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Crop Science 43:350-357 (2003)
© 2003 Crop Science Society of America

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Mapping QTL for Bacterial Brown Spot Resistance under Natural Infection in Field and Seedling Stem Inoculation in Growth Chamber in Common Bean

G. Jung*,a, H. M. Ariyarathneb, D. P. Coynec and J. Nienhuisd

a Department of Plant Pathology, University of Wisconsin-Madison, WI 53706
b Regional Agriculture Research and Development Center, Diyatalawa Road, Bandarawela, Sri Lanka
c Department of Horticulture, University of Nebraska-Lincoln, NE 68583
d Department of Horticulture, University of Wisconsin-Madison, WI 53706

* Corresponding author (jung{at}plantpath.wisc.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial brown spot (BBS), caused by Pseudomonas syringae pv. syringae van Hall (Pss), is an important bacterial disease of common bean (Phaseolus vulgaris L.). The objective of this study was to identify random amplified polymorphic DNA (RAPD) molecular markers linked to quantitative trait loci (QTLs) for BBS resistance. Resistance was assessed by three methods: (i) stem inoculations of plants grown in a greenhouse evaluated by a 1-to-5 rating scale for stem symptoms, (ii) percentage of BBS infected leaves per plot in noninoculated field trials, and (iii) Pss population sizes, on noninoculated field grown plants determined by a leaflet freezing assay (LFA). F8:9 recombinant inbred lines derived from the Mesoamerican cross of Belneb RR-1 (susceptible to BBS) x A 55 (resistant to BBS) were grown in replicated experiments in two years (1996, 1998) in Wisconsin. In addition, two separate experiments were performed in the greenhouse in a randomized complete block design with two replicates for seedling stem inoculation with Pss strain Bs191. One genomic region on linkage group (LG) 2, from a previously published genetic linkage map, was significantly associated with QTLs for BBS resistance measured by three assays over two years. Phenotypic reactions were significantly correlated with the measurement of freezing temperatures as determined by LFA under a favorable environment for the growth of epiphytic bacterial populations. Marker assisted selection for resistance to BBS using molecular markers found during this study may improve selection efficiency for resistance because of the low heritability of the reaction to BBS, and the independent QTLs for resistance to different bacterial diseases. Furthermore, disease screening solely dependent upon natural field inoculations is not reliable because of genotype x environment interactions. The leaflet freezing assay method could be utilized to screen progenies or breeding germplasms, even when no distinct phenotypic disease symptoms are present on the plants.

Abbreviations: BBS, bacterial brown spot • LFA, leaflet freezing assay • LG, linkage group • Pss, Pseudomonas syringae pv. syringae • QTL, quantitative trait loci • RAPD, random amplified polymorphic DNA


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
BACTERIAL BROWN SPOT is one of the important bacterial diseases affecting production of common bean in the USA (Hagedorn and Patel, 1965; Steadman and Schwartz, 1983). On snap beans, the disease reduces crop quality. A relatively small proportion of infected pods may render an entire field unmarketable. Resistant cultivars can provide effective disease control. Bean cultivars–lines with decreased susceptibility to BBS have been developed (Antonius and Hagedorn, 1978; Hagedorn and Rand, 1980), although all commercially acceptable snap bean cultivars retain some level of susceptibility.

Quantitative inheritance patterns for the reaction to Pss have been reported (Hagedorn and Rand, 1975). Antonius (1982) reported that pod reactions to Pss were controlled by 3 to 5 genes and found lower narrow sense heritability estimates for field reactions than in the greenhouse. Seedlings (Antonius, 1982; Lienert and Schwartz, 1993), leaves (Coyne and Schuster, 1969; Antonius, 1982), and pods (Antonius, 1982; Cheng et al., 1988) have been used to evaluate the resistance to BBS in beans. Seedling inoculations require less time and space, but seedling reactions in a greenhouse may not always agree with those in the adult plants in the field. This may be due to plant age, or to differences in plant bacterial interactions under different conditions. The probability of bacterial brown spot disease in the field is a function of the population sizes of Pss on healthy leaves approximately a week earlier (Lindemann et al., 1984; Rouse et al., 1985). Only when population sizes of Pss become very large [>105 colony-forming units (cfu) per leaflet], does disease become likely.

There are differences in Pss population sizes associated with different bean cultivars (Hirano et al., 1987) and it is likely that host genotype plays a major role in determining the sizes of these populations. Population sizes of Pss correlated significantly with disease on inbred backcross lines (Kmiecik et al., 1990); therefore, selecting for lines that diminish numbers of Pss should diminish the likelihood of disease through avoidance of the pathogen. Further, because factors that affect population sizes of the bacteria associated with healthy tissue may be completely different from those that affect lesion formation. Thus, genes that have a major effect on disease in the field may go undetected in laboratory assays in which bacteria are injected into the plant. Such assays completely avoid the need for the bacteria to grow in association with healthy tissue before population sizes become large enough to cause lesions.

Although measurement of bacterial population sizes is very laborious and time consuming, the ice nucleating property of Pss can be used as a more rapid method to estimate population sizes of this bacterium on a population of leaves (Hirano et al., 1987). Ice nucleation provided a significantly accurate estimate of population size to be predictive of BBS disease (Hirano et al., 1987). Therefore, the objective of this research was to identify loci for resistance to BBS in common beans by three different assay methods: (i) disease reactions from stem inoculated plants in the greenhouse, (ii) BBS symptoms on field grown plants, and (iii) the leaflet freezing assay on field grown plants. The heritability of the population size of Pss determined by the leaflet freezing assay was also estimated.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
Seventy-eight F8:9 RI (recombinant inbred) lines from the Mesoamerican gene pool cross of Belneb RR-1 (USDA/Nebraska) (Stavely et al., 1989) x A 55 (Centro Internacional de Agricultura Tropical, Cali, Colombia, CIAT) were used. The population was developed by a single-seed descent breeding method. Belneb RR-1 is a Great Northern breeding line susceptible to BBS with a type III plant habit. A 55 is a black-seeded bean resistant to BBS with a type II plant habit.

Stem Inoculation
Seeds from 78 RI lines and parents were planted in black plastic containers (8.5 cm long and wide, and 6.5 cm deep), two plants per container. Twelve containers were then placed in each of the black plastic trays containing equal parts of sand, sphagnum peat moss, vermiculite, and sharpsburg silty clay loam soil. Plants were grown to seedling stage in the greenhouse at the University of Nebraska, Lincoln, NE. A randomized complete block design (RCBD) with two replicates (two plants per replicate) was used in two separate experiments initiated on 4 Jan. 1996 and 4 April 1997. Pss strain Bs191 (source: Dr. A.K. Vidaver, Dep. of Plant Pathology, UNL) was grown on King's B broth medium (King et al., 1954) for 48 h at 25°C. Cells were suspended in 12.5 mM potassium phosphate buffer (pH 7.1) to 0.1 O.D. (Bausch and Lomb Spectronic 20 spectrophotometer at 640 nm). The suspension was diluted with phosphate buffer at a final concentration of 1 x 106 CFU/mL and was used for inoculations. Stems of 12-d-old seedlings were inoculated with a syringe (16 gauge) at two sites, 1 cm apart, starting about 1 cm below the node of primary leaves (Saettler, 1971). The syringe was pushed through the stem, then withdrawn slowly to deposit about 0.01 mL of bacterial suspension into the stem. The plants were then moved to growth chambers maintained at 22 ± 2°C and a photoperiod of 12 h day/night. Stem reactions to Pss were rated as follows: 1 = no symptoms; 2 = cracking and minor splitting of stems; 3 = splitting with water soaking 1 to 3 mm; 4 = water soaking 4 to 6 mm; 5 = extensive water soaking > 7 mm.

Field Experiment
Experiments were conducted in 1996 and 1998, at the University of Wisconsin-Madison, Agricultural Research Stations in Hancock, and Arlington, WI, respectively. Sixty-two and 78 RI lines including parents, were planted 28 May 1996 and 24 June 1998, respectively, in a RCBD with four replications. Two test rows (15 plants per row) were alternated with two rows of the susceptible cultivar Eagle, the latter serving as a source of inoculum. The ice nucleation temperature of each leaflet was measured as described in the next section (Vali, 1971). A visual estimation of the percentage of leaves diseased per line, which were typically distinguished from other foliar diseases of bean by its characteristics roughly circular, dark brown lesions with narrow yellow haloes, was made 7 d after the leaflet freezing assay.

Estimate of Pss Population Sizes
In the same field experiments, 15 leaflets from the top of the plant canopy were randomly collected from each plot representing a line. The samples from each plot were placed in a paper bag and transported to the laboratory in a cooler. Each of the leaflets was submerged in a 16 mm test tube containing 9.0 mL of sterile, ice nucleus free potassium phosphate buffer (0.01 M, pH 7.0). Ice nucleation temperature was determined with an ice nucleation spectrometer modeled after that described by Vali (1971). Each 16-mm test tube was immersed in a controlled temperature bath and held in a specially modified tube rack. In this rack, a thermocouple within a silicone rubber well was held against the outside of the tube. Temperatures of each of 192 thermocouples were scanned every 12 s with a CR7 datalogger (Campbell Scientific, Logan, UT) and transmitted to a computer. Freezing events were recognized as a temperature rise associated with the release of the latent heat of fusion as supercooled water within a tube froze. When cooling rates are maintained at or below 0.04°C per minute, this method provides an estimate that is within 0.04°C of the temperature at which freezing occurred within the tube. Bath temperatures are measured at several locations, as well as with the thermocouples associated with all tubes that have not yet frozen. Cooling rates are monitored with the aid of the computer and are controlled manually by adjusting the ratio of coolant that is recirculated within the bath to that that is pumped through a bath cooler.

Heritability Estimation
Heritability of the tendency to carry relatively large Pss population sizes using the leaflet freezing assay was estimated from a linear model computed with JMP 3.1 software (SAS Institute, 1995) incorporating data from the RI lines scored in both 1996 and 1998. Factors were genotype, year, and genotype x year which were treated as random effects. The formula of Knapp et al. (1985) was used to calculate confidence intervals for heritability estimates using computed mean squares on a line mean basis.

Construction of Genetic Map and Detection of QTLs for BBS Disease Resistance
The construction of a genetic linkage map, and the locations of QTLs for resistance to the bacterial diseases, halo blight (caused byPseudomonas syringae pv. phaseolicola: Psp) and common bacterial blight, in the above RI population was previously reported by Ariyarathne et al. (1999). Briefly, to summarize, the previous results, 90 out of 174 mapped markers (170 RAPDs, two SCARs, and two phenotypic markers) grouped into 11 linkage groups are shown in Fig. 1. The resulting linkage map spanned 755 centimorgans (cM) in size. Nine of the 11 linkage groups identified in this mapping population were aligned to the integrated maps of Freyre et al. (1998) and Vallejos et al. (1999). This same linkage map was used here to identify genomic regions significantly associated with QTLs for resistance to BBS.



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  		Fig. 1. RAPD marker linkage map previously constructed using F8:9 recombinant inbred lines derived from a common bean cross Belneb RR-1 x A 55. The gene and marker names are given on the right, and the length in cM shown on the right bottom of each linkage group. Markers significantly (*P < 0.05; **P < 0.01; ***P < 0.001) associated with resistance to Pseudomonas syringae pv. syringae (Pss) are indicated by boxes. The associated resistance to Pss is indicated in the boxes, with a line extending from the boxes indicating the confidence interval for interval mapping. The abbreviations of disease reactions to Pss are as follows; STEM = resistance to stem inoculations in growth chamber; BBS = plant resistance to natural infection in the field; LFA = resistance to natural infection in the field using the leaflet freezing assay to indirectly measure bacterial population sizes.

 
The method of interval mapping with MAPMAKER-QTL 2.0 (Lander and Botstein, 1989) was used to detect the most likely location of QTLs and to estimate their genetic effects. The logarithm of odds (LOD) score of 2.0 was used as the threshold for QTL detection (Lander and Botstein, 1989). F-tests of single-factor ANOVA for each pairwise combination of the resistance traits and marker locus were also used to determine if significant variation in trait expression was associated with differences in marker-locus genotypic classes. Markers significant at P < 0.05 in single-factor ANOVA were used for stepwise multiple regression analysis to find possible QTLs linked to disease traits. The QTLs significant at P < 0.05 in stepwise regression were considered linked with RAPD markers and used for the model (Miklas et al., 1996). If several markers in a linkage group were significantly (P < 0.05) associated with a trait by single-factor ANOVA, and were highly correlated, then only the most significant markers were included for multiple regression analysis. Correlations between markers within linkage group were estimated by PROC CORR (SAS Institute, 1989). A relatively high P value (P = 0.05) was used for detection of individual QTL and for stepwise regression analysis with the understanding that this may increase the experimental Type I error rate. However, lower stringency of detection is recommended as a way to reduce the probability of committing Type II errors (Edwards et al., 1992). In addition, we report all QTL objectively in terms of their statistical significance (P value). This allowed us to derive as much information from our study as possible while remaining statistically responsible. All statistical analyses were conducted by means of Statistical Analysis System (SAS Institute, 1989).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The stem reactions to Pss in the growth chamber were not normally distributed (Fig. 2A). The distribution of the percentages of leaves diseased per line was skewed toward resistance (Fig. 1B). An approximately normal distribution of median ice nucleation temperatures among RI lines was noted (Fig. 2C). The freezing assay indirectly estimates the population size of Pss associated with leaves of each line. The difference in the ice nucleation temperatures between the parents was significant in both years (P < 0.01, 1996; P < 0.05, 1998) (Table 1). The lower ice nucleation temperatures (-3.44) of A 55 indicated that A 55 carried lower population sizes of Pss than Belneb RR-1 (-3.00) (Fig. 2C). A low heritability of 0.38 for ice nucleation temperatures among F8:9 lines was estimated.



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  		Fig. 2. Frequency distributions and means of numbers of F8:9 recombinant inbred lines derived from the cross Belneb RR-1 x A 55 for disease reactions and population size of Pseudomonas syringae pv. syringae (Pss) in common bean; (A) mean ratings of stem reactions to Pss strain BS 191 based on two experiments (1 = resistant and 5 = susceptible) in a growth chamber test; (B) mean percentages of the leaves diseased per field plot based on 1996 and 1998 data; (C) mean ice freezing temperatures based on 1996 and 1998 data; this is an indirect measure of Pss population sizes associated with field-grown bean leaves.

 

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  		Table 1. Means and ranges of freezing temperatures (°C) of individual leaflets observed for F8:9 recombinant inbred lines (RILs) of Belneb RR-1 x A 55 population screened in 1996 and 1998 for Pseudomonas syringae pv. syringae population sizes using the leaflet freezing temperatures in the field at Hancock, and Arlington, Wisconsin, respectively.

 
However, the original data collected from the three assay methods were used to estimate the locations and the effects of QTLs associated with each of these traits. Data deviating from a normal distribution were used previously to estimate QTLs for other traits, such as disease resistance (Nienhuis et al., 1987; Paterson et al., 1991; Jung et al., 1996, 1997). The continuous distribution of the above traits, and data from a past study (Antonius, 1982) supports multiple gene control for resistance to Pss.

Transgressive segregation was observed for resistance and susceptibility, because the BBS phenotypic data and leaflet freezing temperatures indicated that some lines were more resistant or susceptible than their parents. This suggests that both parents may have loci controlling resistance to BBS. Transgressive segregation can be exploited in breeding programs to enhance the expression of a trait over both parents through recombination of favorable alleles.

The phenotypic correlation between BBS reactions recorded in the field and the bacterial population data obtained from the leaflet freezing assay in 1996 was moderately high (r = 0.61, P < 0.01), while the correlation between BBS disease in the field and disease reaction by the stem inoculation method was low (Table 2). A significant negative correlation was detected between leaflet freezing temperatures in 1996 and stem inoculation reactions (r = -0.33, P < 0.05).


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  		Table 2. Phenotypic correlations for reactions to stem inoculations (STEM) tested in a growth chamber, for reactions to bacterial brown spot (BBS), and for leaflet freezing assay (LFA) temperatures related to Pseudomonas syringae pv. syringae population sizes using F8:9 recombinant inbred lines derived from the common bean cross Belneb RR-1 x A 55.

 
The high correlation between disease reactions in the field in 1996 when there is ample disease and leaflet freezing temperatures suggests that it would be more efficient to use field evaluation of lines for disease than the leaflet freezing assay. However, in 1998, disease incidence in the field was low due to the lack of intense rain during the growing season, so it was very difficult to select resistant lines solely on the basis of phenotypic readings. Thus, the leaflet freezing assay could provide a useful method to select lines resistant to BBS when the environmental factors are not conducive to the development of disease symptoms in the field.

QTLs Controlling Resistance to BBS as Measured by Stem Inoculation
Nine genomic regions were significantly (P < 0.05) associated with QTLs for the BBS reactions to stem inoculation on the basis of single-factor ANOVA (Table 3). Two genomic regions on LGs 7 and 9 contained QTLs for BBS resistance to stem inoculation at a highly significant level (P < 0.001), along with closely linked QTLs for field BBS resistance and LFA (Fig. 1). The other 7 QTLs were either detected at low significance levels (P < 0.05), or distantly linked to QTLs for BBS resistance and LFA (Fig. 1). Those low significant associations might be due to chance events and require additional experiments for verification of independent QTL. We did not detect any QTL for resistance to stem inoculation in LG 4 indicating that LG 4 was specific to the other assay methods or was not detected by stem inoculation because of experimental variations or genotype x environment interaction. The low or negative correlations obtained between field trial data and stem inoculation reactions suggest that screening lines with the seedling stem method may not be very useful to identify BBS resistance that is useful in the field. Antonius (1982) also reported low correlation (r = 0.19) between reactions of stems and leaves in the field, and indicated that stem assays can only be used for preliminary screening and not for final selection of resistant lines.


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  		Table 3. Summary of single-factor ANOVA and stepwise multiple regression analyses of RAPD molecular markers for detection of QTL associated with resistance to stem inoculation with Pseudomonas syringae pv. syringae strain Bs191 in F8:9 recombinant inbred lines derived from the common bean cross Belneb RR-1 x A 55.

 
Four markers significantly associated with QTLs for BBS resistance in stem inoculation were retained in the final model by stepwise multiple regression (Table 3). These four markers, BC2271500 (LG 7), U10900 (LG 9), AL8900 (LG 5), and R2430 (LG 3), explained 24, 14, 5, and 5% of the phenotypic variation of this trait, respectively.

QTLs Controlling Resistance to BBS in the Field
Six genomic regions in 1996 and four regions in 1998 were significantly associated (P < 0.05) with the field resistance to BBS recorded as percentage of diseased leaves on the basis of single-factor ANOVA (Table 4). Two genomic regions on LGs 2 and 3 containing QTLs affecting disease reactions were detected in both years, although the same markers were not significant. The reason for a larger number of significant markers associated with QTLs for BBS in 1996 than in 1998 may be due to unfavorable environmental conditions reducing bacterial population sizes and thus resulting in less disease in 1998. However, the similar results, a wide variation in the genetic loci associated with quantitative traits with low heritability were reported in other crops (Beavis et al., 1991; Paterson et al., 1991). Other factors such as genotype sampling variation within an environment and genotype x environmental interaction may also result in inconsistency of QTL location and effect between two years.


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  		Table 4. Summary of the single-factor ANOVA and stepwise regression analyses of RAPD molecular markers and phenotypic data for detection of QTL associated with field resistance to bacterial brown spot. F8:9 recombinant inbred lines derived from the common bean cross Belneb RR-1 x A 55 were grown at Hancock and Arlington, WI in 1996 and 1998, respectively.

 
Four QTLs controlling BBS resistance, found by stepwise regression in 1996, and were linked to markers, Y41600, R20400, AD41150, and O121550, located in LGs 9, 3, 7, and 2, explained 20, 12, 6, and 8% of the phenotypic variation of the trait, respectively. Besides these four QTLs, the marker HB-7 on LG 4 was significant (P < 0.01) by single-factor ANOVA but not by stepwise regression. This marker was linked to a QTL for resistance to halo blight detected in a previous study using interval mapping (Ariyarathne et al., 1999). Only one QTL on LG 1 explaining 17% of the variation in disease reaction was detected by stepwise regression in 1998.

QTLs Controlling Population Sizes of Pss
Five genomic regions were significantly (P < 0.05) associated with QTLs for the ice nucleation temperatures (an indirect measure of the number of Pss associated with leaflets) by single-factor ANOVA in each year (Table 5). Three markers included in the final model of multiple regression explained 41% of total phenotypic variation in ice nucleation temperatures in 1996. These QTLs were located in LGs 2, 3, and 9. However, one marker on LG 2 explained 10% of the phenotypic variation of ice nucleation temperatures by multiple regression in 1998. Two of the most significant markers on LGs 2 and 4 associated with QTLs for leaflet freezing temperatures, were detected in both years. Although it is normal for population sizes of Pss to vary substantially with time during a growing season, relative population sizes across bean cultivars were much less variable. Thus, we expected the relative ice nucleation temperatures (associated with population sizes of Pss) to remain fairly constant, but the absolute temperatures at which the lines caused ice nucleation to vary with the weather. For this reason, the ice nucleation assay should produce relatively consistent data for comparison of lines, regardless of the amount of disease present. These predictions are borne out by the results presented by the comparison of Tables 4 and 5. Although there was insufficient disease to provide a good estimate of relative resistance in 1998, the leaflet freezing assay allowed us to identify QTLs on the same two linkage groups in both years. Since the same QTLs for both leaflet freezing assay and BBS for field resistance were detected in each year, it should be feasible to use the leaflet freezing assay screening method to select BBS resistance in common beans in years less favorable for disease expression in the field. Also the value of the molecular markers associated with QTLs for BBS resistance for use in marker-assisted selection needs to be investigated.


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  		Table 5. Summary of the single-factor ANOVA and stepwise regression analyses of RAPD molecular markers and phenotypic data for detection of QTL associated with relative Pseudomonas syringae pv. syringae population sizes using a leaflet freezing assay. F8:9 recombinant inbred lines derived from the common bean cross Belneb RR-1 x A 55 were grown in Hancock and Arlington, WI in 1996 and 1998, respectively.

 
Comparisons of QTLs for BBS Resistance by Three Assay Methods
Methods for estimating percentages of BBS diseased leaves and the leaflet freezing assay generally identified similar locations of QTLs depending on year tested. Four QTLs in LGs 2, 3, 4, and 9 were identified for both BBS resistance and the leaflet freezing assay in 1996, but two QTLs in LG 2 and 8 were detected for both traits in 1998. The QTL in LG 8 detected in 1998 was not observed in 1996. Whether that QTL is only expressed under particular environmental conditions, or was due to chance events cannot be determined here. Markers associated with QTLs for BBS resistance need to be confirmed in other untested segregating populations derived from crosses to the resistant parent A 55 and other resistant source. The discrepancy in the locations and effects of QTLs in both years suggest that high selection intensities, large population sizes, and use of multiple environments will be required to provide information on the merits of those QTLs for BBS in other segregating populations.

The identified QTLs explained 46% of the phenotypic variation for BBS, and 41% of the variation for the leaflet freezing assay data in 1996. Moderate phenotypic correlations between the data recorded by the two methods also support similar QTLs detected by both methods. QTLs in LGs 2, 8, and 9 also control the reactions in stem inoculation. A QTL in LG 2 controls both seedling stem reactions and two BBS reactions estimate of 1996 and 1998. A QTL region in LG 9 controls reaction in stems of seedlings and adult plant reaction in the field of 1996. The same region contains QTL for reactions in seedling stems and bacterial population size estimated indirectly using the leaflet freezing assay in 1996. However, these common regions containing QTLs controlling stem reactions only explain a low percentage of the phenotypic variation. This was confirmed by low correlations between seedling stem reactions to Pss with adult plant in the field and the leaflet freezing assay data.

QTLs on LGs 7 and 9 detected by the stem inoculation assay and BBS disease in the field may be more typical resistance, indicating a decreased probability of infection given a particular inoculum dose. On the other hand, QTLs such as the one on LG 4, detected by leaflet freezing assay and disease incidence in the field, but not by stem inoculation, may be due to a factor that affects Pss population sizes in association with healthy leaves.

Relationship with QTLs for Resistance to Other Pathogens and with Other Traits
The RAPD marker, O12900 on LG 2 associated with QTL for seedling stem and field reactions estimated by two methods also explained 11% of the phenotypic variation of halo blight reactions to strain 83-Sc2A (Ariyarathne et al., 1999). In addition, this genomic region was previously reported to contain resistance to common bacterial blight in pods as well as the I gene, which confers resistance to the Beancommon mosaic virus (BCMV). Therefore, the genomic region might contain common QTLs with pleiotropic effects on these traits, or different tightly linked QTLs may be involved. The position of the QTL controlling both field reactions to Pss and leaf reactions to Psp are strongly suggestive that a single QTL may control both bacterial disease reactions. The phenotypic marker, Hb-7 locus for the hypersensitive reactions to Psp strain 83-Sc2A was also significantly associated with QTL for field reactions to Pss on LG 4 (Ariyarathne et al., 1999).

In conclusion, two QTLs in LGs 2 and 4 were consistently and significantly associated with resistance to both BBS and LFA. Their value in marker-assisted breeding of snap beans needs to be determined in other populations. The leaflet freezing assay appears to be a useful method to screen and select lines resistant to BBS in environments not conducive to the development of typical BBS symptoms. The reactions to stem inoculations of seedlings are not predictive of plant reaction under natural infection in the field. This may be due to a difference in disease reaction between the seedlings and adult stages of growth. More likely, however, it reflects the importance of traits that affect success of P. syringae as it grows in association with plants in the field. This latter aspect of the plant-bacterial interaction is omitted from greenhouse assays. Marker-assisted selection for resistance to BBS may improve selection efficiency, due to low heritabilities of reactions to BBS reported by Antonius (1982) and found here for the leaflet freezing assay.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Susan Hirano and Christen Upper in the Dep. of Plant Pathology, Univ. of Wisconsin and Dr. Gary Yuen in the Dep. of Plant Pathology and Dr. Donald Lee in the Dep. of Agronomy, Univ. of Nebraska for their suggestions and critical review of the manuscript. We also thank Michell Sass for technical support.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The senior author was a Post-Doctoral researcher at the Department of Horticulture, University of Wisconsin-Madison when research was conducted under project WIS 03430, and also NE projects 20-036 and 20-0421. Also Published as Paper No 12984, Journal Series, Nebraska Agricultural Research Division. We acknowledge financial support from the CRIS Hatch grant #0152801.

Received for publication December 13, 2001.


   REFERENCES
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 ABSTRACT
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F. Navarro, P. Skroch, G. Jung, and J. Nienhuis
Quantitative Trait Loci Associated with Bacterial Brown Spot in Phaseolus vulgaris L.
Crop Sci., July 30, 2007; 47(4): 1344 - 1353.
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