Microbial population and diversity on the exoskeletons of four insectspecies associated with gorse (Ulex europaeus L.)

Emmanuel Yamoah,1* E Eirian Jones,1 Richard J Weld,1† David M Suckling,2 Nick Waipara,3

Graeme W Bourdôt,4 Alvin K W Hee1 and Alison Stewart1

1National Centre for Advanced Bio-Protection Technologies, PO Box 84, Lincoln University, Lincoln, New Zealand.2HortResearch, PO Box 51, Lincoln, New Zealand.3Landcare Research, Private Bag 92170, Auckland, New Zealand.4AgResearch Ltd, PO Box 60, Lincoln, New Zealand.

Abstract Fungi and bacteria on the external surfaces of four gorse-associated insect species: gorse seed weevilApion ulicis Förster (Coleoptera: Apionidae), light brown apple moth Epiphyas postvittana Walker(Lepidoptera: Tortricidae), gorse pod moth Cydia ulicetana Denis and Schiffermüller (Lepidoptera:Tortricidae) and gorse thrips Sericothrips staphylinus Haliday (Thysanoptera: Thripidae), were recov-ered by washing and plating techniques. The isolates were identified by morphology and polymerasechain reaction (PCR) restriction fragment length polymorphism and sequencing of internally tran-scribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used toassess fungal diversity by direct amplification of ITS sequences from the washings of the insects. Allinsect species carried Alternaria, Cladosporium, Corallomycetella, Penicillium, Phoma, Pseudozyma spp.and entomopathogens. Ninety-four per cent of the 178 cloned amplicons had ITS sequence similarityto Nectria mauritiicola (syn. Corallomycetella repens). E. postvittana carried the largest fungal spores(spore mean surface area of 126 mm2) and the most fungal colony forming units per insect. Methylo-bacterium aquaticum and Pseudomonas lutea were isolated from all four insect species. P. fluorescens wasthe most abundant bacterium on the lepidopteran insects. This study presents the diversity of microbialtaxa on insect exoskeletons, and provides the basis for developing a novel mycoherbicide deliverystrategy for biological control of gorse using insects as vectors of a plant pathogen.

Key words biological control, Fusarium tumidum, gorse, insect microflora.

INTRODUCTION

Gorse (Ulex europaeus L.) is an economically importantpasture and forest plantation weed in New Zealand and Aus-tralia. In developing a novel biocontrol strategy for gorse, wesuggest the use of insects to pick up and transport conidia of afungal pathogen, Fusarium tumidum to the targeted gorseplants. Part of this strategy requires using pheromones toattract specific insect vectors to a bait station where they willbe loaded with F. tumidum conidia. As the attraction strategywill target specific vectors, it is important to select insectspecies that possess a high capability to transport the pathogento the weed and that do not naturally carry microflora thatmight inhibit the efficacy of the mycoherbicide. Significantinhibition of fungal growth by the bacterium Stenotrophomo-nas maltophilia has been reported (Kerr 1996). For this reason,a study of the external microflora of selected insect specieswas undertaken.

There are two factors that could impede the successful deliv-ery of F. tumidum spores by insect vectors to gorse. The first isthe relatively large size of F. tumidum conidia (Broadhurst &Johnston 1994) which may limit the number the insects cancarry. The size and the number of microbes naturally carried bypotential insect vectors are therefore of interest. Secondly,F. tumidum produces mycotoxins (Morin et al. 2000) whichmay be detrimental to certain insect species. Hence, insectspecies naturally carrying Fusarium spp. (especially, F. tumi-dum) may be more adaptable to vectoring this pathogen.

Several insect species are naturally abundant on gorse, andare well distributed in both the North and South Islands of NewZealand. Two of these insect species; gorse seed weevil Apionulicis Förster (Coleoptera: Apionidae) and gorse pod mothCydia ulicetana Denis and Schiffermüller (Lepidoptera: Tortri-cidae) are well-established biocontrol agents (Hill et al. 2000).These two insect species, along with the light brown apple mothEpiphyas postvittana Walker (Lepidoptera: Tortricidae) whichis abundant on gorse (Suckling et al. 1998), and the gorse thripsSericothrips staphylinus Haliday (Thysanoptera: Thripidae)have been selected as potential insect vectors for this biocontrolstrategy.

*yamoah@xtra.co.nz†Present address: Lincoln Ventures Limited, PO Box 133, Lincoln, New

Zealand.

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© 2008 The AuthorsJournal compilation © 2008 Australian Entomological Society doi:10.1111/j.1440-6055.2008.00655.x

There are no specific reports detailing the natural microf-lora of gorse-associated insects, and the few studies on insectmicroflora have focused on micro-organisms within the gut.Reports suggest that insects and other arthropods such asticks generally harbour multiple microbial taxa (Haynes et al.2003; Rosa et al. 2003; Yoder et al. 2003). For example, Aure-obasidium pullulans, Candida sp., Pseudozyma and Rhodot-orula spp. have all been isolated from the external surfacesof the stingless bees, Tetragonisca angustula Latreille(Hymenoptera, Meliponinae) and Melipona quadrifasciataLep. (Hymenoptera, Apidae) (Rosa et al. 2003). The mycof-lora of gorse-inhabiting insect species is likely to includehost plant (gorse) epiphytes, entomopathogens and a randomassortment of unrelated air-borne taxa.

The study of insect microflora requires an efficient andreliable method of identifying both culturable and uncultur-able microorganisms as it has been reported that only a smallfraction (<1%) of naturally occurring micro-organisms can becultured by standard techniques (Amann et al. 1995). As aresult, the standard techniques of culturing natural populationscan underestimate microbial biodiversity (Van Tuinen et al.1998). Polymerase chain reaction restriction fragment lengthpolymorphism (PCR-RFLP) of internally transcribed spacer(ITS) and 16S rDNA sequencing have proven to be suitableand rapid methods for taxonomic studies of fungi and bacteria,respectively (Viaud et al. 2000; Jang et al. 2003; Li et al.2005). Viaud et al. (2000) used these techniques to study thediversity of cultured and non-cultured fungi in soils. In thisstudy, the microbial populations and size of fungal spores onthe exoskeletons of A. ulicis, C. ulicetana, E. postvittana andS. staphylinus were determined. The fungal species diversitywas identified by morphology and PCR-RFLP and sequencingof ITS and 16S rDNA. In addition, a culture-independenttechnique (direct PCR) was used to assess fungal diversity bydirect amplification of ITS sequences from the washings of theinsects. The culturable bacterial diversity was determined bysequencing of the 16S rDNA. The overall aim of this studywas to gain an insight into the microflora naturally carried bythe studied insects and to identify an insect species whichmight be suitable as a vector of F. tumidum for biologicalcontrol of gorse.

MATERIALS AND METHODS

Natural microflora on insects (Sampling 1)

Apion ulicis, C. ulicetana, and E. postvittana were collectedfrom gorse plants growing at three distinct sites. Site 1 was apine plantation containing a high density of gorse plants, andis situated about 30 km north of Christchurch (43°32′S,172°37′E). Site 2 was on the West Coast (42°7′S, 171°52′E) ofthe South Island of New Zealand. The main vegetation in thisarea was matured, native rainforest. Site 3 was along the slopeson the Tai Tapu Hills (about 30 km south-east ofChristchurch). S. staphylinus was collected from a fourth siteat Lincoln (about 20 km south of Christchurch) because of its

unavailability at the other sites in three separate samplingsover a period of 2 years. Sites 1, 3 and 4 are in Canterbury.C. ulicetana and E. postvittana were collected with a sweepnet and sticky trap with attractants. A. ulicis and S. staphylinuswere shaken off the gorse plants onto white paper spread intrays beneath the plants. All insects were kept in a refrigeratorafter trapping and assessed as soon as possible (within2 weeks). Live insects required for determining the location ofspores on the insects were kept in a cold cabinet at 10°C.

Insect washing and plating techniques

Bacteria and fungi on the surfaces of A. ulicis, C. ulicetana,E. postvittana and S. staphylinus were recovered by washingthe insects and plating the washings on agar medium. ForA. ulicis, C. ulicetana and E. postvittana, 15 individual insectswere sampled for each replicate. For the smaller insect species,S. staphylinus, 100 individuals were sampled. The insectswere placed in sterile Universal bottles in 3 mL of 100 mmolpotassium phosphate buffer (pH 7.0) + 0.01% Tween 80Analar® and shaken for 3 min on a Griffin flask shaker. Eachinsect species was washed separately. There were three repli-cate samples for each insect species per site. Each of thewashed insect samples was diluted up to 100-fold. A 100 mLaliquot of each dilution series (0, 10-1 and 10-2) was platedonto Petri dishes containing Nutrient agar (NA; Oxoid)amended with 200 mg/mL cyclohexamide for total bacterialcounts. Potato dextrose agar (PDA; Oxoid) amended with250 mg/mL chloramphenicol was used for fungal counts.There were two NA and four PDA replicate plates for eachdilution. Fungal cultures were incubated at two different tem-peratures (15 and 25°C) for 7–12 days and bacterial cultures at28°C for 3 days under a 12 h photoperiod after which thecolony forming units (CFU) were counted and expressed asCFU per insect. Voucher specimens have been deposited in theLincoln University culture collection. One millilitre of eachwashed sample was stored at -80°C for subsequent molecularanalysis.

For the plating technique, a total of 36 insects of each of thefour insect species were rolled across the surface of PDA orNA for fungal and bacteria counts, respectively, using steriletweezers. Twenty-four insects of each species were assessedfor fungal counts and 12 for bacterial counts at one insect peragar plate. Insects were removed from the plate after plating,and the plates were incubated as previously described. TheCFU were counted after incubation and expressed as CFU perinsect.

Fungal spore size and insect species bodylength measurements

The size of fungal spores isolated from the insects was deter-mined using a DP12 digital camera system connected to a lightmicroscope. Images were analysed using AnalySIS® software.This program measured the surface area of approximately 60spores from each sporulating fungal isolate from three repli-cate slides. Fifteen isolates with the largest spore sizes from

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each insect species were selected, and the mean spore size wasdetermined. The size of each insect species was determined bymeasuring the body length of 10 insects.

Natural microflora on insects (Sampling 2)

The study of the natural microflora of the insect species wasrepeated using only C. ulicetana and E. postvittana, collectedfrom site 3. The insects were washed in potassium phosphatebuffer as previously described and the washing plated ontoPDA amended with 10 mg/mL chlorotetracycline for fungalcounts and NA medium amended with 200 mg/mL cyclohex-imide for bacterial counts. The plates were incubated asdescribed previously. Based on differing colony morphology,15 bacterial isolates from C. ulicetana and 10 from E. postvit-tana were selected and identified by sequencing of 16S rDNA.Distinct fungal cultures were selected (based on colony mor-phology) and identified by either ITS rDNA sequencing or bymorphology.

Morphological identification of microbes

Representative fungal colonies were subcultured (based ondistinct colony morphology) onto low strength potato carrotagar (PCA) and hay agar (HA) and incubated at 25°C under a12 h photoperiod to produce pure sporulating cultures. PCAcontained 20 g potato, 20 g carrot and 15 g agar per litre ofdistilled water, while HA contained 20 g hay and 15 g agar perlitre of distilled water. Using standard taxonomic keys, fungalcultures were morphologically identified to genus level. Gramreaction tests and colony morphology were used for primaryassessment of bacterial diversity.

DNA extraction and amplification of ITSrDNA and 16S rDNA

DNA of culturable fungi and bacteria were extracted withChelex 100 Resin or with the PowerSoilTM DNA Isolation Kitusing manufacturer’s procedure. Genomic DNA of fungal iso-lates from the washings of the insects was extracted with thePowerSoilTM DNA Isolation Kit. The ITS regions and the 5.8SrDNA gene of each fungus were amplified by PCR usingprimers PN3 (5′-CCGTTGGTGAACCAGCGGAGGGATC-3′), which hybridises to conserved sites at the 3′ end of the18S subunit (Neuvéglise et al. 1994) and PN34 (5′-TTGCCGCTTCACTCGCCGTT-3′), which hybridises to conservedsites at the 5′ in the 28S region (Raffin et al. 1995). Theuniversal ITS primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)(White et al. 1990) which amplify the same region as PN3/PN34 primers were used for amplifying DNA samples whichthe PN3/PN34 primer combination did not amplify well. EachPCR amplification of 15–30 ng of DNA sample was carriedout in a total volume of 50 mL containing 10 mmol Tris HCl(pH 8.0), 1.5 mmol MgCl2, 50 mmol KCl, 0.2 mmol each ofdATP, dCTP, dGTP, 1.5 U of HotMasterTM Taq DNA poly-merase and 0.4 mmol of each primer in a PTC200 thermalcycler. Control amplifications included all reagents except

DNA to test for contamination of reagents. The PCR cyclingprotocol was one cycle of 94°C for 2 min, 35 cycles of 94°Cfor 1 min, 50°C for 1.5 min, 65°C for 1 min and one cycle of72°C for 7 min.

For amplification of bacterial 16S rDNA, each 25 mL PCRreaction was carried out with 10–20 ng of bacterial genomicDNA as template, 10 mmol Tris HCl (pH 8.0), 1.5 mmolMgCl2, 50 mmol KCl, 0.2 mmol each of dATP, dCTP, dGTP,1.5 U of HotMasterTM Taq DNA polymerase, 0.4 mmol offorward primer (B16S-5′) 5′-CATGGCTCAGATTGAACGCTGGCG-3′ and reverse primer (B16S-3′) 5′-CCCCTACGGTTACCTTGTTACGAC-3′. Primer B16S-5′ correspondsto positions 18–41, and primer B16S-3′ corresponds to posi-tions 1494–1517 of the Escherichia coli numbering system(Chen et al. 1996). Control amplifications were as previouslydescribed. The thermocycle program was: 95°C for 5 min,followed by 35 cycles of 95°C for 40 s, 55°C for 40 s, 72°C for2 min and a final extension at 72°C for 10 min (Chen et al.1996). The annealing temperature was raised from 55 to 62°Cwhen non-specific bands were produced with 55°C annealing.Amplified DNA was detected by electrophoresis on a 1%(wt/vol) agarose gel in 1¥ TAE buffer (40 mmol Tris, 4 mmolsodium acetate, 1 mmol EDTA). The gels were stained withethidium bromide, and the PCR products were visualised witha UV transilluminator.

Restriction fragment length polymorphism ofamplified ITS rDNA and 16S rDNA

The amplified ITS products were digested independently withfour restriction enzymes: Hin6I, MboI, BsuRI and HinfI. Foreach reaction, 75 ng of amplified DNA was digested in 20 mLtotal volume. Each reaction contained 2.5 U restrictionenzyme, and was incubated for 2 h at 37°C as per manufac-turer’s instructions. The restriction fragments were size frac-tionated by electrophoresis in 1¥ TAE buffer through 2%agarose gels at 80 V for 90 min. The lengths of the restrictionfragments were estimated by comparison against a 50 bp DNAladder. The amplified 16S rDNA products were digested withfour restriction endonucleases: EcoRI, BsuRI, AluI and Hin6I.For each reaction, 75 ng of amplified 16S rDNA was digestedin 20 mL total volume. Each reaction contained 2.5 U restric-tion enzyme, and was incubated for 3 h at 37°C as per manu-facturer’s instructions. The restriction fragments were sizefractionated as described previously. The 1 Kb-plus DNAladder was used for the estimation of the lengths of the restric-tion fragments. Samples with identical RFLPs for all restric-tion enzymes were identified as belonging to the same RFLPgroup. Representative samples of each RFLP group weresequenced.

Cloning of fungal ITS amplicon

Polymerase chain reaction products amplified from DNAextracted from the washing of the insects were cloned into thebacterial plasmid pGEM-T Easy using the manufacturer’sligation system. The ligated plasmids were transformed into

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E. coli strain INVaF′ by electroporation. Each individualclone obtained was added directly to a PCR mix, and eachcloned ITS was amplified by PCR and then digested withrestriction enzymes. All PCR amplifications were as describedpreviously using PN3/PN34 primers. The restriction enzymedigestion was by four restriction enzymes: Hin6I, MboI, BsuRIand HinfI, as described for the fungal ITS rDNA. ITS cloneswith identical RFLP patterns for all restriction enzymes weregrouped together. Representative samples from each RFLPgroup were sequenced after plasmid extraction from the clonesby the Wizard® Plus SV Minipreps DNA purification kit as permanufacturer’s instructions.

Sequencing

Sequencing reactions were carried out by the dideoxy chaintermination method using the ABI PRISMTM Dye TerminatorCycle Ready Reaction kit with AmpliTaq® DNA Polymeraseand the ABI PRISMTM 3100 Genetic Analyser. All PCR prod-ucts were purified using the Quantum Prep® PCR Kleen Spincolumns as per manufacturer’s instructions. The NanoDropspectrophotometer was used to quantify DNA of all samplesprior to sequencing. The two sequences obtained were alignedusing SequencherTM and the sequences deduced were com-pared with ITS rDNA or 16S rDNA gene sequences in theGenBank database using the BLAST search program for theclosest matching organism (Altschul et al. 1997).

Location of microbes on the surfaces ofthe insects

Scanning electron microscope (SEM) techniques were used todetermine the location of microbes on the body of the insects.Six insects per species from each site were examined byplacing them on aluminium stubs with double-sided stickytape and coating with gold palladium at 1.2 kEv at 20 mA ona Polaron 5000 coater. A Leica S440 SEM was used toexamine all external parts of each insect species.

Statistical analysis

The experiment was set up as a 3 ¥ 3 factor Completely Ran-domised Design consisting of three insect species (A. ulicis,C. ulicetana and E. postvittana) sourced from three sites.There were three replicates. Counts of CFU were log trans-formed to satisfy the assumption of normality for anova andto stabilise the variance. The data were analysed by anovausing the GenStat statistical package and compared withS. staphylinus data as this species was sourced from only onesite. Mean separation was based on Fisher’s protected leastsignificant difference tests at the P < 0.05 level.

RESULTS

Colony forming units and fungal spore size(Sampling 1)

Among the insect species, E. postvittana generally carried thehighest fungal CFU/insect and S. staphylinus the lowest as

determined by the mean CFUs obtained by the washing anddirect plating techniques (Table 1). More surface microbeswere recovered from all insect species using the washing tech-nique than was obtained by the direct plating technique. Forboth washing and plating results, insects sourced from site 2 inthe West Coast carried more fungal CFU than those sourcedfrom the other two sites in Canterbury. The temperature ofincubating the fungal cultures (15 and 25°C) did not have anysignificant effect on the CFU obtained from either washing orplating (data not shown). All insect species carried more bac-teria than fungi (P < 0.05). C. ulicetana carried the most bac-terial CFU/insect while S. staphylinus carried the least.E. postvittana, the largest insect species studied, carried thelargest fungal spores averaging 126 mm2 in surface area. Thesmallest fungal spores with a mean size of 15 mm2 were recov-ered from S. staphylinus which is the smallest insect species.The mean size of fungal spores recovered from C. ulicetanawas 63 mm2 and from A. ulicis was 38 mm2. The mean size offungal spores recovered from the insect species correlatedlinearly with their body length (R2 = 85%).

Fungal isolates recovered from insect species

Based on fungal morphology on the isolation plates, the mostprevalent isolates in both samplings were Cladosporium spp.(accounting for about 50%), yeasts (37%), Penicillium spp.(2.4%), Phoma herbarum (2.4%) and Alternaria spp. (2%).These fungal groups were recovered from all four insectspecies. The remaining fungal species occurred rarely(<0.5%).

Most of the fungal cultures were identified by ITS polymor-phism. ITS sequencing of representative RFLP groups from120 cultured isolates obtained from sampling 1 showed thatAlternaria spp., Cladosporium spp. (C. cladosporioides andC. herbarum), P. herbarum, Penicillium spp. and yeasts were

Table 1 Microbial population (log10 CFU/insect) recovered fromthe surfaces of Cydia ulicetana, Apion ulicis and Epiphyas postvit-tana sourced from three sites in the South Island and Sericothripsstaphylinus collected from Lincoln (South Island) (n = 6)

Treatment FungiLog10 CFU/insect

BacteriaLog10 CFU/insect

Washing Plating Washing Plating

Insect spp.C. ulicetana 2.27 1.15 4.09 1.42A. ulicis 2.30 1.28 2.67 1.56E. postvittana 2.63 1.31 3.38 0.90S. staphylinus† 1.03 0.22 2.03 0.54

SiteSite 1 2.17 1.33 2.73 1.17Site 2 2.52 1.39 3.83 1.37Site 3 2.51 1.02 3.59 1.34LSD 0.275 0.103 0.385 0.180Interaction (P) 0.030 <0.001 <0.001 0.010

†Data for S. staphylinus was excluded from the anova. Mean separa-tion was based on Fisher’s protected least significant difference (LSD)tests at the P < 0.05 level.

CFU, colony forming units.

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present on all four insect species (Table 2). Acremoniumstrictum, Aureobasidium pullulans, Beauveria bassiana(telemorph Cordyceps bassiana), Pithomyces chartarum andSclerotinia sclerotiorum were isolated from all insect speciesexcept S. staphylinus. Pseudozyma fusiformata was the mostabundant yeast on all the insect species. F. lateritium was themost common Fusarium species on the lepidopteran insects,and was isolated in both samplings. The other Fusariumspp. were F. tricinctum and Gibberella pulicaris (anamorphF. sambucinum) but these were only isolated from E. postvit-tana. Eleven fungal species were isolated from both lepi-dopteran insects in both samplings. Apiospora montagnei,Drechslera biseptata and Fusidium sp. were recovered fromC. ulicetana while Botrytis cinerea and Paecilomyces sp. wererecovered from E. postvittana in sampling 2 only.

Cloned amplicon of ITS obtainedby direct PCR

Digestion of ITS amplified products of 47 transformed clonesobtained from A. ulicis, 51 from C. ulicetana, 43 fromE. postvittana and 37 from S. staphylinus produced eightRFLP groups (Table 3). Ninety-four per cent of the 178 clonedamplicons had ITS sequence similarity (i.e. closest matchedorganism in the GenBank database) to Nectria mauritiicola(syn. Corallomycetella repens), and this fungus was commonon all insect species. Two uncultured fungi (with ITS sequencesimilarity to GenBank accession # DQ421003 andAM260859) were identified by this method. Except for S. sta-phylinus, all insect species carried the uncultured fungus withITS sequence similarity to GenBank accession # DQ421003.

Bacterial isolates recoveredfrom insect species

In sampling 1, the 16S rDNA of 34 (E. postvittana), 32(A. ulicis), 29 (C. ulicetana) and 26 (S. staphylinus) isolateswere amplified. Digestion of the amplified 16S rDNA with aset of restriction enzymes grouped the 121 isolates into 33RFLP groups. Bacterial isolates from C. ulicetana andE. postvittana comprised 12 and 13 genera, respectively. Iso-lates from A. ulicis constituted 14 bacterial genera while iso-lates from S. staphylinus were from eight genera (Table 4).Pseudomonas fluorescens and Stenotrophomonas maltophiliaaccounted for about 41% of the 29 isolates recovered fromC. ulicetana with Moraxella osloensis being isolated from thisinsect species sourced from all three sites. Nine bacterialspecies were isolated from both lepidoptera. P. fluorescens,P. lutea and Actinobacterium sp. were identified on E. postvit-tana from all three sites. P. fluorescens was the most commonbacterium on E. postvittana (accounting for about 18% of the34 isolates) while M. osloensis was the dominant bacterium onA. ulicis (16% of the 32 isolates). Methylobacterium aquati-cum and Pseudomonas spp. were recovered from A. ulicisfrom all three sites. M. aquaticum, Providencia rustigianii andE. coli accounted for approximately 77% of all bacterial iso-

lates recovered from S. staphylinus. Most of the 121 bacterialisolates (72%) were gram negative.

Four bacterial species (Arthrobacter chlorophenolicus, Cur-tobacterium flaccumfaciens, P. lutea and Rhodococcus sp.)were isolated from both lepidopteran insects in sampling 2.Four bacterial species belonging to the genera Bacillus,Curtobacterium, Methylobacterium and Pseudomonas wererecovered from C. ulicetana in both samplings while Erwinia,Methylobacterium, Pseudomonas and Rhodococcus spp. wererecovered from E. postvittana from both samplings.

Location of microbes on insects

Scanning electron micrographs of the external parts of theinsects (Fig. 1) revealed no specific area where the microbesare located. Microbes were found on the entire surfaces ofS. staphylinus and A. ulicis, but were restricted to the legs,antennae and ventral abdomen of the moths. Microbes wererarely found on the scales of the moths.

DISCUSSION

The two lepidopteran insects carried more microbes than theother insect species. Insects sourced from site 2 carried themost external microflora probably because of higher rainfall atsite 2 in the West Coast (2148 mm in 2004; National Instituteof Water & Atmospheric Research, New Zealand (NIWA)Instrument Systems) relative to the sites in Canterbury(675 mm). High rainfall favours the growth, development andsporulation of most micro-organisms. Other factors such asrelative humidity might also contribute to this. The sites didnot differ in the mean annual temperature for the same period(NIWA). The number and size of fungal spores carried by eachinsect species appear to be largely dependent on insect size.Larger insects, such as E. postvittana, may, therefore, have thegreatest capacity to carry high numbers of F. tumidum spores.As high numbers of F. tumidum conidia have been shown to bea prerequisite for successful infection of gorse (Yamoah 2007),such insects with high spore carrying capacity may be moresuitable for this biocontrol strategy with small insects such asS. staphylinus being less suitable.

Results from this study indicate that while the insectsharbour multiple microbial taxa on their exoskeleton, mostfungi isolated from the insects were gorse epiphytes (Johnstonet al. 1995) with few air-borne and soil-borne organisms andentomopathogens. Some of the most common air-borne fungalspecies on the insects were Acremonium spp., Alternaria alter-nata, Cladosporium spp., Penicillium spp. and Aspergillusspp. E. postvittana, C. ulicetana and A. ulicis carried the ento-mopathogenic fungi Beauveria spp., Cordyceps bassiana andVerticillium lecanii. Other entomopathogens were Paecilomy-ces spp. which were recovered from E. postvittana and S. sta-phylinus. Reports have shown that entomopathogens parasitiseinsects and, as a result, regulate their natural population(Deacon 2006). In particular, Beauveria spp. are obligateparasitic fungi used in biological control of insect pests

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Table 2 Comparison of internally transcribed spacer (ITS) sequences obtained from fungal isolates recovered from Cydia ulicetana,Apion ulicis, Epiphyas postvittana and Sericothrips staphylinus in Sampling 1 (S1) and from C. ulicetana and E. postvittana in sampling2 (S2)

Closest match in C. ulicetana E. postvittana A. ulicis S. staphylinus % ITSsimilarity

GenBankaccession no.GenBank database S1 S2 S1 S2 S1 S1

Acremonium strictum + + + + + – 99 AY138846Acrodontium crateriforme + – + – – – 94 AY843112Alternaria alternata + + + + + + 99 AY154682Alternaria triticina + – + – + + 95 AY154695Aphanocladium aranearum – – + – – – 100 AF455489Apiosporina morbosa – – + – + – 99 AF493984Arthothelium spectabile + – – – – – 94 AF138814Arthrinium sacchari – + + + – – 99 AF455478Aureobasidium pullulans + + + + + – 99 AY185811Beauveria bassiana† + + + + – – 99 AJ560686Beauveria brongniartii – – – – + – 97 AB027381Chaetomium globosum – – + – – – 99 AY429056Cladosporium cladosporioides + + + + + + 100 AY463365Cladosporium herbarum + + + + + + 100 AF455517Claviceps purpurea – + – – – – 99 AB160991Cordyceps bassiana† + – + – + – 100 AB079126Drechslera biseptata – + – – – 100 AY004787Drechslera dematioidea + – – – – 99 AY004790Foliar endophyte of Picea glauca – – + – – – 98 AY566890Fusarium lateritium + + + + – – 100 AF310979Fusarium tricinctum – – + – – – 100 AF008921Gibberella pulicaris – – + – – – 99 AY188921Lewia infectoria + – – – – – 99 AF455512Metschnikowia pulcherrima + nd + nd + – 96 AY301026Mucor hiemalis f. corticola – – + – – + 99 AY243950Nectria mauritiicola‡ – – + – – – 98 AJ558115Paraphaeosphaeria michotii + – + – – – 98 AB096264Penicillium cecidicola + nd – nd – – 99 AY787844Penicillium chrysogenum + nd – nd – – 99 AY373903Penicillium citreonigrum – nd – nd – + 99 AY157489Penicillium pinophilum – nd – nd – + 100 AB194281Phaeosphaeriaceae sp. – – + – – – 96 AY465459Phoma herbarum + + + + + + 98 AY337712Phomopsis sp. PHAg – – + – – – 96 AY620999Pithomyces chartarum + + + – + – 99 AY433807Pseudozyma fusiformata + + + + + + 99 AB089366Rhodotorula mucilaginosa + nd – nd – + 99 AF444614Sclerotinia sclerotiorum + + + – + – 100 AF455526Sporobolomyces ruberrimus + nd + nd – – 100 AF444581Talaromyces intermedius + – – – – – 98 L14524Tranzscheliella hypodytes – – – – – + 99 AF045867Valsa sordida + – – – – – 98 AB188679Apiospora montagnei – + – – – – #Aspergillus niger + + + – + – #Botrytis cinerea – – – + – – #Epicoccum purpurascens + + + + + – #Fusidium sp. – + – – – – #Paecilomyces spp. – – – + – + #Pestalotiopsis guepinii + – – – – – #Trichoderma sp. + – – – – + #Verticillium lecanii + – + – + – #

†Cordyceps bassiana (anamorph Beauvaria bassiana); ‡syn Corallomycetella repens.+, isolated from insect species; –, not isolated from insect species; #, Isolates were identified by morphology only; nd, not determined, only classified

as yeast in sampling 2; Penicillium species not identified to species level in sampling 2.

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(Cisneros et al. 1999–2000; Mander et al. 2006). Apart fromE. postvittana, all insect species used for this study are classi-cal biological control agents of gorse. Hence, it is important tomaintain high populations of these species on the weed. Ento-mopathogens isolated from the insect species in this studywere of low prevalence, and therefore, may pose only a limitedthreat to the establishment of these insect species.

The most common yeast on the insects was P. fusiformata.Other yeasts included Metschnikowia pulcherrima, Rhodot-orula mucilaginosa and Sporobolomyces ruberrimus. Someyeast spp. have antifungal effects (Yao et al. 2004), andmay be involved in a mutualistic relationship with insects(Rosa et al. 2003) thereby, protecting them against infection.R. mucilaginosa has been reported to be common on a wide

Table 3 Similarities of sequences of internally transcribed spacer (ITS) gene fragments obtained by the polymerase chain reactionculture-independent approach from Cydia ulicetana, Epiphyas postvittana, Apion ulicis and Sericothrips staphylinus in sampling 1

Closest match inGenBank database

C. ulicetana E. postvittana A. ulicis S. staphylinus % ITSsimilarity

GenBankaccession no.

Acremonium kiliense (1)† + – – – 100 AJ853771Claviceps purpurea (3) + – – – 99 AB160991Beauveria bassiana (1) – + – – 99 AY532052Fusarium lateritium (1) + – – – 100 AF310979Nectria mauritiicola (167) + + + + 100 AJ558114Psathyrella cf. gracilis (1) – + – – 94 AY228352Uncultured fungus (1) – + – – 97 AM260859Uncultured soil fungus (3) + + + – 99 DQ421003

+, isolated from insect species; –, not isolated from insect species. †Figures in parenthesis indicate number of clones.

Table 4 Comparison of 16S rRNA sequences obtained from bacterial isolates recovered from exoskeletons of Cydia ulicetana,Epiphyas postvittana, Apion ulicis and Sericothrips staphylinus in sampling 1 (S1) and from C. ulicetana and E. postvittana in sampling2 (S2)

Closest match in C. ulicetana E. postvittana A. ulicis S. staphylinus % 16Ssimilarity

GenBankaccession no.GenBank database S1 S2 S1 S2 S1 S1

Acidovorax temperans – – + – – – 99 AF078766Actinobacterium sp. + – + – – – 99 AY275511Aeromonas allosaccharophila + – + – – – 99 S39232Agrobacterium rubi – – – – – + 99 AY626394Arthrobacter chlorophenolicus – + – + – – 98 AF102267Bacillus megaterium – – – – + – 100 AJ717381Bacillus pumilus + + + – – – 99 DQ105967Chryseobacterium spp. + – – + – – 99 DQ337556Curtobacterium flaccumfaciens + + – + + + 99 AJ312209Enterococcus mundtii + – – – – – 100 AF061013Erwinia billingiae – – – + – – 100 DQ288876Erwinia carotovora – – + – + – 97 BX950851Erwinia cypripedii – + – – – – 99 AJ233413Escherichia coli – – – – + + 99 DQ182324Frigoribacterium sp. + – – – + + 100 AY439250Methylobacterium aquaticum + + + + + + 98 AJ785572Microbacterium spp. – + – + + – 99 AY635868Moraxella osloensis + – + – + – 99 AF005190Paenibacillus polymyxa – – – – + – 99 AM062684Plantibacter flavus – – – – – + 100 AJ310417Providencia rustigianii – – + – – + 98 AM040489Pseudoclavibacter helvolus + – + – + – 99 X77440Pseudomonas fluorescens + – + – + – 100 DQ207731Pseudomonas lutea + + + + + + 99 AY364537Rhodococcus corynebacterioides – – + – – – 98 NC16SR2Rhodococcus erythropolis – – + – + – 100 AY822047Rhodococcus fascians – – + – – – 99 AY730713Rhodococcus sp. – + – + + – 99 AY660692Serratia proteamaculans – – + – – – 99 AY040208Sphingomonas spp. – – – – + – 100 AY336556Staphylococcus xylosus – – – – + – 99 AF515587Stenotrophomonas maltophilia + – + – – – 100 AY367030

+, isolated from insect species; –, not isolated from insect species.

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range of insects including Diptera and Hymenoptera (Zacchi& Vaughan-Martini 2002). The majority of fungal speciesisolated were anamorphs of ascomycetes which is a reflectionof the host plant epiphyte (Johnston et al. 1995). The single

zygomycete species (Mucor hiemalis f. corticola) was isolatedboth from E. postvittana and S. staphylinus. F. lateritium wasthe most common Fusarium spp., and was consistently iso-lated from the lepidopteran insects from both samplings. Thisfungus together with G. pulicaris (anamorph F. sambucinum;isolated from E. postvittana only) has been reported on gorsein New Zealand (Johnston et al. 1995). The fact that only thelepidopteran insects carried Fusarium spp. indicates that theycould be more adaptable to vectoring F. tumidum spores thanthe other two insect species. F. tumidum was not recoveredfrom any of the insects which may indicate low prevalence ofthe pathogen on gorse.

The fact that C. repens (syn. N. mauritiicola; Rossman et al.1999) was abundant in the direct PCR although rarely isolatedin culture suggests either the insects carried many unviable orunculturable C. repens spores or the molecular technique hada high bias towards amplifying C. repens ITS. A similar asym-metry between direct PCR and culture-based techniques hasbeen reported for another Nectria spp. Using primers similar tothose used in this work, Viaud et al. (2000) reported Nectriavilior (syn. Cosmospora vilior; Rossman et al. 1999) as thedominant cloned amplicon from soil samples although thisspecies was rarely (two out of 67 colonies) isolated in culture.The low diversity of fungal species detected by the direct PCRmethod compared with the high diversity detected by culture-dependent techniques was surprising and suggests significantbias in this study in detection by the direct PCR method. DirectPCR is more likely to detect rDNA sequences that most closelymatch the ITS primers and species most amenable to the DNAextraction method used (Von Wintzingerode et al. 1997).Hence, it is important that these steps be as unbiased as pos-sible. The direct PCR method, however, did detect some uncul-tured fungi. In addition, the culturing technique may haveintroduced its own bias selecting towards fast growing fungiand sporulating fungi. It is, therefore, important that bothculture-dependent and culture-independent techniques be usedin conjunction.

Pseudomonas lutea and M. aquaticum were isolated fromall four insect species and were consistently isolated from bothlepidopteran insects in both samplings. This may indicate aclose association of these bacteria with the insects. Pseudomo-nas spp. are found abundantly as free-living organisms in soils,fresh water and marine environments, and in many othernatural habitats. Methylobacterium spp. can be found mostlyin soils, on leaves and in other parts of plants. Both Erwiniaand Staphylococcus, isolated from A. ulicis, have beenreported on the pea aphid, Acyrthosiphon pisum (Haynes et al.2003). Erwinia, Pseudomonas and Arthrobacter were isolatedfrom both lepidopteran insects and have been reported to becommon on the fruit flies Bactrocera tau and B. cucurbitae(Sood & Nath 2002). It is possible that the wash samples couldbe contaminated by insect faecal material.

The results in this study provide insights into the externalmicroflora of insects that will be useful for employing insectsas vectors of pathogens for biological control programs. Allfour insect species have been shown to carry diverse fungaland bacterial taxa on their exoskeleton with Cladosporium,

(a)

(c)

(b)

Fig. 1. Scanning electron micrographs of external parts of insectspecies showing their surface microflora: (a) compound eye ofSericothrips staphylinus, bar = 20 mm; (b) dorsal part of thorax ofApion ulicis, bar = 3 mm; (c) scales of Epiphyas postvittana,bar = 10 mm.

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Corallomycetella, Pseudozyma and Pseudomonas as the mostabundant. Because of the consistency of isolation of thesemicrobes from the insect species from all sites of collection, itis most likely that these micro-organisms are specifically asso-ciated with the insects. Some of these microbes may antago-nise F. tumidum and warrant further investigation. The fact thatsome of the fungal species recovered from the insects weregorse epiphytes (Johnston et al. 1995) indicates that they maypick up F. tumidum conidia if the pathogen is present in highpopulation on gorse. E. postvittana, the largest insect speciesstudied, carried the most fungal CFU/insect and the largestfungal spores, and may have the greatest capacity for vectoringF. tumidum conidia. Additionally, this insect species naturallycarried Fusarium spp. including F. sambucinum (G. pulicaris).The conidium of this fungus is morphologically similar to thatof F. tumidum and both fungi produce the mycotoxin trichoth-ecene (Desjardins et al. 1987; Morin et al. 2000). Coupledwith the availability of pheromone for attracting the maleinsects (Bellas et al. 1983), E. postvittana may be a suitableinsect vector for delivering F. tumidum conidia on gorse usingthis novel biocontrol strategy. Although this insect species ispolyphagous, and may visit non-target plants, F. tumidum is avery specific pathogen of gorse, broom and a few closelyrelated plant species (Barton et al. 2003). Hence, using thisinsect species to vector F. tumidum in a weed biologicalcontrol program, should not pose a significant threat to plantsof economic importance. The transmission of F. tumidum byE. postvittana to infect gorse is currently being investigated.

ACKNOWLEDGEMENTS

This research was funded by the New Zealand Tertiary Edu-cation Commission. We thank Monika Walter and Kirsty S.H.Boyd-Wilson from HortResearch, Lincoln for assistance inconducting sampling in two experiments. Neil Andrews andAlison Lister are acknowledged for their respective assistancewith regard to the SEM and statistical analyses.

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Accepted for publication 12 January 2008.

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