JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Revegetation of coal mine degraded arid areas: The role of a native woody species under optimum water and nutrient resources.

Ecological restoration of coal mine degraded soils across arid and semi-arid environments worldwide remains particularly challenging. We used a combination of greenhouse and field experiments to assess the potential role of a woody species, Ulmus pumila, in the restoration of degraded soils associated with coal-mining activities in the northwest China. We investigated how various combinations of water-nitrogen-phosphorus (W-N-P) resources affect multiple growth parameters in U. pumila. We found that several plant growth traits significantly improved with W-N applications, regardless of P inputs. Moderate-to-highest W-N-P doses increased net photosynthesis and transpiration rates, water use efficiency, stomatal conductance, chlorophyll and carotenoid contents under greenhouse conditions. A combination of high W together with low N-P applications led to high relative water content and net photosynthetic rates under field conditions. Increasing of N-P doses under W-shortage condition, aided U. pumila to enhance osmotic adjustments by increasing contents of proline and soluble sugar and also boost the activity of superoxide dismutase, peroxidase and catalase in leaf tissues to reduce accumulation of reactive oxygen species and malondialdehyde content in all conditions of greenhouse and field. Our study is the first to assess the optimum W-N-P resources in U. pumila and demonstrate that optimum growth performance could be obtained under W supplements corresponding to 90 mm year-1, N and P at 110 and 45 kg ha-1, respectively, under field condition. These findings can have far reaching implications for vegetation restoration of degraded areas associated with coal-mining activities across arid and semi-arid regions worldwide.

Singular Nuclei Segmentation for Automatic HER2 Quantification Using CISH Whole Slide Images.

Human epidermal growth factor receptor 2 (HER2) quantification is performed routinely for all breast cancer patients to determine their suitability for HER2-targeted therapy. Fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) are the US Food and Drug Administration (FDA) approved tests for HER2 quantification in which at least 20 cancer-affected singular nuclei are quantified for HER2 grading. CISH is more advantageous than FISH for cost, time and practical usability. In clinical practice, nuclei suitable for HER2 quantification are selected manually by pathologists which is time-consuming and laborious. Previously, a method was proposed for automatic HER2 quantification using a support vector machine (SVM) to detect suitable singular nuclei from CISH slides. However, the SVM-based method occasionally failed to detect singular nuclei resulting in inaccurate results. Therefore, it is necessary to develop a robust nuclei detection method for reliable automatic HER2 quantification. In this paper, we propose a robust U-net-based singular nuclei detection method with complementary color correction and deconvolution adapted for accurate HER2 grading using CISH whole slide images (WSIs). The efficacy of the proposed method was demonstrated for automatic HER2 quantification during a comparison with the SVM-based approach.

Investigating Antimicrobial Resistance and ESBL Producing Gene in Klebsiella Isolates among Neonates and Adolescents in Southern Bangladesh.

Background: Multidrug-resistant (MDR) clones of Klebsiella pneumoniae (Kpn) have been increasingly documented in community-acquired and nosocomial infections all around the globe. Extended-spectrum ß-lactamases (ESBLs) are a rapidly evolving group of ß-lactamase enzymes derived from SHV genes by mutations. This research work aimed to investigate and analyze the widespread prevalence of Kpn antibiotic resistance in different areas of the southern part of Bangladesh. Methods: This particular study was executed and implemented by using 501 clinical samples or isolates from two different hospitals in Chattogram. The disk diffusion method was used to detect Kpn's sensitivity to 16 antibiotics in a drug susceptibility test. By using the PCR technique, the widespread prevalence of antibiotic-resistant gene blaSHV-11 was studied. Sequencing along with phylogenetic analysis was utilized to verify isolates with the blaSHV-11 gene. Results: Almost all of the Kpn isolates were spotted to be antibiotic-resistant. These Kpn isolates were resistant to ß-lactams, aminoglycosides, and quinolones at high levels. The spatial analysis displayed that infections involving Kpn were more common in the urban areas (70%) than in the rural areas (30%). Neonates had substantially higher levels (p < 0.001) of resistance to multidrug than other age groups. Cefepime was identified as the most frequent antibiotic-resistant to all age groups (56.68%). The highest numbers of resistant isolates (36.92%) were found in urine samples. The ESBL gene blaSHV-11 was found in 38% isolates. Conclusion: The significant frequency of MDR Kpn harboring ß-lactamases and AMR genes strongly suggests the requirement to develop effective antimicrobial resistance control and prevention measures in Bangladesh.

Drought dynamics of Northwestern Teesta Floodplain of Bangladesh: a remote sensing approach to ascertain the cause and effect.

Drought is an affliction for a region that primarily depends on agriculture as economic activity. Commonly monitoring and characterizing of drought is performed by only analyzing the meteorological aspect, assuming precipitation as the primary source of water. However, in riverine Bangladesh, this can lead to an erroneous conclusion, as there is a multitude of available water sources. Consequently, in this study, vegetation condition (Standard Vegetation Index), soil moisture (Soil Moisture Index), and precipitation (Standard Precipitation Index) are separately investigated from 2003 to 2019, in the Northwestern Teesta floodplain. Subsequently, statistical regression analysis is performed to determine the relationships between different aspects of drought. In addition, information obtained from field visits and expert opinions has also been assimilated. Analysis of vegetation and soil moisture condition presents a progressively improving scenario. However, SPI shows an incessant decline in meteorological drought conditions, especially after 2007. Evidently, regression analysis does not provide any indication of an interrelationship between the studied agricultural and meteorological parameters. Presumably, this absence is instigated because the study area is highly irrigated as the groundwater table is suitably near the surface and the existence of nearby Teesta river allows for the utilization of surface water. Moreover, the cropping pattern is shifting toward crops that require much less water and to places where soil moisture is scarce. Thus, this study addresses the gap in knowledge regarding the nature of agricultural drought and the dynamics of different aspects of drought which will be invaluable for the water management and agricultural policy in the study area as well as other regions with a similar backdrop.

Exogenous Melatonin Modulates the Physiological and Biochemical Mechanisms of Drought Tolerance in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Tartary buckwheat is one of the nutritious minor cereals and is grown in high-cold mountainous areas of arid and semi-arid zones where drought is a common phenomenon, potentially reducing the growth and yield. Melatonin, which is an amphiphilic low molecular weight compound, has been proven to exert significant effects in plants, under abiotic stresses, but its role in the Tartary buckwheat under drought stress remains unexplored. We evaluated the influence of melatonin supplementation on plant morphology and different physiological activities, to enhance tolerance to posed drought stress by scavenging reactive oxygen species (ROS) and alleviating lipid peroxidation. Drought stress decreased the plant growth and biomass production compared to the control. Drought also decreased Chl a, b, and the Fv/Fm ratio by 54%, 70%, and 8%, respectively, which was associated with the disorganized stomatal properties. Under drought stress, H2O2, O2•-, and malondialdehyde (MDA) contents increased by 2.30, 2.43, and 2.22-folds, respectively, which caused oxidative stress. In contrast, proline and soluble sugar content were increased by 84% and 39%, respectively. However, exogenous melatonin (100 µM) could improve plant growth by preventing ROS-induced oxidative damage by increasing photosynthesis, enzymatic antioxidants (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase), secondary metabolites like phenylalanine ammonialyase, phenolics, and flavonoids, total antioxidant scavenging (free radical DPPH scavenging), and maintaining relative water content and osmoregulation substances under water stress. Therefore, our study suggested that exogenous melatonin could accelerate drought resistance by enhancing photosynthesis and antioxidant defense in Tartary buckwheat plants.

Loss-of-function of ASPARTIC PEPTIDASE NODULE-INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen-fixing symbiosis with specific Mesorhizobium loti strains.

The nitrogen-fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix- mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix- mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix- mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE-INDUCED 1 (LjAPN1), encodes a nepenthesin-type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain-specific Fix- phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen-fixing) symbiosis in a rhizobial strain-dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.

Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs.

Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. As root hairs are single-cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single-cell model system to measure the impact of environmental stress. We measured changes in cytosine DNA methylation in single-cell root hairs as compared with multicellular stripped roots, as well as in response to heat stress. Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated than were stripped roots. However, in response to heat stress, both root hairs and stripped roots showed hypomethylation in each context, especially in the CHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons. Taken together, the data indicate that changes in DNA methylation are directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/cells or stress (heat).

Lotus japonicus cytokinin receptors work partially redundantly to mediate nodule formation.

Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.

Lotus japonicus NF-YA1 Plays an Essential Role During Nodule Differentiation and Targets Members of the SHI/STY Gene Family.

Legume plants engage in intimate relationships with rhizobial bacteria to form nitrogen-fixing nodules, root-derived organs that accommodate the microsymbiont. Members of the Nuclear Factor Y (NF-Y) gene family, which have undergone significant expansion and functional diversification during plant evolution, are essential for this symbiotic liaison. Acting in a partially redundant manner, NF-Y proteins were shown, previously, to regulate bacterial infection, including selection of a superior rhizobial strain, and to mediate nodule structure formation. However, the exact mechanism by which these transcriptional factors exert their symbiotic functions has remained elusive. By carrying out detailed functional analyses of Lotus japonicus mutants, we demonstrate that LjNF-YA1 becomes indispensable downstream from the initial cortical cell divisions but prior to nodule differentiation, including cell enlargement and vascular bundle formation. Three affiliates of the SHORT INTERNODES/STYLISH transcription factor gene family, called STY1, STY2, and STY3, are demonstrated to be among likely direct targets of LjNF-YA1, and our results point to their involvement in nodule formation.

Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection.

Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation.

Lotus japonicus SUNERGOS1 encodes a predicted subunit A of a DNA topoisomerase VI that is required for nodule differentiation and accommodation of rhizobial infection.

A symbiotic mutant of Lotus japonicus, called sunergos1-1 (suner1-1), originated from a har1-1 suppressor screen. suner1-1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild-type nodule numbers are reached by suner1-1 at a later point after infection. Using an approach that combined map-based cloning and next-generation sequencing we have identified the causative mutation and show that the suner1-1 phenotype is determined by a weak recessive allele, with the corresponding wild-type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen-fixing nodules.

Xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I do not have identical structures in soybean root and root hair cell walls.

MAIN CONCLUSION: Chemical analyses and glycome profiling demonstrate differences in the structures of the xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I isolated from soybean ( Glycine max ) roots and root hair cell walls. The root hair is a plant cell that extends only at its tip. All other root cells have the ability to grow in different directions (diffuse growth). Although both growth modes require controlled expansion of the cell wall, the types and structures of polysaccharides in the walls of diffuse and tip-growing cells from the same plant have not been determined. Soybean (Glycine max) is one of the few plants whose root hairs can be isolated in amounts sufficient for cell wall chemical characterization. Here, we describe the structural features of rhamnogalacturonan I, rhamnogalacturonan II, xyloglucan, glucomannan, and 4-O-methyl glucuronoxylan present in the cell walls of soybean root hairs and roots stripped of root hairs. Irrespective of cell type, rhamnogalacturonan II exists as a dimer that is cross-linked by a borate ester. Root hair rhamnogalacturonan I contains more neutral oligosaccharide side chains than its root counterpart. At least 90% of the glucuronic acid is 4-O-methylated in root glucuronoxylan. Only 50% of this glycose is 4-O-methylated in the root hair counterpart. Mono O-acetylated fucose-containing subunits account for at least 60% of the neutral xyloglucan from root and root hair walls. By contrast, a galacturonic acid-containing xyloglucan was detected only in root hair cell walls. Soybean homologs of the Arabidopsis xyloglucan-specific galacturonosyltransferase are highly expressed only in root hairs. A mannose-rich polysaccharide was also detected only in root hair cell walls. Our data demonstrate that the walls of tip-growing root hairs cells have structural features that distinguish them from the walls of other roots cells.

Identification of microRNAs and their mRNA targets during soybean nodule development: functional analysis of the role of miR393j-3p in soybean nodulation.

Plant microRNAs (miRNAs) play important regulatory roles in a number of developmental processes. The present work investigated the roles of miRNAs during nodule development in the crop legume soybean (Glycine max). Fifteen soybean small RNA libraries were sequenced from different stages of nodule development, including young nodules, mature nodules and senescent nodules. In order to identify the regulatory targets of the miRNAs, five parallel analysis of RNA ends (PARE) libraries were also sequenced from the same stages of nodule development. Sequencing identified 284 miRNAs, including 178 novel soybean miRNAs. Analysis of miRNA abundance identified 139 miRNAs whose expression was significantly regulated during nodule development, including 12 miRNAs whose expression changed > 10-fold. Analysis of the PARE libraries identified 533 miRNA targets, including three nodulation-related genes and eight nodule-specific genes. miR393j-3p was selected for detailed analysis as its expression was significantly regulated during nodule formation, and it targeted a nodulin gene, Early Nodulin 93 (ENOD93). Strong, ectopic expression of miR393j-3p, as well as RNAi silencing of ENOD93 expression, significantly reduced nodule formation. The data indicate that miR393j-3p regulation of ENOD93 mRNA abundance is a key control point for soybean nodule formation.

Factors associated with child and maternal dietary diversity in the urban areas of Bangladesh.

Dietary diversity is an indicator of nutrition that has been found positively associated with diet quality, micronutrient adequacy, and improved maternal health and child growth. Due to the cultural responsibility of women in providing food at the household level, their status is very important to perform this role. Hence, this study has been conducted on the status of dietary diversity of the mother and child to understand how it relates to various factors of women in urban settings. Data were obtained from 1978 mother-child pairs living in different cities in Bangladesh. The foods taken by the women and children were categorized into 10 and 7 groups to measure women's dietary diversity (WDD) and children's dietary diversity (CDD), respectively. The study found that more than three-fourths of the mothers and half of the children had low dietary diversity. The household wealth holdings and access to resources by the women were found inadequate, while two-thirds of them had the lowest to medium level of nutritional knowledge. The binomial logistic regression model was used to measure the factors influencing WDD and CDD. The findings also indicated that children's dietary diversity was influenced by the mother's age, education, supportive attitude and behavior of husband, and access to and control over resources. While the household wealth index can enhance both child and mother's dietary variety, nutrition knowledge, dietary counseling, and access to and control over resources can improve maternal dietary diversity. This study recommends improving women's socioeconomic status by increasing their wealth and access to resources and enhancing their nutrition knowledge by providing food and nutrition counseling.

Genetic determinants of SARS-CoV-2 and the clinical outcome of COVID-19 in Southern Bangladesh.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has had a severe impact on population health. The genetic determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in southern Bangladesh are not well understood. METHODS: This study aimed to determine the genomic variation in SARS-CoV-2 genomes that have evolved over 2 years of the pandemic in southern Bangladesh and their association with disease outcomes and virulence of this virus. We investigated demographic variables, disease outcomes of COVID-19 patients and genomic features of SARS-CoV-2. RESULTS: We observed that the disease severity was significantly higher in adults (85.3%) than in children (14.7%), because the expression of angiotensin-converting enzyme-2 (ACE-2) diminishes with ageing that causes differences in innate and adaptive immunity. The clade GK (n = 66) was remarkable between June 2021 and January 2022. Because of the mutation burden, another clade, GRA started a newly separated clustering in December 2021. The burden was significantly higher in GRA (1.5-fold) highlighted in mild symptoms of COVID-19 patients than in other clades (GH, GK, and GR). Mutations were accumulated mainly in S (22.15 mutations per segment) and ORF1ab segments. Missense (67.5%) and synonymous (18.31%) mutations were highly noticed in adult patients with mild cases rather than severe cases, especially in ORF1ab segments. Moreover, we observed many unique mutations in S protein in mild cases compared to severe, and homology modeling revealed that those might cause more folding in the protein's alpha helix and beta sheets. CONCLUSION: Our study identifies some risk factors such as age comorbidities (diabetes, hypertension, and renal disease) that are associated with severe COVID-19, providing valuable insight regarding prioritizing vaccination for high-risk individuals and allocating health care and resources. The findings of this work outlined the knowledge and mutational basis of SARS-CoV-2 for the next treatment steps. Further studies are needed to confirm the effects of structural and functional proteins of SARS-CoV-2 in detail for monitoring the emergence of new variants in future.

miR172 regulates soybean nodulation.

Micro-RNAs (miRNAs) play a pivotal role in the control of gene expression and regulate plant developmental processes. miRNA 172 (miR172) is a conserved miRNA in plants reported to control the expression of genes involved in developmental phase transition, floral organ identity, and flowering time. However, the specific role of miR172 in legume nodulation is undefined. Ectopic expression of soybean miR172 resulted in an increase in nodule numbers in transgenic roots and an increase in the expression of both symbiotic leghemoglobin and nonsymbiotic hemoglobin. These nodules showed higher levels of nitrogenase activity. Further analysis revealed a complex regulatory circuit in which miR156 regulates miR172 expression and controls the level of an AP2 transcription factor. The latter, either directly or indirectly, controls the expression of nonsymbiotic hemoglobin, which is essential for regulating the levels of nodulation.

Lotus japonicus ARPC1 is required for rhizobial infection.

Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nck-associated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.

Microbiome analysis revealed distinct microbial communities occupying different sized nodules in field-grown peanut.

Legume nodulation is the powerhouse of biological nitrogen fixation (BNF) where host-specific rhizobia dominate the nodule microbiome. However, other rhizobial or non-rhizobial inhabitants can also colonize legume nodules, and it is unclear how these bacteria interact, compete, or combinedly function in the nodule microbiome. Under such context, to test this hypothesis, we conducted 16S-rRNA based nodule microbiome sequencing to characterize microbial communities in two distinct sized nodules from field-grown peanuts inoculated with a commercial inoculum. We found that microbial communities diverged drastically in the two types of peanut nodules (big and small). Core microbial analysis revealed that the big nodules were inhabited by Bradyrhizobium, which dominated composition (>99%) throughout the plant life cycle. Surprisingly, we observed that in addition to Bradyrhizobium, the small nodules harbored a diverse set of bacteria (~31%) that were not present in big nodules. Notably, these initially less dominant bacteria gradually dominated in small nodules during the later plant growth phases, which suggested that native microbial communities competed with the commercial inoculum in the small nodules only. Conversely, negligible or no competition was observed in the big nodules. Based on the prediction of KEGG pathway analysis for N and P cycling genes and the presence of diverse genera in the small nodules, we foresee great potential of future studies of these microbial communities which may be crucial for peanut growth and development and/or protecting host plants from various biotic and abiotic stresses.

Isolation and Characterization of Bacterial Endophytes from Small Nodules of Field-Grown Peanut.

It is evident that legume root nodules can accommodate rhizobial and non-rhizobial bacterial endophytes. Our recent nodule microbiome study in peanuts described that small nodules can harbor diverse bacterial endophytes. To understand their functional role, we isolated 87 indigenous endophytes from small nodules of field-grown peanut roots and characterized them at molecular, biochemical, and physiological levels. The amplified 16S rRNA genes and phylogenetic analysis of these isolates revealed a wide variety of microorganisms related to the genera Bacillus, Burkholderia, Enterobacter, Herbaspirillum, Mistsuaria, Pantoea, Pseudomonas, and Rhizobia. It was observed that 37% (100% identity) and 56% (>99% identity) of the isolates matched with the amplified sequence variants (ASVs) from our previous microbiome study. All of these isolates were tested for stress tolerance (high temperature, salinity, acidic pH) and phosphate (P) solubilization along with ammonia (NH3), indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate deaminase (ACCD), and siderophore production. The majority (78%) of the isolates were found to be halotolerant, thermotolerant, and acidophilic, and a few of them showed a significant positive response to the production of IAA, NH3, siderophore, ACCD, and P-solubilization. To evaluate the plant growth promotion (PGP) activity, plant and nodulation assays were performed in the growth chamber conditions for the selected isolates from both the non-rhizobial and rhizobial groups. However, these isolates appeared to be non-nodulating in the tested conditions. Nonetheless, the isolates 2 (Pantoea), 17 (Burkholderia), 21 (Herbaspirillum), 33o (Pseudomonas), and 77 (Rhizobium sp.) showed significant PGP activity in terms of biomass production. Our findings indicate that these isolates have potential for future biotechnological applications through the development of biologicals for sustainable crop improvement.