Mohamed F. Mohamed and Dermot P. Coyne
Common bacterial blight, incited by Xanthomonas campestris pv. phaseoli (Smith) Dye (Xcp), is a serious disease of common beans (Phaseolus vulgaris L.). Three experiments were conducted twice in growth chambers at 26 ± 1C under short (10 hours light/14 hours dark) and long (16 hours light/8 hours dark) photoperiods to determine the influence of these photoperiods, flower bud removal, pod development, and pre- and post-inoculation photoperiods on the reaction of common beans to Xcp. In one test, `PC-50' (susceptible; S) flowered earlier and was more susceptible to Xcp under the short photoperiod than under the long photoperiod. BAC-6 (resistant; R) flowered at the same time under both photoperiods but developed rapid leaf chlorosis (RLC) (hypersensitive reaction) under long photoperiods. Flowering and disease reactions to Xcp by XAN-159 (R) were similar under both photoperiods. In a second test, daily removal of flower buds of `PC-50' decreased its susceptibility to Xcp under the short photoperiod. RLC of inoculated leaves of BAC-6 occurred during flowering and pod development under both photoperiods. XAN-159 expressed a high level of resistance to Xcp but showed RLC at later pod development stages. In a third test, the disease reaction of `PC-50' was affected by the particular photoperiod applied post-inoculation but was not influenced by the photoperiod applied before inoculation with Xcp. The implications of these results in breeding beans for resistance to Xcp are discussed.
Zhanyuan Zhang, Dermot P. Coyne, and Amitava Mitra
Factors influencing Agrobacterium tumefaciens-mediated transformation of common beans (Phaseolus vulgaris L.) were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system to develop a repeatable transformation protocol. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains—A2760 and EHA105—were used, with emphasis on the former due to its overall higher infection rate. Eleven common-bean genotypes were compared for susceptibility to strain A2760 or EHA105. The pinto bean `Othello' was used extensively in testing different transformation conditions. Factors significantly affecting transformation rate were Agrobacterium × host interactions, explant maturity, preculture and cocultivation conditions, and selection schemes, based on transient GUS gene expression. The best transformation conditions were the use of susceptible genotypes and explants derived from mature seeds, preconditioning of explants in a medium containing 20 μmol of benzyladenine (BA) in darkness or on a filter paper, dipping explants in high concentrations of Agrobacterium cell suspension (OD650 = 0.8-1.0) followed by a long-term (6-day) cocultivation period on a semisolid agar medium in the presence of cytokinin or 3-day cocultivation on a moistened filter paper, and the use of lethal levels of selective agents. About 4% of explants, or 14% of regenerated shoots or buds, were putatively transgenic, as indicated by GUS blue staining throughout the entire shoot or bud, after explants were transformed with Agrobacterium strain A2760 using an optimized protocol.
Dale T. Lindgren and Dermot P. Coyne
Differences in potato leafhopper (Empoasca fubae Harris) injury symptoms were noted in 22 cultivars or lines of dry beans (Phaseolus vulgaris L.) in a 1991 field trial at North Platte, Neb. Seed yield, biomass, and plant injury symptoms were recorded. The same 22 dry bean cultivars or lines were planted in a split-plot design, with main plots protected (sprayed with insecticide) vs. unprotected (not sprayed) and cultivars or lines as subplots in 1992 and 1993. Significant differences were observed between cultivars/lines for leafhopper injury and yield in all 3 years. `Tacaragua' (black-seeded) and pinto `Sierra' were highly resistant to leafhoppers, with no visual leafhopper injury symptoms in all 3 years. Significant negative correlation coefficients between leafhopper injury symptoms and yield were recorded in the protected (4.50) and unprotected (-0.33) plots in 1993 but only in the unprotected (-0.46) plots in 1992. A cultivar x spray interaction response to leafhoppers occurred in 1992 but not in 1993. The degree of leafhopper injury symptoms varied between years.
Dermot P. Coyne, Lisa Sutton, and Debra Fujimoto
No sources of resistance to Xanthomonas campestris pv. cucurbitae (X.c.c.) in the Cucurbita species have been reported. Cultivars, breeding lines, landraces, and PI lines of 5 Cucurbita species were screened for resistance to X.c.c. in 3 greenhouse tests (GH). A `florist`s frog' was used to inoculate the first fully expanded leaves using a X.c.c. suspension (107 CFU/ml). The disease reaction was recorded as the percentage of inoculated leaf area with necrotic lesions and/or chlorosis. Butternut (b n) breeding lines were also evaluated for reaction to X.c.c. under uniform natural in feetion in 2 field (F) trials. A randomized complete block design was used in all GH and F experiments. C. moschata Nebr. BN PM1-88-8 and C. martinezii had high resistance to X.c.c. All other entries in all tests were susceptible. BN PM-88-8 is an early maturing small stable (no crookneck fruit) near-round BN type squash. The fruit are resistant to black–rot, but the leaves are susceptible to powdery mildew. BN PM-88-8 is ideal for microwave cooking because of its near-round shape permitting more uniform cooking. Release is expected in 1990.
James S. Beaver, James R. Steadman, and Dermot P. Coyne
Field reaction of 25 red mottled bean (Phaseolus vulgaris L.) genotypes to common bacterial blight [Xanthomonas campestris pv. phaseoli (Smith) Dye] was evaluated in Puerto Rico over 2 years. The average disease severity (percent leaf area with symptoms) was similar over years. The determinate red mottled genotypes had almost twice as much disease as indeterminate genotypes. Eight of the indeterminate genotypes had significantly less disease than the mean of the field experiments. These genotypes may serve as useful sources of resistance to common bacterial blight. The size of the chlorotic zone around necrotic lesions varied between growing seasons, showing that environment can influence the expression of common bacterial blight symptoms.
Soon O. Park, Dermot P. Coyne, and James R. Steadman
Bean rust, caused by Uromyces appendiculatus, is a major disease of common bean (Phaseolus vulgaris). The objective was to identify RAPD markers linked to the gene (Ur-7) for specific resistance to rust race 59 using bulked segregant analysis in an F2 segregating population from the common bean cross GN1140 (resistant to rust) × Nebraska #1 (susceptible to rust). A single dominant gene controlling specific resistance to race 59 was found in the F2 and was confirmed in the F3. Seven RAPD markers were detected in a coupling-phase linkage with the Ur-7 gene. Coupling-phase RAPD markers OAA11.500, OAD12.550, and OAF17.900 with no recombination to the Ur-7 gene were found. Three RAPD markers were identified in a repulsion-phase linkage with the Ur-7 gene among the three markers at a distance of 8.2 cM. This is the first report on RAPD markers linked to the Ur-7 gene in common bean. The RAPD markers linked to the gene for specific rust resistance of Middle American origin detected here, along with other independent rust resistance genes from other germplasm, could be used to pyramid multiple genes into a bean cultivar for more-durable rust resistance.
Haytham Z. Zaiter, Dermot P. Coyne, and James R. Steadman
Sixteen Alubia lines (15 with long, straight hairs and one with short, hooked hairs on trifoliolate leaves) derived from single-plant selections made in an Alubia landrace (Argentine) were used to evaluate the relation of abaxial leaf pubescence to reaction to rust in a greenhouse experiment. The pinto cultivar UI-114 (short, hooked hairs) was used as a susceptible check. One plant per pot, replicated six times, in a randomized complete-block design was used. The primary leaves and the sixth trifoliolates of all plants from 12- and 50-day-old plants, respectively, were inoculated with a water suspension of urediniospores (105 cells/ml) of rust isolate US-NP85-10-1. Pustule size and rust intensity were assessed 14 days later. No rust pustules were observed on the sixth trifoliolate leaves of the pubescent (long, straight hairs) Alubia lines, but large pustules were observed on the primary leaves (short, hooked hairs) of all Alubia lines and pinto `UI-114'. as well as on the sixth trifoliolate leaf of A-07-2 and pinto `UI-144' (the latter two with short, hooked hairs).
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
A new in vitro protocol was developed for multiple bud induction and plant regeneration from embryonic axis explants of four common bean (Phaseolus vulgaris L.) and two tepary bean (P. acutifolius A. Gray) lines. The explants were prepared from two embryo sizes, 3 to 4 mm and 5 to 7 mm, corresponding to pods collected after 15 and 25 days from flowering, respectively. The embryonic axis was cultured on Gamborg's B5 basal medium with 0, 5, 10, or 20 μm BA in combinations with 0, 1, or 2 μm NAA. The cultures were maintained at 24 to 25C under continuous light or incubated in darkness for 2 weeks followed by continuous light before transfer to the secondary B5 medium (0 or 2 μm BA or 2 μm BA plus 4 μm GA3). Adventitious roots or a single shoot with roots formed on the explants cultured on media without plant growth regulators. Multiple buds were induced on all BA media, but more were produced with 5 or 10 μm for most lines. Dark incubation greatly enhanced multiple bud initiation. Shoot buds were not produced on media containing NAA alone or in combinations with BA. On the secondary medium, six to eight shoots per explant for common bean and up to 20 shoots per explant from tepary bean were observed after 3 weeks. Mature, fertile plants were produced from these shoots. Chemical names used: benzyladenine (BA); 1-naphthaleneacetic acid (NAA); gibberellic acid (GA3).
Mohamed F. Mohamed, Dermot P. Coyne, and Paul E. Read
Plant regeneration has been achieved in two common bean lines from pedicel-derived callus that was separated from the explant and maintained through successive subcultures. Callus was induced either on B5 or MS medium containing 2% sucrose and enriched with 0.5 or 1.0 mg thidiaznron/liter alone or plus various concentrations of indoleacetic acid. The presence of 0.07 or 0.14 g ascorbic acid/liter in the maintenance media prolonged the maintenance time. Up to 40 shoot primordia were observed in 4-week-old cultures obtained from 40 to 50 mg callus tissues on shoot-induction medium containing 1-mg benzyladenine/liter. These shoot primordia developed two to five excisable shoots (>0.5 cm) on medium with 0.1-mg BA/liter. A histological study confirmed the organogenic nature of regeneration from the callus tissues. The R2 line from a selected variant plant showed stable expression of increased plant height and earlier maturity. Chemical names used: ascorbic acid, N- (phenylmethyl)-1H-pnrin-6-amine [benzyl-adenine, BA], 1H-indole-3-acetic acid (IAA), N- phenyl-N'-1,2,3-thiadiazol-5-ylurea [thidiazuron, TDZ].