Interactive Effects of Inoculation with Two Isolates Providencia vermicola and Raoultella planticola and Two Levels of Mineral Fertilizers on Wheat Yield Increase

The aim of this study was to evaluate the efficiency of bio fertilizers produced from local isolates of Providencia vermicola and Raoultella planticola in field trials conducted during 2021-2022 at the Faculty of Agriculture, Al-Muthanna University, Bandar, Iraq, Using a completely randomized design with three replicates, microbial bio fertilizers were considered as the first factor, which included the use of single and double bacterial inoculums consisting of the above bacteria, while the second factor included two levels of fertilizers (0 and 50%) fertilizer recommendation. The experiment also included the use of two comparative treatments, the first with a full recommendation (160, 100 and 100 kg H-1) of nitrogen, phosphorus and potassium, and the second a negative comparison treatment without the use of biological or chemical fertilizers. Characteristics of 1000-grain weight, grain yield, biomass and percentage of above-ground elements (N, K, P) at 50% flowering stage. The results showed the superiority of the double fertilization treatment (P.vermicola and R.planticola) in the characteristics of grain yield and the percentage of elements (nitrogen, potassium and phosphorus) over all bio fertilization treatments, as rates were recorded (7.21 μg H-1, 3.51%, 2.27%), 0.73%). The treatment with half the recommended amount of fertilizer was superior to the treatment with no added fertilizer on all the characteristics studied, as it recorded (48.5 g, 6.76 μg H-1, 20.88 μg H-1, 3.05%, 2.33%, 0.70 %), and treatment Showing an overlap between (P.vermicola and R.planticola), half of the fertilizer recommendations were significantly better than (4.24%, 2.96%, 0.81%) in terms of percentage of NPK elements.


Introduction
Bio fertilizers are an important part of nutritional supplements that must be taken care of Because chemical fertilizers and organic fertilizers cannot meet the nutritional needs of crops Intensive agricultural systems and low fertility levels, coupled with the continued heavy use of chemical fertilizers, with their high economic costs and negative impacts of health and environmental damage, therefore require a supporting and attributable process to increase soil productivity through the use of microorganisms [1].1259 (2023) 012113 IOP Publishing doi:10.1088/1755-1315/1259/1/012113 2 Bio fertilizers promote plant growth by ensuring the availability of micro and macronutrients, and the secretion of some hormones and acids as plant growth regulators, as well as the secretion of some antibiotics, which help to resist some endemic diseases in the soil, in addition to inducing stress resistance Production of sex and siderophores.In turn, it benefits the plant and its production by increasing the content of essential proteins, amino acids and vitamins [2].Also, the use of bio fertilizers limits the large quantities of mineral fertilizers added to 50% of the recommended amount sometimes, which leads to obtaining a clean product of high quality and health security, as it helps to increase production [3].To reduce the amount of chemical fertilizers added to crops, this study achieved this by evaluating the efficiency of bio fertilizers produced from locally isolated P. vermicola and R. planticola, and using them individually and bilaterally, and using half Fertilization recommendations for wheat growth and yield in trials.

Preparation of Bacterial Inoculum
Bacterial isolates of Providencia vermicola, and Raoultella planticola were prepared from our bio bank and sub-cultivated in nutrient broth medium at 28°C for 48 hours which presented the inoculum densities of 0.75 × 10 6 and 0.79 × 10 6 colony-forming units, respectively.The biological inoculum including 100 ml of the bacterial suspension was added to sterilized peat moss inside thermal bags.Then, the bags were rubbed to ensure homogeneous distribution of the suspension with the carrier, peat moss, and then incubated again at a temperature of 28 ° C for 48 hours.The bacterial inoculum and its carrier were considered as microbial bio-fertilizer that submitted to the field experiments.

The Experiment Involved Two Factors
Bio fertilizers (First factor) included three levels with the following symbols: without a inoculum (P0), Inoculation with Providencia vermicola )P1), Inoculation with Raoultella planticola (P2), Inoculation with Providencia vermicola and Raoultella planticola (P3), While the second factor included two levels of chemical fertilization, which: Without chemical fertilization (F0( and 50% of the fertilizer recommendation (F1).Wheat seeds of an endemic variety, were sterilized with 1% sodium hypochlorite solution for three minutes and washed with distilled water for 30 minutes to get rid of the sterile solution, and then the seeds were air dried for inoculating them with bacteria [4].The bags were opened and their contents were mixed with the seeds moistened with a solution of sterilized Arabic gum at a concentration of 10% to ensure the adhesion of the bio-fertilizer to the seeds.This process was carried out away from sunlight and direct lighting and left for half an hour to ensure homogeneous adherence of the biofertilizer to the seeds before planting them in the field.Experiment was conducted in Al-Bandar fields, College of Agriculture, Al-Muthanna University, in the season 2021/2022 and the physicochemical characteristics of its soil were presented in Table 1.In this context, the air-dried soil sample was pulverized and then filtered through a 2 mm sieve to perform soil analysis before planting.The soil pH was measured in a soil: water suspension at a ratio of 1:1 using a pH-meter and the electrical conductivity (EC) was measured as described previously.The organic matter was also estimated according to the well-known method of [5].The available nitrogen (ammonium and nitrate) extracted with a potassium chloride solution was estimated by the microcalcium device and the available phosphorus was extracted and estimated spectrophotometerically at a wavelength of 882 nm.Magnesium, calcium sulfate, carbonates, bicarbonates, and chlorides were measured as reported previously [6].The land has been prepared from the operations of plowing, leveling and modification, and several soil samples were taken and mixed to achieve a composite sample that represents the entire field 7 .Iraqi variety of wheat seeds were planted on 11/18/2021, and the field was divided into three sectors, each sector containing 17 treatments.The fertilizers were added to the treatments of half of the fertilizer recommendation and the full recommendation for each of the nitrogen fertilizers in the form of urea (46% N), with two batches, the first at planting and the second at the elongation stage, noting that the fertilizer recommendation is 160 kg N. H -1 .Phosphate fertilizer was also added in one quantity when planting in the form of triple superphosphate fertilizer (20% P), noting that the fertilizer recommendation is 100 kg P -1 .Potassium fertilization was carried out in one batch when planting in the form of potassium sulfate (50% K 2 O), noting that the fertilizer recommendation is 100 kg K. H -1 .The crop was harvested after full maturity on 04/26/2022.

Statistical Analysis
A factorial experiment was used within the complete sectors and the randomized complete block design (RCBD) used in the experiment, as the total number of transactions was 51 treatments.The GenStat Release 12.1 was used to analyze the trial data.

Effect of Bio Fertilizer and the Level of Mineral Fertilizer on the Weight of 1000 Grains (gm) of Wheat
The results in Table (2) show the effect of fertilization level on the average thousand-grain weight.If the secondary fertilizer F1 level is better than the F0 level, the thousand-grain weight is 48.5 g, while the average value recorded by F0 is 38.2 g , This may be attributed to the role of nitrogen uptake through the bud system in increasing the efficiency of starch production, conversion of starch to sugars, and increased transfer of soluble protein to the grain, thus reflected in an increase in the weight of the grain.
The results showed in Table (2) the effect of the binary interaction between the type of biological fertilization and chemical fertilization on the weight of one thousand grains , The results indicated that the inoculated treatments with the level of chemical fertilization were superior to the inoculated treatments with the same levels of the corresponding chemical fertilization in the supplementary treatment, The results also showed that a binary interaction treatment was recorded between (P.vermicola bacteria and R. planticola) with half of the fertilizer recommendations up to a weight of 54 g for F1.An increase rate of 23.46% was recorded from the treatment for the complete proposal and an average of 41.33 g was recorded, The reason may be that the effect of bio-fertilizers leads to faster emergence, more synthetic metabolites, prolonging the filling period and increasing the thousand-grain weight.Grains, as one of the yield characteristics, affect the increase in thousand-grain weight in addition to increasing the plant's processing nutrient efficiency.This result is consistent with [7,8].

The Effect of Bio Fertilizer and the Level of Mineral Fertilizer on the Grain Yield µg.H -1 of Wheat
The results shown in Table (3) show that if the bio fertilizer treatment outperforms the non-pollinated treatment, if the two overlapping treatments (P3) had the highest grain yield of 7.21 µg H -1 compared to the two single pollination treatments P2 and P1 without Significant difference since they recorded rates of 6.52 µg H -1 and 6.39 µg H -1 respectively, This may be due to the role of the bacterial species used in bio fertilizer preparation (R. planticola and P. vermicola) in regulating the production of growth hormones [9,10].Which is due to its important role in increasing the various growth indicators and yield components of plants by increasing cell division and elongation, and then expanding the root hairs, which in turn will increase the surface area of the roots Hence, the absorption of nutrients is greater, which reflects positively on increasing the various indicators of vegetative growth and the formation of dry matter, and then increasing the grain fullness and weight gain [11].
The results in the same table show the effect of the level of chemical fertilization on the grain yield of the wheat plant if the fertilization treatment at the second level (F1) was superior to the first level (F0) if it recorded the highest grain yield of 6.76 µg H1-, with an increase rate of 21.01% over the F0 treatment, which recorded an average of 5.34 µg H -1 , The reason for this may be due to the increase in available phosphorus in the soil and thus absorbed by the plant to meet the need of the wheat crop for this nutrient for its important role in the metabolic processes of the plant and the formation of energy compounds as well as its significant effects on the process of flowering and grain formation [12].
For the two-sided interaction of bio fertilizer inoculation and mineral fertilizer on grain yield, the results showed that the interaction of P3 with secondary fertilization F1 was superior to all treatments when grain yield reached 7.70 µg H -1 6.10% increase compared to the full fertilization treatment, with an average of 7.23 µg H -1 , This may be due to the role of bio fertilizer by providing part of the nutrients that directly or indirectly affected plant growth and the length of the growing season and increased the efficiency of the vital processes of the plant, which increased the growth of the root and shoot system of the plant and increased the total chlorophyll content and this made the plant more vital in absorbing Nutrients are then transported to storage locations in the grain.Some researchers have also pointed out that the interaction between bio fertilizers and chemical fertilizers increases the food yield of some crops due to the increased activity of microorganisms in the rhizosphere, leading to an increased readiness of some nutrients in the form of insoluble compounds, especially phosphorus compounds, which lead to Reduced use of chemical fertilizers [13,14].Table 3.The effect of bio fertilizer and the level of mineral fertilizer on grain yield (µg H -1 ) of wheat.4) indicate the effect of adding bio fertilizer on the biological yield, as the results showed that there was a significant increase in the biological yield of the vaccinated treatments, whether single or double, compared to the control treatment (without vaccination), Compared with comparative treatment P0, treatment P3 had the highest yield of 22.06 µg H -1 , with a growth rate of 32.579%, and comparative treatment P0 had the lowest yield of 14.88 µg H -1 , followed by treatments P2 and P1, which recorded 21.729 µg H1-20.088µg H -1 , respectively, Perhaps this is due to the fact that bio fertilizers increase the root system of the plant, as they secrete some substances that stimulate plant growth, enzymes, some vitamins such as vitamin B12, and some antibiotics that kill harmful microbes in the soil, which leads to an increase in the efficiency of root absorption of nutrients in the soil, including nitrogen, which helped To increase the vegetative growth, the number of branches and the area of the flag leaf, which in turn increased the biological yield of [15].As shown in Table ( 4), the level of mineral fertilizers has a clear effect, and if the fertilization level F1 exceeds the level F0, this in turn leads to a significant difference at the 5% probability level, as it gives the highest value for biomass of 20.88 µg H -1 .Nitrogen content in plants, by increasing its availability and uptake, leads to increased life processes as it is one of the components of enzymes, proteins and chlorophyll.It is involved in all enzymatic processes and reactions, photosynthesis, and works to increase the fertilization of branches with spikes.This result is consistent with [16].For the two-sided interaction of vigor inoculum type and mineral fertilizer level in biological crops, the results showed that the interaction treatment P1 with fertilization level F1 was 24.792 µg H -1 , which is a 32.92% increase over the recommended fertilizer if the highest grain yield was recorded, The average was 16.63 µg H -1 , which may be due to the use of biological fertilizers and half the recommended fertilizers leading to increased plant growth through a variety of mechanisms, including increased auxin regulation and increased nutrient preparation in the soil, or through their use as bio resistance agent, which in turn helps to promote plant growth [17].

Effects of Biological and Mineral Fertilizers on Nitrogen Content in Shoots of 50% Flowering Wheat
The results in Table (5) show that, regardless of the type of biological inoculation, the addition of biological fertilizers has a significant effect on the amount of nitrogen absorbed by plants if the results show that the inoculated treatment is superior to the non-inoculated treatment, If the results indicate that the inoculation treatment P3 is superior to all inoculation treatments, if it recorded the highest amount of absorbed nitrogen amounting to 3.51%, with an increase rate of 46.44% from the control treatment P0, which recorded a rate of 1.882%,The reason for this may be attributed to the fact that R. planticola and P. vermicola are able to fix nitrogen in free form [18,19] and store it in cells, thus 1259 (2023) 012113 IOP Publishing doi:10.1088/1755-1315/1259/1/0121136 benefiting the plant.After cell death, perhaps The indirect method they use.Bacteria, such as reducing the degree of soil interaction or the secretion of growth regulators and some vitamins, somehow affect the processing of nutrients, including nitrogen [20].The results shown in the above table also indicated the effect of the level of chemical fertilization on the absorbed amount of nitrogen, as there was a significant increase in the amount of nitrogen absorbed by increasing the level of chemical fertilization, as the F1 level was superior to the first level, F0, which recorded 3.05%, with an increase of 29.18% over the F0 treatment.This increase may be attributed to the role of the added mineral fertilizers in increasing the readiness of nutrients in the soil solution, and then their absorption by the roots of plants, and thus increasing their concentration in the vegetative system of the plant, This result is consistent with the results reached by [21,22], who found that the percentage of nitrogen uptake by plants increased with the addition of mineral fertilizers.On the other hand, the results shown in Table (5) show the effect of biological inoculum type and chemical fertilizer on the amount of N uptake by plants, if it shows the superiority of the interaction between P3 × F1 treatment, as recorded 4.24%, compared to Fertilizer recommendations recorded an increase of 11.87% to 3.79%, which may be due to bacterial interference, which played a role in increasing the efficiency of nutrient uptake, thereby increasing the efficiency of fertilizer use [23].

Effects of Biological and Mineral Fertilizers on Potassium Content in Shoots of 50% Flowering Wheat
The results in Table (6) show the effect of the type of biological inoculant on the amount of potassium uptake by plants , If the results indicated the superiority of the P3 treatment, as it recorded a rate of 2.27%, while the P1 treatment recorded the lowest rate of 1.61%, and this result agreed with what was reached [13,24], who found the superiority of double bacterial inoculation over single inoculation in the concentration of potassium absorbed by the plant.The results of the table showed that the use of mineral fertilizer led to an increase in the concentration of potassium in the vegetative part (%), as treatment F1 gave the highest rate of 2.33%, Perhaps the reason for the superiority of mineral fertilizer treatment in potassium concentration is due to the role of nitrogen, where nitrogen is linked to potassium in a positive relationship of overlap, which is characterized by the speed of the process of transport and absorption, and thus entering into the construction of chlorophyll, nucleic and amino acids, and the formation of enzymes responsible for various vital processes, and this was reflected in the efficiency of photosynthesis and the efficiency of nutrient absorption Including potassium, water, improving plant growth and energy compounds, and this result agreed with [25,26].The results showed in Table (6) the effect of the interaction between the type of biological inoculum and chemical fertilization on the amount of potassium absorbed by the plant, If the results show that the overlapping treatment P3 × F1 outperformed the fully recommended fertilization treatment by 2.96%, with a growth rate of 2.81%, recording a growth rate of 5.07%, The reason may be due to the efficiency of the microorganisms used in the experiment to secrete growth regulators and organic acids that dissolve and release phosphorus and potassium from their unprepared sources, which leads to an increase in their readiness, This was confirmed by [3], who indicated the importance of bio fertilizers in providing part of the important nutrients for the plant such as nitrogen, phosphorus and potassium, as well as increasing its readiness in addition to its secretion of growth regulators.

Effects of Bio Fertilizer and Mineral Fertilizer Levels on Percent Phosphorous in Shoots of Wheat Plants with 50% Flowering
The results in Table (7) show that, compared with no addition of biological fertilizers (comparison treatment), the inoculation of biological fertilizers has a significant impact on the amount of phosphorus uptake.The highest concentration of phosphorus absorbed by plants is 0.73%, which is an increase of 0.59% compared with the control treatment P0. 19.18%,This is consistent with [1], who demonstrated that the use of double bio pollen resulted in increased phosphorus uptake by plants.The results also indicated that there was a significant increase in the amount of phosphorus absorbed when increasing the level of chemical fertilization, if the second level F1 was significantly superior to the first level F0 of the fertilizer recommendation if it recorded 0.70%, with an increase of 11.43% over the comparison treatment , the reason may be due to the effect of urea added to the soil and the liberation of hydrogen ions after the conversion of urea into nitrate and the increase in the availability of nutrients and then the increase in phosphorus absorbed by the plant, and this result is consistent with [27].We noted that the interaction coefficient between the biological inoculum type and the secondary fertilizer was significantly superior in terms of the amount of P uptake by plants, as the interaction treatment F1×P3 recorded the highest P uptake rate of 0.81%, This may be due to the role of integrated fertilization, represented by adding half of the specified amount of nitrogen and phosphorus with bio fertilization, which yields results for using the full recommendation of chemical fertilizers for nitrogen and phosphorus, but at lower economic costs while maximizing production [28,29].

Conclusion
Through field trials, we found that using a dual bio fertilization treatment (P.vermicola and R. planticola) had the best results in terms of grain yield characteristics and percentages of elements (N, K and P).Fertilization treatments, while semi-recommended fertilization treatments were superior in all traits studied, while (P.vermicola and R. planticola) interaction and semi-recommended fertilization treatments were significantly superior in the percentage of NPK elements.

Table 1 .
The chemical, physical and biological characteristics of the study soil before planting.

Table 2 .
The effect of bio fertilizer and the level of mineral fertilizer on the weight of one thousand grains (gm) of wheat.

Table 4 .
The effect of bio fertilizer and the level of mineral fertilizer on biological yield (µg H -1 ) of wheat.

Table 5 .
Effects of bio fertilizer and mineral fertilizer levels on percent nitrogen in shoots of wheat plants with 50% flowering.

Table 6 .
Effects of bio fertilizer and mineral fertilizer levels on percent potassium in shoots of wheat plants with 50% flowering.

Table 7 .
Effects of bio fertilizer and mineral fertilizer levels on percent phosphorous in shoots of wheat plants with 50% flowering.