Comm. Appl. Biol.

Sci, Ghent University, 2014

PROTECTION EFFICACY AND MODES OF ACTION OF

TWO RESISTANCE INDUCERS ON WHEAT AGAINST
SEPTORIA TRITICI BLOTCH

M. ORS1, 4, A. SIAH2, B. RANDOUX1, S. SELIM3, G. COULEAUD4, C.

MAUMENE4, K. SAHMER5, Ph. REIGNAULT1, P. HALAMA2.

Unit de Chimie Environnementale et Interactions sur le vivant (UCEIV), GIS PhyNoPi, Universit du Littoral Cte dOpale, Universit Lille-Nord de France,
C.C. 80699, F-62228, Calais Cedex, France.
2
Laboratoire Biotechnologie et Gestion des Agents Pathognes en agriculture, GIS PhyNoPi,
Institut Suprieur dAgriculture,
48 Boulevard Vauban, F-59046 Lille cedex, France
3
Biotechnology and Plant Pathology Platform, GIS PhyNoPi, Institut Polytechnique LasalleBeauvais, 19 rue Pierre Waguet, BP 30313, F-60026 Beauvais Cedex, France
4
Arvalis-Institut du Vgtal, Station exprimentale de Boigneville, F-91720 Boigneville, France.
5
Groupe ISA, Equipe Sols et Environnement, Laboratoire Gnie Civil et goEnvironnement
(LGCgE) Lille Nord de France (EA 4515), 48 Boulevard Vauban, 59046 Lille Cedex, France

ABSTRACT
Septoria tritici blotch caused by Mycosphaerella graminicola is one of the most devastating foliar
diseases of wheat. Disease control relies heavily on fungicides, but frequent development of
fungal resistance and the negative impact of their extensive use on the environment and human health increasingly compromise this control strategy. Plant resistance inducers could be an
alternative to conventional fungicides to control in a more durable manner this pathogen. Here,
we tested in the greenhouse two resistance inducers (FSOV7 and FSOV10) on two wheat cultivars, Alixan (susceptible) and Altigo (resistant), against M. graminicola. FSOV7 conferred a
significant protection level on both cultivars, while FSOV10 conferred a significant protection
level on the resistant cv. Altigo only. Furthermore, the modes of action of the two inducers
were examined using cytological, biochemical and molecular approaches. In planta, investigation of the infection process showed that FSOV10 significantly reduced fungal spore germination, whereas FSOV7 did not. An association of protection efficacy with a decrease of fungal
biomass and fungal -1, 4-endoxylanase and protease activities was observed in both cultivars.
However, no association of plant peroxidase activity with protection efficacy was recorded,
whatever the cultivar and the resistance inducer. A RT-qPCR assay revealed significant inductions of the expression of genes involved in different defense pathways; further statistical analyses should determine which genes are associated with the observed protection efficacies. This
study showed significant inducer-cultivar interactions on wheat against M. graminicola and
allowed us to investigate the modes of action on wheat of the two studied resistance inducers.
Keywords: Mycosphaerella graminicola, Zymoseptoria tritici, Wheat, Resistance inducers.

2
INTRODUCTION
Septoria tritici blotch (STB) is one of the most devastating foliar wheat ( Triticum aestivum L.)
diseases. It is caused by the hemibiotrophic fungus Mycosphaerella graminicola (anamorph:

Zymoseptoria tritici). This disease is currently controlled mainly by the use of fungicides. However, this use is becoming increasingly controversial because of their impact on both the environment and human health. One alternative to chemical control is breeding for new and, if
possible, fully resistant wheat cultivars. Such a strategy requires knowledge concerning wheat
defense mechanisms. The infectious process of M. graminicola is characterized by a latent
period during which the germ tube grows on the leaf surface, penetrates through the stomata
or directly via the epidermic cell wall, and colonizes the intercellular space between mesophyll
cells. After approximately two weeks, the fungus switches to necrotrophic growth associated
with the increase of the fungal biomass and the formation of pycnidia in colonized substomatal
cavities (Duncan and Howard, 2000; Kema et al., 1996). It has been shown that the production
of plant cell wall-degrading enzymes (CWDEs) by the fungus, such as -1, 4-endoxylanase, is
correlated with symptoms, the sporulation level (Douaiher et al., 2007a, 2007b; Siah et al.,
2010), and is highly associated with resistance level of wheat cultivar (Tian et al., 2009). In
recent studies, the early expressions of PR1- and PR2- (-1,3- glucanase) encoding genes have
been suggested to explain wheat resistance to M. graminicola (Adhikari et al., 2007; Ray et al.,
2003; Yang et al., 2013a, 2013b). It has also been reported that wheat peroxydase activity is
more important in a resistant cultivar than in a susceptible one in infectious conditions (Shetty
et al., 2003). The aim of our study was to investigate the protection efficacy and the modes of
action of two resistance inducers (RIs) named FSOV7 and FSOV10 on two wheat genotypes
differing in resistance levels against M. graminicola (Alixan, susceptible and Altigo, resistant).
We used cytological, biochemical and molecular approaches to determine the effect of both RIs
on (i) the infectious process and the pathogenicity of the fungus and on (ii) the induction of
resistance in both genotypes during the early biotrophic phase.

RESULTS AND DISCUSSION

Protection efficacy
The protection efficacy conferred by FSOV7 and FSOV10 on Alixan and Altigo cultivars against
STB was evaluated by quantifying the percentage of leaf lesion area (symptoms) and pycnidial
density (Figure 1). As expected, the levels of symptoms and sporulation in the untreated inoculated plants (control) were significantly more important on Alixan (susceptible cultivar) compared to Altigo (resistant cultivar). Results showed a significant reduction of symptoms and

Comm. Appl. Biol. Sci, Ghent University, 2014

sporulation by FSOV7 on both cultivars, whereas FSOV10 reduced significantly the pycnidial
density on Altigo only.

60

% of leaf area with

pycnidia

40

20

nTI
FSOV7
FSOV10
a

A
Pycnidial density

80

nTI
FSOV7
FSOV10

100

4
3

B
a

0
Alixan

Altigo

Alixan

Altigo

Figure 1. Efficacy of FSOV7 and FSOV10 on symptoms (A) and sporulation (B) at 21 days after
inoculation on Alixan and Altigo wheat cultivars, compared to non treated inoculated control
(nTI). Means tagged with the same letter are not significantly different using the Tukey test at

P = 0.05.
Infectious process and pathogenicity

The level of host colonization at 21 dai, especially when it is measured through the rates of
colonized substomatal cavities and substomatal cavities bearing pycnidia (Figure 2), turned out
to be associated with the obtained protection efficacy (Figure 1). The reduction of colonization
was observed on Alixan plants treated with FSOV7 and on Altigo plants treated with FSOV7 and
FSOV10 (Figure 2A). The percentage of colonized substomatal cavities with pycnidia is
significantly lower in untreated Altigo plants. A significant reduction of pycnidia was observed
only on Alixan plants treated with FSOV7. Our results therefore indicate that the protection
efficacy conferred by the inducers is the result of an inhibition of host tissue colonization

100

60

80

ab
bc
cd

40
20

50
% of colonized
substomatal cavities
with pycnidia

% of substomatal cavities
with colonization

required for disease establishment.

40
30

nTI
FSOV7
FSOV10

20
10

0
Alixan

Altigo

Alixan

Altigo

Figure 2. Efficacy of FSOV7 and FSOV10 on substomatal colonization (A) and pycnidia formation (B) at 21 dai on the two wheat cultivars studied compared to non treated inoculated
control (nTI). Means tagged with the same letter are not significantly different using the Tukey
test at P = 0.05.
The -1, 4-endoxylanase activity involved in M. graminicola pathogenicity was measured in the
susceptible cultivar Alixan according to a time course throughout the infectious process. As

4
expected, the production of -1, 4-endoxylanase increased significantly after 17dai with the
beginning of sporulation in the non treated inoculated control (Figure 3). In plants treated with
FSOV7, a production of -1, 4-endoxylanase activity was observed at 21dai but the production
amounts were significantly less important than in the non treated inoculated control. With
FSOV10, this activity increased after 17dai, reaching the same level than in non treated inoculated control (figure 3). In Altigo, no difference between these different conditions could be
recorded because of the low sporulation rate obtained on this cultivar (data not shown).

-1,4-endoxylanase
activity
(mU.mg-1 total proteins)

0,4

nT nI
nT I
FSOV7 inoculated
FSOV10 inoculated

0,3

0,2

0,1

bc
c

1dai

5dai

0
11dai

17dai

21dai

Figure 3. Time course of -1, 4-endoxylanase activity production in cv. Alixan treated with
FSOV7 or FSOV10 and inoculated with T01193 (FSOV7 inoculated and FSOV10 inoculated
respectively) and compared to non treated and non inoculated (nTnI) and non treated and
inoculated (nTI) controls. Means tagged with the same letter are not significantly different using
the Tukey test at P = 0.05.

Induced defenses.

Peroxydase (PO) activity was measured on Alixan at different time points after treatment with
FSOV7 or FSOV10, and after fungal inoculation performed at 48h after treatment (hat) (figure
4). FSOV10 induced a significant change in PO activity, on the contrary to FSOV7 which did not
athough it conferred a protection on Alixan.,In contrast, FSOV10 didnt protect Alixan but induced PO activity in this cultivar. The induction of PO activity could therefore not be considered
as a protection marker at least in the Alixan cultivar.
nTnI
nTI
FSOV7
FSOV7 inoc.
FSOV10
FSOV10 inoc.

Peroxydase activity
(nKat. mg-1 total protein)

70
60
50

ab
ab
abc

40

cde

30

cdefghijkl

bcde
cdefg

cdefgh

20

fghijkl
hijkl

10

fghijkl

hijkl

ijkl
kl

0
0hat

12hat

24hat

48hat

54hat

kl
60hat

kl
72hat

120hat

Comm. Appl. Biol. Sci, Ghent University, 2014

Figure 4. Time course of peroxydase (PO) activity on cv. Alixan treated with FSOV7 and
FSOV10 in infectious and non-infectious conditions (FSOV7, FSOV7 inoculated, FSOV10 and
FSOV10 inoculated). The arrow at 48hat means the occurrence of inoculation. nTnI, non treated and non inoculated; nTI, non treated and inoculated. Means tagged with the same letter are
not significantly different using the Tukey test at P = 0.05.
The induced mechanisms were also investigated at the molecular level. The expression of the
pathogenesis-related protein encoding genes PR1 and PR2 was quantified by RT-qPCR at 12, 24
and 48 hat with FSOV7 and FSOV10 in non infectious conditions (Figure 5). While a significant
protection level conferred by FSOV7 was recorded in Alixan and Altigo, the expression of PR1
wasnt significantly induced and only the expression of PR2 was induced in Alixan and not in
Altigo at 48h after treatment by FSOV7. However, the expression of PR1 at 48hat was more
important in plants treated with FSOV10 than in non treated plants in both cultivars and this
induction was significantly more important in Altigo than in Alixan. The expression of PR2 was
significantly more important in Altigo treated with FSOV10 at 24hat, and at 48hat in Alixan. In
their studies, Adhikari et al. (2007) and Shetty et al. (2009) observed an early peak of PR1 and

PR2 expression in a resistant cultivar after inoculation with M. graminicola. This rapid induction
of PR1 and PR2 expression could therefore be associated with the resistance level. In our work,
early elicitation of PR1 and PR2 was observed in the resistant cultivar treated with FSOV10. We
recorded a more important induction of PR1 at 48hat and a earlier induction of PR2 in Altigo
than in Alixan, which could be associated to the protection efficacy recorded in this cultivar,
resulting from the induced defense mechanisms.

Expression level

35
30
25

Alixan nT
Alixan FSOV7
Alixan FSOV10
Altigo nT
Altigo FSOV7
Altigo FSOV10

20
b

15
10
5

cd

cd
cdcd cdcd

cd
cdcdcdcdcd

c
cd

16
14
Expression level

40

cdcd

12
10
8
6
4
2

Alixan nT
Alixan FSOV7
Alixan FSOV10
Altigo nT
Altigo FSOV7
Altigo FSOV10

b
b
c

cde
de dededede

de

cd
cde
de

cde
de

de e

0
12hat

24hat

48hat

12hat

24hat

48hat

Figure 5. Relative gene expression of PR1 (A) and PR2 (-1,3- glucanase) (B) in Alixan and
Altigo leaves treated with FSOV7 and FSOV10 in non-infectious condition. nT, non-treated
control. Means tagged with the same letter are not significantly different using the Tukey test at
P = 0.05.

6
MATERIEL AND METHODES
Protection efficacy assay
Two plant resistance inducers were used in this study: FSOV7 (microbial extract-based product)
and FSOV10 (polysaccharide-based product). The protection efficacy of these two inducers
against M. graminicola was assessed in the greenhouse on three-week-old plants of the susceptible Alixan and the resistant Altigo wheat cultivars. The treatments were performed 48 h before
inoculation with the M. graminicola strain T01193). Each pot was treated with 30 mL of a solution containing each product at the following concentrations: FSOV7 at 8.72 L/mL and FSOV10
at 26.18 L/mL. Fungal inoculum preparation and plant cultivation were performed according to
Siah et al.(2010). Three pots of 12 plants were used for each condition. The disease severity
was scored at 21 days after inoculation (dai) by evaluating the percentage of third leaf area
covered by lesions (chlorosis and necrosis) bearing pycnidia. The sporulation intensity was
evaluated by scoring the pycnidium density according to the following scale: 0= absence of
pycnidia, 1= 1 to 20 %; 2= 20 to 40 %; 3= 40 to 60 %; 4= 60 to 80 % and 5= 80 to 100 % of
diseased leaf area containing pycnidia.

Cytological analyses
The effect of each product on spore germination and host colonization was investigated at 1
and 21 dai, respectively, according to the protocol described by Siah et al. (2010).

Xylanase and peroxidase activities

Total proteins extraction and quantification of xylanase activity were done at 1, 5, 11, 17 and
21 dai using 2.5 g fresh leaf tissue according to Siah et al. (2010), with the use of beach tree
xylan as a substrate. Extraction and quantification of peroxidase (PO) activity were performed
at 1, 5, 11, 17 and 21 dai using 0.1 g fresh leaf tissue according to (Reignault et al., 2001).

RNA extraction and gene expression quantification by RT-qPCR.

Wheat leaves were sampled at 12, 24 and 48 hours after treatment (hat) with FSOV7 and
FSOV10. and immediately stored at -80 C until use. Total RNA was extracted from 80 mg of
plant tissue using RNeasy Plant Mini Kit (Qiagen, The Netherlands). Reverse transcription of
total RNA was carried out using QuantiTect Reverse Transcription Kit (Qiagen, USA) according
to the manufacturers protocol. Real-time qPCR was performed using ABI Prism 7300 detection
system (Applied Biosystems, USA). The primers pairs (PR1, HQ848391 and PR2, DQ090946)
were designed using the Primer Express program and were tested for secondary structure
using AmplifX program. The tub6 (U76897) and GAPDH (AF251217) genes were used as
housekeeping genes. The cycling conditions were: denaturation (95 C for 3 min); amplification

Comm. Appl. Biol. Sci, Ghent University, 2014

and quantification repeated 40 times (95 C for 3 s, 60 C for 30 s). Expression ratios for each
cDNA were calculated for each time point, relative to the non-treated control (nT) at the same
time using the 2-Ct calculation method described by (Livak and Schmittgen, 2001), where Ct
= [Ct Target (Sample) - Ct Reference (Sample)] - [Ct Target (Control) - Ct Reference (Control)]
and Ct Reference = geometrical mean (Ct GAPDH : Ct Tub6). The expression level of target
genes in non-treated control samples used for relative expression is considered to be 1.

Statistical analysis

Performed experiments involved distinct and independent series of data and several factors:
day of the kinetics, inoculated or non-inoculated conditions and two independent experiments.
We performed multi-factorial ANOVA to compare the data from each condition (Alixan or Altigo,
inoculated or control), and independent experiments were considered as random factors. The
Tukey test was used to compare the means obtained in each inoculated cultivars and to check
statistical significance (P 0. 05). For enzymatic assay, data were submitted to a logarithmic
transformation. Statistical analyses were carried out with the statistical program Xlstat
(Addinsoft, Paris, France).

REFERENCES
Adhikari, T.B., Balaji, B., Breeden, J., and Goodwin, S.B. (2007). Resistance of wheat to Mycosphaerella
graminicola involves early and late peaks of gene expression. Physiol. Mol. Plant Pathol. 71, 5568.
Cohen, L., and Eyal, Z. (1993). The histology of processes associated with the infection of resistant and
susceptible wheat cultivars with Septoria tritici. Plant Pathol. 42, 737743.
Douaiher, M.-N., Nowak, E., Durand, R., Halama, P., and Reignault, P. (2007a). Correlative analysis of
Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the
importance of xylanase and polygalacturonase. Plant Pathol. 56, 7986.
Douaiher, M.-N., Nowak, E., Dumortier, V., Durand, R., Reignault, P., and Halama, P. (2007b). Mycosphaerella graminicola produces a range of cell wall-degrading enzyme activities in vitro that vary with
the carbon source. Eur. J. Plant Pathol. 117, 7179.
Duncan, K.E., and Howard, R.J. (2000). Cytological analysis of wheat infection by the leaf blotch pathogen Mycosphaerella graminicola. Mycol. Res. 104, 10741082.
Kema, G.H.J., Yu, D., Rijkenberg, F.H.J., Shaw, M.W., and Baayen, R.P. (1996). Histology of the pathogenesis of Mycosphaerella graminicola in wheat. Phytopathology 86, 777786.
Livak, K.J., and Schmittgen, T.D. (2001). Analysis of Relative Gene Expression Data Using Real-Time
Quantitative PCR and the 2CT Method. Methods 25, 402408.
Ray, S., Anderson, J.M., Urmeev, F.I., and Goodwin, S.B. (2003). Rapid induction of a protein disulfide
isomerase and defense-related genes in wheat in response to the hemibiotrophic fungal pathogen
Mycosphaerella graminicola. Plant Mol. Biol. 53, 741754.

8
Reignault, P., Cogan, A., Muchembled, J., Lounes-Hadj Sahraoui, A., Durand, R., and Sancholle, M.
(2001). Trehalose induces resistance to powdery mildew in wheat. New Phytol. 149, 519529.
Shetty, N.P., Kristensen, B.K., Newman, M.-A., Mller, K., Gregersen, P.L., and Jrgensen, H.J.L. (2003).
Association of hydrogen peroxide with restriction of Septoria tritici in resistant wheat. Physiol. Mol.
Plant Pathol. 62, 333346.
Siah, A., Deweer, C., Duyme, F., Sanssen, J., Durand, R., Halama, P., and Reignault, P. (2010). Correlation of in planta endo-beta-1,4-xylanase activity with the necrotrophic phase of the hemibiotrophic
fungus Mycosphaerella graminicola. Plant Pathol. 59, 661670.
Tian, S.M., Weinert, J., and Zhao, Q.H. (2009). Correlation between cell wall-degrading enzymes in
wheat leaves infected by Septoria tritici and disease severity. Can. J. Plant Pathol. 31, 387392.
Yang, F., Melo-Braga, M.N., Larsen, M.R., Joergensen, H.J.L., and Palmisano, G. (2013a). Battle through
signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics. Mol. Cell. Proteomics mcp.M113.027532.
Yang, F., Li, W., and Jrgensen, H.J.L. (2013b). Transcriptional Reprogramming of wheat and the hemibiotrophic pathogen Septoria tritici during two phases of the compatible interaction. PLoS ONE 8, e81606.