Uptake of 15N-urea and phosphates in Triticum aestivum with Pseudomonas putida and Rhizophagus irregularis endophytes of calcareous soil weeds

In calcareous soil, the growth and production of Triticum aestivu m depends on the availability of phosphates, which in turn reduces the uptake of nitrogen in the form of urea, which causes volatilization and partial leaching of both fertilizers, contributing to the greenhouse effect, and warming global. An alternative ecological solution for T. aestivum is to inoculate Pseudomonas putida and Rhizophagus irregularis with endophytes that increase phosphorus uptake such as P 2 O 5 and urea. The objective of this research was to analyze the uptake and distribution of 15 N-urea in T. aestivum with P. putida and R. irregularis fertilized with 50% urea and P 2 O 5 . In this sense, P. putida and R. irregularis isolated from roots of Resenda luteola and Arista purpurea native to the calcareous soil of northeastern Mexico. In T. aestivum with these endophytes, acid and alkaline phosphatase activity in root and stem, N (nitrogen) uptake from total urea (Nt) and yield (Y). The experimental data were analyzed by ANOVA/Tukey (P<0.01). The results showed a positive response of T. aestivum to P. putida and R. irregularis with 60 kg/ha of urea and 40 kg/ha of P 2 O 5 , equivalent to the 50% recommended for this region of Mexico. It was evidenced that in T. aestivum, P. putida, and R. irregularis endophytes of desert weeds improved the uptake of urea and P 2 O 5 to 50% by phytohormones that optimized nitrogen with phosphatases, soil phosphate and that from the applied fertilizer. The Nt in stem and the yield of T. aestivum with P. putida and R. irregularis reached statistically different numerical values to those registered in T. aestivum with 120 kg/ha of urea and 80 kg/ha of P 2 O 5 without P. putida and R. irregularis at 100% (relative control), consequently the performance of T. aestivum in calcareous soils. It is avoided by the generation of greenhouse gases, the contamination of surface water, by using the beneficial interaction of endophytes with weeds with T. aestivum, as well as global warming.


Introduction
In the northeast of Mexico, the soil is of calcareous origin, they have an alkaline pH with high salinity [1] , which limits the absorption of nitrogenous fertilizers (urea) and PO4-3 (phosphates). In specific calcareous soil pH controls the relative availability of the two ionic chemical forms of phosphate. The H 2 PO 4 ion is favored by a pH less than 7, while the divalent ion HPO 4 = is above pH 7. When the pH is alkaline, the phosphate in the form of apatite (Fe2Ca3(PO4)2Ca3), hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), and carbonatoapatite (Ca 3 (PO 4 ) 2 CaCO 3 ) causes a drastic decrease in the production of Triticum aestivum (wheat) [2] . Besides that, it causes volatilization problems of nitrogenous fertilizers as well as the leaching of nitrogenous fertilizers and phosphates, which contributes to the generation of greenhouse gases and contamination of surface and deep waters. While evolutionarily wild plants have these interactions as prokaryotic endophytic microorganisms, such as the eukaryotic genus, that allow them to face this problem of the availability of phosphates [3] [4] , especially in calcareous soil, since phosphates are necessary for obtaining energy and synthesis of nucleic acids, endophytic prokaryotes with acid and alkaline phosphatase enzymes, and endotrophic mycorrhizal fungi with these enzymes and by translocation of phosphates (3)(4)(5). In general, nitrogenous fertilizers such as urea and phosphorus such as P 2 O 5 (triple superphosphate) are applied to the soil in order to be effectively absorbed by the roots of T. aestivum [5] [6] . For this reason, there are ecological solution alternatives such as weeds that grow successfully in adverse environments such as nutritional stress and tolerance to salinity, since they are associated with endophytic microorganisms that solubilize calcium phosphate complexes and less aluminum or iron phosphate compared to mycorrhizae [7] [6] . Therefore, in this work, Pseudomonas putida was isolated and selected, an endophyte due to the synthesis of phytohormones that improves the radical uptake of minerals with nitrogen (NH 4 + ) [8] [9] [10] [11] and a mycorrhizal fungus: Rhizophagus irregularis that synthesizes alkaline phosphatases for the solubilization of phosphorus [12] [13] , recovered from 2 weeds such as Reseda luteola and Aristida purpurea adapted to the alkalinity and salinity associated with T. aestivum. [14] [15] . The objective of this work was to analyze the uptake and distribution of 15 [16] that observed under the microscope, arbuscules and vesicles were observed inside the roots of both weeds, were cut, ground and treated with 0.1 g/l kanamycin to inhibit bacterial growth. The spores of R. irregularis were separated, sieved, and decanted following the technique proposed by [17] . comparison of means (P ≤ 0.01) [19] .
Sowing, inoculation, and fertilization of Triticum aestivum in the field.
This part of the research was carried out in a field of the UAAAN, Saltillo, Coahuila, Mexico, at coordinates 25° 25" 41' north longitude and 100° 57" 57' west longitude, with an altitude of 1742 m. The soil was classified as silty-sandy according to its physicochemical properties as shown in Table 1 [20] . The soil was then prepared in yield plots of 4.0 m 2 (2x2 m) with five rows each, spaced 30 cm apart, and a useful plot of 1.0 m 2 . The seeds of T. aestivum variety: Pavón F-76 used in this trial were provided by UAAAN, were soaked with 10% sucrose, then mixed with 40 g of P. putida and 0.2 g of R. irregularis: G1 and G2. The seeds inoculation with P. putida and R. irregularis were sown by the broadcast method at a density of 150 kg seed/ha per treatment based on the experimental design shown in Table 2. The fertilization rates were 0, 60, and 120 kg/ha of urea and 0, 40, and 80 kg/ha of P 2 0 5 , equivalent to 0, 50, and 100% of the recommended rates for the region, applying 0, 60 and 120 of urea and 0, 40 and 80 kg/ha of P 2 0 5 . Both fertilizers were divided into three equal parts and applied manually in bands during the critical growth stages of T. aestivum: tillering, anthesis, and beginning of grain filling [21] .  [20] .
The response variables to determine the effect of P. putida and R. irregularis: G1 and G2 on T. aestivum the uptake of 15 N-urea, the plots corresponding to the treatments were fertilized with the stable isotope of nitrogen known as 15 N-urea, with a 10% excess of 15 N atoms in seven 1.0 m 2 microplots. In addition to inoculation, the T. aestivum plots were fertilized with 60 kg/ha of urea and 0 and 40 kg/ha of P 2 O 5 . For isotopic analysis, 20 T. aestivum seedlings were collected from each plot, and the grain was separated and ground to a particle size of 1.0 mm. They were analyzed by mass spectrometry at the CINVESTAV unit, Irapuato, Guanajuato, Mexico, and the percentage of 15 N derived from urea was calculated [22] . The percentage of total nitrogen (Nt) was also quantified by the Kjeldahl method and grain yield was estimated in Ton/Ha.
Labeling of Pseudomonas putida on Triticum aestivum in the field conditions.
To evaluate the colonization of P. putida on T. aestivum roots, it was labeled by the antibiotic resistance method. For this, P. putida was grown in a 250 mL flask with 50 mL of nutrient broth (Bioxon) at 30°C/24 h at 150 rpm. It was then seeded on nutrient agar to determine its resistance to 12 antibiotics Gram Negative multidisc (Senofi). As well as its tolerance to 200 µg/mL of trimethoprim-sulfamethoxazole (Bactrin, Roche), to ensure its recovery from field-grown T. aestivum inside of roots.
Detection of acid and alkaline phosphatase in T. aestivum inoculated with P. putida and R. irregularis.
To determine the ability to solubilize phosphatase, according to [23] method was used, P. putida was activated in PO halo was detected around the colony that indicated the release of PO 4 3by phosphatases [6] . Quantitative analysis of  [23] , the following was prepared: a 0.9%-0.1% detergent saline solution as absolute control; the roots and the stem of T.aestivum inoculated and uninoculated with P. putida and R. irregularis were taken at flowering 60 days after planting, then were disinfected with 1% chlorine for 3 min, rinsed 5 times with sterile water, then with 70% alcohol for 3 min, were rinsed 7 times with sterile water, were transferred to a sterile mortar, were macerated with 10 mL of sterile 0.9%-1% detergent saline solution, 5mL was taken, 45mL of sterile distilled water was added with 20mL of buffer adjusted to pH 5.5 for the determination of acid phosphatase and pH 9.0 for the alkaline, the mixture was homogenized at 800 rpm/30 seconds, then 3mL of the suspension was used to 1mL of p-nitrophenyl phosphate (0.025 M) were left for 3h/37°C, centrifuged at 2000rpm/10min at 0.5mL of the 4.5mL of NaOH (0.5M) was added to the supernatant, the pnitrophenol released was measured in a spectrophotometer at 410nm, which indirectly detected the release of PO 4 3from p-nitrophenyl phosphate by acid phosphatases /alkaline of P. putida and R. irregularis in T. aestivum.
The experimental data were analyzed by the statistical test ANOVA/ Tukey HSD P<0.01 with Statgraphics Centurion XVI.I [19] . In the effectiveness test of P. putida and R. irregularis coded as G1 and G2 in T. aestivum fertilized with 60 kg/ha of urea and 40 kg/ha of P 2 O 5 equivalent to 50% of the recommended dose, induced an increase in total dry weight (TDW), similar to that registered in T. aestivum uninoculated and fertilized with 120 kg/ha of urea and 80 kg/ha of P 2 O 5 Kg/ha equivalent to 100% referred to as relative control (data not shown).
Nt content in the grain of T. aestivum inoculated with P. putida and R. irregularis Figure 2 shows the amount of Nt in the grain of T. aestivum with P. putida and R. irregularis (G1) with 60 kg/ha of urea and 40 kg/ha of P 2 O 5 equivalent to 50%, there is registered 3.31%, a numerical value statistically different compared to 2.58% of Nt in grain of T. aestivum uninoculated, fertilized with 120 kg/ha of urea and 80 kg/ha of P 2 O 5 equivalent to 100% or relative control (RC). As well as with 2.59% Nt in T. aestivum grain with P. putida and R. irregularis (G2) fertilized with urea and P 2 O 5 at 50%. In the grain of T. aestivum with P. putida and R. irregularis at 50% urea and phosphate doses, the nitrogen concentration was equal to or higher than the nitrogen concentration in T. aestivum with the 100% urea dose and phosphates recommended for that agricultural zone of northeastern Mexico. (120 and 80 kg/ha respectively); Treatment (T) 1: T. aestivum with P. putida and R. irregularis (G1) with urea and P2O5 at 0%; T2: T. aestivum with P. putida and R. irregularis (G2) with urea and P2O5 at 0%; T3: T. aestivum with P. putida and G1 with urea at 50% and P2O5 at 0%; T4: T. aestivum with P. putida and G2 with urea at 50% and P2O5 at 0%; T5: T. aestivum with P. putida and G1 with urea at 0% and P2O5 at 50%; T6: T. aestivum with P. putida and G2 with 0% urea and 50% P2O5; T7: T. aestivum with P. putida and G1 with urea and P2O5 at 50%; T8: T. aestivum with P. putida and G2 with urea and P2O5 at 50%.
* numerical values with the same letters between bars do not differ statistically (ANOVA-Tukey, P ≤ 0.01).      where this problem is critical [14] . Since endophytic microorganisms have an advantage compared to rhizobacteria isolated from domestic plants, their ability to interact intimately with the diversity of wild or domestic plant species including their adaptability to adverse environments: stress caused by lack of soluble PO 4 -3 as well as other basic minerals for plant nutrition [26] [27] . Therefore, wild plants growing in soils such as calcareous benefit from these endophytic microorganisms, which are able to enhance the uptake of basic nutrients: inorganic nitrogen, PO 4 -3 , and other microelements to deal with water stress and salinity. In Saltillo, Coah, Mexico. Temperatures between 8 and 10°C in the morning, between 29 and 35°C at midday, and in the afternoon are frequent, with higher temperatures the nitrogen and phosphorus cycle can be altered [28] . Wild plants and weeds are harmful to conventional agriculture, but persistent and successful growth in nature despite efforts for its control and/or elimination is an option to isolate endophytic plant growth-promoting microorganisms such as P. putida isolated from A. purpurea and R. irregularis: G1 and G2 from R. luteola both have the ability to positively interact with both wild and domestic plants given the evolutionary versatility of its genome reason why are import tool to enhance in T. aestivum the uptake of urea and P 2 O 5 in calcareous soil [1][2][5] [7] .
In Figure 2, it is supported that the combination of P. putida and R. irregularis was sufficient to improve the efficiency of root uptake of urea into the soil by a hormonal effect that increased the root uptake surface [9] [15] . While in grain of T.
aestivum uninoculated, fertilized with urea and P 2 O 5 at 100%, a relatively low concentration of % Nt was determined, indicating that the root system was unable to uptake urea from the soil, phosphates in alkaline soils react to generate low solubility phosphorus compounds, which remain for a long time in less soluble forms, little available to plant roots [6][3][12] [13] .
In this sense, the alkaline phosphatases of P. putida, as well as the various strategies of R. irregularis, allow the translocation of insoluble phosphates, and the optimization of reduced soluble phosphates as was evident in treatments 3, 7, and 8 since the activity of alkaline phosphatases increases the capacity of T. aestivum roots to uptake not only the insoluble phosphate from the soil, but also the phosphate that is applied as fertilizer, while R. irregularis inside the root system of T aestivum does it through a synergistic action with P. putida and simultaneously by the mechanism of translocation of insoluble phosphates from the soil, it also optimizes the uptake of phosphorous fertilizer and urea at 50% of the recommended value, to improve the health of T.aestivum. In addition to avoiding the release of greenhouse gases, as well as contamination of surface and underground water [7][3] [14][16] [23] .
In Figure 3, based on these results, it is estimated that one way to solve urea uptake in alkaline soil is not necessary to increase the dose of urea and P 2 O 5 , as observed in this work, but due to inoculation of the seed with endophytic P. putida that increase the root system to improve urea uptake and with R irregularis that solubilize P 2 O 5 due phosphatases both microorganisms are working synergistically inside the root system of T. aestivum [11] [4] . The positive effect on the yield of T. aestivum with P. putida and R. irregularis indicates that both contribute to a higher uptake of urea and P 2 O 5 by alkaline phosphatases inside the root system of T. aestivum to maintain an acceptable yield in comparison to T. aestivum used as an RC [10] [15] . The effect of P. putida and R. irregularis has been reported in nature, due to ecological and/or symbiotic interactions: root-microorganism, as in the case of calcareous soils that are deficient in these essential elements for agricultural crops like T. aestivum. Based on the above, there is evidence that P. putida a genus and species of phosphate-solubilizing endophytic bacteria interact with R. irregularis a vesicular-arbuscular mycorrhiza or VAM that induces a synergistic relationship with the root of plants for a better use of poorly soluble P [25][28] [29] . The results obtained in this work demonstrated that it is possible that the phosphate solubilized by the phosphatase of P. putida is effectively absorbed by the roots of T. aestivum through a biological or mycorrhizal bridge that captures the insoluble phosphate and translocates it from the soil including the root and includes the N of urea, [21][22] [29] . This was demonstrated with phosphorus 32 ( 32 P), with phosphate solubilizing, that associated with R. irregularis increased the effective uptake of nitrogenous and phosphorous fertilizers reduced to 50% [23][24] [25] .
In Figure 4, the increase in the uptake of NdF labeled as 15 N-urea in T. aestivum with P. putida indicated a positive activity of alkaline phosphatases and phytohormones inside of root system [11][26] [27] that induced a more abundant root system that increased urea uptake [27] [29] . These results support that an adequate selection of P. putida and R. irregularis endophytic maximizes the radical uptake of nitrogen fertilizer as urea by T. aestivum. Simultaneously phosphatase activity of R. irregularis, inside of the root system P 2 O 5 solubilization occurred to solubilize inorganic no soluble phosphates [6] [12] .
These data supported the need to correctly select a combination of endophytic bacteria and endomycorrhizal fungi working in a synergistic action on urea and P 2 O 5 uptake [4][8] [16] [30] .
In the root system of T. aestivum inoculated with P. putida, a bacterial density between 4.0X10 4 and 7.0X10 7 CFU/g root was registered, reported as sufficient to exert a positive effect on root uptake of urea by T. aestivum [23] [31] . It is also reported that P. putida is competitive with the native microbial population of calcareous soil, due to its adaptation to this alkaline environment. In this sense, P. putida was isolated from A. purpurea, which similarly to T. aestivum is a gramine, and this could lead to the adaptability and population development of P. putida [32] [33] .