Biological Control 70 (2014) 1–8

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Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications Annette Reineke a,⇑, Monika Bischoff-Schaefer a, Yvonne Rondot a, Sandhya Galidevara b, Jacqueline Hirsch a, K. Uma Devi b a b

Geisenheim University, Institute of Phytomedicine, Von-Lade-Str. 1, D-65366 Geisenheim, Germany Andhra University, Department of Botany, 530 003 Visakhapatnam, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Three SSR markers confidentially

discriminated between a world-wide collection of Beauveria bassiana isolates.  Detection thresholds differed depending on the environment of the PCR assay.  SSR markers detected a commercialized B. bassiana strain in different substrates up to 14 weeks after inoculation.

a r t i c l e

i n f o

Article history: Received 25 March 2013 Accepted 29 November 2013 Available online 6 December 2013 Keywords: Beauveria bassiana Microsatellites Monitoring Soil samples Entomopathogenic fungi

a b s t r a c t Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Entomopathogenic fungi are a very diverse and ubiquitous group of natural enemies of arthropods. As such they have ⇑ Corresponding author. Fax: +49 6722 502410. E-mail address: [email protected] (A. Reineke). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.11.012

attracted increased attention in recent times as potential microbial biocontrol agents to be used in integrated pest management programs in an inoculative or inundative manner (Hajek and Delalibera, 2010; Jaronski, 2010). More than 130 commercial products based on entomopathogenic fungi have been developed in the past, with around two-thirds of them consisting of conidial preparations of the two most widely studied entomopathogens, Beauveria

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bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorokin. (both Ascomycota: Hypocreales) (Jackson et al., 2010; Jaronski, 2010). However, despite these numbers, fungus-based mycoinsecticides do not account for a substantial part of the US or European biopesticide market (Jaronski, 2010). Aspects on stability of achieved control levels, costs, product quality and shelf-life, as well as persistence of the fungal propagules in the environment are among the chief reasons for this limited use. The latter is mainly influenced by an array of biotic and abiotic factors such as temperature, solar radiation, moisture or heavy rainfall (Meyling and Eilenberg, 2007). Accordingly, a couple of recent studies have stressed the importance to not only focus on the interaction between an entomopathogenic fungus and its host insect, but also to identify critical environmental constraints and to understand fungal ecology and multi-trophic relationships in semi-natural or anthropogenic habitats (Bruck, 2010; Hesketh et al., 2010; Jackson et al., 2010; Meyling and Hajek, 2010; Meyling and Eilenberg, 2007; St. Leger, 2008). In the case of M. anisopliae, studies by Bruck and Donahue (2007) have shown that fungal propagules can be incorporated in different types of potting media at the time of planting and persist in the potting medium for one or more growing seasons, allowing control of soil-borne insects like larvae of the black vine weevil Otiorhynchus sulcatus. In addition, some M. anisopliae isolates are known to be rhizosphere competent, and insects like O. sulcatus larvae feeding on roots which were colonized with an entomopathogenic fungus showed high levels of fungal infection (Bruck, 2005). Furthermore, recent studies have proved that B. bassiana can endophytically colonize a wide array of plant species, while still maintaining its entomopathogenic habit (Akello et al., 2008; Gurulingappa et al., 2010; Ownley et al., 2010; Quesada-Moraga et al., 2009; Tefera and Vidal, 2009). Together these aspects again stress the importance of understanding the ecology and persistence of fungal entomopathogens in various environments such as the soil or plant (Bruck, 2010; Vega et al., 2009). As a prerequisite for studying the ecology and persistence of entomopathogenic fungi in the soil or other environments as well as their interactions with microbial communities present in the respective habitat, a method allowing strain-specific identification of the particular fungal isolate is required. When applying classical, elaborate and time consuming cultivation-based techniques, a strain-specific identification is often not possible due to a lack of consistent and definite morphological characteristics, which might as well be influenced by culture medium or other physiological conditions (Hussain et al., 2010; Oliveira et al., 2011). Hence, cultivation-independent molecular genetic techniques have been increasingly applied to monitor entomopathogens in the environment (Enkerli and Widmer, 2010). Those PCR-based techniques usually allow a highly specific and high-throughput detection and quantification of the targeted fungal species or strain directly in the respective environment. For the entomopathogenic fungus Metarhizium spp. clade 1, a diagnostic PCR-based method was recently developed, which also allows quantification in complex bulk soil DNA samples (Schneider et al., 2011). For an entomopathogen of aphids, Pandora neoaphidis (Entomophtoromycota: Entomophthorales), Fournier et al. (2008) developed specific PCR primer pairs and applied them to monitor the presence of this fungus in bulk soil DNA. However, this approach did not allow a strain-specific identification of different P. neoaphidis isolates. Castrillo et al. (2003) have developed a marker specific for B. bassiana strain GHA based on a random amplified polymorphic DNA (RAPD) fragment and quantified its abundance using qPCR in various environmental samples such as leaf, bark and soil samples (Castrillo et al., 2008). Schwarzenbach et al. (2007) applied markers based on microsatellites (simple sequence repeats, SSRs) to detect Beauveria brongniartii strains in bulk soil DNA extracts. SSR markers are often

highly polymorphic thus allowing a precise genotyping of different fungal strains and can be multiplexed during the reaction increasing the accuracy of identification and reducing both time and costs of the assays (Selkoe and Toonen, 2006). These SSR markers have been isolated in a variety of entomopathogenic fungi, among them B. bassiana (Rehner and Buckley, 2003), B. brongniartii (Enkerli et al., 2001), Paecilomyces fumosoroseus (Dalleau-Clouet et al., 2005) and M. anisopliae (Enkerli et al., 2005). The intention of the present study was to apply five of the B. bassiana specific SSR markers previously published by Rehner and Buckley (2003) to monitor the presence of a commercialized B. bassiana isolate (ATCC 74040, active ingredient in the product NaturalisÒ) in various environments. For this purpose, these SSR markers were evaluated regarding their discriminative power and sensitivity and were subsequently applied to monitor the presence of B. bassiana isolate ATCC 74040 in three commercially available potting substrates. 2. Materials and methods 2.1. Fungal material Throughout the present study, the commercial product NaturalisÒ (Intrachem, Italy) was used in the experiments. It was formulated as an oily fluid and contained 69.1 g/L B. bassiana isolate ATCC 74040 as an active ingredient with a concentration of at least 2.3  107/ml viable B. bassiana conidia. In addition, for SSR analysis, 17 B. bassiana strains were obtained from several culture collections, including B. bassiana strain GHA, which is the active ingredient in several mycoinsecticides registered worldwide, e.g. product BotaniGardÒ (Table 1). All B. bassiana strains including a suspension of product NaturalisÒ were grown on Beauveria selective medium (BSM, (Strasser et al., 1996)) to obtain pure reference DNA for molecular analysis. 2.2. DNA extractions For DNA extraction, mycelium was directly collected from fungal colonies grown on BSM plates and cells were mechanically disrupted using the Precellys tissue homogenizer (Peqlab Biotechnologies, Erlangen). Fungal DNA was extracted using Power Soil DNA Isolation Kit (Mobio Laboratories, USA) according to the manufacturer’s instructions. DNA from 250 mg of soil samples was extracted in the same way using Precellys tissue homogenizer for soil particle lysis and Power Soil DNA Isolation Kit. Genomic DNA from 5 to 10 mg about 3 weeks old plant leaves (grapevine, Vitis vinifera L. cv. Riesling, plants were about 8 weeks old) was extracted using the MasterPure DNA Purification Kit (Biozym Scientific GmbH, Hessisch Oldendorf) according to the manufacturer’s instructions. We used grapevine as a model plant in this study since we currently apply strain-specific molecular markers to monitor endophytic establishment of B. bassiana in grapevine plants (Rondot and Reineke, 2013). 2.3. Amplification of microsatellite markers Five B. bassiana specific microsatellite primers were used, called Ba01, Ba02, Ba08, Ba12 and Ba13 (Rehner and Buckley, 2003). To allow a fluorescent labelling of the generated PCR products, three primers were incorporated in the PCR reactions according to the method described by Schuelke (2000): a SSR-specific forward primer with an universal M13(21) tail at it’s 50 -end, an unlabelled SSR-specific reverse primer, and a fluorescently labelled CY5 universal M13(21) primer, which will incorporate the fluorescent dye into the PCR product (Schuelke, 2000). PCR amplifications were

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Table 1 Beauveria bassiana strains and their origins used for determination of allele number and sizes of five SSR loci. Locus names are according to Rehner and Buckley (2003). The discriminatory power (DL) of each locus was calculated according to Tessier et al. (1999). Straina

ATCC74040 GHA (syn. ATCC74250) NRRL22865 NRRL20699 NRRL22864 NRRL3108 NRRL20698 ARSEF326 ARSEF3041 ARSEF739 ARSEF1149 ARSEF1166 ARSEF1314 ARSEF1316 ARSEF2860 ITCC1253 ITCC913 ITCC4688 No of alleles per locus DL

Original host

Origin

Anthonomus grandis (Coleoptera) Diabrotica undecim-punctata (Coleoptera) Unknown Unknown Glischrochilus quadri-signatus (Coleoptera) Ostrinia nubilalis (Lepidoptera) Dysdercus koenigii (Hemiptera) Chilo plejadellus (Lepidoptera) Reticulitermes flavipes (Isoptera) Diabrotica paranoense (Coleoptera) Helicoverpa armigera (Lepidoptera) Helicoverpa armigera (Lepidoptera) Helicoverpa virescens (Lepidoptera) Helicoverpa virescens (Lepidoptera) Schizaphis graminum (Homoptera) Musca domestica (Diptera) Unknown Spodoptera litura (Lepidoptera)

Allele size (bp) of locus

(ex Naturalis) Texas, USA ex BotaniGard Iowa, USA Illinois, USA Illinois, USA Unknown Lima, Peru Queensland, Australia Toronto, Canada Brazil Cordoba, Spain Cordoba, Spain La Minière, France La Minière, France Idaho, USA Mumbai, India Netherlands Bangalore, India

Ba01

Ba02

Ba08

Ba12

Ba13

117 117 126 129 113 103 109 99 113 115 105 105 105 NAb 107 121 121 121 11 0.886

152 145 281 135 156 156 135 138 138 140 164 164 164 164 130 140 140 140 8 0.852

255 243 215 215 260 218 215 215 276 215 215 215 213 215 215 260 260 260 6 0.685

231 222 204/293 204/293 218/305 222 226 314 218 222/310 212/300 212/300 212/300 212/300 218/307 231/319 231/319 231/319 13 0.903

216 168 190 168 168 176 168 171 168 173 168 168 168 168 168 176 176 176 6 0.630

a ATCC, American Type Culture Collection; NRRL, Agricultural Research Service Culture Collection; ARSEF, Agricultural Research Service Collection of Entomopathogenic Fungi; ITCC, Indian Type Culture Collection. b No amplification product obtained in several rounds of replicates, presence of a null allele.

set up in a total volume of 20 ll consisting of 90 ng DNA, 10 reaction buffer, 10 pmol of each primer, 1.5 mM MgCl2, 0.2 mM dNTPs and 0.5 U of Phire Hot-Start DNA Polymerase (Biozym Scientific GmbH, Hessisch Oldendorf). PCRs were performed at the following conditions: initial denaturation at 98 °C for 30 s, followed by 35 cycles of 98 °C for 5 s, 60 °C for 15 s and 72 °C for 15 s, followed by a final extension at 72 °C for 1 min and storage at 8 °C. PCR products were analyzed for SSR sizes by capillary electrophoresis on a Beckman GenomeLab GeXP DNA Genetic Analysis System. Except for PCR products of B. bassiana isolate ATCC 74040 and B. bassiana reference strains, reactions were multiplexed prior to electrophoretic separation according to their respective band size with primers Ba02, Ba08 and Ba12 in one pool (Fig. 1) and primers Ba01 and Ba13 in a second pool. For capillary electrophoresis, 0.5 ll of each PCR product, mixed with 30 ll sample loading solution (Beckman) and 0.5 ll of a 400 bp size standard (Beckman) were used. All PCR reactions were repeated twice to check for reproducibility of amplified microsatellite markers.

In order to compare the efficiency of the five SSR markers to differentiate among B. bassiana genotypes the discriminatory power (DL) was calculated for each locus (Tessier et al., 1999). The DL value represents the probability that two randomly chosen isolates show different allelic patterns at the same microsatellite locus, and thus are distinguishable from one another. Allele frequencies for each locus were calculated using Arlequin software (Excoffier et al., 2005). 2.4. Sensitivity assays To test the sensitivity of the five SSR markers for detection of B. bassiana isolate ATCC 74040 in various environments, different sensitivity assays were performed: (i) Total B. bassiana DNA: Genomic DNA was extracted from fungal mycelia of a pure culture of B. bassiana isolate ATCC 74040 grown on an agar plate as described in Section 2.1 and 2.2 and obtained DNA templates were diluted tenfold with concentrations ranging from 100 ng/ll to 0.1 fg/ll. (ii)

7000

255 6000

231

Dye Signal (rfu)

5000 4000 3000 2000

152 1000 0 100

150

200

250

Size (nt) Fig. 1. Multiplexed amplification products obtained with microsatellites markers Ba02 (152 bp), Ba08 (255 bp) and Ba12 (231 bp) in a DNA pool extracted from substrate ED73 previously inoculated with 3% NaturalisÒ (active ingredient B. bassiana isolate ATCC 74040).

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Spiking of a soil DNA pool: Total genomic DNA was extracted from an unsterilized commercially available substrate (ED73, see Section 2.5) and 50 ng soil DNA was spiked by the addition of 1 ll each of tenfold serial dilutions of DNA of B. bassiana isolate ATCC 74040 (from 100 ng/ll to 1 pg/ll). (iii) Inoculation of soil: 100 ll of a conidial suspension of B. bassiana isolate ATCC 74040 was added at various concentrations (tenfold serial dilutions of 5  108 conidia/ml to 50 conidia/ml) to 50 mg unsterilized substrate ED73 and total genomic DNA was extracted as described in Section 2.2. (iv) Spiking of plant DNA: Total genomic DNA was extracted from 5 to 10 mg grapevine V. vinifera leaves as described in Section 2.2 and 50 ng plant DNA was spiked by the addition of 2 ll each of tenfold serial dilutions of B. bassiana isolate ATCC 74040 DNA (from 100 ng/ll to 1 pg/ll). (v) Inoculation of plant leaves: 50 ll of a conidial suspension of B. bassiana isolate ATCC 74040 was added at various concentrations (tenfold serial dilutions of 5  108 conidia/ml to 50 conidia/ml) to 5–10 mg fresh grapevine V. vinifera leaves and total genomic DNA was extracted subsequently using MasterPure Extraction Kit as described in Section 2.2. Amplification of microsatellite markers was performed as described in Section 2.3 using 2 ll of each DNA solution in PCR reactions (total volume of 20 ll). An aliquot (15 ll) of the complete PCR reaction was loaded on a 2% agarose gel and gels were checked for presence or absence of respective SSR products. For sensitivity assays (iii) and (v) the DNA mass concentration per conidia was estimated assuming a genome size of 33.7 Mb for B. bassiana (Xiao et al., 2012) and using the following formula (Dolezˇel et al., 2003): DNA content (in pg) in one conidia equals the genome size (in bp) divided by 0.978 x 109 bp/pg. 2.5. Inoculation of B. bassiana in potting substrates Presence of B. bassiana isolate ATCC 74040 was monitored via SSR markers in three commercially available substrates i.e. ED73 (Patzer, Sinntal, Germany; high clay content, pH 5.9), Eurohum (Patzer, Sinntal, Germany; 30% wood fibre, pH 5.8) and Lignostrat-Bio (Hawita, Vechta, Germany; bark humus, pH 5.5–6.1). For inoculation with B. bassiana, 36 L of the respective substrate were filled into a plastic tray and were sprayed with 1 L of 3% NaturalisÒ (6.9  105 conidia/ml) or with water as a control. After careful mixing, substrates were covered with black plastic foil to avoid evaporation and were incubated for 4 d at 18 °C in the greenhouse. Accordingly, an aliquot of 2 L of inoculated or control substrates was filled into plastic pots (17 cm diameter), with three pots as replicates per substrate and treatment. Pots were installed in the greenhouse and were watered regularly by drip irrigation. Temperatures in the greenhouse were between 16 °C and 23 °C during the experiment. Substrates were incubated for a total duration of 14 weeks. From each substrate and replicate, approximately 5 g soil samples were taken with sterile spatulas, which were plunged at a depth of 7–10 cm into the pots. Samples were taken before inoculation with B. bassiana as well as 1, 2, 6, 10 and 14 weeks after the inoculation of soil and were submitted to SSR marker analysis via capillary electrophoresis.

inoculation and were filled in separate plastic containers. Three replicates were set up for each sample and time point. Ten larvae of Galleria mellonella L. (greater wax moth) in their penultimate instar were placed in each plastic container and baiting was performed in darkness at 20–22 °C. Containers were inverted daily to assure contact of the larvae with the soil samples. After 8 days the total number of dead/and or mycosed larvae were counted and differences in the number of dead G. mellonella larvae in soil samples from control and B. bassiana-inoculated ED73 substrate at each time point were tested for statistical significance using a Mann–Whitney-U test and the software Statistica version 7.1 (StatSoft, 2005). Differences in the infectivity of the substrates collected at different time points after inoculation with B. bassiana were tested using a Friedman ANOVA in Statistica version 7.1 (StatSoft, 2005). 3. Results 3.1. Specificity and size of diagnostic SSR markers in B. bassiana isolates Genotype characteristic profiles were amplified with each of the five SSR markers for the commercially available B. bassiana isolates ATCC 74040 and GHA, as well as for a representative world-wide sample of 16 additional B. bassiana reference strains (Table 1). Some B. bassiana isolates of the world-wide collection had two alleles at locus Ba12. We assume that these two alleles are the consequence of duplication/multiplication events involving large regions containing the respective microsatellite amplified by marker Ba12. The stable size difference of around 88 bp in all isolates showing two alleles supports this assumption. Among the 18 different B. bassiana isolates tested, three strains from an Indian (ITCC) and two strains from the USDA culture collection (ARSEF1149 and ARSEF1166) shared the same alleles at all five SSR loci, respectively. None of the B. bassiana isolates tested showed the same SSR profiles as the commercialized isolates ATCC 74040 from product NaturalisÒ or GHA from product BotaniGardÒ. The discriminatory power (DL) of the five SSR markers applied here was different, with loci Ba01, Ba02 and Ba12 showing high indices around 0.9, respectively, while the DL of markers Ba08 and Ba13 was considerably lower with values around 0.65 (Table 1). This indicates that when using markers Ba01, Ba02 or Ba12 two B. bassiana samples displaying the same SSR profile are with a probability of more than 90% indeed identical. When bulk soil DNA extracted from all types of substrates analysed in the present study was used as template for diagnostic SSR reactions a couple of additional peaks, in particular in the lower molecular weight range below 90 bp, were sometimes visible in the chromatograms. These additional fragments were probably amplified from other microorganisms present in the DNA pool. Because they never had the same size as the five B. bassiana isolate ATCC 74040 specific SSR markers, and because none of the B. bassiana isolates studied here showed a strain specific amplification product below 90 bp, we excluded these fragments from further analysis.

2.6. Galleria bait method 3.2. Sensitivity of B. bassiana isolate specific SSR markers Infectivity of B. bassiana conidia applied to ED73 substrate was assessed through the Galleria bait method (Zimmermann, 1986). ED73 was inoculated with B. bassiana by mixing a 3% NaturalisÒ solution with substrate before filling inoculated substrate into respective pots as described in Section 2.5. Water was used as a control. Pots were installed in the greenhouse under the conditions described above and were watered regularly. 130 g of samples from the NaturalisÒ treated and control substrates were collected with sterile spatulas 7, 14, 28 and 48 days, respectively, after

Sensitivity of the five B. bassiana isolate specific SSR markers varied depending on the environment of the PCR assay. If pure genomic fungal B. bassiana DNA was used as a template for PCR assays, a sensitivity threshold of 0.1 pg DNA was evident in an agarose-gel based assay for all five SSR markers (Table 2). In the presence of total genomic soil DNA, the sensitivity was different depending on the SSR marker used, ranging from 1 ng (for marker Ba02) to 1 pg (for markers Ba01 and Ba08) B. bassiana DNA. If a B.

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bassiana conidial suspension was added to an unsterilized substrate and total genomic DNA was extracted subsequently, detection limits of B. bassiana conidia were also influenced by the marker used, with a threshold level of 5  107 conidia/ml for marker Ba13 down to 50 conidia/ml for marker Ba08 (Table 2, Suppl. 1A for marker Ba02). This equals DNA amounts of approximately 170 ng to 2 pg B. bassiana DNA. In case of plant (grapevine) DNA as a background for subsequent SSR marker detection, sensitivity levels were lower than for soil DNA, with thresholds of 100 pg B. bassiana DNA for all markers except for Ba08, which showed a threshold level of 10 pg (Table 2, Suppl. 1B for marker Ba02). If a B. bassiana conidial suspension was added to grapevine leaf material and total genomic DNA was extracted subsequently, detection limits of B. bassiana conidia were between threshold levels of 5  105 and 5  104 conidia/ml (Table 2, Suppl. 1B for marker Ba02), corresponding to 2 ng to 170 pg B. bassiana DNA.

3.3. Monitoring and infectivity of B. bassiana isolate ATCC 74040 in different potting substrates No B. bassiana specific SSR markers were amplified in total DNA extracted from the three different potting substrates prior to artificial inoculation with B. bassiana isolate ATCC 74040 (data not shown), indicating that no B. bassiana conidia were initially present in the respective substrate. In bulk DNA extracted from all three types of potting substrates inoculated with B. bassiana isolate ATCC 74040, respective SSR markers were amplified at least until 10 weeks after inoculation (Table 3). While primers Ba01 and Ba13 amplified diagnostic SSR fragments even up to 14 weeks after application of B. bassiana to the respective substrates, detection with primer Ba12 was possible up to 6 (ED73) or 10 weeks (Lignostrat, Eurohum) after inoculation. Galleria bioassays revealed that B. bassiana fungal conidia applied via mixing with ED73 substrate were present and infective until termination of the experiment (Fig. 2). There was no significant difference in infectivity of B. bassiana conidia over the time course of the experiment (7, 14, 28 and 48 days, respectively, after inoculation of substrates with fungal conidia, p = 0.69). Mortality of G. mellonella larvae was significantly higher in all soil samples inoculated with B. bassiana and at each time point after inoculation than in the control substrate samples (Mann–Whitney-U test: day 7 Z = 1.99, p = 0.046; day 14 Z = 2.02, p = 0.043; day 28 Z = 2.09, p = 0.037; day 48 Z = 2.02, p = 0.043). Eight days after start of the Galleria bioassays, 100% of the larvae were dead in the B. bassiana inoculated samples, while a maximum of 6% of the larvae were dead after incubation in the control samples (Fig. 2).

4. Discussion In the entomopathogenic fungus B. bassiana, microsatellite markers have been predominantly applied so far to study the genetic structure of different fungal populations (Estrada et al., 2007; Meyling et al., 2009; Rehner and Buckley, 2003; Wang et al., 2003). Here, we tested a set of previously published SSR markers for their discriminative power of different B. bassiana strains and used these markers to monitor the presence of a commercialized B. bassiana strain (ATCC 74040, active ingredient in the product NaturalisÒ) in different soil environments. Three of the five SSR markers analysed displayed a high level of discriminatory power in a subset of B. bassiana isolates from a world-wide collection, while two SSR markers did not fulfil the threshold index of 0.90, which is regarded as an acceptable level of confident discrimination (Hunter and Gaston, 1988). The two commercialized B. bassiana were sufficiently discriminated from all other isolates included in the present study. However, the given subset of 16 B. bassiana strains from a world-wide collection does probably not fully represent the overall diversity of B. bassiana and the calculated discriminatory power of the respective SSR markers might therefore be different if several isolates are analysed which have been obtained e.g. from the same field environment. Yet, in general, SSR markers are considered to have a high discriminatory power and have accordingly been used for isolate specific identification in other entomopathogenic fungi before. Enkerli et al. (2004) used six SSR markers specific for B. brongniartii and identified some of the applied strains up to 14 years after their application to different field sites. For the same fungus (B. brongniartii) and using the same set of SSR markers Schwarzenbach et al. (2007) have shown for the first time that a cultivation-independent and strain specific monitoring of an entomopathogen in bulk soil DNA using SSR markers is possible. However, the way of amplification of SSR markers as conducted in our and the above mentioned studies does not allow a quantification of B. bassiana conidia present in the respective samples. This would have been possible through the application of a strain specific real-time PCR (qPCR) assay, which has recently been developed for B. bassiana strain GHA (Castrillo et al., 2008), or for Metarhizium spp. using clade 1 specific primers (Schneider et al., 2011). Based on results of an agarose-gel based screening of SSR markers, sensitivity levels of the five B. bassiana isolate ATCC 74040 specific markers were dependent on the background genomic DNA of the respective PCR assay, with the lowest detection limit being evident for all five SSR markers in case pure genomic fungal DNA was used as a PCR template. Markers Ba08 and Ba01 showed the highest level of sensitivity if the assay was performed in the presence of soil DNA extracts. However, detection limits were different for all

Table 2 Sensitivity thresholds of PCR assays using five B. bassiana SSR markers for detecting B. bassiana strain ATCC 74040 in various environments.

a

SSR marker

Environment/Sensitivity threshold B. bassiana DNAa

B. bassiana DNA mixed with soil DNAb

B. bassiana conidia mixed with soilc

B. bassiana DNA mixed with plant DNAd

B. bassiana conidia mixed with plant leavese

Ba01 Ba02 Ba08 Ba12 Ba13

0.1 pg 0.1 pg 0.1 pg 0.1 pg 0.1 pg

1 pg 1 ng 1 pg 100 pg 10 pg

5  105 conidia/ml 1.74 ng 5  104 conidia/ml 0.174 ng 50 conidia/ml 1.74 pg 5  103 conidia/ml 17.4 pg 5  107 conidia/ml 174 ng

100 pg 100 pg 10 pg 100 pg 100 pg

5  105 conidia/ml 5  105 conidia/ml 5  104 conidia/ml 5  105 conidia/ml 5  104 conidia/ml

1.74 ng 1.74 ng 174 pg 1.74 ng 174 pg

Serial DNA dilutions of B. bassiana DNA from 100 ng/ll to 0.1 fg/ml were included in the assays. 50 ng soil DNA was spiked with 1 ll of serial dilutions (100 ng/ll to 1 pg/ll) of B. bassiana DNA. c 100 ll of serial dilutions of B. bassiana conidial suspensions from 5  108 conidia/ml to 50 conidia/ml were mixed with 50 mg unsterilized substrate ED73 before total DNA extraction. d 50 ng grapevine leave DNA was spiked with 2 ll of serial dilutions (100 ng/ll to 1 pg/ll) of B. bassiana DNA. e 50 ll of serial dilutions of B. bassiana conidial suspensions from 5  108 conidia/ml to 50 conidia/ml were mixed with 5–10 mg grapevine leaves before total DNA extraction. b

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Table 3 Presence or absence of five different SSR markers specific for B. bassiana isolate ATCC 74040 in samples from three different types of potting substrates during one to 14 weeks after inoculation of substrates with B. bassiana. SSR marker

Substrate

Weeks after inoculation 1

2

6

10

14

Ba01

ED73 Lignostrat Eurohum

+++ +++ +++

++ +++ ++

++ ++ ++

+++ +++ +++

++ ++ +++

Ba02

ED73 Lignostrat Eurohum

+++ +++ +++

+++ +++ +++

+ + +++

++ +++ +++

  +

Ba08

ED73 Lignostrat Eurohum

+++ +++ +++

+++ +++ +++

++ + +++

+ +++ +++

  +

Ba12

ED73 Lignostrat Eurohum

+++ + +++

++  ++

+ + +

 ++ +

  

Ba13

ED73 Lignostrat Eurohum

+++ +++ +++

++ +++ ++

++ ++ ++

+ ++ ++

+ + +++

+++ = SSR fragment of the expected size present in three independent replicates. ++ = SSR fragment of the expected size present in 2 out of 3 independent replicates. + = SSR fragment of the expected size present in 1 out of 3 independent replicates.  = SSR fragment of expected size not present in three independent replicates.

Number of dead G. mellonella larvae

10

Mean Mean±SE Mean±SD

8

6

4

2

0 Co 7

Co 14

Co 28

Co 48

Nat 7

Nat 14

Nat 28

Nat 48

Treatment (days after inoculation) Fig. 2. Boxplot of the number of dead Galleria mellonella larvae after 8 days in bioassays using soil samples inoculated with 3% NaturalisÒ (Nat) or water (Co) collected 7, 14, 28 and 48 days, respectively, after the treatment. Three replicates which 10 larvae per replicate were set up for each treatment and time point.

five SSR markers if soil DNA was present in the reactions, indicating that the SSR markers might have varying levels of susceptibility to PCR inhibitors like humic substances commonly encountered in soil DNA extracts (Braid et al., 2003; Smalla et al., 1993). In addition, sensitivity thresholds were approximately similar for three of the five SSR markers (Ba02, Ba08, Ba12) if B. bassiana conidia were directly added to unsterilized soil substrate and DNA was isolated subsequently. This indicates that DNA extraction efficiency of the method we used was acceptable and that a loss of detection sensitivity of these SSR markers is not to be expected if the test is applied for detection of B. bassiana conidia in bulk soil DNA extracts. Overall, these values are in accordance with detection limits reported in other cultivation independent PCR-based diagnostic assays for specific detection of B. bassiana (Castrillo et al., 2008) or other entomopathogenic fungi like M. anisopliae (Entz et al., 2005) or B. brongniartii (Schwarzenbach et al., 2007) in soil samples. Furthermore, presence of plant DNA decreased the sensitivity of SSR marker assays about tenfold, in particular if plant DNA was spiked with B. bassiana DNA. However, as we used DNA extracted

from grapevine leaves in our assays, these values might be different in the presence of DNA from other plant species. Grapevine is known to be rich in phenolic compounds and polysaccharides, which might inhibit subsequent PCR reactions and optimizations of the respective protocols used for DNA extraction could increase the sensitivity of the assay (Lemke et al., 2011). In addition, we used different DNA extraction kits for isolation of DNA from soil and plant samples, respectively, which could have different efficiencies in removing putative PCR inhibitors having an influence on PCR sensitivity. The apparent loss of sensitivity depending on the particular environment or sample type was not the same for the five markers tested. For example, markers Ba01 and Ba08 both had a sensitivity threshold of 1 pg if soil DNA was spiked with B. bassiana DNA, but threshold levels for both markers were different in all other environments tested (Table 2). The same was true for the different substrates tested, with substrate Eurohum apparently having the lowest inhibition effect on amplification of markers Ba02, Ba08, Ba13 (Table 3). At this point we can only speculate about possible mechanisms influencing this obvious difference in

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detection sensitivities of the individual SSR markers, which is however a well known general phenomenon of SSR markers (Selkoe and Toonen, 2006). One possible explanation might be that individual SSR markers are present in multiple copies in the B. bassiana genome which could influence detection sensitivity for a given marker; however, copy numbers of the five SSR markers used here remains to be tested. We applied these five SSR markers to track the presence of B. bassiana isolate ATCC 74040 in three different types of commercially available potting substrates. Strain specific products were amplified with all five primer pairs up to 10 weeks and with two primers up to 14 weeks after application of B. bassiana to the respective substrate. However, as the pure detection of DNA of the applied B. bassiana isolate in the substrate samples does not necessarily implicate viability and virulence of fungal conidia against putative target insects, our cultivation-independent approach was combined with a traditional baiting technique. Application of the Galleria bait method to one of the inoculated substrate types accordingly confirmed the infectivity of the B. bassiana isolate of at least up to 6 weeks after its application to the respective substrate. However, a more detailed picture on infectivity of fungal conidia over time would have been gained with a modified Galleria bait assay and an independent rearing of individual G. mellonella larvae. In our assay, 10 G. mellonella larvae were incubated together per replicate which could increase the likelihood of infection rates within one container, resulting in higher mortality rates than actually induced by the fungal inoculum present in the containers. Overall, in accordance with studies by Bruck and colleagues (Bruck, 2005, 2006, 2007; Bruck and Donahue, 2007) results of our study suggest that an incorporation of a B. bassiana based commercial biopesticide into different potting media used in containerized plant production could provide an alternative strategy for controlling soil-based insect pests. In case B. bassiana isolate ATCC 74040 is applied to field soils for the purpose of biological control of pest insects we recommend a two-step monitoring approach using the set of SSR markers evaluated here: First, a sufficient number of soil DNA samples obtained from the respective field should be analysed with SSR markers prior to the application of the fungal isolate to the field: This step should exclude the possibility that any indigenous fungal species or strains are present in the respective soil sample which show the same marker profile as the respective commercialized isolate. Accordingly, profiles of at least three of the five SSR markers evaluated here should be assessed for a certain period of time after the fungus has been applied to the field in a high-throughput multiplex reaction. We propose that an analysis of profiles of SSR markers Ba01, Ba08 and Ba12 showing the highest sensitivity in our assays should be sufficient for correct strain identification. For the development of effective integrated pest management strategies based on entomopathogenic fungi, the determination of field persistence of such a mycoinsecticide is an important prerequisite, as it obviously influences timing and frequency of applications. In addition, for registration purposes, the risks concerning the persistence of biological control agents have to be evaluated in order to assess their potential to spread and become established in the environment (Scheepmaker and Butt, 2010). In this regard molecular markers specific for commercialized strains of entomopathogenic fungi like the SSR markers applied in this study are important tools to track and identify a particular fungal strain in complex environmental samples and may have widespread applications in future molecular ecology studies of entomopathogens. Acknowledgments We thank Dustin Kulanek, Mirjam Hauck and Benno Gottwald for help in the laboratory and for performing the Galleria bioassay.

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This study was in part financially supported by the Federal Ministry of Food, Agriculture and Consumer Protection, Germany and a grant of the German Research Foundation (DFG) and the Department of Science and Technology (DST) New Delhi.

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