Antimicrobial Activity of Chrysoeriol 7 and Chochlioquinone 9, White-Backed Planthopper-Resistant Compounds, Against Rice Pathogenic Strains

Simple Summary This study is an important contribution to the development of biopesticide materials for controlling major pathogens in rice. Chrysoeriol 7 and cochlioquinone 9, which repel the white-backed planthopper, were extracted from rice and their antimicrobial activity was investigated. The results show they are effective in limiting the growth of pathogens of the genera Fusarium, Cladosporium and Pythium. This shows that they have great potential as an alternative to chemical pesticides and is also thought to be helpful in development of eco-friendly agriculture. Abstract As environmental damage caused by chemical pesticides appears worldwide, eco-friendly agriculture is increasing, and finding eco-friendly pesticide materials has become very important. Chrysoeriol and cochlioquinone, two flavonoids, act as an antibacterial and antioxidant, and increase the resistance of rice to the white-backed planthopper (WBPH). In this experiment, chrysoeriol 7 (C7) and cochlioquinone 9 (C9) were extracted from rice inoculated with the WBPH using MeOH, and cultivars with high extraction efficiency were selected. In addition, the antimicrobial activity of C7 and C9 against various pathogens causing disease in rice was tested. The results show that C7 has antifungal activity against Fusarium graminearum and Pythium graminicola, and C9 show antifungal activity against Cladosporium herbarum, Cladosporium cladosporioides, Gibberella zeae, Fusarium graminearum and Pythium graminicola. When both substances were treated at a concentration of 1000 ppm, they showed high inhibition rates of 62.3% and 36.2% against P. graminicola, respectively. After that, a phylogenetic tree was created to clarify the relationship between the microorganisms whose growth was inhibited and divided into three groups. This result can contribute to the study of biopesticide materials that can control pests and pathogens.


Introduction
Rice (Oryza sativa L.) is the main food crop for more than 50% of the world's population, making it one of the most important crops in the world. The largest yield of rice is obtained from Asia, where about 94% of the world's supply is grown [1]. In a report on agricultural prospects by the OECD, FAO and other international organizations, the world's population will reach about 9.6 billion by 2050 and demand for food will increase by more than 60% [2]. To meet the needs of the rapidly growing population, a significant increase in rice production is needed in the coming decades.
In natural environments, however, rice is easily attacked by pests, nematodes and pathogens such as viruses, bacteria and fungi [3]. These attacks cause rice diseases and result in significant yield

WBPH Breeding and Plant Material
Fifty male and female WBPHs were obtained from the National Agricultural Science Research Institute of the Rural Development Administration (RDA). The breeding cage was made of an acrylic plate (50 × 50 × 40 cm) and a 100-µm mesh net was used on the back for ventilation and water supply. The insect nursery was maintained at a temperature of 27 • C ± 1 • C, a humidity of 60 to 70%, a luminous intensity of 2000 lux and over a 16-h cycle. Chucheong, one of the most preferred rice cultivars for WBPHs, was used for their food. Twenty to twenty-five grams of seeds were sterilized using a Spotak pesticide, then soaked in an incubator at 33 • C for 3 days and washed with clean water every morning. After sowing into a 21 cm × 14 cm × 5 cm plastic container and supplying sufficient moisture, the plants were placed in dark conditions at 28 • C for 2 to 3 days, and then 2 or 3 leaf seedlings grown in a greenhouse for 1 to 2 weeks were used as feed. The food was changed every week, which helps the next generation of WBPHs to be sustained ( Figure 1). When the WBPHs were at their 2 or 3 instar, they were inoculated into the plant material, namely, Cheongcheong, Nagdong and TN1.

WBPH Breeding and Plant Material
Fifty male and female WBPHs were obtained from the National Agricultural Science Research Institute of the Rural Development Administration (RDA). The breeding cage was made of an acrylic plate (50 × 50 × 40 cm) and a 100-μm mesh net was used on the back for ventilation and water supply. The insect nursery was maintained at a temperature of 27 °C ± 1 °C, a humidity of 60 to 70%, a luminous intensity of 2000 lux and over a 16-h cycle. Chucheong, one of the most preferred rice cultivars for WBPHs, was used for their food. Twenty to twenty-five grams of seeds were sterilized using a Spotak pesticide, then soaked in an incubator at 33 °C for 3 days and washed with clean water every morning. After sowing into a 21 cm × 14 cm × 5 cm plastic container and supplying sufficient moisture, the plants were placed in dark conditions at 28 °C for 2 to 3 days, and then 2 or 3 leaf seedlings grown in a greenhouse for 1 to 2 weeks were used as feed. The food was changed every week, which helps the next generation of WBPHs to be sustained ( Figure 1). When the WBPHs were at their 2 or 3 instar, they were inoculated into the plant material, namely, Cheongcheong, Nagdong and TN1.
Cheongcheong, Nagdong and TN1 were harvested at the Kyungpook National University Experimental Field at Gunwi in Korea in 2018. Cheongcheong is a resistant cultivar, and Nagdong and TN1 are cultivars sensitive toward the WBPH. Sixty grams of seeds were sterilized with 500 μL of Spotak pesticide (HANKOOKSAMGONG, Seoul, South Korea ) in 1 L of water at 33 °C for 3 days in an incubator and washed with clean water every morning. After sowing in a plastic container (21 × 14 × 5 cm) and supplying sufficient moisture, the plants were placed in dark conditions at 28 °C for 2 to 3 days and then grown in the greenhouse. The 50 WBPHs of the 2nd or 3rd instar were inoculated into Cheongcheong, Nagdong and TN1 for a week. In the breeding cage, healthy rice at the seedling stage was provided as food for the white-backed plant hoppers (WBPHs) every week. The WBPH hatches from eggs after 5-8 days and has a nymph period of 13 to 15 days. After that, it becomes an adult and is active for 12-15 days. At this time, if new food is provided, the adult insects move to the new food. The total life cycle of the WBPH is 32 to 35 days, and the sucked rice turns yellow and dies over time. In the breeding cage, healthy rice at the seedling stage was provided as food for the white-backed plant hoppers (WBPHs) every week. The WBPH hatches from eggs after 5-8 days and has a nymph period of 13 to 15 days. After that, it becomes an adult and is active for 12-15 days. At this time, if new food is provided, the adult insects move to the new food. The total life cycle of the WBPH is 32 to 35 days, and the sucked rice turns yellow and dies over time.
Cheongcheong, Nagdong and TN1 were harvested at the Kyungpook National University Experimental Field at Gunwi in Korea in 2018. Cheongcheong is a resistant cultivar, and Nagdong and TN1 are cultivars sensitive toward the WBPH. Sixty grams of seeds were sterilized with 500 µL of Spotak pesticide (HANKOOKSAMGONG, Seoul, South Korea) in 1 L of water at 33 • C for 3 days in an incubator and washed with clean water every morning. After sowing in a plastic container (21 × 14 × 5 cm) and supplying sufficient moisture, the plants were placed in dark conditions at 28 • C Biology 2020, 9, 382 4 of 15 for 2 to 3 days and then grown in the greenhouse. The 50 WBPHs of the 2nd or 3rd instar were inoculated into Cheongcheong, Nagdong and TN1 for a week.

Bacterial and Fungal Pathogens
Two bacteria and 11 fungi causing rice disease were obtained from the Agricultural and Microbiology Department of the National Academy of Agricultural Science, RDA ( Table 1). The freeze-dried bacteria were suspended in 5 mL of Luria-Bertani (LB) broth and cultured at 30 • C and 130 rpm for 3 days in an incubator. The freeze-dried fungi were suspended by adding 0.3-0.5 mL of sterile distilled water, and then the suspended samples were inoculated with 0.1 mL in potato dextrose agar (PDA) solid medium using a pipette and cultured at 25 • C in an incubator for one week. After culturing, the growth and contamination of the strains were confirmed, and the purely cultured strains were used for the experiment.

Extraction and Separation of Compounds
The leaves of Cheongcheong, Nagdong and TN1 inoculated with the WBPHs were cut and ground using a mortar with liquid nitrogen. Thirty-five grams of each leaf was used. Then, 35.0 g of plant sample and 350 mL of 70% methanol were added in a 1000 mL Erlenmeyer flask and shaken overnight in an incubator at 20 • C, 130 rpm. A filter paper (Whatman Grade 2 Qualitative Filter Paper Diameter: 15.0 cm; pore size: 8 µL) was fixed in the funnel and inserted into the Erlenmeyer flask. The sample was poured onto filter paper and this filtering was repeated twice.
The sample was washed using an equal volume of n-hexane: equal amounts of sample and n-hexane were added in a separation glass, shaken for few minutes, and then left until the layers were completely separated before collecting the bottom layer of the solution. The samples were concentrated using a rotary evaporator. The concentration was carried out under conditions of a water bath temperature of 30 • C, a cooling solution at −3 • C to 5 • C and a speed of 6-8. The samples were concentrated until the volume was about 10 mL and then concentrated more using a heating block. They were heated at 60 • C until the sample in each tube was about 0.3 mL.
One hundred and twenty-five grams of silica gel 60 were poured into a glass column (diameter 10 mm × 250 mm) using a funnel. The silica gel was first washed with 20% methanol, and then all of the concentrated sample was added in until it was soaked in silica gel. We poured the 20 mL eluent into a column and waited until the solution had fallen. The eluent gradient was 4% methanol and 20% acetone. We collected the solution in the tube until all samples had come down. We dried the collecting eluent with a heating block at 50 • C. After drying, the compound in some tubes was checked by TLC silica gel 60F 254 plates (Merck, KGaA, Darmstadt, Germany) with a mobile phase (Chloroform:Methanol:1-Butanol:Water = 4:5:6:4) and exposed in a UV eliminator (Bio-rad, USA).
The silica matrix containing the compound was cut to separate C7 and C9. The compounds were then separated from the silica matrix using a small column and eluted by 20 mL of 4% methanol in Biology 2020, 9, 382 5 of 15 100% acetone to get the purified compounds. The purity of compounds was checked by TLC silica gel 60F 254 plates.

LC/MS for Identification
We used LC/MS to analyze the materials, with an MSQ Plus Single Quadrupole Mass Spectrometer (Thermo Fisher Scientific, San Diego, CA Waltham, MA, USA). The infusion concentration was a 1:1000 sample dilution using 50% methanol in 0.1% formic acid and the flow rate was 50 µL/min.

Antimicrobial Activity Test of the Compounds
The separated compounds were dried using a heating block, dissolved with distilled water at 3 concentrations (100 parts per millions (ppm), 500 ppm and 1000 ppm), and then 0.1 mL was applied to the medium. For the bacteria, the liquid LB medium was suspended in distilled water to have a 1.0 OD value at 600 nm using a UV spectrophotometer, and then 0.1 mL of the bacteria was dropped into the LB medium using a pipette and cultured at 30 • C in dark conditions. For the fungi, a part of the subcultured mycelium (0.5 × 0.5 cm) was transplanted to the PDA medium and cultured at 25 • C in dark conditions. After inoculation, the growth over 1 and 2 weeks was confirmed, and the diameter of the colony was measured. According to Kim et al. (2012), the following formula (1) was used for the calculation of the inhibition rate (%) [32].

Statistical Analysis
All experiments were replicated at least three times, and the statistical analysis was performed using the SPSS program (IMMSPSS Statistics, version 22, IBMSPSS Statistics, version 22, Redmond, WC, USA).

Phylogenetic Analysis
The sequences were aligned using Clustal W multiple alignment. Bacterial 16S and fungal ITS sequences were entered in the MEGA X program (Philadelphia, PA, USA) for construction of the phylogenetic trees. The phylogenetic tree was constructed using the distance method, neighbor-joining. Branch support was given using 1000 bootstrap replicates.

Extraction Efficiency by Variety
After separation of C7 and C9, 1.7 mg, 3.6 mg and 12.1 mg of C7 and 9.0 mg, 4.1 mg and 14.0 mg of C9 were obtained in Cheongcheong, Nagdong and TN1, respectively. The highest amount of both C7 and C9 were obtained from TN1, as confirmed by TLC.

Phylogenetic Analysis
The sequences were aligned using Clustal W multiple alignment. Bacterial 16S and fungal ITS sequences were entered in the MEGA X program (Philadelphia, PA, USA) for construction of the phylogenetic trees. The phylogenetic tree was constructed using the distance method, neighborjoining. Branch support was given using 1000 bootstrap replicates.

Extraction Efficiency by Variety
After separation of C7 and C9, 1.7 mg, 3.6 mg and 12.1 mg of C7 and 9.0 mg, 4.1 mg and 14.0 mg of C9 were obtained in Cheongcheong, Nagdong and TN1, respectively. The highest amount of both C7 and C9 were obtained from TN1, as confirmed by TLC.

Antimicrobial Activity Test of C7 and C9
In the antimicrobial activity test of C7, the inhibition of growth of F. graminearum and P. graminicola were significantly different according to the concentration of the C7 treatment (p < 0.05) (Figure 3). At 1 week after inoculation, the inhibition rates of F. graminearum were 10.9% when treated with 100 ppm, 26.6% when treated with 500 ppm and 28.7% when treated with 1000 ppm. It was confirmed that, as the treatment concentration of C7 increased, the growth of the fungi decreased; all media were overcultured at 2 weeks. The inhibition rates of P. graminicola were 22.6%, 48.54% and 62.27%, showing high inhibitory effects at 1 week. However, at 2 weeks after inoculation, the inhibition rates were very low, 0.2%, 1.9% and 5.1% (Tables 2 and 3). In the antimicrobial activity test of C9, the inhibition of growth of C. herbarum, C. cladosporioides, G. zeae, F. graminearum and P. graminicola were significantly different according to the concentration of the C9 treatment (p < 0.05)

Antimicrobial Activity Test of C7 and C9
In the antimicrobial activity test of C7, the inhibition of growth of F. graminearum and P. graminicola were significantly different according to the concentration of the C7 treatment (p < 0.05) (Figure 3). At 1 week after inoculation, the inhibition rates of F. graminearum were 10.9% when treated with 100 ppm, 26.6% when treated with 500 ppm and 28.7% when treated with 1000 ppm. It was confirmed that, as the treatment concentration of C7 increased, the growth of the fungi decreased; all media were overcultured at 2 weeks. The inhibition rates of P. graminicola were 22.6%, 48.54% and 62.27%, showing high inhibitory effects at 1 week. However, at 2 weeks after inoculation, the inhibition rates were very low, 0.2%, 1.9% and 5.1% (Tables 2 and 3). In the antimicrobial activity test of C9, the inhibition of growth of C. herbarum, C. cladosporioides, G. zeae, F. graminearum and P. graminicola were significantly different according to the concentration of the C9 treatment (p < 0.05) (Figure 3). At 1 week after inoculation, the inhibition rates of C. herbarum were 5.0%, 9.9% and 10.1%, respectively. At 2 weeks, the inhibition rates were 2.3%, 4.3% and 8.0%, showing a weak inhibitory effect. The inhibition rates of C. cladosporioides were 12.4%, 14.3% and 15.5% at 1 week and 3.6%, 6.4% and 12.0% at 2 weeks, respectively. The inhibition rates of G. zeae were 7.6%, 8.1% and 24.6%. With the 1000 ppm treatment, the inhibition rate increased significantly, and at 2 weeks it was overcultured. The inhibition rates of F. graminearum were 9.7%, 11.3% and 20.7% at 1 week, respectively, and overcultured in 2 weeks. The inhibition rates of P. graminicola were 26.4%, 27.3% and 36.2% at 1 week, respectively, showing a relatively high inhibitory effect. The inhibition rates at 2 weeks were 20.0%, 44.8% and 52.4%, which were higher than those at Week 1 (Tables 4 and 5).
The growth of F. graminearum and P. graminicola was inhibited by both C7 and C9. The antifungal activity against F. graminearum was slightly higher in C7, and the antifungal activity against P. graminicola was higher in C9. Neither C7 nor C9 had an effect on the bacteria A. avenae subsp. avenae and X. campestris pv. campestris.

Phylogenetic Tree
We obtained approximately 1500 bp 16S rRNA PCR amplification products from the 2 bacteria and 500-600 bp ITS region PCR amplification products from the 11 fungi (Figure 4a). The PCR amplified products were analyzed through the BLAST program of NCBI, and a neighbor-joining phylogenetic

Phylogenetic Tree
We obtained approximately 1500 bp 16S rRNA PCR amplification products from the 2 bacteria and 500-600 bp ITS region PCR amplification products from the 11 fungi (Figure 4a). The PCR amplified products were analyzed through the BLAST program of NCBI, and a neighbor-joining phylogenetic tree was created using the Mega X program. As expected, the bacteria and fungi were divided into two categories, phylogenetic and eukaryotic. G. zeae and F. graminearum grouped with the Fusarium genus with more than 99% identical nucleotide sequences. C. herbarum and C. cladosporioides showed a very high homology of 98.2% to the same Cladosporium genus, and the bootstrap value was very high at 99%. G. zeae, F. graminearum, C. herbarum, and C. cladosporioides could be grouped together by the Ascomycota phylum, but showed low homology of 64-65.8% and a bootstrap value of 65%. P. graminicola, which showed inhibitory effects by C7 and C9, did not belong to the Ascomycota phylum, and the homology was only 30.7-34.3%, showing a distant relationship (Figure 4b).
Biology 2020, 9, x FOR PEER REVIEW 11 of 17 tree was created using the Mega X program. As expected, the bacteria and fungi were divided into two categories, phylogenetic and eukaryotic. G. zeae and F. graminearum grouped with the Fusarium genus with more than 99% identical nucleotide sequences. C. herbarum and C. cladosporioides showed a very high homology of 98.2% to the same Cladosporium genus, and the bootstrap value was very high at 99%. G. zeae, F. graminearum, C. herbarum, and C. cladosporioides could be grouped together by the Ascomycota phylum, but showed low homology of 64-65.8% and a bootstrap value of 65%. P. graminicola, which showed inhibitory effects by C7 and C9, did not belong to the Ascomycota phylum, and the homology was only 30.7-34.3%, showing a distant relationship (Figure 4b).

Discussion
Chemical pesticides are widely used around the world to prevent pests and pathogens from attacking crops, and they have become a major cause of environmental pollution. Chemical pesticides are very efficient and appear to act quickly, but there are problems, such as residual toxicity and increased resistance by pests. Biological pesticides are alternatives to these organic synthetic pesticides. After the Rio summit agreement to replace 20% of the world's pesticides with biological pesticides, there is a clear movement to expand the availability of biological resources by developing new natural pesticides worldwide [33]. Among the currently discovered plant-derived biological pesticides, pyrethrin, which is eluted from pyrethrum flowers, has an insecticidal component and is mainly used to control moths and aphids [34]. Leaf extracts of Piper betle, Ocimum sanctum, Nyctanthes arbor-tristis and Citrus limon are known to inhibit the growth of pathogens of rice in vivo [35].
Chrysoeriol is an organic compound known as a 3 -O-methylated flavonoid and has a structure in which a methoxy group is attached to the c3 atom of the flavonoid backbone. It is abundantly present in many plants, fruits and medicinal herbs. Chrysoeriol is used in research on neuroprotection [36], anti-inflammatory, anticancer [37], antibacterial [38] and antioxidant [39] effects [40]. Cochlioquinone A is a major component of the bioactive pigments isolated from Bipolaris leersia and D. saccharhi. It is anti-angiogenic and has the inhibitory effects of diacylglycerol kinase and diacylglycerol acyltransferase. Cochlioquinone B is a sesquiterpene metabolite, an inhibitor of NADH ubiquinone reductase, and a phytotoxic agent that inhibits root growth of finger millet and rice at a 100 ppm concentration [26]. Cochlioquinone D is a physiologically active natural product that inhibits root growth of Lolium multiflorum [41]. Cochlioquinone 9 is distinguished from cochlioquinone A in that C26 (methyl group) is not bound to C14 but is bound to C15.
In separating chrysoeriol and cochlioquinone from rice inoculated with WBPHs, it is possible to extract a large amount of material faster than when not inoculated, increasing the efficiency and reducing the cost [42]. The highest amount of C7 and C9 was extracted from TN1, a susceptible cultivar, at the seedling stage. However, the amount of material can be different depending on various factors such as the growth time, the period of inoculation and the extraction method. According to Park et al. (2014), there is a large difference in damage between resistant rice cultivars and susceptible cultivars after 25 days of inoculation [31]. In this experiment, compounds were extracted under a single condition, so further experimentation is required to determine under which conditions the C7 and C9 content of the resistant cultivars will be higher compared to other varieties. In the LC/MS analysis of the separated material in the present study, C7 showed molecular weights of 267.36 and 381.37 m/z, and C9 showed molecular weights of 381.38 and 480.40 m/z. This is consistent with the molecular weights of C7 and C9, as previously known [31]; the authors reported that the molecular weight of Chrysoeriol 7 was 267.33, 341.50, 353.42 and 381.50 m/z, and that of Cochlioquinone 9 was 381.50 and 469.42 m/z. Since the molecular weight of the material separated in this experiment almost matched, it was judged to be the same material.
Looking at the results of the inhibition rate against the pathogens when C7 and C9 were treated with varying concentrations, when C7 was treated, the inhibition rate against F. graminearum at 1 week was 10.9, 26.6 and 28.7%, respectively, and the inhibition rate significantly increased when treated with 500 ppm. However, when processing 1000 ppm, it increases very little. The inhibition rate against P. graminicola was 22.6%, 48.54% and 62.27%, showing high inhibitory effects, and the inhibition rate was also greatly increased in proportion to the concentration of the compound. However, at 2 weeks after inoculation, the inhibition rate was very low (Table 2). When looking at the inhibition rate when C9 was treated, when 500 ppm was used in C. herbarum and C. cladosporioides at 1 week, the inhibition rate was 9.9% and 14.3%, which was the most efficient compared to the other concentration. At 2 weeks, both showed weak inhibitory effects overall. In G. zeae, the inhibition rate was 24.6% when treated with 1000 ppm at 1 week, which was higher than when 500 ppm was used. After 1 week, the inhibition rate of F. graminearum and P. graminicola increased significantly to 20.7% and 36.2%, respectively, when 1000 ppm was used, and the inhibition rate of P. graminicola further increased after 2 weeks (Table 3). C7 has an inhibitory effect against two pathogens, F. graminearum and P. graminicola, and C9 has an inhibitory effect against a wider range of fungi, including F. graminearum and P. graminicola, but the inhibitory effect was higher in C7. The most efficient control concentration was different, depending on the type of pathogen when the compounds were treated, but the most efficient treatment concentration for simultaneous control of several fungal pathogens was 500 ppm.
The bacterial 16S rRNA and the fungal ITS region were used to investigate the genetic similarity between microbes whose growth was inhibited by C7 and C9 through the phylogenetic tree. Most of the 16S rRNA sequences are quite conserved, although high sequence diversity appears in some sections. While there is little diversity between homogeneous species, diversity appears between other species, so it is used to identify prokaryotes [43]. The length of the ITS region of the fungus is not long, so it can be easily analyzed. It has been widely used for systematic analysis because it can be used even among nearby taxa due to its rapid mutation rate [44]. As a result of systematic analysis, microorganisms were largely divided into three groups. G. zeae and F. graminearum cause scab in rice and produce fungal toxins that affect rice safety and are toxic to the immune system and digestive system [45]. The outbreak of mycotoxins caused by contamination of F. graminearum has been reported steadily around the world, and it is estimated that 25% of the world's grain production, including rice, is contaminated with mycotoxins [46]. In Korea, scab disease caused by F. graminearum occurred widely in the southern regions during the harvesting season in 1963, resulting in a reduction of yield of 40-60% in some regions and 80-100% in severe regions [47]. C. herbarum and C. cladosporioides are species in the genus Cladosporium, which causes spotting symptoms on crops, causes allergies to livestock and promotes asthma. In addition, each pathogen causes false blast and ear blight, which directly damages the ear of rice, not only reducing the quantity but also the quality of the harvest [48]. P. graminicola is a plant pathogen that infects grains such as barley, wheat, rice and beans. In rice, it causes blight [49]. Damping-off caused by P. graminicola is a disease that affects the early growth of the seedling, infringing on the branch of the hypocotyl and causing the seedling to fall. In the end, it falls and dies. When a lot of damping-off occurs, the seedlings are insufficient, which causes great damage to the planting [50]. However, we found no antimicrobial effect of C7 and C9 on P. ultimum, which has 83.7% homology with P. graminicola, so this needs to be confirmed.
Research using microorganisms, plant extracts and natural enemies is being carried out to develop biological pesticides to replace chemical pesticides and develop eco-friendly agriculture. However, the production of plant extracts is small; thus, the amount that can be used for pesticides in areas with high demands is extremely limited. The extracts can be effective and could be used as biological pesticides and antibacterial agents if a mass extraction method and an automatic analysis system for substances are established in the future.

Conclusions
In this study, in order to overcome the environmental damage caused by chemical pesticides, we tried to find eco-friendly biopesticide material that can control pests and pathogens. Chrysoeriol 7 and cochlioquinone 9, which are insect-resistant compounds, were extracted from rice and their antimicrobial activity against major pathogens causing disease in rice was examined. The results showed that chrysoeriol 7 had antifungal activity against Fusarium graminearum and Pythium graminicola and cochlioquinone 9 had antifungal activity against Cladosporium herbarum, Cladosporium cladosporioides, Gibberella zeae, Fusarium graminearum and Pythium graminicola. These results indicate that they are effective in limiting the growth of some pathogens and show their potential as alternatives to harmful chemical pesticides. These findings can also contribute to the basic research of eco-friendly materials to use as pesticides in rice farming.