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Article

The Effect of the Nitrogen-Fixing Bacteria and Companion Red Clover on the Total Protein Content and Yield of the Grain of Spring Barley Grown in a System of Organic Agriculture

by
Anna Płaza
1,
Alicja Niewiadomska
2,
Rafał Górski
3,*,
Robert Rudziński
3 and
Emilia Rzążewska
1
1
Institute of Agriculture and Horticulture, Faculty of Agrobioengineering and Animal Husbandry, Siedlce University of Natural Sciences and Humanities, B. Prusa 14, 08-110 Siedlce, Poland
2
Department of Soil Science and Microbiology, Poznań University of Life Sciences, Szydłowska 50, 60-656 Poznań, Poland
3
Faculty of Engineering and Economics, Vocational State School of Ignacy Mościcki in Ciechanów, Narutowicza 9, 06-400 Ciechanów, Poland
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(7), 1522; https://doi.org/10.3390/agronomy12071522
Submission received: 18 May 2022 / Revised: 21 June 2022 / Accepted: 24 June 2022 / Published: 24 June 2022
(This article belongs to the Special Issue Nitrogen Cycle in Farming Systems)
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Versions Notes

Abstract

:
Field research was conducted in Poland from 2019–2021 to determine the effect of the bacteria Azospirillum lipoferum Br17 and Azotobacter chroococcum, as well as companion red clover on the total protein content and yield in the grain of spring barley cultivated in a system of organic agriculture. Two factors were examined in the field experiment: I. bacterial formulations: 1—control, 2—nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum), 3—nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum) + phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis), and 4—nitrogen-fixing bacteria (Azotobacter chroococcum) + plant growth-promoting rhizobacteria (PGPR) (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens); II. companion crop: control without a companion crop, red clover, and red clover + Italian ryegrass. In spring barley grain, the total protein content was determined and the total protein yield was calculated. The obtained study results demonstrated that the growing season conditions significantly affected the total protein content and yield in the spring barley grain. The highest total protein content was recorded in the grain of spring barley following an application of nitrogen-fixing bacteria (Azotobacter chroococcum) combined with PGPR (Bacillus subtilis, Pseudomonas fluorescens) and grown with companion red clover.

1. Introduction

Spring barley grain produced in organic agriculture stands out due to its higher content of valuable protein compared with conventionally produced barley [1,2,3]. As a result, it is a high-value raw material for the production of groats and flakes used as functional foods. The protein content of spring barley grain is dependent upon nitrogen inputs. In organic farming, natural and green manures from leguminous crops are the main nitrogen source for crop plants. The aim of this research was to demonstrate that the total protein content and yield in the grain of spring barley produced in a system of organic agriculture may be influenced by appropriate agrotechnological practices using innovative fertilisation methods based on bacterial formulations. The bacteria Azotobacter and Azospirillum are characterised by an ability to biologically convert nitrogen into ammonia which is essential for crop plants to synthesise the amino acids making up their protein [4,5,6]. In the research, nitrogen-fixing bacterial inoculants were used, whose effect on the total protein content and yield have not been studied previously in the grain of spring barley cultivated in Poland under the conditions of organic agriculture, which also boosts the biological activity and fertility of soil. The inoculants contain two bacterial species Azospirillum lipoferum and Azotobacter chroococcum which make nitrogen available to plants. The high nitrogen-fixing potential of these bacteria is due to their ability to produce the enzyme nitrogenase, the property increasing the effectiveness of nitrogen uptake by cereals. The bacteria assimilate N2 only to meet the needs of their cell metabolism so they do not release the fixed nitrogen into the environment. The nitrogen reaches the soil environment only after the death of the bacterial cells. Due to this, the plant rhizosphere, where the quantity and availability of nutrients and energy-supplying components is much higher, is a good place for the development of Azotobacter spp. [7,8,9]. New technologies using biofertilizers need to be introduced to ensure crop yield stability. Currently, this issue is receiving attention from researchers around the world [4,10,11,12]. Biofertilizers promote plant growth by providing nutrients including biologically bound nitrogen or increasing the availability of insoluble nutrients in the soil and synthesizing substances that stimulate plant growth [10,13]. Biofertilizers are economically and ecologically attractive means of improving crop quality and quantity [12]. They are cheaper and improve crop growth and quality by stimulating direct or indirect release of plant hormones [11]. Plant growth-promoting rhizobacteria (PGPR) improve root development and plant growth through solubilization of insoluble P and excretion of plant growth-promoting hormones [13]. Rhizospheric and endophytic bacteria are free-living bacteria that are voluntarily associated with the rhizosphere and within plant roots, and directly correlate with improved plant growth and yield [14,15]. In recent years, the use of beneficial microorganisms in cereals has revealed their positive effects on cereal yield and quality under adverse environmental conditions [16,17].
Cultivation of legumes as companion crops provides another nitrogen source for cereals. The renascence of intercropping with leguminous crops is undoubtedly associated with the present-day tendencies in agriculture whose objective is to promote organic agriculture, which is occurring worldwide. Here, an important role is ascribed to mixtures of leguminous plants and grasses. The plants complement each other as deeply rooted legumes can take up water and minerals from the subsoil whereas the root system of grasses and cereals makes use of the surface soil layer and the nitrogen released to the soil by the leguminous crop. The best understood effect of legumes on soil is its enrichment in nitrogen fixed by the nodulating bacteria Rhizobium which live in symbiosis with leguminous plants [18,19,20,21]. There is a noticeable lack of research pertaining to this topic in Poland. The present work was an attempt to at least partially fill in this gap as its objective was to determine the effect of the bacteria Azospirillum lipoferum and Azotobacter chroococcum, as well as a companion crop of red clover, on the total protein content and yield of the grain of spring barley cultivated in an organically-managed system of farming.

2. Materials and Methods

2.1. Research Methodology

A field experiment was carried out in Poland from 2019–2021 on an organic farm in the village of Wyłazy near Siedlce. The soil of the experimental site was Stagnic Luvisol. The soil reaction was neutral (pH in KCl 6.1) and the organic carbon content was 1.05% a.d.m. The content of available mineral elements in the soil was as follows: P, 8.3 mg⸱100 g−1 soil; K, 12.1 mg⸱100 g−1 soil; and Mg, 4.2 mg⸱100 g−1 soil.

2.2. Agrotechnological Practices

The experimental design was a split-block arrangement with three replicates. Two factors were investigated: I. bacterial formulations: control (without bacterial formulations), inoculant containing nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum), simultaneous inoculation with nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum) and phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis), and nitrogen-fixing bacteria (Azotobacter chroococcum) + plant growth-promoting rhizobacteria (PGPR) which also protect the crop against fungi (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens); II. companion crop: control (spring barley grown in a pure stand without a companion crop, red clover, and red clover + Italian ryegrass.
In the autumn, goat manure was applied at a rate of 15 t∙ha−1 to be ploughed down before the winter. In the spring, in early April, the spring barley with the companion crops were sowed on the same day. The seeding rates of spring barley, red clover, and red clover + Italian ryegrass mixture were as follows: 160, 16, and 9 + 15 kg∙ha−1, respectively. The sowing of the spring barley was carried out with a grain drill with a row spacing of 12.5 cm and a depth of 5–6 cm. The companion crops were then sown in the barley rows, 1–2 cm deep with a row spacing of 12.5 cm. The spring barley seed was purchased from the organic farm “STARY FOLWARK”, and the seeds of the red clover and Italian ryegrass also came from organic cultivation and were purchased from the seed company DSV Polska Sp. z o.o. (DSV Poland Ltd., Węgrowiec, Poland). The seed was certified by AGROBIOTEST Sp. z o.o (AGROBIOTEST Ltd., Warsaw, Poland). The bacteria Azospirillum lipoferum were applied twice in the growing season. Firstly, barley grain was treated with an inoculant suspension (100 mL∙15 kg−1 grain), and later inoculant spraying (the inoculant rate of 1 L/150 L water∙ha−1) was performed at the emergence stage (BBCH 10–15). The bacteria Azotobacter chroococcum were applied as two inoculant sprayings performed during the growing season (the first application time ‘0’ on the day of sowing, the second application—the scale BBCH 29–30). The inoculant rate was 1 L/250 L wody∙ha−1. The inoculant based on phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis) was also applied twice (the first application time ‘0’ on the day of sowing, the second application—the scale BBCH 29–30) and the rate was 1 l of inoculant/150 l water∙ha−1. An application of the inoculant containing PGPR which promote crop plant growth and protect plants against fungi (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens) was made twice in the growing season (the first application time ‘0’ on the day of sowing, the second application—the scale BBCH 29–30). The rate of the inoculant was 1 L/250 L water∙ha−1.

2.3. Origin and Preparation of Bacterial Inoculates

The bacterial species used for inoculation came from the collection of the Department of Soil Science and Microbiology of the Poznań University of Life Sciences. They were isolated from under cultivated plants, on selective medium, and then genetically identified on the basis of a fragment of the 16S rRNA gene sequence. Azotobacter chroococcum was isolated on Jensen medium [22], Azospirillum lipoferum from the maize rhizosphere on DAS medium [23], Bacillus megaterium var. phosphaticum on calcium phosphate solubilizing Ca3(PO4)2 [24], Bacillus subtilis on Bacillus ChromoSelect agar, Bacillus amyloliquefaciens on starch medium [24], Arthrobacter agilis on medium as described by Hagedorn and Holt [25], and King B medium [26] was used for the isolation of Pseudomonas fluorescens. The isolates obtained for the study were stored in tubes on agar slants, in a refrigerator at 8 °C. Before setting up the field experiment, in order for the strains to regain vitality and metabolic activity, the isolates selected for the study were passaged several times onto prepared agar slants with an appropriate medium for the given bacterial species. Then, for each barley inoculation date, liquid cultures of selected inocula were established in 100 mL flasks (five replicates). For this purpose, the three-day-old stock cultures of bacteria were made into a suspension by adding 5 mL of saline to each tube on a slant. Subsequently, the microbial cultures were scraped using an eyedropper and the resulting 0.5 mL bacterial suspension was inoculated into 100 mL of liquid medium. The obtained liquid cultures were incubated at 28 °C, on a shaker at 70 rpm min−1, for 48 h in the case of cultures of Bacillus subtilis, Arthrobacter agilis, Pseudomonas fluorescens, Bacillus megaterium var. phosphaticum, and Bacillus amyloliquefaciens, whereas in the case of Azospirillum lipoferum the incubation time was 86 h, and 72 h in the case of Azotobacter chroococcum.

2.4. Determination of Mutual Interactions between the Bacteria Used in the Construction of Inoculates

In order to select bacterial strains for the composed inoculants and to test their compatibility, the mutual interaction between the selected bacterial strains was determined using the ring method [27]. The experiment was based on 24 experimental variants (five replicates each):
  • Azotobacter chroococcum on Azospirillum lipoferum
  • Azospirillum lipoferum on Azotobacter chroococcum
  • Bacillus megaterium var. phosphaticum on Arthrobacter agilis
  • Arthrobacter agilis on Bacillus megaterium var. phosphaticum
  • Azotobacter chroococcum on Arthrobacter agilis
  • Arthrobacter agilis on Azotobacter chroococcum
  • Azospirillum lipoferum on Arthrobacter agilis
  • Arthrobacter agilis on Azospirillum lipoferum
  • Azotobacter chroococcum on Bacillus megaterium var. phosphaticum
  • Bacillus megaterium var. phosphaticum on Azotobacter chroococcum
  • Azospirillum lipoferum on Bacillus megaterium var. phosphaticum
  • Bacillus megaterium var. phosphaticum on Azospirillum lipoferum
  • Azotobacter chroococcum on Bacillus subtilis
  • Bacillus subtilis on Azotobacter chroococcum
  • Azotobacter chroococcum on Bacillus amyloliquefaciens
  • Bacillus amyloliquefaciens on Azotobacter chroococcum
  • Azotobacter chroococcum on Pseudomonas fluorescens
  • Pseudomonas fluorescens on Azotobacter chroococcum
  • Azospirillum lipoferum on Bacillus subtilis
  • Bacillus subtilis on Azospirillum lipoferum
  • Azospirillum lipoferum on Bacillus amyloliquefaciens
  • Bacillus amyloliquefaciens on Azospirillum lipoferum
  • Azospirillum lipoferum on Pseudomonas fluorescens
  • Pseudomonas fluorescens on Azospirillum lipoferum
In the first step, liquid cultures were established for all tested strains on their characteristic liquid media in 100 mL flasks to multiply the tested bacterial species. Subsequently, for each of the above-mentioned experimental variants, 2% agar in an amount of 5 mL was poured onto previously described sterile Petri dishes. After it had solidified, sterile rings (diameter 10 mm) were applied in a sterile manner, using tweezers, three per plate. After placing them, the previously prepared mixture of dissolved medium specific for the given bacterial species was poured out, with an appropriate dilution (10 × 105) in an amount of 10 mL, so that it did not enter inside the rings. After cooling it down, again under sterile conditions, the rings were removed from the plates. In two of the holes, 0.1 mL of the prepared bacterial suspension was applied and in the third hole, saline was placed as a control. After 1 h, the plates were placed in a thermostat at 28 °C and incubated, without inversion, for 72 h. After this time, the interactions between the tested bacteria were read. Analysis of the interactions between bacterial strains showed a lack of antagonistic interactions, as evidenced by the lack of brightening (halo) around the wells for all tested bacteria except for the interactions of Azospirillum lipoferum and Pseudomonas fluorescens (Figure 1). Hence, for our facility using bacteria that biologically reduce molecular nitrogen from the air (Azotobacter chroococum) with PGPR bacteria that are growth promoters of crop plants and protect them from fungi (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens), only Azotobacter spp. bacteria were selected without the Azospirillum lipoferum strain.
The non-use of Azospirillum for co-inoculation with PGPR group bacteria in our experiment was also due to data provided by the literaturę. Among others, Khorshidi et al. [28] and Couillerot et al. [29] proved in their studies that the presence of Azospirillum in an inoculum with Pseudomonas results in lower phytostimulation.

2.5. Chemical Analyses

The spring barley was harvested in late July. During the harvest, grain samples were collected from each plot to determine the total protein content by means of the Kjeldahl method. The total protein yield was calculated by multiplying protein content and the grain yield.

2.6. Statistical Analysis

Data for each characteristic studied were analysed by means of ANOVA suitable for the split-block arrangement. Comparison of means for significant sources of variation was achieved by means of Tukey test at a significance level of p ≤ 0.05. All the calculations were performed in Statistica, version 12.0, (Hamburg, Germany) and MS Excel (Redmond, WA, USA).

3. Results

The total protein content in the spring barley grain was significantly influenced by the conditions throughout the growing season, test bacterial formulations and their interaction (Table 1).
The highest total protein content in the spring barley grain was recorded in 2020 when both precipitation and temperature distributions were optimal during the growing season. It was significantly lower in 2019 and the lowest in 2021. The applied bacterial formulations significantly affected the total protein content in the spring barley grain. The highest concentration of total protein was recorded in the spring barley grain following an application of the inoculant containing a combination of nitrogen-fixing bacteria (Azotobacter chroococcum) and plant growth-promoting bacteria which also protect crops against fungi (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens). A significantly lower total protein content was recorded in the grain of the spring barley grown in plots co-inoculated with nitrogen-fixing bacteria (Azospirillum lipoferum, Azotobacter chroococcum) and phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis). The lowest total protein content was found in spring barley grain following an application of just nitrogen-fixing bacteria (Azospirillum lipoferum, Azotobacter chroococcum). However, also in this case, the total protein content in the spring barley grain was significantly higher compared with the control grain produced without an application of bacterial formulations. The total protein content in the spring barley grain varied significantly due to the presence of companion crops (Table 2).
The highest concentration of total protein was recorded in the grain of the spring barley accompanied by red clover, it being significantly lower when the companion crop was a mixture of red clover and Italian ryegrass, and the lowest in the control unit without companion crops. An interaction between the study years and companion crops was confirmed. The highest concentration of total protein was recorded in the grain of the spring barley grown in 2020 and accompanied by red clover, and a mixture of red clover and Italian ryegrass, it being the lowest in the spring barley grain harvested in 2021 in the control unit without a companion crop.
A significant interaction between the experimental factors was also confirmed. The highest total protein content was recorded in the grain of the spring barley following an application of nitrogen-fixing bacteria (Azotobacter chroococcum) with PGPR (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens), as well as nitrogen-fixing bacteria (Azospirillum lipoferum, Azotobacter chroococcum) with a mixture of bacteria releasing phosphorus to crop plants (Bacillus megaterium var. phosphaticum, Arthrobacter agilis) and red clover. By contrast, the lowest concentration of total protein was found in the control unit without bacterial inoculants or companion crops (Table 3).
Statistical analysis demonstrated a significant effect of the conditions prevailing in the growing season and test bacterial formulations on total protein yield in the spring barley grain (Figure 2).
The highest total protein yield in the spring barley grain was produced in the conducive year 2020, it being significantly lower in 2021, and the lowest in 2019. Furthermore, bacterial formulations significantly affected the total protein yield in the spring barley grain. The highest yield was recorded following an application of nitrogen-fixing bacteria in combination with PGPR. A lower yield was harvested in the units treated with a combination of nitrogen-fixing bacteria (Azospirillum lipoferum, Azotobacter chroococcum) and phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis). In the plots where only nitrogen-fixing bacteria had been applied, the yield of total nitrogen in the spring barley grain was the lowest although still higher than the figure for the control unit without an application of bacterial formulations. Cultivation with companion crops significantly improved the total protein yield in the spring barley grain (Figure 3).
The highest total protein yield was produced in the grain of the spring barley accompanied by red clover. It was significantly lower for the mixture of red clover and Italian ryegrass, and the lowest for the grain of control the spring barley grown in pure stand. An interaction was confirmed and it revealed that the highest total protein yield was produced in 2020 for the spring barley grown with red clover, it being the lowest in 2019 for the control grain of the spring barley grown without companion crops. The bacterial formulations interacted with companion crops in terms of the total protein yield in the spring barley grain (Figure 4).
The highest total protein yield was observed in the grain of the spring barley grown in plots amended with nitrogen-fixing bacteria combined with PGPR where either red clover or a mixture of red clover and Italian ryegrass was a companion crop. The yield was the lowest in the control unit where neither biological formulations had been applied nor companion crops had been grown.

4. Discussion

In an organic agriculture system, natural fertilisers, green manures from leguminous plants, and nitrogen-fixing bacteria inoculants are nitrogen sources for crop plants. This research demonstrated that both total nitrogen content and yield in spring barley grain were significantly affected by the test bacterial formulations. The bacteria Azotobacter spp. And Azospirillum lipoferum are capable of biological nitrogen fixation by reducing the nitrogen to ammonia which is necessary for the synthesis of amino acids which form protein in plants. A considerable potential of nitrogen fixation by these bacteria is due to their ability to produce the enzyme nitrogenase, which makes it possible to improve nitrogen acquisition efficiency of cereal crops [4,5,6,8,9]. In the present work, protein content and yield following an application of only nitrogen-fixing bacteria (Azospirillum lipoferum and Azotobacter chroococcum) were lower compared with the combined application of nitrogen-fixing bacteria and phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis), or PGPR. The ability of phosphorus-releasing bacteria to take up the soluble form of mineral phosphorus is of importance in crop growing as well. In microbiological processes of phosphorus conversion, the element is released in amounts which are in excess of microbe demand. As a result, a substantial amount of the element can be utilised by plants. Another important property is an ability of microorganisms to convert phosphorus mineral compounds from poorly to easily soluble in water. Microorganisms which are capable of releasing readily available phosphorus forms from various non-soluble compounds are called Phosphate Solubilising Bacteria (PSB), that is strains releasing phosphorus compounds. Additionally, many of these bacteria synthesise other substances such as siderophores, auxins, cytokinins, and vitamins, which improves plant growth enormously due to increased efficiency of phosphorus uptake by plants [30,31]. The genera Pseudomonas and Bacillus are the most numerous group of microorganisms enhancing plant growth (PGPR). They positively affect plant development through direct and indirect stimulation of their growth. The direct stimulation is, among other reasons, due to supplying plants with minerals, synthesising phytohormones affecting plant growth (that is auxins, gibberellins, cytokinins) and reducing the level of ethylene which has an adverse effect on root development. On the other hand, indirect stimulation manifests itself as the biological control of phytopathogens (Bacillus subtilis, Pseudomonas fluorescens, lactic acid bacteria). PGPR are capable of producing phytohormones, thus, they affect plant growth by increasing their endogenous pool of growth regulators. Such regulators include gibberellins produced by the bacteria Bacillus subtilis and Bacillus cereus which influence dormancy survival, seed germination and stem elongation, induce flowering and flower development, improve pollen liveability, stimulate fruit development and root growth, and induce the growth of root hairs. Inoculation of plants with PGPR microorganisms, in particular Pseudomonas bacteria, makes it possible to alleviate losses due to abiotic factors, and contribute to enhanced plant yields. Plant inoculation with P. fluorescens bacteria increases biomass yield and alkaloid content even under stress conditions during drought. Utilisation of inoculants containing growth promoters results in an increase in the amount of biomass and water content, reduction of water losses by leaves, and an increase in the level of proline and sugars supporting plants during drought [32,33,34,35,36,37,38]. The above has been confirmed in the present work which demonstrated that the highest total protein content and yield in spring barley grain were produced following a combined application of nitrogen-fixing bacteria (Azotobacter chroococcum) and PGPR (Bacillus subtilis, Pseudomonas fluorescens) particularly in 2019 where the precipitation sum was the lowest. Nowadays, in Poland there are recurrent droughts during the vegetation period of plants, which translates into a decrease in the grain yield of cereals, including spring barley, especially in organic farming. In order to ensure the stability of plant yields, new technologies using biofertilisers should be introduced. Currently, this topic is receiving attention from researchers all over the world [4,10,11,12]. Biofertilizers promote plant growth by providing nutrients including biologically bound nitrogen or increasing the availability of insoluble nutrients in the soil and synthesizing substances that stimulate plant growth [10,13]. Biofertilizers are economically and ecologically attractive means of improving crop quality and quantity [12]. They are cheaper and improve crop growth and quality by stimulating direct or indirect release of plant hormones [11]. PGPR improve root development and plant growth through solubilization of insoluble P and excretion of plant growth-promoting hormones [13]. Rhizospheric and endophytic bacteria are free-living bacteria that are voluntarily associated with the rhizosphere and within plant roots, and directly correlate to improved plant growth and yield [14,15]. In recent years, the use of beneficial microorganisms in cereals has revealed their positive effects on cereal yield and quality under adverse environmental conditions [16,17]. This is consistent with the results of our own studies.
In the experiment reported here, it was demonstrated that spring barley accompanied by red clover or a mixture of red clover and Italian grass produced superior total protein content and yield. This was due to the fact that leguminous plants which live in symbiosis with the nodulating bacteria of the genus Rhizobium release nitrogen into the soil environment; the nitrogen is then taken up by cereals, which improves total protein content and yield in grain [18,19,20,39]. Taking the above into consideration, it should be recommended to organic growers that they cultivate spring barley with legumes grown as companion crops and their mixtures with grasses in the presence of bacteria, in particular PGPR or phosphorus-releasing bacteria. Spring barley grain from these combinations was characterised by high total protein content, as well as protein yield, which is the resultant of multiplying the total protein content and grain yield. The grain yield was the highest with these interventions and therefore the total protein yield was also the highest. Thus, the spring barley grain from such a crop is a valuable raw material for the production of functional food such as groats and flakes with high protein content [1,3,40].

5. Conclusions

The growing season conditions significantly affected the total protein content and yield in the spring barley grain. The highest total protein content was observed in the grain of the spring barley accompanied by red clover and following an application of nitrogen-fixing bacteria (Azotobacter chroococcum) and PGPR (Bacillus subtilis, Pseudomonas fluorescens). Organic spring barley grown with either red clover or a mixture of red clover and Italian ryegrass accompanied by a combined application of nitrogen-fixing bacteria (Azotobacter chroococcum) and PGPR contributed to the highest total protein yield in barley grain.

Author Contributions

Conceptualization, A.P. and A.N.; methodology, R.G. and R.R.; software, R.G.; validation, E.R.; formal analysis, E.R.; investigation, A.P. and R.G.; resources, A.N. and R.R.; data curation, E.R.; writing—original draft preparation, A.P. and R.G.; writing—review and editing, A.P. and R.G.; visualization, A.P. and A.N.; supervision, A.P. and R.G.; project administration, A.P. and R.G.; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Higher Education, grant number 29/20/B.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 12 01522 g001 550
Figure 1. Antagonistic interaction between Azospirillum lipoferum Br17 and Pseudomonas fluorescens.
Figure 1. Antagonistic interaction between Azospirillum lipoferum Br17 and Pseudomonas fluorescens.
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Figure 2. Total protein yield in spring barley grain according to test bacterial formulations from 2019–2021, kg∙ha−1. 1 For explanations see Table 1. 2 Means for the bacterial formulations and years by the same capital letter (A, B, C) do not differ significantly at p < 0.05. ±, standard deviation.
Figure 2. Total protein yield in spring barley grain according to test bacterial formulations from 2019–2021, kg∙ha−1. 1 For explanations see Table 1. 2 Means for the bacterial formulations and years by the same capital letter (A, B, C) do not differ significantly at p < 0.05. ±, standard deviation.
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Figure 3. Total protein yield in spring barley grain according to companion crops from 2019–2021, kg∙ha−1. 1 Values in years for the interaction followed by the same small letter (a, b) do not differ significantly at p < 0.05, means for the companion crops by the same capital letter (A, B) do not differ significantly at p < 0.05. ±, standard deviation.
Figure 3. Total protein yield in spring barley grain according to companion crops from 2019–2021, kg∙ha−1. 1 Values in years for the interaction followed by the same small letter (a, b) do not differ significantly at p < 0.05, means for the companion crops by the same capital letter (A, B) do not differ significantly at p < 0.05. ±, standard deviation.
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Figure 4. Total protein yield in spring barley grain according to test bacterial formulations and companion crops (means across 2019–2021), kg∙ha−1. 1 For explanations see Table 1. 2 Values in bacterial formulations for the interaction followed by the same small letter (a, b, c) do not differ significantly at p < 0.05. ±, standard deviation.
Figure 4. Total protein yield in spring barley grain according to test bacterial formulations and companion crops (means across 2019–2021), kg∙ha−1. 1 For explanations see Table 1. 2 Values in bacterial formulations for the interaction followed by the same small letter (a, b, c) do not differ significantly at p < 0.05. ±, standard deviation.
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Table
Table 1. Total protein content in spring barley grain according to test bacterial formulations in g∙kg−1 d.m.
Table 1. Total protein content in spring barley grain according to test bacterial formulations in g∙kg−1 d.m.
Bacterial Formulations 1 (A)Years (Y)Means
201920202021
1105.7 ± 2.5 c 2125.0 ± 4.5 c93.8 ± 2.3 c108.2 ± 12.8 C
2111.5 ± 5.0 b127.7 ± 2.4 b96.5 ± 2.2 b111.9 ± 13.4 B
3112.5 ± 5.3 ab128.7 ± 2.5 ab97.5 ± 2.7 ab112.9 ± 13.3 B
4114.1 ± 5.6 a130.2 ± 2.4 a98.6 ± 3.0 a114.3 ± 13.5 A
Means111.0 ± 5.7 B127.9 ± 3.9 A96.6 ± 3.1 C-
1 1—control; 2—nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum); 3—nitrogen-fixing bacteria (Azospirillum lipoferum Br17, Azotobacter chroococcum) + phosphorus-releasing bacteria (Bacillus megaterium var. phosphaticum, Arthrobacter agilis); 4—nitrogen-fixing bacteria (Azotobacter chroococcum) + PGPR (Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens). 2 values in columns for the interaction followed by the same small letter (a, b, c) do not differ significantly at p < 0.05. Means for the bacterial formulations in a column followed by the same capital letter (A, B, C) do not differ significantly at p < 0.05. Means for the years in verse followed by the same capital letter (A, B, C) do not differ significantly at p < 0.05. ±, standard deviation.
Table
Table 2. Total protein content in spring barley grain according to companion crops in 2019–2021, g∙kg−1 d.m.
Table 2. Total protein content in spring barley grain according to companion crops in 2019–2021, g∙kg−1 d.m.
Companion Crops (B)Years (Y)Means
201920202021
Control104.9 ± 2.1 c 1125.2 ± 4.3 c93.6 ± 2.1 c107.9 ± 13.1 C
Red clover113.3 ± 5.7 a128.9 ± 2.6 a98.3 ± 2.8 a113.5 ± 13.1 A
Red clover + Italian ryegrass111.2 ± 3.0 b127.9 ± 2.5 b96.6 ± 2.1 b111.9 ± 13.3 B
1 values in columns for the interaction followed by the same small letter (a, b, c) do not differ significantly at p < 0.05. Means for the companion crops in a column followed by the same capital letter (A, B, C) do not differ significantly at p < 0.05. ±, standard deviation.
Table
Table 3. Total protein content in spring barley grain according to test bacterial formulations and companion crops (means across 2019–2021), g∙kg−1 d.m.
Table 3. Total protein content in spring barley grain according to test bacterial formulations and companion crops (means across 2019–2021), g∙kg−1 d.m.
Bacterial Formulations 1 (A)Companion Crops (B)
ControlRed CloverRed Clover + Italian Ryegrass
1105.9 ± 12.0 b 2109.3 ± 13.8 a109.3 ± 12.8 a
2108.2 ± 13.1 c114.9 ± 13.5 a112.6 ± 13.4 b
3108.9 ± 13.1 c116.3 ± 13.1 a113.5 ± 13.1 b
4110.1 ± 13.4 c118.1 ± 13.3 a114.6 ± 13.2 b
1 For explanations see Table 1. 2 Values in verse for the interaction followed by the same small letter (a, b, c) do not differ significantly at p < 0.05. ±, standard deviation.
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Płaza, A.; Niewiadomska, A.; Górski, R.; Rudziński, R.; Rzążewska, E. The Effect of the Nitrogen-Fixing Bacteria and Companion Red Clover on the Total Protein Content and Yield of the Grain of Spring Barley Grown in a System of Organic Agriculture. Agronomy 2022, 12, 1522. https://doi.org/10.3390/agronomy12071522

AMA Style

Płaza A, Niewiadomska A, Górski R, Rudziński R, Rzążewska E. The Effect of the Nitrogen-Fixing Bacteria and Companion Red Clover on the Total Protein Content and Yield of the Grain of Spring Barley Grown in a System of Organic Agriculture. Agronomy. 2022; 12(7):1522. https://doi.org/10.3390/agronomy12071522

Chicago/Turabian Style

Płaza, Anna, Alicja Niewiadomska, Rafał Górski, Robert Rudziński, and Emilia Rzążewska. 2022. "The Effect of the Nitrogen-Fixing Bacteria and Companion Red Clover on the Total Protein Content and Yield of the Grain of Spring Barley Grown in a System of Organic Agriculture" Agronomy 12, no. 7: 1522. https://doi.org/10.3390/agronomy12071522

APA Style

Płaza, A., Niewiadomska, A., Górski, R., Rudziński, R., & Rzążewska, E. (2022). The Effect of the Nitrogen-Fixing Bacteria and Companion Red Clover on the Total Protein Content and Yield of the Grain of Spring Barley Grown in a System of Organic Agriculture. Agronomy, 12(7), 1522. https://doi.org/10.3390/agronomy12071522

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