Australian Journal of Basic and Applied Sciences, 2(1): 42-52, 2008

ISSN 1991-8178

Effect of Water Stress in Addition to Potassiomag

Application on Mungbean

Tawfik, K.M.

Botany Department, Womens College, Ain Shams University, Cairo, Egypt.

Abstract: A pot experiment was conducted to study the effect of extension of irrigation interval
(2, 5, and 10 days) on growth, yield and metabolic changes in mungbean (Vigna radiata L.) var.
VC 1000 in addition to potassiomag application. Generally, fresh, dry weights and yield were
significantly reduced under water stress treatments. Treatment with K biofertilizer to some extent
mitigated the effect of drought stress. The greatest vegetative growth was obtained for plants irrigated
every two days and treated with potassiomag, while the greatest seed yield was obtained for plants
irrigated every five days and treated with potassiomag. Osmoprotectants such as total soluble sugars,
proline and glycine betaine increased in plants subjected to water stress. It could be concluded, that
to maximize mungbean yield, irrigation should be extended through all phenological stages, specially
the flowering and the pod-filling stages.

Keywords: Mungbean; W ater stress; K-biofertilizer

INTRODUCTION

To cope with the increasing food requirements and as drought is a major stress which adversely affects
plant growth and productivity; it is important to develop stress tolerant crops (Mahajan and Tuteja 2005). Plant
can respond and adapt to water stress by altering their cellular metabolism and invoking various defence
mechanisms (Bohnert and Jensen, 1996).
W ater stress reduces plant growth and yield. However, water stress that exists at the reproductive stage
severely affects grain yield of mungbean more than its occurrence at other stages (Thomas et al., 2004). In
addition, the time of flowering and maturity was shortened under stress compared to well-watered conditions.
Leport et al., (2006) found that pod production of chickpea was more affected by early podding water stress
than by late podding water stress.
Tolerance to abiotic stresses is very complex at the cellular levels of the whole plant (Foolad et al., 2003
a, b; Ashraf and Harris, 2004). This is in part due to the complexity of interactions between stress factors and
various molecular, biochemical and physiological phenomena affecting plant growth and development
(Zhu, 2001).
One of the most common stress responses in plant overproduction of different types of compatible organic
solutes (proline, GB, ABA, soluble sugars and inorganic ions like K + ) (Serraj and Sinclair, 2002). These are
of low molecular weights highly soluble compounds that are nontoxic at high cellular concentrations
and protect plants against stress, including contribution to cellular osmotic adjustment, detoxification of
reactive oxygen species, protection of membrane integrity and stabilization of enzymes proteins (Bohnert and
Jensen, 1996).
W ater stress is considered principal environmental factor limiting growth and yield (Sangakkara et al.,
2001). Potassium fertilizer mitigates the impact of water stress in legume plants. Potassium increased mungbean
shoot growth to a greater extent under sub-optimal moisture conditions. The plant water relations and
photosynthetic rates of mungbean were improved by potassium and it has been concluded that application of
potassium fertilizer could be considered a significant factor in overcoming soil moisture stress. Jouany et al.,
(1996) had shown that the average wheat yield increase for K fertilized plots in comparison to the control
significantly responded to K fertilization.
The present study aims to investigate the effect of different irrigation intervals and different timing of
induced water stress on growth, yield and metabolic products. The objective of the study also aims to
determine the response of addition K biofertilizer ameliorating the negative effect of drought on growth, yield
and solute accumulation in mungbean plants.

Corresponding Author: Tawfik, K. M., Botany Department, Womens College, Ain Shams University, Cairo, Egypt.

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M ATERIALS AND M ETHODS

A pot experiment was carried out on mungbean (Vigna radiata L.) var. VC 1000 obtained from
Agriculture Research Centre, Ministry of Agriculture, Giza, Egypt. Seeds were sterilized with 1.5% chlorox,
washed with water and planted in pots of 50 cm diameter and 30 cm depth, where its soil surface area was
0.2m 2 ; each pot contained 30 kg soil. The soil characteristics was as follows: sandy loam in texture, sand,
80%; silt, 15.5%; clay, 4.5%; pH, 7.8; EC, 0.4 dSm -1 and organic matter 0.45%.

Irrigation Intervals:
First Experim ental Design:

C Irrigation every two days, soil moisture content depleted from 100% to 65% of field capacity.
C Irrigation every five days, soil moisture content depleted from 100% to 56% of field capacity.
C Irrigation every ten days, soil moisture content depleted from 100% to 45% of field capacity.

Twelve plants after seedling thinning were left to grow in each pot. Each treatment was replicated trice.
Plants were harvested at 30, 60, 90 days and after pod maturity. Three replicates were used for determination
of fresh and dry weights. Yield components (no. of pods/10 plants, yield/10 plants and weight of 1000 seed)
were determined at the end of the experiment (140- 160 of sowing date).
Interactive effect of K-biofertilizer potassiomag commercial product (Agriculture Research Centre): A set
of plants was supplied with 50 g potassiomag/pot to study its interactive effect with soil water depletion.

Withholding of Water Irrigation:

Second Experim ental Design:
A set of mungbean (Vigna radiata L.) var. VC 1000 plants were subjected to withholding of water
irrigation in order to study the effects on growth and yield. Irrigation withholding was maintained at three
different phenological stages for ten days once at the vegetative (20 days old), the flowering (50 days old) and
the seed setting stage (80 days old). The plants were rewatered and irrigated every two days.
W ater use efficiency (W UE) (g seed/ l water) was calculated for plants in the first experimental design.
For air temperature, atmospheric relative humidity; diffusion resistance and transpiration rate of leaf were
recorded using diffusion porometer (LI Cor-1600). Daily measurements were commonly recorded at 8 am, 12
pm. and 4 pm. on the third leaf from the shoot apex of three plants for each treatment. Photosynthetic
pigments were deduced according to M etzner et al., (1965). Total soluble carbohydrates were determined
according to Dubois et al., (1956). Proline content was determined according to Bates et al., (1973). Glycine
betaine content was carried out according to Grieve and Grattan (1983). Abscisic acid content was determined
according to Kelen et al., (2004). Total isoflavonoids was extracted according to Hertog et al., (1992) and
determined according to M eda et al., (2005). Mineral content NPK were determined according to A.O.A.C.
(1995). Total nitrogen was determined by usual Kjeldahl method according to A.O.A.C. (1995) and the crude
protein content was calculated by multiplying the total nitrogen by 6.25. Protein electrophoresis, SDS
polyacrylamide gel electrophoresis was performed in 10% acrylamide slab gels according to Laemmli (1970).
For gel analysis, Gels were photographed, scanned and analyzed using Gel Doc 2000 Bis Rad system.
The studied parameters were treated statistically using the one-way analysis of variance as described by
Snedecor and Cockran (1969). The means were compared by LSD using SPSS version 10.

RESULTS AND DISCUSSIONS

Plant Growth Response to Extension of Irrigation Water Intervals:

Growth Criteria:
Drought stress caused an observed adverse action on plant growth and productivity. Fresh and dry weights
were significantly reduced under water stress (Fig. 1). Growth criteria were higher for plants subjected to
extension of irrigation interval for two days compared to five and ten days extension. Plants irrigated every
ten days produced only 36.4% and 38.1% fresh and dry weights, respectively, compared to the control at 90
days old. Plants treated with potassiomag and irrigated every two days recorded, 119.5% and 121.7% fresh
and dry weights, respectively, compared to the control at 90 days old, while fresh and dry weights of plants
irrigated every ten days were reduced to 51.4% and 56.1%, respectively, compared to the control. These
observations indicated that addition of potassiomag biofertilizer mitigated the harmful effect of water stress.

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 Aust. J. Basic & Appl. Sci., 2(1): 42-52, 2008

Fig. 1: Fresh and dry wt. of mungbean plants grown under three irrigation intervals, at 30, 60 and 90
days old.

The present results agree with Schupper et al., (1998), Thomas et al., (2004), Ashraf and Ibram (2005) and
Slama et al., (2006) who stated that shoot fresh and dry weight of two leguminous plants, Phaseolus vulgaris
and Sesbania aculeata, decreased significantly due to water deficit. A reduction in vegetative growth of plants
under drought, in particular shoot growth, reduced cyclin-dependent kinase activity results in slower cell
division as well as inhibition of growth (Schupper et al., 1998).

Yield and Yield Com ponents:

Plants irrigated every five days produced a significant increase in yield and number of pods per ten plants
as compared to irrigation every two and ten days reaching 139.2% yield / plant as compared to the control.
Also addition of potassiomag produced the highest yield 165% of yield of nonfertilized control plants at the
previously mention irrigation interval (Fig. 2).
Concerning the yield component number of pods per plant, it has shown significant increase under
extension of water irrigation to five days than two and ten days and a significant rise with potassiomag than
the control. The yield component weight of 1000 seeds exhibited narrow variations between the three irrigation
water intervals. Comparable review of results were obtained by Haqqani and Pandey (1994) who showed that
water stress decreased yield, pod number and 1000 seed weight of mungbean but higher yield was obtained
by biofertilizer application. Thomas et al., (2004) reviewed that mungbean plants in the rain shelter and rainfed
treatments attained maturity earlier than the well-watered treatment. Jouany et al., (1996) found that K-
fertilization gave higher yield than the control.

Water Use Efficiency (WUE):

Fig. (3) illustrated that W UE was higher for plants under extension of irrigation to five days than that of
two and ten days. W UE values of all biofertilized treatments were higher than those of the unfertilized plants.
These results are in parallel to those of Hati et al., (2006) who showed that application of inorganic fertilizer
and farmyard manure increased seed yield of soybean and W UE 103% and 76%, respectively, over the control.
The study has also indicated that an integrated supply of nutrients through organic and inorganic sources could
be an effective practice of nutrient management for increasing W UE and yield of soybean.

Daily March of Diffusion Resistance and Transpiration Rate:

Daily march of diffusion resistance and transpiration rate together with air temperature and relative
humidity under the three irrigation intervals was investigated (Fig. 4). Plants records were taken immediately
the day after plants had been rewatered and for the three extension of irrigation treatments two, five and ten
days and recorded data was compared between the three selected treatments. Under the prevailing conditions,
the diffusion resistance showed a tendency to rise from records of rewatered plants to plants subjected to two
and five days withholding irrigation to finally plants subjected to ten days withholding irrigation. The diffusion
resistance is an expression of stomatal behavior and higher values are expression of stomatal closure.
Corresponding to higher records of diffusion resistance transpiration rate declined as it obviously appeared in
case ten days withholding irrigation.

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Increase in temperature results in acute water deficit condition in plants and dry air mass, such atmospheric
changes cause an increase in the vapour pressure gradient which enhances water loss from the soil.
Ashraf and Ibram (2005) stated that transpiration rate (E) and stomatal conductance (g s ) decreased
significantly under water deficit conditions. The first response of all plants to acute water deficit is the closure
of their stomata to prevent the transpirational water loss (Mansfield and Atkinson 1990). Closure of stomata
may result from direct evaporation of water from the guard cells (hydropassive closure) or may also be
metabolically dependent and involve processes that result in reversal of the ion fluxes that cause stomatal
opening (hydroactive closure). This process seems to be regulated by abscisic acid (ABA). Environmental
conditions that increase the rate of transpiration promote ABA accumulation and lead to reduction in stomatal
conductance (W ilkinson and Davies 2002).

Metabolites:
Photosynthetic Pigm ents:
Extension of irrigation to five days exhibited the highest values of chlorophyll a, b, a + b and carotenoid
content (mg/g fresh wt.), followed by plants irrigated every two days then by plants irrigated every ten days
(Table 1). Plants fertilized by potassiomag had the highest values of all photosynthetic pigment content than
the unfertilized plants. The present results are comparable to those reported by Ashraf (1994) and Garg et al.,
(1998).

Total Soluble Carbohydrates, Proline and Glycine Betaine (GB):

W ater stress has induced rise of total soluble carbohydrates, proline and GB (Fig.5). A similar trend was
obtained by Ashraf and Ibram (2005), Slama et al., (2006) and Ashraf and Foolad (2007) who found that
osmoprotectants such as proline and glycine betaine were increased under drought stress. Drought tolerance
of Sesbania aculeata was found to be associated with high accumulation of proline and GB in nodules.
Production of osmolytes is a general way to stabilize membranes and maintain protein conformation at low
leaf water potentials, and osmolytes play a major role in osmotic adjustment and also protect the cells by
scavenging ROS (Pinhero et al., 2001).
The concentration of soluble sugars in general increases under a stress condition (Pinheiro et al., 2001).
Recent studies have reported the accumulation of simple sugars such as glucose and fructose following an
increase in the invertase activity in the leaves of the drought challenged plants (Trouverie et al., 2003).

Abscisic Acid Content (ABA):

The effect of the water stress on ABA content in mungbean leaves under the three water intervals studied
is shown in Fig. (5). W ater stress has led to significant increase in the content of ABA. Plants treated with
potassiomag had higher ABA content than control with relatively slight variation due to extending the drying
cycle duration. Fig. 4, previously referred to clearly shows stomatal closure in reaction to water deficiency.
The role of ABA in triggering stomatal closure is rather indisputable.
Zhang and Outlaw (2001) reported that stressing Vicia faba roots could change ABA concentration at the
guard cell apoplast. Jiang and Zhang (2002) found that water stress induced ABA accumulation, triggers the
increased generation of ROS, which, in turn, leads to the up-regulation of the antioxidant defence system. Both
ABA and ROS are thought to be involved in the cellular signalling process as secondary messengers to induce
antioxidant defences under water stress (Guan et al., 2000).

Fig. 2: Yield and yield components of mungbean plants grown under three irrigation intervals.

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Fig. 3: W ater use efficiency of mungbean plants grown under three irrigation intervals.

Fig. 4: Daily march of diffusion resistance, transpiration rate, air temperature and air relative humidity of
mungbean plants grown under the three irrigation intervals.

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Table 1: Photosynthetic pigm ents of m ungbean plants grown under the three irrigation water intervals
30 days 60 days 90 days
Irrigation interval -------------------------------------------- -------------------------------------------- ---------------------------------------------
Chl. a Chl.b Chl. a+b Carot. Chl. a Chl.b Chl. a+b Carot. Chl. a Chl.b Chl. a+b Carot.
2 days Control 27.1 21.8 48.9 8.5 34.1 30.0 64.1 11.1 32.3 44.6 76.9 15.5
+ potassiom ag 29.5 25.7 55.2 15.0 42.1 38.2 80.3 13.0 49.8 41.1 90.9 16.7
5 days Control 29.3 20.9 50.2 10.7 37.1 31.7 68.8 12.3 55.3 50.6 105.9 18.6
+ potassiom ag 34.0 26.9 60.9 14.5 49.5 44.8 94.3 13.9 59.6 54.1 113.7 18.0
10 days Control 28.9 22.6 51.5 9.9 26.6 24.5 51.1 11.7 31.3 30.3 61.6 20.7
+ potassiom ag 35.8 29.2 65.0 11.1 28.3 23.9 52.2 12.4 57.1 48.3 105.2 16.1
LSD at 5% 2.85 3.47 6.52 1.76 3.44 2.98 8.46 1.32 3.52 3.34 7.58 1.69

Jiang and Zhang (2002) stated that there is interrelationship among water stress induced abscisic acid
(ABA) accumulation, the generation of reactive oxygen species (ROS), and the activities of several antioxidant
enzymes in maize leaves.
Increasing evidence indicates that one mode of ABA action may be related to its role in the oxidative
stress in plant cells. It has been documented that ABA can cause an increased generation of O 2 (Zhang et al.,
2001) and induce the expression of antioxidant genes (Kaminaka et al., 1999).
Reddy et al., (2004) showed a knowledge of sensing and signalling pathways, including ABA-mediated
changes in response to drought stress, is essential to improve crop management.

Total Isoflavonoids:
Plants irrigated every two days produced the highest amount of total isoflavonoids. Potassiomag fertilizered
plants produced slightly higher total isoflavonoids than the unfertilized plants. Reduction of isoflavonoid
content with decreased water supply by extension of irrigation water to two and ten days was also
maintained (Fig. 5).

Fig. 5: Metabolic products of mungbean plants grown under the three irrigation intervals.

There is a growing interest in phytoestrogens, particularly isoflavones due to recent findings suggesting
that these agents act as cancer-protective agents (Adrian et al., 1994) as shown in many cell and animal
models and properties often connected with cancer prevention such as antioxidant radical scavenging. Stephen
et al., (2003) showed that several legumes e.g. mungbean sprouts are a source of phytoestrogens with high
levels of estrogenic activity. Mungbean sprouts are used as green salads or in tofu burgers.

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NPK Contents:
In addition to the previously recorded progressive reduction of dry weight production of mungbean plants
due to extension of irrigation intervals compared to the values of N and K (g/100g dry wt.) were higher in
plants irrigated every ten days than in plants irrigated every five and two days. Generally N and K contents
were higher in fertilized than nonfertilized plants. W hile P content was higher in plants irrigated every five
days than plants irrigated every two and ten days (Fig. 6). Nandwal et al., (1998) stated that the highest N
and K percentage were observed in leaves of stressed mungbean plants.
Mahajan and Tuteja (2005) said that K + is one of the essential elements and is required by the plant in
large quantities. K + is required for maintaining the osmotic balance, it is an essential co-factor for many
enzymes and it has a role in opening and closing of stomata.

Fig. 6: N.P.K of mungbean plants grown under the three irrigation intervals.

Fig. 7: SDS-PAGE protein banding pattern of mungbean leaves grown under the three irrigation intervals.

Table 2: Presence (1) absence (0) of SD S-PAGE protein banding pattern of m ungbean leaves grow n under the three irrigation intervals.
Control +Potassiom ag
Band N o. M. W. ------------------------------------------- ------------------------------------------- Rf.
2d 5d 10d 2d 5d 10d
1 140.173 1 1 1 1 1 1 0.055
2 120.907 1 1 1 1 1 1 0.102
3 105.102 1 1 1 1 1 1 0.138
4 93.521 0 0 0 1 1 1 0.168
5 70.668 1 1 1 1 1 1 0.24
6 50.568 1 1 1 1 1 1 0.326
7 38.51 1 1 1 1 1 1 0.396
8 28.098 0 0 0 1 1 1 0.477
9 25.197 1 1 1 1 1 1 0.505
10 20.342 1 1 1 1 1 1 0.56
11 10.295 1 1 1 1 1 1 0.735
12 7.425 0 0 0 1 1 1 0.819
13 6.16 1 1 1 1 1 1 0.867
Total 10 10 10 13 13 13

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Total Crude Protein Content:

Plants irrigated every ten days gave the highest amount of crude protein content followed by plants
irrigated every five and two days (Fig. 5). Fertilized plants gave higher values of crude protein than
nonfertilized plants. K application enhanced the crude protein content (Nandwal et al., 1998). A similar record
was obtained by Ahmed (2004) who found that drought increased protein percentage.

SDS-PAGE Protein Analysis:

A maximum of 13 bands were detected with molecular weight ranging from 6.16 KDa to 140.17 KDa
(Fig. 7 and Table 2). All bands were detected as monomorphic bands except bands No. 4, 8 and 12 which
were recorded as polymorphic bands. All these bands did not exhibit a specific trend with drought treatments.
The polymorphic bands appeared with all fertilized plants as compared with nonfertilized plants.
Sakova et al., (1997) found no substantial differences between drought stressed and control plant patterns
for SDS-protein in Vicia sativa.

Effect of Withholding Irrigation for Ten Days on Growth and Yield:

W ithholding irrigation for ten days time was practiced once at 30, 60 or 90 days old plants. By 90 days
old plants, it was noticed that plants which were exposed to ten days drying cycle at the vegetative stage (V)
expressed the highest growth (fresh and dry wts.) and yield values than those exposed at flowering (F) or pod
setting (p) stages indicating the suppressive effect of this treatment. Plants fertilized by potassiomag showed
generally higher values than unfertilized plants for all parameters studied (Fig. 8).
Thomas et al., (2004) had studied the effect of different timing of water stress on mungbean at different
stages, which resulted in variability in biomass.
Dybing et al., (1986) and Liu et al., (2003) recorded that as a major field crop, soybean yield was highly
affected by drought stress, particularly when drought occurs during flowering and early pod development. The
yield loss caused by drought stress was mainly due to an increased rate of flower and pod abortion (Liu et
al., 2003), the early stage of pod development was characterized by active cell division in the young ovules
and was marked by rapid pod expansion and might be also related to a low level of metabolic activities within
the developing pods.

Fig. 8: Effect of timing of withholding irrigation for ten days on growth and yield.

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 Aust. J. Basic & Appl. Sci., 2(1): 42-52, 2008

Analogous observations on mungbean have been presented by Thomas et al., (2004) who recorded that
the yield was reduced by 25% when water was excluded during vegetative growth, whereas yield was reduced
by 59% when water stress was imposed at flowering stage. Also, De Costa et al., (1999) recorded that seed
yield of mungbean was significantly greater in treatments which included irrigation during the pod-filling and
flowering stages, while the treatment which received irrigation only during the vegetative stage had
significantly lower seed yield. Therefore, irrigation is critical during pod-filling and flowering stages.

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