biology

Article
Foliar Spray or Soil Drench: Microalgae Application Impacts on
Soil Microbiology, Morpho-Physiological and Biochemical
Responses, Oil and Fatty Acid Profiles of Chia Plants under
Alkaline Stress
Samah M. Youssef 1, *, Rasha S. El-Serafy 2, * , Kholoud Z. Ghanem 3 , Abeer Elhakem 4
and Azza A. Abdel Aal 5

1 Horticulture Department, Faculty of Agriculture, Fayoum University, Fayoum 63514, Egypt

2 Horticulture Department, Faculty of Agriculture, Tanta University, Tanta 31527, Egypt
3 Department of Biological Science, College of Science and Humanities, Shaqra University, Shaqra,
Riyadh 11961, Saudi Arabia
4 Department of Biology, College of Sciences and Humanities, Prince Sattam Bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia
5 Soil Microbiology Department, Soils, Water and Environment Research Institute,
Agricultural Research Center, Giza 12619, Egypt
* Correspondence: [email protected] (S.M.Y.); [email protected] (R.S.E.-S.)

Simple Summary: Chia is an important medicinal plant and is a rich source of omega-3 and omega-6
Citation: Youssef, S.M.; El-Serafy, fatty acids. Alkaline soil inhibits the growth and productivity of all crops, including chia. Microalgae
R.S.; Ghanem, K.Z.; Elhakem, A.; are a diverse group of photosynthetic microorganisms that can be used in modest doses to stimulate
Abdel Aal, A.A. Foliar Spray or Soil the growth and productivity of numerous crops in both normal and stressed conditions. Microalgae
Drench: Microalgae Application supplementation by two application methods (foliar spray and soil drench) resulted in an increase in
Impacts on Soil Microbiology, the growth and productivity of chia plants cultivated under alkaline stress conditions, and caused an
Morpho-Physiological and
increase in the antioxidant levels in the chia seeds, although soil drenching gained the superiority
Biochemical Responses, Oil and Fatty
in this respect. The oil content was increased following microalgae application with an increase in
Acid Profiles of Chia Plants under
omega-3 proportion. Chia plants showed different responses to foliar and drenching applications.
Alkaline Stress. Biology 2022, 11, 1844.
Microalgae would be a potential and eco-friendly approach for enhancing agricultural productivity
https://doi.org/10.3390/
biology11121844
in alkaline environments. Our findings also suggest that Arthrospira platensis supplementation via
the soil drenching technique should be used in the future to enhance plant growth and productivity
Academic Editors: Chengliang Sun
under alkaline soil conditions.
and Weiwei Zhou

Received: 26 October 2022 Abstract: Alkaline soil inhibits the growth and productivity of chia plants (Salvia hispanica L.). Mi-
Accepted: 14 December 2022 croalgae as biofertilizers have been reported to induce alkalinity tolerance and enhance yield and
Published: 17 December 2022 quality. However, limited information is known concerning the influence of microalgae application
Corrected: 31 January 2023 on medical plants, including chia. Our experiments were performed to evaluate the effect of mi-
Publisher’s Note: MDPI stays neutral croalgae strains of Arthrospira platensis, Chlorella vulgaris, Nostoc muscorum, and Anabaena azollae with
with regard to jurisdictional claims in two application methods, foliar spray and soil drench, on morpho-physiological and biochemical
published maps and institutional affil- parameters, yield, seed and oil quality, and fatty acid profiles of chia plants cultivated under alka-
iations. line soil conditions, as well as the on soil microbial activity. The results obtained reveal that both
application methods positively influenced the growth and productivity of chia plants. However,
the foliar application showed significant differences in the herb’s fresh and dry weights and leaf
pigments, whereas the drenching application caused more effect than the foliar spray application at
Copyright: © 2022 by the authors.
the reproductive stage. Untreated chia plants showed a slight decline in the growth, productivity,
Licensee MDPI, Basel, Switzerland.
and antioxidant level with an increase in Na content. However, microalgae applications significantly
This article is an open access article
ameliorated these impacts as they induced an enhancement in the growth, leaf pigments, total protein
distributed under the terms and
conditions of the Creative Commons
and carbohydrate contents, nutrient content, seed and oil yields, as well as an increase in linolenic
Attribution (CC BY) license (https:// and linoleic fatty acids, with a reduction in saturated fatty acids, namely, palmitic and lauric acid.
creativecommons.org/licenses/by/ Soil drenching generated an improvement in the soil microbial activity and caused a reduction in the
4.0/).

Biology 2022, 11, 1844. https://doi.org/10.3390/biology11121844 https://www.mdpi.com/journal/biology

Biology 2022, 11, 1844 2 of 23

pH. The treatment of A. platensis with drenching application resulted in higher seed and oil yield,
with an increase of 124 and 263.3% in seed and oil yield, respectively.

Keywords: microalgae; Salvia hispanica; saturated fatty acids; linolenic acid; DPPH; cyanobacteria

1. Introduction
Salvia hispanica L., commonly known as chia, is a member of the Lamiaceae family
and is native to the mountains of Guatemala and Mexico [1]. Currently, chia is grown in
Argentina, Australia, Bolivia, Colombia, Guatemala, Mexico, Peru, and Bolivia [2]. Today,
Mexico is acknowledged as the largest chia grower in the world [3]. Chia is a herbaceous
annual plant that can reach a height of 1 m, has oppositely oriented leaves, and yields white
or purple flowers. Chia seeds are utilized commercially as a whole or ground up to make
flour, mucilage, oil seeds, and other products [4]. Furthermore, it is used as a spice in a
variety of foods, including milk, yogurt, salad dressings, soups, and baked products, as well
as fruit juices, due to their nutritional value. The seeds are rich in dietary fiber, minerals,
proteins, vital fatty acids, carbohydrates, and polyphenolic substances [5]. Chia seed oil
is a sustainable source of polyunsaturated fatty acids omega-3 (58–64%) and omega-6,
the essential fatty acids for human health [6], and antioxidants, including tocopherols,
phytosterols, carotenoids, and phenolic compounds such as chlorogenic acid, caffeic acid,
myricetin, quercetin, and kaempferol [7]. These compounds protect consumers from a
variety of diseases, and have positive effects on human health [8]. However, chia is a crop
that is known to be susceptible to several forms of stress [9].
Foliar feeding is a technique of supplying plants with required nutrients by spraying
an aqueous solution directly onto the leaves, which absorb these nutrients through their
stomata and cuticles [10,11]. Foliar spray might be helpful when plants are lacking in a
certain nutrient, but is not a substitute for healthy soil. In addition, foliar spray application
avoids nutrient leaching from the soil and induces a quick response in the plant [11,12]. The
soil drenching technique, on the other hand, is the process of supplying diluted products
directly to the base of the plant, and has a slow impact because the chemicals applied
require time to be absorbed by the roots and transported to the stems, branches, and leaves.
Soil alkalinization is a worldwide challenge that lowers agricultural quality and crop
yields. Alkaline soil has a pH greater than 7.5, high CO3 −2 /HCO3 - , sufficient Na levels to
limit crop growth, and/or high exchangeable sodium content and/or exchange capacity
(15% or more) [13]. Additionally, alkaline soil is characterized by its poor structure, surface
crusting and cracking, low water holding capacity, organic matter and clay content, and
loss of nutrients by leaching or deep percolation [14,15]. The main effects of alkali stress
on crop production are ion toxicity and osmotic stress, but many studies report that high
pH is actually more hazardous to plants than saline soil [16]. Alkaline soil can prevent
seeds germinating, harm the structure of root cells, and restrict nutrient uptake, causing
a reduction in agricultural yields [15,17]; biofertilization can be a useful strategy to cope
with soil alkalinization. Biofertilization is a necessary strategy for environmentally friendly,
sustainable farming methods [18–20]. Microalgae have attracted great attention due to their
potentially extensive application in agriculture as biofertilizers. Microalgal biofertilizers
may be employed in crop production to improve agricultural sustainability [21].
Microalgae are a diverse group of photosynthetic microorganisms that can be used in
modest doses to stimulate the growth and development of numerous crops in both normal
and stressed conditions. They can be used in concert with synthetic fertilizers to control
plant growth, protect crops, increase yields, and support plant tolerance to environmental
stresses [22]. Microalgae are renewable, sustainable, and economical sources of bioactive
pharmaceutical products, biofuels, and food ingredients [23,24]. Microalgae are microscopic
single cells that can be either eukaryotic, such as green algae (Chlorophyta), or prokaryotic,
such as cyanobacteria (chloroxybacteria). Cyanobacteria have been successfully applied as
Biology 2022, 11, 1844 3 of 23

biofertilizers in soil restoration techniques for alkaline and calcareous soils [25]. Microalgae
have the capacity to promote plant growth, immobilize heavy metals in the soil, link
soil particles into stable aggregates, and lessen the likelihood of erosion [25]. Microalgae
biofertilization for rice plants is related to their ability to fix nitrogen, and has other
beneficial effects for both plants and soil [24]. Chlorella sp. cell soil application with
irrigation water enhanced the chlorophyll level and dry weights in cucumber, rice, eggplant,
and lettuce crops [26].
To our best knowledge, the effects on Salvia hispanica L. treated with foliar spray and
soil drenching application methods using different microalgae strains under alkaline soil
conditions are reported here for the first time. The aim was to evaluate the differential
impact of different microalgae strains, used in the field under alkaline conditions, on
chia plants under different application modes. To achieve this goal, we evaluated the
influence of C. vulgaris, N. muscorum, A. platensis, and A. azollae strains on the growth and
productivity of chia, including photosynthetic pigments, antioxidant activity, yield, and
yield components. Additionally, we assessed the effects of microalgae strains on the seed
quality (oil content and fatty acid composition) and leaves, as well as soil-related traits.

2. Materials and Methods

2.1. Location and Soil Analysis
◦
In a private nursery in El Fayoum Governorate, Egypt, between latitude 29 190 30.100 N
◦
and longitude 30 510 15.800 E, two experiments were performed during the cropping seasons
of 2019/2020 and 2020/2021, in the period of November to July for both seasons. Prior to
the experiment, the soil physiochemical parameters were evaluated in accordance with the
methods described by Page et al. [27] and Klut [28]. The soil biological parameters were
assessed using the technique of Allen [29] and Allen and Stanier [30], and the results are
presented in Table 1. The soil class was identified as alkaline based on the soil characteristics
according to Oster and Ayawardane [31].

2.2. Plant Material and Cultivation

Chia seeds were acquired from the Faculty of Pharmacy’s Experimental Farm at Cairo
University. The seeds were sown in trays on 5 October for both seasons. The symmetrical
seedlings with 2–3 pairs of truly extended leaves and a length of 10 to 15 cm were planted
out in the open field in plots on 20 November for both seasons. The experimental plot had
a total area of 4.8 m2 (8 m in length and 0.6 m in width), and roughly 0.3 m in inter-seedling
distance within each row.

2.3. Microorganisms Source and Culture

The green alga C. vulgaris and three cyanobacteria, namely, N. muscorum and A. platen-
sis, as well as an A. azollae strain, which was isolated from Azolla pinnata [23], were obtained
from the Microbiology Department, Soils, Water and Environment Research Institute,
Egypt. Both N. muscorum and A. azollae were grown on nitrate-free medium (Blue Green
“BG”11 medium) according to Rippka et al. [32], while A. platensis was cultivated on Zarrouk
medium according to Zarrouk [33]. Chlorella vulgaris was cultured in a Bold medium [34].
The cultures were incubated in a growth chamber under continuous illumination (24 h
light, 2000 lux) and a temperature of 25 ◦ C ± 2 ◦ C for all strains except the mesophilic
alga A. platensis, which was grown at 35 ◦ C ± 2 ◦ C. After 30 days of incubation, the algal
suspension was used for the field experiments. To determine algal biomass, 100 mL of the
algal culture was taken after 30 days of incubation, centrifuged at 10,000 rpm for 10 min,
and the supernatant was discarded. The pellet was centrifuged for 10 min at 10,000 rpm
after passing through three phases of resuspension in distilled water to wash it. The cleaned
pellet was weighed, air dried; then, the dry weight was recorded and calculated as g/L.
For the field experiments, 1 g/L of dried algal biomass was used.
Biology 2022, 11, 1844 4 of 23

Table 1. The physiochemical and biological properties of the experimental soil before cultivation.

Soil Characteristics Value

Particle Size Distribution (%) 2019/2020 2020/2021
Coarse sand 7.39 7.42
Fine sand 40.51 40.48
Silt 18.89 18.92
Clay 33.21 33.18
Texture class Sandy clay Loam
pH 7.94 7.92
ECe (dS m–1 ) 3.77 3.75
Soluble cations (mmolc L−1 )
Ca++ 5.13 5.10
Mg++ 4.12 4.10
Na+ 28.22 28.08
K+ 0.23 0.22
Soluble anions (mmolc L−1 )
CO3 – 16.82 16.84
HCO3 – 3.15 2.95
Cl– 16.28 16.30
SO4 – 1.45 1.41
SAR 13.44 13.37
ESP% 15.65 15.58
Available Nutrients (%)
N 0.17 0.18
P 0.20 0.22
K 0.29 0.30
Biological (CFU g–1 soil)
Total bacterial count 7.15 × 107 8.20 × 107
Total cyanobacterial count 6.14 × 102 6.89 × 102
SAR, sodium adsorption ratio; ESP, exchangeable sodium percentage.

2.4. Treatments and Experimental Design

The experimental configuration had a split-plot system built on a randomized com-
plete block design (RCBD) with three replicates. The application method and microalgae
inoculations were distributed at random across the main and subplots, respectively. The
experiments consist of ten different treatments, applied as interactions of two application
methods (foliar spray and soil drench) and microalgae treatments (untreated control, A.
platensis, C. vulgaris, N. muscorum, and A. azollae). The algal growth suspension was used
for foliar and soil drenching application at a rate of 50 L ha− 1 , divided into two equal
doses [35]. The treatments were performed twice at 40 and 60 days after seedlings were
planted. Other agricultural practices (fertilization, irrigation, and weeding) were carried
out exactly as per Souza and Chaves [36].

2.5. Morpho-Physiological Traits

Seven plants aged 90 days were collected at random from each replicate to determine
the fresh and dry weights of the herb (g/plant). Total chlorophyll was assessed from the
top third and fourth leaves using a chlorophyll meter (SPAD-502, Minolta Company, Tokyo,
Biology 2022, 11, 1844 5 of 23

Japan) [37]. The dimethylformamide (DMF) technique was used to estimate total carotene
content (mg/mm2 ) [38].

2.6. Yield and Yield Components

At the harvesting stage, seven plants were collected to determine the number of
inflorescence plant− 1 , seed yield plant− 1 (g), weight of 1000 seeds (g), and seed yield ha− 1
(t). The oil content in the chia seeds was determined following the same steps described in
the method of AOAC [38]. Then, oil percent and oil yield ha− 1 (L) were calculated. Oil (%)
was expressed using the following formula:

Oil (%) = (Weight of extracted oil (g) ÷ sample of seed weight (g)) × 100. (1)

2.7. Gas–Liquid Chromatography (GLC)

Gas–Liquid chromatography (GLC) (Hewlett-Packard Corporation, GA, USA) was
utilized to identify the fatty acid composition of the oil samples [39]. Methylated fatty
acids were fed into the HP6890 Series GC system using a column of type DB-23
(60 m × 0.32 mm × 0.25 µm). Nitrogen was used as the carrier gas, flowing at a rate of
1.5 mL/min and having a splitting ratio of 1:50. The temperatures of the injector and flame
ionization detector (FID) were 250 ◦ C and 280 ◦ C, respectively. The following were the
temperature settings: 150 ◦ C to 210 ◦ C at a rate of 5 ◦ C per minute, followed by a 25 min
hold at 210 ◦ C. Peaks were located by contrasting the obtained retention durations with
standard methyl esters.

2.8. Biochemical Analysis

Total protein and carbohydrate contents, as well as total phenols, total flavonoids,
and DPPH levels in chia seeds were estimated. Total protein was assessed using the semi-
micro Kjeldahl technique [39]. Total carbohydrates were measured colorimetrically as
previously described by Alshallash et al. [40] using the phenol–sulfuric acid technique [41].
For extracting total phenolics and DPPH in the seeds, 1 g of seed samples was mixed with
10 mL of ethanol (60%) and left for 3 h under shaking conditions at room temperature;
then, the mixture was centrifuged at 3000× g for 15 min. Total phenolics were estimated by
the Folin–Ciocalteu colorimetric technique [42]. Briefly, 0.1 mL of the extract was diluted
with 2 mL of distilled water, and then 0.5 mL of the Folin–Ciocalteu reagent was mixed.
A 2 mL solution of sodium carbonate (20%) was added to each tube. Evaluating the test
solutions’ absorbances at 650 nm was conducted in comparison to the reagent used as a
blank. Total flavonoids determination was assessed using the Shi et al. [43] method with
minor modification according to Zhao et al. [44]. Briefly, 1.5 mL of the ethanol extract was
mixed with 4.5 mL of distilled water and 1 mL of 5% sodium nitrite solution in a 25 mL tube.
Subsequently, 1 mL of 10% aluminium nitrate solution was added to the mixture after 6 min.
Following the addition of 10 mL of 4% sodium hydroxide solution after 6 min, 25 mL of
60% aqueous ethanol was added. The mixture’s absorbance at 510 nm was measured after
it had been reacting for 15 min in comparison to a blank that contained 5 mL of extraction
solvent. The capacity of seed extract to scavenge free radicals was evaluated using the
DPPH (2,2-diphenyl-1-picrylhydrazyl) technique [45]. Next, 33 µL of the ethanol extract
and 1.3 mL of the diluted-in-methanol (0.024 mg/mL) DPPH solution were combined. The
absorbance at 515 nm was measured following 30 min of dark incubation.

2.9. Nutrients Determinations

Macroelements, i.e., phosphorus (P), potassium (K+ ), and calcium (Ca2+ ), as well as
micronutrients, i.e., iron (Fe2+ ), magnesium (Mg2+ ), zinc (Zn2+ ), and sodium (Na+ ), were
measured in the seeds. The chlorostannous molybdophosphoric blue color technique
applied in the sulphuric acid method was used to determine P content [46]. The K and Na
levels were measured using a flame photometer (Gallenkamp Company, London, UK) [47].
Biology 2022, 11, 1844 6 of 23

The Ca and micronutrient (Fe, Mg, and Zn) contents were assessed using an atomic
absorption spectrophotometer (Perkin-Elmer, Model 3300, Woodbridge, Canada) [48].

2.10. Soil pH and Microbial Count

During harvesting, when applying the microalgae as a drenching application, the pH
of the soil was estimated using the techniques of Page et al. [27]. The bacterial and total
cyanobacteria count were determined according to Allen [29] and Allen and Stanier [30],
as follows: mixed soil samples from different sites in each treatment were collected, us-
ing a knife, from a depth of 0–10 cm and placed into a plastic container under sterile
conditions. The dilution plate method was used to calculate the total count of bacteria.
The total bacterial count was determined on Thornton’s medium, amended with 50 mg
of pentachloronitrobenzene, 40 mg of actidione, and 35 mg of pimaricin, then incubated
at 30 ◦ C. The total cyanobacterial count was calculated by mounting tenfold serial soil
suspension-dilutions in triplicate onto an agar-coated BG11 medium. The MPN tubes were
incubated at 25 ◦ C in the presence of light (50 µmol photon m− 2 s− 1 ).

2.11. Statistical Analysis

Prior to performing two-way analysis of variance (ANOVA), the homogeneity and
Shapiro–Wilks normality test of error variance for all variables were investigated. The
results showed that all the data were homogeneous enough to support additional ANOVA
testing. The combined analysis was carried out for all variables utilizing ANOVA based on
the split-plot system in RCBD using the InfoStat computer program (version 2020b). Using
Duncan’s multiple range as a post hoc test (DMRT) at p ≤ 0.05, the variable means were
compared [49]. Mean values are presented as mean ± standard error.

3. Results
3.1. Morpho-Physiological Traits
The herb weights and leaf pigment values presented in Table 2 reveal that chia plants
exposed to foliar application with different microalgae strains showed significant differ-
ences in the herb fresh and dry weights, and in leaf pigments, relative to the soil drenching
technique. Herb fresh and dry weights as well as total chlorophyll and carotenoids of
foliar-sprayed plants exhibited 4.26, 4.76, 3.81, and 16.28% increases as compared with
plants treated with soil drenching, respectively. Microalgae strains also considerably af-
fected the vegetative parameters, viz., herb fresh and dry weights and total chlorophyll
and carotenoids, and treatment with A. platensis significantly resulted in the highest values,
followed by C. vulgaris and N. muscorum. By the mean of the interaction treatments, the
foliar spray with A. platensis was the most effective treatment, when compared to other
treatments, as the plants significantly revealed the highest values in this respect, while the
lowest values were produced by untreated control plants.

3.2. Yield and Yield Components

The results presented in Table 3 show the yield and yield component responses of chia
plants cultivated under alkaline conditions to microalgae treatments with two application
methods. The yield and yield component traits were impacted by season, method, microal-
gae, and their interactions (Table 3, Figure 1). Number of inflorescence plant−1 showed
a different trend when compared with other yield component traits (1000 seed weight,
seed and oil yields), as chia plants that received foliar spray with microalgae showed a
significant increase in their inflorescence number plant−1 relative to plants treated with soil
drenching (Table 3). Foliar-sprayed plants with A. platensis showed the highest number of
inflorescence plant−1 , although the differences with the rest treatments were not significant.
Biology 2022, 11, 1844 7 of 23

Table 2. Herb fresh and dry weights and total chlorophyll and carotenoid contents of chia plants
cultivated under alkaline stress conditions in response to different application methods, microalgae
strains, and their interaction.

Herb Fresh Weight Herb Dry Weight Total Chlorophyll Total Carotenoids
Treatment
(g) (g) (SPAD) (mg mm−2 )
Season (S)
SI 400.2 ± 0.07 b 101.2 ± 2.32 b 61.3 ± 2.91 b 0.39 ± b
SII 414.3 ± 0.05 a 106.3 ± 2.21 a 65.8 ± 2.90 a 0.47 ± a
Methods
Spray 417.2 ± 3.11 a 105.0 ± 2.41 a 62.9 ± 2.88 a 0.43 ± 0.04 a
Drench 399.4 ± 3.14 b 100.3 ± 2.71 b 60.5 ± 2.67 b 0.36 ± 0.05 b
Microalgae
Control 323.1 ± 0.90 d 80.5 ± 0.83 d 36.5 ± 0.22 e 0.23 ± 0.01 d
Arthrospiraplatensis 489.0 ± 3.40 a 123.3 ± 1.85 a 77.1 ± 0.67 a 0.68 ± 0.02 a
Chlorella vulgaris 438.6 ± 3.54 b 110.7 ± 1.63 b 72.1 ± 0.69 b 0.50 ± 0.03 b
Nostoc muscorum 394.2 ± 3.38 c 99.6 ± 0.84 c 63.9 ± 1.15 c 0.28 ± 0.01 c
Anabaena azollae 396.8 ± 3.08 c 99.4 ± 0.87 c 59.0 ± 0.46 d 0.28 ± 0.01 c
Methods × Microalgae
Control 323.0 ± 1.17 g 81.1 ± 0.42 g 36.7 ± 0.03 i 0.23 ± 0.01 g
A. platensis 505.2 ± 2.87 a 127.3 ± 0.72 a 78.2 ± 0.61 a 0.73 ± 0.01 a
Spray C. vulgaris 453.0 ± 1.27 c 114.3 ± 0.32 c 73.6 ± 0.37 c 0.58 ± 0.01 c
N. muscorum 401.5 ± 0.89 e 101.4 ± 0.22 e 66.3 ± 0.60 e 0.30 ± 0.01 e
A. azollae 403.5 ± 0.91 e 101.2 ± 0.55 e 59.8 ± 0.30 g 0.29 ± 0.01 e
Control 323.2 ± 1.64 g 80.0 ± 1.72 g 36.1 ± 0.39 i 0.22 ± 0.01 g
A. platensis 472.8 ± 1.77 b 119.2 ± 0.47 b 75.9 ± 0.70 b 0.63 ± 0.01 b
Drench C. vulgaris 424.2 ± 2.10 d 107.0 ± 0.53 d 70.6 ± 0.28 d 0.43 ± 0.01 d
N. muscorum 386.9 ± 1.68 f 97.7 ± 0.41 f 61.6 ± 0.88 f 0.26 ± 0.01 f
A. azollae 390.2 ± 1.54 f 97.5 ± 0.39 f 58.3 ± 0.61 h 0.26 ± 0.01 f
p-value
S <0.001 *** <0.001 *** 0.004 ** <0.001 ***
Methods 0.002 ** 0.008 ** 0.006 ** <0.001 ***
Microalgae <0.001 *** <0.001 *** <0.001 *** <0.001 ***
Methods × Microalgae <0.001 *** <0.001 *** 0.006 ** <0.001 ***
** and *** indicate differences at p ≤ 0.01 and ≤ 0.001 probability levels, and “ns” indicates non-significant
difference. Mean values sharing the same lowercase letter for methods, microalgae, and their interaction in the
same column do not differ significantly at p ≤ 0.05 by DMRT. Mean values are presented as mean ± standard
error.

Regarding the traits of 1000 seed weight, seed yield plant−1 , and seed yield ha−1 , the
plants exposed to the drenching application presented higher values than the plants treated
with the foliar spray method, as the former method resulted in 2.69% higher 1000 seed
weight and 10% increases in both seed yield plant−1 and seed yield ha−1 . Chia plants
subjected to A. platensis showed a significant increase in the seed yield plant−1 (52%),
1000 seed weight (51.4%), and seed yield ha−1 (52.3%) as compared with those of non-
microalgae-treated plants. Concerning the N. muscorum or A. azollae strains, they did
not differ significantly for all yield parameters except 1000 seed weight. All interaction
treatments showed a significant increase in all seed traits as compared with untreated
Biology 2022, 11, 1844 8 of 23

Biology 2022, 11, 1844 8 of 23

controls. However, the soil drenching method with A. platensis was the most efficient of all
interactions.

Figure
Figure 1.
1. Seed yield ha
Seed yield ha −−11 (t)
(t) (a),
(a), oil
oil %
%−1(b),
−1
(b),and
andoil
oilyield
yield ha
ha−1(L)
−1
(L)(c)
(c)ofofchia
chiaplants
plantscultivated
cultivated under
under
alkaline stress conditions in response to different application methods, microalgae strains, and their
alkaline stress conditions in response to different application methods, microalgae strains, and their
interaction. The bars with different letters are significantly different at p ≤ 0.05 by DMRT. Mean
interaction. The bars with different letters are significantly different at p ≤ 0.05 by DMRT. Mean
values are presented as mean ± standard error.
values are presented as mean ± standard error.
Concerning oil percent and oil yield, drenching treatment significantly increased oil
Concerning oil percent and oil yield, drenching treatment significantly increased oil
percent and oil yield traits, as it induced a 4.49 and 14.72% increase as compared with
percent and oil yield traits, as it induced a 4.49 and 14.72% increase as compared with
foliar treatments, respectively (Figure 1). The highest oil yield of chia seeds was obtained
foliar treatments, respectively (Figure 1). The highest oil yield of chia seeds was obtained
by the A. platensis microalgae strain; among the microalgae strains, N. muscorum‐ and A.
by the A. platensis microalgae strain; among the microalgae strains, N. muscorum- and A.
azollae‐treated plants showed the lowest oil percent and oil yield, with a non‐significant
azollae-treated plants showed the lowest oil percent and oil yield, with a non-significant
difference
difference between
between them.
them. On
On the
the other
other hand,
hand, untreated
untreated plants
plants significantly
significantly recorded
recorded the
the
lowest
lowest values in this respect (22.4% and 119 L for oil percent and yield, respectively). The
values in this respect (22.4% and 119 L for oil percent and yield, respectively). The
soil
soil drenching
drenching treatment
treatment with
with A.A. platensis
platensis outperformed
outperformed interactions,
interactions, and
and the interaction
the interaction
treatments significantly increased oil characteristics as compared to untreated controls.
treatments significantly increased oil characteristics as compared to untreated controls.
Biology 2022, 11, 1844 9 of 23

Table 3. Number of inflorescence plant −1 , seed yield plant−1 , and 1000 seed weight of chia plants
cultivated under alkaline stress conditions in response to different application methods, microalgae
strains, and their interaction.

Treatment No. Inflorescence Plant−1 Seed Yield Plant−1 (g) 1000 Seed Weight (g)
Season (S)
SI 16.3 ± 1.31 b 14.56 ± 0.76 b 1.80 ± 0.16 b
SII 19.5 ± 1.22 a 16.78 ± 0.64 a 1.89 ± 0.12 a
Methods
Spray 17.9 ± 1.50 a 14.40 ± 0.86 b 1.81 ± 0.11 b
Drench 15.2 ± 1.38 b 16.00 ± 0.1.11 a 1.86 ± 0.11 a
Microalgae
Control 9.2 ± 0.83 d 9.59 ± 0.03 d 1.08 ± 0.03 e
Arthrospiraplatensis 24.4 ± 0.95 a 19.99 ± 0.69 a 2.22 ± 0.01 a
Chlorella vulgaris 20.3 ± 0.83 b 18.32 ± 0.45 b 2.13 ± 0.02 b
Nostoc muscorum 14.8 ± 0.71 c 14.19 ± 0.37 c 1.93 ± 0.02 c
Anabaena azollae 13.9 ± 0.75 c 13.91 ± 0.47 c 1.81 ± 0.03 d
Methods × Microalgae
Control 10.2 ± 0.62 a 9.56 ± 0.03 f 1.08 ± 0.00 g
A. platensis 26.2 ± 0.64 a 18.5 ± 0.05 b 2.19 ± 0.00 b
Spray C. vulgaris 21.5 ± 1.33 a 17.3 ± 0.09 c 2.09 ± 0.01 c
N. muscorum 16.2 ± 0.52 a 13.51 ± 0.30 e 1.92 ± 0.03 d
A. azollae 15.2 ± 0.58 a 13.07 ± 0.54 e 1.75 ± 0.00 f
Control 8.2 ± 0.52 a 9.61 ± 0.05 f 1.08 ± 0.01 g
A. platensis 22.5 ± 0.86 a 21.49 ± 0.36 a 2.25 ± 0.01 a
Drench C. vulgaris 19.2 ± 0.60 a 19.30 ± 0.17 b 2.16 ± 0.02 b
N. muscorum 13.4 ± 0.47 a 14.87 ± 0.39 d 1.94 ± 0.01 d
A. azollae 12.6 ± 0.86 a 14.75 ± 0.32 d 1.87 ± 0.01 e
p-value
S 0.013 * <0.001 *** <0.001 ***
Methods 0.017 * <0.004 ** 0.021 *
Microalgae <0.001 *** <0.001 *** <0.001 ***
Methods × Microalgae 0.804 ns 0.149 ns 0.004 **
*, **, and *** indicate differences at p ≤ 0.05, ≤ 0.01, and ≤ 0.001 probability levels, and “ns” indicates non-
significant difference. Mean values sharing the same lowercase letter for methods, microalgae, and their interaction
in the same column do not differ significantly at p ≤ 0.05 by DMRT. Mean values are presented as mean ± standard
error.

3.3. Fatty Acid Profile

The results presented in Table 4 reveal that all treatments modified the bioactive
ingredient proportion of chia oil. Under alkaline stress conditions, the interaction between
different methods and microalgae showed twenty compounds in the extracted oil, and all
interactions resulted in an increase in all components of the oil. Total saturated fatty acids
(ranging from 6.51 to 11.81%) contained mainly lauric, myristic, pentadecanoic, palmitic,
margaric, stearic, docosanoic, and tetracosanoic acids, with a predominant proportion of
palmitic acid (C16:0). The highest percentage of total saturated fatty acids (7.68%) was
found in the oil of chia plants supplied with C. vulgaris soil drench. Palmitoleic, oleic,
arachidic, and gadoleic fatty acids accounted for most of the total monounsaturated fatty
Biology 2022, 11, 1844 10 of 23

acids (ranging from 7.01 to 20.52%). Anabaena azollae soil-drenching-treated plants contained
the highest percentage (15.59%) of oleic acid (C18:1 n-9). Total polyunsaturated fatty acids
ranged from 72.42 to 86.23%, with the majority of linoleic (C18:2 n-6), α-linolenic (C18:3
n-3), and γ-linolenic (C18:3 n-6) acids, whereas plants treated with Arthrospira platensis soil
drenching had the highest percentage of this fatty acid (86.2%). Linoleic (C18:2 n-6) and
γ-linolenic (C18:3 n-6) acid constituted the group of n-6, whilst n-3 was represented by
α-linolenic acid (C18:3 n-3). The ratio of n-6 to n-3 was determined to be in the range of
0.30 to 0.40. Plants treated with Arthrospira platensis foliar spray had the greatest n-6: n-3
ratio (0.40).

3.4. Biochemical Traits

The total protein, total carbohydrate, total phenolic, total flavonoid, and DPPH con-
tents of chia plants in response to microalgae supplementation in two different application
methods under alkaline stress conditions are presented in Table 5. The drenching method
considerably outperformed the spray method for total protein, total carbohydrate, total
phenolic, total flavonoid, and DPPH contents by 5.39, 3.88, 5.80, 12.1, and 2.61%, respec-
tively. Furthermore, microalgae strains significantly outperformed the untreated controls
in terms of total carbohydrate, total phenolic, total flavonoid, and DPPH contents (21.2,
109.9, 139.6, and 16.8% for A. platensis, and 11.2, 91.5, 101.2, and 12.5% for C. vulgaris, re-
spectively). In contrast, application of N. muscorum and A. azollae achieved the highest total
protein content in chia seeds in comparison to the control and the other microalgae strains.
The chemical composition and antioxidant content of chia seeds were both considerably
raised by all interactions between the application techniques and microalgae. In compar-
ison to the rest of the interactions, the A. platensis soil drenching treatment substantially
(p ≤ 0.01) induced an increase in total carbohydrates (24.9%), total phenolics (114.7%), total
flavonoids (152.6%), and DPPH (18.1%), while application of the N. muscorum or A. azollae
soil drenching resulted in an increase in the total protein content in seeds by 56.8 and 57.4%,
respectively. Foliar-sprayed plants with A. platensis were placed in the second rank in this
respect, as they exhibited 40.1%, 43.7 mg 100 g−1 , 22.3 mg 100 g−1 , and 70.2 µg mL−1 for
total carbohydrates, total phenolics, total flavonoids, and DPPH. Among all microalgae
applications, foliar application with N. muscorum recorded the lowest values in this respect.

3.5. Nutrient Content

The results in Table 6 show that microalgae supplementation with two methods of
application had a significant impact on the uptake and accumulation of K+ , Ca2+ , Fe2+ ,
Mg2+ , Zn2+ , and Na+ in comparison to untreated plants. Mineral content of K+ , Ca2+ , Fe2+ ,
Mg2+ , and Zn2+ in chia seeds collected from soil-drenching-treated plants were substantially
higher than those obtained from foliar-sprayed plants by 6.75, 11.4, 7.79, 12.3, and 6.33%,
respectively, while Na+ content in the seeds was lower by 5.47%. In contrast, P content in
seeds did not differ significantly between both methods. Statistical significance variations
between the microalgae treatments were noted for P, K+ , Ca2+ , Fe2+ , Mg2+ , Zn2+ , and Na+ .
The highest macro- and micronutrient levels were obtained by A. platensis-treated plants. In
contrast, Na+ content in seeds was dramatically reduced by the microalgae application as
compared to the control plants. Drenching plus A. platensis was substantially more efficient
than spray with control, resulting in significant increases in the levels of K+ , Fe2+ , Mg2+ ,
and Zn2+ by 82, 78.2, 104.1, and 63.1%, respectively. Meanwhile, foliar-sprayed plants with
N. muscorum exhibited the lowest nutrient content as compared with other microalgae
applications. The lowest Na value was produced by chia plants foliar sprayed with A.
platensis, being 57.8% lower as compared with control plants.
Biology 2022, 11, 1844 11 of 23

Table 4. The interactive effect of different application methods and various microalgae applications
on the fatty acid profile of chia plants cultivated under alkaline stress.

Spray Drench
N. Fatty Acids
(%) A. C. N. A. A. C. N. A.
Control platensis vulgaris muscorum azollae Control platensis vulgaris muscorum azollae
Butyric
1 0.01 nd nd nd nd 0.04 0.07 0.13 Nd nd
(C4:0)
Caproic
2 0.01 nd nd nd 0.07 0.24 0.11 0.06 Nd 0.14
(C6:0)
Caprylic
3 0.01 nd 0.09 nd 0.06 0.16 0.04 0.14 Nd 0.15
(C8:0)
Capric
4 0.05 0.04 0.12 0.19 0.14 0.11 0.08 0.11 Nd 0.21
(C10:0)

5 Undecanoic nd nd nd 4.02 nd nd Nd nd Nd nd
(C11:0)
Lauric
6 0.02 0.04 nd 4.01 0.21 0.11 0.08 0.08 Nd nd
(C12:0)
Myristic
7 0.04 0.07 nd 0.71 0.08 0.06 0.04 0.08 0.05 nd
(C14:0)

8 Pentadecanoic 0.04 0.28 0.09 0.05

nd nd nd nd Nd nd
(C15:0)

9 Palmitic 6.98 5.98 7.01 6.81 6.82 6.34 6.91 7.68 6.89 7.65
(C16:0)

10 Palmitoleic 0.09 0.11 0.17 0.18 0.16

nd nd nd Nd nd
(C16:1)
Margaric
11 nd nd nd nd nd nd Nd 0.06 Nd nd
(C17:0)
Stearic
12 nd 0.24 nd nd nd nd Nd nd Nd nd
(C18:0 n-9)

13 Oleic 11.01 6.78 9.38 7.01 15.59 12.06 5.69 7.6 8.01 7.88
(C18:1 n-9)

14 Linoleic 17.26 20.15 18.19 18.63 17.01 16.01 20.97 19.92 20.03 19.27
(C18:2 n-6)

15 α-linolenic 57.24 60.02 61.71 58.34 59.10 55.47 63.8 61.38 58.01 61.53
(C18:3 n-3)

16 γ-Linolenic 4.05 2.11 0.62 0.94 1.46 1.98 0.57

nd nd Nd
(C18:3 n-6)

17 Arachidic 7.23 0.83 0.66 nd 0.10 8.24 0.45 0.35 7.01 1.52
(C20:0)

18 Gadoleic 0.05 0.82 0.42 0.05 0.14

nd nd Nd Nd nd
(C20:1)
Docosanoic
19 nd 0.94 0.31 nd nd nd 0.07 0.13 Nd 0.68
(C22:0)
Tetracosanoic
20 nd nd nd nd nd nd Nd nd Nd 0.4
(C24:0)
Area total 100%
SFA 7.04 7.31 7.32 11.81 7.20 6.51 7.15 8.03 6.94 8.73
MUFA 18.38 8.43 10.46 7.01 15.80 20.52 6.32 8.25 15.02 9.40
PUFA 74.50 84.22 82.01 76.97 76.73 72.42 86.23 83.28 78.04 81.37
n6/n3 ratio 0.30 0.40 0.33 0.32 0.30 0.31 0.35 0.36 0.35 0.32
Data are shown as percentages of the total methyl esters. SFA, saturated fatty acid; MUFA, monounsaturated fatty
acid; PUFA, polyunsaturated fatty acid; nd, not determined.

3.6. Soil pH and Microbial Count

Results illustrated in Figure 2 show that soil treated with microalgae via the drenching
method resulted in significant differences in the total counts of soil microorganisms (bacteria
and cyanobacteria) and the pH values, as compared to those of non-microalgae-treated
plants. The soil pH value was significantly decreased relative to the control; effects were
greatest with the application of A. platensis, followed by C. vulgaris, N. muscorum, and A.
azollae, via drenching (by 8.44, 6.81, and 5.23%, respectively). Likewise, the most noticeably
increasing bacteria counts was shown in the application of A. platensis, followed by C.
vulgaris and N. muscorum, via drenching. The greatest significant increases in cyanobacteria
counts were observed with the application of A. platensis, followed by N. muscorum or A.
azollae, and then C. vulgaris, as a soil drench.
Biology 2022, 11, 1844 12 of 23

Table 5. Total protein, total carbohydrate, total phenolic, total flavonoid, and DPPH levels in
chia seeds cultivated under alkaline stress conditions in response to different application methods,
microalgae strains, and their interaction.

Total Total
Treatment Total Protein Total Phenolic DPPH
Carbohydrates Flavonoids
(%) (mg 100 g−1 ) (mg 100 g−1 ) (µg mL−1 )
Season (S)
SI 24.1 ± 0.73 b 36.1 ± 0.61 b 34.5 ± 2.06 b 15.7 ± 1.03 b 65.2 ± 0.92 b
SII 25.4 ± 0.98 a 37.5 ± 0.63 a 36.5 ± 2.10 a 17.6 ± 1.01 a 66.9 ± 0.81 a
Methods
Spray 24.2 ± 0.89 b 36.3 ± 0.58 b 34.7 ± 2.04 b 15.8 ± 1.15 b 65.3 ± 0.86 b
Drench 25.2 ± 1.03 a 37.3 ± 0.75 a 36.3 ± 2.20 a 17.5 ± 1.26 a 66.8 ± 0.99 a
Microalgae
Control 18.3 ± 0.06 d 33.9 ± 0.02 d 21.2 ± 0.05 e 9.64 ± 0.09 e 60.7 ± 0.19 d
Arthrospiraplatensis 25.7 ± 0.24 b 41.1 ± 0.46 a 44.5 ± 0.37 a 23.1 ± 0.39 a 70.9 ± 0.35 a
Chlorella vulgaris 23.6 ± 0.23 c 37.7 ± 0.23 b 40.6 ± 0.44 b 19.4 ± 0.46 b 68.3 ± 0.44 b
Nostoc muscorum 28.0 ± 0.34 a 35.7 ± 0.22 c 35.2 ± 0.45 d 15.9 ± 0.48 c 65.2 ± 0.54 c
Anabaena azollae 28.0 ± 0.36 a 35.7 ± 0.22 c 36.0 ± 0.60 c 15.3 ± 0.48 d 65.1 ± 0.39 c
Methods × Microalgae
Control 18.3 ± 0.09 g 33.8 ± 0.04 g 21.1 ± 0.09 i 9.46 ± 0.12 i 60.7 ± 0.29 f
A. platensis 25.1 ± 0.01 d 40.1 ± 0.00 b 43.7 ± 0.03 b 22.3 ± 0.07 b 70.2 ± 0.03 b
Spray C. vulgaris 23.1 ± 0.01 f 37.1 ± 0.01 d 39.7 ± 0.06 d 18.4 ± 0.07 d 67.2 ± 0.03 c
N. muscorum 27.2 ± 0.03 b 35.2 ± 0.04 f 34.2 ± 0.07 h 14.2 ± 0.06 h 64.0 ± 0.46 e
A. azollae 27.2 ± 0.01 b 35.2 ± 0.03 f 34.6 ± 0.05 g 14.8 ± 0.07 g 64.4 ± 0.44 e
Control 18.3 ± 0.10 g 33.9 ± 0.03 g 21.2 ± 0.01 i 9.81 ± 0.04 i 60.8 ± 0.32 f
A. platensis 26.2 ± 0.02 c 42.2 ± 0.03 a 45.3 ± 0.15 a 23.9 ± 0.33 a 71.7 ± 0.21 a
Drench C. vulgaris 24.1 ± 0.01 e 38.2 ± 0.03 c 41.6 ± 0.17 c 20.4 ± 0.17 c 69.4 ± 0.16 b
N. muscorum 28.7 ± 0.10 a 36.2 ± 0.01 e 36.2 ± 0.04 f 16.4 ± 0.10 f 66.3 ± 0.06 d
A. azollae 28.8 ± 0.09 a 36.2 ± 0.02 e 37.3 ± 0.07 e 16.9 ± 0.03 e 65.7 ± 0.31 d
p-value
S <0.001 *** <0.001 *** <0.001 *** <0.001 *** <0.001 ***
Methods 0.004 ** <0.001 *** <0.001 *** 0.002 ** <0.001 ***
Microalgae <0.001 *** <0.001 *** <0.001 *** <0.001 *** <0.001 ***
Methods × Microalgae <0.001 *** <0.001 *** <0.001 *** <0.001 *** 0.011 *
*, **, and *** indicate differences at p ≤ 0.05, ≤ 0.01, and ≤ 0.001 probability levels, and “ns” indicates non-
significant difference. Mean values sharing the same lowercase letter for methods, microalgae, and their interaction
in the same column do not differ significantly at p ≤ 0.05 by DMRT. Mean values are presented as mean ± standard
error.
Biology 2022, 11, 1844 13 of 23

Table 6. Mineral content (P, K, Ca2+ , Fe2+ , Mg2+ , Zn2+ , and Na2+ ) of chia seeds cultivated under alkaline stress conditions in response to different application
methods, microalgae strains, and their interaction.

P K+ Ca2+ Fe2+ Mg2+ Zn2+ Na+

Treatment
(mg g−1 Dry Seed)
Season (S)
SI 3.38 ± 0.35 a 22.9 ± 1.11 b 11.2 ± 0.64 b 7.51 ± 0.35 b 6.72 ± 0.37 b 3.91 ± 0.21 b 2.00 ± 0.14 b
SII 3.41 ± 0.32 a 25.8 ± 1.14 a 13.1 ± 0.54 a 8.39 ± 0.42 a 7.95 ± 0.42 a 4.51 ± 0.18 a 2.13 ± 0.13 a
Methods
Spray 3.37 ± 0.39 a 23.7 ± 1.17 b 11.4 ± 0.54 b 7.70 ± 0.36 b 6.81 ± 0.36 b 3.95 ± 0.17 b 2.12 ± 0.15 a
Drench 3.43 ± 0.28 a 25.3 ± 1.23 a 12.7 ± 0.71 a 8.30 ± 0.45 a 7.65 ± 0.48 a 4.20 ± 0.19 a 2.01 ± 0.15 b
Microalgae
Control 2.15 ± 0.01 c 17.5 ± 0.15 e 9.13 ± 0.02 d 6.07 ± 0.02 d 5.53 ± 0.10 d 3.15 ± 0.01 d 3.08 ± 0.02 a
Arthrospira platensis 4.62 ± 0.23 a 30.7 ± 0.28 a 15.2 ± 1.05 a 10.4 ± 0.15 a 10.1 ± 0.38 a 5.07 ± 0.03 a 1.36 ± 0.05 e
Chlorella vulgaris 3.52 ± 0.19 ab 27.6 ± 0.47 b 13.8 ± 0.31 b 8.88 ± 0.34 b 7.64 ± 0.23 b 4.50 ± 0.17 b 1.87 ± 0.04 d
Nostoc muscorum 2.92 ± 0.04 bc 23.5 ± 0.53 c 11.1 ± 0.26 c 7.34 ± 0.08 c 6.46 ± 0.01 c 3.83 ± 0.04 c 1.94 ± 0.03 c
Anabaena azollae 3.80 ± 0.90 ab 23.2 ± 0.39 d 11.0 ± 0.25 c 7.30 ± 0.09 c 6.44 ± 0.13 c 3.83 ± 0.05 c 2.09 ± 0.02 b
Methods × Microalgae
Control 2.17 ± 0.01 a 17.2 ± 0.04i 9.09 ± 0.02 a 6.06 ± 0.04 g 5.34 ± 0.06 h 3.14 ± 0.01 g 3.03 ± 0.02 a
A. platensis 4.10 ± 0.01 a 30.1 ± 0.01 b 13.6 ± 1.67 a 10.1 ± 0.04 b 9.23 ± 0.06 b 5.01 ± 0.01 b 1.28 ± 0.06 a
Spray C. vulgaris 3.10 ± 0.01 a 26.6 ± 0.16 d 13.1 ± 0.02 a 8.11 ± 0.00 d 7.14 ± 0.04 d 4.11 ± 0.01 d 1.82 ± 0.06 a
N. muscorum 2.84 ± 0.01 a 22.4 ± 0.12 g 10.5 ± 0.05 a 7.13 ± 0.01 f 6.18 ± 0.10 f 3.75 ± 0.03 f 1.90 ± 0.05 a
A. azollae 4.66 ± 1.82 a 22.4 ± 0.26 g 10.5 ± 0.03 a 7.14 ± 0.01 f 6.15 ± 0.04 f 3.75 ± 0.06 f 2.04 ± 0.03 a
Biology 2022, 11, 1844 14 of 23

Table 6. Cont.

P K+ Ca2+ Fe2+ Mg2+ Zn2+ Na+

Treatment
(mg g−1 Dry Seed)
Control 2.13 ± 0.01 a 17.9 ± 0.03 h 9.16 ± 0.04 a 6.08 ± 0.03 g 5.73 ± 0.10 g 3.15 ± 0.02 g 3.12 ± 0.01 a
A. platensis 5.14 ± 0.02 a 31.3 ± 0.01 a 16.9 ± 0.06 a 10.8 ± 0.03 a 10.9 ± 0.01 a 5.12 ± 0.02 a 1.43 ± 0.05 a
Drench C. vulgaris 3.95 ± 0.03 a 28.6 ± 0.21 c 14.4 ± 0.23 a 9.64 ± 0.06 c 8.15 ± 0.04 c 4.88 ± 0.06 c 1.91 ± 0.05 a
N. muscorum 2.99 ± 0.01 a 24.7 ± 0.20 e 11.7 ± 0.07 a 7.47 ± 0.07 e 6.74 ± 0.04 e 3.91 ± 0.01 e 1.99 ± 0.01 a
A. azollae 2.95 ± 0.01 a 23.9 ± 0.36 f 11.6 ± 0.04 a 7.54 ± 0.03 e 6.73 ± 0.01 e 3.92 ± 0.00 e 2.13 ± 0.01 a
p-value
S 0.057 ns <0.001 *** 0.013 * <0.001 *** <0.001 ** <0.001 ** 0.004 **
Methods 0.887 ns 0.011 * 0.046 * 0.002 ** 0.003 ** 0.001 ** 0.034 *
Microalgae 0.007 ** <0.0001 *** <0.001 *** <0.001 *** <0.001 ** <0.001 ** <0.001 **
Methods × Microalgae 0.184 ns <0.001 *** 0.092 ns <0.001 *** <0.001 ** <0.001 ** 0.853 ns
*, **, and *** indicate differences at p ≤ 0.05, ≤ 0.01, and ≤ 0.001 probability levels, and “ns” indicates non-significant difference. Mean values sharing the same lowercase letter for
methods, microalgae, and their interaction in the same column do not differ significantly at p ≤ 0.05 by DMRT. Mean values are presented as mean ± standard error.
 were greatest with the application of A. platensis, followed by C. vulgaris, N. muscorum,
and A. azollae, via drenching (by 8.44, 6.81, and 5.23%, respectively). Likewise, the most
noticeably increasing bacteria counts was shown in the application of A. platensis, fol‐
lowed by C. vulgaris and N. muscorum, via drenching. The greatest significant increases in
cyanobacteria counts were observed with the application of A. platensis, followed by N.
Biology 2022, 11, 1844 15 of 23
muscorum or A. azollae, and then C. vulgaris, as a soil drench.

Figure 2.
Figure pH (a),
2. pH (a), total
total cyanobacteria
cyanobacteria (b),
(b), and
and total
total bacterial
bacterial count
count (c)
(c) of
of soil
soil cultivated
cultivated by
by chia
chia plants
plants
under alkaline stress conditions in response to soil drenching application method with
under alkaline stress conditions in response to soil drenching application method with microalgae microalgae
treatments. The bars with different letters are significantly different at pp ≤
different at 0.05 by
≤ 0.05 by DMRT.
DMRT.

4. Discussion
4. Discussion
4.1. Growth and Yield Attributes
4.1. Growth and Yield Attributes
Climate change and inadequate farming practices have caused agricultural land to
become morechange
Climate alkaline,and inadequatethe
threatening farming practices
sustainability ofhave caused agricultural
environmental quality and land to
food
become
production.moreOne alkaline,
of thethreatening
suggested the sustainability
remediation of environmental
strategies quality
is to use effective and food
microalgae
production. One of the suggested remediation strategies is to
with different application methods. These factors might make microalgae an essential use effective microalgae
with
part ofdifferent application
an agricultural methods. strategy
production These factors might make
for controlling microalgae
alkalinity. Ouran results
essential part
reveal
of an agricultural production strategy for controlling alkalinity.
that all microalgae strains significantly improved chia growth and yield. Microalgae Our results reveal that all
microalgae strains significantly improved chia growth and yield.
can stimulate plant growth through atmospheric nitrogen fixation [50], increasing IAA, Microalgae can stimu‐
late plant growth
gibberellin, through atmospheric
and cytokinin levels in plants nitrogen fixation
[51–53]. [50], increasing
Microalgae are usedIAA, gibberellin,
as biofertilizers
and cytokinin levels in plants [51–53]. Microalgae are used as biofertilizers
because of their high content of bioactive components, including pigments (chlorophyll because of their
high content of bioactive components, including pigments (chlorophyll
a, b, β-carotene, phycobilin, phycoerythrin, and xanthophyll), phenolics, peptides, and a, b, β‐carotene,
phycobilin,
lipids [54], as phycoerythrin, and xanthophyll),
well as high protein phenolics,
content and levels peptides, andpolyamines,
of micronutrients, lipids [54], as well
natural
as high protein content and levels of micronutrients, polyamines, natural
enzymes, carbohydrates, amino acids, and vitamins [55], all of which affect overall plant enzymes, carbo‐
hydrates,
metabolism, amino acids, of
synthesis and vitamins [55],pigments,
photosynthetic all of whichandaffect overallactivity,
enzymatic plant metabolism,
causing an
synthesis
improvement of photosynthetic
in plant growth pigments, and enzymatic
and productivity. activity,microalgae
Furthermore, causing ancontributed
improvement in
to an
plant
increasegrowth and productivity.
in endogenous hormone Furthermore,
content, whichmicroalgae contributed
is responsible to an increase
for branch in en‐
development,
dogenous
postponinghormone content,and
leaf senescence, which is transition
floral responsible forUnder
[56]. branch development,
stress conditions,postponing
microalgal
leaf senescence,
can alter certain and floral transition
biochemical processes [56].
to Under
produce stress conditions,
antagonistic microalgalleading
compounds, can alterto
certain biochemical
plant tolerance [57]. processes
The favorable to produce
seed and antagonistic compounds,
oil yields obtained in theleading
currentto plantmay
study toler‐
be
ance
due to [57].
theThe favorable
positive effectseed and oil yields
of microalgae obtained inorganic
as a high-value the current study may
and growth be due
regulator to
[58].
Additionally,
the positive effectthis of
may be because
microalgae as a microalgae have a and
high‐value organic positive
growtheffect on relative
regulator water
[58]. Addi‐
content and
tionally, thisnutrient
may be status
because [59], increasinghave
microalgae the ability for leaf
a positive metabolism,
effect on relativecell elongation,
water content
and expansion [60], as well as providing effective regulation of primarily the zinc finger
protein-160 [61].
The present report reveals better performances in herb fresh and dry weights, inflo-
rescence number, and leaf pigments by foliar application with A. platensis than with other
strains. The drenching treatments with the same microalgae strain showed a biostimulant
effect on chia plants, having a significant impact on all yield component traits. The bios-
timulating effect of foliar application on the growth has already been shown clearly at the
vegetative stage (Table 2), whereas at the reproductive stage, the drenching application
was more effective than the foliar spray application. These results indicate that each appli-
cation method has quite different mechanisms of action (Figure 3). Regarding the foliar
spray treatment, foliar sprays quickly provide plants with maximal nutrient absorption
and utilization [62]. Additionally, based on literature data, it appears to primarily affect
nitrogen metabolism, with simultaneous increases in citrate synthase activity in plants,
 ostimulating effect of foliar application on the growth has already been shown clearly at
the vegetative stage (Table 2), whereas at the reproductive stage, the drenching applica‐
tion was more effective than the foliar spray application. These results indicate that each
application method has quite different mechanisms of action (Figure 3). Regarding the
Biology 2022, 11, 1844 foliar spray treatment, foliar sprays quickly provide plants with maximal nutrient absorp‐ 16 of 23
tion and utilization [62]. Additionally, based on literature data, it appears to primarily
affect nitrogen metabolism, with simultaneous increases in citrate synthase activity in
plants,may
which which bemay be directly
directly related related to the GS‐GOGAT
to the GS-GOGAT pathway’s pathway’s
synthesis synthesis of α‐ketoglu‐
of α-ketoglutarate as
tarate as a precursor
a precursor [63]. Soil [63]. Soil drenching
drenching application application
provides provides
plants with plants with the nutrients
the required required
nutrients slowly,the
slowly, enhances enhances
nutrientthe nutrient in
availability availability in the
the root zone, androot zone, the
improves andyield
improves the
traits [64].
yield traits [64]. Soil drenching appears to primarily affect the Krebs cycle
Soil drenching appears to primarily affect the Krebs cycle in terms of carbon metabolism, in terms of car‐
bon metabolism,
as seen as seen by the
by the simultaneous simultaneous
increase increase
in citrate synthase in citrate synthase
and malate and malate de‐
dehydrogenase en-
hydrogenase
zymes [56]. These enzymes
results[56].
are These
in line results
with theare in lineobtained
findings with thebyfindings obtainedetby
Mohamadineia al. Mo‐
[65],
hamadineia et al. [65],
Faheed and Abd-El Faheed
Fattah [66],and Abd‐El Fattah
Agathokleous [66],
et al. Agathokleous
[67], Puglisi et al.et al. [67],
[68], Puglisi et
and Suchithra
et al.
al. [69],
[68], andbutSuchithra
disagree with
et al. Li et al.
[69], but[70], who found
disagree with that
Li etfoliar application
al. [70], who found of microalgae
that foliar
extract was of
application more successful
microalgae than was
extract soil drenching in promoting
more successful than soilthe growth and
drenching quality of
in promoting
seeds.
the growth and quality of seeds.

Figure 3. The mechanisms of foliar spray and soil drenching with microalgae supplementation on
Figure 3. The mechanisms of foliar spray and soil drenching with microalgae supplementation on
chia plants cultivated under alkaline stress conditions.
chia plants cultivated under alkaline stress conditions.

4.2.
4.2. Oil
Oil and
and Fatty
Fatty Acid
Acid Profiles
Profiles
The
The current studyalso
current study alsodemonstrates that
demonstrates thethe
that application of different
application microalgae
of different with
microalgae
with both methods caused a considerable increase in the oil percentage and fatty acid
both methods caused a considerable increase in the oil percentage and fatty
composition in chia seeds. These traits were positively correlated with increased growth,
nutrition uptake, and biochemical determination. Chia oil’s fatty acid composition is
made up of a combination of saturated and unsaturated fatty acids, which are further
divided into monounsaturated and polyunsaturated fatty acids depending on the number
of unsaturated bonds. The increase in oil content and fatty acid composition may be credited
to microalgae’s capacity to synthesizes lipids, including a wide range of fatty acids [71],
particularly long-chain polyunsaturated fatty acids from the omega-3 and omega-6 groups
and carotenoids such as β-carotene, astaxanthin, and lutein [72]. Additionally, linoleic,
linolenic, and palmitic acids, which are frequently found in chia oil, made up the majority
of the fatty acids found in the isolated microalgae lipid [73,74].
In addition, the application of microalgae with both methods under alkaline conditions
increased the ratio of omega-6 to omega-3. The difference in the positions of the first double
bond in the carbon chains of fatty acids (n-3 and n-6 PUFA) is the cause of the stark
disparities in their biological roles that may result from the course of their interactions [75].
Biology 2022, 11, 1844 17 of 23

The medicinal effectiveness of microalgae is commonly acknowledged to depend on these

components [76]. Its anti-inflammatory and antioxidant effects are significant [77]. The
microalga and its separated chemicals can have the following effects: anti-neural [78],
anti-liver toxicity [79], anti-diabetes, immunomodulatory [80], cardiovascular disease
prevention, and anticancer [81]. Hence, the significance of the seeds as a result of these
effects in terms of medicine, food, and food supplements is high, helping to improve the
general health of humans.

4.3. Physiological and Biochemical Analysis

Leaf pigment levels in chia plants showed values significantly higher than the re-
spective controls at all sampling treatments. Leaf chlorophyll content is an indicator of
overall plant growth as it is critical for photosynthesis, which provides energy for plant
growth, metabolism, and reproductive activities [82]. Carotenoids are another form of
light harvesting system that also showed an increase in content (Table 2), stimulating the
photosystems’ capability to perform light interception and transfer the energy absorbed
to the reaction centers [82]. Carotenoids are significant antioxidant pigments that play a
crucial role in photosynthesis as well as plant disease defense [51].
Carotenoids make up most of the pigments in the antenna complexes. In plant leaves,
the level of chlorophyll is an indicator of how N is distributed [83]. A significant improve-
ment in protein content occurred in chia plants treated with N. muscorum and A. azollae
rather than other microalgae strains. Nostoc muscorum has the capacity to fix atmospheric
nitrogen and a good source of protein [50]. Additionally, Anabaena is a rich source of
protein and necessary amino acids [84]. Similarly, foliar and soil applications positively
influenced the protein content. This improvement in protein content is probably essential
to support the enhanced growth of microalgae-treated plants [82]. The increase in fresh
and dry leaf weights in treated chia plants was likely related to the increased content
of soluble compounds (pigments and proteins) in the leaves, as evidenced by the strict
relationship between carbon fixation and solar radiation absorption [85]. Likewise, signifi-
cant increases in antioxidants (phenols, flavonoids, and DPPH) were seen in response to
the application of microalgae with different methods under alkaline stress, as compared
to non-microalgae treated (Table 4). This may be attributable to the beneficial effects of
microalgae as a biofertilizer [86], which also led to a rise in the activity of enzymes that help
plants absorb nutrients, including nitrate reductase, dehydrogenase, alkaline or acidic phos-
phatase, and activity of ROS-scavenging enzymes such as catalase [87]. These parameters’
substantial increases could be explained by the fact that they are secondary metabolites [88].
Proteins, carbohydrates, and antioxidants as osmolytes and osmoprotectant compounds
are essential for controlling osmotic pressure during alkaline stress conditions [89,90]. In
addition, using various microalgae application methods to cultivate chia plants under
high-alkalinity conditions, the accumulation of these osmolytes improved the plant’s ability
to withstand alkalinity stress through the biosynthetic regulation of osmolytes and certain
plant hormones [91]. Osmolytes’ beneficial impact on stressed chia plants may be due to
their function in maintaining membrane stability and reducing plant cell physiological
dehydration [92]. A similar pattern of osmolyte accumulation under varied stresses was
reported by Tran et al. [93], Gr et al. [94], Singh et al. [95].

4.4. Nutrient Content

Microalgae improve the soil’s quality, resulting in healthy plants and increased nutri-
ent uptake and accumulation, thus increasing productivity [25]. Alkaline-stressed plants
treated with various microalgae applications via drenching or spraying showed lower Na+
concentrations in seeds than untreated plants, which may indicate that the microalgae pre-
cluded Na+ uptake by roots and prevented their transfer to shoot tissues [96]. Even though
non-selective K+ channels make it simple for Na+ to enter plant cells, K+ is most often used
in the cytoplasm to modify the osmotic pressure under alkalinity-related situations [97].
Contrarily, the significant increases in macro and micronutrient contents observed after
Biology 2022, 11, 1844 18 of 23

a variety of microalgae application methods under alkalinity stress conditions may be

attributable to the membrane’s resilience by promoting the H+ -ATPase activity in the cell
membrane [98], which increases the uptake of K and Ca nutrients and enhances the uptake
of water and photosynthesis [52]. Similarly, Ertani et al. [99] observed that plants treated
with this microalga showed greater accumulation of macro and micro elements. Herein,
the different microalgae application methods successfully mitigated the adverse effects
resulting from alkaline stress on the growth and productivity of chia plants

4.5. Soil pH and Microbial Traits

In this study, all microalgae applications caused an increase in the bacteria and
cyanobacteria count and significantly reduced the pH levels in the experimental soil
(Figure 3). This may be due to a rise in the number of microorganisms in the rhizosphere [100]
and their impacts on the quantity of organic materials in microbial biomass [101], as well as mi-
crobial byproducts that contain extracellular polysaccharides and cell wall fragments [102].
Additionally, the application of microalgae improved soil biological activity since microal-
gae are a continuously replenishing source of carbon [103]. Kapil et al. [104] stated that
cyanobacteria application is a significant strategy in bioremediation of alkaline soil as
it declines the soil pH from 9.2 to 7.5. Among all microalgae applications, C. vulgaris
showed the lowest cyanobacteria count, but with a significant increase relative to untreated
soil. That may be due to their vital role in decreasing the soil pH (Figure 2), as generally,
cyanobacteria prefer neutral pH for optimum growth and establishment [105].
In this study, A. platensis was superior to microalgae as an application spray or drench-
ing method. Arthrospira platensis is a species of photosynthetic cyanobacteria with a high
nutritional content [106], and there has been a growing interest in its biological activity and
bioactive components [54]. Furthermore, A. platensis showed effects similar to those of IAA
and gibberellin, which stimulate root elongation and establishment as well as accelerate
vegetative growth [53,107]. Additionally, it is favorable for a variety of applications in
the life sciences because it is Food and Drug Administration (FDA) certified as Generally
Recognized as Safe (GRAS) [108], and can support special biological functions [109].

5. Conclusions
Microalgae application by the methods of foliar spray and soil drenching is a potential
strategy for enhancing the growth and production of chia plants under alkaline stress
conditions. As a great source of nutrients and growth hormones, the microalgae improved
alkalinity resistance in chia by modulating soil, nutrient homeostasis, and photosynthetic
effectiveness. Furthermore, the application of microalgae via different methods can greatly
enhance seed and oil yields and optimize the plant’s composition in terms of total protein
and carbohydrate contents, non-enzymatic compounds (phenolics and flavonoids), and
DPPH, as well as alter fatty acid profiles. Therefore, applying various microalgae applica-
tions by foliar spray or soil drenching would be a potential and eco-friendly approach for
enhancing agricultural productivity in alkaline environments. Our findings also suggest
that the application of Arthrospira platensis using the soil drenching technique should be
used in the future to enhance plant growth and productivity under alkaline soil conditions.

Author Contributions: Conceptualization, S.M.Y. and A.A.A.A.; methodology, S.M.Y. and A.A.A.A.;
software, S.M.Y. and R.S.E.-S.; K.Z.G. validation, K.Z.G., A E. and A.A.A.A.; formal analysis, S.M.Y.,
R.S.E.-S., K.Z.G.; investigation, S.M.Y., R.S.E.-S., A.A.A.A.; resources, S.M.Y., R.S.E.-S.; data cura-
tion, S.M.Y., R.S.E.-S.; writing—original draft preparation, S.M.Y., R.S.E.-S., A.A.A.A., A.E., K.Z.G.;
writing—review and editing, S.M.Y., R.S.E.-S., A.A.A.A., A.E. and K.Z.G.; visualization, S.M.Y. and
K.Z.G.; supervision, S.M.Y., A.A.A.A. and K.Z.G.; project administration, S.M.Y. and K.Z.G.; funding
acquisition, S.M.Y., R.S.E.-S., A.A.A.A., A.E. and K.Z.G. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Biology 2022, 11, 1844 19 of 23

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.
Acknowledgments: The authors gratefully acknowledge Abdel-Nasser El-Sheshtawy professor in
Environment and Bio-Agriculture Department, Faculty of Agriculture, Al-Azhar University for
scientific revision of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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