Nitrate reduction to ammonium: a phylogenetic, physiological, and genetic aspects in Prokaryotes and eukaryotes.

The microbe-mediated conversion of nitrate (NO3-) to ammonium (NH4+) in the nitrogen cycle has strong implications for soil health and crop productivity. The role of prokaryotes, eukaryotes and their phylogeny, physiology, and genetic regulations are essential for understanding the ecological significance of this empirical process. Several prokaryotes (bacteria and archaea), and a few eukaryotes (fungi and algae) are reported as NO3- reducers under certain conditions. This process involves enzymatic reactions which has been catalysed by nitrate reductases, nitrite reductases, and NH4+-assimilating enzymes. Earlier reports emphasised that single-cell prokaryotic or eukaryotic organisms are responsible for this process, which portrayed a prominent gap. Therefore, this study revisits the similarities and uniqueness of mechanism behind NO3- -reduction to NH4+ in both prokaryotes and eukaryotes. Moreover, phylogenetic, physiological, and genetic regulation also shed light on the evolutionary connections between two systems which could help us to better explain the NO3--reduction mechanisms over time. Reports also revealed that certain transcription factors like NtrC/NtrB and Nit2 have shown a major role in coordinating the expression of NO3- assimilation genes in response to NO3- availability. Overall, this review provides a comprehensive information about the complex fermentative and respiratory dissimilatory nitrate reduction to ammonium (DNRA) processes. Uncovering the complexity of this process across various organisms may further give insight into sustainable nitrogen management practices and might contribute to addressing global environmental challenges.

Impacts of conservation agriculture on crop yield and soil carbon sequestration: a meta-analysis in the Indian subcontinent.

In the quest of achieving sustainable crop productivity, improved soil health, and increased carbon (C) sequestration in the soil, conservation agriculture (CA) is increasingly being promoted and adopted in the Indian subcontinent. However, because some researchers from different regions of the world have reported reduced crop yield under CA relative to agriculture based on conventional tillage (CT), a meta-analysis has been conducted based on published research from India to evaluate the effects of CA on the yield of crops, accumulation of soil organic C as an index of soil health, and C sequestration in the soil in different regions and soil textural groups in the country. The meta-analysis is based on 544 paired observations under CA and CT from 35 publications from India was carried out using Meta Win 2.1 software. The results showed an overall significant (p < 0.05) reduction of 1.15% crop yield under CA compared to CT. Yearwise data showed a reduction of yields under CA from 2009 to 2016, but an increase from 2017 to 2020. Yield reduction was observed in the eastern, north-eastern, and southern regions of India but in western, northern, and north-western regions of the country, an increase was observed under CA rather than CT. Sandy loam and clayey soils exhibited higher crop yield under CA than under CT. Compared to CT, soil organic C content and soil C sequestration under CA increased by 8.9% and 7.3%, respectively. Also, in all the regions and soil textural groups both soil organic C accumulation and soil C sequestration were higher under CA than under CT. Factors such as rainfall, soil depth, available nitrogen (N), and total N significantly influenced the extent of yield increase/decrease and soil organic C accumulation under CA. Overall, results of the meta-analysis suggest that the promotion of CA in India will have to be location-specific taking into consideration the crops, soil attributes, and climatic conditions.

Rice-based integrated farming system improves the soil quality, bacterial community structure and system productivity under sub-humid tropical condition.

Rice-based integrated farming system improves the productivity and profitability by recycling resources efficiently. In the sub-humid tropics, rice production without sufficient nutrient replenishment often leads to soil health and fertility degradation. There has been very limited research on soil health and fertility after adopting a multi-enterprising rice-based integrated farming system (IFS), notably in the rice-fish-livestock and agroforestry system, when compared to a conventional farming system (CS). Therefore, the present study analyzed the dynamics of soil properties, soil bacterial community structure and their possible interaction mechanisms, as well as their effect on regulating soil quality and production in IFS, IFSw (water stagnant area of IFS) and CS. The results indicated that soil nutrient dynamics, bacterial diversity indices (Shannon index, Simpson index, Chao 1, ACE and Fisher index) and system productivity were higher in IFSw and IFS compared to CS. Moreover, relative operational taxonomic units of dominant bacterial genera (Chloroflexi, Acidobacteria, Verrucomicrobia, Planctomycetes, Cyanobacteria, Crenarchaeota and Gemmatimonadetes) were also higher in IFSw and IFS compared to CS. Mean soil quality index (SQI) was highest in IFSw (0.780 ± 0.201) followed by IFS (0.770 ± 0.080) and CS (0.595 ± 0.244). Moreover, rice equivalent yields (REY) and rice yields were well correlated with the higher levels of soil biological indices (SQIBiol) in IFS. Overall, our results revealed that rice-based IFS improved the soil health and fertility and ensuing crop productivity through positive interaction with soil bacterial communities and nutrient stoichiometry leading to agroecosystem sustainability.

Homology modeling and virtual characterization of cytochrome c nitrite reductase (NrfA) in three model bacteria responsible for short-circuit pathway, DNRA in the terrestrial nitrogen cycle.

NrfA is the molecular marker for dissimilatory nitrate reduction to ammonium (DNRA) activity, catalysing cytochrome c nitrite reductase enzyme. However, the limited study has been made so far to understand the structural homology modeling of NrfA protein in DNRA bacteria. Therefore, three model DNRA bacteria (Escherechia coli, Wolinella succinogenes and Shewanella oneidensis) were chosen in this study for in-silico protein modeling of NrfA which roughly consists of similar length of amino acids and molecular weight and they belong to two contrasting taxonomic families (γ-proteobacteria with nrfABCDEFG and ε-proteobacteria with nrfHAIJ operon). Multiple bioinformatic tools were used to examine the primary, secondary, and tertiary structure of NrfA protein using three distinct homology modeling pipelines viz., Phyre2, Swiss model and Modeller. The results indicated that NrfA protein in E. coli, W. succinogenes and S. oneidensis was mostly periplasmic and hydrophilic. Four conserved Cys-X1-X2-Cys-His motifs, one Cys-X1-X2-Cys-Lys haem-binding motif and Ca ligand were also identified in NrfA protein irrespective of three model bacteria. Moreover, 11 identical conserved amino acids sequence was observed for the first time between serine and proline in NrfA protein. Secondary structure of NrfA revealed that α-helices were observed in 77.9%, 73.4%, and 77.4% in E. coli, W. succinogenes and S. oneidensis, respectively. Ramachandran plot showed that number of residue in favored region in E. coli, W. succinogenes and S. oneidensis was 97.03%, 97.01% and 97.25%, respectively. Our findings also revealed that among three pipelines, Modeller was considered the best in-silico tool for prediction of NrfA protein. Overall, significant findings of this study may aid in the identification of future unexplored DNRA bacteria containing cytochrome c nitrite reductase. The NrfA system, which is linked to respiratory nitrite ammonification, provides an analogous target for monitoring less studied N-retention processes, particularly in agricultural ecosystems. Furthermore, one of the challenging research tasks for the future is to determine how the NrfA protein responds to redox status in the microbial cells.

Combined application of ascorbic acid and endophytic N-fixing Azotobacter chroococcum Avi2 modulates photosynthetic efficacy, antioxidants and growth-promotion in rice under moisture deficit stress.

This group has previously reported the role of ascorbic acid (AA) as an antioxidant for survivability and ability to enhancing diazotrophic efficacy in Azotobacter chroococcum Avi2 under hydrogen peroxide (H2O2) stress. However, the present study showed the combined application of AA and Avi2 in drought-susceptible (IR64 and Naveen) and drought-tolerant (Ankit and Satyabhama) rice cultivars to determine their photosynthetic efficacy (chlorophyll fluorescence-imaging), antioxidants, and plant growth-promotion (PGP) under moisture deficit stress (MS, -60 kPa). The results indicated that combined application of AA and Avi2 significantly (p < 0.05) increased the total chlorophyll, relative water content, electrolytic leakage, super oxide dismutase, and catalase activities in all rice cultivars as compared to other MS treatments, whereas stress indicators like proline and H2O2 contents were proportionally increased under MS and their concentration were normalized under combined application of AA and Avi2. Photochemical quenching, non-photochemical quenching, photosynthetic electron transport rate, and the effective quantum efficiency were found to be increased significantly (p < 0.05) in Avi2 + AA as compared to other MS treatments. Moreover, rice roots harbored significantly (p < 0.05) higher copy number of nifH gene in Avi2 + AA treatment followed by Avi2 compared to flooded control and other MS treatments. Combined application of AA and Avi2 also increased the grain yield significantly (p < 0.05) by 7.09 % and 3.92 % in drought-tolerant (Ankit and Satyabhama, respectively) and 31.70 % and 34.19 % in drought-susceptible (IR64 and Naveen, respectively) rice cultivars compared to MS treatment. Overall, the present study indicated that AA along with Avi2 could be an effective formulation to alleviate MS vis à vis enhances PGP traits in rice.

DNRA: A short-circuit in biological N-cycling to conserve nitrogen in terrestrial ecosystems.

This paper reviews dissimilatory nitrate reduction to ammonium (DNRA) in soils - a newly appreciated pathway of nitrogen (N) cycling in the terrestrial ecosystems. The reduction of NO3- occurs in two steps; in the first step, NO3- is reduced to NO2-; and in the second, unlike denitrification, NO2- is reduced to NH4+ without intermediates. There are two sets of NO3-/NO2- reductase enzymes, i.e., Nap/Nrf and Nar/Nir; the former occurs on the periplasmic-membrane and energy conservation is respiratory via electron-transport-chain, whereas the latter is cytoplasmic and energy conservation is both respiratory and fermentative (Nir, substrate-phosphorylation). Since, Nir catalyzes both assimilatory- and dissimilatory-nitrate reduction, the nrfA gene, which transcribes the NrfA protein, is treated as a molecular-marker of DNRA; and a high nrfA/nosZ (N2O-reductase) ratio favours DNRA. Recently, several crystal structures of NrfA have been presumed to producee N2O as a byproduct of DNRA via the NO (nitric-oxide) pathway. Meta-analyses of about 200 publications have revealed that DNRA is regulated by oxidation state of soils and sediments, carbon (C)/N and NO2-/NO3- ratio, and concentrations of ferrous iron (Fe2+) and sulfide (S2-). Under low-redox conditions, a high C/NO3- ratio selects for DNRA while a low ratio selects for denitrification. When the proportion of both C and NO3- are equal, the NO2-/NO3- ratio modulates partitioning of NO3-, and a high NO2-/NO3- ratio favours DNRA. A high S2-/NO3- ratio also promotes DNRA in coastal-ecosystems and saline sediments. Soil pH, temperature, and fine soil particles are other factors known to influence DNRA. Since, DNRA reduces NO3- to NH4+, it is essential for protecting NO3- from leaching and gaseous (N2O) losses and enriches soils with readily available NH4+-N to primary producers and heterotrophic microorganisms. Therefore, DNRA may be treated as a tool to reduce ground-water NO3- pollution, enhance soil health and improve environmental quality.

Non-target effect of bispyribac sodium on soil microbial community in paddy soil.

Bispyribac sodium is frequently used herbicide in the rice field. Though, it has been targeted to kill rice weeds, but its non-target effect on soil microbes in paddy soil was largely unknown. Therefore, in the present study, an attempt was made to assess the non-target effect of bispyribac sodium on alteration of functional variation of soil microbial community and their correlation with microbial biomass carbon (MBC) and soil enzymes. A microcosm experiment set up was made comprising three treatments viz., control (CON) (without application of bispyribac sodium), recommended dose of bispyribac sodium (35 g ha-1) (BS), and double the dose of BS (70 g ha-1) (DBS). Results indicated that the MBC and soil enzyme activities (dehydrogenase, alkaline phosphatase and urease) in BS and DBS-treated soil were significantly (p < 0.05) declined from 1st to 30th day after application as compared to CON. Counts of heterotrophic bacteria, actinomycetes and fungal population were also decreased in BS and DBS-treated soil. The average well color development (AWCD) values derived from Biolog®ecoplates followed the order of DBS ˂ BS ˂ CON. Shannon index value was high (p ≤ 0.05) in CON compared to soil-treated with BS and DBS. Principal component analysis (PCA) showed a clear distinction of the cluster of treatments between CON, BS and DBS. Biplot analysis and heatmap suggested that carboxylic compounds and amino acids showed positive response towards BS-treated soil, whereas phenolic compounds had positive correlation with DBS-treated soil. PCA analysis indicated that oligotrophs was rich in BS-treated paddy soil, whereas copiotrophs and asymbiotic nitrogen fixers were richer in DBS treatment. Overall, the present study revealed that application of recommended dose of BS and its double dose alter the soil microbial population, enzyme activities and functional microbial diversity in paddy soil.

Ascorbic acid formulation for survivability and diazotrophic efficacy of Azotobacter chroococcum Avi2 (MCC 3432) under hydrogen peroxide stress and its role in plant-growth promotion in rice (Oryza sativa L.).

Oxidative stress generates reactive oxygen species which causes cell damage of living organisms and are normally detoxified by antioxidants. Indirect reports signify the damages caused by reactive oxygen species and neutralized by antioxidant, but the direct evidence to confirm this hypothesis is still unclear. To validate our hypothesis, an attempt was made in a diazotrophic bacterium (Azotobacter chroococcum Avi2) as a biological system, and hydrogen peroxide (H2O2) and ascorbic acid were used as oxidative stress and antioxidant supplement, respectively. Additionally, rice plant-growth attributes by Avi2 was also assessed under H2O2 and ascorbic acid. Results indicated that higher concentration of H2O2 (2.5 mM-4.5 mM) showed the complete mortality of Avi2, whereas one ppm ascorbic acid neutralized the effect of H2O2. Turbidity, colony forming unit, DNA quantity, nifH gene abundance, indole acetic acid and ammonia productions were significantly (p < 0.5) increased by 11.93%, 17.29%, 19.80%, 74.77%, 71.89%, and 42.53%, respectively in Avi2-treated with 1.5 mM H2O2 plus ascorbic acid compared to 1.5 mM H2O2 alone. Superoxide dismutase was significantly (p < 0.5) increased by 60.85%, whereas catalase and ascorbate peroxidase activities were significantly (p < 0.05) decreased by 64.28% and 68.88% in Avi2-treated with 1.5 mM H2O2 plus ascorbic acid compared to 1.5 mM H2O2 alone. Germination percentage of three rice cultivars (FR13a, Naveen and Sahbhagi dhan) were significantly (p < 0.5) increased by 20%, 13.33%, and 4%, respectively in Avi2-treated with 0.6 mM H2O2 plus ascorbic acid compared with uninoculated control. Overall, this study indicated that ascorbic acid formulation neutralizes the H2O2-oxidative stress and enhances the survivability and plant growth-promoting efficacy of A. chroococcum Avi2 and therefore, it may be used as an effective formulation of bio-inoculants in rice under oxidative stress.

Cyanobiont diversity in six Azolla spp. and relation to Azolla-nutrient profiling.

MAIN CONCLUSION: Illumina-Miseq®-based cyanobiont diversity and biomass were analyzed in six Azolla spp. Results revealed that 93-98% of total operational taxonomic units (OTUs) belong to Nostacaceae followed by Cylindrospermopsis with about 1-6% OTUs. The taxonomy of Azolla-cyanobiont is a long-term debate within the scientific community. Morphological and biochemical-based reports indicated the presence of Anabaena, Nostoc and/or Trichormus azollae as abundant Azolla-cyanobionts, however, molecular data did not support the abundance of Anabaena and/or Nostoc. To understand furthermore, the cyanobiont diversity in six species of Azolla (A. microphylla, A. mexicana, A. filiculoides, A. caroliniana, A. pinnata and A. rubra) was analyzed based on 16S rRNA Illumina-MiSeq sequencing. Additionally, biomass and nutrient profiling of Azolla spp. were analyzed and correlated with cyanobiont diversity. Illumina-MiSeq data revealed that 99.6-99.9% of total operational taxonomic units (OTUs) belonged to Nostocophycideae (class), Nostocales (order) and Nostacaceae (family). At genus level, the unassigned affiliation (93.4-97.9%) under Nostacaceae family was abundant followed by Cylindrospermopsis OTUs (1.1-6.0%). Interestingly, A. pinnata harboured maximum Cylindrospermopsis OTUs and also recorded higher biomass (40.67 g m-2 day-1), whereas crude protein (25.9%) and antioxidants (76.9%) were recorded to be higher in A. microphylla. Biplot analysis revealed that A. pinnata and its cyanobiont abundance were positively correlated with neutral and acid detergent fibers. Overall, the present findings deepened the understanding about cyanobiont in Azolla and its relations with Azolla nutrient profiling.