Enhanced biological nitrogen fixation in alfalfa through the synergistic interactions between Sinorhizobium meliloti and Priestia aryabhattai

Enhanced biological nitrogen fixation in alfalfa through the synergistic interactions between Sinorhizobium meliloti and Priestia aryabhattai | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced biological nitrogen fixation in alfalfa through the synergistic interactions between Sinorhizobium meliloti and Priestia aryabhattai Rui Liu, Chang Li, Yunjun Zhang, Chunli Liu, Yanning Zheng, Jinai Xue This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5665466/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 May, 2025 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 10 You are reading this latest preprint version Abstract Nitrogen fertilizer is crucial for agricultural output. However, prolonged overuse has resulted in nitrate leaching, and potential soil acidification. Research on microbial fertilizers has become essential to enhance soil conditions and minimize nitrogen fertilizer usage. In alfalfa cultivation, research on efficient compound microbial agents remains limited, therefore, this study concentrates on the investigation of dual microbial combinations. In the screening process, black soil was utilized with alfalfa plants as samples to identify a strain of rhizobacteria, Sinorhizobium meliloti LMGL3-1 , exhibiting nitrogen-fixing capabilities, and Priestia aryabhattai ( Bacillus aryabhattai ) YJHT21, demonstrating phosphorus-solubilizing abilities. The S. meliloti strain demonstrated the ability to symbiotically associate with the alfalfa variety Longmu 806, resulting in the formation of effective nodules containing leghemoglobin, thereby enabling the plants to thrive in the absence of nitrogen fertilizer application. Here, we discovered that the addition of phosphorus-solubilizing P. aryabhattai enhanced plant growth and increased nitrogenase activity of S. meliloti . Moreover, the incorporation of P. aryabhattai resulted in a significant increase in flavonoid production within the root system of alfalfa plants. Consequently, under the influence of the inducer extracted from the root system of quantitatively analyzed plants, the rhizobacteria exhibited enhanced production of metabolites associated with the Nod factor cluster. The current experiment demonstrated that the interaction between the two bacteria significantly enhanced nitrogen fixation, effectively substituting nitrogen fertilizer in alfalfa cultivation with improved efficiency and offered theoretical support for the eco-friendly advancement of microbial compound fertilizers as a substitute for chemical fertilizers. Nitrogen fixation Plant growth Flavonoid Sinorhizobium Priestia Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The use of nitrogen fertilizers has resulted in tremendous growth in agricultural production. However, decades of overuse leading to soil compaction, acidification, reduction of organic matter, and exacerbation of the greenhouse effect are significant environmental concerns (Ahmed et al., 2017 ). The application of mineral nitrogen (N) fertilizers to crops may increase greenhouse gas emissions (Lourenço et al., 2019 ). Moreover, it has also precipitated several detrimental impacts on human health (Ahmed et al., 2017 ). Bio-fertilizers, which consist of beneficial bacteria associated with plant roots, enhance plant growth by augmenting the nutritional availability for the host plant when given to seeds, plants, or soil (Mazid and Khan, 2014 ). The use of bio-fertilizers is considered an effective approach to enhancing soil quality and fostering agricultural sustainability (Bardi and E, 2012 ; Malusá and Vassilev, 2014 ). Previous study was conducted to assess the efficacy of the biofertilizer B. subtilis in mitigating NH 3 volatilization (Sun et al., 2020 ). And another study showed that the bacterial fertilizer including Rhizobium americanum and Bacillus laguerreae was discovered to improve the plant development (Jiménez-Gómez et al., 2021 ). Legumes can establish a root-knot symbiosis with nitrogen-fixing soil bacteria called rhizobia. This inter-association is extremely specialized, with each rhizobia species or strain interacting only with a certain type of legumes, and vice versa (Wang et al., 2012 ). Rhizobia must compete to infect legume roots and initiate a signal transduction process with the host plant, ultimately resulting in nodule development (Terpolilli et al., 2012 ). Previous studies have identified interactions between alfalfa cultivars and Rhizobium meliloti strains on acetylene reduction rates, plant height and shoot, root and whole plant dry weight (Tan and Tan, 1986 ). Therefore, it’s important to find a match bacterium for the alfalfa. S. meliloti is a rhizobacterium frequently associated with alfalfa, and the specific strain identified in the alfalfa sample is S. meliloti . This species exhibits significant genetic and phenotypic variation in natural populations, especially with the symbiotic enhancement of plant development. S. meliloti is internationally dispersed and can be found in various soil types, either as a free-living organism or as a symbiotic partner of leguminous plants, which it prompts to produce nodules. The P. aryabhattai strain of Bacillus spp. was isolated from the black soil of northeastern China. It is a soil-dwelling rhizobacterium distinguished by spherical, somewhat yellowish colonies. The colony's exterior is flat and moist, while the interior is gelatinous, measuring approximately 2.0 to 5.0 mm in diameter. It has a positive Gramme reaction, shows motility, and has the capacity to create endospores. It is a strain of phosphate-solubilizing bacterium demonstrating phosphate-solubilizing activity, with a capacity of 19.83 mg/L. A relevant study has demonstrated that the Bacillus spp. strain comparable to the P. aryabhattai analyzed in our research can enhance Sulphur metabolism in oilseed rape (Baruah et al., 2024 ). Additionally, there are analogous Aryabhattai species; Bacillus sp. MCCC 1K02966. It has been identified as a promising biological control agent (Chen et al., 2021 ). And P. aryabhattai has also proven to be an exceptionally excellent multi-stress tolerant crop growth promoter (Shahid et al., 2022 ). And the similar bacteria strain Bacillus aryabhattai AB211 is a Gram-positive bacterium that can enhance plant development (Bhattacharyya et al., 2017 ). A study was conducted to evaluate the impact of PGPR on nodulation and N2 fixation efficiency in soybean through co-inoculation with Bradyrhizobium diazoefficiens USDA110. The inoculation of Bacillus velezensis S141 with USDA110 in soybean resulted in enhanced nodulation and nitrogen fixation efficiency, evidenced by the development of larger nodules (Sibponkrung et al., 2020 ). Another study was conducted to evaluate the promotional effects of a commercial strain of Rhizobium japonicum and a novel isolate of Bacillus megaterium , both individually and in combination, during the biological initiation of seeds (Miljaković et al., 2022 ). The simultaneous application of S. meloloti LMGL3-1 and P. aryabhattai YJHT21 produced analogous outcomes in the study. Despite comprehensive national and worldwide studies on Rhizobium, research on Bacillus remain limited. Previous studies suggest that the simultaneous application of Rhizobium and Bacillus can improve plant growth. Further investigation is necessary due to the scarcity of literature concerning the interaction between these two bacteria. We ought to replace nitrogen fertilizers with microbial fertilizers to improve soil ecology and attain sustainable agriculture. Here we found that under nitrogen-free conditions, S. meliloti LMGL3-1 and P. aryabhattai YJHT21 combination improved alfalfa plant growth compared to single strains, significantly increased chlorophyll levels, increased the number of nodules, and enhanced nitrogen fixation.P. aryabhattai enhances root flavonoid production, subsequently inducing S. meliloti to generate increased nodulation factor, resulting in improved nitrogen fixation in alfalfa Longmu806. The Prietia aryabhattai YJHT21 strain, isolated from black soil, and S. meliloti LMGL3-1 enhance soil microbial diversity, promote plant development, improve nitrogen absorption and utilization, raise arable land efficiency, and protect the soil environment, hence safeguarding human health. This strategy supports sustainability in agriculture and development. Materials and methods Bacterial selected and growth condition P. aryabhattai was extracted from a black soil sample in northeastern China and subsequently purified and isolated using an organophosphorus solid medium (Monkina Medium). The bacteria were cultured at 30℃ with a rotation speed of 200 rpm for 24 hours. In the context of co-culture, the rhizobium medium (YEM media) was employed for the subsequent culture phase, incubated at 28℃ with 150 rpm for 5-7 days for biological indicators. A soil sample was collected from the 5-20 cm topsoil of farmland in Qiqihar, Heilongjiang, China. The soil was naturally air-dried, with plant debris eliminated, and passed through a 2 mm sieve. Subsequently, combine the soil with saline (1:10) for 6 hours, allow it to equilibrate, and then extract the supernatant. S. meliloti was extracted from the roots of alfalfa Longmu 806 in Hulunbeier, Neimenggu, China, and subsequently purified and isolated using rhizobium medium (YEM media). The bacteria were cultured at 28℃ with agitation at 150 rpm for either 24 hours or 5 to 7 days, depending on the solution utilized for biological indicators. The plant root sample was rinsed with 75% ethanol, the root system's surface was swiftly cauterized, the outer epidermis was removed using sterile distilled water, and the supernatant was collected following grinding. Culture medium Organophosphate solid medium (Monkina Medium): C 6 H 12 O 6 10 g, (NH 4 ) 2 SO 4 0.5 g, KCl 0.3 g, FeSO 4 ▪ 7H 2 O 0.03 g, MgSO 4 ▪ 7H 2 O 0.03 g, CaCO 3 5.0 g, lecithin 0.2 g, distilled water 1000 mL, pH 7.0~7.5. The agar and aqueous media were sterilized in an autoclave at 121℃ for 30 minutes. Rhizobium medium (YEM): C 6 H 14 O 6 10 g, K 2 HPO 4 0.5 g, MgSO 4 0.3 g, NaCl 0.2 g, Yeast 0.4 g, distilled water 1000 mL, pH 6.8. The agar media and aqueous medium were sterilized in an autoclave at 121°C for 30 minutes. Cultivation lines and growth conditions All plants were cultivated at approximately 28°C under a photoperiod of 16 hours of light and 8 hours of darkness. For germination, alfalfa seeds were scarified using sandpaper and subjected to surface sterilization with 1% sodium hypochlorite for 10 minutes. Seedlings were rinsed with sterile water five times and agitated in a constant temperature shaker for twelve hours. The seedlings will next be transferred into 8 cm x 8cm pots packed with 180 g of vermiculite and irrigated with 250 mL of Hoagland Nutrient Solution (full-nitrogen) or Hoagland Nutrient Solution (nitrogen-free) from Hopebio. Two control groups: one with a full-nitrogen solution and the other with a nitrogen-free solution. Three control groups were irrigated with a bacterial solution diluted to 0.08 at OD 660nm after one day of incubation, following a week of growth of the alfalfa. Subsequently, for all groups, after the treatments had been post-inoculated for three weeks, chlorophyll, protein content, nodule count, and nitrogen activity (ARA) were documented. Chlorophyll assay Three weeks post-inoculation, weighed 0.1 g of fresh leaves from each treatment group, cut into pieces and put into a mortar, added a small amount of quartz sand to grind and then added 10 mL of 95% ethanol, and then cleaned and extracted for 36 h in the darkness, and then filtered through filter paper into 25 mL volumetric flasks, and then measured at the wavelengths of 665 nm, 649 nm on spectrophotometer. Ca (mg L-1) =13.95 A 665 -6.88 A 649 ; Cb (mg L -1 ) =24.96 A 649 -7.32 A 665 Chlorophyll (mg g -1 ) =[C*V*100]/G C: chlorophyll content; V: volume; G: weight Protein content assay Three weeks post-inoculation, weighed 0.5 g of plant stems and leaves from each treatment group, ground on ice, added saline, diluted 20 times, centrifuged at 10000 rpm for 5 minutes, and determinates by Bradford's protein concentration assay. BSA as standard curve. (Solarbio Science & Technology Co. Ltd, Beijing, China) Acetylene reduction assay The modulated root from single plants was placed in a 20 mL glass gas chromatography vial. A syringe was used to replace 5 mL air in the vial with 100% acetylene. Samples were incubated at room temperature for 3 hours before GC Detection. Root flavonoid extraction Utilize 10 g of fresh alfalfa Longmu 806 roots, incorporating 40 mL of extracted sterilized distilled water via the ultrasonic extraction method, applying an ultrasonic power of 300 W at 60℃ for 30 minutes. Subsequently, centrifuge at 12000 rpm for 10 minutes at 25℃, collect the supernatant, and perform filtering and sterilization using a 0.45 μm hydrophilic membrane. Flavonoid assay Two days post-inoculation, the control group (non-exogenous bacteria) and the treatment group (added P. aryabhattai bacterial solution diluted to OD 660nm 0.08 after growth stabilization period) were taken and the samples were dried to constant weight, pulverized, sieved and weighed 0.1 g. Add 1 mL, 60% ethanol, extracted by ultrasonic extraction method, ultrasonic power 300 W, 60℃extraction 30 minutes, 12000 rpm, 25℃, centrifugation 10 minutes, take the supernatant, add 60% ethanol fixed to 1 mL. Use flavonoid test kit (Solarbio Science & Technology Co. Ltd, Beijing, China). Nod factor induced extraction and Liquid Chromatography assay P. aryabhattai and S. meliloti were incubated with YEM medium at 28℃, 150 rpm, fermented for 5-7 days, and the root net extract was added to the bacteria solution when OD 660nm 0.8-1.0, and the control was added with sterilized water instead. Continued incubation for 2 days. Four groups: Only S. meliloti purified bacterial liquid was supplemented with non-flavonoid; both S. meliloti and P. aryabhattai purified bacterial liquids were supplemented with non-flavonoid; flavonoids extracted from alfalfa roots, without added bacterial solution, were incorporated into the S. meliloti liquid; flavonoids extracted from alfalfa roots irrigated with P. aryabhattai solution were incorporated into the S. meliloti liquid. 500 mL bacterial solution add 200 mL of n-butanol each, shake, take the upper layer of liquid, store at 4℃ under darkness, evaporate to dryness by rotary evaporator, add 4 mL of 18% acetonitrile, seal and store at 4℃. HPLC analysis was conducted with a C18 reversed-phase column with a flow rate of 1.0 mL min –1 and a Vydac guard column. The detector was set at 214 nm. As a baseline 18% acetonitrile was run through the system for at least 20-30 min prior to injection. The sample was loaded, and isocratic elution was conducted with 18% AcN for 45 min to remove all non-polar light fractions. Thereafter, gradient elution was conducted for 90 min. with 18–82% AcN. The LCO was eluted at 84-86 min of HPLC run time (Compared to the control without the addition of the inducer). Results Strain Phylogenetic tree comparison During the screening of functional bacteria in black soil and alfalfa roots, which is from Northeast China, YJHT21 and LMGL3-1 were isolated. And we found that the introduction of these strains to alfalfa pots resulted in a significant enhancement in the growth of the alfalfa Longmu 806. Similarity between YJHT21 and S. meliloti NBRC 14782 r is 100%. Similarity between LMGL3-1 and P. aryabhattai B8W22 r is 100%. Thus, we were able to identify these two bacterial strains. YJHT21 was identified as P. aryabhattai (Fig. 1 a), LMGL3-1 was identified as S. meliloti (Fig. 1 b). Effects of bacillus interactions on physiological indicators of alfalfa To ascertain whether the dual microbial mixture genuinely promotes plant growth, we assessed their physiological signs. Alfalfa, a perennial herb, attains a height of merely 30–100 cm; hence, chlorophyll synthesis under low light conditions is essential for the plant. Therefore, we focused on determining total chlorophyll and protein, which are essential indicators of the growing condition of alfalfa. Here, we found that the two treatment groups ( S. meliloti + P. aryabhattai and S. meliloti alone) significantly elevated chlorophyll a concentration relative to the two control groups (one receiving full fertilizer and the other devoid of nitrogen fertilizer) (p < 0.0001) (Fig. 2 a). The protein content is markedly increased in the treatment group ( S. meliloti + P. aryabhattai ) relative to the two control groups (p < 0.01). Furthermore, the S. meliloti -only group demonstrated a modest elevation relative to the two control groups (p < 0.05) (Fig. 2 d). Compared to S. meliloti alone, the combination of the two bacteria led to an 18.40% decrease in Chlorophyll an after-bacillus compounding (p < 0.05). The amalgamation of two bacterial strains led to a substantial increase of 76.34% in Chlorophyll b (p < 0.0001) (Fig. 2 b). During that period, we computed and determined that the chlorophyll a/b ratio had markedly decreased. This research demonstrates that the amalgamation of the two bacteria improves alfalfa's capacity and efficacy in absorbing blue-violet light. When employed as fodder or green manure among maize plants, alfalfa frequently displays a reduced height in comparison to surrounding vegetation. Alfalfa exhibits a marked preference for sunlight; hence, enhancing its absorption of blue-violet light substantially promotes its growth. Concurrently, the concentrations of Chlorophyll a + b were markedly increased, signifying that alfalfa efficiently assimilates nitrogen (Fig. 2 c). Our investigation revealed that the application of P. aryabhattai exerted negligible influence on the physiological activities of alfalfa. Thus, we concluded that S. meliloti and P. aryabhattai demonstrate complimentary interactions. So, we hypothesize that the participation of P. aryabhattai in the dual-bacteria combination enhances nitrogen fixation by S. meliloti , and subsequent experiments verified our suspicions. Effects of the addition of P. aryabhattai to alfalfa on the nitrogen fixation of S. meliloti To further confirm the groth-promoting benefits of the dual microbial combination, we proceeded to assess the nitrogen-fixing capability of S. meliloti following the addition of P. aryabhattai . However, the production of Nod factor is contingent upon the specific symbiotic compatibility between alfalfa and rhizobacteria, only those rhizobacteria that are adapted to the alfalfa varieties utilized in this experiment can synthesize Nod factor and consequently stimulate rhizome formation. Luckily, this is evidenced by the tumorous growth observed in alfalfa upon the introduction of S. meliloti . Additionally, it is important to note that under the non-exogenous rhizobacteria environment, alfalfa is unable to develop rhizomes and will not exhibit ARA, and the color of the nodules is notable. Even in the presence of rhizobia, incompatible strains do not induce alfalfa to develop effective nodules, which are characterized by a pink hue. Here, we observed that the control group (no-inoculum) receiving nitrogen initially exhibited robust growth, but thereafter had a significant decline in growth rate, akin to the control groups devoid of nitrogen. Conversely, the plants including S. meliloti exhibit enhanced greenness and vigor, indicating that they have assimilated adequate nutrients (Fig. 3 c). Post-harvest, we compared the nodule counts across several groups, revealing that the group containing S. meliloti successfully induced rhizome formation in alfalfa roots, but the control groups failed to facilitate nodule formation in alfalfa. All treatment groups, except for P. aryabhattai , developed nodules, the majority of which were pink and contained leghemoglobin, facilitating nitrogen fixation (Fig. 3 a). This nitrogen-fixing bacteria effectively supplies nitrogen to alfalfa. In contrast, we observed that the additional S. meliloti plants exhibited significantly improved growth, leading us to conclude that S. meliloti can form symbiotic relationships with specific alfalfa species, resulting in effective nitrogen fixation (Fig. 3 c). To examine if the incorporation of P. aryabhattai with S. meliloti enhances plant growth. We established three groups with exogenous batteries. In comparison to the group S. meliloti + P. aryabhattai and S. meliloti alone, we observed that the combination of the two bacteria resulted in a greater formation of pink nodules (p < 0.01) (Fig. 3 c), and the overall number of nodules also significantly increased. Subsequently, we tested its acetylene reduction activity (ARA). In comparison to the S. meliloti group alone, the addition of P. aryabhattai to S. meliloti resulted in a considerable increase in ethylene production (p < 0.0001) (Fig. 3 b). The combination of two bacteria resulted in a significant enhancement in ARA. Furthermore, P. aryabhattai lacks the capacity for nitrogen fixation; hence, it can be assumed that its incorporation primarily enhances the ARA of S. meliloti , rather than the synergistic effect of dual nitrogen activities. Nodules are generated by the plant root system under nitrogen-deficient conditions, subsequently resulting in the formation of flavonoids that induce Nod factor production. Then, we hypothesize that the inclusion of P. aryabhattai results in the formation of flavonoids, and subsequent experiments verified our suspicions. P. aryabhattai mediates the production of flavonoids in alfalfa To trigger the nodulation process of host plants, S. meliloti produce Nod factors. However, Nod factors cannot be produced only by rhizobia. Flavonoids must be excreted by the alfalfa root system in the absence of nitrogen to stimulate the synthesis of Nod factors by rhizobia. Consequent to the data, we determined that the dual-bacteria combination not only enhances plant growth but also increases ARA levels. We hypothesize that P. aryabhattai influences the secretion of flavonoids by the plant's root system, thus impacting the tumorous potential of S. meliloti . We assessed the flavonoid content of alfalfa root systems under complete fertilization, non-nitrogen fertilization, and P. aryabhattai only. In the absence of non-exogenous bacteria, controls treated with nitrogen fertilizer yielded negligible flavonoid production, whereas controls devoid of nitrogen fertilizer generated some flavonoids (Fig. 4 a). In comparison to the control groups, the flavonoids extracted from alfalfa roots with the addition of P. aryabhattai solution were significantly elevated relative to both control groups (Fig. 4 a). To further evaluate our hypothesis, we have purified and conducted liquid phase analysis of Nod Factor. And based on previous methods of determining Nod factor, we may have detected a peak of Nod factor-like at 84–86 minutes (Lian et al., 2001 ) (Fig. 5c-5f). The result illustrates that the control group exhibited no response, indicating that the lack of flavonoid inducers does not generate Nod factor-like metabolites. In comparison with the two control groups, we observed a duration of 84 to 86 minutes, with no peaks evident in the control plots. While the precise type of Nod factor remained ambiguous and could not be quantified through standard curves, liquid phase analysis revealed that its peak area increases upon the addition of flavonoids extracted from alfalfa roots irrigated with the P. aryabhattai solution (Fig. 4 e, 4 f). Consequently, it is evident that additional metabolites potentially functioning as Nod factors-like can be elicited through flavonoid inducers derived from alfalfa roots. Furthermore, the incorporation of bacillus-infested plant inducers yielded elevated response values. The elevated metabolites, potentially various types of Nod factors, initiate the entire process of tumor growth, resulting in an increased number of nodules and enhanced nitrogen fixation capacity. Discussion The development of bacterial strains as microbial fertilizers for plant application has become a common approach to promote sustainable agriculture and create an environmentally friendly farming ecosystem, thereby replacing chemical fertilizers. This experiment focused on applying a nitrogen-fixing strain of rhizobacteria to alfalfa plants, incorporating a phosphorus-solubilizing bacterium, P. aryabhattai YJHT21, which was isolated from black soil, alongside the rhizobacterium S. meliloti LMGL3-1, sourced from alfalfa samples. This strain of nitrogen-fixing bacterium demonstrated symbiotic compatibility with the alfalfa cultivar Longmu 806, promoting the development of effective nodules that contain leghemoglobin, thereby allowing the plants to thrive without the need for nitrogen fertilizer. The findings of this experiment indicate that the addition of P. aryabhattai YJHT21 significantly improved plant growth and boosted nitrogen activity. The interaction between rhizobacteria and host plants is characterized by the role of flavonoids, which act as essential inducers within the root systems of leguminous plants, triggering the nodulation response. Meanwhile, Nod factors are important metabolites generated by rhizobacteria in reaction to flavonoids. The introduction of P. aryabhattai YJHT21 led to a significant enhancement in flavonoid production in the root systems of alfalfa plants. As a result, the inducer extracts from the quantitatively analyzed root systems led to S. meliloti LMGL3-1 producing a higher amount of Nod factor metabolites. The present experiment revealed that the interactions between the two bacterial species markedly enhanced nitrogen fixation and efficiently replaced nitrogen fertilizer in the cultivation of alfalfa. The secretions produced by P. aryabhattai YJHT21 contributed positively to the soil and the ecosystem, markedly improving and revitalizing degraded land. To examine why a two-bacteria consortium facilitated enhanced growth of alfalfa in the absence of nitrogen fertilizer. We assessed variations in protein content, chlorophyll concentration, ARA, flavonoid synthesis, and Nod factor-like metabolites in alfalfa plants. The protein and chlorophyll content of alfalfa under the dual bacterial complex were dramatically elevated, along with the ARA and nodule count, resulting in enhanced growth of the alfalfa plants and adequate nitrogen availability in their environment. Simultaneously, we discovered that the root system of plants supplemented with P. aryabhattai YJHT21 generated an increased quantity of flavonoids. To reaffirm this finding, we incorporated the gathered flavonoid compounds into the fermented rhizobia sap. Following the extraction of the Nod factor and subsequent HPLC analysis, we observed an increase in the number of peaks corresponding to the Nod factor class. This was a significant factor contributing to the rise in the number of nodules and ARA in the result. Consequently, we deduce that dephosphorylating bacteria can facilitate the synthesis of substantial quantities of flavonoids in the alfalfa root system, thereby enhancing the production of nodulation components by rhizobia for nitrogen fixation. We shall now examine the experimental results alongside recent studies pertaining to strain compounding and Nod factor synthesis. Numerous articles have indicated that strains akin to P. aryabhattai YJHT21, such as strain AB211, exhibit diverse metabolic pathways with a robust central carbohydrate metabolism. This suggests that the bacterium can effectively utilize root sap and other organic substrates as energy sources, demonstrating significant potential as a plant growth promoter (Bhattacharyya et al., 2017 ). And a bacillus like a Bacillus licheniformis strain A2, can enhanced the fresh biomass, total length and root length of groundnut (Goswami et al., 2014 ). And an article indicated that numerous scientists discovered that specific dephosphorylating bacteria could enhance plant development in nutrient-deficient soils derived from desolate lakefront regions. The bacteria were utilized in microcosmic studies of wild plant growth, demonstrating enhanced seed germination and resulting in greater root, shoot, and dry weights of sprouted seedlings compared to the uninoculated control plants after many days. The plant growth-promoting capabilities of the aryabhattai strains are acknowledged as an environmentally sustainable method (Lee et al., 2012 ). In our experiments, the P. aryabhattai YJHT21 strain exhibited a comparable effect, inducing the plant root system to produce increased flavonoids. However, in contrast to the nitrogen-deficient conditions employed, a solitary strain of this bacterium was unable to directly influence seedling root systems and plant growth. We suggest that the ineffectiveness of a single strain in directly influencing plant growth may stem from its classification as an inter-root fungus, specifically adapted to black soil, and its presence in a resource-abundant environment, resulting in no direct impact on alfalfa plants under low nitrogen conditions (Fig. 2 ). S. meliloti is a prevalent rhizobia species associated with alfalfa, with numerous reports indicating its compatibility and symbiotic relationship with alfalfa, resulting in the production of multiple rhizomes (Checcucci et al., 2016 ). S. meliloti is recognized as a symbiotic bacterium species that may establish nitrogen-fixing nodules in the roots of both annual and perennial alfalfa species. Researchers disclosed the comprehensive genome sequence of a strain analogous to the present experiment, S. meliloti strain AK76 (Vladimirova et al., 2022 ). The results indicate that the S. meliloti LMGL3-1 strains examined in this study are compatible with the alfalfa species employed in the present experiment. And there are some researchers identified alfalfa truncatellid nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatellid TFs playing a central role during key steps of the S. meliloti -M. truncatula symbiotic interaction (Baudin et al., 2015 ). This result also provides a theoretical basis for the use of alfalfa Longmu 806 and the S. meliloti LMGL3-1 in our research. Flavonoids are essential signaling molecules in the symbiotic relationship between legumes and their nitrogen-fixing partners, the rhizobia (Glyan'ko, 2015 ). The principal role of flavonoids in this association is to stimulate the transcription of genes responsible for the manufacture of rhizobia signaling molecules known as Nod factors, which are recognized by the plant to facilitate symbiotic root infection (Liu and Murray, 2016 ). Prior studies have investigated the symbiotic association between Bacillus and Mesorhizobium sp. The Ca181 strain displayed an enhancement when co-inoculated with six Bacillus strains, demonstrating a shoot dry weight ratio 1.62 to 1.74 times superior to the Mesorhizobium -inoculated treatment group after 80 days of growth under sterile circumstances (Sivaramaiah et al., 2007 ). There was research showed that co-inoculation of soybean plants with a new PGPR strain LL2012 and the natural symbiont ( Bradyrhizobium japonicum ) altered plant growth parameters and significantly improved nodulation (Masciarelli et al., 2014 ). Also, research found that the Co-existence of Leclercia adecarboxylata strain LSE-1 and Bradyrhizobium sp. Strain LSBR-3 improved the nodule number (Kumawat et al., 2019 ). This result illustrates the effectiveness of employing rhizobacteria to improve crop yield. A recent study has disclosed that plant growth promoting rhizobacteria (PGPR) are ubiquitous soil bacteria residing in plant roots that stimulate plant growth (Fan and Smith, 2021 ). Bacillus spp. are well known rhizosphere residents of many crops and usually show plant growth promoting (PGP) activities (Calvo et al., 2010 ). A Bacillus cereus strain TCU11 which is a PGPR can be exploited as inoculums for improving the phytoremediation efficiency in polluted soils (Bruno et al., 2021 ). Therefore, Specific PGPR strains augment nodulation, nitrogen fixation, and legume growth when inoculated with rhizomes. Instances of PGPR include Azotobacter , Azospirillum , Bacillus , and specifically the P. aryabhattai YJHT21 mentioned in our study. All of which share a segment of a shared microbial habitat at the root-soil interface. The effects of co-inoculation with Rhizobium and PGPR have been noted in many symbiotic and plant growth parameters. The co-inoculation of Rhizobium and Azospirillum increased the number of root hairs and the release of flavonoids by the roots compared to the alone inoculation of Rhizobium . The positive effect of PGPR on the nodulation of Rhizobium -legumes is ascribed to their synthesis of phytohormones (Sibponkrung et al., 2020 ). The conclusions of this article provide similar theoretical support to the findings of the current experiment, suggesting that dual bacterial interactions augment flavonoid production, hence affecting Nod factor production and leading to an increased number of nodules. Our experiment entailed screening the wild-type P. aryabhattai YJHT21 and integrating it with S. meliloti LMGL3-1, which was obtained from alfalfa specimens. These strains are common in the alfalfa cultivation environment. The incorporation of dephosphorylating bacteria from black soil seeks to promote alfalfa growth in dry environments, especially under conditions of low nitrogen availability, thus enhancing the microbial resources and nutrient profile of desolate land. This study seeks to promote sustainable agriculture by utilizing microbial compound bacterial fertilizers instead of traditional chemical nitrogen fertilizers, thereby reducing damage to crops and the soil ecosystem, while providing technical assistance for enhanced land use and the exploitation of unproductive resources. An examination of the existing literature indicates a scarcity of research on the combined study of P. aryabhattai YJHT21 and S. meliloti LMGL3-1, with enquiries into the related mechanisms being constrained. P. aryabhattai YJHT21 and S. meliloti LMGL3-1 can be efficiently integrated for use in alfalfa plants. This creates a fundamental experimental framework for investigating alfalfa plants alongside Bacillus bacteria and rhizobia, reinforcing the established PGPR-plant theoretical model pertinent to applied research. The integration of P. aryabhattai YJHT21 led to elevated flavonoid levels in the plants, hence affecting the synthesis of nod components. Furthermore, the different modifications of Nod fators that are differentially induced are still worthy of further exploration and the signaling mechanisms of their interaction with flavonoids remain unknown. Declarations Acknowledgments This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28030201), the National Natural Science Foundation of China (92351302, 91851102, 32070034, 32270056 and 32300084), and the National Key R&D Program of China (2020YFA0906800 and 2022YFC2105300). Data availability The data used to support the fndings of this study are available from the corresponding author upon request. Funding This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28030201), the National Natural Science Foundation of China (92351302, 91851102, 32070034, 32270056 and 32300084), and the National Key R&D Program of China (2020YFA0906800 and 2022YFC2105300). Author information Authors and Affiliations College of Agriculture, Shanxi Agricultural University, Jinzhong, 030801, China Rui Liu, Chang Li, Yunjun Zhang, Chunli Liu, Jinai Xue State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China Yanning Zheng Contributions R.L. (Rui Liu), J.X. (Jinai Xue), Y.Z. (Yanning Zheng) designed research. R.L. and C.L. (Chang Li) screened strains. R.L. performed the studies of nitrogen fixation; flavonoids and nod factor-like, R.L., C.L. (Chang Li), Y.Z. (Yunjun Zhang) and C.L. (Chunli Liu) carried out the pot experiment. R.L. (Yan Zeng), Y.Z. (Yanning Zheng) analyzed data. R.L. (Rui Liu), J.X., Y.Z. (Yanning Zheng) wrote the paper. All authors contributed to the revision of the manuscript. Corresponding authors Correspondence to Yanning Zheng and Jinai Xue Ethics declarations Conflict of interest The authors declare no conflicts of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Ahmed M, Rauf M, Mukhtar Z, Saeed NA (2017) Excessive use of nitrogenous fertilizers: an unawareness causing serious threats to environment and human health. Environ Sci Pollut Res Int 24(35):26983–26987. https://doi.org/10.1007/s11356-017-0589-7 Bardi L, E M (2012) Drought and nutritional stresses in plant: Alleviating role of rhizospheric microorganisms. Nova Science, New York Baruah M, Gogoi M, Chandra Boro R, Barooah M (2024) Priestia aryabhattai MBM3-Mediated enhancement of sulphur metabolism in Brassica campestris. Curr Microbiol 81(10):316. https://doi.org/10.1007/s00284-024-03844-0 Baudin M, Laloum T, Lepage A, Rípodas C, Ariel F, Frances L, Crespi M, Gamas P, Blanco FA, Zanetti ME, de Carvalho-Niebel F, Niebel A (2015) A phylogenetically conserved group of nuclear factor-Y transcription factors interact to control nodulation in legumes. Plant Physiol 169(4):2761–2773. https://doi.org/10.1104/pp.15.01144 Bhattacharyya C, Bakshi U, Mallick I, Mukherji S, Bera B, Ghosh A (2017) Genome-Guided Insights into the plant growth promotion capabilities of the physiologically versatile Bacillus aryabhattai strain AB211. Front Microbiol 8:411. https://doi.org/10.3389/fmicb.2017.00411 Bruno LB, Anbuganesan V, Karthik C, Tripti, Kumar A, Banu JR, Freitas H, Rajkumar M (2021) Enhanced phytoextraction of multi-metal contaminated soils under increased atmospheric temperature by bioaugmentation with plant growth promoting Bacillus cereus. J Environ Manage 289:112553. https://doi.org/10.1016/j.jenvman.2021.112553 Calvo P, Ormeño-Orrillo E, Martínez-Romero E, Zúñiga D (2010) Characterization of Bacillus isolates of potato rhizosphere from andean soils of Peru and their potential PGPR characteristics. Braz J Microbiol 41(4):899–906. https://doi.org/10.1590/s1517-83822010000400008 Checcucci A, Azzarello E, Bazzicalupo M, Galardini M, Lagomarsino A, Mancuso S, Marti L, Marzano MC, Mocali S, Squartini A, Zanardo M, Mengoni A (2016) Mixed nodule infection in Sinorhizobium meliloti-Medicago sativa symbiosis suggest the presence of cheating behavior. Front Plant Sci 7:835. https://doi.org/10.3389/fpls.2016.00835 Chen W, Wang J, Huang D, Cheng W, Shao Z, Cai M, Zheng L, Yu Z, Zhang J (2021) Volatile organic compounds from Bacillus aryabhattai MCCC 1K02966 with multiple modes against Meloidogyne incognita. Molecules 27(1):103. https://doi.org/10.3390/molecules27010103 Fan D, Smith DL (2021) Characterization of selected Plant Growth-Promoting Rhizobacteria and their Non-Host growth promotion effects. Microbiol Spectr 9(1):e0027921. https://doi.org/10.1128/Spectrum.00279-21 Glyan'ko AK (2015) Signaling systems of Rhizobia (Rhizobiaceae) and leguminous plants (Fabaceae) upon the formation of a Legume-Rhizobium symbiosis (Review). Prikl Biokhim Mikrobiol 51(5):453–464 Goswami D, Dhandhukia P, Patel P, Thakker JN (2014) Screening of PGPR from saline desert of Kutch: Growth promotion in Arachis hypogea by Bacillus licheniformis A2. Microbiol Res 169(1):66–75. https://doi.org/10.1016/j.micres.2013.07.004 Jiménez-Gómez A, García-Estévez I, Escribano-Bailón MT, García-Fraile P, Rivas R (2021) Bacterial fertilizers based on Rhizobium laguerreae and Bacillus halotolerans enhance Cichorium endivia L. Phenolic compound and mineral contents and plant development. Foods 10(2):424. https://doi.org/10.3390/foods10020424 Kumawat KC, Sharma P, Singh I, Sirari A, Gill BS (2019) Co-existence of Leclercia adecarboxylata (LSE-1) and Bradyrhizobium sp. (LSBR-3) in nodule niche for multifaceted effects and profitability in soybean production. World J Microbiol Biotechnol 35(11):172. https://doi.org/10.1007/s11274-019-2752-4 Lee S, Ka JO, Song HG (2012) Growth promotion of xanthium italicum by application of rhizobacterial isolates of Bacillus aryabhattai in microcosm soil. J Microbiol 50(1):45–49. https://doi.org/10.1007/s12275-012-1415-z Lian B, Prithiviraj B, Souleimanov A, Smith DL (2001) Evidence for the production of chemical compounds analogous to nod factor by the silicate bacterium Bacillus circulans GY92. Microbiol Res 156(3):289–292. https://doi.org/https://doi.org/10.1078/0944-5013-00107 Liu CW, Murray JD (2016) The role of flavonoids in nodulation Host-Range specificity: An update. Plants (Basel) 5(3):33. https://doi.org/10.3390/plants5030033 Lourenço KS, Rossetto R, Vitti AC, Montezano ZF, Soares JR, Sousa RM, do Carmo JB, Kuramae EE, Cantarella H (2019) Strategies to mitigate the nitrous oxide emissions from nitrogen fertilizer applied with organic fertilizers in sugarcane. Sci Total Environ 650(Pt 1):1476–1486. https://doi.org/10.1016/j.scitotenv.2018.09.037 Malusá E, Vassilev N (2014) A contribution to set a legal framework for biofertilisers. Appl Microbiol Biotechnol 98(15):6599–6607. https://doi.org/10.1007/s00253-014-5828-y Masciarelli O, Llanes A, Luna V (2014) A new PGPR co-inoculated with Bradyrhizobium japonicum enhances soybean nodulation. Microbiol Res 169(7–8):609–615. https://doi.org/10.1016/j.micres.2013.10.001 Mazid M, Khan TA (2014) Future of bio-fertilizers in indian agriculture: An overview. J Agr Food Res 3(3):10–23 Miljaković D, Marinković J, Tamindžić G, Đorđević V, Tintor B, Milošević D, Ignjatov M, Nikolić Z (2022) Bio-priming of soybean with bradyrhizobium japonicum and bacillus megaterium: Strategy to improve seed germination and the initial seedling growth. Plants (Basel) 11(15):1927. https://doi.org/10.3390/plants11151927 Shahid M, Zeyad MT, Syed A, Singh UB, Mohamed A, Bahkali AH, Elgorban AM, Pichtel J (2022) Stress-Tolerant endophytic isolate Priestia aryabhattai BPR-9 modulates Physio-Biochemical mechanisms in wheat (Triticum aestivum L.) for enhanced salt tolerance. Int J Environ Res Public Health 19(17):10883. https://doi.org/10.3390/ijerph191710883 Sibponkrung S, Kondo T, Tanaka K, Tittabutr P, Boonkerd N, Yoshida KI, Teaumroong N (2020) Co-Inoculation of Bacillus velezensis strain S141 and Bradyrhizobium strains promotes nodule growth and nitrogen fixation. Microorganisms 8(5):678. https://doi.org/10.3390/microorganisms8050678 Sivaramaiah N, Malik DK, Sindhu SS (2007) Improvement in symbiotic efficiency of chickpea (Cicer arietinum) by coinoculation of Bacillus strains with Mesorhizobium sp. Cicer. Indian J Microbiol 47(1):51–56. https://doi.org/10.1007/s12088-007-0010-1 Sun B, Bai Z, Bao L, Xue L, Zhang S, Wei Y, Zhang Z, Zhuang G, Zhuang X (2020) Bacillus subtilis biofertilizer mitigating agricultural ammonia emission and shifting soil nitrogen cycling microbiomes. Environ Int 144:105989. https://doi.org/10.1016/j.envint.2020.105989 Tan GY, Tan WK (1986) Interaction between alfalfa cultivars and Rhizobium strains for nitrogen fixation. Theor Appl Genet 71(5):724–729. https://doi.org/10.1007/bf00263270 Terpolilli JJ, Hood GA, Poole PS (2012) What determines the efficiency of N(2)-fixing Rhizobium-legume symbioses? Adv Microb Physiol 60:325–389. https://doi.org/10.1016/b978-0-12-398264-3.00005-x Vladimirova ME, Muntyan VS, Afonin AM, Muntyan AN, Baturina OA, Dzuybenko EA, Saksaganskaya AS, Simarov BV, Roumiantseva ML, Kabilov MR (2022) Complete genome of Sinorhizobium meliloti AK76, a symbiont of wild diploid Medicago lupulina from the Mugodgary Mountain region. Microbiol Resour Announc 11(3):e0108821. https://doi.org/10.1128/mra.01088-21 Wang D, Yang S, Tang F, Zhu H (2012) Symbiosis specificity in the legume: Rhizobial mutualism. Cell Microbiol 14(3):334–342. https://doi.org/10.1111/j.1462-5822.2011.01736.x Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 May, 2025 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 09 Mar, 2025 Reviews received at journal 30 Jan, 2025 Reviews received at journal 18 Jan, 2025 Reviewers agreed at journal 15 Jan, 2025 Reviewers agreed at journal 14 Jan, 2025 Reviewers agreed at journal 13 Jan, 2025 Reviewers invited by journal 28 Dec, 2024 Editor assigned by journal 20 Dec, 2024 Submission checks completed at journal 20 Dec, 2024 First submitted to journal 17 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5665466","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392955128,"identity":"fec65a75-9a15-4add-9539-ea8ebcc7da84","order_by":0,"name":"Rui Liu","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Liu","suffix":""},{"id":392955131,"identity":"d8dfe472-ca95-401c-a0c0-bf39d6aa6169","order_by":1,"name":"Chang Li","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Li","suffix":""},{"id":392955134,"identity":"56509ced-0e4e-4899-b319-fe420490d779","order_by":2,"name":"Yunjun Zhang","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yunjun","middleName":"","lastName":"Zhang","suffix":""},{"id":392955135,"identity":"c0050a8e-55e4-485a-beb7-81d99a584871","order_by":3,"name":"Chunli Liu","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chunli","middleName":"","lastName":"Liu","suffix":""},{"id":392955136,"identity":"15c48df0-7636-4f6a-b7b2-f539d311ace7","order_by":4,"name":"Yanning Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYDACCQglx0ayFmPStSQ2EK1DfnbzsYdf2+6k94kdYPzwg8Euj6AWxjnH0o1l257ltkknMEv2MCQXE9TCLJFjJi3ZdhikhUGageEAYReySeR/A2lJZwPa8psoLTwSOWySH9sOJwC1sBFni4REmpk0w7nDhm3SiW2WPQbJhLXIz0h+Jvmj7LC8/Ozkwzd+VNgRF9rMvOB4ZAQqNiBGPUjtjz9EqhwFo2AUjIKRCQBG8jXhvOFs+QAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yanning","middleName":"","lastName":"Zheng","suffix":""},{"id":392955137,"identity":"ed09efde-019c-4fbe-ad3c-1375e27cb23b","order_by":5,"name":"Jinai Xue","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jinai","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2024-12-18 02:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5665466/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5665466/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-025-04394-8","type":"published","date":"2025-05-26T15:57:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72209062,"identity":"93099331-9d37-4b09-b999-e0a3a964e35b","added_by":"auto","created_at":"2024-12-23 17:22:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115214,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree. \u003cstrong\u003ea\u003c/strong\u003e Phylogenetic tree of Rhizobium bacteria based on 16S rDNA sequence. The red triangle: \u003cem\u003eS. meliloti\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e Phylogenetic tree of Bacillaceae bacteria based on 16S rDNA sequence. The red triangle: \u003cem\u003eP. aryabhattai.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5665466/v1/448fa6cdf62e9dca095b5959.png"},{"id":72209066,"identity":"c481fc5e-a34f-4433-ab59-55096bc3c33e","added_by":"auto","created_at":"2024-12-23 17:22:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96918,"visible":true,"origin":"","legend":"\u003cp\u003eAlfalfa physiological index.\u003cstrong\u003e a-d\u003c/strong\u003e No inoculum represents the treatment group without exogenous bacteria. N represents Nitrogen fertilizer. \u003cem\u003eS. meliloti\u003c/em\u003e represent \u003cem\u003eS. meliloti LMGL3-1\u003c/em\u003e, and \u003cem\u003eP. aryabhattai\u003c/em\u003erepresent\u003cem\u003e P. aryabhattai YJHT21\u003c/em\u003e. Under the non-nitrogen condition, experimental groups added \u003cem\u003eS. meliloti \u003c/em\u003e+ \u003cem\u003eP. aryabhattai\u003c/em\u003e、\u003cem\u003eS. meliloti\u003c/em\u003e only and \u003cem\u003eP. aryabhattai \u003c/em\u003eonly respectively. \u003cstrong\u003ea-c\u003c/strong\u003e The experimental groups (except the one which add \u003cem\u003eP. aryabhattai\u003c/em\u003e) shows that the high Chlorophy content under the non-nitrogen condition. \u003cstrong\u003ea \u003c/strong\u003eChlorophyII a. \u003cstrong\u003eb \u003c/strong\u003eChlorophyII b. \u003cem\u003eS. meliloti\u003c/em\u003e and \u003cem\u003eP. aryabhattai\u003c/em\u003e apply simultaneously makes the ChlorophyII b increased significantly when it compared to other columns.\u003cstrong\u003e c \u003c/strong\u003eChlorophyII a+b. The experiments groups (except \u003cem\u003eP. aryabhattai\u003c/em\u003e) highly increased when they compare to the control groups. Nitrogen highly absorption was revealed. \u003cstrong\u003ed\u003c/strong\u003e Protein content per plant.\u003cstrong\u003e \u003c/strong\u003eNitrogen absorption was revealed.\u003c/p\u003e\n\u003cp\u003e(*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant.)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5665466/v1/7a755d00a0d6fde756af240d.png"},{"id":72209065,"identity":"05439ff1-efb4-4383-98cc-af4ffa391435","added_by":"auto","created_at":"2024-12-23 17:22:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":407057,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen fixation.\u003cstrong\u003e a-c \u003c/strong\u003eThe treatment groups, excluding the \u003cem\u003eP. aryabhattai\u003c/em\u003e only group, received an injection of 1mL of 0.08 OD620mm \u003cem\u003eS. meliloti\u003c/em\u003e combined with \u003cem\u003eP. aryabhattai\u003c/em\u003e bacterial liquid in total (500μl from each bacterial liquid) or solely \u003cem\u003eS. meliloti \u003c/em\u003ebacterial liquid. The \u003cem\u003eP. aryabhattai\u003c/em\u003e only group was injected with 1mL 0.08 OD620mm \u003cem\u003eP. aryabhattai \u003c/em\u003ebacterial liquid. Control groups were injected with 1mL sterilized distilled water. \u003cstrong\u003ea\u003c/strong\u003e Nodule number. The functionally pink nodule number with pink color and total nodule number with gray color. \u003cstrong\u003eb \u003c/strong\u003eARA per plant. The \u003cem\u003eS. meliloti \u003c/em\u003e+ \u003cem\u003eP. aryabhattai \u003c/em\u003egroup significantly increased as compare with the others. \u003cem\u003eP. aryabhattai\u003c/em\u003e’s addition was revealed to have a pro-nitrogen activity effect. \u003cstrong\u003ec \u003c/strong\u003ePlants appearance and nodules appearance. Functionally pink nodules at the front of the alfalfa roots. \u003cstrong\u003ed\u003c/strong\u003e Mechanistic model of nodule formation. Under low nitrogen conditions, alfalfa was infested with adapted rhizobia, alfalfa roots release flavovoid (the part of them affected by \u003cem\u003eP. aryabhattai\u003c/em\u003e), then, the \u003cem\u003eS. meliloti \u003c/em\u003esense the flavovoid to produce Nod factor. After that root hairs sense the Nod factor and rapidly encapsulate the \u003cem\u003eS. meliloti\u003c/em\u003e, which in turn form a rhizomacurled to form nodule. Conversion of N\u003csub\u003e2\u003c/sub\u003e to ammonium, then release NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e to the soil.\u003c/p\u003e\n\u003cp\u003e(*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant.)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5665466/v1/cac138f13b1456455367548d.png"},{"id":72209063,"identity":"c0495b3e-5707-4a58-9c88-f12bcfbfe2bd","added_by":"auto","created_at":"2024-12-23 17:22:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99505,"visible":true,"origin":"","legend":"\u003cp\u003eFlavonoids and Nod factors-like. \u003cstrong\u003ea \u003c/strong\u003e0.1g dry alfalfa plants’ flavonoid. Non represents non-exogenous bacterial control groups, N represents nitrogen fertilizer. \u003cem\u003eP. aryabhattai \u003c/em\u003erepresents a sample of the plant to which \u003cem\u003eP. aryabhattai \u003c/em\u003ewas added. And the result shows the content of the flavonoid was significantly increased than control groups.\u003cstrong\u003e b\u003c/strong\u003e Chemical modelling of flavonoids. \u003cstrong\u003ec-f\u003c/strong\u003e HPLC of Nod factor. Sample were all the purified bacterial liquid after rotary evaporation. \u003cstrong\u003ec\u003c/strong\u003e Non-flavonoid was added, only \u003cem\u003eS. meliloti\u003c/em\u003e purified bacterial liquid. \u003cstrong\u003ed\u003c/strong\u003e Non-flavonoid was added, only \u003cem\u003eS. meliloti\u003c/em\u003e and \u003cem\u003eP. aryabhattai \u003c/em\u003epurified bacterial liquid. \u003cstrong\u003ee\u003c/strong\u003e Flavonoids extracted from alfalfa roots without added bacterial solution was added in the \u003cem\u003eS. meliloti \u003c/em\u003eliquid. The hollow triangles with peak area represent effective Nod Factor-like peaks. \u003cstrong\u003ef\u003c/strong\u003e Flavonoids extracted from alfalfa roots which is irrigated \u003cem\u003eP. aryabhattai\u003c/em\u003e solution was added in the \u003cem\u003eS. meliloti \u003c/em\u003eliquid. The hollow triangles with peak area represent effect Nod Factor-like peaks. Compare with e, the area of peaks is significantly increased.\u003c/p\u003e\n\u003cp\u003e(*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5665466/v1/e2397799e989eec07009f78f.png"},{"id":83782908,"identity":"4388cbd7-5bd3-4cf6-bb4b-626d0d270fa7","added_by":"auto","created_at":"2025-06-02 16:08:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1638767,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5665466/v1/b7b781e8-8a57-4e3a-9b83-62d35cb8a100.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced biological nitrogen fixation in alfalfa through the synergistic interactions between Sinorhizobium meliloti and Priestia aryabhattai","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe use of nitrogen fertilizers has resulted in tremendous growth in agricultural production. However, decades of overuse leading to soil compaction, acidification, reduction of organic matter, and exacerbation of the greenhouse effect are significant environmental concerns (Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The application of mineral nitrogen (N) fertilizers to crops may increase greenhouse gas emissions (Louren\u0026ccedil;o et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, it has also precipitated several detrimental impacts on human health (Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Bio-fertilizers, which consist of beneficial bacteria associated with plant roots, enhance plant growth by augmenting the nutritional availability for the host plant when given to seeds, plants, or soil (Mazid and Khan, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The use of bio-fertilizers is considered an effective approach to enhancing soil quality and fostering agricultural sustainability (Bardi and E, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Malus\u0026aacute; and Vassilev, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous study was conducted to assess the efficacy of the biofertilizer \u003cem\u003eB. subtilis\u003c/em\u003e in mitigating NH\u003csub\u003e3\u003c/sub\u003e volatilization (Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). And another study showed that the bacterial fertilizer including \u003cem\u003eRhizobium americanum\u003c/em\u003e and \u003cem\u003eBacillus laguerreae\u003c/em\u003e was discovered to improve the plant development (Jim\u0026eacute;nez-G\u0026oacute;mez et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Legumes can establish a root-knot symbiosis with nitrogen-fixing soil bacteria called rhizobia. This inter-association is extremely specialized, with each rhizobia species or strain interacting only with a certain type of legumes, and vice versa (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Rhizobia must compete to infect legume roots and initiate a signal transduction process with the host plant, ultimately resulting in nodule development (Terpolilli et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Previous studies have identified interactions between alfalfa cultivars and \u003cem\u003eRhizobium meliloti\u003c/em\u003e strains on acetylene reduction rates, plant height and shoot, root and whole plant dry weight (Tan and Tan, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Therefore, it\u0026rsquo;s important to find a match bacterium for the alfalfa.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. meliloti\u003c/em\u003e is a rhizobacterium frequently associated with alfalfa, and the specific strain identified in the alfalfa sample is \u003cem\u003eS. meliloti\u003c/em\u003e. This species exhibits significant genetic and phenotypic variation in natural populations, especially with the symbiotic enhancement of plant development. \u003cem\u003eS. meliloti\u003c/em\u003e is internationally dispersed and can be found in various soil types, either as a free-living organism or as a symbiotic partner of leguminous plants, which it prompts to produce nodules. The \u003cem\u003eP. aryabhattai\u003c/em\u003e strain of Bacillus spp. was isolated from the black soil of northeastern China. It is a soil-dwelling rhizobacterium distinguished by spherical, somewhat yellowish colonies. The colony's exterior is flat and moist, while the interior is gelatinous, measuring approximately 2.0 to 5.0 mm in diameter. It has a positive Gramme reaction, shows motility, and has the capacity to create endospores. It is a strain of phosphate-solubilizing bacterium demonstrating phosphate-solubilizing activity, with a capacity of 19.83 mg/L. A relevant study has demonstrated that the Bacillus spp. strain comparable to the \u003cem\u003eP. aryabhattai\u003c/em\u003e analyzed in our research can enhance Sulphur metabolism in oilseed rape (Baruah et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, there are analogous \u003cem\u003eAryabhattai\u003c/em\u003e species; \u003cem\u003eBacillus\u003c/em\u003e sp. MCCC 1K02966. It has been identified as a promising biological control agent (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). And \u003cem\u003eP. aryabhattai\u003c/em\u003e has also proven to be an exceptionally excellent multi-stress tolerant crop growth promoter (Shahid et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). And the similar bacteria strain \u003cem\u003eBacillus aryabhattai\u003c/em\u003e AB211 is a Gram-positive bacterium that can enhance plant development (Bhattacharyya et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA study was conducted to evaluate the impact of PGPR on nodulation and N2 fixation efficiency in soybean through co-inoculation with \u003cem\u003eBradyrhizobium diazoefficiens\u003c/em\u003e USDA110. The inoculation of \u003cem\u003eBacillus velezensis\u003c/em\u003e S141 with USDA110 in soybean resulted in enhanced nodulation and nitrogen fixation efficiency, evidenced by the development of larger nodules (Sibponkrung et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another study was conducted to evaluate the promotional effects of a commercial strain of \u003cem\u003eRhizobium japonicum\u003c/em\u003e and a novel isolate of \u003cem\u003eBacillus megaterium\u003c/em\u003e, both individually and in combination, during the biological initiation of seeds (Miljaković et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The simultaneous application of \u003cem\u003eS. meloloti\u003c/em\u003e LMGL3-1 and \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 produced analogous outcomes in the study. Despite comprehensive national and worldwide studies on Rhizobium, research on Bacillus remain limited. Previous studies suggest that the simultaneous application of Rhizobium and Bacillus can improve plant growth. Further investigation is necessary due to the scarcity of literature concerning the interaction between these two bacteria. We ought to replace nitrogen fertilizers with microbial fertilizers to improve soil ecology and attain sustainable agriculture.\u003c/p\u003e \u003cp\u003eHere we found that under nitrogen-free conditions, \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1 and \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 combination improved alfalfa plant growth compared to single strains, significantly increased chlorophyll levels, increased the number of nodules, and enhanced nitrogen fixation.P. aryabhattai enhances root flavonoid production, subsequently inducing S. meliloti to generate increased nodulation factor, resulting in improved nitrogen fixation in alfalfa Longmu806. The \u003cem\u003ePrietia aryabhattai\u003c/em\u003e YJHT21 strain, isolated from black soil, and \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1 enhance soil microbial diversity, promote plant development, improve nitrogen absorption and utilization, raise arable land efficiency, and protect the soil environment, hence safeguarding human health. This strategy supports sustainability in agriculture and development.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial selected and growth condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. aryabhattai\u0026nbsp;\u003c/em\u003ewas extracted from a black soil sample in northeastern China and subsequently purified and isolated using an organophosphorus solid medium (Monkina Medium). The bacteria were cultured at 30℃ with a rotation speed of 200 rpm for 24 hours. In the context of co-culture, the rhizobium medium (YEM media) was employed for the subsequent culture phase, incubated at 28℃ with 150 rpm for 5-7 days for biological indicators.\u003c/p\u003e\n\u003cp\u003eA soil sample was collected from the 5-20 cm topsoil of farmland in Qiqihar, Heilongjiang, China. The soil was naturally air-dried, with plant debris eliminated, and passed through a 2 mm sieve. Subsequently, combine the soil with saline (1:10) for 6 hours, allow it to equilibrate, and then extract the supernatant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. meliloti\u0026nbsp;\u003c/em\u003ewas extracted from the roots of alfalfa Longmu 806 in Hulunbeier, Neimenggu, China, and subsequently purified and isolated using rhizobium medium (YEM media). The bacteria were cultured at 28℃ with agitation at 150 rpm for either 24 hours or 5 to 7 days, depending on the solution utilized for biological indicators.\u003c/p\u003e\n\u003cp\u003eThe plant root sample was rinsed with 75% ethanol, the root system\u0026apos;s surface was swiftly cauterized, the outer epidermis was removed using sterile distilled water, and the supernatant was collected following grinding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrganophosphate solid medium (Monkina Medium): C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e 10 g, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 0.5 g, KCl 0.3 g, FeSO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e▪ 7H\u003csub\u003e2\u003c/sub\u003eO 0.03 g, MgSO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e▪ 7H\u003csub\u003e2\u003c/sub\u003eO 0.03 g, CaCO\u003csub\u003e3\u003c/sub\u003e 5.0 g, lecithin 0.2 g, distilled water 1000 mL, pH 7.0~7.5. The agar and aqueous media were sterilized in an autoclave at 121℃ for 30 minutes.\u003c/p\u003e\n\u003cp\u003eRhizobium medium (YEM): C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e 10 g, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.5 g, MgSO\u003csub\u003e4\u003c/sub\u003e 0.3 g, NaCl 0.2 g, Yeast 0.4 g, distilled water 1000 mL, pH 6.8. The agar media and aqueous medium were sterilized in an autoclave at 121\u0026deg;C for 30 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultivation lines and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll plants were cultivated at approximately 28\u0026deg;C under a photoperiod of 16 hours of light and 8 hours of darkness. For germination, alfalfa seeds were scarified using sandpaper and subjected to surface sterilization with 1% sodium hypochlorite for 10 minutes. Seedlings were rinsed with sterile water five times and agitated in a constant temperature shaker for twelve hours. The seedlings will next be transferred into 8 cm x 8cm pots packed with 180 g of vermiculite and irrigated with 250 mL of Hoagland Nutrient Solution (full-nitrogen) or Hoagland Nutrient Solution (nitrogen-free) from Hopebio. Two control groups: one with a full-nitrogen solution and the other with a nitrogen-free solution. Three control groups were irrigated with a bacterial solution diluted to 0.08 at OD\u003csub\u003e660nm\u003c/sub\u003e after one day of incubation, following a week of growth of the alfalfa. Subsequently, for all groups, after the treatments had been post-inoculated for three weeks, chlorophyll, protein content, nodule count, and nitrogen activity (ARA) were documented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChlorophyll assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree weeks post-inoculation, weighed 0.1 g of fresh leaves from each treatment group, cut into pieces and put into a mortar, added a small amount of quartz sand to grind and then added 10 mL of 95% ethanol, and then cleaned and extracted for 36 h in the darkness, and then filtered through filter paper into 25 mL volumetric flasks, and then measured at the wavelengths of 665 nm, 649 nm on spectrophotometer.\u003c/p\u003e\n\u003cp\u003eCa (mg L-1) =13.95 A\u003csub\u003e665\u003c/sub\u003e-6.88 A\u003csub\u003e649\u003c/sub\u003e; Cb (mg L\u003csup\u003e-1\u003c/sup\u003e) =24.96 A\u003csub\u003e649\u003c/sub\u003e-7.32 A\u003csub\u003e665\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll (mg g\u003csup\u003e-1\u003c/sup\u003e) =[C*V*100]/G\u003c/p\u003e\n\u003cp\u003eC: chlorophyll content; V: volume; G: weight\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein content assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree weeks post-inoculation, weighed 0.5 g of plant stems and leaves from each treatment group, ground on ice, added saline, diluted 20 times, centrifuged at 10000 rpm for 5 minutes, and determinates by Bradford\u0026apos;s protein concentration assay. BSA as standard curve. (Solarbio Science \u0026amp; Technology Co. Ltd, Beijing, China)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcetylene reduction assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe modulated root from single plants was placed in a 20 mL glass gas chromatography vial. A syringe was used to replace 5 mL air in the vial with 100% acetylene. Samples were incubated at room temperature for 3 hours before GC Detection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRoot flavonoid extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUtilize 10 g of fresh alfalfa Longmu 806 roots, incorporating 40 mL of extracted sterilized distilled water via the ultrasonic extraction method, applying an ultrasonic power of 300 W at 60℃\u0026nbsp;for 30 minutes. Subsequently, centrifuge at 12000 rpm for 10 minutes at 25℃, collect the supernatant, and perform filtering and sterilization using a 0.45 \u0026mu;m hydrophilic membrane.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlavonoid assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo days post-inoculation, the control group (non-exogenous bacteria) and the treatment group (added \u003cem\u003eP. aryabhattai\u0026nbsp;\u003c/em\u003ebacterial solution diluted to OD\u003csub\u003e660nm\u003c/sub\u003e 0.08 after growth stabilization period) were taken and the samples were dried to constant weight, pulverized, sieved and weighed 0.1 g. Add 1 mL, 60% ethanol, extracted by ultrasonic extraction method, ultrasonic power 300 W, 60℃extraction 30 minutes, 12000 rpm, 25℃, centrifugation 10 minutes, take the supernatant, add 60% ethanol fixed to 1 mL. Use flavonoid test kit (Solarbio Science \u0026amp; Technology Co. Ltd, Beijing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNod factor induced extraction and Liquid Chromatography assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. aryabhattai\u003c/em\u003e and \u003cem\u003eS. meliloti\u003c/em\u003e were incubated with YEM medium at 28℃, 150 rpm, fermented for 5-7 days, and the root net extract was added to the bacteria solution when OD\u003csub\u003e660nm\u003c/sub\u003e 0.8-1.0, and the control was added with sterilized water instead. Continued incubation for 2 days. Four groups: Only\u0026nbsp;\u003cem\u003eS. meliloti\u003c/em\u003e purified bacterial liquid was supplemented with non-flavonoid; both\u0026nbsp;\u003cem\u003eS. meliloti\u003c/em\u003e and\u0026nbsp;\u003cem\u003eP. aryabhattai\u003c/em\u003e purified bacterial liquids were supplemented with non-flavonoid; flavonoids extracted from alfalfa roots, without added bacterial solution, were incorporated into the\u0026nbsp;\u003cem\u003eS. meliloti\u003c/em\u003e liquid; flavonoids extracted from alfalfa roots irrigated with\u0026nbsp;\u003cem\u003eP. aryabhattai\u003c/em\u003e solution were incorporated into the\u0026nbsp;\u003cem\u003eS. meliloti\u003c/em\u003e liquid. 500 mL bacterial solution add 200 mL of n-butanol each, shake, take the upper layer of liquid, store at 4℃ under darkness, evaporate to dryness by rotary evaporator, add 4 mL of 18% acetonitrile, seal and store at 4℃. HPLC analysis was conducted with a C18 reversed-phase column with a flow rate of 1.0 mL min\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and a Vydac guard column. The detector was set at 214 nm. As a baseline 18% acetonitrile was run through the system for at least 20-30 min prior to injection. The sample was loaded, and isocratic elution was conducted with 18% AcN for 45 min to remove all non-polar light fractions. Thereafter, gradient elution was conducted for 90 min. with 18\u0026ndash;82% AcN. The LCO was eluted at 84-86 min of HPLC run time (Compared to the control without the addition of the inducer).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStrain Phylogenetic tree comparison\u003c/h2\u003e \u003cp\u003eDuring the screening of functional bacteria in black soil and alfalfa roots, which is from Northeast China, YJHT21 and LMGL3-1 were isolated. And we found that the introduction of these strains to alfalfa pots resulted in a significant enhancement in the growth of the alfalfa Longmu 806. Similarity between YJHT21 and \u003cem\u003eS. meliloti\u003c/em\u003e NBRC 14782\u003csup\u003er\u003c/sup\u003e is 100%. Similarity between LMGL3-1 and \u003cem\u003eP. aryabhattai\u003c/em\u003e B8W22\u003csup\u003er\u003c/sup\u003e is 100%. Thus, we were able to identify these two bacterial strains. YJHT21 was identified as \u003cem\u003eP. aryabhattai\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), LMGL3-1 was identified as \u003cem\u003eS. meliloti\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of bacillus interactions on physiological indicators of alfalfa\u003c/h2\u003e \u003cp\u003eTo ascertain whether the dual microbial mixture genuinely promotes plant growth, we assessed their physiological signs. Alfalfa, a perennial herb, attains a height of merely 30\u0026ndash;100 cm; hence, chlorophyll synthesis under low light conditions is essential for the plant. Therefore, we focused on determining total chlorophyll and protein, which are essential indicators of the growing condition of alfalfa.\u003c/p\u003e \u003cp\u003eHere, we found that the two treatment groups (\u003cem\u003eS. meliloti\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. aryabhattai\u003c/em\u003e and \u003cem\u003eS. meliloti\u003c/em\u003e alone) significantly elevated chlorophyll a concentration relative to the two control groups (one receiving full fertilizer and the other devoid of nitrogen fertilizer) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The protein content is markedly increased in the treatment group (\u003cem\u003eS. meliloti\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. aryabhattai\u003c/em\u003e) relative to the two control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, the \u003cem\u003eS. meliloti\u003c/em\u003e-only group demonstrated a modest elevation relative to the two control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Compared to \u003cem\u003eS. meliloti\u003c/em\u003e alone, the combination of the two bacteria led to an 18.40% decrease in Chlorophyll an after-bacillus compounding (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The amalgamation of two bacterial strains led to a substantial increase of 76.34% in Chlorophyll b (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). During that period, we computed and determined that the chlorophyll a/b ratio had markedly decreased. This research demonstrates that the amalgamation of the two bacteria improves alfalfa's capacity and efficacy in absorbing blue-violet light. When employed as fodder or green manure among maize plants, alfalfa frequently displays a reduced height in comparison to surrounding vegetation. Alfalfa exhibits a marked preference for sunlight; hence, enhancing its absorption of blue-violet light substantially promotes its growth. Concurrently, the concentrations of Chlorophyll a\u0026thinsp;+\u0026thinsp;b were markedly increased, signifying that alfalfa efficiently assimilates nitrogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Our investigation revealed that the application of \u003cem\u003eP. aryabhattai\u003c/em\u003e exerted negligible influence on the physiological activities of alfalfa. Thus, we concluded that \u003cem\u003eS. meliloti\u003c/em\u003e and \u003cem\u003eP. aryabhattai\u003c/em\u003e demonstrate complimentary interactions. So, we hypothesize that the participation of \u003cem\u003eP. aryabhattai\u003c/em\u003e in the dual-bacteria combination enhances nitrogen fixation by \u003cem\u003eS. meliloti\u003c/em\u003e, and subsequent experiments verified our suspicions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the addition of\u003c/b\u003e \u003cb\u003eP. aryabhattai\u003c/b\u003e \u003cb\u003eto alfalfa on the nitrogen fixation of\u003c/b\u003e \u003cb\u003eS. meliloti\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further confirm the groth-promoting benefits of the dual microbial combination, we proceeded to assess the nitrogen-fixing capability of \u003cem\u003eS. meliloti\u003c/em\u003e following the addition of \u003cem\u003eP. aryabhattai\u003c/em\u003e. However, the production of Nod factor is contingent upon the specific symbiotic compatibility between alfalfa and rhizobacteria, only those rhizobacteria that are adapted to the alfalfa varieties utilized in this experiment can synthesize Nod factor and consequently stimulate rhizome formation. Luckily, this is evidenced by the tumorous growth observed in alfalfa upon the introduction of \u003cem\u003eS. meliloti\u003c/em\u003e. Additionally, it is important to note that under the non-exogenous rhizobacteria environment, alfalfa is unable to develop rhizomes and will not exhibit ARA, and the color of the nodules is notable. Even in the presence of rhizobia, incompatible strains do not induce alfalfa to develop effective nodules, which are characterized by a pink hue.\u003c/p\u003e \u003cp\u003eHere, we observed that the control group (no-inoculum) receiving nitrogen initially exhibited robust growth, but thereafter had a significant decline in growth rate, akin to the control groups devoid of nitrogen. Conversely, the plants including \u003cem\u003eS. meliloti\u003c/em\u003e exhibit enhanced greenness and vigor, indicating that they have assimilated adequate nutrients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Post-harvest, we compared the nodule counts across several groups, revealing that the group containing \u003cem\u003eS. meliloti\u003c/em\u003e successfully induced rhizome formation in alfalfa roots, but the control groups failed to facilitate nodule formation in alfalfa. All treatment groups, except for \u003cem\u003eP. aryabhattai\u003c/em\u003e, developed nodules, the majority of which were pink and contained leghemoglobin, facilitating nitrogen fixation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This nitrogen-fixing bacteria effectively supplies nitrogen to alfalfa. In contrast, we observed that the additional \u003cem\u003eS. meliloti\u003c/em\u003e plants exhibited significantly improved growth, leading us to conclude that \u003cem\u003eS. meliloti\u003c/em\u003e can form symbiotic relationships with specific alfalfa species, resulting in effective nitrogen fixation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine if the incorporation of \u003cem\u003eP. aryabhattai\u003c/em\u003e with \u003cem\u003eS. meliloti\u003c/em\u003e enhances plant growth. We established three groups with exogenous batteries. In comparison to the group \u003cem\u003eS. meliloti\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. aryabhattai\u003c/em\u003e and \u003cem\u003eS. meliloti\u003c/em\u003e alone, we observed that the combination of the two bacteria resulted in a greater formation of pink nodules (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and the overall number of nodules also significantly increased. Subsequently, we tested its acetylene reduction activity (ARA). In comparison to the \u003cem\u003eS. meliloti\u003c/em\u003e group alone, the addition of \u003cem\u003eP. aryabhattai\u003c/em\u003e to \u003cem\u003eS. meliloti\u003c/em\u003e resulted in a considerable increase in ethylene production (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The combination of two bacteria resulted in a significant enhancement in ARA. Furthermore, \u003cem\u003eP. aryabhattai\u003c/em\u003e lacks the capacity for nitrogen fixation; hence, it can be assumed that its incorporation primarily enhances the ARA of \u003cem\u003eS. meliloti\u003c/em\u003e, rather than the synergistic effect of dual nitrogen activities.\u003c/p\u003e \u003cp\u003eNodules are generated by the plant root system under nitrogen-deficient conditions, subsequently resulting in the formation of flavonoids that induce Nod factor production. Then, we hypothesize that the inclusion of \u003cem\u003eP. aryabhattai\u003c/em\u003e results in the formation of flavonoids, and subsequent experiments verified our suspicions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP. aryabhattai\u003c/b\u003e \u003cb\u003emediates the production of flavonoids in alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo trigger the nodulation process of host plants, \u003cem\u003eS. meliloti\u003c/em\u003e produce Nod factors. However, Nod factors cannot be produced only by rhizobia. Flavonoids must be excreted by the alfalfa root system in the absence of nitrogen to stimulate the synthesis of Nod factors by rhizobia.\u003c/p\u003e \u003cp\u003eConsequent to the data, we determined that the dual-bacteria combination not only enhances plant growth but also increases ARA levels. We hypothesize that \u003cem\u003eP. aryabhattai\u003c/em\u003e influences the secretion of flavonoids by the plant's root system, thus impacting the tumorous potential of \u003cem\u003eS. meliloti\u003c/em\u003e. We assessed the flavonoid content of alfalfa root systems under complete fertilization, non-nitrogen fertilization, and \u003cem\u003eP. aryabhattai\u003c/em\u003e only. In the absence of non-exogenous bacteria, controls treated with nitrogen fertilizer yielded negligible flavonoid production, whereas controls devoid of nitrogen fertilizer generated some flavonoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In comparison to the control groups, the flavonoids extracted from alfalfa roots with the addition of \u003cem\u003eP. aryabhattai\u003c/em\u003e solution were significantly elevated relative to both control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate our hypothesis, we have purified and conducted liquid phase analysis of Nod Factor. And based on previous methods of determining Nod factor, we may have detected a peak of Nod factor-like at 84\u0026ndash;86 minutes (Lian et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) (Fig.\u0026nbsp;5c-5f). The result illustrates that the control group exhibited no response, indicating that the lack of flavonoid inducers does not generate Nod factor-like metabolites. In comparison with the two control groups, we observed a duration of 84 to 86 minutes, with no peaks evident in the control plots. While the precise type of Nod factor remained ambiguous and could not be quantified through standard curves, liquid phase analysis revealed that its peak area increases upon the addition of flavonoids extracted from alfalfa roots irrigated with the \u003cem\u003eP. aryabhattai\u003c/em\u003e solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Consequently, it is evident that additional metabolites potentially functioning as Nod factors-like can be elicited through flavonoid inducers derived from alfalfa roots. Furthermore, the incorporation of bacillus-infested plant inducers yielded elevated response values. The elevated metabolites, potentially various types of Nod factors, initiate the entire process of tumor growth, resulting in an increased number of nodules and enhanced nitrogen fixation capacity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of bacterial strains as microbial fertilizers for plant application has become a common approach to promote sustainable agriculture and create an environmentally friendly farming ecosystem, thereby replacing chemical fertilizers. This experiment focused on applying a nitrogen-fixing strain of rhizobacteria to alfalfa plants, incorporating a phosphorus-solubilizing bacterium, \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21, which was isolated from black soil, alongside the rhizobacterium \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1, sourced from alfalfa samples. This strain of nitrogen-fixing bacterium demonstrated symbiotic compatibility with the alfalfa cultivar Longmu 806, promoting the development of effective nodules that contain leghemoglobin, thereby allowing the plants to thrive without the need for nitrogen fertilizer. The findings of this experiment indicate that the addition of \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 significantly improved plant growth and boosted nitrogen activity. The interaction between rhizobacteria and host plants is characterized by the role of flavonoids, which act as essential inducers within the root systems of leguminous plants, triggering the nodulation response. Meanwhile, Nod factors are important metabolites generated by rhizobacteria in reaction to flavonoids. The introduction of \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 led to a significant enhancement in flavonoid production in the root systems of alfalfa plants. As a result, the inducer extracts from the quantitatively analyzed root systems led to \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1 producing a higher amount of Nod factor metabolites. The present experiment revealed that the interactions between the two bacterial species markedly enhanced nitrogen fixation and efficiently replaced nitrogen fertilizer in the cultivation of alfalfa. The secretions produced by \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 contributed positively to the soil and the ecosystem, markedly improving and revitalizing degraded land.\u003c/p\u003e \u003cp\u003eTo examine why a two-bacteria consortium facilitated enhanced growth of alfalfa in the absence of nitrogen fertilizer. We assessed variations in protein content, chlorophyll concentration, ARA, flavonoid synthesis, and Nod factor-like metabolites in alfalfa plants. The protein and chlorophyll content of alfalfa under the dual bacterial complex were dramatically elevated, along with the ARA and nodule count, resulting in enhanced growth of the alfalfa plants and adequate nitrogen availability in their environment. Simultaneously, we discovered that the root system of plants supplemented with \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 generated an increased quantity of flavonoids. To reaffirm this finding, we incorporated the gathered flavonoid compounds into the fermented rhizobia sap. Following the extraction of the Nod factor and subsequent HPLC analysis, we observed an increase in the number of peaks corresponding to the Nod factor class. This was a significant factor contributing to the rise in the number of nodules and ARA in the result. Consequently, we deduce that dephosphorylating bacteria can facilitate the synthesis of substantial quantities of flavonoids in the alfalfa root system, thereby enhancing the production of nodulation components by rhizobia for nitrogen fixation.\u003c/p\u003e \u003cp\u003eWe shall now examine the experimental results alongside recent studies pertaining to strain compounding and Nod factor synthesis. Numerous articles have indicated that strains akin to \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21, such as strain AB211, exhibit diverse metabolic pathways with a robust central carbohydrate metabolism. This suggests that the bacterium can effectively utilize root sap and other organic substrates as energy sources, demonstrating significant potential as a plant growth promoter (Bhattacharyya et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). And a bacillus like a \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain A2, can enhanced the fresh biomass, total length and root length of groundnut (Goswami et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). And an article indicated that numerous scientists discovered that specific dephosphorylating bacteria could enhance plant development in nutrient-deficient soils derived from desolate lakefront regions. The bacteria were utilized in microcosmic studies of wild plant growth, demonstrating enhanced seed germination and resulting in greater root, shoot, and dry weights of sprouted seedlings compared to the uninoculated control plants after many days. The plant growth-promoting capabilities of the \u003cem\u003earyabhattai\u003c/em\u003e strains are acknowledged as an environmentally sustainable method (Lee et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In our experiments, the \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 strain exhibited a comparable effect, inducing the plant root system to produce increased flavonoids. However, in contrast to the nitrogen-deficient conditions employed, a solitary strain of this bacterium was unable to directly influence seedling root systems and plant growth. We suggest that the ineffectiveness of a single strain in directly influencing plant growth may stem from its classification as an inter-root fungus, specifically adapted to black soil, and its presence in a resource-abundant environment, resulting in no direct impact on alfalfa plants under low nitrogen conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. meliloti\u003c/em\u003e is a prevalent rhizobia species associated with alfalfa, with numerous reports indicating its compatibility and symbiotic relationship with alfalfa, resulting in the production of multiple rhizomes (Checcucci et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eS. meliloti\u003c/em\u003e is recognized as a symbiotic bacterium species that may establish nitrogen-fixing nodules in the roots of both annual and perennial alfalfa species. Researchers disclosed the comprehensive genome sequence of a strain analogous to the present experiment, \u003cem\u003eS. meliloti\u003c/em\u003e strain AK76 (Vladimirova et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The results indicate that the \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1 strains examined in this study are compatible with the alfalfa species employed in the present experiment. And there are some researchers identified alfalfa truncatellid nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatellid TFs playing a central role during key steps of the \u003cem\u003eS. meliloti\u003c/em\u003e-M. truncatula symbiotic interaction (Baudin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This result also provides a theoretical basis for the use of alfalfa Longmu 806 and the \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1 in our research. Flavonoids are essential signaling molecules in the symbiotic relationship between legumes and their nitrogen-fixing partners, the rhizobia (Glyan'ko, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The principal role of flavonoids in this association is to stimulate the transcription of genes responsible for the manufacture of rhizobia signaling molecules known as Nod factors, which are recognized by the plant to facilitate symbiotic root infection (Liu and Murray, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrior studies have investigated the symbiotic association between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eMesorhizobium sp.\u003c/em\u003e The Ca181 strain displayed an enhancement when co-inoculated with six \u003cem\u003eBacillus\u003c/em\u003e strains, demonstrating a shoot dry weight ratio 1.62 to 1.74 times superior to the \u003cem\u003eMesorhizobium\u003c/em\u003e-inoculated treatment group after 80 days of growth under sterile circumstances (Sivaramaiah et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). There was research showed that co-inoculation of soybean plants with a new PGPR strain LL2012 and the natural symbiont (\u003cem\u003eBradyrhizobium japonicum\u003c/em\u003e) altered plant growth parameters and significantly improved nodulation (Masciarelli et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Also, research found that the Co-existence of \u003cem\u003eLeclercia adecarboxylata\u003c/em\u003e strain LSE-1 and \u003cem\u003eBradyrhizobium sp.\u003c/em\u003e Strain LSBR-3 improved the nodule number (Kumawat et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This result illustrates the effectiveness of employing rhizobacteria to improve crop yield. A recent study has disclosed that plant growth promoting rhizobacteria (PGPR) are ubiquitous soil bacteria residing in plant roots that stimulate plant growth (Fan and Smith, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eBacillus spp.\u003c/em\u003e are well known rhizosphere residents of many crops and usually show plant growth promoting (PGP) activities (Calvo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A Bacillus cereus strain TCU11 which is a PGPR can be exploited as inoculums for improving the phytoremediation efficiency in polluted soils (Bruno et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, Specific PGPR strains augment nodulation, nitrogen fixation, and legume growth when inoculated with rhizomes. Instances of PGPR include \u003cem\u003eAzotobacter\u003c/em\u003e, \u003cem\u003eAzospirillum\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and specifically the \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 mentioned in our study. All of which share a segment of a shared microbial habitat at the root-soil interface. The effects of co-inoculation with \u003cem\u003eRhizobium\u003c/em\u003e and PGPR have been noted in many symbiotic and plant growth parameters. The co-inoculation of Rhizobium and \u003cem\u003eAzospirillum\u003c/em\u003e increased the number of root hairs and the release of flavonoids by the roots compared to the alone inoculation of \u003cem\u003eRhizobium\u003c/em\u003e. The positive effect of PGPR on the nodulation of \u003cem\u003eRhizobium\u003c/em\u003e-legumes is ascribed to their synthesis of phytohormones (Sibponkrung et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The conclusions of this article provide similar theoretical support to the findings of the current experiment, suggesting that dual bacterial interactions augment flavonoid production, hence affecting Nod factor production and leading to an increased number of nodules.\u003c/p\u003e \u003cp\u003eOur experiment entailed screening the wild-type \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 and integrating it with \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1, which was obtained from alfalfa specimens. These strains are common in the alfalfa cultivation environment. The incorporation of dephosphorylating bacteria from black soil seeks to promote alfalfa growth in dry environments, especially under conditions of low nitrogen availability, thus enhancing the microbial resources and nutrient profile of desolate land. This study seeks to promote sustainable agriculture by utilizing microbial compound bacterial fertilizers instead of traditional chemical nitrogen fertilizers, thereby reducing damage to crops and the soil ecosystem, while providing technical assistance for enhanced land use and the exploitation of unproductive resources. An examination of the existing literature indicates a scarcity of research on the combined study of \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 and \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1, with enquiries into the related mechanisms being constrained. \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 and \u003cem\u003eS. meliloti\u003c/em\u003e LMGL3-1 can be efficiently integrated for use in alfalfa plants. This creates a fundamental experimental framework for investigating alfalfa plants alongside \u003cem\u003eBacillus\u003c/em\u003e bacteria and rhizobia, reinforcing the established PGPR-plant theoretical model pertinent to applied research. The integration of \u003cem\u003eP. aryabhattai\u003c/em\u003e YJHT21 led to elevated flavonoid levels in the plants, hence affecting the synthesis of nod components. Furthermore, the different modifications of Nod fators that are differentially induced are still worthy of further exploration and the signaling mechanisms of their interaction with flavonoids remain unknown.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28030201), the National Natural Science Foundation of China (92351302,\u0026nbsp;91851102, 32070034, 32270056 and 32300084), and the National Key R\u0026amp;D Program of China (2020YFA0906800\u0026nbsp;and 2022YFC2105300).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the fndings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28030201), the National Natural Science Foundation of China (92351302, 91851102, 32070034, 32270056 and 32300084), and the National Key R\u0026amp;D Program of China (2020YFA0906800 and 2022YFC2105300).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollege of Agriculture, Shanxi Agricultural University, Jinzhong, 030801, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRui Liu, Chang Li, Yunjun Zhang, Chunli Liu, Jinai Xue\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanning Zheng\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.L. (Rui Liu), J.X. (Jinai Xue), Y.Z. (Yanning Zheng) designed research. R.L. and C.L. (Chang Li) screened strains. R.L. performed the studies of nitrogen fixation; flavonoids and nod factor-like, R.L., C.L. (Chang Li), Y.Z. (Yunjun Zhang) and C.L. (Chunli Liu) carried out the pot experiment. R.L. (Yan Zeng), Y.Z. (Yanning Zheng) analyzed data. R.L. (Rui Liu), J.X., Y.Z. (Yanning Zheng) wrote the paper. All authors contributed to the revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Yanning Zheng and Jinai Xue\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmed M, Rauf M, Mukhtar Z, Saeed NA (2017) Excessive use of nitrogenous fertilizers: an unawareness causing serious threats to environment and human health. Environ Sci Pollut Res Int 24(35):26983\u0026ndash;26987. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-017-0589-7\u003c/span\u003e\u003cspan address=\"10.1007/s11356-017-0589-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBardi L, E M (2012) Drought and nutritional stresses in plant: Alleviating role of rhizospheric microorganisms. Nova Science, New York\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaruah M, Gogoi M, Chandra Boro R, Barooah M (2024) Priestia aryabhattai MBM3-Mediated enhancement of sulphur metabolism in Brassica campestris. Curr Microbiol 81(10):316. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00284-024-03844-0\u003c/span\u003e\u003cspan address=\"10.1007/s00284-024-03844-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaudin M, Laloum T, Lepage A, R\u0026iacute;podas C, Ariel F, Frances L, Crespi M, Gamas P, Blanco FA, Zanetti ME, de Carvalho-Niebel F, Niebel A (2015) A phylogenetically conserved group of nuclear factor-Y transcription factors interact to control nodulation in legumes. Plant Physiol 169(4):2761\u0026ndash;2773. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.15.01144\u003c/span\u003e\u003cspan address=\"10.1104/pp.15.01144\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattacharyya C, Bakshi U, Mallick I, Mukherji S, Bera B, Ghosh A (2017) Genome-Guided Insights into the plant growth promotion capabilities of the physiologically versatile Bacillus aryabhattai strain AB211. Front Microbiol 8:411. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2017.00411\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2017.00411\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBruno LB, Anbuganesan V, Karthik C, Tripti, Kumar A, Banu JR, Freitas H, Rajkumar M (2021) Enhanced phytoextraction of multi-metal contaminated soils under increased atmospheric temperature by bioaugmentation with plant growth promoting Bacillus cereus. J Environ Manage 289:112553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jenvman.2021.112553\u003c/span\u003e\u003cspan address=\"10.1016/j.jenvman.2021.112553\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalvo P, Orme\u0026ntilde;o-Orrillo E, Mart\u0026iacute;nez-Romero E, Z\u0026uacute;\u0026ntilde;iga D (2010) Characterization of Bacillus isolates of potato rhizosphere from andean soils of Peru and their potential PGPR characteristics. Braz J Microbiol 41(4):899\u0026ndash;906. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/s1517-83822010000400008\u003c/span\u003e\u003cspan address=\"10.1590/s1517-83822010000400008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheccucci A, Azzarello E, Bazzicalupo M, Galardini M, Lagomarsino A, Mancuso S, Marti L, Marzano MC, Mocali S, Squartini A, Zanardo M, Mengoni A (2016) Mixed nodule infection in Sinorhizobium meliloti-Medicago sativa symbiosis suggest the presence of cheating behavior. Front Plant Sci 7:835. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2016.00835\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2016.00835\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Wang J, Huang D, Cheng W, Shao Z, Cai M, Zheng L, Yu Z, Zhang J (2021) Volatile organic compounds from Bacillus aryabhattai MCCC 1K02966 with multiple modes against Meloidogyne incognita. Molecules 27(1):103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules27010103\u003c/span\u003e\u003cspan address=\"10.3390/molecules27010103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan D, Smith DL (2021) Characterization of selected Plant Growth-Promoting Rhizobacteria and their Non-Host growth promotion effects. Microbiol Spectr 9(1):e0027921. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/Spectrum.00279-21\u003c/span\u003e\u003cspan address=\"10.1128/Spectrum.00279-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlyan'ko AK (2015) Signaling systems of Rhizobia (Rhizobiaceae) and leguminous plants (Fabaceae) upon the formation of a Legume-Rhizobium symbiosis (Review). Prikl Biokhim Mikrobiol 51(5):453\u0026ndash;464\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoswami D, Dhandhukia P, Patel P, Thakker JN (2014) Screening of PGPR from saline desert of Kutch: Growth promotion in Arachis hypogea by Bacillus licheniformis A2. Microbiol Res 169(1):66\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micres.2013.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.micres.2013.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez-G\u0026oacute;mez A, Garc\u0026iacute;a-Est\u0026eacute;vez I, Escribano-Bail\u0026oacute;n MT, Garc\u0026iacute;a-Fraile P, Rivas R (2021) Bacterial fertilizers based on Rhizobium laguerreae and Bacillus halotolerans enhance Cichorium endivia L. Phenolic compound and mineral contents and plant development. Foods 10(2):424. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods10020424\u003c/span\u003e\u003cspan address=\"10.3390/foods10020424\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumawat KC, Sharma P, Singh I, Sirari A, Gill BS (2019) Co-existence of Leclercia adecarboxylata (LSE-1) and Bradyrhizobium sp. (LSBR-3) in nodule niche for multifaceted effects and profitability in soybean production. World J Microbiol Biotechnol 35(11):172. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-019-2752-4\u003c/span\u003e\u003cspan address=\"10.1007/s11274-019-2752-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee S, Ka JO, Song HG (2012) Growth promotion of xanthium italicum by application of rhizobacterial isolates of Bacillus aryabhattai in microcosm soil. J Microbiol 50(1):45\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12275-012-1415-z\u003c/span\u003e\u003cspan address=\"10.1007/s12275-012-1415-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLian B, Prithiviraj B, Souleimanov A, Smith DL (2001) Evidence for the production of chemical compounds analogous to nod factor by the silicate bacterium Bacillus circulans GY92. Microbiol Res 156(3):289\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1078/0944-5013-00107\u003c/span\u003e\u003cspan address=\"10.1078/0944-5013-00107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu CW, Murray JD (2016) The role of flavonoids in nodulation Host-Range specificity: An update. Plants (Basel) 5(3):33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants5030033\u003c/span\u003e\u003cspan address=\"10.3390/plants5030033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouren\u0026ccedil;o KS, Rossetto R, Vitti AC, Montezano ZF, Soares JR, Sousa RM, do Carmo JB, Kuramae EE, Cantarella H (2019) Strategies to mitigate the nitrous oxide emissions from nitrogen fertilizer applied with organic fertilizers in sugarcane. Sci Total Environ 650(Pt 1):1476\u0026ndash;1486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2018.09.037\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2018.09.037\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalus\u0026aacute; E, Vassilev N (2014) A contribution to set a legal framework for biofertilisers. Appl Microbiol Biotechnol 98(15):6599\u0026ndash;6607. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-014-5828-y\u003c/span\u003e\u003cspan address=\"10.1007/s00253-014-5828-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasciarelli O, Llanes A, Luna V (2014) A new PGPR co-inoculated with Bradyrhizobium japonicum enhances soybean nodulation. Microbiol Res 169(7\u0026ndash;8):609\u0026ndash;615. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micres.2013.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.micres.2013.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazid M, Khan TA (2014) Future of bio-fertilizers in indian agriculture: An overview. J Agr Food Res 3(3):10\u0026ndash;23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiljaković D, Marinković J, Tamindžić G, Đorđević V, Tintor B, Milošević D, Ignjatov M, Nikolić Z (2022) Bio-priming of soybean with bradyrhizobium japonicum and bacillus megaterium: Strategy to improve seed germination and the initial seedling growth. Plants (Basel) 11(15):1927. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants11151927\u003c/span\u003e\u003cspan address=\"10.3390/plants11151927\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahid M, Zeyad MT, Syed A, Singh UB, Mohamed A, Bahkali AH, Elgorban AM, Pichtel J (2022) Stress-Tolerant endophytic isolate Priestia aryabhattai BPR-9 modulates Physio-Biochemical mechanisms in wheat (Triticum aestivum L.) for enhanced salt tolerance. Int J Environ Res Public Health 19(17):10883. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijerph191710883\u003c/span\u003e\u003cspan address=\"10.3390/ijerph191710883\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSibponkrung S, Kondo T, Tanaka K, Tittabutr P, Boonkerd N, Yoshida KI, Teaumroong N (2020) Co-Inoculation of Bacillus velezensis strain S141 and Bradyrhizobium strains promotes nodule growth and nitrogen fixation. Microorganisms 8(5):678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms8050678\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms8050678\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSivaramaiah N, Malik DK, Sindhu SS (2007) Improvement in symbiotic efficiency of chickpea (Cicer arietinum) by coinoculation of Bacillus strains with Mesorhizobium sp. Cicer. Indian J Microbiol 47(1):51\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12088-007-0010-1\u003c/span\u003e\u003cspan address=\"10.1007/s12088-007-0010-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun B, Bai Z, Bao L, Xue L, Zhang S, Wei Y, Zhang Z, Zhuang G, Zhuang X (2020) Bacillus subtilis biofertilizer mitigating agricultural ammonia emission and shifting soil nitrogen cycling microbiomes. Environ Int 144:105989. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envint.2020.105989\u003c/span\u003e\u003cspan address=\"10.1016/j.envint.2020.105989\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan GY, Tan WK (1986) Interaction between alfalfa cultivars and Rhizobium strains for nitrogen fixation. Theor Appl Genet 71(5):724\u0026ndash;729. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/bf00263270\u003c/span\u003e\u003cspan address=\"10.1007/bf00263270\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerpolilli JJ, Hood GA, Poole PS (2012) What determines the efficiency of N(2)-fixing Rhizobium-legume symbioses? Adv Microb Physiol 60:325\u0026ndash;389. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/b978-0-12-398264-3.00005-x\u003c/span\u003e\u003cspan address=\"10.1016/b978-0-12-398264-3.00005-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVladimirova ME, Muntyan VS, Afonin AM, Muntyan AN, Baturina OA, Dzuybenko EA, Saksaganskaya AS, Simarov BV, Roumiantseva ML, Kabilov MR (2022) Complete genome of Sinorhizobium meliloti AK76, a symbiont of wild diploid Medicago lupulina from the Mugodgary Mountain region. Microbiol Resour Announc 11(3):e0108821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mra.01088-21\u003c/span\u003e\u003cspan address=\"10.1128/mra.01088-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Yang S, Tang F, Zhu H (2012) Symbiosis specificity in the legume: Rhizobial mutualism. Cell Microbiol 14(3):334\u0026ndash;342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1462-5822.2011.01736.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1462-5822.2011.01736.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nitrogen fixation, Plant growth, Flavonoid, Sinorhizobium, Priestia","lastPublishedDoi":"10.21203/rs.3.rs-5665466/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5665466/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNitrogen fertilizer is crucial for agricultural output. However, prolonged overuse has resulted in nitrate leaching, and potential soil acidification. Research on microbial fertilizers has become essential to enhance soil conditions and minimize nitrogen fertilizer usage. In alfalfa cultivation, research on efficient compound microbial agents remains limited, therefore, this study concentrates on the investigation of dual microbial combinations. In the screening process, black soil was utilized with alfalfa plants as samples to identify a strain of rhizobacteria, \u003cem\u003eSinorhizobium meliloti LMGL3-1\u003c/em\u003e, exhibiting nitrogen-fixing capabilities, and \u003cem\u003ePriestia aryabhattai\u003c/em\u003e (\u003cem\u003eBacillus aryabhattai\u003c/em\u003e) YJHT21, demonstrating phosphorus-solubilizing abilities. The \u003cem\u003eS. meliloti\u003c/em\u003e strain demonstrated the ability to symbiotically associate with the alfalfa variety Longmu 806, resulting in the formation of effective nodules containing leghemoglobin, thereby enabling the plants to thrive in the absence of nitrogen fertilizer application. Here, we discovered that the addition of phosphorus-solubilizing \u003cem\u003eP. aryabhattai\u003c/em\u003e enhanced plant growth and increased nitrogenase activity of \u003cem\u003eS. meliloti\u003c/em\u003e. Moreover, the incorporation of \u003cem\u003eP. aryabhattai\u003c/em\u003e resulted in a significant increase in flavonoid production within the root system of alfalfa plants. Consequently, under the influence of the inducer extracted from the root system of quantitatively analyzed plants, the rhizobacteria exhibited enhanced production of metabolites associated with the Nod factor cluster. The current experiment demonstrated that the interaction between the two bacteria significantly enhanced nitrogen fixation, effectively substituting nitrogen fertilizer in alfalfa cultivation with improved efficiency and offered theoretical support for the eco-friendly advancement of microbial compound fertilizers as a substitute for chemical fertilizers.\u003c/p\u003e","manuscriptTitle":"Enhanced biological nitrogen fixation in alfalfa through the synergistic interactions between Sinorhizobium meliloti and Priestia aryabhattai","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 17:22:03","doi":"10.21203/rs.3.rs-5665466/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-09T18:01:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-30T09:40:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-18T15:13:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200071944757161560536259388374780803689","date":"2025-01-15T13:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336996197843539778661541852424326426151","date":"2025-01-14T06:28:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216933456147902126765124471855727123735","date":"2025-01-13T12:53:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-29T01:14:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-20T16:47:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-20T14:54:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2024-12-18T02:43:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ddf733a-3da9-4331-a37e-3feb14670902","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:02:37+00:00","versionOfRecord":{"articleIdentity":"rs-5665466","link":"https://doi.org/10.1007/s11274-025-04394-8","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2025-05-26 15:57:44","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2024-12-23 17:22:03","video":"","vorDoi":"10.1007/s11274-025-04394-8","vorDoiUrl":"https://doi.org/10.1007/s11274-025-04394-8","workflowStages":[]},"version":"v1","identity":"rs-5665466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5665466","identity":"rs-5665466","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}