Test Kirinyuh Leaf Extract (Euphatorium odoratum.L) as Biofungicides against Antracnose Disease (Colletotrichum capsici) on Chili Plants (Capsicum Annum L)

Research to determine the effectiveness and concentration of extracts E. odoratumas a biofungicide applied to red chilli (C. Annum L) which is attacked by anthracnose (C.capsici). The research was carried out for 4 months from January to April 2020 at the Microbiology Laboratory of the University of North Sumatra, at the UMA Plant Protection Laboratory and in the Experimental Field of the Faculty of Agriculture, Medan Area University. The research was conducted using experimental methods, namely direct experiments and carried out in 2 stages: in vitro and in vivo. In the in vitro research stage, using a completely randomized non-factorial design (CRD Non Factorial) 9 levels with 1 factor which was repeated 3 replications, namely giving E. odoratum extract consisting of: AF0= negative control (without treatment), AF0 + = Control positive (0.2% synthetic fungicide), AF1 = PDA media with 10% E. odoratum extract, AF2 = PDA media with 20% E. odoratum extract, A3 = PDA media with 30% E. odoratum extract, AF4 = PDA media with 40% E. odoratum extract, AF5 = PDA media with 50% E. odoratum extract, AF6 = PDA media with 60% E. odoratum extract, AF7 = PDA media with 70% E. odoratum extract. In the In vivo stage, the experiment used a non-factoral randomized block design (non factorial shelf) with the treatment factor of E. odoratum extract obtained from the results of experiments in vitro in the laboratory, namely the concentration of 0%, 50%, 60% and 70%, which is the level of The extract concentration was below the effective concentration and the optimum extract concentration (best from the In vitro results) and the extract concentration was above optimal. Treatment replications and the number of treatment samples to be carried out were based on the results of the concentration to be applied and determined the level in a Non-factorial Randomized Block Design (Non-factorial RBD). ResultIn laboratory experiments showed that the extract was administered E. odoratum with a concentration of 60% and 70% significantly suppressed the growth of C. capsici colonies up to 0 cm2. The ability to suppress (inhibit) the growth of C. capsici is more effective than the use of synthetic fungicides. In field experiments, it can be concluded that the application of 70% E. odoratum extract has a very significant effect on fruit production and anthracnose attack intensity.


I. Introduction
The Province of North Sumatra has produced red chili production in the last four years experiencing production fluctuations, namely : 197,411 tons, 2013: 161,933 tons, 2014: 147,812 tons, (National Statistics Agency, 2016. When viewed from the data from the national statistical center agency in 2014, the production of red chili in North Sumatra Province decreased by 14,123 tons or 8.72% compared to 2013. One of the causes of the decline in red chili production was anthracnose. Anthracnose is a disease that attacks red chili plants. This disease is caused by the fungus Colletotrichum capsici. This fungus is stimulated by humid conditions and relatively high temperatures (AVRDC, 1988). According to (Efri, 2010) stated that the losses caused by this disease reached 70%. Until now, the control of this disease is by using chemical pesticides. Thirty percent of pesticides are disposed of to the ground during the dry season and 80% in the rainy season are disposed of in the water (Sabarani, 2008). Larvae infected with fungus spores with concentrations of spores / ml to spores / ml caused the death of Spodoptera litura larvae up to 83% on day 12 after infection with fungus spores (Halawa, 2019). .Excessive use of chemical pesticides has a negative impact on the environment and humans. The natural balance is disturbed and will result in resistant pests and threats to predators, parasites, fish, birds and wildlife.
Alternatives in controlling disease among others include using natural ingredients produced as biofungicides, among others.One of the plants that can be used for the therapy of infectious diseases is the leaves of the Euphatorium odoratum L plant. This plant in Thailand is used as a wound medicine, coagulant, and as an antiseptic (Irobi, 1997), in Nigeria it is used as a therapy for malaria (Rungnapa, 2003), while in Indonesia it is used as a medicine for new wounds, fever, cough, and to stop bleeding (Purwati, 2003). Even so, this plant is still very rarely used by the people of Indonesia because it is considered a nuisance plant that is difficult to eradicate. Inya-agha et al (1987) reported that E. odoratum leaf extract contains tannins, phenols, saponins, and its essential oil contains α-pinene, cadinene, camphore, limonene, β-caryophyllene, cadinol isomers.
From the results of previous research that E. Odoratumshows that these plants have secondary metabolite compounds that are active as antibacterial and protozoa. However, the extract biofungicide testE. Odoratumhas not been done and only as a biobactericide and insecticide. Based on these problems, this study aims to determine the effectiveness and concentration of the extractE. Odoratum as a biofungicide applied to Red Chili (C. Annum L) which is attacked by Anthracnose (C. capsici).

Tools and Materials
This research was conducted for 4 months from January to April 2020 at the Microbiology Laboratory of the University of North Sumatra, the UMA Plant Protection Laboratory and in the Experimental Field of the Faculty of Agriculture, Medan Area University. The materials used in this study include: leavesE. Odoratum, C.capsici fungi culture, aquades, Acrobat (synthetic fungicide), 70% alcohol. The tools used in this research include: Laminar air flow, beaker, measuring cup, tweezers, microscope, object glass, petri dish, label paper, ose needle, handsprayer, analytical scale, micropipette, erlyenmeyer flask, oven, incubator. , hot plate stirrer, autoclave and, camera. This research was conducted in 2 stages, namely In vitro and continued with In vivo.

Method of Implementation
The research was conducted using experimental methods, namely direct experiments and carried out in 2 stages: in vitro and in vivo. In the in vitro research stage, using a completely randomized non-factorial design (CRD Non Factorial) 9 levels with 1 factor which was repeated 3 replications, namely giving E. odoratum extract consisting of: AF0-= negative control (without treatment), AF0 + = Control positive (0.2% synthetic fungicide), AF1 = PDA media with 10% E. odoratum extract, AF2 = PDA media with 20% E. odoratum extract, A3 = PDA media with 30% E. odoratum extract, AF4 = PDA media with E. odoratum extract 40%, AF5 = PDA media with E. odoratum extract 50%, AF6 = PDA media with E. odoratum extract 60%, AF7 = PDA media with E. odoratum extract 70%.
In the In vivo stage, the experiment used a non-factoral randomized block design (non factorial shelf) with the treatment factor of the extract E. odoratum obtained from the results of experiments in vitro in the laboratory, namely the concentration of 0%, 50%, 60% and 70%, which is the level of extract concentration below the effective concentration and the optimum extract concentration (best from the in vitro results) and the extract concentration above optimal. Treatment replications and the number of treatment samples to be carried out based on the results of the concentration to be applied and determining the level in a Non-factorial Randomized Block Design (Non-factorial RBD)

Data analysis
For the analysis of data from focus groups and semi-direct interviews we used Krueger's method known as "long table" which consisted of identifying thematically or by questions, any word transcribed in the verbatim of the focus groups and semi-direct interviews. We chose to base ourselves on the themes, a method used by Seguin J. (2010). Each word was transcribed when it was relevant, it was classified according to the themes that correspond to those of the grid. The analysis therefore looked at the frequency of similar responses to the same question in 35 target women selected for group discussions and 10 semi-direct interviews. The analysis of these data was supported by observations made at the household level.

Observations in the Laboratory a. Provision of C. capsici mushroom
Sampling was carried out in Kuta Bangun Village, Tiga Binanga District, Karo Regency, by looking at chili plants that were exposed to anthracnose, where according to Rahman (2009), the initial symptoms began with the presence of small black spots and slightly curved on the surface of the fruit. Martoredjo (2010) adds further symptoms of fruit shrinking, rotting and falling, spotting round or sunken and developing on immature fruit, ripe from size. Furthermore, Damm et al (2010) added that the disease can infect the chili stalks from causing spots such as irregular spots with dark red color. Seen in Figure 1.

Figure 1. Symptoms of Anthracnose Disease in Chili Fruit
Source: Personal Documentation C. capsici fungi cultures that have been isolated and not contaminated on PDA media are gray with whitish gray mycelium that grows gradually (Fitriani, 2014). It can be seen in Figure A. the conditions of the fungus have not yet experienced complete conidia spread, and in Figure B. it can be seen that the conidia spores of the fungus have fully grown.
The results of the macrocopic identification show that the fungus C. capsici in PDA media in Figure A, it can be seen that the mycelium growth of pure fungal culture colonies has not been seen to be widespread., whereas in Figure B, shows that the fungal culture colonies produce a lot of mycelium, the colonies are white-gray, the colony is dark brown, the growth is slow (3-6 mm in 24 hours), and in old cultures (more than 15 days) black spots appeared on the surface of the C. capsici fungal colonies. From microscopic observation, it was found that the type of Colletotricum fungus, namely C. capsici in Figure 2B, has a cylindrical spore shape with a length of 7-14 µm and a width of 3-5 µm, the spores are not septa with hyaline color. Colletotricum Capsici mushroom mycelium. septa and branch. Mckenzie (2013), states that the conidia C. capsici is a whitish gray curved like a crescent moon and ends tapering at both ends, as seen in Figure 3.In the C. acutatum fungus according to AVCRD (2003), states that the fungus C.acutatum has a cylindrical spore shape, tapered tip, spore size 16.1 x 5.3 um with a growth speed of 6.8 mm per day. Sudirga (2016), also states that C. acutatum fungi colonies are in the form of cylindrical spores, the colony color is white gray to blackish brown. Further Dickman (1993) in Sudirga (2016). Based on the picture above, it is obtained two types of Colletotrichum sp. Fungi, and both of them are carried out in vitro development on a petri dish, the observation results show that the faster growth is Colletotrichum capsici. Rompas (2001) states that the temperature required for C. capsici ranges from 24-30 0 C while C. acutatum grows optimally at 24-26 temperatures 0 C (Rosida, 2013). The average temperature in North Sumatra Province is in accordance with the temperature needed by C. capsici mushrooms in its development process, this is supported by BMKG data (2013), which states that the average temperature in North Sumatra Province ranges from 26.5-28. 5 0 C. Wharton and Uribeondo (2004) and Mongkolporn et al. (2010) in Rosida (2013), showed that of the species tested, C. acutatum was the species that had the greatest virulence to chilies at highland temperatures, while according to Syukur (2007) in Ekstiani (2017), it was stated that more than 90 % anthracnose that infects chilies in the lowlands is caused by the fungus Colletotrichum capsici, because of the optimum temperature range for growth. Based on this statement, a development for the invivo test was carried out, in this case the research in the lowlands was Colletotrichum capsici.

Bioctivity Test a. Growth Percentage of C. capsici mushrooms
Based on the results of variance, it showed that the application of E. odoratum extract and synthetic fungicides significantly affected the growth percentage of C. capsici. The results of the E. odoratum extract test as a biofunctionality are presented in Table 1. Table 1 shows that the percentage growth of C. capsici colonies shows a very significant difference at the level of 99%. The highest area of inhibition was in the AF + (synthetic fungicide) treatment, which was not significantly different in AF6 (60% E. odoratum extract) and AF7 (70% E. odoratum extract) where there was no growth of E.odoratum extract colonies. It was significantly different from the AF5 treatment (50% E. odoratum extract) and very significantly different from the AF4 treatment (40% E. odoratum extract), AF3 (30% E.odoratum extract), AF2 (20% E.odoratum extract), AF1 (10% E. odoratum extract) and AF-(without synthetic fungicide treatment and E. odoratum extract) which were the lowest inhibition for all C.capsici colonies.  Data were collected at three days after inoculation to see signs of early growth marked by white mycelium on all treated media. Treatment of synthetic fungicides and extracts of E. odoratum 60% and E. odoratum 70% in (Figure 4) shows that there is inhibition of E. odoratum extract, so there are no signs of growth marked mycelium. This is because the compounds contained in E. odoratum can inhibit the growth of C. capsici. Where that the content of E. odoratum contains secondary metabolites, including flavonoids, saponins, tannins, alkaloids, and steroids / triterpenoids. This is in accordance with the statement conveyed by Parubak, AS, 2013) which states that flavonoid compounds have a number of uses. First, against plants, namely as a plant regulator, a photosynthetic regulator, and can be used as a vegetable pesticide because it acts as an antimicrobial and antiviral agent. This was also proven in the research of Purwati and Undri Rastuti (2009) which explained that Euphatorium odoratum leaf ethyl acetate extract was based on screening of secondary metabolites containing flavonoids, with secondary metabolites in the ethyl acetate extract of Euphatorium odoratum leaves having antioxidant activity with an inhibitory activity sequence of 0, 15%. Hadi's research (2008) explains that the presence of kirinyuh leaf extract on paper causes anti and causes termites to reduce the amount of food consumed so that termites experience mortality. Kiriyuh leaf extract (Euphatorium odoratum) on paper is also toxic to termites, so it can be used as a control for Coptotermes sp. The concentration of kirinyuh leaf extract on paper which is effective (LC-50) as a termite control agent is at a concentration of 2.Robinson (1995) also states that the tannin compounds contained in E. odoratum are antibacterial, Robinson's statement (1995) is strengthened by research (Nurussakinah, 2010) that the ethanol extract of E. odoratum can be used as antibacterial against Streptococcus mutans and Eschericia coli. While antifungal compounds, one of which is saponins, saponins contained in E. odoratum can be antibacterial and anti-fungal, which according to (Osbourn et al 1996) the presence of saponins can be an indicator of the resistance of a plant species to fungal infection, this statement is strengthened by the statement (Sugianitri, 2011) ) Saponins are surfactants that are polar in shape so that they will break down the fat layer on the cell membrane which in turn causes interference with cell membrane permeability.

b. Extract Inhibition ZoneE. odoratum Against C. capsici Mushrooms.
Based on the results of variance, it showed that the application of E. odoratum extract and synthetic fungicides significantly affected the growth percentage of C. capsici. The results of the E. odoratum extract test as a biofunctionality are presented in Table 2. Based on Table 2, it shows that the treatment of E. odoratum extract can inhibit the growth of C. capsici fungi where the best treatment is at concentrations of AF6 (60%) and AF7 (70%), both 95% confidence level and 99% good at the age of 4-10 DIC . In the observation of 4 DICs, it showed that the application of synthetic fungicides could inhibit the growth of C. capsici fungi. However, at the age of 5-10 HSI C. capsici was able to grow on synthetic fungicides and did not grow on E. odoratum extract and did not cause resistance.  This is in accordance with the statement conveyed by Etmawati (2016), that excessive use of chemicals and exposure for too long will cause the generation of pests or diseases to become resistant to chemicals, in contrast to the treatment of E. odoratum extract which causes C. capsici fungi to not. able to grow or not to cause resistance. This is in line with Untung's (2006) statement, which states that the use of organic pesticides does not cause pests and diseases to become immune. This can be seen in the response curve of the relationship between the administration of E. odoratum extract and the mycelium diameter of the C. capsici fungi aged 8 HSI is presented in Figure 6.
Giving Extract E. odoratum Figure 6. Response curve of the relationship between E. odoratum extract and C. capsici mushroom mycelium diameter at 8 DID From Figure 6, it can be seen that the response curve shape of the relationship between E. odoratum extract and C. capsici mushroom mycelium diameter is linear, with the equation: Y = -0.55991x + 0.51427. Regression coefficient value (R 2 ) = 0.7069 explained that 70% inhibition of the growth of C. capsici fungi mycelium was due to the E. odoratum extract with the best treatment, namely AF6 (60%) and AF7 (70%). This is because the content of secondary metabolic compounds found in E. odoratum can inhibit the growth of the fungal mycelium C. capsici, which according to Naim (2004), which states that secondary metabolic found in plants is self-defense against microorganisms (bacteria, fungi, protozoa) , insects and herbivores. According to (Arifin 2014;Rahayu and Pukan, 1998) that the content of E. odoratum contains secondary metabolites, including flavonoids, saponins, tannins, alkaloids, and steroids / triterpenoids. According to Parubak, AS, (2013), which states that flavonoid compounds have a number of uses. First, for plants, namely as a plant regulator, Robinson (1995) also states that the tannin compounds contained in E. odoratum are antibacterial, Robinson's statement (1995) is strengthened by research (Nurussakinah, 2010) that the ethanol extract of E. odoratum can be used as antibacterial against Streptococcus mutans and Eschericia coli. While antifungal compounds, one of which is saponins, saponins contained in E. odoratum can be antibacterial and anti-fungal, where according to Osbourn et al. 1996, the presence of saponins can be an indicator of the resistance of a plant species to fungal infection, this statement is strengthened by the statement (Sugianitri, 2011 ) Saponins are surfactants that are polar in shape so that they will break down the fat layer on the cell membrane which in turn causes interference with cell membrane permeability.

In Vivo Observations. a. Chili Fruit Production
Based on the results of variance, it showed that the administration of E. odoratum extract and synthetic fungicides significantly affected the production of chili fruit. The results of the test for the variety of E. odoratum as a biofunctional acid are presented in Table 6.Based on Table 6 shows that the best treatment for chili plant production at the 1st and 2nd harvests was found in AF3 treatment (70%) of E. odoratum extract which was not different. significantly with AF2 treatment (60%) of E. odoratum extract compared with AF1 treatment (50%) of E. odoratum extract, AF + synthetic fungicide, and AF-without treatment with either 95% or 99% confidence levels. This is because the content of secondary metabolic compounds found in E. odoratum can inhibit the growth of the fungal mycelium C. capsici, which according to Naim (2004), which states that secondary metabolic found in plants is self-defense against microorganisms (bacteria, fungi, protozoa) , insects and herbivores. According to (Arifin 2014;Rahayu and Pukan, 1998) that the content of E. odoratum contains secondary metabolites, including flavonoids, saponins, tannins, alkaloids, and steroids / triterpenoids. According to Parubak, AS, (2013), which states that flavonoid compounds have a number of uses.  Figure 7 it can be seen that the shape of the response curve of the relationship between E. odoratum extract and chili fruit production is linear, with the equation: Y = -19,898x + 0.9295. The regression coefficient value (R 2 ) = 0.9115 explains that 91.15% good chili fruit production is caused by the best treatment of E. odoratum extract, namely AF6 (60%) and AF7 (70%). According to Ariyanti et al (2012), states that phenolic compounds found in plants will cause lysis of microbial cells as well as the content of phenol compounds in E. odoratum. According to Prindle and Wright (1971) in Ariyanti et al (2012), adding that if the microbial cell is damaged, toxins from the cell can enter and result in reduced essential metabolites needed by microbes, phenolic compounds as antimicrobials are active against vegetative microbial cells. At the time of observation, it shows that the pH is close to normal, namely 6.5 with an average temperature of 28.5 0 -31 0 C. According to Widiastoety (1986), states that to absorb certain nutrients requires a pH ranging from 5.0-6.5, whereas if the pH is too low (<4.5) or the pH is too high (> 7.0) it can inhibit plant growth. This will result in the susceptibility of plants to pests and plant diseases. One of the nutrients needed in the formation of fruit is phosphorus. This is consistent with the statement given by Rubatzky and Yamaguchi (1998), which states that plants require P at all levels of growth and flowering. The role of phosphorus is not only that, according to Rismunandar (1990) in plants, phosphorus is used in the formation of protein, especially in the metabolic transfer of ATP, ADP, photosynthesis, and respiration, and is included in the components of phospholipids. the role of other phosphorus in the formation of roots, accelerate the ripening of fruit and strengthen the body of root plants not susceptible to pests and diseases. In the observation, the average temperature in the field ranged from 28.5 0 -31 0 C, with such a high temperature the C. capsici fungus cannot grow properly, which according to Ekstiani (2017) states that anthracnose disease is widespread due to humid conditions and relatively low temperatures. This is in accordance with Marpaung (2016), which states that for the growth and development of disease-causing microbes in plants, it must be in accordance with the desired temperature conditions in the field.

b. Attack Intensity
The intensity of anthracnose attack is the percentage ratio of the number of fruit harvested to the number of fruits attacked by anthracnose. Based on the results of variance, it showed that the administration of E. odoratum extract and synthetic fungicides significantly affected the intensity of the attack. The results of the assay of the variety of E. odoratum extract as a biofunctional acid are presented in Table 7 which shows that the best treatment of attack intensity at the 1st and 2nd harvests was found in AF3 treatment (70%) of E. odoratum extract which was not different from AF2 treatment ( 60%) E. Odoratum extract was compared with AF1 treatment (50%) E. odoratum extract, AF + synthetic fungicide, and AF-without treatment with either 95% or 99% confidence levels. Note: Numbers followed by the same letter in the same column show insignificant differences at the 95% (lowercase) and 99% (uppercase) levels.
In the AF-treatment, chilies had the highest anthracnose attack intensity value. This was due to the AF treatment-the absence of phenolic compounds, alkaloids, flavonoids, saponins was given so that the intensity of anthracnose attacks was greater than the other treatments. According to Dewi et al (2013), which states that secondary metabolic compounds including phenols, flavonoids, and saponins are compounds that damage cell walls. Dewi et al (2013) added that the cell wall as a component of bacterial cell defense was damaged so that secondary metabolites could enter deeper and disturb other organelles. The response curve of the relationship between giving E. odoratum extract to the production of chili plants at the second harvest is presented in Figure 8.
Administration of E. odoratum extract

Figure 8. Correlation Response Curve Between Giving E. odoratum Extract
Against Attack Intensity at the 2nd Harvest From Figure 8 it can be seen that the shape of the response curve of the relationship between the giving of E. odoratum extract and the percentage of attacks is linear, the equation: Y = -0,2493x + 2,2136. The value of the regression coefficient (R 2 ) = 0.8788 explains that 87.88% good chili fruit production is caused by the best treatment of E. odoratum extract, namely AF6 (60%) and AF7 (70%), indicating that the higher the E. odoratum extract, the lower the fruit who are stricken with anthracnose, this is because the higher the dose, the higher the content of secondary metabolite compounds. This is in accordance with the statement conveyed by Hartati (2012), which states that the weaknesses of essential oil-based pesticides are related to the properties of the essential oil itself which are volatile and unstable or not resistant to sunlight. According to Ariyanti et al (2012), states that phenolic compounds found in plants will cause lysis of microbial cells as well as the content of phenol compounds in E. odoratum. According to Prindle and Wright (1971) in Ariyanti et al (2012), adding that if the microbial cell is damaged, toxins from the cell can enter and result in reduced essential metabolites needed by microbes, phenolic compounds as antimicrobials are active against vegetative microbial cells.

c. Special Potential
The results of the analysis of this study were to determine the best quality of E. odoratum extract, the more E. odoratum extract that was applied to anthracnose-stricken chili plants, the more effective it was in controlling C. capsici fungi that attacked chili plants. From the results of this study, it is hoped that it can become a reference for farmers in controlling anthracnose so as to reduce yield losses. The results of this study can also reduce the environmental impact of pollution from the use of synthetic fungicides in controlling anthracnose disease.

V. Conclusion
1. In laboratory experiments, it can be concluded that the administration of E. odoratum extract with a concentration of 60% and 70% significantly suppressed the growth of C.
capsici colonies up to 0 cm. 2 . The ability to suppress (inhibit) the growth of C. capsici is more effective than the use of synthetic fungicides. 2. In the field experiment, it was concluded that the application of E. odoratum extract had a very significant effect on the parameters of fruit production and the intensity of attacks at the 1st and 2nd harvests.