Effect of bio-fertilizer and spraying of nano-fertilizer and gibberellin on the chemical traits of Aloe vera L.

A factorial experiment with three replications was conducted according to a randomized complete block design (RCBD) to study the effect of bio fertilization at three levels (0, Azotobacter, Mycorrhizae) and spraying with nano fertilizer (Nano Iron) (0, 1500, 3000) mg L-1 and gibberellin GA3 (0, 150) (300) mg L-1 in the chemical traits of Aloe vera L.) in lath house of the Department of Plant Production Techniques / Al-Musayyib Technical College / Middle Euphrates University during the 2021-2022 agricultural season. After the experiment ended, the results were: The treatments of adding azotobacter and spraying with nano iron concentration of 3000 mg L and gibberellin concentration of 300 mg L-1 were significantly superior in all studied traits. It was also caused by the bi-interaction (My-corrhizal + Nano iron 3000 mg L-1), (Mycorrhizal + gibberellin 300 mg L-1) and (Mycorrhizal iron 3000 mg L-1 + Gibberellin 300 mg L-1) and the triple interaction between the study factors (Mycorrhizalgia). + Nano iron 3000 mg L-1 + gib-berellin 300 mg L-1) a significant increase in most of the studied traits


Introduction
Aloe vera, L is one of the medicinal plants belonging to the genus Aloe, which includes more than (300) species, all classified within the Asphodelaceae family.The original home of this plant is in North Africa and Spain.As it is cultivated in hot and dry areas of Asia, Europe and America, the aloe vera plant has been used medicinally to treat various diseases such as mild fever, wounds, burns, digestive disorders, diabetes, cancer and skin diseases 1 .The medically used part of aloe vera is the thick, lubricated leaves from which juice is extracted, which contain anthraquinones derivatives of the following glycosides: Aloin, Aloe Emodin and Barbolin.It also contains 96% water and polysaccharides.In addition to containing quinine, enzymes, plant hormones, amino acids, vitamins and minerals and to increase growth and maximize the active substances in the leaves, modern techniques were used, including adding bio-fertilizers, nano-fertilizers and plant hormones.In recent years, many studies and research have appeared that dealt with the introduction of nanotechnology in the agricultural field, which is called Agro-Nanotechnology. Nano-fertilizers have unique properties due to their small size and large surface area, which leads to an increase in the absorption surface and then an increase in the photosynthesis process and thus an increase in production in the plant 2 .Accordingly, with the use of various nano compounds, including iron, which is one of the micronutrients necessary for plant growth, iron directly enters the formation of cytochromes and some enzymes and in the formation of (Ferredoxin) and has a role in the formation of chlorophyll.However, it does not enter into its composition 3 .Scientific studies have also confirmed the importance of biological, bacterial and fungal fertilizers in increasing agricultural production because of their essential role in improving plant growth by increasing the availability of the necessary elements needed for plant growth, such as nitrogen, which is fixed by azotobacteria and phosphorous, which is prepared by the mycorrhizal fungi, and its reduction in the soil reaction number (pH), which increases the readiness of microelements that plants need As well as the production of growth regulators such as auxins, cytokines and gibberellins 4 Gibberellins are plant hormones that promote growth, affect stem elongation by stimulating cell elongation and overcoming genetic dwarfism, encourage cell division and expansion, regulate carbohydrate permeability and activate nucleic acid formation.The treatment with gibberellin contributes to a more significant shift of nutrients towards the growth sites 5 .

Materials and Methods
A factorial experiment was conducted in the lath house of the Department of Plant Production Techniques / Al-Musauib Technical College from 1/9/2021 to 1/6/ with three replications according to the randomized complete block design (RCBD).To study the effect of bio-fertilization, three levels (without addition, Azotobacter chrococcim, Mycorrhizae Glomus spp) and symbolized by them (E2, E1, E0)) respectively and spraying with nano fertilizer (Nano Iron) (0, 1500, 3000) mg L -1 and symbolized by them ( F2, F1, F0), Gibberellins GA3 (0, 150, 300) mg L -1 and their symbol are G2, G1, G0) respectively in the chemical traits of Aloe vera L.Biofertilizer was added to the soil of seedlings with an amount of 3 cm per pot, which was obtained from the Ministry of Science and Technology.Then the plants were sprayed after irrigation with a bio-fertilizer solution (nano chelate), and the next day, it was sprayed with gibberellin acid in the early morning until complete wetness and by four sprays (15) 10 and 15/11/2021 and 1/4 and 1/5/2022.The service operations of irrigation and weeding were conducted whenever needed.After the experiment was completed, the following measurements were taken: 1-The percentage of carbohydrates in the leaves (%) The percentage of carbohydrates was estimated according to the method (Joslyn, 1970).2-Determination of the percentage of protein in the leaves: The protein content of the leaves was estimated based on their nitrogen content according to the following relationship Percentage of protein in leaves = percentage of nitrogen * 6.25 3-The content of the leaves of the active substance Emodine aloe μg .g-1 The extract was prepared by the method mentioned in the scientific literature.Aloe vera powder (10 g) was soaked in 60% ethanol for 24 h.The solution and residues were isolated by centrifuge with a rotation speed of 4000 rpm.A reddish-brown colloidal aloe vera extract was obtained.Sulfuric acid and chloroform were added to the extract and re-condensed to remove chloroform from the extract.This procedure was carried out three times to obtain the largest amount of extract after evaporation of chloroform; a yellowish brown extract was obtained as a crude extract.The tests were conducted in the laboratories of the Ministry of Science and Technology -Food Research Center to identify the active compounds using highperformance liquid chromatography (HPLC) technology, model (SYKAM) of origin.The carrier phase consisting of (acetonitrile: distilled water) was used at a ratio of (75:25 volume/volume) with a flow rate of 1 ml/min, while the separation column used (C18-ODS) was (25 cm * 4.6 mm), a U.V. ray detector was used to detect About the active substance with a wavelength (220 nm).

4-The content of the leaves of the active substance Cinnamic μg g-1 Detection of cinnamic acid:
The phenolic compounds were extracted from the plant samples by taking a weight of (3 g).They were dried and finely ground and placed in a volumetric vial, and 50 ml of solvents were added to it in ethanol/water (70/30 volume/volume).Then, it was transferred to the ultrasound bath at room temperature for one hour.Then, the samples were filtered, and the solvent was removed by a rotary evaporator and dried at 40 °C; 5 ml of liquid extract was used to determine the active compounds.Dry extracts were stored in glass vials at 4 °C to prevent oxidative damage until analysis.The tests were conducted in the laboratories of the Ministry of Science and Technology -Food Research Center to identify the active compounds using high-performance liquid chromatography (HPLC) technology, model (SYKAM) of origin.The carrier phase consisted of (acetonitrile: distilled water) at a ratio of (70:30 volume/volume) with a 1 ml/minute flow rate.While the separation column was (C18-ODS (25 cm * 4.6 mm), a UV-violet detector was used to detect the active substance with a wavelength of 280 nm).5-The content of the leaves of the active substance Aloin μg g -1 The extract was prepared by the method mentioned in the scientific literature.Aloe vera powder (10 g) was soaked in 60% ethanol for 24 h.The solution and residues were isolated by centrifuge with a rotation speed of 4000 rpm.A reddish-brown colloidal aloe vera extract was obtained.Sulfuric acid and chloroform were added to the extract and re-condensed to remove chloroform from the extract.This procedure was carried out three times to obtain the largest amount of extract after evaporation of chloroform; a yellowish brown extract was obtained as a crude extract.The tests were conducted in the laboratories of the Ministry of Science and Technology -Food Research Center to identify the active compounds using highperformance liquid chromatography (HPLC) technology, model (SYKAM) of origin.Where the carrier phase consisting of (acetonitrile: distilled water) was used at a ratio of (75:25 volume/volume) with a flow rate of 1 ml/min, while the separation column used (C18-ODS) was (25 cm * 4.6 mm), a U.V. ray detector was used to detect About the active substance with a wavelength (220).

The percentage of carbohydrates in the leaves (%)
The results in Table (1) showed the biofertilization with Mycorrhizae and Azotobacter significantly excelled in the carbohydrate content of the leaves, which recorded 13.06 and 11.60%, respectively, compared to the control treatment, which recorded 9.36%.As shown in the Table below, the two treatments of spraying with nano iron concentrations of 3000 and 1500 mg L -1 significantly excelled and gave 11.94 and 11.87%, respectively, compared to the control treatment, which recorded 10.21%,While the two treatments of spraying with gibberellin at a concentration of 300 and 150 mg L -1 were significantly excelled and scored 12.24 and 11.54% compared to the control treatment which recorded 10.24%.The same Table also showed that the interactions between the two treatments had a "significant" effect on the percentage of carbohydrates.The interaction treatment between Mycorrhiza and spraying with nano-iron at a concentration of 3000 mg L -1 excelled at the highest average of 13.74%.It was followed by the treatment (Mycorrhizal + spraying with nano iron 1500 mg L-1), scoring 13.45% compared to the control treatment, which recorded 8.09%".While the bi-interactions between Mycorrhizal biofertilization and spraying with gibberellin at a concentration of 300 mg.L -1 gave the highest average of 14.04%.Compared to the control treatment, which scored 8.36%.The interaction between spraying with nano iron at a concentration of 3000 mg.L -1 and spraying with gibberellin at 300 mg.L -1 recorded the highest rate in the percentage of carbohydrates, amounting to 12.91%.The treatment (spraying with nano iron at a concentration of 1500 mg.L -1 + spraying with gibberellin at 300 mg.L -1 , excelled and gave 12.64% compared to the control treatment, which scored 9.22%.The triple interaction between the study factors led to a significant increase in the percentage of carbohydrates, as the triple interaction treatment (mycorrhizal + spraying with nano iron 3000 mg.L -1 + spraying with gibberellin 300 mg.L -1 ) recorded the highest average of 14.97%.It was followed by the treatment (Mycorrhizalgia + spraying with nano iron 1500 mg.L -1 + spraying with gibberellin 300 mg.L -1 ), which recorded 13.92% compared to the control treatment, which recorded 7.49%.

Bio Fertilization
Spraying with nano iron (mg L-1)

Determination of the percentage of protein in the leaves
The results in Table (2) showed that the biofertilization with Mycorrhizae and Azotobacter significantly excelled in the percentage of protein, and they scored 7.73 and 5.32%, respectively, compared to the control treatment, which recorded 3.23%.The results of the Table also showed that the two treatments of spraying with nano iron concentrations of 3000 and 1500 mg.L -1 significantly excelled and gave 6.59 and 5.13%, respectively, compared to the control treatment, which recorded 4.57%, While the treatment of spraying with gibberellin at a concentration of 300 and 150 mg.L -1 significantly, and recorded 6.16 and 5.09% compared to the control treatment, which recorded 5.03%.The results of the Table also showed that the two interactions in the treatments had a "significant" effect on the percentage of protein.The treatment of the interaction between mycorrhizae and spraying with nano iron at a concentration of 3000 mg.L -1 excelled and gave the highest average of 9.61%.They were followed by the treatment (Mycorrhiza + spraying with nano iron 1500 mg.L -1 ).It recorded 7.40% compared to the control treatment, which recorded 2.46%.The bi-interaction between the biofertilization with Mycorrhizae and spraying with gibberellin at a concentration of 300 mg.L -1 gave the highest average of 8.13% compared to the control treatment, which recorded 3.23%".The interaction between spraying with nano iron at a concentration of 3000 mg.L -1 and spraying with gibberellin at 300 mg.L -1 recorded the highest rate of 7.85% compared to the control treatment of 4.37%.The triple interaction between the study factors showed a significant increase in the percentage of protein, where the triple interaction treatment (mycorrhizal + spray with nano iron 3000 mg.L -1 + spray with gibberellin 300 mg.L -1 ) recorded the highest average of 10.83% compared to control treatment which recorded 1.25 %.

Bio Fertilization
Spraying with nano iron (mg L-1) The content of the leaves of the active substance Emodine aloe μg .g - Table (3) results indicated significant differences in Emodine aloe between Mycorrhizal and Azotobacter biofertilizers and the control treatment, as they recorded 65.35 and 65.04 μg .g-1, respectively.Compared with the control treatment, which recorded 64.03 μg .g - , The results of the Table also showed that the two treatments of spraying with nano iron concentrations of 3000 and 1500 mg L -1 were significant, and they were given 65.49 and 64.97 μg .g-1 respectively.Compared with the control treatment, which recorded 63.96 μg .g-1, the treatment of spraying with gibberellin at a concentration of 300 and 150 mg L -1 was significantly excelled, and it recorded 65.35 and 64.85 μg .g-1, respectively, compared to the control treatment, which recorded 64.23 μg .g-1.The results of the Table also showed that the bi-interaction treatments in the treatments had a significant effect on the leaves' content of the active substance.The treatment of the interaction between Mycorrhiza and spraying with nano iron at a concentration of 3000 mg L -1 excelled, and the highest average was 65.85 μg .g-1.It was followed by the treatment (Mycorrhiza + spraying with nano iron 1500 mg L -1 ), and it recorded 65.45 µg g -1 compared to the control treatment, which recorded 62.99 µg g -1 .Whereas the bi-interactions between Mycorrhizal fertilization and gibberellin spraying at a concentration of 300 mg L -1 recorded the highest average of 65.89 µg g -1 compared to the control treatment, which recorded 63.29 µg g -1 .The interaction between spraying with nano iron at a concentration of 3000 mg L -1 and spraying with gibberellin at a concentration of 300 mg L -1 recorded the highest average of 65.71 μg .g-1 compared to the control treatment, which gave 63.17 μg .g-1 .The triple interaction between the study factors led to a significant increase in the leaves content of the active substance, as the triple interaction treatment (mycorrhizalgia + spraying with nano iron 3000 mg L-1 + spraying with gibberellin 300 mg L -1 ) recorded the highest content of 66. 16

The content of the leaves of the active substance Cinnamic μg .g -1
The results in Table (4) recorded the biofertilization with Mycorrhizae and Azotobacter significantly excelled in the leaves content of the active substance Cinnamic, and they recorded 15.86 and 15.58 μg .g-1, respectively, compared to the control treatment, which recorded 14. μg .g-1 .As shown in the Table below, the treatment of spraying with nano iron concentrations of 3000 and 1500 mg L -1 was significant and gave 16.01 and 15.49 μg .g-1 , respectively, compared to the control treatment, which recorded 14.37 μg .g-1.While the treatment of spraying with gibberellin at a concentration of 300 and 150 mg L -1 significantly and recorded 15.92 and 15.46 μg .g-1 , compared to the control treatment, which recorded 14.49 μg .g - .
The results of the Table also showed that the two interactions in the treatments significantly affected the content of the leaves of the active substance Cinnamic.The interaction treatment between mycorrhizal and spraying with nano-iron at a concentration of 3000 mg L -1 excelled.The highest average was 16.79 μg .g - , followed by the treatment (Mycorrhiza + spraying with nano iron 1500 mg L -1 ) and it recorded 15.71 µg g -1 as measured by the control treatment, which recorded 13.23 µg g -1 .Whereas, the bi-interaction treatments between Mycorrhiza biofertilization and gibberellin spraying at a concentration of 300 mg L -1 gave the highest rate of 16.30 μg .g - compared to the control treatment, which recorded 13.72 μg .g - .The interaction between spraying with nano iron at a concentration of 3000 mg L -1 and spraying with gibberellin at a concentration of 300 mg L -1 recorded the highest average of 16.59 µg g -1 , compared to the control treatment of 13.45 µg g -1 .
The triple interaction between the study factors showed a significant increase in the content of the leaves of the active substance Cinnamic, as the triple interaction treatment (Mycorrhizae + spraying with nano iron 3000 mg L -1 + spraying with gibberellin 300 mg L-1) recorded the highest content of 17.26 μg .g - compared to the control treatment, which recorded 12.51 μg .g -

Bio Fertilization
Spraying with nano iron (mg L-1)

The content of the leaves of the active substance Aloin( μg g -1 )
The results in Table (5) showed that the biofertilization with Mycorrhizae and Azotobacter was significantly superior in the leaves content of the active substance Aloin, and they recorded 125.26 and 124.63 μg .g - , respectively, compared to the control treatment, which recorded 123.65 μg .g - .As shown in the Table below, the two treatments of spraying with nano iron concentrations of 3000 and 1500 mg L -1 were significantly excelled, giving 125.51 and 124.55 μg .g - , respectively, compared to the control treatment, which recorded 123.47 μg .g - .While the two treatments of spraying with gibberellin at a concentration of 300 and 150 mg l-1 were significantly excelled and recorded 125.11 and 124.60 μg .g - , respectively, compared to the control treatment, which recorded 123.82 μg .g - .The results of the Table also showed that the two interactions in the treatments significantly affected the leaves content of the active substance Aloin.The interaction treatment between mycorrhizal and spraying with nano iron at a concentration of 3000 mg L -1 excelled and gave the highest average of 126.23 μg .g - .This was followed by the treatment (Azotobacter + spraying with nano iron 3000 mg L -1 ), which recorded 125.73 μg .g - as measured by the control treatment, which recorded 122.72 μg .g - .Whereas the bi-interactions between Mycorrhiza biofertilization and gibberellin spraying at a concentration of 300 mg, L -1 gave the highest rate of 125.82 μg .g - compared to the control treatment, which recorded 122.97 μg .g - .The interaction treatment between spraying with nano iron at a concentration of 3000 mg L -1 and spraying with gibberellin at a concentration of 300 mg L -1 recorded the highest average of 126.05 μg .g - with the control treatment, which gave 122.82 μg .g - .
The triple interaction between the study factors led to a significant increase in the leaves content of the active substance Aloin, as the triple interaction treatment (Mycorrhizae + spraying with nano iron 3000 mg L -1 + spraying with gibberellin 300 mg L -1 ) recorded the highest content of 126.54 μg .g - , It was followed by the treatment (Mycorrhiza + spraying with nano iron 3000 mg L -1 + spraying with gibberellin 150 mg L -1 ) which amounted to 126.36 μg .g - compared to the control treatment which recorded 122.39 μg .g - .

Bio Fertilization
Spraying with nano iron (mg L-1)

Discussion
The Tables (1-5) results showed that the biofertilization with Mycorrhizae Azotobacter significantly excelled in the chemical traits.The reason for this may be due to the role of biological Fertilization in improving the physical and chemical properties of the soil, which leads to an increase in the availability of nutrients in the soil and ease of absorption by the plant, encouraging the formation of root hairs and increasing the surface of the root system.This leads to an increase in the absorption of water and nutrients, which is reflected in the improvement of the plant's vegetative growth, in addition to the role of bacteria in the process of fixing atmospheric nitrogen.It converts nitrogen from the inactive form (N2) to ammonia or converts it from the form of ammonia to nitrate in order for it to be ready for absorption with the help of the nitrogenase enzyme 6 as well as its role in the production of plant hormones such as auxin, gibberellin and cytokinin that help increase the rate of cleavage.Cells, elongation, size increase, and high intracellular metabolism level.Biofertilizers are important in preparing and absorbing nitrogen and phosphorous elements and increasing their plant content.These elements play a vital role in the construction of chlorophyll pigments that absorb energy and the photosynthesis process.That nitrogen enters into the formation of a protein compound, the providing, which is important in building chlorophyll and cytochromes that carry electrons in photosynthesis and respiration, thus increasing the chlorophyll content in leaves and the process of chlorophyll.Photosynthesis and synthesis of nutrients inside the leaves and increased construction of carbohydrates (table 1) and proteins (table 2).These results agree with the results of 4 for carnation, 5 for the Amaranthus plant, 6 for the rose.The increase in carbohydrates (table 1) as a result of the increase in photosynthesis due to biofertilization, caused an increase in the process of building anthraquinone glycosides as a result of its role in the process of building phenylalanine and tyrosin-1 compounds, which represent the first step in building anthraquinone glycosides through the condensation process of the hydroxyl group in The aglycemic fraction and the Hemiacetal process of the hydroxyl group in the diabetic fraction 7 ,8.This shows that the increase in carbohydrates leads to an increase in the construction of the important anthraquinone glycosides in the construction of secondary compounds such as the active substances Emodine aloe, Cinnamic and Aloin Table (3, 4, 5).The results in tables (1-5) showed the significance of the high concentrations of nano-fertilizer in the chemical properties.The reason may be attributed to the small size of the molecules of this fertilizer and its content of the micro-fertilizer elements important in the growth and development of the plant, as well as in the role of the iron element, which is important in building the chlorophyll molecule and stimulating many The important enzymes related to the process of photosynthesis and thus increasing the nutrients manufactured in the leaves, which are used in building the plant's vegetative system 9 This is consistent with what 10,11 stated that iron plays a role in increasing the speed and outputs of photosynthesis that are used in various growth processes, in addition to that iron is included in the composition of many non-heme compounds, the most important of which is Ferredoxin, which contributes to the transfer of electrons in many vital processes within the plant, including photosynthesis, iron also enters the composition of the enzymes Nitrogenase, aconitase and xanthin oxidase with different functions within the plant 12,13 The increase in vegetative growth and its activity in the manufacture of foodstuffs increases the supply of roots with a quantity of these materials, which leads to an increase in their depth and spread.When spraying with nano iron, the reason for this may be due to the increase in the amount of processed carbohydrates in the leaves (table 1),Which is used in various growth processes, 14 ,15 The reason may also be attributed to the fact that the nano-chelated iron increases the effectiveness of the biochemical conversion processes in the cell membrane, which facilitates the penetration of nutrients into the plant, and this helps to increase cell divisions and encourage growth, and thus provides a continuous demand for nutrients that the plant works to absorb from the soil 16 and the increase in carbohydrates Table (1) as a result of the increase in photosynthesis by nano-fertilizer (nano iron) caused an increase in the process of building anthraquinone glycosides as a result of its role in the process of building some secondary compounds and thus increasing the construction of anthraquinone glycosides important in building secondary compounds Table (3, 4,  5 ) such as the active substances Emodin aloe, Cinnamic and Aloin, and these results are consistent with the study of 17 on the Moringa plant, 18 on the peppermint plant.The cause of spraying with gibberellin to a significant increase in the chemical traits Table (1-5), and the reason for the existence of significant differences when foliar spraying with gibberellic acid may be that the active and active vegetative growth has a great relationship in the content of stored carbohydrates This depends on the continuation of the appropriate conditions for its formation, where the active and active leaves lead to the production of carbohydrates (Table 1), and therefore part of them is stored in the woody branches of plants.As for a reason for the superiority of high concentrations of gibberellin spraying, the significant effect of gibberellic acid levels on the protein content in leaves (Table 2) may be due to the role of gibberellin in improving the traits of vegetative growth and its role in increasing the vital processes within the plant from carbon metabolism, respiration and cell division.The effect of increasing the concentration of medically active substances in the leaves (table 3, 4, 5) may be because the treatment of plants with gibberellin causes an increase in vegetative growth and the number, width and thickness of leaves, which leads to an increase in carton manufacturing and then an increase in the production of secondary compounds within the plant.Alternatively, it may work to increase the construction of some enzymes responsible for forming these compounds 18 These results agreed with 19 on the peppermint plant 17 on the moringa plant.

Conclusion
The study concludes the type of bio-fertilizer, the best concentration of nano-fertilizer and gibberellin, and the interaction between the study factors in the growth and maximization of the active substances of the Aloe vera plant.

Table 3 . Effect of biofertilization and spraying with nano-iron and gibberellin on Alea-emodin.
μg .g-1 compared to With the control treatment, which recorded 62.51 μg .g-1