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Comparing virulence of North American Beauveria brongniartiiand commercial pathogenic fungi against Asian longhorned beetles

Tarryn A. Goble a,⇑, Stephen A. Rehner b, Stefan J. Long a, Sana Gardescu a, Ann E. Hajek a

a Department of Entomology, Cornell University, Ithaca, NY 14853-2601, USAb USDA-ARS, Systematic Mycology and Microbiology Laboratory, 10300 Baltimore Ave, Beltsville, MD 20705, USA

h i g h l i g h t s

� DNA analysis showed strain NBL 851is Beauveria asiatica, unlike NorthAmerican Beauveria brongniartii.� Commercial NBL 851 and

Metarhizium F52 were more effectivethan B. brongniartii in bioassays.� This is the first time B. brongniartii

from North America were evaluatedfor their virulence against an invasiveinsect pest.

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

a r t i c l e i n f o

Article history:Received 31 October 2013Accepted 12 February 2014Available online 22 February 2014

Keywords:Microbial controlEntomopathogenic fungusBeauveria asiaticaMetarhizium brunneumBioassayInoculation technique

a b s t r a c t

In the USA, the development and field application of Beauveria brongniartii (Sacc.) Petch (Hypocreales: Cla-vicipitaceae) to control the invasive Asian longhorned beetle, Anoplophora glabripennis (Motschulsky)(Coleoptera: Cerambycidae) have been hampered because it was unknown whether this fungal species isnative to North America. With the recent confirmation of the occurrence of B. brongniartii in North Americathere is renewed interest in this species, particularly as it is an effective pathogen of cerambycids in Japan.However, based on partial sequences of the nuclear intergenic BLOC region the commercially available B.brongniartii strain NBL 851 (Idemitsu Kosan, Tokyo, Japan) belongs instead to the species Beauveria asiaticaRehner and Humber. Further, bioassays using two inoculation methods confirmed that commercially avail-able strains of B. asiatica (NBL 851) and Metarhizium brunneum (F52) (Hypocreales: Clavicipitaceae) weresignificantly more virulent and resulted in lower median survival times (9.5–7.5 d) of A. glabripennis adultsthan two North American B. brongniartii isolates (ARSEF 6215 and ARSEF 10279) (24–31 d). The virulence ofNorth American B. brongniartii isolates is not well-documented in the literature. To our best knowledge thisis the first account of the virulence of native North American B. brongniartii being evaluated for biologicalcontrol of any invasive insect pest.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Several studies have reported results from bioassays of entomo-pathogenic Hypocreales, particularly the species Beauveria bassiana

(Bals-Criv.) Vuill., Beauveria brongniartii (Sacc.) Petch and Metarhiz-ium brunneum Petch (formerly M. anisopliae (Metsch.) Sorokin)against invasive Asian longhorned beetles, Anoplophora glabripen-nis (Motschulsky) (Coleoptera: Cerambycidae; ALB) (Dubois et al.,2004, 2008; Hajek et al., 2007). The application of M. brunneum(F52) as fungal bands for biological control of A. glabripennis hasbeen tested in the laboratory in the USA (Shanley et al., 2009;

http://dx.doi.org/10.1016/j.biocontrol.2014.02.0061049-9644/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author.E-mail address: tazgoble@gmail.com (T.A. Goble).

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Ugine et al., 2013) and in the field in China (Hajek et al., 2006). Fun-gal bands are made from fibre-material impregnated with culturesof fungal entomopathogens and placed around tree trunks or largebranches where pests become inoculated with conidia whenwalking across the bands (Dubois et al., 2004; Shanley et al.,2009). Fungal bands were first developed as a biological controlmethod against the adult life stages of longhorned beetles in orch-ards in Japan (Higuchi et al., 1997). Investigations in Japan reportedthat B. brongniartii was highly virulent and the most importantpathogen of cerambycids, including A. glabripennis, compared toother hypocrealean fungal species (Higuchi et al., 1997). As a re-sult, B. brongniartii strain NBL 851 was developed for the fungalband product Biolisa Kamikiri (now Idemitsu Kosan Co., Ltd., To-kyo, Japan), which has been commercially available since 1996(formerly produced by Nitto Denko) (Higuchi et al., 1997). In theUSA, fungal bands based on B. brongniartii could not be used as abiological control option against A. glabripennis because it wasthought that this fungal species was not native to North America.This has hampered the investigation, development and field appli-cation in the USA of fungal bands impregnated with B. brongniartiistrains (A.E. Hajek unpublished data).

Dubois et al. (2008) conducted a study investigating thepathogenicity and virulence of Japanese and Chinese isolates of B.brongniartii (NBL 851 and ARSEF 6827 [WU 20], respectively)compared to other hypocrealean entomopathogens including B.bassiana sensu lato (s.l.), Isaria farinosa and Metarhizium anisopliaes.l. Three isolates of M. anisopliae s.l. (Bischoff et al., 2009) (nowknown to include both M. brunneum and M. anisopliae) killed A.glabripennis faster than isolates of B. bassiana and I. farinosa andwere comparably virulent with the Japanese and Chinese B. bron-gniartii isolates. Accordingly, the emphasis for evaluation as a bio-logical control agent shifted to the development and use of M.brunneum F52 for control of A. glabripennis because this fungal spe-cies is also native in North America and strains were previouslyregistered for pest control in the USA (Dubois et al., 2008).

At the time of the Dubois et al. (2008) study however neitherthe Beauveria (Rehner et al., 2011) nor Metarhizium (Bischoffet al., 2009) revisionary monographs had been published, leavingthe taxonomic assignment of the species questionable. Subse-quently, B. brongniartii was confirmed to occur in North America(Humber pers. comm.; Rehner et al., 2011) and this finding re-newed interest in comparing isolates of B. brongniartii native toNorth America with the well-known, virulent commercial isolateof Japanese B. brongniartii, strain NBL 851. However, in an earliermolecular study, Rehner and Buckley (2005) demonstrated thatB. brongniartii consists of multiple phylogenetic partitions, possiblyindicating this taxon is a complex of more than one species. Thelack of a type specimen and clear species concept for B. brongniartiiat that time prevented delineation of the species and the status ofits sister lineages. The uncertain taxonomic status of Biolisa Kamik-iri thus presented a challenge to USA commercialization and use ofthis fungal band product.

An important goal of the present study was to assess the taxo-nomic position of the strains of entomopathogenic fungi used inbioassays in this study: presumptive B. brongniartii strains NBL851 (Japan) and ARSEF 6215 and ARSEF 10279 (North America)were sequenced at the nuclear intergenic BLOC locus andcompared phylogenetically with taxonomically validated Beauveriasequences of Rehner et al. (2011). Further, we compared the rela-tive virulence of strain NBL 851 against the above mentioned na-tive North American isolates of B. brongniartii and thecommercial strain M. brunneum F52, which is currently availablein the USA, using both suspension dip and press (the insect waspressed onto a sporulating fungal culture) bioassays against adultA. glabripennis. These bioassays were performed to ascertain whichfungal species represented the best candidate for biological control

of A. glabripennis. To the best of our knowledge this is the firstinvestigation of the virulence of native North American B. brongni-artii isolates against any invasive insect pest.

2. Materials and methods

2.1. Test insects

A. glabripennis larvae were reared on a cellulose-based diet (asper the Supplementary Appendix of Ugine et al., 2011) in a quaran-tine colony at the Sarkaria Arthropod Research Laboratory (SARL)at Cornell University, Ithaca, NY, USA. Adults were maintained at23 �C in 473-ml clear plastic containers and fed twigs of stripedmaple (Acer pensylvanicum L.) every 4–5 d throughout this study.All beetles used in experiments were 12–26 d (mean ± SD:19 ± 4) post-eclosion when inoculated with various fungal isolates.Average beetle weight was 0.9 ± 0.2 g. The number and size of bio-assays conducted were limited because these beetles have long lifecycles and are expensive to rear.

2.2. Fungal isolates and culturing

M. brunneum F52 (Novozymes Biologicals Inc., Salem, VA; ARSEF7711), the Beauveria NBL 851 strain (Idemitsu Kosan Co., Ltd., To-kyo, Japan) and two isolates of B. brongniartii (ARSEF 6215 and AR-SEF 10279) were obtained as lyophilized samples from the USDAAgricultural Research Service, or from cryogenically frozen sam-ples (�80 �C) (Table 1). In addition, the NBL 851 strain was passedthrough A. glabripennis (ALB) and subcultured from sporulatingcadavers in 2009 and then stored at �80 �C (referred to here asstrain 851 ALB). M. brunneum F52 was grown on PPDA (potato dex-trose agar plus 1% peptone) (Peng et al., 2011; Jaronski and Jackson,2012) while all other isolates were grown on quarter strength Sab-ouraud dextrose agar plus yeast extract (SDAY), supplementedwith 10 ml/L gentamycin and 25 mg/L cycloheximide. All cultureswere grown at 26 �C in the dark until sporulation was evident, usu-ally within 2 weeks.

2.3. Fungal identification by sequencing

Species diagnostic partial sequences between internal primersB22U_deg and B822L_deg of the nuclear intergenic region BLOCwere determined for isolates NBL 851, ARSEF 6215 and ARSEF10279. DNA extraction, PCR amplification, sequencing and dataediting were performed according to Rehner et al. (2011). These se-quences were aligned with corresponding data for 25 Beauveriastrains, focusing on the B. brongniartii species group, including B.amorpha (1; HQ880739), B. bassiana (1; HQ880692), B. asiatica (3;HQ880716–HQ880718), B. australis (3; HQ880719–HQ880721)and B. brongniartii (17; HQ880697–HQ880714) using the multiplesequence alignment program MAFFT (Katoh and Standley, 2013)under the FFT-NS-I option, yielding an alignment of length1524 bp. A search for the maximum likelihood tree and 1000 pseu-doreplicate bootstrap analyses were conducted under the GTR + Gsubstitution model of evolution using RaXML 7.0.4 (Stamatakis,2006) (Fig. 1) and the best likelihood tree is presented in Fig. 1.

2.4. Fungal press bioassays

All bioassays took place in a quarantine facility in the SarkariaArthropod Research Laboratory (SARL) at Cornell University, Ithaca,NY, USA. To inoculate beetles with the five fungal isolates, two16-mm agar plugs were cut from the peripheral areas of sporulat-ing culture plates with a cork borer and placed adjacent to eachother on a 9-cm diameter petri dish (Peng et al., 2011). Beetles

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were inoculated with conidia by holding the thorax of each insectand pressing the ventral surfaces onto the two plugs for 5 s. Afterinoculation, each beetle was placed individually into a 473-mlclear, plastic container with A. pensylvanicum twigs for 1 d(to allowexcess conidia to fall off the beetles). Beetles were then removedand placed into new containers with A. pensylvanicum twigs. Forisolates F52, NBL 851 and 851 ALB a total of 36 A. glabripennisadults (18 males and 18 females), representing three replicates of12 beetles each were exposed per sporulating fungal culture. ForB. brongniartii isolates (ARSEF 6215 and ARSEF 10279), only onereplicate of 12 beetles for each isolate was exposed to fungal cul-tures. Thirty-six control beetles (18 males and 18 females), whichrepresented three replicates, were pressed onto sterile agar cores.Replicates were conducted on separate days to ensure indepen-dence. Inoculated beetles were kept at 23 �C at 16:8 h L:D for50 d, checked daily for mortality and cadavers were checked formycosis. Mycosis is not always overt and beetles that did not ap-pear to be sporulating were broken open to look for fungus insidethe body cavity.

To quantify the density of viable conidia from the fungal cul-tures used for bioassays, three 16-mm diameter cores were re-moved from peripheral areas of each culture plate, homogenizedindividually in 0.05% Tween 80, and conidia were counted usinga haemocytometer (Peng et al., 2011). The percentage of viableconidia was determined by suspending 1 mg of conidia from eachof the fungal isolates in 5-ml of 0.01% aqueous Silwet and immedi-ately spreading a 100-ll aliquot onto each of two 6-cm diameterpetri dishes containing SDAY (Inglis et al., 2012). Dishes weresealed with parafilm and incubated 16 h at 25 ± 1 �C in darkness.The first 100–200 conidia observed at 400� at two arbitrary loca-tions were scored for germination for both of the replicate petridishes. Numbers of conidia per unit area were corrected for viabil-ity by multiplying the mean conidial density by the mean propor-tion germinating. Cultures on several petri dishes were used forstudies and counts of viable conidia from individual plates wereaveraged to determine exposure concentrations. All fungal plugscontained >1 � 108 conidia cm2.

2.5. Fungal dip bioassays

Conidia were dry-harvested from sporulating cultures using asterilized camel’s hair brush (17-mm long bristles) and collectedin 50-ml conical polypropylene centrifuge tubes (Castrillo et al.,2008). Conidia were suspended in sterile deionized water with0.05% Tween 80 and 1 g of 3-mm diameter glass beads and mixedfor 15 min on a wrist-action shaker (Burrell Scientific, Pittsburgh,PA) to produce a homogeneous suspension. Conidial stock concen-trations were determined using a haemocytometer followed by

dilution to a final concentration of 1 � 107 conidia ml�1 in steriledeionized water containing 0.05% Tween 80. The percentage of via-ble conidia was determined as described above (Inglis et al., 2012)and all isolates germinated above 88%.

A freshly prepared 50-ml centrifuge tube containing 15-ml of a1 � 107 conidia ml�1 conidial suspension was vortex-mixed andused to inoculate beetles individually. For each of the five isolates,24 A. glabripennis adults (12 males and 12 females) representingthree replicates of eight beetles each were submerged in the conid-ial suspensions for 10 s. Twenty-four control beetles were dippedin 15-ml of sterile deionized water containing 0.05% Tween 80.Replicates were conducted on separate days to ensure indepen-dence. The tubes were inverted gently to ensure total insect cover-age. After inoculation, each beetle was placed individually into a473-ml plastic container containing A. pensylvanicum twigs for1 d(to allow any excess suspension to dry and to maintain continu-ity between bioassays). Beetles were then removed and placed intonew containers with A. pensylvanicum twigs. Beetles were kept at23 �C at 16:8 h L:D for 50 d, checked daily for mortality and cadav-ers were checked for mycosis as in press bioassays.

2.6. Determining press and dip bioassay doses

The actual dose of conidia received per beetle for both the pressand dip bioassays was assessed. For press conidial inoculations,four A. glabripennis adults (two males and two females) wereexposed to each fungal isolate via plugs as described above andthen placed individually into 473-ml plastic containers with A. pen-sylvanicum twigs for 3 h. Beetles were then removed and rinsedindividually three times in 50-ml centrifuge tubes, each containing15-ml dichloromethane (DCM), as described in Ugine et al. (2013).Similarly, for quantification of dip conidial inoculation, threereplicates of four A. glabripennis adults (six males and six females)were exposed to fungal isolates by dipping beetles in a 1 � 107

conidia ml�1 conidial suspension as described above. Beetles wererinsed in DCM 3 h after exposures, following the same procedure.For both types of bioassays, conidial quantification was undertakenusing a haemocytometer and the conidia acquired per beetle wasdetermined by making two separate counts of the suspension(Ugine et al., 2013).

2.7. Statistical analyses

Statistical analyses were conducted using JMP version 10 (SASInstitute, 2012). Median survival times were estimated separatelyfor press and dip bioassays using Kaplan–Meier analysis. Beetlesurvival in press and dip bioassays was analyzed separately withproportional hazards models which included fungal isolate,

Table 1The origin, host and isolation date of the Hypocrealean fungal isolates investigated in this study.

Fungal species Isolate Origin Host Host stage Isolationdate

Metarhiziumbrunneum

ARSEF 7711 (F-52) b Vienna, Austria Cydia pomonella (Lepidoptera: Tortricidae) Larva 1971

Beauveria asiatica a NBL 851 c Gunma, Japan Psacothea hilaris (Coleoptera: Cerambycidae) Adult 1980Beauveria asiatica a NBL 851 (ALB) d Subcultured at Cornell University,

New York, USAAnoplophora glabripennis (Coleoptera:Cerambycidae)

Adult 2009

Beauveria brongniartii ARSEF 6215 Yellow Barn State Forest, Dryden,New York, USA

Barypeithes pellucidus (Coleoptera: Curculionidae) Adult 1998

Beauveria brongniartii ARSEF 10279 Polk County, Oregon, USA Soil-rhizosphere of grape crop Soil 2009

a See Fig. 1 and explanation in text.b The active ingredient of several commercial products in America, ARSEF 7711 called F52 by Novozymes Biologicals.c The active ingredient of the commercial product Biolisa Kamikiri (Idemitsu Kosan Co., Ltd., Tokyo, Japan).d The isolate was passed through Anoplophora glabripennis prior to bioassays in this study.

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replicate (if appropriate), beetle gender, beetle age and weight asparameters. For numbers of conidia acquired by beetles in pressor dip treatments and the density of viable conidia to which beetleswere exposed on plugs, all non-normally distributed data were log-transformed and subjected to ANOVA. Mixed model ANOVA wasused to compare the isolates, with beetle gender and beetle weight.

3. Results and discussion

3.1. Fungal identification by sequencing

A partial phylogeny of the Beauveria genus, focusing on the B.brongniartii species group inferred from the maximum likelihoodanalysis of partial BLOC sequences, demonstrated 100% bootstrapsupport for the inclusion of strain NBL 851 within the B. asiaticaspecies clade (Fig. 1). B. asiatica is a member of the B. brongniartiis.s. complex and is characterized by having broadly ellipsoid or ob-long conidia, slightly larger than those of B. brongniartii s.s (Rehneret al., 2011). We herewith refer to strain NBL 851 as B. asiatica(Rehner and Humber, sp. nov). Kawakami (1978) originally isolated

strain NBL 851 from a cadaver of the yellow spotted longhornbeetle, Psacothea hilaris Pascoe (Coleoptera: Cerambycidae) andidentified it as B. tenella (Delacroix) Siemaszko, a synonym of B.brongniartii (Higuchi et al., 1997). The species name B. brongniartiiwas later used by researchers in Japan (Shimazu, 1994; Higuchiet al., 1997). Kawakami’s (1978) experiments indicated that strainNBL 851 was highly virulent against various species of longhornedbeetles but relatively benign towards other insects, specificallysilkworms (Kawakami, 1978; Higuchi et al., 1997). It was con-cluded that the specificity of infection of strain NBL 851 towardslonghorned beetles qualified it as a promising candidate for biolog-ical control of cerambycid pests in Japan (Kawakami, 1978).However, Wada et al. (2003) later showed that strains of JapaneseB. brongniartii isolated from P. hilaris (including those isolatedwhen strain NBL 851 was found) and other cerambycids differedin genetic and morphological characteristics from strains of B.brongniartii isolated from scarab beetles (Coleoptera: Scarabaei-dae). RFLP banding patterns separated these B. cf. brongniartii intotwo distinct groups, with one group composed mainly of isolatesfrom longhorned beetles and the other group comprised mainlyof isolates from scarabs (Wada et al., 2003). They observed that

B. amorpha

B. bassiana

B. asiatica

B. brongniartii

B. australis

97

99

100

100

100

70

100

74

100

ARSEF_1564 Italy

ARSEF_4580 Australia

ARSEF_4622 Australia

ARSEF_4362 Japan

ARSEF_4363 Japan

ARSEF_7978 Canada, BC

ARSEF_7376 USA, MD

NBL_851 Japan

ARSEF_4850 Korea

ARSEF_4384 China

ARSEF_4474 China

ARSEF_2271 USA, KY

ARSEF_2831 USA, MD

ARSEF_7058 Canada, BC

ARSEF_6215 USA, NY

ARSEF_4031 Denmark

ARSEF_10279 USA, OR

ARSEF_5510 Norway

ARSEF_7971 Canada, BC

ARSEF_7977 USA, WA

ARSEF_7268 Korea

ARSEF_617 France

ARSEF_979 France

ARSEF_1069 Switzerland

ARSEF_985 Japan

ARSEF_7517 Japan

ARSEF_4598 Australia

ARSEF_2641 Brazil

Fig. 1. A partial phylogeny of the Beauveria genus, focusing on the B. brongniartii s.s species group inferred from the maximum likelihood of partial BLOC sequences (1524 bp),as well as the relevant bootstrap support values. Isolates included in bioassays are highlighted in gray.

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the shape of conidia from isolates obtained from longhorned bee-tles was larger and ovoid and these isolates did not produce thered pigment oosporein (Kawakami, 1978; Wada et al., 2003). Incontrast, those isolates obtained from scarabs had long-ovoid con-idia and produced red pigment in culture media.

Our study provides evidence that previously described B. bron-gniartii strain NBL 851 belongs to the B. asiatica species group.Based on this evidence we propose that the observed differencesin B. brongniartii virulence and host specificity demonstrated dur-ing studies by Kawakami (1978) as well as genetic and morpholog-ical differences observed in groups of B. brongniartii isolated fromcerambycids and scarabs during the study by Wada et al. (2003)were caused by working with two separate fungal species. Thistentatively suggests that a group within the B. brongniartii complexmay be more specific to scarabs while B. asiatica may be more spe-cific towards cerambycids. Further, if B. asiatica is more specific to-wards cerambycids, future biological control efforts againstcerambycids could focus more directly on strains of B. asiatica(especially in Asia) instead of B. brongniartii. Commercializationof B. asiatica in the USA for biological control of A. glabripennismight be difficult as at present, this species is not known fromNorth America (Rehner et al., 2011).

Other fungal isolates in our study, ARSEF 6215 and ARSEF10279, were confirmed as belonging to the B. brongniartii speciescomplex with 99% bootstrap support (Fig. 1). These isolates wereoriginally recovered in the US from a weevil (Coleoptera: Curculi-onidae) and from the rhizosphere of grapes, respectively, and donot fall into the two suggested groups of scarab-specific or ceram-bycid-specific isolates. Therefore, it is hypothesized that the spec-ificity and virulence of these B. brongniartii complex isolates maybe more benign towards A. glabripennis. We suggest that furthercollection and evaluation of host specificity of cerambycid- andscarab-associated pathogenic strains from the B. brongniartii spe-cies complex will be necessary to understand the extent to whichthe proposed specificity pattern exists.

3.2. Dip and press bioassays

Results from both types of bioassays confirmed greater viru-lence of the commercial strain of B. asiatica toward cerambycidscompared to two US strains of B. brongniartii. In press bioassays,in which adult beetles were pressed onto conidia-covered agar,both the original strain of B. asiatica (NBL 851) and the strain re-isolated from A. glabripennis (=851 ALB) did not differ from eachother and killed 50% of the test beetles within 10.5 and 11.5 d,respectively. M. brunneum F52 was significantly more virulent thaneither of the B. asiatica isolates (NBL 851; 851 ALB) against A. glab-ripennis and had a median survival time (ST50) of 9.5 d (Fig. 2A).This might have been due to the fact that more M. brunneum con-idia were retained on beetles compared to either of the two B. asi-atica isolates. However, despite many conidia being acquired bybeetles when pressed onto cultures of the two North American B.brongniartii isolates, virulent isolates were able to kill beetlessignificantly faster .(v2

5 ¼ 70:82, P < 0.0001) than either of the B.brongniartii isolates tested. Median survival times for B. brongniartiiARSEF 10279 and ARSEF 6215 were 24.5 and 33.5 d, respectively,which differed from the untreated controls (Fig. 2A). None of the36 control beetles died during the 50 d monitoring period in pressbioassays. All five fungal cultures were sporulating above 1 � 108

conidia/cm2. However, the actual dose received by beetles whenpressed onto conidia differed significantly (F4,15 = 42.08,P < 0.0001). Beetles acquired the highest doses of conidia when ex-posed to B. brongniartii ARSEF 10279 (4.59 � 107 conidia/beetle),M. brunneum F52 (1.39 � 107 conidia/beetle) and ARSEF 6215(6.43 � 106 conidia/beetle) as compared to both B. asiatica isolates(NBL 851 and 851 ALB; Fig. 2B). Peng et al. (2011) reported similar

dose acquisition with M. brunneum F52 (1.52 � 107 conidia/beetle)immediately after A. glabripennis were exposed by pressing beetlesto fungal cultures sporulating at 2.06 � 108 conidia/cm2. Further,we found a weakly significant correlation between increased doseacquisition and profuse sporulation on the agar culture surface(F1,13 = 6.23, P < 0.026). Shanley et al. (2009) however found astrong correlation between increased conidial concentration, mea-sured as the density of viable conidia/cm2 on impregnated fungalbands, and a decrease in days to death by A. glabripennis.

In dip bioassays, the virulence of M. brunneum F52 as comparedto B. asiatica NBL 851 was not significantly different; the isolateskilled 50% of test beetles in 7.5 and 9.5 d, respectively (Fig. 3A). Du-bois et al. (2008) reported similar median survival times for M.brunneum F52 (6 d) and B. asiatica NBL 851 (9 d) using dip bioas-says at the same conidial concentration against A. glabripennis. Asignificant lag in A. glabripennis mortality following treatment withthe re-isolated 851 ALB strain was observed which had a medianlethal time of 13 d (Fig. 3A). Fewer conidia were acquired and re-tained by beetles exposed to 851 ALB compared to the other fungalisolates (Fig. 3B) which may explain this anomaly. Significantlymore conidia (F4,55 = 35.74, P < 0.0001) were acquired by beetleswhen exposed to strains NBL 851 (9.42 � 105 conidia/beetle) andB. brongniartii ARSEF 10279 (1.13 � 106 conidia/beetle) after beingdipped into conidial suspensions. Overall, the number of conidiaacquired by beetles exposed by dipping was significantly lower(F1,28 = 45.47, P < 0.0001) than when beetles were pressed ontoconidia-covered agar. Despite lower acquired conidia, virulent iso-lates such as M. brunneum F52 and B. asiatica NBL 851 still killedbeetles faster in dip bioassays compared to the press bioassays.During dip bioassays, conidial adhesion may have been affectedby the surfactant (Tween 80) which may have lowered the hydro-phobicity of conidia and therefore lowered the total acquisition ofconidia on some beetles (Holder and Keyhani, 2005; Inglis et al.,2012). However, dip treatments ensured that the entire insectbody, including thinner areas of the cuticle, were sufficiently ex-posed to fungal conidia compared with the press bioassays. Thus,the surfactant may have facilitated placement of more conidia ineasily-penetrated parts of the beetles’ bodies. When insects werepressed onto sporulating surfaces in press bioassays they may haveacquired many conidia but the total surface area for adhesion andsubsequent germination was smaller than with dipping. Further,Butt et al. (2006) suggested that virulent fungal isolates germinatequickly; thus, while there may have been fewer conidia quantifiedfrom M. brunneum F52 and B. asiatica NBL 851 dipped insects, theseisolates may have penetrated the cuticle and caused infection morerapidly.

This was not true of the B. brongniartii isolates, as they took sig-nificantly longer (v2

5 ¼ 111:10, P < 0.0001) to kill beetles when in-sects were dipped rather than pressed. This result rules out thepossibility that the aqueous suspension used to dip beetles mayhave contributed to the hydration of conidia and ‘primed’ themto germinate (Lopes et al., 2013). The two North American B. bron-gniartii isolates were less virulent against beetles and medianlethal times for ARSEF 6215 (31 d) and ARSEF 10279 (40.5 d) werenot statistically significantly different from the untreated controls(Fig. 3A). Only one of the 24 control beetles died during the 50 ddip bioassays. Mortality levels and rates after exposure to hypo-crealean fungi are dose-related (Inglis et al., 2012). This trendwas observed in the median survival times for A. glabripennis ex-posed to weaker B. brongniartii isolates in both types of bioassays.It took longer for beetles to die when they were exposed to diptreatments (1 � 107 conidia/ml�1), where the acquired dose waslower than when beetles were pressed onto sporulating fungal cul-tures (>1 � 108 conidia/ml�1). This trend however was not ob-served for virulent fungal isolates, particularly when acquireddoses of B. asiatica NBL 851 conidia were lower in dip bioassays.

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Yet median survival time for beetles exposed to NBL 851 was sig-nificantly shorter compared to all other isolates except M. brunne-um F52 (Fig. 2A and B). While beetles may have acquired fewerconidia from virulent isolates in dips, those conidia that were re-tained on the body may have infected and grown quickly as a resultof their invasive properties (i.e., host recognition that allows quickadhesion, germination, penetration and colonization; Butt et al.,2006). Conidia of fungal isolates can differ in adhesion but high lev-els of adhesion are not always correlated with lower median sur-vival times, for example high levels of ARSEF 10279 conidia wereretained on beetles but median survival times were high (Fig. 3A,B). Further, although the counts of conidia removed by the DCMprovided an estimate of the total conidia load acquired by beetles,significant variation among beetles (as a result of low samplesizes), inoculation method and intrinsic fungal isolate propertiesmay exist which could skew the final count of conidia acquiredand do not correlate with mortality estimates (Fernandez et al.,2001; Butt et al., 2006; Peng et al., 2011), as was observed withB. brongniartii isolate ARSEF 10279.

The effects of in vitro subculture on the virulence and morpho-logical characteristics of entomopathogenic fungi appear to varyconsiderably among isolates and species (Brownbridge et al.,2001). Some studies report a loss in virulence for some strains ofhypocrealean species such as B. bassiana and Nomuraea rileyi (Far-low) Samson (Schaerffenberg, 1964; Morrow et al., 1989), while inother studies, isolates of B. bassiana and B. brongniartii appear tomaintain virulence (Brownbridge et al., 2001; Strasser and Pern-fuss, 2005). In the present study, B. asiatica NBL 851 strain was

passed through A. glabripennis and sub-cultured from sporulatingcadavers in 2009 (851 ALB). The press bioassay results in this studysuggested that there was increased virulence towards A. glabripen-nis when the re-isolated strain (851 ALB) was compared with theoriginal strain (NBL 851), although differences were not statisti-cally significant. However, in dip bioassays, median survival timeswere significantly greater for beetles exposed to the re-isolatedstrain. The lower acquisition of conidia by beetles may explain thisanomaly but overall results suggest that passaging NBL 851through A. glabripennis did not increase virulence towards theinsect.

Bioassay results showed that strains of B. asiatica (NBL 851) andM. brunneum (F52) are more virulent against A. glabripennis adults,compared with the North America B. brongniartii isolates used inthis study. While pathogenicity data for only two North Americanisolates are reported here, we did investigate three additionalNorth American B. brongniartii isolates (unpublished data) and allcaused lower mortality than the isolates included in the currentstudy. In bioassays using the procedures described above, B. bron-gniartii ARSEF 2271 caused an average of 37% mortality in 50 d inboth press and dip bioassays, ARSEF 6214 averaged 33%, and ARSEF10277 averaged 46% mortality. This further highlights that B. bron-gniartii isolates appear to be less effective than other species ofhypocrealean fungi against A. glabripennis.

4. Conclusion

Molecular analyses conducted here identified strain NBL 851 asB. asiatica and not B. brongniartii as previously reported. Bioassay

Fig. 2. (A) Percentage survival of Anoplophora glabripennis adults over time afterexposure to one of five fungal press treatments or control exposures. (B) Mean (±SE)numbers of conidia retained on beetles 3 h after press treatments. Different lettersrepresent statistically significant differences.

Fig. 3. (A) Percentage survival of Anoplophora glabripennis adults over time afterexposure to one of five fungal dip treatments or control dips. (B) Mean (±SE)numbers of conidia retained on beetles 3 h after dip treatment. Different lettersrepresent statistically significant differences.

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results showed that strains of B. asiatica (NBL 851) and M. brunne-um (F52) are more virulent against A. glabripennis adults comparedto the North America B. brongniartii isolates used in this study.Future research and biological control efforts against this invasiveinsect pest should focus on M. brunneum (F52) and B. asiatica (NBL851), although the current lack of information on naturalprevalence of B. asiatica, and its origin in Asia, will likely limit itsuse in the United States. The North American B. brongniartii isolatesincluded in this study did not show any biological controlpotential.

Acknowledgments

We thank our laboratory assistants and technicians, especiallyMeghan Roblee, Jake Henry and George Lu. The AlphawoodFoundation supported the beetle colony and the Litwin Foundationprovided research support.

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