Bioscience Journal | 2023 | vol. 39, e39053 | ISSN 1981-3163 1 Alisson Marcos FOGAÇA 1 , Aline Garcia DE CASTRO 2 , Eduardo Augusto Agnellos BARBOSA 3 1 Postgraduate Program in Agronomy, Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brazil. 2 Boqueirão Agronegócios, Passo Fundo, Rio Grande do Sul, Brazil. 3 Department of Soil Science and Agricultural Engineering, Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brazil. Corresponding author: Alisson Marcos Fogaça alifogaca@hotmail.com How to cite: FOGAÇA, A.M., DE CASTRO, A.G. and BARBOSA, E.A.A. Physiological and morphological responses of two beans common genotype to water stress at different phenological stages. Bioscience Journal. 2023, 39, e39053. https://doi.org/10.14393/BJ-v39n0a2023- 59855 Abstract Comprehension of the bean responses of beans common under to water deficit is an important tool in agricultural planning, like sowing time, and deficit irrigation management strategies. The study aimed to understand the morpho-physiological responses and yield attributes of two common bean genotypes submitted to water stress at different phenological stages. The study was carried out in a greenhouse, in randomized block scheme with five repetitions. To achieve the objectives deficit irrigation of 25% of crop evapotranspiration was practiced during vegetative (DI-V), flowering (DI-F), and pod filling (DI-PF) stages. A non-deficit irrigated (NDI) and deficit irrigated through vegetative to pod filling stages (DI-VP) treatments were added for comparison. The following morphophysiological responses and yield attributes were evaluated: net assimilation of CO2, stomatal conductance, and leaf transpiration, chlorophyll index, number of trifoliate leaves, chlorophyll index, leaf area, number of grains per plant, number of grains per pod, number of pods per plant, the mass of thousand grains, harvest index, and water use efficiency. The beans genotype under DI-V exhibited acclimation, observed by the relative increment with NDI of 195%, 759%, and 231% of net assimilation of CO2, stomatal conductance, and leaf transpiration, respectively. Plants under treatment DI-PF experienced dis-stress and plastic responses as leaf losses and exhaustion of gas exchanges. Treatment DI-V received 11% less water than NDI and exhibited equal yield, resulting in higher water use efficiency. Yield attributes correlations indicated that yield penalty might be related to pods abortion, which not occurred to plants under DI-V. Keywords: Phaseolus vulgaris L. Physiological traits. Stage-based deficit irrigation. Water use efficiency. Yield attributes. 1. Introduction The common bean (Phaseolus vulgaris L.) is worldwide cultivated, its direct consumption provides micronutrients and proteins, which is crucial for emerging countries (Broughton et al. 2003; Bitocchi et al. 2017). However, it is estimated that 60% of bean production regions are affected by drought, reducing yield by water stress (Beebe et al. 2012). To close yield gaps, breeding programs screen for adapted genotypes that shows superior efficiency in the water use (Darkwa et al. 2016). In Brazil, cultivation comprises smalls and large-scale agricultural systems that part of them adopt irrigation. To overcome water stress constraints and to expand the possibilities to produce dry beans when PHYSIOLOGICAL AND MORPHOLOGICAL RESPONSES OF TWO BEANS COMMON GENOTYPE TO WATER STRESS AT DIFFERENT PHENOLOGICAL STAGES https://orcid.org/0000-0002-7520-7271 https://orcid.org/0000-0002-3157-4045 https://orcid.org/0000-0002-1393-4166 Bioscience Journal | 2023 | vol. 39, e39053 | https://doi.org/10.14393/BJ-v39n0a2023-59855 2 Physiological and morphological responses of two beans common genotype to water stress at different phenological stages prices are high, center-pivot irrigation has become more common, mainly in the central region of the country (Beebe et al. 2012). The total irrigated area in 2020 covers 6.95 million hectares and it is expected an expansion of 4.2 million hectares by 2040 (ANA 2021). The growing water demand for agriculture requires rational and efficient water use since ANA (2021) estimates that 46% of withdrawn water from water bodies are used for irrigation. Highlight, that expansion of agriculture irrigated will intensify conflicts over the multiple water use, as well as its availability for irrigated crops. Irrigation management strategies will be essential to ensure high levels of productivity. Among the strategies to mitigate the effect of low water availability for irrigation, there is regulated deficit irrigation (RDI) is the technique of irrigation that water depth is reduced based on plant responsiveness to mild water stress in order to increase water use efficiency (WUE) with minor penalties to productivity. An approach of RDI is the stage-based deficit irrigation (SBDI) that is the reduction of water depth during a non-critical growth stage of plants (Kirda 2002). For this purpose, a thorough comprehension of morpho- physiological responses of plants to water deficit stress is crucial (Kumar et al. 2017). To strategize SBDI, many factors that affect plant responses shall be taken into account as the species, genotype, agronomic management, climate, and soil conditions (Chai et al. 2016). Moreover, it is difficult to isolate water deficit effects in the field due to the superimposition of other abiotic and biotic stresses, resulting in variability among seasons as observed by Webber et al. (2006). Therefore, experiments in greenhouses, where environmental, water supply and pest control are superior, addresses the demand for investigations of plant responses to irrigation shortage (Chaves et al. 2002). Water deficit promotes several physiological disturbances, such as reduced stomatal conductance (Soureshjani et al. 2019). Dioxide carbon is diffused to the substomatal cavity while stomata are open and enter into the photosynthetic pathway (Chaves et al. 2002). Plants under water stress commonly experience a restriction of gas exchanges by stomatal closure to regulate water losses, trading-off for carbon assimilation (Chai et al. 2016). Adaptive mechanisms are stimulated to cope with water limitations, resulting in eu-stress (elastic or reversible responses), which is indicated as a full recovery of gas exchange constraints when plants are rehydrated. This condition stimulates acclimation, which in turn is a series of complex and synergic adaptations that result in plants that are more resilient. Water stress occurrence during critical growth stages or highly severe water deficit may cause dis-stress that results in plant exhaustion and plastic or irreversible responses, e.g. local death and damage repair, ending in significant yield losses and reduction of WUE (Yordanov et al. 2000; Chaves 2008). However, much is needed to elucidate the physiological basis in relation to the effect of the application of water stress at different phenological stages (Chai et al. 2016); therefore, the trace of gas exchanges and leaf morphology may subsidize understanding the effects of SBDI. Cultivar responses to induced water stress variate in accordance to its water stress tolerance, even for genotypes cultivated in the same geographical region (França et al. 2000; Darkwa et al. 2016; Soureshjani et al. 2019). The application of RDI on two Iranian cultivars to surpass limitations of water source exposed that genotypes increased WUE (Soureshjani et al. 2019). This result contrasts with the ones reported by Webber et al. (2006) for varieties in Uzbekistan. In Brazil, up-to-date information with current cultivars would encourage the technique to disseminate cooperating with deficit-irrigation expansion. To address this gap, the study aimed to understand the morpho-physiological responses and yield attributes of two common bean genotypes submitted to water stress at different phenological stages. 2. Material and Methods The experiment was carried out in a greenhouse at Ponta Grossa State University, Paraná, Brazil (25°5'23.88" S, 50°6'8.25" W, and 975 m above sea level). According to Köppen and Geiger classification, the local climate is characterized as Cfb, mesothermic without dry season (Peel et al. 2007). The greenhouse was covered with EVA film of 150 microns and equipped with an evaporative cooling pad and four exhaust fans set to start a wind speed of 2.5 m s -1 whenever the temperature reaches a threshold of 25 °C. A thermo hygrometer HT 2000 (Perfect Prime, USA) and a Class A Pan was positioned in the center of the greenhouse in order to record the variation of air temperature, relative humidity, and reference evapotranspiration (ETo) during the experimental period. The mean air temperature following the https://doi.org/10.1093/aob/mcf105 https://doi.org/10.1093/aob/mcf105 https://doi.org/10.1007/s13593-015-0338-6 file:///C:/Users/eduar/Downloads/Yordanov https://doi.org/10.1093/aob/mcn125 https://dx.doi.org/10.1007/s13593-015-0338-6 Bioscience Journal | 2023 | vol. 39, e39053 | https://doi.org/10.14393/BJ-v39n0a2023-59855 3 FOGAÇA, A.M., DE CASTRO, A.G. and BARBOSA, E.A.A. standard deviation during the experiment was 26.0±5.5 °C and the absolutes maximum and minimum air temperature during the plant cycle were 44.2 and 14.50 °C, respectively. The ETo through the observational period is presented in Figure 1. Figure 1. Reference evapotranspiration during the experimental period. The growth medium was a Ferralsol sifted on 8-mm mesh, which showed the chemical and physical characteristics presented in Table 1. Based on the standardized recommendation for common bean cultivation in Brazil, it was incorporated 1.91 kg dm -3 of lime into the growth medium to reach 70% of base saturations three months before the experiment. Then, 10 dm 3 of soil was accommodated on a layer of 1.5 kg of gravel and 2 kg of sand, respectively in 12-liter pots. Lastly, the soil was saturated and left to drain in order to the water status of the soil to be near to the field capacity. Table 1. Soil chemical and physical characteristics. pH H+Al Al 3+ Ca 2+ Mg 2+ K + CTC(pH 7,0) ----------------------------------- cmolc dm -3 ----------------------------------- 4,9 6,69 0,1 3,2 1,7 0,29 11,88 P C Sand Silte Clay V m mg dm -3 ------------------------- g kg -1 ------------------------ ----------- % ----------- 6,5 33 158 302 540 43,7 1,9 pH in CaCl2, P in Melich-1; C determined with methodology Walkley-Black; V and m: bases and aluminum saturation, respectively. Two common-bean cultivars (Phaseolus vulgaris L.) extensively cultivated in central and southern Brazil cv. BRS Estilo and cv. IPR Campos Gerais were planted on 72 cell trays. The plants were sown on October 27 and emerged on November 5, 2017. When the primary leaves were fully expanded (September 11, 2017), two plants were transplanted on each pot. At this moment, the base fertilization composed of urea (46-00-00) and MAP+Zn (10-49-00) was applied at the doses of 360 and 40 g pot -1 , respectively. Topdressing fertilization was applied at the dose of 310 g pot -1 of urea 30 days after sowing (DAS). The more vigorous of the two plants transplanted was traced during the experiment. Diseases and insects were controlled by treating seed with Standak® Top at 200 ml a.i. 100 kg seeds -1 . A preventive overhead spray with the fungicide Fox® at 0.5 L ha -1 was done when four trifoliates were fully expanded and three overhead sprays were done to control thrips and aphids with Pirate® and Engeo® Pleno at the doses 0.75 L a.i. ha -1 and 125 mL ha -1 , respectively. Bioscience Journal | 2023 | vol. 39, e39053 | https://doi.org/10.14393/BJ-v39n0a2023-59855 4 Physiological and morphological responses of two beans common genotype to water stress at different phenological stages The pots were grouped in five blocks and treatments were distributed in a factorial scheme of two factors. The first source of variation was the imposition of stage-based deficit irrigation (SBDI) by 25% of ETc during one of the three growth stages: vegetative (DI-V), flowering (DI-F), and pod filling (DI-PF). Additional treatments of no deficit irrigation (NDI) and deficit irrigation during vegetative to pod filling (DI- VP) were considered as controls. The value of 25% of ETc was applied because it promotes severe water stress for the crop, according to a previous study carried out with the genotypes in the same greenhouse (Fogaça and Barbosa, 2020). To take into account genetic diversity, the second source of variation was incorporated and was composed of the cultivars (Cv) IPR Campos Gerais and BRS Estilo, which show a life cycle of near to 90 days. The first treatment started when the fourth trifoliate was completely expanded. Subsequently, when 50% of plants bloomed, treatment DI-F took place. Lastly, the pod filling stage was considered when 50% of plants had at least one pod (Figure 2). Figure 2. Experimental chronology. Black bars represent deficit irrigation during vegetative (DI-V), flowering (DI-F), and pod filling (DI-PF) stages. A treatment with no water deficit (NDI) and another with water deficit during the vegetative to the pod filling stages (DI-VP) were added for comparison. The sowing (S) represents day zero and transplantation (T) occurred 10 days after sowing (DAS). Evaluations of leaf gas exchanges (GE), leaf morphology (LM) and chlorophyll index (Chl) were done as the treatments were imposed. Plants were harvested (H) at 90 DAS. Irrigation management was carried out based on the class A pan method. First, the reference evapotranspiration (ETo, Figure 1) was obtained by measuring the Class A pan evaporation (Epan) positioned in the center of the greenhouse. An empirical coefficient that takes account of the environment influence (Kp) was used in the expression ETo = Epan Kp with a value of 0.75. Then, the crop evapotranspiration (ETc) was calculated by expression ETc = ETo.Kc, being Kc the crop coefficient (Allen et al. 1998). Kc values for beans common were adopted (Heinemann et al. 2009). This coefficient started by 0.5 at the sowing to the emergence of plants, raised to 1.4 linearly until flowering, and after pod filling decreased gradually until 1.1 at the end of the experiment. Finally, ETc was multiplied to the pot area to determine the water volume, which was measured with a graduated cylinder and applied uniformly to the soil. Instantaneous leaf gas exchanges were measured with an infrared gas analyzer (IRGA) model LI- 6400XT (LI-COR, USA) on the center leaf of the fully expanded trifoliate leaf, commonly the third trifoliolate counting from the apical meristem. The internal chamber temperature was set to 25°C, photosynthetic photon flux density set to 1200 μmol photons m -2 s -1 , CO2 concentration set to 400 μmol mol -1 , and airflow set to 400 μmol s -1 . The parameters net assimilation of CO2 (A, µmol CO2 m -2 s -1 ), stomatal conductance (gs, mol H2O m -2 s -1 ), and leaf transpiration (E, mmol H2O m -2 s -1 ) were used for further analysis. Measurements were performed between 10h and 12h. Number of trifoliate leaves, chlorophyll index and leaf area, and of the young fully expanded trifoliolate leaf were recorded in order to understand the effects of water restriction on leaf morphology. Chlorophyll index was recorded with a chlorophyll meter model 1030 (Falker, BR). The third trait was estimated as suggested by Figueiredo et al. (2012) by measuring the length and width of individual leaflets with a pachymeter and applying the following equation: Bioscience Journal | 2023 | vol. 39, e39053 | https://doi.org/10.14393/BJ-v39n0a2023-59855 5 FOGAÇA, A.M., DE CASTRO, A.G. and BARBOSA, E.A.A. LA= ∑(0.575 (L W)) (1) Where LA is the trifoliate leaf area (mm 2 ), L and W are the individual length and width (mm) of the leaflet, respectively. The equations features R 2 = 0.98. At 90 DAS, the aerial part of the plants was harvested. The yield attributes: number of grains per plant (NGPl), number of grains per pod (NGPo), number of pods per plant (NPP) were recorded. The mass of thousand grains (MTG, g) was an extrapolation based on the ratio of the NGPl and the mass o grains per plant (MGP, g plant -1 ) with humidity adjusted to 14%. Then, it was calculated the harvest index (HI, g g -1 ) as a ratio of the MGP and total aerial biomass and the water use efficiency (WUE, g L -1 ) as the ratio of MGP and total irrigated water. All traits at all dates of analysis were subjected to the analysis of variance with stage-based deficit irrigation (SBDI) and cultivars (Cv) as factors. When the probability of F was p≤0.05, SBDI levels were compared by confidence intervals with t distribution (p=0.95) plotted as a time series to enhance visibility, and yield and yield components of cultivars were compared with the Tukey’s Honestly Significant Difference test (p≤0.05). The yield attributes relationship was explored using Pearson correlation. All data analyses were performed in software R (R Core Team, 2018) and plotted using package ggplot2 (Wickham 2016). 3. Results The inspection of factors over gas exchanges and leaf morphology revealed a predominance of single effects (Table 2). From the 71 variables analyzed, only LA on 47 DAS was significantly influenced by the interaction of factors at 0.01