The well-known bacteria genus called as Bacillus thuringiensis (Bt) is recognized for being used in the biological control of insect pests in agriculture. However, there is little information on the effect of Bt as a plant growth promoter and in interaction with Micromonospora echinopora, which, being an endophyte, could improve the response of Phaseolus vulgaris with NH₄NO₃ atreduced at 30%of common recommended without risk of affecting plant health or yield.The objective of this work was to analyze the response of P. vulgaris to Bt and M. echinospora with NH₄NO₃ at 30% of recommended dose. The experiment was conducted under a randomized block design, using the response variables: germination percentage, phenology and biomass. The experimental data were analyzed by ANOVA/Tukey HSD (P<0.05). In the results, it was found that B.thuringiensis induced in P. vulgaris a yield of 3.2Ton/Ha, while with M. echinospora 3.6Ton/Ha was reached, with NF reduced at 30%, while it was shown that B.thuringiensis improved the yield with M. echinospora with 4.0Ton /Ha, values with statistical difference compared to the 2.8 Ton/Ha of P. vulgaris without B.thuringiensis or M. echinospora used as relative control with 100% of the NF.Concluded that B. thuringiensis is not only useful in biological control but that it can be an excellent growth promoter that can be improved with M. echinospora, another option for the sustainable production of P. vulgaris.

Keywords: legume, soil, hyperfertilization, beneficial endophytic bacteria, plant health.

The healthy growth of Phaseolus vulgaris (bean) requires nitrogen fertilizer (NF) commonly as a NH₄NO₃, which at high doses causes loss of soil productiveness due to rapid mineralization of organic matter,^1-3 An natural and ecological alternative solution to optimization of NF in P. vulgaris from seed inoculation with Bacillus thuringiensis and Micromonospora echinospora, genera and species of plant growth-promoting endophytic bacteria (PGPEB)which convert exudates from the seed spermosphere and inside the roots into plant growth promoting substancesor phytohormones that accelerate germination and induce a denser root system for the absorption and optimization of the NF applied at doses lower than those recommended, without affecting healthy plant growth.^4-7 Generally, for P. vulgaris, inoculants based on Rhizobium etli are recommended, a symbiote of this class of legumes, which is dependent on the variety of P. vulgaris and/or the soil. They may not be infective and effective in facilitating healthy growth at doses of NF for sustainable production. Based in some reports that showed that other bacteria could be apply in P. vulgaris and other legumescould be inoculated with B. megaterium⁸ or with actinomycetes likeStreptomyces flavoviridis a.⁹ Therefor the objective of this research was to analyze the response of P. vulgaris to B. thuringiensis and M. echinospora with NH₄NO₃ at 30%.

This research was carried out in the greenhouse of the Environmental Microbiology laboratory, Instituto de Investigaciones Químico-Biológicas (IIQB) of the Universidad Michoacana de San Nicolás de Hidalgo (UMSNH), Morelia, Mich, Mexico, with the average microclimatic conditions were: temperature of 23.2°C, luminosity of 450μmol-m^-2s^-1 and 67% relative humidity. In this trial, a non-sterile soil collected from the municipality of Salvador Escalante Michoacán, Mexico, with a silty clay loam texture with 10.44% organic matter and pH of 5.75 moderately acidic according to NOM-021-RECNAT-2000 was used.¹⁰ 20 mesh and solarized at 70°C/48h to minimize pests and diseases, then 1.0kg of soil was placed in the top container of the Leonard jar (Figure 1), while NF in a mineral solution or water in the reservoir at the bottom, P. vulgaris seeds were disinfected with 5% NaClO/5min and washed 5 times with sterile drinking water, then disinfected in 70% alcohol/ 5min and washed 5 times with sterile drinking water, then B. thuringiensis isolated from soil rich in organic matter was activated in nutrient broth with the following chemical composition (g/L): casein peptone 5; yeast extract 3; distilled water, adjusted the culture medium to pH 6.8±0.2; with the antifungal Tecto®60 (Syngenta), while M. echinospora isolated from nodules of M. sativa was propagated on avocado pit agar with the following chemical composition (g/L): avocado pit 10.0, casein peptone 5.0, yeast extract 1.3, K₂HPO₄ 0.17, KH₂PO₄ 2.61, MgSO₄ 1.5, NaCl 0. 9, CuSO₄ 0.05, bromothymol blue 10% (w/v) 10ppm, trace element solution 1.0mL, detergent solution 2.5mL at 10%, antifungal Tecto®60 1.0mL at 10%, 1.0mL, bacteriological agar 18.0, at pH 7.5, both bacterial genera were incubated at 30°C/72h. Then for every 10 P. vulgaris seeds, 1.0mL of B. thuringiensis was used at a density of 4.6x10⁶CFU/mL and M. echinospora at a density of 3.5x10⁶ propagule forming units/mL. Table 1 describes the experimental design with 2 controls and 6treatments with 6 replicates: P. vulgaris irrigated with water or absolute control (AC); P. vulgaris fed with NFat 100%or relative control (RC); P. vulgaris with B. thuringiensis and/or M. echinospora with 30% reduced NF in a mineral solution with the following chemical composition (g/L): NH₄NO₃ 10.0, K₂HPO₄ 2.5, KH₂PO₄ 2.0, MgSO₄ 1.0, NaCl 0.1, CaCl₂ 0.1, trace FeSO₄, trace element solution 10.0mL, adjusted to pH 6.5-6.8. The NF was applied every third day at 80% field capacity.¹¹ The response variables were: percentage (%) of germination and days of emergence; phenology: plant height (PH), root length (RL); biomass: aerial fresh weight (AFW) and root fresh weight (AFW), and aerial dry weight (ADW) and root dry weight (RDW) at seedling, flowering and physiological maturity stages and yield, the experimental data were validated with the statistical program ANOVA/Tukey HSD P<0.05 with Statgraphics Centurion.¹³

Figure 1 Leonard's jar design.¹²

In Table 2 shows the percentage and days of emergence of P. vulgaris seeds with B. thuringiensis and/or M. echinospora and NH₄NO₃ at 30%, emerged 3days after sowing, or 94% germination a numerical value stadistically different compared to 5days of emergence or 83% of P. vulgaris withoutB. thuringiensis/M. echinospora and NH₄NO₃100% or relative control (RC).

Figure 2 Germination of Phaseolus vulgariswith Bacillus thuringiensis and Micromonospora Echinospora with NH4NO3 reduced at 30%. Relative control (RC) = P. vulgaris without B. thuringiensis/M. echinospora fed withNH4NO3 at 100%. Treatment (T3) =P. vulgaris with B. thuringiensis/M echinospora NH4NO at 30%.

In Table 3 shows at the seedling level stage the positive response of P. vulgaris to M. echinospora with NF at 30%, which registered 42.33cm of PH and 16.16cm of RL, as well as 2.82g of AFW, 2.53g of RFW, 0.31g of ADW and 0. 11g of RDW, numerical values with statistical difference compared to phenology data: 30.83cm of PH and 14.33cm of RL; and those of biomass: 1.08g of AFW, 0.76g of RFW, 0.15g of ADW and 0.04g of RDW of P. vulgaris without B. thuringiensis and M. echinospora with NH₄NO₃ at 100% (RC).

Figure 3 Growth of Phaseolus vulgaris withBacillus thuringiensis and/or Micromonospora echinosporaat seedling level andNH4NO3reduced at 30%. Absolute control (AC) = P. vulgaris withoutB. thuringiensis/M. echinospora irrigated with water; Relative control (RC) = P. vulgaris without B. thuringiensis/M. echinospora fed with NH4NO3 at 100% ; Treatment (T) 1= P. vulgaris withB. thuringiensis + NH4NO3 at 30%; T2= P. vulgaris withM. echinospora +NH4NO3 at 30%; T3= P. vulgaris withB. thuringiensis/M. echinospora + NH4NO3at 30%.

In Table 4 shows the response of P. vulgaris to B. thuringiensis and M. echinospora at flowering level with the NF at 30% reduced dose, which registered 71.33cm of PH and 26.33cm of RL, 5.67g of AFW, 5.64g of RFW, 1.61g of ADW and 0. 32g of RWD, numerical values with statistical difference compared to 59.9cm of PH, 15.5cm of RL, 3.54g of AFW, 0.78g of RFW, 0.32g of ADW and 0.07 g of RDW of P. vulgaris without B. thuringiensis or M. echinospora with NH₄NO₃ at 100% or RC.

Figure 4 Growth of Phaseolus vulgaris to Bacillus thuringiensis and Micromonospora echinospora at flowering level andNH4NO3reduced at 30%. Absolute control (AC) = P. vulgaris without B. thuringiensis/M. echinospora irrigated with water; Relative control (RC) = P. vulgaris without B. thuringiensis/M. echinosporafed with NH4NO3at 100% ; Treatment (T1)=P. vulgaris with B. thuringiensiswith NH4NO3 at 30%; T2= P. vulgaris with M. echinospora + NH4NO3at 30 %, T3= P. vulgaris with B. thuringiensis/M. echinospora +NH4NO3at 30%.

In Table 5 shows at the physiological maturity level the response of P. vulgaris to B. thuringiensis and M. echinospora with NH₄NO₃ reduced to 30%, where it registered 125.5cm of PH and 8.83cm of RL, as well as 8.99g of AFW, 8.35g of RFW, 3.83g of ADW and 0.33g of RDW, these numerical values had statistical difference in relation to: 91.16cm of PH, 23.33cm of RL, 4.42g of AFW, 3.34g of RFW, 1.73g of ADW and 0.05g of RDW of P. vulgaris without B. thuringiensis and M. echinospora with NH₄NO₃ at 100% (RC).

Figure 5 Growth of Phaseolus vulgaris at physiological maturity level withBacillus thuringiensis and Micromonospora echinosporaandNH4NO3reduced at 30%. Absolute control (AC) = P. vulgaris without B. thuringiensis/M. echinospora irrigated with water; Relative control (RC) = P. vulgaris without B. thuringiensis/M. echinospora+ NH4NO3at 100% Treatment (T1) =P. vulgaris withB. thuringiensis + NH4NO3 30%; T2= P. vulgaris with M. echinospora + NH4NO3 30%; T3= P. vulgaris with B. thuringiensis/M. echinospora +NH4NO3at 30%.

In Table 6 shows the response of P. vulgaris in biomass and fruit yield to B. thuringiensis and M. echinospora with NH₄NO₃ at 30% reduced dose, which registered 7.70g of fresh fruit weightand 40g of total fruit weight with 4.0Ton/ha, these values had statistical difference in relation to the 3.15g of fresh fruit weight, 28g of total fruit weight and 2.8Ton/ha yield of P. vulgaris without B. thuringiensis and M. echinospora with NH₄NO₃ at 100% (RC).

Figure 6 Elements of yield of Phaseolus vulgariswith Bacillus thuringiensis and Micromonospora echinosporaandNH4NO3reduced at 30%. Absolute control (AC) = P. vulgaris without B. thuringiensis/M. echinosporairrigated with water; Relative control (RC) = P. vulgaris without B. thuringiensis/M. echinospora fed with NH4NO3 at 100% Treatment (T1) =P. vulgaris with B. thuringiensis+ NH4NO3 at 30%; T2= P. vulgaris withM. echinospora + NH4NO3 at 30%; T3= P. vulgaris withB. thuringiensis/M. echinospora + NH4NO3 at 30%.

The data presented in Table 2 support the response of P. vulgaris seed to B. thuringiensis and M. echinosporawith NH₄NO₃at 30%, which fast emergence time of P. vulgarisseeds which were releasing organic compounds that B. thuringiensis and M. echinospora transformed into phytohormons that accelerated the emergence of root and seedling primordium.14-1 This fact that is confirmed in Figure 2, where B. thuringiensis and M. echinospora on P. vulgaris at the fifth day, after sowing showed the largest size of stem and root primordium, compared to P. vulgaris without B. thuringiensis and M. echinosporaand NH₄NO₃ at 100%.

In Table 3, the numerical data of phenology and biomass of P. vulgaris with M. echinospora with NH₄NO₃ 30% at seedling stage shows that B. thuringiensis and M. echinospora converted some organic compounds from photosynthesis into phytohormons inducing karyokinesisto increasemore density of root hairs to maximize NH₄NO₃ uptake at 30%.^17,18 As it’s been reported by Gopalakrishnan et al.,¹⁹ with C. arietinum to Streptomyces sp at NH₄NO₃ level of soil. Figure 3 demostratethatM. echinospora on P. vulgarisby phytohormones enhanced stem diameter and root density of P. vulgaris compared to P. vulgaris without B. thuringiensis and M. echinospora had lower stem diameter and root density, indicated that NH₄NO₃ at 100% shows that was not efficiently taken up by root of P. vulgaris.

In Table 4, are show values of phenology and biomass of P. vulgaris with B. thuringiensis and M. echinospora with NH₄NO₃ at 30% at flowering level, suggesting that which converted photosynthesis metabolites into phytohormons for P. vulgaris to reach the highest concentration in the apical zone, to improve and rapid stem and leaf growth^20,21 as is observed in Figure 4. The same way as Pérez-Fernández & Alexander,²² in C. arietinumwith B. megaterium that increased RDW by enhancing uptake of NH₄NO₃.

In Table 5, shows the growth of P. vulgaris in terms of the phenology and biomass at physiological maturity due to B. thuringiensis and M. echinospora with NH₄NO₃30%, its reported that organic compounds from photosynthesis transformed into phytohormones that stimulated the generation of vegetative axillary buds that induced root elongation,^22-24 in that sense the root systems were able to exploration soil to uptake and optimized NH₄NO₃ reduced at 30%, even that with healthy plant growth, compared to P. vulgaris without B. thuringiensis and M. echinospora fed with NH₄NO₃ at 100% (RC), the root system grew less as its observed in Figure 5, indicating that the recommended dose was not uptake.

In Table 6, the beneficial effect of B. thuringiensis and M. echinospora on the biomass and pod yield of P. vulgaris with the dose reduced NF to 30%, which support both PGPEB maintained the conversion of photosynthesis derived carbon compounds into phytohormones:,¿ which at higher concentration stimulated the maturation of P. vulgaris pods despite the reduction of NF by up to 30%,^6,25 thus P. vulgaris had healthy growth by enhancing root system formation in the soil for maximum absorption and optimization of the NF applied at 30%.^26-28 The above is confirmed by what is shown in Figure 6, where the positive effect of B. thuringiensis and M. echinospora on P. vulgaris pods compared to those of RC is observed.

It was demonstrated that B. thuringiensis and M. echinospora, however being different genus and species endophytic bacteria are to improve germination and colonize the interior of the roots of ¨P. vulgaris; to increased root uptake of the reduced NF by up to 30%, without compromising the healthy growth of P. vulgaris. In that sense B. thuringiensis and M. echinospora endophytes of wild plants of the L. leucocephala and M. sativa types are an option for sustainable production of P. vulgaris preventing soil lost productivity and pollution due to hyper fertilization.

Conceptualization JMSY and AVM; data curation, JMSY, JLIC and AVM; writing—original draft preparation, JMSY and JLIC; writing—review and editing, JMSY and JLIC; Final version JMSY. All authors have read and agreed to the published version of the manuscript.

To Project 2.7 (2022) of the Scientific Coordination of the Universidad Michoacana de San Nicolas de Hidalgo, Morelia, Mich, México. To Phytonutrimentos de Méxicoand BIONUTRA S. A. de C.V., Maravatío, Michoacán, Mexico

The authors declared no have conflict interest for the study.

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