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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: nvm@kvl.dk (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

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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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