Plant Pathology - 2015 - Imada - Magnesium oxide nanoparticles induce systemic resistance in tomato against bacterial wilt

Plant Pathology (2016) 65, 551–560 Doi: 10.1111/ppa.
12443
Magnesium oxide nanoparticles induce systemic resistance

in tomato against bacterial wilt disease
K. Imadaa, S. Sakaib, H. Kajiharac, S. Tanakad and S. Itod*

a
The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-Minami, Tottori; bUbe Material Industries, 1985
Kogushi Ube, Yamaguchi; cYamaguchi Prefectural Agriculture and Forestry General Engineering Center, 1419 Ouchi-mihori, Yamaguchi;
and dFaculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, Japan
Bacterial wilt is a serious problem affecting many important food crops. Recent studies have indicated that treatment
with biotic or abiotic stress factors may increase the resistance of plants to bacterial infection. This study investigated
the effects of magnesium oxide nanoparticles (MgO NP) on disease resistance in tomato plants against Ralstonia sola-
nacearum, as well as its antibacterial activity. The roots of tomato seedlings were inoculated with R. solanacearum
and then immediately treated with MgO NP; the treated plants showed very little inhibition of bacterial wilt. In con-
trast, when roots were drenched with a MgO NP suspension prior to inoculation with the pathogen, the incidence of
disease was significantly reduced. Rapid generation of reactive oxygen species such as O2˙ radicals was observed in
tomato roots treated with MgO NP. Further O2˙ was rapidly generated when tomato plant extracts or polyphenols
were added to the MgO NP suspension, suggesting that the generation of O2˙ in tomato roots might be due to a reac-
tion between MgO NP and polyphenols present in the roots. Salicylic acid-inducible PR1, jasmonic acid-inducible
LoxA, ethylene-inducible Osm, and systemic resistance-related GluA were up-regulated in both the roots and
hypocotyls of tomato plants after treatment of the plant roots with MgO NP. Histochemical analyses showed that
b-1,3-glucanase and tyloses accumulated in the xylem and apoplast of pith tissues of the hypocotyls after MgO NP
treatment. These results indicate that MgO NP induces systemic resistance in tomato plants against R. solanacearum.
Keywords: induced resistance, magnesium oxide nanoparticles, Ralstonia solanacearum, reactive oxygen species, ROS,
Solanum lycopersicum
(ROS) such as superoxide radicals (O2˙), hydrogen per-

Introduction
oxide (H2O2), and hydroxyl radicals (OH˙) (Foyer
The soilborne vascular pathogen Ralstonia solanacearum et al., 1997). ROS not only limit pathogen ingress but
is a Gram-negative b-proteobacterium that causes bacterial also play a role in activating local and systemic defence
wilt in more than 200 plant species, including diverse and responses such as the induction of pathogenesis-related
important food crops such as tomato, potato, banana and protein (PR) genes (Henry et al., 2013). The plant hor-
ginger (Hayward, 1991). It is difficult to control bacterial mones salicylic acid (SA), jasmonic acid (JA) and ethy-
wilt with chemicals because the bacterium survives for lene (ET) play important roles in defence responses as
years in infested soils and weed hosts. So far, the most signalling molecules (Robert-Seilaniantz et al., 2011) and
effective method for controlling bacterial wilt disease is by are involved in two major pathogen-defence signalling
using resistant cultivars (Hayward, 1991). Recent studies pathways: the SA-mediated pathway and ET/JA-mediated
have raised the possibility of an alternative strategy by pathway, which are stimulated in response to biotrophic
using biotic and abiotic stress agents such as antagonistic and necrotrophic pathogens, respectively (Kunkel &
microbes (Sudisha et al., 2013; Yi et al., 2013), DL-3- Brooks, 2002).
aminobutyric acid (Hassan & Abo-Elyousr, 2013) and sili- Although information on the mechanisms underlying
con (Kurabachew & Wydra, 2014), which have the poten- defence responses against R. solanacearum is still limited,
tial to control bacterial wilt by inducing resistance in host several reports have indicated that SA, JA and ET sig-
plants against R. solanacearum. nalling are involved in resistance responses in tomato. A
Exposure to various biotic and abiotic stresses results transcriptome analysis showed that expression of genes
in the cellular accumulation of reactive oxygen species for JA/ET biosynthesis is increased in the resistant tomato
cultivar LS-89 in response to R. solanacearum infection
(Ishihara et al., 2012). Chen et al. (2009) reported that
*E-mail: shinsan@yamaguchi-u.ac.jp silencing of the genes associated with SA (NPR1, TGA2.2
and TGA1a), JA (COI1) and ET (ACO1/3, EIN2 and
ERF3) signalling led to increases in bacterial proliferation
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552 K. Imada et al.
signalling pathways, R. solanacearum infection induces was carried out using a JEM-2100 microscope (JEOL) at an
the generation of physical barriers, which play an acceleration voltage of 200 kV.
important role in disease resistance. For example,
R. solanacearum infection in the resistant tomato cultivar Bacterial isolate and growth conditions
Cara€ıbo induces the formation of tyloses, which are bal-
loon-like structures in the xylem and parenchyma that Ralstonia solanacearum was isolated from a naturally infected
occlude xylem and adjacent vessels that have been colo- tomato plant grown in Yamaguchi, Japan. A stored stock cul-
ture of the isolate was streaked on triphenyl tetrazolium chloride
nized by the bacterium (Grimault et al., 1994).
(TTC) medium and incubated at 30°C for 48 h (Kelman, 1954).
In addition to studies to elucidate innate plant defence A single colony was selected and cultured at 30°C on rich casa-
responses to bacterial infection, there is increasing inter- mino acids–peptone–glucose (CPG) medium (pH 70) for 48 h
est in externally applied treatments that might aid resis- (Kelman, 1954). Bacterial cells were suspended in sterile water
tance. Magnesium oxide nanoparticles (MgO NP) have and adjusted to approximately 108 colony-forming units
important industrial uses in pharmaceuticals, toxic waste (cfu) mL1 by measuring optical density with a spectrophotome-
remediation, toxic gas removal, paint and semiconduc- ter at 660 nm. The suspension was used to inoculate tomato
tors (Liang & Gay, 1986; Tsuji et al., 1994; Yang & plants.
Lieber, 1996; Bhargava et al., 1998; Hossain et al.,
2014). MgO NP can be prepared with extremely high Antibacterial activity of MgO NP
surface areas and crystal morphologies with numerous
edges/corners and reactive surface sites (Klabunde et al., Ralstonia solanacearum cultures were set up at 108 cfu mL1 in
1996; Stoimenov et al., 2002). Because of the unusual CPG medium containing two concentrations (005, 01%) of
MgO NP. Cultures were incubated at 30°C with constant shak-
characteristics of their structures, MgO NP can produce
ing at 120 rpm. Bacterial concentrations were determined by
ROS on their surfaces (Baird & Lunsford, 1972; Sawai dilution plating on TTC medium after 6 h incubation of the
et al., 1996), a remarkable property that attracts interest bacteria with MgO NP.
as a potential antibacterial treatment: MgO NP can inac-
tivate bacteria by the formation of O2˙ or through
adsorption of negatively charged bacteria on their posi- Plant materials and inoculation method
tively charged surfaces (Koper et al., 2002; Huang et al., Tomato (Solanum lycopersicum) plants (cultivar Momotaro)
2005; Jin & He, 2011). The frequency of contact were grown in small pots (75 cm diameter, 65 cm tall) con-
between bacterial cells and MgO particles determines the taining a mixture of vermiculite and perlite (1:1 mixture) at
bactericidal activity (Sawai et al., 2000). In comparison 25°C under a 12 h photoperiod with a photon flux density of
to other solid bactericides such as TiO2, supported silver 100 lM m2 s1 for 5 weeks. These 5-week old tomato seed-
or supported copper, MgO NP have advantages such as lings were used in all experiments in the present study.
being non-toxic and being easily prepared from readily In one set of experiments, the roots of 5-week-old tomato
seedlings were dipped into a bacterial suspension for 3 min. The
available, economical precursors. Therefore, MgO NP
inoculated seedlings were planted into small pots (as above) con-
have considerable potential for use as a solid bactericidal taining vermiculite and drenched immediately with 50 mL MgO
material (Huang et al., 2005). NP suspension (01 or 1%); the plants were then grown under
Despite their bactericidal activity, MgO NP have not the same conditions as described above. The numbers of
been examined for their utility in the control of bacterial R. solanacearum cells in the hypocotyls and roots of tomato
plant diseases, such as bacterial wilt in tomato. In addi- plants was determined by homogenizing plant samples and
tion, the effect of MgO NP on plants in terms of their spreading them on TTC medium.
potential to induce disease resistance has not been clari- In another set of experiments, each pot was drenched with
fied. As MgO NP form O2˙, which are induced during 50 mL MgO NP suspension (01, 05, 07 or 1%); the plants
the early stages of the plant resistance response, they were then allowed to grow for different periods of time (3–
10 days) under the same conditions. After the MgO NP treat-
may mimic the early stages of this resistance response.
ment, seedling roots were washed in water with shaking to
The aims of this study were to investigate the effects of remove MgO NP. For inoculation with R. solanacearum, the
MgO NP on disease resistance in tomato plants against seedling root was dipped into the bacterial suspension for
R. solanacearum and to identify possible mechanisms for 3 min. The inoculated seedlings were transferred to small pots
MgO NP-induced resistance. containing vermiculite and grown under the same conditions as
described above.
The disease severity was assessed by determining the propor-
Materials and methods tion of wilted leaves using the following scale: 0, no wilt symp-
toms; 1, 1–25% of leaves wilted; 2, 26–50% of leaves wilted; 3,
MgO nanoparticles and microscopic observations 51–75% leaves of wilted; and 4, 76–100% of leaves wilted.
MgO NP (UCM250) were obtained from Ube Material Indus-
tries. In all experiments, MgO NP were suspended in distilled In vivo and in vitro detection of ROS
water, and the suspension was ultrasonicated for 5 min before
use. For transmission electron microscope (TEM) observation, The generation of O2˙ in vivo was detected using p-nitrotetra-
5 mg MgO NP were mixed with 10 mL ethanol (99%). Samples zolium blue (NBT) staining. Roots of 5-week-old tomato seed-
were mounted on hexagonal copper grids. TEM observation lings were dipped in a 1% MgO NP suspension for 6 h and
Plant Pathology (2016) 65, 551–560

MgO-induced systemic resistance 553
incubated in a solution containing 01% NBT and 10 mM a graded series of t-butyl alcohol–ethanol, and then embedded
sodium azide in 50 mM potassium phosphate buffer (pH 78; in paraffin. Transverse sections, 14–18 lm thick, were cut using
NBT) for 20 min. O2˙ production was visualized as a blue for- a rotary microtome. Paraffin was removed in Histo-Clear
mazan deposit within the root tissues and was observed under a (National Diagnostics) and sections were hydrated in a graded
light microscope (BH-2; Olympus). series of ethanol to water.
For in vitro detection of O2˙, formazan deposition was b-1,3-glucanase was detected as described by Ishihara et al.
measured according to the assay described by Grellet Bournon- (2012) with some modifications. Briefly, deparaffinized sections
ville & Dıaz-Ricci (2011) with some modifications. Briefly, 1% were immersed in 15% acetic acid for 15 min and washed twice
MgO NP suspension, solutions of polyphenols (20 lg mL1) in Tris-buffered saline (pH 74; TBS). Sections were incubated
and NBT (as above) were mixed in equal volumes (100 lL for 1 h in blocking solution containing 2% bovine serum albu-
each) and incubated for 30 min at room temperature. The min (BSA) and 005% Tween 20 in TBS, then for 1 h in pri-
polyphenols p-coumaric acid, rutin, quercetin and tannic acid mary antibodies raised against the tobacco b-1,3-glucanase PR-
were obtained from Wako Pure Chemical. After incubation, N (Niki et al., 1998) (1:3000) in TBS-BSA (03% BSA, 005%
lactic acid was added to dissolve the MgO NP. Deposited for- sodium azide in TBS). Afterwards, the sections were washed and
mazan was quantified as described by Grellet Bournonville & incubated with alkaline phosphatase-conjugated anti-rabbit sec-
Dıaz-Ricci (2011). ondary antibodies (Wako Pure Chemical; 1:5000 in TBS-BSA)
Hydrogen peroxide concentrations in vitro were measured for 1 h. For colourimetric detection, sections were incubated
using a Bioxytech H2O2-560 Quantitative Hydrogen Peroxide with NBT and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)
Assay (Oxis International) in accordance with the manufac- (Wako Pure Chemical) for 30 min and observed under an all-in-
turer’s protocol. one fluorescence microscope BZ-9000 (Keyence).
Tylose formation was examined in deparaffinized sections that
had been incubated with 05% calcofluor (Fluorescent brightener
RNA extraction and quantitative reverse transcription 28, Wako Pure Chemical) for 30 min. The sections were then
PCR mounted in 50% glycerol on microscope slides and analysed using
a BZ-9000 microscope at an excitation wavelength of 360 nm
Tomato seedlings (5-weeks old) were drenched with 50 mL
07% MgO NP suspension and grown under the same condi- and using a band-pass filter with 435–485 nm emission.
tions as described above. Roots and upper hypocotyls of the The Vectastain ABC horseradish peroxidase staining kit (rab-
plants were harvested at 12, 24, 72 and 120 h after MgO NP bit IgG) (Vector Laboratories) and primary antibodies against
R. solanacearum (d’Ursel et al., 1999) (1:3000 in TBS) were
treatment, frozen in liquid nitrogen, and stored at 80°C until
use. Total RNA was extracted using Sepasol-RNA I Super G used for immunohistochemical localization of R. solanacearum
(Nacalai Tesque). The total RNA concentration was determined cells in tomato hypocotyl tissues using 3,30 -diaminobenzidine
using a Biospec-nano spectrophotometer (Shimadzu). Reverse tetrahydrochloride (Wako Pure Chemical) as the substrate.
transcription (RT) was performed using 1 lg total RNA in a
20 lL reaction volume with the ReverTra Ace qPCR RT Master Statistical analysis
Mix with gDNA Remover (Toyobo) according to the manufac-
turer’s instructions. The cDNA was diluted (1:1) and 1 lL was The experimental data were represented as standard error of the
used as a template in 20 lL total volume of THUNDERBIRD mean (SEM). Statistical significance of differences between mean
SYBR qPCR Mix (Toyobo). Gene-specific primers were designed values was determined with Student’s t-test. Significant differ-
using PRIMER EXPRESS (Applied Biosystems) to amplify 70– ences at the 005 and 001 probability levels are marked in the
150 bp fragments using sequences in GenBank: pathogenesis-re- figures by single and double asterisks, respectively.
lated protein 1 (PR1, M69247), lipoxygenase A (LoxA,
U09026), osmotin-like protein (Osm, M21346), b-1,3-glucanase
A (GluA, M80604), and Actin as the reference gene (FJ532351). Results
The following primers were used for quantitative RT-PCR: PR1
forward 5ʹ-TGTTGTTTCCCTTTGATGTTGCT-3ʹ, reverse 5ʹ-A TEM analysis of MgO NP
ACCTAAGCCACGATACCATGAA-3ʹ; LoxA forward 5ʹ-AAA
TEM analysis showed that the MgO NPs in the present
CAGAACAGGCCCCGTTA-3ʹ, reverse 5ʹ-GCCTGTAAGTCCA
CCTTCACTTG-3ʹ; Osm forward 5ʹ-TGTACCACGTTTGGAG
study were composed of particles with an average diame-
GACA-3ʹ, reverse 5ʹ-ACCAGGGCAAGTAAATGTGC-3ʹ ter of c. 100 nm (20–200 nm; Fig. 1a). The lattice
(Milling et al., 2011); GluA forward 5ʹ-TTTTGGCCATGCTGA fringes of the MgO NP crystal were observed in the clus-
TGATAAT-3ʹ, reverse 5ʹ-TGCATCGTTTAGCCCTTGTTG-3ʹ; ter of MgO NP (Fig. 1b).
and Actin forward 5ʹ-TGTCCCTATCTACGAGGGTTATGC-3ʹ,
reverse 5ʹ-CAGTTAAATCACGACCAGCAAGA-3ʹ. Real-time
quantitative PCR was performed using a 7300 system (Applied In vitro antibacterial activity of MgO NP
Biosystems). Relative quantification was conducted using the MgO NP, at both concentrations of 005 or 01%, showed
DDCt method (Livak & Schmittgen, 2001). strong antibacterial activity against R. solanacearum cul-
tured in liquid medium (Fig. 2).
Histochemical analysis
Tomato seedlings were subjected to MgO NP treatment (07%) Effect of MgO NP on disease resistance in tomato
and grown as described above. Upper hypocotyls (5 mm sec- against R. solanacearum
tions) were excised at 7 days after MgO NP treatment and fixed
in 5% glutaraldehyde in cacodylate buffer (pH 70). Fixed sam- To determine whether the bactericidal activity of MgO NP
ples were immersed in t-butyl alcohol after dehydration through could alter disease development in tomato plants, seedling

554 K. Imada et al.
(a) (b)
Figure 1 Transmission electron microscope

images of magnesium oxide nanoparticles.
Bars indicate 200 nm (a) and 20 nm (b),
respectively.
observed in seedlings pretreated with 05, 07 and 1%

MgO NP suspension (data not shown).
Pretreatment with MgO NP significantly reduced the
size of the bacterial population in hypocotyls compared
to control plants (Fig. 3c). Although the difference in
population sizes between pretreated and control roots
was not statistically significant, the treated roots consis-
tently had lower bacterial populations (Fig. 3c).
In vivo and in vitro detection of ROS generation

To investigate the potential of MgO NP to generate ROS
in vivo, we analysed O2˙ production in tomato plants
treated with MgO NP. Colourless NBT precipitates as a
blue-purple formazan when reduced by O2˙. Blue-purple
precipitates were greatly increased in tomato roots by
Figure 2 Antibacterial activity of magnesium oxide nanoparticles (MgO
treatment with 1% MgO NP compared to controls
NP) against Ralstonia solanacearum in vitro. Bacterial viability was
determined by dilution plating on triphenyl tetrazolium chloride medium
(Fig. 4a,b). As ROS are generated on the surface of
after 6 h incubation of the bacteria with MgO NP. Data represent the MgO NP (Baird & Lunsford, 1972; Sawai et al., 1996),
average of three biological replicates SE. Data sets marked with attempts were made to detect their production in MgO
asterisks are significantly different (*P ˂ 005, Student’s t test) from the NP suspensions using colourimetric indicators, namely
water-treated control (0%). N.D., not detected. xylenol orange for H2O2 and NBT for O2˙. Neither
H2O2 nor O2˙ was detected in the MgO NP suspensions
(data not shown).
roots were treated with MgO NP immediately after inocu- To test the hypothesis that compounds in the plants
lation with R. solanacearum. The R. solanacearum cells might react with MgO NP to generate ROS, various plant
were expected to be on the surfaces of the roots when they materials, such as total lipids, proteins and polyphenols
were treated with MgO NP, and therefore likely to be in [extracted from 200 mg fresh tomato leaves, dissolved in
contact with the MgO NP at a high frequency. Bacterial 100 lL ethanol (lipids) or phosphate-buffered saline (pro-
wilt symptoms were not suppressed using 1% MgO NP, teins and polyphenols)], were added to MgO NP suspen-
and were slightly suppressed using 01% MgO NP com- sions and then O2˙ production measured using NBT.
pared to untreated controls (Fig. 3a). Formazan deposition was detected when purified polyphe-
To determine whether MgO NP induced disease resis- nols were mixed with the MgO NP (Fig. 4c). Formazan
tance in tomato plants, seedlings were pretreated with deposition appeared to be dependent on the number of
MgO NP for different periods; they were then washed hydroxyl residues in the polyphenol molecule.
with water and inoculated with R. solanacearum. In this
treatment, the bacterial cells were not expected to be in
Expression of defence-related genes in MgO NP-treated
contact with MgO NP as there would have been few NP
plants
on root surfaces. Plants grown in 07% MgO NP for
7 days before inoculation showed significant suppression Changes in expression of defence-related genes were
of bacterial wilt symptoms between 4 days post-inocula- investigated using quantitative RT-PCR. Relative levels
tion (dpi) and 8 dpi (Fig. 3b). Seedlings exposed to MgO of expression of PR1, LoxA, Osm and GluA were
NP for less than 7 days before inoculation showed less assessed in roots and hypocotyls of plants at 12, 24, 72
suppression of wilt symptoms. However, treatments of and 120 h after treatment with 07% MgO NP or water
more than 10 days caused partial necrosis in the roots (control). The level of expression of PR1 in roots
(data not shown). Suppression of disease symptoms was increased at 12 h after treatment and peaked at 72 h

Figure 3 Effect of magnesium oxide

nanoparticles (MgO NP) on disease
resistance in tomato plants against Ralstonia
solanacearum. (a) Progression of bacterial
wilt symptoms in tomato plants treated with
01% MgO NP, 1% MgO NP, or water
(control) immediately after inoculation with
R. solanacearum. Data are means of three
plants SE. (b) Progression of bacterial wilt
symptoms in tomato plants pretreated with a
drench of 07% MgO NP or distilled water
(control) 7 days before inoculation. Data are
means of six plants SE. (c) Bacterial
numbers in hypocotyls and roots of plants,
pretreated 7 days before inoculation with a
drench of 07% MgO NP, at 9 days post-
inoculation with R. solanacearum. Data are
means of six plants SE. Error bars indicate
standard errors from at least two
independent experiments. Data sets marked
with asterisks are significantly different from
control as assessed by a Student’s t test;
*P ˂ 005 and **P ˂ 001. N.D., not detected.
(Fig. 5a); expression in the hypocotyls was also signifi- of fluorescence in the cell walls of root and hypocotyl tis-
cantly increased over the control by 72 h after treatment sues of MgO NP-treated plants compared to controls
(Fig. 5b). Expression of LoxA increased in both roots (Fig. 7). Granular structures were also stained by the cal-
and hypocotyls at 72 h after treatment (Fig. 5c,d). Osm cofluor dye (Fig. 7b,d).
showed a small increase in expression at 12 and at 24 h Ralstonia solanacearum was detected in xylem vessels
in treated roots (Fig. 5e), and increased from 72 h after and the apoplast of pith tissues of untreated controls
treatment in the hypocotyls (Fig. 5f). Expression of GluA (Fig. 8a,b). In contrast, R. solanacearum was absent
in roots increased significantly at 12 h after treatment from the apoplast of pith tissues following exposure of
and then declined (Fig. 5g); in hypocotyls, expression roots to MgO NP (Fig. 8d). Additionally, tylose accumu-
increased at 120 h after treatment (Fig. 5h). The results lation around R. solanacearum was observed in xylem
indicated that expression of all these defence-related vessel tissue of MgO NP-treated plants (Fig. 8c).
genes was enhanced by MgO NP, not only in the roots
(the MgO NP-treated tissue) but also in the hypocotyls
Discussion
(untreated tissue).
The results of the present study demonstrate that MgO
NP have strong antibacterial activity against
Histochemical analysis of tomato hypocotyl tissues
R. solanacearum in vitro. This finding is consistent with
An anti-PR-N (class II acidic b-1,3-glucanase) polyclonal previous studies that showed that MgO NP are effective
antibody was used to detect b-1,3-glucanase in hypocotyl for killing some Gram-positive and Gram-negative bacte-
tissues of tomato plants after treatment of the roots with ria (Koper et al., 2002; Jin & He, 2011). Although the
MgO NP. There was clear evidence of accumulation of mechanism of the bactericidal activity of MgO NP is
b-1,3-glucanase in the hypocotyls of the MgO NP-treated poorly understood, the generation of ROS on the sur-
plants (Fig. 6b,d,f) compared with water-treated plants faces of the MgO NP and the induction of oxidative
(Fig. 6a,c,e). The b-1,3-glucanase signal was especially stress may be involved in the antimicrobial activity. An
prominent in the xylem and apoplast of pith tissues alternative mechanism was recently suggested by Leung
(Fig. 6f). There was a marked increase in granular struc- et al. (2014), who found that membrane damage proba-
tures in the xylem and pith cells at 7 days after treat- bly occurs due to a combination of the attachment of the
ment with MgO NP (Fig. 6d). nanoparticles and effects such as pH change and Mg2+
Calcofluor dye was used to detect tyloses in the hypo- release, even in the absence of ROS production. In the
cotyl tissues of the plants. There was an increased level present study, it was hypothesized that MgO NP might

556 K. Imada et al.
pendent gene expression. For example, the application of

the bioorganic fertilizer Bacillus amyloliquefaciens T-5
reduces the incidence of tomato wilt; additionally, the
expression of marker genes for JA (PIN2) and ET
(PR1b) in the hypocotyls of tomato plants treated with
the fertilizer was significantly higher in response to inoc-
ulation with R. solanacearum (Tan et al., 2013). Silicon
treatment can also inhibit bacterial wilt in tomatoes.
Thus, JA/ET marker genes encoding jasmonate- and
ethylene-responsive factor 3 and 1-aminocyclopropane-1-
carboxylate oxidase are induced upon challenge of sili-
con-treated plants with R. solanacearum (Kurabachew &
Wydra, 2014). Genes involved in ET- and SA-dependent
signalling pathways are activated in a tomato cultivar
that shows increased resistance to R. solanacearum
(Milling et al., 2011). In the present study, the expres-
sion of a selection of genes in the SA-, JA- and ET-sig-
nalling pathways was activated, although in different
patterns, after treatment of the plants with MgO NP.
The results suggest that the mechanisms underlying MgO
NP-induced resistance in tomato may share common fea-
tures with those involved in resistance induced by bio-
organic fertilizer or silicon and with those in resistant
cultivars. A notable feature of MgO NP-induced resis-
tance is that genes involved in the resistance responses
against R. solanacearum were induced in the absence of
the pathogen.
Treatment of plant roots with MgO NP caused a rapid
generation of O2˙. O2˙ generation also occurred when
MgO NP came into contact with tomato plant extract or
polyphenols. These results suggest that the generation of
Figure 4 In vivo and in vitro detection of ROS generation induced by
O2˙ in tomato roots was due to a reaction between
magnesium oxide nanoparticles (MgO NP) treatment. Microscopic
MgO NP and polyphenols in the roots. Polyphenols have
images of formazan deposition after p-nitrotetrazolium blue (NBT)
staining of tomato roots treated with distilled water (control) (a) or 1%
the potential to act as pro-oxidants, and can generate
MgO NP (b). (c) Quantification of formazan formation after addition of radicals as electrophiles under certain conditions (Saki-
NBT to a mixture of various polyphenols and MgO NP. Bar: 50 lm. hama et al., 2002). MgO is a solid base catalyst that can
N.D., not detected. This experiment was repeated three times with act as a mediator of deprotonation (hydrogen atom
similar results. acceptor; Lamb et al., 1988). Therefore, it is proposed
that ROS generation in tomato roots treated with MgO
NP might be phenoxyl radicals produced by deprotona-
suppress bacterial wilt in tomato plants by killing tion of phenolic hydroxyls. In general, a striking increase
R. solanacearum cells. However, MgO NP treatment in ROS occurs as a resistance response in plants to
immediately after R. solanacearum inoculation showed pathogen attack. Rapid generation of ROS following
very little inhibition of disease development, suggesting recognition of R. solanacearum has been observed in the
that suppression of bacterial wilt could not be explained resistant tomato cultivar BT-10 (Mandal et al., 2011).
by the bactericidal activity of MgO NP. In contrast, pre- Thus, the rapid generation of O2˙ or phenoxyl radicals
treatment of tomato roots with MgO NP significantly in tomato roots treated with MgO NP might play a simi-
inhibited development of bacterial wilt, suggesting that lar role in the resistance response of tomatoes against
MgO NP induced resistance in tomato plants against R. solanacearum.
R. solanacearum. Ralstonia solanacearum colonizes the xylem and apo-
Several biotic and abiotic stresses can induce systemic plast of pith tissues in the tomato hypocotyl. Ishihara
resistance against R. solanacearum via phytohormone-de- et al. (2012) reported that in the resistant tomato culti-
Figure 5 Expression patterns of defence marker genes in response to treatment with 07% magnesium oxide nanoparticles (MgO NP). Expression
of PR1 (a, b), LoxA (c, d), Osm (e, f), and GluA (g, h) was assessed in roots (a, c, e, g) and hypocotyls (b, d, f, h) of tomato plants. Error bars
indicate standard errors (n = 4). Data marked with asterisks are significantly different from the distilled water-inoculated control (Student’s t test);
*P ˂ 005; **P ˂ 001.

557
MgO-induced systemic resistance

558 K. Imada et al.
(a) (b)
(c) (d)
Figure 6 Immunohistochemical detection of

b-1,3-glucanase in hypocotyl tissues of
tomato plants following treatment of their
roots with magnesium oxide nanoparticles
(e) (f)
(MgO NP). Hypocotyl sections using a b-1,3-
glucanase antibody 7 days after treatment
with distilled water (a, c, e) or 07% MgO NP
suspension (b, d, f). The bluish-purple colour
indicates a positive reaction. Light
microscopic images of hypocotyl (a, b), pith
(c, d) and xylem (e, f) tissues. Arrows in (d),
b-1,3-glucanase localization in the apoplast
of pith tissues. Arrowheads in (d) and (f),
granular structures. Bars: (a) and (b) = 50
lm, (c) to (f) = 100 lm.
(a) (b)
(c) (d)
Figure 7 Fluorescence microscope detection

of tylose-like structures in hypocotyl tissues.
Calcofluor-stained hypocotyl tissue sections
of tomato plants treated with distilled water
(a, c) or 07% magnesium nanoparticles
(MgO NP) suspension (b, d). Bright-field (a,
b) and monochromatic fluorescent images
(c, d) of pith tissues are shown. Arrowheads,
tylose-like structures. Bar: 50 lm.

(a) (b)
Figure 8 Immunohistochemical detection of

Ralstonia solanacearum in hypocotyl tissues
of tomato plants using an anti-
R. solanacearum antibody 7 days after
treating the roots with distilled water (a, b) or (c) (d)
07% magnesium oxide nanoparticles (MgO
NP) suspension (c, d). The brown colour
(arrows) indicates the location of R.
solanacearum. Colonies were detected in
xylem vessels and apoplast of pith tissues in
water-treated control (a, b); no colonies were
observed in the apoplast of pith tissues in
MgO NP-treated plants (d). Tyloses are
present around bacterial colonies in a xylem
vessel (c) and by cell walls in pith tissue (d)
in MgO NP-treated plants. Bars: 20 lm.
var LS-89, b-1,3-glucanase accumulated in the xylem might be triggered by phenoxyl radicals generated via
and pith tissues surrounding xylem vessels colonized by deprotonation of phenolic hydroxyl residues of polyphe-
R. solanacearum. The authors suggested that a defence nols in the roots treated with MgO NP. MgO NP is
response involving b-1,3-glucanase accumulation might harmless to humans and to the environment, and the
play an important role in preventing bacterial movement cost of the compound is suitable for farmers. Therefore,
towards the outside of the xylem vessels. In the current MgO NP may be a potential agent for the control of
study, a significant increase in b-1,3-glucanase in the bacterial wilt in tomato. Further study will be necessary
xylem and apoplast of pith tissues of hypocotyls was using other tomato cultivars with different susceptibilities
observed in plants treated with MgO NP. This finding to R. solanacearum to determine whether MgO NP is
suggests that the resistance response induced by MgO also effective in these plants. Preliminary results from a
NP was similar to that of LS-89. Tylose accumulation in field study have shown that MgO NP might suppress
xylem and pith tissues in hypocotyls of MgO NP-treated bacterial wilt in tomato cultivars other than Momotaro.
tomatoes was also observed. Tylose formation results
from metabolic changes in vessel-associated parenchyma
cells, causing protrusions into the xylem vessels through Acknowledgements
pits; these protrusions can block the spread of pathogens The authors thank Ichiro Mitsuhara (National Institute
(Talboys, 1972; Grimault et al., 1994). Tylose formation of Agrobiological Sciences, Tsukuba, Ibaraki, Japan) for
is a common defence response in xylem vessels against kindly providing the anti-PR-N antibody and Dr K.
vascular wilt pathogens such as Fusarium oxysporum, Tuchiya (Kyusyu University, Fukuoka, Japan) for kindly
Verticillium albo-atrum and R. solanacearum (Hutson & providing polyclonal antibodies for R. solanacearum.
Smith, 1980; Rahman et al., 1999). In the resistant
tomato cultivar Cara€ıbo, tyloses occlude colonized xylem
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Plant Pathology (2016) 65, 551–560 Doi: 10.1111/ppa.

Magnesium oxide nanoparticles induce systemic resistance

K. Imadaa, S. Sakaib, H. Kajiharac, S. Tanakad and S. Itod*

(ROS) such as superoxide radicals (O2˙), hydrogen per-

ª 2015 British Society for Plant Pathology 551

Plant Pathology (2016) 65, 551–560

Plant Pathology (2016) 65, 551–560

Figure 1 Transmission electron microscope

observed in seedlings pretreated with 05, 07 and 1%

In vivo and in vitro detection of ROS generation

Plant Pathology (2016) 65, 551–560

Figure 3 Effect of magnesium oxide

Plant Pathology (2016) 65, 551–560

pendent gene expression. For example, the application of

Plant Pathology (2016) 65, 551–560

Plant Pathology (2016) 65, 551–560

Figure 6 Immunohistochemical detection of

Figure 7 Fluorescence microscope detection

Plant Pathology (2016) 65, 551–560

Figure 8 Immunohistochemical detection of

Plant Pathology (2016) 65, 551–560

Plant Pathology (2016) 65, 551–560

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