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2009. Plant Management Network. This article is in the public domain.
Accepted for publication 25 April 2009. Published 15 June 2009.


Evaluation of Selected Genotypes of Soybean for Resistance to Phakopsora pachyrhizi


S. Li and L. D. Young, USDA-ARS, Crop Genetics and Production Research Unit, Stoneville, MS 38776


Corresponding author: S. Li. shuxian.li@ars.usda.gov


Li, S., and Young, L. D. 2009. Evaluation of selected genotypes of soybean for resistance to Phakopsora pachyrhizi. Online. Plant Health Progress doi:10.1094/PHP-2009-0615-01-RS.


Abstract

Soybean rust, caused by Phakopsora pachyrhizi Syd. & P. Syd., is one of the most destructive diseases of soybean [Glycine max (L.) Merr.] worldwide. To identify sources of resistance to domestic soybean rust fungus populations for plant breeding, our strategy has been to evaluate soybean lines that were previously identified as resistant to foreign isolates. In this study, two sets of plant introductions (PI) were evaluated using P. pachyrhizi urediniospores collected in Mississippi in 2006. The first set of PIs contained 10 lines previously identified as resistant in Paraguay, four PIs with known single genes for resistance to P. pachyrhizi, and Freedom and Williams 82 as susceptible checks. The second set included 17 lines that were selected based on information from Germplasm Resources Information Network (GRIN) and susceptible Williams 82. PI567102B was one of the most resistant lines to a Mississippi bulk isolate of P. pachyrhizi with the lowest severity rating, no sporulation, and red-brown lesion type.


Introduction

Soybean rust, caused by Phakopsora pachyrhizi Syd. & P. Syd., is one of the most destructive diseases of soybean [Glycine max (L.) Merr.] worldwide. Treatment with fungicide has been used as the first line of defense to minimize the impact of soybean rust. Although there are significant benefits, using fungicide for controlling soybean rust increases production costs, concern of the environmental contamination, and the risk of fungicide resistance development.

Breeding for resistance is a sustainable approach for long-term management and control of soybean rust (2,5) Since soybean rust was not present in the continental USA before 2004 (11), evaluation of United States soybean lines for resistance was conducted only with foreign isolates. It is important to identify resistance to domestic soybean rust fungus populations due to the pathogen races and the variability (1). In this study, two sets of soybean with 33 genotypes were evaluated using P. pachyrhizi urediniospores collected in Mississippi in 2006. The objective of this study was to evaluate selected soybean lines with resistance to a Mississippi bulk isolate. The overall goal was to identify soybean lines that are resistant to both foreign and domestic P. pachyrhizi populations for plant breeding purposes.


Pathogen Isolates and Maintenance

Urediniospores of P. pachyrhizi were collected from numerous pustules on field-collected kudzu [Pueraria lobata (Willd.) Ohwi] leaves on two sites within 1.5 km in Jefferson County, Mississippi in August, 2006. The soybean rust-causal pathogen was confirmed by microscopy, enzyme-linked immunosorbent assay and polymerase chain reactions as previously described (7). Urediniospores were increased on soybean cultivar Williams 82 in the Stoneville, MS Research Quarantine Facility.


Plant Materials

Two sets of plant introductions (PI) were evaluated. The first set of PIs contained 10 soybean genotypes previously identified as resistant in Paraguay (10), four PIs with known single genes for resistance to P. pachyrhizi (3,6) and Freedom and Williams 82 as susceptible checks (Table 1). The second set included 17 soybean genotypes selected based on P. pachyrhizi reaction data in Germplasm Resources Information Network (www.ars-grin.gov) and Williams 82 as susceptible check (Table 2).


Table 1. Set 1 soybean genotypes used for seedling assay, their maturity group, and previously reported reactions to other Phakopsora pachyrhizi isolates

Accession Maturity
group
Paraguay
(Capitán Miranda,
Itapúa,
greenhouse, 2006)
v
Paraguay
(Capitán Miranda,
Itapúa,
field, 2006)
w
FDWSRU
(Ft. Detrick, MD, 2004)x
severity
(%)
lesion
 typey
severity (%) lesion typey severity (1 to 5 scale) lesion typey
PI567099A IX 0.2 RB 1.8 RB 2.7 RB
PI567102B IX 0.2 RB 0.9 RB 3.0 RB
PI587880A VI 0.2 RB 0.2 RB 2.7 RB
PI587886 VI 0.2 RB 0.0 IMM 3.3 RB
PI594723 IX 0.2 RB  N/Az  N/A 3.3 RB
PI594754 IX 0.2 RB 0.0 IMM 2.5 MIX
PI594760B IX 0.2 RB 2.6 RB 3.7 RB
PI594767A IX 0.2 RB 2.4 RB 3.2 RB
PI605779E VIII 0.2 RB 0.9 RB 3.5 MIX
PI605833 IX 0.2 RB 0.1 RB 3.0 RB
PI200492 (Rpp1) VII N/A  N/A 2.2 RB 3.1 TAN
PI230970 (Rpp2) VII  N/A  N/A 3.8 RB 3.0 RB
PI462312 (Rpp3) VIII  N/A  N/A  N/A  N/A  N/A  N/A
PI459025B (Rpp4) VIII  N/A  N/A 4.1 RB 3.5 RB
Freedom V  N/A N/A  N/A N/A N/A N/A
Williams 82 III  N/A  N/A  N/A  N/A N/A N/A

 v Mean soybean rust severity used 1 to 9 scale and data were converted to percentages using the midpoint for each range (10).

 w Maturity group VI and VII plants were evaluated at 106 and 129 days after planting (DAP), respectively. Maturity group VIII and IX plants were evaluated at 129 and 130 DAP, respectively (10).

 x Tested (P2 evaluation) at the USDA-ARS, Foreign Disease-Weed Science Research Unit, MD. The first 10 plant introductions (PIs) were evaluated with four isolates: Brazil 01-1, Paraguay 01-2, Thailand 01-1, and Zimbabwe 01-1 (8), and the four PIs with known resistance genes were evaluated with six isolates: Brazil 01-1, Paraguay 01-2, Thailand 01-1, TW72-1, TW80-2, and Zimbabwe 01-1 (9).

 y TAN = tan lesion; RB = red-brown lesion; MIX = a mixture of TAN and RB lesion types on the same leaf or different plants; IMM = immune or no visible rust lesions;

 z Not available.


Table 2. Set 2 soybean genotypes used for seedling assay, their maturity group, and previously reported reactions to other Phakopsora pachyrhizi isolates.

Accession Maturity
group
Paraguay
(Capitán Miranda, Itapúa,
field, 2006)
w
FDWSRU
(Ft. Detrick, MD,
2004)
x
severity
(%)
lesion typey severity
(1 to 5 scale)
lesion typey
PI417560 0  N/Az N/A 3 TAN
PI437151 II N/A N/A 2.8 MIX
PI398399 V N/A N/A 2.5 TAN
PI605854A V N/A N/A 2.7 MIX
PI594172A VII 1.8 TAN 2.7 RB
PI417012 I N/A N/A 3 MIX
PI548519 I N/A N/A N/A N/A
PI437437A II N/A N/A 1.5 RB
PI561377 II N/A N/A 2 TAN
PI68806 III 2.2 TAN 2 MIX
PI437663 II N/A N/A 1.5 RB
PI547878 III 4.9 TAN 2 MIX
PI567718 IV N/A N/A 2 MIX
PI567522A IV 1.8 TAN 3 MIX
PI423823 V 0.7 TAN 2 MIX
PI407730 III N/A N/A 1.5 RB
PI407859-1 V 0.8 TAN 2 MIX
Williams 82 III N/A N/A N/A N/A

 w Maturity group III and IV plants were evaluated at 100 days after planting (DAP), MG V and MG VII plants were evaluated at 93 and 129 DAP, respectively (10).

 x Tested (P2 evaluation) with four isolates: Brazil 01-1, Paraguay 01-2, Thailand 01-1, and Zimbabwe 01-1 at the USDA-ARS, Foreign Disease-Weed Science Research Unit, MD (8).

 y TAN = tan lesion; RB = red-brown lesion; MIX = a mixture of TAN and RB lesion types on the same leaf or different plants.

 z Not available.


Seed of soybean lines were sown in Jiffy Poly-Pak Pot (Hummert, St. Louis, MO) in (27 × 52 cm) flats that contained 5 × 10 pots. Sun Grow Metro Mix 360 soil (Sun Grow Horticulture Products, Belleview, WA) was used. Each pot contained one seed. Flats were placed in a Conviron growth chamber (Controlled Environments Inc., Pembina, ND) under a 16-h photoperiod with a light intensity of 433 mmol/m²/sec at 25 ± 2°C and watered daily.


Inoculation of Plants

Inoculation was performed on 21-day-old seedlings. Inocula were prepared using freshly collected urediniospores from Williams 82. Spore suspensions in sterile distilled water containing 0.01% Tween-20 (vol/vol) were mixed and filtered through a 100-μm cell strainer (BD Biosciences, Bedford, MA) to remove any debris and clumps of urediniospores. Urediniospores were quantified using a hemocytometer and diluted to a final concentration of 40,000 per mL. Inoculation was at the rate of 1 mL of spore suspension per plant and applied with Preval sprayer (Precision Valve Co., Yonkers, NY). After inoculation, plants were placed in a dew chamber in the dark at 22°C overnight (approximately 16 h) and then moved to Conviron growth chambers where temperatures were maintained at 23°C during the day and 20°C at night under a 16-h photoperiod with a light intensity of 280 mmol/m²/sec.

Experiments to evaluate 33 genotypes were conducted separately due to space limitation in the quarantine facility and the availability of the seed. Each experiment was a randomized complete block design consisting of 16 or 18 genotypes and three replications. Each experimental unit was five plants of each genotype, and each experiment was performed three times. The tests for Set 1 genotypes were performed from September 2006 through July 2007, and Set 2 genotypes were evaluated from November 2007 through June 2008.


Disease Severity, Lesion Types, and Sporulation Assessment

Disease severity, lesion types, and sporulation were assayed at 14 d after inoculation (DAI) on the first trifoliate leaves. A five-point disease severity scale based on lesion density was used (8) with modification of the percentage of infected area, in which 1 = no visible lesions, 2 = a few lesions (1-20% infected area), 3 = a moderate lesion density (21-50% infected area), 4 = a heavy lesion density (51-80% infected area), and 5 = a very heavy lesion density over most of the leaf (81-100% infected area). Lesion types were "TAN," "RB," "MIX," or "IMM" reactions as previously described (1,3) (Fig. 1). TAN lesions were tan in color, RB referred to reddish brown lesion color, MIX reaction was a mixture of TAN and RB lesions on the same leaf or different plants, and IMM was for an immune reaction and lack of obvious symptoms. Sporulation was based on the relative percentage of lesions producing urediniospores on each plant using a 1-to-5 scale where 1 = no sporulation, 2 = less than 25%, 3 = 26 to 50%, 4 = 51-75%, and 5 = 76 to 100% of the lesions sporulating (9).


 
A

 

B
 
C

Fig. 1. Soybean lesion types for a Mississippi bulk isolate of Phakopsora pachyrhizi: (A) tan lesions with profuse sporulation in Williams 82; (B) red-brown lesions in PI459025B; and (C) immune reaction on PI200492 (Rpp1). Upper photos: adaxial view of leaf pictures. Lower photos: abaxial view of leaf micrographs under the Olympus SZX12 dissecting microscope.


Since there were no significant (P ≤ 0.05) line by trial interactions, data from the three tests of each genotype set were pooled for analysis. Analysis of variance was conducted using general linear mixed model procedure (PROC MIXED) of SAS (version 9.1, SAS Institute Inc., Cary, NC). Means were compared with Fisher’s protected least significant difference (LSD) at P ≤ 0.05 unless otherwise stated.


Evaluation of Set 1 Genotypes

Soybean rust severity differed significantly (P ≤ 0.05) by genotype with range of severity from 1.0 to 4.8. No soybean rust lesions were found on PI200492 (Rpp1). Susceptible genotypes Freedom and Williams 82 had the greatest severity (Fig. 2A). Among 10 resistant lines identified in Paraguay, PI567102B had the lowest severity of 1.3 (Fig.2A). PI567099A, PI594723, PI594760B, PI594767A, PI605779E, PI462312 (Rpp3) and PI459025B (Rpp4) had severity < 3.


 

Fig. 2. Mean soybean rust severity (A) and sporulation rating (B) of the Set 1genotypes evaluated against a Mississippi bulk isolate of Phakopsora pachyrhizi. Reactions were immune or no visible rust lesions (IMM) on PI200492; red-brown (RB) lesions (horizontal striped bar); tan (TAN) lesions (solid gray bar); or MIX with a mixture of TAN and RB lesion types on the same leaf or different plants (diagonal striped bar). Means with the same letter are not significantly different by the least significant difference test (P = 0.05).

 

Soybean rust sporulation also differed significantly (P ≤ 0.05) among soybean lines with range from 1.0 to 4.8. PI200492 (Rpp1) had IMM reaction to soybean rust at 14 DAI (Fig. 2B). PI567102B had RB reactions, and none of the lesions sporulated. PI230970 (Rpp2), PI462312 (Rpp3), and PI459025B (Rpp4) also had the RB reaction with sporulation (Fig. 2B). PI605779 had TAN reaction with a sporulation rating of only 2.6 (Fig. 2B).


Evaluation of Set 2 Genotypes

Soybean rust severity for the Set 2 genotypes differed significantly (P ≤ 0.05) by genotypes with range from 2.7 to 4.9. PI407730 had the lowest severity value at 2.7, followed by the PI437663 with severity value of 2.9 (Fig. 3A).

Soybean rust sporulation also differed significantly (P ≤ 0.05) among soybean lines with range from 2.6 to 5.0. PI561377, PI068806, and PI407730 had RB reaction; PI417560, PI437151, and PI398399 had MIX reaction; and 12 genotypes had TAN reactions (Fig. 3B).


 

Fig. 3. Mean soybean rust severity (A) and sporulation rating (B) of the Set 2 genotypes evaluated against a Mississippi bulk isolate of Phakopsora pachyrhizi. Lesion types were red-brown (RB) lesions (horizontal striped bar); tan (TAN) colored lesions (solid gray bar); and a mixture of TAN and RB lesion types on the same leaf or different plants (diagonal striped bar). Means with the same letter are not significantly different by the least significant difference test (P = 0.05).

 

Discussion and Summary

Due to space limitations in the quarantine facility at Stoneville, our strategy to identify soybean resistant to domestic P. pachyrhizi populations was to evaluate soybean lines with some levels of resistance in previous tests with foreign isolates. Results from this study indicated that soybean lines that are resistant to foreign isolates could either be resistant or susceptible to US isolates. It is necessary to screen soybean lines for resistance with US isolates.

The MIX lesion type for some entries may indicate the heterogeneity of the bulk isolate of P. pachyrhizi collected in Mississippi in 2006. PI567102B, one of the partially resistant lines identified in Paraguay, was also one of the most resistant lines to the Mississippi bulk isolate. It had RB reaction, the lowest severity rating, and no sporulation. A breeding population with PI567102B as one of the parents has been developed and collaborative research with soybean breeders is underway to evaluate the segregation of this population for mapping of resistance genes.

Our disease assessments indicated that multiple components of resistance were expressed among soybean genotypes, perhaps including longer incubation and latent periods, fewer lesions per leaf, smaller lesion diameter, lower sporulation index, and lesser leaf area damage. Chakraborty et al. (4) reported mapping of QTL for resistance to soybean rust using a population derived from a PI with a low number of TAN lesions previously described as a susceptible reaction. In this study, PI567099A, PI594723, PI594760B, and PI594767A, had TAN reactions, but their disease severity values were less than 3.0. Since the ability of P. pachyrhizi to overcome single-gene qualitative resistance has been reported (5), development of durable ‘‘less-rusting” and ‘‘slow-rusting” cultivars is one of the options for breeding for resistance to soybean rust.


Acknowledgments

We would like to thank D. Walker for sharing his unpublished data; R. Nelson for providing the seed; J. Ray and R. Smith for developing soybean populations using resistant lines identified in this study for further genetics and mapping studies; G. Hartman, T. Muller, and X. B. Yang for valuable discussion about the screening methods; D. Boykin for assistance in data analysis; and A. Clark, M. Porter, S. Parker, and E. Montgomery for assistance in planting and maintaining plants for experiments.

Trade and manufacturers names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of products to the exclusion of others that may also be suitable.


Literature Cited

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