Physiochemical Changes of Mung Bean [Vigna radiata (L.) R. Wilczek] in Responses to Varying Irrigation Regimes

Abstract

Mungbean is one of the most powerful pulses providing substantial protein for human diets and fixing N to the soil, improving nutritional food security and agricultural sustainability. The production of summer mungbean in the tropics and subtropics is adversely affected by drought due to water scarcity caused by various factors as well as lack of rainfall. Irrigation at different growth phases is not a suitable solution. An environmentally friendly and economically viable answer is a convenient irrigation management option that will be available to farmers together with drought-tolerant genotypes. The study considered to determine the effect of differences between drought-tolerant and drought susceptible genotypes on water productivity response and physiological traits in mung beans. To quantify seed yield-related to irrigation at different growth stages eventually to quickly determine the most appropriate irrigation stage. One water stress tolerant mung bean genotype (BMX-08010-2) and one sensitive genotype (BARI Mung-1) were grown in the field with four different irrigation schedules along with water stress conditions (no irrigation) under rain shelter at Regional Agricultural Research Station, BARI, Ishwardi, Pabna, Bangladesh. The experiment was laid out in split plots with three replications, with irrigation schedules assigned in the main plot and mung bean genotypes assigned in the side plots. Water use efficiency ranged from 3.79 to 4.68 kg ha⁻¹ mm⁻¹ depending on irrigation regime, and mung bean seed yield of mung bean Water stress decreased plant water status, photosynthetic pigment and membrane stability index, and increased proline soluble sugar content. Treatments that received irrigation during two or three phases (I₃ or I₄) gave significantly higher yields than those that received irrigation during only one stage (I₁ and I₂) with the lowest yield. While the yield obtained ranged between 1145.44 kg ha⁻¹ with seasonal irrigation of 277 mm (I₄) and 555.14 kg ha⁻¹ without irrigation (I₀). The flowering stage (I₃) was recorded as the most sensitive growth stage with an 18.15% yield reduction compared to the treatment with triple irrigation (I₄). Also, depending on the irrigation sources, at least two irrigation phases should be provided at the triple leaf stage (I₂, i.e., 20 DAS) and at the flowering stage (I₃, i.e., 35 DAS) to achieve the highest yield. Genotypes that maintained the higher performance of physicochemical traits under water stress provided higher seed yield and promoted drought tolerance. Therefore, these parameters can be used as physiological and biochemical markers to identify and develop superior genotypes suitable for drought-prone environments.

1. Introduction

Mung bean (Vigna radiata (L.) R. Wilczek) is a short-duration leguminous crop, widely grown in Asia, It is a vital component of major rice-based cropping systems, which offers benefits in soil health and farmer’s income [1]. It is used as a cover crop in-between two cereal crops due to its short growing period (80–90 days) [2]. To some extent, it is used as crucial green manure, produces huge biomass (7.16 t ha⁻¹) [3], and supplies a lot of nitrogen into soil ranging from 30 to 251 kg ha⁻¹ [4,5]. In Bangladesh, it is generally grown from March to early May (Kharif-1 season), and the period is mainly characterized by low rainfall along with the high temperature., Its consequences often lead to significant soil water deficit at various growth stages, especially during the post-flowering period. Soil moisture deficiency alters several physiological and biochemical activities such as the production of excess proline and ROS (reactive oxygen species), disrupts plant water relations, reduces nutrient uptake capacity, prevents stem closure and opening, leading to low photosynthesis, reduces chlorophyll content, pigment composition and plant morphological characteristics [6,7]. Consequently, the present yield is significantly below the potential yield.

Various management options avoid drought stress. One of them is proper irrigation scheduling. Crop irrigation requirements depend mainly on the amount of rainfall, solar radiation insulation, crop species, and genotype [8]. However, most of the area in Bangladesh receives most of its precipitation (around 90%) in the monsoon from June to September. In the other months, crop production is hampered due to lack of rain, and the crops need to be irrigated in these periods. Moreover, the average solar radiation insulation level for Bangladesh is pretty high, ranging from 14.4–18 MJ day⁻¹ (4–5 kWh day⁻¹) around the year [8], and the direct solar radiation remains maximum in March–April [9].

There is a scope to increase yield significantly for mung beans through optimum irrigation scheduling. Adequate vegetative growth is a prerequisite for achieving a high seed yield in mung beans. Therefore, the most suitable irrigation stages should be determined in a situation of limited water availability [10]. The proper application of irrigation practices can significantly save irrigation water supplies [11]. One way of achieving these objectives is to exploit the differential sensitivity of mung beans to irrigation at different growth stages. In addition, identifying critical growth stages to water (irrigation) of mung bean is an essential attribute for quantifying the response of growth and yield, which helps to improve water productivity [12]. Therefore, the optimal time and accurate irrigation application can be designed for the expected crop yield.

Conversely, the use of drought-tolerant genotypes is one of the effective strategies to properly survive water stress [11,13,14]. Developing new drought-tolerant varieties is a desirable solution from an economic and ecological point of view. However, genotypic information regarding drought tolerance in summer mung beans is relatively scarce. It is also clear that breeding is difficult and complex, and drought tolerance could be simplified by identifying agronomic traits and morphological or physiological characteristics that are closely related to yield in water-limited environments. Yield improvement in mung beans can be achieved either by using drought-tolerant genotypes or by better crop management, for example, by proper scheduling of irrigation water supply, which could counteract the effect of water stress. However, few attempts have been made to assess the impact of water deficit on physiological and morphological traits, which may help understand mungbean’s drought tolerance. Therefore, the present study attempted to evaluate the response of physicochemical and yield characteristics of mung bean genotypes to irrigation at different growth stages.

2. Results and Discussion

2.1. Soil Moisture Content in Response to Irrigation Regimes in Mung Bean Genotypes

The moisture content (MC) of each irrigation schedule treated plot showed a distinct deviation pattern concerning irrigation timing (Figure 1). Results revealed that the plants of non-irrigated plots suffered a considerable moisture shortage after 30–40 days after sowing (DAS), and the extreme water deficit occurred following 40 DAS. Moreover, the MC goes down below the permanent wilting point (PWP), ranging from 10.51 to 8.73% after 40 DAS. This result indicated that mung beans faced water stress conditions from pre-flowering to the latter growing period. Consequently, the plants under this treatment forwarded to early maturity resulted in a low seed yield of mung bean (640.14 kg ha⁻¹). One stage irrigation at 1st trifoliate leaf stage (20 DAS) obtained sufficient moisture up to 40 DAS, i.e., flowering development does not hamper due to water shortage. However, significant moisture scarcity occurred from 45 DAS to the later growth stage, near about wilting point. The wilting point of the experimental soil is 13%. One stage irrigation at flowering stage (35 DAS) prevailed substantial moisture shortage in between 20 and 35 DAS (17.32 to 15.2% MC), i.e., flowering development hindered due to water shortage but got adequate moisture from initial to 15 DAS, and 35 to 50 DAS (26 to 20% MC). Two-stage irrigation schedules at 1st trifoliate leaf stage (20 DAS) and flowering stage (35 DAS) did not suffer moisture deficiency during the growing period. The maximum MC (29 to 22.3%) regained during 35 to 45 DAS. That’s why it took proper plants growth and development and provided a large seed yield (1117.41 kg ha⁻¹) as compared to one stage irrigation at 20 DAS (963.26 kg ha⁻¹), or 35 DAS (937.52 kg ha⁻¹) as well as no irrigation schedules. The irrigation schedule at three stages of 1st trifoliate leaf stage (20 DAS) + flowering stage (35 DAS) + pod-filing stage (45 DAS) prevailed the MC of 23% during the whole growing period. The MC remained near about the field capacity during 35 to 40 DAS. These results indicated that the available water prevailed throughout the entire growing period in this irrigation schedule. Consequently, the plant growth and development were much higher than other irrigation schedules, which enhanced higher seed yield (1145.44 kg ha⁻¹). These also lengthened the growth duration of 3 to 6 days in BMX 08010-2, and 1 to 7 days in BARI Mung-1 genotype compared to other irrigation schedules.

2.2. Physiological Properties in Response to Irrigation Regimes in Mung Bean Genotypes

2.2.1. Water Use Efficiency

Irrigation treatments influenced the water use efficiency (WUE) of mung bean crops concerning yield. Total water use and WUE during the crop season are shown in

Table 1. Water use efficiency of mung bean under different irrigation schedules.

Treatments	No. of Irrigation	Post-Sowing Irrigation (mm)	Irrigation (mm)	Effective Rainfall (mm)	Soil Water Depletion (mm)	Total Water Use (mm)	Water Use Efficiency (kg ha−1 mm−1)
I0	0	48	0	0 *	121	169	3.79
I1	1	48	74	0	84	206	4.68
I2	1	48	88	0	74	210	4.46
I3	2	48	143	0	58	249	4.49
I4	3	48	183	0	46	277	4.14

I₀ = No irrigation, I₁ = Irrigation at 1st trifoliate leaf stage, I₂ = Irrigation at flowering stage, I₃ = Irrigation at 1st trifoliate leaf stage + flowering stage, I₄ = Irrigation at 1st trifoliate leaf stage + flowering stage + pod-filing stage. * The crop was protected from rainout shelter.

. Total water use (seasonal) of mung beans varied with the different irrigation schedules, and it was between 169 and 277 mm. The maximum seasonal water use (277 mm) was found at three stages of irrigation (I₄) followed (249 mm) by two stages irrigation (I₃), and the lowest (169 mm) was at control (I₀). One stage irrigation treatment of I₁ and I₂ used total water of 206 and 210 mm, respectively. The WUE varied from 3.79 to 4.68 kg ha⁻¹ mm⁻¹ depending on irrigation schedules and seed yield of mung bean. The maximum WUE (4.68 kg ha⁻¹ mm⁻¹) was observed at I₁ with a seed yield of 963.26 kg ha⁻¹. This might be due to corresponding water use that produced moderate seed yield, resulting in maximum effective utilization of water. The lowest WUE (3.79 kg ha⁻¹ mm⁻¹) was recorded at I₀ with a seed yield of 640.14 kg ha⁻¹. One stage irrigation (I₂) and two stages irrigation (I₃) showed almost the similar WUE of 4.46 and 4.49 kg ha⁻¹ mm⁻¹, with the seed yield 937.52 and 1117.41 kg ha⁻¹, respectively. The irrigation schedule of I₄ showed the WUE of 4.14 kg ha⁻¹ mm⁻¹ with a seed yield of 1145.14 kg ha⁻¹. In the earlier study, the maximum WUE was noted with one-time irrigation than 2 or 3 times irrigation in French bean [15].

The lowest rate of soil moisture depletion (SMD) was recorded during the early growing period. The highest was at the later growing period due to minimum air temperature at the initiation period and increment of temperature up to the harvest, respectively—the temperature increment at the later stage of plant growth strongly correlated with the evapotranspiration rate. The SMD ranged from 46 to 121 mm among the irrigation schedules, and the maximum SMD (121 mm) was found at control, while the minimum (46 mm) was at the I₄ followed by the I₃ (58 mm) treatment. Results demonstrated that triplicate irrigation at 20, 35, and 45 DAS (I₄) improved WUE in mung bean, although double irrigation at flowering and pod filling stages gave the highest yield. Single irrigation at the triple leaf stage (20 DAS) should be recommended with very limited water. It has been previously reported that the seasonal water requirement for mung beans is generally about 400 mm for 60–75 days, depending on soil and other environmental factors [16]. Additionally, supplying higher amounts of moisture to the soil through irrigation increases evapotranspiration losses, which has resulted in higher water use in mung beans and also in maize [17]. Similar observations were also made in earlier studies in mung beans [18,19]. Another reported that grain yield of mung bean is strongly influenced by soil moisture stress at flowering and pod filling stage, which ultimately affects water use efficiency [20], and application of sufficient water during flowering and pod development is the most influential factor on yield in mung bean [21].

2.2.2. Chlorophyll Content (mg g⁻¹ FW)

Regarding chlorophyll content, the results exposed that the application of different irrigation levels showed a significant variation in the leaf chlorophyll (Chl) content (

Table 2. Chlorophyll content (mg g⁻¹ FW) of selected mung bean genotypes at DF and DPF under different irrigation schedules.

Treatment	Chlorophyll a		Chlorophyll b		Total Chlorophyll		Chlorophyll a/b Ratio
	DF (40 DAS)	DPF (50 DAS)	DF (40 DAS)	DPF (50 DAS)	DF (40 DAS)	DPF (50 DAS)	DF (40 DAS)	DPF (50 DAS)
Irrigation schedules
I0	1.74b (0) #	1.57c (0) #	0.75b (0) #	0.65b (0) #	2.48b (0) #	2.22c (0) #	2.38a	2.43a
I1	1.87a (7.47)	1.65bc (5.10)	0.84a (12)	0.73ab (12.31)	2.71a (9.27)	2.37bc (6.76)	2.23bc	2.26b
I2	1.79b (2.87)	1.68abc (7.01)	0.75b (0)	0.75a (15.38)	2.54b (2.42)	2.43ab (9.46)	2.34ab	2.23b
I3	1.88a (8.05)	1.77ab (12.74)	0.85a (13.33)	0.81a (24.62)	2.73a (10.08)	2.57ab (15.77)	2.22c	2.20b
I4	1.89a (8.62)	1.79a (14.01)	0.84a (12)	0.81a (24.62)	2.71a (9.27)	2.61a (17.57)	2.25bc	2.21b
LSD (0.05)	0.06	0.13	0.06	0.09	0.10	0.20	0.11	0.14
CV (%)	2.31	5.67	5.54	8.77	2.95	6.30	3.78	4.68
LS	***	*	**	*	**	*	*	*
Genotypes
G1	1.88a	1.74a	0.84a	0.78a	2.72a	2.53a	2.25b	2.23a
G2	1.78b	1.64b	0.77b	0.72b	2.54b	2.35b	2.31a	2.31b
LS	**	**	***	**	***	**	*	*
Interactions
I × G	ns	ns	ns	ns	ns	ns	ns	ns

Where, LS = level of significance; * significant at p = 0.05; ** significant at p = 0.01; *** significant at p = 0.001; I₀ = No irrigation; I₁ = Irrigation at 1st trifoliate leaf stage; I₂ = Irrigation at flowering stage; I₃ = Irrigation at 1st trifoliate leaf stage + flowering stage; I₄ = Irrigation at 1st trifoliate leaf stage + flowering stage + pod-filing stage; G₁ = Genotype BMX-08010-2; G₂ = Genotype BARI Mung-1; DF = Days to flowering; DPF = Days to pod filing; ^# % increase over control (No irrigation), ns = not significant; Data followed by the same letter are not differed significantly by LSD test at p < 0.05.

). The tested mung bean genotypes also showed a remarkable variation in the leaf Chl content under different irrigation levels, and it was reduced with the advancement of crop age. However, the Chl content ranged from 1.74 to 1.89, and 1.57 to 1.79 mg g⁻¹ FW among the irrigation levels at days to flowering (DF) and days to pod filling (DPF) stage. The irrigation treatments of I₃ and I₄ contained the maximum Chl a (1.88 and 1.77, and 1.89 and 1.79 mg g⁻¹ FW), whereas the minimum Chl a was recorded in I₀ treatment (1.74 and 1.57 mg g⁻¹ FW) at DF and DPF stage, respectively. The results revealed that Chl a gradually increased with the increasing irrigation frequencies, and the highest increment of Chl a (8.05 and 12.74; and 8.62 and 14.01%) was recorded with I₃ and I₄ irrigation treatments at DF and DPF, respectively. Between the genotypes, the G₁ (BMX-08010-2) showed superior Chl a content (1.88 mg g⁻¹ FW) to G₂ (BARI Mung-1) (1.78 mg g⁻¹ FW). Similar trends were observed for chlorophyll b as well as total chlorophyll under different irrigation schedules. The genotype BMX-08010-2 gave higher Chl a, Chl b, and total chlorophyll under water deficit conditions than BARI Mung-1. This indicates that the BMX-08010-2 genotype can take up water better than the BARI Mung-1 genotype under water deficit conditions, which is directly related to higher yield. Similar results were also found by Mafakheri et al. [22]. They also observed that the chlorophyll a, chlorophyll b, and total chlorophyll contents in the vegetative and flowering stages were significantly decreased in chickpea cultivars due to drought stress in the vegetative stage.

The Chl a/b ratio also found a significant variation among the irrigation levels as well as selected genotypes (Table 2). The maximum Chl a/b ratio (2.38 and 2.43) was observed in severe water stress (control) at DF and DPF stage, respectively. Between the selected mungbean genotypes, BARI Mung-1 showed a higher Chl a/b ratio (2.31 and 2.31) than BMX-08010-2 (2.25 and 2.23) in respective two growth stages, respectively. Furthermore, the study indicated that Chl b content decreased more than the Chl a among the treatments. Under water stress (no irrigation) conditions, chlorophyll loss might occur due to the damage of the mesophyll chloroplasts, which led to a lower photosynthetic rate.

2.2.3. Relative Water Content

Relative water content (RWC) expresses the amount of water held by the plant tissues. Irrigated plot increased the RWC in plants at both the growth stages as compared to the water deficit plot (Figure 2). It is assumed that water stress reduces the soil water potential resulted in a reduction of the RWC by dehydration at the cellular level and osmotic stress. However, the highest RWC (88.51 and 88.28%) was observed at I₃ and I₄ treatments at DF, and it increased 9.46 and 9.18% over control (no irrigation), respectively. One stage irrigation at 20 DAS (I₁) showed the RWC of 84.36% (4.33% increase over control), while one stage irrigation at 35 DAS (I₂) showed the RWC of 82.81% (2.41% increase over control) at the flowering stage. The reverse scenario was observed at the pod filling stage where I₂ gave the higher RWC of 81.94% (4.53% increase over control), and I₁ gave 80.31% RWC (2.45% increase over control). It might be due to fact that plants in the I₁ plot maintained comparatively higher soil moisture content up to the flowering stage because it received irrigation at 20 DAS, whereas I₂ maintained comparatively higher soil moisture content up to the pod filling stage as it received irrigation at 35 DAS. At the pod filling stage, the treatment I₄ gave the maximum RWC of 89.16% (13.74% higher over control). It received three times irrigation at 20, 35, and 45 DAS, which ensured available soil moisture resulted in more values of RWC. It is well known that the water deficit stress reduces the water availability in soil and consequently reduces the RWC. The reduction in RWC due to water stress was also observed in French beans [23] and mungbean [24].

The genotype BMX-08010-2 showed higher RWC (86.59 and 85.02%) as compared to BARI Mung-1 (83.34 and 82.27%) at the DF and DPF stages, respectively (Figure 3). These results indicated that the BMX-08010-2 genotype maintained a higher amount of water in the plant compared to that of BARI Mung-1. It might give evidence of accumulating a higher amount of osmolites through higher RWC. It is evident that most crops maintain their tissue RWC above 85% during their active growth [25] and generally begin to show tissue damage signs when RWC falls within 60–30% [25,26].

2.2.4. Xylem Exudation Rate (mg hr⁻¹)

Xylem exudation rate (XER) refers to the flow of sap from the cut end of the stem against the gravitational force. The XER under well soil moisture conditions is higher than that under stress conditions. Thus, the XER can be used as a tool to measure the severity of water stress. Water stress drastically reduced the exudation rate in the studied growth stages (Figure 2). Nonetheless, the XER increased with the increasing irrigation schedules in the present study.

Moreover, the time and frequency of irrigation influenced the XER, and it declined with the advancement of growth stages. In addition, the XER was higher in G₁ compared to G₂ under different irrigation levels in both the growth stages. The XER of the G₂ genotype in water deficit condition (I₀) is more affected (28.67 and 17.33 mg hr⁻¹) than that of G₁ (47.33 and 30.00 mg hr⁻¹) at the DF and DPF stages, respectively (Figure 3). These results indicated that the G₂ suffered more due to water stress than the G₁. The maximum XER of a plant means that the plant uptakes more water from the soil media. The decreased water uptake of a plant due to water stress was also observed in mung bean [27] and French bean [28].

2.2.5. Membrane Stability Index (%)

The membrane stability index (MSI) was significantly affected by water stress conditions, and the MSI decreased significantly with increasing stress intensity due to irrigation schedules (Figure 4). However, well-watered plants maintained better MSI than non-irrigated plants. The decrease in membrane stability under water deficit stress may be due to membrane disorganization, which is responsible for the higher leakage of ions from leaves.

BMX-08010-2 genotype had higher MSI than BARI Mung-1 genotype. It was also clearly shown that the highest MSI was recorded under stress (no irrigation) in BMX-08010-2 genotype compared to BARI Mung-1 with the same conditions, not only that BMX-08010-2 genotype gave better MSI result than irrigation treatment. The highest reduction in MSI (11.48%) due to water stress was recorded in BARI Mung-1 under triple irrigation (I4), which is considered as a water stress-sensitive genotype. The genotype BMX-08010-2 was evaluated as a drought-tolerant variety which showed the lowest reduction in MSI (9.25%) under the same conditions (I4). These data also indicate that the BMX-08010-2 genotype was able to tolerate water stress better than the BARI Mung-1 genotype. Thus, the higher MSI reflects the relative tolerance to drought stress [29]. The decrease in MSI under water stress is probably because the overproduction of ROS occurs under water stress conditions, which disrupts the cell membrane by altering its phospholipid and fatty acid compositions [30,31]. Furthermore, MSI is the prime defense in plants under drought stress, and the ability of a plant to maintain membrane stability and integrity would explain its tolerance toward drought [32]. Thus, MSI is considered as the critical indicator of water status under drought stress [33,34].

2.3. Biochemical Properties in Response to Irrigation Regimes in Mung Bean Genotypes

2.3.1. Proline Content (mg g⁻¹ FW)

In adverse environmental conditions, plants accumulate soluble materials with low cellular weight like amino acids, sugars, and some soluble mineral substances named adaptable solutions [35]. These versatile solutions do not interfere with normal biochemical reactions in the cell but act as osmotic protectors during osmotic stress. Not only that, they maintain the tissue’s turgor and can also help protein sustainability and cellular structure [36]. Proline is possibly the most well-known soluble substance. Its accumulation seems to help the plant survive just after undergoing drought stress and re-establish growth after tension resolution. Hence, the accumulation of proline probably has a positive effect on yield. Proline activates more positively, and its assembly acts negatively on the yield under prolonged stress conditions [37]. In the present study, the proline content differed significantly among the irrigation schedules as well as selected genotypes (Figure 5). Increase the stress intensity due to applying different irrigation levels.

The proline accumulation differed correlatively. However, the minimum proline accumulation was recorded by irrigation at all the stages with sufficient moisture content, and the proline accumulation increased with the progress of the crop age. This is because of a decrease in the internal water status of the plant with the advancement of crop age, which could be evident from a reduction in leaf water potential with the crop age. At the flowering and pod filling stage, the highest proline contents of 0.473 and 1.117 mg g⁻¹ FW were recorded underwater stress (no irrigation). The treatment I₁ accumulated more proline (0.382 and 0.777 mg g⁻¹ FW) than I₂ (0.357 and 0.712 mg g⁻¹ FW⁻¹) at 40 and 50 DAS, respectively, although they received one irrigation in the growing season. Plants under I₁ treatment suffered more soil moisture shortage at flowering and afterward than I₂ treatment resulted in higher proline accumulation in I₁. The treatment I₄ (three times irrigations at 20, 35, and 45 DAS) gave the most negligible value of proline (0.590 mg g⁻¹ FW) during the pod filling stage followed by I₃ (0.605 mg g⁻¹ FW) that received two times irrigation (Figure 5).

Regarding tested genotypes, the highest accumulation of prolines (0.399 and 0.847 mg g⁻¹ FW) was recorded in BMX-08010-2 than BARI Mung-1 (0.311 and 0.672 mg g⁻¹ FW) at flowering and pod filling stages, respectively. In interaction effect, the highest proline accumulation (0.54 and 1.31 mg g⁻¹ FW) was recorded with the treatment combination of I₀G₁ (BMX-08010-2 under water stress) than I₀G₂ (0.41 and 0.93 mg g⁻¹ FW), and the lowest (0.24 and 0.52 mg g⁻¹ FW) was with I₄G₂ (BARI Mung-1 under three times irrigation) at flowering and pod filling stage, respectively. The result means that the genotype BMX-08010-2 is more tolerant than the BARI Mung-1 due to producing higher proline under water stress. A similar effect was observed by Stoyanov [38] in beans, where susceptible cultivar showed the highest accumulation of proline, and proline levels were more closely related to the decrease in RWC than in water potential. It is well evident that proline acts as an osmolyte and protects the plant against low water potential by maintaining osmotic regulation in plant organs [39,40]. In addition, proline also plays a significant role as an electron receptor and may promote damage repairability in the plant by increasing antioxidant activity during drought stress [41]. A positive correlation between the level of proline accumulation and drought tolerance intensity exists in drought-stressed plants [42].

2.3.2. Soluble Sugar Content (mg g⁻¹ Dry Weight)

Water stress significantly influenced the soluble sugar (SS) content (Figure 6). Under water stress conditions, the SS plays a vital role in plant growth and development by regulating the carbohydrate metabolism during photosynthesis and is also considered as a crucial osmotic adjustment tool that is widely regarded as an adaptive response to water stress conditions [43]. Accumulation of higher sugars may result from a reduction in the utilization of assimilates induced by water stress concerning inhibition of sucrose synthase or invertase activities in one hand and deterioration of translocation from sources to sink from the other hand [35].

The application of different irrigation levels significantly influenced the total SS content in mung bean genotypes, but the SS content increased considerably under water deficit conditions (Figure 6). However, the highest value of SS (41.78 mg g⁻¹ DW) was recorded under control (no irrigation) condition (I₀), while the least (24.51 mg g⁻¹ DW) was with three times irrigation at 20, 35, and 45 DAS (I₄) followed (25.29 mg g⁻¹ DW) by two times irrigation at 20 and 35 DAS (I₃). One-time irrigation at 20 DAS produced the superior SS content (28.93 mg g⁻¹ DW) to former irrigation at 35 DAS (28.11 mg g⁻¹ DW). It might be due to the plant suffering prolonged water stress I₁ condition than I₂ condition. Our findings pertaining to increasing the SS due to water deficit stress follow the conclusion accounted by Islam et al. [42], who reported that drought stress decreased the physiological functions in plants, thereby increasing total soluble sugar. The genotype BMX-08010-2 accumulated a higher amount of SS (30.77 mg g⁻¹ DW) than BARI Mung-1 (28.67 mg g⁻¹ DW), which indicates that the genotype BMX-08010-2 is more tolerant than the genotype BARI Mung-1. Under drought stress, higher production of SS improves water absorption, reduces the osmotic potential, and consequently increases the stress tolerance of plants [42].

The genotype BMX-08010-2 under control condition (I₀G₁) generated the highest SS content (44.74 mg g⁻¹ DW) followed by the genotype BARI Mung-1 (38.82 mg g⁻¹ DW) with the same condition (I₀G₂), which indicates that the genotype BMX-08010-2 is more tolerant than BARI Mung-1. The least values (24.13 and 24.89 mg g⁻¹ DW) were observed in both the genotypes with the I₄ treatment combination (I₄G₁ and I₄G₂) followed (25.23 and 25.34 mg g⁻¹ DW) by I₃G₁ and I₃G₂, respectively. It has been reported previously that the SS content was significantly higher (46 and 67%) in the non-irrigated plants of Brassica campestris at the flowering and pod filling stage, respectively [44]. It has been suggested that under water deficit stress, the SS can act in two ways as an osmotic agent and as osmoprotectants which are difficult to separate [45,46]. In addition, SS accumulation under water stress also possibly functions to form reserve assimilates for seed filling [37]. The accumulation of simple sugars increases the invertase activity in the leaves of the drought-challenged plants [47], resulting in decreased water potential, which is essential for growth, development, and physiological compatibility [48]. Besides, it acts as stabilization of membranes action [49] as regulators of gene expression [50] and signals molecules [51].

2.3.3. Seed Protein Content (%)

The seed protein content remarkably increased due to the water deficit stress condition (no irrigation) in this study (Figure 6). Furthermore, increasing water deficit stress due to different irrigation levels showed the variation of seed protein contents. However, control treatment (no irrigation) gave the highest seed protein content (26.31%) as compared to irrigated plots, and the I₄ treatment (three times irrigation) gave the minimum seed protein (24.94%) followed (25.03%) by two times irrigation treatment (I₃). The common opinion is that the high N content in plants subjected to water stress is due to the rapid accumulation of free amino acids that are not incorporated into proteins. This finding was significantly closer to the conclusion of [52].

Regarding the genotypes, the elevated seed protein (25.44%) was obtained in BARI Mung-1 than BMX-08010-2 (25.21%). In the case of an interaction effect, the highest seed protein (26.66%) was recorded in a combination of I₀G₂ followed by I₀G₁ (25.96%) and the lowest (24.91%) was in the I₄G₁ combination (Figure 6). The result also revealed that the genotype BMX-08010-2 recorded a minor deviation in seed protein content (0.82–1.05) compared to BARI Mung-1 (1.34–1.69) due to water deficit stress. Therefore, this genotype (BMX-08010-2) can be used as a source of drought tolerance in different breeding programs. The present findings were also in agreement with results in spotted beans [53], red beans [54], in mung bean [55] where they reported that water deficit stress significantly increased the amount of seed protein. In addition, numerically differences in protein content occurred in selected mung bean genotypes, probably due to variation of nitrate assimilation in respective treatments [55,56].

2.4. Crop Harvests in Response to Irrigation Regimes in Mung Bean Genotypes

2.4.1. SeedYield

The seed yield significantly varied (p < 0.05) among the different irrigation schedules (Figure 7). The genotype G₁ showed superior seed yield (613.53–1196.22 kg ha⁻¹) to G₂ (496.74–1094.66 kg ha⁻¹). However, the maximum seed yield of 1196.22 kg ha⁻¹ for G₁, and 1094.66 kg ha⁻¹ for G₂ was recorded under treatment, which received irrigation at three stages (I₄) followed by irrigation at two stages (I₃), which was significantly higher than those received irrigation during only one step (I₁ and I₂ treatments). The lowest seed yield of 613.53 and 496.74 kg ha⁻¹ was shown by the I₀ treatment in both the genotypes, respectively. So, seed yield reduction was higher in G₂ than in G₁ under severe water stress (I₀).The I₄ and I₃ treatments increased above two-fold yields (95 and 120%, and 92 and 113%) over the I₀ treatment in G₁ and G₂, respectively. The result also showed that plants treated with method I₂, which encountered soil moisture deficiency during the vegetative stage, had numerically lower yield than plants treated with process I₁, which experienced soil moisture deficiency after the flowering stage. This may be due to water stress during the vegetative stage, which reduces grain yield through limited plant size, leaf area, and root growth, reducing dry matter accumulation, the number of pods per plant, and low harvest index [57,58]. Nielson and Nelson [59] reported that drought stress at the vegetative stage leads to a reduction in seed yield due to a reduction in the number of pods per plant or the number of seeds per pod. The results clearly showed that the yield of mung bean is very sensitive to irrigation, and considerable yield gain is achieved by irrigating at least two growth stages [10]. An earlier study reported that omitting one irrigation during the growth stages of vegetation, flowering, and pod formation significantly reduced all growth parameters, photosynthetic pigments, yield components, and mung bean [60]. Water stress induced a reduction in seed yield and genotypic variability related to seed yield reduction due to water stress in mungbean [61]. The significant impact of water deficit stress was in agreement with other findings [10,21,62], showing that plants suffer from water deficit stress during the reproductive stage, especially at the flowering and pod formation stage, which significantly reduces grain yield.

2.4.2. Biological Yield

The obtained results divulged that the different irrigation treatments significantly influenced mung bean genotypes’ biological yield (BY). Nevertheless, the highest BY (3410.20 and 3216.99 kg ha⁻¹) recorded with I₄ treatment in G₁ and G₂, respectively, and which was statistically at par with the treatments of I₃, I_2, and I₁ by providing BY of 3374.25, 2893.88, and 2971.82 kg ha⁻¹ in G₁, and 3156.80, 2811.98 and 2848.39 kg ha⁻¹ in G₂, respectively (Figure 7). The lowest BY (2005.79 and 1701.40 kg ha⁻¹) was observed under water stress conditions (I0) in both genotypes. The genotype G₁ gave a higher BY than G₂ under water deficit stress and irrigated treatment conditions. These results disclosed that the G₁ genotype is more tolerant to water stress than the G₂ genotype. The results align with Khan [63], who noticed that irrigation significantly increased biomass production. Sangakara [64] reported that the growth and yield of mung beans considerably increased under adequate soil moisture conditions.

3. Materials and Methods

3.1. Experimental Site andPlant Materials

The experimental research work was conducted under rain shelter from March 2017 to May 2017 in the Kharif-1 season at Regional Agricultural Research Station, Ishurdi, Pabna, Bangladesh. The experimental site is located at 24.03° N, 89.05° E, at an altitude of 16 m above sea level and under subtropical monsoon. One water stress-tolerant (BMX-08010-2):G₁ and one sensitive (BARI Mung-1):G₂ genotypes of mung bean were used and selected from laboratory screening based on their germination, seedling growth behavior, and relative performance under PEG water deficit stress [65].

3.2. Soil Status and Weather Information

The soil of the experimental site belongs to Ishwardi series under the High Ganges River Floodplain soil (Agro-ecological Zone-11) in Bangladesh, and it has been classified as Calcareous Dark Gray Floodplain soil [66]. The soil is characterized by clay loam and is slightly alkaline in nature. The initial soil (0–15 cm depth of the soil profile) was analyzed before sowing of the mung bean crop. The soil’s field capacity, bulk density, and permanent wilting point were 28.5%, 1.41 g/cc, and 13%, respectively. Other soil physical and chemical properties are given in

Table 3. Soil physical and chemical properties of the initial experimental soil.

Items	Soil Texture (Clay Loamy)			pH	OM (%)	N (%)	p (µg mL−1)	K (meq 100 g−1 soil)	S (µg mL−1)	B (µg mL−1)	Zn (µg mL−1)
Initial soil	Sand (18.6%)	Silt (32.0%)	Clay (49.4%)	7.36	1.10	0.06	31.12	0.31	10.75	0.35	1.43
Critical level	-	-	-	-	-	0.12	10.00	0.12	10.00	0.20	0.60

. The seasonal weather data during the study periods of the crops recorded at the meteorological station of Bangladesh Sugar crops Research Institute situated about 400 m from the experimental site are given in

Table 4. Decades wise (10th days mean) weather information during the study period.

Month	Decades	Air Temperature		Mean Relative Humidity (%)	Rainfall (mm)	Sunshine Hour (day−1)
		Max. (°C)	Min. (°C)
March	1	31.0	17.7	72.6	22.0	5.5
	2	29.8	15.9	71.9	12.0	7.4
	3	32.8	22.2	79.6	4.0	6.2
April	1	35.3	24.6	75.5	0.1	6.9
	2	35.3	21.6	71.2	2.8	8.7
	3	33.7	23.6	82.5	11.0	5.5
May	1	34.4	24.4	81.6	1.5	6.9
	2	34.9	24.5	80.9	5.3	7.7
	3	35.9	26.4	80.5	0.5	7.1

.

3.3. Experimental Design and Treatments

The experiment was conducted with five levels of irrigation viz., (i) Control (water deficit stress): I₀, (ii) Irrigation at 1st trifoliate leaf stage (20 DAS): I₁, (iii) Irrigation at flowering stage (35 DAS): I₂, (iv) Irrigation at 1st trifoliate leaf stage (20 DAS) + flowering stage (35 DAS): I₃, (v) Irrigation at 1st trifoliate leaf stage (20 DAS) + flowering stage (35 DAS) + pod-filing stage (45 DAS): I₄, and two mung bean genotypes viz., (i) BMX-08010-2, drought-tolerant: G₁, and (ii) BARI Mung-1, susceptible one: G₂. The experiment was arranged in a split-plot design distributing irrigation levels to the main plots and genotypes to the sub-plots with three replications using plots of 3 m × 3 m size.

3.4. Experimentation

3.4.1. Fertilization

The crop was fertilized with recommended fertilizer rates of 20–17–18–10–2 kg of N-P-K-S-B ha⁻¹ in the form of urea, triple superphosphate, muriate of potash, gypsum, and boric acid, respectively [67]. All the fertilizers were applied during the final land preparation and thoroughly mixed with soil.

3.4.2. Seed Sowing and Crop Management

Provex-200 treated the seeds @ 3.5 g kg⁻¹ seed before 8 h of sowing. The seeds were then sown in line at the rate of 25 kg/ha on 4 March 2017, subjected to Kharif-1 season (summer), and maintained 30 cm apart rows by the technique of continuous seeding. The depth of the seeds was 3–4 cm. After 15 DAS, mung bean plants were thinned out to keep the plant to plant distance of about 6–7 cm. Weeds were controlled by hand racking in between rows of mung bean plants and by hand weeding with a hoe at 15 and 30 DAS. For the protection of the plants from flower thrips, two sprayings were done by Imidachloprid (Imitaf 20 SL) @ 0.5 mL liter⁻¹ of water at 35 DAS (100% flowering stage) and 42 DAS (Peak flowering and 100% podding stage). For pod borer control, spraying was done with Lambda-Cyhalothrin (Karate 2.5 EC/Reeva 2.5 EC) @ 1 mL liter⁻¹ of water at 100% podding and after 2–3 times at seven days interval.

3.5. Imposition of Treatments

In each treatment, a post sowing irrigation (48 mm) was done for proper germination and seedling establishment based on existing soil moisture. In the control treatment (water deficit stress), irrigation was not applied to impose water deficit stress. The rest of the pots were irrigated as per treatment specifications. Irrigation water was done up to field capacity level by estimating soil moisture content. Each treatment plot was made boundary with soil ridge to protect the seepage and percolation of water from one plot to another. Moreover, the distance between the two plots was maintained by 1 m. The net amount of water applied (d) to each irrigation treatment was calculated based on the soil moisture contents at the irrigation time by using the following expression [68]. The estimated water was applied manually in each plot.

$$\mathrm{d}=\frac{\mathrm{FC}-\mathrm{MC}}{100}\times p\times D$$

where FC = Field capacity of the soil (%);

MC = Moisture content of the soil at the time of irrigation (%);

p = Bulk density of the soil (g/cc);

D = Root zone depth (cm).

The depth of the effective rooting zone was considered as 45 cm. It is reported that 70% of total moisture is extracted from the 50% effective root zone depth [68]. The calculated amount of water was applied manually in each treatment plot through flood irrigation.

3.6. Data Collection

3.6.1. Monitoring of Soil Moisture

The soil moisture content of experimental plots was computed gravimetrically using a hand-driven Auger to check the water balance and the predicted groundwater contributions. For this purpose, soil samples were taken from the plant’s effective root zone, which is considered 0–45 cm. Soil sample from each plot at different depths (0–15, 15–30, and 30–45 cm) was collected (in the center of the plot) at 10-day intervals starting from sowing to maturity of the crop. Before and after irrigation, the soil moisture sampling was carried out to verify the depletion of soil moisture at the respective treatment. Duplicate soil samples were taken at each sampling date, immediately packed tightly in loosed aluminum cans, transported to the laboratory, and then weighed. The samples were oven-dried at 105 °C until a constant weight was obtained, then weighed again, and finally, their moisture content was calculated on a dry weight basis.

$$\%\mathrm{soil}\mathrm{moisture}=\frac{{\mathrm{W}}_1-{\mathrm{W}}_2}{{\mathrm{W}}_2}\times 100$$

where,

W₁ = Weight of moist soil;

W₂ = Weight of oven-dry soil.

The initial soil moisture in the experimental plot was calculated to be 27.93%, which was maintained by applying light irrigation (48 mm) based on soil moisture monitoring to ensure emergence (Table 1).

3.6.2. Soil Water Depletion

Soil water depletion in the effective root zone was estimated by the following equation [69]:

$$SWD={\displaystyle \sum }_{i=1}^n\frac{\left(\mathit{Mbi}-\mathit{Mei}\right)}{100}\times BDi\times Di$$

where,

SWD = Soil moisture depletion (mm);

Mbi = Moisture percentage at the beginning of the season in the specific layer of the soil;

Mei = Moisture percentage at the end of the season in the specific layer of the soil;

n = Number of soil layers in the root zone (3);

BDi = Bulk density of specific layer of the soil;

Di = Depth of the specific layer of soil within the root zone (mm).

3.6.3. Water Use Efficiency

The water use efficiency is used to evaluate yield performance and water management practices. The water utilized by the crop was calculated by the following relationship [70]:

$$\mathrm{Water}\mathrm{use}\mathrm{efficiency}\left(\mathrm{WUE}\right)=\frac{\mathrm{Yield}\left({\mathrm{kg}\mathrm{ha}}^{-1}\right)}{\mathrm{Total}\mathrm{water}\mathrm{use}\left(\mathrm{mm}\right)}$$

3.6.4. Estimation of Chlorophyll Content

The chlorophyll content of third trifoliate fresh leaves (0.1 g) was extracted with 10 mL 80% acetone through shaking overnight using an electric horizontal shaker. The optical density of the supernatant was measured by using a UV-visible spectrophotometer at 663 nm and 645 nm wavelength for chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll by the following method described by [71].

3.6.5. Relative Water Content (RWC)

The estimated values of the fresh, pompous, and dry weights of each treatment’s fully expanded uppermost trifoliate leaves were used to calculate RWC [72]. For turgid weight, the leaves were dipped in distilled water for 8 h at room temperature in the dark and weighed after removing excess water from the leaf surface by gently wiping with a paper towel. The leaves were then dried in an oven at 80 °C for 72 h to determine their dry weight (DW). The formulawas presented below:

$$\mathrm{Relative}\mathrm{water}\mathrm{content}\left(\mathrm{RWC}\right)=\frac{\mathrm{Fresh}\mathrm{weight}-\mathrm{Dry}\mathrm{weight}}{\mathrm{Turgid}\mathrm{weight}-\mathrm{Dry}\mathrm{weight}}\times 100$$

3.6.6. Measurement of Xylem Exudation Rate

Clean and dry cotton was considered first. An oblique cut of the stem was made with a sharp knife 5 cm above the base of the plant. The cotton was then placed on the cutting surface. After one hour, the oozing sap was removed from the stem at an average temperature. The cotton was covered with a polythene bag to prevent evaporation. Then the cotton was released with the weight of the sap. The exudation rate was calculated using the following formula [73]:

$$\mathrm{Exudation}\mathrm{rate}=\frac{\left(\mathrm{Weight}\mathrm{of}\mathrm{cotton}+\mathrm{sap}\right)-\left(\mathrm{Weight}\mathrm{of}\mathrm{cotton}\right)}{\mathrm{Time}\left(\mathrm{h}\right)}$$

3.6.7. Estimation of the Membrane Stability Index

The membrane stability index (MSI) was estimated using the protocol developed by [74]. Leaf materials with 100 mg were taken into two sets of test tubes containing 10 mL of double-distilled water. One set was heated at 40°C for 30 min in a water bath, and the electrical conductivity of the solution was recorded (EC1) using an electrical conductivity meter. The second set was boiled at 100°C on a boiling water bath for 10 min, and its conductivity (EC2) was measured as above.

Membrane stability index (MSI) = [1 − (EC1/EC2)] × 100

3.6.8. Estimation of Proline Content of the Third Trifoliate Leaf

The uppermost third trifoliate fresh leaves (0.5 g) was used to determine proline content, and it was estimated using the acid ninhydrin method [75]. The absorbance of the solutions (sample and standard solution) was measured at 520 nm wavelength using a UV-visible spectrophotometer (T60 UV, Japan). The proline content was then calculated from the standard curve and expressed as mg proline g⁻¹ FW.

3.6.9. Estimation of Soluble Sugars

Soluble sugar content in the dry third trifoliate leaf sample at flowering and pod development stages of mungbean with all treatment combinations was determined by using the method described by [76]. The values of soluble sugar content in the respective sample were measured from the standard curve and expressed mg g⁻¹ dry weight.

3.6.10. Crop Harvests

An area of 2.0 m × 1.5 m (3 m²) was harvested from the undisturbed central area of the unit plots. Three pickings of mung bean were done at 60–65, 70–75, and 90–95 DAS. Harvested pods of each plot were tagged and carried to the threshing floor. The pods were sun-dried. Threshing was done manually by beating with a stick. The seeds were dried, cleaned, and weighed. The moisture content of the grain (~10%) was measured by a moisture meter (model F/RMEX). Straw was sun-dried, and the sample was subjected to oven drying at 70 °C for 72 h. Then straw yield was adjusted at 14% moisture content.

3.7. Statistical Analysis

All the collected data on crop characters were subjected to statistical analysis through the computer using the R-stat software program (version 3.1.2) following the basic procedure outlined by [77]. Significant effects of treatments were determined by analysis of variance (ANOVA), and treatments were compared by the Least Significant Difference (LSD) test. Correlation analysis was done to study the relationship between desired variables.

4. Conclusions

The study’s outcome showed that water regimes significantly affected the physiological and biochemical characteristics of mung bean genotype and yield. Different phenological stages are differently sensitive to irrigation regimes. Physiological traits such as WUE, RWC, chlorophyll content, XER, and MSI, and biochemical characteristics such as proline and soluble sugar content showed positive response due to the introduction of the I₄ variant, which received triple irrigation and closely related to I₃, which received double irrigation. Conversely, negative responses were observed under water deficit conditions. BMX 08010-2 genotype showed higher values of RWC, chlorophyll content, WUE, XER, MSI, proline, soluble sugar, and lower protein content than BARI Mung-1 genotype under water stress conditions, indicating that the earlier genotype was more tolerant to water stress than the later one. Significant differences in seed yield were observed due to differences in the timing and frequency of irrigation, and water stress (I₀) proportionally reduced seed yield. Maintaining adequate soil moisture by irrigation during the growing season positively influenced pod filling and ensured a higher yield of mung beans. The yield obtained ranged from 1145.44 kg ha⁻¹ with seasonal irrigation of 277 mm to 555.14 kg ha⁻¹ without irrigation throughout the growing season. The flowering stage was recorded as the most sensitive growth stage with an 18.15% yield reduction compared to treatment I4. Therefore, to overcome the effect of water stress and obtain a higher yield, the field should be irrigated at least during the period from the trifoliate leaf stage to the pod filling stage, which is the most moisture-sensitive stage during the life cycle of mung bean. In addition, if irrigation resources are available, at least two irrigation phases should be provided at the trefoil stage (20 DAS) and at the flowering stage (35 DAS) to obtain the highest yield. The trifoliate stage is the most critical irrigation stage for mung beans.