Sains Malaysiana 47(12)(2018): 3061–3068

http://dx.doi.org/10.17576/jsm-2018-4712-16

 

Optimization of Metabolite Extraction Protocols for Untargeted Metabolite Profiling of Mycoparasitic Scytalidium parasiticum using LC-TOF-MS

(Pengoptimuman Protokol Pengekstrakan Metabolit Tak Bertumpu bagi Profil Metabolit daripada Mikoparasit Scytalidium parasiticum Menggunakan LC-TOF-MS)

CHOON KIAT LIM¹*, RAFIDAH AHMAD², NURUL FADHILAH MARZUKI¹, YIT KHENG GOH¹, KAMALRUL AZLAN AZIZAN², YOU KENG GOH¹ & KAH JOO GOH¹

 

¹Advanced Agriecological Research Sdn Bhd, No. 11 Jalan Teknologi 3/6, Taman Sains Selangor 1, Kota Damansara, 47810 Petaling Jaya, Selangor Darul Ehsan, Malaysia

 

²Metabolomics Research Laboratory, Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 30 Mei 2018/Diterima: 18 September 2018

 

ABSTRACT

Basal stem rot disease of oil palm caused by Ganoderma boninense is one of the most devastating diseases in oil palm plantation resulting in low yield, loss of palm stands and shorter replanting cycle. To-date, there is no effective treatment for Ganoderma infected palms. Control measures, either chemical or cultural approaches, show varying degrees of effectiveness. The application of biological control agents which is environmental-friendly could be an attractive solution to overcome the problem. Earlier, we had isolated a mycoparasite, Scytalidium parasiticum from the basidiomata of Ganoderma boninense. In vitro assay and nursery experiment showed that this fungus could suppress Ganoderma infection and reduce disease severity. However, metabolites which might contribute to the antagonistic or mycoparasitic effect remain unknown. In the current study, optimization of fungal sample processing, extraction, and analytical procedures were conducted to obtain metabolites from the maize substrate colonized by mycoparasitic ascomycetous Scytalidium parasiticum. This technique capable of producing sexual spores in sac-like organs. Untargeted metabolomics profiling was carried out by using Liquid Chromatography Time of Flight Mass Spectrometry (LC-TOF-MS). We found that S. parasiticum in both liquid- and solid-state cultivation gave higher metabolite when extracted with 60% methanol with 1% formic acid in combination with homogenisation methods such as ultrasonication and grinding. The findings from this study are useful for optimisation of metabolite extraction from other fungi-Ganoderma-plant interactions.

 

Keywords: Biocontrol agent; Ganoderma boninense; LC-ToF-MS; metabolomics; oil palm

 

ABSTRAK

Penyakit busuk pangkal batang bagi pokok kelapa sawit yang disebabkan oleh Ganoderma boninense adalah salah satu penyakit yang paling serius di ladang sawit yang mengakibatkan hasil yang rendah, kekurangan pokok sawit yang produktif dan kitaran penanaman semula yang lebih singkat. Sehingga kini, tiada rawatan berkesan untuk pokok sawit yang dijangkiti Ganoderma. Langkah-langkah kawalan, sama ada pendekatan kimia dan teknik pengkulturan, menunjukkan tahap keberkesanan yang berlainan. Penggunaan agen kawalan biologi yang mesra alam boleh menjadi langkah alternatif yang terbaik dalam menangani masalah ini. Scytalidium parasiticum telah dipencilkan daripada basiodamata G. boninense. Kajian in vitro dan nurseri telah menunjukkan bahawa kulat ini boleh merencatkan jangkitan Ganoderma dan mengurangkan kesan yang lebih parah terhadap penyakit ini. Walau bagaimanapun, metabolit yang mungkin menyumbang kepada kesan antagonistik atau mikoparasit masih tidak diketahui. Dalam kajian ini, pengoptimuman pemprosesan sampel, pengekstrakan dan analisis dilakukan untuk mendapatkan metabolit daripada mikoparasit“askomise S. parasiticum yang dikultur dengan substrat jagung. Teknik ini mampu menghasilkan spora seksual dalam organ seperti sac. Pemprofilan metabolomik tidak bertumpu dilakukan dengan Kromatografi Cecair Ion Terbang Spektrometri Jisim (LC-ToF-MS). Kami mendapati bahawa S. parasiticum yang dikultur dalam media cecair dan dalam keadaan pepejal memberikan jumlah metabolit lebih tinggi apabila diekstrak dengan 60% metanol dan 1% asid formik dengan kombinasi kaedah penghomogen seperti ultrasonikasi dan pengisaran. Keputusan kajian ini adalah berguna untuk mengoptimumkan penghasilan metabolit daripada ekstrak interaksi kulat-Ganoderma-sawit.

 

Kata kunci: Agen biokawalan; Ganoderma boninense; kelapa sawit; LC-ToF-MS; metabolomik

RUJUKAN

Agustini, L., Wahyuno, D., Indrayadi, H. & Glen, M. 2014. In vitro interaction between Phlebiopsis sp. and Ganoderma philippii isolates. Forest Pathology 44: 472-476.

Boosalis, M.G. 1964. Hyperparasitism. Annual Review of Phytopathology 2: 363-376.

Chaverri, P., Branco-Rocha, F., Jaklitsch, W., Degenkolb, T. & Samuels, G.J. 2015. Systematics of the Trichoderma harzianum species complex including the identification of commercial biocontrol strains. Mycologia 107: 558-590.

Elfirta, R.R., Falah, S., Andrianto, D. & Lastini, T. 2018. Identification of active compounds and antifungal activity of Toona sinensis leaves fractions against wood rot fungi. Biodiversitas 19: 1313-1318.

Ferreira, J.F.S., Luthria, D.L., Sasaki, T. & Heyerick, A. 2010. Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with Artemisinin against malaria and cancer. Molecules 15: 3135-3170.

Gams, W., Diederich, P. & Põldmaa, K. 2004. Fungicolous fungi. In Biodiversity of Fungi: Inventory and Monitoring Methods, edited by Mueller, G.M., Bills, G.F. & Foster, M.S. USA: Elsevier Academic Press. pp. 343-392.

Goh, Y.K., Marzuki, N.F., Goh, T.K., Tan, S.Y., Goh, Y.K. & Goh, K.J. 2016. Mycoparasitic Scytalidium parasiticum as a potential biocontrol agent against Ganoderma boninense basal stem rot (BSR) in oil palm (Elaeis guineensis). Biocontrol Science and Technology 26: 1352-1365.

Goh, Y.K., Goh, T.K., Marzuki, N.F., Tung, H.J., Goh, Y.K. & Goh, K.J. 2015. Scytalidium parasiticum sp. nov., parasitizing on Ganoderma boninense from oil palm in Malaysia. Mycobiology 43: 107-117.

Gray, N., Heaton, J., Musenga, A., Cowan, D.A., Plumb, R.S. & Smith, N.W. 2013. Comparison of reversed-phase and hydrophilic interaction liquid chromatography for the quantification of ephedrines using medium-resolution accurate mass spectrometry. Journal of Chromatography A 1289: 37-46.

Helfer, W. 1991. Pilze auf Pilzfruchtkorpern. Untersuchungen zur on the c Systematik und Chemie. Libri Botanici 1: 11.

Joana Gil-Chávez, G., Villa, J.A., Fernando Ayala-Zavala, J., Basilio Heredia, J., Sepulveda, D., Yahia, E.M. & González-Aguilar, G.A. 2013. Technologies for extraction and production of bioactive compounds to be used as nutraceuticals and food ingredients: An overview. Comprehensive Reviews in Food Science and Food Safety 12: 5-23.

Kang, H.J., Ahn, K.S., Han, C.W., Jeong, K.H., Park, S.J., Song, I.G., Yun, T. & Min, K.B. 2014. Xylogone ganodermophthora strain with antifungal activity and composition including same for preventing plant diseases, U.S. Patent Application No. 20140220068, The United States Patent and Trademarks Office, Washington, DC, USA.

Kang, H.J., Sigler, L., Lee, J., Gibas, C.F.C., Yun, S.H. & Lee, Y.W. 2010. Xylogone ganodermophthora sp. nov., an ascomycetous pathogen causing yellow rot on cultivated mushroom Ganoderma lucidum in Korea. Mycologia 102: 1167-1184.

Kirschner, R., Arnold, G.R.W. & Chen, C.J. 2007. Cladobotryum semicirculare sp. nov. (Hyphomycetes) from commercially grown Ganoderma tsugae in Taiwan and other Basidiomycota in Cuba. Sydowia 59: 114-124.

Lei, Z., Huhman, D.V. & Sumner, L.W. 2011. Mass spectrometry strategies in metabolomics. Journal of Biological Chemistry 286(29): 25435-25442.

Lim, H., Kim, H.J., Jeong, H. & Park, H.R. 2017. Anti-inflammatory effects of 1-isothiocyanato-7-(methylsulfonyl) heptane by suppressing the NFκ-B signalling pathway. European Journal of Inflammation 15: 57-65.

Lin, C.Y., Wu, H., Tjeerdema, R.S. & Viant, M.R. 2007. Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics. Metabolomics 3: 55-67.

Madla, S., Miura, D. & Wariishi, H. 2012. Optimization of extraction method for GC-MS based metabolomics for filamentous fungi. Microbial & Biochemical Technology 4: 005-009.

Marzuki, N.F., Goh, Y.K., Tung, H.J., Goh, Y.K. & Goh, K.J. 2015. Evaluation of the cultural characteristics and antagonistic activities of Cladobotryum semicirculare against Ganoderma boninense in-vitro. Journal of Oil Palm Research 27: 326-338.

Oh, S.J., Chun, C.S., Lee, J.K. & Kim, H.K. 1998. Occurrence and identification of the fungus causing yellow rot on Ganoderma lucidum. Korean Journal of Mycology 26: 31-38.

Pinu, F.R. & Villas-Boas, S.G. 2017. Extracellular microbial metabolomics: The state of the art. Metabolites 7: 1-15.

Põldmaa, K., Larsson, E. & Kõljalg, U. 1999. Phylogenetic relationships in Hypomyces and allied genera, with emphasis on species growing on wood-decaying homobasidiomycetes. Canadian Journal of Botany 77: 1756-1768.

Rogerson, C.T. & Samuels, G.J. 1993. Polyporicolous species of Hypomyces. Mycologia 85: 231-272.

Srivastava, S.K., Srivastava, S.D. & Chouksey, B.K. 2001. New antifungal constituents from Terminalia alata. Fitoterapia 72: 106-112.

Stupar, M., Grbić, M.Lj., Džamić, A., Unković, N., Ristić, M., Jelikić, A. & Vukojević, J. 2014. Antifungal activity of selected essential oils and biocide benzalkonium chloride against the fungi isolated from cultural heritage objects. South African Journal of Botany 93: 118-124.

 

*Pengarang untuk surat-menyurat; email: limck@aarsb.com.my