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2004 Plant Management Network.
Accepted for publication 5 February 2004. Published 9 March 2004.


Evaluation of Soybean Differentials for Their Interaction with Phytophthora sojae


A. E. Dorrance and H. Jia, Department of Plant Pathology, The Ohio State University, Wooster 44691; and T. S. Abney, USDA-ARS, Department of Botany and Plant Pathology, Purdue University, W. Lafayette, IN 47907


Corresponding author: A. E. Dorrance. dorrance.1@osu.edu


Dorrance, A. E., Jia, H., and Abney, T. S. 2004. Evaluation of soybean differentials for their interaction with Phytophthora sojae. Online. Plant Health Progress doi:10.1094/PHP-2004-0309-01-RS.


Abstract

Soybean lines, each containing a different resistance gene (Rps), are used as differentials to characterize isolates of Phytophthora sojae as physiologic races. Surveys in different soybean production regions have used various sets of soybean differentials thought to carry the same Rps genes. In some instances, isolates of P. sojae have been reported to have different reactions when evaluated in labs using different sets of differentials that were believed to have the same Rps gene. The objective of this study was to compare the consistency of racial classification when three different sets of soybean differentials were challenged with a common set of five races of P. sojae from Ohio and Indiana. Three soybean differential sets (USDA Soybean Germplasm Collection, The Ohio State University, and USDA-ARS Purdue University) were challenged with P. sojae using the hypocotyl inoculation test at OSU and USDA-ARS Purdue. Isolates of races 1, 3, 4, 7, and 25 from Ohio and Indiana had the same reaction on all three sets of soybean differentials for Rps1b, Rps1c, Rps1k, Rps3a, Rps3b, Rps3c, Rps6, Rps7, and on differentials Harlon, Harosoy 12xx, L59-731, and Union for Rps1a. L88-8470 used as a differential for Rps1a and L93-3312 used for Rps1d did not have the expected response. Isolates of races 4 and 25 from Ohio and Indiana responded differently on differentials with the Rps2 gene because this gene was not used previously to characterize races of P. sojae. A similar reaction occurred when differentials with Rps4 and Rps5 were inoculated with isolates of races 1 and 7, respectively. A standardized set of soybean differentials, corresponding to different maturity groups, for thirteen of the fourteen Rps genes is recommended.


Introduction

A set of host differentials is comprised of lines or cultivars of a host that have one or more resistance gene(s) to a specific plant pathogen. Host differentials are used in the classification of races or pathotypes of plant pathogenic bacteria, fungi, and nematodes (11). Individual genotypes within a set of differentials may change periodically as additional resistance genes are identified or as pathogen pathotypes change or adapt to resistance genes that are deployed within a region (20).

Phytophthora sojae (M. J. Kaufmann & J. W. Gerdemann) is a host-specific pathogen with more than 55 described races (1,10,12,15,16,23,25,26,27). Recently, the number of virulence genes that have been identified in each pathogen isolate has increased dramatically, which has made the older number classification system cumbersome (10,12,13,17). Hence, pathotypes or virulence formulas are now used to describe virulence patterns based on reactions on sets of differentials (10,12).

Races of P. sojae were identified in 1965 using soybean cultivars D60-9647, D60-11082, FC31745, Harrel, and Nansemond (19). Ten years later, a standard set of differentials was adopted by plant pathologists and plant breeders (3,15), which included Harosoy, Harosoy63, Sanga, Mack, Altona, PI 103091, and PI 171442. These differentials allowed researchers to place isolates of P. sojae into groups of physiologic races. Subsequently, two sets of soybean differential isolines were developed by backcrossing resistance into the soybean cultivars Williams (25) and Harosoy (5,6) (Table 1). Currently, there are Rps genes that confer resistance to P. sojae at eight loci, of which two are comprised of an allelic series (2,4,8). Over the past twenty years, most characterized strains of P. sojae races have been based on host differentials that contain Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps3a, Rps6, and Rps7 (3). Recent surveys have used host differentials with additional Rps genes (10,12,13,21,23,26).


Table 1. Soybean differentials originating from the USDA Soybean Germplasm collection (USDA-PGC), The Ohio State University (OSU), and USDA-ARS Purdue University (USDA-P) evaluated with five races of Phytophthora sojae.

Rps
gene
Differentiala Background Source of
Rps gene
Seed source
of differential
rps Williams Williams        OSU
Rps1a Harlon Harosoy Blackhawk OSU
Harosoy 12XX Harosoy Blackhawk OSU
L59-731 (PI547677) Harosoy Blackhawk USDA-PGC
Union Williams Mukden USDA-P
L88-8470 (PI591505) Williams Mukden USDA-PGC
Rps1b Harosoy 13XX Harosoy Sanga OSU
Harosoy 13XX Harosoy Sanga USDA-P
L77-1863 (PI547842) Williams Harrell USDA-PGC
Rps1c Williams79 Williams Lee68 OSU
L75-3735 Williams Lee68 USDA-P
L75-3735 (PI547834) Williams Lee68 USDA-PGC
L77-1727 (PI547841) Williams Clark 63
(PI229342)
USDA-PGC
L85-129 (PI547791) Harosoy Higan USDA-PGC
Rps1d Haro16 Harosoy PI 103091 USDA-P
PI 103091 Plant
Introduction
PI 103091 OSU
L99-3312 Williams PI 103091 USDA-PGC
Rps1k Williams82 Williams Kingwa OSU
Williams82 Williams Kingwa USDA-P
L77-1794 (PI547890) Williams Kingwa USDA-PGC
Rps2 L76-1988 (PI547838) Williams CNS USDA-PGC
L82-1449 (PI547788) Williams CNS USDA-PGC
L76-1988 Williams CNS OSU
Rps3a PI171442 Plant
Introduction
PI 171442 USDA-P
L83-570 Williams PI 86972-1 OSU
L83-570 (PI547862) Williams PI 86972-1 USDA-PGC
Rps3b L91-8347 (PI591509) Williams PI 172901 USDA-PGC
L89-1541 (PI591507) Williams PI 82.312N USDA-PGC
L91-8347 Williams PI 172901 USDA-P
PRX-146-36 Harosoy         OSU
Rps3c PRX-145-48 Harosoy PI 340046 OSU
L92-7857 Williams PI 340046 USDA-PGC
Rps4 L85-2352 Williams PI 86050 OSU
L85-2352 Williams PI 86050 USDA-PGC
Rps5 L62-904 Harosoy PI 91160 USDA-PGC
L85-3059 (PI547876) Williams PI 91160 USDA-PGC
L85-3059 Williams PI 91160 OSU
Rps6 Haro 62xx Harosoy           OSU
L89-1581 (PI591511) Williams Altona USDA-PGC
L89-1581 Williams Altona USDA-P
Rps7 Harosoy Harosoy         OSU
Harosoy Harosoy        USDA-P
L93-3258 (PI591512) Williams Harosoy USDA-PGC
Rps1a &
Rps2
L86-493 Williams               
Rps1c &
Rps2
L81-4352 (PI547856) Williams Williams 79
L76-2013
USDA-PGC

a The name of the cultivar or soybean line of each of the differentials along with the accession designation in parenthesis if it is deposited in the USDA Soybean Germplasm Collection in Urbana, IL.


In many cases, soybean cultivars or lines were used as differentials for P. sojae prior to the identification of resistance gene(s) in the line (2,15,19). Subsequently, a previously unidentified Rps gene or two Rps genes were identified in some of these cultivars, i.e., Tracy. For example, Harosoy was regarded as the universal "suscept" in standard differential sets used to characterize races of P. sojae during the 1960s and 1970s, but eventually, P. sojae isolates were discovered that had a resistant interaction with Harosoy (18). Resistance expressed by Harosoy was conferred by the Rps7 gene (2).

Sets of differentials used to classify races of P. sojae are not uniform among all researchers. In some instances, the race classification of the same isolate of P. sojae differed among labs that were using differentials that were believed to carry the same Rps gene. Thus, periodic reassessment of differentials is required to minimize differences between studies as well as to make recommendations for future pathotype evaluations. The objective of this study was to compare the consistency of racial classification when three different sets of the standard soybean differentials were challenged with a common set of five races of P. sojae originating from Indiana and Ohio.


The Differential Sets

Sets of the host differentials were obtained from the USDA-ARS Soybean Germplasm Collection (R. Nelson, University of Illinois), USDA-ARS Purdue University and The Ohio State University (Table 1). Seeds of the soybean lines from the Germplasm Collection were increased at The Ohio State University (OSU), Ohio Agricultural Research and Development Center, Wooster, OH. Increased sets of differentials were then used for subsequent evaluations.


The Experiments

Five races of P. sojae -- race 1 (vir 7), race 3 (1a, 7), race 4 (1a, 1c, 7), race 7 (1a, 3a, 6, 7), and race 25 (1a, 1b, 1c, 1k, 7) -- from both OSU and Purdue were used to evaluate the response of the differentials. The differentials were evaluated at both locations with both sets of isolates using a hypocotyl inoculation technique in laboratory trials (Ohio) and in greenhouse trials (Indiana) (1,4,16,22). The hypocotyl inoculation technique is the preferred method for classifying races of P. sojae because there is no interference with partial resistance or field resistance that is expressed in the roots (Figs. 1 to 3). Eight plants of each differential were inoculated with each isolate. At each location, all differentials were evaluated with the same isolate at the same time, but different isolates were evaluated at different times. The reactions were scored as a resistant reaction (R), intermediate (I), and susceptible (S) based on the number of seedlings killed: 0 to 2; 3 to 5; and 6 to 8 dead, respectively. The trials were repeated at each location. The pathotypes were then compared among trials.



A
 
B

Fig. 1. (A) Rps gene expression is evaluated with the hypocotyl inoculation technique where macerated mycelium of P. sojae is placed in the hypocotyls of 7- to 10-day-old soybean seedlings. Plants are kept in a humid environment for 15 to 24 hours following inoculation. The plants are evaluated 5 to 10 days following inoculation. (B) The presence of a dying or expanded lesion indicates a susceptible or compatible interaction. Resistance response is expressed has a healed wound and healthy seedling.


 

Fig. 2. Inoculum for the hypocotyls inoculation is macerated by placing a P. sojae colony from a 7-day-old culture grown on -strength lima bean in agar at 15 g/liter into a syringe and passing it through once.

 

 
A
 
B
 
 

Fig. 3. (A, B) For laboratory evaluations of the Rps genes, etiolated seedlings are inoculated 7 days after placing on germination paper. The resistance reaction is a necrotic area at the inoculation site; a susceptible interaction is a slowly expanding brown lesion.

 

Comparison of Same Isolates in Ohio and Indiana

Reactions of the same isolates on differentials in trials at Ohio and Indiana were identical with two exceptions. The differential, Haro16 (Rps1d), was resistant to P. sojae OHR7 in the Indiana trial but susceptible to this isolate in the Ohio trial. Also, the differential L85-2353 (Rps4) was resistant to P. sojae INR7 in the Ohio trial but susceptible to this isolate in the Indiana trial (Table 2). Changes in reactions of P. sojae isolates on differentials occur over time. Temperature following inoculation, bacterial contamination, and excessive wounding during inoculation alter this response and affect racial classification.


Table 2. Reaction of ten isolates of P. sojae representing five races from Ohio and Indiana on three sets of differentials from USDA-ARS Soybean Germplasm Collection, Ohio State/OARDC (OSU), and USDA/Purdue University (Purdue) to determine resistance (R) or susceptibility (S).

Rps gene,
Differential
a
P. sojae isolates
Race 1 Race 3 Race 4 Race 7 Race 25
IN OH IN OH IN OH IN OH IN OH
rps
Williams S S S S S S S S S S
Rps1a
Harlon R R S S S S S S S S
Harosoy12xx R R S S S S S S S S
L59-731 R R S S S S S S S S
Union R R S S S S S S S S
L88-8470 R R R R S S R R R R
Rps1b
Haro 13XX R R R R R R R R S S
L77-1863 R R R R R R R R S S
Rps1c
L75-3735 R R R R S S R R S S
Williams 79 R R R R S S R R S S
L77-1727 R R R R S S R R S S
L85-129 R R R R S S R R S S
Rps1d
Haro 16 R R R R R R R R/S* R R
PI103091 R R R R R R R R R R
L99-3312 R R S S S S S S S S
Rps1k
Williams 82 R R R R R R R R S S
L77-1794 R R R R R R R R S S
Rps2
L82-1449 R R R I S R S S S R
L76-1988 R R R R S R S S S R
Rps3a
PI171442 R R R R R R S S R R
L83-570 R R R R R R S S R R
Rps3b
PRX146-36 R R R R R R R R R R
L91-8347 R R R R R R R R R R
L89-1541 R R R R R R R R R R
Rps3c
PRX145-48 R R R R R R S S R R
L92-7857 R R R R R R S S R R
Rps4
L85-2352 S R S R R R R/S* S R R
Rps5
L62-904 R R R R R R R S R R
L85-3059 R R R R R R R S R R
Rps6
Haro 62XX R R R R R R S S R R
L89-1581 R R R R R R S S R R
Rps7
Harosoy S S S S S S S S S S
L93-3258 S S S S S S S S S S
Rps1a,2
L86-493 R R R R S R S S S R
Rps1c,2
L81-4352 R R R R S R R R S R

* Indicates data from USDA-Purdue compared to Ohio State University where the results were not the same for the same isolate in the two locations.


Comparison of Host Differentials

Races 1, 3, 4, 7, and 25 from both Ohio and Indiana had the expected reaction on all lines in the three sets of differentials used for Rps1b, Rps1c, Rps1k, Rps3a, Rps3b, Rps3c, Rps6, and Rps7; and on the differentials Harlon, Harosoy 12xx, L59-731, and Union used for Rps1a (Table 2). However, several differentials did not have the expected response following inoculation with these races of P. sojae. L88-8470, a differential used for Rps1a, had resistant reactions to races 3, 7, and 25 whereas the other four differentials used for Rps1a were susceptible (Table 2). This indicates that Rps1a is not the Rps gene(s) present in L99-8470. L99-3312, a differential used for Rps1d, was susceptible to races 3, 4, 7, and 25 while the other two differentials for Rps1d, Haro 16, and PI 103091 were resistant except Haro 16, inoculated with race OH7 in the Ohio trial. This indicates that L99-3312 has a resistance gene other than Rps1d. All of the isolates used in this study caused resistant reactions on differential Rps3b. Further evaluations with additional isolates classified as different pathotypes are needed to compare susceptible reactions on Rps3b.


Comparison of Isolates from Ohio and Indiana

Differentials that carried the Rps2 gene, L86-493 (Rps1a and Rps2), L81-4352 (Rps1c, Rps2), L82-1449, and L76-1988 were susceptible to isolates of P. sojae races 4 and 25 from Indiana but were resistant to isolates of these same races from Ohio. Differentials with Rps5 were resistant to isolates of race 7 from Indiana but susceptible to the isolate of the same race from Ohio. In addition, the differential for Rps4, L85-2352, was susceptible to the isolate of race 1 from Indiana but resistant to the isolate of the same race from Ohio. Because Rps2, Rps4, and Rps5 were not used for race classification, these differences are due to the differences in virulence of the isolates and not a reflection of the differentials.


Conclusions

The P. sojae populations that are present in the north-central U.S. and Ontario Canada soybean production regions are adapting to many of the commonly deployed Rps genes in commercial cultivars (1,7,10,12,13,17). These genes include Rps1a, Rps1b, Rps1c, and Rps1k. Future pathotype assessment of the P. sojae population within this region should include differentials with as many Rps genes as possible to provide the best recommendations about gene deployment.

Based on the results of this study, the soybean differentials presented in Table 3 are recommended for classifying isolates of P. sojae against Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, and Rps7. This set of differentials was chosen based on the consistent reactions in our trials. More than one differential is recommended for each Rps gene with two exceptions, Rps1k and Rps4. Differentials were chosen to cover more than one maturity group, which will allow scientists to maintain their own differentials but also be able to compare their results with others. Differentials with Rps1k, Rps3a, and Rps4 that are in earlier-maturing backgrounds are needed. There are several plant introductions that have been identified recently as novel sources of Rps genes, including Rps8 in PI399073 (4,9,14). These sources were not evaluated in this study, but they also should be included in differential sets. Periodic reassessment of differentials will be necessary as more Rps genes are identified and deployed to ensure that studies can be compared across the regions.


Table 3. Recommended soybean differentials to assess pathotypes (races) of Phytophthora sojae based on hypocotyl inoculations with P. sojae races 1, 3, 4, 7, and 25.

Rps
gene
Differential Background Maturity
group
Accessiona
rps Williams Williams III PI 548631
Rps1a Harlon   I PI 548571
Harosoy 12xx Harosoy II  
L59-731 Harosoy II PI 547677
Union Williams IV PI 548622
Rps1b Haro13xx Harosoy II  
L77-1863 Williams III PI 547842
Rps1c L75-3735 Williams III PI 547834
L85-129 Harosoy II PI 547791
Rps1d Haro 16 Harosoy II  
PI 103091 Plant Introduction IV PI 103091
Rps1k L77-1794 Williams III PI 547890
Rps2 L82-1449 Harosoy II PI 547788
L76-1988 Williams III PI 547838
Rps3a L83-570 Williams/PI 86972-1 III PI 547862
PI 171442 Plant Introduction V PI 171442
Rps3b L91-8347 Williams III PI 591509
PRX 146-36 Harosoy II  
Rps3c PRX 145-48 Harosoy II  
L92-7857 Williams III PI 591510
Rps4 L85-2352 Williams III PI 547874
Rps5 L62-904 Harosoy II PI 547764
L85-3059 Williams III PI 547876
Rps6 Harosoy 62xx Harosoy II  
L89-1581 Williams III PI 591511
Rps7 Harosoy Harosoy II  
L93-3258 Williams III PI 591512

a Accession designation in the USDA-ARS National Plant Germplasm System (24). Other accessions are available through the Ag-Canada Research Station, Harrow, Ontario.


Acknowledgments

We thank S. Berry and B. Bardall for assistance with field increase of seed as well as P. Lipps, M. Ellis, and an anonymous reviewer for their comments on this manuscript. Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This is a contribution of the Agricultural Research Service, U.S. Department of Agriculture, in cooperation with the Purdue Agricultural Experiment Station, West Lafayette, IN. This research was funded in part by a joint project of the United States Department of Agriculture, the United Soybean Board, and the American Seed Trade Association.


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