Interactions between Scots pine, Ips acuminatus (Gyll.) and Ophiostoma brunneo-ciliatum (Math.): estimation of the critical thresholds of attack and inoculation densities and effects on hydraulic properties in the stem
Ann. For. Sci.
Interactions between Scots pine, Ips acuminatus (Gyll.) and Ophiostoma brunneo-ciliatum (Math.): estimation of the critical thresholds of attack and inoculation densities and effects on hydraulic properties in the stem
Natacha Guérard 0 2
Erwin Dreyer 0
François Lieutier 1 2
0 Unité d'Écophysiologie Forestière, INRA Nancy , 54280 Champenoux , France
1 Laboratoire de Biologie des Ligneux, Université d'Orléans-la-Source , BP 6759, 45067 Orléans Cedex 2 , France
2 Zoologie Forestière, INRA Orléans , Avenue de la Pomme de Pin, BP 20619, 45166, Ardon Cedex , France
- The aggressiveness towards Scots pine (Pinus sylvestris L.) of the association between a bark beetle (Ips acuminatus Gyll) and an Ophiostomatale fungus (Ophiostoma brunneo-ciliatum Math.) was investigated by estimating experimentally with young trees, the critical threshold of attack or inoculation densities. Records of the relationship between natural attack densities by the beetles and survival of trees in a pine stand yielded a critical attack density threshold of about 900 m-2. Experimental mass inoculations of young pines with the fungus, in a forest stand in Central France, demonstrated a weak pathogenicity of this fungal species towards Scots pine. Inoculation densities varying from 200 to 1000 m-2 were used. Damage in the bark or in the sapwood recorded three months after the inoculations, remained rather limited. The length of the induced reaction zones in the bark was small as compared to those obtained with more aggressive fungi, and did not increase with inoculation density. Damage in the sapwood, estimated either visually from the observed sapwood drying, and from resinosis, remained limited, but increased significantly with the inoculation density. The impairment of hydraulic conductivity of inoculated trunk segments was rather large with, at highest densities, a loss of conductivity estimated to about 60%. Nevertheless, due to the fact that the resistance to water transfer in the trunk is much smaller than in other organs (like roots or needles), this increase probably only had a small impact on water relations at whole tree level. It may be concluded that the association I. acuminatus - O. brunneo-ciliatum displays only a weak aggressiveness towards Scots pine, and that high densities of attacks or inoculations (above 1000 m-2) are required in order to reach the critical threshold able to kill trees.
Des inoculations massives de jeunes pins en forêt (Orléans, France) effectuées avec le champignon O. brunneo-ciliatum, à des
densités de points d'inoculation allant de 200 à 1000 m–2 ont confirmé la faible pathogénicité de cette espèce. Les dégâts observés trois
mois après inoculation dans le liber et l’aubier étaient modérés même aux plus fortes densités. La longueur des réactions induites
dans le liber était faible par rapport à celle qui résultait d'inoculations avec d’autres champignons, et est restée insensible à
l'accroissement des densités d'inoculation. Les dégâts dans l’aubier, estimés visuellement (sections d'aubier desséchées et imprégnées de
résine) sont restés modérés mais ont néanmoins augmenté significativement avec la densité d’inoculation. La perte de conductivité
hydraulique a été sensible dabs les segments de troncs inoculés. Elle pouvait atteindre 60 % en réponse aux plus fortes densités
d’inoculation. Toutefois, cette diminution de conductivité locale n'a probablement eu qu'un faible impact sur les relations hydriques à
l’échelle de l’arbre entier, du fait de la faiblesse relative des résistances dans les troncs par rapport à d'autres organes comme les
aiguilles. L’association I. acuminatus - O. brunneo-ciliatum s'est donc révélée peu pathogène pour le Pin sylvestre, et les densités
d'attaques et d'inoculation susceptibles d'entraîner à terme la mort des arbres sont sans doute très élevées par rapport à d'autres
Pinus sylvestris / Ips acuminatus / Ophiostoma brunneo-ciliatum / densité d’inoculation / densité d’attaque / niveau de
résistance / réaction induite / aubier / liber / conductivité hydraulique / scolyte / champignon associé
Bark beetles are one of the major threats to coniferous
forests. These insects use trees as a substrate during their
life cycle. During periods of endemic population levels,
bark-beetles restrict their attacks to weakened or dying
trees. The rapidity with which they develop, however,
helps them reach epidemic population levels as soon as
conditions become favourable, i.e., when sufficient
substrate is available like after heavy windbreak damage or
severe stress episodes that weaken standing trees. The
high population levels allow the beetles to extend their
attacks to healthy and vigorous trees [
3, 18, 29
epidemic gradations, even if they are rather unfrequent,
are nevertheless disastrous for forests. For instance,
300 000 m3 pines had to be cut down between 1983 and
1986 after attacks by Tomicus piniperda and Ips
] and the same amount of Spruce were destroyed
between 1992 and 1993 in North Eastern France after
attacks by Ips typographus. Dendroctonus ponderosae
has been responsible for the death of 80 million pine
trees between 1979 and 1983 in USA and 4.7 million of
m3 of pine per year in the north of America [
Trees are able to develop defence reactions that reject
or isolate the aggressors [
]. Such defence processes
– the flow of pre-existing resin promoted by the
mechanical disruption of resin ducts due to insect
– an induced reaction consisting of an active
accumulation of secondary metabolites (terpenes,
monophenols,...) around the attack point, that limits the
progression of the aggressor; in a second step, the
build-up of wound tissue isolates the reaction zone
from the rest of the tree [
7, 23, 32, 37
Isolated attacks by most bark-beetle species fail on
healthy tree, and only mass attacks can result in insect
establishment and in death of the attacked trees. Several
observations have shown that above a given attack
density, the number of killed trees increases rapidly,
suggesting the existence of a critical threshold of attack density
8, 28, 31, 36
]. A dynamic balance between tree defence
and attack density has been described, in which the
critical threshold of attack density can be used as a
quantitative index for tree resistance. Bark-beetles are
furthermore frequently bearing phytopathogenic fungi
belonging to the group of Ophiostomatales. The bark
beetle associated Ophiosomatales display a weak to
moderate pathogenicity. Their role in this mutualistic
association is probably to amplify the induced reactions
of the trees, thus contributing to exhaust their defence
ability, and therefore facilitating the establishment of the
insect population by decreasing the critical attack density
7, 19, 20
]. In contrast, fungal establishment
has sometimes been claimed to constitute a prerequisite
for successful beetle establishment in pine  and in
It is possible to experimentally estimate this critical
threshold of attack density by promoting controlled and
variable numbers of insect attacks [
]. A much simpler
procedure is to inoculate directly the associated fungus
into the bark, with increasing densities of inoculation
points, and to detect the density above which damage
and possibly tree death can be observed [
]. It has
been shown that the two procedures yield comparable
values of density, thus allowing a comparison of critical
thresholds for a variety of tree species and fungal strains
6, 11, 16
The critical attack or inoculation density threshold has
been shown to vary according to the aggressiveness of
the beetle and the pathogenicity of the fungus strain [
]. It is also modulated by the health status and vigour
of the trees, increasing with tree vigour and productivity
(expressed as the ratio of the width of the latest
increment ring to the sapwood section [
3, 16, 22
changes during the season .
Decline and ultimately death of the trees is the usual
indication that the critical threshold has been reached.
But death of attacked or mass inoculated trees usually
occurs several months after the aggression. It is therefore
more convenient to use indices able to detect whether or
not the fungus or the insect has been able to overcome
the tree resistance. The length of the reaction zones
around inoculation points has sometimes been
considered as a possible indicator of resistance [
], but its
significance has been questioned as it displays only
small variations in response to changing tree-health
conditions or with increasing inoculation densities [
Induced reaction zones occur both in the phloem and
the sapwood in response to bark inoculation. They may
therefore, together with the development of the fungus,
have a negative impact on the water conducting ability
of the sapwood, and result in impaired hydraulic
functions of the tree [
]. There may be several causes of
such impairment. The mechanical occlusion of tracheids
by resin macromolecules or by mycelial strains in
vicinity of the attack points may be one of them. In addition,
air seeding into tracheids and cavitation [
occurs before tracheid occlusion. Cavitation may be
favoured by the presence of the fungal mycelium in the
sapwood, but the precise chain of events reaching from
mycelial spread into the sapwood to the irreversible loss
of hydraulic conductivity is still poorly understood.
There may be several techniques to assess the amount
of sapwood dysfunction. Staining dyes have been
frequently used to evidence functional (stained) sapwood
11, 13, 17, 32
]. Losses of hydraulic conductivity
can also be measured directly in cut stem segments using
pressurised water and measuring the resulting flow
through the stem [
]. Such direct measurements of
losses of hydraulic conductivity in response to mass
inoculations with increasing attack or inoculation
densities could therefore be an efficient method to obtain an
early marker of successful invasion and tree decline.
We investigated the characteristics of Scots pine
responses to attacks by Ips acuminatus (Gyll.) and its
associated fungus Ophiostoma brunneo-ciliatum (Math.).
More than 95% of the insects of this species carry the
fungus within mycangia on the external mandibular
]. I. acuminatus preferentially attacks
tree segments with thin bark. It has been responsible for
damages that were locally very severe in the pine forests
of central and South Eastern France . In this study,
we compared the threshold attack density derived from
observations following natural attacks in a stand in
Southern France, and the critical threshold of inoculation
density, obtained experimentally on young trees in
central France. Inoculation at varying densities were made
on young trees, and sterile inoculations were performed
in parallel in order to separate effects induced by
wounding alone from pathogenic effects of the fungus.
2. MATERIAL AND METHODS
2.1. Natural attacks
The observations were made during March 1989, at
Comps-sur-Artuby (Var, South-Eastern France), on 48
fifteen-year-old Scots pine trees (height: 8.2 ± 0.05 m;
circumference at breast height: 44 ± 0.3 cm, i.e., ca.
14 cm DBH). They were naturally attacked by Ips
acuminatus (Gyll.). Two 50-cm-long stem segments
were collected on each tree, one in the upper third, and
one in the middle of the trunk. The number of individual
galleries (successfull attacks) and of aborted attacks was
recorded on each segment, and related to the health
status of the tree (still living or dead). Two of the trees
presented only half of the stem length still living and a large
blue staining; they were counted as dead. One had only
the upper third declining, and was counted as still alive.
No other intermediate cases were recorded.
2.2. Mass inoculations
During spring 1997, 220, 7 to 8-year-old Scots pine
trees (Pinus sylvestris L.) were selected in a natural
regeneration in the Forest of Rambouillet (Central France;
height: 1 to 2.7 m; dbh: 2–2.5 cm). Inoculation was made
with a 3-week-old monospore culture of Ophiostoma
brunneo-ciliatum (Math.) isolated from I. acuminatus
galleries on bait stems distributed in the pine forests of
south-eastern France. Trees were inoculated in situ
between June 28 and July 7 1997 with inoculation point
densities ranging from 170 to 1 270 m–2, on a belt width
comprised between 16 and 52 cm (figure 1). Inoculations
were made either with sterile malt agar disks, or with
fungus cultures. Five-mm-diameter disks of bark were
punched out down to the cambium. A disk of fungal
culture or sterile malt-agar was inserted in the hole, bringing
the mycelium in contact with the cambial layer. The hole
was sealed again with the removed bark disk.
The impact of the inoculations was estimated 3
months later through records of:
– the length of induced reaction zones in the bark
– the sectional area of blue stained, dried or resin
– the loss of hydraulic conductivity in the inoculated
Inoculation density (m–2)
Twelve control trees, free of any inoculation or
wounding, were harvested to estimate the maximal hydraulic
conductivity. The inoculated trees (sterile or with
fungus) were randomly divided into 2 equivalent groups.
The trees in the first one were used for induced reaction
zone and sapwood measurements, and those in the
second one were used for hydraulic conductivity
Ten induced reaction zones were randomly selected,
excluding the ones close to the border of the inoculation
belts, and their length was recorded. Three stem sections
were cut in each tree within the inoculated belt. Dried,
resin soaked and blue stained areas were redrawn on
transparent paper. Resulting drawings were digitised to
estimate the area of each type of sapwood, with an image
analysis software. The fraction of functional sapwood
(As%) was derived from these estimates.
The technique developed by Sperry et al. [
used to estimate the actual local hydraulic conductivity
of 20 cm long stem segments that were cut from within
the inoculation belt. Deionised, degassed and acidified
water was used at a pressure of 5 kPa obtained from a
water tank placed exactly 0.5 m above the sample, and
the flow through the segment was recorded as the
weighted amount of water recovered at the open end of
the segment after 10 min circulation. Hydraulic
conductivity (K) was calculated as:
F × L
where F is the flow of water (kg s–1), L, the length of the
segment (usually close to 0.2 m) and P the pressure
applied at segment entry (= r g h, with h, height of the
water tank above the segment). Values of K were
standardised to sapwood specific hydraulic conductivity
(Ks, kg m–1 s–1 MPa–1) using the total sapwood cross
sectional area of the sample.
The loss of conductivity is usually expressed with
respect to maximal conductivity measured after
resaturating xylem vessels under a high pressure (0.175 MPa;
]). In our case, due to the potential occurrence of
tracheid occlusion, we estimated maximal conductivity
from the relationship between diameter and actual
conductivity obtained on the 12 healthy trees (figure 2). This
relationship was later used to compute the maximal
hydraulic conductivity of inoculated trees.
2.3. Statistical analyses
Due to the small diameter of the trees, it was not
possible to obtain inoculation densities matching exactly the
target values. Real densities were therefore recomputed
for each tree (figure 1), leading to a continuum of values
that were discretised into 4 groups with homogenous
numbers of trees.
Normalised variance analyses were made using the
GLM procedure of SAS (SAS Institute, Cary, NC),
followed by Scheffe's t-test (or LSD when n < 5), at a
significance level of 0.05. Graphical displays present mean
values ± confidence interval (p = 0.05).
3.1. Natural attacks
Records of natural attacks by the bark beetle Ips
acuminatus resulted in the death of 26 among the
48 selected trees. A very large difference of attack density
was recorded between surviving (around 180 ± 20
attacks m–2) and dead trees (1 060 ± 30 attacks m–2). The
densities on dead trees ranged from 400 up to 1 800 m-2.
The trees were discretised into 9 equal attack-density
classes (0–200; 201–400; ….) and the relative fraction of
dead trees was computed in each class, and represented as
a function of attack density (figure 3). A sigmoid
relationship could be fitted to the data, with following equation:
Mortality (%) =
with D: density (m–2) and D50, density at which half the
trees were killed. The adjusted relationship yielded a
value of D50 of about 510. D95 (95% killed trees) was
close to 850: this value can be regarded as the mean
critical attack density threshold of the stand.
3.2. Mass inoculations
A general ANOVA was conducted to test for the
effects of three factors (presence or absence of the
fungus in the malt agar disk, inoculation density and
inoculation belt width) on four parameters (sapwood specific
hydraulic conductivity, Ks, length of the induced reaction
zones in the phloem, fraction of resin soaked, of dry, and
of healthy sapwood, table I). The inoculation with fungal
strains yielded significant effects with respect to sterile
malt-agar disks, on all measured parameters (with the
exception of dry sapwood). Inoculation density had
significant impacts on all parameters, while the width of the
inoculation belt had none. We therefore skipped the
factor “belt width” from all further analyses and
concentrated on inoculation densities solely.
Sterile inoculations yielded 20 mm long reaction
zones (average value), which length decreased only
slightly to 12 mm at higher inoculation densities
(figure 4a). The length of the induced reaction zones was
very stable when the fungus was used, with no visible
impact of density. The occurrence of such small
differences in reaction zone lengths between sterile and fungal
inoculations confirmed the weak pathogenic power of
O. brunneo-ciliatum, and the non-specific nature of the
induced reaction in the phloem.
Reactions in the sapwood were different. Sterile
inoculations resulted in a small but stable reduction of
healthy sapwood independently of density (figure 4b).
The loss, that never exceeded 10%, was due to resin
soaking (20% of the loss, figure 4c) and to tissue drying
(80% of the loss) in close vicinity of the wounds. The
presence of the fungus led to much more severe effects
on sapwood. One of the trees inoculated at 1 100 m–2
displayed blue-staining, and had correspondingly only 40%
healthy sapwood cross section left. No other tree
presented blue staining. Loss of healthy sapwood increased
significantly with inoculation densities (from 15 to
almost 30%). The contribution of resin soaking to this
loss represented 60%, and was independent of
inoculation density (figure 4c).
Sterile inoculations induced no significant loss of
conductivity; the slight increase at highest density was not
statistically significant. Inoculations with the fungus
resulted in significant losses ranging from around 25% at
lowest densities up to 55% at the highest ones
(figure 4d); the impact of increasing inoculation
densities was significant although small.
The fraction of intact sapwood cross-sectional area
was significantly correlated with the loss of conductivity,
with a non-linear relationship between the two
parameters. The loss of conductivity increased much faster than
the loss of intact sapwood (figure 5).
Our results with either records of natural attacks of
young Scots pines by the bark beetle Ips acuminatus
Gyll. or with mass inoculations of its associated fungus
Ophiostoma brunneo-ciliatum Math. into the bark
confirmed the weak aggressiveness of this bark
beetle-fungus association. The natural attacks allowed us to
estimate the critical attack density threshold at around
850 m–2 (95% dead trees above this density). For the
fungus, inoculations close to 1000 m–2 induced
significant damage to the sapwood of the infected trees (but not
A comparison with the few published data on critical
threshold densities of attacks or inoculations (table II)
yielded the following observations: 1. There is an
agreement between the two techniques: mass inoculation of
the associated fungus or direct attacks by the bark beetle
result usually in close values despite the known
differences in the frequency of association between fungi and
insects (high in I. acuminatus; much lower in the other
species); 2. The association I. acuminatus - O.
brunneociliatum is one of the less pathogenic ones when
compared to others, either on different host species, or even
for Scots pine.
The different markers of susceptibility of the trees to
the fungus behaved very dissimilarly in response to
increasing inoculation densities. The length of the
induced reaction zone in the bark tissues has been
frequently proposed as an index for the resistance of trees
towards attacks [
13, 14, 22
]. It is expected that, below
the threshold inoculation density, long reactions indicate
a low efficiency of the resistance mechanisms. Above
the threshold, the length of the reactions may be reduced
due to a lack of available carbohydrates needed to
accumulate secondary compounds. In addition, this length
has been shown to vary with season and with tree vigour
]. A long reaction zone is expected to reveal an
aggressive fungal strain [
]. In the case of O.
brunneociliatum, the length was close to 20 mm, that is much
lower than those recorded after inoculation with other
fungi (see table III). Moreover, it was only slightly
different from that of reactions induced by wounding alone
(sterile inoculations). This confirms that the induced
reaction is rather unspecific, and that the presence of the
fungus is not increasing its intensity to a significant
degree. The lack of difference in this parameter with
increasing densities up to the threshold density observed
for natural attacks agrees with earlier results [
strengthens the conclusion that reaction zone length in
bark tissues is a poor index for tree resistance .
Damage in the sapwood could be another relevant
criterion for tree resistance, even if it may be argued that
this damage occurs late in the infection cycle, and plays
probably only a minor role in the potential success of
insect installation. The latter is probably strongly relying
on the dynamic balance between rapid responses of
trees, and velocity of fungus propagation in the phloem.
Nevertheless, our results demonstrated clearly that
sapwood damage was a more sensitive indicator than
reaction zone length. Even if this damage remained
rather low when compared to that recorded in similar
trees inoculated with L. wingfieldii at 400 m–2 [
nevertheless displayed a significant increase with
density, and differed largely from that induced by wounding
alone. The latter resulted mainly in a very limited
sapwood drying very close to the wounds and almost no
resinosis. The presence of the fungus resulted in heavy
resinosis, sapwood drying and in one case, blue staining.
In contrast, the loss of hydraulic conductivity
demonstrated even larger dysfunctions in the sapwood than
those derived from direct visual observations. Up to 60%
loss of conductivity was recorded at the highest
densities. This was still largely below the amount of damage
caused by L. wingfieldii [
], confirming again a weak
pathogenicity of O. brunneo-ciliatum.
What could be the impact of such hydraulic
impairment on the water relations of the whole tree? Hydraulic
properties were measured on small segments cut within
the inoculation belt which may explain why against
expectations, belt width had no impact on the measured
loss of conductivity. The loss of conductance at whole
trunk level was probably more affected by the extent of
inoculation or attacks along the stem, but we have no
direct measurement to support this point.
Nevertheless, to discuss the impact on water relations
at whole tree level, one needs to take into account the
relative importance of resistances along the
soil-toneedle water pathway. It may be safely assumed that the
distribution of resistances to water flow is approximately
40% in the extra fascicular pathway in the needles, 10%
in the shoot xylem, 10% in the root xylem and again
40% in the root cortex (Cochard, personal
communication). A rough calculation shows that a 50% increase of
resistance in the sapwood would only result in a 5%
increase in total resistance, which is almost undetectable
with classical techniques like combined records of
transpiration and needle water potential.
The most striking result was the significant but
nonlinear relationship between the amount of damage and
the loss of conductivity; the latter increasing much faster
then the former. Loss of hydraulic functionality was
clearly due to the presence of the fungus, as wounding at
similar densities induced almost no loss. This
discrepancy between anatomical observations and recorded loss of
hydraulic conductivity may imply several explanations:
• a rapid spread of the fungus in the sapwood without
any visible anatomical damage and an embolisation of
the tracheids that can only be detected by conductivity
• the induction of cavitation and embolism at some
distance from the fungal mycelia. It has sometimes been
hypothesised that micro-organisms (or induced
reactions) could emit secondary metabolites able to
decrease the surface tension of xylem sap, and
therefore increase the vulnerability to cavitation. This
hypothesis was put forward for the pine wilt nematode
] and for bark-beetles [
], but is far from being
• the mere fact that anatomical damage is assessed on
2D wood sections, while hydraulic impairment is
recorded on 3D stem segments. The impact of a given
amount of visible damage could change dramatically
depending on the spatial distribution of the lesions; in
our case, inoculation points from successive
inoculation rings were not aligned, but overlapping, and this
distribution probably maximised the conductivity
losses induced by a given extent of cross sectional
Detailed microscopic studies combined with
conductance measurement of whole stems would be required to
answer these questions related to the interplay between
fungal development in the sapwood and induced
Can we conclude from these observations that the
critical threshold of inoculation density was reached in this
experiment? The experiment did not last long enough to
observe tree death. The indirect indices used to
characterise the impact of inoculations (amount of damage in
the sapwood from anatomical and hydraulic points of
view) increased gradually in response to increasing
inoculation densities and did not display the expected
threshold type response (fungus contained in the reaction zones
and low densities, and fungal spread to the whole tree at
densities above the threshold). Could this lead to a
contrasted tree survival with death occurring after several
months only at the highest densities? This question still
remains open, and would need many more informations
on the complex interplay between fungal colonisation,
impairment of watertransport and tree decline.
Nevertheless, it is clear from these experiment, that the
amount of damage in the sapwood displays a larger
variability in response to increasing inoculation densities
than the ones in the bark.
The mutualistic association Ips
acuminatus/Ophiostoma brunneo-ciliatum displayed only a weak
pathogenicity towards young Scots pines, as revealed from the
observed critical threshold of natural attack densities in
pine stands (850 m–2), and from the limited extent of
damage induced by controlled inoculations of the fungus
into the bark. The extent of damage in the sapwood
nevertheless displayed a significant increase with increasing
inoculation densities while the length of reaction zones
in the bark did not. This observations again confirms that
the latter is only a poor index of tree defence ability
against fungal attacks. The highest inoculation densities
(1000 m–2) promoted visible damage in the sapwood and
large losses of hydraulic conductivity. No threshold
response was visible from these observations.
Furthermore, we were unable to predict whether the
inoculated trees would have died after a few months or
recovered from the damage. The question of the critical
threshold inoculation density still remains open.
Acknowledgments: This work was supported by the
European Union Project “Stress and Tree Health” (FAIR
3 CT96-1854), and by a grant of the Région Centre to
N.G. The authors are grateful to “Office National des
Forêts” for providing the Scots pine stand in the forest of
Rambouillet, and to P. Romary and J. Garcia for their
technical help. Helpful comments by two anonymous
reviewers are gratefully acknowledged.
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