Revista Mexicana Ciencias Agrícolas   volume 11   number 1   January 01 - February 14, 2020

DOI: https://doi.org/10.29312/remexca.v11i1.2162

Investigation note

Improving biometric traits of wheat seedlings with the inoculation
of a consortium native of Bacillus

Rosa Icela Robles Montoya1

Luis Abraham Chaparro Encinas1

Fannie Isela Parra Cota2

Sergio de los Santos Villalobos1§

1Technological Institute of Sonora. February 5, 818 South, Col. Centro, Ciudad Obregón, Sonora, Mexico. CP. 85000. 2Experimental Field Norman E. Borlaug-INIFAP. Norman E. Borlaug km 12, Ciudad Obregón, Sonora, Mexico. CP. 85000.

§Corresponding author: sergio.delossantos@itson.edu.mx.

Abstract

Plant growth promoting bacteria (BPCV) represent an alternative for reducing the use of chemical fertilizers and improving their use by agricultural crops. The study determined the ability of a bacterial consortium native to the genus Bacillus to promote the growth of wheat seedlings (Triticum turgidum subsp. durum) under controlled conditions, 30 days after inoculation. The consortium was integrated by strains TRQ8, TE3T, TRQ65 and TSO9. The molecular identification (based on the 16S RNAr gene) of these strains revealed their taxonomic affiliation to Bacillus megaterium TRQ8, B. cabrialesii TE3T, B. paralicheniformis TRQ65 and B. subtilis TSO9. These strains showed the ability to synthesize siderophores, indoles, and phosphate solubilization, as well as tolerance to thermal stress (43.5 °C), water (10% PEG) and saline (5% NaCl). The inoculation (4x107 CFU) of said bacterial consortium to wheat seedlings showed a significant increase (compared to non-inoculated seedlings) in the length of the aerial part (28%), root length (25%), total length (28 %), stem diameter (46%), circumference (50%), dry weight of the aerial part (72%) and the biovolume index (57%). The foregoing evidences the capacity to promote plant growth of said bacterial consortium; therefore, the development of future trials under field conditions is crucial to know the feasibility of the design of a biofertilizer containing the study strains for use in current and future agriculture.

Keywords: agriculture, bioinoculant, BPCV, food security, Yaqui Valley.

Reception date: January 2020

Acceptance date: February 2020

Wheat (Triticum turgidum subsp. durum) is one of the most important crops worldwide (Grijalva-Contreras et al., 2016). At present, the production of this cereal requires the use of large amounts of chemical fertilizers (∼300 kg N ha-1), whose utilization rate is 20 to 35% (Grageda-Cabrera et al., 2018). Which leads to economic and environmental problems, such as: eutrophication, emission of greenhouse gases, and loss of soil fertility (de los Santos-Villalobos et al., 2018; Liu and Xie, 2019). Therefore, the generation of sustainable agro-biotechnological alternatives that allow maintaining or increasing agricultural yields decreasing the negative effects on the environment, human health, and economy of the producer is decisive.

In recent years, interest in the generation of integral agricultural systems has emerged; through the application of plant growth promoting bacteria (BPCV), which are a group of bacteria that have the ability to improve the health and quality of plants, restoring soil fertility (Rojas-Solis et al., 2016; Valenzuela-Aragon et al., 2019). The positive effects shown by BPCV have been strongly associated with various mechanisms of action i) direct, associated with plant growth, such as: the production of phytohormones (auxins, cytokinins, and gibberellins), volatile organic compounds (acetoin, and sulfur derivatives), biological nitrogen fixation, cycling and nutrient solubilization (Moreno-Resendez et al., 2018; Valenzuela-Ruiz et al., 2018); and ii) indirect, associated with crop resistance to phytopathogens, through the production of lipopeptides, antibiotics, or lithic enzymes (Villarreal-Delgado et al., 2018; Villa-Rodríguez et al., 2019).

At present, there is a growing interest in the formulation of these biotechnological products from one or more species or bacterial genera with phyto-stimulant capabilities, called bacterial consortia (Jha et al., 2013). The objective of the study was to evaluate the wheat growth promoting capacity, under controlled conditions, of a native bacterial consortium composed of strains of the genus Bacillus, which were isolated from the Yaqui Valley, Sonora, Mexico (region that contributes 50% to the production of this cereal nationwide).

The bacterial strains under study (TE3T, TRQ8, TRQ65 and TSO9) were isolated (by serial dilutions (1:10), using nutritive agar as a culture medium, at 28 °C) from the rhizosphere of the CIRNO2008 variety wheat crop, grown in commercial plots located in the Yaqui Valley, Sonora, Mexico (27° 35’ 53.14” N and 110° 2’ 53.26” W). These strains were preserved in the Collection of Endophytic and Native Edaphic Microorganisms (www.itson.edu.mx/COLMENA) (de los Santos-Villalobos et al., 2018).

The TE3T, TRQ8, TRQ65 and TSO9 strains were metabolically characterized for traits associated with the promotion of plant growth, such as: phosphate solubilization, indole production, tolerance to thermal, water, and saline and chlorothalonil stress (Valenzuela-Aragón et al., 2019; Díaz-Rodríguez et al., 2019). Thus, TRQ8 was the only strain that showed the ability to produce siderophores (8.1 ±0.8%). In addition, it was observed that certain strains under study showed phosphate solubilization capacity (TE3T, 43.2 ±1.7%; TRQ8, 38.0 ±0.9%; TSO9, 54 ±1%), indole production (TRQ8, 12 ±0.4; TRQ65, 28.8 ±0.9 ppm). Finally, all strains showed the ability to tolerate thermal stress (43.5 °C), water [Polyethylene glycol (PEG), 10%, -0.84 mPa)], saline (NaCl, 5%, 6.8 dS m-1) and chemical (Chlorothalonil).

The molecular identification of the strains under study was performed by sequencing the 16S rRNA gene, based on what was reported by Valenzuela-Aragon et al. (2019). The sequences obtained were edited with the FINCH TV software (Geospiza, Inc.; Seattle, WA, USA; http://www.geospiza.com) and were compared with the BLASTn database of the National Center for Biotechnology Information (NCBI), considering the percentage of maximum identity. The 16S rRNA gene sequences of the strains studied were deposited at the NCBI Genbank (https://www.ncbi.nlm.nih.gov/) and published with the accession number: MH057214 (TE3T), MK493655 (TSO9), MK493638 (TRQ8) and MK493706 (TRQ65).

The DNA sequences obtained were used to construct a phylogenetic tree using the CLC Sequence Viewer software (bio CLC, Aarhus, Denmark), with the Kimura80 evolution model, and the Neighbor-Joining construction model. This construction was done based on 1000 iterations to calculate the percentage of bootstrap. The 16S rRNA gene sequence of Pseudomonas sp. (MH569548) was used as ‘outgroup’. Thus, strain TRQ65 was taxonomically affiliated with Bacillus paralicheniformis (100% coverage and 100% identity) (Valenzuela-Ruiz et al., 2019), TSO9 to B. subtilis (100% coverage and 100% identity) (Villa-Rodríguez et al., 2019), TE3T to B. cabrialesii (100% coverage and 100% identity) (de los Santos-Villalobos et al., 2019) and TRQ8 to Bacillus megaterium (100% coverage and 99.66% identity) (Robles-Montoya et al., 2019). The taxonomic affiliation mentioned above was supported by the value of bootstrap, indicating the percentage at which the configuration of a clade is repeated in 1000 random simulations or iterations (Figure 1).

Figure 1. Phylogenetic tree constructed with the 16S rRNA gene sequences of the strains under study, with the Kimura80 and Neighbor-Joining models, clades were determined with 1 000 iterations (bootstrap). *= outgroup sequence (Pseudomonas sp. MH569548).

The impact of inoculation of the bacterial consortium composed of Bacillus megaterium TRQ8, B. paralicheniformis TRQ65, B. cabrialesii TE3T, and B. subtilis TSO9 on biometric traits of wheat seedlings (Triticum turgidum subsp. durum) CIRNO C2008 variety was studied. For this, pre-inoculums of each bacterial strain were made, inoculating ~104 colony forming units (CFU) mL-1 in 10 mL of sterile nutrient broth (CN, DIBICO®) and incubated at 28 °C with continuous agitation

(120 RPM ) for 24 h. Subsequently, the inoculums were generated by inoculating 1 mL of the pre-inoculum (optical density at 600 nm: 0.5) in 500 mL of CN and incubating under the conditions described above. Finally, the bacterial cultures were centrifuged at 3 500 RCF, for 15 minutes, performing two washes with sterile distilled water (15 lb, 121 °C, 15 min) and the cell density of each strain under study was adjusted to 1*107 CFU mL-1 , with sterile distilled water.

Wheat seeds were superficially disinfected, washing them with commercial soap and soaking them in sterile distilled water (121 °C and 15 lb per 15min) for 24 h, then the seeds were washed with 2% NaCl for 10 min, followed by 3 washes with sterile distilled water. The disinfected seeds were placed in 230 g of a sterile soil mixture (121 °C and 15 lb for 5 days), at a rate of 70:30 (perlite: agricultural soil). The agricultural soil used was collected from commercial plots for wheat production in the Yaqui Valley (hyposodic vertisol, pH 7.74 ±0.05, organic matter <1%, clay texture 40-60%).

A unifactorial design with two treatments was used (two independent repetitions, n= 15 per repetition): i) inoculated seedlings (4*107 CFU, at 0 and 15 days after planting) by the consortium under study vs; and ii) a negative control (seedlings not inoculated and sprinkled with sterile distilled water). The experiment was carried out in a growth chamber (BJPX-A450, BIOBASE) under 70% relative humidity, temperature of 25 °C (day) and 15 °C (night), and a photoperiod of 14 h light/12 h dark, for 30 days. Once the experiment was completed, the biometric variables evaluated in the seedlings were total length, height, root length, dry weight of the shoot and root, stem thickness, and biovolume index (Thilagar et al., 2016).

The bacterial consortium integrated by Bacillus cabrialesii TE3T, B. paralicheniformis TRQ65, B. megaterium TRQ8 and B. subtilis TSO9 showed a significant increase with respect to the control treatment [Fischer LSD (p≤ 0.05), Statgraphics 5.0] on the length of the aerial part (28%), root length (25%), total length (28%), stem diameter (46%), circumference (50%), dry weight of the aerial part (72%) and the biovolume index (57%) (Table 1).

Table 1. Effect of inoculation of a bacterial consortium of the genus Bacillus on wheat seedlings, 30 days after inoculation, under controlled conditions. *significant difference (p< 0.05) Fisher LSD.

The bacterial consortium studied increased biomass production and cell elongation in inoculated wheat seedlings (Table 1), which may be associated with the metabolic capacity of the strains, ie. production of indoles (12-28 ppm), siderophores (8%), phosphate solubilization (38-54%), repression of the growth of phytopathogenic microorganisms, and resistance to stress conditions (Villarreal-Delgado et al. 2018; Valenzuela-Aragón et al., 2019; Díaz-Rodríguez et al., 2019; Villa-Rodríguez et al., 2019). The inoculation of BPCV producing phytohormones leads to an increase in the length and root biomass of plants, promoting the acquisition of nutrients from the soil and promoting plant growth, even under stress conditions (Valenzuela-Ruiz et al., 2018); this beneficial effect is also coupled with a greater bioavailability and mobilization of nutrients towards the plant (Jacoby et al., 2017). The ability to solubilize phosphates, as well as siderophores production, has been associated with the increase in the growth rate and yield in wheat, maintaining this attribute under conditions of chemical stress and decreased dose of inorganic fertilization (Munir et al., 2019).

The results obtained in this research show the compatibility and ecological/functional role in vivo of the metabolic activities detected (and others not described so far) in the strains that make up the bacterial consortium under study (Bacillus cabrialesii TE3T, B. paralicheniformis TRQ65, B. megaterium TRQ8 and B. subtilis TSO9). Thus, the positive effects observed in wheat seedlings inoculated with the developed bacterial consortium can be attributed to the metabolic diversity of the Bacillus strains that integrate it, as well as their ability to respond as an organism to the various environmental stressors, which gives an increased capacity for adaptation (synergistic interactions to provide nutrients and stimulate their growth, degradation of inhibitory compounds, microbial competition, among others), compared to individual strains.

Conclusions

Inoculation of a bacterial consortium formed by strains B. cabrialesii TE3T, B. paralicheniformis TRQ65, B. megaterium TRQ8 and B. subtilis TSO9 significantly increased biometric traits in wheat seedlings related to cell elongation, biomass acquisition and efficient use of the water. This suggests that the metabolic attributes shown by the constituent strains of the consortium have a synergistic effect on the acquisition of nutrients, as well as on the regulation of phytohormones in the plant. The results presented suggest evaluating said bacterial consortium under field conditions and developing a biofertilizer for use in current and future agriculture.

Acknowledgments

The authors are grateful for the funding granted by CONACYT to project 257246, ‘Wheat interaction x plant growth promoting microorganisms: identifying genes with agro-biotechnological potential’. In addition, the masters and doctorate scholarship granted by the National Council of Science and Technology (CONACYT) to Rosa Icela Robles Montoya and Luis Abraham Chaparro Encinas.

Cited literature

de los Santos-Villalobos, S.; Parra-Cota, F. I.; Herrera-Sepúlveda, A.; Valenzuela-Aragon, B. and Estrada-Mora, J. C. (2018). Collection of edaphic microorganisms and native endophytes to contribute to national food security. Rev. Mex. Cienc. Agríc. 9(1):191-202. https://doi.org/10.29312/remexca.v9i1.858.

de los Santos-Villalobos, S.; Robles-Montoya, R. I.; Parra-Cota, F. I.; Larsen, J.; Lozano, P. & Tiedje, J. M. (2019). Bacillus cabrialesii sp. nov., an endophytic plant growth promoting bacterium isolated from wheat ( Triticum turgidum subsp. durum ) in the Yaqui Valley , Mexico. Inter. J. System. Evol. Microbiol. 69(12):3939-3945. https://doi.org/10.1099/ ijsem.0.003711.

Díaz-Rodríguez, A. M.; Parra-Cota, F. I.; Santoyo, G. and de los Santos-Villalobos, S. 2019. Chlorothalonil tolerance of indole producing bacteria associated to wheat (Triticum turgidum L.) rhizosphere in the Yaqui Valley, Mexico. Ecotoxicology. 28(5):569-577. https://doi.org/10.1007/s10646-019-02053-x.

Grageda-Cabrera, O. A.; Vera-Nuñez, J. A. and Peña-Cabriales, J. J. 2018. Efecto de los biofertilizantes sobre la asimilación de nitrógeno por el cultivo de trigo. Rev. Mex. Cienc. Agríc. 9(2):281-289. https://doi.org/10.29312/remexca.v9i2.1071.

Grijalva-Contreras, R. L.; Robles-Contreras, F. and Macías-Duarte, R. 2016. Nitrógeno en trigo y su efecto en el rendimiento y en la concentración de nitratos y potasio en el extracto celular de tallo ( ECT ). Acta Universitaria. 26(5):48-54. https://doi.org/10.15174/au.2016.963.

Jacoby, R.; Peukert, M.; Succurro, A. and Koprivova, A. 2017. The role of soil microorganisms in plant mineral nutrition-current knowledge and future directions. Frontiers Plant Sci. 8(1):1-19. https://doi.org/10.3389/fpls.2017.01617.

Jha, M.; Chourasia, S. and Sinha, S. 2013. Microbial consortium for sustainable rice production. Agroecol. Sustainable Food Systems. 37(3):340-362. https://doi.org/10.1080/ 10440046.2012.672376.

Liu, G. and Xie, H. 2019. Simulation of regulation policies for fertilizer and pesticide reduction in arable land based on farmers’ behavior-using jiangxi province as an example. Sustainability, 11(136):1-22. https://doi.org/10.3390/su11010136.

Moreno-Reséndez, A.; García-Mendoza, V.; Reyes-Carrillo, J. L.; Vasquez-Arroyo, J. and Cano-Ríos, P. 2018. Rizobacterias promotoras del crecimiento vegetal : una alternativa de biofertilización para la agricultura sustentable. Plant growth promoting rhizobacteria: a biofertilization alternative for sustainable agriculture. Rev. Colomb. Biotecnol. 20(1):68-83. https://doi.org/10.15446/rev.colomb.biote.v20n1.73707.

Munir, I.; Bano, A. and Faisal, M. 2019. Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum) in the presence of pesticides. Brazilian J. Biol. 79(1):29-37. https://doi.org/10.1590/1519-6984.172213.

Radhakrishnan, R.; Hashem, A. and Abd-Allah, E. F. 2017. Bacillus: a biological tool for crop improvement through bio-molecular changes in adverse environments. Frontiers in Physiol. 8(1):1-14. https://doi.org/10.3389/fphys.2017.00667

Robles-Montoya, R. I.; Parra Cota, F. I. and de los Santos Villalobos, S. 2019. Draft genome sequence of Bacillus megaterium TRQ8, a plant growth-promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico. 3 Biotech. 9(1):200-205. https://doi.org/10.1007/s13205-019-1726-4.

Rojas-Solís, D.; Hernández-Pacheco, C. E. and Santoyo, G. 2016. Evaluation of Bacillus and Pseudomonas to colonize the rhizosphere and their effect on growth promotion in tomato (Physalis ixocarpa Brot. ex Horm.). Rev. Chapingo Ser. Hortic. 22(1):45-57. https://doi.org/10.5154/r.rchsh.2015.06.009.

Thilagar, G.; Appa, D. J. B.; Podile, R. and Rao, P. 2016. Bacillus sonorensis, a novel plant growth promoting Rhizobacterium in improving growth, nutrition and yield of chilly (Capsicum annuum L .). Proceedings of the National Academy of Sciences, India Section B: Biological Sciences. https://doi.org/10.1007/s40011-016-0822-z.

Valenzuela-Aragon, B.; Parra-Cota, F. I.; Santoyo, G.; Arellano-Wattenbarger, G. L. and de los Santos-Villalobos, S. 2019. Plant-assisted selection: a promising alternative for in vivo identification of wheat (Triticum turgidum L. subsp. Durum) growth promoting bacteria. Plant and Soil. 435(1-2):367-384. https://doi.org/10.1007/s11104-018-03901-1.

Valenzuela-Ruiz, Valeria; Ayala-Zepeda, M.; Arellano-Wattenbarger, G. L.; Parra-Cota, F. I.; García-Pereyra, G.; Aviña-Martínez, G. N. and de los Santos-Villalobos, S. 2018. Las colecciones microbianas y su potencial contribución a la seguridad alimentaria actual y futura. Rev. Latinoam. Rec. Nat. 14(1):18-25.

Villa-Rodríguez, E.; Parra-Cota, F.; Castro-Longoria, E.; López-Cervantes, J. and de los Santos-Villalobos, S. 2019. Bacillus subtilis TE3: A promising biological control agent against Bipolaris sorokiniana, the causal agent of spot blotch in wheat (Triticum turgidum L. subsp. durum). Biological Control. 132(1):135-143. https://doi.org/10.1016/j.biocontrol. 2019.02.012.

Villarreal-Delgado, M. F.; Villa-Rodríguez, E. D.; Cira-Chavéz, L. A.; Estrada-Alvarado, M. I.; Parra-Cota, F. I.; de los Santos-Villalobos, S. 2018. The genus Bacillus as a biological control agent and its implications in the agricultural biosecurity. Rev. Mex. Fitopatol. 33(1):95-130. https://doi.org/10.18781/R.MEX.FIT.1706-5.