Sains Malaysiana 47(12)(2018): 3025–3030

http://dx.doi.org/10.17576/jsm-2018-4712-12

 

Isolation and Identification of Endophytic Fungi from UiTM Reserve Forest, Negeri Sembilan

(Pemencilan dan Pengenalpastian Kulat Endofit dari Hutan Simpan UiTM Negeri Sembilan)

 

SITI NURSYAZWANI MAADON1, SARINI AHMAD WAKID1, IWANA IZNI ZAINUDIN1, LILI SYAHANI RUSLI1, MOHD SYAHRIL MOHD ZAN1, NOR'AISHAH HASAN1, NOR'AISHAH ABU SHAH1 & EMELDA ROSSELEENA ROHANI2*

 

1Faculty of Applied Sciences, Universiti Teknologi MARA, Cawangan Negeri Sembilan,72000 Kuala Pilah, Negeri Sembilan Darul Khusus, Malaysia

 

2Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Received: 30 May 2018 /Accepted: 18 September 2018

 

ABSTRACT

Endophytic fungi are those living inside the host plant without causing any apparent negative effect on the host plant. Two isolates endophytic fungi from leaves and two isolates from root at Universiti Teknologi MARA (UiTM) Reserve Forest, Negeri Sembilan were successfully isolated and identified by morphology and molecular characteristic. Samples were surface sterilized and sub-cultured to obtain a pure culture. Characteristics of the isolates such as colony appearance, mycelial texture, conidia/spores and pigmentation were studied to explore their morphology. Isolates were also subjected to a PCR-based genotyping test. There were noticeable differences in morphological characteristics among the four isolates. Microscopic analysis showed four isolates consist of septa and conidia/spores. The pigmentation result showed that colony in A1leaf samples demonstrated an orange color on potato dextrose agar (PDA) media, colony in A1root demonstrate a black texture in PDA media while hairy colonies in the others two isolates showed a white color on PDA media. Based on molecular analyses the fungal genera showed 99-100% similarity with the related fungi recorded in the GenBank. Both morphology and molecular sequencing of internal transcribed spacer (ITS) regions of endophytic fungi showed that three isolates (A1root, C2leaf, and C3root) were grouped in Basidiomycota while one isolate (A1leaf) belonged to Ascomycota. The endophyte funguses were identified as Daldinia sp. (A1leaf), Polyporales sp. (A1root,) Lentinus sp. (C2leaf,) and Rigidoporus sp. (C3root). Overall, the new discoveries of isolated endophyte fungal have dyeing potential of fungal pigments which offer a viable alternative to natural vegetable and harmful synthetic dyes.

 

Keyword: Endophyte fungi; identification; morphological characteristic

 

ABSTRAK

Kulat endofit yang hidup di dalam tumbuhan perumah tanpa menyebabkan sebarang kesan negatif yang jelas terhadap tumbuhan perumah. Dua pencilan kulat endofit daripada daun dan dua pencilan daripada akar dari Hutan Simpan Universiti Teknologi MARA (UiTM), Negeri Sembilan telah berjaya dipencilkan dan dikenal pasti daripada segi morfologi dan ciri molekul. Sampel disterilkan pada permukaan dan di sub-kultur untuk memperoleh kultur tulen. Ciri-ciri pencilan seperti rupa koloni, tekstur miselium, conidia/spora dan pigmentasi dikaji untuk meneroka morfologi mereka. Pencilan juga dijalankan untuk ujian genotip berasaskan PCR. Terdapat perbezaan yang ketara di dalam ciri-ciri morfologi dalam kalangan empat pencilan. Analisis mikroskopik menunjukkan empat pencilan terdiri daripada septum dan conidia/spora. Keputusan pigmentasi menunjukkan bahawa koloni di dalam sampel A1daun menunjukkan warna jingga pada media dekstrose agar (PDA), koloni di dalam A1akar menunjukkan tekstur hitam di dalam media PDA manakala koloni berbulu di dua lagi pencilan menunjukkan media PDA berwarna putih. Berdasarkan analisis molekul, genus kulat menunjukkan persamaan 99-100% dengan kulat yang berkaitan yang direkodkan di dalam bank Gen. Kedua-dua morfologi dan jujukan molekul di kawasan mentranskripsi penjarak dalaman (ITS) kulat endofit mendedahkan bahawa tiga pencilan (A1akar, C2daun, dan C3akar) dikelompokkan di dalam Basidiomycota manakala satu pencilan (A1daun) tergolong di dalam Ascomycota. Kulat endofit dikenal pasti sebagai Daldinia sp. (A1daun), Polyporales sp. (A1akar,) Lentinus sp. (C2daun,) dan Rigidoporus sp. (C3akar). Secara keseluruhannya, penemuan baru pencilan kulat endofit mempunyai potensi pewarnaan pigmen daripada pigmen kulat yang menawarkan alternatif terkedepan kepada sayur-sayuran semula jadi dan pewarna sintetik yang merbahaya.

 

Kata kunci: Ciri morfologi; kulat endofit; pengenalpastian

REFERENCES

Anchana, A. 2014. Extraction of natural dyes from fungus - an alternate for textile dyeing. Journal of Natural Sciences 4(7): 1-7.

Anderson, I.C., Campbell, C.D. & Prosser, J.I. 2003. Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. Applied and Environmental Microbiology 5: 36-47.

Bałazy, S., Wrzosek, M., Sosnowska, D., Tkaczuk, C. & Muszewska, A. 2008. Laboratory trials to infect insects and nematodes by some acaro- pathogenic Hirsutella strains (Mycota: Clavicipitaceous anamorphs). Journal of Invertebrate Pathology 97: 103-113.

Barnett, H.L. & Hunter, B.B. 1998. Illustrated Genera of Imperfect Fungi, 4th ed. Minnesota: American Phytopathological Society Press.

Barseghyan, G.S. & Wasser, S.P. 2010. Species diversity of hypogeous ascomycetes in Israel. Mycobiology 38(3): 159- 165.

Ellis, M.B. 1971. Dematiaceous Hyphomycetes. 1st ed. England: Commonwealth Mycological Institute. Evidence-Based Complementary and Alternative Medicine.

Guo, L.D., Hyde, K.D. & Liew, E.C.Y. 2000. Identification of endophytic fungi from Livistona chinensis based on morphology and rDNA sequences. The New Phytologist 147(3): 617-630.

Jiaojiao, S., Wattanachai, P. & Kasem, S. 2016. Isolation and identification of endophytic fungi from 10 species palm trees. Journal of Agricultural Technology 12(2): 349-363.

Leslie, J.F. & Summerell, B.A. 2007. The Fusarium Laboratory Manual. Iowa, USA: Blackwell Publishing Ltd.

Liu, L., Liu, S., Jiang, L., Chen, X., Guo, L. & Che, Y. 2008. Chloropupukeananin, the first chlorinated pupukeanane derivative and its precursors from Pestalotiopsis fici. Organic Letters 10(7): 1397-1400.

Lord, N.S., Kaplan, C.W., Shank, P., Kitts, C.L. & Elrod, S.L. 2002. Assessment of fungal diversity using terminal restriction fragment pattern analysis: Comparison of 18S and ITS ribosomal regions. FEMS Microbiology Ecology 42: 327-337.

Lu, Y., Chen, C., Chen, H., Zhang, J. & Chen, W. 2012. Isolation and identification of endophytic fungi from Actinidia macrosperma and investigation of their bioactivities. Evidence-Based Complementary and Alternative Medicine 2012: Article ID. 382742.

Martinez-Klimova, E., Rodríguez-Peña, K. & Sánchez, S. 2017. Endophytes as sources of antibiotics. Biochemical Pharmacology 134: 1-17.

Parthibhan, S., Rao, M.V. & Kumar, T.S. 2017. Culturable fungal endophytes in shoots of Dendrobium aqueum Lindley - An imperiled orchid. Ecological Genetics and Genomics 3(5): 18-24.

Porras-Alfaro, A. & Bayman, P. 2011. Hidden fungi, emergent properties: Endophytes and microbiomes. Annual Review of Phytopathology 49(1): 291-315.

Qiu, M., Xie, R., Shi, Y., Chen, H., Wen, Y., Gao, Y. & Hu, X. 2010. Isolation and identification of endophytic fungus SX01, a red pigment producer from Ginkgo biloba L. World Journal of Microbiology and Biotechnology 26(6): 993-998.

Rodriguez, R.J., White Jr., J.F., Arnold, A.E. & Redman, R.S. 2009. Fungal endophytes: Diversity and functional roles. New Phytologist 182(2): 314-30.

Saad, D.S., Kinsey, G.C., Kim, S. & Gaylarde, C.C. 2004. Extraction of genomic DNA from filamentous fungi in biofilms on water-based paint coatings. International Biodeterioration & Biodegradation 54: 99-103.

Stadler, M., Læssøe, T., Fournier, J., Decock, C., Schmieschek, B., Tichy, H.V. & Peršoh, D. 2014. A polyphasic taxonomy of Daldinia (Xylariaceae). Studies in Mycology 77(1): 1-143.

Velmurugan, P., Kamala-kannan, S., Balachandar, V., Lakshmanaperumalsamy, P., Chae, J. & Oh, B. 2010. Natural pigment extraction from five filamentous fungi for industrial applications and dyeing of leather. Carbohydrate Polymers 79(2): 262-268.

Venugopalan, A. & Srivastava, S. 2015. Endophytes as in vitro production platforms of high value plant secondary metabolites. Biotechnology Advances 33: 873- 887.

Verma, V.C., Kharwar, R.N. & Strobel, G.A. 2009. Chemical and functional diversity of natural products from plant associated endophytic fungi. Natural Product Communications 4(11): 1511-1532.

 

*Corresponding author; email: emelda@ukm.edu.my  

 

 

 

 

 

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