Published in Crop Sci. 43:2071-2076 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Increased Soybean Pubescence Density
Yield and Soybean mosaic virus Resistance Effects
Todd W. Pfeiffer*,a,
Rebecca Peyyalaa,
Quanxing Rena and
Said A. Ghabrialb
a Dep. of Agronomy, Univ. of Kentucky, Lexington, KY, 40546
b Dep. of Plant Pathology, Univ. of Kentucky, Lexington, KY, 40546
* Corresponding author (tpfeiffe{at}uky.edu).
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ABSTRACT
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Soybean mosaic virus (SMV) infection of double-cropped soybean [Glycine max (L.) Merr.] is likely to reduce yield due to soybean flowering date occurring after the start of aphid (Aphis spp.) movement that spreads the virus. Increased pubescence density provides a mechanical barrier to aphid probing that may delay SMV infection until after flowering, a strain-nonspecific resistance mechanism. The objectives of this study were to (i) evaluate the resistance benefit derived from increased pubescence density and the resulting reduction in incidence of SMV infection in soybean double-cropping systems, and (ii) compare the yield of soybean having increased pubescence density with the yield of genetically resistant genotypes of normal pubescence. Thirty soybean lines in three maturity sets with combinations of normal, dense, and extra-dense pubescence and SMV-resistant and -susceptible alleles were grown for 3 yr in late-planted tests at Lexington, KY. Inoculated border rows provided the virus source for natural aphid transmission of SMV. Extra-dense pubescence significantly reduced the incidence of SMV infection at R1 and R6 and produced a delay parameter on disease progress curves where the maximum disease increase was after flowering. In the late maturity set where the incidence of SMV infection on normal-pubescence SMV-susceptible genotypes was greater than 20%, extra-dense pubescence provided the same SMV resistance yield benefit as the Rsv1y resistance allele. The mean yield of extra-dense pubescence genotypes, however, was less than that of normal- and dense-pubescence genotypes.
Abbreviations: SMV, Soybean mosaic virus • IOI, incidence of infection • N, normal pubescence • D, dense pubescence • DD, extra-dense pubescence • R, resistant to SMV • S, susceptible to SMV
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INTRODUCTION
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SOYBEAN MOSAIC VIRUS, a member of the genus Potyvirus in the family Potyviridae, is transmitted nonpersistently by aphids (Abney et al., 1976). The mosaic disease induced by SMV occurs in double-cropped soybean and may cause yield loss (Ren et al., 1997b). Yield losses due to SMV infection depend on the interaction between the timing and incidence level of infection (Ross, 1969; Irwin and Goodman, 1981; Ren et al., 1997a). Infection occurring before flowering causes significant yield reduction, whereas infection by R1 growth stage is necessary to produce a significant yield loss (Ren et al., 1997a). Thus, control strategies that delay SMV infection could limit yield losses.
Genetic resistance to SMV in soybean is currently provided by single dominant alleles. These alleles, found in multiple allelic series at several genes, confer resistance to subsets of SMV strains (Buss et al., 1989; Chen et al., 1991). Although selection for a resistance allele has produced resistant cultivars, several SMV strains have been reported as breaking resistance of some of the alleles. Pubescence density can be an important factor in controlling SMV infection because increased pubescence density can bring a delay in time of infection by acting as a mechanical barrier to aphid probing (Halbert et al., 1981; Irwin and Goodman, 1981; Gunasinghe et al., 1988). Secondary spread of SMV occurs only by aphids feeding on infected plants which result from the seed transmission of SMV from the previous generation. This reduction in aphid probing provided by increased pubescence density offers a non-strain-specific resistance/avoidance mechanism to SMV spread.
Increased pubescence density controlled by the Pd1 and Pd2 alleles produces both positive and negative agronomic effects. Reduced leaf temperature, restricted transpiration water loss, and enhanced photosynthesis due to radiation being reflected lower into the canopy should be beneficial (Garay and Wilhelm, 1983; Specht and Williams, 1985; Zhang et al., 1992). Denser pubescence results in increased vegetative vigor. Densely pubescent lines have a greater root density and a deeper root extension (Garay and Wilhelm, 1983). Both the Pd1 and Pd2 alleles, however, have been shown to reduce seed yield, delay maturity, and increase plant height with a concomitant increase in lodging (Specht et al., 1985; Cooper and Waranyuwat, 1985).
Both the advantages and disadvantages to soybean productivity of increased pubescence density as a means of controlling SMV need to be considered. The objectives of this study were (i) to evaluate the resistance benefit derived from increased pubescence density as a result of reducing SMV infection in soybean double-cropping systems, and (ii) to compare the yield due to the resistance benefit provided by increased pubescence density with the yield of normally pubescent, genetically resistant genotypes.
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MATERIALS AND METHODS
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Thirty SMV-resistant or -susceptible F4–derived soybean lines with different pubescence densities, selected from the cross L79-1815 (Clark-Pd1Pd2) x ‘Hutcheson’ (Buss et al., 1988; Nelson and Bernard, 1991) were used in this experiment (Table 1). Lines with differing pubescence densities were selected visually, and pubescence properties were then analyzed and confirmed according to Ren et al. (2000). The increased pubescence density was derived from Clark-Pd1Pd2, and the resistance to SMV was derived from Hutcheson, which possesses a single dominant allele that responds to SMV strains in a manner similar to the Rsvy1 allele in ‘York’ (SMV-resistant cultivar in the pedigree of Hutcheson; Buss et al., 1988). These 30 genotypes were planted in a split-plot design in 1995, 1996, 1997, and 1998 at the University of Kentucky Experiment Station farm at Lexington, KY (38° N lat). The 1996 experiment was lost to hail damage. Soil type was a Maury silt loam (fine, mixed, semiactive, mesic Typic Paleudalfs). Whole-plot treatments were three maturity sets arranged as a randomized block design with three replications. Subplot treatments were 10 genotypes in each maturity group. The 10 genotypes consisted of two SMV-susceptible normal pubescent lines, two SMV-resistant normal pubescent lines, two SMV-susceptible dense pubescent lines, two SMV-resistant dense pubescent lines, one SMV-susceptible extra-dense pubescent line, and one SMV-resistant extra-dense pubescent line. Only one pair of extra-dense pubescent lines was used in each maturity set because our initial visual selection for extra-dense pubescence density was not confirmed by trichome density counts, resulting in a shortage of extra-dense pubescent lines. The planting dates, 20 June 1995 and 25 June 1997 and 1998, approximated the normal planting date for double-cropped soybean in Kentucky. Plots were planted into a tilled seedbed following a previous wheat (Triticum aestivum L.) crop. The plots were 8 rows, 6 m long, with 0.38 m between rows. Seeding rates were 20 seeds m-1. In 1998, a total of 650 mm irrigation water was applied on 25 August, 3 September, and 14 September.
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Table 1. Pubescence properties, reaction to Soybean mosaic virus (SMV) infection, and maturity of the 30 F4–derived soybean lines used to evaluate SMV resistance and pubescence density.
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To provide the virus source to be transmitted by aphids, all plants in one outside row of each susceptible plot were inoculated at the V2/V3 growth stage with an SMV strain G2 isolate collected in Kentucky and used in previous experiments (Ren et al., 1997a,b). Soybean mosaic virus strain G2 was maintained in susceptible soybean plants in the greenhouse. Inoculum was prepared by grinding the infected leaves in 0.05 M inoculation buffer (0.05 M K2HPO4 + 0.05 M KH2PO4, pH 7.4) at the ratio of 1 g of infected leaves per 5 mL of the inoculation buffer. Carborundum (silicon monocarbide) was added to the mixture. The inoculum was kept on ice before inoculation. All three leaflets of a newly developed leaf, one leaf per plant, were inoculated by rubbing the inoculum mixture onto the leaves with a cheesecloth pad.
The incidence of SMV infection (percentage) was estimated at the R1 and R6 growth stages from 50 sequential plants from the noninoculated row adjacent to the inoculated border row of each plot. A different set of 50 plants was sampled at each growth stage. In 1995, incidence of infection (IOI) was estimated by visual inspection for SMV symptoms in susceptible and resistant normal and dense pubescence genotypes. Direct ELISA (Ghabrial and Schultz, 1983) was used to estimate the IOI for susceptible extra-dense pubescence genotypes. In 1997 and 1998, one uppermost leaf per plant was collected, and the leaf samples were stored frozen (-4°C) in plastic bags before estimating SMV incidence. A tissue immunoblot assay (see below) of the frozen leaves estimated the IOI for susceptible normal, dense, and extra-dense pubescence genotypes in three replications and for resistant normal, dense, and extra-dense pubescence genotypes in one replication.
Combine harvested yield (kilograms per hectare at 130 g kg-1 moisture) was measured on the central four rows of each plot end-trimmed at maturity to 5 m. Plant height (mm) was measured at maturity. Lodging at maturity was recorded with a 1 to 5 score (1 = almost all plants erect, 2 = all plants leaning slightly or a few plants down, 3 = all plants leaning moderately or 25 to 50% of the plants down, 4 = all plants leaning considerably or 51 to 80% of the plants down, 5 = almost all plants down). The data were analyzed by ANOVA for 3 yr combined with the following mixed model: maturity sets, resistant and susceptible genotypes, pubescence density, and entries were considered as fixed effects, while replication and year were considered to be random effects.
The spread of SMV was monitored each year. Three ‘Clark’ isolines with normal (Clark), dense (Clark-Pd2, L75-6648), and extra-dense (Clark-Pd1Pd2, L79-1815) pubescence were grown in a randomized complete block design with two replications in 1995. In 1997 and 1998, normal, dense (Clark-Pd1, L62-1686), and extra-dense pubescence Clark isolines were grown in three replications in a randomized complete block design. R.L. Bernard, USDA and the University of Illinois, developed the isolines (Nelson and Bernard, 1991). Planting dates and plot sizes were the same as in the previously described experiment. These plots were planted in close proximity to the previous experiment where the first row of each susceptible plot was mechanically inoculated at the growth stage V2/V3 with SMV strain G2 to provide the virus source to be transmitted by aphids.
From the V5/V6 growth stage to the R6 growth stage, a 50-leaf sample was collected each week from consecutive plants in the Clark isolines, one uppermost leaf per plant. A different 50 plants were sampled each week. These leaf samples were tested by a tissue immunoblot assay to estimate the incidence of SMV infection of each isoline. Disease progress curves were fitted with an exponential, y = a{1 - exp[-b(t - m)]}, and logistic, y = a/{1 + exp[-b(t - m)]} model, where y is the incidence of SMV infection (percentage of plants infected) averaged across replications; t is days from planting to sampling date; a is the upper asymptote; b is the rate parameter; and m is a location or delay parameter. The better fitting model was used to compare the SMV disease progress curves of the different pubescence density isolines (Gilligan, 1990; Ren et al., 2000).
The tissue immunoblot protocol described by Lin et al. (1990), as modified by Srinivisan and Tolin (1992), was used to monitor SMV infection of susceptible soybean. Briefly, torn leaf tissue was blotted onto nitrocellulose membranes, and the blotted membranes were processed within a few minutes or stored in an envelope at room temperature for up to 4 wk. Following decolorizing, rinsing, and blocking steps, the membranes were transferred to the primary antibody solution (an unfractionated antiserum to SMV diluted 1:10000 in phosphate-buffered saline), and incubated for 30 min with gentle agitation. The membranes were then rinsed and transferred to the secondary antibody solution (goat anti-rabbit IgG-alkaline phosphate conjugate diluted 1:5000 in phosphate-buffered saline; Sigma, St. Louis, MO). Following a rinsing step, the membranes were transferred to a NBT/BCIP substrate solution (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) and left until a blue color developed. The membranes were then dried and the data scored.
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RESULTS AND DISCUSSION
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The 30 F4–derived lines used in this experiment expressed the characteristics for which they were selected. The genotypes in the intermediate maturity set matured 8 d later than the genotypes in the early maturity set and 7 d earlier than the genotypes in the later maturity set (Table 1). Pubescence density was distinct among the three pubescence density classes and similar to previously reported densities (Gunasinghe et al., 1988; Ren et al., 2000): 160 trichomes cm-2 for normal (N) pubescent genotypes (pd1pd1pd2pd2), 1550 trichomes cm-2 for dense (D) pubescent genotypes (Pd1Pd1pd2pd2 or pd1pd1Pd2Pd2), and 3230 trichomes cm-2 for extra-dense (DD) pubescent genotypes (Pd1Pd1Pd2Pd2) (Table 1). No differentiation was made between Pd1Pd1 and Pd2Pd2 dense pubescent genotypes, as the pubescence properties of these two genotypes did not differ previously (Ren et al., 2000). As the pubescence density increased, trichome length shortened and trichome orientation flattened (Table 1).
Yield did not differ significantly among years. When the 3 yr were combined, the 15 SMV-resistant genotypes did not yield significantly more than the 15 susceptible genotypes (Table 2), since the IOI at the R1 growth stage for the susceptible genotypes averaged only 15%. A 20% infection by the R1 growth stage is necessary to produce a significant yield loss (Ren et al., 1997a). The mean IOI at the R6 growth stage for the susceptible genotypes was 39%, which was significantly greater than that for the resistant genotypes.
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Table 2. Yield and incidence of Soybean mosaic virus infection (IOI) at flowering (R1) and mid-pod-fill (R6) of soybean mosaic virus (SMV) resistant and susceptible soybean genotypes averaged across 3 yr (1995, 1997, and 1998) and all pubescence densities.
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The DD genotypes were 5% lower yielding than the N and D genotypes (Table 3). The DD genotypes were not significantly different in height than N and D genotypes, but the DD genotypes lodged more than the other genotypes (Table 3). The DD genotypes had a significantly lower incidence of SMV infection compared with N and D genotypes (Table 3).
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Table 3. Yield, height, and lodging for normal, dense, and extra-dense pubescence soybean genotypes averaged across three years (1995, 1997, 1998) and Soybean mosaic virus (SMV) resistance and susceptibility.
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Since normally pubescent/resistant (NR), densely pubescent/resistant (DR), and extra-densely pubescent/resistant (DDR) genotypes all have the resistance allele preventing SMV infection, we assumed that pubescence density would not provide an SMV-resistance benefit in these genotypes. Thus, the direct effect of the pubescence density on yield was determined from different pubescent density genotypes in the resistant background. On average, across the three maturity sets, DDR genotypes, DR genotypes, and NR genotypes (at 2380 kg ha-1) were not significantly different in yield (Table 4). Some comparisons within maturity sets, however, were significant. In the early maturity set, the two DR genotypes yielded significantly more than the NR genotypes. In the intermediate maturity set, no significant differences in yield due to pubescence density were observed. In the late maturity set, the DR genotypes had similar yield as the NR genotypes, but the one DDR genotype yielded significantly less than the NR genotypes (-315 kg ha-1) and DR genotypes (-328 kg ha-1).
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Table 4. Effect on yield in the Soybean-mosaic-virus-(SMV)-resistant background of increased compared with normal pubescence density soybean lines.
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Across all maturity sets, the resistance allele provided an average of 39, 115, and 9 kg ha-1 increase in yield in the N, D, and DD genotypes, respectively, but these were all nonsignificant. When this experiment was designed, it was hypothesized that the effect of the resistance allele would decrease with increasing pubescence density; this trend was not observed. The resistance effect for yield provided by the resistance allele did vary with pubescence density in the different maturity sets (Table 5). In the early maturity set, the DR genotypes gave a significantly higher yield than the susceptible genotypes with the same pubescence density. This yield difference may be related to the specific lines found in the early maturity resistant and susceptible groups, as a 14% incidence of SMV infection at R1 is normally below the threshold that produces yield reductions (Ren et al., 1997a). In the intermediate maturity set, no significant differences in yield were observed. In the late maturity set, the resistance allele effect for yield decreased with an increase in pubescence density (Table 5). The resistance allele significantly increased yield of N and D genotypes, but provided no yield benefit for the DD genotype. In the late maturity set, where the incidence of SMV infection at R1 on NS and DS genotypes was 30 and 26%, respectively, the SMV-resistance allele provided a yield benefit. In the DD genotype comparison in the late maturity set, the presence of the SMV-resistance allele did not have a yield benefit, as the incidence of SMV infection on the DDS genotype was only 2% (Table 5).
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Table 5. Effect of Soybean mosaic virus (SMV) resistance on yield and incidence of SMV infection (IOI) at flowering (R1) and mid-pod-fill (R6) for genotypes of three different soybean pubescence densities.
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For all pubescence types, the logistic model was the best-fitting model for all 3 yr. The F test for comparison of fitting a single curve (the disease progress curves on all three isolines with different pubescence density have the same parameters) vs. separate curves was highly significant in all 3 yr, indicating that the disease progress curves on the isolines with different pubescence density had different parameters (Fig. 1). The F tests for the effect of varying the upper asymptote a and the effect of varying the delay parameter m, given a varies, were significant in all 3 yr, while the F test for the effect of varying the rate parameter b, given a and m vary, was not significant in any year. The detailed analysis of the 1995 SMV disease progress curves has been reported previously (Ren et al. 2000).
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Fig. 1. Observed data points and the Soybean mosaic virus disease progress curves fitted with the logistic model on normal (Clark), dense (Clark-Pd1 or Clark-Pd2), and extra-dense (Clark-Pd1Pd2) pubescence soybean isolines. (A) 1995, planting date 20 June. (B) 1997, planting date 25 June. (C) 1998, planting date 25 June.
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The delay parameter m (time of the highest rate of disease increase) was generally later for the DD isoline compared with the N and D isolines. In 1995, the DD isoline reached the highest rate of disease increase during mid-August (55 d after planting), while N and D genotypes reached m 10 d earlier. In 1997, N, D, and DD genotypes all reached the maximum rate of disease increase at approximately the same time, 25 August (61 d after planting). In 1998, m occurred 15 August (51 d after planting) for the N and D genotypes. For the DD isoline, where the curve did not reach an upper asymptote, m was not fitted at any date during the growing season.
The logistic model fits disease progress curves for which infection is polycyclic or the pathogen spreads from plant to plant (Campbell, 1998). Soybean mosaic virus infection of soybean is a polycyclic disease since initially infected plants provide the virus source for the later secondary infection of the remaining uninfected plants. Disease progress curves show that increasing pubescence density reduces the maximum incidence of SMV infection (Fig. 1). More importantly, increasing pubescence density delays the progress of infection. If the IOI at R1 remains below 20%, yield reduction due to SMV infection will be minimized (Ren et al., 1997a). In both the Clark isolines (Fig. 1, R1 at 44 to 46 d after planting) and the random SMV-susceptible genotypes (Table 3), extra-dense pubescence provided a mechanical barrier to aphid transmission of SMV that kept the IOI at R1 < 20%.
These results suggest that extra-dense pubescence has the potential to be a valuable trait for double-crop soybean. Extra-dense pubescence can significantly reduce the incidence of SMV infection at R1. At the same time, the extra-dense pubescent SMV-resistant late maturity genotype was significantly lower yielding than the normal pubescent SMV-resistant late maturity genotype (Table 4). Also, extra-dense pubescence reduced yield across all genotypes (Table 3). Continued hybridization with and selection of extra-dense pubescent genotypes should improve yield potential and agronomic characteristics to utilize this non-strain-specific SMV avoidance mechanism.
After this study was completed, the soybean aphid (Aphis glycines Matsumura) has appeared in the USA. The soybean aphid is a much smaller aphid than those that commonly probe soybean in the USA (Grayson Brown, 2002, personal communication). The interrelationships among extra-dense pubescence, the much smaller soybean aphid, and soybean aphid predators must now be assessed. In the meantime, selecting for SMV resistance provided by one of the Rsvx resistance alleles in the normal pubescence density background of current cultivars appears to be the appropriate breeding strategy.
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NOTES
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This research was funded in part by grants from the Kentucky Soybean Promotion Board and the United Soybean Board. This paper (02-06-51) is published with approval of the director of the Kentucky Agric. Exp. Stn.
Received for publication March 25, 2002.
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ABSTRACT
INTRODUCTION
