Spatial and taxonomical overlap of fungi on phylloplanes and invasive alien ladybirds with fungal infections in tree crowns of urban green spaces

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1 FEMS Microbiology Ecology, 92, 2016, fiw143 doi: /femsec/fiw143 Advance Access Publication Date: 27 June 2016 Research Article RESEARCH ARTICLE Spatial and taxonomical overlap of fungi on phylloplanes and invasive alien ladybirds with fungal infections in tree crowns of urban green spaces Andy G. Howe 1,,, Hans Peter Ravn 1, Annette B. Jensen 2 and Nicolai V. Meyling 2 1 Department of Geosciences and Natural Resource Management, University of Copenhagen, Rolighedsvej 23, DK-1958 Frederiksberg C, Denmark and 2 Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Corresponding author: Forest, Nature and Biomass, Kobenhavns Universitet Institut for Geovidenskab og Naturforvaltning, Rolighedsvej 23, DK-1350 Frederiksberg C, Denmark. Tel: ; andy@ign.ku.dk One sentence summary: Leaves of urban trees harbour entomopathogenic fungi up to 13 m in tree crowns and act as a reservoir of fungal infection in invasive alien and native ladybirds. Editor: Ian Anderson Andy G. Howe, ABSTRACT Occurrence of entomopathogenic fungi on phylloplanes in Tilia europaea crowns between 1 and 13 m was assessed in urban parks. Prevalence of fungal infections in ladybirds (Coleoptera: Coccinellidae) collected from Tilia europaea was assessed to determine whether fungi found on phylloplanes also occurred as infections in ladybirds. Isaria spp. was most abundant on phylloplanes (mean colony forming units (CFU) per leaf ± SE, 0.33 ± 0.03) followed by Beauveria spp. (0.22 ± 0.02 CFU per leaf) and Lecanicillium spp. (0.19 ± 0.02 CFU per leaf). Densities of inoculum were higher in inner crowns and decreased with height, although Lecanicillium spp. peaked at 5 7 m. Upper phylloplane surfaces harboured higher densities of Isaria spp. and Beauveria spp. than lower surfaces, whereas Lecanicillium spp. was equally distributed. Most prevalent on ladybirds were Isaria spp. (20.6% Harmonia axyridis; 4.8% natives), Lecanicillium spp. (13.6% H. axyridis; 4.8% natives), with fewer Beauveria spp. infections (2.6% H. axyridis). Molecular identification revealed Beauveria bassiana, B. pseudobassiana, Isaria farinosa and Lecanicillium muscarium among isolates of both tree and ladybird origin. Tilia europaea phylloplanes support a diverse assemblage of entomopathogenic fungal species with a different prevalence in coccinellids compared to their relative abundance in this habitat. Keywords: Phylloplane imprinting; Beauveria; Isaria farinosa; Lecanicillium; urban parks; Harmonia axyridis INTRODUCTION Entomopathogenic fungi are widespread community members of most terrestrial ecosystems below- and aboveground (Lacey et al. 2001; Hajek 2004). In contrast to comprehensive knowledge of entomopathogenic fungi and insect interactions in agricultural systems, limited understanding exists on the effects of naturally occurring pathogens on non-pest species and their ecology in other ecosystems, e.g. semi-natural habitats (Roy et al. 2009; Hesketh et al. 2010). Recent studies from agricultural and semi-natural systems offer valuable insights into Received: 11 April 2016; Accepted: 20 June 2016 C FEMS All rights reserved. For permissions, please journals.permissions@oup.com 1

2 2 FEMS Microbiology Ecology, 2016, Vol. 92, No. 9 aboveground fungal spatial distribution and dynamic pathways into foliar habitats (Hesketh et al. 2010; Meyling et al. 2011; Schneider et al. 2012; Garrido-Jurado et al. 2015). For example, Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) has been isolated from conifer needles up to 1.5 m (Ormond et al. 2010), while aerial deposition and/or insect activity may be responsiblefor B. bassiana isolated from phylloplanes on vegetation up to 2 m in hedgerows (Meyling and Eilenberg 2006; Meyling et al. 2006). Entomopathogenic fungi are recognised as important natural enemies of ladybird beetles (Coleoptera: Coccinellidae) (Roy and Cottrell 2008; Ceryngier et al. 2012). For example, B. bassiana, Isaria farinosa Holmsk and Lecanicillium lecanii (Zimmerman) Zare and Gams (Ascomycota: Hypocreales) contribute to overwintering mortality among adult seven-spot ladybirds Coccinella septempunctata L. (Ceryngier 2000; Ormond et al. 2006). However, the role of entomopathogenic fungi on coccinellid population dynamics beyond agroecosystems remains largely unknown (Roy and Cottrell 2008). Given the context of rapid environmental change associated with habitat degradation, climate change and invasive alien species, knowledge on coccinellid fungi interactions in semi-natural habitat is pertinent (Roy et al. 2009; Hesketh et al. 2010). For example, whether environmental changes potentially alter the impact of entomopathogenic fungi on ecosystem services provided by ladybirds is largely unknown. Intriguingly, the invasive harlequin ladybird Harmonia axyridis Pallas (Coleoptera: Coccinellidae), with origins in Asia, is less susceptible to B. bassiana compared to several native coccinellids in the USA and UK (Cottrell and Shapiro-Ilan 2008; Roy et al. 2008). Although B. bassiana is reported to infect H. axyridis in the species native range (Kuznetsov 1997), reduced susceptibility to the fungus in its introduced range may indicate that H. axyridis benefits to some extent from natural enemy release (Roy et al. 2011). However, entomopathogenic fungi can exploit H. axyridis; one year subsequent to invasion of Denmark, hypocrealean fungi including I. farinosa, L. muscarium (Petch) Zare and Gams, L. lecanii and B. bassiana were isolated from various life stages of this coccinellid collected in late autumn and summer (Steenberg and Harding 2009, 2010). There is, however, a paucity of studies on the extent of spatial niche overlap between entomopathogenic fungi and arboreal coccinellids in semi-natural habitats. In Europe, H. axyridis occupies an array of ecosystems, with particularly dense populations occurring in anthropogenic habitats including urban green spaces, often on lime trees Tilia spp. Malvaceae (Adriaens et al. 2008; Brown et al. 2008; Howe et al. 2016). Few studies have focused on fungal ecology in urban ecosystems (reviewed by Newbound et al. 2010), and investigated potential for interactions between entomopathogenic fungi and coccinellids in these environments (Welch et al. 2001; Steenberg and Harding 2009). Urban habitats are exposed to a disproportionately high number of invasive alien species through pathways associated with trade (van Ham et al. 2013), and can act as sites of establishment for invasive alien species prior to dispersal (Rebele 1994; Alberti 2005). As such, urban environments (in Europe) represent valuable systems for the study of community dynamic effects involving invasive alien species, naturally occurring entomopathogenic fungi and the wider insect community. We assessed the abundance and distribution of fungi of genera with entomopathogenic members on Tilia europaea phylloplanes in order to illuminate whether urban parks represent potential sites of infection for arboreal invasive alien and native coccinellids. The spatial distribution and density of potentially entomopathogenic fungi were achieved using a phylloplane imprinting technique combined with morphological and molecular identification. Furthermore, the prevalence and identity of fungi infecting arboreal invasive alien and native coccinellids co-occurring in the same habitat were evaluated. Although no direct infection experiments were included in this study, these observations are expected to indicate whether fungal occurrence in trees reflects fungal infections in ladybirds. MATERIALS AND METHODS Leaf and bark collection Sampling took place in five recreational parks in Copenhagen, Denmark, over five consecutive days from July 30 to August 3, 2012, in the following order: Ørstedsparken ( N, E), Churchilparken ( N, E), Østre Anlæg ( N, E), Genforeningspladsen ( N, E) and Nørrebroparken ( N, E). In each park, two mature Tilia europaea (15 25 m) were sampled between 9:00 14:00 under sunny, dry conditions (15 C 24 C, RH 70% 80%, wind <5 m s 1 ). Distances between parks were km, and within parks trees were m apart. Parks contained a variety of native and introduced species of trees, shrubs, herbs and flowers, and open recreational spaces with lawns and paths. Access to canopies was attained using single rope technique (SRT), whereby a single rope is positioned in a suitable crotch in a tree and one end is tethered at the base of the tree trunk. The climber ascends the untethered rope end using an ascender (Petzl) supported by a belay device (Grigri). The rope is static in that it does not move over tree limbs during the climb, resulting in minimal friction against bark, eliminating injury to trees, while providing reasonable access within tree crowns. Leaves were sampled at three strata within each tree crown: low (1 2 m), mid (5 6 m), high (10 13 m). At each stratum, leaves were sampled from two crown positions, namely the inner ( 1 m from trunk) and outer ( 1 m from branch tips, maximum 4 m from trunk) crown (Fig. 1). These distances were determined by tree structures and accessibility by SRT. Sampling from low-tohigh and inner-to-outer crown positions ensured minimum disturbance to samples in the upper crown. Fifteen leaves from three to five branches were sampled at each crown position (inner/outer), from each stratum (i.e. 90 leaves per tree, 900 leaves sampled in total). Leaves collected at the low stratum were held at the base of petioles by forceps and cut with scissors and dropped directly into separate clear polyethylene bags (15 35 cm, one leaf per bag). At the mid and high strata, branches were cut either from thegroundusingclippersona4mtelescopepolewheretree structure allowed (mid stratum), or by a single climber in the tree (mid, high strata). Branches (<1 m) from within the canopy were either dropped to collectors on the ground when there was a clear passage through underlying branches or lowered in polyethylene bags (25 50 cm, branches cut to fit) to minimise contact with other branches. Individual leaves were then detached from branches and transferred to polyethylene bags as above. Utensils were rinsed in 70% ethanol and water between heights. Samples were kept in the shade for a maximum of 5 h prior to returning to the laboratory, where leaves were kept at room temperature for no more than 3 h prior to processing. Composite bark samples comprised of three cm sections of trunk were chiselled directly into a single

3 Howe et al. 3 Figure 1. Overview of sampling regime and distribution of entomopathogenic fungi isolated from Tilia europaea and coccinellids (Coleoptera: Coccinellidae) and identified using molecular analysis. Leaves and bark were sampled from three strata within tree crowns: low (1 2 m), mid (5 6 m) and high (10 13 m). Leaves were sampled from inner ( 1 m from trunk) and outer ( 1 m from branch tips, max. 4 m from trunk) crowns. Coccinellids were sampled <3 m from both inner and outer tree crowns. Morphological identification revealed Beauveria spp., Isaria spp. and Lecanicillium spp. on leaves, at all sampled positions, including both phylloplane surfaces (lower/upper) not depicted here. polyethylene zip-lock bag at each stratum from all trees (three composite samples per tree, 30 in total). Bark samples were kept at 4 C in the dark and processed 3 weeks after the last sampling date. Within each tree crown, samples were collected to ensure that a variety of aspects were represented (i.e. leaves from N, S, E and W facing crowns of trees). Isolation of filamentous fungi from phylloplanes and bark A growth medium selective for Beauveria followed that described by Meyling and Eilenberg (2006), with slight modification by adjusting the ph of the agar medium to 6.3 with 1 M HCl prior to autoclaving, and then addition of 2.5 ml of g ml 1 cyclohexamide (Sigma-Aldrich). Leaf petioles were held with sterile plastic forceps when transferred from collection bags to the selective medium in 90 mm diameter triple vented Petri dishes. The lower (abaxial) phylloplane was covered with a thin sheet of wax paper cmandrolled10timeswithapaint roller (35 mm diameter) modified to fit Petri dishes. The paper was then discarded, the leaf inverted with sterilised forceps and the upper (adaxial) phylloplane was processed identically in a new Petri dish. A few of the collected leaves were slightly larger than the Petri dish; leaf margins which overhung dishes were not rolled in the medium. Leaf samples were processed before 17:00 on the day of collection. To isolate fungi from bark samples, we used the method described by Ormond et al. (2010). In short, samples were crushed to ensure a relatively homogenous mix, before 0.3 g was transferred to a test tube with 9 ml of 0.05% sterile Triton X (Sigma- Aldrich). After vortexing for 1 min, 100 μl aliquots were plated onto selective medium in triplicate 90 mm diameter triple vented Petri dishes. In total, 23 control dishes were left open during leaf/bark processing (4 h) in the laboratory to detect the presence of fungal inoculum in the air. A single Isaria farinosa colony forming unit (CFU) was found on a control plate (exposed during phylloplane imprinting; August 2), but deemed unlikely to bias results. The plates were placed in a dark room (23 C), checked on several occasions for signs of emerging CFUs, prior to counting of all CFUs following 3 and 9 weeks incubation for leaf and bark, respectively. Sporulating colonies were identified by morphology under a light microscope (400 magnification) after Humber (2012) and Zare and Gams (2008). A selection of fungal CFUs representing isolates from phylloplanes in different parks and crown strata were isolated on Sabouraud Dextrose Agar (SDA) for storage at 80 C in the culture collection of entomopathogenic fungi at Department of Plant and Environmental Sciences, University of Copenhagen (Table S1, Supporting Information). Collection of coccinellids Coccinellids were collected from Tilia europaea at 15 locations (parks and roadsides) within the inner city area of Copenhagen (Table S2, Supporting Information). Collections took place on sunny days throughout June 2012 (6, 11, 13, 14 and 27) and

4 4 FEMS Microbiology Ecology, 2016, Vol. 92, No. 9 July 2012 (2, 3, 4, 27 and 29). This period was chosen to coincide with first generation larvae/adult Harmonia axyridis and available native cocinellids. As fungi were isolated from phylloplanes immediately after coccinellid collection ceased, phylloplane imprinting is expected to reflect the potential pathogen reservoir of ladybird populations towards the end of the coccinellid sampling period. Insects were collected from T. x europaea up to 3 m by either (1) using a beating tray to dislodge insects from branches before transfer with forceps to 30 ml containers (forceps sterilised with 70% ethanol and water between each insect) or (2) by directly catching individual insects into the containers (insects were not touched). The bottom of containers consisted of approximately 5 mm sterilised 1.5% water agar. Coccinellids were stored for up to an hour in the shade prior to return to the laboratory. Coccinellid larvae other than H. axyridis were not identified to species level, but were assigned as native; native adults were identified to species level following Majerus and Kearns (1989), without opening containers. Coccinellids were stored at ambient temperature in a laboratory (22 C ± 3 C, RH 50% ± 25%, natural light, but not direct sunlight) without food and checked over 3 months for life stage at death and fungal emergence; most coccinellids died within 2 5 weeks. Due to logistic limitations, no supplementary moisture other than the aforementioned water agar solution or food was provided; laboratory reared coccinellids were not stored as mortality controls. Fungi on cadavers were identified following Humber (2012) and selected isolates were obtained on SDA for storage at 80 C. Molecular identification Selected fungal isolates originating from leaves, bark and insects were grown on SDA in darkness in an incubator for 2 4 weeks (23 C). DNA extraction entailed suspending scraped (with sterile inoculation loops) fungal material into sterile flasks containing liquid growth medium (2% peptone, 3% sucrose and 0.2% yeast extract) and incubating at room temperature on a shaker (170 rpm) for 3 days. Fungal material was then filtered under suction, transferred to 20 C for 24 h, prior to freeze-drying at 80 Cfor a further 24 h. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Denmark) following the manufacturer s instructions. Two regions of DNA were amplified by polymerase chain reactions (PCR): (1) the internal transcribed spacer ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster was amplified using the primer pair ITS5/ ITS4 (White et al. 1990) and (2) the 5 end of the translation elongation factor 1-alpha (5 -TEF) region was amplified using the primer pair EF2F/EFjR (Rehner and Buckley 2005; Meyling et al. 2012). The ITS region was amplified for all isolates, whereas 5 -TEF was only amplified for Beauveria spp. PCR conditions and procedures followed Meyling et al. (2012). PCR products were purified with illustra GFX PCR DNA and Gel Band Purification Kit (GE healthcare, UK) following the manufacturer s instructions, prior to evaporation and subsequent sequencing with the aforementioned primers by MWG (Ebersberg, Germany). Sequences were assembled using Geneious version 5.0 (Biomatters) and aligned with reference sequences from authoritative taxonomic literature in GenBank using ClustalW (Zare et al. 2000; Luangsa-ard et al. 2005; Rehner and Buckley 2005; Zare and Gams 2008; Meyling et al. 2009, 2012; Rehner et al. 2011; Table S3, Supporting Information). Sequence alignments for ITS of Isaria spp. and Lecanicillium spp., and for 5 - TEF of Beauveria spp., were analysed in two separate analyses by neighbour joining (NJ) in MEGA6 (Tamura et al. 2013) by complete gap deletion with a maximum composite likelihood model, resampled with 1000 bootstrapped replicates. The two sequence alignments were then analysed separately in Topali v2.5 (Milne et al. 2009), initially using ModelTest to determine the optimal DNA substitution model (Milne et al. 2009). The two phylogenies were then inferred with maximum likelihood (ML) estimation using substitution models GTR+G for the ITS sequence alignment and TrNef+I+G forthe5 -TEF alignment in Topali, both with 1000 bootstrapped replicates. The resulting dendrograms showed comparable topologies with the NJ trees obtained which were therefore used to depict branch support of both analyses. Representative sequences were submitted to GenBank (NCBI) and deposited under accession numbers KX KX (Table S1, Supporting Information). Statistical analysis The densities of CFUs on selective media were analysed for each fungal genus separately as prior analysis of the complete dataset revealed a significant effect of genera. CFU densities were modelled with zero-inflated generalized linear mixed models (GLMM) with negative binomial distribution (variance = μ(1 + μ/k) forisaria spp. and Beauveria spp.; variance = øμ for Lecanicillium spp.) as visualisation of raw data indicated consistent zero inflation. For phylloplanes, we analysed the fixed explanatory effects of stratum (low, mid, high), position in crown (inner, outer) and phylloplane surface (upper, lower), whereas only stratum was analysed for bark samples; interactions between fixed effects were not included. On six leaves, the number of CFUs exceeded 20 (Beauveria spp.: 24, Isaria spp.: 59, 72, 98, 165, Lecanicillium spp.: 32). These outliers were changed to 20 (other high CFU counts ranged from 16 to 19) and models with and without outliers were compared, resulting in similar trends. We chose to include amended values in order to reduce model overdispersion, and zero inflation (instead of omitting these outliers). The distribution of fungi was assessed using frequencies of leaves containing target CFUs and analysed by logistic regression with fixed effects as for CFU density GLMMs. In all GLMMs, random effects were park location and tree at a given location, accounting for heterogeneity between sampling locations. Model reduction was based on backwards elimination with a 5% cut-off level, where P-values correspond to likelihood ratio (LR) tests of the effect of a variable in the presence of other variables present in the model, and is appropriate when ratios of total sample size to number of fixed effects levels and random effects levels are large (Bolker et al. 2009). Statistical analysis was implemented using R version (R Core Team 2013). GLMMs for CFU densities were fitted using the glmmadmb package (Skaug et al. 2013), GLMMs for proportion data using the lme4 package (Bates et al. 2014, 2015). Post hoc pairwise comparisons (adjusted for multiple testing with the single-step method) using the multcomp package (Hothorn et al. 2008) assessed differences between variables for bark and proportion of CFUs, as well as differences between taxa densities on leaves and bark. Frequencies of occurrence by fungi genera on coccinellids were compared using standard χ 2 tests. Figures were produced with ggplot2 (Wickham 2009). RESULTS Fungi in Tilia europaea crowns In total, 1800 phylloplanes were imprinted from 900 Tilia europaea leaves. Filamentous fungi were isolated from 988 of these phylloplanes, of which 482 harboured fungal genera known to

5 Howe et al. 5 Table 1. Results from best GLMMs (negative binomial) examining effects of leaf position in crown (inner/outer), stratum in crown (low, mid, high) and leaf surface (upper, lower) on density of CFU isolated from leaves and bark (stratum only) of Tilia europaea. CFUs isolated from leaves CFUs isolated from bark Genus Variable Estimate SE Pr(< Z ) Estimate SE Pr(< Z ) Beauveria Intercept < Position (outer) <0.001 Stratum (mid) < Stratum (high) < Leaf (upper) <0.01 Isaria Intercept < Position (outer) <0.001 Stratum (mid) Stratum (high) < Leaf (upper) <0.001 Lecanicillium Intercept <0.01 Position (outer) <0.001 Stratum (mid) Stratum (high) <0.001 contain entomopathogenic species: Beauveria spp., Isaria spp. and Lecanicillium spp. (henceforth referred to by genus, unless species name is given). All three genera co-occurred on 16 phylloplanes (3 lower, 13 upper), two genera were found to co-occur on 123 surfaces and single genera were isolated from 343 surfaces. The number of isolated CFUs on leaves (including outliers, hence n = 1800) ranged from 0 to 24 for Beauveria (mean ± SE: 0.22 ± 0.02), 0 to 165 for Isaria (0.33 ± 0.03) and 0 to 32 for Lecanicillium (0.19 ± 0.02). Marginally more Isaria CFUs (mainly Isaria farinosa) than Beauveria were isolated from phylloplanes (pairwise comparison: z = 2.30, P = 0.052), similar densities of Beauveria and Lecanicillium (pairwise comparison: z = 1.52, P = 0.27), but significantly fewer Lecanicilliumthan Isaria (pairwise comparison: z = 2.69, P = 0.018). From 90 bark samples (i.e replicates), the aforementioned three fungal genera cooccurred in 5 samples, while two genera were isolated together in 40 samples. Single genera were obtained from 28 samples, whereas 17 samples were void of any of these focal fungi (other unidentified filamentous fungi grew in 27 samples). The mean ± SE number of CFUs recovered from aliquots of 0.3 g of bark ranged for Beauveria from 0 to 203 (n = 90: 9.05 ± 2.92), Isaria 0 to 489 (41.2 ± 11.55) and from 0 to 4 (0.29 ± 0.09) for Lecanicillium. Similar numbers of Isaria and Beauveria CFUs were isolated (pairwise comparison: z = 2.14, P = 0.08), while significantly fewer Lecanicillium CFUs were isolated (pairwise comparisons: Beauveria:z= 6.58, P < 0.001; Isaria:z= 6.72, P < 0.001). Factors affecting density and distribution of fungi Leaf position in tree crown, stratum in crown and phylloplane surface contributed to explaining CFU densities in tree crowns (Table 1). Consistently, fewer CFUs were isolated from leaves collected in the outer canopy (Fig. 2), echoing a very strong effect of leaf position in crown in all models (LR tests,beauveria: P < 0.001; Isaria: P < 0.001; Lecanicillium: P < 0.001). A significant effect of stratum on CFU density was detected in each model (LR tests, Beauveria: P < 0.001; Isaria: P < 0.01; Lecanicillium: P < 0.001), reflecting a decreasing number of CFUs on leaves with increasing height for Beauveria and Isaria, whereas Lecanicillium CFU densities peaked on leaves between 5 and 7 m (Table 1,Fig.2). A significant effect of phylloplane surface was detected for Beauveria and Isaria, but not Lecanicillium (LR tests, Beauveria: P < 0.01; Isaria: P < 0.001; Lecanicillium: P = 0.8), revealing consistently higher CFU densities on upper phylloplane surfaces for Beauveria and Isaria, but no difference for Lecanicillium (Fig. 2, Table1). Logistic regression revealed a significant effect of leaf position in crown on the proportion of leaves with CFUs for all genera (LR tests, Beauveria: P < 0.001; Isaria: P < 0.001; Lecanicillium: P < 0.001; Table S4, Supporting Information), reflecting consistently higher proportions of leaves with fungi in the inner canopy (Fig. 3; Table S5, Supporting Information). There were significant effects of stratum in all models, although frequencies of occurrence differed between species (LR tests, Beauveria: P < 0.001; Isaria: P < 0.001; Lecanicillium: P < 0.001; Tables S4 and S5, Supporting Information). The lowest proportion of leaves containing Isaria and Lecanicillium CFUs occurred in the high stratum (10 13 m), whereas higher proportions were found at low and mid strata. In contrast, Beauveria CFUs occurred most frequently at 1 2 m, and decreased in the strata above, although the frequency of leaves with CFUs was similar at 5 7 m and m(fig.3; Table S5, Supporting Information). The proportion of leaves with Beauveria and Isaria CFUs was higher on upper than on lower phylloplane surfaces, whereas frequencies of Lecanicillium were similar on both surfaces (LR tests, Beauveria: P = 0.04; Isaria: P < 0.001; Lecanicillium: P = 0.73; Fig. 3, TablesS4andS5, Supporting Information). Analysis of the number of CFUs per 0.3 g of bark revealed a significant effect of stratum for Beauveria (LR test: P < 0.01) and Isaria (LR test: P < 0.001; Table 1). The greatest number of Beauveria CFUs was isolated from bark at 5 7 m, while densities at low and high strata were lower (Fig. 4). In contrast, fewest Isaria were recovered at 5 7 m, while CFU densities of this genus were highest at low and high strata. Curiously, very few Lecanicillium CFUs were isolated from bark which precluded analysis (Fig. 4). Fungal occurrence on coccinellids Seven hundred and ninety-six Harmonia axyridis were collected of which 330 cadavers (41.5%) showed signs of infection by fungi. In comparison, of the 63 natives (predominantly Adalia bipunctata L.) collected, 6 (9.5%) were infected, by Isaria (n = 3) and

6 6 FEMS Microbiology Ecology, 2016, Vol. 92, No. 9 Figure 2. Mean observed densities (+1 SE) of CFU of fungi per leaf surface (lower/upper) at three strata (low: 1 2 m; mid: 5 7 m; high: m) and positions in crowns (inner: 1 m from trunk; outer: 1 m from branch tips) of Tilia europaea (n = 900 leaves). Three genera of fungi are depicted: Beauveria spp., Isaria spp. and Lecanicillium spp. Lecanicillium (n = 3) (Table 2). Among coccinellids, a higher frequency of fungi was detected for H. axyridis compared to combined native species (χ 2 1 = 25.29, P < 0.001). On H. axyridis, the highest proportion of individuals were infected with Isaria (predominantly I. farinosa), although a comparable frequency of Lecanicillium was observed (χ 2 1 = 0.74, P = 0.39). In contrast, the frequency of Beauveria waslessthanbothisaria (χ 2 1 = 10.33, P = 0.001) and Lecanicillium (χ 2 1 = 5.31, P < 0.05) (Table 2). Molecular identification of fungal species Among the 33 fungal isolates identified by morphology as 9 Lecanicillium spp., 12 Isaria spp. and 12 Beauveria spp., characterisation by DNA sequencing revealed significant clusters with five species known to be entomopathogenic (Figs S1 and S2, Supporting Information). Based on NJ and ML analyses of the ITS sequence alignment and authoritative references (Table S3, Supporting Information) with a total of 470 positions, Lecanicillium muscarium was isolated from Tilia europaea leaves at all strata sampled, as well as from larva (including pre-pupa) and adult invasive alien and native coccinellids (Fig. 1; Fig. S1, Supporting Information). Sequences of I. farinosa were found for 10 isolates from tree bark (mid stratum), leaves (all strata) and larvae and adult H. axyridis, whiletwo I. fumosorosea isolates were identified from Tilia europaea phylloplanes (low and mid strata) (Fig. 1; Fig. S1, Supporting Information). Based on NJ and ML analyses of 5 -TEF sequences (total of 721 positions), Beauveria bassiana was identified from tree bark (mid and high strata), leaves (all strata) and a single H. axyridis larva. Beauveria pseudobassiana was identified from a high stratum leaf and H. axyridis larvae (Fig. 1; Fig. S2, Supporting Information). Molecular identification of several samples suggests that entomopathogenic fungi were encountered and infected ladybirds in trees; a number of B. bassiana (leaves, n = 3, all strata; bark,

7 Howe et al. 7 Figure 3. Mean observed frequencies (+1 SE) of leaf surfaces (lower/upper) with CFU at three strata (low: 1 2 m; mid: 5 7 m; high: m) and positions in crowns (inner: 1 m from trunk; outer: 1 m from branch tips) of Tilia europaea (n = 900 leaves). Three genera of fungi are depicted: Beauveria spp., Isaria spp. and Lecanicillium spp. n = 1, high stratum), B. pseudobassiana (leaf, n = 1, high stratum), L. muscarium (leaf, n = 1, mid stratum) and I. farinosa (bark, n = 1, mid stratum) identified from molecular sequences sampled from trees (August 1) and H. axyridis (B. bassiana: fourth instar larva, n = 1; B. pseudobassiana: fourth instar larva, n = 1; L. muscarium: adult, n = 1; I. farinosa: fourth instar larva, n = 1; all coccinellids collected July 27) were collected within a 5-day period, from the same park (Østre Anlæg). DISCUSSION Our results demonstrate that bark and leaves of Tilia europaea in urban green spaces comprise reservoirs of fungal inoculum of taxa of entomopathogenic fungi, of which the density and occurrence within tree crowns differ between fungal taxa. Several studies have revealed the presence of Beauveria inocula on aboveground crop and non-crop vegetation including phylloplanes of hedgerow plants, needles and bark of conifers and Ulmus spp. bark (Doberski and Tribe 1980; Meyling and Eilenberg 2006; Reay et al. 2010; Ormond et al. 2010). Recently, nine entomopathogenic fungi species were isolated from phylloplanes of trees and herbs in different cropping systems in Spain, including arboreal habitats. Co-occurrence of up to four entomopathogenic fungal taxa was recorded on Holm oak (Quercus ilex L., Fagaceae) phylloplanes, with Beauveria bassiana consistently being the dominant community member (Garrido-Jurado et al. 2015). Results from the present study are, however, the first to document inocula of five species of fungi known to be entomopathogenic on bark, leaves and coccinellid hosts in arboreal habitats as high as up to 13 m in Tilia europaea crowns in urban ecosystems. Intriguingly, despite similar densities of Beauveria and Isaria on phylloplanes, both of which were higher than Lecanicillium densities, the proportion of coccinellids infected with

8 8 FEMS Microbiology Ecology, 2016, Vol. 92, No. 9 Figure 4. Mean observed densities (+1 SE) of CFU of fungi isolated from 0.3 g Tilia europaea trunk bark sampled at three strata (low: 1 2 m; mid: 5 7 m; high: m). Three genera of fungi are depicted: Beauveria spp., Isaria spp. and Lecanicillium spp. Bars with different letters above indicate significantly different mean CFU values within each genus; n = 90. Beauveria was significantly less than infections of the two other genera. Moreover, prevalence of Isaria and Lecanicillium on Harmonia axyridis cadavers was similar, even though Lecanicillium was least abundant on phylloplanes and almost absent from trunk bark. This could indicate that Beauveria is less apt at exploiting H. axyridis and native coccinellids as hosts, possibly because these ladybirds have relatively low susceptibility to Beauveria. However, overwintering mortality of Coccinella septempunctata is partly attributed to Beauveria, probably through exposure to fungal inoculum in the leaf litter (Ormond et al. 2006). Moreover, whereas populations of C. septempunctata and Adalia bipunctata in the UK exhibited similar susceptibility to B. bassiana (commercial strain GHA) in laboratory assays, invasive and native H. axyridis were less susceptible (Roy et al. 2008), echoing evidence of reduced susceptibility of invasive H. axyridis compared to a native coccinellid in field and laboratory pathogenicity assays conducted in the USA (Cottrell and Shapiro-Ilan 2003). Roy et al. (2008) suggest that the lack of reports of B. bassiana infection in A. bipunctata and H. axyridis may in part be because coccinellids do not encounter sufficient amount of inoculum in their arboreal habitat, i.e. reduction of a host s ecological susceptibility (Roy and Cottrell 2008). Here we show that these habitats in urban green spaces in Denmark predominantly harbour Isaria spp. and Beauveria spp. In the present urban system, Tilia europaea is a known habitat for H. axyridis and four native coccinellid species (including A. bipunctata) whose spatial distribution in tree crowns (to 13 m) widely overlap those of the five fungal species isolated in the present study (Howe et al. 2016). Although collection of coccinellids was restricted to the lowest stratum (<3 m), molecular identification of Lecanicillium muscarium, Isaria farinosa, B. bassiana and B. pseudobassiana from tree habitats and from H. axyridis, sampled within 5 days of each other from the same park, suggests that coccinellids do encounter significant amounts of inocula in their arboreal habitat (e.g. at Østre Anlæg). In Tilia europaea crowns, H. axyridis larvae and adults are uniformly distributed between 2 and 13 m. In contrast, the density of a potential prey, the lime aphid Eucallipterus tiliae L., decreases with height in Tilia europaea crowns, although the aphid exclusively prefers abaxial phylloplanes (Dixon 1971; Howe et al. 2016). In a given tree, adult coccinellids are potentially more exposed to inoculum given their enhanced capacity for dispersal compared to the less mobile larvae. On the other hand, as larvae are clustered as eggs and early instars (and potentially close to aphid colonies), were larval distribution to overlap with entomopathogenic fungi inoculum, their exposure to infective units may be somewhat patchy in crowns. This may result in increased susceptibility following multiple encounters to conidia at a smaller spatial scale, especially at the low stratum where fungi densities are greatest. Furthermore, acquisition of inoculum from other sources would also be possible (for dispersing adults), e.g. when locating prey on different tree species in a park or when moving between parks, street trees, etc. Thus, we cannot discount that inocula were encountered in other habitats, in particular for coccinellids collected as adults. We draw attention to the few caveats of this study. (1) The relatively short sampling period of the phylloplane imprinting at best provides a snapshot of fungi densities in trees in summer. In addition, phylloplane imprinting and coccinellid collection periods were temporally discrete, thus we are unable to determine the extent to which fungi inoculum densities on phylloplanes relate to infection levels in coccinellids. As fungi inoculum densities on phylloplanes are shown to increase throughout a year (Meyling and Eilenberg 2006; Ormond et al. 2010), coupling temporal densities of fungi inoculum and their distribution in arboreal habitat with fungi prevalence in H. axyridis life stages over the same time period would illuminate the role that entomopathogenic fungi play in regulating populations of invasive H. axyridis and native coccinellids. (2) Noteworthy is that levels of H. axyridis infections here (approximately 41%) may be inflated due to stress from starvation and finite moisture availability during incubation as seen for the ladybird Hippodamia convergens Guerin which showed increased susceptibility to I. fumosorosea infection under stressful conditions (Pell and Vandenberg 2002). (3) Fungi isolates from phylloplanes were not used to test pathogenicity against ladybirds thereby not demonstrating Koch s postulate (Kaya and Vega 2012). However, the identity of fungal taxa recovered from phylloplanes and coccinellids concurs largely with the entomopathogenic fungi previously isolated from H. axyridis cadavers in late autumn-winter and early summer in Denmark 2007/8 (Steenberg and Harding 2009); the exceptions include L. lecanii isolated by Steenberg and Harding (2009), and B. pseudobassiana isolated from H. axyridis in the present study. Similarly, the relative prevalence of entomopathogenic fungi isolated from H. axyridis and native coccinellids here and by Steenberg and Harding (2009) indicate infection by Isaria > Lecanicillium > Beauveria. In contrast, entomopathogenic fungi isolated from H. axyridis collected in late-summer 2009 revealed Beauveria to be most prevalent (Steenberg and Harding 2010); hence, fungi

9 Howe et al. 9 Table 2. Fungi recorded from ladybirds (Coleoptera: Coccinellidae) collected in Tilia europaea (up to 3 m in crowns) during June July Number in brackets denotes the number of mixed infections in which a fungi genus was present. L1 L4 denote first to fourth larval life stages, PP denotes pre-pupa stage. Dead during incubation with signs of 1 fungi Number of individuals infected with Life stage No. of life stage No. dead at incubated life stage N N N As % of total H. axyridis (or native) Beauveria spp. Isaria spp. Lecanicillium spp. Mixed infections H. axyridis L L L (1) 2(1) 1 L (1) 31(2) 12(3) 3 PP (5) 0(5) 5 Pupa Adult (3) 115(26) 90(27) 28 Total H. axyridis (25) 164 (198) 108 (144) 37 % Infected Natives a Larva Adult b Total natives % Infected Percent of single infections of total collected. a A. bipunctata L. (n = 31), Calvia quatuordecimguttata L. (n = 4), Coccinella septempunctata L. (n = 2), A. decempunctata L. (n = 2), Exochomus quadripustulatus L. (n = 2); native larvae not identified to species. b A. bipunctata (one Lecanicillium, oneisaria), C. septempunctata (one Isaria). prevalence in H. axyridis varies both in space and time. Given that habitat overlap and subsequently H. axyridis encountering inoculum, this variability in ecological susceptibility of H. axyridis (and other coccinellids) could reflect variable abiotic conditions favouring fungi species differently (Roy and Cottrell 2008), rather than unanimous support for reduced susceptibility to B. bassiana in H. axyridis (e.g. Roy et al. 2008). An alternate explanation may be found in H. axyridis immune response; Vilcinskas et al. (2013) linked expression of several antimicrobial proteins in H. axyridis to fungal defence. Synthesised coleoptericins, identified in immune-challenged H. axyridis, strongly or completely inhibited germination of the entomopathogenic fungi Metarhizium anisopliae (strain 43) when cultured in vitro (Vilcinskas et al. 2013). It would thus be relevant to investigate whether differential immune responses exist when H. axyridis is challenged by the suite of entomopathogenic fungi it encounters in the field. Only few studies have reported entomopathogenic fungal infections in immature coccinellid life stages under field conditions. Steenberg and Harding (2009, 2010) observed between 11% and 62% infection in dead or dying H. axyridis larvae, which was comparatively higher than infection levels in adults. Additionally, pathogenicity assays using a naturally occurring I. farinosa isolate obtained from a H. axyridis larva reveal 100% mortality of H. axyridis larvae, yet complete lack of susceptibility in adult H. axyridis and A. bipunctata, suggesting differential susceptibility between life stages (Steenberg and Harding 2009). In the present study, more adults than larvae died showing signs of fungal infection. However, as a large proportion of the dead adult H. axyridis exhibiting signs of infection were collected as larvae, our results lend further support that infection takes place during larval stages, but infections are first manifested in the pupae and adults (Steenberg and Harding 2010). Exposure to solar radiation negatively affects persistence of conidia (James et al. 1995; Braga et al. 2002; Zimmermann 2007), thereby potentially influencing the ecological susceptibility of hostinsects. ConidiaofI. fumosorosea isolates have been found to be more susceptible to solar radiation than conidia of isolates of Beauveria spp. and Metarhizium spp. (Fargues et al. 1996). The effect of solar radiation on I. farinosa isolates has not been investigated, but is presumably similar to I. fumosorosea (Zimmermann 2008). Inoculum of Beauveria spp. conidia in inner canopies persisted longer than in outer canopies (Daoust and Pereira 1986; James et al. 1995) and solar radiation presumably contributed to the consistent pattern of reduced density and occurrence of the fungi genera on outer canopy phylloplanes observed in this study. In contrast, higher densities of Beauveria and Isaria on upper phylloplane surfaces do not agree with general trends of degradation by solar radiation. However, since most trees were more than 15 m high, even the high stratum sampled in this study may have afforded enough protection for inoculum to persist. In addition, aerially dispersed propagules are more likely to be deposited on upper surfaces, which may have contributed to higher densities and observed frequencies in the present study (Arnold and Herre 2003). Mechanisms of dispersal for hypocrealean entomopathogenic fungi onto phylloplanes include dispersal by wind, transport by insect hosts and non-hosts, and from sporulating cadavers in the habitat (Hesketh et al. 2010). Results reported here from Tilia europaea reflect a general decrease in CFU density with increasing height for Beauveria and Isaria, whereas Lecanicillium densities peaked at the mid stratum. Very little information exists pertaining to distribution of fungi in arboreal habitats above 2 m. For example, lower microbe densities were found on leaves at 5 m (sampled by robbing Atta leaf-cutting ants returning to nests of their leaf discs) compared to leaves sampled 1 m above the ground in tropical forest canopies (Griffiths and Hughes 2010). Rain splash of conidia from soil is unlikely to reach leaves >1.5 m above soil (Meyling and Eilenberg 2006), but transport of conidia from upper strata

10 10 FEMS Microbiology Ecology, 2016, Vol. 92, No. 9 in tree crowns with rain throughfall may have contributed to the higher densities of conidia at low and mid strata in this study (Inglis et al. 1995, 2000). In contrast, the number of CFUs isolated from bark revealed no trend; Beauveria densities were highest at the mid stratum, Isaria densities lowest at mid stratum, while Lecanicillium were virtually absent from bark. Compared to phylloplane imprinting, this may be due to the different isolation methods used, or relatively lower sample size. This study provides novel ecological insight into the spatial distribution and densities of indigenous, entomopathogenic fungi on phylloplanes and bark in trees in city parks. A substantial proportion of invasive H. axyridis and native coccinellids, including larvae, were exploited by fungi indicating that trees are likely to be reservoirs of pathogen inoculum to arboreal insects. However, the relative abundance of fungi in trees differed to fungal prevalence in the coccinellids sampled, which together with other studies suggests that the contribution of indigenous entomopathogenic fungi towards regulating H. axyridis populations varies from year to year. Future studies pursuing fungus coccinellid interactions in semi-natural systems or invasion processes involving H. axyridis and entomopathogenic fungi would benefit by not only focusing on Beauveria, but also to include interactions with Isaria and Lecanicillium. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. ACKNOWLEDGEMENTS We acknowledge the helpful comments from two anonymous reviewers which improved our manuscript. We kindly thank Louise Lee Munk Larsen for invaluable technical assistance and disposition in the laboratory, Rob Weir, Arthur Miller, Derrick Ndibnu Mudohsen and Ben Howe for lively assistance during field work and phylloplane imprinting., and AGH thanks Malene Fogh Bang for artwork and GC Howe for fruitful inputs. FUNDING AGH was supported by a PhD grant from Copenhagen University, HPR by Villum Fonden. Conflict of interest. None declared. REFERENCES Adriaens T, San M, Maes D. Invasion history, habitat preferences and phenology of the invasive ladybird Harmonia axyridis in Belgium. Biocontrol 2008;53: Alberti M. The effects of urban patterns on ecosystem function. Int Regional Sci Rev 2005;28: Arnold AE, Herre EA. Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia 2003;95: Bates D, Maechler M, Bolker B et al. lme4: Linear mixed-effects models using Eigen and S4. 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