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Plant EcologyAn International Journal ISSN 1385-0237Volume 219Number 5 Plant Ecol (2018) 219:539-548DOI 10.1007/s11258-018-0816-4

Do novel weapons that degrademycorrhizal mutualisms promote speciesinvasion?

Philip Pinzone, Daniel Potts, GaryPettibone & Robert Warren

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Do novel weapons that degrade mycorrhizal mutualismspromote species invasion?

Philip Pinzone . Daniel Potts . Gary Pettibone . Robert Warren II

Received: 8 September 2017 / Accepted: 7 March 2018 / Published online: 23 March 2018

� Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Non-native plants often dominate novel

habitats where they did not co-evolve with the local

species. The novel weapons hypothesis suggests that

non-native plants bring competitive traits against

which native species have not adapted defenses. Novel

weapons may directly affect plant competitors by

inhibiting germination or growth, or indirectly by

attacking competitor plant mutualists (degraded mutu-

alisms hypothesis). Japanese knotweed (Fallopia

japonica) and European buckthorn (Rhamnus cathar-

tica) are widespread plant invaders that produce potent

secondary compounds that negatively impact plant

competitors. We tested whether their impacts were

consistent with a direct effect on the tree seedlings

(novel weapons) or an indirect attack via degradation

of seedling mutualists (degraded mutualism). We

compared recruitment and performance using three

Ulmus congeners and three Betula congeners treated

with allelopathic root macerations from allopatric and

sympatric ranges. Moreover, given that the

allelopathic species would be less likely to degrade

their own fungal symbiont types, we used arbuscular

mycorrhizal (AMF) and ectomycorrhizal (ECM) tree

species to investigate the effects of F. japonica (no

mycorrhizal association) and Rhamnus cathartica

(ECM association) on the different fungal types. We

also investigated the effects of F. japonica and R.

cathartica exudates on AMF root colonization. Our

results suggest that the allelopathic plant exudates

impact seedlings directly by inhibiting germination

and indirectly by degrading fungal mutualists. Novel

weapons inhibited allopatric seedling germination but

sympatric species were unaffected. However, seedling

survivorship and growth appeared more dependent on

mycorrhizal fungi, and mycorrhizal fungi were inhib-

ited by allopatric species. These results suggest that

novel weapons promote plant invasion by directly

inhibiting allopatric competitor germination and indi-

rectly by inhibiting mutualist fungi necessary for

growth and survival.

Keywords Allelochemicals � Mycology � Invasive

species � Fallopia japonica � Polygonum cuspidatum �Reynoutria japonica � Rhamnus cathartica

Introduction

Invasive non-native plants often can outcompete and

displace native species (Levine et al. 2003; Spector

Communicated by William E. Rogers.

Electronic supplementary material The online version ofthis article (https://doi.org/10.1007/s11258-018-0816-4) con-tains supplementary material, which is available to authorizedusers.

P. Pinzone � D. Potts � G. Pettibone � R. Warren II (&)

Department of Biology, SUNY Buffalo State, 1300

Elmwood Avenue, Buffalo, NY 14222, USA

e-mail: hexastylis@gmail.com

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https://doi.org/10.1007/s11258-018-0816-4

Author's personal copy

and Putz 2006). The competitive advantages held by

non-native species likely derive from several mecha-

nisms, such as release from home range enemies,

including specialist consumers, pathogens, and para-

sites (Keane and Crawley 2002; Levine et al. 2003;

Mack et al. 2000; Maron and Vila 2001; Mitchell and

Power 2006). Fewer enemies may give non-native

species a greater competitive ability by exempting

them from the negative burdens enemies impose on

native competitors (Blossey and Notzold 1995; Mallik

and Pellissier 2000; Muller-Scharer et al. 2004;

Rabotnov 1982). Non-native species also may possess

direct advantages by bringing competitive mecha-

nisms to which native competitors have not adapted,

such as novel weapons, including degraded mutu-

alisms (Callaway and Ridenour 2004; Janos 1980;

Smith and Smith 2011, 2012; Stinson et al. 2006;

Vogelsang and Bever 2009).

Plant weapons include allelochemicals that directly

harm competing plants (Duke and Dayan 2006; Hale

and Kalisz 2012). Some phytotoxins disrupt essential

plant processes by targeting photosynthetic structures,

and/or the enzymes involved in respiration (Cipollini

et al. 2012; Dallali et al. 2014; Duke and Dayan 2006).

For example Centaurea maculosa (spotted knapweed)

disrupts calcium signaling in the root meristem of

competitors (Bais et al. 2003). Similar phytotoxins

also can inhibit seed germination and seedling growth

(Inderjit et al. 2008; Jessing et al. 2014; Klionsky et al.

2011; Warren et al. 2017). Plants also may indirectly

inhibit competitors by employing allelochemicals that

attack or deter their mutualist partners (Cantor et al.

2011; Raguso 2008; Stinson et al. 2006). For example,

some phytotoxic chemicals deter competitor repro-

duction by masking or overpowering attractive floral

scents, thereby reducing pollinator visitation (Raguso

2008).

Plants also may release anti-microbial allelochem-

icals belowground that reduce competitor fungal

mutualists. Approximately 90% of terrestrial plants

form mycorrhizal associations (Smith and Read 2008),

and most woody plants (but not all, Klironomos 2003)

require mycorrhizal colonization for germination,

growth, and/or survival (Nantel and Neumann 1992;

Siqueira and Saggin-Junior 2001). Mycorrhizal mutu-

alisms also can increase plant fitness compared to

plants without colonized roots (Janos 1980; Koide and

Dickie 2002). Mycorrhizae increase plant nutrient

acquisition as the fungi ‘scavenge’ for soluble

phosphorus and ‘mine’ for insoluble organic nitrogen

(Lambers et al. 2008; Smith and Smith 2012).

Additionally, with mycorrhizae, plants can allocate

more nutrients, water, and energy towards reproduc-

tive effort (Aguilar-Chama and Guevara 2012; Gange

and Smith 2005; Varga and Kytoviita 2010). Ectomy-

corrhizae (ECM) and arbuscular mycorrhizal fungi

(AMF) have similar functions, but differ morpholog-

ically and evolutionarily (Brundrett 2002). ECM

filaments live within the plant roots, but only in the

extracellular spaces, whereas AMF penetrate the

cortical cells of the plants roots (Malloch et al. 1980;

Smith and Read 2008).

The allelopathic degradation of competitor fungal

mutualisms may provide a decided competitive

advantage (Cantor et al. 2011; Hale and Kalisz 2012;

Schreiner and Koide 1993; Stinson et al. 2007;

Vierheilig et al. 2000), and plants that do not require

mycorrhizal fungi are more likely to use traits that

degrade mycorrhizal fungi (Bais et al. 2003; Stinson

et al. 2007). For example, a Eurasian species, garlic

mustard (Alliaria petiolata), is highly invasive in

North America (NA), and it comes from a lineage of

plants that do not require mycorrhizal fungi (Brundrett

2002; Janos 1980; Smith and Read 2008; Smith and

Smith 2011, 2012; Stinson et al. 2007). In turn, A.

petiolata root exudates (glucosinolates, flavonoids,

and allyl isothiocyanate) can inhibit fungal spore

germination by up to 57% and, as a result, the number

of mycorrhizal soil propagules decrease when A.

petiolata is present (Callaway et al. 2008; Cantor et al.

2011; Herrera et al. 1993; Requena et al. 1996; Stinson

et al. 2007). The lowered mycorrhizal potential in

invaded soils gives A. petiolata a competitive edge

against mycorrhizal dependent individuals (Callaway

et al. 2008; Stinson et al. 2007). Like A. petiolata,

Fallopia japonica (Japanese knotweed) does not form

mycorrhizal mutualisms (Schnitzer and Muller 1998),

and it may have a similar effect on competitor

mycorrhizae. Unlike F. japonica, Rhamnus cathartica

(European buckthorn) is an arbuscular mycorrhizal

AMF-dependent species (Godwin 1943). AMF-de-

pendent plants may still degrade mutualisms, but

presumably only selectively against ECM plant

species.

The goal of this study is to investigate the

allelochemical effect of R. cathartica and F. japonica

on European, Asian, and North American congeners

from two globally distributed tree genera, Betula

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(ECM) and Ulmus (AMF), that are sympatric and

allopatric with R. cathartica and F. japonica. If R.

cathartica and F. japonica allelochemicals act only as

direct novel weapons, we predicted the tree seedling

mycorrhizal communities would remain unaffected,

whereas allopatric seedling recruitment and perfor-

mance would be reduced. Alternately, if R. cathartica

and F. japonica allelochemicals degrade fungal

mutualisms, we predicted that R. cathartica (AMF

host) would reduce ECM colonization in Betula,

whereas non-mycorrhizal F. japonica should reduce

fungal colonization on both Betula and Ulmus.

Methods

Study species

Rhamnus cathartica and F. japonica both contain

potent allelochemicals, including emodin, that appear

to strongly deter herbivores and competitors in their

invaded ranges (Hasan 1998; Izhaki 2002; Sera 2012;

Trial and Diamond 1979; Tsahar et al. 2002). The

effects of R. cathartica (Europe) and F. japonica

(Asia) allelochemical exudates were tested using three

Ulmus congeners and three Betula congeners (S1). All

of the selected tree species have similar moisture and

nutrient requirements (Atkinson 1992; Bu et al. 2008;

Coyle et al. 1982). The three AMF tree species were

Ulmus alata (winged elm; eastern NA), U. parvifolia

(Chinese elm, eastern Asia), and U. minor (field elm;

Europe). The three ECM study tree species were

Betula pubescens (European white birch, Europe), B.

nigra (black birch, NA), and B. davurica (Asian black

birch, Asia). No U. alata or B. davurica seeds

germinated, and these species were not considered

for the rest of the study.

Germination/growth experiment

The 14-week germination/growth experiment (July–

October 2015) was carried out at the Dorsheimer

Laboratory/Greenhouse (State University of New

York at Buffalo, Buffalo, NY). We filled 25-cm-deep

tree seedling planters (n = 140, Stuewe and Sons,

Tangent, Oregon USA) with a nutrient poor, coarse-

textured soil pre-inoculated by the manufacturer with

spores of four generalist arbuscular fungi (Glomus

intraradices, G. mosseae, G. aggregatum, G.

etunicatum) and seven generalist ectomycorrhizal

species (Rhizopogon villosulus, R. luteolus, R. amylo-

pogon, R. fulvigleba, Scleroderma cepa, S. citrinum,

Pisolithus tinctorius).

We planted the four tree species (U. parvifolia, U.

minor, B. pubescens, and B. nigra) using the following

5 soil treatments: (1) R. cathartica roots and fungicide

(n = 5); (2) F. japonica roots and fungicide (n = 5);

(3) R. cathartica roots only (n = 10); (4) F. japonica

roots only (n = 10); and (5) control soils with no root

or fungicide addition (n = 5). This unbalanced facto-

rial design gave us a total of 35 pots for each of the 4

tree species (n = 140 pots total).

The soils were treated before adding tree seeds by

mixing in the fungicide and/or macerated R. cathar-

tica and F. japonica roots in random planters. For

fungicide, we added 14 mg of Captan 50 WP per gram

of soil as suggested by the manufacturer (Bonide

products, Oriskany, NY USA). For allelochemicals,

we added 10 g of macerated R. cathartica or F.

japonica roots. The roots were collected from dense,

monospecific stands of R. cathartica and F. japonica

at the Tifft Nature Preserve (Buffalo, New York,

USA). The roots were washed thoroughly with

deionized water, dried at 60 �C for 5 days, and

pulverized using an industrial blender. Warren et al.

(2017) found that similar application of R. cathartica

root macerations reduced germination and growth in

multiple plant species seedlings.

We based tree seed density for each species on the

germination rates provided by the seed distributor

(Sheffield’s Seed Co. Locke, NY). The greenhouse

had an average daytime temperature of 25 �C. Planters

were watered twice a day and checked weekly for seed

germination. Only a single seedling was left to grow in

each container after multiple germinations. After

14 weeks of growth, the tree seedlings were harvested

and 1 g of live root was prepared for an AMF

colonization assays (n = 5 U. parvifolia and 14 U.

minor). Remaining plant root biomass was cleaned of

soil and placed into a labeled paper bag and dried in a

drying oven at 60 �C for 5 days before weighing.

AMF colonization assay

We used a mycorrhizal staining procedure slightly

modified from Phillips and Hayman (1970). We

placed 1 g of fresh, rinsed Ulmus spp. root per test

tube (upright in a test tube rack). We added 10–15 mL

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of 10% KOH solution into each test tube and placed

the tube into a 100 �C water bath for 25 min during

which the KOH caused the root cells to lyse their

contents. We then rinsed the roots with deionized

water and added a 2% HCl solution to ensure the stain

would fix. The stain was prepared by combining water,

glycerin, and lactic acid in 1:1:1 ratio (v/v/v). Acid

fuchsin was then added to the solution at a concen-

tration of 0.05%. Test tubes containing cleared,

acidified roots with the mycorrhizal stain were refrig-

erated for 24 h. The root material was strained, rinsed,

and stored in deionized water for a week to leach

excess stain from the roots and create a stronger visual

contrast between fungal and root cells.

To quantify arbuscular, vesicular, and hyphal

colonization, we used the objective crosshair tech-

nique (McGonigle et al. 1990). Prepared AMF tree

roots were placed on microscope slides, and focused

using a compound microscope (model CX31, Olym-

pus Corporation, Tokyo, Japan). Two intersecting

perpendicular lines (crosshairs) were drawn on the

eyepiece of the compound microscope. Five random

root segments were selected and, at each of the five

segments, ten fields of view were analyzed, tallying a

total of 50 mycorrhizal observations for each root

sample. For each observation, the field of view was

rotated so that one of the two crosshairs dissected the

root widthwise. Arbuscules (S2) were tallied if they

intersected the crosshair. If a crosshair intersected

more than one arbuscule in a single field of view, it

was still only tallied as a single arbuscule. The same

approach was used for vesicles and hyphae (S2). If a

crosshair overlapped both an arbuscule and a vesicle, a

tally was marked for both. However, given that hyphae

frequently co-occur with the other two fungal struc-

tures, they were not counted when they appeared with

either. Fields of view without any fungal formations

were tallied as mycorrhizae absent.

Data analysis

Plant germination and survival were analyzed as

binomial proportions using generalized linear models

(GLM) assuming binomial error distributions. Tree

seedling species (U. parvifolia, U. minor, B. pub-

escens, and B. nigra), allelopathic species (R. cathar-

tica and F. japonica), and fungicide were analyzed as

categorical treatments. Germination was calculated by

counting the number of seedlings emerged by week 6

from the total planted. Survivorship was calculated as

week 14 survivors from those germinated week 6.

Given that the biomass data (g) were highly skewed

and could not include numbers below zero, growth

was analyzed using a GLM with a Poisson error

distribution. The mycorrhizal data (arbuscules, vesi-

cles, hyphae) all were analyzed using GLM models

with a binomial proportion (presence of fungal

structure/50 samples). The coefficients for the fitted

GLM models were estimated using analysis of

deviance (ANODEV) with Chi-square tests. ANO-

DEV is a maximum likelihood approach used with

GLMs fit using an analysis of variance (ANOVA)

model with a Chi-square test. Comparisons between

the reduced model and full model, which includes all

predictors, are made using scaled deviance. The model

output produces a table with rows corresponding to

each of the parameters with an additional top row for

the null model.

Collinearity was tested using the variance inflation

function in the package ‘car’ (Fox and Weisberg

2011). The data also were checked for overdispersion,

(u[ 1.5) and corrected when needed using quasi-

error distributions. All data were analyzed using R

statistical software (R Core Team Version 3.3.2 2016).

Pearson’s correlation coefficient was used to

examine correlation among the three fungal indicators

(arbuscules, hyphae, and vesicles) and plant growth

(biomass). Based on the correlation results, a linear

regression model was used to test the relationship

between mycorrhizal vesicles and plant biomass.

Results

Germination

Overall, tree seedling germination (mean ± SE) was

low (26 ± 4%). Betula nigra and B. pubescens both

had germination rates of 9 ± 5%. Ulmus parvifolia

(31 ± 7%) and U. minor (57 ± 8%) had the highest

germination rates. A root treatment 9 tree species

interaction term indicated a species-specific effect of

root treatments on tree germination (Table 1, Fig. 1).

Both Betula spp. were unaffected by R. cathartica and

F. japonica root macerations (Fig. 1a); however, U.

minor (Europe) germination dropped with allopatric

(F. japonica; Asia) root treatments and was unaffected

by sympatric (R. cathartica; Europe) root treatments

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(Fig. 1b). Similarly, U. parvifolia (Asia) germination

dropped with allopatric (Europe) root treatments and

was unaffected by sympatric (Asia) root treatments.

Tree seedling germination was unaffected by the

fungicide treatment, and there was no fungicide 9 root

treatment interaction effect.

Survivorship and growth

Once a seed germinated, 71% of the seedlings lived to

harvest at 14 weeks. Seedling survival decreased with

the fungicide treatment, but survivorship did not

otherwise differ between tree species, root treatment,

or interactions (Table 2). Seedling biomass was

greater for Ulmus than Betula spp. (Table 3, Fig. 2a),

and declined in all species with the addition of root

macerations (Fig. 2b).

Mycorrhizal colonization

The mycorrhizal parameters (arbuscules, vesicles, and

hyphae) were moderately correlated among one

Fig. 1 Interaction plot for

tree species 9 root treatment

impacts on germination. The

interaction indicated that the

effects of the individual

species root treatments were

species specific on tree seed

germination. Betula spp.

appeared unaffected by

treatments, but these effects

may have been masked by

low germination rates (a).

For Ulmus species,

allopatric root macerations

inhibited germination,

whereas sympatric

allelopathic species showed

little effect (b)

Table 1 Analysis of deviance of tree seed germination as a

function of root treatment, fungicide, tree species, and

interactions

Coefficient df Deviance Res. deviance p value

Tree species 3 32.428 127.19 \ 0.001

Root treatment 2 4.025 123.16 0.133

Fungicide 1 1.900 121.26 0.168

Root 9 tree 6 11.657 109.6 0.070

Root 9 fungicide 1 0.437 109.17 0.508

Table 2 Analysis of deviance of tree seedling survival as a

function of root treatment, fungicide, tree species, and

interactions

Coefficient df Deviance Res. deviance p value

Tree species 3 4.723 39.593 0.193

Root treatment 2 0.467 39.125 0.791

Fungicide 1 3.471 35.654 0.062

Root x tree 4 7.009 28.645 0.135

Root x fungicide 1 2.448 26.196 0.117

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another (r = 0.40–0.50), and of the three, vesicle

presence correlated strongest with plant biomass

(r = 0.64). Plant biomass increased (Esti-

mate = 0.026, SE = 0.174, t-value = 3.584,

p value = 0.002; r2 = 0.29) with increased vesicle

presence (Fig. 3).

Given that the Betula germination rates were so

low, mycorrhizal analysis only was conducted on the

AMF Ulmus species. Arbuscular presence decreased

in the presence of R. cathartica root treatments for

both Ulmus species (Table 4, Fig. 4a), but fungicide

and fungicide 9 root treatment had no effect. Vesicle

presence decreased with both root treatments (Fig. 4b)

and decreased with fungicide (Table 5). Fungal

hyphae decreased with fungicide but were unaffected

by root treatments and root treatment 9 fungicide

(Table 6).

Discussion

Our results suggest contingently effective allelopathy

that directly inhibits plant germination and indirectly

inhibits growth and survival by reducing fungal

mutualists. Moreover, for some species, these effects

depended on whether the allelopathy and tree species

co-occur. Some tree species resisted possibly familiar

allelopathic weapons from sympatric species; the

same weapons inhibited seed germination when

introduced to allopatric tree species. These results

are consistent with the novel weapons hypothesis,

suggesting that species co-evolve compensatory

mechanisms to resist competitive weapons in sym-

patric communities. Once established, however, early

tree seedling survivorship was unaffected by the root

treatments; however, both allopatric and sympatric

root treatments inhibited seedling growth. The impact

of allelopathic species appeared indirect through the

degradation of Ulmus symbiotic fungi. Unexpectedly,

R. cathartica, an AMF host, appeared to have more

impact on Ulmus, an AMF host, than did F. japonica,

which hosts no mycorrhizal fungi. The root treatment

suppression of mycorrhizal colonization was consis-

tent with the degraded mutualism hypothesis as both

allelopathic plant root macerations suppressed AMF

vesicles. Overall, these results support both novel

weapons and degraded mutualisms hypotheses.

Interactions between root treatment and the initial

life stages of Ulmus species showed a direct compet-

itive mechanism from allelopathic species. The ger-

mination of European U. minor was unaffected by

sympatric R. cathartica, but reduced by allopatric F.

Fig. 2 Tree seedling growth (biomass at end of 14-week experiment). Ulmus grew much more than Betula (a), and both allopathic root

macerations (Rhamnus cathartica and Fallopia japonica) inhibited seedling growth in all tree species (b)

Table 3 Analysis of deviance of tree seedling growth as a

function of root treatment, fungicide, tree species, and

interactions

Coefficient df Deviance Res. deviance p value

Tree species 3 1226.840 4481.8 0.078

Root treatment 2 1738.610 2743.2 0.008

Fungicide 1 73.730 2669.4 0.523

Root x tree 3 107.970 2564.5 0.900

Root x fungicide 1 81.840 2482.6 0.500

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japonica root exudates. Similarly, the germination of

Asian U. parvifolia was reduced by allopatric R.

cathartica allelochemicals, but unaffected by the

exudates of a sympatric F. japonica. We did not

examine root maceration effects on sympatric or

allopatric mycorrhizal fungi, but Callaway et al.

(2008) found that A. petiolata had much greater

inhibitory effect on mycorrhizas in invaded NA soils

than in European soils where it is native. We found

that mycorrhizal arbuscule and vesicle abundance on

roots decreased with root treatments, and the

decreased vesicle abundance corresponded with a

Fig. 3 Ulmus dry mass

increase as a function of

vesicle colonization (R2 =

0.43)

Fig. 4 Arbuscule (a) and vesicle (b) colonization on Ulmus roots in soils treated with Rhamnus cathartica or Fallopia japonica root

macerations

Table 5 Analysis of deviance of vesicle colonization on Ul-

mus roots as a function of root treatment, fungicide, and

interactions

Coefficient df Deviance Res. deviance p value

Root treatment 2 5.158 22.684 0.075

Fungicide 1 7.476 15.207 0.006

Root x fungicide 1 0.134 15.072 0.713

Table 4 Analysis of deviance of arbuscular colonization on

Ulmus roots as a function of root treatment, fungicide, and

interactions

Coefficient df Deviance Res. deviance p value

Root treatment 2 48.149 161.48 0.050

Fungicide 1 19.954 141.53 0.116

Root x fungicide 1 21.642 119.88 0.101

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decrease in tree seedling growth. We also found that

fungicide decreased vesicle and hyphae abundance in

roots, and fungicide also corresponded with a decrease

in seedling survival.

Both R. cathartica and F. japonica contain the

secondary compound emodin, which is a potent

allelochemical in 17 Eurasian plant families (Izhaki

2002) that limits the germination, growth, and survival

of both native and non-native species (Hasan 1998;

Inoue et al. 1992; Klionsky et al. 2011; Sera 2012;

Serniak 2016). We found that arbuscular and vesicular

colonization were reduced in Ulmus sp. with the

addition of R. cathartica root exudates, but only

vesicular formation was reduced when Ulmus sp.

interacted with F. japonica exudates. These results

contradicted our hypothesis that the exudates of an

allopatric AMF host (R. cathartica) would not target

AMF mycorrhizae (Ulmus also is an AMF host),

suggesting that degraded mutualism attack may not

depend on fungal mutualist type. Indeed, in a meta-

analysis of native/non-native plant interactions with

mycorrhizal fungi, Bunn et al. (2015) found that

association with non-native plants reduced mycor-

rhizal fungi in native plants. Given that native and

non-native species, and different plant functional

types, host very different AMF species, the negative

allelopathic effect may be because each group hosts

different arbuscular fungi species.

Our data suggest that the invasion success of two

plants, F. japonica and R. cathartica, may depend on

how allopatric flora and associated fungal mutualists

respond to their phytotoxins. Our results are consistent

with the novel weapons and associated degraded

weapons hypotheses, though the effects were not

consistent across all tree seedling species, suggesting

that, rather than a single magic bullet, invading plants

may employ a multi-prong allelopathic attack on

native plants.

Acknowledgements We thank Dr. James Berry at

the University of Buffalo for use of the Dorsheimer

Laboratory/Greenhouse. We also thank two anonymous

reviewers for helpful comments that improved the manuscript.

Data accessibility The data generated and analyzed for the

current study are available in the SUNY Buffalo State Digital

Commons [http://digitalcommons.buffalostate.edu].

Author contributions PP conceived the ideas and designed

methodology; PP collected the data; PP and RW analyzed the

data; PP and RW led the writing of the manuscript. All authors

contributed critically to the drafts and gave final approval for

publication.

References

Aguilar-Chama A, Guevara R (2012) Mycorrhizal colonization

does not affect tolerance to defoliation of an annual herb in

different light availability and soil fertility treatments but

increases flower size in light-rich environments. Oecologia

168:131–139

Atkinson MD (1992) Betula Pendula Roth (B. Verrucosa Ehrh.)

and B. Pubescens Ehrh. J Ecol 80:837–870

Bais HP, Vepachedu R, Gilroy S, Callaway RM, Vivanco JM

(2003) Allelopathy and exotic plant invasion: from mole-

cules and genes to species interactions. Science

301:1377–1380

Blossey B, Notzold R (1995) Evolution of increased competitive

ability in invasive nonindigenous plants—a hypothesis.

J Ecol 83:887–889

Brundrett MC (2002) Coevolution of roots and mycorrhizas of

land plants. New Phytol 154:275–304

Bu R, He HS, Hu Y, Chang Y, Larsen DR (2008) Using the

LANDIS model to evaluate forest harvesting and planting

strategies under possible warming climates in Northeastern

China. For Ecol Manag 254:407–419

Bunn RA, Ramsey PW, Lekberg Y (2015) Do native and

invasive plants differ in their interactions with arbuscular

mycorrhizal fungi? A meta-analysis. J Ecol

103:1547–1556

Callaway RM, Ridenour WM (2004) Novel weapons: invasive

success and the evolution of increased competitive ability.

Front Ecol Environ 2:436–443

Callaway RM, Cipollini D, Barto K, Thelen GC, Hallett SG,

Prati D, Stinson K, Klironomos J (2008) Novel weapons:

invasive plant suppresses fungal mutualists in America but

not in its native Europe. Ecology 89:1043–1055

Cantor A, Hale A, Aaron J, Traw MB, Kalisz S (2011) Low

allelochemical concentrations detected in garlic mustard-

invaded forest soils inhibit fungal growth and AMF spore

germination. Biol Invasions 13:3015–3025

Cipollini K, Titus K, Wagner C (2012) Allelopathic effects of

invasive species (Alliaria petiolata, Lonicera maackii,

Ranunculus ficaria) in the Midwestern United States.

Allelopath J 29:63–75

Table 6 Analysis of deviance of hyphae colonization on Ul-

mus roots as a function of root treatment, fungicide, and

interactions

Coefficient df Deviance Res. deviance p value

Root treatment 2 2.987 128.68 0.224

Fungicide 1 51.533 77.15 \ 0.001

Root x fungicide 1 1.079 76.07 0.298

546 Plant Ecol (2018) 219:539–548

123

Author's personal copy

Coyle BF, Sharik TL, Feret PP (1982) Variation in leaf mor-

phology among disjunct and continuous populations of

river birch (Betula nigra). Silvae Genet 31:122–125

Dallali S, Lahmayer I, Mokni R, Marichali A, Ouerghemmi S

(2014) Phytotoxic effects of volatile oil from Verbena spp.

on the germination and radicle growth of wheat, maize,

linseed and canary grass and phenolic content of aerial

parts. Allelopath J 34:95–105

Duke SO, Dayan FE (2006) Modes of action of phytotoxins

from plants. In: Reigosa MJ, Pedrol N, Gonzalez L (eds)

Allelopathy: a physiological process with ecological

implications. Springer, Dordrecht

Fox J, Weisberg S (2011) An R companion to applied regres-

sion. Sage, Thousand Oaks

Gange AC, Smith AK (2005) Arbuscular mycorrhizal fungi

influence visitation rates of pollinating insects. Ecol

Entomol 30:600–606

Godwin H (1943) Rhamnaceae. J Ecol 31:66–92

Hale AN, Kalisz S (2012) Perspectives on allelopathic disrup-

tion of plant mutualisms: a framework for individual- and

population-level fitness consequences. Plant Ecol

213:1991–2006

Hasan HAH (1998) Studies on toxigenic fungi in roasted

foodstuff (salted seed) and halotolerant activity of emodin-

producing Aspergillus wentii. Folia Microbiol 43:383–391

Herrera MA, Salamanca CP, Barea JM (1993) Inoculation of

woody legumes with selected arbuscular mycorrhizal fungi

and rhizobia to recover desertified mediterranean ecosys-

tems. Appl Environ Microbiol 59:129–133

Inderjit Seastedt TR, Callaway RM, Kaur J (2008) Allelopathy

and plant invasions: traditional, congeneric, and bio-geo-

graphical approaches. Biol Invasions 10:875–890

Inoue M, Nishimura H, Li HH, Mizutani J (1992) Allelochem-

icals from Polygonum sachalinense Fr. Schm. (Polygo-

naceae). J Chem Ecol 10:1833–1840

Izhaki I (2002) Emodin—a secondary metabolite with multiple

ecological functions in higher plants. New Phytol

155:205–217

Janos DP (1980) Mycorrhizae influence tropical succession.

Biotropica 12:56–64

Jessing KK, Duke O, Cedergreen N (2014) Potential ecological

roles of artemisinin produced by Artemisia annua L.

J Chem Ecol 40:100–117

Keane RM, Crawley MJ (2002) Exotic plant invasions and the

enemy release hypothesis. Trends Ecol Evol 17:164–170

Klionsky SM, Amatangelo KL, Waller DM (2011) Above- and

belowground impacts of European buckthorn (Rhamnus

cathartica) on four native forbs. Restor Ecol 19:728–737

Klironomos J (2003) Variation in plant response to native and

exotic arbuscular mycorrhizal fungi. Ecology

84:2292–2301

Koide RT, Dickie IA (2002) Effects of mycorrhizal fungi on

plant populations. Plant Soil 244:307–317

Lambers H, Raven JA, Shaver GR, Smith SE (2008) Plant

nutrient-acquisition strategies change with soil age. Trends

Ecol Evol 23:95–103

Levine JM, Vila M, D’Antonio CM, Dukes JS, Grigulis K,

Lavorel S (2003) Mechanisms underlying the impacts of

exotic plant invasions. R Soc 270:775–781

Mack RN, Simberloff D, Lonsdale WM, Evans H, Clout M,

Bazzaz FA (2000) Biotic invasions: causes, epidemiology,

global consequences, and control. Ecol Appl 10:689–710

Mallik AU, Pellissier F (2000) Effects of Vacciniummyrtillus on

spruce regeneration: testing the notion of coevolutionary

significance of allelopathy. J Chem Ecol 26:2197–2209

Malloch DW, Pirozynski KA, Raven PH (1980) Ecological and

evolutionary significance of mycorrhizal symbioses in

vascular plants (a review). Proc Natl Acad Sci

77:2113–2118

Maron JL, Vila M (2001) When do herbivores affect plant

invasion? Evidence for the natural enemies and biotic

resistance hypotheses. Oikos 95:361–373

McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA

(1990) A new method which gives an objective measure of

colonization of roots by vesicular-arbuscular mycorrhizal

fungi. New Phytol 115:495–501

Mitchell CE, Power AG (2006) Disease dynamics in plant

communities. In: Collinge S, Ray C (eds) Disease ecology:

community structure and pathogen dynamics. Oxford

University Press, New York

Muller-Scharer H, Schaffner U, Steinger T (2004) Evolution in

invasive plants: implications for biological control. Trends

Ecol Evol 19:417–422

Nantel P, Neumann P (1992) Ecology of ectomycorrhizal-ba-

sidiomycete communities on a local vegetation gradient.

Ecology 73:99–117

Phillips JM, Hayman DS (1970) Improved procedures for

clearing roots and staining parasitic and vesicular-arbus-

cular mycorrhizal fungi for rapid assessment of infection.

Trans Br Mycol Soc 55:158–161

R Core Team Version 3.3.2 (2016) R: A language and envi-

ronment for statistical computing. R Foundation for Sta-

tistical Computing, Vienna, Austria

Rabotnov T (1982) Importance of the evolutionary approach to

the study of allelopathy. Sov J Ecol 12:127–130

Raguso RA (2008) Wake up and smell the roses: the ecology and

evolution of floral scent. Annu Rev Ecol Evol Syst

39:549–569

Requena N, Jeffries P, Barea JM (1996) Assessment of natural

mycorrhizal potential in a desertified semiarid ecosystem.

Appl Environ Microbiol 62:842–847

Schnitzer A, Muller S (1998) Ecology and biogeography of

highly invasive plants in Europe: giant knotweeds from

Japan (Fallopia japonica and F-sachalinensis). Revue

d’ecologie 53:3–38

Schreiner RP, Koide RT (1993) Antifungal compounds from the

roots of mycotrophic and non-mycotrophic plant-species.

New Phytol 123:99–105

Sera B (2012) Effects of soil substrate contaminated by knot-

weed leaves on seed development. Pol J Environ Stud

21:713–717

Serniak LT (2016) Comparison of the allelopathic effects and

uptake of Fallopia japonica phytochemicals by Raphanus

sativus. Weed Res 56:97–101

Siqueira J, Saggin-Junior OJ (2001) Dependency on arbuscular

mycorrhizal fungi and responsiveness of some Brazilian

native woody species. Mycorrhiza 11:245–255

Smith SE, Read D (2008) Mycorrhizal symbiosis, 3rd edn.

Academic Press, New York

Plant Ecol (2018) 219:539–548 547

123

Author's personal copy

Smith SE, Smith FA (2011) Roles of arbuscular mycorrhizas in

plant nutrition and growth: new paradigms from cellular to

ecosystem scales. Annu Rev Plant Biol 62:227–250

Smith SE, Smith FA (2012) Fresh perspectives on the roles of

arbuscular mycorrhizal fungi in plant nutrition and growth.

Mycologia 104:1–13

Spector T, Putz FE (2006) Biomechanical plasticity facilitates

invasion of maritime forests in the southern USA by

Brazilian pepper (Schinus terebinthifolius). Biol Invasions

8:255–260

Stinson KA, Campbell SA, Powell JR, Wolfe BE, Callaway RM

(2006) Invasive plant suppresses the growth of native tree

seedlings by disrupting belowground mutualisms. PLoS

Biol 4:727–731

Stinson K, Kaufman S, Durbin L, Lowenstein F (2007) Impacts

of garlic mustard invasion on a forest understory commu-

nity. Northeast Nat 14:73–88

Trial H Jr, Diamond JB (1979) Emodin in buckthorn: a feeding

deterrent to phytophagous insects. Can Entomol

111:207–212

Tsahar E, Friedman J, Izhaki I (2002) Impact on fruit removal

and seed predation of a secondary metabolite, emodin, in

Rhamnus alaternus fruit pulp. Oikos 99:290–299

Varga S, Kytoviita M-M (2010) Gender dimorphism and myc-

orrhizal symbiosis affect floral visitors and reproductive

output in Geranium sylvaticum. Funct Ecol 24:750–758

Vierheilig H, Bennett R, Kiddle G, Kaldorf M, Ludwig-Muller J

(2000) Differences in glucosinolate patterns and arbuscular

mycorrhizal status of glucosinolate-containing plant spe-

cies. New Phytol 146:343–352

Vogelsang KM, Bever JD (2009) Mycorrhizal densities decline

in association with nonnative plants and contribute to plant

invasion. Ecology 90:399–407

Warren RJ II, Labatore AC, Candeias M (2017) Allelopathic

invasive tree (Rhamnus cathartica) alters native plant

communities. Plant Ecol 218:1233–1241

548 Plant Ecol (2018) 219:539–548

123

Author's personal copy