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a Plant, Soil and Entomological Sciences Dep., Univ. of Idaho, Kimberly Research & Extension Center, 3793 North 3600 East, Kimberly, ID 83341-5076
b Dep. of Bioagricultural Sciences & Pest Management, Colorado State Univ., Fort Collins, CO 80523-1177
c Mision Biologica de Galicia, Carballeira 8, 36143 Salcedo, Pontevedra, Spain
* Corresponding author (singh{at}kimberly.uidaho.edu).
ABSTRACT
Interspecific breeding lines (IBL) VCW 54 (Reg. No. GP-276, PI 655531) and VCW 55 (Reg. No. GP-277, PI 655532) of common bean (Phaseolus vulgaris L.) resistant to white mold [WM; caused by Sclerotinia sclerotiorum (Lib.) de Bary] were jointly developed at the University of Idaho-Kimberly Research and Extension Center and Colorado State University, Fort Collins, CO. The Agricultural Experiment Stations of Idaho and Colorado released VCW 54 and VCW 55 on 10 Dec. 2008. Both IBL were derived from congruity backcrossing between small-seeded tropical black bean cultivar ICA Pijao and P. coccineus L. accession G 35172. ICA Pijao has an indeterminate upright growth habit Type II and resistance to bean common mosaic virus and some races of rust pathogen, and tolerance to bean golden mosaic virus (BGMV). G 35172 has large purple-mottled seeds, an indeterminate climbing growth habit Type IV, and resistance to BGMV. G 35172 also is variable for WM reaction. VCW 54 has scarlet flowers, purplish black seed, and the highest WM resistance. VCW 55 has white with pink-striped flower, black seed, and an intermediate level of WM resistance. Both IBL also have growth habit Type II, small seeds, and are late maturing, requiring 
100 d from planting to maturity in southern Idaho. White mold resistance from VCW 54 and VCW 55 should be pyramided with WM resistance from across Phaseolus species and introgressed into common bean cultivars using appropriate crosses, population sizes, and disease screening and selection methods.
Abbreviations: BCMV, bean common mosaic virus • BGMV, bean golden mosaic virus • BGYMV, bean golden yellow mosaic virus • CIAT, Centro Internacional de Agricultura Tropical • DPI, days post inoculation • IBL, interspecific breeding line(s) • PDA, potato dextrose agar • QTL, quantitative trait locus/loci • WM, white mold
White mold (WM) is among the most devastating and widely distributed diseases of both green and dry common bean (Phaseolus vulgaris L.) in the United States and Canada. Crop losses average 30% in the central high plains of the United States, with individual field losses as high as 92% (Schwartz et al., 1987). For every percentage increase in WM incidence, seed yield was reduced by an average of 12 kg ha–1 in pinto (the largest market class of dry bean in North America) and by 23 kg ha–1 in navy bean in rainfed production systems in North Dakota (del Río et al., 2004). White mold incidence is increasing in the western United States with the increasing use of overhead sprinkler irrigation. The necrotrophic fungus is endemic and seed transmitted, and it produces sclerotia that survive in soil for years. It can be spread from field to field by internally infected seed, sclerotia mixed with seed, contaminated soil on farm equipment, and irrigation runoff water, in addition to wind-blown ascospores (Steadman and Boland, 2005).
Adequate control of WM using fungicides and other disease management strategies has been difficult to achieve (del Río et al., 2004; Schwartz and Steadman, 1989), especially on the susceptible indeterminate prostrate growth habit Type III (Singh, 1982) dry bean cultivars that predominate in the western United States. Moreover, wide-row spacing and low plant populations, reduced irrigation and fertilizer, and use of upright cultivars with open plant canopy may reduce WM severity and incidence; however, they also reduce dry bean yield and the economic return to producers (Park, 1993; Saindon et al., 1995).
Plant architectural traits associated with WM resistance or avoidance in the field have been identified (Kolkman and Kelly, 2002). Nonetheless, it is often difficult to discriminate between the physiological resistance and plant architectural avoidance of WM because both are confounded under field conditions (Miklas and Grafton, 1992).
False positives for physiological resistance may result due to reduced disease severity in open plant canopies of upright genotypes, especially when planted in wide-row spacing. Moreover, often it is difficult to achieve high and uniform WM pressure in the field year after year to permit adequate evaluation of germplasm, segregating populations, families, breeding lines, and cultivars.
Dry and green bean germplasm with mostly partial physiological resistance to WM have been reported (Steadman et al., 2001). The highest levels of physiological WM resistance occur in the scarlet runner bean (P. coccineus L.), a member of the secondary gene pool of the common bean (Gilmore et al., 2002). The scarlet runner bean hybridizes with the common bean without embryo rescue, particularly when common bean is used as the female (Manshardt and Bassett, 1984). However, tropical and subtropical germplasm from the scarlet runner bean are highly photoperiod sensitive and poorly adapted in the western United States. Thus, direct field screening of the scarlet runner bean germplasm for identification of WM resistance alleles and quantitative trait loci (QTL) is difficult and cumbersome.
Abawi et al. (1978) and Schwartz et al. (2006) reported a single dominant gene controlling resistance to WM in P. vulgaris/P. coccineus interspecific populations. In contrast, Myers and Stotz (2002) found the F1 between resistant (PI 255956) and susceptible (PI 153209) P. coccineus to be susceptible to WM, whereas the F1 between other susceptible (‘Woven Pole’) and resistant (PI 255956) accessions segregated into 3:2 resistant:susceptible ratio (Myers and Stotz, 2002). The latter could be because of possible escapes in the F1 (as supported by the occurrence of only recessive WM resistance segregation in the F2), or PI 255956 could have been variable for WM reaction. Furthermore, two or more recessive WM resistance genes segregated in the F2 of both populations.
Hunter et al. (1982) reported a group of F5 interspecific breeding lines (IBL), and Miklas et al. (1998) reported four IBL, I9365-3, I9365-5, I9365-31, and 92BG-7, derived from P. vulgaris/P. coccineus interspecific populations that possessed moderate to high levels of WM resistance. Resistance of the latter group of IBL has been more effective than P. vulgaris germplasm in multilocation greenhouse and field tests (Steadman et al., 2001). The objectives of this study were (i) to develop WM resistant IBL VCW 54 (Reg. No. GP-276, PI 655531) and VCW 55 (Reg. No. GP-277, PI 655532) by congruity backcrossing (i.e., backcrossing alternately to either parent) between ‘ICA Pijao’ and P. coccineus accession G 35172, and (ii) to compare the reaction of the two IBL with known sources of WM resistance (A 195, G 122, I9365-25, ‘ICA Bunsi’, VA 19, 92BG-7) and susceptible (‘Othello’) checks.
Materials and Methods
Parental Germplasm and Development of Interspecific Breeding Lines
VCW 54 and VCW 55 were developed by congruity backcrossing between ICA Pijao and the scarlet runner bean accession G 35172. ICA Pijao is a small-seeded (<25 g 100 seed weight–1) black bean cultivar from Colombia. ICA Pijao possesses an upright erect growth habit Type II (Singh, 1982), the I gene resistance to bean common mosaic virus (BCMV, an aphid-vectored potyvirus), resistance to some races of Uromyces appendiculatus (Pers.) Ung. (the cause of common bean rust), and tolerance to bean golden mosaic virus (BGMV, a whitefly-vectored geminivirus) and bean golden yellow mosaic virus (BGYMV, a whitefly-vectored geminivirus). ICA Pijao is also insensitive to long photoperiod (>12 h daylength) occurring at higher latitudes and is a noncarrier of the Dl-1 and Dl-2 incompatibility genes (Singh and Gutiérrez, 1984). ICA Pijao is known to facilitate interspecific hybridization with the tepary bean, P. acutifolius A. Gray, a member of the tertiary gene pool of the common bean (Mejía-Jiménez et al., 1994; Parker and Michaels, 1986). G 35172 possesses a high level of resistance to BGMV and BGYMV (CIAT, 1986) and was variable for white mold resistance in the greenhouse modified straw-test (Terán et al., 2006). G 35172 has extremely large seeds (>70 g 100 seed weight–1) of different colors and an aggressive climbing growth habit Type IV (Singh, 1982). G 35172 is highly photoperiod sensitive such that it would not flower during the summer months in Idaho.
The single cross (ICA Pijao/G 35172) and congruity backcross (ICA Pijao/G 35172//ICA Pijao/3/G 35172) between ICA Pijao and G 35172 were made at the Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, in 1988–1989. In the single cross, ICA Pijao was used as the female. Three to five staggered plantings of ICA Pijao were made to coincide with flowering of the G 35172 used as male. Approximately 50 F1 seeds were produced for the interspecific single-cross hybrid. Three staggered plantings of ICA Pijao and the F1 were made for the congruity backcrossing. The resulting F1 from the congruity backcross (
125 seeds) was advanced four generations, using a single-pod bulk within each congruity-backcross F1–derived family.
Thirty-one IBL derived from the congruity-backcross F1 were introduced in Idaho from CIAT in 1998–1999. Only a very few plants within 28 of 31 IBL were relatively insensitive and produced seed when first grown at the University of Idaho Parma Research and Extension Center, Parma, ID, in 2000 (Singh et al., 2007b). In 2000 and 2001, plants within each IBL were harvested in bulk, using a single pod from each plant. No selection for WM resistance or any other trait was practiced.
Screening for White Mold Resistance
Greenhouse Screening in Colorado
From 2002 to 2006, all IBL were screened in the greenhouse at the Colorado State University, Fort Collins, CO, and in the field in southern Idaho. In the greenhouse, an average of 12 plants were screened from each IBL without replication from 2002 to 2004 and with two replicates (each with 12 plants) in 2005 and 2006. Each plant was inoculated with a 2-d-old mycelial plug from a colony of S. sclerotiorum isolate CO-S20 (from a pinto bean plant infected in northern Colorado in 1996) grown in the dark on potato dextrose agar (PDA) medium (1.5% PDA) at 22°C. The original CO-S20 isolate was maintained in the refrigerator at 4°C as PDA-produced sclerotia. A sclerotium was surface disinfested with 0.6% sodium hypochlorite for 30 s and transferred aseptically to PDA to initiate a supply of fresh mycelium for each greenhouse experiment. The straw-test (Petzoldt and Dickson, 1996) was used and WM scored at 7 d postinoculation (DPI) from 2002 to 2004 and 14 DPI in 2005 and 2006 on a 1 to 9 scale, where 1 = symptomless, and 9 = severely diseased or dead.
Greenhouse Screening in Idaho
The surviving IBL in Colorado were also screened using the modified petiole-test (del Río et al., 2000) in 2004 and the straw-test in 2005 and spring 2006 in the greenhouse at the University of Idaho, Kimberly Research and Extension Center, Kimberly, ID. Sclerotinia sclerotiorum isolate CO-S20 also was used for all greenhouse tests as described above and by Schwartz et al. (2006). A randomized complete block design with two replicates was used. Each plot consisted of six plants, two plants grown in a 15-cm-diameter pot, and 3 pots genotype–1. Furthermore, White mold was scored at 14 DPI on a similar 1 to 9 scale, and only plants with score of 
4 were harvested individually within each IBL.
Field Screening in Idaho
A naturally infested field with a history of WM disease was used each year. For the field screening in 2002 and 2003, each plot consisted of a single row 3.5 m long spaced 0.56 m apart without replicates. A randomized complete block design with four row plots with two replicates in 2004 and 2005 and three replicates in 2006 was used. Approximately 50 seeds were planted in each row. The nursery was inoculated three times (early-, mid-, and late-flowering stages) with ascospores (supplied by Dr. Boosalis, University of Nebraska, Lincoln) at 106 spores mL–1 in 2003 (Boosalis et al., 2000), ascospores and mycelial culture in 2004, and only mycelial culture in 2005 and 2006. The mycelial fragment inoculation procedure consisted of the following. A mycelial suspension (approximately 106 fragments mL–1) of S. sclerotiorum grown on PDA in the lab at 22°C for 48 h was applied in 234 L tap water ha–1 with a backpack solo sprayer and flat fan nozzle directed onto the upper third of the flowering plants after 8:00 p.m. (H. Schwartz and K. Otto, unpublished). Reaction to WM was recorded on a plot basis on a 1 to 9 scale, where 1 = no visible WM symptoms on leaves, stem, and pods, and 9 = severely diseased or dead plants.
Each year, both field data from Idaho and greenhouse data from Colorado and Idaho were considered together, with only individual plants from the IBL with combined 1 to 4 WM scores selected. Nonetheless, VCW 54 and VCW 55 were variable for WM reaction when screened in subsequent trials in the greenhouse and field in 2006 (Singh et al., 2007b). Therefore, toward the end of 2006, VCW 54 and VCW 55 were subjected to a more rigorous greenhouse screening. A modified straw-test (Terán et al., 2006) combined with disease scoring at 28 DPI was used. Thus, finally true-breeding WM-resistant VCW 54 and VCW 55 were developed for a comparative study.
Comparison of VCW 54 and VCW 55 with ICA Pijao and Other Sources of White Mold Resistance
Greenhouse Trials in Colorado and Idaho in 2007
VCW 54 and VCW 55 along with ICA Pijao and resistant (ICA Bunsi, I9365-25, G 122) and susceptible (Othello) checks were compared in the greenhouse in Colorado and Idaho in spring 2007 and again in the greenhouse and field in Idaho in the summer–fall of 2007. The modified straw-test (Terán et al., 2006) was used in the greenhouse. A randomized complete block design with three replicates was used. Each replication consisted of two plants grown in a 15-cm-diameter pot, and 3 pots genotype–1. The mycelial plug from a 48-h-old culture of S. sclerotiorum in an eppendorf tip (1 mL volume) was allowed to stay on the tip of the inoculated cut stem below the fifth or sixth node until the plant died, matured, or eppendorf tip dropped automatically. Two or three portable humidifiers and two to three times per day wetting of the greenhouse floor were used to maintain high humidity in the greenhouse in Idaho. Thus, relative humidity in the greenhouse was kept above 80% and temperature fluctuated between 16 and 22°C. Reaction to WM was scored (1–9 scale) on a single-plant basis 28 DPI followed by verification of resistance reaction at maturity.
Field Trial in Idaho in 2007
The field trial was conducted late summer–early fall (mid-June to October) in the field at the University of Idaho Parma Research and Extension Center in 2007. Each plot consisted of a single row 3 m long with three replicates. An average of 50 seeds were planted per plot. One row of highly susceptible pinto Othello was planted between plots. Three mycelial inoculations were made using a power-driven backpack solo sprayer at the beginning, middle, and late flowering stages after 8:00 pm. Moreover, postinoculation field plots were kept moist by extra gravity (20 h run once a week) and solid-set sprinkler irrigations (1 h run three times per day until disease evaluations were made). White mold reaction (1–9 scale) was recorded on a plot basis near physiological maturity (green or purple pods turning tan, brown, or light purple). All genotypes were also characterized for their growth habit, flower color, and seed characteristics.
Greenhouse Trials in Colorado and Idaho in 2008
In 2008, VCW 54, VCW 55, ICA Pijao, and resistant (VA 19, 92BG-7, A195, G122, ICA Bunsi) and susceptible (Othello) checks again were compared in the greenhouse in Colorado and Idaho. The experimental design and number of plants per plot were the same as in the greenhouse experiments in 2007. However, four replicates and two inoculations were made in 2008. In the first inoculation, one mycelial plug was used in the eppendorf tip to inoculate each plant. In the second inoculation, 1 wk later, the same plants were inoculated at three additional points each with three mycelial plugs. Similar to 2007, reaction to WM was scored on a single-plant basis 28 DPI followed by verification of resistance reaction at harvest maturity.
Despite a highly severe disease pressure in 2007, WM scores in the field at Parma tended to be lower than greenhouse scores (probably because of additional effects of plant architectural avoidance). A field experiment, therefore, was not conducted in 2008. Each greenhouse and field data set was analyzed separately using PROC-GLM procedure of SAS statistical procedure (SAS Institute, 2004). Also, the mean WM score for each genotype and the least significant difference (LSD at P = 0.05) were determined.
Results and Discussion
Comparison of VCW 54 and VCW 55 with ICA Pijao and Other Sources of White Mold Resistance in Race Mesoamerica
Pinto Othello had susceptible mean WM scores ranging from 7.3 to 9.0 in all evaluation environments (Tables 1
and 2
). Furthermore, entire rows of Othello planted as WM spreader in the field at Parma were killed by the time pods had fully developed (i.e., R8 stage). Thus, the susceptible reaction of Othello was consistent with our previous observations. Moreover, because Othello is an early-maturing, high-yielding, broadly adapted cultivar in the western United States, noninoculated plots also served as a standard for comparing maturity and adaptation of VCW 54 and VCW 55.
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Thus, ICA Pijao could not be considered completely susceptible to WM, and like ICA Bunsi, ICA Pijao may carry some QTL imparting low to intermediate levels of resistance. ICA Bunsi had an intermediate WM score in the greenhouse at Fort Collins in 2007 (Table 1) and when inoculated with one mycelial plug in 2008 (Table 2). In contrast, it had a susceptible reaction in Idaho in the greenhouse in 2007 and 2008 and in the field in 2007. Thus, researchers using ICA Bunsi or its derived genotypes should expect little progress in improving WM resistance for production regions with severe WM incidence, irrespective of breeding methods used. Both ICA Bunsi and ICA Pijao were developed by the Colombian National Program before dry bean breeding was initiated at CIAT in the late 1960s and early 1970s. Moreover, unlike prostrate growth habit Type III of ICA Bunsi, ICA Pijao has an upright growth habit Type II, is high yielding, widely adapted, and possesses resistance to some races of the rust pathogen and partial resistance to BGMV and BGYMV.
VCW 54 and VCW 55 had significantly (P = 0.05) lower overall mean disease scores than ICA Pijao across three greenhouse and field environments in 2007 (Table 1) and their overall mean WM scores also were lower than ICA Pijao in 2008 (Table 2). This would suggest that P. coccineus G 35172 contributed complementary additive genes/QTL for WM resistance to VCW 54 and VCW 55. This also would justify a continued search for germplasm accessions from P. coccineus with yet higher levels of WM resistance and introgress that into common bean cultivars. Thus, in addition to resistance to BGMV and BGYMV (CIAT, 1986; Osorno et al., 2007), G 35172 has high levels of WM resistance.
Comparison of VCW 54 and VCW 55 with White Mold Resistance in Race Nueva Granada
Large-seeded breeding lines A 195 (Singh et al., 2007a) and VA 19 and germplasm accession G 122 belong to the Andean common bean race Nueva Granada. G 122 was included in all comparative greenhouse and field trials in 2007 and 2008. A 195 and VA 19 were included in 2008 trials, but not in 2007 due to lack of seed. The overall mean WM scores of G 122 across all greenhouse and field environments was significantly (P = 0.05) higher than that of VCW 54 and VCW 55 at 28 DPI in 2007 (Table 1). G 122 also had a significantly higher overall mean disease score than that of VCW 54 across inoculation methods and greenhouse environments in 2008 (Table 2). A 195 and G 122 had the same overall mean WM score (5.4) and VA 19 had a score of 6.0 in 2008. A 195 (Singh et al., 2007a) and VA 19 (Dr. P. Miklas, personal communication, 2009) derive WM resistance from large-seeded light red kidney cultivar Red Kloud developed at Cornell University, Ithaca, NY.
Comparison of VCW 54 and VCW 55 with Other White Mold Resistant Interspecific Breeding Lines Derived from P. coccineus
White mold–resistant IBL compared with VCW 54 and VCW 55 included I9365-25 in trials in 2007 and 92BG-7 in 2008. These genotypes are believed to derive their WM resistance from P. coccineus (Dr. P. Miklas, personal communication, 2002). The overall mean WM scores of VCW 54 and VCW 55 were significantly (P = 0.05) lower than that of I9365-25 in 2007 (Table 1). Miklas et al. (1998) had released four other IBL derived from P. coccineus. Of the four IBL, 92BG-7 had the highest WM resistance; therefore, 92BG-7 was included in the greenhouse tests in 2008.
When VCW 54 and VCW 55 were compared with 92BG-7 and six other genotypes in the greenhouse in Colorado and Idaho in 2008, the mean squares due to locations, inoculation methods, and genotypes were highly significant (P = 0.01, Table 3 ). Also, the interaction between locations and genotypes was significant (P = 0.05). This further would justify testing of genotypes using diverse screening methods and environments to detect broad-spectrum resistance (Steadman et al., 2001). The mean WM scores in both inoculation methods were significantly lower in Colorado than in Idaho (Table 2). Also, the difference between the two methods were significant only in Colorado. Thus, researchers interested in identifying and breeding for higher levels of WM resistance may consider using two or more inoculations of the same plant using two or more mycelial plugs each time. To save resources and time, however, it may be advisable to wait 12 to 14 d after the first inoculation, discard the susceptible genotypes, and only inoculate presumably resistant plants a second time.
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Flower Color, Growth Habit, Maturity, and Seed Characteristics of VCW 54 and VCW 55
In addition to differences in the levels of WM resistance, VCW 54 and VCW 55 could also be unique because of their combination of flower and seed color (Table 1). For example, VCW 54 had light scarlet flower color with purplish black seed. On the other hand, the standard of VCW 55 flower was white with light pink stripe, and seed was black. In common bean, we have seldom observed such combinations of flower and seed color. Because VCW 54 and VCW 55 are sister lines developed under similar screening and selection methods, it is very likely that the majority of their genes/QTL for white mold resistance are the same. Nonetheless, they will probably give different recombinants in regards to flower and seed color when crossed with other common bean genotypes. In the common bean, all black-seeded germplasm accessions, breeding lines, and cultivars that we know of possess purple flower color as exemplified by ICA Pijao. When such germplasm is crossed with cultivars of cranberry, dark and light red kidney, pink, pinto, and other colored market classes, seldom is it possible to recover desired seed color from biparental crosses.
VCW 54 and VCW 55 were late maturing (100 d) and had small seed and an upright growth habit Type II, similar to ICA Pijao (Table 1). Often late maturity combined with upright growth habit and resistance to lodging (associated with stay-green stem characteristic) seem to be associated with field avoidance of WM. Given the WM reaction of VCW 54 and VCW 55 in the greenhouse and field in 2007 under extremely severe disease pressure, it is likely that, in addition to the physiological WM resistance, the two IBL may also possess some architectural avoidance traits to lower WM scores in the field (Kolkman and Kelly, 2002; Park, 1993; Saindon et al., 1995). Furthermore, while VCW 55 had no apparent fertility problems and behaved as a normal common bean, VCW 54 was partially sterile, and hence, seed set was sparse and delayed. Despite more than 10 generations of selection, it was not possible to overcome the sterility. In our limited experience, however, we have been able to select against this partial sterility in crosses of VCW 54 with other common bean genotypes.
Availability
A small quantity of seed of VCW 54 and VCW 55 for research purposes is available from the corresponding author for the first five years. If VCW 54 and VCW 55 are used for research or contributes to the development of a breeding line or cultivar, appropriate acknowledgment of the researchers and institutions responsible for development and evaluation of VCW 54 and VCW 55 would be highly appreciated.
Acknowledgments
This research was supported by the USDA-ARS National Sclerotinia Initiative Grant No. 58-5442-2-256 "Introgressing White Mold Resistance from the Secondary Gene Pool of Common Bean" from 2002 to 2009. The authors also thank Marie Dennis and Richard Hayes for management of the greenhouses at Kimberly, ID, and to Craig Robinson for field plot management at Parma, ID.
Footnotes
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 November 11, 2008.
References
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