Soybean Root Nodules Research Articles

Enhanced biochemical properties of soybean root nodule asparaginase through plant molecular farming compared to bacterial enzyme for cancer treatment

Asparaginase is a therapeutic enzyme used as an anticancer agent and is typically produced through microbial fermentation using organisms such as Escherichia coli and Erwinia chrysanthemi. However, this method faces challenges, including potential enzyme contamination during production, allergic reactions to the enzyme, and stability issues requiring stringent control measures. An innovative solution is the application of plant molecular farming, utilizing Rhizobium root symbiosis for asparaginase production. The objective is to optimize nodule development for asparaginase yield, characterize the enzyme's properties, and evaluate its anticancer efficacy against microbial enzyme. In our study, we established soybean root cultures and inoculated them with Bradyrhizobium japonicum to form root nodules. We evaluated eukaryotic asparaginase production at different incubation times. We purified asparaginase from the root nodule cultures and compared its physicochemical properties and anticancer activity with microbial asparaginase. Results showed that asparaginase reached maximum activity in root nodule cultures 10 days after rhizobium inoculation in the culture media. The root nodule asparaginase exhibited a high content of alpha helices and beta sheets and a low random coil. It demonstrated higher stability and activity across different pH levels and temperatures than Escherichia coli asparaginase. Additionally, root nodule asparaginase displayed better catalytic parameters and stability over time than E. coli asparaginase. Thus, root nodule asparaginase is superior to E. coli asparaginase as an anticancer agent. This ensures the root nodule asparaginase can effectively target cancer cells, enhancing the overall therapeutic outcome. This provides a renewable, cost-effective, and environmentally friendly alternative to traditional enzyme production methods.

Effects of the combination of biochar and organic fertilizer on soil properties and agronomic attributes of soybean (Glycine max L.)

This research aimed to investigate the impacts of a combination of rice husk biochar and organic fertilizer on the physical and chemical properties of soil, the population of soil bacteria, the relative chlorophyll content of leaves, the development of soybean root nodules, and yield components under strongly acid soil conditions. A greenhouse and pot experiment was designed using a randomize complete block design with factorial 2 × 3 treatments and three replications. The experimental treatments comprised two rates of biochar (35 and 70 g/pot) and three rates of organic fertilizer (70, 105, and 140 g/pot). After 100 days of amendment of strongly acidic soils, the results showed that application of treatments B35F70 and B70F140 increased soil pH by 16.80% compared to the control group (CK). On the other hand, treatments B35F140 and B70F105 resulted in an increase of soil electrical conductivity by 66.67% compared to CK. In addition, after 100 days of amendment with treatments B35F105, B35F105, B35F140, B70F105, B70F70, B70F70, and B35F140, organic matter, available phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), and zinc (Zn), organic matter, available phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), and zinc (Zn), significantly increased when compared to the control group (CK). Treatment B35F140 increased relative leaf chlorophyll content and soybean seed weight per plant by 60.76% and 100.56%, respectively when compared to the CK. Furthermore, treatment B35F70 produced 125% more root nodules than CK. Moreover, each amended strongly acid soil resulted with a significant upsurge in total soil bacteria compared to the CK. Overall, statistics proved that a combination of biochar and organic fertilizer improved soil properties and soybean agronomic attributes.

Characterization of Root Hair Curling and Nodule Development in Soybean-Rhizobia Symbiosis.

Soybean plants form symbiotic nitrogen-fixing nodules with specific rhizobia bacteria. The root hair is the initial infection site for the symbiotic process before the nodules. Since roots and nodules grow in soil and are hard to perceive, little knowledge is available on the process of soybean root hair deformation and nodule development over time. In this study, adaptive microrhizotrons were used to observe root hairs and to investigate detailed root hair deformation and nodule formation subjected to different rhizobia densities. The result showed that the root hair curling angle increased with the increase of rhizobia density. The largest curling angle reached 268° on the 8th day after inoculation. Root hairs were not always straight, even in the uninfected group with a relatively small angle (<45°). The nodule is an organ developed after root hair curling. It was inoculated from curling root hairs and swelled in the root axis on the 15th day after inoculation, with the color changing from light (15th day) to a little dark brown (35th day). There was an error between observing the diameter and the real diameter; thus, a diameter over 1 mm was converted to the real diameter according to the relationship between the perceived diameter and the real diameter. The diameter of the nodule reached 5 mm on the 45th day. Nodule number and curling number were strongly related to rhizobia density with a correlation coefficient of determination of 0.92 and 0.93, respectively. Thus, root hair curling development could be quantified, and nodule number could be estimated through derived formulation.

A novel electrochemical sensor based on Pb0.95Ti0.05O-decorated nitrogen self-doped biochar for highly sensitive and selective detection of carvacrol

Carvacrol has widely used in the field of medicine and sterilization. Thus, developing reliable, efficient, and rapid method for the carvacrol detection is meaningful. In this study, we fabricated a carvacrol sensor based on Pb0.95Ti0.05O decorated with nitrogen self-doped biochar (Pb0.95Ti0.05O/NC), which was synthesized by hydrothermal and carbonization of soybean root nodules, respectively. The prepared sensor exhibited better electrochemical performances than that of PbO, Pb0.95Ti0.05O, NC. The fabricated Pb0.95Ti0.05O/NC sensor exhibited a wide detection range from 0.1 to 190 µM and a low detection limit of 0.054 µM. The excellent electrochemical performances were attributed to the increased electron density and electron mobility after the introduction of Ti doping and nitrogen doped carbon. Mechanistic studies revealed that the electrochemical oxidation of carvacrol involves one electron and was limited by adsorption process. The sensor demonstrated good stability, anti-interference, reproducibility, and good repeatability. Moreover, the sensor displayed excellent recovery rates ranging from 92.7% to 102.9% when applied to the detection of carvacrol in real samples, making it a promising candidate for practical applications in the sensitive detection of carvacrol.

Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process

The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized. Using single-nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens–infected cells in the mature soybean nodule, including those actively involved in nitrogen fixation and those engaged in senescence. Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein that controls rhizobial infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

Biohydrogen utilization in legume-rhizobium symbiosis reveals a novel mechanism of accelerated tetrachlorobiphenyl transformation

Symbiosis between Glycine max and Bradyrhizobium diazoefficiens were used as a model system to investigate whether biohydrogen utilization promotes the transformation of the tetrachlorobiphenyl PCB77. Both a H2 uptake-positive (Hup+) strain (wild type) and a Hup- strain (a hupL deletion mutant) were inoculated into soybean nodules. Compared with Hup- nodules, Hup+ nodules increased dechlorination significantly by 61.1 % and reduced the accumulation of PCB77 in nodules by 37.7 % (p < 0.05). After exposure to nickel, an enhancer of uptake hydrogenase, dechlorination increased significantly by 2.2-fold, and the accumulation of PCB77 in nodules decreased by 54.4 % (p < 0.05). Furthermore, the tetrachlorobiphenyl transformation in the soybean root nodules was mainly testified to be mediated by nitrate reductase (encoded by the gene NR) for tetrachlorobiphenyl dechlorination and biphenyl-2,3-diol 1,2-dioxygenase (bphC) for biphenyl degradation. This study demonstrates for the first time that biohydrogen utilization has a beneficial effect on tetrachlorobiphenyl biotransformation in a legume-rhizobium symbiosis.

Complete genome sequence of Bradyrhizobium ottawaense strain MIAE 01942 isolated from soybean nodules grown in antibiotic-amended soil.

Bradyrhizobium ottawaense MIAE 01942 is a symbiotic nitrogen-fixing bacterium isolated from the root nodules of soybeans grown in agricultural soils amended with veterinary antibiotics. The genome consists of a single 8.45 Mb circular chromosome that harbors genes involved in nitrogen fixation, denitrification, and antibiotic and metal resistance.

Ciceribacter sichuanensis sp. nov., a plant growth promoting rhizobacterium isolated from root nodules of soybean in Sichuan, China.

The fast-growing rhizobia-like strains S101T and S153, isolated from root nodules of soybean (Glycine max) in Sichuan, People's Republic of China, underwent characterization using a polyphasic taxonomy approach. The strains exhibited growth at 20-40°C (optimum, 28°C), pH 4.0-10.0 (optimum, pH 7.0) and up to 2.0% (w/v) NaCl (optimum, 0.01%) on Yeast Mannitol Agar plates. The 16S rRNA gene of strain S101T showed 98.4% sequence similarity to the closest type strain, Ciceribacter daejeonense L61T. Major cellular fatty acids in strain S101T included summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. The predominant quinone was ubiquinone-10. The polar lipids of strain S101T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethyl ethanolamine, phosphatidyl ethanolamine, amino phospholipid, unidentified phosphoglycolipid and unidentified amino-containing lipids. The DNA G + C contents of S101T and S153 were 61.1 and 61.3mol%, respectively. Digital DNA-DNA hybridization relatedness and average nucleotide identity values between S101T and C. daejeonense L61T were 46.2% and 91.4-92.2%, respectively. In addition, strain S101T promoted the growth of soybean and carried nitrogen fixation genes in its genome, hinting at potential applications in sustainable agriculture. We propose that strains S101T and S153 represent a novel species, named Ciceribacter sichuanensis sp. nov., with strain S101T as the type strain (= CGMCC 1.61309T = JCM 35649T).

Occurrence of diverse plant growth promoting bacteria in soybean [Glycine max (L.) Merrill] root nodules and their prospective role in enhancing crop yield

Root nodules are the small nitrogen fixing factories housing endophytic bacteria. In this study, seventeen endophytic bacteria were isolated from the root nodules of soybean, which were subjected to the in vitro testing of plant growth-promoting characteristics. Bacterial isolates were able to produce indole acetate ranging from 0.7 to 18.3 μg ml−1. Five isolates were able to solubilize inorganic phosphate, in which NSB15 exhibited maximum solubilization potential. All bacterial isolates showed growth on nitrogen-free mineral media, except NSB7 and NSB11. Bacterial isolates were able to form biofilm, therein NSB10 displayed a maximum efficiency of 2.6 at OD570 nm. Seven isolates were positive for cellulase activity and five for pectinase activity. Based on the in vitro characterization, five efficient endophytic bacteria were phylogenetically identified by 16 S rRNA sequence analysis. NSB5 showed 99.52% identity with different species of Pseudomonas, and NSB9 showed 99.68% sequence identity with Bradyrhizobium. NSB15 and NSB16 showed maximum sequence identity with Comamonas terrigena, and NSB17 showed 100% sequence identity with Pseudomonas indica. This study represents the first international report of C. terrigena and P. indica in soybean host. In a pot experiment, inoculation of NSB9 and NSB17 significantly increased plant dry weight by 29% and 35%, respectively, compared to control. In a field trial, inoculation of NSB9, NSB15 and NSB17 significantly improved grain yield by 30%, 31% and 45%, respectively, compared to control. Conclusively, Bradyrhizobium sp. NSB9, C.terrigena NSB15 and P.indica NSB17 could be used as biofertilizers to improve the yield of soybean in a sustainable manner.

Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Visualization and Quantitative Evaluation of Functional Structures of Soybean Root Nodules via Synchrotron X-ray Imaging.

The efficiency of N2-fixation in legume-rhizobia symbiosis is a function of root nodule activity. Nodules consist of 2 functionally important tissues: (a) a central infected zone (CIZ), colonized by rhizobia bacteria, which serves as the site of N2-fixation, and (b) vascular bundles (VBs), serving as conduits for the transport of water, nutrients, and fixed nitrogen compounds between the nodules and plant. A quantitative evaluation of these tissues is essential to unravel their functional importance in N2-fixation. Employing synchrotron-based x-ray microcomputed tomography (SR-μCT) at submicron resolutions, we obtained high-quality tomograms of fresh soybean root nodules in a non-invasive manner. A semi-automated segmentation algorithm was employed to generate 3-dimensional (3D) models of the internal root nodule structure of the CIZ and VBs, and their volumes were quantified based on the reconstructed 3D structures. Furthermore, synchrotron x-ray fluorescence imaging revealed a distinctive localization of Fe within CIZ tissue and Zn within VBs, allowing for their visualization in 2 dimensions. This study represents a pioneer application of the SR-μCT technique for volumetric quantification of CIZ and VB tissues in fresh, intact soybean root nodules. The proposed methods enable the exploitation of root nodule's anatomical features as novel traits in breeding, aiming to enhance N2-fixation through improved root nodule activity.

Quality parameters and root nodules of soybean as influenced by weed management practices

Quality parameters and root nodules of soybean as influenced by weed management practices

COMPLEX INOCULATION OF SOYBEANS WITH NODULE BACTERIA BRADYRHIZOBIUM JAPONICUM AS A MEASURE TO OPTIMIZE SYMBIOTIC NITROGEN FIXATION

Objective. Establish effective mixed inoculants for soybeans based on new active strains of Bradyrhizobium japonicum with different intensity and dynamics of nitrogen-fixing activity to improve nodulation processes and functional activity of legume-rhizobia symbiosis. Methods. Microbiological, physiological, statistical, gas chromatography. Legume-rhizobia systems based on Glycine max L. (Merill) soybean, variety Samorodok, were studied using recombinant strains of B. japonicum: B199, B201, Д47, Д48, Д52, Д60, Д63. Monoinoculation of soybean with B. japonicum 634б served as the control. Results. Mixed inoculation of B. japonicum ensured positive ontogenetic dynamics of root nodule formation in soybean. The indicator of the number of root nodules in experimental plants was 1.2–1.9 times higher versus the control plants from Day 18 to 45 of soybean vegetation. When soybeans were inoculated with B. japonicum 634б + B201 and 634б + Д 52, the mass of nodules at Day 45 of vegetation increased by 30.0–35.3 % versus the control and was the highest (1.10–1.15 g/plant) among the studied variants, as a result of the strengthening effect of complex bacterization. A positive synergistic effect of the combined use of strains was obtained — intensification of the nitrogen-fixing activity of soybean root nodules. In the phase of budding/beginning of flowering and full flowering under combined inoculation with active strains of B. japonicum 634б + Д47, 634б + Д52 and 634б + В201, nitrogen fixation of soybean nodules increased by 10.7–25.5 % (at Day 32), by 13.2–30.9 % (at Day 38), and was also 2.3–2.8 times higher in the phase of full flowering (Day 45 of soybean vegetation) versus the similar indicator of control plants. In the phase of full flowering of plants (Day 45 of vegetation), under mixed inoculation with strains of B. japonicum 634б + Д47, 634б + В201, 634б + Д52 and В201 + Д52, the aboveground mass of plants exceeded that of control plants by 8.3–17.2 % variant, which was probably provided by improved conditions of their nitrogen nutrition, due to enhanced assimilation of N2 by root nodules. The most developed root system was in soybeans inoculated with strains B. japonicum 634б and B. japonicum 634б + Д52, B. japonicum 634б + В201 and B. japonicum Д48 + Д52. Conclusion. Symbiotic features of active recombinant strains of B. japonicum and their variability in functional activity at different stages of soybean ontogenesis should be taken into account when creating complex nitrogen-fixing bacterial preparations for soybeans with the effect of microbiological synergy.

Peculiarities of soybean-rhizobial systems subject to different levels of water supply fol-lowing treatment with succinic acid and epibrassinolide

All around the world, one of the leading – according to area of cultivated fields – oleic crops is soybean, which has a high demand for moisture. Given the significance of this crop and negative impact of drought on its yield, integrated research of the influence of insufficient water supply on the intensity of physiological-biochemical processes in those plants is necessary for identifying and understanding the drought-tolerance mechanisms of soybean, as well as symbiotic systems created with its participation, and also for search for ways to adapt it to this stressor. Therefore, our objective was determining the specifics of formation and functioning of the symbiotic systems of soybean and Bradyrhizobium japonicum, following treatment with succinic acid (0.01 g/L) and 24-epibrassinolide (0.00001 g/L), subject to different levels of watering. Our studies revealed that pre-sowing treatment of the seeds with a solution of 24-epibrassinolide with their subsequent inoculation with B. japonicum Т21-2 resulted in the most pronounced stimulation of formation and functioning of the symbiotic systems of soybean in the optimal growing conditions. At the same time, during water shortage, the intensity of nitrogen fixation was the highest in the plants grown from seeds that had been successively treated with the acid and the inoculant. We confirmed that water deficit led to significant increase in the overall content of phytohormones of cytokinin nature in the soybean root nodules, depending on the way the seeds were treated. However, the largest pool of cytokinins was seen in the plants that had been treated with succinic acid against the background of both optimal and insufficient water supply. Treatment of the seeds with 24-epibrassinolide caused significant excess of content of zeatin riboside over the content of zeatin during the flowering stage, whereas in the stage of pods formation it led to an opposite effect – excess of zeatin over zeatin riboside. Fourteen days-long water deficit decreased the content of chlorophylls in the leaves and grain productivity of the plants of all variants of the experiment. The use of growth regulators managed to alleviate the negative impact of stress and protect the pigment complex from ruination. Treatment of the seeds with solutions of succinic acid and 24-epibrassinolide provided the growth of soybean grain productivity regardless on water-supply level. The most efficient was 24-epibrassinolide. Therefore, use of 24-epibrassinolide for pre-sowing treatment of the soybean seeds provided formation of effective symbiotic systems with high nitrogen-fixing activity and caused a number of specific changes in the pattern of accumulation of free and complex forms of cytokinins in the root nodules of those plants. At the same time, the treatment provided the highest concentration of photosynthesis pigments in the soybean leaves, and as a result produced the greatest increase in grain productivity of plants of all the variants, regardless of levels of water supply. In turn, use of succinic acid produced the highest level of nitrogen-fixing activity in the case of the lowest number of root nodules in the conditions of insufficient water supply, and also caused significant accumulation of cytokinins in the nodules, compared with other studied variants against the background of both optimal and insufficient water supply. Therefore, it did result in increase in soybean grain productivity, but this was lower than in the plants treated with 24-epibrassinolide.

Co-Inoculation with Bradyrhizobium and Humic Substances Combined with Herbaspirillum seropedicae Promotes Soybean Vegetative Growth and Nodulation

The effect of humic substances (HSs) in combination with or without plant-growth-promoting bacteria on soybean vegetative growth and root nodulation was examined in this study. Seeds were inoculated with Bradyrhizobium japonicum SEMIA 5079 in the presence of HSs from leonardite and Herbaspirillum seropedicae HRC54. Additional HSs and H. seropedicae application at the substrate surface was conducted at the V3 stage. The experiment was carried out in a greenhouse using pots filled with a top layer of an Oxisol soil, and plants were harvested at the R1 stage. The HS and H. seropedicae treatments significantly promoted plant shoot and root growth. The number and weight of soybean nodules were higher in the treated plants when compared to a control. The plant nodulation process was affected by the treatments that included activities of malate dehydrogenase (MDH), nitrate reductase (NR) and plasma membrane H+-ATPase (MHA). At low concentrations, the HSs and H. seropedicae increased the nodule quantity, size and weight, favoring plant growth. Combining humic substances and plant-growth-promoting bacteria (PGPB) could be a promising approach to promoting soybean nodulation and increasing crop production.

Expanded Coverage of Phytocompounds by Mass Spectrometry Imaging Using On-Tissue Chemical Derivatization by 4-APEBA.

Probing the entirety of any species metabolome is an analytical grand challenge, especially on a cellular scale. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a common spatial metabolomics assay, but this technique has limited molecular coverage for several reasons. To expand the application space of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for the confident identification of several dozen elusive phytocompounds. Overall, this new OTCD method enabled the annotation of roughly 280 metabolites, with only a 10% overlap in metabolic coverage when compared to analog negative ion mode MALDI-MSI on serial sections. We demonstrate that 4-APEBA outperforms other derivatization agents by providing: (1) broad specificity toward carbonyls, (2) low background, and (3) introduction of bromine isotopes. Notably, the latter two attributes also facilitate more confidence in our bioinformatics for data processing. The workflow detailed here trailblazes a path toward spatial hormonomics within plant samples, enhancing the detection of carboxylates, aldehydes, and plausibly other carbonyls. As such, several phytohormones, which have various roles within stress responses and cellular communication, can now be spatially profiled, as demonstrated in poplar root and soybean root nodule.

Erratum for the Research Article "Light-induced mobile factors from shoots regulate rhizobium-triggered soybean root nodulation," by T. Wang et al.

Erratum for the Research Article "Light-induced mobile factors from shoots regulate rhizobium-triggered soybean root nodulation," by T. Wang et al.

Role of phosphorus and molybdenum on root growth, nodulation and nutrient uptake of soybean [Glycine max (L.) Merr.

A field investigation was carried out during kharif 2015 to study the response of soybean to phosphorus and molybdenum nutrition. Results revealed that the interaction effect of phosphorus application (60 kg P2O5/ha) and molybdenumseed treatment @ 6 g/kg seed recorded significantly higher vigourindex-I (2305), vigour index- II (90), root length (21.26 cm), root volume (12.70 cm3), effective nodules/plant (37), nodule dry weight (50.73 mg), oilcontent (19.43 %) and molybdenum uptake by seed (4.10 g/ha). The leghaemoglobin content was highest (249 mg/g fresh weight) with P application @ 60 kg/ha and it was unaffected due to Mo seed treatment and interaction of P and Mo. Among the P levels, protein content and seed yield were significantly higher (41 % and 1784 kg/ha) with 60 kg/ha and with Mo seed treatment @ 6 g/kg seed (40.2 % and 1572 kg/ha).

Respon Pertumbuhan dan Hasil Kedelai dengan Aplikasi Pupuk Organik dan Rhizobium

&lt;p&gt;Soybean are basic need and the main source of vegetable protein for most of the Indonesian population. Organic matter and &lt;em&gt;Rhizobium &lt;/em&gt;are improvement efforts to optimize soybean growth between durian plants. The purpose of the research was to determine the use of &lt;em&gt;Rhizobium&lt;/em&gt; and organic fertilizers on the growth and yield of soybeans between durian plants in LubukKelik village, Bangka. The experiment used split plot and RAL design. The main plot was &lt;em&gt;Rhizobium&lt;/em&gt; applications consist of without &lt;em&gt;Rhizobium&lt;/em&gt; (P0) and &lt;em&gt;Rhizobium&lt;/em&gt; application (P1). The sub plot was the use of organic fertilizers, which consists of chicken manure (K1) and empty oil palm fruit bunches compost (K2). The results showed that the types of organic fertilizer application not significantly affected soybeans growth dan yield in between durian plants. Empty fruit bunches compost provided the best growth on soybean plants. The best yield of soybean in between durian plants are shown by empty fruit bunches treatment. &lt;em&gt;Rhizobium&lt;/em&gt; application significantly affected growth parameters of soybean and effective root nodules. Soybean growth parameters with &lt;em&gt;Rhizobium&lt;/em&gt; application was better than without &lt;em&gt;Rhizobium&lt;/em&gt; application. Interaction between organic fertilizers and &lt;em&gt;Rhizobium&lt;/em&gt; significantly not affected soybeans growth dan yield. Combination of empty oil palm fruit bunches compost with &lt;em&gt;Rhizobium&lt;/em&gt; is treatments that provide the best growth of soybean.&lt;/p&gt;

Development of High-Quality Nuclei Isolation to Study Plant Root-Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean.

Single-nucleus RNA sequencing (sNucRNA-seq) is an emerging technology that has been rapidly adopted and demonstrated to be a powerful tool for detailed characterization of each cell- and sub cell-types in complex tissues of higher eukaryotes. sNucRNA-seq has also been used to dissect cell-type-specific transcriptional responses to environmental or developmental signals. In plants, this technology is being utilized to identify cell-type-specific trajectories for the study of several tissue types and important traits, including the single-cell dissection of the genetic determinants regulating plant-microbe interactions. The isolation of high-quality nuclei is one of the prerequisite steps to obtain high-quality sNucRNA-seq results. Although nuclei isolation from several plant tissues is well established, this process is highly troublesome when plant tissues are associated with beneficial or pathogenic microbes. For example, root tissues colonized with rhizobium bacteria (nodules), leaf tissue infected with bacterial or fungal pathogens, or roots infected with nematodes pose critical challenges to the isolation of high-quality nuclei and use for downstream application. Therefore, isolation of microbe-free, high-quality nuclei from plant tissues are necessary to avoid clogging or interference with the microfluidic channel (e.g., 10× Genomics) or particle-templated emulsion that are used in sNucRNA-seq platforms. Here, we developed a simple, effective, and efficient method to isolate high-quality nuclei from soybean roots and root nodules, followed by washing out bacterial contamination. This protocol has been designed to be easily implemented into any lab environment, and it can also be scaled up for use with multiple samples and applicable to a variety of samples with the presence of microbes. We validated this protocol by successfully generating a barcoded library using the 10× Genomics microfluidic platform from tissue subjected to this procedure. This workflow was developed to provide an accessible alternative to instrument-based approaches (e.g., fluorescent cell sorting) and will expand the ability of researchers to perform experiments such as sNucRNA-seq and sNucATAC-seq on inherently heterogeneous plant tissue samples.