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a Dep. of Crop, Soil, and Environmental Sciences, Univ. of Arkansas, Fayetteville, AR 72701
b Dep. of Plant Pathology, Univ. of Arkansas, Fayetteville, AR 72701
* Corresponding author (pchen{at}uark.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: DAI, days after inoculation • ELISA, enzyme-linked immunosorbent assay • SMV, Soybean mosaic virus • STN, stem-tip necrosis • TMV, Tobacco mosaic virus • TSWV, Tomato spotted wilt tospovirus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three independent loci, Rsv1, Rsv3, and Rsv4, have been reported so far for SMV resistance in soybean in the United States. Nine resistance alleles (Rsv1, Rsv1-t, Rsv1-y, Rsv1-m, Rsv1-k, Rsv1-r, Rsv1-h, Rsv1-s, Rsv1-n) have been identified at the Rsv1 locus in PI 96983, Ogden, York, Marshall, Kwanggyo, ‘Raiden’, ‘Suweon 97’, LR1, and PI 507389 (Kiihl and Hartwig, 1979; Chen et al., 1991, 1994, 2001, 2002; Ma et al., 1995, 2003). Two alleles have been identified at the Rsv3 locus in soybean lines OX 686 (Buzzell and Tu, 1989) and L29 (Buss et al., 1999).
Susceptible soybean cultivars exhibit typical mosaic symptoms when infected with SMV. However, some resistant soybean cultivars often develop necrotic symptoms when infected with certain SMV strains. The necrotic reaction is often observed when soybean cultivars that are resistant to less virulent SMV strains are infected by more virulent strains (Cho and Goodman, 1979, 1982; Chen et al., 1994; Ma et al., 2003). The necrotic reaction is also an expression of resistance genes in the heterozygous state (Kiihl and Hartwig, 1979; Buss et al., 1989, Chen et al., 1994; Liao et al., 2002).
Expression of the necrotic reaction to virus infection is affected by environmental conditions (Cho et al., 1977; Chen et al., 1994, 2001; Liao et al., 2002). High temperature has been reported to induce a switch from a necrotic reaction to mild systemic symptoms. A continuous high temperature of 33°C caused systemic infection in Capsicum Chinese PI 152225 by Tomato spotted wilt virus (TSWV), while all the inoculated plants grown in the greenhouse as controls at lower temperatures of 18 to 24°C reacted hypersensitively (necrotic local lesions) to TSWV (Roggero et al., 1996). Tobacco breeding line L8 was resistant (necrotic reaction on the inoculated leaves and the absence of systemic infection) to Tobacco ringspot virus at 24°C, but became systemically infected (faint oak leaf and chlorotic ring patterns) at 35°C (Hendrix, 1972). Tu (1989) reported that bean plants developed tip necrosis between 16 and 24°C when infected with Bean yellow mosaic virus, but plants exhibited yellow mosaic symptoms and little or no tip necrosis at 28 and 32°C. Samuel (1931) first reported that at the high temperature of 35°C Tobacco mosaic virus (TMV) produced chlorotic lesions and a systemic mottle in Nicotiana glutinosa instead of the necrotic local lesions produced at lower temperatures. Kassanis (1952) found that at 36°C Nicotiana glutinosa inoculated with TMV gave chlorotic local lesions instead of necrotic ones, and the plants became systemically infected.
Tu and Buzzell (1987) reported that STN is a temperature-dependent reaction of soybean to infection by SMV. However, there is no information about the effect of temperature on the expression of STN in soybean plants carrying different resistance genes when infected with different SMV strains. The objective of this study was to investigate the effect of different temperature regimes on symptom expression in soybean cultivars carrying different resistance genes when inoculated with SMV strains G1 and G7. This information will lead to a better understanding of the soybean genotype x SMV strain interaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twelve seeds were planted in each pot, and plants were thinned to eight per pot for inoculation. Plants were inoculated at the unifoliolate stage at the same time. For each genotype, three pots of plants were inoculated with leaf sap from healthy plants as a control and three pots of plants were inoculated with each virus strain (G1 or G7) for each temperature treatment. After inoculation, the plants were maintained in one of six growth chambers at constant temperatures of 10, 15, 20, 25, 30, and 35°C. For each temperature, the pots for all genotypes inoculated with G1 or G7 and the healthy control were completely randomized. All growth chambers were programmed for a 12-h photoperiod and were illuminated with cool-white fluorescent lamps supplemented with incandescent lamps (with light intensity of 180 µmol m–2 s–1). On the basis of the different symptoms expressed between 30 and 35°C, further experiments were conducted in which two sets of inoculated plants were maintained at constant temperatures of 32 and 33°C. After observing the symptom expression at 32 and 33°C, another two sets of inoculated plants were maintained at alternate 12-h temperatures of 33°C day/20°C night and 35°C day/20°C night. Plants were monitored for symptom expression on a regular basis for 6 to 8 wk.
Virus Strains
SMV-G1 and SMV-G7 were provided by Dr. Sue Tolin of Virginia Tech and maintained by mechanical inoculation in Essex soybean in the greenhouse. Virus strain identity was verified by inoculation of a set of differential soybean genotypes: V94-5152, L29, PI 96983, PI 507389, York, Essex, and Suweon 97 (Cho and Goodman, 1979; Chen et al., 2002). These soybean genotypes were maintained in the greenhouse at 25°C after inoculation and their reactions to SMV are shown in Table 1. Differential soybean genotypes were provided by Dr. G.R. Buss of Virginia Tech.
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Test for Virus Infection
Leaf samples of test plants were assayed for SMV by Protein-A ELISA (Edwards and Cooper, 1985) using anti-SMV rabbit polyclonal antibodies. The middle leaflet from young, fully expanded, uppermost trifoliolate leaves was collected from each plant in each pot and mixed as one sample per pot for ELISA (three pots were sampled for each strain x genotype temperature treatment) when symptoms developed at each temperature (about V3 stage for most temperatures). For plants expressing STN symptoms, the middle leaflet from uppermost trifoliolate leaves (local necrotic lesions already developed, but the trifoliolate leaves were still green) was collected. Leaf extracts were prepared using a tissue extractor (leaf and bud press, Erich Pollahne, West Germany). Samples of the leaf extracts were tested at a dilution of 1:10 (v/v) in PBS-Tween [0.01 M sodium phosphate, 0.15 M sodium chloride, 0.1% (v/v) Tween 20, pH 7.2]. The ELISA values were determined spectrophotometrically 30 min after substrate addition at a wavelength of 405 nm with a microplate reader (Model 7250, Cambridge Technology Inc., Cambridge MA). Samples were considered positive for SMV if ELISA values were three times or more than those of healthy plant extracts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 20, 25, and 30°C, symptoms in V262 developed more rapidly than at lower temperatures (10 and 15°C), and STN was expressed in V262 at 14, 8, and 8 DAI with G1, respectively. Virus was detected by ELISA in plants exhibiting STN (Table 2). Local necrotic lesions developed initially in both unifoliolate leaves and upper trifoliolate leaves (Fig. 1e), and eventually the upper trifoliolate leaves became completely necrotic (Fig. 1f). At 35°C, V262 developed mild mosaic symptoms instead of necrosis after inoculation with G1, and virus was detectable by ELISA. No necrosis was observed at 35°C in V262 up to 42 DAI with G1.
V94-3971 was resistant to G1 at all temperatures and no virus was detected at the V3 stage by ELISA (Table 2). Essex exhibited mosaic symptoms after inoculation with G1 at all temperatures, and virus was detected at the V3 stage in all the inoculated plants. Mosaic symptoms in Essex developed sooner at 25 to 30°C than at lower and higher temperatures.
Effect of Temperature on Symptom Expression in Soybean Plants Inoculated with G7
After inoculation with G7, V94-3971 (Rsv1) developed STN at 10, 15, 20, 25, 30°C that was first observed at 44, 41, 14, 8, and 8 DAI, respectively (Table 3; Fig. 1b,d). Mosaic symptom rather than STN developed in V94-3971 at 35°C. V262 and Essex showed mosaic symptoms at all the temperatures tested when inoculated with G7, and virus was detected in all inoculated plants. Similar to results with G1, symptoms developed sooner in all isolines at 25 to 30°C than at 10 to 20°C and 35°C.
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Effect of Alternating Temperatures on Symptom Expression in Soybean Plants Inoculated with SMV
Since soybeans grow in the field where the temperature fluctuates between day and night, we attempted to determine the impact of alternating temperatures on the expression of necrosis in soybean infected by SMV. Inoculated plants of three soybean isolines were kept in growth chambers set at 33°C day/20°C night and 35°C day/20°C night. At the alternate temperature of 33°C day/20°C night, STN was observed in V262 (Fig. 3a) 7 DAI with G1 and symptoms were more severe at 14 DAI (Fig. 3b). Local necrotic lesions were found on inoculated leaves of V94-3971 (Fig. 3c) 7 DAI with G7 and STN developed at 14 DAI (Fig. 3d; Table 5).
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The responses of the three soybean isolines to the two SMV strains were consistently uniform within each temperature treatment tested in our experiments. All the plants of each genotype in a treatment developed the same symptoms on the same day since they were planted and inoculated at the same time. All the plants of the three pots of V94-3971 were resistant to G1 at all temperatures and exhibited STN from 10 to 32°C and 33/20°C. All plants of the three pots of V262 expressed mosaic symptoms after inoculation with G7 at all temperatures and exhibited STN from 10 to 32°C and 33/20°C, 35/20°C. All the plants of Essex expressed mosaic symptoms following inoculation with G1 and G7 at all temperatures. All the inoculated plants in the three pots of V262 and V94-3971 shifted to mosaic symptoms at high temperatures of 33 and 35°C when infected with G1 and G7, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Temperature had a marked effect on symptom expression in SMV-infected soybeans. The optimum temperature for symptom development in SMV-infected plants appeared to be 25 to 32°C. Lower temperatures (10–15°C) tended to slow down plant growth and delay viral symptom expression. Virus was not detected in the inoculated plants at low temperatures at 15 DAI (data not shown), indicating that low temperature inhibited virus replication and movement as well as the growth and development of host plants. Similar results have been reported in other virus–host systems. Dawson et al. (1975) reported that low temperatures of 5 and 12°C inhibit TMV synthesis and no TMV synthesis could be detected 15 DAI at 5°C. Only minute amounts of TMV could be detected 15 DAI at 12°C.
High temperatures (33–35°C) also delayed symptom development in SMV-infected soybeans (Tables 1–3). Similar results have been reported in other virus-host systems. Banana plants infected with Banana streak badnavirus showed more severe symptoms and had significantly higher virus titers at 22°C than plants that grew at 28 to 35°C (Dahal et al., 1998).
V94-3971 was resistant to G1 at all temperatures tested. No symptoms of virus infection and no detectable virus were observed in the inoculated plants, demonstrating that the expression of the resistant reaction of the Rsv1 gene to G1 was not affected by temperature. However, temperature has been reported to alter the host resistance in other host–pathogen systems. The perennial ryegrass (Lolium perenne L.) cultivar Carrick was resistant to crown rust at 10°C but susceptible at 25°C (Roderick et al., 2000). Wheat cultivar/lines VPM1 (Pm4b) and Yuma/*Cc (Pm4a) were more resistant to powdery mildew at 25°C postinoculation temperature than at 15°C. However, two other wheat cultivars/lines Kavkaz (Pm8) and Axminster/8*Cc (Pm1) were consistently resistant at both temperatures. Therefore, there are differences in temperature sensitivity among powdery mildew resistance genes when interacting with corresponding powdery mildew pathogenicity genes. The effect of temperature on host reaction was significant on VPM1 (Pm4b) and Yuma/*Cc (Pm4a), but not on Kavkaz (Pm8) and Axminster/8*Cc (Pm1) (Ge et al., 1998). In our research, the reaction of the soybean isoline V94-3971 (Rsv1) to SMV-G1 was very stable from 10 to 35°C.
At 10, 15, 20, 25, 30, and 32°C, all the plants of V262 infected with G1 and V94-3971 infected with G7 developed STN. However, at 33 and 35°C, all the plants developed mosaic symptoms instead of STN (Table 1–3, Fig. 2), indicating that high temperature (
33°C) inhibits the expression of necrosis. The expression of necrosis associated with SMV resistance genes in soybean is temperature dependent. Tu and Buzzell (1987) found that at 20 and 24°C, the majority of the plants of soybean line OX 686 with the Rsv3 gene developed STN when they were inoculated with an SMV strain similar to the American Type Culture Collection SMV strain PV94. The relationship of the SMV strain used by Tu and Buzzell (1987) to G1 and G7 is unclear. However, at 28 and 32°C, nearly all the inoculated plants in their study developed typical mosaic symptoms. The discrepancy between our results and those by Tu and Buzzell (1987) in the threshold temperature for the switch from a necrotic reaction to a mosaic reaction is probably due to the use of different soybean genotypes and SMV strains. Cho and Goodman (1979) found that necrosis was more conspicuous on the soybean differentials (cultivars Clark, Rampage, Davis, York, Marshall, Ogden, Kwanggyo, Buffalo) inoculated with the seven SMV strains (G1–G7) at 30 ± 1°C than on plants at 24 ± 1°C, but no qualitative difference in reactions was noted at both temperatures. This result agrees with our observation that symptom expression is not altered between 24 and 30°C.
Stem-tip necrosis in V262 (Rsv1-n) appeared 7 and 8 DAI with G1 at alternate day/night temperatures of 33/20°C and 35/20°C, respectively. STN in V94-3791 (Rsv1) appeared 14 DAI with G7 at 33/20°C, and only necrotic local lesions developed in the inoculated leaves and upper leaves at 35/20°C. Evidently, the necrosis expression in the V262 (Rsv1-n) x SMV-G1 combination was not affected by the alternate day/night temperatures treatment. However, the STN response was delayed in V94-3971 at 33/20°C and altered to local necrotic lesions mixed with mosaic symptoms at 35/20°C, indicating that the necrosis expression in the V94-3971 (Rsv1) x SMV-G7 combination was affected by elevated daytime temperature. Similar temperature-dependent responses have been reported by Adams and Jones (1986), in which they demonstrated that systemic mosaic symptoms in potato cultivars were obvious at 10 to 20°C for Potato virus X (PVX) isolate DX, and at 10 and 15°C for PVX isolate B. Viral antigen accumulation was greatest at 15 and 20°C with isolate B and at 25°C with isolate DX, and no symptoms appeared and no viral antigen was detected at 30°C. In our research, SMV-G7 had a lower alternate temperature threshold (33/20°C) for STN expression in V94-3971 than SMV-G1 (35/20°C) for STN expression in V262. Temperature sensitivity for symptom expression by SMV in soybean appears to be genotype and strain dependent. It is also worth noting that the same daytime temperature results in different responses to SMV when alternating with a low night temperature as compared with a constant day and night temperature (Tables 2–5). For example, V262 inoculated with G1 exhibited mosaic symptom at 33 and 35°C, but STN at 33/20 and 35/20°C. Apparently, the exposure of inoculated plants to a lower night temperature led to expression of the gene for STN, which could otherwise be turned off and switched to a mosaic reaction at a constant high temperature. An understanding of the temperature dependence of gene expression will be a valuable tool for future research on gene expression, resistance mechanisms, and soybean genotype x SMV strain interactions.
Received for publication May 10, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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