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© 2013 Plant Management Network.
Accepted for publication 20 January 2013. Published 18 March 2013.

Identification of Soybean Accessions Resistant to Macrophomina phaseolina by Field Screening and Laboratory Validation

Alemu Mengistu and Prakash Arelli, USDA-ARS, Crop Genetics Research Unit, Jackson, TN 38301; Jason Bond, Southern Illinois University, Carbondale, IL 62901; Randall Nelson, USDA-ARS, Soybean/Maize Germplasm, Pathology, and Genetics Research Unit, Urbana, IL 61801; John Rupe, University of Arkansas, Fayetteville, AR 72701; Grover Shannon and Allen Wrather, University of Missouri, Portageville, MO 63873

Corresponding author: Alemu Mengistu.

Mengistu, A., Bond, J., Nelson, R., Rupe, J., Shannon, G., Arelli, P., and Wrather, A. 2013. Identification of soybean accessions resistant to Macrophomina phaseolina by field screening and laboratory validation. Online. Plant Health Progress doi:10.1094/PHP-2013-0318-01-RS.


Charcoal rot of soybean, caused by Macrophomina phaseolina (Tassi) Goidanich, has been a problem for soybean farmers in the United States for many years. However, recently its incidence and severity has increased in midwestern and north-central states. Most management strategies, including chemical and biological methods and other cultural options, have been ineffective in controlling this disease. Furthermore, soybean is often planted every other year or in monoculture. Although genetic resistance would be the most sustainable control strategy, resistant commercial soybean cultivars are presently unavailable. The objective of this test was to identify resistant accessions from the USDA soybean germplasm collection in Maturity Groups (MG) 00 to VII by field screening in five environments. A total of 628 accessions were evaluated using established methods for reaction to this disease in Missouri and Illinois (2008 to 2009), and in Tennessee (2009). In 2010, accessions with disease severity reactions of 1 to ≤ 2 were selected for further evaluation in Tennessee. Among the 45 accessions identified as resistant and moderately resistant (1 to ≤ 2), ten were further assessed using colony forming units recovered from ground root and stem tissues which was then converted to colony forming unit index (CFUI) and validated their resistance using this bioassay. All ten had CFUI levels ranging from 0 to 18.2, which were significantly lower (P ≤ 0.05) than the moderately resistant accession, DT97-4290, with 31.8. Four selected accessions, PI594302 (MG VII), PI567562A (MG IV), PI506764 (MG VII), and PI567334 (MG VI), had significantly lower (P ≤ 0.05) CFUI of 0, 0.8, 0.8, and 1.5, respectively. This is the first report of soybean accessions with better levels of resistance than the standard released germplasm, DT97-4290. These accessions can serve as additional sources of charcoal rot resistance in future breeding programs.


Charcoal rot (CR) of soybean (Glycine max L. Merr.) is a common disease in many countries of the world. The pathogen that causes CR, Macrophomina phaseolina (Tassi) Goidanich (MP), has the capability to attack a wide range of plants, including economic crop plants such as corn (Zea mays L.), cotton (Gossypium hirsutum L.), and soybean (21). Charcoal rot suppresses soybean yield in stress environments and was ranked sixth among economically important diseases for the top eight producing countries in 2006 (20). The disease ranked fourth among economically important soybean diseases in the United States from 1996 to 2007 (19) and was exceeded only by soybean cyst nematode (Heterodera glycines Ichinoche), Phytophthora root and stem rot (Phytophthora sojae), and seedling diseases (including Pythium spp., Rhizoctonia solani Kuhn, P. sojae, and Fusarium spp.) (19).

Soybean can be infected by M. phaseolina (MP) soon after emergence, but symptoms of the disease are most often observed in the upper tap root and lower stem at the R7 to R8 growth stages (10). In the US, the fungus rarely kills seedlings, but high seedling mortality has been reported in the tropics (15). Symptoms of CR are usually greater on plants stressed by high heat and drought, especially when drought occurs during the reproductive stages of growth (Fig. 1A). According to Mengistu et al. (10), the combination of stress and the presence of MP caused much higher yield loss on soybeans than drought alone. It is not known whether this loss is due to synergistic or additive effects. However, recent research (10) found that yield loss due to CR ranged from 6 to 33% in irrigated environments, suggesting that it can be an important disease even in an environment where there is adequate moisture. This result is in agreement with a previous report that showed that even though CR severity was reduced under irrigation it did still has damaging effect (7). There was a total yield reduction when CFU levels in root and stem tissues were high under non-irrigated conditions (10).

Fig. 1. Severely infected soybean with Macrophomina phaseolina showing: (A) above-ground symptoms; (B) evaluation systems used to identify resistance based on root and stem severity on the scale 1 to 5, where 1 = no microsclerotia visible in tissue and 5 = vascular tissue darkened due to high numbers of microsclerotia both inside and outside of the stem and root tissues; and (C) colony forming unit of M. phaseolina on semi-selective media. Figs. 1A and 1C were adopted from Mengistu, et al. (11) and Mengistu et al. (8), respectively.

Cultural methods, such as reduced plant populations and use of cultivar blends, are somewhat useful to reduce damage avoiding mid- to late-season soybean drought stress (1). Tillage has also been reported to impact soil population density of MP (17). In an attempt to reduce the soil population of MP below damaging levels, Francl found that a 3-year rotation with cotton caused the soil population density of MP to decline (6). Mueller et al. (12) found that a 3-year rotation with corn reduced soybean root colonization by MP. There are contradictory reports on the effects of planting date on population density of MP. Wrather et al. (18) reported that planting date did not affect the soil population density of M. phaseolina; however, Smith and Carvil (16) determined that the population density of MP in soybean root and lower stem tissue at the R7 growth stage was greater for a June than May planting, but plants from the May planting were still infected. Mengistu et al. (9) estimated MP population density using CFU recovered from infected stem and root tissue and concluded that it provided a better estimate of population density of MP on the effects of tillage and cover crops than estimates made from soil. Additional studies found that the population density of M. phaseolina increased slowly during the season from the V5 to R6 growth stages and then rapidly from the R6 to R7 growth stages, and suggested that R7 is the critical time for evaluation of cultivars for resistance (10). Recently, Paris et al. (13) released a MG V genotype rated as moderately resistant; additional MG V genotypes with moderate resistance have been identified by Mengistu et al. (11).

Most of the previous evaluations for CR resistance were focused on soybean accessions adapted for production in the mid-southern US and did not include early maturity groups (MG) adapted for the northern part of the US. Charcoal rot has been reported in Wisconsin (2), North Dakota (3), Iowa (22), and Minnesota (5), reflecting an expansion in the range of MP with a record of epidemics of the disease at several locations bringing the problem to the forefront of soybean production in the Upper Midwest (3). Since no reliable greenhouse technique that provides comparable results to that of field tests is yet available, screening of accessions through field testing remains the most viable option. The objective of this study was to identify soybean accessions resistant to MP in MG 00 to VII based on field screening in five environments, with field and laboratory confirmation of selected accessions in Tennessee.

Experimental Field Plots

Non-irrigated field trials were established at the University of Missouri-Delta Center near Portageville, MO, and at Southern Illinois University near Carbondale, IL, in 2008 and 2009, as well as at the USDA-ARS facility at the University of Tennessee, West Tennessee Research and Education Center in Jackson in 2009 and 2010. The soil at the Missouri site is classified as Dundee silt loam (fine-silty, mixed, active, thermic Typic Endoaqualfs). The soil at the Illinois site is classified as Meadowbank silt loam (fine-silty, mixed, superactive, mesic Typic Argiudolls), while the soil at the Tennessee site is classified as a Dexter fine-silty loam (mixed, active, thermicultic hapludalfs). The plots in Missouri and Illinois were rotated with grain sorghum (Sorghum bicolor L. Moench), whereas plots at the Tennessee site were established in a field previously planted to soybean in a no-till cropping system. Both of these cropping systems were used to enhance the population density of natural inoculum of MP. Prior to planting each year in Missouri and Illinois, fields were disked twice, and 75- to 96-cm row beds were formed. The top 10 cm of the beds were pushed off just prior to planting to form a flat-top ridge. The herbicides imazaquin at 0.02 kg a.i./ha (Scepter 70 DG, BASF) and alachlor at 0.4 kg a.i./ha (Intrro, Monsanto) were pre-plant incorporated into the flat-top ridges.

A total of 628 soybean accessions from the USDA soybean germplasm collection (Table 1) including three controls (Croton and Pharaoh, susceptible MG III and V, respectively, and DT97-4290, moderate resistant, MG V) were tested for their reaction to natural inoculum of MP in Missouri, Illinois, and Tennessee. No known resistant soybean accessions or cultivars were available as controls for other MGs. The accessions originated from China, Japan, North and South Korea, Nepal, Russia, United States, Germany, Romania, Hungary, Slovakia, Zambia, and Morocco, with the largest number from China. Also, accessions in this test were selected for relatively high seed yield and differences among phenotypic descriptors available in the Germplasm Resource Information Network ( The MG of these accessions ranged from 00 to VII with 3 accessions in MG 00, 10 in MG 0, 36 in MG I, 69 in MG II, 308 in MG III, 134 in MG IV, 54 in MG V, 11 in MG VI, and 3 in MG VII (Table 1). Nearly 50% of the accessions tested were in MG III (Fig. 2). Planting dates for all the tests were 10 to 20 May at Missouri, 20 May to 1 June at Illinois, and 5 to 20 May at Tennessee. The test sites were treated with a post-emergence directed application of bentazon at 0.56 kg a.i./ha (Basagran 4 SC, Arysta). Plots were non-irrigated, cultivated once, and hand-weeded each year, as necessary.

Table 1. Reactions of soybean genotypes in MG 00 to VII to Macrophomina phaseolina evaluated in field studies at Missouri, Illinois, and Tennessee during 2008 to 2009.


Fig. 2. The number of soybean accessions evaluated within each maturity group in one to five environments in Missouri, Illinois, and Tennessee in 2008 through 2009.


Ten seeds of each accession were planted by hand in hills in the center of a ridge. Hills were spaced 0.6 m apart, and plots were arranged in a randomized complete block design with two replications. Each accession was planted in one to five environments with some accessions planted in 2, 3, 4, and 5 environments. Only accessions planted in 3 to 5 environments from 2008 to 2009 with disease severity values of 1 to ≤ 2 were further evaluated in 2010 in Tennessee to validate their resistance using colony forming units (CFU). In 2010, each accession was planted in single 3.1-m-long row in a randomized complete block design with three replications. The same CR susceptible and resistant controls were used as mentioned above.

Disease Ratings for Evaluation of Resistance

Reaction of the 628 accessions to MP was evaluated at the R7 (4) growth stage using the root and stem severity rating (RSS) (8) in 2008 and 2009. Five to ten plants were gently uprooted. Stem and taproots of each plant were split longitudinally (Fig. 1B) and the severity of discoloration was rated on scale of 1 to 5; where 1 = no discoloration and 5 = highly discolored). The scale for RSS was divided into four classifications: resistant (values of 1), moderately resistant (values > 1 and ≤ 2), moderately susceptible (values > 2 and < 3), and susceptible (values 3 to 5), as described by Mengistu et al. (8).

Accessions selected as R and MR in 2008 and 2009 were further evaluated in the field in 2010 in Tennessee and only accessions that had rating between 1 to ≤ 2 were processed for CFU using the protocols described by Mengistu et al. (8). In the field, all plant samples within a row were carefully uprooted and separated into two groups with those exhibiting symptoms and non-symptomatic plants for determination of CFU (Fig. 1C). The lower stem sections and roots including lateral and fibrous roots of non-symptomatic plants were excised just below the cotyledonary node. These samples were thoroughly washed and rinsed in water to remove soil and air dried. The combined root and stem sections from each plot were ground with a Wiley Laboratory Mill 50/60HZ, single phase, 1HP (Model 4, 3375-E15, Thomas Scientific, Swedesboro, NJ) and passed through a 28-mesh screen (600-μm openings). The mill was thoroughly cleaned between samples with air using a suction device. For each sample, 0.005 g of ground tissue was placed in a Warring blender with 100 ml of 0.525% NaOCl mixed for 3 min and then collected over a 45-μm pore-size sieve. The triturated tissue was washed with sterile distilled water and then added to 100 ml of autoclaved Acidified Potato Dextrose Agar (Difco Laboratories, Detroit, MI) amended with rifampicin (100 mg/liter) and tergitol (0.1 ml/liter) that had been cooled to 60°C and poured 20 ml into each petri dish (11). After 3 days of incubation at 30°C, MP CFUs were counted and converted to CFU per gram of root and stem tissues. Using protocols proposed by Mengistu et al. (8), Colony Forming Init Index was calculated by dividing the CFU for each selected accession by the accession with the highest CFU (Croton).

Statistical Analysis

Analysis of variance using SAS 9.3 (SAS Institute Inc., Cary, NC) was performed on disease severity of 628 accessions in 9 MGs planted in 5 environments with each accession planted in at least one environment and some planted in 2, 3, 4, and 5 or combination of environments. The data were combined across all environments because of uneven data sets and analysis of variance was performed using general linear mixed model with environment, and environment * accessions, and MG as random effects. Maturity group and accessions within MG were considered fixed effect. Least square means were obtained from the analysis of variance described above. Colony forming unit data were log10 transformed, since there were zero values in the CFU data. The data was back transformed after analysis and CFUI was calculated.

Environmental Data

Air temperature and precipitation data for the growing season were obtained from the 5 environments (Fig. 3. A-E). Crop water deficits, resulted from hot and dry conditions during the growing season generally developed in June, July, and August. According to Mengistu et al. (11), these are critical months for MP infection. During these three months in 2009, there were at least 5 days that temperatures exceeded 35°C in Illinois, 7 days in Tennessee, and 7 days in Missouri. The corresponding total precipitation was 32, 36, and 15 cm of rain in Illinois, Tennessee, and Missouri, respectively, for the same months. During the growing season in 2008, temperature in Illinois was 26°C, lower by 4°C than in 2009. In Missouri, the temperature was 31°C in 2008. Precipitation from June through August was 10 and 6 cm for Illinois and Missouri, respectively. In 2010, there were 16 days when temperature exceeded 35°C and precipitation was 46 cm during the months of June, July, and August in Tennessee. The temperatures were 31, 31, and 32°C for Illinois, Missouri, and Tennessee, respectively, and remained approximately the same as the 30-year average ( Thus, the environmental conditions in all locations were favorable for MP infection.


Fig. 3. Total precipitation (cm) and maximum air temperature (°C) shown for the months of April through October for Missouri in 2008 (A) and 2009 (B); for Illinois in 2008 (C) and 2009 (D), and for Tennessee in 2009 (E) and 2010 (F). Disease severity rating on a scale of 1 to 5 was recorded in the five environments (A-E). The CFU determination was made in 2010 (Fig 1 F). A dashed line at 35°C across the graph compares the temperature differences among years.

Identifying Soybean Accessions with Charcoal Rot Resistance

Ninety-three percent of the accessions evaluated showed moderately susceptible to susceptible reactions (Table 1). The least square means showed that no R or MR accessions were present in MG 00, but there was 1 in MG 0, 4 in MG I, 3 in MG II, 13 in MG III, 7 in MG IV, 9 in MG V, 5 in MG VI, and 3 in MG VII for a total of 45 accession with R or MR reactions in 1 to 5 environments. This data indicates that a majority of these accessions were susceptible to CR. Ten of the 45 R and MR accessions that were tested in 3 to 5 environments were further evaluated in Tennessee (Table 2). These accessions had RSS values between 1 to ≤ 2. This classification was used in separating differences between resistant and susceptible accessions (8, 13). These ten accessions also had significantly lower CFUI (P ≤ 0.05), ranging from 0 to 18.2, than the MR control DT97-4290, with 31.8. The two susceptible controls, Croton and Pharaoh, had CFUI of 100 and 77.3, respectively. The four top accessions, PI594302 (MG VII), PI567562A (MG IV), PI506764 (MG VII), and PI567334 (MG VI) had significantly lower (P ≤ 0.05) CFU of 0, 0.8, 0.8, and 1.5, respectively (Table 2). The fact that CFUs were detected in non-symptomatic accessions (severity of 1) indicates that absence of visual vascular discoloration symptoms does not necessarily mean absence of infection. This result is consistent with a previous report that indicated that CFUs provide a more precise measurement for determining resistance (8). Variation in the rate of colonization by MP among soybean genotypes has been reported in several studies (7,11,14,16). Differences in disease severity may be due to varying levels of soil inoculums, variation in environmental condition, or differences in aggressiveness of MP pathotypes at different locations.

Table 2. Soybean accessions in MG II through MG VII evaluated at Jackson, TN, in 2010 with total number of plants evaluated and percent plants with resistance and the corresponding CFUIv.

MG Accessions Original
of plants
% of
plants with
VII PI594302 Fukuoka,
39 1.6 73 0.0 G
IV PI567562A Shandong,
38 1.0 92 0.8 G
VII PI506764 Kyushu,
27 1.6 65 0.8 G
VI PI567334 Gansu,
33 1.5 50 1.5 G
V PI567335B Gansu,
29 1.0 71 2.3 FG
V PI567343 Gansu,
23 1.4 74 2.3 EFG
V PI567349A Gansu,
44 1.2 71 4.5 EF
V PI567303B Gansu,
28 1.1 68 5.3 E
II PI567429A Shanxi,
50 1.4 70 16.8 D
VI PI221717 Transvaal,
South Africa
27 1.4 55 18.2 D
V DT97-4290w Germplasm
release, USA
45 2.4 60 31.8 C
V Pharaohx Cultivar,
50 3.1 0 77.3 B
III Crotonx Cultivar,
38 2.8 0 100.0 A
LSD (P ≤ 0.05) 2.8

 v CFUI (colony forming unit Index/g of ground stem and root tissues) is obtained by dividing the CFU of each accession by the CFU for the accession with the highest CFU (Croton). The CFUI values followed by the same letter are not significantly different from each other at P ≤ 0.05.

 w DT97-4290 is a moderately resistant genotype check for MG V.

 x Croton and Pharaoh are susceptible checks for MG III and V, respectively.

 y Disease severity was based on root and stem severity rating of 1 = resistant and 5 = susceptible. Mean comparisons are based on least significant difference values.

 z Percent of plants with resistance refers to plants that did not show visual vascular discoloration and no presence of sclerotia.

The ten R and MR accessions were tested in a non-irrigated environments, where both MP and environmental stress were present, yet no accession produced symptoms of wilting or incomplete pod filling (data not shown), which are typically observed under hot and dry soil conditions (15). This suggests that these accessions may carry traits for both drought tolerance and CR resistance. In addition, the ten accessions tested represent a wide range of maturity groups, from early (MG IV) to late types (MG VII), which will increase their utility to soybean breeders across a wide geographical area in the US. However, more accessions in MG 00 through II need to be screened to identify CR resistant accessions for use in Midwestern and Northern states. Thirty-five of the 45 accessions with MR reactions may have value and need to be further tested in multiple environments. The diversity of sources for the ten resistant accessions makes it more likely that the genetic control of resistance will be different among these accessions, making them useful in developing soybean cultivars with improved resistance to MP.


The authors thank Mrs. Debbie Boykin, USDA-ARS Mid-South Area Statistician, for assistance in data analysis. This study was supported in part by the University of Missouri Agriculture Experiment Station and Southern Illinois University. The authors thank the United Soybean Board and the Tennessee Soybean Promotion Council for the financial support from soybean check-off dollars. This research was also funded by United States Department of Agriculture, Agricultural Research Service project number 6401-21000-002-00D. We wish to thank Jason Deffenbaugh, Jamie Jordan, Tara Sydboten, Joyce Elrod, Cory Cross, and Kent Fothergill for their efforts in this project.


The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.

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