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2006. Plant Management Network. This article is in the public domain.
Accepted for publication 9 November 2005. Published 4 January 2006.

Evaluation of Soybean Germplasm for Resistance to Phakopsora pachyrhizi

M. R. Miles, USDA-ARS, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, IL 61801; R. D. Frederick, USDA-ARS, Foreign Disease-Weed Science Research Unit (FDWSRU), Ft. Detrick, MD 21702; and G. L. Hartman, USDA-ARS and the Department of Crop Sciences, National Soybean Research Center, 1101 W. Peabody Drive, University of Illinois, Urbana 61801

Corresponding author: G. L. Hartman. ghartman@uiuc.edu

Miles, M. R., Frederick, R. D., and Hartman, G. L. 2006. Evaluation of soybean germplasm for resistance to Phakopsora pachyrhizi. Online. Plant Health Progress doi:10.1094/PHP-2006-0104-01-RS.

Abstract

Phakopsora pachyrhizi, the causal fungus of soybean rust, was discovered in the continental U.S. in November 2004. The presence of this disease in the U.S. may have an impact on soybean (Glycine max) production, as the current commercial varieties are considered to be susceptible, and the use of one or more applications of fungicides will add additional costs to production. One objective of the USDA-ARS research on soybean rust is to identify soybean germplasm with resistance to the disease. There are over 16,000 soybean accessions in the USDA Germplasm Collection located at the University of Illinois. These accessions were evaluated in a two-tiered inoculation program using a mixture of four P. pachyrhizi isolates in Biosafety Level 3 containment greenhouses the FDWSRU. In the first round of evaluations, 16,595 accessions were rated for rust severity. Of these, 3,215 accessions, based on low visual rust severity or the presence of a red-brown reaction, were selected for a second round of evaluation. After the second round of replicated evaluations of the 3,215 accessions, 805 were selected for further evaluation, again based on low mean visual severity or the presence of a red-brown reaction. Some of these selected accessions have the potential to provide soybean rust resistance genes that may be useful for incorporation into commercial soybean cultivars.

Introduction

Soybean rust, Phakopsora pachyrhizi, may drastically reduce yields and/or increase production costs for U.S. producers. Yield losses have been significant in some countries in Africa, Asia, and South America (6,12,15). The risk to U.S. soybean producers was reviewed in 2003 (9), and now has become more imminent since the first report of soybean rust in the continental U.S. occurred in November, 2004 (11).

Specific resistance to P. pachyrhizi is known. Four single dominant genes identified as Rpp₁, Rpp₂, Rpp₃, and Rpp₄ have been reported, and a recent symposium review cites the original sources of the references for these named genes (4). These four genes condition resistance to a limited set of P. pachyrhizi isolates. There are three reactions associated with soybean rust (1). The two resistant reactions include an "immune" (no visible signs or symptoms) and a red-brown reaction (RB). The susceptible reaction is tan (TAN) (Fig. 1). The immune reaction, where no visible symptoms are observed, has only been reported with Rpp₁ when inoculated with certain isolates, including India 73-1 (1). These single gene sources have not been durable when used in commercial cultivars and are not effective when challenged with additional isolates of P. pachyrhizi (3).

Fig. 1. Soybean rust reacton types: TAN = fully susceptible reaction, RB = resistant reddish brown lesions with defined margins, and immune = no visible symptoms (1).

Partial resistance to P. pachyrhizi, also referred to as rate reducing resistance, is also known in soybean (14). Lines with partial resistance in field evaluations were rated as moderately resistant, since fewer lesions developed on plants throughout the season (2,14). In greenhouse inoculation studies, host-pathogen combinations that resulted in RB reactions tended to have longer latent periods, lower rates of increase in pustule number over time, and smaller lesions compared with susceptible interactions that resulted in a TAN reaction (1,7). Identification and utilization of partial resistance in breeding programs has been limited and more research is needed to fully utilize partial resistance traits.

Since the report of soybean rust in Hawaii in 1994 (5), the USDA-ARS has renewed its support for soybean rust research. The USDA-ARS FDWSRU at Ft. Detrick, Frederick, MD, has been the focal point of identifying resistant soybean germplasm. There are over 16,000 soybean accessions in the USDA Germplasm Collection located at the University of Illinois. The objective of this work was to evaluate these soybean accessions for resistance to P. pachyrhizi under controlled conditions in the FDWSRU Biosafety Level 3 containment greenhouses (8). The overall goal of these evaluations was to identify accessions that may provide new sources of resistance. Part of these data were previously reported (10).

Plant Propagation and Growth Conditions

Seeds for all soybean accessions were obtained from R. L. Nelson, USDA Germplasm Collection located at the University of Illinois. Two seeds of each accession were glued to pre-labeled 10-cm wooden pot labels with washable white school glue (Elmers Products, Columbus, OH). Seeds were planted in a single cell by placing the "seed on a stick" into one cell of a flat (27 × 52 cm) containing 6 × 12 cells filled with Sunshine LC1 mix (Sun Grow Horticulture Products, Belleview, WA). Plants were thinned to one plant per cell after germination. Inoculation sets contained 10 to 18 flats each and were inoculated and incubated together in dew chambers. Cells on the outside edge of the flats were planted with a susceptible public cultivar (‘Ina,’ ‘Maverick,’ or ‘Pana’) as a border.

Rust Isolates and Inoculation Methods

Four P. pachyrhizi isolates, Thailand (TH01-1), Brazil (BZ01-1), Paraguay (PG01-2), and Zimbabwe (ZM01-1), all collected in 2001, were used as a mixture in all studies. The isolates were sent to R. Frederick at the FDWSRU under the appropriate APHIS permit by the following individuals: TH01-1, Srisuk Poonpolgul, Thailand Department of Agriculture, Bangkok, Thailand; BZ01-1, José Tadashi Yorinori, EMBRAPA SOJA, Londrina, Brazil; PG01-2, Wilfrido Morel Paiva, Centro Regional de Investigacón Agrícola, Capitán Miranda, Paraguay; and ZM01-1, Clive Levy, Commercial Farmers Union of Zimbabwe, Harare, Zimbabwe. After arrival at FDWSRU, urediniospores of each isolate were inoculated on the soybean cv. ‘Williams.’ Individual isolates were separately increased on Williams, and all urediniospores collected from each isolate were stored in liquid nitrogen. Prior to inoculation, urediniospores were removed from liquid nitrogen tank, heat shocked at 40°C for 5 min, and hydrated by incubating over water in an enclosed petri plate overnight. Inoculum was prepared by combining equal weights of the four isolates in distilled water with 0.1% Tween 20, vigorously mixing, and then filtering the mixture through a 53 µ nylon screen. Urediniospores were quantified using a hemocytometer and diluted in distilled water with 0.1% Tween 20 to a final concentration 60,000 per ml for inoculation.

Seedlings were inoculated when plants were 14 to 18 days old and evaluated 14 days later. Plants were atomized with 40 ml spore concentrate per flat at 20 psi until runoff several successive times, and placed in dew chambers at 20 to 25°C overnight. Plants were removed from dew chambers and placed in the greenhouse at 20 to 25°C under a 16-h photoperiod and watered from below. Supplemental light was provided by 1000 watt Metalarc lights (Sylvania, Danvers, MA) spaced 0.6 m apart 1.2 m above the bench. Susceptible cultivars (Dwight or Ina) were randomly placed within each set, with at least one susceptible cultivar per every sixth flat. The cultivars were used to evaluate the success of the inoculation and to provide a visual reference for disease severity assessments.

Germplasm Evaluations

Disease severity was evaluated on the first trifoliolate leaf for most accessions; however, the unifoliate leaf was evaluated for a few accessions that were slower in growth. A disease severity scale of 1 to 5, based on lesion density, was used where 1 = no visible lesions, 2 = few scattered lesions present, 3 = moderate number of lesions on at least part of the leaf, 4 = abundant number of lesions on at least part of the leaf, and 5 = prolific lesion development over most of the leaf (Fig. 2). The presence of TAN, RB, or mixed lesions was also recorded. The TAN lesion was considered a susceptible reaction, while the RB or lack of lesions was considered resistant (1). The mixed reaction was recorded when both RB and TAN lesions were observed on the same leaf, and were considered an RB reaction for part of the data summary.

Fig. 2. Scale used for the visual assessment of soybean rust in the preliminary evaluations of the germplasm in the USDA Soybean germplasm collection. 1 = no visible lesions, 2 = few scattered lesions present, 3 = moderate number of lesions on at least part of the leaf, 4 = abundant number of lesions on at least part of the leaf, and 5 = prolific lesion development over most of the leaf.

Preliminary evaluation one (P1). A total of 16,595 soybean accessions were evaluated as single seedlings in inoculation sets of approximately 1,000 accessions each. Soybean rust severity ranged from one to five with the majority of accessions having a disease severity rating of three or four (Fig. 3). The RB lesion type was recorded in 1,237 accessions with 9 accessions (1%) rated as a one, 170 accessions (14%) rated as a two, 558 accessions (45%) rated as a three, 410 accessions (33%) rated as a four, and 90 accessions (7%) rated as a five. A total of 3,215 accessions (19%) were selected for a second evaluation. Those that were advanced to the next level had severity scores of two or less or had RB lesions at any disease severity. All other accessions were not re-evaluated except for a few accessions that had a disease severity of three and appeared to have low spore-producing TAN lesion types.

Fig. 3. Frequency distribution of 16,595 soybean accessions that had rust severities of 1, 2, 3, 4 and 5 with either a TAN or RB lesion type in the first preliminary evaluation.

Preliminary evaluation two (P2). The P2 evaluation was done in inoculation sets of 300 to 340 accessions each that were replicated three times. Entries were randomized within each replication using a randomized complete block design. If germination was low or if greenhouse conditions did not permit normal disease development, inoculation sets or subsets within an inoculation set were repeated. In the P2 evaluation, severity ranged from 1 to 5 with 58% of the accessions rated between 3.0 and 3.9 (Fig. 4). RB lesions were recorded on a total of 535 accessions (17%) distributed across all severities. The greatest number of accessions with a RB lesion occurred within a severity range of 3.0 to 3.9, but the ratios of RB to TAN were greater at lower severity ratings.

Fig. 4. Frequency distribution of 3,215 soybean accessions with either TAN or RB lesion type in the first preliminary evaluation (additional data is presented of 697 severity ratings due to duplication of some accessions in more than one set.

There were 805 accessions selected as potential sources of resistance from the P2 evaluation. These accessions had a mean severity of 2.7 or less, or had an RB lesion recorded from two of three plants evaluated (see Appendix, Table 1). Since the accessions were evaluated in several P2 sets that were inoculated and evaluated in different time periods, no comparison among them was made, although in tabular format the accessions are ranked by severity. The distribution of the selected lines ranged from 1 to 4.3 in severity (Fig. 5). Among these accessions, 486 (60%) had RB or mixed lesion types and 319 (40%) accessions had the TAN lesion type. Of the accessions where the RB lesion type was reported, 321 (66%) had a severity greater than 3.0, while most of the selected accessions with the TAN lesion type had severity ratings from 2.0 to 2.9.

Fig. 5. Frequency distribution of 805 soybean accessions with either TAN or RB lesion type in the second preliminary evaluation.

Among the selected accessions, the number of plants evaluated differed, with some accessions having only one or two seedlings evaluated. Although attempts were made to re-evaluate accessions in sets with poor germination or poor disease development, not all accessions produced three plants. Rather than discard potential sources of resistance, those accessions with mean severities of 2.5 or less, and those accessions with RB reactions were selected for further evaluation.

Conclusions

In our study, a two-tiered preliminary screen of 16,595 soybean accessions of the USDA Germplasm Collection has identified accessions that have resistance to soybean rust in seedling evaluations. Some of these accessions had RB lesions, which have been associated with single gene resistance (3), while others had TAN lesions but had low visual rust severity. The accessions with low rust severities may be sources of partial resistance, that may limit infection and/or lesion development, or may be the result of an incompatible reaction similar to that reported in Rpp₁ when challenged with a limited number of isolates.

In the P2 evaluation, three seedlings per accession were evaluated within each inoculation set. However, many accessions have data from more than three seedlings, while other accessions have data from only one or two seedlings. If germination within a set was poor, or if greenhouse conditions did not permit normal development of disease, sub-sets within each inoculation set were re-planted and re-evaluated. Accessions with data from fewer than three seedlings from the P2 screen were included for further evaluation if severities were low (less than 2.5) or if they had the RB lesion type. Even though the rust severity was low, further evaluation is needed before these lines can be identified as superior to others on the list.

The accessions selected in these preliminary evaluations were inoculated with a mixture of four P. pachyrhizi isolates. These isolates were among the most recent in the soybean rust collection at the FDWSRU, and they were selected to represent a diverse geographic distribution covering three continents in an attempt to challenge the accessions with a broad array of virulence. Since a mixture of isolates was used, none of the accessions selected in this study have been evaluated for race-specific resistance. Race-specific resistance to P. pachyrhizi in soybeans is well documented (3), but it has not been considered durable when these lines were deployed in the field. Single P. pachyrhizi isolate evaluations will need to be done on these accessions in order to evaluate race specificity.

The P1 and P2 preliminary evaluations were done using seedling screens. Under field conditions, soybean rust severity increases after flowering; therefore all the accessions selected in these seedling screens should be evaluated in the field to identify differences in adult plant resistance before they can be considered useful in a breeding program. There is no information correlating severity in a seedling screen with adult plant resistance to soybean rust. Previous resistance sources were identified in field screens in Taiwan and India (1) and confirmed using a single isolate in greenhouse evaluations (1,4). A set of 776 accessions has been identified from the P2 screen. These accessions were planted, by maturity group, in field trials at several locations across the U.S. in 2005. If soybean rust develops at these sites and the appropriate data is collected, this will be the first evaluation of adult plant resistance in these materials.

The commercial soybean cultivars currently in use throughout the U.S. soybean production areas may be moderately to very susceptible to soybean rust based on a limited number of cultivars that have been tested in containment (13). In the absence of high levels of genetic resistance to the pathogen, producers may need to rely on fungicides to protect the crop. In order to reduce this expense, incorporation of rust resistance into commercial soybean cultivars from sources identified in this and other research is imperative. The sources of resistance identified in this research may provide the resistance genes needed for future development of soybean cultivars with soybean rust resistance.

Disclaimer and Acknowledgments

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 the product to the exclusion of others that may also be suitable.

We are grateful to Randy Nelson and his staff for providing soybean seed; Theresa Herman and Tara Lynch for organizing the sets of seed sent to containment; and to Craig Austin, JoAnn Garrett, Sheila Diepold, and Christine Stone for their technical assistance with the seedling evaluations at the FDWSRU. This research was in part supported by the United Soybean Board as USB Projects 2229, 3217, and 4217.

Literature Cited

1. Bromfield, K. R. 1984. Soybean Rust. Monograph No. 11. American Phytopathological Society, St. Paul, MN.

2. Hartman, G. L. 1995. Highlights of soybean rust research at the Asian Vegetable Research and Development Center. Pages 19-28 in: Proceedings of the Soybean Rust Workshop, 9-11 Aug. 1995, J. B. Sinclair and G. L. Hartman, eds. College of Agricultural, Consumer, and Environmental Sciences, National Soybean Research Laboratory, Urbana, IL.

3. Hartman, G. L., Bonde, M. R., Miles, M. R., and Frederick, R. D. 2004. Variation of Phakopsora pachyrhizi isolates on soybean. Pages 440-446 in: Proceedings of VII World Soybean Research Conference, IV International Soybean Processing and Utilization Conference, III Congresso Mundial de Soja (Brazilian Soybean Conference). F. Moscardi, C. B. Hoffman-Campo, O. Ferreira Saraiva, P. R. Galerani, F. C. Krzyzanowski and M. C. Carrão-Panizzi, eds. Embrapa Soja.

4. Hartman, G. L., Miles, M. R., and Frederick, R. D. 2005. Breeding for resistance to soybean rust. Plant Dis. 89:664-666.

5. Killgore, E., and Heu, R. 1994. First report of soybean rust in Hawaii. Plant Dis. 78:1216.

6. Levy, C. 2004. Epidemiology and chemical control of soybean rust in South Africa. Plant Dis. 89:669-674.

7. Marchetti, M. A., Uecker, F. A., and Bromfield, K. R. 1975. Uredial development of Phakopsora pachyrhizi in soybeans. Phytopathology 65:822-823.

8. Melching, J. S., Bromfield, K. R., and Kingsolver, C. H. 1983. The plant pathogen containment facility at Frederick, Maryland. Plant Dis. 67:717-722.

9. Miles, M. R., Frederick, R. D., and Hartman, G. L. 2003. Soybean rust: Is the U.S. crop at risk? Online. APSnet Feature, American Phytopathological Society, June 2003.

10. Miles, M. R., Morel, W., Yorinori, J. T., Ma, Z.-H., Poonpolgul, S., Hartman, G. L., and Frederick, R. D. 2004. Preliminary report of Asian soybean rust reaction on soybean accessions planted in Brazil, China, Paraguay and Thailand with seedling reactions from greenhouse screens in the United States. (Abst.) Documentos 228: Abstracts of contributed papers and posters VII World Soybean Research Conference, IV International Soybean Processing and Utilization Conference, III Congresso Mundial de Soja:162.

11. Schneider, R. W., Hollier, C. A., Whitam, H. K., Palm, M. E., McKenny, J. M., Hernandez, J. R., Levy, L., and Devries-Paterson, R. 2005. First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States. Plant Dis. 89:774.

12. Sinclair, J. B., and Hartman, G. L. 1999. Soybean rust. Pages 25-26 in: Compendium of Soybean Diseases, G. L. Hartman, J. B. Sinclair and J. C. Rupe, eds. American Phytopathological Society, St. Paul, MN.

13. VIPS. 2006. Varietal Information Program for Soybeans. Online. IL Soybean Assoc. and Coll. of Agric., Consum. and Environ. Sci., Univ. of IL.

14. Wang, T. C., and Hartman, G. L. 1992. Epidemiology of soybean rust and breeding for host resistance. Plant Prot. Bull. 34:109-124.

15. Yorinori, J. T., Paiva, W. M., Frederick, R. D., Costamilan, L. M., Bertagnolli, P. F., Hartman, G. L., Godoy, C. V., and Nunes Jr., J. 2005. Epidemics of soybean rust (Phakopsora pachyrhizi) in Brazil and Paraguay from 2001 to 2003. Plant Dis. 89:675-677.

Appendix

Table 1. Soybean rust severity and lesion type for accessions selected as potential resistance sources from the preliminary one (P1) and preliminary two (P2) evaluations based on plants inoculated as seedlings with a mixture of Phakopsora pachyrhizi isolates at the USDA-ARS Foreign Disease-Weed Science Research Unit Biosafety Level 3 containment greenhouses