Australian Dryland Wheat Growth and Yield Are Positively Impacted by a Methylobacterium symbioticum Biostimulant Under Reduced Nitrogen Supply

Abstract

Enhancing nitrogen use efficiency (NUE) in cereal crops is a major challenge for dryland systems that rely heavily on synthetic nitrogen (N) inputs. Microbial biostimulants have recently emerged as promising alternatives for cost-effective N inputs in wheat through foliar colonization and endophytic biological N fixation. Methylobacterium symbioticum strain SB23 (also known as BlueN or Utrisha N) is a pink-pigmented, obligately aerobic, Gram-negative, facultative methylotrophic bacterium demonstrated to potentially reduce N chemical fertilization and improve yields in various crops. A field trial consisting of large replicated 2.3 ha plots of Australian Prime Hard (APH) wheat cv. Rockstar was established in south central New South Wales, Australia, to evaluate the foliar application of M. symbioticum strain SB23 under both standard and reduced N regimes for winter wheat maturing in late spring. Application of the SB23 biostimulant significantly increased wheat leaf chlorophyll concentration at 30 and 60 days after application (DAA) and promoted biomass accumulation at 60, 90 and 120 DAA in contrast to the untreated control, with the strongest positive response under reduced N input. Specifically, the 75% N + biostimulant treatment improved biomass by up to 23% and grain yield by 14% relative to the reduced-N control, demonstrating potential supplemental fertility without yield loss. Correlation analyses revealed that mid-season chlorophyll was strongly associated with biomass and carbon assimilation (r = 0.87 and 0.84, respectively), while biomass at 60 DAA was highly correlated with grain spike weight (r = 0.81), suggesting a strong association of improved crop vigor and yield with inoculation. At harvest, SB23 enhanced biomass nitrogen accumulation and nitrogen use efficiency, with the 75%N + biostimulant treatment achieving the highest plant N uptake (25% above the reduced-N control) and the greatest partial factor productivity of nitrogen (51.8 kg grain kg⁻¹ N applied), while both 100%N treatments showed the lowest efficiency. Collectively, these findings suggest that Methylobacterium symbioticum SB23 improves NUE through enhanced crop performance thereby providing a supplementary N source and delivering a cost–benefit advantage of approximately A$170 ha⁻¹ under reduced N application.

1. Introduction

Global food demand is projected to increase substantially over the coming decades as the world population continues to grow, requiring intensified crop production on existing farmland [1,2]. Australia currently plays a key role in maintaining global food security through export of agricultural products, particularly cereal crops. Wheat production makes up around 14% of Australia’s global agricultural trade, serving as an important supplier to wheat-dependent countries [3,4]. The 2030 Road Map put forth by the Australian National Farmers’ Federation seeks to enhance the Australian agricultural economy by targeting an increased output worth over AUD 100 B by 2030 [5].

Intensified crop production typically relies heavily on synthetic fertilizers to enhance crop yields and secure the global food supply. In Australian grain production, nitrogen inputs have increased substantially in recent decades, with urea (68%) and ammoniated phosphates (12%) widely applied across rainfed cropping systems [6,7]. Despite these inputs, nitrogen use efficiency (NUE) in cereal production remains relatively low, typically averaging 40–44% [8]. Dependence on synthetic N fertilizers has increased to account for nearly 30% of total input expenses [9], and even then, high-yielding cropping seasons can still lead to negative N balances [10]. Additionally, overuse of N additives can promote environmental degradation in the form of leaching, greenhouse gas emissions, and soil degradation [11,12].

Amid these challenges, microbial biostimulants have recently emerged as promising options due to their ease of application, eco-friendly nature and potential to improve crop yields while reducing N application rates. These biostimulants can modulate plant phytohormones—including auxins, cytokinins, gibberellins, abscisic acid, and ethylene—which regulate plant development and stress signaling pathways [13,14]. They also enhance photosynthetic capacity by increasing pigment concentration, improving carbon and nitrogen metabolism, and enhancing chlorophyll content and light-use efficiency [15,16]. Furthermore, microbial biostimulants stimulate the production of secondary metabolites such as phenolics, flavonoids, terpenes, carotenoids, and saponins, which contribute to improved nutrient uptake, stress tolerance, plant defense, and ultimately higher grain yield and crop productivity [13,17]. Notably, plant growth-promoting rhizobacteria and phyllobacteria enhance N acquisition in plants through biological N fixation (BNF) with either foliar, soil, or seed-based applications [18,19]. In addition, endophytic and epiphytic associations with N-fixing bacteria in the phyllosphere can result in improved N availability in planta, thus enhancing crop productivity [20,21].

Biostimulants containing free-living or associative nitrogen fixers offer a supplementary N source in broadacre cropping systems. Genera such as Azospirillum, Azotobacter, Bacillus, Clostridium, and Rhodospirillum contribute substantially to the global N cycle, fixing 30–50% of total N in non-leguminous crops [22,23]. Azospirillum species, notably A. brasilense and A. diazotrophicus, have enhanced NUE and reduced N fertilizer use by up to 25% in maize and wheat, with yield increases of 18% and increased grain N content of 11.8% under semi-arid conditions [24,25]. Similarly, Azotobacter species have been reported to increase wheat yield by up to 60%, enabling a reduction of 40 kg N ha⁻¹ in urea application under a variety of environmental conditions [26,27].

An emerging group of special interest is the methylotrophic bacteria, particularly the genus Methylobacterium, which are increasingly recognized for enhancing NUE in cereal crops [28,29,30]. Metagenomic studies indicate that certain Methylobacterium strains promote crop growth under N-deficient conditions [31,32], occur in diverse environments [33], and associate with more than 70 plant species [34], although only about 30% are capable of fixing atmospheric N in the phyllosphere [20]. These bacteria can enhance photosynthesis by delaying leaf senescence and prolonging photosynthetic activity, as evidenced by increased stomatal conductance and PSII efficiency in wheat under reduced nitrogen fertilization [30]. These bacteria also promote chlorophyll retention, improving photosynthetic capacity in maize and peach trees, which supports higher biomass and grain productivity even with reduced nitrogen inputs [29,35,36]. Hormonal regulation is influenced by the bacterium’s production of cytokinins, especially trans-Zeatin, which stimulates cell division, nutrient allocation, and stress tolerance; this hormonal modulation also reduces ethylene levels via ACC-deaminase activity, contributing to delayed senescence and improved nitrogen metabolism [30,37,38]. Secondary metabolites produced by Methylobacterium, including amino acids, carotenoids, and antimicrobial compounds, enhance plant metabolism and stress resilience under adverse conditions [39,40].

Methylobacterium symbioticum strain SB23, a recently identified species, has gained attention for its ability to reduce synthetic N requirements by up to 50% in rice, maize, and wine grapes without compromising yield across various climates [41]. In Portugal, M. symbioticum SB23 reduced synthetic N use by 40% in orange trees and improved tomato yields with lower N inputs [42,43]. Similar benefits have been observed in maize and strawberries in Spain, maize in Mexico, and apples under limited water availability in Germany [29,44,45].

However, despite this potential, the broader adoption of microbial biostimulants, particularly in broadacre cereal crops, remains limited. Following their recent introduction into some cropping systems, challenges have arisen that include regulatory complexity for registration, difficulties associated with product formulation, inconsistent field performance, cost, and a prevailing reliance on synthetic N fertilizers [46,47,48]. Currently, only a small number of promising strains ever reach commercialization; however, their performance has been noted to vary substantially between lab, greenhouse, and field conditions—further complicated by differences in cultivar, soil type, and environmental factors [49,50].

Recent field-based results with M. symbioticum SB23 in agronomic crops have also been mixed. While enhanced nutrient uptake and growth and development were noted in crops such as rice [51], oregano and lettuce [52,53], and nut trees [54], the impacts on yield and N uptake in many crops have often been variable. For example, M. symbioticum improved N concentration in planta but had moderate effects on overall growth in turfgrass [55], while hydroponic tomato studies demonstrated improved root development and seed vigor without measurable improvement in nitrogen uptake or yield [56]. In cultivated olive (Olea europaea L.), it enhanced N recovery without significantly affecting yield or N content [57]. In wheat, a small-scale field trial in Italy demonstrated enhanced N metabolism and photosynthetic activity but no significant growth or yield increase [30], suggesting the potential of M. symbioticum SB23 to support factors other than those involved in direct yield enhancement.

Most reported studies to date have been conducted under semi-favorable to favorable conditions in Europe, South America, or North America, with limited evaluation in water-limited, low-input environments like Australian drylands [42,43,44,58]. In Australia, the deployment of Methylobacterium-based products such as Utrisha N (Corteva Agriscience, Wagga Wagga, NSW, Australia) and N-LEAF (De Sangosse, Alexandria, NSW, Australia) has recently commenced. Such foliar formulations, compatible with common agrochemicals, are claimed to contribute up to 30 kg N ha⁻¹ through continuous atmospheric N fixation [59,60]. However, their efficacy in Australian dryland wheat systems remains largely untested. The unique phyllosphere environment of wheat, coupled with climatic, soil, and agronomic variability, necessitates robust field-scale trials to assess the true potential of these biostimulants [61,62,63].

Building on recent advances in microbial biostimulant research, this study evaluates the agronomic, physiological, nitrogen use efficiency, and economic impacts of a Methylobacterium symbioticum SB23-based foliar biostimulant (Utrisha N; Corteva Agriscience, Wagga Wagga, NSW, Australia) under large-scale dryland wheat production in Australia. From a practical standpoint, Australian growers face increasing pressure to reduce nitrogen inputs due to rising fertilizer costs, low nitrogen use efficiency, and heightened climatic risk, while maintaining yield and profitability. This research addresses a critical knowledge gap by assessing whether M. symbioticum SB23 can improve crop performance and nitrogen productivity under reduced nitrogen supply without compromising yield. Conducted in a replicated, commercial-scale paddock experiment at Englefield Plains (Viridis Ag Pty Ltd., Junee Reefs, NSW, Australia), this study specifically examines the effects of foliar SB23 application on (i) wheat physiological responses following application, (ii) biomass accumulation and yield components, (iii) nitrogen partitioning and nitrogen use efficiency indices, and (iv) on-farm economic returns, thereby providing field-relevant evidence to inform nitrogen management decisions in Australian dryland wheat systems.

2. Materials and Methods

2.1. Experimental Site and Design

In 2024, a field experiment was conducted at a commercial broadacre farm producing dryland wheat (Viridis Ag Pty Ltd., Albury, New South Wales (NSW), Australia.) on Englefield Plains, Junee, NSW, Australia (34°42′4.2″ S, 147°46′26.9″ E; 297 m above sea level). The geographic location of the experimental site and the spatial arrangement of treatment plots within the paddock are shown in Figure 1. The trial area covered 24 ha and was previously cropped with hybrid canola. The site featured a red Kandosol soil with a pH ranging from 4.8 to 5.5, 1.2% organic matter, and a cation exchange capacity of 10.1 cmol(+) kg⁻¹. Soil nutrient analysis of the 0–40 cm profile showed that root-zone mineral nitrogen (NO₃⁻–N + NH₄⁺–N) was 63.8 kg N ha⁻¹, while Colwell phosphorus, potassium, and sulfur concentrations were 47.1, 413.6, and 11.9 mg kg⁻¹, respectively. The site receives a long-term mean annual rainfall of approximately 530 mm, typical of dryland cropping systems in southern NSW, Australia. During the 2024 wheat growing season (April–December), total rainfall was 336 mm. Meteorological data including rainfall and temperature were obtained from the nearest station (ID: 073019, Junee Treatment Works, Junee, NSW, Australia) of the Australian Bureau of Meteorology and are reported in Figure 2 and Figure 3.

The experiment was conducted using a randomized complete block design (RCBD) with five replications, arranged as a two-factor factorial. The factors included N rate, applied either at 100% of the locally recommended rate of N for a high-yield goal or at 75% of the recommended high-yield rate (equivalent to the optimum-yield goal), and foliar biostimulant application (with (MB+) or without (MB−)). This factorial arrangement resulted in four treatment combinations: 100%N-MB: 100% recommended N without biostimulant; 100%N+MB: 100% recommended N with biostimulant; 75%N-MB: 75% recommended N without biostimulant; and 75%N+MB: 75% recommended N with biostimulant. Each block contained all four treatments; treatments were randomly allocated within blocks to reduce the influence of spatial variability. Each treatment plot measured 72 m × 36 m and was bordered by buffer plots planted with the corresponding wheat variety. To avoid microbial cross-contamination, a 36 m buffer zone separated adjacent treatment plots, and a 72 m space between replications. Each plot represented a single experimental unit and constituted one biological replicate (n = 5 per treatment). Subsampling within plots was conducted for physiological, biomass, and nitrogen measurements and represents technical replication. This design enabled the evaluation of the main effects of nitrogen rate and biostimulant application, as well as their interaction, on the measured plant and soil response variables.

2.2. Crop Establishment and Input Management

Australian Prime Hard (APH) wheat (Triticum aestivum L.) cv. Rockstar, a widely grown mid–slow-maturing cultivar in southern New South Wales with high yield potential, resistance to key foliar and stem diseases, and stable performance under commercial N management and foliar biostimulant application, was selected as a suitable model for field-scale evaluation under dryland conditions. The crop was sown using an Ausplow Deep Blade System (DBS) tyne seeder at 30 cm row spacing and a seeding rate of 70 kg ha⁻¹, with 60 kg ha⁻¹ monoammonium phosphate (MAP) applied at sowing. All plots received a uniform basal application of 23 kg N ha⁻¹ at sowing, supplied as urea (46% N). N treatments were defined exclusively on a N basis. The full N rate (100% N) was 115 kg N ha⁻¹, while the reduced rate (75% N) was 86.3 kg N ha⁻¹. For the 100% N treatment, the remaining N was applied in two equal top-dressings of 46 kg N ha⁻¹ at 21 and 51 days after sowing (DAS). In contrast, for the 75% N treatment, the final top-dressing was reduced to 17.3 kg N ha⁻¹ to achieve the target total N rate. The microbial biostimulant M. symbioticum SB0023/3T or SB23 (Utrisha N™, Corteva Agriscience), formulated with wettable powder (with a viable cell count of 3 × 10⁷ CFU/g), was applied to the relevant treatment plots as a foliar spray at a rate of 333 g/ha using 3 bar pressure during the maximum tillering stage (Zadoks 29) at 51 DAS.

2.3. Crop Protection Management, Environmental Conditions, and Harvest

A detailed list of all commercial crop protection products used in the field experiment, including active ingredients and formulation types, is provided in the Supplementary Mterials section (Table S1). Weeds were managed using Sakura (118 g/ha) and Triallate Gold (1.6 L/ha) as pre-emergent herbicides on 7 May, followed by Paradigm (25 g/ha) and MCPA LVE 570 (650 mL/ha) as post-emergents at 41 DAS. Pest and disease control involved applications of Soprano 500 (65 mL/ha, before 22 days of sowing), Radial Opti (165 mL/ha,91 DAS), and a combination of AC Mightyzole 420 (115 mL/ha) and Prothio 420 (27 mL/ha,115 DAS). The crop experienced moderate frost stress during the early vegetative phase, occurring primarily in late June to early July, as assessed using the visual scoring criteria outlined in the GRDC Frost Identification Guide for Cereals [64]. Frost severity was classified as moderate, based on field observations of limited tissue damage across the site. Although these frost events may have constrained the overall yield potential for the season, their spatially uniform occurrence across the experimental area ensured that relative treatment comparisons among nitrogen rate and foliar biostimulant treatments were not confounded. Harvest was performed on 10 December 2024 via direct heading using a John Deere automated S-7 series header.

2.4. Bacterial Colonization Verification

To verify the viability and identity of the applied microbial inoculant, M. symbioticum SB23 product in the form of Utrisha N powder was obtained from Corteva Agriscience Australia Pty Ltd., (Chatswood, NSW, Australia). Serial dilutions of the Utrisha N powder were plated on both nutrient agar and a nitrogen-free selective microbial medium (Minimum Salt Methanol Medium, MMM-nN) following the composition described by Pascul [41], in brief: methanol, 20.0 mL; NaNO₃, 0.5 g; MgSO₄·7H₂O, 0.5 g; K₂HPO₄, 1.0 g; FeSO₄·7H₂O, 0.01 g; CaCl₂·2H₂O, 0.01 g; KCl, 0.5 g; vitamin solution, 1.0 mL; micronutrient solution, 2.0 mL; Agar, 12 g in 1000 mL of distilled water; pH 7.20. Colony-forming units (CFUs) were counted, and individual colonies were isolated for morpho-molecular confirmation. Isolated colonies were subjected to genomic DNA extraction using a Qiagen bacterial DNA kit (Qiagen, Hilden, Germany). The 16S rRNA gene was amplified using universal primers [65], and purified products were sequenced (Next-Generation Sequencing, NGS) at the Australian Genome Research Facility (AGRF). Taxonomic classification was performed using VSEARCH and a Naive Bayes classifier against reference databases (SILVA, GTDB, and RefSeq + RDP), as implemented in the AGRF analysis pipeline. This verification was conducted solely to confirm the presence of the microbial strain of interest in the commercial product and our genomic and microbiological data is not presented.

2.5. Soil Sampling and Analysis

Soil sampling was conducted at two time points—prior to biostimulant application and at crop harvest. Eighteen cores (0–15 cm depth) were collected per treatment plot using a zigzag pattern and composited. Samples were analyzed for physicochemical properties, including soil moisture (gravimetric method), organic carbon (Walkley-Black), nitrate-N, ammonium-N, phosphorus (Colwell), potassium (Colwell), sulfur (KCl-40), pH (CaCl₂), and electrical conductivity. Analyses were performed by the CSBP Soil and Plant Laboratory (Bibra Lake, WA, Australia).

2.6. Chlorophyll Measurement

Leaf chlorophyll content was measured at three key developmental stages according to the Zadoks scale [66]: tillering (Z29), heading (Z55), and dough development (Z85). At each stage, ten fully expanded second-upper leaves from the main tillers were randomly collected from plants located in the center of each treatment plot and immediately stored at 4 °C in a cool box for analysis. Chlorophyll content was then extracted using 80% acetone following the method described by Wellburn [67] and Lichtenthaler [68]. Briefly, approximately 150 mg of leaf tissue was chopped, ground with chilled 80% acetone using a mortar and pestle, and the homogenate was then centrifuged at 3000 rpm for 3 min followed by 4500 rpm for 5 min. The pellet was re-extracted until colorless, and the pooled supernatant was then adjusted to 25 mL in a volumetric flask at room temperature. Absorbance readings were taken at 663, 646, and 710 nm using a Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Chlorophyll concentrations were calculated using the following equations:

Chlorophyll a (Ca) = 12.25 × A663 − 2.79 × A646

Chlorophyll b (Cb) = 21.50 × A646 − 5.10 × A663

Total Chlorophyll (Ca + Cb) = 7.15 × A663 + 18.71 × A646

Absorbance values were corrected using the reading at 710 nm to account for sample turbidity and background scattering.

Chlorophyll content per unit dry weight was calculated as

Chlorophyll concentration in leaf (µg/mL) = (C_extract × V_extract) ÷ DW_leaf

where C_extract is the chlorophyll concentration (µg/mL), V_extract is the final volume (25 mL), and DW_leaf is the dry weight of the leaf (g).

2.7. Biomass and Yield Components

Aboveground biomass was sampled at 0, 30, 60, 90, and 120 days after biostimulant application (DAA). At each sampling time, two biomass subsamples were collected from randomly selected locations within each plot to ensure representative sampling and to avoid border effects. All samples were oven-dried at 65 °C to constant weight and expressed on an area basis. At physiological maturity, final aboveground biomass was collected using 0.25 m² quadrats positioned at randomly selected mid-plot locations. Two subsamples per plot were collected to quantify yield components. In addition, key agronomic traits—plant height, spike length, grains per spike, and thousand-seed weight (TSW)—were determined using 20 randomly selected plants per plot. Dry matter allocation was assessed by separating harvested plant material into individual components (leaf blade, leaf sheath, stem, spike, and grain), which were then oven-dried at 65 °C to constant weight prior to analysis. The total nitrogen, carbon, and sulfur content in plant tissues was measured using a LECO TruMac^® CNS analyzer (LECO Corporation, St. Joseph, MI, USA) at Charles Sturt University.

2.8. Grain Yield and Quality

Grain yield (with 13% moisture) was determined in each plot utilizing a John Deere S7 series combine harvester (John Deere, Moline, IL, USA). Grain quality parameters, including protein and starch concentrations, were estimated using a John Deere HarvestLab 3000™ near-infrared (NIR) sensor (John Deere, Moline, IL, USA) mounted on the harvester. The sensor operates within the near-infrared spectral range (approximately 950–1650 nm) and quantifies grain constituents based on calibrated spectral reflectance models. These calibration models were developed against reference laboratory analyses, including enzymatic starch assays and combustion-based nitrogen determination, and the predicted values were validated against laboratory measurements. This approach enabled rapid, non-destructive, and real-time assessment of grain quality during harvesting.

2.9. Nitrogen Use Efficiency and Partitioning Indices

Harvest index (HI) and nitrogen harvest index (NHI) were calculated to quantify the partitioning of biomass and nitrogen to grain at maturity. Harvest index was calculated as the ratio of grain yield to total aboveground biomass at harvest, while nitrogen harvest index was calculated as the ratio of grain nitrogen uptake to total aboveground plant nitrogen content. Nitrogen use efficiency indices were calculated to assess nitrogen input efficiency and internal nitrogen utilization. Partial factor productivity of nitrogen (PFPₙ) was calculated as the ratio of grain yield to the amount of fertilizer nitrogen applied (kg grain kg⁻¹ N applied). Nitrogen utilization efficiency (NUtE) was calculated as the ratio of grain yield to total aboveground plant nitrogen uptake (kg grain kg⁻¹ plant N). All indices were calculated on a plot basis using harvest and plant nitrogen data.

2.10. Statistical Analysis

All experimental data were subjected to analysis of variance (ANOVA) using the R statistical software (version 4.3.2). The experimental design was fitted according to the factorial randomized complete block framework, with replication included as a blocking factor. Treatment effects were partitioned into the main effects of N rate (N), microbial biostimulant (MB), and their interaction (N × MB). Normality of residuals was tested using the Shapiro–Wilk test, and homogeneity of variance was checked through visual inspection of residual plots. When ANOVA indicated significant treatment effects (p ≤ 0.05), mean separation was performed using the least significant difference (LSD) test, as implemented in the Agricolae package in R. Regression and correlation analyses were further conducted, where appropriate, to explore relationships among physiological, agronomic, and biochemical traits.

3. Results

3.1. Effect of N Rates and MB Application on Physiological Traits of Wheat

3.1.1. Leaf Chlorophyll Content (mg/g)

Leaf chlorophyll concentration was monitored at multiple time points following foliar application of Methylobacterium symbioticum (0, 30, and 60 days after application, DAA) to evaluate the interactive effects of nitrogen supply and biostimulant treatment (Figure 4). At 0 DAA, chlorophyll levels were similar across treatments, indicating comparable initial physiological status prior to biostimulant application.

By 30 DAA, a significant main effect of biostimulant application was detected (p < 0.05), with MB-treated plants exhibiting higher chlorophyll concentrations than their corresponding non-treated controls under both nitrogen rates. Both MB-treated combinations recorded approximately 21% and 23% higher total chlorophyll concentrations, respectively, compared with their corresponding MB- treatments, with the strongest response observed under the reduced nitrogen (75%N) condition.

This positive effect of MB application persisted to 60 DAA (p < 0.05), when chlorophyll content in MB-treated plants remained elevated, recording approximately 19% and 6% higher concentrations under 75%N and 100%N conditions, respectively, relative to their non-treated controls. Overall, these results indicate that foliar M. symbioticum application enhanced chlorophyll retention during early to mid-season growth, particularly under reduced nitrogen supply, while detailed treatment means are presented in Figure 3.

3.1.2. Biomass Production (kg/ha)

Biomass production was assessed at multiple time points following foliar application of Methylobacterium symbioticum (0, 30, 60, 90, and 120 days after application, DAA) to evaluate temporal responses to nitrogen rate and biostimulant treatment (Figure 5). At 0 DAA, biomass did not differ among treatments, indicating uniform early crop establishment prior to the expression of treatment effects. By 30 DAA, a significant main effect of biostimulant application was observed (p < 0.05), with MB-treated plots accumulating 6–14% greater biomass than their corresponding non-treated controls across both nitrogen levels. From 60 DAA onwards, this positive effect became more pronounced (p < 0.05), particularly under reduced nitrogen supply (75%N), where MB application resulted in a 20% increase in biomass relative to the non-treated control, indicating an early enhancement of vegetative and reproductive growth.

At 90 DAA, a significant interaction between nitrogen rate and biostimulant application was observed (p < 0.05), with the 75%N+MB treatment recording the highest biomass accumulation and the 75%N-MB treatment the lowest, corresponding to a 14% increase in biomass under MB application at the reduced nitrogen rate. This interaction persisted through to 120 DAA, when biomass reached its maximum across treatments, with the 75%N+MB treatment showing an approximate 23% increase over its corresponding control, whereas both 100%N treatments remained intermediate and statistically comparable.

3.2. Effect of N Rates and MB Application on Agronomic and Yield Traits

The two N application rates alone did not significantly influence many wheat growth parameters other than biomass and chlorophyll content, as leaf sheath weight, leaf blade weight, and stem weight remained statistically comparable between 100%N and 75%N (

Table 1. Agronomic traits of wheat at harvest in response to two individual factors, N rates and MB (Methylobacterium symbioticum) application and interaction of both factors.

Treatments	Plant Height (cm)	Spike Length (cm)	Leaf Sheath wt. (kg ha−1)	Leaf Blade wt. (kg ha−1)	Stem wt. (kg ha−1)	1000-Seed wt. (g)
Factor 1 (N Rates)
100%N	75.1 ns	9.4 ns	895.0 ns	798.7 ns	2027.3 ns	46.3 ns
75%N	75.3 ns	9.3 ns	877.8 ns	743.2 ns	2026.4 ns	45.7 ns
Factor 2 (Microbial biostimulant)
MB−	72.8 b	9.0 b	900.1 ns	760.8 ns	2027 ns	44.6 b
MB+	77.6 a	9.7 a	827.6 ns	781.1 ns	2074.2 ns	47.5 a
LSD (5%)	4.3	0.6	111.2	67.3	284.9	2.7
Interaction (Factor 1 × 2)
100%N-MB	73.3 ab	9.0 ns	919.9 ns	813.2 a	1982.6 ns	45.1 ns
100%N+MB	76.8 ab	9.7 ns	875.2 ns	784.1 ab	2070.2 ns	47.4 ns
75%-MB	72.3 b	8.9 ns	880.4 ns	708.36 b	2071.9 ns	44.0 ns
75%+MB	78.3 a	9.7 ns	870.0 ns	778.08 ab	2078.2 ns	47.5 ns
LSD (5%)	6.0	0.8	157.3	95.2	402.9	3.8

100%N: 100% of the locally recommended N rate for high-yield goal; 75%N: 75% of the recommended N rate; foliar biostimulant application (with, MB+ or without, MB−); treatment combinations: 100%N-MB: 100% recommended N without biostimulant; 100%N+MB: 100% recommended N with biostimulant; 75%N-MB: 75% recommended N without biostimulant; and 75%N+MB: 75% recommended N with biostimulant. Lowercase letters indicate significant differences at p < 0.05; ns = not significant.

). In contrast, MB application had a consistent positive effect on several traits. Plants receiving MB showed significantly greater height (77.57 cm compared with 72.80 cm in the control), longer spike length (9.71 cm compared with 8.96 cm), and greater 1000-seed weight (47.45 g compared with 44.57 g; p ≤ 0.05). However, MB application did not significantly affect leaf sheath weight, leaf blade weight, or stem weight.

A significant N × MB interaction was observed for leaf blade weight (p ≤ 0.05). The highest leaf blade weight occurred under 100%N-MB (813.2 kg ha⁻¹), followed by 100%N+MB (784.1 kg ha⁻¹) and 75%N+MB (778.1 kg ha⁻¹), while the lowest value was recorded under 75%N-MB (708.4 kg ha⁻¹). In contrast, N × MB interactions were not significant for plant height, spike length, leaf sheath weight, stem weight, or 1000-seed weight. For these traits, interaction means ranged from 72.26 to 78.34 cm (plant height), 8.92 to 9.72 cm (spike length), 870.0 to 919.9 kg ha⁻¹ (leaf sheath weight), 1982.6 to 2078.2 kg ha⁻¹ (stem weight), and 44.04 to 47.48 g (1000-seed weight), none of which differed significantly at the 5% level.

Spike Dry Weight (t ha⁻¹) and Yield (t ha⁻¹)

The interaction of N rate and MB application on spike dry weight and grain yield are presented in Figure 6. For spike dry weight, the highest value was recorded under the 75%N+MB treatment (6.42 t ha⁻¹), which was clearly superior to the 75%N-MB combination (5.57 t ha⁻¹), the lowest among all treatments. In contrast, the 100%N-MB (6.12 t ha⁻¹) and 100%N+MB (6.10 t ha⁻¹) treatments produced intermediate spike weights that did not differ statistically from either the highest or the lowest treatments.

A similar pattern was evident for grain yield. The highest yield was obtained under the 75%N+MB treatment (4.46 t ha⁻¹), which was significantly greater than the 75%N-MB combination (3.90 t ha⁻¹), the least productive treatment and the lower-N control without SB23. The 100%N+MB (4.28 t ha⁻¹) and 75%N+MB (4.46 t ha⁻¹) treatments achieved yield increases of 1.4% and 14.4%, respectively, compared with their corresponding controls, 100%N-MB (4.22 t ha⁻¹) and 75%N-MB (3.90 t ha⁻¹). The two intermediate treatments, 100%N-MB and 100%N+MB, remained statistically comparable to both the highest and lowest yielding treatments.

3.3. Effect on Grain Quality

Starch (%) and Crude Protein (%)

Grain starch was significantly higher in 75%N+MB compared to other treatments as illustrated in Figure 7. For starch concentration, the highest value was observed under the 75%N+MB treatment (60.14%), which was significantly greater than the 75%N-MB combination (59.00%). The 100%N-MB (59.54%) and 100%N+MB (59.88%) treatments were intermediate, showing no statistical difference from either the highest or lowest values. Relative to their respective controls, the 100%N+MB and 75%N+MB treatments produced modest increases of 1.0% and 1.9%, respectively.

For protein concentration, the highest value was recorded under the 75%N-MB treatment (13.54%), which was statistically superior to both the 100%N+MB (12.68%) and 75%N+MB (12.50%) combinations. The 100%N-MBtreatment (13.00%) was intermediate and did not differ significantly from either group.

3.4. Effect on Biomass Nutrient Accumulation and Nitrogen Partitioning Efficiency

3.4.1. N Accumulation (kg ha⁻¹)

N rate and MB treatment interacted to influence tissue C and N accumulation at harvest (Figure 8). The highest N accumulation occurred under the 75%N+MB treatment (153 kg ha⁻¹), which was significantly greater than the corresponding control at the same N level, 75%N-MB (122 kg ha⁻¹). The 100%N-MB (143 kg ha⁻¹) and 100%N+MB (146 kg ha⁻¹) treatments occupied intermediate positions and did not differ statistically from either the highest or lowest values. Relative to its control (75%N-MB), the 75%N+MB treatment achieved a 25% increase in biomass N accumulation.

3.4.2. C Accumulation (kg ha⁻¹)

A similar interaction pattern was observed for tissue C accumulation (Figure 7). The highest C accumulation occurred under the 75%N+MB treatment (4335 kg ha⁻¹), which was significantly greater than the corresponding control, 75%N-MB (3906 kg ha⁻¹). The 100%N-MB (4165 kg ha⁻¹) and 100%N+MB (4151 kg ha⁻¹) treatments showed intermediate values and were statistically comparable to both the highest and lowest treatments. Relative to its control, the 75%N+MB treatment achieved an approximate 16% increase in biomass C accumulation.

3.4.3. Biomass and Nitrogen Partitioning

Reducing N input from 100% of the locally recommended high-yield rate to 75% of the recommended rate (corresponding to the optimum-yield goal), either with (MB+) or without (MB−) foliar M. symbioticum application, did not alter the proportional allocation of biomass or nitrogen to grain. Harvest index (HI) and nitrogen harvest index (NHI) were not significantly influenced by nitrogen (N) rate, foliar biostimulant application, or their interaction (Figure 9). Across treatments, HI values ranged from 0.36 to 0.38, while NHI values ranged from 0.64 to 0.77, with all treatment combinations sharing the same statistical grouping (p > 0.05; LSD).

3.4.4. Nitrogen Use Efficiency Indices

Nitrogen use efficiency indices showed contrasting responses to N rate and foliar biostimulant application (Figure 9). Nitrogen utilization efficiency (NUtE) varied among treatments (p ≤ 0.05; LSD). The reduced N treatment with biostimulant (75%N+MB) showed the highest NUtE (37 kg grain kg⁻¹ N uptake), representing a clear improvement and bringing it to a level comparable with both 100%N treatments, whereas 75%N-B recorded the lowest NUtE (34 kg grain kg⁻¹ N uptake). In contrast, partial factor productivity of nitrogen (PFPn) was significantly influenced by N rate and biostimulant application (Figure 9). The highest PFPn was observed under the 75% N+MB treatment, reaching 51.8 kg grain kg⁻¹ N applied, which was significantly greater than all other treatments (p ≤ 0.05; LSD). The 75% N-MB treatment also exhibited elevated PFPn (45.4 kg grain kg⁻¹ N applied), exceeding both 100% N treatments. Both 100% N treatments (high-yield goal), irrespective of biostimulant application, showed the lowest PFPn values (36.8–37.2 kg grain kg⁻¹ N applied) and did not differ significantly from each other. These results demonstrate that reducing fertilizer N input from the recommended high-yield rate to 75% of that rate substantially increased N input productivity, with the greatest benefit occurring when reduced N supply was combined with foliar M. symbioticum application.

3.5. Effect on Soil Nutrient Status

Post-harvest soil nutrient concentrations varied numerically among treatments, although none of the differences were statistically significant (

Table 2. Post-harvest soil nutrient concentrations under different N and MB treatment combinations.

Treatment Combinations	Ammonium N (kg ha−1)	Nitrate N (kg ha−1)	Phosphorus (kg ha−1)	Potassium (kg ha−1)	Sulfur (kg ha−1)	Organic Carbon (%)
100%N-MB	12.8 ns	60.9 ns	92.0 ns	742.2 ns	18.3 ns	1.13 ns
100%N+MB	12.1 ns	59.3 ns	123.3 ns	817.5 ns	19.4 ns	1.15 ns
75%N-MB	11.3 ns	56.2 ns	104.9 ns	715.7 ns	13.9 ns	1.01 ns
75%N+MB	10.9 ns	56.9 ns	85.8 ns	799.1 ns	16.5 ns	0.91 ns

Treatment combinations: 100%N-MB: 100% recommended N without biostimulant; 100%N+MB: 100% recommended N with biostimulant; 75%N-MB: 75% recommended N without biostimulant; and 75%N+MB: 75% recommended N with biostimulant. ns = not significant.

). Ammonium-N was slightly higher under the high-input urea regime (100%N), with values of 12.8 mg kg⁻¹ in the 100%N-MB treatment and 12.1 kg ha⁻¹ in the 100%N+MB treatment. In contrast, the reduced-input treatments (75%N) maintained lower residual ammonium, ranging from 11.3 kg ha⁻¹ under 75%N-MB to 10.9 kg ha⁻¹ under 75%N+MB. Nitrate-N showed a similar pattern, with higher concentrations under 100%N (60.9 kg ha⁻¹ for 100%N-MB and 59.3 kg ha⁻¹ for 100%N+MB) compared with the 75%N treatments (56.2 kg ha⁻¹ for 75%N-MB and 56.9 kg ha⁻¹ for 75%N+MB).

For other soil nutrients, phosphorus was highest under the 100%N+MBtreatment (123.3 kg ha⁻¹) and lowest under 75%N+MB (85.8 kg ha⁻¹). Potassium concentrations were generally higher in MB-treated plots, particularly in 100%N+MB (817.5 kg ha⁻¹) and 75%N+MB (799.1 kg ha⁻¹), compared with their respective controls, 100%N-MB (742.2 kg ha⁻¹) and 75%N-MB (715.7 kg ha⁻¹). Sulfur peaked under the 100%N+MB treatment (19.4 kg ha⁻¹) but declined to 13.9 kg ha⁻¹ under 75%N-MB. Soil organic carbon also showed a numerical increase under 100%N+MB (1.15%) compared with 75%N+MB (0.91%).

3.6. Correlation Matrices Among Physiological, Biomass, and Yield Parameters of Wheat

The correlation matrices revealed that M. symbioticum application strengthened key physiological–yield linkages compared to that of synthetic N alone (Figure 10b). Under biostimulant treatment, chlorophyll indices were more predictive of later growth and yield. For instance, chlorophyll content at 60 DAA (Chl60) exhibited strong positive correlations with multiple biomass parameters (Figure 10a). Chl60 was closely associated with biomass at 60 DAA (r = 0.87), biomass at 90 DAA (r = 0.96), and biomass at 120 DAA (r = 0.93), indicating that higher chlorophyll levels at mid-growth stages translated into greater vegetative accumulation throughout the season. In addition, Chl60 was strongly correlated with carbon accumulation at 120 DAA (BioC120; r = 0.84), confirming that enhanced photosynthetic activity during mid-season supported improved carbon assimilation and storage, whereas these relationships were notably weaker under synthetic N. Similarly, biomass at 90 DAA exhibited a closer association with spike weight under biostimulant application (r = 0.81) than with synthetic N alone (r = 0.70), indicating that inoculation sustained mid-season vigor and translated it more effectively into reproductive growth.

3.7. Relationship Between Chlorophyll Concentration, Biomass, and Spike Weight

The relationships among mid-season physiological traits and subsequent spike development highlighted the beneficial role of the biostimulant under constrained nitrogen supply (Figure 11 and Figure 12). The association between biomass at 60 DAA and spike weight at 120 DAA indicated that the strongest positive relationship occurred under the 75%N+MB treatment (R² = 0.62), followed by 100%N-MB (R² = 0.34) and 100%N+MB (R² = 0.25), whereas 75%N-MB showed only a weak relationship (R² = 0.17). Similarly, chlorophyll concentration at 60 DAA was strongly associated with spike weight under 100%N+MB (R² = 0.78) and 75%N+MB (R² = 0.72), while weaker associations were observed under 75%N-MB (R² = 0.42) and 100%N-MB (R² = 0.24). Collectively, these relationships indicate that M. symbioticum SB23 strengthens the functional coupling between mid-season photosynthetic capacity and reproductive sink development through increased biomass accumulation and subsequent spike development, particularly under reduced nitrogen supply.

4. Discussion

The application of the nitrogen-fixing bacterium M. symbioticum SB23 biostimulant to Australian dryland winter wheat at the Zadoks 29 growth stage generated promising results in a large dryland field experiment evaluating nitrogen management in Australia. In the present study, foliar inoculation, when combined with reduced N inputs, improved chlorophyll content, biomass accumulation, and grain yield. The most consistent benefits were observed under treatment combination corresponding to the 75%N+biostimulant treatment, where both biomass and yield exceeded the reduced N control by approximately 20% and 14%, respectively. These findings suggest that M. symbioticum SB23 can partially offset fertilizer reduction, decreasing reliance on synthetic N while sustaining crop performance. Similar outcomes have been reported in spring wheat, maize, and rice, in American or European systems where M. symbioticum application under suboptimal N regimes maintained or improved growth and yield, underscoring its potential across cereals [29,30,35,41,69].

4.1. Biostimulant Application Improved Physiological Performance

The application of M. symbioticum SB23 significantly enhanced chlorophyll concentration and biomass accumulation in wheat under reduced N input. These results suggest that foliar inoculation supports photosynthetic efficiency and carbon assimilation during critical growth stages. Increases in chlorophyll content at 30 and 60 DAA were strongly associated with subsequent biomass accumulation at 60–120 DAA and carbon assimilation at harvest, particularly in the 75% N+MB treatment. Such outcomes are consistent with earlier studies in maize, rice, and horticultural crops, where M. symbioticum inoculation enhanced photosynthetic activity and nitrogen assimilation [30,41].

Consistent with these findings, M. symbioticum has been shown to enhance chlorophyll retention and delay senescence, contributing to a prolonged stay-green phenotype [30,35,36]. In our study, inoculated wheat maintained higher chlorophyll levels at mid-growth stages, which translated into greater vegetative accumulation and sustained crop vigor under N-limited conditions. Similar improvements in chlorophyll and photosynthetic efficiency as detected by SPAD and NDVI have been reported in wheat [30] and maize [29], where inoculation reduced chlorophyll decline under lowered N supply, effectively compensating for nutrient stress.

The underlying mechanisms associated with enhanced vegetative wheat growth and yield may involve the bacterium’s capacity to supply fixed N in the form of ammonium directly to host tissues, bypassing the plant’s energy-intensive nitrate reductase-mediated nitrate reduction pathway and improving NUE [29]. In addition, M. symbioticum possesses ACC-deaminase that may result in reduced ethylene production, delayed senescence, and prolonged photosynthetic activity during later growth stages [35]. Collectively, these results indicate that M. symbioticum SB23 enhances mid-season photosynthetic capacity and delays functional senescence, thereby strengthening source–sink relationships and improving nitrogen use efficiency under reduced N supply.

4.2. Yield Response Under Reduced Nitrogen Inputs

Grain yield was potentially maximized in the 75% N+M. symbioticum treatment combination under dryland production conditions, which resulted in 14% yield increase over the reduced-N control. This suggests that this biostimulant not only compensated for reduced fertilizer input but also provided measurable gains in productivity in yield, thereby resulting in enhanced NUE. These results are consistent with recent findings in maize, grapevine, and strawberry, where M. symbioticum inoculation enabled 25–50% reductions in synthetic N without yield penalties [29,41,43,44,69]. Notably, Santos and Bundt [69] reported up to a 53% yield increase in maize under 50% N reduction, while Bolla et al. [35] similarly observed delayed senescence, improved chlorophyll retention, and greater aboveground N accumulation, all contributing to yield stability under lower N inputs.

In contrast, benefits we observed under full N supply were less pronounced, with only marginal improvements in yield or biomass. Similar observations were reported by Valente et al. [30] and Rodrigues et al. [57,70], who found minimal or inconsistent effects of inoculation under optimal fertilization, suggesting reduced bacterial contribution when external N was abundant. Collectively, these results reinforce the concept that M. symbioticum acts as a supplementary rather than additive N source, with the greatest efficacy expressed under moderate N stress where its N-fixation or uptake capacity complements reduced fertilizer applications.

4.3. Effects on Grain Quality and Nutrient Accumulation

Grain quality responses to M. symbioticum were modest at best in this dryland field trial. Starch concentration was marginally higher in MB-treated wheat, while protein content remained greater in non-inoculated plots under reduced N, consistent with the dilution effect commonly observed in cereals, where enhanced carbohydrate accumulation offsets grain protein concentration [10]. Similar results were reported by Valente et al. [30], who found that inoculation with M. symbioticum did not increase grain protein concentration but altered its composition, improving glutenin-to-gliadin and HMW-to-LMW ratios, thereby enhancing dough stability. In maize, Bolla et al. [35] also observed small but positive shifts in grain protein/N content (+5%), suggesting that M. symbioticum may also influence nitrogen partitioning and quality traits rather than crude protein levels.

Similarly, the nutrient accumulation patterns we observed in the mature wheat crop reinforce this interpretation. Biomass N and C were significantly greater in MB-treated wheat under reduced N, consistent with observations in maize [35,69], tomato [56], and olive [57], where inoculation improved total N recovery even when tissue N concentrations remained unchanged. Together, these results suggest that one benefit of M. symbioticum may be associated with enhanced whole-plant nutrient capture and assimilation efficiency, particularly under suboptimal N availability. Another potential effect of M. symbioticum application under consideration is the hormonal stimulation of plant growth, particularly through the presence of increased auxins and cytokinins, which are characteristic of plant growth-promoting methylotrophs [37,71,72]. In addition, some strains produce siderophores and other secondary metabolites that can indirectly enhance nutrient uptake and plant performance [20]. Increased auxin production has been suggested as another possible mode of action of this Gram-negative bacterium, thereby contributing to the regulation of plant vegetative growth [73,74]. However, field data to support this concept is limited, suggesting that further experimentation must be conducted to explore this possibility.

4.4. Nitrogen Use Efficiency Pathways Under Foliar M. symbioticum Application

The contrasting responses of nitrogen use efficiency indices observed in this study indicate distinct efficiency pathways associated with foliar M. symbioticum application compared with conventional soil-applied N fertilization. The higher partial factor productivity of nitrogen (PFP_n) under biostimulant application reflects improved grain yield per unit of applied nitrogen, demonstrating enhanced input efficiency under reduced fertilizer supply. Similar increases in PFP_n have been reported in cereals and other crops inoculated with Methylobacterium spp. or related plant growth-promoting bacteria, where yield was maintained or improved despite reductions in mineral nitrogen inputs [30,35,69].

In addition to improved input efficiency, nitrogen utilization efficiency (NUtE) also responded positively to biostimulant application under reduced N supply. The higher NUtE observed in the 75% N+ biostimulant treatment indicates that the crop converted absorbed N into grain yield more efficiently when foliar M. symbioticum was applied. This improvement likely reflects enhanced physiological functioning of the crop, including improved N uptake dynamics, sustained photosynthetic activity, and delayed leaf senescence—responses commonly associated with Methylobacterium spp. Previous studies have shown that foliar application of M. symbioticum can maintain higher chlorophyll content (SPAD), improve photosystem II efficiency, and prolong photosynthetic activity under reduced N supply. These effects have been linked to ACC-deaminase activity, reduced ethylene production, and improved N metabolism, thereby supporting enhanced N assimilation and crop performance [53,54]. Similar improvements in NUtE have also been reported in cereal and other cropping systems where microbial or plant-derived biostimulants enhanced N acquisition and assimilation under reduced N inputs [75,76].

Despite these improvements in nitrogen use efficiency indices, harvest index (HI) and nitrogen harvest index (NHI) remained stable across treatments, indicating that reductions in N input from 100% of the locally recommended high-yield rate to 75% of the recommended optimum-yield rate, with or without biostimulant application, did not disrupt source–sink partitioning. This conservation of biomass and N allocation to grain is consistent with previous reports showing that wheat harvest indices are relatively resilient to moderate N reductions when yield penalties are avoided [77,78].

Although biological nitrogen fixation was not directly quantified in the present study, the increased biomass N accumulation and maintenance of grain yield under reduced fertilizer input are consistent with a supplementary N contribution mediated through foliar colonization. M. symbioticum has been shown to fix atmospheric N in the phyllosphere and provide reduced N forms, predominantly ammonium, that can be assimilated directly by plant tissues [29,30,41].

By comparison, the 100% N treatments showed lower partial factor productivity of nitrogen (PFP_n) due to the substantially greater fertilizer inputs required to achieve similar yield levels. This pattern reflects the well-documented decline in N input efficiency as fertilizer rates increase, where higher N supply often leads to diminishing returns in N productivity and economic performance in cereal systems [79,80]. In contrast, the combination of reduced N supply (75% of the recommended rate) with foliar M symbioticum application resulted in both higher NUtE and higher PFP_n, indicating that the crop utilized absorbed N more efficiently while also producing greater grain yield per unit of applied fertilizer. These responses suggest that the biostimulant promoted a more efficient N use pathway characterized by improved N acquisition and physiological utilization under reduced fertilizer input, allowing grain yield to be maintained while enhancing overall N productivity.

4.5. Soil Nutrient Dynamics

Although post-harvest soil nutrient concentrations did not differ significantly among treatments, numerical increases in available potassium and sulfur under MB+ treatment suggest potential interactions between microbial inoculants and soil nutrient cycling. While residual N levels remained comparable, the improved plant uptake observed in biomass measures suggests that M. symbioticum enhances in planta assimilation rather than altering soil mineral pools. This finding also supports the proposed mechanism of phyllospheric N fixation contributing directly to plant metabolism [20,61]. Clearly additional studies focused on the primary and secondary modes of action of this biostimulant in both monocots and dicots are required to determine the mechanisms associated with biostimulation in higher plants.

4.6. Cost Benefits of Biostimulant Application with M. symbioticum

Applying M. symbioticum SB23 at the optimum-yield N rate (75% of the recommended high-yield rate) produced the highest grain yield, adding 0.56 t ha⁻¹ (a 14.4% increase) over the 75%N control (

Table 3. Economic benefit of foliar Methylobacterium symbioticum SB23 application under reduced nitrogen input in dryland wheat.

Treatment Comparison	Grain Yield Difference (t ha−1)	Yield Change (%)	Gross Value of Additional Grain (A$ ha−1)	Fertilizer N Saving (kg Urea ha−1)	Value of Fertilizer Saving (A$ ha−1)	Biostimulant Cost (A$ ha−1)	Net Economic Benefit (A$ ha−1)
75%N+MB vs. 75%N-MB	+0.56	+14.4	196	0	0	27	169
75%N-MB vs. 100%N-MB	+0.24	+6.1	84	62	43	27	127

Grain value calculated using a farm-gate wheat price of A$350 t⁻¹ (2025). Fertilizer savings calculated using a urea price of A$700 t⁻¹ (2024), corresponding to a reduction of 62 kg urea ha⁻¹ when nitrogen input was reduced from 100% to 75% of the locally recommended high-yield rate. The biostimulant cost for M. symbioticum SB23 was A$27 ha⁻¹. No additional application cost was assumed, as the biostimulant can be co-applied with routine crop protection sprays.

). At a farm-gate wheat price of A$350 t⁻¹ in 2025, this additional grain is worth A$196 ha⁻¹, and after accounting for the A$27 ha⁻¹ biostimulant cost, the net economic benefit is A$169 ha⁻¹. Remarkably, this reduced-N treatment also outperformed the conventional 100%N (recommended rate for high-yield) fertilizer strategy, yielding 0.24 t ha⁻¹ more grain (worth A$84 ha⁻¹) despite using 25% less N. Furthermore, reducing the N rate from 100% to 75% saved 62 kg urea ha⁻¹, providing an additional A$43 ha⁻¹ at a urea price of A$700 t⁻¹ in 2024. Combined, these yield gains and fertilizer savings deliver a total economic advantage of A$127 ha⁻¹ after accounting for the biostimulant cost. If SB23 is co-applied with routine pesticide or insecticide sprays—possible due to its broad chemical compatibility—the grower avoids additional spray costs, further enhancing profitability.

Beyond economic gains, reducing urea inputs also delivers important environmental benefits, and the use of M. symbioticum SB23 can be considered one of the recognized strategies [81,82,83] reported earlier for mitigating risks such as nitrate leaching, nitrous oxide emissions, and ammonia volatilisation—key environmental concerns in Australian dryland cereal production systems [7,11,12].

5. Conclusions

This study demonstrates that foliar application of Methylobacterium symbioticum can enhance chlorophyll retention, biomass accumulation, and nitrogen assimilation in wheat, particularly under reduced N fertilization. The reduced N treatment applied with M. symbioticum SB23 foliar spray consistently outperformed the reduced-N control, achieving significant gains in biomass, nutrient recovery, and yield. These results clearly suggest that M. symbioticum could provide a supplementary nitrogen source, partially compensating for reduced fertilizer inputs and improving NUE without compromising productivity. While benefits were modest under the actual rates of N application within a context of N balance, the consistent improvements observed at reduced N rates underscore the suitability of this biostimulant for low- to moderate-input systems in dryland crop production. The strong correlations between mid-season chlorophyll, biomass accumulation, and subsequent yield further highlight its role in sustaining physiological activity including early season growth and development of cereal crops, later impacting harvestable yield and quality. Overall, M. symbioticum shows promise as an environmentally sustainable strategy to reduce reliance on synthetic N in dryland wheat systems. However, further replicated experiments across multiple seasons and environments are required to validate these findings, particularly under low N availability and stress-prone conditions, and also to develop robust recommendations for its use in cereal production.