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Evaluation of the pathogenicity of Aschersonia aleyrodis on Bemisia tabaci in

the laboratory and greenhouse

Article · December 2016

DOI: 10.1080/09583157.2016.1274878

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BIOCONTROL SCIENCE AND TECHNOLOGY, 2017
VOL. 27, NO. 2, 210–221
http://dx.doi.org/10.1080/09583157.2016.1274878

RESEARCH ARTICLE

Evaluation of the pathogenicity of Aschersonia aleyrodis on

Bemisia tabaci in the laboratory and greenhouse
Can Zhang, Shaukat Ali*, Peter Dennis Musa, Xing-Min Wang and Bao-Li Qiu
Key Laboratory of Bio-Pesticide Innovation and Application, Engineering Technology Research Centre of
Agricultural Pest Biocontrol Guangdong Province, College of Agriculture, South China Agricultural University,
Guangzhou, People’s Republic of China

ABSTRACT ARTICLE HISTORY

The entomopathogenic fungus Aschersonia aleyrodis (Webber) is a Received 4 July 2016
promising fungal species against whiteflies. In this work, the Returned 4 December 2016
pathogenicity of A. aleyrodis isolate Aa005 against MEAM1 Bemisia Accepted 15 December 2016
tabaci (Gennadius) was evaluated under laboratory and
KEYWORDS
greenhouse conditions. The bioassay results indicated that the Entomopathogenic fungi;
percentage of larval mortalities was concentration and age biological control;
dependent. A. aleyrodis showed high pathogenicity against Aschersonia aleyrodis; Bemisia
second and third instars and pupae with LC50 values of 7.93 × 106, tabaci
1.08 × 107, and 1.56 × 107 conidia mL−1, respectively. The median
lethal time (LT50) was lower (4.60 days) for second instars and was
the highest (6.17 days) for pupae when inoculated with a
concentration of 1 × 107 conidia mL−1. Weekly sampling of
immatures showed that the per cent mortality caused by
A. aleyrodis at a conidial concentration of 1 × 107 conidia mL−1 was
71.21% in small nymphs, 69.31% in large nymphs and 53.36% in
pupae. The dispersion index (DI) and Lloyd’s Index of Patchiness
(LIP) values indicated that the infected immatures had a tendency
to aggregate. The study demonstrated that A. aleyrodis isolate
A005 is an effective biocontrol agent for B. tabaci control under
laboratory and greenhouse conditions.

Introduction
The sweet potato whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), is a
major pest of cash crops in different tropical and subtropical regions of the world
(Oliveira, Henneberry, & Anderson, 2001). Since the 1980s, the B. tabaci Middle East-
Asia Minor 1 (MEAM1) cryptic species (previously known as ‘B biotype’) has drastically
increased worldwide; this increase is attributed to the development of Global Trade
(IUCN, 2004). B. tabaci damages its host plants directly through feeding on phloem sap
and produces honey dew, which decrease the rate of leaf photosynthesis (Huang, Ali,
Ren, & Wu, 2010). It also affects plants indirectly by acting as a vector of various plant
viruses (Alegbejo, 2000). B. tabaci is a serious pest in China and is widely distributed in

CONTACT Bao-Li Qiu baileyqiu@scau.edu.cn Key Laboratory of Bio-Pesticide Innovation and Application, Engin-
eering Technology Research Centre of Agricultural Pest Biocontrol Guangdong Province, College of Agriculture, South
China Agricultural University, Guangzhou 510642, People’s Republic of China
*
Shaukat Ali is the joint first author.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
 BIOCONTROL SCIENCE AND TECHNOLOGY 211

29 provinces (Ahmed et al., 2009; Shen et al., 2010). The management of heavy whitefly
infestations is very difficult, and pesticides are mainly used to suppress whitefly popu-
lations (Liang, Tian, Biondi, Desneux, & Gao, 2012). However, injudicious use and
overuse of synthetic chemicals have resulted in the development of pest resistance to pes-
ticides (He et al., 2013). Apart from this, the harmful effects of these chemicals on non-
target organisms and environmental awareness by the public have promoted the develop-
ment of alternatives to insecticides for B. tabaci control, such as biological control (Huang
et al., 2010).
Organisms belonging to different taxa (bacteria, viruses, fungi, nematodes and proto-
zoa) are being used for microbial control of different pests and, among them, more
than 700 species of fungi are known to be pathogenic against different insect species
(Ali, Ren, & Huang, 2014). Entomopathogenic fungi belonging to genus Aschersonia
have been identified for the control of scale insects and whiteflies (Meekes, Fransen, &
van Lenteren, 2002; Meekes, van Voorst, Joosten, Fransen, & van Lenteren, 2000; Qiu,
Song, Mao, Tu, & Guan, 2013).
Aschersonia aleyrodis is a promising fungal species that has shown considerable viru-
lence against different species of whiteflies (Meekes et al., 2000). A. aleyrodis is considered
a prospective fungus for whitefly management because of its wide tolerance to relative
humidity, long persistence on leaf surfaces, compatibility with other natural enemies
and their ability of infect plant-sucking insects by penetrating the insects following
direct or secondary contact (Fransen & Van Lenteren, 1994; Qiu et al., 2013). The
species that attack whitefly mainly infect the nymphal stage (Fransen, 1995). The speci-
ficity of this fungus to whitefly has attracted considerable attention for its possibility to
act as a potential microbial agent against B. tabaci. This indicates that more research is
required on this pathogen.
In this study, we evaluated the pathogenicity of one isolate of A. aleyrodis as a potential
microbial control agent against MEAM1 B. tabaci. First, the potential isolate of
A. aleyrodis was used as a pathogen to infect the developmental stages and determine
the lethal and sub-lethal responses of MEAM1 B. tabaci; this step identified which devel-
opmental stages were susceptible to the pathogen under laboratory conditions. After that,
post-released measures were carried out in the greenhouse at timely intervals to determine
the distribution pattern of fungal spores on whitefly immatures as a way to determine the
transmission patterns of fungal diseases. It is hoped that this research will offer in-depth
knowledge on the exploitation of this fungus as a microbial control agent against MEAM1
B. tabaci.

Materials and methods

Source and cultivation of the fungal isolate
This research was carried out at the Key laboratory of Biopesticides Innovation and
Application, Engineering Technology Research Centre of Agricultural Pest Biological
Control, South China Agricultural University, Guangzhou. The strain Aa005 of
A. aleyrodis was derived from naturally occurring epizootic populations of citrus whitefly
Dialeurodes citri (Ashmead) from mandarins (Citrus reticulata Blanco), which were
collected from Guangdong Province, China. The inoculum for the experiment was
212 C. ZHANG ET AL.

produced following the method of Ali, Wu, Huang, and Ren (2010). A conidial suspension
(1 × 108 conidia mL−1) was used as a stock solution for the different studies, and lower
concentrations were prepared by serial dilution.

Insects and host plants

The stock of MEAM1 whiteflies were collected in Guangzhou from cotton plants and
reared at the Engineering Research Centre of Biological Control, Ministry of Education,
South China Agricultural University. The MEAM1 B. tabaci were identified by using
mitochondrial COI (mtCOI) sequencing described by Ahmed et al. (2010) and were main-
tained on eggplants in a greenhouse. The plants were cultured in 18.0 cm pots and kept in
cages to avoid any premature infestation from whiteflies or other insects until use. The
rearing conditions were set at 26 ± 1°C, 70 ± 10% R.H., and a photoperiod of 12 h of
light: 12 h of darkness.

Bioassays on pathogenicity in the laboratory

Effects of A. aleyrodis spores on whitefly probing and reproduction
The effects of A. aleyrodis spores on probing and reproduction were investigated in a
choice and no-choice bioassay by placing two differently or equally treated eggplants
(four leaves per plant) in bioassay cages. In the choice bioassay, one half of the leaves
were treated with A. aleyrodis Aa005 spores at a concentration of 1 × 107 conidia mL−1,
and the other half was sprayed with surfactant 0.05% Tween 80 as a control. The leaves
in the no-choice assay had the same treatment with 0.05% Tween 80. Thirty newly
emerged B. tabaci adults were then released into each cage for 48 h and maintained at
standard experimental conditions for probing and egg production. The experiment was
conducted twice with eight replications.

Effects of A. aleyrodis on egg hatchability and larval development

The effects of A. aleyrodis spores on egg hatchability and larval development were deter-
mined on detached leaves in Petri dishes (9.0 cm diameter). For homogenous egg pro-
duction, a micro-cage (3.5 cm diameter, 3 cm high) was attached to the lower surface of
the leaves of eggplant plants and approximately 3–5 B. tabaci adults were released with
an aspirator into the micro-cage for egg production. A cohort of 50 eggs on the leaves
of the host plant were demarcated with indelible ink and sprayed to run-off with the fol-
lowing five different concentrations of A. aleyrodis isolate Aa005: 1 × 104, 1 × 105, 1 × 106,
1 × 107, and 1 × 108 conidia mL−1 and a control treated with 0.05% Tween 80. From pre-
vious knowledge on egg development on this host plant, the leaves were detached 2 days
prior to hatching and placed in 9.0 cm Petri dishes with moistened filter paper laid at the
bottom of the Petri dish. A small amount of distilled water was added daily to prevent
desiccation and the dishes were incubated under the standard experimental conditions
of 25 ± 1 °C and 80 ± 10% R.H. The newly hatched nymphs were recorded and their pos-
itions marked with indelible ink after settling on the leaves. Their development and sur-
vival were monitored for 4 days, which was based on a preliminary test on the
developmental period using this host.
 BIOCONTROL SCIENCE AND TECHNOLOGY 213

Concentration–response relationship
This trial was conducted on the leaves of ‘eggplant’ Solanum melongena L. Homogenous
cohorts for the second, third and pupal stages were reared in a similar fashion as men-
tioned in the previous trial. For easy monitoring of the individuals in a cohort (n = 30),
five immatures per leaf were finely demarcated with indelible ink on one eggplant leaf.
Each stage was subjected to varying concentrations of A. aleyrodis isolate Aa005 spores,
namely, 1 × 104, 1 × 105, 1 × 106, 1 × 107, and 1 × 108 conidia mL−1 and a control treated
with 0.05% Tween 80. The leaves bearing the cohort for each stage were sprayed for
30 s to run-off using an atomiser sprayer. Thereafter, the inoculated leaves with immatures
were incubated at 26 ± 1°C for 5 days. The mortality after 5 days was noted, and the exper-
iment was replicated 6 times. The LC50 (the concentration required to kill 50% of the
nymphs tested) was calculated. Moreover, the isolate Aa005 was tested against three
instars of B. tabaci (N = 30) at the same concentration of 1 × 107 conidia mL−1 on eggplant
plants as mentioned above to determine the Lethal Time LT50 (the time required to kill
50% of the nymphs tested). The experimental set-up and protocol were the same as
above, and the percentage of mortality was recorded daily for 8 days. The diagnostic fea-
tures for fungal infections can be easily identified by its orange colour and fluffy appear-
ance with hyphal outgrowth, and natural death can be identified from a light black
appearance with a flat surface. Live nymphs can be easily distinguished by their opaque-
ness and are greenish-white and shiny depending on their developmental stages.

Evaluation of the infestation incidence in the greenhouse

The infestation incidence and the manner in which the pathogen spread among B. tabaci
immatures with time and in space in a greenhouse situation were explored. The infected
immatures were sampled weekly on eggplant leaves to determine the spread of fungal
spores (infestation incidence) on the immatures reared in a greenhouse at South China
Agricultural University. The Lloyd’s index of patchiness (LIP) was used to quantify the
distribution pattern of spores on the nymphs.

Crop maintenance and inoculation

Eggplant seeds were nursed in a square metal tray for 2 weeks and then established in
potted containers and placed in bioassay cages (60 cm × 60 cm × 60 cm) to exclude
foreign infestations from other insects. Prior to being transferred, the greenhouse was
thoroughly disinfected to exclude any foreign insects that might have harboured in the
greenhouse (the greenhouse was cleared out and kept empty for 10 days before transfer).
The potted seedlings were placed in two rows at a distance of 30 cm from each other. The
seedlings were allowed to grow for another 1 week after being transferred to the green-
house and were then sprayed with A. aleyrodis isolate Aa005 containing 1 × 107 conidia
mL−1 to run-off.

Whitefly infestation and sampling

Approximately 1000 B. tabaci adults collected from the stock colony were released into the
greenhouse containing the inoculated plants for 72 h for egg production. Thereafter, the
plants were transferred to another greenhouse for subsequent development of immatures
and population build-up for 2 weeks. The eggplant leaves were sampled at weekly intervals
214 C. ZHANG ET AL.

for six consecutive weeks to assess the proportion of immatures stages infested with
A. aleyrodis spores after being sprayed with Aa005.
On each sampling date, 10 leaves (one leaf per plant) were randomly excised, bagged
and transported to the laboratory for processing. For easy sampling, two perpendicular
lines were drawn to divide the leaf into four quadrants. A square cardboard with an
area of 1.0 cm2 was cut out and placed over the leaf surface bearing the immatures.
Both infected and uninfected immature stages (small larva, large larva and pupa) were
counted under a microscope at magnification (×20) within the demarcation of the card-
board. In this way, all immatures on the leaf were sampled. Infection was identified by the
presence of Aschersonia spores, where the width of germinating tubes corresponded to the
length of the conidia.

Dispersion models
The dispersion statistics, the parameter k of the negative binomial distribution, and LIP
were computed for each sample date. Lloyd’s mean crowding can be computed from
the following expression:
X ∗ = m + (S2 /m) − 1,
X∗
LIP = ,
m
where m is the mean of infected immatures sampled and S2 is the variance.

Data collection and analysis

For the collected data, the mean values were computed and compared using a least signifi-
cant difference (LSD) test at P = .05 in SAS version 9.1 (SAS Institute, 2004). A T-test and a
one-way analysis of variance (ANOVA) test were also applied to evaluate any significant
differences for each treatment. For the greenhouse evaluation, the mean values were sub-
jected to an ANOVA test to determine the effect of sample week (time) on infection of the
immatures.

Results
Pathogenicity of A. aleyrodis in the laboratory
Effects of A. aleyrodis on B. tabaci adults on probing behaviour and reproduction
Whitefly adults are mobile and usually found on the undersurface of leaves. Unlike the
immature stages, they may tend to move away when they come in contact with a plant
protection treatment. The effects of the antagonist on whitefly probing and reproductive
behaviour were determined in a choice and no-choice experiment, and the results are pre-
sented in Table 1. In the choice experiment, one half of the leaves were treated with
A. aleyrodis spores, while the other half of the leaves were treated with 0.05% Tween 80
used as the control. Leaves were confined in a bioassay cage. The prophylactic treatment
with the fungus had no determined effect on the number of whitefly adults probing the
leaves nor on the number of eggs produced (t = −0.287; P = .787). Similarly, in the no-
choice experiment with no prophylactic treatment of the leaves, no significant differences
 BIOCONTROL SCIENCE AND TECHNOLOGY 215

Table 1. Effects of A. aleyrodis spores on B. tabaci probing location and reproduction on eggplant.
Location of probing and reproduction
Parameters Choice No-choice
Adults (M ± SE) 12.80 ± 0.72a 13.30 ± 0.80a 11.30 ± 0.52a 10.80 ± 0.46a
Eggs (M ± SE) 85.30 ± 7.61a 89.10 ± 8.71a 90.20 ± 8.61a 88.00 ± 9.37a
Note: T-test shows no significant difference in probing and reproductive behaviour of B. tabaci at a 5% level of significance
on choice and no-choice bioassays; (N = 30) adults.

were observed in the number of adults probing the leaves nor the number of eggs pro-
duced (t = −0.149; P = .884). A comparison of the results between the choice and no-
choice experiments showed no significant results (t = −0.153; P = .783).

Effects of A. aleyrodis on egg hatchability and larval development

The efficacy of A. aleyrodis against B. tabaci eggs and their hatching was observed in
detached leaves assays. The per cent hatchability for examined B. tabaci eggs was all
above 70%, though the control test showed the highest value (Table 2). No significant
differences were observed in the mean values of eggs hatched. There was no trend in
hatchability with A. aleyrodis isolate Aa005 concentrations, indicating that the number
of eggs hatched is independent of the concentration. These values clearly demonstrated
that there was no antagonistic effect with the use of A. aleyrodis as a pathogen to
control B. tabaci eggs. The per cent mortality, as a result of A. aleyrodis spores, was
high among the hatched larva, indicating pathogenic effects on the B. tabaci crawlers.
Mortality increased with increasing concentration with the highest mortality being at
76% for 1 × 108 conidia mL−1 (Table 2). In contrast, the control resulted indicated the
lowest mortality value (2.20%), further indicating that the mortality of crawlers is due
to A. aleyrodis spores.
Three stages of B. tabaci, namely, second and third instar and the immature pupa
stages, were evaluated against different concentrations of A. aleyrodis isolate Aa005 to
determine a concentration–mortality response. The results of the trial are displayed in
Table 3. The results indicated an increase in the mortality of the immatures as the concen-
tration was increased, and the highest mortality was recorded for the second instar stage
(69.38%). At the highest concentration (1 × 108 conidia mL−1), the cumulative mortality
values were 69.38%, 51.83% and 32.31 for the second and third instar and pupa stages,
respectively. For the lowest spore concentrations (1 × 104 conidia mL−1), the cumulative
mortality recorded after 8 days of treatments were 15.54%, 9.21% and 0.68% for second
and third instar and pupa stages, respectively. The second instars were highly pathogenic,

Table 2. Effects of A. aleyrodis spores on the hatchability of B. tabaci and larval development.
Treatment (conidia mL−1) Hatched eggs (M ± SE) Hatchability (%) Larval mortality (%)
1 × 104 40.15 ± 1.49a 80.33 ± 0.87a 20.60 ± 3.35a
1 × 105 35.00 ± 1.99a 71.33 ± 0.48 a 36.40 ± 5.24b
1 × 106 39.83 ± 0.70a 79.66 ± 0.34 a 59.00 ± 2.30c
1 × 107 38.83 ± 1.99a 77.66 ± 0.33 a 62.40 ± 4.41cd
1 × 108 31.83 ± 1.66a 75.66 ± 0.45 a 76.00 ± 2.77d
Ck (Tween 80) 41.63 ± 0.84a 82.10 ± 0.88 a 2.20 ± 0.08e
F; d.f; P 23.65; 5; .061 19.71; 5; .064 27.89; 5; <.01
Notes: Means in the same columns with the same letters are not significantly different from each other (Tukey’s P < .05).
A cohort of 50 eggs was treated per concentration.
216 C. ZHANG ET AL.

Table 3. Cumulative mean mortality of B. tabaci instars reared on eggplant plants 5 days after the
treatment with A. aleyrodis isolate Aa005.
B. tabaci instars
Spore concentration (conidia mL−1) Second Third Pupa
4
1 × 10 15.54 ± 1.33a 9.21 ± 1.31a 0.68 ± 0.01a
1 × 105 34.55 ± 2.49b 19.37 ± 2.84b 4.21 ± 0.09a
1 × 106 42.17 ± 2.81c 29.88 ± 2.96c 14.39 ± 1.26b
1 × 107 57.21 ± 3.09d 43.29 ± 3.31d 25.40 ± 2.28b
1 × 108 69.38 ± 4.28e 51.83 ± 4.89e 32.31 ± 3.48c
F; d.f; P 32.65;4; <.001 26.93;4;<.001 18.54;4; <.001
Notes: Means in the same columns with the different letters are significantly different from each other (Tukey’s P < .05).

while the pupae were less pathogenic. For the second and the third instars, significant
differences in mortality were observed at each concentration level.

Median lethal concentration (LC50) and median lethal time (LT50)

The median lethal concentration (LC50) and median lethal time (LT50) are important
parametric indices used for evaluating and comparing the effectiveness and toxicity of
microbial pathogens such as entomopathogenic fungi against target insects. The results
showed that A. aleyrodis in this study was pathogenic to the immatures of B. tabaci,
and higher concentrations were required to reach 50% mortality in the pupae than in
the second and third instars (Table 4). The isolate Aa005 showed high pathogenicity on
second and third instars and pupae with LC50 values of 7.93 × 106, 1.08 × 107, and
1.56 × 107 conidia mL−1, respectively. The median lethal time (LT50) was lower for
second instars (4.60 days) and higher for pupae (6.17 days) (Table 5) when inoculated
with a concentration of 1 × 107 conidia mL−1.

Evaluation of the infestation incidence of A. aleyrodis on B. tabaci in the

greenhouse
Mean per cent infection of B. tabaci immatures
The results from weekly samples of B. tabaci immatures and the per cent infected with
A. aleyrodis pathogens are presented in Table 6. The population of B. tabaci immatures
increased with an increase in time, indicating a timely build-up of the population.
There is a corresponding increase in infected immatures with the pathogen for the
small and large larva across the weeks. The weekly samples of total immatures across
the weeks were 718, 849, 927, 1295, 1730 and 2053, respectively. These values indicated
a build-up of the pest population as the weeks progressed. When the mean per cent of
infected immatures was regressed on the total number of immatures sampled, a positive
correlation was observed between the number of immatures per week and number of

Table 4. Median lethal concentration (LC50) values required for the mortality of B. tabaci immatures
treated with A. aleyrodis (Aa005).
Instars Mortality function LC50 conidia mL−1) r2
6
Second instars Y = 0.04 + 0.63x 7.93 × 10 0.966
Third instars Y = 0.04 + 0.36x 1.08 × 107 0.964
Pupa Y = 0.05 + 0.32x 1.56 × 107 0.932
Notes: Lethal concentration of A. aleyrodis isolates required for 50% mortality of B. tabaci immatures. Each batch (N = 30) of
immatures was assayed with 1 × 106 inoculum of A. aleyrodis.
 BIOCONTROL SCIENCE AND TECHNOLOGY 217

Table 5. Median lethal time (LT50) values required for the mortality of B. tabaci immatures treated with
A. aleyrodis (Aa005).
Instars Mortality function LT50 (days) r2
Second instars Y = 0.07 + 6.5x 4.60 0.984
Third instars Y = 0.02 + 6.3x 4.93 0.925
Pupa Y = 0.10 + 4.2x 6.17 0.966
Notes: Median time required for the mortality of B. tabaci immatures treated with A. aleyrodis isolates. Each batch (N = 30)
of immatures was assayed with a concentration of 1 × 107 conidia mL−1.

infections for small larva and large larva. On the contrary, there was no corresponding
increase in the pupal stage. These results indicated that the infection of immatures is
dependent on the build-up of the peak population in the greenhouse.
From Table 6, the values indicated a trend of immature infestation with A. aleyrodis
spores in the order of small larva > large larva > pupa. The highest and lowest mean
values of infected immatures (71.21 and 7.35) were recorded on small larva and pupa,
respectively. An analysis of variance test at P = .05 was performed to determine any
effects of weekly sampling on the infection of A. aleyrodis on B. tabaci immatures and
a significant difference in the mean values of infection in small instars (F = 17.32; df =
5, P < .001) was found. Similarly, the mean values of large instars infected with the
fungal spores demonstrated a significant difference across sampling weeks (F = 12.48;
df = 5, P = .004) at P = .05. In contrast, the pupae showed no significant difference in
the mean values of infection (F = 2.45; df = 5, P = .069). An ANOVA test for the effects
of the insect stage on fungal infection demonstrated a significant difference at the .05
level (F = 14.32; df = 17, P = .002). These results noted that the fungal infection tended
to be influenced by both time and insect stage.

Distribution pattern of fungal spores on B. tabaci immatures

The dispersion index (DI), which describes the degree of aggregation of infected imma-
tures, was computed for each week, and the values were between 8.26 and 0.25 (Table
7). A DI value > 1 showed a tendency of aggregation, and a DI value < 1 showed random-
ness or uniform distribution. No infection of the pupae was observed in the first week of
sampling. In both the small and large instars, the DI for the first and second weeks were
higher than the subsequent weeks, revealing an initial infestation of fungal spores on the
immatures that tended to aggregate, serving as source of inoculums for subsequent
infestation.
Lloyd’s mean crowding index (X*) and LIP were computed to define the measures of
aggregation of the infected immatures of B. tabaci. A general trend of increase across

Table 6. Mean per cent infection of B. tabaci immatures with the A. aleyrodis pathogen.
Mean per cent infection (M ± SE)
Sample dates (week) Density of immatures (N ) Small larva Large larva Pupa
First 718 18.25 ± 1.05e 16.32 ± 1.95e 0
Second 849 32.95 ± 2.38d 29.50 ± 2.33d 7.35 ± 0.46d
Third 927 45.28 ± 3.96c 41.27 ± 2.87c 15.35 ± 1.23c
Fourth 1295 57.40 ± 4.22bc 52.31 ± 3.03b 32.27 ± 2.15b
Fifthh 1730 62.22 ± 4.54b 65.37 ± 4.85a 43.31 ± 3.62ab
Sixth 2053 71.21 ± 6.78a 69.31 ± 5.20a 53.36 ± 4.19a
Notes: Weekly samples of the population of B. tabaci immatures and the per cent infected with the A. aleyrodis pathogen.
N = the total number of immatures sampled weekly. The means were compared by LSD at a 5% level of significance.
218 C. ZHANG ET AL.

Table 7. B. tabaci immatures infected with A. aleyrodis spores.

Insects stage Sampling dates (week) DI LIP
Small larva First 6.50 1.30
Second 4.09 1.09
Third 3.91 1.06
Fourth 4.08 1.05
Fifth 3.27 1.04
Sixth 3.44 1.03
Large larva First 8.26 1.44
Second 6.14 1.17
Third 1.63 1.02
Fourth 3.38 1.05
Fifth 3.97 1.05
Sixth 3.68 1.04
Pupa First – –
Second 4.97 1.54
Third 0.95 1.00
Fourthh 1.13 1.00
Fifth 0.35 0.98
Sixth 0.25 0.99
Notes: Descriptive DIs of the infestation patterns of B. tabaci immatures infected with A. aleyrodis in a greenhouse exper-
iment at weekly sampling dates. Immatures were tested in three groups: small larva (first and second instars, average
body length: 317 μm), large larva (third and early fourth instars, average body length: 636 μm) and pupa (average
body length: 758 μm). DI = dispersion index is computed as S 2/m. LIP = X*/m

the weeks was observed in all three immature stages. The computed range of LIP in the
current study ranged from 0.98 to 1.54, respectively (Table 7). These values indicated
that the infected immatures tended to aggregate.

Discussion
An effective biological control agent should demonstrate the potency to infect all stages of
the target insects of all categories, namely, the feeding and non-feeding stages, as well as the
mobile and immobile stages. In the case of B. tabaci, adults are the only mobile stage,
although the newly hatched nymphs, referred to as crawlers, can also exhibit some move-
ment, which is only a few millimetres prior to settling on the leaves. Therefore, adult white-
flies have a tendency to escape crop protection treatments. The effect of A. aleyrodis spores
on B. tabaci probing behaviour and reproduction, which was investigated in a choice and
no-choice experiment, indicated no deterrent effect on B. tabaci probing and reproduction
behaviour. This was evident from the results obtained in this investigation, where there
was no significant difference in the choice experiment based on a T-test. A study
carried out by Wraight et al. (2000) on greenhouse whitefly T. vaporariorum to evaluate
the effectiveness of A. aleyrodis indicated less than 2% mycosis on the adults. Similarly,
Meekes et al. (2002) used isolates of A. aleyrodis against whiteflies and observed low
mycosis for eggs and adults. These instances indicate a lower susceptibility of whitefly
adults to the pathogen. The findings of the investigation conducted by Lacey, Kirk,
Millar, Mercadier, and Vidal (1999) also indicated that B. tabaci adults are unresponsive
to fungal spores. This could apparently be a desirable strategy where the adults, as the
only active stage with contact to spores, pick up fungal spores and transmit them to
other locations and to other target pests, hence facilitating large scale epizootic spread.
Insect developmental stage has been considered as one of the most important factors
that influences the pathogenicity of fungal pathogens (Qiu et al., 2013). In this study,
 BIOCONTROL SCIENCE AND TECHNOLOGY 219

when B. tabaci eggs were treated with A. aleyrodis spores prior to hatching, the results
show a high percentage of hatchability (>75% of eggs hatched into larva). The results
revealed that the eggs were less infected with the pathogen even at higher concentrations.
No significant differences were observed between the percentage of eggs hatched in the
fungal treatment and the control; however, it was observed that the nymphs hatching
from the eggs reared on the treated leaves were highly susceptible to the pathogen. This
was revealed in the low percentage of survivors, which varied significantly across the con-
centration treatments.
In the incubation trial, a similar trend was observed where the per cent eggs hatched
was significantly higher, but there was significantly low survivorship for the first
instars, indicating a high susceptibility of the first nymphs to the fungal spores.
A. aleyrodis were observed to be pathogenic to the first nymphs but less pathogenic to
eggs in both trials. In our study, the smaller immatures experienced high mortality com-
pared to the older instars, which is consistent with previous research (Fransen, Winkel-
man, & van Lenteren, 1987; Qiu et al., 2013). The older instars of greenhouse whitefly
were less susceptible to A. aleyrodis, and the adults were seldom infected.
The parametric indices median lethal concentration (LC50) and median lethal time
(LT50) are extensively used as measuring indices to determine the effectiveness of microbes
such as fungal pathogens. These indices are quite useful for comparing the effectiveness of
fungal isolates, as well as for determining the most vulnerable stage of the insect. The con-
centration–response relationship of B. tabaci immatures to the pathogens proportionately
measured insect mortality, where mortality increases proportionately with an increase in
concentration. The findings in this investigation indicated that susceptibility of the insect
stages to the pathogen is in the order of second instar > third instar > pupa. The LT50
values correspond to exposure time of the insects to the pathogen, and this was dependent
on the concentration. The LT50 for the instars were in the order of second instar < third
instar < pupa. This implies that when the pathogen at the same concentration is applied
to these insect stages, it takes less time to achieve high mortality at the second instar
stage than the third instar or pupa stages. Many researchers have employed median
values to predict effectiveness or pathogenicity against insect pests. A similar study
carried out by Bajwa, Abood, and Ibrahim (2016), using isolates of Beauveria bassiana
on Atteva sciodoxa, showed that the LC50 was age related, indicating that the LC50 for
younger nymphs are less than older nymphs.
The possible success of any biocontrol agent in the field can be judged relative to its
performance under greenhouse conditions (Faria & Wraight, 2001). Although green-
houses are subjected to human intervention, many factors such as the presence of stabil-
ized environmental conditions, regular sanitation and the possibility of avoiding pest
invasion can lead to the success of microbial pathogens in the greenhouse (Dowell & Stein-
berg, 1990). In this work, the bioassay of A. aleyrodis at the conidial concentration (1 ×
107 conidia mL−1) against the immatures of B. tabaci under greenhouse conditions had
a per cent mortality of 71.21% on young nymphs, 69.31% on old nymphs and 53.36%
on pupae. Our results are similar to Malekan, Hatami, Ebadi, Akhavan, and Radjabi
(2015), who observed that the percentages of T. vaporariorum mortality caused by
B. bassiana and L. muscarium were 63.73% and 62.49% on young nymphs and 71.68%
and 87.13% on old nymphs, respectively.
220 C. ZHANG ET AL.

The infestation incidence of the fungal pathogen can be used as a measure to determine
the rate of epizootics in the insect population after an inoculated release of the fungal
pathogen. It gives a measure of the extent to which the fungal pathogen is spreading
into the population, and this is an indicative parameter for monitored pathogens in bio-
logical control programmes (Hajek & St. Leger, 1994). The aggregation pattern displayed
in the transmission of the fungal infection in the greenhouse can be explained partly as a
result of the fact that whitefly smaller immatures were reduced immediately following the
pathogen application. The densities of small immatures infected with the pathogen were
initially higher, creating a source of inoculation for subsequent infestations. Similarly, the
infection of large nymphs built as time progressed. Consequently, only a few healthy small
immatures passed on to the large nymph stage. Thus, with time and in space, more eggs
entered into the small nymphal stage, but only a few small nymphs entered into the large
nymph stage, eventually ending up with a high density of infected smaller nymphs
(Gridin, Geschtoyt, Raccah, & Barash, 2000).
This study has demonstrated that A. aleyrodis isolate A005 is an effective biocontrol
agent for B. tabaci control under laboratory and greenhouse conditions. Further research
on the mass production and formulation of this isolate is required for its use as a bio-pes-
ticide in integrated pest management programmes of B. tabaci.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research was funded by grants from the Science and Technology Programme of Guangzhou,
P.R. China (201509010023, 201604020180).

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