Comparative evaluation of two Metarhizium anisopliae isolates on various media for controlling mortality of maize weevil (Sitophilus zeamais)

Avaliação comparativa de dois isolados de Metarhizium anisopliae em diferentes meios de cultura para o controle da mortalidade do gorgulho do milho (Sitophilus zeamais)

Abstract

This study aimed to evaluate the effect of two culture media, Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA), on the growth, spore density, viability, and effectiveness of two isolates of the entomopathogenic fungus Metarhizium anisopliae obtained from the Indonesian Pest Forecasting Institute (BBPOPT) and the Indonesian Institute for Seed and Plantation Protection (BBPPTP). The experiment was arranged in a Completely Randomized Factorial Design (CRFD) with four treatments and five replications. The results showed that the isolate of M. anisopliae from BBPOPT grown on SDA medium (a2m1) produced the highest spore density (1.76 × 10⁸ conidia/ml) and viability (81.25%), as well as the highest maize weevil mortality of 44% at 14 days after application. In contrast, the isolate from BBPPTP cultured on PDA showed the lowest density and viability. Thus, the combination of the BBPOPT isolate with SDA medium was the most effective treatment in improving inoculum quality and biological control efficacy against Sitophilus zeamais.

Keywords:
Metarhizium anisopliae; SDA; PDA; viability; mortality; Sitophilus zeamais

Resumo

Este estudo teve como objetivo avaliar o efeito de dois meios de cultura, Ágar Dextrose de Batata (PDA) e Ágar Dextrose de Sabouraud (SDA), no crescimento, densidade de esporos, viabilidade e eficácia de dois isolados do fungo entomopatogênico Metarhizium anisopliae, obtidos do Instituto Indonésio de Previsão de Pragas (BBPOPT) e do Instituto Indonésio de Proteção de Sementes e Plantações (BBPPTP). O experimento foi conduzido em um Delineamento Fatorial Inteiramente Casualizado (DFIC) com quatro tratamentos e cinco repetições. Os resultados mostraram que o isolado de M. anisopliae de BBPOPT cultivado em meio SDA (a2m1) produziu a maior densidade de esporos (1,76 × 10⁸ conídios/ml) e viabilidade (81,25%), bem como a maior mortalidade do gorgulho do milho de 44% aos 14 dias após a aplicação. Em contrapartida, o isolado de BBPPTP cultivado em PDA apresentou a menor densidade e viabilidade. Assim, a combinação do isolado BBPOPT com meio SDA foi o tratamento mais eficaz na melhoria da qualidade do inóculo e eficácia do controle biológico contra Sitophilus zeamais.

Palavras-chave:
Metarhizium anisopliae; SDA; PDA; viabilidade; mortalidade; Sitophilus zeamais

1. Introduction

The maize weevil (Sitophilus zeamais) is one of the main pests in corn storage warehouses, capable of causing yield losses of up to 80% and 100% grain damage in storage.Sitophilus zeamaislays its eggs in corn kernels before harvest and in storage, and its life cycle finishes inside the kernels, leading to damaged grains. Control using insecticides can lead to pest resistance, residues, and environmental impacts, thus necessitating biological control methods such as entomopathogenic fungi (Nwosu, 2018).

Common control methods to suppress pest populations involve synthetic chemical insecticides due to their rapid results and ease of application. However, imprudent use of synthetic chemical insecticides can lead to negative impacts such as resistance, pest resurgence, and environmental pollution. One effort to reduce the side effects caused by chemical insecticides is to use biological agents, one of which is entomopathogenic fungi (Nasution et al., 2021; Al-Saedi et al., 2025).

Entomopathogenic fungi are fungi that are parasitic to insects. Several advantages can be gained from utilizing entomopathogenic fungi, including a relatively short life cycle and the ability to form spores resistant to environmental influences. There are more than 700 species of entomopathogenic fungi that can infect insect pests. One such entomopathogenic fungus isMetarhizium anisopliae(Widiarti et al., 2019; Kambarov et al., 2024; Nazori et al., 2024).

Metarhizium anisopliaeis the most widely used entomopathogenic fungus for controllingOryctes rhinocerospests.M. anisopliaeis not only saprophytic but also possesses parasitic capabilities against several insect orders such as Coleoptera, Lepidoptera, Homoptera, Hemiptera, and Isoptera. Research by (Nasution et al., 2021) usingM. anisopliaeinfected toO. rhinoceroslarvae at a spore density of 1.81 x 10⁸ spores/ml caused a larval mortality rate of up to 94%.M. anisopliaehas a high reproductive capacity, a short life cycle, can form long-lasting spores in nature even under unfavorable conditions, is safe, selective, easy to produce, and has a low likelihood of pest resistance (Manurung et al., 2012).

The propagation ofM. anisopliaecan be done in two ways: using liquid media or solid media. Liquid media significantly influence fungal morphology, growth, structure, and reproduction. Liquid media rich in essential nutrients such as carbohydrates, proteins, lipids, and minerals will provide the necessary nutrition for the fungi to grow and develop. Liquid media provide nutrients that are more easily absorbed by the fungi because they are already in a dissolved form, allowing the fungi to grow quickly and efficiently. Furthermore, in liquid media, environmental parameters such as temperature, pH, and humidity are easier to control and precisely regulate. This allows for optimal growth conditions to maximize fungal production. Liquid media with complete amino acids provide an optimal nutritional environment for increasing spore viability and high density in fungi (Jackson et al., 1985; Hussein et al., 2025; Mavlyanova et al., 2024; Inayata et al., 2023).

The entomopathogenic fungusMetarhizium anisopliaeis one of the biological agents widely used as a "mycoinsecticide" with a good ecological safety record for non-target organisms. However, the performance of the fungus in the field is influenced by environmental factors such as UV radiation, humidity, temperature, and leaf surface conditions (phylloplane), so the success of control depends on the quality of the propagules (conidia) and appropriate application strategies.

Spore quality is highly influenced by the growth medium. Various studies show that the nutritional composition of the medium can affect growth rate, sporulation, and even the surface properties of conidia related to cuticular penetration. This, of course, significantly affects virulence against host pests (Shah and Butt, 2005; Ongdash et al., 2024; Moro et al., 2023). Comparisons of common media such as PDA (Potato Dextrose Agar) and SDA/SDAY (Sabouraud Dextrose Agar / +Yeast extract) are often used to assess the morphological and physiological responses ofMetarhizium(Ibrahim et al., 2002).

Producing adequate quantities and good quality inoculum is one important component to support the development ofM. anisopliaefungus as a biological agent. Therefore, a suitable propagation method is needed to support the increasing demand forM. anisopliaefungus as a raw material for bioinsecticides. The same isolate propagated on PDA and SDA yields different conidial qualities, as shown by the speed of mortality (LT₅₀) ofS. zeamaisin bioassays. The interaction between isolates on several agar media also has different efficacy capabilities againstS. zeamaisadults (Teshome and Tefera, 2009; Puspitasari et al., 2022).

Therefore, this study will use several isolates of the fungusMetarhizium anisopliaefrom the Indonesian Pest Forecasting Institute (BBPOPT) and the Indonesian Institute for Seed and Plantation Protection (BBPPTP) that have not been tested on various types of agar media such as PDA and SDA media. This will be followed by applying the isolates to maize weevils (Sitophilus zeamais) and testing their mortality. This will determine the suitability of each isolate on each agar medium used for conidial density, viability, and mortality against pests.

2. Material and Methods

The experiment was using an experimental method at the Laboratory of the Plantation Protection Agency, Badnung, West Java, Indonesia, from March to May 2025. This experiment consists of 2 stages, namely: the first stage to determine the quality of conidia originating from 2 isolates and the second stage to test the two isolates against the mortality of test insects as S. zeamais. The tools used in this experiment as a digital scale, beaker glass, stirring rod, stove, petri dish, loop needle, Bunsen burner, cork borer, test tube, micropipette, glass funnel, test tube rack, Laminar Air Flow (LAF), autoclave, erlenmeyer flask, vortex, hemocytometer, hand counter, syringe, object glass, cover glass, scalpel, microscope, jar container and hand sprayer. The materials used as pure culture of Metarhizium anisopliae fungus from Center for Plant Pest Forecasting (BBPOPT) Karawang, West Java and Center for Plantation Seed and Protection (BBPPTP) Surabaya, East Java, sterile distilled water, Potato Dextrose Agar (PDA) media, Sabouraud Dextrose Agar (SDA) media, 70% alcohol, methylated spirits, toothpicks, cotton, tissue, cracked corn and distilled water.

First experiment was used a Completely Randomized Design with factorial pattern (CRFD), consists of two factors: the first factor is agar media (A), consisting of PDA medium (a₁) and SDA medium (a₂); the second factor is theMetarhizium anisopliaefungus isolates (M) obtained from BBPOPT (m₁) and BBPPTP (m₂) with five replications. The inoculated results will be observed the Conidial density (conidia/ml) and the conidial viability (%). Second experiment was used a Completely Randomized Design (CRD) with 4 treatments and 5 replications each. Each treatment will contain 20 maize weevils. They will be sprayed with theMetarhizium anisopliaefungus from the first experiment, and their mortality will be observed for 14 days with observations every 2 days. Observational data were statistically tested with Anova and Duncan's Multiple Range Test at a 5% significance level.

2.1. Propagation of Metarhizium anisopliae fungal isolates on various agar media

The Metarhizium anisopliae fungal isolates used in this experiment were propagated on PDA and SDA media. PDA is a commonly used medium for growing various types of fungi and has the ability to support the growth of fungal mycelia (Devi et al., 2018). The PDA used was instant PDA, prepared by weighing 42 grams of PDA and dissolving it in 1 liter of distilled water in a beaker glass. The solution was boiled using a double-boiling method. The boiled medium was then transferred to Erlenmeyer flasks and sterilized using an autoclave at 121°C for 45 minutes at 1 atm pressure. The sterile medium was then poured into Petri dishes, approximately ± 10 ml per dish, under aseptic conditions.

SDA is a medium commonly used for fungal growth. SDA with 4% glucose has been shown to produce good growth for fungi and is considered one of the best culture media. SDA medium was prepared by weighing 65 grams of SDA medium. This was then dissolved in 1 liter of distilled water in a beaker glass. The mixture was stirred while heating until the medium dissolved. The boiled medium was then transferred to Erlenmeyer flasks and sterilized using an autoclave at 121°C for 45 minutes at 1 atm pressure. The sterile medium was then poured into Petri dishes, approximately ± 10 ml per dish, under aseptic conditions.

The solidified PDA and SDA media could be directly inoculated withM. anisopliaefungus in a Laminar Air Flow (LAF) hood, which had previously been sterilized using 70% alcohol and UV light for 15 minutes. The inoculation process involved transferring agar pieces colonized withM. anisopliaemycelia and spores to fresh PDA media using an inoculating loop. The inoculated media were then incubated for ± 7-14 days or until theMetarhizium anisopliaefungus grew evenly on the medium surface.

2.2. Growth ofMetarhizium anisopliaefungus on agar media

The observed parameters included fungal growth, as indicated by the average incubation period. The average incubation period was observed for all treatment combinations by noting the day on which fungal growth first appeared on the agar medium, calculated from the time of inoculation (Gusnawaty et al., 2017).

2.3. Conidial density and conidial viability ofMetarhizium anisopliaefungus

Testing was performed on severalMetarhizium anisopliaefungal isolates with different agar media to determine conidial density and viability. Samples to be tested were randomly selected for each fungus grown on each agar medium. The conidial density of each entomopathogenic fungal isolate was calculated using a Hemocytometer. Seven-day-old fungal isolates were diluted twice by adding 10 mL of distilled water, then one drop was placed on the Hemocytometer, observed, and counted under a microscope.

After obtaining the data on the number of conidia in each counting area, the number of conidia/ml was calculated using the following formula (Equation 1):

$\text{S}=\frac{\overline{X}}{\text{L}\times \text{t}\times \text{d}}\times {10}^3$ (1)

Where:

S: Density (conidia/ml)

$\overline{X}$: Average number of conidia in both upper and lower counting fields

L: Counting area 0.04 mm²

T: Counting depth 0.1 mm

D: Dilution factor

10³: Volume of suspension counted (1 ml = 10³ mm³).

The conidial viability test was a continuation of the density test. For the conidial viability test, PDA medium was first prepared in Petri dishes, and then the agar was cut using a cork borer of 0.5 cm. Agar pieces were taken using a sterile scalpel and placed on a sterile object glass, three pieces as replicates. The conidial suspension at 10⁻² dilution was vortexed again for 3 minutes and drawn using a 1 ml syringe. Each agar piece was inoculated with one drop of conidial suspension and then covered with a cover glass. The object glass was placed in a Petri dish, supported by two toothpicks. Two rolls of moist cotton were also placed inside the Petri dish. The Petri dish was sealed with cling wrap and incubated in a dark place. Spore viability was observed 16 hours after incubation using a microscope at 400x magnification. During observation, the number of germinated and non-germinated conidia was counted for each agar piece. According to Shah and Butt (2005), conidia are considered germinated if the germ tube has reached ½ the length of the conidia. The percentage of germination was calculated using the following formula (Equation 2):

$\text{Viability}=\frac{\text{Σ germinating conidia}}{\text{Total conidia observed}}\text{x}100\%$ (2)

2.4. Preparation of treatment media

The containers used were jars with a diameter of 15 cm and a height of 15 cm as habitats for the insects during the study. They were thoroughly cleaned and dried. Each jar was filled with 100 g of cracked corn as food for the maize weevils. A total of 25 such jars were prepared. Then, 20 maize weevil adults were introduced into each jar. Each jar was then covered.

2.5. Application ofMetarhizium anisopliaefungus on maize weevils

The application was performed only once by spraying the entomopathogenicMetarhizium anisopliaefungus according to the treatment onto the adult maize weevils and the medium. TheMetarhizium anisopliaefungus was applied using a hand sprayer with 20 ml per jar, concurrently with pest introduction. For the control, only sterile distilled water was sprayed onto the pests. Subsequently, the number of dead maize weevil adults was observed daily.

3. Result and Discussions

3.1. Quality test ofMetarhizium anisopliaefungus on PDA and SDA agar media spore density

The statistical test results showed an interaction between the twoMetarhizium anisopliaefungal isolates tested on different agar media regarding spore density (Table 1).

Table 1
Test of Two Isolates of M. anisopliae Fungus on Different Agar Media Against Spore Density (10⁸/ml).

Agar Media	Isolate From
	m1 = BBPOPT		m2 =BBPPPT
a1 = PDA medium	0.36	a	0.38	a
	A		B
a2 = SDA mediun	1.76	a	1.12	B
	A		B

• Remarks: The same lowercase letters in the horizontal direction and the same uppercase letters in the vertical direction show a significant difference according to the Duncan Multiple Distance Test at the level of 5%.

From Table 1 showed that the spore density ofMetarhizium anisopliaeinoculated on different agar media was highly significant in a₂m₁ (Metarhizium anisopliaeBBPOPT isolate with SDA agar medium). The highest average spore density (1.76x10⁸/ml) was found in a₂m₁ (Metarhizium anisopliaeBBPOPT isolate with SDA agar medium) and the lowest (0.36x10⁸/ml) in a₁m₁ (Metarhizium anisopliaeBBPOPT isolate with PDA agar medium), because SDA agar medium can provide the nutrients needed byM. anisopliaefor conidia formation, thereby accelerating colony formation, as stated by Moro et al. (2023) that the medium used to grow entomopathogenic fungi largely determines the rate of colony formation and the number of conidia during growth. Fungal growth images are shown in Figure 1.

Figure 1
Incubation period of isolates to show spore density in fungi. (A) 2 days incubation, (B) 4 days incubation, (C) 6 days incubation, (D) 8 days incubation, (E) 10 days incubation, (F) 12 days incubation, (G) 14 days incubation.

The spore population ofM. anisopliaein a₂m₂ (Metarhizium anisopliaeBBPPTP isolate with SDA agar medium) was also higher than a₁m₂ (Metarhizium anisopliaeBBPPTP isolate with PDA agar medium) and a₁m₁ (Metarhizium anisopliaeBBPOPT isolate with PDA agar medium), because SDA medium also capable of enhancing spore formation. The difference in spore density betweenMetarhizium anisopliaeisolates (BBPOPT and BBPPTP) is determined by the ability of colony growth. This occurs because physiological diversity among strains affects hyphal elongation rate, nutrient utilization efficiency, and adaptation to medium conditions.

PDA, with its potato extract content, provides complex carbon sources and growth factors that promote hyphal elongation, while SDA has osmolarity/C:N ratio and pH that are not always optimal for every entomopathogenic isolate. In the BBPOPT isolate, this difference is clearly visible because the spore density of a₂m₁ is higher than a₂m₂. In theM. anisopliaeBBPPTP isolate, the difference in spore density between a₁m₁ and a₁m₂ is not very significant, indicating a relatively flat medium response for that isolate. From the spore density results, it can be seen that the highest average counts are in a₂m₁ and a₂m₂, both of which used SDA agar medium for their growth. Overall, SDA medium significantly enhanced spore production in both isolates, indicating that nutrient composition and osmotic conditions play a critical role in regulating sporulation in M. anisopliae.

3.2. Viability ofMetarhizium anisopliaefungus

The statistical test results showed no interaction between the twoMetarhizium anisopliaefungal isolates on different agar media regarding conidial viability (Table 2)

Table 2
Test of Two Isolates of M. anisopliae Fungus on Different Agar Media for Conidial Viability (%).

Treatment	Viability (%)
Agar Media
a1 = PDA medium	63.89 a
a2 = SDA medium	66.63 a
Isolate From
m1 = BBPOPT	73.75 b
m2 = BBPPPT	56.77 a

• Remarks: The average value followed by the same letter in the same column shows an unreal difference according to the Duncan Multiple Distance Test at the level of 5%.

The viability test results (Table 2) showed that the viability ofMetarhizium anisopliaeconidia differed among combinations of isolates and agar media. Agar media is not significant to conidia viability but isolate from BBPOPT have a significantly with 73.75% conidial viability higher than isolate from BBPPPT with 56.77% conidial viability. This indicates that these three combinations were equally capable of producing well-germinated conidia.

In general, these results show that differences in isolates and agar media affect the viability ofM. anisopliaeconidia. PDA and SDA media, with their different nutrient availability, can influence the conidial germination rate. This aligns with the opinion of Safavi et.al (2007) that nutritional factors are one of the main determinants in conidial biosynthesis and viability. Shah and Butt (2005) also emphasized that the availability of energy sources and environmental culture factors play a role in determining the viability of entomopathogenic fungal conidia, as shown in Figure 2.

Figure 2
Development of the fungal isolateM. anisopliae(A), progressing to a collection of conidia (conidioma) (B), and a conidioma that has already formed hyphae (C).

From a field application perspective, treatments with high conidial viability have the potential to be more effective in infecting target insects. Conversely, isolates with low viability will result in reduced pest mortality. Therefore, selecting a combination of media and isolates that yield high viability is crucial to ensure the success of M. anisopliae as a biological control agent in Integrated Pest Management (IPM) programs.

3.3. Test of two isolates of the fungus Metarhizium anisopliae on several agar media against the mortality of corn powder beetles (Sitophilus zeamais)

At the beginning of the observation period (2–6 Days after Application), beetle mortality was still very low (0–2%) and did not differ significantly between treatments, as indicated by the same letter in the Duncan test results. This is normal, as entomopathogenic fungi require time to penetrate the insect cuticle, colonize internally, and produce the toxin that causes death (Table 3).

Table 3
Mortality of S. zeamais after application of several isolates of the fungus M. anisopliae.

Treatment	Mortality of S. zeamais
	2 DAA				4 DAA				6 DAA				8 DAA				10 DAA				12 DAA				14 DAA
	Mean (%) ± SE				Mean (%) ± SE				Mean (%) ± SE				Mean (%) ± SE				Mean (%) ± SE				Mean (%) ± SE				Mean (%) ± SE
Control	0.00	±	0.00	a	0.00	±	0.00	a	0.00	±	0.00	a	0.00	±	0.00	b	0.00	±	0.00	b	1.00	±	1.00	c	1.00	±	1.00	c
a1m1	0.00	±	0.00	a	1.00	±	1.00	a	2.00	±	1.22	a	6.00	±	1.87	a	12.00	±	2.55	a	23.00	±	2.00	a	32.00	±	3.39	a
a1m2	0.00	±	0.00	a	0.00	±	0.00	a	1.00	±	1.00	a	4.00	±	1.00	b	9.00	±	1.87	b	20.00	±	3.16	b	31.00	±	3.32	a
a2m1	0.00	±	0.00	a	2.00	±	1.22	a	3.00	±	2.00	a	11.00	±	2.45	a	21.00	±	4.00	a	32.00	±	3.39	a	44.00	±	4.00	a
a2m2	0.00	±	0.00	a	0.00	±	0.00	a	2.00	±	1.22	a	6.00	±	1.00	a	12.00	±	2.55	a	17.00	±	3.39	b	21.00	±	4.30	b

Starting at 8 DAA, beetle mortality increased in the a₂m₂ (6.00%) and was higher than the control (0.00%) and some other treatments. At 10 DAA, the a₂m₁ (21.00%) showed higher mortality than another treatment. This indicates that conidia in certain isolates germinate more quickly, penetrate the cuticle, and cause infection. The result of fungal infection M. anisopliae on S. zeamais are shown on Figure 3.

Figure 3
Fungal Infection M. anisopliae on S. zeamais.

The increase in mortality was more pronounced at 12 and 14 days after planting. The a₂m₁ treatment resulted in the highest mortality (32% and 44%), significantly different from the control and another treatments. The a₁m₁ and a₁m₂ treatments also showed relatively high mortality (around 20–32%), while the a₁m₂ treatment showed lower mortality. This indicates differences in pathogenicity potential between isolates and the influence of culture media on fungal virulence.

The significant differences between treatments indicate that the combination of isolate and culture media plays a significant role in influencing the virulence of M. anisopliae. A nutrient-rich medium can increase the production of conidia and secondary metabolites, thereby accelerating infection of test insects (Safavi et al., 2007; Shah and Butt, 2005). Furthermore, differences in the origin of fungal isolates also contribute to variations in pathogenicity due to fungal genetic factors.

These results align with those of Widiarti et al. (2019) and Manurung et al. (2012), who found that M. anisopliae isolates from different sources exhibited varying levels of mortality in test insects. The high mortality rate in the best treatment demonstrates the potential for this entomopathogenic fungus to be used as an effective biological control agent within an IPM program. The results showed that treatment of M. anisopliae isolates in different media significantly affected corn mealybug mortality. The best treatment resulted in the highest mortality rate and was significantly different from the other treatments and the control. This demonstrates that the appropriate combination of isolates and culture media can enhance the effectiveness of entomopathogenic fungi as biological control agents. Therefore, M. anisopliae has great potential for use in IPM programs as an environmentally friendly alternative to chemical insecticides.

4. Conclusion

• TheMetarhizium anisopliaefungal isolate from BBPOPT grown on SDA agar medium showed the highest colony growth with an average of 1.76x10^8/ml fungal spores.

• The combination of the isolate from BBPOPT on SDA medium was the best treatment, while the two treatment combinations of isolates from BBPPTP did not differ significantly and were the lowest on two types of agar media.

• From the viability test results, theMetarhizium anisopliaefungal isolate from BBPOPT grown on SDA agar medium also showed significantly different results from other treatments, reaching 81.25% of its average value.

• The highest mortality ofSitophilus zeamaiswas achieved by theMetarhizium anisopliaefungal isolate from BBPOPT on SDA agar medium, reaching 44%.

4.1. Recommendations

Further research can be conducted to find the medium with the best and most optimal concentration to produce a high level of virulence against pests. Some studies show that certain media can decrease or increase conidia virulence, but this depends on the isolate's agar medium and its environmental growth conditions. This is highly needed to enhance the use of biological control agent media in pest management.

Data Availability Statement

Research data is only available upon request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  20 July 2026
• Date of issue
  2026

History

• Received
  02 Feb 2026
• Accepted
  31 Mar 2026