Abstract

Introduction. Common beans (Phaseolus vulgaris L.) in Central America and the Caribbean are often produced on low fertility soils which reduces crop yield. Bean breeding programs need to identify genotypes that have superior adaptation to these conditions. Objective. Identify Mesoamerican bean germplasm lines with superior adaptation to low soil fertility. Materials and methods. The performance of twenty-seven Mesoamerican bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation (BASE) 120 panel were evaluated in an unfertilized oxisol at Isabela, Puerto Rico over five growing seasons (four-year period from 2015-2018). The lines were inoculated with a mixture of Rhizobium etli and R. tropici to promote symbiotic nitrogen fixation (SNF). Results. Four lines produced mean seed yields >1,200 kg ha-1 and had estimates of nitrogen derived from the atmosphere (NDFA) >50 %. Greater nodule number was positively correlated with % NDFA, later maturity and seed yield. The heat and drought tolerant small red cultivar ‘Rojo Chortí’ and the heat tolerant white cultivar ‘Verano’ had among the smallest apparent C isotope discrimination values suggesting greater water use efficiency. Among the elite lines in the trial, root rot damage was minimal and the basal root growth angles were intermediate (40-60 %), which favored the uptake of water and soil nutrients. Conclusion. Mesoamerican bean lines with superior seed yield and enhanced symbiotic nitrogen fixation in a low fertility soil were identified. Many of these lines also possess resistance to other biotic and abiotic factors that limit bean seed yield in Central America and the Caribbean.

Introduction

The production of Mesoamerican (black, small red, and white) beans (Phaseolus vulgaris L.) in Central America and the Caribbean (CAC) is threatened by several diseases and pests including Bean golden yellow mosaic virus, Bean common mosaic virus, Bean common mosaic necrosis virus, rust caused by Uromyces appendiculatus (Pers.: Pers.) Unger, common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli and leafhoppers (Empoasca spp.) (Beaver et al., 2003; Rosas et al., 2000; Rosas, 2011). Considerable progress has been made within the region in the development and release of Mesoamerican bean cultivars having enhanced levels of disease resistance to these and other biotic constraints (Beaver et al., 2018a, 2018b; Porch et al. 2014; Rosas et al., 2004). Bean producers in Central America and the Caribbean also face numerous abiotic yield constraints including terminal drought, high temperatures, and infertile soils (Miklas et al., 2006). Beans in Latin America and the Caribbean are often produced by farmers with limited resources on degraded soils that are deficient in N (Beebe et al., 2012). Low levels of soil P can reduce nodule numbers and increase the proportion of nodules that are ineffective for symbiotic N2 fixation (SNF) (Pereira & Bliss, 1987).

The combination of enhanced SNF with the capacity to better acquire or utilize soil N should improve adaptation of beans to unfertile soils. Bean genotypes that combine greater SNF and nitrogen use efficiency would reduce both dependence on N fertilizer and potential contamination of N in the environment (Akter et al., 2018).

Among the grain legumes, common bean has the lowest levels of SNF with an average percentage of nitrogen derived from the atmosphere (NDFA) of 40 % (Herridge et al., 2008). Nitrogen fixation in common bean can be limited by energy supply to the nodules (Graham et al., 2003). Assuming that additional factors such as low soil P are not limiting, bean lines in low N soils that have greater %NDFA may have the capacity to partition greater amounts of photosynthate to roots. This may lead to a temporary deficiency in the availability of photosynthates for growth and development of the aerial portion of the plant (Oldroyd & Leyser, 2020). Chlorotic leaves are a common symptom of soil N deficiency. A positive correlation has been reported between leaf N content and SPAD scores in bean leaves at the V4 stage (vegetative) of development (Abrahão et al., 2013). A positive association between SPAD at flowering and %NDFA was reported in field trials conducted in Ontario, Canada (Farid et al., 2017).

Although efforts to improve symbiotic nitrogen capacity of beans have been successful, the time and cost of phenotypic screening limits routine selection for this complex trait (Kamfwa et al., 2015). Indirect selection for enhanced symbiotic nitrogen fixation might be possible when breeding lines are evaluated in low N soils. Because bean breeding programs in Central America and the Caribbean often screen lines on research stations and farms having poor soil fertility, some selection of bean lines having improved SNF or greater acquisition of nutrients from the soil may have occurred (Graham et al., 2003). Under drought conditions, bean genotypes that combine high seed yield and larger Δ13C values often have deeper roots and lower water use efficiency and may represent the “water user” response to drought stress (Polania et al., 2016a; Sanz-Saez et al., 2019).

This study evaluated the performance of a group of elite Mesoamerican bean breeding lines and cultivars that were selected, in large part, for disease resistance in an unfertilized oxisol at Isabela, Puerto Rico having low levels of soil N. The objective of this research was to identify elite Mesoamerican bean germplasm with superior adaptation to low soil fertility. This germplasm can be used as parents for the continued genetic improvement of Mesoamerican beans for Central America and the Caribbean.

Materials and methods

The performance of twenty-seven elite lines of common bean (Phaseolus vulgaris L.) from the Bean Abiotic Stress Evaluation (BASE) 120 panel (Oladzad et al., 2019) was evaluated at the Isabela Substation of the Agricultural Experiment Station of the University of Puerto Rico over a four-year period (2015 to 2018). The Substation is located on the northwestern coastal plain of Puerto Rico at 18.468 N, -67.042 W at an altitude of 128 m. The average annual minimum and maximum temperatures at the Isabela Experimental Substation are 22.2 and 27.8 °C, with an average annual rainfall of 1,630 mm.

The soil where the field trials were performed is a Coto Clay, a very fine, kaolinitic, isohyperthermic Typic Eutrustox. Soil samples were taken from sites where the BASE 120 trials were conducted. Soil sampling consisted of taking samples at different points in the experimental sites using a zig-zag sampling pattern. A soil sampling tube was used to take sub-samples at a depth of 30 cm in the soil. The top 5 cm of soil was removed to eliminate plant material that could contaminate the sample. The subsamples were mixed to prepare a composite sample for each site. The soil chemical analyses were performed at AgSource Harris Laboratories in Lincoln, NE, to determine the availability of soil nutrients (Table 1). Soil pH was measured in a 1:1 soil:water ratio; soil organic matter was measured by loss on ignition; available phosphorus was extracted with Bray 1 if pH was ≤ 7.2, followed by quantification with Inductively Coupled Plasma Spectroscopy (ICP) and exchangeable cations (potassium), using ammonium acetate extraction followed by quantification with ICP.

Table 1 Results from soil tests from the sites where the tests were carried out with bean (Phaseolus vulgaris L.) lines of the Bean Abiotic Stress Evaluation panel (BASE 120) trials. Isabela, Puerto Rico, from 2015 to 2018. Cuadro 1. Resultados de los análisis de suelo de los sitios donde se realizaron los ensayos con líneas de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abióticos del Frijol (BASE 120). Isabela, Puerto Rico, entre 2015 y 2018. Table 1 Results from soil tests from the sites where the tests were carried out with bean (Phaseolus vulgaris L.) lines of the Bean Abiotic Stress Evaluation panel (BASE 120) trials. Isabela, Puerto Rico, from 2015 to 2018.

The trials were conducted at the Isabela Substation during five growing seasons. The trials were planted in June and November 2015, June 2016, June 2017, and June 2018. In order to evaluate the ability of bean genotypes to nodulate and establish symbiotic nitrogen fixation in a soil having low N fertility, no fertilization was applied to the BASE 120 trials.

Except for the June 2017 planting date, field trials included 118 common bean genotypes and two tepary bean (Phaseolus acutifolius L.) lines from the BASE 120 panel (Tables 2 and 3). The June 2017 planting included 27 elite lines from the BASE 120 panel. Entries in the BASE 120 panel included elite bean breeding lines and cultivars from Zamorano University in Honduras, the International Center for Tropical Agriculture (CIAT), the University of Puerto Rico, USDA-ARS Tropical Agriculture Research Station and Michigan State University.

Table 2 Seed type, pedigree, and traits of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico, over five planting dates between 2015 to 2018. Cuadro 2. Tipo de semilla, pedigrí y características de líneas élite de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abióticos del Frijol (BASE 120) evaluadas en Isabela, Puerto Rico, durante cinco fechas de siembra, entre 2015 y 2018. Table 2 Seed type, pedigree, and traits of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico, over five planting dates between 2015 to 2018. Porch et al. (2012); Granadino-Espinal & León-Gonzalez (2016); Granadino-Espinal & León-Gonzalez (2016); Instituto de Ciencia y Tecnología Agrícolas (2010); Beaver et al. (2011); Beaver et al. (2014); Beaver et al. (2018); Rosas et al. (2016)

Table 3 Seed type, pedigree, and traits of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico, over five planting dates between 2015 to 2018. Cuadro 3. Tipo de semilla, pedigrí y características de líneas élite de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abióticos del Frijol (BASE 120) evaluadas en Isabela, Puerto Rico, durante cinco fechas de siembra entre 2015 y 2018. Table 3 Seed type, pedigree, and traits of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico, over five planting dates between 2015 to 2018. Beaver et al. 2018a; Beaver et al., 2011; Centro Nacional de Tecnología Agropecuaria y Forestal, 2005; Chaves-Barrantes, 2015; Liao et al., 2001; Porch et al., 2014; Rosas, 2015; Rosas et al., 2019; Villar-Sánchez et al., 2010

Although no fertilizer was applied in the elite BASE 120 trials, the lines were inoculated with two strains of Rhizobium; R. etli (CIAT 632) and R. tropici (CIAT 899), that was prepared at the Juana Diaz Station Laboratory. During the June 2015 planting date, a liquid suspension of inoculum at a concentration of 107 rhizobia cells per mm was applied first directly to the seeds in the row and, after emergence, to the base of the seedlings using a backpack sprayer. The estimated amount of liquid inoculant was based on the volume recommended for 50 kg of seeds and 14 seeds per m. The inoculation for the other growing seasons was performed using a solid inoculant. This peat-based inoculant contained the same two strains of Rhizobium. The procedure consisted of applying 25 g of Rhizobium inoculant and PREMAX (bacterial protector) per 1 kg of seeds. The inoculant was applied to each genotype by stirring the inoculant and the seed of each genotype in a plastic container. The inoculant had a concentration of 1x10 9 rhizobia g-1 peat. When using this inoculation technique, it was necessary to ensure that the seeds were evenly covered by the inoculant. The inoculated seed were stored at room temperature, overnight and planted the following day.

The entries from the BASE 120 trials (Tables 2 and 3) were planted in a randomized complete block design with five replications. The experimental units were single 3 m rows with 0.76 m spacing between experimental units. The seeding rate was 14 seeds per m. The field trials received supplementary aerial irrigation to avoid drought stress. Weeds were controlled manually. The trials were monitored twice a week to detect the presence and incidence of pests and diseases. Preventive measures were taken in cases where a high density of pests was found. No fungicides were applied to the field trials.

Nodulation was evaluated during flowering (approximately 45 days after planting) using two plants per experimental unit. Plant root crowns were carefully extracted at about 0.75 m from the end of each plot. Root crowns were washed to remove the excess soil. Nodule numbers were evaluated by assigning values from 1-9 using the CIAT scale, where 1 represented a plant with >81 nodules, 3 had 41-80 nodules, 5 had 21-40 nodules, 7 had 10-20 nodules, and 9 had <10 nodules per plant (van Schoonhoven & Pastor-Corrales, 1987). The chlorophyll content was measured approximately 45-50 days after planting using a Minolta Chlorophyll Meter SPAD-502 on three randomly chosen central leaves of plants from each experimental unit.

Seed samples from each of three replications of the BASE 120 trials conducted at the Isabela Substation in 2016, 2017, and 2018 were used to estimate percentage of N derived from the atmosphere (%NDFA) and apparent C isotope discrimination (Δ13C). Approximately 5 seed of each sample were dried until a constant weight was achieved at 70 °C, and then were ground at the USDA-ARS Tropical Agriculture Research Station using a Wiley mini-mill (Thomas Scientific, Swedesboro, New Jersey, USA), passed through a #40 mesh sieve, resulting in a fine powder. The 4.2 mg samples were packaged in 5×8 mm tin capsules (D1008, EA Consumables, Pennsauken, NJ) and shipped to the University of California, Davis Stable Isotope Facility in 96-well plates.

The 15N natural abundance method (Unkovich et al., 2008) was used to calculate the %NDFA estimates. The white bean R-99 was used as the non-N-fixing reference line.

%NDFA = [(Δ 15N R99 - Δ 15N BASE 120 line) / (Δ 15N R99-B)]*100

Where B represents the Δ15N of the bean line grown under N-free conditions and relies on symbiotic nitrogen fixation for all N requirements. In this study, the variable B was assigned the value of 0 because the R99 reference line does not nodulate. The entries in the BASE 120 trial and the R99 reference bean line have similar patterns of phenological development and seed size. In a N-free trial conducted by Heilig et al. (2017), no significant differences in Δ15N were found among flowering plants of ‘Puebla 152’, ‘Zorro’, ‘Medalist’, and PR0443-151. Constant B values for seed of 2.44 0/00 for bean genotypes having type II and 2.88 0/00 for genotypes having type III growth habits were used for greenhouse studies conducted by CIAT in Cali, Colombia (Polania et al., 2016b). The use of a constant B value, however, would not change the ranks of %NDFA estimates among the entries. The size of the B value is less important when %NDFA estimates are low (<50 %) (Unkovich, 2008). The 15N natural abundance method using seed tissue is well-suited for use by a breeding program to screen for superior SNF (Polania et al., 2016b).

Carbon isotope discrimination (Δ13C, ‰) estimates were calculated using the following equation:

Δ13C = (Δ13Catmosphere − Δ13Csample) / (Δ13Csample + 1)

Where Δ13Catmosphere (−8 ‰) is the C isotope composition of CO2 in the atmosphere (Farquhar et al., 1989) and Δ13Csample is the C isotope composition of the seed sample.

Data collected from the 27 lines were used for the analyses. Statistical analysis was performed using a GLIMMIX model from SAS/STAT 14.3 (SAS Institute, Cary, NC) to analyze main effects and interactions and to compare the least squares means. Planting dates and bean genotypes were considered as fixed effects whereas replication was considered a random effect within planting dates. A 95 % probability level was used to establish statistical significance. Pearson correlations were calculated to study associations between least squares means of seed yield and other traits measured in this study.

Results

Soil pH in the fields at the Isabela Substation where the BASE 120 trials were conducted ranged from 5.7 to 6.6 and soil organic matter ranged from 3.9 to 4.4, which are adequate values for normal bean growth and development (Table 1). Three of the planting dates had soil nitrate levels <10 ppm and a fourth date had 11 ppm of soil nitrate. Results from soil samples taken for the June 2018 planting had a sub-optimum level of P. The soil samples taken for the other planting dates had medium to high levels of P and K for dry bean production.

The pedigrees in Tables 2 and 3 show that a diverse group of Mesoamerican parents was used to develop the elite breeding lines in this study. The pedigrees, however, include very few parents from other bean races and no Andean bean parents.

Fourteen of the 27 lines produced mean seed yields >1,000 kg ha-1 in un-fertilized trials planted at the Isabela Substation over five growing seasons (Table 4). In order to adapt to existing cropping systems in CAC, the elite lines have Type II and III growth habits and reached harvest maturity from 73.4 to 87.0 days after planting (Table 5).

Table 4 Least square means of seed yield of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico over five growing seasons from 2015 to 2018. Cuadro 4. Medias ajustadas de rendimiento de semillas de líneas élite de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abióticos del Frijol (BASE 120), evaluadas durante cinco fechas de siembra en Isabela, Puerto Rico entre 2015 y 2018. Table 4 Least square means of seed yield of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico over five growing seasons from 2015 to 2018.

Tabla 5 Least square means of seed yield, SPAD scores, nodulation scores, root rot scores, basal root growth angle (BRGA), and harvest date of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico. 2015-2018. Tabla 5. Medias ajustadas de rendimiento de semilla, lecturas de pudrición de la raíz, BRGA y fecha de cosecha de líneas élite de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abiótico del Frijol (BASE 120), evaluadas en Isabela, Puerto Rico. 2015-2018. Tabla 5 Least square means of seed yield, SPAD scores, nodulation scores, root rot scores, basal root growth angle (BRGA), and harvest date of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico. 2015-2018.

Significant genotype x season (G×E) interactions for seed yield were observed for most bean lines. One of the exceptions was the line SB2-170 that combined a mean seed yield >1,000 kg ha-1 and non-significant G×E across environments. The black bean cultivar ‘Hermosa’ produced among the highest mean seed yields although nodulation scores were poor and %NDFA estimates were low.

Good root health is needed to efficiently absorb available soil nutrients and water. Mean root rot scores were low (<3) suggesting that the lines had good root health in these trials, thus this was not a factor limiting nutrient uptake (Table 5). Mean basal root growth angle (BRGA) ranged from 54.1 to 64.6 % (Table 5). G×E for BRGA, except for TARS-MST1 and PR1483-105, was non-significant for the higher yielding lines. The eight highest-yielding lines also had non-significant G×E for SPAD, %NDFA, and root rot scores (Table 6).

Table 6 Least square means of seed yield, SPAD scores, nodulation scores, and % nitrogen derived from the atmosphere (%NDFA) of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico, from 2015 to 2018. Tabla 6. Medias ajustadas de rendimiento de semilla, lecturas de SPAD, lecturas de nodulación y (% nitrógeno derivado de la atmósfera (% NDFA) de líneas élite de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abióticos del Frijol (BASE 120), evaluadas en Isabela, Puerto Rico, entre 2015 y 2018. Table 6 Least square means of seed yield, SPAD scores, nodulation scores, and % nitrogen derived from the atmosphere (%NDFA) of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120) evaluated in Isabela, Puerto Rico, from 2015 to 2018.

SPAD scores ranged from 37.1 to 50.1 (Table 6). Five lines had nodulation scores <6.0, although the five lines with the highest mean seed yields had nodulation scores >6.0 (Table 6). In contrast, four of the five lines with the highest seed yields had %NDFA >50 % (Table 6).

Seed yield and harvest date were not correlated. Nodulation scores were negatively correlated with harvest date (-0.48*) but positively associated with seed yield (0.39*). Thus, the later maturing lines and cultivars in the study tended to have greater nodule numbers although a few of the highest yielding lines had fewer nodules. Nodule number, however, does not reflect the effectiveness of nodules to fix nitrogen. In this study, nodule number and %NDFA were positively associated reflected by a negative correlation (-0.40*) between nodulation scores (1 ≥81 and 9 <10 nodules/plant) and %NDFA estimates. %NDFA was not significantly associated with days to maturity or seed yield. There was a negative correlation (-0.53**) between SPAD scores and %NDFA.

F Tests for comparing season means sliced by lines for bean lines having superior %NDFA were not significant, suggesting that this trait was stable across environments.

The small red bean, ‘Rojo Chortí’, consistently had the lowest Δ13C CID values over the three growing seasons (Table 7). The black bean lines B12724, PR1165-3, and ‘Sankara’ had among the largest least squared means for Δ13C across growing seasons. The black bean lines TARS-MST1 and B12724 were ranked in the top 6 in seed yield over 5 growing seasons and showed high Δ13C values. The white bean cultivar ‘Verano’ and the small red breeding line TARS-LFR1 had among the lowest Δ13C values across seasons. Both lines were selected in Puerto Rico for adaptation to higher temperatures when daily water demand is the highest, thus lower Δ13C values in this study may be associated with water use efficiency.

Table 7 Least square means of carbon isotope discrimination of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120), evaluated at Isabela, Puerto Rico, over three growing seasons. 2016-2018. Tabla 7. Medias ajustadas de discriminación de isótopos de carbono de líneas élite de frijol (Phaseolus vulgaris L.) del Panel de Evaluación de Estreses Abióticos del Frijol (BASE 120), evaluadas en Isabela, Puerto Rico, durante tres fechas de siembra. 2016-2018. Table 7 Least square means of carbon isotope discrimination of elite bean (Phaseolus vulgaris L.) lines from the Bean Abiotic Stress Evaluation panel (BASE 120), evaluated at Isabela, Puerto Rico, over three growing seasons. 2016-2018.

Discussion

Soil and climatic conditions were suitable in this study to identify Mesoamerican bean lines having superior adaptation to low soil fertility and moderate levels of heat tolerance. Daytime temperatures >30 °C and nighttime lows >20 °C can reduce bean seed yield (Beebe et al., 2012). On the northern coast of Puerto Rico, from the months of June to October, mean daytime maximum temperatures normally range from 30-31 °C and nighttime minimum temperatures are often >25 °C (National Centers for Environmental Information, 2020). Therefore, lines in the four BASE 120 trials planted in June were exposed to moderate levels of heat stress throughout the growing season. Given the projections for climate change in Central America and the Caribbean, it would be appropriate for bean breeders in the region to routinely screen bean lines under warmer than currently normal temperatures.

The oxisol at the Isabela Substation has proven to be a useful site for screening beans for adaptation to low soil fertility (Dorcinvil et al., 2010). In four of the five BASE 120 trials soil nitrate levels were sub-optimum. In Colorado, USA soils having nitrate levels <11 are recommended to be fertilized for dry bean production at a rate of 67.25 kg ha-1 of N (Davis & Brick, 2009).

Indirect selection for increased symbiotic nitrogen fixation can be successful on low N soils that are not fertilized (Bliss, 1993). Bean breeders in Central America and the Caribbean should regularly evaluate the performance of bean lines in unfertilized field trials.

Seed yield from the most promising lines from the BASE 120 trial were as great or greater than the mean seed of beans in Central America and the Caribbean (Food and Agriculture Organization, 2019). Several of the lines with superior performance in the unfertilized trials planted at Isabela, Puerto Rico possess additional traits of economic importance. SB2-170, a line that combined superior seed yield and non-significant GxE across environments, was selected for broad adaptation through a shuttle breeding program between the USDA-ARS in Puerto Rico and the University of Nebraska (McClean et al., 2011). Another line that combined a mean seed yield >1,000 kg ha-1 and non-significant G×E across growing seasons was the small red TARS-LFR1. This line was selected in Puerto Rico for resistance to root rot and common bacterial blight, and for SNF and adaptation to low fertility soils (Porch et al., 2014).

In soils that are deficient in N, root growth and development, and SNF are favored to expand the capacity to acquire nutrients (Oldroyd & Leyser, 2020). The ability of Hermosa to produce superior seed yield, while having poor nodulation scores and low %NDFA, suggests that this black bean cultivar has the capacity to better utilize available soil nutrients. Beans can use different mechanisms to improve acquisition and efficiency of use of soil N (Dorcinvil et al., 2010). Increased efficiency of nutrient acquisition or use would contribute sustainably to increased bean seed yield (Beebe et al., 2012).

Mean BRGA of the lines in this study were intermediate in magnitude. Soybean [Glycine max L. (Merr.)] plants with roots having intermediate (40-60 %) BRGA were reported to be well-suited for acquiring both soil water and nutrients (Zhao, 2004).

SNF is sensitive to abiotic stresses including drought, low soil fertility and high temperatures (Beebe et.al., 2012). However, differences between bean genotypes in their SNF response to drought (Devi et al., 2013) and higher temperature (Fernández-Toledo et al., 1997) have been reported. Four of the five highest-yielding bean lines had three-year averages of %NDFA > 50 % which is greater than estimates of %NDFA for common bean in most trials conducted in different regions of the world where this grain legume is produced (Peoples, 2009). One study reported a mean %NDFA of 45.5 % in a subset of 259 lines of the Andean Diversity Panel evaluated in Michigan, USA (Kamfwa et al., 2015). In Ontario Canada, Wilker et al. (2019) obteined a mean %NDFA among Middle American bean lines to be 62.2 %. However, daylengths are longer, nights are cooler and net Fs may be greater at higher latitudes, which may favor SNF.

Pedigrees of the BASE 120 lines show that a large amount of genetic diversity was used to develop the elite Mesoamerican race bean lines in this study. It was possible to identify lines in this study that combined disease resistance with superior SNF. Breeding lines and cultivars that combine superior symbiotic nitrogen fixation and resistance to diseases including anthracnosis, common blight and BCMV have been identified (Wilker et al., 2019).

Although some of the elite lines in this study did not perform well in the low N soil at Isabela, Puerto Rico, they possess traits that may be useful as parents to develop cultivars for CAC. For example, the small red line SER 118 has yielded well and expressed greater yield efficiency in soils in Colombia having moderate levels of low P stress (Beebe et al., 2008) and terminal drought (Chaves-Barrantes et al., 2018). SEF 16 has resistance to BGYMV and has performed well in terminal drought and greater than optimum night temperatures (Chaves-Barrantes, 2015).

Carbon isotope discrimination (Δ13C) provides an indirect measure of water use efficiency in bean plants (Beebe et al., 2013). There were significant differences for Δ13C among entries and season × entry least square means were significant for all genotype × season combinations. Reduced SNF under drought associated with lower water use efficiency has been reported (Fenta et al., 2020). ‘Rojo Chortí’ was released in El Salvador (Parada-Cardona et al., 2015) and in Honduras (Rosas et al., 2019) as a heat and drought tolerant small red bean cultivar. Greater water use efficiency was associated with bean genotypes having lower Δ13C values and greater harvest index (Sanz-Saez et al., 2019). The Δ13C values reported on Mesoamerican beans planted in field trials in Ontario, Canada in 2011 and 2012 were greater than the Δ13C values in this study (Farid & Navabi, 2015). The lowest Δ13C estimate was 24.58 in Canada and the highest Δ13C value in this study was 22.4. Except for one location, where a negative association between Δ13C and %NFDA has been reported (Farid & Navabi, 2015).

This study identified elite Mesoamerican bean lines that possess unique combinations of traits that could be recombined in a breeding program to produce cultivars with superior ability to cope with biotic and abiotic constraints facing bean producers in Central America and the Caribbean. For example, the white bean cultivar ‘Bella’ had the highest overall seed yield and resistance to BGYMV, BCMNV, and common bacterial blight (Beaver et al., 2008). ‘Bella’ also produced more nodules and had estimates of %NDFA >50 %. The small red cultivar ‘Rojo Chortí’ combines BGYMV and BCMV resistance and tolerance to heat. Its low Δ13C suggests that ‘Rojo Chortí’ may also have characteristics that favor better adaptation to drought. Plants possess different mechanisms to adapt to soils deficient in nutrients (Oldroyd & Leyser, 2020). The black bean line ‘Hermosa’ produced superior seed yield despite having poor nodulation and a low estimated %NDFA. This line may possess characteristics that contribute to more efficient acquisition or utilization of available soil nutrients.

Lines of diverse genetic background were identified in this study that had superior SNF, thus additional cycles of recombination and selection may produce progenies with improved SNF characteristics. Using parents having different traits that were expected to contribute to symbiotic nitrogen fixation, recurrent selection in a controlled environment for increased nodule number of common bean lines was successful (Pereira et al., 1993). Recurrent selection to increase seed yield and shoot and seed N accumulation in a low N soil in Minnesota was also effective (Elisondo-Barron et al., 1999). Much of the progress was attributed to enhanced SNF. The authors recommended advancing lines to at least the F4 generation to increase the frequency of lines having desirable combinations of traits and to allow evaluation in replicated trials. Most breeding programs in Central America and the Caribbean have low N sites that could be used to routinely screen bean advanced generation breeding lines for greater SNF. Another study reported that both shoot dry weight and shoot N and seed yield and seed N have been reported to be highly correlated (r >0.9) (Elisondo-Barron et al., 1999). Therefore, evaluating bean lines for seed yield, biomass and days to maturity in a low N soil may provide an adequate preliminary screening for SNF. Indirect measures of biomass production using canopy reflectance indices (Gutiérrez-Rodríguez et al., 2006) would facilitate the screening of bean lines for this trait.

Conclusions

This study identified elite Mesoamerican bean lines from the Bean Abiotic Stress Evaluation (BASE 120) panel with superior performance and enhanced symbiotic nitrogen fixation (SNF) in an unfertilized soil at Isabela, Puerto Rico. The pedigrees show that a wide array of genetic diversity was used to develop the elite lines in the BASE 120 panel and that many of these lines possess resistances to several of the most important biotic and abiotic factors limiting bean seed yield in Central America and the Caribbean (CAC). Bean breeders in CAC should be able to make use of this genetic diversity to continue to make significant progress developing improved Mesoamerican beans cultivars.

The occasional assembly, distribution and evaluation of regional nurseries containing elite Mesoamerican bean lines from different breeding programs could represent a valuable resource for bean breeding programs in Central America and the Caribbean. A regional nursery could also include the best sources of resistance to major biotic and abiotic constraints from other races and gene pools of the common bean. As technologies such as the molecular SNP assays become more accessible and cost-effective, Mesoamerican bean breeding programs should be able to utilize marker-assisted selection to introgressive genes of economic importance into Mesoamerican bean breeding lines.