Published in Crop Sci. 44:1195-1199 (2004).
© 2004 Crop Science Society of America
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CROP BREEDING, GENETICS & CYTOLOGY
Estimation of Combining Ability for Resistance to Fusarium Stalk Rot in Grain Sorghum
Tesfaye Tesso,
Larry E. Claflin and
Mitchell R. Tuinstra*
Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS 66506
* Corresponding author (drmitch{at}ksu.edu).
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ABSTRACT
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Fusarium stalk rot (caused by Fusarium spp.) is an important disease of grain sorghum [Sorghum bicolor (L.) Moench]. The objectives of this study were to identify genetic sources of Fusarium stalk rot resistance and estimate combining ability. Six sorghum R-lines, representing commercial parent lines and potential sources of stalk rot resistance, Tx2737, Tx435, SC33, SC35, SC599 and SC1158, and three standard seed parents, ASA3042, AWheatland, and ARedlan, were intercrossed in a Design II mating scheme. The inbred parent lines and corresponding hybrids were evaluated in field trials at Manhattan and Hesston, KS, in 1999 and 2000. Three randomly selected plants in each plot were artificially inoculated in the pith tissue of the basal stalk with liquid cultures of F. proliferatum at 14 d after flowering. One additional plant in each plot was inoculated with distilled water as a control. At 28 d after inoculation, plants were harvested and scored for disease severity. Significant differences in disease reactions were noted among parent lines and hybrids. Among hybrids, significant male and female parent effects and interactions were detected. SC599 consistently had very low mean disease scores, followed by SC1158. Hybrids produced with SC599 were also highly resistant, indicating that genes for resistance had a dominant mode of inheritance in this genotype. SC599 represents an excellent source of resistance to F. proliferatum for use in breeding programs.
Abbreviations: GCA, general combining ability
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INTRODUCTION
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STALK ROT HAS BEEN a priority disease of grain sorghum in various parts of the world (Thakur et al., 1996). Besides its direct effect on yield, stalk rot induces lodging that causes further reduction in yield and quality of grains and increased problems with harvest (Mughogho and Pande, 1984). Significant yield losses due to stalk rot have been reported in Africa (Frowd, 1980; Omar et al., 1985; Hulluka and Esele, 1992), India (Khune et al., 1984; Seetharama et al., 1987), Australia (Trimboli and Burgess, 1982), and the USA (Reed et al., 1983; Jardine and Leslie, 1992). The disease is generally characterized by degradation of pith tissue at or near the base of the stalk and is associated with senescence of stalk pith cells (Reed et al., 1983).
Stalk rots are caused by various species of fungal organisms, and infection is enhanced under conditions of environmental stress (Bramel-Cox et al., 1988). Two or more causal pathogens usually occur together, making identification of the primary pathogen difficult. The most important stalk rotting organisms, however, are Macrophomina phaseolina (Tassi) Goidanich, the causal organism of charcoal rot, and a number of Fusarium species, the pathogens responsible for Fusarium stalk rot (Edmunds and Zummo, 1975; Bramel-Cox et al., 1988; Wildermutch et al., 1997). Fusarium stalk rot is generally less virulent than charcoal rots and it has been given less attention. However, in certain regions, Fusarium stalk rots are more important than charcoal rot. In Kansas, where over 40% of the sorghum in the USA is produced, Fusarium species are the predominant causal agents of stalk rot disease (Jardine and Leslie, 1992). Jardine and Leslie (1992) indicated that the average yield loss caused by this disease in Kansas is about 4%, while the loss at specific locations can reach as much as 50%. Recent reports from South Central Kansas experiment stations support this estimate, indicating that stalk-rot-induced lodging of up to 60% is common among high yielding hybrids and the incidence can be even higher under stressful conditions. When converted to monetary terms, the annual economic loss associated with this disease in Kansas surpasses $15 million per year. Despite this economic impact, little work has been done to overcome the problem.
The objectives of this study were (i) to identify genetic sources of Fusarium stalk rot resistance and estimate their combining ability to assess their potential for use in breeding programs, and (ii) to determine the mode of inheritance of the stalk rot resistance characters for each of the resistance sources.
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MATERIALS AND METHODS
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Test Genotypes
Nine parent lines and 18 F1 hybrids were planted in field trials near Manhattan and Hesston, KS. Trials were conducted in 1999 and 2000 at both locations. The female parents were stalk rot susceptible lines ASA3042, AWheatland, and ARedlan. They are standard seed parents used in commercial hybrid seed production in the USA. Their F1 hybrids with standard males generally are susceptible to stalk rot and, consequently, are vulnerable to lodging. The six male parents used in the study were Tx2737, Tx435, SC33, SC35, SC599, and SC1158. Tx2737 and Tx435 are standard male lines used in the hybrid seed industry and their hybrids represent commercial checks with intermediate stalk rot resistance characteristics. SC33, SC35, and SC599 are converted landrace varieties reported to be resistant to stalk rot. The stalk rot reaction of SC1158 is unknown, but was included in the test because it was nonsenescent under late-season drought conditions, a trait often associated with stalk rot resistance.
Field trials near Manhattan were conducted on Smolan Silty Clay Loam soils and trials in Hesston were conducted on Ladysmith Silty Clay Loam soils. The experiments were conducted in randomized complete block designs with four replicates. The seeds were planted in single-row plots (6.5 x 0.76 m) at a population of 129000 plants ha–1. Seeds were treated with N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (Captan, Drexel Chemical Co., Memphis, TN) at the label rate. Cultural practices included application of 41 kg N ha–1 before planting and 0.24 L Quinclorac ha–1 plus 0.68 kg Atrazine ha–1 applied post-emergence for weed control. Flowering date was recorded for each plot when approximately 50% of the plants were at half-bloom. At this time, four plants with uniform bloom were randomly selected in each plot and tagged for artificial inoculation at a later time.
Inoculum Preparation
Pure cultures of F. proliferatum (strain KSLM) were obtained from the collection of L.E. Claflin, Kansas State University. This strain was originally cultured from diseased sorghum stalks collected from a severely infected sorghum field in central Kansas. Pathogenicity tests showed that the strain was highly virulent on sorghum. Cultures were initiated on potato dextrose agar. Small sections (2–3 mm2) of the fungal mat were placed in 500-mL Erlenmeyer flasks containing potato dextrose broth (DIFCO, Detroit, MI). Cultures were grown on a shaker (60 rpm) for approximately 2 d at 24°C to produce conidia. Conidia were separated from the mycelial mass by straining the culture suspension through four layers of cheesecloth. Conidial concentrations were ascertained with a hemacytometer. Spore concentrations were adjusted to 5 x 104 conidia mL–1 using 10 mM (pH 7.2) phosphate-buffered saline. The suspension was kept on ice until inoculation.
Field Inoculation
Inoculations were performed with an Idico filler-plug gun (Forestry Suppliers, Inc., Jackson, MS) equipped with a stainless steel needle similar to that described by Toman and White (1993). At 14 d after flowering, three of the tagged plants from each plot were artificially inoculated with 5 x 104 conidia in the pith of the stalk approximately 10 cm above the soil surface. One plant in each plot was inoculated with distilled water as a control. On 28 d after inoculation, plants were harvested and split longitudinally to score disease severity. The scoring was done by measuring the length of the visible lesion in the pith of the stalk and by counting the number of diseased nodes crossed by the lesion.
Statistical Analysis
Analyses of variance and combining ability were performed per established methods (Hallauer and Miranda, 1988) with the PROC GLM and PROC MIXED procedures of SAS (SAS Institute, 1989). The combined ANOVA was performed by considering blocks and environments and corresponding interactions as random effects and the remaining variables as fixed effects. Tests of significance for entry, inbred, inbred vs. hybrid, hybrid, male parent, female parent, and male by female effects for all traits were made by comparison with their respective environmental interaction. The Design II fixed model (Model I) of the ANOVA (Hallauer and Miranda, 1988) provided independent estimates of general combining ability (GCA) for both male and female parents. The GCA estimates of each parental line were calculated as the difference between the grand mean and the marginal hybrid mean of each line. Significance of the GCA effect for each line was tested using a two-tailed t test procedure in SAS after rearranging the data. These results were confirmed by manual calculation of the standard error for GCA for both males and females following the procedure described by Cox and Frey (1984).
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RESULTS
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Significant differences were observed in lesion length and number of diseased nodes (Table 1). Although significant location and location-by-entry interaction effects were detected (Table 1), entry performance across environments was generally very consistent and interaction effects were primarily the result of a change in scale of disease severity between locations (Tables 2, 3).
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Table 1. Mean squares from the combined analysis of variance for Fusarium stalk rot resistance of inbred lines and their hybrids following artificial stalk inoculation with F. proliferatum at four location–years.
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Table 2. Average disease lesion lengths for entries inoculated with F. proliferatum and control treatments in four location–years.
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Table 3. Average number of diseased nodes for entries inoculated with F. proliferatum and control treatments in four location–years.
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The average lesion lengths for entries inoculated with the pathogen from the combined analysis ranged from 5.4 cm in the line SC599 to 46.1 cm in the hybrid ARedlan x SC33 (Table 2). Similar results were observed in the individual experiments in 1999 and 2000. In 1999, the longest lesions were recorded in hybrids of SC33 (ARedlan x SC33, AWheatland x SC33, and ASA3042 x SC33) while the smallest lesions were observed in SC599 (Table 2). Results from experiments conducted in 2000 were similar. Hybrids produced with SC33 had the longest lesions, while SC599 and its crosses consistently were the most resistant (Table 2). Differences in lesion length among entries in the control treatment were also observed. The trends in disease scores among entries in the control treatment were generally similar to those observed following inoculation with the pathogen; however, differences in the control treatment were quite small (Table 2). These infections were probably caused by secondary infection resulting from entry of soil- or air-borne conidia through the wound site.
The average number of diseased nodes resulting from inoculation with the pathogen was least in SC599 and greatest in the female ASA3042; a pattern similar to that observed for lesion length (Table 3). Among hybrids, the average number of diseased nodes was significantly higher in crosses involving SC33, but hybrids produced using SC599 had the fewest diseased nodes in all test environments (Tables 3). No significant differences among entries for number of diseased nodes were observed in the control treatment (Table 3).
Partitioning of the entry effect into parent line and hybrid effects indicated significant differences between the parent line vs. hybrid groups for lesion length (Table 1). The hybrids generally expressed more severe stalk rot than inbred parent lines (Tables 2). Significant differences among parent lines and among hybrids were also detected (Table 1). Among parent lines, the lowest scores for lesion length and number of diseased nodes were observed in SC599, while the highest scores were recorded in ASA3042, ARedlan, and SC33 (Tables 2 and 3). Similarly among hybrids, the lowest stalk rot ratings were recorded in the crosses of SC599 and the highest ratings were observed in hybrids produced using SC33 (Tables 2 and 3). Trends for performance of parent lines and hybrids were remarkably similar across locations.
Further partitioning of the hybrid effect into male and female components revealed that the majority of variability among hybrids for lesion length and number of diseased nodes was associated with the male parent effect (Table 1). The female parent effect and male x female interaction effects were significant for lesion length but were much smaller in magnitude than the male parent effect in hybrids (Table 1). Several of the males expressed significant GCA effects for disease resistance (Table 4). Male lines including SC33, SC35, SC599, SC1158, and Tx2737 had significant positive or negative GCA effects for lesion length or number of diseased nodes (Table 4). SC599 stands out among the male lines with the highest GCA for resistance, and hybrids produced with SC33 were generally the most susceptible. These genotypes consistently expressed similar disease reactions across all test environments. Among the females, only ARedlan had significant GCA effects for lesion length, indicating greater-than-average susceptibility (Table 4).
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Table 4. Average hybrid performance and general combining ability (GCA) estimates for Fusarium stalk rot resistance of male and female parent lines tested at Manhattan and Hesston, KS, in 1999 and 2000.
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DISCUSSION
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Growth environment significantly affected the development and severity of Fusarium stalk rot between experiments (Table 1). This may be because of variation in weather conditions among test environments. In 1999, the Manhattan location received adequate rainfall throughout the growing season, while intermittent drought stress was encountered at Hesston. Early drought conditions at Hesston were alleviated during grain development. The weather in 2000 was generally characterized by prolonged drought and high temperature stress at both locations; a condition less suitable for Fusarium stalk rot development. Fusarium stalk rot is generally most severe in later growth stages when wet, cool conditions follow hot, dry weather (Odvody and Dunkle, 1979; Zummo, 1984). The lack of adequate late-season moisture may have led to an overall lower mean disease score in 2000 and resulted in the variation in disease reactions between test environments. Variation in disease severity across environments resulted in a change in the scale of the differences noted among genotypes. This change of scale effect resulted in significant location interaction effects (Table 1). It should be noted, however, that although significant location interaction effects were detected, the inoculation assay used in this study provided remarkably consistent rankings of genotypes across environments.
Significant differences in lesion lengths were noted between the parent line and hybrid groups (Table 1). The average lesion length for hybrids was generally higher than for parental lines (Table 2). This is consistent with reports that severity of stalk rot infection is often higher among high-yielding hybrids than among inbred lines (Seetharama et al., 1991). These differences probably result from increased sink demand on stalk carbohydrate reserves during grain development. A reduction in carbohydrate reserves may lead to tissue senescence and provide an opportunity for root and stalk rot infection (Dodd, 1980).
Essentially no quantitative studies have been conducted in sorghum that relate differences in host-plant resistance following artificial inoculation with resistance under natural conditions. In part, this reflects the difficulty of screening for stalk rot resistance under field conditions because of the highly variable nature of stalk rot epidemics in these environments. Furthermore, relatively few stalk inoculation studies have been conducted to identify sets of lines or hybrids with differential disease reactions. The use of contrasting hybrids identified in this study may provide the genetic materials needed to address this question.
SC33, SC35, and SC599 have been reported to be staygreen genotypes, a trait thought to be associated with drought tolerance and stalk rot resistance (Bramel-Cox et al., 1988; Tenkouano et al., 1993; Tao et al., 2000). Staygreen lines maintain a greater proportion of assimilates in their stems at maturity (Seetharama et al., 1991). Bramel-Cox et al. (1988) reported that a staygreen line derived from SC33 was highly resistant to M. phaseolina. In a study by Tenkouano et al. (1993), the staygreen genotypes B35, a close relative of SC35, SC599, and their hybrids showed high levels of resistance to M. phaseolina. It was originally hypothesized that the staygreen trait might provide protection to an array of stalk rot pathogens in sorghum; however, these studies indicate that only SC599 expresses high levels of resistance to F. proliferatum. This suggests that resistance to F. proliferatum and M. phaseolina may be regulated by different genes. SC599 represents an excellent potential source of resistance to the two major stalk rot diseases in sorghum, charcoal and Fusarium stalk rots.
The greatest proportion of the variation in stalk rot resistance among hybrids was determined by male parent effects. These findings were not unexpected, since the male parents, unlike the female testers, included genotypes having variable reactions to stalk rot disease. Comparisons of the hybrid mean disease reactions among male parents indicated that SC599 consistently expressed the highest degree of resistance to F. proliferatum infection, followed by SC1158 and Tx2737 (Table 4). The greatest susceptibility to infection was detected in hybrids produced with SC33. These genotypes had lesions more than four-fold longer than hybrids produced with SC599. SC599 hybrids had a combined mean lesion length of 9 cm that crossed less than 2 nodes as opposed to SC33 hybrids that had lesions > 40 cm long and crossed more than 4 nodes (Table 4). The female GCA effects were significant in the combined analysis as well as individual location analyses for lesion length but not for number of nodes crossed. Although significant, the magnitude of the variation among female parents was very small in comparison with the male parent effect. The SCA effects also were generally very small.
Comparison of individual GCA effects for males revealed that SC599 had the highest significant GCA effect for resistance based on lesion length and number of nodes crossed. Hybrids produced with SC599 were consistently resistant to F. proliferatum across locations and years and its hybrids performed nearly as well as the parental line per se. This indicates dominant or incompletely dominate gene action for Fusarium stalk rot resistance in this particular genetic background.
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CONCLUSIONS
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Fusarium stalk rot in grain sorghum, despite its complexity and interaction with environment, can be minimized by developing resistant hybrids through breeding. SC599 consistently expressed a high degree of resistance to F. proliferatum infection and, hence, could be considered an excellent source of resistance for use in breeding programs. Moreover, the resistance genes in this particular line seem to have a dominant or incompletely dominant mode of inheritance. This pattern of inheritance should simplify incorporation of desirable genes into the required genetic backgrounds. Segregation analyses need to be performed to estimate the number of genes that are conditioning resistance in this line.
Lines such as SC33 and SC35 have been widely used as potential sources of resistance to stalk rot disease because of their staygreen characteristics. In the present study, these lines and their hybrids were average or highly susceptible to F. proliferatum infection, suggesting that resistance to M. phaseolina and F. proliferatum are most likely controlled by different genes. If this is confirmed in future studies, independent screening programs will need to be developed for both classes of stalk rot organisms.
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NOTES
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Contribution No. 03-418-J from the Kansas Agric. Exp. Stn.
Received for publication July 1, 2003.
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REFERENCES
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ABSTRACT
INTRODUCTION
