materials

Article
Surface Modification of Carbon Nanotubes with
an Enhanced Antifungal Activity for the Control
of Plant Fungal Pathogen
Xiuping Wang 1 , Zilin Zhou 2 and Fangfang Chen 2, * ID

1 College of Life Science and Technology, Hebei Normal University of Science and Technology,
Qinhuangdao 066000, China; wangxiuping0721@163.com
2 CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden,
Chinese Academy of Sciences, Wuhan 430074, China; zhouzilin16@mails.ucas.ac.cn
* Correspondence: chenff@wbgcas.cn

Received: 3 November 2017; Accepted: 28 November 2017; Published: 30 November 2017

Abstract: The addition of surface functional groups to multi-walled carbon nanotubes (MWCNTs)
expands their application in engineering, materials, and life science. In the study, we explored the
antifungal activities of MWCNTs with different surface groups against an important plant pathogenic
fungi Fusarium graminearum. All of the OH-, COOH-, and NH2 -modified MWCNTs showed
enhanced inhibition in spore elongation and germination than the pristine MWCNTs. The length
of spores decreased by almost a half from 54.5 µm to 28.3, 27.4, and 29.5 µm, after being treated
with 500 µg· mL−1 MWCNTs-COOH, MWCNTs-OH, and MWCNTs-NH2 separately. Furthermore,
the spore germination was remarkably inhibited by surface-modified MWCNTs, and the germination
rate was only about 18.2%, three times lower than pristine MWCNTs. The possible antifungal
mechanism of MWCNTs is also discussed. Given the superior antifungal activity of surface modified
MWCNTs and the fact that MWCNTs can be mass-produced with facile surface modification at low
cost, it is expected that this carbon nanomaterial may find important applications in plant protection.

Keywords: MWCNTs; surface modification; antifungal activities; plant protection

1. Introduction
Carbon nanotubes (CNTs) are considered one of the most popular types of nanomaterials with
unique morphologies and surface properties and have been intensively studied for various applications
in bionanotechnology, including drug and gene delivery, tissue engineering, plant-technology [1,2],
and other biomedical applications [3–8]. In recent years, CNTs have been found to have
an active antibacterial activity and garnered a significant research interest around the use of
nanotechnology-based approaches for agricultural system and plant protection [9–12]. A nanotube
filter covered with a thin layer of single-walled carbon nanotubes (SWCNTs) are demonstrated
to be effective in removing viral and bacterial pathogens [13–15]. Pristine SWCNTs dispersed in
a biocompatible surfactant solution exhibited strong bactericidal activity against both gram-positive
and gram-negative bacteria [16]. Lately, an exceptional application of CNTs in controlling plant
pathogens in biological science has been described [17]. From the toxicological point of view,
single-walled carbon nanotubes have higher antimicrobial properties than multi-wall carbon nanotubes
(MWCNTs) [18]. At the same time, our previous studies verified that CNTs displayed superior
inactivation effects on the copper-resistant plant pathogenic microorganisms Ralstonia solanacearum,
Fusarium graminearum, and F. oxysporum [19,20]. These findings implied that CNTs may be applied to
phytopathogen control in plant protection because of their superior antimicrobial activity.

Materials 2017, 10, 1375; doi:10.3390/ma10121375 www.mdpi.com/journal/materials

Materials 2017, 10, 1375 2 of 11

However, a crucial step toward the application of CNTs in the plant science is to regulate their
impacts on biological systems. The use of CNTs is currently considered with apprehension owing to
their yet undefined safety profile and their potential environmental and health risks, especially given
their structural resemblance to asbestos fibers [21]. It has been proven that surface functional groups
on multiwalled CNTs (MWCNTs) reduce their immune perturbations in mice and in macrophages and
improve the colloidal properties of the CNT dispersions without changing their inherent antibiosis
performance [22,23]. For example, well-functionalized CNTs are safe to animal cells, while CNTs
without functionalization show severe toxicity to animal or human cells even at low doses [24].
Moreover, several studies have shown that the surface functional groups of CNTs are the critical
factors that affect the overall antibacterial effects of CNTs [25,26]. In contrast to the focused studies on
antibacterial properties of CNTs, researches related to antibiosis activities of CNTs in the treatment of
plant fungal infections are underemphasized. However, more than 80% of plant diseases are caused by
pathogenic fungi. There are relatively few reports about the influence of surface chemistry and length
of CNTs on fungi growth and propagation. Therefore, it is critical to study the antifungal properties of
CNTs in plants to explore their potential antifungal activity.
In many cases, placing functionalized covalent and non-covalent chemical groups onto the surface
of nanotubes can improve their biological performance [27]. For example, sugar with a terminal
amino group used to modify SWCNTs can control their aqueous solubility and biological activity
in binding assays with pathogens [28]. In additions, studies showed that SWCNTs with surface
functional groups of –OH and –COOH displayed very strong bactericidal activity against both
gram-positive and gram-negative bacteria, while similar functional groups of MWCNTs did not
display antimicrobial action to either type of microbial cells [29]. Other studies have documented that
functionalized SWCNTs (f-SWCNTs) have lower cytotoxicity to mammalian cells than SWCNTs [30,31].
Herein, we study the antifungal properties of CNTs against pathogenic fungal diseases with special
emphasis on the effects of CNT surface chemistry modification and length on their antifungal activity.
First, we studied the effects of different surface functional groups (–OH, –COOH and –NH2 ) of
MWCNTs on the antifungal activity against F. graminearum, which causes head blight or scab in
wheat. Second, we also discussed the potential antifungal mechanism of surface modified MWCNTs.
MWCNTs were selected in this study, because they can be produced in mass-scale and low-cost.
To the best of our knowledge, this is the first report to address this question. The results of this study
will advance the application of MWCNTs as antimicrobial agents in plant protection.

2. Results

2.1. Surface Modification Effects of MWCNTs on Spore Length

Fungal spores are specialized reproductive structures and play an important role in the
dissemination of diseases [32]. As shown in Figure 1, the average length of normal spores is
about 68.5 µm, and was reduced to 54.5, 28.3, 27.4, and 29.5 µm after being treated with MWCNTs,
MWCNTs-COOH, MWCNTs-OH, and MWCNTs-NH2 , respectively. The spore length was affected
by MWCNTs—it was one-fifth shorter than the control group—and was significantly affected after
it was treated with functional MWCNTs modified by the –OH group—It was almost three-fifths
shorter than the control group on average, and one-half shorter than the group treated with pristine
MWCNTs. Previous studies demonstrated that water is a major factor required for spore germination
in the resumption of cellular metabolism and growth, and after water uptake by spore, swelling can
significantly increase the volume of spores [32]; accordingly, the length of spore will be increased.
In our experiment, the length of spores did not increase after being treated with CNTs, indicating that
spores did not absorb water normally, and thus they could not germinate normally.
Materials 2017, 10, 1375 3 of 11
Materials 2017, 10, 1375 3 of 11

Materials 2017, 10, 1375 3 of 11

FigureFigure 1. Effects
1. Effects of multi-walledcarbon
of multi-walled carbon nanotubes
nanotubes (MWCNTs)
(MWCNTs)modified
modified with different
with groups
different groups
(500 μg−· 1
mL −1) on the spore length. Error bars represent the standard deviation (N = 4). Black asterisk
(500 µg·mL ) on the spore length. Error bars represent the standard deviation (N = 4). Black asterisk
Figure 1. Effects of multi-walled carbon nanotubes (MWCNTs) modified with different groups
indicated
indicated significant
significant differences
differences withinconsisting
within group group consisting of Control
of Control (CK), MWCNTs, (CK), MWCNTs,
MWCNTs-COOH,
(500 μg·mL−1) on the spore length. Error bars represent the standard deviation (N = 4). Black asterisk
MWCNTs-COOH, MWCNTs-OH, and MWCNTs-NH2 based on analysis of variance using the GLM
MWCNTs-OH,
indicated and MWCNTs-NH
significant 2 based
differences on analysis ofconsisting
variance using the GLM(CK),procedure with SAS
procedure with SAS system. GLM,within
Generalgroup
Linear Model; SAS, of Statistics
Control AnalysisMWCNTs,
System.
system. GLM, General
MWCNTs-COOH, Linear Model;
MWCNTs-OH, SAS, Statistics
and MWCNTs-NH Analysis System. Red asterisk indicated significant
2 based on analysis of variance using the GLM
Red asterisk indicated significant difference between MWCNTs, MWCNTs-COOH, MWCNTs-OH,
difference between
procedure with MWCNTs,
SAS MWCNTs-COOH,
system. GLM, General MWCNTs-OH,
Linear Model; and
SAS,MWCNTs-NH 2 , determined
Statistics Analysis System. as
and MWCNTs-NH 2, determined as described above. * p < 0.05; ** p < 0.01.
described
Red above. p < 0.05;significant
asterisk *indicated ** p < 0.01.difference between MWCNTs, MWCNTs-COOH, MWCNTs-OH,
2.2. Surface Modification2,Effects
and MWCNTs-NH determined as described
of MWCNTs above.
on Spore * p < 0.05; ** p < 0.01.
Germination
2.2. Surface Modification Effects of MWCNTs on Spore Germination
Spore germination
2.2. Surface represents
Modification Effects a pivotal
of MWCNTs step Germination
on Spore in the colonization of new environments by
filamentous
Spore fungi [33].
germination As shownainpivotal
represents Figure 2, we in
step studied the effects of of
the colonization pristine
new MWCNTs
environments and by
Spore germination represents a pivotal step in the colonization of new environments by
modified
filamentous MWCNTs
fungi fungi
[33]. As (surface
shown modified
in Figure by –OH,
2, we 2, –COOH
studied and
the effects –NH 2) on F. graminearum spore
of pristine MWCNTs and modified
filamentous [33]. As shown in Figure we studied the effects of pristine MWCNTs and
MWCNTsgermination.
(surface F. modified
graminearum by spores
–OH, were
–COOH incubated
and in dispersions
–NH ) on F. of MWCNTs,spore
graminearum MWCNTs-OH,
germination.
modified MWCNTs (surface modified by –OH, –COOH 2 and –NH2) on F. graminearum spore
MWCNTs-COOH, and MWCNTs-NH2 at the concentration from 62.5 to 500 μg·mL−1 for 5 h.
F. graminearum
germination.spores were incubated
F. graminearum sporesinwere
dispersions
incubated of inMWCNTs,
dispersions MWCNTs-OH,
of MWCNTs, MWCNTs-COOH,
MWCNTs-OH,
MWCNTs caused a dose-dependent inhibitory effect on F. graminearum −1 spore germination. When
and MWCNTs-NH
MWCNTs-COOH, 2 at and
the MWCNTs-NH
concentration2 at fromthe 62.5 to 500 µgfrom
concentration · mL62.5
the spore germination rate reached 98.1% in the control, it was only 55.2% in the MWCNT group.
for 5 h.μg·mL
to 500 MWCNTs−1 for 5caused
h.
MWCNTs caused
a dose-dependent a dose-dependent
inhibitory effect on F. inhibitory effect
graminearum spore on F. graminearum When
germination. spore germination.
the spore When
germination
Spore germination decreased by more than 30% at the highest dose of MWCNTs-COOH, -OH, and
the spore
rate reached germination
98.1% in theμg·mL rate−1 reached
control, 98.1%55.2%
in the control, it was only 55.2% in the MWCNT group.
-NH2 tested (500 ). itFigure
was only S1 showsinthe the MWCNT
corresponding group. Spore germination
photomicrograph of decreased
spore
by moreSpore germination
than 30% at thedecreased
highest by more
dose than 30%
of MWCNTs-COOH,at the highest dose of MWCNTs-COOH,
-OH, and that -NHMWCNTs,
tested (500-OH,
µg and
·mL−1 ).
germination after treatment with MWCNTs, which visually confirmed 2 especially
-NH2 tested (500 μg·mL−1). Figure S1 shows the corresponding photomicrograph of spore
Figure S1 shows
modified the corresponding
MWCNTs, can effectivelyphotomicrograph
restrain the spore of spore germination
germination. This result after treatment
suggests that thewith
germination after treatment with MWCNTs, which visually confirmed that MWCNTs, especially
functional MWCNTs with –COOH, –OH, and –NH groups have
MWCNTs, which visually confirmed that MWCNTs, especially modified MWCNTs, can effectively
2 stronger antifungal activity than
modified MWCNTs, can effectively restrain the spore germination. This result suggests that the
pristine
restrain ones.
the spore From Figure
germination. 2, MWCNTs
This result with
suggests–OH thatgroups showed highest antifungal
the functional MWCNTs with –COOH, activity on–OH,
functional MWCNTs with –COOH, –OH, and –NH 2 groups have stronger antifungal activity than
spore germination among the three groups.
and –NH 2 groups
pristine ones.have
Fromstronger
Figure 2,antifungal
MWCNTs activity
with –OH than pristine
groups ones.highest
showed From Figure 2, MWCNTs
antifungal activity onwith
–OH groups showed highest
spore germination amongantifungal activity on spore germination among the three groups.
the three groups.

Figure 2. Effects of different functional groups of MWCNTs on the spore germination. Spores were
germinated in distilled water at 28 °C in darkness at different concentrations of MWCNTs,
Figure 2. Effects
2. Effects of different
of different functional
functional groupsofofMWCNTs
MWCNTs on on the spore
sporegermination. Spores were
Figure
MWCNTs-COOH, MWCNTs-OH andgroups
MWCNTs-NH the
2 dispersions. germination.
Germination Spores
was evaluated afterwere
germinated in distilled water at 28 °C in darkness at different concentrations of MWCNTs,
incubation
germinated in for 5 h. Error
distilled water at 28 ◦ C
bars represent thein
standard deviation
darkness (N = 4). concentrations of MWCNTs,
at different
MWCNTs-COOH, MWCNTs-OH and MWCNTs-NH2 dispersions. Germination was evaluated after
MWCNTs-COOH, MWCNTs-OH and MWCNTs-NH2 dispersions. Germination was evaluated after
incubation for 5 h. Error bars represent the standard deviation (N = 4).
incubation for 5 h. Error bars represent the standard deviation (N = 4).
Materials 2017, 10, 1375 4 of 11

2.3. Surface Modification Effects of MWCNTs on Germination Pattern of Spores

Materials 2017, 10, 1375 4 of 11
F. graminearum typically has a bipolar germination pattern in which one germ tube emerges from
2.3. Surface
each apical cell ofModification
the spore Effects of MWCNTs
[32]. After treated on with
Germination
MWCNTs, Patternthere
of Spores
was a difference in the branching
Materials 2017, 10, 1375 4 of 11
pattern of the germ tubestypically
F. graminearum from conidia. The number
has a bipolar of germ
germination tubes
pattern originating
in which one germfrom theemerges
tube terminal cells
was significantly
from
2.3. Surface reduced
each apical cell of(pthe
Modification < 0.05),
spore
Effects especially
[32].
of MWCNTs whenwith
Afterontreated treated
Germination MWCNTs,
Pattern byof surface
there was
Spores functional MWCNTs
a difference in the with
–COOH, branching
–OH, pattern
and –NH of the germ tubes
groups at from conidia. The
concentration of number
500 µg · mL −1 . As
of germ tubes originating
shown in from the
Figure 3, 7.8% of
F. graminearum2 typically has a bipolar germination pattern in which one germ tube emerges
terminal cells was significantly reduced (p < 0.05), especially when treated by surface functional
the germfromtubeseachemerged
apical cell from interstitial
of the spore [32].cells
Afterin control
treated group,
with MWCNTs, but this
therenumber increased
was a difference to 35.0%,
in the
MWCNTs with –COOH, –OH, and –NH2 groups at concentration of 500 μg·mL−1. As shown in
42.3%, 44.6%, and
branching 38.5%,
pattern respectively,
of the germ tubes in MWCNTs-treated
from groups.
conidia. The number Also,
of germ modified
tubes
Figure 3, 7.8% of the germ tubes emerged from interstitial cells in control group, but this number
MWCNTs
originating from the showed
enhanced terminal cells was to
teratogenicity significantly
F. reducedFigure
graminearum. (p < 0.05),
S2 especiallythe
displays when treated by surface
representative functionalimages
microscopic
increased to 35.0%, 42.3%, 44.6%, and 38.5%, respectively, in MWCNTs-treated −1groups. Also,
MWCNTs with –COOH, –OH, and –NH2 groups at concentration of 500 μg·mL . As shown in
of sporemodified
germinationMWCNTs after treatment
showed withteratogenicity
enhanced different surface functional Figure
to F. graminearum. MWCNTs, confirming
S2 displays the that
Figure 3, 7.8% of the germ tubes emerged from interstitial cells in control group, but this number
a largerrepresentative
proportion ofmicroscopic
interstitial images
germ of spore
tubes germination
occurred when after treatment
exposed
increased to 35.0%, 42.3%, 44.6%, and 38.5%, respectively, in MWCNTs-treated groups. Also,
to with different
surface functionalsurface
MWCNTs.
As shownfunctional
in FigureMWCNTs,
S2, longconfirming
and normal that germ
a larger proportion
tubes couldtoof
beF.interstitial
observed germ tubes occurred
visually whensamples.
modified MWCNTs showed enhanced teratogenicity graminearum. FigureinS2thedisplays
control the
exposed to surface functional MWCNTs. As shown in Figure S2, long and normal germ tubes could
However, most germ microscopic
representative tubes of the spores
images of immersed in surface
spore germination afterfunctional
treatment withMWCNTsdifferent(500 µg·mL−1 )
surface
be observed visually in the control samples. However, most germ tubes of the spores immersed in
developed functional
from MWCNTs,
the side confirming that a−1 larger proportion of interstitial germ tubes occurred when
of spores.
surface functional MWCNTs (500 μg·mL ) developed from the side of spores.
exposed to surface functional MWCNTs. As shown in Figure S2, long and normal germ tubes could
be observed visually in the control samples. However, most germ tubes of the spores immersed in
surface functional MWCNTs (500 μg·mL−1) developed from the side of spores.

Figure 3. Effects of MWCNTs on the pattern of germ tube. The number of interstitial germ tubes was
Figure 3. Effects of MWCNTs on the pattern of germ tube. The number of interstitial germ tubes was
counted after 6 h of incubation at 28 °C in water (control) or water containing 500 μg·mL−1 MWCNTs.
countedError
afterbars
6 h represent
of incubation at 28 ◦ C in water (control) or water containing 500 µg· mL−1 MWCNTs.
the standard deviation (N = 4). Where appropriate, statistical significance is
Figure
Error bars 3. Effectsthe
represent of MWCNTs on the pattern of germ tube. The number of interstitial germ tubes was
indicated: ** p< 0.01. standard deviation (N = 4). Where appropriate, statistical significance is
counted after 6 h of incubation at 28 °C in water (control) or water containing 500 μg·mL−1 MWCNTs.
indicated: ** p < 0.01.
Error Modification
2.4. Surface bars represent the standard
Effects deviation
of MWCNTs (N = 4). Where
on Persistence appropriate,
of Antifungal statistical significance is
Activity
indicated: ** p< 0.01.
2.4. Surface We
Modification Effects
next examined the of MWCNTs onantifungal
time-dependent Persistence of Antifungal
behavior Activity
of pristine MWCNTs and functional
MWCNTs.
2.4. SurfaceMore effectiveEffects
Modification and durable inhibitory
of MWCNTs activity toward
on Persistence spore Activity
of Antifungal germination was observed at
We24next
and examined the time-dependent
48 h in the groups antifungal
treated with functional MWCNTs behavior
versusofpristine
pristine MWCNTs
nanotube. and functional
As shown in
MWCNTs. We next
More examined
effective andthedurable
time-dependent antifungal
inhibitory activitybehavior
towardof spore
pristinegermination
MWCNTs andwas functional
observed at
Figure 4, after treatment with functional MWCNTs, the germination rate was only about 25% even at
MWCNTs. More effective and durable inhibitory activity toward spore germination was observed at
24 and 48
48h.h The
in the groups
groups treated
treated with functional
with pristine MWCNTs had MWCNTs versus
a germination ratepristine
of >70% atnanotube.
24 h. As shown in
24 and 48 h in the groups treated with functional MWCNTs versus pristine nanotube. As shown in
Figure 4,Figure
after treatment with with
4, after treatment functional MWCNTs,
functional MWCNTs,the the germination rate
germination rate waswas
onlyonly
about about 25%ateven at
25% even
48 h. The48groups treatedtreated
h. The groups with with
pristine MWCNTs
pristine MWCNTs had
hadaagermination
germination raterate
of of >70%
>70% at 24ath.24 h.

Figure 4. Effects of different functional MWCNTs on the germination rate at (A) 24 h and (B) 48 h,
respectively. Error bars represent the standard deviation (N = 4). Where appropriate, statistical
significance is indicated: ** p < 0.01.
Figure 4.Figure 4. Effects
Effects of different
of different functional
functional MWCNTson
MWCNTs on the
the germination
germination raterate
at (A)
at 24
(A)h 24
andh(B)
and48 (B)
h, 48 h,
respectively. Error bars represent the standard deviation (N = 4). Where appropriate, statistical
respectively. Error bars represent the standard deviation (N = 4). Where appropriate, statistical
significance is indicated: ** p < 0.01.
significance is indicated: ** p < 0.01.
Materials 2017, 10, 1375 5 of 11
Materials 2017, 10, 1375 5 of 11
Materials 2017, 10, 1375 5 of 11
2.5. Length Effect of MWCNTs on Antifungal Activity
2.5. Length
2.5. We also
Length Effect of
of MWCNTs
examined
Effect MWCNTs on Antifungal
Antifungal
the influence
on Activity length on their antifungal activity. Three different
of MWCNTs
Activity
lengths of MWCNTs-COOH were chosen as a model for this study. Figure 5 shows the germination
We also
We also examined
examined the the influence
influence of of MWCNTs length on
MWCNTs length on their
their antifungal
antifungal activity.
activity. Three
Three different
different
rate of spores that were treated with different lengths of MWCNTs-COOH at different
lengths of
lengths of MWCNTs-COOH
MWCNTs-COOHwere werechosen
chosenasas a model
a model forfor
thisthis study.
study. Figure
Figure 5 shows
5 shows the germination
the germination rate
concentrations for 5 h. As shown in Figure 5, there is no significant difference in the germination rate
rate of spores that were treated with different lengths of MWCNTs-COOH
of spores that were treated with different lengths of MWCNTs-COOH at different concentrations for 5 h. at different
of spores treated with MWCNTs-COOH1 (~50 μm in length), MWCNTs-COOH2 (10–30 μm in
concentrations
As for 5 5,
shown in Figure h. there
As shown
is no in Figure 5,difference
significant there is noinsignificant difference
the germination rateinofthe germination
spores rate
treated with
length), and MWCNTs-COOH3 (0.2–2 μm in length) at the same weight concentration.
of spores treated1 (~50
MWCNTs-COOH with µmMWCNTs-COOH 1 (~50 μm in (10–30
in length), MWCNTs-COOH 2
length),
µmMWCNTs-COOH 2 (10–30 μm in
in length), and MWCNTs-COOH 3
length), and MWCNTs-COOH (0.2–2 μm in
(0.2–2 µm in length) at the same weight concentration.
3 length) at the same weight concentration.

Figure 5. Antifungal effects of different length of MWCNTs-COOH on the germination rate of spores.
Error bars represent the standard deviation (N = 4). Where appropriate, statistical significance is
Figure 5.
Figure 5. Antifungal
Antifungal effects
effects ofof different
different length
length of
of MWCNTs-COOH
MWCNTs-COOH on on the
the germination
germination rate
rate of
of spores.
spores.
indicated: * p < 0.05; ** p < 0.01.
Error bars represent the standard deviation (N = 4). Where appropriate, statistical significance
Error bars represent the standard deviation (N = 4). Where appropriate, statistical significance is is
indicated: ** pp << 0.05;
indicated: 0.05; ** pp << 0.01.
**can 0.01.
A brief summary be drawn for the antifungal activity of MWCNTs: (1) the surface
chemistry of MWCNTs plays an important role in their antifungal activity; (2) the functional
A brief summary can be drawn for the antifungal activity of MWCNTs: (1) the surface
MWCNTsA briefshow
summary can beantifungal
stronger drawn foreffects
the antifungal activitysamples
than pristine of MWCNTs: (1) theofsurface
regardless surface chemistry
coating
chemistry of MWCNTs plays an important role in their antifungal activity; (2) the functional
of MWCNTs
groups (–OH,plays an important
–COOH, or –NH2role ); (3)in the
their antifungal
length activity; (2)
of MWCNTs doesthenot
functional MWCNTs
significantly affectshow
their
MWCNTs show stronger antifungal effects than pristine samples regardless of surface coating
stronger antifungal
antifungal activity. effects than pristine samples regardless of surface coating groups (–OH, –COOH,
groups (–OH, –COOH, or –NH2); (3) the length of MWCNTs does not significantly affect their
or –NH2 ); (3) the length of MWCNTs does not significantly affect their antifungal activity.
antifungal activity.
2.6. The Antifungal Mechanism of Surface Modification MWCNTs
2.6. The Antifungal Mechanism of Surface Modification MWCNTs
2.6. The
To Antifungal Mechanism the
better understand of Surface Modification
antifungal mode MWCNTs
of MWCNTs, we examined how MWCNTs
To better
interacted understand
with spores usingthe TEM.
antifungal mode6B–E,
In Figure of MWCNTs, we examined
a few clusters of pristinehow MWCNTs
MWCNTs or interacted
functional
with To
sporesbetter understand
using TEM. In the
Figure antifungal
6B–E, a few mode
clustersof MWCNTs,
of pristine we
MWCNTs examined
or how
functional MWCNTs
MWCNTs
MWCNTs accumulated around the spores. The tubular MWCNTs could be observed lying beside
interacted with
accumulated sporesthe
around using TEM.The In Figure 6B–E, a few clusters beofobserved
pristine MWCNTs or functional
the spores (Figure 6G–J).spores.
These results tubular MWCNTs
indicate that the could
contact between lyingsporesbeside
and theCNTs spores
is a
MWCNTs
(Figure accumulated
6G–J). These around
results indicatethe spores.
that the The
contacttubular
between MWCNTs
spores could
and CNTsbe observed
is a necessarylying beside
condition
necessary condition for the inactivation of spores. Moreover, as shown in Figure 6, the spores in
the the
for spores (Figure 6G–J). TheseMoreover,
results indicate thatinthe contact between spores and group
CNTs is a
control inactivation of spores.
group and MWCNTs-treated groups ashad
shownbasically Figure 6, the
complete sporesstructure,
cellular in control indicatingandthe
necessary
MWCNTs-treatedcondition for the inactivation of spores. Moreover, as shown in Figure 6, the spores in
integrity of the cellgroups
wall. Ithad basicallythat
implicates complete cellular
disrupting thestructure, indicating
cell wall with CNTs the integrity
is not a major of the cell
cause of
control
wall. It group and that
implicates MWCNTs-treated
disrupting the groups
cell wall had
withbasically
CNTs complete
is not a cellular
major causestructure,
of spore indicating
inactivation.the
spore inactivation.
integrity of the cell wall. It implicates that disrupting the cell wall with CNTs is not a major cause of
spore inactivation.

Figure 6. Cont.
Materials 2017, 10, 1375 6 of 11
Materials 2017, 10, 1375 6 of 11

Figure 6.
Figure 6. TEM
TEM images
images of
of spores
spores after
after incubation
incubation with
with deionized
deionized (DI)
(DI) water
water and
and MWCNTs.
MWCNTs. In In the
the
upper row are microscopic images of spores treated without (A) and with
upper row are microscopic images of spores treated without (A) and with MWCNTs, MWCNTs-COOH, MWCNTs,
MWCNTs-COOH,
MWCNTs-OH, MWCNTs-OH,(B–E).
and MWCNTs-NH and The
MWCNTs-NH 2 (B–E). The lower row shows partial
lower row shows partial magnification images of spores
2
treated without (F) and with MWCNTs, MWCNTs-COOH,and
magnification images of spores treated without (F) with MWCNTs,
MWCNTs-OH, MWCNTs-COOH,
and MWCNTs-NH 2 (G–J).
MWCNTs-OH,
The and MWCNTs-NH
red arrows indicate 2 (G–J). The red arrows indicate MWCNTs around the spores and
MWCNTs around the spores and the magnified location of B–E.
the magnified location of B–E.
3. Discussion
3. Discussion
The volume of spore increases dramatically during swelling, which is essentially caused by water
The volume of spore increases dramatically during swelling, which is essentially caused by
absorption [34]. This procedure is under the influence by temperature, culture solution, and protein
water absorption [34]. This procedure is under the influence by temperature, culture solution, and
nutrients. In this study, all the treatment experiments were carried out in distilled water at 28 ◦ C,
protein nutrients. In this study, all the treatment experiments were carried out in distilled water at
so the influences of temperature are excluded. Therefore, the enhanced inhibition of spore elongation
28 °C, so the influences of temperature are excluded. Therefore, the enhanced inhibition of spore
is dominated by additional functional groups on the surface of MWCNTs which causes the significant
elongation is dominated by additional functional groups on the surface of MWCNTs which causes
decrease in spore size.
the significant decrease in spore size.
Noticeably, the MWCNTs have only mild inhibition on spore germination, but the modified
Noticeably, the MWCNTs have only mild inhibition on spore germination, but the modified
MWCNTs show superior activity against F. graminearum spores. This is likely due to the marked
MWCNTs show superior activity against F. graminearum spores. This is likely due to the marked
difference in dispersity of the functional MWCNTs and pristine MWCNTs. Generally, the MWCNTs
difference in dispersity of the functional MWCNTs and pristine MWCNTs. Generally, the MWCNTs
dispersions are unstable and containing larger aggregates which effectively block the interaction with
dispersions are unstable and containing larger aggregates which effectively block the interaction
spores. The dispersibility of MWCNTs can be improved by the functional groups on their surface [35].
with spores. The dispersibility of MWCNTs can be improved by the functional groups on their
When carboxyl, hydroxyl, amino, and epoxy groups are introduced onto the surface of the MWCNTs,
surface [35]. When carboxyl, hydroxyl, amino, and epoxy groups are introduced onto the surface of
they facilitate the formation of much more stable dispersions versus the hydrophobic pristine carbon
the MWCNTs, they facilitate the formation of much more stable dispersions versus the hydrophobic
planes [36]. Thus, the MWCNTs surface modified with –OH, –COOH, and –NH2 groups can form small
pristine carbon planes [36]. Thus, the MWCNTs surface modified with –OH, –COOH, and –NH2
and stable dispersions, which offers more opportunities to interact with spores than pristine MWCNTs.
groups can form small and stable dispersions, which offers more opportunities to interact with
Previous studies suggested that charge effect is a very important factor that affects the
spores than pristine MWCNTs.
antibacterial efficiency of CNTs [24]. Here, the pH values of these incubation buffers are 7.0.
Previous studies suggested that charge effect is a very important factor that affects the
At this pH, MWCNTs-OH are neutrally charged, while MWCNTs-COOH are negatively charged and
antibacterial efficiency of CNTs [24]. Here, the pH values of these incubation buffers are 7.0. At this
MWCNTs-NH2 are positively charged. However, the antifungal activity of functional MWCNTs shows
pH, MWCNTs-OH are neutrally charged, while MWCNTs-COOH are negatively charged and
no significant difference between these three surface functional groups, indicating that the charge
MWCNTs-NH2 are positively charged. However, the antifungal activity of functional MWCNTs
effect is not a crucial factor for antifungal efficiency of MWCNTs. It is distinct with findings obtained
shows no significant difference between these three surface functional groups, indicating that the
from bacterial cells, in which charge effects of MWCNTs change the interaction between bacterial
charge effect is not a crucial factor for antifungal efficiency of MWCNTs. It is distinct with findings
cells and MWCNTs [24]. Normally, both the gram-positive and gram-negative cell walls have overall
obtained from bacterial cells, in which charge effects of MWCNTs change the interaction between
negative charges [24]. According to previous reports [15,37], the antibacterial mechanisms of CNTs are
bacterial cells and MWCNTs [24]. Normally, both the gram-positive and gram-negative cell walls
mainly due to destruction of cell wall integrity by direct contact with CNTs. Therefore, we suspect
have overall negative charges [24]. According to previous reports [15,37], the antibacterial
that the distinct performance of MWCNTs as antimicrobial against fungal spores or bacterial cells is
mechanisms of CNTs are mainly due to destruction of cell wall integrity by direct contact with
attributed to the difference between their cell walls.
CNTs. Therefore, we suspect that the distinct performance of MWCNTs as antimicrobial against
Meanwhile, we observed that a larger proportion of interstitial germ tubes occurred when
fungal spores or bacterial cells is attributed to the−difference between their cell walls.
exposed to MWCNTs at the dose of 500 µg· mL 1 MWCNTs. The results are consistent with the
Meanwhile, we observed that a larger proportion of interstitial germ tubes occurred when
work of Harris et al. [32]. They have found that spores germinate preferentially from the lateral
exposed to MWCNTs at the dose of 500 μg·mL−1 MWCNTs. The results are consistent with the work
end under certain conditions, such as high glucose or other adverse environmental conditions.
of Harris et al. [32]. They have found that spores germinate preferentially from the lateral end
These results suggest that MWCNTs is an unfavorable factor for the growth and development of
under certain conditions, such as high glucose or other adverse environmental conditions. These
spores. It was shown that the pattern of germination requires the presence of functional microtubules,
results suggest that MWCNTs is an unfavorable factor for the growth and development of spores.
which may be responsible for the transport of key polarity factors for chitin deposition to specific sites.
It was shown that the pattern of germination requires the presence of functional microtubules,
Thus, in our experiments, we speculate that the regulatory system for chitin deposition to specific
which may be responsible for the transport of key polarity factors for chitin deposition to specific
sites. Thus, in our experiments, we speculate that the regulatory system for chitin deposition to
specific sites may be disturbed after treated by MWCNTs [38]. Similar phenomena have been
Materials 2017, 10, 1375 7 of 11

sites may be disturbed after treated by MWCNTs [38]. Similar phenomena have been previously
observed on other carbon nanomaterials. For instance, when F. graminearum spores were exposed
to increasing concentrations of graphene oxide (GO), more interstitial germ tubes were noted and
then the length of germ tube was inhibited, and GO treatment can remarkably reduce macroconidia
viability ultimately [39].
A notable difference between the pristine MWCNTs and functional MWCNTs is the lack of
dispersibility of pristine MWCNTs in most solvents owing to strong inter-tube van der Waals forces,
and this has been an obstacle for their effective use in antifungal applications. However, this may be
largely overcome by surface modification of the nanotube backbone, allowing the functional MWCNTs
well dispersed in water to form a homogeneous solution. It is much more stable even after several
days of storage [39]. These results suggest that the stability of the MWCNTs dispersions is critical to
their antifungal activities.
One prior study on the length effects of SWCNTs on bacteria showed that the antimicrobial
activity of SWCNTs increased with increasing length of SWCNTs [39]. In contrast to this observation,
the present study (Figure 5) did not indicate any length effects of the MWCNTs on their antifungal
activity, at least in the length range discussed here. One possible explanation is the inherent
composition differences between fungi spores and bacteria. Fungal spores are usually dozens of
microns long and have chitin cell walls, while bacteria are only a few microns long and have cell walls
made of cellulose [40]. These size and morphology differences result in different interactions between
spores or bacteria and CNTs. Due to smaller size of bacteria, CNTs are easily intertwined and packaged
with them and damages their cell walls. The larger fungal spores are more resistant to damage because
the CNTs only adhere to the spore rather than integrating with the cell wall. Furthermore, the main
difference between the cell walls of fungi and bacteria is that the cell wall of fungi is mainly composed
of chitin and cellulose, while the cell wall of bacteria is mainly composed of peptide polysaccharides.
Fungal spores are more resistant to mechanical damage, because the main composition of their cell
walls is chitin and cellulose, which are stronger than peptide polysaccharides. Thus, CNTs probably
cannot damage the spore cell walls. This hypothesis was further verified by TEM imaging.
This finding is also in good agreement with our prior work in which the damage and disruption
of the cell wall is not a dominating mechanism to interpret the antifungal activity of MWCNTs.
The possible antifungal mechanism is the blockage of water channel imposed by surface-adsorbed
MWCNTs [19]. During the exposure of spores to MWCNTs, the MWCNTs seemed to be more inclined
to form aggregates with the largest involvement of spores. The MWCNTs aggregates absorbed on
the spore surface and blocked the water channel. This caused a substantial loss of water involved in
germination. The proposed mechanism could also be applied to interpret the effects of MWCNTs on
spore length.
The plant disease Fusarium head blight (FHB) is caused by the fungal pathogen F. graminearum
and is a serious plant disease world widely affecting wheat, corn, etc. [41]. Currently, the wheat
industry has few effective materials for FHB control. In this work, we demonstrated that surface
modified MWCNTs showed enhanced inhibition in spore germination. During a growth cycle, once the
spore germination is inhibited or stopped, the spore can no longer form mature mycelium, and finally
interrupts pathogen reproduction and terminates the infection cycle [31].

4. Materials and Methods

4.1. Chemicals
MWCNTs with –OH, –COOH, and –NH2 surface functional groups were purchased from
ChengDu Organic Chemicals Co., Ltd. Chinese Academy of Science (Chengdu, China) and used
as received. The physical dimensions, purity, and functional groups are summarized in Table 1.
All CNTs suspensions were obtained by sonication for 30 min using a bath sonicator (Elamsonic,
S60H, ELMA-Tech, Mosbach, Germany) at 37 kHz under 550 W without any dispersant.
Materials 2017, 10, 1375 8 of 11

Table 1. Purity, functional groups, and physical dimensions of MWCNTs.

Items Purity (%) Functional Groups Content (%) ID (nm) OD (nm) Length (µm)
MWCNTs-OH >95 3.70 3–5 8–15 ~50
MWCNTs-NH2 >95 0.45 3–5 8–15 ~50
MWCNTs-COOH1 >95 3.82 3–5 8–15 ~50
MWCNTs-COOH2 >95 2.56 2–5 <8 10–30
MWCNTs-COOH3 >95 3.86 2–5 <8 0.2–2
This table is provided by ChengDu Organic Chemicals Co., Ltd.

4.2. Fungal Strains

F. graminearum was obtained from the State Key Laboratory of Agricultural Microbiology of
Huazhong Agricultural University (Wuhan, China). F. graminearum spores were obtained as described
previously [19]. Briefly, spores incubated in 3% green bean soup liquid medium for 5 days were
harvested by centrifugation at 3500 rpm for 5 min. After filtering through gauze, the spores were
washed twice with sterile distilled water and adjusted to 5 × 105 spores mL−1 .

4.3. Spore Germination and CNTs Treatment

For spore germination studies, F. graminearum spores were treated with CNTs according to
a previous report [19]. Briefly, an 80 µL suspension of spores was mixed with 80 µL of different types
of MWCNTs (–OH, –COOH, and –NH2 ) in the tubes to obtain MWCNTs at a final concentration
of 62.5, 125, 250, and 500 µg·mL−1 , respectively. Control samples containing 80 µL suspensions
of spores were mixed with 80 µL DI water. The 30 µL mixture with a different concentration of
MWCNTs was transferred onto a concave slide for further incubation at 28 ◦ C for 5 h in complete
darkness. Five concave slides were prepared for each treatment, and the mean germination values
were calculated based on five measurements. Photomicrographs were taken with a digital camera
connected to a Leica microscope. For each treatment, one hundred spores were used to measure the
average spore length. The spore germination rate was calculated as follows:

Rgerm = Ngerm /Ntotal × 100%.

Rgerm represents the spore germination rate (%); Ngerm represents the number of germinated
spores; Ntotal is the total number of spores.

4.4. Morphological Observation by TEM

The morphological changes of spores were further investigated using a transmission electron
microscopy (TEM) (FEI, Brno, Czech Republic). The spore preparation for morphological study was
according to our previous report [19].

4.5. Statistical Analysis

Each treatment was performed in four replicates, and the data for all figures were represented
as mean values ± SE (standard errors). Statistical analysis used SAS 8.1 software, and the statistical
significance was determined by a p value < 0.05 (or < 0.01) in a Student’s t-test.

5. Conclusions
In summary, we studied the effects of MWCNT surface modification on their antifungal activity.
It was demonstrated that MWCNTs functionalized with –COOH, –OH, and –NH2 groups had a more
effective and durable inhibitory antifungal activity than pristine nanotubes. In addition to having
an improved solubility, it is likely that the functional groups conjugated to the nanotubes favors
interaction with spores, which endows enhanced antifungal activity to the surface modified MWCNTs.
Besides this, the effect of length was also studied and was found that it is not a noticeable factor in
Materials 2017, 10, 1375 9 of 11

antifungal activity. Taken together with our preliminary results, this work clearly shows the great
potential of these easily produced carbon materials in eliminating a severe agricultural problem.

Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/10/12/1375/s1,

Figure S1: Microscopic images of MWCNTs on germination rate of spores, Figure S2: Microscopic images of
MWCNTs on germination pattern of spores.
Acknowledgments: All the authors gratefully acknowledge the support for this research by the PhD Research
Startup Foundation of Hebei Normal University of Science and Technology (2013YB005) and the National Natural
Science Foundation of China (31501680 and 31600240).
Author Contributions: Xiuping Wang and Fangfang Chen conceived and designed the experiments;
Xiuping Wang performed the experiments; Zilin Zhou assisted with interpretation of the materials; Xiuping Wang
and Fangfang Chen analyzed the data and wrote the manuscript. All authors supervised the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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