ORIGINAL PAPER

Residual efficacy of thiamethoxam, Beauveria bassiana (Balsamo)Vuillemin, and diatomaceous earth formulation againstRhyzopertha dominica F. (Coleoptera: Bostrychidae)

Waqas Wakil • Tahira Riasat • Muhammad Ashfaq

Received: 31 July 2011 / Accepted: 9 December 2011 / Published online: 6 January 2012

� Springer-Verlag 2012

Abstract The residual efficacy of diatomaceous earth

alone and in combination with Beauveria bassiana

(Balsamo) Vuillemin or a neonicotinoid insecticide thia-

methoxam against Rhyzopertha dominica (Coleoptera:

Bostrychidae) was studied under laboratory conditions. The

mortality of adult R. dominica was decreased over the

9 months of storage period and the combined application of

the test materials enhanced the mortality rates compared with

alone treatments. The greatest mortality was observed in the

combination of DE with thiamethoxam. Progeny suppres-

sion was decreased with the extended storage period. The

maximum rate of mycosis and sporulation in the cadavers of

R. dominica was observed where B. bassiana was applied

alone at the lowest-dose rate. The results of this study indi-

cate that all three control measures may provide safety for an

extended period of time against R. dominica.

Keywords Residual efficacy � Thiamethoxam �B. bassiana � Diatomaceous earth � R. dominica

Introduction

Most of the economically important stored grain insect

pests have developed resistance against generally used

grain protectants all around the world (Subramanyan and

Hagstrum 1996). In general, the tendency to increase

insect pest resistance to insecticides is related with fre-

quent application at increased dose rates, as a result the

chemical residues on grain commodities serve as a source

of resistance development in the insects. The process of

resistance development can be effectively delayed by

using integrated pest management (IPM) strategies

focusing on the use of biological control methods and the

application of safer and selective insecticides. The

insecticidal potential of entomopathogenic fungi as a

substitute to conventional grain protectants has received

considerable attention during the last few years with

particular reference of stored product insect management

(Michalaki et al. 2006; Wakil and Ghazanfar 2010; Wakil

et al. 2011). Entomopathogenic fungi have been shown to

have good residual persistence that is generally consid-

ered as the limitation in the use of customary grain pro-

tectants (Stathers et al. 2002). Various earlier studies

proposed that Beauveria bassiana (Balsamo) Vuillemin

(Hyphomycetes) could be utilized successfully against

several stored grain insect species (Adane et al. 1996;

Meikle et al. 2001; Lord 2009).

Diatomaceous earth (DE) is another alternative for

insect control in grain and can be effective against a broad

range of stored product insects (Fields and Korunic 2000;

Subramanyam and Roesli 2000; Wakil et al. 2010). The

DEs are desiccant inert dusts that act by destroying the wax

layer of insect cuticles and cause the death due to water

loss (Ebeling 1971). DEs are non-toxic and provide long-

term protection of the produce against insect pests (Vayias

et al. 2006). Moreover, they do not affect the baking

quality of grain products and are equally effective as a

surface or admixed grain treatment.

Thiamethoxam is a second generation neonicotinoid

with systemic, contact, and stomach insecticidal activities

A contribution to the special issue on Recent Advances in Stored-

Product Protection.

Communicated by C. G. Athanassiou.

W. Wakil (&) � T. Riasat � M. Ashfaq

Department of Agricultural Entomology, University

of Agriculture, Faisalabad, Pakistan

e-mail: arid1972@yahoo.com; waqaswakeel@hotmail.com

123

J Pest Sci (2012) 85:341–350

DOI 10.1007/s10340-011-0408-8

(Meienfisch et al. 2001). It has been effectively used as

seed treatment against a wide range of economically

important insect pests on an array of crops such as wheat,

barley, sorghum, corn, and cotton (Hofer and Brandl 1999;

Lawson et al. 1999). The insecticidal activity of this mol-

ecule has also been evaluated against stored grain insect

species either as seed treatment (Yue et al. 2003) or as a

grain protectant (Arthur et al. 2004).

Protection of commodities for extended time periods of

storage is a basic purpose of any grain management pro-

gram. Previous studies indicated the long-term efficacy of

DE, entomopathogenic fungi, and some traditional grain

protectants. For instance, Athanassiou et al. (2005) repor-

ted residual efficacy of Insecto, SilicoSec, and PyriSec

against Sitophilus oryzae L. on wheat and barley for up to

450 days. Similarly, the efficacy of Metarhizium anisopliae

(Metschnikoff) Sorokin (Deuteromycotina: Hyphomyce-

tes) alone or in combination with Protect-It was effective

for the period of 5 months against S. oryzae and Rhyzop-

ertha dominica F. on wheat and maize (Athanassiou et al.

2008). Good residual effects of various traditional grain

protectants have also been reported against several stored

grain pests by numerous researchers (Collins and Cook

1998; Arthur 1999; Kljajic and Peric 2009).

The literature available on the residual effectiveness of

different grain protectants is lacking the efficacy assess-

ment of the treatment effect of DE formulation either with

B. bassiana or low-toxicity insecticides. In this study, we

investigated the residual effect of DE alone and in the

combined treatment with B. bassiana and a novel insecti-

cide, thiamethoxam, against R. dominica for the storage

period of 9-months. In addition to adult mortality, progeny

production was assessed and the mycosis and sporulation

on the beetles was also examined.

Materials and methods

Test insects

Two-week-old adult R. dominica used in the bioassays

were obtained from an insect culture raised on whole wheat

under laboratory conditions for up to four generations at

25 ± 2�C and 70 ± 5% r.h.

Test commodity

Infestation free, untreated wheat grain (var. Inqilab-91)

were used in the bioassays. The moisture contents of the

grain prior to the experimentation were 11.5% determined

by the Dickey-John moisture meter (Dickey-John Multi-

grain CAC II; Dickey-John Co., USA).

Test grain protectants

DE formulation used in the tests was SilicoSec (Biofa

GmbH, Munsingen, Germany), a fresh water DE contain-

ing 92% SiO2, 3% Al2O3, 1% Fe2O3, and 1% Na2O. The

B. bassiana was isolated from an infected insect (Cocci-

nella septempunctata L. Coccinellidae: Coleoptera) by

single spore method (Choi et al. 1999). The fungus was

identified on morphological characters using the key

(Barnett and Hunter 1998) and was deposited in the IPM

Laboratory of Department of Agricultural Entomology,

University of Agriculture, Faisalabad (Pakistan). It was

further sub-cultured on potato dextrose agar by incubating

at 20 ± 5�C and 70% r.h., and the conidia were harvested

using a sterilized scalpel after 14 days. The conidia were

suspended in sterile 0.05% Tween-80, enumerated with a

hemocytometer and adjusted to achieve concentrations of

1.5 9 106 and 1.5 9 108 conidia/ml. Prior to the initiation

of the tests, the germination rate of B. bassiana conidia was

determined as [90%.

The formulation of thiamethoxam used in the experi-

ments was Actara (Syngenta Pakistan Limited, Karachi,

Pakistan), a wettable granule formulation containing 25%

active ingredient.

Radial growth test

Thiamethoxam was tested at three-dose rates for the

assessment of its inhibitory effect on the radial growth of

B. bassiana. The fungus was grown on Sabouraud dextrose

agar (SDA) plates for 7 days at 25�C. Then 3-mm diameter

cores of fungus were taken from the developed cultures and

placed singly upset down in the middle of Petri dishes

containing SDA with 0, 0.25, 0.5, and 0.75 ppm of thia-

methoxam solution. The potential inhibitory effect of

insecticide on fungal growth was determined by surface

radial growth that was measured for 6 days on alternate

days. Each treatment was replicated six times and the

measurements were made along two radii at right angles to

each other drawn on the bottom of each dish (Russell et al.

2010).

Grain treatment

There were nine treatments: the DE alone (200 ppm);

thiamethoxam alone (0.5 ppm); B. bassiana alone

(1.5 9 106 and 1.5 9 108 conidia/kg of wheat); DE with

thiamethoxam; DE with the low dose of B. bassiana; DE

with the high dose of B. bassiana; thiamethoxam with the

low dose of B. bassiana; and thiamethoxam with the high

dose of B. bassiana. The wheat lots of 1 kg were prepared

in plastic jars. The thiamethoxam was dissolved in water to

prepare solution containing 0.5 ppm and then sprayed onto

342 J Pest Sci (2012) 85:341–350

123

grains spread in plastic trays by using a hand sprayer.

Before the addition of DE in insecticide treated grains, the

trays were kept in incubators set at 30�C and 70 ± 5% r.h.

for 48 h (Kavallieratos et al. 2009) to normalize the

moisture contents. The DE and B. bassiana alone or in

combination with thiamethoxam were admixed with the

grain. An additional lot of wheat grain was left untreated to

serve as control. All the lots were placed in plastic jars and

shaken manually for 3–4 min to have uniform distribution

in the grain. These jars were kept at 30�C and 70 ± 5% r.h.

in incubators during the entire period of trials.

Bioassays

The residual effectiveness of the treatments was deter-

mined for 9 months (March to November, 2010) by con-

ducting bioassays. On the initial day and every 30 days

thereafter, four samples of 70 g were taken from each jar

(treated and control) to perform the bioassays. Each 70 g

wheat sample was placed in a small jar (11 cm height and

6.5 cm diameter) followed by the introduction of 50 adult

beetles. The jars were set in incubators at 30�C and

70 ± 5% r.h. The humidity was maintained by using sat-

urated NaCl solution (Greenspan 1977). The data for

mortality count was taken after 48 h, 7, and 14 days of

exposure with the successive removal of dead adults after

each count. After the last mortality count, all the dead and

alive adults were removed from the grains and the jars were

further incubated at the same conditions for further 46 days

to record the emergence of F1 adults. The whole procedure

was replicated thrice with new materials each time inde-

pendently to avoid pseudo-replication phenomenon.

Mycosis and sporulation

The mycosed R. dominica cadavers after each mortality

count were kept in sterilized Petri plates and refrigerated at

4�C. These cadavers were surface sterilized with sodium

hypochlorite solution (0.05%) for 2–3 min followed by

three washings in sterilized distilled water. The cadavers

were placed on SDA plates that were maintained at

25 ± 2�C and 75% r.h. for 7 days. The dead insects with

white fungal growth on their body were determined under

the stereo microscope. The rate of sporulation (number of

conidia/ml) from the mycosed cadavers was determined by

mixing in 20-ml distilled water with a drop of Tween-80 in

beaker (Riasat et al. 2011). After thorough mixing of the

solution, the number of conidia/ml was counted by using

hemocytometer.

Statistical analysis

Mortality counts were corrected by using Abbott’s (1925)

formula. All data were analyzed by repeated measure

ab

b

a

b

a

a

ca

ba

a

a

0

1

2

3

4

5

6

7

6D4D2D

Days

Rad

ial g

rwot

h/da

y (m

m)

0.25 ppm 0.50 ppm 0.75 ppm ControlFig. 1 Radial growth rate (mm/

day ± SE) of B. bassiana on

SDA amended with 0.25, 0.5,

and 0.75 ppm thiamethoxam

(Thi) (within each day means

followed by the same letter are

not significantly different; HSD

at P = 0.05)

Table 1 ANOVA parameters of main effects and interactions for mortality levels of R. dominica adults in each exposure interval (for each

exposure interval total df = 269)

S.O.V. df 48 h 7 days 14 days

F P F P F P

Storage periods 9 377.51 \0.01 735.39 \0.01 403.63 \0.01

Treatments 8 254.12 \0.01 217.79 \0.01 218.33 \0.01

Storage periods 9 treatments 72 19.25 \0.01 7.94 \0.01 8.69 \0.01

J Pest Sci (2012) 85:341–350 343

123

Ta

ble

2M

ean

mo

rtal

ity

(±S

E)

of

R.d

om

inic

aad

ult

sex

po

sed

for

48

ho

nw

hea

ttr

eate

dw

ith

on

e-d

ose

rate

of

DE

(20

0p

pm

),th

iam

eth

ox

am(T

hi)

(0.5

pp

m),

two

-do

sera

tes

of

B.b

ass

ian

a(F

1:

1.5

91

06;

F2

:1

.59

10

8co

nid

ia/k

g),

and

thei

rre

spec

tiv

eco

mb

inat

ion

sin

10

sto

rag

ep

erio

ds

con

du

cted

fro

m0

to2

70

day

sp

ost

-gra

intr

eatm

ent

(wit

hin

each

sto

rag

ep

erio

dm

ean

sfo

llo

wed

by

the

sam

ele

tter

are

no

tsi

gn

ifica

ntl

yd

iffe

ren

t;H

SD

test

atP

=5

%)

Tre

atm

ents

Sto

rag

ep

erio

ds

(day

s)

03

06

09

01

20

15

01

80

21

02

40

27

0

DE

12

.11

±2

.13

de

19

.30

±1

.43

de

16

.15

±3

.11

d1

2.4

9±

1.6

1d

e7

.56

±2

.54

cde

5.1

5±

2.2

1cd

1.3

3±

1.1

5c

0.0

0±

1.1

5a

0.0

0±

0.0

0a

0.0

0±

0.0

0a

Th

i3

8.0

2±

1.5

7c

40

.49

±2

.00

c3

3.5

9±

2.8

7b

c2

5.4

7±

2.9

2b

c1

8.1

1±

1.7

3b

c7

.41

±2

.67

cd2

.51

±1

.99

c0

.47

±1

.35

a0

.15

±1

.12

a0

.17

±1

.24

a

F1

5.1

6±

2.0

3e

9.0

1±

1.1

6e

11

.07

±2

.08

de

7.6

3±

1.6

9e

4.4

1±

1.2

9e

2.2

3±

2.1

5d

0.2

8±

0.0

5d

0.1

2±

2.1

1a

0.4

9±

1.3

6a

0.0

0±

0.0

0a

F2

10

.36

±2

.47

de

16

.51

±0

.86

de

18

.85

±1

.48

d1

4.3

5±

2.6

5cd

e1

0.5

7±

1.3

7cd

e9

.67

±2

.56

bcd

4.5

9±

2.1

6b

c2

.53

±1

.15

a0

.17

±0

.26

a0

.00

±0

.00

a

DE

?T

hi

71

.37

±2

.23

a7

0.6

9±

2.8

0a

54

.08

±3

.37

a4

3.1

9±

3.0

8a

35

.02

±2

.49

a2

1.6

1±

3.6

4ab

13

.59

±1

.64

a7

.29

±2

.24

a1

.69

±1

.11

a0

.15

±1

.31

a

DE

?F

19

.15

±2

.13

de

14

.52

±1

.43

de

12

.82

±2

.20

d9

.62

±1

.42

de

6.5

1±

2.1

5d

e3

.28

±1

.83

d2

.03

±0

.77

c0

.65

±1

.18

a0

.00

±0

.00

a0

.00

±0

.00

a

DE

?F

21

7.1

2±

2.8

0d

25

.44

±3

.30

d2

3.4

0±

2.3

9cd

20

.31

±1

.56

cd1

7.0

8±

1.3

1b

cd1

2.1

0±

2.3

6b

cd7

.48

±0

.19

abc

3.1

9±

1.2

7a

1.1

8±

1.2

4a

0.0

0±

0.0

0a

Th

i?

F1

42

.54

±2

.47

c4

6.6

2±

3.0

7b

c4

1.6

8±

3.1

0ab

32

.57

±2

.78

ab2

4.4

9±

1.9

9ab

17

.54

±0

.40

abc

6.6

2±

1.1

5ab

c3

.55

±0

.99

a0

.00

±0

.00

a0

.66

±1

.25

a

Th

i?

F2

54

.36

±0

.96

b5

7.4

2±

2.1

8b

52

.62

±2

.66

a4

0.3

6±

3.4

2a

31

.64

±3

.47

a2

5.2

7±

3.2

7a

11

.52

±2

.43

ab5

.44

±1

.43

a2

.37

±1

.62

a0

.00

±0

.00

a

Ta

ble

3M

ean

mo

rtal

ity

(±S

E)

of

R.

do

min

ica

adu

lts

exp

ose

dfo

r7

day

so

nw

hea

ttr

eate

dw

ith

on

e-d

ose

rate

of

DE

(20

0p

pm

),th

iam

eth

ox

am(0

.5p

pm

),tw

o-d

ose

rate

so

fB

.b

ass

ian

a(F

1:

1.5

91

06;

F2

:1

.59

10

8co

nid

ia/k

g),

and

thei

rre

spec

tiv

eco

mb

inat

ion

sin

10

sto

rag

ep

erio

ds

con

du

cted

fro

m0

to2

70

day

sp

ost

-gra

intr

eatm

ent

(wit

hin

each

sto

rag

ep

erio

dm

ean

sfo

llo

wed

by

the

sam

ele

tter

are

no

tsi

gn

ifica

ntl

yd

iffe

ren

t;H

SD

test

atP

=5

%)

Tre

atm

ents

Sto

rage

per

iods

(day

s)

030

60

90

120

150

180

210

240

270

DE

62.0

0±

1.9

5de

68.6

7±

1.7

3cd

65.6

7±

1.7

3cd

57.6

7±

1.9

7b

51.2

8±

1.9

6ab

43.1

8±

1.4

2ac

d35.1

8±

1.4

2bcd

31.3

2±

2.1

9bc

26.5

2±

1.9

3bc

21.5

6±

1.6

1bc

Thi

87.0

6±

2.1

6ab

85.4

9±

3.3

0ab

74.6

0±

2.6

5ab

c61.7

9±

3.6

2ab

47.7

0±

2.5

3bc

38.7

5±

3.2

0bcd

24.6

2±

2.8

6def

15.5

7±

1.7

8de

10.4

3±

1.2

7de

4.2

6±

3.2

6e

F1

43.1

8±

1.5

2f

41.4

3±

1.8

2e

46.6

7±

1.9

5e

35.0

6±

2.6

0d

26.3

5±

4.1

1d

22.1

0±

2.2

1e

17.0

4±

2.0

2f

12.6

8±

0.8

2e

5.4

9±

1.9

4e

2.5

9±

1.9

1e

F2

72.0

3±

2.5

6cd

75.3

4±

2.4

1bc

71.4

3±

2.0

3bc

65.8

2±

2.9

1ab

54.4

6±

2.8

2ab

46.2

1±

2.5

0ab

c41.1

7±

2.1

9ab

c36.4

7±

2.4

8ab

30.3

3±

2.4

9ab

23.3

5±

3.6

7ab

DE

?T

hi

95.3

5±

1.6

9a

91.6

5±

2.0

6a

84.1

0±

2.5

6a

73.0

9±

1.9

7a

58.3

8±

1.9

6ab

47.3

4±

2.6

8ab

c36.7

4±

1.7

5bc

19.1

6±

2.1

5de

13.4

5±

1.7

8de

7.5

6±

2.9

2de

DE

?F

154.0

7±

1.9

8e

57.3

4±

2.5

7d

53.3

4±

2.4

8e

42.4

1±

1.7

7cd

34.1

4±

3.7

9cd

29.0

8±

1.9

7de

21.8

8±

2.4

6ef

17.6

0±

2.3

6de

12.2

5±

1.5

6de

9.2

±1.3

9cd

e

DE

?F

281.2

1±

2.4

8bc

83.0

6±

2.4

1ab

80.2

7±

1.7

3ab

71.3

3±

2.2

8a

63.2

5±

3.6

4a

54.0

1±

2.4

4a

50.1

6±

1.9

2a

43.3

0±

3.6

1a

38.1

1±

1.1

2a

34.3

2±

2.8

4a

Thi

?F

165.3

4±

1.7

2d

61.6

8±

2.6

6d

58.0

1±

2.4

1de

53.6

7±

2.6

7bc

45.1

3±

3.1

3bc

37.6

9±

2.9

5cd

32.5

5±

2.5

1cd

e25.1

6±

2.6

7cd

19.1

4±

2.0

1cd

12.2

5±

2.8

8bcd

e

Thi

?F

292.3

0±

2.4

7a

87.2

1±

2.5

8ab

83.6

2±

2.0

4a

74.2

9±

2.4

9a

64.0

6±

2.5

0a

51.2

5±

1.5

6ab

46.3

5±

2.7

5ab

34.4

2±

1.1

6ab

c23.0

7±

2.2

4bc

18.2

1±

1.9

1

344 J Pest Sci (2012) 85:341–350

123

analysis using General Linear Model of SAS (1999). The

dose rates and storage periods were the main effects while

adult mortality was the response variable. For progeny

production, the number of emerged adults was the response

variable whereas the storage periods and the dose rates

were the main effects. The means were separated with the

Tukey–Kramer test (HSD) at a = 0.05 (Sokal and Rohlf

1995).

Results

Radial growth test

The dose rates significantly affected the B. bassiana growth

on SDA plates for each incubation day (D2: F3,11 = 10.8,

P B 0.01; D4: F3,11 = 5.72, P = 0.02; D6: F3,11 = 5.45,

P = 0.02). The differences in mm growth by low- and

medium-dose rates of thimethoxam (0.25 and 0.5 ppm)

were not significantly different from that of control and the

highest-radial growth was observed after 6 days of incu-

bation (Fig. 1).

Adult mortality after 48 h exposure

The main effects and their associated interaction were sig-

nificant (Table 1). The adult mortality generally decreased

with increasing storage period. During the first bioassay, the

lowest mortality (5.16%) of the beetles was at the lowest-

dose rate of B. bassiana, whereas the greatest mortality

(71.37%) was in the grains treated with the combined

application of DE and thiamethoxam. At 60 days, the mean

mortality percentage was either increased or remained same

for all treatments with the exception of thiamethoxam alone

for which the mortality rapidly decreased with increasing

storage period (Table 2). The addition of DE increased

mortality with both thiamethoxam and B. bassiana. For

combined treatments, B. bassiana mixed with thiamethoxam

gave higher mortalities than B bassiana mixed with DE.

Adult mortality after 7 day exposure

The main effects and their associated interaction was sig-

nificant (Table 1). There were significant differences

among all treatments but mortalities decreased with the

extending storage period. The highest-adult mortalities

were at 0.5 ppm of thiamethoxam alone or in combination

with 200 ppm of DE during at 30 days of storage

(Table 3). The mortality with DE alone showed little var-

iation up to 60 days of storage and then declined succes-

sively after each additional storage period. Almost the

same trend was followed by the other treatments except for

thiamethoxam alone and its combined treatments for which Ta

ble

4M

ean

mo

rtal

ity

(±S

E)

of

R.

do

min

ica

adu

lts

exp

ose

dfo

r1

4d

ays

on

wh

eat

trea

ted

wit

ho

ne-

do

sera

teo

fD

E(2

00

pp

m),

thia

met

ho

xam

(0.5

pp

m),

two

-do

sera

tes

of

B.

ba

ssia

na

(F1

:

1.5

91

06;

F2

:1

.59

10

8co

nid

ia/k

g),

and

thei

rre

spec

tiv

eco

mb

inat

ion

sin

10

sto

rag

ep

erio

ds

con

du

cted

fro

m0

to2

70

day

sp

ost

-gra

intr

eatm

ent

(wit

hin

each

sto

rag

ep

erio

dm

ean

sfo

llo

wed

by

the

sam

ele

tter

are

no

tsi

gn

ifica

ntl

yd

iffe

ren

t;H

SD

test

atP

=5

%)

Tre

atm

ents

Sto

rage

per

iods

(day

s)

030

60

90

120

150

180

210

240

270

DE

83.4

8±

2.5

3cd

80.1

0±

2.4

8de

82.3

1±

2.5

5ab

c74.2

4±

3.7

8bcd

72.7

2±

1.8

3ab

67.3

5±

2.6

2ab

63.1

9±

2.1

3a

57.2

1±

2.6

355.9

8±

3.4

3ab

54.4

7±

2.9

5ab

Thi

96.3

1±

2.1

5ab

92.8

6±

3.0

1ab

c86.0

4±

2.1

9ab

72.3

2±

1.8

4cd

61.1

2±

2.6

9bc

49.1

1±

2.7

6cd

35.6

4±

3.4

7cd

26.7

2±

3.0

118.3

5±

3.2

7d

12.3

3±

1.3

9e

F1

64.4

2±

2.4

0e

60.3

4±

2.3

3f

58.2

1±

2.1

0d

47.1

4±

1.6

9e

42.3

3±

2.3

0d

38.3

8±

2.4

9d

32.2

8±

4.1

4d

29.0

8±

3.3

625.2

8±

3.2

0cd

23.7

0±

3.4

7de

F2

89.0

3±

2.6

2ab

c87.2

3±

1.5

2bcd

85.1

0±

3.1

8ab

80.1

7±

2.4

6ab

c78.2

0±

2.7

2a

73.4

2±

2.9

5a

71.0

6±

2.6

6a

66.1

6±

1.7

463.5

1±

3.0

2ab

57.7

5±

3.6

4a

DE

?T

hi

100.0

0±

0.0

0a

100.0

0±

0.0

0a

92.5

0±

2.0

8a

87.2

9±

2.5

7a

65.0

1±

2.2

9bc

59.7

8±

2.7

3bc

46.2

7±

2.6

5bc

43.2

9±

2.6

435.5

2±

2.9

0c

31.5

2±

3.8

5cd

DE

?F

175.0

9±

3.3

8de

73.7

2±

2.6

5e

70.2

7±

2.1

7cd

62.3

2±

2.4

5d

57.2

1±

2.4

0c

51.2

4±

3.7

0cd

42.6

9±

3.1

2bcd

38.0

8±

2.8

132.4

8±

1.9

7cd

27.6

1±

0.3

3d

DE

?F

292.3

4±

2.0

4ab

c90.1

9±

1.4

3ab

cd88.0

6±

3.1

6ab

83.4

2±

2.3

8ab

c81.0

6±

2.6

4a

78.0

0±

2.9

6a

75.5

8±

2.4

6a

72.6

1±

2.6

069.7

6±

2.4

1a

64.0

0±

3.0

2a

Thi

?F

186.3

7±

2.7

9bcd

83.5

5±

2.5

0cd

e79.1

2±

2.5

1bc

67.1

7±

2.9

0d

63.7

1±

2.1

7bc

55.1

5±

1.8

6bc

47.4

0±

2.6

2b

41.3

7±

2.5

133.4

5±

2.6

3c

29.6

3±

2.9

5cd

Thi

?F

298.0

2±

1.4

8a

95.4

4±

1.7

9ab

90.0

1±

2.2

3ab

86.4

6±

2.8

9a

82.4

1±

2.9

2a

74.0

7±

2.0

6a

68.0

7±

2.4

0a

60.8

2±

3.1

654.3

4±

3.3

1b

42.8

4±

2.5

1bc

J Pest Sci (2012) 85:341–350 345

123

decline initiated after 30 days (Table 3). Here again, the

combination treatments were the most effective compared

with the individual treatments.

Adult mortality after 14 day exposure

The main effects and their associated interaction was sig-

nificant (Table 1). The adult mortality rates were higher in

all treated units than after 7 days of exposure but the

decreasing trend of mortality with storage period was same.

The adult mortality reached 100% for DE and thiameth-

oxam together at 0 and 30 days of storage while the lowest

mortality at the beginning of storage (64.42%) was in the

grain treated with lowest-fungal dose rate (Table 4). Like

48 h and 7 day exposure intervals, there was a very gradual,

steady decline in mortality with DE with successive storage

periods resulting in[50% mortality even after 150 days of

storage not only in the units treated with DE alone but also

where it was mixed with B. bassiana.

The slopes for adult mortality levels determined by

regression equations during storage period were not similar

for the different treatments. For all treatments, the decline

in mortality levels with increased storage time was

observed, with coefficients between 0.96 and 0.99 (Fig. 2).

Progeny production

There were significant differences for main (storage periods:

F9,299 = 555.31, P B 0.01; treatments: F9,299 = 1566.40,

P B 0.01) and their associated interaction (F81,299 = 10.23,

P B 0.01). The progeny emergence was highly suppressed

in treated grain compared with untreated. The fewest

emerged adults were in the grain treated with combined

application of DE ? thiamethoxam or thiamethoxam with

higher dose rate of B. bassiana. On the other hand, the sup-

pression was reduced gradually and more F1 adults were

recorded with extended storage period (Table 5).

Mycosis and sporulation

The highest mycosis (93.25%) and sporulation (287.25

conidia/ml) was at 30 days for the treatments where

B. bassiana was applied at the dose rate of 1.5 9 106

conidia/kg. The mycosis and sporulation declined with the

prolonged storage, the lowest rates of mycosis (4.33%) and

sporulation (28.14%) were during the last sample period in

the grain with thiamethoxam mixed with B. bassiana.

The decreased rate of mycosis and sporulation with

increased storage time was also significant in all regression

models (Figs. 3, 4). The slopes for mycosis determined by

regression equations during the storage period were similar for

the conidiation. Among different treatments, the observed

difference for mycosis with coefficients was between 0.93 and

0.99, while in case of conidiation variation between 0.87 and

0.99 was observed.

Discussion

The prolongation of protection is very important consid-

eration when insect management tactics are to be designed

for stored products. The findings of our experiments indi-

cate that DE, B. bassiana, and thiamethoxam can be

applied successfully against R. dominica but their efficacy

varies depending upon the dose rates and exposure interval.

The decline in the efficacy of DE and entomopathogenic

fungi occurs slowly compared with thiamethoxam. Among

the advantages of DE, the most important one is the inert

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9

Storage period (months)

Mor

talit

y (%

)

DE Thi F1 F2 DE+Thi DE+F1 DE+F2 Thi+F1 Thi+F2

Fig. 2 Mean mortality of R. dominica adults exposed for 14 days on

grain treated with DE, thiamethoxam, B. bassiana, and their respective

combinations for 9 months. Lines represent linear regression equations

fitted to the data for each treatment (DE: y = -3.599x ? 88.902,

R2 = 0.9653; Thi: y = -10.279x ? 111.62, R2 = 0.989; F1:

y = -4.8846x ? 68.985, R2 = 0.9761; F2: y = -3.4808x ? 94.312,

R2 = 0.9889; DE ? Thi: y = -8.7389x ? 114.19, R2 = 0.965;

DE ? F1: y = -5.7074x ? 84.465, R2 = 0.989; DE ? F2: y =

-3.0102x ? 95.962, R2 = 0.992; Thi ? F1: y = -6.7502x ? 95.739,

R2 = 0.9927; Thi ? F2: y = -6.0417x ? 108.38, R2 = 0.9759)

346 J Pest Sci (2012) 85:341–350

123

nature and stability in the treated materials. In this study,

DE provided a good control of the test species and

remained stable even after 270 days of storage. The grad-

ual decrease in its effectiveness may be attributed primarily

to the environmental conditions, specifically the relative

humidity during the storage period and secondarily to the

grain oil absorption by DE particles (Subramanyam and

Roesli 2000). The DE particles absorb moisture from the

air when in contact for prolonged period of time with high-

relative humidity which reduces its efficacy (Fields and

Korunic 2000; Arthur 2002). It has been demonstrated that

DE products (Dryacide and Protect-It) were more effective

at 50% than at 60% r.h. (Stathers et al. 2004); similarly,

Vayias and Athanassiou (2004) also reported reduced

effectiveness of SilicoSec at 65% than at 55% r.h. Some

earlier studies have shown reduced performance of DE

over time (Stathers et al. 2002; Athanassiou et al. 2005).

McGaughey (1972) exhibited that aged deposits of Perma-

Guard (DE formulation) were less effective than fresh

deposits, and the results were attributed to oil absorption by

DE particles.

At least in laboratory studies, B. bassiana has been

utilized with success against R. dominica (Rice and Cog-

burn 1999; Batta 2005; Lord 2001, 2005, 2009). In the

current study, the integration of B. bassiana of DE and the

neonicotinoid, thiamethoxam showed their potential for

long-term grain protection. The presence of DE in grains

not only increased the levels of adult mortality but also

enhanced the residual effectiveness of B. bassiana. This

was more obvious at higher fungal concentrations. The

reduced efficacy of B. bassiana applied at the low-dose rate

with DE could be due to the ‘‘critical conidial concentra-

tion’’ needed to exhibit the additive effect (Vassilakos et al.

2006). Persistence is also a desirable characteristic for

microbial insecticides (Thomas and Jenkins 1997) for

adaptation as insect pest management tools. We found that

the test fungus at its high-application rate not only proved

effective but relatively more stable compared with DE

alone. Similar to DE, there was a gradual decrease in

fungal efficacy with increasing storage period. Athanassiou

et al. (2008) reported a steady decline in the efficacy of

M. aniospliae against S. oryzae and R. dominica over

period of 6 months. Similarly, Moore et al. (2000) noted

conidial viability decreases over time and these findings are

in accordance with our results. The reduction in mortality

was associated with a reduction in mycosis, as expected.

This is the first report of integration of thiamethoxmam

with DE and B. bassiana for the management of R. dom-

inica. The thiamethoxam yielded the highest-mortality

rates up to 30 days of storage, but the effectiveness sharply

declined after that period and caused the least mortality

among all treatments at 270 days. The results indicate that

thiamethoxam is less stable than DE and B. bassiana. TheTa

ble

5M

ean

nu

mb

er(±

SE

)o

fR

.d

om

inic

ao

ffsp

rin

gp

rod

uce

din

wh

eat

trea

ted

wit

ho

ne-

do

sera

teo

fD

E(2

00

pp

m),

thia

met

ho

xam

(0.5

pp

m),

two

-do

sera

tes

of

B.

ba

ssia

na

(F1

:1

.59

10

6;

F2

:1

.59

10

8co

nid

ia/k

g),

and

thei

rre

spec

tiv

eco

mb

inat

ion

sin

10

sto

rag

ep

erio

ds

con

du

cted

fro

m0

to2

70

day

sp

ost

-gra

intr

eatm

ent

(wit

hin

each

sto

rag

ep

erio

dm

ean

sfo

llo

wed

by

the

sam

e

lett

erar

en

ot

sig

nifi

can

tly

dif

fere

nt;

HS

Dte

stat

P=

5%

)

Tre

atm

ents

Sto

rage

per

iods

(day

s)

030

60

90

120

150

180

210

240

270

DE

3.1

6±

0.3

0cd

5.4

1±

1.0

1bc

4.5

8±

1.3

7cd

e7.4

1±

1.5

8bc

9.2

5±

1.8

0cd

10.1

6±

1.2

5de

12.0

8±

2.1

6fg

14.5

8±

2.5

6ef

18.8

3±

1.2

4ef

20.2

5±

2.2

6ef

Thi

0.0

0±

0.0

0d

0.0

0±

0.0

0c

3.5

0±

1.8

2cd

e9.2

5±

2.4

4bc

13.0

8±

1.2

4bc

19.4

1±

2.2

6b

26.1

6±

4.2

3b

30.5

0±

1.5

4b

36.0

8±

2.6

7b

43.1

6±

5.6

8b

F1

8.2

5±

1.2

3b

6.5

8±

2.1

5b

11.2

5±

0.9

4b

12.0

8±

1.7

3b

19.0

0±

1.3

2b

22.5

0±

1.2

7b

24.7

5±

3.7

5bc

27.2

5±

1.2

7bc

32.4

1±

1.9

6bc

36.7

5±

5.2

3bc

F2

2.0

8±

1.1

5cd

5.2

5±

1.5

2bc

3.7

5±

1.4

4cd

e8.0

0±

1.6

4bc

10.5

8±

1.2

0cd

9.5

8±

1.7

6e

15.1

6±

3.2

5ef

17.1

6±

2.4

7ef

16.7

5±

0.8

7ef

18.0

8±

1.9

4f

DE

?T

hi

0.0

0±

0.0

0d

0.0

0±

0.0

0c

0.0

0±

0.0

0e

2.4

1±

1.8

4c

5.0

8±

1.3

6d

12.4

1±

2.1

1cd

e18.2

5±

1.3

4cd

ef19.0

8±

3.2

1de

24.5

8±

3.4

1cd

e28.2

5±

2.3

6cd

e

DE

?F

15.4

1±

1.6

2bc

4.3

3±

1.1

6bc

7.3

3±

1.3

7bcd

11.5

8±

0.9

1b

12.2

5±

0.7

2c

16.2

5±

1.5

8bcd

22.4

1±

2.2

5bcd

26.2

5±

1.8

9bcd

29.4

1±

3.5

6bcd

34.1

6±

2.0

7bcd

DE

?F

20.0

0±

0.0

0d

1.1

6±

0.7

2bc

3.1

6±

1.1

5cd

e5.7

5±

1.8

5bc

4.8

3±

1.1

5d

7.5

8±

1.6

7e

8.4

1±

2.6

5g

11.0

8±

0.9

4f

13.3

3±

2.4

3f

16.3

3±

3.1

2f

Thi

?F

12.1

6±

2.0

4cd

3.4

1±

1.5

5bc

8.0

8±

1.2

0bc

10.4

1±

0.8

0bc

15.4

1±

0.8

2bc

18.3

3±

2.4

5bc

20.0

8±

3.4

5bcd

e25.1

6±

4.2

2bcd

28.2

5±

4.7

6bcd

31.0

8±

2.6

5cd

Thi

?F

20.0

0±

0.0

0d

0.0

0±

0.0

0c

1.5

8±

1.1

6de

3.0

8±

1.5

8c

5.1

6±

1.3

0d

8.2

5±

1.2

8e

17.5

0±

2.6

6def

20.0

8±

1.9

4cd

e21.0

8±

2.1

7de

25.4

1±

2.1

3def

Contr

ol

42.5

8±

4.2

0a

50.6

5±

2.6

7a

52.0

8±

2.3

9a

55.1

6±

1.1

6a

64.4

1±

1.0

8a

73.0

8±

6.2

1a

74.0

8±

4.3

5a

85.1

6±

5.3

5a

86.1

6±

7.2

6a

92.2

5±

6.2

8a

F199

143

136

89.2

205

228

196

209

150

144

PB

0.0

1B

0.0

1B

0.0

1B

0.0

1B

0.0

1B

0.0

1B

0.0

1B

0.0

1B

0.0

1B

0.0

1

J Pest Sci (2012) 85:341–350 347

123

presence of residues sometimes is advantageous for long-

term protection of stored commodity. Thiamethoxam

degrades rapidly as suggested by Soliman (2011) who

found that initial deposits of thiamethoxam on cowpea

pods showed the least residual activity of any of the tested

insecticides. The combined treatment of thiamethoxam

with DE and B. bassisna has not been reported for stored

products. de Oliveira et al. (2003) found that thiamethoxam

at its lower application rates (half of the field recom-

mended) caused no inhibitory effect on radial growth of B.

bassiana conidia, and Neves et al. (2001) reported that

thiamethoxam along with certain other neonicotinoid

insecticides had no effect on the conidial germination,

conidiogenesis and vegetative growth of B. bassiana.

Similar observations have been made during the current

research and the simultaneous presence of thiamethoxam

with B. bassiana also increased the adult mortality com-

pared with their respective individual treatments during

initial storage periods.

The suppression of progeny production is as important a

parameter as the adult mortality in order to minimize grain

damage by insect pests. During current studies, each of

three control strategies been tested suppressed the progeny

production of R. dominica effectively. Entomopathogenic

fungi and DEs are slow acting insecticides particularly

compared with certain chemical insecticides, therefore,

insects may recover and move to less treated layers of grain

and thus may continue to cause the grain damage. The

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9

Storage period (months)

Myc

osis

(%

)

F1 F2 DE+F1 DE+F2 Thi+F1 Thi+F2

Fig. 3 Mycosis in cadavers of R. dominica adults treated with B.bassiana alone and mixed with DE and thiamethoxam for 9 months.

Lines represent linear regression equations fitted to the data for each

treatment (F1: y = -5.6832x ? 97.375, R2 = 0.9856; F2:

y = -5.7937x ? 89.762, R2 = 0.965; DE ? F1: y = -6.2995x ?

87.779, R2 = 0.9978; DE ? F2: y = -7.0615x ? 83.989, R2 =

0.9784; Thi ? F1: y = -6.648x ? 70.473, R2 = 0.9802; Thi ? F2:

y = -6.0947x ? 59.091, R2 = 0.936)

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6 7 8 9

Storage period (months)

Spor

ulat

ion

(con

idia

/ml)

F1 F2 DE+F1 DE+F2 Thi+F1 Thi+F2

Fig. 4 Sporulation of R. dominica cadavers treated with B. bassianaalone and mixed with DE and thiamethoxam for 9 months. Linesrepresent linear regression equations fitted to the data for each treatment

(F1: y = -14.427x ? 306.19, R2 = 0.9925; F2: y = -15.045x ?

282.35, R2 = 0.8701; DE ? F1: y = -14.127x ? 229.04, R2 =

0.9895; DE ? F2: y = -12.877x ? 188.34, R2 = 0.9868; Thi ? F1:

y = -11.704x ? 166.87, R2 = 0.9946; Thi ? F2: y = -11.983x ?

145.51, R2 = 0.9976)

348 J Pest Sci (2012) 85:341–350

123

reduced number of emerged adults in combination com-

pared with separate treatments during initial storage peri-

ods may be correlated with the higher mortality

percentages in the tested treatments.

The results of this study suggested that B. bassiana can

be used effectively with DE and thiamethoxam against

R. dominica by exhibiting additive effect, but despite the

high effectiveness of thiamethoxam, it is not capable of

long-term protection of stored wheat. Thiamethoxam has

been used as a seed treatment for various field crops. The

grain protection ability of thiamethoxam has been discussed

by Arthur et al. (2004) who suggested it as a candidate for

wheat and maize seed protection. Some other insecticides

commonly used for field crops, urban, and veterinary insect

pests control have also been evaluated for their possible

effect as grain protectants. For example, fipronil, an insec-

ticidal pyrazol commonly used against the insect pests of

field crops, was also tested for the control of stored product

insects and found to be highly effective (Kavallieratos et al.

2010). The results presented here indicate that further

studies are justified to reduce the application rates of thia-

methoxam so that it could be used in post-harvest IPM

programs and registered as grain protectant.

Acknowledgments We would like to thanks Prof. Dr. Christos G.

Athanassiou, University of Thessaly, Volos, Greece and Dr. Jeffrey C.

Lord, USDA-ARS, Center for Grain and Animal Health Research,

Manhattan, Kansas, USA for helpful comments on the draft of the

manuscript. This publication is the product of research project funded

by the Higher Education Commission (HEC), Islamabad, Pakistan

(074-2407-Av4-128).

References

Abbott WS (1925) A method of computing the effectiveness of an

insecticide. J Econ Entomol 18:265–267

Adane K, Moore D, Archer SA (1996) Preliminary studies on the use

of Beauveria bassiana to control Sitophilus zeamais (Coleoptera:

Curculinoidae) in the laboratory. J Stored Prod Res 32:105–113

Arthur FH (1999) Knockdown, mortality and progeny production of

lesser grain borers (Coleoptera: Bostrichidae) and rice weevils

(Coleoptera: Curculionidae) exposed for short intervals on wheat

treated with cyfluthrin. J Econ Entomol 92:1198–1205

Arthur FH (2002) Survival of Sitophilus oryzae (L.) on wheat treated

with diatomaceous earth: impact of biological and environmental

parameters on product efficacy. J Stored Prod Res 38:305–313

Arthur FH, Yue B, Wilde GE (2004) Susceptibility of stored-product

beetles on wheat and maize treated with thiamethoxam: effects

of concentration, exposure interval, and temperature. J Stored

Prod Res 40:527–546

Athanassiou CG, Kavallieratos NG, Economou LP, Dimizas CB,

Vayias BJ, Tomanovic S, Milutinovic M (2005) Persistence and

efficacy of three diatomaceous earth formulations against

Sitophilus oryzae (Coleoptera: Curculionidae) on wheat and

barley. J Econ Entomol 98:1404–1412

Athanassiou CG, Kavallieratos NG, Vayias BJ, Tsakiri JB, Mikeli NH,

Meletsis CM, Tomanovic Z (2008) Persistence and efficacy of

Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina:

Hyphomycetes) and diatomaceous earth against Sitophilus oryzae(L.) (Coleoptera: Curculinoidae) and Rhyzopertha dominica (F.)

(Coleoptera: Bostrichidae) on wheat and maize. Crop Prot

27:1303–1311

Barnett HL, Hunter BB (1998) Illustrated genera of imperfect fungi.

APS Press, Minnesota

Batta YA (2005) Control of the lesser grain borer (Rhyzoperthadominica (F.), Coleoptera: Bostrichidae) by treatments with

residual formulations of Merarhizium anisopliae (Metschnikoff)

Sorokin (Deuteromycotina: Hyphomycetes). J Econ Entomol

41:221–229

Choi YW, Hyde KD, Ho WH (1999) Single spore isolation of fungi.

Fungal Div 3:29–38

Collins DA, Cook DA (1998) Periods of protection provided by

different formulations of pirimiphos-methyl and etrimos, when

admixed with wheat against four susceptible storage beetle pests.

Crop Prot 17:521–528

de Oliveira CN, Neves PMOJ, Kawazoe LS (2003) Compatibility

between the entomopathogenic fungus Beauveria bassiana and

insecticides used in coffee plantations. Sci Agric 60:663–667

Ebeling W (1971) Sorptive dusts for pest control. Ann Rev Entomol

16:123–158

Fields PG, Korunic Z (2000) The effect of grain moisture content and

temperature on the efficacy of diatomaceous earths from

different geographical locations against stored-product beetles.

J Stored Prod Res 36:1–13

Greenspan L (1977) Humidity fixed points of binary saturated

aqueous solutions. J Res Natl Bur Stand A 81:89–96

Hofer D, Brandl F (1999) Cruiser/Cruiser performance features of

thiamethoxam as a seed treatment in worldwide cotton. Proc

Beltwide Cotton Conf Memphis 2:1101–1104

Kavallieratos NG, Athanassiou CG, Vayias BJ, Mihail SB, Tomano-

vic Z (2009) Insecticidal efficacy of abamectin against three

stored-product insect pests: influence of dose rate, temperature,

commodity, and exposure interval. J Econ Entomol 102:

1352–1359

Kavallieratos NG, Athanassiou CG, Vayias BJ, Betsi PCC (2010)

Insecticidal efficacy of fipronil against four stored-product insect

pests: influence of commodity, dose, exposure interval, relative

humidity and temperature. Pest Manag Sci 66(6):640–649

Kljajic P, Peric I (2009) Residual effects of deltamethrin and

malathion on different populations of Sitophilus granarius (L.)

on treated wheat grains. J Stored Prod Res 45:45–48

Lawson DS, Dunbar DM, White SM, Ngo N (1999) Actara 25 WG:

control of cotton pests with a new neonicotinoid insecticide,

thiamethoxam. Proc Beltwide Cotton Conf Memphis 2:1106–1109

Lord JC (2001) Desiccant dusts synergize the effect of Beavueriabassiana (Hyphomycetes: Moniliales) on stored grain beetles.

J Econ Entomol 94:367–372

Lord JC (2005) Low humidity, moderate temperature, and desiccant

dust favor efficacy of Beauveria bassiana (Hyphomycetes:

Moniliales) for the lesser grain borer, Rhyzopertha dominica(Coleoptera: Bruchidae). Biol Control 34:180–186

Lord JC (2009) Beauveria bassiana infection of eggs of stored-

product beetles. Entomol Res 39:155–157

McGaughey WH (1972) Diatomaceous earth for confused flour beetle

and rice weevil control in rough, brown and milled rice. J Econ

Entomol 65:1427–1428

Meienfisch P, Huerlimann H, Rindlisbache A, Gsell L, Dettwiler H,

Haettenschiller J, Sieger J, Walti M (2001) The discovery of

thiamethoxam: a second generation of neonicotinoid. Pest

Manag Sci 57:165–176

Meikle WG, Cherry AJ, Holst N, Hounna B, Markham RH (2001) The

effects of an entomopathogenic fungus Beauveria bassiana(Balsamo) Vuillemin (Hyphomycetes), on Prostephanus trunca-tus (Horn) (Col.: Bostrichidae), Sitophilus zeamais Motschulsky

J Pest Sci (2012) 85:341–350 349

123

(Col.: Curculinoidae) and grain losses in stored maize in the Benin

Republic. J Invertebr Pathol 77:198–205

Michalaki MP, Athanassiou CG, Kavallierateos NG, Batta YA,

Balotis GN (2006) Effectiveness of Metarhizium anisopliae(Metschinkoff) Sorokin applied alone or in combination with

diatomaceous earth against Tribolium confusum Du Val larvae:

influence of temperature, relative humidity and type of com-

modity. Crop Prot 25:418–425

Moore D, Lord JC, Smith SM (2000) Pathogens. In: Subramanyam

BH, Hagstrum DW (eds) Alternatives to pesticides in stored-

product IPM. Kluwer Academic, Dordrecht, pp 193–227

Neves PMOJ, Hirose E, Tchujo PT, Moino A Jr (2001) Compatibility

of entomopathogenic fungi with neonicotinoid insecticides.

Neotrop Entomol 30:263–268

Riasat T, Wakil W, Ashfaq M, Sahi ST (2011) Effect of Beauveriabassiana mixed with diatomaceous earth on mortality, mycosis

and sporulation of Rhyzopertha dominica on stored wheat.

Phytoparasitica 39:325–331

Rice WC, Cogburn RR (1999) Activity of the entomopathogenic

fungus Beauveria bassiana (Deuteromycota: Hyphomycetes)

against three coleopteran pests of stored grain. J Econ Entomol

92:691–694

Russell CW, Ugine TA, Hajek AE (2010) Interaction between

imidacloprid and Metarhizium brunneum on adult Asian long-

horned beetles (Anoplophora glabripennis (Motschulsky))

(Coleoptera: Cerambycidae). J Invertebr Pathol 105:305–311

SAS Institute (1999) SAS/STAT user’s guide, 8th edn. SAS Institute,

Cary

Sokal RR, Rohlf FJ (1995) Biometry, 3rd edn. Freedman and

Company, New York

Soliman MMM (2011) Persistence of new insecticides and their

efficacy against insect pests of cowpea. Aust J Basic Appl Sci

5:82–89

Stathers TE, Mvumib BM, Golob P (2002) Field assessment of the

efficacy and persistence of diatomaceous earths in protecting

stored grain on small-scale farms in Zimbabwe. Crop Prot 21:

1033–1048

Stathers TE, Denniff M, Golob P (2004) The efficacy and persistence

of diatomaceous earths admixed with commodity against four

tropical stored product beetle pests. J Stored Prod Res 40:113123

Subramanyam BH, Roesli R (2000) Inert dusts. In: Subramanyam B,

Hagstrum DW (eds) Alternatives to pesticides in stored-product

IPM. Kluwer Academic, Boston, pp 321–380

Subramanyan BH, Hagstrum DW (1996) Resistance measurement

and management. In: Subramanyam BH, Hagstrum DW (eds)

Integrated management of insects in stored products. Marcel

Dekker, New York, pp 331–397

Thomas MB, Jenkins NE (1997) Effects of temperature on growth of

Metarhizium flavoviride and virulence to the variegated grass-

hopper, Zonocerus variegatus. Mycol Res 101:1469–1474

Vassilakos TN, Athanassiou CG, Kavallieratos NK, Vayias BJ (2006)

Influence of temperature on the insecticidal effect of Beauveriabassiana in combination with diatomaceous earth against

Rhyzopertha dominica and Sitophilus oryzae on stored wheat.

Biol Control 38:270–281

Vayias BJ, Athanassiou CG (2004) Factors affecting the insecticidal

efficacy of the diatomaceous earth formulation SilicoSec against

adults and larvae of the confused flour beetle, Tribolium confusumDu Val (Coleoptera: Tenebrionidae). Crop Prot 23:565–573

Vayias BJ, Athanassiou CG, Kavallieratos NG, Tsesmeli CD,

Buchelos CTh (2006) Persistence and efficacy of two diatoma-

ceous earth formulations and a mixture of diatomaceous earth

with pyrethrum against Tribolium confusum Jacquelin Du Val

(Coleoptera: Tenebrionidae) on wheat and maize. Pest Manag

Sci 62:456–464

Wakil W, Ghazanfar MU (2010) Entomopathogenic fungus as a

biological control agent against Rhyzopertha dominica F.

(Coleoptera: Bostrychidae) on stored wheat. Arch Phytopathol

Plant Prot 43:1236–1242

Wakil W, Ashfaq M, Ghazanfar MU, Riasat T (2010) Susceptibility

of stored-product insects to enhanced diatomaceous earth.

J Stored Prod Res 46:248–249

Wakil W, Riasat T, Ghazanfar MU, Kwon YJ, Shaheen FA (2011)

Aptness of Beauveria bassiana and enhanced diatomaceous

earth (DEBBM) for control of Rhyzopertha dominica F. Entomol

Res 41:233–241

Yue B, Wilde GE, Arthur F (2003) Evaluation of thiamethoxam and

imidacloprid as seed treatments to control European corn borer

and Indian meal moth (Lepidoptera: Pyralidae) larvae. J Econ

Entomol 96:503–509

350 J Pest Sci (2012) 85:341–350

123