Biological Control 31 (2004) 320–328

www.elsevier.com/locate/ybcon

Efficacy of fiber bands impregnated with Beauveria brongniartiicultures against the Asian longhorned beetle, Anoplophora

glabripennis (Coleoptera: Cerambycidae)

Thomas Dubois,a,1 Zengzhi Li,b Hu Jiafu,b,2 and Ann E. Hajeka,*

a Department of Entomology, Cornell University, Ithaca, NY 14853-0901, USAb Department of Forestry, Anhui Agricultural University, Hefei, Anhui, 233036, China

Received 27 January 2004; accepted 30 March 2004

Available online 14 May 2004

Abstract

Use of 50� 500mm non-woven fiber bands impregnated with cultures of entomopathogenic fungi was investigated for control of

Anoplophora glabripennis. Fungal bands produced as a commercial pest control product and obtained from industry contained

Beauveria brongniartii NBL 851 while lab-produced bands contained B. brongniartii WU 20 or Metarhizium anisopliae VD 1. In the

laboratory, adult longevity decreased when adult A. glabripennis were forced to walk for 5 s on bands impregnated with B. bron-

gniartii NBL 851 and for 25 s on B. brongniartii WU 20 and M. anisopliae VD 1 bands. A field experiment was conducted over a 31-

day period in Huaiyuan, Anhui, China, by attaching bands impregnated with B. brongniartii NBL 851 or WU 20 around the trunks

of willows (Salix sp.) at two sites infested with A. glabripennis. Treatments were compared directly, by monitoring adult longevity,

and indirectly, by counting oviposition scars and exit holes per tree to quantify relative oviposition per female. Adults collected from

trees treated with B. brongniartii WU 20 died more quickly than adults from the trees treated with B. brongniartii NBL 851 or

controls. At the site with higher A. glabripennis population densities, relative oviposition per female was significantly lower for both

fungal treatments compared with the control treatment. Between fungal treatments, B. brongniartii NBL 851 had a 5 day earlier

negative effect on oviposition per female than B. brongniartii WU 20.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Anoplophora glabripennis; Asian longhorned beetle; Beauveria bassiana; Beauveria brongniartii; Metarhizium anisopliae; Entomopath-

ogenic fungi; Biological control

1. Introduction

The Asian longhorned beetle, Anoplophora glabrip-

ennis (Motschulsky) (Coleoptera: Cerambycidae) has

been found infesting trees in New York City (Haack

et al., 1996), Chicago (Poland et al., 1998), and Toronto

areas. Due to the enormity of problems caused by thisinvasive beetle in China where it is native, it poses an

enormous threat to urban and rural North American

* Corresponding author. Fax: 1-607-255-0939.

E-mail address: aeh4@cornell.edu (A.E. Hajek).1 Present address: International Institute of Tropical Agriculture,

Eastern and Southern Africa Regional Center, Kampala, Uganda.2 Present address: General Station of Forest Pest Control, State

Administration of Forestry, Shenyang, Liaoning, 110034, China.

1049-9644/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.biocontrol.2004.03.011

forests (Haugen, 2000). To date, no effective methods

for controlling A. glabripennis are available (Nowak

et al., 2001) and biological control might provide a

viable option.

Fungi are the most common pathogens reported from

Cerambycidae (A.E.H., unpubl. data). For control, en-

tomopathogenic fungi have been successfully introducedexperimentally into the habitat of tree-boring Ceram-

bycidae by: (1) foliar sprays (Shimazu et al., 1983), (2)

application of wheat bran impregnated with fungal

cultures on the ground in the vicinity of infested trees

(Ishiishikawa et al., 1988; Kawakami and Shimane,

1986) or inside infested tree trunks (Shimazu et al.,

1995), or (3) release of bark beetles contaminated with

conidia (Nobuchi, 1993). To increase infection rate,Higuchi et al. (1997) developed a product specifically

T. Dubois et al. / Biological Control 31 (2004) 320–328 321

matching a behavioral characteristic of some tree-boringCerambycidae, namely the tendency to wander on the

tree bark after emergence (Iba, 1993). The product de-

veloped by Higuchi et al. (1997) consists of 50� 500mm

non-woven fiber bands in which B. brongniartii is grown,

producing conidia on the surface. The bands are wrap-

ped around the trunk or a branch of the tree and adult

beetles become contaminated and field studies have

demonstrated high levels of infection for at least 30 daysafter placement in the field (see Higuchi et al., 1997).

Based on the success of this approach, Beauveria bron-

gniartii has been commercialized in non-woven fiber

bands for control of Psacothea hilaris (Pascoe), a wide-

spread pest of fig and mulberry in Japan, and Anoplo-

phora chinensis (F€orster) (¼Anoplophora malasiaca

Thomson), a serious pest of citrus in Japan (Higuchi,

1999). The product, named Biolisa Kamikiri, is pro-duced by Nitto Denko (Ibaraki, Osaka, Japan) and has

many additional advantages. The bands can be stored in

sealed polyethylene bags for over 400 days at 5 �C, arebiodegradable because wood pulp is used to fabricate

the bands, and B. brongnartii in the bands retains its

infectivity for at least 30 days under field conditions with

periodic rainfall and temperatures up to 35 �C (Higuchi

et al., 1997). Because P. hilaris exhibits behavioral pat-terns similar to A. glabripennis (Dubois et al., unpub-

lished data; Higuchi et al., 1997; Hua et al., 1992), we

investigated use of fiber bands impregnated with fungi

pathogenic to A. glabripennis for control.

Anoplophora glabripennis is a difficult pest to control

for numerous reasons. The fact that A. glabripennis can

attack and kill apparently healthy trees makes this spe-

cies a serious pest. A. glabripennis is known to attack>24 species of trees (Yang et al., 1995) but concern is

greatest regarding maples (Acer spp.) in the US and

poplars (Populus spp.) and willows (Salix spp.) in China.

In Anhui Province, China, where this species is native,

this species has 1–2 generations per year based on the

timing of adult emergence (Fan et al., 1997). Adult fe-

males cut a small impression in the tree bark and lay an

egg under the bark; sites for oviposition are varied,ranging from tree trunks to branches of 3 cm diameter

or less. Young larvae develop just under the bark but

later larvae develop in the xylem. Most adults emerge

from June-August but emergence is not synchronous

and can extend well after August. Adults can be long-

lived, with individuals collected in the field during our

studies living up to 86 days after collection (A.E.H.,

unpubl. data), and females often lay eggs over at least 5weeks (Keena, 2002). After emergence, the large rela-

tively heavy-bodied adults feed on the phloem–cambium

of twigs, walking throughout tree canopies and on tree

trunks; laboratory studies have shown that adults do not

readily fly when on a good-quality host (Keena and

Major, 2001). For a period after emergence and before

oviposition begins adults feed and wander on bark; the

goal with these bands is that oviposition would be pre-vented when pre-ovipositional beetles contact fungal

bands and die before laying eggs. In addition, studies

with bands have shown that fungal inoculum can be

transferred from a contaminated beetle to an uncon-

taminated beetle during mating (Tsutsumi and Yama-

naka, 1995).

To evaluate use of bands, mortality of A. glabripennis

was investigated in the laboratory by exposing adults tobands for various lengths of time. The use of fungal

bands was investigated in the field to compare efficacy of

the Biolisa Kamikiri product with the efficacy of lab-

produced bands of an isolate, B. brongniartii WU 20,

that was equally virulent during laboratory bioassays

(Dubois, 2003). Field studies were conducted over a 31-

day period in east-central China. Adult beetles were

collected at regular intervals to assess longevity and in-fection. In addition, relative oviposition per female was

calculated over time using counts of oviposition scars

and exit holes per tree.

2. Materials and methods

2.1. Fungi

Biolisa Kamikiri bands contained cultures of B. bron-

gniartii NBL 851, originally isolated from P. hilaris in

Japan.B. brongniartiiWU20 (ARSEF 6827) was isolated

from the cadaver of an A. glabripennis adult obtained

from a field population in Wuhe, Anhui, China. Meta-

rhizium anisopliaeVD 1 (ARSEF 7234) was isolated from

an adult reared at the USDA-FS Quarantine Facility inAnsonia, Connecticut, from an infested log from Chi-

cago, IL. The two strains obtained from A. glabripennis

cadavers were grown in 60-mm petri dishes containing

Sabouraud dextrose agar medium (10 g peptone, 40 g

dextrose, and 15 g agar per 1000ml) supplemented with

2% w/w yeast (SDAY) and 0.2% v/v gentamycin (Goettel

and Inglis, 1997), harvested, and stored as conidia in 10%

glycerol at )80 �C until band fabrication.

2.2. Band fabrication

All bands used in the experiments measured

50� 500mm. Initial conidial density on bands was

quantified by blending three 20–25 cm2 pieces of bands

and counting conidia using a hemocytometer. Non-

woven fiber bands obtained from Nitto Denko werecomposed of wood pulp (density of 61 kg/m3 and water

absorption capacity of 1600%) and had an initial co-

nidial density of 4.25� 108 � 3.90� l07 conidia/cm2.

Bands were stored at 4 �C until field application and had

been produced less than 1 month previously.

Bands were impregnated withM. anisopliae VD 1 and

B. brongniartii WU 20 at Anhui Agricultural University,

322 T. Dubois et al. / Biological Control 31 (2004) 320–328

Hefei, Anhui Province, China, directly prior to use. Forlab production, the exact same non-woven fiber material

used for Biolisa Kamikiri could not be obtained.

Therefore, laboratory-produced bands were made using

non-woven polyester fiber material, with an estimated

density of 32 kg/m3 and a water absorption capacity of

approximately 1350%. To produce bands, methods have

been described in detail in Dubois (2003). In general, the

fungus was grown on SDAY for 14 days at 27 �C. Oneliter of medium (10 g peptone, 10 g yeast extract, and

40 g dextrose per 1000ml) was inoculated with ca. 0.1 g

conidia and incubated on a rotary shaker for 2 days at

170–175 rotations per min at 25 �C. Four liters of me-

dium (see recipe above) were then inoculated with the

1 liter of incubated medium containing fungal cells and

incubated on a rotary shaker under the same conditions.

Nineteen liters of medium was sterilized and cooled tobelow 40 �C, and then transferred in a sterilized room to

a sterile 60 liter plastic container. The 5 liters of incu-

bated medium containing fungal cells was subsequently

added to the container. Bands were dipped in the mix-

ture and then placed horizontally on an iron rack

(1.5m� 0.8m). The rack was covered with a plastic

sheet, underneath which a warm mist humidifier was

operated for at least 11 days. Bands were then allowedto dry overnight by removing the plastic sheet, turning

off the warm mist humidifier and opening windows. This

procedure yielded P50 M. anisopliae VD 1 bands from

25 liters total, requiring a total of 16 days. Conidial den-

sity of these bands was 1.65� 108 � 2.38� 107 conidia/

cm2. A larger batch of B. brongniartii WU 20 bands was

produced (P250 bands) using a final volume of 110 liters,

requiring use of a 7 and then a 50 liter fermenter. B.brongniartii WU 20 bands averaged 1.12� 108 � 7.00�106 conidia/cm2. Lab-produced bands were stored at 4 �Cfor a maximum of 1 week prior to field application.

2.3. Band exposure bioassay

The effect of length of exposure (5, 25, 50, and 250 s)

to the three types of bands was investigated in the lab-oratory. Adult beetles were collected from field popu-

lations in Huaiyuan during the 2 weeks prior to the

bioassay and kept individually in closed 650ml

(170� 80mm) polypropylene containers with ventilated

lids, and supplemented with willow (Salix sp.) twigs.

Thirteen beetles were used for each exposure length and

fungal treatment. Untreated adults were allowed to walk

on polyester bands that were not impregnated withfungus. Insects of approximately the same age and size

were distributed equally among treatments, and all

treatments had equal sex ratios of 4 females: 9 males.

Adults were allowed to walk for 5, 25, 50, or 250 s on

vertically held bands at 22.5� 2.5 �C. To optimize

chances for infection, after the bioassay adults were

immediately placed in closed 650ml polypropylene

containers with a saturated cotton plug. Twenty fourhours later, three holes were punched in container lids

and freshly cut willow twigs were provided. Thereafter,

adults were moved to a clean container, provided with

fresh twigs every 2 days and monitored daily until death.

Dead adults were maintained at 100% RH and moni-

tored for fungal growth for 10–14 days. Insects were

scored as infected when their cadavers exhibited fungal

growth characteristic of B. brongniartii or M. anisopliae.Speed of walking for beetles was calculated for the

adults that walked greater than 500mm during this

study.

2.4. Location and design of field experiments

The field experiment was duplicated at two sites with

A. glabripennis-infested willow trees (Salix sp.), one oneach side of the Huai River, Huaiyuan. Site A had

higher A. glabripennis densities and was close to the

city while site B had lower population densities and

was relatively undisturbed. At Site A, trees were

17.9� 0.5 cm in diameter and at Site B, trees were

19.4� 0.5 cm in diameter. Each of the sites was divided

for three treatments: one area for a control and two

areas in which trees were treated with bands impreg-nated with either B. brongniartii WU 20 or B. bron-

gniartii NBL 851. Each treatment was separated by at

least 122.5m and consisted of 100 trees, ca. 11 m in

height, and spaced at 3.8� 0.1m. On each tree in the

fungal treatments, a band was hung at a height of

2.5m on 2 July (site A) and 6 July (site B), 2001, re-

spectively. The date of band application was set as �day0.� For each treatment, the 100 trees were divided intoa core of 40 trees and an outer ring of 60 trees.

Throughout the period that bands were evaluated on

trees at these sites, the average maximum temperature

was 34.5� 0.5 �C, with maximum temperatures over

35 �C on 45% of the days.

2.5. Evaluation of field trials

In the field experiments, the effect of B. brongniartii

bands against A. glabripennis adults was monitored in

two different ways. Every 5 days, adults were collected

in the outer ring of 60 trees, from 3 to 28 days after

band application. To collect adults, trees were scouted

from approximately 06:00 to 18:00 h using ladders, and

adults were carefully collected anywhere on the trees.

Adults were difficult to find so up to 10 beetles werecollected per treatment area during each sample period.

Collected adults were kept individually in 650ml

polypropylene containers with lids punched with three

small holes to allow ventilation. Adults were fed every

2 days and monitored daily. Cadavers were placed at

100% RH to promote fungal growth. Cadavers could

not be collected in the field because they were virtually

T. Dubois et al. / Biological Control 31 (2004) 320–328 323

never found since they were scavenged and removedrapidly, often by ants.

Each treatment was also assessed indirectly using the

inner core of 40 trees to calculate the relative oviposition

per female. For each of the 40 trees of the inner core, the

height and age (old, unknown age, or new) of every exit

hole and oviposition scar were recorded every 5 days,

from 4 days before bands were hung ()4), until 31 days

after bands were hung, up to a height of 3.5m. Re-cordings were made without marking the trees, so as not

to interfere with the natural behavior of adults. There-

fore, the time of appearance of each oviposition scar and

exit hole over the 36-day period was calculated. The

total oviposition scars and exit holes per tree was cal-

culated after each 5-day interval in the 36-day period,

and, assuming negligible dispersal among trees, used to

calculate the oviposition per female on the ith day foreach tree Eq. (1):

ðOi þ 1Þ0:5� ðEi�5 þ 1Þ ðfor i ¼ 1; 6; 11; 16; 21; 26; 31Þ: ð1Þ

Eq. (1) represents the quotient of the sum of all ovipo-

sition scars (O) over the sum of all exit holes (E) per tree.

The denominator is shifted backwards in time by 5 days,

because female A. glabripennis have a pre-oviposition

period of P5 days (Smith et al., 2001), and multiplied by

0.5, because, with a sex ratio of 1:1 (see Section 3), only

half of the exit holes counted resulted from emerging

females. At each site, initial oviposition per female Eq.(1) differed significantly among the treatments (see Sec-

tion 3). To be able to compare oviposition per female

over time among treatments, Eq. (1) was divided by its

value on day 1, yielding the relative oviposition per

female on the ith day for each tree Eq. (2). For succes-

sive sample dates, values for oviposition scars and exit

holes were cumulative:

Oi þ 1

0:5� ðEi–5 þ 1Þ

� �� O1 þ 1

0:5� ðE�4 þ 1Þ

� ��1

ðfor cumulative i ¼ 1; 6; 11; 16; 21; 26; 31Þ: ð2Þ

2.6. Statistics

Anoplophora glabripennis longevity in the band ex-

posure bioassay was examined using survival analysis.

The survivor function was regressed as a generalized

gamma distribution and, for each of the treatments, the

slope was estimated as a function of the control treat-

ment. Exponentiated slopes yielded the expected relative

ratio (hazard) of the survival time for each of thetreatments as a function of the controls. Omitting the

control treatment allowed for investigation of the effects

of fungal treatment (Allison, 1995). Infection was ana-

lyzed using logistic regression. Likelihood-ratio tests

were performed to investigate the effect of treatment on

infection, and, within the fungal treatments, the effectsof time on band.

Longevity of adults collected during field studies was

square-root transformed for ANOVA, followed by Tu-

key�s studentized range test for post hoc comparisons.

Infection was analyzed using logistic regression, with

disintegrated cadavers or cadavers attacked by flies or

ants treated as missing data because we hypothesized

that the fungus would have less chance to develop nor-mally in these. Data for different sites were merged to

analyze infection due to low sample sizes. Likelihood-

ratio tests were performed to investigate the effects of

size and sex of the beetles, field site, time since fungal

application, and treatment on infection.

To study the effect of bands on female oviposition,

Friedman�s two-way non-parametric ANOVA was used

to compare the initial (on the first day) and final (on the31st day) numbers of oviposition scars and exit holes per

tree among treatments and sites. Oviposition per female

on day 1 was natural log-transformed for ANOVA,

followed by Tukey�s studentized range test for post hoc

comparisons. Relative oviposition per female was nat-

ural log-transformed to obtain normally distributed

data and was analyzed as a mixed model. Trees were

considered random effects, whereas the field site, treat-ment, and time were fixed effects. Because the quadratic

time factor was also included in the mixed model, time

was centered around zero prior to analysis to avoid

correlation with the linear time factor. After each 5 day

interval and for each of the field sites, relative oviposi-

tion per female was compared among the treatments,

using a Bonferroni correction. For each of the treat-

ments and field sites, mean relative oviposition per fe-male was regressed by time, using both linear and

quadratic functions, while holding the intercept constant

at one (SAS Institute, 1985).

3. Results

3.1. Band exposure bioassay

Beetles required an average of 13.1� 1.5 s to cross

50mm of band. Survival time after exposures differed

between controls and each of the fungal treatments

(v2 P 13:41; df ¼ 1; P 6 0:0003) (Table 1), and among

the three fungal treatments (v2 P 6:98; df ¼ 1;

P 6 0:0083). For all fungal treatments, beetles walking

on bands impregnated with conidia for 25, 50, or 250 sdied significantly faster than beetles walking on control

bands. The only group of beetles exposed to bands for

5 s that died faster than controls were those exposed to

B. brongniartii NBL 851 bands. B. brongniartii NBL 851

bands had significantly higher conidial densities than

laboratory-produced bands of the other two isolates

(F ¼ 130:32; df ¼ 2; P < 0:0001).

Table 1

Survival time regression statistics of field-collected A. glabripennis adults inoculated by walking on non-woven fiber bands impregnated with

B. brongniartii and M. anisopliae in the laboratory

Time on

band (s)

Median survival time (days� SE) Hazard ratio (95% confidence interval)a

Control Fungal treatmentb Fungal treatmentb ;c

NBL 851 WU 20 VD 1 NBL 851 WU 20 VD 1

5 9.9� 1.3 5.7� 0.8 5.7� 0.8 8.0� 1.0 0.76 (0.58–1.00)� 0.91 (0.70–1.19) 1.03 (0.81–1.30)

25 10.0� 1.4 5.8� 0.9 5.8� 0.9 8.1� 1.2 0.53 (0.53–0.53)�� 0.73 (0.73–0.73)�� 0.73 (0.73–0.73)��

50 10.3� 1.4 5.9� 1.0 5.9� 1.0 8.3� 1.2 0.47 (0.47–0.47)�� 0.59 (0.59–0.59)�� 0.76 (0.76–0.77)��

250 9.0� 1.3 5.1� 0.8 5.1� 0.8 7.3� 1.1 0.62 (0.61–0.62)�� 0.69 (0.69–0.69)�� 0.85 (0.85–0.85)��

aHazard (relative risk) ratios compare exponentiated slopes of individual treatments versus controls.b Strain VD 1 is M. anisopliae; strains NBL 851 and WU 20 are B. brongniartii.c �Significantly different from the control group at a ¼ 0:05 and ��significantly different from the control group at a ¼ 0:001.

324 T. Dubois et al. / Biological Control 31 (2004) 320–328

Among the fungal-treated beetles, infection levels did

not differ between the control and the fungus treatments

(v2 ¼ 0:05; df ¼ 1; P ¼ 0:82; see Section 4). Among

fungus-treated beetles, infection did not depend on

fungal treatment or time on the bands (v2 tests;

P > 0:05) and equaled 15% (n ¼ 206).

3.2. Effects of bands on collected adults during field

studies

More adults were collected at site A (79) than in site B

(30). Because an almost equal number of males (51) and

females (58) were collected, it was assumed that the sex

ratio in the field was approximately 1:1, and the latter

assumption was used for constructing Eqs. (1) and (2).Longevity of adult A. glabripennis did not differ by

field site (F ¼ 2:44; df ¼ 1; P ¼ 0:12) and therefore sites

were merged for analyses (Table 2). Adult A. glabrip-

Table 2

Longevity of A. glabripennis adults from two different sites containing

trees treated with bands impregnated with B. brongniartii culturesa ;b

Treatment n Mean (�SE)

Control 42 11.7� 0.8 a

NBL 851 20 8.6� 0.9 ab

WU 20 47 8.6� 0.9 b

aMeans in the same column followed by different letters are sig-

nificantly different (Tukey�s studentized range test and logistic regres-

sion, P < 0:05).bResults for field sites are pooled.

Table 3

Total oviposition scars and exit holes per tree produced by A. glabripennis

pregnated with B. brongniartii culturesa

Site Treatment Oviposition scars per tree

On day 1 On d

A Control 3.5� 0.9 a 13.4�NBL 851 1.8� 0.7 a 7.3�WU 20 16.1� 3.7 b 46.7�

B Control 3.0� 0.8 a 9.7�NBL 851 1.3� 0.7 b 6.8�WU 20 1.5� 0.9 b 4.5�

aMeans in the same column within each site followed by different letters

ennis collected from untreated trees lived longer than

those collected from B. brongniartiiWU 20-treated trees.

At both sites, a much lower number of adults was col-

lected in the B. brongniartii NBL 851 treatments than in

the two other treatments, which probably reduced the

statistical power of tests. In consequence, statistical

differences in longevity between the beetles collected inthe untreated and the B. brongniartii NBL 851 treat-

ments were not detected although longevity of the Bi-

olisa Kamikiri-treated insects was numerically lower

(Table 2). Longevity was independent of time of col-

lection (F ¼ 0:47; df ¼ 2; P ¼ 0:63) or sex (F ¼ 107;

df ¼ 1; P ¼ 0:30).Although fewer A. glabripennis adults collected from

untreated trees became infected than those collectedfrom fungus-treated trees, percent infection did not

differ significantly among treatments (v2 ¼ 5:27; df ¼ 2;

P ¼ 0:072), ranging from 11.4% in controls to 47.1% for

Biolisa Kamikiri.

3.3. Effects of bands on female oviposition

The numbers of oviposition scars were greater at siteA than B (F P 17:81; df ¼ 1; P < 0:0001), both at the

beginning (on the first day) and at the end (on the 31st

day) of the experiment (Table 3). Although the number

of exit holes per tree at the beginning of the experiment

did not differ between field sites (F ¼ 0:03; df ¼ 1;

P ¼ 0:97), the number of exit holes per tree was higher

adults in two different sites containing trees treated with bands im-

Exit holes per tree

ay 31 On day 1 On day 31

1.9 a 1.2� 0.3 a 2.6� 0.5 a

2.0 b 0.8� 0.2 a 1.5� 0.3 a

7.3 c 2.2� 0.7 a 6.2� 1.2 b

1.3 a 1.1� 0.2 a 2.2� 0.3 a

1.7 b 1.2� 0.2 a 2.2� 0.4 a

1.4 b 0.7� 0.2 a 1.5� 0.3 a

are significantly different (Tukey�s studentized range test, P < 0:05).

T. Dubois et al. / Biological Control 31 (2004) 320–328 325

(F ¼ 3:28; df ¼ 1; P < 0:039) in site A (3.5� 0.5 exitholes/tree) than site B (2.0� 0.2 in initial exit holes/tree)

at the end of the experiment. A. glabripennis population

densities were also not equal across plots within sites.

For example, for site A, the numbers of initial oviposi-

tion scars and final exit holes were highest in the WU 20

plot. There were also inequalities in oviposition scar

densities within site B, with greater initial oviposition in

the control compared with treatments. Therefore, thecalculations to adjust by exit holes at a site were clearly

necessary.

Relative oviposition per female increased over time for

all plots (F ¼ 96:11; df ¼ 1; P < 0:0001). However, the

increase in relative oviposition per female differed among

field sites (F ¼ 6:22; df ¼ 2; P ¼ 0:013). There was a

significant interaction between time and treatment

(F ¼ 5:12; df ¼ 2; P ¼ 0:0061), demonstrating thatslopes of relative oviposition per female over time differed

among treatments. In site A, relative oviposition per

female followed a linear relationship as a function of

time for the control (r2 ¼ 0:99; df ¼ 1; P < 0:0001) andB. brongniartii NBL 851 (r2 ¼ 1:00; df ¼ 1; P < 0:0001)treatment, increasing 0.052� 0.002 and 0.025� 0.001 per

day, respectively. Relative oviposition per female fol-

lowed a quadratic relationship (r2 ¼ 1:00; df ¼ 2;P < 0:0001) for the B. brongniartii WU 20 treatment

(relative oviposition per female¼ 1+ (0.061� 0.004)

� time ) (0.0012� 0.0002)� time2). Therefore, a treat-

ment effect was seen slighly earlier for Biolisa Kamikiri

than B. brongniartii WU 20. From day 16 onward at site

A, relative oviposition per female was lower (tP 3:19;df ¼ 834; P 6 0:0015) in the Biolisa Kamikiri treatment

than in the control but relative oviposition per female atsite A was only lower (tP 3:55; df ¼ 834; P 6 0:0004) intheB. brongniartiiWU20 treatment than the control from

Fig. 1. Cumulative relative oviposition (mean� SE) per A. glabripennis

female in control versus plots with trees with two treatments of fungal-

impregnated non-woven fiber bands in Huaiyuan, Anhui, China (site

A) over 31 days.

day 21 on (Fig. 1). At field site B, relative oviposition perfemale followed a linear relationship (r2 ¼ 0:99, 0.97,

and 0.99, for the control, B. brongniartii NBL 851, and

B. brongniartii WU 20 treatment, respectively; df ¼ 1;

P < 0:0001) as a function of time for all treatments.At site

B, treatments did not differ among each other at any time

(�0:026 t6 2:53; df ¼ 834; P P 0:012).

4. Discussion

Field studies demonstrated a decrease in adult lon-

gevity associated with application of non-woven fiber

bands impregnated with cultures of entomopathogenic

fungi that were hung on tree trunks. Studies with beetles

caged with fungal bands have shown that decreased

adult longevity is accompanied by declining ovipositionby infected females before death (Dubois et al., 2004).

The decrease in relative oviposition performance docu-

mented during this field trial therefore is due to both

decreased oviposition before death of infected females

and decreased female longevity. Use of fungal-impreg-

nated bands is a novel method for application of ento-

mopathogenic fungi. This technique is especially

appropriate because it takes advantage of the tendencyof these beetles to wander on tree trunks during the pre-

ovipositional period.

Beauveria brongniartii only had an effect on A. glab-

ripennis adults in site A, with a quicker effect of de-

creasing population increase for Biolisa Kamikiri than

B. brongniartiiWU 20 bands. Laboratory bioassays with

these strains have demonstrated that both are highly

infective and virulent (Dubois, 2003). Conidial densitieswere higher in Biolisa Kamikiri bands but, because the

methods for making bands and the band substrates

themselves were different, at this time we cannot deter-

mine whether the effects seen were due to isolate alone.

Fungal treatments had no effect on increase in A. glab-

ripennis populations in site B. Of course, the population

densities differed between sites, with much lower popu-

lations at site B. Compared to the site A controls, theincrease in relative oviposition per female over time was

lower for the site B controls. Site A was situated within

the city perimeter, next to a busy road, and divided into

small private vegetable gardens that were constantly

frequented by local people. Site B was more remote and

not frequented by local people. We believe that local

natural enemies of A. glabripennis were inhibited in site

A, but were undisturbed in site B, thus masking thefungal effect in the latter site by reducing the increase in

relative oviposition per female over time in the control

treatment. Based on local observations, it was believed

that woodpeckers such as Dendrocopos major L., as

reported by Gao and Li (2001), fed extensively on

A. glabripennis larvae in site B. At both sites, it is

possible that effects of fungal bands would have been

326 T. Dubois et al. / Biological Control 31 (2004) 320–328

greater if temperatures during the exposure period hadnot been so high. Higuchi et al. (1997) found that con-

stant temperatures of P35 �C resulted in fast declines in

densities of viable conidia on Biolisa Kamikiri bands.

Bands impregnated with both B. brongniartii strains

were active for at least 31 days against adult A. glab-

ripennis in the field, as illustrated by the independence of

adult longevity and time of collection from fungal

treatments at both sites and the lower slopes of relativeoviposition per female in the fungal treatments than in

the control treatment in site A. This finding is similar to

that of Higuchi (1999), who found that insecticidal ac-

tivity of bands impregnated with B. brongniartii NBL

851 against P. hilaris is maintained for 30 days in the

field. We therefore concur with Higuchi (1999), and

conclude that B. brongniartii NBL 851 impregnated

bands should be applied at the start of beetle emergence,especially taking into account that A. glabripennis

exhibits an obligatory pre-oviposition period (Higuchi

et al., 1997; Smith et al., 2001).

Anoplophora glabripennis survival decreased when

adults were present for 25 s on bands impregnated with

all fungal strains, and when present for 5 s on bands

impregnated with B. brongniartii NBL 851. Survival was

not shortened more when adults were allowed for longeron the bands. Assuming adults walk in a straight vertical

line on the tree surface, one can extrapolate from this

laboratory study that adults would need to cross a

50mm wide band of Biolisa Kamikiri hung around the

stem of a tree once for mortality. However, bands of the

other two fungal treatments would need beetles to cross

them two times to be exposed to sufficient conidia for

mortality.Fungus did not grow from many cadavers of beetles

collected from fungal treatments in the field or from

many cadavers that resulted from the bioassay. Kawa-

kami (1978) and Shimazu (1994) also found that

B. brongniartii did not grow from many cadavers of the

cerambycids P. hilaris and M. alternatus exposed to

fungal bands. We speculate that contaminants may be

preventing mycelial outgrowth of B. brongniartii fromthe cadavers of insects killed by this fungus. Addition-

ally in this study, growth of fungi from cadavers may

not have occurred because in some cases, the relative

humidity inside cups declined because cotton plugs ac-

cidentally dried out. Therefore, relative humidity inside

cups dropped below 95%, and this has been shown to

prevent fungal outgrowth from dead Rhodnius prolixus

(St�al) infected by Beauveria bassiana (Luz and Fargues,1998).

Unexpectedly, B. brongniartii and M. anisopliae also

grew from some of the untreated cadavers. This could

be due to contamination in the laboratory or exposures

to these fungi in the field. In agreement with findings of

Higuchi et al. (1997), we consider it unlikely that

conidia from B. brongniartii-impregnated bands drifted

into the control treatments in high enough quantities toinfect many A. glabripennis adults in the control

treatment. Extensive migration of A. glabripennis

among treatments is also unlikely. Although A. glab-

ripennis is capable of flights of over 1 km, abundance of

host material per beetle is negatively correlated with

dispersal (Bancroft and Smith, 2001). Also, Huang

(1991) and Huang and Zhou (1992) showed that a

homogenous young plantation where trees are planted3–5m apart prevents adult A. glabripennis dispersal.

Because A. glabripennis population densities were rel-

atively low and trees were densely planted in the

present experiments, we hypothesize that A. glabripen-

nis did not disperse among treatments and that Hy-

phomycetes would not have been carried along.

Therefore, it seems most likely that these fungi were

already present in the field before bands were applied,explaining some infections among adults collected from

the control treatments. Infection in control treatments

was also demonstrated in field experiments using cages

carried out at the same location in China in 2001

(Dubois et al., 2004).

Assessing the impact of fungus-impregnated bands

against adult A. glabripennis field populations using the

direct sampling method was difficult, since A. glabrip-

ennis adults sometimes occured high in the tree where

they were difficult to detect and collect, and researchers

are forced to devise other methods to assess efficacy of

control methods. For instance, in the only study per-

formed thus far with A. glabripennis, Zhang et al. (1998)

used sticky tape or a nylon net hung immediately above

the band on each tree to trap adults. The latter tech-

nique might impede natural behavior, since adult A.

glabripennis are reported to move up and down along

the tree stem in laboratory studies (Keena and Major,

2001). Therefore, this latter technique is likely to distort

results, since using bands impregnated with fungal cul-

tures was especially developed to exploit the tendency of

Cerambycidae to walk along the stems of trees after

emergence (Higuchi et al., 1997).

In conclusion, we present results from initial uncagedstudies investigating control of these long-lived tree-

dwelling beetles using fungal-impregnated non-woven

fiber bands hung around trees. Bioassays demonstrated

that adult beetles died more quickly than controls after

walking on bands for 5–25 s. Our design for field trials

suffered from lack of replication due to limited avail-

ability of A. glabripennis-infested sites and relatively low

densities of A. glabripennis. Despite these obstacles, fieldtrials demonstrated that adults collected from fungal-

treated plots died more quickly than controls and fewer

eggs were laid in fungal-treated plots compared with

controls. These results suggest that this novel applica-

tion method holds promise for controlling this difficult

pest and these studies provide a framework for further

investigations.

T. Dubois et al. / Biological Control 31 (2004) 320–328 327

Acknowledgments

We are greatly indepted to Chris Caudill, Jan Nyrop,

John Losey, John Vandenberg, and Francoise Vermey-

len for advice on modeling and statistical aspects of this

study. In the USA, we thank Monica Bertoia, Callie

Eastburn, James McNeil, and Micheal Wheeler for their

technical assistance. Technical assistance in China was

provided by Bo Huang, Chunru Li, Bin Wang, SibaoWang, Jingzhu Xiu, Xingtuan Zhang, and Zhenglong

Zhang. Yugu Chen, Meizhen Fan, Yiming Fang, Fen-

gliang Li, Shao Liu, Wenlang Liu, Zhenggang Song,

Meili Tao, Songqang Zhang, and Wuwei Zhang helped

greatly with coordination and logistics. We would have

been unable to carry out the experiments without the

dedicated help of Shaomin Hong and Xingming Wang.

Toshio Higuchi and Nitto Denko were so kind to pro-vide us with Biolisa Kamikiri. Chinese literature was

translated by Li Zhang. Mitsaku Shimazu provided

helpful insights regarding band fabrication. Comments

from three anonymous reviewers improved this paper

significantly. Studies were funded in part by an USDA-

ARS cooperative agreement and USDA-FAS grants.

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