Triticum aestivum requires nitrogen fertilizer (NF) as NH₄NO₃ for healthy growth, however its excessive application causes loss of soil fertility. An alternative to decrease the amount of NH₄NO₃ and to optimize is to inoculate T. aestivum seeds with Xanthobacter autotrophicus or/and Bacillus thuringiensis well known asendophytic plant growth promoting bacteria (EPGPB). Both genera and species of EPGPB are able transform metabolic compounds from seeds and roots into phyto hormones, which can be optimized by crude extract carbon nano particles (CECNPs) to enhance the growth of radical system and to improve uptake at 50% dose of NH₄NO₃ Response variables were: germination percentage, phenology, and biomass to seedling stage. All experimental data were validated by ANOVA/Tukey HSD P<0.05. Seeds of T. aestivum were inoculated with X. autotrophicus and/or B. thuringiensis, plus CECNPs at 50% NH₄NO₃. Results showed a positive response of T. aestivum seeds to X. autotrophicus and/or B. thuringiensis, with 10 ppm CECNPs, and 50% NH₄NO₃ enhanced germination percent of 93% in comparison with 73% of T. aestivum when its seeds were not inoculated with these EPGPB fed only with 100% dose of NH₄NO₃ (relative control); the same positive response of T. aestivum to X. autotrophicus and B. thuringiensis improved by CECNPs at seeding stage compared to T. aestivum used as a relative control.

Keywords: soil, nitrogen fertilizer, wheat, EPGPB, CECNPs, phytohormons

The healthy growth of Triticum aestivum (wheat) requires nitrogen fertilizer (NF) such as NH₄NO₃,¹ but applied in excess leads to cause of soil productivity due to accelerate mineralization of the organic matter.² An alternative ecological solution to this problem related to NF, is to inoculate seeds of T. aestivum with X. autotrophicus and B. thuringiensis both genera and species are endophytic plant growth promoting bacteria (EPGPB), its beneficial effect can be optimized with a crude extract of nano particles (CECNPs) at 50% dose of NH₄NO_3.Due to X. autotrophicus and B. thuringiensis are able to transform seed exudates and metabolic compounds from photosynthesis of roots into phytohormones, to improve germination and to induce accelerated and dense root formation that optimizes uptake reduced dose of NH₄NO₃, without detrimental effects on the healthy growth of T. aestivum.^3-7 Currently, there is scarce information on the optimized of the beneficial effect of X. autotrophicus and B thuringiensis to maximize the capacity of radical absorption and optimization at 50% of NH₄NO₃ this beneficial activity of EPGPB can been hancing by applying a CECNPs, to improve the germination and plant growth, as reported by Srivastava & Rao,⁸ whom treated Zea mays seeds with 5 ppm of multi-walled carbon nano tubes (MWCNTs), where up to 80% germination was recorded compared to 60% of Z mays without the MWCNTs irrigated only with water. While Joshi et al.,⁹ analyzed the effect of T. aestivum with 8 ppm nano particles of carbon, increased aerial fresh weight of 1.3 g and 0.09 g of root fresh weight in comparison with aerial fresh weight of 0.8 g and 0.5 g of root fresh weight obtained for treatment with only 100% NH₄NO₃ dose. In that sense the aim of this research work was to analyze Triticum aestivum with X. autotrophicus and/or B. thuringiensis, enhance by CECNPs, at 50% of NH₄NO₃.

This research at the greenhouse of the Environmental Microbiology laboratory - Instituto de Investigaciones Químico-Biológicas at Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Mich., México. The studies were performed under greenhouse environmental conditions with average values: T = 23.2°C, luminosity = 450 µmol•m^-2•s^-1, relative humidity = 67%. The agricultural soil was collected from 19°37’10” north latitude and 101°16’41.00” west longitude, with an altitude of 2013 meters above sea level, at a temperate climate zone called “Uruapilla” municipality of Morelia, Michoacán, Mexico, on the Morelia-Pátzcuaro highway. The soil was sieved with a mesh No. 20 and was exposed to sun light for 40 h to prevent pests and diseases,¹⁰ the physicochemical properties are shown in Table 2.

1. Preparation of crude extract of crude extract carbon nano particles from Albizia pluriforma

Leaves of A. pluriforma were collected from UMSNH campus in Morelia, Mich, México. The leaves were disinfected by immersion in a 0.5% NaClO for 1 min and were rinsed with sterile deionized H₂O. The leaves were cut into 5 cm pieces and dried in an oven at 80°C for 12 h, 30 g were suspended in 300 mL deionized H₂O, heated at 70°C for 30 min and filtered through a Whatman No.1 paper. The filtrate was centrifuged at 4000 rpm for 10 min and then placed in a fridge at 4°C. Subsequently for the characterization of the CECNPs a JEOL JSM-6400 scanning electron microscope (SEM) from the Institute of Research in Metallurgy and Materials of the UMSNH, Morelia, Mich, Mexico was used to characterize the size and morphology of the nano particles synthesized from A. pluriforma according to the following procedure: dried at 100°C/12 h, then a gold film was applied to the CECNPs to allow the sample to be conductive so that the size of the nanoparticles could be analyzed. To determine the qualitative and quantitative composition of CECNPs was performed by energy dispersive spectroscopy (EDS), for this the CECNPs was dried, then analyzed using a JEOL-JSM-7600F EDS field emission microscope.

2. Triticum aestivum inoculated with Xanthobacter autotrophicus and Bacillus thuringiensis plus a crude extract of carbon nano particles and NH₄NO₃ at 50%

T. aestivum seeds had a positive response to X. autotrophicus and B. thuringiensis, enhancing with CECNPs at 50% of NH₄NO₃. T. aestivum seeds were disinfected with 0.2% NaClO for 5 min, rinsed 6 times with sterile tap water and disinfected then with ethanol 70% (v/v) for 5 min. The seeds were rinsed 6 times with sterile tap water and inoculated with X. autotrophicus and/or B. thuringiensis.¹⁰ A suspension of saline solution 0.85% of NaCl (wt/v) of 1.0 mL de X. autotrophicus and/or B. thuringiensis in a relation 1:1 (v/v) was used for every 10 seeds of T. aestivum, followed by treatment with 1.0 mL of CECNPs at 10 or 20 ppm concentration, 0.85% NaCl and 0.5% (wt/v) of commercial detergent “La Corona”^MR. The treated seeds were stirred at 200 rpm for 30min at 28°C to generate an homogeneous dispersion of CECNPs, then sown in a container with 100 g of soil (Table 2), fed with a mineral solution (MS) with recommended dose of NF as NH₄NO₃ for T.aestivum as nitrogen source of following chemical composition (g/L): NH₄NO₃ 10; K₂HPO₄, 2.5; KH₂PO₄, 2.0; MgSO₄ 0.5; NaCl, 0.1; CaCl₂, 0.1; FeSO₄ 1.0; and a microelements solution (g/L): H₃BO₃ 2.86; ZnSO₄•7H₂O 0.22; MgCl₂•7H₂O 1.8, at pH = 6.8. The MS was applying to feed T. aestivum using a volume of 5 mL every 3 days per month to assure the field capacity at 80%. The test was carried out according to Table 1, which shows the experimental design of random blocks with 6 treatments, 2 controls and 10 repetitions, with the following conditions: Absolute control (AC), T. aestivum irrigated with water only; Relative Control (RC): T. aestivum fed with100% NH₄NO₃ without X. autotrophicus and/or B. thuringiensis and all treatments: T. aestivum with X. autotrophicus and/or B. thuringiensis, plus 10 or 20 ppm CECNPs, at 50% NH₄NO₃.The variables-responses of this test were germination percentage (%) between four and ten days after sowing, phenology: plant height (PH) and root length (RL); and the biomass: aerial fresh weight/root fresh weight (AFW/RFW) and aerial dry weight/root dry weight (ADW/RDW) at seedling were analyzed at 30 days after sowing. The results were validated by ANOVA/Tukey HSD P<0.05% using the Statgraphics Centurion software.¹¹ 

Table 2 shows the physicochemical properties of soils, with analysis according to the Mexican regulation NOM-021-RECNAT-2000. The soil is slightly acidic (pH = 5.75), with high content of organic matter of 10.44%, with texture: clay 31.08%, slit 42.00%, sand 26.92%, and classified as clay-loam soil. It contains a medium concentration of total N of 0.32%, 244.16 ppm NO₃^-, and 219.34 ppm PO₄^-.

Figure 1 shows the SEM micrographs providing the morphology and size of the CECNPs nano particles synthesized from A. pluriforma, which presented spherical shapes with variable size, where some tended to be dispersed while others formed aggregates (Figure 1a). Additionally, EDS analysis provided a qualitative and quantitative status of the elements that may be involved in the formation of these nano particles. The elemental profile of the CECNPs recorded an atomic percentage of carbon of 50.98%, being the main element. There was also presence of oxygen up to 30.41% atomic and some traces of magnesium, phosphorus, chlorine and potassium, confirming the formation of carbon nano particles (Figure 1b).¹²

Figure 1 Morphology and qualitative/quantitative analysis of CECNPs from Albizia pluriforma:
a. SEM micrographs and
b. EDS composition.

Table 3 shows 73% of germination of T. aestivum seeds for the relative control, 93% of germination of T aestivum seeds with B. thuringiensis and X. autotrophicus with 10 ppm CECNPs and 50% NH₄NO₃, while increasing the CECNPs concentration to 20 ppm causes a decrease in germination to 86%. This suggests that water uptake by the seeds induced the activity of α-amylase that hydrolyzes amylose with the release of organic acids, amino acids and sugars,¹² that both B. thuringiensis and X. autotrophicus transformed in gibberellin-type phytohormons which were enhancing by the CECNPs accelerated the interruption of dormancy in the seeds.^9,14-16

Table 4 shows the phenology at seedling stage of T. aestivum with X. autotrophicus, B. thuringiensis, 20 ppm CECNPs and 50% of NH₄NO₃, where it registered 20,5 cm plant height (PH) and 16,16 cm root length (RL), numerical values with statistical difference compared to the 18,85 cm PH and 10,83 cm RL of T. aestivum without inoculating X. autotrophicus, B. thuringiensis, 20 ppm CECNPs with 100% of NH₄NO₃ as a nitrogen fertilizer (NF) or relative control (RC). Related to biomass of T. aestivum inoculated with X. autotrophicus, B. thuringiensis, plus 20 ppm CECNPs and 50% of NH₄NO₃ registered 0.34 g aerial fresh weight (AFW), 0.17 g root fresh weight (RFW), 0.07 g aerial dry weight (ADW) and 0.036 g root dry weight (RDW). All these numerical values showed statistical difference compared to T. aestivum without X. autotrophicus and/or B. thuringiensis, not treated with CECNPs at 100% of NH₄NO₃ or relative control (RC) with following values: 0.21 g AFW, 0.11 g RFW, 0.02 g DAW and 0.020 g RDW of It is possible that CECNPs improved the activity of X. autotrophicus and/or B. thuringiensis, converting organic compounds of the root and photosynthesis metabolism into phytohormons that induce accelerate growth of hole roots system,^2,17 enhancing by CECBPs that maximizing the radical absorption of 50% of NH₄NO₃.^18-21 In Table 4 indicate that the positive responds of T. aestivum to B. thuringiensis and/or X. autotrophicus plus CECNPs enhancing dry aerial weight and dry root weighare higher than values observed for T. aestivum or relative control, which suggest that CECNPs improving the uptake of NH₄NO₃ as a nitrogen fertilizer without the risk of losing soil productivity and might prevent pollution of the environment caused by the irrational use of nitrogen fertilizer.

 T. aestivum had a positive response to X. autotrophicus and B. thuringiensis enhanced by ECNPCs (T1-T8) on seedling stage of growth are visually evident (Figure 2). A largest stem diameter is observed in T. aestivum with X. autotrophicus and/or B. thuringiensis plus ECNPCs this fact suggests that X. autotrophicus and B. thuringiensis they convert compounds of the photosynthesis metabolism into phytohormons such as auxin to induce an accelerated formation of dense roots in this way the root system enhancing the exploration capacity increased the exploration capacity of the soil improved by CECNPs for the optimization of 50% of NH₄NO₃ in comparison to T. aestivum without any EPGPB, fed with 100% of NH₄NO₃regard as a relative control showing that normally T.aestivum not inoculated with EPGPB was unable to effectively absorb of NH₄NO₃ for better growth causing soil lost fertility and environmental pollution.^9,13,17

Figure 2 Response of Triticum aestivum seedling to Xanthobacter autotrophicus and Bacillus thuringiensis enhanced with a crude extract carbon nano particle and 50% NH₄NO_3.
*n=10
AC = T. aestivum pure water
RC = T. aestivum without X. autotrophicus and 100% NH₄NO₃
T1 = B. thuringiensis, 10 ppm crude extract carbon nano particles (CECNPs), 50% of NH₄NO₃
T2 = X. autotrophicus, 10 ppm CECNPs, 50% of NH₄NO₃
T3 = B. thuringiensis/ X. autotrophicus, 10 ppm CECNPs, 50% of NH₄NO₃
T4 = 10 ppm CECNPs, 50% of NH₄NO₃
T5 = B. thuringiensis, 20 ppm crude extract CECNPs, 50% of NH₄NO₃
T6 = X. autotrophicus, 20 ppm CECNPs, 50% of NH₄NO₃
T7 = B. thuringiensis/ X. autotrophicus, 20 ppm CECNPs, 50% of NH₄NO₃
T8 = 20 ppm CECNPs, 50% of NH₄NO₃.

The positive response of T. aestivum to X. autotrophicus or mixed with B. thuringiensis enhances the uptake of 50% NH₄NO₃ without the risk of affecting the healthy plant growth. Future studies will be performed with purified crude extract carbon nano particles to figure out which carbon nanostructure is responsible for enhancing the health growth of T. aestivum. It is expected that the ongoing studies might lead us to combinations in which lower doses of nitrogen fertilizer could be applied without compromising healthy growth of T. aestivum and thus avoiding soil productivity and environmental pollution.

We acknowledge support from a Mexico Innovation Fund grant provided by the David Rockefeller Center for Latin American Studies at Harvard University (2020-2022). To the project 2.7 (2022) from CIC-UMSNH, BIONUTRA S. A. de C.V., Maravatío, Mich., México.

The author declares there is no conflict of interest.

1. Araujo V, Amarilla D, Melgarejo M, et al. Efecto del fraccionamiento del nitrógeno en las características agronómicas del trigo. Revista de la Sociedad Científica del Paraguay. 2020;25(1):41–48.
2. Martínez Viera R, Dibut B, Yoania R. Efecto de la integración de aplicaciones agrícolas de biofertilizantes y fertilizantes minerales sobre las relaciones suelo-planta. Cultivos Tropicales. 2010;31(3):27–31.
3. Martínez Trujillo MA, García-Rivero M. Revisión: Aplicaciones ambientales de microorganismos inmovilizados. Revistamexicana de ingenieríaquímica. 2012;11(1):55–73.
4. Zahid M. Isolation and identification of indigenous plant growth promoting rhizobacteria from Himalayan region of Kashmir and their effect on improving growth and nutrient contents of maize (Zea mays). Frontiers in Microbiology. 2015;6:207.
5. Singh M, Singh PP, Patel AK, et al. Enumeration of Culturable Endophytic Bacterial Population of Different Lycopersicum esculentum Varieties. Int J Curr Microbiol App Sci. 2018;7(2):3344–3352.
6. Sanchez Yañez JM, Ocampo J, Nocera D, et al. The México Innovation Fund (MIF). Progress Report. Year 1. Field Test a Living Biofertilizer for Crop Growth in México. 2019.
7. Sanchez Yañez JM, Castro VJA, Nocera D, et al. The México Innovation Fund (MIF). Progress Report. Field Test a Living Biofertilizer for Crop Growth in México. 2020.
8. Srivastava A, Rao DP. Enhancement of seed germination and plant growth of wheat, maize, peanut and garlic using multiwalled carbon nanotubes. European Chemical Bulletin. 2014;3(5):502–504.
9. Joshi A, Kaur S, Dharamvir K, et al. Multi‐walled carbon nanotubes applied through seed‐priming influence early germination, root hair, growth and yield of bread wheat (Triticum aestivum L.). Journal of the Science of Food and Agriculture. 2018;98(8):3148–3160.
10. Sánchez Yáñez JM. Breve Tratado de Microbiología Agrícola, Teoría y Práctica. Mich: México; 2007.
11. Walpole RE, Myers RH, Myers SL, et al. Probabilidad y estadística para ingeniería y ciencias. Norma: México; 2012;162:
12. Abdelmoteleb A, Valdez-Salas B, Ceceña-Duran C, et al. Silver nano particles from Prosopis glandulosa and their potential application as bio control of Acinetobacter calcoaceticus and Bacillus cereus. Chemical Speciation & Bioavailability. 2017;29(1):1–5.
13. Camelo M, Vera SP, Bonilla RR. Mecanismos de acción de las rizo bacterias promotoras del crecimiento vegetal. Ciencia & Tecnología Agropecuaria. 2011;12(2):159–166.
14. Liu C, Sakimoto KK, Colón BC, et al. Ambient nitrogen reduction cycle using a hybrid inorganic–biological system. Proceedings of the National Academy of Sciences. 2017;114(25):6450–6455.
15. Shojaei TR, Salleh MAM, Tabatabaei M, et al. Applications of nanotechnology and carbon nano particles in agriculture. In Synthesis, Technology and Applications of Carbon Nano materials. Elsevier. p. 247–277.
16. Ali M, Sobze JM, Pham TH, et al. Carbon nano particles functionalized with carboxylic acid improved the germination and seedling vigor in upland boreal forest species. Nanomaterials. 2020;10(1):176.
17. Jordán M, Casaretto J. Hormonas y reguladores del crecimiento: auxinas, giberelinas y citocininas. In: Squeo FA, Cardemil L, Fisiología Vegetal. 2006:1–28.
18. DeRosa MC, Montréal C, Schnitzer M, et al. Nanotechnology in fertilizers. Nature nanotechnology. 2010;5(2):91.
19. Chung H, Son Y, Yoon TK, et al. The effect of multi-walled carbon nanotubes on soil microbial activity. Ecotoxicology and environmental safety. 2011;74(4):569–575.
20. Lahiani MH, Dervishi E, Chen J, et al. Impact of carbon nanotube exposure to seeds of valuable crops. ACS applied materials & interfaces. 2013;5(16):7965–7973.
21. Lira Saldivar RH, Méndez Argüello B, Santos Villarreal GDL. Potencial de la nanotecnología en la agricultura. Acta universitaria. 2018;28(2):9–24.
22. Norma oficial mexicana NOM-021-SEMARNAT-2000. Que establece las especificaciones de fertilidad, salinidad y clasificación de suelos, estudio, muestreo y análisis. México: DOF Secretaria de Gobernación; 2013.