Leaf concentrations of Cu, Fe, Mn, and Zn of soybean affected by cobalt and molybdenum application mode and
Considering the main current limitations and potential of biological fixation of N2 (BNF) in soybean crop and benefits attributed to various crops by inoculation with Azospirillum brasilense (diazotrophic bacteria with free life), with emphasis on larger development of the root system and consequently greater absorption of water and nutrients, we can infer that co‐inoculation with both microorganisms of Bradyrhizobium sp. and A. brasilense can improve the crop performance in an approach that meets the current demands of agricultural, economic, and environmental sustainability. Thus, important researches are needed to evaluate the nutritional status, production components, and the soybean yield affected by cobalt and molybdenum application mode and co‐inoculating seeds with bradyrhizobia and A. brasilense. We found that seed inoculated with A. brasilense and application of cobalt and molybdenum provided higher N concentration in leaf and mass of 100 grains, with a positive impact on the grain yield of soybean, with an increase of 1007 kg ha‐1 of grain, equivalent to 18.4% more than the control (only inoculated with rhizobia). This research demonstrated that co‐inoculation with Bradyrhizobium sp. and A. brasilense associated with the application of cobalt and molybdenum is beneficial for nutrition and soybean yields.
- diazotrophic bacteria
- mineral fertilization
- nutritional status
- foliar diagnosis
The soybean is the major source of vegetable protein, an essential component in the production of animal feed, in addition to increasing use for human consumption .
This explains why the soybean plant is very demanding on nitrogen (N). It is estimated that 80 kg of N is needed to produce 1000 kg of soybean grains. Therefore, to obtain high yields, the biological fixation of N2 (BNF) should be as efficient as possible [2–7].
The process of BNF in Brazil is responsible for nitrogen accumulated by plants; it represents about 200 kg ha-1 N , which is no longer applied via mineral fertilizers. It reduces the cost of production .
In addition, the use of selected and efficient bradyrhizobia inoculant and cobalt (Co) and molybdenum (Mo) nutrition contributes decisively in the BNF . Cobalt and molybdenum are essential for BNF . The first B12 vitamin is essential for the processing of BNF and other parts of the molybdoenzymes, used in absorption and metabolism of nitrogen . The application of Mo and especially Mo + Co increases BNF .
In Brazil, soybean generally responds positively to fertilization with Mo in soils of low fertility and in fertile soils depleted of Mo due to long‐term cropping. The micronutrient can be supplied by seed treatment. However, the toxicity of Mo sources to
Considering the main current limitations and potential of BNF in soybean crop and benefits attributed to various crops by inoculation with
Bacteria promoters of plant growth (BPPG) correspond to a group of beneficial microorganisms to plants due to the ability to colonize the surface of roots, rhizosphere, phyllosphere, and internal plant tissues [16, 17]. The BPPG can stimulate plant growth in several ways. The most relevant are BNF capacity , increase in nitrate reductase activity when the BPPG grows endophytically plants , production of hormones such as auxins, cytokinins, gibberellins, and ethylene, and a variety of other molecules , phosphate solubilization , and act as biological control agent of pathogens . In general, it is believed that the benefit of BPPG to plant growth is caused by a combination of all these mechanisms .
Based on the above information and the lack of research about the interaction between co‐inoculation with
2. Materials and methods
The experiment was conducted in the 2014/2015 season in an experimental area that belongs to the UNESP Engineering Faculty located in Selvíria, MS/Brazil, with the following geographical coordinates, 20o22′S and 51o22′W and an altitude of 335 m. The experimental area soil was classified as Distroferric Red Oxisol with clay texture (the granulometric analysis indicated values of particle size of 420, 50 kg-1, and 530 g of sand, silt, and clay, respectively), according to Embrapa (2013) , which has been cultivated with annual cultures over 27 years, with the last 10 years in the direct tillage system. Before soybean sowing, corn was cultivated in the area. The annual average temperature was 23.5°C, the annual average pluvial precipitation was 1370 mm, and the annual average relative air humidity was between 70% and 80%.
The experimental design was carried out in a randomized blocks with six treatments and four replications. The treatments were as follows: (1) control (without soybean inoculation with
In all treatments, the inoculation with Rhizobium was performed in seeds at a dose of 200 ml ha-1 (strains: SEMIA 5019 (
Chemical properties of the soil in the tillable layer were determined before 2014, before the soybean experiment began. The methods proposed by Raij et al.  provided the following results: 10 mg dm‐3 of P (resin), 5 mg dm-3 of S‐SO4, 22 g dm-3 of organic matter (OM), pH(CaCl2) of 5.3, 2.4 mmolc dm-3 of K+, 21.0 mmolc dm-3 of Ca2+, 18.0 mmolc dm-3 of Mg2+, 28.0 mmolc dm‐3 of H+Al, 3.2 mg dm-3 of Cu, 22.0 mg dm-3 of Fe, 24.2 mg dm-3 of Mn, 1.2 mg dm-3 of Zn (diethylenetriaminepentaacetic acid (DTPA)), 0.16 mg dm-3 of B (hot water), and 60% base saturation. Based on soil analysis and soybean crop fertilization recommendation , the fertilization was done in the seed furrows with 96 kg P2O5 ha-1 (in the form of triple superphosphate) and 70 kg ha-1 K2O (in the form of potassium chloride).
The seeds were treated with the fungicide Thiram + Carbendazim at a dosage of 30 + 70 g active ingredient (a.i.) per 100 kg seed, respectively, after drying the seeds, and were inoculated with Rhizobium, and depending on the treatment the seed was inoculated with
The experiments were conducted in a no‐tillage system. The area was irrigated by a central pivot sprinkler system when necessary. The water coverage was 14 mm over a period of around 72 h. The control of weeds, pests, and diseases prevention was carried out when necessary in soybean crop. The plants were harvested 120 days after soybean emergence.
Concentrations of N, P, K, Ca, Mg, S, Cu, Fe, Mn, and Zn were measured in soybean plant leaves. The third upper trifoliate leaves (30 plants) in the flowering soybean plants (R2 stage) were collected according to the methodology described in Ambrosano et al. . The determination of nutrients was carried out as described by Malavolta . The leaf chlorophyll index (LCI) was determined indirectly after application of the treatments and when the plants were in the flowering (R2 stage), in 10 plants per plot through readings in the third upper trifoliate leaves, using a digital chlorophyll CFL 1030 Falker (Falker Agricultural Automation, Porto Alegre, Brazil).
The leaf area of 10 leaves per plot was measured using the software ImageJ 1:45 (2011), according to the methodology described by Bauermann . At the time of harvest, 10 soybean plants representing were collected for counting the number of grains per pod, grains per plant, and mass of 100 grains. The mass was determined on a precision scale of 0.01 g and corrected for 13% moisture (wet basis). The soybean was harvested from the plants in the useful area of each plot and grain yield was calculated after mechanical threshing. Data were transformed into kg ha-1 and corrected for 13% moisture (wet basis). The results of all the evaluations were subjected to analysis of variance and the Tukey test at 5% probability to compare the averages of treatments, using the Sisvar program.
3. Results and discussion
The seed inoculated with
|‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ g kg-1 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐|
|Control||48.65 b||4.01 a||19.12 a||9.70 a||5.67 ab||3.06 a|
|Co, Mo seed||50.91 ab||3.75 a||19.98 a||8.94 a||4.55 b||3.08 a|
|Co, Mo + Azos seed||56.21 a||4.16 a||20.42 a||8.64 a||4.73 ab||3.16 a|
|Azos foliar||49.00 b||4.75 a||19.92 a||9.51 a||5.63 ab||3.36 a|
|Co, Mo foliar||55.95 a||4.01 a||18.38 a||8.74 a||5.32 ab||3.35 a|
|Co, Mo + Azos leaf||54.30 ab||4.28 a||18.70 a||8.99 a||5.83 a||3.64 a|
Increases in total nitrogen biologically fixed by plant through symbiosis with rhizobia, associated with
The treatments in this research provided similar leaf concentrations of P, K, Ca, and S (Table 1). However, there was a higher concentration of Mg in the leaves when Co and Mo and
The leaf chlorophyll index (LCI) and leaf Fe and Cu concentrations were not affected by treatments (Table 2). This can be explained by adequate leaf N concentrations obtained for soybean crop. Zuffo et al.  also observed that the use of
|‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ mg kg-1 ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐|
|Control||43.79 a||9.00 a||156.33 a||90.00 ab||47.67 b|
|Co, Mo seed||44.12 a||8.33 a||163.33 a||71.00 b||50.00 ab|
|Co, Mo + Azos seed||44.52 a||8.67 a||189.67 a||70.33 b||47.67 b|
|Azos foliar||43.56 a||10.33 a||212.33 a||85.00 ab||53.33 a|
|Co, Mo foliar||44.23 a||9.33 a||191.00 a||84.67 ab||46.00 b|
|Co, Mo + Azos leaf||44.15 a||10.67 a||184.00 a||97.67 a||50.00 ab|
The results are different from those found by other authors using corn plants, who found that the LCI was higher in the treatments with diazotrophs than in the treatments without inoculation. Corn plants that were inoculated with
Leaf application of Co and Mo and foliar inoculation with
The leaf area of soybean was greater in treatment with the application of
|Treatments||Leaf area (cm2)||Grains per pod||Grains per plant||Mass of 100 grains (g)||Grains yield (kg ha-1)|
|Control||64.05 ab||3.00 a||176.30 a||14.68 b||5550 b|
|Co, Mo seed||62.90 b||2.55 b||145.20 a||15.88 ab||6083 ab|
|Co, Mo + Azos seed||68.35 ab||2.78 ab||155.67 a||16.10 a||6557 a|
|Azos foliar||77.80 a||2.65 b||156.90 a||14.80 ab||5355 b|
|Co, Mo foliar||69.75 ab||2.80 ab||185.07 a||14.60 b||5685 ab|
|Co, Mo + Azos leaf||67.50 ab||2.65 b||138.47 a||14.88 ab||5602 ab|
The control treatment provided greater number of grains per pod, and the number of grains per pod did not differ between treatment with Co and Mo of the leaf and treatment of inoculation of the seed with
Seed inoculated with
These results may be due to several mechanisms, which are the anticipation in the BNF of the nodes, an increase in the dry weight of nodes, promoting the occurrence of nodulation heterologous through the increased formation of hair root and secondary roots, an increase in infection sites, inhibition of plant pathogens and production of phytohormones and influences in the partition of dry matter between the roots and shoots . Yet, pondering Hungria et al. , these results caused by co‐inoculation bacteria promoters of plant growth and Rhizobia appear to be under the influence of specific signals among bacterial genotypes involved and the genotype of the host plant. It is important to do more related studies on the response of the co‐inoculation depending on the genotypes, aiming at the development of more responsive genotypes.
In an important research by Campos et al. , they concluded that there are no Mo and Co effects on nodulation in soil with established
4. Final consideration
Leaf application of Co and Mo and foliar inoculation with
Seed inoculated with
This research demonstrated that co‐inoculation with
Ignácio VL, Nava IA, Malavasi MM, Gris EP. Influence of foliar fertilization with manganese on germination, vigor and storage time of RR soybean seeds. Revista Ceres. 2015; 62:446–452. doi: 10.1590/0034‐737X201562050004
Figueiredo MVB, Martinez CR, Burity HA, Chanway CP. Plant growth‐promoting rhizobacteria for improving nodulation and nitrogen fixation in the common bean ( Phaseolus vulgarisL.). World Journal of Microbiology and Biotechnology. 2008; 24:1187–1193. doi: 10.1007/s11274‐007‐9591‐4
Vieira Neto SA, Pires FR, Menezes CCE, Menezes JFS, Silva AG, Silva GP, Assis RL. Forms of inoculant application and effects on soybean nodulation. Revista Brasileira de Ciência do Solo. 2008; 32:861–870 (in Portuguese with abstract in English). doi: 10.1590/S0100‐06832008000200040
Zilli JE, Marson LC, Marson BF, Gianluppi V, Campo RJ, Hungria M. Soybean inoculation by spraying Bradyrhizobiumover plants. Pesquisa Agropecuária Brasileira. 2008; 43:541–544 (in Portuguese with abstract in English). doi: 10.1007/s11104‐009‐0262‐0
Hungria M, Campo RJ, Souza EMS, Pedrosa FO. Inoculation with selected strains of Azospirillum brasilenseand A. lipoferumimproves yields of maize and wheat in Brazil. Plant and Soil. 2010; 331:413–425. doi 10.1007/s11104‐009‐0262‐0
Rodrigues M, Arf O, Barbieri MKF, Portugal JR, Rodrigues RAF. Inoculation with Azospirillum brasilenseand application of plant growth regulator in irrigated wheat in the cerrado. In: Reunião da Comissão Brasileira de Pesquisa de Trigo e Triticale; July 2012; Londrina – PR. Londrina: IAPAR; 2012. CD ROM (in Portuguese).
Bulegon LG, Rampim L, Klein J, Kestring D, Guimarães VF, Battistus AG, Inagaki AM. Components of production and yield of soybean inoculated with Bradyrhizobiumand Azospirillum. Revista Terra Latinoamericana. 2016; 34:169–176 (in Portuguese with abstract in English).
Zilli JE, Gianluppi V, Campo RJ, Rouws RC, Hungria M. In‐furrow inoculation with Bradyrhizobiumalternatively to seed inoculation of soybean. Revista Brasileira de Ciência do Solo. 2010; 34:1875–1991 (in Portuguese with abstract in English). doi: 10.1590/S0100‐06832010000600011
Albareda M, Rodríguez‐Navarro DN, Temprano FJ. Soybean inoculation: Dose, N fertilizer supplementation and rhizobia persistence in soil. Field Crops Research. 2009; 113:352–356. doi: 10.1016/j.fcr.2009.05.013
Sfredo GJ, Oliveira MCN. Soybeans, molybdenum and cobalt. Documents 322. Londrina: Embrapa Soja; 2010. 36 p (in Portuguese).
Taiz L, Zeiger E. Vegetal physiology. 5nd ed. Porto Alegre: Artmed; 2013. 918 p (in Portuguese).
Novais RF, Alvarez V. VH, Barros NF, Fontes RLF, Cantarutti RB, Neves JCL. Soil fertility. Viçosa ‐ MG: Brazilian Society of Soil Science; 2007. 1017 p (in Portuguese).
Campo RJ, Albino UB, Hungria M. Importance of molybdenum and cobalt to the biological nitrogen fixation. nitrogen fixation: from molecules to crop productivity. Current Plant Science and Biotechnology in Agriculture. 2008; 28:597–598.
Campo RJ, Araujo RS, Hungria M. Molybdenum‐enriched soybean seeds enhance N accumulation, seed yield, and seed protein content in Brazil. Field Crops Research. 2009; 110:219–224. doi: 10.1016/j.fcr.2008.09.001
Hungria M, Nogueira MA, Araujo RS. Co‐inoculation of soybeans and common beans with rhizobia and azospirilla: strategies to improve sustainability. Biology and Fertility of Soils. 2013; 49:791–801. doi:10.1007/s00374‐012‐0771‐5
Davison J. Plant beneficial bacteria. Nature Biotechnology. 1988; 6:282–286. doi:10.1038/nbt0388‐282
Kloepper JW, Lifshitz R, Zablotowicz RM. Free‐living bacterial inocula for enhancing crop productivity. Trends in Biotechnology. 1989; 7:39–43. doi:10.1016/0167‐7799(89)90057‐7
Huergo LF, Monteiro RA, Bonatto AC, Rigo LU, Steffens MBR, Cruz LM, Chubatsu LS, Souza EM, Pedrosa FO. Regulation of nitrogen fixation in Azospirillum brasilense. In: Cassán FD, Garcia SI, editors. Azospirillumsp.: cell physiology, plant interactions and agronomic research in Argentina. Argentina: Asociación Argentina de Microbiologia; 2008. p. 17–35.
Cassán F, Sgroy V, Perri GD, Masciarelli O, Luna, V. Producción de fitohormonas por Azospirillumsp. Aspectos fisiológicos y tecnológicos de la promoción del crecimiento vegetal. In: Cassán FD, Garcia SI, editors. Azospirillumsp.: cell physiology, plant interactions and agronomic research in Argentina. Argentina: Asociación Argentina de Microbiologia; 2008. p. 61–86.
Perrig D, Boiero L, Masciarelli O, Penna C, Cassán F, Luna V. Plant growth promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and their implications for inoculant formulation. Applied Microbiology and Biotechnology. 2007; 75:1143–1150. doi:10.1007/s00253‐007‐0909‐9
Rodriguez H, Gonzalez T, Goire I, Bashan Y. Gluconic acid production and phosphate solubilization by the plant growth‐promoting bacterium Azospirillumspp. Naturwissenschaften. 2004; 91:552–555. doi: 10.1007/s00114‐004‐0566‐0
Correa OS, Romero AM, Soria MA, Estrada, M. Azospirillum brasilense‐plant genotype interactions modify tomato response to bacterial diseases, and root and foliar microbial communities. In: Cassán FD, Garcia SI, editors. Azospirillumssp.: cell physiology, plant interactions and agronomic research in Argentina. Argentina: Asociación Argentina de Microbiologia; 2008. p. 87–95.
Dobbelaere S, Vanderleyden J, Okon Y. Plant growth‐promoting effects of diazotrophs in the rhizosphere. Critical Reviews in Plant Sciences. 2003; 22:107–149. doi.org/10.1080/713610853
Bárbaro IM, Centurio MAPC, Gavioli EA, Sarti DGP, Bárbaro Júnior LS, Ticelli M, Miguel FB. Analysis of soybean cultivars in response to the inoculation and application of cobalt and molybdenum. Revista Ceres. 2009; 56:342–349 (in Portuguese with abstract in English).
Empresa Brasileira de Pesquisa Agropecuária ‐ Embrapa. National Center for Soil Research. Brazilian system of soil classification. 3nd ed. Brasília: Embrapa; 2013. 353 p (in Portuguese).
Raij B. van, Andrade JC, Cantarella H, Quaggio JA. Chemical analysis to evaluate the fertility of tropical soils. Campinas: IAC; 2001. 285 p (in Portuguese).
Ambrosano EJ, Tanaka RT, Mascarenhas HAA, Raij B. van, Quaggio JA, Cantarella H. Legumes and oilseeds. In: Raij B. van, Cantarella H, Quaggio JA, Furlani AMC, editors. Recommendations liming and fertilization for the State of São Paulo. Campinas: IAC; 1997. 285 p (Boletim técnico, 100) (in Portuguese).
Malavolta E, Vitti GC, Oliveira SA. Evaluation of the nutritional status of plants: principles and applications. 2nd ed. Piracicaba: Brazilian Association for Research of Potash and Phosphate; 1997. 319 p (in Portuguese).
Bauermann G. Leaf area measurement. 2009. Available from: http://www.imagesurvey.com.br.[Accessed: 2015‐02‐15]
Zuffo AM, Rezende PM, Bruzi AT, Oliveira NT, Soares IO, Neto GFG, Cardillo BES, Silva LO. Co‐inoculation of Bradyrhizobium japonicumand Azospirillum brasilensein the soybean crop. Revista de Ciências Agrárias. 2015; 38:87–93.
Galindo FS, Teixeira Filho MCM, Buzetti S, Santini JMK, Alves CJ, Nogueira LM, Ludkiewicz MGZ, Andreotti M, Bellotte, JLM. Corn yield and foliar diagnosis affected by nitrogen fertilization and inoculation with Azospirillum brasilense. Revista Brasileira de Ciência do Solo. 2016; 40:e0150364. doi:10.1590/18069657rbcs20150364
Kappes C, Arf O, Arf MV, Ferreira JP, Dal Bem EA, Portugal JR, Vilela RG. Seeds inoculation with diazotrophic bacteria and nitrogen application in side‐dressing and leaf in maize. Semina: Ciencias Agrárias. 2013; 34:527–538 (in Portuguese with abstract in English). doi: 10.5433/1679‐0359.2013v34n2p527
Quadros PD, Roesch LFW, Silva PRF, Vieira VM, Roehrs DD, Camargo FAO. Field agronomic performance of maize hybrids inoculated with Azospirillum. Revista Ceres. 2014; 61:209–218 (in Portuguese with abstract in English). doi:10.1590/S0034‐737X2014000200008
Okon Y, Vanderleyden J. Root‐associated Azospirillumspecies can stimulate plants. Applied and Environment Microbiology. 1997; 6:366–370. citeulike:6806747
Bashan Y, Holguin G, De‐Bashan LE. Azospirillum‐plant relations physiological, molecular, agricultural, and environmental advances (1997‐2003). Canadian Journal of Microbiology. 2004; 50:521–577. doi:10.1139/w04‐035
Hungria M. Inoculation with Azospirillum brasilense: innovation in performance at low cost. Documents, 325. Londrina: Embrapa Soja; 2011. 37 p (in Portuguese).
Hungria M, Campo RJ, Mendes IC. The importance of biological nitrogen fixation process for the soybean crop: essential component of competitivity of Brazilian products. Documents, 283. Londrina: Embrapa Soja; 2007. 80 p (in Portuguese).