Crop Science 42:1818-1823 (2002)
© 2002 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Analysis of Combining Ability for Ergot Resistance in Grain Sorghum
J. D. Reeda,
M. R. Tuinstra*,a,
N. W. McLarenb,
K. D. Kofoidc,
N. W. Ochandaa and
L. E. Claflina
a Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS, 66506
b ARC-Grain Crops Institute, Private Bag X1251, Potchefstroom, 2520, Republic of South Africa
c Kansas State Univ. Agric. Res. Center, Hays, KS, 67601
* Corresponding author (mtuinstra{at}bear.agron.ksu.edu)
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ABSTRACT
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Ergot caused by Claviceps africana Frederickson, Mantle & de Milliano has become an important constraint to F1 hybrid seed production in North and South America. Identification and utilization of sources of host-plant resistance would contribute to the effective control of this disease. A Design II mating scheme was used to test the combining ability of four reported sources of ergot resistance in sorghum [Sorghum bicolor (L) Moench]—IS8525, IS14131, IS14257, and IS14357. Male-sterile hybrids of these accessions and a susceptible check, TxARG1, were produced using five cytoplasmic-genetic male-sterile seed parents—A3Tx430, A3Tx436, A3Tx7000, A3KS70, and A3Tx2737. Parent lines and hybrids were planted in three replications at three locations and were evaluated for ergot resistance following artificial inoculation at flowering. Differences in ergot severity and ergot breakdown point (EBP) were used to quantify differences in resistance. The combined analysis of variance showed that the expression of ergot resistance was not stable with significant entry x location interaction. The analyses from two locations showed significant differences among hybrids, while the third location showed no differences. Regression analyses were performed to determine the relationship between weather variables at flowering and observed ergot severity. These analyses indicated a strong relationship between maximum daily temperature and ergot severity. Ergot infection was higher in cooler environments regardless of genetic background. IS8525 appeared to have the highest expression of ergot resistance in male-sterile genetic backgrounds on the basis of EBP and ergot severity ratings; however, the expression of resistance was only effective within a limited temperature range.
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INTRODUCTION
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ERGOT CAUSED by C. africana is a fungal disease of sorghum infecting unfertilized ovaries (Frederickson et al., 1994). Ergot epidemics in seed and grain production fields can lead to yield loss, harvesting difficulties, and reduced seed quality (Bandyopadhyay et al., 1998). Ergot can be particularly devastating in hybrid seed production fields because of the use of male-sterile seed parents, which are highly susceptible to the disease. The recent and global spread of ergot has forced hybrid sorghum seed producers and researchers to change cultural practices and management strategies to avoid financial losses (Bandyopadhyay et al., 1998). Fungicides represent one control option, but a more economical method, such as genetic resistance, would be useful for maintaining low seed costs.
Significant genetic variability exists within sorghum. The African continent is believed to be the origin of both sorghum (Doggett, 1970) and the North and South American isolates of C. africana (Paoutová et al., 2000). Given the close relationship and similar geographic origin of these species, the possibility of host-plant resistance in the sorghum gene pool exists. Genetic resistance to sorghum ergot (C. africana and C. sorghi Kulkarni, Seshadri & Hegde) has been reported (Sundaram, 1971; Khadke et al., 1978; Frederickson et al., 1994; Tegegne et al., 1994; Dahlberg et al., 1998; Ramundo et al., 1999), but, in most cases, resistance appears to be a pollen-mediated disease escape. While this resistance mechanism is useful in fertile hybrids, it cannot be used in seed parent nurseries (Frederickson et al., 1994; Bandyopadhyay et al., 1998). Ergot resistance must be functional in male-sterile backgrounds to be considered effective for use in seed parent lines.
Dahlberg et al. (1998) first confirmed nonpollen-based ergot resistance in sorghum in IS8525. Intermediate resistance was expressed in male-sterile hybrids of IS8525 inoculated with the pathogen. Ramundo et al. (1999) identified IS14131 and IS14257 as two additional sources of resistance that also expressed intermediate levels of resistance in male-sterile backgrounds (Reed et al., 2002). While male-sterile hybrids of these accessions were shown to be more resistant than male-sterile controls, a more comprehensive evaluation of the combining ability and genotype x environment interactions of these accessions is needed. Ramundo et al. (1999) also identified IS14357 as another potential source of resistance, but this accession was not tested in male-sterile backgrounds.
Host-pathogen x environment interactions play an important role in determining ergot severity in a genotype at a given location. Minimum temperature 23 to 27 d before flowering, and maximum temperature and maximum relative humidity 1 to 5 d after anthesis, are significantly correlated with increased ergot severity (McLaren and Wehner, 1990; McLaren, 1992; McLaren and Flett, 1998). Thus, if ergot severity measurements are not taken on dates representing variable climatic conditions, false conclusions about the resistance of a genotype can be drawn. Combining ability studies for ergot resistance can allow for an effective measure of parent line usefulness under specified conditions, but these studies will not provide a reliable measure of ergot resistance without adequate sampling of environments. To measure ergot resistance of a genotype accurately, it must be quantified across variable environments.
McLaren (2000) developed a method to quantify ergot resistance of a sorghum genotype on the basis of previously determined relationships between environmental conditions and observed ergot severity. This method makes use of the relationship between observed and predicted ergot severity to predict an Ergot Breakdown Point (EBP) specific to a genotype. Genotypic EBPs standardize genotypic responses to variable environments and can be used to compare ergot resistance levels across genotypes.
IS8525, IS14131, IS14257, and IS14357 appear to have some level of ergot resistance not related to efficient fertilization. An analysis of combining ability in male sterile hybrids is needed before these putative sources of ergot resistance can be effectively used in sorghum improvement programs. The objectives of this study were to evaluate the combining ability of IS8525, IS14131, IS14257, and IS14357 for ergot resistance in male-sterile genetic backgrounds and to quantify that resistance on the basis of EBP as described by McLaren (1992).
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MATERIALS AND METHODS
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The hybrids for this study were developed by intercrossing five A3 cytoplasmic-genetic male-sterile sorghum seed parents (A3Tx430, A3Tx436, A3Tx2737, A3Tx7000, and A3KS70) with four ergot resistant and one susceptible pollinator lines (IS8525, IS14131, IS14257, IS14357, and TxARG1) using a Design II mating scheme (Comstock and Robinson, 1952). These pollinator lines do not possess fertility restoration genes, therefore the hybrids resulting from these crosses are male sterile. This genetic system allows the F1 hybrids to be evaluated for ergot resistance independent of male-fertility characteristics.
The parent lines and hybrids were evaluated for ergot severity at Guayanilla, Puerto Rico, Puerto Vallarta, Mexico, and Bethlehem, Cedara, and Potchefstroom, South Africa, under artificial inoculation in 2000-2001. Entries tested at each location included the 10 parent lines and 25 hybrids from the Design II mating scheme, as well as the five maintainer lines (male fertile) of the A3 testers used in these experiments. These entries were planted in a randomized complete block design with three replications in Puerto Rico and Mexico. Each entry was planted over several dates at the three South Africa locations to provide a wide range of environmental conditions during and before flowering. Each South Africa location was considered a replication in a randomized complete block design. Entries were grown in single-row plots (5.08 x 0.76 m) with 100 seeds per row.
Inoculum was prepared as a spore suspension of C. africana in water (approximately 1 x 106 spores mL-1) as described by Tegegne et al. (1994). Nine random heads in each plot were tagged to indicate flowering and initial inoculation date and were spray inoculated with the spore suspension until run off. These heads were inoculated every other day until anthesis was complete. Approximately 1 mo after initial inoculation, ergot severity, quantified as the percentage of infected florets per panicle, was rated for each inoculated head in the plot.
In Puerto Rico and Mexico, weather data were collected within experimental plots with a relative humidity–temperature data logger. South African weather data were collected at First Order weather stations located within 400 m of the trials at Bethlehem and Cedera and 600 m of the trials at Potchefstroom. Pentad values (mean of 5 consecutive days) of maximum temperature, minimum temperature, and relative humidity were collected at each location through the duration of the experiments.
Mean ergot severity values were calculated on a plot mean basis in each experiment. Analyses of variance (ANOVA) for ergot severity were conducted by means of the PROC MIXED procedure of SAS (SAS, 1990). Analyses of variance and estimates of combining ability were carried out by established methods (Hallauer and Miranda, 1988; McIntosh, 1983). The three locations were considered random effects and entries were considered fixed effects. Tests of significance for entry, inbred, inbred vs. hybrid, hybrid, male general combining ability (GCA), female GCA, and male by female specific combining ability (SCA) for ergot severity were carried out by testing these mean squares against their respective environmental interaction. Each environmental interaction term was tested against the pooled error mean square. GCA estimates were calculated according to Hallauer and Miranda (1988) and standard errors for GCA effects were calculated by methods established by Cox and Frey (1984) where SEGCA = {MSml[(m - 1)/mflyr]}1/2 or {MSfl[(f - 1)/mflyr]}1/2.
The predisposition variable in the multiple regression model described by McLaren and Flett (1998) attempts to take into account the effects of preflowering cold-induced sterility on male-fertile plants. Because male-sterile plants are already 100% predisposed to ergot infection in terms of pollen-mediated resistance, the resistance of male-sterile hybrids in this study was quantified on the basis of a regression of observed ergot severity vs. mean maximum temperature 1 to 5 d after anthesis. Nonlinear regression analyses were used to determine the relationship between mean observed ergot severity across all locations and mean maximum temperature 1 to 5 d after anthesis for all male-sterile hybrids associated with a particular male parent. Since highly susceptible male-sterile rather than male-fertile hybrids were evaluated in this study, differences in disease resistance were quantified by an EBP of 60% rather than 5% as originally described by McLaren (2000).
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RESULTS
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Mean ergot severity ratings were analyzed across locations to determine the significance of location and location interaction effects. The combined analysis indicated that each of the main effects was highly significant (Table 1)
. Significant entry x location interaction effects indicated that differences in ergot resistance were influenced by location. A separate analysis of each location was conducted to determine the basis of this interaction effect.
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Table 1. Mean squares from the combined analysis of variance for ergot (Claviceps africana) severity for inbred lines and their hybrids under artificial inoculation at three locations.
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The mean squares for analyses of variance of individual experiments are presented in Table 2
. Significant differences among entries were observed at each location. The entry effects were partitioned to determine the magnitude and significance of variation between and among parent lines and hybrids. At each location, the analyses of variance indicated significant differences among inbred parent lines (Table 2). Among inbreds, most of the variation was associated with the male vs. female effect. The differences among males and females were large and highly significant at each location as shown in the comparisons of mean ergot severity scores in Table 3
. The average ergot severity ratings for the male parents ranged from 7 to 9% depending on the location. In contrast, the ergot severity ratings for females ranged from 66 to 85%. The highest ergot severity ratings were observed in South Africa for both male and female parents. The analysis of inbred vs. hybrid effects (Table 2) revealed significant differences between the inbred and hybrid groups. The analysis of entry means indicated that the hybrids as a group were much more susceptible to ergot infection than the inbred lines, particularly the male parent lines (Table 3).
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Table 2. Mean squares from the analyses of variance for ergot severity among parent lines and hybrids under artificial inoculation in Puerto Rico, Mexico, and South Africa.
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Average ergot severity scores for hybrids ranged from 42 to 86% depending on the location. The average ergot severity scores for hybrids in Puerto Rico and Mexico were 42 and 43%, respectively; however, the scores in South Africa were much higher with an average rating of 86% (Table 3). Significant differences among hybrids were observed in Puerto Rico and Mexico, but these differences were not evident in South Africa. In Puerto Rico and Mexico, differences among hybrids were primarily related to male parent GCA effects; some specific combining ability effects were noted in Mexico (Table 2).
Estimates of GCA for ergot severity were calculated to determine the stability of hybrid performance for male and female parents across locations (Table 4)
. Significant differences in GCA among male parents were observed in Puerto Rico and Mexico. TxARG1 was the susceptible male parent check and had a high positive GCA value for ergot severity at these locations, indicating that hybrids produced using TxARG1 tended to be more susceptible to ergot infection than other male parents in these environments. IS8525 and IS14131 had high negative GCA values in Puerto Rico, indicating that hybrids produced from these parents were more resistant to ergot at this location (Table 4). IS14357 had a high negative GCA value for ergot severity in Mexico, which shows that its hybrids were more resistant at this location. There were no consistent patterns in GCA effects for male parents across all locations; however, TxARG1 was more susceptible than average and IS8525 was more resistant than average on a numerical basis at all locations (Table 4). There was little variation in combining ability for ergot resistance among the female parent lines evaluated in this test (Table 4).
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Table 4. Estimates of general combining ability for ergot severity under artificial inoculation in Puerto Rico, Mexico, and South Africa.
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The range in mean maximum temperature at anthesis at each location and the observed mean ergot severity scores for all hybrids are reported in Table 5
. The temperature regimes at each location differed and increased ergot severity was correlated with cooler daytime temperatures. The relationship between mean maximum temperature 1 to 5 d after flowering and observed ergot severity appeared to be nonlinear. Regression analyses of ergot severity for hybrids with each male parent yielded the models presented in Fig. 1
. These models accounted for 39 to 61% of the variation in observed ergot severity. Most of the resistant parent lines showed good levels of resistance in male-sterile hybrids at temperatures above 28°C, but resistance was not effective under cooler conditions, particularly below 25°C (Fig. 1). The male-sterile hybrid EBPs (60%) summarized by common male pollinator parent are presented in Table 6
. The EBPs for hybrids produced from IS8525 and IS14131 were lowest with values of 27.66 and 28.16, respectively. The 60% EBP for hybrids produced from IS8525 was nearly 1.76°C lower than for TxARG1.
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Table 5. Range in maximum temperature 1 to 5 d post-anthesis and mean ergot scores of all male-sterile hybrids at three locations.
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Fig. 1. Relationship between mean maximum temperature 1 to 5 d after flowering and observed ergot severity in all male-sterile hybrids with the common male parent (a) IS8525, (b) IS14257, (c) IS14357, (d) IS14131, and (e) TxARG1.
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Table 6. Ergot Breakdown Points (60%) for male-sterile hybrids summarized by common male parent line calculated using the relationship between mean maximum temperature 1 to 5 days after flowering and observed ergot severity in all male-sterile hybrids with a common male parent.
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DISCUSSION
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This study of genetic resistance to ergot confirmed previous studies involving IS8525 and IS14131. Dahlberg et al. (1998) found low levels of infection in IS8525 and intermediate infection in male-sterile hybrids for this line when tested in Puerto Rico. Reed et al. (2002) observed high levels of resistance in IS14131 and moderate resistance in male-sterile hybrids produced using this line in a field study in Puerto Rico and in a greenhouse study. Similar patterns and levels of resistance were observed for these accessions in this study; however, these sources of resistance were not stable across diverse environments.
Ergot susceptibility is greatly influenced by male-fertility characteristics. Ergot only attacks unfertilized ovaries and male-fertile entries are typically more resistant to ergot than male-sterile entries because of pollen-mediated escape (Futrell and Webster, 1965). Other characteristics including protogynous florets and stigma drying time can also influence ergot resistance (Dahlberg et al., 2002). In this study, the largest differences in ergot severity were generally associated with comparisons between male-fertile and male-sterile entries or groups of entries. This was fully consistent with all reports and has previously been documented (Bandyopadhyay et al., 1998).
Environmental conditions can also have a tremendous effect on ergot resistance. In this study, lines and hybrids expressed greater susceptibility to ergot in cool environments. This can be explained, in part, by the effect of environment on male-fertility characteristics of sorghum. Previous studies have shown that variations in temperature and humidity can influence pollen production and viability (Artschwager and McGuire, 1949; Brooking, 1979). Models have been developed that accurately predict susceptibility of sorghum lines and hybrids to ergot infection on the basis of low temperature effects on male fertility (McLaren and Wehner, 1990). Cooler environments are also more conducive to the growth and survival of C. africana with an optimal temperature of approximately 19°C (Bandyopadhyay et al., 1998). The increased infection of male-sterile hybrids in cooler environments observed in this study was probably explained by the fact that these environments are more conducive to growth and survival of C. africana than warmer environments.
The combining ability analyses in this study indicated that while ergot resistance is heritable and can be transmitted to male-sterile progeny, this resistance is not stable across environments. This instability may, in part, be explained by the fact that resistance is not maintained under cooler conditions; however, even in environments with similar temperature profiles, such as Puerto Rico and Mexico, the expression of resistance tended to be variable. The bases for these interaction effects were not clear.
The expression of resistance in terms of EBP provides a slightly different measure of performance as it provides an indication of the temperature range in which resistance is effective. The EBPs for the sorghum lines evaluated in this test were lower than the EBP for the susceptible check; however, the differences were small. Taken as a whole, the ergot severity data and the EBP models suggest that IS8525 might be the most useful source of ergot resistance in male-sterile seed parent lines. It is important to note that in cool environments, fungicide applications might be needed for additional protection since genetic control is limited under these conditions. Cultural management to maximize pollen flow will also continue to be important in the control of this disease because of the effectiveness of rapid pollination in control of ergot in sorghum.
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CONCLUSIONS
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Combining ability analyses revealed that the ergot resistance trait was not stable across variable environments and significant location interactions occurred. The expression of ergot resistance in male-sterile hybrids was only useful within a limited temperature range during flowering. IS8525 appears to be the best source of resistance for use in sorghum seed parent lines.
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NOTES
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Contribution No. 02-223-J from the Kansas Agric. Exp. Stn.
Received for publication January 17, 2002.
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REFERENCES
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ABSTRACT
INTRODUCTION
