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Peer Reviewed

© 2012 Plant Management Network.
Accepted for publication 20 December 2012. Published 21 March 2012.

Evaluation of Soybean Genotypes for Resistance to Three Seed-borne Diseases

Alemu Mengistu and P. A. Arelli, USDA-ARS, Jackson, TN 38301; Nacer Bellaloui, USDA-ARS, Stoneville, MS 38766; J. P. Bond, Southern Illinois University, Carbondale, IL 62901; G. J. Shannon and A. J. Wrather, University of Missouri, Portageville, MO 63873; J. C. Rupe and P. Chen, University of Arkansas, Fayetteville, AR 72701; C. R. Little, Kansas State University, Manhattan, KS 66506; C. H. Canaday and M. A. Newman, University of Tennessee, Jackson, TN 38301; and V. R. Pantalone, University of Tennessee, Knoxville, TN 37996

Corresponding author: Alemu Mengistu.

Mengistu, A., Arelli, P. A., Bellaloui, N., Bond, J. P., Shannon, G. J., Wrather, A. J., Rupe, J. C., Chen, P., Little, C. R., Canaday, C. H., Newman, M. A., and Pantalone, V. R. 2012. Evaluation of soybean genotypes for resistance to three seed-borne diseases. Online. Plant Health Progress doi:10.1094/PHP-2012-0321-02-RS.


Seed-borne diseases of soybeans caused by Phomopsis longicolla (Phomopsis seed decay), Cercospora kukuchii (purple seed stain), and M. phaseolina (charcoal rot) are economically important diseases that affect seed quality. Commercial cultivars marketed as resistant to all three diseases are not available. Reactions of 27 maturity group (MG) III, 30 early MG IV, 33 late MG IV, and 53 MG V genotypes were evaluated for resistance to these pathogens during the 2006 to 2008 growing season in the same field that had been in no-till production, not irrigated, and naturally and artificially infested. There was great variation in seed infection among genotypes and years, indicating the value of screening genotypes over multiple years. Some genotypes were resistant to these pathogens in one, two, or in all three years. Genotypes, DP 3478 (early MG IV), and RO1-769F (MG V) were resistant and DG4460 was moderately resistant to P. longicolla infection across three years. Genotypes AG3705 and FFR3990 (MG III) and DC20300, DC7816, Stoddard, and Ozark (MG V), were resistant to C. kukuchii infection during all three years. Ten genotypes in MG III, eight in early MG IV, seven in late MG IV, and 14 in MG V had no seed infection by M. phaseolina in all three years. These results indicate that seed infection comparison to these pathogens among genotypes should be made over several years, or false conclusions about resistance to any of the three pathogens may be made when disease is assessed for limited period of time. The genotypes identified as having resistance to each or combinations of the seed-borne diseases across the three years could be useful as a source for resistance in improving soybean seed quality.


Many of the pathogens of economic importance are associated with soybean [Glycine max (L.) Merr.] seeds. Among these are three pathogens: Phomopsis longicolla, T. W. Hobbs, which causes Phomopsis seed decay (PSD); Cercospora kukuchii Mastsumoto & Tomoyasu, which causes purple seed stain (PSS) and Cercospora blight (27); and Macrophomina phaseolina (Tossi) Goidanich which causes charcoal rot (CRT) (8). Depending on the year and environmental conditions, these diseases may produce significant losses in seed quality. Yearly soybean yield losses in the United States due to PSD and PSS were estimated to be 8.3 × 105 tons from 2003 to 2005 (37); however, loss estimates for seed-borne charcoal rot are not available.

Phomopsis seed decay (Fig. 1A) has become increasingly important in the mid-southern production region of the United States since farmers began planting MG III and IV cultivars in April rather than planting MG VI and VII cultivars in May or June. In this "Early Soybean Production System" (10), seed develops during hot and humid weather conditions which enhances PSD development (1,3,5,8,16,17,20,21,25,36,38). Seed heavily infected by P. longicolla are moldy, have lower test weight, greater numbers of split seeds (11), and exhibit reduced germination (8,9).

The incidence of PSS has varied over years, geographical areas of the United States, and soybean genotype (8,13,27). Roy and Abney (31) reported that the highest incidence of PSS (Fig. 1B) occurred from controlled inoculation at full bloom or early pod stages, but they also found a high percentage of PSS when inoculations were made at the beginning of seed development (R5) (6). The incidence of PSD was reduced in the presence of PSS (7,31). PSS begins when C. kikuchii conidia from overwintering infections are rain splashed onto leaves, stems, and pods during periods of high moisture and warm temperatures (15). Once pods become infected, the fungus grows through the hilum and into the seed coat (33).

Charcoal rot is a major disease of economic significance in soybeans (Fig. 1C) resulting in reduced yield and seed quality (8,34) and is more severe during drought and high temperature (22,24). M. phaseolina may move from infected roots to stems and to seed causing low germination and seedling rots (8,29).


Fig. 1. Symptoms of seed infected with: (A) Phomopsis longicolla; (B) Cercospora kukuchii (image courtesy of Loren J. Giesler); and (C) Macrophomina phaseolina growing from infected seed.


Resistance to PSD among some plant introductions (PIs) and some breeding lines have been reported (7,26,35). Unfortunately, there are few reports of soybean with resistance to C. kukuchii (13). Moderate resistance to the root and stem phase of infection by M. phaseolina has been identified (24). However, resistance to seed infection has not been described.

The majority of soybean acreage is produced in non-irrigated environments that provide less soil moisture, especially at critical times during the growing season resulting in lower yields and lower seed qualities and may favor greater incidence of charcoal rot. Periods of high humidity, frequent precipitation, and warm temperatures during pod development may favor PSD and PSS. Faced with unpredictable rainfall (too little or too much) on dry land farms, soybean genotypes with reduced susceptibility to seed infection by PSD, PSS, and by charcoal rot would be beneficial to farmers. By combining genes for different traits, plant breeders have developed new varieties and breeding lines to meet specific abiotic and biotic constraints (28,30). Since there are no known cultivars with resistance to these pathogens, it is important to compare the level of resistance among soybean genotypes developed by both public and private breeders. The objective of this study was to evaluate the reaction of 143 soybean genotypes in MG III, IV, and V to P. longicolla, C. kukuchii, and M. phaseolina. Identification of soybean genotypes resistant to seed-borne diseases will be valuable to soybean breeders in planning future selections.

Experimental Field Plots

Field plots were established from 2006 to 2008 at the West Tennessee Research and Education Center in Jackson, TN, to evaluate the reaction of 27 MG III, 30 early IV, 33 late IV, and 53 V genotypes to P. longicolla, C. kukuchii, and M. phaseolina. Plots were established in a no-till and non-irrigated field with soybean planted the previous three years, and soybean genotypes had developed symptoms of PSD, CRT, and PSS. In addition, the field was inoculated with charcoal rot-infected millet seed dropped with soybean seed in the furrow at a rate of 0.5 g per 30.5 cm of row. Such soil inoculation was necessary to minimize plot to plot variability. The soil was a Dexter fine-silty loam (mixed, active, thermicultic hapludalfs). The soybean genotypes were selected based on their commercial use, high soybean yield in state variety trials and good performance in breeding trials. Within each MG the experimental design was a randomized complete block with three replications. Among the 53 genotypes in MG V, five were plant introductions (PIs).

All seeds were treated with Apron Maxx + Moly (a.i. 0.8 ml/kg of seed) prior to planting. Planting dates were 13 May, 3 May, 3 and 7 May for 2006, 2007, and 2008, respectively. The experiments were conducted in the same area each year and each genotype was planted in four 6.1 m rows with an Almaco 4-row cone planter (Model #AJ4RP2). Weeds were controlled at pre-planting application of Roundup Weathermax, pre-emergent application of Canopy 75 DG, and post-emergence application of Reflex 2LC and Select 2E labeled herbicides.

Harvest dates for MG III genotypes were 21 September for 2006 and 2007, and 9 September for 2008. Harvest dates for early MG IV genotypes were 10 October, 11 October, and 9 September for 2006, 2007, and 2008, respectively. Harvest dates for late MG IV genotypes were 6 October in 2006 and 25 September in 2007 and 2008. Harvest dates for MG V soybeans were 10, 11, and 2 October in 2006, 2007, and 2008, respectively. Two center rows of each plot were harvested just after the R8 growth stage (6), when pods on all plants were mature. Samples of soybean seeds (500 g) from each plot were collected, dried under forced air at 32°C to 12% uniform seed moisture.

Environmental Data

Air temperature, precipitation (Fig. 2), soil temperatures, and water potential were measured during the growing season (Table 1). The water potential was determined with a Watermark soil moisture sensor and gypsum moisture block using a Watchdog Weather Station (Spectrum Technologies, Inc., Plainfield, IL). The soil water potential sensors were placed within rows at 15 cm depth and attached to a micro-logger. Sensors were placed in a representative area of the field to record soil water potential on a daily basis. Temperature sensors were also attached to a micro-logger to measure soil temperature at 5 and 10 cm depths.


Fig. 2. Total precipitation and the maximum air temperatures for each month of April through October for 2006 (A), 2007 (B), and 2008 (C) at Jackson, TN. A dashed line at 35°C across the graph compares the temperature differences between years.


Table 1. Mean monthly soil temperature at 5- and 10-cm depth and soil water potential for May through October in 2006-2008 at Jackson, TN.

Year Month Soil temperatures (°C) at depths Soil water

5.1 cm 10 cm
2006 May 23.4 21.5  −70.0
June 28.2 26.3  −76.0
July 30.0 28.0 −136.5
August 31.2 29.4 −145.8
September 25.9 25.0 −180.5
October 20.0 19.1 −153.7
2007 May 27.2 24.3  −80.5
June 30.0 27.1  −91.1
July 31.0 29.0 −136.1
August 33.2 30.7 −200.0
September 28.7 27.1 −111.0
October 22.9 22.0  −54.6
2008 May 24.5 22.1  −55.0
June 30.5 28.1  −81.0
July 32.2 29.9  −92.5
August 30.6 28.8 -163.8
September 27.4 26.0 -164.5
October 25.0 22.9 -137.7

Seed Assays and Resistance Measurements

Phomopsis seed decay susceptible checks were selected based on the highest level of infection detected for each MG. The percent seed infection index (PSII), a measure of relative resistance compared to the most susceptible check in each year, was calculated based on a prior study (25) by dividing the percent seed infection for each genotype averaged across replicates by the percent seed infection of the most susceptible genotype multiplied by 100. The index is used to standardize and compare cultivar reactions each year by removing the overall year-to-year variability in disease levels. The genotypes were categorized based upon their reaction: resistant (R) = 1 to 10%; moderately resistant (MR) = 11 to 20%; moderately susceptible (MS) = 21 to 30%; and susceptible (S) >30%. Since no known resistant soybean genotypes were available there were no standard checks for CRT or PSS were used. The resistance reaction to CRT and PSS were categorized as 0% = R, 1 to 3% = MR, 3 to 5% = MS, and >5% = S. Reaction of genotypes was determined based on the maximum infection value in any of the three years.

One hundred seeds from each replication were disinfested in 0.25% NaOCl for 60 sec, blotted and plated on acidified potato dextrose agar (APDA) (Difco Laboratories, Detroit, MI), and incubated for 7 days at 24°C. While M. phaseolina and C. kukuchii were easily identifiable when cultured on APDA, P. longicolla required further steps to confirm and validate its identity. Putative colonies of P. longicolla were verified by growing isolates on APDA. Identification was based on a single-monoconidial isolate from ten representative cultures using morphological and cultural characteristics (12,18,22,23). Each isolate was examined for sporulation, dimensions of conidia, pattern of stroma, and the presence or absence of beta-conidia and perithecia (2,12,18).

Data Analysis

All data were analyzed using Proc GLIMIX (SAS Procedures Guide, Version 8, 1999; SAS Institute Inc., Cary, NC). Mean comparisons were made using Fisher’s least significant difference test. Analysis of variance on seed infection by P. longicolla, C. kukuchii, and M. phaseolina indicated that there was a genotype by year interaction (P < 0.05). Therefore, data on seed infection for genotypes is presented separately for each year.

Environmental Conditions

The daily maximum air temperatures and precipitation in the 2006, 2007 and 2008 growing seasons at the Jackson, TN, location are shown in Figure 2. When comparing the August maximum air temperature for the three years, the average maximum air temperature for 2007 was 37°C and was 4°C higher than the temperatures for the corresponding month in 2006 and 6°C higher in 2008. The 37°C air temperature in August of 2007 was also the highest recorded for any month in all the three years. The soil temperatures of 33 and 30°C were also the highest recorded for the month of August in 2007 at 5 and 10 cm depths, and were 1 to 11°C and 1 to 10°C higher than in any other month during the three years, respectively (Table 1). This condition combined with the lowest water potential recorded in 2007 (-200 kPa) was conducive for charcoal rot infection and severity. On the other hand, the average precipitation for September and October in 2007 was 64% and 74% higher than the same months in 2006 and 2008.

Reaction of Soybean Genotypes to Seed-Borne Diseases

Phomopsis seed decay susceptible controls for each year and MG were used to compare percent seed infection among genotypes. The following genotypes were used as standard controls: The cultivars AG3705 had seed infection of 44%, NKS37N4 had 39%, and Armor39P7 had 33% in MG III; Excel8427 had seed infection of 13%, DC032033 had 28%, and Trisler4254 had 27% in early MG IV; Crows4817 had seed infection of 24% and Armor47G7 had 44% and 17% for late MG IV; Excel8509 had seed infection of 7%, DT9916864 had 80%, and DG5160 had 19% seed infection for MG V in 2006, 2007, and 2008, respectively. No controls were used for seed infection of PSS and CRT.

Maturity Group III. The mean PSII for the 27 MG III genotypes was 27, 42, and 47% in 2006, 2007, and 2008, respectively (Table 2). The mean PSII for 2007 and 2008 were 36% and 44% greater than for 2006. There was no genotype that was resistant across the three years. However, in 2006 and 2008, two genotypes, FFR3990 and Magellan, were rated as moderately resistant (MR). Croton was the only genotype considered MR across the three years with a mean PSII of 14%. Two genotypes in 2006, 27 in 2007, and six in 2008 had no PSS infection. Only AG3705 and FFR3990 had no PSS infection across the three years. For CRT seed infection 96, 48, and 74% of the genotypes had no infection in 2006, 2007, and 2008, respectively. Thirty seven percent of the genotypes had no seed infection of charcoal rot across the three years.

Table 2. Percent of seed infected with three seed-borne diseases of soybeans in MG III genotypes grown during the 2006-2008 season at Jackson, TN, in a non-irrigated and no-till environments.

Early Maturity Group IV. The mean PSII for the 30 genotypes was 36, 49, and 24% in 2006, 2007, and 2008, respectively (Table 3). There was no genotype that was immune to Phomopsis infection but only one genotype, DP3478 had a mean value of 9.8% PSII across the three years. Phomopsis infected 62, 82, and 41% of all genotypes in 2006, 2007, and 2008, respectively. The mean PSS seed infection was 7.0% for 2006, 0% for 2007, and 4.0% for 2008, respectively. Only one genotype GHH4534 had no seed infection from C. kukuchii across the three years. There was no seed infection by M. phaseolina in 2006, but the mean seed infection was 1.7 and 0.3% infection in 2007 and 2008, respectively. Overall, eight genotypes had no seed infection from M. phaseolina across the three years.

Table 3. Percent of seed infected with three seed-borne (PSII, Phomopsis seed infection index; PSS; PSS, purple seed stain; CRT, charcoal rot) diseases of soybeans in early MG IV genotypes grown in 2006-2008 in Jackson, TN, in a non-irrigated and no-till environments.

Late Maturity Group IV. All the genotypes in this MG were susceptible to P. longicolla. The mean PSII within the 33 genotypes was 29, 44, and 29% in 2006, 2007, and 2008, respectively (Table 4). However, there was no genotype that was moderately resistant across the three years. All genotypes in this MG were infected with C. kukuchi in 2006. There was no genotype that was resistant across all the three years. No seed infection was detected from M. phaseolina in 2006 but the average infection was 2.6 and 0.4% in 2007 and 2008, respectively. Seven of the 33 genotypes (21%) exhibited no seed infection from M. phaseolina across all three years.

Table 4. Percent of seed infected with three seed-borne diseases of soybeans in Late MG IV genotypes grown during the 2006-2008 growing seasons at Jackson, TN, in a non-irrigated and no-till environment.

Maturity Group V. The mean PSII within the 53 genotypes was 12, 36, and 6% in 2006, 2007, and 2008, respectively (Table 5). Forty-one and 39% of the genotypes had no Phomopsis seed infection in 2006 and 2008, respectively. However, none of the genotypes in 2007 were free of infection and only 3% had a MR reaction. PI587585B, RO1-581F, and RO1-769F had a MR reaction across three years with an average PSII of 14, 7, and 2%, respectively. Most genotypes in this MG were susceptible to PSS, but 38, 40, and 38% of them showed no detectable seed infection in 2006, 2007, and 2008, respectively. However, four genotypes, DC20300, DC7816, Stoddard, and Ozark, showed no infection across the three years. Infection by M. phaseolina was not detected in all the genotypes in 2006 but in 2007 and 2008, 57 and 42% of the genotypes were infected. Fourteen of the 53 genotypes (26%) in this MG did not have infection from M. phaseolina across the three years.

Table 5. Percent of seed infected with three seed-borne diseases of soybeans in MG V genotypes grown during 2006-2008 growing seasons at Jackson, TN, in a non-irrigated and no-till environments.

Discussion and Conclusion

The reason for greater infection of seed by P. longicolla in 2007 compared to 2006 was due to periods of uninterrupted rain at maturity making it impossible for harvest at maturity. Based on the relative resistance criterion using the PSII was compared to the most susceptible check in each year, the rainfall in September (160 mm) and October (228 mm) resulted in severe infection from P. longicolla. The average precipitation for September and October 2007 was 68 mm, which was 64 and 74% higher than the same period in 2006 and 2008. These two months coincided with the maturity dates for most of the late maturing genotypes, particularly MG Vs. Such environmental conditions are essential to measure the level of resistance in order to capture a high level of disease pressure. In the absence of such environmental conditions, it is suggested that screening and evaluation of soybean genotypes for P. longicolla seed infection be performed under irrigation and delayed harvesting (25).

The average temperature in 2007 dropped by 5.5°C from May to August, but continued to stay warm at an average temperature of 30°C, with high humidity September (Fig. 2) making it conducive for C. kukuchii infection of seed in MG V genotypes (Table 1). This agrees with earlier studies (15,31,32) that indicated favorable conditions during early pod development (R2 to R3) (6) may result in latent infections of pods (31,32) followed by the fungus producing the fungus producing cercosporin (19) causing the characteristic purple discoloration of infected seed (4). With the exception of one genotype in late IV, there was no seed infection from PSS in MGIII through late MG IV in 2007. Most of the MG III and IV genotypes have passed the susceptible growth stage during this period. The absence of PSS infection for these MGs may be associated with very low rainfall in July and August. Early drought in 2007 followed by continuous rains in September and October may have set up the conditions for symptom expression of PSS in MG V genotypes. The dry and high temperature environment in 2007 had elevated the soil temperature making it conducive for seed infection with CRT.

Protectant seed treatments may be one alternative management options for use during planting. However, infection of seed occurs at later growth stage of growth and is unprotected. The data on Phomopsis seed infection index (PSII), PSS, and CRT indicates the need for evaluating soybean genotypes over several years. Cultivar selection for resistance is one of the most important decisions that producers make to maximize productivity. However, decisions made about a cultivar for its disease performance in one year may not indicate its predicted performance in another year as demonstrated in this test. By comparing the level of resistance from a wide range of environments, growers may have the best opportunity of predicting the next year’s performance for a variety choice. The genotypes with low or no seed infection for each of the three diseases across the three-years may be considered as resistance sources to improve seed quality.

Acknowledgment and Disclaimer

The authors thank Debbie Boykin for help with data analysis and J. Deffenbaugh and J. Jordan for technical assistance. This research was funded by the USDA Agricultural Research Service, project number 6401-21220-002-00D; The Tennessee Soybean Promotion Board; the United Soybean Board; the North Central Soybean Research Program; and contribution no. 11-234-J from the Kansas Agricultural Experiment Station, Manhattan, KS.

Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture (USDA). USDA is an equal opportunity provider and employer.

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