Journal of Plant Diseases and Protection, 117 (1), 3338, 2010, ISSN 1861-3829.
Eugen Ulmer KG, Stuttgart
Different effects of the insectpathogenic fungus Beauveria bassiana (Deuteromycota) on the
bark beetle Ips sexdentatus (Coleoptera: Curculionidae) and on its predator Thanasimus
formicarius (Coleoptera: Cleridae)
Unterschiedliche Wirkung von Beauveria bassiana auf den Borkenkfer Ips sexdentatus (Coleoptera: Curculionidae) und
dessen Ruber Thanasimus formicarius (Coleoptera: Cleridae)
B.M. Steinwender1,2,*, H.W. Krenn2 & R. Wegensteiner1
1 University of Natural Resources and Applied Life Sciences, Institute for Forest Entomology, Forest Pathology and Forest Protection, Vienna,
Austria
2 University of Vienna, Department of Evolutionary Biology, Vienna, Austria
* Corresponding author, e-mail bernhardt.steinwender@boku.ac.at
Received 5 February 2009; accepted 14 September 2009
Abstract
In our study, Beauveria bassiana was tested on the bark beetle
Ips sexdentatus (Coleoptera: Curculionidae) and its predator
Thanasimus formicarius (Coleoptera: Cleridae). Infection ex-
periments were conducted in the lab at 20C and long day
conditions. B. bassiana, isolated from a different bark beetle
species Ips typographus, was grown on malt extract agar.
I. sexdentatus was collected from infested pine log sections
and T. formicarius from pheromone baited traps. To check for
insect pathogens that would influence the infection experi-
ments significantly, a pathogen analysis was conducted. In-
sect pathogens were found in different frequencies (multiple
infections were also noted): the ascomycete Metschnikowia cf.
typographi (29.8%), the protozoan Gregarina cf. typographi
(36.0%) as well nematodes in the hindgut (38.5%) and nem-
atodes in the haemolymph (16.0%). Adult I. sexdentatus were
inoculated with two different concentrations of conidia sus-
pension (1 106, 1 107 conidiospores ml1) and dry conidia,
stripped off from infected bark beetle cadavers. T. formicarius
was inoculated with conidia suspension (1 107 conidiospores
ml1), dry conidia and a very high amount of dry conidia. The
experiments showed that B. bassiana killed up to 93.7% of the
inoculated I. sexdentatus within 8 days, but no T. formicarius
at these doses and only seven out of 15, inoculated with the
highest amount of conidiospores.
Key words: entomoptahogenic fungus, entomopathogens,
side effects, wood pest
Zusammenfassung
In dieser Studie wurde die Wirkung von Beauveria bassiana
auf den Borkenkfer Ips sexdentatus (Coleoptera: Curculioni-
dae) und dessen Ruber Thanasimus formicarius (Coleoptera:
Cleridae) getestet. Alle Infektionsversuche wurden im Labor
bei 20C und Langtagbedingungen durchgefhrt. Der B.-bas-
siana-Stamm wurde von einer anderen Borkenkferart (Ips
typographus) isoliert und auf Malzextraktagar gezogen. I. sex-
dentatus wurde von befallenen Kiefernstmmen, T. formica-
rius aus Pheromon bekderten Borkenkferfallen abgesam-
melt. Um einen entscheidenden Einfluss anderer Insekten-
pathogene auf die Infektionsversuche auszuschlieen, wurde
eine begleitende Pathogenanalyse durchgefhrt. Dabei wurde
der Ascomycet Metschnikowia cf. typographi (29.8%), der Ein-
zeller Gregarina cf. typographi (36.0%) und Nematoden,
sowohl im Enddarm (38.5%) als auch in der Haemolymphe
(16.0%), gefunden (diese Pathogene/Parasiten traten auch
kombiniert auf). Adulte I. sexdentatus wurden entweder mit
einer von zwei unterschiedlich konzentrierten Konidiensus-
pensionen (1 106, 1 107 Konidiosporen ml1), oder mit
trockenen Konidien, die direkt von toten, verpilzten Kfern
J.Plant Dis.Protect. 1/2010
auf die Versuchstiere abgestreift wurden, inokuliert. T. formi-
carius wurde entweder mit der hher konzentrierten Koni-
diensuspension (1 107 Konidiosporen ml1), mit trockenen
Konidien bzw. einer extrem hohen Konzentration von trocke-
nen Konidien inokuliert. Die Ergebnisse zeigen, dass dieser
B.-bassiana-Stamm innerhalb von 8 Tagen bis zu 93.7% der
damit inokulierten I. sexdentatus ttet. Die gleiche Dosierung
aber fhrte zu keinem einzigen toten T. formicarius. Deshalb
wurde die Dosierung nochmals erhht und erst damit sieben
von 15 dieser Insekten gettet.
Stichwrter: Holzschdling, Insektenpathogene, insekten-
pathogener Pilz, Nebenwirkungen
1 Introduction
Usually, bark beetles (Coleoptera: Curculionidae, Scolytinae)
ensure the regeneration of the forest by eliminating old and
sick trees and accelerating their decomposition. For managed
spruce forests, bark beetles may be problematic when they
occur in a high abundance, so that they do not only kill old
and severely sick trees, but also slightly weakened ones. They
develop under the bark of their host trees and usually feed on
the phloem during their development/maturation. After that,
they emerge and spread to find other trees appropriate for
breeding. There they mate and lay their eggs in special niches
under the bark. If there is enough breeding material, espe-
cially after extreme weather events (snow, hail, storm) or
after forest fire, they can cause high losses to the forestry. As
bark beetles live most of their life cycle in the phloem, the start
of a bark beetle attack is difficult to detect, and the treatment
with synthetic insecticides is inefficient too, due to the insuf-
ficient control of the insects under the bark. Additional to this
negative point, there are also side effects known on other,
non-target insects, when chemicals are used. Until now, no
systemic insecticides are known, that would be able to effi-
ciently target the bark beetle in the phloem. Therefore, there
is an urgent need to search for adequate natural enemies, such
as predators, parasitoids and pathogens (SCHWERDTFEGER 1981;
ALTENKIRCH et al. 2002).
Ips sexdentatus (Coleoptera: Curculionidae) (Boerner) is
known to attack pine trees and is categorized as very aggressive
in Germany and Spain (GRGOIRE and EVANS 2004). In some
parts of Turkey and Georgia its damage is disastrous (Yamman
pers. comm.). Also in Austria this species was recorded of
increasing importance (PERNY 2003). Furthermore, I. sexden-
tatus is known to transfer blue-stain fungi, which results in a
dramatic value loss of the infested wood (FERNANDEZ 2004).
Beauveria bassiana (Balsamo) Vuillemin (Ascomycota:
Hypocreales) is a well known insect pathogenic fungus that
has a wide host range within the insects (FENG et al. 1994;
BUTT et al. 2001; INGLIS et al. 2001; ZIMMERMANN 2007). It is
34 Steinwender et al.: Effects of Beauveria bassiana on Ips sexdentatus and Thanasimus formicarius
also known to infect a number of bark beetle species (NOVK
and SAMINKOV 1967; WULF 1983; LUTYK and SWIEZYNSKA
1984; WEGENSTEINER and WEISER 1996; WEGENSTEINER 2000).
B. bassiana can often be found during autumn on beetle
cadavers in old bark beetle galleries. This common natural
occurrence obtrudes it as one of the most promising candi-
dates for the biological control of bark beetles. Ips typographus
L. (WEGENSTEINER 1992; KREUTZ et al. 2004a), Polygraphus poli-
graphus (WEGENSTEINER 2000) and very recently I. sexdentatus
(DRAGANOVA et al. 2007) have been subject to different bio-
assays with B. bassiana, which indicates the interest in this
subject. The idea behind the use of B. bassiana for bark beetle
control is not only based on the fact that a pathogen endemic
in Austria will be used, but also that inoculated bark beetles
will introduce the conidiospores into their breeding systems
(galleries) before they die and provide a new source of fresh
conidiospores for conspecifics and offspring. So the main
barrier that exists between the bark beetles and their efficient
control, the bark, could be bypassed. KREUTZ et al. (2004b)
suggest the use of pheromone baited traps combined with dry
conidiospores to catch and inoculate bark beetles, allow them
to escape afterwards and die elsewhere to spread the spores.
WEGENSTEINER (2004) suggests the treatment of logs with
conidia suspension, where the beetles would be inoculated by
touching the surface of the treated logs. Unfortunately the
problem concerning the use of B. bassiana in the field is not
only the unknown efficiency on the targeted pest, but also
possible side effects on non-target insects and the lack of data
in this field of pest control.
The bark beetle predator Thanasimus formicarius L. (Co-
leoptera: Cleridae) could be one of these non-targets. It is a
predator specialised on bark beetles, but within this group, it
is a generalist. During its larval stage it hunts bark beetle
larvae in their galleries and as an adult it predates adult bark
beetles on the bark. HEIDGER (1994) found that an adult T. for-
micarius can kill and consume up to five adult bark beetles a
day (depending on their size) and that a larva consumes dur-
ing its development about 42 Ips typographus larvae. T. formi-
carius is often mentioned in connection with bark beetle
control (SCHWERDTFEGER 1981; HEIDGER 1994; ALTENKIRCH et al.
2002; KENIS et al. 2004; WARZE and GRGOIRE 2003; WARZE et
al. 2006) and is therefore a perfect object for investigations
about possible side effects of B. bassiana.
There is only little known about pathogens of I. sexdentatus
(WEGENSTEINER et al. 2007; YAMAN 2007), and even less about
the effects of entomopathogenic fungi except for one prelimi-
nary test (DRAGANOVA et al. 2007). All these facts were the rea-
son to investigate the effects of the entomopathogenic fungus
B. bassiana on the beetles I. sexdentatus and T. formicarius.
The present investigation is a laboratory approach that
shows the effects of B. bassiana on the bark beetle I. sexdenta-
tus and on the bark beetle predator T. formicarius to estimate
the impact of this entomopathogenic fungus on these insects.
It is the first step on the way to develop a method for biological
control of bark beetle.
2 Material and methods
The inoculation experiments were conducted in the labora-
tory from spring to autumn 2006. All manipulations for the
experiments including the B. bassiana conidia were conduct-
ed in a laminar flow sterile working bench (Prettl, Pfullingen,
Germany).
Up to this time no B. bassiana field infection was described
from I. sexdentatus. The B. bassiana culture was established
with an isolate from I. typographus (collected in summer 2005
in Rothwald, Lower Austria) to provide sufficient and fresh
B. bassiana conidiospores for the inoculation experiments.
The entomopathogenic fungus was identified with a light
microscope due to its characteristic zigzag grown conidiog-
enous cells and its small, spherical conidiospores. Afterwards
the dead beetle was rolled over 2% malt extract agar plates
[MEA; 1000 ml aqua dest.: 20 g malt extract (Diamalt) + 16 g
agar powder 2521 (W. Behrend & Co., Hamburg, Germany) +
0.1 g streptomycinsulfate] which were stored in an incubator
at a temperature of 20C ( 2C) and with a relative humidity
(RH) higher than 70%. After 4 days incubation, the resulting
colony forming units were examined and the most promising
and pure parts of them transferred to other 2% MEA plates
(same mixture, but without streptomycinsulfate). The result-
ing culture was again confirmed as B. bassiana under a light
microscope. The culture was duplicated and if required (every
month) transferred to new MEA plates. One of the plates was
sent to the fungal library of the fungal collection of Institut
Pasteur (Paris), accepted and stored (number UMIP 2621.07).
To prepare the conidia suspension for the experiments, a
B. bassiana plate was flooded with sterile water, agitated and
after that the fluid sucked off and a drop of Tween 80 added
to avoid agglutination of spores. Conidia concentration was
determined in a haemocytometer (Brker-Trk, LO-Labor-
technik, Friedrichsdorf, Germany). Germination tests were
conducted with the suspended conidia using agar covered
microscopical slides. Twenty-four hours later, the germination
process was stopped by adding lactophenolic blue solution
(Merck, Darmstadt, Germany) and the germination rate was
determined under a normal light microscope at magnification
of 200 x.
I. sexdentatus infested pine log sections were brought from
the field to the lab and incubated in breeding chambers at
21C ( 2C) under long day conditions (light : dark = 16 : 8 h).
Emerging beetles were collected every day and separated into
old adults (dark brown to black) and young adults (light
brown). To ensure that there were no diseases influencing the
results of the inoculation experiments, a pathogen analysis
was conducted with 400 emerging beetles. In addition, gen-
der of the beetles was noted. For re-inspection and archival
storage the native smears were first dried, then fixed with a
drop of methanol and stained with Giemsa staining fluid
(Merck; one drop per 1 ml aqua dest.).
For inoculation, beetles were dipped for 3 seconds into
conidia suspensions with a concentration of 1 107 or 1 106
conidiospores ml1. After that, 10 beetles were put into one
Petri dish in each case, fed with bark and checked daily. Dead
beetles were removed and put individually into Petri dishes
and transferred into high humidity glass boxes. After the fun-
gus penetrated the cuticle and produced conidia, the conid-
iospores were checked under a light microscope. Inoculation
experiments were conducted in an incubator at 20 1C)
under long day conditions and high humidity (> 70%). For the
higher concentrated suspension, 210 I. sexdentatus were used
and for the lower concentrated one 150. For the other part of
the I. sexdentatus inoculation experiments, dry conidia from
lab infected I. sexdentatus were used. The bark beetles last
distal sternits were inoculated by stripping conidia directly off
from cadavers with sporulating B. bassiana (n = 190). After in-
oculation, the beetles were treated the same way as described
before. Beetles of the untreated control group were incubated
at the same conditions (n = 200).
T. formicarius were attracted by bark beetle pheromones
into Theysohn bark beetle traps baited with Ipsowit or
Trypowit (both: Witasek, Feldkirchen, Austria). Some more
T. formicarius were collected directly from logs in the forest.
They were kept singly in Petri dishes and fed with one adult
I. typographus a day. The feeding behaviour and consumption
rate was listed before and during the experiments.
T. formicarius was inoculated by the same methods as
I. sexdentatus, but only one conidia suspension concentration
(107 conidiophoores ml1) (n = 30) was used. Due to the fact
that after this inoculation and the inoculation of the last distal
sternits with dry conidia (n = 30) none of the beetles died
because of B. bassiana it was not sure if they could be infected
with this strain of B. bassiana. It was decided to use an even
higher dose of dry conidia by covering the whole ventral side
J.Plant Dis.Protect. 1/2010
 Steinwender et al.: Effects of Beauveria bassiana on Ips sexdentatus and Thanasimus formicarius
of the beetles with dry conidia (n = 15). The latter inoculation
method therefore was named extreme, the prior normal.
However, it was not possible to calculate the number of inoc-
ulated conidia. Beetles of the untreated control group (n = 30)
were incubated at the same conditions (20 1C); long day
conditions).
For calculation of the arithmetic mean and the standard
deviation the program Microsoft Excel 2002 was used. The
chi-square test was used to check differences in the frequency
of the pathogens found in I. sexdentatus, homogeneity test
(Kolmogoroff and Smirnoff in SACHS 1997) was used to deter-
mine whether mortality of I. sexdentatus was different or not.
In addition t-test was used to check differences in T. formi-
carius mortality and prey consumption.
As a part of preliminary tests and investigations, the dissec-
tion of 400 I. sexdentatus (246 females, 154 males), brought
evidence of the ascomycete Metschnikowia cf. typographi
(29.8%) and the protozoan Gregarina cf. typographi (36.0%)
in the cells of the mid-gut epithelium and the mid-gut lumen,
respectively, as well as nematodes in the hindgut (38.5%) and
in the haemolymph (16.0%). Some of these pathogens
appeared also as mixed infections. All these pathogens were
identified as known bark beetle pathogens with no immediate
effect on this beetle group and therefore the use of this bark
beetle source for the inoculation experiments was approved.
To determine the number of inoculated conidia per beetle
using the two different conidia suspensions, 10 I. sexdentatus
were weighed on a micro-scale (Mettler Toledo MT5, Mettler
Toledo, Vienna, Austria, d = 0.1 g), before and after they
were dipped for 3 seconds into sterile water. One mg weight
difference between each measurement was equivalent for 1 l
fluid on the beetles surface. The weighing resulted in an
average weight difference of 5.3 mg equivalent for 5.3 l.
That means about 5,300 conidiospores for the 1 106 conid-
iospores ml1 and 53,000 conidiospores for the 1 107 conid-
iospores ml1 concentrated suspension inoculating one beetle.
Four repetitions of the germination test resulted in a mean
germination rate of 92.3% (SD: 1.5%).
For preliminary virulence tests, 50 I. typographus were used
from the laboratory stock. I. typographus was inoculated with
5 107 conidiospores ml1 concentrated conidia suspension
and afterwards treated in the same way as described with
I. sexdentatus. The first six beetles died after 3 days. On the
5th day, 60% of the beetles were already dead. The last one
died on day 9. LT50 was 4.7 days. Altogether 92% of the tested
I. typographus were killed by B. bassiana.
3 Results
3.1 Ips sexdentatus inoculation tests
The B. bassiana inoculated I. sexdentatus died at different
times post inoculation, depending on whether they were inoc-
100
90
80
70
60
% 50
40
30
20
10
0 tion with dry conidia (DC) or with
1 2 3 4 5 6 7 8
Days
J.Plant Dis.Protect. 1/2010
35
ulated with dry conidia or different concentrations of conidia
suspension (Fig. 1). Each experiment was stopped, after all
beetles died (except beetles from the control group that was
stopped after 30 days). Not all of the beetles inoculated with
B. bassiana died because of the entomopathogenic fungus.
I. sexdentatus inoculated with dry conidia died faster than
beetles inoculated with the suspensions 1 107 and 1 106
conidiospores ml1. A comparison of freshly emerged, young
adults with old adults of the same type of inoculation showed
no significant differences, so the results of young and old
beetles were put together in Figure 1. Till day 4, the cumula-
tive mortality (caused by B. bassiana) was almost 0, after day
four, mortality progress was different. The first I. sexdentatus
inoculated with dry conidia died on the 2nd day, the first
beetles (n = 2) inoculated with the 1 107 conidiospores ml1
conidia suspension died on the 3rd day, the first beetles (n = 7)
inoculated with the lower concentration died on the 5th day.
The first beetles (n = 4) of the control group died on the 6th
day, but they where not infected with B. bassiana; none of the
beetles of the untreated control group died on a fungal infec-
tion. B. bassiana caused highest mortality (93.7%) on the
beetles inoculated with dry conidia on the 8th day, followed by
the higher suspension group (93.3% mortality) on 9th day
and the lower suspension group (82.3% mortality) after 13
days (Fig. 1). The four different mortality curve progressions
were significantly different (P > 0.01).
Most beetles of the group inoculated with dry conidia died
during day 5 and day 6 (LT50 = 5.2). During these two days,
74.7% of all B. bassiana infected I. sexdentatus of this group
died. 42.9% of the group inoculated with the higher concen-
trated suspension died on day 6, which was much more than
on day 5 (10.5%) and day 7 (23.3%). The LT50 of this group
was 5.8 days. For the group inoculated with the lower concen-
trated suspension, there is no distinct mortality peak
because I. sexdentatus killed by B. bassiana are nearly evenly
distributed from day 6 to day 9 with values between 15.3%
and 18.0%. The LT50 was 8.0 days.
3.2 Thanasimus formicarius inoculation tests
Together 75 T. formicarius were tested with conidia suspen-
sion, dry conidia normal or dry conidia extreme. Further
30 beetles were used for the untreated control group
(Table 1). None of the T. formicarius (n = 30) that were inocu-
lated with the conidia suspension died because of B. bassiana.
Their mean life span was 157 days. The 30 beetles inoculated
with the normal dose of dry conidia showed a mean life span
of 152 days, with no deaths caused by B. bassiana. From 15
T. formicarius that were inoculated with the extreme dose of
B. bassiana eventually 7 died. The first died after 24 days, the
last after 48 days. The last T. formicarius of this group died
after 108 days, but not because of B. bassiana. Their mean life
span was 52 days. The beetles of the control group had a mean
DC
1 10 * 7
1 10 * 6
Cont.
Fig. 1: Cumulative Beauveria
bassiana caused mortality of Ips
sexdentatus (in %) after inocula-
9 10 B. bassiana conidia suspension
(1 107, 1 106) as well as the
untreated control group (Cont.).
36 Steinwender et al.: Effects of Beauveria bassiana on Ips sexdentatus and Thanasimus formicarius
Table 1: Mean life span (MLS) of Thanasimus formicarius after inoculation with Beauveria bassiana; B. bassiana caused mortality
and mean number of daily consumed Ips typographus by T. formicarius during inoculation experiments, inoculated with an
extreme or normal amount of dry conidia (DC) or with conidia suspension (Susp.) 1 107 conidiospores ml1, and the control
group. Values of a column with different letters (a, b) are significantly different (P < 0.05) and standard deviation (SD) is added in
parentheses
Number of tested
beetles
DC extreme 15
DC normal 30
Susp. 1 107 conidiospores ml1 30
Control 30
life span of 188 days, without any death caused by B. bassiana.
There was a significant difference (P < 0.05) between the
mean life span of the extreme inoculated group and all other
groups (Table 1).
The mean number of consumed I. typographus a day
(I.t./d) during 50 days (post inoculation) differed significant-
ly. The control group fed on 0.73 I.t./d, and beetle consump-
tion was therefore significantly different (P < 0.05) from the
groups inoculated with conidia suspension (0.62 I.t./d), with
dry conidia normal (0.61 I.t./d) and dry conidia extreme
(0.53 I.t./d) (Table 1).
4 Discussion
In our I. sexdentatus experiments, the reason for the signifi-
cant differences in B. bassiana caused mortality between two
different concentrations of conidia suspension and dry conidia
as well as the untreated control group, must be the number of
inoculated conidiospores per beetle. Beetles inoculated with
the 1 106 conidiospores ml1 suspension are estimated to get
into contact with approximately 5,300 conidiospores. The
number of spores in the fluid layer on the beetle after dipping
it into the 1 107 conidiospores ml1 suspension must be 10
times higher ( 53,000). Due to the fact that it was not possi-
ble to determine the amount of conidia that was stripped off
during the inoculation process with dry conidia, it can only be
estimated. KREUTZ et al. (2004b) dipped I. typographus with its
ventral side into Boverol powder. They numbered the
amount of conidia that was transferred to a single beetle 1.0
3.0 105. Due to the fact that I. sexdentatus is bigger in size
than I. typographus, but that only the distal part of its ventral
abdomen was brought into contact with dry conidia, the num-
ber of conidiospores per beetle can be estimated (compared
with KREUTZ et al. 2004b) at least 7.5 104 1.0 105, which
would be the highest amount of inoculated conidiospores in
our experiments.
The result of these experiments can be summarized: The
higher the amount of inoculated conidiospores, the faster and
the more beetle die. The same effect of B. bassiana was deter-
mined on other bark beetle species like I. typographus (WEGEN-
STEINER 1992; KREUTZ et al. 2004a) or P. poligraphus (WEGEN-
STEINER 2000). The experiments showed also that the number
of inoculated conidiospores is more important than the higher
humidity on the cuticle provided by the conidia suspension.
FENG et al. (1994) priorizes the relative humidity on the
insects cuticula to the overall relative humidity. Our results
confirm this theory.
The inoculation experiments with I. sexdentatus showed
that there was no significant difference between freshly
emerged young adults and old adults, if they were inoculated
with B. bassiana. Even though the cuticle of the young beetles
was visibly brighter, and at least the inclusion of special pig-
ments (e.g. melanin) in the exocuticle (HOFFMANN 1995) not
MLS (days) Beetles killed by Mean of daily consumed
Beauveria bassiana Ips typographus
52 ( 24)a 7 0.56 ( 0.14)a
151 ( 68)b 0 0.61 ( 0.17)a
157 ( 85)b 0 0.62 ( 0.11)a
188 ( 78)b 0 0.73 ( 0.15)b
yet finished, this did not seem to have an impact on the infec-
tion success. Apparently other substances incorporated later
into the cuticle do not result in a higher impermeability of the
cuticle against germination tubes of B. bassiana.
KREUTZ (2001) reports significant differences between
young and old adult I. typographus. His experiments (inocula-
tion with conidia suspension 1 107 conidiospores ml1)
showed that the older beetles died in average after 2.8 days,
whereas the younger ones died after 5.1 days. In addition he
recorded a mortality caused by B. bassiana significantly high-
er on old adults (98%) compared to young adults (88%), but
no explanation was given. It is possible that I. typographus
(compared to I. sexdentatus) possesses one factor (some cuti-
cle inclusion or a special layer), that appears in young beetles
but is abrased in older ones. It is also possible that the tested
beetles had an additional disease that weakened the old
I. typographus more than the young ones, because of the
disease progress.
DRAGANOVA et al. (2007) tested three different B. bassiana
strains on I. sexdentatus. Two of them were isolated from
Lepidoptera larvae, one directly from I. sexdentatus. The latter
killed the tested I. sexdentatus most rapidly and showed the
highest mortality rate with 96%. The two Lepidoptera strains
caused a mortality rate of 89% and 90%. So the B. bassiana
strain (isolated from I. typographus) used for our experiments
showed a slightly lower mortality than the I. sexdentatus strain
with 93.8% (dry conidia) and 93.3% mortality (higher con-
centrated conidia suspension), but a higher one than the two
Lepidoptera strains. Such results can be expected, because the
strain isolated directly from an I. sexdentatus should be more
specific on infecting the same species, than the one isolated
from other Ips ssp. or from another insect order. The com-
parison of the LT50 values of DRAGANOVA et al. (2007) leads to
a different conclusion: The I. sexdentatus strain (LT50 = 4.3)
and the two Lepidoptera strains (LT50 = 4.6/4.7) seem to be
faster than our tested I. typographus strain (LT50 = 5.2/5.8).
This difference can be explained by the different inoculation
methods that were used. While I. sexdentatus in our experi-
ments where inoculated during a short time (submersion or
powdery contact), DRAGANOVA et al. (2007) inoculated the
bark beetles via contact on a layer of filter paper (with 1 ml,
1 108 conidiospores ml1 concentrated conidia suspension
per Petri dish/filter paper) for a period of 24 hours. So most
probably the beetles got into contact with much more conid-
iospores during this time and therefore died faster.
Until now, there is no natural B. bassiana infection reported
of T. formicarius or even of any other Cleridae. KREUTZ (2001)
submerged 10 T. formicarius into Boverol powder. He report-
ed nine deaths without any information about the time factor.
At the beginning of our experiments it was suspected that
T. formicarius could be relative easily infected with B. bassi-
ana. When after the inoculation of 30 beetles with a normal
amount of dry conidia and the inoculation of 30 beetles with
the conidia suspension 30 days passed and there were no dead
J.Plant Dis.Protect. 1/2010
 Steinwender et al.: Effects of Beauveria bassiana on Ips sexdentatus and Thanasimus formicarius
T. formicarius, 15 beetles were inoculated with the extreme
amount of conidia, in order to check whether it is at least
possible to infect T. formicarius with a high dosage of this
strain. The seven dead beetles proofed that it is possible, but
only with a very high amount of conidia. It is remarkable that
the first T. formicarius did not die before day 24 and that the
last beetle that was killed by B. bassiana died on day 48. There
is one comparable long incubation period reported by ZIMMER-
MANN (2007) for Scarabaeus sp. larvae (24 weeks).
The rating of the amount of consumed I. typographus a day
shows that the control group consumed significantly more
bark beetles a day than the T. formicarius that were inoculated
with conidiospores (even if they did not die because of
B. bassiana). This difference could be a hint for the effects of
the humoral system. It is known that infected insects produce
antifungal substances, detoxicating proteins and inhibitors of
protease (VILCINSKAS and GTZ 1999). It may be possible that
these reactions reduce the activity of the inoculated predator
and with it its food consumption.
The main reason for the big differences in the effects of
B. bassiana on I. sexdentatus and T. formicarius is probably the
strain specificity (ZIMMERMANN 2007). A lot of investigations
focused on insect pathogenic fungus strains that are very
effective against a certain group of insects (BOURASSA et al.
2001; COTTRELL and SHAPIRO-ILAN 2003; MAKOTO and TAKAFUMI
2005; ROY et al. 2008). Therefore it is plausible that the tested
B. bassiana strain is highly virulent against bark beetles (espe-
cially I. typographus and I. sexdentatus), but only low virulent
against T. formicarius.
Until now, there were only a few investigations that
checked both, the intended effects of B. bassiana on the pest
insect and compared them with the unintended side effects
on their predators (or parasitoids). BOURASSA et al. (2001)
demonstrated that the investigated predator Teretrisoma
nigrescens (Coleoptera: Histeridae) showed a remarkable
lower mortality ( 50%) than its prey Prostephanus truncatus
(Coleoptera: Bostrichidae) (> 95%). It was concluded that the
reason for this difference was the specificity of the tested
fungus.
TODOROVA (2000) found three of 10 B. bassiana isolates that
were highly virulent on the two pest insects Leptinotarsa
decemlineata (Crysomelidae) and Myzus persicae (Aphididae),
but killed only a few of their predator, Coleomegilla maculata
(Coccinellidae).
Beside the argument of strain specificity, there could be
another reason for different effects on pest insects and their
predators: The predators` survival is depending on its ability
to find, to overcome and to consume its prey as quick as pos-
sible. If the prey is ill (or near to death) because of a certain
pathogen infection, its agility and mobility, sensory capacity,
alertness and resistance will probably be less than that of
their healthy conspecifics. So a predator would more easily
catch an ill (and therefore handicapped) prey than a healthy
one, but it would also get into contact with the preys patho-
gens. Predators being extremely sensitive to these pathogens
could get infected and suffer severe strength losses because of
a disease and may even die because of a prey, which was
easier to catch. So it is possible, that a tolerance for pathogens
of the prey coevolved, to make it less dangerous to feed on
moribund prey. To support this hypothesis, investigations of
insects that feed on moribund or fresh dead insects should be
conducted.
The B. bassiana strain (UMIP 2621.07) showed a germina-
tion rate of 92.3% ( 1.5%). Compared with the 50 different
B. bassiana strains tested by POSADA and VEGA (2005), the re-
sult is slightly lower than their best one with 93.8% ( 2.7%)
but higher compared to the other 49 strains. KREUTZ et al.
(2004a) reached with his two isolates germination rates
between 99% and 100%, and Boverol (Fytovita, Ostrozska
Lhota, Czech Republic) resulted in 92%. So the germination
rate of our B. bassiana strain that was tested on I. sexdentatus
and T. formicarius is suggested to be sufficiently high.
J.Plant Dis.Protect. 1/2010
37
The inoculation experiments with I. typographus also helped
to compare the effect of our B. bassiana strain with other
strains on the same bark beetle species. Experiments with
B. bassiana on I. typographus by WEGENSTEINER (1992) resulted
in a mean life span (MLS) of 4.8 days and an infection rate of
> 95%. KREUTZ et al. (2004a) achieved with different strains a
MLS from 3.6 days to 6.3 days and mycosis between 71% and
99%. Our B. bassiana strain is comparable with a LT50 of 4.7
days and an infection rate of 92% and can be suggested at
least sufficient virulent.
We showed with our experiments, that it is possible to cause
a high percentage of B. bassiana deaths among the targeted
pest insects, the bark beetle I. sexdentatus, whereas the
non-target, the predator T. formicarius, is lesser or even not
affected. These results are an important step towards the
development of efficient measures in bark beetle control with
B. bassiana and its selective use.
Acknowledgements
We want to thank Andrea Stradner for her support during the
experiments, Dipl.-Ing. Peter Kritsch for running the I. typo-
graphus breeding at the Institute, Roman Wanjek for helping
check the bark beetle traps in the field, Of Ing. Gustav
Schwarz and Of Ing. Reinhard Kornfeld who provided areas
for the bark beetle traps and Dr. Bernard Papierok and his
team at the Institute Pasteur in Paris, who checked and
archived the B. bassiana strain that quickly.
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