Abeed et al.

BMC Plant Biology (2022) 22:559

https://doi.org/10.1186/s12870-022-03935-9

RESEARCH Open Access

Impact of sewage water irrigation on Datura

innoxia grown in sandy loam soil
Amany H. A. Abeed1*, Mohammed Ali2, Mamdouh A. Eissa3 and Suzan A. Tammam1

Abstract
Background: A potential solution for recycling and reusing the massively produced sewage water (SW) is to irrigate
certain plants instead of highly cost recycling treatment. Although the extensive and irrational application of SW may
cause environmental pollution thus, continual monitoring of the redox status of the receiver plant and the feedback
on its growth under application becomes an emergent instance. The impact of SW, along with well water (WW) irriga-
tion of medicinal plant, Datura innoxia, was monitored by some physio-biochemical indices.
Results: The SW application amplified the growth, yield, minerals uptake, and quality of D. innoxia plants compared
to the WW irrigated plants. The total chlorophyll, carotenoid, non-enzymatic antioxidants, viz. anthocyanin, flavonoids,
phenolic compounds, and total alkaloids increased by 85, 38, 81, 50, 19, and 37%, respectively, above WW irrigated
plants. The experiment terminated in enhanced leaf content of N, P, and K by 43, 118, and 48%, respectively. Moreover,
stimulation of carbon and nitrogen metabolites in terms of proteins, soluble sugars, nitrate reductase (NR) activity,
and nitric oxide (NO) content showed significant earliness in flowering time. The SW application improved not only
Datura plants’ quality but also soil quality. After four weeks of irrigation, the WW irrigated plants encountered nutrient
deficiency-induced stress evidenced by the high level of proline, ­H2O2, and MDA as well as high enzyme capabilities.
Application of SW for irrigation of D. innoxia plant showed the improvement of secondary metabolites regulating
enzyme phenylalanine ammonia-lyase (PAL), restored proline content, and cell redox status reflecting high optimal
condition for efficient cellular metabolism and performance along the experiment duration.
Conclusions: These evidences approved the benefits of practicing SW to improve the yield and quality of D. innoxia
and the feasibility of generalization on multipurpose plants grown in poor soil.
Keywords: Alkaloids, Datura innoxia, nitrate reductase, Phenylalanine ammonia lyase, Proline, Sewage water

Background elevation in water demand from other sectors, including

In 1966, resources of renewable water were 2189 ­m3/ industrial and municipal water supply. SW represents a
capita/year, which will minimize to 500 ­ m3/capita/ continuous disposable source of water [3, 4]. Vast efflu-
year by 2025 [1], obligatory, it requires us to rational- ent quantities are produced as a consequence of large-
ize consumption. The agriculture prerequisites surpass scale industrialization and urbanization [5]. At present,
80 % of the total water demand [2]. The development of Egypt generates an estimated 5.5–6.5 billion cubic meter
Egypt’s economy intensely relies on its capacity to man- (BCM) of SW per year, accounted as 2.5% of water
age and preserve its water resources to face the predicted resources in Egypt [5]. Of that amount, only 0.7 BCM
annually is utilized in agriculture, chiefly in direct as well
*Correspondence: dramany2015@aun.edu.eg
as indirect reuse in desert areas via mixing with the water
1
of agricultural drainage [6]. Since 1980, this practice has
Department of Botany and Microbiology, Faculty of Science, Assiut
University, Assiut 71516, Egypt increased as tremendous potential relevance to Egypt.
Full list of author information is available at the end of the article SW is primarily rich in organic matter (OM), macro- and

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Abeed et al. BMC Plant Biology (2022) 22:559 Page 2 of 15

micronutrients, dissolved minerals, and irrigation with in medicine, where its leaves contain tropane alka-
SW consequently increases soil fertility and nutrient loids with pharmacological activities and significant
content [7, 8]. However, the over-application of SW may medicinal properties such as atropine, scopolamine,
cause chemical pollution problems, especially for edible and hyoscyamine that are used as parasympathicolitics
crop plants, due to accumulation of heavy metals (HMs) as having the ability to suppress the parasympathetic
[8] and increase sodicity as well as soil salinity [9]. More- nerve activity [13, 14]. Plants grow more luxuriously
over, when SW is continually utilized as a sole source of when levels of N fertilizer increase in the medium and
irrigation water, toxic chemicals and excessive nutrients alkaloid synthesis extent paralleled plant overall growth
could be applied to plants, which would impose undesir- [15]. Some previous studies indicated that SW irriga-
able and toxic effects on plant productivity and quality. tion can enhance plant growth, yield, and quality while
Despite all the aforementioned issues, SW reuse is neces- increasing nutrient availability [4, 9, 16]. This practice
sary to design future water policies. This makes it man- became popular in countries that faced water scarcity
datory to critically evaluate the effects of this practice on especially Egypt. However, the extensive and irrational
agriculture, human health, and the environment [7]. use of SW for irrigation practices imposes significant
The Egyptian government is rising significant concerns disadvantages including plant growth and performance
about human health from SW-irrigated crops; thus, ined- disorders due to the highly loaded HMs per unit of time
ible plants (grasses and forest trees) have been replaced and/or the ionic stress. Hence, the impact of sewage
compared to crops grown in control, where SW was water irrigation should be monitored keeping in view
not previously applied [5]. Using SW in the agriculture for how long SW application can be effective and safely
sector might solve the expected energy crisis [10]. Oil- operational. Some physio-biochemical indices can be
accumulating plants (sunflower, corn, olive, and cas- monitored being a mirror of the plant performance
tor) and carbohydrate-accumulating plants (wheat, rice, efficacy under SW irrigation. The current study aimed
corn, sugarcane, and potatoes) that might be cultivated to address the impact of SW practiced on Datura via
for hydrogen and methane production, or bioethanol or following up some growth parameters in terms of plant
biodiesel production, trees grown for greenery at hotels dry weight (DW), branches, and a number of flowers,
and touristic villages, nursery plant, fiber crops, indus- besides pigments content, secondary metabolites con-
trial oil crops, wood trees, fodder/ feed crops, trees for tent, nutritional profile, and redox status in terms of
city’s green belts and roads or high ways afforestation, all stress markers and membrane damage traits, as well as
seem to be most profitable alternatives [5]. The private malondialdehyde (MDA) as a lipid peroxidation marker,
sector and land reclamation are cultivating the afore- ­H2O2, and its quenching enzymes. Moreover, the study
mentioned plants in several country sites, particularly in of antioxidant response in addition to metabolites
the south, which are deliberately constructed to convert in the inedible commercially valuable plant, Datura
low-quality land (e.g., sand) and low-quality water (e.g., innoxia, may collectively help donating new reference
sewage) into valuable resources [10]. In Egypt, many information for underlining the possibility of practic-
areas currently lack the high capability for agriculture ing SW and the feasibility of generalization on large-
due to poor nutritional availability and high calcium car- scale plants. Thus, plant growth assessments and the
bonate content [11]. Therefore, it is necessary to explore characterization of enzymatic as well as non-enzymatic
different available practices, assess their potentiality for antioxidant responses were parallel conducted. This
viable agriculture, and plan appropriate sustainable land research also investigated the changes in soil physico-
use. Accordingly, SW application has a double privilege, chemical parameters as a secondary objective.
where it is a costless alternative irrigation water source
and achieves environmentally SW disposal or recycling
management as well as improving the quality of the low- Experimental results
grade soil with poor input of OM by increasing soil nitro- Physical and Chemical Characteristics of Sewage Water
gen, phosphorus, and potassium. Data shown in Table 1 revealed that pH and EC con-
Recently, the growth of medicinal plants such as centration in SW were higher (7.4 and 0.80 ds m ­ − 1,
Datura received high awareness nationally [12]. Many respectively) than that of WW (7.2 and 0.51 ds ­m− 1,
species of Datura were cultivated for secondary metab- respectively). Moreover, almost all measured nutrients
olites production. Datura innoxia, a perennial plant determined in the SW samples collected from the Arab
with inedible parts, is a commercially important plant Elmadabegh sewage line were higher than the corre-
for having a broad range of heavy market demand sponding criteria of the WW (tap water). However, the
for bioactive metabolites due to their extensive use micronutrients and HMs in the SW herein are relatively
low and within the limits of standard concentrations.
Abeed et al. BMC Plant Biology (2022) 22:559 Page 3 of 15

Table 1 Properties of well water and sewage water used in Table 2 Physico-chemical characteristics of soil used in the
irrigation study
Properties Well water Sewage water Soil properties Value

pH 7.20 ± 0.43 7.41 ± 0.22 Sand (g/kg) 76 ± 0.57

EC (dS/m) 0.51 ± 0.04 0.80 ± 0.05 Clay (g/kg) 14 ± 0.31
Na (mg/L) 0.66 ± 0.05 1.22 ± 0.07 Silt (g/kg) 10 ± 0.22
K (mg/L) 0.21 ± 0.01 0.54 ± 0.03 Texture class Sandy loam
Mg (mg/L) 0.69 ± 0.01 1.81 ± 0.08 pH 8.02 ± 0.08
Ca (mg/L) 2.76 ± 0.07 5.1 ± 0.10 EC (dS/m) 1.85 ± 0.02
CO3 (mg/L) 0.4 ± 0.01 0.9 ± 0.02 Organic carbon (g/kg) 15 ± 0.12
HCO3 (mg/L) 2.2 ± 0.03 4.9 ± 0.11 Total nitrogen (mg/kg) 500 ± 0.86
Cl (mg/L) 1.61 ± 0.02 4.2 ± 0.06 Organic matter (g/kg) 10.9 ± 0.21
Fe (mg/L) 18 ± 0.92 83 ± 0.83 Total phosphorus (P) (mg/kg) 293 ± 3.21
Zn (mg/L) 2 ± 0.09 2 ± 0.10 Na (mg/L) 1.69 ± 0.05
Cu (mg/L) 1.2 ± 0.07 2.4 ± 0.11 K (mg/L) 0.56 ± 0.01
Cd (mg/L) nd 0.07 ± 0.002 Mg (mg/L) 0.62 ± 0.02
Pb (mg/L) 0.9 ± 0.01 1.5 ± 0.07 Ca (mg/L) 3.11 ± 0.11
Mn (mg/L) 16 ± 0.66 22 ± 0.77 CO3 (mg/L) 5.59 ± 0.15
V Volume, EC Electrical conductivity, dS/m decisiemens per metre, mg Milligram, HCO3 (mg/L) 7.34 ± 0.21
g Gram, kg Kilogram, L Liter, nd Not detected. Each value is an average of 4 Cl (mg/L) 3.03 ± 0.22
replicates
Available Fe (mg/kg) 3.85 ± 0.20
Available Zn (mg/kg) 1.96 ± 0.08
Physical and Chemical Characteristics of the Soil Available Cu (mg/kg) 0.75 ± 0.013
Physico-chemical characteristics of the soil were inter- Available Mn (mg/kg) 1.93 ± 0.06
preted in Table 2. The results showed that the used soil Available Cd (mg/kg) Nd
was slightly alkaline (pH 8.02) with sandy loam textural Available Pb (mg/kg) Nd
grade having little concentration of OM (11 g/kg) and V Volume, EC Electrical conductivity, dS/m decisiemens per metre, g Gram, kg
poor in nitrogen (260 mg/kg) and phosphorus content Kilogram, nd Not detected
(2.5 mg/kg).
Data depicted in Table 3 revealed that the soil pH Visibly, the application of SW irrigation had a poten-
reduced with SW irrigation, reaching a value of 6.9. tial impact on Datura innoxia’s morphological feature in
Whereas, the soil concentrations of OM, organic car- terms of branching and flowering criterion. Early branch-
bon (OC), N, and P recorded higher values by SW irri- ing and flowering resulted within the 2nd and 4th week,
gation of 5.9, 11.3, 0.33, and 0.28 g/Kg, respectively. respectively, submitted the highest number of branches
Obviously, for Fe, Zn, Cu, and Mn, SW treatments (4 branches/plant) and flowers (7 flowers/plant) by the
increased soil micronutrients as well as macronutri- end of the experiment. In contrast, the WW irrigated
ents. However, the HMs (Cd and Pb) were not affected plants exhibited branch initiation at 5th week and floral
by SW irrigation. initiation at 7th week with final relatively low branch and
flower number (2 branches and two flowers/plant).
SW application positively influenced the photosyn-
Growth parameters and physio‑biochemical alternations thetic pigment synthesis in terms of the total chlorophyll
of Datura innoxia Plant Datura innoxia Dry Weight, and carotenoids over the corresponding values recorded
Photosynthetic Pigments, No. of Flowers and Lateral in WW irrigated plants (Table 4) on the 10th week, with
Branches, Primary Metabolites and Proline Content Under a percent increase of 102 and 38% for total chlorophyll
Sewage Water Irrigation and carotenoids, respectively.
The obtained results in Table 4 revealed that, by the end Unlike the chlorophyll fluctuation, proline showed
of the experiment duration, the SW application signifi- heterogeneous activities, and it was kept the baseline of
cantly (p < 0.0001) increased plant biomass in terms of control and restored by SW application along the study
DW with a percent increase amounted to 163%, while duration while it was promoted considerably by the end
plants irrigated with WW increased by only 26% when of the experiment with WW irrigation with a percent
compared to corresponding starter values (1.33 and 1.35, increase of 433% over the corresponding starter value
respectively). (1.5 mg/g DW). The displayed data in Table 4 showed
Abeed et al. BMC Plant Biology (2022) 22:559 Page 4 of 15

Table 3 Chemical characteristics of soil used in the study at the their content kept low fluctuation and diminished at the
end of experiment end of the experiment under WW application with per-
Properties Irrigated by well water Irrigated cent increase of only 54 and 120%, respectively.
by sewage
water
Datura innoxia Secondary Metabolites: Anthocyanins,
pH 7.90 ± 0.12 6.90 ± 0.11 Flavonoids, Phenolics and Alkaloids and Activity of Their
EC (dS/m) 0.31 ± 0.01 3.50 ± 0.10 Mediated Enzyme, Phenylalanine ammonia lyase, Under
Organic matter (g/kg) 9.80 ± 0.15 13 ± 0.34 Sewage Water Irrigation
Organic carbon (g/kg) 11 ± 0.21 16.33 ± 0.44 SW irrigation noticeably increased secondary metabo-
Total nitrogen (N) (mg/Kg) 240 ± 0.76 433 ± 0.88 lites in terms of anthocyanins, flavonoids, phenolic
Total phosphorus (P) (mg/kg) 220 ± 0.77 311 ± 0.98 compounds, and alkaloids recording final values with
Na (mg/L) 0.56 ± 0.02 11.22 ± 0.42 a percent increase of 81, 50, 19, and 37%, respectively,
K (mg/L) 0.21 ± 0.007 5.14 ± 0.12 when compared to the corresponding values of WW
Mg (mg/L) 0.52 ± 0.01 8.41 ± 0.14 irrigated plants (Table 4). This augmentation in second-
Ca (mg/L) 2.06 ± 0.11 14.10 ± 0.43 ary metabolites accumulation significantly (P < 0.0001)
CO3 (mg/L) 0.35 ± 0.01 2.90 ± 0.11 increased over time. Data depicted in Table 4 demon-
HCO3 (mg/L) 1.60 ± 0.10 7.90 ± 0.15 strated that the interaction between water irrigations and
Cl (mg/L) 1.61 ± 0.10 11.21 ± 0.46 time had a substantial impact (P < 0.0001) on secondary
Fe (mg/L) 18 ± 0.66 70 ± 0.55 metabolites content.
Zn (mg/L) 2 ± 0.11 12 ± 0.40 The secondary metabolites-mediating enzyme, PAL,
Cu (mg/L) 1.20 ± 0.09 1.81 ± 0.09 reached the maximal (122 μmol/mg protein/min) on
Cd (mg/L) nd Nd the 10th week with a percent increase of 110% over the
Pb (mg/L) 0.09 ± 0.002 0.13 ± 0.02 starter value (Fig. 4d), whereas the maxima under WW
Mn (mg/L) 9 ± 0.22 18 ± 0. 43 irrigation were on the 6th week recording value of only
V Volume, EC Electrical conductivity, dS/m decisiemens per metre, mg Milligram, 81 μmol/mg protein/min which tended to decrease to
g Gram, kg Kilogram, L Liter, nd Not detected. Each value is an average of 4 reach a value of 70 μmol/mg protein/min.
replicates

Datura innoxia Stress Markers and Membrane Damage

that the interaction between water application and time Criteria Under Sewage Water Irrigation
significantly impacted DW, branch and flower number, Some more indicative physio-biochemical parameters,
total chlorophyll, and proline content of Datura plants such as hydrogen peroxide ­(H2O2, a stress marker) and
(P < 0.0001). membrane damage were evaluated as the levels of lipid
The data presented in Figs. (1a, b, c) denoted that SW peroxidation (malondialdehyde), as well as variations
irrigation influenced the primary metabolites of Datura in enzymatic antioxidant capacities of Datura innoxia
plants. Amino acids, protein, and soluble sugars content leaves, were monitored over a period of ten weeks for
were accumulated by the irrigation of SW and reached insight evaluation of the performance of Datura plants
the maximum values in plant leaves after the 5th week of under different irrigations. Data in Fig. (3 a-b) revealed
irrigation with percent increases amounting to 32, 105, that irrigation of SW did not affect hydrogen peroxide
and 59%, respectively, over the corresponding values ­(H2O2.). Conversely, MDA concentration was reduced
recorded for WW irrigated plants and maintained the considerably submitted the lowest values (7.04 μmol/g
highest values up to the end of the experiment; thereby, FW) at the 6th and the 7th week.
improving final plant growth.
Datura innoxia Specific Antioxidant Enzymes Activity Under
Datura innoxia Nitric Oxide (NO) and Nitrate Reductase (NR) Sewage Water Irrigation
Activity Under Sewage Water Irrigation The enhanced related scavenger enzymes viz. After four
NO content of the leaves of SW-irrigated plants was weeks of irrigation, CAT, APX, and GST were concomi-
elevated across the time of application with a maximum tant with inducing cellular reactive oxygen species under
percent increase of 166% over the corresponding starter WW application (Fig. 4a, b, c). The contrast was observed
value (50 nmol/g FW) (Fig. 2a). Likewise, NR enzyme for SW irrigated plants where constant ­H2O2 and MDA
activity (Fig. 2b) was maximized by SW irrigation to be content were kept around the control values as well as
maximally recorded with percent increase of 470 over not affected by the screened activities of CAT and APX.
the corresponding starter value (23 μmol ­NO2 g/h) while However, GST had a divergent trend. It was activated in
Table 4 Measurements of different growth parameters of Datura innoxia plant under two treatments: well water (WW) or sewage water (SW) for 10 weeks. Each value represents
a mean value of four replicates ±SE. The observations were recorded on 2 randomly selected plants on plot mean basis analysis per replication per treatment for each correspond
week
Water source Weeks DW BN FN TC Car An Fl Ph Al Pro

SW SV 1.33 ± 0.05 – – 1.22 ± 0.05 1.83 ± 0.02 0.43 ± 0.03 51.00 ± 1.47 9.50 ± 0.21 0.90 ± 0.01 1.71 ± 0.05
Abeed et al. BMC Plant Biology

1st 1.57 ± 0.01 – – 1.72 ± 0.02 1.89 ± 0.04 0.50 ± 0.02 58.00 ± 0.71 10.00 ± 0.71 1.31 ± 0.04 3.25 ± 0.25
2nd 1.86 ± 0.02 – – 1.98 ± 0.04 1.97 ± 0.02 0.56 ± 0.01 66.00 ± 1.47 14.00 ± 0.41 1.77 ± 0.03 3.50 ± 0.29
3rd 1.89 ± 0.04 – – 2.20 ± 0.11 1.99 ± 0.03 0.70 ± 0.01 71.00 ± 0.91 18.00 ± 0.41 1.98 ± 0.05 4.25 ± 0.25
4th 2.11 ± 0.08 1.00 ± 0.25 2.00 ± 0.25 2.50 ± 0.18 2.60 ± 0.13 0.78 ± 0.00 77.00 ± 0.91 23.00 ± 0.41 2.50 ± 0.20 2.75 ± 0.25
5th 2.65 ± 0.05 2.00 ± 0.25 3.00 ± 0.25 2.89 ± 0.11 2.65 ± 0.05 0.82 ± 0.02 80.00 ± 0.91 29.00 ± 0.91 2.87 ± 0.07 3.00 ± 0.41
(2022) 22:559

6th 2.77 ± 0.10 2.00 ± 0.48 3.00 ± 0.25 3.03 ± 0.02 2.78 ± 0.10 0.89 ± 0.02 87.00 ± 0.58 34.00 ± 0.41 3.33 ± 0.14 1.70 ± 0.10
7th 2.89 ± 0.03 3.00 ± 0.25 4.00 ± 0.25 3.40 ± 0.14 2.89 ± 0.35 0.94 ± 0.02 92.00 ± 1.29 39.00 ± 0.71 3.72 ± 0.08 2.00 ± 0.41
8th 3.05 ± 0.05 3.00 ± 0.25 4.00 ± 0.25 3.63 ± 0.09 3.44 ± 0.11 0.97 ± 0.06 96.00 ± 1.08 44.00 ± 0.41 4.00 ± 0.15 1.63 ± 0.14
9th 3.30 ± 0.15 4.00 ± 0.25 5.00 ± 0.41 3.71 ± 0.10 3.63 ± 0.09 1.11 ± 0.06 112.00 ± 1.22 53.00 ± 1.08 4.51 ± 0.16 1.50 ± 0.02
10th 3.50 ± 0.18 4.00 ± 0.25 7.00 ± 0.41 3.72 ± 0.13 4.55 ± 0.21 1.50 ± 0.02 123.00 ± 1.22 57.00 ± 1.47 5.93 ± 0.17 1.70 ± 0.04
WW SV 1.35 ± 0.05 – – 1.24 ± 0.04 1.81 ± 0.04 0.44 ± 0.02 51.50 ± 1.76 9.00 ± 0.41 0.90 ± 0.01 1.54 ± 0.04
1st 1.45 ± 0.06 – – 1.51 ± 0.02 1.84 ± 0.05 0.47 ± 0.01 52.50 ± 1.76 10.00 ± 0.41 0.96 ± 0.02 1.54 ± 0.04
2nd 1.61 ± 0.06 – – 1.70 ± 0.04 1.93 ± 0.02 0.49 ± 0.01 52.25 ± 1.03 10.00 ± 0.71 1.11 ± 0.03 1.83 ± 0.05
3rd 1.64 ± 0.03 – – 1.75 ± 0.02 2.19 ± 0.12 0.51 ± 0.02 57.00 ± 0.82 11.00 ± 0.41 1.63 ± 0.14 2.04 ± 0.05
4th 1.69 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 1.79 ± 0.02 2.24 ± 0.20 0.56 ± 0.01 62.50 ± 1.76 19.25 ± 0.48 1.88 ± 0.03 3.25 ± 0.48
5th 1.72 ± 0.03 1.00 ± 0.25 0.00 ± 0.00 1.83 ± 0.02 2.73 ± 0.11 0.59 ± 0.03 68.50 ± 0.65 20.00 ± 0.82 1.99 ± 0.07 4.25 ± 0.25
6th 1.75 ± 0.02 2.00 ± 0.29 0.00 ± 0.00 1.92 ± 0.02 2.81 ± 0.16 0.60 ± 0.02 67.50 ± 0.65 26.25 ± 0.85 2.21 ± 0.23 6.00 ± 0.41
7th 1.71 ± 0.02 2.00 ± 0.00 2.00 ± 0.25 1.90 ± 0.04 2.88 ± 0.3 0.64 ± 0.01 66.25 ± 1.55 32.50 ± 1.04 2.21 ± 0.24 6.00 ± 0.41
8th 1.71 ± 0.02 2.00 ± 0.25 2.00 ± 0.29 1.83 ± 0.03 2.97 ± 0.18 0.77 ± 0.01 70.00 ± 0.41 36.25 ± 1.03 3.43 ± 0.10 7.00 ± 0.41
9th 1.70 ± 0.03 2.00 ± 0.25 2.00 ± 0.41 1.80 ± 0.04 3.03 ± 0.33 0.79 ± 0.01 77.75 ± 1.60 41.50 ± 1.19 3.83 ± 0.05 6.25 ± 0.48
10th 1.70 ± 0.04 2.00 ± 0.25 3.00 ± 0.25 1.83 ± 0.05 3.33 ± 0.15 0.83 ± 0.02 82.25 ± 1.60 48.25 ± 0.63 4.30 ± 0.18 8.00 ± 0.71
L.S.D.(0.05) 0.13 0.41 0.43 0.15 0.33 0.06 2.45 1.51 0.25 0.63
P Value < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0032 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001
Source df Mean Square
DW BN FN TC Car An Fl Ph Al Pro
Weeks 10 1.393** 12.632** 21.257** 2.147** 3.741** 0.375** 2064.309** 1881.096** 13.933** 6.964**
Water Sources 1 14.378** 15.557** 70.920** 21.721** 1.078** 1.166** 7640.909** 804.045** 12.768** 78.001**
Weeks x Water Sources 10 0.805** 1.832** 5.170** 1.018** 0.342** 0.066** 298.109** 25.895** 0.487** 19.836**
Error 66 0.017 0.170 0.186 0.023 0.111 0.002 6.000 2.296 0.060 0.394
SV Starter value, DW Dry weight (g/plant), BN Branch number (branches/plant), FN Flowers/plant), TC Total chlorophyll (mg/g FW), Car Carotenoids (mg/g FW), An Anthocyanin (μmol/g FW), FL Flavonoids (mg/g FW), Ph
Phenolics (mg/g FW), Al Alkaloids (mg/g FW), Pro Proline (mg/g FW)
Page 5 of 15
Abeed et al. BMC Plant Biology (2022) 22:559 Page 6 of 15

Fig. 2 Datura innoxia leaves content of nitric oxide, NO (a) and

nitrate reductase activity, NR (b) as influenced by duration of well and
sewage water irrigation (weeks). The data are averages of 4 replicates
± SE

Mn) in Datura leaves manifested in SW irrigated plants

compared to WW irrigated ones (Table 5), with a per-
cent increase, accounted by 43, 118, 48, 26, and 34% for
nitrate, phosphate, K, Mg, and Ca, respectively, and 100,
71, 144 and 27% for Na, Fe, Zn, and Mn, respectively,
when compared to WW irrigated plants.

Discussion
Water resource scarcity is a vital problem in several
localities in Egypt; thus, recycling and the reuse of alter-
Fig. 1 Datura innoxia leaves content of primary metabolites; amino native resources is a potential solution for getting rid of
acids (a), proteins (b), and soluble sugars (c) as affected by duration of
that massively produced SW. Furthermore, irrigating of
well and sewage water irrigation (weeks). The data are averages of 4
replicates ± SE certain plants is a good substitution for highly cost recy-
cling treatment; however, the over-use on land for irri-
gation practices imposes considerable hazards causing
chemical pollution problems. However, the plant growth
the case of SW application (Fig. 4c) as a defensive mecha-
and physio-biochemical parameters evaluated in the cur-
nism alarming the cell.
rent study for along ten weeks showed an enrichment
response rather than a toxic one, which can be due to the
Datura innoxia Leaf Nutritional Profile Under Sewage Water SW proprieties normally varying according to its locality
Irrigation and the source from which it is produced [17]. Addition-
Noticeable accumulation of macro- (Nitrate, Phosphate, ally, pH value is highly effective in the mobility and bio-
K, Mg, and Ca) and micronutrients (Na, Fe, Zn, and availability of various minerals [18]. As shown in Table
Abeed et al. BMC Plant Biology (2022) 22:559 Page 7 of 15

by [20], thus expected to be not harmful and to fulfill SW

reuse standards in irrigation [19]. Contrarily, some pre-
vious studies reported high contents of some elements
and HMs in SW [18], demonstrating the accumulation
of these metals in plants as well as soil with continual
SW usage in irrigation. Soil’s physicochemical proper-
ties indicated the poor quality of the used soil from Wadi
El- Assiuty, aligning with the results of Attia et al. [11],
who revealed that the presence of C ­ aCO3 (counted as
32% of total salt in the soil) caused soil alkalinity in many
regions of Wadi El- Assiuty and the OM, total nitrogen
and total phosphorus content ranged between 0.019–
0.73%, 30–370 mg/kg and 2.2–10.7 mg/kg, respectively.
This finding may be because of ammonium nitrification
as well as organic compound oxidation [17]. The current
study indicated that after sewage irrigation, soil alkalinity
decreased. These results are in accordance with Balkhair
and Ashraf [21], who reported that SW treatment
decreased pH. The increment in soil EC herein by the
application with SW rather than WW could be ascribed
to the original high level of total dissolved salts (TDS) of
the sewage water as similarly reported by Mohammad
and Mazahreh [22]. The current results revealed that irri-
gation with sewage water increased soil organic matter
which is ascribed directly to the contents of the organic
compounds and nutrients in the sewage water applied.
Also, these results also agreed with those reported by da
Silva et al. [23] who stated that sewage water irrigation
considerably increased the soil OM, EC, N, and OC and
diminished the soil pH. Several researchers attributed
the accumulation of OM, N, and P in the soil with SW
application to the original contents of these nutrients in
the SW applied [24]. Furthermore, SW can result in K,
P, and N in amounts equal to 4, 10, and 8 times the for-
Fig. 3 Datura innoxia leaves content of hydrogen peroxide, ­H2O2 (a) age fertilizer requirements [25]. Similar results by Demir
and malondialdehyde, MDA (b) as influenced by duration of well and and Sahin [26] indicated that SW treatments increased
sewage water irrigation (weeks). The data are averages of 4 replicates soil fertility by elevating the micro-and macronutrients
± SE
as achieved in the present work (Table 3). Contrarily,
Mohammad and Mazahreh [22] and Mojid et al. [27]
revealed an elevation in soil Fe and Mn with SW irriga-
(3) EC concentration in sewage water was higher (0.80 tion and no response regarding to Zn and Cu in the soil..
ds/m) than that of well water (0.51 ds/m), this is usually Regarding the most environmental hazards elements viz.
due to Cl and Na ion accumulation in SW but still within Pb and Cd [28], the current study revealed that Pb and
the recommended range. It was recommended that water Cd were not affected by SW irrigation. Similar results
with values of EC greater than 300 ds/m is unsafe for irri- obtained by Mohammad and Mazahreh [22], who men-
gation [17]. The SW herein has a considerable amount of tioned that irrigation with SW had no substantial impact
carbon, potassium, and magnesium, essential nutrients on the soil Pb and Cd concentrations.
for improving plant growth, soil fertility, as well as levels Ten-week period was the limit of our study as the fur-
of productivity. These results were confirmed by the data ther application of WW completely stressed the plants
reported by Chopra and Pathak [19]. However, in this grown in poor desert soil in terms of high values of ROS,
research, HMs, as well as micronutrient concentrations alarming antioxidant capacity and decreasing primary
in the SW, were considerably low, and they were within metabolites manufacturing. Interestingly, SW irrigation
the limits of standard concentrations and recommended exhibited substantial plant liveliness than WW irrigated
Abeed et al. BMC Plant Biology (2022) 22:559 Page 8 of 15

Fig. 4 Alternations in the capacities of enzymatic antioxidant of

Datura innoxia leaves; CAT, catalase (a), APX, ascorbate peroxidase
(b) and GST, glutathione-S-transferase (c) and the activities of PAL,
phenylalanine ammonia lyase (d) as influenced by duration of well
and sewage water irrigation (weeks). The data are averages of 4
replicates ± SE

plants, in addition to safely promoting characteristics of

plants evaluated and continued for the 10 weeks. In this
respect, the most substantial dry matter production and
chlorophyll accumulation were recorded by SW appli-
cation. This growth enhancement impact is compatible
with Urbano et al. [29]. Some mineral ions in the irrigated
SW, e.g., Cu and Mn, stimulate the two photosystems.
­Cu2+ enhances the rate of total electron transport from
water to NADP. M ­ n2+ is essential for PSII (­O2 evolving
system). In addition, there is also a direct interaction
between ferredoxin and copper in the reducing site of PSI
[30]. Consequently, the stimulatory effect of SW on total
chlorophyll could be ascribed to the SW impact, result-
ing in enhancing the rate of chlorophyll a and b biosyn-
thesis. The elevated total chlorophyll content is parallel
with the enhancing Datura plant biomass. Teixeira et al.
[31] indicated a close correlation between biomass acqui-
sition and chlorophyll content. These results are compat-
ible with El-Okkiah [32], who found that the application
of SW considerably increased protein and total carbo-
hydrates content in Faba bean compared with WW irri-
gated plants. The manifested accumulation of nitrogen
compounds in terms of amino acids and proteins in this
study can be attributable to soluble, organic, or inorganic
substances in the SW, that may collectively trigger growth
[30]. Soluble sugar accumulation may indicate that SW-
irrigated Datura plant leaves had the highest concentra-
tion of photo-assimilates, accelerating the transition from
the vegetative to the flowering stage; consequently, flow-
ering jointed with the triggering of photo-assimilate pro-
duction in the green area, which was the primary source
until reaching the reproductive. Chen and Chu [33] dem-
onstrated that the content of nutrient components in SW
had a decisive influence on Ottelia acuminata flowering,
and the total number of flowers increased as nitrogen
content in SW increased. The early flowering was sup-
ported by highly upregulated nitrogenous metabolism
of the leaves, where proteins and amino acids accumula-
tion as a result of SW irrigation suggest that the leaves
are well-constructed and have an elevated metabolic effi-
ciency [34]. Moreover, this nitrogen and carbon metab-
olite stimulation may provide the organic components
necessary for forming new branches, where the most
substantial carbohydrates, protein, and amino acids are
linked to a greater number of branches per plant [34].
In the present study, sewage irrigated plants successfully
Abeed et al. BMC Plant Biology (2022) 22:559 Page 9 of 15

Table 5 Leaf nutritional composition at the end of the the current study, the activation of NR via SW irrigation
experiment as affected by well and sewage water irrigations was concomitant with the elevation of NO, which shoul-
Parameter (mg/g Well water Sewage water P Value ders an essential role in plant immune signaling besides
dw) enhancing whole plant development [39]. Conclusively,
SW irrigation might contribute to the activation of NR,
NO3 23 ± 1.29 33 ± 1.38 0.0016
thus influencing nitrate assimilation by supporting NR
PO4 0.87 ± 0.04 1.9 ± 0.06 < 0.0001
and its substrate (NO) uptake. Free amino acids, as well
Na 11 ± 0.91 22 ± 1.11 0.0002
as proteins, were augmented due to the enrichment effect
K 23 ± 1.78 34 ± 1.11 0.0017
of SW irrigation upon catalyzing nitrogen assimilation
Mg 4.6 ± 0.23 5.81 ± 0.41 0.0382
enzyme NR [38].
Ca 3.5 ± 0.21 4.7 ± 0.29 0.0122
Another healthy effect stimulated in SW irrigated
Fe 7 ± 1.11 12 ± 1.11 0.0189
Datura was the enhancement of secondary metabolisms
Zn 0.9 ± 0.03 2.2 ± 0.44 0.0229
and augmented production of highly valuable secondary
Cu 0.2 ± 0.01 0.4 ± 0.05 0.0024
metabolites that imparted the valuable medicinal prop-
Mn 78 ± 0.85 99 ± 3.57 0.0012
erties and quality of Datura plant, viz. anthocyanins,
Cd nd nd –
flavonoids, phenolic compounds, and alkaloids. All of
Pb nd nd –
them meaningfully increased across the duration of cul-
P-values: P < 0.05 indicates significance, P < 0.01 indicates highly significance tivation owing to SW. It was suggested that SW irrigation
improved de novo nitrogenous components synthesis,
thus increasing the production of secondary metabolites
submitted a nourished formation of lateral branches compared to control [40].
accounted by two folds that of WW irrigated plants. Song Regarding Datura innoxia since it is a factory of inter-
and Lee [35] reported that SW application caused shoot est gained alkaloids, it is noteworthy that the increase of
increase determined by new branch formation. In addi- alkaloids due to SW irrigation was concomitant with the
tion, this stimulation of carbon and nitrogen metabolites low proline level along the study duration. This finding
might accomplish the supplies of organic components could be since ornithine, the precursor of tropane alka-
required for forming new branches where the highest loid, and the proline have the same precursor, namely:
increase of proteins, carbohydrates, and amino acids cor- glutamic acid [41] that healthy directed to the pathway
responded to more branches per plant [34]. In the pre- of ornithine production rather than accumulation of pro-
sent study, sewage irrigated plants successfully submitted line, the stress damage indicator. Ornithine was further
a nourished formation of lateral branches accounted by profitably transported to torpane alkaloid, evident with
two folds that of WW irrigated plants. the high content of the total alkaloids in Datura plants
Hence, the exacerbation of proline under WW irriga- irrigated with SW [41]. Furthermore, the augmentation in
tion across time was combined with the decrease in solu- the production of secondary metabolites was witnessed
ble protein generation. This apparent proline production by enhancing PAL activities that reached the maximal on
was not always advantageous; rather, it may have been the 10th week with a percent increase of 110% over the
a negative consequence of extended WW irrigation of starter value (Fig. 4d), indicating that SW irrigation effec-
nutrient-poor soil. Göring and Thien [36] indicated that tively upregulated the production of secondary metabo-
the proline content of plants increased at mineral nutri- lites, along with elevated PAL activity. This result is
ent deficiency, and in case of limited soil nutrition with mainly because PAL is an enzyme that synthesizes a pre-
prolonged WW and poor irrigation, plants may face cursor for the formation of different secondary metabo-
nutrient deficiency-induced stress. Thus, this accumu- lites and is a vital regulator between secondary as well
lation in proline content was considered an indicator of as primary metabolism [37]. In contrast, the increased
stress damage and/or stress resistance [36]. It might also amino acid content may have improved the availability of
act as a storage for organic nitrogen, which, upon stress phenylalanine (Phe) as an elite substrate for PAL, mak-
reduction, could be converted into a variety of nitroge- ing more Phe available for the formation of secondary
nous molecules [37]. The stabilization of proline content, metabolites [37]. Contrarily, a low inducible PAL activity
combined with exacerbation in soluble protein content rate jointed with diminishing amino acid accumulation
under SW application, may reflect optimum conditions was pronounced under WW application, particularly in
for effective cellular metabolism and performance with the last week of the experiment indicating down-regula-
SW irrigation. tion of the secondary metabolites production.
Numerous studies indicated that NR substantially con- Whereas plants with WW irrigation exhibited elevated
tributes to plants’ NO biosynthesis [38]. Therefore, in levels of ­H2O2 that were concomitant with decreasing
Abeed et al. BMC Plant Biology (2022) 22:559 Page 10 of 15

membrane integrity and stability evidenced by high MDA uptake in the shoot system, thereby not only improved
concentration, which may be another main reason for soil properties by enriching with essential nutrients but
suppressing the growth rate of Datura plants under WW amplified the quality of D. innoxia plants [49] as well.
irrigation compared to SW irrigated plants. As stress Consistent with our results, Bedbabis et al. [50] sug-
indicators, the enhanced levels of these toxic molecules gested that SW application significantly increases K, P,
­(H2O2 and MDA) indicated that plants under prolonged N, and HMs (Mn and Zn) concentrations in the olive
WW irrigation might encounter nutrient deficiency after leaves, thereby upgrading the olive property. It should be
the fourth week and thus undergo nutrient deficiency- noted that SW lessens the toxicity of some elements in
induced stress. Tewari et al. [42] reported that plants the soil, as suggested by Demir and Sahin [26]. Therefore,
could undergo Zn deficiency-induced oxidative stress the growth of Datura plant in the presence of SW indi-
when poorly irrigated. As a response to this stress, plants cated suspected resistance against HMs accumulated in
may modify nutrition and metabolites in order to estab- the soil.
lish defense mechanisms on account of growth. Further-
more, the overproduction of anthocyanins, flavonoids, Conclusion
phenolic compounds, and alkaloids may partially explain The ten-week monitoring period was the timeframe/limit
the diminished lipid peroxidation in plants supplied with of our study as the further application of WW completely
nutritive SW. Consequently, increased membrane integ- stressed the plants grown in poor desert soil in terms of
rity than the WW irrigated plants experiencing nutrient high values of ROS and alarming antioxidant molecules
deficiency-induced stress. These valuable compounds and the dramatically diminished primary metabolites’
display various functions jointed to antioxidant charac- content. Optimistically, according to the revealed results
teristics as well as the capacity to trap free oxygen radi- regarding healthy growth and improved medicinal prop-
cals, thus stabilizing membranes by diminishing their erty of Datura innoxia, jointed with a well-furnished
fluidity, ultimately limiting free radical diffusion and metabolic profile and positive antioxidative changes in
reducing membrane lipid peroxidation [37]. response to SW irrigation, it can be deduced that SW
The constant ­H2O2 and MDA content maintained near could be safely reused for Datura cultivation while at the
the control values reflects the high optimal condition and same time to provide poor soil with adequate amend-
maintained membrane function for effective cellular per- ments. Hence, the soil was fertilized by the nutrient
formance as well as metabolism that can be attributed to favorable to plant performance and development rather
the stabilized cell redox status [43], leading to a health- than be diseased. Nevertheless, the existence of high
ier growth of SW irrigated Datura. Moreover, activating concentrations of HMs and traces of some toxic com-
GST as a defensive mechanism alarming the cell may be pounds lead to the instance for continual monitoring of
due to some xenobiotic agrochemical loaded in the deliv- the redox status of the receiver plant and the feedback on
ered SW, which may trigger wide disciplines of antioxi- its growth. In that case, it will have priority for irrigation
dants and metabolic pathways that cumulatively improve purposes. Thus, experiencing the crises of water scarcity
leaf physiological status in Datura plants. Hence, SW and costive fertilizers, sewage might be effectively gener-
irrigation positively influenced the growth and physi- alized for irrigation multipurpose plants.
ological parameter as well as valuable secondary metabo-
lites production of Datura. Materials and Methods
However, SW irrigation manifested a noticeable accu- Water and soil samples collection
mulation of macro- (Nitrate, Phosphate) and micronutri- The experiment was conducted over ten weeks, from
ents (Na, K, Mg, Ca, Fe, Zn, Mn) in Datura leaves plants March to mid-May 2019. Two water samples were col-
compared to WW irrigated ones. The contents were still lected, one from Arab Elmadabegh, Assiut, Egypt
within the critical limits recorded for the metals’ phyto- (27°12 N and 31°09 E), where the most extensive sewage
toxicity [24]. Nitrogen (N) and phosphorous (P) are the line in Assiut governorate [51] (source of SW), another
main elements of plant nutrition [44] and are fundamen- one was collected from tape water (source of WW) at the
tal to plant development, growth, crop yield, and adapta- botanical farm of Botany and Microbiology Department,
tion [45]. In this study, N and P levels increased in plant Faculty of Science (42 “and 28° 59’ 23 “E and latitude 25°
leaves (in the form of nitrate and phosphate) after SW 45` 06 “and 25° 53’34 “N). As described in the follow-
application. Amâncio and Stulen [46] stated that nitrogen ing section, the two water samples were analyzed for
content is one of the vital factors influencing crop growth their physicochemical characteristics (Table 1) and were
and determining the quantity and quality of crop yields. directly kept in the dark bottles under cooling (4 °C) for
Furthermore, Mohammad and Ayadi [47] and Hernán- further usage in irrigation. Soil samples were collected
dez-Pérez et al. [48] stated that SW increased nutrient from the surface soil at 0–25 cm soil depth from Wadi
Abeed et al. BMC Plant Biology (2022) 22:559 Page 11 of 15

Al-Assiuty (31°18′ and 31°48′ E and 27°10′ and 27°45′ respectively; relative humidity (34–42%) and reference
N), a part of the eastern desert east of Assiut city that evaporation (4.65–5.48 mm).
has been recorded as a poor desert soil area in Assiut, At the beginning of the experiment, using watering can,
Egypt [11]. The samples were air dried, ground to pass the dry soil received the WW and SW in field capacity at
through 2 mm sieve, and stored in plastic bottles before the rate of 180 ml/kg, soil with polyethylene bags to avoid
usage. Soil samples were analyzed for the physicochemi- soil treatment leaching. During cultivation, the frequency
cal characteristics two times, one prior to the start of the of SW was maintained once a week as per the plant water
lab experiment and another at the end of the experiment, requirement (compensating the lost water via evapora-
and plant harvesting results were represented in Tables 2 tion and maintaining the moisture level at field capacity).
and 3, respectively. Along with experiment duration, soil water content was
sustained in field capacity by weight method via the addi-
tion of WW day by day if required.
Physical and chemical analysis of the samples The harvest of all treatments was scheduled at 7-day
For both water and soil, soluble Ca and Mg concentra- intervals and performed by picking up the whole plant,
tions were measured using the EDTA titration method, root and shoot, from the soil. At the end of each week
and Na and K were estimated using a flame photom- and for ten weeks, the observations were recorded on 2
eter. OC for soil was evaluated by adopting the method randomly selected plants per replication per treatment
of Jackson [52]. Soil total nitrogen was determined fol- for all indices. For the chemical analysis, 2 randomly
lowing the procedure of Singh et al. [8]. Soil phosphorus selected plants were handled by blending as one sample.
was determined using Olsen extraction (0.5 M N ­ aHCO3)
[53]. Cation exchange capacity (CEC) was as described
by Jackson [52]. Free calcium carbonate ­(CaCO3) was Growth Parameters and Physio‑Biochemical indices
estimated by calcimeter method [54]. Available micro- Analysis of Datura innoxia Plant
nutrients and HMs were estimated as per the procedure Growth Analysis
described by Singh et al. [8]. The pH of water and soil (1:1 Fresh weight (FW) of harvested plants was determined
suspension) was estimated according to McNeal [55]. immediately and then cleaned via thoroughly rinsed
Electrical conductivity was measured using a conductiv- with distilled water to be oven dried at 60 °C to constant
ity meter (Orion, EA 940 USA). Soil texture was analyzed weight for two days to evaluate DW. The number of
as the method described by Piper [54]. Water and soil branches and flowers per plant was also calculated.
samples were examined for different physical and chemi-
cal characteristics as per the standard procedure depicted
in Tables (1, 2, and 3). Chlorophyll Content
Chlorophyll was extracted from 0.5 g of fresh leaves sus-
pended in 5 ml of 95% ethyl alcohol at 60–70°C in a water
Growth Condition and Treatments
bath. Absorbance readings were taken with a spectropho-
The experiment was performed during spring 2019.
tometer (Unico UV-2100 spectrophotometer). Chloro-
Plants were gathered from the botanical garden of the
phyll was estimated as mg/g FW at 663 and 644 nm using
Faculty of Agriculture, cut into 160 uniform/same-
equations of Lichtenthaler [56].
sized plantlets of approx. 25 g. Plantlets were randomly
divided into two equal groups; WW and SW irrigations.
Afterward, plantlets were washed with fresh water and
Nitric Oxide Content and Nitrate Reductase Activity
weighed before being transplanted into the pots. The
NO content was quantified according to Ding et al. [57]
pots (60 cm in diameter and 45 cm in depth, filled with
and Hu et al. [58] and expressed as nmoles/g FW. Leaves
50 kg desert soil collected from Wadi Al-Assiuty) were
were incubated in a buffer of acetate (pH = 3.6), and the
organized in a completely random arrangement with
leaves tissue was then separated by centrifugation and re-
four replicates for each group, and plantlets were trans-
extracted by charcoal, then centrifuged again, the super-
planted at the rate of 20 plantlets/pot. Immediately, they
natant was mixed with Greiss reagent and read at 540 nm.
were transported to a greenhouse at the Department of
NR activity expressed as micromoles of ­ NO2 g/hr.
Botany and Microbiology, Faculty of Science, Assiut Uni-
was estimated by adopting the described method of
versity, receiving natural light (transmitted through glass
Downs et al. [59], in which leaves were soaked in potas-
panels) under ambient sunlight, the temperature rang-
sium phosphate buffer (pH 7.5) and ­KNO3. The resultant
ing between 27 and 38 and 12–15 °C at day and night,
Nitrite was detected by adding naphthyl-ethylenediamine
dihydrochloride and sulfanilamide. Absorbance readings
Abeed et al. BMC Plant Biology (2022) 22:559 Page 12 of 15

at 540 nm were taken with a spectrophotometer (Unico was added, the total volume was made up to 10 ml, and
UV-2100 spectrophotometer). absorbance was measured at 415 nm. A routine quanti-
fication method for analysis of the total alkaloidal con-
Determination of Primary Metabolites tent spectrophotometrically. The yellow-colored complex
First, 1 ml of Stannus Chloride reagent was combined formed followed at 435 nm based on Dragendorff ’s rea-
with 0.5 ml of the water extract, and then the tubes were gent (DR) described by Sreevidya and Mehrotra [68].
heated in a water bath for 20 minutes and then cooled.
The plant water extract was made by steeping 0.5 g of dry Oxidative Stress Indicators
leaves in 10 ml of distilled water for 1 hour at 95 °C. The The content of hydrogen peroxide (­H2O2; μmol/g FW)
extinction of violet color was measured at 570 nm using was measured spectrophotometrically in the leaves.
the aforementioned reagents and distilled water instead Fresh leaves were ground in cold acetone (5 ml). After-
of the extract of the plant sample [60]. Soluble protein ward, 3 ml of the acetone extract was added to 1 ml of
was assayed according to Lowry et al. [61]. In the previ- titanium dioxide (0.1%) in ­H2SO4 (20%) before centri-
ous water extract of free amino acid, 0.1 ml of plant water fuging the mixture at 6000 rpm for 15 min. The yellow
extract was added to 5 ml of the alkaline reagent solution. color developed was measured at 415 nm [69]. Malon-
Afterward, 0.5 ml of diluted Folin-Ciocalteu’s reagent (1: dialdehyde as a lipid peroxidation marker (MDA; μmol/
2 v/v) was added. After 20 min, the extinction against the g FW) was quantified utilizing the protocol of Madhava
appropriate blank was measured at 750 nm utilizing a Rao and Sresty [70]. Fresh leaves were homogenized in
spectrophotometer. The water-soluble sugars were esti- trichloroacetic acid (TCA) (0.1%) and then centrifuged
mated by the method of anthrone–sulfuric acid according at 10,000 rpm for 10 min. One ml of the supernatant was
to the method of Fales [62] and Schlegel [63]. In addition, mixed with a TCA-TBA reagent. The mixture was heated
30 mg of dry leaves were taken and extracted in 3 ml dis- for 20 min in a water bath at 90°C and then cooled rapidly
tilled ­H2O, which was blended with 4.5 ml anthrone rea- on an ice bath. The resultant mixture was centrifuged for
gent and boiled in a water bath for 5 min before cooling 15 min at 10,000 rpm, and the absorbance of the super-
down on an ice bath. The absorbance of the developed natant was spectrophotometrically monitored at 532 nm.
blue-green color was determined at 620 nm using a Unico
UV-2100 spectrophotometer. Proline was determined Enzyme Extraction and Quantification for Antioxidant
in dry leaves. Leaves tissue was ground in 6 ml sulfosali- Activities
cylic acid (3%) before the centrifugation of the mixture. Plant samples (four replicates from each treatment)
Then, the outcome supernatant was mixed with 2 ml of were extracted via homogenizing leaf samples in
glacial acetic acid as well as 2 ml of ninhydrin. The reac- 0.1 M phosphate buffer (pH 7.4) containing 10 mM
tion mixture was extracted with 4 ml toluene to quantify β-mercaptoethanol, 1 mM EDTA, and 1% polyvinylpyr-
at 520 nm [64]. rolidone. The homogenates were centrifuged at 10,000 g
for 25 min, and the supernatant was used for the assays.
The activities of catalase (CAT; EC 1.11.1.6) and ascor-
Determination of Secondary Metabolites bate peroxidase (APX; EC 1.11.1.11), glutathione peroxi-
Determination of anthocyanin pigments was done dase (GPX/EC.1.11.1.9), glutathione-S-transferase (GST;
according to the method described by Dawood and EC 2.5.1.18), and (PAL; EC 4.3.1.5) were assayed follow-
Abeed [65] on acidified methanol (1% HCl v/v) extract of ing the method of Abeed et al. [71], Flohé and Günzler
fresh leaves that were hydrolyzed at 80 °C for 30 minutes [72], Habig et al. [73], and Sykłowska-Baranek et al. [74],
to the absorbance obtained corresponding to anthocya- respectively.
nidins was spectrophotometrically detected at 520 nm.
Determination of phenolic content was according to Determination of Leaf Element Composition
Kofalvi and Nassuth [66] using the Folin-Ciocalteu’s phe- Potassium and sodium concentrations were measured
nol reagent. Subsequently, 100 μl of the methanol extract utilizing the flame emission technique (Carl-Zeiss DR
was diluted to 1 ml with distilled water and mixed with LANGE M7D flame photometer) according to Abeed
0.5 ml of Folin-Ciocalteu’s reagent (2 N) and 2.5 ml of and Dawood [75]. Nitrate content was determined fol-
­Na2CO3 (20%). The absorbance of the developed color lowing the protocol of Cataldo et al. [76]. Phosphorus
was measured at 725 nm with a Unico UV-2100 spectro- content was spectrophotometrically measured by the
photometer. The methanolic extract of fresh leaves was methods of Fogg and Wilkinson [77]. The Ca, Mg, Fe, Zn,
utilized to analyze flavonoids by the method by Harborne and Mn contents were determined with atomic absorp-
and Williams [67]. Five ml distilled water and 3 ml ­AlCl3 tion (Shimadzu- model AA-630-02) in acid-digestion
(1:10) were added. After 5 min, 2 ml 1 M ­CH3- COOK extract (2:1 ­HNO3:HClO4 mixture), as described by Eissa
Abeed et al. BMC Plant Biology (2022) 22:559 Page 13 of 15

Fig. 5 Summarization of the harvest intervals and data collected throughout the study from transplanting to finalizing. WW; well water, SW; sewage
water

and Abeed [78]. Summarization of the harvest intervals Faculty of Agriculture, Assiut University, Assiut, Egypt, for production and
direction of the statistical issues.
and data collected throughout the study from transplant-
ing to finalizing is provided in Fig. 5. Authors’ contributions
Amany H. A. Abeed and Suzan A. Tammam: speculation, data curation, formal
analysis, writing review. Amany H. A. Abeed, Mohammed Ali and Mamdouh A.
Statistical Analysis Eissa: validation, writing-original draft and editing the manuscript. All authors
The analysis of variance for completely randomized read and approved the final manuscript.
design (CRD) was carried out using Costat (CoHort
Funding
software, Monterey, CA, USA) with two main treat- Open access funding provided by The Science, Technology & Innovation
ments, WW and SW. The observations were recorded on Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
plot mean basis analysis [79]. Means were compared by (EKB). The authors have no relevant financial or non-financial interests to
disclose.
revised Least Significant Difference (R LSD) at a 5% level
of significant [80]. Availability of data and materials
All the data is in the published article.

Abbreviations
WW: Well water; SW: Sewage water; FW: Fresh weight; DW: Dry weight; SV:
Declarations
Starter value; BN: Branch number; FN: Flower number; TC: Total chlorophyll;
Ethics approval and consent to participate
Car: Carotenoids; An: Anthocyanin; FL: Flavonoids; Ph: Phenolics; Al: Alkaloids;
This study uses plant materials and does not utilize transgenic technology,
Pro: Proline; NO: Nitric oxid; NR: Nitrate reductase; MDA: Malondialdehyde;
neither involves endangered or protected species. We complied with all
ROS: Reactive oxygen species; H2O2: Hydrogen peroxide; CAT​: Catalse; APX:
relevant institutional, national and international guidelines and the appropri-
Ascorbate peroxidase; GPX: Glutathione peroxide; GST: Glutathione-s-trans-
ate permissions were obtained from Faculty of agriculture, Assiut University,
ferase; PAL: Phenylalanine ammonialyase.
Egypt, for obtaining Datura plantlets. This study was supported by the Depart-
ment of Botany & Microbiology, Faculty of Science, Assiut University, including
Acknowledgements
handling this plant and processing the experiment.
The authors are very grateful for Dr. Nemmat A. Husein— Assistant profes-
sor of Mycology, Botany and Microbiology Dept., Faculty of Science, Assiut
Consent for publication
University (nemmh​ussein@​gmail.​com), for her revision and critical reading of
Not applicable.
this research article and Mohamed Tharwat Said (Said MT) (mthar​wat@​aun.​
The authors confirm that the manuscript has been read and approved by all
edu.​eg), Professor of crop production and Physiology, Agronomy Department,
authors. The authors declare that this manuscript has not been published and
Abeed et al. BMC Plant Biology (2022) 22:559 Page 14 of 15

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