Cyanobacteria like Arthrospira platensis and Nostoc muscorum are known to be beneficial for plant development. They can improve plant growth, yield, proximate content (protein and carbohydrate), and stimulate plant tolerance to abiotic stresses like herbicides. In the present study, the impacts of brominal herbicide (Bh) alone or in combination with natural cyanobacterial or synthetic tryptophan treatments were investigated on the morphological and physiological parameters of wheat plants. The Bh treatment caused a reduction in all the estimated morphological and physiological parameters of the plants. The combination of Bh and cyanobacterial or tryptophan treatments could significantly increase the plant length, fresh and dry weights, and yield parameters (spike length, spike weight, number of spikes per plant, number of grains per spike, weight of 1000 grains, and productivity). In addition, pigments, carbohydrates, and protein content was also reduced in response to the Bh treatment, while the antioxidant enzyme activity and lipid peroxidation increased. Priming wheat grains in cyanobacterial aqueous extract and tryptophan before cultivation reversed the toxic effects of Bh application, amplified extra antioxidant ability, and decreased the lipid peroxidation of the plants. Finally, the combination of cyanobacterial and tryptophan as treatments for priming wheat grains before being sown in the soil should help to strengthen the defense systems of the wheat plant to tolerate the adverse effects of species-specific Bh upon application.
Hinweise
Data availability
All data generated or analyzed during this study are included in this published article.
Introduction
Wheat is the third-largest crop behind rice and corn in total world production (Shewry and Hey 2015). Wheat accounts for 20% of the world’s food calories, with the primary uses being in bread production, foodstuffs, and livestock and fowl feed. Industrial applications include the production of starch, alcohol, and oil. Approximately 95% of the total wheat production comes from common bread wheat (Snape and Pánková 2007), which serves as the main food source for 30% of the human population. Triticum aestivum L. is a hexaploid species with 21 pairs of chromosomes, which is rich in protein, carbohydrates, and minerals. In the last ten years, universal wheat production has been insufficient to meet requests as declared by FAO (2013). Furthermore, as the global population grows, researchers and growers will need to increase wheat production by approximately 70% to meet future demand (Singh et al. 2017a). In Egypt, the wheat plant (T. aestivum L.) is one of the essential cereal crops that is being used for food, is cultivated in larger areas of Egypt (Kareem et al. 2021), and plays a critical role as the basic food necessity for people. Growing food demands have primarily increased the production of several agrochemicals such as herbicides, fungicides, and insecticides to achieve high and good quality crop yields. Agrochemicals improved crop plants by killing weeds and pests but simultaneously caused pollution to the main environmental components, soil, water, and air, not to mention serious problems for human health. Herbicides cause unfavorable effects on crop plants, such as inhibiting seed germination, reducing shoot and root growth, and accelerating oxidative stress (Halliwell 2006). In the present study, brominal herbicide (Bh) was used as a post-emergence herbicide for controlling weeds in the field of the wheat plant. Bh inhibits photosynthetic system II (PSII). These PSII inhibitors can stop energy production and negatively affect plant respiration. Disrupting electron transport causes superoxide ions (O−2) to be released, destroying cell membranes and inhibiting chlorophyll formation, leading finally to plant death (Forouzesh et al. 2015).
Biological degradation, removal, or accumulation of these chemicals is a process called bioremediation, which was established by many organisms, including cyanobacteria. A large number of cyanobacterial species, such as Nostoc linckia, Oscillatoria salina, Plectonema terebrans, N. muscorum, Oscillatoria animalis, Aphanocapsa sp., Synechococcus sp., and Phormidium foveolarum, have beneficial applications in the remediation of many toxicants, including heavy metals, pesticides, crude oil, phenanthrene, naphthalene, catechol, and xenobiotics contaminated soil and water (Fioravante et al. 2010; Kumar et al. 2016; Singh et al. 2016). Furthermore, cyanobacteria can produce an unlimited number of substances such as growth-promoting regulators, vitamins, amino acids, fatty acids, and exopolysaccharides, all of which can enhance the growth and development of the plant (Sood et al. 2011).
Anzeige
L‑tryptophan is an essential amino acid with an indole ring. It is a precursor of auxin, and its agricultural application increases the level of auxin in plants (Palego et al. 2016). Many studies have reported that the application of tryptophan improves crop growth, productivity, and quality (El-Awadi and Hassan 2011; Hussein et al. 2014; Mustafa et al. 2016).
The purpose of this study was to examine the influence of priming wheat (T. aestivum L.) grains in cyanobacterial aqueous extract of A. platensis and N. muscorum as protective treatments against the toxic action of Bh. In addition, the effects of the cyanobacterial extract were compared with those of tryptophan, a synthetic treatment usually used for stimulating the tolerance of wheat plants under Bh treatment.
Materials and Methods
Isolation, Identification, and Purification of Cyanobacterial Species
Arthrospira platensis (A. platensis) and Nostoc muscorum (N. muscorum) were isolated from various local municipal water bodies. Both cyanobacterial species were isolated and purified according to the method described by Stein (1973). The two selected axenic species were identified using the morphological and taxonomic features of the purified isolates as described by Desikachary (1959). The two species were further confirmed as Arthrospira platensis (Gomont) and Nostoc muscorum (Calothrix C. Agardh ex Bornet and Flahault) using the AlgaeBase website (http://www.algaebase.org).
Preparation of Cyanobacterial Aqueous Extract
Biomass of cyanobacterial species was obtained by centrifuging the culture at 4000 rpm (Fisher CenterificTm Centrifuge) for 15 min, and the pellets (biomass) were washed twice with autoclaved growth medium followed by distilled water (Rogers and Burns 1994). The biomass was collected by centrifugation and dried in a freeze dryer for 12 h. Finally, the dried powders were stored at −20 °C until used. Later, 100 mL of distilled water was added to the obtained pellet, and the obtained suspension was shaken for 48 h at 25 °C on a shaker (VS-8480) for stimulation to extract secondary metabolites from the cells. Supernatants were collected by centrifugation after extraction, as previously mentioned, and dried in a freeze dryer for 12 to 15 h until a constant weight was obtained.
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Gas Chromatography-Mass Spectroscopy (GC-MS) for the Aqueous Extracts of A. platensis and N. muscorum
The chemical composition of A. platensis and N. muscorum aqueous extract (1%) was determined using a GC/MS instrument (Trace GC-ISQ mass spectrometer, Thermo Scientific, USA). The instrument was equipped with an A3000 autosampler and a TG-5MS capillary column of 30 m in length, 0.25 mm internal diameter, and 0.25 μm film thickness. The temperature program was set from 50 to 280 °C at a rate of 10 °C/min. A mass spectrometer was set in electron ionization mode at 70 eV, source temperature at 200 °C, interface temperature at 220 °C, and the injector temperature was 220 °C. A diluted sample of 1 μL was injected in splitless mode and a mass scan of 50–600 amu was performed. Helium was used as a carrier gas with a 1 mL/min flow rate (El-Kareem et al. 2016). The components were identified tentatively by comparing their relative retention times and mass spectra with WILEY (Wiley Registry of Mass Spectral Data, 9th Edition Version 1.02) and NIST 05 (NIST/EPA/NIH mass spectral library version 2.0d) mass spectral databases.
Treatments and Conditions of the Experiment
The wheat grains (T. aestivum L. Giza 171) were obtained from the Main Crops Improvement Station in Kafr El-Sheikh City, Egypt. Selected wheat grains were primed for 12 h in aqueous cyanobacterial extract (1%) and were also primed with a 100 ppm tryptophan solution (0.01 g in 100 mL dH2O) for 12 h (Osman et al. 2021). Primed grains were sown in pots (35 cm in diameter and 30 cm in depth) containing equal amounts of loamy soil. The soil analyses (pH, electrical conductivity (EC), field capacity, nitrogen (N), and phosphorous (P) content) were determined according to the methods described by Black et al. (1965). The soil properties are shown in Table 1.
Table 1
Physical and chemical properties of the cultivated soil
Soil
Clay: sand (2:1 w/w)
pH
7.3
EC
0.8 (m mohs cm−1)
Field capacity
57%
N‑content
2.6 (mg/g dry weight)
P‑content
2.4 (mg/g dry weight)
The pots were kept in a greenhouse (Botany Department, Faculty of Science, Tanta University) under normal conditions of daylight and temperature. The recommended fertilization program by the Ministry of Agriculture of ammonium nitrate and superphosphate was added to the surface area of a hectare. Bh was applied to plants after 55 days of cultivation as a foliar application (post-emergence herbicide) using the recommended dose of (100 L/hectare). All Bh treatments were carried out in the early morning. The amount of the applied Bh per pot was calculated according to its surface area and relative to the surface area of a hectare. The design of the pot experiment in the greenhouse was divided into five groups (in triplicates), which represent the different priming treatments at the recommended dose of (100 L/hectare) (Table 2).
Table 2
The design of the pot experiment in the greenhouse
Group
Treatments
1
Control plants: grains primed in distilled water and not sprayed by Bh
2
Positive control plants: grains primed in distilled water and sprayed by Bh
3
Treated plants: grains primed in tryptophan solution and sprayed by Bh
4
Treated plants: grains primed in N. muscorum aqueous extract and sprayed by Bh
5
Treated plants: grains primed in A. platensis aqueous extract and sprayed by Bh
Measurements of Growth and Yield Characteristics
Five plants were chosen from each treatment after seventy days of sowing and their growth parameters such as shoot length, root length, shoot (fresh and dry wt), and root (fresh and dry wt) were measured. Furthermore, the harvested wheat plant samples (120 days old) were spread over clean sheets of paper until dry. Yield parameters including the number of spikes per plant, spike length (cm), the weight of a spike (g), the number of grains per spike, and the weight of 1000 grains (g) of wheat plants were estimated. In addition, the productivity of wheat grains (weight of the yielded grains per pot (g)) was calculated.
Biochemical Analysis
Determination of Total Chlorophyll and Carotenoids Content
The chlorophyll pigments were determined according to the protocol described by Metzner et al. (1965). Briefly, 0.1 g of leaf tissue was homogenized with 5 mL of cold acetone (85%) and stored at 4 °C overnight. The absorbance of the solution was then measured at 663 nm, 644 nm, and 452 nm using an ultraviolet spectrophotometer (Unico2000, Canada), and the total chlorophyll content was calculated using the standard formulas given by Metzner et al. (1965).
Determination of Total Soluble Carbohydrates and Proteins Content
The carbohydrate content was determined spectrophotometrically using the phenol-sulfuric acid method at 490 nm (Dubois et al. 1956), and glucose was used as standard sugar. In addition, protein content was measured spectrophotometrically using the Bradford (1976) method at 595 nm with bovine serum albumin as a standard.
Determination of the Antioxidant Activities
The leaves of wheat plants were ground in 2.5 mL of cold phosphate buffer (pH 7.4) mixed with 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT). Later, the homogenate was centrifuged at 4000 rpm (Sigma Laborzentrifugen1K15) for 15 min. The supernatant was used for the determination of antioxidative (enzymatic and non-enzymatic) activities as follows:
a)
The enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST), and glutathione peroxidase (GPX) were assessed using the methods described by Nishikimi et al. (1972); Aebi (1984); Pabst et al. (1974); and Paglia and Valentine (1967), respectively.
b)
Non-enzymatic GSH content was detected by the method described by Ellman (1959). The intensity of the formed yellow color was measured at 420 nm. A calibration curve using GSH as a standard was prepared, and GSH enzyme activity was expressed as μmol/g fresh weight (FW).
Determination of Lipid Peroxidation
Lipid peroxidation was determined using the method described by Yoshioka et al. (1979). The formation of pink-colored malondialdehyde (MDA) was measured at 535 nm using a spectrophotometer against a blank containing water instead of samples. The MDA concentration was expressed as μmol/g FW of the sample after preparation of a calibration curve using 1,1,3,3-tetra methoxy propane as a standard.
Quantification of Total Phenolics and Flavonoid Content in the Harvested Wheat Grains
a)
Total phenolic content was estimated using the Folin-Ciocalteu reagent, as described by Jindal and Singh (1975). The total phenolic content was expressed in terms of mg of gallic acid equivalent/g dry weight (DW) of the sample.
b)
Total flavonoids were determined by the method described by Zhishen et al. (1999). The total flavonoid content was expressed as mg Quercetin (standard) equivalent/g DW of the sample.
Minerals Analysis in the Harvested Wheat Grains
The digestion method was used for the determination of the mineral elements in the wheat grains (Allen et al. 1974). Fine powdered grains (0.5) were mixed with 2 mL of perchloric acid (30%) and 4 mL of nitric acid and heated gently until the whole mixture turned into a clear solution. The solution was then diluted to a constant volume and used for the determination of N, P (spectrophotometrically), and the mineral content of sodium (Na+), potassium (K+), calcium (Ca2+), and magnesium (Mg2+) by using the Atomic Absorption Spectrophotometer (A.A.S., Perkin Elmer 2380).
Statistical Analysis
All results were evaluated as means ± standard deviation of five replicates (n = 5). Graphpad Prism 9.01 (Graphpad Software, San Diego, USA) was used for performing the statistical analysis of the obtained data. The pot experiment results were analyzed using a one-way analysis of variance with a significant difference of p < 0.05.
Results
GC-MS Analysis of A. platensis and N. muscorum Aqueous Extract (1%)
A. platensis Aqueous Extract
The GC-MS chromatogram (Fig. 1) of A. platensis aqueous extract showed several bioactive compounds which mainly contained 9‑octadecenoic acid (E)-, methyl ester (45%), hexadecanoic acid, methyl ester (34%), tetradecanoic acid, methyl ester (3.2%), and heptadecanoic acid, methyl ester (2.05%). The biological activities of these compounds are presented in Table 3.
Fig. 1
GC-MS chromatogram of A.platensis aqueous extract
Table 3
GC-MS analysis of A.platensis aqueous extract
RT
Compound Name
PA %
MF
Biological activity
19.14
Tetradecanoic acid, methyl ester
3.20
C15H30O2
Antimicrobial activities, anti-inflammatory
22.44
1,3,7-Trimethyl-1H-purine‑2,6(3H,7H)-dione
7.51
C8H10N4O2
Caffeine compound; antibacterial, antioxidant, and antiviral activities
23.29
Hexadecanoic acid, methyl ester
23.51
C17H34O2
Antioxidants, antibacterial, antifungal, nematicide, pesticides, ameliorate the effects of different toxic agents
23.39
Hexadecanoic acid, methyl ester
8.38
C17H34O2
Antioxidants, antibacterial, antifungal, nematicide, pesticides, ameliorate the effects of different toxic agents
24.52
Hexadecanoic acid, methyl ester
2.11
C17H34O2
Antioxidants, antibacterial, antifungal, nematicide, pesticides, ameliorate the effects of different toxic agents
RT Retention Time, PA Peak Area, MF Molecular Formula
×
N. muscorum Aqueous Extract
The GC-MS chromatogram (Fig. 2) of N. muscorum aqueous extract showed various bioactive compounds, such as 9‑octadecenoic acid (Z)-, methyl ester (58.35%), 9,12,15-octadecatrienoic acid (8.61%), pentadecanoic acid, 14-methyl-, methyl ester (8.48%), cyclooctasiloxane, hexadecamethyl (2.75%), cyclooctasiloxane, hexadecamethyl (2.75%), cyclononasiloxane, octadecamethyl- (2.28%), and cycloheptasiloxane, tetradecamethyl (0.75%). The biological activities of these compounds are presented in Table 4.
RT Retention Time, PA Peak Area, MF Molecular Formula
×
Morphological and Yield Measurements
The morphological changes that occurred in wheat plant samples collected at the vegetation stage are presented in Table 5. The results indicated that Bh treatment induced a significant decrease in shoot length (19.6%), root length (34.94%), FW (shoot 34.38% and root 47.86%), and DW (shoot 41.5% and root 47.73%) as compared to the values recorded for the control plants. The harmful impact of Bh was predominant on roots compared to shoots for all the measured growth parameters. However, the coapplication of Bh with various treatments ameliorated the toxicity of brominal on the studied growth parameters. The highest amelioration was detected for A. platensis+ Bh treatment, recording 17.23% for shoot length, 25.35% for root length, FW (shoot 29.78% and root 40.98%), and DW (shoot 14.84% and root 39.13%). The lowest values recorded in response to the T + Bh treatment were 7.37% for shoot length, 6.4% for root length, FW (shoot 12.42% and root 21.31%), and DW (shoot 5.16% and root 13.04%) in comparison to the Bh treatment (Table 5).
Table 5
Effect of different treatments on wheat plant growth parameters
Treatment
Length (cm)
Length (cm)
Fresh weight (g)
Dry weight (g)
Shoot
Root
Shoot
Root
Shoot
Root
Control
72.60 ± 5.5a
25.47 ± 1.4a
9.57 ± 0.7a
1.17 ± 0.06a
2.65 ± 0.3a
0.44 ± 0.06a
Bh
58.37 ± 3.47e
16.57 ± 1.2d
6.28 ± 0.4e
0.61 ± 0.01d
1.55 ± 0.2d
0.23 ± 0.02d
T + Bh
62.67 ± 4.4d
17.63 ± 1.15c
7.06 ± 0.3d
0.74 ± 0.04c
1.63 ± 0.08c
0.26 ± 0.03c
N. muscorm + Bh
65.00 ± 3.5c
18.30 ± 1.26c
7.72 ± 0.5c
0.84 ± 0.03b
1.69 ± 0.05c
0.29 ± 0.02b
A. platensis + Bh
68.43 ± 4.2b
20.77 ± 1.15b
8.15 ± 0.4b
0.86 ± 0.07b
1.78 ± 0.1b
0.32 ± 0.01b
Data are presented as the mean ± SD (n = 5). Different small subscript letters for each column indicated a significant difference between treatments at p < 0.05
Furthermore, the post-emergence spraying of Bh treatment caused a significant reduction in spike length (28.09%), spike weight (47.22%), number of spikes per plant (32.5%), number of grains per spike (35.27%), the weight of 1000 grains (39.57%), and productivity (73.61%) as compared to the control plants (Table 6). A combination of Bh with different treatments effectively ameliorated all of these yield parameters in wheat plants. The T + Bh treatment resulted in the lowest recovery of different yield parameters, i.e., spike length (7.11%), spike weight (22.11%), number of spikes per plant (23.33%), number of grains per spike (11.17%), the weight of 1000 grains (22.22%), and productivity (67.06%). However, the highest recovery was recorded with A. platensis + Bh treatment, i.e., spike length (16.28%), spike weight (52.6%), number of spikes per plant (37.04%), number of grains per spike (27.25%), the weight of 1000 grains (46.57%), and productivity 1.55-fold. Hence, the recovery effect of treatments could be arranged in the following order: A. platensis + Bh > N. moscorum + Bh > T + Bh treatments.
Table 6
Effect of different treatments on yield parameters of wheat plants
Treatment
Spike length (cm)
Spike weight (g)
No. of spike/plant
No. of grains/spike (g)
Weight of 1000 grains (g)
Productivity (g/pot)
Control
16.23 ± 1.2a
1.80 ± 0.05a
4.00 ± 0.3a
56.70 ± 6.2a
70.00 ± 10a
15.88 ± 2.1a
Bh
11.67 ± 1.6d
0.95 ± 0.01e
2.70 ± 0.15d
36.70 ± 5.4e
42.30 ± 5.5d
4.19 ± 0.3e
T + Bh
12.50 ± 1.3c
1.16 ± 0.01d
3.33 ± 0.2c
40.80 ± 3.8d
51.70 ± 6.2c
7.00 ± 0.6d
N. muscorm + Bh
13.23 ± 1.1b
1.28 ± 0.03c
3.50 ± 0.22c
42.60 ± 5.6c
55.00 ± 7.3c
8.20 ± 1.1c
A. platensis + Bh
13.57 ± 0.9b
1.45 ± 0.05b
3.70 ± 0.14b
46.70 ± 4.2b
62.00 ± 9.4b
10.70 ± 1.3b
Data are presented as the mean ± SD (n = 5). Different small subscript letters for each column indicated a significant difference between treatments at p < 0.05
Biochemical Analyses of Pigments, Carbohydrates, and Protein Content
Fig. 3 shows the effect of Bh alone or in combination with tryptophan and two cyanobacteria extracts on the content of the photosynthetic pigments (chlorophyll: chl a and chl b), carotenoids, and total pigments of wheat leaves. Treatment with only Bh caused a significant reduction in the contents of chl a (65.2%), chl b (66.5%), carotenoids (61.3%), and total pigments (62.86%) in comparison to control plants. However, combined treatments with Bh could effectively ameliorate the toxic action of Bh on the content of the pigments. The highest improvement was observed for A. platensis + Bh treatment by 1.13 fold for chl a, 1.05-fold for chl b, 88.89% for carotenoids, and 90.63% for total pigments. The induced pigment content in response to different treatments could be arranged in the following order: A. platensis + Bh > N. moscorum + Bh > T + Bh treatments over the recorded Bh values and the control values.
Fig. 3
a–d Effect of different treatments on Chl a (a), Chl b (b), carotenoids (c), and total pigments (d) contents of wheat plants. Data are presented as the mean ± SD (n = 5), different small subscript letters for each column indicate a significant difference between treatments at p<0.05
×
Furthermore, spraying of plants with only Bh significantly reduced the total carbohydrates and total protein content in wheat plants by 44.7 and 45.3%, respectively, in comparison to their values in control plants (Fig. 4). However, a combination of Bh with different treatments showed a significant recovery in the total carbohydrates and total protein content compared to those treated with Bh only. The recovery values could be arranged in the following sequence: 41.49 and 58.62% for carbohydrates and protein content, respectively, for A. platensis + Bh treatment; followed by 39.46 and 20.69% for carbohydrates and proteins, respectively, in the case of N. moscorum + Bh treatment; and finally 38.09 and 13.79% for carbohydrates and proteins, respectively, for T + Bh treatment.
Fig. 4
a,b Effect of different treatments on carbohydrates (a) and proteins (b) content of wheat plants. Data are presented as the mean ± SD (n = 5), different small subscript letters for each column indicate a significant difference between treatments at p<0.05
×
Antioxidant Enzymes Activity
Fig. 5 shows the antioxidative activity of SOD, CAT, GPX, GST, and GSH content. The Bh application alone caused a slight increase in the SOD activity by 5.04% as compared to the control. The combination of Bh with different treatments resulted in an increase in SOD content compared to Bh alone treatment. The highest recorded value was 1.14-fold for A. platensis + Bh treatment (Fig. 5a). Similarly, the application of Bh induced a marked increase in the activity of CAT (32.56%) and GST (39.53%) enzymes (Fig. 5b, d). The maximum enzyme content and activity were recorded for GSH (76.68%) and GPX (88.85%) due to Bh application (Fig. 5e, c). Similarly, CAT, GPX, GST activity, and GSH content were enhanced in wheat plants on treatment with T + Bh, N. moscorum + Bh, and A. platensis + Bh. The A. platensis + Bh treatment recorded the highest increases in CAT (10.53%), GPX (19%), GST (1.38-fold), and GSH (1.23-fold) in comparison with their recorded values with the Bh treatment alone. However, the lowest values for CAT (4.39%), GPX (13.4%), GST (38.89%), and GSH (56.27%) were established with T + Bh treatment (Fig. 5). Moreover, all the natural and chemical treatments stimulated significantly higher enzyme activity and content than that of the Bh alone treatment. The stimulating effect of different antioxidant enzyme contents with all treatments could be arranged as follows: GST > GSH > SOD > GPX > CAT.
Fig. 5
a–e Effect of priming grains for 12 h in different solutions on the antioxidant activities and GSH contents of the wheat seedlings. SOD (a), CAT (b), GPX (c), GST (d), and GSH (e). Data are presented as the mean ± SD (n = 5). Different small subscript letters for each column indicated a significant difference between treatments at p < 0.05
×
Oxidative Stress as Lipid Peroxidation
MDA content was quantified as an indication of lipid peroxidation. Fig. 6 revealed that Bh treatment alone caused a significant increase in MDA content, reaching 60.4% over that estimated for the control plants. Interestingly, the combination of Bh and different treatments caused a significant reduction in the accumulation of MDA content. The reduction in MDA content was arranged in the following order: 19.66% for A. platensis + Bh treatment, followed by 14.38% for N. moscorum + Bh treatment, and the least in T + Bh treatment, with a 12.68% recovery percentage.
Fig. 6
Effect of different safener treatments on the MDA content of wheat plants. Data are presented as the mean ± SD (n = 5). Different small subscript letters for each column indicate a significant difference between treatments at p<0.05
×
Secondary Metabolites Content in the Grains of the Harvested Wheat Plants
Phenolics Content
Fig. 7a shows that the phenolic compound content in the wheat grains decremented by 13.49% in response to the Bh application. The application of different treatments caused a significant enhancement in phenolic content. The maximum enhancement was verified for A. platensis + Bh by 6.57%, followed by N. muscorum + Bh (4.52%) and T + Bh (3.27%) compared to the Bh treatment alone.
Fig. 7
a,b Effect of different treatments on phenolics (a) and flavonoids (b) content of wheat plants. Data are presented as the mean ± SD (n = 5), different small subscript letters for each column indicate a significant difference between treatments at p<0.05
×
Flavonoids Content
Fig. 7b shows that the amount of flavonoids compound in the grains decreased by 15.67% in response to the Bh application. Safener treatments caused a notable recovery in the flavonoid content. The maximum increase was shown in response to A. platensis + Bh (9.6%), followed by N. moscorum + Bh (5.18%), and the least in T + Bh (4.42%) treatment as compared to the Bh treatment alone.
Mineral Content in the Grains of the Harvested Wheat Plants
The content of essential nutrient elements N, P, K, Na, K/Na, Ca, and Mg in the grains of the harvested wheat plants was estimated (Fig. 8 and Table 7). Fig. 8 shows that the N and P content decreased in response to Bh application by 27.82 and 50.47%, respectively, compared to control grains. The application of different treatments in combination with Bh caused a significant restoration in the N and P contents, and these increases could be arranged in the following order:
a)
For N content: A. platensis + Bh (19.79%) > N. moscorum + Bh (11.46%) > T + Bh (6.56%) (Fig. 8a).
b)
For P content: A. platensis + Bh (57.73%) > N. moscorum + Bh (37.85%) > T + Bh (19.87%) (Fig. 8b).
Fig. 8
a,b Effect of different safener treatments on the contents of Nitrogen (a) and Phosphorus (b) nutrient elements in the grains of the harvested wheat plants. Data are presented as the mean ± SD (n = 5). Different small subscript letters for each column indicate a significant difference between treatments at p<0.05
Table 7
Effect of Bh application alone or in combination with different treatments on the minerals content of grains of the harvested wheat plants
Treatment
Minerals ions (mg/g DW)
K
Na
K/Na
Ca
Mg
Control
12.90 ± 0.4a
14.57 ± 0.25a
0.88 ± 0.03a
7.63 ± 0.25a
3.37 ± 0.3a
Bh
8.40 ± 0.1b
18.60 ± 0.6b
0.45 ± 0.01b
3.70 ± 0.2b
1.52 ± 0.1b
T + Bh
9.14 ± 0.5c
17.87 ± 1.2c
0.50 ± 0.03b
4.40 ± 0.46c
1.83 ± 0.12c
N. moscorom + Bh
10.87 ± 1.1d
16.30 ± 0.9d
0.66 ± 0.05c
5.13 ± 0.15d
1.87 ± 0.14c
A. platensis + Bh
11.50 ± 1.5d
16.04 ± 0.69d
0.720 ± 0.1c
6.80 ± 0.4e
2.40 ± 0.4d
Data are presented as the mean ± SD (n = 5). Different small subscript letters for each column indicate a significant difference between treatments at p<0.05
×
Furthermore, the treatment with Bh caused a significant reduction in the contents of K, Ca, and Mg by 34.88%, 51.5%, and 54.9%, respectively, compared to the content of the control grains. Alternatively, the content of Na was amplified by 27.66% over the control (Table 7). Different treatments caused a significant improvement in the contents of K, Ca, and Mg, while the content of Na was reduced. These treatment effects are arranged in the following order: A. platensis + Bh > N. moscorum + Bh > T + Bh. The maximum increase in the content of K, Ca, and Mg were noted in response to A. platensis + Bh treatment with 36.90%, 83.78%, and 57.89%, respectively, compared to the control contents. Also, the main reduction in Na content was detected with A. platensis + Bh treatment by 13.76% (Table 7).
Correlation Between Growth, Biochemical, and Yield Parameters
The correlation between growth, biochemical, and yield parameters of the wheat plants under different treatments was determined (Fig. 9). All measured parameters showed a negative correlation due to the Bh application. On the contrary, the same parameters showed a positive correlation in response to different safener combinations with Bh. A notable exception was MDA, which exhibited a negative correlation with all measured parameters (Fig. 9).
Fig. 9
Correlogram showing the correlation analysis of growth, biochemical, and yield parameters under different Bh treatments
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Discussion
Cyanobacteria are very beneficial organisms due to their ability to produce many biological compounds such as hormones, vitamins, amino acids, and fatty acids (Alam et al. 2014). Several studies have been conducted to investigate the biostimulant properties of compounds secreted by cyanobacteria. Cyanobacterial biomass, filtrates, and/or extracts contain a wide diversity of bioactive secondary metabolites that have a positive influence on alleviating abiotic stresses in crops (Van Oosten et al. 2017; Múnera-Porras et al. 2020; Gonçalves 2021). In the present study, we examined aqueous extracts of two cyanobacteria, A. platensis, and N. muscorum, in addition to tryptophan, as they can induce the ability of wheat plants to tolerate Bh toxicity. The GC-MS analysis for A. platensis and N. muscorum extracts showed a higher content of fatty acid compounds. Studies have shown that microalgae and cyanobacteria can accumulate more than 70% of fatty acids per DW (Singh et al. 2017b). Furthermore, cyanobacteria contain different classes of lipids and fatty acids according to their nature, which varies from those found in animals and plants (Ryckebosch et al. 2012). Palmitic (hexadecanoic acid) and oleic acids (octadecenoic acid) are the most prevalent fatty acids produced by various strains of cyanobacteria such as Arthrospira sp., Nostoc sp., and Anabaena sp. (Kneeland et al. 2013). They have many biological activities such as antioxidant, antibacterial, antifungal, nematocidal, and pesticidal effects (Gołeȩbiowski et al. 2013). Fatty acids reserved by the cells of crop plants encourage oxidative stress mediation, which has a vital role in protecting plants against cytotoxic or genotoxic effects of different toxic agents, including herbicides (Lewinska et al. 2015). This report might explain the obtained results of this study concerning the increased antioxidant enzyme content of the primed wheat grains.
Herbicides (as xenobiotics) are frequently used to control the growth of weeds. They are different in structure, but they all cause side effects on crop plants. Herbicide application increases ROS formation, leading to oxidative stress, cellular damage, and changes in antioxidant activity (Lukatkin 2002). The results of the present study showed that the application of Bh alone induced reductions in all morphological growth and yield parameters, pigments, carbohydrates, and proteins of wheat plants. Similar results were obtained by Yoon et al. (2011), who demonstrated that paraquat herbicide-induced oxidative stress and ultrastructural changes in squash (Cucurbita spp.). The combination of nicosulfuron + isoxadifen-ethyl herbicides at different concentrations of 37.5, 75, 150, and 300 mg kg−1 could decrease shoot length by 38.8, 32.9, 25.7, and 24.8%, respectively. Furthermore, at the same previous concentrations, a decrease in DW of 79.1, 74.5, 75.9, and 72.1%, respectively, was observed in maize plants (Sun et al. 2017). Additionally, Brazier-Hicks et al. (2020) noted that the application of clodinafop herbicide in wheat and rice fields caused extreme crop damage.
Several studies have reported the passive effect of herbicide application on the biochemical parameters of plants, which is in agreement with our results. Nacheva et al. (2012) found that the application of the herbicides pendimethalin and napropamide reduced the pigment content in the leaves of Prunus domestica plants. Kumar (2012) demonstrated that wheat grains grown in different concentrations (0, 50, 100, 200, 400, 800, and 1200 ppm) of herbicides 2, 4‑D, and isoproturon had decreased the content of carbohydrates and proteins after 72 h of treatment in comparison to the control. Moreover, Gomes et al. (2019) found that the use of Roundup herbicide interrupted the growth of maize seeds, declined root elongation, and encouraged ROS accumulation in seeds. The application of Granstar herbicide in barley fields caused a drop in all yield parameters, such as the number of spikes per plant, spike length, spike weight, the number of grains per spike, and the weight of 100 grains (Abo-Shady et al. 2019). Furthermore, many studies have also reported that greater damage is observed from herbicides at later crop stages than at the early growth stages of the crop (Sciumbato et al. 2014; Hale et al. 2019; Oseland et al. 2021).
The combination of Bh with other natural or synthetic treatments could reduce the negative effects resulting from Bh spraying. These results were in harmony with other studies reporting that using cyanobacteria extracts can directly stimulate plant growth and development through the improvement of plant germination rate and characteristics, such as an increase in the shoot and root length, leaf area, and higher nutritional content (El Arroussi et al. 2016; Osman et al. 2021). Essa et al. (2015) described a significant enhancement of seed germination, shoot length, lateral roots, spike length, grain weight, protein content, micronutrients, and the endogenous phytohormone pool of sorghum durra var. aegyptiacum and Helianthus annuus L. var. Giza 102 plants after pre-priming their grains in Anabaena oryzae, Nostoc ellipsosporum, and Synechococcus sp. filtrates before cultivation. Additionally, Mógor et al. (2018) used A. platensis biomass as a stimulator for lettuce seedling growth in an organic matter-rich field. Furthermore, Dineshkumar et al. (2019) reported that Chlorella vulgaris and Arthrospira platensis increased growth performance at the early stage of maize (Zea mays L) in addition to increasing seed germination rates. Similarly, L‑tryptophan acts as a precursor of auxin synthesis (Zhao 2010), which regulates different physiological processes in plants. L‑tryptophan has a beneficial role in stimulating crop growth and refining productivity under different stress conditions (Hanc and Tuncer 2020; Bulgari et al. 2015). Tryptophan is used in many ways, such as seed priming (Bulgari et al. 2015), foliar spray (El-Awadi and Hassan 2011), and soil application (Muneer et al. 2009).
Environmental abiotic stresses can reduce crop yields by 50% (Ningombam et al. 2021). Abiotic stresses cause ROS generation, which leads to oxidative damage, peroxidation of lipids, oxidation of proteins, inhibition of enzymes, and destruction of DNA and RNA (Sharma and Dubey 2005). Crop plants activate complex antioxidant defense systems composed of several enzymatic and non-enzymatic antioxidants to improve the damage introduced by ROS (Rady and Hemida 2016; Rady et al. 2016). In this study, the antioxidant enzymes, including SOD, CAT, GPX, GST activity, and GSH content, were increased in response to Bh treatment alone, with further increments shown by using combinations of Bh and different treatments (A. platensis + Bh, N. moscorum + Bh, and T + Bh). GSH is a non-enzymatic molecule that is the key to the antioxidant defense system. Glutathione reacts with oxygen (O2), hydroxyl, and hydrogen peroxide (H2O2) and plays a vital role as an electron donor through ROS detoxification and oxidation to glutathione disulfide (GSSG) (Sharma et al. 2012). Glutathione not only participates in detoxification processes but also controls the antioxidant enzymes. The SOD is usually reflected as the first line of defense against ROS in the detoxification process and alters O2 to H2O2, which is converted to H2O by CAT or H2O2 and may enter the ascorbate-glutathione cycle. Ascorbate-glutathione detoxification of ROS can be triggered by activating glutathione peroxidase or glutathione-S-transferase enzymes or by activating GST enzymes to degrade xenobiotics. In addition, the glutathione is converted into GSSG throughout ROS detoxification, and this GSSG is used again for GSH formation by the activation of the glutathione reductase enzyme (Gaafar and Seyam 2018).
Many researchers mention the reduction in the antioxidant activities of various crop plants in response to different herbicide applications (El-Tayeb and Zaki 2009; Foyer and Shigeoka 2011). The use of chlorotoluron and phenyl urea herbicides encouraged oxidative stress in wheat plants, resulting in the accretion of O2 and H2O2 in leaves and peroxidation of the lipids of the plasma membrane in plants (Xiao et al. 2008). In the same perspective, Badr et al. (2013) found that the application of metosulam herbicide to broad bean (Vicia faba) plants reduced peroxidase activity and induced the activities of catalase and ascorbate peroxidase. This implies that the combination of different treatments and Bh boosted more activation of the plant antioxidant defense system, which can protect wheat from oxidative injury caused by Bh treatment. These results were in agreement with Osman et al. (2016), who reported that priming of bean seeds in A. platensis suspension caused enhancement in the antioxidant enzymes, which led to the amelioration of the harmful effects due to fusillade super herbicide application. Further, Brito et al. (2022) noted that the cyanobacterium Oculatella lusitanica resolves salinity stress harmful effects on lettuce plant size and root FW by stimulating the non-enzymatic antioxidant system (proline, H2O2, and GSH). Similarly, MDA content is an indicator of lipid peroxidation and is a non-enzymatic oxidative stress marker when accumulated in plant cells (de Souza et al. 2017). ROS has been linked to herbicide toxicity and causes the loss of membrane integrity via lipid peroxidation, which is considered the most destructive molecular process in all organisms (Gill and Tuteja 2010). The development of lipid peroxidation content was also documented as a response to atrazine (Nemat Alla and Hassan 2006), glyphosate (Sergiev et al. 2006), and imazethapyr herbicides (Zabalza et al. 2007). According to the obtained results, pre-applied safener treatments significantly reduced the accumulation of MDA caused by the Bh application. Cyanobacterial treatments with A. platensis + Bh, N. moscorum + Bh, and T + Bh could alleviate the MDA production and recover the cell lipid peroxidation to levels near those of the control. In agreement with these results, Babu et al. (2015) found that using Anabaena torulosa, Anabaena laxa, Anabaena azollae, Anabaena oscillarioides, and Calothrix sp. caused an increased activity of defense enzymes and decreased the level of MDA. Also, Osman et al. (2016) reported that treatment of broad bean seeds with A. platensis culture suspension reduced the lipid peroxidation induced by using fusillade herbicide. Verma and Prasad (2021) reported that using Nostoc muscorum and Anabaena sp reduced the content of MDA induced by cadmium stress and augmented the growth and antioxidant defense system enzymes, which enriched the fertility of soil under the condition of metal contamination in a rice field.
Furthermore, concerning the reduction in the content of the biochemical constituents of wheat plants, Bh treatment decreased the content of the essential minerals in the harvested wheat grains. These results indicate that herbicides can cause a reduction in the ion uptake process (Rachoń and Szumiło 2009). Also, Kraska (2011) showed that the application of atrazine ranging from 10 to 100 ppm occasioned a reduction in the K and Na contents of wheat plants. Furthermore, Abo-Shady et al. (2019) found that the contents of N, K, Ca, Mg, and Fe were reduced by 31.5, 7.8, 55.8, 25.1, and 40.8%, respectively, due to Granstar application in barely planted fields in comparison to control.
Secondary plant metabolites are divided into several families, such as phenolics, terpenes, steroids, alkaloids, and flavonoids (Kessler and Kalske 2018). They serve several functions, including plant growth, development, defense, environmental stress responses, and responses to environmental stresses (Yang et al. 2018). Hence, lessening their content causes poor responses and weak stimulation of the defense mechanisms under different stress conditions (Isah 2019). Therefore, using biostimulants from cyanobacteria and microalgae could be very beneficial in enhancing growth, yields, quality, nutrient uptake, production of secondary metabolites of crop plants, and stimulating tolerance to abiotic stresses (Bulgari et al. 2015; Yakhin et al. 2017). The application of microalgal and cyanobacterial extracts can provide safety against abiotic stresses in different plants (Guzmán-Murillo et al. 2013). In the present study, the combination of Bh and different safener treatments reversed the damaging effect of the herbicide. Many studies have reported the involvement of greater nutrient uptake, elevated biomass, and larger crop yields when microalgae are used in combination with different environmental stresses (Stadler et al. 2006; Fareed and Abd-El Fattah 2008). In similar studies, Abd El-Baky et al. (2010) reported that the extracts of Spirulina spp. and Chlorella spp. enhanced wheat tolerance to salinity and improved the antioxidant ability and protein content of the grains produced by treating plants with algal extracts. Also, Abo-Shady et al. (2019) described how primed grains in the cyanobacterial suspension of Nostoc muscorum before cultivation ameliorated the drawback effect induced by Granstar herbicide on all yield parameters of the barley plants harvested from the grains. The improvement effect of biostimulants is different from one crop to another. The use of cyanobacteria or their extracts, as well as tryptophan amino acids, may improve crop growth and promote crop protection against stress factors, such as herbicide use (Toribio et al. 2020; Gonçalves 2021).
Conclusion
For wheat plants grown under herbicide stress, priming grains in cyanobacterial aqueous extract before cultivation proved to be a useful tool for stimulating growth progress, physiological processes, and yield. A. platensis+ Bh was the most effective natural treatment compared to Bh alone or in combination with N. moscorum + Bh or the synthetic T + Bh treatments. The current complementarity of A. platensis+ Bh treatment enriched the level of physiological, biochemical, and antioxidant defense systems, leading to the tolerance of wheat plants against Bh toxicity. Consequently, the study suggested priming crop grains with cyanobacterial extracts to provide additional protection against species-specific herbicides. More research should be conducted to screen cyanobacterial species, extracts, and biochemical components to select the most potent algal species as grain or seed safener agents.
Conflict of interest
M. E.-A.H. Osman, A.M. Abo-Shady, R.M. Gaafar, G.A. Ismail and M.M.F. El-Nagar declare that they have no competing interests.
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