Comparative pathogenicity of four
entomopathogenic fungal species against
nymphs and adults of citrus red mite on the
citrus plantation
Muhammad Qasim, Jiang Ronliang,
Waqar Islam, Habib Ali, Khalid Ali
Khan, Chandra Kanta Dash, Zakia
A. Jamal & Liande Wang
International Journal of Tropical
Insect Science
e-ISSN 1742-7592
Int J Trop Insect Sci
DOI 10.1007/s42690-020-00263-z
1 23
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https://doi.org/10.1007/s42690-020-00263-z
ORIGINAL RESEARCH ARTICLE
Comparative pathogenicity of four entomopathogenic fungal species
against nymphs and adults of citrus red mite on the citrus plantation
Muhammad Qasim 1,2 & Jiang Ronliang 1 & Waqar Islam 3 & Habib Ali 4 & Khalid Ali Khan 5 & Chandra Kanta Dash 6 &
Zakia A. Jamal 7 & Liande Wang 1
Received: 27 March 2020 / Accepted: 14 September 2020
# African Association of Insect Scientists 2020
Abstract
Panonychus citri (citrus red mite) is a devastating pest of citrus orchards. The conventional chemical acaricides have been
strongly forbidden for the management of agricultural insect pests in China. Therefore, we evaluated the susceptibility of adult
and nymphs P. citri in laboratory against eight isolates of four fungal species, Akanthomyces lecanii, Metarhizium anisopliae,
Beauveria bassiana and Aschersonia aleyrodis. Each citrus seedling having 40 adults (2-d-old) and nymphs (on separate plants)
were sprayed with isolates at the concentration of 104 ~ 108 conidia mLˉ1 whereas controlled seedlings were sprayed with 0.02%
Tween-80. After 9 days of fungal exposure, the four fungal isolates caused more than 50% mortality of mites, such as; 85.6%,
87.9%, 64.6% and 79.7% by A. lecanii (V3450), B. bassiana (BFZ0409), M. anisopliae (MFZ0706) and A. aleyrodis
(AsG0910), respectively. The nymphal mites were less susceptible to applied fungi compared to adults. The LC50s of the tested
isolates were determined by the fitted time-concentration-mortality relationships, which declined over days after spray. LT50s
were decreased with a high concentration of isolates. After the 9-d inoculation, two isolates of B. bassiana (BFZ0409 and D1344)
and one isolate of A. lecanii (V3450) were highly effective at the minimal dose of LC50 of 104 conidia mLˉ1 and are promising
candidates to control mites, as compared to other tested fungal isolates.
Keywords Citrus seedlings . Time-concentration-mortality . Panonychus citri . Metarhizium anisopliae . Beauveria bassiana .
Aschersonia aleyrodis
Introduction
Mites belonging to Arachnida class (Ruppert et al. 2004) consist of four stages, i.e. egg, larva, nymph and adult. Nymphs
(protonymph, deutonymph, and tritonymph) and adult are
most feeding and damaging stages. There are more than
50,000 species of mites, which are predatory, parasitic, saprophagous, herbivores, necrophagous, fungivores, coprophagous as well as phoretic species (Dhooria 2016).
Mites that attack citrus plantations worldwide include
Panonychus citri McGregor, Eotetranychus kankitus Ehara,
Polyphagotarsonemus latus Banks, Tetranychus kanzawai
* Muhammad Qasim
cmqasimgill@zju.edu.cn
3
College of Geography, Fujian Normal University, Fuzhou 350007,
People’s Republic of China
* Liande Wang
wang_liande@126.com
4
Department of Agricultural Engineeirng, Civil Engineering Block,
Khwaja Fareed University of Engineering and Information
Technology (KFUEIT), Abu Dhabi Rd, Rahim Yar Khan, Punjab,
Pakistan
5
Research Center for Advanced Materials Science (RCAMS); Unit of
Bee Research and Honey Production; Biology Department, Faculty
of Science, King Khalid University, Abha 61413, Saudi Arabia
6
Faculty of Agriculture, Sylhet Agricultural University, Sylhet 3300,
Bangladesh
7
Biology Department, Faculty of Science Yanbu, Taibah University,
Al-Sharm, Yanbu, El-Bahr 46429, Saudi Arabia
1
2
State Key Laboratory of Ecological Pest Control for Fujian and
Taiwan Crops, Key Laboratory of Biopesticide and Chemical
Biology, MOE, Fujian Agriculture and Forestry University,
Fuzhou 350002, People’s Republic of China
Ministry of Agricultural and Rural Affairs Key Laboratory of
Molecular Biology of Crop Pathogens and Insects, Institute of Insect
Sciences, College of Agriculture & Biotechnology, Zhejiang
University, Hangzhou 310058, China
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Kishida, Phyllocoptruta oleivora Ashmead, as well as
Brevipalpus yothersi Baker (Al-Azzazy 2016; Vechia et al.
2018; Zhou et al. 1999). Among them, Panonychus citri (citrus red mite) (CRM) is more harmful to fresh citrus shoots
(Haiyuan 1996; Li 1990). Adult and nymph nurture and survive on the delicate plant leaves through sucking (Kranz et al.
1977), and develop lighter grey spots on leaves, that may
hinder the process of photosynthesis (Kennett et al. 1999).
High infestations cause early leaf dropping as well as shoot
dieback, and weaken the plant vigor, along with feeding and
damaging the fresh fruits (Jamieson et al. 2005; Kranz et al.
1977), and destroys the citrus plantation in China (Li et al.
1990; Yang et al. 2009).
A considerable number of acaricides have been used to
control mites in China. Therefore, mite pests―including
P. citri―become resistant to various insecticides due to
long-term usage (Gerson and Cohen 1989; Meng et al.
2000). It is easy for CRM to become resistant to insecticides,
and since the 1970s, citrus mites have become resistant to
organophosphates and organochlorines, e.g. dipterex, dimethoate and chlorodifon (Gerson and Cohen 1989). Likewise,
P. citri has developed resistance to pyridaben, abamectin
and dicofol up to 11.2, 13.4 and 23 fold, respectively, in
China (Meng et al. 2000). Therefore, conventional chemical
acaricides have been strongly forbidden for the management
of agricultural insect pests in China (Yu 2001). However,
many other alternative control measures are being considered
against mites in the world (Idrees et al. 2016). Predators and
entomopathogens are being used as biological agents to control mites on broad-spectrum (Chandler et al. 2000; Jamieson
et al. 2005; Paz et al. 2007; Poinar Jr and Poinar 1998; Shi and
Feng 2004; Van der Geest et al. 2000). Therefore, entomopathogenic fungi are being applied as biopesticides for the
management of mites (Chandler et al. 2000).
Entomopathogenic hyphomycetes infecting citrus mites
have been described, especially on citrus plantation such as
Beauveria bassiana, Akanthomyces (Lecanicillium) lecanii
(Kepler et al. 2017), Metarhizium anisopliae and Cordyceps
(Isaria) fumosorosea (Kepler et al. 2017) are well known microbial agents (Martins et al. 2016; Roberts and Leger 2004)
and are being applied in pests control (Qasim et al. 2018;
Wraight et al. 2000). These entomopathogens strongly respond against various mite pest across the globe in different
ecosystems (Chandler et al. 2000). Recently, it has been observed that entomopathogenic fungi infect the various stages
of spider mites (Alves et al. 2002; Shi et al. 2008a; Wekesa
et al. 2005, 2006) as well as ectoparasitic mites (Shaw et al.
2002). Some other fungal isolates had been exploited against
CRM, which caused significant mortality of mites. Meira
geulakonigii (Boekhout, Scorzetti, Gerson & Sztejnberg)
was applied on seedlings of sour orange at the rate of 2 ×
108 conidia mLˉ1, which caused 75% mortality of P. citri
within 1 week (Sztejnberg et al. 2004). While Paz et al.
(2007) claimed that M. geulakonigii caused 63% mortality
of P. citri with the dose of 1 × 108 conidia mLˉ1 within a week.
Furthermore, M. argovae (Boekhout, Scorzetti, Gerson &
Sztejnberg) and Acaromyces ingoldii (Boekhout, Scorzetti,
Gerson & Sztejnberg) resulted in the death of 59% and 58%
CRM population, respectively. Whereas, Puspitarini et al.
(2011) reported that more than 40% population of P. citri
collected from natural citrus plantation was infected with
Hirsutella sp. in Indonesia.
The present research was conducted to test the lethal response of P. citri to the four hypocrealean fungi with a complementary log-log (CLL) model, and this model was selected
to confirm an apparent trend of fungal toxicity under the binary effect of concentration and time. Moreover, the comparative effectiveness of all isolates was assessed at different
concentrations as well as fungal exposure.
Material and methods
Source of fungal isolates and conidia preparation
Eight isolates of four species were used in this study, i.e. A.
(L.) lecanii (Vl6063, V3450, Vp28 and V09); B. bassiana
(D1344, BFZ0409); M. anisopliae (MFZ0706) and
A. aleyrodis (AsG0910), and all of the isolates were procured
from different labs (Table 1). All fungal isolates were cultured
on the Petri plates of Sabouraud dextrose agar (SDA), under
the conditions of 25 ± 1 °C, 80 ± 5% R.H., and 14:10 Light:
Dark (L:D) photoperiod. Conidia of all isolates were collected
separately according to the method of Ye et al. (2005). Then
the suspension was prepared into universal flasks, having
3 mm glass beads, by using 10 mL deionized water as well
as 0.02% (v/v) Tween®-80 (Fluka). After that, suspensions
were homogenized by shaking tubes on vortex for 5 min,
and concentration was determined by using a Neubauer hemocytometer (model 1103) (Goettel and Inglis 1997).
However, conidial viability of all isolates was observed before
each bioassay, according to Wang et al. (2004). The conidial
viability of suspensions was consistently high in all bioassays
being greater than 98.3 ± 0.52%.
CRM rearing
Colonies of P. citri were established from individuals collected from citrus seedlings (Citrus sinesis Osbeck) in a greenhouse (25 ± 2 °C) of Fujian Agriculture and Forestry
University, Fuzhou, China and reared on citrus plants. After
that, bioassays were done against both stages of mite, nymph
and adult. For this purpose, 20 healthy adult females were
collected from the population, and kept separately on fresh
seedlings for 24 h, to get a homogeneous batch of eggs, and
at last uniform aged nymphs. The vigorous nymphs (24 h after
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Table 1 Geographical location
and original host of fungi in
present study
Species
Fungal isolate
Geographical location
Original host
Akanthomyces lecanii
A. lecanii
A. lecanii
A. lecanii
Beauveria bassiana
Vl6063
V3450
Vp28
V09
BFZ0409
Halifax, Canada
Guangzhou, China
Guangzhou, China
Hefei, China
Fuzhou, China
Trialeurodes vaporarioum
Bemisia tabaci
Pseudococcus sp.
Noplophora chinesis
Plutella xylostella
B. bassiana
Aschersonia aleyrodis
Metarhizium anisopliae
D1344
AsG0910
MFZ0706
DSMZ, Germany
Guangzhou, China
Fuzhou, China
Unknown
Dialeurodes citri
Blattella germanica
hatching from eggs of uniform age in the same growth chamber) were transferred onto detached citrus twigs with two
leaves on a sponge containing a rhizocaline (100 g mLˉ1) for
bioassays. To collect particular aged adults for further experiment, 50 quiescent deutonymphs (second stage of nymph of
mite) were taken from the seedlings and transferred onto detached citrus twigs with two leaves on a sponge containing a
rhizocaline of 100 g mLˉ1, under the conditions of 25 ± 1 °C,
12:12 L: D as well as 80 ± 5% RH. After that, nymphs and
adult females were collected under a stereo-microscope
(Nikon SX-45-TR) and then transferred onto new twigs of
healthy citrus leaves, and these twigs were maintained the
glass pot (12 × 9 cm).
Bioassays
We assayed for the biocontrol potential of eight isolates from
four fungal species mentioned above against the nymphs and
adults of P. citri in a lamp-chimney-caged seedling bioassay
system. A 3.0 mL spore suspension (1.0 × 104 to 1.0 × 108
conidia mLˉ1) (of each fungal species) was sprayed into a
chamber having infested twigs with a gas sprayer (Preval
Sprayer, NY, USA, Vapor Pressure 4.018, Vapor Density
1.8) as fungal treatment whereas 3.0 mL 0.02% (v/v)
Tween®-80 was sprayed on controlled twigs. After that, the
top of lamp-chimney-cage was covered with a water-proof
mesh film up to initial 24 h to maintain relative humidity,
for the conidial germination. While inner wet filter paper lined
on the Petri dish was changed daily (surviving mites on the
filter paper dropping from the twigs were transferred onto the
leaves of the twigs again). All observations (counting of dead
and alive for nymph and adult) were done daily for 9 days
with the help of a 10-fold hand magnifier. Later on, these mite
cadavers were transferred into moist Petri dishes for fungal
growths up to 2–3 days and followed by the verification of
fungal infection, under the stereomicroscope (Nikon SX-45TR) at 50X magnifications. Then the particular individuals,
having fungal outgrowths, were counted as dead as a consequence of the tested isolates. All bioassays were repeated for
five times with 40 female adult or nymphal mites for every
treatment or blank control in each repeat.
Data analysis
The serial time-concentration-mortality was designed according to the description of Preisler and Robertson (1989) as well
as Robertson and Preisler (1992). Data were analyzed by
using the complementary log-log model (CLL model), and
cumulative mortality was estimated (Nowierski et al. 1996;
Wang et al. 2004). Controls were adjusted according to the
model, described by Christensen and Chen (1985), and
Robertson and Preisler (1992). Similarly, the particular
formulae of Robertson and Preisler (1992) were used for
the values of LC50 (or LC90) whereas LT50 values were
estimated by linear interpolation (Feng et al. 1998;
Nowierski et al. 1996). Mortality was adjusted according to the method of Nowierski et al. (1996).
The procedures, including modeling, estimation of time
and concentration-effect parameters for the CLL models, test
for goodness of fit, and estimation of virulence indices (LC50)
using the parameters were analyzed using DPS data processing system software (Feng et al. 1998; Tang and Feng 2007).
Results
CRM infected by fungal pathogens
Early mycosis-caused deaths of CRM (nymphs and adults)
began from 3-d inoculation, and the infected nymph and female adults became lethargic before death. The mycosed dead
bodies indicated subtle fungal out-growths on the confined
citrus leaves, perhaps, due to less humidity. However, all
nymphs and adults became well infected after being transferred into moist petri dishes, within 3 days. All fungal infected mites (adult and nymphs) produced proper mycelia and
conidia in the petri dishes, and, which mean all of eight strains,
were capable of infecting both stages of the mites.
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Mortality of CRM by fungal isolates
The cumulative mite mortalities by different concentrations of
all fungal isolates are presented in Figs. 1 and 2. The trends of
the observed mite mortalities were depended on both concentration and time. After 4 days of fungal application, there was
considerable mortality of both stages (nymph and adults).
After the ninth day of fungal application at the concentration
of 108 against nymphal mite population, maximum mortality
was 70.7% by BFZ0409 and D1344 (B. bassiana), whereas
least mortality was 46.5% by AsG0910 (A. aerlodis) (Fig. 2).
Similarly, after 9-d fungal exposure at the highest concentration against adult mites, BFZ0409 (B. bassiana) caused maximum mortality (87.9%), followed by V3450 (A. lecanii) with
85.6% mortality. The least mortality (51.9%) was presented by Vl6063 (A. lecanii). On the other hand, after
9-d of observation in the controlled treatment, the maximum mortality was 5.4% and 6.9% for nymphal and
adult mites, respectively (Fig. 1).
The results of time-concentration-mortality modeling of
nymph and adult female mites infected by eight isolates simulated by CLL model are shown in Figs. 1 and 2. The t-tests
Fig. 1 Relationship between percentage mortality, spores concentration, and time for the female mites of Panonychus citri. a Vl6063; b V3450; c Vp28;
d V09; e BFZ0409; f D1344; g AsG0910; h MFZ0706
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Fig. 1 (continued)
for all parameters estimated were significant (P < 0.01).
^ (a grouped Pearson’s χ2, i.e.
Hosmer–Lemeshow statistic C
modified Pearson’s χ2 by Nowierski et al. (1996) for the heterogeneity of the goodness of fit were non-significant for all
eight fungal isolates (P < 0.05, Table 2). The data of the eight
fungal isolates against the nymphs and females was fitted well
to the CLL model. The slope values (β), the parameters from
the maximum likelihood estimation in the CLL model indicated the rate of the proportion of CRM mortality as a function
of log (spores concentrations of each suspension). Significant
relationships between proportion mortality and log dose were
found in all eight fungal isolates considered (P < 0.01,
Table 2). The mortality and treatment had a strong relationship, as illustrated by the magnitude of the slope values (P =
0.05 by DMRT, Table 3). The fitted parameter β represented
the slope values of the fitted curve, with the range from 0.15 to
0.49 and 0.16 to 0.36 against nymphal and adult mites, respectively. Isolates with a larger magnitude of slope values caused
CRM mortality at a faster rate. Two isolate of A. lecanii (Vp28
(β = 0.49) and V3450 (β = 0.36)) were found to have the most
substantial magnitude of slope value among all strains, examined for the impact on nymphal and adult mites, respectively
(Table 2). The fitted parameters indicated that the concentration and time affected the efficiency of the tested isolates. The
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Fig. 2 Relationship between percentage mortality, spores concentration, and time for the nymphal mites of Panonychus citri. a Vl6063; b V3450; c
Vp28; d V09; e BFZ0409; f D1344; g AsG0910; h MFZ0706
estimated parameters of 4-d spaying (γ4) were the largest for
most tested isolates (Vl6063, Vp28, V09, BFZ009), indicating
the estimate of latent periods for these microbial agent tested
(Christensen and Chen 1985).
Based on the cumulative relationships of the fungal
isolates against the mites determined by the fitted β and
γj, the values of LC50s and associated confidence 95%
intervals were computed as a function of the post-spray
days (Tables 3 and 4). After 9 days of fungal exposure to
adult mites, two isolates of B. bassiana (BFZ0409 and
D1344) and one isolate of A. lecanii (V3450) showed
the highest virulence at the least LC50 value (104 conidia
mLˉ 1 ). On the other hand, two isolates of A. lecanii
(Vl6063 and V09) showed the least virulence at higher
LC50s of 106 conidia mLˉ 1, respectively. However, at
the same fungal and time exposure, the LC50s values were
higher for all isolates against nymphal mites. For example, least LC50 value was 3.25 × 105 conidia mLˉ1 for
BFZ0409 (B. bassiana), and higher LC 50 value was
6.59 × 108 conidia mLˉ1 for MFZ0706 (M. anisopliae).
The estimated LT50s were reduced with the increment of
concentrations of fungal concentration (Tables 3 and 4). For
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Fig. 2 (continued)
example, the LT50s were computable at the concentration of
105 conidia mLˉ1, only for BFZ0409. The two B. bassiana
isolates showed rapid mortality of 4.4d-5.0d at the high concentration of 108 conidia mLˉ1. For four tested A. lecanii isolates, the estimates ranged from 5.3d-6.5d, V09 exhibited the
slowest mortality with 6.5d at the concentration of 108 conidia
mLˉ 1 . The isolates of A. aleyrodis (AsG0910) and
M. anisopliae (MFZ0706) showed intermediate mortality.
The equivalent slopes for concentration effects, the virulence indices (LC50s) and LT50s indicated that two isolates of
B. bassiana (BFZ0409 and D1344) were the most virulent and
high efficient isolates against CRM, followed by one isolate of
A. lecanii (V3450). The others were the intermediate in virulence and efficacy.
Discussion
In the current study, we assessed the potential of eight isolates
from four fungal species against CRM on citrus seedlings. Our
results proved the effectiveness of all tested isolates against
CRM females, but LC50s and LT50s determined by the fitted
Table 2
Parameters estimated from the simulation of bioassay of eight isolates against Panonychus citi by the CLL model
Isolates/ adults
Parameters(Mean ± SE) a
Slope
^ test b
C
γ4
γ5
γ6
γ7
γ8
γ9
(β ± SE)
^
C
d.f
P value
Vl6063
V3450
Vp28
−4.18 ± 0.37
−4.86 ± 0.32
−3.76 ± 0.33
−2.31 ± 0.30
−4.54 ± 0.32
−2.27 ± 0.28
−2.69 ± 0.31
−4.23 ± 0.31
−3.13 ± 0.31
−3.61 ± 0.36
−4.02 ± 0.30
−2.88 ± 0.31
−3.88 ± 0.38
−3.76 ± 0.30
−3.36 ± 0.34
−4.06 ± .41
−4.26 ± 0.32
−3.47 ± 0.35
−7.75 ± 2.03
−3.54 ± 0.29
−4.72 ± 0.53
0.16 ± 0.05
0.36 ± 0.04
0.16 ± 0.04
0.234
1.641
1.292
8
8
8
0.999
0.990
0.996
V09
BFZ0409
D1344
AsG0910
MFZ0706
Isolates/ nymph
Vl6063
V3450
Vp28
V09
BFZ0409
D1344
AsG0910
MFZ0706
−3.99 ± 0.35
−4.18 ± 0.30
−4.64 ± 0.33
−4.40 ± 0.33
−4.38 ± 0.32
−2.66 ± 0.31
−3.28 ± 0.28
−3.45 ± 0.29
−4.28 ± 0.33
−4.63 ± 0.33
−3.43 ± 0.33
−4.50 ± 0.33
−3.24 ± 0.29
−3.08 ± 0.29
−3.72 ± 0.30
−4.02 ± 0.37
−3.63 ± 0.29
−4.09 ± 0.33
−3.48 ± 0.31
−3.55 ± 0.30
−3.69 ± 0.35
−3.94 ± 0.30
−3.52 ± 0.30
−3.42 ± 0.31
−3.45 ± 0.30
−3.97 ± 0.38
−3.58 ± 0.29
−3.64 ± 0.31
−3.81 ± 0.33
−3.75 ± 0.32
−3.63 ± 0.35
−3.04 ± 0.27
−3.22 ± 0.30
−4.52 ± 0.40
−4.53 ± 0.38
0.19 ± 0.05
0.31 ± 0.04
0.27 ± 0.04
0.27 ± 0.04
0.31 ± 0.04
2.185
4.438
3.957
2.498
0.720
8
8
8
8
8
0.975
0.816
0.861
0.962
0.999
−5.91 ± 0.48
−5.03 ± 0.39
−5.81 ± 0.48
−3.57 ± 0.36
−4.25 ± 0.33
−5.42 ± 0.43
−4.96 ± 0.41
−4.94 ± 0.46
−4.07 ± 0.41
−5.59 ± 0.43
−5.68 ± 0.47
−2.55 ± 0.33
−3.91 ± 0.33
−4.28 ± 0.38
−4.84 ± 0.41
−3.12 ± 0.38
−4.50 ± 0.42
−4.85 ± 0.39
−5.99 ± 0.49
−3.37 ± 0.35
−3.96 ± 0.33
−4.04 ± 0.38
−3.75 ± 0.37
−3.60 ± 0.39
−5.38 ± 0.47
−4.59 ± 0.38
−6.86 ± 0.55
−4.09 ± 0.40
−3.76 ± 0.32
−5.23 ± 0.44
−4.24 ± 0.39
−4.34 ± 0.43
−5.87 ± 0.51
−4.85 ± 0.39
−6.44 ± 0.53
−3.49 ± 0.37
−4.01 ± 0.34
−4.59 ± 0.40
−4.26 ± 0.39
−5.20 ± 0.52
−6.49 ± 0.61
−5.68 ± 0.46
−7.03 ± 0.61
−4.53 ± 0.46
−3.57 ± 0.32
−4.66 ± 0.40
−4.75 ± 0.43
−5.35 ± 0.55
−6.89 ± 0.69
−4.46 ± 0.38
−5.31 ± 0.47
−4.30 ± 0.46
−3.16 ± 0.30
−5.71 ± 0.51
−5.60 ± 0.52
−5.83 ± 0.64
0.37 ± 0.06
0.38 ± 0.05
0.49 ± 0.07
0.15 ± 0.05
0.26 ± 0.04
0.33 ± 0.05
0.29 ± 0.05
0.21 ± 0.06
2.488
6.337
6.740
2.877
12.680
0.256
2.979
0.427
8
8
8
8
8
8
8
8
0.962
0.61
0.565
0.942
0.123
0.999
0.936
0.999
a
b
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γ3
A subscript associated with γ denotes the specific day after spray. All t-test were significant at P < 0.01 in each modeling for estimated parameters
^ test) χ2 <χ2 0.05 = 15.51 with d.f. =8 in each CLL modeling regression
Goodness-of-fit statistic (C
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Table 3
Time-specific LC50s and concentration-specific LT50s of isolates against the female adult of Panonychus citri
Fungal isolates LC50 / conidia mLˉ1
(95% CI / conidia mLˉ1)
V3450
Vp28
V09
BFZ0409
D1344
AsG0910
MFZ0706
a
day 4
day 5
day 6
day 7
day 8
day 9
1 × 105 1 × 106 1 × 107 1 × 108
1.53 × 1011
(4.39 × 1010–5.30 × 1011)
1.30 × 1010
(3.92 × 109–4.33 × 1010)
2.51 × 1010
(1.56 × 109–4.03 × 1011)
6.74 × 1010
(4.42 × 109–1.03 × 1012)
1.77 × 108
(4.00 × 107–7.79 × 108)
3.00 × 1010
(2.89 × 109–3.11 × 1011
1.96 × 108
(1.06 × 108–3.61 × 108)
3.12 × 108
(1.44 × 108–6.76 × 108)
3.79 × 108
(6.53 × 107–2.22 × 109)
1.56 × 109
(2.41 × 108–1.02 × 1010)
4.34 × 107
(1.24 × 107–1.51 × 108)
1.03 × 108
(3.02 × 107–3.49 × 108)
2.70 × 107
(1.72 × 107–4.25 × 107)
1.94 × 107
(1.15 × 107–3.27 × 107)
6.94 × 106
(2.51 × 106–1.92 × 107)
2.89 × 108
(6.44 × 107–1.30 × 109)
3.32 × 106
(1.28 × 106–8.64 × 106)
2.02 × 107
(7.67 × 106–5.34 × 107)
7.00 × 106
(4.82 × 106–1.02 × 107)
1.71 × 106
(1.11 × 106–2.64 × 106)
9.35 × 105
(3.73 × 105–2.34 × 106)
3.82 × 107
(1.25 × 107–1.16 × 108)
7.87 × 105
(3.09 × 105–2.00 × 106)
2.09 × 106
(9.48 × 105–4.61 × 106)
2.50 × 106
(1.78 × 106–3.50 × 106
5.61 × 105
(3.58 × 105–8.79 × 105)
1.93 × 105
(6.77 × 104–5.48 × 105)
1.02 × 107
(4.01 × 106--2.54 × 107)
1.49 × 105
(5.21 × 104–4.28 × 105)
4.17 × 105
(1.81 × 105–9.60 × 105)
2.43 × 106
(1.74 × 106–3.41 × 106)
9.03 × 104
(5.28 × 104–1.54 × 105)
1.26 × 105
(4.16 × 104–3.83 × 105)
1.97 × 106
(8.94 × 105–4.33 × 106)
1.67 × 104
(4.33 × 103–6.46 × 104)
5.71 × 104
(2.02 × 104–1.61 × 105)
–
–
6.7
5.3
8.9
7.6
6.3
5.4
–
7.0
5.9
5.3
–
–
8.0
6.5
8.2
6.8
5.6
4.4
8.7
7.5
6.3
5.0
1.40 × 1012
(8.79 × 1010−2.24 × 1013)
1.14 × 1011
(2.96 × 1010–4.39×101)
2.46 × 108
(7.47 × 107–8.13 × 108)
4.90 × 108
(2.30 × 108-1.05 × 109)
1.18 × 107
(5.41 × 106–2.57 × 107)
1.40 × 107
(8.78 × 106–2.22 × 10
1.11 × 106
(5.68 × 105–2.18 × 106)
1.02 × 106
(6.85 × 105–1.52 × 106)
3.04 × 105
(1.46 × 105–6.35 × 105)
2.33 × 105
(1.49 × 105–3.64 × 105)
1.72 × 105
–
(7.85 × 104–3.77 × 105)
1.29 × 105
–
(8.02 × 104–2.09 × 105)
7.4
6.1
5.3
7.0
6.1
5.4
No computable LT50s for the given concentrations (conidia mLˉ1 ) if not given
Author's personal copy
Vl6063
LT50 (Days) a
Table 4
Time-specific LC50s and concentration-specific LT50s of isolates against the nymph of Panonychus citri
Fungal isolates LC50 / conidia mLˉ1
(95% CI / conidia mLˉ1)
day 4
day 5
5.17 × 109
9
V3450
Vp28
V09
BFZ0409
D1344
AsG0910
MFZ0706
a
day 6
3.12 × 108
10
(1.51 × 10 –1.77 × 10 )
9.34 × 1010
(9.77 × 109–8.93 × 1011)
3.68 × 109
(5.42 × 108–2.50 × 1010)
1.29 × 1012
(3.66 × 109–4.55 × 1014)
3.59 × 1011
(1.01 × 1010–1.28 × 1013)
1.55 × 1011
(6.56 × 1010–3.66 × 1011)
1.64 × 1013
(9.22 × 1011–2.92 × 1014)
1.45 × 1012
(4.90 × 1011–4.27 × 1012)
8
day 7
1.30 × 108
8
(1.36 × 10 –7.16 × 10 )
3.16 × 109
(6.58 × 108–1.52 × 1010)
7.77 × 108
(1.70 × 108–3.55 × 109)
1.97 × 1010
(2.99 × 108–1.29 × 1012)
7.24 × 109
(5.84 × 108–8.98 × 1010)
1.29 × 109
(7.72 × 108–2.16 × 109)
8.99 × 109
(1.88 × 109–4.31 × 1010)
1.47 × 1010
(7.05 × 109–3.05 × 1010)
day 8
8.02 × 107
8
(6.38 × 1007–2.65 × 10 )
2.18 × 108
(7.44 × 107–6.37 × 108)
4.60 × 108
(1.15 × 108–1.85 × 109)
3.70 × 109
(1.09 × 108–1.26 × 1011)
2.96 × 108
(5.25 × 107–1.67 × 109)
4.80 × 108
(3.07 × 108–7.51 × 108)
7.21 × 108
(2.29 × 108–2.27 × 109)
2.80 × 109
(1.52 × 109–5.15 × 109)
7
6.28 × 107
8
(4.18 × 10 –1.54 × 10 )
4.99 × 107
(2.14 × 107–1.16 × 108)
2.28 × 108
(6.79 × 107–7.66 × 108)
2.66 × 108
(2.13 × 107–3.32 × 109)
4.50 × 107
(1.19 × 107–1.69 × 108)
1.00 × 108
(7.07 × 107–1.42 × 108)
1.10 × 108
(4.67 × 1072.62 × 108)
1.49 × 109
(8.50 × 108–2.62 × 109)
7
8
(3.38 × 10 –1.17 × 10 )
2.91 × 107
(1.35 × 107–6.27 × 107)
1.61 × 108
(5.23 × 107–4.96 × 108)
1.16 × 108
(1.25 × 107–1.08 × 109)
4.51 × 106
(1.68 × 106–1.22 × 107)
3.03 × 107
(2.28 × 107–4.02 × 107)
4.21 × 107
(2.03 × 107–8.72 × 107)
8.96 × 108
(5.30 × 108–1.52 × 109)
day 9
1 × 105 1 × 106 1 × 107 1 × 108
5.35 × 107
–
–
–
6.5
–
–
8.7
6.5
–
–
–
8.3
–
–
–
8.2
–
8.6
7.4
6.6
–
–
–
7.0
–
–
–
7.1
–
–
–
–
(2.93 × 107–9.77 × 107)
6.23 × 106
(3.36 × 106–1.15 × 107)
3.33 × 107
(1.45 × 107–7.62 × 107)
4.38 × 107
(6.59 × 106–2.91 × 108)
3.25 × 105
(1.18 × 105–8.92 × 105)
2.08 × 107
(1.59 × 107–2.71 × 107)
2.88 × 107
(1.46 × 107–5.70 × 107)
6.59 × 108
(3.98 × 108–1.09 × 109)
Author's personal copy
Vl6063
LT50(Days a)
No computable LT50s for the given concentrations (conidia mLˉ1 ) if not given
Int J Trop Insect Sci
Author's personal copy
Int J Trop Insect Sci
cumulative relationships varied considerably. All isolates
were sufficiently virulent to both stages of CRM, although,
all isolates presented significantly different mortality ranges.
Adult females were more susceptible to all isolates as compared to nymphs. The reason may be some conidial spores
drop off with ecdyses as molting. Therefore, with the time,
nymphicidal activities of these isolates varied considerably
with the complicated situation for their variation of enzymes
and germination potency. All fungal entomopathogens could
be applied in the greenhouse and field conditions, through
various application methods. These entomopathogens could
be spread directly in the target area, as spore coatings, spore
bags, spore containing media or indirectly by spreading the
fungal-infected insect bodies (Farenhorst and Knols 2010;
Pilz et al. 2011; Stafford and Allan 2014).
Numerous entomopathogenic fungi, including B. bassiana,
have been assessed for the management of several mites, such
as Tetranychus urticae (Bugeme et al. 2014; Ullah and Lim
2015) and T. cinnabarinus (Erler et al. 2013). Tehri et al.
(2015) described that B. bassiana reduced more than 60% population of T. urticae on okra in the field conditions. Likewise, the
efficiency of B. bassiana was also evaluated against T. urticae at
the rate of 1 × 108 conidia mLˉ1 on bean and cucumber, which
caused mortality of 50% mite population in the greenhouse
(Seyed-Talebi et al. 2014). Moreover, the pathogenicity of
B. bassiana was explored against eggs and adults of T. urticae
on Okra by Krishna and Bhaskar (2013), which caused mortality
only 7 % of both stages. Moreover, B. bassiana was also presented 92% mortality against CRM adults in laboratory bioassays within 5 days at the rate of 1 × 108 conidia mLˉ1 (Alves et al.
2005). Variation in the potency of B. bassiana may be affected
by several factors, such as temperature, humidity, experimental
conditions, the concentration of used dose as well as plant variety. The pathogenicity of B. bassiana (ARSEF 2860) against
P. citri has been documented up to 90% after 20 days of fungal
application with a high dose of 1.2 × 1013 conidia haˉ1 in the field
conditions (Shi and Feng 2006). But according to our findings,
B. bassiana (BFZ0409) caused quick mortality of CRM, and
killed half population within 5 days with a dose of 1 × 108 conidia mLˉ1 on citrus seedlings in controlled conditions.
The susceptibility of different mites to various fungal species is much attractive for the management of the mites in a
diverse environment. In the current study, we observed that
both stages of CRM were significantly susceptible to all tested
fungal isolates. Likewise, Aguirre and Krugg (2014) described that adults of CRM were more susceptible to
A. lecanii as well as B. bassiana at the concentration of 106
conidia mLˉ1, as both fungal species significantly reduced the
adult population of mites by 71% within 2 weeks. Therefore,
the results of the current study were in accordance with the
findings of Aguirre and Krugg (2014), because two isolates
(Vp28 and V3450) of A. lecanii killed more than half populations of nymphs and adults within 9 days by 108 conidia mLˉ1.
Similarly, A. lecanii has much potential to inhibit the growth
of different mites, like Tetranychus urticae Koch (Amjad et al.
2012) and Dendrolaelaps sp. (Bałazy et al. 2008).
Similarly, the lethal potential of M. anisopliae was also
appealing against several insect pests, as well as mite pests.
M. anisopliae is much effective against several pest mites, like
B r ev i p a l p us p h oe n i c i s ( M a ga l h ã es e t al . 20 0 5) ,
Mononychellus tanajoa (Barreto et al. 2004), T. urticae
(Bugeme et al. 2015), T. evansi (Maniania et al. 2016),
T. truncates and T. turkestani (Shi et al. 2008a, 2008b).
Likewise, it was observed in our current work that P. citri
had faced 63% and 44% mortality of adult and nymphs, respectively, by M. anisopliae at 108 conidia mLˉ1 within 9 days
of exposure, which shows that our findings are in accordance
with the reports of García and Krugg (2015), who described
that M. anisopliae caused up to 83% mortality of CRM in lab
conditions under the fungal exposure for 2 weeks. Moreover,
Aschersonia aleyrodis also has a significant potential to control various insect pest, like whitefly (Zhang et al. 2017).
However, there is one report by Tamai et al. (2002) who
described that A. aleyrodis had presented minute potential to
inhibit the growth of T. urticae. Beyond all fungal explorations against CRM, A. aleyrodis still uncovered for its pathogenicity. We observed in the current research that A. aleyrodis
(AsG0910) caused 70% and 52% mortality of adults and
nymphs of CRM within a week, respectively, after application
of 108 conidia mLˉ1.
Conclusion
In conclusion, two isolates of B. bassiana (BFZ0409, D1344)
and one isolate of A. lecanii (V3450) were highly virulent
against both stages of CRM at the lowest doses, and therefore,
these isolates could be recommended as promising candidates
for the management of CRM. Whereas, other five isolates
were less effective against both stages of CRM. Thus,
employing these isolates into integrated management of mites
could assist synthetic acaricides in the citrus orchards and
avoid some predator mites susceptible to mycosis. However,
the potency of these isolates is still needed to be evaluated in
field conditions, especially, the compatibilities with some
phytoseiid predators before field application.
Acknowledgements We thank Miss Qiuping Jiang and Mr.
Zhisheng Liang for help in the experiment. This research was
supported jointly by research grants funded by the National Key
Projects of R & D of China (2018YFD0201502); Key projects of
Science and Technology of Fujian Province (2016 N0005); and
Research Fund for the International Collaborative Program
(KXGH17004), grant (CXZX2017211), (CXZX2018101),
(CXZX2019008S) and (KF2015068) from FAFU. We also acknowledge the support of a grant RCAMS/KKU/08/20 under
Research Center for Advanced Materials Science (RCAMS) at
King Khalid University, Saudi Arabia.
Author's personal copy
Int J Trop Insect Sci
Compliance with ethical standards
Conflict of interest All authors declare no conflict of interest, and are
agree to proceed the article in the International J. Tropical Insect Science.
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