Healthy growth of Zea mays requires NH₄NO₃   as nitrogen fertilizer (NF), and its uptake is important to avoid loss of the NF. An alternative solution to enhance the root uptake capacity of Z. mays of NF at a dose to supply Z. mays demand without compromise its health; with beneficial entophytic genera and species of Stenocereus queretaroensis of the type Burkholderia vietnamiensis and Gluconacetobacter diazotrophicus. The objectives of this research were: a) to select from the interior of roots of Stenocereus queretaroensis: B. vietnamiensis and G. diazotrophicus, b) to analyze the growth of Z. mays with B. vietnamiensis and G. diazotrophicus and NF at 50%. B. vietnamiensis and G. diazotrophicus were recovered from the roots of S. queretaroensis and inoculated on Z. mays seed with NF. Using the response variables: percentage of emergency, phenology and biomass to seedling and flowering, the experimental data were analyzed by ANOVA-Tukey (P ≤ 0.05). The percentage of emergency, phenology, and biomass at seedling and flowering of Z. mays with B. vietnamiensis and G. diazotrophicus at 50% of NH₄NO₃, registered numerical values with statistical difference compared to those obtained in Z. mays without B. vietnamiensis and G. diazotrophicus only with NF at 100% or relative control (RC). This supports that B. vietnamiensis and G. diazotrophicus, entophytes of S. queretaroensis, invading the interior of Z. mays roots, converted metabolites related to root physiology into phytohormones that allowed maximum root uptake of NH₄NO₃ at 50%.

 Keywords: soil, xerophytic plant, domestic crop, chemical fertilization, endophytic bacteria

The healthy growth of Zea mays (maize) demands NH₄NO₃ as nitrogen fertilizer or NF.^1,2 This NF normally is not uptake by root system at 100% dose, due to several problem related with complex soil-root-microorganisms system in contrast not absorbed NF could caused loss fertility, or surface and groundwater contamination.³ An  ecological alternative solution to optimize  reduced dose of the NF in Z. mays is to treat its seeds with: Bacillus subtilis,⁴ Paenibacillus polymixa,⁵ or Pseudomonas putida⁶ including Burkholderia cepacia,⁷ all of them are able to convert exudates organic  compounds from the seed or/and the root  into phytohormons,^8,9 to enhance the growth of an extensive and dense root system for  reduced dose of NF.^10,11 In the literature it has been shown that some genera and species of those previously mentioned are specific for certain varieties of Z. mays, which can be relatively displaced by native soil rhizobacteria that colonize the roots of this cereal, without an evident beneficial effect on the uptake and optimization of NF reduced to 50% of what is recommended. An alternative solution to enhance and uptake  NF at 50% is the selection of genera and species of endophytic bacteria that promote plant growth since they are competitive and effective in enhancing  NF at 50%, without compromising healthy of Z. mays.^12–14 This endophytic  bacteria can be to recover  from xerophytes such as Stenocereus queretaroensis  called “pitayo” in Spanish;¹⁵ a cactus adapted to water stress due to low humidity, drastic changes in temperature; chemical related to the limitation of essential mineral salts of N (nitrogen), PO₄^-3 (phosphates) and K (potassium): which includes biological ones such as root phytopathogens, given that S. queretaroensis grows successfully without human protection in this environment.^16,17 The hypothesis of this research is that certain endophytic genera and species such as Burkholderia vietnamiensis^18,19  and Gluconacetobacter diazotrophicus^20,21  recovered from inside of the  roots of S. queretaroensis  could be benefical for domestic crop such as Z. mays, in enhancing uptake of NF at 50% through B. vietnamiensis and G. diazotrophicus, by invading the root system of Z. mays, since they would avoid competition with other microorganisms in the rhizosphere and the soil, by living inside of root system to convert organic compounds into phytohormons,^21–23 which maximize the radical uptake capacity in NF reduced at 50%, and even that healthy growth of Z. mays. Therefore, the objectives of this research were: a) to select from inside S. queretaroensis roots: B. vietnamiensis and G. diazotrophicus, b) to analyze the growth of Z. mays with B. vietnamiensis and G. diazotrophicus and the NH₄NO₃ at 50%.

This research was carried out in the Environmental Microbiology Laboratory of the Biological Chemical Research Institute of the Michoacán University of San Nicolás de Hidalgo, the average microclimatic conditions were: temperature of 23.2°C, luminosity of 450 μmol・m-2s-1, relative humidity of 67%.

Origin and preparation of the soil

Table 1 shows the main soil properties of the experiment: one of the sodium lateritic type; Clay texture, poor in the concentration of organic matter with 4.57% and total N (nitrogen), with a slightly acidic pH of 6.64. This soil was collected from an agricultural plot called “La cajita” of the Tenencia Zapata in the municipality of Morelia, Mich., on km 5 of the Morelia-Pátzcuaro highway, México, solarized at 75^oC/48 h to minimize pest problems and diseases.²⁴ Then a 1.0 Kg of soil was placed in the upper container of the Leonard jar, while in the lower part reservoir the NF or water, both parts were connected by a 25 cm long cotton strip for capillarity movement of the liquid to the ground (Figure 1).

Figure 1 Diagram of the Leonard jar. (Selection of endophytic Burkholderia vietnamiensis and Gluconacetobacter diazotrophicus from roots of Stenocereus queretaroensis).

Isolates of endophytic B. vietnamiensis and G. diazotrophicus were recovered from inside the root of S. queretaroensis; 5-cm sections were taken and washed with sterile  water, disinfected with 70% alcohol/2.5 min, then with 10% NaClO/2.5 min; then they were macerated in a mortar with 10.0 mL of a saline solution (NaCl 0.85%) Roma™ detergent 0.01% (SSD), from which 1.0 mL was taken, and striked in Pseudomonas cepacia agar, azaleic acid with and without tryptamine (PCAAA) with the following composition (gL-1): tryptamine 0.2; azaleic acid 2.0; K₂HPO₄ 4.0; KH₂PO₄ 4.0; yeast extract 0.02; MgSO₄ 0.2; pH adjusted to 6.7; PCAAA was incubated at 28°C/72h; while to recover G. diazotrophicus, sucrose yeast extract agar (SYEA) was used with the following composition (gL-1): sucrose 100.0; K₂HPO₄ 2.0; KH₂PO₄ 2.0; NaCl 1.0; MgSO₄ 3.0; yeast extract 1.0; 10 mL bromothymol blue at 2.0% (p/v) (Cavalcante & Dobereiner, 1988) with 10 mL/L of the trace element solution with the following composition (gL-1): H₃BO₄ 2.86; ZnSO₄ 0.22; MnCl₂ 1.81; K₂MnO₄ 0.09; the pH was adjusted to 5.7; both  PCAAA and SYEA were incubated at 30°C/72h;²⁴ when round bright translucent colonies suspicious for B. vietnamiensis were detected, they were again growth in PCAAA to obtain an axenic culture, then short Gram-negative bacilli were registered by Gram stain; microscopic morphological characteristics common in B. vietnamiensis: while for G. diazotrophicus in the SYEA, bulging and bright round colonies with a yellow intracellular pigment were observed, while Gram-negative coccobacilli were detected under the microscope, which according to the literature belong to these genus.^25–28

Growth of Zea mays with Burkholderia vietnamiensis and Gluconacetobacter diazotrophicus endophytes and 50% of NF. Z. mays seeds were disinfected with NaClO at 3% (v/v)/5 min, washed six times with sterile water, then with alcohol at 70% (v/v)/5 min, washed six times with sterile water, later in plastic bags of 250 g for every 10 Z. mays seeds, were inoculated with 1.0 mL of B. vietnamiensis and G. diazotrophicus in a concentration that was adjusted to 1 x 10⁶ CFU/mL, obtained by viable count on plate in PCAAA and SYEA; when both were inoculated, a 1:1 ratio was used; after the inoculation of the seeds with B. vietnamiensis and G. diazotrophicus were shaken for 1 h to ensure the entry of B. vietnamiensis and G. diazotrophicus to the seeds of Z. mays, then five seeds of cereal s were sown in each Leonard Jar. Z. mays with or without B. vietnamiensis and G. diazotrophicus; according to the experimental design in Table 2, randomized blocks with two controls, three treatments and six replicates: Z. mays without B. vietnamiensis and G. diazotrophicus irrigated only with water or absolute control (AC); Z. mays without B. vietnamiensis and G. diazotrophicus fed with the NH₄NO₃ at 100% or relative control (RC); Z. mays inoculated individual/in consortium with B. vietnamiensis and G. diazotrophicus with NF at 50%in a mineral solution for this cereal with the following chemical composition (gL-1): NH₄NO₃ 12.0, KH₂PO₄ 3.0, K₂HPO₄ 3.5, MgSO₄ 1.5, CaCl₂ 0.1, FeSO₄ 0.5 mL/L, and a 1.0 mL/L trace element solution, pH adjusted to 7.0, in distilled water, while NH₄NO₃ or NF was reduced to 50% equivalent to 6 g L-1.²⁴

The growth response variables of Z. mays with B. vietnamiensis and G. diazotrophicus were: the percentage of emergence (%), at the seedling and flowering level based on phenology: plant height (PH) and root length (RL); in the biomass: aerial fresh weight (AFW) and radical (RFW); with aerial dry weight (ADW) and radical (RDW). The experimental data obtained were subjected to ANOVA analysis of variance using Tukey’s comparative test of means (P ≤ 0.05), for which the statistical program Statgraphics Centurion²⁹ was used.

 Evidence of the presence of B. vietnamiensis and G. diazotrophicus in the stem and roots of Zea mays

The sensitivity profile of B. vietnamiensis and G. diazotrophicus was obtained before and after inoculating Z. mays. Using the Kirby and Bauer method for genera and species of Gram negative bacteria: Ampicillin, Cefotaxime, Ceftazidime, Cefuroxime, Pefloxacin, Tetracycline and Trimethoprim-Sulfamethoxazole, Cephalotin, Dicloxacin, Erythromycin, Gentamicin and Penicillin.³⁰ Both B. vietnamiensis and G. diazotrophicus isolated from S. queretaroensis were grown on nutrient agar and on PCAAA and SYEA; As a reference, the sensidiscs were placed before inoculating the Z. mays seed, then they were incubated at 32°C/48 h, and the inhibition halos were measured according to the Kirby-Bauer method. Later, when Z. mays was inoculated with B. vietnamiensis and G. diazotrophicus and the cereal reached the level of seedling and flowering, the following were taken: 1.0 g of leaves, stem and/or roots each were disinfected and macerated as described in item b), then 1.0 mL of leaves, stem and root with B. vietnamiensis and G. diazotrophicus; it was grown with a sterile swab in nutrient agar, PCAAA and SYEA, then the antibiotic sensidisks were placed and incubated from 24 to 48 h/30oC. Inhibition halos were then measured according to the Kirby-Bauer method,^31,32 and the sensitivity pattern of individual B. vietnamiensis and G. diazotrophicus was obtained to compare with the antibiotic sensitivity profile of each before inoculating the seed of Z. mays. Finally, to confirm that they belonged to B. vietnamiensis and G. diazotrophicus, specific biochemical tests were used to identify them as members of these bacterial groups.^33–35

Table 3 shows the results of the emergence percentage of the Z. mays seeds treated with B. vietnamiensis and G. diazotrophicus and NH₄NO₃ at 50%,  with  95% of emergence, which indicates that  B vietnamiensis and G. diazotrophicus converted exudates from the  seed, known as spermosphere effect, such as tryptophan into phytohormons to accelerate starch hydrolysis,  to end embryo dormancy and to accelerate the rate of stem and root primordium emergence, as detected in Z. mays only treated with B. vietnamiensis or with G. diazotrophicus and FN at  50%.^9,36 These percent emergence values were statistically different than 84% Z. mays without B. vietnamiensis or either G. diazotrophicus fed only of the NF at 100% used as relative control (RC).

In Table 4, the growth of Z. mays with G. diazotrophicus and 50% NF is presented for seedling, where 17.5 cm of PH and 13.0 cm of RL were registered. Numerical values ​​without statistical difference with those observed in Z. mays with B. vietnamiensis and G. diazotrophicus plus  NF at 50%, according to various reports it is shown that these endophytic genera interact efficiently with wild  hosts, compared with those that inhabit the phyllosphere and/or the rhizosphere of a wide variety of domestic plants.³⁷ What supports, B. vietnamiensis and G. diazotrophicus after germination colonized and penetrated the roots of Z. mays through different mechanisms some known and other not yet discovered that occur during plant,³⁸ inside the plant conduction system they converted some low molecular weight carbon compounds derived from the metabolism of photosynthesis into phytohormons,³⁹ which accelerated and improved the functioning of the root system to maximize uptake of NF to 50%, without compromising the healthy growth of Z. mays.²⁰ shown in an increase in the numerical values ​​of the phenology that were statistically different from those registered when Z. mays grew without B. vietnamiensis and G. diazotrophicus and the NF at 100% or relative control (RC); which the following  results 13.3 cm PH and 9.1 RL. The growth of Z. mays with B. vietnamiensis and G. diazotrophicus and the NF at 50% based on the biomass registered 1.4 g of AFW and 0.37 g of RFW, while 0.23 g of DAW and 0.09 g DRW, the above indirectly demonstrates that both B. vietnamiensis and G. diazotrophicus were inside the roots, in the conduction system, transformed compounds  from photosynthesis into phytohormons, to induce the greatest uptake of NH₄NO₃ and  to optimize the dose at 50%.^12,23,40 These numerical values ​​of the biomass were statistically different with the 0.9 g of AFW and the 0.24 g of RFW, with the 0.08 g of DAW and with the 0.02 g of DRW of Z. mays used as RC, which shows that based on the healthy growth of Z. mays, the positive effect of B. vietnamiensis and G. diazotrophicus in optimizing NH₄NO₃ at 50%.

Table 5 shows the growth of Z. mays with B. vietnamiensis individually or in combination with G. diazotrophicus and 50%  of  NH₄NO₃ flowering; there, they registered 92.17 cm of PH and 28.12 cm of RL; while in Z. mays with G. diazotrophicus and  50%, of NH₄NO₃ 90.93 cm AP and 26.95 cm RL were obtained. These numerical data indicate that B. vietnamiensis and G. diazotrophicus, after colonizing inside of the root system, transformed organic compounds from root physiology into phytohormons,²² inducing a dense and extensive root system, the roots of Z. mays had a higher exploration capacity to uptake NH₄NO₃ at 50% without compromising the healthy growth of Z. mays.^19,21,41 The numerical values ​​of the phenology of Z. mays treated with B. vietnamiensis and G. diazotrophicus were statistically different compared to the 74.41 cm RL and 21.05 cm RL of Z. mays without B. vietnamiensis and G. diazotrophicus and the NF at 100% or relative control (RC). Concerning the biomass of the growth of Z. mays with B. vietnamiensis and G. diazotrophicus and the NF at 50%, they registered 31.03 g of AFW and 8.91 g of  DRW, as well as 5.97 g of DAW and 1.23 g DRW; like Z. mays with G. diazotrophicus and  NF at 50%. This suggests that both B. vietnamiensis and G. diazotrophicus endophytes of S. queretaroensis, by invading the interior of the roots of Z. mays, converted  metabolites related to root physiology into phytohormones that induced the maximum radical uptake for the optimization of NF at 50%.^40–43 These numerical values ​​of the biomass of Z. mays with B. vietnamiensis and G. diazotrophicus and the NF at 50% were statistically different with the 26.06 g of AFW and the 2.85 g of RFW, with the 2.91 g of ADW and with the 0.91 g of DRW from Z. mays used as RC,  that fact suggests that B. vietnamiensis as G. diazotrophicus when they are inside the root transformed organic compounds producing during root physiology  into phytohormones that activated and improved the capacity of the root system for maximum uptake and optimization  of NH₄NO₃  at 50% , and even that the healthy growth of Z. mays.^15,19,21

In Table 6, the sensitivity profile of B. vietnamiensis and G. diazotrophicus endophytes of S. queretaroensis is shown to demonstrate that healthy root, stem and leaf growth of Z. mays seedling and flowering level, according to the Kirby and Bauer method, registered sensitivity to: Cefotaxime, Tetracycline, Trimethoprim-Sulfamethoxazole, Cefalotin, Erythromycin, in contrast resistant to: Ampicillin, Ceftazidime, Cefuroxime, Pefloxacin, Gentamicin, and Penicillin. This sensitivity profile was compared to that obtained for B. vietnamiensis and G. diazotrophicus before inoculating Z. mays seed. This suggests that B. vietnamiensis and G. diazotrophicus invaded the xylem of Z. mays to transform organic compounds from root physiology into phytohormones,^43,44 which allowed the maximum uptake of NF to 50% for healthy growth.⁴⁵

Table 7 shows the biochemical profile of B. vietnamiensis and G. diazotrophicus endophytes of S. queretaroensis, based on a prototype strain and related literature; there, it is reported that the genus and species of B. vietnamiensis is a short bacillus Gram negative, a facultative aerobe that synthesizes catalase and oxidase; so it has an oxidative metabolism, it uses sugars: esculin, glucose, which do not ferment, but organic acids: sodium citrate; it is a solubilizer of PO₄^-3 by the generation of acid and alkaline phosphatases; They reduce NO₃ to NO₂. B. vietnamiensis fixes N₂, grows in a mineral medium without any form of combined N;^46,47 selectively uses tryptamine as a source of N; convert tryptophan into indole acetic acid and induces a dense and larger root system, which allowed Z. mays to uptake NH₄NO₃ at 50%.^5,48,49 Gluconoacetobacter diazotrophicus of S. queretaroensis is an osmophilic endophytic genus and species similar to the prototype strain of G. diazotrophicus of S. officinarum (sugar cane). It has an oxidative metabolism of sugars: glucose, fructose and sucrose; uses citrate as the sole source of carbon and energy; by glucose fermentation it generates ethanol, lactate and acetic acid;⁵⁰ fixes N₂ in facultative endophyte association, grows in a culture medium without any source of organic or inorganic N, in anaerobiosis reduces NO₃ to NO₂; transform L-tryptophan into auxin, a phytohormone that generates a dense radical system that in Z. mays improved radical uptake of NH₄NO₃ and optimized the dose reduced at 50%.^51–54

We acknowledge support to the Project 2.7 (2022) from CIC-UMSNH also to Phytonutrimentos de México and BIONUTRA, S.A  de CV Maravatío, Michoacán, México.

The authors state that there is no conflict of interest.

To Project 2.7 (2022) from CIC-UMSNH and to Phytonutrients de México and BIONUTRA, S.A: de CV Maravatio, Michoacán, México.

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