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Occurrence and distribution of soil borne entomopathogenicfungi within a single organic agroecosystem
Nicolai V. Meyling *, Jrgen Eilenberg
Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
Received 10 December 2004; received in revised form 14 October 2005; accepted 24 October 2005
Available online 20 December 2005
Abstract
By baiting soil samples with larvae ofGalleria mellonelladetailed surveys of the occurrences of entomopathogenic fungi were conducted
over two consecutive years in the soil of an organically farmed field (17.1 ha) and the associated hedgerow. Samples were collected at specific
points (at distances of 25 m) based on Geographical Information Systems (GIS) and sample point coordinates were relocated by Global
Positioning System (GPS). In the agricultural field soil Beauveria bassianawas the most common fungus whilePaecilomyces fumosoroseus
was most common in soil from the hedgerow. Significant clustering ofB. bassianain the agricultural field was found in one of the two years.
High and low densities ofB. bassianawere subsequently confirmed within selected areas by reducing distances between sample points. The
results demonstrated the suitability of the sampling method for identifying distribution patterns of soil borne entomopathogenic fungi and the
importance of large sample sizes to describe local biodiversity of the fungi in the soil environment.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Entomopathogenic fungi; Beauveria bassiana; Galleria bait method; GIS; GPS; SADIE; Sustainable agriculture
1. Introduction
Microbial assemblages in agricultural soils are important
for ecosystem services in sustainable agricultural systems,
including pest control (Altieri, 1999). High populations of
beneficial soil borne organisms are characteristics of healthy
soils (Magdoff, 2001). The soil environment constitutes an
important reservoir for a diversity of entomopathogenic
fungi, which can contribute significantly to the regulation of
insect populations (Keller and Zimmerman, 1989). Many
species belonging to Hypocreales (Ascomycota) inhabit the
soil for a significant part of their life cycle at northernlatitudes. Of these, Beauveriaspp.,Metarhizium anisopliae
(Metschnikoff) Sorokin and Paecilomyces spp. are espe-
cially common (Keller and Zimmerman, 1989). Conversion
from conventional to organic farming generally increases
the diversity and activity of soil microorganisms over time
(Mader et al., 2002). There is evidence for higher population
levels of entomopathogenic fungi in soils of organically
farmed fields as opposed to conventionally farmed fields in
Norway (Klingen et al., 2002).
Knowledge of local species composition and distribution
is important if the indigenous populations of entomopatho-
genic fungi in the soil are to be managed in ways to facilitate
the control of pest insect populations within the agroeco-
system. Most studies of the occurrence and biodiversity of
entomopathogenic fungi in soils have focused on differences
in species composition between areas defined by habitat
types (e.g. arable soils, semi-natural habitats, etc.) on
regional or national scales, where several localities withsimilar types of habitat have been considered together
(Steenberg, 1995; Vanninen, 1996; Bidochka et al., 1998;
Klingen et al., 2002; Keller et al., 2003). In most of these
previous studies, relatively few soil samples were collected
arbitrarily at each locality and at a single time point only.
In the present study, the occurrence and spatial
distribution of entomopathogenic fungi in the soil of a
single organically grown agroecosystem was for the first
time investigated using a high precision sampling scheme
www.elsevier.com/locate/ageeAgriculture, Ecosystems and Environment 113 (2006) 336341
* Corresponding author. Tel.: +45 3528 2666; fax: +45 3528 2670.
E-mail address: [email protected] (N.V. Meyling).
0167-8809/$ see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.agee.2005.10.011
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based on Geographical Information Systems (GIS). Several
hundreds of samples were collected from this single arable
field and the associated hedgerow. The implementation of
GIS allowed for continued sampling of the exact same grid
over two seasons (2001 and 2002). In 2003, the aim was to
evaluate if the method reliably described the distribution of
selected soil borne entomopathogenic fungi.
2. Materials and methods
The study site (17.1 ha) was located at Taastrup, 20 km
west of Copenhagen, Denmark (558400N, 128180E) on an
experimental research farm, Bakkegarden. A hedgerow
consisting mainly of hawthorn (Crataegus monogyna L.)
and poplar (Populus sp.) with herbaceous vegetation
dominated by nettles (Urtica dioica L.) and grasses
(Poaceae) lined the field to the southeast. The soil at the
field site was formed on calcareous glacial till from the
Weichselian Glaciation and is classified as a Typic Argiudoll
by the American soil taxonomy system (Soil Survey Staff,
1999), equivalent to a sandy loam.
In 2000, the cultivation of the field was converted from
conventional to organic farming practice, and a sampling
grid based on GIS covering the entire field was imple-
mented. Points in the grid were oriented northsouth and
were located 25 m apart. The points could subsequently be
located in the field by Global Positioning System (GPS) by a
Trimble AgGPS1 214 high-accuracy receiver linked to a
Real-Time Kinematic (RTK) base station, which allows
location of points with a precision of 12 cm (http://
www.trimble.com).In 2001, the investigated area was divided into three
separate rectangular sub-fields, each 4.5 ha. In between the
fields were areas with permanent grass. Soil sampling in
2001 was done prior to the sowing of crops. In 2002, the sub-
fields were cropped from west to east with: (1) undersown
peabarley intercropping, (2) undersown spring barley and
(3) clover-grass, respectively. The study area was com-
pletely covered by 274 sampling points in the sampling grid.
Each soil sample (n= 270 in 2001; n= 274 in 2002) was
taken in relation to one of the GIS points. The sampling in
spring (AprilMay 2001) was done as a part of the initial
characterisation of the field. All soil samples were taken to a
depth of 30 cm using an automatic core sampler (2 cm
diameter) mounted on a small tractor. In each point, between
25 and 30 cores were collected within an area of
approximately 0.5 m2. These soil cores, representing each
point, were mixed together in individual polyethylene bags.
Sampling in September 2002 was done as follows: at each
point, 25 cores were collected to a depth of 10 cm using a
manual core sampler (12 mm diameter). The cores were
evenly distributed over a 5 5 square grid (total area of
0.25 m2). The 25 cores from each sample point were mixed
together in separate polyethylene bags. The core sampler
was rinsed in water, 70% ethanol and water, respectively,
between consecutive sampling points. Additionally, 70 soil
samples (each comprised of 25 cores) were collected from
the hedgerow lining the southeastern border of the field on 6
September 2002 and 20 September 2003, respectively. The
samples in the hedgerow were dispersed 5 m apart along a
transect in the middle of the hedgerow. The same collection
procedure as described above for 2002 was used for allsamples in the hedgerow.
In 2003, additional soil samples were collected in the
field at sample points that were selected based on occurrence
of entomopathogenic fungi in the previous years. Specifi-
cally, points at which Beauveria bassiana (Balsamo)
Vuillemin was found in both 2001 and 2002 (positive
points) as well as points where no fungi were found in either
of the years (negative points) were selected. This yielded 35
positive and 33 negative points, respectively, that were
resampled in September 2003.
In order to elucidate the distance between sample points
thatwereappropriate for reliable evaluation of the distribution
pattern of entomopathogenic fungi in the field, 150 sample
points were selected as follows: between selected GIS points
two 25 m 25 m sampling grids were established, each
consisting of 5 m 5 m cells (n= 25). One such 625 m2 grid
was located between four points that had yieldedB. bassiana
in both 2001 and 2002 (high density area) while the other
grid was placed in between four points where no entomo-
pathogenic fungi had been found in either of the years (low
density area). In both grids, one sample, consisting of 25 soil
cores as describedabove for2002 collections,was taken in the
middle of each of the 25 cells. In addition, two randomly
selected 25 m2 cells within each grid were divided into
1 m 1 m sub-cells (n= 25). One sample of 25 cores wastaken from the middle of each of these 1 m2 sub-cells. Thus,
each of the two areas gave 25 samples from the large 625 m2
grid (distance between sampling points = 5 m) and 50
samples from the two small 25 m2 cells (distance between
sampling points = 1 m). All soil samples were stored in a
refrigerated room at 45 8C for one to four months until
further processing.
In the laboratory, each bag containing soil was
thoroughly mixed and homogenised by hand. The soil
was then transferred from the bag to a 155 ml transparent
plastic cup leaving 1 cm of free air at the top. If the soil was
too dry it was moistened with tap water to obtain equal levels
of humidity during baiting.
Entomopathogenic fungi were isolated from soil samples
by the Galleria bait method (Zimmermann, 1986). The
wax moth Galleria mellonella L. (Lepidoptera: Pyralidae)
came from a continuous reared colony maintained in
constant darkness at 20 8C. Larvae of third or fourth instar
(approximately four weeks after hatching) were used for
baiting the soil samples. Prior to baiting, the larvae were
immersed in 56 8C water for 15 s to minimise their ability to
produce silk webbing in the soil (Woodring and Kaya, 1988).
Each soil sample was baited with 10 larvae and the cups
were sealed with perforated lids and incubated in the dark in
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closed cardboard boxes at ambient room temperature (20
25 8C). During the first two weeks of baiting the cups were
frequently shaken, inverted and left upside down.
Once a week the soil was inspected for dead larvae.
Cadavers were transferred individually to 30 ml medicine
cups and washed three times in demineralised water. Each
medicine cup was provided with moist filter paper, sealedwith a lid and incubated at room temperature. Incubated
larvae were inspected for presence of external fungal
growth. The fungi were identified morphologically both by
low magnifying stereomicroscope (40 magnification) of
cadavers and by preparing slides for light microscopy (400
magnification).
Analyses were made of frequencies of occurrence of the
different species of entomopathogenic fungi between the
surveyed areas and years by standard x2 tests. Odds ratios
were calculated when more than two groups were included.
Larval mortality in each soil sample for all fungi and each
fungus species, respectively, was modelled for field and
hedgerow soils in 2001, 2002 and 2003 by logistic regression
(link = logit) in PROC GENMOD in SAS (SAS Institute
Inc., 1999) using habitat type and year as class variables.
The analyses were adjusted for overdispersion and
differences between proportions were identified by the
CONTRAST option after the final models were found. The
number of weeks for larvae to die was compared for 2002
between field and hedgerow soils by fitting a generalised
linear model using PROC GLM (SAS Institute Inc., 1999).
The spatial distribution of the fungi was analysed within
the field for 2001 and 2002, as the exact position of each
sampling point in the field was known. This was done using
the software programme Spatial Analysis of DistanceIndices (SADIE), which is freely available for download
athttp://www.rothamsted.ac.uk/pie/sadie/. The method used
information of the positions of the samples in two-
dimensional space as well as the count values of the
samples. In this study, the values in each sample ranged
between 0 and 10. Notations below are based onPerry et al.
(1999). The SADIE programme compared the observed data
set with a large number of permutated randomisations of
similar values. For each sample unit a dimensionless
clustering index was identified based on the actual data and
outcome of the randomisations. For each unit with count
larger than average (patch unit) an outflow index, v i, was
calculated and for each unit with count smaller than average
(gap unit) an inflow index,vj, was found. A test for overallclustering was performed for the entire data set by
calculating an average index of vi and vj, respectively.
This was compared with the values of the randomisations.
Thus, tests for both patches and gaps were made
independently (Perry et al., 1999). By convention, clustering
indices >1.5 indicated that the sampling units were
members of a patch while clustering indices less than
1.5 were interpreted as belonging to a gap area. The spatial
locations of these indices identified patches (aggregations of
units with large clustering indices) and gaps (aggregations of
units with small clustering indices) (Perry et al., 1999).
3. Results
The agricultural field soil most frequently harboured B.
bassianawhile soil from the hedgerow most often contained
Paecilomyces fumosoroseus (Wise) Brown and Smith
(Table 1). However, B. bassiana was also common in
hedgerow soil in both 2002 and 2003. In the field soil,
Metarhizium flavoviride Gams and Rozsypal was more
frequently isolated than M. anisopliae. M. anisopliae was
not found in the soil of the hedgerow andM. flavovirideonly
occurred there in three samples in September 2002. While P.
fumosoroseus very rarely was isolated from the field soil,this habitat often contained Paecilomyces farinosus (Holm
ex S.F. Grey) Brown and Smith, and the frequencies of the
latter species were not significantly different between years
and habitat types (Table 1). Rare entomopathogenic fungi
isolated from the field soil were Conidiobolus coronatus
(Constantin) Batko and Lecanicillium lecanii (Zimmer-
mann) Gams & Zare (Table 1). Additionally, isolation
N.V. Meyling, J. Eilenberg / Agriculture, Ecosystems and Environment 11 3 (2006) 336341338
Table 1
Frequencies of occurrence (% positive samples) of entomopathogenic fungi in soil samples from field in spring 2001 and September 2002, and hedgerow in
September 2002 and September 2003
Fungus species Field Hedgerow x2 P
2001 (n= 270) 2002 (n= 274) 2002 (n= 70) 2003 (n= 70)
Beauveria bassiana 29.3 42.0 (1.75)a 54.3 (2.87) 62.9 (4.09) 34.478
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following the sampling of selected areas of the field in
September 2003 yielded a few isolates of Hirsutella
nodulosa Petch (3.3% occurrence; 5/150 samples) from
G. mellonella larvae.
Significant effects of the interaction between habitat type
and year of sampling on the number of larvae that died of all
fungi and specific fungus species, respectively, in the baited
soil samples was found by fitting logistic regression models
(see Table 2 for test statistics). Considering all entomo-
pathogenic fungi, more G. mellonella larvae died from
infection in the hedgerow soil than in soil from the field
(Table 2). This was especially due to infections of P.
fumosoroseus, but more larvae also died fromB. bassianain
the hedgerow soil compared to the field soil. In the field soil,
significantly more larvae died fromB. bassiana in samples
from autumn 2002 than in samples from spring 2001
(Table 2). Most larvae died from infections ofP. farinosusin
the field soil in 2001 compared to the field soil in 2002 as
well as hedgerow soils in both years.TheGalleriabait method yielded more than one fungus
species in some baited samples. From the field soil in 2001,
11.9% of the samples gave two species of entomopatho-
genic fungi and 0.7% gave three species. In 2002, two
species of fungi were isolated from 13.5% of the baited soil
samples and 1.1% of the samples gave three species. These
frequencies were not significantly different between years
(x2 = 0.466; d.f. = 1; P = 0.4950). Two species of fungi
were isolated from 45.6% of the soil samples from the
hedgerow in 2002, and 5.7% of the samples gave three
species. In 2003, 34.3% of the samples yielded two species
while three species were found in 4.3% of the samples.
These frequencies were not significantly different between
years (x2 = 2.338; d.f. = 1; P= 0.1263). However, the
frequencies of samples with two or more species of
entomopathogenic fungi were different between the
samples from the field and hedgerow soil (x2 = 72.479;
d.f. = 1; P < 0.0001).
The time for larvae to die from fungal infections in
2002 was shorter in the soil from hedgerows when
compared to field soil. Larvae in the hedgerow soil died
within a mean (95% confidence limits) of 1.7 (1.56; 1.78)
weeks, while G. mellonella larvae in samples from the
field soil died of infections within 2.5 (2.48; 2.60) weeks.
These means were found to be significantly different
(F1,343= 183.25; P < 0.0001) by PROC GLM (SAS
Institute Inc., 1999).
The analysis of spatial distribution of entomopathogenic
fungi within the field was restricted to only B. bassiana as
the other species occurred too infrequently for a reliable
analysis. In 2001, no significant clustering with respect to
patches (average vi 1:097; P = 0.2023) or gaps (average
vj 1:085; P = 0.2348) was found when compared with
5967 randomisations. The distribution pattern ofB. bassiana
over the whole field surface could therefore not be
distinguished from that of a random distribution. In contrast,
the clustering of B. bassiana in 2002 was found to be
significant both with regard to patches (average vi 1:772;
P= 0.0003) and gaps (average vj 1:686; P= 0.0012).
Some of this clustering was associated with the cropping
system. In undersown spring barley in 2002, 63.4% of the
sampling points (n= 73) were gap units (vj < 1:5)
compared to 11.6% in peabarley intercropping (n= 69).This former frequency was significantly higher than the
latter (x2 = 41.66; d.f. = 1; P < 0.0001). In contrast, 24.6%
of the peabarley intercropping sampling points were
patch units (vi > 1:5) while 4.1% of the sampling points
in undersown spring barley were patch units. These
frequencies were also significantly different (x2 = 12.35;
d.f. = 1; P = 0.0004). The area with clover-grass (n= 72)
contained 31.9% gap units and no patch units.
The additional sampling in September 2003 in specific
points showed that although positive points yielded a
slightly higher frequency of B. bassiana (68.5%; n= 35)
compared to negative points (54.0%; n= 33) there was no
significant difference between the two categories in 2003
(x2 = 1.415; d.f. = 1; P= 0.234).
Sampling at reduced distances in two selected areas
between original sampling points gave different occurrences
of entomopathogenic fungi in September 2003. When
distances between sampling points were 5 m (n= 25) the
frequency of occurrence of all entomopathogenic fungi in
the high density area was 84%. In contrast, it was 36% in
the low density area. These frequencies were significantly
different (x2 = 12.00; d.f. = 1; P= 0.0005). Of all isolated
fungi, B. bassiana occurred in 68% of the samples in the
highdensity area while the species was found in 16% of the
N.V. Meyling, J. Eilenberg / Agriculture, Ecosystems and Environment 113 (2006) 336341 339
Table 2
Mean numbers [95% confidence limits] of G. mellonella larvae that died from infections of fungi during the baiting of the soil samples
Field Hedgerow F3,680* P
2001 2002 2002 2003
All fungi 1.64 [1.41; 1.89] a 1.70 [1.15; 1.95] a 6.41 [5.79; 7.00] b 5.89 [5.25; 6.49] b 120.03
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samples in the low density area. Again, there was
significant difference between these frequencies
(x2 = 13.88; d.f. = 1; P= 0.0002). Reducing distances
between sampling points further to 1 m (n= 50) confirmed
the results. The frequency of occurrence of all entomo-
pathogenic fungi in the high density area was 72% while it
was 38% in the low density area (x2
= 11.68; d.f. = 1;P= 0.0006). In the high density area 54% of samples
containedB. bassianawhile the frequency ofB. bassianain
the low density area was 20% (x2 = 12.39; d.f. = 1;
P= 0.0004).
4. Discussion
TheGalleriabait method (Zimmermann, 1986) has been
found to be a very sensitive method for detection of
entomopathogenic fungi in soil samples (Keller et al., 2003).
The species detected in the present study were within the
expected range based on previous studies performed at
similar latitudes using bait insects (Steenberg, 1995;
Chandler et al., 1997). The representation of B. bassiana
and P. farinosus in agricultural and hedgerow soils
corresponded well with earlier investigations (Steenberg,
1995; Vanninen, 1996; Chandler et al., 1997). Similar to the
present study,Steenberg (1995)foundP. fumosoroseusmost
commonly in Danish hedgerows and in the UK, Chandler
et al. (1997) also isolated the species most often from
hedgerow soils. In Poland, however, Mietkiewski et al.
(1998) isolated P. fumosoroseus frequently from soils
originating from rye fields by baiting with G. mellonella
whereas this species was almost absent from the agriculturalsoil of the present study. The present findings indicated a
relatively high density ofP. fumosoroseus in the soil of the
hedgerow habitat. Additionally,B. bassianawas common in
the hedgerow and several soil samples from this habitat
yielded more than one fungus species. This observation,
together with the shorter mortality time for bait larvae in
hedgerow soil, suggest that higher densities of entomo-
pathogenic fungi were present in the hedgerow habitat than
in the agricultural field.
Interestingly, the species H. nodulosa was found on a
fewG. mellonellalarvae in 2003. The only previous record
of isolation of aHirsutellaspecies from soil by bait insects
has been ofHirsutella jonesii(Speare) Evans and Samson
in Palestine (Ali-Shtayeh et al., 2003). It was surprising to
find low frequencies ofM. anisopliae, since this species has
generally been recognised as common in agricultural soils
(Vanninen, 1996; Bidochka et al., 1998) and even in Danish
agricultural fields (Steenberg, 1995). Although M. flavo-
viridehas been documented very rarely in other studies of
entomopathogenic fungi in soil, it was quite common in the
field soil at the investigated site. Steenberg (1995)found
only one larva infected with M. flavoviride while
Mietkiewski et al. (1997) detected the species at very
low frequencies in arable soils from southern UK using G.
mellonella as bait larvae. In the present study, M.
flavoviride was locally abundant while M. anisopliae
was locally rare. Thus, knowledge of the local species
composition of entomopathogenic fungi in the soil is
necessary when evaluating the potential for this group of
natural enemies as a reservoir for controlling pest insects in
a specific agroecosystem.Resampling at selected points in 2003 underscored the
importance of collecting a sufficient number of soil
samples for detection of entomopathogenic fungi in the
soil environment. The occurrence of B. bassiana was
dynamic and not persistent at specific points. However,
collecting several samples within two selected areas in
2003 confirmed the high and low densities, respectively, of
B. bassianaobserved in 2002. This indicates that the initial
sampling scheme of distances of 25 m identified distribu-
tion and clustering of B. bassiana within the field quite
reliably and that this distance was suitable for conducting a
whole field survey. The observed high and low density
areas were persistent in time until the following autumn.
This suggests that high densities of B. bassiana persisted
after establishment within an area. Permanent persistence
of B. bassiana at high densities in the soil depends on
interactions with the surrounding environment. Several
abiotic factors have been demonstrated to influence the
persistence of B. bassiana in soil. For instance, high soil
humidity and temperature reduced conidial survival and
infectivity in laboratory tests (Lingg and Donaldson, 1981)
while cultural practices, such as reduced tillage regimes,
enhanced B. bassiana levels in the soil (Bing and Lewis,
1993; Hummel et al., 2002a). Organic matter content and
biological activity of the soil adversely affected persis-tence ofB. bassiana due to antagonistic effects of other
soil microorganisms (Lingg and Donaldson, 1981; Fargues
and Robert, 1985; Keller and Zimmerman, 1989).
Vanninen et al. (2000)showed that augmentedB. bassiana
conidia persisted poorly in Finnish soils compared to M.
anisopliae. Furthermore, Gottwald and Tedders (1984)
found that B. bassiana grew and proliferated well from
infected host insects in the soil. This suggests that B.
bassianarely on repeated infections of susceptible hosts to
maintain high density levels in soils (Fargues and Robert,
1985), as demonstrated for Beauveria brongniartii
(Saccardo) Petch (Kessler et al., 2004).
Crop diversification and crop rotation systems can
influence insect populations, both pests and beneficial
species (Hummel et al., 2002b; Hooks and Johnson, 2003).
Occurrence of soil dwelling insects in the cropping system
may facilitate the high levels ofB. bassianaobserved in the
present study, primarily associated with the peabarley
intercropping. Through specific management strategies
providing optimal conditions for the entomopathogenic
fungi in the soil these natural enemies of insects can be
included in the suppression of pests in a conservation
biological control strategy (Landis et al., 2000; Eilenberg
et al., 2001).
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Acknowledgements
Michael Nrremark provided the GPS equipment used in
this study. Hanne Lipczak Jacobsen assisted with advice
during the initiation of the fieldwork at Bakkegarden.
Charlotte Nielsen, Susanne Vestergaard and Sren Navntoft
helped with soil sampling and Christina Wolsted providedvaluable technical assistance. Cezary Tkaczuk and Stanis-
aw Baazy kindly identifiedH. nodulosa. We thank Stephen
A. Rehner for correcting the English. The Royal Veterinary
and Agricultural University funded a Ph.D. grant for NVM
and provided the field facilities at Bakkegarden.
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