Bioassay test of Beauveria bassiana alginate pellet formulation on mortality of Spodoptera frugiperda in the laboratory

The attack level of Spodoptera frugiperda greatly influences the production of maize in the field. The use of insecticides for control certainly has many negative impacts so that they switch to more environmentally friendly controls. One of them uses the entomopathogenic fungus (EPF) Beuvaria bassiana which has the ability to aggressively paralyze insect larvae and imago that are affected by its spores. The B. bassiana can be mass produced in both solid and liquid media. The aim of this study was to test the effectiveness of the B. bassiana alginatepellet formulation on spore density and mortality of Spodoptera frugiperda test larvae in the laboratory. The efficacy test of the formulation on the test insects was carried out by dipping the test larvae feed into the B. bassiana alginate pellet suspension in each treatment using 50third instar of larvae. Corn had an effect on the spore density of B. bassiana alginate pellets, the highest spore density with conidia density of 2.35x108 conidia/mlwas found in the wheat flour treatment as well as with the highest percentage of mortality of Spodoptera frugiperda larvae, namely on day 6-9 DAA (days after application) in a row of 60%, 68% and 70%. In addition, it can be concluded that the alginate pellet formulation made from the active entomopathogenic fungus Beauveria bassiana is effective in controlling the population of Spoduptura frugiperda larvae.


Introduction
Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) or better known as fall armyworm (FAW) isone of the most important maize noctuid pests in the world.FAW is known to be a migratory pest that is polyphagous and can attack around 353 host plant species from 76 plant families [1].This pest originally appeared in the neotropical region of the Americas but over time began to spread throughout the world through various intermediary media such as wind, humans, animals and the distribution of food contaminated by these pest eggs [2].In Indonesia, this pest was first detected in September 2019 in farmers' maize plantations in South Sulawesi Province, specifically in Gowa District (Botononompo) and Takalar District (Sandrobone).The losses caused by this pest attack are quite large and very detrimental to farmers, therefore farmers immediately take control measures in the field.Until now, synthetic pesticides are still widely used by farmers as the main choice to control Spodoptera frugiperda because they are easier to find in the market.However, this has actually caused resistance in FAW to several types of insecticides such as pyrethroids, spinosad, and organophosphates [3].In addition, the use of synthetic insecticides has many negative impacts on human health and the environment if used continuously at high doses and not in accordance with 1230 (2023) 012107 IOP Publishing doi:10.1088/1755-1315/1230/1/012107 2 health protocols [4].Therefore, a more sustainable and environmentally friendly alternative to S. frugiperda control in the field is needed, namely by using biological agents such as fungi and fungi.Entomopathogenic fungi are known as heterotrophic organisms that can live as parasites on insects [5].Entomopathogenic fungi work by infecting insects and then penetrating the cuticle layer of the insect, unlike the mechanism of action of viruses and bacteria which must be swallowed by the host insect.Entomopathogenic fungi can infect target insects by entering the host insect's body through the skin, digestive tract, spiracles, and other openings.In addition, the Beauveria bassiana fungus can also be transmitted vertically into the host body through the transmission of blastospores that are able to survive in the host body until the egg phase produced by the host [6].
EPF-based control is an alternative biological control that is an option for controlling the FAW pest itself in the field.However, further exploration is needed to obtain this biological control agency.Exploration is important to do in order to get the right and desired biological control agents, one of which is EPF which can be the first step in applying biological control techniques in the field.EPF itself can be obtained from naturally infected insects in nature, soil, or parts of the rhizosphere of plant roots [7].In addition, topical (direct contact) application of EPF, B. bassiana (Deuteromycotina: Hyphomycetes) has been shown to kill more than 80% of S. frugiperda larvae [8].A research study proved that the treatment of B. bassiana with a dilution concentration of 10 8 could cause the highest larval mortality of 64%, with the lowest percentages of pupal and imago development, namely 36% and 12% [9].
Conidia of the entomopathogenic fungus B. bassiana that can be applied can be in the form of suspension (not formulated), granules, and pellets, and all three show quite significant control results on their target insects [10].In addition, conidia of entomopathogenic fungi can be grown on artificial media such as Potato Dextorse agar (PDA) and glucose yeast agar (GYA).The content of the artificial media can affect the growth and development of B.bassiana and Metarhizium anisopliae [11].Some additives such as tapioca starch, wheat flour at a storage temperature of 5°C are effective in maintaining the viability of B. bassiana conidia for at least two months in storage [12].Corn flour contains carbohydrates (73.7 g) which is lower than wheat flour (77.3 g) and contains higher fat, phosphorus, iron, vitamin B1, and beta carotene pigment which functions as a precursor of vitamin A and antioxidants [13].Wheat flour has a water content of 14%, protein content of 8-12% ash content of 0.25-1.60%,and wet gluten of 24-36% [14].
Based on the description of the background above, it is necessary to conduct research on the bioassay of B.bassiana with the treatment of the formulation against S.frugiperda in the laboratory.

Preparation of Beauveria bassiana isolate
The B. bassiana isolates used were obtained from the Laboratory of Plant Pests, Hasanuddin University.The purified isolates were then multiplied on PDA (Potato Dextorse Agar) media.The media used for the pure propagation of entomopathogenic fungi.The composition of the media was made of 200 g of potatoes, 17 g of agar, 20 g of sugar, 4 capsules of chlormphenicol, 100 mL of distilled water.Then the ingredients were mixed and stirred evenly and then put in a 1000 mL Erlenmeyer then covered with aluminum foil and sterilized in an autoclave.

Propagation of Spodoptera frugiperda larvae
The larvae of Spodoptera frugiperda were collected from corn plantations in Takalar Regency and then brought to the Pest Laboratory for reared, then the eggs produced by the imago were transferred into several small containers filled with young corn feed until they hatched and reached the third instar larval stage which is ready to be tested.At the time of larvae propagation, observations were also made on the stages of larvae, pupae and adult.

Propagation of Beauveria bassiana on corn media
The corn media used is cornstarch (white) soaked which has been air-dried and then put into the media in the form of small clear plastic and then sterilized using an autoclave for two hours.To grow B. bassiana in corn media, the rejuvenated B. bassiana culture was taken in a Petri dish.Then transfer the B. bassiana in the Petri dish to a plastic filled with sterile corn, then store in a room with a temperature of 25-28 o C for two weeks.After B. bassiana has grown evenly in the corn medium, then pulverized using a blender machine until the corn media turns into flour.

Making Beauveria bassiana alginate pellets
Beauveria bassiana isolate which has been grown on corn rice media as much as 15 g, then added 4 g of sodium alginate and sterile distilled water with a volume of 100 mL then stirred slowly for five minutes until the ingredients are evenly distributed.The suspension that has been stirred is dripped into a 2% CaCl 2 solution using a sterile pipette as much as 10 mL to form pellets.After being stored, the bio-insecticide solution formed into pellets and then rinsed with sterile water and dried on filter paper for 3-4 days.After the pellets were dry, they were weighed and then stored in a cover glass at ideal room temperature and humidity (room temperature 24-28ºC and humidity 50-70 %).Likewise with treatments cornstarch to Maizena Flour made as in treatment control with the addition of media for each treatment 2 g of bran, wheat flour, tapioca flour and cornstarch.

Application of Beauveria bassiana alginate pellets
Application of B.bassiana alginate pellets to test 3rd instar larvae control treatment, rice bran, wheat flour, tapioca flour and cornstarch were weighed at 2 g per treatment, then crushed separately using a mortar until it became fine powder.The pellets were then placed in containers according to each treatment and mixed into 20 mL of distilled water each.Each pellet suspension was then homogenized using a vortex for 20 seconds.Each container contains 1 test larvae of S. frugiperda using five treatments and five replications.Each replicate uses 10 test larvae that are ready to be applied.The application method uses the immersion method, which is applied by dipping the testlarvae feed (baby corn) into the B. bassiana alginate pellet suspension/solution with each of these treatments.Observations were made every 24 hours for nine observations day after application (one-nine DAA), then observed and recorded the number of dead larvae and live larvae.While the larvae that are still alive are still being observed until they reach the adult.

Parameter observation of plants
The spore density of Beauveria bassiana was observed by dissolving B. bassiana alginate pellets with each solution treatment, take each suspension using a micron pipette and drop it on a spore counter (hemocytometer) and then count the number of spores.This observation was carried out only once, namely 1 week after the manufacture of the alginate pellets.
The spore density of B. bassiana can be calculated using the as shown in equation ( 1) (1) C = density of spores per ml of solution, t = number of spores in the sample box observed, d = dilution factor, n = number of sample boxes, 0.25 = correction factor using sample boxes, small scale in Hemocytometer, Observation of the mortality of test larvae of Spodoptera frugiperda the as shown in equation (2).
P = % mortality, n = number of test larvae that died, N = initial number of test larvae.

Data analysis
Research data were analyzed descriptively and quantitatively, as well as analysis of variance using Ms.
Excel and Minitab V.20.The results of the analysis of variance were significantly different, followed by Fisher's postoch test at the 95% confidence level.Observations that have been made show that the wheat flour treatment (Fungi B. bassiana (Cornstarch + alginate + wheat flour) has the number of spores observed in the hemocytometer and a high spore density compared to other treatments.The nutritional ingredients contained in wheat flour such as protein, starch, vitamins, fats/oils and the most important substances have a large percentage of content so that the B. bassiana spores formed in pellets can retain their nutrition so that the spores remain active.This could be because the media or materials used for the growth of entamopagenic fungi determine the rate of formation of new colonies/isolates and the number of conidia or spores during the growth of the fungus [15].

Observation of Mortality of Spodoptera frugiperda larvae
Potential of B. bassiana in killing Spodpotura larvae is very interesting to study where we know that the use of B. bassiana has many advantages including being easy to infect target insects (pests), does not kill non-pest insects, has many strains, can grow in vitro and safe for the environment.In addition, this fungus can produce toxins or poisons that can cause aggressive paralysis of insect larvae and imago that are affected by this fungus.Various types of these poisons that have been isolated include: beauvericine, beauverolide, isorolide and dyes and oxalic acid so that they can cause aggressive paralysis in insects [17].
The results of the Beuveria bassiana mortality test on the test larvae of Spodoptera frugiperda in all treatments showed that there was the highest mortality percentage of all treatments.Treatment control had the highest percentage of mortality from day 3 of DAA to day 5 of DAA, the highest in mortality of 48%.Furthermore, from the 6th day of DAA to the 9 th day of DAA, the cornstarch treatment (Fungi B. bassiana (corn rice) + alginate + wheat flour) had the highest percentage of mortality, the highest mortality was 70%.This happened at control from the start which showed a very high mortality, possibly because the spore density was still high so that the test larvae died faster.Several properties of EPF affect its ability to induce herbivorous insect mortality, namely, spore production and viability [17].This is also in line with a study which showed that B. bassiana with ICIPE 676 type could cause moderate mortality of 30% in second instar larvae [18].Beauveria bassiana mortality test against Spodoptera frugiperda in all treatments for 9 days after application (DAA), the results are obtained in the following table.42 a Note: The numbers followed by the same letter in the same column did not differ markedly in the BNT test level 0.05 (5%).Day 6 after application (DAA) showed very high mortality rates in almost all treatments.This was due to the treatment of the test material in the pathogenicity stage of the toxin originating from the test material to the test larvae so that at that time there was a significant mortality rate.It can be seen from the table that treatments control and cornsyrach have a high spore density so they also have the best average mortality rate compared to other treatments.The two treatments also had a mortality rate that was almost close to the mortality value.This is because the density of the applied fungal spores greatly determines the mortality of insects affected by Beuvaria basiana fungal spores [19].The higher the spore density, the higher themortality rate of the tested insects.Optimal spore density for pest control depends on the type of insect to be controlled.This is in line with studies using the application of B. bassiana which can increase larval mortality by demonstrating a negative effect on survival, duration, imago longevity, and fecundity in pests Coccinella undecimpunctata L. and Hippodamia variegate L. [20].Beauveria bassiana can be isolated by taking it from the body parts of infected target insects so that the level of virulence is high against the mortality of the insect pests tested.In addition, the high and low mortality of insect pathogenic fungi is also influenced by several factors, namely spore production, spore growth and spore viability of the fungus itself [20].The behavior of the test larvae of Spodoptera frugiperda was highly visible after the administrationof the test material in each treatment.When after being treated, the larvae eat feed that has been mixed with a pellet solution containing B. bassiana spores and then the spores that have entered the larva's body will grow into hyphae threads throughout the larva's body tissues so that they can damage internal organs and nerves.Parts of the digestive organs and so forth.As a result, the larvae will move slowly followed by a lack of appetite, their bodies appear limp, lethargic, stiff, movement is slow, the larval body's metabolism is disrupted, and damage to other important nerves can inhibit the growth and life cycle of the larvae.To control the larvae which generally hide in the young leaves of corn plants in the field, fungi or spores are needed which can colonize plant tissue or fungi which can attach to plant tissue which are a food source for S. frugiferda larvae [21].Endophytic fungi from B.bassiana usually associate with their host plants so that they can stimulate plant growth but can also suppress the growth of pest attacks that attack these plants [22].

Percentage of mortality of S. frugiperda larvae
The percentage of S. frugiperda larvae mortality in all treatments did not reach 100%.Likewise with the percentage of mortality of larvae (52%) and pupae (9%) if the total is only 61% (Figure 4).This is caused by the temperature and humidity factors that are not supportive in maintaining the quality of B. bassiana alginate pellets so that the spores in it gradually no longer survive.A study that has been conducted also shows that the percentage of insect mortality does not reach 100% due to environmental factors (humidity and temperature) which cannot be tolerated for the growth of the fungus B. bassiana [23].Humidity obtained during research usually ranges from 70.6-79.3and the temperature is between 28.6-32 o C. Different concentrations of conidia will result in different mortality, the higher the concentration of conidia, the higher the mortality (and can reach 100%) depending on the formulation used.These results are also supported by a study which found that a single application of B. bassiana reduced fourth instar Lycorma delicatula nymphs by 48% after 14 days.Application of B. bassiana to adult L. delicatula in the same park resulted in 43% mortality after 14 days [23][24].In addition, the application of Beuvaria bassiana can cause death of Nezara viridula by 66.67% [5] Live (Larva to adult) Died (Larva) Died (Adult)

Conclusion
The addition of formulation ingredients such as Cornstarch, wheat flour, tapioca flour and corn flour has in effect on the spore density of Beauveria bassiana alginate pellets, depending on the composition of the nutritional ingredients of the formulation.The treatment was the addition of wheat flour which had a high spore density of 2.35 x 10 8 spores per ml.Treatment control had high mortality of Spodoptera frugiperda larvae on day 3 -5 DAA respectively 24%, 38% and 48%, while treatment P3 (wheat flour) had high mortality of Spodoptera fr ugiperda larvae on day 6 -9 consecutive DAAs of 60%, 68% and 70%.

Figure 4 .
Figure 4. Percentage of mortality of larvae, pupae to imago of S. Frugiperda if accumulated in all treatments.

Table 1 .
3.1.Observation of B. bassiana spore densityFrom the results of observations one week after the manufacture of Beauveria bassiana alginate pellets (1time observation), the results are as shown in the following table 1: Calculation of the number of spores observed and spore density in each treatment.

Table 2 .
Average mortality of 3rd instar larvae of Spodoptera frugiperda larvae after application of B. bassiana atvarious concentration in laboratory.
. Percentage of S. Frugiperda larvae developing into pupae.Percentage (%) pupae develop into imago after being infected Beauveria bassiana on larvae instar 3.