IOBC-WPRS Bulletins, 89: 303-307

From the lab to the field scale: dose-dependent efficacy and mode of
action of resistance inducers in wheat against Septoria tritici leaf
blotch
Marie-Eva Ors1,2,3,4, Batrice Randoux1, Ali Siah2, Sameh Selim3, Fabienne Boizet2,4,
Cline Ade4, Gilles Couleaud4, Claude Maumen4, Patrice Halama2 and Philippe
Reignault1
1
Unit de Chimie Environnementale et Interactions sur le Vivant (UCEIV), GIS PhyNoPi,
Universit du Littoral Cte dOpale, C.S. 80699, F-62228, Calais cedex, France.
2
Laboratoire Biotechnologie et Gestion des Agents Pathognes en agriculture (BioGAP), 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
4
Arvalis-Institut du vgtal, station exprimentale de Boigneville, F-91720 Boigneville France
Abstract The use of resistance inducers in crop protection is one of the most promising strategies that
could be considered as either alternative or complementary to the use of conventional fungicides.
However, the protection efficacy conferred by such inducers is often compromised in field conditions
since their activity depends on many environmental factors. Here, we investigated the dose effect of
four resistance inducers (referred as FSOV2, FSOV4, FSOV7 and FSOV10) on the protection of
wheat against Mycosphaerella graminicola, the causal agent of Septoria tritici leaf blotch (SLB). Our
investigations were performed on the susceptible wheat cultivar Alixan in both laboratory and field
conditions. At the laboratory scale, each inducer was used on its own at four different doses. At the
field level, each inducer was used at a single dose, on its own or in mixture with other inducers, and in
association with the Cherokee fungicide (50 g/L cyproconazole + 62,5 g/L propiconazole + 375 g/L
chlorothalonil). A significant protection level was observed for all products, but the efficacy obtained
clearly increased with the dose used. Further investigations revealed that all inducers exhibited at the
highest dose only a direct in vitro effect on the fungus. A dose-dependant effect on host penetration
and on the induction of peroxidase (PO) activity involved in plant defence mechanisms was
highlighted for FSOV7 and FSOV10.
Key words: Mycosphaerella graminicola, wheat, plant resistance inducers, elicitors.

Introduction
Septoria tritici leaf blotch (SLB) caused by Mycosphaerella graminicola (anamorph:
Zymoseptoria tritici) is one of the most devastating foliar diseases on wheat. Since no fully
resistant wheat cultivar is available, the use of azoles and strobilurins has been the main
strategy to control this disease, leading during the last decade to the emergence and rapid
widespread of resistances to these fungicides (Siah et al., 2010a). In addition, in the current
context of growing concern about the extensive use of fungicides, alternative strategies
allowing the reduction of their applications are developed by farmers. Stimulation of plant
defense mechanisms using plant resistance inducers is one of the most interesting strategies.
However, their efficacy still seems compromised in the field since their activity depends on
many conditions including environment, wheat cultivars and applied doses. We therefore
aimed in the present study at characterizing the dose effect of four resistance inducers
1

(referred as FSOV2, FSOV4, FSOV7 and FSOV10) on the induction of wheat resistance
against M. graminicola at both laboratory and field scales.
First, the protective effect of all resistance inducers against SLB was assessed in laboratory
(controlled or semi-controlled) and field conditions on the susceptible wheat cultivar Alixan
at different doses. Furthermore, the dose effect of these products was assessed either on in
vitro fungal growth (spore germination and mycelial growth), on host penetration or on the
induction of peroxidase (PO) activity known to be involved in reactive oxygen species
defense pathway (Reignault et al., 2001; Shetty et al., 2003).

Material and methods

Doses of resistance inducers
Four plant resistance inducers were used in this study: FSOV2 (mineral-ion based inducer),
FSOV4 (polysaccharide-based inducer), FSOV7 (microbial-extract-based inducer) and
FSOV10 (polysaccharide-based inducer). In laboratory conditions, FSOV2, FSOV7 and
FSOV10 were tested at four doses (D1, D2, D3 and D4), while FSOV4 was tested only at two
doses (D1 and D4). D4 was the lowest dose and corresponds to 3L/ha for FSOV2, 0,5L/ha for
FSOV4, 5L/ha for FSOV7 and 1.5Kg/ha for FSOV10. The other doses were calculated as
follows: D1 (highest dose) = 63D4, D2 = (D1+D4)/2 and D3 = (D2+D4)/2. In the field, each
inducer was tested at the D4 concentration in association with the Cherokee fungicide at 0.7
L/ha (35% of the homologated dose) and in mixture or not with another inducer (Table 1).
Protection efficacy assay at the laboratory scale
The protection efficacy of the inducers against M. graminicola was assessed in controlled and
semi-controlled conditions on wheat cv. Alixan. In controlled conditions, tests were
performed on wheat plantlets (2nd fully expanded leaf) in a growth chamber in staggered rows
under day-night cycle 16/8 h at 20/16 C with 80 % relative humidity. Light intensity was 56
E.m-2.s-1 for 16 h.day-1. Plantlets were first pretreated with 15 mL of solution containing
FSOV7 or FSOV10 at D4, 48 h before inoculation with the T01193 M. graminicola strain
using 30 mL of spore suspension (106 spores/mL).
In semi-controlled conditions, experiments were performed in the greenhouse on three-weeks
old plants pretreated with FSOV2, FSOV7 or FSOV10 at D1, D2, D3 and D4 or with FSOV4
at D1 and D4, 48 h before inoculation. Three pots of 12 plants each were used for each
condition. Fungal inoculum preparation and plant growth conditions were performed
according to Siah et al. (2010b). 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.
Protection efficacy assay at the field scale
The protection efficacy of the inducers in the field was assessed on Alixan wheat plants
grown in randomized blocs. Three blocks were used for each condition. The treatments were
performed at D4 on growth stage (GS) Z30 with each inducer on its own or in mixture with
another inducer and on GS Z32 to Z37 in mixture with Cherokee at 0.7 L/ha (35% of the
homologated dose). The disease severity obtained in each condition was measured by
determining the area under the disease progress curve (AUDPC).

Cytological analyses, peroxidase activity and biocide effect evaluation

The effect of each inducer on host penetration was investigated at 5 dai, according to Siah et
al. (2010b). Quantification of PO activity was performed at 0, 1, 3, 5 and 11 dai using 0.1 g of
fresh leaf tissue, according to Reignault et al. (2001). The direct effect of the products on
spore germination and mycelial growth was assessed in vitro on T01193 using PDA plates
amended with different concentrations of each inducer, according to Siah et al. (2010a).

Results and discussion

Dose-dependant protection efficacy conferred by resistance inducers
Protection efficacy of FSOV2, FSOV4, FSOV7 and FSOV10 against M. graminicola was
assessed at different doses in both laboratory and field conditions. In laboratory conditions,
the protection conferred by the inducers varied with the dose used (Table 1). The obtained
efficacies vary with the applied inducer. In addition, each inducer applied on its own
conferred a protection at D1 but not at D4 and the efficacy of the inducers decreased with the
doses that were applied (Table 1). Indeed, FSOV7 remained highly effective at D1, D2 and
D3 and not at D4. The efficacy of FSOV2 was reduced at D2 and D3 and could not be
observed at D4. As for FSOV10, the protection was strongly reduced at D2 and could not be
observed at D3 and D4. These results therefore indicate that the protection efficacy of these
inducers against SLB is dose-dependent even in laboratory conditions.
On the contrary, under field conditions, all inducers except FSOV4 led to a protection in the
field at D4 when tested alone or when combined to another inducer. Such a difference
between laboratory and field conditions could be explained by the combination of the
inducers with the fungicide Cherokee at the field level. A synergic effect between this
triazole and some resistance inducers could explain such a better efficacy against SLB in the
field at D4 since similar results were obtained in other field experiments (data not shown).
Moreover, the use of FSOV4 in combination with FSOV2 or FSOV10 in the field led to a
higher protection efficacy when compared to these inducers applied on their own (Table 1). In
addition to the greater efficacy of FSOV10 when used in combination with FSOV4 (Table 1),
such a combination also improved the crop yield (data not shown). The use of other
combinations of resistance inducers will therefore be considered in our future investigations.

Table 1. Protection levels obtained with FSOV2, FSOV4, FSOV7 and FSOV10 in laboratory
and field conditions at D1, D2, D3 and/or D4 doses.
Laboratory conditions
Products
FSOV2
FSOV4
FSOV7
FSOV10
FSOV2+4
FSOV10+4

D1

D2
+++
+
+++
+++
nt
nt

D3
++
nt
+++
+
nt
nt

Field conditions
D4

++
nt
+++
nt
nt

nt
nt

D4+Cherokee 0.7L/ha
++
+
++
+++
++

Under controlled conditions: +++: efficacy > 50%, ++: efficacy from 30 to 50%, +: efficacy < 30%, -:
no efficacy compared to untreated plants, nt: non-tested.

Under field condition: +++: efficacy equivalent to high reference (Cherokee 1.4L/ha, >50%), ++:
middle efficacy, +: efficacy equivalent to low reference (Cherokee 0.7L/ha, <30%)

Dose-dependent direct biocide effect of the resistance inducers

At the D1 dose, all inducers exhibited a direct in vitro biocide effect on M. graminicola spore
germination and fungal mycelial growth. However, this effect was not observed for FSOV7 at
D2 and significantly decreased for FSOV2 and FSOV4 at D2 and D3. Only FSOV10 showed
a slight biocide effect at D4 (data not shown).
Dose-dependent effect of FSOV7 and FSOV10 on host penetration
Rates of host penetration events at 5 dai in plants were compared between plants pretreated
with FSOV7 and FSOV10 at D3 in semi-controlled conditions and D4 in controlled ones. The
percentage of both direct and stomatal penetrations did not differ between plants treated or not
with FSOV7 or FSOV10 at D4. On plants treated at D3, both types of penetration events were
reduced on plants treated with FSOV7, whereas only stomatal penetrations were reduced with
FSOV10 (data not shown).
Dose-dependent effect of FSOV7 and FSOV10 on peroxidase activity
Peroxidase (PO) activity was measured in plants pretreated with FSOV7 and FSOV10 at D3
(semi-controlled conditions) and D4 (controlled conditions). The induction of PO activity
took place at 1 dai in plants treated at D3 with both inducers but only on plants treated with
FSOV10 at D4 (Figure 1). Although PO activity at 1 dai was higher on plants treated with
FSOV10 with both doses compared to the other treatments, this inducer didnt protect wheat
against SLB (Figure1, Table 1). This result indicates that PO activity, which is known
involved in wheat defence mechanisms (Randoux et al., 2010), could not be considered as a
standard marker of induced protection in wheat against M. graminicola.

(A)

(B)

Figure 1. Peroxidase activity amounts in cv. Alixan plants pretreated or not with FSOV7 or FSOV10
at D4 in controlled conditions (A) and D3 in semi-controlled conditions (B).

Acknowledgements
This research was supported by FSOV and Arvalis-Institut du Vgtal.

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