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BY 4.0 license Open Access Published by De Gruyter Open Access October 29, 2019

The use of microorganisms as bio-fertilizers in the cultivation of white lupine

  • Hanna Sulewska , Karolina Ratajczak EMAIL logo , Alicja Niewiadomska and Katarzyna Panasiewicz
From the journal Open Chemistry


The agricultural usability of bio-fertilizers, particularly including microbiological seed vaccines meet the recommendations for integrated protection/cultivation applicable in Poland. Combined vaccination seeds with Rhizobium bacteria together with endophitic bacteria from the group of Plant Growth Promoting Rhizobacteria (PGPR), increase the efficiency of biological nitrogen fixation and therefore stimulate the development and yielding of legume crops. This hypothesis was verified in a series of field experiments with white lupine conducted between 2016 and 2018 at Złotniki, Poland. The one-factor experiment consisted of different variants of inoculation including: seeds uninoculated, inoculated with nitragine, nitroflora, Pseudomonas fluorescens strain, Bacillus subtillis strain and seeds co-inoculated with Rhizobium from nitragine or nitroflora + Pseudomonas fluorescens, or + Bacillus subtillis. The experiment showed a positive response of white lupine to all tested seed vaccinations. The highest seed yield was found after seed inoculation with Rhizobium bacteria (from nitragine) and with co-inoculation Rhizobium with Pseudomonas fluorescens. The results indicated that plant height, the development of aboveground parts and roots as well as root nodules and the setting of pods and seeds on the plant increased significantly after seed co-inoculation of Rhizobium derived from the preparation of nitragine with Pseudomonas fluorescens, as compared to the control object without vaccination. A generally positive correlation was found between the number of root nodules, dry mass of nodules and yield, and an especially significant strength of this relationship was found in variant with co-inoculation Rhizobium with Pseudomonas fluorescens.

1 Introduction

In modern agriculture, cultivation methods with low energy intensity should be used which, by reducing water and soil pollution, protect the natural environment. The application of environmentally friendly practices is dictated by European Union law (EU Directive 2009/128) and recommendations for integrated protection/cultivation have been applicable in Poland since 2014. In addition, for environmental reasons, it is appropriate to introduce bio-fertilizer for crops or natural stimulators for plant growth and development. Fertilizers containing live microorganisms are available, which when in contact with the surface of the plant or soil are able to colonize the rhizosphere or the interior of the plant, thus accelerating its growth [1]. The vast majority of previous studies show that these bacteria affect the regulation of plant growth through the production of plant hormones, enzymes that reduce hormone production in the host plant or stimulate it to produce signaling substances (ex. flavonoids) for other symbionts [2]. These compounds usually affect the development of the root and its morphology, causing an increase in the surface through which nutrients are absorbed or when the symbiont has another symbiosis of the host (the so-called “auxiliary” effect of the bacteria) [3]. This synergistic action encourages research into the possibility of increasing the effectiveness of biological nitrogen fixation through the simultaneous use of the strain characteristic for macrosymbionta and endophytes included in the so-called plant growth microbes PGPR (Plant Growth Promoting Rhizobacteria). A typical strain for legume plants are bacteria of the Rhizobiaceae family that encourage nitrogen fixation which makes 122 million tons of nitrogen [4] per year in the biosphere scale, while depending on the ecosystem, the amount of nitrogen fixation by symbiotic microorganisms can range from 30-150 kg N•ha-1 in legumes grown for grain or from 50-250 kg N•ha-1 in cultivation for green fodder [5]. In turn, the microorganisms that stimulate plant growth include bacteria from various phylogenetic groups, of which the most numerous groups are Pseudomonas and Bacillus [6]. So far, their growth-stimulating effects have been proven thanks to the synthesis of phytohormones in soybean plants [7] and pine [8] (cytokinins), alder [9] (gibberellins) or rapeseed [10] thanks to ACC deaminase, degrading the precursor of ethylene biosynthesis, adversely affecting plant rooting [3]. In the situation of feed protein deficit in Europe and Poland [11], use of bacteria from various groups in the form of so-called “consortia of microorganisms” is becoming increasingly important in seeking the possibility of increasing the yield of plants, especially legumes, which are a valuable source of this ingredient. The phenomenon of biological nitrogen fixation (diazotrophy) by bacteria from the family Rhizobiaceae is well known and used in the cultivation of legumes. There are products on the market containing active strains of rhizobium bacteria intended for the inoculation of seeds of a specific species before sowing, in order to increase the biological nitrogen fixation, which allows reducing the mineral fertilization of crops with this macroelement. In addition, both symbiotic N2 fixation in root nodules and inorganic nitrogen uptake by the roots are important for legumes not only for obtaining high yields but also to increase quality traits, especially proteins or other features, as for example TSW. Moreover, the newest research has indicated even the spermidine content in soybean [12]. Thanks to seed inoculating, it is possible to reduce fertilization costs and reduce environmental impacts.

Therefore, the aim of the study was to evaluate the effects of inoculation and co-inoculation of seeds with rhizobium and endophytic bacteria from the PGPR group in the cultivation of white lupine. It was assumed that Rhizobium bacteria derived from commercial vaccines can form a synergistic system with selected PGPR bacteria: Bacillus subtillis and Pseudomonas fluorescens, and co-inoculation will increase the efficiency of the biological nitrogen fixation.

2 Experimental part

2.1 Materials

Two preparations available on the market were used in the experiment, containing live strains of rhizobial bacteria (Rhizobium) under the name nitragina (produced by the Institute of Soil Science and Fertilization - National Research Institute) and nitroflora (Mykoflor company) and Bacillus subtillis and Pseudomonas fluorescens inoculants from the collection of Department of General and Environmental Microbiology, University of Life Sciences in Poznań. Immediately prior to sowing, the seeds were inoculated according to the label with a properly prepared suspension of rhizobia bacteria with nitragine and nitroflora, and then after the seeds had dried, with endophytes of Pseudomonas fluorescens and Bacillus subtillis (108•ml-1 of liquid culture). The study used lupine seeds (Lupinus albus L.) of the ‘Butan’ variety registered in 2000, originating from HR Smolice Spółka z o.o. The IHAR Group, Branch in Przebędowo. This variety is characterized by a high yielding potential (in 2004 - 2006 an average of 3.36 t•ha-1) (according to COBORU data) and is the first thermoneutral variety [13]. The seeds were sown on a light soil with a granulometric composition of sandy clay, classified as a typical podzolic soil, formed from light sandy clay sands, embedded in a shallow layer of light clay [14]. The weather course in the years of the study was presented as mean values of the Sielianinov [15] hydrothermal indicator (K) (Figure 1), calculated according to the formula K = (P·10)/(T·L), where K - hydrothermal Sielianinow factor, P - total monthly precipitation, T - average temperature of the month, L– number of the days of the month, based on meteorological data registered in the Experimental Station. Interpretation of the indicator: K> 1.5 - excess moisture for plants, K = 1.0-1.5 - optimal humidity, K = 0.5-1.0 - insufficient humidity for plants, K <0.5 - humidity below required for plants (drought).

Figure 1 Plant water supply hydrothermal coefficient according to Sielianinov (K) in 2016-2018.
Figure 1

Plant water supply hydrothermal coefficient according to Sielianinov (K) in 2016-2018.

2.2 Field experiment

Field experiments were conducted in 2016-2018 at the Department of Agronomy of the University of Life Sciences in Poznań, in the fields of the Research and Education Center Gorzyń, branch in Złotniki (52°29’N, 16°50’E). The experiments were run as one-factor in four replicates with 9 factor objects: 1) uninoculated seeds (control), 2) seeds inoculated with nitragine, 3) seeds inoculated with nitroflora, 4) seeds inoculated with Pseudomonas fluorescens strain, 5) seeds inoculated with Bacillus subtillis strain, 6) seeds co-inoculated with Rhizobium from nitragine + Pseudomonas fluorescens, 7) seeds co-inoculated with Rhizobium from nitragine + Bacillus subtillis, 8) seeds co-inoculated with Rhizobium from nitroflora + Pseudomonas fluorescens, 9) seeds co-inoculated with Rhizobium from nitroflora + Bacillus subtillis. The agrotechnical and cultivation treatments were carried out in accordance with the principles of good agricultural and experimental practice for this species [16]. During the vegetation of plants, non-destructive measurements of plant condition parameters were performed on each plot: leaf area index (LAI) using the SunScan Canopy Analysis System (Delta-T Devices, England) and the chlorophyll content indicator by means of a portable device the Hydro N-tester optical instrument (Minolta, Japan). In the phase of full plant maturity, 20 plants were randomly taken from each plot and the number of pods and seeds on the main and lateral shoots, along with the number and mass of seeds per plant were determined. In addition, the height of plants, the number of branches, the mass of aboveground parts and roots as well as the number of root nodules and their mass were determined. During the harvest, the seed yield, moisture content, and the weight of one thousand seeds were evaluated, and then the yield was converted to 15% H2O. Soil samples were collected after white lupine harvest. Soil pH and the content of macronutrients in soil were assessed. Soil analyses and the content of macronutrients in plants and lupine seeds were carried out in the Laboratory Accredited by Polish Centre of Accreditation (PCA) according to AB 1270 and standard methods were used. A soil reaction value of 1 mole KCl•dm-3 was measured by potentiometry method (PN-ISO 10390:1997) and the content in the soil of macronutrients in the form of: nitrate nitrogen, ammonium nitrate and total nitrogen soluble by colorimetric method (PN-ISO 14255:2001), phosphorus with the Egner-Rhiem method (PN-R-04023:1996), potassium and calcium by flame photometry (PB-1-2013), magnesium by atomic absorption spectrometry (PN-R-04020:1994+Az1:2004). The organic carbon was determined by the titration method (Tiurin) (PB-4-2015). According to the accumulation of mineral composition in seeds and white lupine plants the macroelements uptake by plants was calculated. The values of the assessed features were subjected to analysis of variance (ANOVA) for single factorial experiments, and then a synthesis was made from the years of research, using the Statistica 12.0 software (StatSoft, Poland). To assess the significance of differences between object-related averages, the Duncan’s test was used at the significance level of p <0.05. Principal component analysis (PCA) was used to visualize multidimensional dependences between traits.

Ethical approval: The conducted research is not related to either human or animal use.

3 Results and discussion

The yield of white lupine ‘Butan’ was on average at the level of 3.6 t•ha-1, so by 0.06 t•ha-1 it exceeded the average yield of seeds of registered lupine varieties, which confirms the correctness of choosing the variety for cultivation. Yielding results of lupines are largely determined by weather conditions. Particularly good conditions occurred in 2016 and 2017, characterized by an even supply of plants in water from April to July, including during the critical period of pods setting. In turn, in 2018, the drought occurring from May to June could certainly reduce the yield of lupine (Figure 1). In addition, the lupine yield was influenced by the seed inoculating variants used (Table 1). In the synthesis from years of research it was proved that the use of each of the tested variants of seed inoculation contributed to the increase in yield. The yield was particularly increased due to the influence of nitragine and co-inoculation of nitragine with Pseudomonas fluorescens, and compared to the control it

Table 1

Influence of vaccination treatment in white lupine seeds on yield, its moisture and root nodule.

CharactersSeed yield, t•ha-1Thousand seed weight, gMoisture, %Root nodules
Objectsnumber, pc.•plant-1dry mass, g
13.15 d347.616.228.9 e0.405 e
23.89 a351.216.333.4 de0.558 b
33.60 bc353.016.143.8 abc0.565 b
43.59 bc363.117.149.5 ab0.632 a
53.61 bc362.517.041.1 bcd0.476 d
63.82 a351.916.652.0 a0.500 cd
73.66 b358.516.746.6 abc0.550 bc
83.58 bc356.416.438.6 cd0.598 ab
93.46 c350.316.844.1 abc0.477 d
  1. 1- Control-without vaccination, 2- Nitragine, 3- Nitroflora, 4- Pseudomonas, 5-Bacillus, 6- Nitragine +Pseudomonas, 7- Nitragine + Bacillus, 8- Nitroflora + Pseudomonas, 9- Nitroflora + Bacillus -lack of homogeneous groups means no significant differences at the level of p <0.05, a, b-homogeneous groups (Duncan’s test, p <0.05), CV- coefficient of variation, SD- standard deviation, min -max - minimum-maximum

was respectively 0.74 and 0.67 t•ha-1. Literature reports, that the positive effect of nitragine on the yields of white lupine [17] has been proven so far, the weight of a thousand seeds and the more intensive nodulation and the greater germination power of yellow lupine seeds [18]. In addition, in field and laboratory studies, the effect of co-inoculation of bacteria Rhizobium and Bacillus on biological nitrogen fixation has been demonstrated for this species [19]. Previous studies by Niewiadomska and Swędrzyńska [20] on the effect of simultaneous inoculation of alfalfa with the strains of Sinorhizobium meliloti and endophytic bacteria Herbaspirillum frisingense showed a positive effect of such inoculation on the process of symbiosis and the yielding of alfalfa. Subsequent studies on alfalfa and clover showed the beneficial effect of simultaneous inoculation of plants with the Rhizobium, Sinorhizobium and Azospirillum strains respectively on plant yield and photosynthesis efficiency and the process of biological nitrogen fixation [21]. In field conditions, co-inoculation of Rhizobium and Pseudomonas proved to be more effective in stimulating the yield and components of yielding of common bean, compared to a single inoculation of Rhizobium [22].

The weight of one thousand lupine seeds after seed inoculation with Pseudomonas fluorescens increased by 15.5 g compared to controls, however, this difference was not confirmed statistically. The moisture content of lupine seeds was on average 16.5% and was not dependent on the inoculation variant. The most root nodules were formed by lupine plants, in which the co-inoculation with Rhizobium from nitragine and Pseudomonas fluorescens was applied, it should be emphasized that each of the inoculation variants contributed to the improvement of root nodules forming. In studies by Mascarelli et al. [23] in a synergistic interaction bacteria of the species, Bacillus enhanced the ability to colonize the root and increase the number of nodules formed by Bradyrhizobium in soybean plants, the seeds of which were simultaneously inoculated with the strain Bacillus amyloliquefaciens and the symbiotic bacteria like Bradyrhizobium japonicum. Also, in the studies of Subramaniania et al. [24] the beneficial effect of simultaneous co-inoculation of Rhizobium with Bacillus megaterium on the growth of plants and root nodules as well as the level of nitrogenase activity and thus the total protein content in soybean plants has been demonstrated.

The root nodules in our own studies weighed an average of 0.5 g and each of the tested seed inoculation variants led to an increase in their mass, and the greatest effect was noted after the Pseudomonas fluorescens seed inoculation. Seed inoculation significantly influenced plant height and the highest of the tested variants was recorded after co-inoculation of nitragine with Pseudomonas fluorescens (48.2 cm) (Table 2). In earlier studies, nitragine inoculation also significantly stimulated the growth of yellow lupine plants [25]. White lupine plants produced on average three branches, with the most branched ones observed after co-inoculation of Rhizobia from nitroflora with Bacillus subtillis. In addition, the plants of this combination achieved the highest values of the leaf greenness index and the mass of the aboveground part, which were higher by 162 SPAD units and 9.4 g respectively compared to the control. A similar increase in the leaf greenness index was observed in maize plants of which the seeds were co-inoculated with bacteria of the species Rhizobium and Azospirillum brasilense [26].

Table 2

Influence of vaccination treatment in white lupine seeds on height, number of branches, leaf area index, chlorophyll content index and mass of aboveground plant part and roots.

CharactersPlant height, cmNumber of bran-ches, pc.LAISPADMass, g
Objectsaboveground plant partroots
143.6 d2.9 d5.62 bc662.0 e13.4 c0.90 c
244.6 cd2.9 d5.51 bc712.2 d15.1 c0.87 c
343.5 d3.1 bcd5.71 bc744.7 c15.4 c1.01 bc
446.4 abc3.0 d6.51 a697.4 d15.1 c0.92 c
547.1 ab3.2 abcd5.32 c760.7 bc15.4 c0.92 c
648.2 a3.4 abc5.89 b770.5 b21.2 ab1.18 a
744.6 bcd3.5 ab6.09 a771.2 bc19.0 b1.25 a
844.9 bcd3.0 cd6.33 a773.0 bc14.9 c0.97 bc
946.4 abc3.7 a5.94 b823.7 a22.8 a1.10 ab
  1. 1- Control-without vaccination, 2- Nitragine, 3- Nitroflora, 4- Pseudomonas, 5-Bacillus, 6- Nitragine +Pseudomonas, 7- Nitragine + Bacillus, 8- Nitroflora + Pseudomonas, 9- Nitroflora + Bacillus; -LAI - leaf area index (m2 leaves/m2 surface), SPAD- chlorophyll content index, designation as in Table 1

The largest leaf area in the field, expressed in the values of the leaf area index (LAI), was recorded after the application of seed inoculation of Pseudomonas fluorescens. The use of co-inoculation Rhizobium from nitragine with Pseudomonas fluorescens significantly increased the mass of aboveground parts and roots of lupine plants by 7.8 and 0.28 g, respectively, in relation to the control (Table 2). In addition, these plants have formed more pods and seeds, with a higher weight (Table 3). Similarly, Dileep et al. [27] showed that the co-inoculation of Pseudomonas and Rhizobium in pea stimulated plant height, root length and dry weight. In addition, these authors showed a wide range of fungicidal activity of this co-inoculation against specific pathogens for peas. The beneficial role of Pseudomonas in the created “consortia of microorganisms” is mainly attributed to the ability of these strains to share nutrients, mainly phosphorus, necessary to initiate symbiosis. These microorganisms largely stimulate the growth of side roots and root hairs, thanks to which the root surface is increased, and thus the possibility of adhesion of microorganisms, hence they can be of great importance for yielding leguminous plants and increasing the efficiency of biological nitrogen fixation [6, 28]. Currently, only a few bio-fertilizers are available in the form of Rhizobium co-inoculants along with Bacillus subtilis or Azospirillum brasilense, thanks to which it is possible to increase the yield of mainly soya, depending on the preparation by 4-30% compared to a single inoculation [29].

Table 3

Influence of vaccination treatment in white lupine seeds on the weight of seeds and pods per plant, seed share in the whole plant mass and the number of pods and seeds per plant.

CharactersMass, gSeeds share in the whole plant mass, %Number of pods per plant, pc.Number of seeds per plant, pc.
Objectsseeds per plantpods per plant
16.23 d9.3 c46.45.9 d17.8 d
27.09 cd10.7 bc47.67.2 bcd20.3 cd
37.62 bc11.7 b49.87.4 bc22.8 bc
47.08 cd10.9 bc47.16.4 cd19.8 cd
57.45 bcd10.8 bc48.37.2 cd21.7 c
610.0 a15.3 a47.19.3 a27.8 a
78.69 ab13.9 a45.99.3 a27.3 a
86.98 cd10.9 bc47.37.1 cd20.8 cd
99.38 a15.4 a43.18.6 ab26.1 ab
  1. designation as in Table 1

White lupine was cultivated on soil, which was characterized by slightly acidic pH in 1 mol KCl=5,34, indicating the need for liming, in which usually Azotobacter, which is quite sensitive to soil properties, was not occurring [30]. Seed inoculation treatments did not affect significantly the pH value of experimental light soil. The content of phosphorus, potassium, magnesium and calcium in the soil of the control object before starting the experiment was very high (177.2 mg P•kg-1), high (116.5 mg K•kg-1), high (94.5 mg Mg•kg-1) and very low (164.0 mg Ca•kg-1), respectively (Table 4). The content of these elements in the soil after the seed inoculation treatment significantly decreased on the object with a co-inoculation of nitragine with Pseudomonas fluorescens and a co-inoculation of nitroflora with Bacillus subtillis (Table 4).

Table 4

Soil reaction and content of nutrients before and after experiments.

pH in 1 mol KCl5.34 a5.34 a5.32 a5.30 a5.30 a5.31 a5.29 a5.30 a5.33 a5.34 a
Phosphorus P (mg•kg-1)177.2 a165.5 a163.1 b164.4 ab164.3 ab164.1 ab160.5 c162.4 bc164.3 ab160.9 c
Potassium K (mg•kg-1)116.5 a80.6 a72.8 b77.1 ab76.6 ab76.3 ab62.9 c69.6 bc77.0 ab63.6 c
Magnesium Mg (mg•kg-1)94.5 a89.1 a87.9 b88.6 ab88.5 ab88.4 ab86.3 c87.4 bc88.6 ab86.3 c
Calcium Ca (mg•kg-1)164.0 a154.1 a151.9 b153.1 ab152.9 ab152.9 ab148.8 c150.8 bc153.1 ab148.9 c
Organic carbon (%)0.59 a0.59 a0.59 a0.59 a0.59 a0.59 a0.59 a0.59 a0.59 a0.59 a
Percent of caries1.0 a1.0 a1.0 a1.0 a1.0 a1.0 a1.0 a1.0 a1.0 a1.0 a
  1. 0-Control soil sample, 1- Control-without vaccination, 2- Nitragine, 3- Nitroflora, 4- Pseudomonas, 5-Bacillus, 6- Nitragine +Pseudomonas, 7- Nitragine + Bacillus, 8- Nitroflora + Pseudomonas, 9- Nitroflora + Bacillus; a, b-homogeneous groups (Duncan’s test, p <0.05)

The accumulation of N, P, K, Mg and Ca in white lupine plants was highest after vaccination treatment, however a significant increase was found on the object with the co-inoculation of nitragine with Pseudomonas fluorescens and the co-inoculation of nitroflora with Bacillus subtillis (Table 5). The highest accumulation of macronutrients in the seed was found after the inoculation treatment with nitragine, compared to other objects. Moreover, in our research the variation in the amount of the elements taken by plants results from the size of the seed yield, similar to studies conducted on winter wheat made by Nogalska et al. [31]. Co-inoculation of nitragine with Pseudomonas fluorescens and co-inoculation of nitroflora with Bacillus subtillis significantly increased the uptake N, P, K, Mg and Ca by white lupine plants. Adesemoye et al. [32] indicated that the application of inoculants (plant growth-promoting rhizobacteria, arbuscular mycorrhizal fungi or their combinations) can lead to a reduction in the buildup of N, P and K from fertilizers and without removing the plants, plant nutrients may get back into the biogeochemical cycle through decomposition. Similarly, as in our studies, significantly higher amounts of N, P, K were removed from the plots with inoculants.

Table 5

The influence of vaccination treatment on accumulation of mineral composition in plants and seeds of white lupine and their uptake (kg•ha-1).

accumulation inuptakeaccumulation inuptakeaccumulation inuptakeaccumulation inuptakeaccumulation inuptake
1194.1 c195.8 c389.8 b34.6 c18.2 c52.8 b132.3 c29.9 c162.2 b41.3 c4.0 c45.3 b21.7 c2.9 c24.6 b
2236.6 bc233.1 a469.6 ab42.2 ab21.7 a63.9 ab161.3 ab35.6 a196.9 ab50.4 ab4.7 a55.1 ab26.4 ab3.5 a29.9 ab
3213.1 bc211.5 bc424.6 ab38.0 bc19.7 bc57.7 ab145.3 bc32.3 bc177.6 b45.4 bc4.3 bc49.7 b23.8 bc3.2 bc26.9 b
4216.7 bc211.2 bc427.9 ab38.7 bc19.6 bc58.3 ab147.8 bc32.3 bc180.0 b46.1 bc4.3 bc50.4 b24.2 bc3.2 bc27.3 b
5216.9 bc217.9 ab434.8 ab38.7 bc20.3 ab59.0 ab147.9 bc33.3 ab181.1 b46.2 bc4.4 ab50.6 b24.2 bc3.3 ab27.5 b
6303.5 a228.6 ab532.1 a54.2 a21.2 ab75.4 a206.9 a34.9 ab241.8 a64.6 a4.6 ab69.3 a33.9 a3.4 ab37.3 a
7260.7 ab219.4 ab480.1 ab46.5 ab20.4 ab66.9 ab177.8 ab33.5 ab211.3 ab55.5 ab4.5 ab60.0 ab29.1 ab3.3 ab32.4 ab
8212.9 bc215.9 ab428.8 ab38.0 bc20.1 ab58.1 ab145.2 bc33.0 ab178.1 b45.3 bc4.4 ab49.7 b23.8 bc3.2 ab27.0 b
9302.9 a209.3 bc512.2 a54.1 a19.5 bc73.5 a206.5 a32.0 bc238.5 a64.5 a4.3 bc68.8 a33.8 a3.1 bc36.9 a
  1. 1- Control-without vaccination, 2- Nitragine, 3- Nitroflora, 4- Pseudomonas, 5-Bacillus, 6- Nitragine +Pseudomonas, 7- Nitragine + Bacillus, 8- Nitroflora + Pseudomonas, 9- Nitroflora + Bacillus, a, b-homogeneous groups (Duncan’s test, p <0.05)

The activity of useful microflora delivered in the form of seed inoculation in the rhizosphere may have contributed to increasing the efficiency of collection and absorption of minerals, which are usually difficult to access in the soil, entrapped in the soil absorption complex. In research conducted on soil after 100 years of the cultivation of cereal plants, Siebielec et al. [33] showed that soil pH and organic carbon had a strong impact on soil enzymatic activity and the structure of the microorganism population. As a result of the intensification, simplification of the crop rotation and the change in the method of fertilization in agriculture, there also appeared to be changes in the activity of microorganisms in the soil. In the future, it may be effective to introduce microorganisms into the soil, to maintain the balance, because over about 70% of all soil processes are conditioned by their activity [34].

The dependences between the agronomic traits in selected experimental and control objects were illustrated by means of principal component analyses (PCA) for the synthesis of years (Figure 2, 3). On the object with seed co-inoculation (Rhizobium+Pseudomonas fluorescens) the first component explained 42.75% of the dependences, whereas the second component explained 30.72% of the dependences. In total they explained 73.47% of variability. On the control object without inoculation they explained 52.57% and 25.02% of variability, respectively, i.e. 77.59% of total variability. Both on the control and co-inoculation Rhizobium with Pseudomonas objects there were strong dependences between yield, number of root nodules, nodule dry mass and root mass. The correlation coefficients between the above-listed traits were r=0.65; 0.84; 0.55 and r=0.82; 0.94; 0.90, respectively on the control and Rhizobium+Pseudomonas objects. However, the correlation between yield and root mass on the control was not statistically proven. The yield increase caused by inoculation in tested variants, especially with Rhizobium+Pseudomonas could be attributed to an increase in root development, which allows for better mineral and water uptake.

Figure 2 Dependences observed on control object without seeds inoculation.
Figure 2

Dependences observed on control object without seeds inoculation.

Figure 3 Dependences observed after inoculation seeds with Rhizobium + Pseudomonas fluorescens.
Figure 3

Dependences observed after inoculation seeds with Rhizobium + Pseudomonas fluorescens.

4 Conclusion

Based on the synthesis from three years of field experiments, it was shown that all tested variants of white lupine seed inoculation stimulated plant height, number of root nodules, green leaf index value, mass of aboveground part and number of pods and seeds in the plant, resulting in yield seeds. Among the tested rhizobium and endophytic bacteria PGPRs intended for the inoculation of white lupine seeds, Rhizobium bacteria from the nitragine preparation used exclusively or in the form of co-inoculation together with Pseudomonas fluorescens proved to be the most effective in stimulating the seed yield. In addition, co-inoculation with bacteria of the species Rhizobium derived from the preparation of nitragine with Pseudomonas fluorescens stimulated plant growth, the development of aboveground parts, roots, root nodules and the formation of pods and seeds on the plant. Therefore, according to the obtained results, the optimal for white lupine treatment was Rhizobium+Pseudomonas.

There is a need to bring new combinations to the market. In this respect, attention should be paid to the potential use of Rhizobium and Pseudomonas fluorescens as the so-called consortium of microorganisms to stimulate the growth of white lupine plants in balanced plant production systems.


This research is financed by the Ministry of Agriculture and Rural Development of the Republic of Poland in the framework of the Multi-annual Program ‘Increasing the use of domestic feed protein for the production of high quality animal products in conditions of balanced development’ implemented at the Department of Agronomy of the University of Life Sciences in Poznan.

  1. Conflict of interest

    Authors state no conflict of interest.


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Received: 2019-01-17
Accepted: 2019-05-10
Published Online: 2019-10-29

© 2019 Hanna Sulewska et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 Public License.

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