Azospirillum inoculation of massai grass seeds and nitrogen fertilization: effects on forage development

Azospirillum inoculation of massai grass seeds and nitrogen fertilization: effects on forage development | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 February 2026 V1 Latest version Share on Azospirillum inoculation of massai grass seeds and nitrogen fertilization: effects on forage development Authors : Josimari Paschoaloto 0000-0001-9517-3817 [email protected] , Arthur Santos , Patrícia Monteiro , Geângelo Rosa , Viviane Santos , and Henrique Perez Authors Info & Affiliations https://doi.org/10.22541/au.177067318.89919612/v1 99 views 55 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The demand for sustainable livestock production has intensified interest in biological strategies that enhance forage development while reducing reliance on chemical fertilizers. This study evaluated the effects of seed inoculation with Azospirillum brasilense and four nitrogen (N) fertilization rates (0, 50, 100, and 150 kg N ha⁻¹) on the growth of Massai grass (Megathyrsus maximus cv. Massai) in a greenhouse experiment using a 2 × 4 factorial design. Plant height, tiller number, fresh and dry masses of shoots and roots were measured 60 days after sowing. Nitrogen fertilization significantly increased plant height and tiller number, with the greatest response observed at 100 kg N ha⁻¹ . Inoculation did not improve shoot growth under high N conditions but consistently stimulated root biomass across all N rates. The interaction between inoculation and nitrogen suggests that microbial benefits were more evident at moderate N levels, where A. brasilense may contribute to improved nutrient acquisition and efficient resource allocation. Enhanced root development in inoculated plants may increase soil exploration capacity, potentially improving water and nutrient uptake under field conditions. Combining moderate nitrogen fertilization with A. brasilense inoculation could be an effective strategy to increase forage productivity and nitrogen use efficiency in tropical pasture systems, thereby supporting more sustainable beef production. These findings offer practical insights for integrating microbial inoculants into forage management practices. Azospirillum inoculation of massai grass seeds and nitrogen fertilization: effects on forage development ABBREVIATIONS A. brasilense , Azospirillum brasilense ; C4, plants with C4 photosynthetic pathway; cv., cultivar; g, gram; ha⁻¹, per hectare; kg, kilogram; N, nitrogen. Abstract The demand for sustainable livestock production has intensified interest in biological strategies that enhance forage development while reducing reliance on chemical fertilizers. This study evaluated the effects of seed inoculation with Azospirillum brasilense and four nitrogen (N) fertilization rates (0, 50, 100, and 150 kg N ha⁻¹) on the growth of Massai grass ( Megathyrsus maximus cv. Massai) in a greenhouse experiment using a 2 × 4 factorial design. Plant height, tiller number, fresh and dry masses of shoots and roots were measured 60 days after sowing. Nitrogen fertilization significantly increased plant height and tiller number, with the greatest response observed at 100 kg N ha⁻¹. Inoculation did not improve shoot growth under high N conditions but consistently stimulated root biomass across all N rates. The interaction between inoculation and nitrogen suggests that microbial benefits were more evident at moderate N levels, where A. brasilense may contribute to improved nutrient acquisition and efficient resource allocation. Enhanced root development in inoculated plants may increase soil exploration capacity, potentially improving water and nutrient uptake under field conditions. Combining moderate nitrogen fertilization with A. brasilense inoculation could be an effective strategy to increase forage productivity and nitrogen use efficiency in tropical pasture systems, thereby supporting more sustainable beef production. These findings offer practical insights for integrating microbial inoculants into forage management practices. 1 Introduction Livestock production in Brazil is predominantly pasture-based, and Megathyrsus maximus is widely cultivated owing to its high forage yield potential. Massai grass is noted for its vigorous tillering capacity, good nutritional profile, and adaptability to soils of intermediate fertility (EMBRAPA, 2001). Fertilizers, particularly nitrogen, can improve forage production (Lopes et al., 2020; Motta et al., 2024). However, excessive nitrogen fertilizer applications can severely affect soil health and water quality. Furthermore, nitrogen-based fertilizers can generate potent greenhouse gases such as nitrous oxide, exacerbating climate change. In addition, runoff from fertilized areas can introduce nitrates into groundwater and surface water, putting human health at risk. This runoff often stimulates the excessive growth of aquatic vegetation and algae, which can reduce oxygen levels in water bodies and endanger aquatic life (Bodirsky et al., 2014; Craswell, 2021). Given the significant nitrogen losses caused by volatilization, leaching, and rapid decomposition of organic matter, as well as the high economic and environmental costs of synthetic N fertilizers, biological nitrogen fixation has emerged as a logical and sustainable alternative for meeting crop nitrogen demands (Figueiredo et al. 2016). As a result, nitrogen fixing microorganisms have been used to sustainably meet the need for this nutrient. Azospirillum brasilense is currently one of the most extensively investigated and commercially used plant growth-promoting bacteria. Studies show that it can fix atmospheric N 2 and promote plant growth by synthesizing phytohormones. These free-living nitrogen-fixing bacteria demonstrate versatile carbon and nitrogen metabolism while maintaining low energy requirements. This adaptability allows them to establish themselves effectively in the competitive rhizosphere environment (Steenhoudt and Vanderleyden, 2000). Inoculation with diazotrophic bacteria such as Azospirillum brasilense is a promising sustainable alternative that promotes biological nitrogen fixation and plant growth through phytohormone production (Hungria et al., 2016; Freitas et al., 2019). Recent research suggests that A. brasilense plays an important role in improving nitrogen use efficiency and forage resilience in tropical environments (Castro et al., 2025; Silva, 2025). Furthermore, meta-analyses and field trials have shown that inoculation can partially replace nitrogen fertilization and preserve productivity while reducing environmental impacts (Barbosa et al., 2022; Nakatani et al., 2024; Jalal et al., 2022; Nascimento et al., 2025). The objective of this study was to evaluate forage development and biomass production of Megathyrsus maximus cv. Massai as a function of application of N doses combined with inoculation of Azospirillum brasilense . 2 Materials and Methods The experiments were conducted in a greenhouse covered with a 50% shade screen. This greenhouse was located in the experimental area of Instituto Federal Baiano, Campus Bom Jesus da Lapa, Bahia State, Brazil (13°15′33″ S, 43°32′49″ W). According to Köppen’s classification, the regional climate is tropical savanna (Aw), characterized by hot and dry conditions, with an average annual temperature of 27.0 °C and 679 mm of precipitation. Soil samples were collected from the arable layer (0–20 cm depth) of the experimental area. The soil, classified as sandy clay loam, was sieved through a 5‑mm screen and placed in plastic pots with a capacity of 0.7 dm³. The soil had a pH of 6.9, high base saturation (98.1%), and required no liming or fertility adjustment before the experiment. The forage grass used was Megathyrsus maximus cv. Massai, in which seed dormancy was broken, as described by Meschede et al. (2004). Commercial product containing AbV5 and AbV6 strains at a concentration of 1.8 × 10⁸ CFU mL⁻¹, was used for Azospirillum brasiliense inoculation treatments. For seed inoculation, 10 mL of the product was diluted in 150 mL of water and sprayed onto 500 g of seeds. Ten seeds were sown directly into each pot at an average depth of 2.5 cm. Germination started approximately four days after sowing. Thinning was performed 15 days after sowing, leaving only one Panicum maximum cv. Massai plant per pot. Irrigation was regularly performed throughout the experimental period to maintain soil moisture levels suitable for plant development. Nitrogen topdressing was divided into four applications. The first application was made at sowing, followed by applications at 15, 25, and 35 days after sowing, using urea as the nitrogen source, depending on the treatment. Forage evaluation samples were collected 60 d after sowing. For urea application, the appropriate doses (2.3, 4.5, and 6.7 g) were diluted in 300 mL of distilled water. Subsequently, 20 mL of each solution was added to each pot. In the control treatment, 30 mL of distilled water was added. Plant height (cm) was measured using a graduated ruler from the base of the plant to the tip of the longest leaf. The number of tillers, fresh mass of shoots and roots, and dry mass of shoots and roots were also recorded. Samples were dried in a forced-draft oven at 65 °C for 72 h until a constant weight was achieved (Malavolta, 2006), and the dry mass was then determined. The experiment had a completely randomized design with a 2 × 4 factorial scheme. Treatments included two levels of inoculation (Massai with and without Azospirillum brasilense inoculation) and four nitrogen fertilization rates (0, 50, 100, and 150 kg N ha⁻¹) and six replicates each. Nitrogen was supplied as urea (45%). Data analyzed using the PROC MIXED procedure in SAS 9.4 (SAS Institute, 2017). Data were analyzed using analysis of variance at a 5% probability of error; when significant, means were compared using Tukey’s test for qualitative factors (inoculation). Regression analysis was used when a significant effect was found for quantitative components (nitrogen levels). The criteria for choosing the model were the coefficient of determination (R 2 ) and a p-value of ≤ 0.05. 3 Results and Discussion Nitrogen fertilization increased plant height and tiller number in Megathyrsus maximus cv. Massai, confirming that canopy development in tropical C4 grasses is strongly responsive to mineral N supply (Table 1). N rates (kg ha −1 ) 0 50 100 150 Overall mean Standard error I N I × N Plant height (cm) Inoculation 83.66 94.3 95.25 92.58 91.58 1.0546 0.279 <0.0001 0.0572 No inoculation 80.33 88 94.41 95.69 89.61 Overall mean 82 91.42 94.83 94.13 Tiller number Inoculation 13.16 20.16 26.5 21.33 20.29 1.0974 0.1004 <0.0001 0.2127 No inoculation 13 28.33 24.33 24.66 22.58 Overall mean 13.08 24.25 25.42 23 Fresh shoot mass Inoculation 20.36 44.16 56.36 47.53 42.10 B 3.0791 0.001 <0.0001 0.0287 No inoculation 21.13 57.85 59.96 77.16 54.03 A Overall mean 20.75 51 57.66 62.85 47.11 Shoot dry mass Inoculation 5.92 11.37 12.51 12.35 10.49 B 0.6622 0.0008 <0.0001 0.0032 No inoculation 5.9 13.08 14.41 19.21 12.91 A Overall mean 12.41 12.71 15.78 Fresh mass of roof Inoculation 17.13 18.6 20.2 18.76 18.67 A 0.2466 <0.0001 <0.0001 0.0265 No inoculation 14.45 16.75 17.22 17.76 16.40 B Overall mean 15.79 17.67 18.71 17.97 Dry mass of roof Inoculation 5.46 6.55 7.86 6.85 6.68 A 0.2369 <0.0001 <0.0001 0.0273 No inoculation 2.56 4.38 5.13 5.41 4.37 B Overall mean 4.01 5.46 6.5 6.13 Leaf senescence Inoculation 1.83 2.5 2.83 4.33 2.87 A 0.1857 0.0132 <0.0001 0.0141 No inoculation 1.16 2.33 2.83 1.66 2.00 B Overall mean 1.5 2.41 2.83 3 I = inoculation effect; N = nitrogen rate effect; I × N = inoculation × nitrogen rate interaction; Means followed by different letters in the columns are statistically different by Tukey test at 0.05 probability level The response up to 100 kg N ha⁻¹ followed by stabilization at 150 kg N ha⁻¹ is consistent with a diminishing-return pattern in which additional N no longer increases leaf elongation and tiller initiation proportionally once key sinks (e.g., active meristems and leaf area expansion rate) approach saturation. From a management standpoint, this plateau supports the interpretation that N rates above greenhouse conditions, and that defining an “agronomic optimum” is more informative than reporting only “significant increases”. These findings confirm the strong dependence of this tropical forage grass on the mineral nitrogen supply, which is consistent with Guimarães et al. (2023), who reported linear increases in the aerial biomass of Megathyrsus maximus under increasing nitrogen rates. There was a significant interaction between inoculation and nitrogen dose in terms of offshoot biomass. Uninoculated plants produced 77.2 g of fresh shoot biomass at 150 kg N ha⁻¹, whereas inoculated plants yielded only 47.5g under the same conditions. Uninoculated plants produced 19.2 g of dry shoot mass at 150 kg N ha⁻¹, compared to 12.3 g in inoculated plants. This pattern is biologically plausible because the growth-promotion effect of Azospirillum brasilense often depends on environmental context and plant nutritional status. Under high mineral N, plants can meet N demand directly from fertilizer, potentially reducing the relative benefit of plant growth–promoting pathways and shifting the limiting factor from N supply to other constraints (e.g., pot volume, light, or internal carbon partitioning). In addition, under high N availability, host plants may allocate less carbon to associative interactions, and bacterial effects mediated by hormones (auxin/cytokinin balance) can modify source–sink relations, sometimes favoring root sinks over shoot accumulation. This “context dependency” is widely recognized for Azospirillum –plant associations and helps explain why inoculation effects are often more consistent at low-to-moderate N than at excessive N supply (Dobbelaere et al., 2003). These findings suggest that Azospirillum brasilense inoculation does not improve shoot growth when mineral nitrogen availability is high, supporting Hungria’s (2023) assertion that inoculation benefits are more pronounced under moderate nitrogen supply, where bacterial activity contributes to improved nutrient-use efficiency. In contrast, the inoculation consistently stimulated root development at all nitrogen levels, indicating that the most stable response in Massai grass was root system stimulation. Fresh root biomass averaged 18.7g in inoculated plants compared with 16.4g in uninoculated plants, whereas dry root biomass was 6.7 g with inoculation and only 4.4g without inoculation. Azospirillum brasilense induces morphophysiological changes, including phytohormone (such as auxins) synthesis, which promotes root branching and expansion (Martínez-Martínez et al., 2023). A larger root system improves the plant’s ability to absorb water and nutrients while also increasing its resistance to abiotic stressors. In practical pasture systems, a more robust root system can be agronomically valuable beyond immediate shoot yield, because it may improve stand persistence and resilience to intermittent water stress, and can contribute to greater nutrient capture efficiency (e.g., reduced N losses by better uptake synchronization). Furthermore, inoculated plants had more senescent leaves (2.9 leaves) than uninoculated plants (2.0 leaves), indicating a potential physiological trade-off in resource allocation in which increased investment in root growth occurs alongside faster leaf turnover. This does not necessarily imply reduced performance; leaf turnover can reflect a dynamic canopy adjustment, especially when roots expand and compete for assimilates. Overall, nitrogen fertilization was the primary driver of shoot growth and biomass accumulation, whereas inoculation with A. brasilense consistently increased root system development. Combining moderate nitrogen rates (approximately 100 kg N ha⁻¹) with inoculation can improve forage productivity, nitrogen use efficiency, and sustainability in tropical livestock production systems. This is caused by the interplay of nitrogen-promoting shoot characteristics and inoculation, which improves root architecture. However, because this experiment was conducted in small pots under greenhouse conditions, root confinement and microclimatic uniformity may amplify allocation patterns. Therefore, field validation should test whether inoculation affects leaf lifespan, forage nutritive value, and canopy structure under grazing or cutting management, where regrowth dynamics and nutrient cycling differ substantially from pot conditions (Leite et al., 2019; Heinrichs et al, 2020). CONCLUSION Nitrogen fertilization was the main driver of shoot growth in Panicum maximum cv. Massai, whereas inoculation with Azospirillum brasilense consistently increased root biomass. The combined use of inoculation and a recommended dose of 100 kg N ha⁻¹ represents a promising approach to increase forage productivity, optimize nitrogen use efficiency, and support more sustainable livestock production in tropical systems. References Barbosa, O. D., Silva, R. V., & Pereira, J. F. (2022). Biological inoculation and nitrogen use efficiency in tropical forages: A meta-analysis. Grass and Forage Science , 77(3), 425–438. https://doi.org/10.1111/gfs.12516 Bodirsky, B. L., et al. (2014). Nitrogen fertilizers and climate change: Assessing the interactions. Nature Climate Change , 4(5), 427–431. https://doi.org/10.1038/nclimate2193 Castro, A. C., Ribeiro, L. S., Nogueira, M. A., & Hungria, M. (2025). 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Critical Reviews in Microbiology , 26(2), 69–90. https://doi.org/10.1080/10408410091159189 Information & Authors Information Version history V1 Version 1 09 February 2026 Copyright This work is licensed under a Creative Commons Attribution 4.0 International License Keywords forage management grazing management nitrogen plant nutrition symbiosis Authors Affiliations Josimari Paschoaloto 0000-0001-9517-3817 [email protected] Instituto Federal de Educação Ciência e Tecnologia Baiano - Campus Bom Jesus da Lapa, BA, Brazil View all articles by this author Arthur Santos Instituto Federal de educação, Ciência e Tecnologia Baiano - Campus Bom Jesus da Lapa View all articles by this author Patrícia Monteiro Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG), Campo Experimental de Acauã, Minas Gerais, Brasil View all articles by this author Geângelo Rosa Instituto Federal de Educação Ciência e Tecnologia Baiano - Campus Bom Jesus da Lapa, BA, Brazil View all articles by this author Viviane Santos Instituto Federal de educação ciência e Tecnologia Baiano, Campus Santa Inês View all articles by this author Henrique Perez Universidade Estadual de Maringá View all articles by this author Metrics & Citations Metrics Article Usage 99 views 55 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Josimari Paschoaloto, Arthur Santos, Patrícia Monteiro, et al. 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