published: 28 September 2015
Lomonosov Moscow State University,
Consiglio Nazionale delle Ricerche,
University of Torino, Italy
College of Life Sciences, Fujian
Agriculture and Forestry University,
Fuzhou 350002, China
This article was submitted to
Plant Biotic Interactions,
a section of the journal
Frontiers in Plant Science
Received: 30 July 2015
Accepted: 11 September 2015
Published: 28 September 2015
Song Y, Chen D, Lu K, Sun Z
and Zeng R (2015) Enhanced tomato
disease resistance primed by
arbuscular mycorrhizal fungus.
Front. Plant Sci. 6:786.
Enhanced tomato disease resistance
primed by arbuscular mycorrhizal
Yuanyuan Song1,2, Dongmei Chen1,KaiLu
1, Zhongxiang Sun1and Rensen Zeng1,2 *
1College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China, 2State Key Laboratory of Conservation
and Utilization of Subtropical Agro-Bioresources, College of Agriculture, South China Agricultural University, Guangzhou,
Roots of most terrestrial plants form symbiotic associations (mycorrhiza) with soil- borne
arbuscular mycorrhizal fungi (AMF). Many studies show that mycorrhizal colonization
enhances plant resistance against pathogenic fungi. However, the mechanism of
mycorrhiza-induced disease resistance remains equivocal. In this study, we found
that mycorrhizal inoculation with AMF Funneliformis mosseae signiﬁcantly alleviated
tomato (Solanum lycopersicum Mill.) early blight disease caused by Alternaria solani
Sorauer. AMF pre-inoculation led to signiﬁcant increases in activities of β-1,3-glucanase,
chitinase, phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX) in tomato
leaves upon pathogen inoculation. Mycorrhizal inoculation alone did not inﬂuence the
transcripts of most genes tested. However, pathogen attack on AMF-inoculated plants
provoked strong defense responses of three genes encoding pathogenesis-related
proteins, PR1,PR2, and PR3, as well as defense-related genes LOX,AOC, and PA L ,in
tomato leaves. The induction of defense responses in AMF pre-inoculated plants was
much higher and more rapid than that in un-inoculated plants in present of pathogen
infection. Three tomato genotypes: a Castlemart wild-type (WT) plant, a jasmonate
(JA) biosynthesis mutant (spr2), and a prosystemin-overexpressing 35S::PS plant were
used to examine the role of the JA signaling pathway in AMF-primed disease defense.
Pathogen infection on mycorrhizal 35S::PS plants led to higher induction of defense-
related genes and enzymes relative to WT plants. However, pathogen infection did not
induce these genes and enzymes in mycorrhizal spr2 mutant plants. Bioassays showed
that 35S::PS plants were more resistant and spr2 plants were more susceptible to
early blight compared with WT plants. Our ﬁnding indicates that mycorrhizal colonization
enhances tomato resistance to early blight by priming systemic defense response, and
the JA signaling pathway is essential for mycorrhiza-primed disease resistance.
Keywords: defense priming, arbuscular mycorrhizal fungus, induced disease resistance, systemic defense
responses, jasmonate pathway, tomato
Frontiers in Plant Science | www.frontiersin.org 1September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
In response to various abiotic stresses and biotic attack by
herbivorous insects or pathogens, plants have evolved an
array of sophisticated strategies to protect themselves against
these agents. One strategy is the ability of plant root systems
to form mycorrhizal associations, which are mutualistic and
reciprocally beneﬁcial symbiotic relationships between plant
roots and some speciﬁc soil-borne fungi. Mycorrhizal fungi
are the most important symbionts for the majority of plant
species in terrestrial ecosystems (Smith and Read, 2008). It has
been estimated that over 90% of land plants form arbuscular
mycorrhizas (AM) with fungi belonging to the phylum
Glomeromycota (Brundrett, 2002). The presence of widespread
extra radical mycelium networks of mycorrhizal fungi in soils
helps plants to acquire nutrients and water in soils which plant
roots can not reach. Mycorrhizal associations facilitated the
plant colonization on land (Redecker et al., 2000). Mycorrhizas
also inﬂuence plant physiology (Smith et al., 2010)andsoil
structure (Rillig and Mummey, 2006;Fellbaum et al., 2012), as
well as a series of important ecosystem processes, including plant
diversity, nutrient cycling, and ecosystem productivity (van der
Heijden et al., 1998;Vogelsang et al., 2006;Cheng et al., 2012).
Numerous studies have proven that arbuscular mycorrhiza
fungi (AMF) enhance plant resistance against various pathogens
(Harrier and Watson, 2004;Pozo et al., 2005;Bi et al., 2007).
Mycorrhiza colonization of onion (Allium cepa)byFunneliformis
mosseae (syn. Glomus mosseae) signiﬁcantly alleviated the pink
root disease caused by Pyrenochaeta terrestris (Saﬁr, 1968).
The verticillium wilt was signiﬁcantly reduced in cotton plants
colonized by AMF, F. mosseae,G. versiforme, and Sclerocystis
sinuosa (Liu, 1995). Mycorrhizal colonization improved tomato
resistance to an array of diseases caused by Erwinia carotovora
(García-Garrido and Ocampo, 1988), Fusarium oxysporum
f. sp. lycopersici (Akköprü and Demir, 2005), Phytophthora
nicotianae var. parasitica (Cordier et al., 1996), P. parasitica
(Cordier et al., 1998), and Pseudomonas syringae (García-Garrido
and Ocampom, 1989). Mycorrhizal symbiosis also enhanced
tomato resistance to foliar disease of early blight (Fritz et al.,
2006). Common mycorrhizal networks between tomato plants
conferred protection of neighbors against early blight (Song et al.,
2010). Use of AMF provides a sustainable alternative for crop
disease management (Liu et al., 2007;Elsen et al., 2008). However,
the underlying mechanism of AMF-induced disease resistance
remains elusive. A signiﬁcant transcriptional reprogramming
occurs in host plant upon mycorrhizal colonization (López-Ráez
et al., 2010;Jung et al., 2012). The induction of plant defenses
during mycorrhization plays a vital role in mycorrhiza-enhanced
resistance (Pozo and Azcón-Aguilar, 2007;Jung et al., 2012).
Colonization or infection by certain beneﬁcial microbes or
necrotizing pathogens provokes a speciﬁc physiological state in
plants called “priming” (Hao et al., 2012;Aimé et al., 2013). The
primed state in plants can also be induced by various natural
and artiﬁcial compounds, such as β-aminobutyric acid (BABA),
jasmonic acid (JA), and salicylic acid (SA) (Jakab et al., 2001;
Worrall et al., 2012). The primed plants show quicker and/or
stronger induction of various cellular defense responses following
exposure to either pathogens herbivore insects, or abiotic stress
(Kuc, 1987;Ton et al., 2006;Jung et al., 2009;Slaughter et al.,
2012;Ye et al., 2013). Recent studies demonstrate that the defense
priming in Arabidopsis thaliana plants can be transferred to
their progeny, conferring better protection from pathogen attack
(Slaughter et al., 2012). Tomato plants grown from seeds treated
with JA and BABA displayed enhanced resistance against insect
herbivory and powdery mildew disease (Worrall et al., 2012).
The objectives of this study are to investigate the eﬀects of
pre-inoculation of tomato plants with Funneliformis mosseae on
resistance to early blight disease caused by Alternaria solani, as
well as on defense responses in pre-inoculated and un-inoculated
tomato plants. We hypothesizedthatAMFpre-colonization
primes tomato plants and initiates host defense response upon
subsequent pathogen attack. In general, SA signaling triggers
resistance against biotrophic and hemibiotrophic pathogens,
whereas the JA pathway activates resistance against necrotrophic
pathogens (Glazebrook, 2005;Robert-Seilaniantz et al., 2011).
Since the pathogen A. solani exhibits a necrotrophic lifestyle, we
examined the role of the JA pathway in AMF-induced priming
in tomato by using transgenic tomato plants that overexpress
the prosystemin gene (35S::PS) and plants with a mutation in
the JA biosynthetic pathway (spr2). In tomato plants, systemic
induction of JA-dependent defense responses is mediated by
an 18-amino-acid peptide signal called systemin (Howe and
Ryan, 1999). Tomato transgenic line 35S::prosystemin (35S::PS)
that overexpress prosystemin, the systemin precursor, exhibit
constitutive expression of several JA-regulated defensive proteins
including proteinase inhibitors and polyphenol oxidase (Chen
et al., 2006).
Materials and Methods
Tomato plants (S. lycopersicum Mill. cv. Jin Bao) were inoculated
with mycorrhizal fungus Funneliformis mosseae (syn. G. mosseae)
Gerdemann & Trappe BEG 167. A. solani Sorauer (ACCC36110)
was inoculated to cause tomato early blight disease. Two tomato
plants were cultivated in a rectangular pot (24 cm in length, 18 cm
in height, 12 cm in width). To examine eﬀects of mycorrhizal
colonization on pathogen infection and tomato defense response,
we designed four treatments (CK, As, Fm, and Fm+As): (1) CK:
control plants without AMF and pathogen inoculation; (2) As:
plants inoculated with A.solani only; (3) Fm: plants inoculated
with F. mosseae only; (4) Fm+As: plants inoculated with
F. mosseae and later challenged with A.solani. For mycorrhizal
inoculation, the sand substrate (100 g) containing the inoculum
of F. mosseae was applied to each plastic pot in treatments Fm and
Fm+As before sowing. Leaves of tomato plants were harvested
18, 65, 100, and 140 h after pathogen inoculation for real-time
RT-PCR and enzymatic analysis.
Plant and Fungal Materials
Tomato seeds were sterilized with H2O2(10%) for 10 min and
rinsed ﬁve times with sterile distilled water. The seeds were then
sowed in autoclaved sand-soil mixture (1:1).
Frontiers in Plant Science | www.frontiersin.org 2September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
The inocula of F. mosseae were propagated by using corn
plants (Zea mays L.) cultured in autoclaved sand (Chellappan
et al., 2002). A mixture of corn roots and rhizospheric sand from
trap cultures containing approximate 35 AMF propagules per
gram was used for AM inoculation.
The pathogen was cultured for 6 day on potato dextrose broth
with 100 mg/l streptomycin sulfate, at 28◦Cindarknessandon
a shaker at 150 rpm. Then the fungal culture was centrifuged
at 1000 g, re-suspended in sterilized distilled water, and re-
centrifuged. The concentration of AMF spores was measured and
adjusted to 106conidia per milliliter using a hemacytometer.
To reveal the role of the jasmonate (JA) signaling pathway
in mycorrhiza-induced systemic priming of disease resistance
against A. solani, both overexpressing 35S::prosystemin and
defective spr2 mutant lines, as well as their corresponsive wild-
type (WT) tomato plants, were used to compare their diﬀerential
responses to A. solani infection after mycorrhizal colonization
by AMF F. mosseae. Tomato cv Castlemart was used as the
WT parent, the mutant line suppressor of prosystemin-mediated
responses2 (spr2) was derived from cv Castlemart (Li et al., 2003).
The 35S::PS transgenic plants were developed from the seeds
collected from a 35S::prosys/35S::prosys homozygous line (Chen
et al., 2006) that was backcrossed ﬁve times using the recurrent
parent cv Castlemart.
To determine mycorrhizal colonization on tomato disease
resistance, a bioassay was carried out to compare the disease
incidence and disease severity index (see deﬁnitions below)
between non-mycorrhizal and mycorrhizal tomato plants. The
brown loam soil collected from the campus of South China
Agricultural University in Guangzhou (China) contained 1.52%
organic matter, 0.789 g/kg total N, 0.42 g/kg total P, 1.76 g/kg
total K, 35.93 mg/kg available N, 1.30 mg/kg available P, and
37.14 mg/kg available K with a pH of 4.68. The soil autoclaved
at 121◦C for 2 h was mixed with sterilized sand at a ratio of
2:1. The mixture was used as culture medium of tomato plants.
The inocula (225 g) of AMF F. mosseae were incorporated into
the obtained mixture (1.5 kg) for mycorrhizal inoculation. The
same amount of mixture (1.5 kg) and sterilized sand (225 g)
was applied to each non-mycorrhizal control pot. The control
pots were watered with a soil ﬁltrate obtained by shaking non-
pasteurized rhizospheric sand with sterilized water then ﬁltering
it through a Watman No 1 ﬁlter, to exclude possible eﬀect of
other soil microorganisms. The ﬁltrate contained the natural soil
microbial population without AMF inocula.
Two pre-germinated tomato seeds were transplanted into
each plastic pot with the growth substrate. Ten days later, the
seedlings were thinned to one plant per pot. The plants were
grown in a growth chamber at 25 ±1◦Cwitha16hphotoperiod,
150 Md/m2/s PAR and 60% relative humidity. The seedlings were
watered daily and fertilized every 5 days with 50 mL of Hoagland
nutrient solution (5 ml 1 M KNO3,5ml1MCa(NO
2PO4, 1 ml 46 mM H3BO3,1ml
11 mM MnCl2,1ml1mMZnSO
4, 1 ml 3.5 mM CuSO4and
1 ml 17.7 mM FeEDTA in one liter water). Thirty-ﬁve days
after transplanting, tomato leaves in each pot were carefully
sprayed with 30 ml of a conidia suspension (106conidia/ml) of
A.solani. All plants were covered with an air-tight plastic bag
during pathogen infection to maintain the high relative humidity
facilitating spore germination.
The incidence and severity of tomato early blight were
measured 10 d post pathogen inoculation. Disease incidence
was indicated by percentage of diseased tomato leaves. Disease
severity was estimated using a Disease Index (DI) calculated
from disease grades 0–5 (Sriram et al., 1997), using the following
DI =Sum of individual ×leaf ratings
Maximum disease score ×Number of leaves sample ×100 (1)
Individual leaf ratings in the formula refer to disease grade
of each leaf of tomato. The maximum disease score refers to
the maximum disease grade observed during the entire period
of the experiment. Fifty root samples (1 cm in length) were
collected from each plant, cleaned and stained to examine AM
colonization by the ink-vinegar staining method (Vierheilig et al.,
1998;Mukerji et al., 2002).
The experiment setups for enzyme assays and real-time RT-PCR
analysis were the same as those for disease bioassays. PAL activity
was determined as the rate of the conversion of L-phenylalanine
to trans-cinnamic acid at 290 nm. Leaf samples (0.2 g) were
harvested from the diﬀerent treatment conditions (CK, Fm, As,
and Fm+As) and ground using liquid nitrogen and homogenized
in 1 ml ice cold 0.05 M sulfate buﬀer, pH 8.8 containing 7 mM
2-mercaptoethanol and 0.1 g insoluble polyvinylpyrrolidone.
The homogenate was centrifuged at 12000 gfor 20 min. The
supernatant was used for enzyme analysis. PAL activity was
determined spectrophotometrically (Edwards and Kessmann,
Lipoxygenase (LOX) activity was measured as conjugated
diene formation (Macri et al., 1994). Leaf samples (0.2 g) were
ground using liquid nitrogen and extracted with 1 ml ice-cold
0.5 M TRIS-HCl buﬀer (pH 7.6) and centrifuged at 12 000 g
for 15 min at 4◦C. The supernatant was kept at 4◦Cuntilused.
The substrate contained 1.6 mM linoleic acid and 0.5% (v/v)
Tween 20 in 0.1 M phosphate buﬀer (pH 7.6). The reaction
was initiated by the addition of 0.2 ml crude extract in 4.8 ml
of the substrate. Diene formation was followed as increase of
absorbance at 234 nm.
Leaf samples (0.1 g) were ground in liquid nitrogen and
extracted with 2 ml 0.05 M sodium acetate buﬀer (pH
5.0) and centrifuged at 12 000 gfor 15 min at 4◦C. The
supernatant was used for the enzyme assay of β-1,3-glucanase
and chitinase. β-1,3-Glucanase activity was assayed by the
laminarindinitrosalicylic acid method (Pan et al., 1991). The
chitinase activity was analyzed as described (Boller and Mauch,
Real-time RT-PCR Analysis
Diﬀerential expression of selected genes was veriﬁed by real time
-polymerase chain reaction (RT-PCR) using the RNA samples
Frontiers in Plant Science | www.frontiersin.org 3September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
isolated from tomato leaves obtained from the four treatments.
The total RNA was extracted and isolated as described by Kiefer
et al. (2000), with slight modiﬁcation. Fresh leaves (0.2 g) were
ground with a mortar and pestle in liquid nitrogen, and the
powdered tissue transferred to a 2 ml Eppendorf tube, then
1000 μl TRIzol reagent (Invitrogen) was added and mixed. After
incubation for 8–10 min on ice, 200 μl chloroform was added
and mixed. Following 5 min incubation at room temperature,
the mixture was centrifuged at 12,000 gfor 15 min at 4◦C.
The supernatant was transferred to a 1.5 ml Eppendorf tube
and 500 μl isoamylalcohol was added, followed by vortexing at
room temperature for 10 min and centrifugation at 13,000 g
for another 10 min at 4◦C. The supernatant was discarded and
the pellet was washed with 1 ml 75% ethanol (v/v), dissolved
in 30 μlRNAsefreewaterandkeptat−80◦Cuntilused.RNA
integrity was checked on a denaturing agarose gel electrophoresis;
the concentration was determined spectrophotometrically before
The expression patterns of defense-related genes (PAL ,LOX,
AOC,PR1,PR2,andPR3) in diﬀerent treated tomato leaves
were analyzed by using Real Time-PCR. The primers for target
genes PAL,LOX,AOC,PR1,PR2,andPR3 were designed
by Primer 3.0 software (Applied Biosystems, http://fokker.wi.
mit.edu/primer3/input.htm) based on tomato mRNA sequences
deposited in GenBank. The gene-speciﬁc primer sequences used
are listed in Tab l e 1 .Ubi3 (Accession No. X58253) was used
as a reference. Proteinase inhibitor II (Pin2) gene was chosen
because it is typical jasmonic acid responsive gene systemically
induced upon wounding (Farmer and Ryan, 1992). Real-time
PCR reactions were carried out with 0.2 μl (0.15 μM) of
each speciﬁc primers, 1 μl (50 ng) cDNA, and 12.5 μlof
the SYBR green master mix (Quanti Tech SYBR Green kit,
Qiagen, Gmbh Hilden, Germany), and the ﬁnal volume was
made up to 25 μlwithRNase-freewater.Inthenegative
control, cDNA was replaced by RNase free water. The reactions
were performed on a DNA Engine Opticon 2 Continuous
Fluorescence Detection System (MJ Research Inc., Waltham, MA,
US). The program used for real-time PCR was 3 min initial
denaturation at 95◦C, followed by 35 cycles of denaturation
for 20 s at 95◦C, annealing for 20 s (PAL:57
◦C; LOX: 56.9◦C;
AOC: 56.5◦C; PR1: 55.4◦C; PR2:56
◦C; Pin2: 60.0◦C;
◦C) and extension for 20 s at 72◦C. The ﬂuorescence
signal was measured immediately after incubation for 2 s at 75◦C
following the extension step, which eliminates possible primer
dimer detection. At the end of the cycles, melting temperatures
of the PCR products was determined between 65 and 95◦C.
The speciﬁcity of amplicons was veriﬁed by melting curve
analysis and agarose gel electrophoresis. Three independent
biological replicates for each treatment were used for qRT-PCR
For each treatment, three replicates were maintained in
a completely randomized design. SAS 8.0 (SAS Institute,
Cary, North Carolina) package for windows was used for
statistical analysis. The data were analyzed with a one-
way analysis of variance with the signiﬁcant diﬀerences
among means identiﬁed by Tukey’s multiple range test
Induction of Disease resistance by Mycorrhizal
Inoculation of tomato plants with the AMF, F. mosseae,
led to a signiﬁcant decrease in disease incidence and
disease severity of early blight compared to the control
plants without mycorrhizal inoculation (Ta b l e 2 ). Disease
incidences and indices were reduced in mycorrhizal plants
by 54.3% and 72.8%, respectively, 10 d after pathogen
inoculation. Mycorrhizal plants had signiﬁcantly fewer
disease symptoms than non-mycorrhizal plants (Figure 1).
Furthermore, disease development in AMF-inoculated plants
TABLE 1 | Speciﬁc primer for real-time PCR.
Gene Accession No. Primer sequence (5to 3) PCR product size
LeLOX U13681 F: 5-ATCTCCCAAGTGAAACACCACA-3
LeAOC AW624058 F: 5-CTCGGAGATCTTGTCCCCTTT-3
LePR1 DQ159948 F: 5-GCCAAGCTATAACTACGCTACCAAC-3
LePR2 M80604 F: 5-GGACACCCTTCCGCTACTCTT-3
LePR3 Z15140 F: 5-AACTATGGGCCATGTGGAAGA-3
LePIN2 X94946 F:5-AATTATCCATCATGGCTGTTCAC-3
R: 5- CCTTTTTGGATCAGATTCTCCTT-3
LePAL AW035278 F: 5-CTGGGGAAGCTTTTCAGAATC-3
LeUBI3 X58253 F: 5- TCCATCTCGTGCTCCGTCT -3
Frontiers in Plant Science | www.frontiersin.org 4September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
TABLE 2 | Mycorrhizal colonization rates, disease incidences, and indices
of tomato plants inoculated with either Funneliformis mosseae,Alternaria
solani, or both.
F. mosseae and
63 ±4.2 a
40.1 ±5.3 b
44.5 ±2.6 a
17.2 ±0.8 b
60.3 ±1.7 a
55.1 ±1.5 b
Four sets of bioassays were independently carried out and three pots per treatment
were set up for each set of bioassays. Values are means ±SE. Signiﬁcant
differences (P <0.05 using Tukey post hoc test) among treatments in the same
column are indicated by different letters.
was signiﬁcantly slower. Microscopic observation showed that
the mycorrhizal infection rate was 55.1% in the inoculated plants
(Tab l e 2 ).
Induction of Defense-related Enzymes by
To examine eﬀects of mycorrhizal colonization on defense
responses in host plants in presence of pathogen infection
tomato plants were subjected to four treatments: (1) CK: control
without fungal inoculation; (2) As: inoculation with A.solani;
(3) Fm: inoculation with F. mosseae;(4)Fm+As: inoculation
with both F. mosseae and A.solani. Four defense-related
enzymes, including PAL, LOX, chitinase, and β-1,3-glucanase
were analyzed in the leaves of tomato plants. Mycorrhizal pre-
inoculation signiﬁcantly enhanced activities of the four enzymes
intheleavesuponpathogeninfection(Figures 2A–D). The
activities of all tested enzymes were signiﬁcantly higher in
treatment Fm+As after the pathogen inoculation and reached a
maximum at 65 h. The activity of β-1,3-glucanase was increased
by 34.7, 33.3, and 28.8%, respectively, relative to those in
treatments CK, As, and Fm 65 h post the pathogen inoculation
(Figure 2A). However, the activity of β-1,3-glucanase did not
diﬀer signiﬁcantly between the other treatments (CK, As, and
Fm) (Figure 2A).
The enzymatic activity of LOX in treatment Fm+As was
signiﬁcantly higher after pathogen inoculation (Figure 2B). LOX
activity in treatment Fm+As increased by 48.1, 32.2, and 99.5%
at 18 h after pathogen inoculation compared to treatments CK,
As, and Fm, respectively, and increased by 38.1, 37.8, and 68.1%
at 65 h after pathogen inoculation, respectively.
Although mycorrhization led to some increase in PAL activity
in treatment Fm, the PAL induction was more pronounced
in treatment Fm+As. The PAL activity in treatment Fm+As
was, on average, higher by 104.3, 74.9, and 79.5% than that of
treatment CK, As, and Fm, respectively, at 65 h after pathogen
inoculation (Figure 2C). In contrast, the diﬀerence in PAL
activity among treatments CK, As, and Fm were less variable. In
particular, the PAL activity was not signiﬁcantly diﬀerent among
treatment CK, As, and Fm at 18 and 65 h following pathogen
Chitinase activity in mycorrhizal pre-inoculated plants
(Fm+As) was signiﬁcantly higher at 18, 65, and 100 h after the
FIGURE 1 | Disease symptoms of early blight in leaves of tomato
plants with or without mycorrhizal colonization by Funneliformis
mosseae. The photos were taken 10 days after pathogen inoculation by
Alternaria solani. Three treatments included: (1) CK: control plants without
pathogen and mycorrhizal inoculation; (2) As: plants inoculated with A.solani
only; (3) Fm+As: plants inoculated with both F. mosseae and A.solani.
pathogen inoculation (Figure 2D). It displayed increases of 44.1,
62.1, and 55.1% in treatment Fm+As compared to treatments
CK, As, and Fm, respectively, at 65 h after pathogen inoculation.
Transcript Induction of Defense-related Genes
by Mycorrhizal Colonization
To determine whether mycorrhizal colonization enhances the
disease resistance and defense response by inducing transcription
of defense-related genes (Pozo et al., 2005), the expression
patterns of the six genes (PAL,LOX,AOC (encoding allene oxide
cyclase for JA biosynthesis), PR1,PR2, and PR3)wereanalyzed
by using real-time RT-PCR from tomato leaves 18, 65, 100, and
140 h post pathogen inoculation. Mycorrhizal pre-inoculation of
tomato plants with F. mosseae and later pathogen inoculation
with A.solani (treatment Fm+As) induced accumulation of
PAL ,LOX,AOC,PR1,PR2, and PR3 transcripts over basal
levels present in the leaves of un- inoculated control (CK), sole
A.solani inoculation (As), and F. mosseae colonization (Fm)
treatments 18, 65, 100, and 140 h after pathogen inoculation
(Figure 3). The expression levels of PAL, LOX, and AOC were
induced approximately 3.0, 7.1, and 18.8-fold at 100 h, and
by 4.1, 5.3, and 5.8-fold at 140 h post pathogen inoculation,
respectively, in response to dual inoculation with the AMF and
the pathogen (Fm+As) relative to the non-mycorrhizal control
(CK) (Figures 3A–C). Mycorrhizal pre-colonization induced
transcripts of PR1,PR2, and PR3 by 20.4, 35.5, and 47.7-
fold at 65 h, by 8.0, 37.7, and 22.9-fold at 100 h, respectively
(CK) (Figures 3D–F). Pathogen infection alone (treatment As)
induced transcripts of the six genes in the leaves of non-
preinoculated tomato plants, but the induction was much less
and slower compared with that in mycorrhizal and pathogen-
infected plants (treatment Fm+As). Mycorrhizal colonization
(treatment Fm) alone did not induce gene expression of PA L
and PR3 (Figures 3A,F). Although sole mycorrhization up-
regulated transcripts of PR1,PR2, and AOC at 100 and
140 h, the induction was even weaker than that by pathogen
Frontiers in Plant Science | www.frontiersin.org 5September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
FIGURE 2 | Activity levels of defense-related enzymes in tomato leaves in response to mycorrhizal colonization and pathogen infection. The tomatoes
were pre-inoculated with mycorrhizal fungus Funneliformis mosseae and later inoculated with A. solani, the causal agent of early blight disease of tomato. Four
defense-related enzymes are β-1,3-glucanase (A), lipoxygenase (LOX) (B), phenylalanine ammonia-lyase (PAL) (C), and chitinase (D). Four treatments included: (1)
CK: control plants without pathogen and mycorrhizal inoculation; (2) As: plants inoculated with A.solani only; (3) Fm: plants inoculated with F. mosseae only; (4)
Fm+As: plants inoculated with both F. mosseae and A.solani. Values are means ±SE from three sets of independent experiments with three pots per treatment for
each set of experiments. Signiﬁcant differences among treatments were tested at P=0.05 by Tukey post hoc test.
Role of Jasmonate Signaling Pathway in
AMF-induced Disease Resistance
Three tomato genotypes: a WT plant, a JA biosynthesis mutant
(spr2), and a prosystemin-overexpressing 35S::PS plant, were
used to identify the role of the JA signaling pathway in
mycorrhiza-induced disease resistance against A.solani.The
plants of the three genotypes were subjected to the same
four treatments as above. There was no signiﬁcant diﬀerence
in activities of the four defense-related enzymes in control
plants of the three genotypes (Figures 4A–D). However, the
three genotypes showed large diﬀerences in activities of the
four defense-related enzymes in response to pathogen infection
(As) and dual inoculation with the pathogen and mycorrhizal
fungus (Fm+As). The 35S::PS plants showed higher induction
of enzymatic activities in A.solani-inoculated plants compared
to the other two genotypes. Most importantly, mycorrhizal
pre-inoculated 35S::PS plants showed the highest induction of
enzymatic activities (Figures 4A–D). β-1,3-Glucanase activity
in treatment Fm+As was increased by 1083.7, 291.3, and
495.5% at 100 h post pathogen inoculation compared with
that in treatments CK, As, and Fm, respectively (Figure 4Ac).
Similarly, chitinase activity in treatment Fm+As was increased
by 795.2, 161.5, and 498.8% at 65 h compared with the
other three treatments (Figure 4Db). Similar trends were
observed for LOX and PAL activities (Figures 4B,C). The
LOX and PAL activities were increased in mycorrhizal pre-
inoculated 35S::PS and WT plants after pathogen inoculation,
but 35S::PS plants exhibited signiﬁcantly higher LOX and
PAL activities as compared to WT plants (Figures 4B,C).
The 35S::PS plants showed 48.5, 125.0, 56.0, and 111.4%
higher PAL activity in treatment Fm+As than that of WT
plants 18, 65, 100, and 140 h after pathogen inoculation,
respectively (Figure 4Ca–d). In contrast, the four tested
enzymes were not induced in the spr2 plants in response
to pathogen inoculation (As) and dual inoculation (Fm+As)
Mycorrhizal pre-inoculation on 35S::PS and WT tomato
plants resulted in strong induction of transcripts of defense-
related genes (PAL,LOX,Pin2,PR1,PR2, and PR3)upon
pathogen attack (treatment Fm+As) (Figures 5A–F). The
highest induction of the six defense-related genes was found
in the mycorrhizal 35S::PS plants. While no induction of
PAL expression was found with pathogen inoculation alone
(As) or AMF inoculation alone (Fm), dual inoculation
with the AMF, and pathogen induced PAL 24.0- and 10.2-
fold in 35S::PS and WT plants, respectively, 65 h after
pathogen inoculation (Figure 5Ab). Similarly, AMF pre-
inoculation and later pathogen infection induced PR1 18.3-,
6.5-, and 17.5-fold in 35S::PS plants as compared to that in
treatments CK, As, and Fm, respectively, 100 h after pathogen
inoculation (Figure 5Dc). Although pathogen infection
alone induced PR1 transcripts in 35S::PS and WT plants, the
induction was signiﬁcantly lower relative to treatment Fm+As
(Figure 5D). No induction was found in the spr2 plants in
response to pathogen inoculation (As) or dual inoculation
(Fm+As) (Figures 5A–F). Similar inductions of PR3 and
Frontiers in Plant Science | www.frontiersin.org 6September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
FIGURE 3 | Transcripts of defense-related genes in tomato leaves in response to mycorrhizal colonization and pathogen infection. The tomates were
pre-inoculated with mycorrhizal fungus Funneliformis mosseae and later inoculated with A. solani, the causal agent of early blight disease of tomato. Quantitative real
time RT-PCR was used to detect the transcripts of six defense-related genes encoding PAL (A),LOX (B), allene oxide cyclase (AOC)(C), pathogen- related proteins
(PR1)(D),β-1,3-glucanase (basic type PR-2)(E), and chitinase (PR-3)(F). Four treatments included: (1) CK: control plants without pathogen and mycorrhizal
inoculation; (2) As: plants inoculated with A.solani only; (3) Fm: plants inoculated with F. mosseae only; (4) Fm+As: plants inoculated with both F. mosseae and A.
solani. Values are means ±SE from three sets of independent experiments with three pots per treatment for each set of experiments. Signiﬁcant differences
(P<0.05 using Tukey post hoc test) among treatments in a group are indicated by different letters above bars.
Pin2 transcripts were observed in 35S::PS and WT plants,
but there was no induction in the JA-deﬁcient spr2 mutant
Bioassays showed that mycorrhizal pre-inoculation on
35S::PS and WT tomato plants signiﬁcantly reduced disease
incidence and disease severity of early blight relative to
AMF un-inoculated control plants (Table 3). Mycorrhizal
colonization led to 17.6 and 15.6% reductions in the
disease incidence and disease severity, respectively, in WT
plants, and 19.7 and 20.4% reductions, respectively, in
35S::PS plants. In contrast, mycorrhizal inoculation did
not aﬀect the disease incidence and severity of the spr2
mutant plants. On the other hand, the spr2 plants had the
lowest mycorrhizal colonization rate and the highest disease
incidence and severity among the three tomato genotypes
(Tab l e 3 ).
In last two decades, early blight has become a major disease
of tomato in many parts of China (Dong et al., 2015). This
study shows that tomato early blight can be alleviated through
mycorrhizal inoculation, which is consistent with previous
ﬁnding by Fritz et al. (2006). The enhanced disease resistance was
not due to improved phosphorus nutrient, though the underlying
mechanism was not clear (Fritz et al., 2006). Mycorrhizal fungi
are ideal biocontrol agents because they are natural soil-borne
biota and can establish stable and long lasting mutualistic
symbiosis with the roots of most vascular plant species, including
most crops (Smith and Read, 2008). Mycorrhizal associations
beneﬁt not only plant nutrient absorption (Smith and Read,
2008), but also plant resistance to diverse abiotic stresses (Ruiz-
Lozano et al., 1996) and soil-borne fungal pathogens (Harrier and
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Song et al. Disease resistance primed by mycorrhiza
FIGURE 4 | Levels of defense-related enzymes in leaves of tomatoes with mycorrhizal colonization and pathogen infection. Wild-type (WT) and mutant
plants (35S::PS and spr2) of tomato were pre-inoculated with mycorrhizal fungus Funneliformis mosseae and later inoculated with A. solani, the causal agent of early
blight disease of tomato. Four defense-related enzymes are β-1,3-glucanase (A),LOX(B),PAL(C), and chitinase (D). Enzymatic activities were analyzed 18 (a), 65
(b), 100 (c) and 140 h (d) after pathogen inoculation. Four treatments included: (1) CK: control plants without pathogen and mycorrhizal inoculation;(2)As:plants
inoculated with A.solani only; (3) Fm: plants inoculated with F. mosseae only; (4) Fm+As: plants inoculated with both F. mosseae and A.solani. Three tomato
genotypes included: (1) WT: wild type plant; (2) 35S::PS: Prosystemin-overexpressing 35S::PS plant; (3) spr2: JA biosynthesis mutant plant. Values are means ±SE
from three sets of independent experiments with three pots per treatment for each set of experiments. Signiﬁcant differences among treatments were tested at
P=0.05 by Tukey post hoc test.
Watson, 2004;Bi et al., 2007). More interestingly, AM symbiosis
also enhances plant resistance against foliar pathogens such as
fungal pathogens [e.g., Botrytis cinerea (Pozo et al., 2010;Fiorilli
et al., 2011)andA. solani (Fritz et al., 2006)], bacteria [e.g.,
Xamantomonas campestris (Liu et al., 2007) and viruses (e.g.,
Tomato yellow leaf curl Sardinia virus (Maﬀei et al., 2014)].
Induction of pathogenesis-related (PR) proteins is believed an
indicator of plant induced defense responses. Accumulation of
chitinase and β-1,3-glucanase has been associated previously with
induced systemic resistance in tomato to A.solani (Lawrence
et al., 1996)andFusarium oxysporum (Pozo et al., 2002).
Basic isozymes of chitinase and β-1,3-glucanase inhibit A.solani
in vitro (Lawrence et al., 1996). Early blight-resistant tomato
lines possess higher levels of chitinase and β-1,3-glucanase than
susceptible genotypes (Lawrence et al., 2000). PR genes have
been frequently used as marker genes for systemic acquired
resistance in many plant species (Mitsuhara et al., 2008). Our
study showed that mycorrhizal pre-inoculation in tomato roots
systemically induced both enzyme activities of chitinase and
β-1,3-glucanase, and transcripts of the genes PR1,PR2, and
PR3 encoding PR proteins in the leaves of tomato. Pozo et al.
(2002) found that F. mosseae colonization in tomato plants
reduced both local and systemic disease symptoms caused by
Phytophthora parasitica infection, as well as provoked local and
systemic induction of chitinase, β-1,3-glucanase and superoxide
Our study showed that mycorrhizal inoculation itself did
not aﬀect most enzyme activitiesandonlyslightlyinduced
transcripts of AOC, PR1, and PR2.However,uponpathogen
attack AMF pre-inoculation strongly induced defense responses
of all six tested genes and four defense-related enzymes in
tomato plants. Based on the results that plants inoculated
Frontiers in Plant Science | www.frontiersin.org 8September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
FIGURE 5 | Transcripts of defense-related genes in leaves of tomatoes with mycorrhizal colonization and pathogen infection. WT and mutant plants
(35S::PS and spr2) of tomato were pre-inoculated with mycorrhizal fungus Funneliformis mosseae and later inoculated with A. solani, the causal agent of early blight
disease of tomato. Quantitative real time RT-PCR was used to detect the transcripts of six defense-related genes encoding the PAL (A),LOX (B),(Pin2)(C),
pathogen-related proteins (PR1)(D),β-1,3-glucanase (basic type PR-2)(E), and chitinase (PR-3)(F). Transcript levels were quantiﬁed 18 (a), 65 (b), 100 (c), and
140 h (d) after pathogen inoculation. Four treatments included: (1) CK: control plants without pathogen and mycorrhizal inoculation; (2) As: plants inoculated with A.
solani only; (3) Fm: plants inoculated with F. mosseae only; (4) Fm+As: plants inoculated with both F. mosseae and A.solani. Three tomato genotypes included: (1)
WT: wild type plant; (2) 35S::PS: Prosystemin-overexpressing 35S::PS plant; (3) spr2: JA biosynthesis mutant plant. Values are means ±SE from three sets of
independent experiments with three pots per treatment for each set of experiments. Signiﬁcant differences among treatments were tested at P=0.05 by Tukey post
Frontiers in Plant Science | www.frontiersin.org 9September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
TABLE 3 | Mycorrhizal colonization rates, disease incidences, and indices of AMF-inoculated and un-inoculated tomato plants inoculated by AMF
Funneliformis mosseae and pathogen A. solani.
Microbial inoculation Tomato genotype Disease incidence (%) Disease index (%) Mycorrhizal colonization (%)
F. mosseae and A. solani WT 60.2 ±1.3 cd 36.8 ±1.3 c 40.6 ±4.1 a
35S::PS 54.5 ±4.4 d 28.5 ±1.7 d 41.2 ±1.2 a
spr2 86.4 ±2.1 a 53.4 ±1.4 a 14.0 ±1.9 b
A. solani WT 73.1 ±3.1 b 43.6 ±2.4b 0c
35S::PS 67.9 ±3.6 bc 35.8 ±1.2c 0c
spr2 89.6 ±1.4 a 56.2 ±1.4a 0c
Three tomato genotypes included: (1) WT: wild type plant; (2) 35S::PS: Prosystemin-overexpressing 35S::P S plant; (3) spr2: JA biosynthesis mutant plant. Four sets of
bioassays were independently carried out and three pots per treatment were set up for each set of bioassays. Values are means ±SE. Signiﬁcant differences (P <0.05
using Tukey post hoc test) among treatments in the same column are indicated by different letters.
with both F. mosseae and A.solani had less disease damage,
higher levels of defense-related enzymatic activities and gene
expression than the controls, or pathogen or mycorrhizal
inoculations alone, we suggest that mycorrhizal colonization on
tomato can prime plant defense responses against early blight
Most studies on defense priming focus on priming signals
triggered by herbivore-induced volatile compounds (Ton et al.,
2006;Heil and Silva Bueno, 2007;Ramadan et al., 2011).
Some studies show that priming of plant defense can also be
triggered by certain beneﬁcial micro-organisms (Pozo et al.,
2005;van Hulten et al., 2006;van Wees et al., 2008), including
AMF (Pozo and Azcón-Aguilar, 2007;Pozo et al., 2009;Jung
et al., 2012). Mycorrhizal pre-inoculation results in signiﬁcantly
higher production of PR-1a and basic β-1,3-glucanases in
tomato plants upon Phytophthora infection (Cordier et al.,
1998;Pozo et al., 1999;Maldonado-Bonilla et al., 2008). RNA-
seq-based transcriptome analysis showed that mycorrhization
led to the transcriptional changes in categories of signaling,
hormone metabolism, biotic, and abiotic stresses, and several
diﬀerentially expressed genes were related to systemic defense
priming (Cervantes-Gámez et al., 2015). Our study conﬁrms that
priming is an important mechanism operating in mycorrhiza-
induced disease resistance.
Plant disease resistance is tightly manipulated through
an interconnected network of signaling pathways of JA and
SA. PAL is the key enzyme involved in the biosynthesis
of the signal molecule, SA (Mauch-mani and Slusarenko,
1996). SA accumulates in cells undergoing hypersensitive
response and it is essential for local and systemic resistance
response (Gaﬀney et al., 1993;Makandar et al., 2012).
Induction of PAL activity is a reliable indicator of plant
resistance expression (Mauch-mani and Slusarenko,
1996). An increase in of PAL activity indicates that, upon
pathogen attack, mycorrhizal colonization initiates SA
signaling pathways and increase accumulation of phenolic
The JA signaling pathway has been demonstrated to play
a vital role in mediating plant defense responses to chewing
herbivore insects (Howe and Jander, 2008;Bosch et al., 2014)and
necrotrophic pathogens (Glazebrook, 2005;Robert-Seilaniantz
et al., 2011). External application of methyl JA primes Arabidopsis
defense against caterpillar herbivory (Rasmann et al., 2012).
Since A. solani is a necrotrophic pathogen, we examined
the roles of the JA pathway in AMF-induced priming in
tomato. LOX, AOC, and AOS (allene oxide synthase) are
three important enzymes in JA biosynthesis (Hause et al.,
2002;Schaller et al., 2005). Stronger and quicker induction of
LOX and AOC in mycorrhizal plants suggest that mycorrhizal
colonization can also provoke the JA pathway, which thereby
increases broad-spectrum disease resistance (De Vos et al.,
Use of JA biosynthesis (spr2) mutant and prosystemin-
overexpressing 35S::PS plants revealed that the JA signaling
pathway mediated AMF-primed defense in tomato plants.
Mycorrhizal 35S::PS plants had signiﬁcantly higher levels of
defense-related enzyme activity and gene expression than
mycorrhizal WT plants and non-mycorrhizal 35S::PS plants in
response to A. solani infection (Figures 4 and 5). Although
pathogenic infection alone induced enzymatic activities and gene
transcripts in WT plants, the induction was lower than that
of pathogen-infected mycorrhizal plants. However, AMF pre-
inoculation and pathogenic infection did not lead to induction
of defense-related enzymes and genes in spr2 plants. The
mycorrhizal 35S::PS plants were the most resistant to early blight
and mycorrhizal spr2 plants were the most susceptible (Table 3).
Non-mycorrhizal 35S::PS plants showed similar level of disease
resistance to mycorrhizal WT plants. These results suggest that
the JA pathway is required for AMF-induced systemic priming
of defense against A. solani. Rasmann et al. (2012) showed that
herbivory in the previous generation primed Arabidopsis and
tomato for enhanced insect resistance, and Arabidopsis mutants
that were deﬁcient in JA perception did not exhibit inherited
resistance, demonstrating that the JA pathway is required in
mother plants for priming resistance in the next generation.
Low mycorrhizal colonization rate (Ta b l e 3 ) may suggest that
JA signalings are necessary for establishment of mycorrhizal
Pre-inoculation of tomato plants with F. mosseae enhanced
tomato resistance to early blight. Root colonization by AMF
systematically induced the defense-related enzymes and genes
in the leaves of tomato upon pathogen challenge. Our
Frontiers in Plant Science | www.frontiersin.org 10 September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
results suggest that mycorrhizal-induced disease resistance
in tomato is associated with priming for an eﬃcient
activation of defense responses upon pathogen attack. The
AMF-induced primed responses were systemic and the JA
pathway is required for such responses. Since most land
plants have symbiotic association with mycorrhizal fungi
(Brundrett, 2002), use of mycorrhizal fungi as defense priming
elicitors may be an important evolutionary strategy for plant
defense against pathogens and it may serve as an important
alternative for management of crop disease in sustainable
This research was supported by the Natural Science Foundation
of China (31470477, 31100286), National 973 project of China
(2011CB100400), One Hundred Talents Program of Fujian
Province of China (2014) and Talent Program of Fujian
Agriculture and Forestry University. We acknowledge Prof.
Chuanyou Li of Institute of Genetics and Developmental Biology
of the Chinese Academy of Sciences for providing us with
transgenic tomato lines, and Prof. Suzanne Simard and Dr. Simon
Zebelo for improving the language.
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Conﬂict of Interest Statement: The authors declare that the research was
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