Journal of Invertebrate Pathology 148 (2017) 102–109

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Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Laboratory evaluation of the effect of Beauveria bassiana on the predatory MARK

mite Phytoseiulus persimilis (Acari: Phytoseiidae)
⁎
Mohammad Shaef Ullaha,b, Un Taek Lima,
a
Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea
b
Department of Entomology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: Tetranychus urticae Koch (Acari: Tetranychidae), a major pest of many agricultural crops, is mainly controlled
Spider mites with chemical acaricides. However, predatory mites and entomopathogens have been proposed as alternative
Phytoseiids control agents. In this study, the effect of the BotaniGard® GHA strain of Beauveria bassiana on the survival,
Entomopathogenic fungus longevity, fecundity, and egg hatch rate of the predatory mite Phytoseiulus persimilis Athias-Henriot (Acari:
Interactions
Phytoseiidae) were studied under laboratory conditions. When B. bassiana was applied directly to P. persimilis
Biological control
eggs at a concentration of 1 × 108 conidia/ml, corrected hatchability was less than 5%, and the corrected
Life table
mortality of nymphs and adults was not significantly different from control 10 days after treatment. Phytoseiulus
persimilis nymphs that hatched from treated eggs showed no significant change in their development time, adult
female longevity, hatch rate, survival rates over time, or offspring sex ratio. However, significant negative effects
on fecundity and life table parameters (net reproductive rate, intrinsic rate of natural increase, mean generation
time, finite rate of increase, and doubling time) were found when B. bassiana was applied to the adult stage.
Spraying B. bassiana at 1 × 108 conidia/ml on newly emerged adults of P. persimilis caused 44% reduction in the
oviposition period, 26% in adult longevity, and 63% in fecundity. Due to these negative effects, B. bassiana
should be used with careful adjustment of application timing (first spray B. bassiana and then release P. persi-
milis) to supplement biological mite control systems using P. persimilis.

1. Introduction 2007). However, different degrees of success have been obtained in

different crops and conditions (Zhang and Sanderson, 1995; Kim et al.,
The spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is 1997; Opit et al., 2003; Naher and Haque, 2007). In addition to the use
an important pest of ornamentals and vegetables, causing extensive of predatory mites, growers have recently adopted the use of com-
economic losses in greenhouse and open-field crops (Grbić et al., 2011). mercial microbial products such as entomopathogenic fungi to control
The economic damage to crops caused by spider mites can increase populations of T. urticae (Alves et al., 2002; Chandler et al., 2005; Shi
rapidly due to its rapid developmental rate (Ullah et al., 2012). Che- and Feng, 2009; Alma et al., 2007). The entomopathogenic fungus
mical acaricides have been the primary strategy for controlling spider Beauveria bassiana (Balsamo) Vuillemin is among these (Leger et al.,
mites, but as resistance to acaricides has spread, biological control has 1986). Beauveria bassiana applied against aphids, leafhoppers, and
become a more widely used alternative (Gerson and Weintraub, 2007). whiteflies has been shown to be effective in laboratory and greenhouse
Predatory mites such as Phytoseiulus persimilis Athias-Henriot (Acari: trials (Faria and Wraight, 2001; Feng et al., 2004; Hatting et al., 2004;
Phytoseiidae) are more selective to spider mites and safer to environ- Pu et al., 2005). Successful control of spider mites may be achieved by
ment than chemical pesticides, making them more compatible with applying entomopathogenic fungi that are more toxic to the pest than to
other control measures and an important element of integrated mite its natural enemies (Gonzalez et al., 2016). Because releases of P. per-
management programs (Skirvin et al., 2002; Shi and Feng, 2004; Alma similis may also be an effective alternative for spider mite control in
et al., 2007; Avery et al., 2008; Vergel et al., 2011). field crops, identification of a selective mycopesticide that is compa-
Phytoseiulus persimilis, a specialist predator of spider mites, has been tible with this predator is important. The combined application of B.
successfully employed for regulating populations of T. urticae in bassiana with predatory mites has been found to reduce the density of T.
greenhouse crops (Cote, 2002; Skirvin and Fenlon, 2001; Oliveira et al., urticae by as much as 98% in greenhouse tomatoes (Chandler et al.,

⁎
Corresponding author at: Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea.
E-mail address: utlim@andong.ac.kr (U.T. Lim).

http://dx.doi.org/10.1016/j.jip.2017.06.006
Received 18 July 2016; Received in revised form 7 April 2017; Accepted 15 June 2017
Available online 16 June 2017
0022-2011/ © 2017 Elsevier Inc. All rights reserved.
M.S. Ullah, U.T. Lim Journal of Invertebrate Pathology 148 (2017) 102–109

2005). However, compatibility of predatory mites and en- control tests of B. bassiana showed 90% conidial viability. Conidial
tomopathogenic fungi can be a critical issue in the field conditions suspension was prepared at a concentration of 1 × 108 conidia ml−1
(Ghazy et al., 2016). Several researchers have found B. bassiana having diluted with distilled water just before assay.
an adverse effect on the biological life history parameters of predators
fed on infected prey (Agboton et al., 2013; Wu et al., 2015), and Seiedy 2.2. Effect of B. bassiana on P. persimilis survival
et al. (2012) found that feeding B. bassiana-contaminated prey (T. ur-
ticae) to P. persimilis caused undesirable effects on the predator’s life 2.2.1. Eggs
history parameters. Beauveria bassiana sprayed on adults of P. persimilis Beauveria bassiana conidia were assayed using a direct spray method
resulted in ca. 43% mortality (Duso et al., 2008; Vergel et al., 2011) and to evaluate their ovicidal effect on P. persimilis eggs. Four 5-day-old
significant reduction in fecundity (Duso et al., 2008). However, inter- adult female P. persimilis were taken from the stock predator culture and
actions between arthropods and entomopathogenic fungus are complex, transferred on a kidney bean leaf (4 × 4 cm2 leaf disc) infested with
and the sub-lethal effects of B. bassiana on life history parameters of P. mixed stages of T. urticae. Those adult predators were allowed to lay
persimilis are not yet studied when sprayed on eggs and adults sepa- eggs for 24 h and then removed, and the eggs laid were collected and
rately. used for bioassay. There were four discs (2 × 2 cm2 leaf disc), each
The aim of this study was therefore to assess the effect on P. persi- with 15 predator eggs from a cohort of the population was kept for
milis life table parameters after direct application of the B. bassiana ovicidal effect test. Subsequently, 1 × 108 conidia ml−1 suspension of
based commercial product BotaniGard® ES to either eggs or adult pre- B. bassiana was sprayed onto the leaf disc (2 × 2 cm2 leaf disc) with a
datory mites. As a preliminary assay, we examined the hatch rate of P. hand sprayer (4 ml in 2 × 2 cm2 leaf disc). In order to standardize the
persimilis eggs sprayed with B. bassiana and subsequent life history use of the sprayer to deliver a consistent dose, five consecutive sprays
parameters of surviving mites at three different RHs. This information were performed on Petri dishes (50 mm diameter, 15 mm depth), each
will help define the degree of compatibility of these two biological in triplicate, and the deposits were quantified (5.0 ± 0.02 ml). No
control agents and determine the possibility of combined applications significant differences were obtained (data not shown), and so hand
of both agents in spider mite biological control programs. application was used in this manner in the main experiment. For a
control, pure distilled water was sprayed on eggs of P. persimilis present
2. Material and methods on leaf discs. After air drying, the sprayed spider mite eggs were held at
one of three RHs (55, 75, and 95%), as this variable often affects spore
2.1. Sources of T. urticae, P. persimilis, and entomopathogenic fungi germination and hence infection rates. The three RHs were achieved by
dissolving Mg(NO3)2·6H2O, NaCl, and K2SO4 in distilled water in de-
2.1.1. Mites siccators (140 mm diameter, Scienceware®, Wayne, New Jersey, USA)
Tetranychus urticae obtained from Insect Ecology laboratory, to prepare 55, 75, and 95% RH, respectively (Rockland, 1960). Tem-
Andong National University, Republic of Korea in 2014. Laboratory perature and RH were measured using a data logger (U10-001; Onset
stocks of T. urticae were maintained on bean leaf discs (ca. 16 cm2) of Computer Corporation, Cape Cod, MA). All treated and control Petri
common bean, Phaseolus vulgaris L., placed on water-saturated poly- dishes were held at 24.7 ± 0.05 °C in an incubator, and the number of
urethane mats in plastic Petri dishes (90 mm diameter, 20 mm depth) eggs hatched was recorded daily until there was no change for three
and held at 23.0–26.9 °C, 38–61% RH, and a photoperiod of 16L: 8D h consecutive days. Later, the eggs were individually examined under an
in a growth chamber (DS-11BPL, Dasol Scientific Co., Ltd, Suwon, inverted microscope (40× magnification, Leica microsystems, GmbH
Republic of Korea) for more than one year before the present study. Wetzlar, Germany) for verification of fungal infection. Finally, all un-
Mixed stages of T. urticae were used to rear P. persimilis. hatched eggs were transferred to moist chambers for three days to de-
tect any fungal outgrowth, as evidence of egg mortality due to fungal
2.1.2. Phytoseiulus persimilis infection.
Phytoseiulus persimilis was collected from the Dongbu Farm Ceres
Company in 2014. Laboratory stocks of P. persimilis were maintained 2.2.2. Protonymphs and newly eclosed adults
using all stages of T. urticae (green form) reared on bean leaf discs (ca. Protonymphs and newly eclosed adults of P. persimilis collected from
16 cm2) placed on water-saturated polyurethane mats in plastic Petri the predator colony were sprayed with a 1 × 108 conidia ml−1 sus-
dishes (90 mm diameter, 20 mm depth) for more than one year in a pension of B. bassiana strain GHA onto the leaf disc with a hand sprayer
rearing chamber (23.0–26.9 °C, 38–61% RH, and a 16L: 8D h photo- (4 ml in 2 × 2 cm2 leaf disc), and their survival was assessed. In se-
period). The leaf discs were renewed as necessary. parate trials, for each stage, mites were exposed to the fungal pathogen
through direct spray and then transferred to plastic Petri dishes (50 mm
2.1.3. Fungal pathogen and preparation of conidial suspension diameter, 15 mm depth) lined with freshly excised bean leaf discs and
The entomopathogenic fungus tested was obtained as the commer- held at 24.7 ± 0.05 °C, a photoperiod of 16L: 8D h, and one of three
cial product BotaniGard® ES (Beauveria bassiana, GHA strain, Arysta RHs. Mixed stages of T. urticae were provided on leaf discs as food. After
LifeScience, Tokyo, Japan). As a viability test, subcultures were grown air drying, the leaf discs with treated protonymphs or adults of P. per-
on Sabouraud Dextrose Agar (SDA) in Petri dishes and maintained in similis were held under one of three RHs (55, 75, and 95%) and counted
the dark at 25 ± 1 °C for 10–14 days. Conidia were harvested from daily. Mortality was recorded daily for 10 days after application. For
surface cultures by scraping and were then suspended in 10 ml of sterile each life stage there were five replicates for each level of humidity, with
distilled water containing 0.05% Triton X-100 using universal bottles 10 predators per replicate (total of 50 mites exposed per life stage per
containing glass beads. Conidial suspensions were vortexed for 5 min, RH level). The presence of fungal mycelia on dead mites was regarded
and spore concentrations were determined using a haemocytometer as an indication of mycosis. Controls consisted of predators treated with
(Neubauer-improved haemocytometer, Lauda-Königshofen, Germany). distilled water. As no statistical differences were observed in rates of
Conidial viability was determined before the bioassay by spread-plating mortality among the three levels of RH for either egg hatch or nymphal
0.1 ml of conidial suspension titrated to 1 × 104 conidia ml−1 on SDA or adult survival, we used 75.0 ± 2.0% RH only for all subsequent
plates (Seiedy et al., 2012). Plates were incubated at 25 ± 1 °C, and experiments.
the percentage germination was determined after 24 h from 100-spore
counts by placing a sterile microscope coverslip on each plate and 2.3. Life table study of P. persimilis with B. bassiana applied at the egg stage
counting germinants under a microscope (100× magnification, Nikon,
Eclipse E200, Japan). Each plate was replicated four times. Quality To determine the effects of exposure (during the egg stage only) on

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M.S. Ullah, U.T. Lim Journal of Invertebrate Pathology 148 (2017) 102–109

∞
the life history parameters of P. persimilis over two generations (P1 and
F1) a cohort of predator mite eggs were treated with
R0 = ∑ (l x m x ).
X =0
1 × 108 conidia ml−1 suspension of B. bassiana strain GHA and then
allowed to hatch. In each treatment, number of eggs tested ranged from The intrinsic rate of increase (rm) was calculated using the Lotka-Euler
35 to 39. One egg in each leaf disc was transferred using a moistened equation with age indexed from zero (Tuan et al., 2014) as
red marten's hair brush. The developmental times of the immature ∞
stages from hatching eggs were then measured for treated and un- ∑ e−r (X + 1) (l x m x ) = 1.
treated controls. Adults developed from survived nymphs were paired, X =0

and their reproductive output and longevity were measured. Finally, for
the F1 generation of progeny, we calculated the egg hatch rate, the
survival rate of immature stages, and the sex ratio of F1 adults. From
these data, life history parameters were calculated and compared for
control and treated mite cohorts.

2.3.1. Immature development time

To determine the effect of exposure to B. bassiana during the egg
stage on the developmental times of the immature stages of P. persimilis,
inseminated adult females obtained from stock cultures were trans-
ferred individually onto a leaf disc of common bean (2.0 × 2.0 cm2).
Females of P. persimilis were allowed to lay eggs on the leaf disc, but one
egg per leaf disc was kept for the development study. Eggs were sprayed
with a concentration of 1 × 108 conidia ml−1 and incubated at Fig. 1. Hatch rate of Phytoseiulus persimilis eggs treated with either 1 × 108 conidia mL−1
24.7 ± 0.05 °C, a photoperiod of 16L: 8D h, and 75.0 ± 2.0% RH. To of B. bassiana or a water control, at 55, 75, and 95% RH.

provide food sources for the immature P. persimilis, 20–30 females of T.

urticae were allowed to lay eggs on the leaf disc for 2–3 days before we
transferred immature P. persimilis into the leaf disc. Developmental
stages were checked twice each day (12 h intervals) until all mites
became adults. The presence of exuviae on the leaf disc was regarded as
proof of moulting. Control mite eggs were treated with distilled water
and observed as described above.

2.3.2. Reproduction and adult longevity

When females of P. persimilis from the above described cohort
(treated as eggs with B. bassiana) reached the adult stage, females were
paired singly with one adult male from the colony. This male was kept
on the disc for the total experimental period. The leaf discs were ob-
served at 12 h intervals to determine the pre-oviposition period. The
number of eggs laid by each P. persimilis female was observed and re-
corded daily under a stereomicroscope (40× magnification) to de-
termine oviposition period, total number of eggs laid per female, eggs
laid per female per day, post-oviposition period, and female longevity
until all females were dead.

2.3.3. Hatch rate of F1 mite eggs and the survival rate and sex ratio of F1
nymphs and adults
Egg hatch rate, the survival rate of F1 immature stages, and the
proportion, in the F1 generation, of female offspring were assessed
under the same conditions as described above.

2.3.4. Life history parameters

Parameters from the experiments described above for mite cohorts
either exposed or not exposed to B. bassiana in the egg stage of the P1
generation were subsequently used to calculate the age-specific survival
rate (lx) and fecundity (mx) for both generations of the treatment and
control cohorts. The eggs (F1) obtained from each female were then
incubated until adulthood to assess the above-mentioned parameters.
The life history raw data of individuals for each treatment were
analysed by using TWOSEX-MSChart program (Chi, 2012), based on the
age-stage, two-sex life table analysis (Chi and Liu, 1985) and method
described by Chi (1988). The survival rate (Sxj) (x = age, j = stage),
which is the probability that a newly laid egg will survive to age x and
stage j, and fecundity fxj, which is the number of hatched eggs produced
by female adult at age x, were calculated. The net reproductive rate (R0)
is defined as the total number of offspring that an individual can pro- Fig. 2. Survival of nymphs of Phytoseiulus persimilis over 10 days following treatment with
duce during its lifetime (Tuan et al., 2014) and is calculated as either 1 × 108 conidia mL−1 of B. bassiana or a water control at 55, 75, and 95% RH.

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ln (2)
tD = .
rm
The mean and standard errors of population parameters were calcu-
lated using the bootstrap method (Huang and Chi, 2013).

2.4. Life table study of P. persimilis spraying them with B. bassiana on the
adult stage

To examine the effect on life history parameters of treating P. per-

similis mites in the adult stage with B. bassiana, newly eclosed adult P.
persimilis were inoculated by spraying with them a
1 × 108 conidia ml−1 suspension of B. bassiana onto the leaf disc with a
hand sprayer (4 ml in 2 × 2 cm2 leaf disc) at 24.7 ± 0.05 °C with
75.0 ± 2.0% RH. The leaf discs with treated or control mites (sprayed
with water) were observed at 12-h intervals to determine the pre-ovi-
position period of mites. The number of eggs laid by a female P. persi-
milis was observed and recorded daily under a stereomicroscope to
determine oviposition period, total number of eggs laid per female, eggs
laid per female per day, post-oviposition period, and female longevity
until death. These parameters were used to calculate the age-specific
survival rate (lx) and fecundity (mx) for both the treated and control
cohorts of P. persimilis adults.

2.5. Statistical analyses

Data for mortality were corrected for levels of mortality in controls

using Abbot’s formula (Abbott, 1925) and were normalized using arc-
sine transformation. Developmental time, oviposition period, adult
longevity, fecundity, and life table parameters were logarithmic (ln)
transformed before analysis. Proportional data (hatch rate, survival
rate, and offspring sex ratio) were arcsine transformed before the
analysis. Two-way analysis of variance (ANOVA) was used for data on
developmental time to measure the effect of B. bassiana and mite sex,
while one-way ANOVA was used to test for significant differences
(P < 0.05) in female longevity, pre-oviposition period, oviposition
period, post-oviposition period, fecundity, survival, and offspring sex
ratio of P. persimilis. Data were analysed with a Generalized Linear
Fig. 3. Survival of adult Phytoseiulus persimilis over 10 days following inoculation with
Model (GLM) and Tukey’s student range test as a post hoc analysis using
either 1 × 108 conidia mL−1 of B. bassiana or a water control at 55, 75, and 95% RH.
SAS (SAS Institute, 2011). Age-stage survival rate and fecundity of P.
persimilis treated with B. bassiana or distilled water were estimated
The mean generation time (tG) represents the period that a popu-
using the computer program TWOSEX-MSChart (Chi, 2012).
lation requires to increase to R0-fold of its size as time approaches in-
finity and the population settles down to a stable age-stage distribution
3. Results
(Tuan et al., 2014). Mean generation time is calculated as
lnR 0 3.1. The effect of B. bassiana on egg hatchability and survivability of
tG = .
r immatures of P. persimilis
The finite rate of increase (λ) is a multiplication factor of the original
population at each time period. The decimal part of the finite rate of No significant differences on egg hatchability were observed after
increase corresponds to the daily rate of increase, expressed as a per- spraying with B. bassiana at 1 × 108 conidia ml−1 suspension among
centage (Maia et al., 2000). The rate of increase is expressed as the three RHs (χ2 = 1.59, df = 5, P = 0.90) (Fig. 1).
For protonymphs, the corrected mortalities were 6.4, 8.3, and 6.4%
λ = e rm.
10 days after application of B. bassiana spores at 55, 75, and 95% RH,
Doubling time (tD) is the time span necessary for doubling the initial respectively (Fig. 2). For mites treated as newly emerged adults, the
population. It is calculated from the exponential growth model (Maia corrected mortalities were 2.1, 8.3, and 8.5% 10 days after application
et al., 2000), expressed as of B. bassiana at 55, 75, and 95% RH, respectively (Fig. 3). No

Table 1
Developmental time (egg to adult) of Phytoseiulus persimilis with and without Beauveria bassiana treatment of mites as eggs at 75% RH.

Treatment Sex N Egg Larvae Protonymph Deutonymph Egg to adult

B. bassiana ♀ 39 1.83 ± 0.04a 0.58 ± 0.03a 0.72 ± 0.04a 0.91 ± 0.05a 4.04 ± 0.05a
♂ 22 1.75 ± 0.05a 0.55 ± 0.05a 0.73 ± 0.05a 0.84 ± 0.05b 3.86 ± 0.07b
Control ♀ 35 1.81 ± 0.04a 0.57 ± 0.03a 0.73 ± 0.06a 0.93 ± 0.04a 4.03 ± 0.04a
♂ 21 1.71 ± 0.06a 0.55 ± 0.03a 0.73 ± 0.07a 0.74 ± 0.07b 3.88 ± 0.09b

Mean values differ significantly at P < 0.001 (***) (ANOVA). Durations followed by the same letters within a column are not significantly different at the 5% level.

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M.S. Ullah, U.T. Lim Journal of Invertebrate Pathology 148 (2017) 102–109

2.87 ± 0.05b
3.21 ± 0.05a
Eggs/Female/
significant differences in mortality were observed among the three RHs
for either nymphs (χ2 = 4.93, df = 5, P = 0.42) or adults (χ2 = 4.38,

Pre-oviposition and oviposition period, adult longevity (days), total eggs per female, and eggs per female per day (number) of Phytoseiulus persimilis with or without treatment of mite eggs and adults with Beauveria bassiana at 75% RH.
df = 5, P = 0.50).

Day

21.47 ± 0.85b
53.97 ± 1.15a
3.2. Life table study of P. persimilis when B. bassiana was applied during the
egg stage

Total eggs/
Female
3.2.1. Immature development time
The developmental times of P. persimilis life stages derived from B.

17.71 ± 0.64b
Adult longevity

20.86 ± 0.22a
bassiana-treated eggs were significantly influenced by sex but not by
treatment with B. bassiana (for egg-to-adult, B. bassiana F = 0.00,
df = 1, 116, P = 0.996; sex F = 7.81, df = 1, 116, P = 0.006; B. bas-
siana × sex F = 0.01, df = 1, 116, P = 0.917) (Table 1). The duration
of the egg stage was 1.8 days for P. persimilis for both females and males
Post-oviposition

when treated as eggs with B. bassiana, the same (1.8 and 1.7 days, males
1.5 ± 0.15b
4.7 ± 0.36a

and females) as in the control (B. bassiana P = 0.563; sex P = 0.006; B.

bassiana × sex P = 0.865). The durations of the larval, protonymph,
period

deutonymph, and egg-to-adult periods were not significantly different

between controls and mites treated as eggs with B. bassiana (for larvae,
B. bassiana F = 0.00, df = 1, 116, P = 0.983; sex F = 0.86, df = 1,
11.43 ± 0.61b
18.19 ± 0.28a

116, P = 0.357; B. bassiana × sex F = 0.04, df = 1, 116, P = 0.833;

Oviposition

Mean values differ significantly at P < 0.001 (***) (ANOVA). Values followed by the same letters within a column are not significantly different at the 5% level.

for protonymphs, B. bassiana F = 1.35, df = 1, 116, P = 0.249; sex

period

F = 2.89, df = 1, 116, P = 0.092; B. bassiana × sex F = 2.20, df = 1,

116, P = 0.140; for deutonymphs, B. bassiana F = 0.31, df = 1, 116,
Spraying on adult stage

P = 0.579; sex F = 5.92, df = 1, 116, P = 0.002; B. bassiana × sex

Pre-oviposition

1.18 ± 0.09b
1.58 ± 0.09a

F = 1.88, df = 1, 116, P = 0.173).

period

3.2.2. Reproduction and adult longevity

Pre-oviposition periods of P. persimilis adults derived from eggs
30
32
N

treated with B. bassiana were significantly affected by treatment

(F = 4.78, df = 1, 71, P < 0.003), but no significant differences were
2.87 ± 0.05b
3.21 ± 0.05a
Eggs/Female/

observed in lengths of the oviposition period (F = 2.38, df = 1, 71,

P = 0.127), the post-oviposition period (F = 2.61, df = 1, 71,
P = 0.111), or female adult longevity (F = 0.78, df = 1, 71,
Day

P = 0.382) (Table 2). The total number of eggs/female of P. persimilis

58.13 ± 2.67b
69.30 ± 1.57a

was higher in the control (69.3) than in the B. bassiana treatment (58.1)
Total eggs/

(F = 13.16, df = 1, 71, P < 0.001). Number of eggs/female/day of P.

persimilis were also significantly reduced by B. bassiana spray
Female

(F = 794.23, df = 1, 71, P < 0.001) (Table 2).

23.95 ± 0.91a
24.45 ± 0.52a
Adult longevity

3.2.3. Hatch rate, survival rate, and sex ratio of F1 offspring of mites from
B. bassiana-treated egg stage
There were no significant differences in the hatchability (F = 0.18,
df = 1, 71, P = 0.672), immature survivorship (F = 0.42, df = 1, 71,
P = 0.517), or sex ratio of F1 offspring of mites from B. bassiana-treated
Post-oviposition

2.26 ± 0.19a
1.76 ± 0.16a

eggs (F = 0.24, df = 1, 71, P = 0.627) between the control and B.

bassiana treatments (data not shown). The lx, mx, and lxmx (expected
period

number of female offspring at age x) curves at both conditions showed

an asymmetrical pattern (Fig. 4). The highest mx value or the oviposi-
tion rate at age x (age-specific oviposition × proportion of females) of
20.46 ± 0.93a
21.67 ± 0.50a

P. persimilis was 1.5 on the 13th and 14th day of adulthood in the B.
Oviposition

bassiana treatment and in the control, respectively (F = 0.25, df = 1,

period

71, P = 0.619).
Spraying on egg stage

Pre-oviposition

1.03 ± 0.09b
1.23 ± 0.07a

3.2.4. Life history parameters

The population growth parameters of P. persimilis were superior in
the control than in the B. bassiana treatment (Table 3). R0, rm, and λ of
period

P. persimilis were significantly higher in the control than in the B.

bassiana treatment (R0 F = 466.98, df = 1, 1999, P < 0.001; rm
39
33
N

F = 2504.82, df = 1, 1999, P < 0.001; λ F = 2509.26, df = 1, 1999,

P < 0.001). Consequently, mean generation time and doubling time
B. bassiana
Control

were significantly longer in the B. bassiana treatment than in the control

Table 2

(T F = 5944.84, df = 1, 1999, P < 0.001; Dt F = 2484.36, df = 1,

1999, P < 0.001).

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Fig. 4. Age-specific survival rate (lx), fecundity (mx), and maternity

rates (lxmx) with or without application of Beauveria bassiana on eggs
of Phytoseiulus persimilis held at 25 °C and 75% RH.

Table 3
Life history parameters of Phytoseiulus persimilis with and without application of Beauveria bassiana to mite eggs at 75% RH.

Treatment N Net reproductive rate (R0) Intrinsic rate of increase (rm) Mean generation time (tG) Finite rate of increase (λ) Doubling time (tD)

B. bassiana 39 33.75 ± 0.12b 0.2978 ± 0.0004b 11.80 ± 0.01a 1.3470 ± 0.0005b 2.33 ± 0.01a
Control 33 37.87 ± 0.14a 0.3273 ± 0.0004a 11.08 ± 0.01b 1.3874 ± 0.0006a 2.12 ± 0.01b

Mean values differ significantly at P < 0.001 (***) (ANOVA). Values followed by the same letters within a column are not significantly different at the 5% level.

3.3. Life table study of P. persimilis when B. bassiana was applied during the population (Ullah and Lim, 2015). Environmental conditions for the
adult stage bioassays were highly conducive for the development of infection
(Shipp et al., 2003), making it likely that any deleterious effects on
Treatment of P. persimilis and B. bassiana on adults resulted in the development, survival, or adult longevity of the fungus would have
longer pre-oviposition (F = 11.96, df = 1, 61, P < 0.001) and post- been evident.
oviposition periods (F = 65.47, df = 1, 61, P < 0.001) compared to When using phytoseiid predators as supplementary biological con-
the control (1.3 and 3.1-fold higher, respectively) (Table 2). The ovi- trol agents, it is essential to consider the compatibility of pathogens and
position period of adult mites treated with B. bassiana decreased to predators. Here, we used a GHA strain of B. bassiana that is highly
37.2% of that of the control (F = 83.54, df = 1, 61, P < 0.001). Fe- virulent to T. urticae (Ullah and Lim, 2015), but hatch rate and survival
male adult longevity was longer in the control than in the B. bassiana of P. persimilis were not significantly different at a concentration of
treatment (F = 23.14, df = 1, 61, P < 0.001). The total number of 1 × 108 conidia/ml under three different RHs, e.g., 55, 75, and 95%
eggs/female (F = 411.36, df = 1, 61, P < 0.001) and eggs/female/ RH.
day (F = 231.74, df = 1, 61, P < 0.001) were both reduced sig- Direct application of B. bassiana on egg stage did not significantly
nificantly compared to controls when B. bassiana was sprayed on newly affect the developmental time of P. persimilis. The sex ratio of the pro-
emerged adult female of P. persimilis (Table 2). Age-specific fecundity geny was also unaffected and female-biased in all treatments. However,
was higher in younger females sprayed with B. bassiana compared to fecundity of P. persimilis was negatively affected by B. bassiana spray on
fecundity of older females. Age-specific fecundity in the control was either egg or adult stage, and was reduced by 17% and 60%, respec-
also higher compared to that of adults treated with B. bassiana. The age- tively. This reduced fecundity could be categorized as a sublethal effect.
specific survival rate and fecundity curves showed asymmetrical pat- The fungus might not be transmitted transovarially, but they might
terns in both treatments (Fig. 5). survive a prolonged time even without the presence of the host (Roy
et al., 2006). These results are supported by other studies by Ludwig
4. Discussion and Oetting (2001) and Duso et al. (2008) who also found decreased
fertility of P. persimilis after the application of B. bassiana. Our results
These bioassays were designed to simulate worst-case scenarios that showed that spraying B. bassiana on egg stage did not affect the ovi-
could occur in field applications by exposing predators to concentra- position and post-oviposition period much, whereas spraying on adult
tions of B. bassiana sufficient to cause high mortality in a T. urticae stage did lengthen the pre-oviposition and post-oviposition periods.

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M.S. Ullah, U.T. Lim Journal of Invertebrate Pathology 148 (2017) 102–109

Fig. 5. Age-specific survival rate (lx), fecundity (mx), and maternity

rates (lxmx) with or without spraying of Beauveria bassiana on adults of
Phytoseiulus persimilis held at 25 °C and 75% RH.

However, this likely reflects time spent by the mites in grooming off (2012) also reported that life table parameters of P. persimilis were
conidia and probably was not an outcome of any physiological effect on strongly affected by feeding on prey infected with B. bassiana. Another
females. study on life table parameters showed that B. bassiana has indirect
In general, entomopathogenic fungus when sprayed directly on negative effects on the population dynamics of Neoseiulus barkeri
different predators or when predators were fed contaminated prey may Hughes by influencing its prey (Frankliniella occidentalis Pergande
affect fecundity. Both oviposition and survival of the predatory mite larvae), thus suggesting an element of risk in the combined use of B.
Typhlodromalus aripo De Leon were reduced significantly when they bassiana and N. barkeri (Wu et al., 2015). An important difference be-
consumed the entomopathogenic fungus (Neozygites tanajoa Delalibera tween this study and ours is that Wu et al. (2015) used infected prey (T.
Jr.) in the form of infected cassava green mites, Mononychellus tanajoa urticae or F. occidentalis) as a food source while we directly sprayed the
(Bondar) (Agboton et al., 2013). Seiedy et al. (2012) reported that entomopathogenic fungus onto the predator and provided mixed stages
longevity and fecundity were lower when P. persimilis fed on B. of uninfected T. urticae for food. Applying entomopathogenic fungi di-
bassiana-treated T. urticae for 24–72 h. Direct application of two en- rectly onto predatory mites has an undesirable effect on fertility table
tomopathogenic fungi, B. bassiana and Paecilomyces fumosoroseus parameters and reduces the intrinsic rate of natural increase of the
(=Isaria fumosorosea) (Wize) Brown & Smith, was found to reduce the predator population. Although the entomopathogenic fungi are well
fecundity of Neoseiulus californicus (McGregor) significantly, but not the known for their capacity to cause dramatic epizootics that rapidly re-
fecundity of P. persimilis (Vergel et al., 2011). However, Wekesa et al. duce pest populations (Pell et al., 2010), it also causes unexpected ef-
(2007) reported that the fecundity of the predatory mite Phytoseiulus fects on arthropod natural enemies through changes in behaviour of
longipes Evans was not affected by feeding on either Tetranychus evansi pests or natural enemies or through changes in pest densities or di-
Baker & Pritchard or T. urticae that were infected with Neozygites flor- versity (Roy et al., 2006). Therefore, predator-mediated side effects
idana (Weiser & Muma). Predatory mites both in laboratory and should be taken into account when applying entomopathogenic fungus
greenhouse trials have shown less vulnerability to some isolates of the against a specific pest species (Gonzalez et al., 2016).
entomopathogenic fungi B. bassiana (Duso et al., 2008; Vergel et al., In conclusion, while there was only a minimal effect of B. bassiana
2011). Mortality on adult P. persimilis caused by B. bassiana was ca. 43% spray on the egg stage of P. persimilis, this entomopathogen did reduce
(Duso et al., 2008; Vergel et al., 2011) whereas mortality caused by P. the fecundity of P. persimilis. With careful adjustment of application
fumosoroseus was 31% (Vergel et al., 2011). In addition, the same en- timing (first spray B. bassiana and then release P. persimilis), Beauveria
tomopathogenic fungus generated 10–14% mortalities of adult N. cali- bassiana could be used to supplement biological mite control systems
fornicus under laboratory conditions (Castagnoli et al., 2005; Vergel using P. persimilis without significant disruption to the predatory mite.
et al., 2011).
In this study, P. persimilis was monitored over their entire life span
Acknowledgements
and suffered no significant mortality from B. bassiana spray as eggs, but
did experience reduced fecundity if treated as adults. Seiedy et al.
This work was supported by Korea Institute of Planning and

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M.S. Ullah, U.T. Lim Journal of Invertebrate Pathology 148 (2017) 102–109

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