Antimicrobial Activity of Endophytic Fungi Isolated from Physalis angulata L. Plant

Some endophytic fungi live in plant tissue and does not caused any damage on their host plant. The endophytic fungi could produce antimicrobial secondary metabolites. Some endophytic fungi have been isolated from a medicinal plant Physalis angulata, i.e: Penicillium verrucosum, Colletotrichum alienum, Fusarium subglutinans, Aspergillus nidulans, Mycelia sterilia 1, Mycelia sterilia 2, and Rhizoctonia sp. The purpose of this research are: 1) to examine the antimicrobial activity of each endophytic fungi species toward Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and, 2) to determine the endophytic fungi species that have highest antimicrobial activity. Each endophytic fungi isolates were inoculated in Potato Dextrose Broth (PDB) medium and shaked in 120 rpm, during 7 x 24 hours, then the broth culture centrifugated in the rate of 3000 rpm for 10 minutes. The supernatants from each endophytic fungi were treat to the bacteria to know the antimicrobial activity by agar diffusion method. Afterwards the antibaterial effect of each endophytic fungi species were measured. Then the highest antimicrobial effect were determined. The research results shows that: 1) each endophytic fungi species have antimicrobial activity towards B. subtilis, E. coli, S. aureus, and; 2) Penicillium verucossum secondary metabolites have the highest antimicrobial activity.


Introduction
Some research about antimicrobial activity of medicinal plants have been done. People try to find an alternative material from nature for antibiotic substance. It is because some pathogenic bacteria was resistance against synthetic antibiotic. Medicinal plants could produce antibacterial secondary metabolites, i. e: flavonoid, alkaloid, tannin, there for it is potentially to use as antibiotic sources [1]. Some endophytic fungi could produce secondary metabolites that have characters; antifungal, antibacterial, antiinflamatory, antiviral, antitumor; and the compounds belonging to: flavonoids, alkaloids, terpenoids, and steroids [2] [3].
Some endophytic fungi species live in symbiotic mutualism interaction with meditional plants as the host plant. The endophytic fungi found in roots, stem, and leaf tissues of potato plants [4]. Altough the endophytic fungi live in the host plants it does not cause any damage, but the fungi could produce some antimicrobial secondary metabolites. The last research results shows that some endophytic fungi species live in Hedychium acuminatum tissue, it was: Fusarium oxysporum, Rhizoctonia sp., Fusarium solani, Fusarium semitectum, Colletotrichum alienum, Colletotrichum ti, Colletotrichum aotearoa, Colletotrichum gloesporoides, and Aspergillus parasiticus. These fungi species have an ability to produce antimicrobial secondary metabolites as well as the host plant produce, i.e: alkaloid, flavonoid, terpenoid, and tannin [5]. It was also proved that the secondary metabolites in liquid culture of each endophytic fungi species isolated from H. acuminatum have antibacterial effect towards Staphylococcus aureus, and Bacillus subtilis [6].
Physalis angulata is another sort of medicinal plant. This plant could produce some secondary metabolities i. e: alkaloid, flavonoid, saponin, polifenol, terpenoid. People use this plant for infectious disease. Some endophytic fungi live in this plant tissue. In the preliminary research some endophytic fungi species have been isolated from Physalis angulata plant tissues, i.e: Penicillum verrucosum, Colletotrichum alienum, Fusarium subglutinans, Aspergillus nidulans, Mycelia sterilia 1, Mycelia sterilia 2, and Rhizoctonia sp. The purpose of this research are: 1) examine the antimicrobial activity of each endophytic fungi species towards Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, 2) determine the endophytic fungi species isolated from P. angulata plant that have the highest antimicrobial activity. The procedure is written as follows.

Preparation for Endophytic Fungi Liquid Cultures
The preparation of endophytic fungus liquid culture was conducted obtain the secondary metabolites produced by each endophytic fungi species [7]. Each endophytic fungus isolates inoculated on PDA plate medium containing chloramphenicol (100 mg/L) and incubated in 26°-27°C for 7-24 hours. Then each endophytic fungus species culture were cutted into five pieces of 1×1 cm each. The endophytic fungus pieces were inoculated in 100 mL PDB medium and shaked at 120 rpm for 7×24 hours in 26°-27°C. Afterwards the liquid were centrifugated at the rate of 3000 rpm for 10 min. The supernatant of each endophytic fungus species of 20 µL each were used for antimicrobial examination in vitro [8].

Examination of Endophytic Fungi Liquid Cultures Antimicrobial Effect
The examination of each endophytic fungus liquid culture antimicrobial effect againts Staphylococcus aureus, Eschericia coli, and Bacillus subtilis was done by the agar diffusion methods. Each bacteria species were inoculated on NB medium, standardized with MC Farland 0,5 (1,5 x 10 8 cfu/mL), the result of the incubation hour in 18 hours. The NA medium were prepared and cutted with sterile cork borrer to make wells. Then the bacteria culture were inoculated on NB medium and incubated at 37°C for 18 hours. Afterwards the bacteria liquid cultures were inoculated on NA medium and the wells were filled with 20 µL supernatant of each fungus spesies, then incubated at 37°C for 1 x 24 hour. The positive control used 5 mg/5 mL chloramphenicol and the negative control used PDB medium. The antimicrobial effect of each endophytic species against S. aureus, E. coli and B. subtilis was determined by measuring the growth inhibition zone around the wells on NA medium. The data were analyzed to know whether any differences in the antimicrobial activity of each endophytic fungi species by Anova and continued by Duncan's to determine the endophytic fungi species that have the highest antibacterial activity compared with the other species based on the growth inhibition zone diameter size.

Detection of Secondary Metabolites Content of Each Endophytic Fungi Species
The flavonoid, alkaloid, tannin, and saponin content in the liquid cultures of each endophytic fungi species were analyzied by spectrophotometry.

Result and Discussion
The secondary metabolites of endophytic fungi antimicrobial activity could be examine based on the diameter of inhibition zone around the well contained with the endophytic fungi liquid cultures on the NA medium. The NA medium have been inoculated with B. subtilis, E. coli and S. aureus culture. The research result showed that secondary metabolites of each endophytic fungi species isolated from P. angulata, i.e: Penicillium verrucosum, Colletotrichum alieum, Fusarium subglutinans, Aspergillus nidulans, Mycelia sterilia 1, Mycelia sterilia 2, and Rhizoctonia sp. have an antimicrobial activity toward E. coli, S. aureus, and B. subtilis (Figure 1) The data were analized with ANOVA and continued with Duncan 5% to know which endophytic fungi liquid culture that have the highest antibacterial activity towards E. coli, B. subtilis, and S. aureus. There are differences of growth inhibition zone diameter between each endophytic fungi species toward B. subtilis, E. coli, and S. aureus. P. verrucossum liquid culture produce the biggest growth inhibition zone diameter compare with the other species used in this research toward B. subtilis and S. aureus ( Table 1).
The clear zone around the well contains of each endophytic fungi secondary metabolities proved that there are an inhibition activity to the bacteria growth (Figure 1), It is proved that the fungi secondary metabolites in the well diffused to the medium and inhibited the bacterial growth. The reserch results proved that each endophytic fungi species could produced the four antibacterial componds although the endophytic fungi species have already isolated from Physalis angulata as the host plant that could also produce the same secondary metabolites.  The secondary metabolites content of each endophytic fungi species isolated from P. angulata plant were analiyzes ( Table 2). Each endophytic fungi species could produce antimicrobial secondary metabolites that protected P. angulata as the host plant from pathogenic bacteria infection. This fact is advantage for P. angulata [9] [10].
Each endophytic fungi species isolated from P. angulata plant liquid cultures could produce flavonoid, alkaloid, tannin, and saponin as well as in the host plant. There are differences content of the secondary metabolites produced by each endophytic fungi species. Penicillium verrucosum is the most potential species to produce its secondary metabolites in the highest content compared with the others species.
The four secondary metabolites produced by endophytic fungi species take a role in the bacteria colony growth inhibition. Alkaloid could inhibite some enzyme activity, i.e: esterase, RNA polymerase, DNA polymerase, and celluler respiration enzyme [11]. Flavonoid take a role in bacteria cell membrane damaged and inhibite the cellular metabolism enzymes activity [12]. Besides that, OHion in phenol compound could bound to H + ion in hydrogen bound at protein cell wall structural protein and cause protein denaturation [13]. Then it cause bacteria cell wall and cell membrane damaged. The bacteria cell membrane semipermeability will be decreased and caused the cell water and enzymes removed from the cell. It caused metabolism and the cell growth inhibition. Tannin could dissolve the lipid layer on bacteria cell wall and also could denaturate the structural protein in bacteria cell membrane [14] [15]. This case caused cell wall structure damaged and also decrease the cell membrane semipermeability. Then the nutrients and enzymes removed from the cell and caused celluler metabolims and cell growth inhibition.
This researh result proved that the liquid culture of each endophytic fungi species contains antibacterial secondary metabolites have antimicrobial activity towards B. subtilis, E. coli, and S. aureus. P. verrucosum liquid culture have the highest antibacterial activity compared with the other endophytic fungi species isolated from P. angulata plant. The liquid culture of P. verrucosum also contains flavonoid, alkaloid, tannin, saponin in the highest content. Based on this research result people could use this endophytic fungi liquid cultures as an antibiotic source. It is an alternative way besides use the P. angulata plant parts extract.

Conclusion
The conclusions are: 1) each endophytic fungi species isolated from P. angulata plant have antimicrobial activity toward B. subtilis, E. coli, and S. aureus; 2) Penicillium verrucosum secondary metabolites in liquid culture have the highest antimicrobial activity. For the suggestion, it is need to make the next research to prove that secondary metabolites produced by the endophytic fungi species isolated from another parts of P. angulata plant also have an antibacterial effect to E. coli, S. aureus, and B. subtilis.