PHYTOCHEMICAL ANALYSIS OF Mucuna pruriens L. LEAVES EXTRACT AND ITS INHIBITION TEST AGAINST Curvularia lunata (WAKK). BOED. THAT CAUSED LETTUCE LEAF SPOT

Curvularia lunata is a pathogenic fungi that attack various plant species including lettuce. Its control needs to be carried out in an environmentally friendly manner, namely using botanical fungicides. M. pruriens is a legume plant that is widespread in Indonesia that has a potential as a botanical fungicide, beside as a traditional medicine. The purpose of this study was to study the chemical content of M. pruriens leaves and its potency to inhibit the growth of C. lunata . The determination of the compounds was carried out by Gas Chromatography-Mass Spectrophotometry and the inhibition test was carried out using diffusion well, and agar dillution method. The result showed that the crude extract of M. pruriens leaves contained ethyl isothiocyanate that act as an antifungal with AUC of 20,78%. The MIC of the extract was 0,9%. The extract at 5% concentration was capable to inhibit C. lunata growth, mycelium mass, and conidia production respectively 38,33%; 67,14%; 99,88% compared to control.


INTRODUCTION
Curvularia lunata is a pathogenic fungus that attacks various types of plants (Garcia-Aroca et al., 2018), including lettuce.
The highlands climatic conditions that have high humidity are optimal conditions for pathogenic fungi to grow.C. lunata is one of the important pathogens that cause leaf spots in lettuce especially in nurseries and causes extensive losses (Baiyee et al., 2018).The first report on lettuce was in Thailand in 2018 (Pornsuriya et al., 2018).C. lunata spreads through the soil and seeds (Akinbode, 2010;Watanabe, 2010).The application of synthetic fungicides is the most effective control technique for controlling C. lunata.
Long-term application of mancozebbased fungicides will cause C. lunata to be resistant against this fungicide (Susanto and Prasetyo, 2013).Therefore, environmentally friendly control is needed, namely with botanical fungicides.Mucuna pruriens is a legume plant that is widespread in Indonesia (Mulyani et al., 2016).M. pruriens is often used as a traditional medicine traditional (Sathiyanarayanan and Arulmozhi, 2007), in addition, M. pruriens also has the potential to be a botanical fungicide because it has antifungal properties (Nidiry et al., 2011).Nidiry et al. (2011) in their study state that M. pruriens seeds extractive was able to inhibit Colletotrichum sp. and Fusarium solani.Khan et al. (2008) also state that M. pruriens seed extract from various solvent showed antifungal activity against Aspergillus niger.Rayavarapu et al. (2011) found that the whole plant of M.
The aim of this study was to determine the content of compounds in the leaf extract of M. pruriens and its potential in inhibiting the growth of C. lunata.

MATERIAL AND METHOD
The equipments used in this study were and then poured into a sterile petri dish.

Pathogen isolation and identification
Symptomatic lettuce leaves were taken and sterilized using 70% alcohol on the part between the symptomatic and healthy part.
The leaves were cut by 0.5 cm x 0.5 cm and then placed on a solid PDA medium and incubated for 5 days at a temperature of 25 o C.
The growing fungus then purified.Once pure, isolates were observed under a microscope and identified based on their morphology following the Barnett and Hunter (1998) identification key.

Inhibition test assay
The inhibition test against the growth of C. lunata was carried out by agar dilution method.The design used was a Complete Randomized Design with four replicate.The observed variables were colony growth, mycelium mass, and the number of conidia.
The concentration of the extract tested was 0.1%; 0,2%; 0,5%; 1%; 2%; 5%; and 0% as a control.A colony of C. lunata with 5 mm in diameter was placed in the center of a solid PDA medium that already contains the extract.Observations were made as the control fungus fully grew to the petri.For colony growth assay, the diameter of the colony was measured, then the percentage of colony growth inhibition of the extract is calculated by equation (1).
For the mass of mycelium assay, the mycelium of C. lunata was cleaned from the media by pouring heated 10% HCl, then filtered using filter paper.The mycelium then dried at a temperature of 80 o C until its weight constant, then weighed.For the amount of conidia formed, conidia were harvested by pouring 20 mL of aquades, then dredged with an ose needle so that a conidia suspension obtained.The number of conidia was calculated using a haemacytometer.

Data analysis
The data obtained were quantitatively analyzed using analysis of variance (ANOVA) at a level of 5% and then analyzed using the Least Significant Difference (LSD) test with a level of 5%.

RESULT AND DISCUSSION
Maceration of 0.5 kg of simplisia leaves of M. pruriens produced a rendement of 4.13%, which is 20.63 g of green-yellow-black crude extract.The crude extract obtained then analyzed using GC-MS and tested its antifungal activity.The results of the analysis of the content of compounds in the leaf extract of M. pruriens using GC-MS showed that there were 22 compounds contained in the extract shown in Figure 1.et al., 1988).lunata are presented in Table 3.     (Kara & Soylu, 2020;Kurt et al., 2011;Otoni et al., 2014;Park et al., 2013;Smolinska et al., 2003;Wu et al., 2011;Yang et al., 2021).
Gluthatione, one of natural antioxidant that produced by living organism (Aguirre et al., 2006;Pócsi et al., 2004), often become the target of isothiocyanate group compounds.

Figure 1
Figure 1 Chromatogram of M. pruriens leaves extract

Figure 2
Figure 2 MIC of M. pruriens leaves crude extract against C. lunata inhibitory power of M. pruriens leaves crude extract against the growth of C. lunata colonies are shown in Figure3.

Figure. 3
Figure. 3 Inhibitory power of M. pruriens leaves crude extract against C. lunata colonies growth

Table 1 .
Primary compunds found in M. pruriens leaves extract

Table 3
Inhibitory power of M. pruriens leaves crude extract against C. lunata Analysis Of Mucuna Pruriens L. Leaves Extract And Its Inhibition Test Against Curvularia Lunata (Wakk).Boed. Phytochemical