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Published online 6 May 2005
Published in Crop Sci 45:1092-1095 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP BREEDING, GENETICS & CYTOLOGY

Characterization of Resistance to Brown Stem Rot of Soybean in Five Accessions from Central China

M. E. Patzoldt, S. R. Carlson and B. W. Diers*

Dep. of Crop Sciences, Univ. of Illinois, 1101 W. Peabody Dr., Urbana, IL 61801

* Corresponding author (bdiers{at}uiuc.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Brown stem rot (BSR) of soybean [Glycine max (L.) Merr.], caused by Phialophora gregata (Allington & Chamberlain) W. Gams f. sp. sojae Kobayashi, Yamamoto, Negishi, and Ogoshi, can be effectively controlled with genetic resistance. Although three BSR resistance genes have been described, they all map to the same genetic region on linkage group (LG) J. In a previous screening of plant introductions (PIs) for BSR resistance, PI 567296B, PI 567323A, PI 567479, PI 567535B, and PI 567544 from central China were identified as having a high level of resistance. The objective of this study was to determine if resistance in these PIs maps to the same region on LG J where BSR resistance genes were previously identified. To map the resistance genes in these accessions, each PI was crossed to the susceptible cultivar Century 84 and 94 F2:3 lines were developed from each cross. Lines were evaluated for BSR resistance in the greenhouse and screened with molecular markers from LG J. In all five populations, a quantitative trait locus (QTL) conferring BSR resistance was mapped to the same region on LG J where resistance has already been reported. These results suggest that the BSR resistance genes identified in these five PIs from central China are likely allelic with previously reported BSR resistance genes.

Abbreviations: BSR, brown stem rot • CAPS, cleaved amplified polymorphic sequence • cM, centimorgan • LG, linkage group • LOD, likelihood of odds • MG, maturity group • PI, plant introduction • QTL, quantitative trait locus/loci • SSR, simple sequence repeat • TBE, tris-borate


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
BROWN STEM ROT of soybean results in browning of stem pith tissue and in some cases interveinal chlorosis and necrosis of leaves. In conditions favoring BSR symptom development, yield losses of up to 38% have been reported (Gray, 1972). This disease consistently ranks as one of the most important soybean diseases in northern U.S. soybean production areas (Gray and Grau, 1999; Wrather et al., 2003). Brown stem rot can be effectively controlled with genetic resistance (Bachman et al., 1997), and three dominant, independently segregating loci that confer BSR resistance in soybean have been reported using classical genetic studies. These genes include Rbs1, which was identified in the germplasm line L78-4094 (Hanson et al., 1988), Rbs2 from PI 437833 (Hanson et al., 1988), and Rbs3 from PI 437970 (Willmot and Nickell, 1989). The Rbs1 gene in L78-4094 was inherited from PI 84946-2, an accession collected from South Korea. Plant introduction 437833 and PI 437970 were collected from northeastern China.

Although BSR resistance has been shown to be controlled by major genes, this resistance is typically quantitative in segregating populations (Bachman et al., 2001; Klos et al., 2000; Lewers et al., 1999; Patzoldt et al., 2005). The quantitative nature of this resistance has led researchers to map BSR resistance genes as QTL. This alleviates the need to distinguish between resistant, segregating, and susceptible lines when the data are continuous.

All three of the previously described genes were mapped to a region on LG J that includes the simple sequence repeat (SSR) markers Satt431 and Satt244, and the cleaved amplified polymorphic sequence (CAPS) marker 21E22.sp2 (Bachman et al., 2001; Cregan et al., 1999; Klos et al., 2000; Lewers et al., 1999). In addition, Patzoldt et al. (2005) mapped a major BSR resistance gene originating from PI 88788 to the same region on LG J. The mapping of BSR resistance to the same 20-cM region brings into question whether these sources actually have resistance genes in common. An alternative explanation was provided by Bachman and Nickell (2000), who proposed that BSR resistance may be caused by an interaction between a resistance gene on LG J and genes on other linkage groups.

Plant introductions from the USDA Soybean Germplasm Collection have been valuable sources of genetic diversity in soybean improvement. Evaluations of PIs from central and southern China have resulted in the identification of a large number of accessions with resistance to BSR (Bachman et al., 1997; Bachman and Nickell, 1999). Bachman et al. (1997) identified 64 putatively resistant accessions from central China in greenhouse evaluations. We selected the five most resistant PIs from this study for our evaluations. These PIs could be valuable sources of new resistance genes if they are shown to carry genes that are different than those currently found in cultivars. The objective of this research was to determine whether the genes responsible for BSR resistance in five PIs from central China map to the LG J region where the previously reported BSR resistance genes map.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The PIs evaluated in this study originated from central China and were identified as BSR resistant by Bachman et al. (1997). PI 567296B and PI 567323A were collected from Gansu province and were classified as maturity group (MG) IV and MGII accessions, respectively. PI 567479 is a MG IV accession collected from the Shanxi province. Both PI 567535B and PI 567544 are MG IV accessions collected from the Shandong province. Each PI was crossed to the MG II cultivar Century 84 (Walker et al., 1986), which is susceptible to BSR. F1 seed from the crosses were planted and the F1 plants were harvested individually at maturity. F2 plants were grown and each plant was harvested and threshed individually to form a total of 94 F2:3 lines from each cross.

The F2:3 lines were grown in a greenhouse and evaluated for BSR resistance using the root dip inoculation method as outlined in Patzoldt et al. (2003). Briefly, ten seedlings were grown in sand to the V2 growth stage (Fehr et al., 1971). Five uniform plants were selected and their roots gently washed with water before being dipped into a 50 mL inoculum solution containing conidia and mycelial fragments of P. gregata f. sp. sojae isolate Oh2. Plants were then transplanted into steamed 1:1 sand: soil mixture and the remaining inoculum was poured onto the roots before being covering with the soil mixture. An experimental unit consisted of one pot containing five plants. Growing conditions included a 14-h photoperiod, weekly fertilization, and watering as needed.

Each population was screened in a single separate greenhouse test. If resistance to BSR in any of the five populations was not found to be associated with LG J markers, a second greenhouse test would be undertaken at that time. Within a test, pots were arranged in a completely random design with each line in the population grown without replication in a single pot. Each test also contained three replications of all check genotypes and the PI parent of the population being tested. The checks included the three resistance sources L78-4094 (Rbs1), PI 437833 (Rbs2), PI 437970 (Rbs3), and the susceptible cultivars Century 84 (the susceptible parent) and Colfax. Six to eight weeks after inoculation, when the plants had reached the R2 to R4 growth stage (Fehr et al., 1971), the five plants in each pot were rated for both foliar and stem BSR symptoms. Foliar ratings were taken by determining the percentage of nodes with symptomatic leaves on each plant. Stem ratings were taken by splitting each stem longitudinally and determining the percentage of nodes that had symptomatic browning. For both foliar and stem data, percentages were then assigned values from the HB Scale (Horsfall and Barratt, 1945; Mengistu and Grau, 1987; Patzoldt et al., 2005) and later converted to weighted percentages using Elanco products conversion tables (Division of Eli Lilly & Co., Indianapolis, IN). Means across the five plants in each plot were then calculated and used in further data analyses.

The parents of the populations were tested with a total of 51 SSR and CAPS markers spanning the length of LG J to identify polymorphisms. Ten to 15 markers gave clear polymorphisms between the parents of any given population and these were used to evaluate the populations. Parental and population DNA was extracted from young leaf tissue collected from 8 to 10 plants from each genotype. DNA was extracted according to Keim and Shoemaker (1988) with modifications outlined in Patzoldt et al. (2005) and was genotyped with polymorphic genetic markers from LG J. Simple sequence repeat markers from the USDA LG J consensus map (Song et al., 2004) and the CAPS markers (Klos et al., 2000) also from LG J were used. Simple sequence repeat markers were amplified using polymerase chain reaction (PCR) according to Cregan et al. (1999). Amplified samples were run on 3% Metaphor agarose or on 6% non-denaturing acrylamide gels (Wang et al., 2003). Polymerase chain reactions for the CAPS markers were performed according to the protocol outlined in Patzoldt et al. (2005). Samples were stored at 4°C until run on 2% agarose gels in 0.5 x TBE. Gels were stained with ethidium bromide and bands were visualized under UV light.

Genotypic data were analyzed with JoinMap v3.0 (VanOoijen and Voorrips, 2001) to form a genetic map of LG J for each population. Linkage map information was combined with phenotypic data and interval mapping of resistance QTL was accomplished with MapQTL v4.0 (VanOoijen et al., 2002). LOD score values over 2.4 were considered significant for the interval mapping analysis.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The PI parent of each population showed significantly (P < 0.05) greater resistance than Century 84 (Table 1), with the exception of the foliar ratings in the test of the PI 567323A x Century 84 population. Among the PI parents, PI 567479 and PI 567544 exhibited the lowest foliar or stem symptoms while PI 567323A showed the greatest symptoms (Table 1). The disease scores were averaged across all five tests, and Century 84 had an average foliar score of 51% and a stem score of 49% (Table 2). In the test of the PI 567323A x Century 84 population, Century 84 had both low foliar and stem ratings. It is unclear why these ratings were low, especially in the light of the wide segregation of resistance in the population. The average disease scores for Century 84 were similar to the susceptible check cultivar Colfax, which had a foliar score of 39% and a stem score of 43%. The resistant check genotypes all exhibited a low level of disease with foliar scores ranging from 2 to 4% and stem symptoms ranging from 1 to 4% (Table 2).


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  		Table 1. Brown stem rot (BSR) resistance ratings from separate greenhouse tests of five populations of lines and parents.

 

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  		Table 2. Brown stem rot (BSR) disease ratings of the check genotypes across five separate greenhouse experiments conducted in Urbana, IL.

 
In all five populations, phenotypic segregation was observed for both foliar and stem BSR symptoms (Table 1) and these data were used to conduct interval mapping with the molecular markers from LG J. Significant QTL for both stem and foliar symptom resistance were mapped to LG J in all five populations (Table 3). Lines homozygous for the PI alleles for markers on LG J exhibited less stem browning and foliar symptoms than lines homozygous for Century 84 alleles in all populations. The LOD peaks from interval mapping in each population were near the marker Satt244, indicating that these five PIs may have resistance genes at the same locus (Table 3). The segregation of Satt244 was tested with a Chi-square analysis and the segregation of this marker fit expectations in all populations except PI 567296B x Century 84. In this population, there was an overrepresentation of individuals that were homozygous for the allele from Century 84.


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  		Table 3. Likelihood of odds (LOD) peak scores, distances to the LOD peaks from the simple sequence repeat marker Satt244, means of genotypic classes, and R2 values obtained from interval mapping analysis of molecular markers on linkage group (LG) J associated with brown stem rot (BSR) resistance.

 
The R2 values for the resistance QTL in the populations ranged from 15 to 51 (Table 3). The lowest values are less than previously reported R2 values for BSR resistance QTL (Bachman et al., 2001; Klos et al., 2000; Lewers et al., 1999; Patzoldt et al., 2005) and are likely the result of our nonreplicated testing of lines in the populations. Brown stem rot screening of lines in the populations were not replicated because BSR resistance in all five populations mapped to LG J after the first replication of testing. Repeating the experiments may have given a better estimate of the resistance QTL effect found in each PI. However, the focus of this study was to determine if resistance in these PIs mapped to the same region of LG J that is already known to provide BSR resistance.

The region on LG J near Satt244 where a major BSR resistance QTL was mapped in each of our populations is the same region where all other BSR resistance QTL have been reported (Bachman et al., 2001; Klos et al., 2000; Lewers et al., 1999; Patzoldt et al., 2005). There are a few explanations for these results. It is possible that these BSR resistance sources all possess the same resistance gene, or that a cluster of resistance genes exists in this region with different sources having resistance alleles at different genes. Alternatively, these sources could potentially have different resistance alleles at one locus. Further research is needed to show the relationship of genes from these sources.

It is surprising that all mapped BSR resistance genes have been located to the same region on LG J. The mapping of resistance genes from several sources to a single region is not typical in disease resistance studies in soybean. For example, eight Phytophthora rot (caused by Phytophthora sojae Kaufmann & Gerdemann) resistance loci with a total of 14 resistance alleles have been identified in soybean germplasm (Grau et al., 2004). Although fewer resistance genes have been identified for other soybean diseases, resistance genes are commonly found at more than one genetic region when breeders have made concerted efforts to find and map resistance genes (Grau et al., 2004).

The discovery of the common map location of these BSR resistance genes was unexpected because the five PIs in this study were collected from central China, which differs geographically from where accessions carrying previously mapped BSR resistance genes originated. PI 437833 (Rbs2) and PI 437970 (Rbs3) were both collected from northeast China. The Rbs1 gene in L78-4094 was inherited from PI 84946-2, an accession collected from South Korea. PI 88788 was collected from northeastern China near the Liao River in the Kaiyuan district of the Liaoning province (Bernard et al., 1987).

The five PIs that we evaluated could possess additional resistance QTL elsewhere in the genome. No attempt was made to map additional QTL since our data suggest that only the LG J resistance alleles were needed to recover the resistance level of the PI parent of each population. Lines in the populations which were homozygous for the PI allele on LG J showed resistance levels equal to, or greater than, the PI parent (Tables 1 and 3). In populations developed by crossing PI 567323A and PI 567535B with Century 84, where the PI means were greater than the means of the homozygous PI class in the population, these means were not significantly different. In addition, the almost complete resistance of lines in the homozygous resistant category shows that there is little variability among lines in this group.

Additional studies should be done to determine which Rbs genes are present in the PIs and if any of these accessions contain novel alleles at the Rbs loci. If new alleles are found, these accessions may yet prove useful for future breeding efforts. Additional efforts also are needed to further screen the soybean germplasm collection for novel BSR resistance genes.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Research supported in part by the United Soybean Board and the Illinois Soybean Program Operating Board.

Received for publication July 2, 2004.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 


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Crop Science 2005 45: xiii. [Full Text]  




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