A fungal pathogen in time and space: the population dynamics of Beauveriabassianain a conifer forest

The fungal entomopathogen Beauveria bassiana is ubiquitous in below-ground systems; however, there is a dearth of information on the above-ground diversity, temporal and spatial distribution of this fungus. Therefore, we assessed its occurrence in a conifer forest ( Pseudotsuga monziesii and Pinus nigra var. maritima ) using selective media to isolate B. bassiana from soil, branch and bark samples collected in October 2005, March and June 2006. Fungal density was the highest at all locations in October, declining in March and June, and absent from conifer branches in June. This above-ground decline most likely resulted from more extreme environmental conditions compared with those below ground. Molecular analyses (ISSR-PCR) indicated that B. bassiana is genetically diverse, comprising both distinct microhabitat-speciﬁc and seasonal isolates. The occurrence of dissimilar above- and below-ground isolates suggests that B. bassiana occupies various overlapping niches in these systems. distribution, and of B. the the and of of the identifying on the


Introduction
The globally ubiquitous anamorphic genus Beauveria (Ascomycota: Hypocreales) is thought to have a broad host range and has received considerable attention as a potential biological control agent of pest insects. However, the wealth of literature on the role of such fungal entomopathogens in controlling pest insects is countered by a lack of basic ecological understanding on aspects such as their distribution in natural habitats (Meyling et al., 2009;Roy et al., 2009Roy et al., , 2010Vega et al., 2009;Hesketh et al., 2010). Despite the potential economic importance of Beauveria, research into its fundamental ecology is scarce and its phylogeny and taxonomy remain debatable (Rehner & Buckley, 2005;Rehner et al., 2006). Indeed, it is appropriate to consider Beauveria bassiana in the broadest sense as 'B. bassiana sensu lato' (Rehner & Buckley, 2005;Rehner et al., 2006); here, we will refer to B. bassiana as a taxonomical entity (morphospecies), but with the understanding that Beauveria comprises two distinct clades that cannot be separated morphologically.
It is generally assumed that genetic groups of B. bassiana have coevolved with particular host taxa (Couteaudier & Viaud, 1997;Berretta et al., 1998;Gaitan et al., 2002); yet recent studies suggest that B. bassiana also includes generalist ento-mopathogens (Wang et al., 2003;Rehner & Buckley, 2005). Environmental factors are speculated to determine the patterns of distribution of B. bassiana because particular phylogenetic associations with the insect host appear to be singularly absent (Bidochka et al., 2002;Meyling et al., 2009). Little is known about the community ecology of B. bassiana in natural (or semi-natural) habitats, which would be invaluable in further developing our understanding of the ecology of this fungus. If B. bassiana is widely distributed above ground, then we might expect that there are separate genetic groups of isolates adapted to the different environmental conditions associated with foliage and bark compared with soil. Genotypes could, however, also vary across foliage types as well as between foliage and soil environments. There is a precedence for genotype selection in relation to habitat type, for example, Bidochka et al. (2002) identified distinct genetic groups associated with the soil from three different (forest, agricultural and arctic) Canadian habitats. Genetic groups isolated from Arctic and forested habitats grew at lower temperatures relative to those from the agricultural habitat, which also proved tolerant of UV exposure (Bidochka et al., 2002).
A number of studies have demonstrated the prevalence of B. bassiana on above-ground vegetation (Doberski & Tribe, 1980;Reay et al., 2008), with elm bark representing the first report of B. bassiana on vegetation (Doberski & Tribe, 1980). Posada & Vega (2005) isolated B. bassiana as an endophyte of cocoa seedlings (Theobrama cacao), while  isolated the pathogen from hedgerow vegetation. More recently, Reay et al. (2008) surveyed entomopathogenic fungi from soil, bark and insect frass in New Zealand Pinus radiata forest sites and reported that the density of Beauveria species varied both within and between sites and substrates. These authors identified three Beauveria species using molecular analyses: B. bassiana, Beauveria malawiensis and Beauveria caledonica; the latter was uniquely isolated from several substrates including mycosed bark beetles, soils, bark and insect frass.
The application of modern molecular techniques has identified genetic clades of B. bassiana, and indeed, many other fungal species (Castrillo et al., 2003;Atkins & Clark, 2004;Rehner & Buckley, 2005). DNA-based methods provide insights into fungal ecology that are not possible via morphological characters; indeed, some DNA-based techniques can (1) distinguish individual isolates, (2) monitor and track isolates during laboratory and field studies and (3) assess their distribution, persistence and potential for genetic exchange. Castrillo & Brooks (1998) used RAPD-PCR to demonstrate the genetic proximity between B. bassiana isolates from similar habitats (habitat selection), detecting a variation both between different regions and among isolates collected from the same insect host. Further isolate correlation with the host and geographical origin has been demonstrated using both AFLP-PCR (Aquino de Muro et al., 2005) and inter simple sequence repeat (ISSR)-PCR (Wang et al., 2005;Estrada et al., 2007).
The primary objective of this work was to assess the population ecology of B. bassiana on temporal and spatial scales. We describe a field study conducted between October 2005 and June 2006 to investigate the distribution, density and genetic diversity of B. bassiana from the soil, conifer bark and branches in a forest in south-east England. In addition to providing insights into the spatial and temporal dynamics of B. bassiana, this study tests the sensitivity of the ISSR-PCR technique in identifying genetic groups. Previous works on the genetic diversity of B. bassiana have been conducted on a broad geographical scale comparing isolates from different areas within and between countries (Bidochka et al., 2002;Aquino de Muro et al., 2005;Estrada et al., 2007); thus, a key aim of this study was to focus on the differences between B. bassiana isolates over restricted spatial and temporal scales.
Samples were taken from Douglas fir and Corsican pine on three dates: 15 October 2005, 18 March 2006 and 24 June 2006. The dates were chosen to represent three seasons within Britain: autumn, spring and summer. Twelve samples were taken at each sampling date from four locations (soil, bark, low branch and mid branch) from three trees of a similar size (c. 3 m high), yielding a total of 48 samples from each species at each sampling date and 288 samples across the entire study. Trees were not marked for resampling, but randomly sampled on each visit, and so it is unlikely that the same trees were sampled more than once.
Individual soil samples (approximately 50 g) were collected, using a stainless-steel trowel (alcohol washed between samples), from four randomly selected positions around the base of each tree beneath the moss and humus layer at a depth of 2 cm. The soil was placed in 180 Â 229-mm polyethylene food bags for transport back to the laboratory.
The terminal 5 cm of a branch, with needles, was removed from a lower (o 1 m) and a mid section (o 1.5 m) of the tree using surgical scissors, which were alcohol washed between samples. The terminal section was selected as the most convenient section to remove. 3 Â 2 Â 0.2-cm sections of bark were collected from the trunk 1 m above the soil.
All samples were placed in individual 50-mL disposable plastic tubes (29 Â 115 mm) and stored in the shade for no more than 3 h until return from the field to the laboratory, where they were stored for no more than 6 h at 5 1C before processing.
To isolate B. bassiana from the soil samples, a subsample of 1 g of soil was taken from the field sample and suspended in 9 mL of sterile 0.03% Tween 80 (Fisher Scientific, Loughborough, UK) and vortexed for 1 min. Triplicate 100-mL aliquots of this dilution were streaked out onto oatmeal dodine agar (ODA; Chase et al., 1986). The plates were placed in a dark (nonilluminated) incubator at 22 1C and the number of CFUs was recorded after 1 week. To isolate B. bassiana from the tree samples, the above process was repeated with a 2-cm section of distal branch or 0.3 g of bark (approximately 1/4th of the field sample). Bark weight was used as opposed to a surface area because the bark sample comprised both the inner and the outer bark.

Molecular characterization
The isolates for molecular characterization were obtained by randomly selecting CFUs (one per plate) from 144 of the initial 288 ODA plates (ensuring, where possible, equal numbers of each sample type, although CFUs were not present in all samples). Each CFU was streaked out onto a second Population dynamics of a fungal entomopathogen ODA plate and placed in a dark incubator at 22 1C for 8 days. A CFU (assumed to be single conidium isolate), again randomly selected from each plate, was placed on a fresh ODA plate and maintained in a dark incubator at 22 1C for 2-4 weeks. This incubation regime yielded sufficient fungal growth for DNA extraction from a total of 144 isolates.
DNA was extracted by suspending fungal material scraped with a sterilized microbiological loop from a sporulating single conidium isolate in 100 mL of 0.03% Tween 80. The DNA was then extracted using a DNEasy Tissue Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions.
Screening for molecular diversity among the isolates was via ISSR-PCR using one of four primers (Table 1) in each reaction. Each 20 mL PCR comprised 2 mL buffer [750 mM Tris-HCl (pH 8.8 at 25 1C), 200 mM (NH 4 ) 2 SO 4 ], 2 mL 100 mM dNTPs (ABgene, UK), 4.7 mL sterile-distilled water, 4 mL primer, 4 mL Q solution (Qiagen), 0.1 mL Taq polymerase (Qiagen) and 2 mL DNA. PCR was performed on a TC-412 thermocycler (Techne). The PCR programme for primer 6 was composed of an initial 3 min at 94 1C, followed by 35 cycles at 94 1C for 1 min, 63 1C for 2 min and 72 1C for 6 min, and finalized by 7 min at 72 1C. The PCR programmes for primers 7, I and D were identical, except for the annealing temperatures, which were 52, 47.8 and 58 1C, respectively. The PCR programmes were run using a manual hot start: 1.2 mL of MgCl 2 (25 mM) was added to each PCR when the reaction temperature reached 94 1C. Amplified PCR products were run on a 2% agarose gel, containing 1 mL of ethidium bromide (25 mg mL À1 ), and photographed in UV light using a Canon digital camera. Reproducibility was established for each primer using a sample of 20 isolates before full screening was undertaken.

Data analysis
The statistical software R (version 2.8.1) (www.r-project.org) was used for the generalized linear models (GLM). GLMs were used to analyse (1) the frequency of B. bassiana across all positions (soil, bark, low and mid branches), (2) the CFU density data obtained for soil and bark samples and (3) the CFU count data for branch samples (mid and low positions). The values for bark and soil were compared by converting the number of CFUs to density per gram of substrate. Samples from branches at low and mid locations were directly comparable as number of CFUs per 2 cm of branch. Full models with all interactions were fitted for both data sets and nonsignificant factors were eliminated successively until a best-fit model was achieved. The quasibinomial function was used for the frequency of occurrence of B. bassiana and the quasi-Poisson function was used for the CFU density and count data. GLM-derived coefficients were compared using ANOVA.
Gels were scored manually for band size and the resulting binary (presence/absence of amplified bands) data matrices were analysed using the MULTIVARIATE STATISTICAL PACKAGE (MVSP) (Kovach, 1999). Our original primary MS 'EXCEL' spreadsheet defined the data, in terms of gel bands, via the five discrete parameters of 'tree' (Corsican pine/Douglas fir), 'date' (October/March/June), 'substrate' (bark/low foliage/ mid foliage/soil), 'aspect' (north/east/south/west) and 'replicate' (1-6). The primary dataset was totalled by 'band', and all '0'-scoring bands were removed. The residual band data were analysed in MVSP via principal components analysis (PCA), which applied Kaiser's rule (Legendre & Legendre, 1983) to transposed and centred data under default 'advanced settings' with scatter plots shown as variable loadings.

Cluster analysis
Gel band matrices were further analysed using FREE TREE (Hampl et al., 2001), where 10 000 bootstrap replicates of trees generated via the Jaccard coefficient and neighbourjoining algorithms were produced for each of the four ISSR primers, and then pooled to produce a tree using the programme TREEVIEW (Page, 1996).

Spatiotemporal distribution of B. bassiana
Beauveria bassiana was isolated from the soil and foliage of both conifer species (Table 2) Douglas fir mean ( AE SE) B. bassiana CFU densities ranged from 32.6 ( AE 4.5) to 148.9 ( AE 15.5) per gram of soil and 1.7 ( AE 0.8) to 28.8 ( AE 14.8) per gram of bark. Douglas fir mean ( AE SE) CFU number ranged from 0.0 ( AE 0.0) to 42.3 ( AE 9.9) per 2 cm low branch and from 0.0 ( AE 0.0) to 10.8 ( AE 3.8) per 2 cm mid branch.
The density of B. bassiana on the two tree species was similar throughout the study ( Fig. 1a and b). There was, however, a significant difference between sampling periods, with a pronounced decline in density in June compared with the previous months (F 2, 140 = 26.0; P o 0.001; Fig. 1a and b). The fungal density was higher in the soil compared with the bark (F 1, 142 = 33.2; P o 0.001; Fig. 1a and b). There was a significant interaction between tree species and sampling date (F 2, 138 = 10.0; P o 0.001; Fig. 1a and b), which relates to the more rapid decline in all March samples, with the exception of the soil samples taken from the base of the Corsican pine in which the fungal density was similar in October and March samples.
There was a significant difference in the number of CFUs of B. bassiana per 2 cm of branch on the different sampling dates (F 1, 141 = 83.6; P o 0.05; Fig. 2a and b). There was also a significant interaction between tree species and branch position on the tree (low or mid) (F 1, 138 = 30.2; P o 0.001; Fig. 2a and b). The fungus could only be isolated from  branches in October and on the Corsican pine, the mid branch had a higher number of CFUs than the low branch, and on the Douglas fir, the pattern was reversed.

Molecular characterization
One hundred and fifty-eight polymorphic ISSR bands were scored for presence/absence across the 144 isolates. Initial PCA analysis of the complete data extracted 78% of potential variance from 32 axes. This, together with the random scatter plot derived from the 19.1% variance extracted by the first two axes, suggests a substantial interaction between the five sample parameters of tree species, sample date, substrate, aspect and replicate. To overcome this problem of parameter interaction, we sorted the data into the 10 pairedparameter combination data sets and again removed all '0'scoring bands. PCAs of the residual scoring bands identified four significant groupings that extracted substantially more variance from fewer axes (Table 3). One implies that tree species is significant and yet three (e.g. Figs 3 and 4) identify sample month as significant in terms of consistent 'March', 'June', 'October' PCA groupings, but no clearly defined complementary signal in either 'aspect' (Fig. 3) or 'tree' (Fig. 4). This suggests the presence of potentially genetically distinct isolates on different trees, although more convincingly in summer (June) compared with autumn (October) and winter (March). The cluster analysis indicated variability among the 144 isolates (Fig. 5) that were habitat specific (38% soil, 31% foliage and 31% bark isolated). Each habitat type included isolates from both tree species.

Discussion
Our isolation of B. bassiana from conifer branches and bark samples confirms the few records of this fungus on foliar material (Doberski & Tribe, 1980;Reay et al., 2008;Meyling et al., 2009). Despite the frequent records of B. bassiana from both soil (Bidochka et al., 1998;Glare & Inwood, 1998;Sun & Liu, 2008) and insect hosts (Lipa et al., 1975;Gaitan et al., 2002), little is known about its wider occurrence in terrestrial ecosystems and especially on foliage (Reay et al., 2008). All locations sampled (above and below ground) yielded B. bassiana, but the density of this fungus was greater in the soil than any of the tree samples and the greatest in October.  also found that B. bassiana was more abundant in September (autumn) compared with May and July (summer) samples.  The spatial and temporal dynamics of B. bassiana within an ecosystem are dependent on both abiotic and biotic factors. Furthermore, the movement of B. bassiana inoculum could follow a number of ecosystem pathways Hesketh et al., 2010) including dispersal via air currents and insect hosts (Hajek, 1997) or rain splash from the soil to above-ground vegetation (Bruck & Lewis, 2002a). The latter is possibly confirmed by the observed decrease in conidia density on branches at high locations. However, the movement of host insects can be important in the dispersal of this fungus.  demonstrated the ability of nettle insects (Anthocoris nemorum) to disperse the conidia of B. bassiana from soil or sporulating cadavers into the nettle canopy. The ability of sap beetles (Coleoptera: Nitidulidae) to carry a specific strain of B. bassiana to overwintering sites has also been demonstrated (Bruck & Lewis, 2002b;Dowd & Vega, 2003). Host insects could also play a role in dispersing the fungus from the soil to the trees, and indeed between trees, within this conifer forest. Many insects are present in this conifer system throughout the year, with ladybirds (Coleoptera: Coccinellidae) being particularly abundant in this system, and B. bassiana is a major mortality factor of the seven-spot ladybird, Coccinella septempunctata L. (Majerus, 1994;Cottrell & Shapiro-Ilan, 2003;Roy et al., 2008). Sevenspot ladybirds, C. septempunctata, overwinter in the soil, moving into the trees in the spring (Majerus, 1994). Sevenspot ladybirds have been shown to move an obligate aphid fungal pathogen between aphid colonies (Roy et al., 2001). It is feasible that the dispersal of B. bassiana is facilitated through the movement of this and other insect hosts, although this requires further study.
We observed clear temporal trends in both tree and soil data with decreasing conidial density (more so in trees than in soil) from October to June. Our molecular data show separate clustering of October, March and June isolates, suggesting seasonal genetic differentiation. The fungal communities of plant surfaces are exposed to different, and more variable, environmental factors, such as UV flux, desiccation and thermal regime, than those in the soil. The detrimental effects of UV light on the conidial survival of entomopathogenic fungi are well established Morley-Davies et al., 1996;Fargues et al., 1997), and moisture content is thought to be the principal underlying cause of decreasing B. bassiana density in the soil (Sandhu et al., 1993) because B. bassiana conidia require high levels of humidity to germinate. Although summer temperatures are favourable for conidium production, it is likely that conidium germination and hyphal growth are limited by suboptimal humidity.
This study has shown that the DNA profiling technique ISSR-PCR is a powerful tool for differentiating between B. bassiana isolates collected from a single ecosystem.
Previous work has shown that B. bassiana isolates cluster at the geographic scale of a country (Wang et al., 2003;Aquino de Muro et al., 2005); here, we report on isolates clustering within a single ecosystem. This confirms that B. bassiana isolates correlate with spatial and temporal factors, with some being ubiquitous within the habitat throughout the year and others restricted to certain locations or time periods. This could reflect the facultative nature of B. bassiana as an insect pathogen, with survival driven primarily by adaptation to habitat factors. Furthermore, the simultaneous occurrence of some isolates in the soil and on trees suggests that the pathogen is moving between above-and below-ground environments. Meyling et al. (2009) found similar results in a study on an arable field/ hedgerow agroecosystem and, as here, noted (1) considerable genetic diversity among isolates and (2) both ubiquitous and habitat (field vs. hedgerow)-specific isolates. The results presented in this paper indicate that fluctuating environmental factors within a single habitat can drive genetic differentiation from both a spatial (above and below ground) and a temporal perspective as suggested by Bidochka et al. (2002).
The results of this investigation contradict the conventional opinion that B. bassiana is principally a soil fungus; identification of isolates above and below ground suggests that this taxon occupies various overlapping niches. Meyling et al. (2009) propose a metapopulation structure for fungi occupying an agroecosystem and we envisage that the B. bassiana conifer forest discussed here represents a metapopulation in which local extinction and colonization are driven by the presence of suitable hosts, favourable abiotic conditions and dispersal events.