Nitrogen-fixing Ability Research Articles

Advancements in Genetic Enhancement: CRISPR/Cas-Mediated Genome Editing in Leguminous Crops

Legumes play a crucial role in human nutrition and sustainable agriculture due to their high protein content and health-promoting phytochemicals. To accelerate genetic gain in yield, stress resilience, and nutritional quality, extensive efforts are underway. Recent advancements in genomic resources have paved the way for the application of cutting-edge breeding technologies like genomic selection and genome editing in legume crops. This review focuses on the latest advancements in CRISPR/Cas9-based gene editing technology, specifically tailored for improving traits in legume crops. While successful gene-editing methods have been established for crops like soybean, cowpea, and chickpea, challenges remain, particularly in overcoming the recalcitrance of certain legumes to gene transfer and regeneration in vitro. Strategies to enhance transformation and regeneration rates through modifications in culture methods and DNA delivery are discussed. Despite the potential benefits of gene editing in legume breeding, regulatory barriers pose significant challenges. A comparison of regulatory environments in different regions sheds light on the importance of favourable regulations and public acceptance for realizing CRISPR's potential in enhancing global food security. Additionally, leguminous crops are vital for their nutritional properties, nitrogen-fixing abilities, and contribution to sustainable agriculture. Traditional breeding methods focused on increasing yield, while recent advancements in genome editing techniques, notably CRISPR-Cas technology, have revolutionized the improvement of agronomic traits in legumes. This chapter provides insights into the application of genome editing tools for enhancing traits such as stress tolerance, yield, and seed content in grain legumes. Furthermore, it discusses the challenges and prospects associated with enhancing grain legumes using molecular breeding techniques. Genome editing has been successfully employed in diverse legumes, including model species like Medicago and widely cultivated crops such as soybean and chickpea, offering exciting opportunities for enhancing agronomic characteristics.

IAA-producing plant growth promoting rhizobacteria from Ceanothus velutinus enhance cutting propagation efficiency and Arabidopsis biomass.

Climate-induced drought impacts plant growth and development. Recurring droughts increase the demand for water for food production and landscaping. Native plants in the Intermountain West region of the US are of keen interest in low water use landscaping as they are acclimatized to dry and cold environments. These native plants do very well at their native locations but are difficult to propagate in landscape. One of the possible reasons is the lack of associated microbiome in the landscaping. Microbiome in the soil contributes to soil health and impacts plant growth and development. Here, we used the bulk soil from the native plant Ceanothus velutinus (snowbrush ceanothus) as inoculant to enhance its propagation. Snowbrush ceanothus is an ornamental plant for low-water landscaping that is hard to propagate asexually. Using 50% native bulk soil as inoculant in the potting mix significantly improved the survival rate of the cuttings compared to no-treated cuttings. Twenty-four plant growth-promoting rhizobacteria (PGPR) producing indole acetic acid (IAA) were isolated from the rhizosphere and roots of the survived snowbrush. Seventeen isolates had more than 10µg/mL of IAA were shortlisted and tested for seven different plant growth-promoting (PGP) traits; 76% showed nitrogen-fixing ability on Norris Glucose Nitrogen free media,70% showed phosphate solubilization activity, 76% showed siderophore production, 36% showed protease activity, 94% showed ACC deaminase activity on DF-ACC media, 76% produced catalase and all of isolates produced ammonia. Eight of seventeen isolates, CK-6, CK-22, CK-41, CK-44, CK-47, CK-50, CK-53, and CK-55, showed an increase in shoot biomass in Arabidopsis thaliana. Seven out of eight isolates were identified as Pseudomonas, except CK-55, identified as Sphingobium based on 16S rRNA gene sequencing. The shortlisted isolates are being tested on different grain and vegetable crops to mitigate drought stress and promote plant growth.

Advancements in Biofertilizers for Pulse Crops: From Single-Strain Inoculants to Microbiome-Based Technologies

The application of biofertilizers in pulse crop agriculture has garnered significant attention as an eco-friendly and sustainable alternative to conventional chemical fertilizers. This comprehensive review aims to elucidate the progression of biofertilizer technologies from traditional single-strain inoculants to contemporary microbiome-based strategies. Pulse crops, such as beans, lentils, chickpeas, and peas, are pivotal in global agriculture due to their high protein content, nitrogen-fixing ability, and role in improving soil health. However, optimizing their productivity necessitates effective nutrient management, where biofertilizers play a crucial role. Initially, biofertilizer development concentrated on single-strain inoculants, predominantly Rhizobium species, which establish symbiotic relationships with leguminous plants to enhance nitrogen fixation and improve crop yields. Despite their proven benefits, single-strain inoculants often exhibit variable performance influenced by soil conditions, climatic factors, and interactions with indigenous soil microbiota. This variability highlights the need for more consistent and resilient biofertilizer solutions. Recent advances in microbiome research have paved the way for the development of microbiome-based biofertilizers. These advanced formulations leverage the synergistic interactions among diverse microbial communities, including bacteria, fungi, and archaea, to provide a more comprehensive and stable enhancement of soil fertility and plant growth. Microbiome-based approaches recognize the complexity of plant-microbe-soil interactions, offering improved nutrient cycling, increased plant stress resilience, and better adaptation to various environmental conditions. The benefits of microbiome-based biofertilizers are manifold, encompassing enhanced nutrient availability, improved plant health, and sustainable soil management. However, their development and application pose several challenges, including understanding the intricate dynamics of microbiome interactions, developing precise formulation and application methods, and navigating regulatory landscapes. Addressing these challenges requires multidisciplinary research and innovation, integrating genomics, metagenomics, and advanced data analytics to unravel the complexities of soil microbiomes. This includes the potential for personalized biofertilizer formulations tailored to specific soil and crop conditions, enhancing the efficacy and scalability of biofertilizer technologies.

Metabolic relationships between marine red algae and algae-associated bacteria

Mutualistic interactions between marine phototrophs and associated bacteria are an important strategy for their successful survival in the ocean, but little is known about their metabolic relationships. Here, bacterial communities in the algal sphere (AS) and bulk solution (BS) of nine marine red algal cultures were analyzed, and Roseibium and Phycisphaera were identified significantly more abundantly in AS than in BS. The metabolic features of Roseibium RMAR6-6 (isolated and genome-sequenced), Phycisphaera MAG 12 (obtained by metagenomic sequencing), and a marine red alga, Porphyridium purpureum CCMP1328 (from GenBank), were analyzed bioinformatically. RMAR6-6 has the genetic capability to fix nitrogen and produce B vitamins (B1, B2, B5, B6, B9, and B12), bacterioferritin, dimethylsulfoniopropionate (DMSP), and phenylacetate that may enhance algal growth, whereas MAG 12 may have a limited metabolic capability, not producing vitamins B9 and B12, DMSP, phenylacetate, and siderophores, but with the ability to produce bacitracin, possibly modulating algal microbiome. P. purpureum CCMP1328 lacks the genetic capability to fix nitrogen and produce vitamin B12, DMSP, phenylacetate, and siderophore. It was shown that the nitrogen-fixing ability of RMAR6-6 promoted the growth of P. purpureum, and DMSP reduced the oxidative stress of P. purpureum. The metabolic interactions between strain RMAR6-6 and P. purpureum CCMP1328 were also investigated by the transcriptomic analyses of their monoculture and co-culture. Taken together, potential metabolic relationships between Roseibium and P. purpureum were proposed. This study provides a better understanding of the metabolic relationships between marine algae and algae-associated bacteria for successful growth.

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress

BackgroundWhite clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored.ResultsIn this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana.ConclusionThese findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Nitrospirillum viridazoti sp. nov., an Efficient Nitrogen-Fixing Species Isolated from Grasses.

A group of Gram-negative plant-associated diazotrophic bacteria belonging to the genus Nitrospirillum was investigated, including both previously characterized and newly isolated strains from diverse regions and biomes, predominantly in Brazil. Phylogenetic analysis of 16S rRNAand recA genes revealed the formation of a distinct clade consisting of thirteen strains, separate from the formally recognized species N. amazonense (the closest species) and N. iridis. Comprehensive taxonomic analyses using the whole genomes of four strains (BR 11140T = AM 18T = Y-2T = DSM 2788T = ATCC 35120T, BR 11142T = AM 14T = Y-1T = DSM 2787T = ATCC 35119T, BR 11145 = CBAmC, and BR 12005) supported the division of these strains into two species: N. amazonense (BR 11142T and BR 12005) and a newly proposed species (BR 11140T and BR 11145), distinct from N. iridis. The phylogenomic analysis further confirmed the presence of the new Nitrospirillum species. Additionally, MALDI-TOF MS analysis of whole-cell mass spectra provided further evidence for the differentiation of the proposed Nitrospirillum species, separate from N.amazonense. Analysis of chemotaxonomy markers (i.e., genes involved in fatty acid synthesis, metabolism and elongation, phospholipid synthesis, and quinone synthesis) revealed that the new species highlights high similarity and evolutionary convergence with other Nitrospirillum species. This new species exhibited nitrogen fixation ability in vitro, it has similar NifHDK protein phylogeny position with the closest species, lacked denitrification capability, but possessed the nosZ gene, enabling N2O reduction, distinguishing it from the closest species. Despite being isolated from diverse geographic regions, soil types, and ecological niches, no significant phenotypic or physiological differences were observed between the proposed new species and N. amazonense. Based on these findings, a new species, Nitrospirillum viridazoti sp. nov., was classified, with the strain BR 11140T (DSM 2788T, ATCC 35120T) designated as the type strain.

Microbial inoculation alters rhizoplane bacterial community and correlates with increased rice yield

Microbial inoculants are recognized as environmentally friendly methods to promote plant growth and improve soil properties. However, the effects of inoculation on the rhizosphere and rhizoplane community structure of plants remain poorly documented and need further investigation. Rhodopseudomonas palustris (R. palustris) strain has nitrogen fixing ability and Bacillus subtilis (B. subtilis) strain is a plant growth-promoting rhizobacterium (PGPR). In this study, we investigated the effects of single and co-inoculation with R. palustris and B. subtilis on the increase of rice yield as well as on the microbial communities in the rhizosphere and rhizoplane of rice through potting experiments, respectively. The results showed that inoculation significantly increased rice yield and seed setting rate, with co-inoculation raising the yield by up to 13.7%. Inoculation influenced both rhizosphere and rhizoplane community structures and functions, amplifying the differences between them. The most significant changes were brought about by the combined inoculation treatment. Co-inoculation with R. palustris and B. subtilis had a synergistic effect. The profound alterations of rhizoplane bacterial community structures and functions were proved to be positively correlated with rice yield and seed setting rate (r = 0.59–0.76, p < 0.05). These results provide new ideas for the investigation of the potential microbiological mechanisms of microbial co-inoculation in practical agricultural applications.

Smart multifaceted potential microbial inoculant isolated from rhizospheric soils of Bergenia ciliata and possible role in developing green biosynthesized nanoparticles

Nanotechnology employs miniscule yet potential nanoparticles and has immense applications in various fields of scientific research. This has revolutionized technological advancements in multiple spheres and recently touched the field of plant biology and sustainable agriculture. Beneficial rhizobacteria isolated from plants may prove efficacious as they are able to impart plant growth. From the medicinal plant Bergenia ciliata, one isolate BC-I-15 was characterized in this study and displayed growth promoting traits. 16s rDNA sequencing and molecular characterization confirmed the isolate as Pseudomonas sp. (NCBI accession number- OQ891764) which showed similarity with other Pseudomonads in phylogenetic tree. The isolate exhibited oxidative stress tolerance assay, nitrogen fixing ability, IAA, calcite and phosphate solubilization etc. But such microbial bioinoculants suffers from few limitations and can be enriched when their nanoparticle counterparts are developed for efficient function. The citation network analysis showed good and encouraging relationships between terms like ‘Nanoparticles, Microbes, Sustainable agriculture’. So, when such beneficial microbes in bulk form, or biofilm and exopolysaccharides (EPS) secreting, secondary metabolites etc. parts thereof are used as starting material for nanoparticle formation, then such nanoparticles possess intrinsic and enriched capacity to impart stress tolerance to plants and impart qualities in sustainable agriculture. The results of the present study, further aids in the efficient utilization of the selected beneficial BC-I-15 rhizobacteria and their future exploration in developing biogenic nanoparticles for active agents in sustainable agriculture.

Comparative Analyses of Acacia Plastomes to Detect Mutational Hotspots and Barcode Sites for the Identification of Important Timber Species

The genus Acacia is a crucial source of industrial papermaking and timber, possessing significant economic value. Due to their nitrogen-fixing ability, rapid growth, and high tolerance to stress, Acacia species have become integral to short-term industrial timber forests, particularly in southern China and various other regions worldwide. Nevertheless, comparative genomic analyses of Acacia species remain limited, impeding the development of molecular markers for identifying and distinguishing between distinct Acacia species as well as distinguishing their wood counterparts from other tree species. To address this gap, we generated plastomic sequences from eight species commonly cultivated in Chinese plantation forests and compared them with existing data. Our findings revealed a generally conserved structure and gene content within the plastid genomes of Acacia. Notably, the dN/dS ratio of genes from distinct functional groups varied, particularly between ribosomal proteins and photosynthesis genes. Furthermore, phylogenetic analyses and sequence comparisons suggest that the introduction of Acacia to China may have been less diverse than previously thought or that interspecies introgression has occurred more frequently than previously documented. This study offers a valuable starting point for future research in this area and for improving timber quality through marker-assisted breeding.

Pseudomonas chlororaphis IRHB3 assemblies beneficial microbes and activates JA-mediated resistance to promote nutrient utilization and inhibit pathogen attack.

The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as well as plant responses to environmental stimuli. In our previous work, one typical PGPR strain, Pseudomonas chlororaphis IRHB3, isolated from the soybean rhizosphere, had positive impacts on soil-borne disease suppression and growth promotion in the greenhouse, but its biocontrol mechanism and application in the field are not unclear. In the current study, IRHB3 was introduced into field soil, and its effects on the local rhizosphere microbiome, disease resistance, and soybean growth were comprehensively analyzed through high-throughput sequencing and physiological and molecular methods. We found that IRHB3 significantly increased the richness of the bacterial community but not the structure of the soybean rhizosphere. Functional bacteria related to phosphorus solubilization and nitrogen fixation, such as Geobacter, Geomonas, Candidatus Solibacter, Occallatibacter, and Candidatus Koribacter, were recruited in rich abundance by IRHB3 to the soybean rhizosphere as compared to those without IRHB3. In addition, the IRHB3 supplement obviously maintained the homeostasis of the rhizosphere microbiome that was disturbed by F. oxysporum, resulting in a lower disease index of root rot when compared with F. oxysporum. Furthermore, JA-mediated induced resistance was rapidly activated by IRHB3 following PDF1.2 and LOX2 expression, and meanwhile, a set of nodulation genes, GmENOD40b, GmNIN-2b, and GmRIC1, were also considerably induced by IRHB3 to improve nitrogen fixation ability and promote soybean yield, even when plants were infected by F. oxysporum. Thus, IRHB3 tends to synergistically interact with local rhizosphere microbes to promote host growth and induce host resistance in the field.

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Phasey Bean (Macroptilium lathyroides) in China.

The forage legume phasey bean Macroptilium lathyroides (L.) Urb. is an annual or short-lived perennial of the family Fabaceae. It is native to the tropical and subtropical areas from North to South America, and is naturalized throughout the tropics and subtropics of the world (Tobisa and Nakano 2019). It is mainly used for forage, green manure, and slope protection (Silva et al. 2018). In addition, the nitrogen fixation ability of this plant can also improve the soil. In February 2022 and January 2023, powdery mildew symptoms were observed on 70% of M. lathyroides plants on the Hainan Medical University campus (19° 58' 53″ N; 110° 19' 47″ E) in Haikou, Hainan Province, China. Powdery mildew colonies covered the leaf surfaces and stems of affected plants, causing discoloration and defoliation. Mycelia were superficial and hyphal appressoria were nipple-shaped. Conidiophores (n =30) were unbranched, cylindrical, 100 to 233 × 8 to 15 µm, and produced three to five immature conidia in chains with a crenate outline. Foot cells (n =30) were cylindrical, straight or sometimes curved at the base, and 36 to 56 µm long. Conidia (n =100) were ellipsoid-ovoid to doliiform, 24 to 34 ×13 to 20 m (length/width ratio = 1.5 to 2.3), with well-developed fibrosin bodies, and produced germ tubes from the lateral position. Based on these morphological characteristics, the pathogen was provisionally identified as Podosphaera xanthii (Braun and Cook 2012). The teleomorph was not observed. A specimen was deposited in the Hainan Medical University Plant Pathology Herbarium as HMML-23. To confirm the identification, genomic DNA was extracted from mycelium, conidiophores, and conidia using a fungal DNA kit (Omega Bio-Tek, USA). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990) and sequenced directly. The resulting 575-bp sequence was deposited in GenBank (accession no. OR240256). A BLASTn search in GenBank of this sequence showed 100% similarity with the ITS sequences of P. xanthii isolates from China (MT242593, MK439611 and MH143483), Korea (MG754404), Vietnam (KM260704), Japan (MZ604267), and Puerto Rico (OP882310). Additionally, the 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O´Donnell 1993; accession no. OR240255). This region shared 100% similarity with P. xanthii isolates (MK357436, LC371333, OP765401, and MZ604267) as well. To confirm pathogenicity, five healthy potted plants of M. lathyroides were inoculated by gently pressing a powdery mildew-infected leaf onto 15 young leaves. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 24 to 30°C, 70% relative humidity, with a 16-h photoperiod. After 7 days, inoculated leaves showed powdery mildew symptoms whereas no symptoms were observed on control plants. The fungal colonies observed on inoculated plants were morphologically identical to those found on the originally infected leaves collected from Hainan Province. Based on the morphological characteristics and molecular identification, the fungus was identified as P. xanthii. This fungus has been reported causing powdery mildew on M. atropurpureum in Thailand (Meeboon et al. 2016). In the United States phasey bean powdery mildew caused by Erysiphe fallax has been previously reported (Poudel and Zhang 2019). To our knowledge, this is the first record of P. xanthii infecting M. lathyroides in China. Over the past 50 years of introduction, phasey bean has become one of the main leguminous forages for establishing artificial mixed seeding grasslands in southern China. We are concerned that the pathogen could become a threat to the widespread planting of M. lathyroides in the future.

Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and ɩ-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Soybean-mediated suppression of BjaI/BjaR1 quorum sensing in Bradyrhizobium diazoefficiens impacts symbiotic nitrogen fixation.

The acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR quorum sensing (QS) system orchestrates diverse bacterial behaviors in response to changes in population density. The role of the BjaI/BjaR1 QS system in Bradyrhizobium diazoefficiens USDA 110, which shares homology with LuxI/LuxR, remains elusive during symbiotic interaction with soybean. Here this genetic system in wild-type (WT) bacteria residing inside nodules exhibited significantly reduced activity compared to free-living cells, potentially attributed to soybean-mediated suppression. The deletion mutant strain ΔbjaR1 showed significantly enhanced nodulation induction and nitrogen fixation ability. Nevertheless, its ultimate symbiotic outcome (plant dry weight) in soybeans was compromised. Furthermore, comparative analysis of the transcriptome, proteome, and promoter activity revealed that the inactivation of BjaR1 systematically activated and inhibited genomic modules associated with nodulation and nitrogen metabolism. The former appeared to be linked to a significant decrease in the expression of NodD2, a key cell-density-dependent repressor of nodulation genes, while the latter conferred bacterial growth and nitrogen fixation insensitivity to environmental nitrogen. In addition, BjaR1 exerted a positive influence on the transcription of multiple genes involved in a so-called central intermediate metabolism within the nodule. In conclusion, our findings highlight the crucial role of the BjaI/BjaR1 QS circuit in positively regulating bacterial nitrogen metabolism and emphasize the significance of the soybean-mediated suppression of this genetic system for promoting efficient symbiotic nitrogen fixation by B. diazoefficiens.IMPORTANCEThe present study demonstrates, for the first time, that the BjaI/BjaR1 QS system of Bradyrhizobium diazoefficiens has a significant impact on its nodulation and nitrogen fixation capability in soybean by positively regulating NodD2 expression and bacterial nitrogen metabolism. Moreover, it provides novel insights into the importance of suppressing the activity of this QS circuit by the soybean host plant in establishing an efficient mutual relationship between the two symbiotic partners. This research expands our understanding of legumes' role in modulating symbiotic nitrogen fixation through rhizobial QS-mediated metabolic functioning, thereby deepening our comprehension of symbiotic coevolution theory. In addition, these findings may hold great promise for developing quorum quenching technology in agriculture.

Strategy of increasing protein production based on lupin and nano disintegrator technology

The article considers the problem of providing food to the population. To solve this problem, it is proposed to master the process of protein production from lupin. The cultivation of lupin will allow the development of the northern territories of the country and will make it possible to accelerate the development of the northern sea route. It has been established that of all leguminous crops, lupin has the most nitrogen-fixing ability, which will allow further use of arable land for sowing other crops and reduce the amount of nitrogen fertilizers used. It is noted that lupine feed is difficult to digest. To solve this problem, high-temperature grain processing has been developed. This technology improves the process of feeding poultry and animals, but has disadvantages such as: high-temperature processing of lupin grain contributes to the destruction of a number of amino acids and reduces the effect of the vitamin complex; significant additional costs are required for hydrothermal exposure. To eliminate these shortcomings, a new lupin processing process is proposed using a disentangulatory technology. To improve the disintegrator technology, a nanodesintegrator process is proposed by using microturbines instead of electric motors, which increases the effect of grinding the structure and activating biological systems by 19.2 times.

No effect of invasive tree species on aboveground biomass increments of oaks and pines in temperate forests

Prunus serotina and Robinia pseudoacacia are the most widespread invasive trees in Central Europe. In addition, according to climate models, decreased growth of many economically and ecologically important native trees will likely be observed in the future. We aimed to assess the impact of these two neophytes, which differ in the biomass range and nitrogen-fixing abilities observed in Central European conditions, on the relative aboveground biomass increments of native oaks Quercus robur and Q. petraea and Scots pine Pinus sylvestris. We aimed to increase our understanding of the relationship between facilitation and competition between woody alien species and overstory native trees. We established 72 circular plots (0.05 ​ha) in two different forest habitat types and stands varying in age in western Poland. We chose plots with different abundances of the studied neophytes to determine how effects scaled along the quantitative invasion gradient. Furthermore, we collected growth cores of the studied native species, and we calculated aboveground biomass increments at the tree and stand levels. Then, we used generalized linear mixed-effects models to assess the impact of invasive species abundances on relative aboveground biomass increments of native tree species. We did not find a biologically or statistically significant impact of invasive R. pseudoacacia or P. serotina on the relative aboveground biomass increments of native oaks and pines along the quantitative gradient of invader biomass or on the proportion of total stand biomass accounted for by invaders. The neophytes did not act as native tree growth stimulators but also did not compete with them for resources, which would escalate the negative impact of climate change on pines and oaks. The neophytes should not significantly modify the carbon sequestration capacity of the native species. Our work combines elements of the per capita effect of invasion with research on mixed forest management.

Organik ve kimyasal gübre ile kullanılmak üzere mikroorganizma izolasyon ve karakterizasyonu

Isolation and Characterization of Microorganisms for Use with Manure and Chemical Fertilizers&#x0D; Levent Değirmenci1, Özge Kaygusuz İzgördü2, Cihan Darcan3&#x0D; ORCID: 0000-0001-6608-0398; 0000-0002-3652-4266; 0000-0003-0205-3774&#x0D; 1 Corresponding author: Department of Chemical Engineering, Bilecik Şeyh Edebali University, 11210, Bilecik/Turkey Tel: +90 228 2141548, e-mail: levent.degirmenci@bilecik.edu.tr &#x0D; 2 Biotechnology Application and Research Center, Bilecik Şeyh Edebali University, 11210, Bilecik, Turkey 3 3Department of Molecular Biology and Genetics, Bilecik Şeyh Edebali University, 11210, Bilecik, Turkey&#x0D; &#x0D; Abstract&#x0D; A microbial consortium with the ability to utilize nitrogen of certain chemical fertilizers were established via a series of tests. Results indicated four strains, 3 of which were identified as Bacillus cereus sp and an Alcaligenes faecalis sp. which was the only non-Bacillus member of the consortium. The members of the consortium showed no antagonistic activity against each other implying their successful utilization as components of a commercial fertilizer.&#x0D; Keywords: IAA, siderophore, Bacillus spp., nitrogen fixation, ammonia/nitrite oxidation, MALDI-TOF MS&#x0D; &#x0D; Organik ve Kimyasal Gübre ,le Kullanılmak Üzere Mikroorganizma İzolasyon ve Karakterizasyonu&#x0D; &#x0D; Özet &#x0D; Farklı kimyasal gübrelerin içeriğinde yer alan azotu bitkinin kullanımına sunabilen mikroorganizma karışımı bir dizi testler sonucunda elde edilmiştir. Sonuçların değerlendirilmesiyle mikroorganizma karışımına üç Bacillus Cereus sp. suşu ile karışımın Bacillus sp. olmayan tek üyesi Alcaligenes faecalis sp. seçilmiştir. Mikroorganizma karışımında yer alan suşların birbirlerine karşı antagonistik etki göstermediği dolayısıyla karışımın mikrobiyal gübre olarak ticarileştirilmesinin mümkün olduğu da elde edilen sonuçlar arasındadır. &#x0D; Anahtar Kelimeler: IAA, siderophore, Bacillus spp. azot fiksasyonu, amonyum/nitrit oksidasyonu, MALDI-TOF MS&#x0D; 1. Introduction &#x0D; Chemical fertilizer is gaining an increasing attention and its harmful effects on environment possesses a serious threat [2]. Heavy metal accumulation is another problem during utilization of chemical fertilizers [2].The variation of soil pH, another problem encountered due to excessive fertilizer utilization, has negative impact on phosphorus uptake [3]. Plant growth promoting bacteria when added to soil, operate via certain mechanisms facilitating acquisition of already existing resources in soil [4]. Siderophore producing bacteria also acts as biocontrol agent capable of decreasing phytopathogen population in the flora [4–7].&#x0D; Certain procedures were developed and/or modified in the present study to obtain a microbial consortium which would act as an enhancer during plant growth. &#x0D; 2. Materials and Method&#x0D; &#x0D; 2.1. Semi-selective isolation procedure as starting-point&#x0D; &#x0D; Semi-selective isolation procedures were conducted according to [12]. &#x0D; 2.2. Standard tests applied to determine the potential of isolates as PGPB.&#x0D; &#x0D; Nitrogen fixation ability, IAA production, phosphate solubility siderophore production were conducted according to [13-18]. &#x0D; 2.3. Specified tests to determine nitrogen evaluation potential of PGP isolates&#x0D; &#x0D; Nitrogen evaluation potential was determined according to [19]. &#x0D; 2.4. Biochemical tests conducted to determine Bacillus sp. in accordance with MALDI-TOF MS analyses evaluated for validation. &#x0D; &#x0D; Biochemical tests were evaluated based on positive results for both caseinase activity and starch hydrolysis [20-22].&#x0D; MALDI-TOF MS analyses applied for selected strains and an isolate with positive activity towards siderophore production.&#x0D; 3. Results &#x0D; IAA production, Nitrogen fixation performance, Ammonifier selection, phosphate solubility, siderophore production ability, urea degradation, ammonium oxidation and nitrite oxidation abilities of strains were evaluated to establish a microbial consortium. &#x0D; 4. Conclusion and discussion&#x0D; • Media selection could serve as a selection criterion to assemble a certain group of microorganisms.&#x0D; • Tests conducted for isolates could be diversified to establish a consortium with modified properties.&#x0D; • Test procedures could be modified to facilitate establishment of microbial consortium.&#x0D; • Strains with different properties could serve as members of consortium which was the case in our study. &#x0D; • Identification in species level was possible to a certain extent. MALDI-TOF MS analysis presented a brief idea on strains and similar results would have been obtained in the case of applying its costlier alternatives. Hence a relatively simpler procedure is concluded to be better for initial investigation.&#x0D; • Evaluation of results led to investigation of certain prospects including determination of physicotolerant activity of strains and management of Fusarium wilt disease as the topics of future investigations.

Physiological Response of Blackgram Varieties to Foliar Nutrition and Growth Regulators under Partial Shade in Coconut Orchard in Kerala

Background: Coconut is one of the major plantation crops in Kerala which are planted at a wider spacing of 7.6 m ´ 7.6 m giving an ample opportunity for growing intercrops. To enhance soil fertility and productivity of crops inclusion of legumes is a viable option. Blackgram, though a shade sensitive crop performs well in partially shaded coconut gardens due to fast growing nature. The unique ability of biological nitrogen fixation, soil amelioration, carbon sequestration, low water requirement and capacity to withstand extreme drought, pulses have remained an integral component of coconut based cropping system. Since blackgram is a shade sensitive crop, to increase the productivity of blackgram in coconut garden, foliar spray of nutrients and growth regulators is a viable option. Under, shaded or stressed situations, nutrients and plant growth regulators provide optimum vegetative growth by regulating plant growth and architecture. The primary objective of the present study was to investigate the impact of foliar nutrition and growth regulator application on the physiology and yield of different blackgram varieties. Methods: A field trial was conducted at College of Agriculture, Vellayani, Kerala, India during rabi 2020 and summer 2020-21. To find the performance of five blackgram varieties. The treatments consisted of five performing blackgram varieties under partial shade viz; Sumanjana, DBGV 5, VBN 5, VBN 6, CO 6 and foliar sprays of nutrients (19:19:19 NPK, sulphate of potash) and plant growth regulators [naphthalene acetic acid (NAA) and salicylic acid (SA)] alone and in combinations in split plot design at 45 and 60 days after sowing (DAS). Result: The study identified Sumanjana and DBGV 5 as shade adaptive varieties with suitable physiological traits. In both seasons, a higher stomatal conductance and lower stomatal index were observed, indicating a more efficient allocation of resources towards the photosynthetic machinery. Foliar spray of 19:19:19 (1%) and NAA at 40 mg/L and SA at 100 mg/L at 45 and 60 DAS resulted in higher seed yield, dry matter production and also higher net income and benefit cost ratio for these two varieties.

Pseudomonas fluorescens with Nitrogen-Fixing Function Facilitates Nitrogen Recovery in Reclaimed Coal Mining Soils.

Coal mining has caused significant soil nitrogen loss in mining areas, limiting reclamation and reuse in agriculture. This article studies the effects of organic fertilizer, inorganic fertilizer, and the combined application of Pseudomonas fluorescens with the ability of nitrogen fixation on soil nitrogen accumulation and composition in the reclamation area of the Tunlan Coal Mine from 2016 to 2022 under the conditions of equal nitrogen application, providing a scientific basis for microbial fertilization and the rapid increase in nitrogen content in the reclaimed soil of mining areas. The results showed that as the reclamation time increased, the nitrogen content and the composition and structure of the soil treated with fertilization rapidly evolved toward normal farmland soil. The soil nitrogen content increased most rapidly in the presence of added P. fluorescens + organic fertilizer (MB). Compared to other treatments (inorganic fertilizer (CF), organic fertilizer (M), and P. fluorescens + inorganic fertilizer (CFB)), MB increased total nitrogen (TN) to normal farmland soil levels 1-3 years earlier. The comprehensive scores of MB and CFB on the two principal components increased by 1.58 and 0.79 compared to those of M and CF treatments, respectively. This indicates that the combination of P. fluorescens and organic fertilizer improves soil nitrogen accumulation more effectively than the combination of P. fluorescens and inorganic fertilizer. In addition, the application of P. fluorescens increases the content of unknown nitrogen (UN) in acid-hydrolysable nitrogen (AHN) and decreases the content of amino acid nitrogen (AAN) and ammonia nitrogen (AN). However, there was no significant effect on the content of ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3--N) in soil-mineralized nitrogen (SMN). When combined with inorganic fertilizer, the contribution of SMN to TN increased by 14.78%, while when combined with organic fertilizer, the contribution of AHN to TN increased by 44.77%. In summary, the use of P. fluorescens is beneficial for nitrogen recovery in the reclaimed soil of coal-mining areas. The optimal fertilization method under the experimental conditions is the combination of P. fluorescens and organic fertilizer.

Comparative analysis of nitrogen content and its influence on actinorhizal nodule and rhizospheric microorganism diversity in three Alnus species.

Alnus spp. (alder) are typical nonleguminous nitrogen-fixing trees that have a symbiotic relationship with Frankia. To explore the differences in nitrogen-fixing microorganisms between three alders (A. cremastogyne, A. glutinosa, and A. formosana) with different chromosome ploidies, the community structure and compositional diversity of potential nitrogen-fixing microorganism in root nodules and rhizosphere soil were comparatively analyzed using 16S rRNA and nitrogenase (nifH) gene sequencing. The nitrogen contents in the root nodules and rhizosphere soil were also determined. The results showed that the contents of total nitrogen and nitrate nitrogen in the root nodules of the three alders are significantly higher than those in the rhizosphere soils, while the ammonium nitrogen content show the opposite trend. The family, genus, and species levels showed obviously differences between root nodules and rhizosphere soils, while there were no significant differences at the classification level between the three alders. At the phylum level, the dominant phyla from 16S rRNA and nifH gene data in the root nodules and rhizosphere soil of the three alders are phylum Actinomycetota and phylum Pseudomonadota, respectively. The LEfSe results showed that there are significant differences in the dominant groups in the root nodules and rhizosphere oil of the three alders. The relative abundances of dominant groups also showed obvious differences between the root nodules and rhizosphere soils of three alders. The relative abundances of Frankia and unclassified_Frankia in root nodules are obviously higher than those in rhizosphere soils, and their relative abundances in A. glutinosa root nodules are significantly higher than those in A. cremastogyne and A. formosana at the genus and species levels. The diversity of potential nitrogen-fixing microorganism from 16S rRNA and nifH gene data in the A. glutinosa root nodules and rhizosphere soils are all higher than those in A. cremastogyne and A. formosana. The results of functional prediction also showed that the OTUs for nitrogen fixation, nitrate respiration, and ureolysis in A. glutinosa root nodules are higher than those in the other two alders. Redundancy analysis revealed that the total nitrogen content mostly affects the Frankia community. Overall, there are significant differences in the community composition and structure of potential nitrogen-fixing microorganism in the root nodules and rhizosphere soils between the three alders. A. glutinosa showed a relatively stronger nitrogen fixation capacity than A. formosana and A. cremastogyne. The results help elucidates how the community structure and nitrogen-fixing ability of potential nitrogen-fixing microorganism differ between alder species and serve as a reference for applying Frankia to alder plantations.