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

doi:10.21203/rs.3.rs-643885/v1

PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-643885/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 24 Jun, 2021

You are reading this latest preprint version

Aims

Bacillus, a gram-positive bacterium, has multiple beneficial traits which help the plants in nutrients 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 species.

Methods

A. artemisiifolia monoculture, mixture of A. artemisiifolia and S. viridis and S. viridis monoculture were designed in the field experiment. Bacillus diversity in their rhizospheres was analyzed using 16S rRNA and their effects on the competitive growth of A. artemisiifolia and S. viridis were tested in greenhouse experiment.

Results

The Shannon index, species richness, and evenness index of Bacillus diversity in the rhizosphere soil of A. artemisiifolia in the monoculture treatment were lower than in the mixture treatment. The relative abundance of Bacillus megaterium in the rhizosphere soil of A. artemisiifolia was higher than that in the rhizosphere soil of S. viridis. Whether Bacillus in the rhizosphere soil of A. artemisiifolia or B. megaterium inoculation enhanced the relative competitiveness of A. artemisiifolia and inhibited that of S. viridis by altering their carbon, nitrogen, and phosphorus concentrations.

Conclusions

A. artemisiifolia invasion influenced Bacillus communities, especially B. megaterium. The higher abundance of B. megaterium in A. artemisiifolia rhizosphere creates higher levels of the available nutrient than that in native S. viridis.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Bacillus

rhizosphere soil

Ambrosia artemisiifolia

competitive growth nutrient

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 efficiency and photosynthetic energy use efficiency 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 benefit 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 fixation, 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 beneficial 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 beneficial traits which directly or indirectly aids plants to acquire nutrients (Saxena et al. 2019). Different Bacillus spp. had different ability to fix nitrogen, solubilize and mineralize phosphorus (Goswami et al. 2016; Pramanik et al. 2019). Moreover, root exudates are among the important factors that influence 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 specific 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 Zhang et al. (2018), A. artemisiifolia invasion changes the rhizosphere microbial community in its rhizosphere soil. Specifically, 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 (Zhang et al. 2018). 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 co-occurring native species; and (ii) the assembly of specific Bacillus species benefits 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 specific Bacillus species in its rhizosphere soil. Lastly, the role of specific 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.

Experiment I: Comparative analysis of the effect of Bacillus from the rhizosphere soil of A. artemisiifolia and S. viridis on the competitive growth of A. artemisiifolia

Experimental design

A long-term field experiment was established at the Langfang Experimental Station, Chinese Academy of Agricultural Science (CAAS), Beijing, China (39° 30ʹ 42ʺ N, 116° 36ʹ 07ʺ E), about 10 km southeast of Beijing. Our experimental design was as described by Zhang et al. (2018). S. viridis is an annual C₄ 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 five replicates. Without tilling, the plots were hand weeded to leave bare soil before sowing. The soil was watered to maintain a soil field capacity of 40%. The seeds were sterilized in 10% H₂O₂ for 2 min, rinsed with sterile dH₂O for 2 min, and then rinsed with dH₂O five 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 reflect the mean. We visually estimated the mean and chose plants that seemed to be typical in each treatment. In each treatment, five 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 five-point sampling. Three plots were sampled for each of the five 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. Briefly, 1 g of sample soil was added to 9 mL sterile distilled water in a sterilized conical flask. 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 filtered through a membrane filter with a diameter of 0.22 µm to remove allelochemicals and other organic compounds. The Bacillus remained on the membrane and was rinsed five 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 10⁸cfu/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 five 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 field 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 10⁸cfu/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).

CRCI = arcsine [(X-Y)/max (X, Y)], 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 modification. Briefly, 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 purified 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 amplification and sequencing The 16S rRNA gene was commercially sequenced to identify the isolated Bacilli. The full-length gene was amplified 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 final extension at 72°C for 8 min. The amplified 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 identification 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.

Bacillus community diversity The relative abundance (RA) of Bacilli in our entire sample (including non-Bacillus DNA amplified by the Bacillus primers) was calculated as:

RA = A/N×100%, where A indicates the number of sequences of one Bacillus phylotype, and N indicates the total number of sequences.

The Shannon index (H’), species richness (D), and evenness index (E) were calculated as additional measures of Bacillus diversity. The formulae included:

p _i = n_i/N,

where S represents the total number of Bacillus phylotypes, n_i represents the number of Bacillus phylotype i, and N represents the number of all Bacillus phylotypes.

D = (S − 1)/lnN

E = H’/lnS

Experiment III: Effect of specific Bacillus species ( Bacillus megaterium ) on the competitive growth of A. artemisiifolia

B. megaterium inoculation

B. 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 × 10⁸ 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: 5×10⁸cfu/mL, C2: 15×10⁸cfu/mL, C3: 30×10⁸cfu/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 (K₂Cr₂O₇-H₂SO₄) 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 NaHCO₃ was measured by the molybdenum blue method, while available potassium (AK) extracted by 1 M CH₃COONH₄ was measured using the flame photometry method (Shi et al. 2018).

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. Specifically, 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 flattened 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.

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 significantly 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 identified 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).

Effect of B. megaterium on the competitive growth of A. artemisiifolia

Biomass and CRCI

There was no significant 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 significantly 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).

Total carbon, nitrogen, and phosphorus concentrations of A. artemisiifolia and S. viridis

B. megaterium had a dose-dependent effect on total carbon, nitrogen, and phosphorus concentrations of A. artemisiifolia and S. viridis (Fig. 6, Table S2). There was no significant difference between C0 and C1 treatments (Carbon: A: P = 0.081; S: P = 0.108; Nitrogen: A: P = 0.537; S: P = 0.1; Phosphorus: A: P = 0.183; S: P = 0.331). However, in the monoculture of both C2 and C3 treatments, the total carbon, nitrogen, and phosphorus concentrations of A. artemisiifolia or S. viridis were significantly higher than in C0 treatment (C2: Carbon: A: P < 0.001; S: P < 0.001; Nitrogen: A: P < 0.001; S: P < 0.001; Phosphorus: A: P < 0.001; S: P < 0.001. C3: Carbon: A: P < 0.001;S: P < 0.001; Nitrogen༚A: P < 0.001; S: P < 0.001; Phosphorus: A: P < 0.001; S: P < 0.001). Compared to the monoculture, the total carbon, nitrogen, and phosphorus concentrations of A. artemisiifolia in the mixture increased (Carbon: A: F = 55.737; P < 0.001; Nitrogen: A: F = 92.315; P < 0.001; Phosphorus: A: F = 23.285; P < 0.001). However, the concentrations in S. viridis decreased in both uninoculated and inoculated treatments (Carbon: S: F = 46.598; P < 0.001; Nitrogen: S: F = 96.474; P < 0.001; Phosphorus: S: F = 58.443; P < 0.001).

Available nitrogen and phosphorus concentrations in the soil of different treatments

No significant difference in available nitrogen and phosphorus concentration in the soil was revealed between the C0 and C1 treatments (Phosphorus: A: P = 0.181; S: P = 0.628; A:S: P = 0.454; Nitrogen: A: P = 0.337; S: P = 0.292; A:S: P = 0.153)(Table S2). However, in both C2 and C3 treatments, the total carbon, nitrogen and phosphorus concentrations of A. artemisiifolia or S. viridis were significantly higher than in C0 treatment (C2: Phosphorus: A: P < 0.001; S: P < 0.001; A:S: P < 0.001; Nitrogen: A: P < 0.001; S: P < 0.001; A:S: P < 0.001; C3: Phosphorus: A: P < 0.001; S: P < 0.001; Nitrogen: A: P < 0.001; S: P < 0.001). The total carbon, nitrogen, and phosphorus concentrations of A. artemisiifolia in the mixture increased compared to the monoculture, while S. viridis concentrations decreased in both uninoculated and inoculated treatments (Phosphorus: S: F = 59.462; P < 0.001; Nitrogen: S: F = 9.865; P < 0.001).

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 significantly 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 significantly higher than all the others (P < 0.001). It was also found that the density of soil B. megaterium in the mixture treatment was significantly 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.

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 influenced 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 fixation 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 influencing the performance of invasive species (Funk and Vitousek 2007; Luo et al. 2019). Invasiveness of the exotic species is associated with significant 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 beneficial 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 fixation, and phosphorus solubilization mobilization (Wang et al. 2020). In the study, Bacillus diversity in the rhizosphere soils of A. artemisiifolia and S. viridis differed significantly. Bacillus diversity in the rhizosphere soil decreased with the invasion of A. artemisiifolia. Meanwhile, the study also showed that some specific 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 modification by plants may be self-beneficial 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; Sardans et al. 2017; Zhou et al. 2019). For example, Sardans et al. (2017) 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 influence resource availability. For example, the litter of invasive Ageratina adenophora is rich in terpene. Soil microbial communities benefit from the release of nitrogen from decomposing litter (Bajpai and Inderjit 2013), which facilitates the growth of A. adenophora. Our findings reveal that A. artemisiifolia employs specific Bacillus species in its rhizosphere soil, different from that of the native species. The specific 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 finding implies that the increased relative abundance of B. megaterium in the rhizosphere soil of A. artemisiifolia can modify the resources and create a self-beneficial 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 of interest

None

Acknowledgements

This research was funded by the National Natural Science Foundation of China, Grant No. 31972343; Hebei National Natural Science Foundation, Grant No. C2019201059; College of Life Science, Institute of Life Science and Green Development, Hebei University.

Bajpai D, Inderjit (2013) Impact of nitrogen availability and soil communities on biomass accumulation of an invasive species. AoB Plants 5:plt045. https://doi.org/10.1093/aobpla/plt045

Baruch Z, Goldstein G (1999) Leaf construction cost, nutrient concentration, and net CO₂ assimilation of native and invasive species in Hawaii. Oecologia 121:183–192. https://doi.org/10.1007/s004420050920

Chase JM, Leibold MA (2003) Ecological niches: linking classical and contemporary approaches. Chicago University Press, Chicago

Chikerema SM, Pfukenyi DM, Hang’ombe BM, L’Abee-Lund TM, Matope G (2012) Isolation of Bacillus anthracis from soil in selected high-risk areas of Zimbabwe. Appl Environ Microb 113:1389–1395. https://doi.org/10.1111/jam.12006

Daehler CC (2003) Performance comparisons of co-occurring native and alien invasive plants: implications for conservation and restoration. Annu Rev Ecol Evol Syst 34:183–211. https://doi.org/10.1146/annurev.ecolsys.34.011802.132403

Dai ZC, Fu W, Wan LY, Cai HH, Wang N, Qi SS, Du LL (2016) Different growth promoting effects of endophytic bacteria on invasive and native clonal plants. Front Plant Sci 7:706. https://doi.org/10.3389/fpls.2016.00706

Davidson AM, Jennions M, Nicotra AB (2011) Do invasive species show higher phenotypic plasticity than native species and if so, is it adaptive? A meta-analysis. Ecol Lett 14:419–431

https://doi.org/10.1111/j.1461-0248.2011.01596.x

Du EW, Chen X, Li Q, Chen FX, Xu HY, Zhang FJ (2020) Rhizoglomus intraradices and associated Brevibacterium frigoritolerans enhance the competitive growth of Flaveria bidentis. Plant soil 453:281 – 95. https://doi.org/10.1007/s11104-020-04594-1

Dybzinski R, Tilman D (2007) Resource use patterns predict long-term outcomes of plant competition for nutrients and light. The Am Nat 170:305–318. https://doi.org/10.1086/519857

Ehrenfeld JG (2003) Effects of exotic plant invasions on soil nutrient cycling processes. Ecosystems 6:503–523. https://doi.org/10.1007/s10021-002-0151-3

Feng YL (2008) Photosynthesis, nitrogen allocation and specific leaf area in invasive Eupatorium adenophorum and native Eupatorium japonicum grown at different irradiances. Physiol Plant 133:318–326. https://doi.org/10.1111/j.1399-3054.2008.01072.x

Fraterrigo JM, Balser TC, Turner MG (2006) Microbial community variation and its relationship with nitrogen mineralization in historically altered forests. Ecology 87:570–579. https://doi.org/10.1890/05-0638

Funk JL, Vitousek PM (2007) Resource-use efficiency and plant invasions in low-resource systems. Nature 446:1079–1081. https://doi.org/10.1038/nature05719

Ghiani A, Aina R, Asero R, Bellotto E, Citterio S (2012) Ragweed pollen collected along high-traffic roads shows a higher allergenicity than pollen sampled in vegetated areas. Allergy 67:887–894

https://doi.org/10.1111/j.1398-9995.2012.02846.x

Gibbons SM (2017) Microbial community ecology: function over phylogeny. Nat Ecol Evol 1,0032. https://doi.org/10.1038/s41559-016-0032

Gonza´lez AL, Kominoski JS, Danger M, Ishida S, Iwai N, Rubach A (2010) Can ecological stoichiometry help explain patterns of biological invasions? Oikos 119:779–790. https://doi.org/10.1111/j.1600-0706.2009.18549.x

Goswami D, Thakker JN, Dhandhukia PC, Tejada MM (2016) Portraying mechanics of plant growth promoting rhizobacteria (PGPR): a review. Cogent Food Agric 2:1127500. https://doi.org/10.1080/23311932.2015.1127500

Harpole WS (2006) Resource-ratio theory and the control of invasive plants. Plant Soil 280:23–27. https://doi.org/10.1007/s11104-005-2951-7

Huangfu CH, Li HY, Chen XW, Liu HM, Yang DL (2015) The effects of exotic weed Flaveria bidentis with different invasion stages on soil bacterial community structures. Afr J Biotechnol 14:2636–2643. https://doi.org/10.5897/AJB2015.14658

Huber (2011) Robust Statistics. In: Lovric M (ed) International Encyclopedia of Statistical Science. Springer, Berlin

Inderjit, Cahill JF (2015) Linkages of plant–soil feedbacks and underlying invasion mechanisms. AoB Plants 7. https://doi.org/10.1093/aobpla/plv022

Isaac RA, Johnson WC (1983) High speed analysis of agriculture samples using inductively coupled plasma-atomic emission spectroscopy. Spectrochim Acta 38B:277–282. https://doi.org/10.1016/0584-8547(83)80124-4

Iwaoka C, Imada S, Taniguchi T, Du S, Yamanaka N, Tateno R (2018) The impacts of soil fertility and salinity on soil nitrogen dynamics mediated by the soil microbial community beneath the halophytic shrub Tamarisk. Microb Ecol 75:985–996. https://doi.org/10.1007/s00248-017-1090-z

Kourtev PS, Ehrenfeld JG, Haggblom M (2003) Experimental analysis of the effect of exotic and native plant species on the structure and function of soil microbial communities. Soil Biol Biochem 35:895–905. https://doi.org/10.1016/S0038-0717(03)00120-2

Luo X, Xu XY, Zheng Y, Guo H, Hu SJ (2019) The role of phenotypic plasticity and rapid adaptation in determining invasion success of Plantago virginica. Biol Invasions 21:2679–2692. https://doi.org/10.1007/s10530-019-02004-x

Nelson DW, Sommers LE (1972) Determination of total nitrogen in plant material. Agron J 65:109–112. https://doi.org/10.2134/agronj1973.00021962006500010033x

Oksanen L, Sammul M, Magi M (2006) On the indices of plant-plant competition and their pitfalls. Oikos 112:149–155. https://doi.org/10.1111/j.0030-1299.2006.13379.x

Ozaslan C, Onen H, Farooq S, Gunal H, Akyol N (2016) Common ragweed: an emerging threat for sunflower production and human health in Turkey. Weed Biol Manag 16:42–55. https://doi.org/10.1111/wbm.12093

Pattison RR, Goldstein G, Ares A (1998) Growth, biomass allocation and photosynthesis of invasive and native Hawaiian rain-forest species. Oecologia 117:449–459. https://doi.org/10.1111/wbm.12093

Parker JD, Torchin ME, Hufbauer RA, Lemoine NP, Alba C, Blumenthal DM, Bossdorf O, Byers JE, Dunn AM, Heckman RW (2013) Do invasive species perform better in their new ranges? Ecology 94:985–994. https://doi.org/10.1890/12-1810.1

Parker SS, Harpole WS, Seabloom EW (2019) Plant species natural abundances are determined by their growth and modification of soil resources in monoculture. Plant Soil 445:273–287. https://doi.org/10.1007/s11104-019-04299-0

Pramanik P, Goswami AJ, Ghosh S, Kalita C (2019) An indigenous strain of potassium-solubilizing bacteria Bacillus pseudomycoides enhanced potassium uptake in tea plants by increasing potassium availability in the mica waste-treated soil of North-east India. J Appl Microbiol 126:215–222. https://doi.org/10.1111/jam.14130

Rodríguez-Caballero G, Caravaca F, Diaz G, Torres P, Roldan A (2020) The invader Carpobrotus edulis promotes a specific rhizosphere microbiome across globally distributed coastal ecosystems. Sci Total Environ 719:137347. https://doi.org/10.1016/j.scitotenv.2020.137347

Saxena AK, Kumar M, Chakdar H, Anuroopa N, Bagyaraj DJ (2019) Bacillus species in soil as a natural resource for plant health and nutrition. J Appl Microbiol 128:1583–1594. https://doi:10.1111/jam.1450

Sardans J, Alonso R, Janssens IA, Carnicer J, Vereseglou S, Rillig MC, Fernandez-Martinez M, Sanders TGM, Penuelas J (2017) Foliar and soil concentrations and stoichiometry of nitrogen and phosphorous across European Pinus sylvestris forests: relationships with climate, N deposition and tree growth. Functional Eco 30:676–689. https://doi.org/10.1111/1365-2435.12541

Sardans J, Bartrons M, Margalef O, Gargallo-Garriga A, Janssens IA, Ciais P, Obersteiner M, Sigurdsson BD, Chen HYH, Penuelas J (2017) Plant invasion is associated with higher plant–soil nutrient concentrations in nutrient-poor environments. Glob Chang Biol 23:1282–1291. https://doi.org/10.1111/gcb.13384

Shakeel M, Rais A, Hassan MN, Hafeez FY (2015) Root associated Bacillus sp. improves growth, yield and zinc translocation for Basmati Rice (Oryza sativa) varieties. Front Microbiol 6:1286. https://doi.org/10.3389/fmicb.2015.01286

Shea K, Chesson P (2002) Community ecology theory as a framework for biological invasions. Trends Ecol Evol 17:170–176. https://doi.org/10.1016/S0169-5347(02)02495-3

Shi Y, Li YT, Xiang XJ, Sun RB, Yang T, He D, Zhang KP, Ni YY, Zhu YG, Adams JM (2018) Spatial scale affects the relative role of stochasticity versus determinism in soil bacterial communities in wheat fields across the North China Plain. Microbiome 6:27. https://doi.org/10.1186/s40168-018-0409-4

Song Z, Zhang RH, Fu WD, Zhang T, Yan J, Zhang GL (2017) High-throughput sequencing reveals bacterial community composition in the rhizosphere of the invasive plant Flaveria bidentis. Weed Res 57:204–211. https://doi.org/10.1111/wre.12250

Sun YY, Zhang QX, Zhao YP, Diao YH, Gui FR, Yang GQ (2020) Beneficial rhizobacterium provides positive plant-soil feedback effects to Ageratina Adenophora. J Integr Agri 19:2–10. https://doi.org/10.1016/S2095-3119(20)63234-8

Suding KN, Larson JR, Thorsos E, Steltzer H, Bowman WD (2004) Species Effects on Resource Supply Rates: Do They Influence Competitive Interactions? Plant Ecol 175:47–58. https://doi.org/10.1023/B:VEGE.0000048093.92118.27

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. https://doi.org/10.1093/molbev/msr121

Tariq M, Hameed S, Malik KA, Hafeez FY (2007) Plant root associated bacteria for zinc mobilization in rice. Pak J Bot 39:245–253

Wang Z, Yu ZX, Solanki MK, Yang LT, Xing YX, Dong DF, Li YR (2020) Diversity of sugarcane root-associated endophytic Bacillus and their activities in enhancing plant growth. J Appl Microbiol 128:814–827. https://doi.org/10.1111/jam.14512

Xu and Qiang (2004) Catalog of invasive species in China. China environmental science press, Beijing

Zhang FJ, Li Q, Yerger EH, Chen X, Shi Q, Wan FH (2018) AM fungi facilitate the competitive growth of two invasive plant species, Ambrosia artemisiifolia and Bidens pilosa. Mycorrhiza 28:703–715. https://doi.org/10.1007/s00572-018-0866-4

Zhang N, Wang DD, Liu YP, Li SQ, Shen QR, Zhang RF (2014) Effects of different plant root exudates and their organic acid components on chemotaxis, biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains. Plant Soil 374:689–700. https://doi.org/10.1007/s11104-013-1915-6

Zhou Y, Staver AC (2019) Enhanced activity of soil nutrient-releasing enzymes after plant invasion: a meta-analysis. Ecology 100:e02830. https://doi.org/10.1002/ecy.2830

Slide10.png
Fig. S1 Neighbor-joining tree showing phylogeny of representative Bacillus DNA sequences isolated from root and reference sequences from GeneBank. Fungal sequences from this study are identified by their GenBank sequences beginning with MK463590. Closely aligned sequences from GenBank are identified by the species name or accession number. Scutellospora alterata is used as the out-group (left panel). Relative abundance of each AMF type detected from root sampled from different treatments (right panel). Treatments: A (A.artemisiifolia monoculture), A(M) (A. artemisiifolia in the mixture of A. artemisiifolia and S. viridis), S (S. viridis monoculture), S(M) (S. viridis in the mixture of A. artemisiifolia and S. viridis).

PDF

Version 1

posted 24 Jun, 2021

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Experimental design

Soil sampling

Bacillus innoculation design

Measurements

Experimental design

Available nutrients

Statistical analysis

Results

Biomass and CRCI

Total carbon, nitrogen, and phosphorus concentrations of A. artemisiifolia and S. viridis

Available nitrogen and phosphorus concentrations in the soil of different treatments

The density of B. megaterium in different treatments

Correlation of the density of B. megaterium with plant growth indicators

Discussion

Declarations

References

Supplementary Files

Status:

Version 1