A review of application of natural products as fungicides for chili

Anthracnose disease in chillies is a serious problem for farmers. So far, synthetic fungicides have been used as solution for the treatment of this disease. However, the side effects of synthetic fungicides to public health and environment raised awareness on alternative fungicides derived from natural resources. This paper aims to review plants that are potential as an alternative to fungicides for chili plantation, fabrication of test solutions, in vitro and in vivo fungicide test. Many plants were investigated as alternatives to plant-based fungicide. The utilization of leaves as samples including rhizomes, roots, tubers, weevils, seeds, fruit, flowers and other parts of the plant. The extract fabrication method used as a fungicide test include: maceration method, gradual fractionation method, and decoction method. The maceration method is the method most widely used to extract fungicidal active compounds from plants. Some studies that carried out in vitro tests were unable to compare with synthetic fungicides so it was not possible to determine their effectiveness for plant-based fungicide for chillies when compared to synthetic fungicides. In vitro extract of 80% alcohol and 10%/60% n-hexane of pacar cina (Aglaia odorata L.) leaves can be compared with the performance of propineb 0.2%. In addition, the 60% and 70% kirinyuh (Chromolaena odorata L.) leaf extracts were also able to match Acrobat 0.2% performance in vitro. Based on the in vivo test, suren (Toona sureni Merr) leaf extract and nut bulbs can be used as an alternative to vegetable / natural fungicides to help overcome the problem of anthracnose in chilies.


Introduction
One of the goals of the Sustainable development goals (SDGs) is to achieve food security and declare as sustainable agriculture. Chili is one of the food commodities whose production must be increased in order to realize food security in Indonesia. Every year, there are increased in demand for chilies which is in line with the growth in population and the development of food industry that require the chilies as raw material (Subagyono et al., ). In addition, there is always increase in the price of chili in particular month due to low productivity of chili harvest. The decrease in chili productivity can be caused by pests and plant diseases (Warisno Dahana, ). The pests attack the plants and causes chilies suffered severe damage and crop failure. The pests that can attack chili plants include: peach aphids, thrips pests, mites, fruit fly pests, and fruit borer pests. On the other hand, chili plant diseases include: anthracnose, phytophthora rot, fusarium wilt, cercospora leaf spot, bacterial wilt, yellow virus, mosaic disease (Piay et al., ). Therefore, control of plant pest organisms must be done in order to increase the production of chilies (Badan Pusat Statistik Republik Indonesia, ).
Some plants that have the potential to be used as natural pesticides include: tembelekan/cherry pie (Lantana camara), jarak tintir/coral plant (Jatropha multifida), pacar cina/chinese rice (Aglaia odorata L.), mengkudu/noni (Morinda citrifolia L.), mimba/neem (Azadirachta indica A. Juss.), kenikir/compositae (Cosmos caudatus Kunth.), sirih/betel (Piper batle L.), awar-awar (Ficus septica) and others. Basically, natural pesticides do not only come from plants, but also from bacteria, viruses, and fungi (Novizan, ). The purpose of this paper is to review: ) plants that have the potential as an alternative natural fungicide for chili, ) fabrication of solution for in vitro and in vivo test, ) in vitro test as fungicide for chili, and ) in vivo test as fungicide for chili.

Potential plants as alternative fungicide for chili
Many plants have been investigated on the potential as an alternatives to plant-based / natural fungicides for chili. Table shows the names and parts of the plant and the method tested for fungicide. The part of plants that is widely used in research on finding alternative natural fungicides is the leaves. Few studies have used parts of rhizomes, roots, tubers, weevils, seeds, fruit, flowers or all parts of a plant (combination of flowers, leaves, stems, roots, and seeds). Betel leaf is a part of the plant that has been investigated both in vitro and in vivo. The researchers only used one plant type separately to determine its potential as natural fungicide. Only few researchers have combined plants, for example: mixture of betel and tobacco leaf (Oktarina et al., ), (Anjani, ), (Nur Rohmah, ) and mixture of kenikir/compositae (Cosmos caudatus Kunth.) and betel (Maimunah et al., ). In general, fungicide test methods used in many studies are divided into categories, namely: ) in vitro and ) in vivo. There are researchers who only focus on using in vitro test methods or in vivo test methods. In addition, the researchers also used both test methods in combination . In the in vitro test method, many types of fungi that cause Anthracnose disease in chilies are used, for example: Collectotrichum capsici, Colletotrichum gloesporioides and Colletotrichum acutacum mushrooms. Several parameters that can be observed in the in vitro test include: percentage of inhibition, diameter of fungal colony growth, zone of inhibition, spore growth, spore germination and percentage of spore density. On the other hand, in the in vivo test more parameters can be observed which include: anthracnose disease severity, intensity of fungal attack on chilies, percentage of disease incidence, effectiveness of fungicides, diameter of chili spots, incubation period of fungi in chilies, plant height, number of fruit and the weight of the chilies. In this in vivo test, the success of the research is strongly influenced by environmental factors, for example: temperature, humidity and rainfall (Suwastini et al., ).

Preparation of extract
The preparation of test solutions for chilies fungicide was summarized in Table . In general, the method of extracts preparation used can be classified into types, namely: ) maceration method, ) graded fractionation method, and ) decoction method.

. Maceration method
This method are most widely used to extract active compounds from certain plants for fungicide. Plants were prepared in the powder or flour form are added with a solvent and then are soaked for a designated time. The filtrate is separated from the dregs and the maceration process can be continued with new solvent until color filtrate is clear. The filtrate is concentrated using rotary evaporator with temperature control according to the type of solvent used until concentrated extract is free solvent (K Ngibad, ), (Khoirul Ngibad, ), (Wibowo et al., ). The solvents used in the extract preparation for test fungicide on chilies, including: water solvent (ultrapure water) and organic solvents ( % methanol, methanol, % ethanol, % ethanol, ethanol, ethyl acetate, and n-hexane). The usage of solvents in the maceration process is expected to be able to extract the large fraction of possible fungicidal active compounds. In addition, there are differences in the ratio of sample weight and volume of solvent used between researchers, ranging from : to : . The greater the ratio of solvent volume and sample weight will maximize the extract or active fungicidal compound produced. However, it is necessary to pay attention to the effectiveness of usage the solvent volume. .

Stratified fractionation method
Practices of the graded fractionation method have been carried out, for example: the fine powder of Chinese henna leaves was fractionated in stages using filter made of various sizes of paralon to form funnel containing activated charcoal as filter and adsorption of nonpolar compounds. The liquid-liquid solvent extraction method used cold distilled water. Then, it was followed by solution of alcohol or n-hexane with concentrations of , , , , , , , , and %, respectively (Efri et al., ). Then, babadotan/goatweed (Ageratum conyzoide) leaf powder was placed into simple fractionation tool, then the filtered residue was collected and air-dried. The filtrate or crude extract was added with methanol solvent then was collected and air-dried to obtain the methanol fraction of the babadotan leaf extract. In the same way, to get ethyl acetate and n-hexane extract (Wulandari et al., ). The water solvent is expected to be able to extract the active polar fungicide compound which is polar. Decreasing the level of polarity starting from methanol, ethyl acetate, and nhexane solvents is expected to be able to separate the active fungicide compounds based on their polarity level. .

Decoction method
Decoction method has been used to extract the fungicidal active compounds found in betel leaf. Samples were boiled in water with ratio of : for hour. The extract are filtered and sterilized using autoclave at temperature of °C to obtain sterile betel leaf extract (Trisnawati et al., ). The boiling process of the Cassia alata Linnaeus sample which was blended with water was carried out for minutes (Arneti Sulyanti, ). This decoction method is rarely used because it is feared that the active fungicidal compounds present in the sample could be damaged by heat treatment.

In vitro test as fungicide for chili
The review results of research related to in vitro fungicide test are summarized in Table . The concentration of the test solution was carried out in various ways. For example, the concentration of mixture of betel leaf and tobacco extract with concentration of % was made by mixing ml of PDA (Potato Dextrosa Agar) and ml of mixture of betel and tobacco extracts (Nur Rohmah, ). Another technique was found in preparation of kenikir leaf extract test solution which is done by mixing the extract with Tween as emulsifier with ratio of : (w / v) and diluted using sterile distilled water to get concentration of %, %, %, and % (Amelia et al., ). In other cases, the suren concentrated extract was assumed to be % concentration then the concentrated extract was diluted using distilled water into several concentrations ( %, %, and %) (Andriyani et al., ). Besides water, methanol was also used as solvent to make test solution for the Curcuma sp. rhizome with concentration of -ppm (Sari et al., b). The synthetic fungicide control used by several researchers in in vitro tests included: propineb %, . % propineb, azoxistrobin, diphenoconazole, benomyl, anthracol, . % acrobat and . % carbendazim. The usage of synthetic fungicide controls is very useful as comparison against the plants being studied. Many studies do not use synthetic fungicide controls so that the potential of these plants is less known when compared to synthetic fungicide controls. On the other hand, the most widely used fungi for in vitro tests are Colletotrichum capsici and then Colletotrichum gloeosporioides. The percentage of inhibition of fungal mycelium (%): . -. (Lestari et al., ) Betel and Tobacco % with concentration ratio  Some of the parameters used in the in vitro test include: colony diameter, percentage of colony inhibition, density / number of spores, and colony area. Colony diameter is measured by making vertical and horizontal lines perpendicular to each other at the bottom of the petri dish as vertical and horizontal diameters.
Then, the colony diameter is calculated using formula (Andreas et al., ) : With : D = diameter of horizontal colony D = diameter of vertical colony Observations are made by measuring the diameter of the growth of C. capsici colonies. The measurement of inhibition using the formula: ( ) With : DH = Inhibition (percent) a = diameter of C. capsici colony (mm) (negative control) b = diameter of C. capsici colony (mm) (treatment) Spore density was determined by taking ml of spore suspension from isolate propagation treatment. Furthermore, the spore density was calculated using hemocytometer that had been dropped by the suspension under a double lens (binocular), which is one type of lens from a light microscope with a magnification of times. (Herlinda et al., ). The spore density was calculated using Gabriel Riyatno formula ( ) (Gabriel Riyanto, ): = 0, 25 10 6 ( ) With: C = spore density per ml of solution t = total number of spores in the sample box observed n = number of sample boxes ( large x small boxes) . = correction factor for the use of a small-scale sample box on the haemacytometer Colony area was measured using millimeter plotting paper by depicting the colony area on plastic glass (Liswarni Edriwilya, ). The plants studied as an alternative to natural fungicides for chili have the ability to inhibit the growth of anthrax-causing fungi in chilies by in vitro study, which include: Colletotrichum capsici, Colletotrichum gloeosporioides, and Colletotrichum acutacum. However, many in vitro studies do not compare with synthetic fungicides. So, it is not possible to know the effectiveness of the performance of natural fungicides for chilies when compared to synthetic fungicides. Based on Table , it can be seen that % alcohol extract and % n-hexane extract and % Chinese henna leaves is similar with the . % propineb performance by in vitro study. In addition, the % and % kirinyuh leaf extracts were also able to match . % acrobat performance by in vitro study.

In vivo test as fungicide for chili
In effort to find alternatives natural fungicides, the researchers focused not only on in vitro studies but also in vivo studies of var-ious plants with certain concentrations as shown in Table . This in vivo test was directly applied to chili plants to be treated with natural fungicides with test conditions appropriate to the actual environment in chili farm.  Anthracnose disease in chilli is characterized by the appearance of blackish brown spots that will expand into soft rot with black dots in the middle which are collection of seta and conidia of C. capcisi fungi. The attack of C. capsici fungi begins by attaching the spores to the fruit and then the spores will germinate. Furthermore, through the fungal hyphae inject the fruit tissue and take nutrients in it so that it can interfere with metabolism and even cause cell death. The more severe the disease attack, the more extensive the rotting area on the fruit will be, this is due to damage to the fruit tissue and even cell death which ultimately results in the fruit experiencing dry rot or shrinking. (Andriyani et al., ). Disease severity is the surface area of chilies that shows symptoms of disease. Disease severity can also be interpreted as the part of the plant affected by disease or the disease area of the sample plant. Determination of the percentage of disease severity can be calculated by the formula as follows (Suwastini et al., ): Several studies have also identified other parameters in the in vivo test, for example: fruit weight, mycelium dry weight, spot diameter, yield / number of red chilies, number of fruits, effectiveness and level of fungicidal ability, incubation period of anthracnose disease, morphometry of cayenne pepper, period incubation, percentage and fresh weight of healthy cayenne pepper affected by anthracnose disease, when the early symptoms of anthracnose disease appeared in red chilies, and the height of chili plants. The plants studied for chilies had the effectiveness of being used as a natural fungicide. However, many in vivo studies do not compare with synthetic fungicides. So, it is not possible to know the effectiveness of the performance of natural fungicides for chilies when compared to synthetic fungicides. Table shows that suren leaf extract and nut bulbs can be used as alternatives to natural fungicides to help overcome the problem of anthracnose in chilies.

Conclusion
This paper reviews the potential plants as an alternative to chili fungicides, the preparation of test solutions, in vitro and in vivo fungicide tests. The part of the plant that is widely studied as fungicide for chilies is the leaves, while the parts of the plant that are rarely used as samples are the parts of the rhizome, roots, tubers, weevils, seeds, fruit, flowers or all parts of the plant. The methods of extract preparation used as fungicide test include: maceration method, stratified fractionation method, and decoction method. The plants studied had the ability to inhibit the growth of the Colletotrichum capsici, Colletotrichum gloeosporioides, and Colletotrichum acutacum. The % alcohol extract and % and % n-hexane extract of Chinese henna leaves can be equal with the performance of . % propineb by in vitro study. In addition, the % and % kirinyuh leaf extracts were also able to match acrobat . % performance by in vitro study. Two parameters that are often observed in the in vivo test are the percentage of anthracnose disease incidence and the percentage of anthracnose disease severity. Suren and nut bulbs leaf extract can be used as alternative to natural fungicides to help overcome the problem of anthracnose in chilies.

Declaration of competing interest
The authors declare no known competing interests that could have influenced the work reported in this paper.