Isolation, molecular characterization, and virulence assays of entomopathogenic fungi of Oryctes rhinoceros larvae from Asahan origin

Palm oil is the most important plantation commodity in Indonesia. One factor that can suppress oil palm productivity in the field is due to pest attacks, one of which is the attack of the Rhinoceros beetle called Oryctes rhinoceros. This study aims to isolate entomopathogenic fungi found in Oryctes rhinoceros larvae at oil palm plantations in Tanjung Alam Village, Asahan, North Sumatra. The results showed that the fungus isolates had conidia that were greenish and could cause mummification which varied for 7-24 days. The results of molecular identification by PCR (polymerase chain reaction) using ITS1-ITS4 primers showed that the isolates of the entomopathogenic fungi were Metarhizium majus and Metarhizium anisopliae species with DNA bands measuring around 500 base pairs (bp).


Introduction
Palm oil is one of the country's large foreign exchange depositors.For this reason, the productivity of oil palm in Sumatra and Kalimantan needs to be optimized.One of the obstacles in oil palm cultivation is pests.Pests can be defined as insects or mammals that disturb and or destroy plants either economically or aesthetically [1].Oryctes rhinoceros or rhinoceros beetle is one of the pests that often attack coconut farms and also oil palm plantations.This insect can reduce crop yields and cause young seedlings to die in seedbeds [2].
To control the insect population, the farmer is using synthetic pesticides.Even synthetic pesticides can be toxic, but it's helping farmers very much [3].However, synthetic pesticides are substances that are not friendly to the environment resulting in the emergence of pest resistance and resurgence.So it is not recommended to be used more widely.
The most appropriate method to control Oryctes rhinoceros is a biological method, by using bioinsecticides.Bioinsecticide is an environmentally friendly pesticide because the compounds contained in it are easily decomposed in nature or with natural ingredients [4].
In the field, indigenous isolates are sometimes found capable to become pathogens of insects.One of them is the isolate of the fungus Metarhizium sp.This fungus is known to be able to attack and cause death in Oryctes rhinoceros.To ensure the entomopathogen we need to identify the fungus.
At least two methods to identifying fungus such as morphological observations with a key determination approach, and a molecular approach with polymerase chain reaction.Polymerase Chain Reactions (PCR) is an enzymatic method used for DNA amplification in vitro.DNA amplification by PCR can be successful when using oligonucleotide primers or refractions called amplimers.This 1308 (2024) 012028 IOP Publishing doi:10.1088/1755-1315/1308/1/012028 2 DNA primer serves to initiate the synthesis of DNA chains.PCR is possible to multiply a DNA fragment.The primary design was built based on a known DNA sequence or from the intended protein sequence.DNA or protein sequence data can be obtained from the GenBank database [5].The internal region of transcribed spacers (ITS) is a region of DNA sequences that do not encode functional proteins and is located in the ribosomal RNA (rRNA) region [6].The purpose of this study was to determine the ability of a fungal indigenous pathogen originating from Asahan in infecting Oryctes rhinoceros and identifying using a molecular approach by using Polymerase Chain Reaction (PCR).

Place and Time of Research
Field-scale research was conducted from August 2020 to February 2021.Larvae samples were collected and semi-field tested at the Oil Palm Estate located in Tanjung Alam Village, Asahan, North Sumatra.Meanwhile, fungal DNA preparation and PCR activities were conducted at the Molecular Laboratory of the Oil Palm Research Center, Bogor Unit, Jl.Taman Kencana No. 1, Bogor.

Isolation of fungal from the Field
Larvae were collected from the field, then larvae that showed mummification symptoms were separated and observed for conidia that surrounded the surface of the larvae.The difference in conidial color served as a specific differentiator for fungus species.The larvae showing mummification symptoms were sent to the Indonesian Oil Palm Research Institute (IOPRI) laboratory in Bogor.In the IOPRI laboratory, the larvae with mummification symptoms were surface sterilized by soaking in a 70% alcohol solution for 30 seconds.Next, the larval body was longitudinally split, then a small piece of tissue was taken from inside the body using tweezers and grown on Potato Dextrose Agar (PDA) (200 g potato, 20 g sucrose, 17 g agarose, 1 L equates were autoclaved at 121 °C and 1 atm for 15 minutes).

Fungal DNA Isolation
Hyphae of the insect fungus that grew 7 days on the surface of PDA media were taken with sterile tweezers from the Petri dish, placed on aluminum foil, weighed, and labeled.Then, placed hyphae on sterile mortar and poured slowly liquid nitrogen sparingly.Afterward, the sample was crushed on the mortar, until dried, then the sample was placed on the tube and marked as A1, A2, B1, B2, C1, and C2.DNA extraction methods are followed as developed by [7].Then electrophoresis bath was prepared and filled with 0.5X TBE buffer.The gel was put in the bath until submerged.The loading buffer is prepared on a parafilm layer, every sample was taken as much as 5 and then mixed with 2 μl loading buffer.The samples were inserted into electrophoresis wells.The electrophoresis device is run by connecting a power supply.

Polymerase Chain Reaction (PCR)
A twenty point eight μl of nuclease-free water (NFW) was taken using a micropipette and then inserted into a tube.Then the red Mix DNA polymerase (Bioline) is inserted as much as 20 μl, ITS 1 as much as 1.6 μl, and ITS 4 as much as 1.6 μl into the tube containing NFW, then flips several times.PCR was performed on 3 samples of fungus Samples of 1, 2, and 3 @100 ng were inserted as much as 1 μl into the new tube.The mix that has been made was inserted as much as 9 μl into the tube containing the sample.The extracted DNA was amplified using forward primer ITS1 (5'-TCCGTAGGTGAACCTTGCGG-3') and reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3').ITS1 and ITS4 primers will amplify DNA with molecular weight in the range of 500 bp [8].The cycles used in this PCR process are 35 cycles.The process cycle of PCR is as follows.Pre-denatured at 95°C for 3 minutes; denaturation at 95°C for 15 seconds; annealing at 58°C for 15 seconds; extension at 72°C for 15 minutes; and post-extension at 72°C for 7 minutes [9].
For making agarose gel, as much as 0.2 g agarose was put into Erlenmeyer and poured by 20 ml TBE 0.5X then heated using a microwave until it dissolves.One μl Peq green DNA dye was then added and stirred until homogeneous.After that agarose solution was poured into the mold and waited 30 minutes until solid.PCR samples were inserted 5 μl into the wells in the agarose.The electrophoresis starts at 220 V for 30 minutes, the gel was taken and visualized with UV light using a gel doc to photograph and view its DNA band.

Virulency Assay Test
The testing was carried out by placing 50 larvae of O. rhinoceros instar 2 and instar 3 each into container 20 L called "jirigen" that can be opened and closed.The jirigen is also given empty fruit bunch (efb) litter and organic litter around oil palm plantations up to one-third of the volume of jirigen (Figure 1).The test was carried out with as many as 5 repetitions so that as many as 250 larvae were needed.Jirigen containing O. rhinoceros larvae was then inoculated with 400 ml of inoculum solution each, which is a solution made by destroying 8 larvae infected with Metarhizium in 1000 ml of water or the equivalent of 10 5 spores per ml.The observation was held on 7, 11,15, 19, and 24 days after inoculation.In every observation, we measure the infected percentage of larvae.
Figure 1 Preparation of virulency assay test such as selecting the larvae, gathering the organic material, and placing both into the container.

Results and Discussion
3.1 Fungal isolation from the infected body of Oryctes rhinoceros larva O. rhinoceros were multiple on piles of organic matter that are undergoing weathering process.Imago will scrape the shoots of oil palm plants so that they can inhibit plant growth.If the attack damages the growing point, it will be able to kill the plant.Controls technique that is widely used by farmers generally uses synthetic insecticides.Pest control that prioritizes the use of synthetic insecticides is ineffective, unsustainable, and results in various negative impacts [10].The negative impact of the use of synthetic insecticides such as resistance, pest resurgence, killing of natural enemies, increasing of residue on yield, environmental pollution, and health problems for users [10].
From the observation, we conclude the targeted fungus is Metarhizium spp.Biologically, controlling O. rhinoceros is carried out using the entomopathogenic fungus Metarhizium anisopliae [11].Metarhizium anisopliae fungus is known to effectively control insect pest larvae.M. anisopliae fungi can control various species of insect pests both living in the soil and the plant canopy.The wide range of hosts indicates that this fungus has the potential to infect various species of insect pests [12].The use of entomopathogenic fungi has been applied overseas as an alternative to environmentally friendly pest control to reduce the negative impact of the use of chemical pesticides [13].The entomopathogenic fungus M. anisopliae can be isolated from soil and infected insects.In the soil, this fungus is saprophytic [13].
Insect larvae infected with M. anisopliae will show symptoms of attack, change in color, and black rickshaws on the cuticle look like traces of fungal penetration (Figure 2).If environmental conditions are favorable, the surface of the larval body will appear as mycelia.The affected larva usually secretes a reddish liquid from its mouth continuously.After death, at first, the body is soft and within 5 hours becomes stiff (mummy).A day later his body was covered with mycelia.Colonies of Metarhizium obtained from the field show different colony colors (Figure 3).The first isolate showed a bright green color, while the 2nd isolate showed a grayish-green color.Microscopic observations show conidiophores that grow upright, hyaline, and branched.In addition, it was observed that conidia are produced in the form of chains.Conidia are cylindrical to slightly oval, hyaline, and one-celled (Figure 4).Conidiophores can reach a length of 75 μm, stacked -stacked and enclosed by apical conidia measuring 6-9.50 reams x 1.50-3.90reams, branched, clustered to form a dense, loose mass [14].Mycelium in Metarhizium has partitions, is colorless, and conidia grow upright.Fungi have partitioned mycelium, and branched conidiophores filled with spores, called conidia, which are oval-round in shape [15].

DNA Isolation, PCR, and Molecular Identification Results
After the DNA isolation process, then we measure DNA quantity using Nanodrop.The quantity of extracted DNA was tested using Nanodrop to see its concentration and purity.DNA concentration measurements with Nanodrop were carried out at a wavelength of 260 nm, while proteins were measured at a wavelength of 280 nm [16].The results are shown in Table 1.Based on the results of measuring the concentration and purity of DNA, the highest DNA concentrations were obtained, namely sample 1, sample 3, and sample 2 with values of 3090.6 ng / μl, 2272.1 ng / μl, and 1661.8 ng / μl.Meanwhile, the highest DNA purity was obtained respectively, namely sample 1, sample 3, and sample 2 with values of 2.07, 2.12, and 2.16.DNA purity is determined by calculating the absorbance ratio at A260 to A280 (Ratio A260:A280).DNA molecules are said to be pure if their absorbance ratio ranges from 1.8 -2.0 [17].If the value of the A260/A280 ratio is less than 1.8, then this indicates that the resulting DNA isolate still contains contaminants in the form of phenols, and allegedly solvents used was too much.Meanwhile, if the value of the A260/A280 ratio is more than 2.0, then the resulting DNA isolate still contains contaminants in the form of proteins and other compounds [18].Measuring the DNA concentration of all sample isolates obtained a relatively high concentration.[19], explained that this high concentration is due to the entire DNA, obtained through the manual DNA isolation method, in the sample nothing is wasted as a result of the screening process.Based on the results of DNA quality readings, the DNA bands obtained are in the form of thin lines in sample 1 and sample 3 isolates (Figure 5).However, the DNA bands obtained in the sample 2 isolate are very thin lines.This is related to the relatively low concentration of DNA obtained.The higher the concentration of DNA obtained, the thicker and brighter the band formed [20].From the PCR results the DNA band were thick and clear.Marker DNA using a 1 kb marker plus ladder.This marker contains 13 double-stranded DNA fragments and is suitable for measuring double-stranded DNA from 250 bp to 10,000 bp.The 1 kb plus ladder is a unique combination of several exclusively ingested plasmids with appropriate restriction enzymes and PCR products used as molecular weight standards for electrophoresis.PCR was successful, and a band appeared (Figure 6).The primer amplified DNA with a molecular weight of around 500 bp.After sequencing, blasting, and identifying we concluded that sample 1 was Metarhizium majus with an identical percentage of as much as 83.45%.Sample 2 has been identified as Metarhizium anisopliae with an identical percentage of as much as 80.89%.Both sample 1 and sample 2 are coming from the Asahan field, taken from larvae that were suspected infected by Metarhizium.sample 3 was identified as Fusarium torulose which is a laboratory collection (Table 2)

Virulency testing in semi-field in Asahan
The results of semi-field testing in the Asahan district showed that Metarhizium infection continued to increase slowly at every observation starting from observation 7 days after inoculation to observation 24 days after inoculation (Figure 7 and Figure 8).The result of 5 repetitions shows the same trend.At 7 days the average infection of all 5 repetitions is 14.44 %.At 11 days of observation, the infection rate was 28.71%.On day 15 observation, the average infection was as high as 46.91%.On the 19th day after inoculation, the average infection progressed by 70.36%, and on the last observation on the 24th day, the average infection was 91.2%.The observation was stopped on day 24 because one of the repetitions had reached infection of 100% of the population.The resulting linear regression equation is Y = 14.027X+24.865with an R 2 value of 0.9634.R 2 value close to 1 indicates a strong and reliable level of data acceptability.

Conclusion
Based on the research conducted, the following conclusions can be drawn isolation of the Metarhizium from the asahan field from the infected larvae was successful and resulted in 2 isolate of green and grayish-gray green in color.Morphological and molecular biological identification processes have been successfully carried out, and a DNA band result of ∞ 500 bp has been obtained using ITS 1 and ITS 4 primers.We conclude 2 isolates of Metarhizium namely Metarhizium majus and Metarhizium anisopliae as indigenous species from Asahan field.The virulence test was in every observation, resulting linear regression equation is Y = 14.027X+24.865with an R 2 value of 0.9634.

4 Figure 2
Figure 2 Oryctes rhinoceros larvae from Asahan field infected by Metarhizium with conidia over the entire surface of the body (A) and (B).

Figure 3
Figure 3 Color of Metarhizium colony isolated from infected larvae from the field, light green/sample 1 (A) and grayish gray green/sample 2 (B).

Figure 7 .Figure 8 .
Figure 7. Graphic of Growth of infection of Metarhizium test on larvae instar 2-3 in semifield testing at Asahan

Table 1 .
Measurement of concentration and purity of M. anisopilae DNA samples

Table 2 .
Molecular identification result of 3 isolates of the fungus