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DAFTAR PUSTAKA Abdollahzadeh, J., Javadi, A., Mohammadi Goltapeh DAFTAR PUSTAKA Abdollahzadeh, J., Javadi, A., Mohammadi Goltapeh, E., Zare, R., and Phillips, A.J.L. 2010. Phylogeny and Morphology of Four New Species of Lasiodiplodia from Iran. Persoonia 25, 2010: 1–10. doi:10.3767/003158510X524150. Alejandra P., Saul R., Felix J. L., Barajas A. S., dan Molar R. L. 2015. Lasiodiplodia theobromae in Agricultural Crops in Mexico: Taxonomy, Host, Diversity and Control. Revista Mexicana de Fitopatologia. Volume 33, No. 1. Ali, S., Asman, A., Shao, J., Balidion, J.F., Strem, M.D., Puig, A.S., Meinhardt, L.W., and Bailey, B.A. 2019. Genome and Transcriptome Analysis of The Latent Pathogen Lasiodiplodia theobromae, An Emerging Threat to The Cacao Industry. Genome, gen-2019-0112.R1. Alves, A., Crous, P.W., Correia, A., dan Phillips, A.J.L. 2008. Morphological and Molecular Data Reveal Cryptic Speciation in Lasiodiplodia theobromae. Fungal Diversity 28: 1-13. Bogoude, B.A.D., Slippers, B., Wingfield, M.J., dan Roux, J. 2010. Botryosphaeriaceae Assosiated with Terminalia cappa in Cameroon, South Africa and Madagascar. University of Pretoria, South Africa. Carris, L. M., C. R. Little and C. M. Stiles. 2012. Introduction to Fungi. The Plant Health Instructor. DOI:10. 1094/PHI-I-2012-0426-01. Coutinhoa, I. B. L., Freire, F. C. O., Lima, C. S., Lima, J. S., Goncalves, F. J. T. Machadod, A. R., Silvae, A. M. S. and Cardoso, J. E. 2017. Diversity of Genus Lasiodiplodia associated with Perennial Tropical Fruit Plants in Northeastern Brazil. Plant Pathology, 66: 90–104, Doi: 10.1111/ppa.12565. Denman S, Crous PW, Taylor JE, Kang JC et al. 2000. An overview of the taxonomic history of Botryosphaeria, and a re-evaluation of its anamorphs based on morphology and ITS rDNA phylogeny. Studies in Mycology 45, 129–140. Ditjenbun. 2013. Pedoman Teknis Penanganan Pasca Panen Tanaman Kakao. Kementan, Jakarta. Dou, ZP., He, W., dan Zhang Y. 2017. 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Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications. Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White. Academic Press Inc., New York. Pp. 315-322. Winter, P.C. 2005. Polymerase Chain Reaction (PCR). Encyclopedia of Life Science. DOI:10.1038/npg.els.0005339. 34 LAMPIRAN Lampiran 1. Empat belas isolat cendawan yang diidentifikasi dari tanaman kakao pada media PDA a. Isolat Hasil Pemurnian b. Isolat Hasil Perbanyakan 35 Lampiran 2. Komposisi Reagen PCR a. PCR Mix (25 µl) Komposisi : 2X Taq Plus PCR Master Mix 12,5µl Primer ITS4 1µl Primer ITS 5 1µl ddH2O 5,5µl Template DNA 5µl b. Gel Agarose 2% Komposisi : Serbuk Agarose 2 g TBE Buffer 100 mL Ethidium Bromida 8µl Lampiran 3. Kit ekstraksi (TIANamp Genomic DNA kit ) yang digunakan dalam ekstraksi DNA 36 Lampiran 4. Alur Kerja Ekstraksi DNA Sampel (30 mg) 200 µl Buffer GA Gerus, sentrifius pada 11.200 G/1 menit 20 µl Proteinase K Vortex, inkubasi pada suhu 56⁰C/10 menit 200 µl Buffer GB Vortex, inkubasi pada suhu 70⁰C/10 menit 200 µl Etanol Pindahkan ke spin column, sentrifius pada 13.400 G/30 detik 500 µl Buffer GD Sentrifius pada 13.400 G/30 detik 600 µl Buffer PW Sentrifius pada 13.400 G/30 detik 600 µl Buffer PW Pindahkan ke tube, sentrifius pada 13.400 G/2 menit 200 µl Buffer TE Inkubasi 2 menit Sentrifius 2 menit 37 Lampiran 5. Alur Kerja Amplifikasi DNA dengan PCR Sampel DNA Dibuatkan campuran reagen, yaitu 2X Taq Plus PCR Master Mix 12,5 µl, Primer ITS4 1 µl, Primer ITS 5 1 µl, dan ddH2O 5,5 µl. 20 µl campuran reagen PCR Dimasukkan ke dalam mikro tube 5 µl template DNA Dimasukkan ke dalam mikro tube yang berisi reagen PCR Amplifikasi dengan PCR Diamplifikasi sebanyak 35 siklus. Setiap siklus terdiri dari: 0 Predenaturasi pada suhu 94 C selama 3 menit Denaturasi pada suhu 940C selama 30 detik Annealing pada suhu 520C selama 30 detik Extending pada suhu 720C selama 1 menit. Post Extending pada suhu 720C selama 5 menit. Produk PCR 38 Lampiran 6. Alur Kerja Elektroforesis Produk PCR Produk PCR Dibuatkan gel agarose 2% dengan komposisi: Serbuk Agarose 2 g TBE Buffer 100 ml Ethidium Bromida 8µl 5 µl produk PCR Dipipet dengan pipet mikron dan dimasukkan ke dalam masing-masing sumuran Gel agarose 2% Elektroforesis Dielektroforesis dengan arus listrik 70 V selama 30 menit. Gel hasil elektroforesis diletakkan di atas uv transluminator dan difoto untuk dokumentasi. Pita DNA 39 Lampiran 7. Foto Proses Kerja Proses PCR Proses Elektroforesis 40 Lampiran 8. Hasil Sekuensing Isolat LasioKB (Lasiodiplodia theobromae) 41 Lampiran 9. Hasil Sekuensing Isolat LasioKK1 (Lasiodiplodia brasiliensis) 42 Lampiran 10. Hasil Sekuensing Isolat LasioKK2 (Lasiodiplodia theobromae) 43 Lampiran 11. Hasil Sekuensing Isolat LasioKK3 (Tidak teridentifikasi) 44 Lampiran 12. Hasil Sekuensing Isolat LasioKL1 (Lasiodiplodia citricola) 45 Lampiran 13. Hasil Sekuensing Isolat LasioKL2 (Lasiodiplodia theobromae) 46 Lampiran 14. Hasil Sekuensing Isolat LasioKL3 (Lasiodiplodia citricola) 47 Lampiran 15. Hasil Sekuensing Isolat LasioKL4 (Lasiodiplodia citricola) 48 Lampiran 16. Hasil Sekuensing Isolat LasioKL5 (Lasiodiplodia theobromae) 49 Lampiran 17. Hasil Sekuensing Isolat LasioKL6 (Lasiodiplodia theobromae) 50 Lampiran 18. Hasil Sekuensing Isolat LasioKL7 (Lasiodiplodia theobromae) 51 Lampiran 19.
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