Reduced tillage and foliar application of Methylobacterium symbioticum synergistically improve yield and nutrient uptake in organic black barley

Abstract

Sustainable nitrogen management remains a major challenge in organic farming, where plough-based tillage is commonly used to incorporate fertilizers and control weeds, limiting the adoption of reduced tillage systems. Foliar nitrogen-fixing bacteria may offer an alternative nitrogen source that supports simplified soil management while maintaining crop productivity. This study evaluated the effects of soil tillage (ploughing vs. reduced tillage), foliar biofertilisation with Methylobacterium symbioticum (single and double application), and sowing density (350 and 450 seeds m⁻²) on the productivity and nutrient dynamics of organically grown black barley (Hordeum vulgare var. rimpaui). A field experiment was conducted during two contrasting growing seasons (2023–2024) on an organic farm in west-central Poland. Biometric traits, grain yield and its components, and the content and uptake of nitrogen, phosphorus, and potassium were assessed. Weather conditions strongly modified crop responses. Tillage system and biofertilisation exerted the greatest influence on most parameters, whereas sowing density had a weaker effect. The highest grain yield (3.08 t ha⁻¹) was obtained under reduced tillage combined with higher sowing density and a single application of M. symbioticum. Biofertilisation under reduced tillage enhanced grain number per ear, shoot dry matter, and macronutrient uptake, while the absence of biofertiliser increased tissue N and K concentrations but reduced grain size. These results indicate that foliar nitrogen-fixing bacteria can support reduced tillage strategies in organic black barley production. Further research should focus on long-term soil effects and performance under diverse climatic conditions to refine recommendations for organic growers.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-47850-7.

Introduction

In recent years, significant attention has been directed towards the utilisation of reduced (simplified) tillage methods, a pivotal element in the realm of sustainable agriculture¹. Reduced tillage, also termed conservation tillage, is hypothesised to exert a less detrimental effect on the physical and biological characteristics of soil than conventional tillage. The extant evidence suggests that simplified soil cultivation may have a positive impact on various aspects of soil health, including its structure, organic matter content, and microbial activity². Furthermore, it has been demonstrated that conservation tillage has the potential to reduce the risk of erosion, water evaporation and nutrient losses during leaching or air emission³. It is important to note that this tillage method does not appear to have any negative effect on plant productivity⁴. Furthermore, evidence suggests that the adoption of reduced tillage techniques is associated with a decline in fuel consumption, resulting in a reduction in production resource outputs (leading to financial savings) and machinery CO2 emissions⁵. Nevertheless, in organic farming, plough-based soil cultivation is often considered obligatory as it assists in the diminution of weeds, pests and fungous infestations and, most importantly, the implementation of organic and natural fertilisers in the soil profile⁶. The latter appeared to be of pivotal significance, given the challenges associated with the effective management of nutrients in organic farming systems, particularly when the most crucial essential macroelement for plants—nitrogen—is the primary limiting factor for proper plant growth and development and finally yielding in those farms⁷. Research into finding solutions for achieving better plant nutritional status in organic farms has been ongoing for years.

There has been a recent increase in the interest surrounding the utilisation of biofertilisers based on microbes, which have the potential to supply plants with essential macro- and micronutrients⁸. Bacteria capable of fixing nitrogen appear to be particularly significant in organic farming, where such applications are permitted. Among biological biofertilisers, Methylobacterium symbioticum merits particular attention due to its ability to colonise the leaf surface rapidly without exerting any detrimental influence on the soil microbiota⁹. M. symbioticum is a Gram-negative, rod-shaped, aerobic (facultative methylotrophic) bacterium¹⁰. These organisms inhabit the phyllosphere and have the capacity to proliferate in the presence of compounds with a single carbon atom, such as methanol, which may function as a source of energy for their enzymatic activity. During this process, they transform atmospheric nitrogen, thanks to the nitrogenase complex, into a form that is subsequently available to plants, thereby decreasing the necessity for nitrogen fertiliser¹¹. Recent studies demonstrated the potential for M. symbioticum to supply 25–50% of the nitrogen necessary for plant cultivation without compromising their productivity¹². The capacity of phyllosphere bacteria to perform nitrogen fixation and provide it to plants indicates the possibility of reducing the usage of ploughing as a practice that aims to provide nutrients to plants from natural sources. Consequently, this could potentially lead to the adoption of reduced tillage practices in the context of organic agriculture.

The employment of nitrogen biofertilisers, which are based on micro-organisms, in the sphere of organic farming may hold considerable promise, particularly when considered in conjunction with the frequent selection of specific species for cultivation within such agricultural frameworks. In the context of organic farming, the cultivation of ancient grains has become a prevalent practice, owing to the fact that these grains are reputed to enhance resilience in low-input agricultural settings when compared to conventional cereals¹³. Ancient grains are believed to generally exhibit lower nutritional demands, including reduced reliance on nitrogen^14,15, which suggests that M. symbioticum based products may provide an important supply of this macroelement. Furthermore, the aforementioned grains possess enhanced disease and insect resistance, as well as superior resilience under less favourable weather conditions^16,17. An example of a species belonging to the group of primitive cereals is Hordeum vulgare var. rimpaui, which is distinguished by its black grains, a consequence of the presence of the phytomelanin pigment¹⁸. This barley genotype has been shown to contain substantial levels of numerous health-promoting compounds, including soluble and insoluble fibre, vitamins, macro- and micronutrients, carotenoids, polyunsaturated fatty acids, and polyphenols. Furthermore, it has been demonstrated to exhibit a high degree of antioxidant activity^19,20. The extant research has also indicated that black barley is capable of yielding satisfactory levels within the context of organic farming²¹.

Regardless of the farming system, final productivity and yield are influenced by several agrotechnical factors. In addition to the abovementioned fertilisation and tillage practices, sowing density may also have an effect on them²². In organic farming, sowing density is of crucial importance, as it influences a wide range of factors. It can affect weed infestation, alter the incidence of diseases and pests, and modify the availability of nutrients and water¹⁶. Furthermore, the density of sowing has the capacity to influence intraspecific competition for soil resources, light and space¹⁵. This agrotechnical element may be of particular relevance in the context of barley cultivation, given this species’s notable capacity for tillering. This trait has the potential to counterbalance the comparatively lower sowing density by producing a greater quantity of generative tillers²³. Nonetheless, the effect of sowing density on the biometric parameters of cereals, their nutrient management and yield and its components vary and is shaped by multiple factors, including weather and site conditions, as well as agrotechnical practices, farming systems and genetic traits^16,20–24.

To date, there have been no studies conducted on the potential application of products derived from free-living N-fixing bacteria in organic farming, particularly in conjunction with reduced tillage practices or varying sowing densities. Moreover, there is a paucity of research on organically cultivated black barley in relation to reduced tillage, biological nitrogen fertilisation and differing plant population density, particularly with regard to parameters such as biometric parameters, NPK management and yield and its components. However, the utilisation of M. symbioticum in organic farming, which is set to become increasingly significant in the near future, necessitates research into its role as a potential source of problematic nutrients within the organic system. The adoption of this alternative nitrogen supply may also facilitate the implementation of simplified, more sustainable tillage practices within this farming system. Due to its good performances in low-input cultivation, black barley exhibits considerable potential for adoption on organically managed farms. Additionally, this barley may contribute to higher biodiversity on fields and serve as an excellent source of high-nutritional food. Therefore, the research was undertaken to evaluate the response of biometric parameters, yield and its components, and the content of NPK and their uptake by H. v. rimpaui, to the different tillage (ploughing and reduced), biofertilisation (single or dual M. symbioticum application and control) and varying sowing density (350 and 450 grain per 1m²) under varied hydrothermal conditions of the growing seasons. It was hypothesised that the use of M. symbioticum would have a favourable impact on biometric parameters, yield and its components, the content and uptake of NPK by H. v. rimpaui, and that the tillage methods, sowing density management and hydrothermal conditions during the growing season would modify the effects.

Materials and methods

Plant material

The subject of this research was Hordeum vulgare L. var. rimpaui cv. ‘Kaptur’, a form that produces grains with black-colored husks, fruits, and seed coats. This genotype is further distinguished by the reduction of the awns into hooded form. H. v. rimpaui is a two-row spring barley. The seed was obtained through the process of own multiplication. The authors’ institution is the breeder of this variety and holds exclusive rights to it.

Site description and experiment design

The study was based on a field experiment located in certificated organic farm in Wolkowo, Greater Poland Voivodeship, west-central Poland (52° 09′17″ N, 16° 29′54″ E). The field experiment was conducted over two growing seasons of black barley (2023–2024), in a split-plot design with four replicates with soil tillage practices as the main plot factor, sowing density as the subplot factor, and methods of stimulating plant nitrogen nutrition as the sub-subplot factor. The soil at the experimental sites was categorised as belonging to the Luvisol group (LV, according to the IUSS WRB) and was described as sandy loam. The soil exhibited a slight acidity (pH of 6.5), and the content of available forms of macroelements in the soil was 18.4 mg K₂0 100 g⁻¹ (high), 10.1 mg MgO 100 g⁻¹ (very high), and 37.1 mg P₂O₅ 100 g⁻¹ (very high). The content of organic carbon in the soil was 1.12%, and mineral nitrogen (N-NH₄ + N-NO₃) was 70.1 kg N ha⁻¹. The forecrop for barley was potatoes.

The soil tillage practices encompass traditional ploughing and reduced tillage. In the autumn period, preceding the barley growing season, traditional ploughing was performed to a depth of 25 cm, or alternatively, reduced tillage was employed using a chisel plough to a depth of 25 cm. In both years, spring tillage focused on two applications of a tillage unit, consisting of a disc harrow and a string roller. H. v. rimpaui was sown at a depth of 3–4 cm, with a row spacing of 12.5 cm, in the second or third ten-day period of March in 2023 and 2024, respectively.

The sowing density was established at either 350 or 450 seeds per square meter [no per m²]. The seeding rate was determined using the current seed parameters (thousand grain weight and germination capacity) and the theoretical field emergence indicator. Prior to sowing, the seeds were treated with a natural preparation called Chito, which is a natural chitosan. Pre-sowing, plants were fertilised with phosphate rock powder at a rate of 40 kg ha⁻¹ P₂O₅, kainite at a rate of 90 kg ha⁻¹ K₂O. The weeding harrowing was performed at the 3–4 leaf stage, diagonally to the rows, in both years, for weed control. The methods used to stimulate plant nitrogen nutrition included a single foliar application of a biofertiliser based on nitrogen-fixing bacteria at full tillering, and a double-application variant in which the first dose was applied at full tillering and the second dose at the stem elongation stage. In both treatments, a dose of 333 g ha⁻¹ of the microbiological biofertiliser BlueN (Methylobacterium symbioticum), with the addition of Cropvit FeMo (0.2 l ha⁻¹), was used. On this certified organic farm, no pesticides were used to control diseases and pests. The harvesting of the black barley took place in mid-July in both years and was carried out using a plot harvester.

Meteorological data

The climatic conditions of the region in which the field is located are categorised as temperate transitional, ranking among the warmest and driest in Poland. The average yearly temperature from the long-term period (1991–2020) was 9.1 °C, and the annual sum of precipitation was 544.7 mm. The overview of long term (1991–2020) average air temperature and precipitation, during the period corresponding to the vegetation period of black barley, in the region is shown in Fig. S1 (Supplementary Materials, Fig. S1).

Weather conditions (temperature and precipitation) during the black barley growing season varied in the years of study. It was noticeably cooler in the period from 11 March to 10 April, as well as throughout May and the third ten-day period of June 2023 compared to the corresponding time in 2024. During the remaining periods, the temperature was similar in both years (Fig. 1). In 2023, the lowest average daily air temperature during the vegetation period of black barley occurred in the period from 1 to 10 April and amounted to 4.36 °C, but in the following year, the lowest temperature was recorded during the second ten-day period of March, with an average of 6.65 °C. The warmest period during the growth of H. v. rimpaui was recorded between 21st to 30th July in both years, with an average temperatures of 19.6 °C and 21.2 °C, respectively.

Fig. 1.

The weather conditions at the experimental site. The meteorological data was obtained from the IUNG Experimental Station in Wielichowo.

In 2023, during the growing season of the black barley (from mid-March to mid-July), the total amount of rainfall was 150.2 mm. The total amount of precipitation during the barley vegetation period in the following year, from the third ten-day period in March to the mid-July, was 214.8 mm (Fig. 1). However, the distribution of precipitation exhibited variability across the years under consideration. In the period commencing in the second ten-day period of March and continuing for the subsequent two months, the time of the greatest plant development and biomass production, the total precipitation in the first year of study was found to be almost 1.8 times greater in comparison with the second year. Conversely, during the period of final grain filling, ripening, and drying (VI – VII) rainwater availability was found to be higher in the second year.

The Sielianinov index was used to estimate the meteorological condition during the black barley growing season (mid-March to mid-July)²⁵. Sielianinov index (k), alternatively referred to as hydrothermal coefficient, quantifies the relationship between precipitation and evapotranspiration and characterizes the moisture conditions for plant growth. The present study employed classification and calculation methodologies for the Sielianinov coefficient (k) as described in the work of Szczepanek et al.¹⁷. The k index is described as follows:

• Extremely dry or very dry—k ≤ 0.7;

• Dry or quite dry—0.7 < k ≤ 1.3;

• Optimal or quite moist—1.3 < k ≤ 2.0;

• Humid, very humid, or extremely humid—k > 2.0.

Plant parameters

In the flowering stage of H. v. rimpaui (BBCH 59), plant samples were collected from a sowing row strip length corresponding to an area measuring 0.5 m² (4 m under ploughing and reduced tillage) to determine the length of roots (RL) and generative tillers (GTL), the number of generative tillers per plant (GTN), the dry matter of shoots (DM_shoot), and the content of macronutrients in plants (NC_shoot, PC_shoot, KC_shoot). The collection of plants was conducted through excavation with a spade, without the separation of the below- and above-ground biomass, with the objective of differentiating each plant from the others. Additionaly, at a similar stage of black barley growth development, flag-leaves were collected from 50 plants at each plot in order to determine the nitrogen content (NC_leaf). Root and shoot lengths were measured from the base of the stem, where in the case of the latter, the length was measured to the top of the ear (excluding reduced awns). The procedure was performed on 30 plants from the sample, using a ruler. In plants that developed multiple generative tillers, the measurements were taken for each individual tiller and then averaged to provide a mean value for the single plant. Subsequently, the barley shoots were separated from the roots, dried in a laboratory dryer, and then ground into particles measuring up to 0.5 cm. A 150–200 g laboratory sample of the grounded, dried biomass was collected and finely ground into fractions of approximately 20 μm. The total nitrogen, phosphorus and potassium content of each dried biomass sample was then analysed using the Kjeldahl method [PN-A-04018]²⁶, the vanadium–molybdenum method²⁷ and flame photometry²⁸, respectively. The nitrogen content of flag leaves was determined with the Kjeldahl method [PN-A-04018]. The nutrient uptake by the plants was calculated on the basis of their concentration (NC_shoot, PC_shoot, KC_shoot), and the dry matter content of shoots (DM_shoot). This was then transferred to the total nutrient uptake in kilograms per hectare (NU_shoot, PU_shoot and KU_shoot).

At the full maturity of barley (BBCH 89), the number of ears per 1 m² (EN) was determined in four replications of each plot. Thereafter, 30 ears were randomly collected from each plot in order to ascertain the number of grains per ear (GN). Following the harvesting process using the Winterstaiger Classic harvester (manufactured by Winterstaiger in Ried im Innkreis, Austria), the grain yield and moisture levels were measured and then converted to a constant humidity level of 14%. The thousand grain weight (TGW) was determined on grain samples with 14% humidity, based on Polish Norm [PN-68/R-74017]²⁹. A mechanical grain sorter, type SŻK (Sadkiewicz Instruments, Bydgoszcz, Poland), was utilised to sort the grain of H. v. rimpaui according to three distinct dimensions: exceeding 2.5 mm, 2.2–2.5 mm, and under 2.2 mm. To this end, 100 g of grain samples was utilised, with the weight of each fraction being determined to calculate the share of grain from 2.5 mm and 2.2–2.5 mm dimensions (GF1 > 2.5 mm [%]; GF2 2.2–2.5 mm [%]).

Statistical analysis

The data that were obtained were utilised for the calculation of fundamental statistical descriptors, including the mean and standard deviation. The normality of the distribution was confirmed through the application of the Shapiro–Wilk test. In order to ascertain the influence of multiple factors (interaction between soil tillage, biofertilisation and sowing density) on the variables under investigation, a multivariate analysis of variance (MANOVA) was conducted. Tukey’s HSD (honestly significant difference) method was employed to assess the differences between mean values at the level of significance at α = 0.05. Pearson’s correlation coefficient was utilised to ascertain the relationships between: (1) analysed biometric parameters, yield and its components, and grain parameters; and (2) dry matter, nitrogen, phosphorus and potassium content and nitrogen, phosphorus and potassium uptake by H. v. rimpaui. Furthermore, a principal component analysis (PCA) was performed on black barley, with the objective of identifying any correlations between soil tillage, biofertilisation, sowing density, as well as years of study, and the analysed parameters. All statistical calculations (MANOVA, Pearson’s correlation, and PCA) were performed using the Statistica 13.0 PL package (Statsoft, Poland). The graphs illustrating the weather conditions, grain yield of black barley and N content in the shoot and in flag-leaf of barley were generated using Grapher 21 (Golden Software, Golden, CO, USA).

Results

Plant biometric parameters

Experimental factors (soil tillage, sowing density and biofertiliser) had different effects on individual variables (Supplementary Materials, Tab. S1, S2 and Tables 1 and 2) in the first and second study year. In 2023 the generative tillers of H. v. rimpaui were longer in reduced tillage. Moreover, a significantly higher number of generative tillers per plant was also determined in this tillage method. In the same year, barley not treated with biofertiliser exhibited longer roots than those treated with BlueN twice. In turn, a single application of the BlueN had a stimulatory effect on generative tiller length. This plant trait was also influenced by sowing density; generative tillers were longer at a lower density (350 no of grains per m²). No influence of experimental factors on the number of ears per m2 was demonstrated in the first study year (Table 1).

Table 1.

Plant biometric parameters of H. v. rimpaui in 2023.

Treatments		RL [cm]	GTL [cm]	GTN [no plant−1]	EN [no m−2]
Soil tillage	Ploughing	9.59 ± 1.03a	55.5 ± 5.21b	1.32 ± 0.405b	559.1 ± 61.3a
	Reduced	9.03 ± 1.34a	58.4 ± 5.66a	1.52 ± 0.344a	558.9 ± 76.7a
Sowing density [no of grains m−2]	350	9.23 ± 1.14A	58.7 ± 5.54A	1.51 ± 0.380A	540.1 ± 76.8A
	450	9.39 ± 1.30A	55.2 ± 5.14B	1.33 ± 0.378A	577.9 ± 54.7A
Biofertiliser	BlueN 1x	9.08 ± 1.42xy	59.7 ± 4.83x	1.55 ± 0.392x	588.4 ± 77.4x
	BlueN 2x	8.97 ± 1.25y	55.5 ± 6.00y	1.39 ± 0.397x	557.6 ± 56.7x
	Control	9.87 ± 0.717x	55.7 ± 5.07xy	1.32 ± 0.352x	531.0 ± 61.8x

RL, root length; GTL, tiller length; GTN, number of generative tillers; EN, ears number. ^{a – b}, ^{A – B}, ^{x – y}, values with different letters significantly differ at p < 0.05. The sign ± refers to SD.

Table 2.

Plant biometric parameters of H. v. rimpaui in 2024.

Treatments		RL [cm]	GTL [cm]	GTN [no plant−1]	EN [no m−2]
Soil tillage	Ploughing	13.5 ± 1.54a	61.7 ± 3.65a	1.57 ± 0.314a	356.9 ± 53.2a
	Reduced	12.0 ± 1.43b	58.7 ± 4.48b	1.31 ± 0.232b	361.2 ± 29.6a
Sowing density [no of grains m−2]	350	12.5 ± 1.55A	60.3 ± 4.96A	1.59 ± 0.298A	353.3 ± 47.1A
	450	12.9 ± 1.75A	60.2 ± 3.67A	1.29 ± 0.225B	364.8 ± 38.0A
Biofertiliser	BlueN 1x	12.1 ± 1.15y	60.8 ± 3.87x	1.46 ± 0.245x	373.0 ± 39.0x
	BlueN 2x	13.4 ± 2.14x	60.1 ± 5.22x	1.40 ± 0.353x	349.1 ± 51.5x
	Control	12.6 ± 1.29xy	59.8 ± 3.95x	1.46 ± 0.312x	355.1 ± 34.8x

The abbreviations are defined under the Table 1.

In 2024, soil tillage had a pronounced effect on both root length and tiller length, which were longer under ploughing. Furthermore, ploughing resulted in a substantial increase in the number of generative tillers per plant (Table 2). In contrast to the results observed in 2023, during the second year of the study, the sequential application of BlueN led to an augmentation in the length of the roots of H. v. rimpaui. In 2024, the sowing density had a significant impact solely on the number of generative tillers, which was found to be higher when barley was sown in smaller density. In accordance with the 2023, the number of ears per m² was not influenced by any treatment (Table 2).

The results from both study years demonstrate that the root length (cm), tiller length (cm), number of generative tillers (no per plant¹) and ear number (no per m²) were not influenced by the interaction of experimental factors (Supplementary Materials, Table S2).

Grain yield, and grain parameters

An analysis of the grain yield and yield components indicated that their response to experimental factors varied across the study years (Supplementary Materials, Tables S1, S2 and Tables 3 and 4). In 2023, the reduced tillage had a stimulatory effect on the grain number per ear, thousand grain weight, as well as on the share of grain with a diameter > 2.5 mm. Barley grain sown at a lower density (350 grains per 1 m²) was characterized by a higher thousand grain weight. Furthermore, the proportion of GF1 was significantly higher, while the proportion of GF2 was significantly lower, in comparison to barley sown at a higher density. In 2023, both BlueN applications resulted in higher grain yield than the control, with the best results achieved with the single dose application (Table 3).

Table 3.

Grain yield, and grain parameters of the H. v. rimpaui in 2023.

Treatments		GN [no ear−1]	GY [t ha−1]	TGW [g]	GF1 > 2.5 mm [%]	GF2 2.2–2.5 mm [%]
Soil tillage	Ploughing	16.6 ± 1.06b	2.70 ± 0.170a	35.7 ± 1.97b	46.3 ± 4.12b	38.2 ± 2.38a
	Reduced	18.0 ± 1.13a	2.66 ± 0.251a	37.6 ± 1.71a	49.6 ± 6.03a	37.0 ± 3.62a
Sowing density [no of grains m−2]	350	17.6 ± 1.16A	2.68 ± 0.159A	37.9 ± 1.37A	50.7 ± 5.13A	36.4 ± 3.04B
	450	17.1 ± 1.40A	2.68 ± 0.260A	35.3 ± 1.86B	45.2 ± 4.15B	38.9 ± 2.65A
Biofertiliser	BlueN 1x	17.6 ± 1.50x	2.86 ± 0.177x	36.2 ± 1.81x	46.7 ± 4.97x	38.2 ± 2.91x
	BlueN 2x	17.5 ± 1.10x	2.69 ± 0.127y	37.1 ± 1.94x	48.7 ± 5.60x	37.3 ± 3.54x
	Control	16.9 ± 1.22x	2.49 ± 0.150z	36.6 ± 2.45x	48.4 ± 5.65x	37.4 ± 2.90x

GN, grain number; TGW, thousand grain weight; GY, grain yield; GF1, grain fraction with a diameter > 2.5 mm; GF2, grain fraction with a diameter 2.2–2.5 mm. ^{a – b}, ^{A – B}, ^{x – z}, values with different letters significantly differ at p < 0.05. The sign ± refers to SD.

Table 4.

Grain yield, and grain parameters of the H. v. rimpaui in 2024.

Treatments		GN [no ear−1]	GY [t ha−1]	TGW [g]	GF1 > 2.5 mm [%]	GF2 2.2–2.5 mm [%]
Soil tillage	Ploughing	15.2 ± 0.765 a	1.81 ± 0.112 a	28.6 ± 0.707 b	19.2 ± 1.83 b	49.5 ± 1.40 a
	Reduced	14.8 ± 1.06 a	1.75 ± 0.133 a	29.7 ± 1.57 a	26.5 ± 3.48 a	47.8 ± 1.61 b
Sowing density [no of grains m−2]	350	15.5 ± 0.557 A	1.78 ± 0.142 A	29.3 ± 1.36 A	23.1 ± 3.95 A	49.1 ± 1.65 A
	450	14.5 ± 0.954 B	1.78 ± 0.109 A	29.0 ± 1.30 A	22.6 ± 5.24 A	48.2 ± 1.73 B
Biofertiliser	BlueN 1x	15.4 ± 0.676 x	1.84 ± 0.109 x	28.9 ± 1.25 xy	22.8 ± 4.05 x	49.0 ± 1.49 x
	BlueN 2x	15.0 ± 1.15 xy	1.74 ± 0.122 y	29.8 ± 1.29 x	23.3 ± 6.00 x	48.8 ± 2.07 x
	Control	14.7 ± 0.794 y	1.76 ± 0.126 y	28.7 ± 1.23 y	22.4 ± 3.66 x	48.2 ± 1.60 x

The abbreviations are defined under the Table 3.

In 2024, as was in the 2023, reduced tillage contributed to the production of barley grains with a higher thousand-grain weight, as well as a significantly larger proportion of grains with a diameter above 2.5 mm (Table 4). In that year, the plants cultivated at a density of 350 grains per m² exhibited an increase in the number of grains per ear and proportion of grain between 2.2 and 2.5 millimeters in diameter. The application of BlueN was found to have an impact on GN, GY and TGW. A single application of BlueN has been shown to exert a stimulatory effect on the grain number per ear (GN) and grain yield (GY), while a sequential application to promote the grain weight (TGW) (Table 4).

In the first year of the study, significant interaction was observed between the soil tillage, sowing density, and biofertiliser, with respect to grain yield (Supplementary Materials, Table S2, Fig. 2). It can be noted that the single application had the greatest positive effect on the barley yield, especially when plants were sown in greater density under reduced tillage.

Fig. 2.

Grain yield of black barley: (A) in 2023; and (B) in 2024. ^{a – e}, values with different letters significantly differ at p < 0.05.

Relationship of plant and grain features, and grain yield

A statistically significant, strong (r > 0.6) and very strong (r > 0.8), positive correlation was observed between each of the following pairs of variables: EN (no per m²), GY (t ha⁻¹), GN (no per ear), TGW (g) and GF1 > 2,5 mm (%). In contrast, all of these variables demonstrated a strong or very strong negative correlation with RL (cm) and GF2 2,2–2,5 mm (%), with RL and GF2 exhibiting a strong positive correlation with each other (Fig. 3).

Fig. 3.

Heatmap for Pearson’s correlation coefficients between analysed biometric parameters, yield and its components, and grain parameters. RL, root length; GTL, tiller length; GTN, number of generative tillers; EN, ears number; GN, grain number; TGW, thousand grain weight; GY, grain yield; GF1, grain fraction with a diameter > 2.5 mm; GF2, grain fraction with a diameter 2.2–2.5 mm. Correlation significantly important at p < 0.05.

Dry matter, nitrogen content and nitrogen uptake

Over the two-year study period, the experimental factors exerted divergent influences on the shoot dry matter, N content and N uptake (Supplementary Materials, Table S1, S2 and Tables 5 and 6).

Table 5.

Dry matter, nitrogen content and nitrogen uptake by H. v. rimpaui in 2023.

Treatments		DMshoot [g m−2]	NCleaf [g kg−1]	NCshoot [g kg−1]	NUshoot [kg ha−1]
Soil tillage	Ploughing	491.4 ± 60.5b	29.6 ± 4.21a	10.5 ± 1.13a	52.0 ± 9.89b
	Reduced	527.8 ± 81.9a	31.1 ± 2.60a	10.8 ± 0.773a	57.1 ± 9.01a
Sowing density [no of grains m−2]	350	504.5 ± 77.7A	31.2 ± 2.41A	10.9 ± 0.921A	54.9 ± 9.74A
	450	514.7 ± 70.5A	29.5 ± 4.27A	10.5 ± 0.998A	54.2 ± 9.87A
Biofertiliser	BlueN 1x	566.7 ± 65.5x	30.1 ± 5.10x	10.7 ± 0.745x	60.6 ± 7.37x
	BlueN 2x	512.2 ± 48.8y	29.9 ± 2.44x	10.6 ± 1.18x	54.4 ± 9.38xy
	Control	449.9 ± 54.9z	31.1 ± 2.53x	10.8 ± 0.984x	48.7 ± 8.82y

DM_shoot, shoots dry matter; NC_leaf, nitrogen content in flag-leaf; NC_shoot, nitrogen content in shoots; NU_shoot, nitrogen uptake by shoots. ^{a – b}, ^{x – z}, values with different letters significantly differ at p < 0.05. The sign ± refers to SD.

Table 6.

Dry matter, nitrogen content and nitrogen uptake by H. v. rimpaui in 2024.

Treatments		DMshoot [g m−2]	NCleaf [g kg−1]	NCshoot [g kg−1]	NUshoot [kg ha−1]
Soil tillage	Ploughing	437.2 ± 86.1a	21.4 ± 2.44a	6.57 ± 0.466a	28.9 ± 6.87a
	Reduced	408.2 ± 87.1a	21.2 ± 1.27a	6.34 ± 0.531b	24.1 ± 6.19a
Sowing density [no of grains m−2]	350	418.8 ± 90.3A	21.5 ± 1.51A	6.67 ± 0.259A	26.2 ± 6.77A
	450	426.6 ± 85.3A	21.1 ± 2.28B	6.24 ± 0.604B	26.8 ± 7.14A
Biofertiliser	BlueN 1x	445.9 ± 122.1x	21.7 ± 0.825y	6.69 ± 0.343x	30.0 ± 9.03x
	BlueN 2x	398.6 ± 72.3x	22.7 ± 1.79x	6.55 ± 0.469x	26.1 ± 4.99xy
	Control	423.6 ± 48.5x	19.5 ± 1.36z	6.12 ± 0.532y	23.4 ± 4.36y

The abbreviations are defined under the Table 5.

In 2023, a significant influence of soil tillage and biofertiliser on the shoots dry matter (DM_shoot) and nitrogen uptake by shoots (NU_shoot) (Table 5) was found. The highest values of the DM_shoot and the NU_shoot were observed under reduced tillage. A single application of biofertiliser (BlueN 1x) resulted in the greatest accumulation of shoots dry matter (DM_shoot) and to significantly higher nitrogen uptake by shoots (NU_shoot) in comparison to control. The employed sowing density (350 and 450 grains per m²) did not exert any influence on the DM_shoot, NC_leaf, NC_shoot and NU_shoot during the first study year. It is noteworthy that neither the nitrogen content of the flag leaves (NC_leaf) nor that of the shoots (NU_shoot) was influenced by soil tillage, sowing density, or biofertiliser.

In 2024 the most influenced variables by the experimental factors were NC_leaf and NC_shoot (Table 6). Shoots of barley grown under ploughing showed higher NC than those under reduced tillage. The density of 350 grains per m² resulted in the highest concentration of N in both flag leaves and shoots. In 2024, both BlueN application regimes resulted in higher NC_leaf and NC_shoot than in the control. Furthermore, it is notable that the highest NC_leaf were achieved after sequential application of biofertiliser (Table 6). In addition, the single dose resulted in enhanced nitrogen uptake by barley shoots in comparison to the control.

It is important to emphasise the noteworthy findings that emerged from the observed interaction between the applied soil tillage treatments, sowing density, and biofertiliser, with respect to nitrogen content both in the leaves and shoots (Supplementary Materials, Tab. S2, Figs. 4 and 5). It has been demonstrated that, in the second year of the study, the sequential application of BlueN contributed to the highest concentration of NC_leaf when barley was sown in a density of 450 grains per m² under ploughing. The efficacy of this application regime to elevate the NC_leaf was also demonstrated in barley grown under reduced tillage, but in smaller density (Fig. 4B).

Fig. 4.

N content in flag-leaf of black barley: (A) in 2023; and (B) in 2024. ^{a – f} – values with different letters significantly differ at p < 0.05.

Fig. 5.

N content in shoots of black barley: (A) in 2023; and (B) in 2024. ^{a – d}, values with different letters significantly differ at p < 0.05.

The analysis of three-factor interaction revealed some significant influences of them on nitrogen content in shoots in 2023; nevertheless, more substantial influences were identified in 2024 (Fig. 5). It has been observed that the application of biofertiliser in 2024 has generally led to an enhancement in nitrogen (N) levels in the shoots, with the exception of barley sown in a reduced tillage and smaller sowing density (350 no grains per m²).

Phosphorus and potassium content and uptake

Throughout the experimental period, the phosphorus (PC_shoot) and potassium content (KC_shoot) and their uptake (PU_shoot; KU_shoot) by barley shoots exhibited greater sensitivity to the experimental factors in the first study year (Supplementary Materials, Tab. S1, S2 and Tables 7 and 8). In 2023, soil tillage exclusively influenced the content of potassium in shoots, with a higher K content found under ploughing (Table 7). Moreover, it was observed that a density of 350 grains per 1m² was more conducive to the K content (KC_shoot) and K uptake (KU_shoot) by barley than a higher density (450 no grains per m²). The application of biofertiliser was found to have a positive effect on PC_shoot, PU_shoot and KU_shoot. A single application of BlueN increased these parameters by 4.78%, 31.8%, and 40.5%, respectively, when compared to the control. The double application of BlueN resulted in the following increases: 8.53%, 23.5% and 17.4%, respectively (Table 7).

Table 7.

Phosphorus content, phosphorus uptake, potassium content and potassium uptake by H. v. rimpaui in 2023.

Treatments		PCshoot [g kg−1]	PUshoot [kg ha−1]	KCshoot [g kg−1]	KUshoot [kg ha−1]
Soil tillage	Ploughing	3.09 ± 0.295a	15.2 ± 2.66a	33.0 ± 11.3a	145.2 ± 35.7a
	Reduced	3.04 ± 0.205a	16.0 ± 2.43a	27.5 ± 4.76b	163.6 ± 64.3a
Sowing density [no of grains m−2]	350	3.08 ± 0.296A	15.5 ± 2.71A	33.7 ± 10.2A	171.1 ± 63.2A
	450	3.05 ± 0.205A	15.7 ± 2.44A	26.9 ± 6.12B	137.7 ± 31.9B
Biofertiliser	BlueN 1x	3.07 ± 0.199xy	17.4 ± 1.64x	32.1 ± 8.84x	181.8 ± 55.6x
	BlueN 2x	3.18 ± 0.306x	16.3 ± 2.19x	29.7 ± 10.8x	151.9 ± 55.0xy
	Control	2.93 ± 0.180y	13.2 ± 1.67y	29.0 ± 7.23x	129.4 ± 31.4y

PC_shoot, phosphorus content in shoots; PU_shoot, phosphorus uptake by shoots; KC_shoot, potassium content in shoots; KU_shoot, potassium uptake by shoots. ^{a – b}, ^{A – B}, ^{x – y}, values with different letters significantly differ at p < 0.05. The sign ± refers to SD.

Table 8.

Phosphorus content, phosphorus uptake, potassium content and potassium uptake by H. v. rimpaui in 2024.

Treatments		PCshoot [g kg−1]	PUshoot [kg ha−1]	KCshoot [g kg−1]	KUshoot [kg ha−1]
Soil tillage	Ploughing	2.97 ± 0.332a	12.7 ± 3.01a	10.8 ± 0.996a	47.4 ± 10.9a
	Reduced	2.33 ± 0.167b	9.14 ± 2.26b	10.8 ± 1.06a	41.5 ± 11.1a
Sowing density [no of grains m−2]	350	2.69 ± 0.481A	11.0 ± 3.57A	10.9 ± 1.00A	42.9 ± 11.4A
	450	2.55 ± 0.258B	10.9 ± 2.87A	10.7 ± 1.05A	46.0 ± 11.1A
Biofertiliser	BlueN 1x	2.60 ± 0.293x	11.7 ± 3.85x	10.7 ± 1.03x	47.9 ± 14.6x
	BlueN 2x	2.61 ± 0.331x	10.5 ± 2.61x	10.5 ± 0.664x	41.8 ± 8.46x
	Control	2.65 ± 0.526x	10.6 ± 3.09x	11.2 ± 1.19x	43.6 ± 9.50x

The abbreviations are defined under the Table 7.

In 2024, ploughing tillage elevated the PC_shoot and PU_shoot. Furthermore, a smaller sowing density (350 no grains per m²) resulted in an increase in PC_shoot. In the experiment conducted in 2024 no significant impact of biofertiliser on the PC_shoot, PU_shoot, KC_shoot and KU_shoot were found (Table 8).

Relationship of dry matter, NPK content and uptake

The statistical analysis revealed a significant positive correlation for each of the pairs of variables analysed: DM_shoot, NC_leaf, NC_shoot, NU_shoot, PC_shoot, PU_shoot and KU_shoot. As can be observed, DM_shoot exhibited very strong (r > 0.8) or close to very strong correlation with the uptake of macronutrients, particularly nitrogen (NU_shoot) and phosphorus (PU_shoot). A comparable degree of strength of relationship was also observed between the content of each N, P and K and their uptake by shoots (Fig. 6).

Fig. 6.

Heatmap for Pearson’s correlation coefficients between dry matter, nitrogen, phosphorus and potassium content and nitrogen, phosphorus and potassium uptake by H. v. rimpaui. DM_shoot, shoots dry matter; NC_leaf, nitrogen content in flag-leaf; NC_shoot, nitrogen content in shoots; NU_shoot, nitrogen uptake by shoots; PC_shoot, phosphorus content in shoots; PU_shoot, phosphorus uptake by shoots; KC_shoot, potassium content in shoots; KU_shoot, potassium uptake by shoots. Correlation significantly important at p < 0.05.

Principal component analysis (PCA)

To evaluate the correlation between the type of soil tillage (ploughing; reduced), sowing density (350 or 450 no of grains per m²), biofertiliser application (BlueN 1x; BlueN 2x; Control) and analysed parameters of H. v. rimpaui, a principal component analysis (PCA) was performed (Fig. 7). The biplot PCA explained 72.64% of the first principal component (PC1) and 12.12% of the second principal component (PC2) from 84.76% of the total variance (Fig. 7). The results of PCA showed that analysed variables had greater impact on the PC1 variance. The results of the Principal Component Analysis (PCA) demonstrate that the year in which the study was conducted exerted a more substantial influence on the results obtained than the implementation of any given factor. In the second year of the study, root length (RL) and tiller length (GTL), as well as the proportion of GF2, were found to be most strongly shaped by ploughing tillage (Fig. 7). In 2023, the interaction of experimental factors exerted a divergent influence on the studied variables. It can be observed that ploughing and no biofertiliser application exerted the greatest influence on NC_leaf, NC_shoot, KC_shoot, EN, TGW and G1 > 2.5 mm. Moreover, the present study has demonstrated that the reduced tillage, in combination with the application of BlueN, has a tendency to influence the parameters of GY, GN, GTN, SDM, PC_shoot and the uptake of macroelements (NU_shoot, PU_shoot, KU_shoot).

Fig. 7.

Biplot principal component analysis (PCA) of analysed factors and parameters of H. v. rimpaui.

P, ploughing tillage; R, reduced tillage; 350/450, no of grains/m² (sowing density); C, control; B1, BlueN 1x; B2, BlueN 2x; 23, 2023; 24, 2024. RL, root length; GTL, generative tiller length; GTN, number of generative tillers; EN, ear number; GN, grain number; TGW, thousand grain weight; GY, grain yield; GF1 > 2.5 mm, share of grain fraction with a diameter > 2.5 mm; GF2 2.2–2.5 mm, share of grain fraction with a diameter 2.2–2.5 mm; DM_shoots, shoots dry matter; NC_leaf, nitrogen content in flag-leaf; NC_shoot, nitrogen content in shoots; NU_shoot, nitrogen uptake by shoots; PC_shoot, phosphorus content in shoots; PU_shoot, phosphorus uptake by shoots; KC_shoot, potassium content in shoots; KU_shoot, potassium uptake by shoots.

Discussion

Soil tillage, sowing density, and biofertilisation influenced biometric parameters, ear number, grain yield, yield components, and grain parameters of H. v. rimpaui. Furthermore, these traits were significantly modified by varying weather conditions during the growing seasons.

In the present study, the barley roots were longer in 2024 than in 2023. This phenomenon may be attributable to increased root growth under periodic soil water deprivation, driven by limited rainfall in 2024 (Fig. 1)³⁰. The development of longer roots under ploughing in 2024 may also be attributable to variations in soil water availability. The predominant viewpoint is that conventional ploughing results in greater water loss than reduced tillage, which may promote root elongation³¹. Moreover, in the first study year, when the plants experienced reduced exposure to stress induced by water deficiency, increased root length was observed in the control (without the application of biofertiliser), which can be attributed to the plants’ desire to increase the nitrogen absorption surface, in contrast to treatments where this nutrient was more available through the BlueN application³². In our study, root length was strongly negatively correlated with most biometric parameters (Fig. 3), which obtained the lowest values in a year characterized by suboptimal weather patterns (dry spring with Sielnaninov index k < 1.3). It follows from the above that this root elongation was not enough to ensure efficient plant growth.

As demonstrated in Tables 1 and 2, GTL and GTN are contingent on weather conditions, which, in turn, might influence other factors such as soil tillage, sowing density, and biofertilisation. As posited by some researchers, the RTL or GTN may be modulated indirectly through soil tillage, which influences factors such as temperature, water availability, macronutrient availability, and biological activity, all of which directly affect plant growth^33,34. However, it has been established that the number of tillers developed by cereals during tillering is primarily determined by genotypic traits, hormones, and enzyme activity^35,36. The activity of abscisic acid (ABA) is a pivotal factor in the process of tiller formation in plants. The activity of ABA is also contingent upon the presence of soil microbes or soil organic matter (SOM)³⁷, which in turn is subject to modulation by tillage methods. Nonetheless, the extant data and the study conducted have demonstrated discrepancies in how plants respond to ST with respect to GTL or GTN (Tables 1 and 2). Consequently, further research is required to ascertain this matter. In the present study, longer tillers and more tillers per plant were observed at a density of 350 grains per m², which is in accordance with the findings of Liao et al.³⁶. An elevated plant density has been shown to result in an intensification of shadow at the stem that is involved in the process of main-stem elongation and tiller formation³⁶.

It is widely accepted that nitrogen plays a critical role in plant development. The form and dosage of that nutrient carry significant importance on many parameters such as length or number of tillers³⁸. In our study, the single application of nitrogen-fixing bacteria promoted tiller elongation by ensuring better nutrition with this macronutrient.

In the present research, no influence of the factors on the number of ears (EN per m²) was observed, although discrepancies were detected between growing seasons. This finding is consistent with the hypothesis that this trait is significantly sensitive to meteorological conditions³⁹. It is noteworthy, however, that the EN has a strong positive correlation with GN, TGW, GF1 share and, most importantly, GY (Fig. 3). The number of ears per plant, in combination with the number of ear-fertile florets and grain set after pollination, is a key factor in determining the eventual GN⁴⁰.

In the present research study, the experimental factors have been found to exert a divergent influence on the grain number per ear obtained in both years (Tables 3 and 4). The weather conditions are considered to be a primary element that shapes the grain number of cereals, with a direct influence on pollination, the stages of grain development, and their filling^39,41. This may provide a rationale for the observed differences in the present research.

The impact of sowing density or N status (referred to as BlueN in this study) has been previously explored in several research works^42,43. The findings of this study are consistent with the extant data, which demonstrate that optimal plant density or nitrogen fertilisation can have a positive effect on the number of grains per ear. The impact of soil tillage on ear parameters, particularly under organic farming, remains a subject that has not been extensively researched to date. In our study, we found that under optimal weather conditions, reduced tillage resulted in an augmentation in the number of grains per ear. It can be hypothesized that these practices exerted a positive influence on soil physico-chemical and microbiological properties, which in turn influenced the development of grains⁴⁴. There is a widely accepted belief that a notable negative correlation exists between GN and TGW, and that relationship⁴⁰. Nonetheless, during our research, we obtained divergent results. The correlation between these variables was strong yet positive (Fig. 3). It may be plausible to attribute this to the specific properties of the tested barley species (H. v. var. rimpaui).

The grain weight (TGW) is a crucial component in determining plant yield, and since the inception of plant breeding, breeders have sought to enhance it. The implementation of contemporary breeding techniques in the cultivation of cereal cultivars has led to specific changes in their genomic structure, thereby facilitating the increased grain weight, while simultaneously decreasing the number of grains per ear^45,46. The subject of the present study, H. v. rimpaui, is an ancient grain that has not been subjected to any form of breeding. Consequently, specific components of grain yield may be related differently from common cultivars, thus presenting a valuable opportunity for detailed research on this topic. It was observed that grain weight (TGW) and the share of grain fractions with a diameter > 2.5 mm (GF1) were strongly positively correlated (Fig. 3), and were notably higher in 2023. This finding suggests a significant dependence of grain development on weather conditions³⁹.

Notwithstanding the study season, reduced tillage has been shown to enhance grain size significantly (Tables 3 and 4). This attribute, akin to ear number, may depend on soil physical and chemical characteristics, which are shaped by various tillage practices^44,47. In the second year of the study, the TGW was significantly influenced by biofertilisation, and a similar trend was observed in the first year, especially with the dual application of BlueN. The process of grain filling is contingent upon various factors, including the plant’s capacity to accumulate and redistribute nitrogen, a capacity which fluctuates throughout the various stages of plant growth⁴⁸. The present study suggests that the term and the number of applications of nitrogen-fixing bacteria may be pivotal in ensuring the most active nitrogen management in plants (Tables 3 and 4). This, in turn, has a stimulatory effect on grain filling.

The mean grain yield (GY) in the first year was approximately 1 t ha⁻¹ higher than in 2024 (Tables 3 and 4). This finding is consistent with those reported by Nowak et al.²², who observed that weather conditions significantly influence yields of ancient barley. The findings of our study confirmed the primacy of the influence of three factors on grain yield (so-called yield components)⁴⁹: number of ears (EN), grain number per ear (GN), and thousand-grain weight (TGW) (Fig. 3). It is worth noting that the influence of the factors on GY was comparable across both years of the study, but that the obtained outcomes did not entirely align with the results for the GY components (Tables 3 and 4).

In both years, the highest grain yield was achieved with a single application of BlueN. The impact of Methylobacterium symbioticum (BlueN) on barley, particularly the ancient form, has not been comprehensively studied. Nevertheless, it is plausible to posit that the advantage of single over double treatment may be attributable to the bacterium’s enhanced capacity to colonize aboveground biomass in reduced N availability, as in the case of only a single application, facilitated by biochemical processes associated with ACC enzymes^9,50. The activity of M. symbioticum in the plants also depends on the amount of carbon required for their proper functioning. The lower yield of black barley following dual application could be attributed to the increased competitiveness of the applied bacterium for carbon sources derived from plants¹¹.

In the year that was distinguished by more favourable weather conditions, the influence of the interaction between soil tillage x sowing density x fertilisation on barley grain yield was observed. A solitary application obtained a superior yield; however, under reduced tillage conditions, higher yield was observed at a plant density of 450 no per m², as opposed to 350 no per m², when employing ploughing techniques (Fig. 2). This finding is consistent with a recent viewpoint that plant growth-promoting bacteria may exert divergent effects on plant growth under exact agronomic management^9,51. This indicates that further research is required to facilitate a more complete understanding of the phenomenon. In our study, the dry matter content of shoots was higher in 2023, which was more optimal for plant development (Tables 5 and 6). This indicates that the yield of aboveground biomass of Hordeum species is primarily contingent upon prevailing weather patterns⁵². In the first year of the study, reduced tillage was found to increase shoot dry matter content. This phenomenon may be attributed to the hypothesis proposed by Krauss et al.⁶ in which they suggested that reduced tillage in organic farming, by virtue of its ability to enhance soil physical conditions, nutrient distribution, and to effect shifts in soil biological communities may have a beneficial impact on aboveground biomass production.

The application of M. symbioticum also led to an enhancement in dry matter content of barley shoots (Tables 5 and 6). This may be attributable not only to their capacity for N-fixation but also to their role as a source of cytokinin, which play a pivotal role in various plant processes, such as shoot growth and nutrient uptake⁵³. This ability to fix nitrogen is significant, given that nitrogen constitutes the most essential nutrient for all plants. It has considerable effects on plant growth, plant development, hormonal and metabolic activity, photosynthesis and ultimately on yield and its quality⁵⁴.

Aboveground biomass is the primary reservoir of nitrogen, with the flag-leaf constituting a significant pool of this nutrient. The intensity and duration of N remobilisation from the flag leaf have been demonstrated to exert a significant influence on the protein content and grain filling of cereals⁵⁵. This observation provides a rational basis for the monitoring of NC in those leaves. In 2023, the impact of soil tillage and biofertilisation on the nitrogen uptake by plants (NU_shoot) was predominantly associated with the observed influence of these factors on dry matter of shoot (DM_shoot), as no effect of these factors was observed on nitrogen content (NC) in leaf and shoot (Tables 5 and 6). This hypothesis is further substantiated by the observation of a high and positive correlation between the nitrogen uptake by plants (NU_shoot) and the biomass of barley (DM_shoot) (Fig. 6).

In the second year of the study, the applied treatments had more pronounced effects on the content of nitrogen in both the whole shoot biomass and flag and sub-flag leaves. A higher N content was observed in the shoots of barley under ploughing compared to those under reduced tillage, probably due to the longer roots (Table 2), which facilitated the uptake of nitrogen from the soil profile⁵⁶. It was observed that at higher sowing density (450 plants per m²), the N concentration in both shoots and leaves was smaller than that at lower density. This is most likely the ‘dilution effect’^42,57.

In the second year of our study, the augmented NC was observed in leaves and shoots following biofertilisation (Table 6). Methylobacterium symbioticum employed in this study are can thrive in the phyllosphere of their host plants and are capable of fixing and converting atmospheric N₂ into a form that is subsequently available to plants, thus increasing the quantity of N to support their growth⁹.

Furthermore, the present study demonstrated that the NC_leaf and NC_shoot were influenced by the soil tillage x sowing density x fertilisation interaction; especially in 2024 (Figs. 4 and 5). BlueN application (particularly dual) resulted in enhanced nitrogen concentration in plants when barley was sown at 450 no per m² under ploughing. However, this increase also occurred in reduced tillage, when barley was sown at 350 grains per m². Given the paucity of research conducted on this subject, further investigation is warranted to elucidate the observed phenomena.

As previously observed for other variables, the effects of the analysed treatments on phosphorus and potassium content and uptake differed in both years of the study (Tables 7 and 8). The availability of both nutrients, their uptake and final concentration are depend on many aspects such as environmental conditions, tillage practices, soil properties, and microbial activity⁵⁸. Phosphorus is the second most vital nutrient for plants. It is imperative for the transfer of energy (ATP) and signal transmission, the construction of nucleic acids and phospholipids, and various other processes⁵⁹. In 2023, a positive impact of biofertilisation on the P content in shoots was observed, which may be attributable to the ability of some Methylobacterium spp. to make inorganic phosphate forms of P available to the plant¹¹.

In the second year of study, the higher P content in shoots was under ploughing (Table 8). P is immobile in the soil profile, and its vertical distribution is contingent, among other factors, on soil mixing, which is minimal in reduced tillage⁶⁰. Given the longer root system of barley under ploughing in 2024 and the probable more even P distribution, it can be hypothesised that those had a beneficial impact on P uptake and its concentration in shoots. A comparative analysis of similar relationships can be found in the study conducted by Yakahashi et al.⁶¹ and Szczepanek et al.⁶². Similarly, as in the case of nitrogen, the phosphorus content in shoots was also higher at a smaller sowing density (350 no m²). It is probably due to reduced competition among plants for available P with less plant biomass⁶³. The significant correlation between PU_shoot and both DM_shoot and PC_shoot indicates a strong interdependence among these variables (Fig. 6).

Potassium (K) is another significant nutrient that plays a pivotal role in crop yield, and stress resistance⁶⁴. The impact of treatments on KC and KU was identified during the first year of the study (Table 7). This finding is likely attributable to the more equitable distribution of precipitation, which has a notable influence on non- and exchangeable K⁺ sources for plants⁶⁵. Under ploughing K content in shoots was higher than under reduced tillage. Ploughing contributes to redistribution of K within the soil and improves its accessibility to plants⁶⁶. In turn, the increased concentration of K in barley sown in lower density may be explained by the same rationale as that for N and P concentration⁶³.

Conclusions

The present study has demonstrated that the varying weather conditions have exerted different influences on organically cultivated H. v. rimpaui. Biofertilisation and soil tillage had the strongest effect on yield, its components, and macroelement content and uptake, while sowing density had the weakest. According to PCA analysis biofertiliser applied under reduced tillage most strongly affected GN, GY, DM_shoot, PC_shoot and macroelement uptake. In contrast, the lack of BlueN under both tillage methods influenced plant N and K concentrations (NC_leaf, NC_shoot, KC_shoot), EN, and grain size (TGW, G1 > 2.5 mm).

Under favorable weather conditions for barley, reduced tillage increased GN, TGW, GF1, and GTN per plant (with 8.4%, 5.3%, 7.1%, and 15.2% increase compared with ploughing, respectively). The highest grain yield was achieved with reduced tillage, at a sowing density of 450 no of grain per m², after a single application of biofertilizer (3.08 t ha⁻¹). Under unfavorable weather conditions (dry spring), ploughing stimulated root (+ 12.5% ) and shoot elongation (+ 5.1%) and increased the number of shoots per plant (+ 19.8%). However, by increasing the proportion of fine grain (GF2; + 3.6%), it contributed to a reduction in TGW, which was 3.7% lower than that observed in reduced tillage. This phenomenon may indicate that plough-induced soil drying promotes deeper rooting but smaller grain formation, ultimately reducing yield.

The outcomes of the study demonstrate that satisfactory productivity can be maintained at lower densities (with mean yield in favourable year ranging 2.68 t ha⁻¹), which also reduces pressure from pests, diseases, lodging, and competition, while lowering seed requirements. The present study is consistent with the current trend of increased use of reduced-tillage techniques, which may offer a viable alternative in organic systems that are traditionally based on conventional tillage methods. One of the most significant challenges confronting organic farming, namely the limited availability of nitrogen, can be mitigated by the utilisation of innovative products comprising microorganisms such as N-fixing bacteria. The findings of this study demonstrate that M. symbioticum generally has a stimulatory effect on barley yield (+ 14.9% under favourable conditions and + 4.5% in a dry year), and aboveground biomass (DM; an average increase of 26% under favourable weather conditions and + 5.3% in a dry year). This underscores its potential value in enhancing plant performance in diverse climates. However, the results indicate that N-fixing bacteria yielded the best results when applied to barley cultivated under reduced tillage in optimal conditions. Although this effect was not observed under suboptimal weather conditions, the absence of yield reduction is itself advantageous, thereby supporting the feasibility of simplified cultivation practices in organic systems.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, K.B. and M.S.; Methodology, K.B. and M.S.; Software, K.B.; Validation, K.B. and M.S.; Formal analysis, K.B. and M.S.; Investigation, K.B., M.S. and M.P.; Resources, K.B. and M.S; Data curation, K.B., M.S. and M.P.; Writing—original draft, K.B. and M.S.; Writing—review & editing, K.B., M.S., and M.P.; Visualization, K.B.; Supervision, K.B. and M. S. All authors have read and agreed to the published version of the manuscript.

Funding

The source of funding: European Agricultural Fund for Rural Development: Europe investing in rural areas. The publication is co-financed from the European Union funds under the COOPERATION of the Rural Development Programme for 2014–2020. The Managing Authority of the Rural Development Programme for 2014–2020 is the Minister of Agriculture and Rural Development. Project no 00066.DDD.6509.00175.2022.11.

Data availability

The data presented in this study are available on request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.