Legacy nicotine stimulates soil nitrification and available nitrogen in long-term tobacco cropping system via microbial priming and nitrogen supply

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Li, Guitong Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9136135/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The legacy effects of nicotine, alkaloid from tobacco, on soil bacteria and nutrient cycling functions are not fully understood. In this microcosm study using soil from a decade-long tobacco monoculture field. Nicotine exhibited a persistent influence on nitrification for 42 days via direct nicotine-N supply and priming nitrifier activity. Gross nitrification rates and available nitrogen (NH 4 + and NO 3 − ) content were significantly higher at 100 mg kg − 1 nicotine than those at 10 mg kg − 1 . Nicotine significantly altered soil bacterial dynamics (contribution = 0.22) and increased the abundance of Intrasporangiaceae and Bryobacter . Changes in nitrification rates were positively correlated with increases in ammonia-oxidizing bacteria (AOB)- amoA copy numbers. Phylogenetic analysis revealed dominant AOB Operational Taxonomic Units (OTUs) affiliated with the genus Nirosospira , closely related to ‘ Nitrosospira sp. Np 39 − 19’ (99% identity). Dominant ammonia-oxidizing archaea (AOA) include Nitrosopumilaceae (> 70% of sequences) and Nitrosopumilus . This study enhances understanding nicotine’s role in microbial function shifts and suggests potential strategies for rhizodeposition-based nitrogen management in tobacco fields. nicotine legacy effect nitrification ammonia oxidizing archaea ammonia oxidizing bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Highlights Nicotine stimulation on nitrification lasted for 42 days via direct N supply and nitrifier priming. Nicotine enriched specific bacterial taxa like I ntrasporangiaceae and Bryobacter . Ammonia-oxidizing bacteria (AOB) was more responsive to nicotine than ammonia-oxidizing archaea (AOA). AOB ( Nitrosospira ) dominated the nicotine-induced nitrification response. Dominant AOA genera were Nitrosopumilaceae and Nitrosopumilus . 1. Introduction Soil legacy effects refer to the influences of prior soil conditions on subsequent soil functions (Nannipieri et al., 2023 ; Yang et al., 2024 ; Zou et al., 2026 ). Such effects reflect “soil memory”, often resulting from microbial adaptation to environmental changes such as temperature (Targulian et al., 2019; Lian et al., 2022 ), drought (Bennett et al., 2021 ), and grazing intensity (Matson et al., 2013; Ren et al., 2024 ). Legacies can also arise from soil chemical and biological processes mediated by rhizodeposition (Semchenko et al., 2021 ; Han et al., 2023 ). While legacy impacts on soil microbiome diversity and composition are well documented (Turner et al., 2013 ; Ling et al., 2022 ), less is known about rhizodeposit-mediated legacies on nutrient cycling and their agronomic utility. A better understanding of these mechanisms could inform strategies for soil health management, crop rotation design, and the development of bio-simulants that mimic positive plant legacy effects. In tobacco ( Nicotiana tabacum L.) cultivation excessive agrochemical inputs, including nitrogen fertilizers and biochemicals such as nicotine, can induce long-lasting negative legacies (Postigo et al., 2023 ; Wang et al., 2024 ; Huang et al., 2025 ; Cao et al., 2026 ). Nicotine (C 10 H 14 N 2 ), a major alkaloid in tobacco wastes (2%–8% of leaf dry biomass) and root exudates, accumulates in soil at concentrations of 10–20 mg kg − 1 in the root zone (Lisuma et al., 2019 ). Annual tobacco processing generates approximately one million tons of solid waste with nicotine content averaging 18 g kg⁻¹dry weight (Wang et al., 2010 ; Liu et al., 2023 ; Wang et al., 2024 ). These nicotine residues in field need more than 8 months to degradation (approximately 90%); however, it is legacy effect may persist in soil for over a year (Ma et al., 2018 ). Nicotine exerts both positive and negative legacy effects on soil microbial communities (Rivera et al., 2023 ), is also readily absorbed by crop roots and translocated to edible tissues, poses a growing threat to food safety. Conventional management often focuses on mitigating negative impacts, such as reduced microbial diversity and environmental pollution. However, the potential to harness positive legacies via rhizodeposit-mediated microbial functions remains underexplored. Notably, tobacco cultivation has been associated with increased total soil N but decreased availability of phosphorus (P), potassium (K), and sulfur (S) (Rivera et al., 2023 ). Nicotine in the rhizosphere may enhance plant uptake of N and zinc (Zn) while reducing P and K availability (Lisuma et al., 2019 ). Wild-type tobacco plants subjected to nitrogen deficiency exhibited a reduction in nitrogen use efficiency (NUE) accompanied by an increase in nicotine accumulation (Farooq et al., 2014 ). Lisuma et al. ( 2019 ) reported, before tobacco established, soil nicotine (0.01 mg kg − 1 ) was negligible, while increased to 2.71 mg kg − 1 of harvesting unfertilized tobacco, to be more upon to 8.69 mg kg − 1 in soil of fertilized tobacco, results showed significant interactions among fertilizer application and soil nicotine content. These interactions suggest that nicotine may legacy soil nitrogen availability for subsequent crops (Wu et al., 2001 ), yet the persistence and mechanisms of nicotine effects on nitrogen cycling remain unclear. This study aimed to investigate the persistence of nicotine rhizodeposits, their legacy effects on soil microbiome composition, and their role in fast-cycling nitrogen pools. Modern agricultural systems often exhibit high nitrification activity, leading to low nitrogen use efficiency (Beeckman et al., 2018 ). Nitrification, a key step in nitrogen cycling, is influenced by root exudates that can either stimulate or inhibit nitrifier activity. For example, legume exudates show little inhibitory effect, whereas cereals like sorghum produce biological nitrification inhibitors (Coskun et al., 2017a ). Soybean isoflavones stimulate Nitrosomonas (Basalirwa et al., 2020 ), highlighting the potential for rhizodeposit-mediated nitrification regulation, while sorghum ( Sorghum bicolor ) presented high capacity in biological nitrification inhibitors (Subbarao et al., 2013 ). These examples highlight the potential for rhizodeposit-mediated soil nitrification, which exploited by the application of bioactive compounds that act as nitrification inhibitor or augmenter against or improve specific agrochemicals as part of an integrated nitrogen management strategy. Nicotine, with its heterocyclic structure, serves as a useful model for studying tobacco-derived effects on soil processes. Understanding the persistence of nicotine’s impact is essential for evaluating its legacy contribution to soil nitrogen dynamics and developing nitrogen management strategies in monocropping systems. Here, we conducted a factorial microcosm experiment with soil from a long-term tobacco field to disentangle the rhizodeposit-induced legacy effects of nicotine on nitrogen availability. We assessed interactions between tobacco soil biota and chemical factors, defining rhizodeposit-mediated legacy as persistent changes in the soil microbiome following nicotine exposure. We hypothesized that AOA and AOB communities would respond differently to varying nicotine concentrations, influencing their relative contributions to nitrification for tobacco culturing. 2. Materials and methods 2.1. Experimental details and treatments Soil was collected from a 10-year tobacco field in Heishi Town, Bijie City, Guizhou Province, China (104.00°N, 26.76°E). Classified as Mollic Gleysols (FAO), its chemical properties are listed in Table 1 . Soil was pre-incubated at 25°C for 7 days at 60% water-holding capacity. Nicotine (99% purity) was provided by Bijie Tobacco Company. Table 1 Characterization of chemical properties of soils. Items Soil Available K (mg kg − 1 ) 196.4 Total P (g kg − 1 ) 1.00 Conductivity (cmol kg − 1 ) 0.36 NH 4 + (mg kg − 1 ) 15.8 ± 1.1 NO 3 − (mg kg − 1 ) 26.5 ± 1.7 pH 5.40 Total organic carbon (g kg − 1 ) 16.3 ± 1.4 Total N (g kg − 1 ) 1.27 ± 0.06 15 N atom percent excess (atom%) 0.37 A 3 × 2 microcosm experiment included three nicotine concentrations (0, 10, 100 mg kg⁻¹ dry soil) and two nitrogen levels (0 and 50 mg N kg⁻¹). Preliminary tests with different nicotine dosages (0.1, 1.0, 10, 100 mg kg − 1 dry soil), and no nicotine (as control) carried out to comparing and selecting nicotine concentrations, showed that 0.1 and 1.0 mg kg⁻¹ nicotine had no significant effect on soil NH₄⁺ and NO₃⁻ compared to the control. Based on Preliminary tests data, for comparison, selected three nicotine concentrations (0, 10, 100 mg kg − 1 dry soil) to analyzing nicotine legacy effect, i . e . control, NT10, NT100. Nicotine solutions were applied daily for 10 days, mixed into fresh soil (2.0 kg dry weight equivalent), and incubated in the dark at 25°C. Soil samples were destructively collected at 1, 3, 7, 14, 28, and 42 days, sieved (2 mm), and divided for DNA extraction (− 80°C) and chemical analysis (4°C). ¹⁵N labeling (10 atom%) was used to track N dynamics. Each treatment had 18 replicates. 2.2 Soil chemical properties NH 4 + and NO 3 − were extracted with 2.0 M KCl (1:4 soil: solution) and analyzed by flow injection (QuikChem 8500). Soil organic carbon was determined according to the Walkley Black method, and total N by the Kjedahl digestion, pH in a 1:2 soil-water suspension, and conductivity in 1:2 soil-KCl. Residual nicotine was measured spectrophotometrically at 602 nm. 2.3 Gross nitrification rates Gross nitrification rates were measured with ¹⁵NO₃ − . Soil subsamples (20 g dry weight) received 2.0 mL of K 15 NO₃ solution (10 atom% ¹⁵N, 50 mg N kg⁻¹), was gradually (separate 10 times) added and carefully mixed, creating a final soil water content of 0.1 kg kg − 1 . Immediately following the soil mixture, one-part subsamples (three replications) were harvested and extracted with 2.0 M KCl (1:4 of soil: solution by mass) to determine NO 3 − concentration ( Q 1 ) and 15 N enrichment at time-0 ( A 1 ). Then other samples (three replications for each treatment) placed in 200 mL Mason jars with lids containing butyl rubber septa and with 1.0 mL water at bottom of the jar to minimize loss of moisture from soil. The Mason jars placed in a constant temperature (25℃) incubator in the dark for 24 h, and detected soil NO 3 − -N ( Q 2 ) and 15 N enrichment ( A 2 ) at time-24 h. The δ 15 N of NO 3 − -N detected with sequential diffusion method. Briefly, 25 mL KCL extraction solution added into the regent bottle with screw cap (100 mL). Ammonia traps (filter pack) were prepared by taking 1 cm diameter GF/D filter (What man #1823010) on the central of 2.5 cm diameter Teflon membranes (10 um pore-size; Millipore LCWP 02500), and 25 µL of 2 M H 2 SO 4 were pipetted onto the center of the GF/D filter, then PTFE tape was immediately sealed with tweezers. Immediately put the prepared filter pack and 3 g L − 1 reagent grade MgO (combusted at 450℃ for 4 h) into bottle and sealed, and incubated in a custom-made shaker (140 rpm) at 25℃ for 7 days. After incubation, took the filter pack into desiccator with silica gel and an open container of concentrated sulphuric acid (to remove trace ammonia) for 1–2 days, then the small filter was stored in individual vials for detecting the δ 15 N of NH 4 + . The incubation solutions were then kept shaking for 2 days to allow the NH 3 in the bottle vented. After then 0.2 g Devarda’s Alloy were added into the solution, and the filter pack were suspended and incubated as above, finally obtaining the samples for δ 15 N of NO 3 − detection. The total N concentration and δ 15 N of soil were analyzed by an Elemental Analyzer System (Vario PYRO cube; Elementar Analysensysteme GmbH) coupled to an Isotope Mass Spectrometer (Isoprime 100; Elementar Analysensysteme GmbH). The gross nitrification rate calculated based on differences of NO 3 − -N and δ 15 N between pre-incubated soil and incubated soil for 24 h, the equation was according to Davidson et al. (1991) and Zhang et al. ( 2015 ) as below: F =[( Q 1 - Q 2 )×ln ( A 1 / A 2 )]/[ t ×ln ( Q 1 / Q 2 )] (1) where, F (mg kg − 1 d − 1 ) is gross nitrification rate, t is the incubation time (here is 24 h); Q 1 and Q 2 are NO 3 − concentration (mg kg − 1 ) at time-0 and time-24 h, respectively; while A 1 and A 2 are the initial abundance of 15 N (atom %) and 15 N (atom %) of soil after 24 h incubation. 2.4. Soil DNA extraction and qPCR analysis DNA was extracted from 0.5 g soil using the FastDNA Spin Kit (MP Biomedicals). The 25-µL reaction mixture used (containing 10 µL of 10-fold diluted soil DNA) for soil DNA extraction. DAN extracts were quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), then used as PCR templates. Primer pair 338F (5’-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) used for amplifying V3 and V4 regions of bacterial 16S ribosomal RNA (rRNA) genes, then the amplified products were purified by AMPure XP beads. Quantitative PCR of AOA and AOB amo A genes measured using the SsoAdvanced SYBR Green Supermix and a CFX CONNECT Real-Time PCR Detection System (Bio-Rad laboratories, Hercules, CA, USA). Primers amoA 19F and amoA 643R used to quantify AOA amoA gene abundance, while primers amoA 189F and amoA 2R used to quantify AOB amoA gene abundance (Habteselassie et al., 2013 ). Amplifications carried out as follows: an initial denaturation step of 95℃ for 10 min, 40 cycles of 95℃ for 45 s, 60℃ for 1 min for AOB or 58℃ AOA, and 72℃ for 45 s, and a final extension step of 72℃ for 10 min. Reaction efficiencies greater than 90%, and R 2 >0.97. 2.5. Statistical analysis Repeated-measures ANOVA (SPSS 21.0) assessed treatment effects on soil properties and amoA copy numbers. Two-way ANOVA evaluated nicotine and nitrogen effects on nitrification rates, soil microbial diversity, AOA- amoA and AOB- amoA relative abundance. Linear mixed-effects models tested interactions between nicotine and time on diversity indices (indexes of Shannon, Simpson and Chao 1) p < 0.05 were considered statistically significant. 3. Results 3.1. Effects of nicotine on soil bacterial communities Approximately 80,000 high-quality 16S rRNA sequences were obtained per sample. Bacterial composition shifted with nicotine treatment and incubation time (Fig. 1 A). Dominant phylum included Sphingomonas , Pseudarthrobacter , MND1 , Intrasporangiaceae and Bryobacter (Fig. 1 A and B). Among these, relative abundance of Streptomyces and Intrasporangiaceae increased under nicotine treatment, while Bradyrhizobium and MND1 decreased (Fig. 2 ). Pseudarthrobacter became the overwhelmingly dominant genus in the 100 mg kg⁻¹ nicotine treatment by day 14 (Fig. 1 A). Variance partitioning indicated nicotine explained 22% of bacterial community variation (Fig. 1 C). Nicotine correlated more strongly with NH₄⁺ than NO₃⁻ (Fig. 1 D). In addition, some nitrifiers e . g . OUT 23 ( Nitrospira ) presented a negative correlation with nicotine. 3.2. Soil inorganic N and gross nitrification Nicotine and N fertilization significantly affected inorganic N and gross nitrification (Fig. 3 ). Nicotine's primming of soil nitrification is dosage-dependent: an initial inhibition phase (< 100 mg kg⁻¹) is followed by a time-dependent recovery period (7–42 days). The 100 mg kg⁻¹ treatment showed higher NH₄⁺, NO₃⁻, and gross nitrification, peaking at day 14 for NH₄⁺ and nitrification. Specifically, the 100 mg kg − 1 nicotine treatment increased soil NH 4 + in the first 14 days followed with a decrease, while the other two nicotine treatments decreased NH 4 + in the first 14 days and finally equalized to the control after 21 days. Nitrate increased with incubation time and residual nicotine concentrations (Fig. 3 B). Gross nitrification rates were higher in fertilized soils and increased with nicotine dosage. In addition, Nicotine degraded rapidly within 3 days and returned to baseline by day 42. 3.3 Abundance and structure of AOA and AOB Initial amoA copy numbers were 6.9×10 4 g⁻¹ (AOA) and 7.3×10 4 g⁻¹ (AOB) (Fig. 4 A and B). Both AOA- amoA and AOB- amoA numbers were significantly affected by nicotine dosage and incubation time. Nicotine influenced amoA expression both in AOA and AOB in a dosage-dependent manner: on day 3, a priming effect was observed at low concentration (10 mg kg⁻¹), while inhibition occurred at high concentration (100 mg kg⁻¹). This initial phase was followed by a sustained period (days 7–42) which priming effects continued to depend on nicotine concentration. Additionally, the AOB/AOA amoA gene copy number ratio ranged from 0.4 to 1.5, consistently higher in nicotine-treated groups than in controls (Fig. 4 ). Structure of AOB and AOA communities revealed by analysis of amoA genes in each sample (Fig. 5 and Fig. 6 ). Totals of 6.2×10 4 and 2.3×10 5 sequence reads obtained for AOB and AOA in controls, respectively, with 2.6×10 3 and 2.2×10 3 unique OTUs respectively (90% identity cut off). Phylogenetic analysis showed that dominant AOB OTUs were affiliated with Nirosospira (≈ 99% relative abundance) (Fig. 5 ). Relative abundance of AOB OTUs affected by culture period and nicotine dosage. For example, Nitrosomonas increased at days 14 and 42 in control and NT10 but only at day 14 in NT100. AOA communities were dominated by Nitrosopumilaceae (> 70%) and Nitrosopumilus . Nicotine initially increased Nitrosopumilus but decreased Nitrosopumilaceae , by day 42, inhibition ceased and abundance rose with nicotine concentration. Figure 6 shows that 100 mg kg − 1 nicotine presented the most negative relevance with AOA community on day 42, with the relevant index of -2.5, and presented a negative relevant index of -1.2 with AOB. Note Nitrosomonas not detected in the samples at initial day and 3-day. Interactions of nicotine and incubation time presented significant effect on AOB and AOA diversities ( P < 0.01), AOB was more response to nicotine legacy than AOA (Fig. 7 and Table 2 ). Results of LMMs reflect that sampling time presented a main-factor effect on nitrifiers diversity ( P < 0.01), while nicotine presented a main-factor effect only on AOB diversity, i . e . Shannon and Simpson indexes (Table 2 ). In general, diversity of AOB was higher than that of AOA. On day 14, both Simpson and Shannon indexes increased in the 10 mg kg − 1 nicotine treatment, while decreased in 100 mg kg − 1 treatment; however, on day 42, these two indexes improved in 100 mg kg − 1 treatment but decreased in 100 mg kg − 1 treatment. Chao 1 index was mainly influenced by incubation time, as increased in AOB and decreased in AOA along with the incubation time. PCoA results show that AOA structure was different between nicotine treatments and incubation times, mainly defined AOA structures as revealed by weighted UniFranc difference matrices, as the community of nicotine treatments on day 3 and day 42 respectively approached together, while the community of nicotine treatments on day 14 merged with the control (Fig. 8 ). NT100 at 42 days differed significantly from other treatments, indicating a concentration-dependent legacy effect. Phylogenetic analysis revealed AOB sequences clustering with Nitrosospira sp. Np 39 − 19 and N. multiformis ATCC 25196 (Fig. 9 ). There were four sequences clustering with Nitrosospira multiformis ATCC 25196. Similar with AOB, AOA communities also divided into two branches, there were four OTU sequences posed a 100% match score with MF324845 in this experiment. Another branch was clustering with Nitrososopumilus ureiphilus strain PS0 and Nitrosarchaeum koreeense MY1. Table 2 Significance analysis ( p -values) of influences of nicotine treatments, sampling time and impact on the microbial diversity in the linear mixed-effect models. Dependent variables Nicotine treatment Sampling time Treatment × Time F value p -value F value p -value F value p -value AOB Shannon 8.356 0.000 103.2 0.000 58.1 0.000 Simpson 14.07 0.000 56.77 0.000 29.82 0.000 Chao 1 1.688 0.187 45.04 0.000 28.74 0.000 AOA Shannon 1.598 0.205 87.45 0.000 26.88 0.000 Simpson 0.062 0.940 81.64 0.000 21.113 0.000 Chao 1 0.581 0.560 29.170 0.000 21.737 0.000 4. Discussion 4.1 Tobacco nicotine legacy effect on soil microbes and environmental safety Legacy effects, especially on crop growth, are often attributed to the presence and relative balance between beneficial and pathogenic organisms (Matson et al., 2013; Huang et al., 2025 ; Zou et al., 2026 ) and finally influence the pedosphere safety. Persistent legacy effects have been reported that the population of soil bacteria and fungi decreased significantly, for example, Santhanam et al. ( 2015 ) observed increased plant mortality due to sudden wilt disease in soils with a 15-year tobacco history, indicating long-term alterations in soil microbial communities. In this study, nicotine explained 22% (Fig. 1 C) of the variation in soil microbial structure and diversity, suggesting it strongly modulates the balance between beneficial and pathogenic taxa. Although nicotine generally reduced bacterial and fungal abundance, certain taxa such as Intrasporangiaceae (family level) increased in relative abundance under nicotine treatment. This enrichment implies nicotine-tolerant or nicotine-degrading capabilities, consistent with reports that Intrasporangiaceae can degrade methylbenzene by using isotopic probe technology (Ben Israel et a., 2021). Similarly, Pseudomonas tolerates high nicotine concentrations under a pH range between 6.5 and 7.0 (Wang et al., 2018 ). In tobacco soils, Bradyrhizobium is a common rhizobacterium (Zhu et al., 2023 ), however, nicotine accumulation slightly suppressed its abundance (Fig. 1 D), indicating nicotine potential toxicity to this beneficial genus. In otherwise, persistent legacy effects on nitrogen cycling have been reported various nitrification inhibitors, ( e . g . sorgoleone, sakuranetin, caffeic acid, ferulic acid), with effects persisting decades after plant removal (Coskun et al., 2017b ). Our results show that nicotine’ legacy on microbes extended beyond 6-weeks, which may constrain nitrogen fixation in subsequent crops. Given the cost and practicality limitations of large-scale soil remediation, agronomic strategies focusing on crop selection and breeding have emerged as a sustainable frontline defense. In future, the strategic use of low-accumulating crop varieties presents a practical, economically viable, and environmentally sound approach to safeguard the food chain from soil nicotine contamination (Xiang et al., 2020a ; Xiang et al., 2020b ). Nicotine released into soil can be mineralized, contributing to soil nitrogen pools. However, its acidic nature may alter structure and soil ecological health (Lisuma et al., 2019 ). Farooq et al. (Rivera et al., 2023 ) proposed nicotine-induced increases in soil total N could result from suppression of ammonia-oxidizing bacteria (AOB) such as Nitrosomonas , Nitrococcus and Nitrobacter . Here, nicotine was positively correlated with Nitrospira and Nitrosomonadaceae (Fig. 1 D). Thus, nicotine likely affects N cycling not only via direct N mineralization but also through modulation of key functional microbial populations. Further studies should clarify the temporal dynamics of nicotine’s legacy on soil N-cycling functions during tobacco cultivation and in subsequent crop rotations. 4.2 Nicotine legacy effect on soil nitrification and fast-cycling N pools While legacy effects on carbon dynamics have received attention, the coupling between C and N cycles during decomposition implies that N-related legacies warrant equal scrutiny (Walke et al., 2019; Schiedung et al., 2023 ). Cong et al. (Cong et al., 2014 ) attributed soil legacies partly to altered N availability, via conducting a laboratory experiment of single or litter mixing with soils previously cultivated single plant species or mixtures. The mechanism underlying such legacies, whether driven primarily by shifts in soil biota or in soil chemistry, remains debated. Evidence supports both pathways: some studies emphasize chemical changes (Mooshammer et al., 2022 ), whereas others highlight microbial community restructuring (Ayres et al., 2006 ). Nicotine-degrading bacteria, such as Arthrobacter and Pseudomonas , metabolize nicotine via pyridine and pyrrolidine pathways, using it as both C and N source. Under field conditions, nicotine from incorporated tobacco residues is typically fully degraded within 60 days, with nitrification capacity restored (Ma et al., 2018 ). Our findings indicate that nicotine influences nitrification through two complementary mechanisms: (1) direct input of nicotine-derived N (chemical pathway), and (2) stimulation of nitrifier activity (biotic pathway). Containing 17% N, nicotine treatments elevated soil NH₄⁺ levels, with the highest concentration observed at 100 mg kg⁻¹ (Fig. 3 ). Correspondingly, gross nitrification rates were higher in nicotine-treated soils. This stimulatory effect persisted for about 6 weeks, aligning with nicotine degradation kinetics. Net changes in soil N also depends on nicotine-induced shifts in microbial diversity, which may promote N loss via organic N mineralization. Nitrification rates increased with nicotine concentration, suggesting selective stimulation of nitrifier populations. Soil nitrification rates varied among nicotine treatments and increased with nicotine concentrations, indicating this increase may be due to the presence of certain nicotine causing higher nitrifier abundance. However, in the absence of fertilizer, N availability was a limiting factor for nicotine’s legacy on nitrification. Additionally, the increased nitrification rate in nicotine treatments related with the higher soil N availability for decomposer community may explain the accelerated soil fast-cycling N pools (NH 4 + and NO 3 − ) in tobacco monoculture fields. These results suggested that association of nicotine legacy effects with soil nitrification rates is prevalent not only with nicotine concentrations but also with soil N conditions. Furthermore, the soil legacy effect may predominantly act on priming the mineralization of organic matter. 4.3 AOA and AOB response to nicotine and contribution to nitrification Nicotine exhibits a concentration-dependent priming effect on nitrifying microorganisms, on day 3, slightly stimulate soil nitrogen mineralization at low concentration (10 mg kg⁻¹) whereas significantly inhibit the ammonia oxidation process at high concentration (100 mg kg⁻¹, Fig. 4 ). Thereafter, the priming responses remained consistently dependent on nicotine concentration from day 7 to day 42. Specifically, nitrification rates significantly correlated with amoA -AOB copy numbers, but not with amoA -AOA over two weeks after nicotine exposure (Fig. 4 ). Nicotine effects on AOA abundance occurred in hysteresis, i . e ., 6 weeks after nicotine cultivation. This is consistent with several previous studies, as reviewed by Nannipieri et al. ( 2023 ). Legacy effects of rhizodeposition on soil microbial communities may persist for several months to several years, especially in monocropping soil (Matson et al., 2013). Recently, Yang et al. ( 2016 ) supposed that the nitrification activity was mainly due to AOB, as AOB abundance being easier changed by ammonium fertilizer than AOA abundance. Nitrification causes NO 3 - leaching and N 2 O emission, which resulted in the fertilizer loss environmental risk (Wrage et al., 2001 ). In our study, gross nitrification rates range from 20 to 70 mg N kg -1 d -1 . Since soil AOA and AOB are the rate-limiting step of ammonia oxidation to nitrite, they can be used to reflect the legacy effect of nicotine on soil nitrification (Schleper et al., 2010 ; Norton et al., 2011). In the present study, amplicon pyrosequencing revealed that the community composition of AOB was significantly altered by nicotine of N added treatments, while AOA community was less responsive to N treatment (Figs. 4 and 5 ). Overall, we observed higher abundance of AOB community compared to AOA in nicotine treatments, it is similarly to observations of Yang et al. ( 2016 ) in agricultural soils. At the first 3 days, 10 mg kg -1 nicotine improved the abundance of AOB and AOA, but decreased at 100 mg kg -1 , suggesting nicotine legacy effect on nitrifiers was not only by supplying carbon, but probably also by influencing enzyme activity of nitrifiers, e . g . AMO and HAO (O'Sullivan et al., 2016 ). After two weeks, copy numbers of AOB increased in nicotine superimposed ammonium-fertilizer treatments but not in the control, and resulted in a higher ratio of AOB to AOA, this legacy effect of nicotine lasted for more than 6 weeks (Fig. 2 D). Likely, published study shown that following the return of tobacco residues to the field, nicotine content in soil decreases to an extremely low level of 0.009–0.098 µg/g within 40 days (Ma et al., 2018 ). As shown in this study, AOB- amoA OTUs are affiliated with Nitrosopira occupied 99% of OTUs, which commonly detected in agricultural soils (Chu et al., 2007 ; Habteselassie et al., 2013 ; Yang et al., 2016 ). The results indicate dominance of Nitrosopira in tobacco soil and potential enhancement of nicotine on soil nitrification by priming Nitrosopira (Fig. 1 D and Fig. 4 ). The AOB communities were mainly affiliated with Nitrosospira sp. Np 39 − 19, a finding consistent with other studies that have shown the dominance of this group of AOB in a number of agricultural soils (Habteselassie et al., 2013 ; Jiang et al., 2014 ; Li et al., 2018 ). Tourna et al. ( 2010 ) revealed Nitrosospira sp. Np 39 − 19 often outcompetes other Nitrosospira under high ammonium conditions. Another abundant AOB OTUs was classified into Nitrosospira multiformis ATCC 25196 subcluster, which is often abundant in soils. Ammonia-oxidizing bacteria (AOB), such as Nitrosomonas and Nitrosospira , have been known for some considerable time but have generally found to be inactive in acidic conditions (Li et al., 2018 ). In this study, we observed that AOA also plays a role in nitrification potential, especially in the control and NT10 treatments. Since AOA may have a higher affinity for ammonia than AOB (Yang et al., 2016 ), we expected AOA community might dominate in-situ nitrification, and this hypothesis was disapproved by findings that the addition of nicotine did not inhibit the gross nitrification rate in whole soil samples. Li et al. (Li et al., 2018 ) discovered that in cropped soil AOB dominantly contributed to the nitrification activity under saturated ammonium conditions, while AOA activity dominated without ammonium addition. This is consistent with other studies that AOA dominate nitrification activity when ammonia produced by mineralization of soil organic matter. AOA amoA OTUs all affiliated with Nitrosopumilaceae and Nitrosopumilus , which commonly detected in deep-sea and marine (Metcalf et al., 2012 ; Abdulaziz et al., 2020 ; Garritano et al., 2023 ). Most of AOA amoA gene sequences were distributed into two clades as one cluster hold a 100% matching score with MF324845, while the other was clustering with Nitrososopumilus ureiphilus strain PS0 and Nitrosarchaeum koreeense MY1, suggested that these three clusters often outcompete other AOA- amoA under high nicotine conditions. Collectively, our results reveal for the first time that high genomic diversity of the class Nitrosopumilaceae across soil systems and provide novel insights into their adaptive mechanisms and evolutionary histories. 4.4 Functional gene and metabolic pathway validation: nicotine effects on microbial nitrogen transformation functions Beyond shifts in microbial abundance and composition, the functional impacts of nicotine on nitrogen-cycling microorganisms require validation at the genetic and metabolic levels. Recent advances in metagenomics and metatranscriptomics have provided deeper insights into soil nitrogen cycling processes. For example, Zhao et al. ( 2021 ) reported that in tobacco rhizosphere soil, AOB amoA (especially Nitrosospira cluster 8a) gene transcription levels were significantly higher than those of AOA and positively correlated with nitrification rates, supporting AOB’s functional dominance in nicotine-affected systems. Our observed increase in AOB amoA gene copies aligns with elevated nitrification; however, future studies should incorporate amoA mRNA quantification or proteomic assays to verify whether nicotine directly enhances AOB ammonia oxidation activity. Nicotine-degrading microbes such as Pseudomonas and Arthrobacter often carry nicotine dehydrogenase gene clusters ( nic genes), whose expression may interact with nitrogen cycling pathways. Liu et al. ( 2022 ) demonstrated that Pseudomonas putida S16 upregulates nicA (encoding nicotine oxidase) under NH₄⁺-rich conditions while suppressing amoA expression, suggesting potential substrate competition between nicotine degradation and ammonia oxidation. The enrichment of Intrasporangiaceae and Pseudarthrobacter in the nicotine treatments warrants further investigation, via qPCR or stable isotope probing, to assess whether these taxa functionally participate in nicotine degradation and how this influences nitrogen transformation. Additionally, nicotine may affect denitrification functional genes (e.g., nirS , nosZ ) and greenhouse gas emissions. Chen et al. ( 2020 ) found that tobacco straw incorporation increased nirS gene abundance and N₂O emissions. Whether nicotine similarly influences denitrifier activity and N₂O flux remains to be tested through combined functional gene quantification and gas chromatography. In summary, integrated multi-omics approaches (metatranscriptomics, proteomics) coupled with isotope tracing ( e.g. , ¹³C-nicotine, ¹⁵N-ammonium) are needed to directly verify nicotine’s effects on microbial functional activity, nitrogen pathway regulation, and legacy mechanisms in tobacco soils. Declarations Conflict of 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. Author Contribution Lin Zhang did the conceptualization, validation, investigation, figures drawing, data curation, wrote the main manuscript text and reviewed the manuscript.Qing X. Li did the manuscript review.Guitong Li did the supervision and manuscript review. Acknowledgement We would like to thank Bijie Tobacco Company of Guizhou province for supplying the nicotine standard. Henan Dabieshan National Field Observation and Research Station of Forest Ecosystem (2023XYQN09) also acknowledged. This work was financially supported in part National Natural Science Foundation of China (NO. 32301444) and the USDA (HAW5032-R). Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Ayres E, Dromph KM, Bardgett RD (2006) Do plant species encourage soil biota that specialize in the rapid decomposition of their litter? 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J Environ Sci 34:10–19 Zhu K, Zhu W, Zhang W, Liu J, Ding C (2023) Characteristics of phyllosphere microbial communities associated with three different plants in the semi-arid areas of northwest liaoning. J Soil Sci Plant Nutr 23:2066–2079 Zhao T, Liu J, Wang S, Li X, Bao Z, Long H, Wu J, Chen L, Yang Z, Shen Z, Li R, Shen Q, Ran W (2021) Nitrosospira cluster 8a plays a predominant role in the nitrification process of a tobacco-planted soil with high nitrogen content. J Soils Sediments 21:2591–2604 Additional Declarations No competing interests reported. Supplementary Files floatimage1.jpeg Graphical Abstract Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-9136135","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":610093783,"identity":"8c5cf975-fd56-4abc-ac45-babcecc54e91","order_by":0,"name":"Lin Zhang","email":"","orcid":"","institution":"Henan University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhang","suffix":""},{"id":610093784,"identity":"e4c12f98-881a-4514-80ec-4cb809b7c175","order_by":1,"name":"Qing X. Li","email":"","orcid":"","institution":"University of Hawaiʻi at Mānoa","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"X.","lastName":"Li","suffix":""},{"id":610093785,"identity":"72a31ba3-3e38-4589-8f6d-2573b0e4f4d5","order_by":2,"name":"Guitong Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPghlA6F4iNHCxsAMotJI13KYFC386w8+Lvh1Xp5/RgLjg7dtDPLmBLVIPGY2ntl323DGjQRmw7ltDIY7GwhqOcwmzdtzO4HhRgKQ0caQYHCAOC3nEuRvJLD/Jk4LfzObNM+PAwkGQFuYibSF2diYtyHZcOOZh82Sc85JGG4gpIWf/+DDxzx/7OTljicf/PCmzEaeoC0MEgkMDIxtIBZjA4hLSD3IGpChf4hQOApGwSgYBSMXAACzEjo0RK0wQQAAAABJRU5ErkJggg==","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Guitong","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-03-16 09:55:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9136135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9136135/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105317577,"identity":"e9e8d75c-ed57-4829-956e-2ffeae18da3f","added_by":"auto","created_at":"2026-03-24 16:40:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":378179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotine effect on soil bacterial community during the incubation period. \u003c/strong\u003eDominant communities composition (A) and relative abundance (B); Variance partitioning canonical correspondence analysis (VPA analysis; C), the different color circle present different factors (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and nicotine) and number respect the factor or there interact effect on soil bacterial community; D) is the correlation hot map analysis (blue respect the positive correlation and red respect the negative correlation; the ducker the color is, the correlation is more high; * means \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** means \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/9757a0ef6158658ac94f8d27.jpeg"},{"id":105317700,"identity":"a3d8a200-57a0-4b93-af5d-c5f7a3772dcb","added_by":"auto","created_at":"2026-03-24 16:40:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamics of dominant bacterial phylum under the nicotine treatments across the three culturing times.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/806b0de455a3b83e88c1eb62.jpeg"},{"id":105317572,"identity":"8e0e909c-a3b9-4ba6-9d11-de21e636917e","added_by":"auto","created_at":"2026-03-24 16:40:36","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":179749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamics of soil available nitrogen (NH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e, A; NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e, B), potential nitrification rate (C) and nicotine degradation (D) in different nicotine doses treatments. \u003c/strong\u003eNT0, NT10, and NT100 standard for nicotine at concentrations of 0, 10, and 100 mg/kg, respectively.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/b2dcd88d499479cb7212ec4b.jpeg"},{"id":105317635,"identity":"1aa1da37-67b9-4d31-8242-2bfb3fae0921","added_by":"auto","created_at":"2026-03-24 16:40:42","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":188514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCopy numbers of AOB-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eamoA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and AOA-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eamoA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in the different pulse-nicotine treatments.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/2549eda15e40acf4abb8a156.jpeg"},{"id":105317573,"identity":"3227e296-9d05-4f42-9f48-cc704d647af3","added_by":"auto","created_at":"2026-03-24 16:40:36","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative abundances (%) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eamoA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genus from AOB (A) and AOA (B) during the study, determined by 16S rRNA Amplicon sequencing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: \u003cem\u003eNitrosomonas\u003c/em\u003enot detected in the samples at initial day and 3-day.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/96385ca8e022a82b42a296db.jpeg"},{"id":105317729,"identity":"46cc9584-eb78-4a77-8a0e-403a47c6e379","added_by":"auto","created_at":"2026-03-24 16:41:04","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":356024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative abundance of nitrifiers (AOB, A; AOA, B) and NMDS (Non-metric Multidimensional Scaling) analysis (AOB, C; AOA, D) of different nicotine treatments during culturing periods.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/dbaeda45075c0cd2ab1a55ca.jpeg"},{"id":105317650,"identity":"2b2f1bdf-fec5-4399-aae4-965e0ec813bc","added_by":"auto","created_at":"2026-03-24 16:40:49","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":344929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCommunity diversity of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/66358c6697e6a0684f48714b.jpeg"},{"id":105317746,"identity":"b7d3d3ec-a4d7-463b-b6ad-a38dedab3757","added_by":"auto","created_at":"2026-03-24 16:41:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":186535,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCommunity differences of AOA and AOB analyzed by Principal coordinate analysis (PCoA) based on the abundance of all AOA and AOB \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eamoA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene OTUs (weighted UniFrac).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/1503a29568c3440023e50c4a.png"},{"id":105317673,"identity":"78921db2-3aa8-4218-88a7-7bcbd9ebcd16","added_by":"auto","created_at":"2026-03-24 16:40:53","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":124424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeighbor-joining phylogenetic trees of bacterial and archaeal \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eamoA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene sequences reflecting the relationship between sequences obtained from the nicotine treated soil and reference organisms.\u003c/strong\u003e Sequences clustered using a 90% identity cutoff, and the number of sequences of each cluster shown in parenthesis. Accession numbers for the reference sequences also shown in parenthesis. Bootstrap values (1000 replicates) higher than 50% indicated at branch points. Branch lengths correspond to sequence differences as indicated by the scale bar. (A) Bacterial \u003cem\u003eamoA\u003c/em\u003e, and (B) archaeal \u003cem\u003eamoA\u003c/em\u003e phylogenetic trees.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/793a3235de9e823478d4690a.jpeg"},{"id":106680290,"identity":"4529786a-d204-43d0-bf91-4b2e9cc1ba36","added_by":"auto","created_at":"2026-04-11 14:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3600497,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/318b54b1-9e0e-4309-9d34-80827451cd3a.pdf"},{"id":105317563,"identity":"b1b92731-2ad1-46a5-a9f5-51c5178f79cc","added_by":"auto","created_at":"2026-03-24 16:40:33","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":285987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9136135/v1/ba4d5d0aaee133940c16ceff.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Legacy nicotine stimulates soil nitrification and available nitrogen in long-term tobacco cropping system via microbial priming and nitrogen supply","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eNicotine\u0026nbsp;stimulation on nitrification lasted for 42 days via direct N supply and nitrifier priming.\u003c/li\u003e\n \u003cli\u003eNicotine\u0026nbsp;enriched specific bacterial taxa like\u0026nbsp;\u003cem\u003eI\u003c/em\u003e\u003cem\u003entrasporangiaceae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBryobacter\u003c/em\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAmmonia-oxidizing bacteria (AOB) was more responsive to nicotine than ammonia-oxidizing archaea (AOA).\u003c/li\u003e\n \u003cli\u003eAOB (\u003cem\u003eNitrosospira\u003c/em\u003e) dominated the nicotine-induced nitrification response.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDominant AOA genera were \u003cem\u003eNitrosopumilaceae\u003c/em\u003e and \u003cem\u003eNitrosopumilus\u003c/em\u003e.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eSoil legacy effects refer to the influences of prior soil conditions on subsequent soil functions (Nannipieri et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zou et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Such effects reflect \u0026ldquo;soil memory\u0026rdquo;, often resulting from microbial adaptation to environmental changes such as temperature (Targulian et al., 2019; Lian et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), drought (Bennett et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and grazing intensity (Matson et al., 2013; Ren et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Legacies can also arise from soil chemical and biological processes mediated by rhizodeposition (Semchenko et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While legacy impacts on soil microbiome diversity and composition are well documented (Turner et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ling et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), less is known about rhizodeposit-mediated legacies on nutrient cycling and their agronomic utility. A better understanding of these mechanisms could inform strategies for soil health management, crop rotation design, and the development of bio-simulants that mimic positive plant legacy effects.\u003c/p\u003e \u003cp\u003eIn tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e L.) cultivation excessive agrochemical inputs, including nitrogen fertilizers and biochemicals such as nicotine, can induce long-lasting negative legacies (Postigo et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Nicotine (C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e), a major alkaloid in tobacco wastes (2%\u0026ndash;8% of leaf dry biomass) and root exudates, accumulates in soil at concentrations of 10\u0026ndash;20 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the root zone (Lisuma et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Annual tobacco processing generates approximately one million tons of solid waste with nicotine content averaging 18 g kg⁻\u0026sup1;dry weight (Wang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These nicotine residues in field need more than 8 months to degradation (approximately 90%); however, it is legacy effect may persist in soil for over a year (Ma et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nicotine exerts both positive and negative legacy effects on soil microbial communities (Rivera et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), is also readily absorbed by crop roots and translocated to edible tissues, poses a growing threat to food safety. Conventional management often focuses on mitigating negative impacts, such as reduced microbial diversity and environmental pollution. However, the potential to harness positive legacies via rhizodeposit-mediated microbial functions remains underexplored. Notably, tobacco cultivation has been associated with increased total soil N but decreased availability of phosphorus (P), potassium (K), and sulfur (S) (Rivera et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nicotine in the rhizosphere may enhance plant uptake of N and zinc (Zn) while reducing P and K availability (Lisuma et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Wild-type tobacco plants subjected to nitrogen deficiency exhibited a reduction in nitrogen use efficiency (NUE) accompanied by an increase in nicotine accumulation (Farooq et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Lisuma et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported, before tobacco established, soil nicotine (0.01 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was negligible, while increased to 2.71 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of harvesting unfertilized tobacco, to be more upon to 8.69 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in soil of fertilized tobacco, results showed significant interactions among fertilizer application and soil nicotine content. These interactions suggest that nicotine may legacy soil nitrogen availability for subsequent crops (Wu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), yet the persistence and mechanisms of nicotine effects on nitrogen cycling remain unclear. This study aimed to investigate the persistence of nicotine rhizodeposits, their legacy effects on soil microbiome composition, and their role in fast-cycling nitrogen pools.\u003c/p\u003e \u003cp\u003eModern agricultural systems often exhibit high nitrification activity, leading to low nitrogen use efficiency (Beeckman et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nitrification, a key step in nitrogen cycling, is influenced by root exudates that can either stimulate or inhibit nitrifier activity. For example, legume exudates show little inhibitory effect, whereas cereals like sorghum produce biological nitrification inhibitors (Coskun et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Soybean isoflavones stimulate \u003cem\u003eNitrosomonas\u003c/em\u003e (Basalirwa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), highlighting the potential for rhizodeposit-mediated nitrification regulation, while sorghum (\u003cem\u003eSorghum bicolor\u003c/em\u003e) presented high capacity in biological nitrification inhibitors (Subbarao et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These examples highlight the potential for rhizodeposit-mediated soil nitrification, which exploited by the application of bioactive compounds that act as nitrification inhibitor or augmenter against or improve specific agrochemicals as part of an integrated nitrogen management strategy. Nicotine, with its heterocyclic structure, serves as a useful model for studying tobacco-derived effects on soil processes. Understanding the persistence of nicotine\u0026rsquo;s impact is essential for evaluating its legacy contribution to soil nitrogen dynamics and developing nitrogen management strategies in monocropping systems.\u003c/p\u003e \u003cp\u003eHere, we conducted a factorial microcosm experiment with soil from a long-term tobacco field to disentangle the rhizodeposit-induced legacy effects of nicotine on nitrogen availability. We assessed interactions between tobacco soil biota and chemical factors, defining rhizodeposit-mediated legacy as persistent changes in the soil microbiome following nicotine exposure. We hypothesized that AOA and AOB communities would respond differently to varying nicotine concentrations, influencing their relative contributions to nitrification for tobacco culturing.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.1. Experimental details and treatments\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSoil was collected from a 10-year tobacco field in Heishi Town, Bijie City, Guizhou Province, China (104.00\u0026deg;N, 26.76\u0026deg;E). Classified as Mollic Gleysols (FAO), its chemical properties are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Soil was pre-incubated at 25\u0026deg;C for 7 days at 60% water-holding capacity. Nicotine (99% purity) was provided by Bijie Tobacco Company.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of chemical properties of soils.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSoil\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable K (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e196.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal P (g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConductivity (cmol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal organic carbon (g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal N (g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN atom percent excess (atom%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA 3 \u0026times; 2 microcosm experiment included three nicotine concentrations (0, 10, 100 mg kg⁻\u0026sup1; dry soil) and two nitrogen levels (0 and 50 mg N kg⁻\u0026sup1;). Preliminary tests with different nicotine dosages (0.1, 1.0, 10, 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry soil), and no nicotine (as control) carried out to comparing and selecting nicotine concentrations, showed that 0.1 and 1.0 mg kg⁻\u0026sup1; nicotine had no significant effect on soil NH₄⁺ and NO₃⁻ compared to the control. Based on Preliminary tests data, for comparison, selected three nicotine concentrations (0, 10, 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry soil) to analyzing nicotine legacy effect, \u003cem\u003ei\u003c/em\u003e.\u003cem\u003ee\u003c/em\u003e. control, NT10, NT100. Nicotine solutions were applied daily for 10 days, mixed into fresh soil (2.0 kg dry weight equivalent), and incubated in the dark at 25\u0026deg;C. Soil samples were destructively collected at 1, 3, 7, 14, 28, and 42 days, sieved (2 mm), and divided for DNA extraction (\u0026minus;\u0026thinsp;80\u0026deg;C) and chemical analysis (4\u0026deg;C). \u0026sup1;⁵N labeling (10 atom%) was used to track N dynamics. Each treatment had 18 replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Soil chemical properties\u003c/h2\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were extracted with 2.0 M KCl (1:4 soil: solution) and analyzed by flow injection (QuikChem 8500). Soil organic carbon was determined according to the Walkley Black method, and total N by the Kjedahl digestion, pH in a 1:2 soil-water suspension, and conductivity in 1:2 soil-KCl. Residual nicotine was measured spectrophotometrically at 602 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Gross nitrification rates\u003c/h2\u003e \u003cp\u003eGross nitrification rates were measured with \u0026sup1;⁵NO₃\u003csup\u003e\u0026minus;\u003c/sup\u003e. Soil subsamples (20 g dry weight) received 2.0 mL of K\u003csup\u003e15\u003c/sup\u003eNO₃ solution (10 atom% \u0026sup1;⁵N, 50 mg N kg⁻\u0026sup1;), was gradually (separate 10 times) added and carefully mixed, creating a final soil water content of 0.1 kg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Immediately following the soil mixture, one-part subsamples (three replications) were harvested and extracted with 2.0 M KCl (1:4 of soil: solution by mass) to determine NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration (\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e) and \u003csup\u003e15\u003c/sup\u003eN enrichment at time-0 (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e). Then other samples (three replications for each treatment) placed in 200 mL Mason jars with lids containing butyl rubber septa and with 1.0 mL water at bottom of the jar to minimize loss of moisture from soil. The Mason jars placed in a constant temperature (25℃) incubator in the dark for 24 h, and detected soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e) and \u003csup\u003e15\u003c/sup\u003eN enrichment (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e) at time-24 h. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N detected with sequential diffusion method. Briefly, 25 mL KCL extraction solution added into the regent bottle with screw cap (100 mL). Ammonia traps (filter pack) were prepared by taking 1 cm diameter GF/D filter (What man #1823010) on the central of 2.5 cm diameter Teflon membranes (10 um pore-size; Millipore LCWP 02500), and 25 \u0026micro;L of 2 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were pipetted onto the center of the GF/D filter, then PTFE tape was immediately sealed with tweezers. Immediately put the prepared filter pack and 3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e reagent grade MgO (combusted at 450℃ for 4 h) into bottle and sealed, and incubated in a custom-made shaker (140 rpm) at 25℃ for 7 days. After incubation, took the filter pack into desiccator with silica gel and an open container of concentrated sulphuric acid (to remove trace ammonia) for 1\u0026ndash;2 days, then the small filter was stored in individual vials for detecting the \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. The incubation solutions were then kept shaking for 2 days to allow the NH\u003csub\u003e3\u003c/sub\u003e in the bottle vented. After then 0.2 g Devarda\u0026rsquo;s Alloy were added into the solution, and the filter pack were suspended and incubated as above, finally obtaining the samples for \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e detection. The total N concentration and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN of soil were analyzed by an Elemental Analyzer System (Vario PYRO cube; Elementar Analysensysteme GmbH) coupled to an Isotope Mass Spectrometer (Isoprime 100; Elementar Analysensysteme GmbH). The gross nitrification rate calculated based on differences of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN between pre-incubated soil and incubated soil for 24 h, the equation was according to Davidson et al. (1991) and Zhang et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) as below:\u003c/p\u003e \u003cp\u003e \u003cem\u003eF\u003c/em\u003e=[(\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e-\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e)\u0026times;ln (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e/\u003cem\u003eA\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e)]/[\u003cem\u003et\u003c/em\u003e\u0026times;ln (\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e/\u003cem\u003eQ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e)] (1)\u003c/p\u003e \u003cp\u003ewhere, \u003cem\u003eF\u003c/em\u003e (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) is gross nitrification rate, \u003cem\u003et\u003c/em\u003e is the incubation time (here is 24 h); \u003cem\u003eQ\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e and \u003cem\u003eQ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e are NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at time-0 and time-24 h, respectively; while \u003cem\u003eA\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e are the initial abundance of \u003csup\u003e15\u003c/sup\u003eN (atom %) and \u003csup\u003e15\u003c/sup\u003eN (atom %) of soil after 24 h incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Soil DNA extraction and qPCR analysis\u003c/h2\u003e \u003cp\u003eDNA was extracted from 0.5 g soil using the FastDNA Spin Kit (MP Biomedicals). The 25-\u0026micro;L reaction mixture used (containing 10 \u0026micro;L of 10-fold diluted soil DNA) for soil DNA extraction. DAN extracts were quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), then used as PCR templates. Primer pair 338F (5\u0026rsquo;-ACTCCTACGGGAGGCAGCA-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;) used for amplifying V3 and V4 regions of bacterial 16S ribosomal RNA (rRNA) genes, then the amplified products were purified by AMPure XP beads. Quantitative PCR of AOA and AOB \u003cem\u003eamo\u003c/em\u003eA genes measured using the SsoAdvanced SYBR Green Supermix and a CFX CONNECT Real-Time PCR Detection System (Bio-Rad laboratories, Hercules, CA, USA). Primers \u003cem\u003eamoA\u003c/em\u003e 19F and \u003cem\u003eamoA\u003c/em\u003e 643R used to quantify AOA \u003cem\u003eamoA\u003c/em\u003e gene abundance, while primers \u003cem\u003eamoA\u003c/em\u003e 189F and \u003cem\u003eamoA\u003c/em\u003e 2R used to quantify AOB \u003cem\u003eamoA\u003c/em\u003e gene abundance (Habteselassie et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Amplifications carried out as follows: an initial denaturation step of 95℃ for 10 min, 40 cycles of 95℃ for 45 s, 60℃ for 1 min for AOB or 58℃ AOA, and 72℃ for 45 s, and a final extension step of 72℃ for 10 min. Reaction efficiencies greater than 90%, and \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e \u0026gt;0.97.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.5. Statistical analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eRepeated-measures ANOVA (SPSS 21.0) assessed treatment effects on soil properties and \u003cem\u003eamoA\u003c/em\u003e copy numbers. Two-way ANOVA evaluated nicotine and nitrogen effects on nitrification rates, soil microbial diversity, AOA-\u003cem\u003eamoA\u003c/em\u003e and AOB-\u003cem\u003eamoA\u003c/em\u003e relative abundance. Linear mixed-effects models tested interactions between nicotine and time on diversity indices (indexes of Shannon, Simpson and Chao 1) \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effects of nicotine on soil bacterial communities\u003c/h2\u003e \u003cp\u003eApproximately 80,000 high-quality 16S rRNA sequences were obtained per sample. Bacterial composition shifted with nicotine treatment and incubation time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Dominant phylum included \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003ePseudarthrobacter\u003c/em\u003e, \u003cem\u003eMND1\u003c/em\u003e, \u003cem\u003eIntrasporangiaceae\u003c/em\u003e and \u003cem\u003eBryobacter\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Among these, relative abundance of \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eIntrasporangiaceae\u003c/em\u003e increased under nicotine treatment, while \u003cem\u003eBradyrhizobium\u003c/em\u003e and MND1 decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003ePseudarthrobacter\u003c/em\u003e became the overwhelmingly dominant genus in the 100 mg kg⁻\u0026sup1; nicotine treatment by day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Variance partitioning indicated nicotine explained 22% of bacterial community variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Nicotine correlated more strongly with NH₄⁺ than NO₃⁻ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, some nitrifiers \u003cem\u003ee\u003c/em\u003e.\u003cem\u003eg\u003c/em\u003e. OUT 23 (\u003cem\u003eNitrospira\u003c/em\u003e) presented a negative correlation with nicotine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Soil inorganic N and gross nitrification\u003c/h2\u003e \u003cp\u003eNicotine and N fertilization significantly affected inorganic N and gross nitrification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Nicotine's primming of soil nitrification is dosage-dependent: an initial inhibition phase (\u0026lt;\u0026thinsp;100 mg kg⁻\u0026sup1;) is followed by a time-dependent recovery period (7\u0026ndash;42 days). The 100 mg kg⁻\u0026sup1; treatment showed higher NH₄⁺, NO₃⁻, and gross nitrification, peaking at day 14 for NH₄⁺ and nitrification. Specifically, the 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nicotine treatment increased soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the first 14 days followed with a decrease, while the other two nicotine treatments decreased NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the first 14 days and finally equalized to the control after 21 days. Nitrate increased with incubation time and residual nicotine concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Gross nitrification rates were higher in fertilized soils and increased with nicotine dosage. In addition, Nicotine degraded rapidly within 3 days and returned to baseline by day 42.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Abundance and structure of AOA and AOB\u003c/h2\u003e \u003cp\u003eInitial \u003cem\u003eamoA\u003c/em\u003e copy numbers were 6.9\u0026times;10\u003csup\u003e4\u003c/sup\u003e g⁻\u0026sup1; (AOA) and 7.3\u0026times;10\u003csup\u003e4\u003c/sup\u003e g⁻\u0026sup1; (AOB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Both AOA-\u003cem\u003eamoA\u003c/em\u003e and AOB-\u003cem\u003eamoA\u003c/em\u003e numbers were significantly affected by nicotine dosage and incubation time. Nicotine influenced \u003cem\u003eamoA\u003c/em\u003e expression both in AOA and AOB in a dosage-dependent manner: on day 3, a priming effect was observed at low concentration (10 mg kg⁻\u0026sup1;), while inhibition occurred at high concentration (100 mg kg⁻\u0026sup1;). This initial phase was followed by a sustained period (days 7\u0026ndash;42) which priming effects continued to depend on nicotine concentration. Additionally, the AOB/AOA \u003cem\u003eamoA\u003c/em\u003e gene copy number ratio ranged from 0.4 to 1.5, consistently higher in nicotine-treated groups than in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStructure of AOB and AOA communities revealed by analysis of \u003cem\u003eamoA\u003c/em\u003e genes in each sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Totals of 6.2\u0026times;10\u003csup\u003e4\u003c/sup\u003e and 2.3\u0026times;10\u003csup\u003e5\u003c/sup\u003e sequence reads obtained for AOB and AOA in controls, respectively, with 2.6\u0026times;10\u003csup\u003e3\u003c/sup\u003e and 2.2\u0026times;10\u003csup\u003e3\u003c/sup\u003e unique OTUs respectively (90% identity cut off). Phylogenetic analysis showed that dominant AOB OTUs were affiliated with \u003cem\u003eNirosospira\u003c/em\u003e (\u0026asymp;\u0026thinsp;99% relative abundance) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Relative abundance of AOB OTUs affected by culture period and nicotine dosage. For example, \u003cem\u003eNitrosomonas\u003c/em\u003e increased at days 14 and 42 in control and NT10 but only at day 14 in NT100. AOA communities were dominated by \u003cem\u003eNitrosopumilaceae\u003c/em\u003e (\u0026gt;\u0026thinsp;70%) and \u003cem\u003eNitrosopumilus\u003c/em\u003e. Nicotine initially increased \u003cem\u003eNitrosopumilus\u003c/em\u003e but decreased \u003cem\u003eNitrosopumilaceae\u003c/em\u003e, by day 42, inhibition ceased and abundance rose with nicotine concentration. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nicotine presented the most negative relevance with AOA community on day 42, with the relevant index of -2.5, and presented a negative relevant index of -1.2 with AOB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003e \u003cem\u003eNitrosomonas\u003c/em\u003e not detected in the samples at initial day and 3-day.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInteractions of nicotine and incubation time presented significant effect on AOB and AOA diversities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), AOB was more response to nicotine legacy than AOA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Results of LMMs reflect that sampling time presented a main-factor effect on nitrifiers diversity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while nicotine presented a main-factor effect only on AOB diversity, \u003cem\u003ei\u003c/em\u003e.\u003cem\u003ee\u003c/em\u003e. Shannon and Simpson indexes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In general, diversity of AOB was higher than that of AOA. On day 14, both Simpson and Shannon indexes increased in the 10 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nicotine treatment, while decreased in 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatment; however, on day 42, these two indexes improved in 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatment but decreased in 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatment. Chao 1 index was mainly influenced by incubation time, as increased in AOB and decreased in AOA along with the incubation time. PCoA results show that AOA structure was different between nicotine treatments and incubation times, mainly defined AOA structures as revealed by weighted UniFranc difference matrices, as the community of nicotine treatments on day 3 and day 42 respectively approached together, while the community of nicotine treatments on day 14 merged with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). NT100 at 42 days differed significantly from other treatments, indicating a concentration-dependent legacy effect. Phylogenetic analysis revealed AOB sequences clustering with \u003cem\u003eNitrosospira\u003c/em\u003e sp. Np 39\u0026thinsp;\u0026minus;\u0026thinsp;19 and \u003cem\u003eN. multiformis\u003c/em\u003e ATCC 25196 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). There were four sequences clustering with \u003cem\u003eNitrosospira multiformis\u003c/em\u003e ATCC 25196. Similar with AOB, AOA communities also divided into two branches, there were four OTU sequences posed a 100% match score with MF324845 in this experiment. Another branch was clustering with \u003cem\u003eNitrososopumilus ureiphilus\u003c/em\u003e strain PS0 and \u003cem\u003eNitrosarchaeum koreeense\u003c/em\u003e MY1.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSignificance analysis (\u003cem\u003ep\u003c/em\u003e-values) of influences of nicotine treatments, sampling time and impact on the microbial diversity in the linear mixed-effect models.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eDependent variables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eNicotine treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSampling time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eTreatment \u0026times; Time\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAOB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShannon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e103.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSimpson\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e29.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChao 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.688\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e28.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAOA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShannon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e87.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e26.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSimpson\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e81.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e21.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChao 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e21.737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e4.1\u003c/em\u003e Tobacco nicotine legacy effect on soil microbes and environmental safety\u003c/h2\u003e \u003cp\u003eLegacy effects, especially on crop growth, are often attributed to the presence and relative balance between beneficial and pathogenic organisms (Matson et al., 2013; Huang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zou et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2026\u003c/span\u003e) and finally influence the pedosphere safety. Persistent legacy effects have been reported that the population of soil bacteria and fungi decreased significantly, for example, Santhanam et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed increased plant mortality due to sudden wilt disease in soils with a 15-year tobacco history, indicating long-term alterations in soil microbial communities. In this study, nicotine explained 22% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) of the variation in soil microbial structure and diversity, suggesting it strongly modulates the balance between beneficial and pathogenic taxa. Although nicotine generally reduced bacterial and fungal abundance, certain taxa such as \u003cem\u003eIntrasporangiaceae\u003c/em\u003e (family level) increased in relative abundance under nicotine treatment. This enrichment implies nicotine-tolerant or nicotine-degrading capabilities, consistent with reports that \u003cem\u003eIntrasporangiaceae\u003c/em\u003e can degrade methylbenzene by using isotopic probe technology (Ben Israel et a., 2021). Similarly, \u003cem\u003ePseudomonas\u003c/em\u003e tolerates high nicotine concentrations under a pH range between 6.5 and 7.0 (Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In tobacco soils, \u003cem\u003eBradyrhizobium\u003c/em\u003e is a common rhizobacterium (Zhu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), however, nicotine accumulation slightly suppressed its abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating nicotine potential toxicity to this beneficial genus. In otherwise, persistent legacy effects on nitrogen cycling have been reported various nitrification inhibitors, (\u003cem\u003ee\u003c/em\u003e.\u003cem\u003eg\u003c/em\u003e. sorgoleone, sakuranetin, caffeic acid, ferulic acid), with effects persisting decades after plant removal (Coskun et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). Our results show that nicotine\u0026rsquo; legacy on microbes extended beyond 6-weeks, which may constrain nitrogen fixation in subsequent crops. Given the cost and practicality limitations of large-scale soil remediation, agronomic strategies focusing on crop selection and breeding have emerged as a sustainable frontline defense. In future, the strategic use of low-accumulating crop varieties presents a practical, economically viable, and environmentally sound approach to safeguard the food chain from soil nicotine contamination (Xiang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Xiang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNicotine released into soil can be mineralized, contributing to soil nitrogen pools. However, its acidic nature may alter structure and soil ecological health (Lisuma et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Farooq et al. (Rivera et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) proposed nicotine-induced increases in soil total N could result from suppression of ammonia-oxidizing bacteria (AOB) such as \u003cem\u003eNitrosomonas\u003c/em\u003e, \u003cem\u003eNitrococcus\u003c/em\u003e and \u003cem\u003eNitrobacter\u003c/em\u003e. Here, nicotine was positively correlated with \u003cem\u003eNitrospira\u003c/em\u003e and \u003cem\u003eNitrosomonadaceae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Thus, nicotine likely affects N cycling not only via direct N mineralization but also through modulation of key functional microbial populations. Further studies should clarify the temporal dynamics of nicotine\u0026rsquo;s legacy on soil N-cycling functions during tobacco cultivation and in subsequent crop rotations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2 \u003cem\u003eNicotine legacy effect on soil nitrification and fast-cycling N pools\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWhile legacy effects on carbon dynamics have received attention, the coupling between C and N cycles during decomposition implies that N-related legacies warrant equal scrutiny (Walke et al., 2019; Schiedung et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cong et al. (Cong et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) attributed soil legacies partly to altered N availability, via conducting a laboratory experiment of single or litter mixing with soils previously cultivated single plant species or mixtures. The mechanism underlying such legacies, whether driven primarily by shifts in soil biota or in soil chemistry, remains debated. Evidence supports both pathways: some studies emphasize chemical changes (Mooshammer et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), whereas others highlight microbial community restructuring (Ayres et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Nicotine-degrading bacteria, such as \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, metabolize nicotine via pyridine and pyrrolidine pathways, using it as both C and N source. Under field conditions, nicotine from incorporated tobacco residues is typically fully degraded within 60 days, with nitrification capacity restored (Ma et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur findings indicate that nicotine influences nitrification through two complementary mechanisms: (1) direct input of nicotine-derived N (chemical pathway), and (2) stimulation of nitrifier activity (biotic pathway). Containing 17% N, nicotine treatments elevated soil NH₄⁺ levels, with the highest concentration observed at 100 mg kg⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Correspondingly, gross nitrification rates were higher in nicotine-treated soils. This stimulatory effect persisted for about 6 weeks, aligning with nicotine degradation kinetics. Net changes in soil N also depends on nicotine-induced shifts in microbial diversity, which may promote N loss via organic N mineralization. Nitrification rates increased with nicotine concentration, suggesting selective stimulation of nitrifier populations. Soil nitrification rates varied among nicotine treatments and increased with nicotine concentrations, indicating this increase may be due to the presence of certain nicotine causing higher nitrifier abundance. However, in the absence of fertilizer, N availability was a limiting factor for nicotine\u0026rsquo;s legacy on nitrification. Additionally, the increased nitrification rate in nicotine treatments related with the higher soil N availability for decomposer community may explain the accelerated soil fast-cycling N pools (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) in tobacco monoculture fields. These results suggested that association of nicotine legacy effects with soil nitrification rates is prevalent not only with nicotine concentrations but also with soil N conditions. Furthermore, the soil legacy effect may predominantly act on priming the mineralization of organic matter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3 AOA and AOB response to nicotine and contribution to nitrification\u003c/h2\u003e \u003cp\u003eNicotine exhibits a concentration-dependent priming effect on nitrifying microorganisms, on day 3, slightly stimulate soil nitrogen mineralization at low concentration (10 mg kg⁻\u0026sup1;) whereas significantly inhibit the ammonia oxidation process at high concentration (100 mg kg⁻\u0026sup1;, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Thereafter, the priming responses remained consistently dependent on nicotine concentration from day 7 to day 42. Specifically, nitrification rates significantly correlated with \u003cem\u003eamoA\u003c/em\u003e-AOB copy numbers, but not with \u003cem\u003eamoA\u003c/em\u003e-AOA over two weeks after nicotine exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Nicotine effects on AOA abundance occurred in hysteresis, \u003cem\u003ei\u003c/em\u003e.\u003cem\u003ee\u003c/em\u003e., 6 weeks after nicotine cultivation. This is consistent with several previous studies, as reviewed by Nannipieri et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Legacy effects of rhizodeposition on soil microbial communities may persist for several months to several years, especially in monocropping soil (Matson et al., 2013). Recently, Yang et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) supposed that the nitrification activity was mainly due to AOB, as AOB abundance being easier changed by ammonium fertilizer than AOA abundance.\u003c/p\u003e \u003cp\u003eNitrification causes NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e leaching and N\u003csub\u003e2\u003c/sub\u003eO emission, which resulted in the fertilizer loss environmental risk (Wrage et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In our study, gross nitrification rates range from 20 to 70 mg N kg\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e. Since soil AOA and AOB are the rate-limiting step of ammonia oxidation to nitrite, they can be used to reflect the legacy effect of nicotine on soil nitrification (Schleper et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Norton et al., 2011). In the present study, amplicon pyrosequencing revealed that the community composition of AOB was significantly altered by nicotine of N added treatments, while AOA community was less responsive to N treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Overall, we observed higher abundance of AOB community compared to AOA in nicotine treatments, it is similarly to observations of Yang et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in agricultural soils. At the first 3 days, 10 mg kg\u003csup\u003e-1\u003c/sup\u003e nicotine improved the abundance of AOB and AOA, but decreased at 100 mg kg\u003csup\u003e-1\u003c/sup\u003e, suggesting nicotine legacy effect on nitrifiers was not only by supplying carbon, but probably also by influencing enzyme activity of nitrifiers, \u003cem\u003ee\u003c/em\u003e.\u003cem\u003eg\u003c/em\u003e. AMO and HAO (O'Sullivan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). After two weeks, copy numbers of AOB increased in nicotine superimposed ammonium-fertilizer treatments but not in the control, and resulted in a higher ratio of AOB to AOA, this legacy effect of nicotine lasted for more than 6 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Likely, published study shown that following the return of tobacco residues to the field, nicotine content in soil decreases to an extremely low level of 0.009\u0026ndash;0.098 \u0026micro;g/g within 40 days (Ma et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As shown in this study, AOB-\u003cem\u003eamoA\u003c/em\u003e OTUs are affiliated with \u003cem\u003eNitrosopira\u003c/em\u003e occupied 99% of OTUs, which commonly detected in agricultural soils (Chu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Habteselassie et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The results indicate dominance of \u003cem\u003eNitrosopira\u003c/em\u003e in tobacco soil and potential enhancement of nicotine on soil nitrification by priming \u003cem\u003eNitrosopira\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The AOB communities were mainly affiliated with \u003cem\u003eNitrosospira\u003c/em\u003e sp. Np 39\u0026thinsp;\u0026minus;\u0026thinsp;19, a finding consistent with other studies that have shown the dominance of this group of AOB in a number of agricultural soils (Habteselassie et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Tourna et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) revealed \u003cem\u003eNitrosospira\u003c/em\u003e sp. Np 39\u0026thinsp;\u0026minus;\u0026thinsp;19 often outcompetes other \u003cem\u003eNitrosospira\u003c/em\u003e under high ammonium conditions. Another abundant AOB OTUs was classified into \u003cem\u003eNitrosospira multiformis\u003c/em\u003e ATCC 25196 subcluster, which is often abundant in soils. Ammonia-oxidizing bacteria (AOB), such as \u003cem\u003eNitrosomonas\u003c/em\u003e and \u003cem\u003eNitrosospira\u003c/em\u003e, have been known for some considerable time but have generally found to be inactive in acidic conditions (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we observed that AOA also plays a role in nitrification potential, especially in the control and NT10 treatments. Since AOA may have a higher affinity for ammonia than AOB (Yang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), we expected AOA community might dominate in-situ nitrification, and this hypothesis was disapproved by findings that the addition of nicotine did not inhibit the gross nitrification rate in whole soil samples. Li et al. (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) discovered that in cropped soil AOB dominantly contributed to the nitrification activity under saturated ammonium conditions, while AOA activity dominated without ammonium addition. This is consistent with other studies that AOA dominate nitrification activity when ammonia produced by mineralization of soil organic matter. AOA \u003cem\u003eamoA\u003c/em\u003e OTUs all affiliated with \u003cem\u003eNitrosopumilaceae\u003c/em\u003e and \u003cem\u003eNitrosopumilus\u003c/em\u003e, which commonly detected in deep-sea and marine (Metcalf et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Abdulaziz et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Garritano et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Most of AOA \u003cem\u003eamoA\u003c/em\u003e gene sequences were distributed into two clades as one cluster hold a 100% matching score with MF324845, while the other was clustering with \u003cem\u003eNitrososopumilus ureiphilus\u003c/em\u003e strain PS0 and \u003cem\u003eNitrosarchaeum koreeense\u003c/em\u003e MY1, suggested that these three clusters often outcompete other AOA-\u003cem\u003eamoA\u003c/em\u003e under high nicotine conditions. Collectively, our results reveal for the first time that high genomic diversity of the class \u003cem\u003eNitrosopumilaceae\u003c/em\u003e across soil systems and provide novel insights into their adaptive mechanisms and evolutionary histories.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Functional gene and metabolic pathway validation: nicotine effects on microbial nitrogen transformation functions\u003c/h2\u003e \u003cp\u003eBeyond shifts in microbial abundance and composition, the functional impacts of nicotine on nitrogen-cycling microorganisms require validation at the genetic and metabolic levels. Recent advances in metagenomics and metatranscriptomics have provided deeper insights into soil nitrogen cycling processes. For example, Zhao et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that in tobacco rhizosphere soil, AOB \u003cem\u003eamoA\u003c/em\u003e (especially \u003cem\u003eNitrosospira\u003c/em\u003e cluster 8a) gene transcription levels were significantly higher than those of AOA and positively correlated with nitrification rates, supporting AOB\u0026rsquo;s functional dominance in nicotine-affected systems. Our observed increase in AOB \u003cem\u003eamoA\u003c/em\u003e gene copies aligns with elevated nitrification; however, future studies should incorporate \u003cem\u003eamoA\u003c/em\u003e mRNA quantification or proteomic assays to verify whether nicotine directly enhances AOB ammonia oxidation activity.\u003c/p\u003e \u003cp\u003eNicotine-degrading microbes such as \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eArthrobacter\u003c/em\u003e often carry nicotine dehydrogenase gene clusters (\u003cem\u003enic\u003c/em\u003e genes), whose expression may interact with nitrogen cycling pathways. Liu et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that \u003cem\u003ePseudomonas putida\u003c/em\u003e S16 upregulates \u003cem\u003enicA\u003c/em\u003e (encoding nicotine oxidase) under NH₄⁺-rich conditions while suppressing \u003cem\u003eamoA\u003c/em\u003e expression, suggesting potential substrate competition between nicotine degradation and ammonia oxidation. The enrichment of \u003cem\u003eIntrasporangiaceae\u003c/em\u003e and \u003cem\u003ePseudarthrobacter\u003c/em\u003e in the nicotine treatments warrants further investigation, via qPCR or stable isotope probing, to assess whether these taxa functionally participate in nicotine degradation and how this influences nitrogen transformation.\u003c/p\u003e \u003cp\u003eAdditionally, nicotine may affect denitrification functional genes (e.g., \u003cem\u003enirS\u003c/em\u003e, \u003cem\u003enosZ\u003c/em\u003e) and greenhouse gas emissions. Chen et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that tobacco straw incorporation increased \u003cem\u003enirS\u003c/em\u003e gene abundance and N₂O emissions. Whether nicotine similarly influences denitrifier activity and N₂O flux remains to be tested through combined functional gene quantification and gas chromatography. In summary, integrated multi-omics approaches (metatranscriptomics, proteomics) coupled with isotope tracing (\u003cem\u003ee.g.\u003c/em\u003e, \u0026sup1;\u0026sup3;C-nicotine, \u0026sup1;⁵N-ammonium) are needed to directly verify nicotine\u0026rsquo;s effects on microbial functional activity, nitrogen pathway regulation, and legacy mechanisms in tobacco soils.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \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 \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLin Zhang did the conceptualization, validation, investigation, figures drawing, data curation, wrote the main manuscript text and reviewed the manuscript.Qing X. Li did the manuscript review.Guitong Li did the supervision and manuscript review.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Bijie Tobacco Company of Guizhou province for supplying the nicotine standard. Henan Dabieshan National Field Observation and Research Station of Forest Ecosystem (2023XYQN09) also acknowledged. This work was financially supported in part National Natural Science Foundation of China (NO. 32301444) and the USDA (HAW5032-R).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAyres E, Dromph KM, Bardgett RD (2006) Do plant species encourage soil biota that specialize in the rapid decomposition of their litter? 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J Soil Sci Plant Nutr 23:2066\u0026ndash;2079\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao T, Liu J, Wang S, Li X, Bao Z, Long H, Wu J, Chen L, Yang Z, Shen Z, Li R, Shen Q, Ran W (2021) Nitrosospira cluster 8a plays a predominant role in the nitrification process of a tobacco-planted soil with high nitrogen content. J Soils Sediments 21:2591\u0026ndash;2604\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"nicotine, legacy effect, nitrification, ammonia oxidizing archaea, ammonia oxidizing bacteria","lastPublishedDoi":"10.21203/rs.3.rs-9136135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9136135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe legacy effects of nicotine, alkaloid from tobacco, on soil bacteria and nutrient cycling functions are not fully understood. In this microcosm study using soil from a decade-long tobacco monoculture field. Nicotine exhibited a persistent influence on nitrification for 42 days via direct nicotine-N supply and priming nitrifier activity. Gross nitrification rates and available nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e) content were significantly higher at 100 mg kg\u003csup\u003e− 1\u003c/sup\u003e nicotine than those at 10 mg kg\u003csup\u003e− 1\u003c/sup\u003e. Nicotine significantly altered soil bacterial dynamics (contribution = 0.22) and increased the abundance of \u003cem\u003eIntrasporangiaceae\u003c/em\u003e and \u003cem\u003eBryobacter\u003c/em\u003e. Changes in nitrification rates were positively correlated with increases in ammonia-oxidizing bacteria (AOB)-\u003cem\u003eamoA\u003c/em\u003e copy numbers. Phylogenetic analysis revealed dominant AOB Operational Taxonomic Units (OTUs) affiliated with the genus \u003cem\u003eNirosospira\u003c/em\u003e, closely related to ‘\u003cem\u003eNitrosospira\u003c/em\u003e sp. Np 39 − 19’ (99% identity). Dominant ammonia-oxidizing archaea (AOA) include \u003cem\u003eNitrosopumilaceae\u003c/em\u003e (\u0026gt; 70% of sequences) and \u003cem\u003eNitrosopumilus\u003c/em\u003e. This study enhances understanding nicotine’s role in microbial function shifts and suggests potential strategies for rhizodeposition-based nitrogen management in tobacco fields.\u003c/p\u003e","manuscriptTitle":"Legacy nicotine stimulates soil nitrification and available nitrogen in long-term tobacco cropping system via microbial priming and nitrogen supply","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 16:38:45","doi":"10.21203/rs.3.rs-9136135/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92308fc5-804a-4b6c-90cd-6b46b97ecf41","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-11T14:09:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 16:38:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9136135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9136135","identity":"rs-9136135","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}