RESEARCH ARTICLE | FEBRUARY 06 2024

Pathogenicity evaluation of Metarhizium anisopliae isolates

against sugarcane white grub Lepidiota stigma (Coleoptera:
Scarabaeidae) 
Nur Asbani  ; I. Gusti Agung Ayu Indrayani

AIP Conf. Proc. 2957, 090008 (2024)

https://doi.org/10.1063/5.0184324

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08 February 2024 10:51:55

 Pathogenicity Evaluation of Metarhizium anisopliae Isolates
Against Sugarcane White Grub Lepidiota stigma
(Coleoptera: Scarabaeidae)
Nur Asbania) and I Gusti Agung Ayu Indrayani

Research Center for Horticultural and Estate Crops, National Research and Innovation Agency (BRIN), Cibinong
Science Center, Jalan Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16915, West Java, Indonesia.
a)
Corresponding author: nura035@brin.go.id, nurasbani@gmail.com

Abstract. Damage caused by sugarcane white grub, Lepidiota stigma, significantly decreased sugarcane yield and may
cause a total yield loss if the grub is not controlled immediately. Until now, farmers have relied on chemical insecticides
to control the grub, which may be harmful to the environment and non-target organisms. Alternatively, Metarhizium
anisopliae, which is known for its effectiveness and wide host spectrum, can be applied against the grub. This research
was conducted at the Insect Pathology Laboratory of the Indonesian Sweeteners and Fiber Crops Research Institute from

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March to August 2020. The purpose of the study was to obtain the highest pathogenicity of M. anisopliae isolates against
sugarcane white grub. Treatment consisted of nine isolates, e.g. JT-1, JT-2, JT-17, BL-4, BL-8, BL-9, BL-10, BL-18,
BL-20, and control (untreated). This study used a single concentration of conidia of 1.5x109 conidia/mL, which was
equivalent to 1 g of dry conidia. The treatment was arranged in a complete randomized design (CRD) with three
replications. Parameters observed were white grub mortality and mycosis. The results showed that the JT-2 and BL-20
isolates caused high mortalities of white grub within a range of 80‒90%. All tested isolates also caused mycosis against
the white grub within 60‒90%. To determine the best between the two isolates, more tests are needed to find out their
level of virulence against white grub by estimating LC50. Further investigation of M. anisopliae pathogenicity and
virulence involving tri-trophic levels, namely the grub, fungus, and plant rhizosphere, needs to be done.

INTRODUCTION

Grub, a root feeder, significantly reduces sugarcane yield, especially when grown on sandy soils. The yield loss
due to the grub may reach more than 50% or even a total loss if the control is not taken as early as possible. A grub
species in Java and Sumatra, Lepidiota stigma, causes significant loss of sugarcane (Coleoptera: Scarabaeidae) [1,2].
The lack of effective, efficient, and sustainable grub control causes this pest always appear to damage sugarcane
crops.
So far, sugarcane farmers still rely on chemical insecticides to control the grub. In addition, it is common
practice to mix several brands without knowing their compatibility. Inappropriate and intensive use of chemical
insecticides may pollute the environment, including soil, water, and air; ultimately, it harms the human health and
sustainability of sugarcane production. Furthermore, the high price and its increasing price may reduce the profit of
sugarcane cultivation. The negative effects of the insecticide have led to the finding of alternative methods of
control measures.
The use of entomopathogenic fungi, Metarhizium anisopliae, to control sugarcane grub has been known since
the beginning of sugarcane cultivation in Indonesia. However, this alternative control has not been applied as
expected due to several technical and nontechnical constraints. The technical constraints include: (1) the limited of
M. anisopliae strains with high pathogenicity to sugarcane grubs and (2) M. anisopliae handling has not fully been
understood. In addition, the nontechnical obstacles include the lack of support from the government and
dissemination efforts of its use to farmers.

Proceedings of the 1st International Conference on Food and Agricultural Sciences (ICFAS) 2022
AIP Conf. Proc. 2957, 090008-1–090008-6; https://doi.org/10.1063/5.0184324
Published by AIP Publishing. 978-0-7354-4817-9/$30.00

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 M. anisopliae is known as a soil biota that has the potential to control insect pests both inside and on the soil
surface [3,4]. Many studies have been carried out on the fungus in the control of sugarcane grubs [5,6]. Most of the
research results show that the fungus is very effective in killing various species of grubs. Furthermore, a commercial
product had been produced that effectively reduces up to 50% of the sugarcane greyback beetle Dermolepida
albohirtum and the sugarcane white grub Antitrogus consanguineous in Australia [7].
In addition, a pathogenicity trial in the laboratory showed that M. anisopliae was more effective in killing the
grub of Leucopholis lepidophora by 62% at 45 days after treatment at a dose of 2x108 conidia/mL compared to other
fungi, such as Beauveria bassiana and B. brongniartii which only reached 55% and 59% of mortality [8]. Another
laboratory test also showed a high level of pathogenicity of the indigenous fungus M. anisopliae and commercial
products against the species of Chiloloba acuta grub, which was able to achieve mortality of 97.8% and 89%,
respectively [9]. The test results indicate that both native and commercial products of M. anisopliae have sufficient
effectiveness and pathogenicity.
Several new isolates of the M. anisopliae fungus had been collected from various soil samples located in East
Java and Bali. The level of pathogenicity of these isolates to sugarcane larvae of L. stigma is unknown, so a
pathogenicity screening test is necessary. This study aimed to obtain isolates of M. anisopliae having a high level of
pathogenicity against L. stigma.

MATERIALS AND METHODS

The study was carried out at the Insect Pathology Laboratory of the Indonesian Sweetener and Fiber Crops
Research Institute, Malang, Indonesia, from March to August 2020. It mainly consisted of two steps, i.e.,
preparation and pathogenicity.

Preparation

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The isolates of M. anisopliae were collected from East Java (JT) and Bali (BL), namely: JT-1, JT-2, JT-17, BL-
4, BL-8, BL-9, BL-10, BL-18, BL-20. The fungus was isolated from soil samples using Tenebrio molitor larvae as
sentinel/baiting insects [10,11]. Only cadavers showing M. anisopliae infection were collected; then, the fungus was
isolated and propagated on potato dextrose agar (PDA) media and incubated at 25ºC for 25 days. Subsequently, the
conidia were harvested, and the density was determined with a hemocytometer under a microscope [12].
The fungus requires soil as a transmission medium, and the grub also needs soil to be living. To imitate its
natural habitat conditions, the sandy soil from the sugarcane field in Asembagus, Situbondo, East Java, was used in
this investigation as a suitable habitat for the grub and fungus. To prevent infection of insects caused by non-tested
M. anisopliae, the soil was first sterilized using hot air at 100oC for 24 hours. The sterile soil medium was allowed
to reach room temperature before being used in the study.
In this experiment, the third instar larvae of L. stigma were used. They were collected from the grub-infested
sugarcane field in Asembagus District, Situbondo, East Java, collected around April–May, and at the third instar,
they were easier to collect because they were close to the soil surface. To find the sugarcane grub, the clumps are
slowly dismantled using a hoe; then the grubs are collected in a plastic tube filled with soil and pieces of fresh carrot
as feed for the grub. From each sugarcane clump, 1–2 grubs were commonly found, or even more if the level of
damage to the sugarcane was very high. The grubs were taken and reared in the laboratory feeding them fresh
carrots every 2 days until they were treated with the fungus.

Pathogenicity

In this study, nine isolates of the fungus were tested, namely: JT-1, JT-2, JT-17, BL-4, BL-8, BL-9, BL-10, BL-
18, BL-20, and Control. Each treatment was arranged in a completely randomized design (CRD) with three
replications. A concentration of 1.5x109 conidia/mL of the fungus was used, or equivalent to 1 g of dry conidia. To
prepare the suspension, the conidia were dissolved in 10 mL of water and added to 0.05% Tween 80 solution. Each
treatment consisted of 10 grubs which were placed individually in a 100 mL pot 5 cm in diameter and 7 cm in height
filled with 60 g of sterile sand. The suspension of the fungus was poured into each sterile soil medium and then
stirred to evenly mix. Each plastic pot was filled with 60 g of a soil and fungus mixture before being infested with a
third larva instar and a piece of ±2 cm2 of fresh carrot as feed for the grub, then closed slightly to maintain the air
circulation in the pot. The soil was kept moist, allowing the fungus-infected grubs.

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 The parameters were grub mortality and mycosis or the presence of M. anisopliae fungal colonies in the infected
grub. Mortality was observed from one day after treatment and the next observations were every 7 days until the
grubs entered the pupal stage, while mycosis was observed during the development of fungal colony in the infected
grubs or cadaver.

Data Analysis

Analysis of variance (ANOVA) was applied to the mortality and mycosis data. If the ANOVA was significant at
p<0.05, then the post hoc test of the least significance difference (LSD) procedure was tested at p<0.05 to compare
the pathogenicity among isolates. Additionally, probit analysis was used to determine the median lethal dose (LT50).
The ANOVA and the post hoc test were calculated with MS Excel, and the LT50 was analyzed with the Polo Plus
1.0.

RESULTS AND DISCUSSION

A cadaver infected with M. anisopliae was initially covered with white mycelia and turned a dark green color
when conidia formed (Fig. 1). The result showed that the L. stigma mortality due to M. anisopliae infection was
significantly higher than the control, and all of the isolates of the fungus caused high mycosis incidence (Fig. 2). The
mortality varied among the isolates indicating that each isolate had a different level of pathogenicity. The JT-2 and
BL-20 isolates resulted in higher pathogenicity, with mortalities of 80% and 90%, respectively, than the rest, with
mortality of 60‒80%. Jackson et al. [13] stated that each isolate of the M. anisopliae has different biological
characteristics due to differences in temperature and humidity requirements that affect the pathogenicity and
virulence against a given host. Regarding pathogenicity, Ortiz-Urquiza and Keyhani [14] stated that the level of
virulence of pathogenic fungi and host resistance is strongly influenced by the strain, host range, and virulence,
which can be restored through the insect passage mechanism.

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FIGURE 1. Symptoms of cadavers of L. stigma infected by M. anisopliae

The mycosis of the isolates ranged from 60% to 90%, indicating that all fungal isolates were able to adapt well
and make their host body a suitable source of nutrients for their development. The success of mycosis is largely
determined by the interaction between the fungal conidia and the cuticle of the host. Ortiz-Urquiza and Keyhani [14]
stated that the insect cuticle is the starting point for contact with fungi, as well as a barrier between fungi and insects.
The activity of the fungal inoculum on the cuticle surface will determine the interaction, whether it will lead to
mycosis or otherwise lead to infection failure so that mycosis does not occur because the insect's defense system
works effectively.
Mycosis is a further process of fungal infection that confirms the cause of mortality in host insects [15]. Mycotic
grub is usually indicated by aggressive fungal growth in the cuticle. In nature, the incidence of mycosis is very

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beneficial and becomes part of the horizontal transmission mechanisms from the infected hosts to healthy hosts.
Considering that the M. anisopliae is very persistent in the soil, the chance of contact with the host will also be very
high. If the occurrence of this mycosis occurs in the L. stigma larvae in the field, the accumulation of fungal conidia
produced from the grub with mycosis will be a potential source of infection for the next generation of L. stigma.
Furthermore, field observations showed that the L. stigma beetle has the behavior of laying eggs in the same place
for several generations, thus facilitating contact between the fungus and its new host generation.

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FIGURE 2. Mortality and mycosis of L. stigma larvae due to M. anisopliae infection. Different uppercase
and lowercase letters on the histogram show statistical differences among isolates in terms of mortality and mycosis, respectively
(LSD-test, p< 0.05)

The rate of mycosis increased linearly with the mortality rate of L. stigma grub (Fig. 2). It depends on the degree
of fungal infection. The more severe the infection rate, the higher the chance of developing mycosis. Environmental
conditions in the laboratory that can be controlled allow this linearity between mortality and mycosis. In addition,
soil sterilization is one of the efforts to homogenize and remove the influence of various factors that could mask the
activity of the treatment.
Various soil biota lives in the soil with their roles. Under conditions of sufficient food and nutrient availability,
soil biota can live in a balance between biota. On the other hand, if there is a lack of nutrients, certain biota can
change their behavior to become predators of other biotas. One of the soil biotas that acts as a predator of insect
pathogenic fungi is the amoeba group[16]. The predatory activity of such an amoeba group is an important factor in
the mycoses development, which the high activity hinders an epizootic in the pest population. As a result, the fungi
become less effective to control the pest population. However, human intervention may change this situation with
some techniques, such as releasing a reasonable amount of the entomopathogenic on the inundative release of the
entomopathogen.
The mortality of the grub in most isolates started after 14 days after treatment (DAT), then steadily increased to
56 DAT and did not increase again at 63 days after treatment (Fig. 3). All isolates showed high pathogenicity at 56
DAT, causing grub mortality of 60‒90%. Similar to other entomopathogenic fungal species, M. anisopliae is also a
slow-infecting pathogen because of its long incubation period. During the incubation period, the fungus completes
its developmental stages within the host's body, such as attachment of conidia to the host's integument (adhesion),
germination (germination), formation of the germ tube (appressorium), penetration into the host's internal organs
(penetration), growth (extrusion), and formation of conidia (sporulation)[17,18].

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 FIGURE 3. Cumulative mortality of L. stigma after application of several M. anisopliae isolates

The median lethal time LT50 of all isolates tested was roughly between 36‒51 days (Table 1). The LT50 of JT-2,
BL-4, and BL-20 was low, roughly within 37‒39 DAT, while the rest was more than 45 DAT. These median lethal
times are longer than LT50 reported by Indrayani & Ridhawati [19] previously that the fungus could result in 29 days
of LT50 at the half concentration of this study. The lethal time indicates the fungus virulence [20], which the low
LT50 means that they could kill sooner than the high one and vice versa. However, a lower LT50 was reported from

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India that was carried out on three species of sugarcane grubs, namely Cyclocephala spp., Phyllophaga spp., and
Popillia japonica at a concentration of 107 conidia/mL, achieved at 152.7 DAT [6]. Another study showed that the
LT50 against the Leucopolis lepidophora grub was 45 days at a concentration of 108 conidia/ml [8]. The natural
enemy of the sugarcane grub species outside of Asia is so diverse, especially entomopathogenic fungi, which also
tend to be more host species-specific. There are also quite several sugarcane grubs in Indonesia, but L. stigmas are
one of the most destructive species, especially in Sumatra and Java. In other countries, such as India, several species
of grub have been reported to damage sugarcane crops, including Holotrichia serrata, Adoretus versutus, Anomala
ruficapilla, and Leucopolis lepidophora, but the first species is the most dominant and most important [21].
Similarly, in several African countries, the attack of the grub of Adoretus emarginatus and Heteronychus licas is
quite high [22], but the population can be effectively controlled using the M. anisopliae [23].

TABLE 1. Median lethal time (LT50) of M. anisopliae isolates

Isolate LT50 (day) 95% Limit LT50 (lower-upper)
JT-1 46.7 43.5‒50.7
JT-2 36.7 34.3‒39.3
JT-17 50.6 47.8‒54.2
BL-4 38.9 36.3‒41.7
BL-8 51.2 48.4‒54.7
BL-9 50.1 47.2‒53.5
BL-10 45.8 41.6‒51.4
BL-18 46.7 43.3‒50.8
BL-20 37.2 35.7‒38.7

The framework of our study was based only on two trophic levels, namely the fungus, and grub. Meanwhile,
there is a more complex interaction that occurs naturally involving tri-trophic levels of interaction among the fungus,
pest, and plant. Interaction between the fungus and plant in the soil rhizosphere, which is mediated by root exudates,
may activate and promote fungus persistence [24]. Therefore, the interactions may increase the fungus virulence and
resulting an epizootic.

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 CONCLUSION

Two isolates of M. anisopliae, namely JT-2 and BL-20, are potential as biological control agents against white
sugarcane grub, L. stigma, which caused mortality of 80‒90%. All tested isolates were effective in causing mycosis
in the grub, with a range of 60‒90% of the grub infected with the fungus M. anisopliae experiencing mycosis.
Further study involving the tri-trophic levels is needed to understand the interaction among the fungus, plant
rhizosphere, and grub.

ACKNOWLEDGMENTS

The authors would like to thank Herlin, who assisted in laboratory work and collected the grubs and soils from
the sugarcane field.

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