BACKGROUND
the predominantly entomopathogenic fungal genus metarhizium  has a global distribution  and several species have been intensely researched for their pest control potential.
discordantly, limited focus has been given to the assessment of natural occurrence and distribution of this widespread fungal genus.
knowledge of the compositions of entomopathogenic fungal communities and structure of their populations is important to understand their ecological function and contribution to host regulation and potential for conservation biological control  <cit> .
evaluation of fungal diversity and community structure depends heavily on criteria for species identification.
with few morphological distinct features, species referred to as m. anisopliae and variants thereof in the literature could belong to several different species if no explicit molecularly based identification has been conducted.
a revised taxonomy of the m. anisopliae lineage based on a multigene phylogeny resolved nine species within what could be considered m. anisopliae sensu lato if based solely on morphology  <cit> .
recently, the taxonomy of the whole genus metarhizium was revised leading to the inclusion of other genera as well as elevation of some of the former m. flavoviride variants to species level  <cit> .
kepler and rehner  <cit>  further specified additional genomic regions for species identification.
molecularly based evaluation of the structure of metarhizium communities should therefore be included in studies of natural occurrence and distribution to provide full recognition of diversity.
in temperate climatic regions, metarhizium spp.
are predominantly isolated from the soil environment  <cit> .
surveys of entomopathogenic fungi from soil samples conducted at different geographical locations have shown that m. anisopliae s.l.
can be abundantly isolated from managed ecosystems .
however, few studies have evaluated metarhizium community structure using explicit molecular characterization.
in denmark, meyling et al.
<cit>  isolated m. anisopliae s.l.
from an agricultural field and steinwender et al.
<cit>  subsequently showed that the metarhizium community was composed of several species, predominantly m. brunneum followed by m. robertsii.
in the soil environment, naturally occurring metarhizium spp.
may also be isolated from roots of different plants  <cit> .
some level of plant association was indicated by a predominance of m. robertsii found on roots of herbaceous plants as compared to m. brunneum, which was mostly recovered from roots of woody plants  <cit> .
sampling roots of different plants from an ecosystem might therefore inform about the metarhizium diversity of an area and reveal potential habitat associations related to plants growing at the site.
it was recently shown that similar m. brunneum and m. robertsii multilocus genotypes can be repeatedly isolated from roots and soil samples at the same site  <cit> .
however, the structure of the entomopathogenic fungal community in the soil can differ markedly between agricultural fields within relatively close proximity and characterization of a single site may not provide a general distribution pattern.
for example, meyling et al.
<cit>  reported of high frequency of occurrence of m. anisopliae s.l.
at a site in denmark, while another danish agricultural field revealed a low occurrence of m. anisopliae s.l.
<cit>  using same isolation methods.
at the latter site, m. flavoviride was isolated more frequently than m. anisopliae s.l.
indicating location specific entomopathogenic fungal communities.
the aim of the current study was to examine the natural occurrence and diversity of metarhizium spp.
isolates obtained from roots and root associated soil of different common crops at geographically separated agricultural fields in denmark.
fields with oil seed rape, winter wheat as well as permanent grassland were each sampled from three distinct geographical locations.
using such selection criteria, we expected to reveal potential crop and/or location associations of the metarhizium populations from these three distinct geographical sites.
the study resulted in a predominance of m. flavoviride isolates which was contrary to our expectations to isolate m. brunneum most frequently based on recent evidence  <cit> .
metarhizium flavoviride has not previously been studied for within-species diversity, thus the intraspecific variability was examined using amplified fragment length polymorphism  markers to evaluate diversity and potential habitat and/or geographical associations.
RESULTS
metarhizium distribution and species diversity
a total of  <dig> metarhizium isolates were acquired from the soil samples collected at nine agricultural fields .
fourteen of the isolations were obtained using selective media; the remaining  <dig> isolates were attained using standard soil baiting with tenebrio molitor larvae.
the overall distribution of isolates by area and crop type was not homogenous .
a total of  <dig> isolates were from the fårevejle area,  <dig> isolates from skibby and  <dig> isolates from tåstrup; the resulting frequency distribution by area was significantly heterogeneous .
similarly, significantly different frequency distributions were found when compared by crop type .table  <dig> total number of metarhizium spp.
isolations from soil samples collected at three locations in denmark
m. brunneum
m. flavoviride
m. majus
each location is represented by three crop types.
three different species were identified and isolates for each are presented and summed.
the numbers represent isolates obtained both by insect baiting using tenebrio molitor larvae and by plating of root homogenate on selective agar media
of the  <dig> isolates,  <dig> were morphologically identified as m. flavoviride based on their characteristic bright green colony color and conidial dimensions.
the remaining  <dig> isolates were morphologically classified as belonging to m. anisopliae s.l.
the m. flavoviride isolates were collected from all fields except the uncultivated pasture in fårevejle , which had more moist soil than at the other areas.fig.
<dig> map depicting geographical location of the three sampled localities at the island of zealand, denmark.
map of europe is inserted.
at each locality, three fields were sampled for  <dig> root systems and associated soil of each of the respective crops, i.e.
oilseed rape, winter wheat and uncultivated pasture.
inserted pie charts indicate number of metarhizium flavoviride isolates obtained from each field.
areas of pie charts indicate relative contribution of each locality to the total number of isolates characterized.
maps were created from free maps available at d-maps.com
to verify species identity,  <dig> of the isolates collected were sequenced for the intergenic region mzfg543igs located between an atp synthase gamma chain and nad binding protein encoding genes and phylogenetically analyzed.
species branches of the resulting phylogenetic tree  were supported by both bayesian and maximum-likelihood analyses with  <dig> isolates clustering with m. brunneum.
the m. brunneum isolates were distributed among three separate clades with two clades represented by isolates from the undeveloped field at skibby, while eight isolates representing all the three geographical areas clustered with a clade previously found in denmark .
the 116 m. flavoviride isolates that were sequenced for mzfg543igs all shared the same sequence which was similar to isolates from france .
the single isolate of m. majus originated from plating of root homogenate on selective agar medium collected at the undeveloped field at skibby.fig.
<dig> phylogenetic tree of metarhizium spp.
based on sequence alignment of the nuclear intergenic region mzfg543igs.
tree based on bayesian analysis is shown with branch support for both bayesian and maximum-likelihood analyses; bayesian  / maximum-likelihood .
<dig> metarhizium spp.
isolates representing three species were sequences for mzfg543igs.
colored symbols indicate location and crop from which the isolate was acquired
intraspecific variation of m. flavoviride
amplified fragment length polymorphism  analysis was performed using  <dig> of the m. flavoviride isolates collected in this study as well as  <dig> isolates collected from different geographical locations, including: jutland, denmark , Årslev, denmark   <cit> , poland , and southern sweden .
the aflp analysis produced  <dig> polymorphic loci; however, based on the scoring criteria and inconsistency between dual replicates, only  <dig> repeatable and consistent loci were selected for inclusion in the analysis.
using the unweighted pair group method with arithmetic mean  analysis, intraspecific variation among the m. flavoviride isolates was quite high identifying  <dig> haplotypes among the  <dig> analyzed isolates, of which  <dig> were shared among 2– <dig> isolates from different areas while the remaining were unique .
however, only three significant aflp haplotype clusters could be identified by bootstrapping and thus area or crop associated population structure could not be established.fig.
<dig> dendrogram based on  <dig> aflp loci from  <dig> metarhizium flavoviride isolates.
the dendrogram was generated with the phylip package using dice coefficient with  <dig> bootstrap replicates.
bolded branches represent significant pairwise clusters based on bootstrapping.
colored symbols indicate location and crop from which the isolate was acquired
discussion
the majority of metarhizium related research has favored species of the m. anisopliae complex; in contrast m. flavoviride are often reported as being infrequently isolated  and this species has not been studied in detail.
meyling and eilenberg  <cit>  have reported significant occurrence of m. flavoviride in denmark within an agroecosystem located close to the fields sampled in tåstrup in the present study.
however, studies at another location in denmark revealed a predominance of m. brunneum isolates using similar isolation methods  <cit> .
in the present study we surveyed three geographically separate areas and found m. flavoviride to be most abundant at all three areas, although at different frequencies of occurrence, indicating that the relatively high frequency of m. flavoviride reported by meyling and eilenberg  <cit>  was unlikely to be a one-off event.
clearly the species composition of metarhizium communities is not ubiquitous or random at the studied geographical scales, and further in-depth ecological studies are necessary to identify the driving forces that determine metarhizium spp.
distribution in nature.
the predominance of m. flavoviride observed in the present study was unexpected; our initial intention was to analyze the within-species diversity using microsatellites or simple sequence repeat  markers as done previously  <cit> ; these markers have been shown to be highly suitable for explicit multilocus genotyping  <cit> .
however, the ssr markers developed for m. anisopliae s.l.
were observed to have a high frequency  of null alleles when tested on m. flavoviride isolates and no polymorphism  <cit> , and were therefore unsuitable for diversity evaluation within this species.
thus, to characterize the intraspecific variability within m. flavoviride and evaluate the diversity we chose to use aflp markers.
diversity assessment using aflp markers has been used previously for other metarhizium spp.
isolate collections  <cit> .
inglis et al.
<cit>  used aflp markers to determine the diversity within m. anisopliae s.l.
from western canada, and they were able to identify several distinct haplotypes.
it is important to note however that this was done prior to the bischoff et al.
<cit>  taxonomic revision and the material evaluated by inglis et al.
<cit>  has later been split into separate species, predominantly m. brunneum .
fernandes et al.
<cit>  used aflp markers to evaluate inter- and intra-species variability among isolates of several metarhizium species; they found that the method revealed high level of diversity but only clear clustering within m. acridum was seen.
reliable species identification placed within an authoritative molecular phylogeny is important when applying general markers such as aflp to evaluate intra-specific diversity of metarhizium spp.
in contrast to the relatively few m. brunneum isolates sequenced in the current study, all m. flavoviride isolates shared the same sequence at the selected genomic intergenic region, even among spatially distant sampled isolates.
this indicates that m. flavoviride may not be composed of phylogenetically distinct clades as is evident for m. brunneum  <cit>  and m. robertsii  <cit> .
in the present data set m. brunneum was also found to be represented by three distinct clades, all co-occurring at the skibby site.
aflp characterizations of the m. flavoviride isolates included only repeatedly reproduced markers since reproducibility is known to be a challenge with amplified fragments from restriction enzyme markers  <cit> .
the relatively low number of loci in the aflp analysis is likely to have resulted in the limited statistical support for the haplotype groupings.
however, the upgma analysis clearly revealed diversity within the species indicating that the isolates are not of the same immediate clonal origin, but also that m. flavoviride isolates from other countries shared haplotypes with some of the isolates of the present study.
this could tentatively indicate that all the analyzed samples originated from a single diverse population of clonal lineages with little local adaptation.
the lack of host specialization and the ability to subsist as a pathogen or saprophyte may result in a reduced rate of adaption, due to limited selection pressure in particular habitats and the advantage of maintaining broad host and habitat ranges.
future development of species-specific markers  would however improve the ability to evaluate m. flavoviride diversity and determine more evidently any potential habitat associations.
in line with other studies, a single species of metarhizium was found to be the most prevalent in a given area  <cit> , however it is unclear what factors contribute to this high frequency in the community.
metarhizium spp.
are able to acquire nutrients as an insect pathogen or as a plant root associate, which makes it reasonable to suggest that an aptitude at either life strategy would favor a particular species.
it has been observed that an isolate of m. flavoviride  had lower virulence than m. brunneum or m. robertsii isolates towards t. molitor larvae  <cit>  suggesting that m. flavoviride may possess relatively low virulence towards at least some insects.
however, meyling et al.
<cit>  reported of relatively frequent natural mycosis by m. flavoviride in aboveground weevils suggesting that the fungus plays a role in the regulation of some insect populations.
at the site investigated by meyling et al.
<cit>  m. brunneum was most frequently isolated by insect baiting of soil samples, but absent as mycosis in aboveground hosts.
we implemented two standard methods for the isolation of entomopathogenic fungi in this study, viz., a soil bait method with t. molitor and an in vitro selective media method.
of the two, the soil bait method was most effective in recovering isolates of m. flavoviride.
the selective media method used was also employed by wyrebek et al.
<cit>  to isolate metarhizium spp.
from roots of different plants.
although wyrebek et al.
<cit>  did not report of the isolation of m. flavoviride from roots, behie and bidochka  <cit>  reported that m. flavoviride was able to associate with plant roots in the rhizosphere and transfer nitrogen to the plant from insect cadavers.
however, in the present study we sequenced the same isolate  used by behie and bidochka  <cit>  which appeared to be m. pemphigi , and it thus remains undetermined if m. flavoviride can associate with plant roots.
the reduced number of isolates of m. flavoviride collected on selective media from roots in the present study might indicate that, while present in the soil environment, m. flavoviride was not associating closely with the roots of the crop plants, but neither were other metarhizium species.
further studies are needed to confirm these observations and investigate what might be driving the m. flavoviride predominance at the investigated field sites.
however, the current data and that of meyling et al.
<cit>  indicate that entomopathogenicity is an important ecological trait for m. flavoviride occurrence and distribution in agroecosystems.
CONCLUSIONS
the present study revealed that m. flavoviride was the predominant metarhizium species in the soil environment at all three agricultural localities investigated in denmark each represented by three fields, and that the fungal isolates were mainly obtained using t. molitor baiting.
aflp analysis of the m. flavoviride isolates revealed a high level of intra-specific diversity within m. flavoviride, with some widespread aflp haplotypes also occurring in other countries.
this is the first report of an in-depth analysis of the molecular diversity within a large isolate collection of the entomopathogenic fungal species m. flavoviride which could lay the foundations for new research of this little investigated fungus.

 the predominantly entomopathogenic fungal genus metarhizium has a global distribution and several species have been intensely researched for their pest control potential. discordantly, limited focus has been given to the assessment of natural occurrence and distribution of this widespread fungal genus. knowledge of the compositions of entomopathogenic fungal communities and structure of their populations is important to understand their ecological function and contribution to host regulation and potential for conservation biological control. evaluation of fungal diversity and community structure depends heavily on criteria for species identification. with few morphological distinct features, species referred to as m. anisopliae and variants thereof in the literature could belong to several different species if no explicit molecularly based identification has been conducted. a revised taxonomy of the m. anisopliae lineage based on a multigene phylogeny resolved nine species within what could be considered m. anisopliae sensu lato if based solely on morphology. recently, the taxonomy of the whole genus metarhizium was revised leading to the inclusion of other genera as well as elevation of some of the former m. flavoviride variants to species level. kepler and rehner further specified additional genomic regions for species identification. molecularly based evaluation of the structure of metarhizium communities should therefore be included in studies of natural occurrence and distribution to provide full recognition of diversity. in temperate climatic regions, metarhizium spp. are predominantly isolated from the soil environment. surveys of entomopathogenic fungi from soil samples conducted at different geographical locations have shown that m. anisopliae s.l. can be abundantly isolated from managed ecosystems. however, few studies have evaluated metarhizium community structure using explicit molecular characterization. in denmark, meyling et al. isolated m. anisopliae s.l. from an agricultural field and steinwender et al. subsequently showed that the metarhizium community was composed of several species, predominantly m. brunneum followed by m. robertsii. in the soil environment, naturally occurring metarhizium spp. may also be isolated from roots of different plants. some level of plant association was indicated by a predominance of m. robertsii found on roots of herbaceous plants as compared to m. brunneum, which was mostly recovered from roots of woody plants. sampling roots of different plants from an ecosystem might therefore inform about the metarhizium diversity of an area and reveal potential habitat associations related to plants growing at the site. it was recently shown that similar m. brunneum and m. robertsii multilocus genotypes can be repeatedly isolated from roots and soil samples at the same site. however, the structure of the entomopathogenic fungal community in the soil can differ markedly between agricultural fields within relatively close proximity and characterization of a single site may not provide a general distribution pattern. for example, meyling et al. reported of high frequency of occurrence of m. anisopliae s.l. at a site in denmark, while another danish agricultural field revealed a low occurrence of m. anisopliae s.l. using same isolation methods. at the latter site, m. flavoviride was isolated more frequently than m. anisopliae s.l. indicating location specific entomopathogenic fungal communities. the aim of the current study was to examine the natural occurrence and diversity of metarhizium spp. isolates obtained from roots and root associated soil of different common crops at geographically separated agricultural fields in denmark. fields with oil seed rape, winter wheat as well as permanent grassland were each sampled from three distinct geographical locations. using such selection criteria, we expected to reveal potential crop and/or location associations of the metarhizium populations from these three distinct geographical sites. the study resulted in a predominance of m. flavoviride isolates which was contrary to our expectations to isolate m. brunneum most frequently based on recent evidence. metarhizium flavoviride has not previously been studied for within-species diversity, thus the intraspecific variability was examined using amplified fragment length polymorphism markers to evaluate diversity and potential habitat and/or geographical associations.  metarhizium distribution and species diversity a total of metarhizium isolates were acquired from the soil samples collected at nine agricultural fields. fourteen of the isolations were obtained using selective media; the remaining isolates were attained using standard soil baiting with tenebrio molitor larvae. the overall distribution of isolates by area and crop type was not homogenous. a total of isolates were from the fårevejle area, isolates from skibby and isolates from tåstrup; the resulting frequency distribution by area was significantly heterogeneous. similarly, significantly different frequency distributions were found when compared by crop type.table total number of metarhizium spp. isolations from soil samples collected at three locations in denmark m. brunneum m. flavoviride m. majus each location is represented by three crop types. three different species were identified and isolates for each are presented and summed. the numbers represent isolates obtained both by insect baiting using tenebrio molitor larvae and by plating of root homogenate on selective agar media of the isolates, were morphologically identified as m. flavoviride based on their characteristic bright green colony color and conidial dimensions. the remaining isolates were morphologically classified as belonging to m. anisopliae s.l. the m. flavoviride isolates were collected from all fields except the uncultivated pasture in fårevejle, which had more moist soil than at the other areas.fig. map depicting geographical location of the three sampled localities at the island of zealand, denmark. map of europe is inserted. at each locality, three fields were sampled for root systems and associated soil of each of the respective crops, i.e. oilseed rape, winter wheat and uncultivated pasture. inserted pie charts indicate number of metarhizium flavoviride isolates obtained from each field. areas of pie charts indicate relative contribution of each locality to the total number of isolates characterized. maps were created from free maps available at d-maps.com to verify species identity, of the isolates collected were sequenced for the intergenic region mzfg543igs located between an atp synthase gamma chain and nad binding protein encoding genes and phylogenetically analyzed. species branches of the resulting phylogenetic tree were supported by both bayesian and maximum-likelihood analyses with isolates clustering with m. brunneum. the m. brunneum isolates were distributed among three separate clades with two clades represented by isolates from the undeveloped field at skibby, while eight isolates representing all the three geographical areas clustered with a clade previously found in denmark. the 116 m. flavoviride isolates that were sequenced for mzfg543igs all shared the same sequence which was similar to isolates from france. the single isolate of m. majus originated from plating of root homogenate on selective agar medium collected at the undeveloped field at skibby.fig. phylogenetic tree of metarhizium spp. based on sequence alignment of the nuclear intergenic region mzfg543igs. tree based on bayesian analysis is shown with branch support for both bayesian and maximum-likelihood analyses; bayesian / maximum-likelihood. metarhizium spp. isolates representing three species were sequences for mzfg543igs. colored symbols indicate location and crop from which the isolate was acquired intraspecific variation of m. flavoviride amplified fragment length polymorphism analysis was performed using of the m. flavoviride isolates collected in this study as well as isolates collected from different geographical locations, including: jutland, denmark, Årslev, denmark, poland, and southern sweden. the aflp analysis produced polymorphic loci; however, based on the scoring criteria and inconsistency between dual replicates, only repeatable and consistent loci were selected for inclusion in the analysis. using the unweighted pair group method with arithmetic mean analysis, intraspecific variation among the m. flavoviride isolates was quite high identifying haplotypes among the analyzed isolates, of which were shared among 2– isolates from different areas while the remaining were unique. however, only three significant aflp haplotype clusters could be identified by bootstrapping and thus area or crop associated population structure could not be established.fig. dendrogram based on aflp loci from metarhizium flavoviride isolates. the dendrogram was generated with the phylip package using dice coefficient with bootstrap replicates. bolded branches represent significant pairwise clusters based on bootstrapping. colored symbols indicate location and crop from which the isolate was acquired discussion the majority of metarhizium related research has favored species of the m. anisopliae complex; in contrast m. flavoviride are often reported as being infrequently isolated and this species has not been studied in detail. meyling and eilenberg have reported significant occurrence of m. flavoviride in denmark within an agroecosystem located close to the fields sampled in tåstrup in the present study. however, studies at another location in denmark revealed a predominance of m. brunneum isolates using similar isolation methods. in the present study we surveyed three geographically separate areas and found m. flavoviride to be most abundant at all three areas, although at different frequencies of occurrence, indicating that the relatively high frequency of m. flavoviride reported by meyling and eilenberg was unlikely to be a one-off event. clearly the species composition of metarhizium communities is not ubiquitous or random at the studied geographical scales, and further in-depth ecological studies are necessary to identify the driving forces that determine metarhizium spp. distribution in nature. the predominance of m. flavoviride observed in the present study was unexpected; our initial intention was to analyze the within-species diversity using microsatellites or simple sequence repeat markers as done previously; these markers have been shown to be highly suitable for explicit multilocus genotyping. however, the ssr markers developed for m. anisopliae s.l. were observed to have a high frequency of null alleles when tested on m. flavoviride isolates and no polymorphism, and were therefore unsuitable for diversity evaluation within this species. thus, to characterize the intraspecific variability within m. flavoviride and evaluate the diversity we chose to use aflp markers. diversity assessment using aflp markers has been used previously for other metarhizium spp. isolate collections. inglis et al. used aflp markers to determine the diversity within m. anisopliae s.l. from western canada, and they were able to identify several distinct haplotypes. it is important to note however that this was done prior to the bischoff et al. taxonomic revision and the material evaluated by inglis et al. has later been split into separate species, predominantly m. brunneum. fernandes et al. used aflp markers to evaluate inter- and intra-species variability among isolates of several metarhizium species; they found that the method revealed high level of diversity but only clear clustering within m. acridum was seen. reliable species identification placed within an authoritative molecular phylogeny is important when applying general markers such as aflp to evaluate intra-specific diversity of metarhizium spp. in contrast to the relatively few m. brunneum isolates sequenced in the current study, all m. flavoviride isolates shared the same sequence at the selected genomic intergenic region, even among spatially distant sampled isolates. this indicates that m. flavoviride may not be composed of phylogenetically distinct clades as is evident for m. brunneum and m. robertsii. in the present data set m. brunneum was also found to be represented by three distinct clades, all co-occurring at the skibby site. aflp characterizations of the m. flavoviride isolates included only repeatedly reproduced markers since reproducibility is known to be a challenge with amplified fragments from restriction enzyme markers. the relatively low number of loci in the aflp analysis is likely to have resulted in the limited statistical support for the haplotype groupings. however, the upgma analysis clearly revealed diversity within the species indicating that the isolates are not of the same immediate clonal origin, but also that m. flavoviride isolates from other countries shared haplotypes with some of the isolates of the present study. this could tentatively indicate that all the analyzed samples originated from a single diverse population of clonal lineages with little local adaptation. the lack of host specialization and the ability to subsist as a pathogen or saprophyte may result in a reduced rate of adaption, due to limited selection pressure in particular habitats and the advantage of maintaining broad host and habitat ranges. future development of species-specific markers would however improve the ability to evaluate m. flavoviride diversity and determine more evidently any potential habitat associations. in line with other studies, a single species of metarhizium was found to be the most prevalent in a given area, however it is unclear what factors contribute to this high frequency in the community. metarhizium spp. are able to acquire nutrients as an insect pathogen or as a plant root associate, which makes it reasonable to suggest that an aptitude at either life strategy would favor a particular species. it has been observed that an isolate of m. flavoviride had lower virulence than m. brunneum or m. robertsii isolates towards t. molitor larvae suggesting that m. flavoviride may possess relatively low virulence towards at least some insects. however, meyling et al. reported of relatively frequent natural mycosis by m. flavoviride in aboveground weevils suggesting that the fungus plays a role in the regulation of some insect populations. at the site investigated by meyling et al. m. brunneum was most frequently isolated by insect baiting of soil samples, but absent as mycosis in aboveground hosts. we implemented two standard methods for the isolation of entomopathogenic fungi in this study, viz., a soil bait method with t. molitor and an in vitro selective media method. of the two, the soil bait method was most effective in recovering isolates of m. flavoviride. the selective media method used was also employed by wyrebek et al. to isolate metarhizium spp. from roots of different plants. although wyrebek et al. did not report of the isolation of m. flavoviride from roots, behie and bidochka reported that m. flavoviride was able to associate with plant roots in the rhizosphere and transfer nitrogen to the plant from insect cadavers. however, in the present study we sequenced the same isolate used by behie and bidochka which appeared to be m. pemphigi, and it thus remains undetermined if m. flavoviride can associate with plant roots. the reduced number of isolates of m. flavoviride collected on selective media from roots in the present study might indicate that, while present in the soil environment, m. flavoviride was not associating closely with the roots of the crop plants, but neither were other metarhizium species. further studies are needed to confirm these observations and investigate what might be driving the m. flavoviride predominance at the investigated field sites. however, the current data and that of meyling et al. indicate that entomopathogenicity is an important ecological trait for m. flavoviride occurrence and distribution in agroecosystems.  the present study revealed that m. flavoviride was the predominant metarhizium species in the soil environment at all three agricultural localities investigated in denmark each represented by three fields, and that the fungal isolates were mainly obtained using t. molitor baiting. aflp analysis of the m. flavoviride isolates revealed a high level of intra-specific diversity within m. flavoviride, with some widespread aflp haplotypes also occurring in other countries. this is the first report of an in-depth analysis of the molecular diversity within a large isolate collection of the entomopathogenic fungal species m. flavoviride which could lay the foundations for new research of this little investigated fungus. 
the objective was therefore to evaluate molecular diversity and resolve the potential population structure of m. flavoviride. this study represents the first in-depth analysis of the molecular diversity within a large isolate collection of the species m. flavoviride. species of the genus metarhizium are widespread in soils and have recently been reported to associate with plant roots, but the species m. flavoviride has so far received little attention and intra-specific diversity among isolate collections has never been assessed. the aflp analysis confirmed a high level of intra-specific diversity within the species and lack of apparent association patterns with crop or location indicates limited ecological specialization. in the present study m. flavoviride was found to be abundant among metarhizium spp. knowledge of the natural occurrence and community structure of entomopathogenic fungi is important to understand their ecological role. aflpentomopathogenic fungisoil baitingrhizosphere competencepopulation ecologyissue-copyright-statement© the author 2015 further characterization of intraspecific variability within m. flavoviride was done by using amplified fragment length polymorphisms to evaluate diversity and potential crop and/or locality associations. a high level of diversity among the m. flavoviride isolates was observed, indicating that the isolates were not of the same clonal origin, and that certain haplotypes were shared with m. flavoviride isolates from other countries. however, no population structure in the form of significant haplotype groupings or habitat associations could be determined among the analyzed m. flavoviride isolates. isolates obtained from roots and root-associated soil of winter wheat, winter oilseed rape and neighboring uncultivated pastures at three geographically separated locations in denmark. of the metarhizium isolates obtained, morphological data and dna sequencing revealed that belonged to m. flavoviride, to m. brunneum and one to m. majus. the relatively infrequent isolation of m. flavoviride directly from crop roots suggests low dependence of root association for the species.
 knowledge of the natural occurrence and community structure of entomopathogenic fungi is important to understand their ecological role. species of the genus metarhizium are widespread in soils and have recently been reported to associate with plant roots, but the species m. flavoviride has so far received little attention and intra-specific diversity among isolate collections has never been assessed. in the present study m. flavoviride was found to be abundant among metarhizium spp. isolates obtained from roots and root-associated soil of winter wheat, winter oilseed rape and neighboring uncultivated pastures at three geographically separated locations in denmark. the objective was therefore to evaluate molecular diversity and resolve the potential population structure of m. flavoviride.  of the metarhizium isolates obtained, morphological data and dna sequencing revealed that belonged to m. flavoviride, to m. brunneum and one to m. majus. further characterization of intraspecific variability within m. flavoviride was done by using amplified fragment length polymorphisms to evaluate diversity and potential crop and/or locality associations. a high level of diversity among the m. flavoviride isolates was observed, indicating that the isolates were not of the same clonal origin, and that certain haplotypes were shared with m. flavoviride isolates from other countries. however, no population structure in the form of significant haplotype groupings or habitat associations could be determined among the analyzed m. flavoviride isolates.  this study represents the first in-depth analysis of the molecular diversity within a large isolate collection of the species m. flavoviride. the aflp analysis confirmed a high level of intra-specific diversity within the species and lack of apparent association patterns with crop or location indicates limited ecological specialization. the relatively infrequent isolation of m. flavoviride directly from crop roots suggests low dependence of root association for the species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aflpentomopathogenic fungisoil baitingrhizosphere competencepopulation ecologyissue-copyright-statement© the author 2015 