Effect of Mycorrhizae, Azotobacter and Vermicompost tea on Nitrogen, Phosphorus, and Potassium (NPK) Concentrations in Soil and Cucumber Plants (Cucumis sativus)

An experiment was carried out inside a greenhouse (sandy loam soil) at the College of Agriculture / University of Anbar to investigate the effect of Vesicular-Arbuscular Mycorrhizaee, Azotobacterchroococcum and Vermicompost tea on the concentration of NPK macronutrients in soil and cucumber plants. A factorial experiment consisting of two factors was designed for this purpose: the first factor is a combination of mycorrhizae (M) with 35 g plant-1, azotobacter (A) bacteria 16 ml plant-1 with a microbial density of 2.2 x 109 cfu ml-1 and phosphate Rock (R) with 40 g plant-1, added with half of the mineral fertilizer recommendation each individually with its interactions, The second factor is the addition of three concentrations (0, 5, 10%) of vermicompost tea sprayed on the plant. The experiment was designed with a completely randomized block design (RCBD) with three replications. Cucumber seeds were planted on 15/9/2022 and service operations were conducted for the crop and the experiment continued until 15/12/2022. Concentrations of nutrients were measured in the soil after harvesting the crop as well as in the leaves of the plant. The results of the research proved that the use of mycorrhizae, azotobacter bacteria and phosphate Rock with half the mineral recommendation (MAR) and spraying vermicompost tea at a concentration of 10% are the best in increasing the concentration of nutrients in the soil and plant and revealed significant differences compared to mycorrhizae or azotobacter or vermicompost tea individually, so this treatment gave the highest concentration of available nitrogen (AN) (56.37 mg kg-1 soil), the highest available phosphorus (AP) (16.34 mg kg-1 soil), the highest available potassium (230.3 mg kg-1 soil), and gave the highest percentage of nitrogen in the leaves (NIL) (2.80%), The highest percentage of phosphorus in the leaves (PIL) (0.40%), the highest potassium in the leaves (KL) (1.95%), followed by the treatment of adding mycorrhizae with phosphate Rock and half the mineral recommendation (MR) with vermicompost tea at a concentration of 10%, then the treatment of adding mycorrhizae with azotobacter bacteria with half the mineral recommendation (AR) with 10% of vermicompost tea, then the treatment of mycorrhizae with phosphate Rock plus half the fertilizer recommendation (MR) with 10% of vermicompost tea, then the treatment of mycorrhizae with azotobacter plus half of the fertilizer recommendation (MA) with 10% of vermicombe tea. Then the two treatments of mycorrhizae (M) and azotobacter (A) separately, while finally came the treatment of chemical fertilization (S), which gave the lowest values to the mentioned properties in the soil and plants.


Introduction
Bio-fertilizers as a modern technology for agriculture in the world are environmentally friendly and economically viable, while preserving the soil in its natural position and biodiversity.Bio-fertilizers improve plant growth and yield by increasing the absorption of nutrients, as well as increasing the availability of these nutrients in the soil.This is done by fixing atmospheric nitrogen and increasing the solubility of phosphates, potassium compounds and other nutrients necessary for plant growth, as well as the production of enzymes, plant growth regulators and bio-control that improve plant growth and yield, There are many types of these bio-fertilizers such as nitrogen-fixing bacteria such as Rhizobia that promote the growth of legumes and other plants as well as Azotobacter free living bacteria.There are other organisms that increase the availability of phosphorus and many other nutrients in the soil, including root fungi (mycorrhizae), which is a secondary root of the plant that works to increase the absorption of water and nutrients through the extension of its hyphae to several meters, as well as for its secretion of growth hormones and enzymes and its ability to bio-control diseases.The use of microbial bio-fertilizers is therefore an effective and viable option [1,2].Organic fertilizers are very important for microorganisms and soils alike.Vermicompost is one of the best organic fertilizers recently, which has a very important impact on increasing plant growth and yield, improving soil properties and creating a healthy and safe ecosystem for food processing.An extract of vermicompost called vermicompost tea can be added to the soil or sprayed on the plant and is considered a supplement to mineral fertilization as it contains nutrients necessary for plant growth and is also used in biological resistance as an alternative to chemical pesticides that negatively affect public health and the environment, giving ideal properties in terms of the quality of the yield and the original taste desired by the consumer free of environmental and health pollutants [3].Cucumber plant Cucumis sativus L belongs to the Cucurbitaceous family, which is one of the important vegetable crops in most countries of the world, including Iraq, and it is widespread, especially in protected agriculture, as it has nutritional, economic and health value [4].Cucumbers are grown in the protected environment under tunnels, greenhouses and glass, grow quickly and give a good yield, but it is a stressful vegetable for the soil as it consumes large amounts of nutrients, including N, P, and K, and thus requires quantities of mineral fertilizers that are added to the soil or with irrigation water or sprayed on the plant to supply it with the necessary nutrients [5].This research aims to find out the effect of using bio-fertilizers Vesicular-Arbuscular Mycorrhizaee (VAM) and Azotobacter bacteria as a combination with phosphate Rock with vermicompost tea (spray on the plant) and its efficiency in increasing the concentration of nutrients remaining in the soil after harvesting the crop as well as increasing its concentration in the leaves of the cultivated cucumber plant.

Materials and Methods
An experiment was conducted in a plastic house measuring 9 x 44 m at the site of the College of Agriculture / University of Anbar for the fall season 2022 inside a greenhouse with dimensions to know the effect of biofertilizer Mycorrhizaee and Azotobacter as a combination with phosphate Rock) with the addition of vermicompost tea (spray on the plant) in the concentration of the major elements NPK in the soil after planting and in the leaves of the cucumber plant during the planting season (flowering stage).The soil used in the greenhouse is sandy loam with high water permeability and poor in nutrient content, the soil was plowed twice and adjustment and leveling operations were carried out, then soil samples were taken randomly for multiple sites of the surface layer to a depth of 0-30 cm, chemical and physical analyzes of the soil were carried out as shown in Table (1).
Table 1.Some chemical and physical properties of greenhouse soil before planting. S: 100% complete fertilizer recommendation for cucumber plant.

Vermicompost Tea
It was sprayed on the vegetative part of the plant in the following concentrations:  V0: without addition (control). V1: addition of 5% Vermicompost Tea. V2: addition 10% Vermicompost Tea.Vermicompost tea was prepared from solid vermicompost in a ratio of 1:10 (water: vermicompost) according to the method described in [6,7].Table 2 shows the properties of prepared vermicompost tea.
Table 2. Properties of vermicompost tea used in the study.Total fungi 2.2x 10 3 Cfu g -1 The experiment design was the randomized completely blocks (RCBD) with three replicates.The cucumber plant seeds planted on 9/15/2022.The last crop harvest was on 12/152022/, as the experiment was terminated.The concentrations of N, P, and K macronutrients in the soil after harvesting as well as their concentration during the flowering phase in the leaves of the plant were measured.The results were analyzed statistically by analysis of variance, F test and the value of the least significant difference (LSD) below the probability level of 0.05 according to [8].

Total Available Nitrogen (TAN) in the Soil (mg kg -1 )
It is clear from the table (3) that there are significant differences between the treatments of combinations, as the triple combination MAR gave the highest rate of TAN of 47.64 mg kg -1 soil with significant difference from all other combinations.It was followed by the combination MR which recorded 39.18 mg kg -1 soil with an increase of 21.59%, followed by the combination MA and AR with 36.70 and 35.27 mg kg -1 soil.It is also found that the treatment of mycorrhizae M gave a significantly higher rate of available nitrogen (AN) than the treatment of the addition of azotobacter (A) which recorded 33.24 and 31.32 mg kg -1 soil respectively, while the treatment of the full mineral recommendation (S) gave the lowest rate of AN of all the treatments, recording 29.17 mg kg -1 soil and was surpassed by the triple combination MAR by an increase of 63.3%.The table shows the effect of vermicompost tea concentrations on the concentration of AN, and the concentration of 10% gave the highest concentration of AN of 40.63 compared to concentrations of 0 and 5%, which recorded 31.60 and 36.04 mg kg -1 soil respectively.It is clear from Table 3 that there are significant differences for most of the interaction treatments between bio-fertilizers with phosphate Rock and vermicompost tea concentrations.The interaction between the combination MAR and the concentration of vermicompost tea gave 10% the highest rate of AN recording 56.37 mg kg -1 soil, significantly superior to all other interaction treatments.The highest interaction treatment (MAR with a concentration of 10%) gave a significant increase of 126.29% compared to the lowest interaction treatment which is the full fertilizer recommendation (S) with a concentration of 0% which recorded the lowest rate of AN which was 24.91 mg kg -1 soil.

Available Phosphorus in the Soil (AP) (mg kg -1 soil)
Table 4 shows the effect of bio-fertilizer combination with phosphate rock and vermicompost tea concentration in available phosphorus (AP) in soil after harvesting (mg kg -1 soil), it is clear from the table that there are significant differences between the treatments of the combinations, as the triple combination MAR gave the highest rate of AP of 15.31 mg kg -1 soil and a significant difference from all other combinations, it was followed by the combination MR, which recorded 13.71 mg kg -1 soil with an increase of 11.67%, then the two combinations MA and AR recorded 12.85 and 12.48 mg kg -1 soil, and biologically mycorrhizae M higher than azotobacter with a phosphorus rate of 12.18 and 11.17 mg kg -1 soil respectively.The whole mineral recommendation (S) treatment gave the lowest AP rate of all treatments, recording 9.61 mg kg -1 soil, which was outperformed by the triple combination with half of the MAR fertilizer recommendation, an increase of 59.3%.  4 that there are significant differences in the interaction treatments between bio-fertilizer with phosphate rock and vermicompost tea concentrations in AP in soil, the interaction between the combination MAR and the concentration of vermicompost tea 10% gave the highest rate of AP recording 16.34 mg kg -1 soil, significantly superior to all other interaction treatments.The highest interaction treatment (MAR with a concentration of 10%) gave a significant increase of 87.3% compared to the lowest interaction treatment which is the full fertilizer recommendation (S) with a concentration of 0% which recorded the lowest rate of AP which was 8.72 mg kg -1 soil.

Available Potassium (AK) in Soil (mg kg -1 soil)
Table 5 shows the impact of Bio-combinations combinations with phosphate rock and vermicompost tea concentration in the available potassium (AK) in soil after harvesting (mg kg -1 soil), it is clear from the table that there are significant differences between the treatments of combinations of potassium concentration in the soil, the triple combination MAR gave the highest level of AK at 217.0 mg kg - 1 soil and a significant difference from all other combinations, followed by the combination MR and MA which recorded 206.7 and 203.7 mg kg -1 soil.Then the AR combination gave AK of 201.2 mg kg - 1 soil, and the treatment of mycorrhizae M gave a significantly higher AK than the treatment of azotopacter A which recorded 198.0 and 195.6 mg kg -1 soil respectively, while the treatment of the full mineral recommendation (S) gave the lowest rate of AK compared to the other combinations recorded 185.4 mg kg -1 soil which was exceeded by the triple combination with half of the fertilizer recommendation MAR by an increase of 17.04%.The effect of vermicompost tea concentrations on AK concentration is illustrated by the table, and the concentration of 10% gave the highest rate of AK of 207.9 compared to 0 and 5% concentrations of 195.2 and 200.2 mg kg -1 soil respectively.Table 5 shows that there are significant differences in the interaction treatment between Biocombinations with phosphate rock and vermicompost tea concentrations.The interaction between the combination MAR and the concentration of vermicompost tea 10% gave the highest rate of AK recording 230.3 mg kg -1 soil significantly superior to all other interaction treatments, the interaction treatment of the highest treatment (MAR with a concentration of 10%) gave a significant increase of 27.7% compared to the lowest interaction treatment which is the full fertilizer recommendation (S) with a concentration of 0% which recorded the lowest rate of AK which was 180.3 mg kg -1 soil.

Nitrogen in Leaves (NIL) (%)
Table 6 shows the effect of bio-fertilizer combination with phosphate rock and the concentration of vermicompost tea in the proportion of nitrogen in the leaves (NIL) beginning of flowering (%).It is clear from the table that there are significant differences between the treatment of the combinations, as the triple combination MAR gave the highest rate of NIL by 2.65% and a significant difference from all other combinations, followed by the combination MR which recorded 2.52%, then the combination MA and AR recorded 2.46% and 2.42%, and the treatment of mycorrhizae M gave a higher rate of NIL significantly higher than the treatment of adding azotobacter (A), which recorded 2.53 and 2.27% respectively.While the full mineral recommendation (S) treatment gave the lowest NIL among all the treatments, recording 2.05%, which was surpassed by the triple combination with half of the fertilizer recommendation MAR with an increase of 29.2%.The table shows the effect of vermicompost tea concentrations on nitrogen concentration in the leaves (NIL), and the concentration of 10% gave the highest nitrogen rate of 2.53% compared to the concentrations of 0 and 5%, which recorded a nitrogen percentage in the leaves of 2.24 and 2.40% respectively.The table shows that there are significant differences in the interaction treatments between bio-combinations with phosphate rock and vermicompost tea concentrations in the percentage of NIL, the interaction between the combination MAR and the concentration of vermicompost tea 10% gave the highest NIL recording 2.80%, significantly superior to all other interaction treatments.The interaction treatment of the highest treatment (MAR with a concentration of 10%) gave a significant increase of 49.7% compared to the lowest interference treatment which is the full fertilizer recommendation (S) with a concentration of 0% which recorded the lowest rate of NIL for all treatments which was 1.87%.

Phosphorus in Leaves (PIL) (%)
Table 7 shows the effect of bio-fertilizer combination with phosphate rock and vermicompost tea concentration on phosphorus content in leaves (PIL) (%).It is clear from the table that there are significant differences between the treatment of the combinations, The triple combination MAR gave the highest percentage of phosphorus in the leaves (PIL) of the plant recording 0.358% and a significant difference from all other combinations, followed by the combination MR which recorded 0.328%, then the two combination MA and AR by 0.293 and 0.269%.7 that there are significant differences for some interaction treatments between bio-combinations with phosphate rock and vermicompost tea concentrations in the average of PIL.
The interaction between the combination MAR and the concentration of vermicompost tea 10% gave the highest rate of PIL recording 0.401%, significantly superior to all other interaction treatments.The highest interaction treatment (MAR with a concentration of 10%) gave a significant increase of 131.79% compared to the lowest interaction treatment which is the full fertilizer recommendation (S) with a concentration of 0% which recorded the lowest rate of phosphorus in the leaves was 0.173%.

Potassium Content in Leaves (KIL) (%)
Table 8 shows the effect of bio-fertilizer combination with phosphate rock and vermicompost tea concentration on the percentage of potassium in leaves (KIL) (%).It is clear from the table that there are significant differences between the treatment of the combinations, as the triple combination MAR gave the highest KIL, recording 1.83%, with a significant difference from all other combinations, followed by the combination MR and MA, which recorded 1.70% and 1.66%, then the combination AR which gave 1.64%.While the treatment of mycorrhizae M gave a significantly higher rate of potassium compared to the treatment of azotobacter A, which recorded 1.59 and 1.55% respectively.The full mineral recommendation (S) treatment gave the lowest potassium rate of all the treatment recorded at 1.43% and was surpassed by the triple combination with half of the fertilizer recommendation MAR by an increase of 27.9%.The table shows the effect of vermicompost tea concentrations on the KIL percentage.The concentration of 10% gave the highest KIL of 1.73% and a significant increase of 13 and 6.1% compared to the concentrations of 0 and 5%, which recorded 1.53 and 1.63% respectively.It is clear from Table 8 that there are significant differences for some interaction treatment between bio-combinations with phosphate rock and vermicompost tea concentrations.The interaction between the combination of MAR and the concentration of vermicompost tea 10% gave the highest KIL rate recording 1.95%, significantly superior to all other interaction treatments.The interaction treatment of the highest treatment (MAR with a concentration of 10%) gave a significant increase of 47.7% compared to the lowest interaction treatment which is the full fertilizer recommendation (S) with a concentration of 0% which recorded the lowest KIL which was 1.32%.

Discussion
It is clear from Tables 3, 4, 5, 6, 7 and 8 that the addition of bio-fertilizers represented by mycorrhizae M and azotobacter A bacteria with phosphate rock R and the use of vermicompost tea V sprayed on the plant by adding half of the fertilizer recommendation of the cucumber plant (whether added together as a triple or double combination or single each individually) gave a significant increase in the concentration of nitrogen, phosphorus and potassium in the soil and leaves of the plant compared to the full mineral recommendation for cucumbers, and the triple combination MAR significantly outperformed the concentration of nutrients on binary combinations, which in turn also significantly outperformed unilateral treatment, The triple combination MAR with a concentration of 10% vermicompost tea gave the best results for the mentioned properties compared to the binary combinations MR, MA and AR, which in turn outperformed the single treatment M and A, and the concentration of vermicompost tea exceeded 10% in all properties giving a significant increase compared to the concentration of 5% or concentration 0%.Many studies have indicated that mycorrhizae infects the plant host and works to stimulate plant growth and a clear increase in the availability of nutrients, i.e. an acceleration in the relative growth rate of the plant [9].The reason for the increase in the raise in the availability of nutrients in the soil and then the increase in their concentration in the plant and the subsequent improvement in plant growth and its total vegetative and root group, and then its yield when infected with mycorrrhiza comes through many factors, the most important of which are: The role of mycorrhizae in encouraging the absorption of nutrients, especially phosphorus, by exploring areas beyond the reach of the root, as the mushroom hyphes extend far outside the root system of the plant, which leads to an increase in the volume of exploited soil and thus Increase the absorption of water and nutrients necessary for plant growth, whether macro or micronutrient, as well as that mycorrhizae encourages physiological responses to the plant such as root branching and the secretion of enzymes and organic acids that indirectly enhance the absorption of nutrients, especially phosphorus, and this explains the increased availability of phosphorus from phosphate rock and increase plant growth and production when using mycorrhizae with it [10], Also, the bacteria associated with the decomposition of vermicompost fertilizer improve growth and increase the nutrient content of the soil, which increases the effectiveness of the absorption of roots, and this in turn was reflected in increasing the effectiveness of photosynthesis and storage of carbohydrates in the plant and then the components of the yield and this is consistent with [11].
On the other hand, the bio-fertilizers and their use provide a large part of the nutrients necessary for the plant such as nitrogen, phosphorus and potassium in addition to the production of some plant growth regulators (like auxines), as it has the ability to produce some important antibiotics that contribute to the resistance to many pathogens in the soil [12].Furthermore, the bio-fertilizers have an important roles in the production of enzymes as well as decomposition of organic wastes in the soil which increase the availability of soil nutrients [13].The usage of bio-fertilizers wither nitrogenic or phosphoric led to an increase of soil nutrition in addition to the improvement of chemical, physical, IOP Publishing doi:10.1088/1755-1315/1259/1/0120109 and biological properties of soil and thus enhance the quality and quantity of plants' growth and production [14,].The addition of vermicopost tea sprayed on the vegetative system causes an increase in the permeability of the stomata, which leads to an increase in the absorption of macro-and micronutrients, vericompost tea stimulates the growth and elongation of cells, which is reflected in increasing the root system, increasing the amount of nutrients absorbed, and increasing the vegetative system because it contains plant growth regulators such as auxins, cytokines, abcisic and gibberellins [3].All these specifications gave the plant better growth and higher production due to the increased availability of nutrients in the soil and then increased their concentration in the plant, which was evident through the use of vermicompost tea, especially at the highest concentration (10%) and was more influential and positive when interacting with the bio-fertilizers used in this research [16].

Conclusions
We conclude that the triple combination MAR represented by the addition of mycorrhizae M fungus and azotobacter A bacteria with phosphate rock R and the use of vermicompost tea sprayed on the plant with half of the fertilizer recommendation of the cucumber plant has significantly exceeded in increasing the availability of macronutrients (NPK) in the soil after planting, as well as significantly outperformed in increasing the proportion of the mentioned nutrients in the leaves of the plant during the growth period (the stage of the beginning of flowering) on all bilateral combinations between the experimental factors, namely MR, MA and AR, it also significantly outperformed the single treatment, which are M and A, and the triple combination MAR with a concentration of 10% vermicompost tea gave the best results for the mentioned properties compared to the binary combinations MR, MA and AR, which in turn outperformed the single treatments M and A, and the concentration of vermicompost tea exceeded 10% in all properties, giving a significant increase compared to the concentration of 5% or concentration 0%.

Table 3 .
Bio-fertilizer combination effect with phosphate Rock and vermicompost tea concentrate in available nitrogen (AN) (mg kg -1 soil).

Table 4
also shows the effect of vermicompost tea concentrations in AP and the concentration of 10% gave the highest concentration of AP of 13.72 compared to concentrations of 0 and 5% recorded 11.01 and 12.59 mg kg -1 soil respectively.It is noticed from Table

Table 4 .
Bio-fertilizer combination effect with phosphate Rock and vermicompost tea concentrate in available phosphorus (AP) (mg kg -1 soil).

Table 5 .
Bio-combinations effect with phosphate rock and vermicompost tea concentrate in available potassium (AK) (mg kg -1 soil).

Table 6 .
Bio-combinations effect with phosphate rock and vermicompost tea concentrate in the nitrogen percentage in leaves (NIL) (%).
While the full mineral recommendation (S) treatment gave the lowest phosphorus rate of all the treatment, recording 0.203%, exceeded by the triple combination with half of the fertilizer recommendation MAR with an increase of 76.3%.Table 7 also shows the effect of vermicompost tea concentrations on the PIL percentage, the concentration of 10% gave the highest PIL rate of 0.320% and a significant increase of 35 and 12% compared to the concentrations of 0 and 5%, which recorded a PIL ratio of 0.237 and 0.285% respectively.It is clear from Table It is also clear from the table that the treatment of mycorrhizae M gave the highest rate of phosphorus compared to azotobacter A,

Table 7 .
Bio-combination Effect with Phosphate Rock and Vermicompost Tea Concentrate in Phosphorus in Leaves (PIL) (%).

Table 8 .
Effect of bio-combination with phosphate rock and vermicompost tea concentration in potassium in leaves (KIL) (%).