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SOIL BIOLOGY
Nitrogen use by plants and nitrogen flows after application of standard and biomodified nitrogen fertilizers on barley
Alexey Zavalin1, Vladimir Chebotar2, Alexey Alferov1,
Lyudmila Chernova1, Elena Shcherbakova2, and Elena Chizhevskaya2
1All-Russian Scientific Research Institute of Agrochemistry named after D. N. Pryanishnikov, ul. Pryanishnikova, 31a, Moscow, 127434, Russian Federation 2All-Russia Research Institute for Agricultural Microbiology, Shosse Podbel'skogo, 3, Saint Petersburg, 196608, Russian Federation
Address correspondence and requests for materials to Vladimir Chebotar, vladchebotar@rambler.ru
Abstract
Citation: Zavalin, A., Chebotar, V., Alferov, A., Chernova, L., Shcherbakova, E., and Chizhevskaya, E. 2021. Nitrogen use by plants and nitrogen flows after application of standard and biomodified nitrogen fertilizers on barley. Bio. Comm. 66(4): 283-289. https://doi.org/10.21638/ spbu03.2021.401
Authors' information: Alexey Zavalin, Dr. of Sci. in Agricultural Sciences, Professor, Head of Laboratory, orcid.org/0000-0001-7717-877X; Vladimir Chebotar, PhD, Head of Laboratory, orcid.org/0000-0001-9762-989X; Alexey Alferov, Dr. of Sci. in Biology, Senior Researcher, orcid.org/0000-0001-7988-6809; Lyudmila Chernova, PhD, Senior Researcher, orcid.org/0000-0001-8424-1175; Elena Shcherbakova, PhD, Junior Researcher, orcid.org/0000-0002-7871-034X; Elena Chizhevskaya, PhD, Senior Researcher, orcid.org/0000-0002-7715-8696
Manuscript Editor: Alla Krasikova, Department of Cytology and Histology, Faculty of Biology, Saint Petersburg State University, Saint Petersburg, Russia
Received: October 13, 2020;
Revised: August 10, 2021;
Accepted: August 16, 2021.
Copyright: © 2021 Zavalin et al. This is an open-access article distributed under the terms of the License Agreement with Saint Petersburg State University, which permits to the authors unrestricted distribution, and self-archiving free of charge.
Funding: The study was partly supported by the Russian Foundation for Basic Research, Grant no. 18-016-00048 A. The work of V. K. Chebotar and E. P. Chizhevskaya was supported by the project "Development of potato breeding and seed production in the Russian Federation" of the Federal Scientific and Technical Program for Agricultural Development for 2017-2025.
Ethics statement: This paper does not contain any studies involving human participants or animals performed by any of the authors.
Competing interests: The authors have declared that no competing interests exist.
The aim of our study was to assess the efficiency of application of biomodified nitrogen fertilizers for barley, to reveal the sources of nitrogen used for biomass formation with the use of the 15N stable isotope, and to study nitrogen flows in the system of fertilizers-soil-plants-atmosphere. We demonstrated in a model experiment the ability of the plant growth-promoting bacteria Bacillus subtilis Ch-13 to move from the granules of mineral fertilizers to plant roots and to colonize them effectively. The effectiveness of biomodified nitrogen fertilizers for barley, Nur variety, was assessed in a microfield trial. After the application of biomodified nitrogen fertilizers, the accumulation of 15N in the plants increased by 2-5%, its incorporation in the soil decreased and gaseous losses were decreased by 7% as compared with the use of the usual forms of fertilizers. The application of biomodified nitrogen fertilizers can be used in agricultural practice as a novel technology to regulate nitrogen flows in the system of fertilizers-soil-plants-atmosphere.
Keywords: PGPB, nitrogen fertilizers, biomodified fertilizers, nitrogen isotope, barley biomass, nitrogen flows
Introduction
About 3 million tons of the active ingredient (NPK) of mineral fertilizers are annually used in Russian agriculture, which corresponds to 40 kg of the active ingredient (NPK) per 1 ha of crops. Less than half of the nutrients introduced with mineral fertilizers is actually used by plants for yield formation (Zavalin and Sokolov, 2019). Increasing the efficiency of mineral fertilizers is important both economically and ecologically (Ohkama-Ohtsu and Wasaki, 2010). One of the ways to achieve this is to use microbial preparations based on plant growth-promoting bacteria for treatment of granules of mineral fertilizers (Hassan et al., 2019).
The mechanism of their action is based on the fact that the microorganisms used for pelleting of granules of mineral fertilizers increase the availability of nutrients contained in mineral fertilizers and mobilize their reserves in soil; they produce amino acids, vitamins, hormones and organic acids promoting plant growth and enhancing its immune defences; and they synthesize substances blocking the development of phytopathogenic microorganisms (Adesemoye and Kloepper, 2009; Chebotar et al., 2009; Bhat 2019). After treatment with bacteria, a "biocapsule" is formed on the surface of pelleted mineral fertilizers. It has several functions at the same time: it fertilizes, protects and stimulates.
Such a combined beneficial effect makes it possible to achieve a considerable increase in the yield of agricultural crops and thus, in the pay-off of mineral
fertilizers (Kozhemyakov et al., 2015; Chebotar et al., 2016b). Such microorganisms enhance plant resistance to phytopathogens (Barraquio et al., 1997; Rothballer et al., 2007; Chebotar et al., 2015) and their ability to synthesize phytohormones (Bloemberg and Lugten-berg, 2001), stimulate root system growth thus improving mineral nutrition (Okon and Vanderleyden, 1997; Bertrand et al., 2000; Ruby and Raghunath, 2011; Chebotar et al., 2016a), and regulate the rate of water uptake (Dobbelaere et al., 2001; Shaposhnikov et al., 2011). Plant growth-promoting bacteria (PGPB) Bacillus subtilis Ch-13, used in Russia for the production of the microbiological fertilizer Extrasol, stimulated plant growth, producing phytohormones — auxin derivatives (Chebotar et al., 2009). Inoculation with Bacillus subtilis Ch-13 reduced plant stress induced by heavy metals exposure and salinity (Pishchik et al., 2009).
The aim of our study was: 1) to assess the efficiency of application of nitrogen fertilizers biomodified by the Bacillus subtilis Ch-13 for barley (Hordeum vulgare L.), and 2) to reveal the sources of nitrogen used for biomass formation with the use of the 15N stable isotope and to study nitrogen flows in the system of fertilizers-soil-plants-atmosphere.
Materials and methods
Gnotobiotic system for plants
A 15-cm-long tube with holes on the surface was placed in a glass beaker with calcined moistened sand. Granules of mineral fertilizer Nitroammophoska (3 g per beaker) treated with a sterile mineral carrier (negative control) and granules treated with the microbiological powder fertilizer on the basis of Bacillus subtilis Ch-13 (50x103 CFU/g) were placed into the tube. Barley seeds (Hordeum vulgare L.) of the universal Nur variety were used for the study. Seeds (40-50) were sterilized for 20 minutes in 70 % ethanol, followed by 35 min in a sterilizing mixture containing 20 ml of sodium hypochlorite, 50 ml of sterile water, and 1 ml of 10 % SDS. The seeds were then washed thrice with sterile water for 3 min, placed into Petri dishes with tryptic soy nutrient agar (TSA, Difco Laboratories, MI, USA) and placed in an incubator at 28 °C for 24 hours to check their sterility. Sterile seeds were transferred into Petri dishes with sterile moistened filter paper and germinated in an incubator at 28 °C for 24 hours. Sterile plant seedlings were transferred to the system and grown for 3 days. Then the gnotobiotic system was disassembled under sterile conditions, and the seedlings were washed from the sand in sterile water. The experiment was performed in three replications with five plants in each beaker.
Plants roots from each variant were used to isolate bacteria that had colonized the root surface. The plant
roots were additionally washed with sterile 0.85 % NaCl solution, ground in a mortar with 2 ml of 0.85 % NaCl solution, and used for preparation of serial dilutions. Aliquots of 100 ^l of the cell suspension were plated on tryptic soy agar plates. Plates were incubated at 28 °C for 3 days. Colonies derived from cell suspension were identified and subjected to Amplified Fragment Length Polymorphism (AFLP) analysis.
Identification of bacterial strains
Total genomic DNA of all Bacillus strains was isolated using the lysozyme-sodium dodecyl sulfate method (Laguerre et al., 1992). The following primers were used for PCR amplification of 16S rRNA gene: (5'-3') fD1 (AGAGTTT-GATCCTGGCTCAG) and rD1 (CTTAAGGAGGT-GATCCAGCC). PCR amplification was performed according to standard protocol (Weisburg et al., 1991). The obtained PCR fragments were isolated from agarose gel (Onishchuk et al., 2015) and sequenced using the ABI PRISM 3500xl (Applied Biosystems, Waltham, MA, USA). The sequences were compared with the sequence of the 16S rRNA gene of the strain Ch-13 B. subtilis, available in the GenBank database (Accession Number: MW050985).
AFLP analyses
Genomic DNA of bacterial strains was digested with EcoRI and MseI and ligated to EcoRI and Msel adapters, as described by Vos et al. (1995). Three sets of primers were used in separate PCRs: (5'-3') EcoRI-0 (GACTGCGTACCAATTC) and MseI-A (FAM-GAT-GAGTCCTGAGTAAA), EcoRI-0 and MseI-CA (FAM-GATGAGTCCTGAGTAACA), EcoRI-0 and MseI-GC (FAM-GATGAGTCCTGAGTAAGC). All PCRs were performed with the following temperature profile: de-naturation for 2 min at 72 °C, 20 cycles of denaturation (20 s at 94 °C), annealing (30 s at 55 °C) and extension (2 min at 72 °C). The results of PCRs were analyzed using the sequencer ABI PRISM 3500xl according to the manufacturer's instructions (AFLP™ Microbial Fingerprinting [Applied Biosistems, USA]).
Microfield trial
The effectiveness of biomodified nitrogen fertilizers, ammonium nitrate (Nan) and urea (Nu) for barley (universal Nur variety) was assessed in a microfield trial in bottomless pots in 2017-2019. Soil moisture was sustained at a level of 60-70 % of the total field water holding capacity. We used Soddy Retisol (Loamic) soil (IUSS Working Group WRB. 2015) with the following characteristics: humus content = 1.98-2.04 %; pHkci = 5.1-5.2; total N content (according to Kjeldahl) 0.11-0.12 %; content of mobile forms of P205 and K20 = 58-67 and 153-161 mg/kg, respectively. The pots were filled with
AFLP analyses by three sets of PCRs primers: a — primers EcoRI-0 and Msel-A, b — primers EcoRI-0 and Msel-CA, c — primers EcoRI-0 and Msel-GC; lines: 1-3 strains isolated from the barley roots grown in "gnotobiotic system" (variants 1-3, respectively), 4 — strain Ch13 B. subtilis (positive control), 5 — strain 1.5A B. pumilus (negative control).
soil and nitrogen fertilizers were applied there in the form of salts: ammonium nitrate — Nan (15NH415NO3) 47.29 atomic %, and urea — Nu (CO(15NH4)2) with an enrichment of 47.85 atomic %, both at rates of 96 and 193 mg/pot, which corresponds to N45 kg and N90 kg per hectare. Two forms of nitrogen fertilizers were added separately to each pot in four replications (4 pots).
Double superphosphate and potassium chloride (P) were also applied to all pots at rates equivalent to P60K60 per hectare. The experiment was conducted in six replications. In the tillering, tubing, and earing phases, the weight of plants was measured and soil samples were taken in one replication. In the dead-ripe phase, the biomass of plants was measured in three replications.
Biomodification of nitrogen fertilizers
For biomodification, granulated mineral fertilizers were treated with microbiological powder fertilizer on the basis of Bacillus subtilis Ch-13 (MF) at a rate of 10 g/kg of the fertilizer to achieve a cell number not less than 50x103 CFU/g (Chebotar et al., 2017).
Agrochemical analysis
Total nitrogen and its isotopic composition in the plants and in the soil were measured using Delta V mass spectrometer (Thermo Fisher Scientific, USA). Phosphorus
and potassium in plants were measured using infrared spectroscopy.
Statistic analysis
The data in the trial were assessed by Fisher's least significant difference (LSD) method (ANOVA).
Results
Colonization of barley roots by the PGPB B. subtilis Ch-13 in a model experiment
A gnotobiotic system was used to study the colonization of the barley roots by the strain B. subtilis Ch-13. Bacteria were isolated from the roots of barley seedlings and identified as belonging to the type B. subtilis by the 16S rRNA method. AFLP analyses demonstrated that all isolates belonged to the strain B. subtilis Ch-13 (Figure). The number of cells of B. subtilis Ch-13 on the barley roots in the variant with biomodified Nitroammophoska was 5.19x106 CFU per plant. In the control with Nitro-ammophoska, B. subtilis Ch-13 was not detected. Our experiments with the use of the "gnotobiotic system" showed that the strain B. subtilis Ch-13 could effectively colonize the barley roots, moving from the tube with biomodified Nitroammophoska to the plant seedlings.
Microfield trial
The effect of biomodified fertilizers on the barley yield.
The agronomical efficiency of nitrogen fertilizers was assessed based on the grain and the straw weight and other production parameters such as height of plants, productive tilling capacity, number of plants, head length, thousand-kernel weight and protein content in grain. When Nan was applied at a rate of N45, the grain weight of barley statistically significantly increased as compared with the PK control, while the application of the biomodified Nan at this rate had no positive effect (Table 1). The use of standard Nu at a rate of N45 increased the grain weight as compared with the PK background. The application of its biomodified form at the same rate statistically significantly increased the grain weight as compared with the standard Nu.
Increasing the rates of the usual forms of nitrogen fertilizers had a positive effect on the grain weight, which was associated with improved nitrogen nutrition of the plants. The application of biomodified Nan at a rate of N90 had a positive effect on the increase of the barley grain weight (Table 1).
After the application of standard and biomodified nitrogen fertilizers on barley, the straw weight increased up to two times as compared with the PK control. A positive effect was observed after the use of biomodified Nu at both rates and the use of biomodified Nan at a rate of N90.
We revealed differences in the barley yield structure under standard and biomodified nitrogen fertilizers (Table 1). Owing to improved nitrogen nutrition associ-
ated with nitrogen fertilizers, the head length of barley increased from 4.5 to 5.2-6.1 cm and showed a tendency to grow in the case of application of biomodified forms of nitrogen fertilizer. The content of raw protein in the barley grain increased after the application of standard nitrogen fertilizers. This same effect from their biomodified forms was registered only in the case of Nu (at both rates), the added protein in the yield being 1 % (Table 1).
Accumulation of nutrition elements in barley. Accumulation of nitrogen, phosphorus and potassium in the plants and the proportion of different sources of nitrogen in the formation of barley biomass have been analysed. Owing to the improved nitrogen nutrition, the biomass of grain and straw increased, while the accumulation of nitrogen, phosphorus and potassium in the plants increased considerably (Table 2). Using the stable isotope 15N, we identified the proportion of nitrogen from the soil, nitrogen from the fertilizer, biological (fixed from atmosphere) nitrogen and "extra" (mineralization of organic matter in the soil) nitrogen in the formation of barley biomass (grain + straw) (Table 2). In the experiment without the application of nitrogen fertilizers, the biomass of barley was formed only at the expense of soil nitrogen (100 %). In the experiment with the application of Nan and Nu, the biomass accumulated not only the soil nitrogen but also nitrogen from fertilizers, identified based on the 15N isotope (Table 2). A positive effect was observed after the use of biomod-ified Nu at both rates and the use of biomodified Nan at a rate of N90. It is interesting to note that biomodification of mineral fertilizers considerably increased the proportion of "biological" nitrogen in the proportion of
Table 1. Barley productivity after application of usual and biomodified nitrogen fertilizers
Variant Grain weight, g/pot Straw weight, g/pot Height of plants, cm Productive tilling capacity, number of plants Head length, cm Thousand-kernel weight, g Protein content in grain,%
1. PK (background -P) 15.0a 12.9a 44.7a 1.66a 4.5a 42.6a 8.3a
2. P + MF 15.7a 14.0a 48.5b 1.55a 5.4a 45.0a 8.9b
3. P + Nan45 19.2b 17.5b 50.0b 1.79a 5.2a 46.7b 8.7b
4. P + Nan45 + MF 19.1b 16.2b 51.3b 1.88a 5.4a 46.8b 8.2a
5. P + Nu45 18.3b 16.6b 49.5b 1.72a 5.5a 48.1b 8.3a
6. P + Nu45 + MF 21.3b 19.8b 50.3b 1.76a 5.8b 50.0b 9.1b
7. P + Nan90 23.3b 23.2b 55.2b 1.93a 5.7b 46.4b 9.2b
8. P + Nan90 + MF 26.0b 27.0b 54.6b 2.04b 6.4b 45.9b 9.0b
9. P + Nu90 22.1b 21.1b 50.9b 2.04b 5.3 46.6b 8.7b
10. Nu90 + MF 24.3b 25.3b 52.2b 2.04b 6.1b 47.7b 9.2b
LSD as 2,7 3,1 5,7 2,8 0,28 1,1 2,6
LSD (Least Significant Difference)
Letters by values denote significant differences among the treatments in a trial as assessed by Fisher LSD (ANOVA). Different letters indicate significant differences (P < 0.05).
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Table 2. Accumulation of nutrition elements in barley and the proportion of different sources of nitrogen in the formation of barley biomass
Variant Accumulation in plants, mg/pot (average values for 2017-2019 years) Proportion of nitrogen sources in plants, % (average values for 2017-2018 years)
N P2O5 K2O N soil 15N fertilizer N biological N "extra"
1. PK (background -P) 313a 157a 355a 100 - - -
2. P + MF 407b 206b 353a 77 - 23 -
3. P + Nan45 447b 264b 417a 70 9 - 21
4. P + Nan45 + MF 473b 234b 414a 66 9 20 5
5. P + Nu45 489b 233b 412a 64 11 - 25
6. P + Nu45 + MF 534b 267b 498b 59 11 18 12
7. P + Nan90 564b 307b 542b 56 15 - 29
8. P + Nan90 + MF 609b 326b 628b 51 16 15 18
9. P + Nu90 604b 328b 541b 52 18 - 30
10. P + Nu90 + MF 686b 352b 641b 45 17 14 24
LSD 0505 49 32 64
Letters by values denote significant differences among the treatments in a trial as assessed by Fisher LSD (ANOVA). Different letters indicate significant differences (P < 0.05).
Table 3. Flows of 15N nitrogen from fertilizers during barley cultivation, % of the applied amount (average values for 2017-2018years)
Variant Utilized by plants Fixed in soil Losses
1* 2 3 4 1 2 3 4 1 2 3 4
Nan45 11 22 35 42 81 63 47 36 8 15 18 22
Nan45 + MF 10 25 40 45 85 63 46 35 5 12 14 20
Nu45 20 28 42 57 78 69 47 28 2 3 11 15
Nu45 + MF 26 34 45 59 73 64 44 27 1 2 11 14
Nan90 13 19 39 45 75 62 40 30 12 19 22 25
Nan90 + MF 18 24 41 46 70 58 38 31 12 18 21 23
Nu90 25 30 41 57 64 56 43 25 11 14 16 18
Nu90 + MF 27 32 45 59 66 59 45 30 7 8 10 11
*Note: 1 — tillering, 2 — booting, 3 — ear emergence, 4 — dead-ripe grain.
nitrogen sources in plants, due to the enhanced activity of native nitrogen-fixing bacteria in the barley rhizos-phere (Table 2).
Flows of 15N nitrogen from fertilizers during barley cultivation. We studied the flows of nitrogen from fertilizers in the agroecosystem during barley vegetation by determining the amount of 15N in the plants and in the soil (Table 3). After application of fertilizers at a rate of N90, the use of 15N by plants increased by 1012 % as compared to N45, and a greater biomass of barley was formed. When biomodified nitrogen fertilizers were applied, the plants accumulated 15N more inten-
sively than in the case of standard nitrogen fertilizers. The accumulation was greater by 2-5 % in the tillering phase, by 3-5 % in the booting phase and by 2-5 % in the ear emergence phase (Table 3). A more intensive accumulation of 15N in different phases after the application of biomodified Nan and Nu as compared with the usual forms can be explained by the activity of PGPB B. subtilis Ch-13 on the plant roots. The application of biomodified nitrogen fertilizers on barley decreased the incorporation of 15N in the soil by 2-5 % and decreased the gaseous losses of nitrogen by 2-7 % as compared with the usual forms.
Discussion
Microbial inoculants are promising components for integrated solutions to agro-environmental problems because inoculants possess the capacity to promote plant growth, enhance nutrient availability and uptake, and support the health of plants (Vessey, 2003; Han and Lee, 2005; Adesemoye et al., 2008). We demonstrated in a model experiment the ability of the strain B. subtilis Ch-13 to move from the granules of mineral fertilizers to plant roots and to colonize them effectively. Thus PGPB B. subtilis Ch-13 can improve the growth and productivity of plants (Chebotar et al., 2009). The specific mechanism involved in PGPB-elicited enhanced nutrient uptake was proposed by Adesemoye and Kloepper (2009). It was proposed that PGPB promoted the growth of the plant and increased the root surface area or the general root architecture, better roots then released higher amounts of C in root exudates, the increase prompted more microbial activity, and the cycle of events made more N available for plants' uptake. The effect of nitrogen fertilizers biomodified with PGPB B. subtilis Ch-13 was demonstrated in a microfield trial with barley. We revealed differences in the barley yield and production parameters under standard and biomodified nitrogen fertilizers. An increase in the protein content is considered as a positive effect for fodder grain. At the same time, increased protein content associated with the use of nitrogen fertilizers corresponds to a higher brewing quality of the barley grain (Zavalin, 2000). When nitrogen fertilizers are applied, the mineralization of organic matter in the soil resulted in the formation of "extra" nitrogen (Zavalin et al., 2015; Chebotar et al., 2017), which is used by plants. Biomodification of mineral fertilizers with PGPB B. subtilis Ch-13 can considerably decrease the proportion of gaseous losses of nitrogen, thus improving the efficiency of fertilizers.
Conclusion
The application of biomodified nitrogen fertilizers in all the vegetative phases increased the accumulation of 15N in the barley plants, decreased its incorporation in the soil and reduced gaseous losses as compared with the usual forms. The application of biomodified nitrogen fertilizers can be used in agricultural practice as a novel technology to regulate nitrogen flows in the system of fertilizers-soil-plants-atmosphere and can improve the efficiency of fertilizers.
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