Shifts in the Efficiency of ¹⁵N-Ammonium Sulfate Fertilization to Sugarcane Varieties Inoculated with Diazotrophic Bacteria

Abstract

Nitrogen is an essential plant nutrient, but N fertilization contributes to greenhouse gas emissions through its production and application as well as nitrous oxide emissions when applied to soil. Diazotrophic bacteria, known to modify root architecture and increase nutrient uptake, have been proposed as a potential strategy to improve nitrogen use efficiency (NUE) in sugarcane cultivation. The objective of this study was to evaluate the efficiency of N use from ¹⁵N ammonium sulfate applied to different sugarcane varieties inoculated with diazotrophic bacteria. The experiment was conducted in pots filled with soil (100 kg pot⁻¹). The sugarcane varieties tested were RB867515 and RB92579. A commercial diazotrophic bacteria inoculant for sugarcane was used. The experimental design was a randomized block design with four replicates. The treatments were as follows: control without inoculation, inoculation with five strains of bacteria, with or without nitrogen fertilization. The evaluations were performed on different parts of the plant. At 360 days after transplanting, the accumulation of N, fresh mass, dry mass, and the proportion of excess ¹⁵N were determined. In the studied sugarcane varieties, the efficiency of ¹⁵N fertilizer use was 60%, with no influence from inoculation.

1. Introduction

Among grasses, sugarcane (Saccharum sp.) is renowned for its high accumulation of N, requiring annual N fertilizer applications, which generally vary from 30 to 60 kg ha⁻¹ in plant cane and over 100 kg ha⁻¹ in ratoons [1,2,3]. In Brazil, the sugarcane crop is the country’s second-largest N consumer, with doses of N applied in recent years averaging 56 kg ha⁻¹ and expected to increase by 1.9 kg ha⁻¹ over the next few years [3,4].

The efficiency of nitrogen fertilization depends on the variety, soil, and especially management, which is associated with microbial interaction that helps in the availability of N in the soil. The inoculation of diazotrophic bacterial strains can provide benefits to the plant through different mechanisms of action, such as the production of phytohormones, phosphate solubilization, and biological nitrogen fixation [1,5,6]. Compounds produced by secondary metabolism favor germination, increase the speed of sprouting of the buds, enhance the growth of the root system, and, as a result, lead to greater accumulation of water and nutrients, resulting in a greater gain in dry matter, N content, and sucrose concentration, in addition to biological nitrogen fixation (BNF) [6,7,8,9,10].

The recovery of N fertilizer by sugarcane is generally between 10 and 40%, values considered low, which demonstrates the possibility of significant N losses in the sugarcane production system [11,12,13,14,15]. Therefore, increasing the efficiency of N use in sugarcane and other cereal production systems with the use of diazotrophic bacteria, which enable the plant to absorb the applied fertilizer more efficiently, is an attractive strategy in the effort to reduce the applied dose [16,17].

However, information is lacking on the processes involved in the effects of inoculants containing diazotrophic bacteria on the recovery of N from the fertilizer. In this study, the use of ¹⁵N as a tracer is of great importance, as it can produce reliable results in terms of the potential to improve fertilizer efficiency. The use of isotopic techniques is of great relevance in studies of plant nutrients in the sugarcane crop due to the complexity of its biogeochemical cycle, which involves three main sources of N: biological N₂ fixation, mineralization of organic matter from the soil, and nitrogen fertilization.

The hypothesis raised in this study is that inoculation with diazotrophic bacteria positively influences the efficiency of N use in sugarcane crops. The objectives were to determine the accumulation of dry mass, total N, and to determine the efficiency of nitrogen fertilizer use in sugarcane.

2. Materials and Methods

2.1. Plant Material and Experimental Conditions

The experiment was conducted in the experimental area of Embrapa Agrobiologia in Seropédica, Rio de Janeiro, Brazil (22°44′38″ South, 43°42′28″ West and 26 m altitude). The region’s climate, according to the Köppen classification, is Aw-tropical with a dry winter—with dry winters and hot and rainy summers. The average annual temperature is 25 °C, with annual precipitation of 1300 mm. Data on precipitation, irrigation water used and temperature data during the experimental period are displayed in Figure 1.

The experiment was carried out in pots containing 100 kg of soil, placed in the field. The soil classified as Dystrophic Red Argisol (WRB/FAO) or Typic Hapladult (USDA, Soil Taxonomy) was collected at a depth of 0 to 40 cm at the Embrapa field station and presents the following attributes: texture (g kg⁻¹) clay, 164; silt, 36; sand, 800; pH (H₂O), 5.6; C, 10 g kg⁻¹; N, 1.1 g kg⁻¹; available P (Mehlich I), 1.42 mg dm⁻³; and Ca²⁺, Mg²⁺, K⁺, H⁺ Al³⁺, respectively, 1.81; 1.19; 0.14; 4.29 cmolc dm⁻³.

The soil was amended with 55 mg P kg⁻¹ of soil (equivalent to 110 kg P ha⁻¹) as single superphosphate, 105 mg K kg⁻¹ of soil (equivalent to 210 kg K ha⁻¹) as potassium sulfate. As a source of micronutrients, 50 mg kg⁻¹ of soil of fritted trace elements (FTE BR-12) were added (equivalent to 100 kg ha⁻¹) with a composition of (g kg FTE⁻¹—18 B, 8 Cu, 30 Fe, 20 Mn, 1 Mo, 90 Zn). In addition, powdered gypsum was applied at a dose of 250 mg kg⁻¹ soil (equivalent to 500 kg ha⁻¹).

After thorough mixing, 100 kg of soil was transferred to opaque plastic containers with cylindrical shape and dimensions of 0.40 m in diameter × 0.80 m in height (total capacity of 100 dm⁻³). To facilitate water drainage from the pots, 10 holes were made in the base of the pots and filled to a depth of 10 cm with gravel (4.8 to 9.5 mm), covered with an acrylic blanket. In the field, the pots were arranged in a 1.2 × 1.2 m grid in a level outdoor area without shade.

The experimental design was randomized blocks with four replicates. The component factors of the treatments were as follows: inoculation with diazotrophic bacteria (with and without inoculation) and N treatments (with and without 45 kg ha⁻¹ of ammonium sulfate) in the different parts of the sugarcane plant (root, green leaf, senescent leaf, and lower, middle, and upper part of the stalk).

Sugarcane was planted with the commercial varieties RB867515 and RB92579, chosen for their excellent adaptation to the diverse soil and climate conditions of Brazil, and for their great representation in the national agricultural scenario, standing out for their high productivity. The seedlings were produced by the pre-sprouted seedling system, made from pieces of single-node stem (“minisetts”), following the production system described by Landell et al. [18]. The seedlings were obtained from healthy plants grown in a germplasm field, cultivated in the same type of soil used in the experiment with pots. The selected canes had their nodes removed, and from this process, seedlings were produced through the pre-sprouted seedling (MPB) system, grown in a commercial substrate rich in nutrients. This method consists of the use of mini-sprouted stems (nodes) with individual buds, according to the production protocol described by Landell et al. [18]. Before planting, the mini-cuttings underwent a short heat treatment, being immersed in a water bath at 52 °C for 30 min [19,20]. After this treatment, the mini-cuttings were inoculated with diazotrophic bacteria. The bacterial strains were chosen due to their high BNF capacity, especially in sugarcane. Additionally, they promote plant growth and reduce the use of nitrogen fertilizers [21,22]. A peat inoculant was used which contained the five strains of diazotrophic bacteria recommended for the crop (Gluconacetobacter diazotrophicus—PAL-5^T [23]; Herbaspirillum seropedicae—BR11335 [24]; Herbaspirillum rubrisubalbicans—BR11504 [25]; Paraburkholderia tropica—BR11366T [26]; and Nitrospirillum amazonense—BR11145 [27]. The final inoculant was prepared by diluting 50 g of peat containing each strain in 2.5 L of distilled water (1/50 w/v). The inoculant was prepared in Dyg’s culture medium following the methodology described by Baldani et al. [28]. The final bacterial population was approximately 10⁷ cells mL⁻¹ [29]. At the time of inoculation, the inoculant was diluted in distilled water at a ratio of 1:100 or 1:50 (v/v). The mini-stems were then immersed in the inoculant solution for thirty minutes.

The seedlings were transplanted into the designated pots in the field, with one seedling per pot. After transplanting, the sugarcane plants were cultivated for one year. Senescent leaves were collected and stored throughout the growth cycle. At harvest, the aerial part of each plant was separated into green leaves, senescent leaves, and the upper, middle, and lower portions of the stalks. All collected material was weighed, and subsamples were taken and dried in a forced-air oven at 65 °C for more than 72 h. To collect the roots, the pots were vertically sectioned using an electric saw. The roots were then separated from the soil by washing them with running water through a 1.0 mm mesh sieve.

Twenty days after transplanting, following the modifications already made to the pots, nitrogen treatments (45 kg N ha⁻¹) were applied in the form of ammonium sulfate, labeled with 2% excess ¹⁵N atoms (4.25 g per pot). The fertilizer was applied around the plant inside the pot and covered immediately after application.

2.2. Calculation of Nitrogen Derived from Fertilizers and Evaluation of Use Efficiency

All plant material was ground in a Wiley knife mill and subsequently ground to a fine powder using a roller mill [30]. The determination of N content was carried out using the semi-micro Kjeldahl method [31]. The ¹⁵N enrichment was determined with an automated continuous-flow isotope ratio mass spectrometer consisting of a Finnigan Delta V mass spectrometer coupled to the output of a Carlo Erba EA 1108 total C and N analyzer (Finnigan MAT, Bremen, Germany) in the ‘John Day Stable Isotope Laboratory’ at Embrapa Agrobiologia (Seropédica, RJ, Brazil). With the values of atom % ¹⁵N excess, the N derived from the fertilizer (% Ndff) was determined, which were calculated according to the procedure described by the International Atomic Energy Agency [32]:

$$\mathrm{N}\mathrm{d}\mathrm{d}\mathrm{f}={\displaystyle \frac{\mathrm{A}\mathrm{t}\mathrm{o}\mathrm{m}\mathrm{o} 15\mathrm{N} \mathrm{e}\mathrm{x}\mathrm{c}\mathrm{e}\mathrm{s}\mathrm{s} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{s}}{\mathrm{A}\mathrm{t}\mathrm{o}\mathrm{n} 15\mathrm{N} \mathrm{e}\mathrm{x}\mathrm{c}\mathrm{e}\mathrm{s}\mathrm{s} \mathrm{i}\mathrm{n} \mathrm{f}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{r}}}\times 100$$

With the value of % Ndff, the N content of the plant sample (%N), and the dry mass of the plants (MSP) in g per pot, the amounts of N derived from the fertilizer (QNDFF), in g per pot, were calculated by following equation:

$$\mathrm{Q}\mathrm{N}\mathrm{D}\mathrm{F}\mathrm{F}=\mathrm{M}\mathrm{S}\mathrm{P}\times {\displaystyle \frac{\mathrm{N}}{100}}\times {\displaystyle \frac{\mathrm{N}\mathrm{d}\mathrm{d}\mathrm{f}}{100}}$$

Thus, the efficiency of N fertilizer use by plants (NUE%) was calculated by the following equation:

$$N\mathrm{U}\mathrm{E}={\displaystyle \frac{\mathrm{Q}\mathrm{N}\mathrm{D}\mathrm{D}\mathrm{F}}{\mathrm{Q}\mathrm{u}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{o}\mathrm{f} \mathrm{N} \mathrm{a}\mathrm{p}\mathrm{p}\mathrm{l}\mathrm{i}\mathrm{e}\mathrm{d} \mathrm{p}\mathrm{e}\mathrm{r} \mathrm{p}\mathrm{o}\mathrm{t}}}\times 100$$

2.3. Statistical Analyses

Statistical analyses were performed using ASSISTAT software, Version 7.7, from the Department of Agricultural Engineering at the Federal University of Campina Grande (UFCG), located in Campina Grande, Paraíba, Brazil [33], to verify the normality and homogeneity of error variances using the Shapiro–Wilk and Bartlett tests. Since the varieties were planted at an eight-day interval, each variety was treated separately, with the following treatments: fertilizers (with or without fertilizer), inoculation (with or without inoculation). and plant parts. Data were subjected to analysis of variance and, when there was a significant effect of the treatments by the F test, the means were compared using the Tukey test (p < 0.05).

3. Results

3.1. Stem Production, N Accumulation, and Dry Matter

Neither inoculation with diazotrophic bacteria nor the addition of N fertilizer influenced the production of stem fresh mass, total plant dry mass and total N accumulation in the RB92579 and RB867515 varieties (

Table 1. Fresh mass of stems, dry matter, and total N accumulation in uninoculated and inoculated sugarcane plants; unfertilized and fertilized with ¹⁵N-labeled ammonium sulfate.

Treatments	Sugarcane Varieties
	RB92579	RB867515
Fresh weight of stems (kg pot−1)
Non-inoculated	10.4 a (±1.0)	10.5 a (±2.1)
Inoculated	10.7 a (±1.7)	9.5 a (±1.1)
No N fertilizer	10.9 a (±1.4)	10.6 a (±1.8)
+N fertilizer	10.2 a (±1.4)	9.4 a (±1.3)
F test
Inoculant (I)	0.3 ns	2.1 ns
Fertilizer (F)	1.6 ns	3.8 ns
Interaction I × F	0.1 ns	0.3 ns
CV (%)	12	14
Whole plant dry matter (kg pot−1)
Non-inoculated	6.2 a (±0.6)	5.7 a (±0.9)
Inoculated	6.2 a (±0.9)	5.5 a (±0.5)
No N fertilizer	6.1 a (±0.8)	5.8 a (±0.9)
+N fertilizer	6.3 a (±0.7)	5.5 a (±0.7)
F test
Inoculant (I)	0.0 ns	0.8 ns
Fertilizer (F)	0.2 ns	3.5 ns
Interaction I × F	0.0 ns	0.0 ns
CV (%)	15	9
Whole planta N accumulation (g N pot−1)
Non-inoculated	9.8 a (±2.2)	10.5 a (±2.8)
Inoculated	9.7 a (±1.1)	10.0 a (±0.8)
No N fertilizer	9.8 a (±1.6)	10.1 a (±2.5)
+N fertilizer	9.8 a (±1.8)	10.4 a (±1.4)
F test
Inoculant (I)	0.3 ns	0.2 ns
Fertilizer (F)	0.0 ns	0.8 ns
Interaction I × F	0.1 ns	0.2 ns
CV (%)	13	19

Means followed by the same letter in the column do not differ statistically by the Tukey test, at p<0.05. Note: ns: not significant; significant by the F test, at p < 0.05.

). The average value of total stem fresh mass was 10.5 and 10.0 kg pot⁻¹ for RB92579 and RB867515. Total plant dry matter values ranged from 6.2 to 5.6 kg pot⁻¹ and the total N accumulation in the plant was 9.8 and 10.3 g pot⁻¹ in the RB92579 and RB867515 varieties, respectively (Table 1).

In general, the treatments under study showed no consistent or stable significant influence on most variables associated with crop yield in any of the varieties, despite the fact that the soil used as a substrate was poor in available N. These results indicate that sugarcane is indeed a crop adapted to soils poor in available N.

As for the ¹⁵N enrichment, the inoculation of diazotrophic bacteria was influenced only in the RB92579 variety (

Table 2. ¹⁵N enrichment (atom % ¹⁵N excess) of different parts of two varieties of sugarcane fertilized with ¹⁵N-labeled ammonium sulfate inoculated, or not, with diazotrophic bacteria.

Treatments	Sugarcane Varieties
	RB92579	RB867515
15N enrichment of different parts of the sugarcane plant
Roots	0.081 a (±0.01)	0.079 a (±0.01)
Basal portion of the stalk	0.090 a (±0.01)	0.076 a (±0.01)
Middle portion of the stalk	0.090 a (±0.00)	0.072 a (±0.00)
Upper portion of the stalk	0.084 a (±0.01)	0.080 a (±0.01)
Flag leaves	0.084 a (±0.01)	0.077 a (±0.01)
Senescent leaves	0.083 a (±0.00)	0.075 a (±0.01)
F test—Inoculation (I)	10.2 *	1.1 ns
F test—Part of the plant (PP)	2.5 *	0.6 ns
F test—Interaction I × PP	0.6 ns	2.7 *
CVI %	13	6
CVpp %	8	11
Weighted mean whole plant
Non-inoculated	0.085 b (±0.00)	0.078 a (±0.01)
Inoculated	0.091 a (± 0.00)	0.076 a (±0.01)
CV %	5	3
Weighted mean shoot
Non-inoculated	0.080 b (±0.01)	0.078 a (±0.01)
Inoculated	0.090 a (±0.00)	0.073 a (±0.01)
CV %	5	5

Means followed by the same letter in the same column do not differ statistically by the Tukey test, at p < 0.05. Note: ns: not significant; *: significant by the F test, at p < 0.05.

). Applying ¹⁵N labeled fertilizer, there were no differences in the values of excess ¹⁵N in the different parts of the sugarcane plant in the varieties under study (Table 2).

On the other hand, regarding the effect of the interaction between inoculation and sugarcane plant parts in RB867515, there was a significant effect of inoculation on the ¹⁵N excess of the leaves (

Table 3. Effect of the interaction of inoculation and plant part for the RB867515 sugarcane variety.

Treatment	Varieties RB867515
	Non-Inoculated	Inoculated
15N enrichment of different parts (Atom % 15N excess)
Roots	0.076 aA (±0.01)	0.080 aA (±0.01)
Basal portion of the stalk	0.070 aA (±0.00)	0.082 aA (±0.01)
Middle portion of the stalk	0.070 aA (±0.00)	0.075 aA (±0.01)
Upper portion of the stalk	0.085 aA (±0.01)	0.075 aA (±0.01)
Flag leaves	0.085 aA (±0.01)	0.069 bA (±0.00)
Senescent leaves	0.077 aA (±0.00)	0.075 aA (±0.00)

Means followed by the same letter, lowercase in the same column and uppercase in rows, do not differ statistically by the Tukey test, at p < 0.05.

).

3.2. Efficiency of ¹⁵N Fertilizer Use Inoculated with Diazotrophic Bacteria

Regarding N derived from nitrogen fertilizer (Ndff), only in the RB92579 variety did inoculation favor the recovery of N fertilizer, with values ranging from 4.9 and 5.5% for the non-inoculated and inoculated treatments, respectively (

Table 4. Values of N derived from fertilizer (Ndff), amount of N derived from fertilizer (QNDFF), and efficiency of use of ¹⁵N fertilizer (NUE) in sugarcane varieties inoculated, or not, with diazotrophic bacteria.

Treatments	Sugarcane Varieties
	RB92579			RB867515
	Non- Inoculated	Inoculated	CV (%)	Non- Inoculated	Inoculated	CV (%)
Values
Ndff (%)	4.9 b (±0.30)	5.5 a (±0.37)	13	4.7 a (±0.34)	4.6 a (±0.44)	6
QNDFF *	0.47 a (±0.07)	0.50 a (±0.05)	13	0.48 a (±0.07)	0.48 a (±0.05)	16
NUE (%)	59 a (±0.09)	61 a (±0.06)	13	60 a (±0.09)	59 a (±0.06)	16

Means followed by the same letter in the line do not differ statistically by the Tukey test, at p < 0.05. * (g N pot⁻¹).

).

It was observed that the values of QNDFF had no influence of inoculation in any of the varieties, with average values of 0.47 and 0.50 g N pot⁻¹ for all treatments, and with an efficiency of use of ¹⁵N fertilizer of 60%, regardless of the varieties studied (Table 4).

4. Discussion

4.1. Plant Growth, Nitrogen Concentration, and Dry Biomass Accumulation

The response of sugarcane varieties in terms of dry mass productivity and nitrogen (N) accumulation can vary significantly. Each variety has specific genetic characteristics that influence nutrient absorption efficiency, leading to differences in yield [34,35]. Since N is an essential nutrient for plant growth and development, its deficiency negatively affects stalk and dry mass productivity [36,37]. The variety RB92579 exhibited a high capacity for N absorption and accumulation [30]. The results obtained for this variety confirm the existence of significant differences among sugarcane genotypes regarding N absorption and utilization capacity in response to inoculation [7,31].

However, the response of sugarcane varieties to inoculation with diazotrophic bacteria may present inconsistencies when compared to previous studies [1,21,34,38]. These variations are primarily attributed to differences in soil and cultivation conditions. In the present study, the fact that the experiment was conducted in pots in the field may have directly influenced the results, suggesting that controlled experimental conditions can impact the expression of N use efficiency.

Therefore, the primary objective of this study was to evaluate N use efficiency, with a focus on the ¹⁵N enrichment technique, which enables a more precise analysis of available N utilization [39]. Although values for N accumulation and growth were obtained, these results reflect the specific conditions of the study and may differ from those observed in long-term field trials. Comparisons with such studies may lead to discrepancies, as environmental variables and agricultural practices can vary significantly, influencing N accumulation and plant growth. However, it is important to highlight that conducting ¹⁵N studies under field conditions—without the use of pots and over multiple years—would be extremely costly and financially unfeasible, making large-scale research in this area particularly challenging.

The ¹⁵N enrichment observed in the flag leaves of the RB867515 variety in response to inoculation suggests that diazotrophic bacteria may have positively influenced N absorption, translocation, and utilization, potentially enhancing the plant’s access to additional forms of N and optimizing fertilizer use [8]. Furthermore, inoculation may have affected the interaction between nitrogen and other essential nutrients, such as phosphorus, potassium, and sulfur, which play complementary roles in plant metabolism [40]. These effects contributed to the greater accumulation of ¹⁵N in the flag leaves, which are crucial for vegetative growth, as the increase in N rate consistently increases plant biomass [41]. If the inoculant promotes organic matter decomposition or increases microbial activity in the soil, the plant could have greater N availability, which would also explain the increase in isotopic enrichment. This finding suggests that the plant was able to access and utilize soil-derived N more efficiently [39].

The increase in nutritional efficiency resulting from inoculation likely led to greater N absorption and utilization, as reflected in the higher ¹⁵N content in the flag leaves. These results indicate improved N use efficiency, whether from fertilizer or soil sources, with the potential to enhance the growth and productivity of the RB867515 sugarcane variety. Thus, the findings of this study are crucial for understanding how the interaction between inoculation and nitrogen use efficiency can impact productivity, particularly under controlled growing conditions.

4.2. Impact of ¹⁵N Fertilizer on Nitrogen Use Efficiency and Contribution to Sugarcane

The increase in Ndff in response to inoculation in the RB92579 variety can be explained by mechanisms that enhance the efficiency of nitrogen fertilizer use. Inoculation with diazotrophic bacteria, for example, contributes to improving the root structure of sugarcane, promoting better nutrient absorption [16,42]. These bacteria aid in the expansion of the root system, allowing the plant to explore a larger area of soil and absorb nutrients more efficiently [16,20].

Additionally, diazotrophic bacteria increase the efficiency of nitrogen fertilizer use by providing an additional source of biologically fixed N₂, allowing the plant to more effectively utilize both Ndff and N from biological nitrogen fixation (BNF). As a result, inoculation stimulates plant growth, which increases the demand for nutrients, especially nitrogen. This increased demand is likely directly related to the greater absorption capacity provided by a more vigorous root system.

A more vigorous root system, combined with an increase in the number of leaves and stems, enhances Ndff absorption, as the plant becomes more efficient at capturing nutrients [43]. Thus, inoculation not only improves the use of the applied fertilizer but also optimizes the use of available N in the soil. However, to better understand this dynamic, it is essential to evaluate nitrogen use efficiency (NUE), a critical indicator of the plant’s ability to convert absorbed nutrients into biomass.

The NUE ranged from 59 to 61% and 60 to 59% in the non-inoculated and inoculated treatments for RB92579 and RB867515, respectively (Table 4). The high NUE values are due to the efficiency being dose-dependent, possibly because the plants in this study grew in a limited soil volume, where the soil was better explored by the sugarcane roots. This result highlights the importance of experimental conditions when interpreting the response of varieties to nitrogen.

In sugarcane, studies evaluating the efficiency of N recovery from fertilizer have generally recorded low levels, typically ranging from 10 to 40% [14]. According to Joris et al. [44], N recovery from fertilizer can reach values of around 45%, and in soils fertilized with N in previous cycles, N recovery does not exceed 30%. In this study, NUE was not influenced by inoculation with diazotrophic bacteria in either variety (Table 4). Pereira et al. [9] studied the influence of inoculation with diazotrophic bacteria on NUE in sugarcane plants (first and second ratoon) and also found no effect of inoculation in the RB92579 variety. These findings suggest that the response to inoculation may depend on multiple factors, including plant growth stage and environmental conditions.

In some situations, a varietal effect on NUE is observed at certain stages of plant growth. Kölln et al. [35] found that, under controlled conditions during the initial development phase, RB867515 responded to inoculation but was not efficient in recovering N fertilizer. These results underscore the importance of considering genetic variability and environmental interactions when interpreting the effects of inoculation.

The search for greater efficiency in nitrogen fertilizer use is highly relevant. In general, the efficiency of nitrogen fertilizer use in the sugarcane crop evaluated was not influenced by diazotrophic bacteria. Although variations in responses and interactions between bacteria and sugarcane varieties were observed, several groups of bacteria showed a significant contribution when inoculated, either by their ability to fix nitrogen from the air or by improving nitrogen recovery efficiency due to their strong plant growth-promoting effects. Therefore, understanding the mechanisms of interaction between sugarcane varieties and diazotrophic bacteria remains essential for optimizing nitrogen use efficiency and reducing dependence on synthetic fertilizers [16,21,38,42,45].

5. Conclusions

The efficiency of N fertilizer recovery by sugarcane plants in the studied varieties was 60% for the conditions evaluated. When a plant receives ¹⁵N fertilizer as an additional nitrogen source, the enrichment with ¹⁵N tends to be uniform. This suggests that, in studies of ¹⁵N fertilizer recovery in sugarcane, sampling can be performed in different parts of the plant. However, in the experimental conditions of this study, inoculation positively influenced the enrichment with ¹⁵N only in the entire plant and in the flag leaf of the RB92579 variety, with no significant effect on the other parts of the plants evaluated in the two studied varieties.

Although the results show that inoculation does not have an effect on all parts of the plant, future studies may improve understanding of the relationship between ¹⁵N enrichment and plant growth, promoting better use of fertilizers and, consequently, reducing possible losses in agricultural production.