Bacillus Benets the Growth of Ambrosia Artemisiifolia by Increasing Available Nutrient Levels

help the acquisition, either directly or indirectly. However, the mechanisms that mediate the positive or negative impact of Bacillus on exotic or native species are poorly understood. Our objective was to determine whether the quantitative and/ or qualitative differences in the Bacillus community present on the exotic Ambrosia artemisiifolia and the native Setaria viridis provide a competitive advantage to the invader over native


Introduction
Invasive plant species often outcompete native species when colonizing new ranges. In principle, the growth and expansion of a plant species in a diverse community of competing organisms are dependent on two components: its ability to absorb nutrients from the soil and its impact on the available soil resources (Chase and Leibold 2003). According to some studies, invasive plant species have a higher nutrient acquisition capacity than native species. This difference has been attributed to the fact that the photosynthetic nitrogen use e ciency and photosynthetic energy use e ciency of invasive species are higher than that of native species in their invaded range (Pattison et al. 1998; Baruch and Goldstein 1999;Feng et al. 2008). Some studies also pointed out that invasive species adapt faster to different levels of nutrient environments and have a higher ability to modify resources than the native species, thus their successful invasion (Davidson et al. 2011;Parker et al. 2013;Luo et al. 2019). Upon the successful invasion of a new range, the invasive plant species may exploit unused resources (Shea and Chesson 2002) or modify resources to bene t themselves, or weaken the growth of other species (Chase and Leibold 2003;Parker et al. 2019). Therefore, it is crucial to investigate the differences in invasive and native plant species potential to modify resources during an invasion.
Soil microbial communities are essential in the soil nutrient cycle (Fraterrigo et al. 2006;Iwaoka et al. 2018). The rhizosphere microbiome is involved in important processes, such as nitrogen xation, mobilization of phosphorus, and alteration of other nutrients (Rodríguez-Caballero et al. 2020). Moreover, invasive plants can alter the microbial community in the rhizosphere and accumulate some bene cial microorganisms in their rhizospheres, such as Bacillus, arbuscular mycorrhizal fungi and Pseudomonas (Ehrenfeld 2003;Kourtev et al. 2003;Gibbons et al. 2017). Bacillus is one of the rhizosphere-promoting bacteria genera known as a natural plant nutrition resource (Saxena et al. 2019). Besides, members of the genus Bacillus are known to have multiple bene cial traits which directly or indirectly aids plants to acquire nutrients (Saxena et al. 2019). Different Bacillus spp. had different ability to x nitrogen, solubilize and mineralize phosphorus (Goswami et al. 2016;Pramanik et al. 2019). Moreover, root exudates are among the important factors that in uence those abilities (Tariq et al. 2007;Zhang et al. 2014;Shakeel et al. 2015;Wang et al. 2020). The difference between the root exudates of invasive and native species may enable the invasive species to recruit speci c Bacillus species in the rhizosphere of the invasive plant species. The bacteria help the invading species exploit more availble resources in the soil and eventually become the dominant plant species. However, only a few studies have investigated the interaction between exotic species and Bacillus species (Dai et al. 2016;Sun et al. 2020). Therefore, understanding the differences in Bacillus communities and function between native and exotic populations would help predict the ecological roles of Bacillus communities in plant invasion.
The exotic species Ambrosia artemisiifolia, commonly known as ragweed, belongs to the family Asteraceae and is widely distributed in China (Xu and Qiang 2004). Its invasion poses a serious threat to biodiversity and agricultural production (Ozaslan et al. 2016). In addition, it often causes human health problems due to its allergenic pollen (Ghiani et al. 2012). According to , A. artemisiifolia invasion changes the rhizosphere microbial community in its rhizosphere soil. Speci cally, when A. artemisiifolia grows with native Setaria viridis (L.) Beauv. (Poaceae) the increased AMF colonization in A. artemisiifolia and the decreased AMF colonization in S. viridis provide a competitive advantage to the invasive species over the native species . Previous studies have shown that Bacillus spp. is often found in the rhizosphere soil of invasive plants (Huangfu et al. 2015;Song et al. 2017). To test the role of Bacillus spp in the invasion of A. artemisiifolia we hypothesized that: (i) the Bacillus community in the A. artemisiifolia rhizosphere differs from that in the rhizosphere of cooccurring native species; and (ii) the assembly of speci c Bacillus species bene ts the invasive species than the native species. Then, we conducted three sets of experiments to test our hypotheses. First, Bacillus was separately isolated from the rhizosphere soil of A. artemisiifolia and S. viridis to compare their effect on the competitive growth of A. artemisiifolia in a greenhouse experiment. Then a comparison of Bacillus diversity was performed to determine whether A. artemisiifolia recruits speci c Bacillus species in its rhizosphere soil. Lastly, the role of speci c Bacillus species in the competitive growth of A. artemisiifolia was evaluated in a greenhouse experiment. The present study results provide a broader understanding of the functional role of Bacillus in promoting the invasion of A. artemisiifolia.  . S. viridis is an annual C 4 monocotyledonous species that is widely distributed in areas invaded by A. artemisiifolia. The experimental plots (3 m × 2 m) were prepared in 2008, with a 1 m isolation zone to prevent edge effects. Three treatments used in the experiment included: (1) S. viridis monoculture (S), (2) an equal mixture of A. artemisiifolia and S. viridis (1:1; A:S), and (3) A. artemisiifolia monoculture (A). Each treatment had ve replicates. Without tilling, the plots were hand weeded to leave bare soil before sowing. The soil was watered to maintain a soil eld capacity of 40%. The seeds were sterilized in 10% H 2 O 2 for 2 min, rinsed with sterile dH 2 O for 2 min, and then rinsed with dH 2 O ve times for 5min. In May 2008, 100 seeds were sown in each plot to begin the experiment (for the mixture (A:S), we used 50 A. artemisiifolia seeds and 50 S. viridis seeds). No fertilizers were applied, and the plant species composition in each plot was maintained by manually removing the weeds throughout the experiment period annually. Each spring of every year, all plants were pulled out of the soil, and the plots were reseeded to ensure that the target plants (A. artemisiifolia and S. viridis) would be exposed to the desired treatment conditions every year.

Soil sampling
After eleven years, the abundance of each plant species had changed, so we reduced the number of replicates to three, with similar plant aerial cover. In 2019, the cover of A. artemisiifolia and S. viridis in the monocultures was 99% and 93%, respectively, and 76% and 20%, respectively, in the mixed treatment. We followed Huber (2011) to identify the outliers of plants and thinned samples to obtain a sample that would robustly re ect the mean. We visually estimated the mean and chose plants that seemed to be typical in each treatment. In each treatment, ve plants were selected per species and pooled together for subsequent index determination. We brushed off the closely adhered soil, which serves as rhizosphere soil, after shaking off the loosely bound soil. The rhizosphere soil from the three treatments was pooled per plot per plant species. The control soil was collected after the removal of the surface soil using vepoint sampling. Three plots were sampled for each of the ve treatments, yielding a total of 15 soil samples (3 treatments × 5 replicates = 15). The soil samples were collected and sieved (< 0.4 mm). For the Bacillus diversity analysis, 6 g of the soil samples was stored at -80°C, while 10 g was stored at 4°C in sterile containers for the Bacillus inoculum experiment.
Bacillus isolation from the rhizosphere soil Bacillus was isolated from the rhizosphere soil of A. artemisiifolia and S. viridis, separately. Brie y, 1 g of sample soil was added to 9 mL sterile distilled water in a sterilized conical ask. The suspension was homogenized and heated in a hot water bath at 90°C for 10 min. After 12 h, 4 mL of supernatant was aspirated and ltered through a membrane lter with a diameter of 0.22 µm to remove allelochemicals and other organic compounds. The Bacillus remained on the membrane and was rinsed ve times with 5mL of sterile distilled water. The Bacillus suspension was cultured on the beef extract peptone liquid medium while shaking (180 rpm) for 24 h at 37°C. The optical density of the suspension was adjusted to approximately 1.0 (O.D at 600 nm) by diluting it with sterile distilled water. The population count of Bacillus was maintained at 10 8 cfu/mL.

Bacillus innoculation design
Three treatments were used to determine the effect of the Bacillus isolated from the rhizosphere of A. artemisiifolia and S. viridis on the competitive advantage they provide to the former. (1) Monoculture of A. artemisiifolia (A), (2) monoculture of S. viridis (S), and (3) an equal mixture of A. artemisiifolia and S. viridis (A:S). Each treatment was further divided into three levels: (1) C, uninoculated treatment; (2) AA, inoculated with Bacillus isolated from the rhizosphere of A. artemisiifolia; and (3) SV, inoculated with Bacillus isolated from the rhizosphere of S. viridis. Seeds of A. artemisiifolia and S. viridis were obtained from the Langfang Experimental Station, surface sterilized with 75% ethanol, and washed with sterile distilled water ve times before sowing in pots (10 × 10 × 12 cm). The soil in the box was composed of sandy clay and vermiculite (v/v, 1:1). The sandy clay was collected near the experimental eld site from an open area that had not been covered with vegetation for the previous three years. Vermiculite was obtained from Baisheng Plant and Flower Co., Ltd., Baoding, China. Basic soil properties included: pH (w/v water = 1:5) of 8.20, organic matter content of 14.29 g/kg, available nitrogen of 52.18 mg/kg, and available phosphorus of 3.4 mg/kg. Seeds were planted in pots with 1 kg soil, and the respective bacterial suspensions (10 mL 10 8 cfu/mL) were added to initiate the experiment. The bacterial suspension was added once a month. After germination, each pot was weeded to contain only four plants (four per species in the monocultures and two per species in the mixed treatment in a substitution design). Each treatment was replicated ten times (3 × 4 treatments × 10 replicates = 120 pots). The pots were placed in a greenhouse for 90 days under a 10-h light: 14-h dark photoperiod at 25°C, arranged in a completely randomized design.

Measurements
The plants grown under the different treatments were harvested after 90 days. The roots were washed free of soil and then oven-dried at 80°C for 48 h to collect growth index data. The entire plant, including aboveground biomass and root biomass, was used to determine dry biomass. The corrected index of relative competition intensity (CRCI) was calculated as described by Oksanen et al. (2006).
, where X is the average biomass of individual plants grown without competition and Y is the biomass which grown in competition.
Experiment II: Comparative analysis of the Bacillus diversity in the rhizosphere soil of A. artemisiifolia and S. viridis

Bacillus isolation
We isolated Bacilli from the soil samples as described by Chikerema et al. (2012), with modi cation. Brie y, soil slurry was prepared by suspending 1 g of soil in 9 mL sterile water. The suspension was homogenized and cultured for 24 h at 25°C and 180 rpm in an incubator (BSD-100, Shanghai Boxun Industry & Commerce Co., Ltd). The soil suspension was serially diluted after being heated in a water bath at 90°C for 10 min. Appropriate dilutions were cultured on beef paste with a peptone medium (10 g peptone, 3 g beef paste, 5 g sodium chloride, and 1000 mL distilled water. pH: 7.2-7.4, 121°C for 30 min) using the spread-plate method. The plates were cultivated at 30°C for 36 h. Based on the number of Bacilli and their separation, 10 − 3 g/mL of the suspension was used to isolate the bacteria. All the colonies in the plates were picked and puri ed on nutrient-agar plates.
Analysis of Bacillus diversity DNA extraction A single colony was picked from the plate and transferred into a 1.5 mL microfuge tube containing a liquid medium (10 g peptone, 3 g beef paste, 5 g sodium chloride, and 1000 mL distilled water. pH: 7.2-7.4, 121°C for 30 min). The homogenized bacterial culture was precipitated by centrifugation at 28600 × g for 10 min at 4°C. The supernatant was discarded, and 400 µL 10× TE (1 M Tris-HCl: 0.5 M EDTA = 5:1) was added to the tube. The cells were homogenized twice in a bead-beater (Mini-Bead Beater 8™, Biospec Products, Bartlesville, OK, USA) at 4.5 speeds (200 rpm) for 45 s then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was discarded, and 400 µL 10× TE was added to eliminate the protein. After centrifugation, the supernatant was removed, and 400 µL 10× TE and 25 µL lysozyme (50 mg/mL) was added to the tube. The cells were then incubated for 12 h at 37°C. Thereafter, 100 µL 10 % SDS was added, and the cells were homogenized twice in a bead-beater at 4.5 speeds (200 rpm) for 45 s. An aliquot (400 µL) of phenol-chloroform-isoamyl alcohol 25:24:1 (Sigma, Milan, Italy) was added and centrifuged at 28600 × g for 10 min at 4°C. The supernatant was transferred to a 1.5-mL tube, and 400 µL ice-cold (− 20℃) chloroform-isoamyl (24:1) was added, followed by centrifugation at 28600 × g for 10 min at 4°C. The supernatant was transferred into a 1.5-mL tube, and 0.5 mL ice-cold absolute ethanol (100%) was added to the tube. After incubation for 1 h at − 20°C, DNA was precipitated by centrifugation at 28600 × g rpm for 10 min at 4°C. The pellet was then washed with 200 µL 70 % ethanol and centrifuged. The supernatant was removed, and the pellet was then air-dried. The NanoDrop 2000 ultra-microspectrophotometer was used to determine the quality and concentration of DNA.
16S rRNA gene ampli cation and sequencing The 16S rRNA gene was commercially sequenced to identify the isolated Bacilli. The full-length gene was ampli ed from the bacterial DNA using universal primers F27 (5′-AGAGTTTGATCCTGGCTCAG-3′) and R1492 (5′-GGTTACCTTGTTACGACTT-3′). A PCR mixture of 50 µL volume was conducted, comprising 2 µL Bacillus DNA, 0.6 µL deoxynucleotide triphosphate, 3 µL PCR buffer, 0.3 µL each of reverse and forward primer, 0.3 µL Es Taq DNA polymerase, and 23.5 µl distilled water. The PCR reaction was carried out in a T100 thermocycler (BIO-RAD, USA). PCR conditions included: 30 cycles at 94°C for 1 min, 60°C for 45 s, and 72°C for 1 min, followed by a nal extension at 72°C for 8 min. The ampli ed PCR product (1500 bp) was separated via gel electrophoresis on 1% (w/v) agarose gel. The sequencing was performed commercially by the General Biosystems (Anhui, China).
Phylogenetic analyses The obtained 16S rDNA sequences were assembled using DNAstar (Lasergene, Madison, WI, USA). Sequences were imported into EZBioCloud (http://www.ezbiocloud.net) and subjected to a BLAST homology search with reference to a known 16S rDNA sequence. Species-level identi cation was performed based on a 16S rDNA sequence similarity of ≥ 97% to that of a prototype strain sequence in the GenBank. Clustal W (2.0.10) in BioEdit was used to perform multiple sequence alignment for the sequence and several near-margin sequences. Sequences were aligned with other published Bacilli sequences, and neighbor-joining phylogenetic analyses were done using MEGA v5.05 (Tamura et al., 2011). Distances in the neighbor-joining tree were computed using the default parameters. Phylogenetic trees were generated under maximum parsimony, neighbor-joining with Jukes-Cantor correction, and a maximum likelihood algorithm with 1000 bootstrap replicates. The sequences reported in the experiment were deposited in the EzTaxon database (https://www.ezbiocloud.net/) under the accession numbers MW759418-MW759434. . megaterium strains were isolated from the rhizospheres of A. artemisiifolia. The previously refrigerated (-20°C) Bacillus was thawed in a water bath at 30°C for 90 s. An aliquot of the bacterial suspension was spread in a petri dish of beef paste with a peptone medium. The petri dish was cultivated at 37°C for 12-24 h. Subsequently, a single colony was inoculated into 1 mL liquid medium at 30°C for 24 h. Then, 1 mL B. megaterium suspension was inoculated into 100 mL liquid medium at 30°C on a rotary shaker for 24 h at 180 r/min to attain a spore concentration of 1 × 10 8 colony-forming units (CFU) mL − 1 .

Experimental design
Three treatments, including A. artemisiifolia monoculture (Am), S. viridis monoculture (Sm), and an equal mixture of A. artemisiifolia and S. viridis (A:S), were used to determine the effects of B. megaterium on the competitiveness of A. artemisiifolia over S. viridis. The seeds were added to pots with 1 kg of soil. After seed germination, each pot was thinned out to four plants (4 individuals in the monocultures and two species each in the mixed treatments in a substitution design). Four densities of B. megaterium (C0: 0, C1: 5×10 8 cfu/mL, C2: 15×10 8 cfu/mL, C3: 30×10 8 cfu/mLof B. megaterium) were tested. The bacterial suspensions were added monthly, as described by Du et al. (2020). Meanwhile, the uninoculated treatment (C0) was treated with 1 mL sterile water and 100 mL liquid medium. Each treatment had 10 replicates (3 × 3 treatments × 10 replicates = 90 pots). The pots were placed in a greenhouse for 90 days under a 10 h photoperiod at 25°C in a randomized design. No fertilizer was used, and each pot was regularly weighed to maintain the soil water content at 25%.

Plant growth parameters
A. artemisiifolia and S. viridis grown in different treatments were harvested 90 days after germination. The soil was washed from the roots, and the plants were oven-dried at 80°C for 48 h to collect growth index data. The whole plant, including aboveground and root biomass, was used to determine dry biomass. Total dry plant weight data of A. artemisiifolia and S. viridis were recorded. The corrected index of relative competition intensity (CRCI) was used to quantify the effect. Samples weighing 20 mg each of A. artemisiifolia and S. viridis leaves and stems were analyzed to determine the total carbon content. The carbon content was measured using the potassium dichromate-concentrated sulfuric acid (K 2 Cr 2 O 7 -H 2 SO 4 ) oxidation method. Dry A. artemisiifolia and S. viridis matter (2 g each) were digested in a mixture of concentrated perchloric and nitric acids (v:v = 1:6) to analyze total nitrogen and phosphorus contents. The nitrogen content was determined using the micro-Kjeldahl method (Nelson and Sommers 1972), while the phosphorus content was measured using inductively coupled plasma spectroscopy (Isaac and Johnson 1983). Each experiment was replicated ten times.

Available nutrients
Available phosphorus (AP) extracted by 0.5 M NaHCO 3 was measured by the molybdenum blue method, while available potassium (AK) extracted by 1 M CH 3 COONH 4 was measured using the ame photometry method ).

B. megaterium density
The density of B. megaterium was determined to compare the growth of B. megaterium among different treatments. This was done for each fresh soil sample using serial dilution techniques on agar plates with nutrient broth medium. Speci cally, when plants were harvested, a 1 g soil sample was collected from the rhizosphere soil and transferred to a tube, and 9 mL distilled water was added. The suspension was shaken to homogeneity at 200 r/min for 24 h, then heated in a hot water bath at 90°C for 10 minutes.
After 12 h standing, the supernatant was serially diluted from 10 − 2 to 10 − 5 , then up to 0.1 mL was pipetted from each aseptic dilution using a attened micropipette and added to nutrient agar plates. The plates were incubated at 37°C for 12 h. The density of B. megaterium was estimated by counting the single colonies. The Bacillus concentration was determined based on the number of colonies and colony separation in the 10 − 3 dilution. Then the cfu/1 g dry weight of soil (cfu/g DWs) was calculated according to the volume dilution. Each treatment was replicated thrice.

Statistical analysis
One-way ANOVA was performed to test the differences in Bacillus diversity between the A. artemisiifolia and S. viridis rhizosphere soils in different treatments, as well as the effect of Bacilli isolated from the rhizosphere soil of A. artemisiifolia or S. viridis on the CRCI of each species. Two-way ANOVA was used to analyze the effect of competition and Bacillus on plant growth parameters, concentrations of available nitrogen and phosphorus in the soil, and the density of Bacillus. All the analyses were performed using IBM SPSS Statistics 19.0 (IBM Corp., Armonk, NY, USA). The Tukey Honest procedure was applied to compare means between treatments.

Results
Comparative analysis of the effect of Bacillus from the rhizosphere soil of A. artemisiifolia and S. viridis on the competitive growth of A. artemisiifolia Compared to the monoculture of A. artemisiifolia, the total dry biomass of A. artemisiifolia in the mixture treatment increased (F = 108.495, P < 0.001) (Fig. 1). When Bacillus from the rhizosphere soil was inoculated, the biomass of A. artemisiifolia was signi cantly higher than that of the uninoculated treatment (F = 99.723, P < 0.001). Furthermore, the biomass of A. artemisiifolia inoculated with Bacillus from A. artemisiifolia soil was higher than that from S. viridis soil (P < 0.001). However, the biomass of S. viridis inoculated with Bacillus from A. artemisiifolia soil was lower than that from S. viridis soil (P < 0.001). CRCI of A. artemisiifolia further showed that Bacillus in the rhizosphere soil of A. artemisiifolia enhanced its competitive growth (Fig. 2).

Bacillus diversity in rhizosphere soil
The Bacillus diversity varied among the different treatments. For instance, seven and eleven Bacillus phylotypes were identi ed in the rhizosphere soil of A. artemisiifolia in A and A:S. Meanwhile, the rhizosphere soil of S. viridis in S and A:S had ten and six Bacillus phylotypes, respectively (Table S1). The Shannon index, species richness, and evenness index of Bacillus diversity in the rhizosphere soil of A. artemisiifolia in A treatment was lower than the A:S treatment (all, p < 0.05). However, Bacillus in the rhizosphere soil of S. viridis in S treatment was higher than that in A:S treatment (all, p < 0.05)( Table 1). Bacillus megaterium, Bacillus aryabhattai, and Bacillus bingmayongensis were the dominant species in all the soil samples, with B. megaterium having the highest relative abundance in the rhizosphere soil of A. artemisiifolia in A (Fig. 3).

Biomass and CRCI
There was no signi cant change in the biomass of A. artemisiifolia between the monoculture and mixed treatment (with S. viridis) in the uninoculated (control) treatments (P = 0.228). Conversely, the competition with A. artemisiifolia decreased the biomass of S. viridis (F = 136.15; P < 0.001) (Fig. 4, Table S2). In both plant species, the biomass increased with the increase of the density of B. megaterium (P < 0.05), showing a dose-dependent relationship. Our analysis showed that the biomasses of C2 and C3 inoculation treatments were signi cantly higher than the uninoculated treatment (all, P < 0.001). Compared to the monoculture, competition had different effects on the biomass of A. artemisiifolia and S. viridis in the inoculation with C2 and C3 B. megaterium; the biomass of A. artemisiifolia increased (F = 65.091; P < 0.001), while that of S. viridis decreased (F = 136.15; P < 0.001). CRCI results further showed inoculation with C2 and C3 B. megaterium concentrations enhanced the relative competitiveness of A. artemisiifolia (F = 12.996; P < 0.001) while decreasing that of S. viridis (F = 7.569; P = 0.002) (Fig. 5).

The density of B. megaterium in different treatments
A dose-dependent increase in soil B. megaterium levels was observed when grown in monoculture (A or S) or mixture (A: S). For the A. artemisiifolia monoculture, the density of B. megaterium in C2 and C3 treatments was signi cantly higher than the C0 treatment (F = 35.944, P < 0.001). Meanwhile, the density of B. megaterium in C3 treatments of S. viridis monoculture was signi cantly higher than all the others (P < 0.001). It was also found that the density of soil B. megaterium in the mixture treatment was signi cantly higher than in the A. artemisiifolia (F = 49.849, P < 0.001) and S. viridis (F = 47.811, P < 0.001) monocultures.

Correlation of the density of B. megaterium with plant growth indicators
The density of B. megaterium was positively correlated with the growth indicators of A. artemisiifolia in all the treatments (Table S3). In S. viridis, the density of B. megaterium was positively correlated with the biomass, total carbon, nitrogen, and phosphorus content in the monoculture but was not correlated with the growth indicators of S. viridis in the mixture.

Discussion
We aimed to determine whether Bacillus in the rhizosphere soil of A. artemisiifolia enhances the competitive growth of the exotic species. Rhizosphere soils were inoculated with Bacillus to provide an advantage to the invasive species over the native S. viridis. Our study shows that the effects of Bacillus in the rhizosphere soil of A. artemisiifolia and in the rhizosphere soil of S. viridis on plant growth are different. For instance, Bacillus in the rhizosphere soil of A. artemisiifolia enhanced the competitive growth of A. artemisiifolia. In contrast, Bacillus in the rhizosphere soil of S. viridis did not have any effect, which suggesting that A. artemisiifolia assembles the Bacillus community in the rhizosphere soil during its invasion to facilitate its growth.
Nutrient acquisition capacity is one of the important factors that affect the competitive ability of invasive species (Daehler 2003;González et al. 2010;Sardans et al. 2016). Plant nutrient acquisition capacity is generally in uenced by two factors, namely, (i) the concentrations of available nutrients in the soil and (ii) the ability of plants to absorb nutrients (Du et al. 2020). The ability of plants to absorb nutrients is an important factor in competitive plant growth. A. artemisiifolia and S. viridis belong to Asteraceae and Poaceae families, respectively, and they have different carbon xation and nutrient absorption abilities. When A. artemisiifolia grows alongside S. viridis without Bacillus inoculation, the competition promotes the growth of A. artemisiifolia by increasing its photosynthetic product, nitrogen, and phosphorus absorption. However, the growth of S. viridis is inhibited by reducing photosynthetic product, nitrogen, and phosphorus absorption. The different abilities to absorb nutrients led to their difference in competitive ability. Meanwhile, soil nutrient availability is another key factor in uencing the performance of invasive species (Funk and Vitousek 2007;Luo et al. 2019). Invasiveness of the exotic species is associated with signi cant changes in plant-soil elemental composition and stoichiometry (Daehler 2003;González et al. 2010). One of the invasion hypotheses proposes that the changes in soil microbial communities caused by invasive plants can result in positive plant-soil feedback by accumulating bene cial microorganisms in the rhizosphere (Inderjit and Cahill 2015). Bacillus species are prominent soil inhabitants that increase plant growth by various mechanisms, such as the production of growth-stimulating phytohormones, nitrogen xation, and phosphorus solubilization mobilization (Wang et al. 2020). In the study, Bacillus diversity in the rhizosphere soils of A. artemisiifolia and S. viridis differed signi cantly. Bacillus diversity in the rhizosphere soil decreased with the invasion of A. artemisiifolia. Meanwhile, the study also showed that some speci c Bacillus such as B. megaterium, B. aryabhattai, and B. bingmayongensis dominated in its rhizosphere soil. The ability of B. megaterium to modify soil available nutrients was demonstrated in a B. megaterium inoculation experiment. The increase in available nitrogen and phosphorus concentrations in the soil and the increase in nitrogen and phosphorus concentrations in plants suggest that B. megaterium in the rhizosphere soil of A. artemisiifolia increases the concentration of available nutrients in the soil. Furthermore, the higher density of B. megaterium in the rhizosphere soil of A. artemisiifolia provides more available nutrients in the soil. We posit that A. artemisiifolia assembles more B. megaterium than native S. viridis to increase the level of available nutrients in its rhizosphere soil.
Resource modi cation by plants may be self-bene cial because the competitive ability of a species is directly related to its ability to modify growth-limiting resources (Dybzinski and Tilman 2007;Harpole 2006). Subsequently, these effects can promote positive or negative feedbacks (Suding et al. 2004). Some research showed that the invasive plant species might increase the concentrations of available nutrients during its invasion (Bajpai and Inderjit 2013;Zhou et al. 2019). For example,  reported that the concentrations of soluble nitrate and Olsen-P in soils under invasive plants are 117% and 21% higher, respectively, than in soils under native plant communities. Besides, Zhou et al. (2019) found that invaded soils had higher nutrient stocks and soil microbial biomass than uninvaded soils. Meanwhile, the decomposition of litter in the soil community can in uence resource availability. For example, the litter of invasive Ageratina adenophora is rich in terpene. Soil microbial communities bene t from the release of nitrogen from decomposing litter (Bajpai and Inderjit 2013), which facilitates the growth of A. adenophora. Our ndings reveal that A. artemisiifolia employs speci c Bacillus species in its rhizosphere soil, different from that of the native species. The speci c Bacillus species (e.g., B. megaterium) increases the levels of available nutrients. Furthermore, as the density of B. megaterium increased, the competition between A. artemisiifolia and S. viridis further enhanced the relative competitiveness of A. artemisiifolia while decreasing that of S. viridis. This nding implies that the increased relative abundance of B. megaterium in the rhizosphere soil of A. artemisiifolia can modify the resources and create a self-bene cial effect on A. artemisiifolia. We conclude that during its invasion, A. artemisiifolia assembles B. megaterium to create higher levels of the available nutrient in its rhizosphere soil than the native S. viridis, promoting its nitrogen and phosphorus absorption to enhance its competitive growth.

Declarations
Declarations of interest None Figure 1 Effects of competition and inoculum on the biomass of A. artemisiifolia and S. viridis Notes: C, control; AA: Bacillus from the rhizosphere soil of A. artemisiifolia in the monoculture SV: Bacillus from the rhizosphere soil of S. viridis in the monoculture. Different higher case letters indicate signi cant differences between in the mixture and in the monoculture treatments at P < 0.05. Different lower case letters indicate signi cant differences among inoculation of different Bacillus at P < 0.05 (n=10).

Figure 2
Page 19/22 Effects of inoculum on CRCI of A. artemisiifolia and S. viridis Notes: C, control; AA: Bacillus from the rhizosphere soil of A. artemisiifolia SV: Bacillus from the rhizosphere soil of S. viridis . Different higher case letters indicate signi cant differences between in the mixture and in the monoculture treatments at P < 0.05. Different lower case letters indicate signi cant differences among inoculation of different Bacillus at P < 0.05 (n=10).

Figure 3
Relative abundance of Bacillus species in different treatments soil Notes: C, control; A: Bacillus from the rhizosphere soil of A. artemisiifolia in the monoculture S: Bacillus from the rhizosphere soil of A. artemisiifolia in the monoculture; A/A:S, Bacillus from the rhizosphere soil of A. artemisiifolia in the mixture; S/A:S, Bacillus from the rhizosphere soil of S. viridis in the mixture (n=3).

Figure 4
Effects of different concentration of B. megaterium on biomass of A. artemisiifolia and S. viridis. Notes: C0, control; C1: 5×108cfu/mL of B. megaterium C2: 15 108cfu/mLof B. megaterium C3: 30× 108cfu/mLof B. megaterium. Different higher case letters indicate signi cant differences between in the mixture and in the monoculture treatments at P < 0.05. Different lower case letters indicate signi cant differences among different concentrations of B. megaterium at P < 0.05(n=10).