MATERIALS AND METHODS

Study area: This research was carried out at the Laboratory of Pests and Plant Penyakits, Department of Pests and Plant Penyakits, Faculty of Pertanian, Sam Ratulangi University, Manado. Penelitian is held for 8 months, from March to October, 2021.

The research used a randomized design of complete and as a treatment, was a dose of the pathogen concentration of M. anisopliae. The study was conducted in two stages, namely the study of the effect of M. anisopliae concentration on imago mortality of S. oryzae and the study of the effective concentration dose (LC₅₀) of the pathogen M. anisopliae on imago mortality of S. oryzae.

Maintenance of test insects: Rice samples that have been attacked or show symptoms of S. oryzae attacks were collected from traditional markets in Manado City and Minahasa Regency and under laboratory to be rearing as the propagation of rice test insects that were scattered g or showed that S. oryzae attacks were put in jars of 1 kg each and covered with white azahi cloth. The rice jars were left for 1.5 to 2 months until the pest S. oryzae in the rice multiplied into many. The test insect S. oryzae is ready to be applied with a microbial insecticide, namely the culture of the pathogen M. anisopliae on rice media.

Preparation of M. anisopliae pathogen culture on rice media: The rice is steamed until half cooked and then packed in transparent plastic bags per 20 g. Pure culture of M. anisopliae was added in each rice pack. After the pathogen M. anisopliae has experienced full growth 2-3 weeks after the inoculation culture, the culture is ready to be applied.

Effect of dose concentration of M. anisopliae pathogen culture on imago S. oryzae mortality: The treatment consisted of 5 levels of dose concentration of M. anisopliae pathogen culture with 3 repeats, namely A = 10% concentration (pathogen culture 10 g/100 cc aquades), B = Concentration 20% (pathogen culture 20 g/100 cc aquades, C = Concentration 30% (pathogen culture 30 g/100 cc aquades, D = concentration 40% (pathogen culture 40 g/100 cc aquades, E = Concentration 50% (pathogen culture 50 g/100 cc aquades and K = Control (pathogen concentration 0 g/100 cc aquades.

A total of 25 Sitophilus oryzae imago on each petri dish according to the number of treatments and replays. It is also a small jar that has been filled with pest-free rice according to the number of treatments and tests. Evenly disburse the solution of the pathogen M. anisopliae on the test insects according to the level of treatment concentration. Imago S. oryzae is applied with a solution of the pathogen M. anisopliae in each filled treatment jar of 15 g of pest-free rice each and then a lid with a white azahi cloth. Observations after application with an observation time interval of 2 days and observations were made 5 times. The same procedure was also for the implementation of research on the effective concentration dose (LC₅₀) of the pathogenic culture of M. anisopliae in pest control of S. oryzae rice powder.

Effective concentration dose research (LC₅₀) of M. anisopliae pathogenic culture in pest control of S. oryzae rice powder: Three levels of dose of M. anisopliae pathogen culture concentration with three repeats, namely A = Concentration of 20% (pathogen culture 20 g/100 cc aquades), B = Concentration 30% pathogen culture 30 g/100 cc aquades), D = Concentration 40% (pathogen culture 40 g/100 cc aquades) and K = Control (pathogen concentration 0 g/100 cc aquades.

Observation: The observed things were 1) growth progression of M. anisopliae in S. oryzae test insects until the test insect dies and 2) imago S. oryzae mortality for each concentration of M. anisopliae pathogens due to the application of M. anisopliae pathogens. Observations of the growth development of M. anisopliae in the test insect were carried out from when the test insect died until the hyfa or fungal mycelia grew on the surface of the test insect’s body. The observations made were macro.

Observations of test insect mortality due to the treatment of M. anisopliae began to be carried out the second day after application. Observation of S. oryzae mortality was carried out five times and the observation interval was two days. Calculation of S. oryzae pest mortality due to the application of the pathogen M. anisopliae using the formula:

Data analysis: Data on the results of penelitian, namely S. oryzae mortality due to the application of the pathogen M. anisopliae were analyzed using the statistical analysis program SPSS 21.0. Calculate the effective dose of culture solution of M. anisopliae culture (LC₅₀) using probit analysis.

RESULTS

Development of the pathogen Metarhizium anisopliae (Metch.) on the test insect S. oryzae: Imago S. oryzae’s behavior after applying the pathogen M. anisopliae is that insects stop/do not want to eat, are on the material’s surface, are less active/stay silent and die. After the insect dies, two days later, there is a change in color, namely the body's surface becomes pitch black and on the cuticle, a black rickshaw is seen as a trace of mold penetration. Imago S. oryzae, who was attacked by M. anisopliae in the laboratory, can be followed in Fig. 1 (a and b). Figure 1 showed that after the imago S. oryzae dies, the initial symptoms were the appearance of hyfa or white M. anisopliae fungal mycelia on the sutura or wing boundaries on the abdomen, the boundary between the abdomen and thoracic and the base of the legs. The subsequent development of mycelia or fungal hypha will cover the entire body surface and change color from white to muscardine green.

Effect of dose concentration of M. anisopliae pathogen culture on imago S. oryzae mortality: Data from the research on the application of the effect of the pathogen concentration of M. anisopliae on the mortality of the rice powder pest S. oryzae gave quite satisfactory results. The study’s results on the effect of the pathogen concentration of M. anisopliae on the mortality of the rice powder pest S. oryzae were shown in Table 1.

Table 1 showed that all treatments showed marked differences in control except for treatment A, which was a concentration of 10%. The dose of M. anisopliae pathogen culture concentration of 50% gave the highest mortality result of 89.33%, followed by the concentration of M. anisopliae pathogen 40% with a mortality of 72.0%, pathogen concentration M. anisopliae 30% with mortality 57.33%, pathogen concentration M. anisopliae 20% with mortality 13.33% and lowest at the pathogen concentration M. anisopliae 10% with a mortality of 1.33%. This study showed that the fungus M. anisopliae could be used to control the pest of S. oryzae rice powder.

The development of imago S. oryzae mortality from the first observation to the fifth observation showed that the increase in the dose of the pathogen concentration of M. anisopliae was directly proportional to the mortality of imago S. oryzae (Fig. 2).

Fig. 1(a-b):

Imago S. oryzae attacked by M. anisopliae pathogen in the laboratory, (a) Imago S. oryzae healthy and (b) Imago S. oryzae stricken M. anisopliae

Fig. 2:

Graph of the development of mortality of the rice powder pest S. oryzae at varying dose levels of the culture concentration of the pathogen M. anisopliae from the first observation to the fifth observation, A: Concentration of 10% (pathogenic culture 10 g/100 cc aquades), B: Concentration of 20% (pathogen culture 20 g/100 cc aquades), C: Concentration of 30% (pathogenic culture 30 g/100 cc aquades), D: Concentration of 40% (pathogen culture 40 g/100 cc aquades), E: Concentration 50% (pathogen culture 50 g/100 cc aquades and F: Control (pathogen culture 0 g/100 cc aquades)

Figure 2 showed that the higher the culture concentration of the pathogen M. anisopliae, the higher the mortality of S. oryzae rice powder. At the first observation, there had been no mortality in the pest S. oryzae and mortality began to occur on the second observation (4th day after application) and increased steadily on the third, fourth and fifth observations. Increased mortality of rice powder pest S. oryzae occurs due to an increase in the number of spores at each increased dose of concentration of the culture of the pathogen M. anisopliae. An increase always follows the increase in spores in the toxins contained in each increased dose of the culture concentration of the M. anisopliae pathogen.

Effective concentration dose (LC₅₀) of M. anisopliae pathogenic culture in pest control of S. oryzae rice powder: The results of the probit test of effective concentration dose (LC₅₀) of the pathogenic culture of M. anisopliae in the control of S. oryzae rice powder pests were shown in Table 2. The results of the probit analysis showed that the effective concentration dose (LC₅₀) of the M. anisopliae pathogenic culture in pest control of S. oryzae rice powder was 31.885%. Lethal Concentration 50 (LC₅₀) is a substance that can cause 50% of deaths when exposed to a population of an organism.

Typically, this parameter is important regarding the toxicity of a substance or chemical substance related to water or contained in 100 cc or 1000 cc of water. The LC₅₀ refers to the lethal concentration of a substance that can cause 50% death when exposed to a population of test organisms. The unit of measurement for LC₅₀ is in milligrams per cubic meter or ppm.

DISCUSSION

The initial symptoms of S. oryzae pests after being applied with the pathogen M. anisopliae are insects that stop eating, are on the surface of the material, less active/more silent. After the insect dies, two days later, there is a change in color, namely the body’s surface becomes pitch black and on the cuticle, a black rickshaw is seen as a trace of mold penetration. The appearance of hyphae or mycelia of the fungus M. anisopliae is white on the sutura or the boundaries of the wings on the abdomen, the boundary between the abdomen and thoracic and the base of the legs. The subsequent development of mycelia or fungal hypha will cover the entire body surface and change color from white to mustard green.

The initial symptoms of insects attacked by the fungus M. anisopliae do not want to eat, so the body becomes weak¹⁸. It lacks orientation, over time, it is silent and dies. The insect changes color and on the cuticle, a black rickshaw is visible as a trace of penetration of the fungus. If environmental conditions are favorable, white mycelia will appear on the surface of his body. The affected larva usually secretes a reddish discharge from its mouth continuously. After death, the body begins to become soft and, within 5 hrs, becomes stiff (mummy). A day later, his body was covered with mycelia. Dead larvae attacked by the fungus M. anisopliae will later harden and stiffen. The skin of the larvae will be covered with white flour that will change color to dark green¹⁹.

The color of all isolates of M. anisopliae fungi macroscopically at the beginning of white growth, then turns into a dark green color²⁰. Microscopically hyaline spores, cylindrical in shape and form chains. Spores of M. anisopliae enter the body of an insect through the skin. Spores that have entered the insect body begin to form hyphae ranging from the tissues of the epidermis until the entire tissues of the insect's body are filled with hyphae. Once the host is killed the group of hyphae will form primary and secondary spores, depending on weather conditions, as the weather favors the spores appearing on the insect's cuticle^20,21. According to Athifa et al.²² that infection and the spread of spores are influenced by several factors, namely wind, humidity and host solids. Strong winds and high humidity can help spread spores and the even distribution of infection throughout individuals in the host population²². Entomopathogenic microorganisms widely used to control pest attacks are microorganisms from the entomopathogenic fungi group¹⁸. Fungi widely used as bioinsecticides are from the genus Metarhizium and Beauveria, namely M. anisopliae and B. bassiana. The M. anisopliae and B. bassiana were found to have attacked many invading pests of the orders Hemiptera and Coleoptera. Some entomopathogenic fungus species that can be considered as biological insecticides are B. bassiana, M. anisopliae, Verticillium lecanii and Hirsutella thompsonii²³. The fungus M. anisopliae can infect insects of the order groups of Orthoptera, Coleoptera, Hemiptera, Lepidoptera and

Hymenoptera. M. anisopliae could be used to control the pest Oryctes rhinoceros. The M. anisopliae carried by imago O. rhinoceros can infect and kill the larvae of O. rhinoceros²³. The research results of Kastilong et al.²³ showed that the application of M. anisopliae PH04 fungi could reduce the population of Lepidiota stigma larvae in sugarcane plantations and increase crop yields by more than 60%. The fungus can survive on the farm for more than six months, potentially controlling L. stigma in the long term.

This study also showed that the increase in dose concentration of the pathogen M. anisopliae was directly proportional to the mortality of imago S. oryzae. The higher the culture concentration of the pathogen M. anisopliae, the higher the mortality of S. oryzae rice powder. Increased mortality of rice powder pest S. oryzae occurs due to an increase in the number of spores at each increased dose of concentration of the culture of the pathogen M. anisopliae. An increase always follows the increase in spores in the toxins contained in each increased dose of the culture concentration of the M. anisopliae pathogen.

The increase in the culture concentration of the pathogen Metarhizium anisopliae (Metch.) is directly proportional to the mortality of the larvae of Hexamitodera semivelutina Hell. on Clove plants. The more high concentrations of M. anisopliae pathogenic cultures, the higher the spore content in the pathogen's culture so that the pathogenic culture's killing power also increases and causes the mortality of test insects to increase²⁴.

The research results by Wamiti et al.²⁵ showed that Metarhizium sp. infects the host through four stages: Inoculation, pasting, penetration and digestion. The first stage is the inoculation of contact between the propagules of the fungus and the insect's body. The second stage is the process of pasting and germination of propagules of fungi on the integuments of insects. The third stage is penetration and invasion. Fungi penetrating through the integuments can form sprout tubes (appressorium). The morphological configuration of the integument strongly influences the penetration point. Breakout is carried out mechanically or chemically by secreting enzymes and toxins. The fourth stage is digestion at the end of penetration and the formation of blastospores which then circulate into the hemolymph and form secondary hyphae to attack other tissues. So that in general, all tissues and body fluids are used up by fungi, so insects die with hardened bodies.

The LC₅₀ stands for lethal concentration i.e., the parameter for various chemical compounds that describe the amount of substance in a unit volume of water (100 cc or 1000 cc) that can cause death. These two parameters measure the lethal character of a substance when exposed to a population and cause the death of 50% of that population. The LC₅₀ is a concentrated dose administered once (single) or several times in 24 hrs of a substance that is statistically expected to kill 50% of test animals²⁶. The LC₅₀, commonly abbreviated as LC₅₀, is a calculation to determine an extract's or compound's activeness. The LC₅₀ means at what concentration the extract can kill 50% of the test organisms that can be estimated by graphs and calculations or at a specific observation time²⁷. The LC₅₀ is the concentration at which the extract solution can cause the death of the test organism population up to 50%.

CONCLUSION

The increase in the concentration of the pathogenic culture M. anisopliae is directly proportional to the mortality of the rice powder pest S. oryzae. The higher the culture concentration of the pathogen M. anisopliae, the higher the mortality of S. oryzae rice powder. The effective concentration dose (LT₅₀) of the M. anisopliae pathogenic culture to control the pest of S. oryzae rice powder, which is 31.885%, is a dose of M. anisopliae culture concentration which can cause the mortality of S. oryzae rice powder pests to be 50%.

SIGNIFICANCE STATEMENT

Rice is the main food ingredient in Asia, including Indonesia. Sitophilus oryzae L., becomes a post-harvest pest that reduces the quantity and quality of rice. This research was conducted to provide alternative pest control for Sitophilus oryzae L. with biological control using the entomopathogenic fungus Metarhizium anisopliae (Metch.). This study found the Entomopathogen Metarhizium anisopliae (Metch.) to be effective in controlling Sitophilus oryzae L. Further research can be carried out on a field scale.

ACKNOWLEDGMENT

It was conveyed thanks to Lembaga Research and Community Service Sam Ratulangi University for supporting the implementation of this research. Expressed his gratitude to the Plant Pests and Diseases Laboratory for helping us with this research.