Persistence of conidia and potential efficacy of Beauveria bassiana against pinhole borers in New Zealand southern beech forests

Forest Ecology and Management 246 (2007) 232–239 www.elsevier.com/locate/foreco

Persistence of conidia and potential efficacy of Beauveria bassiana against pinhole borers in New Zealand southern beech forests Stephen D. Reay a, Celine Hachet b, Tracey L. Nelson b, Michael Brownbridge b, Travis R. Glare b,* b

a Silver Bullet Forest Research, Auckland, New Zealand Biocontrol & Biosecurity, AgResearch, PO Box 60, Lincoln, New Zealand

Received 30 March 2006; received in revised form 2 April 2007; accepted 4 April 2007

Abstract Three native species of pinhole borer Treptoplatypus caviceps, Platypus apicalis and Platypus gracilis (Curculionidae: Platypodinae) are occasional pests of indigenous forests in New Zealand. These species predominantly attack ‘southern beech’, usually colonising fallen logs or stumps, but populations can reach densities which threaten healthy trees when large amounts of breeding material are available. The larvae live in tunnels which penetrate deep into the trees. Few control processes or agents are known in New Zealand. A search for naturally occurring biological enemies of pinhole borer resulted in the isolation of several insect-pathogenic fungi, the majority of isolates being Beauveria bassiana. Several strains of B. bassiana were isolated from pinhole borer in South Island southern beech forests, as determined by sequencing of the ITS-5.8s region of rDNA. Previous research demonstrated that B. bassiana is pathogenic to both pinhole borer larvae and adults in the laboratory. This paper reports on further investigations into the practical use of B. bassiana as a biopesticide for localised control of pinhole borer outbreaks. Aqueous suspensions of conidia were applied to logs and persistence was examined in a southern beech forest. B. bassiana colony-forming units were isolated up to 7 months after application, with better survival on the sides of logs than either the top or bottom. It was also demonstrated that conidia could penetrate several centimetres into the tunnels. In the field there was some pinhole borer mortality, as evidenced by cessation in frass production and a reduction in pinholes, but the level was too low to be considered acceptable for control purposes. # 2007 Elsevier B.V. All rights reserved. Keywords: Platypus spp. Treptoplatypus sp.; Beauveria bassiana; Biopesticide; Persistence; Biological control

1. Introduction There are three species of pinhole borer native to New Zealand indigenous forests, Platypus apicalis (White), Platypus gracilis (Broun) and Treptoplatypus caviceps (Broun) (Curculionidae: Platypodinae) (Milligan, 1979). These species predominantly attack ‘southern beech’ (Nothofagus spp.), but will attack other native and exotic trees. The beetles usually colonise dead standing and windthrown trees, and larvae and adults tunnel into trees allowing the ingress of fungi deep into the wood. Three types of fungi are associated with these insects. Firstly, ambrosia fungi, which pinhole borer depend on for food and are not

* Corresponding author. E-mail address: [email protected] (T.R. Glare). 0378-1127/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2007.04.005

pathogenic to the trees (Kirkendall et al., 1997). Secondly, pathogenic fungi (Sporothrix sp.) are carried by the beetles and can infect and kill live trees when attacked. Finally, other fungi that appear to be associated (although not necessarily a strict association) with the insects, and include sapstain fungi. The galleries produced by the insects and wood staining caused by fungi damage harvested timber and reduce its market value (Audino et al., 2005). At high population densities, Platypus/ Treptoplatypus spp. can kill mature host trees (McCracken, 1994); internal tunnelling combined with wood decay caused by the fungi compromise the xylem, thereby weakening the tree’s stem which will break under conditions of extreme stress. These trees are then open to colonisation by more beetles (Faulds, 1977; Audino et al., 2005). The insect is difficult to control as it lives deep within the wood. Populations are currently regulated by silvicultural

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practices. Biological control would be an attractive option in New Zealand, both to support sustainable forestry and to allow these beetles to be controlled in indigenous forests. Few parasites and predators have been recorded attacking Platypodinae, and do not appear to be effective control agents (Milligan, 1979; Glare et al., 1993). Milligan (1979) reported an un-described parasitic nematode from all three native species but its significance in population decline is unknown. Several microbial pathogens have been found (Milligan, 1979; McCracken, 1994). Biocontrol with fungi has been successfully tested against various bark beetles (Kreutz et al., 2004) and has appealing characteristics for use against pinhole borers, such as specificity for the target pest, high virulence and low environmental impact. While P. apicalis, P. gracilis and T. caviceps are native to New Zealand and large-scale felling of native forest for commercial sale has ceased, the need may arise for localised control of these beetles in areas where southern beech debris is high e.g., in selectively logged areas, where felling takes place for other purposes (roading, mining, etc.), or when large-scale natural disturbances occur (windfall, earthquake, etc.). Two mycopathogens have been recorded from pinhole borer. Beauveria sp. was ‘‘regularly found infecting dead or unhealthy larvae and adults’’ (McCracken, 1994). More recently strains of B. bassiana, B. brongniartii and Metarhizium anisopliae (Deuteromycetes: Hyphomycetes) isolated from southern beech forest soil or non-pinhole borer hosts were tested against pinhole borer larvae and adults (Glare et al., 2002). Of the 10 isolates tested, all killed the beetles and sporulated on cadavers, indicating the potential of these common entomopathogenic fungi as control agents. Adults exposed to sporulating fungal cultures could also transfer a lethal dose of inoculum to larvae (Glare et al., 2002). In this paper, we report on the isolation of B. bassiana from pinhole borer and their local environment. Molecular

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characterization of recovered Beauveria sp. and Metarhizium sp. strains is undertaken to allow future distinction between applied and naturally occurring strains. The potential use of B. bassiana for the biocontrol of pinhole borers is investigated, and aspects relating to the practical application of the fungus to logs in a native forest are discussed. 2. Materials and methods 2.1. Fungal isolates and culturing Infected pinhole borer were found during field surveys and field trials in southern beech forests in the South Island of New Zealand. Fungi were isolated directly from cadavers onto a semi-selective medium consisting of potato dextrose agar (PDA-Merck) containing streptomycin sulphate (Sigma: 350 mg/l), tetracycline hydrochloride (Sigma: 50 mg/l) and cycloheximide (Sigma; 125 mg/l). Isolates were accessioned into the AgResearch Insect Pathogen Culture Collection (Lincoln). Isolates from the collection were used in bioassays and transfer experiments with pinhole borer (Table 1). All isolates were cultured on PDA and incubated at 20–25 8C for 2– 3 weeks until conidia were produced. For tests of efficacy against pinhole borer in logs, conidia were harvested into sterile aqueous 0.01% Triton X-100 (BDH) and the concentration was adjusted to 109 conidia/ml unless otherwise stated. In all cases, concentration was verified using a haemocytometer and conidial viability was determined by serial dilution in 0.1 M phosphate buffer and plating onto PDA, with colony forming units counted 4–7 days after incubation at 25 8C. 2.2. DNA isolation and analysis Fungal hyphae were prepared by spread plating spores onto PDA plates overlaid with colourless cellophane. Plates were

Table 1 Isolates of Beauveria spp. and Metarhizium sp. used in this study Isolate no. a

Genus and species

Source

Host

Genbank accession number

Date recovered or received

F265

DQ385618

4 April 1997

– –

DQ385620 DQ385615

20 November 1997 11 March 1999

F361

B. bassiana

–

DQ385614

11 March 1999

F363

B. bassiana

–

DQ385616

11 March 1999

F501 F502 F531 F547

B. bassiana B. bassiana Beauveria cf. bassiana Beauveria cf. bassiana

P. gracilis T. caviceps P. gracilis –

DQ385621 DQ385617 DQ385619 DQ407710

11 11 16 23

F548

Beauveria cf. bassiana

–

DQ407711

23 December 2005

F530

M. flavoviride var. novazealandicum

Soil isolation; collected from the West Coast, NZ Isolated from BotaniGardTM (Mycotech, USA) Soil isolation from logged southern beech forest, West Coast, NZ Soil isolation from un-logged southern beech forest, West Coast, NZ Soil isolation from un-logged southern beech forest, West Coast, NZ Log collected from Reefton, West Coast, NZ Cragieburn, Arthurs Pass, NZ Maruia, Lewis Pass, NZ Pinhole borer frass on southern beech logs, west Coast, NZ Pinhole borer frass on southern beech logs, West Coast, NZ Cragieburn, Arthurs Pass, NZ

–

F305 F359

Beauveria nr brongniartii (C. scarabaeicola) B. bassiana B. bassiana

Platypus sp.

DQ385622

November 2003

a

AgResearch culture accession number.

July 2003 July 2003 January 2004 December 2005

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grown at 25 8C for 24–48 h, or until a fine layer of hyphae was observed. Microcentrifuge tubes and pestles were cooled in liquid nitrogen. Small amounts of hyphae were added to the frozen tubes and immediately ground into a fine powder. Tubes were removed from the liquid nitrogen and 750 ml of extraction buffer and 750 ml of phenol:chloroform added. The contents were mixed and tubes were incubated on ice for 30 min. After incubation, tubes were centrifuged for 5 min at 13,000  g. The upper phase was transferred to a new tube and 0.6 of the volume of isopropanol added. Tubes were centrifuged for a further 15 min at 13,000  g and the supernatant removed. The resulting pellet was air-dried at 37 8C for 10 min and then resuspended in 300 ml of TE buffer. Three hundred microliters of phenol:choloroform was added and the tube was centrifuged for 5 min at 13,000  g. The upper phase was transferred to a new tube and two volumes of 100% ethanol were added before centrifuging again for 15 min at 13,000  g. The supernatant was aspirated and the pellet air-dried at 37 8C before finally being resuspended in 100 ml of TE. The concentration of the DNA used in the PCR reactions was determined empirically and ranged from a 1 to 10 dilution of the initial isolation. Amplifications of ITS1-5.8sITS2 and 28s fragments were performed using primers TW81 (50 -GTTTCCGTAGGTG AACCTGC) and AB28 (50 -ATATGCTTAAGTTCAGCGGGT) (Curran et al., 1994). PCR reactions were performed in 25 ml volumes containing 0.4 mM of each primer (Invitrogen), 200 mM dNTPs (Innovative Sciences), 2.5 ml reaction buffer, 2.5 mM MgCl2, 2 ml DNA and Taq (0.7U/reaction) (Roche, Expand HiFidelity). Amplifications were carried out in a Perkin-Elmer 480 thermal cycler using 30 cycles of 1 min at 94 8C, 1 min at 54 8C, 2 min at 72 8C. Positive and negative (dH2O) controls were included in each PCR run. PCR products were cleaned up using Eppendorf Perfect Prep Gel Cleanup Kit and sequenced directly (Waikato DNA Sequencing Facility, The University of Waikato, New Zealand or AWCGS Sequencing Facility, Massey University, New Zealand). All sequences from isolates used that are held in the permanent culture collection have been submitted to Genbank. Sequences were aligned using DNAMAN (Lynnon BioSoft, Quebec, Canada) and ClustalX (Thompson et al., 1997). Phylogenetic analysis using Bayesian inference was conducted using the programme MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Models of nucleotide substitution that best fit the data were selected using the Akaike Information Criterion (see Posada and Buckley, 2004) in MrModelTest v2 (Nylander, 2005) implemented in PAUP*4.0b10 (Swofford, 2002). The model selected for the ITS1-5.8s-ITS2 dataset was GTR + G, which is a general time reversible model (Rodrı´guez et al., 1990; Yang et al., 1994) with a gamma-shaped rate variation across sites, Dirichlet distribution and a proportion of invariable sites. Two runs of four chains, saving trees every 100 generations were conducted. After 3,500,000 generations the two runs had converged close to the same value (determined by when the standard deviation of split frequencies fell below 0.005) and the

first 25% of trees were discarded as burn-in. The consensus tree, with the posterior probabilities for each split and mean branch lengths, was visualised using Treeview 1.6.6 (Page, 1996). 2.3. Infection of beetles in artificial tunnels in southern beech logs Pinhole borer adults and larvae were recovered from southern beech logs collected in South Island forests. Short log sections were dissected in the laboratory and larvae and adults removed from tunnels. Beetles were identified using the descriptions of Milligan (1979) and were used immediately. Fifteen holes 1.9 mm in diameter (for T. caviceps) and seven holes 1 mm in diameter (for P. gracilis) were drilled into each of 14 southern beech log sections (60 cm  25 cm diameter) to a depth of 30 mm. Two milliliters of B. bassiana F361 containing 109 conidia/ml 0.01% Triton X-100 was applied by spraying onto the bark of each of five log sections. After the surface had dried, 22 beetles (15 T. caviceps and 7 P. gracilis) were positioned on the logs adjacent to the artificial galleries (treatment A). A further five logs were sprayed with F361 conidia after 22 beetles (15 T. caviceps and 7 P. gracilis) had already been placed inside the artificial galleries (treatment B). For the control, four southern beech logs were each sprayed with 2 ml 0.01% Triton X-100 after 20 beetles (14 T. caviceps and 6 P. gracilis) had been located in the artificial galleries. Logs were kept moist for the duration of the experiment (17 days) by misting with water every day, and were maintained at a constant 20 8C. The presence of B. bassiana in frass deposited at the entrance of the tunnels was determined by spread plating suspensions prepared from frass samples onto semi-selective PDA. The presence of B. bassiana on tunnel walls was assessed by scraping the sides of the tunnels with a wire loop and streaking onto PDA antibiotic medium. Frass production was recorded daily as an indicator of beetle activity and health, and all cadavers (dead beetles often fell from the logs) were collected and incubated in 100% humidity to promote fungal outgrowth and confirm infection with B. bassiana. After 17 days, logs were destructively sampled and any remaining beetles removed from the tunnels. If the beetles had died, the location of the cadaver was noted, and then observed for symptoms of infection by B. bassiana. Beetles that died within 48 h of placement in the logs (no frass produced) were excluded from the final data. 2.4. Use of Beauveria bassiana to protect logs from pinhole borer attack and damage in the field An area of mixed red beech (Nothofagus fusca)/silver beech (Nothofagus menziesii) forest near the Lewis Pass, South Island, New Zealand was used as the experimental site. An area 40 m in diameter was selectively harvested during winter 2003. The majority of trees greater than 40 cm diameter in this area were felled and the merchantable sections of logs removed for processing. The remaining log sections and un-merchantable trees were utilized in this trial.

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In spring (October) 2003, 38 of the remaining logs were selected and randomly assigned to each of two treatments. None of the logs showed evidence of fresh pinhole borer attack at this time. One month later (November), logs were either sprayed with B. bassiana (F361) conidia (109 conidia/ml 0.01 Triton X-100) or 0.01% Triton X-100 only (control). Sprays were applied at a rate of approximately 1 l/2.5 m2. Isolate F361 was used in this experiment as it had not been recovered on-site and had a low natural incidence; furthermore, it was possible to differentiate the strain using molecular techniques from other ‘background’ Beauveria strains recovered. However, it was not selected on the basis of its virulence against pinhole borer. Logs were sprayed using a hand held garden sprayer, which was adjusted to deliver a fine mist coating to all surfaces. At this time all logs were assessed for evidence of pinhole borer attacks (galleries), and each existing gallery was marked with a map pin. Logs were sprayed four more times thereafter (total five sprays) at approximately monthly intervals until autumn (March 2004). One month after the final spray (April 2004), when colonisation activity appeared to have ceased, each gallery was recorded, marked, and all visible frass removed using a fine haired brush; a different brush was used for logs from each treatment. The persistence of B. bassiana on the treated logs was assessed by taking 6.25 cm2 bark samples using a chisel, which was pre-sterilized with 70% ethanol. In December 2003, following the first spray treatment; a single bark sample was taken from the side of each log. In October 2004, (7 months after the final treatment), eight bark samples (two from each side, top, and bottom of the logs) were taken from every second log. In April 2005, one bark sample was taken from the side of every log. Each sample was placed in a plastic bag and taken back to the laboratory for processing as described above to determine the concentration of B. bassiana on the bark. During spring (October 2004), all marked holes were revisited and the presence of frass recorded. Frass is not ejected from the time adults complete tunnelling (following egg laying) until larval frass is produced, a period of approximately 2–6 months (McCracken, 1994). The assumption was made that the proportion of live nests with no frass being produced would be similar for both treatments. During autumn (March 2004) a second field trial was established to investigate the persistence of Beauveria on logs treated with a single spray. Eighteen felled logs were randomly located in an area in close proximity to the trial described above and treated as above using B. bassiana (F361) sprayed at 109 conidia/ml or 0.01% Triton X-100 only (control). Approximately 6 months (September 2004) and 1 year later (April 2005) one bark sample was taken from the side of each log as described above. Persistence (cfu) data were analysed using analyses of variance (Minitab, Minitab Inc., State College, PA, USA). Data collected on the proportion of galleries with frass were arcsine transformed before being analysed using ANOVA.

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3. Results 3.1. Isolation and identification of fungi from pinhole borer and the environment Pathogenic fungi were successfully isolated from dead pinhole borer and other source materials collected in the field (Table 1). Morphological examination based on conidia morphology and sporulation structures identified most isolates as B. bassiana. One isolate, F530, originally identified as M. anisopliae, was later re-classified as Metarhizium flavoviride var. novazealandicum. To confirm species and determine genotype, all Beauveria sp. isolates were subjected to molecular characterization based on sequence of the ITS-5.8s region of nuclear ribosomal DNA. The sequence comparisons with known isolates of Beauveria sp. (e.g. Rehner and Buckley, 2005) demonstrated that all isolates were B. bassiana, but belonged to several, clearly distinct genotypes (Fig. 1), suggesting that there is little adaptation to pinhole borer as a host for this common entomopathogenic fungus. However, two isolates from locations over 100 km apart (Reefton and Craigburn) were very similar in ITS-5.8s sequences. Isolate F530 aligned with the genotype M. flavoviride var. novazealandicum as defined by Driver et al. (2000). In the Maruia field trials, B. bassiana was isolated directly from both control (LognC in Fig. 1) and F361-treated (LognT in Fig. 1) logs. Ten of these isolates were analysed by ITS-5.8s sequencing to determine if they were identical to the applied strain (F361) or were ‘natural’ strains. B. bassiana from untreated logs were generally unrelated to the F361 isolate (with the exception of samples from two logs). On treated logs, all but one of the B. bassiana isolates analysed were homologous to F361; however, it is likely that treated logs would also have been contaminated with ‘natural’ strains (albeit at a lower level than the applied strain) and as selections for analyses were based on phenotypic characteristics of colonies on a general isolation medium, such results are not unexpected (Fig. 1). Isolates from West Coast southern beech forest soil (F359, F361 and F363) were all similar, while a single isolate from soil (F265) aligned with a clade (E) defined by Rehner and Buckley (2005) as Cordyceps scarabaeicola, although F265 isolate has not produced any sexual structures in culture or on insects. Two further isolates recovered from frass produced by pinhole borer at Reefton (F547 and F548), were very similar to F531 (from P. gracilis), and align with Rehner and Buckley’s (2005) classification as B. cf. bassiana (clade C). 3.2. Infection of beetles in artificial holes in southern beech logs Data from these trials were intended to provide a simple ‘proof of concept’ for the following field applications. The sample size used was deemed too small for a robust statistical analysis, but the trial served to demonstrate two ways in which beetles may acquire a fungal infection, and data on the location of cadavers after death. Nearly 39% of the untreated beetles

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Fig. 1. Relationship between Beauveria spp. and Metarhizium sp. associated with platypodine beetles based on sequence comparison of ITS1, 5.8s and ITS2 region of rDNA. B. bassiana isolates from Platypus spp. (starred) compared with B. bassiana from West Coast soil, frass, logs in treated and untreated plots, and sequences from Rehner and Buckley (2005) (ARSEF = R: R32 = AY532017; R156 = AY531985; R344 = AY532023; R792 = AY532048; R813 = AY532052; R816 = AY532053; R843 = AY532055; R1431 = AY531980; R1685 = AY531990; R4474 = AY532027). AF139851 is M. flavoviride var. novazealandicum (Driver et al., 2000). Posterior probabilities for each split are given.

were alive after 17 days, while none of the beetles in treated tunnels survived (Table 2, Fig. 2). Approximately 90% of beetles placed in galleries prior to the logs being treated were killed by B. bassiana, while approximately 70% of beetles placed on logs after they were treated with B. bassiana were infected with the fungus at the end of the study. Early death (days 2–5) may have been the result of mechanical injury when placing the beetles in the tunnels. B. bassiana was isolated from samples collected from the tunnel walls after plating onto semi-selective PDA, with a mean number of colonies per agar plate of 47 and 33, for logs sprayed

after beetles were placed in galleries and those sprayed before beetles were positioned, respectively. The mean number of colonies per agar plate for control logs was 0.6 (B. bassiana was detected in only one control gallery). Viable colony-forming units on agar media indicated that spores had either survived passage through the beetle gut or were contaminating frass in the tunnel walls.

Table 2 Survival of pinhole borer placed on treated and untreated logs in the laboratory Treatment

Control Logs treated prior to beetles being placed in galleries Logs treated after beetles were placed in galleries

Percentage survival (%) a 7 days (%)

13 days (%)

17 days (%)

84.6 76.9

61.5 38.5

38.5 0

76.5

17.6

0

a survival on days 7 and 13 determined by production of fresh frass between sampling dates.

Fig. 2. Relative survival of platypodine beetles and cause of death in untreated logs (control), treatments A (bark sprayed with B. bassiana F361 before putting beetles onto southern beech logs) and treatment B (bark sprayed with F361 after putting beetles into the tunnels), and location of cadaver (in/out of tunnels) if beetles died during the trial.

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Fig. 3. Mean number of pinhole borer galleries (per 100 cm2) on treated and untreated southern beech logs, 1 and 5 months after application of B. bassiana (bars = S.E.).

3.3. Use of B. bassiana to protect logs from pinhole borer attack and damage in the field

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Fig. 4. The proportion of platypodine beetle galleries with frass in spring 2004, 7 months after application of B. bassiana F361 (bars = S.E.).

3.3.2. Persistence of Beauveria sp. on logs In Trial one, 6.8  108 Beauveria sp. cfus/m2 were detected on treated logs 1 month after the first application but prior to the second spray, which was significantly higher than on untreated logs (F (1,37) = 41.91, P < 0.001) (Table 3). Seven months after the fifth treatment, Beauveria sp. levels were still significantly higher on treated than on untreated logs (F (1,17) = 25.03, P < 0.001) (Table 3). At this time there was also a significant difference in Beauveria sp. concentration with respect to the

location of the bark sample on the original log (i.e. top, bottom, side) for treated (F (2,24) = 3.87, P = 0.04) and control (F (2,23) = 4.88, P = 0.02) logs (Table 4). More colony forming units were found on bark sampled from the sides than the top or bottom of treated logs. On control logs, more colony forming units were found on the bottom of logs. One year after the final spray, there was no significant difference in the level of Beauveria sp. on the bark with respect to treatment (F (1,37) = 0.40, P = 0.53) (Table 3). Levels of Beauveria sp. detected dropped around 300 times between the first and last sampling. The levels of Beauveria sp. on control logs ranged between 9.8  105 and 1.4  107 cfu/m2 (Table 3) with the lowest level recorded at the mid sampling time point, suggesting a random distribution of natural occurrence rather than a steady decline like that observed for the treated logs. For logs treated only once, levels of Beauveria sp. recovered were still higher on treated logs (1.0  107  8.3  106 cfu/m2) compared with untreated control logs (6.0  105  7.1  105 cfu/m2) 6 months after treatment (F (1,15) = 9.49, P = 0.008). Approximately 1 year after treatment the concentration on treated logs was 1.0  106  1.5  106 cfu/m2 and 2.5  105  4.8  105 cfu/m2 for untreated logs, and was not significantly different (F (1,15) = 1.35, P = 0.27). This represented a 10-fold decrease in concentration over a 6-month period. Although sequencing of the ITS-5.8s region was only done on a proportion of the Beauveria spp. isolated, in general, those fungi recovered from control logs were different to the applied F361 strain. In contrast, all of the Beauveria sp. recovered from treated logs were identical to F361.

Table 3 Persistence of B. bassiana propagules on logs treated five times

Table 4 Differences in B. bassiana concentration (cfu/m2) on southern beech bark with respect to surface orientation at 7 months after treatment

Time

Treatment

3.3.1. Colonisation by pinhole borer The number of galleries observed (all three species combined) during spring 2003 was not significantly different between treated and untreated (control logs) (F (1,37) = 0.60, P = 0.44) (Fig. 3). Five months later the number of galleries had increased, on average, 10-fold on all logs, while there were nearly 20% fewer holes in Beauveria sp. treated logs than the controls. However, the difference between the number of galleries in treated and untreated logs was not significant (F (1,37) = 1.30, P = 0.26) (Fig. 4). Seven months later (the following winter) there was a significant difference in the number of galleries which produced frass between treated and untreated logs (F (1,37) = 5.77, P = 0.02) (Fig. 4). At this time, 34% of treated logs had galleries with frass at the entrance compared with 56% of holes on untreated logs.

1 month after 1st treatment 7 months after final treatment 12 months after final treatment

Treated

Control

B. bassiana cfu/m2

S.D.

B. bassiana cfu/m2

S.D.

6.8  108

4.5  108

1.4  107

2.1  10 7

3.5  107

2.1  107

9.8  105

1.1  10 6

2.4  106

4.2  106

6.2  106

2.5  10 7

Surface

Mean B. Bassiana (cfu/m2) 7

S.D.

Beauveria

Top Side Bottom

1.5  10 b 3.5  107 a 1.4  107 b

1.6  107 2.9  107 1.6  107

Control

Top Side Bottom

8.9  104 a 9.8  105 a 2.1  106 b

2.6  105 1.6  106 2.4  106

Means in each treatment with the same letter are not significantly different (P > 0.05).

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4. Discussion The results of this and a previous study (Glare et al., 2002) demonstrate that B. bassiana can infect both P. apicalis, P. gracilis and T. caviceps in the field and is the most common mycopathogen within these New Zealand species. Molecular characterisation and the analysis of genotypes of Beauveria sp. infecting pinhole borer, in pinhole borer frass, and in soils around pinhole borer breeding sites suggests there is little specificity in strains attacking the beetles. The only other fungal pathogen found in our surveys was M. flavoviride var. novazealandicum and this infection occurred in only one beetle. This variety of Metarhizium is found in New Zealand and Australia and is active at lower temperatures than other Metarhizium spp. strains (Driver et al., 2000). The use of B. bassiana as a mycoinsecticide in high risk situations may offer a method of controlling localized pinhole borer infestations. Effective control requires use of a virulent strain which shows good environmental persistence and the ability to penetrate and survive in tunnels formed by the insects. In the present study we examined persistence of conidia applied on logs and penetration into tunnels, as well as the efficacy of conidia applied to logs to reduce pinhole borer attack. The field application trials successfully demonstrated proof of concept, despite little evidence of control at levels that could be considered to be economically viable. We have demonstrated that B. bassiana is able to persist in the field for an extended period following application, and that the fungus can kill beetles after application to logs. While field applications did not stop damaging populations of the insect building up, they did have an impact on colonisation and beetle survival; furthermore, the current study was only a preliminary investigation using unformulated conidia. The F361 B. bassiana strain used was selected on the basis of its unique genotype which allowed the fungus to be differentiated from background Beauveria sp. in the field. In the laboratory, beetles and larvae have been shown to be very susceptible to attack by Beauveria spp. (Glare et al., 2002) and it may be possible to select a more active strain through further bioassays. The re-isolation of the applied isolate from logs in the field, as well as cadavers from these treated logs, several months after treatment indicates the ability of B. bassiana to persist and infect for an extended period. B. bassiana propagules persisted on logs for well over 4 months in all situations, although there was a steady decline in recoverable colonies over time. However, it does not appear that persistence of infective conidia will be the limiting factor in the utilization of B. bassiana as a biopesticide, providing sufficient inoculum is initially applied. If a more efficacious strain is subsequently identified, formulation and application techniques must be devised to enable these microbes to be effectively used in a pest management strategy. Formulation for greater persistence or efficacy has been used to overcome limitations of entomopathogenic fungi in a number of situations (Glare, 2004; Brownbridge, 2006). The field life and persistence of the applied strains can be considerably extended using formulation techniques, for example using a biopolymer coating that

protects conidia from UV radiation, as well as making them more rain-fast. While chemical control of pinhole borers is ineffective, microbial agents in biopesticide formulations can infect all beetle life stages, even deep inside logs. The ability of conidia of B. bassiana to penetrate into tunnels could be crucial to the success of the fungus as a biopesticide, given that larvae do not leave the galleries, and adults spend significant periods in them. We demonstrated that conidia survive and could be recovered from galleries in laboratory experiments. This penetration may be due to transmission on beetles entering the galleries, or conidia entered the tunnels during spraying. Glare et al. (2002) demonstrated the importance of adults in transmitting conidia to other adults and larvae. This transmission could greatly increase the infection potential to offspring populations. Colonisation is initiated by males who first bore galleries into logs before attracting females. P. apicalis and P. gracilis release aggregation pheromones that attract both males and females of these species, resulting in mass attacks on suitable hosts (Milligan et al., 1988). T. caviceps males produce a sex pheromone to attract females, and do not induce mass attacks (McCracken, 1994). Following mating, the females continue construction by tearing wood fibres from the tunnel face. The fibres are then passed back and ejected from the tunnel entrance by males (Milligan, 1979; McCracken, 1994). Repeated excursions to the gallery entrance by male beetles increases the likelihood of exposure to and acquisition of fungal propagules, leading to infection and potential transmission to other individuals within the gallery. However, it is possible that infection does not occur rapidly enough to stop initial pinholes being formed, or that penetration into the tunnels is limited. It was interesting that B. bassiana was detected in frass around the artificial tunnels, suggesting that beetles come into contact with B. bassiana, repeatedly. This activity may increase the potential efficacy of treatments applied post colonisation. Under such a scenario, logs could be treated after the initial colonisation has occurred, and viable colonies with offspring may be killed by conidia transmitted into the galleries as male beetles become contaminated as they clear frass from the galleries. If either parent is killed in the early stages of gallery development, then the gallery fails. If males die during later stages of gallery construction few larvae develop to maturity (McCracken, 1994). Cadaver position could be significant in terms of recycling of infection in the environment. During the field experiments we observed galleries in treated logs that were blocked by mycosed male cadavers. In such cases, any subsequent emerging beetles would likely become infected or trapped. In contrast, in the laboratory experiments, B. bassiana infected cadavers were usually found outside the galleries. This may have been an artifact of the shallow holes, but if found in the field it would reduce potential transmission to larval populations. However, it is difficult to determine the extent of this phenomenon in field populations. Behavioral responses have been noted in other insects infected with fungal pathogens in the field (e.g. Gilliam et al., 1983; Krasnoff et al., 1995).

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Biological control of pinhole borer would be an attractive option in New Zealand, both to support sustainable forestry and to allow sustainable control strategies to be implemented in indigenous forests. Availability of a fungal-based biopesticide has the advantage of providing a short-term management tool for use in outbreak situations, that will have minimal sideeffects on non-target invertebrates (Brownbridge, 2006). Furthermore, as the fungus appears to have limited persistence, targeted applications to logs within a small area are unlikely to displace native micro-flora. Several Beauveria-based products are available around the world for other pest species, such as scarabs (e.g. MELOCONT; Strasser, 1999; Brownbridge, 2006), indicating the potential of this fungus for pinhole borer control. In the current study we successfully demonstrated that all three New Zealand species of pinhole borer are susceptible to entomopathogenic fungi and that some strains could be utilized in a control strategy. Future research will concentrate on the development and evaluation of formulations to improve persistence on logs (especially under the rainy conditions experienced in New Zealand’s southern beech forests), penetration into pinholes, and overall efficacy against platypodine beetles. Acknowledgements We would like to thank John Dronfield and David Norton for their valuable assistance during this study, Dr Cor Vink, AgResearch, for instruction in running Mr Bayes, and Philippa Gerard for comments on a previous version of this manuscript. This study was funded by the Foundation for Science, Research and Technology, Sustainable Indigenous Forestry Programme C09X0308. References Audino, P.G., Villaverde, R., Alfaro, R., Zerba, E., 2005. Identification of volatile emissions from Platypus mutatus (=sulcatus) (Coleoptera: Platypodidae) and their behavioral activity. J. Econ. Entomol. 98, 1506–1509. Brownbridge, M., 2006. Entomopathogenic fungi: status and considerations for their development and use in integrated pest management. Recent Res. Dev. Entomol. 5, 27–58. Curran, J., Driver, F., Ballard, J.W.O., Milner, R.J., 1994. Phylogeny of Metarhizium: analysis of ribosomal DNA sequence data. Mycol. Res. 98, 547–552. Driver, F., Milner, R.J., Trueman, J.W.H., 2000. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol. Res. 104, 134–150. Faulds, W., 1977. A pathogenic fungus associated with Platypus attack on New Zealand Nothofagus species. NZ J. For. Sci. 7, 384–396. Gilliam, M., Taber, S., Richardson, G.V., 1983. Hygienic behavior of honey bees in relation to chalkbrood disease. Apidologie 14, 29–39. Glare, T.R., 2004. Biotechnological potential of entomopathogenic fungi. In: Arora, D.K. (Ed.), Fungal Biotechnology in Agricultural, Food, and

239

Environmental Applications, Mycology Series, vol. 21. Marcel Dekker, NY, pp. 79–90. Glare, T.R., O’Callaghan, M., Wigley, P.J., 1993. Checklist of naturallyoccurring entomopathogenic microbes and nematodes in New Zealand. NZ J. Zool. 20, 95–120. Glare, T.R., Placet, C., Nelson, T.L., Reay, S.D., 2002. Potential of Beauveria and Metarhizium as control agents of pinhole borers (Platypus spp.). NZ Plant Prot. 55, 73–79. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755. Kirkendall, L.R., Kent, D.S., Raffa, K.R., 1997. Interactions among males, females and offspring in bark and ambrosia beetles: the significance of living in tunnels for the evolution of social behaviour. In: Choe, J.C.,Cuespi, B.J. (Eds.), The Evolution of Social Behaviour in Insects and Arachnids. Cambridge University Press, pp. 181–215. Krasnoff, S.B., Watson, D.W., Gibson, D.M., Kwan, E.C., 1995. Behaviorial effects of the entomopathogenic fungus, Entomophthora muscae on its host Musca domestica: postural changes in dying hosts and gated pattern of mortality. J. Insect Physiol. 41, 895–903. Kreutz, J., Zimmermann, G., Vaupel, O., 2004. Horizontal transmission of the entomopathogenic fungus Beauveria bassiana among the spruce bark beetle, Ips typographus (Col., Scolytidae), in the laboratory and under field conditions. Biocontrol Sci. Technol. 14, 837–848. McCracken, I.J., 1994. The Platypus pinhole borers and management of beech forest: a review of present knowledge. Unpublished report, Forest Research Institute, 25 pp. Milligan, R.H., 1979. Platypus apicalis White Platypus caviceps Broun Platypus gracilis Broun (Coleoptera: Platypodidae). Forest Timber Insects NZ 37, 15. Milligan, R.H., Osbourne, G.O., Ytsma, G., 1988. Evidence for an aggregation pheromone in Platypus gracilis (Broun) (Coleoptera: Platypodiae). J. Appl. Entomol. 106, 20–24. Nylander, J.A.A., 2005. MrModeltest 2.2 Department of Systematic Zoology. Uppsala University, Uppsala. Page, R.D.M., 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comp. Appl. Biosci. 12, 357–358. Posada, D., Buckley, T.R., 2004. Model selection and model averaging in phylogenetics: advantages of akaike information criterion and Bayesian approaches over likelihood ratio tests. System. Biol. 53, 793– 808. Rehner, S.A., Buckley, E., 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97, 84–98. Rodrı´guez, F., Oliver, J.F., Marı´n, A., Medina, J.R., 1990. The general stochastic model of nucleotide substitution. J. Theoret. Biol. 142, 485–501. Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Strasser, H., 1999. Beurteilung der Wirksamkeit des biologischen Pflanzenschutzpra¨parates MELOCONT-Pilzgerste zur Maika¨ferbeka¨mpfung. Forderungsdienst 5, 158–164. Swofford, D.L., 2002. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882. Yang, Z., Goldman, N., Friday, A., 1994. Comparison of models for nucleotide substitution used in maximum-likelihood phylogenetic estimation. Mol. Biol. Evol. 11, 316–324.