1. Introduction
Rice (
Oryza sativa L.) is widely cultivated around the world and is primarily consumed as a basic food source in Asia, Latin America, the Caribbean, and increasingly more in Africa [
1]. The global consumption of rice should increase by 1.1% YoY in the coming decade, with Asian countries responsible for 70% of the projected increase, in large part driven by population growth as opposed to an increase in per capita consumption. In the various regions, only in Africa is significant growth in per capita consumption of rice projected [
1]. The forecasted global rice production in 2022 is of the order of 512.6 million tonnes (beneficiary base) [
2], and it is expected that rice productivity will increase by 12% due to technological and cultivation practice improvements, particularly in middle-income countries [
1].
In Brazil, rice cultivation irrigated by flooding is cultivated in 1.3 million hectares of lowlands, and produces ±10,700 million tonnes of the cereal annually [
3]. Flood-irrigated rice, which is concentrated in southern Brazil, provides ±70% of national production. In the southern region, one of the challenges is to increase the profitability and quality of irrigated rice, as well as reduce the risk of environmental contamination due to the high use of chemical inputs, mainly pesticides and synthetic fertilizers. In rice-growing areas of Malaysia, nitrogen fertilizers are used extensively in rice cultivation to meet the growing demands of the crop. However, the excessive use of chemical fertilizers in recent decades has led to soil toxicity by contamination with toxic heavy metals, as well as affects on the health of rice plants [
4]. Commercially available fertilizers are blended with a range of trace metals, which are introduced into the soil along with the application of fertilizers [
5]. Among inorganic fertilizers, phosphate fertilizers are the major source of contaminants, as they may contain traces of Cd, Pb, As, Cr, fluorine (F), strontium (Sr), thorium (Th), uranium (U), and zinc (Zn) [
6]. In the south of Brazil, where some crops of irrigated rice have been cultivated for more than 100 years, a study revealed that elements (Cd, Cr, and Pb) quantified in the soil and in the crops were within the limits allowed by Brazilian legislation, with the exception of the Cd levels in the crop waters [
7].
Therefore, the use of biological fertilizers to reduce chemical fertilizers is one of the most effective steps toward sustainable agriculture [
8,
9].
In the State of Rio Grande do Sul (RS), about one million hectares are cultivated with irrigated rice (due to flooding) that reaches a productivity of more than 8 tonnes per hectare as a result of the technification of the crop and favorable environmental conditions. In rice farming irrigated by flooding, the application of chemical nitrogen fertilizers is essential to achieve high yields. It is noteworthy, however, that there is great variability in the results inherent to agronomic efficiency, which rarely exceed 50% of the applied dose, resulting in limits to productivity and increases the cost of production [
10]. Previous reports by Fageria and Baligar [
11] already highlighted that the efficiency of N recovery by the flooded rice is around 40% in lowland soil. In this situation, the rational use of nitrogen fertilization is essential to not only increase recovery efficiency, but also to increase crop productivity and reduce the production costs and risks of environmental pollution. Additionally, excessive nitrogen fertilization is usually applied to maximize yields in irrigated rice fields in China, with the N recovery efficiency being around 30 to 35%, resulting in serious diffuse agricultural pollution [
12]. Agroecosystem modeling processes can be applied to assess the impacts of nitrogen management alternatives on agricultural production and the environment at municipal, state, regional, federal, continental, and global scales [
13], since the optimization of agronomic application of nitrogen fertilizers depends on agroclimatic variables, as well as soil, crop, and nutrient management [
14]. Thus, mitigating measures are studied to reduce nitrogen fertilization and maintain and/or increase the productivity of agricultural crops, such as the use of bio-stimulants based on
Ascophyllum nodosum extracts [
15]. In addition, the increase in the cost of these fertilizers in the world market and the gap between supply and demand constitute difficulties, which is added to the possibility of gaseous losses (N
2O) by leaching and superficial runoff. In irrigated rice, N losses due to ammonia volatilization resulting from the use of urea vary from 15 (saturated soil) to 22% (moist soil) of the applied nitrogen when the interval between application of this fertilizer and the beginning of irrigation is 10 days [
16]. In rice fields with wheat straw incorporation, the application of controlled-release nitrogen fertilizer in place of the conventional one can notably increase rice grain yield and improve the efficiency of N use, but have little effect on greenhouse gases, due to its stimulation in the emission of N
2O [
17]. Recently, Veçozzi et al. [
18] found that the technology for increasing N use efficiency with the use of controlled-release nitrogen fertilizers, as a potential alternative to reduce N losses, was similar to the behavior of uncoated urea, and did not increase the release time of the nutrient in irrigated rice cultivation. Thus, it does not constitute a management practice to minimize N losses from the irrigated rice crop. Furthermore, the outcome of the use of controlled-release nitrogen fertilizer depends on the environmental conditions of a given region, which can result in the variation of N release characteristics and synchronization with the demand of the rice crop [
19]. Zheng et al. [
20] report that to avoid soil degradation caused by the abundant use of chemical fertilizers and to promote high efficiency in the use of nitrogen, the combination of biochar and controlled-release nitrogen fertilizer can be applied as an effective way to achieve high yield, high fertilization efficiency, and sustainable rice production in northeast China.
In this context, addressing the loss of N in agricultural production and proposing realistic mitigation strategies based on efforts aimed at materializing associative N fixation in non-legume plants [
21], especially in cereals such as rice, corn, and wheat, are some of the approaches that should be further investigated [
22]. In this sense, it is important to note that, in flooded soil, the root-soil interface is the site of nitrogen fixation, and the bacteria sustaining this activity under anaerobic conditions are heterotrophic diazotrophs, with the rhizosphere being composed of microsites where this process occurs [
23].
In addition, there are other problems associated with the use of N, especially when high doses are applied in the initial phase of the crop, which include promoting excessive plant growth, causing self-shading of leaves and increasing susceptibility to fungal diseases, especially to brusone [
24]. The provision of high doses of nitrogen can increase the susceptibility of rice plants to brusone. This phenomenon is called nitrogen-induced susceptibility, which causes infections by the fungus
Magnaporthe oryzae (syn.
Pyricularia oryzae) [
25]. When doses above 60 kg ha
−1 of nitrogen are supplied without the application of fungicides, they cause an increase in brusone severity in rice panicles, a reduction in the percentage of the whole and vitreous grains, and an increase in plastered grains and plastered area [
26].
Biological nitrogen fixation (BNF) is one of the technological alternatives for reducing the use of chemical nitrogen fertilizer (CNF) in this crop and is part of the Low Carbon Agriculture Program of the Ministry of Agriculture, Livestock and Supply (MAPA). The inoculation of rice plants with plant growth-promoting rhizobacteria can significantly increase rice production, thus reducing the need for nitrogen fertilizers and contributing to sustainable rice production and to reduced environmental problems [
8]. Considering that diazotrophic endophytic bacteria may bring about a potential 20–30% increase in rice production [
27] and interact positively with irrigated rice genotypes in lowlands (TB) [
28,
29,
30], it is necessary to define the effectiveness of nitrogen doses to complement the benefit of BNF. In addition, there are strains (
Azospirillum brasilense Ab-V5 and Ab-V6) that benefit the accumulation of N and promote the growth of grasses, increasing the biomass of the aerial part by 16.8%, with inoculation by the spraying of seeds and leaves of
Brachiaria (
Urochloa spp.) [
31], and the N content by 25% in the species of
Urochloa brizantha and
Urochloa ruziziensis [
32].
In this study, the agronomic efficiency of the Ab-V5 and Ab-V6 strains of Azospirillum brasilense, recommended by MAPA (Normative Instruction N° 13 of 03/24/2011) for maize (Zea mays L.), wheat (Triticum aestivum L.), and rice (Oryza sativa L.), as well as for co-inoculation with rhizobia in soybean (Glycine max (L.) Merr.) and common bean (Phaseolus vulgaris L.), on the development of rice cultivars irrigated by flooding in the lowland agroecosystem is examined. The efficiency is studied to obtain conclusive information about the possibility of reducing nitrogen fertilization with the inoculation of A. brasilense in the soil and climate conditions of Rio Grande do Sul.
4. Discussion
In this work, we demonstrated that
A. brasilense in rice, besides the agronomic potential for saving about 30% of N, involves the employment of an input of biological nature that benefits plant root growth promotion and increases resistance to biotic and abiotic stresses. Additionally, plant growth-promoting diazotrophic bacteria can improve the ability of rice plants to assimilate N from the soil, and represents a viable economic and environmental strategy to improve pasture production as well [
31]. Another fact is that a high N supply to rice plants does not allow for optimal photosynthetic rates and increases the likelihood of photoinhibition due to an imbalance of sunlight absorption and utilization [
44]. Another study reported that regardless of the inoculation method—in seeds or by foliar application—
A. brasilense strains Ab-V5 and Ab-V6 promoted corn plant growth, which was a reflection of the application of cells and metabolites that promoted both the synthesis of phytohormones and the induction of plant defense-related genes [
45].
We also highlight the alignment of BNF in rice with the Sustainable Development Goals (SDGs) of the United Nations Sustainable Development Summit and the United Nations (UN) Sustainable Rice Platform (SRP), with action for innovation in the relations between producers and the Brazilian government regulatory bodies. Concerns about the N economy, efficiency, and impact on the environment have renewed interest in exploring alternative or supplemental sources of N for sustainable agriculture [
46]. BNF in irrigated rice constitutes an alternative source in organic production systems and a supplementary source to reduce nitrogen fertilization in chemical production systems. It also presents itself as a “bio-economy” practice to replace fossil and non-renewable resources.
In the experimental field,
A. brasilense. (strains Ab-V5 + Ab-V6) combined with a reduced nitrogen fertilizer dose using seed inoculation with a liquid formulation promoted an average increase of 30% in rice production when compared with the absence of nitrogen fertilizer. On the other hand, in the 2014/2015 season experiment, plants with no nitrogen fertilizer and inoculant showed higher shoot dry mass production. This result was associated with climatic factors, such as temperature and solar radiation, which occurred in this season and were favorable to productivity [
47]. Although, the productivity variable tends to decrease with the absence of nitrogen coverage application, which is in agreement with Scivittaro and Machado’s observations [
48] that nitrogen requirements are higher in the tillering and reproductive phases, and it is in this last phase that the plant presents greater efficiency in the absorption of N for grain production.
A similar result was found with the highest agronomic efficiency of inoculation of rice seeds with the liquid inoculant based on
A. brasilense (strains Ab-V5 + Ab-V6) in experiments carried out in the municipalities of Toledo, Palotina, Cascavel, and São Miguel do Iguaçu in the state of Paraná, Brazil [
49].
Field studies indicate that associative N
2 fixation can potentially contribute amounts of N (>30–40 kg N ha
−1 yr
−1) to the nutrition of plants that are important in tropical agriculture, including sugarcane (
Saccharum sp.) and forage grasses (
Panicúm maximum,
Brachiaria sp. And
Leptochloa fusca), when cultivated in non-inoculated and N-deficient soils. Data from pot experiments indicate that rice can naturally benefit from associative N
2 fixation and that inoculation responses may occur due to N
2 fixation [
50].
In our work, the practice of inoculating rice seeds with
Azospirillum allowed for a reduction of 30 kg of N ha
−1 and, consequently, contributed to reducing the emission of greenhouse gases. The agronomic efficiency (AE) in the use of N by irrigated rice was obtained by the difference between the grain yield in the nitrogen treatments (nitrogen fertilizer 120 kg of N ha
−1), treatments with
A. brasilense, and nitrogen fertilizer (90 kg of N ha
−1), and treatments with the absence of nitrogen fertilization and inoculant, divided by the amount of N applied, according to the following formula [
51]:
where PG
cf = grain yield with fertilizer, PG
sf = grain yield without fertilizer, and QN
a = amount of nutrient applied, all expressed in kg·ha
−1.
The results of the AE of 32 kg grains kg−1 of N applied with A. brasilense and 90 kg of N ha−1 and of 22 kg grains kg−1 of N applied with 120 kg of N ha−1, demonstrate an economic productivity that is 45% higher for grains with the inoculation of rice seeds with A. brasilense.
In our work, in five consecutive agricultural seasons, average yields above 12,000 kg ha
−1 in the BRS Pampa cultivar were observed with the combined use of
A. brasilense and reduced mineral nitrogen fertilization. The productivity differential of the combination of 90 kg of N ha
−1 (covering fertilization) with the inoculation in relation to the treatment of 120 kg of N ha
−1 was 1.5%, which corresponded to about four bags ha
−1. Considering that the price of paddy rice (50 kg bag) was BRL 83.82 (May 2021) [
52], there was a gain of BRL 335.28 ha
−1 with the difference in bags obtained with the use of
A. brasilense (strains Ab-V5 + Ab-V6). Regarding the cost, with the average price of 1 kg of nitrogen fertilizer (urea source) in October 2022 in Brazil (US
$0.802; CONAB; 1 US
$ = R
$5.36), which corresponds to US
$802 per tonne of urea (US
$1.78 kg of N), the farmer saving 30 kg of N ha
−1 saves US
$53.4 ha
−1. As the cost of inoculation is about US
$2 ha
−1, the savings would be over US
$51 ha
−1.
In the validation fields, A. brasilense combined with nitrogen fertilization with a 25% reduction in the coverage fertilization (90 kg of N ha−1) and with the cover fertilization adopted by the producer showed agronomic efficiency in obtaining high productions of rice grains. The highest yield was obtained with the cultivar BRS Pampeira (12,180 kg ha−1), which resulted from the use of A. brasilense combined with a 20% reduction in nitrogen fertilization (119.2 kg of N ha−1), which resulted in an increase of 8.6 bags ha−1 compared to the control treatment (without the reduction of the nitrogen fertilization of cover).
In economic terms, the inoculation technology of rice seeds with
A. brasilense (strains Ab-V5 and Ab-V6) for different cultivars increased the net profit per hectare with the adoption of the inoculant combined with nitrogen fertilization for: BRS Pampeira (R
$318.08), BRS Pampa (BRL 2025.91), and IRGA 424RI (BRL 677.11) [
53]. The economic viability analysis was based on the change in management (adoption of the use of inoculant instead of nitrogen fertilization adopted by the producer) using the partial budgeting technique. The price of rice considered was US
$ 6.51 (1 US
$ = R
$ 5.36) sc
−1 of 50 kg during the 2017/18 harvest (03/2018), and US
$ 7.45 (1 US
$ = R
$ 5.36) sc
−1 of 50 kg, during the 2018/19 harvest (03/2019). The prices of nitrogen fertilizer (urea) were US
$ 0.150 (1 US
$ = R
$ 5.36) kg
−1, the commercial inoculant was US
$ 1.86 (1 US
$ = R
$ 5.36) vial
−1 of 10 mL, and the inoculant additive was US
$ 1.30 (1 US
$ = 5.36) ha
−1, in effect at the sowing time of the 2017/18 (10/2017) and 2018/19 (10/2018) harvest.
The results of these validations demonstrate that, in lowland agroecosystems, it is possible to obtain higher-than-average yields of rice irrigated by flooding in RS with the practice of inoculating seeds with an inoculant containing
A. brasilense. Similar results with the inoculation of grain-producing grasses with
A. brasilense, in different soil and climatic conditions in Brazil, have been previously reported for rice [
49], corn and wheat [
40], and wheat [
54].
We also highlight the importance of BNF in irrigated rice for systems with official government certifications, such as Organic Rice Production, which needs alternatives for nitrogen supply, given the high demand of the nutrient for this cereal and the fact that the use of fertilizers obtained through industrial processing would not be allowed [
55].
Thus, the results of our research demonstrate that it is possible to use a low-carbon and low-cost technology to improve the profitability of irrigated rice crops, which contributes to the sustainability of rice farming activities in Brazil and respects the environment. The use of inoculants combined with the reduction of nitrogen fertilization in irrigated rice, associated with other management practices, can reduce the emission of greenhouse gases and mitigate global climate change. In a review of plant growth-promoting microorganisms (MPCV) and their derived compounds, Naamala and Smith [
56] highlighted the need for research to make the use of MPCV technology more effective in developed and developing countries, with the aim to increase plant growth and to reduce greenhouse gas emissions from the agricultural sector. In this sense, in work carried out in the field with genotypes of
Brachiaria spp., Hungria et al. [
32] found that
A. brasilense strains Ab-V5 and Ab-V6 increased N accumulation in the biomass, which were equivalent to a second application of 40 kg ha
−1 of fertilizer N. At the same time, they proved that gains of 0.103 Mg C ha
−1 were due to inoculation, which corresponded to 0.309 Mg CO
2 eq. ha
−1. Thus, the authors concluded that inoculation with
A. brasilense may represent a key component in the pasture recovery program and help sequester CO
2 from the atmosphere. In turn, Veçozzi et al. [
57], in a study carried out with greenhouse gas emissions associated with controlled-release nitrogen fertilizer and urea in irrigated rice cultivation, determined that the management of nitrogen fertilization influenced the partial global warming potential. The determined values corresponded to 13,946 kg CO
2 eq. ha
−1 for urea, 14,297 kg CO
2 eq. ha
−1 for the controlled release nitrogen fertilizer applied by surface broadcast, and 11,683 kg CO
2 eq. ha
−1 for controlled release nitrogen fertilizer applied locally in the sowing furrow.
Nitrogen use efficiency (NUE) by rice use is necessary for environmental conservation and agricultural sustainability. Genetically modified crops and integrated management practices are the most effective biotechnological methods for enhancing NUE [
58]. The development of new cultivars that utilize N more efficiently through conventional breeding, as well as through the use of CRISP-based gene editing, such as manipulation of the flavone biosynthetic pathway, must be considered as a viable strategy for the induction of biological fixation in rice and a reduction in the use of inorganic nitrogen fertilizers [
59].
Thus, we emphasize that although the use of the inoculant Azospirillum brasilense (strains Ab-V5 and Ab-V6) in irrigated rice does not completely replace the application of mineral nitrogen fertilizers when aiming at high yields, it may reduce the amount demanded of this nutrient and, thus, reduce the global warming potential.