initially, and then turned necrotic and eventually died. To confirm the hypothesis that virus replication is limited in the necrotic reaction, we used LIIA to detect virus antigen in situ in a leaf. By LIIA, SMV in inoculated unifoliolate leaves of mosaic and necrotic plants could be visualized at the site of inoculation within 6 days after inoculation (DAI). In the mosaic reactions of Lee 68 to G4 or G5 and York to G5 viral protein was detected along the midrib and some side veins within 9 DAI, indicating the replication and movement of the virus (Table 3). The color intensity and area of the leaves containing virus increased with time, with maximum signal being detected at 18–20 DAI at a distance of 5–6 mm either side of the midrib and along several lateral veins. In the necrotic reactions of York to G4, or Kwanggyo to G5, virus was detected only exactly on or immediately surrounding a necrotic area on the leaf. Virus was sometimes detected even on a very small
necrotic lesion, indicating that virus replicated to a detectable level within a few cells before the necrotic area developed in the leaf. At longer times and as necrosis developed along the midrib and in trifoliolate leaves, virus was detected in the areas of necrosis only. No virus was detectable in inoculated unifoliolate and upper trifoliolate leaves of resistant plants (Kwanggyo inoculated with G4). This assay was more sensitive than ELISA in detecting virus in necrotic plants and should prove useful for further study of mixed infections. There was no substantial difference in plant reactions to separate- and mixed-double inoculations (Table 4). The G1 and G4 combination did not induce 100% necrotic plants with either type of double inoculation, which is comparable to the single inoculation of York with G4 alone. Thus, G4 was predominant when introduced together with G1 to York plants. The strain-differentiating York plants inoculated with inoculum from mix-infected (G1+G4) plants also exhibited necrotic reactions with a few escapes, as observed on York plants inoculated singly with G4. Therefore, the strain isolated from the double-inoculated (G1+G4) plants was identified as G4. Necrotic lesions were observed on G4-inoculated unifoliolate leaves of York plants and results of LIIA had shown that G4 is localized in these lesions. When G4 and G1 were inoculated to separate unifoliolate leaves of the same York plant, lesions were only present on the leaf inoculated with G4 but not on the other leaf inoculated with G1. Both unifoliolate leaves of York plants showed necrotic lesions when inoculated with the mixed inoculum of G4 and G1. In the tests of strain combinations of G1 (avirulent) and a mosaic-inducing strain (G5, G6, or G7), the inoculated York plants showed mosaic symptoms as observed with single inoculation by the mosaic strains, indicating that G1 did not interfere with the mosaic strains (Table 4). Strains recovered from the mixinoculated plants were identified as the respective mosaic-inducing strains. Upon inoculation of necrotic strain (G4) and a mosaic strain (G5, G6 or G7), mosaic
Table 3 Distribution of Soybean mosaic virus in spot-inoculated soybean leaves detected by leaf imprinting immunoassay 9 days after inoculation Cultivar (genotype)
SMVstrain
Phenotypic reactiona
Blotting patternb
SMV local detectionc
SMV movementd
York (Rsv1-y) Kwangyyo (Rsv-k) Lee 68 (rsv)
G4 G5 G4 G5 G4 G5
N M R N M M
LSL AVM NVD LSL AVM AVM
+ +++ + +++ +++
+ +++ + +++ +++
a
R=resistant (symptomless), N=necrosis (systemic necrosis), M=mosaic. LSL=localized spots and lesions, AVM=along veins and midrib; NVD=no virus detected. c +=virus detected, = no virus detected, +++ indicates higher titer of SMV detected, based on the intensity of immuno-reaction. d +=virus moved from inoculated site, = no virus movement, +++ indicates higher titer of SMV detected. b
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Table 4 Reaction of York soybean to separate double and mixed double inoculations with two Soybean mosaic virus strains Separate double inoculationa SMV Inoculum
Reaction on York
Avirulent and necrotic strains G1+G4 N/Rc Avirulent and mosaic strains G1+G5 M G1+G6 M G1+G7 M Necrotic and mosaic strains G4+G5 M G4+G6 M G4+G7 M Two mosaic strains G5+G7 M
Mixed double inoculationb
Recovery onto
Strain recovered
York
PI 96983
N/Rc
—d
G4
M M M
— — N
M M M M
Reaction on York
Recovery onto
Strain recovered
York
PI 96983
N/Rc
N/Rc
—d
G4
G5 G6 G7
M M M
M M M
— — N
G5 G6 G7
— — N
G5 G6 G7
M M M
M M M
— — N
G5 G6 G7
N
G7
M
M
N
G7
a
Two unifoliolate leaves of a test plant were inoculated separately with two different SMV strains. Both unifoliolate leaves were inoculated with mixed inoculum from two SMV strains. c R=resistant (symptomless), N=necrosis, M=mosaic, N/R=most plants were necrotic and mixed with a few resistant plants (escapes). d —=not tested, inoculation will not differentiate the two strains. b
Table 5 ELISA and reaction of PI 96983 soybean to mixed double inoculation with two Soybean mosaic virus strains Strains
Reactiona
A405 nmb
Strains
Reactiona
A405 nmb
G1+G2 G1+G3 G2+G3 G1+G4 G2+G4 G3+G4 G1+G6 G2+G6 G3+G6 G4+G6 Uninoculated Mock inoculated
R R R R/N R/N R/N R R R R R R
0.111 0.105 0.057 0.166 0.108 0.118 0.078 0.087 0.078 0.074 0.077 0.011
G1+G7 G2+G7 G3+G7 G4+G7 G6+G7 G1+G7A G2+G7A G3+G7A G4+G7A G6+G7A G7+G7A LSD0.05
N N N N N M M M M M M
0.134 0.129 0.161 0.102 0.187 0.198 0.269 0.459 0.350 0.307 0.445 0.083
R N/R M M
0.087 0.142 0.316 0.445 0.094
Pooled data Two avirulent strains Avirulent and necrotic strains Avirulent and mosaic strains Necrotic and mosaic strains LSD0.05 a
R=resistant (symptomless), N=necrosis, S=mosaic, R/N=most of resistant mixed with a few necrotic plants, N/R=most plants were necrotic and mixed with a few resistant plants (escapes). b Mean values of absorbance at A405 nm, determined by four replications (two samples from each of two pots), readings were taken 1 h after addition of the substrate.
strains became predominant and caused mosaic symptoms on York with no systemic necrosis. The strains recovered from the mix-inoculated plants were the corresponding mosaic strains. SMV-G7 predominated over G5 when both were introduced to York plants by either inoculation method because only G7 was recoverable. This is consistent with the suggestion that G7 is more virulent than G5 (Cho and Goodman, 1979).
Thus, no apparent phenotypic synergism was indicated by the double inoculation of two mosaic strains. In the complementation tests of avirulent strains on PI 96983, G6 combined with other strains (G1–G4) caused no symptoms and no virus was detected by ELISA, suggesting that G6 did not complement any of the four avirulent strains (Table 5). A few necrotic plants were observed when G4 was mix-inoculated with
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G1, G2, or G3, and relatively low ELISA values were obtained from those plants, indicating that there was limited virus accumulation in the necrotic plants, especially with the G1+G4 inoculation. Plants inoculated with G1+G2, G1+G3 and G2+G3 remained healthy and showed an ELISA value similar to the healthy controls. No mosaic plants were observed for mixed inoculations with any two of the five avirulent strains (G1–G4 and G6). Thus, complementation among avirulent strains was not phenotypically demonstrable in our system. PI 96983 plants inoculated with the necrosis-inducing strain (G7) and any avirulent strains (G1–G4 or G6) all showed stem–tip necrosis and gave ELISA values numerically higher than the negative controls. However, the differences were not statistically different except for G1+G4, G3+G7, and G6+G7 combinations (Table 5). This was to be expected since the virus titer in necrotic tissue, as shown by LIIA, was very low (Table 3). The necrotic symptoms and ELISA values from the mix-inoculated plants were comparable to those from the single inoculation of G7 (Tables 2 and 5), indicating that avirulent strains were unable to interfere with or complement G7 when they were mixed and present in the same plant, and that the strain causing necrosis was G7 alone. The strain combinations of G7A and an avirulent strain (G1–G4 and G6) resulted in mosaic symptoms on PI 96983, as expected from single inoculation of G7A. However, ELISA showed higher amount of virus in PI 96983 plants inoculated with G3+G7A than in plants inoculated with G1+G7A, G2+G7A, G4+G7A or G6+G7A. It appears that G7A replication was somewhat unaffected in PI 96983 plants by G1, G2, G4, or G6, but was enhanced by G3. The mix-inoculation of a necrotic (G7) and a mosaic strain (G7A) induced mosaic symptoms and gave rise to higher ELISA values than single inoculation with either strain, indicating that the replication of the mosaic strain was somewhat elevated by the necrotic strain. All mixed inoculations involving a mosaic strain resulted in significantly higher virus titer than inoculation combinations of avirulent and necrotic strains. In our test for double inoculations of an avirulent strain and a mosaic or necrotic strain, mosaic or necrotic symptoms were always observed and the virulent (symptom-causing) strain was recovered. However, the possibility that the avirulent strain co-existed with virulent strains in the mix-inoculated plants but was not visible phenotypically on symptomatology cannot be ruled out. Isolation from a population of lesions on Topcrop bean, as was done with G2 and G7A by Mansky and coworkers (1990), would be needed to confirm the hypothesis. Detection of the avirulent strains in the mix-infected plants would require SMV strain specific antibody which was not available to us. G7 and G7A together induced mosaic symptoms instead
of stem–tip necrosis on PI 96983, indicating that the mosaic strain G7A predominated over the necrotic strain G7 when both strains were present in the same plants. A higher ELISA value (0.445) was obtained from the mix-inoculated (G7+G7A) PI 96983 plants than from G7 or G7A-single-inoculated PI 96983 plants (0.179 and 0.25), suggesting that the two strains might have interacted synergistically when inoculated together. In the combined data analysis, plants inoculated with a necrotic and a mosaic strain had highest ELISA value. Plants inoculated with an avirulent and a mosaic strain gave the second highest ELISA value, which is significantly higher than that of plants inoculated with an avirulent and a necrotic strain or plants inoculated with two avirulent strains (Table 5). The implications of this work are that a soybean cultivar recognized as resistant to one strain of SMV may be mosaic or necrotic to another strain. When multiple strains with different pathogeniecity and/or virulence occur in a field situation, the dominance relationship among them will be in the order of severe mosaic strains > mild mosaic strains > necrotic strains > avirulent strains. We suggest that strain identity and stability be monitored in virus research to ensure that the biological properties of the virus strain(s) used for screening soybean germplasm are known and that the strains are representative of the virus which the commercial soybean will ultimately encounter. In selecting soybean varieties for production areas with potential threat of multiple SMV strains, it is highly recommended that lines with genes such as Rsv1-h for resistance to multiple strains be utilized (Chen et al., 2002). As for soybean breeding research, reliance on a single source of resistance would result in genetic uniformity and potential vulnerability. Gene pyramiding to incorporate resistance genes at different loci from various sources into a single soybean line might be desirable for developing cultivars with durable resistance to multiple strains of SMV.
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Reaction of soybean to single and double inoculation with different Soybean mosaic virus strains Pengyin Chena,*, Glenn R. Bussa, Sue A. Tolinb b
a Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Received 18 July 2003; received in revised form 12 February 2004; accepted 17 February 2004
Abstract Two soybean genotypes [Glycine max (L.) Merr.] were inoculated either singly with eight Soybean mosaic virus (SMV) strains, or doubly with combinations of two strains, to examine the interactions between strains and determine if complementation, interference, or synergism is evident. Inoculated plants were monitored for symptom development and assayed for strain identity. Virus replication and movement were also monitored by enzyme-linked immunosorbent assay and by a leaf imprint immunoassay. Plants with resistance genes inoculated with two avirulent SMV strains remained healthy and virus was not detected by immunoassays, indicating no apparent complementation between avirulent strains to break resistance. Virulent necrosis-inducing or mosaic-inducing strains, in the presence of an avirulent strain, induced necrosis or mosaic symptoms, respectively, and only the virulent strains were recoverable from the mix-inoculated plants. A necrotic strain inoculated together with a mosaic strain resulted in mosaic symptoms and only the mosaic strain was recovered. Inoculation with two mosaic strains gave rise to mosaic symptoms and only the more virulent mosaic strain was recovered. Thus, following mixed inoculation, the interaction appears to be that of interference. Mosaic strains predominate over necrotic strains, and both predominate over avirulent strains. There is no evident complementation among avirulent strains or phenotypic synergism between two mosaic strains. r 2004 Elsevier Ltd. All rights reserved. Keywords: Glycine max; Viral interaction; Symptom expression; Pathogenicity; Complementation; Interference; Synergism
1. Introduction Soybean mosaic virus (SMV; genus Potyvirus; family Potyviridae) occurs in soybean [Glycine max (L.) Merr.] worldwide and causes significant yield loss and seed quality reduction (Hill, 1999). The virus is seedtransmitted and also readily sap- and aphid-transmissible. Various SMV isolates have been found to differ in pathogenicity and symptom expression on soybean (Cho and Goodman, 1979; Conover, 1948; Ross, 1969, 1975). A variety of symptoms ranging from mild mosaic to severe necrosis have been observed in various soybean cultivars (Cho et al., 1977; Cho and Goodman, 1979; Conover, 1948; Hill, 1999; Ross, 1969, 1975). Cho and Goodman (1979) developed a classification system for SMV isolates based on their virulence on *Corresponding author. Tel.: +1-479-575-7564; fax: +1-479-5757465. E-mail address: [email protected] (P. Chen). 0261-2194/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2004.02.009
eight soybean cultivars and assigned 98 isolates to seven strain groups, designated G1–G7. The reactions of these cultivars to SMV strains were described as resistant (symptomless), necrotic (localized and/or systemic necrosis), or susceptible (mosaic). Two additional isolates, G7A (Buzzell and Tu, 1984) and C14 (Lim, 1985), were later reported and used in SMV-resistance studies. A necrotic strain, SMV-N, occurred in Korea, causing an outbreak of severe necrosis on the cultivar Kwanggyo and four other leading cultivars possessing SMV-resistance genes (Cho et al., 1977). The infected plants produced virtually no seeds. Another necrotic strain was reported in Canada causing stem-tip necrosis on cultivar Columbia (Tu and Buzzell, 1987) and its derivatives. Takahashi and coworkers (1980) reported five SMV strains (A–E) in Japan. In addition, six SMV strains have been identified and characterized in China (Gai et al., 1989). However, the pathotypic relationships among the SMV strains from different countries are unknown.
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of soybean cv. York. All eight strains were verified for their pathotype identities by inoculation to a set of differential soybean genotypes consisting of ‘Lee 68’, ‘York’, ‘Marshall’, ‘Kwanggyo’, ‘Ogden’, and ‘PI 96983’ (Table 1) prior to the inoculation experiment. Two SMV-resistant soybean genotypes, PI 96983 (Rsv1) and York (Rsv1-y) (Chen et al., 1991, 1994), were used in the experiments. Soybean seeds were planted in 15-cm pots (12–15 seeds/pot) containing composite soil (60% Metro Mix 360 and 40% top soil). Eight to 12 plants were maintained in each pot (experiment unit) under greenhouse conditions described previously (Chen et al., 1991). Each inoculation treatment was assigned to a pot with 8–12 plants and replicated twice. Inoculation was performed at the unifoliolate leaf stage (V1) (Chen et al., 1991). Uninoculated plants and mock inoculated plants served as checks. The inoculation treatments included single inoculation, separate double inoculation, and mixed double inoculation. The single inoculation was conducted by rubbing both unifoliolate leaves of each test plant with inoculum of only one SMV strain. The separate double inoculations were performed by inoculating one unifoliolate leaf of each plant with one strain, and the other unifoliolate with a second strain. Mixed double inoculations were done by inoculating both unifoliolate leaves of each plant with mixed inoculum (equal volume) from two SMV strains. The strain combinations for separateand mixed-double inoculation included all possible combinations of any two of the eight SMV strains, except for the combination of G5 and G6 that are similar in pathogenicity and cannot be differentiated by the soybean genotypes used for double inoculation. The inoculum of each SMV strain was prepared separately from leaves showing typical mosaic symptoms 2–3 weeks after inoculation of the virus-maintenance plants. Infected trifoliolate leaves were homogenized in a chilled mortar and pestle in 0.01 M sodium phosphate buffer (pH 7.0) at a ratio of 1 g infected tissue per 10 ml buffer. The inoculum prepared for each strain was used for single and separate double inoculations. For the mixed double inoculation, equal
The worldwide occurrence of multiple strains of SMV has epidemiological importance for soybean production, and has the potential to jeopardize the value of resistant cultivars and affect exchange of soybean germplasm. For example, resistance to a mild strain in a resistant cultivar can be overcome by a more virulent strain resulting in mosaic or necrotic symptoms (Cho and Goodman, 1979; Cho et al., 1977). It is not yet known whether a mosaic or necrotic stain will be predominant if both are present in a soybean production region. In addition, stains with different virulence can be spread with germplam exchange worldwide causing epidemic problems since SMV is highly seed-transmissible. The necrotic reaction, once occurring, causes stem tip necrosis and bud blight which eventually lead to total plant death. It is thus critical to determine the interactions and dominance relationships among various SMV strains in a single plant. This investigation was initiated to examine which, if any, of the possible interactions (complementation, interference, or synergism) occur upon simultaneous inoculation of two SMV strains to soybean cultivars having SMV-strain-specific resistance genes.
2. Materials and methods The SMV strains used in this study were G1–G7 and G7A. The G1 strain, designated SMV-VA previously, was originally isolated from ‘Lee’ soybean in Virginia and is analogous to Cho and Goodman’s (1979) G1 (Hunst and Tolin, 1982). Strains G2–G7 and G7A were obtained from Dr. R. M. Goodman in 1984, then at the University of Illinois. Cultures of these SMV strains have been deposited in the American Type Culture Collection (Rockville, MD) as PV-571 (SMV-G1/VA, a G1 isolate from Virginia), PV-572 (SMV-G4), PV-573 (SMV-G5), PV-612 (SMV-G6), PV-613 (SMV-G7), and PV-614 (SMV-G7A) (Chen et al., 1994). Strains G1–G4 were mechanically transmitted and individually maintained on soybean cv. Lee 68 in the greenhouse. Other strains (G5–G7 and G7A) were maintained separately by successive inoculations on greenhouse-grown plants
Table 1 Differential reactions of soybean genotypes with different resistance alleles to Soybean mosaic virus strains identified in the US Cultivar/line
Lee 68 York Marshall Kwanggyo Ogden PI 96983 a
Genotype
rsv Rsv1-y Rsv1-m Rsv1-k Rsv1-t Rsv1
Reaction to SMV strainsa G1
G2
G3
G4
G5
G6
G7
G7A
M R R R R R
M R N R R R
M R N R N R
M N R R R R
M M R N R R
M M N N R R
M M N N N N
M M M N M M
R=resistant (symptomless), N=necrotic (systemic necrosis), M=mosaic.
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volumes of inoculum from each of two SMV strains were taken and mixed well in a test tube. The inoculum was kept on ice until inoculation was completed. Inoculum was applied to the 75% expanded unifoliolate leaves using a pestle dipped in the inoculum (one dip per leaflet, approximately 100 ml inoculum per leaflet) to rub the entire leaf surface. The leaves for inoculation were pre-dusted with 600-mesh carborundum as abrasive before inoculation and rinsed with tap water after inoculation. Buffer (mock) inoculation and/or noninoculation of York and PI 96983 plants served as controls. Spot inoculation for tracking the virus replication and movement in York, Kwanggyo, and Lee 68 soybean was done by applying inoculum of G4 or G5 with a glass rod onto one or two 3–4 mm diameter sites on an unifoliolate leaf. York is necrotic to G4 and mosaic to G5, whereas Kwanggyo is resistant to G4 and necrotic to G5. Lee 68 is mosaic to both G4 and G5. These genotype and stain combinations allow the detection and tracking of virus activities and comparisons among different reactions. Inoculated plants were monitored for symptom development on a regular basis and notes were taken for 4 weeks after inoculation. Plants were classified as resistant (R) if no symptoms developed, necrotic (N) if stem tip necrosis or systemic necrotic lesions or systemic vein, stem, and petiole necrosis occurred, and mosaic (M) if systemic mosaic symptoms were observed. When mosaic or necrotic symptoms were observed on plants inoculated with two SMV strains involving G4–G6, or G7, trifoliolate leaf samples were taken from those symptomatic plants to produce inoculum for inoculation of another set of York or PI 96983 plants, which would differentiate the two strains (Table 1). ELISA was conducted according to Lister (1978) using trifoliolate leaf samples taken from PI 96983 plants inoculated with one or two SMV strains. Each sample was replicated twice and values of absorbance at 405 nm were determined 1 h after adding the substrate. Data for ELISA were analyzed using the general linear model procedure (PROC GLM) in SAS (SAS Institute In., Carry, NC). Fisher’s protected least significant difference (LSD) at P ¼ 0:05 was used to compare mean ELISA values of different inoculation treatments. Leaf imprint immunoassay (LIIA) was conducted according to methods described previously (Holt, 1992; Lin et al., 1990; Mansky, 1990; Mansky et al., 1990) with modifications to detect virus movement and replication at exact locations in the leaves. The leaf was imprinted onto a hardened filter paper (Schleicher & Schuell #8) rather than nitrocellulose membranes. The paper was first immersed in 5% Triton X-100 (Sigma Laboratories) to remove the residual green color caused by the leaf imprinting (Srinivasan and Tolin, 1992), and then rinsed in 1X KPS-T buffer (0.1 M K2HPO4, 0.75 M NaCl, and 0.05% Tween-20, pH 7.4). Blocking was with
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4% non-fat dry milk in 1X KPS-T buffer and 0.5% bovine serum albumin (BSA) (Sigma Laboratories). Following incubation with SMV-specific whole serum from rabbit (primary antibody) (Hunst and Tolin, 1982) in 1X KPS-T buffer, and then in goat anti-rabbit antibody (secondary antibody) (Hunst and Tolin, 1982) conjugated to alkaline phosphatase (Sigma Laboratories), color was developed in nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl-phosphate (BCIP) (Zymed Laboratories). The results were analyzed visually using the leaf imprints and compared with photographs of leaves before blotting.
3. Results and discussion York exhibited a resistant reaction to single inoculation of SMV strains G1–G3, but was mosaic to strains G5–G7 and G7A. PI 96983 was infected only when inoculated with G7 and G7A and showed necrosis and mosaic symptoms, respectively (Table 2). The inoculation results for strains G1–G7 are consistent with those obtained by Cho and Goodman (1979, 1982). However, in our tests York did not give 100% necrotic plants when inoculated with G4. The non-symptomatic York plants were apparently result of escapes from the virus infection. Mixed reactions of resistance and necrosis from other soybean genotype SMV strain combinations were also observed on occasion in genetic studies (Chen et al., 1991, 1994). It also appears that the necrotic reaction is somewhat affected by environmental conditions. Tu and Buzzell (1987) found that the Table 2 Reaction of York and PI 96983 soybean to single inoculation with different Soybean mosaic virus strains SMV strain
G1 G2 G3 G4 G5 G6 G7 G7A Uninoculated Mock inoculated LSD0.05
Phenotypic reactiona
A405 nmb
York
PI 96983
PI 96983
R R R N/R M M M M R R
R R R R/N R R N M R R
0.074 0.055 0.060 0.060 0.062 0.078 0.179 0.250 0.077 0.011 0.019
a R=resistant (symptomless), N=necrosis (systemic necrosis), M=mosaic, N/R=most plants (two pots of 8–12 plants for each inoculation) were necrotic and mixed with a few resistant plants (escapes), R/N=most plants were resistant and mixed with a few necrotic plants. b Mean values of absorbance at A405 nm, determined by four replications (two samples from each of the two pots), readings were taken 1 h after addition of the substrate.
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development of stem tip necrosis on cultivar Columbia was temperature dependent; inoculated plants developed necrosis at 20–24 C, but not at 28–32 C. Similar thermosensitive responses have been reported in the Tobacco mosaic virus (TMV) tobacco N gene interaction system (Kassanis, 1952). PI 96983 plants inoculated singly with strains G1–G6 remained healthy and no virus was detected in trifoliolate leaves by ELISA (Table 2). Cho and Goodman (1979, 1982) also found that symptomless plants inoculated with SMV did not contain virus, as determined by indexing to Topcrop bean. Positive results were obtained by ELISA for necrotic and mosaic PI 96983 plants inoculated with SMV-G7 and G7A, respectively. However, the ELISA values were higher for the mosaic plants than that for the necrotic plants. Apparently, virus replication and movement were limited in the necrotic response. Tu and Buzzell (1987) were able to isolate SMV from necrotic tissue of Columbia soybean, but did not estimate the virus concentration. We observed that plants exhibiting the stem–tip necrosis reaction usually showed mosaic symptoms on the first trifoliolate leaf initially, and then turned necrotic and eventually died. To confirm the hypothesis that virus replication is limited in the necrotic reaction, we used LIIA to detect virus antigen in situ in a leaf. By LIIA, SMV in inoculated unifoliolate leaves of mosaic and necrotic plants could be visualized at the site of inoculation within 6 days after inoculation (DAI). In the mosaic reactions of Lee 68 to G4 or G5 and York to G5 viral protein was detected along the midrib and some side veins within 9 DAI, indicating the replication and movement of the virus (Table 3). The color intensity and area of the leaves containing virus increased with time, with maximum signal being detected at 18–20 DAI at a distance of 5–6 mm either side of the midrib and along several lateral veins. In the necrotic reactions of York to G4, or Kwanggyo to G5, virus was detected only exactly on or immediately surrounding a necrotic area on the leaf. Virus was sometimes detected even on a very small
necrotic lesion, indicating that virus replicated to a detectable level within a few cells before the necrotic area developed in the leaf. At longer times and as necrosis developed along the midrib and in trifoliolate leaves, virus was detected in the areas of necrosis only. No virus was detectable in inoculated unifoliolate and upper trifoliolate leaves of resistant plants (Kwanggyo inoculated with G4). This assay was more sensitive than ELISA in detecting virus in necrotic plants and should prove useful for further study of mixed infections. There was no substantial difference in plant reactions to separate- and mixed-double inoculations (Table 4). The G1 and G4 combination did not induce 100% necrotic plants with either type of double inoculation, which is comparable to the single inoculation of York with G4 alone. Thus, G4 was predominant when introduced together with G1 to York plants. The strain-differentiating York plants inoculated with inoculum from mix-infected (G1+G4) plants also exhibited necrotic reactions with a few escapes, as observed on York plants inoculated singly with G4. Therefore, the strain isolated from the double-inoculated (G1+G4) plants was identified as G4. Necrotic lesions were observed on G4-inoculated unifoliolate leaves of York plants and results of LIIA had shown that G4 is localized in these lesions. When G4 and G1 were inoculated to separate unifoliolate leaves of the same York plant, lesions were only present on the leaf inoculated with G4 but not on the other leaf inoculated with G1. Both unifoliolate leaves of York plants showed necrotic lesions when inoculated with the mixed inoculum of G4 and G1. In the tests of strain combinations of G1 (avirulent) and a mosaic-inducing strain (G5, G6, or G7), the inoculated York plants showed mosaic symptoms as observed with single inoculation by the mosaic strains, indicating that G1 did not interfere with the mosaic strains (Table 4). Strains recovered from the mixinoculated plants were identified as the respective mosaic-inducing strains. Upon inoculation of necrotic strain (G4) and a mosaic strain (G5, G6 or G7), mosaic
Table 3 Distribution of Soybean mosaic virus in spot-inoculated soybean leaves detected by leaf imprinting immunoassay 9 days after inoculation Cultivar (genotype)
SMVstrain
Phenotypic reactiona
Blotting patternb
SMV local detectionc
SMV movementd
York (Rsv1-y) Kwangyyo (Rsv-k) Lee 68 (rsv)
G4 G5 G4 G5 G4 G5
N M R N M M
LSL AVM NVD LSL AVM AVM
+ +++ + +++ +++
+ +++ + +++ +++
a
R=resistant (symptomless), N=necrosis (systemic necrosis), M=mosaic. LSL=localized spots and lesions, AVM=along veins and midrib; NVD=no virus detected. c +=virus detected, = no virus detected, +++ indicates higher titer of SMV detected, based on the intensity of immuno-reaction. d +=virus moved from inoculated site, = no virus movement, +++ indicates higher titer of SMV detected. b
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Table 4 Reaction of York soybean to separate double and mixed double inoculations with two Soybean mosaic virus strains Separate double inoculationa SMV Inoculum
Reaction on York
Avirulent and necrotic strains G1+G4 N/Rc Avirulent and mosaic strains G1+G5 M G1+G6 M G1+G7 M Necrotic and mosaic strains G4+G5 M G4+G6 M G4+G7 M Two mosaic strains G5+G7 M
Mixed double inoculationb
Recovery onto
Strain recovered
York
PI 96983
N/Rc
—d
G4
M M M
— — N
M M M M
Reaction on York
Recovery onto
Strain recovered
York
PI 96983
N/Rc
N/Rc
—d
G4
G5 G6 G7
M M M
M M M
— — N
G5 G6 G7
— — N
G5 G6 G7
M M M
M M M
— — N
G5 G6 G7
N
G7
M
M
N
G7
a
Two unifoliolate leaves of a test plant were inoculated separately with two different SMV strains. Both unifoliolate leaves were inoculated with mixed inoculum from two SMV strains. c R=resistant (symptomless), N=necrosis, M=mosaic, N/R=most plants were necrotic and mixed with a few resistant plants (escapes). d —=not tested, inoculation will not differentiate the two strains. b
Table 5 ELISA and reaction of PI 96983 soybean to mixed double inoculation with two Soybean mosaic virus strains Strains
Reactiona
A405 nmb
Strains
Reactiona
A405 nmb
G1+G2 G1+G3 G2+G3 G1+G4 G2+G4 G3+G4 G1+G6 G2+G6 G3+G6 G4+G6 Uninoculated Mock inoculated
R R R R/N R/N R/N R R R R R R
0.111 0.105 0.057 0.166 0.108 0.118 0.078 0.087 0.078 0.074 0.077 0.011
G1+G7 G2+G7 G3+G7 G4+G7 G6+G7 G1+G7A G2+G7A G3+G7A G4+G7A G6+G7A G7+G7A LSD0.05
N N N N N M M M M M M
0.134 0.129 0.161 0.102 0.187 0.198 0.269 0.459 0.350 0.307 0.445 0.083
R N/R M M
0.087 0.142 0.316 0.445 0.094
Pooled data Two avirulent strains Avirulent and necrotic strains Avirulent and mosaic strains Necrotic and mosaic strains LSD0.05 a
R=resistant (symptomless), N=necrosis, S=mosaic, R/N=most of resistant mixed with a few necrotic plants, N/R=most plants were necrotic and mixed with a few resistant plants (escapes). b Mean values of absorbance at A405 nm, determined by four replications (two samples from each of two pots), readings were taken 1 h after addition of the substrate.
strains became predominant and caused mosaic symptoms on York with no systemic necrosis. The strains recovered from the mix-inoculated plants were the corresponding mosaic strains. SMV-G7 predominated over G5 when both were introduced to York plants by either inoculation method because only G7 was recoverable. This is consistent with the suggestion that G7 is more virulent than G5 (Cho and Goodman, 1979).
Thus, no apparent phenotypic synergism was indicated by the double inoculation of two mosaic strains. In the complementation tests of avirulent strains on PI 96983, G6 combined with other strains (G1–G4) caused no symptoms and no virus was detected by ELISA, suggesting that G6 did not complement any of the four avirulent strains (Table 5). A few necrotic plants were observed when G4 was mix-inoculated with
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G1, G2, or G3, and relatively low ELISA values were obtained from those plants, indicating that there was limited virus accumulation in the necrotic plants, especially with the G1+G4 inoculation. Plants inoculated with G1+G2, G1+G3 and G2+G3 remained healthy and showed an ELISA value similar to the healthy controls. No mosaic plants were observed for mixed inoculations with any two of the five avirulent strains (G1–G4 and G6). Thus, complementation among avirulent strains was not phenotypically demonstrable in our system. PI 96983 plants inoculated with the necrosis-inducing strain (G7) and any avirulent strains (G1–G4 or G6) all showed stem–tip necrosis and gave ELISA values numerically higher than the negative controls. However, the differences were not statistically different except for G1+G4, G3+G7, and G6+G7 combinations (Table 5). This was to be expected since the virus titer in necrotic tissue, as shown by LIIA, was very low (Table 3). The necrotic symptoms and ELISA values from the mix-inoculated plants were comparable to those from the single inoculation of G7 (Tables 2 and 5), indicating that avirulent strains were unable to interfere with or complement G7 when they were mixed and present in the same plant, and that the strain causing necrosis was G7 alone. The strain combinations of G7A and an avirulent strain (G1–G4 and G6) resulted in mosaic symptoms on PI 96983, as expected from single inoculation of G7A. However, ELISA showed higher amount of virus in PI 96983 plants inoculated with G3+G7A than in plants inoculated with G1+G7A, G2+G7A, G4+G7A or G6+G7A. It appears that G7A replication was somewhat unaffected in PI 96983 plants by G1, G2, G4, or G6, but was enhanced by G3. The mix-inoculation of a necrotic (G7) and a mosaic strain (G7A) induced mosaic symptoms and gave rise to higher ELISA values than single inoculation with either strain, indicating that the replication of the mosaic strain was somewhat elevated by the necrotic strain. All mixed inoculations involving a mosaic strain resulted in significantly higher virus titer than inoculation combinations of avirulent and necrotic strains. In our test for double inoculations of an avirulent strain and a mosaic or necrotic strain, mosaic or necrotic symptoms were always observed and the virulent (symptom-causing) strain was recovered. However, the possibility that the avirulent strain co-existed with virulent strains in the mix-inoculated plants but was not visible phenotypically on symptomatology cannot be ruled out. Isolation from a population of lesions on Topcrop bean, as was done with G2 and G7A by Mansky and coworkers (1990), would be needed to confirm the hypothesis. Detection of the avirulent strains in the mix-infected plants would require SMV strain specific antibody which was not available to us. G7 and G7A together induced mosaic symptoms instead
of stem–tip necrosis on PI 96983, indicating that the mosaic strain G7A predominated over the necrotic strain G7 when both strains were present in the same plants. A higher ELISA value (0.445) was obtained from the mix-inoculated (G7+G7A) PI 96983 plants than from G7 or G7A-single-inoculated PI 96983 plants (0.179 and 0.25), suggesting that the two strains might have interacted synergistically when inoculated together. In the combined data analysis, plants inoculated with a necrotic and a mosaic strain had highest ELISA value. Plants inoculated with an avirulent and a mosaic strain gave the second highest ELISA value, which is significantly higher than that of plants inoculated with an avirulent and a necrotic strain or plants inoculated with two avirulent strains (Table 5). The implications of this work are that a soybean cultivar recognized as resistant to one strain of SMV may be mosaic or necrotic to another strain. When multiple strains with different pathogeniecity and/or virulence occur in a field situation, the dominance relationship among them will be in the order of severe mosaic strains > mild mosaic strains > necrotic strains > avirulent strains. We suggest that strain identity and stability be monitored in virus research to ensure that the biological properties of the virus strain(s) used for screening soybean germplasm are known and that the strains are representative of the virus which the commercial soybean will ultimately encounter. In selecting soybean varieties for production areas with potential threat of multiple SMV strains, it is highly recommended that lines with genes such as Rsv1-h for resistance to multiple strains be utilized (Chen et al., 2002). As for soybean breeding research, reliance on a single source of resistance would result in genetic uniformity and potential vulnerability. Gene pyramiding to incorporate resistance genes at different loci from various sources into a single soybean line might be desirable for developing cultivars with durable resistance to multiple strains of SMV.
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