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Enhanced Tomato Disease Resistance Primed by Arbuscular Mycorrhizal Fungus

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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 PAL, 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.
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ORIGINAL RESEARCH
published: 28 September 2015
doi: 10.3389/fpls.2015.00786
Edited by:
Vitaly Dzhavakhiya,
Lomonosov Moscow State University,
Russia
Reviewed by:
Raffaella Balestrini,
Consiglio Nazionale delle Ricerche,
Italy
Luisa Lanfranco,
University of Torino, Italy
*Correspondence:
Rensen Zeng,
College of Life Sciences, Fujian
Agriculture and Forestry University,
Fuzhou 350002, China
rszeng@fafu.edu.cn
Specialty section:
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
Citation:
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.
doi: 10.3389/fpls.2015.00786
Enhanced tomato disease resistance
primed by arbuscular mycorrhizal
fungus
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,
China
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
Introduction
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
Experimental Design
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 28Cindarknessandon
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.
Bioassay
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 121C 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 ±1Cwitha16hphotoperiod,
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
3)2,1ml
1MMgSO
4,2ml1MKH
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
formula:
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).
Enzyme Assays
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,
1992).
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 4C. The supernatant was kept at 4Cuntilused.
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 4C. 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,
1988).
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 4C.
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 4C. The supernatant was discarded and
the pellet was washed with 1 ml 75% ethanol (v/v), dissolved
in 30 μlRNAsefreewaterandkeptat80Cuntilused.RNA
integrity was checked on a denaturing agarose gel electrophoresis;
the concentration was determined spectrophotometrically before
further use.
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 95C, followed by 35 cycles of denaturation
for 20 s at 95C, annealing for 20 s (PAL:57
C; LOX: 56.9C;
AOC: 56.5C; PR1: 55.4C; PR2:56
C; PR3:58
C; Pin2: 60.0C;
Ubi3:58
C) and extension for 20 s at 72C. The fluorescence
signal was measured immediately after incubation for 2 s at 75C
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 95C.
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
analyses.
Statistical Analysis
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
(P<0.05).
Results
Induction of Disease resistance by Mycorrhizal
Colonization
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
R: 5-TCATAAACCCTGTCCCATTCTTC-3
109 bp
LeAOC AW624058 F: 5-CTCGGAGATCTTGTCCCCTTT-3
R: 5-CTCCTTTCTTCTCTTCTTCGTGCT-3
115 bp
LePR1 DQ159948 F: 5-GCCAAGCTATAACTACGCTACCAAC-3
R: 5-GCAAGAAATGAACCACCATCC-3
139 bp
LePR2 M80604 F: 5-GGACACCCTTCCGCTACTCTT-3
R: 5-TGTTCCTGCCCCTCCTTTC-3
81 bp
LePR3 Z15140 F: 5-AACTATGGGCCATGTGGAAGA-3
R: 5-GGCTTTGGGGATTGAGGAG-3
128 bp
LePIN2 X94946 F:5-AATTATCCATCATGGCTGTTCAC-3
R: 5- CCTTTTTGGATCAGATTCTCCTT-3
254 bp
LePAL AW035278 F: 5-CTGGGGAAGCTTTTCAGAATC-3
R:5-TGCTGCAAGTTACAAATCCAGAG-3
150 bp
LeUBI3 X58253 F: 5- TCCATCTCGTGCTCCGTCT -3
R:5-GAACCTTTCCAGTGTCATCAACC-3
144 bp
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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.
Microbial
inoculation
Disease
incidence (%)
Disease
index (%)
Mycorrhizal
colonization (%)
Non-inoculation control
F. mosseae
A. solani
F. mosseae and
A. solani
0c
0c
63 ±4.2 a
40.1 ±5.3 b
0c
0c
44.5 ±2.6 a
17.2 ±0.8 b
0c
60.3 ±1.7 a
0c
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
Mycorrhizal Colonization
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
inoculation.
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
infection.
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)
(Figures 4A–D).
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
(Figures 5C,F).
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 ).
Discussion
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
Frontiers in Plant Science | www.frontiersin.org 7September 2015 | Volume 6 | Article 786
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
dismutase.
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
hoc test.
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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
disease.
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
compounds.
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.,
2005).
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
association.
Conclusion
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
agriculture.
Acknowledgments
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.
References
Aimé, S., Alabouvette, C., Steinberg, C., and Olivain, C. (2013). The endophytic
strain Fusarium oxysporum Fo47: a good candidate for priming the defense
responses in tomato roots. Mol. Plant-Microbe Interact. 26, 918–926. doi:
10.1094/MPMI-12-12-0290-R
Akköprü, A., and Demir, S. (2005). Biological control of Fusarium wilt in tomato
caused by Fusarium oxysporum f. sp. lycopersici by AMF Glomus intraradices
and some Rhizobacter. J. Phytopathol. 153, 544–550. doi: 10.1111/j.1439-
0434.2005.01018.x
Bi, H. H., Song, Y. Y., and Zeng, R. S. (2007). Biochemical and molecular responses
of host plants to mycorrhizal infection and their roles in plant defence.
Allelopathy J. 20, 15–28.
Boller, T., and Mauch, F. (1988). Colorimetric assay for chitinase. Methods
Enzymol. 161, 430–435. doi: 10.1016/0076-6879(88)61052-4
Bosch, M., Wright, L. P., Gershenzon, J., Wasternack, C., Hause, B., Schaller, A.,
et al. (2014). Jasmonic acid and its precursor 12-oxophytodienoic acid control
different aspects of constitutive and induced herbivore defenses in tomato.
Plant Physiol. 166, 396–410. doi: 10.1104/pp.114.237388
Brundrett, M. (2002). Coevolution of roots and mycorrhizas of land plants. New
Phytol. 154, 275–304. doi: 10.1046/j.1469-8137.2002.00397.x
Cervantes-Gámez, R. G., Bueno-Ibarra, M. A., Cruz-Mendívil, A., Calderón-
Vázquez, C. L., Ramírez-Douriet, C. M., Maldonado-Mendoza, I. E., et al.
(2015). Arbuscular mycorrhizal symbiosis-induced expression changes in
Solanum lycopersicum leaves revealed by RNA-seq analysis. Plant Mol. Biol. Rep.
23, 1–14. doi: 10.1007/s11105-015-0903-9
Chellappan, P., Christy, S. A. A., and Mahadevan, A. (2002). “Multiplication of
arbuscular mycorrhizal fungi on roots,” in Techniques in Mycorrhizal Studies,
edsK.G.Mukerji,C.Manoharachary,andB.P.Chamola(Dordrecht:Kluwer
Academic Publishers), 285–297.
Chen, H., Jones, A. D., and Howe, G. A. (2006). Constitutive activation of
the jasmonate signaling pathway enhances the production of secondary
metabolites in tomato. FEBS Lett. 580, 2540–2546. doi: 10.1016/j.febslet.2006.
03.070
Cheng, L., Booker, F. L., Tu, C., Burkey, K. O., Zhou, L. S., Shew, H. D., et al.
(2012). Arbuscular mycorrhizal fungi increase organic carbon decomposition
under elevated CO2.Science 337, 1084–1087. doi: 10.1126/science.1224304
Cordier, C., Gianinazzi, S., and Gianinazzi-Pearson, V. (1996). Colonisation
patterns of root tissues by Phytophthora nicotianae var. parasitica related
to reduced disease in mycorrhizal tomato. Plant Soil 185, 223–232. doi:
10.1007/BF02257527
Cordier, C., Pozo, M., Barea, J., Gianinazzi, S., and Gianinazzi-Pearson, V.
(1998). Cell defence responses associated with localized and systemic
resistance to Phytophthora parasitica induced in tomato by an arbuscular
mycorrhizal fungus. Mol. Plant-Microbe Interact. 11, 1017–1028. doi:
10.1094/MPMI.1998.11.10.1017
De Vos, M., Van Oosten, V. R., Van Poecke, R. M. P., Van Pelt, J. A., Pozo, M. J.,
Mueller, M. J., et al. (2005). Signal signature and transcriptome changes of
Arabidopsis during pathogen and insect attack. Mol. Plant-Microbe Interact. 18,
923–937. doi: 10.1094/MPMI-18-0923
Dong, Y., Chen, Y., and Zhang, H. (2015). Progresses of the microbial control of
tomato early blight. Chin.Agric.Sci.Bull.31, 111–115.
Edwards, R., and Kessmann, H. (1992). “Isoflavonoid phytoalexins and their
biosynthetic enzymes,” in Molecular Plant Pathology: A Practical Approach,Vol.
2, eds S. J. Gurr, M. J. MePherson, and D. J. Bowles (Oxford: Oxford University
Press), 45–62.
Elsen, A., Gervacio, D., Swennen, R., and de Waele, D. (2008). AMF-induced
biocontrol against plant parasitic nematodes in Musa sp.: a systemic effect.
Mycorrhiza 18, 251–256. doi: 10.1007/s00572-008-0173-6
Farmer, E. E., and Ryan, C. A. (1992). Octadecanoid precursors of jasmonic acid
activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell 4,
129–134. doi: 10.2307/3869566
Fellbaum, C. R., Gachomo, E. W., Beesetty, Y., Choudhari, S., Strahan, G. D.,
Pfeffer, P. E., et al. (2012). Carbon availability triggers fungal nitrogen uptake
and transport in arbuscular mycorrhizal symbiosis. Proc.Natl.Acad.Sci.U.S.A.
109, 2666–2671. doi: 10.1073/pnas.1118650109
Fiorilli, V., Catoni, M., Francia, D., Cardinale, F., and Lanfranco, L. (2011). The
arbuscular mycorrhizal symbiosis reduces disease severity in tomato plants
infected by Botrytis cinerea.J. Plant Pathol. 93, 237–242.
Fritz, M., Jakobsen, I., Lyngkjær, M. F., Thordal-Christensen, H., and Pons-
Kühnemann, J. (2006). Arbuscular mycorrhiza reduces susceptibility of
tomato to Alternaria solani.Mycorrhiza 16, 413–419. doi: 10.1007/s00572-00
6-0051-z
Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Scott, U., et al. (1993).
Requirement of salicylic acid for the induction of systemic acquired resistance.
Science 261, 754–756. doi: 10.1126/science.261.5122.754
García-Garrido, J. M., and Ocampo, J. A. (1988). Interaction between
Glomus mosseae and Erwinia carotovora and its effects on the growth
of tomato plants. New Phytol. 110, 551–555. doi: 10.1111/j.1469-8137.1988.
tb00295.x
García-Garrido, J. M., and Ocampom, J. A. (1989). Effect of VA mycorrhizal
infection of tomato on damage caused by Pseudomonas syringae.Soil Biol.
Biochem. 21, 165–167. doi: 10.1016/0038-0717(89)90027-8
Glazebrook, J. (2005). Contrasting mechanisms of defense against biotrophic
and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227. doi:
10.1146/annurev.phyto.43.040204.135923
Hao, Z. P., Fayolle, L., van Tuinen, D., Chatagnier, O., and Li, X. L. (2012).
Local and systemic mycorrhiza-induced protection against the ectoparasitic
nematode Xiphinema index involves priming of defence gene responses in
grapevine. J. Exp. Biol. 63, 3657–3672. doi: 10.1093/jxb/ers046
Harrier, L. A., and Watson, C. A. (2004). The potential role of arbuscular
mycorrhizal (AM) fungi in the bioprotection of plants against soil-borne
pathogens in organic and/or other sustainable farming systems. Pest Manag.
Sci. 60, 149–157. doi: 10.1002/ps.820
Hause, B., Maier, W., Miersch, O., Kramell, R., and Strack, D. (2002). Induction
of jasmonate biosynthesis in arbuscular mycorrhizal b arley roots. Plant Physiol.
130, 1213–1220. doi: 10.1104/pp.006007
Heil, M., and Silva Bueno, J. C. (2007). Within-plant signaling by volatiles leads to
induction and priming of an indirect plant defense in nature. Proc. Natl. Acad.
Sci. U.S.A. 140, 5467–5472. doi: 10.1073/pnas.0610266104
Howe, G. A., and Jander, G. (2008). Plant immunity to insect herbivores. Annu.
Rev. Plant Biol. 59, 41–66. doi: 10.1146/annurev.arplant.59.032607.092825
Howe, G. A., and Ryan, C. A. (1999). Suppressors of systemin signaling identify
genes in the tomato wound response pathway. Genetics 153, 1411–1421.
Frontiers in Plant Science | www.frontiersin.org 11 September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
Jakab, G., Cottier, V., Toquin, V., Rigoli, G., Zimmerli, L., Métraux, J. P., et al.
(2001). β-Aminobutyric acid-induced resistance in plants. Eur. J. Plant Pathol.
107, 29–37. doi: 10.1023/A:1008730721037
Jung, H. W., Tschaplinski, T. J., Wang, L., Glazebrook, J., and Greenberg,
J. T. (2009). Priming in systemic plant immunity. Science 324, 89–91. doi:
10.1126/science.1170025
Jung, S. C., Martinez-Medina, A., Lopez-Raez, J. A., and Pozo, M. J. (2012).
Mycorrhiza- induced resistance and priming of plant defenses. J. Chem. Ecol.
38, 651–664. doi: 10.1007/s10886-012-0134-6
Kiefer, E., Heller, W., and Ernst, D. (2000). A simple and efficient protocol for
isolation of functional RNA from plant tissues rich in secondary metabolites.
Plant Mol. Biol. Rep. 18, 33–39. doi: 10.1007/BF02825291
Kuc, J. (1987). “Plant immunization and its applicability for disease control, in
Innovative Approaches to Plant Disease Control, ed. I. Chet (New York, NY:
Wiley & Sons), 255–273.
Lawrence, C. B., Joosten, M. H. A. J., and Tuzun, S. (1996). Differential induction of
pathogenesis-relatedproteins in tomato by Alternaria sol ani and the association
of a basic chitinase isozyme with resistance. Physiol. Mol. Plant Pathol. 48,
361–377. doi: 10.1006/pmpp.1996.0029
Lawrence, C. B., Singh, N. P., Qiu, J., Gardner, R. G., and Tuzun, S. (2000).
Constitutive hydrolytic enzymes are associated with polygenic resistance of
tomato to Alternaria solani and may function as an elicitor release mechanism.
Physiol. Mol. Plant Pathol. 57, 211–220. doi: 10.1006/pmpp.2000.0298
Li, C. Y., Liu, G. H., Xu, C. C., Lee, G. I., Bauer, P., Ling, H. Q., et al. (2003).
The tomato Suppressor of prosystemin-mediated responses 2 gene encodes a
fatty acid desaturase required for the biosynthesis of jasmonic acid and the
production of a systemic wound signal for defense gene expression. Plant Cell
15, 1646–1661. doi: 10.1105/tpc.012237
Liu, J. Y., Maldonado-Mendoza, I., Lopez-Meyer, M., Cheung, F., Town, C. D.,
and Harrison, M. J. (2007). Arbuscular mycorrhizal symbiosis is accompanied
by local and systemic alterations in gene expression and an increase in
disease resistance in the shoots. Plant J. 50, 529–544. doi: 10.1111/j.1365-
313X.2007.03069.x
Liu, R. J. (1995). Effect of vesicular-arbuscular mycorrhizal fungi on verticillium
wilt of cotton. Mycorrhiza 5, 293–297. doi: 10.1007/BF00204965
López-Ráez, J. A., Verhage, A., Fernández, I., García, J. M., Azcón-Aguilar, C.,
Flors, V., et al. (2010). Hormonal and transcriptional profiles highlight
common and differential host responses to arbuscular mycorrhizal fungi and
the regulation of the oxylipin pathway. J. Exp. Bot. 61, 2589–2601. doi:
10.1093/jxb/erq089
Macri, F., Braidot, E., Petrussa, E., and Vianello, A. (1994). Lipoxygenase activity
associated to isolated soybean plasma membranes. Biochim. Biophys. Acta 1215,
109–114. doi: 10.1016/0005-2760(94)90098-1
Maffei, G., Miozzi, L., Fiorilli, V., Novero, M., Lanfranco, L., and Accotto, G. P.
(2014). The arbuscular mycorrhizal symbiosis attenuates symptom severity and
reduces virus concentration in tomato infected by Tomato yellow leaf curl
Sardinia virus (TYLCSV). Mycorrhiza 24, 179–186. doi: 10.1007/s00572-013-
0527-6
Makandar, R., Nalam, V. J., Lee, H., Harold, N. T., Dong, Y. H., and Shah, J. (2012).
Salicylic acid regulates basal resistance to Fusarium head blight in wheat. Mol.
Plant-Microbe Interact. 25, 431–439. doi: 10.1094/MPMI-09-11-0232
Maldonado-Bonilla, L. D., Betancourt-Jiménez, M., and Lozoya-Gloria, E.
(2008). Local and systemic gene expression of sesquiterpene phytoalexin
biosynthetic enzymes in plant leaves. Eur. J. Plant Pathol. 121, 439–449. doi:
10.1016/j.plantsci.2014.04.008
Mauch-mani, B., and Slusarenko, A. J. (1996). Production of salicylic acid
precursors is a major function of phenylalanine ammonia-lyase in the
resistance of Arabidopsis to Peronospora parasitica.Plant Cell 8, 203–212. doi:
10.1105/tpc.8.2.203
Mitsuhara, I., Iwai, T., Seo, S., Yanagawa, Y., Kawahigasi, H., Hirose, S., et al.
(2008). Characteristic expression of twelve rice PR1 family genes in response
to pathogen infection, wounding, and defense-related signal compounds. Mol.
Genet. Genomics 279, 415–427. doi: 10.1007/s00438-008-0322-9
Mukerji, K. G., Manoharachary, C., and Chamola, B. P. (2002). Techniques in
Mycorrhizal Studies. Dordrecht: Kluwer Academic Publishers.
Pan, S. Q., Ye, X. S., and Kuc, J. (1991). Association of beta-1,3-glucanase
activity and isoform pattern with systemic resistance to blue mould in tobacco
induced by stem injection with Peronospora tabacina or leaf inoculation with
tobacco mosaic virus. Physiol. Mol. Plant Pathol. 39, 25–39. doi: 10.1016/0885-
5765(91)90029-H
Pozo, M. J., and Azcón-Aguilar, C. (2007). Unraveling mycorrhiza-induced
resistance. Curr. Opin. Plant Biol. 10, 393–398. doi: 10.1016/j.pbi.2007.
05.004
Pozo, M. J., Azcón-Aguilar, C., Dumas-Gaudot, E., and Barea, J. M. (1999). β-1,3-
Glucanase activities in tomato roots inoculated with arbuscular mycorrhizal
fungi and/or Phytophthora parasitica and their possible involvement in
bioprotection. Plant Sci. 141, 149–157. doi: 10.1016/S0168-9452(98)00
243-X
Pozo, M. J., Cordier, C., Dumas-Gaudot, E., Gianinazzi, S., Barea, J. M., and
Concepción, A. A. (2002). Localized versus systemic effect of arbuscular
mycorrhizal fungi on defence responses to Phytophtora infection in tomato
plants. J. Exp. Bot. 53, 525–534. doi: 10.1093/jexbot/53.368.525
Pozo, M. J., Jung, S. C., López-Ráez, J. A., and Azcón-Aguilar, C. (2010). “Impact
of arbuscular mycorrhizal symbiosis on plant response to biotic stress: the
role of plant defence mechanisms,” in Arbuscular Mycorrhizas: Physiology and
Function, eds H. Koltai and Y. Kapulnik (Amsterdam: Springer), 193–207.
Pozo, M. J., Van Loon, L. C., and Pieterse, C. M. J. (2005). Jasmonates-
signals in plant-microbe interactions. J. Plant Growth Regul. 23, 211–222. doi:
10.1007/BF02637262
Pozo, M. J., Verhage, A., García-Andrade, J., García, J. M., and Azcón-Aguilar, C.
(2009). “Priming plant defence against pathogens by arbuscular mycorrhizal
fungi, in Mycorrhizas-Functional Processes and Ecological Impact,edsC.
Azcón-Aguilar, S. Gianinazzi, J. M. Barea, and V. Gianinazzi-Pearson (Berlin:
Springer-Verlag), 123–135.
Ramadan, A., Muroi, A., and Arimura, G. (2011). Herbivore-induced maize
volatiles ser ve as priming cues for resistance against post-att ack by the specialist
armyworm Mythimna separata.J. Plant Interact. 6, 155–158.
Rasmann,S.,DeVos,M.,Casteel,C.L.,Tian,D.,Halitschke,R.,Sun,J.Y.,etal.
(2012). Herbivory in the previous generation primes plants for enhanced insect
resistance. Plant Physiol. 158, 854–863. doi: 10.1104/pp.111.187831
Redecker, D., Kodner, R., and Graham, E. (2000). Glomalean fungi from
the Ordovician. Science 289, 1920–1921. doi: 10.1126/science.289.5486.
1920
Rillig, M. C., and Mummey, D. L. (2006). Mycorrhizas and soil structure. New
Phytol. 171, 41–53. doi: 10.1111/j.1469-8137.2006.01750.x
Robert-Seilaniantz, A., Grant, M., and Jones, J. D. G. (2011). Hormone crosstalk
in plant disease and defense: more than just jasmonate-salicylate antagonism.
Annu. Rev. Phytopathol. 49, 317–343. doi: 10.1146/annurev-phyto-073009-
114447
Ruiz-Lozano, J. M., Azcón, R., and Gómez, M. (1996). Alleviation of salt stress by
arbuscular-mycorrhizal Glomus species in Lactuca sativa plants. Physiol. Plant
98, 767–772. doi: 10.1034/j.1399-3054.1996.980413.x
Safir, G. (1968). The Influence of Vesicular Mycorrhiza on the Resistance of
Onion to Pyrenochaeta terrestris.The Influence of Vesicular Mycorrhiza on the
Resistance of Onion to Pyrenochaeta terrestris. MS. thesis, Unive rsity of Illinois,
Urbana.
Schaller, F., Schaller, A., and Stintz, A. (2005). Biosynthesis and metabolism
of jasmonates. J. Plant Growth Regul. 23, 179–199. doi: 10.1007/s00344-004-
0047-x
Slaughter, A., Daniela, X., Flors, V., Luna, E., Hohn, B., and Brigitte, M. M. (2012).
Descendants of primed Arabidopsis plants exhibit resistance to biotic stress.
Plant Physiol. 158, 835–843. doi: 10.1104/pp.111.191593
Smith, S. E., Facelli, E., Pope, S., and Smith, F. A. (2010). Plant performance
in stressful environments: interpreting new and established knowledge of the
roles of arbuscular mycorrhizas. Plant Soil 326, 3–20. doi: 10.1007/s11104-009-
9981-5
Smith, S. E., and Read, D. J. (2008). Mycorrhizal Symbiosis, 3nd Edn. London:
Academic Press.
Song, Y. Y., Zeng, R. S., Xu, J. F., Li, J., Shen, X., and Yihdego, W. G. (2010).
Interplant communication of tomato plants through underground common
mycorrhizal networks. PLoS ONE 5:e13324. doi: 10.1371/journal.pone.00
13324
Sriram, S., Raguchander, T., Vidhyasekaran, P., Muthukrishnan, S., and
Samiyappan, R. (1997). Genetic relatedness with special reference to virulence
among the isolates of Rhizoctonia solani causing sheath blight in rice. J. Plant
Dis. Prot. 104, 260–271.
Frontiers in Plant Science | www.frontiersin.org 12 September 2015 | Volume 6 | Article 786
Song et al. Disease resistance primed by mycorrhiza
Ton, J., D’Alessandro, M., Jourdie, V., Jakab, G., Karlen, D., Held, M., et al. (2006).
Priming by airborne signals boosts direct and indi rect resistance in maize. Plant
J. 49, 16–26. doi: 10.1111/j.1365-313X.2006.02935.x
van der Heijden, M. G. A., Klironomos, J. N., Ursic, M., Moutoglis, P., Streitwolf-
Engel, R., Thomas, B., et al. (1998). Mycorrhizal fungal diversity determines
plant biodiversity, ecosystem variability and productivity. Nature 365, 69–72.
van Hulten, M., Pelser, M., van Loon, L. C., Pieterse, C. M. J., and Ton, J. (2006).
Costs and benefits of priming for defense in Arabidopsis.Proc. Natl. Acad. Sci.
U.S.A. 103, 5602–5607. doi: 10.1073/pnas.0510213103
van Wees, S. C. M., van der Ent, S., and Pieterse, C. M. J. (2008). Plant immune
responses triggered by beneficial microbes. Curr. Opin. Plant Biol. 11, 443–448.
doi: 10.1016/j.pbi.2008.05.005
Vierheilig, H., Coughlan, A. P., Wyss, U., and Piché, Y. (1998). Ink and vinegar,
a simple staining technique for arbuscular-mycorrhizal fungi. Appl. Environ.
Microbiol. 64, 5004–5007.
Vogelsang, K. M., Reynolds, H. L., and Bever, J. D. (2006). Mycorrhizal
fungal identity and richness determine the diversity and productivity of
a tallgrass prairie system. New Phytol. 172, 554–562. doi: 10.1111/j.1469-
8137.2006.01854.x
Worrall, D., Holroyd, G. H., Moore,J. P., Glowacz, M., Croft, P., Taylor, J. E., et al.
(2012). Treating seeds with activators of plant defence generates long-lasting
priming of resistance to pests and pathogens. New Phytol. 193, 770–778. doi:
10.1111/j.1469-8137.2011.03987.x
Ye, M., Song, Y. Y., Long, J., Wang, R. L., Baerson, S. R., Pan, Z. Q.,
et al. (2013). Priming of jasmonate-mediated antiherbivore defense responses
in rice by silicon. Proc. Natl. Acad. Sci. U.S.A. 110, E3631–E3639. doi:
10.1073/pnas.1305848110
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
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Frontiers in Plant Science | www.frontiersin.org 13 September 2015 | Volume 6 | Article 786
... In the present work, two groups of root associated-fungi, arbuscular mycorrhizal fungi (AMF) and entomopathogenic fungi (EPF), were studied for their effects on tomato plant resistance against a phytopathogen and on plant growth under greenhouse conditions. AMF are obligate mutualistic symbionts colonizing plant roots (Thygesen et al., 2004;Fritz et al., 2006;Song et al., 2015;Mustafa et al., 2017;Bidellaoui et al., 2019;Ravnskov et al., 2020). They improve plant nutrient acquisition and abiotic stress tolerance, but AMF can also protect the plant against attack by necrotrophic pathogens and arthropod herbivores through the induction of systemic resistance (Pozo and Azcón-Aguilar, 2007). ...
... However, we did not find protection against B. cinerea by the AMF inoculation alone, rather a slight increase in disease severity in one experimental repetition, and no additional protective effects were observed by co-inoculation with EPF. This is in contrast to previous studies reporting of AMF inoculated tomato plants showing protection against B. cinerea infections (Sanchez-Bel et al., 2016;Sanmartin et al., 2020b) and against other fungal phytopathogens in different plant hosts (Thygesen et al., 2004;Fritz et al., 2006;Song et al., 2015;Mustafa et al., 2017;Bidellaoui et al., 2019;Ravnskov et al., 2020). ...
Article
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Many fungi live intimately associated with plants and may benefit or harm the host plant. Improved knowledge of such interactions is needed for increasing plant health and crop productivity by implementation of fungal inoculants. Co-inoculations of different beneficial fungi offer the possibility to understand complex plant-microbe interactions that may by functionally complementary for improved plant production and protection. Here, we studied the individual and combined effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae with three isolates of entomopathogenic fungi (EPF), representing Metarhizium brunneum, M. robertsii and Beauveria bassiana, on protection against the foliar phytopathogen Botrytis cinerea and on plant growth. Seedlings of tomato (Solanum lycopersicum L. var. Moneymaker) were inoculated in the substrate with AMF or EPF alone and in dual combinations under greenhouse conditions. Inoculation with the different EPF isolates reduced lesion sizes of B. cinerea on inoculated tomato leaves, but only in the experimental repetition that showed highest level of disease severity. The AMF F. mosseae had no additional effect on B. cinerea lesion size in combinations with EPF. In the experimental repetition with least disease severity, the AMF treatment led to limited increase of B. cinerea lesion sizes. In general, F. mosseae caused an increase in plant biomass, and the co-inoculations of AMF and EPF did in some combinations increase plant growth. Below-ground interactions between AMF and EPF were observed, as the presence of AMF in the roots was associated with a decrease of EPF root colonization densities. However, AMF colonization rates were unaffected by EPF presence. The study indicated a functional complementarity between EPF and AMF by suppressing phytopathogens and increasing plant growth, respectively. However, it further revealed the challenge of obtaining consistent results of plant-microbe-phytopathogen interactions, which must be overcome for future implementation of beneficial fungi as inoculants in plant production.
... An important number of genes related to hormone signalling are involved in the enhanced resistance or tolerance of mycorrhizal plants to infection by F. virguliforme [27]. The systems of mycorrhizal plants play an active role in the disease resistance process, and plants with mycorrhizae are generally more resistant to soilborne pathogens than plants that lack them [28,29]. The induction of defense responses in mycorrhizal plants was much higher and more quickly than that in non-mycorrhizal plants when infected by pathogens [29]. ...
... The systems of mycorrhizal plants play an active role in the disease resistance process, and plants with mycorrhizae are generally more resistant to soilborne pathogens than plants that lack them [28,29]. The induction of defense responses in mycorrhizal plants was much higher and more quickly than that in non-mycorrhizal plants when infected by pathogens [29]. However, it is unclear how the physiological and molecular responses of mycorrhizal and nonmycorrhizal prevent the infection of apple seedlings with F. solani. ...
Article
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Background Apple (Malus domestica Borkh.) is an important economic crop. The pathological effects of Fusarium solani, a species complex of soilborne pathogens, on the root systems of apple plants was unknown. It was unclear how mycorrhizal apple seedlings resist infection by F. solani. The transcriptional profiles of mycorrhizal and non-mycorrhizal plants infected by F. solani were compared using RNA-Seq. Results Infection with F. solani significantly reduced the dry weight of apple roots, and the roots of mycorrhizal apple plants were less damaged when the plants were infected with F. solani. They also had enhanced activity of antioxidant enzymes and a reduction in the oxidation of membrane lipids. A total of 1839 differentially expressed genes (DEGs) were obtained after mycorrhizal and non-mycorrhizal apple plants were infected with F. solani. A gene ontogeny (GO) analysis showed that most of the DEGs were involved in the binding of ADP and calcium ions. In addition, based on a MapMan analysis, a large number of DEGs were found to be involved in the response of mycorrhizal plants to stress. Among them, the overexpressed transcription factor MdWRKY40 significantly improved the resistance of the apple ‘Orin’ callus to F. solani and the expression of the resistance gene MdGLU by binding the promoter of MdGLU. Conclusion This paper outlines how the inoculation of apple seedlings roots by arbuscular mycorrhizal fungi responded to infection with F. solani at the transcriptional level. In addition, MdWRKY40 played an important role in the resistance of mycorrhizal apple seedlings to infection with F. solani.
... JAs involved in plant responses to biotic and abiotic stresses (Wasternack and Hause, 2002), also play an essential for mycorrhiza-primed disease resistance (Song et al., 2015). In the AMF developmental stage upon the formation of the perpetration apparatus (PPA), fungal hyphae enter the plant host cell, accompanied by high-frequency calcium spiking (Sieberer et al., 2012). ...
... The AM colonization in tomato plants is influenced by JA through modification and expression of genes involved in carbohydrate partitioning (Tejeda-Sartorius et al., 2008). The positive role of JA in Glomus fasciculatum of JA-deficient tomato lines spr-2 and def-1 found lower AM colonization, which restores by application of methyl jasmonates (Song et al., 2013(Song et al., , 2014(Song et al., , and 2015. In addition, exogenous application of JA increased the expression of genes, and CKs levels in potato plants (Miransari, 2010). ...
Chapter
Arbuscular mycorrhizal fungi (AMF), the most common association form a symbiotic association with more than 80% of land plants. During AMF symbiosis, fungal hyphae and root cells exchange signal molecules. Phytohormones are biostimulants that stimulate host roots and influence AMF development. Phytohormones regulate the presymbiotic stage of AMF from the early recognition of events up to the formation of arbuscular, hyphal branching and consequently influence arbuscular mycorrhizal (AM) intra and extra-radical hyphae and spore formation. The crucial roles of numerous phytohormones, such as auxins, cytokinins, gibberellins, etc., in the modulation of AM symbiosis, have been known. However, new generation phytohormones such as strigolactones, salicylates (salicylic acid), brassinosteroids, and jasmonic acid are involved in the recognition and regulation of fungal growth are needed to be explicated. The hypothesis is that some phytohormones promote AMF formation in situ and consequently the synergistic effect may stimulate plant growth. In this chapter, we provided the current status of the role of these plant hormones on AMF and associated mechanisms involved in the regulation of AM symbiosis.
... In this study, it is also observed that the induced activity of PAL was found in plants inoculated with bacterial strains and was statistically significant in contrast to other treatments. The induced PAL activity in tomato is reported upon treatment with Bacillus thuringiensis [48] and Funneliformis mosseaae [60]. ...
Article
Wilt disease, caused by Fusarium oxysporum. f. sp. lycopersici, is a global threat to tomato production that needs to be addressed seriously. The current research envisages the use of two self-compatible Bacillus strains, Bacillus tequilensis PKDN31 and Bacillus licheniformis PKDL10, in a combinatorial approach. The spent supernatant of liquid cultures from strains PKDN31 and PKDL10 showed in vitro antifungal activity against Fusarium sp. attaining an inhibition percentage of 95.33% and 96.54%, respectively. The bacterial isolates lytic activity against Fusarium oxysporum was evaluated by scanning electron microscopic analysis and lytic enzyme production of amylase, lipase, protease and β-1,3 glucanase. Furthermore, PKDN31 and PKDL10 produced siderophores and had root colonizing ability that enhanced the biocontrol efficiency. Combined in vivo inoculation of Bacillus tequilensis PKDN31 and Bacillus licheniformis PKDL10 on tomato seeds revealed that the strains could induce systemic resistance in tomato against Fusarium oxysporum. f. sp. lycopersici by increasing defence enzymes such as β-1,3 glucanase, polyphenol oxidase, peroxidase, phenylalanine ammonia-lyase, chitinase, and total phenol accumulations. Pot culture experiments also proved the biocontrol efficacy of the above dual culture supplementation as this treatment displayed a better growth as well as defense against Fusarium challenge compared to the controls. The obtained results suggest that rhizobacterial isolates could be employed as systemic resistance inducers and biocontrol agents in tomato plants to protect against Fusarium wilt disease.
... On the other hand, the AMF-plant interaction could favor the resistance of the plant to soil phytopathogens through the competition for space and nutrients, induced systemic resistance and the establishment of other antagonistic microorganisms in the mycorrhizosphere (Battini et al., 2017;Cameron et al., 2013). In this sense, there have been reports of induced systemic resistance and reduced nematode populations (Elsen et al., 2008;Vos et al., 2012); control of and reduction of early blight in tomato through increased activity of glucanase and chitinase (Song et al., 2015). In addition, bioprotection of chili pepper against Verticillium spp. ...
Article
Chili pepper (Capsicum annuum L.) is an important horticultural crop in Mexico in terms of both diet and culture. However, chili pepper production is associated with excessive use of agrochemicals both pesticides and mineral fertilizers causing adverse environmental and human health problems. Hence, alternative biological disease control measures and crop nutrition strategies are needed. Arbuscular mycorrhizal fungi (AMF) that naturally associate with roots of most crop plants including chili pepper are known to promote plant nutrition and root health, represent a biological alternative to reduce the use of agrochemicals in chili pepper production. In three greenhouse pot experiments, we examined the response of five chili pepper genotypes to single and combined inoculation with the AMF Rhizophagus irregularis and the root pathogen P. capsici in terms of plant growth response and root disease development. Main results showed that inoculation with R. irregularis caused differential plant growth promotion in the five chili pepper genotypes examined, but had no effect on the disease development caused by P. capsici. Single inoculation with P. capsici resulted in differential root disease development in the chili pepper genotypes. Most interesting, a Chilaca landrace genotype not only showed resistance against P. capsici, but on the contrary also resulted in plant growth promotion. In conclusion, our work show that the examined chili pepper genotypes responded differentially to single inoculation with R. irregularis and P. capsici, but dual inoculation had no effect on the level of root disease caused by P. capsici. The observed resistance to P. capsici in the Chilaca landrace could be a source of resistance to the pathogen in plant breeding programs and used directly by chili pepper growers that could also benefit from pre-inoculating chili pepper transplants with AMF before planting in terms of plant growth promotion.
... This phenomenon, called mycorrhiza-induced resistance (MIR), is generally associated with the priming of the plant immune system, resulting in faster and/or enhanced activation of the JA-regulated defences (Jung et al., 2012;Martinez-Medina et al., 2016;Pozo & Azcón-Aguilar, 2007). The defence priming associated with AM symbiosis provides the plant with a cost-effective mechanism of protection against pathogens and pests (Jacott et al., 2017;Sanmartín et al., 2020;Song et al., 2013Song et al., , 2015. ...
Article
Full-text available
Arbuscular mycorrhizal (AM) symbiosis modulates plant‐herbivore interactions. Still, how it shapes the overall plant defense strategy and the mechanisms involved remain unclear. We investigated how AM symbiosis simultaneously modulates plant resistance and tolerance to a shoot herbivore, and explored the underlying mechanisms. Bioassays with Medicago truncatula plants were used to study the effect of the AM fungus Rhizophagus irregularis on plant resistance and tolerance to Spodoptera exigua herbivory. By performing molecular and chemical analyses, we assessed the impact of AM symbiosis on herbivore‐triggered phosphate (Pi)‐ and jasmonate (JA)‐related responses. Upon herbivory, AM symbiosis led to an increased leaf Pi content by boosting the mycorrhizal Pi‐uptake pathway. This enhanced both plant tolerance and herbivore performance. AM symbiosis counteracted the herbivore‐triggered JA burst, reducing plant resistance. To disentangle the role of the mycorrhizal Pi‐uptake pathway in the plant´s response to herbivory, we used the mutant line ha1‐2, impaired in the H+‐ATPase gene HA1, which is essential for Pi‐uptake via the mycorrhizal pathway. We found that mycorrhiza‐triggered enhancement of herbivore performance was compromised in ha1‐2 plants. AM symbiosis thus affects the defense pattern of M. truncatula by altering resistance and tolerance simultaneously. We propose that the mycorrhizal Pi‐uptake pathway is involved in the modulation of the plant defense strategy. This article is protected by copyright. All rights reserved.
... Second, mycorrhizal fungi can induce defense responses on the host plant, including improved leaf chemical defenses against herbivores and pathogens (Cameron et al. 2013;Minton et al. 2016;Kaling et al. 2018;Vishwanathan et al. 2020;Frew et al. 2021). This defense response tends to be stronger for EM than for AM tree species (Gange et al. 2005;Vishwanathan et al. 2020), but have also been observed in AM plants (Li et al. 2010;Shrivastava et al. 2015;Song et al. 2015). Finally, tree species associating with AM and EM fungi often have different leaf functional traits. ...
Article
Full-text available
Tree mycorrhizal type plays an important role in promoting plant species diversity and coexistence, via its mediating role in conspecific negative density dependence (CNDD), i.e., the process by which an individual’s performance is impaired by the density of conspecific plants. Previous findings suggest that ectomycorrhizal (EM) tree species are generally less susceptible to CNDD than arbuscular mycorrhizal (AM) tree species, due to the chemical and physical protection that EM fungi provide their host with. We examined how CNDD effects on leaf herbivory, seedling growth, and survival differ between AM and EM seedlings of ten tree species collected over 3 years in an old-growth temperate forest in northeastern China. We found that AM and EM seedlings differed in how conspecific density affected their leaf herbivory, seedling growth, and survival. Specifically, AM seedlings leaf herbivory rates significantly increased with increasing conspecific seedling and adult density, and their growth and survival rates decreased with increasing conspecific adult density, these patterns were, however, absent in EM seedlings. Our work suggests that AM seedlings have a performance disadvantage relative to EM seedlings related to the negative effects from conspecific neighbors. We highlight the importance of integrating information on seedling leaf herbivory, seedling growth, to provide further understanding on potential mechanisms driving differences in CNDD between AM and EM tree seedlings.
... Wang et al. (2018) reported that WRKY70 modulated by Bacillus cereus triggered induced systemic resistance through activating SA signaling pathway in Arabidopsis leaves. In the same trend, Song et al. (2015) revealed that mycorrhizal colonization by defense priming increases the resistance of tomato plants to early blight. To our knowledge, this is the first report about the induction of UDP by Streptomyces strains in tomato plants probably to detoxify the fungal pathogen. ...
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