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a Dep. of Agronomy, Univ. of Kentucky, Lexington, KY 40546
b Dep. of Plant Pathology, Univ. of Kentucky, Lexington, KY 40546
* Corresponding author (dtekrony{at}ca.uky.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: SMV, Soybean mosaic virus • FS, full seed • YP, yellow pod • HM, harvest maturity • R-NI, SMV-resistant isolines (L78-434, L78-379) not inoculated with SMV • S-NI, S-V4, S-V8, S-R2, SMV-susceptible cultivars (Clark, Williams) not inoculated with SMV, or inoculated with SMV at the V4, V8, or R2 growth stage • Std, standard • AA, accelerated aging • BC, bulk conductivity
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Soybean mosaic, caused by Soybean mosaic virus (SMV), is probably the most common soybean viral disease. It is seedborne, and is carried over between production seasons by seeds (Gardner and Kendrick, 1921; Ross, 1969; Hill et al., 1980), and within seasons by aphids (Homoptera: Aphididae) in a nonpersistent manner (Abney et al., 1976; Halbert et al., 1981). Embryonic infection with SMV is a prerequisite for seed transmission. The rate of seed transmission varies from 0 to 68%, but is most often close to 10%, depending on the host genotype, virus strain and time of infection (Shepherd, 1972). Although seedcoat infection in the absence of embryonic infection does not lead to seed transmission, SMV antigen can be detected in >99% of seeds collected from SMV-infected plants (Bossenec and Maury, 1978; S.A. Ghabrial, 1997, unpublished). Therefore, detection of seedcoat infection, which is independent of host genotype and virus strain, serves as a good indicator of SMV infection of the individual plants and provides reliable estimates of SMV incidence in the field (S.A. Ghabrial, 1997, unpublished).
Infection with SMV has been associated with reduced yield (Irwin and Goodman, 1981; Ross, 1987; Ren et al., 1997), with the magnitude of yield loss depending largely on the growth stage at which infection occurred, incidence of infection, degree of cultivar susceptibility, virulence of the virus, and environmental conditions (Ross, 1987; Ren et al., 1997).
Ross (1977) found a higher incidence of Phomopsis sojae S.G. Lehman [syn. Phomopsis phaseoli (Desmaz.) Sacc.] seed infection in SMV-susceptible plants than in SMV-resistant plants. Earlier SMV infections (before or during full bloom) increased the incidence of P. sojae seed infection compared with SMV infections that occurred later during pod development (Hepperly et al., 1979). Stuckey et al. (1982), using a mild strain of SMV, did not consistently identify significant increases in Phomopsis spp. seed infection as a result of SMV infection. These previous studies, however, did not include SMV-resistant isolines as fundamental controls, or monitor the extent of SMV transmission by aphid-vectors, the soybean growth stage at which infection occurred, or the incidence of SMV infection. Thus, the association between SMV and Phomopsis spp. seed infection is not completely understood and warrants further attention.
This study examined the hypothesis that infection by SMV predisposes soybean seeds to Phomopsis spp. infection. Using SMV-resistant isolines, we investigated the effects of SMV on Phomopsis spp. seed infection in relation to (i) the developmental stage at which plants were inoculated with SMV, (ii) the accumulation of SMV in seedcoats, (iii) the stage of seed development, and (iv) seed quality.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Each genotype was planted at a rate of 25 seeds m-1 in four row plots, 6 m long with 0.76-m spacing between rows in a randomized complete block design with three replications. The SMV-resistant isolines were not inoculated with SMV (R-NI). During 1995, all SMV-susceptible plants were mechanically inoculated with SMV (G2 strain) at the R2 (Fehr and Caviness, 1977) growth stage (S-R2). In 1996 treatments were the two noninoculated resistant isolines (R-NI) and the two susceptible cultivars either noninoculated (S-NI), or mechanically inoculated at the V4, V8, or R2 growth stage (Fehr and Caviness, 1977) referred to as the S-V4, S-V8, and S-R2 treatments, respectively. The 1997 treatments were similar except the V8 growth stage inoculation was omitted. The SMV inoculum was prepared by grinding young, SMV-infected soybean leaves in 0.05 M potassium phosphate buffer (pH 7.4) at a ratio of 1:5 (w/v) in a blender (Ren et al., 1997). Infected leaves were obtained from infected plants that were grown in the greenhouse. Carborundum was added to inoculum and the mixture was rubbed onto all three leaflets of a newly developed leaf.
To provide a source of Phomopsis spp. inocula, Phomopsis-infested stems and residue, collected from the previous seasons' soybean fields, were scattered on the soil surface between plants and rows at R2 (Garzonio and McGee, 1983). Additionally, a Phomopsis spp. spore suspension, prepared from cultures grown on acidified (pH 4.5) Potato Dextrose Agar (aPDA) for 14 to 21 d, was atomized (Kmetz et al., 1979; Spilker et al., 1981; Rupe and Ferriss, 1987) onto plants at R5. Plants were irrigated overhead, as needed to minimize moisture stress and maintain a favorable environment for disease infection.
To provide a pool of pods at the same stage of development, a minimum of 80 pods per plot (at one pod per plant, from three soybean rows) were marked with acrylic paint (Egli, 1999) when they reached maximum length and contained three seeds just starting to swell (3–4 mm in diameter). During 1995, marked pods were randomly selected from plants; however, in 1996 and 1997, marked pods were collected from plants that were either SMV symptomless [noninoculated plants (R-NI and S-NI)], or SMV symptomatic (McGee, 1992; Sinclair, 1992) [SMV-inoculated plants (S-V4, S-V8, S-R2)]. Ten (1995, 1996) or 20 (1997) marked pods were harvested from each plot at three stages of seed maturation. Harvests were made at (i) full seed (FS), when the pods were dark green and the seeds were green and immature but completely filled the locular cavity; (ii) yellow pod (YP), where the pods were 
90% yellow, and the seeds were yellow and at a moisture content of 550 g kg-1; and (iii) harvest maturity (HM), where the pods were brown, and the seeds were yellow and at a moisture content of 
140 g kg-1.
Seeds were immediately removed from sampled pods and stored at 10°C until laboratory evaluations were made. One carpel from each pod was immediately evaluated for Phomopsis spp. infection after 14 d on blotter paper at 25°C under continuous light (McGee and Sweets, 1989). Seeds were dissected into two halves (each consisting of a cotyledon and its seedcoat-half). One seed-half was evaluated for Phomopsis spp. infection, after 14 d on aPDA at 25°C under continuous light (TeKrony et al., 1984), and the other seed-half was evaluated for the accumulation of SMV antigen in the seedcoat, using the direct form of ELISA (Ghabrial and Schultz, 1983). Since the incidence of Phomopsis spp. is not influenced by the position of the seed within the pod (Kmetz et al., 1978; Tomes et al., 1985; Roy and Abney, 1988), infection was determined only on seeds in the apical position in the pod in 1995 and on pooled apical and basal seeds in 1996 and 1997. To determine the influence of seed position within the pod on SMV accumulation, seeds were sorted according to their apical or basal position in the pod and evaluated separately for SMV in 1995 and 1996, and pooled in 1997.
A composite harvest was made from all treatments at harvest maturity (<140 g kg-1 seed moisture) by mechanically threshing plants from three end-trimmed rows from each plot. All plants were harvested from each plot in 1995, while in 1996 and 1997 only SMV-symptomless plants were harvested from noninoculated (R-NI and S-NI) plots and, SMV-symptomatic plants were harvested from SMV-inoculated (S-V4, S-V8, S-R2) plots. SMV-symptomatic and symptomless plants were identified using marked pods as a guide. One hundred seeds from each plot were evaluated for Phomopsis spp. infection on a PDA, and the levels of SMV accumulation in seed coats from 50 seeds were quantified by ELISA as described by Copeland (1998). The percentage of SMV-infected embryos (SMV seed transmission) was determined by field planting three replicates of 100 seeds from each plot and recording the number of SMV-symptomatic seedlings. Accuracy of this visual estimate was confirmed by ELISA. Four replicates of 50 seeds from each plot were tested for germination and vigor by standard germination (Association of Official Seed Analysts, 1998), accelerated aging (International Seed Testing Association, 1995), and bulk conductivity (Loeffler et al., 1988) tests.
Data obtained by sampling pods at various stages of seed maturation were analyzed as a factorial treatment structure (replication x genotype x treatment x seed maturation stage) in a randomized complete block design with repeated measures by PROC MIXED of SAS. During 1995 and 1996, the seed's locular position was included as an additional factor in the treatment structure. The covariance structure used for all response variables was either variance components or heterogenous compound-symmetry. PROC CORR and PROC REG of SAS were used for correlation and simple linear regression analysis, respectively.
Data obtained from the composite harvest at harvest maturity were analyzed as a factorial treatment structure (replication x genotype x treatment) in a randomized complete block design by PROC GLM of SAS, and differences were determined by the Least Significant Difference (LSD) procedure.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Noninoculated SMV-resistant (R-NI) plants reached the YP and HM stages at approximately the same time as the noninoculated SMV-susceptible (S-NI) plants (Table 1). On the other hand, SMV-susceptible plants inoculated with the G2 strain of SMV (S-V4, S-V8, S-R2) extended the time for the FS to YP period of development between 1 and 9 d longer than R-NI and S-NI plants (Table 1). Early SMV inoculations at V4 extended the time from FS to YP by 8 or 9 d compared with S-NI plants, whereas late inoculations at R2 extended the period only 1 or 6 d in 1996 and 1997, respectively.
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50% less SMV antigen in their seedcoats than at YP, while seeds at the YP stage had 1.6 times more SMV antigen in their seedcoats than at HM. At FS in 1996 and 1997, and at YP and HM in 1997, seeds harvested from S-V4 plants had 1.2 to 1.7 times more SMV antigen in their seedcoats than seeds from S-V8 and S-R2 plants.
Phomopsis spp. Infection
All pods harvested from SMV-inoculated plants exhibited visual signs (McGee, 1992) of Phomopsis spp. infection (data not shown). The incidence of Phomopsis spp. seed infection was consistently low ( 2%) at FS, with primary infection occurring at or after YP. The highest levels of infection occurred at HM for both inoculated and noninoculated susceptible plants (Fig. 1b). The incidence of Phomopsis spp. seedcoat infection, at and after YP, ranged from 2 to 19% in R-NI, 2 to 22% in S-NI, and 8 to 59% in SMV-inoculated plants (Fig. 1b). Generally, a higher incidence of Phomopsis spp. infection occurred in the seedcoats than the cotyledons (data not shown). Inoculation of SMV-susceptible plants with SMV resulted in up to a 12-fold increase in the incidence of Phomopsis spp. seedcoat infection compared with noninoculated plants. Although inoculation at V4 yielded 1.4 times more Phomopsis spp. seed infection than inoculation at R2 (except for YP in 1996), this difference was not significant at either the YP or HM harvests.
Relationship between SMV and Phomopsis spp. Seed Infection
There was a positive and highly significant linear relationship between the incidence of Phomopsis spp. seed infection and the concentration of SMV antigen in seedcoats at YP and HM in 1997 and HM in 1996 (Fig. 2)
. This relationship was not significant at YP in 1996.
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10%) as when plants were inoculated at R2. Susceptible plants inoculated with SMV increased the incidence of Phomopsis spp. seed infection in seedcoats and cotyledons (Fig. 1b). The presence of low levels of SMV in noninoculated plants (S-NI) provided evidence of natural aphid transmission in the field and also resulted in Phomopsis spp. seed infection (S-NI vs. R-NI, Table 2). The level of Phomopsis spp. infection of seedcoats harvested from SMV-inoculated plants ranged between 43 and 71% and was significantly higher than infection of S-NI seeds.
Seed quality tests indicated that seeds from R-NI and S-NI plants were significantly higher in the standard germination, germination following accelerated aging (AA) and lower in bulk conductivity (BC) than seeds from SMV-inoculated plants (Table 2). Following SMV inoculation, as the incidence of Phomopsis spp. seed infection increased, the standard germination decreased 29 to 51 percentage points compared with seeds from S-NI plants. Likewise, the seed vigor (AA and BC) declined two-fold for seed harvested from SMV-inoculated plants.
At harvest maturity, across all genotypes and SMV-inoculation treatments there was a significant, positive correlation between the duration of the FS-YP phase and the incidence of Phomopsis spp. seed infection in 1996 (r = 0.56, n = 30) and 1997 (r = 0.65, n = 24). A significant correlation was not found between the duration of the FS-HM or YP-HM phase and Phomopsis spp. infection. Each year, highly significant negative correlations (-0.96 r 
-0.86; n = 30, 24 in 1996 and 1997, respectively) were found between the incidence of Phomopsis spp. seed infection and seed quality variables. As the incidence of Phomopsis spp. seed infection increased, the BC increased, while the germination (standard, and following AA) decreased. Thus, seed infection by Phomopsis spp. was a major factor influencing seed quality.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The SMV-resistant isolines used in this study both have the dominant Rsv1 SMV resistance allele, which confers resistance to SMV strains G1 to G6, whereas the susceptible cultivars have the recessive rsv1 allele and are susceptible to all SMV strains. In Kentucky, SMV-resistant genotypes did not show symptoms of SMV, nor was SMV detected in those plants which were mechanically inoculated under greenhouse conditions with either the prevailing strain of SMV (G2), or with other strains (G1–G6) collected in Kentucky. Likewise, the SMV-resistant cultivars remained SMV free when exposed to natural infection for three consecutive growing seasons under conditions where SMV was rapidly spreading in the field (S.A. Ghabrial and T.W. Pfeiffer, 1995, unpublished data). In our study, noninoculated SMV-resistant plants were SMV symptomless and consistently SMV free. Using the sensitive serological ELISA technique, we detected a low level of SMV in seedcoats from seeds harvested from noninoculated SMV-susceptible cultivars, which was possibly due to natural spreading of the virus by aphids in the field. A significantly higher level of SMV was however detected in seeds harvested from SMV-inoculated plants. Thus, both the noninoculated SMV-susceptible and resistant plants provided suitable experimental controls.
A positive and significant relationship was observed between the accumulation of SMV in seedcoat tissues and the incidence of Phomopsis spp. seed infection. The mechanism by which SMV-infected plants were predisposed to Phomopsis spp. seed infection is, however, not completely understood. Noninoculated susceptible plants matured at the same time as SMV-resistant plants, while SMV-inoculated susceptible plants matured later and had a higher incidence of Phomopsis spp. seed infection than the noninfected (control) plants, in agreement with Gardner and Kendrick (1921) and Tu (1989). The delay in maturation was due to an extension of the linear phase of seed development (full seed to yellow pod), and the length of this phase correlated positively and significantly to Phomopsis spp. seed infection, in agreement with Tu (1989) and Vaughan et al. (1989). It may be speculated that infection by SMV extended seed maturation, placing the pods and seeds in favorable, warm, and wet conditions, thereby providing a greater opportunity for pods and seeds to become infected or colonized by Phomopsis spp. However, the higher levels of Phomopsis spp. seed infection of SMV-infected plants could not be directly related to more rainfall events during the extended period of seed maturation (Koning, 1999). Balducchi and McGee (1987) showed that 2 to 3 d of high relative humidity (>95%) at or after growth stage R7 could result in 90% seed infection by Phomopsis; however, in our data there were no differences in the relative humidities experienced by different seed lines receiving different treatments in the 3 to 4 d following YP. Furthermore, when a direct comparison could be made in 1996 between those plants inoculated with SMV at R2 and noninoculated plants, the days required for the FS to YP stage of development were nearly identical (20 vs. 19 d, respectively) (Table 1); however, the levels of Phomopsis spp. seed infection were 5-fold greater for SMV-infected plants.
An important aspect of seed infection, is the effect of the pathogen on seed quality, with seed vigor being an important component of seed quality. In this study, SMV infection was directly associated with an increase in SMV seed transmission and Phomopsis spp. seed infection. As noted previously (Bowers and Goodman, 1979; Tu, 1989, 1992; Ren et al., 1997), early infection with SMV (before R2) increased the percentage of SMV seedling transmission (a negative seed quality factor in seed used for planting) significantly compared with later infection (at R2). Infection by SMV and Phomopsis spp. clearly reduced seed quality. Seeds from SMV-inoculated susceptible plants had significantly higher levels of SMV and Phomopsis spp. seed infection, and lower seed quality and vigor (lower germination following AA and higher conductivity) than seeds from SMV-resistant or noninoculated susceptible plants. Ross (1977) and Hepperly et al. (1979) also showed reductions in germination when SMV and Phomopsis spp. infection occurred; however, they did not measure the relationship of these two pathogens to seed vigor.
These data confirmed the reports by Ross (1977) and Hepperly et al. (1979) that SMV infection increases Phomopsis spp. seed infection, but were not in complete agreement with Stuckey et al. (1982) who, using a mild strain of SMV, found little effect on Phomopsis spp. seed infection. They reported that a consistently significant increase in Phomopsis spp. seed infection only occurred in plants that were doubly infected with SMV and Bean pod mottle virus (BPMV). These authors explained the differences between their results and those of Hepperly et al. (1979), who used a severe strain of SMV, on the basis of the virus strain used. The G2 strain of SMV used in our studies had a much higher level of virulence than the mild strain used in earlier experiments by Stuckey et al. (1982). The levels of BPMV were not measured in our field studies, however a significant increase in Phomopsis spp. seed infection also occurred following SMV inoculation (G2 strain, V8 stage) where natural infection by either SMV and BPMV was prevented in an insect-free environment (Koning et al., 1999). Thus, there was little doubt that an increase in the accumulation of SMV in seedcoats of seeds harvested from SMV-susceptible plants clearly resulted in an increase in the incidence of Phomopsis spp. seed infection.
In conclusion, the results obtained during this investigation clearly support the hypothesis that infection by SMV predisposes soybean seeds to Phomopsis spp. seed infection, and results in the loss of seed quality and vigor. In areas favorable to Phomopsis spp. infection, and when SMV infection is likely, measures that reduce or prevent the spread of the virus (i.e., the use of SMV-resistant cultivars), especially during the early stages of plant development, may promote the production of high vigor and quality seeds.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication May 20, 2000.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  G. Koning, D. M. TeKrony, S. A. Ghabrial, and T. W. Pfeiffer Soybean mosaic virus (SMV) and the SMV Resistance Gene (Rsv1): Influence on Phomopsis spp. Seed Infection in an Aphid Free Environment Crop Sci., January 1, 2002; 42(1): 178 - 185. [Abstract] [Full Text] [PDF]
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) and harvest maturity (
) in a) 1996 (n = 60) and b) 1997 (n = 120). Data averaged across apical and basal seeds, and regressed across all the SMV-inoculation treatments received by two SMV-susceptible cultivars (Clark, Williams) and their SMV-resistant isolines (L78-434, L78-379).
.