© 2005 Plant Management Network.
A Temperature-Controlled Water Bath Method for Evaluating Soybean Reaction to Sudden Death Syndrome (SDS)
R. Y. Hashmi, Post-Doctoral Researcher, J. P. Bond, Assistant Professor, M. E. Schmidt, Associate Professor, and J. H. Klein, Assistant Scientist, Department of Plant, Soil and Agricultural Systems - Center of Excellence for Soybean Research, Teaching and Outreach, College of Agricultural Sciences, Southern Illinois University, Carbondale 62901
Hashmi, R. Y., Bond, J. P., Schmidt, M. E., and Klein, J. H. 2005. A temperature-controlled water bath method for evaluating soybean reaction to Sudden Death Syndrome (SDS). Online. Plant Health Progress doi:10.1094/PHP-2005-0906-01-RS.
Many greenhouse screening methods have been tested to evaluate soybean genotypes for reaction to sudden death syndrome (SDS) caused by Fusarium solani f. sp. glycines. These methods generally have proven disappointing in that results are not consistent among methods or do not correlate with field reaction. In the present study, SDS foliar symptom severity among 24 soybean genotypes was compared using three inoculation methods in the greenhouse. The pathogen inoculum was either mixed (seedbed mixing) or layered (seedbed layering) in the soil medium prior to planting seed and then kept on a greenhouse bench or the inoculum was layered in the soil medium and kept in a temperature control water bath. The water bath method was similar to the layering method with the addition of precise temperature control. The water bath method was superior to the other methods in consistency of SDS symptoms among genotypes among trials and in agreement with SDS field reaction. When disease severity data obtained in the greenhouse were regressed with foliar disease data obtained in field trials, R2 values were 0.56, 0.60, and 0.81 for the seedbed mixing, seedbed layering, and water bath methodS, respectively. The improved ability to predict field response using the water bath method likely results from precise control of the temperature in the rhizosphere. The water bath method described herein will increase the efficiency of selection for highly adapted SDS resistant cultivars by reducing the number of genotypes that must be evaluated under field conditions.
Sudden death syndrome (SDS) is a season-long root disease of soybean (Glycine max L.) with foliar symptoms beginning in late vegetative and early reproductive stages of the plant growth. This disease has been found in most soybean growing regions of the United States and the world (16,24). The causal organism of SDS has been described as Fusarium solani f. sp. glycines Roy (FSG), to indicate its host specificity on soybean (15). Annually, yield losses resulting from SDS can be sporadic. However, yield loss can be severe and depends upon plant growth stage at infection, host genotype, and the environment (9,18,19). Current strategies for management of this disease include planting resistant cultivars and cultural practices (3).
The pathogen infects the roots early in seedling development (13) with fungal colonization mainly restricted to the taproot (17). Diagnostic root symptoms include brown discoloration of vascular tissue with the pith remaining white (20) (Fig. 1). Foliar symptoms of SDS include interveinal, chlorotic spots beginning at or near the flowering stage of plant growth. These spots usually coalesce and become necrotic during the pod-filling period leaving only the veins and the region adjacent to the major leaf veins green (Fig. 2). As disease severity increases, extensive defoliation occurs leaving the petioles upright and attached (Fig. 3) (20). Foliar symptoms may result from phytotoxic compound(s) produced by the fungus in roots and translocated to leaves (1,5,6). The pathogen is frequently isolated from the root system and lower stem, but not from the leaves (15,17).
The environment plays a critical role in symptom expression, making consistent selection of resistant soybean genotypes difficult under field conditions. When environments are optimal for disease development, partially resistant cultivars may appear to be susceptible to SDS (12). However, several cultivars have consistently exhibited low incidence and severity of SDS in a range of field environments (2,4,22).
Selection of resistant cultivars has relied on field testing of germplasm across several environments in time and space. Although very expensive and time-consuming, this has proven to be the most successful method for evaluating disease reaction. Several other methods have been evaluated to determine soybean reaction to SDS including growth chambers, aeroponic chambers in greenhouses, and conventional greenhouse methods (6,7,8,10,11,12,14,22). These methods employ a variety of inoculation strategies including pathogen-colonized tooth picks, grafting soybean resistant and susceptible genotypes, placing cut seedlings in fungal culture filtrate, or placing infested sorghum kernels on roots or in potting media. Two commonly employed greenhouse methods involve mixing infested sorghum kernels in potting medium (seedbed mixing method) or layering infested kernels in potting medium (seedbed layering method). The limitation of these greenhouse methods is that results are inconsistent among cultivars and across trials and often do not correlate with foliar disease ratings obtained in field trials. Soil moisture and temperature are critical factors in the expression and development of foliar symptoms of SDS (21,23). While air temperatures can be managed in the greenhouse environment, great fluctuations in temperature can occur in the potting medium. A greenhouse water bath would allow for precise management of soil temperatures while also providing consistent growing conditions for the plant. The objectives of this research were to compare the SDS reaction of 24 soybean genotypes in the field with the reaction of these genotypes in the greenhouse using two standard inoculation methods and a method using precise control of soil temperature with a water bath.
Selection of Soybean Genotypes
In 2000, 24 soybean genotypes were chosen as a set of differentials to make it possible to compare various resistance evaluation methods in both field and greenhouse environments (Table 1). These genotypes were selected because their range of reaction to SDS ranges from susceptible to resistant, and their maturity group ranges from III to V. Selected genotypes were a subset of several hundred tested in Southern Illinois University at Carbondale’s Commercial SDS Variety Field Testing program. Field reaction to disease was determined as a foliar disease index (DX) as described by Njiti et al. (13). Briefly, DX is a function of a disease incidence (DI) score, representing a percentage of plants in a plot expressing symptoms, and a disease severity (DS) score, rated on a 1 to 9 scale. The DS score is assigned as 1 = 0 to 10% chlorosis or 1 to 5% necrosis; 2 = 10 to 20% chlorosis or 6 to 10 % necrosis; 3 = 20 to 40% chlorosis or 10 to 20% necrosis; 4 = 40 to 60% chlorosis or 20 to 40% necrosis; 5 = >60% chlorosis or >40% necrosis; 6 = up to 33% defoliation; 7 = up to 66% defoliation; 8 = > 66% defoliation; and 9 = plant death. The DX score represents a scale of 0 to 100, as DX = (DI × DS) /9. Only those genotypes assayed in three or more environments where the mean DX for the susceptible check cultivars was 20 or higher were in this set of differentials.
Table 1. Soybean genotypes selected based on disease reaction to sudden death syndrome and relative maturity.
x Genotypes were selected based on maturity group and SDS response in replicated field disease evaluations conducted by SIUC,1998 to 2004. Genotypes represents resistant (< 8.8), intermediate (15.4 to 28.0), and susceptible (> 35.2) classes.
y Environment includes both multiple locations and years (1998 to 2004).
z Mean foliar disease index (DX) averaged across locations. Foliar disease index was evaluated on a 0 to 100 scale and represents a composite of disease severity and disease incidence.
Based on field DX, this set of genotypes represents three classes of reaction to SDS: resistant, susceptible, and intermediate (Table 1). The resistant class is represented by Egyptian (DX = 8.8) and all genotypes having a lower DX rating. The susceptible class is represented by Pioneer 3981 (DX = 35.2) and genotypes with a higher DX rating. The remaining genotypes are classified as intermediate (DX range 15.4 to 28.0).
Kernels of white sorghum (Sorghum bicolor L. Moench) were soaked in water for 12 h. After soaking, 250 g of kernels were transferred to 500-ml flasks and autoclaved twice with a 24-h interval between cycles. Each flask was infested with 7 to 10 plugs (8 mm in diameter) of actively growing fungal hyphae on potato dextrose agar (PDA). Plugs were mixed thoroughly with sorghum kernels and incubated at 24°C under fluorescent light for 14 days. Flasks were hand shaken on alternate days to ensure uniform distribution of fungal inoculum and to prevent clumping. For each method and trial, infested kernels were added to soil at a rate resulting in 7,500 to 8,000 cfu/g soil as determined by standard plate counting.
Seedbed Layering and Seedbed Mixing Methods
For the seedbed layering method, 150 g of soil medium were added to each of the 12 planting slots of Nu-Tray plastic trays (Hummert International, St. Louis, MO) measuring 44.5 × 32.5 × 8.0 cm. The soil medium was a mixture of steam pasteurized soil:sand:pro-mix (Premier Horticulture Inc., Quakertown, PA) in a 1:1:1 ratio. Ten infested sorghum kernels were placed on the top of the soil which were then covered with an additional 100 g of soil medium. For the seedbed mixing method, the infested sorghum kernels were mixed thoroughly with the 250 g of soil medium and placed into each of the slots of the same type planting tray as above. Seed of each genotype were surface disinfested in NaOCl (0.053%) for 3 min. Two seeds were planted to a depth of 2.5 cm into each slot, and, following emergence, seedlings were thinned to one seedling per planting slot. Trays were maintained on greenhouse benches under supplemental lighting from sodium lamps (14 h light:10 h dark). Soil moisture was maintained near saturation by hand watering.
Water Bath Method
The water bath method developed was a modification of the seedbed layering method previously described. The water bath consisted of a box measuring 243.8 × 76.2 × 33.0 cm that was lined with 6 mil plastic sheeting (Fig. 4). The box was completely filled with water, and the water temperature was maintained at 24°C (± 0.5°C) with a chilling unit (Model # 2095; Forma Scientific Inc., Marietta, OH) that circulates chilled antifreeze through copper tubes submerged in the water at the bottom of the box. Each water bath contained 24 plastic buckets (22 cm × 20 cm diameter) that were suspended in the water bath. Eighteen polyvinyl chloride (PVC) tubes (20.3 cm × 2.5 cm in diameter) open at both ends were stacked vertically and maintained in position by filling the plastic buckets with the previously mentioned soil medium. The tubes were partially filled to a height of 15.5 cm with 300 g of the soil medium. Fifteen infested sorghum seeds were placed on top of the soil medium column and then covered with an additional 2.5 cm of soil medium. Two surface disinfested seeds of each soybean genotype were placed in individual tubes and covered with an additional 1.5 cm of soil medium. Seedlings were thinned to one per tube 1 week after emergence. The water bath was maintained in the greenhouse with light and air temperatures as described in the seedbed layering and mixing experiment.
Experimental Design and Data Analysis
The seedbed layering and mixing experiments were run in a greenhouse separate from the water bath experiments. The experiments were conducted in the same greenhouse range albeit in separate but adjacent houses. Experiments for the techniques were run concurrently. The seedbed experiment consisted of a factorial arrangement of genotypes and soil infestation method in a completely randomized design. Treatments in the water bath experiment consisted of genotypes in a completely randomized design. All treatments were replicated 12 times with each replication consisting of a single seedling as described above. Three trials were initiated on 6 November 2002, 18 April 2003, and 10 June 2003. Foliar disease severity was recorded for each seedling 21 to 28 days after emergence (Fig. 5). Disease severity was measured on a scale of 1 to 9 as described under Selection of Soybean Genotypes. Data for each experiment were analyzed separately. All data were tested for normality and subjected to analysis of variance, pair wise correlations, Spearman’s rank correlation, and regression analysis using SAS Version 8.0 (SAS Institute, Inc., Cary, NC). All differences are significant at P < 0.05 unless otherwise noted. Means were separated using Fishers’ Protected LSD test.
Comparison of Methods
In the seedbed mixing and layering experiments, there were significant differences in SDS severity between methods of inoculation, among genotypes, and trials (P < 0.001). Across trials, the layering method consistently resulted in a greater foliar disease severity with a lower variance compared to the seedbed mixing method (Table 2). However, the magnitude of the difference between methods was different across trials, hence a significant method by trial interaction (P < 0.01). A significant genotype by trial interaction also occurred (P < 0.001). This interaction was the result of rank changes for those genotypes with similar reaction and not the result of major rank changes for resistant or susceptible genotypes. Correlations in genotype performance were obtained between trials within methods with coefficients ranging from 0.45 to 0.77. A stronger correlation was found between methods across trials, with coefficients ranging from 0.82 to 0.95. When combining data across trials for the two seedbed methods, the layering method provided the best agreement with field disease ratings (R2= 0.60 versus R2 = 0.56). These results are in agreement with those of Njiti et al. (12) who found a similar agreement with field DX rating (R2 values ranged from 0.40 to 0.60) and also indicated that variation in genotype performance across trials could not be controlled.
Table 2. Mean and rank of soybean genotypes for SDS foliar disease index (DX) from field trials and greenhouse trials using three inoculation methods.w
w Rank was assigned based on field foliar disease index (DX) and greenhouse foliar disease severity of the genotypes within each method.
x Mean foliar disease index (DX) averaged across locations. Foliar disease index was evaluated on a 0-100 scale and represents a composite of disease severity and disease incidence.
y Disease severity was measured on a scale of 1 to 9.
z Least significant difference according to Fishers’ LSD test P < 0.05.
In the water bath experiment, significant differences occurred among genotypes and trials (P < 0.001). A greater consistency in genotypic performance was observed across trials in that there was no genotype by trial interaction. The water bath method provided the best correlation between the foliar disease severity ratings and the field foliar disease ratings (R ranging from 0.82 to 0.93 for independent trials) than the other inoculation methods. When combining data across trials, over 80% of the variation in field reaction was accounted for by the water bath results (Fig. 6).
When ranking genotypes from resistant to susceptible and comparing to the field DX rank, the layering and mixing methods were less consistent than the water bath method (Table 2). The Spearman’s rank correlations were r = 0.60 for the layering method, r = 0.59 for the mixing method, and r = 0.84 for the water bath method. Eleven genotypes were classified as resistant in the field. The layering method ranked eight of these genotypes as resistant and three as intermediate, while the mixing method ranked nine as resistant and two as intermediate. In contrast, the water bath method ranked ten of these genotypes as resistant. Eight genotypes were classified as intermediate based on field ratings. The layering method ranked three as resistant, the mixing method ranked four as resistant, and the water bath method ranked one as resistant. Five genotypes were classified as susceptible in the field. The layering and water bath methods each ranked one of these genotypes as intermediate, and the mixing ranked one of these genotypes as resistant. Greater resolution was exemplified by the water bath method in that this method provided a greater range between the most resistant and most susceptible genotypes (range = 28, 26, and 24 for the water bath, layering, and mixing methods, respectively) with a smaller LSD value.
When evaluating germplasm for resistance under greenhouse conditions, results can vary across trials depending on ambient temperatures and photoperiod experienced during the time trials are conducted. For both experiments, the mean greenhouse ambient temperature range across the temporal span of each trial was 18 to 30°C, 21 to 33°C, and 23 to 36°C, for the 6 November 2002, 18 April 2003, and 10 June 2003 trial, respectively. For all methods, the agreement with field disease ratings decreased as the greenhouse ambient temperature range increased. However, the water bath method was the only method that maintained a constant temperature (24°C) for the soil medium, and this was likely the reason that consistent data were obtained. Soil temperature is a critical factor for promoting fungal infection, colonization, and symptom production (21,23).
A set of differentials has been identified that can be used to evaluate and compare current and future germplasm for SDS reaction. Seed of this germplasm set is maintained by SIUC and can be obtained by contacting the corresponding author.
Both the seedbed layering method and the mixing method provided satisfactory results in delineating resistant from susceptible genotypes. However, as indicated in this study, a single trial would probably not be sufficient for accurate prediction of field reaction and should be repeated. The results of the seedbed experiment demonstrated a greater variation among trials than between methods. The increased disease pressure obtained by the layering method may indicate its advantage over the mixing method in minimizing the probability of “escapes” in genotype assessment. The consistency of the seedbed methods across trials and their agreement with field reaction were similar.
The water bath method proved to be most consistent across trials and provided better agreement with field reaction than the other methods. In addition, it proved to be superior for identification of resistant genotypes, allowed fewer escapes, and provided greater separation among genotypes of similar classification. The advantage of this method over others is precise management of soil temperature. Though either layering or mixing methods can facilitate selection of SDS resistant genotypes, a more consistent and field-reliable result would be attained by using the temperature-controlled water bath method. Evaluation of the efficiency of this method for selection of resistant genotypes from breeding populations segregating for resistance is in progress.
This project was funded by the Illinois Soybean Program Operating Board and the North Central Soybean Research Program. We thank J. S. Russin, W. P. Bond, A. K. Gregor, and two anonymous reviewers for critical review of this manuscript.
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