Screening of beans (Phaseolus vulgaris L.) genotypes for drought tolerance

Drought is an important factor limiting crop yield and food insecurity globally. Sixty- percent of the bean production areas are prone to drought and subsequently result in eighty-percent of yield reduction. It is emerging as a rising threat to farming communities in Bhutan. Limited studies on crop drought tolerance done in Bhutan. Six bean genotypes (Orey serbu, Orey regtang, Orey brokchilu, Yadhipa orey, Kerongree orey, Brokopali) chose to ascertain drought tolerance. The study was a factorial experiment with Completely Randomized Design (CRD) with six treatments and three replications. Genotypes were subjected to drought stress after 50% flowering till the start of pod formation. The drought condition induced for ten days during the pod formation and the irrigation resumed till harvesting. Means of water-stressed genotypes compared to their corresponding non- stressed with an All-pairwise comparison using the Bonferroni test at the significant level p<.05. The growth (leaf area, relative water content, root length, shoot weight, maturity) and yield parameters (the number of pods, pod length, the number of seeds number per pod, seed weight) determined during the time of harvest. There were significant differences (p<.05) in all the parameters measured under stress and non-stress conditions. Water-stress decreases plant growth and development of all the bean genotypes. With regards to drought susceptibility index (DSI), Orey serbu(1), Orey regtang(1), Yadhipa orey(1), and Kerongree orey(1) had the lowest DSI value. The lesser the susceptible indices, the greater is the tolerance level to the drought and vice versa.


Introduction
Common beans (Phaseolus vulgaris L.) consume as a staple and low-cost protein source in the underdeveloped countries where protein malnutrition is prevalent [13]. [12] stated that the genus Phaseolus originated from the Mesoamerican regions comprises five domesticated species: Phaseolus vulgaris (common bean), Phaseolus dumosus, Phaseolus coccineus (runner beans), Phaseolus acutifolius (tepary beans), and Phaseolus lunatus (Lima bean). Dry beans are a staple food in Latin American, Eastern, and South African countries [2]. Similarly, beans are the source of dietary carbohydrates and micronutrients for more than 300 million people from Eastern Africa and Latin America [50,9]. India produced 6,75,188 metric tons [MT] of green beans, followed by Bangladesh (137495 MT) in 2017 [21Error! Reference source not found.]. As per [16], Bhutan produced 1,475 MT of beans in 2016 and 5,273 MT in 2017 [17]. Tsirang has the highest harvested area (475 acres), and Bumthang the least harvested area (3 acres) [17].
Bean production is adversely affected by both biotic and abiotic factors [23], and the major abiotic factor affecting crop production worldwide is water stress [28]. Water-stress is ranked second after pests and diseases in reducing the agricultural grains. Sixty-percent of the bean production areas are prone to the drought that drains an eighty-percent of yield [24,41]. The 60% of beans production in the world occurs in the agricultural land prone to water deficit, lack of irrigation system, and the dry periods may result in an 80% yield reduction [41]. According to [17], the drought (4% of total households) and the insufficient irrigation supply (27% of total households) are emerging as a threat to agriculture in Bhutan. Beans are the most important, directly consumed food legume, and it is one of the most important cash crops after potato grown by every Bhutanese farmer. Almost every Bhutanese household consumes beans and remains as highly demanded in the market; however, the study by [49] reported that the drought gets severer in the future due to continuous anthropogenic activities. This study aimed to identify the drought-tolerant bean genotypes, which would help maintain the production and to meet the demand in the Bhutanese market, and provide the basis for the development of drought-tolerant hybrids in the future.

Study site, experimental design, stress treatment, and irrigation schedule
The greenhouse of the agriculture farm of College of Natural Resources (CNR), Lobesa, was used to perform this study in August 2019. It is at an elevation of 1450 meters above sea level between 27° 30' 1'' N and 89° 52' 42'' E of Greenwich. The area experiences an annual rainfall of 500 mm to 1500 mm with temperatures ranging from 5℃ to 30℃. It lies in the dry subtropical region and experiences hot and humid summers during the monsoon months of June, July, and August. The bean varieties used for the experiment were Yadhipa orey (13), Orey serbu(27), Kerongree orey (7), Orey regtang (6), Orey brokchilu (15), and Bropali (25). Used completely randomized design (CRD) for this factorial experiment. There were six treatments (six bean genotypes) and three replications (three plants per replica) and classed treatments into the water-stressed and nonstressed groups. Used a total of 108 potted plants during the experiment, and the distance between the pots was 30 cm within rows and 40 cm between rows [4]. For media, used 3 kg of topsoil to fill 4,416 cm3 volume plastic pots. Subjected genotypes to drought stress after 50% flowering and lasted for ten days, and resumed irrigation till harvesting similar to [4,8]. Weighed pots following one-day intervals [15] and applied the water by calculating the required amount of water using the formula; the amount of water = the weight of pot at field capacity -pot weight at field condition and maintained the soil moisture near field capacity (FC) throughout the experiment [8].

Data collection and analyses
Recorded the weather data using Datalogger (temperature and relative humidity) and recorded both growth and yield attribute data during physiological maturity that defines the number of days for 75% to 90% of pods to lose their green pigmentation [48]. Collected data on leaf area, the number of pod per plant, weight of seed per plant, shoot dry weight, root dry weight, and calculated drought susceptibility index (DSI).
Calculated drought Susceptibility index (DSI) using the following formula:

Effect of drought stress on leaf area (dm 2 )
The significant difference in leaf area was recorded between the treatments group (stress and non Wilting, shedding, and curling of leaves, closure of stomata, and reduction of cell enlargement could be the reason for the reduction in leaf area under the stress condition. During the onset of water stress, it inhibits cell elongation in the leaf, and the lower leaf area leads to less water uptake from the soil, and transpiration is reduced [20]. Under the non-water stressed condition genotypes, Orey serbu had recorded significantly higher in leaf area (M = 0.75 dm 2 , SD ± 0.01).
The result coincided with [42,15], who observed that plants grown under water deficit conditions showed a lower leaf area than plants grown under control conditions.
[43] argued that the leaf area is related to plant metabolism, dry matter production, and yield.

Effect of drought stress on pod number
There findings are in line with [13], who reported that under the high moisture stress during the reproductive stage, the plants are exposed to floral abortion resulting in low yield. Other authors [7,44] also reported that water-stress imposed during the flowering and pod setting causes

Effect of drought stress on seed weight (g)
There was a highly significant difference in seed weight both between treatment and within  Figure 3]. However, Brokpali recorded the highest seed weight (M = 5.94g, SD ± 0.59). The reduction in seed weight/yield under the water stress was may be due to a decrease in carbohydrate assimilation that might have lead to less pod number. Under the non-water stress condition, Brokpali had the highest average seed weight of (M= 14.51g, SD ± 1.51). The reduction in a legumes seed yield under the stress condition was due to the lower number of pods per plant [27,38,30,7]. The reduction in the seed yield and the number of pods is due to the detrimental effects of drought on pod set and grain filling [14].

Effect of drought stress on dry weight (g) of the shoot
The study revealed a significant difference in shoot weight between the treatment group [F (1, condition, plant shoot dry weight decreased significantly than non-stress [ Figure 4]. It could be due to limited photosynthate assimilation and the limited amount of water absorbed by roots. Orey serbu was observed the highest shoot weight of (M = 6.91 g, SD ± 0.14) under water stress.
For non-water stress conditions, Brokpali had the highest shoot weight (M= 14.61 g, SD ± 0.29).
A similar finding was investigated by [35,10] there is more reduction in shoot growth than root growth under stress. According to [46], both shoots and roots are the most affected parts of plants and key components of the adaptation to drought at the morphological level.

Figure 4: Shoot weight (g)
Drought stress severely reduces stem growth and the ability to intercept solar radiation [1].
Among the genotypes, in stress conditions, Orey serbu, Brokpali, and orey regtang showed significantly higher shoot dry weight than the other genotypes. According to [11], a positive correlation (r = 0.53**) exists between shoot and root dry weight in optimal irrigation. However, no correlation (r =0.11) exists under drought stress. It could be due to the genotypic difference in terms of root adjustment to stress.

Effect of drought stress on root dry weight (g)
Significant difference (p <.05) was observed in the root weight both between the treatment and

Yield components and Biomass accumulation in beans are affected by drought stress [31];
however, few genotypes under the stress condition seem to perform better (Brokpali (1.84 g), Orey regtang (1.62 g), and Yadhipa orey (1.60 g)). It could mean that these genotypes have better soil water acquiring efficiency compared to other genotypes. According to [18], the number of roots decreases with a decrease in water potential. Moreover, reduction in lateral roots directly affects the root biomass [39]. The root characters (biomass, length, density, and depth) are the main drought avoidance traits that contribute to the final yield under terminal drought environments [25].

Drought intensity (D) and Drought susceptibility Index (DSI)
Determined the drought susceptibility index (DSI) on relative water content, shoot weight, seed weight, root weight, leaf area, pod number, pod length, seed number, and root length for six water-stressed genotypes. The Drought susceptibility index (DSI) for [

Scoring and ranking of genotypes based on drought susceptibility indices
Ranked the water-stress toleration of six bean genotypes [ Table 2]; the scoring and ranking were such that the least susceptible genotypes scored 1, and the highest susceptible genotypes score 6 [ Table 4]. Values of Drought Susceptibility Index (DSI) less than 1.0 indicate toleration to drought. Values of DSI equal to 0.0 indicate maximum possible drought tolerance (no effect of drought on yield). Four genotypes (Orey serbu, Yadhipa orey, Orey regtang, and Kerongree orey) had the least DSI (rank 1st) and have a greater drought tolerance level. Orey brokchilu had the highest DSI (ranked 6th), followed by Brokpali (ranked 5th) and more susceptible to drought conditions.