Invasion by the weed Conyza canadensis alters soil nutrient supply and shifts microbiota structure

Journal Pre-proof Invasion by the weed Conyza canadensis alters soil nutrient supply and shifts microbiota structure Hai-Yan Zhang, Priscila Goncalves, Elizabeth Copeland, Shan-Shan Qi, Zhi-Cong Dai, Guan-Lin Li, Cong-Yan Wang, Dao-Lin Du, Torsten Thomas PII:

S0038-0717(20)30036-5

DOI:

https://doi.org/10.1016/j.soilbio.2020.107739

Reference:

SBB 107739

To appear in:

Soil Biology and Biochemistry

Received Date: 16 August 2019 Revised Date:

23 January 2020

Accepted Date: 2 February 2020

Please cite this article as: Zhang, H.-Y., Goncalves, P., Copeland, E., Qi, S.-S., Dai, Z.-C., Li, G.-L., Wang, C.-Y., Du, D.-L., Thomas, T., Invasion by the weed Conyza canadensis alters soil nutrient supply and shifts microbiota structure, Soil Biology and Biochemistry (2020), doi: https://doi.org/10.1016/ j.soilbio.2020.107739. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

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Invasion by the weed Conyza canadensis alters soil nutrient supply and

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shifts microbiota structure

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Hai-Yan Zhanga,b, Priscila Goncalvesc, Elizabeth Copelandc, Shan-Shan Qi a,d,e,f, Zhi-

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Cong Daia,c,d,e,*, Guan-Lin Lid,e, Cong-Yan Wangd,e, Dao-Lin Dua,d,e,*, Torsten Thomasc

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a

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212013, PR China b

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c

d

Institute of Environment and Ecology, Academy of Environmental Health and Ecological Security, Jiangsu University, Zhenjiang 212013, PR China

e

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Centre for Marine Science and Innovation, School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, 2052, Australia

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Changzhou Environmental Monitoring Center, Puqian Street 149, Changzhou 213000, PR China

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Institute of Agricultural Engineering, Jiangsu University, Xuefu Road 301, Zhenjiang

School of the Environment and Safety Engineering, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, PR China

f

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Ecology and Evolution Research Centre, School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, 2052, Australia

H.Y. Zhang and P. Goncalves contributed equally to this work. *

Corresponding author. E-mail addresses: [email protected] (Z. C. Dai), [email protected] (D. L. Du).

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Abstract

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Modifications in soil fertility and microbiota structure driven by invasive plants can initiate a

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self-promoting mechanism that facilitates their invasion process. This study aimed to resolve

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how the progression of invasion affects the chemical, biochemical and microbial properties of

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soil using Conyza canadensis, a widespread and noxious invasive farmland weed, as a model.

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Different stages of the invasion process were simulated by growing C. canadensis and a non-

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invasive crop, Lactuca sativa, at different relative densities. Increasing invasion levels (i.e.

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increasing invader relative densities) resulted in altered properties of the soil, with an overall

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increase in nutrient supply and enzymatic activities as invasion intensified. Threshold

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changes in available nitrogen, organic matter and catalase activity in the soil were identified

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at invasion levels of 69%, 50% and 47%, respectively. Increasing invasion levels also

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affected the structure of the soil microbiota, with substantial changes occurring in the relative

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abundance for a number of bacterial and fungal taxa, including some that are relevant to

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nutrient cycling. Such changes in soil abiotic and biotic composition driven by C. canadensis

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might lead to positive plant-soil feedbacks that could promote the establishment and spread

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of the invasive weed.

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Keywords: Plant invasion · Soil microbiota · Chemical properties · Biochemical properties ·

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Microbial communities

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1. Introduction

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Invasive weeds have been considered one of the major threats to the structure and functioning

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of global ecosystems (Lövei, 1997; van der Putten et al., 2007; D'Antonio et al., 2017),

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leading, for example, to substantial economic losses in agriculture (Duncan et al., 2004). The

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progression of the invasion process is known to be strongly related to soil properties as well

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as to the composition and functional capabilities of soil microbial communities (van der

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Putten et al., 2007; Jordan et al., 2008; Lorenzo et al., 2013; Dai et al., 2016; Kong et al.,

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2017). Belowground microbial communities play important roles in soil nutrient cycling and

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provision of essential plant nutrients (Trognitz et al., 2016). Successful invasion by weed

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species has been associated with the establishment of plant-soil feedback mechanisms

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(Rodriguez-Echeverria et al., 2013). Plant-soil feedback has been widely studied in both

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natural and agricultural systems, and it occurs when plants directionally alter the soil abiotic

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and biotic properties that influence their growth, with consequent effects on the plant

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community dynamics and agricultural systems (Mariotte et al., 2018; Bennett and

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Kliromomos, 2019). Plants can associate with a range of bacterial and fungal mutualists,

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which are all important drivers of plant-soil feedback (Mariotte et al., 2018). Besides,

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invasive plants may influence microbial community structure through allelopathic root

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secretions or litter decomposition (Ehrenfeld et al., 2001), for many exudates are selective for

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specific soil microbial groups mediating biochemical and nutrient acquisition processes

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(Kremer, 2014). Evidence also shows that some invasive species are able to establish more

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positive feedback interactions with the soil microbiota and seem to have greater dependence

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on these associations than non-invasive crops do (Massenssini et al., 2014; Trognitz et al.,

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2016).

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Successful invasion requires that the invasive species moves from its original

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ecosystem into a new habitat, where it propagates and establishes itself outside its native

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range, followed by a typically rapid spread to eventually form a monoculture through the

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displacement of the native species. Stages of this invasion process can be characterized by

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different densities and ratios of native to invasive species. However, current studies on soil

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properties and microbiota in response to plant invasion have focused only on three stages of

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the invasion process: (1) monoculture of native species (i.e. no invasion), (2) native and

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invasive plants mixed in the habitat (usually at equal proportions), and (3) monoculture of

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invasive species (i.e. invader monoculture) (Lorenzo et al., 2010; Lazzaro et al., 2014; Qin et

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al., 2014; Kong et al., 2017). An understanding of the impacts of different ratios of invader to

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native species has however been missing, thus precluding the identification of critical

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invasion thresholds, where soil resource or ecological processes do not respond linearly to

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invasion.

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Conyza canadensis (commonly known as horseweed) is a globally distributed

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invasive weed, which has been shown to cause considerable impacts on the structure,

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biodiversity and functioning of many ecosystems, including huge economic losses in

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agriculture by overgrowing into a dense monoculture and preventing regeneration of other

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species (Wiese et al., 1995; Queiroz et al., 2012). Worldwide, C. canadensis has been

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affecting more than 40 main crops, invading orchards, vineyards, hay crops, pastures,

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rangeland and field crops such as corn, soybean and cotton, particularly where conservation

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tillage or no-till systems are used (Bruce and Kells, 1990; Buhler, 1992; Wiese et al., 1995;

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Holm et al., 1997; Weaver, 2001). In China, C. canadensis encroaches a large number of

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varied agricultural fields from the northeast to the south (Weber et al., 2008). Several studies

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have identified the factors that contribute to the spread of C. canadensis into new habitats and

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regions, and these include genetic diversity (Main et al., 2004; Circunvis et al., 2014),

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evolutionary adaptation (Nandula et al., 2006; Shah et al., 2015) and allelopathic effects

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(Djurdjević et al., 2011; Queiroz et al., 2012; Zhang, 2017). The density of C. canadensis has

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also been found to be significantly correlated with soil properties (Shontz and Oosting, 1970),

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and the herbicide resistance of this species has been associated with biotic components in the

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soil (Schafer et al., 2013). These results indicate that soil abiotic and biotic factors are

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important for the invasion success of C. canadensis. However, the dynamics and impacts

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resulting from the interplay between this weed and soil nutrient availability and cycling as

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well as its associated microbial communities have not been fully explored.

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In this study, we investigated the effects of the invasion process on the abiotic and

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biotic properties of soil using C. canadensis as a model due to its ecological and economic

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significance. The relative abundance of invasive species can be used as an indicator of

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invasion level since it reflects the contribution of the species to a community, and, as such,

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the extent or severity of weed invasion in an area, besides being independent of scale and

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comparable across regions and ecosystems (Catford et al., 2012). Here, progressive levels of

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invasion were simulated by growing the invasive weed C. canadensis with a non-invasive

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species at different relative abundances, spanning from 0 to 100% with 25% increments. We

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hypothesize that these different invasion levels affect the soil chemical and biochemical

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properties as well as alter the structure of the soil microbial communities. Such changes could

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then trigger positive feedbacks to support the establishment and dominance of C. canadensis.

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Based on these hypotheses, we focused this study on addressing three main questions: (1)

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How do the abiotic and biotic properties of soil change as invasion by C. canadensis

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intensifies? (2) Are there threshold changes in soil properties dependent on the relative

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density of C. canadensis? (3) Do such changes support the notion of plant-soil feedback as a

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driver of invasion by C. canadensis?

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2. Materials and Methods

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2.1. Study design

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Seeds of the weed Conyza canadensis were collected from a vegetable field near the campus

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of Jiangsu University (32º12'17"N, 119º30'17"E), Zhenjiang, China, in September 2015, and

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stored at 4°C. As the non-invasive species, seeds of the crop Lactuca sativa (common lettuce)

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were purchased from a local merchant company in Zhenjiang, China. The study was

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conducted from May to September 2016.

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Seeds of C. canadensis and L. sativa were sterilized with 5% sodium hypochlorite for

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10 min and rinsed 10 times with sterile distilled water. The soil (loam texture, pH = 8.2) was

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obtained from a vegetable field near the campus of Jiangsu University, being sieved and

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thoroughly mixed before seeding. C. canadensis and L. sativa were cultivated in plastic pots

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(39 × 27 × 9 cm) containing 8 kg of soil and watered with 200 mL sterilized distilled water

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every two days. Seedlings of C. canadensis and L. sativa with similar sizes (height of

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approximately 5 cm) were used in the experiment.

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To explore the effects of invasion by C. canadensis on soil abiotic and biotic

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properties, different ratios of C. canadensis to L. sativa were used as a proxy to represent

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successive levels of invasion. A total of four seedlings were grown in each pot, as follows

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(Fig. 1): (1) No invasion stage included four seedlings of L. sativa (0% invader relative

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density); (2) Early stage of invasion included three seedlings of L. sativa and one seedling of

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C. canadensis (25% invader relative density); (3) Intermediate stage of invasion included two

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seedlings of L. sativa and two seedlings of C. canadensis (50% invader relative density); (4)

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Dominant stage of invasion included one seedling of L. sativa and three seedlings of C.

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canadensis (75% invader relative density); (5) Displacement of native/crop species included

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four seedlings of C. canadensis (100% invader relative density). Soil samples were also

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placed in pots without plants (bare soil; negative control). Treatments were set up in triplicate

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and pots were randomly placed in a greenhouse under natural light. Other weeds that very

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rarely and spontaneously emerged were removed by hand.

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2.2. Soil sampling and nutrient assessment

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Samples of soil were collected from each pot after four months of plant growth for analysis

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of chemical and biochemical properties and microbial community composition. Soil samples

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(113 to 122 g from 5 cm of top layer; 5 cm core diameter) were randomly collected from

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each plot after removal of surface debris (Qin et al., 2014). Samples were homogenized and

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stored at -80°C in aseptic sealed bags until further processing.

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The chemical characterization of the soil was performed following the methods

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described by Bao (2000) (methodological details were in Table S1). Briefly, soil organic

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matter (SOM) content was measured by the potassium dichromate volumetric method, total

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nitrogen (TN) content was quantified using the Kjeldahl method, total phosphorus (TP)

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content was assessed by spectrophotometry using ammonium molybdate, while total

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potassium (TK) content was determined by flame photometry. The concentrations of

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available nitrogen (AN), available phosphorus (AP) and available potassium (AK) were

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analyzed following the methods of Conway, spectrophotometry and flame photometry,

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respectively.

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The biochemical characterization of the soil involved the quantification of the

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activities for the enzymes urease, invertase, catalase and alkaline phosphatase (ALP). Urease

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activity was determined spectrophotometrically at 578 nm and expressed as mg of NH4+-N

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produced per gram of dried soil in 24 h (Nannipieri et al., 1980). Invertase activity was

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quantified spectrophotometrically at 508 nm using the 3,5-dinitrosalicylic acid method and

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expressed as mg of glucose produced per gram of dried soil in 24 h (Ohshima et al., 2007).

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Catalase activity was measured using the potassium permanganate titration method and

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expressed as mg of KMnO4 produced per gram of dried soil in 30 min (Guan, 1986). ALP

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activity was determined spectrophotometrically at 410 nm using the p-nitrophenyl phosphate

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salt method and expressed as mg of p-nitrophenol released per gram of fresh soil (Tabatabai,

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1994).

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2.3. DNA extraction and amplicon sequencing

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Total DNA was extracted from 250 mg of homogenized soil sample using the PowerSoil

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DNA Isolation Kit according to the manufacturer’s protocol (MoBio Laboratories, Inc.,

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USA). DNA quality was determined by visualization on a 0.8% (w/v) agarose gel. The V3-

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V4 region of the bacterial 16S rRNA gene was amplified using the primer pair 338F (5’-

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ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’).

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The internal transcribed spacer-1 (ITS1) gene region of fungi was amplified using the primers

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ITS1F

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GCTGCGTTCTTCATCGATGC-3’). The amplified products were examined for integrity

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and correct fragment sizes by running on a 1.8% (w/v) agarose gel. The amplicons were

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sequenced using the Illumina HiSeq 2500 platform (2 × 250 bp; Illumina Inc., San Diego,

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USA) by BioMarker Technologies Co., Ltd., Beijing, China.

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Sequencing data were processed to remove low quality bases and the paired end reads were

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assembled using FLASH (version 1.2.7) (Magoc and Salzberg, 2011). Sequences shorter than

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50 bp or containing low-quality nucleotides (minimum Phred score of 30) were removed

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using Trimmomatic (version 0.33) (Bolger et al., 2014). Chimeric sequences were removed

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using UCHIME (version 4.2) (Edgar et al., 2011). Sequences with similarities greater than

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97% were grouped into operational taxonomic units (OTUs) using UCLUST implemented in

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QIIME (version 1.8.0) (Edgar, 2010). Taxonomic assignment of bacterial sequences was

(5’-CTTGGTCATTTAGAGGAAGTAA-3’)

8

and

ITS1R

(5’-

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performed using the SILVA database (version 132) (Quast et al., 2013), while fungal

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sequences were classified using the UNITE database (version 7.2) (Koljalg et al., 2013).

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2.4. Statistical analyses

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Statistical analyses were performed using the R software (version 3.5.1) (R Core Team,

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2018). The effects of weed invasion on soil chemical and biochemical properties as well as

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on soil microbial community structure were assessed using univariate and multivariate

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analyses. Alpha-diversity indexes, including OTU richness, Chao1, Shannon and Inverse

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Simpson, were calculated for both bacterial and fungal communities. Calculation of alpha-

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diversity metrics, rarefaction curves and distance-based analysis (PCoA and PERMANOVA

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using Bray-Curtis distance) were conducted using the R package ‘vegan’ (Oksanen et al.,

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2019). Linear regression models were used to test the effect of invasion levels on the

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different chemical and biochemical soil parameters as well as on the alpha- and beta-diversity

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of the microbial communities. One-way ANOVA followed by Tukey post-hoc comparisons

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tests were performed to compare the response of the univariate abiotic and biotic parameters

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among the different invasion levels. Data that did not fit a linear regression model (non-linear

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response to invasion levels) were further analyzed using locally estimated scatterplot

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smoothing (LOESS) regression. Invasion thresholds were determined by fitting piecewise

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linear regressions to chemical, biochemical and microbial data (using the R package

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‘segmented’). Vector fitting to ordinations (function ‘envfit’ from the R package ‘vegan’)

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was used to identify the soil abiotic properties that best predicted bacterial and fungal

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community structures. Multivariate negative binomial models were used to identify the

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bacterial and fungal sequences most affected by the gradient of invasion (R package

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‘mvabund’) (Wang et al., 2019). Statistical significance was set at p < 0.05 for all statistical

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tests.

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3. Results

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3.1. Chemical and biochemical characterization of soil

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The gradient of invasion by C. canadensis resulted in modifications in the nutrient and

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enzymatic composition of the soil, including changes in the concentration of TN, AN, AP and

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SOM, as well as in the urease, invertase, ALP and catalase activities (Fig. 2). Increasing

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invasion levels (i.e. increasing invader relative densities) correlated in a linear, positive way

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with soil TN amount (R2 = 0.38, p = 0.015), and urease (R2 = 0.50, p = 0.003) and invertase

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activities (R2 = 0.70, p < 0.001) (Fig. 2). However, the majority of the chemical and

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biochemical parameters showed non-linear responses to increasing invasion levels. AN and

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AP contents and ALP activity were found to remain stable or slightly reduced as invasion

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level reached 75%, after which they increased up to 2-fold (p = 0.004), 5-fold (p < 0.001 )

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and 1.7-fold (p = 0.006), respectively (Fig. 2). SOM content (p = 0.045) and catalase activity

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(p < 0.001) decreased at 50% invasion level , but then increased in soil samples when

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dominated by C. canadensis (75% invasion levels) (p = 0.035 for SOM content and p < 0.001

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for catalase activity), reaching values similar or higher to those of the no invasion stage (Fig.

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2). Piecewise regression identified invasion levels of 69%, 50% and 47% as critical

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thresholds for AN, SOM and catalase, respectively, at which the parameters abruptly shifted

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in direction or magnitude (Fig. 2). No significant differences in soil TK (Fig. 2), AK and TP

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contents were found among the invasion levels (p > 0.05).

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Changes in soil abiotic factors driven by C. canadensis resulted in a clear separation

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among the invasion levels in an ordination plot (Fig. 3), which demonstrates that 0% and

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25% levels are more similar to each other, while 50% and 75% invasion levels move

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sequentially away from the non-invaded/low-invaded states through changes mainly in SOM

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and AK contents, and urease and invertase activities. The C. canadensis monoculture stage

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was found to be the most distinct group and this was mainly driven by changes in ALP

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activity, and in the amount of AP, AN and TP in soil (Fig. 3).

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3.2. Changes in the diversity and structure of soil microbial communities

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Sequencing of microbial communities generated a total of 1.01 million bacterial sequences

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(average of 56,239 sequences per sample) and 1.11 million fungal sequences (average of

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61,496 sequences per sample). These sequences were clustered into 2,376 and 273 distinct

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operational taxonomic units (OTUs) for bacteria and fungi, respectively. Rarefaction curves

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(Fig. S1) and Goods coverage index (min = 99.64 and max = 99.74 for bacteria; min = 99.96

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and max = 99.99 for fungi) revealed nearly complete sampling of the soils’ microbial

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communities with the given sequencing effort.

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The richness and diversity of soil bacterial communities did not change significantly

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in response to the increasing invasion levels of C. canadensis (p > 0.05) (Fig. 4A). However,

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we observed a decrease in richness (p = 0.017) and diversity (p = 0.035 for Chao1, p = 0.005

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for Inverse Simpson and p = 0.014 for Shannon) of fungal communities as the relative

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density of C. canadensis increased from 0 to 25% (Fig. 4B). At higher invasion levels

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(invader relative density of 50 to 100%), richness (p = 0.006) and diversity (p > 0.05 for

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Shannon and Inverse Simpson, and p = 0.011 for Chao1) of fungal communities were found

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to be similar or slightly higher to the non-invasion stage (Fig. 4B).

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Beta-diversity analysis showed that the invasion by C. canadensis significantly

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altered the bacterial and fungal structure of the soil (PERMANOVA, p = 0.001) (Fig. 5). Bare

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soil (i.e. soil samples kept in pots under the same environmental conditions for the duration

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of the experiment) exhibited a distinctive microbial community structure when compared to

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soil samples where any seedling was present. Increasing relative densities of C. canadensis

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were accompanied by more pronounced changes in microbial community structure. Soils

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with no to low levels of invasion (0 and 25% invasion levels) had similar community

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structure for bacteria and fungi. At invasion levels higher than 50%, soil exhibited a similar

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structure for bacterial communities (Fig. 5A), but a divergent structure for fungal

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communities (Fig. 5B). Vector fitting analyses (using ‘envfit’ function from the R package

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‘vegan’) revealed that differences in microbial structure across the gradient of C. canadensis

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densities were strongly correlated with the activity of the enzymes invertase (R2 = 0.49, p =

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0.025 for bacterial communities, and R2 = 0.54, p = 0.012 for fungal communities) and ALP

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(R2 = 0.71, p = 0.002 for fungal communities) (Fig. 6 and Table S2).

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Differences in microbial community structure across the gradient of invasion by C.

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canadensis were investigated at various levels of taxonomic classification. Soil samples were

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found to be dominated by bacteria from the phyla Proteobacteria, Acidobacteria,

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Actinobacteria and Bacteroidetes, which together accounted for ~73% of the bacterial

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community, regardless of the invasion level (Fig. S2). Compared with the non-invasion stage,

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soils with elevated relative densities of C. canadensis (50 to 100% invasion levels) were

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enriched in bacteria from the phyla Actinobacteria (p = 0.023) and Chloroflexi (p = 0.011). In

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contrast, dominance of the invasive weed led to a drop in the abundance of Planctomycetes (p

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= 0.005) (Fig. 7A).

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The fungal community of the soil was dominated by members of the phyla

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Chytridiomycota and Ascomycota, which together made up ~70% of the fungi present in the

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samples (Fig. S2). Similar to what was observed for the bacterial communities, fungal

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community structure also changed in response to the gradient of C. canadensis invasion. The

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presence of C. canadensis (25% relative density) resulted in a decrease in the abundance of

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fungi from the phylum Ascomycota (2.2-fold) (p = 0.002) (Fig. 6B). Increasing invasion

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levels (at 75% invasion level) led to a reduction of Chytridiomycota (up to 1.8-fold) (p =

12

281

0.003) compared with 25% invasion level. In addition, fungi from the phylum

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Glomeromycota were found to be enriched (up to 6-fold) (p = 0.001) at 75% invasion level

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compared with no-invasion stage (Fig. 7B).

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A total of 725 bacterial OTUs and 48 fungal OTUs were found to have their

285

abundances significantly affected by the relative density of C. canadensis. The abundances of

286

many of these OTUs were clearly distinct, particularly between soils from no- to low-invaded

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states (0 to 25% invasion levels) and those from higher invasion levels (50 to 100% invasion

288

levels) (Fig. S3 and Fig. S4). Differentially abundant OTUs in response to C. canadensis

289

invasion included bacteria from the families Sphingomonadaceae (p = 0.003 for OTU20360

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and p < 0.001 for OTU27857) and Nitrosomonadaceae (p = 0.005) (both from phylum

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Proteobacteria)

292

Glomeromycota) (p < 0.001) and Thelebolaceae (phylum Ascomycota) (p = 0.012) (Fig. S3).

(Fig.

S3)

and

fungi

from

the

families

Glomeraceae

(phylum

293 294

4. Discussion

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Increasing ratios of the invasive weed C. canadensis to L. sativa resulted in pronounced

296

changes in soil nutrient composition, with an overall increase in nutrient content and

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enzymatic activities. Specifically, urease and invertase activities and levels of TN increased

298

linearly with increments in the relative density of C. canadensis, whereas AN, AP, ALP, TK,

299

SOM and catalase activity showed non-linear responses. The non-linear responses were in

300

some cases characterized by clear thresholds, where the parameters (i.e. AN, SOM and

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catalase) changed dramatically in magnitude and/or direction of response. An evident shift in

302

the response of these parameters was observed when ratios of the weed C. canadensis to L.

303

sativa were between 47% and 69%. Such threshold at particular ratios might represent a key

304

event in the invasion process and would support the concept of ‘threshold of potential

13

305

concern’ (TPC), in which any increase in density beyond the intermediate stage of invasion

306

could present a major threat to biodiversity as well as to ecosystem services and provisions

307

(Foxcroft, 2009). Our findings suggest that the invasive weed C. canadensis, particularly at

308

relative densities higher than 50%, is capable of inducing major alterations in the supply of

309

nutrients with potential downstream effects on soil ecosystem function, which could

310

ultimately facilitate its spread and dominance into the new habitat.

311

In addition to changes in soil nutrient and enzymatic activities, increasing relative

312

densities of C. canadensis triggered shifts in the soil microbiota. We observed a decrease in

313

the diversity of fungal communities at 25% versus 0% of C. canadensis, which could be

314

considered to reflect a comparison of initial invasion with non-invasion stages. Interestingly,

315

despite a drop in alpha-diversity, the fungal community structure has not been changed

316

significantly between those two invasion stages (see Fig. 5B). This observation suggests that

317

the invasion by C. canadensis causes mainly subtle reduction in the evenness of the

318

community and/or in the presence of low abundant members. At invader to native/crop ratios

319

reflecting later stages of the invasion process (e.g. 50-100% invasion levels), bacterial and

320

fungal communities showed different trajectories both in terms of diversity (in Fig. 4) and

321

structure (in Fig. 5).

322

Levels of AN and AP in soil were found to be slightly reduced when C. canadensis

323

was present at relatively low densities (25-50%). This depletion in soil nutrient might be

324

attributed to high uptake by the weed, and/or intense competition with soil microorganisms at

325

the early to intermediate stages of invasion (Zhang et al., 2009). However, once the

326

environment becomes dominated by C. canadensis (75-100% invasion levels), the contents of

327

these nutrients returned or surpassed those observed at the non-invasion stage (0%). This

328

series of sequential modifications in soil abiotic factors could represent a competitive trait for

329

invasive plants, as previously reported for the invasive Australian acacia Acacia dealbata.

14

330

Although areas with a partial coverage of A. dealbata (transition zone between native and

331

invaded vegetation) show lower exchangeable P content compared to non-invaded areas, the

332

concentration of this nutrient increased considerably in areas dominated by the invasive plant,

333

reaching values much higher than those observed in non-invaded areas (Lorenzo et al., 2010).

334

Our study revealed that such changes in nutrient supply are likely to be associated with the

335

interactions between microbial community members and C. canadensis and were correlated

336

with increasing weed relative density (Fig. 8). The ability of invasive plants to selectively

337

alter the soil microbiota through their root exudates and mucilage-derived substances or by

338

litter decomposition has been reported in a number of species (Ehrenfeld et al., 2001;

339

Kulmatiski et al., 2008; Zhao et al., 2014; Trognitz et al., 2016), whereas increasing invasion

340

levels might enhance this effect. Modifications in the structure of the soil microbiota can lead

341

to directional transformations of the chemical and biochemical properties of soil, which is

342

instrumental in regulating plant-plant interactions and, consequently, underpin plant

343

composition and diversity (Bever et al., 2010)..

344

A number of studies have demonstrated that microbial community structures in soil

345

change with plant invasion (Wolfe and Klironomos, 2005; Rodrigues et al., 2015; Kong et al.,

346

2017). Rodrigues et al. (2015) showed that the abundance of several specific bacterial and

347

fungal taxa (belonging to the phyla Proteobacteria, Acidobacteria, Actinobacteria and

348

Ascomycota) were increased with the increasing invasion levels, while Kong et al. (2017)

349

found a drop in Planctomycetes and Actinobacteria. We observed an increased abundance of

350

Actinobacteria and Chloroflexi, and depletion of members of Planctomycetes response to

351

invasion by C. canadensis in our study (compared with no- and low-invaded states). Bacteria

352

from the phylum Actinobacteria are important soil saprophytes capable of breaking down

353

litter in the process of decomposition, thereby affecting remineralization and nitrogen cycling

354

(Jiang et al., 2014). Similarly, the group of photosynthetic bacteria from the phylum

15

355

Chloroflexi (OTU37023, OTU38067 and OTU4941 in Fig. S4), which was found at higher

356

relative abundance in treatments dominated by C. canadensis, can contribute to soil

357

fertilization and promote plant growth (Hug et al., 2013). High invasion levels also correlated

358

with a low relative abundance of some bacteria from the families Nitrosomonadaceae and

359

Nitrospiraceae (OTU37725 and OTU8328), which are known to oxidize ammonium and

360

nitrite, respectively, under aerobic conditions (Daims, 2014; Prosser et al., 2014).

361

Along with some bacterial taxa, mycorrhizal fungi are important decomposers in the

362

soil converting recalcitrant organic materials into accessible forms, and contributing to

363

recycle both N and P (Rodriguez et al., 2004; Wenke, 2008; He et al., 2009). Fungi from

364

phylum Glomeromycota (e.g. OTU7201), which harbors many arbuscular mycorrhizal fungi

365

forming symbiotic relationships with plants (Rodriguez et al., 2004), were found at higher

366

relative abundance in soil dominated by C. canadensis. This finding could potentially

367

indicate that symbiotic interaction facilitated invasion by C. canadensis, as previously

368

reported for the myco-heterotrophic plant Thismia sp. (Merckx et al., 2017). Moreover, the

369

family Thelephoraceae (e.g. OTU8536; phylum Basidiomycota), which can form

370

ectomycorrhizae with other fungal members (Haug et al., 2005), was also enriched at high

371

levels of invasion. Yagame and Yamato (2013) found that the ectomycorrhizal formation by

372

Thelephoraceae fungi significantly improved the growth of Cephalanthera falcate. The

373

changes in bacterial and fungal community structure that we observed at invasion levels of

374

50-75% may have contributed to the elevated soil TN content, and the replenishment of AN

375

and AP supply after the enhanced uptake of these nutrients for the growth and establishment

376

of C. canadensis (Fig. 8). Such changes in nutrient cycling might support the positive plant-

377

soil feedback, promoting the spread of the weed species, while suppressing the growth of the

378

crop population.

16

379

The effects of invasive species on the microbial community structure of soil have also

380

been evaluated in previous studies by measuring enzymatic activities (Kourtev et al., 2002;

381

Allison et al., 2006; Souza-Alonso et al., 2014). For instance, the phosphomonoesterase

382

activity has been considered a relevant driver of bacterial communities in the rhizosphere

383

(Rodríguez-Caballero et al., 2017). Nannipieri et al. (2011) showed the close relationship

384

between alkaline phosphomonoesterase and the microbial community structures. Similarly, in

385

our study, soil microbial structure strongly correlated with invertase and ALP activities

386

across the gradient of C. canadensis densities. Enzymatic activities can be seen as measures

387

of nutrient cycling (Lagomarsino et al., 2008). Urease activity was found to be higher with

388

increasing invasion levels, and this might have supported the increased TN content in

389

treatments dominated by C. canadensis, as urease is involved in N cycling (May and

390

Douglas, 1976). The enhanced invertase activity would result in higher SOM (Chase et al.,

391

1962; Chen et al., 2016), since this enzyme is directly related to carbon cycling. However, we

392

did not find such a direct correlation between invertase activity and SOM in the current

393

study. The lowest SOM content observed at 50% invasion level might associate with the

394

reduced abundance of some soil decomposers (e.g. OTU26157; phylum Chytridiomycota, p =

395

0.008) observed at that stage relative to the non-invasion stage. For instance,

396

Chytridiomycota play a crucial role in the decomposition process of cellulose, hemicellulose

397

and lignin, contributing to the regulation of carbon cycle (Barr, 2001; Gadd, 2004; Arellano

398

et al., 2009). In contrast, the relatively high SOM content in treatments dominated by C.

399

canadensis (above 50% invasion level), similar in values to those of the no- to low-invaded

400

states, might be related to the enrichment of bacteria from the family Sphingomonadaceae

401

(e.g. OTU20360, p <0.001 and OTU27857, p = 0.037). Members of this family are

402

commonly found in nutrient-enriched soils and are known for their capacity to utilize a wide

403

variety of carbon sources (Glaeser and Kämpfer, 2014). At 50% invasion level, soil also

17

404

exhibited the lowest catalase activity across the gradient of invasion (Fig. 8), which is

405

contrary to the previous study of Qin et al (2014).

406

In summary, our study shows that the invasive weed C. canadensis induces major

407

changes in both abiotic and biotic components of the belowground ecosystem. The triggering

408

of such changes may represent important functional traits for invasiveness by promoting

409

positive feedbacks that support the ecological dynamics and successful colonization of C.

410

canadensis. The understanding of plant-soil feedback mechanisms involving invasive species

411

can guide towards weed management and sustainable agriculture of crop species. The

412

impacts driven by C. canadensis invasion on soil nutrients and enzymatic activities also

413

indicate that a relative density of around 50% might represent a critical invasion threshold (i.e.

414

‘threshold of potential concern’, TPC) for this species. Identifying critical invasion thresholds

415

and understanding the biogeochemical implications associated with invasive weeds are key

416

steps towards mitigating the impact of invasive plants on soil biodiversity.

417 418

Competing interests

419

The authors declare no competing interests in relation to the work described.

420 421

Acknowledgements

422

This work was supported by the State Key Research Development Program of China

423

(2017YFC1200100), the National Natural Science Foundation of China (31600326,

424

31570414, and 31770446), the Natural Science Foundation of Jiangsu (BK20150503), the

425

China Postdoctoral Science Foundation (2017T100329), the Jiangsu University Research

426

Foundation (15JDG032), the Jiangsu Collaborative Innovation Center of Technology and

427

Material of Water Treatment, the Priority Academic Program Development of Jiangsu Higher

18

428

Education Institutions (PAPD), and Study Abroad Scholarship of Jiangsu Province & Jiangsu

429

University and the Australian Research Council.

430 431

Author contributions

432

Z.C. Dai, S.S. Qi, D.L. Du and H.Y. Zhang designed the research; H.Y. Zhang, S.S. Qi, G.L.

433

Li and C.Y. Wang performed the experiments; P. Goncalves and E. Copeland performed

434

bioinformatic and statistical analyses; H.Y. Zhang, P. Goncalves and Z.C. Dai wrote the first

435

draft; H.Y. Zhang, P. Goncalves, Z.C. Dai and T. Thomas reviewed the manuscript.

436 437

Appendix A. Supplementary data

438

Supplementary data (Figures S1-S4 and Table S1) to this article can be found online at Soil

439

Biology and Biochemistry’s website.

440

19

441

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Wiese, A.F., Salisbury, C.D., Bean, B.W., 1995. Downy brome (Bromus tectorum), Jointed

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goatgrass (Aegilops cylindrica) and horseweed (Conyza canadensis) control in fallow.

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Weed Technol 9, 249–254.

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Wolfe, B.E., Klironomos, J.N., 2005. Breaking New Ground: Soil Communities and Exotic Plant Invasion. Bioscience 55, 477–487.

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Yagame, T., Yamato, M., 2013. Mycoheterotrophic growth of Cephalanthera falcata

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(Orchidaceae) in tripartite symbioses with Thelephoraceae fungi and Quercus serrata

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(Fagaceae) in pot culture condition. Journal of Plant Research 126, 215–222.

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Zhang, H.Y., Qi, S.S.,Dai, Z.C., Zhang, M., Sun, J.F.,Du, D.L., 2017. Allelopathic potential

650

of flavonoids identified from invasive plant Conyza canadensis on Agrostis stolonifera

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and Lactuca sativa. Allelopathy J 41, 223–238.

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652

Zhao, J., Cheng, C., Gu, X., Liu, B., 2014. Effects of root exudates from invasive plant

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(Mirabilis jalapa) on soil microenvironment under different land-use types. Advanced

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655

30

656

Figure legends

657

Fig. 1 Experimental design used to assess the effects of invasion by the weed Conyza

658

canadensis on soil properties and microbial community structure. We simulated a gradient of

659

invasion, spanning from 0 to 100% (with 25% increments), by manipulating the relative

660

densities of invasive (C. canadensis) and non-invasive species (the crop Lactuca sativa).

661

Each treatment was set up in triplicate, and soil samples were collected from each pot after

662

four months of plant growth.

663

Fig. 2 Effect of invasion by Conyza canadensis on soil chemical and biochemical properties.

664

Soil nutrient and enzymatic levels show different patterns of response to the gradient of

665

invasion (0 to 100% invasion levels, with 25% increments), including linear and non-linear

666

relationships. Solid lines represent linear or LOESS fits and grey areas denote the 95%

667

confidence intervals. Vertical dotted grey lines indicate breakpoints in response identified by

668

piecewise regressions. TN: total nitrogen (unit: g/kg); AN: available nitrogen (unit: mg/kg);

669

AP: available phosphorus (unit: mg/kg); ALP: alkaline phosphatase; TK: total potassium

670

(unit: g/kg); SOM: soil organic matter (unit: %). Units for enzymatic activity are described in

671

Materials and Methods.

672

Fig. 3 PCA biplot integrating the nutrient and enzymatic levels of soil under different relative

673

densities of the weed Conyza canadensis. Length and color saturation of the arrows indicate

674

the contribution of that parameter to the divergence between treatments (i.e. parameters

675

represented by longer and darker arrows have a stronger contribution to group separation).

676

Fig. 4 Alpha-diversity of microbial communities associated with the different levels of

677

invasion by the weed Conyza canadensis (0 to 100% invasion levels, with 25% increments).

678

Alpha-diversity indexes were calculated for bacterial (A) and fungal (B) communities. Solid

679

lines represent LOESS fits and grey areas indicate 95% confidence intervals.

31

680

Fig. 5 Effect of invasion by Conyza canadensis on soil microbiome composition. Principal

681

coordinate analysis (PCoA) of soil bacterial (A) and fungal (B) communities at the different

682

invasion levels by C. canadensis. Ordinate separation was constructed based on Bray-Curtis

683

dissimilarity matrices and statistical differences between groups were assessed by

684

PERMANOVA.

685

Fig. 6 Correlation between microbial community structure (points and ellipses) and soil

686

abiotic factors (arrows). Non-metric dimensional scaling (NMDS) plots display the soil

687

abiotic variables found to be significantly correlated with bacterial and fungal communities

688

(p < 0.05, R2 > 0.4).

689

Fig. 7 Soil microbial communities strongly associated with the different stages of invasion by

690

Conyza canadensis. Plots display the relative abundance of sequences at the phylum level for

691

bacterial (A) and fungal (B) datasets. Solid lines represent LOESS fits and grey areas indicate

692

95% confidence intervals.

693

Fig. 8 Summary of the major changes in soil abiotic and biotic properties induced by

694

different relative densities of the invasive weed Conyza canadensis. Increasing relative

695

densities of C. canadensis (in combination with decreasing relative densities of Lactuca

696

sativa) are used as a proxy for the successive stages of invasion. The color associated with

697

the different parameters represents the fold-change in concentration or abundance, relative to

698

the non-invaded state (0% invasion level), where blue boxes indicate a decrease in the

699

abundance of a particular parameter (i.e. down-regulation), while red boxes reflect an

700

increase (i.e. up-regulation). Statistical analysis was performed between each displayed

701

invasion level (25-100%) and the non-invaded stage (0%). The boxes with (*) indicated a

702

statistically significant difference at p < 0.05.

32

Gradient of invasion (invader relative density)

Soil

0%

25%

Non-invasive crop Lactuca sativa

50%

75%

100%

Invasive weed Conyza canadensis

TN

1.8 1.6 1.4 1.2 1.0

Urease

Invertase

1.8

2.0

1.5

1.0

1.2 AN

AP

6.0

80.0

ALP 10.0

60.0

4.0

40.0

2.0

6.0

20.0

0.0

4.0

TK

40.0

SOM

35.0 30.0 25.0

8.0

0

25

50

75 100

4.0 3.5 3.0 2.5 2.0 1.5

Catalase

6.5 6.0 5.5 5.0

0

25

50

75 100

Invader relative density (%)

0

25

50

75 100

PCA2 (21.6%)

2

AP AN TP

AK

0%

100% ALP

25%

Catalase

0

TK

TN

50%

SOM Urease

−2

75%

−2

0

Invertase

PCA1 (28.8%)

2

4

Invader relative density 0%

25%

50%

75%

100%

(A)

Richness

2300

Chao1 2300

2200

2200

2100

2100

2000

2000

1900 Shannon

Inverse Simpson

6.8

400

6.7 300

6.6 6.5

200

6.4 6.3

0

25

50

75

100

0

25

50

75

100

Invader relative density (%) (B)

Richness

Chao1

190

200

170

180

150

160 Shannon

Inverse Simpson 20

3.5

15

3.0

10

2.5

5 0

25

50

75

100

0

25

Invader relative density (%)

50

75

100

(A)

(B) 0.1 0% 0.0

100%

100%

25%

PCoA2 (13.5%)

PCoA2 (18.3%)

75%

50%

−0.1

−0.2

0.1 50%

Soil 0.0 0% 25%

−0.1 Soil

p = 0.001 −0.1

0.0

p = 0.001 0.1

0.2

−0.2

PCoA1 (31.1%) Soil

75%

0%

−0.1

0.0

0.1

PCoA1 (19.7%) 25%

50%

75%

100%

0.2

(B)

ALP

NMDS2

NMDS2

(A) Invasion level

Invasion level

50%

Invertase

0%

Invertase

25%

100%

50%

100% 75%

75% 25%

0%

NMDS1

NMDS1

Invader relative density 0%

25%

50%

75%

100%

Relative abundance (%)

(A) Actinobacteria

Chloroflexi

8.0

15.0

Planctomycetes 2.0

7.0

12.5

1.5

6.0

10.0

1.0

5.0

7.5

0.5

4.0 0

25

50

75

100

0

25

50

75

100

0

25

50

75

100

Invader relative density (%) Relative abundance (%)

(B) Ascomycota

35.0

Chytridiomycota 70.0

30.0

30.0

60.0

25.0

20.0

50.0

20.0

10.0

40.0

15.0

30.0

10.0 0

25

50

75 100

Glomeromycota

0.0 0

25

50

75 100

Invader relative density (%)

0

25

50

75 100

Highlights Different invasion levels of Conyza canadensis were simulated in pot trials. Nutrients contents and enzyme activities increased with the higher invasion levels. Invasion thresholds were identified for soil nitrogen, organic matter and catalase. Invasion levels affected some microbes related to nutrient-cycling. Positive plant-soil feedback might be involved in the spread of weed C. canadensis.

Declaration of interests

The authors declare that there are no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.