A laboratory evaluation to determine the compatibility of microbiological control agents with the pollinator Bombus terrestris.
ABSTRACT This study was undertaken to identify any potential adverse side effects of the use of seven microbiological control agents (MCAs) on the bumblebee, Bombus terrestris L., in the context of combined use in integrated pest management (IPM). AQ10 (Ampelomyces quisqualis), Binab-T-vector (Hypocrea parapilulifera + T. atroviride; 1/1), Prestop-Mix (Gliocladium catenulatum J1446), Serenade (Bacillus subtilis QST713), Trianum-P (Trichoderma harzianum T22), Botanigard (Beauveria bassiana GHA) and Granupom (Cydia pomonella granulovirus), comprising five biofungicides and two bioinsecticides, were investigated. Bumblebee workers were exposed under laboratory conditions to each MCA at its maximum field recommended concentration (MFRC) via three different routes of exposure: dermal contact and orally via either treated sugar water or pollen.
The tested MCAs were found to be safe for workers of B. terrestris, with the exception of Botanigard and Serenade. Exposure to Botanigard via contact at its MFRC caused 92% mortality after 11 weeks, while the 1/10 MFRC killed 46% of exposed workers. For Serenade, topical contact and oral delivery via sugar water resulted in 88 and 100% worker mortality respectively. With lower concentrations (1/2, 1/5 and 1/10 MFRC) the toxicity decreased, but the effect depended on the route of exposure. In addition to lethal effects, nests were also evaluated for sublethal effects after treatment with the seven MCAs at their respective MFRCs over 11 weeks. In these bioassays, only Botanigard and Serenade gave rise to a significant (P < 0.05) decrease in drone production. Sublethal effects on foraging behaviour were also evaluated, and only Botanigard at its MFRC delivered via treated sugar water induced negative effects.
The results demonstrated that most of the MCAs tested can be considered safe for use in combination with B. terrestris, based on the International Organisation for Biological Control of Noxious Animals and Plants (IOBC) classification. However, some can be harmful, such as the biofungicide Serenade and the bioinsecticide Botanigard. Therefore, it is recommended that all should be tested before use in combination with pollinators. In this context, it is also advisable that these MCAs should be evaluated in more realistic field situations for the assessment of potentially deleterious effects on foraging behaviour.
- SourceAvailable from: G. Christopher Cutler[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: Pollinator exposure to pesticides is a concern in agricultural systems that depend on pollinators for crop production. However, not all pesticides elicit toxic effects, and response to a pesticide will vary depending on dose and exposure route. The effects of biopesticide formulations of Bacillus subtilis and Beauveria bassiana and of the tetramic acid insecticide spirotetramat on the common eastern bumblebee, Bombus impatiens, were evaluated. Microcolonies of bees were exposed to field-rate or lower concentrations, and data were collected over 60 days. RESULTS: When ingested, field rates of spirotetramat caused high mortality after 10 days, and B. subtilis significantly reduced drone production, number of days to oviposition and number of days to drone emergence. Converse to effects observed following ingestion, topical applications of B. subtilis at concentrations less than the recommended field rate resulted in a hormetic response, with significantly increased drone production. Topical application of spirotetramat and oral or topical application of B. bassiana had no effects on bees. CONCLUSIONS: Spirotetramat and B. subtilis can induce adverse effects on B. impatiens, but hormetic effects following B. subtilis treatment can also occur, depending on exposure route. Additional experiments are required to determine whether similar toxic or hormetic effects occur under more realistic field conditions. © 2012 Society of Chemical Industry.Pest Management Science 11/2012; · 2.74 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: To date, there are no validated internal reference genes for the normalization of RT-qPCR data from virus infection experiments with pollinating insects. In this study we evaluated the stability of five candidate internal reference genes: elongation factor-1-alpha (ELF1α), peptidylprolyl isomerase A (PPIA), 60S ribosomal protein L23 (RPL23), TATA-binding protein (TBP) and polyubiquitin (UBI), in relation to Israeli acute paralysis virus (IAPV) infection of Bombus terrestris. We investigated the stability of these genes: in whole bodies and individual body parts, as well as in whole bodies collected at different time intervals after infection with IAPV. Our data identified PPIA as the single, most-optimal internal reference gene and the combination of PPAI-RPL23-UBI as a fully-sufficient multiple internal reference genes set for IAPV infection experiments in B. terrestris.Journal of Invertebrate Pathology 10/2013; · 2.67 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Anthropogenic changes of the environment influence the distribution and abundance of pollinators such as bumblebees and have been proposed as one of the main causes in their worldwide decline. In order to evaluate the impact of expanding anthropogenic landscapes on supporting pollinator potential, reliable tools are needed. Bombus terrestris is one of the most abundant bumblebee species in Europe, and these bumblebees are known as generalist pollinators of not only wild flowers in nature but also of crops in agriculture. For more than two decades, these bumblebees have been commercially mass reared for biological pollination in greenhouses. In this project, we placed commercial hives of the bumblebee B. terrestris containing one queen and 40 workers, in three different locations in the region of Ghent (Belgium), and the performance of these hives was followed during a 4-week period in spring 2012. In parallel, we determined the floral richness and diversity index in the chosen study sites. The sites consisted of a rich urban environment with patchy green areas opposed to an urban environment with poor landscape metrics; a third rural study site showed average positive landscape metrics. The results demonstrated that the hive biomass and numbers of workers increased significantly in the rich compared to the poor environment, providing a mechanism to discriminate between study sites. In addition, the bumblebee-collected pollen showed that the flowering plants Salix spp. and Rosaceae/Prunus spp. are dominant food sources in all anthropogenic environments during early spring. Finally, the results are discussed in relation to the optimization of the experimental setup and to the use of commercial bumblebee hives in assessing local pollinator support within any given environment.Environmental Monitoring and Assessment 12/2013; · 1.68 Impact Factor
Received: 7 December 2008Revised: 3 February 2009Accepted: 4 February 2009Published online in Wiley Interscience: 12 May 2009
(www.interscience.wiley.com) DOI 10.1002/ps.1778
A laboratory evaluation to determine the
compatibility of microbiological control agents
with the pollinator Bombusterrestris
VeerleMommaerts,a∗Guido Sterk,bLucien HoffmanncandGuy Smagghea,d
BACKGROUND: This study was undertaken to identify any potential adverse side effects of the use of seven microbiological
control agents (MCAs) on the bumblebee, Bombusterrestris L., in the context of combined use in integrated pest management
catenulatum J1446), Serenade(Bacillus subtilis QST713), Trianum-P(Trichoderma harzianum T22), Botanigard(Beauveria
bassiana GHA) and Granupom(Cydia pomonella granulovirus), comprising five biofungicides and two bioinsecticides, were
investigated. Bumblebee workers were exposed under laboratory conditions to each MCA at its maximum field recommended
Exposure to Botanigardvia contact at its MFRC caused 92% mortality after 11 weeks, while the 1/10 MFRC killed 46% of
exposed workers. For Serenade, topical contact and oral delivery via sugar water resulted in 88 and 100% worker mortality
respectively. With lower concentrations (1/2, 1/5 and 1/10 MFRC) the toxicity decreased, but the effect depended on the route
theirrespectiveMFRCsover11 weeks.Inthesebioassays,onlyBotanigardandSerenadegaverisetoasignificant(P < 0.05)
decrease in drone production. Sublethal effects on foraging behaviour were also evaluated, and only Botanigard at its MFRC
delivered via treatedsugar water induced negative effects.
CONCLUSION: The results demonstrated that most of the MCAs tested can be considered safe for use in combination with
B.terrestris, based on the InternationalOrganisation for Biological Control of Noxious Animals and Plants (IOBC) classification.
However, some can be harmful, such as the biofungicide Serenadeand the bioinsecticide Botanigard. Therefore, it is
MCAs should be evaluated in more realistic field situations for the assessment of potentially deleterious effects on foraging
c ? 2009Society of ChemicalIndustry
Keywords: Bombus terrestris; bumblebee; Binab-T-vector; Prestop-Mix; Botanigard; AQ10; Serenade; Granupom; Trianum-P; biological
control; sublethal effects; survival; foraging behaviour
Important field and greenhouse crops such as tomato, sweet
pepper, apple, pear and strawberry are under constant attack
from pathogenic fungi and insects. In spite of the significant ef-
forts that have been undertaken to improve pathogen control
with disease prediction models, procedures may still fail when
infection pressure is high. Moreover, the limited use of resistant
cultivars,the developmentofresistantpathogenpopulations, hu-
man health concerns and the increasingly constrained availability
of synthetic pesticides have driven the search for alternatives.1,2
Antagonistic microorganisms could provide a long-term solution
that iscompatible with integrated pest management(IPM)strate-
by the production of enzymes and antibiotics. Biofungicides such
as Binabproducts that are based on a combination of two an-
(Hyprocreales, Hypocreaceae) and Trichoderma atroviride Karst.
Gliocladium catenulatum Gilman & Abbott J1446 (Hyprocreales,
∗Correspondence to: VeerleMommaerts, Laboratory of Cellular Genetics,
a Laboratory of Cellular Genetics, Department of Biology, Free University of
c Department of Environment and Agro-biotechnologies (EVA), Centre de
d Laboratory of Agrozoology, Department of Crop Protection, Ghent University,
PestManagSci 2009; 65: 949–955www.soci.org
c ? 2009 Society of Chemical Industry
www.soci.org V Mommaerts etal.
Bionectriaceae), have been used to control grey mould caused
by the fungus Botrytis cinerea Pers.: Fr. in several economically
important greenhouse vegetables and fruits, while the biofungi-
cide AQ10, a formulation of the picnidial fungus Ampelomyces
quisqualis Ces.: Schlidt (Pleosporales, Leptosphaeriaceae), has
been used in IPM to control several species of powdery mildew
fungi.3,4In addition to fungi, several bacterial strains have been
evaluated for their capacity to control pathogens. Goodexamples
(JM Reade) Honey, the causal agent of mummy berry disease of
blueberries, and Bacillus subtilis QST713 for the control of the
pathogenic fungus Septoria glycines Hemmi on soybeans.5,6Fur-
thermore, over the last decade, extensive research has evaluated
methods for the biological control of pests. Predators, parasitic
nematodes and entomopathogenic fungi such as Botanigard
[Beauveria bassiana (Bals.-Criv.) Vuill. GHA] (Hyprocreales, Cordy-
cipitaceae) have been extensively used for the control of pest
insects such as thrips, grasshoppers and locusts, Colorado potato
beetle and Lepidoptera.7–13Alongside fungi, entomopathogenic
viruses have also shown good potential.14–16In orchards, a com-
mercial preparation of the Cydia pomonella granulovirus is in use
to control the codling moth, Cydia pomonella L., the most serious
pest of apple orchards worldwide.17
Bumblebees such as Bombus terrestris L. are important polli-
nators, and they are used worldwide in combination with honey
bles and fruitcrops in greenhousesand orchards. The advantages
of natural pollination include improved fruit quality, higher fruit
weight and less malformed fruit.18Therefore, pollination must be
guaranteed. In practice, bees can come into contact with applied
MCAs via a number of different routes of exposure. Firstly, when
MCAs are sprayed while bees are foraging, and secondly by a sec-
deposits of MCA inoculum. Unfortunately, only limited data are
on bumblebees. Bombus terrestris and Bombus pratorum L. have
effects have been recorded from the pollinator Bombusimpatiens
Cresson after treatment with B.bassiana.20By contrast, high mor-
tality was reported in B. terrestris after treatment with B. bassiana
related organismsand are also strongly affected by the MCA used
and how it is applied. As a consequence, it is recommended that,
before a new biological strategy can be implemented, an assess-
ment of the potential risks towards beneficial insects, such as
pollinators, should be performed.
The objectives of this study were to investigate the lethal
and sublethal effects of a selection of seven MCAs when
bumblebees are exposed via contact and ingestion. In this
study, five biological fungicides were investigated – AQ10(A.
quisqualis), Binab-T-vector(H. parapilulifera + T. atroviride; 1/1),
Prestop-Mix(G. catenulatum J1446), Trianum-P(Trichoderma
harzianum T22) and Serenade(B. subtilis QST713), and two bi-
ological insecticides – Botanigard(B. bassiana) and Granupom
(C. pomonella granulovirus). Additionally, for the MCAs that re-
sulted in no adverse effects on survival and reproduction, their
impact on bumblebee foraging behaviour was evaulated in the
on the compatibility of MCAs used for the control of major plant
pathogens and pest insects with the bumblebee B.terrestris.
Seven microbiological control agents, comprising five fungi, one
bacterium and one virus, were tested at their maximum field
recommended concentration (MFRC) (Table 1). Each product was
stored in accordance with the manufacturer’s guidelines.
Microbiological control agents
All experiments were performed with worker bumblebees ob-
tained from a continuous mass rearing programme (Biobest NV,
Westerlo, Belgium)and conducted under standardised laboratory
pollen (Soc. Coop. Apihurdes, Pinofranqueado-C´ aceres, Spain) as
energy and protein source respectively.22
side effects of MCAs on bumblebee workers
Newly emerged workers were collected from the bumblebee
colony, and five were placed in artificial plastic nest boxes
(15 × 15 × 10 cm).22Four artificial nests were exposed for each
were exposed to the MCAs at their respective MFRCs via three
different routes: via contact by topical application, and orally via
treated sugar water and via treated pollen. For each treatment,
worker mortality was scored after 72 h and on a weekly basis
during a period of 11 weeks.
For the contact treatments, the MFRC of each MCA (Table 1)
was prepared in water. Individual bees were topically treated
with 50 µL of this aqueous solution on their dorsal thorax with
a micropipette. For the oral treatments, bumblebee workers
were continuously exposed to 500 mL of sugar water that was
dosed with the MCA, or to pollen sprayed until saturation with
the MCA in water. The treated sugar water and pollen were
bumblebees were exposed to tap water via topical treatment,
untreated sugar water and water-treated pollen; here, mortality
should be zero as in the untreated controls. In addition, as
positive controls, workers were also treated with a neonicotinoid
insecticide formulation, imidacloprid 200 g L−1SL (Confidor;
Bayer CropSciences, Monheim, Germany), at its MFRC (200 mg
should be scored.
In the different artificial nests, worker survival was evaluated
daily for the first 3 days post-treatment, and then on a weekly
basis for a period of 11 weeks. The treatments were scored in
accordance with the International Organisation for Biological
Control of Noxious Animals and Plants (IOBC) classification: less
than 25% effect = 1, non-toxic; 25–50% effect = 2, weakly toxic;
4, highly toxic.23Subsequently, the adverse sublethal effects on
reproduction were monitored on a weekly basis for 11 weeks by
scoring the numbers of drones produced per nest. In all cases,
dead workers were collected and incubated at room temperature
to determine the presence of the respective MCAs.
with the MCAs that exerted lethal effects in the experiments
described above, and investigated the side effects of a series of
dilutions of the MFRC (1/1, 1/2, 1/5, 1/10) in a manner similar to
that described for the MFRC.
Insect bioassays with microhive colonies to assess the
c ? 2009 Society of Chemical Industry PestManagSci 2009; 65: 949–955
Effect of microbiological control agents on B.terrestriswww.soci.org
colony-forming units (CFU) g−1, and their respectiveMFRC in % formulation and correspondingCFU L−1or OB L−1
Overview of the seven commercial MCAs tested, comprising five biofungicides and two bioinsecticides, their formulation type and
(type of microorganism)
(CFU g−1formulated product)
formulation in water
Hypocreaparapilulifera + T.atroviride; 1/1 (fungi)
Gliocladium catenulatum J1446 (fungus)
Bacillussubtilis QST713 (bacterium)
WG; 5 × 109
WP; 1 × 105
WP; 1 × 108
WP; 5 × 109
WP; 1 × 109
35 × 107CFU L−1
1.25 × 105CFU L−1
7.5 × 108CFU L−1
7.5 × 109CFU L−1
6 × 108CFU L−1
Beauveriabassiana GHA (fungus)
Cydiapomonella granulovirus (virus)
SE; 2 × 1010
SC; 2.2 × 1013
2.5 × 1010CFU L−1
6.6 × 1012OB L−1
aWP = wettable powder;WG = water-dispersiblegranules; SE = suspo-emulsion;SC = suspensionconcentrate.
Only those MCAs that did not show harmful effects on survival
and reproduction when evaluated in the experiment described
in Section 2.3.1 were tested. As in Section 2.3.1, newly emerged
workers were collected from the bumblebee colony, and five
workers were placed in an artificial plastic nest box (15 × 15 ×
10 cm). The experimental set-up to assess effects on foraging
behaviour consisted of two artificial nests.24Briefly, the two
artificial nest boxes (A and B) were connected by a tube of
approximately20 cmlengthand2 cmdiameter.Inonebox(A) the
workers constructed their nest. After 2 weeks, when third- and
were allowed a training period of 2 days to forage for untreated
food in box B. Afterwards, the sugar water in box B was replaced
with sugar water treated with the MCAat itsMFRC as describedin
Section 2.3.1. Four replicates were performed for each treatment,
and each experiment was repeated twice. For the water controls,
tap water replaced the MCAs in all treatments; here, mortality
control, workers were also treated with imidacloprid (Confidor)
in bumblebees.24Worker survival and drone production were
scored on a weekly basis, in a similar manner to that described in
Section 2.3.1, over a period of 9 weeks.
Unless otherwise stated, data were analysed by one-way analysis
of variance (ANOVA), and means ± SEM were separated using a
post hoc Tukey–Kramer test (P = 0.05) in SPSS 15.0 (SPSS Inc.,
None of the seven MCAs tested at their MFRC exhibited lethal
effects against the workers during the first 72 h following
treatment, regardlessofthedifferentroutesofexposure (data not
nests that were treated with Botanigardand Serenade(Fig. 1),
and, as a consequence, these MCAs were scored as 4 (highly
Lethal effects of MCAs on worker survival
toxic) in accordance with the IOBC classification. For Botanigard,
92 ± 3% of the workers were killed following topical treatment.
In addition, when the concentration was reduced to 1/2, 1/5
and 1/10 of the MFRC, the mean worker mortality still reached
59 ± 1%, 41 ± 6% and 46 ± 4%, respectively, after 11 weeks. The
presence of B. bassiana GHA mycelium on the bodies of workers
was also confirmed when cadavers were inspected under the
microscope. For Serenade, exposure via contact and via drinking
treated sugar water resulted in high mortality, with 88 ± 1% and
100± 0% respectivelyafter 11 weeks. When the B.subtilisQST713
concentrations were reduced, it was clear that the toxicity of 1/2
MFRC after topical contact did not cause significant mortality
(20 ± 0%), although the oral treatment with 1/10 MFRC in sugar
water remained highly toxic (class 4) as it killed 79 ± 3% of the
= 1’ in accordance with the IOBC classification after 11 weeks of
exposure. Forimidacloprid,all treatednestsshowed100%worker
In nests exposed by contact to AQ10, Binab-T-vector,
Granupom, Prestop-Mixand Trianum-Pat their respective
MFRCs, the production of drones after 11 weeks was not signif-
icantly different (P > 0.05) from the control nests treated with
water (Table 2). These MCAs can be considered harmless accord-
ing to the IOBC classification. By contrast, Serenadeat its MFRC
resulted in total loss of reproduction owing to high worker mor-
tality (88%). As a consequence, the numbers of workers per nest
were too low to build up a nest and to produce drones. Even
at the lower concentrations of 1/2, 1/5 and 1/10 MFRC, strong
detrimental effects were induced by Serenade, with drone pro-
duction recorded as only 29%, 36% and 58% of that observed in
the control nests (34.3 ± 5.6 drones; data not shown). Similarly,
Botanigardwas detrimental for nest reproduction, and 1/2, 1/5
and 1/10 MFRC caused reductions of 69%, 63% and 65%, respec-
tively, when compared with the control nests (34.3 ± 5.6 drones;
data not shown).
Exposure of worker bumblebees via sugar water treated
Mixand Trianum-Pat their respective MFRCs for 11 weeks
resulted in reproductive rates that were not significantly different
(P > 0.05) from those observed in the controls (Table 2) and
can be classified harmless according to the IOBC classification.
Sublethal effects of MCAs on reproduction
PestManagSci 2009; 65: 949–955
c ? 2009 Society of Chemical Industry
www.soci.org V Mommaerts etal.
Figure 1. Toxicity of the seven tested MCAs against adult worker
bumblebees after 11 weeks when exposed at their respective MFRCs
by topical contact or orally via treated sugar water or treated pollen. Data
toxic; 4 = highly toxic.
By contrast, Serenadeapplied at 1/1, 1/2, 1/5 and 1/10 MFRC
resulted in very low percentages of only 0%, 0%, 4% and 5%,
respectively, of the drone production observed in the controls,
this being a consequence of the severe worker mortality that
occurred at these concentrations.
Pollen treatment with AQ10, Binab-T-vector, Granupom,
Prestop-Mix, Serenadeand Trianum-Pcaused no negative
Here, adverse effects were only observed when workers were fed
production of males was significantly (P < 0.05) lower: 27.3 ± 0.5
in the treated nests as opposed to 32.5 ± 0.8 in the controls.
Although significant, this reduction of 16% is harmless according
to the IOBC classification (class 1).
Botanigard, Prestop-Mixand Trianum-Pat their respective
MFRCs, worker mortality was 5%, 20%, 3%, 18% and 10% respec-
tively. Thus, based on the IOBC classification, all the MCAs tested
Sublethal effect of MCAs on bumblebee behaviour
9 weeksof exposure to AQ10, Binab-T-vector,
terrestris by the seven MCAs tested when treated at their respective
MFRCs via topical contact or oral exposure via treated sugar water or
treated pollen over a period of 11 weeks
Mean number of drones (± SEM)a
MCA Contact Sugar waterPollen
30.0 (±1.3) a
34.5 (±3.8) ab
1.1 (±1.1) c
36.5 (±1.5) b
30.0 (±1.0) a
0.0 (±0.0) c
30.3 (±2.0) a
31.3 (±0.5) ab
29.9 (±1.6) a
28.9 (±0.1) a
30.1 (±1.6) a
36.3 (±2.3) b
28.9 (±1.6) a
0.0 (±0.0) c
27.6 (±0.1) a
32.1 (±2.4) ab
28.9 (±1.9) bc
28.6 (±0.3) bc
27.3 (±0.5) c
35.9 (±1.1) a
32.4 (±0.6) ab
29.9 (±0.6) bc
29.3 (±1.3) bc
32.5 (±0.8) ab
aData are expressed as mean numbers of drones per nest (± SEM),
based on four artificial nests per treatment and five workers per nest,
and with the experiment repeated twice. ANOVA resulted in three
groupsforcontactexposure(F = 150.121,df=63,P < 0.001),inthree
groupsforsugarwaterexposure(F = 113.122,df=63,P < 0.001)and
in three groups for pollen exposure (F = 10.317, df = 63, P < 0.001).
Values per route of exposure that are followed by a different letter
(a to c) are significantly different (post hoc Tukey–Kramer test with
P = 0.05).
here were classified as safe (class 1) as they showed no toxicity
was also the case for nests treated with imidacloprid at 1/10000
In addition, the authors examined whether a reduction in
foraging by workers may result in a reduced nest reproductive
rate, i.e. numbers of drones, through increasedlarval mortality. As
shown in Table 3, none of the MCAs tested at its MFRC caused
larval mortality or exerted detrimental effects on the production
of drones, with the exception of Botanigard. Here, significantly
(P < 0.05) lower numbers of drones were counted after 9 weeks:
13.4 ± 2.9 as opposed to 28.4 ± 2.9 in the control group treated
with water. This reduction of 53% corresponds to an IOBC class of
nest−1was measured in the imidacloprid treatment at 1/10000
and the building up of the nest and their travel times were much
longer when compared with workers in the control nests.
with B.terrestrisunder laboratory conditions. In the present study,
effects on reproduction and foraging behaviour were observed
for the biological fungicides based on the fungi T. atroviride +
H.parapilulifera(Binab T-vector) and T.harzianum T22 (Trianum-
via the three different routes of exposure utilised. These fungi
are known as specific mycoparasites of plant-pathogenic fungi
such as B. cinerea. Therefore, it is very likely that such products
are safe for use in close proximity to B. terrestris colonies. The
present data are in agreement with previous work that has also
reported no detrimental side effects when honey bees have been
c ? 2009 Society of Chemical IndustryPestManagSci 2009; 65: 949–955
Effect of microbiological control agents on B.terrestris www.soci.org
of Bombus terrestris by the five MCAs tested, when treated at their
respective MFRC via oral exposure through drinking treated sugar
water over a period of 9 weeks
Sublethal effects on the foraging behaviour in workers
Mean number of dead
larvae per nest
18.9 (±1.6) bc
19.8 (±1.2) c
7.8 (±5.8) ab
12.9 (±0.9) bc
16.8 (±4.5) bc
10.9 (±0.4) abc
0.0 (±0.0) a
Mean number of
drones per nest
27.8 (±1.8) a
31.4 (±1.9) a
13.4 (±2.9) b
29.1 (±0.6) a
31.2 (±1.2) a
28.4 (±2.9) a
5.6 (±0.1) c
aThe data are expressed as the mean numbers of dead larvae or
drones per nest (± SEM), based on four artificial nests per treatment
and five workers per nest, and with each experiment repeated twice.
ANOVA resulted in three groups for larval mortality (F = 8.120, df =
52, P < 0.001) and three groups for drone production (F = 49.014, df
= 53, P < 0.001). Values per route of exposure that are followed by a
test with P = 0.05).
used in combination (as vectors) with the fungi T. harzianum T22
Trichoderma formulations, such as Binab-TF-WPand Binab-TF-
exist two exceptions: the fungi T. harzianum strains 101645 and
206040 show insecticidal activity against larvae of the mealworm
Tenebrio molitor L.28The latter information clearly indicates the
need to test MCAs, and their different strains, for detrimental
effects against beneficial insects and pollinators before they are
used in practical crop protection.
The present insect bioassays demonstrated that contact and
oral exposure to AQ10, containing the fungus A. quisqualis,
and Granupom, a suspension concentrate of C. pomonella
of B.terrestris. To date, no information is available in the literature
Granupom, Sterk etal.23reported no mortality by direct contact
on worker bumblebees, which is in agreement with the present
study, but there was no evaluation of the impact of oral exposure
or of any potential effects on nest and colony development. The
results of the present study have demonstrated that the MCAs
investigated are probably compatible with B. terrestris, although
Serenade, a wettable powder formulation of the bacterium
B.subtilis QST713, was toxic for worker bumblebees after contact
treatment with 50 µL of the MFRC. In contrast, Dedej etal.5
reported no toxicity against honey bees by contact with a
dry Serenadeformulation containing the bacterium B. subtilis
QRD132, and this was at relatively similar doses per pollinating
insect. As a consequence, it can be hypothesised that the toxicity
of Serenademay depend on the B. subtilis strain (QST713 being
more toxic than QRD132), the pollinator species (bumblebees
being more susceptible than honey bees) or the type of exposure
(wet treatment being more toxic than dry treatment). Feeding
bumblebees on treated sugar water with Serenade(QST713)
at MFRC and 1/10 MFRC caused significant bumblebee worker
mortality, as seen in the present experiment, while contact
exposure to Serenade(QST713) at the MFRC in dry conditions
had no deleterious impact (Mommaerts V and Smagghe G,
unpublished data). Similarly, Ngugi etal.29found no deleterious
effects of Serenade(QRD132) on pollination by honey bees in
blueberry when applied as a dry formulation. The results suggest
that it is possible that a biopesticide based on the bacterium
but further testing is necessary before definitive conclusions can
For the insecticidal biopesticide Botanigard
B.bassiana GHA, severe worker mortality was observed after con-
tact treatment of bumblebee workers, but not via the ingestion
of treated sugar water or treated pollen. To explain the high con-
tact toxicity of the fungus B. bassiana, Ekesi etal.30reported that
environmental conditions such as temperature play an important
role in determining the pathogenicity observed, while Fernan-
des etal.31demonstrated that germination of B. bassiana isolates
bassianamyceliumisableto growoverawidetemperature range
≥37◦C. However, dormant conidia tolerate higher temperatures
owing to higher amounts of saturated fatty acids, decreasing cell
membrane permeability, and the higher trehalose and mannitol
concentrations, protecting against denaturation of proteins and
membranes.33,34Also, Liu etal.35reported that the temperature
limit for growth of B. bassiana conidia lies between 45 and 50◦C.
Recently, with the use of an infrared camera, Mommaerts etal.27
determined that the body temperature for worker bees of B. ter-
to survive and grow on the bumblebee body, resulting in death
of the insect. In contrast, Al-Mazra’awi etal.20reported that B.
American bumblebee, B. impatiens, in a vector system to control
In the same year, Al-Mazra’awi etal.36made a similar report that
honey bees were not affected when exposed to dormant conidia
via walking through a drypowder formulation of B.bassianaconi-
dia. Similarly, B. bassiana strains isolated from varroa mites have
been used in a powder formulation in France to treat honey bee
hives against varroasis without any negative impacts on colony
health.37,38In contrast, in the present tests the bumblebees were
treated via contact with a water suspension of the conidia, and
this may explain the high worker toxicity observed. Al-mazra’awi
etal.20and Fernandes etal.31also reported negative side effects
on B. impatiens and A. mellifera when the surfactant Tween 80
was added to Botanigard, leading to good germination. In this
it is strongly indicated that the side effects of MCAs under these
can be recommended.
In the context of modern and safe crop protection strategies,
to considering the easy-to-observe effects on mortality, while less
attention is paid to sublethal effects, which may be detrimental
towards pollination and subsequently bee populations. In this
context, Thompson and Maus39suggested that sublethal studies
on honey bees should only be performed if the effects are
PestManagSci 2009; 65: 949–955
c ? 2009 Society of Chemical Industry
www.soci.org V Mommaerts etal.
proven to be ecologically relevant. Secondly, they concluded
that data of higher-tier tests on honey bees should make such
additional testing for sublethal effects unnecessary, as they cover
etal.40also reported that not only mortality but also sublethal
effects must be considered when evaluating the impact of
pesticides on pollinators, as impairment of foraging behaviour
can have a serious impact on nest development. This was
demonstrated in experiments where the reproductive capacities
and quality.41,42In addition, as demonstrated in the present
experiments, MCAs may pose significant sublethal effects on nest
reproduction and foraging behaviour of bumblebee workers. As
shown forBotanigard, there wasa significantreduction in drone
production and foraging. Typically, the nests of workers treated
the untreated controls. Also, the authors believe that the present
experimental set-up with two nests connected by a ‘foraging
connection tube’ allows the assessment of sublethal effects
whereby a decrease in foraging behaviour results in lower nest
reproduction. In addition, it should be considered that MCAs can
Maccagnani etal.43showed that honey bees were responsible for
a secondary colonisation of the bacterium B. subtilis BD170 in
blueberry. Similarly, the yeast Candida sake E583 and bacteria
Bacillus spp. wereable to growin nectar, andthe bacterial growth
rate depended on the nectar sugar concentrations; there was
a negative correlation between growth and osmotic pressure.44
Also, as seen with the B. subtilis strain QST713 as used in this
study, a doubling in colony-forming units (CFU) mL−1in artificial
that, under practical conditions, pollinators may be exposed to
MCAs when visiting flowers to collect pollen, and could transport
MCAs back to their nest. Therefore, it is suggested that full risk
assessments of (bio)pesticides on bees/bumblebees should take
into account, in addition to the lethal effects, the more subtle
sublethal side effects before drawing conclusions about their
suitability for use in IPM programmes. These implications require
the development of standardised methods to assess sublethal
effects in the laboratory and the field.
help in the experiments, and to Dr Howard Bell (CSL, York, UK) for
his careful editing. They also thank Bio-innovation (Helsingborg,
Sweden) for the kind gift of Binab-T-vector, Koppert (Berkel
en Rodenrijs, the Netherlands) for Trianum-P, Verdera (Helsinki,
Finland) for Prestop-Mix, Agraquest (Davis, CA) for Serenade,
for Granupom and Emerald BioAgriculture Crop (Okemos, MI) for
Botanigard. This research was funded by the Research Council of
VUB (Brussels, Belgium) and a PhD fellowship of the Luxembourg
Ministry for Culture, Higher Education and Research.
1 B´ elanger RR and Benyagoub M, Challenges and prospects for
integrated control of powdery mildews in the greenhouse. Can
JPlantPathol 19:310–314 (1997).
2 Dianez F, Santos M, Blanco P and Tello JC, Fungicide resistance
in Botrytis cinerea isolates from strawberry crops in Huelva
(southwesternSpain). Phytoparasitica 30:529–534 (2002).
3 Prokkola S and Kivijarvi P, Effect of biological sprays on the incidence
of grey mould, fruit yield and fruit quality in organic strawberry
production. AgricFoodSci 16:25–33 (2007).
4 Elad Y, Kirshner B, Yehuda N and Sztejnberg A, Management of
powdery mildew and gray mold of cucumber by Trichoderma
harzianum T39 and Ampelomyces quisqualis AQ10. Biocontrol
5 Dedej S, Delaplane KS and Scherm H, Effectiveness of honey bees in
to suppressmummyberry disease.BiolControl 31:422–427 (2004).
6 ManteconJD, Efficacy of chemical and biological strategies for
controlling the soybean brown spot (Septoria glycines). Cien Inv
Agr 35:211–214 (2008).
7 JacobsonRJ, Chandler D, Fenlon J and Russel KM, Compatibility of
Beauveria bassiana (Balsamo) Vuillemin with Amblyseius cucumeris
Oudemands (Acarina: Phytoseiidae) to control Frankliniella
occidentalis Pergande (Thysanoptera: Thripidae) on cucumber
plants.BioconSciTechnol 11:391–400 (2001).
8 Georgis R,Koppenh¨ oferAM,
Grewal PS, etal, Successes and failures in the use of parasitic
nematodesfor pest control. BiolControl 38:103–123 (2006).
9 Xu X, Borgemeister C and Poehling HM, Interactions in the biological
and the two-spotted spider mite Tetranychus urticae Koch by the
predatory bug Orius insidiosus Say on beans. Biol Cont 36:57–64
10 Ugine TA,Wraight SPandSanderson JP,Effectsofmanipulatingspray-
application parameters on efficacy of the entomopathogenic
Frankliniella occidentalis, infesting greenhouse Impatiens crops.
BiocontSciTechnol 17:193–219 (2007).
11 Jaronski ST and Goettel MS, Development of Beauveria bassiana for
control of grasshoppers and locusts, in Microbiological Control
of Grasshoppers and Locusts, ed. by Goettel MS and Johnson DL.
Ottawa, Canada, Vol. 171, pp. 225–237 (1997).
12 Poprawski TJ, Greenberg SM and Ciomperlik MA, Effect of host plant
on Beauveria bassiana and Paecilomyces fumosoroseus-induced
mortality of Trialeurodes vaporariorum (Homoptera: Aleyrodidae).
EnvironEntomol 29:1048–1053 (1997).
13 Vandenberg JD, Shelton AM, Wisley WT and Ramos M, Assessment
of Beauveria bassiana sprays for control of diamondback moth
(Lepidoptera: Plutellidae) on crucifers. J Econ Entomol 91:624–630
14 Jaques R, Hardman J, Laining J and Smith R, Orchard trials in Canada
Entomophaga 39:281–292 (1994).
15 CossentineJE and Jensen LB, Persistence of a commercial codling
BritColumbia 101:87–92 (2004).
16 Arthurs SP, Lacey LA and Fritts R, Optimizing the use of the codling
moth granulovirus: effects of application rate and spraying
frequence on control of codling moth larvae in Pacific Northwest
apple orchards. JEconEntomol 98:1459–1468 (2005).
17 Barnes MM, Tortricids in pome and stone fruits, codling moth
occurrence,hostraceformationand damage,in TortricidsPests,ed.
byvan der Geest LPSand Evenhuis HH.Elsevier SciencePublishers,
Amsterdam, the Netherlands, pp. 313–327 (1991).
18 Kovach J, Petzoldt R and Harman G, Use of honey bees and
bumble bees to disseminate Trichoderma harzianum 1295–22 to
strawberries for Botrytis control. BiolCont 18:235–242 (2000).
19 Goettel MS,Poprawski TJ,Vandenberg JD,Li ZandRoberts DW,Safety
to nontarget invertebrates of fungal biocontrol agents, in Safety
ofMicrobial Insecticides, ed. by Laird M, LaceyLA and Davidson EW.
CRC Press, Boca Raton, FL, pp. 209–229 (1990).
20 Almazra’awi MS, Shipp L, Broadbent B and Kevan P, Biological control
(Thysanoptera: Thripidae) by Bombus impatiens (Hymenoptera:
Apidae) vectored Beauveria bassiana in greenhouse sweet pepper.
BiolCont 37:89–97 (2006).
21 Hokkanen H,Zeng QQandMenzler-HokkanenI,Assessingtheimpact
of Metarhizium and Beauveria on bumblebees, in Progress in
Biological Control: Environmental Impacts of Microbial Insecticides,
Needs and Methods for Risk Assessment, ed. by Hokkanen H and
Lacey LA, B´ elair G,Duncan LW,
c ? 2009 Society of Chemical Industry PestManagSci 2009; 65: 949–955
Effect of microbiological control agents on B.terrestriswww.soci.org
Vol. 1, pp. 63–72 (2004).
22 Mommaerts V, Sterk G and Smagghe G, Hazards and uptake of chitin
synthesis inhibitors in bumblebees Bombus terrestris. Pest Manag
Sci 62:752–758 (2006).
23 Sterk G, Heuts F, Merck N and Bock J, Sensitivity of non-target
Proc 1st Internat Symp Biological Control of Arthropods, Honolulu,
Hawaii, 14–18 January, pp. 306–313 (2002).
24 Mommaerts V, Reynders S, Sterk G and Smagghe G, Development
of a bioassay to assess sublethal side-effects on the foraging
behaviour of bumblebees. Proc 15th Belgian Congress on Zoology,
Liege, Belgium, 30–31 October,p. 121 (2008).
25 Van der Steen JJM, Langerak CJ, Van Tongeren CAM and Dik AJ,
Aspects of the use of honeybees and bumblebees as vector
of antagonistic micro-organisms in plant disease control, in
Proc Netherlands Entomol Soc Meeting, ed. by Jan Bruin.
Uitgeverij Nederlandse Entomologische Vereniging, Amsterdam,
the Netherlands,pp. 41–46 (2003).
26 Shaffir S,Dag A,Bilu A,Abu-Toamy MandElad Y,Honeybeedispersal
of the biocontrol agent Trichoderma harzianum T39: effectiveness
EurJPlantPathol 116:119–128 (2006).
27 Mommaerts V,Platteau G, Boulet J,
Trichoderma-based biological control agents are compatible with
the pollinator Bombus terrestris: a laboratory study. Biol Cont
28 Shakeri J andFoster HA,Proteolyticactivityandantibioticproduction
by Trichoderma harzianum in relation to pathogenicity to insects.
EnzymeMicrobTechnol 40:961–968 (2007).
29 Ngugi HK, Dedej S, Delaplane KS, Savelle AT and Scherm H, Effect
of flower-applied Serenade biofungicide (Bacillus subtilis) on
pollination-related variables in rabbit eye blueberry. Biol Cont
30 Ekesi S, Maniania NK andAmpong-Nyarko K,Effectoftemperatureon
and Beauveria bassiana on Megalurothrips sjostedti. Biocont Sci
Technol 9:177–185 (1999).
31 Fernandes EKK, Rangel DEN, Moares AML, Bittencourt VREP and
Roberts DW, Cold activity of Beauveria and Metarhizium, and
thermotolerance of Beauveria. JInvertebrPathol 98:69–78 (2008).
32 Fargues J, Goettel MS, Smits N, Quedraogo A and Rougier M, Effect of
temperature on vegetative growth of Beauveria bassiana isolates
from different origins. Mycologia 89:383–392 (1997).
Sterk Gand Smagghe G,
33 Guerzoni ME,Lanciotti RandCocconcelliPS,Alterationincellularfatty
acid composition as a response to salt, acid, oxidative and thermal
stresses in Lactobacillus helveticus. Microbiology 147:2255–2264
34 Rangel DEN, Anderson AJ and Roberts DW, Growth of Metarhizium
anisopliae on non-preferred carbon sources yields conidia with
increasedUV-B tolerance. JInvertebrPathol 93:127–134 (2006).
35 Liu HP, Skinner M, Brownbridge M and Parker BL, Characterization
of Beauveria bassiana and Metarhizium anisopliae isolates for
management of tarnished plant bug, Lygus lineolaris (Hemiptera:
Miridae). JInvertebrPathol 82:139–147 (2003).
36 Almazra’awi MS, Shipp L, Broadbent B and Kevan P, Dissemination
of Beauveria bassiana by honey bees (Hymenoptera: Apidae) for
Entomol 35:1569–1577 (2006).
37 Meikle WG, Mercadier G, Holst N, Nansen C and Girod V, Duration
and spread of an entomopathogenic fungus, Beauveria bassiana
(Deuteromycota:Hyphomycetes),used totreat varroa mites(Acari:
Varroidae) in honey bee (Hymenoptera: Apidae) hives. J Econ
Entomol 100:1–10 (2007).
38 Meikle WG, Mercadier G, Holst N, Nansen C and Girod V, Impact of a
treatment of Beauveria bassiana (Deuteromycota: Hyphomycetes)
mites (Acari: Varroidae). Apidologie 39:247–259 (2008).
39 Thompson HM and Maus C, The relevance of sublethal effects in
honey bee testing for pesticide risk assessment. Pest Manag Sci
40 Desneux N, Decourtye A and Delpuech JM, The sublethal effects of
pesticides on beneficial arthropods. Annu Rev Entomol 52:81–106
41 G´ enissel A,Aupinel P,Bressac C,Tasei J-N and Chevrier C,Influenceof
pollen origin on Bombusterrestris micro-colonies.EntomolExpAppl
42 Hoover SE, Higo HA and Winston ML, Worker honeybee ovary
development: seasonal variation and the influence of larval and
adult nutrition. JCompPhysiolB 176:55–63 (2006).
43 Maccagnani B,Giacomello F,Fanti M,Gobbin D,Maini SandAngeli G,
of Bacillussubtilis on apple flowers. Biocontrol 54:123–133 (2009).
44 Pusey PL, Effect of nectar on microbial antagonists evaluated for use
in control of fire blight of pome fruits. Phytopathology 89:39–46
PestManagSci 2009; 65: 949–955
c ? 2009 Society of Chemical Industry