Classical and Molecular Approaches to Breeding Horticultural Plants for Disease Resistance: Introduction to the Colloquium

Diseases of horticultural crops have always been a concern of crop producers, processors, merchandisers, and consumers because of reductions in yield and product quality. Excessive chemical application to control diseases of some crops has been a health concern in some countries. Some diseases pose a major constraint, or even a threat, to the production of a crop in a region. Plant breeders, in association with plant pathologists, have had notable successes in breeding disease-resistant varieties, thus ensuring the economic production of a crop. There is now an increased need to breed for disease resistance of horticultural crops because of a loss of chemicals for disease control, environmental and human health concerns, the need to enhance economic competitiveness of horticultural producers and to reduce the genetic vulnerability of crops to pathogens, and an emphasis on using sustainable crop production systems. It is timely to review new concepts and strategies in breeding for disease resistance in horticultural crops in view of the new technologies that have been developed along with the continuing role of classical breeding. This colloquium was organized to review recent developments in classical and molecular approaches to breeding diverse horticultural plants for resistance to viruses, bacteria, and fungi. The reviews embody concepts, methods, strategies, and examples of current developments and successes that will interest breeders of all horticultural crops. A comprehensive classical breeding program for disease resistance involving cooperation between breeders and pathologists encompasses most or all of the following areas: knowledge of the biology, strain, variation and virulence, and epidemiology of the pathogen; development of rapid inoculation methods with repeatable results; use of sets of differential varieties/lines (where available) to provide information on strain variation; easily applied and understood disease rating scales; identification of sources of resistance; knowledge of the genetics of resistance; development of effective selection and breeding schemes for resistance; and widespread evaluation of new lines along with controls in production areas to determine merit for release. Many graduate students in plant breeding are now heavily involved in using the tools of molecular biology and tissue culture (molecular markers, gene tagging, molecular mapping, transformation methods, and regeneration of specific tissues into intact plants) in the laboratory. They also need to be well versed in the disciplines associated with classical breeding programs. The new tools need to become a part of the total plant breeding program. At this stage, breeders need to combine molecular and classical approaches in enhancing germplasm and developing crop varieties with desirable horticultural traits along with disease resistance. The goal of plant breeding is to enhance the welfare of a society through genetic improvement of crops, while at the same time improving breeding methods and advancing knowledge in attaining that goal. This perspective needs to be imparted to graduate students, administrators of land-grant institutions, and funding agencies because of the continued erosion of plant breeding programs. It is imperative for the future well-being of humanity that we continue to train plant breeders to meet the stated goal

Photoperiod Sometimes Influences Common Bacterial Blight Disease of Common Beans

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 postinoculation 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

Resistance gene analog polymorphism (RGAP) markers co-localize with disease resistance genes and QTL in common bean

Resistance (R) genes containing nucleotide-binding site (NBS)-leucine rich repeats (LRR) are the most prevalent types of R gene in plants. The objective of this study was to develop PCR-based R-gene analog polymorphism (RGAP) markers for common bean (Phaseolus vulgaris L). Twenty degenerate primers were designed from the conserved kinase-1a (GVGKTT) and hydrophobic domains (GLPLAL) of known NBSLRR type R-genes and from EST databases. Sixty-six of the 100 primer combinations tested yielded polymorphism. Thirty-two RGAP markers were mapped in the BAT 93/Jalo EEP558 core mapping population for common bean. The markers mapped to 10 of 11 linkage groups with a strong tendency for clustering. In addition, the RGAP markers co-located, on six linkage groups, with 15 resistance gene analogs (RGAs) that were previously mapped in other populations of common bean. The distance between the priming sites in NBS-LRR type R-genes is around 500 bp. Of the 32 RGAP markers, 19 had sizes larger and 13 less than 500 bp. RGAP markers mapped close to known R-genes on B11, and to QTLs for resistance on B1, B2, B6, B7, B8, B10, and B11. RGAP appears to provide a useful marker technique for tagging and mapping R-genes in segregating common bean populations, discovery of candidate genes underlying resistance QTL, and future cloning of R-genes in common bean

A Radiation-induced Mutant with Resistance to Common Bacterial Blight Disease in Common Beans

The leaf reaction of the Phaseolus vulgaris L. germplasm—UNECA (M6 mutant derived from the cultivar Chimbolito, Costa Rica), ‘Chimbolito’, BAC-6 (Brazil), XAN- 159 (Centro Internacional de Agricultura Tropical, Cali, Colombia), and ‘PC-50’ (Domican Republic)—to Xanthomonas campestris pv. phaseoli strain V4S1 (Dominican Republic) were determined in two replicated trials conducted in a greenhouse in Lincoln, Neb. (Feb.– Mar. and July–Aug. 1993). ‘PC-50’ and ‘Chimbolito’ were susceptible to Xcp strain V4S1 in both tests. UNECA, BAC-6, and XAN-159 had similar levels of resistance to Xcp in the July to August trial. However, in the February to March trial, the resistance of UNECA was greater than that of BAC-6 but less than that of XAN-159

Field Reaction of Landrace Components of Red Mottled Common Bacterial Blight

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

‘Lakota’ Winter Squash, A Cultivar Derived from Native American Sources in Nebraska

‘Lakota’ is a novel, smooth- and thinskinned, small-fruited, early maturing, ovoidshaped winter squash [Cucurbita maxima (Duch.)] (Fig.1). Plants produce fruit exhibiting various degrees of green and orange variegated patterns, along with some solid green and orange fruit. ‘Lakota’ was released because of its novel decorative value and good baking quality. A similar winter squash is not available commercially. Origin Seeds of the winter squash population from which ‘Lakota’ was selected were donated to the Univ. of Nebraska, Lincoln, by A.G., who originally received the seed from the late Martha Newman, Alliance, Neb. By examining the Quarter Master Reports (1820), A.G. was able to determine that the original squash landrace was grown by Native Americans living along the Missouri Valley and that this squash also was grown in gardens by troops stationed at Fort Atkinson in northeastern Nebraska; subsequently, troops stationed at the later-developed Fort Robinson (1870 to 1875) in northwestern Nebraska obtained seeds of this squash landrace from Fort Atkinson, grew it in their gardens, and stored it in cellars for use during the winter (U.S. Army Quarter Master Reports for Fort Robinson, 1870). Newman, living on a homestead in northwestern Nebraska, apparently received seeds of the squash landrace from her brother, Alfred Iossi, who was a civilian employee at Fort Robinson (the late Martha Newman, personal communication). Eventually, the Newman family moved to Alliance in western Nebraska, brought the seeds with them, and raised the squash each year. Although this squash has excellent baking quality (personal observation), it was never introduced into commerce. Fruit of the original landrace were described as being elongated (75 to 85 cm long, 20 to 25 cm in diameter) and cylindrical, with dark green and orange variegated skin patterns. Although the fruit shape is nearly similar, it is smaller than the C. maxima banana type (Casttetter and Erwin, 1927). The Winnebago Indians of eastern Nebraska also grew a C. maxima landrace with similarly shaped fruit, but the fruit were smaller, warty, and dark green. ‘Winnebago’, which was derived from this landrace, was introduced by Oscar H. Will Co. in the 1920s (Casttetter and Erwin, 1927; Tapley et al., 1937). In Lincoln, only one of 200 plants grown from the seed donated by A.G. produced fruit resembling the original description (elongated and cylindrical with mottled orange and green skin color pattern). The segregation pattern of fruit shape suggested that the original landrace was outcrossed to hubbard squash because many fruit were ovoid and some were ovoid with pointed ends, similar to those of hubbard squash. Casttetter and Erwin (1927) described hubbard squash as “nearly spherical, tapering into a neck at the stem end and to [a] curved, pointed projection at the blossom end.” According to Newman, her population also deviated from the original type, suggesting that the original landrace was outcrossed in her garden in Alliance during the intervening years (personal communication). The variation in fruit shape, color, and size in the population grown at Lincoln provided an opportunity to select for a novel, decorative, small, ovoid squash of good baking quality

Rust Reaction and Pubescence in Alubia Beans

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)

Expression of Human Lactoferrin cDNA Confers Resistance to \u3ci\u3eRalstonia solanacearum\u3c/i\u3e in Transgenic Tobacco Plants

A construct containing a human lactoferrin cDNA was used to transform tobacco (Nicotiana tabacum) using an Agrobacterium-mediated DNA-transfer system to express this human protein in transgenic plants. Transformants were analyzed by Southern, Northern, and Western blots to determine integration of the cDNA into the plant genome and lactoferrin gene expression levels. Most transgenic plants demonstrated significant delays of bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. Quantification of the expressed lactoferrin protein by enzyme-linked immunosorbent assay in transgenic plants indicated a significant positive relationship between lactoferrin gene expression levels and levels of disease resistance. Incorporation of the lactoferrin gene into crop plants may enhance resistance to other phytopathogenic bacteria as well

Medium pH and Leaf Nutrient Concentration Influence Rust Pustule Diameter on Leaves of Dry Beans

Nine bean cultivars/lines (Phaseolus vulgaris L.) were grown in three soils/ rooting media at pH values of 7.9, 6.5, and 5.8 in greenhouse, growth chamber, and field experiments to evaluate the leaf reaction of the plants to a Nebraska bean rust [Uromyces appendiculatus (Pers.) Unger var. appendiculatus] isolate US85-NP-10-1. Significant differences were observed for rust pustule diameter between cultivars/lines grown in the three growth media. Plants grown in the medium at pH 5.8 showed significantly larger rust pustule diameters than those of plants grown at pH 6.5 or 7.9. A significant interaction occurred between growth medium and cultivars/lines for the rust reaction. Concentrations of Cl and Mn in leaves were positively correlated with rust pustule diameter. In contrast, concentration of K in leaves was negatively correlated with rust pustule diameter. Plant breeders attempting to improve beans for rust resistance must consider the growth medium pH in evaluating intensity and severity of rust symptoms on leaves