Total chlorophyll concentration was determined in A. annua leaves using the [16] method: approximately 0.5 g of fresh leaves were ground and homogenized with 4 ml of 80% acetone. The resulting homogeneous mixture was filtered using filter paper (Wattman No. 4), and the filtrate, constituting the crude chlorophyll extract, was collected in a test tube for quantitative analysis. The optical densities of these extracts were read using a spectrophotometer at 652 nm against 80% acetone, and the concentrations were determined using the [16] equation before being adjusted to the levels in mg. g⁻¹of fresh matter.

Each month during the cultivation period, leaf samples were collected, weighed to determine fresh mass (FM), then immediately soaked in demineralized water and kept in the dark before being reweighed to determine turgor mass (TM). They were then placed in an incubator at 85˚C for 48 h and reweighed upon removal to determine dry mass (DMS). The values obtained for each sample were used to determine the relative water contents (RWC) (%) using the formula [(FM − DM)/(TM − FM)] × 100 [17].

Mineral elements were extracted from the leaves using the mineralization method proposed by [10]. This method consists of drying the sample in an oven at 105˚C for 24 hours, calcining it at a high temperature (550˚C) in a muffle furnace for 4 hours, and adding a mineral acid, followed by gentle heating after the ash has cooled. The resulting solution is used to determine the phosphorus, sodium, calcium, magnesium, zinc, and iron contents.

Phosphate ions are determined based on the principle of the formation of the yellow phospho-vanado-molybdate complex, measurable by molecular absorption spectrophotometry at 430 nm in the presence of the nitro-vanado-molybdate reagent [10]. Thus, 6 ml of distilled water and 2 ml of nitro-vanado-molybdate reagent are added to each test tube containing 2 ml of the previously prepared extract. The mixture is homogenized, incubated for 1 hour, and the optical density is read using a UV-VI spectrophotometer at 430 nm against the blank.

The principle for extracting K⁺, Na⁺, and Ca²⁺ ions is based on capturing light emissions from their excitation on heated leaf samples [10]. 5 g of the previously obtained leaf powder sample is diluted in 20 mL of 2N nitric acid; the resulting solution is transferred into a 50 ml volumetric flask, and the volume is adjusted to the mark with distilled water. The solution is filtered, and the optical densities are read using a flame photometer.

Mg²⁺, Zn²⁺, and Fe²⁺ ions are determined using the atomic absorption spectrometry method [10], based on the theory of atomic energy quantification. Thus, 5 g of the sample and 20 mL of 2 N nitric acid were added to a 50 mL volumetric flask, and the solution was adjusted with distilled water to the fill line. The optical densities were then read using an atomic absorption spectrophotometer. The contents of these mineral elements were automatically obtained using the Spectra Manager software, which calculated the concentration in mg/L of the extract run through the spectrophotometer from the calibration curve.

The extraction was performed according to the modified protocol of [18]; Approximately 0.5 g of fresh adult leaves of A. annua were cold-ground in 2 ml of hydrogen chloride. The resulting mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant is collected for measurement, which is based on the reduction of the Folin-Ciocalteu reagent in an alkaline medium due to the oxidation of phenolic compounds, producing a blue color whose absorption at 760 nm is proportional to the phenolic compound content in the extract. Thus, to a volume of 100 µl of the extract obtained previously, add 2.5 ml of distilled water, 10 µl of Folin-Ciocalteu and 0.5 ml of 20% Na₂CO₃. Incubate the mixture obtained in a water bath at 40˚C for 20 minutes and read the DO values using a spectrophotometer at 720 nm relative to distilled water. The phenolic compound content is expressed in mg. g⁻¹ of fresh matter by reference to the calibration curve established with hydrochloric acid (0.1 mg. ml⁻¹).

The extraction was performed according to the modified protocol of [19]. Approximately 0.5 g of freeze-dried leaves were ground and transferred to test tubes with 4 ml of a 70% acetone and 2% acetic acid (V/V) mixture. After homogenization, the mixture was incubated for one hour in the dark and centrifuged at 4500 rpm for five minutes. The first supernatant, which constitutes the crude flavonoid extract, was collected. Quantification of total flavonoids was determined using the modified colorimetric method [20]. Thus, 2.5 ml of distilled water and 500 µl of 5% sodium nitrite were added to each test tube containing 100 µl of extract. The mixture was incubated in the dark for 6 minutes. Then, 15 µl of 10 % aluminum trichloride and 500 µl of 1 M sodium hydroxide were added. The DO were read using a spectrophotometer at 510 nm against the blank. The flavonoid content was expressed in milligrams per liter of quercetin equivalent extract (mg EQ.ml⁻¹) of fresh material and converted to the levels in mg. g^-1 of fresh material.

Total protein extraction was performed according to the modified protocol of [21]. Approximately 0.5 g of fresh leaves was cold-ground in 2 ml of phosphate buffer. The resulting mixture was vortexed for 10 minutes, incubated, and centrifuged at 3000 rpm for 10 minutes at 7˚C. The collected supernatant constituted the crude protein extract. Successively introduce 2 μl of Molarite pH phosphate buffer, 10 μl of extract, and 2000 μl of Bradford reagent into a test tube, and, after 30 minutes of incubation at room temperature, the ODs are read at 595 nm using a spectrophotometer against distilled water. Protein quantities are expressed in mg. g⁻¹ of fresh matter by reference to the calibration curve established with BSA (Bovine Serum Albumin) 0.1 mg.ml⁻¹.

Data presented as (mean ± standard deviation) with (n = 5) were analyzed descriptively using GraphPad Prism 8 software; comparison of means was performed by analysis of variance (ANOVA) using the Student-Newman-Keuls test at the 5% threshold. The results obtained are represented in the form of graphs and tables using Microsoft Excel 2013 software and the Simultaneous multiple correlation comparisons using the Bonferroni method.

The data from the soil substrate analysis of the cultivation site are shown in Table 1. The analysis reveals that this soil has a clayey texture and an acidic pH. These characteristics are unsuitable for growing A. annua [22]. Indeed, the ideal pH range for optimal plant production of this plant species is between 5.0 and 6.4 [23]. Nevertheless, its average fertility (CEC 11.35 meq/100g of soil) and very low available phosphorus content (6.48 mg.kg⁻¹) and high C/N ratio demonstrate a slow organic matter mineralization and a low total nitrogen level [24]. The exchangeable cations and average cation exchange capacity in this field show that the clay-humic soil complex is dominated by calcium and potassium ions, with high levels, in contrast to calcium, magnesium, and sodium.

Table 1.Physicochemical characteristics of the soil.

Growth parameters, fresh biomass, and water content assessed in the different treatments varied significantly (p ≤ 0.05) over time (Table 2). Analysis showed that plants fertilized with ENTO + PW exhibited a significant increase (p ≤ 0.05) in the number of secondary branches of the five basal branches, the average number of axillary buds, average plant height, and stem collar diameter at month 3 compared to the positive control (Cont + PW). Similar observations were made with AMF compared to the control, and in the average number of roots from month 1 compared to the negative control. Similarly, the application of AMF resulted in a significant increase (p < 0.05) in the root collar diameter, the average number of secondary branches of the last five branches of the plants at month 3, and the average number of primary roots at the same period, compared to the negative control (cont.). While the highest plant height compared to the negative control was observed in the plot treated with Entomocompost (ENTO) compared to the negative control.

Similarly, the average fresh weight of the leaves as well as the water contents increased significantly (p ≤ 0.05) in all treatments compared to the negative and positive controls.

Table 2. Growth parameters and biomass assessed in A. annua plants in the applied treatments.

Control (Cont.); Arbuscular mycorrhizal Fungal (AMF); Entomocompost (ENTO); Control + Purified water (Cont. + PW); Arbuscular Mycorrhizal Fungi + Purified water (AMF + PW) and Entomocompost + Purified water (ENTO + PW). Values are the averages of five repetitions.

with ENTO + PW, the fresh biomass increased significantly (p ≤ 0.05) at month 2 compared to the negative control and the positive control. In the sub-block of plants watered only with purified water (positive control), there was no significant difference in fresh weight between the plants of the treated batches and those of the negative control. However, the contribution of AMF and ENTO in the culture medium led to a significant increase (p ≤ 0.05) of this parameter after month 1 compared to the control. The increase in the size of A. annua plants over time would be the result of the physiological response due to the supply of necessary nutrients through AMF on the one hand and watering with purified water (PW) on the other. This corroborates the results of [25] who showed that other biofertilizers, namely chicken droppings and cow purse, lead to an increase in the growth parameters of A. annua. Thus, fertilization of plants with AMF would contribute to the increase in the volume of hydromineral absorption following the development of a network of fungal hyphae that colonize the soil and offer a larger absorption surface to the plant [26]. These beneficial soil microorganisms also provide favorable conditions, namely a favorable pH, for plant development [27]-[29]. The results presented by plants treated with ENTO + PW followed by those treated with ENTO on these different parameters could be explained by the richness of this fertilizer in mineral elements, namely N, P, and K, essential for plant growth [30][31], which contributes to water balance and chlorophyll synthesis, which is explained by an increase in the total chlorophyll content in the leaves of A. annua as well as fresh biomass.

The slowdown in growth observed at month 1 in A. annua plants is thought to be due to the time taken to recognize the symbiotic partners (plants-AMF). The plant wastes energy feeding these non-productive microorganisms. From month 2 onwards, the microorganisms then become beneficial to the plant because the AMF would have already sufficiently branched the plant roots for better hydromineral nutrition, complementing the texture, nature, and pH of the soil, which play an important role in plant growth [27][29]. However, the decrease in the number of plant roots over time in the different treatments could be explained by root renewal, as was the case with ferns [32].

The results of plant biofertilization on total chlorophyll content assessed in A. annua leaves after harvest are presented in Figure 1. Analysis of this figure shows that: in plants watered with purified water, total chlorophyll content increased significantly (p ≤ 0.05) at month 1 in plants fertilized with AMF compared to the positive control and at month 2 in plants fertilized with AMF and ENTO compared to the two controls. Similarly, total chlorophyll content in A. annua leaves decreased over time.

The peak in chlorophyll levels in month 3 could be explained by the fact that young Artemisia plants are full of numerous non-senescent adult leaves that perform optimal photosynthesis during growth. However, over time, flowering and fruiting lead to a decline in photosynthetic activity due to leaf aging and the inhibition of new leaf growth [33]. The higher chlorophyll content during the first month and its decline from month 2, unlike the variation in phenolic compounds, could be explained by the fact that during the first month, the sugars produced primarily supported the fungi (AMF) in establishing the symbiosis [34]. Then, during the second and third months, the protein biosynthesis pathway was favored to the detriment of the phenylalanine ammonia lyase (PAL) pathway.

Figure 1. Leaf chlorophyll content of A. annua (µg. g^-1 of FW) after applying the purified water (PW) irrigation. Control (Cont.); Arbuscular mycorrhizal Fungal (AMF); Entomocompost (ENTO); Control + Purified water (Cont + PW); Arbuscular Mycorrhizal Fungi + Purified water (AMF + PW) and Entomocompost + Purified water (ENTO + PW). Values are the averages of five repetitions.

The different treatments applied showed significant effects (p < 0.05) on the water content of A. annua plants in the field compared to the controls (Figure 2). Indeed, the addition of purified water resulted in a significant decrease (p < 0.05) in relative water content in both AMF fertilized and ENTO-fertilized plots. This could be explained by a high dry biomass following an accumulation of reserves in the leaves and an enrichment of the cytoskeleton [35] because, the location of the experimental site in an urban area result in a high CO₂ content available inside the cells which consequently leads to the accumulation of synthesized organic compounds.

Figure 2. Leaf relative water content (%) of A. annua applying the purified water (PW) irrigation. Control (Cont.); Arbuscular mycorrhizal Fungal (AMF); Entomocompost (ENTO); Control + Purified water (Cont + PW); Arbuscular Mycorrhizal Fungi + Purified water (AMF + PW) and Entomocompost + Purified water (ENTO + PW). Values are the averages of five repetitions.

The mineral composition of A. annua leaves after three months of field cultivation is shown in Table 3. Analysis of this table shows that ENTO fertilization promotes calcium absorption, while CMA fertilization also promotes iron and zinc absorption. Furthermore, the addition of purified water to ENTO-enriched media promotes the absorption of Ca, Mg, Fe, P, and K, and limits that of Na. Fertilization with CMA and ENTO promotes the absorption of mineral elements in A. annua.

Mineral content is higher in plants irrigated with purified water. AMF promotes the absorption of mineral elements by these plants. These results corroborate those of [26], who showed that mycorrhizae allow plants to benefit from better phosphorus absorption thanks to the network of fungal hyphae that colonize the soil and provide a larger absorption surface with the substrate.

The high calcium content in plants fertilized with ENTO + PW could be explained primarily by the composition of the purified water, which is rich in mineral elements, and by the fact that ENTO provides nutrients that are easily assimilated by these plants to the soil [36]. Thus, these variations in peak levels of essential mineral elements are explained by the nature of the fertilizer applied and the soil.

Table 3. Mineral content of A. annua leaves according to the treatments applied in the presence of purified water.

Cont (Control), Cont (-) (Negative control), AMF (Arbuscular Mycorrhize Fungal), ENTO (Entomocompost), EW (Epure Water). PO 4 3 − (Phosphore). Na⁺ (Sodium). K⁺ (Potassium). Zn²⁺ (Zinc). Ca²⁺ (Calcium). Mg²⁺ (Magnesium) & Fe²⁺ (Fer).

The results of total phenolic compound contents evaluated in adult A. annua leaves are presented in Figure 3. Analysis of this figure shows that in plants watered with purified water (PW), the phenolic compound content increased significantly (p ≤ 0.05) at months 1 and 2 in plants enriched with AMF compared to the controls, as well as in plants fertilized with ENTO at month 1 under the same conditions compared to the positive control and at month 3 of the same treatment compared to both controls. The high phenolic compound contents would protect plants against abiotic stresses generated by soil acidity, which has an acidic pH [33], although corresponding to the crop requirements; These results corroborate those of [34] who showed that by fertilizing X. sagittifolium with poultry manure and NPK fertilizer, the polyphenol content increased in the leaves which were resistant against biotic and abiotic attacks.

Figure 3.Total phenolic compound content of the A. annua plant (mg/g of FW) applying the purified water (PW) irrigation. Control (Cont.); Arbuscular mycorrhizal Fungal (AMF); Entomocompost (ENTO); Control + Purified water (Cont + PW); Arbuscular Mycorrhizal Fungi + Purified water (AMF + PW) and Entomocompost + Purified water (ENTO + PW). Values are the averages of five repetitions.

The results of flavonoid content assessed in A. annua leaves are presented in Figure 4 and its analysis shows that: In the sub-block of plants watered with purified water (PW), flavonoid content increased significantly (p < 0.05) at month 2 in media fertilized with AMF compared to the two controls. This was also the case for months 2 and 3 respectively, with the maximum obtained at month 3 (19.49 ± 5.68 of FWM). In media fertilized with ENTO, a significant increase (p < 0.05) in leaf flavonoid content was observed at month 3 compared to the positive control. Similarly, the addition of purified water (PW) leads to a significant increase (p < 0.05) in the flavonoid content at months 2 and 3 in the media fertilized with ENTO compared to the two controls with the maximum content 19.58 ± 2.80 µg. g⁻¹ of FWM at month 3. Overall, the results show that the total flavonoid contents of the leaves in A. annua are very low during the first two months of growth for all treatments applied. Flavonoids are constituents of phenolic compounds that are involved in the regulation of plant cell growth [37]. The results show that these contents are lower than those of polyphenols and proteins. However, they increase over time. This could be explained by the slow and progressive mineralization in the media fertilized with ENTO and the complete establishment of the root-AMF exchange network.

Figure 4.Total flavonoid content of the A. annua plant (µg/g of FW) applying the purified water (PW) irrigation. Control (Cont.); Arbuscular mycorrhizal Fungal (AMF); Entomocompost (ENTO); Control + Purified water (Cont + PW); Arbuscular Mycorrhizal Fungi + Purified water (AMF + PW) and Entomocompost + Purified water (ENTO + PW). Values are the averages of five repetitions.

The results of the soluble protein content of A. annua L. leaves evaluated are presented in Figure 5. Analysis shows that the addition of purified water (PW) leads to a significant increase (p < 0.05) in this content at month 3 in media enriched with CMA compared to the two controls. In media fertilized with ENTO, a significant increase (p < 0.05) in protein content is observed at month 3 compared to the controls. In contrast, the addition of purified water (ENTO + PW) leads to a significant decrease (p < 0.05) in soluble protein content at month 3. Generally, this content increases over time in the leaves of plants watered with purified water. Metabolically, the higher soluble protein contents at month 3 obtained in ENTO+ CMA-enriched media would be due to the increase in the plant’s nutrient absorption surface (CMA). Similar results are obtained by the work of [38] who showed protein accumulation in the leaves of corn fertilized with nitrogen fertilizer.

The lower levels of proteins, flavonoids and phenolic compounds during the first month and higher levels from month 2 onwards could be explained by the fact that during the first month the sugars produced by photosynthetic activity rather maintained the fertilizers (AMF) which would be entirely dependent on them in order to allow the establishment of the symbiosis [34] then during the second and third months it was the protein biosynthesis pathway which was favored to the detriment of the phenylalanine ammonia lyase (PAL) pathway, demonstrating the effect of correlations between parameters assessed on plants.

Figure 5. Soluble proteins content of the A. annua plant (µg/g of FW) applying the purified water (PW) irrigation. Control (Cont.); Arbuscular mycorrhizal Fungal (AMF); Entomocompost (ENTO); Control + Purified water (Cont + PW); Arbuscular Mycorrhizal Fungi + Purified water (AMF + PW) and Entomocompost + Purified water (ENTO + PW). Values are the averages of five repetitions.

The expression of the correlation of parameters assessed on A. annua L. plants after purified water (PW) irrigation in each treatment is presented in Figure 6. Its analysis reveals a significantly (p < 0.05) negative correlation (r = 0.57) between proteins and chlorophylls on the one hand and between proteins and flavonoids (r = 0.45) on the other hand (Figure 6).

Figure 6. Correlation diagram of the parameters assessed on A. annua plants after purified water (PW) irrigation in each treatment.

Otherwise, in the PAL pathway, a significantly (p < 0.05) positive correlation (r = 0.54) is observed between flavonoids and phenols. Similarly, a strongly positive correlation is observed between the fresh and dry masses of the samples, confirming active chlorophyll synthesis and accumulation of reserves in the leaves with an enrichment of the cytoskeleton. Although this study shows the good performance of Artemisia annua L. under organic fertilization in the field, it was conducted over only one cycle and its level of soil depletion remains to be evaluated.