Plant-Microbe Symbiosis

The Rhizobia-Legume symbiosis is crucial for sustainable agriculture, yet the host range compatibility of root-nodulating bacteria (RNB) from wild legumes with crop legumes remains underexplored. This study investigated the potential of three RNB isolates from wild Crotalaria tetragona, Crotalaria mysorensis, and Leucaena leucocephala—namely Burkholderia territorrii (MOKCS15), Rhizobium tropici (LUMCR3), and Ensifer fredii (LUMLL12)—to enhance growth and symbiotic efficiency in four edible crop legumes: Cajanus cajan, Glycine max, Phaseolus vulgaris, and Phaseolus lunatus. Experiments evaluated individual isolates and various RNB consortia. Individual isolates demonstrated varied compatibility. MOKCS15 broadly nodulated C. cajan, P. lunatus, and P. vulgaris (34–53 nodules/plant), significantly enhancing growth and biomass. LUMCR3 successfully nodulated Phaseolus species, while LUMLL12 showed limited effectiveness on G. max and P. lunatus with underdeveloped nodules. Notably, RNB consortia generally outperformed single strains, leading to superior growth and biomass in P. lunatus, P. vulgaris, and C. cajan. For instance, consortium RC-1 nodulated all tested crop legumes, with P. vulgaris forming 28–30 nodules. G. max consistently showed limited or ineffective nodulation, likely due to dominance by Bradyrhizobia. Symbiotic efficiency (RSE%) varied, with MOKCS15 yielding the highest RSE% (161%) in C. cajan and RC-4 achieving 159% in P. vulgaris. A strong positive correlation (0.94) was observed between flavonoid content, nodule formation, and symbiotic efficiency. This study highlights the significant potential of wild native Rhizobia, both as single strains and in consortia, to serve as effective bioinoculants. These findings offer valuable insights for developing sustainable agricultural practices by leveraging diverse rhizobial resources.

Maize (Zea mays L.) is the most widely produced crop in the world, and conventional production requires significant amounts of synthetic nitrogen fertilizer, which has negative economic and environmental consequences. Maize landraces from Oaxaca, Mexico, can acquire nitrogen from nitrogen-fixing bacteria that live in a mucilage secreted by aerial nodal roots. The development of these nodal roots is a characteristic traditionally associated with the juvenile vegetative stage of maize plants. However, mature Oaxacan landraces develop many more nodes with aerial roots than commercial maize varieties. Our study shows that Oaxacan landraces develop aerial roots during the juvenile and adult vegetative phases and even during early flowering under greenhouse and field conditions. Surprisingly, the development of these roots was only minimally affected by soil nitrogen and ambient humidity. These findings are an essential first step in developing maize varieties to reduce fertilizer needs in maize production across different environmental conditions.

•Rhizobia and non-rhizobial endophytes synergistically enhance alfalfa growth
• Functional redundancy of synthetic microbial communities to predict success in field trials
• Effective endophytic inoculants do not significantly alter the native rhizosphere biodiversity

The integration of microbial nitrogen (N2) fixation with photochemical processes using inorganic light-absorbing nanomaterials is a burgeoning field in sustainable energy production. Here, we explore the synergistic combination of inorganic semiconductor nanowires (NWs) with whole-cell microorganisms to create an inorganic-bacterial biohybrid system. Specifically, we employ Cu2O@TiO2 NWs with a core/shell structure to harness sunlight and generate photoexcited electrons. Azotobacter vinelandii, serving as a biocatalyst, adsorbs onto these NWs and facilitates the reception of photoexcited electrons, thereby enhancing the efficiency of the photoelectrochemical N2 fixation reaction (PEC-NRR). The biohybrid system achieves an impressive ammonia (NH3) yield of (1.49 ± 0.05) × 10-9  mol s-1 cm-2 (5.36 ± 0.18 μmol h-1 cm-2). The enhancement in NH3 synthesis within the Cu2O@TiO2 NWs/A. vinelandii biohybrid is attributed to the increased concentrations of nicotinamide adenine dinucleotide-hydrogen (NADH) and adenosine 5’-triphosphate (ATP), as well as the overexpression of N2-fixing genes like nifH and nifD within the nitrogenase enzyme complex. This study underscores the potential of inorganic-bacterial biohybrid systems in solar-chemical conversion, paving the way for more diverse and functional approaches to harnessing solar energy for sustainable chemical production.

Introduction: Although the mulched drip irrigation system combined with high nitrogen input (240∼310 kg ha-1) in Xinjiang, China, frequently achieves record-high soybean yields (6855 kg ha-1), this practice is not conducive to symbiotic nitrogen fixation and compromises agricultural sustainability.

Methods: Under the mulched drip irrigation, this study evaluation four nitrogen application treatments (N0: 0 kg ha-1, N120: 120 kg ha-1, N180: 180 kg ha-1, and N240: 240 kg ha-1) were evaluated over two consecutive growing seasons to investigate their effects on nodule morphological and physiological traits, stem ureide content, and the percentage of nitrogen derived from the atmosphere (%Ndfa) during the reproductive growth stage.

Results: The application of 180 kg ha-1 nitrogen significantly increased nodule number, nodule dry weight, nodule sucrose content, and nodule starch content, while improving soybean yield and nitrogen agronomic use efficiency. Conversely, the application of nitrogen exceeding 180 kg ha-1 inhibited nitrogenase activity, suppressed leghemoglobin synthesis, disrupted the glutamine synthetase/glutamate synthase metabolic pathway, and reduced ureide translocation from nodules to stems, leading to significant accumulation of ureides in nodules. Correlation and path analyses indicated that nitrogenase activity, leghemoglobin content, urate oxidase activity, and stem ureide content were significantly positively correlated with %Ndfa, whereas nodule ureide content showed a significant negative correlation with %Ndfa. Stem ureide content exhibited a strong direct positive effect on %Ndfa (path coefficient = 0.95), confirming its validity as a robust indicator for assessing SNF capacity.

Discussion: In conclusion, mulched drip irrigation, applying 180 kg ha-1 nitrogen at the beginning pod stage (R3) effectively enhances root nodulation, promotes carbohydrate allocation to nodules, sustains symbiotic nitrogen fixation activity, and ultimately increases soybean yield and nitrogen use efficiency. Thus, under mulched drip irrigation system, applying the correct rate of nitrogen fertilizer is beneficial for enhancing soybean yield and mitigating environmental risks, which holds significant importance for promoting sustainable agricultural development.

Soil microorganisms are a key protection goal in the European Union (EU) pesticide regulatory framework. Arbuscular mycorrhizal fungi (AMF) were identified as good proxies for assessing pesticides toxicity on the soil microbiota. This could involve ecotoxicity testing at the different life stages of AMF. We evaluated the effects of five pesticides (pyraclostrobin, fludioxonil, hymexazol, etridiazole, glyphosate) and a transformation product (AMPA), with distinct mode of action, on the development and functionality of Rhizophagus irregularis at the asymbiotic and symbiotic phase using a spore germination assay and a gnotobiotic AMF-host plant system (AMF-sandwich test), respectively. Based on arbuscular colonization in the AMF-sandwich test, fludioxonil was the most toxic (EC50 0.085 mg/L) followed by glyphosate (EC50 2.58 mg/L) and pyraclostrobin (EC50 9.22 mg/L), while etridiazole, hymexazol, and AMPA showed EC50 values higher than the highest tested concentration. However, for glyphosate and pyraclostrobin negative effects on symbiosis functioning were observed at lower concentrations than for colonization, as depicted by the expression of plant marker genes and/or P-uptake, suggesting the establishment of non-functional arbuscular symbiosis. The high toxicity of fludioxonil (EC50 0.03 mg/L) and the low toxicity of AMPA (EC50 > 432 mg/L) on R. irregularis was verified also for the asymbiotic phase via spore germination assay. Comparative tests showed differences in the toxicity of pure active substances and commercial formulations of fludioxonil and pyraclostrobin on the AMF-sandwich test. We propose that the AMF-sandwich system together with the spore germination test could be used as a toolbox for Tier-I assessment of pesticides toxicity on AMF.

Introduction: Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes mimicking the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging.

Methods: Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla. Genomic analysis of the individual strains was performed to reveal encoded plant growth-promoting traits, including genes for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters, and some predicted traits were validated by laboratory phenotypic assays.

Results: The SynCom demonstrates strong stability both in vitro and in planta. Most strains encoded multiple plant growth-promoting functions, and several of these were confirmed experimentally. The presence of osmoprotectant and ion transporter genes likely contributed to the observed resilience of Brachypodium to drought stress, where plants amended with the SynCom recovered better than those without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria.

Discussion: Our results demonstrate that trait-informed construction of synthetic communities can yield stable, functionally diverse consortia that enhance plant resilience under drought. Preferential colonization near root tips points to active, localized plant–microbe signaling as a component of stress-responsive recruitment. This stable SynCom provides a scalable platform for probing mechanisms of plant-microbe interaction and for developing microbiome-based strategies to improve soil and crop performance in variable environments.

Background and aims
Flavonoids produced by legumes play a crucial role in initiating and regulating nodulation, serving as key signaling molecules in the legume-rhizobium symbiosis. However, the specific roles of individual flavonoids in the symbiotic process of Medicago truncatula remain poorly understood.

Methods
Here, we performed transcriptomic and flavonoid metabolomic analyses of Sinorhizobium meliloti 1021 (Sm1021)-inoculated and mock-inoculated M. truncatula roots at 8 hours post-inoculation (hpi) to investigate the early responses to rhizobial infection.

Results
Our results revealed that flavonoid biosynthesis pathways were significantly enriched in Sm1021-inoculated roots compared to mock-inoculated roots at 8 hpi. Flavonoid metabolome profiling identified 98 differentially accumulated flavonoids between Sm1021-inoculated and mock-inoculated roots at 8 hpi. Three key flavonoids (2,4,2′,4′-tetrahydroxy-3′-prenylchalcone, methylnissolin, and kaempferol) were identified based on high variable importance in projection (VIP) scores and network analysis. Notably, the isoflavone methylnissolin strongly induced the expression of key nodulation genes of Sm1021. Molecular docking analysis indicated that methylnissolin potentially bound to three NodD proteins of Sm1021. Co-inoculation of Sm1021 with methylnissolin promoted nodulation of M. truncatula under nitrogen-limited conditions.

Conclusion
This study provides the first evidence that methylnissolin acts as a crucial inducer of rhizobial nodulation genes, enhancing nitrogen fixation efficiency in M. truncatula. Our findings highlight the pivotal role of flavonoids, particularly isoflavones, in the regulation of the legume-rhizobium symbiotic process.

Plant Growth Promoting Bacteria (PGPB) have been widely shown to improve growth and stress management in plants. Identifying and isolating PGPB are critical steps toward understanding the role of increasing crop yields and growth in complicated ecosystems. However, the techniques available for culturing microorganisms remain limited. Various studies have shown that single microorganisms can exert beneficial effects on plants. Still, it is increasingly evident that when a microbial consortium-two or more interacting microorganisms is involved, additive or synergistic results can be expected. This review article provides an overview of the isolation, cultivation, and application of PGPB. We summarize and discuss the advantages and limitations of novel techniques for screening PGPB, including selective media, high-throughput screening (HTS), and gene editing. The chapter introduces how microbial consortiums are constructed through function combination, machine learning (ML), or artificial intelligence (AI). Compared to conventional techniques, these approaches offer higher precision in selecting compatible and synergistic strains, enabling the development of more stable and efficient PGPB communities. Although promising, such methods are still in the early stages of development and face challenges such as limited datasets, high computational demands, and the complexity of accurately modeling microbe–microbe and microbe–plant interactions. Besides, the discussion concludes with an overview of the commercialization of PGPB, which is expected to facilitate the development of sustainable agriculture.

Background
Symbiotic nitrogen-fixation between bacteria called rhizobia and leguminous plants is a critical aspect of sustainable agriculture. Complex, two-way communication governs the invasion of plant roots and the formation of nodules in which the rhizobia reduce N2 to bioavailable ammonia. Research has uncovered many of the genes required for the symbiosis; however, engineering the symbiosis to function with alternative hosts such as cereal crops necessitates the establishment of a core set of symbiotic players.

Results
We examined the symbiotic relevance of the genes on the 1.68 Mb pSymB chromid of the model rhizobium Sinorhizobium meliloti. By employing a strain in which pSymB was removed, we used a gain-of-function approach to assess a select group of known symbiotic regions totalling 261 kb (15.5%) of pSymB. This gene set enabled symbiotic N2-fixation with alfalfa with a high degree of plant genotype-dependent variation in which nodules often senesced prematurely. We demonstrate that additional regions lacking canonical symbiosis genes are important for the efficient formation of symbiosis with the plant host. These regions appear to contain auxiliary symbiotic loci whose genes encode products with quasi-essential functions for the symbiosis and that are redundant in nature. We further established a 673-kb pSymB genome that engages consistently in N2-fixation with alfalfa with 45% efficiency.

Conclusions
The reduction of the pSymB genome showcases the complexity and nuance of its involvement in the N2-fixing symbiosis.

Nitrogen (N) is essential for plant growth, yet excessive fertilizer use contributes to environmental degradation. Actinorhizal trees like Alnus glutinosa form symbiotic relationships with nitrogen‐fixing bacteria of the genus Frankia, reducing reliance on synthetic fertilizers. However, distinguishing between soil‐derived and symbiotically fixed nitrogen remains a challenge. This study investigates the potential of NIR spectroscopy as a nondestructive tool for differentiating N sources in A. glutinosa . Seedlings were grown in sterilized soil under controlled conditions with and without Frankia inoculation, and across a gradient of NH4NO3 fertilization (0–20 mM). We measured leaf chlorophyll, nitrogen content, biomass, and NIR reflectance (330–1100 nm) of the third fully expanded leaf. principal component analysis (PCA) and partial least squares (PLS) regression revealed that spectral signatures significantly differed between inoculated and uninoculated plants, particularly in the visible range around 555 nm. Despite similar leaf chlorophyll levels, Frankia‐inoculated plants and those fertilized with 20 mM NH4NO3 exhibited spectral differences that could otherwise not be detected by SPAD measurements. PLS regression explained up to 54.8% of spectral variance based on nitrogen source, even in the absence of unique spectral peaks. These findings highlight the potential of NIR spectroscopy for rapid, in vivo and in vitro assessment of symbiotic N‐fixation in trees, offering a novel and more precise approach than SPAD measurements.

This study developed Klebsiella oxytoca W6-nasR2, a bioluminescent bioreporter containing the nasR2 promoter fused to the luxCDABE operon for detecting bioavailable nitrogen. The 267-bp nasR2 open reading frame (ORF) encodes an 88-amino acid protein with regulatory elements for nitrogen metabolism and stress responses, including NtrC/σ54, FNR-type motifs, GlnR, and SOS motifs. COBALT analysis identified conserved nitrate-regulation motifs (DLRQQLVDRIDGQQPC, FDSFQALAEAPQT). The nasR2 promoter, with -10 (TTTTCA) and -35 (CATATT) boxes, coordinates nitrogen metabolism and stress adaptation. Predicted phosphorylation sites at serine residues 49 and 75 likely enhance nasR2 stability and sensitivity, making it a robust nitrate reductase. Wild-type K. oxytoca W6 reduced 92.39% of 1.2 g NO₃-N/L in 27 h, with peak nitrate reductase activity (600 U/mL) observed at 168 h during extended enzymatic monitoring. The W6-nasR2 bioreporter exhibited a 186-fold increase in bioluminescence in response to peptone, with optimal activity at pH 6–7, retaining ~ 56% activity across pH 4–10. Strongest responses were observed with organic nitrogen sources (yeast extract > peptone > casein > skim milk), moderate responses with inorganic nitrogen (NaNO₃ > NH₄Cl > NaNO₂), and amino acids (lysine > asparagine). Urea and NaCN showed minimal or inhibitory effects. Field applications detected bioavailable nitrogen in municipal wastewater (45,000–62,000 CPS), industrial wastewater (41,000–42,000 CPS), desert soil (4,000 CPS), and agricultural soil (25,000 CPS), correlating with total nitrogen levels of 0.045–0.058 mg/L. These findings highlight the utility of the W6-nasR2 bioreporter for monitoring bioavailable nitrogen in real-world environments and position NasR2 as a potential novel nitrate reductase.

•Harnessing AM fungi offers nature-based solution to ecological restoration.
•Establishing an ecological support system (EES) can optimize AM fungal functionality.
•ESS integrates plant selection, soil conditioning and microbial facilitation.
•Emphasized the importance of further research on hyphosphere microbes