Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles




Abstract. Junaid M, Purwantara A, Guest G. 2021. Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles. Biodiversitas 22: 2838-2843. Understanding the biology of the fastidious Basidiomycete Ceratobasidium theobromae occupying host-tissue is essential for plant disease management. Direct pathogen DNA extraction from infected plant tissue avoids the need for isolation in artificial media. We report a modified DNA isolation protocol to obtain total plant DNA designed to overcome DNA extraction and isolation problems caused by infected petioles rich in polysaccharides and phenolic substances as a primary source of gummosis. This study examined and compared total plant DNA isolated from petioles high in polysaccharides and polyphenolic compounds using two methods: conventional CTAB lysis buffer and Kits (the standard method), and a new modified CTAB protocol to address these PCR inhibitors. The modified method resulted in higher quality and quantity of C. theobromae crude DNA and amplified PCR product. The modified method produced large quantities of clear, transparent, aqueous DNA-containing lysate (crude DNA) with a clear separation between the upper crude DNA and organic waste layers. Mean DNA absorbance was 1.80, and the lowest DNA yield was 836.6 ng/µl. With the standard method, the blurred, viscous lysate obtained showed signs of gummosis, with poor separation between layers of crude DNA, polysaccharides, protein, and organic waste layers and low yield. Gel electrophoresis indicated poor quality DNA extract. We conclude that this modified method will be valuable for genetic diversity and disease studies in a range of previously challenging plant tissues and their pathogens.


Afifah, E. N., et al. (2019). "Metabolomics Approach for The Analysis of Resistance of Four Tomato Genotypes (Solanum lycopersicum L.) to Root-Knot Nematodes (Meloidogyne incognita)." Open Life Sciences 14(1): 141-149.
Agrios G (2012) Plant Pathology. Burlington: Elsevier Science p 48-63.
Allen GC, Flores-vergara M et al (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 1 (5) 2320-5.
Castro-Moretti, F. R., et al. (2020). "Metabolomics as an Emerging Tool for the Study of Plant–Pathogen Interactions." Metabolites 10(2): 52.
Croteau, R., et al. (2000). Natural Products (Secondary Metabolites). B. Buchanan, W. Gruissem and R. Jones, American Society of Plant Physiologies: 1250-1318.
Doyle JJ (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 19 11-5.
Gómez, J. D., et al. (2020). "Leaf metabolic profiles of two soybean genotypes differentially affect the survival and the digestibility of Anticarsia gemmatalis caterpillars, Plant Physiology and Biochemistry" Plant Physiology and Biochemistry 15: 196-212.
Henderson DC, Hammond J. CKC (2013) Isolation of Nucleic Acids from a Diversity of Plants Using CTAB and Silica Columns. Journal of Molecular Biotechnology. 53 (5) 109-17.
Junaid, M., & Guest, D. (2021). Modified culture assay to obtain a diversity of hyphal structures of Ceratobasidium theobromae-VSD pathogen on cocoa. BIODIVERSITAS, 22(4), 1879-1886. doi:10.13057/biodiv/d220434
Junaid, M. (2018). Geographic distribution of “old” and “new” symptoms of vascular streak dieback (VSD) disease of cocoa in Sulawesi. University of Sydney.
Junaid M, Purwantara A et al (2020) First report of vascular streak dieback symptom of cocoa caused by Ceratobasidium theobromae in Barru District, South Sulawesi. IOP Conf. Ser.: Earth Environ. Sci. 486 012170.
Lima MRM Felgueiras ML. et al (2017) Differential phenolic production in leaves of Vitis vinifera cv. Alvarinho affected with esca disease. Plant Physiology and Biochemistry. 112 45-52.
Pichersky E and Gang DR (2000) Genetics and biochemistry of secondary metabolites in plants: An evolutionary perspective. Trends Plant Sci. p 205.
Pontes, J. G. M., et al. (2016). "Metabolomics by NMR Spectroscopy in Plant Disease diagnostic: Huanglongbing as a Case Study." ChemistrySelect 1(6): 1176-1178.
Porebski S Bailey LG et al (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter. 15 (4) 8-15.
Santoso TI Miftahudin M et al (2017) Analysis of Secondary Metabolites as Potential Phytoalexins, Their Secretion Sites and Proposed Resistance Markers to Vascular Streak Dieback in Theobroma cacao L. Journal of Pelita Perkebunan. 33 (4) 10-23.
Samuels, GJ Ismaiel, et al (2012) Vascular streak dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy. Fungal biology 116 (1) 11-23.
Strobel, G. (2017). Natural Products from Endophytic Microbes: Historical perspectives, Prospects, and Guidance Chemical Biology of Natural Products D. J. Newman, G. M. Cragg and P. Grothaus. Baton Rouge, USA, CRC Press.
White TJ Bruns T et al (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. 18 (1) 315-322.