INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
Efficacy of Bacillus thuringiensis and Mineral Oil against
Phyllocnistis citrella Stainton (Lepidptera: Gracillariidae)
B. AMIRI BESHELI1
Laboratory of Toxicology, Department of Plant Protection, Faculty of Agricultural Science, Mazandaran University, P.O.
Box-578, Sari, Iran
Corresponding author’s e-mail: firstname.lastname@example.org
A laboratory experiments was conducted to examine the efficacy of Bacillus thuringiensis for control of citrus leafminer,
Phylocnistis citrella Stainton, in isolated citrus leaves in Mazandaran province, Iran. Several preparations of Bt tested at the
concentrations of 0.5, 1, 3 and 6 g per liter of water with or without mineral oil in these bioassays. In all tests, the bacterial
suspensions or mineral oils were applied on the leaves of citrus with second or third instars of leafminer larvae in a leaf-dip
bioassay. For all concentrations of Bt treatments, with the exception of 0.5% the number of larval mortality was significantly
(P < 0.01) decreased compared to un-treated control group. The larval mortality positively correlated with Bt concentrations.
The number of larvae per leaf, were significantly (P < 0.01) reduced by treatment with Bt plus mineral oil. Treatment with Bt
plus mineral oil reduced the CLM larvae numbers to a level not significantly different from treatment with Bt alone. These
data suggest that the activity of Bt is enhanced, probably due to increased penetration of Bt into leaf cuticles by treatment of
Key Words: Phyllocnistis citrella; Bacillus thuringiensis; Mineral oil; Bioassay
INTRODUCTION on the environment (Jyoti & Brewer, 1999). Since CLM is
protected inside the mine it is suggested that the mineral oils
The citrus leafminer (CLM), Phyllocnistis citrella used as a surfactant would reduce the surface tension and
Stainton originated in Asia but now is distributed increase the penetration of the Bt suspension through the
throughout the five continents where citrus is grown. The leaf epidermis. Petroleum oil reduced infestation by
CLM is an important pest of citrus and related species of preventing oviposition and there is a negative effect
Rutaceae family and some related ornamental plants. The between the number of mines/leaf and concentration of oil
CLM mines leaves, surface tissue of young shoots, stems (Dias et al., 2005).
and less frequently the fruit. The lamina of mined leaves The first record of CLM from southern part of Iran
dries and rolls, reducing leaf area and photosynthetic dates back to 1961, but in the northern of Iran its presence
activity of the plant. Although citrus leaf-miner causes was noted for the first time in September 1994. Since then,
indirect damage to young leaves, which predisposes them to it has shown a dramatic increase and widespread dispersal.
infection by canker, controlling citrus leaf-miner is also a Almost all commercial varieties are affected but data on
vital component of canker management (Belasque et al., economic losses are not available. The pest has about 5 - 9
2005). Several insecticides are used against this pest, but generations during the year, with peak periods in early
these may involve un-desirable effects on the environment, during summer and autumn. Preliminary field trials with
including interference in control of the pest by natural selected insecticides indicate the superiority of Dimilin
enemies. Biological control is the best option for controlling (diflubenzuron) over diazinon, Zolone (phosalone) and
this pest. The effective control of CLM is very complicated, Ekamet (etrimfos) in controlling of CLM in the northern
because of its high migration ability from outside of Iran, but it was not totally effective (Jafari, 1996). No
orchards and the high fertility of CLM. It has been proven information on other methods of control or on indigenous
that citrus leaf epidermis provides substantial protection for parasitoids is available. The aim of the present study was to
CLM and the difficulty of direct contact of chemical to the evaluate the toxicity of the commercial formulation of Bt
larval body (Pena, 1997). with and without mineral oil on second and third in star
It has been shown that the CLM has also a long history larvae of the CLM in laboratory conditions.
of resistance to many insecticides and development of
resistance against the chemicals sometimes makes it MATERIALS AND METHODS
difficult to obtain enough control (Mafi & Ohbayashi,
2006). Bacillus thuringiencis (Bt) subsp Krustaki is the Laboratory bioassays. The toxicity of insecticides against
bacterial insecticide most widely used for controlling CLM was conducted under laboratory condition using
Lepidoptera larvae population (Broderick et al., 2000). It is commercial Bt pesticides with or without mineral oil at
safe for many non-target insects and has a minimal impact research station of Sari Agricultural and Natural Resources
BESHELI / Int. J. Agri. Biol., Vol. 9, No. 6, 2007
Table I. The effect of different concentrations of Bt (0.5, dependent variable using the ANOVA. If a significance
1, 3 & 6 gram per liter of water) on percentage of CLM effect of variables was calculated, the means were
larvae mortality (Mean ± sd) contrasted by Duncan’s multiple range tests.
Treatment Mean comparison RESULTS
c b a
Control 8.49±1.5 Analyses of variance indicated significant differences
0.5 35.40±7.5 35.40±7.5 among Bt treatments (P < 0.01). The results clearly
1 40.07±5.3 40.07±5.3 demonstrated that the efficacy of Bt against CLM increased
3 46.96±5.7 46.96±5.7
6 61.16±7.5 with increasing Bt concentration (Table I). The comparison
a, b, c, Means did not followed by the same letters in rows are between different post treatment times showed significant
significantly different (P<0.01). differences (P < 0.05) among the periods of the post
treatments (Table II). Post-treatment for 96, 72 and 48 h
Table II. The effect of different post treatment time of were more effective than 24 h on pest mortality. There was
Bt on percentage of CLM larvae mortality (Mean ± sd) no significant difference between interaction of Bt and post
Post treatment time (h) Mean comparison treatment time on CLM larvae mortality. Results for
b a different concentrations of Bt plus MO indicated significant
24 16.81±3.8 differences among treatments. The results showed that the
48 35.37±4.64 35.37±4.64 treatment of Bt plus MO increased the mortality of CLM
96 54.10±7.15 larvae at higher concentration of Bt (Table III). The
a, b, Means did not followed by the same letters in rows are significantly comparison between different post treatment times showed
different (P<0.01). significant differences (P < 0.05) among the periods of the
University in 2006. The insecticides and respectively post treatments on CLM larvae mortality (Table IV). No
concentrations used were Bt (0.5, 1, 3 & 6 g L-1 of water) in statistically differences were observed in CLM larvae
experiment 1, different percentage (0.1, 0.2, 0.3, 0.5) of mortality between Bt treated groups in comparison with
mineral oil (MO) in experiment 2 and BT (0.5, 1, 3 & 6 g L- their counterparts Bt plus MO groups (Table V).
) + 0.5% MO in experiment 3. In each experiment a control DISCUSSION
group was run using sterile water. The leaf-dip bioassays
were devised to test the toxicities of Bt pesticide. In assay, The effect of insecticides in citrus orchards against the
only leaves with actively feeding second or third in star leaf- CLM is difficult to achieve the maximum CLM larval
miner larvae were completely excised with petioles from mortality and it is not very sufficient, because several
citrus Thomson trees and used for bioassays. To keep the generations of CLM are usually overlapping and the CLM
leaves turgescent during the bioassay, each petiole was larvae are protected by a cuticular layer of the leaves in the
covered by wet cotton. Leaves were dipped separately for serpintine mine and the pupal stage is also protected by the
approximately 10 seconds into each treatment. Air-dried for rolled leaf margins (Raga et al., 2001). The results of
approximately 2 h and placed at the bottom of the plastic present study clearly demonstrated that the efficacy of Bt
petridishes (9 cm diameter × 2.5 cm high). These dishes and Bt plus MO against CLM increased with the increasing
were lined with a wet filter paper and covered with a plastic Bt concentration. It is known that the larval mortality varies
lid. The experiment for each treatment was replicated four with spray volume suggesting that oil reduced infestation by
times along with distilled water treated as a control group. acting as an oviposition deterrent (Liu et al., 2001).
After 24, 48, 72 and 96 h of post-treatment the numbers of Pesticides may be applied to protect new flushes of
live and dead larvae for each replicate were counted under a growth when the leaves are most vulnerable to CLM
stereo-micro-scope. Variable measured per replicate of each damage. However, the best foliar insecticides confer only 2
treatment were the average number of mines per leaf larval weeks of leaf miner infestations (Michaud & Grant, 2003).
mortality (the proportion of larvae that were dead). These data showed that the Bt and Bt plus MO are active
Statistical analysis. The experiment was conducted in a against the leaf-miner demonstrating that these bio-
completely randomized design using factorial arrangements pesticiedes penetrate into leaf mines, thereby killing the
of treatments (four replications for each treatment). larvae. Recently, the toxicity of different insecticides to the
Normality of the data was assessed using probability plots. citrus leaf-miner and its parasitoids was evaluated under
The normal distributed was approximated for the number of laboratory conditions in Japan (Mafi & Ohbayashi, 2006).
dead larvae per leaf when these data were reciprocally They found that the percentage corrected mortality of eggs
transformed using: of the citrus leaf-miner exposed to insecticides (dipping
method bioassay) ranged from 3 to 44%, but all the
y . insecticides tested showed almost over 90% mortality to the
100 first in star larvae of citrus leaf-miner.
Mortality data were corrected using Abbott's formula It has been demonstrated that using petroleum oil
(Abbott, 1925). The analysis of data was performed on each spray residues reduced infestations of CLM by preventing
EFFICACY OF BT AND BT PLUS MINERAL OIL FOR CONTROL OF CITRUS LEAF-MINER / Int. J. Agri. Biol., Vol. 9, No. 6, 2007
Table III. The effect of different concentrations of Bt Studies show that neonicotinoid, pyrethroid and
(0.5, 1, 3 & 6 gram per liter of water) plus MO (0.5%) growth regulator insecticides have a significant, negative
on percentage of CLM larvae mortality (Mean ± sd) impact on some predators, which are appearing to be the
most important biological control agents of leaf-miners.
Treatments Mean comparison Depending on the rate of insecticide used the number and
c b a timing of applications and the level of coverage of the tree.
0.5 53.38±3.4 Thus, it is necessary to be aware of the effect of these
1 54.72±5.6 54.72±5.6 pesticides on beneficial insects including parasitoid and
3 56.25±4.8 56.25±4.8 predators for these reasons it is better to use biopesticides
6 63.13±5.5 such as Bt and/or Bt plus MO (Villanueva-Jiménez et al.,
a, b, c, Means did not followed by the same letters in rows are
2000; Grafton & Gu, 2003). Sometimes the indirect damage
significantly different (P<0.01)
of CLM is very important. Mining of immature foliage by
Table IV. The effect of different post treatment time of the larvae can lead to reduced growth rates, yield and mined
Bt plus MO (0.5%) on percentage of CLM larvae surfaces serve as foci for the establishment of diseases such
mortality (Mean ± sd) as citrus canker, Xanthomonas citri. In the absence of citrus
canker, citrus leaf-miner is a serious pest of rapidly growing
Post treatment time (h) Mean comparison
immature or pruned trees. But in presence of citrus canker, it
24 42.6±10 is a major pest of both immature and mature trees (Liu et al.,
48 59.27±9.6 59.27±9.6 2001). Therefore it is important to select less toxic
72 64.82±8.3 64.82±8.3 chemicals against the natural enemies in order to expect
both the activity of natural enemies and control effect of
a, b, Means did not followed by the same letters in rows are significantly
insecticides for suppressing the infestation of CLM. The
higher activity of Bt in Bt plus MO treated groups at the
Table V. Mean comparison between the effect of Bt and present study may be due to increased penetration of Bt
Bt plus MO (0.5%) on percentage of CLM larvae through the mine by helping of MO.
mortality (Mean ± sd) For better understanding it is necessary to investigate
the third generation pesticides such as growth regulators in
Treatments BT BT+MO
combination with mineral oil, microbial and fungi
0 8.49±1.51 8.49±1.51
0.5 35.40±7.60 56.04±3.46 insecticides to get much more suitable results in the field
1 38.22±5.62 53.73±5.65 conditions. However, more field studies will need to be
3 46.96±5.72 54.24±4.91 performed to understand the effect of Bt and Bt plus MO
6 61.16±7.56 63.21±5.61 against P. citrella and to determine the optimum timing of
oviposition is related to the concentration of oil in sprays the multiple application.
and timing of spray (Beattie et al., 1995). Both of REFERENCES
Abamectin and Lufenuron pesticides along with petroleum Abbott, W.S., 1925. A method of computing the effectiveness of an
oil provided a significant increase in CLM larval activity insecticide. J. Econ. Entom., 18: 265–7
(Raga et al., 2001). However, the efficacy of petroleum- Beattie, G.A.C., Z.M. Liu, D.M Watson, A.D. Clift and L. Jiang, 1995.
derived spray oils used as oviposition deterrents to control Evaluation of petroleum pray oils and polysaccharides for control of
Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae). J.
citrus leaf-miner is related to timing of spray application the Australian Entom., 34: 349–53
amount of oil deposited on sprayed surfaces (dose) and the Belasque, J., J.R. Parra-Pedrazzoli, A.L. Rodrigues, J. Neto, P.T.
persistence of oil molecules on sprayed surfaces or efficacy Yamamoto, M.C.M. Chagas, J.R. Parra, B.T. Vinyard and J.S.
is also related to increasing molecular weight of oil Hartung, 2005. Adult citrus leafminers (Phyllocnistis citrella) are not
efficient vectors for Xanthomonas axonopodis pv. citri. Pl. Dis., 89:
molecules as reflected by nCy values and therefore 590–4
persistence of oil molecules on sprayed surfaces (Liu et al., Broderick, N.A., R.M. Goodman, K.F. Raffa and J. Handelsman, 2000.
2001). Therefore the petroleum oils alone or combine with Synergy between Zwitttermicin A and BT subsp Krustaki against
microbial agent as emulsifier, which has synergist and less gypsy moth (Lepidoptera:Lymanteridae). Env. Entom., 29: 101–7
Dias, C., P. Carsia, N. Simoes and L. Oliveira, 2005. Efficacy of Bacillus
harmful effect for the environment recommended for using thguringiencis Against Phyllocnistis citrella (Lepidoptera:
in IPM program (Khyami & Ateyyat, 2002). In our Phyllocnitidae). J. Econ. Entom., 98: 1880–3
experiment, by comparing the activity of the commercial Grafton, C.E. and P. Gu, 2003. Conserving Vedalia Beetle, Rodolia
formulation between Bt and Bt plus MO against the CLM, cardinalis (Mulsant) (Coleoptera:Coccinellidae), in Citrus: A
continuing challenge as new insecticides gain registration. J. Econ.
we observed that the CLM larval mortality was higher (not Entom., 96: 1388–98
statistically) in Bt plus MO treated groups than the Bt alone. Jafari, M.I., 1996. Report of the Workshop on Citrus Leaf-miner
Several research groups have shown that, the application of (Phyllocnistis citrella) and its Control in the Near East, p: 33. Safita
Abamectin in combination with petroleum oil provides the (Tartous), Syria, 30 September-3 October
Jyoti, J.L. and G.J. Brewer, 1999. Median lethal concentration and efficacy
most synergistic effect to control of the Helicoverpa of BT against banded sunflower moth (Lepidoptera:Tortricidae). J.
armigera and CLM (Wang et al., 2005). Econ. Entom., 92: 1289–91
BESHELI / Int. J. Agri. Biol., Vol. 9, No. 6, 2007
Khyami, H. and M. Ateyyat, 2002. Efficacy of Jordanian isolates of Pena, J.E., R. Duncan and H. Browning, 1996. Seasonal abundance of
Bacillus thguringiencis against the citrus leaf-miner Phylocnistis Phyllocnistis citrella (Lepidoptera: Gracillariidae) and its parasitoids
citrella. Int. J. Pest Manag., 48: 297–300 in south Florida citrus. Env. Entom., 25: 698–702
Liu, Z., G. Beatie, M. Hodgkinson and L. Jiang, 2001. Influence of Raga, A., M.E. Satol, M.F. Souza and R.C. Siloto, 2001. Comparison of
petroleum derived spray oil aromaticity, equivalent n-paraffin carbon spray insecticides against citrus leaf-miner. Arq. Inst. Biol., 68: 77–
number and emulsifiers concentration on oviposition of citrus 82
leafminer Phyllocnistis citrella Stainton. J. Australian Entom., 40: Villanueva-Jiménez, J.A., M.A. Hoy and F.S. Davies, 2000. Field
193–6 evaluation of integrated pest management-compatible pesticides for
Mafi, S.A. and N. Ohbayashi, 2006. Toxicity of insecticides to the citrus the citrus leafminer P. citrella and its parasitoid Ageniaspis citrocola.
leafminer, Phyllocnistis citrella and its parasitoids, Chrysocharis J. Econ. Entom., 93: 357–67
pentheus and Sympiesis striatipes (Hymenoptera: Eulophidae). Appl. Wang, Q., J. Cheng, Z.M. Liu, S.G. Wu, X.P. Zhao and C.X. Wu, 2005.
Entom. Zool., 41: 33–9 Influence of insecticides on toxicity and cuticular penetration of
Michaud, J.P. and A.K. Grant, 2003. IPM-compatibility of foliar abamectin in Helicoverpa armigera. Insect Sci., 12: 109
insecticides for citrus: Indices derived from toxicity to beneficial
insects from four orders. J. Insect Sci., 3: 1–8 (Received 13 February 2007; Accepted 12 March 2007)