154 J. Trop.

J. Trop. Plant Plant

Pests Pests
Dis. Dis.
ISSN:
Vol. 24, No. 1411-7525
2 2024: 158–161
Vol. 24, No. 2, September 2024 E-ISSN: 2461-0399
Pages: 154–161 DOI : 10.23960/j.hptt.224154-161

RESEARCH PAPER

Development of Bacillus thuringiensis-based liquid and paste formulations for

controlling invasive pest species Spodoptera frugiperda J. E. Smith
Achmad Djunaedy1, Syaiful Khoiri1, Dheananda Fyora Hermansyah Azari1, Zahratus Syamsiyah1, Gita
Pawana1, Dita Megasari2, & Giyanto3

Manuscript received: 6 October 2023. Revision accepted: 23 December 2023. Available online: 12 June 2024.

ABSTRACT

Spodoptera frugiperda J.E. Smith (Spodoptera: Noctuidae) is an invasive pests of maize that has been reported around
the world. Control efforts using biological agents continue to be developed, including the use of entomopathogen bacteria
such as Bacillus thuringiensis. To boost the efficacy and efficiency of biological control, formulations are required. The
objective of this study was to develop biopesticide formulations and evaluate their efficacy. The research was carried out by
formulating B. thuringiensis strain BtJ2 (1010 cfu mL-1) in liquid and paste formulations. The effectiveness of the formulations
was evaluated using the feed dipping method. The results showed that paste formulations at a concentration of 10% caused
100% mortality, whereas the liquid formulation resulted in 85% mortality. The LC90 for the paste formulation was 6.66%,
while the LC90 for the liquid formulation was 12.90%. Both the liquid and paste formulations had similar effects on mortality
and viability. Based on the LC90 and LT90, the paste formulation was more efficient and faster in killing S. frugiperda than
the liquid formulation. The results of this study provide recommendations that B. thuringiensis as a bioinsecticide is better
formulated in a paste than in a liquid form.
Key words: entomopathogen, fall armyworm, formulation, mortality

INTRODUCTION egg stage lasts 2–3 days and the larval period 14–30
days. The larval stage of this pest prefers leaves and
Spodoptera frugiperda J.E. Smith is a highly sensitive shoots, particularly buds, and develops into a
destructive invasive pest of maize that has been plant tissue chewer (He et al., 2020). Pupae formation
documented all over the world (Milano et al., 2008; occurs on the ground and lasts 8–30 days (da Silva et
Shylesha et al., 2018; Sisay et al., 2019; Bajracharya et al., 2016; Sharanabasappa et al., 2018). The short life
al., 2020; Cokola et al., 2020; Ashok et al., 2021; Yan et cycle and high reproductive capasity make this pest
al., 2022). The incidence and intensity of S. frugiperda one of the most destructive.
attacks can reach 100% (Sisay et al., 2019; Megasari In general, synthetic pesticides such as carbaryl,
& Khoiri, 2021). S. frugiperda attacks maize crops and diazinon, cypermethrin, methyl parathion, and
causes significant losses (Kebede & Shimalis, 2019). In methomyl have been used to control S. frugiperda
African and European countries, the economic losses (Paredes-Sánchez et al., 2021). However, several
caused by this pest amount to 20.6 million tonnes per studies have reported that pest resistance builds up
year, with a total loss of US$ 2.5-6.2 billion (FAO & quickly, making synthetic insecticides ineffective
CABI, 2019). (Kumela et al., 2019). Pest resistance is induced by
This pest’s imago can lay up to 2000 eggs. The mutations in genes that confer resistant to pyrethroids,
organic phosphates, and carbamates (Boaventura et
Corresponding author: al., 2020). Therefore, alternative control methods are
Syaiful Khoiri (syaiful.khoiri@trunojoyo.ac.id) needed. One example is utilizing the entomopathogenic
bacterium, B. thuringiensis. The use of B. thuringiensis
1
Program study of Agroecotechnology, Department of as a biological agent is environmentally friendly
Agricultural Science and Technology, Faculty of Agriculture,
because its crystal protein is selective (Arsi et al.,
Universitas Trunojoyo Madura, Bangkalan, Indonesia 69162
2
Program study of Agrotechnology, Faculty of Agriculture, 2019).
Universitas Pembangunan Nasional “Veteran” Jawa Timur, Previously, B. thuringiensis strains were found
Surabaya, Indonesia 60294 to be efficient biological controls in prior investigations
3
Departmen of Plant Protection, Faculty of Agriculture, IPB (Pinto et al., 2012; Gazali et al., 2017; de Oliveira et
University, Bogor, Indonesia 16680 al., 2022). However, B. thuringiensis must be prepared
Djunaedy et al. Development of Bacillus thuringiensis-based liquid and paste 155

for large-scale manufacturing. Formulations can Institute), Malang, Indonesia. All chemicals used in
help sustain population density while also increasing this research were of analytical grade and purchased
the efficacy of active compounds (Fravel et al., from Sigma (St. Louis, MO), Merck (Darmstadt,
1998; Yulensri, 2020). Furthermore, the appropriate Germany), and HiMedia (Maharashtra, India).
formulation can aid in availability, mass distribution,
storage, transportation, packaging, application, and Preparation of S. frugiperda Larvae. The larvae of S.
marketing (Fravel et al., 1998; Wardati & Erawati, frugiperda were maintained at the Laboratory of Plant
2015). Protection and Environment, Universitas Trunojoyo
Based on this, two B. thuringiensis formulations, Madura. The rearing box used were cages made of
liquid and paste, were created. The liquid formulation insect netting, measuring 50 cm × 50 cm × 50 cm, and
is the simplest and has the benefit of being more stable the larvae were fed baby beans.
in terms of color, scent, and appearance, as it does
not alter from the start of manufacturing (Wardati & Preparation of Formulation. Preparation of B.
Erawati, 2015). The paste formulation was developed thuringiensis strain BtJ2 began with re-culture isolation
for comparison. The purpose of this study was to from cold storage, followed by quadrant streaking on
determine the toxicity of liquid and paste formulations tryptic soy agar (TSA) and incubation at 37 ºC for
with the active ingredient B. thuringiensis in killing 24 hours. Novel liquid and paste formulations were
S. frugiperda, estimate lethal concentration and lethal created by combining the B. thuringiensis culture
time, and compare viability. The findings of this study with additional components (Table 1). The active
provide recommendations for the development of the component was B. thuringiensis. Glycerol acts as
B. thuringiensis formulation. a protector, preventing cell damage and preserving
bacterial viability during storage (Stevenson et al.,
MATERIALS AND METHODS 2017). Sodium nitrate serve as a nitrogen source,
citric acid is an organic acid compound (Zhang et al.,
Research Site. This research was carried out at the 2013), molasse is a carbon source, and TiO2 acts as
Laboratory of Plant Protection and Environment, Major an adjuvant, protecting cells and making them more
of Agroecotechnology, Department of Agricultural resistant to UV light.
Science and Technology, Faculty of Agriculture,
Universitas Trunojoyo Madura, Indonesia. Bioassays. The toxicity test was carried out by bioassay
using the feed dipping method (Balfas & Willis, 2009).
Materials. The B. thuringiensis strain BtJ2 was an The test feed used 5 cm pieces of baby beans, which
isolated collection from the Plant Protection and were prepared by dipping them into the formulation
Environment Laboratory. S. frugiperda was provided according to the treatment. The formulations used
by Balai Penelitian Tanaman Pemanis dan Serat (The were two weeks old. Next, the beans were dried for
Indonesian Sweetener and Fiber Crops Research one hour (Turhadi et al., 2020). Second instar larvae
Table 1. Composition of liquid and paste formulation
Ingredient Liquid formulation Paste formulation
Cell culture 50 mL 50 mL
TiO2 40 mL (in acetic acid 0.6%) 39 g
Sodium nitrate (NaNO3) 1% 2.4 g -
Citric acid (C6H8O7) 0.1% 0.24 g -
Glycerol 20% 58.82 mL 15 mL
Sterile-Aquadest 98.54 mL -
Talc powder - 225 g
Molase - 15 g
Tween 80 - 6.5 mL
CMC - 5g
Xanthan gum - 7.5 g
156 J. Trop. Plant Pests Dis. Vol. 24, No. 2 2024: 158–161

were fasted for 24 hours, then placed into plastic vials (Umaru & Simarani, 2020).
with two larvae per vial and fed according to treatment
(Asmaliyah et al., 2010). This bioassay consisted of RESULTS AND DISCUSSION
five concentration treatments: 0% (control), 0.5%,
1%, 5%, and 10%. The treatment concentrations Insecticidal Effect of the Formulations on S.
were determined to facilitate the calculation of frugiperda. Based on the results of observations, it
LC and LD. In the control group, green beans were can be seen that in the liquid formulation, the highest
dipped in distilled water only. Each experimental mortality was 85% at a concentration of 10%, and the
unit consisted of ten larvae and was repeated four lowest was 18% at a concentration of 0.5%. In the
times. The feed was changed every day without any paste formulation, the highest mortality was 100% at a
treatment. Observations were made every day for three concentration of 10%, while the lowest mortality was
days. Every two hours for 72 hours, larval mortality 0% at a concentration of 0% (Table 2). This indicates
was recorded and documented. After the first three that the paste formulation was more effective in killing
days, observations were made once a day. Mortality S. frugiperda larvae.
observation continued until one concentration resulted Salaki & Watung (2022) classify virulence
in 100% larval death. Mortality was calculated using levels based on the mortality value of the test insects:
the formula below (Hidayati et al., 2013). high virulence if the mortality value is above 50%,
n
M = N # 100% moderate virulence if the mortality value is 30%–
<50%, low virulence if the mortality value is <30%,
M = Larvae mortality (%); and no virulence if the mortality value is 0%. Both
n = The number of dead larvae; formulations showed high virulence starting from a
N = The number of treated larvae. 5% concentration. A previous study reported that the
mortality value increases if the treatment is given to
Formulation Effect on Viability. The viability test the larvae for a long period (Zulfiana et al., 2017).
was performed by combining 1 mL of the compound Factors that affect virulence include pH, temperature,
with 9 mL of distilled water. Following that, dilution humidity, and light (Motta, 2021).
were performed up to 10-8. Then, 1 mL of the diluted The results of the probit analysis for concentration
suspension solution was added to 9 mL of warm nutrient estimation showed that the liquid formulation required
agar and vortexed. The medium was then poured into a concentration of 5.83% to kill 50% of the larvae,
the petri dishes. Observations were made after 24 and 12.9% to kill 90% of the larvae. Meanwhile, the
hours to calculate the bacterial population, which can paste formulation required a concentration of 4.308%
be computed using the method below (Habazar et al., to cause 50% mortality and 6.61% to cause 90% (Table
2015). 3).
PB = A # C The results of linear regression analysis with
the equation Y = a + bX showed that for the liquid
PB = The population of bacteria (cfu mL-1);
formulation, the value of a is -0.2857 and the value of
A = The colony number of bacteria;
b is 1.7857, with an R2 value of 0.928 (in equation Y =
C = The dilution factor.
-0.2857 + 1.7857X). Meanwhile, the linear regression
for the paste formulation showed a value of a as -4.294
Data Analysis. Mortality data were analyzed using
and a value of b as 6.770, with an R2 value of 0.939
probit analysis to estimate the LC50, LC90, LT50, and
(in equation Y = -4.294 + 6.770X). The two regression
LT90 values, utilizing IBM SPSS Statistics software
equations indicated that the concentration variable (X)
Table 2. Comparison of mortality effects on S. frugiperda between liquid and paste formulation
Liquid formulation Paste formulation
Concentration (%)
Mortality (%) Virulence level Mortality (%) Virulence level
0 (control) 0 - 0 -
0.5 18.00 Low 27.50 Low
1 25.00 Low 30.00 Low
5 53.00 High 77.50 High
10 85.00 High 100 High
Djunaedy et al. Development of Bacillus thuringiensis-based liquid and paste 157

affected the larval mortality variable (Y). faster than liquid formulation in killing larvae. One
The results of the probit analysis for estimating of the purpose formulations was enhance activity of
the time of death showed that the liquid formulation microbial agents. The density of the suspension was
required 65.32 hours to kill 50% of the larvae and not an important factor (Bharti & Ibrahim, 2020). This
165.63 hours to kill 90% of the larvae. In contrast, the result may be caused by composition of formulation.
paste formulation required 50.115 hours to kill 50% of Tamez-Guerra et al. (2000) tested 80 formulation and
the larvae and 63.701 hours to kill 90% of the larvae determined optimal combinations of ingredients such
(Table 4). as corn flours, lignin, and pregelatinized corn flour
The results of linear regression analysis with (PCF). The addition of PCF increase the effectifity.
the equation Y = a + bX showed that for the liquid
formulation, the value of a is -4.5 and the value of b Mortality Symptoms. There were no different
is 2.5, with an R2 value of 0.98 (in equation Y = -4.50 symptoms between the liquid and paste formulations.
+ 2.50X). Meanwhile, for the paste formulation, the Symptoms of poisoning caused by the application
value of a is -20.913 and the value of b is 12.302, of a bioinsecticide paste formulation with active B.
with an R2 value of 0.542 (in equation Y = -20.913 + thuringiensis began with paralysis of the S. frugiperda
12.302X). These two regression equations indicated larvae, commonly known as the knockdown effect.
the concentration variable (X) affects the larval death Poisoned larvae initially became yellow-brown
time variable (Y). The closer the R (coefficient of with a soft texture and exhibited passive movement.
determination) value is to 1, the stronger the effect of The symptoms of death from the observed after the
the independent variable (X) on the dependent variable application included physical changes in the larvae.
(Y) (Muckoya et al., 2020). Initially, the body of the S. frugiperda larva, which
This study showed that paste formulation was light brown with active movements, turned green-
Table 3. The results of the probit analysis to estimate the lethal concentration formula (LC50,90) on larval mortality
Probability of concentration (%)
Mortality (%)
Liquid formulation Paste formulation
50 5.83 4.31
55 6.30 4.50
60 6.82 4.70
65 7.40 4.91
70 8.07 5.15
75 8.85 5.42
80 9.82 5.74
85 11.08 6.13
90 12.90 6.66

Table 4. The results of the probit analysis to estimate the lethal time formula (LT50,90) based on larval
Estimation of lethal time (hours)
Mortality (%)
Liquid formulation Paste formulation
50 65.32 50.11
55 71.56 51.30
60 78.51 52.54
65 86.41 53.86
70 95.59 55.28
75 106.59 56.85
80 120.34 58.66
85 138.62 60.84
90 165.63 63.70
158 J. Trop. Plant Pests Dis. Vol. 24, No. 2 2024: 158–161

brown to dark brown, and then black-brown, with its factors, and increase the activity of the agent on the
body size decreasing. The texture of the body became target host (Leland & Behle, 2004).
soft (Figure 1). These symptoms were caused by the
presence of parasporal crystals (Cry protein) produced CONCLUSION
by B. thuringiensis and the attachment of protoxins to
receptors in the digestive tract, assisted by protease The results showed that paste formulation at
enzymes, which then led to proteolysis (Schünemann a concentration of 10% could cause 100% mortality,
et al., 2014; Zulfiana et al., 2017). whereas the liquid formulation resulted in 85%
mortality. The LC90 in the paste formulation was
The Bacterial Viability. A bacterial viability test 6.66%, while the LC90 in the liquid formulation it
was carried out to determine the survival ability of was 12.90%. Both liquid and paste formulations had
bacteria during storage in the formulations. Based similar effects on mortality and cell viability. Based
on the viability test, the number of bacterial colonies on the LC90 and LT90, the paste formulation is more
after storage for 8 weeks, in both the liquid and paste efficient and faster in killing S. frugiperda compared to
formulations, did not decrease and remained at a the liquid formulation.
concentration of 1010 cfu mL-1 (Table 4). This indicated
that the bacteria could survive during storage. Both ACKNOWLEDGMENTS
formulations showed stable characteristics in storage.
The right formulation of biological agents can This study was supported in part by the Institution
maintain the stability of the agent during storage and of Research and Community Services (LPPM),
distribution, increase the persistence of the agent in the Universitas Trunojoyo Madura, under contract number
field, facilitate the application of these products in the 2929/UN46.4.1/PT.01.03/2021.
field, protect the agent from unfavorable environmental

B1 B2

A C1 C2
Figure 1. Effect of B. thuringiensis BtJ2-base formulation treatment on S. frugiperda larvae. A. Healthy larvae in
the control treatment; B. Symptoms of larval death due to liquid formulation treatment; C. Symptoms
of larval death due to paste formulation treatment. 1. After 24 hours; 2. After 48 hours.
Table 4. Viability of B. thuringiensis strain BtJ2 in the formulation over 8 weeks
Population of B. thuringiensis strain BtJ2 (x 1010 cfu mL-1)
Storage period (Weeks)
Liquid formulation Paste formulation
0 3.64 1.74
4 3.65 1.74
8 3.65 1.79
Djunaedy et al. Development of Bacillus thuringiensis-based liquid and paste 159

FUNDING terhadap serangga hama Spodoptera litura

Fabricus (Lepidoptera: Noctuidae) [Toxicity
This study was funded in part by the Institution assay of Nicolia atropurpurea leaf extract
of Research and Community Services (LPPM), against armyworm Spodoptera litura]. Jurnal
Universitas Trunojoyo Madura, under contract number Penelitian Hutan Tanaman. 7(5): 253–263.
2929/UN46.4.1/PT.01.03/2021. https://doi.org/10.20886/jpht.2010.7.5.253-263
Bajracharya ASR, Bhat B, & Sharma P. 2020. Spatial
AUTHORS’ CONTRIBUTIONS
and seasonal distribution of fall armyworm,
Spodoptera frugiperda (J.E. Smith) in Nepal.
AD and SK conceived and planned the
J. Plant Prot. Soc. 6: 192–201. https://doi.
experiment. G carried out the isolation and
org/10.3126/jpps.v6i0.36486
pathogenicity test of B. thuringiensis. DM managed
rearing and identification of S. frugiperda. DFHA and Balfas R & Willis M. 2009. Pengaruh ekstrak tanaman
ZS collected bioassay data and also performed analysis obat terhadap mortalitas dan kelangsungan
data. SK and GP interpreted the data. SF prepared hidup Spodoptera litura F. (Lepidoptera,
the manuscript. The authors provided responses and Noctuidae) [Effect of medicinal plant extract
comments on the research flow, data analysis, and on mortality and survival of Spodoptera litura
interpretation as well as the manuscript’s structure. All F. (Lepidoptera, Noctuidae)]. Bul. Littro. 20(2):
authors have read and approved the final manuscript. 148–156.
Bharti V & Ibrahim S. 2020. Biopesticides: production,
COMPETING INTEREST formulation and application systems. Int. J.
Curr. Microbiol. App. Sci. 9(10): 3931–3946.
The authors declare that there is no competing https://doi.org/10.20546/ijcmas.2020.910.453
interest regarding the publication of manuscripts.
Boaventura D, Martin M, Pozzebon A, Mota-Sanchez
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