Online conference undip Indonesia

1. EXPLORATION OF SPONGE-
ASSOCIATED FUNGI FROM
MANGROVE ECOSYSTEM

2. Mangrove ecosystem
1. Organic matters
2. Salinity
3. pH
4. Temperature
5. Visibility

3. Sponge-Associated Fungi
Liu et al., 2018
Penicillilactone A
Liu et al. 2018. 10.1080/14786419.2018.1452012
Selvam et al. 2014. 10.13005/bbra/1298
Extracellular enzyme
Selvam et al., 2014
Research objectives
1. To isolate sponge-associated fungi from mangrove ecosystem
2. To screen the antimicrobial activity and its secondary metabolite
3. To screen the production of extracellular enzyme
4. To identify the potential isolates

4. Unidentified sponges
Mangkang Mangrove Project

5. Unidentified sponges
Fungal Isolation
• Tapping method (Kjer et al., 2010; Sibero et al., 2018)
Screening of antimicrobial activity
• Agar plug diffusion method (Balouiri et al., 2016)
• Anti-MDR (ESBL E. coli, MRSA, S. enterica ser. Typhi)
• Dermatophyte (Candida albicans, Trichophyton rubrum dan Malassezia furfur )
Screening of extracellular enzyme
• Cellulose (CMC + 1% Congo red)
• Amylase (Starch + Iodin)
• Protease (Skim Milk Agar, HiMedia)
Characterization of potential isolates
• DNA barcoding (ITS region)
• Detection of BGC (NRPS, PKS-I, PKS-II)
• Detection of enzyme genes
Characterization of secondary metabolite
• HPLC (Allegiant)
• CH3CN  0-3 min: 15%; 25-29 min: 85%; 29-32 min: 15%Kjer et al. 2010. DOI: 10.1038/nprot.2009.233
Balouiri et al. 2016.
https://doi.org/10.1016/j.jpha.2015.11.005

6. SPONGE ASSOCIATED-FUNGI

7. ISOLATE
PATHOGENS ENZYME
ESBL MRSA SET CA TR MF A P S
SPMKF 1 - - - - - - √ - -
SPMKF 2 - - - - - - - - -
SPMKF 3 - - - - - - - - -
SPMKF 4 - √ - - - - - - -
SPMKF 5 - √ - - - - - - -
SPMKF 6 - - - - - - √ - -
SPMKF 8 √ - √ √ - - √ - -
SPMKF 9 - - - - - - - - -
ANTIMICROBIAL ACTIVITY AND ENZYME ACTIVITY
ESBL E. coli
C. albicans
S. enterica ser. Typhi

8. FUNGAL CHARACTERISTICS
Xylariomycetidae
Dothideomycetidae
Hypocreomycetidae

9. FUNGAL CHARACTERISTICS
Denaturation : 95oC, 60’
Annealing : 55oC, 60’
Extension : 72oC, 60’
30 cycles
(A: Marker, B: NRPS in SPMKF 4, C: NRPS in SPMKF 5, D: NRPS in SPMKF 8, E: amylase genes in SPMKF 1,
F: amylase genes in SPMKF 6, G: amylase genes in SPMKF 8)

10. min0 5 10 15 20 25 30
mAU
-400
-200
0
200
400
600
800
1000
METABOLITE PROFILE
nm300 400 500 600 700
mAU
0
200
400
600
800
1000
1200
Rt 20.221 nm300 400 500 600 700
mAU
0
200
400
600
800
1000
1200
nm300 400 500 600 700
mAU
0
100
200
300
400
500
600
Rt 22.854
Rt 23.119
Column Microsorb (RP-C18)
Conc. 1 mg/mL
Pump speed 1 mL/min
HPLC Condition
 Buffer 0.1% Formic acid
 CH3CN
S. enterica ser. Typhi
Cultivated in MEB (7 h, 200 r.p.m, 30oC)
Extracted in 1-Butanol

11. • There were 8 isolates from sponges associated with mangroves
• SPMKF 1 & SPMKF 6 only performed the activity of amylase, SPMKF 4 & 5 only
performed anti-MRSA while SPMKF 8 showed antimicrobial activity against ESBL
E. coli, C. albicans and S. enterica ser. Typhi and activity of amylase
• SPMKF 1 was identified as Cladosporium tenuissimum
• SPMKF 4 was identified as Eutypella sp.
• SPMKF 5 was identified as Lasiodiplodia theobromae
• SPMKF 6 was identified as Fusarium keratoplasticum
• SPMKF 8 was identified as Fusarium solani
• α-Amylase gene was identified in SPMKF 1, 6 & 8 while there was only SPMKF 8
showed positive result of NRPS gene
• There was antibacterial activity at fraction of 21, 23 and 24 from fungus F. solani
SPMKF 8
CONCLUSIONS

12. 1. Optimization of PCR condition to detect
the BGC and α-Amylase genes
2. Purification and characterization of the
enzymes
3. Isolation and elucidation of single lead
compounds from F. solani SPMKF 8 using
bioguided isolation method
FURTHER PLAN

13. ACKNOWLEDGEMENT

14. Thank you 
THANK YOU