differential occurrence of suberized sheaths in canes of grapevines suffering from black dead arm,...
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ORIGINAL PAPER
Differential occurrence of suberized sheaths in canes of grapevinessuffering from black dead arm, esca or Eutypa dieback
Pierrette Fleurat-Lessard • Andree Bourbouloux •
Florence Thibault • Eric Menard • Emile Bere •
Christophe Valtaud • Gabriel Roblin
Received: 23 July 2012 / Revised: 24 January 2013 / Accepted: 5 February 2013 / Published online: 20 February 2013
� Springer-Verlag Berlin Heidelberg 2013
Abstract Compared to healthy canes of Ugni-Blanc
grapevines, structural modifications were observed in
August in wood of growing canes showing foliar symptoms
induced by esca and Eutypa Dieback. The observed
changes appeared attenuated in canes of the current season
sampled in December. In contrast, Black Dead Arm (BDA)
did not induce significant modifications compared to
healthy canes. A seasonal pattern in suberization was
observed in control canes since a first suberized sheath
occurred in mid-July at the interface xylem ring/pith and a
second sheath was built up in August till December at the
interface primary phloem/cortex. The same pattern was
observed in BDA-attacked vines. In contrast, these con-
tinuous structural barriers were not formed in July and
August in canes of the current season in grapevines
attacked by esca or Eutypa dieback, but restored in the
canes observed in December. These structural
modifications were quantified and these events were dis-
cussed in the scope of plant physiology and pathogenicity
of the implied fungi.
Keywords Black dead arm � esca � Eutypa dieback �Grapevine � Histology � Suberin
Introduction
Fungi colonizing the trunk of grapevines induce wood
diseases that are responsible for extended damage in
vineyards in major grape-producing areas around the world
(Chiarappa 1959; Carter et al. 1983). In Black Dead Arm
(BDA), the bark tissue of the diseased parts becomes dis-
coloured to dark-brown and formed large vertical streaks
which extended from trunk until canes. In addition, yellow-
orange areas at the edges of the streaks may also be
recorded (Larignon et al. 2001). The nature of the infecting
agents in this disease remains controversial, although it is
not recognized that members of Botryosphaeriaceae (in
particular Botryosphaeria dothidea, Neofusicoccum par-
vum and Diplodia seriata) (Larignon et al. 2001; Auger
et al. 2004) are involved in the spreading of the disease (for
review, see Urbez-Torres 2011). Esca is a complex disease
involving successive fungal infections and, in particular,
Phaeomoniella chlamydospora and Phaeoacremonium
aleophilum have been associated with the disease (Lari-
gnon and Dubos 1997). The xylem-inhabiting fungi pro-
duce two types of necrotic areas in the wood of the trunk.
On one hand, black spots, observed in cross-sections, are
sparsely distributed in the woody tissues around the pith, in
which the deuteromycete Phaeomoniella chlamydospora
can be isolated with high frequency. On the other hand, in
a pink-brown area, located in the central zone, the
Communicated by R. Hampp.
P. Fleurat-Lessard (&) � A. Bourbouloux � F. Thibault �C. Valtaud � G. Roblin
Universite de Poitiers, Equipe de Physiologie Moleculaire
du Transport des Sucres, EBI CNRS-UMR 7267,
Batiment Botanique B31, 3, rue Jacques Fort,
86022 Poitiers, France
e-mail: [email protected]
E. Menard
Station Viticole du Bureau National Interprofessionnel
du Cognac, 69 Rue de Bellefonds, 16101 Cognac, France
e-mail: [email protected]
E. Bere
Universite de Poitiers, Image UP, Service de Microscopie
Electronique et Photonique, Pole Biologie Sante, Batiment B36,
1, rue Georges Bonnet, 86022 Poitiers, France
e-mail: [email protected]
123
Trees (2013) 27:1087–1100
DOI 10.1007/s00468-013-0859-z
deuteromycete Phaeoacremonium spp., in particular
P. aleophilum has been found with high frequency (Mugnai
et al. 1999; Valtaud et al. 2009). Esca infection includes
other pathogenic fungi namely the basidiomycete Fomiti-
poria mediterranea, most often isolated in white-rotted
wood corresponding to the last stage of wood degradation
(Tabacchi et al. 2000; Fischer 2006). A new definition of
esca was proposed by Surico (2001, 2009) for the syn-
dromes into which this disease has been fragmented. Thus,
when damage in wood and leaf symptoms occurred toge-
ther, the term ‘‘esca proper’’ was retained. For Eutypa
dieback, the case is easier since the causal agent was soon
identified being the ascomycete Eutypa lata. Ascospores
infect the xylem tissue and then colonize adjacent lignified
parts in trunk (Moller and Kasimatis 1978). The spreading
of the fungus results in a characteristic brown and wedge-
shaped necrosis (Dubos 1994).
Hyphae of pathogenic fungi develop in vessel elements
that contain high amount of water and nutrients, but also in
fibres and in rays, well-adapted for the spreading of these
pathogens that find nutritional sources due to the high storage
of starch from August until February. In addition, the high
wall damage observed in fibres in which large cavities are
caused by Eutypa lata (Rudelle et al. 2005) and esca fungi
(Valtaud et al. 2009) indicate that fungi are able to liberate
cellulose, hemicellulose and pectin from lignin incrustation,
and therefore, can find nutrients from xylem walls.
In current canes of the year, in which only one xylem
ring occurs (Fig. 1a, b), symptoms characteristic of the
diseases can be detected successively along the foliate
period: Eutypa Dieback in June, BDA in July and esca in
August (Valtaud et al. 2011). However, these diseases are
insidious because the first visible foliar symptoms may
develop several years after the planting of grapevines and
vary from 1 year to another on the same stock. In BDA,
brown spots appear generally in June at the leaf margin and
in intraveinal position, leading to necrosis invading the
entire blade. In the mild form of esca, the leaves in summer
present chlorotic intraveinal areas that later become
necrotic. In the case of Eutypa Dieback, symptoms occur at
the end of spring as dwarfed and withered shoots and, later,
as marginal chlorosis leading to leaf necrosis and dying of
inflorescence. A severe form of these diseases is charac-
terized by a sudden and rapid wilting of the leaves leading
to the death of an arm or the entire stock. These symptoms
are induced at distance from the trunk area, where fungi are
located, indicating that molecular signals, produced by the
pathogens and flown in the transpiration stream, spread
from the trunk to the canes (Mahoney et al. 2003; Octave
et al. 2009; Fleurat-Lessard et al. 2010).
Disease symptoms are spatially and temporally removed
from the initial infection of the vine that makes their
relationship understanding more difficult (review by
Mundy and Manning 2011). Following the pathogen attack,
defence mechanisms are built up by the plant and in par-
ticular, physical barriers are produced to limit the pathogen
invasion. Thus, in Eutypa Dieback and esca, tyloses
occlude the vessels in the trunk, therefore, restricting the
vertical spreading of sap and microorganisms (Octave et al.
2006a; Valtaud et al. 2009). Tylosis has also been observed
in the brown band occurring in the arms in case of BDA
(unpublished data). Defence mechanisms are triggered in
trees following pathogen injury and, notably, the barrier
constituted by tyloses can be reinforced by a suberin
deposit (Rioux et al. 1995). Suberin is a hydrophobic
polymer composed of an aliphatic and aromatic domain
(Kolattukudy 1981), observed at various plant-environment
interfaces particularly in the bark, and in coat of the seeds,
but also in the periderm of roots and shoots and in the
endodermis and hypodermis of roots. It is also laid natu-
rally in protective continuous boundaries, for example in
abscission areas, in bundle sheaths of certain leaves and in
the Caspary band (see reviews by Kolattukudy 2001;
Bernards 2002). Suberization occurs as thin layers suc-
cessively deposited on the internal wall border of cells in
which cytoplasm next degenerates. The chemical compo-
sition of suberin has been determined (reviews by Bernards
2002; Kolattukudy 2001; Franke et al. 2005) and the
molecular mechanism of its biosynthesis and deposition
began to be documented (Beisson et al. 2007; Hofer et al.
2008; Franke et al. 2009; Molina et al. 2009; Soler et al.
2011). According to its lipidic wax nature, suberin forms
apoplastic barriers limiting gas and water loss, controlling
nutrient and water transport, and also protecting against
pathogens spreading by sealing the infected tissues.
Pi
CPPh
CaPi
a b
Xy XyCP
CPPh
Ph
R
Fig. 1 a Transverse sections in a current cane showing a thin cortical
parenchyma (CP) around one growth ring with ordered phloem (Ph)
and xylem (Xy) tissues. b Tangentially bands of fibres (white arrow)
in phloem; many vessels spread in primary (black arrow) and
secondary xylem (star); black-stained starch in numerous and wide
(R) rays enlarged in older phloem and joining pith (Pi); (Ca)
cambium. Ethanol (70 %) fixative, 15 lm-thick sections, a left, Fasga
staining; a right, and b lugol staining, Zeiss Orthoplan (bar in
a 750 lm, in b 150 lm
1088 Trees (2013) 27:1087–1100
123
The aim of the present study was to describe and
quantify structural modifications that occurred in the vas-
cular apparatus in Vitis vinifera cv. Ugni blanc on current
canes from vines showing foliar symptoms of BDA, esca
and Eutypa Dieback. In particular, the observations were
focused on the occurrence and damage of the suberized
sheaths at pith/xylem interface and at the phloem external
border during the development of the vascular apparatus in
the course of an annual cycle.
Materials and methods
Plant material
Observations were mainly carried out on field-grown Ugni-
blanc grapevines grown in the Cognac area. The used RSB
1-109 rootstocks resulted from Vitis riparia and Vitis
berlandieri crossing, well-adapted for both water deficit
and humidity. Sampling in canes of the year occurred on
15 year-old numbered stocks during 4 years (2004, 2005,
2006, 2007) every 2 weeks from May until September
(foliate season) and then every month in the remaining
period. According to the schedule previously carried out
(Valtaud et al. 2011), each year, 12 current cane fragments
were harvested at each stage from six vines chosen at
random over 30 vines defined by no apparent disease foliar
symptoms (i.e. controls), and others showing the charac-
teristic foliar symptoms of BDA, esca or Eutypa Dieback,
described above in the introduction. Observations were
done in the apical part (at 50 cm from the apex) of 1.5 until
2.5 m long canes. The observation of seasonal variations in
suberin occurrence in canes of the year was extended in
2006 and 2007 to Cabernet and Folle Blanche stocks grown
in the vineyards of Service de Protection des Vegetaux
(Poitiers) and to Chasselas and Muscat de Hambourg
stocks grown in the University of Poitiers.
Cuttings, 10 cm long, sampled in January on field-
grown Ugni-blanc vines defined as controls, were inocu-
lated with P. chlamydospora and P. aleophilum by insert-
ing a 5-mm diameter plug of mycelium in a hole (5 mm in
diameter, 20 mm deep) drilled at their top (Octave et al.
2009). These inoculated, and the non-inoculated, cuttings
were grown in greenhouses.
Histocytochemistry and microscopy
Sampling occurred in the Cognac area on vines already
used to study cytophysiological variations that occurred in
leaves (Valtaud et al. 2009, 2011) in relationship with esca,
BDA and Eutypa Dieback diseases. At 50 cm below the
apical part of current canes of the year, fragments (6 cm
long and 0.5–0.75 cm wide) were collected in glass vials
and immersed in 70 % ethyl alcohol. This fixative was
applied for at least a week, but allowed the storage of
samples for several months. Pieces (0.5 cm high) excised
in the middle part of each fragment were cross-sectioned,
using a 2125 RM Leica microtome. The sections (15 lm
thick), obtained in the entire thickness of the canes, were
collected in dry conditions and immediately stained, or
kept one night in the refrigerator. Ten fragments, 6 cm
long, of inoculated- and non-inoculated (control) cuttings
were processed according to this treatment (alcohol fixa-
tion and microtomy).
To detect lignin, at least 6 cross-sections were immersed
in embryo dishes that contained 2 ml of Fasga solution
(Tolivia and Tolivia 1987), � diluted in distilled water.
The reaction occurred one night at room temperature in a
sealed vial to prevent evaporation. The ‘‘Fasga’’ stock
solution contained for 65 ml: 3 ml of 1 % safranin (1 g
safranin ? 1 g sodium acetate ? 75 ml 100 % ethyl
alcohol ? 25 ml distilled water ? 2 ml formaldehyde),
11 ml of 0.5 % alcian blue 8GX (500 mg alcian
blue ? 100 ml 100 % ethyl alcohol), 30 ml of 99 %
glycerol, 1 ml of pure acetic acid and 20 ml of distilled
water. This reaction reveals the distribution of the
red-coloured lignified elements and the blue-coloured
pectocellulose elements. After staining, the sections were
carefully washed in distilled water and laid on glass slides
in a drop of glycerol/distilled water (50 % v/v) mixture.
After adding a coverslip, the sections were observed under
a Zeiss Axioplan microscope equipped with a Kappa
camera to obtain numerized pictures.
In the same conditions, starch detection occurred on the
cross-sections after 10 min immersed in the commonly
employed Lugol solution (2 g potassium iodide and 1 g
Iodine dissolved in 200 ml of distilled water) and were
directly observed. To detect suberin the Wiesner reaction
(Adler et al. 1948, cited by Pomar et al. 2002) occurred as
follows: a freshly prepared 2 % (p/v) phloroglucin solution
in 95 % alcohol was applied for 10 min on the sections,
and after solution removal, a drop of 50 % (v/v) HCl was
added on the sections laid on glass slides. After 5 min
cover slips were added and the sections were immediately
observed, since this staining is transient. As phloroglu-
cinol–HCl presents an affinity for lignin, a pink colour
develops on cinnamaldehyde residue precursors of both
syringyl and guaiacyl lignin monomers. Using UV light
excitation, obtained with a 365–397 nm filter (Zeiss Axi-
oplan microscope and Olympus MVX10 microscope),
lignin autofluorescence was masked with phloroglucinol–
HCl, whereas suberin was highly fluorescent blue. This
protocol is considered to be specific for suberized tissues
(Biggs 1987).
Trees (2013) 27:1087–1100 1089
123
Quantification and data analysis
Measurements were carried out on magnified pictures of
cross-sectioned canes. The surface area occupied by xylem
vessels was taken on the entire surface of vascular bundles.
The surface area of suberized cells in the external sheath and
that of lignified fibre groups were calculated in delimited
phloem areas (Fig. 4a). Similarly, the surface area of suber-
ized cells in the internal sheath was calculated in areas
delimited at the border of primary xylem and pith (Fig. 4b).
In each disease, the measurements were carried on at least 32
pictures, using Image J software. In each picture, the ratio
between the surface area of vessels or that of sheaths and the
corresponding delimited surface areas was calculated. These
surface area ratios were compared between the different
diseases. After testing for normality (calculations and Gauss
curve), values were subjected to the analysis of variance,
followed by the Fischer’s and Duncan’s tests to check the
differences in means. Calculations occurred according to
classical theories and corresponding tables using Excel
software. Differences were considered to be statistically
significant at P \ 0.05. Other details are given in the legends.
Results
Compared anatomy in canes of the year sampled
in August and December, showing BDA, esca
or Eutypa Dieback
The healthy canes of the current season showed a pseudo
axial symmetry with a thin cortical parenchyma and a
vascular ring five- to six-fold wider (Fig. 1a), but charac-
terized by an irregular thickness all around the cane. The
xylem ring, 2 or 3 times thicker than the phloem, contained
a large number of vessels, the largest of which occupied the
median part. The lugol reaction showed abundant starch in
uniseriate and in wide rays spread from phloem border
until pith (Fig. 1b).
In the upper part of the control canes sampled in August,
the Fasga staining (Fig. 2a) allowed to see a cap of lignified
fibres at the top of each vascular bundle. The phloem was
made up of tangential bands of lignified red fibres, alternately
with cellulosic dark-blue-coloured areas of parenchyma cells
associated with sieve elements. The cambium generated a
secondary xylem, 2 or 3 times thicker than the phloem which
consisted of only one single ring with wide vessels (133.1 ±
3.4 lm) in the initial wood generated in spring and smaller
(23.8 ± 1.9 lm) in that formed in summer. The primary
xylem formed a triangle in which the vessels showed an
ordered layout. They were surrounded by parenchyma cells
with slightly lignified walls. In grapevines infected with
BDA, structural features in canes were similar (Fig. 2b) to
that in control. The cambium generated tangential bands of
lignified fibres in phloem and in xylem wide vessels
(140.1 ± 3.1 lm) and then narrower (23.8 ± 15 lm) ones,
as in the control. Tyloses sometimes occurred. By contrast, in
case of esca disease (Fig. 2c) the differentiation of phloem
and xylem was highly modified. The cap of external phloem
fibres with thick walls was present but damage in secondary
phloem was shown well, notably by the reduction of lignified
fibres in which the tangential bands were no longer recog-
nizable. The initial wood was made up of wide vessels
(99.3 ± 2.4 lm) full of tyloses while the final wood con-
tained smaller vessels (34.4 ± 2.3 lm) arranged in no reg-
ular order. In the most damaged areas, the vessels presented
irregular shapes and some were surrounded by thick-walled
fibres. The uniseriate rays were numerous between the
existing wide rays. In the metaxylem, thick-walled fibres
occurred. The protoxylem vessels were grouped at the cru-
shed xylem pole and surrounded by parenchyma cells with a
lignified wall on the edge of the pith. In case of Eutypa
Dieback (Fig. 2d), large vessels (80.1 ± 2.5 lm) occurred in
initial wood and smaller (30.5 ± 2.4 lm) in final wood.
Damage was similar to that in esca, in particular for tan-
gential phloem fibre groups. Regarding wood organization,
measurements taken in the entire vascular bundles have
shown that the surface area occupied by all vessel elements in
xylem tissue corresponded to a ratio of 35 ± 0.06 % in
controls, 37 ± 0.07 % in BDA disease, whereas only
24 ± 0.04 % in esca and Eutypa Dieback.
In December, phloem observed in controls (Fig. 3a)
consisted of blocks of living lignified fibres alternating
with blocks of sieve elements, companion cells and
parenchyma, as observed in August (Fig. 2a). Ray cells
were thick-walled in the xylem, but thin-walled in the
phloem. While early wood consisted of a broad zone of
xylem lignified tissue with wider vessels, late wood that
was initiated in the grapevine at the end of the foliate
season formed a narrow zone. This zone was stronger than
the early wood due to the large volume of wall material
contained in the abundant fibres that surrounded some
narrow vessels (Fig. 3a). In December BDA-attacked
stocks (Fig. 3b) presented a structure similar to that
observed in control stocks. By contrast, in esca (Fig. 3c) or
Eutypa Dieback (Fig. 3d), the structure of canes showed
similarities in the two kinds of diseases, but differed con-
siderably in December from that observed in August. In
particular, well-organized groups of fibres occurred in
phloem and many xylem fibres were regularly scattered
next to the thin cambium zone and around the narrow
vessel elements in the final wood. In addition, 2 or 3 layers
of thick-walled parenchyma cells were observed along the
internal border of primary xylem at pith interface.
The decrease in surface area of groups of phloem fibres
observed in summer in esca and Eutypa diseases and
1090 Trees (2013) 27:1087–1100
123
the higher values of this surface area in December are
sustained by the measurements shown in Fig. 4c. The
comparison of these data indicates that the structure of the
canes varies in the course of a yearly period and,
more importantly, that modification in this structure
induced by the fungal pathogens implicated in esca and
Eutypa Dieback also varied, according to the month of
observation.
Seasonal pattern of suberin deposition
In the experimental scheme described above, observations
were focused on the occurrence of suberin in the current
canes of the year of infected grapevines, according to the
role ascribed to this component as a barrier to pathogen
movement (Kolattukudy 2001).
Young growing canes sampled in control stocks from
the cognac vineyards did not contain suberized sheaths in
May (Fig. 5a, b) and June (Fig. 5c, d). At mid-July, any
suberisation occurred in the phloem area (Fig. 5e), but an
‘‘internal’’ suberized sheath that included 2 or 3 layers of
large cells was observed at the pith margin (Fig. 5f). Next,
in August (Fig. 5g, h) and December (Fig. 5i, j), in addi-
tion to the ‘‘internal’’ sheath, an ‘‘external’’ sheath con-
taining at least four layers of highly suberized cells was
formed at the primary phloem border under the supra
phloemic fibre groups. As shown in longitudinal sections of
canes, these sheaths are continuous, thus, building up two
suberized walls around the vascular ring (Fig. 5k). The
external sheath (Fig. 5m) was formed by regularly aligned
parallelepipedic cells of small sizes (33.3 ± 4.9 lm on
tangential side and 19.7 ± 4.4 on radial side) (Fig. 6a). At
difference, the internal sheath (Fig. 5l), which is at least 6
times thicker than the external sheath (see Fig. 5g–j), was
formed by large isodiametric cells (95.8 ± 4.4 lm)
(Fig. 6d). As observed in TEM, alternate electron opaque
and electron translucent lamellae of suberin, that were
deposited on the inner part of the pectocellulose wall,
occupied a larger area in the external (Fig. 6b, c) than in
the internal sheath (Fig. 6e, f).
BDAcontrol esca Eutypa Dieback
OPFOPF
OPF
V
V
V
V
PFPF PF
R
R
R
R
R
RR
R
Ca
Ty
Ty
Mx
MxMxMx
PiPiPi
Pi
Px
Px
Px Px
XFXF XF
XF
Ty
Ca
Ca
Ca
a
b c d
Fig. 2 Structural features in vascular apparatus of current canes
sampled in August: comparison in control and in vines infected by
intraxylem fungi. a Control cane: outermost groups of phloem fibres
(OPF) with lignified wall and large lumen; in phloem strands of cells
(white square) alternate with groups of lignified fibres (PF); large
rays (R); cambium area (Ca). Ring of secondary xylem with vessels
(V) bordered by VACs (arrow) and many thick-walled xylem fibres
(XF), metaxylem (Mx) and protoxylem (Px), xylem pole surrounded
by small parenchyma cells contiguous with the large vacuolated cells
of pith (Pi). b In BDA disease, cane anatomical features as in control.
c In esca disease, damage in phloem which fibre groups are small-
sized or absent (white star). In secondary xylem small-sized vessels
occur in groups (circle) near cambium (Ca); abundant tylosis (Ty) in
vessels of secondary and primary xylem; crushed protoxylem vessels
surrounded by large parenchyma cells. d In Eutypa Dieback disease,
damage as in esca. Ethanol (70 %) fixative, 15 lm-thick cross-
sections, Fasga staining, Zeiss Orthoplan (bar 100 lm)
Trees (2013) 27:1087–1100 1091
123
Occurrence or damage in suberized sheaths in BDA,
esca or Eutypa Dieback diseases
According to the previous data, the reported observations
made on diseased vines are restricted to three characteristic
periods, namely mid-July, August and December. In mid-
July, the ‘‘internal’’ suberization was similar in controls
(Fig. 7a, b) and in vinestocks in which BDA symptoms
occurred (Fig. 7c, d). In canes where esca developed, any
suberin deposit, occurred in phloem (Fig. 7e), and only a
Control Eutypa DiebackescaBDA
R
R RR R
R
R
V
V V
V
PiPi
Pi
Pi
Px Px
CaCa
Ca
XFXF
XF
PF PF PF
MxMx
MxMx
XF
PF
MxPx
Px
CaLW LW LW
LW
a b c d
Fig. 3 Structural features in vascular apparatus of current canes
sampled in December: comparison in control and in vines infected by
intraxylem fungi. a Control cane: phloem consists of lignified fibres
groups (PF) alternating with groups of cellulose walled cells (white
square) as in August (see Fig. 2a); early wood consists of a broad
zone of lignified tissue with wide vessels and late wood (LW) forms a
narrow zone, with many fibres (XF) and few vessels, that abuts
cambium area (Ca). b BDA disease: structural features as in August
(Fig. 2b). c Esca and d Eutypa Dieback disease: recovery of August
damage (see Fig. 2c, d) since groups of phloem fibres (PF) alternate
with cellulose cells, as in control, and late wood (LW) consists of a
narrow zone of xylem fibres with some vessels; absence of tyloses in
secondary and primary xylem. Other legends as in Fig. 2 (bar
100 lm)
C BDA ESC EUT
a b c a a d a d20
10
Fib
re a
rea
(%
)
External suberized sheath
Internal suberized sheath
Primary xylem
Pith
Phloem fiber groups400 nm
600 nm
700 nm
a
b
c
Fig. 4 Scheme of the surface areas delimited in cane cross sections to
measure the spread of suberin sheath and phloem fibre groups at
external phloem border (a) and suberin sheath at primary xylem/pith
interface (b). c Variation in surface area of phloem fibre groups. In
summer (white bars): high decrease in esca and Eutypa Dieback,
slight decrease in BDA. In winter (black bars): no effect in esca and
Eutypa Dieback, very slight decrease in BDA. Measurements
obtained using Image J software, calculations as mentioned in
‘‘Materials and methods’’; each bar represents the mean ± SE from 4
vines, 8 current canes 94 years; n = 32. Values with the same letters
are not statistically different, according to Fisher’s and Duncan’s test
at P \ 0.05
1092 Trees (2013) 27:1087–1100
123
faint perimedullary deposit was observed (Fig. 7f) and a
similar behaviour was also observed in canes showing
Eutypa Dieback symptoms (Fig. 7g, h). In August, the
periphloemic and perimedullary sheaths detected in con-
trols (Fig. 8a, b) also occurred with the same pattern in
case of BDA (Fig. 8c, d). By contrast, these barriers
became thinner or completely damaged in esca disease
(Fig. 8e, f) and in Eutypa Dieback (Fig. 8g, h). Abundant
tyloses, thick walls in fibres and ray walls were not
suberized. In December, the suberized perimedullary and
phloem sheaths were present in controls canes (Fig. 9a, b)
and BDA infected stocks (Fig. 9c, d) with the same pattern,
as those observed in August. These suberized areas, which
were absent in August in canes of vines showing esca or
Eutypa Dieback disease, now occurred in December
(Fig. 9e–h), indicating that a suberin deposit happened
after fall and along the winter period.
To give support to these structural observations, a
quantitation of suberin surface area was done in the
external and in the internal sheaths, leading to the com-
parison of values between summer (August) and winter
(December). In August, the external sheath area was
decreased in BDA disease, but more highly in esca and
Eutypa Dieback (Fig. 10a). By contrast, in December, this
sheath was not decreased in BDA and esca, but slightly in
Eutypa Dieback (Fig. 10a). In August, the internal sheath
decrease was very slight in BDA, whereas large in esca and
Eutypa Dieback (Fig. 10b). By contrast, in December, the
internal sheath in BDA was similar to that in control
and only a low diminution occurred in esca and Eutypa
Dieback (Fig. 10b).
Modification of suberin sheaths in cuttings infected
with P. chlamydospora and P. aleophilum
Suberin sheaths were observed in cutting cross-sections
made at 4 cm under the infection hole where fungi were
inoculated. In controls, suberin was detected all along the
observation period of 7 months, in the external (Fig. 11a,
c, e, g) as well as in the internal (Fig. 11b, d, f, h) sheath.
By contrast, modifications occurred in cuttings infected
with P. chlamydospora and P. aleophilum. The external
sheath observed till March (Fig. 11i) was still fluorescent
but thinner in May (Fig. 11k), and was absent in August
(Fig. 11m). Damage in the internal sheath (Fig. 11j) was
expressed earlier, since its fluorescence was faint as soon as
Controls inJuneMay Mid-July August December
a b c e g i
d f h j
OPFOPF
OPFOPF
Xy
PiPi Pi Pi
PxPxPx
Px
Px
Ph
Ph Ph
Ph Ph
k l
OPF
Pi
OPFPh
Px
Pi
VR VR
m
Pi
Fig. 5 Detection of suberin sheaths during a yearly period in control
canes observed under visible light (a) and under UV light (b–m). a–
j Cross-sections. a Structure of a 30 cm long growing cane observed
in May. b No suberin detected. c, d Observation in June of a 80 cm
long cane: no suberin detected, respectively, at phloem (Ph) and pith
(Pi) border; Px, protoxylem. e, f Observation in Mid-July in a 120 cm
long cane: no suberin at phloem border but presence of a blue ring
detected at pith border (white arrow). g, h Observation in August:
occurrence of an external suberin sheath (red arrow) between
outermost phloem fibres (OPF) and primary phloem and an internal
suberin sheath at pith border (white arrow). i, j Observation in
December: occurrence of the two suberized sheaths, respectively, at
the external (red arrow) and the internal part of the vascular ring
(white arrow). k–m Longitudinal sections : sheaths are continuous
around the vascular ring (VR) of control canes sampled in December
(k). The internal sheath at pith border includes large cells (l) and the
external sheath includes two or three layers of small-sized cells (m).
Ethanol (70 %) fixative, phloroglucin staining (a) and UV light
observation (b–m); (a–j) Zeiss Orthoplan, (k–m) Olympus MVX10
(bar in a–j, l, m 100 lm, in k 1.8 mm)
Trees (2013) 27:1087–1100 1093
123
May (Fig. 11l), and absent in August (Fig. 11n). In addi-
tion, suberin deposition was more strongly affected fol-
lowing a selective infection with P. aleophilum than with
P. chlamydospora (data not shown).
Generalization of the phenomenon of suberin
occurrence and damage in phloemic and perimedullary
sheath
To give insight on the physiological significance of con-
tinuous sheath boundaries in grapevine, we checked the
generality of the variations of suberin occurrence by car-
rying out observations on stocks of other grapevine varie-
ties, namely Cabernet, Folle Blanche, Chasselas and
Muscat de Hambourg. Thus, the two suberized sheaths
developed along the foliate period in the control canes of
the year, according to the same pattern as previously
described in the case of Ugni blanc. Similarly, sampling on
stocks in which Eutypa Dieback or esca infection was
precisely detected using immunological tests (Octave et al.
2009; Fleurat-Lessard et al. 2010) showed that these bar-
riers were also absent in mid-July and August, but present
in December in these cultivars (data not shown). These
observations indicate that the suberization pattern in vines
and its modification by wood pathogens are general pro-
cesses not depending on the observed cultivar.
Discussion
Functional significance of continuous suberized sheaths
and seasonal regulation of suberization
Several data deal with phloemic (periderm) suberized
sheaths that give phellogen in various species, including
grapevine, whereas the descriptions of a suberized tissue at
pith margin (Rioux and Baayen 1997) are rare. As shown in
grapevine (Fig. 5), this latter sheath represents an ana-
tomical barrier formed sooner (mid-July) than the phloemic
deposit which develops thereafter from August. This
development occurs in parallel with the summer months in
which the climate conditions are less favourable for plants
in terms of humidity and high temperature leading in
extreme conditions to embolism and hydric stress. Suberin
deposits in cells of the external zone may intervene by
reducing water loss. Thus, additional suberin deposition
has been shown to occur in roots observed in drought
conditions, leading to an increased resistance to water
movement (North and Nobel 1994; Steudle and Peterson
1998). ABA would play a role in the triggering of the
processes, since it has been shown that this hormone gives
rise to a suberization-inducing factor (Espelie and Kol-
attukudy 1985; Lulai et al. 2008) and to increase suberin
synthesis (Soliday et al. 1978).
d e f
a b c
W
W
W
W
OPF
XP
cy
cy
ESC
ISC
Fig. 6 Detail in suberin structure. In the external sheath 3 to 4 layers
of small-sized suberized cells (esc) (a); alternate opaque and
translucent layers (arrow) of suberin in the inner wall part (b, c);
damaged cytoplasm (cy). In the internal sheath large suberized cells
(isc) (d); suberin layers (arrow) along the inner wall part (e); detail of
dark lamellar suberin deposit (f). TEM views in b, c, e, f; ml, middle
lamellae; OPF, outer lignified phloem fibres; w, pectocellulose wall;
XP, xylem parenchyma (bar in a, d, 40 lm, in b, e, 2 lm, in c, f,0.5 lm)
1094 Trees (2013) 27:1087–1100
123
Differential effect of the various wood-inhabiting fungi
on the vascular apparatus and suberized sheaths
The scheme of COmpartmentalization of Decay In Trees
(CODIT) (Shigo 1984) occurred in the xylem of new year
canes sampled in stocks presenting esca and Eutypa Die-
back: the wall 1 with the presence of tyloses (and gels), the
wall 2, represented by fibres with thick walls that slow
down the radial circulation and the wall 3 with the presence
of numerous rays uni or pluriseriate, creating discontinuous
barriers inhibiting the tangential circulation. In these 3
walls, which are considered as reaction zones, we have not
or have rarely observed suberin in canes of the year,
whereas this polymer was present in the trunk of the vines
suffering from esca or Eutypa Dieback. This suberin
deposit occurred frequently in the axial parenchyma and in
the rays forming, therefore, a boundary around the areas
infected by the fungi. In various species, suberized cells
surrounded invaded xylem areas, therefore, preventing
fungal propagation (Kolattukudy 1984), in particular,
tyloses both lignified and suberized constitute a well-
adapted system to seal xylem vessels (Pearce and Hollo-
way 1984; Rioux et al. 1995). In the current canes of the
year of vines, showing esca or Eutypa Dieback symptoms,
cambium damage occurred and vascular member differ-
entiation was modified. In these canes the wall 4, consid-
ered to be the most effective (Shigo 1984), was not formed.
This barrier zone matches to cambium suberization, the
importance of which has been shown in Ulmus americana
L. infected by Ophiostoma ulmi. The rapid attack of the
cambium makes the tree incapable of defending itself in
building this wall (Rioux and Ouellette 1989, 1991).
Different grapevine responses according to the disease
are evidenced by the data reported here, since pathogens
involved in BDA did not act with the same intensity in the
foliate part as those implicated in esca and Eutypa Dieback.
In the later diseases, deep and extended modifications were
induced in the general structural organization in wood
h
c
Control BDA esca Eutypa Dieback
b
e
d f
ga
OPFOPF OPF
OPF
PhPhPhPh
Xy Xy Xy Xy
PiPiPi Pi
Px
Px Px
Px
Mid - July
Fig. 7 Comparative occurrence of suberin sheaths in canes of the
year sampled in mid-July in controls and in vines infected by
intraxylem fungi. a, b Suberin deposit at pith border and no deposit at
phloem border in control canes. c, d Suberin deposit at pith border
and no deposit at phloem border in canes of BDA -infected vines. e,
f Faint deposit at pith border and no deposit at phloem border in canes
of esca- infected vines. g, h No suberin deposit in canes of vines
presenting Eutypa Dieback. Legends as in Fig. 5 (bar 100 lm)
Trees (2013) 27:1087–1100 1095
123
August
esca Eutypa DiebackControl BDA
a c e g
b d f h
PhPhPhPh
Xy XyXyXy
OPF OPFOPF
Px
PxPxPx
Pi PiPiPiFig. 8 Comparative occurrence of suberin sheaths in canes of the
year sampled in August in controls and in vines infected by
intraxylem fungi. a, b Continuous ring of blue-coloured suberin
sheaths at border of phloem (red arrow) and pith (white arrow) in
control canes. c, d Suberin sheaths in canes of vines showing BDA
symptoms. No suberin in canes of vines showing (e, f) esca, or (g, h)Eutypa Dieback disease. Legends as in Fig. 5 (bar 100 lm)
Control BDA esca Eutypa DiebackDecember
a
b
ec
d f h
gOPFOPF
PhPhPh Ph
OPF
Xy XyXy Xy
Px Px Px
Px
Pi Pi Pi PiFig. 9 Comparative occurrence of suberin sheaths in canes of the
year sampled in December in controls and in vines infected by
intraxylem fungi. Occurrence of the external (red arrow) and the
internal (white arrow) blue-coloured sheaths (a, b) in control canes,
and in canes of vines showing (c, d) BDA, or (e, f) esca, or (g,
h) Eutypa Dieback disease. Legends as in Fig. 5 (bar 100 lm)
1096 Trees (2013) 27:1087–1100
123
tissues. In particular, cambium is highly damaged with a
marked change in the vascular differentiation. Subse-
quently, changes must happen in the circulation of nutri-
ents, particularly in the partitioning of photoassimilates
following disorders in the phloem structure. Indeed, wide
phloem rays separate islets of disorganized phloem tissue
without tangential bands of lignified fibres and without
suberization at primary phloem border. In addition, the
transport of nutrients from the source leaves towards the
sinks (namely xylem in the trunk) can be modified since the
differentiation of xylem members and their structure are
deeply changed. More particularly in esca and Eutypa
Dieback, the association between abundant tyloses and the
decrease (at least 10 %) in total surface area of conducting
elements may considerably slow down xylem sap translo-
cation in current canes. This difference in the respective
fungal action was also found more precisely at the level of
suberization location. In our opinion, this is of significance
since it has been argued that suberin and suberization play
a major role in plant defence against pathogens by limiting
the spread of the infection (Kolattukudy 2001). In canes
from vinestocks showing esca or Eutypa Dieback symp-
toms, both suberized sheaths were highly damaged during
the foliate period. Consequently, circulation between cor-
tex, vascular apparatus and pith might occur allowing an
easier solute diffusion, in particular the metabolites
excreted by the pathogens.
Several works reported that a fungal attack elicited the
deposition of suberin in the walls of cells located around
the site of penetration. By considering the inhibition of
suberin deposition in canes of vines infected with esca and
Eutypa Dieback, one can think that the mechanism
underlying the suberization is modified in these diseases.
The impact of the implied fungi at distance from their
location on the vascular apparatus of canes and on the
suberized boundaries should be questioned but, pertinently,
the involvement of various kinds of metabolites secreted by
fungi and transported in the transpiration stream should be
considered (Mahoney et al. 2003; Octave et al. 2006b;
Luini et al. 2010). First, fungal metabolites may prevent
both synthesis and/or deposit of suberin in the sheaths of
the canes by acting either indirectly, through modifications
of plant metabolism, or directly, through an intrinsic
enzymatic activity. For instance, eutypine and related
compounds inhibited energetics of the cells as uncouplers
and agents destroying plasma membrane integrity (Desw-
arte et al. 1996; Amborabe et al. 2001; Kim et al. 2004). In
addition, polypeptides secreted by the fungal pathogens in
esca and Eutypa Dieback, interestingly were able to modify
activity of enzymes intervening in plant defence processes
(e.g. NADPH oxidase, phenylalanine ammonia lyase)
(Octave et al. 2006b; Luini et al. 2010) and to exhibit
enzyme activities having wood-degrading capacities
(Schmidt et al. 1999; Bruno and Sparapano 2006; Santos
et al. 2006; Valtaud et al. 2009). Secondly, a direct dam-
aging effect on suberin sheaths already formed cannot also
be excluded since in cuttings infected with P. chlamydos-
pora and P. aleophilum for 7 months these sheaths (and
more particularly the internal) were damaged (Fig. 11).
However, as noted in other fungi species, Rosellinia des-
mazieresii (Ofong and Pearce 1994) and Mycena meliigena
(Schultz et al. 1996), the modalities of biodegradation of
suberin barriers in canes by vascular fungi are yet
unknown. Few reports deal with the degradation of this
complex substrate. Its enzymatic modifications would
intervene to allow the penetration of pathogens such as
Armillaria mellea in the roots of forest trees (Swift 1965)
and Fusarium solani f. sp. pisi (Zimmermann and Seem-
uller 1984). To this concern, the action of a suberinase
produced by Nectria haematococca, identical to cutinases,
C BDA ESC EUT
30
20
10
0
30
20
10
0
Su
ber
in a
rea
(%
)
a
b
a b a c a d e d
b f b b g h g h
Fig. 10 Variation in surface area of suberin sheaths in the current
canes of the year. Comparison of values in external (a) and internal
(b) sheaths, according to BDA- or esca- or Eutypa Dieback disease, in
reference with apparently healthy control, and season. In August
(white bars) small decrease in BDA, in external sheath only (a); high
decrease in esca and Eutypa Dieback in both sheaths (a, b). In
December (black bars) slight decrease in Eutypa Dieback in external
sheath (a) and in esca and Eutypa Dieback in internal sheath (b).
Measurements obtained using Image J software on cane cross-
sections, calculations as mentioned in ‘‘Materials and methods’’. Y-
axis: % of suberin in areas shown in Fig. 4a, b; each bar represents
the mean ± SE from 4 vines, 8 current canes 94 years; n = 32.
Values with the same letters are not statistically different, according
to Fisher’s and Duncan’s test at P \ 0.05
Trees (2013) 27:1087–1100 1097
123
was mentioned (Fett et al. 1999). Indeed, the suberin-
induced enzymes showed catalytic properties similar to
cutin-hydrolyzing enzymes previously isolated from dif-
ferent fungi. The in vitro degradation of raspberry suberin
by Fusarium solani f. sp. pisi was observed (Zimmermann
and Seemuller 1984), when Fusarium was grown in a
medium supplemented with 0.5 % suberin, and monomers
like fatty alcohols and acids with chain-lengths from C16
to C26, as well as C16 and C18 x-hydroxyacids, could be
identified as products. However, the enzymatic nature of
the process was not clearly demonstrated and the nature of
the enzymes that release the aromatic components from the
polymer is unknown. The biochemical mechanism in this
process might be similar to that used in lignin degradation.
Such a type of enzyme activity has not yet been checked
for the fungi involved in esca and Eutypa Dieback, which
would now be a line of further research. In this scope, a
comparison of the metabolites excreted, respectively, by
the pathogens involved in esca/Eutypa Dieback and BDA
would be also fruitful in the explanation of the seasonal
differences observed.
Acknowledgments This work was supported by the firm ‘‘CLS
Remy-Cointreau’’, 20 Rue Societe Vinicole, BP 37, 16102 Cognac
Cedex and CNRS (FRE 3091, Contrat 781263). The authors are
grateful to Gisele Thery and Bruno Merceron for their technical
assistance in ‘‘Image UP’’ (service de microscopie et d’imagerie),
University of Poitiers. They also thank Mrs Tracey Barnes for
improving the English of this manuscript.
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