Isolation and characterization of Beauveria spp. associated with exotic bark beetles in New Zealand Pinus radiata plantation forests

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Isolation and characterization of endophytic Beauveria spp. (Ascomycota:Hypocreales) from Pinus radiata in New Zealand forests

S.D. Reay a, M. Brownbridge b,*, B. Gicquel b, N.J. Cummings c, T.L. Nelson b

a Silver Bullet Forest Research, Auckland, New Zealandb AgResearch Ltd., Lincoln Research Centre, Private Bag 4749, Christchurch 8140, New Zealandc Canterbury University, Christchurch, New Zealand

a r t i c l e i n f o

Article history:Received 4 December 2009Accepted 8 March 2010Available online 11 March 2010

Keywords:EndophytesPinus radiataBeauveria spp.Hylastes aterHylurgus ligniperdaBiological control

a b s t r a c t

New Zealand Pinus radiata and associated exotic and native trees were surveyed for presence of Beauveriabassiana. Samples were collected from trees located in 33 sites in four geographically distinct areas ofNew Zealand. Needle samples were taken at all sites, while seed samples were taken from a single treeat one site and four root samples were collected from two sites where P. radiata seedlings were present.The bulk of the needle samples were from P. radiata, but exotic and indigenous trees growing in the samevicinity were also periodically sampled. After processing plant material from 167 trees, 21 fungal isolatesresembling Beauveria spp. were recovered; DNA sequence analysis of a terminal region of the EF1-a locusshowed that these isolates all classified as B. bassiana Clade A. The pathogenicity of these isolates againstHylurgus ligniperda adults and Tenebrio molitor larvae was confirmed in laboratory bioassays. This studydemonstrated widespread occurrence of B. bassiana as endophytes of pine in New Zealand. This associa-tion may provide new opportunities to regulate insect pests of pine using a biological control agent.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Endophytes may be broadly defined as microbes that live inhealthy plant tissue (Hyde and Soytong, 2008). Commonly, theseare bacteria and fungi that that have no effect on, or a beneficialrelationship with their host, including the ability to naturally con-fer resistance to pests and diseases (Backman and Sikora, 2008). Adiverse range of fungal endophytes have been reported from foli-age, seeds and roots of coniferous trees, including spruce (Piceaspp.), fir (Pseudotsuga spp., Abies spp.) and pine (Pinus spp.) (Carrolland Carroll, 1978; Johnson and Whitney, 1989; Hata and Futai,1996; Hata et al., 1998; Guo et al., 2003; Hoff et al., 2004; Ganleyand Newcombe, 2006; Stefani and Bérubé, 2006; Sokolski et al.,2007) and the hardwood Carpinus caroliniana Walter (Americanhornbeam) (Bills and Polishook, 1991). Hypocrealean fungi havebeen documented in these surveys (Stefani and Bérubé, 2006),including the insect pathogen Beauveria bassiana (Balsamo) Vuille-min (Bills and Polishook, 1991; Ganley and Newcombe, 2006).

Beauveria bassiana is a recognized endophyte that occurs natu-rally in, or has been successfully introduced into a diverse range ofplant species, including maize (Bing and Lewis, 1991; Wagner andLewis, 2000; Cherry et al., 2004), tomato (Ownley et al., 2004), co-

coa (Posada and Vega, 2005; Vega et al., 2008), coffee (Posada et al.,2007; Vega et al., 2008), bananas (Akello et al., 2007), date palm(Gómez-Vidal et al., 2006), American hornbeam (Bills and Polis-hook, 1991), and opium poppy (Quesada-Moraga et al., 2006,2009). In several instances colonization of plant tissues by the fun-gus has provided protection against insect damage, or has inhibitedinsect development and establishment (Bing and Lewis, 1991;Cherry et al., 2004; Ownley et al., 2004; Akello et al., 2008; Vegaet al., 2008; Quesada-Moraga et al., 2009).

Ganley and Newcombe (2006) isolated B. bassiana from Pinusmonticola Dougl. ex D. Don but the wider prevalence of the fungusin pines has not been documented, and the potential utilization ofsuch endophytic relationships to mitigate insect pests in plantationforestry has not been investigated. Pinus radiata D. Don is the pre-dominant species grown in plantation forests in New Zealand.Feeding damage caused by cryptic insect pests such as the intro-duced bark beetle Hylastes ater (Paykull) (Curculionidae: Scolyti-nae) can periodically lead to significant mortality of newlyplanted pine seedlings (Reay et al., 2002, 2005). Hylurgus ligniperda(F.) (Curculionidae: Scolytinae) is another exotic bark beetle pestthat is currently only a minor pest of pines in New Zealand butcauses considerable damage to regenerative forests in Chile (Bain,1977; Mausel et al., 2006). Entomopathogenic fungi can be impor-tant natural mortality factors of bark beetles, although their overallimpact on beetle populations is estimated to be relatively low(Balazy, 1968). Fungi in the genus Beauveria have been commonlyisolated from bark beetles and their associated habitat, including

1049-9644/$ - see front matter � 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.biocontrol.2010.03.002

* Corresponding author. Present address: Vineland Research and InnovationCentre, 4890 Victoria Ave. N., PO Box 4000, Vineland Station, Ont., Canada L0R 2E0.Fax: +1 905 562 0084.

E-mail address: [email protected] (M. Brownbridge).

Biological Control 54 (2010) 52–60

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those present in New Zealand (Wegensteiner, 2004; Glare et al.,2008; Reay et al., 2008). Metarhizium flavoviride var. pemphigi Dri-ver & Milner and Hirsutella guignardii (Maheu) Samson, Rombach &Seifert were recently recovered from H. ater cadavers, but have lowprevalence within the bark beetle population (Brownbridge et al.,2010).

As a first step in the process to assess the role endophyticentomopathogens may play in insect pest management in NewZealand pines, a survey was undertaken to establish whetherBeauveria spp. are present as endophytes in P. radiata in New Zea-land, to determine their prevalence and distribution, to character-ize any isolated endophytic Beauveria spp. associated with P.radiata, and to determine whether isolated endophytes are path-ogenic to a model insect (Tenebrio molitor L.) and a target pest (H.ligniperda).

2. Materials and methods

2.1. Collection of needles, seeds and roots

Asymptomatic foliage, seeds and roots were sampled from arange of trees and stand types in New Zealand from February 2008through January 2009. In total, 33 sites were visited in a non-system-atic sampling strategy in four regions of New Zealand: Auckland (7sites), central North Island (8 sites), Canterbury (15 sites), and theWest Coast (South Island) (3 sites). Sites were selected when P. rad-iata was present. The number of trees sampled in each site variedfrom one to six and was chosen arbitrarily. At sites where there weremixed stands, foliar samples were taken from other tree species,including P. contorta Douglas ex Louden (Pinaceae), P. coulter D.Don (Pinaceae), P. nigra Arnold (Pinaceae), P. pinaster Ait. (Pinaceae),Pseudotsuga menziesii (Mirb.) Franco (Pinaceae), Picea obovata Ledeb.(Pinaceae), Sequoia sempervirens (Lamb ex D. Don) Endl. (Taxodia-ceae), Larix sp. (Pinaceae), Podocarpus totara G. Benn. ex D. Don(Podocarpeceae), Pseudowintera colorata (Raoul) Dandy (Wintera-ceae), Melicytus ramiflorus J.R. Forst. & G. Forst. (Violaceae), Pittospo-rum tenuifolium Sol. ex Gaertn. (Pittosporaceae), Hedycarya arboreaJ.R. Forst. & G. Forst. (Monimiaceae), Coprosma rotundifolia A. Cunn.(Rubiaceae), and Kunzea ericoides (A. Rich.) Joy Thomps. (Myrtaceae).

In total, 167 trees were sampled. Of these 132 were from thegenus Pinus, with the majority (125) being P. radiata. Trees sam-pled were found as single specimens, in hedgerows in rural areas,small forest blocks (<5 ha) or large plantation forests. At the time ofsampling all trees appeared in good health. Needle samples weretaken from every pine, while four root samples and one seed sam-ple were collected.

Needles (approx. 6–10 cm length) were sampled from the low-er canopy of trees by removing one short branch (approx. 10 cm)and placing it into a labeled sterile plastic bag, which was storedin a refrigerated bin until it could be processed (within 48 h).Whenever possible, second and/or third year needles were prefer-entially selected as these have been found to contain a greaterproportion of fungal endophytes (Johnson and Whitney, 1992).While we acknowledge the limitations of this sampling scheme,particularly in determining whether Beauveria can exist as anendophyte in other tree tissues, e.g., in the cambium layer, theease with which foliage can be collected enabled a wider diversityand number of samples to be taken and processed. Four coneswere sampled from one tree (seeds were later removed for pro-cessing) and roots were sampled from four seedlings and weresimilarly held until they could be processed. Needles were alsotaken from trees where cones and roots were sampled. For non-coniferous species up to six leaves (including leaf stalk) were re-moved from lower branches and placed into a sterile plastic bagas described above.

2.2. Isolation of fungal endophytes

From each branch six needles (or leaves) were arbitrarily se-lected. Four seeds were arbitrarily selected from each of four cones,and one 5 cm root sample was selected from each of four seedlingroot masses. The needle, seed and root samples were surface ster-ilized by soaking in ethanol (96%) for 1 min, followed by 10% so-dium hypochlorite (NaOCl) for 5 min, before rinsing twice insterile distilled water for 1 min. To confirm the efficiency of thesurface sterilization methods, 100 ll of the second rinse waterwas spread onto quarter-strength potato dextrose agar (PDA;Merck, Darmstadt, Germany) and plates were incubated at 20 �Cfor 14 days. The absence of fungal or bacterial growth on the med-ium confirmed the reliability of this sterilization procedure. Thismethod has previously been shown to effectively kill epiphytic fau-na (Arnold et al., 2000; Schulz et al., 1997).

After sterilization each needle or root was cut into three sec-tions, and each seed was cut into thin (�1 mm) sections. Sampleswere placed onto quarter strength PDA containing antibiotics(streptomycin sulfate 350 mg/l�1, Sigma, St. Louis, MO, USA; tetra-cycline hydrochloride 50 mg/l�1, Sigma, St. Louis, MO, USA; andcycloheximide 125 mg/l�1, Sigma, St. Louis, MO, USA). Plates weresealed with Parafilm and incubated at 20 �C for 10–14 days. Devel-oping colonies that appeared to be Beauveria sp. were then subcul-tured onto new PDA-antibiotic plates which were incubated at20 �C. The identity of Beauveria spp. colonies was confirmed onthe basis of their general morphological characteristics by visualexamination of sporulating cultures under a dissecting microscope(at 40�). A representative selection of isolates was deposited in theAgResearch Insect Pathogen Culture Collection (Lincoln, New Zea-land). Culture collection numbers for the isolates and their geo-graphic and host tree origins are given in Table 1. A B. bassianaendophyte isolated from Pinus monticola (Ganley and Newcombe,2006) was included for comparison in the sequencing analysisand bioassays.

2.3. DNA isolation, PCR and sequencing

All Beauveria spp. isolates were grown by spread-plating conidiaonto PDA overlaid with colorless cellophane. Plates were incubatedat 20 �C for 24–48 h, or until a fine layer of hyphal growth was vis-ible across the surface of the cellophane.

Microcentrifuge tubes and pestles were placed in liquid nitro-gen. Small amounts of hyphae were added to the tubes using asterile spatula, frozen and immediately ground into a fine powder.The tubes were removed from the liquid nitrogen and DNA was ex-tracted from the ground hyphae using a DNeasy Plant Kit (Qiagen,Hilden, Germany).

The concentration of DNA used in the PCR reactions was deter-mined empirically and ranged from 1 to a 10� dilution of the ini-tial isolation. Amplification of a terminal region of the elongationfactor 1-alpha (EF1-a) locus was performed using the primers1577F (50-CARGAYGTBTACAAGATYGGTGG) and 2218R (50-ATGACACCRACRGCRACRGTYTG) (Rehner and Buckley, 2005). PCR reac-tions were performed in 25 ll volumes containing 0.4 lM of eachprimer (Invitrogen, Carlsbad, CA, USA), 200 lM dNTPs (InnovativeSciences, Dunedin, New Zealand), 2.5 ll reaction buffer, 2.5 mMMgCl2, 2 ll DNA and Taq (0.7 U/reaction) (Roche, Expand HiFideli-ty, Basel, Switzerland). Amplifications were carried out in a PerkinElmer 480 thermal cycler using 30 cycles of 1 min at 94 �C, 1 min at58 �C and 2 min at 72 �C. Positive and negative distilled water con-trols were included in each PCR run. PCR products were cleanedusing an Eppendorf Perfect Prep Gel Cleanup Kit (Hamburg, Ger-many) and sequenced directly (Canterbury Sequencing and Geno-typing Facility, University of Canterbury, New Zealand). Allsequences have been deposited in GenBank (Table 1).

S.D. Reay et al. / Biological Control 54 (2010) 52–60 53

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Sequences were aligned using ClustalX (Thompson et al., 1997).Phylogenetic analysis using Bayesian inference was conductedusing MRBAYES version 3.1.2 (Huelsenbeck and Ronquist, 2001;Ronquist and Huelsenbeck, 2003). Models of nucleotide substitu-tion that best fitted the data were selected using the Akaike Infor-mation Criterion (see Posada and Buckley, 2004) in MrModelTestv2 (Nylander, 2004) implemented in PAUP*4.0b10 (Swofford,2002). For EF1-a, the model selected was GTR+I+G, which is a gen-eral time reversible model (Tavaré, 1986; Rodríguez et al., 1990;Yang et al., 1994) with a gamma-shaped rate variation across sitesand a proportion of invariable sites. Two runs of four chains, savingtrees every 100 generations were conducted. After 5,000,000 gen-erations the two runs had converged close to the same value(determined when the standard deviation of split frequencies fellbelow 0.005) and the first 25% of trees were discarded as burn-in. The consensus tree, with the posterior probabilities for eachsplit and mean branch lengths, was visualized using Treeview1.6.6 (Page, 1996).

2.4. Bioassays

To confirm the entomopathogenicity of selected Beauveria spp.isolates, laboratory bioassays were carried out using field-collectedH. ligniperda adults and commercially supplied T. molitor larvae(BioSuppliers, Auckland, New Zealand). Bark beetles are only avail-able for a limited period each year which restricted the number ofassays that could be carried out using these insects; therefore bio-assays were also carried out using a model test insect, T. molitor.Hylurgus ligniperda were collected from under the bark of P. radiatastumps (from a single post-harvest site in Riverhead Forest, Auck-land) and held at 10 �C in the laboratory for up to 1 week prior tobioassay.

A total of 21 Beauveria isolates were selected and tested againstinsects; selections were made to provide representatives from arange of geographic locations where samples were collected. Notall isolates were tested against both H. ligniperda and T. molitor.All isolates were cultured on PDA and incubated at 20–25 �C for2–3 weeks. Conidia were harvested into sterile aqueous 0.01% Tri-ton X-100 and suspensions prepared for the assays. Conidial viabil-ity was confirmed by spread-plating 0.1 ml of a 107 suspensiononto PDA (without antibiotics); plates were incubated at 25 �Cand germination rate (based on germ tube formation) determinedat 20 h (Goettel and Inglis, 1997). Viability was >95% for all batchesof conidia used in the bioassays.

For the T. molitor assays, batches of five larvae were pre-sortedinto separate plastic dishes (55 mm diam.) with snap-top lids. Fil-ter papers (55 mm diam.) (Advantec, Selangor Darul Ehsan, Malay-sia) were placed in a second series of plastic containers and dosedwith 0.5 ml of a 5 � 106 conidia/ml suspension, five replicate con-tainers per isolate. For the controls, 0.5 ml 0.01% Triton only wasapplied to the filter paper. Batches of larvae were randomly as-signed to each treated container (5 larvae/container, total 25 pertreatment replicate). Containers were arranged in a complete ran-domized block design. Assays were undertaken in two series tohelp facilitate the processing of the large number of isolates. Forthe first series of bioassays, each experiment was repeated threetimes using freshly-produced conidia. A total of 75 individual lar-vae were used for each isolate tested. For the second series of bio-assays, each experiment was repeated two times using freshly-produced conidia. A total of 50 individual larvae were used for eachisolate tested.

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contents were then poured through a sterile nylon filter and thebeetles were transferred to individual wells lined with1 cm � 1 cm squares of filter paper (Advantec, Selangor Darul Eh-san, Malaysia) in 24-well cell culture plates (BD Falcon™, San Jose,CA, USA). For the control, beetles were immersed in 5 ml 0.01% Tri-ton only. The 24-well plates were enclosed in plastic bags contain-ing a paper towel moistened with sterile distilled water tomaintain beetles under high ambient humidity. For each isolate,three vials of 12 beetles each were individually treated but theexperiment was only replicated once due to limited beetle avail-ability. Trays were arranged in a complete randomized design.

All experiments were maintained at 20 ± 2 �C. Data were col-lected every 2 days for 14 days; filter papers or paper towels werere-moistened using sterile distilled water as-needed. At eachobservation, any T. molitor cadavers were removed from the trea-ted arena to prevent any cross-contamination of survivors andplaced into Petri dishes lined with moist filter paper to promotefungal outgrowth and sporulation. Cadavers were observed every2–3 days thereafter for a further 10 days. Infection (vs. contamina-tion) was considered to have occurred when fungal outgrowth wasdetected from joints in the exoskeleton, indicating that it origi-nated from inside the host; cadavers were held until conidiationoccurred, allowing confirmation of infection by B. bassiana. Hylur-gus ligniperda cadavers were left in their individual wells, but sim-ilarly examined to confirm that mortality occurred as a result ofmycosis.

We acknowledge limitations to the assay system used. For in-stance, insects were not fed during the bioassay, which could haveincreased their susceptibility to the pathogen. In addition, T. moli-tor larvae were essentially exposed to Beauveria-contaminated fil-ter paper for the duration of the assay. However, as the primarypurpose was not to determine the relative virulence of the isolatesbut to confirm that they were true insect pathogens, the systemwas considered adequate for this purpose. This method has provenutility (Glare et al., 2008; Reay et al., 2008), and the low controlmortality also suggested that the system did not overly stress theinsects.

To confirm the significance of the mortality levels obtained fol-lowing exposure to the pathogens, mortality data were statisti-cally analyzed by inputting the treatment mean mortalities foreach of the three bioassays into an analysis of variance for a ran-domized complete block design, assuming nine treatments andthree blocks (=bioassays) for the first series of bioassays, and 16treatments and two blocks (=bioassays) for the second series ofbioassays. An unrestricted least significant difference (LSD) proce-dure was then used to compare each strain with the control (Sa-ville, 1990).

3. Results

3.1. Survey of Beauveria endophytes

A total of 21 fungal colonies morphologically resembling Beau-veria spp. developed from the plated samples. Colonies were onlyconsidered to have originated from the source material if theygrew from the cut end of a needle or root, or from a cut surfaceof a seed. Pure cultures were prepared from each of these and wereincluded in the sequencing analysis. Sequences obtained from theEF1-a region were compared with representative sequences fromRehner and Buckley (2005) and Reay et al. (2008) (Table 1). Usinga 517 bp segment of the terminal end of the EF1-a gene, the 21 iso-lates from this study were found to align with B. bassiana, and clus-tered with isolates from Rehner and Buckley (2005) (Fig. 1). Therewas only slight variation in the B. bassiana isolates recovered in thecurrent study; all fell into B. bassiana Clade A (Rehner and Buckley,

2005). Of these, 19 were isolated from needle samples (18 P. radi-ata, 1 Larix sp.) and one each from a single P. radiata seed and P.radiata root sample (Table 1). When Beauveria was recovered fromroot or seed samples, the fungus was not isolated from needlessampled from the same source trees, indicating localized infec-tions. Interestingly, the B. bassiana isolated as an endophyte byGanley and Newcombe (2006; GenBank Accession No.GU236996) aligned with those from New Zealand. The relationshipof B. bassiana endophytes from other plant species with those re-ported herein remains to be determined.

3.2. Bioassays

The pathogenicity of the Beauveria spp. strains collected duringthe survey was confirmed in bioassays against both H. ligniperdaadults and T. molitor larvae. All of the fungi tested caused signifi-cantly higher levels of insect mortality than the control treatmentand most insects treated with conidial suspensions were dead10 days after exposure. For the first series of bioassays against T.molitor, mean control mortality was 7% and ranged between 96%and 100% for isolates E9, E97, F624, F641, F646, F647, F648 andF668 at day 10 (LSD = 8) (Fig. 2). For the second series of assays,mean T. molitor control mortality was 38% at day 10 and 100%for all 15 isolates tested (LSD = 28). For the endophyte isolates as-sayed against H. ligniperda adults, mean control mortality was 8%,and 67% (E94), 92% (E83, E85, E106) and 100% for the remainingisolates (E84, E87, E89, E97, E107, E110, E112, E115, E119A,E134A, F647).

4. Discussion

Our study is the first systematic survey documenting Beauveriabassiana as an endophyte of P. radiata in New Zealand. Although arelatively small number of trees were sampled, the samples weretaken from a diverse range of sites spread across a wide geograph-ical area and demonstrate a new host-plant association for thefungus.

All isolates recovered from pines were pathogenic to both H. lig-niperda and T. molitor in laboratory bioassays. Beauveria bassianahas been isolated from numerous insects, with apparent crypticdiversification to specific habitats and hosts (Hajek, 1997; Glareand Inwood, 1998; Rehner and Buckley, 2005; Rehner et al.,2006a,b; Meyling and Eilenberg, 2007; Quesada-Moraga et al.,2007; Zimmermann, 2007). The fungus has previously been iso-lated from bark beetles and although their susceptibility to B. bas-siana has been demonstrated in laboratory bioassays, no epizooticshave been reported in nature (Doberski, 1981; Wegensteiner,1996; Prazak, 1997; Kreutz et al., 2000, 2004a,b). Reay et al.(2008) recovered Beauveria spp. from pine bark, frass collectedfrom bark beetle galleries and from the bodies of both H. aterand H. ligniperda adults. This latter observation demonstrates thecapacity for insect-mediated movement of B. bassiana in a pine for-est, with potential for transmission of conidia into a host tree. Thefungus is ubiquitous in soil and may also enter pines through theroots, a route which has been used to successfully inoculate severalplant species (Posada and Vega, 2005; Quesada-Moraga et al.,2009; Tefera and Vidal, 2009). However, colonization of pines viathe root system under natural conditions is likely to be inhibitedby various biotic and abiotic factors.

Molecular characterization of the Beauveria isolates by PCRamplification and sequence analysis of the EF1-a gene proved tobe a valuable tool in resolving fungal identity. All of the Beauveriafungi isolated in our present study were classified as B. bassianaClade A in the scheme proposed by Rehner and Buckley (2005).The fungi were predominantly recovered from P. radiata needles,

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though one isolate was recovered from needles of another exotictree species (Larix sp.); while few P. radiata root and seed sampleswere collected, B. bassiana was infrequently isolated from thesesource materials. Beauveria was not isolated from native or other

exotic species sampled. Inclusion of more native species in a surveywould be required to determine whether B. bassiana is also endo-phytic in these trees, combined with more sensitive detectiontechniques.

Fig. 1. Consensus tree of phylogenies from terminal partial sequence of elongation factor EF1-a determined in the Bayesian likelihood analysis. Posterior probability valuesare shown for each branch. Sequences of isolates starting with R are from Rehner and Buckley (2005); those starting with F are from the AgResearch Culture Collection, NZ,and include some recovered from pine trees; isolates prefaced with E are all from NZ Pinus radiata; F624 was isolated from P. monticola (Ganley and Newcombe, 2006).

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Reay et al. (2008) previously isolated three species of Beauveria(B. bassiana, B. caledonica Bissett & Widden and B. malawiensis Reh-ner & Aquino de Muro) from bark beetles (H. ligniperda, H. ater) andtheir associated habitat (i.e., soil, bark, frass) in New Zealand. Beau-veria caledonica was the predominant species isolated in their sur-vey but was not detected in any of the sample materials collectedin the present study. Beauveria bassiana Clade A was the sole spe-cies detected as an endophyte in New Zealand pines. The Beauveriaisolate (F624) recovered by Ganley and Newcombe (2006) was in-cluded for comparison in our current study. The PCR analysis con-firmed that this isolate also classified as B. bassiana Clade A.Despite being isolated from P. monticola in North America, it isidentical to the New Zealand isolates, based on the EF1-a gene.Similarly, the B. bassiana isolates recovered from H. ater and H. lig-niperda in New Zealand aligned with those obtained from pines,suggesting a relationship between isolates in Clade A and bark bee-tles associated with pines. However, use of other regions of theDNA for analysis, e.g., the intron region of EF1-a and/or the Bloc lo-cus (Rehner et al., 2006a; Meyling et al., 2009), will enable greaterdiscrimination among this series of isolates and will provide moredetail on their variability and ecological relationships.

The ability of B. bassiana to colonize pine tissues may provide aunique way of regulating cryptic species such as bark beetleswhich are difficult to control, either biologically or chemically,using orthodox application techniques. Endophytic isolates of B.bassiana have previously been shown to reduce insect damage,probably as a result of in planta production of insecticidal metabo-lites, by triggering host-plant defenses, or as a result of feedingdeterrence/antibiosis (Bing and Lewis, 1991; Cherry et al., 2004;Ownley et al., 2004; Akello et al., 2008; Backman and Sikora,2008; Vega et al., 2008; Quesada-Moraga et al., 2009). Some iso-lates have also demonstrated anti-microbial activity and can pro-vide protection against infection by plant pathogens (Ownleyet al., 2004, 2010). As endophytes, the fungi are in a protected envi-ronment where they are not exposed to abiotic and biotic factorsthat can limit efficacy when fungi are applied to foliage or the soil(Brownbridge, 2006; Jaronski, 2007, 2010). However, protection ofpine seedlings against bark beetles such as H. ater and H. ligniperdawould be reliant upon colonization of the cambium layer such thatbeetles feeding on these tissues would be exposed to the fungus

and/or its metabolites. Cambium samples were not collected andanalyzed in the current study so the natural incidence of B. bassi-ana in this layer was not determined. Colonization of the cambiumlayer may be inhibited by anti-fungal compounds that are presentin these tissues, e.g., a- and b-pinenes, but B. bassiana has been re-ported from within the bark of C. caroliniana Walt. (Bills and Polis-hook, 1991), demonstrating that this association can occur.Colonization of pine needles could protect against other beetlepests, e.g., Hylobius abietis L., which feed on seedling foliage.

Other fungal endophytes have been reported from Pinus species(Hata and Futai, 1996; Hata et al., 1998; Deckert and Peterson,2000) and are common in coniferous trees (Stefani and Bérubé,2006; Sokolski et al., 2007). It is likely that P. radiata also hosts arange of fungal and bacterial endophytes. Opportunities to utilizethe (potentially) protective properties of endophytic B. bassianawill depend on its ability to successfully compete with these mi-crobes and establish in P. radiata. Introduction into ‘sterile’ P. rad-iata cells in tissue culture may provide a way of introducing B.bassiana into pine in a minimally competitive environment and iscurrently being evaluated. This method has previously been usedto inoculate tissue culture banana (Akello et al., 2007).

While colonization by entomopathogenic endophytes offers ameans of naturally protecting pine seedlings, endophytic microor-ganisms may also be used as vehicles to introduce novel genes intoplants. For example, some have been modified to express insecti-cidal proteins and anti-fungal compounds with a view to their pro-duction in planta, providing another way in which these organismscan be used to enhance insect and disease resistance (Haapalainenet al., 1998; Li et al., 2007; Backman and Sikora, 2008).

The overall aim of our present study was to document theoccurrence of B. bassiana as an endophyte in radiata pine in NewZealand, to confirm their status as entomopathogens against amodel test insect and a potential target pest, and to characterizethe isolated Beauveria by DNA sequence analysis. Studies are nowunderway to assess different methods of inoculating pines with se-lected B. bassiana isolates, e.g., to P. radiata tissue culture, via rootsand seed coatings, and to determine the extent (roots, cambiumlayer, needles) and duration of colonization prior to evaluating ef-fects on insect pests and diseases. Colonization of specific tissueswill ultimately determine their utility for insect pest management

Fig. 2. Mean cumulative mortality of Tenebrio molitor larvae after inoculation with Beauveria bassiana Clade A in three separate assays (N = 75). Bars = standard error.

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but this study has provided a suite of isolates for inclusion in theseevaluations. Successful establishment of entomopathogenic endo-phytes in pine and other conifers may provide a novel means ofregulating a variety of economically-important insect specieswhich currently cannot be effectively managed using conventionalcontrol methods.

Acknowledgments

We thank Andrew Soreley and Simon Rapley (Rayonier NewZealand), Ngaro Tumai (Hancock Forest Management), Dr. Cor Vink(AgResearch) for instruction in running Mr. Bayes, and Dr. GeorgeNewcombe, Dept. Forest Resources, University of Idaho, Moscow,USA, for providing the Beauveria bassiana isolate from Pinus monti-cola. This study was funded by the Foundation for Science, Re-search and Technology, Ecosystem Bio-Protection ProgrammeLINX0304. The authors also thank Dr. Travis Glare, Lincoln Univer-sity, NZ, and Dr. Fernando Vega, USDA-ARS, Beltsville, USA, for theirhelpful comments on earlier versions of this manuscript.

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