1. Introduction
Nitrogen (N) is an ecological factor that determines plant productivity in natural and agricultural ecosystems. In the second half of the 20th century, the widespread use of N as a fertilizer was an important component of the green revolution, along with the use of improved varieties and advances in irrigation techniques and crop protection [
1]. Furthermore, the costs of using industrially synthesized N in agriculture have been increasing, mainly because the energy consumption in its manufacture remains high [
2]. In addition, the use of N fertilizers tends to have low N-use efficiency. On a global scale, it is estimated that no more than 50% of N applied to crops is recovered in harvests, with the remainder lost to the environment [
3]. N loss from agricultural fields in its inorganic form has a major impact on water contamination, contributing to its eutrophication with harmful algal blooms [
4,
5]. Significant amounts of N can also be lost from the soil through denitrification, contaminating the atmosphere with important greenhouse gases such as N oxides [
5,
6].
Organic amendments, in turn, were, for centuries, the main way to maintain soil fertility. In addition to supplying N and other nutrients after their mineralization by soil microorganisms, organic amendments have the potential to enhance overall plant growth conditions by promoting aeration in clay soils, water holding capacity in sandy soils, and, in general, soil biological activity, which is the driving force behind nutrient cycling [
7,
8]. In recent decades, the specialization of agricultural activity and the mechanization of farming practices have greatly reduced the availability of animal manure in vast regions of the world [
5,
9]. In addition, waste from agro-industrial activities, municipal solid waste, and other organic materials, although they have been valued as organic amendments [
10,
11], are available for agriculture also in limited quantities, which leads farmers to find other ways to fertilize their crops.
Biological N fixation has the potential to enhance soil fertility and decrease the requirement for external inputs. Organisms capable of fixing N from the atmosphere, collectively called diazotrophs, are those possessing a nitrogenase complex, which allow them to separate the two N atoms of the dinitrogen molecule (N
2) with the formation of ammonia [
12,
13]. N-fixing organisms can live in terrestrial or aquatic ecosystems, be aerobic or anaerobic, heterotrophic, or photosynthetic, and can be found free-living or in association with higher plants, sometimes forming a highly specific symbiotic relationship with the host plant [
12,
13].
Regarding biological N fixation, symbiotic associations play a major role in N fixation in agricultural fields, accounting for about 50% of total biological fixation in terrestrial ecosystems [
3]. Nodulated legumes, in association with bacteria belonging to the Rhizobiaceae family, may have access to atmospheric N in sufficient quantities so that they do not need to receive N fertilizers to reach high productivity levels. In addition, N fixed by legumes can be available for other non-legume crops when integrated in intercropping [
9,
14,
15] or in rotation with legumes [
16,
17]. The symbiotic relationship between the aquatic fern of the genus
Azolla and the cyanobacterium
Anabaena azollae is also a system with a high capacity for N fixation, with the fern commonly used as green manure in rice (
Oryza sativa L.) cultivation [
18,
19,
20]. High N fixation in agricultural fields can also be achieved from the relationship between some tropical grasses and N-fixing microorganisms, which may or may not be endophytic associations; perhaps the most striking example is the relationship between sugarcane (
Saccharum officinarum L.) and some N fixers, such as
Gluconoacetobacter diazotrophicus and
Azospirillum brasilense, which are able to satisfy almost all sugarcane N requirements [
18,
21,
22].
Even so, in agricultural fields, access to relevant amounts of atmospheric N is restricted to nodulated legumes and few other species, as mentioned above. Many important crops, such as lettuce (
Lactuca sativa L.), do not have access to biologically fixed N. Recently, a commercial plant biostimulant (BlueN
®, SYMBORG BUSINESS DEVELOPMENT S.L.U., Murcia, Spain), containing a N-fixing microorganism (
Methylobacterium symbioticum) that promises to be able to thrive in the phyllosphere of most plants, was launched on the market. The bacterium
M. symbioticum sp. Nov. (strain SB0023/T) was isolated from
Glomus iranicum var.
tenuihypharum spores [
23]. Species of the genus
Methylobacterium are ubiquitous in nature and can be present in a wide range of environments, including soil, air, water, and plants [
24,
25,
26]. Other bacteria of the genus
Methylobacterium have been recognized as capable of fixing N in interactions with plants.
M. nodulans and
M. radiotolerans, for instance, are reported to be N-fixing bacteria, but by forming nodules on legume roots [
27]. Regarding
M. symbioticum, a recent study showed that the application of the inoculant resulted in decreases of 50% and 25% in the amount of N that would be required for maize and strawberry, respectively, accompanied by an increase in production compared to treatments of equivalent rates of nitrate-N applied, but without the application of the inoculant [
28].
In fact, the possibility of a microorganism having the ability to fix N living in the phyllosphere of most plants, without an apparent specific relationship with the host plant, and being able to be applied as a foliar spray, gives it an unlimited potential for use in agriculture. Thus, it is of extraordinary importance that more studies emerge to evaluate the fixation capacity of this inoculant across a wide range of crops and cultivation conditions. The objective of this study was to evaluate the capacity of N fixation by M. symbioticum, commercial inoculant BlueN®, when applied to lettuce, measuring DMY, and estimating the apparent N recovery and apparent N fixation from four lettuce growing cycles. The hypotheses raised were about whether the bacterium can supply N to the crop in relevant amounts during its cropping cycle and whether the amount of fixed N depends on the nutritional status of lettuce, for which the commercial product was applied to plants subjected to different rates of mineral N-fertilizer.
2. Materials and Methods
2.1. Establishment of Pot Experiments
Four independent pot experiments were conducted during the 2021 and 2022 spring/summer seasons in Bragança, northeastern Portugal. The region benefits from a Mediterranean climate, concentrating its precipitation in winter (October to March) and presenting hot and dry summer months (June to September). The average air temperature is 12.3 °C, and the annual precipitation is 758.3 mm [
29]. The four growing seasons began and ended in June–July 2021, August–October 2021, May–June 2022, and August–October 2022, respectively.
The experiment was arranged as a factorial, with the application of plant biostimulant (Yes and No) and four N rates (0, 25, 50, and 100 kg ha
−1 of N) and four replicates (4 pots) of each treatment. N was split into two applications, half the rate at planting and the other half at phenological stage 43, with “30% of expected head size reached” [
30]. Each pot received the fertilization corresponding to a single plant, assuming a typical commercial-lettuce planting density of 140,000 plants ha
−1. Thus, each pot received 0, 0.179, 0.357, or 0.714 g N, which corresponds to the field applications of 0, 25, 50, or 100 kg ha
−1 of N. Ammonium nitrate 27% N (50% NH
4+, 50% NO
3–) was the fertilizer used. The pots also received phosphorus (P) and potassium (K) applied at planting, at rates corresponding to 50 kg ha
−1 of P
2O
5 (as superphosphate, 18% P
2O
5) and K
2O (as potassium chloride, 60% K
2O). The plant biostimulant was applied at the time of N side-dressing application. The commercial product (Blue N
®) contains 3 × 10
7 colony-forming units (CFUs g
−1) of
M. symbioticum. The leaf spray was prepared at the concentration recommended by the manufacturer, 333 g ha
−1, diluted in 80 to L of water. Once again, it was considered that 1 ha represents 140,000 lettuces, with each lettuce receiving a fraction corresponding to the individual dose (2.37 mg, 1.5 mL water). The foliar spray was applied with a small household sprayer used to care for indoor plants, wetting the adaxial and abaxial sides of the leaves.
The pots (0.160 m mean diameter, 0.135 m height) were filled with 3 kg of dried (40 °C) soil, sieved through a 2 mm mesh, and obtained from the 0–0.20 m layer of a plot that had been fallow for a year. The soil was a Regosol [
31] of colluvial origin, with a sandy clay loam texture (soil separates: 242, 217, and 541 g kg
−1 clay, silt, and sand, respectively). It contained 11.7 g kg
−1 of organic carbon (C) (Walkley–Black), had a pH
(H2O) of 6.8, and P and K levels (Egnér–Riehm) of 85.7 mg kg
−1 (P
2O
5) and 94.0 mg kg
−1 (K
2O), respectively. The cation exchange capacity (ammonium acetate) was 17.9 cmol
+ kg
−1. The pots, both treated and untreated with the inoculant, were kept at 50 m from each other to prevent any potential cross-contamination.
2.2. Crop Management
The seedlings of lettuce (cv. Summer Wonder) were prepared in propagation trays with a square top measuring 0.025 × 0.025 m
2 and 0.04 m deep. A suitable commercial substrate for seed germination (Siro Agro 1
®) (Leal e Soares, S.A., Mira, Portugal) was used. It was prepared with pine bark, blond peat (
Sphagnum sp.), coconut peat, and NPK fertilizer (13:40:13) (1 kg m
3). The substrate had a granulometry of 0–8 mm, pH (CaCl
2) 5.0–6.0, and a conductivity of 150 to 200 µs cm
−1. The seedlings were transplanted approximately three weeks after sowing, at phenological stage 13, with the 3
rd true leaf unfolded [
30].
After planting the lettuces, the pots were surrounded with a wooden plank structure to prevent solar radiation from falling directly on the sides of the pots and excessively increasing the temperature at the level of the rhizosphere. The spatial arrangement of the pots was regularly changed to ensure they received uniform radiation exposure.
During the growing season, the lettuces were watered as many times as necessary. Considering that the treatments resulted in different lettuce growth, leading to different amounts of water transpired, and that some environmental variables, namely temperature and radiation, also lead to different water consumption, the amounts of water applied to each pot were managed daily by observing the apparent hydration level of the soil and the lettuces. After planting, the emergence of weeds was also monitored, which were promptly eliminated in the cotyledonary phase.
2.3. Crop Harvest, Sampling, and Laboratory Analysis
The plants were harvested by cutting them close to the ground. Some dirt on basal leaves was removed by gently washing them with water. Then, lettuces were dried in an oven at 70 °C until a constant weight was reached and ground (1 mm mesh) for laboratory analysis.
Lettuce tissues were subjected to elemental chemical analysis and a determination of nitrate concentration. Elemental tissue analyses were performed by Kjeldahl (N), colorimetry (B and P), flame emission spectrometry (K), and atomic absorption spectrophotometry (Ca, Mg, Cu, Fe, Zn, and Mn) methods after the nitric digestion of the samples [
32]. Nitrate concentrations in lettuce tissues were determined according to Baird et al. [
33] by UV-vis spectrophotometry in a water extract (dry matter/water, 1:50
m/
v).
Initial soil samples were analysed for pH (soil: solution, 1:2.5), cation-exchange capacity (ammonium acetate, pH 7.0), organic C (wet digestion, Walkley–Black method) and extractable P and K (Egner–Riehm method, ammonium lactate extract). Soil separates (clay, silt, and sand) were determined by the Robinson pipette method. These analytical procedures are described in detail in Van Reeuwijk [
34].
2.4. Data Analysis
Statistical analysis was performed using SPSS Statistics software (version 25, IBM SPSS, Armonk, NY, USA). The data were firstly tested for normality and homogeneity of variances using the Shapiro–Wilk test and Bartlett’s test, respectively. After ANOVA examination, the means of the N treatments with significant differences (α < 0.05) were separated by a Tukey HSD test (α = 0.05).
Apparent N recovery (ANR) was used as an index of the N-use efficiency of the soil-applied N. The ANR was estimated according to the following equation: ANR (%) = 100 × [(N recovered in the fertilized treatments—N recovered in the control treatment)/N applied as a fertilizer].
Apparent N fixation (ANF) was used as an index of the effectiveness of N fixation by the microorganism. The ANF was determined by the difference between N recovered by plants that were and were not treated with the N-fixing microorganism separately for each rate of N applied to the soil: ANF = N recovered in treated plants—N recovered in untreated plants.
5. Conclusions
In a study where a strong response to applied N to the soil occurred, as typically observed in N fertilization trials, and where the response to applied N reduced N-use efficiency, the plant’s response to the application of M. symbioticum to the shoots was weak for any of the N rates applied to the soil, but particularly for N0 and N25 treatments, even considering that four lettuce growing cycles were conducted. In biological systems, high N fixation typically occurs when there is a high degree of specificity between the microorganism and the host plant. Therefore, achieving a high capacity for fixation across a wide range of plant species is unlikely.
Certainly, the inoculant has the potential to be used in agriculture, providing benefits to farmers, but likely not across all crops. It seems necessary to establish a much better understanding of more specific conditions under which the benefits can be quantitatively more effective, reducing the risk of failure in agricultural fields. This is because it is an input factor with associated acquisition and application costs. Moreover, if the technology fails, it can lead to a loss of crop productivity due to N deficiency, reducing farmers’ profits.
As far as we know, these are the first independent results of the use of this inoculant. It is important that more studies emerge in the coming years for a more accurate assessment of the potential use of this commercial product.