RESEARCH ARTICLE
Relationships between Methylobacteria
and Glyphosate with Native and Invasive Plant
Species: Implications for Restoration
Irina C. Irvine,1,2,3 Marti S. Witter,1 Christy A. Brigham,1 and Jennifer B. H. Martiny2
Abstract
After removing invasive plants, whether by herbicides or
other means, typical restoration design focuses on rebuilding native plant communities while disregarding soil microbial communities. However, microbial–plant interactions
are known to influence the relative success of native versus
invasive plants. Therefore, the abundance and composition
of soil microorganisms may affect restoration efforts. We
assessed the effect of herbicide treatment on phytosymbiotic pink-pigmented facultative methylotrophic (PPFM)
bacteria and the potential consequences of native and invasive species establishment post-herbicide treatment in the
lab and in a coastal sage scrub (CSS)/grassland restoration
site. Lab tests showed that 4% glyphosate reduced PPFM
abundance. PPFM addition to seeds increased seedling
length of a native plant (Artemisia californica) but not
an invasive plant (Hirschfeldia incana). At the restoration site, methanol addition (a PPFM substrate) improved
native bunchgrass (Nassella pulchra) germination and size
by 35% over controls. In a separate multispecies field
experiment, PPFM addition stimulated the germination of
N. pulchra, but not that of three invasive species. Neither
PPFM nor methanol addition strongly affected the growth
of any plant species. Overall, these results are consistent
with the hypothesis that PPFMs have a greater benefit to
native than invasive species. Together, these experiments
suggest that methanol or PPFM addition could be useful in improving CSS/grassland restorations. Future work
should test PPFM effects on additional species and determine how these results vary under different environmental
conditions.
Key words: herbicide, invasive species, methanol, Methylobacterium, methylotrophic bacteria, PPFM.
Published 2011. This article is a U.S. Government work and is in the public domain
in the USA.
doi: 10.1111/j.1526-100X.2011.00850.x
rebuilding functioning plant communities. Soil microbial communities are rarely considered, but may potentially play an
important role in plant recovery and ecosystem restoration
(Harris 2009). For example, native whole-soil inoculum added
during restoration of a cheatgrass-dominated montane site
increased native perennial cover and reduced cheatgrass cover
(Rowe et al. 2009). Similarly, Requena et al. (1997) found
particular combinations of arbuscular mycorrhizal fungi, Rhizobium, and plant-growth-promoting bacteria that were best
for success of a woody legume commonly used in restoration.
Despite the potential role of microbes in restoration, the
effects of glyphosate applications on microbial communities
remain poorly understood and current evidence is equivocal
(Weidenhamer & Callaway 2010). If herbicides do alter the
long-term abundance or composition of soil microorganisms,
these changes may affect the relative success of native and
invasive plant species during restoration via differential effects
on plant fitness and competition (Callaway et al. 2008; Bains
et al. 2009; Bever et al. 2010). For instance, native seeding
and the addition of activated charcoal can alter plant-soil
feedbacks, the soil microbial community, and reduce invasive
species success (Kulmatiski 2011). Similarly, changes in
the density and composition of mycorrhizal fungi can affect
Restoration Ecology Vol. 21, No. 1, pp. 105–113
105
Introduction
Land managers commit considerable energy and funds to prevent, detect, and control invasive plants each year. When
invasive plants dominate large areas, herbicide use is often
the best option for removal to begin native plant community restoration. One of the most widely used herbicides is
glyphosate [N -(phosphonomethyl)glycine], the active ingredient in RoundUp (Monsanto, St. Louis, MO, U.S.A.), which is
primarily used to control crop weeds. It is also commonly used
in habitat restoration as it is generally rapidly inactivated in
soil and has a low toxicity to mammals (McComb et al. 2008),
although concerns about effects on nontarget organisms persist
(Relyea 2005; Eker et al. 2006; Neumann et al. 2006).
Once non-native species have been removed by glyphosate
or other means, typical restoration design is focused on
1 Santa Monica Mountains National Recreation Area (U.S. National Park Service),
401 West Hillcrest Drive, Thousand Oaks, CA 91360, U.S.A.
2 Department of Ecology & Evolutionary Biology, University of California, Irvine,
CA 92697, U.S.A.
3 Address correspondence to I. C. Irvine, email Irina_Irvine@NPS.gov
JANUARY 2013
PPFMs and Glyphosate in Restoration
the competitive success of invasive species over natives
(Stinson et al. 2006).
Much of the work on microbes and restoration has focused
on a subset of the soil microbial community: mycorrhizal
fungi, nitrogen-fixers and pathogens (Vitousek & Walker 1989;
Morris et al. 2007; Mangla et al. 2008). Another group that
may be useful for restoration is the pink-pigmented facultative methylotrophic bacteria (PPFMs) within the genus Methylobacterium. PPFMs are widespread symbionts associated with
the roots, leaves, and seeds of most terrestrial plants, but
also free-living in air, water, and soil. PPFMs can utilize
toxic C1 compounds generated by growing plants during cell
division, such as methanol (Trotsenko et al. 2001). In agricultural systems, PPFMs can affect seed germination, crop yield,
pathogen resistance (Kalyaeva et al. 2001; Madhaiyan et al.
2004, 2006), and drought stress tolerance (Pospisilova et al.
2005). The mechanisms by which PPFMs affect plants include
excretion of growth hormones (Doronina et al. 2002; Madhaiyan et al. 2005), ureases (Holland & Polacco 1992), and
osmoprotectants (sugars and alcohols) (Trotsenko et al. 2001).
We do not know how PPFMs might differentially affect native
and invasive plant species.
Given their interactions with plants, we suggest that PPFMs
may be useful for restoration, depending on their response to
glyphosate application and their relative effects on native versus invasive species. We focused on California coastal sage
scrub (CSS) dominated by drought-deciduous species adapted
to a Mediterranean-type climate. Specifically, we asked:
(1) Does glyphosate reduce PPFM abundance or growth?
(2) In the presence of glyphosate (or after glyphosate treatments in the field), do PPFM or methanol (a PPFM substrate)
additions improve plant germination and growth? (3) Do the
results from (2) differ for native and invasive species in monocultures and mixtures?
Methods
Restoration Site
Cheeseboro Canyon is an 872-ha park located in the Santa
Monica Mountains National Recreation Area (SMMNRA, U.S.
National Park Service) in Los Angeles County, California
(34◦ 09 51 N, 118◦ 43 21 W). Multiple wildfires, grazing, and
open-field agriculture have resulted in landscape-level changes
and type conversions in much of the park. Invasive annual
grasses (Bromus and Avena spp.), mustards (Brassica and
Hirschfeldia spp.), and thistles (Centaurea, Salsola, and Silybum spp.) dominate the area. The site has a history of variable
restoration success with most efforts failing to meet management objectives. Beginning in January 2008, a 10-ha site was
mowed and treated with 2% glyphosate removing annual vegetation, though woody perennial natives remained (Fig. 1a).
In vitro Tests of PPFM Sensitivity to Glyphosate
PPFMs from CSS soil near the restoration site were enriched
using nitrate mineral salts (NMS) media with methanol
106
(a)
(b)
Figure 1. Cheeseboro Canyon restoration site and experiment block
design: (a) restoration site with fenced block exclosures, October 2009,
(b) diagram of one block of the field experiment. Treatments were
randomized in each block and species were randomized within the
monoculture plots.
(1.0% vol/vol) as the sole carbon source and cycloheximide
(100 mg/L) to prevent fungal growth. In prior work, the most
common PPFM isolated from this habitat was Methylobacterium extorquens (I. Irvine, unpublished data). The sensitivity
of PPFMs to five concentrations of glyphosate was tested (1, 2,
4, 10, and 41%). One, two, and four percent solutions are commonly used in foliar spray applications, whereas 10 and 41%
are used in cut-stump methods (likely only occurring in soils as
a result of accidental spills). A log growth phase PPFM culture
was spread on NMS media. Sterile filter paper disks (0.5 cm
diameter), dipped in glyphosate or a sterile water control, were
placed in the center of the inoculated agar plates and incubated
at 30◦ C. On day 14, glyphosate sensitivity was measured as
the diameter of the zone of clearing. A one-way analysis of
variance (ANOVA) was used to test whether PPFM sensitivity
differed among glyphosate levels. All statistical analyses were
performed in JMP (8.01) except where noted.
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PPFMs and Glyphosate in Restoration
The effect of glyphosate on PPFM growth in soil was
also tested because glyphosate can be rapidly inactivated by
an ion exchange with soil particles (Hensley et al. 1978).
Surface soil from an intact CSS stand in the SMMNRA was
homogenized and sieved. For each glyphosate concentration,
0.7 mL was sprayed onto 2 g soil (N = 5 per treatment).
Soils were incubated for 24 hours at room temperature. PPFM
abundance was estimated with the most probable number
(MPN) technique; a dilution series from each sample was
plated in sterile microtiter plates and incubated at 30◦ C. Wells
with pink growth after 14 days were scored as positive for
PPFMs. A one-way ANOVA was used to test for differences
in abundance estimates among glyphosate concentrations.
In vitro Seed Germination Experiment
In the lab, a 2 × 2 factorial design was used to test the effects
of 2% glyphosate and PPFMs (an enriched culture of the
PPFM community of CSS soil) on seed germination of a
native perennial CSS shrub, Artemisia californica, and a common invasive annual mustard, Hirschfeldia incana. Twentyfive seeds were placed in a sterile petri dish lined with filter
paper and the seeds were evenly sprayed with 0.5 mL of
the different treatments or a sterile water control (N = 10
plates/species/treatment). The seeds were covered, incubated
in the dark at 20◦ C, and kept moist by adding sterile water
throughout the experiment. The number of germinated seeds
(i.e., radical emergence) was counted daily. On the final day
of the experiment, the seedling length and number of seeds or
seedlings visibly infected with fungi were also recorded. The
effects of glyphosate and/or PPFMs on seed germination rates
were tested with repeated-measures two-way ANOVAs separately for each species. Treatment effects on seedling length
and fungal infection were tested with two-way ANOVAs and
Tukey’s HSD post hoc tests.
PPFM Abundance at the Restoration Site
Soil samples were collected on 7 January 2009, from the
top 2 cm in the Cheeseboro Canyon restoration area and
an adjacent control area (30 m away) that had never been
treated with glyphosate. The control area was covered by
dead invasive annual grasses (Bromus diandrus and Avena
spp.), and its soil type, aspect, and slope were similar to
the restoration site. The MPNs of PPFMs were estimated as
described above for three subsamples (2 g) of each of five
samples (approximately 100 g). The samples were dry at the
time of collection and stored at 4◦ C for 14 days prior to
processing.
Nassella pulchra Field Experiment
The entire restoration site was drill-seeded with locally collected seeds from the native perennial bunchgrass, N. pulchra
(460 seeds/m2 ), on 12 January 2009. To test whether the germination and growth of N. pulchra could be improved by
adding methanol, five randomly distributed paired plots (1 m2 )
JANUARY 2013
Restoration Ecology
were established within the site 3 days after seeding. A 20%
methanol solution or a water control was sprayed onto the
soil (approximately 780 mL/plot) immediately and again on
28 March.
On 6th May, the number and size (widest basal diameter
of 10 randomly selected individuals) of N. pulchra in the
plots were recorded. On 30th July, the surviving plants were
counted and measured. A mixed-model ANOVA (fixed factors:
methanol treatment; random factor: block) was used to test for
the effects of methanol addition.
Multispecies Field Experiment
Using a second field experiment at the same site, we tested
whether the addition of methanol and/or PPFMs in glyphosatetreated soil would differentially affect the growth and competitive ability of multiple native and invasive plant species. We
implemented a randomized block design with four treatments:
PPFM addition, methanol addition, PPFM plus methanol addition, and control. Five blocks (4.9 × 2.3 m) were fenced and
bird-netted to exclude herbivores and granivores. To eliminate some of the existing seed bank, each block was watered
beginning 60 days before the experiment started and emergent seedlings were killed with 2% glyphosate 21 days after
watering. The final herbicide treatment was 30 days before the
start of the experiment on 22 October 2009. Any additional
seedlings were hand-pulled on 15 October 2009.
Within each treatment, 25 × 25 cm subplots were seeded
with one of three native perennial or three invasive annual
species (randomly distributed). A larger (71 × 71 cm) mixedspecies subplot was also seeded within each treatment
(Fig. 1b). The total number of subplots in the experiment
was 140 (4 treatments × [6 monocultures + 1 mixed] ×
5 blocks). The native perennial species were: A. californica
(CSS shrub), N. pulchra (CSS/grassland bunchgrass), and
Salvia leucophylla (CSS shrub). The invasive annual species
were: B. diandrus (grass), Centaurea melitensis (forb), and
H. incana (forb). The species were selected because they are
abundant in Cheeseboro Canyon and are often the focus of
restoration or removal efforts. The invasive and native species
are confounded by life history, because native annual CSS
species have a patchy distribution in the SMMNRA and perennial invasive species are rare in Cheeseboro Canyon (although
they occur elsewhere in the SMMNRA, it was inadvisable
to introduce them to this area). Seeds were collected near the
restoration site in late 2008 and 2009. Germination rates of the
seed stock were determined in the SMMNRA nursery prior to
the start of the experiment and appropriate amounts of seed
were added to each plot to yield approximately 25 plants/plot.
One day before planting, the PPFM treatment seeds were
inoculated with a PPFM-enriched culture isolated from soil
of the control area of the restoration site. This culture was
washed twice, resuspended in sterile water, and sprayed on the
seeds. To estimate pre-experiment PPFM abundance, soil was
collected in each of the mixed species plots prior to seeding
as described above. A 20% methanol solution (approximately
780 mL/plot) was applied to soil of the appropriate plots after
107
Results
Sensitivity of PPFMs to Glyphosate
All glyphosate concentrations reduced PPFM abundance and
higher concentrations produced the largest zones of clearing
(p < 0.0001, Fig. 2a). Glyphosate also significantly reduced
PPFM abundance in soils at concentrations above 4% (p =
0.040, Fig. 2b). At low concentrations in soil, glyphosate
appears to inhibit growth rather than induce mortality. No
PPFMs grew in the initial dilution (0.35%) of the 10%
glyphosate treatment, but did grow in subsequent dilutions,
suggesting that the higher concentration of glyphosate inhibited PPFM growth, while a further 10-fold dilution was low
enough that viable cells grew. No PPFMs grew in the 41%
glyphosate treatment at any dilution, consistent with cell
mortality.
Effects of PPFMs and Glyphosate on in vitro Germination
In the laboratory, seed germination of the native, Artemisia
californica, gradually increased over time, whereas most
non-native Hirschfeldia incana seeds germinated by day 5
(Fig. 3a). Germination was unaffected by the presence of
PPFMs or glyphosate for either species (p > 0.05). Seedling
length of A. californica increased with PPFM addition
108
4
P<0.0001
3
2
1
co
250
41
10
4
Percent glyphosate
P=0.040
225
200
175
150
125
100
75
50
25
41
10
4
2
ro
co
nt
1
0
l
(b)
2
1
0
nt
ro
l
(a)
Estimated PPFM abundance
(mean #cells/g soil + 1SE)
seeding and again to soil and seedlings 21 days later. The
entire experiment was watered regularly to maintain moist
soils.
Germination and the presence/absence of each species were
recorded daily for the first 2 weeks of the experiment until
most of the seedlings emerged and every few days thereafter
until late November. At the end of the experiment (103 days),
the percent cover of each species was recorded and the
whole plants including root systems were harvested, dried,
and weighed. Soil samples to estimate PPFM abundance were
collected; however, because many were accidentally destroyed,
PPFM abundance is not reported.
Mixed-model three-way ANOVAs were used to test for
treatment effects on percent cover, number of individuals,
total dry weight, shoot weight, root weight, and shoot-toroot ratio for each plant species, with methanol addition,
PPFM addition, and plant competition (mixture vs. monoculture) as fixed factors and block as a random factor. All
variables were ln-transformed to improve normality with the
exception of the number of individuals (untransformed) and
percent cover (fourth root-transformed excluding zero values). Treatment effects on first day to germination were
tested with a time-to-event analysis using a survival analysis procedure (PROC LIFETEST) and proportional hazards
model (PROC PHREG) with SAS statistical software (version 8e). Block 5 was eliminated from this analysis because
of extremely low germination. In the remaining four blocks,
for two plots where no seedlings had emerged by day 26, a
value of 26 was assigned and they were treated as censored
observations.
Zone of clearing with glyphosate
(mean cm diam. + 1SE)
PPFMs and Glyphosate in Restoration
Percent glyphosate
Figure 2. In vitro glyphosate-sensitivity assays: (a) zones of PPFM
clearing in glyphosate assays in non-soil media, F5,24 = 45.73,
p < 0.0001, (b) PPFM abundance estimates (MPN) in soil samples,
F5,24 = 2.791, p = 0.040 (one-way ANOVA).
(p < 0.0001); the length of H. incana seedlings was unaffected (p = 0.305, Fig. 3b).
Fungal infections on H. incana and A. californica seeds and
seedlings were affected by glyphosate and PPFM addition in
different ways (Fig. 3c). Both glyphosate and PPFMs reduced
the number of A. californica seeds and seedlings infected
by fungus (p = 0.0005). In contrast, glyphosate increased
H. incana fungal infection, whereas PPFMs reduced infection
(p < 0.0001).
PPFM Abundance at the Restoration Site
At the Cheeseboro Canyon site, PPFMs were on average
eight times less abundant in 2% glyphosate-treated soil (mean
3,690 cells/g soil ± 5,420 SE) compared to nearby control
soil (30,184 cells/g soil ± 5,610, F1,27 = 11.53, p = 0.0021).
Within the glyphosate-treated area where the multispecies
experiment was located, there were no differences in PPFM
abundance among blocks (9,351 cells/g soil ± 2,695).
Effect of Methanol on Nassella pulchra in the Field
Methanol application (20%) generally improved N. pulchra
seed germination and size at the restoration site. Three-anda-half months after the first treatment was applied (May), the
Restoration Ecology
JANUARY 2013
PPFMs and Glyphosate in Restoration
Seeds Germinated
(mean cumulative per day)
25
20
Control
Glyphosate
PPFM
Gly+PPFM
H. incana
P=0.484
15
10
5
4
A. californica
P=0.854
0
4
5
6
7
8
9 10 11 12 13 14 15
Day of Germination
Mean # Individuals
(N. pulchra seedlings/m2 + 1SE)
(a)
(a)
100
P=0.046
80
60
40
20
0
control
30
a a
a
a
H. incana
A. californica
a
20
b
10
c
d
15
10
ab
H. incana
A. californica
bc
a
c
5
b
c
d
Gl Con
yp tr
ho ol
sa
t
Gl PP e
y+ FM
PP
FM
0
Figure 3. In vitro germination experiment: (a) average cumulative seeds
germinated by day, A. californica: F3,36 = 0.259, p = 0.854; H. incana:
F3,36 = 0.835, p = 0.484 (repeated-measures ANOVA), (b) average
seedling length measured from root tip to cotyledon tip on the last day of
the experiment, two-way ANOVA by species: H. incana: F3,36 = 1.254,
p = 0.305; A. californica: F3,35 = 111.645, p < 0.0001, with significant
interaction of glyphosate × PPFM addition, (c) average number of
fungal infections of seeds and seedlings counted on the last day of the
experiment, two-way ANOVA by species: H. incana: F3,36 = 10.79,
p < 0.0001; A. californica: F3,35 = 7.660, p = 0.0005 (N = 250
seeds/species). Letters represent results from Tukey’s post hoc tests.
mean number of seedlings increased by 35% (p = 0.046) and
seedling size increased by 32% (p < 0.001) with methanol
addition compared to control plots (Fig. 4a & 4b). Three
months later (July), the number of individuals was not significantly different between treatments, apparently due to
JANUARY 2013
40
control
20% MeOH
Restoration Ecology
P=0.025
30
20
P<0.0001
10
0
May 2009
a
Gl Con
yp tr
ho ol
sa
t
Gl PP e
y+ FM
PP
FM
Fungal infections
(mean # individuals + 1SE)
(c)
Gl Con
yp tr
ho ol
sa
t
Gl PP e
y+ FM
PP
FM
Gl Con
yp tr
ho ol
sa
t
Gl PP e
y+ FM
PP
FM
0
20% MeOH
(b)
Mean Seedling Size
(N. pulchra basal width mm + 1SE)
Seedling Length
(mean mm + 1SE)
(b)
July 2009
Figure 4. Methanol effects on Nassella pulchra (field experiment):
(a) average number of N. pulchra individuals/m2 , 111 days after
drill-seeding, F1,8 = 5.519, p = 0.046; (b) average plant size (basal
width in millimeter) in May 2009: F3,96 = 13.91, p < 0.0001; and July
2009: F1,63 = 5.649, p = 0.025 (one-way ANOVA; N = 10 plots).
heavy herbivory at the entire restoration site; in total, 88%
of the seedlings were lost from the plots from May to July.
However, those plants that did survive were 28% larger on
average in the methanol treatment compared to the controls
(p = 0.0205).
Effects of PPFMs and Methanol on Multiple Species in the Field
The responses to methanol and PPFM addition were speciesspecific. Similar to the previous experiment, PPFM addition
stimulated earlier germination of native N. pulchra in monoculture plots by 5 days compared to control plots (χ 2 =
4.8371, p = 0.0279; Fig. 5). In contrast, day of first germination of the three invasive species was unaffected by PPFMs
(p = 0.2245–0.6178). The native shrubs, Salvia leucophylla
and A. californica, did not germinate in most of the plots
(N < 10 individuals total), so could not be considered further. Methanol addition did not significantly affect day of first
germination for any species.
There were few main effects of methanol and PPFM addition on the number of individuals or biomass of any species
(Table 1), except for the invasives, Centaurea melitensis,
109
PPFMs and Glyphosate in Restoration
First Day to Germination
(mean # days + 1SE)
20
15
Control
MeOH
PPFM
MeOH+PPFM
**
10
5
0
B. diandrus
C. melitensis
H. incana
N. pulchra
Figure 5. Day of first germination in the multispecies field experiment.
Average first day to germination by combining mixed and monoculture
plots in all treatments, χ 2 = 4.4447, p = 0.035 (time to event analysis
with proportional hazards model and survival analysis). *Indicates
significant treatment effect.
where methanol increased shoot biomass per individual by
13% (p = 0.0498), and H. incana, where PPFMs reduced total
biomass by 36% (p = 0.0439). Neither methanol nor PPFM
addition affected the percent cover of any species (results not
shown).
Germination and growth of the three invasive species were
greater in mixtures compared to monoculture (Table 1, Fig. 5).
Bromus diandrus germinated 2 days sooner (χ 2 = 5.14, p =
0.0234) and had twice as many individuals (p = 0.0020) that
grew nearly two times larger in mixture versus monoculture
(p < 0.0001). Similarly, H. incana shoot weight per individual
increased by 30% (p = 0.0092) and C. melitensis individuals
by 40% (p = 0.0378) in mixtures relative to monocultures.
Biomass of N. pulchra did not change when grown in mixtures
(p = 0.1332).
In four cases, the effect of methanol or PPFM addition
depended on the competitive environment (Table 1). Methanol
increased H. incana biomass (p = 0.0168) and shoot weight
Table 1. Summary of the mixed-model results.
Total Dry Weight (g)
Species and Source of Variation
B. diandrus
Methanol (M)
PPFMs (P)
Competition (C)
M×P
M×C
P×C
M×P×C
H. incana
M
P
C
M×P
M×C
P×C
M×P×C
C. melitensis
M
P
C
M×P
M×C
P×C
M×P×C
N. pulchra
M
P
C
M×P
M×C
P×C
M×P×C
No. of Individuals
Shoot Weight/Individual (g)
Shoot:Root Weight
df
F -ratio
df
F -ratio
df
F -ratio
df
F -ratio
1,30
1,30
1,30
1,30
1,30
1,30
1,30
0.00
0.15
26.91∗∗∗
0.07
0.00
0.43
2.26
1,30
1,29
1,29
1,30
1,30
1,30
1,29
0.62
0.04
10.13∗∗
0.25
0.15
0.41
0.26
1,29
1,29
1,29
1,29
1,29
1,29
1,29
1.13
0.79
4.55∗
0.36
0.62
0.07
0.34
1,31
1,31
1,31
1,31
1,32
1,31
1,31
0.01
1.12
0.24
0.00
1.69
0.00
0.59
1,23
1,23
1,23
1,23
1,23
1,23
1,23
3.01
4.55∗
1.55
0.21
6.66∗
0.24
0.62
1,24
1,24
1,24
1,24
1,24
1,23
1,24
2.25
0.02
0.33
0.01
0.15
0.02
0.19
1,23
1,23
1,23
1,23
1,23
1,23
1,23
0.19
2.22
8.09∗∗
0.51
4.52∗
0.03
1.65
1,22
1,21
1,21
1,21
1,21
1,21
1,21
1.06
0.79
0.71
1.21
0.66
1.93
0.58
1,28
1,28
1,28
1,28
1,29
1,28
1,28
0.29
1.17
0.88
0.19
0.03
0.02
0.08
1,27
1,28
1,28
1,28
1,31
1,27
1,27
0.34
0.16
6.97∗
0.03
0.02
0.31
0.70
1,28
1,28
1,28
1,28
1,29
1,28
1,28
4.21∗
2.84
1.05
2.62
0.41
0.10
0.42
1,26
1,26
1,26
1,25
1,26
1,25
1,25
0.37
15.38∗∗∗
7.45∗
0.09
0.18
5.95∗
5.29∗
1,33
1,33
1,33
1,33
1,34
1,34
1,34
0.10
2.40
2.44
2.37
1.28
0.00
0.10
1,34
1,34
1,34
1,34
1,34
1,34
1,35
0.03
2.16
2.98
3.23
0.93
1.07
0.17
1,33
1,33
1,33
1,33
1,33
1,33
1,34
0.27
2.22
1.09
0.05
0.82
0.85
0.58
1,33
1,34
1,34
1,34
1,34
1,34
1,34
0.07
7.44∗
1.65
2.45
3.35
4.84∗
0.86
For each plant species (invasive Bromus diandrus, Hirshfeldia incana, Centaurea melitensis, and native Nassella pulchra), the model tests the effects of methanol (M), PPFMs
(P), competition (monoculture versus mixture plots) (C), and their interactions (denoted by “×”) on plant growth parameters. The biomass responses on a per individual basis
(total, root, or shoot weight per individual) were very similar; therefore, only the results for shoot weight/individual are shown. The variables reported below were ln-transformed.
For readability, the denominator d.f. is rounded to the nearest integer.
∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.0001.
110
Restoration Ecology
JANUARY 2013
PPFMs and Glyphosate in Restoration
Shoot to Root Ratio
(mean dry wt. g + 1SE)
15
monocultures
mixed species
C. melitensis
*
* *
10
5
N. pulchra
*
0
no
PPFMs
with
PPFMs
no
PPFMs
with
PPFMs
Figure 6. Shoot-to-root ratio comparing N. pulchra and C. melitensis.
Dry weight means from the multispecies field experiment, N. pulchra:
F1,34 = 4.84, p = 0.035; C. melitensis: in competition without PPFMs,
F1,25 = 7.45, p = 0.011; with PPFMs in competition and monoculture,
F1,26 = 15.38, varies p < 0.0001–0.022, results of mixed-model
three-way ANOVA, see also Table 1. *Indicates significant treatment
effect.
per individual (p = 0.0446) in the mixed plots, but not in
monoculture. PPFM addition increased shoot:root biomass for
N. pulchra (p = 0.0101), but this was driven by an increase
in the mixed plots (p = 0.0347; Fig. 6). Similarly, PPFMs
increased shoot:root ratio of C. melitensis (p = 0.0006),
but the increase was substantially greater in monocultures
than in mixtures (p = 0.0220; Fig. 6). The shoot:root ratio
of C. melitensis was also affected by a complex interaction between methanol, PPFMs, and plant competition (p =
0.0299).
Discussion
Soil PPFM abundance can be markedly reduced by a typical restoration site preparation of 2% glyphosate foliar
applications, potentially affecting plant restoration success.
Glyphosate directly reduced PPFM abundance through cell
death or growth inhibition in the lab. Consistent with this
pattern, we observed an order of magnitude fewer PPFMs in
the glyphosate-treated soils compared to nearby control soils,
which may be either a direct chemical effect or indirectly
caused by plant removal. Further work is needed to confirm
this observation at additional sites and ecosystem types and to
determine the mechanism by which glyphosate affects PPFMs.
Plant species in this study were differentially affected by
PPFM and/or methanol addition, a pattern that has also been
observed among crop plants (e.g. Ramírez et al. 2006). Here
we assume that methanol addition stimulates plant growth at
least in part through PPFMs. However, methanol application
may also affect plant growth directly (e.g. Nonomura & Benson 1992), and the relative contribution of these mechanisms
remains untested (Tavassoli & Galavi 2011).
In most (but not all) cases, our results were consistent
with the hypothesis that PPFMs have a greater benefit to
native over invasive species, suggesting that PPFMs may be
JANUARY 2013
Restoration Ecology
useful to include in restoration of sites where recolonization by invasives may be a problem. For example, the native
bunchgrass, Nassella pulchra, had positive germination and
growth responses to methanol and/or PPFM addition in the
field, whereas the invasive species had mixed responses, either
unaffected (B. diandrus), negatively affected (Hirschfeldia
incana), or positively affected (Centaurea melitensis). Nevertheless, the benefit of PPFMs to N. pulchra will only be
useful for restoration if it can overcome the negative effects of
competition with the invasives, for example, by shifting native
germination earlier in the season to counteract priority effects
of invasive species (Dyer & Rice 1999; Abraham et al. 2009).
While N. pulchra generally responded positively to
methanol and PPFM addition, the responses depended on the
experiment. In the N. pulchra experiment, methanol dramatically improved N. pulchra germination and growth. In the
multispecies experiment, methanol had no effect and PPFM
inoculation decreased the time to germination but did not
affect biomass. The functioning of the PPFM–plant interaction
could depend on abiotic conditions—in the first experiment,
the plants were under ambient drought-stressed conditions,
whereas in the multispecies experiment plants were regularly watered. Mirakhori et al. (2009) similarly found that
methanol addition to soybeans increased growth and yield
only when plants were drought-stressed. Alternatively, it is
possible that differences in PPFM composition or abundance
occurred between the experiments and resulted in different
treatment responses. For instance, methanol may stimulate different PPFMs compared to culturing and direct inoculation.
More generally, soil microbial communities can vary with
drought (Hawkes et al. 2010) and seasonality (Smalla et al.
2001), which may be an important consideration in restoration
efforts.
The success of invasive annuals was substantially improved
in mixtures compared to monocultures, which was unrelated to PPFM inoculation. However, soil microbes often
alter the interactions between native and invasive plants (e.g.
Klironomos 2002; Callaway et al., 2004). For instance, in
other experiments with C. melitensis, growth of the invasive
was nearly 5-fold greater in the presence of N. pulchra, but
only when the fungal community was intact (Callaway et al.
2003). Thus, disentangling these microbe-mediated interactions could be important for using PPFMs in areas where
invasives are still present.
Native perennials and invasive annuals may differ in their
response to PPFMs and/or methanol in part because of life history differences. We have observed, for instance, that PPFM
abundance in root zone soil varies among annual/biennial and
perennial species (both native and invasive) in CSS habitat
(I. Irvine, unpublished data). Other studies in similar California grassland systems have found species-specific interactions
of native and invasive grasses with mycorrhizal communities
that were partly dependent on annual versus perennial lifestyle
(e.g. Hausmann & Hawkes 2010). Whether this dichotomy
will help or hinder developing PPFMs as a restoration tool
for invaded ecosystems depends on the underlying drivers of
those interactions.
111
PPFMs and Glyphosate in Restoration
We conclude that PPFM and/or methanol addition in a
glyphosate-treated area might impact the success of CSS/
grassland restoration. Incorporating the soil microbiota into
our broader restoration toolkit could provide useful strategies
for restoring native plant communities (Richter & Stutz 2002;
Powell et al. 2009; Pringle et al. 2009; Zubek et al. 2009).
Although we have begun to elucidate a potential role for some
microbial taxa (mycorrhizal fungi, N-fixers, PPFMs), the vast
diversity of soil remains underexploited. More work will be
needed to identify both broad patterns and mechanisms for the
effects of PPFMs and other microorganisms on plants before
this can be a broadly useful management tool.
Implications for Practice
• Glyphosate and subsequent plant removal may reduce
PPFM abundance in soils.
• Under certain conditions, foliar methanol application
(20%) can increase Nassella pulchra germination and
size and should be tested on other natives in the field.
• Native soil microbial amendments including PPFMs
should be considered prior to native plant restoration after invasives have been removed and/or postglyphosate treatment.
• PPFM inoculation of native seed might improve germination and growth and could protect against some fungal
pathogens.
Acknowledgments
The authors thank Edith Allen, Anthony Amend, China
Hanson, Kristin Matulich, and two anonymous reviewers
for their thoughtful comments on prior drafts. We are also
grateful to John Orrock for statistical analysis advice, and
Joseph Algiers and Stephanie Raymond for invaluable field
assistance. For their laboratory expertise, we thank Claudia
Weihe, Stephanie Chen, Peris Bentley, Amanda Lee, and
Torben Intemann. The final manuscript was greatly improved
by the excellent editing of Christine Hawkes. This project was
funded in part by the National Science Foundation (MCB0701494) and the Newkirk Center for Science and Society.
LITERATURE CITED
Abraham, J. K., J. D. Corbin, and C. M. D’Antonio. 2009. California native
and exotic perennial grasses differ in their response to soil nitrogen,
exotic annual grass density, and order of emergence. Plant Ecology
201:445–456.
Bains, G., A. S. Kumar, T. Rudrappa, E. Alff, T. E. Hanson, and H. P. Bais.
2009. Native plant and microbial contributions to a negative plant-plant
interaction. Plant Physiology 151:145–2151.
Bever, J. D., I. A. Dickie, E. Facelli, J. M. Facelli, J. Klironomos, M.
Moora, M. C. Rillig, W. D. Stock, M. Tibbett, and M. Zobel. 2010.
Rooting theories of plant community ecology in microbial interactions.
Trends in Ecology & Evolution 25:468–478.
Callaway, R. M., B. E. Mahall, C. Wicks, J. Pankey, and C. Zabinski. 2003.
Soil fungi and the effects of an invasive forb on native versus naturalized
grasses: neighbor identity matters. Ecology 84:129–135.
112
Callaway, R. M., G. C. Thelen, S. Barth, and P. W. Ramsey. 2004. Soil fungi
alter interactions between the invader Centaurea maculosa and North
American natives. Ecology 85:1062–1071.
Callaway, R. M., D. Cipollini, K. Barto, G. C. Thelen, S. G. Hallett, D.
Prati, K. Stinson, and J. Klironomos. 2008. Novel weapons: invasive
plant suppresses fungal mutualists in America but not in its native Europe.
Ecology 89:1043–1055.
Doronina, N. V., E. G. Ivanova, and Y. A. Trotsenko. 2002. New evidence for
the ability of methylobacteria and methanotrophs to synthesize auxins.
Microbiology 71:116–118.
Dyer, A. R., and K. J. Rice. 1999. Effects of competition on resource availability and growth of a California bunchgrass. Ecology 80:2697–2710.
Eker, S., L. Ozturk, A. Yazici, B. Erenoglu, V. Romheld, and I. Cakmak. 2006.
Foliar-applied glyphosate substantially reduced uptake and transport of
iron and manganese in sunflower (Helianthus annuus L.) plants. Journal
of Agricultural and Food Chemistry 54:10019–10025.
Harris, J. 2009. Soil microbial communities and restoration ecology: facilitators
or followers? Science 325:573–574.
Hausmann, N. T., and C. V. Hawkes. 2010. Order of plant host establishment
alters the composition of arbuscular mycorrhizal communities. Ecology
91:2333–2343.
Hawkes, C. V., S. N. Kivlin, J. D. Rocca, V. Huguet, M. A. Thomsen, and
K. B. Suttle. 2010. Fungal community responses to precipitation. Global
Change Biology 17:1637–1645.
Hensley, D. L., D. S. N. Beuerman, and P. L. Carpenter. 1978. The inactivation of glyphosate by various soils and metal salts. Weed Research
18:287–291.
Holland, M. A., and J. C. Polacco. 1992. Urease-null and hydrogenase-null
phenotypes of a phylloplane bacterium reveal altered nickel metabolism
in two soybean mutants. Plant Physiology 98:942–948.
Kalyaeva, M. A., N. S. Zacharchenko, N. V. Doronina, E. B. Rukavtsova, E. G.
Ivanova, V. V. Alekseeva, Y. A. Trotsenko, and Y. I. Bur’yanov. 2001.
Plant growth and morphogenesis in vitro is promoted by associative methylotrophic bacteria. Russian Journal of Plant Physiology
48:514–517.
Klironomos, J. N. 2002. Feedback with soil biota contributes to plant rarity
and invasiveness in communities. Nature 417:67–70.
Kulmatiski, A. 2011. Changing soils to manage plant communities: activated
carbon as a restoration tool in ex-arable fields. Restoration Ecology
19:102–110.
Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, S. Seshadri, H. Y.
Chung, J. C. Yang, S. Sundaram, and T. M. Sa. 2004. Growth promotion and induction of systemic resistance in rice cultivar Co-47 (Oryza
sativa L.) by Methylobacterium spp. Botanical Bulletin of Academia
Sinica 45:315–324.
Madhaiyan, M., S. Poonguzhali, H. S. Lee, K. Hari, S. P. Sundaram, and
T. M. Sa. 2005. Pink-pigmented facultative methylotrophic bacteria
accelerate germination, growth and yield of sugarcane clone Co86032
(Saccharum officinarum L.). Biology and Fertility of Soils 41:350–358.
Madhaiyan, M., B. V. S. Reddy, R. Anandham, M. Senthilkumar, S.
Poonguzhali, S. P. Sundaram, and T. M. Sa. 2006. Plant growthpromoting Methylobacterium induces defense responses in groundnut
(Arachis hypogaea L.) compared with rot pathogens. Current Microbiology 53:270–276.
Mangla, S., Inderjit, and R. M. Callaway. 2008. Exotic invasive plant accumulates native soil pathogens which inhibit native plants. Journal of Ecology
96:58–67.
McComb, B., L. Curtis, C. Chambers, M. Newton, and K. Bentson. 2008.
Acute toxic hazard evaluations of glyphosate herbicide on terrestrial
vertebrates of the Oregon coast range. Environmental Science and
Pollution Research 15:266–272.
Mirakhori, M., F. Paknejad, F. Moradi, M. Ardakani, H. Zahedi, and P. Nazeri.
2009. Effect of drought stress and methanol on yield and yield components of Soybean Max (L 17). American Journal of Biochemistry and
Biotechnology 5:162–169.
Restoration Ecology
JANUARY 2013
PPFMs and Glyphosate in Restoration
Morris, W. F., R. A. Hufbauer, A. A. Agrawal, J. D. Bever, V. A.
Borowicz, G. S. Gilbert, J. L. Maron, C. E. Mitchell, I. M. Parker, A. G.
Power, M. E. Torchin, and D. P. Vazquez. 2007. Direct and interactive
effects of enemies and mutualists on plant performance: a meta-analysis.
Ecology 88:1021–1029.
Neumann, G., S. Kohls, E. Landsberg, K. S. O. Souza, T. Yamada, and
V. Romheld. 2006. Relevance of glyphosate transfer to non-target plants
via the rhizosphere. Journal of Plant Diseases and Protection 20:
963–969.
Nonomura, A. M., and A. A. Benson. 1992. The path of carbon in photosynthesis: improve crop yields with methanol. Proceedings of the National
Academy of Sciences 89:9794–9798.
Pospisilova, J., M. Vagner, J. Malbeck, A. Travniakova, and P. Batkova. 2005.
Interactions between abscisic acid and cytokinins during water stress and
subsequent rehydration. Biologia Plantarum 49:533–540.
Powell, J. R., R. G. Campbell, K. E. Dunfield, R. H. Gulden, M. M. Hart, D. J.
Levy-Booth, et al. 2009. Effect of glyphosate on the tripartite symbiosis
formed by Glomus intraradices, Bradythizobium japonicum, and genetically modified soybean. Applied Soil Ecology 41:128–136.
Pringle, A., J. D. Bever, M. Gardes, J. L. Parrent, M. C. Rillig, and
J. N. Klironomos. 2009. Mycorrhizal symbioses and plant invasions.
Annual Review of Ecology Evolution and Systematics 40:699–715.
Ramírez, I., F. Dorta, V. Espinoza, E. Jiménez, A. Mercado, and H. PeñaCortés. 2006. Effects of foliar and root applications of methanol on
the growth of Arabidopsis, tobacco, and tomato plants. Journal of Plant
Growth Regulation 25:30–44.
Relyea, R. A. 2005. The lethal impact of RoundUp on aquatic and terrestrial
amphibians. Ecological Applications 15:1118–1124.
Requena, N., I. Jimenez, M. Toro, and J. M. Barea. 1997. Interactions between
plant-growth-promoting rhizobacteria (PGPR), arbuscular mycorrhizal
fungi, and Rhizobium spp. in the rhizopshere of Anthyllis cytisoides, a
model legume for revegetation in Mediterranean semi-arid ecosystems.
JANUARY 2013
Restoration Ecology
New Phytologist 136:667–677.
Richter, B. S., and J. C. Stutz. 2002. Mycorrhizal inoculation of big sacaton:
implications for grassland restoration of abandoned agricultural fields.
Restoration Ecology 10:607–616.
Rowe, H. I., C. S. Brown, and M. W. Paschke. 2009. The influence of soil
inoculum and nitrogen availability on restoration of high-elevation
steppe communities invaded by Bromus tectorum. Restoration Ecology
17:686–694.
Smalla, K., G. Wieland, A. Buchner, A. Zock, J. Parzy, S. Kaiser, N. Roskot, H.
Heuer, and G. Berg. 2001. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent
enrichment and seasonal shifts revealed. Applied and Environmental
Microbiology 67:4742–4751.
Stinson, K. A., S. A. Campbell, J. R. Powell, B. E. Wolfe, R. M.
Callaway, G. C. Thelen, S. G. Hallett, D. Prati, and J. N. Klironomos.
2006. Invasive plant suppresses the growth of native tree seedlings by
disrupting belowground mutualisms. PLoS Biology 4:e140.
Tavassoli, A., and M. Galavi. 2011. Effect of foliar application of methanol on
efficiency, production and yield of plants - a review. Indian Journal of
Agricultural Research 45:1–10.
Trotsenko, Y. A., E. G. Ivanova, and N. V. Doronina. 2001. Aerobic methylotrophic bacteria as phytosymbionts. Microbiology 70:623–632.
Vitousek, P. M., and L. R. Walker. 1989. Biological invasion by Myrica faya
in Hawai’i: plant demography, nitrogen fixation, ecosystem effects.
Ecological Monographs 59:247–265.
Weidenhamer, J. D., and R. M. Callaway. 2010. Direct and indirect effects of
invasive plants on soil chemistry and ecosystem function. Journal of
Chemical Ecology 36:59–69.
Zubek, S., K. Turnau, M. Tsimilli-Michael, and R. Strasser. 2009. Response
of endangered plant species to inoculation with arbuscular mychorrizal
fungi and soil bacteria. Mychorriza 19:113–123.
113