Journal of Invertebrate Pathology 93 (2006) 121126

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Dispersal of Beauveria bassiana by the activity of nettle insects

Nicolai V. Meyling a,, Judith K. Pell b, Jrgen Eilenberg a
a
Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
b
Plant and Invertebrate Ecology Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

Received 21 March 2006; accepted 25 May 2006

Available online 14 July 2006

Abstract

Recent studies have shown that the entomopathogenic fungus Beauveria bassiana occurs naturally on the phylloplanes of several
plants, including nettles. Insects could, by their activity, be contributing to this inoculum by dispersing it from other sites. The potential of
nettle aphids Microlophium carnosum and their predator Anthocoris nemorum to disperse conidia of B. bassiana from soil to nettles and
from sporulating cadavers in the nettle canopy was investigated in laboratory experiments. In petri dish assays, aphids showed potential
to distribute B. bassiana from soil to nettle leaves. Predators dispersed inoculum from both soil and cadavers to nettle leaves in petri
dishes. In microcosms, aphids did not disperse B. bassiana from the soil or from cadavers conWned in the canopy, but A. nemorum were
able to transfer inoculum from soil into the nettle canopy and to distribute conidia from cryptic cadavers. In some instances, infections
were initiated in aphids and predators as a consequence of dispersal.
2006 Elsevier Inc. All rights reserved.

Keywords: Entomopathogenic fungi; Hypocreales; Urtica dioica; Microlophium carnosum; Aphididae; Anthocoris nemorum; Anthocoridae; Soil;
Phylloplane

1. Introduction et al., 1997; Roy et al., 1998, 2001). Regarding hypocrealean

The cosmopolitan entomopathogenic fungus Beauveria
bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales)
infects insects from most orders and the fungus is ubiquitous
in soil (Keller and Zimmerman, 1989). Recently, propagules
of B. bassiana were documented to occur frequently on
phylloplanes of hedgerow vegetation and this new aspect of
B. bassiana distribution raises the question of dispersal path-
ways to the foliage (Meyling and Eilenberg, 2006). Besides
dispersal by wind currents (Shimazu et al., 2002) and rain
splash from soil surfaces (Bruck and Lewis, 2002b) insects
could potentially contribute to the distribution of fungus
inoculum. Aphid predators are known to disperse conidia of
the aphid entomopathogen Pandora neoaphidis (Remaudire
and Hennebert) Humber (Zygomycota: Entomophthorales)
thereby enhancing infection rates in aphid populations (Pell
*
Corresponding author. Fax: +45 3528 2670.
E-mail address: nvm@kvl.dk (N.V. Meyling).
entomopathogenic fungi, Paecilomyces fumosoroseus (Wize)
Brown and Smith was dispersed by the ladybird Hippodamia
convergens Guerin (Coleoptera: Coccinellidae) to Russian
wheat aphids Diuraphis noxia Kurdjumov (Homoptera:
Aphididae) in laboratory experiments (Pell and Vandenberg,
2002). Likewise, B. bassiana infections were initiated in Euro-
pean corn borers Ostrinia nubialis Hbner (Lepidoptera:
Crambidae) through dispersal by the fungivorous beetle
Carpophilus freemani Dobson (Coleoptera: Nitidulidae)
(Bruck and Lewis, 2002a). Within the soil environment,
which is a well-known reservoir of B. bassiana inoculum
(Keller and Zimmerman, 1989), the fungus can be dispersed
and vectored by collembolans (Dromph, 2001, 2003). Dis-
persal of B. bassiana by insect activity has been developed
and exploited for pest management using the auto-dissemi-
nation strategy (Meadow et al., 2000; Dowd and Vega, 2003;
Vickers et al., 2004).
We hypothesised that fungal inoculum on phylloplanes
could originate from the soil as well as from fungus infected
cadavers conWned in cryptic places within the nettle canopy

0022-2011/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2006.05.010
122 N.V. Meyling et al. / Journal of Invertebrate Pathology 93 (2006) 121126
such as leaves rolled by lepidopteran larvae. Laboratory
studies were conducted on the potential of insects to dis-
perse B. bassiana inoculum from soil to phylloplanes and
within the canopy from cryptic sources of inoculum. We
selected the stinging nettle system, Urtica dioica L. (Urtica-
ceae), since earlier studies showed that nettle plants har-
boured the greatest number of B. bassiana propagules
compared with other hedgerow plants (Meyling and Eilen-
berg, 2006). Nettle aphids Microlophium carnosum (Buk-
ton) (Homoptera: Aphididae) and the predator Anthocoris
nemorum (L.) (Heteroptera: Anthocoridae) are common
insects on nettles in Northern Europe (Davis, 1973) and A.
nemorum is one of the most important predators of M. car-
nosum (Perrin, 1976). The dispersal potential of these spe-
cies was investigated in petri dish and microcosm
experiments where the sources of B. bassiana were either
soil inoculated with conidia or sporulating cadavers placed
in cryptic positions in the nettle canopy.
2. Materials and methods
2.1. Insects and plants
For experiments 2.4, 2.5 and 2.7, adult A. nemorum were
collected by sweep netting nettles in April and May at
Rothamsted Research, Hertfordshire, UK, and insects were
maintained and reared as described by Meyling and Pell
(2006) in a controlled environment room at Rothamsted
Research. In experiments using Weld collected adults they
were used within 3 weeks of collection. Laboratory reared
adults were used in experiments 24 weeks after adult eclo-
sion. Nettle plants were grown from seed in a glasshouse.
The plants for microcosm experiments were 3 weeks old
while detached leaves for petri dish experiments were
removed from 5 to 7 weeks old plants. Nettle aphids, M.
carnosum, were reared on 46-week-old nettle plants in
cages in the insectary at Rothamsted Research (18 C, L16:
D8).
For experiment 2.6, adult A. nemorum were collected on
nettles in September at Bakkegrden, Northeast Zealand,
Denmark, and kept in ventilated plastic jars at 20 C, L16:
D8, in a climate cabinet for up to 3 weeks prior to use. For
this experiment nettle plants were grown outside in Den-
mark in large pots under ambient conditions and pea
aphids, Acyrthosiphon pisum (Harris) (Homoptera: Aphidi-
dae), were reared on broad bean, Vicia faba L. (Fabaceae),
in cages placed in a glasshouse at 2023 C, L16: D8.
2.2. Suspensions of B. bassiana conidia
The B. bassiana isolate KVL 03-90 was originally iso-
lated from an adult A. nemorum collected on stinging nettle
at Bakkegrden, Denmark, in June 2002. Stock cultures on
Sabouraud dextrose agar (SDA) (Oxoid Ltd, Basingstoke,
Hampshire, UK) were stored at 5 C and subcultures onto
SDA made for use in experiments as required. Only single
transfers from stock cultures were made. Subcultures were
incubated in darkness at 25 C. Conidia suspensions were
made from sporulating plates, 35 weeks after subculturing,
by adding 0.03% Tween 80 (Acros Organics, Morris Plains,
New Jersey, USA) to the petri dish and scraping the sur-
face. The suspensions were Wltered through four layers of
muslin and the conidia concentration determined by count-
ing a diluted sample in a Neubauer bright-line haemocy-
tometer at 400 magniWcation. For all experiments,
suspensions containing 1 108 conidia per ml were used.
The germination rate of conidia from each suspension was
assessed by counting the proportion of germinated conidia
after incubation on SDA at 25 C for 24 h. Germination
was always over 95%.
2.3. B. bassiana-infected aphid cadavers
Cadavers of pea aphids, A. pisum, were prepared by dip-
ping single leaXets of broad bean into 30 ml 1 108 conidia
per ml suspension. The petiole of each leaXet was embedded
in 3% water agar in the bottom of a 30 ml medicine cup.
Two or three second instar pea aphid nymphs were trans-
ferred to every inoculated leaXet. The aphids were kept for
5 days at 20 C in a climate cabinet; by this time the aphids
had died from fungal infection. Cadavers were transferred
to petri dishes and incubated at 25 C in darkness and
under humid conditions for 56 days until they sporulated
profusely and could be used in experiments.
Cadavers of nettle aphids, M. carnosum, were prepared
by dipping single detached nettle leaves as described above.
The nettle leaves were air-dried on tissue paper and then
embedded abaxial side facing up in 2% water agar in 9 cm
petri dishes. Ten to Wfteen large nymphs of M. carnosum
were placed on each leaf and then incubated at 18 C,
L15.5 : D8.5, for 89 days until aphids died due to infection.
Dead, infected aphids were transferred to petri dishes and
incubated at 25 C in darkness and under humid conditions
for 56 days until they sporulated profusely and could be
used in experiments.
2.4. Petri dish experiment to quantify dispersal of B.
bassiana from soil to leaves
Soil from a commercial potting medium mix (75%
medium grade peat, 12% sterilised loam, 3% medium grade
vermiculite and 10% 6 mm screened, lime free grit, Peters-
Weld Products, Leicester, U.K.) was sterilised in an auto-
clave at 115 C and 10 psi. for 20 min. This soil was used in
all experiments involving dispersal of B. bassiana from soil
to leaves. The water content as wet weight (w.w.) of the soil
was determined by weighing 10 samples and then reweigh-
ing them after drying in an oven at 100 C overnight. A sus-
pension of 1 108 conidia per ml was prepared and added
to 100 g of soil which was mixed by hand in a clean plastic
bag to reach a level of 1 107 conidia per g of dry soil. Dis-
tilled water was subsequently added to reach a water con-
tent of 50% w.w. Control soil was prepared by adding an
amount of sterile 0.03% Tween 80 similar to the volume of
 N.V. Meyling et al. / Journal of Invertebrate Pathology 93 (2006) 121126
conidia suspension and then distilled water to also achieve
50% w.w.
Ten gram of soil were placed in the base of a 9 cm petri
dish. A 5 cm high cylinder made from an acetate sheet was
placed into the soil inside the petri dish base to form an
arena. The inside of the cylinder was painted with Fluon
(Asahi Glass Fluoropolymers UK Ltd, Lancashire, UK) to
prevent insects from climbing the sides. For both inocu-
lated soils and control soils each of three insect treatments
were compared: (1) one adult M. carnosum; (2) 10 adult M.
carnosum; (3) one adult Weld collected A. nemorum female.
Three replicates of each combination of soil and insect
treatments were prepared and the experiment was repeated
on four occasions. All experiments were made under ambi-
ent conditions in the laboratory (2125 C).
The insects remained in the arenas for 1 h and were then
transferred to clean 9 cm petri dishes each containing a
detached nettle leaf. Each insect was placed on the base of
the dish next to the leaf and left for 1 h. After this time, the
insects were removed, kept at 18 C, L16: D8, for two weeks
and mortality due to infection recorded. Aphids were kept
on nettle leaves embedded in 2% water agar, while each
A. nemorum was incubated in a ventilated petri dish with
some 2% water agar and fed eggs of Ephestia kuehniella
Zeller (Lepidoptera: Pyralidae) (Nutrimac, Biobest, Bel-
gium). To recover B. bassiana conidia, both adaxial and
abaxial surfaces of the leaves in the dishes were pressed
onto a selective medium in 9 cm petri dishes. The selective
medium contained antibiotics and fungicides as described
by Meyling and Eilenberg (2006). The petri dishes were
incubated in the dark at 25 C for two weeks and then the
numbers of colony forming units (CFU) were recorded.
2.5. Microcosm experiment to quantify dispersal of B.
bassiana from soil to leaves
In a microcosm experiment, the petri dish experiment
was scaled up. Groups of three 3-week-old nettle plants
were repotted into seed trays (length: 36 cm, width: 23 cm,
height: 8 cm) in autoclaved soil to which 100 g of sterile soil
inoculated with B. bassiana (as described previously) was
spread over the soil surface of each tray. Four treatments
were applied to the inoculated soil: (1) no insects (control);
(2) 30 adult nettle aphids; (3) three laboratory reared
A. nemorum, two adults, male and female, and one fourth
instar nymph; (4) 30 adult nettle aphids [as in (2)] and three
laboratory reared A. nemorum [as in (3)] added. Aphids
were placed on the plants 1 h before the inoculated top soil
was spread while predators were placed on the plant imme-
diately after the top soil had been spread. The seed trays
were covered with a transparent ventilated plastic hood
(height: 12 cm) to produce an enclosed microcosm and
incubated in a climate room at 23 C, L16: D8, for 48 h. The
experiment was repeated on three occasions and on each
occasion two replicates were made of each treatment.
After 48 h the hood was removed from each microcosm
and the insects were recovered and kept in petri dishes at
123
18 C, L15.5 : D8.5. Mortality was assessed daily and myco-
sis identiWed. Two of the three nettle plants in each micro-
cosm were cut at the base and from each plant one leaf in
each of four strata was removed and placed individually in
petri dishes. The strata were denoted 14 relating to the
position on the plant with leaf 1 being uppermost and leaf
4 closest to the soil surface. The adaxial and abaxial sur-
faces of each of the leaves were pressed onto selective
medium plates under sterile conditions and the plates were
incubated as described above. The numbers of CFUs per
leaf surface were estimated after 2 weeks.
2.6. Petri dish experiment to quantify dispersal of
B. bassiana from cadavers to leaves
This experiment was similar to experiment 2.4 except that
B. bassiana-infected aphid cadavers were the inoculum
source. Here, the experimental arena consisted of a 35 mm
petri dish in which a nettle leaf disc (diameter D 35 mm) was
placed, abaxial side facing up, in the base of the dish. Four
treatments (Wve replicates of each treatment) were estab-
lished: (1) leaf alone (control); (2) one dead third instar pea
aphid nymph, freshly killed by freezing; (3) one B. bassiana-
infected sporulating pea aphid cadaver; (4) one freeze-killed
third instar pea aphid nymph placed adjacent to one B. bas-
siana-infected sporulating pea aphid cadaver. Prior to exper-
imentation adult, Weld collected A. nemorum were starved
individually for 24 h at 20 C. One individual predator was
placed into each of the arenas for 1 h at 20 C in a climate
cabinet during the photophase. Afterwards each A. nemo-
rum was transferred to a 9 cm petri dish containing a clean
nettle leaf and incubated for 1 h at 20 C. Consumption of
uninfected aphid prey items in treatments (2) and (4) was
recorded. The experiment was repeated on six occasions.
Each A. nemorum was placed individually in medicine
cups, anaesthetised with CO2, placed in 400 l sterile 0.05%
Triton X (Research Organics, Cleveland, Ohio, USA) in an
Eppendorf tube and shaken for 45 s at 2500 rpm on a vortex
mixer. 200 l of this suspension was plated on selective
medium, incubated in darkness at 23 C and the numbers of
CFUs assessed once a week for two weeks. Adaxial and
abaxial surfaces of each nettle leaf from the 9 cm arenas
were pressed onto selective medium. Leaf impressions were
incubated in darkness at 23 C and the numbers of CFUs
assessed after two weeks.
Each sporulating aphid cadaver that had been used as an
inoculum source was placed individually in 10ml of 0.05% Tri-
ton X and shaken for 30 s at 2500rpm on a vortex mixer. Hun-
dred micro litres of the suspension was plated on selective
medium and germination percentages were estimated after
three days under a microscope. Germination levels were >95%.
2.7. Microcosm experiment to quantify dispersal of B.
bassiana from cadavers to leaves
A microcosm experiment equivalent to the one with
inoculated soil (experiment 2.5) was conducted using sporu-
124 N.V. Meyling et al. / Journal of Invertebrate Pathology 93 (2006) 121126
lating cadavers as the source of B. bassiana. Three nettle
plants were placed in a seed tray as described previously.
One randomly selected leaf, from leaf stratum 3, on a single
plant was rolled into a tube and Wtted with a paper cuV with
a diameter of 89 mm to mimic a rolled leaf gall. One
B. bassiana-infected and sporulating cadaver of M. carno-
sum was placed inside the tube on a small piece of 2% water
agar. Then insects were introduced as described previously
(aphids were added 1 h before introduction of the cadaver),
two replicates of each insect treatment were prepared and
the whole experiment repeated on three occasions. After
48 h in the climate room the leaf containing the cadaver was
enclosed in a petri dish and removed from the plant. Leaves
from the two plants not harbouring the cadaver were sam-
pled as described above and pressed onto selective medium.
The cadavers were placed in 10 ml of 0.03% Tween 80, vor-
texed for 3 min and 100 l sampled and spread onto a plate
of selective medium. Germination of conidia was assessed
after incubation for 3 days in darkness at 25 C. Germina-
tion levels were always >92%.
2.8. Data analyses
All statistical analyses were performed using SAS (SAS
Institute Inc., 1999). The data were not normally distrib-
uted and transformations failed to normalise the data and
homogenise variances. As a consequence, numbers of
CFUs per nettle leaf in the petri dish and microcosm exper-
iments were analysed by non-parametric methods and pair-
wise comparisons were made between the medians using
Median scores tests in PROC NPAR1WAY. DiVerences in
frequencies of occurrence of B. bassiana on adaxial and
abaxial leaf surfaces, proportions of infected insects as well
as the recovery of aphids in the large scale experiments
were tested by standard 2 tests.
3. Results
3.1. Petri dish experiment to quantify dispersal of B.
bassiana from soil to leaves
No signiWcant diVerences between recovered CFUs on
adaxial or abaxial leaf surfaces were found among the
insect groups (2 D 1.9634; df D 2; P D 0.3747). In all three
insect categories, the median numbers of CFUs were signiW-
cantly greater on recipient leaves when the insects had the
opportunity to forage on soil containing B. bassiana com-
pared to controls (Fig. 1). When individual aphids were
incubated on inoculated soil surfaces 41.7% became
infected by B. bassiana after incubation. The proportion of
infected aphids in the treatment with batches of 10 aphids
on inoculated soil was 20%, but the two proportions were
not signiWcantly diVerent (2 D 2.9873; df D 1; P D 0.0839).
No A. nemorum predators were infected after one hour on
inoculated soil. In 19.4% of control treatments 12 CFUs
were counted on plates, but medians remained 0 for all con-
trols in the three pair-wise experiments (Fig. 1).
Median CFUs (+95 percentile) per leaf
50
45
40
35
30
25
20
15 ***
10 ***
5 **
0
C T C T C T
1 aphid 10 aphids 1 predator
Fig. 1. Median numbers of colony forming units CFUs (+95 percentile)
recovered per nettle leaf in petri dish experiments where insects prior to
contact with the leaf had the opportunity to contact B. bassiana inocu-
lated soil for 1 h. Median Scores non-parametric tests were made between
pair-wise comparisons of controls (C) and inoculated soil (T) in the three
insect categories which were one adult nettle aphid (1 aphid), 10 adult net-
tle aphids (10 aphids) or one adult female A. nemorum (1 predator). Sig-
niWcance levels are indicated as if 0.01 < P < 0.001 and if
P < 0.0001.
3.2. Microcosm experiment to quantify dispersal of B.
bassiana from soil to leaves
SigniWcantly more aphids were recovered in the aphid
only treatment (90.6%) compared to the aphid + predator
treatment (76.1%) (2 D 13.520; df D 1; P D 0.0002). In one
microcosm with aphid-only treatment a single aphid died
of B. bassiana infection (3.6%). No predators became
infected.
Isolation of B. bassiana from leaves after 48 h revealed
that limited numbers of CFUs could be recovered when
conidia were inoculated onto soil. The numbers ranged
from 0 to 20 per leaf and all medians equalled 0 except in
the aphid + predator treatment at the leaf 4 stratum imme-
diately above the soil surface where the median was 1 CFU
per leaf. The data from this latter treatment were almost
signiWcantly greater than the control (|Z| D 1.8663; df D 1;
P D 0.0620). CFUs of B. bassiana were present in signiW-
cantly larger numbers on the leaf 2 stratum when preda-
tors had been present alone compared to the control
(|Z| D 2.1448; df D 1; P D 0.032). Also, more CFUs were
recovered in this stratum when predators had been present
with aphids compared to the control (|Z| D 2.1448; df D 1;
P D 0.032). The remaining treatments were not signiWcantly
diVerent from each other in any of the leaf strata (P > 0.05,
tests not presented).
3.3. Petri dish experiment to quantify dispersal of B.
bassiana from cadavers to leaves
There was no signiWcant diVerence in the numbers of
CFUs (and therefore deposition of conidia) from abaxial
compared to adaxial leaf surfaces (2 D 0.6102; df D 1;
P D 0.4347). The median numbers of recovered CFUs per
leaf (i.e. combining abaxial and adaxial for a given leaf)
 N.V. Meyling et al. / Journal of Invertebrate Pathology 93 (2006) 121126
Median CFUs (+95 percentile) per leaf
400
350
300
250
200
150
100
50
a a
0 This study demonstrates that both the aphid M. carno-
Control, Control,
no aphid 1 aphid
Fig. 2. Median numbers of colony forming units CFUs (+95 percentile)
recovered per nettle leaf in petri dish experiments deposited by individual
A. nemorum adults that prior to transfer had been conWned in leaf arenas
either alone (control, no aphid), with one freeze-killed aphid (control, 1
aphid), one aphid cadaver sporulating with B. bassiana (cadaver, no
aphid) or one aphid cadaver sporulating with B. bassiana and one freeze-
killed aphid (cadaver, 1 aphid). SigniWcance groups, indicated by diVerent
letters, were established by multiple pair-wise comparisons using Median
Scores non-parametric tests (P < 0.05).
were signiWcantly smaller in the control treatments com-
pared to the cadaver treatment both with (|Z| D 5.3634;
df D 1; P < 0.0001) and without a freeze-killed aphid prey
item (|Z| D 5.8851; df D 1; P < 0.0001). There were no signiW-
cant diVerences in numbers of CFUs among the control
treatments (|Z| D 0.4618; df D 1; P D 0.6442) or among the
cadavers treatments (|Z| D 1.0242; df D 1; P D 0.3058;
Fig. 2). In the two cadaver treatments, the numbers of
deposited CFUs and the numbers of remaining CFUs on
the cuticle of the predators were positively correlated (Pear-
son correlation coeYcient: No aphid treatment D 0.8314,
P < 0.0001; Dead aphid treatment D 0.8260, P < 0.0001).
3.4. Microcosm experiment to quantify dispersal of B.
bassiana from cadavers to leaves
SigniWcantly more aphids were recovered in the aphid
only treatment (96.7%) compared to the aphid + predator
treatment (85.6%) (2 D 13.720; df D 1; P D 0.0002). In two
aphid + predator microcosms containing cadavers, 51.9%
and 4.0% of the recovered aphids died of infections by B.
bassiana, respectively. In the latter, one adult A. nemorum
also died of infection.
Isolation of B. bassiana from leaves after 48 h revealed
that limited numbers of CFUs could be recovered in the
control treatment or when only aphids were present on net-
tles, ranging between 0 and 2 CFUs per leaf with all medi-
ans equal to 0. In the treatments including predators all
medians were also equal to 0, but the number of CFUs per
leaf ranged between 0 and 176. In the two upper leaf strata
signiWcantly more B. bassiana CFUs were recovered per
leaf when predators had been present together with aphids
compared to the control (leaf 1: |Z| D 2.4602; df D 1;
P D 0.0139; leaf 2: |Z| D 2.1982; df D 1; P D 0.0279) and
compared to the aphid only treatment (leaf 1: |Z| D 2.4602;
df D 1; P D 0.0139; leaf 2: |Z| D 2.1982; df D 1; P D 0.0279).
125
In addition, signiWcantly more CFUs were recovered per
b leaf in the aphid + predator treatment compared to the
b aphid only treatment in the leaf 3 stratum (|Z| D 2.4602;
df D 1; P D 0.0139). The remaining treatments were not sig-
niWcantly diVerent from each other in any of the leaf strata
(P > 0.05, tests not shown).
4. Discussion
Cadaver, Cadaver,
no aphid 1 aphid sum and its predator A. nemorum were able to disperse
inoculum from soil to nettle leaves. For M. carnosum, how-
ever, this was only found in petri dish experiments and not
when scaled up to microcosms. Furthermore, A. nemorum
distributed B. bassiana conidia from secluded cadavers
within the upper nettle canopy.
The soil environment is recognised as a reservoir of
B. bassiana (Keller and Zimmerman, 1989) and CFUs of
B. bassiana on plant surfaces could originate from the soil.
Numbers of CFUs recovered when inoculum originated
from soil surfaces were comparable to levels encountered
on nettle leaves in the Weld (Meyling and Eilenberg, 2006).
Aphids often fall from plants to escape predators and para-
sitoids (Chau and Mackauer, 1997; Losey and Denno,
1998a,b) and if they return to the plant they could poten-
tially carry fungal inoculum to the leaves of the host plant.
It was evident from the microcosm experiments that the
presence of the predators reduced the number of recovered
aphids. Anthocoris nemorum is known to cause aphids to
drop (Evans, 1976). Predators alone were able to signiW-
cantly elevate the numbers of CFUs of B. bassiana on
higher nettle leaves in microcosm experiment 2.5. In treat-
ments with aphids and predators in combination, signiW-
cantly larger numbers of CFUs were found on leaves in the
leaf 2 stratum and marginally signiWcantly so (P D 0.062)
in the leaf 4 stratum when compared to the control. This
dispersal could be due to the activity of both aphids and
predators. However, aphids are mostly observed to be
infected by entomophthoralean fungi in the Weld (Pell et al.,
2001). Aphids are unlikely to contribute signiWcantly to the
deposition of B. bassiana inoculum on leaf surfaces if this
originates from the soil environment.
It is possible that A. nemorum occasionally dwell on the
ground in the Weld. The predator is unable to detect and
thus avoid B. bassiana inoculum in soil (Meyling and Pell,
2006) and the species is naturally infected with the fungus
in the Weld thus encountering inoculum of B. bassiana.
However, in the present study no predators were infected
from soil inoculations while aphids often became infected
after contacting contaminated soil. If B. bassiana is encoun-
tered by A. nemorum on the soil in the Weld the predators
may disperse conidia to host plants but only infrequently
acquire infections.
Another possible source of conidia could be sporulating
cadavers conWned in secluded places in the canopy. Though
we have not observed B. bassiana-infected cadavers on
plants in the Weld, tunnelling insects succumb from
126 N.V. Meyling et al. / Journal of Invertebrate Pathology 93 (2006) 121126
infections of B. bassiana inside plants (Bruck and Lewis,
2002a). Furthermore, nettle canopies contain rolled leaves
etc. in which insects dwell. The predator A. nemorum dis-
persed inoculum of B. bassiana from hidden cadavers in
microcosms while aphids did not. The distributions of
conidia on the leaf strata further suggest that A. nemorum
preferentially dwelled in the upper parts of the nettle plant
during the experiment and would enter hiding places such
as rolled leaves. Behavioural studies have shown that
A. nemorum rapidly withdraw from B. bassiana-infected
cadavers (Meyling and Pell, 2006), but this study shows
that, even though they are likely to only contact inoculum
brieXy, they apparently become contaminated upon such
encounters and distribute the inoculum on the host plant.
Infections of B. bassiana were occasionally initiated in
aphids and predators following dispersal from cadavers by
predators. Potentially, A. nemorum could vector the fungus
to other insects that would not otherwise contact inoculum.
Targeted dispersal of entomopathogenic fungi through vec-
toring has been demonstrated to increase infection levels in
other insect-pathogen systems (Roy et al., 2001; Bruck and
Lewis, 2002a).
The ability of nettle insects to disperse inoculum of
B. bassiana through their activity remains to be investi-
gated under Weld conditions in order to evaluate the signiW-
cance of the mechanism in natural habitats, but insects are
potential contributors of the newly documented occurrence
of B. bassiana on phylloplanes of nettles.
Acknowledgments
We thank Jason Baverstock, Paresh A. Shah and Helen
E. Roy for valuable discussions on experimental set-up and
appropriate equipment. N.V.M. was supported by a PhD
grant from The Royal Veterinary and Agricultural Univer-
sity. J.K.P. is funded by the Department for Environment,
Food and Rural AVairs of the UK (Defra). Rothamsted
Research receives grant-aided support from the Biological
and Biotechnology Research Council of the UK (BBSRC).
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