Stimulation of tree defenses by Ophiostomatoid fungi can explain attack success of bark beetles on conifers
Ann. For. Sci.
Stimulation of tree defenses by Ophiostomatoid fungi can explain attack success of bark beetles on conifers
Annie Yt 1
0 INRA, Unité de Zoologie Forestière , 2163 avenue de la Pomme de Pin, CS 40001, Ardon 45075 Orléans Cedex 2 , France
1 Université d'Orléans, Laboratoire de Biologie des Ligneux et des Grandes Cultures UPRES EA 1207 , rue de Chartres, BP 6759, 45067 Orléans Cedex 2 , France
• Our aim is to present why the hypothesis, that Ophiostomatoid fungi play an important role in the establishment of most bark beetle species on living conifers, is valuable. • After summarizing knowledge about the relationships of bark beetles with conifers and fungi, we conclude that controversy results from misinterpretations when using fungal pathogenicity to demonstrate the role of Ophiostomatoid fungi in beetle establishment on host trees. • We demonstrate that fungal pathogenicity is not the right parameter to appreciate the role of fungus in beetle establishment on host trees. We argue that artificial low density inoculations that allow the appreciation of fungus ability to stimulate tree defenses and thus to help beetles in overcoming tree resistance must be used in complement to mass inoculations. In both cases, results must be expressed in terms of tree defense stimulation rather than in terms of tree killing. (i) Fungal species stimulating tree defenses are generally not those that grow the best in the sapwood. (ii) We argue that beetle development in the phloem, fungal invasion of the sapwood and phloem, and tree death, occur after tree defenses are exhausted, and that any fungus present in the beetle gallery could thus potentially invade the sapwood after defense exhaustion.
pathogen / forest tree / physiological stress / blue stain fungi / host resistance
arbre forestier /
stress physiologique /
champignon du bleuissement /
résistance de l’arbre
Résumé – La stimulation des défenses de l’arbre par les champignons Ophiostomatoïdes peut
expliquer le succès des attaques de Scolytes sur conifères.
• Notre objectif est de présenter les raisons de la validité de l’hypothèse selon laquelle les
champignons jouent un rôle important dans l’installation de la plupart des espèces de Scolytes sur conifères
• Après avoir résumé les connaissances sur les relations des Scolytes avec les conifères et les
champignons, nous concluons que la controverse résulte d’interprétations erronées lorsque l’on utilise le
pouvoir pathogène des champignons pour démontrer le rôle des Ophiostomatoïdes dans l’installation
des insectes sur les arbres hôtes.
• Nous démontrons que le pouvoir pathogène n’est pas le paramètre correct pour apprécier le rôle du
champignon dans l’installation des Scolytes sur les arbres hôtes. Nous soutenons que des inoculations
artificielles à faible densité, qui permettent d’apprécier la capacité du champignon à stimuler les
défenses de l’arbre et à ainsi aider le Scolyte à surmonter la résistance de celui-ci, doivent être utilisées
en complément des inoculations massives. Dans les deux cas, les résultats doivent être exprimés en
termes de stimulation des défenses de l’arbre plutôt qu’en termes de mortalité de l’arbre.
(i) les espèces de champignons qui stimulent les défenses de l’arbre ne sont généralement pas celles
qui présentent la meilleure croissance dans l’aubier.
(ii) nous soutenons que le développement de l’insecte dans le phloème, l’invasion de l’aubier et du
phloème par le champignon, et la mort de l’arbre, interviennent après épuisement des défenses
de l’arbre, et que tout champignon présent dans les galeries de l’insecte pourrait donc
potentiellement envahir l’aubier après épuisement de ces défenses.
• Nous concluons que la stimulation des réactions de défense de l’arbre à la fois dans le phloème et
l’aubier superficiel représente un bénéfice réel apporté par les champignons aux Scolytes pendant la
première phase de leur installation (surmonter la résistance de l’arbre).
• En ce qui concerne l’origine des associations Scolytes – champignons attaquant les arbres vivants
et considérant leur fonctionnement général basé sur une stimulation des défenses de l’arbre, nous
développons trois hypothèses :
(i) toute espèce de Scolyte serait aidée dans son installation sur une espèce d’arbre donnée en
développant une association, même lâche, avec une espèce de champignon appartenant à la flore
Ophiostomatoïde de cette espèce d’arbre ;
(ii) la nécessité d’un très faible niveau de résistance de l’arbre pour autoriser l’extension fongique
dans le végétal est la pression de sélection qui a conduit les champignons à développer leur
capacité intrinsèque de stimulation des défenses de l’arbre, à travers leur capacité à croître dans le
phloème. Cette association peut être complétée par des espèces fongiques antagonistes contrôlant
l’extension des espèces précédentes dans les tissus de l’arbre ;
(iii) les espèces de Scolytes utilisant la stratégie de surmonter la résistance de l’arbre sont associées
à un complexe fongique dont les espèces assurent trois fonctions eu égard aux relations entre les
Scolytes et les arbres : 1– stimuler les défenses de l’arbre dans le phloème et l’aubier superficiel,
2– croître dans l’aubier après que la résistance de l’arbre ait été vaincue, and 3– contrôler
l’extension des deux catégories précédentes dans le phloème. L’apport de nutriments à la progéniture
du Scolyte peut représenter une quatrième fonction.
• Nous proposons que les associations Scolytes – Ophiostomatoïdes puissent être classées, en se
basant sur la fréquence et la complexité de l’association et en prenant en compte l’agressivité de
l’insecte. Nous montrons qu’il existe une étroite correspondance entre l’agressivité des insectes et la
capacité de leur principale espèce fongique associée à stimuler les défenses de l’arbre hôte.
• Nous concluons en suggérant que la plupart des espèces de champignons envahissant l’aubier
pourraient être des “tricheurs” qui ont profité de l’efficacité des relations entre les Scolytes et les espèces
fongiques stimulatrices des défenses de l’arbre.
Bark beetles (Coleoptera: Scolytinae, Curculionidae)
represent a diversified group of insects gathering a little more than
6 000 described species worldwide and at least 225 genera
(Knizek and Beaver, 2004)
, specialized in exploiting woody
plants (hardwood trees and shrubs, and conifers) and
exhibiting a high diversity of associations with micro-organisms
(Harrington, 2005; Kirisits, 2004; Paine et al., 1997; Six, 2003;
Whitney, 1982, among others)
. All species bore galleries in
their host, where larval development and most often adult
maturation take place. However, with the exception of the species
involved in Dutch elm disease, bark beetles of hardwood trees
do not generally cause much damage. Conifer bark beetles
on the contrary are directly responsible for frequent
extensive and dramatic damage to forests, making them the most
important forest pests in the temperate zones
. Understanding how their attacks succeed is thus
essential, and their relationships with host trees have been
documented in a variety of situations. Moreover, their frequent
relationships with fungi have also stirred up many studies
aiming at determining the role of these micro-organisms in the
interactions between trees and bark beetles.
We believe that fungi play an important role in bark beetle
establishment on conifers, a hypothesis first proposed by
et al. (1967)
that has raised a lot of
controversy, and our objective in this paper is to present and
justify our positioning and our views regarding this hypothesis.
We re-examine the relationships between trees and bark
beetle – associated fungi, to propose that fungi effectively play a
role in beetle establishment on trees, based on the stimulation
of tree defenses and not on tree killing. We then propose
hypotheses to explain why and how the beetle-fungi relationships
interfere in this context. We finally propose different types of
beetle-fungus associations based on our hypotheses.
Before proceeding however, we will first summarize what is
known about relationships between bark beetles and conifers
on the one hand, and between bark beetles and their associated
micro-organisms on the other.
2. RELATIONSHIPS OF BARK BEETLES
WITH CONIFERS AND FUNGI
2.1. Bark beetles and conifers
Although some conifer bark beetles species develop inside
the sapwood, the vast majority are phloem borers. Four groups
of conifer bark beetle species can be distinguished based on
the type of relationship they maintain with their hosts. The
“true parasites” can develop and reproduce on healthy trees
without killing them and this group seems to contain only two
species, which both live on spruces, the European
Dendroctonus micans and the North American Dendroctonus
punctatus. The “near-obligate parasites”
(Raffa et al., 1993)
regular tree-killers able to kill healthy trees over large areas
during periods of outbreaks and which survive on subdued or
dying trees when their population is at a low level. The group
is composed of a small number of species, amongst which are
Dendroctonus ponderosae, Dendroctonus brevicomis,
Dendroctonus frontalis, attacking North American pines and Ips
typographus attacking spruces in Europe. Following its
massive outbreaks in extended areas of spruces in North-West
America in the 1990’s, Dendroctonus rufipennis can also be
considered a near-obligate parasite. The “facultative parasites”
(Raffa et al., 1993)
usually colonize fallen or weakened trees
but are occasionally able to colonize healthy trees and to cause
significant damage, especially when there are circumstances
favorable to a considerable increase of their populations on
weakened trees (important storm felling for example). Species
such as Ips pini on pines in North America, or Ips
acuminatus and Ips sexdentatus on pines and Pityogenes
chalcographus on spruces in Europe, belong to this group. The
(Raffa et al., 1993)
comprises the large majority
of species which never colonize and reproduce on healthy
trees. They usually develop on logs and dead trees,
eventually killed by species of the other groups, but a few species
are able to attack very weakened living trees. Although this
latter group represents around 95% of the total bark beetles
species in the temperate zones and plays an important
ecological role, few studies have been devoted to them because of
their very low economical interest outside of those vectoring
tree pathogens. The near-obligate and the facultative parasites
together make up the quasi-totality of species directly causing
damage to trees.
Besides the saprophytic species, all groups must face a
certain level of tree resistance before succeeding in establishing
in their host. Conifer resistance to bark beetle attacks involves
multiple passive or active defense mechanisms which
overlap and complete each other
(Berryman, 1972; Christiansen
et al., 1987; Franceschi et al., 2005; Lieutier, 2002; 2004;
Paine et al., 1997)
. Among these mechanisms, the
hypersensitive reaction which develops in response to beetle attacks
has been demonstrated to play an essential role in the
resistance of many conifer species to many bark beetle species
(Christiansen et al., 1987; Lieutier, 2002; Paine et al., 1997;
Raffa, 1991; Raffa and Berryman, 1982a; 1982b; Safranyik
et al., 1975)
. It develops around each point of attack in both the
phloem and the superficial sapwood and is visible as a
longitudinal elliptical resin impregnated zone associated with more
or less extended cell necrosis
(Berryman, 1972; Reid et al.,
. Such a zone is considerably enriched with terpenes and
neosynthesized phenolic compounds
(Bois and Lieutier, 1997;
Brignolas et al., 1995; Delorme and Lieutier, 1990; Långström
et al., 1992; Lieutier et al., 1991a; Paine et al., 1987; Raffa and
Berryman, 1982a; Russell and Berryman, 1976)
to stop the aggressors. Resin flow released by a section of resin
ducts can constitute another important resistance mechanism,
at least for beetles of which the females bore transversal egg
(Berryman, 1972; Lieutier, 2002)
Depending on how they cope with tree defense mechanisms
when they establish on their host, it has been suggested that
bark beetle species attacking living conifers fit in with one or
the other of two strategies
(Lieutier, 1992; 2002)
nearobligate and facultative parasites would use the strategy of
overcoming host resistance (or exhausting host defenses). The
insects stimulate the syntheses of defensive compounds and
thus increase the energy demand until the tree becomes
unable to mobilize energy rapidly enough
(Christiansen et al.,
. At this moment, the tree’s defenses are exhausted and
beetle establishment succeeds. The stimulation of these
syntheses is achieved basically through beetle aggregation on the
(Berryman, 1972, 1976; Raffa, 2001; Raffa and
Berryman, 1983a; Wood, 1982)
, which multiplies the points
of aggression and thus increases the mechanical stress
inducing and stimulating the tree’s hypersensitive response
et al., 1995)
. Above a critical threshold of attack density
(Berryman, 1976; Raffa, 2001; Raffa and Berryman, 1983a;
Safranyik et al., 1975; Thalenhorst, 1958)
, tree resistance is
depleted to such a low level that it is not able to stop
aggressors anymore, finally resulting in beetle establishment and tree
death. If it allows limiting interspecific competition because a
low number of beetle species are able to attack living trees,
such a strategy however can lead to intraspecific competition
(Raffa, 2001; Raffa et al., 1993)
. The opposite is true for the
strategy of avoiding host defenses, which would be used by
the true parasites. Beetles behave in order to minimize the
development of the tree’s hypersensitive reaction. They thus
do not need to aggregate and they attack their hosts
individually. They also bore transversal galleries to avoid stimulating
the hypersensitive reaction too much, but this behavior causes
the cutting of numerous resin ducts, which implies high
beetle tolerance to constitutive resin (Lieutier, 2002). In such a
strategy which allows avoiding both interspecific and
intraspecific competition, tree resistance is not depleted and the tree
stays alive after attack success and during beetle brood
2.2. Bark beetles and their associated micro-organisms
Among the numerous species of micro-organisms
(nematodes, fungi, yeasts, bacteria) described as associated with
bark beetles, fungi have been the subject of most studies
2003; Whitney, 1982)
. Associations with Basidiomycetes,
mainly from the genus Entomocorticium, have been
described in D. ponderosae, Dendroctonus jeffreyi, D.
brevicomis, D. frontalis, I. typographus and Ips avulsus
(references in Six, 2003; and Kirisits, 2004)
but most bark beetle
associated fungi are Ascomycetes, which have indeed been
isolated from practically all species where they have been
looked for. Bark beetle associated Ascomycetes mostly
belong to the genera Ophiostoma, Ceratocystis and
Ceratocystiopsis and their related asexual form Leptographium. These
genera form, with few other genera, the morphologically
homogenous group of Ophiostomatoid fungi
(Kirisits, 2004; Six,
2003; Wingfield et al., 1993)
, which are often referred to as
“blue-stain”, “black-stain” or “sap-stain” fungi, because the
melanized hyphae of most species give a bluish-grey color
to the sapwood they colonize, mostly in conifers
2005; Kirisits, 2004)
Functional relations between bark beetles and
Ophiostomatoid fungi correspond to various degrees of interactions
including antagonism, commensalism and mutualism,
facultative or obligatory
(Klepzig and Six, 2004; Raffa and Klepzig,
1992; Six, 2003; Six and Klepzig, 2004)
. Considering bark
beetle species attacking living trees, the most important benefit
that Ophiostomatoid fungi usually gain from the associations
is transport by the insect, on which they completely depend
for their dissemination
(Klepzig and Six, 2004; Six, 2003)
Facilitation in entering the available hosts through wounding
made by the beetles and limited interspecific competition is
also an advantage for fungi associated with beetles attacking
living trees, compared to fungi developing on dead substrates.
Physical relationships are also very diverse. Fungal spores can
be carried by beetles externally on the cuticle or inside more
or less complex mycangia
(Harrington, 2005; Six, 2003; Six
and Klepzig, 2004)
which, depending on beetle species and
according to the classification of
, can be simple
cuticular pits, pocket or tube sacs of varying depth in the
integument, or setal brush, each of these structures being eventually
associated with glandular cells. Transportation within the
mycangia would also give fungi a protection from UV light and
(Klepzig and Six, 2004; Six, 2003)
The existence of mycangia, which has arisen several times
independently in the Scolytinae
, demonstrates that
bark beetles also have real benefits from the association. The
nature of the benefits however is not always very clear. Their
associations with Ophiostomatoid fungi seem to bring bark
beetles diverse benefits depending on both beetle and fungal
(Harrington, 2005; Paine et al., 1997; Six, 2003)
Nutrition is certainly important since, except for the true parasitic
species, bark beetles larvae feed on dying or dead trees, that is
on nutritionally poor substrates. Considering the near-obligate
and facultative parasitic species, it is thought that fungi can
improve larval diet through modifying the substrate or providing
complementary nutrients such as vitamins, proteins or sterols
(Klepzig and Six, 2004; Six, 2003)
. Evidence for a role of
mycangial basidiomycetes in helping D. frontalis to meet its
nitrogen needs has been found (Ayres et al., 2000). Similarly, the
presence of the Ophiostomatoid species Grosmannia clavigera
and Ophiostoma montium can increase the nitrogen levels in
the phloem of trees attacked by D. ponderosae by 40%,
probably through their ability to restitute nitrogen from the sapwood
to the phloem
(Bleiker and Six, 2007)
, and all larval stages and
the adults of the insect can feed on the fungi
(Adams and Six,
. More generally, mycophagy on Ophiostomatoid fungi,
or improvement of larvae development in relation to the
presence of such fungal species, has been reported on several
(references in Six, 2003; Six and Klepzig, 2004; and
, and Six and
speculated that all Dendroctonus species, except the true parasitic
ones, would feed on fungi. According to
however, fungal feeding does not seem obligatory for the
completion of the life cycle of bark beetles, and fungi would only
supplement larval diet, leading to shorter larval galleries and
thus reducing both intraspecific and interspecific competition.
Based on their nutritional relations with fungi and the
localization of their galleries, bark beetles species have been
traditionally divided into three groups, the xylomycetophagous
species which bore galleries in the sapwood and feed
exclusively on symbiotic fungi, the phloeomycetophagous species
whose galleries are located in the phloem and which feed on
both phloem and fungi, and the phloeophagous species which
feed only on the phloem
(Francke-Grosmann, 1967; Kirisits,
Apart from the nutritional aspect, another important benefit
that conifer bark beetles could gain from the association would
be the help brought by Ophiostomatoid fungi to beetle species
attacking living trees, in the success of their attacks and
establishment on trees. The reality of this benefit is the subject of
the present paper.
Following the pioneer work of
Reid et al. (1967)
first model of conifer-bark beetle-fungus interactions proposed
, blue stain fungi have been said to be
responsible for the development of the hypersensitive reaction,
and to be essential for beetle establishment on conifers through
helping to overcome host resistance and killing the tree, both
beetles and fungi contributing simultaneously to tree death
. Establishment is said to begin (i.e.
beetles bore galleries and begin to lay eggs) when host
resistance stops and death is assured
(Berryman, 1982; Coulson,
1979; Wood, 1982)
. However, these early propositions have
often been misinterpreted as it is necessary for the tree to
be already killed by fungi for beetle establishment to begin.
Then, studies developed to prove the essential role of
associated fungi in beetle establishment on trees aimed at
demonstrating the pathogenicity of these fungi for the beetle host
trees, mainly by using artificial mass inoculations supposed to
mimic bark beetle attacks
(Christiansen, 1985a; Guérard et al.,
2000; Horntvedt et al., 1983; Kim et al., 2008; Kirisits, 1998;
Krokene, 1996; Krokene and Solheim, 1998; Neal and Ross,
1999; Ross and Solheim, 1997; Solheim et al., 1993; Solheim
and Långström, 1991; Yamaoka et al., 1995)
supposing that a high beetle aggressiveness could be due to a high
fungal pathogenicity. However, as results accumulated it soon
became appearant that no correlation existed between fungal
pathogenicity and beetle aggressiveness
2005; Lieutier, 2002; Paine et al., 1997)
. This has led several
authors to deny any role of Ophiostomatoid fungi in beetle
establishment on conifers
(Harrington, 1993b, 2005; Klepzig
and Six, 2004; Six, 2003)
The reasons for controversy seem to result from
misinterpretations. Indeed, studies using fungal pathogenicity to
demonstrate the role of Ophiostomatoid fungi in beetle
establishment have been based on three kinds of arguments:
– assertion: tree death is a prerequisite for beetle
– observations: most bark beetle species vector staining
fungi and the sapwood of bark beetle killed trees is stained;
– experiments: experimental demonstrations that fungi
isolated from aggressive beetles are able to kill trees after
artificial mass inoculations supposed to mimic bark beetle
However, all these arguments do not lead to the conclusion that
fungi are responsible for killing the bark beetle attacked trees
before beetle attacks succeed. They do not even prove that
fungi are involved in the tree killing process at all
. Indeed it is possible that fungi invade the sapwood
after the tree has been killed by other factors. Moreover, artificial
mass inoculations certainly mimic natural bark beetle attack
very badly, as the conditions of fungal introductions differ
largely between the two situations (see below). In addition, it
has already been reported that trees can be successfully
colonized and killed by bark beetles without blue stain fungi
(Bridges et al., 1985; Whitney and Cobb, 1972)
that fungi would not be responsible for killing trees attacked
by bark beetles would not prove either that they do not play
any role in beetle establishment. One may even question the
necessity that the trees be killed for beetle establishment to
3. RELATIONSHIPS BETWEEN TREES AND BARK
BEETLE – ASSOCIATED FUNGI AND REALITY
OF THE ROLE OF THE FUNGI IN BEETLE
ESTABLISHMENT ON TREES
3.1. Definition and quantification of beetle aggressiveness and fungal pathogenicity
Raffa and Smalley (1988)
, aggressiveness is
“used to denote the relative degree of vigor characterizing
trees that can be colonized by a Scolytid species”. Thus
aggressiveness is synonymous with the ability of the beetle to
become established on a living tree. Consequently, bark
beetle aggressiveness can be considered to be highest for the true
parasitic species living permanently on healthy trees,
intermediate for near obligate and facultative parasites, and very low
to inexistent for the saprophytic species. For the aggregative
species using the strategy of overcoming host resistance, the
level of aggressiveness can be more precisely quantified by
the attack density above which trees are killed, i.e. the level
of the critical threshold of attack density
Christiansen et al., 1987; Lieutier, 2004; Paine et al., 1997;
Raffa and Berryman, 1983a)
, aggressiveness being inversely
related to threshold level. Such a threshold has been quantified
for some bark beetle species and, although it is influenced by
genetic and environmental factors affecting the trees’ level of
resistance, it can be used to rank these species according to
their aggressiveness. For example, the critical threshold of
attack density, expressed in number of attacks per m2 of bark,
was estimated, depending on tree vigor, between 50 and 120
for D. ponderosae on lodgepole pine
(Raffa and Berryman,
1983a; Raffa, 2001; Waring and Pitman, 1983)
, around 75 for
D. rufipennis on Picea engelmanii
, around 45
for S. ventralis on Abies grandis
(Raffa and Berryman, 1982a)
and between 200 and 400 for I. typographus on Norway spruce
(Mulock and Christiansen, 1986), while on Scots pine, that
of I. acuminatus was around 850 (Guérard et al., 2000) and
that of Tomicus piniperda during its trunk attacks around 400
(Långström and Hellqvist, 1993; Långström et al., 1992)
Fungal pathogenicity is the ability to kill a tree, and the
level of pathogenicity corresponds to the fungal virulence. In
the case of bark beetle associated fungi, it has often been
measured by the critical threshold of inoculation density, which
is the density of artificial inoculations above which trees are
(Christiansen et al., 1987; Lieutier, 2004; Paine et al.,
1997; Raffa and Berryman, 1983a)
. A low threshold indicates
a high level of pathogenicity. In such artificial mass
inoculations, fungal cultures have generally been introduced at the
cambium level, in holes previously bored with a puncher. As
tree death is generally thought to result from disruption of
water transport in the sapwood, tree death caused by fungal
mass inoculations has been estimated by the fading of the
foliage or, most often, by measuring sapwood occlusion or
sapwood invasion by the fungus (percentage of occluded or blue
stained sapwood in transversal bole sections), after
harvesting the trees a few months after inoculations
1985a; 1985b; Christiansen et al., 1987; Croisé et al., 1998a;
Solheim et al., 1993, among others)
. In some cases, tree death
has been determined by measuring sapwood specific hydraulic
conductivity of bole sections (Guérard et al., 2000).
Sometimes, the percentage of dead phloem or cambium was also
(Krokene and Solheim, 1998)
. The same methods have
also often been used to compare the resistance level of
different trees to the same fungus or the effect of a treatment on
(Brignolas et al., 1998; Christiansen and Glosli,
1996; Sallé et al., 2008; Solheim et al., 1993, among
since the critical threshold of inoculation density depends
on genetic and environmental factors affecting the trees’ level
of resistance. Critical thresholds of inoculation density have
been determined for various fungi. Expressed in the number
of inoculations per m2 of bark, it is for example 100 to 200
for Ceratocystis polonica on Norway spruce
. It is 300 to 400 for Leptographium wingfieldii
et al., 1998a; Langström et al., 1993; Solheim et al., 1993)
400 to 800 for Ophistoma minus
(Langström et al., 1993;
Solheim et al., 1993)
, and more than 1000 for Ophiostoma
brunneo-ciliatum (Guérard et al., 2000), all on Scots pine. In
many cases however, fungal pathogenicity has been tested on
seedlings with few inoculations, which is difficult to compare
with mass inoculations on adult trees.
3.2. Relationships between beetle aggressiveness and fungal pathogenicity
Fungi associated with conifer bark beetles appear to have
diversified levels of pathogenicity but all of them, even those
associated with aggressive tree killing bark beetles, need
high inoculation densities to kill a tree. In contrast, classical
Ophiostomatoid pests not specifically associated with conifer
bark beetles, such as Ophiostoma ulmi and Ophiostoma
novoulmi in elms, Ceratocystis fagacearum in oaks, Ceratocystis
fimbriata in various woody plants, Leptographium wageneri
in conifers, can kill a tree with only a few inoculations. With
this in mind, it may be of interest to mention that most of
these fungi are invasive species, thus corresponding to recent
tree-fungus associations. Such a comparison leads to the
statement that conifer bark beetles are associated with moderately
pathogenic fungi that have a very little capacity to colonize
living trees, as already concluded by several authors
1993a; 2005; Lieutier, 2002; Paine et al., 1997; Raffa and
Klepzig, 1992, among others)
In these conditions, one may wonder if a relationship
between beetle aggressiveness and fungus pathogenicity exists
among blue stain fungi associated with conifer bark
beetles. Table I presents the relationships between conifer
beetle species and Ophiostomatoid fungal species supposed, for
most of them, to play a role in beetle establishment. Beetle
species are grouped according to their level of aggressiveness
and fungi to their level of pathogenicity. Because the critical
threshold of attack density has been determined for very few
species only, bark beetle aggressiveness has been estimated
according to the importance of damage on living trees,
especially on healthy trees, mainly by using information from
Furniss and Carolin (1977)
in North America, and
Grégoire and Evans (2004)
in Europe. References
for fungal pathogenicity are indicated in Table I. Clearly, even
when considering only Ophiostomatoid fungi associated with
conifer bark beetle, there is no relation between beetle
aggressiveness and fungus pathogenicity, a conclusion already
proposed by Harrington
and Paine et al. (1997).
There are three main possible explanations to such an
1. Fungi do not play any role in beetle establishment and the
association is not a real one, except in some cases when
fungi provide complementary nutrition to the larvae;
2. Fungal pathogenicity is not the right parameter to
appreciate the benefit brought by the fungus to beetle
establishment on conifers (independently of possible food
3. Early tree death due to fungi is not a prerequisite for beetle
We think that each of these explanations could be valid,
depending on the attack strategy used by the beetle species. Both
explanations 1 and 3 are valid for beetles using the strategy of
avoiding tree defenses. In such a strategy, the tree is not killed
and, in order to minimize the development of the
hypersensitive reaction (see Sect. 2.1), the association with a fungus able
to stimulate it, must be avoided
(Lieutier, 1992; 2002)
beetles use the strategy of overcoming tree resistance, we think
that both explanations 2 and 3 are correct and that fungi
effectively do play a role in beetle establishment. In the following
part of this paper, we will concentrate on this latter strategy
and try to explain why explanations 2 and 3 are valid. We will
then report on results related to the modalities of fungal impact
on tree defenses, a point of importance to appreciate the real
role of fungi.
3.3. Fungal pathogenicity is not the right parameter to appreciate the benefit brought by fungi to beetle establishment on trees
The crucial aspect in the beetle strategy to overcome host
resistance is to exhaust tree defenses or at least to considerably
lower them so that the tree cannot put up effective resistance
to the aggressors and cannot impede beetle establishment and
larval development. In this strategy, everything stimulating the
tree’s defense reactions accelerates the tree’s energy
expenditure and thus lowers the critical threshold of attack
(Lieutier, 1992; 2002)
. Introducing fungi able to strongly
stimulate the tree defensive reactions, into the beetle galleries,
would thus be of great help for beetle populations, since it
would allow them to establish on trees at a lower population
level than without fungi
(Berryman, 1972; 1976; Christiansen
et al., 1987; Franceschi et al., 2005; Lieutier, 2002; Paine et al.,
1997; Raffa and Berryman, 1983a)
. Consequently for
establishment, the beetle population doesn’t need fungus to kill the
tree but to help in overcoming tree resistance through rapidly
stimulating host defenses (by causing extensive reactions) in
each place where it can be inoculated
(Lieutier, 2002; Paine
et al., 1997)
. In this context, there is no reason for an a priori
relationship between fungus ability to stimulate tree defense
reactions when introduced into the tree by a beetle, and its
References related to the level of fungal pathogenicity or the association with beetle species: 1 =
; 2 = Ben Jamaa et al. (2007); 3 =
Christiansen (1985a); 4 = Croisé et al. (1998a); 5 =
Filip et al. (1989)
; 6 =
; 7 =
Guérard et al. (2000)
; 8 =
Highley and Tattar
; 9 = Hornvedt et al. (1983); 10 =
Kim et al. (2008)
; 11 =
; 12 =
); 13 =
; 14 =
Kirisits et al. (2000)
Klepzig et al. (1995)
; 16 = Krokene and
; 17 = Långström et al. (1993); 18 = Lee et al. (2006); 19 = Lieutier et al. (1989b); 20 =
Lieutier et al. (1990); 21 =
Lieutier et al. (2005)
; 22 =
; 23 =
a); 24 =
b); 25 =
Neal and Ross (1999)
; 26 =
Owen et al. (1987)
; 27 =
Rane and Tattar (1987)
; 28 =
Redfern et al. (1987)
; 29 =
; 30 =
Ross and Solheim (1997)
; 31 =
; 32 =
Six and Bentz (2003)
; 33 = Solheim (1992b); 34 = Solheim (1993b); 35= Solheim (1995); 36 = Solheim and Krokene (1998a); 37 =
Solheim and Krokene (1998b); 38 =
Solheim and Långström (1991)
; 39 =
Solheim and Safranyik (1997)
; 40 =
Solheim et al. (1993)
; 41 =
et al. (2001)
; 42 = Viiri and
; 43 = Whitney and Cobb (1972); 44 =
Yamaoka et al. (1995)
ability to kill a tree, especially when the latter is determined
through the critical threshold of artificial inoculation density.
In fact, mass inoculations allow a comparison of the
pathogenicity levels of different fungi or the level of
resistance of different trees to be made, but they certainly mimic
bark beetle attacks very badly. With artificial mass
inoculations, every wound is inoculated with the fungus, which is
often far from being the case during a beetle mass attack, as
the frequency of beetle contamination is rarely 100% and can
even be extremely low in certain populations: see for
, Viiri and
and Sallé et al.
(2005) for I. typographus with C. polonica,
Bridges et al.
for D. frontalis with O. minus, Lieutier et al. (1989b)
Solheim and Långström (1991)
for T. piniperda with L.
wingfieldii, Lee et al. (2006) for D. ponderosae with O.
montium and G. clavigera. The number of spores introduced by a
beetle in its gallery may also differ largely from that contained
in an inoculated disc of sporulated culture (see below). Gallery
excavation by beetles contributes to fungal spread inside the
host phloem, which is not the case for artificial inoculations
(Raffa and Klepzig, 1992). Moreover, it has been reported that
the pattern of host colonization by fungi after artificial mass
inoculations differs from that observed following beetle mass
(Parmeter et al., 1992)
Since the important factor is the ability of the fungi to
stimulate tree defenses, results of inoculation experiments aiming
at appreciating the role of the fungus in beetle establishment
must be expressed in terms of tree defense stimulation rather
than in terms of killing trees. In such a way, mass
inoculations can be useful to assess the stimulation ability of various
fungi or to examine the mechanisms of defense exhaustion,
when approaching the critical threshold as done for example in
the case of G. clavigera on P. contorta
(Raffa and Berryman,
. In addition to mass inoculations, low density
inoculations (usually less than 20 inoculations per tree) far below the
critical threshold of inoculation density, are also very useful
to evaluate fungus ability to stimulate tree defenses, especially
because the tree stays alive and can better exhibit the intensity
of its response to aggression. Such inoculations allow
understanding basic mechanisms of tree defenses and the processes
leading to their stimulation, prior the alterations due to high
(see for example Brignolas et al., 1995;
Franceschi et al., 2005; Kim et al., 2008; Lieutier et al., 1995,
. Moreover, different fungal species and strains can be
compared on the same trees, thus limiting variations due to
(Rice et al., 2007a)
. It has also been suggested that
taking into account the speed of reaction development is of
importance for the interpretation of the results from low density
(Wallin and Raffa, 2001)
. The two approaches
thus complement each other. However, interpretations of
results from artificial inoculations must be done carefully. In all
cases, adult trees, i.e. comparable to trees attacked by the
beetles, must be used. Fungus stimulation capability must also
be estimated after a delay comparable to that involved for the
issue (success or failure) of beetle attacks to be determined,
which is often within a few days (from 4–5 to 20) depending
on beetle aggressiveness and weather conditions
et al., 1988; Lieutier, 2002; Payne, 1980; Raffa and Berryman,
. The physiological status of the tree must also be taken
into account since it can condition the phloem response in
(Croisé and Lieutier, 1993; Croisé et al., 1998b;
Sallé et al., 2008; see also references in Paine et al., 1997;
and Lieutier, 2004)
. Modalities of fungal impact on tree
defenses should also be considered when interpreting the data
(see Sect. 3.6).
Direct comparisons between phloem reaction zone length
after low density inoculations and fungus pathogenicity
after mass inoculation (or sapwood invasion after beetle
attacks) have shown that pathogenicity is not related to fungus
ablity to stimulate tree defenses
(Krokene and Solheim, 1997;
1998; 1999; Solheim, 1988; 1992a; 1992b)
. As an example,
in Figure 1, Norway spruce trees were inoculated with four
fungal species, either at a low density (see left part of the
figure) or at a high density (see right part of the figure)
and Solheim, 1999)
. Among all assayed fungal species, only
C. polonica was really pathogenic (as indicated by its ability to
stain the sapwood after mass inoculations), whereas all species
were able to stimulate the phloem reaction significantly after
low density inoculations (as indicated by the extension of the
phloem reaction zone). It thus clearly appeared that the
pattern of fungal ability to stimulate the phloem reaction differed
largely from the pattern of fungal pathogenicity. Another
significant example is Ambrosiella symbioticum, a fungus
associated with S. ventralis. It strongly stimulates A. grandis phloem
reactions after low density inoculations
(Raffa and Berryman,
1982a; Wong and Berryman, 1977)
but is unable to penetrate
the sapwood after mass inoculations (Filip et al., 1989). It
should be noted however that, when comparing isolates from
the same species, a correlation between reaction zone length
after low inoculation density and fungal pathogenicity can be
(Lieutier et al., 2004; Plattner et al., 2008; Sallé et al.,
Moreover, the fungus invading the sapwood after a beetle
attack is not necessarily the one that plays a major role in
stimulating tree defenses, even after high inoculations densities. In
Figure 2, five different blue stain species are compared
regarding their ability to stimulate the phloem hypersensitive
reaction of Norway spruce after high density inoculation
, and their ability to grow into the sapwood after a
successful natural infestation by I. typographus
Whereas O. penicillatum induced more extensive phloem
reaction zone than C. polonica and O. bicolor, it stayed largely
behind these latter two species during sapwood invasion. The
pattern of the two fungal behaviors thus completely differed.
The pattern of fungal growth also largely differs between
the phloem and the sapwood. In Figure 3 for example, four
blue stain species were compared for their ability to grow
in the tissues of Douglas fir after low density inoculations
(Solheim and Krokene, 1998b)
. Ophiostoma pseudotsugae
and O. europhioides extended much more than L. abietinum
and C. rufipenni in the phloem, whereas no difference between
species was observed in the sapwood although the two
latter species tended to grow deeper. Clearly and as already
reported, various blue stain species mainly infest the phloem and
show a slow spread in the sapwood, whereas the opposite is
true for other species (Solheim, 1992b; Solheim and Krokene,
Low density inoculations
High density inoculations
Phloem reaction zone length (mm) after 5 weeks Bluestained sapwood (%) after 15 weeks 40 30
1998a,b; Uzunovic and Webber, 1998). Such different
behaviors may be explained by referring to oxygen availability in
tree tissues, the ability to invade sapwood being possibly
related to high tolerance to oxygen deficiency
1992a; Solheim and Krokene, 1998a)
Figure 3 also shows that for all species, stimulation of
tree defenses concerned both the phloem and the
superficial sapwood. Between fungi comparisons related to reaction
zone extension in the phloem show a tendency similar to that
of fungus extension in the phloem. In the superficial
sapwood however, C. rufipenni induced a more extended
reaction zone than O. pseudotsugae and O. europhioides, while
the reaction zone induced by L. abietinum was more extended
than that of O. pseudotsugae. One can thus think that during
a beetle attack, both fungal categories certainly interfere in
the stimulation of tree defenses, those growing better in the
phloem stimulating mainly phloem defenses, and those
growing better in the sapwood stimulating mainly superficial
sapwood defenses. Meanwhile, as it appears in Figure 3 related
to Douglas fir, stimulation of tree defenses seems mainly a
% blue stained sapwood
phloem phenomenon, especially when considering only the
fungal species associated with the Douglas fir beetle (and thus
excluding C. rufipenni). Evaluation of energy expenditure in
building tree defenses is needed in each tissue to really
compare the role of fungal species in exhaustion of tree defenses.
In addition, a possible interference of other micro-organisms
such as bacteria should also be taken into account
et al., 2006)
. However, it is worth mentioning that
stimulation of sapwood defenses concerns only the superficial part
of this tissue. This is an additional reason for not using
fungal pathogenicity and sapwood invasion to make conclusions
about the role of fungi in beetle establishment.
3.4. Prior tree death due to fungi is not a prerequisite for beetle establishment
This statement is, in some ways, the consequence of the
lack of relationship between fungus ability to stimulate tree
reaction and fungus pathogenicity. As long as tree resistance
is not overcome (below the critical threshold of attack density),
it contains the aggressors. Fungi can extend into the sapwood
only very little or not at all while beetles cannot establish in
the phloem. As soon as tree defenses are exhausted (above the
threshold), nothing impedes the development of the
aggressors and, even if a low level of resistance may still persist,
phloem and sapwood are invaded rapidly. As an example,
Figure 4 shows that, during a natural attack by I. typographus on
Norway spruce, no blue stain was observed in the sapwood
until a certain number of beetle attacks was reached but this
tissue was invaded rapidly above that threshold
. If, as generally thought, tree death effectively results
from disruption of water transport in the sapwood, the trees
were still alive when the threshold was reached. We thus think
that beetle establishment begins after tree resistance is
Number of beetle attacks
come, i.e. before tree is killed. Paine et al. (1997) have
already concluded that trees are overcome well in advance of
fungal growth in sapwood or changes in tree moisture status.
By comparing the timing of increase in stem circumference
with that of variations in sap flow velocity of loblolly pines
naturally colonized by D. frontalis,
Wullschleger et al. (2004)
also reached the conclusion that disruption of water balance
was not a prerequisite for beetle oviposition and larval
development. In these conditions, sapwood (and phloem) invasion
would simply be the unavoidable consequence of the
considerable depletion of tree resistance. Considering that beetles are
often associated with a complex group of fungi rather than
with a single fungal species (see below, Sect. 4.1), at the
moment when tree resistance is overcome, any present fungus,
even with a low level of pathogenicity, depending on substrate
characteristics and on its competitive ability, could invade the
sapwood successfully and thus possibly be involved in tree
death. Similarly, any insect present could also succeed in
establishing in the phloem. Death would then result from all
invaders acting together
(Berryman, 1972; Lieutier, 2002; Paine
et al., 1997)
, in addition to the sacrifice of tissues through the
induced reactions (Lieutier, 2002), and would occur after the
completion of the critical interactions between the tree and its
(Franceschi et al., 2005; Lieutier, 2002; Paine et al.,
The fastest growing species in the sapwood may have a
dominant role in tree death, in agreement with the
Paine et al. (1997)
that competition with other fungi
could have been a driving force of pathogenicity. However,
the presence of a fungus in the sapwood does not prove that
it is responsible for tree death since sapwood at this moment
can be colonized by any present pathogen. Several studies
have concluded that fungi are not the primary factor of
mortality in trees attacked by bark beetles
(Nebeker et al., 1993;
Parmeter et al., 1992; Stephen et al., 1993)
Hobson et al.
have even reported that during attacks by D.
brevicomis on Pinus ponderosa, Ophiostoma species apparently
invade the sapwood after the tree has died. In addition, there
are several examples where trees were killed without fungus.
That was reported in the case of D. frontalis
Bridges et al., 1985; Hetrick, 1949)
. Similarly, pines have been
killed by D. ponderosae, although G. clavigera, the only D.
ponderosae-associated fungus able to kill trees after mass
inoculation, was absent
(Six and Klepzig, 2004)
3.5. Fungi do play a role in beetle establishment on trees
We think that the beetle strategy of being associated with
moderately pathogenic fungi is really in operation in case
of the strategy of exhausting tree defenses, and that it acts
through the ability of the fungi to lower the critical
threshold of beetle attack density by stimulating tree reactions to
(Franceschi et al., 2005; Lieutier, 2002; Paine et al.,
. This association is even a necessity for successful
beetle attacks in trees with high resistance level. It has been
suggested that nutrition is very likely an important benefit for bark
(Adams and Six, 2007; Bleiker and Six, 2007; Klepzig
and Six, 2004; Six, 2003)
. We believe that stimulation of the
tree defense reactions is also a real benefit brought by the
fungi to the beetles. Following Paine et al. (1997) and
, beetle establishment is suggested to proceed in two
successive steps: (1) overcoming tree resistance (exhausting
tree defenses by stimulation) in both the phloem and the
superficial sapwood; (2) Invasion of tree tissues by beetles (in the
phloem) and fungi (in the sapwood and phloem). Experiments
with beetle attacks at low density have demonstrated that fungi
are effectively able to stimulate tree reaction when introduced
by a beetle into the tree. Figure 5 gives an example for I.
sexdentatus on Scots pine, after beetle attraction to the trees with
(Lieutier et al., 1995)
. Phloem reaction
zones were significantly more extended in front of galleries
with Ophiostoma than in front of those without Ophiostoma.
The observation that D. frontalis attacks without fungi
succeed at higher densities than attacks with fungi
(Bridges et al.,
1985; Whitney and Cobb, 1972)
is also in agreement with the
role of fungi in helping overcoming tree resistance.
3.6. Modalities of fungal impact on tree defenses
Several parameters can affect the role of the fungi during a
beetle mass attack, which must be taken into account before
concluding about the role that is or isn’t played by a fungus
in beetle establishment. These factors have already been
mentioned as invalidating the use of artificial mass inoculations
followed by measuring fungus pathogenicity to appreciate the
benefit brought by the fungus to the beetle. They also
condition the utilization of the results from artificial inoculations (at
low or high densities) performed to appreciate beetle ability to
stimulate tree defenses. They are the frequency of beetle
contamination, the diversity of fungal species, the between-isolate
variability in a fungal species, and the level of fungus load of
a contaminated beetle.
– The frequency of beetle contamination by a fungus can
vary largely between localities (even at a relatively short
distance) as well as with time in a same locality, and such huge
variations certainly affect the role of the fungus in beetle
In Europe, C. polonica has been reported as a common
associate of I. typographus in several localities
1989; Kirisits, 2001; Siemaszko, 1939; Solheim, 1986)
was however found less frequently and even very rarely in
(Harding, 1989; Jankowiak, 2005; Kirisits, 2001;
Mathiesen-Käärik, 1953; Sallé et al., 2005; Solheim, 1993b;
Viiri, 1997; Viiri and Lieutier, 2004)
. Similar situations have
been observed in North America with the associations between
D. rufipennis and C. rufipenni
(Six and Bentz, 2003; Six and
or L. abietinum (Aukema et al., 2005), between
D. ponderosae and G. clavigera or O. montium
(Six and Bentz,
2007; Six and Klepzig, 2004)
, and between D. frontalis and
(Bridges et al., 1985)
. The consequences, for
beetle population dynamics, of huge variations in the frequency
of fungal contamination have already been evoked through the
effects on beetle nutrition and reproduction
(Six and Bentz,
2007; Six and Klepzig, 2004)
. Certainly such variations
interfere in beetle population dynamics also through effects on
helping beetle establishment on trees.
– Additionally, the diversity of fungal species associated
with a beetle population must also be considered. There are
many examples of multiple associations in bark beetle species
(Lee et al., 2006; Six, 2003; Six and Paine, 1999; as
examples for North America, Kirisits; 2004, and references therein
. In forests of southern Poland, the frequency of
I. typographus beetles carrying O. polonicum with was 5.6%,
while it was 54.8% for O. penicillatum, 30.8% for O. bicolor,
27% for O. ainoae, 23.3% for O. piceaperdum and 20.3%
for O. piceae (Jankowiak, 2005). In these conditions, even if
O. polonicum appears the most pathogenic and even if it is
able to strongly stimulate tree responses after artificial
inoculations, one may doubt its dominant role in beetle
establishment (and even in sapwood invasion) in the natural conditions
of these forests.
Raffa and Smalley (1988)
mentioned that “the
relative abundance and proportions of various components of
the fungal flora could have a strong effect on the colonization
success”. To appreciate the role of fungi in the establishment
of a beetle population, it may thus be more important to
consider the total percentage of beetles carrying blue stain fungi
rather than the percentage of beetles associated with the
fungus which is most efficient in stimulating tree defenses. The
frequencies of the associations of D. ponderosae with G.
clavigera and O. montium can vary largely, one or the other fungal
species being the most prevalent depending on localities and
(Adams and Six, 2007; Bleiker and Six, 2009; Six and
probably in relation to differential performances
of each species under different temperature or moisture
(Bleiker and Six, 2009; Rice et al., 2008; Six and Bentz,
. However, as the two fungal species play a similar role
in beetle nutrition
(Adams and Six, 2007)
, they are believed
to complement each other, and the prevalence of one or the
other is said to assure the insect a correct feeding substrate
any time in all habitats of its geographic range
Six, 2009; Rice et al., 2008; Six and Bentz, 2007)
complementarities could be suggested between fungal species
involved in the stimulation of tree defenses, in this case and in
the case of other beetle species. Variations of associations
between D. frontalis and its symbiotic fungi, as well as between
beetle-associated-mites and fungi, have also been reported to
occur in relation to temperature (Hofstetter et al., 2007).
– The ability to stimulate tree defenses can also vary among
isolates within the same fungal species. A comparison
between 15 isolates of L. wingfieldii collected on the same date
within the same forest revealed large variations in their
ability to stimulate phloem tree defenses and to grow into the
phloem of Scots pine, after artificial low density inoculations
(Lieutier et al., 2004)
. The locality had no effect and,
owing to the dispersal capabilities of the vector T. piniperda,
it was concluded that all isolates were very likely to coexist
throughout the forest. They thus certainly coexist also within
the same beetle population. Similar results have been obtained
among isolates from different localities, with O. bicolor and
O. piceaperdum associated with I. typographus (Sallé et al.,
2005) and with G. clavigera associated with D. ponderosae
(Plattner et al., 2008; Rice et al., 2007a)
. In the latter
association, variations among isolates in their adaptation to cold
temperature have also been reported (Rice et al., 2008). Such
phenomena could have evident consequences regarding the role of
the associated fungus in stimulating tree defenses and
consequently in beetle attack success.
– The level of fungus load carried by an individual
beetle must also be considered to appreciate the real role of an
associated fungus in stimulating tree defenses in the event of
natural beetle attacks. Experiments with artificial low density
inoculations have demonstrated that resin concentration in the
phloem hypersensitive reaction zone that develops around a
wound is positively related to the number of spores introduced,
and that a minimum number of spores must be present to cause
mg resin / g fresh phloem in the first 25 mm of the phloem reaction zone, 5 weeks after inoculation
Numberof spores in sterile water
not significantly different from
sterile water alone
significantly different from
sterile water alone
inoculation of a 5 mm diameter
diskof malt agar culture
a significant response
(Lieutier et al., 1988; 1989a)
example (Fig. 7), this minimum number was higher than 104 spores
for L. wingfieldii, a fungus vectored by T. piniperda, whereas
it was 100 spores only for O. brunneo-ciliatum, vectored by
I. sexdentatus. Certainly a contaminated I. sexdentatus beetle
can introduce 100 spores of O. brunneo-ciliatum in its gallery
when attacking a tree, but it is not certain that a
contaminated T. piniperda beetle carries a sufficiently high number
of L. wingfieldii spores to introduce at least 104 of them in its
gallery. A 5 mm diameter disc of a 3-week-old sporulated agar
culture of L. wingfieldii or O. brunneo-ciliatum (that is
comparable to the discs commonly used in most experiments
using artificial inoculations) contains at least 106 spores
et al., 1989a)
and induces a tree response comparable to that
obtained with such a high number of spores (Fig. 7). This
could explain why L. wingfieldii, although strongly
stimulating tree reactions and having a high pathogenicity with
(Croisé et al., 1998a; Lieutier et al., 1989b;
Solheim et al., 1993; Solheim and Långström, 1991)
, is not
able to stimulate the phloem hypersensitive reaction during
T. piniperda trunk attacks
(Lieutier et al., 1989b; 1995)
thus not able to help this beetle in its establishment on the
trunk. On the contrary, O. brunneo-ciliatum can play a real
role during I. sexdentatus attacks on the same tree species
(Lieutier, 1995; Lieutier et al., 1989a; 1989b; 1995)
artificial inoculations of fungal culture to appreciate fungus
ability to stimulate tree defenses may thus lead in some cases
to overestimate the role of the fungus during natural beetle
4. WHY AND HOW BEETLES-FUNGI
RELATIONSHIPS INTERFERE IN THE
STRATEGY OF EXHAUSTING TREE DEFENSES
Here we discuss beetle – fungi relationships on a wider
temporal scale, by proposing functional and evolutionary
hypotheses for the role of Ophiotomatoid fungi in beetle establishment
through stimulating tree defenses. We propose: (1) that any
beetle species can develop an association with a fungus species
belonging to the Ophiostomatoid flora of its host tree, and can
take advantage of this association for its establishment; (2) that
the selection pressure that has led fungi to develop their
ability to stimulate tree defenses is the necessity of a considerably
low level of tree resistance for fungus extension into the tree.
Meanwhile, we replace the stimulating role of the
Ophiostomatoid fungi in the general context of conifer-bark beetle-fungi
relationships, leading us to present our views on the general
functioning of these relationships and finally to hypothesize
that beetle species are associated with fungal complexes, of
which species assume various roles regarding their
relationships with beetles and trees.
4.1. Origin of the associations and existence of fungal complexes
Beetle-associated fungal flora is generally similar between
two conifer species belonging to the same genus, even
attacked by beetle species from different genera. Conversely,
there are often large differences between the beetle-associated
fungal floras of conifers belonging to different genera, even
attacked by closely related beetle species. As examples in
Europe, the fungal flora associated with I. typographus, I.
duplicatus and I. amitinus on spruce resembles that of P.
chalcographus (also attacking spruce) much more than that associated
with I. sexdentatus and I. acuminatus on pines, which on the
contrary resembles that of O. proximus (also attacking pines)
. This suggests that the host tree has more
importance than the beetle in speciation of Ophiostomatoid fungi
(Harrington, 1993b; Kirisits, 2004)
. Phylogenetic studies of
the Ceratocystis species that develop on conifers have also
concluded that adaptation of these fungi to trees is older than
their adaptations to bark beetles
(Harrington and Wingfield,
. In these conditions, it is logical to suppose that
fungal ability to stimulate tree defenses is also adapted to the
We thus first hypothesize that any beetle species would be
helped in its establishment on a given tree species by
developing an association, even loosely, with a fungus species
belonging to the Ophiostomatoid flora of that tree species. In
addition, because several fungi often cohabit in a tree, we suppose
that the association could be developed with various species in
the same beetle population, each fungal species playing a
comparable or complementary role, even they aren’t as efficient.
Such a comparable and complementary role has already been
suggested for G. clavigera and O. montium in their feeding
association with D. ponderosae (see Sect. 3.6). The beetle
population would thus be associated with a fungal complex. For
I. typographus as an example, a similar background of fungal
species is generally found, C. polonica, O. anoiae, O. bicolor,
O. penicillatum and O. piceaperdum being the most common
and ecologically important species
varying in proportion depending on locality and time (see above
Sect. 3.6). In addition, competition certainly occurs between
the different fungal species of a complex in a bark beetle
(Bleiker and Six, 2009; Klepzig et al., 2001a; 2001b)
efficiency of the beetle fungus associations in beetle
population establishment would then depend on several components
acting complementarily: the intrinsic ability of the
beetleassociated fungi to stimulate tree defenses, the percentage of
contaminated beetles in the attacking population, the level of
individual inoculum, and the possible competitive interactions
between fungal species. As each of these components can be
affected by environmental factors and substrate variability, it is
not surprising that the nature of the dominant beetle-associated
fungal species and the efficiency of the insect-fungus
associations vary considerably in space and time, especially for
beetle species with large geographic distribution and high
dispersal capabilities. Moreover, diversified environmental
conditions favor a considerable mixing between beetle individuals
and associated fungal flora, certainly resulting in limiting
selective pressures, which in return would favor the coexistence
of different fungal species in a same beetle population.
4.2. Why fungi stimulate tree defenses
According to the above hypothesis, the beetle-fungus
associations can be weak and variable, which proves that certainly
no coevolution has occurred between beetles and
. Being basically adapted to
trees, blue stain fungi have then adapted to transportation by
beetles that live on these trees because they benefit from being
easily disseminated and then introduced, through wounding,
into a suitable host
(Harrington, 1993a; 2005; Kirisits, 2004;
. However, as said above, fungus extension in
sapwood is allowed only if tree resistance is depleted or at least
We thus hypothesize that the necessity of a considerably
low level of tree resistance for fungus extension into the tree is
the selection pressure that has led fungi to develop their
intrinsic ability to stimulate tree defenses. In healthy vigorous trees
attacked by very aggressive beetles, fungi must have
developed a high ability to stimulate tree defenses. This selection
could have been induced, directly or indirectly, through the
ability of the fungus to grow into the phloem. Indeed, in a
context of interspecific competition, growth speed is a crucial
element for a fungus, since in both dead and living trees it
conditions fungus access to resources like food or ability to produce
fruiting bodies in the galleries of dispersing beetles, and fungal
extension is positively related to phloem reaction zone length
after low density inoculations
(Ben Jamaa et al., 2007;
Lieutier et al., 1989b; 2004; 2005; Wong and Berryman, 1977)
That aggressive beetle species develop more frequently in
living trees than moderately or low aggressive species would thus
have lead, for the former, to more specific associations with
fungi able to grow rapidly in living trees, and thus to strongly
stimulate tree reactions. Moreover, in the context of an
association with bark beetles for transportation, it must be noted
that developing the ability to stimulate tree defenses to
invade a tree is a better strategy for a fungus than developing its
own pathogenicity, because this latter choice would have led
to competition with the insect for the tree tissues (see below).
4.3. The necessity of controlling fungus extension
Being associated at a high frequency with a fungus that
extends quickly in the tree is a risk for beetle progeny if
fungus extension is not controlled after tree defenses are
(Harrington, 1993a; Lieutier, 2002; Six and Klepzig,
2004, among others)
. There are several examples indeed where
phloem invaded by fungi is unsuitable for beetle larvae, such
as in the case of O. minus for D. frontalis
Klepzig et al., 2001a; 2001b)
, O. montium for D. ponderosae
(Six and Paine, 1998)
or O. ips for I. avulsus
in Klepzig and Six, 2004)
. These observations raised the
question of how a fungus that is antagonist to the beetle larval
development can be maintained in a beetle population at a high
level of association, instead of being strongly counter-selected.
Klepzig et al. (2001a) and Six and
that an explanation could be the benefits brought by such fungi
to the phoretic mites carried by the beetles. Certainly also, the
benefit brought by the fungus in stimulating tree defense
reactions is a valid reason.
For the beetle, equilibrium must be maintained between
benefit from stimulating tree defenses and antagonism to
larval development. Dendroctonus frontalis has achieved such an
equilibrium by developing an association with a mycangial
species of the genus Entomocorticium, antagonist to O.
minus in addition to bringing nutrients to beetle larvae
et al., 2001a; 2001b)
. Similar associations, eventually with
other micro-organisms such as bacteria
(Cardoza et al., 2006)
certainly also exist in other bark beetle species. In the fungal
complexes associated with bark beetles, there is often one
relatively virulent blue stain species, while others are less
(Kirisits et al., 2000; Kirisits, 2004; Lee et al., 2006;
Lieutier et al., 1989b; Six and Klepzig, 2004; Solheim, 1988)
The role of each of them is ignored. Possibly antagonisms
comparable to those described in the D. frontalis complex
also exist, resulting in beetle brood protection and nutrition.
Phloeomycetophagous species with sac mycangia should be
(Klepzig and Six, 2004; Six, 2003; Six and Klepzig,
, amongst which are D. ponderosae
(Six and Paine,
1998; Whitney and Farris, 1970)
, D. jeffreyi
(Six and Paine,
, D. brevicomis
(Paine and Birch, 1983)
acuminatus (Francke-Grosmann, 1967). Beetles with pit mycangia
may also be concerned especially if pits have glands, such as
I. sexdentatus (Levieux et al., 1991). However, in this later
species, no mycangial fungus has been identified and
fungifree insects develop the same way as fungi-contaminated
beetles (Colineau and Lieutier, 1984).
Klepzig and Six (2004)
have suggested that, on poor substrates like dead trees or trees
that die rapidly after attack, beetles need complementary food
(mainly nitrogen and sterols) and could get it from associated
fungi even if they are not mycangial. Possibly and similarly to
the mycangial fungi, non mycangial fungi that serve as
additional food may also serve as antagonists to blue stain fungi
after tree resistance is overcome.
4.4. Multiple roles of associated fungal complexes, a final hypothesis
As a consequence of the discordance between fungal
pathogenicity and beetle aggressiveness, of the tree killing
process proposed above, and of our above comments and
hypotheses on relationships between beetles and fungi, we
finally hypothesize that a beetle species using the strategy of
overcoming tree resistance is generally associated with a
fungal complex, of which species could assume four roles
regarding relations with beetles and trees: (1) to stimulate tree
defenses in the phloem and superficial sapwood, (2) to grow
into the sapwood after tree resistance is overcome, (3) to
control phloem extension of the first other two categories, and
(4) to bring nutrients to the beetle progeny, at least for the
phloeomycetophagous beetles. Depending on beetle species
and host trees, as well as environmental factors and beetle
population levels, the nature and the relative abundance of the
beetle associated fungal species can vary (see Sects. 3.6 and 6,
references included), and thus the above roles could be played
by different fungal species, or the same fungal species could
be involved in several roles, which could have consequences
for beetle population dynamics
(Hofstetter et al., 2006)
As suggested by
Paine et al. (1997)
, multiple and complex
interactions among beetle-associated fungi and between fungi
and beetles thus certainly exist. Their complexity is even
increased, at least in some cases, by additional interactions with
other partners such as nematodes, bacteria and mites
et al., 2006; 2008; Klepzig et al., 2001a; 2001b)
interfering in beetle establishment and brood development in trees,
these complex interactions must play an important role in the
beetle population dynamics and outbreaks.
5. TYPES OF BEETLE-FUNGUS ASSOCIATIONS
There is a large diversity of types of bark beetle –
Ophiostomatoid fungus associations, from absent or very simple to
very elaborate. In the following section, we tentatively classify
these associations by using the above considerations. Among
beetle species able to develop on living trees, we are able to
define three main groups while indicating the possible levels
of beetle aggressiveness within them. For species using the
strategy of overcoming tree resistance, we also tentatively
distinguish fungal species mainly stimulating tree defenses from
those rather specialized in invading the sapwood after tree
defenses are exhausted. In these cases, we present known
examples when fungus roles have been observed, or predictions
when fungus roles are unknown.
5.1. Group 1 – Beetle species not using the strategy
of exhausting tree defenses and without an effective
association with Ophiostomatoid fungi
This group gathers beetle species from both ends of the
– Group 1a is composed of highly aggressive beetles
attacking living trees and succeeding in establishing and
developing in their hosts without killing them, by using the strategy
of avoiding tree defenses. They are not associated with fungi
because they must avoid stimulating tree defenses. Typical
examples are D. micans and D. punctatus on spruces. D. micans
carries O. canum but at a very highly variable frequency (0.5
to 90 %) and the fungus does not play any role in the success of
(Lieutier et al., 1992)
corresponding to no real association.
– Group 1b gathers the very secondary beetle species that
are able to attack living trees but with very low or
without defense ability. They do not need to exhaust tree
defenses and thus have not developed fungal associations for
establishing in trees. Some secondary beetles, that are weakly
or even frequently associated with more or less pathogenic
black stain fungi, also belong to this group. They are
however passive vectors, apparently unaffected by the presence
of the fungus, and their associations are rather commensal
(Klepzig and Six, 2004)
. For example, D. terebrans attacks
only very weak pines, although it has already been found to
be highly associated (up to 100% of the beetles) with L.
terebrantis, a fungus which, in addition to its high pathogenicity
(Harrington and Cobb, 1983)
, is able to induce violent tree
(Rane and Tattar, 1987; Wingfield, 1983)
occasionally, Hylastes and Hylurgus carry L. serpens, L.
procerum or L. wageneri
(Harrington, 1993a; Jacobs and
Wingfield, 2001; Klepzig et al., 1991)
. Hylurgops are also vectors
of L. procerum, and D. valens can carry L. serpens and even L.
(Klepzig et al., 1995; Jacobs and Wingfield, 2001)
In all these situations however, the insect doesn’t need fungus
to be present in order to establish itself on trees and certainly
only plays the role of a vector for the pathogen, making the
association not a true one
(Klepzig et al., 1991)
. The fact that
fungus has no role to play in beetle establishment on trees,
however, does not discard the possibility that associations with
fungi have been developed to bring nutrients to the insect, at
least at the larval stage
(Eckhardt et al., 2004; Klepzig and Six,
5.2. Group 2 – Beetles species using the strategy of exhausting tree defenses but very loosely associated with fungi
Beetle species of this group attack living trees but only a
very low proportion of individuals carry fungi and/or the fungi
are unable to stimulate tree defenses when introduced into the
tree by a beetle. The strategy of exhausting tree defenses is
used but with little success because the fungal association is
not effective enough to stimulate tree defenses. The best
example seems to be T. piniperda on pines during the reproduction
phase of its life cycle when it attacks trunks. It is associated
with two blue stain fungi, L. wingfieldii rather pathogenic and
O. minus moderately pathogenic, but the latter species has a
very low and variable frequency, while the former is unable
to stimulate tree defenses when introduced into the tree by
a beetle (see above Sect. 3.6)
(Lieutier et al., 1989a; 1989b,
. Probably Cryphalus abietis on fir
in Kirisits, 2004)
and Pityogenes quadridens on Scots pine
(Mathiesen-Käärik, 1953; in Kirisits, 2004)
also belong to this
group because of the low frequency of their associated fungi.
In this group, stimulation of tree defenses is only due to beetle
tunneling activity resulting, as observed during T. piniperda
reproductive attacks, in a high critical threshold of attack
(Långström and Hellqvist, 1993; Långström et al., 1992)
and a very moderate or low aggressiveness
Hellqvist, 1988; Lieutier et al., 1995)
5.3. Group 3 – Beetle species using the strategy of exhausting tree defenses and highly associated with blue stain fungi
Beetle species attack living trees, with a high percentage of
individuals (sometimes up to 100%) carrying blue stain fungi.
Within this group it is possible to distinguish beetle species
according to their level of aggressiveness, while comparing it
with the ability of their associated blue stain fungi to
stimulate tree defenses (Tab. II). The problem is however that, in
no situation, is the ability of the fungus to stimulate tree
defenses after introduction into the host tree by a beetle known.
We are thus using the fungus stimulating ability after artificial
low density inoculations, and making predictions in situations
where this ability is still unknown.
In Table II, considering species where data on fungus
ability to stimulate tree defenses is available, the highly aggressive
beetle species, D. frontalis, D. ponderosae, D. jeffreyi, D.
pseudotsugae, S. ventralis and I. typographus
(Furniss and Carolin,
1977; Grégoire and Evans, 2004)
are all frequently associated
with Ophiostomatoid fungi amongst which at least one species
is able to highly stimulate tree phloem defenses (references in
Tab. II). This is the same for I. cembrae in Scotland, its area
(Redfern et al., 1987)
. inversely, I. acuminatus,
I. sexdentatus, O. erosus and I. pini, known to be moderately
or weakly aggressive
(Chararas, 1962; Furniss and Carolin,
1977; Grégoire and Evans, 2004)
, are all associated with
fungal species that stimulate the phloem response only
moderately or weakly (references in Tab. II). There is thus a close
correspondence between beetle species aggressiveness and the
ability of their main associated fungal species to stimulate tree
The stimulating ability of the fungi associated with the
other beetle species presented in Table II is not known but
we can make the following predictions. Since D. brevicomis
is known to be a very aggressive beetle for pines
and Carolin, 1977)
, O. brevicomi certainly stimulates pine
defenses very strongly during attacks by D. brevicomis.
Similarly, D. rufipennis is a highly aggressive beetle species on
spruce (Dumerski et al., 2001) and is highly and frequently
associated with L. abietinum
(Aukema et al., 2005; Reynolds,
1992; Six and Bentz, 2003)
. We predict that L. abietinum
strongly stimulates spruce defenses when introduced by D.
rufipennis in its host. Conversely, the aggressiveness of I.
cembrae for the European larch is usually only moderate in
(Grégoire and Evans, 2004; Redfern et al.,
, its area of origin. Certainly, C. laricicola or another
highly associated fungal species stimulates larch defenses only
moderately in that area.
At this stage of the discussion, one may wonder if a relation
exists between beetle aggressiveness and the ability of the
fungal complex or other organisms to control blue stain extension
in the phloem. Indeed, when a possibility of controlling
fungus extension exists, it should allow beetles to tolerate fungal
species which are particularly able to grow well in the phloem
and thus be able to strongly stimulate tree reactions. However
the lack of sufficient knowledge on the existence of
antagonistic species does not allow the validity of this speculation to be
6. SPECULATIVE CONCLUSION
Bark beetles and fungi maintain very complex and multiple
interactions, making it difficult to propose a general model of
beetle - fungus relationships. Bark beetles vary considerably
in their dependence on fungal complexes for nutrition and
establishment on trees, according to both their strategy of
establishment and the physiological state of their usual host tree.
For the species using the strategy of exhausting tree defenses,
however, to be associated at a minimum frequency with fungi
References regarding fungus ability to stimulate tree defenses: 1 = Ben Jamaa et al. (2007); 2 =
Cook et al. (1986)
; 3 =
Cook and Hain (1987)
; 4 =
Krokene and Solheim (1997)
; 5 = Krokene and Solheim (1999); 6 = Lieutier et al. (1989b; 7 =
Lieutier et al. (1990)
; 8 = Lieutier et al. (1991b); 9 =
Lieutier et al. (2005)
; 10 =
Paine and Stephen (1987)
; 11 =
Raffa and Smalley (1988)
; 12 =
Redfern et al. (1987)
; 13 = Rice et al. (2007a); 14 = Rice
et al. (2007b); 15=
Ross et al. (1992)
; 16 =
; 17 = Solheim and Krokene (1998a); 18 = Solheim and Krokene (1998b); 19 =
able to naturally stimulate tree defenses is a necessity for an
efficient attack. The association, even loosely, with
Ophiostomatoid blue stain fungi allows that.
For the aggressive beetle species, one may speculate that
no association existed at the origin, except in some cases for
nutrition, which is currently the case for the saprophytic and
very secondary beetles of group 1b. The conquest of living
trees would have then diverged in two directions, one towards
avoiding tree defenses without developing a fungal
association (today group 1a), the other towards exploiting tree
defenses, with the progressive help of fungi. In this latter
situation, the association would have begun first as occasional,
possibly comparable to the current situation for T. piniperda
in group 2. Then, fungi would have been selected for their
ability to grow in the phloem and thus to stimulate tree
defenses (group 3). But this step would not allow selecting fungi
that were particularly efficient in stimulation because such
efficient fungi would grow too fast in the phloem after tree
defenses had been exhausted. The final step would thus have
been to develop novel associations with additional fungi (or
other micro-organisms) able to control the phloem extension
of the former after defense exhaustion, thus allowing the
development of associations with fungi that were fast growing in
the phloem and very efficient in stimulating tree defenses. As
already mentioned, the most active sapwood invading species
are most often not those that have helped the beetle in
previously exhausting tree defenses. In such a context, most
sapwood invading fungi might simply be “cheaters” which have
taken advantage of the increasing efficiency of the
relationships between the beetles and the fungi stimulating tree
defenses. The rather high level of pathogenicity of some of them
would result only from competition with other sapwood
invaders. Such a scenario of building beetle-fungi associations
based on primary association with fungal species specialized
in stimulating tree defense, completed later with the arrival of
pathogenic “cheaters”, can be interestingly compared with the
suggestion that weakly pathogenic fungi are “established
associates” whereas pathogenic fungi are incidental late arriving
(Six, 2003; Six and Paine, 1999; Six and Klepzig,
Moving from group 1b to groups 2 and then 3 gives the
appearance of moving from very simple relationships
(nutritional only) to more and more elaborate relationships
(nutritional plus stimulation plus eventual control of extension)
between beetles and fungal complexes. However, these
successive groups do not reflect the phylogeny of bark beetles, at
least when referring to the Dendroctonus genus
Farrell, 1998; Six and Klepzig, 2004)
. The above speculated
process of building beetle-fungi associations would have thus
evolved independently for the different beetle species of a
same group. This is in agreement with the conclusion that no
coevolutionary process has occurred between beetles and their
Acknowledgements: We thank two anonymous reviewers for their
very constructive and stimulating remarks on an earlier version of
this paper. We also thank Caroline Sarré (University of Orleans) for
proofreading the paper.
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