Response to infection by Fusarium graminearum in the rachis of a resistant and a
susceptible barley cultivar
S. Shea Miller, Mehri Hadinezhad, Denise Chabot and Eva M. Watson
Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, ON, K1A 0C6
Fig. 1. Cross sections through
the rachis node at the base of
the inoculated floret, 5 dpi. F =
vasculature derived from floret;
R = rachis vasculature.
Fusarium head blight (FHB), caused by Fusarium
graminearum, is a serious problem in cereals in North
America, as well as globally. Infection causes dramatic yield
losses and impairs the quality of the grain that is produced,
because of the presence of mycotoxins, and also because of
changes in grain functionality. One widely accepted strategy
to control FHB is the use of resistant varieties. Our previous
results in wheat (1,2) and barley (3) have indicated that a
component of resistance is expressed in the rachis,
particularly in the node at the base of the floret, where the
fungus is slowed or prevented from progressing further into
the head. In this study, we have used a strain of F.
graminearum transformed to express green fluorescent
protein (GFP-Fg) to follow the infection process in a resistant
(Chevron) and a susceptible (Chapais) cultivar (cv) of barley.
In addition, results of chemical analysis of the rachis are
presented.
a) Chevron, mock inoculated.
Note autofluorescence of the
cell walls in and surrounding
the vascular bundles in both
regions (arrowheads), and that
the lumen of the vessels is
clear. b) Chapais, Fusarium
inoculated. Note that the
vessels derived from the floret
(upper part of the node) are full
of green mycelium (arrows).
c) Chevron, Fusarium
inoculated. Note decrease in
autofluorescence in floret
vascular cell walls (arrows), as
well as the presence of
pigmented deposits
(arrowheads) in the node..
Materials & methods
Plants were grown in growth cabinets (20 °C day, 15 °C
night, 16 h of light per day) in 4 inch peat pots. Upon
flowering (still in the flag leaf sheath) heads were point
inoculated in two lateral spikelets from the same node using
GFP-Fg for microscopy, or a wild-type F. graminearum for
chemical analysis. Several nodes were inoculated on the
same head for chemical analysis. Heads were harvested at
day 5 days post inoculation (dpi), then fixed and
cryosectioned for microscopy.
For chemical analysis,
heads were harvested at 4 dpi, and the rachis freeze dried
and ground. The chemical composition of the resulting
material was evaluated by sequential gravimetric analysis,
and carbohydrate profile analysis using HPLC-ELSD.
Gravimetric analysis
Barley rachis, Chevron and Chapais cultivars,
mock and Fusarium inoculated
Hot water
fraction
Lignin fraction
Hemicellulose
fraction
Cellulose
fraction
Results & discussion
ChP_F
Hot water, 19.4
lignin, 11.8
Cellulose, 44.0
Cellulose, 49.6
lignin, 14.9
Hemicellulose,
28.2
Hemicellulose,
24.5
ChV_H
ChV_F
Hot water,
15.9
Cellulose,
45.9
Hot water,
18.8
Cellulose,
42.0
lignin, 12.2
lignin, 23.2
Hemicellulose
, 26.1
Hemicellulose
, 20.5
Fig. 2. Chemical composition of barley rachis. ChP, Chapais;
ChV, Chevron; _H, mock inoculated; _F, Fusarium inoculated.
3.5
Fructose
Glucose
Sucrose
3
2.5
2
1.5
1
0.5
0
ChP_H
Gravimetric analysis
 In all treatments, cellulose was the fraction with the
highest content (Fig. 2).
 The content of lignin in the infected samples of Chevron
(23.2 ± 1.2 %, w/w dried rachis tissue) was significantly
higher than in Chapais (12.2 ± 1.2 %) which could reflect
cell wall fortification in Chevron as a defence against
FHB.
 Sucrose was the predominant free sugar in the water
soluble fraction of all treatments (Fig 3.), being nearly
twice the content of fructose or glucose.
 The fungus moved from the base of the infected floret into
the vascular bundles of the node, and down into the rachis.
Typically, the infection travelled farther, faster in the rachis in
Chapais than in Chevron.
 In Chapais, green fluorescence in the vascular bundles of the
inoculated node which are derived from the floret (Fig. 1b,
upper half of the node) shows the extensive colonization by
the fungus, compared with the node of a water-inoculated
plant (Fig. 1a).
 The sugar composition of the cellulose fraction was
similar for all treatments (Fig. 4), glucose being the main
component with small amounts of xylose and fructose
which could be from hemicellulose contamination and
hydrolysis byproducts.
 Chevron appeared visually to be more affected by the fungus
in the inoculated node as evidenced by loss of fluorescence
in the vascular bundles, and the presence of dark coloured
deposits blocking some of the vascular bundles (Fig. 1c);
however, less fungus was visible, and progression of the
hyphae into the rachis was restricted.
 The content of glucose in the cellulose fraction was
higher in the rachis of Fusarium inoculated Chevron
compared to Chapais, suggesting stronger cell wall
structure in the resistant cultivar.
ChP_F
ChV_H
ChV_F
Fig. 3. Sugar profile of water soluble fraction of barley rachis
Xylose,
7.7
ChP_H
 In both cultivars, while the content of hemicellulose and
cellulose decreased after inoculation with Fusarium
compared to the mock inoculated samples, indicating
some cell wall degradation by fungal enzymes, the lignin
and water soluble components increased.
 The total content of free sugars (sucrose, glucose and
fructose) was significantly higher in the Fusarium
inoculated Chapais (5.3 ± 0.2 %, w/w dried rachis tissue)
compared to Chevron (4.5 ± 0.2 %), suggesting greater
fungal degradation of the cell wall and stored fructans in
the susceptible cultivar.
Microscopy
Hot water,
10.3
ChP_H
Sugar concentration (% w/w of dried rachis)
Introduction
ChP_F
Glucose, 83.7
Glucose, 83.8
Fructose,
10.3
ChV_H
ChV_F
Glucose,
84.3
Glucose,
82.5
Fig. 4. Sugar composition of cellulose fraction of barley rachis
Conclusions
 Microscopy showed cell wall changes, particularly in the
vascular bundles of the inoculated floret in Chevron,
compared to the susceptible cv Chapais, which are
reflected in the gravimetric results.
 Chemical composition analysis of the rachis demonstrated
significantly higher lignification in response to Fusarium
infection in the resistant barley compared to the
susceptible cultivar.
References
1. Miller et al (2004). Can. J. Plant Pathol. 26: 453-463
2. Miller et al (2011) Botany 80:301-311
3. Miller, S.S. 2003. Proceedings , 3rd CWFHB (Winnipeg, MB) p. 86