Title Science and Technology Indonesia e-ISSN:2580-4391 p-ISSN:2580-4405 Vol. 3, No. 4, October 2018 Research Paper Potential of Anabaena Azollae Extract as Antimicrobial Agent For Paddy Crop Disease Nuni Gofar1*, Andi Diana2, Eka Setianingsih2 1Department of Soil Science, Faculty of Agriculture, Sriwijaya University, Indonesia 2Department of Agroecotechnology, Faculty of Agriculture, Sriwijaya University, Indonesia *Corresponding author: nigofar@unsri.ac.id Abstract The research objective was to test antimicrobial activity from compound produced by A. azollae which symbiosis with Azolla sp. toward microbial test of X. oryzae and Pyricularia oryzae as cause of disease on paddy crop. Sampling of Azolla which symbiosis with A. azollae was done in Azolla cultivation pond at Faculty of Agriculture, Sriwijaya University. The available Azolla was consisted of two types, i.e. Azolla pinnata and Azolla mycrophiylla. The extraction of A. azollae from Azolla leaves was done by method of Ultrasound Assisted Extraction (UAE) using ethyl ester solvent and maseration method using methanol solvent. Test media for bacteria and fungus respectively are Mueller Hilton Agar (MHA) and Sabouraud Dextrose Agar (SDA). The diameter of produced clear zone is an indication of extract’s inhibitory power toward bacteria or fungus. The different of inhibitory zone diameter is compared by using t-test. Analysis of active compounds on Anabaena azollae extract was done by using HPLC. Ethyl acetate or methanol extract of A. azollae which symbiosis with A. pinnata or A. microphylla was capable to inhibit the growth of X. oryzae bacterium and P. oryzae fungus. The dominant compounds containing within extract with probability more than 60% and area of more than 10% are consisted of phytol, hexadecanoate acid and 2-(tert-butyl)-4,6-dimethyl phenol. Keywords Anabaena azolae, Azolla microphylla, Azolla pinnata, antimicrobe, rice crop Received: 14 September 2018, Accepted: 16 October 2018 https://doi.org/10.26554/sti.2018.3.4.183-188 1. INTRODUCTION Rice (Oryza sativa L.) is the main food commodity in Indonesia because the staple food for most of Indonesia population is rice. The blast disease caused by Pyriculari aoryzae Cav. fungus and leaf blight disease caused by Xanthomonas oryzae bacterium are important diseases that attack paddy crop. These diseases strike during wet season or humid dry season, especially on constantly �ooding paddy �eld. Azolla is water fern plant that frequently found in paddy �eld area and it symbiosis with Anabaena azollae that capable to �x N2 and subsequently convert N2 into nitrogen available for crop (Muniappan et al., 2016). In addition to contribute nitrogen, A. azollae also produce plant growth hormone and antimicrobia compound (Renuka et al., 2018). Sianobakter is a source of several compound having antimi- crobial activity such as alkaloid, aromatic compound, depipep- tide cyclic, peptide cyclic, undecapeptides cyclic, siclopane, extracellulair pigment, lipid acid, linear peptide, lipopeptide, nucleoside, phenol, macrolide, polyketides, polyphenyl ethers, por�noide and terpenoid so that its extract can be used as an- timicrobia (Swain et al., 2017; Salman and M.M, 2016). These bioactive compounds can inhibit the growth of soil induced pathogen bacteria such as X. oryzae bacterium (Abraham et al., 2015) and fungi such as P. oryzae that cause blast disease on rice crop (Blahova et al., 2013). This research will conduct test in term of antibacterial and antifungal activities from compound produced by A. azollae which symbiosis with Azolla sp. on X. oryzae and P. oryzae that frequently cause disease on rice crop. It is expected that results of this research will provide an alternative biological control for blast and leaf blight diseases on rice crop by using Azolla sp. as well as N2 contributor for crop. 2. EXPERIMENTAL SECTION This research was conducted from November 2017 to April 2018. Sampling of Azolla which symbiosis with A. azollae was taken from Azolla cultivation pond at Faculty of Agriculture, Sriwijaya University. There are two types of Azolla, i.e. Azolla pinnata and Azolla mycrophiylla. Extraction of A. azollae from leaves of Azolla pinnata and Azolla mycrophiylla was done by using Ultrasound Asisted Extraction (UAE) with ethyl ester solvent and maseration method with methanol solvent. Reju- venation of X. oryzae bacterium and P. oryzae fungus was done by using tilt agar medium. Analysis of active compounds from https://doi.org/10.26554/sti.2018.3.4.183-188 Gofar et. al. Science and Technology Indonesia, 3 (2018) 183-188 extraction yield was done by using HPLC. 2.1 Extraction of Anabaena azollae Azolla that had been air dried subsequently is blended to pro- duce powder. In maseration method, 300 mL of methanol solvent is added into erlenmeyer containing 50 g dry biomass of azolla. This mixture is incubated for 7 days until the sol- vent’s color change into dark green. After 7 days, supernatan is collected and dried by using Rotatory evapourator. In Ultra- sound Asisted Extraction method, 40 mL ethyl acetate solvent is added into erlenmeyer containing 32 g dry biomass of azolla. This mixture is sonicated for 5 minutes, then it is shaked for 15 minutes by using shaker and subsequently it is centrifused at speed of 4000 rpm for 10 minutes. Supernatan is sepa- rated and collected within erlenmeyer. Biomass within pellete is added again with 40 ml ethyl acetate solvent. The extrac- tion steps are repeated several times until all metabolites in biomass had been extracted which indicated by the change of cell biomass color into white color. The collected supernatan is subsequently dried by using Rotatory evaporator. 2.2 Preparation of Bacteria and Fungi Pure isolates of X. oryzae bacterium and P. oryzae fungus are cultured on tilt agar media by taking colonies from stock of bacterium or fungus by using ose needle and it was scratched on tilt agar media. Tilt agar media is incubated at 37°C for 24 hours. Subsequently, colonies of bacterium or fungus from tilt agar media are taken by using sterile ose needle and each colony is put into test tube containing 10 mL Nutient Broth (NB) media. 2.3 Preparation of Test Media Test media for bacterium used Mueller Hilton Agar (MHA) media with composition as follows: 6% peptone, 17.5% casein, 1.5% starch and 10% agar. Twenty �ve grams (25g) of MHA media is dissolved in 1000 mL aquadest and sterilized using autoclave at 121°C for 15 minutes. Test media for fungus used Sabouraud Dextrose Agar (SDA) media with composition as follows: 10 g mycological peptone, 40 g glucose 40 g and 15 g agar. SDA media with magnitude of 6.5 g is dissolved with 100 mL aquadest within erlenmeyer and subsequently put on hot plate stirrer to homogenize the solution in order to produce clear solution. Media is sterilized in autoclave at temperature of 121°C for 20 minutes. 2.4 Test of Antibacterial and Antifungal Activities Test of antibacterial and antifungal capabilities was done by using disk paper di�usion method. Sterile media of MHA and SDA are respectively poured into petri dish with thickness of ± 0,5 cm and waited until condense at room’s temperature. Suspension of bacterium and fungus respectively with magni- tude of 0.1 mL are innoculated using spread plate technique in surface of MHA media for bacterium and in surface of SDA media for fungus. Disk from sterile �lter paper with diameter of 5 mm is saturated with ethyl acetate or methanol extract of A. azollae which life within leaves of A. pinnata and A. mycro- phylla. Filter paper is put in center of agar surface that had been innoculated with bacterium or fungus, then it is covered and put in upside down position within incubator at temperature of 37°C for 24 hours. Diameter of produced clear zone is an indication of extract’s inhibitory power toward bacterium or fungus. Inhibitory zone diameter is measured by using vernier caliper. The di�erent of produced inhibitory zone diameter are compared by using t-test. Analysis of active compounds from ectraction yield was done by using HPLC. 3. RESULTS AND DISCUSSION 3.1 Test of Antibacterial and Antifungal Activities Figure 1 showed the development of growth inhibitory zone for bacterium or fungus by antimicrobial compounds from ethyl acetate or methanol extract of A. azollae which life within leaves of A. pinnata and A. mycrophylla. An extract has potential as antimicrobia if it produces mi- crobial growth inhibitory zone during the test. The magnitude of produced inhibitory zone is a�ected by antimicrobal com- pound activity on this extract. Salman and M.M (2016) showed that A. azollae can produce toxic compound as antibacterial agents such as alkaloid, neurotoxins and anatoxin. Rossana et al. (2006) had reported that antifungal compound produced by A. azollae which life in leave tissue of Azolla sp. are phenol, �avonoid, alkaloid, terpenoid, glycoside and saponin. Alkaloid compound has inhibitory mechanisms by disturbing peptido- glycan constituent components within cells so that cell wall layers are not fully developed resulting in death of cells (Ju- liantina, 2008). Gunawan (2009) had stated that base group containing nitrogen which is available within alkaloid com- pound reacts with amino acids that compile cell wall and DNA of bacterium and fungus. This reaction results in the change of structure and arrangement of amino acids which in turn produce the change of genetic equilibrium in DNA chains and damage which promote lysis and death of cells on bacterium. The capability di�erences of ethyl acetate or methanol ex- tract of A. azollae from leaves of A. microphylla and A. pinnata in inhibiting the growth of X. oryzae bacterium and P. oryzae fungus is shown in Table 1. Tabel 1 showed capability e�ect of A. azolae extract from leaves of A. microphylla and A. pinnata on activitities of X. orizae bacterium and P. oryzae fungus. There was signi�cant di�erent between inhibitory zone diameter of bacterial activity by A. azollae extract from leaves of A. microphylla and A. pinnata using the same solvent, either ethyl acetate or methanol. How- ever, there was no signi�cant di�erent between inhibitory zone diameter of P. oryzae fungus activity by A. azollae extract from leaves of A. microphylla and A. pinnata using the same solvent, either ethyl acetate or methanol. Tabel 2 showed test results of capability di�erences of A. azollae extract from leaves of A. microphylla and A. pinnata us- ing di�erent solvents toward X. oryzae bacterium and P. oryzae fungus activities. There was signi�cant di�erent between in- hibitory zone diameter toward X.oryzae bacterium and P.oryzae © 2018 The Authors. Page 184 of 188 Gofar et. al. Science and Technology Indonesia, 3 (2018) 183-188 Figure 1. Development of growth inhibitory zone for X. oryzae bacterium or P. oryzae fungus on agar media Table 1. Capability di�erence of A. azolae extract from A. microphylla and A. pinnata leaves with the same solvent Extraction source of A. azollae Solvent Inhibitory zone diameter (mm) t-calculated t-table 0.05 On activity of X.oryzae bacterium Azolla microphylla Ethyl acetate 13 3.45* 2.13 Azolla pinnata Ethyl acetate 15 Azolla microphylla Methanol 10 2.59* 2.13Azolla pinnata Methanol 11.5 On activity of P. oryzae fungus Azolla microphylla Ethyl acetate 16.25 046tn 2.78 Azolla pinnata Ethyl acetate 15.58 Azolla microphylla Methanol 22.25 235tn 2.78Azolla pinnata Methanol 21.33 © 2018 The Authors. Page 185 of 188 Gofar et. al. Science and Technology Indonesia, 3 (2018) 183-188 Figure 2. Chemical structures for three compounds within A. azollae extracted from A. pinnata and A. microphylla leaves fungus activities due to treatment of A. azollae extract from leaves of A. microphylla and A. pinnata using di�erent solvent. Table 2 showed that four A. azollae extracts had capability in inhibiting the growth of bacterium and fungus. This in accordance to the study results by Nabakishore et al. (2015) which showed that extract from A. azollae can inhibit the growth of Staphylococcus aureus. A. azollae extract with methanol solvent had lower antibacterial activity than that of using ethyl acetate solvent. This in accordance to the study results by Moshi and Moshi and Mbwambo (2005) which showed that semi polar extract (ethyl acetate) can inhibit E. coli and B. anthracic bacteria with higher inhibitory diameter than that of polar extract (methanol). On the other hand, antifungal activity from extract with methanol solvent was higher than that of using ethyl acetate solvent. Methanol that has high polarity is capable to dissolve most of compounds having antifungal property. Solvent with low polarity such as ethyl acetate draws antifungal active extract in less quantity than that of ethanol and methanol mixture or methanol alone (Ismail et al., 2004). 3.2 The compounds available in A. azolae extract Factors which cause di�erences in pathogen inhibitory power are variation and concentration of secondary metabolites con- tains within extract and the produced fraction. Table 3 showed compounds available in A. azolae extract which life within leaves of A. pinnata and A. mycrophylla using ethyl acetate and methanol solvents. Widiana (2012) had reported that one of factors that a�ect antimicrobia in inhibiting microbia growth is concentration of antimicrobial substance. The higher the concentration of extract containing antibacteria, the faster the killing process of pathogen. Microorganisms have di�erent tenacity to antimicrobia. The higher the number of microbial cells, the longer the treatment time required to kill all microbia. Table 3 showed that dominant compounds within A. azollae extract with probability higher than 60 % and area of more than 10 % are consisted of phytol, hexadecanoic acid and 2-(tert- butyl)-4,6-dimethyl phenol. Figure 2 showed that chemical structures for three compounds in Table 3. In accordance to the published papers by Rossana et al. (2006); Salman and M.M (2016); Swain et al. (2017), the above compounds (hexadecanoat acid, phytol, and phenol) had antimcrobial activity. Further research is needed to determine the possibility of compounds that contains in A. azollae extract can be applied as biocide. 4. CONCLUSIONS Ethyl acetate or methanol extract of A. azollae which symbio- sis with A. pinnata or A. microphylla was capable to inhibit the growth of X. oryzae bacterium and P. oryzae fungus. The domi- nant compounds containing in extract of A. azollae with proba- bility higher than 60 % and area of more than 10 % are consisted of phytol, hexadecanoat acid and 2-(tert-butyl)-4,6-dimethyl phenol. 5. ACKNOWLEDGEMENT This research is part of Profession Grant Research with title of Application of Functional Microbia for Food and Organic Feed which was funded through PNPB Unsri for �scal year of 2018. REFERENCES Abraham, G., R. Yadav, , and G. Kaushik (2015). Antimi- crobial activity and identi�cation of potential antimicrobial compounds from aquatic pteridophyte, Azolla microphylla Kaulf. Indian Journal of Experimental Biology, 53(4); 232– 235 Blahova, L., O. Adamovsky, L. Kubala, L. Svihalkovd, R. Zounkova, and L. Blaha (2013). The isolation and char- acterization of lipopolysaccharides from Microcystis aerugi- nosa, a prominent toxic water bloom forming cyanobacteria. Toxicon, 76; 187–196 Gunawan, I. (2009). 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Probability, area and retention time of compound formed from A. azollae extracted from A. pinnata and A. microphylla leaves Extraction sources Compound formed Probability (%) Area (%) Retention time (min- utes) A. pinnata with ethyl acetate solvent Phytol 74.09 15.88 19.56 Hexadecanoic acid 68.27 16.01 18.74 A.microphylla with ethyl acetate solvent Phytol 73.68 11.89 19.55 Hexadecanoic acid 77.57 15.29 18.26 A. pinnata with methanol solvent 2-(tert-butyl)-4,6- dimethyl phenol 95.81 17.05 22.1 Hexadecanoic acid 76.23 12.67 18.25 A. microphylla with methanol solvent 2-(tert-butyl)-4,6- dimethyl phenol 69.69 10.62 19.55 Hexadecanoic acid 60.22 14.84 18.5 © 2018 The Authors. Page 187 of 188 Gofar et. al. Science and Technology Indonesia, 3 (2018) 183-188 (Wulfen) Lamouroux on the growth of human pathogen yeasts. Brazilian of Biology and Technology, 49; 915–921 Salman, J. and W. M.M (2016). Activity of cynophyta algal extracts(Anabaena azolla) against some species of fungi in local habitats. Mesopotemia Environmental Journal, 3(1); 1–9 Swain, S. S., S. K. Paidesetty, and R. N. Padhy (2017). Antibac- terial, antifungal and antimycobacterial compounds from cyanobacteria. Biomedicine & Pharmacotherapy, 90; 760–776 Widiana, R. (2012). Konsentrasi hambat minimum (KHM) ekstrak daun teh (Camellia sinensis L.) pada Escherichia coli dan Salmonella sp. Jurnal Pelangi, 4(2) © 2018 The Authors. Page 188 of 188 INTRODUCTION EXPERIMENTAL SECTION Extraction of Anabaena azollae Preparation of Bacteria and Fungi Preparation of Test Media Test of Antibacterial and Antifungal Activities RESULTS AND DISCUSSION Test of Antibacterial and Antifungal Activities The compounds available in A. azolae extract CONCLUSIONS ACKNOWLEDGEMENT