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Microbial Ecology ISSN 0095-3628Volume 66Number 3 Microb Ecol (2013) 66:608-620DOI 10.1007/s00248-013-0249-5

Effects on Diversity of Soil FungalCommunity and Fate of an ArtificiallyApplied Beauveria bassiana Strain AssessedThrough 454 Pyrosequencing

Jacqueline Hirsch, Sandhya Galidevara,Stephan Strohmeier, K. Uma Devi &Annette Reineke

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SOIL MICROBIOLOGY

Effects on Diversity of Soil Fungal Community and Fateof an Artificially Applied Beauveria bassiana StrainAssessed Through 454 Pyrosequencing

Jacqueline Hirsch & Sandhya Galidevara &

Stephan Strohmeier & K. Uma Devi & Annette Reineke

Received: 22 June 2012 /Accepted: 17 May 2013 /Published online: 5 June 2013# Springer Science+Business Media New York 2013

Abstract The entomopathogenic fungus Beauveria bassi-ana is widely used as a biological control agent (BCA) forinsect pest control, with fungal propagules being eitherincorporated into the potting media or soil or sprayed di-rectly onto the foliage or soil. To gain a better understandingof entomopathogenic fungal ecology when applied as aBCA to the soil environment, a case study using tag-encoded 454 pyrosequencing of fungal ITS sequences wasperformed to assess the fate and potential effect of anartificially applied B. bassiana strain on the diversity of soilfungal communities in an agricultural field in India. Resultsshow that the overall fungal diversity was not influenced byapplication of B. bassiana during the 7 weeks of investiga-tion. Strain-specific microsatellite markers indicated both anestablishment of the applied B. bassiana strain in the treatedplot and its spread to the neighboring nontreated controlplot. These results might be important for proper risk as-sessment of entomopathogenic fungi-based BCAs.

Introduction

Fungal entomopathogens are used worldwide as microbialbiocontrol agents (BCA) against arthropod pests [1]. Of the~130 commercially available products based on entomopa-thogenic fungi, about two thirds consist of conidial prepara-tions of the two most widely studied entomopathogens: Beau-veria bassiana (Bals.-Criv.) Vuilleman and Metarhizium ani-sopliae (Metschn.) Sorokin (both Ascomycota: Hypocreales)[2–4]. Fungal propagules can be incorporated into the pottingmedia or soil at the time of planting [5] or sprayed directlyonto the plant or soil. The entomopathogen B. bassiana isknown to infect a wide range of insects [6, 7]. It also exists asan endophyte inside the plant or as a saprotroph in the soil [8].While the interactions between entomopathogenic fungi andtheir host insects are quite well studied [9, 10], aspects offungal ecology regarding putative interactions between theentomopathogen and the soil microbiota including indigenousfungal communities have been rarely assessed [11–13]. How-ever, as soil fungi are involved in many key processes in soilecosystem functioning like decomposing organic matter or asmycorrhizal symbionts of plants [14], any effect exerted bythe application of an entomopathogenic fungus to the structureand diversity of indigenous fungal communities in the soilmight have important implications for various ecological pro-cesses and functional soil biodiversity. Consequently, theseaspects should be taken into account during the process of riskassessment required for registration of the respective entomo-pathogenic fungal-based commercial product.

In the past, selective media were used to study the impactof an application of entomopathogens like B. bassiana on soilmicroorganisms [11]. Since many soil microorganisms arefastidious and their morphological determination is often dif-ficult, cultivation-independent approaches have been appliedsubsequently [15, 16]. Different DNA fingerprinting techni-ques like denaturing gradient gel electrophoresis (DGGE) or

Jacqueline Hirsch and Sandhya Galidevara Both authors contributedequally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s00248-013-0249-5) contains supplementary material,which is available to authorized users.

J. Hirsch :A. Reineke (*)Institute of Phytomedicine, Geisenheim University, Von-Lade-Str. 1,65366 Geisenheim, Germanye-mail: annette.reineke@hs-gm.deURL: www.hs-geisenheim.de

S. Galidevara :K. U. DeviDepartment of Botany, Andhra University, 530 003Visakhapatnam, India

S. StrohmeierSMS-Development, Ortsstr. 6, 69226 Nussloch, Germany

Microb Ecol (2013) 66:608–620DOI 10.1007/s00248-013-0249-5

Author's personal copy

temperature gradient gel electrophoresis (TGGE) and single-strand conformation polymorphism (SSCP) or traditionalmetagenomic approaches with clone library-based techniqueshave been used to define community structure of soil micro-biota [12, 16]. However, these community profiling techni-ques are time-consuming and costly, especially when taxo-nomic affiliations of respective organisms are analyzed. In thiscontext, next-generation sequencing technologies like 454pyrosequencing represent new, cost-efficient, and fast strate-gies to depict microbial diversity without the need for cultur-ing the respective organisms [17]. The internal transcribedspacer (ITS) regions in the 18S, 5.8S, and 28S ribosomalRNA gene cluster of fungi have been analyzed successfullyin metagenomic studies [18] and are regarded as validatedDNA barcode markers for the taxonomic classification offungi [19–21]. In the present study, we report the applicationof multitag 454 pyrosequencing of fungal ITS-1 sequences forcharacterizing the fungal community structure in an agricul-tural field in India and for assessing both the fate and potentialeffects of an artificially applied B. bassiana strain on diversityof soil fungal communities.

Materials and Methods

Study Site, Fungal Treatment, and Sample Collection

Experiments were conducted from October to December2010 in a cultivated agricultural field near Visakhapatnam(Andhra Pradesh, India) with a standing crop of 4-week-oldchili plants. Chili was chosen as a crop in this study, as it isfrequently attacked by insect pests like Spodoptera lituraand Helicoverpa armigera (Lepidoptera: Noctuidae) [22]that are, at the same time, potential targets for the appliedB. bassiana isolate [6, 23]. No naturally occurring entomo-pathogenic fungal epizootics have been documented in theselected field over the last 15 years and the last artificialintroduction of Beauveria sp. was done in 1996 (RameshKongara—cultivator of the field, personal communication).In addition, no insecticides or fungicides have been appliedin the field over the last 2 years (Ramesh Kongara, personalcommunication). The field is fertilized with sun-dried cowdung. Within the field, two 50-m2 plots ~15 m apart wereselected for the experiment. One of them was treated (T)once with B. bassiana strain ITCC 4688 (Indian Type Cul-ture Collection, IARI, Delhi, India) and the other was left asan untreated control (C). The experimental design was notreplicated on the given plot. Before the application of B.bassiana to the (T) plot, seven soil cores (approximately 4×4×15 cm depth) were collected separately every 3 m on a27-m2 subplot (size of 9×3 m) in each plot. B. bassianaisolate ITCC 4688 was collected originally from infectedcotton bollworm larvae (H. armigera) in Andhra Pradesh,

India [23]. Mass culture of B. bassiana strain ITCC 4688was done in the laboratory. The starter culture was initiatedby inoculation of 1 ml of aqueous conidial suspension (107

conidia per milliliter) in 100 ml of Sabouraud's dextroseyeast (SDY) broth medium, followed by incubation at 25±2 °C with gentle agitation for 72 h. Subsequently, sterilizedrice bags (with a capacity of 200 g rice with 30 % moistureand 2 % sunflower oil) were inoculated with 15 ml of a 3-day-old blastospore suspension. The inoculated rice bagswere incubated for 14 days at 27 °C by which time thefungus grew and sporulated extensively. Germination ofconidia was analyzed in the laboratory and was found tobe more than 90 % over an incubation time of approximate-ly 16 h. For application of the fungus to the treated (T) plot,200 g of rice containing sporulated B. bassiana strain ITCC4688 was suspended in 30 l water with 2 ml Tween 80 togive a final concentration of ~109 conidia per milliliter. Thewhole suspension was dispensed manually onto the soil andplants in the (T) plot, resulting in a concentration of approx-imately 3×1013 conidia per 50 m2. For assessment of effectsof this B. bassiana strain on indigenous soil fungal commu-nity structure, soil samples were collected in each plot asdescribed above at weekly intervals for a duration of7 weeks. No samples were collected in the sixth week dueto heavy rain fall. For our experiment, we used a cultivatedagricultural field. Accidentally, cow dung slurry flowedfrom an adjoining cattle shed into a part of the treatmentplot. Therefore, from the fourth week after B. bassianaapplication onwards, only five soil samples were taken fromthe unaffected parts of the treatment plot. A total of 92 soilsamples were collected over the whole duration of thesurvey from the (C) and (T) plots. The collected soil sam-ples were transported to the laboratory (distance of ~28 km)in an ice chest (8 °C) and stored at 4 °C (for a maximumduration of 48 h) or frozen at −20 °C until further process-ing. Soil parameters such as pH (6.9), organic matter(0.74 mg/kg), and clay content (44 %) were determinedcommercially (Lotus Granges India Ltd., Visakhapatnam,India). Rainfall and temperature data for the duration ofthe experiment were obtained from the local Mandal Reve-nue Office (Anandapuram, India) and Cyclone WarningCentre (Visakhapatnam, India) (Online Resource 1).

DNA Isolation, ITS Amplification, and Pyrosequencing

Soil samples were homogenized independently and genomicDNA was extracted from each of the 92 samples usingPowerSoil® DNA Isolation Kit (Süd-Laborbedarf GmbH,Gauting, Germany) according to the manufacturer's instruc-tions. The variable region of the ITS-1 was amplified withfungal-specific primers as described by Buée et al. [18],which were modified for multitag 454 GS-FLX ampliconpyrosequencing by adding a four-base library “key”

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sequence (TCAG) and a multiplex identifier (MID) tagsequence specific to each soil sample. PCR amplificationswere set up in a total volume of 30 μl consisting of 2–6 μl ofundiluted soil DNA (~10–35 ng/μl DNA), 15 pmol primers,and 15 μl GeNei™ Red Dye PCR Master Mix (2×) (GeNei,Bangalore, India). The PCR reactions were performed at94 °C for 4 min, followed by 30 cycles of 30 s at 94 °C,55 °C for 1 min and 72 °C for 90 s, and a final elongation at72 °C for 10 min. An aliquot of 4 μl of each amplificationproduct was analyzed for correct size (~400 bp) on a 1 %agarose gel. The remaining 26 μl of PCR product waspurified for 454 pyrosequencing analysis with HiYieldPCR Clean-up/Gel Extraction Kit (Süd-Laborbedarf GmbH,Gauting, Germany). In total, 92 fungal PCR products, tag-encoded according to sampling date and plot, were pooled atequimolar concentrations and 454 pyrosequencing was per-formed commercially on a Roche (454) FLX Genome Se-quencer (LGC Genomics GmbH, Berlin, Germany).

Sequence Editing and Taxonomy-Dependent Analysis(MEGAN)

Clipping and sorting of 454 sequence reads by MID tagswas done by LGC Genomics GmbH (Berlin, Germany).Individual sequences were evaluated using BLASTn2.2.25+ with word length of 28 against NCBI nt database.Data was imported to MEGAN version 4.64.2 (MEtaGe-nome ANalyzer [24]) for similarity-based phylotyping.Parameters for the Lowest Common Ancestor (LCA) as-signment algorithm were set as follows: “min support 1”allowing support of a taxon by a single read, “min score 35,”“top percent 10,” “win score 0.0,” and “min complexity0.3.”

Sequence Editing and Analysis by Operational TaxonomicUnit Clustering

Processing, quality filtering, and clustering 454 reads intooperational taxonomic units (OTUs) was performed usingthe online pipeline CLOTU [25]. Sequences with a length of<150 bp and ambiguous bases as well as barcode and primersequences were trimmed by the software. Identical sequen-ces (duplicates) were removed before clustering to reduceredundancy in the data set. Sequences were clustered andassigned to OTUs using the CD-HIT package implementedin CLOTU with a threshold of 97 % sequence similarity andwith at least 75 % of sequence coverage. For taxonomicannotation, one representative sequence from each OTUwas submitted to BLASTn for a comparison against theNCBI nonredundant nucleotide database. All OTUs notdefined as fungi according to BLASTn search were exclud-ed from further data analysis. Remaining OTUs per soil

sample were used for rarefaction analysis and calculationof diversity and richness indices as described below.

Statistical Analysis of Pyrosequencing Reads

Rarefaction analysis as well as fungal diversity (Shannon)and richness (ACE, Chao1) indices at family, species, andOTU levels were computed using PAST [26] and EstimateS8.20 [27], respectively. All reads that were not defined asfungi were excluded from these computations. Singletonswere removed from the data set, except for calculation ofrichness estimators as well as for rarefaction analysis. Priorto the application of B. bassiana, similarities in fungaldiversity between the plots were assessed through a t test(p value threshold of 0.05) based on Shannon diversityindices (species and OTU levels) [28]. The effect (if any)of B. bassiana application on fungal diversity and commu-nity structure was assessed through a comparison of themean of Shannon diversity indices at OTU and specieslevels, respectively, of samples collected in the control andtreated plots subsequent to treatment (C1–C7 versus T1–T7)using a one-way analysis of similarities (ANOSIM) imple-mented in PAST [26]. ANOSIM creates a test statistic of R,which indicates if differences between samples exist. Inter-pretation of R values is according to [29] with the followingcategories: separated R>0.75, clearly different R>0.5, andbarely separable R<0.25. Prior to ANOSIM, nonmetricmultidimensional scaling (NMDS) of the samples was donebased on the Bray–Curtis dissimilarity matrix using analgorithm implemented in PAST [26].

Microsatellite Analysis to Track the Applied B. bassianaStrain

As the ITS-1 gene region is not suitable for strain-specificidentification of an artificially applied B. bassiana isolate,three microsatellite (simple sequence repeats, SSR) markers(Ba01, Ba08, and Ba13; [30]) were used in order to verifythe presence of B. bassiana isolate ITCC 4688 in the re-spective soil samples. The allele sizes of the these SSR lociin B. bassiana strain ITCC 4688 were previously deter-mined as being 121, 260, and 176 bp, respectively (Reinekeet al. in preparation). To allow fluorescent labeling andmultiplexing of the PCR products, a M13(−21) tail wasplaced at the 5′ end of each forward primer and a fluores-cently labeled CY5 or IRD700 universal primer M13(−21)was added to PCR reactions according to the method de-scribed by Schuelke [31]. PCR amplifications were set up ina total volume of 15 μl consisting of 90–100 ng DNA, 10×reaction buffer with 1.5 mM MgCl2, 5 pmol of each primer,0.5 μl 100× BSA, 0.2 mM dNTPs, and 0.5 U of Dream TaqPolymerase (Fermentas, St. Leon-Rot, Germany). PCR reac-tions were performed at the following conditions: 94 °C for

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5 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for45 s and 72 °C for 45 s, and a final extension step at 72 °Cfor 10 min. Each PCR product was checked for successfulamplification on a 2 % agarose gel and analyzed subse-quently for size of SSR alleles via capillary electrophoresisin a multiplex analysis on a GenomeLab GeXP DNA Ge-netic Analysis System (Beckman Coulter GmbH, Krefeld,Germany).

Results

Analysis of 454 Pyrosequencing Reads Basedon Taxonomy-Dependent Analysis (MEGAN)

In the 92 soil samples analyzed in the present study, a totalof ~63,000 reads were sequenced via 454 pyrosequencing.After clipping, 29,109 reads were available for analysis withMEGAN. Most (97 %) of the sequence reads were assignedand only around 3 % lacked a taxonomic annotation orshowed no hits in MEGAN (Table 1). Of the assigned reads,69 % were classified as belonging to the kingdom Fungi.The most dominant phyla were Ascomycota and Basidio-mycota (Table 2). Excluding singletons, 54 fungal specieswere identified by collapsing the phylogenetic tree at spe-cies level (Table 3). Among them, neither B. bassiana norCordyceps sp. (the genus to which the teleomorph of B.bassiana belongs) were detected (Table 3). However, whenthe tree was collapsed at family level, in the samples col-lected prior to treatment, four sequences were assigned tothe family Cordycipitaceae in the C0 plot while none weredetected in the T0 plot (Online Resource 2). In the samplescollected after treatment, the number of reads showing ho-mology to Cordycipitaceae increased both in the control andtreated plots with a massive increase observed in the treatedplot in samples collected 2 weeks after treatment (T2)(Online resource 2).

Analysis of 454 Pyrosequencing Reads Based on OTUClustering

After filtering and trimming of the obtained 63,000 se-quence reads, 39,263 unique reads were clustered into atotal of 2,227 OTUs (excluding 4,000 singletons) usingsoftware CLOTU (Table 1, Online resource 3, sheet 1).More than half (1,164, 53 %) of OTUs showed no hitsagainst the NCBI database and less than 3.6 % (81) of OTUswere not assigned to fungal taxa (Table 1). In accordancewith results from MEGAN analysis, Ascomycota was thepredominant fungal phylum represented by 75 % of detectedOTUs followed by Basidiomycota with 12 % (Table 2).

A few reads homologous to Cordyceps bassiana (tele-omorph of B. bassiana) were detected in both the controland treated plots prior to application of B. bassiana isolateITCC 4688 (Table 4, Online resource 3, sheet 2). Subse-quent to treatment, a massive increase in number of readshomologous to B. bassiana and Cordyceps spp. wasdetected in the treatment plot in samples collected 1 and2 weeks after treatment (T1 and T2) (Table 4, Online re-source 3). Furthermore, from the third week onwards, B.bassiana and Cordyceps spp. were nearly equally repre-sented in both the control and treatment plots (Table 4,Online resource 3, sheet 2).

The number of fungal species identified based on OTUclustering using software CLOTU far exceeded thosedetected through taxonomy-dependent analysis usingMEGAN. Species identified through CLOTU were not in-clusive of all species detected in MEGAN analysis. Forexample, reads of Fibulobasidium murrhardtense and Asco-bolus crenulatus were detected in abundance in MEGANanalysis, while these species were not found in data analysiswith CLOTU (Tables 3 and 4). On the opposite, B. bassianareads were not detected in MEGAN analysis while they

Table 1 Details of ITS-1 454 sequence reads obtained from 92 soilsamples collected at weekly intervals over 7weeks from a chili field inIndia. Results of taxonomy-dependent analysis (MEGAN) and OTU-based clustering (CLOTU) are shown

Number of reads(MEGAN)

Number of clusters(CLOTU)

Assigned 28,318 (97.3 %) 982 (44.0 %)

Unassigneda 483 (1.7 %) 81 (3.5 %)

No hits 308 (1 %) 1,164 (52.5 %)

Total 29,109 2,227

a Reads were not assigned to any group, while clusters were grouped asunclassified fungi

Table 2 Distribution of species and OTUs representing different fun-gal phyla as assessed from ITS-1 454 sequence reads excluding single-tons obtained from 92 soil samples before and after the application ofB. bassiana

Phylum No. species(MEGAN)

No. OTUs(CLOTU)

Ascomycota 35 (64.8 %) 796 (74.9 %)

Basidiomycota 9 (16.6 %) 134 (12.6 %)

Unclassified fungi 0 (0 %) 81 (7.6 %)

Blastocladiomycota 3 (5.6 %) 21 (2.0 %)

Fungi incertae sedis 1 (1.9 %) 14 (1.3 %)

Zygomycota 0 (0 %) 4 (0.4 %)

Chytridiomycota 4 (7.4 %) 6 (0.6 %)

Glomeromycota 2 (3.7 %) 7 (0.7 %)

Total 54 1,063

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Table 3 Abundance of fungal ITS-1 454 sequence reads excludingsingletons in 92 soil samples in an agricultural field in India. Taxa wereassigned by MEGAN by collapsing the tree at species level according

to the Lowest Common Ancestor (LCA) parameter values. C = controlplot and T = treatment plot, and numbers refer to weeks after applica-tion of B. bassiana isolate ITCC 4688 to the treated plot

Fungal species C0 T0 C1 T1 C2 T2 C3 T3 C4 T4 C5 T5 C7 T7 Total

Blastocladiomycota

Allomyces arbuscula 10 3 14 0 8 4 4 5 2 0 4 0 8 4 66

Allomyces macrogynus 0 0 2 2 0 2 0 0 0 0 0 0 0 0 6

Blastocladiella emersonii 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2

Chytridiomycota

Gaertneriomyces spectabile 0 0 0 0 0 0 4 0 3 0 0 0 0 0 7

Olpidium brassicae 5 0 0 0 0 0 0 0 0 0 0 0 0 0 5

Rhizophlyctis rosea 9 2 4 2 0 25 13 2 9 3 12 3 12 4 100

Spizellomyces dolichospermus 2 0 0 0 0 0 2 0 0 0 0 0 0 0 4

Ascomycota

Acremonium alcalophilum 11 6 10 5 10 22 35 14 29 5 15 4 11 6 183

Anthostomella conorum 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2

Aphanoascus pinarensis 0 0 0 0 0 3 3 0 2 0 0 0 0 0 8

Arnium gigantosporum 0 0 0 0 0 0 0 0 0 0 0 0 3 0 3

Ascobolus crenulatus 12 8 7 11 9 11 2 2 10 3 10 3 0 0 88

Ascotaiwania sawada 0 0 0 0 0 0 0 0 0 0 2 0 0 0 2

Aspergillus flavipes 3 0 0 0 0 0 0 0 0 0 0 0 0 0 3

Aspergillus penicillioides 0 3 0 2 0 0 0 0 0 4 5 2 17 10 43

Bagnisiella examinans 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2

Campylocarpon fasciculare 0 0 0 0 0 2 0 2 0 0 0 0 0 2 6

Cephaliophora tropica 18 8 53 21 35 18 65 25 74 12 73 14 63 25 504

Cercophora sparsa 0 0 2 4 2 0 0 0 3 3 3 0 0 0 17

Chaetomium atrobrunneum 3 0 0 0 0 2 2 0 0 6 0 0 2 0 15

Cladophialophora modesta 7 0 0 0 0 0 0 3 0 0 7 4 3 3 27

Cladorrhinum bulbillosum 0 9 3 0 0 33 0 0 0 0 0 0 4 0 49

Cladorrhinum samala 0 4 20 7 4 3 33 8 29 8 14 8 10 2 150

Cochliobolus lunatus 0 2 0 0 2 0 0 5 0 0 0 0 0 0 9

Edenia gomezpompae 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2

Helicoma isiola 0 3 0 0 0 0 0 0 0 0 0 0 0 0 3

Hydropisphaera erubescens 0 0 0 0 0 0 0 0 0 0 0 0 2 0 2

Letendraea helminthicola 0 0 0 0 0 0 0 0 0 4 0 0 0 0 4

Microascus trigonosporus 0 0 4 0 0 0 0 0 0 0 0 0 0 0 4

Penicillium citreonigrum 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2

Penicillium pimiteouiense 0 0 0 0 5 0 0 0 0 0 0 0 0 0 5

Phialosimplex chlamydosporus 2 2 0 0 0 0 0 2 0 0 0 0 2 0 8

Podospora cupiformis 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2

Preussia terricola 0 0 0 0 2 2 2 0 0 0 0 0 0 0 6

Rhytidhysteron rufulum 0 0 0 0 0 0 0 0 0 0 3 0 0 0 3

Scedosporium aurantiacum 2 6 2 4 5 3 3 0 2 3 0 2 0 0 32

Scytalidium thermophilum 2 0 0 2 2 0 0 0 0 0 0 0 0 0 6

Spiromastix warcupii 7 15 13 9 9 14 8 11 12 7 11 11 12 3 142

Stachybotrys elegans 3 0 0 0 0 2 0 0 0 0 2 0 0 0 7

Trichocladium pyriforme 0 0 0 0 0 0 3 0 0 0 0 0 0 0 3

Xylogone sphaerospora 0 3 0 0 3 3 0 0 0 3 4 0 2 0 18

Xylomyces elegans 0 0 0 2 0 2 0 0 2 0 2 0 0 0 8

Basidiomycota

Amanita nauseosa 6 2 8 2 4 10 4 2 3 2 4 6 11 7 71

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were identified through OTU-based clustering (Tables 3 and4, Online resource 3).

Assessment of Effect of B. bassiana on Soil FungalCommunity Diversity and Richness

Prior to the application of B. bassiana to the treatment plot, nosignificant differences in the diversity (Shannon index) offungal communities between control and treatment plots wereevident, neither at the species nor at the OTU levels (t=1.96;df=105 at species and t=0.48; df=1,669 at OTU level,p<0.05). Samples collected at different time intervals in bothplots (control and treatment) after application of B. bassianato the treatment plot fitted consistently to the ordination plot inNMDS computation (Fig. 1), also indicating the suitability ofpooling of data from each plot (C1 to C7, T1 to T7) forcomparison through ANOSIM. At species and OTUlevels, R values for diversity indices were 0.029 and0.207, respectively (p<0.05). These values indicate thatthe control and treated plots are overlapping or barelyseparable regarding their assemblage of fungal commu-nities and that there is, thus, little or no effect on fungalcommunity structures due to the artificial application ofB. bassiana to the treated plot.

The Chao1 and ACE mean richness estimates at all threetaxonomic levels (species, family, and OTU) were highcompared to the observed richness (Table 5). The Shannondiversity indices of all samples ranged from 1.56 to 2.73,2.37 to 2.99, and 5.16 to 5.69 at species, family, and OTUlevels, respectively (Table 5). Shannon diversity indicesincreased by a value of 3 when the calculations werebased on OTU numbers compared to taxonomy-dependent analysis based on MEGAN results. Rarefactioncurves revealed that the number of species, families, and

OTUs increased with the number of sequences sampled,with none of the curves reaching saturation (Fig. 2),indicating that further sampling would have revealedadditional fungal diversity.

Strain-Specific Identification of B. bassiana Strain ITCC4688 Using SSR Markers

As a strain-specific identification of members of the familyCordycipitaceae was not possible on the basis of theobtained ITS sequences, B. bassiana ITCC 4688 strain-specific SSR markers were amplified from the same soilDNA samples as used for pyrosequencing. SSR profilestypical of B. bassiana strain ITCC 4688 were not amplifiedprior to the treatment in both the control and treated plots.However, alleles with the respective size were evident insoil samples collected in the treated plot starting from thefirst week after application until the end of the experiment7 weeks later (T1 to T7; Table 6). In DNA isolated from thecontrol plot, a few samples started to show minor peaks ofthe respective allele size 2 weeks after B. bassiana ITCC4688 application (C2), with a more prominent amplificationbeing evident during the following weeks also in samplesfrom the control plot (Table 6). SSR marker Ba08 amplifiedalleles of the respective size in soil samples from the treat-ment plot only 1 and 2 weeks after B. bassiana ITCC 4688strain application, confirming previous observations on alower sensitivity of this marker for amplification of respec-tive sequences from bulk soil DNA samples (Reineke et al.in preparation). In addition, a few other alleles wereamplified from all samples, mainly in the lower molec-ular weight range, which are likely the result of cross-amplification of DNA of other microorganisms presentin the respective samples.

Table 3 (continued)

Fungal species C0 T0 C1 T1 C2 T2 C3 T3 C4 T4 C5 T5 C7 T7 Total

Entorrhiza aff. fineranae 2 0 0 0 3 0 0 0 3 0 0 0 4 0 12

Ganoderma lucidum 0 0 6 0 3 2 0 0 0 0 2 0 0 0 13

Fibulobasidium murrhardtense 12 21 11 16 26 17 17 24 32 117 16 26 18 10 363

Laetisaria arvalis 0 0 0 0 0 0 0 0 0 0 6 0 0 0 6

Pluteus longistriatus 0 0 0 0 0 0 2 0 0 0 0 0 0 0 2

Thanatephorus cucumeris 2 2 0 18 0 0 0 0 3 0 5 0 2 4 36

Tricholoma giganteum 2 0 0 0 0 0 0 0 0 0 0 0 0 0 2

Waitea circinata 0 0 0 0 0 2 0 0 0 0 0 0 0 0 2

Fungi incertae sedis

Endogone lactiflua 0 2 0 0 0 5 0 0 0 0 0 0 0 0 7

Glomeromycota

Glomus intraradices 0 0 0 3 0 0 0 0 0 0 0 3 0 0 6

Glomus mosseae 0 0 0 0 0 4 0 0 0 5 0 0 0 0 9

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Table 4 List of 46 consistently and abundantly found fungal OTUs as detected in analysis with the programCLOTU from 92 soil samples in an agriculturalfield in India. C = control plot and T = treatment plot, and numbers refer to weeks after application of B. bassiana isolate ITCC 4688 to the treated plot

Fungal species C0 T0 C1 T1 C2 T2 C3 T3 C4 T4 C5 T5 C7 T7

Blastocladiomycota

Allomyces arbuscula 17 0 17 2 14 12 2 4 2 0 8 0 13 2

Ascomycota

Acremonium spp. 83 0 2 0 4 7 7 2 0 2 0 5 2 0

Acremonium alcalophilum 10 4 22 2 18 24 42 12 28 2 25 5 13 8

Aspergillus spp. 6 0 0 2 0 5 4 0 8 0 0 0 0 5

Aspergillus penicillioides 2 3 0 0 0 0 0 0 0 0 0 0 16 4

Beauveria bassiana 0 0 0 0 0 16 4 4 2 0 4 0 5 0

Bionectria spp. 6 0 12 7 6 3 2 0 8 2 2 4 6 3

Cephaliophora tropica 22 22 83 25 51 19 99 28 99 23 94 17 90 31

Chaetomium spp. 0 5 2 0 0 5 0 0 9 0 17 2 2 2

Chaetomium nigricolor 30 36 25 16 33 48 26 29 24 20 33 40 35 30

Cladophialophora modesta 14 0 0 0 0 2 2 2 0 0 5 6 3 2

Cladorrhinum spp. 0 3 2 0 0 22 0 0 0 0 0 0 2 0

Cladorrhinum samala 5 16 30 15 6 8 49 15 48 9 27 6 23 7

Cochliobolus spp. 0 0 3 5 2 0 9 4 2 2 0 2 2 0

Cochliobolus lunatus 8 14 8 8 20 17 28 28 10 12 17 4 15 8

Cordyceps spp.a 2 3 2 43 5 163 38 29 39 35 23 36 63 46

Curvularia spp. 3 2 0 0 5 2 5 2 4 14 4 2 4 01

Emericella spp. 10 5 2 8 5 10 2 2 5 0 2 0 8 2

Emericilla rugulosa 4 4 17 0 11 8 10 5 2 3 7 5 6 10

Engyodontium album 0 2 3 2 9 3 0 6 2 5 9 2 2 5

Fusarium spp. 45 73 93 57 138 128 244 226 100 119 153 63 204 86

Fusarium larvarum 0 6 0 2 0 2 0 4 3 10 4 6 0 0

Fusarium oxysporum 13 27 4 26 14 12 18 17 21 10 4 11 11 3

Fusarium solani 7 14 14 7 12 13 17 20 18 8 8 11 11 9

Humicola spp. 51 65 44 43 49 89 63 50 86 45 47 43 51 42

Microsphaeropsis spp. 10 15 13 35 18 29 16 25 14 15 20 17 8 10

Monacrosporium megalosporum 8 8 6 12 15 6 10 2 28 15 12 9 8 7

Ochroconis spp. 9 7 6 5 15 10 15 0 11 6 5 4 9 3

Paecilomyces spp. 15 40 22 18 12 71 12 24 25 15 18 20 11 12

Phoma spp. 25 16 54 58 43 92 92 55 83 46 42 16 152 54

Plectosphaerella spp. 0 0 2 0 10 0 17 0 2 0 2 0 14 0

Pseudallescheria boydii 6 2 2 0 12 3 8 6 14 5 0 0 0 0

Pycnidiophora spp. 10 2 2 7 6 3 9 3 25 4 2 3 4 0

Scedosporium apiospermum 0 10 2 3 4 3 2 2 22 0 2 4 0 0

Sordaria spp. 39 89 60 89 88 82 104 82 120 35 82 33 91 27

Spiromastix warcupii 20 18 19 14 18 16 5 15 11 13 10 14 11 5

Thielavia spp. 27 44 24 22 22 23 52 22 32 11 9 22 20 54

Westerdykella spp. 6 4 7 2 7 12 0 7 19 4 18 4 7 0

Zopfiella spp. 28 8 42 0 59 36 46 16 55 10 62 4 23 2

Basidiomycota

Amanita manicata 7 2 12 2 6 10 3 2 3 2 6 7 12 7

Conocybe dumetorum 0 0 84 0 0 0 0 0 0 0 0 0 0 0

Disciseda candida 18 12 7 2 6 5 5 6 8 0 8 4 7 3

Rhizoctonia spp. 0 2 12 71 9 46 2 3 2 17 18 125 0 0

Rhizoctonia solani 8 4 0 88 0 0 0 13 11 0 19 0 20 10

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Discussion

We assessed the composition of soil fungal communities viatag-encoded 454 pyrosequencing to obtain insights on theeffects of artificial application of the entomopathogenicfungus B. bassiana on indigenous fungal species presentin the soil. The plot chosen for this experiment was anagricultural field in the tropical savannah climate zone(Aw zone according to Köppen–Geiger climate classifica-tion, [32]) of India, which was cultivated according toconventional small-scale Indian farming standards and wasplanted with a standing crop of chili during this experiment.Conditions of the Aw climate zone are favorable for spreadand establishment of entomopathogenic fungi, and annualepizootics of these fungi are known to occur regularly [32].Moreover, we considered it to be important to perform sucha trial under managed conditions with as much practicalrelevance for farmers as possible. Accordingly, in this study,we obtained both a first insight in fungal communitiesassociated with this type of agricultural practice in the givengeographic location, and we were able to assess the fate andthe dynamics of spread of a fungal biocontrol agent appliedartificially to this field. As there are no true replicates of ourexperimental design in the given agricultural field, our studycan be considered as a “proof-of-concept” for these partic-ular questions.

C. bassiana (a teleomorph of B. bassiana) was detectedalbeit in low concentration in both the control and treatedplots prior to artificial application of B. bassiana to thetreatment plot. After application, a remarkable increase inthe number of sequence reads homologous to B. bassianaand C. bassiana were observed in samples from both thecontrol and treatment plots, with the highest number of B.bassiana reads detected in samples from the treated plotcollected 2 weeks after application. Specific SSR markerswere amplified from the same DNA samples to confirm thatthese reads belong to the applied B. bassiana strain ITCC4688. Based on amplification of all three SSR markers, anSSR profile identical to the applied B. bassiana strain wasobserved in the treatment plot only in samples collected 1 and2 weeks after treatment (T1 and T2). In the samples collectedsubsequently in both the treatment and control plots, one (T3to T5 and C2 to C5) or two (C7 and T7) of the three SSRmarkers were not amplified. Template quantity and/or effi-ciency of amplification of different SSR loci are reported toaffect the results of SSR profiling in samples with high se-quence diversity [33]. Together, both the results obtained viapyrosequencing as well as via SSR marker analysis indicate anatural spread of the applied B. bassiana isolate from thetreatment to the control plot. As water plays an important rolein the movement of fungal pathogens [4, 34–36], we supposethat several rainfall events from October till December 2010

Table 4 (continued)

Fungal species C0 T0 C1 T1 C2 T2 C3 T3 C4 T4 C5 T5 C7 T7

Fungi incertae sedis

Mortierella spp. 0 3 19 0 0 2 0 2 0 6 0 11 10 17

Mortierella wolfii 3 0 0 0 6 5 23 0 5 9 17 0 24 2

a All OTUs are homologues to HQ630968.1, which is identified by the authors as Cordyceps bassiana (Online resource 3, sheet 2)

Stress = 0.062(a) Stress = 0.061(b)

Fig. 1 Nonmetric multidimensional scaling (NMDS) plot of OTU (a)-or species (b)-based clustering of data from the fungal ITS-1 regionobtained from soil samples of a chili field treated with the

entomopathogenic fungus B. bassiana. C1 to C7 and T1 to T7 repre-sent samples from control and treated plots, respectively, collected atweekly intervals after treatment

Fate of Beauveria bassiana Strain 615

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Tab

le5

Shann

ondiversity

indexandChao1

andACErichness

estim

atorsof

fung

altaxa

ondifferentlevels(species,fam

ily,and

OTU)in

soilsamples

incontrol(C)andtreatm

ent(T)plotsbefore

andaftertheapplicationof

B.ba

ssiana

isolateITCC46

88to

achili

field.

Num

bers

referto

weeks

afterB.ba

ssiana

applicationto

thetreatedplot.Singleton

swereexclud

edforcompu

tatio

nof

Shann

onindex

Soilsamples

C0

T0

C1

T1

C2

T2

C3

T3

C4

T4

C5

T5

C7

T7

Species

Observed

3230

2831

4245

3624

3123

4620

3824

Shann

on2.73

2.66

2.31

2.42

2.37

2.66

2.12

2.14

2.08

1.56

2.38

2.13

2.40

2.17

ACE

40.25

3537

.16

4610

270

.66

64.5

33.16

46.16

33.5

111

29.33

57.12

46

Chao1

40.28

36.25

40.1

49.75

120.12

75.25

72.1

36.1

50.6

47.5

130.5

3661

.14

60

Fam

ily

Observed

4135

2938

4238

4333

3427

4427

4429

Shann

on2.87

2.83

2.74

2.82

2.88

2.60

2.87

2.80

2.82

2.37

2.89

2.79

2.99

2.67

ACE

67.25

54.5

5044

.87

5341

62.5

36.5

35.87

3452

.66

3260

.541

Chao1

78.5

63.16

5046

.64

56.4

42.08

71.16

37.57

36.57

39.25

54.56

39.5

6849

.25

OTU

Observed

1,01

994

81,03

997

21,21

31,23

41,26

31,00

11,22

01,03

01,29

090

11,20

975

0

Shann

on5.47

5.47

5.42

5.5

5.69

5.64

5.6

5.45

5.66

5.43

5.67

5.46

5.65

5.16

ACE

2,42

3.18

2,09

2.94

2,43

1.36

2,24

8.8

2,63

7.72

2,72

4.23

2,91

2.35

2,38

6.25

2,66

0.09

2,60

8.11

3,19

6.38

1,86

0.06

2,54

3.07

1,71

2.68

Chao1

2,43

4.57

2,10

2.15

2,44

2.22

2,25

9.78

2,64

7.03

2,73

4.06

2,92

3.27

2,39

7.78

2,66

9.49

2,62

1.42

3,20

9.55

1,86

8.11

2,55

1.61

1,72

3.27

616 J. Hirsch et al.

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(Online Resource 1) may have favored the dispersal of B.bassiana conidia. In addition, wind, arthropods, and agricul-tural cultivation practices are effective dispersal mechanismsof entomopathogenic fungal conidia [37]. We assume thatsuch factors favored the dispersal of B. bassiana conidia fromthe treatment to the control plots and contributed to thisapparent spread of B. bassiana in the field. Such a naturalspread and establishment of B. bassiana is in agreement withthe concept of classical biological control [1]. In classical

biological control, small amounts of inoculum of entomopath-ogens are intentionally released and are expected to naturallyincrease in population density and get permanently estab-lished. Including fungal entomopathogens in a classical bio-logical control approach is, for sure, of interest for small-scalefarmers, where such a strategy represents a long-lasting andcost-efficient avenue for environmentally friendly insect pestcontrol. However, our molecular approach does not detectviability and virulence of the B. bassiana fungal propagules

0

5

10

15

20

25

30

35

40

45

50

No. of sequences

No

. of

ob

serv

ed s

pec

ies

05

101520253035404550

1 21 41 61 81 101 121 141 161 181 201 221

1 41 81 121 161 201 241 281 321 361 401 441 481 521 561

No. of sequences

No

. of

ob

serv

ed f

amili

es

0

250

500

750

1000

1250

1500

3001200110011

No. of sequences

No

. of

ob

serv

ed O

TU

s

C0 T0 C1 T1 C2 T2 C3 T3 C4 T4 C5T5 C7 T7

(a)

(b)

(c)

Fig. 2 Rarefaction curvesillustrating observed number offungal species (a), families (b),and OTUs (c) in soil samplescollected from a chili fieldtreated with Beauveriabassiana. C control plot, Ttreatment plot; numbers refer toweeks after application of B.bassiana isolate ITCC 4688 tothe treated plot

Table 6 Amplification of strain-specific alleles of three SSR loci(Bao1, Ba08, and Ba13) of B. bassiana strain ITCC 4688 in soilDNA samples from a chili field in India. Presence (+) or absence (−)

of alleles of the expected size is shown. C = control plot and T =treatment plot, and numbers refer to weeks after application of B.bassiana isolate ITCC 4688 to the treated plot

SSR marker (size) C0 T0 C1 T1 C2 T2 C3 T3 C4 T4 C5 T5 C7 T7

Ba01 (121 bp) − − − + + + + + + + + + + +

Ba08 (260 bp)) − − − + − + − − − − − − − −

Ba13 (176 bp) − − − + + + + + + + + + − −

Fate of Beauveria bassiana Strain 617

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in the plots. Cultivation-independent methods such as SSRmarkers and 454 pyrosequencing will also amplify any DNAfrom dead fungal cells or senescent conidia. A combination ofmolecular methods and baiting techniques such as the Galle-ria bait method [38] would help to detect the infectivity of theapplied fungi-based BCA.

A second goal of our study was to address the questionwhether an artificial application of a microbial BCA causesa shift in the diversity of the indigenous fungal communityin the treatment plot either indirectly due to competition fornutrients or directly due to suppression or antibiosis. Duringthe 7 weeks of our investigation, no effect of B. bassiana,applied at a concentration of 3×1013 conidia per 50 m2, wasevident on the diversity of indigenous fungal communities.Similar observations were reported by Schwarzenbach et al.[39] in their assessment of the effect of an application of a B.brongniartii-based BCA on soil fungal community struc-tures in a controlled environment (soil microcosms). In thisstudy [39], only small effects on fungal community struc-tures were evident and the authors assumed that smalleffects caused by fungal BCAs to soil fungal communitiesmay be difficult to detect in the field due to high ecosystemvariation and fast compensation effects.

Predicted richness estimates at species, family, and OTUlevels were high (more than double at OTU level) comparedto the observed richness, indicating the presence of highlydiverse fungal communities in the selected field. Fungibelonging to Glomeromycota and Chytridiomycota wereprobably underestimated in the present study as the appliedITS-1 primers originally have been designed for amplifica-tion of Dikaryotic fungi [18]. Moreover, approximately31 % of the analyzed reads in MEGAN lacked a deeptaxonomic resolution or were assigned to Cercozoa or Vir-idiplantae. This indicates a certain nonspecificity of theapplied primers. A large proportion of the ITS-1 sequencereads in this study were, however, found to have no hits todatabase entries or were assigned to unclassified fungi.

In the present study, we used two different approaches ofanalyzing our data set, i.e., taxonomy-dependent analysis usingthe program MEGAN as well as taxonomy-independent anal-ysis based on OTU clustering. Both approaches yielded con-siderable differences regarding fungal species composi-tion in the respective plots, which can be explained bythe different types of algorithms implemented in bothapproaches. Taxonomy-dependent analysis depends onentries in reference sequence databases such as the NCBItaxonomy and might, thus, have poor performance ifsequences of many novel or nonculturable organisms arepresent in the given data set. Taxonomy-independent algo-rithms cluster sequences within a data set into OTUs, based onpairwise identity cutoffs [40, 41]. Accordingly, OTU-basedapproaches overcome limitations associated with taxonomy-dependent analysis, in particular if a given sequence is not

sufficiently represented by a reference sequence in the taxon-omy outline [42].

The SSR markers chosen for tracking the ITCC 4688strain of B. bassiana used in this study were found usefulonly when the density of the applied strain was considerablyhigh. For registration purposes of fungal based biocontrolagents, any risks concerning the persistence of the appliedfungal inoculum have to be evaluated in order to assess theorganism's potential to spread and to become established inthe environment [43]. In addition, registration authorities inthe European Union require information on long-term non-target effects such as potential competitive displacement ofsoil microorganisms as well as information on the naturalbackground level of a particular entomopathogenic fungus.Both requirements may be achieved by using multitag 454pyrosequencing as obtained sequence reads give a compre-hensive description of the fungal diversity [18, 44], and readabundance allows a quantification of the applied fungus andthe present soil fungal community with some limitations[45]. A more detailed insight into the dynamics and inter-actions of entomopathogenic fungi like B. bassiana withother microorganisms present in the soil is crucial for abetter understanding of factors influencing fungal survivaland persistence and for estimating success rates of applica-tions of these organisms for biological insect pest control. Acombination of new molecular methods like 454 pyrose-quencing and classical approaches like bait methods repre-sent powerful tools to acquire a more thorough knowledgeon the ecology of fungal entomopathogens.

Acknowledgments We thank Ramesh Kongara for providing theexperimental sites and Ravi Kanth Reddy Sathi, Swapna Guntupalli,and Suman Keerthi for field and laboratory assistance as well as MartinPfannkuchen and Surendra Kumar for advice in bioinformatic analysis.We are grateful to the German Research Foundation (DFG, projectnumber: RE 1444/4-1) and the Department of Science and Technology(DST, project number: INT/FRG/DFG/P-07/2008) New Delhi for fi-nancial support.

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