Effectiveness test the entomopathogenic fungus Metarhizium anisopliae (Metch) at various concentrations against Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) larvae in the laboratory

Spodoptera frugiperda is one of the most important major pests of maize in the world. Armyworms are polyphagous, the main target is corn, pests can move to other hosts if there is no primary host so that these pests can survive and maintain their populations throughout the year. S.frugiperda is also considered a serious insect pest because it reproduces in several generations per year and adults can fly up to 100 km per night, giving it the potential to spread widely in a short time. This study aim to determine the effectiveness of the entomopathogenic fungus Metarhizium anisopliae (match) at various concentrations against S. Frugiperda. This study was conducted in laboratory conditions, using a completely randomized design using four treatments and five replications. Each replicates used 20 test larvae. the treatments were: control, concentration of M. anisopliae 107, M. anisopliae 108, and of M. anisopliae 109. The larvae used as test larvae were instar 2 larvae, and observations were made every day to see the mortality of the test insects and record the symptoms that appeared in larvae after the application of M. anisopliae. The results showed that there was an effect of mortality on the test insects seen in treatment 108 showing the highest treatment of 9.15 days after application compared to all treatments. at 14 days post-application. The results of this study indicate the need for proper development for the purpose of effective pest control in crop cultivat.


Introduction
Corn is one of the most important crops in the tropics and comprises wheat and rice, a major proportion of the daily food intake of the population.World maize production is estimated at 1,291 million tonnes in 2016.In Indonesia, maize provides almost half of the calories consumed in both rural and urban areas becoming a staple food in many parts of the country.The lion's share of maize production is associated with small-scale farmers and is a direct source of household livelihoods [1].The armyworm Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae)) is one of the major pests of maize crops that is very important in the world [2].This pest originated in the Americas and spread to various other continents, including Asia.This pest is a strong flier with a high level of adaptability.adaptability, S. frugiperda is able to spread rapidly.S. frugiperda is polyphagous with a wide range of host plants from various families, especially Po. S. frugiperda is polyphagous with a wide host plant range from various families, especially Poaceae, which consists of corn, sorghum, and rice, while soybean, cotton, and other monoculture crops are the most common host plants.other monoculture crops are alternative host plants [3].This pest can move to other hosts if there is no main host.So this pest can survive longer and can even maintain its population throughout the year.There are several reports on armyworm infestation in Indonesia, but not much is known about more comprehensive reports [4].
Since 2019 the S. frugiperda insect has entered Indonesia.Its existence was first discovered in West Pasaman Regency, West Sumatra.These insects enter without being followed by their natural enemies and directly attack the corn plants.so the existence and development of the population need to be watched out for.S. frugiperda has spread to several other areas, even to areas that are centers of corn production.S. frugiperda is also considered a serious insect pest because it reproduces in several generations per year and adults can fly up to 100 km per night [5], giving it the potential to spread widely in a short time.This pest attacks the growing point of the plant which can fail the formation of young shoots/leaves of the plant.Larvae have high feeding ability.The larvae will enter the plant parts and actively eat there, so if the population is still small it will be difficult to detect [6] and the severity level in corn plants ranges from 26.50 to 100% [7].
S. frugiperda larvae voraciously feed on foliage and cause extensive damage to seedlings, with pest infestation sometimes resulting in total crop losses.The larvae of S. frugiperda feed on the vegetative and reproductive tissues of maize plants and thereby either directly or indirectly reduce yields and crop quality [8].This pest causes damage to several host plants (maize, peanuts, cotton, soybeans, and forage), maize is the main agricultural crop favored by S. Frugiperda.The most serious damage produced by this pest is the continued consumption of young shoots which reduces the photosynthetic area of the plant.These pests are generally controlled using synthetic insecticides.However, the resistance of FAW to many insecticides, such as pyrethroid, spinosad, and organophosphate insecticides has occurred.In addition, insecticide application has a negative impact on human health and the environment.Alternative control methods that are more sustainable and environmentally friendly against S. Frugiperda are urgently needed [4].
Currently, the most widely used method to reduce the population of S. frugiperda is spraying chemical insecticides, but despite its fast action, the larvae have developed resistance as an effect of this control method, and cause environmental pollution.Moreover, S. frugiperda larvae continue to feed inside the shoots of plants reducing contact with the insecticides used for their control [6].The demand for organically grown food requires methods that utilize non-chemical inputs for pest control to reduce the side effects of pesticides that hazardous to public health and the environment.Pest management involving biocontrol agents is considered prominent and has been considered an important strategy in reducing insect populations.One of them is utilizing entomopathogenic fungi [9].
Entomopathogens are natural microbial control biological agents that can effectively control pest populations.Several entomopathogenic fungi have been used to control insect pests from various insect orders, one of which is the Lepidoptera order.The use of synthetic insecticides above the economic threshold and exceeding the usage limit can result in the re-emergence of new pests, environmental degradation, and the development of resistance, but it can also cause harmful effects on human health.The consequences of adverse effects of chemical insecticides emphasize the need to develop alternative control strategies that are more environmentally friendly.In addition, resistance to insecticides limits control strategies, while insect pests are unable to develop resistance to entomopathogenic fungi, making them an effective weapon in controlling pest populations [10].Entomopathogenic fungi are widely used as biological agents against various pests in agriculture and forestry that are attacked by plant pests and aquatic invertebrates.Actually, the use of entomopathogens in pest control dates back about 100 years.This is considered because the use of natural insecticides has been established as one of the most promising pest control options for breeding important insects or other arthropods [11].
Entomopathogenic fungi have the potential as a biological agents to control pest attacks.One is Metarhizium anisopliae recently, the full potential and many advantages of this practice reach application on a commercial scale using M. anisopliae this fungus releases toxins (M.anisopliae produces destruction) which correlates with its etiopathogenesis [12] This fungus is used as a biological control agent for insect pests.Fungus propagation can be done using various media [13].The full potential and many advantages of this practice achieve application on a commercial scale by using M. anisopliae with the ability to synthesize antagonistic compounds.It causes infection by growing through the insect's body and releasing extracellular cuticle hydrolyzing enzymes (proteases and chitinases) [14].And fungi are biological and general control agents of various types of insects.has been widely exploited [15].
This study aims to determine the effectiveness of the fungus entomopagen M. anisopliae (match) at various concentrations against the armyworm S. frugiperda.Until now, there has not been much information regarding the effectiveness of the fungus M. anisopliae at various concentrations on the armyworm S. frugiperda.The absence of a report on the results of this study makes this research important because it can be an alternative solution for controlling the main pests of maize that is environmentally friendly and practical in its application.

Experiment design and implementation
The design used was a completely randomized design (CRD) using four treatments and five replications.Each replicates used 20 test larvae.The larvae used as test larvae were 2nd instar larvae, while the treatments were: Control, M. anisopliae concentration 10 7 , M. anisopliae concentration 10 8 , M. anisopliae concentration 10 9.

Rearing of Spodoptera frugiperda
The S. frugiperda insect, which was used in this study, was obtained from a corn plantation located in Kec.Manggala Makassar City in the form of larvae, then the samples were rearing in the Hasanuddin University Pest Laboratory.S. frugiperda larvae were reared in plastic containers and fed baby corn.Then when it is about to enter the pupal stage, the insect is transferred into a plastic container filled with sterile sand (which has been baked) which has been given a little water.After the insect enters the imago stage, the image is moved in a cage by hand.In a cage containing male and female imago and given cotton that has been dripped with 10% honey as nutrition for the image, this cotton is placed at the top of the cage by hanging using thread, besides that in the cage also given fresh corn leaves for laying imago eggs.After the images produce eggs, the eggs produced are reared to obtain larval uniformity until they reach the second instar larval stage which is ready to be tested.

Rearing of Metarhizium anisopliae
M. anisopliae isolates were obtained from the Maros Food Crop Protection and Horticulture Center.On September 16, 2020, the M. anisopliae isolate was subcultured on potato dextrose agar (PDA) media in a petri dish.The fungi were multiplied until the test insects were ready to be applied, while the age of the fungi used during application was 4 days after purification, then when the concentration of the conidia suspension was prepared by adding sterile distilled water to a petri dish containing the culture of the M. anisopliae fungus.The cup is shaken and then with the help of a small sterile brush, the conidia can be removed.The suspension is filtered and then put into Erlenmeyer.Then prepared test tubes filled with sterile distilled water 9 ml each and then dilutions of 10 7, 10 8 , and 10 9 were carried out.Then the results of these dilutions were taken using a micron pipette and then dripped onto the hemocytometer and covered with a cover glass.Then the spore density was calculated using a hemocytometer under a (1) Information: S: number of spores/ml t: total number of spores in the observed sample box n: the number of sample boxes observed 0.25: is a correction factor using small-scale sample boxes in Haemocytomeyer.

Application of Metarhizium anisopliae
M. anisopliae suspension, applied using the topical application method.The test insects were placed in small containers containing baby corn feed.Each container contained 1 test larvae, dripped with one drop for each concentration treatment or equivalent to (45.45 µl).For the insect control treatment, it is only dripped with distilled water.Observations were made every day to see the mortality of the test insects and record the symptoms that appeared in the larvae after the application of M. anisopliae.

Koch's postulate test
From the results of applying the fungus M. anisopliae to the dead S. frugiperda pest, then re-isolation was carried out on PDA media, the purpose of which was to do re-isolation of the entomopathogenic fungus to prove whether the dead insects were caused by the fungus M. anisopliae.

Observation parameters
Observations were made by counting the number of insects that died for each treatment and recording the symptoms that appeared in the larvae after the application of M. anisopliae.Observation of total larval mortality started after one day of application (24 hours after application) until the last day of observation, Meanwhile, after the application the insects are still alive until they change stadia, the observation continues until the insects die.Calculation of the average percentage of larvae that develop into pupa and imago.

Data analysis
The analyzed data is processed using fingerprint analysis (ANOVA) to test the treatment to be given.if there is a real or very real effect, then proceed with further tests using the smallest real difference (BNT) further test at the level of 5%.And the purpose of data transformation is to change the scale of data measurement into other forms to meet the assumptions of SQRT analysis (original data + 0.5).Note: Numbers followed by different letters in the same column mean significantly different on the 0.05 LSD test.

Percentage of mortality of S. frugiperda larvae
From table 1 it can be seen that the 2 observations showed larval mortality in treatments 10 8 and 10 9 .The 5-14 observations, showed an increased mortality rate from each treatment up to 14 has, and the highest mortality rate was in treatment 10 8 followed by treatments 10 7 and 10 9 which were significantly different from the control treatment.This was due to differences in the density of conidia from each treatment which could affect the percentage of mortality of the test larvae.The symptoms of the test insects or the behavior of the larvae since exposure to M. anisopliae are starting with slow movements, decreased appetite, changing color to pale, then a few days later black, and finally, the larvae change color and become stiff.
The mycelium that grows in the host's body will attack the tissue, if it has been attacked, the host will die, but the fungus will continue to grow.The fungus forms new conidia on the host's body (saprophytic phase) [16].Other studies using cigarette butts will be toxic and attack the nervous system in spodoptera larvae, and soursop leaf extract act as an acetogenin which is toxic to mitochondria.The high mortality rate that occurred in this study was because the larvae used were second-instar larvae which had very thin cuticles, making it easier for the fungus to infect.The younger the instars used, the higher the mortality rate of the larvae.In addition to being influenced by the growth medium, the virulence level, and the frequency of application, the effectiveness of the fungus is also largely determined by the age of the insect instars [17].Conidia attach to and germinate on the cuticle and penetrate the body of the insect.After entering the hemocoel, mycelia grows and spreads throughout the body then forms hyphae and produces blastospores.Host mortality often occurs due to a combination of fungal toxins, physical obstruction of blood circulation, nutrient depletion, and organ invasion.)[18].
The data showed that the density of conidia affected the mortality of the test larvae.Low mortality occurs when the density of conidia applied is high so that the density of conidia given must be appropriate for controlling armyworms with optimal function, if it is excessive (such as conidia density 10 9 ), there will be a decrease in effectiveness, presumably this is related to disturbed feeding activity of larvae.This can be seen from the low percentage of larval mortality from other treatments.This is presumably because each biological agent has a different optimal limit of pathogenicity so if it is applied 1230 (2023) 012109 IOP Publishing doi:10.1088/1755-1315/1230/1/0121096 to insects and exceeds the optimum limit there will be a decrease in effectiveness, for example, there is competition in nutrients and space between these biological agents.The concentration of entomopathogenic fungi must be determined precisely to obtain optimal control results and the frequency of application needs to be known precisely so that the pest population is below the control threshold value.There are several pathogenic factors possessed by entomopathogenic fungi, including toxins contained by the fungus, conidia germination speed, conidia germination power, growth, sporulation, and conidia size [19].
Pathogens sprayed on plants, besides being able to enter the larvae through the sensitive skin of the larvae, are also directly consumed by the larvae through the leaves they eat.Treatment with a concentrated suspension containing more conidia and the development of the fungus in the body of the larvae will be faster and cause the eating activity of the larvae to be disrupted.Germination Once the conidia adhere to the host cuticle, they germinate in the presence of exogenous carbon and nitrogen sources, the latter of which is preferably used.Trehalase, which utilizes trehalose commonly found in host hemolymph, can be observed during early germination.Trehalase activity is thought to supply glucose for energy production [20].

S. frugiperda Larvae Developing into Pupae and Imago
Observations that have been obtained from the 7th day of HSA or starting from the formation of pupae show that the development of S. frugiferda larvae into pupae and imago has different population levels.The percentage of the pupal population with the highest average compared to other treatments was in the control treatment with a value of 50%.The existence of larvae that can still form pupae because the larvae have the ability to prevent infections caused by entomopathogenic fungi.Besides that, due to the mobility of the insect and the molting event that entered the next phase, not all of the applied entomopathogenic fungal conidia reached the target, while the formed pupae showed deformed symptoms.The symptoms that arise in the pupae due to M. Anisopliae are the color of the pupa becomes black and the body becomes soft.The pupae that are formed are not normal, varying as there are half pupae where on the abdomen the pupa is formed intact but on the candidate the head of the pupa does not change, it remains the shape of the larva's head.In the control treatment the pupae were completely formed from other treatments.In treatments 10 7 , 10 8 , 10 9 , many pupae and larvae were found that failed to pupate and then died [21].The percentage of pupae that become imago can be seen in the picture above, the lowest population level is found in treatment 108 with a value of 12%.In the 14th observation HSA S. Frugiferda entered the imago stage, the highest observed results were found in the control treatment with a value of 48%, and in treatment 10 8 it was the lowest treatment in population, namely with a value of 8%.At the stage of formation of pupae into imago, it was found that the highest population of pupae that formed into imago was found in the control treatment when compared to the three treatments.Pupae that do not turn into imago do not show signs of life so that the pupa is declared to have failed to form an imago.Success in the process of development from larva to pupa and from pupa to imago is related to the concentration level of the fungus applied to S. Frugiperda larvae (Figure 2) [21].From observations, it can be seen that the dead larvae turn black.Besides that, it can also be seen that the body of the larvae shrinks and hardens.This is because, during the developmental phase of the fungus, it starts by attacking the tissue and ends with the formation of the reproductive organs so that the body of the infected larvae shrinks.when the insect dies, the saprophyte development phase of the fungus starts with tissue invasion and ends with the formation of reproductive organs that grow inside the body of the larvae which suck up the liquid until they run out so that the larvae die with a hardened body like a mummy.[17] The fungus M. anisopliae produces a cyclic peptide poison called Destruxin, this compound is composed of five amino acids namely Proline, Isoleucine, Methyl-Valin, Methyl-Alanine, and beta-alanine.Destruxin has effects that cause functional abnormalities of the middle stomach, hemocyte, malpighian tubules, and muscle tissue in the host [22] which correlates with its etiopathogenesis [12].M. anisopliae invades the host's body and absorbs fluids from the host's body, the fungus grows out of the host's body and produces conidia so that the host's body becomes hard (mummification).This is due to the start of the action of the toxin produced by the fungus.The toxin

Results of reisolation of S. frugiferda larvae after application of M. Anisopliae fungus
The collected cadaver was then isolated and purified on Potato Dextrose agar PDA media.Then the results of the purification (Figure 4.) were then identified to find out whether the insects that died were a result of the effects of the treatment given.The results of the isolation and purification of infected cadavers were then identified macroscopically and microscopically.macroscopic characteristics; a and b M. anisopliae fungi, namely the color of the colony is yellow-green and yellowish-white and the shape of the colony is not flat on the edge or surfacea.then identified microscopically.Hyphae and spores of M. anisopliae were also detected on microscopic identification with 400x magnification; c, c1, c2.The morphology of the hyphae and conidia of M. anisopliae is cylindrical and single-celled, hyaline colored, and spherical [22].

Conclusion.
The results showed that the effectiveness test of the entomopathogenic fungus M. anisopliae at various concentrations on the armyworm S. frugiperda was able to kill the test larvae, namely treatment 10 7 and the most appropriate or having the most significant effect, namely treatment 10 8.So it is recommended to use this concentration.

Figure 1 .
Figure 1.The percentage of larvae becoming pupae.
and absorbs the body cell fluids of the larvae, causing the larvae to dry out and die.

Table 1 .
Average Mortality Percentage of S. frugiperda after application of M. anisopliae at various concentrations (after transformation).