Antagonistic activity of phylloplane yeasts from Moringa oleifera Lam. leaves against Aspergillus flavus UNJCC F-30 from chicken feed

  • Dalia SukmawatiEmail author
  • Marsha Hanin Andrianto
  • Zico Arman
  • Nuniek Ina Ratnaningtyas
  • Shabrina Nida Al Husna
  • Hesham Ali El-Enshasy
  • Daniel Dailin
  • Ahmed Atta Kenawy
Research Article


Aspergillus flavus is widely known as an aflatoxin-producing fungus that frequently contaminates feed and affects livestock, which leads to severe health problem for animal and human. Biological agents have been proven to prevent this contamination since they can produce metabolites which have antagonistic activity. In this study, phylloplane yeasts isolated from Moringa oleifera leaf have shown an ability to inhibit the growth of Aspergillus flavus UNJCC F-30 collected from chicken feed. This research was conducted in three stages: (1) yeast isolation (leaf washing and direct method), followed by (2) antagonistic test using dual culture method, and (3) molecular identification using D1/D2 region of 26S rDNA. In the first stage, 38 yeast isolates have been succesfully obtained. These isolates were of different colors: peach pigment (60.5%), the non-pigmented yeast (26.5%), cream (10%), and orange (3%). Antagonistic activity against A. flavus UNJCC F-30 was tested based on growth, sporulation, and the presence of clear zones. Screening result showed that 12 yeast isolates are capable of inhibiting A. flavus UNJCC F-30. Among them, 4 isolates with the code K4, K10, K15, and K26 showed the highest antagonist ability. Molecular identification resulted that the 4 isolates show a similar identity with Aureobasidium pullulans UWFP 993 (100%), Aureobasidium melanogenum QCC:M017/17 (99%), Aureobasidium melanogenum QCC:M017/17 (100%) and Rhodotorula taiwanensis CBS:11729 (99%), respectively. Isolate K10 exhibited the highest percentage of inhibition activity among all isolates which is potential for application as biocontrol agent against A. flavus. As A. pullulans is a common yeast found on leaf surfaces of many Indonesian flora, therefore it can be considered as safe and alternative to reduce fungal contamination from A. flavus in feed chicken.


Antangonistic Asperillus flavus Yeast Morinaga oleifera Aureobasidium 



The present research work was supported by Direktorat Riset dan Pengabdian Masyarakat Direktorat Jenderal Penguatan Riset dan Pengembangan Kementrian Riset, Teknologi dan Pendidikan Tinggi Hibah Penelitian Riset Terapan No: 23/SP2H/DRPM/LPPM UNJ/III/2019, grand on behalf of Dalia Sukmawati (2019–2020). We express deep gratitude and appreciation to the Department Biology Universitas Negeri Jakarta Research Grant for supporting. We also appreciate member of Microbiology Lab. for all expertise assistance and technical contributions on the research projects, and also Universitas Negeri Jakarta Culture Collection (UNJCC) for the use of the facilities and availability of isolates. We also acknowledge the support of MOHE and UTM-RMC through HICOE Grant No. R.J130000.7846.4J262. We hope this research could be a starting point for every institution to do further study and findings to give more contribution to the research related area.


  1. Alemu F (2016) Isolation of Pseudomonas fluorescents species from rhizospheric soil of healthy faba bean and assessed their antagonistic activity against Botrytis fabae(chocolate spot diseases). Int J Sci Technol Soc 4(2):25–34CrossRefGoogle Scholar
  2. Ashiq S, Hussain M, Ahmad B (2014) Natural occurrence of mycotoxins in medicinal plants: a review. Fungal Genet Biol 66:1–10PubMedCrossRefGoogle Scholar
  3. Bbosa GS, Kitya D, Odd J, Okeng JO (2013) Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis. Health 5(10A):14–34CrossRefGoogle Scholar
  4. Bencheqroun S, Bajji M, Sebastien M, Jaafari S, Jijakli M (2007) In vitro and in situ study of postharvest apple blue mold biocontrol by Aureobasidium pullulans: evidence for the involvement of competition for nutrients. Postharvest Biol Technol 46:128–135. CrossRefGoogle Scholar
  5. Bhatnagar-Mathur P, Sunkara S, Bhatnagar-Panwar M, Waliyar F, Sharma KK (2015) Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination in crops. Plant Sci 234:119–132. PubMedCrossRefGoogle Scholar
  6. Chang CW, Wang PH (2002) Six Rhodotorula species from Taiwan. Fungal Sci 17:23–26Google Scholar
  7. Chen X, Grenier B, Applegate TJ (2013) Aflatoxins in poultry. Purdue University Department of Animal Sciences. Accessed 4 Apr 2017
  8. Chen Y, Cheng N, Xu Y, Huang K, Luo Y, Xu W (2016) Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor. Biosens Bioelectron 81:317–323PubMedCrossRefGoogle Scholar
  9. Di Francesco A, Ugolini L, Lazzeri L, Mari M (2015) Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens. Biol Control 81:8–14CrossRefGoogle Scholar
  10. Dimakopoulou M, Tjamos SE, Antoniou PP, Pietri A, Battilani P, Avramidis N, Markakis EA, Tjamos EC (2008) Phyllosphere grapevine yeast Aureobasidium pullulans reduces Aspergillus carbonarius (sour rot) incidence in wine-producing vineyards in Greece. Biol Control 46:158–165CrossRefGoogle Scholar
  11. Ehrlich KC, Cotty PJ (2004) An isolate Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyktide synthase gene. J Microbiol Biotechnol 65(4):473–478CrossRefGoogle Scholar
  12. Ehrlich KC, Yu J, Cotty PJ (2005) Aflatoxin biosynthesis gene clusters and flanking regions. J Appl Microbiol 99(3):518–527PubMedCrossRefGoogle Scholar
  13. Elkhateeb WA (2018) Where to Find? A report for some terrestrial fungal isolates, and selected applications using fungal secondary metabolites. Biomed J Sci Tech Res. CrossRefGoogle Scholar
  14. Fatima T, Sajid MS, Jawad-ul-Hassan M, Siddique RM, Iqbal Z (2014) Phytomedicinal value of Moringa oleifera with special reference to antiparasitics. Pak J Agric Sci 51(1):251–262Google Scholar
  15. Frisvad JC, Hubka V, Ezekiel CN, Hong SB, Nováková A, Chen AJ (2019) Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins. Stud Mycol 93:1–63. PubMedCrossRefGoogle Scholar
  16. Fuglie LJ (2001) Combating malnutrition with Moringa. The miracle tree: the multiple attributes of Moringa. CTA Publication, Wageningen, pp 117–136Google Scholar
  17. Gonzalez V, Tello ML (2011) The endophytic mycota associated with Vitis vinifera in central Spain. Fungal Divers 47:29–42. CrossRefGoogle Scholar
  18. Hejri AL, Jinap S, Radu S (2013) Occurrence of aflatoxins and aflatoxigenic Aspergillus in peanuts. J Food Agric Environ 11(3,4):228–234Google Scholar
  19. Hospenthal DR, Murray CK, Beckius ML, Green JA, Dooley DP (2002) Persistence of pigment production by yeast isolates grown on CHROMagar Candida media. J Clin Microbiol 40:4768–4770PubMedPubMedCentralCrossRefGoogle Scholar
  20. Jeff-Agboola YA, Awe LB (2016) Antifungal and phytochemical screening of some Nigerian medicinal plant extracts against toxigenic Aspergillus flavus. Cogent Food Agric 2(1):1210556Google Scholar
  21. Kaewwichian R, Limtong S (2014) Nakazawaea siamensisf.a., sp. nov., a yeast species isolated from phylloplane. Int J Syst Evol Microbiol 64:266–270. PubMedCrossRefGoogle Scholar
  22. Korsten L, De Jager ES, De Villiers EE, Lourens A, Kotzé JM, Wehner FC (1995) Evaluation of bacterial epiphytes isolated from avocado leaf and fruit surfaces for biocontrol of avocado postharvest diseases. Plant Dis 79:1149–1156CrossRefGoogle Scholar
  23. Liu GL, Wang DS, Wang LF, Zhao SF, Chi ZM (2011) Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11. Fungal Genet Biol 48:904–913PubMedCrossRefGoogle Scholar
  24. Makene VA (2014) Identification of non-albicans Candida yeasts associated with Vulvovaginal Candidiasis in Tanzania using a combination of multiplex PCR and DNA sequence divergence of the 26S LSU rDNA. Scholars Acad J Biosci 2(2):124–131Google Scholar
  25. Mari M, Martini C, Guidarelli M, Neri F (2012) Postharvest biocontrol of Monilinia laxa, Monilinia fructicolaand Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains. Bioll Control 60(2):132–140. CrossRefGoogle Scholar
  26. Megeed AA (2013) Antagonistic activities of some fungal strains against the toxigenic Aspergillus flavus isolate and its aflatoxins productivity. J Pure Appl Microbiol 7:169–178Google Scholar
  27. Mirzwa-Mróz E, Wińska-Krysiak M, Dzięcioł R, Miękus A (2014) Characteristics of Aureobasidium pullulans (de Bary et Löwen-thal) G. Arnaud isolated from apples and pears with symptoms of sooty blotch in Poland. Acta Sci Pol Hortorum Cultus Ogrodnictwo 13:13–22Google Scholar
  28. Moliné M, Flores M, Libkind D, Dieguez M, Farías M, van Broock M (2010) Photoprotection by carotenoid pigments in the yeast Rhodotorula mucilaginosa: the role of torularhodin. Photochemical & photobiological sciences. Photochem Photobiol Sci Off J Eur Photochem Assoc Eur Soc Photobiol 9:1145–1151. CrossRefGoogle Scholar
  29. Mostafa AA, Al-Rahmah A, Megeed AA, Rushdy S, Hatamleh A (2013) Antagonistic activities of some fungal strains against the toxigenic Aspergillus flavus isolate and its aflatoxins productivity. J Pure Appl Microbiol 7:169–178Google Scholar
  30. Nachtigall R, Dechen R (2006) Seasonality of nutrients in leaves and fruits of apple trees. Sci Agric 63(5):493–501. CrossRefGoogle Scholar
  31. Nair SC, Bhagobaty RK, Nampoothiti K, Kalaigandhi V, Menon KRK (2014) Detection of aflatoxin production by fungi in spice samples using HPLC and direct visual cultural methods. Innov Romanian Food Biotechnol 14:1–12Google Scholar
  32. Niessen L, Bechtner J, Fodil S, Taniwaki MH, Vogel RF (2018) LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection. Int J Food Microbiol 266:241–250. PubMedCrossRefGoogle Scholar
  33. Nieuwenhuijzen EJ, Houbraken J, Meijer M, Adan O, Samson R (2016) Aureobasidium melanogenum: a native of dark biofinishes on oil treated wood. Anton Leeuw Int J Gen 109:1–25. CrossRefGoogle Scholar
  34. Pereira VL, Fernandes JO, Cunha SC (2014) Mycotoxins in cereals and related foodstuffs: a review on occurrence and recent methods of analysis. Trends Food Sci Technol 36(2):96–136. CrossRefGoogle Scholar
  35. Pietsch C, Burkhardt-Holm P (2015) Feed-borne exposure to deoxynivalenol leads to acute and chronic effects on liver enzymes and histology in carp. World Mycotoxin J 8(5):619–627. CrossRefGoogle Scholar
  36. Punnapayak H, Sudhadham MS, Prasongsuk Pichayangkura S (2003) Characterization of Aureobasidium pullulans isolated from airborne spores in Thailand. J Ind Microbiol Biotechnol 30:89–94PubMedCrossRefGoogle Scholar
  37. Rosa-Magri MM, Tauk-Tornisielo SM, Ceccato-Antonini SR (2011) Bioprospection of yeasts as biocontrol agents against phytopathogenic molds. Braz Arch Biol Technol 54(1):1–5. CrossRefGoogle Scholar
  38. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  39. Schmit JP, Lodge DJ (2004) Classical methods and modern analysis forstudying fungal diversity. Mycol Ser 23:193Google Scholar
  40. Shepherd RW, Wagner GJ (2007) Phylloplane proteins: emerging defenses at the aerial frontline. Trends Plant Sci 12(2):51–56. PubMedCrossRefGoogle Scholar
  41. Sibounnavong PK, Soytong CC, Divina Sofrio PK (2009) In vitro biological activities of Emericella nidulans, a new fungal antagonist against Fusarium oxysporum f. sp. lycopersici. J Agr Sci Technol 5(1):75–84Google Scholar
  42. Sjamsuridzal W, Oetari A, Kanti A, Saraswati R, Nakashima C, Widyastuti Y, Ando K (2010) Ecological and taxonomical perspective of yeasts in Indonesia. Microbiol Indonesia 4:60–67CrossRefGoogle Scholar
  43. Sukmawati D (2016) Antagonism mechanism of fungal contamination animal feed using phylloplane yeasts isolated from the bintaro plant (Cerbera manghas) Bekasi in Java, Indonesia. Int J Curr Microbiol App Sci 5:63–74. CrossRefGoogle Scholar
  44. Sukmawati D, Miarsyah M (2017) Pathogenic activity of Fusarium equiseti from plantation of citrus plants (Citrus nobilis) in the village Tegal Wangi, Jember Umbulsari, East Java, Indonesia. Asian J Agric Biol 5(4):202–213Google Scholar
  45. Sukmawati D, Ariyanti O, Dian H, Mega A, Wellyzar S (2015) Identification of phylloplane yeasts from paper mulberry (Broussonetia papyrifera (L.) L’Hér. ex Vent.) in Java, Indonesia. J Microbiol 11(4):324–340Google Scholar
  46. Sukmawati D, Setyaningsih A, Rahayu S, Rustam Y, Moersilah M, Wahyudi P, Husna SNA (2018) Isolation and characterization of aflatoxigenic Aspergillus spp. from maize of livestock feed from Bogor. In: IOP conference series: materials science and engineering, vol 434(1), p 012105CrossRefGoogle Scholar
  47. Tamura K, Stecher G, Peterson D, Filipski Kumar S (2013) MEGA 6:Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. PubMedPubMedCentralCrossRefGoogle Scholar
  48. Ungureanu C, Ferdes M (2012) Evaluation of antioxidant and antimicrobial activities of torularhodin. Adv Sci Lett 5:1–4CrossRefGoogle Scholar
  49. Varga J, Baranyi N, Chandrasekaran M, Vágvölgyi CS, Kocsubé S (2015) Mycotoxin producers in the Aspergillus genus: an update. Acta Biol Szeged 59:151–167Google Scholar
  50. Wu F, Narrod C, Tiongco M, Liu Y (2011) The health economic of aflatoxin: global burden of disease. Working paper 4. International food policy research instituteGoogle Scholar
  51. Yu J, Deepak B, Kenneth CE (2002) Aflatoxin biosynthesis—a review. Mycology 19:191–200Google Scholar
  52. Zhang D, Spadaro D, Garibaldi A (2010) Selection and evaluation of new antagonists for their efficacy against postharvest brown rot of peaches. Postharvest Biol Technol 55:174–181CrossRefGoogle Scholar
  53. Zhao Y, Gao F, Li J, Yi Z, Warren A (2012) Phylogenetic analyses on the tintinnid ciliates (Protozoa, Ciliophora) based onmultigene sequence data. Acta Protozool 51:319–328Google Scholar

Copyright information

© Indian Phytopathological Society 2020

Authors and Affiliations

  • Dalia Sukmawati
    • 1
    • 2
    Email author
  • Marsha Hanin Andrianto
    • 1
  • Zico Arman
    • 1
  • Nuniek Ina Ratnaningtyas
    • 3
  • Shabrina Nida Al Husna
    • 4
  • Hesham Ali El-Enshasy
    • 5
    • 6
  • Daniel Dailin
    • 5
  • Ahmed Atta Kenawy
    • 6
  1. 1.Department of Biology, Faculty of Mathematics and Natural SciencesUniversitas Negeri JakartaRawamangun, Jakarta TimurIndonesia
  2. 2.Univeritas Negeri Jakarta Culture Collection (UNJCC), Faculty of Mathematics and Natural SciencesUniversitas Negeri JakartaRawamangunIndonesia
  3. 3.Faculty of BiologyUniversitas Jenderal SoedirmanPurwokerto, Jawa TenggahIndonesia
  4. 4.Department of Microbiology, School of Life Sciences and TechnologyInstitut Teknologi BandungBandungIndonesia
  5. 5.Institute of Bioproduct DevelopmentUniversiti Teknologi Malaysia (UTM)Skudai, Johor BahruMalaysia
  6. 6.City of Scientific Research and Technology ApplicationsNew Burg Al Arab, AlexandriaEgypt

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