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. 2021 Feb 9;16(2):e0246685. doi: 10.1371/journal.pone.0246685

Invasive species allelopathy decreases plant growth and soil microbial activity

Tongbao Qu 1,*, Xue Du 1, Yulan Peng 1, Weiqiang Guo 1, Chunli Zhao 1, Gianalberto Losapio 2
Editor: Raffaella Balestrini3
PMCID: PMC7872269  PMID: 33561161

Abstract

According to the ‘novel weapons hypothesis’, invasive success depends on harmful plant biochemicals, including allelopathic antimicrobial roots exudate that directly inhibit plant growth and soil microbial activity. However, the combination of direct and soil-mediated impacts of invasive plants via allelopathy remains poorly understood. Here, we addressed the allelopathic effects of an invasive plant species (Rhus typhina) on a cultivated plant (Tagetes erecta), soil properties and microbial communities. We grew T. erecta on soil samples at increasing concentrations of R. typhina root extracts and measured both plant growth and soil physiological profile with community-level physiological profiles (CLPP) using Biolog Eco-plates incubation. We found that R. typhina root extracts inhibit both plant growth and soil microbial activity. Plant height, Root length, soil organic carbon (SOC), total nitrogen (TN) and AWCD were significantly decreased with increasing root extract concentration, and plant above-ground biomass (AGB), below-ground biomass (BGB) and total biomass (TB) were significantly decreased at 10 mg·mL-1 of root extracts. In particular, root extracts significantly reduced the carbon source utilization of carbohydrates, carboxylic acids and polymers, but enhanced phenolic acid. Redundancy analysis shows that soil pH, TN, SOC and EC were the major driving factors of soil microbial activity. Our results indicate that strong allelopathic impact of root extracts on plant growth and soil microbial activity by mimicking roots exudate, providing novel insights into the role of plant–soil microbe interactions in mediating invasion success.

Introduction

Invasive species degrade the environment, reduce biodiversity and alter ecosystem processes, threatening livelihoods worldwide [1]. The mechanisms underlying successful plant invasion is a long-standing topic which is debated in ecology [2–5]. The theory of ‘novel weapon hypothesis’ (NWH) states that plant invasive success depends upon the ability to release novel phytochemicals into the invaded community [6, 7]. According to the NWH, invasive success depends on plant biochemical weapons, including allelopathic and antimicrobial root exudates, that directly inhibit native plant growth or mediate new plant–soil microbial interactions. Today, allelopathic effects of invasive species on co-occurring native plants are widely recognized as the major factors responsible for successful invasion in plant communities [8]. Despite the large number of previous studies investigating the allelopathic potential of invasive plants [9, 10], it is unclear about the role soil properties and microbial community play in mediating the impact of invasive species on plants. Addressing the effects of alien invasion on soil biota may improve our understanding of the key role soil organisms play in ecological systems, mediating biotic interactions.

It is important to notice that aboveground and belowground systems are not closed and isolated compartment, but they are rather linked together, exchanging energy and materials. Soil microbes play a crucial role in soil nutrient cycling and soil health [11]. Invasive plants can outcompete and displace native species thanks to allelopathy, which changes soil conditions via underground root exudates [12]. Invasive species root allelochemicals released into the soil can change soil properties, in turn affecting the composition and diversity of soil microbial community [13, 14]. Therefore, it is of great significance to study the effects of root allelochemicals from invasive plants on the soil physical and chemical properties and soil microorganisms in order to improve our understanding of plant invasion mechanisms. Yet, we have a limited knowledge of the relative contribution of direct and indirect allelopathic effects between invasive and other species.

Rhus typhina L. (Anacardiaceae) is a perennial shrub that make clones through production of underground root buds and forms large, dome-shaped canopies of a single genet, with progressively younger ramets developing around the periphery [15]. As an invasive shrub in most areas of China, R. typhina is classified as one of the most destructive invading exotic species and poses a great threat to terrestrial ecosystems due to his great ability to adapt and reproduce fast and its resistance to eradication once is established [16–18]. Tagetes erecta L. (Compositae) is a widespread ornamental plant and is commonly known as Marigold, native to Mexico [19]. It is found that there are patterns of negative effects grown in conjunction with garden trees where may co-occur with R. typhina, which could be allelopathic effects impacted by R. typhina. Recent studies highlight that R. typhina impacts both native plant communities and T. erecta by inhibiting seed germination and seedling growth [20, 21]. These evidences the potential allelopathy of R. typhina and its broad impact on biodiversity and local environmental conditions. At present, however, it remains unclear whether and how R. typhina impacts soil microorganisms through root extracts, in turn affecting plant growth.

Here, we examine the allelopathic effects of the invasive shrub R. typhina on the cultivated plant T. erecta via changes in soil conditions and microbial communities. We compare soil properties, metabolic activity and microbial carbon utilization at increasing concentration of R. typhina root extracts and examine the effects of changing soil conditions on T. erecta growth. We hypothesize that 1) R. typhina root extracts inhibit plant growth; 2) R. typhina root extracts decrease soil conditions; 3) The reasons of inhibition are the extracts negatively impact soil conditions and rhizophere soil microbial carbon source utilization.

Materials and methods

Root extracts of R. typhina

Healthy, fully developed and fresh roots of R. typhina were collected from Changchun (43°52’ N, 125°21’ E), China in August 2017. The region has a temperate continental humid climate with an average annual air temperature of 4.6°C and annual precipitation ranges from 600 mm to 700 mm. Annual freezing period is 5 months with an average 140–150 frost-free days.

1000 g of roots were taken from the plants of R. typhina and washed, air dried for 48 h and then powdered in a grinder until 98% of the powder could pass through 10 mm mesh sieve. The samples were extracted with deionized water, shaken at 50 rpm-1 for 48 h, then concentrated by rotary evaporation, and finally dried in a freeze dryer. The obtained substance was 24.96 g, and the extraction rate was 2.5%. The concentration of the extract solution was set to 2.5, 5, 7.5 and 10 mg·mL-1 according to previous experimental data, then kept in brown bottles at 4°C for subsequent processing [22].

Experimental design and soil sample collection

Seeds of T. erecta were collected on the campus of Jilin Agricultural University, Changchun, China in October 2016. The selected seeds were soaked and disinfected with 0.3% KMnO4 solution for 5 min, then rinsed with distilled water 3–5 times. We initially sowed ten seeds of T. erecta per pot (18 cm diameter×21 cm height) in a climate-controlled incubator at 25°C with diurnal lighting (light intensity was set at 2200 Lux on a 14:10 h light–dark cycle). After germination, we kept five plants in each pot. We use a mix of garden soil, turfy soil and perlite by volume 6: 3: 1. Pots were randomly assigned to treatments (i.e., control, 2.5, 5, 7.5 and 10 mg·mL-1 of root extracts). Pots were watered with 20 mL of different concentrations of root extracts every day, or 20 mL of distilled water for the control groups. Each treatment was replicated three times, for a total of n = 15 pots and n = 75 plants.

After 60 days, plant and soil samples were collected for analyses. Soil samples were screened through sieves with 2 mm mesh and divided into two parts after removal of coarse roots and stones. One part was air dried and used for physico-chemical analyses whereas the other one used for EcoPlates (Biolog, Hayward, CA, USA) and microbial activity analyses. All samples were stored at 4°C before analysis.

Growth of T. erecta

We measured plant height, root length, aboveground biomass (AGB) and underground biomass (BGB). AGB was measured by clipping standing plant materials to 1 cm above ground level.

Soil physico-chemical properties

We measured the following soil properties: pH, electrical conductivity (EC), water content (SWC), soil organic carbon (SOC), total nitrogen (TN). The soil pH and EC of the samples were measured using a digital pH-meter (CP-401, ELMETRON) and electric conductivity meter (DDSJ-308A); SWC was measured by the oven-drying method; SOC was measured using a Mebius method by the Walkley-Black acid digestion; TN was determined with an autoanalyzer (Foss 2100, FOSS Kjeltec®) using the Kjeldahl method following vitriol digestion.

Community level physiological profiles (CLPP)

Community-level physiological profiles (CLPP) is a good method used for measured microbial activity of rhizosphere soil, which were measured with Biolog Eco-platesTM (Biolog Inc., Hayward, CA, USA), which contains 31 types of carbon sources and one substrate-free control. Soil samples (10 g d.w.) were firstly shaken (5 min, 180 r·min-1) after dilution with 90 mL of distilled sterile water (pH 7) for 20 min at 20°C. Then aliquot (150 μL) were inoculated into wells of 96 Biolog Eco-plates and incubated at 25°C. The rate of utilization was showed by the reduction of the tetrazolium, a redox indicator dye that changes from colorless into purple. Lastly, substrate utilization was monitored by measuring light absorbance at 590 and 750 nm. The first measurement was made immediately after inoculation, and the subsequent ones were read every 12 h for a total of 240 h. The readings for individual substrates were corrected for background absorbance by subtracting the absorbance of the control (water) well and one of the absorbance of the first reading.

Average well color development (AWCD) was determined as follows: AWCD = Σ(C590 –C750)/31. C590 and C750 are optical density value in 590 nm and 750 nm from each well, corrected subtracting the blank well (inoculated with distilled water) values from each plate well. Diversity indices of AWCD were calculated as Shannon index (H = -Σ(Pi×lnPi)), Pielou evenness index (E = H/Hmax = H/lnS), and Simpson diversity index (D = 1-ΣPi2). Pi = C590-C750/∑(C590—C750), S were calculated as the number of oxidized carbon substrates (Qu et al. 2020).

According to the change curve of carbon source utilization over time, incubation at 180 h was used to calculate AWCD, H, E and D since it was the highest average well color development (AWCD) that was most consistent between treatments. The categorized carbon substrates were grouped into six guilds–carbohydrates, amino acids, carboxylic acids, polymer, phenolic acids and amine.

Statistical analysis

The experiment has been repeated three times. Differences in plant growth, soil physico-chemical properties and microbial activity between the different concentration extracts were tested by one-way ANOVA. Tukey’s tests were conducted separately to for post hoc analysis of differences between specific factor levels. Redundancy analysis (RDA) was conducted to link the use of particular carbon substrates to soil properties and to compare CLPPs. Monte Carlo permutations (MCP) with 999 permutation were used to test the significance of different factors along the first axis.

Data analysis was done with the SAS Statistical Software and by Canoco.

Results

Growth of T. erecta

R. typhina root extracts significantly reduced, root length, AGB, BGB and TB of T. erecta compared to the control group at 10 mg mL-1 (Table 1 and Fig 1). Plant height firstly increased then decreased, and root length significantly decreased with extract concentrations increasing (Table 1). There were no significant different AGB, BGB and TB among different concentration below 10 mg mL-1. At highest concentration (10 mg mL-1), plant height was reduced by 7.38% (P < 0.05), root length decreased by 13.13%, and plant AGB, BGB and TB significantly decreased by 27.7%, 22.9% and 26.5% than control, respectively (P < 0.05).

Table 1. Growth of Tagetes erecta plants in response to different concentrations of Rhus typhina root extract.

Concentration Plant height Root length AGB BGB TB
(mg·mL-1) (cm) (cm) (g) (g) (g)
Control 16.52±0.29 bc 16.30±0.36 a 18.72±1.99 a 6.23±1.09 a 24.95±3.47 a
2.5 19.14±0.84 a 15.32±0.62 bc 17.87±1.02 a 6.55±0.39 a 24.42±1.34 a
5 17.82±1.53 ab 15.64±0.41 b 16.86±1.03 a 5.78±0.51 a 22.44±0.54 a
7.5 15.94±0.48 c 15.20±0.30 bc 16.73±1.23 a 6.46±0.58 a 23.20±0.69 a
10 15.30±0.85 c 14.16±0.54 c 13.53±0.08 b 4.80±0.22 b 18.33±0.29 b
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Note. Value ± SE with ANOVA results (n = 3). Different letters represent significant differences between different concentration (P < 0.05; Tukey’s test). AGB: aboveground biomass, BGB: belowground biomass, TB: total biomass.

Fig 1. Growth of T. erecta at different R. typhina root extract concentration after 60 days.

Fig 1

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CK, 2.5, 5, 7.5, 10 represent different extract concentrations of 0, 2.5, 5, 7.5, 10 mg·mL-1, respectively.

Soil physicochemical properties

Root extracts significantly increased soil EC and pH, and significantly decreased SOC and TN (Table 2). No significant effect was found for SWC and C/N (Table 2). Soil pH increased with extract concentration increasing, and the highest pH and EC were at concentration of 10 mg·mL-1 and 5 mg·mL-1 with changes of 11.5% and 50.6%, respectively (P < 0.05). SOC and TN significantly decreased with increasing extract concentrations, and the lowest SOC and TN were at 10 mg·mL-1 concentration, with average change of 25.4% and 22.6%, respectively (P < 0.05).

Table 2. Soil physicochemical responses to different concentrations of R. typhina root extract.

Concentration EC pH SWC SOC TN C/N
(mg·mL-1) (μS·cm-1) (%) (g·kg-1) (g·kg-1)
Control 86.73±0.25 e 6.08±0.02 cd 20.58± 1.67 a 68.93±2.68 a 5.22±0.11 a 13.19±0.50 a
2.5 94.30±0.10 d 5.92±0.06 d 16.43± 2.82 a 59.17±0.51 b 4.65±0.03 b 12.72±0.18 a
5 130.60±0.10a 6.18±0.03bc 16.01± 0.93a 62.79±0.34 b 4.95±0.11 a 12.69±0.31 a
7.5 127.70±0.36b 6.32±0.01b 16.73± 1.64a 54.59±0.18 c 4.34±0.17 c 12.60±0.55 a
10 99.27±0.15c 6.78±0.13 a 19.57±2.00 a 51.44±1.72 c 4.04±0.04 d 12.75±0.35 a
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Note. Value ± SE with ANOVA results (n = 3). Different letters represent significant differences among different concentrations (P < 0.05; Tukey’s test). EC: electrical conductivity, SWC: soil water content, TN: total nitrogen, SOC: soil organic carbon and C/N: C: N ratio.

Community level physiological profiles of soil microbial communities

After 12 h inoculation, a first substrate conversion was indicated by beginning color development in the microplate wells. AWCD then rapidly increased from 12–180 h incubation followed by clear differentiation among different treatments. After 180 h we observed a steady, saturating increase (Fig 2). Higher AWCD values were maintained in controls compared to all extract concentrations until the end of the incubation period of 240 h (Fig 2). At the peak (i.e., 180 h), results indicate that AWCD decreased by 15.20%, 5.71%, 23.46% and 28.56% at 2.5, 5, 7.5, and 10 mg·mL-1 concentrations compared to control, respectively (all P < 0.05, Fig 2).

Fig 2. Variation in average well color development (AWCD) of soil microbes at different R. typhina root extract concentration over time.

Fig 2

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CK, 2.5, 5, 7.5, 10 represent different extract concentrations of 0, 2.5, 5, 7.5, 10 mg·mL-1, respectively. Values represent means ± standard deviation.

All 6 groups of carbon source were metabolized in Biolog Eco-plates in different concentrations of extracts (Fig 3), indicating different functional diversity of soil microbiota. The extracts from R. typhina roots significantly reduced the carbon source utilization of carbohydrates, carboxylic acids and polymers, and significantly enhanced phenolic acid (all P < 0.05) compared to control. Amino acids and amines firstly increased and then decreased with the increasing of extract concentrations (P < 0.05). The carbon source mostly used in controls was polymers and the least was phenolic acid, same patterns of response for phenolics acids than aminoacids. Among the treatments, phenolic acids were the most common, especially at 5 mg·mL-1, while amines were the least used, especially at 10 mg·mL-1 (P < 0.05).

Fig 3. Substrate utilization patterns of different carbon source by soil microbial community after 180 h of incubation in the Biolog Eco-plates at different R. typhina root extract concentrations.

Fig 3

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Values represent means ± standard deviation (n = 15). Significant differences between carbon sources utilization are indicated with capital letters (A-C; P < 0.05); significant differences between treatments of concentration are indicated with lowercase letters (a-c; P < 0.05).

Diversity of soil microbial carbon utilization

Shannon index and Simpson diversity index firstly significantly increased and then significantly decreased and Pielou evenness index decreased gradually with the increasing of extract concentrations (P < 0.05) (Fig 4). Shannon index, Pielou evenness index and Simpson diversity index reached lowest values at 10 mg·mL-1 extract, decreasing by 10.74%, 2.24% and 1.80% as compared to control, respectively (P < 0.05).

Fig 4. Diversity and evenness indices of soil microbial communities.

Fig 4

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H’: Shannon index; D: Simpson diversity index; E: Pielou evenness index. Values represent means ± standard deviation (n = 15). Significant differences between treatments are indicated with lowercase letters (a-c; P < 0.05).

Redundancy analysis (RDA) of soil carbon utilization

We report results of RDA in biplot graph with response variables (31 kinds of soil microbial carbon utilization) and explanatory variables (soil physicochemical characteristics) (Fig 5A). Eigenvalues for the first, second, third and fourth axes were 0.5201, 0.2927, 0.0345, respectively. Axis 1 significantly explained most of the variation in soil microbial carbon utilization (P <0.001). All the soil physicochemical characteristics could explain 85.20% of soil microbial carbon utilization variation (Monte Carlo permutation test with 999 permutation, P <0.001). Conditional effects show that the main factors driving soil microbial carbon utilization were pH (32.3%, P = 0.008), TN (29%, P = 0.002) and EC (19.7%, P = 0.004).

Fig 5.

Fig 5

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RDA based on AWCD and soil properties (left) and six carbon source utilization and soil properties(right) among different extract concentration treatments. Symbols indicate concentrations (circle: 0 g‧mL-1, triangle: 2.5 mg‧mL-1, diamond: 5 mg‧mL-1, square: 7.5 mg‧mL-1, star: 10 mg‧mL-1). SWC: soil water content, EC: electrical conductivity, TN: total nitrogen, SOC: soil organic carbon and C/N: C: N ratio.

RDA analysis based on carbon sources and soil physicochemical characteristics under different concentrations of extracts was performed through the graph of triplots (Fig 5B). Eigenvalues for the first, second, third and fourth axes were 0.5792, 0.2411 and 0.0845, respectively. Axis 1 explained most of the variation in soil microbial carbon utilization (P = 0.002). All the soil data together could explain 91.9% of soil microbial carbon utilization variation (Monte Carlo permutation test with 999 permutation, P <0.001). Conditional effects showed that the main factors affecting soil microbial carbon utilization were pH (51.8%, P = 0.002), SOC (22.4%, P = 0.002) and EC (14%, P = 0.002). According to soil pH, the order of affecting soil microbial categorized carbon utilization by concentration of extracts was 10, 7.5, 2.5, CK and 5 mg·mL-1. Results also showed that soil pH mainly decreased microbial utilization to Amino acids and Amines, and SOC mainly enhanced utilization of carboxylic acids, polymers and carbohydrates, and EC mainly positively affected utilization of phenolic acid (Fig 5B).

Discussion

Our findings of allelopathic effects of invasive species on plant growth and soil microbial activity shed new light on the poorly understood mechanisms underlying the role plant–soil interactions play in shaping invasion success. Invasive species root extracts decrease plant biomass, soil SOC and TN, increase soil EC and pH, and alter microbial carbon source utilization. Taken together, our results indicate how allelopathy impacts both plant and soil community further facilitates the successful invasion.

Consistent with previous studies [23], our results show that root extracts of R. typhina may result in dramatic changes in soil community activity by alter soil properties, and in turn to alter the growth of other plants. According to the NWH, invasive species disturb native species growth via root allelopathy [6], altering soil microbial communities [24, 25]. There is increasing evidence that plant–soil interactions can promote successful invasions of alien species and drive plant–plant interactions [5, 26–28]. Chemical allelopathy is suggested as a fundamental mechanism by which invasive plant species displace native ones [12]. Some studies found that successful invaders suppress neighboring plant species growth through allelopathy, via altering soil community’s composition [23, 29]. Allelochemicals produced by alien invaders may disrupt regular decomposing processes in soil community when they are released into the novel ecosystems [30]. Our study, conducted in controlled growth chambers, provides evidence that allelopathic effects change with concentration of root extracts. In particular, soil microbial community functional diversity, as characterized by CLPP, is sensitive to root extracts.

The different patterns of microbe carbon utilization indicate the variability in soil microbial metabolic potential in response to root extracts. R. typhina roots extracts decrease the AWCD, diversity and evenness, and inhabit on soil microbial carbon utilization activity. AWCD reflects single carbon source utilization ability and metabolic activity of soil microbe, and the lower the value, the lower the metabolic activity of soil microorganism [31, 32]. All six groups of substrate utilization are altered by root extracts, with carbohydrates, carboxylic acids and polymers decreasing and phenolic acid increasing in treated soils. This indicates that soil microbial communities feed less on carbohydrates, carboxylic acids and polymers but more on phenolic acid in soils with root extracts. Hydroxybenzoic and hydroxycinnamic acids are the two major types of phenolic acids produced by R. typhina, which may enhance phenolic acid consumption of soil microbial communities.

Our results show that R. typhina root extracts increase soil pH and EC, while decrease SOC and TN, in accordance with previous studies [33, 34]. Increase in pH may be caused by a higher uptake of nitrate as the preferential nitrogen source [35, 36]. By altering soil pH and changing the microbial community activity, invasive plants can modify the native plant nutrient uptake [23, 37]. Previous experiments indicate that invasive plants increase utilization of nutrients with allelopathic substance that also inhibit native plant growth [38, 39]. According to this evidence, our study suggests that changes in soil pH, TN, SOC and EC are the main factors shifting soil microbial activity.

In conclusion, our study suggests that R. typhina can increase its fitness via allelopathic biochemicals that modify the EC, soil pH, SOC, TN and its microbial community metabolic activity, ultimately inhibiting the growth of T. erecta. We highlight that interactions between plants and soil microbes are critical for the successful invasion of alien plant species.

Acknowledgments

We thank Liang Jin and Jianfeng Zhang for providing valuable comments on this manuscript.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported by the Department of Science and Technology of Jilin Province (20190303078SF). GL was supported by the Swiss National Science Foundation (P2ZHP3_187938).

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Decision Letter 0

Raffaella Balestrini

3 Nov 2020

PONE-D-20-31348

Invasive species allelopathy decreases plant growth and soil microbial activity

PLOS ONE

Dear Dr. Qu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The aim of the work is to demonstrate the allelopathic effect of the plant Rhus typhina on Tagetes erecta and soil microbial population. Authors use a water extract from R. typhina roots. The extract was obtained after grinding the dried roots, incubating the root powder in water for 48 h under shaking, concentrating the extract in a rotary evaporator and freeze drying. The extract was then used to prepare solutions at different concentrations. Solutions were used to water pots containing plants. All the following analyses (plant growth, soil properties, microbial community diversity) were done on soil and plants sampled from these pots.

The work is well described; however, I have a big concern about the pertinence of using the root extract.

Author should explain how this root extract added to the soil can mimic what R. typhina is really releasing in the soil during its growth. In fact, the extract contains the molecules that water could extract from the whole root, but there is no proof that those molecules are the ones that R. typhina roots exudate in the soil. In the introduction, authors cite the “novel weapon hypothesis” and say that “Our results support the ‘novel weapons hypothesis’”. The cited hypothesis is proposed by Callaway and Ridenour (2004) who say “that some exotics transform from native weaklings to invasive bullies by exuding biochemicals that are highly inhibitory (allelopathic) to plants or soil microbes in invaded communities”. The hypothesis is based on molecules released as exudates. Also, the cited works in the discussion are about root exudates, rhizospheric soil, leaf leachates. Authors describe the effects on T. erecta growth and on soil bacteria, but those effect are due to molecules extracted from roots non exudated by roots. There is a big difference between root exudates and root extract.

Also, the choice of the control is not convincing. In my opinion, a more appropriate control would be not water but root extract from a plant that do not have an allelopathic effect on T. erecta, like one historically living in the same habitat. Authors should be sure that the effect on soil microbial activity is due specifically to R. typhina molecules and not to a general shift in microbial population due to the addition to the soil of any root extract.

In some points of the discussion authors over-interpret their data:

lines 202-203: they state “Taken together, our results indicate how allelopathy impacts both plant and soil community, revealing that plant–soil microbe interactions can mediate invasion success”. I agree on the first part on the sentence, since root extract do have an effect on plant growth and on microbial population. However, data do not help us understanding whether the effect on plant is direct or mediated by microbes.

Line 204: “our results show that plants have the potential to alter soil”: this sentence is misleading: written like this means that plants, in their active growth, can alter soil, but results are on root extract not on exudates.

Lines 206-207. “This study suggests that invasive species can increase their own fitness relative to other plant species by modifying soil conditions and microbial community” and Lines 238-239 “are the main factors shifting soil microbial activity and mediating the impact of invasive plants on the growth of other plants.” Again, there is not proof in the work that effect on plants is either direct or mediated by other factors.

Citation of literature is sometimes not appropriate

Line 209: Tian et al 2017: this work is about the effect of N fertilisation on soil microbial community, not on allelopathy

Lines 226-228: “These results are partially in agreement with previous studies indicating that root extracts serve as soil carbon substrates for specific soil microbial communities (Fan et al. 2010, Wu et al. 2013, Li et al. 2017)”. None of these cited papers deal about root extracts: they describe works on exudates and rhizospheric soil.

Minor points

Line 41: either remove “is”, or add “which” before “is”

Line 64: Substitute “As” with “It is” or rephrase the sentence

Line 98: “five plant” modify in “five plants”

Line 137: remove “in”

Line 138: change “concentration extracts” to “extract concentrations”

Lines 146-147: according to Table 1, plant height is not significantly different from control at 10 mg/mL.

Line 164: table 2 shows soil properties data not AWCD.

Line 230: Olchowik et al. 2012 demonstrated the presence of tannins in leaves: there is no indication of their presence in roots

Reviewer #2: The manuscript Invasive species allelopathy decreases plant growth and soil microbial activity aims at demonstrating the capaicty of the invasive shurb R. typhina to inihibit other plants growth to invade the soil. Data reported support the hypothesis that R. typhina roots extracts inhibited T. erecta growth via altering the physico-chemical soil conditions, as well as the microbial community.

The manuscript is well written and presented in intellegible fashion. Beside I am not English mother-tongue, it seems that the manuscript has been written in standard English.

The conclusion are supported by the reported data.

However more information on the conditions in which T. erecta has been grown during the experimental condition,such as light humidity, temperature, photoperiod.

More information about the use Community-level physiological profiles (CLPP) should be provided for reader who are not familiar with such method.

**********

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Reviewer #1: No

Reviewer #2: Yes: Biancaelena Maserti

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PLoS One. 2021 Feb 9;16(2):e0246685. doi: 10.1371/journal.pone.0246685.r002

Author response to Decision Letter 0

5 Dec 2020

1.We revise the manuscript and ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

2.All relevant data are within the paper.

3.We amend our list of authors on the manuscript to ensure that each author is linked to an affiliation.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (22.3KB, docx)

Decision Letter 1

Raffaella Balestrini

25 Jan 2021

Invasive species allelopathy decreases plant growth and soil microbial activity

PONE-D-20-31348R1

Dear Dr. Qu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Raffaella Balestrini

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors took in consideration all my concerns and replied accordingly.

Minor points

lines 25 and 209: change "in particularly" with "in particular"

Reviewer #2: The authors responded to all my previous comments and now the manuscript could be published.

I reccomen to change to lower case, the initial character of roots at line 22: roots, instead Roots.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Biancaelena Maserti

Acceptance letter

Raffaella Balestrini

27 Jan 2021

PONE-D-20-31348R1

Invasive species allelopathy decreases plant growth and soil microbial activity

Dear Dr. Qu:

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on behalf of

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PLOS ONE

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