S238

Document heading doi: 10.1016/S1995-7645(14)60239-X

Screening and study of antifungal activity of leaf litter actinomycetes isolated from Ternate Island, IndonesiaArif Nurkanto*, Heddy JulistionoResearch Center for Biology, Indonesian Institute of Sciences (LIPI), Jl. Jakarta-Bogor KM 46 Cibinong, West Java-16911, Indonesia

Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

*Corresponding author: Arif Nurkanto, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Jl. Jakarta-Bogor KM 46 Cibinong, West Java-16911, Indonesia. E-mail: arif.nurkanto@lipi.go.id Foundation Project: Supported by Research Center for Biology, Indonesia Institute of Sciences (Grant. No. 3400.003.002/028).

1. Introduction

Ternate Island (Moluccas) is one of eastern Indonesian islands located in Wallace biogeographical region. These regions are meeting-point of the biota of two major geographical regions: Australia and Oriental region. Ternate has a large number of species that are unique to the area and is recognized as a biodiversity hotspot[1]. The diversity of microbes of Ternate Island is expected to be more higher than that of plants. This is based on the prediction of ratio of unique species and vascular plant of 6:1[2]. Despite the richness of fauna and flora of Ternate Island, there is only a few information about microbial diversity. Microbial diversity of the island is, therefore, intriguing to study. Actinomycetes

belong to a microbial group that is still interesting to explore until now due to their potency to produce active substances against drug resistance microbes[3]. In agriculture, actinomycetes are promising to be used for biocontrol against fungal pathogen of plant[4], and bacterial pathogen in aquaculture[5]. Fish pathogenic fungi is also a problem in aquaculture[6]. Fungal diseases candidiasis and aspergillosis are infections caused by fungi, leading to increasing morbidity and mortality[7]. Thus, actinomyctes having activity against harmful fungi are useful not only for agriculture and aquaculture but also for human health. In an ecosystem, leaf litter is an important source of microbes including actinomycetes[8]. Moreover, it is also reported that actinomycetes could be antagonist to decomposer fungi including fast growing genera Mucor, Penicillium, Trichoderma and moderately slow-growing genera Cladosporium and Mortierella[9]. The aim of this research is to obtain information about actinomycetes abundance from leaf litters in Ternate

ARTICLE INFO ABSTRACT

Objective: To characterize abundance of leaf litter actinomycetes from Ternate Island and to assess the antifungal activity of actinomycetes isolates against Candida albicans, Saccharomyces cerevisiae (S. cerevisiae), and Aspergillus niger.Methods: Actinomycetes were isolated from leaf litter of Durio species, Syzygium aromaticum, Piper betle, Myristica fragrans, or Pandanus species and unknown plants. Actinomycetes isolates were cultured in a liquid medium. Bioactive compounds were extracted and tested against fungal using Beury-Kirby method with modification. Minimum inhibitor concentration and cell leakages were conducted. Actinomycetes that produced the highest antifungal activity were indentified using molecular sequence data in 16S rRNA gene.Results: Out of 50 selected isolates, two isolates MG-500-1-4 and SR-2-2 has highest activity against S. cerevisiae. Concentration of material containing nucleic acids, proteins, Ca+ and K+ ions and morphological observations indicated that extracts of MG-500-1-4 and SR-2-2 caused cell leakage and invagination of S. cerevisiae cells. Based on 16S rRNA gene identification, MG-500-1-4 and SR-2-2 isolates are similar to Streptomyces misakiensis and Streptomyces tricolor respectively.Conclusions: Ternate Island contains interesting biodiversity of actinomycetes that has potential use in agriculture, fisheries, and human health to reduce problem of fungal pathogen.

Contents lists available at ScienceDirect

Article history:Received 20 Feb 2014Received in revised form 1 Mar, 2nd revised form 7 Mar, 3rd revised form 12 Mar 2014 Accepted 26 Apr 2014Available online 19 Aug 2014

Keywords:Ternate islandWallace biogeographicActinomycetesAntifungi

Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243 S239

Island and to reveal their potential activity against yeast Candida albicans (C. albicans), Saccharomyces cerevisiae (S. cerevisiae) and filamentous fungi Aspergilus niger (A. niger).

2. Material and methods

2.1. Sample site of actinomycetes isolation

Leaf litter samples were obtained from Ternate Island, mainly in Gamalama Mountain, Northern Moluccas, Indonesia. Sites for sampling had been taken according to the altitude and canopy of plantation (monoculture) or vegetation (heterogenic mixture) of 5 species: Durio species, Syzygium aromaticum (S. aromaticum) , Piper betle (P. betle), Myristica fragrans, or Pandanus species and others (mixture of unknown plant species) respectively.

2.2. Isolation of actinomycetes

2.2.1. Fermentation and extracts preparation Actinomycetes isolates were cultured in a liquid medium as follows. A loopful of mature slant culture was inoculated into a 50 mL culture tube containing 10 mL Actino medium No. 1 (Daigo, Japan) of the seed medium consisting of 5 g polypeptone, 3 g yeast extract and 1 L water, pH 7.2. The inoculated tubes were shaken on a rotary shaker (130 r/min) at 28 ˚C for 7 d. About 3 mL of the seed culture were transferred into a 500 mL flask containing 250 mL of the production medium consisting of glucose with same composition. The inoculated flasks were shaken on a rotary shaker (130 r/min) at 28 ˚C for 7 d. Then, 250 mL of ethyl acetate: methanol (4:1) was added to the flasks and they were shaken for 1 h. Mycelium was removed by filtration and centrifugation. The resulting supernatant without water was evaporated until dry powder. The powder extracts were resolved using acetone and used for bioassay.

2.2.2. Antifungal assay Three strains of fungi/yeast tested in this screening were C. albicans NBRC 1594, S. cerevisiae NBRC 10217 and A. niger. Fungi and yeasts were cultured on fresh yeast malt agar. The inhibitory effect of the extract obtained from leaf litter actinomycetes was tested by a modified Beury-Kirby method with paper discs (5 mm diameter). Sterile paper discs were placed on the surface of assay plates, and 20 µL of extract were pipetted onto each disc and incubated at 35 °C. The inhibition zones around each

disc were measured after 24 h.

2.2.3. Identification and phylogenetic analysis Identif ication and phylogenetic analysis were conducted just for active actinomycetes isolates. Isolated strains were cultivated in 5 mL of Actino medium No. 1 broth (ISP2). Pellets were collected during early log phase growth. DNA extracted was conducted with GES method. About 0.5 µL DNA from extraction process was used as a temple for polymerase chain reaction (PCR) amplification of an approximately 1500 base segment of the 16S rDNA gene. The PCR primers were 20F (5’-GATTTTGATCCTGGCTCAG-3’ ) and 1500 R (5’-GTTACCTTGTTACGACTT-3’). The resulting PCR products were purified with polyethylene glycol precipitation[10]. The purified PCR products were sequenced on an ABI 3130 Genetic Analyzer (Applied Biosystems Inc., Foster, California) with the BigDye Terminator version 3.1 sequencing method. The sequencing primer used was primer 20F (5’-GATTTTGATCCTGGCTCAG-3’). The sequences were then assembled with BioEdit. The 16S rDNA sequences inferred from the 16S rDNA sequences determinated as described above were aligment with 16S rDNA sequences from selected species from several related genera. Sequences of the reference strains were obtained from NCBI gene bank and were chosen based on a hight similarity rank with the strain from the actinobacteria along with a sequence to serve as an outgroup. Approximately 800 bases were included in the phylogenetic analysis, which was performed with the Clustal X 8.13 version and NJ plot win 95 computer program.

2.2.4. Minimum inhibitor concentration (MIC) MIC value was determined based on broth micro dilution method[11,12]. Fungi were cultivated in Sabouraud broth at 35 °C, respectively until microbial concentration reached approximately 1伊105 CFU/mL to 5伊105 CFU/mL. We conducted 3 times repetition. Agar diffusion method for confirm MIC value was done. We used commercial antibiotic, such as antibacterial and antifungal, to compared the results.

2.2.5. Fungal cell leakages: protein, nucleic acid and ion Cell leakage analysis was carried out based on measurement of content of material absorbing at 260 nm or 280 nm according to Lunde and Kubo[13], and ion K+ and Ca+ according to Prashar method[14], with some modifications. Yeast grew up in Saboroad medium for 24 h. About 10 mL suspension was added with Tween-80 and centrifuged at 3 500 r/min for 20 min. Supernatant

Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243S240

was discarded, pellets were washed twice with phosphate buffer. Cell in phosphate buffer were treated with control (0 MIC), 1 MIC and 2 MIC. Solvent without extract were used for control treatment. Treatment 1 MIC and 2 MIC used in this research mean one time and twice concentration of extract, respectively. After treatment, incubation was conducted for 12 h. Pellets cell and supernatant were separated using centrifugation (3 500 r/min for 20 min). Pellets cell was then observed using scanning electron microscope (SEM) (after SEM procedure preparation). Nucleic acid and protein leakages were investigated using UV/VIS (Shimadzu, Japan) at 260 nm and 280 nm. Ion Ca+ and K+ leakages were detected in supernatant using atomic absorption spectroscopy (GBC Scientific Equipment, Sens AA).

3. Results

3.1. Abundance of leaf litters actinomycetes

Nine leaf litter samples were collected from different altitude ranging from 17 to 1463 m above sea level with variation substract or vegetation (Table 1).

Table 1Abundance of leaf litter actinomycetes from various type of ecosystem and different altitude.Samples code GPS coordinate Altitude (m) pH Substract or vegetation Colony abundance

(CFU/g) 伊 104

MG.10 00°47’462’’ N

127°18’072’’ E

17 7.0 Durio sp. 46.00依4.95

LGN 00°46’ N

127°20’969’’ E

250 7.0 S. aromaticum 0.00依0.00

SR-1 -- 250 6.8 P. betle L 1.75依0.35

SR-2 -- 250 6.8 P. betle L 11.50依14.14

MG.500 00°47’001’’ N

127°21’325’’ E

514 6.5 Myristica fragrans 31.80依22.50

MG.750 00°47’252’’ N

127°21’118’’ E

742 6.9 S. aromaticum 75.00依9.80

MG.1 000.1 00°47’764’’ E

127°20’749’’ E

1 000 6.8 Hetero-culture 1.25依0.35

MG.1 250 00°47’705’’ N

127°20’864’’ E

1 253 6.0 Pandanus sp. 27.50依19.80

MG.1 500 00°47’881’’ N

127°20’686’’ E

1 463 6.9 Hetero-culture 85.00依7.07

3.2. Screening of antifungal activity of actinomycetes

We then selected 50 actinomycetes isolates based on morphotype characters (color of colony, pigment production, spore chain) and different ecosystem source. These isolates were cultivated for extracts preparation. Extracts obtained were used for screening of bioactivity against A. niger, C. albicans and S. cerevisiae by using paper disk method, the resluts are shown in Table 2.

Table 2Antifungal activity of metabolite produced by selected actinomycetes isolates.Isolates Clear zone diameter (mm)

C.albicans A. niger S. serevisiaeMG-10-1-L-1 - - -MG-10-1-L-2 - - -MG-10-1-L-3 - - -MG-10-1-L-4 - - -MG-10-1-L-5 - - -MG-10-1-L-6 - - -MG-10-1-L-7 - - -MG-10-1-L-8 - - -MG-10-1-L-9 - - -MG-500-1-1 - - -MG-500-1-2 - - -MG-500-1-3 - - -MG-500-1-4 - - 30.0MG-1500-1-1 - - -MG-750-1 - - -MG-500-2-1 - - -MG-500-2-2 - - -MG-500-2-3 - - -MG-500-2-4 - 11.5 -SR-1-1 - 15.0 -SR-1-2 - 13.0 -MG-1250-2-1 - - -MG-1250-2-2 - - -MG-1250-2-3 - - -MG-1250-2-4 - - -MG-1500-1-2 - - -MG-1500-1-3 - - -MG-1500-1-4 - - -MG-1500-1-5 - - -MG-1500-1-6 - - -MG-10-1-L-10 - - -MG-1250-2-5 - - -MG-1250-2-6 - - -MG-1250-2-7 - - -SR-2 -1 - - -SR-2 -2 - 17.0 20.5MG-100-1-S-1 - - -MG-100-1-S-2 - - -MG-100-1-S-3 - - -MG-500-2-5 - - -MG-500-2-6 - - -MG-500-2-6 - - -MG-500-1-5 - - -MG-500-1-6 - - -MG-500-1-7 - - -MG-500-1-8 - - -MG-500-1-9 - - -MG-500-1-10 - - -MG-500-1-11 - - -MG-500-1-12 - - -

MIC from extracts produced by MG-500-1-4 and SR-2-2 were observed (Table 3). MG-500-1-4 has high activity against S. cerevisiae; about 128 times than commercial antifungal (nystatin and kabicidin). However, anti-filamentous fungal activity of MG-500-1-4 isolate is less than that of nystatin and kabicidin and not active against C. albicans. Antifungal activity of SR-2-2 isolate is lower than that of MG-500-1-4, nystatin and kabicidin.

Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243 S241

Table 3MIC value of most active compound and commercial antifungal against three fungi.Microbe tested Extracts (µg/mL) Comercial antifungal (µg/mL)

MG-500-1-4 SR-2-2 Nystatin KabicidinC. albicans - - 32 32

S. cerevisiae 0.5 128 64 64

A. niger >128 >128 16 64

3.3. Effects of extract of MG-500-1-4 and SR-2-2 isolate on S. cereviseae cells integrity

Since S. cereviseae is more sensitive, we focused more on S. cereviseae response treated with the active extracts. Nucleic acid and protein content in supernantan of yeast cells treated with MG-500-1-4 (Figure 1) or SR-2-2 (Figure 2) increased significantly compared to control. Control, 1 MIC (0.5 µg/mL) and 2 MIC (1 µg/mL) were treated for MG-500-1-4 extract. Treatment for SR-2-2 extract in this investigation was control, 1 MIC (128 µg/mL) and 2 MIC (256 µg/mL).

Figure 1. Protein and nucleic acid leak from S. cereviseae after treatment with MG-500-1-4 extract.

260 nm 280 nm

0 MIC 1 MIC 2 MIC

0.07

0.06

0.05

0.04

0.03

0.02

0.01

0

Optic

al d

ensit

y (O

D)Op

tical

den

sity

(OD)

Figure 2. Prorein and nucleic acid leak from S. cereviseae after treated with SR-2-2 extract.

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

280 nm260 nm

0 MIC 1 MIC 2 MIC

In addition, we evaluated ion leakages (Ca+ and K+) in S. cereviseae treated with MG-500-1-4 or SR-2-2 extract. As presented in Figure 3 and 4, content of Ca+ and K+ ion in supernatant of treated cells was higher than that of control. There was no significant difference of ions concentration between 1 MIC and 2 MIC treatment. These data support our suggestion that the extracts altered cell membrane and caused cell leakage.

Figure 3. Ca+ and K+ leak from S. cereviseae after treatment with MG-500-1-4 extract.

25

20

15

10

5

0

K+Ca+

0 MIC 1 MIC 2 MIC

Tota

l ion

leak

ages

( µg/

mL)

Figure 4. Ca+ and K+ leak from S. cereviseae after treatment with SR-2-2 extract.

0 MIC 1 MIC 2 MIC

25

20

15

10

5

0

Ca+ K+

Tota

l ion

leak

ages

( µg/

mL)

In relation with membrane cell damage in S. cerevisiae cells treated with active extracts, morphological change was also observed by using SEM (Figure 5 and 6).

A B C

Figure 5. Morphological observation using SEM from S. cereviseae after treatment with MG-500-1-4 extract (A: 0 MIC/control; B: 1 MIC; C: 2 MIC); 5 000伊 magnification.

A B C

Figure 6. Morphological observation using SEM from S. cereviseae after treatment with MG-500-1-4 extract (A: 0 MIC/control; B: 1 MIC; C: 2 MIC); 5 000伊 magnification.

3.4. Indentification of MG-500-1-4 and SR-2-2 isolates Two isolates, MG-500-1-4 and SR-2-2, were identified based on 16S rRNA gene. Both of them belong to Streptomyces genera. MG-500-1-4 has similarities with Streptomyces misakiensis (S. misakiensis) (99%) and SR-2-2 has similarities

Arif Nurkanto and Heddy Julistiono/Asian Pac J Trop Med 2014; 7(Suppl 1): S238-S243S242

with Streptomyces tricolor (S. tricolor) (99%) (Figure 7). MG-500-1-4 has bootstrap value 76 and SR-2-2 has bootstrap value 84. Isolates that collected in this research were deposited at Indonesia Culture Collection as open access isolates.

4. Discussion

The results indicated that there were no specific correlation between colony abundance of actinomycetes and the altitude. No actinomycetes were isolated in monoculture leaf litter of S. aromaticum. The absence of actinomyctes living on leaf of S. aromaticum, might be caused by toxicity of the plant substances against actinomycetes shown by

Cai and Wu[15]. Ecosystem with heterogen vegetation gave the most abundance of actinomycetes. Complex nutrient came from different sources of leaf litter in an ecosystem with different plants and may ensure a better condition for actinomycetes growing. In our study, there were only 10% of actinomycetes isolates have activity against S. cerevisiae and A. niger. No extracts could inhibit C. albicans. However, some of them have the highest activity especially against S. cerevisiae. MG-500-1-4 and SR-2-2 are isolates that have highest activity. These two isolates were then selected for extracts scaling up and further investgation. Nucleic acid and protein content in supernatant of yeast cells treated with MG-500-1-4 or SR-2-2 increased significantly compared to control. However, in cell treated

S. tricolor0.005

Figure 7. Phylogenetic positions of selected actinomycetes isolates based on partial sequences of 16S rDNA. The tree was constructed by Neighbour-Joining method.

SR-2-2Streptomyces anulatus

Streptomyces rubiginosohelvolusStreptomyces tanashiensis

Streptomyces anulatus

84

15

9

40

64

12

Streptomyces californicus100 Streptomyces globisporus

Streptomyces roseochromogenusStreptomyces celluloflavus

74

Streptomyces lohii67

6799

Streptomyces griseus

Streptomyces globisporus

Streptomyces avidinii98

Streptomyces virginiae

99

79 51 Streptomyces colombiensisStreptomyces sporoverrucosusStreptomyces sporoverrucosus

44Streptomyces aureus

17

44

84

63

86

100

79

Streptomyces tauricusStreptomyces tauricus

Streptomyces umbrinusStreptomyces ederensis

Streptomyces kunmingensisStreptomyces avermitilis

Streptomyces coelicolor

Streptomyces nodosusStreptomyces albovinaceus

Streptomyces rimosusStreptomyces aburaviensis

Streptomyces tsukiyonensisKitasatospora kifunensis

Kitasatospora azaticaStreptomyces purpeofuscus

S. misakiensisMG-500-1-4

Kitasatospora arboriphila

78

34

6367

100

32

9

99 41

5792 30

76

9047

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with MG-500-1-4, there was no significant difference between 1 MIC and 2 MIC treatments. Higher content of nucleic acids and protein in supernatant indicated cell leakages and cell wall alteration[13,16]. This visual observation on yeast cells treated with toxic substance may be helpful to understand cells damage mechanism. In this research, we observed oval shaped cells with apparent smooth cell surfaces on untreated yeast. After exposure to MIC of MG-500-1-4 and SR-2-2 extracts 1 or 2 times, invaginations of cells were observed. The changes in morphology of the yeast cells in response to polyene antifungal or osmotic pressure are consistent with the findings of other researchers[17,18]. They reported invagination of plasma membrane upon examining the freeze fracture images and outside envelope. Ternate has high abundance of leaf litter actinomycetes. Several isolates produced bioactive substances against fungi. Two bioactive extracts of MG-500-1-4 and SR-2-2 have highest activity against S. cerevisiae. MG-500-1-4 extract inhibited S. cereviseae better than nystatin and kabicidin did. Treatment with these two extracts effected nucleic acid, protein and ion leakages. Morphological change of cells after treatment with MG-500-1-4 and SR-2-2 extracts were also observed. Based on 16S rRNA gene identification, MG-500-1-4 and SR-2-2 isolates have similarity with S. misakiensis and S. tricolor respectively.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

Funding to carry out the project was provided by regular governmental annual fund every fiscal year from Research Center for Biology, Indonesia Institute of Sciences (Grant No. 3400.003.002/028). Authors would like to thank Professor Ibnu Maryanto for managing the Ternate’s expedition and Dian Matrika Widyasih for assistance during laboratory analyses.

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