Published online 16 January 2008
Published in Crop Sci 48:14-24 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Frogeye Leaf Spot of Soybean: A Review and Proposed Race Designations for Isolates of Cercospora sojina Hara
M. A. R. Miana,*,
A. M. Missaouib,d,
D. R. Walkerb,e,
D. V. Phillipsc and
H. R. Boermab
a USDA-ARS and Dep. of Horticulture and Crop Science, The Ohio State Univ., 1680 Madison Ave., Wooster, OH 44691
b Dep. of Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602-7272
c Dep. of Plant Pathology, Univ. of Georgia–Griffin Campus, Griffin, GA 30223
d current address: Monsanto Co., 1551 Hwy. 210, Huxley, IA 50124
e current address: USDA-ARS Soybean Germplasm, Pathology & Genetics Unit, Urbana, IL 61801
* Corresponding author (mian.3{at}osu.edu).
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ABSTRACT
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Frogeye leaf spot (FLS), caused by Cercospora sojina K. Hara, is a common disease of soybean [Glycine max (L.) Merr.] in most soybean-growing countries of the world. Significant yield losses of soybean (10–60%) have been attributed to FLS under hot and humid growing conditions. The FLS in the southern United States has been kept under control by planting resistant cultivars. Cercospora sojina is a dynamic pathogen with extensive virulence or race diversity. The current number of C. sojina isolates at the University of Georgia collection is 93. Assigning race designations to this large number of isolates has been difficult because of the lack of a well-defined set of differentials. To facilitate future studies of the genetics of soybean resistance to FLS and the identification and comparison of existing and future races of C. sojina, we have reassessed both the soybean differentials and the C. sojina races. Based on the reactions of 93 isolates of C. sojina on 38 putative soybean differentials, we propose a core set of 12 differentials and 11 races that represent the major diversity among the 93 C. sojina isolates. The definition of these 12 differentials and 11 races should provide the foundation for the identification and comparison of additional soybean resistance genes and new races of C. sojina.
Abbreviations: AFLP, amplified fragment length polymorphism • AUDPC, area under disease progress curve • BSA, bulked segregate analysis • cM, centimorgan • FLS, frogeye leaf spot • LAD, leaf area damaged • LG, linkage group • NIL, near-isogenic line • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication August 4, 2007.
Frogeye Leaf Spot of Soybean: A Review and Proposed Race Designations for Isolates of Cercospora sojina Hara
M. A. R. Miana,*,
A. M. Missaouib,d,
D. R. Walkerb,e,
D. V. Phillipsc and
H. R. Boermab
a USDA-ARS and Dep. of Horticulture and Crop Science, The Ohio State Univ., 1680 Madison Ave., Wooster, OH 44691
b Dep. of Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602-7272
c Dep. of Plant Pathology, Univ. of Georgia–Griffin Campus, Griffin, GA 30223
d current address: Monsanto Co., 1551 Hwy. 210, Huxley, IA 50124
e current address: USDA-ARS Soybean Germplasm, Pathology & Genetics Unit, Urbana, IL 61801
* Corresponding author (mian.3{at}osu.edu).
Frogeye leaf spot (FLS), caused by Cercospora sojina K. Hara, is a common disease of soybean [Glycine max (L.) Merr.] in most soybean-growing countries of the world. Significant yield losses of soybean (10–60%) have been attributed to FLS under hot and humid growing conditions. The FLS in the southern United States has been kept under control by planting resistant cultivars. Cercospora sojina is a dynamic pathogen with extensive virulence or race diversity. The current number of C. sojina isolates at the University of Georgia collection is 93. Assigning race designations to this large number of isolates has been difficult because of the lack of a well-defined set of differentials. To facilitate future studies of the genetics of soybean resistance to FLS and the identification and comparison of existing and future races of C. sojina, we have reassessed both the soybean differentials and the C. sojina races. Based on the reactions of 93 isolates of C. sojina on 38 putative soybean differentials, we propose a core set of 12 differentials and 11 races that represent the major diversity among the 93 C. sojina isolates. The definition of these 12 differentials and 11 races should provide the foundation for the identification and comparison of additional soybean resistance genes and new races of C. sojina.
Abbreviations: AFLP, amplified fragment length polymorphism • AUDPC, area under disease progress curve • BSA, bulked segregate analysis • cM, centimorgan • FLS, frogeye leaf spot • LAD, leaf area damaged • LG, linkage group • NIL, near-isogenic line • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat.
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INTRODUCTION
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FROGEYE LEAF SPOT (FLS) is caused by a fungal pathogen, Cercospora sojina K. Hara, and is a common disease of soybean [Glycine max (L.) Merr.] in most soybean-growing countries in the world (Athow and Probst, 1952; Bernaux, 1979; Akem et al., 1992; Ma, 1994). Frogeye leaf spot was first reported on soybean in Japan in 1915 (Lehman, 1928) and in the United States in 1924 (Melchers, 1925). The disease is most common in the southern U.S. soybean production region but is regularly present in parts of the midwestern United States (Athow and Probst, 1952; Grau et al., 2004). Recently, FLS has moved further northward in the United States. Yang et al. (2001b) confirmed the presence of FLS in Iowa, and Mengistu et al. (2002) reported it in Wisconsin. In 2005 FLS was found in Ohio (Anne Dorrance, personal communication, 2005). It is possible that a combination of warm winter temperatures and cultivation of susceptible soybean cultivars has contributed to the appearance of FLS in these northern states. The practice of conservation tillage that leaves pathogen-infested plant debris on the soil surface may also be a factor in the increased occurrence of this disease. With such an increase in its incidence in the United States, FLS is receiving renewed attention from the soybean research community.
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THE PATHOGEN AND SYMPTOMS
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Cercospora sojina K. Hara is currently recognized as the causal agent of FLS, although earlier literature reported C. daizu as the causal agent of this disease (Athow, 1987). The primary and secondary inocula are hyaline conidia of 5 to 7 µm x 39 to 70 µm, which are produced on leaf and stem residues or infested seeds. Conidia are formed on tips of conidiophores and are pushed aside as the conidiophores continue to grow. The size and shapes of conidia and conidiophores may vary depending on the substrate on which the fungus grows. Conidia can germinate on a leaf surface within an hour of deposition in the presence of water at 25 to 30°C, producing one to a few germ tubes from the end cells and sometimes from the intervening cells. In culture media, conidia frequently germinate to form short germ tubes that may produce secondary conidia (Phillips, 1999).
The FLS disease primarily occurs on foliage of soybean, even though seeds, pods, and stems can also become infected (Sinclair and Backman, 1989). The lesions are circular to angular spots with diameters ranging from 1 to 5 mm (Grau et al., 2004). The lesions initially appear as dark water-soaked spots that sometimes have lighter centers (Fig. 1A
). These spots eventually develop into brown spots surrounded by narrow dark reddish brown margins. However, with ideal conditions for invasion and high inoculum levels adjacent lesions may coalesce to form larger irregular spots (Phillips, 1999) (Fig. 1B). Eventually, lesions may cover more than 30% of the leaf surface, giving the appearance of blight and causing premature defoliation. Stem lesions are less common and generally appear late in the season. The long narrow lesions (two to four times longer than wide) can cover up to half of the stem surfaces. Lesions on pods are circular to elongate and slightly sunken, with a reddish brown color. As the lesions age, they become brown to light gray with narrow dark-brown borders. The fungus can penetrate through the pod walls and infect the maturing seeds in the pods (Phillips, 1999). Symptoms on seeds appear as conspicuous light to dark gray or brown areas that can range from specks to large blotches covering the entire seed coat (Bisht and Sinclair, 1985). The seed coats of infected seeds often crack or flake.
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Figure 1. Characteristic frogeye leaf spot lesions resulting from Cercospora sojina infection of soybean leaves. (A) The lesions start as dark water-soaked spots and develop into well-defined lesions with light centers and dark borders; also shown is the head of an artificial frog. (B) Several adjacent lesions may coalesce and form larger irregular spots.
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DISEASE CYCLE, EPIDEMIOLOGY, AND MANAGEMENT
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The fungus overwinters in infected seeds and in infested soybean residues. The pathogen has not been confirmed to cause disease on any other crop or weed host. Heavily infested discolored seeds have poor germination, and the percentage of germination may be inversely related to the extent of discoloration of the seed surface (Sherwin and Kreitlow, 1952; Phillips, 1999). The seedlings from infected seeds are usually weak and stunted, with lesions on the cotyledons. Spores produced on infected cotyledons become inoculum that may infect young leaves. The lesions do not appear for nearly 2 wk after invasion of the host tissue, so they are not observed on young expanding leaves. For plants grown in warm (25–30°C) and humid conditions (>90% relative humidity), sporulation can occur profusely within 48 h of the appearance of visible symptoms. In areas or seasons with infrequent dews or rainfalls, the occurrence of FLS is sporadic, but it can become severe in seasons with frequent rainfall. Young expanding leaves are highly susceptible, while fully expanded leaves are more resistant to invasion. The lesions that develop on older leaves become progressively smaller and less uniform than lesions that develop on younger leaves. Even with artificial inoculation of plants grown at 25 to 30°C and saturated atmosphere, very few lesions develop on fully expanded leaves. Leaves that expand during periods of weather unfavorable for invasion by the fungus remain relatively disease free (Phillips, 1999).
Initially, seed infection occurs through direct contact with pod lesions, but infection may spread from seed to seed as they mature in the field. Uninfected seeds may become contaminated with spores or mycelium during harvest. Conidia can be carried short distances by air currents and rain splashes. Under favorable conditions, secondary infection of leaves, stems, and pods continue throughout the soybean growing season (Laviolette et al., 1970).
In addition to the planting of resistant cultivars, crop rotation and plowing under crop residues will reduce disease incidence (Grau et al., 2004; Phillips, 1999). Cultivars with resistance to all known FLS races in the United States are currently available. Residue from soybean crops should be plowed deep into the soil and a 2-yr rotation with other crops should be used. Good-quality seeds free of the pathogen should be used and treated with a fungicide. Fungicides applied at the late-flowering and beginning seed (R2-R5) growth stages (Fehr and Caviness, 1977) will protect against C. sojina infection (Grau et al., 2004).
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SEED YIELD LOSS FROM FROGEYE LEAF SPOT
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The seed yield loss of soybean from FLS is mainly due to reduction in photosynthetic leaf area by necrotic lesions and/or premature defoliation resulting in reduced seed weight (Dashiell and Akem, 1991). Yield reductions in the range of 10 to 60% due to FLS have been reported (Akem and Dashiell, 1994; Dashiell and Akem, 1991; Laviolette et al., 1970; Bernaux, 1979; Ma, 1994; Mian et al., 1998). Mian et al. (1998) compared the performance of four pairs of FLS susceptible vs. resistant near-isogenic lines (NILs) grown in five states (South Carolina, Georgia, Florida, Mississippi, and Alabama) in the southern United States. Yield losses for the four susceptible soybean cultivars were as high as 31% compared with the respective resistant NILs. The authors also investigated the relationships of seed yield to two disease parameters: the area under the disease progress curve (AUDPC) and the percentage of leaf area damaged (LAD) at the end of the growing season (R7 stage of development) (Fehr and Caviness, 1977). The AUDPC values were found to be more useful than LAD data in explaining the yield loss due to FLS. This was expected because the yield loss from FLS is mainly a result of reduced photosynthetic area, and the longer the lesions are present, the greater the reduction in photosynthesis. Evaluations of FLS-resistant and FLS-susceptible soybean breeding lines in USDA Uniform Tests indicated yield losses varying from 10% at Quincy, FL (Hartwig and Edwards, 1989), to 30% at Tallassee, AL (Hartwig, 1990).
Mwase and Kapooria (2001) surveyed the incidence and severity of FLS of soybean during the 1997–1998 growing season in agroecological region II of Zambia. Seed yield loss of soybean cultivars due to FLS were evaluated by comparing seed yield of soybean plots that received two applications of benomyl (benlate) with plots that did not receive the fungicide. The average seed yield loss from FLS in untreated plots ranged from 30 to 37% for the cultivars in the study. The yield losses from FLS were mainly due to a reduction in seed size. Differences in weather conditions and amount of inoculum were major factors influencing variation in incidence and severity of the disease among locations.
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RACES OF CERCOSPORA SOJINA AND GENETIC RESISTANCE IN SOYBEAN
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In the United States, the use of resistant cultivars reduced the incidence of FLS to a great extent until the late 1950s, when C. sojina race 2 appeared (Athow et al., 1962). In the mid-1960s, C. sojina races 3 and 4 were discovered in North Carolina (Ross, 1968), and race 5 was identified in 1978 (Phillips and Boerma, 1981). Most cultivars were susceptible to race 5 of C. sojina, although resistant cultivars were also available. Three single genes conditioning resistance to C. sojina are currently recognized by the Soybean Genetics Committee. Rcs1 in ‘Lincoln’ was the first gene found that conferred resistance to race 1 of C. sojina (Athow and Probst, 1952), Rcs2 for resistance to race 2 was identified in ‘Kent’ (Athow et al., 1962), and Rcs3 from ‘Davis’ was found to condition resistance to race 5 and to all other known races of C. sojina in the United States (Boerma and Phillips, 1983; Phillips and Boerma, 1982) as well as to all known Brazilian isolates (Yorinori, 1992). Other dominant genes for resistance to race 5 were found in ‘Ransom’, ‘Stonewall’, and ‘Lee’ in 1993, and each of these genes was nonallelic to Rcs3 and to each other (Pace et al., 1993). These are not considered to be important sources of resistance, however, because currently, race 5 is not seen as an economic threat to soybean in the United States (Baker et al., 1999). Another single dominant gene (reported as nonallelic to Rcs3) from the cultivar Peking was found later that provided resistance against many isolates of C. sojina (Baker et al., 1999).
Damage from FLS is considered to be a major constraint to soybean production in the Heilongjiang province of China. Many studies on resistance of soybean to FLS have been conducted in China (Liu and Huang, 1986; Zhang et al., 1990). Huo et al. (1988) reported 11 races of C. sojina in China. Among these, races 1, 7, and 10 were considered the major ones. The gene symbol Rcsc7 was assigned to a dominant gene for conditioning resistance to Chinese race 7 (Zou et al., 1999). Because the allelism between Rcsc7 and other resistance genes is not known, the Rcsc7 gene symbol has not been officially approved by the Soybean Genetics Committee.
Gravina et al. (2004) measured the reaction to C. sojina as a quantitative rather than qualitative trait. They created F1 plants from the diallel mating of seven soybean cultivars (Bossier, Cristalina, Davis, Kent, Lincoln, Paraná, and Uberaba). The F1 plants and parents were evaluated for their reactions to C. sojina race 4 using a multivariate variable developed from five soybean reaction characteristics (infection degree, mean lesion diameter, percent of lesioned leaf area, lesions per square centimeter, and disease index). Although the importance or need for this multivariate variable to quantify the disease reaction was unclear, they did report that Davis, Cristalina, and Uberaba were basically free of C. sojina infection.
While deployment of cultivars with genetic resistance to FLS is the most cost effective and environmentally benign method of controlling this disease, the effectiveness of resistance genes depends on the population dynamics of pathogen races. Because pathogens often overcome resistance provided by single genes (R-gene), the widespread use of a few resistance genes may lead to the evolution of new pathogenic races that can overcome the resistance genes in use (Browning and Frey, 1969; National Academy of Sciences, 1972). The appearance of new virulent C. sojina races, such as race 2 in the late 1950s (Athow et al., 1962), races 3 and 4 in the mid 1960s (Ross, 1968), and race 5 in the late 1970s (Phillips and Boerma, 1981), and the associated breakdown of the deployed FLS resistance have demonstrated the need to identify multiple sources of genetic resistance to this pathogen.
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MOLECULAR MAPPING OF FROGEYE LEAF SPOT RESISTANCE GENES
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Conventional approaches to breeding for disease and pest resistance require development of segregating populations derived from crosses between resistant germplasm sources and susceptible but otherwise desirable and productive cultivars. The resistant genotypes need to be selected in early generations by screening large segregating populations either at natural disease "hot-spots" or in disease nurseries under artificially controlled environments. Screening of a large number of segregating soybean plants in early generations for FLS resistance requires greenhouse facilities and technical skills for C. sojina culture maintenance, preparation of inoculum, inoculation techniques, and scoring of plants for disease expression. Marker-assisted selection for this trait would therefore be an attractive alternative because it would greatly reduce the need for early generation phenotypic screening.
Although only the Rcs3 gene has been located on the soybean genetic map, several researchers have identified unmapped DNA markers associated with resistance to several races of C. sojina (see http://soybase.org/). The Rcs3 gene was mapped on soybean linkage group (LG) J using simple sequence repeat (SSR) and restriction fragment length polymorphism (RFLP) markers (Mian et al., 1999). A combination of bulked segregate analysis (BSA) (Michelmore et al., 1991) and comparisons of NILs (Muehlbauer et al., 1988) was used for mapping this gene. The Rcs3 gene was mapped near a known resistance gene cluster on soybean LG J. The gene was mapped within a 2-centimorgan (cM) interval between SSR markers Satt244 and Satt547. Several disease resistance genes, including Rbs1, Rbs2 (Bachman et al., 2001), and Rbs3 (Webb, 1997) for resistance to brown stem rot (causal agent Phialophora gregata), have been mapped within 10 cM from the Rcs3 gene (Bachman et al., 2001). Another resistance gene cluster of Rps2, Rmd, and Rj2 was mapped at a distance of approximately 20 cM from the Rcs3 gene (Polzin et al., 1994). Rps2 conditions resistance to root and stem rot (causal agent Phytophthora sojae), Rmd conditions resistance to powdery mildew (causal agent Microsphaera diffusa), and Rj2 controls Bradyrhizobia japonicum–mediated nodulation.
Filho et al. (2002) used the BSA technique to screen three segregating F2 populations (susceptible ‘Bossier’ x resistant ‘Parana’, ‘Cristalina’, and ‘Uberaba’) with random amplified polymorphic DNA (RAPD) (Williams et al., 1990) markers for tagging and identifying the gene(s) for FLS resistance in these cultivars. The RAPD markers linked to the FLS resistance in these populations were in turn linked to SSR markers Satt431 and Satt547 in the same region of LG J in which Rcs3 was mapped by Mian et al. (1999). From these results, Filho et al. (2002) inferred that the FLS resistance gene in Parana, Cristalina, and Uberaba was at the Rcs3 locus. These authors found that the segregation ratios of two of the F2 populations (Cristalina x Bossier and Parana x Bossier) were consistent with a single dominant gene model (3-resistant to 1-susceptible progeny) for FLS resistance. The F2 population from the cross of Uberaba x Bossier, however, had a segregation ratio of 13:3, indicating that at least two loci were controlling the FLS resistance in this population. Cordeiro (1986) reported that two loci controlled C. sojina race 4 resistance in a cross between ‘Santa Rosa’ and Bossier. Santa Rosa is a progenitor of Uberaba.
In the literature, there is some confusion regarding the FLS resistance gene(s) in ‘Peking’. Baker et al. (1999) reported a 15:1 resistant to susceptible ratio of F2 plants from a cross of Davis and Peking when progeny were inoculated with a mixture of field-collected C. sojina isolates. They concluded that Peking contained a single dominant gene that segregated independent of Rcs3. Yang et al. (2001a) mapped a FLS resistance gene from Peking to the same genomic region on soybean LG J that contains Rcs3. In this study, the F2 plants and parents were inoculated in the field with an unidentified isolate of C. sojina. The resistance gene in Peking was mapped in a 2.1-cM interval between Satt244 and an amplified fragment length polymorphism (AFLP) marker, AACCTA178, positioned 1.1 cM from Satt244 and 1.0 cM from AACCTA178. While AFLP marker AACCTA178, a 178-nucleotide fragment, was present in Peking, this fragment was absent in Davis. Also, Satt244 amplified a 195-nucleotide fragment in Peking as opposed to the 154-nucleotide fragment amplified in Davis. Based on this evidence, Yang et al. (2001a) suggested that the Peking resistance gene was linked, but at a different locus than Rcs3, and that both are located within the same gene cluster.
A different interpretation of these data is that Peking contains a different resistance allele than Davis at the Rcs3 locus and a second FLS resistance gene at an independent locus. Reconciling the results from these studies requires the assumption that the plants in each study were inoculated with different isolates of C. sojina. Given that the initial study was undertaken with a mixture of isolates of C. sojina and that the inoculum source was not clearly described in the second study, it is likely that the C. sojina isolates differed. If these assumptions are correct, the 15:1 resistant to susceptible ratio among F2 plants from the Davis and Peking population reported by Baker et al. (1999) may have resulted from segregation of the Rcs3 allele from Davis (Peking's unique allele at the Rcs3 locus or a tightly linked gene did not provide resistance to this isolate) and a resistance allele from Peking at an independent locus. In the F2 population of Peking and Lee evaluated by Yang et al. (2001a), the Peking allele at the Rcs3 locus conditioned resistance to the isolate of C. sojina used as inoculum, and the Peking allele at the independent locus did not provide resistance. This provides a logical explanation for the results of the two studies and illustrates the importance of knowing the specific race of the pathogen and the specific allele at a resistance locus when conducting genetic studies with a host and a pathogen.
Zou et al. (1999) identified two dominant markers and one codominant RAPD marker that were linked to the FLS resistance gene Rcsc7. (Note that although the Rcsc7 gene symbol was assigned by the authors of the publication, the authors did not conduct allelism tests to Rcs1, Rcs2, or Rcs3 and the gene symbol has not been approved by the Soybean Genetics Committee).
A number of the inheritance and resistance gene mapping studies described above demonstrate the need for researchers to understand both the soybean genotype and the C. sojinia race under study to properly elucidate the specific soybean resistance gene(s) involved in a soybean plant's reaction to C. sojina.
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A NEW CLASSIFICATION OF C. SOJINA ISOLATES
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In a recently published soybean monograph, Grau et al. (2004) referred to 12 races of C. sojinia reported from various states in the United States and indicated that more are likely present. In addition, they indicated that 22 races have been reported in Brazil (Yorinori, 1992) and 14 races in China (Ma and Li, 1997). Grau et al. (2004) point out that different sets of soybean differential cultivars were used to identify the C. sojina races in the United States, Brazil, and China. There is a lack of a universally accepted set of soybean differential cultivars for the classification of C. sojinia isolates into races to identify, designate, and compare races of this pathogen. Thus, it is impossible to properly conduct genetic studies on soybean resistance to FLS. To address this void, we have attempted to identify a new set of soybean differential cultivars and revise the C. sojinia race designations with the purpose of providing a starting point both to advance characterization of C. sojinia races and to allow identification of additional FLS resistance genes in soybean.
In the past 25 yr, one of the authors of this paper (D.V. Phillips) has systematically collected C. sojina isolates from around the world. These include 71 isolates from the United States, 15 isolates from Brazil, and 7 isolates from China (Table 1
). In total, 93 C. sojina isolates have been analyzed for their reaction on 38 putative soybean differential cultivars (Missaoui et al., 2007). This research was conducted by various individuals over a long time period, and there was a wide range in the number of replications of the various C. sojina isolate–soybean differential evaluations. In addition, these 3534 isolate–differential combinations have never been included in a single experiment because of logistical issues and the requirement for the 93 isolates to possess an adequate level of virulence at the same time.
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Table 1. Collection of 93 Cercospora sojina isolates at the University of Georgia, used in a new classification system of races of this soybean pathogen.
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To facilitate future studies of the genetics of soybean resistance to FLS and the identification of existing and future races of C. sojina, we felt there was a need to reassess both the set of soybean differential cultivars and C. sojina races. Although there are risks associated with this type of reassessment, we believe that a conservative approach to this issue would allow designation of a standard set of soybean differential cultivars for the classification of C. sojina isolates into distinct races and facilitate studies of both the genetics of FLS resistance in soybean and the virulence diversity of C. sojina.
Greenhouse Screening for Frogeye Leaf Spot Resistance
The following protocol was used to determine the FLS reactions of the 38 soybean differential cultivars that were inoculated with 93 isolates of C. sojina. This protocol has been used for FLS screening in our breeding program for the past 25 yr. Soybean plants from each of the 38 differential cultivars were evaluated in the greenhouse at the University of Georgia– Griffin Campus, Griffin, GA. The plants were grown in 10-cm plastic pots on a greenhouse bench. At the V2–V3 stage of development (Fehr and Caviness, 1977), the seedlings were inoculated with the various C. sojina isolates. Cultures of each C. sojina isolate were maintained and the inoculum was produced on a medium composed of equal parts of soybean stem agar and lima bean (Phaseolus lunatus L.) agar. Conidial suspensions were made by flooding colonies of the fungus growing on agar in petri plates with sterile water and then lightly scraping the colonies to dislodge conidia. The suspensions were passed through several layers of cheesecloth to remove large mycelial fragments. Conidial suspensions from agar cultures were adjusted to a concentration of 4 x 104 to 6 x 104 spores mL–1. One trifoliolate leaf per plant was inoculated by atomizing a conidial suspension (
0.3 mL) onto the upper leaf surface. The inoculated plants were enclosed in clear plastic bags for 48 h to maintain a high relative humidity. Disease ratings were made 14 d after inoculation. The FLS reaction was scored as a qualitative trait (i.e., susceptible vs. resistant). Plants that showed numerous, predominately large lesions with light centers and dark margins were classified as susceptible. Plants that showed no lesions or only flecks or predominately small lesions without clearly differentiated light centers were classified as resistant. Plants classified as resistant were reinoculated on younger leaves to eliminate possible escapes.
Data Analyses
The reaction (resistance and susceptibility) of each soybean differential cultivar to the 93 races was coded as 1 for a resistant reaction or 0 for a susceptible reaction. The resulting matrix of binary characters was used to generate a matrix of Jaccard similarity values using SYSTAT 11 (version 11.0, SYSTAT, Richmond, CA). The Jaccard index is commonly used to compare associations limited to absence or presence data, and the Jaccard coefficient is defined as the number of variables that are coded as 1 for both states divided by the number of variables that are coded as 1 for either or both states (Jaccard, 1908; Falouss, 1989; Wolda, 1981). Jaccard distances were calculated from the similarity matrix by subtracting from 1 and were used for cluster analysis using Ward's minimum-variance criteria. Ward's method applies an analysis of variance approach for the evaluation of distances between clusters and attempts to minimize the sum of squares of any two hypothetical clusters that can be formed at each step (Ward, 1963). A dendrogram showing the various clusters (Fig. 2
and 3
) was produced by SYSTAT 11.
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Figure 2. Ward minimum variance cluster analysis of 38 soybean differentials grouped based on their reaction (resistance and susceptibility) to 93 isolates of Cercospora sojina. Distances represent semipartial R2 values. These are equal to the between cluster sum of squares divided by the corrected total sum of squares and correspond to the decrease in the proportion of variance accounted for as a result of joining the two clusters. The bolded and underlined cultivars represents those that were selected as the differentials representing each group.
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Figure 3. Ward minimum variance cluster analysis of 93 isolates of Cercospora sojina grouped based on the reaction (resistance and susceptibility) of the isolates on 10 soybean differential cultivars. Numbers below the dendrogram refer to semipartial R2 values, which correspond to the decrease in the proportion of variance taken into account, because of joining the two clusters. Numbers in parentheses refer to the specific clusters.
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Cluster Analyses and Proposed Classification of C. sojina Isolates
Resistance or susceptibility to the 93 isolates of C. sojina was initially used to group the 38 differentials into clusters of similarity (Fig. 2). From each cluster, a representative soybean differential cultivar was selected on the basis of its previously known reaction (resistance or susceptibility) to known races of C. sojina and the extent of its contribution to the ancestry of the major North American public cultivars (Gizlice et al., 1994). Initially, a total of 10 differentials were selected (Table 2
, Fig. 2). Lincoln, Kent, and Davis were chosen because they are sources of the Rcs1, Rcs2, and Rcs3 resistance genes, respectively. Peking, ‘CNS’, ‘Palmetto’, ‘Tracy’, and ‘S-100’ are all ancestors of cultivars developed for the southern United States, whereas ‘Richland’ was an ancestor of many cultivars developed for northern growing regions. ‘Blackhawk’ was included because of its universal susceptibility to FLS. These differentials were also chosen in part because, as a set, they span the range of reactions from highly susceptible (Blackhawk) to virtually immune (Davis) when challenged with the 93 isolates of C. sojina in the University of Georgia collection.
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Table 2. Resistance index of 12 soybean differential cultivars to 93 isolates of Cercospora sojina and their selection criteria.
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The reactions (resistance or susceptibility) of the 10 selected differentials to the 93 C. sojina isolates were used to cluster these isolates using the SYSTAT 11 software as described earlier (Fig. 3). A semipartial R2 of 0.605 and a distance of 0.17 were used as the cutoff threshold for the delimitation of the clusters. Grouping of the 93 isolates resulted in 13 major clusters ranging in size from 3 to 25 isolates (Fig. 3, Table 3
). Clusters 2, 10, and 11 contained only U.S. isolates. Cluster 1 was composed of 25 isolates, containing 24 U.S. and 1 Brazilian isolates. Clusters 9 and 12 each contained two U.S. and two Brazilian isolates. Both Clusters 6 and 13 contained isolates from China and the United States, while the remaining clusters (3, 4, 5, 7, and 8) contained isolates from the United States, Brazil, and China.
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Table 3. Geographic distribution of the 93 Cercospora sojina isolates among the 13 clusters based on the reaction of the isolates on 10 soybean differential cultivars.
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Of the 93 isolates, 67 were collected in the southern United States. Of these, 36 (54%) were grouped into Cluster 1 or 2 (Table 3). The relatively high number of southern U.S. isolates was a result of the higher incidence of FLS in this region during the past 30 yr. Of the seven isolates from China, each one grouped into a different cluster, indicating a high level of diversity among these seven isolates. The 15 Brazilian isolates were grouped into eight clusters, with clusters 4, 7, 8, 9, and 12 each containing two or more isolates.
To establish a contemporary race structure for C. sojina and confirm the repeatability of the reaction types on the potential set of soybean differential cultivars (Table 2), we attempted to select one representative C. sojina isolate from each of the 13 clusters (with the exception of clusters 4 and 8, from which we included two isolates). Based on our previous experience with these isolates and the initial group of 38 soybean differential cultivars, we also included ‘Hood’ and Lee as potential differentials in our confirmation experiment. Based on this experiment, we were successful in identifying virulent C. sojina isolates to represent clusters 1 through 8 and cluster 12 (Fig. 3). These nine clusters include 75 of the 93 C. sojina isolates in the University of Georgia collection. Several attempts to identify a virulent isolate to represent cluster 9, 10, 11, and 13 were unsuccessful. The observed loss of virulence or decline in aggressiveness of these isolates was one of the compelling reasons to initiate this study.
The successful differentiation of the remaining 11 C. sojina isolates into 11 distinct races required the inclusion of soybeans Hood and Lee as differential cultivars (Table 4
). Although the soybean differential cultivars Davis, Peking, and Kent have the same reaction for these 11 races, they are retained as differentials because Davis is the source of Rcs3 gene, Kent is the source of Rcs2 gene, and Peking either has a different allele at the Rcs3 locus than Davis or another FLS resistance gene that is tightly linked to the Rcs3 locus.
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Table 4. Proposed races of Cercospora sojina based on their reaction on 12 soybean differential cultivars. Positive signs represent compatible reactions (susceptibility), and negative signs represent incompatible reactions (resistance).
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We have initiated the new race structure with race 5. Since this race was described in the literature by Phillips and Boerma (1981), it has been maintained at the University of Georgia as isolate S5. There are no known existing cultures of races 1 to 4. Therefore, we made the decision to begin the new race structure with the known race 5. Any arbitrary assignment of an existing C. sojina isolate to the previously described races 1 to 4 would result in potential problems with their use in soybean genetic studies or in relating new research with these races to the previous literature.
We have described the reactions of 12 soybean differential cultivars to 11 C. sojina isolates and proposed their designations as race 5 to 15 (Table 4). For each of these 11 races, the specific isolate(s) and the cluster it represents are listed. For example, isolate S5 from cluster 5 is the original C. sojina isolate described as race 5 by Phillips and Boerma (1981), and the isolate S127 from cluster 2 was designated race 15. The specific isolates were selected on the basis of their virulence on Blackhawk and their ease of sporulation. This study has identified 11 unique isolates, designated races as 5 to 15, to represent the virulence diversity present in the University of Georgia C. sojina collection. These 11 C. sojina races will be deposited in the American Type Culture Collection and maintained as a working collection by the senior author (R. Mian, Wooster, OH).
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Received for publication August 4, 2007.
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