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Citation: Ramadan, K.M.A.;
El-Beltagi, H.S.; El-Mageed, T.A.A.;
Saudy, H.S.; Al-Otaibi, H.H.;
Mahmoud, M.A.A. The Changes in
Various Physio-Biochemical
Parameters and Yield Traits of Faba
Bean Due to Humic Acid Plus
6-Benzylaminopurine Application
under Deficit Irrigation. Agronomy
2023,13, 1227. https://doi.org/
10.3390/agronomy13051227
Academic Editor: Małgorzata
Szczepanek
Received: 29 March 2023
Revised: 24 April 2023
Accepted: 25 April 2023
Published: 26 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
agronomy
Article
The Changes in Various Physio-Biochemical Parameters and
Yield Traits of Faba Bean Due to Humic Acid Plus
6-Benzylaminopurine Application under Deficit Irrigation
Khaled M. A. Ramadan 1,2 , Hossam S. El-Beltagi 3, 4, * , Taia A. Abd El-Mageed 5, Hani S. Saudy 6,* ,
Hala Hazam Al-Otaibi 7and Mohamed A. A. Mahmoud 2
1Central Laboratories, Department of Chemistry, King Faisal University, Al-Ahsa 31982, Saudi Arabia;
kramadan@kfu.edu.sa
2Department of Agricultural Biochemistry, Faculty of Agriculture, Ain Shams University, Hadayek Shobra,
Cairo 11241, Egypt; mohamed_mahmoud1@agr.asu.edu.eg
3Agricultural Biotechnology Department, College of Agriculture and Food Sciences, King Faisal University,
Al-Ahsa 31982, Saudi Arabia
4Biochemistry Department, Faculty of Agriculture, Cairo University, Gamma St., Giza 12613, Egypt
5Soil and Water Department, Faculty of Agriculture, Fayoum University, Fayoum 63514, Egypt;
taa00@fayoum.edu.eg
6Agronomy Department, Faculty of Agriculture, Ain Shams University, 68-Hadayek Shoubra,
Cairo 11241, Egypt
7Food and Nutrition Science Department, Agricultural Science and Food, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; hhalotaibi@kfu.edu.sa
*Correspondence: helbeltagi@kfu.edu.sa (H.S.E.-B.); hani_saudy@agr.asu.edu.eg (H.S.S.)
Abstract:
Implementing the deficit irrigation pattern has become a major strategy in crop production
systems. However, using less water than is required to irrigate crops is associated with changes
in plant physiology and lower productivity. Therefore, the current research aimed to assess the
integrated effect of humic acid and cytokinin on faba bean under water deficit. Under two irrigation
levels (full irrigation, FI and deficit irrigation, DI), two humic acid treatments (without addition,
H
0
and with addition of 10 kg ha
−1
, H
10
) and two cytokinin concentrations (without spray, C
0
and
spraying with 25 mg L
−1
, C
25
), faba bean growth, physiology, and productivity were evaluated. The
experiment was implemented for two winter seasons of 2019/20 and 2020/21 and performed in a
split–split plots design with three replicates. The findings revealed that under low water supply (DI),
H
10
plus C
25
was the most efficient treatment for enhancing faba bean growth. All physiological faba
bean traits estimated under DI showed remarkable increases with the application of H
10
plus C
25
in
both seasons. The increases in proline, catalase, and total soluble sugars under DI due to H
10
plus
C
25
were 31.4 and 31.8%, 51.9 and 55.1% as well as 43.8 and 46.6%, in the first and second seasons,
respectively. There was no significant difference between FI
×
H
10
plus C
25
and DI
×
H
10
plus C
25
in
phosphorus content in both seasons. FI
×
H
10
plus C
25
and DI
×
H
10
plus C
25
in the second season
produced a similar number of pods plant
−1
and seed yield of faba bean. Conclusively, the combined
application of humic plus cytokinin achieved physiological and nutrient homeostasis, adjusting the
biochemical compounds in faba bean under water deficit.
Keywords:
drought stress; faba bean yield; osmo-protectants; physiological homeostasis; seed
nutrient contents; water use efficiency; chemometrics
1. Introduction
Faba bean (Vicia faba L.), as a member of the Fabaceae family, has seeds rich in protein,
minerals, and vitamins [
1
]. Unfortunately, the yield obtained from stressed faba bean plants
had undesirable properties both in terms of quantity and quality [2–4].
Agronomy 2023,13, 1227. https://doi.org/10.3390/agronomy13051227 https://www.mdpi.com/journal/agronomy
Agronomy 2023,13, 1227 2 of 16
It is well documented that drought causes changes in plant physiology [
5
] and bio-
chemical constituents [
6
,
7
]. Furthermore, water deficit disrupts nutrient homeostasis in
plants. Thus, lower crop yield and quality are obtained under drought conditions [
8
]. Sev-
eral physiological and biochemical indices are associated with drought tolerance in plants.
Plants respond and become acclimatized to drought stress by modulating many physiolog-
ical, biochemical, and molecular aspects [
9
]. Under drought, the metabolic activity in plant
cells is influenced by the relative water content (RWC), which decreases in drought-affected
plant tissues [
10
]. Further, the membrane stability index (MSI) is a physiological indicator
for drought tolerance, since a reduction in cell membrane stability refers to reactive oxygen
species (ROS)-generated oxidation of lipid peroxidation [
11
]. Furthermore, photo-oxidation
and disintegration of chlorophyll, expressed in the chlorophyll stability index (CSI), are
features reflect strongly drought-affected plant status, correlating with crop yield [
12
]. Ad-
ditionally, drought tolerance in plants is positively correlated with maintaining a high level
of enzymatic and non-enzymatic antioxidants [
13
–
15
]. Herein, proline as a non-enzymatic
antioxidant and catalase as an enzymatic antioxidant can scavenge and/or suppress the
production of ROS in plant organelles under oxidative stresses [10,16].
Humic acid is involved in numerous organic complexes and has various active chem-
ical groups [
17
,
18
]. In these compounds, the abundance of humic acid improves the
availability of nutrients in soil and mineral uptake by plants [
19
–
22
]. Applications of humic
acid serve the plant via increasing root growth, stimulating soil microorganisms, increasing
water holding, or soil aggregation [
19
,
23
]. Consequently, humic acid-treated plants had
better root growth, hence productivity, than non-treated plants [
17
,
24
]. Further, drought
can be effectively overcome by the exogenous application of plant growth regulators to
motivate plant tolerance to various abiotic stresses [
25
]. The increases associated with the
hormonal products for plant tolerance to various stresses can be attributed to stimulating
plants’ detoxifying potential and adjusting physiological behavior [26,27].
Furthermore, growth regulators can mitigate the adverse impacts of drought by
increasing and upregulating antioxidant-based enzymes and osmo-protectants, reducing
the peroxidation of lipids [
28
,
29
]. Cytokinins, as distinctive growth regulators, have
diverse roles in plant development, involving cell growth and differentiation [
30
]. Further,
reports have alluded the significance of cytokinins as moderators of cellular readjustment
responses to drought [
31
,
32
]. Cytokinin compound application alleviated osmotic stress
by delaying leaf senescence and reducing physiological deterioration [
33
,
34
]. There is
copious evidence showing that cytokinins assist in better plant growth under osmotic stress
conditions, eventually leading to improvements in crop yield [
35
–
37
]. Despite the clear
role of humic substances and growth regulators on plant growth and development, the
interactive effect of humic acid and cytokinin on faba bean under water deficiency requires
further investigation.
In this work, we hypothesize that humic acid plus cytokinin can increase physiological
balance and improve the quantity and quality of faba bean seeds. Therefore, this study
aimed to assess the changes in growth, physiological status, biochemical compounds,
nutrient content and yield traits of faba bean due to humic acid and cytokinin interaction
under full and deficit irrigation.
2. Materials and Methods
2.1. Experimental Site Description
At a private farm in the El Fayoum region of Egypt (latitudes 29
◦
06
0
and 29
◦
35
0
N,
longitudes 30
◦
26
0
and 31
◦
05
0
E, and altitude:
−
3 m.a.s.l.), field trials were conducted over
two succeeding seasons (2019/20 and 2020/21). Additionally, the soil’s primary physio-
chemical properties were assessed in accordance with Klute and Dirksen and Page [
38
,
39
].
The soil is a loamy sand texture containing sand (75.4%), silt (12.5%), and clay (12.1%), with
a bulk density of 1.54 g cm
−3
, a pH of 7.66, an electrical conductivity of saturation extract,
ECe, of 5.24 dSm
−1
, and a cation exchange capacity of 12.3 cmol kg
−1
, as well as the follow-
ing amounts of nutrients: calcium carbonate (4.2%), organic carbon (1.06%), available N,
Agronomy 2023,13, 1227 3 of 16
(57.2 mg kg
−1
soil), available P (4.4 mg kg
−1
soil), available K (52.1 mg kg
−1
soil) and
available Zn (0.78 mg kg−1soil). The experimental site was located in an arid region with
moderate winters and rare precipitation.
2.2. Agronomic Management and Treatments
Faba bean healthy seeds (Vicia faba. L., cultivar Sakha 1) were sown on October 15
and 20 in 2019 and 2020 and harvested on April 21 and 27 in 2020 and 2021, respectively.
Treatments involved the combination of irrigation levels, humic acid, and cytokinin spray.
Two irrigation levels based on crop evapotranspiration (Etc), full irrigation (FI, 100% of
Etc), and deficit irrigation (DI, 80% of Etc) were applied. There were two rates of humic
acid (without H
0
and with the application of 10 kg ha
−1
, H
10
) as well as foliar spray
with synthetic cytokinin, 6-benzylaminopurine (without spraying, plants were sprayed
with distilled water C
0
, and spraying with 25 mg L
−1
, C
25
). Plants were treated with cy-
tokinin twice at 30 and 45 days after sowing. Humic acid was added once during planting
and it was mixed well with the appropriate amount of sand (~200 kg), and then evenly
distributed over the top layer of the soil and mixed in the rhizosphere zone. Irrigation
levels were allocated in main plots, while humic acid was distributed in the sub-plots.
Finally, the cytokinin levels were decreased in the sub-sub-plots. A total of nine treatments
were replicated three times via a randomized complete split–split plot block design, re-
sulting in a total of 24 experimental plots. The experimental plots were 12.8 m
2
in size
(0.8
×
16 m), with two planting rows; the rows were 1 m in width, with 15 cm spacing be-
tween each plant. A drip irrigation system was utilized, and 2 drip lines were placed 30 cm
apart in every elementary test plot. Irrigation treatments began after the full germination
stage. Phosphorus (P) and potassium (K) fertilizers were added at planting at a rate of
75 kg P ha
−1
in the form of calcium superphosphate (15.5% P
2
O
5
) and 120 kg K ha
−1
in
the form of potassium sulfate (48% K
2
O), respectively. Nitrogen (N) fertilizer was added
once as a starter dose at planting at a rate of 48 kg N ha
−1
in the form of ammonium nitrate
(33.5% N).
2.3. Irrigation Water Applied
According to the FAO Penman–Monteith equation, the daily reference evapotranspira-
tion (Eto) was calculated using the following formula [39]:
Etc =Eto ×Kc (1)
where Etc is the crop water requirement (mm d−1) and Kc is the crop coefficient.
The irrigation water applied (IWA) per bed was calculated according to the following
equation:
IWA =
Etc ×A×Ii
Ea ×1000 (2)
where IWA is the irrigation water applied (m
3
), A is the plot area (m
2
), Ii is the irrigation
period (day), and Ea is the irrigation efficiency (%).
2.4. Measurements
2.4.1. Water Status and Photosynthetic Capacity
At 75 days after sowing, the relative water content (RWC%) and the membrane
stability index (MSI%) were assessed [
40
,
41
]. To assess the photosynthetic efficiency,
the performance index, and chlorophyll fluorescence were determined according to
Clark et al. [
42
] and Maxwell and Johnson [
43
] by Handy PEA, Hansatech Instruments
(Ltd., Kings Lynn, London, UK). Additionally, leaf greenness (SPAD value) was determined
using a chlorophyll meter (SPAD502, KONICAMINOLTA. Inc., Tokyo, Japan).
Agronomy 2023,13, 1227 4 of 16
2.4.2. Free Proline Content, Total Soluble Sugars and Enzyme
The free proline content and total soluble sugars (TSS) (mg g
−1
FW) of fresh faba bean
leaves were extracted and quantified utilizing procedures described previously [
44
,
45
].
Plant cells were extracted following the technique of Bradford [
46
] for use as a crude
enzyme extract to measure CAT content. The CAT activity (EC 1.11.1.6) was established
using the approach published by Aebi (Burgdorf, Switzerland) [47].
2.4.3. Growth Traits
At the end of the growing season, ten plants were randomly obtained from every
experimental plot and assessed for their growth characteristics. Plant height was recorded
as well as the number of leaves and branches plant
−1
. Total leaf area plant
−1
, was measured
using a digital plan meter, Planix 7 (Sokkia Co., Ltd. Atsugi, Kanagawa, Japan). Shoot dry
weight plant−1was recorded after oven-drying at 70 ◦C until constant weight.
2.4.4. Leaf Mineral Contents
To assess the contents of N, P, and K, faba bean leaves were dried and grounded to
form a powder. The digestion process was performed for the dried samples with a mixture
consisting of HClO
4
and H
2
SO
4
(at 1:3 v/v, respectively). N content was assessed using
micro-Kjeldahl equipment (Ningbo Medical Instruments Co., Ningbo, China [
48
]. Molyb-
denum blue, diluted H
2
MoO
7
S, and 8% (w/v) NaHSO
3
-H
2
SO
4
were used as standard
reagents for quantifying P [
49
]. K contents were measured using a Perkin-Elmer Model
52-A Flame Photometer (Waltham, MA, USA) Jackson [50].
2.4.5. Yield and Yield Components
At harvesting stage, 10 plants were randomly selected from each plot and utilized to
determine yield components, i.e., the number of pods per plant and 100-seed weight. Seeds
of all plants per plot were utilized to determine seed yield (t ha−1).
2.4.6. Water Use Efficiency
According to Fernández et al. [
51
], water use efficiency (WUE) was computed using
the formula given below:
WUE =
Seed yield kg ha−1
Water applied m3ha−1(3)
2.5. Statistical Analysis
Data were statistically evaluated following Gomez and Gomez [
52
] with analysis of
variance procedures in the GenStat statistical package (version 11) (VSN International Ltd.,
Oxford, UK). Data for each growing season were subjected to two-way analysis of variance
(ANOVA). The Duncan multiple range test, at a 0.05 probability level, was utilized to com-
pare treatment means. Further, data preparation for chemometric analysis was according to
Mahmoud et al. and Mahmoud and Magdy [
53
,
54
]. Agglomerative hierarchical clustering
(AHC) and principal component analysis (PCA) were used in XLSTAT 2022
®
(Addinsoft,
Paris, France).
3. Results
3.1. Growth Response
Growth of faba bean significantly responded to the combinations of humic acid
and cytokinin in the 2019/20 and 2020/21 seasons (Table 1). The maximum values for
all growth traits were more pronounced with FI
×
H
10
plus C
25
, statistically equal in
FI
×
H
10
plus C
0
and FI
×
H
0
plus C
25
as well as DI
×
H
10
plus C
25
and DI
×
H
0
plus C
25
in number of branches plant
−1
in the first season. Under FI, the combinations of H
10
plus
C
25
(for all traits), H
10
plus C
0
(for number of branches plant
−1
and dry matter plant
−1
)
Agronomy 2023,13, 1227 5 of 16
and H
0
plus C
25
(for leaf area and dry matter plant
−1
) exhibited the maximum values in
the second season. Furthermore, under DI, H
10
plus C
25
was the most efficient treatment
for enhancing faba bean growth, significantly similar to H
10
plus C
0
in all growth traits,
in both seasons, except plant height and leaf area in the first season. Compared to the
counterpart treatment (DI
×
H
0
plus C
0
H
0
), the DI
×
H
10
plus C
25
treatment increased
plant height (by 22.3 and 23.5%), number of leaves plant
−1
(by 21.2 and 12.8%), number of
branches plant
−r
(by 50.0 and 35.1%), leaf area (by 22.2 and 23.6%) and dry matter plant
−n
(by 22.4 and 23.5%) in the first and second seasons, respectively.
Table 1.
Faba bean growth as influenced by humic acid plus cytokinin treatments under irrigation
regimes in the 2019/2020 and 2020/2021 seasons.
Season Irrigation
Regime Treatments Plant Height
(cm)
Number of
Leaves Plant−1
Number of
Branches Plant−1
Leaf Area
(dm2)
Dry Matter
Plant−1(g)
2019/2020
FI
H0C087.1 ±0.88 d97.0 ±1.16 d5.56 ±0.11 b189.7 ±0.94 g49.7 ±0.81 f
C25 90.7 ±0.33 c101.3 ±0.88 c5.89 ±0.22 ab 233.3 ±1.3 b56.9 ±1.1 bc
H10 C095.7 ±0.33 b106.6 ±0.37 b6.33 ±0.33 a227.6 ±0.79 c58.0 ±0.20 b
C25 103.0 ±0.58 a114.9 ±0.64 a6.00 ±0.11 ab 245.0 ±1.37 a62.5 ±0.35 a
DI
H0C075.2 ±0.67 e73.7 ±2.6 g4.00 ±0.00 c178.6 ±1.58 h45.61 ±0.40 g
C25 86.6 ±0.67 d83.3 ±1.8 f6.00 ±0.33 ab 205.5 ±1.58 f52.3 ±0.40 e
H10 C090.3 ±0.33 c87.6 ±0.32 e5.67 ±0.32 b214.4 ±0.79 e54.8 ±0.20 d
C25 92.0 ±0.58 c89.3 ±0.56 e6.00 ±0.33 ab 218.3 ±1.37 d55.8±0.35 cd
2020/2021
FI
H0C084.5 ±2.02 de 90.3 ±3.38 d4.3 ±0.33 c184.1 ±2.65 ef 48.2 ±0.92 d
C25 94.7 ±4.33 bc 93.8 ±0.87 c4.6 ±0.32 bc243.3 ±10.3
ab 59.5 ±3.5 ab
H10 C096.7 ±0.33 b108.8 ±0.32 b5.6 ±0.33 a
230.0
±
0.97
bc
58.6
±
0.20
abc
C25 104.0 ±0.53 a116.9 ±0.56 a6.0 ±0.31 a247.4 ±1.37 a63.1 ±0.5 a
DI
H0C073.7 ±0.88 f78.3 ±0.33 e3.7 ±0.34 d174.8 ±2.1 f44.7 ±0.53 d
C25 80.3 ±2.7 e83.5 ±6.7 de 4.3 ±0.33 c190.7 ±6.3 e48.7 ±1.6 d
H10 C089.3 ±0.33 cd 86.7 ±0.37 cde 4.6 ±0.29 bc 212.0 ±0.97 d54.2 ±0.20 c
C25 91.0 ±0.58 bc 88.3 ±0.64 cd 5.0 ±0.33 b216.0 ±1.4 cd 55.2 ±0.35 bc
Each value indicates the mean
±
standard error (n= 3). Mean values in each column followed by the same
lower-case letter in each column are not significantly different according to the Duncan test (p
≤
0.05). FI, full
irrigation; DI, deficit irrigation (80% of crop evapotranspiration); H
0
and H
10
: without and with the application of
10 kg ha−1of humic acid, respectively. C0and C25 : without and with 25 mg L−1of cytokinin, respectively.
3.2. Physiological Response
The physiological changes in faba bean due to humic acid plus cytokinin under
irrigation regimes are presented in Table 2. Under FI or DI, H
10
plus C
25
or C
0
in both
seasons, in addition to FI
×
H
0
plus C
25
(for Fv/Fm in the second season), resulted
in the maximum increases in SPAD and Fv/Fm (except DI
×
H
10
plus C
0
for SPAD in
the first season). Moreover, FI
×
H
10
plus C
25
was the effective practice for improving
the performance index, the relative water content, and the membrane stability index,
significantly similar to H
10
plus C
0
for the relative water content in both seasons. It
should be noted that all physiological parameters of faba bean measured under DI showed
distinctive improvements with the application of H
10
plus C
25
in both seasons. Herein,
under DI, H
10
plus C
25
increased SPAD, Fv/Fm, the performance index, the relative water
content and the membrane stability index by approximately 1.50 and 1.50, 1.10 and 1.10, 3.76
and 3.32, 1.18 and 1.17 and 1.32 and 1.6 fold, in the first and second seasons, respectively,
compared to H0plus C0.
3.3. Biochemical Compounds
Humic plus cytokinin had a significant effect on proline (Figure 1), catalase (Figure 2),
and total soluble sugars (Figure 3) in both seasons of 2019/20 and 2020/21. FI ×H10 plus
C
25
resulted in the maximum value of proline, surpassing that of FI
×
H
0
plus C
0
by 47.9
and 48.4% in the first and second seasons, respectively. Moreover, H
10
plus C
25
resulted
Agronomy 2023,13, 1227 6 of 16
in the highest values of catalase and total soluble sugars whether with FI or DI in both
seasons, except catalase under DI in the second season. The increases in proline, catalase,
and total soluble sugars under DI due to H
10
plus C
25
amounted to 31.4 and 31.8%, 51.9
and 55.1%, as well as 43.8 and 46.6%, in 2019/20 and 2020/21, respectively.
Table 2.
Physiological response of faba bean as influenced by humic acid plus cytokinin treatments
under irrigation regimes in the 2019/2020 and 2020/2021 seasons.
Season Irrigation
Regime Treatments SPAD Fv/Fm Performance
Index
Relative Water
Content
Membrane
Stability Index
2019/2020
FI
H0C044.6 ±0.80 b0.79 ±0.01 c3.1 ±0.02 f78.34 ±1.7 c39.0 ±1.3 d
C25 46.0 ±1.4 b0.82 ±0.00 b5.0 ±0.12 de 80.7 ±0.66 bc 55.1 ±1.6 b
H10 C050.7 ±0.60 a0.83 ±0.03 ab 6.2 ±0.87 d82.8 ±0.34 ab 54.7 ±1.8 b
C25 51.5 ±0.23 a0.84 ±0.01 a12.2 ±0.33 a84.1 ±0.55 a61.9 ±1.6 a
DI
H0C033.8 ±1.1 d0.76 ±0.01 d2.6 ±0.06 f67.1 ±0.43 e34.0 ±1.1 f
C25 40.0 ±0.573 c0.78 ±0.00 c4.8 ±0.48 e74.8 ±1.2 d37.7 ±0.54 e
H10 C046.2 ±0.53 b0.83 ±0.01 ab 8.2 ±1.2 c75.5 ±0.79 d41.3 ±0.88 d
C25 50.8 ±0.33 a0.84 ±0.01 a9.8 ±0.78 b79.3 ±0.54 c45.0 ±0.58 c
2020/2021
FI
H0C043.4 ±0.40 d0.80 ±0.01 b2.9 ±0.06 f79.6 ±1.6 c36.0 ±0.89 f
C25 45.9 ±1.3 cd 0.82 ±0.01 ab 4.9 ±0.50 e81.6 ±1.3 bc 57.1 ±1.1 b
H10 C050.5 ±0.81 ab 0.82 ±0.01 ab 5.9 ±0.33 de83.7 ±0.79 ab 56.7 ±0.88 b
C25 52.1 ±0.57 a0.83 ±0.01 a12.8 ±0.58 a85.1±1.5 a63.9 ±0.58 a
DI
H0C033.3 ±1.3 e0.76 ±0.01 d2.8 ±0.20 f68.2 ±1.4 e29.3 ±0.33 g
C25 48.5 ±1.2 bc 0.79 ±0.01 c6.3 ±0.80 cd 74.7 ±1.2 d39.7 ±0.33 e
H10 C051.6 ±1.1 a0.84 ±0.01 a7.6 ±0.25 c77.3 ±1.7 d43.3 ±0.86 d
C25 50.1 ±0.55 ab 0.84 ±0.01 a9.3 ±0.82 b80.0 ±0.54 c47.0 ±1.1 c
Each value indicates the mean
±
standard error (n= 3). Mean values in each column followed by the same
lower-case letter in each column are not significantly different according to the Duncan test (p
≤
0.05). FI, full
irrigation; DI, deficit irrigation (80% of crop evapotranspiration); H
0
and H
10
: without and with the application of
10 kg ha−1of humic acid, respectively. C0and C25 : without and with 25 mg L−1of cytokinin, respectively.
Agronomy 2023, 13, x FOR PEER REVIEW 6 of 16
Season Irrigation
Regime Treatments SPAD Fv/Fm Performance
Index
Relative Water
Content
Membrane Sta-
bility Index
2019/2020
FI
H
0
C
0
44.6 ± 0.80
b
0.79 ± 0.01
c
3.1 ± 0.02
f
78.34 ± 1.7
c
39.0 ± 1.3
d
C
25
46.0 ± 1.4
b
0.82 ± 0.00
b
5.0 ± 0.12
de
80.7 ± 0.66
bc
55.1 ± 1.6
b
H
10
C
0
50.7 ± 0.60
a
0.83 ± 0.03
ab
6.2 ± 0.87
d
82.8 ± 0.34
ab
54.7 ± 1.8
b
C
25
51.5 ± 0.23
a
0.84 ± 0.01
a
12.2 ± 0.33
a
84.1 ± 0.55
a
61.9 ± 1.6
a
DI
H
0
C
0
33.8 ± 1.1
d
0.76 ± 0.01
d
2.6 ± 0.06
f
67.1 ± 0.43
e
34.0 ± 1.1
f
C
25
40.0 ± 0.573
c
0.78 ± 0.00
c
4.8 ± 0.48
e
74.8 ± 1.2
d
37.7 ±0.54
e
H
10
C
0
46.2 ± 0.53
b
0.83 ± 0.01
ab
8.2 ± 1.2
c
75.5 ± 0.79
d
41.3 ± 0.88
d
C
25
50.8 ± 0.33
a
0.84 ± 0.01
a
9.8 ± 0.78
b
79.3 ± 0.54
c
45.0 ± 0.58
c
2020/2021
FI
H
0
C
0
43.4 ± 0.40
d
0.80 ± 0.01
b
2.9 ± 0.06
f
79.6 ± 1.6
c
36.0 ± 0.89
f
C
25
45.9 ± 1.3
cd
0.82 ± 0.01
ab
4.9 ± 0.50
e
81.6 ± 1.3
bc
57.1 ± 1.1
b
H
10
C
0
50.5 ± 0.81
ab
0.82 ± 0.01
ab
5.9 ± 0.33 d
e
83.7 ± 0.79
ab
56.7 ± 0.88
b
C
25
52.1 ± 0.57
a
0.83 ± 0.01
a
12.8 ± 0.58
a
85.1± 1.5
a
63.9 ± 0.58
a
DI
H
0
C
0
33.3 ± 1.3
e
0.76 ± 0.01
d
2.8 ± 0.20
f
68.2 ± 1.4
e
29.3 ± 0.33
g
C
25
48.5 ± 1.2
bc
0.79 ± 0.01
c
6.3 ± 0.80
cd
74.7 ± 1.2
d
39.7 ± 0.33
e
H
10
C
0
51.6 ± 1.1
a
0.84 ± 0.01
a
7.6 ± 0.25
c
77.3 ± 1.7
d
43.3 ± 0.86
d
C
25
50.1 ± 0.55
ab
0.84 ± 0.01
a
9.3 ± 0.82
b
80.0 ± 0.54
c
47.0 ± 1.1
c
Each value indicates the mean ± standard error (n = 3). Mean values in each column followed by the
same lower-case leer in each column are not significantly different according to the Duncan test (p
≤ 0.05). FI, full irrigation; DI, deficit irrigation (80% of crop evapotranspiration); H
0
and H
10
: without
and with the application of 10 kg ha
−1
of humic acid, respectively. C
0
and C
25
: without and with 25
mg L
−1
of cytokinin, respectively.
3.3. Biochemical Compounds
Humic plus cytokinin had a significant effect on proline (Figure 1), catalase (Figure
2), and total soluble sugars (Figure 3) in both seasons of 2019/20 and 2020/21. FI × H
10
plus
C
25
resulted in the maximum value of proline, surpassing that of FI × H
0
plus C
0
by 47.9
and 48.4% in the first and second seasons, respectively. Moreover, H
10
plus C
25
resulted in
the highest values of catalase and total soluble sugars whether with FI or DI in both sea-
sons, except catalase under DI in the second season. The increases in proline, catalase, and
total soluble sugars under DI due to H
10
plus C
25
amounted to 31.4 and 31.8%, 51.9 and
55.1%, as well as 43.8 and 46.6%, in 2019/20 and 2020/21, respectively.
Figure 1. Proline content of faba bean as influenced by humic acid plus cytokinin treatments under
irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the mean ± standard
error (n = 3). Mean values in each bar followed by the same leer are not significantly different
according to the Duncan test (p ≤ 0.05). FI, full irrigation; DI, deficit in irrigation (80% of crop
Figure 1.
Proline content of faba bean as influenced by humic acid plus cytokinin treatments under
irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the mean
±
standard
error (n= 3). Mean values in each bar followed by the same letter are not significantly different
according to the Duncan test (p
≤
0.05). FI, full irrigation; DI, deficit in irrigation (80% of crop
evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid,
respectively. C0and C25: without and with 25 mg L−1of cytokinin, respectively.
Agronomy 2023,13, 1227 7 of 16
Agronomy 2023, 13, x FOR PEER REVIEW 7 of 16
evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid, re-
spectively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
Figure 2. Total soluble sugars (TSS) content of faba bean as influenced by humic acid plus cytokinin
treatments under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the
mean ± standard error (n = 3). Mean values in each bar followed by the same leer are not signifi-
cantly different according to the Duncan test (p ≤ 0.05). FI, full irrigation; DI, deficit in irrigation
(80% of crop evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of
humic acid, respectively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
Figure 3. Catalase (CAT) activity of faba bean as influenced by humic acid plus cytokinin treatments
under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the mean ± stand-
ard error (n = 3). Mean values in each bar followed by the same leer are not significantly different
according to the Duncan test (p ≤ 0.05). FI, full irrigation; DI, deficit in irrigation (80% of crop evap-
otranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid, respec-
tively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
3.4. Nutrient Contents
As shown in Table 3, the nutrient content of faba bean markedly changed based on
the combinations of humic acid and cytokinin in the 2019/20 and 2020/21 seasons. In this
respect, the application of FI × H
10
plus C
25
resulted in the highest values of nitrogen, phos-
phorus and potassium in both seasons. However, the difference between FI × H
10
plus C
25
and DF × H
10
plus C
25
in terms of phosphorus content was not significant in both seasons.
Figure 2.
Total soluble sugars (TSS) content of faba bean as influenced by humic acid plus cytokinin
treatments under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates
the mean
±
standard error (n= 3). Mean values in each bar followed by the same letter are not
significantly different according to the Duncan test (p
≤
0.05). FI, full irrigation; DI, deficit in irrigation
(80% of crop evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of
humic acid, respectively. C0and C25: without and with 25 mg L−1of cytokinin, respectively.
Agronomy 2023, 13, x FOR PEER REVIEW 7 of 16
evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid, re-
spectively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
Figure 2. Total soluble sugars (TSS) content of faba bean as influenced by humic acid plus cytokinin
treatments under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the
mean ± standard error (n = 3). Mean values in each bar followed by the same leer are not signifi-
cantly different according to the Duncan test (p ≤ 0.05). FI, full irrigation; DI, deficit in irrigation
(80% of crop evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of
humic acid, respectively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
Figure 3. Catalase (CAT) activity of faba bean as influenced by humic acid plus cytokinin treatments
under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the mean ± stand-
ard error (n = 3). Mean values in each bar followed by the same leer are not significantly different
according to the Duncan test (p ≤ 0.05). FI, full irrigation; DI, deficit in irrigation (80% of crop evap-
otranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid, respec-
tively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
3.4. Nutrient Contents
As shown in Table 3, the nutrient content of faba bean markedly changed based on
the combinations of humic acid and cytokinin in the 2019/20 and 2020/21 seasons. In this
respect, the application of FI × H
10
plus C
25
resulted in the highest values of nitrogen, phos-
phorus and potassium in both seasons. However, the difference between FI × H
10
plus C
25
and DF × H
10
plus C
25
in terms of phosphorus content was not significant in both seasons.
Figure 3.
Catalase (CAT) activity of faba bean as influenced by humic acid plus cytokinin treatments
under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the mean
±
standard
error (n= 3). Mean values in each bar followed by the same letter are not significantly different
according to the Duncan test (p
≤
0.05). FI, full irrigation; DI, deficit in irrigation (80% of crop
evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid,
respectively. C0and C25: without and with 25 mg L−1of cytokinin, respectively.
3.4. Nutrient Contents
As shown in Table 3, the nutrient content of faba bean markedly changed based on
the combinations of humic acid and cytokinin in the 2019/20 and 2020/21 seasons. In this
respect, the application of FI
×
H
10
plus C
25
resulted in the highest values of nitrogen,
Agronomy 2023,13, 1227 8 of 16
phosphorus and potassium in both seasons. However, the difference between FI
×
H
10
plus C
25
and DF
×
H
10
plus C
25
in terms of phosphorus content was not significant in both
seasons. Compared to their counterpart control treatments, the increases in phosphorus
content due to H
10
plus C
25
application under FI and DI was 1.52 and 2.24 fold in the first
season and 1.46 and 2.17 fold in the second season, respectively.
Table 3.
Leaf nutrient contents of faba bean as influenced by humic acid plus cytokinin treatments
under irrigation regimes in the 2019/2020 and 2020/2021 seasons.
Season Irrigation
Regime Treatments Nitrogen % Phosphorus % Potassium %
2019/2020
FI
H0C016.59 ±0.30 d3.87 ±0.10 e14.47 ±0.46 c
C25 19.52 ±0.24 b5.19 ±0.16 bc 17.74 ±0.50 b
H10 C018.36 ±0.25 c4.87 ±0.06 cd 15.79 ±0.45 c
C25 21.49 ±0.24 a5.87 ±0.15 a20.15 ±0.51 a
DI
H0C09.82 ±0.59 g2.50 ±0.29 g12.46 ±0.92 d
C25 15.15 ±0.53 e4.60 ±0.31 d15.67 ±0.33 c
H10 C011.92 ±0.59 f3.28 ±0.28 f14.12 ±0.60 cd
C25 17.49 ±0.52 cd 5.60 ±0.30 ab 17.76 ±0.33 b
2020/2021
FI
H0C017.69 ±0.30 c4.11 ±0.06 d15.56 ±0.45 de
C25 20.51 ±0.23 b5.51±0.15 ab 18.83 ±0.51 bc
H10 C019.66 ±0.20 b5.01 ±0.10 bc 17.09 ±0.46 cd
C25 21.88 ±0.20 a6.03 ±0.16 a21.45 ±0.50 a
DI
H0C010.81 ±0.58 f2.67 ±0.28 f13.96 ±0.91 e
C25 16.14 ±0.52 d4.73 ±0.30 c17.17 ±0.30 cd
H10 C012.81 ±0.58 e3.43 ±0.30 e15.77 ±0.60 de
C25 18.38 ±0.53 c5.80 ±0.30 a19.41 ±0.30 b
Each value indicates the mean
±
standard error (n= 3). Mean values in each column followed by the same
lower-case letter in each column are not significantly different according to the Duncan test (p
≤
0.05). FI, full
irrigation; DI, deficit irrigation (80% of crop evapotranspiration); H
0
and H
10
: without and with the application of
10 kg ha−1of humic acid, respectively. C0and C25 : without and with 25 mg L−1of cytokinin, respectively.
3.5. Yield Traits and Water Use Efficiency
The number of pods plant
−1
, the weight of 100 seeds and the seed yield of faba bean
showed significant changes in response to humic acid and cytokinin applications in the
2019/20 and 2020/21 seasons (Table 4). The most effective practice for increasing all yield
traits in both seasons was the application of H
10
plus C
25
under FI. In the first season,
FI
×
H
0
plus C
25
showed similar values for the number of pods plant
−1
and DI
×
H
10
plus C
25
showed similar values for seed yield to that of FI
×
H
10
plus C
25
. FI
×
H
10
plus C
25
and DI
×
H
10
plus C
25
produced a similar number of pods plant
−1
and seed
yield in the second season. It must be pointed out that the application of H
10
plus C
25
under DI achieved resulted in increases of 30.8 and 46.7% in the number of pods plant
−1
,
19.8 and 17.2% in the weight of 100 seeds and 20.1 and 23.1% in the seed yield compared to
H
0
plus C
0
in the first and second seasons, respectively. Concerning water use efficiency
(WUE), Figure 4shows that the addition of H
10
either with C
0
or C
25
under DI resulted
in the maximum values of WUE in both seasons, surpassing that of the other treatments.
FI
×
H
0
plus C
0
was the least effective practice, resulting in the lowest values of WUE in
both seasons.
Agronomy 2023,13, 1227 9 of 16
Table 4.
Yield parameters of faba bean as influenced by humic acid plus cytokinin treatments under
irrigation regimes in the 2019/2020 and 2020/2021 seasons.
Season Irrigation
Regime Treatments Number of
Pods Plant−1
Weight of 100
Seeds (g)
Seed Yield
(t ha−1)
2019/2020
FI
H0C012.3 ±0.67 de 80.7 ±0.47 c4.10 ±0.08 ef
C25 17.6 ±0.89 ab 87.6 ±2.50 b4.40 ±0.03 cd
H10 C015.7 ±0.67 bc 89.7 ±0.82 b4.53 ±0.04 bc
C25 18.3 ±0.67 a94.6 ±0.42 a4.78 ±0.02 a
DI
H0C012.0 ±0.51 e73.7 ±0.64 d3.98 ±0.02 f
C25 14.9 ±0.48 c79.0 ±0.60 c4.27 ±0.03 de
H10 C014.3 ±0.33 cd 86.7 ±0.74 b4.53 ±0.04 bc
C25 15.7 ±0.67 bc 88.3 ±2.33 b4.78 ±0.13 a
2020/2021
FI
H0C013.7 ±1.33 cd 82.0 ±0.50 d4.05 ±0.13 d
C25 17.0 ±0.67 ab 90.6 ±2.70 b4.33 ±0.07 b
H10 C017.3 ±0.58 ab 87.0 ±0.50 bc 4.57 ±0.09 ab
C25 19.7 ±0.88 a96.8 ±0.77 a4.67 ±0.07 a
DI
H0C012.0 ±1.00 d76.2 ±0.61 e3.76 ±0.03 e
C25 15.0 ±1.00 bc 84.3 ±1.67 cd 4.13 ±0.03 cd
H10 C016.6 ±0.35 b87.0 ±0.58 bc 4.57 ±0.07 ab
C25 17.6 ±0.74 ab 89.3 ±0.33 b4.63 ±0.09 a
Each value indicates the mean
±
standard error (n= 3). Mean values in each column followed by the same
lower-case letter in each column are not significantly different according to the Duncan test (p
≤
0.05). FI, full
irrigation; DI, deficit irrigation (80% of crop evapotranspiration); H
0
and H
10
: without and with the application of
10 kg ha−1of humic acid, respectively. C0and C25 : without and with 25 mg L−1of cytokinin, respectively.
Agronomy 2023, 13, x FOR PEER REVIEW 9 of 16
Table 4. Yield parameters of faba bean as influenced by humic acid plus cytokinin treatments under
irrigation regimes in the 2019/2020 and 2020/2021 seasons.
Season Irrigation
Regime Treatments Number of Pods
Plant
−1
Weight of 100
Seeds (g)
Seed Yield
(t ha
−1
)
2019/2020
FI
H
0
C
0
12.3 ± 0.67
de
80.7 ± 0.47
c
4.10 ± 0.08
ef
C
25
17.6 ± 0.89
ab
87.6 ± 2.50
b
4.40 ± 0.03
cd
H
10
C
0
15.7 ± 0.67
bc
89.7 ± 0.82
b
4.53 ± 0.04
bc
C
25
18.3 ± 0.67
a
94.6 ± 0.42
a
4.78 ± 0.02
a
DI
H
0
C
0
12.0 ± 0.51
e
73.7 ± 0.64
d
3.98 ± 0.02
f
C
25
14.9 ± 0.48
c
79.0 ± 0.60
c
4.27 ± 0.03
de
H
10
C
0
14.3 ± 0.33
cd
86.7 ± 0.74
b
4.53 ± 0.04
bc
C
25
15.7 ± 0.67
bc
88.3 ± 2.33
b
4.78 ± 0.13
a
2020/2021
FI
H
0
C
0
13.7 ± 1.33
cd
82.0 ± 0.50
d
4.05 ± 0.13
d
C
25
17.0 ± 0.67
ab
90.6 ± 2.70
b
4.33 ± 0.07
b
H
10
C
0
17.3 ± 0.58
ab
87.0 ± 0.50
bc
4.57 ± 0.09
ab
C
25
19.7 ± 0.88
a
96.8 ± 0.77
a
4.67 ± 0.07
a
DI
H
0
C
0
12.0 ± 1.00
d
76.2 ± 0.61
e
3.76 ± 0.03
e
C
25
15.0 ± 1.00
bc
84.3 ± 1.67
cd
4.13 ± 0.03
cd
H
10
C
0
16.6 ± 0.35
b
87.0 ± 0.58
bc
4.57 ± 0.07
ab
C
25
17.6 ± 0.74
ab
89.3 ± 0.33
b
4.63 ± 0.09
a
Each value indicates the mean ± standard error (n = 3). Mean values in each column followed by the
same lower-case leer in each column are not significantly different according to the Duncan test (p
≤ 0.05). FI, full irrigation; DI, deficit irrigation (80% of crop evapotranspiration); H
0
and H
10
: without
and with the application of 10 kg ha
−1
of humic acid, respectively. C
0
and C
25
: without and with 25
mg L
−1
of cytokinin, respectively.
Figure 4. Wat er u se efficiency (WUE) of faba bean as influenced by humic acid plus cytokinin treat-
ments under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates the mean ±
standard error (n = 3). Mean values in each bar followed by the same leer are not significantly
different according to the Duncan test (p ≤ 0.05). FI, full irrigation; DI, deficit in irrigation (80% of
crop evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of humic acid,
respectively. C
0
and C
25
: without and with 25 mg L
−1
of cytokinin, respectively.
3.6. Chemometric Methods
Agglomerative hierarchical clustering (AHC) and principal component analysis
(PCA) were utilized to present a collective understanding of the obtained data. With AHC
(Figure 5), samples were clustered into two groups based on their dissimilarities. The first
Figure 4.
Water use efficiency (WUE) of faba bean as influenced by humic acid plus cytokinin
treatments under irrigation regimes in the 2019/20 and 2020/21 seasons. Each value indicates
the mean
±
standard error (n= 3). Mean values in each bar followed by the same letter are not
significantly different according to the Duncan test (p
≤
0.05). FI, full irrigation; DI, deficit in irrigation
(80% of crop evapotranspiration); H
0
and H
10
: without and with the application of 10 kg ha
−1
of
humic acid, respectively. C0and C25: without and with 25 mg L−1of cytokinin, respectively.
3.6. Chemometric Methods
Agglomerative hierarchical clustering (AHC) and principal component analysis (PCA)
were utilized to present a collective understanding of the obtained data. With AHC
(Figure 5), samples were clustered into two groups based on their dissimilarities. The first
cluster contained the untreated FI and DI samples together with DI
×
H
0
plus C
25
. The rest
of the samples were grouped into the second cluster.
Agronomy 2023,13, 1227 10 of 16
Agronomy 2023, 13, x FOR PEER REVIEW 10 of 16
cluster contained the untreated FI and DI samples together with DI × H
0
plus C
25
. The rest
of the samples were grouped into the second cluster.
Figure 5. ACH clustering of the DI and FI samples.
According to the PCA biplot (Figure 6), two principal components can explain 87.65%
of the variation (76.1% and 11.5 aributed to F1 and F2, respectively). Thereby, F1 can
differentiate between the two irrigation systems regardless of their treatment method, ex-
cept for DI × H
0
plus C
0
in the second season grouped with the FI samples. On the other
hand, F2 can differentiate between the untreated and treated samples with humic acid or
cytokinin regardless of their irrigation system, except for DI × H
0
plus C
25
in both seasons.
Further, two major regions can be observed, marked in light purple and light green, indi-
cating the samples that were clustered in the AHC test.
Figure 6. PCA biplot of the DI and FI samples.
It was clear that the DI samples treated with H
10
plus C
0
or H
10
plus C
25
in both seasons
were correlated with the performance index, seed yield, WUE, TSS, Fv/Fm, SPAD, proline,
and CAT as they were placed in the same quadrant (+F1/+F2). On the other hand, the FI
samples treated with humic acid and/or cytokinin were correlated with the leaves, pods,
Figure 5. ACH clustering of the DI and FI samples.
According to the PCA biplot (Figure 6), two principal components can explain 87.65%
of the variation (76.1% and 11.5 attributed to F1 and F2, respectively). Thereby, F1 can
differentiate between the two irrigation systems regardless of their treatment method,
except for DI
×
H
0
plus C
0
in the second season grouped with the FI samples. On the
other hand, F2 can differentiate between the untreated and treated samples with humic
acid or cytokinin regardless of their irrigation system, except for DI
×
H
0
plus C
25
in both
seasons. Further, two major regions can be observed, marked in light purple and light
green, indicating the samples that were clustered in the AHC test.
Agronomy 2023, 13, x FOR PEER REVIEW 10 of 16
cluster contained the untreated FI and DI samples together with DI × H
0
plus C
25
. The rest
of the samples were grouped into the second cluster.
Figure 5. ACH clustering of the DI and FI samples.
According to the PCA biplot (Figure 6), two principal components can explain 87.65%
of the variation (76.1% and 11.5 aributed to F1 and F2, respectively). Thereby, F1 can
differentiate between the two irrigation systems regardless of their treatment method, ex-
cept for DI × H
0
plus C
0
in the second season grouped with the FI samples. On the other
hand, F2 can differentiate between the untreated and treated samples with humic acid or
cytokinin regardless of their irrigation system, except for DI × H
0
plus C
25
in both seasons.
Further, two major regions can be observed, marked in light purple and light green, indi-
cating the samples that were clustered in the AHC test.
Figure 6. PCA biplot of the DI and FI samples.
It was clear that the DI samples treated with H
10
plus C
0
or H
10
plus C
25
in both seasons
were correlated with the performance index, seed yield, WUE, TSS, Fv/Fm, SPAD, proline,
and CAT as they were placed in the same quadrant (+F1/+F2). On the other hand, the FI
samples treated with humic acid and/or cytokinin were correlated with the leaves, pods,
Figure 6. PCA biplot of the DI and FI samples.
Agronomy 2023,13, 1227 11 of 16
It was clear that the DI samples treated with H
10
plus C
0
or H
10
plus C
25
in both
seasons were correlated with the performance index, seed yield, WUE, TSS, Fv/Fm, SPAD,
proline, and CAT as they were placed in the same quadrant (+F1/+F2). On the other hand,
the FI samples treated with humic acid and/or cytokinin were correlated with the leaves,
pods, and number of branches plant
−1
, leaf area, the weight of 100 seeds, plant height, dry
matter, the membrane stability index, and the relative water content as they were placed in
the same quadrant (+F1/−F2).
4. Discussion
Plants exposed to water deficit exhibited changes in physio-biochemical status [
55
]
and nutrient content [
56
], hence a reduction in growth and yield potential [
57
]. How-
ever, combined applications of humic acid and cytokinin mitigated the negative effects of
drought, as evidenced in this research through the improvements in growth and physiology
of faba bean. Plants under drought stress commonly close the stomata to reduce loss of
water via transpiration [
58
]. However, stomatal closure led to a reduction in CO
2
inflow. In
contrast, well-watered plants ensured CO
2
delivery through the stomatal apparatus [
58
].
Environmental stresses, specifically drought, adversely influenced plant pigments [
59
,
60
],
particularly chlorophyll b [
61
]. Drought caused a decline in chlorophyll pigments and
also accounted for reduced photosynthesis [
62
]. As a plant response to drought stress,
plants develop a degree of drought tolerance through modulating gene functions that
increase antioxidant defensive actions while reducing plant growth [
63
]. The reducing in
soil moisture under drought stress adversely affected plant pigments and photosynthetic
reactions, causing significant declines in crop growth and yield [
64
,
65
]. Accordingly, our
findings revealed that supplying faba bean with low water (DI) without the exogenous
application of humic acid plus cytokinin caused a reduction in SPAD, Fv/Fm, the relative
water content, and the membrane stability index, hence reducing growth and yield.
To counteract the harms of drought, several actions should be adopted. In this respect,
the defensive mechanisms of plants need to be exogenously equipped through specific
compound applications. Amending the nutritional status of agricultural lands in favor of
plant growth is crucial to increasing crop productivity [
3
]. In this context, soil structure,
microorganism growth, plant growth and yield attributes were increased with humic acid
application [
66
]. The uptake of several macro- and micronutrients was increased with
humic acid supply [
67
,
68
]. By accelerating the rate of nutrient uptake, humic acid resulted
in increases in plant growth, chlorophyll and protein content [
69
] and the photosynthetic
rate [
70
]. Since humic acid increases micro- and macro-elements, activates enzyme, protein,
sugar and vitamin synthesis, and alters the permeability of cell membranes [
71
–
73
], in
addition to its high chelating potential [
72
], it increases crop yield. Accordingly, humic
materials had a significant impact on plant growth and productivity under both normal
and stress conditions [17,18].
The exogenous application of cytokinins during stress resulted in improvements in
the membrane and chlorophyll stability indices, photosynthetic pigments, leaf relative
water and soluble sugar content [
74
,
75
]. A range of processes related to plant growth and
development, i.e., cell division, nutrient mobilization, tissue differentiation, the production
of anthocyanin and retarding senescence, are influenced by cytokinins [
30
,
76
]. Additionally,
cytokinins are significant to nitrogen and sulfur complements [
77
], causing inhibition
of nitrate and sulphate uptake by plant roots [
78
,
79
]. Cytokinin increased the activity
of superoxide dismutase, ascorbate peroxidase and catalase as well as ROS scavenging,
and protected the cell membrane under abiotic stress [
75
]. The high concentration of
cytokinin during osmotic stress resulted in several benefits such as a reduction in abscisic
acid effects [
80
–
85
], a change in nutrient balance [
86
], and improvement in photosynthetic
efficiency [
87
,
88
], hence leaf senescence was delayed [
89
]. The antagonistic work between
abscisic acid and cytokinin led to the dominance of cytokinin, mediating the adverse effect
of drought while regulating the developmental mechanisms in plants [
90
,
91
]. Recently, it
has been documented that cytokinin is effective in alleviating stress through maintaining ion
Agronomy 2023,13, 1227 12 of 16
balance [
92
]. Furthermore, at a molecular level, cytokinin improves photosynthesis under
drought by adjusting the activity of proteins related to stomatal conductance, chlorophyll
content and activation of rubisco [93].
Accordingly, our research work has provided insight into favorable changes by humic
acid plus cytokinin for plants under drought. In this regard, providing drought-stressed
faba bean plants with humic acid plus cytokinin maintained the appropriate water status,
cell membrane stability, photosynthetic pigments and capacity (SPAD and Fv/Fm), as well
as inducing osmo-protectants, particularly proline, catalase activity and nutrient absorption,
as shown in Figure 7. Thus, humic acid–cytokinin-treated plants showed higher growth
and yield in addition to nutrient content than non-treated plants.
Agronomy 2023, 13, x FOR PEER REVIEW 12 of 16
Accordingly, our research work has provided insight into favorable changes by hu-
mic acid plus cytokinin for plants under drought. In this regard, providing drought-
stressed faba bean plants with humic acid plus cytokinin maintained the appropriate wa-
ter status, cell membrane stability, photosynthetic pigments and capacity (SPAD and
Fv/Fm), as well as inducing osmo-protectants, particularly proline, catalase activity and
nutrient absorption, as shown in Figure 7. Thus, humic acid–cytokinin-treated plants
showed higher growth and yield in addition to nutrient content than non-treated plants.
Figure 7. Illustration of the changes in physio-biochemical parameters in faba bean plants under
deficit irrigation (DI) due to application of humic acid plus cytokinin for enhancing drought toler-
ance.
5. Conclusions
The use of a water-deficit strategy in crop irrigation, especially in arid and semi-arid
regions, is a dire need for rationalizing the use of agricultural water. However, reduced
water supply is associated with drought damages, which affect crop yield and quality.
This research proved the complementary role of humic acid and cytokinin in ameliorating
drought impacts by conserving water, nutrient balance and the photosynthesis apparatus
of faba bean. Thus, the application of humic acid (10 kg ha
−1
) and cytokinin (25 mg L
−1
) is
advisable for faba bean production, especially under drought stress conditions.
Author Contributions: Conceptualization, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and
M.A.A.M.; methodology, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; soft-
ware, T.A.A.E.-M., H.S.S. and M.A.A.M.; validation, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S.,
H.H.A.-O. and M.A.A.M.; formal analysis, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and
M.A.A.M.; investigation, K.M.A.R., H.S.E.-B. and H.S.S.; resources, T.A.A.E.-M., H.S.S. and H.H.A.-
O.; data curation, T.A.A.E.-M., H.S.S. and M.A.A.M.; writing—original draft preparation K.M.A.R.,
H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; writing—review and editing, K.M.A.R.,
H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; visualization, K.M.A.R., H.S.E.-B.,
T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; supervision, K.M.A.R., H.S.E.-B. and H.S.S.; project
administration, K.M.A.R., H.S.E.-B. and H.S.S.; funding acquisition, K.M.A.R., H.S.E.-B. and
H.H.A.-O. All authors have read and agreed to the published version of the manuscript.
Figure 7.
Illustration of the changes in physio-biochemical parameters in faba bean plants under
deficit irrigation (DI) due to application of humic acid plus cytokinin for enhancing drought tolerance.
5. Conclusions
The use of a water-deficit strategy in crop irrigation, especially in arid and semi-arid
regions, is a dire need for rationalizing the use of agricultural water. However, reduced
water supply is associated with drought damages, which affect crop yield and quality.
This research proved the complementary role of humic acid and cytokinin in ameliorating
drought impacts by conserving water, nutrient balance and the photosynthesis apparatus
of faba bean. Thus, the application of humic acid (10 kg ha
−1
) and cytokinin (25 mg L
−1
) is
advisable for faba bean production, especially under drought stress conditions.
Author Contributions:
Conceptualization, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and
M.A.A.M.; methodology, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; software,
T.A.A.E.-M., H.S.S. and M.A.A.M.; validation, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O.
and M.A.A.M.; formal analysis, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.;
investigation, K.M.A.R., H.S.E.-B. and H.S.S.; resources, T.A.A.E.-M., H.S.S. and H.H.A.-O.; data
curation, T.A.A.E.-M., H.S.S. and M.A.A.M.; writing—original draft preparation K.M.A.R., H.S.E.-B.,
T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; writing—review and editing, K.M.A.R., H.S.E.-B.,
T.A.A.E.-M., H.S.S., H.H.A.-O. and M.A.A.M.; visualization, K.M.A.R., H.S.E.-B., T.A.A.E.-M., H.S.S.,
H.H.A.-O. and M.A.A.M.; supervision, K.M.A.R., H.S.E.-B. and H.S.S.; project administration,
K.M.A.R., H.S.E.-B. and H.S.S.; funding acquisition, K.M.A.R., H.S.E.-B. and H.H.A.-O. All authors
have read and agreed to the published version of the manuscript.
Agronomy 2023,13, 1227 13 of 16
Funding:
This research work was supported and funded by the Deputyship for Research and
Innovation, Ministry of Education in Saudi Arabia (Project number INSTR001).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are available within this manuscript.
Acknowledgments:
The authors extend their appreciation to the Deputyship for Research and
Innovation, Ministry of Education in Saudi Arabia for funding this research.
Conflicts of Interest: The authors declare no conflict of interest.
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