Abstract
This study aimed to investigate the utilization of a buffer solution, containing Tris, HCl, EDTA, NaCl, and glycerol (THENG), in combination with a thermal shock process, as a novel DNA extraction method (THENG-tsp) for endophytic fungi. Three mg of fungal mycelium was added to 100 μL of THENG buffer and incubated in a water-bath (83 ± 2 °C) for 35 min. The vials were subsequently frozen at -20 °C for 15 min and then subjected to centrifugation (11,200 m/s2; 6 min). The resulting supernatant was used as a pure DNA source. In addition, the DNA of endophytic fungi was extracted using the cetyl trimethylammonium bromide (CTAB) method. DNA quality was evaluated based on light intensity and distance (in pixels) of bands observed on a 0.8% agarose gel after electrophoresis. The results indicate that the application of the THENG-tsp method improved the average band quality of all endophytic fungi by 7.86%. More precisely, bands corresponding to endophytic fungi belonging to the Basidiomycota and Ascomycota phyla exhibited an average increase in band quality of 13.50% and 8.57%, respectively, compared to the CTAB method. The application of the THENG-tsp method also resulted in improved band quality for Alternaria spp. and Fusarium spp., with average increases of 8.47% and 28.40%, respectively, compared to the CTAB method. Hence, the THENG-tsp method is considered appropriate for DNA extraction from endophytic fungi in laboratory settings due to its minimal requirement of initial fungal mycelium, time efficiency, and the absence of the use of potentially harmful chemical compounds.
Graphical abstract
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Raw data of the current study are available from the corresponding author on reasonable request. The ITS genome sequence of the endophytic fungi used in this research are available at Nucleotide database of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Also, comprehensive and more complete information about the endophytic fungi used in this research are available online (https://doi.org/10.1093/jambio/lxac015; https://doi.org/10.7717/peerj.8309).
Abbreviations
- APU-LIPG:
-
Area percentage under the light intensity-pixels graph
- CTAB:
-
Cetyl-trimethyl ammonium bromide extraction protocol
- PCR:
-
Polymerase chain reaction
- PDA:
-
Potato Dextrose Agar
- PDB:
-
Potato Dextrose Broth
- THENG:
-
Buffer solution comprising Tris, HCl, EDTA, NaCl, and glycerol
- THENG-tsp:
-
Application of THENG buffer in conjunction with a particular thermal shock procedure as an innovative DNA extraction method
References
Akbari Oghaz N, Hatamzadeh S, Rahnama K et al (2022) Adjustment and quantification of UV–visible spectrophotometry analysis: an accurate and rapid method for estimating Cladosporium spp. spore concentration in a water suspension. World J Microbiol Biotechnol. 38(10):183. https://doi.org/10.1007/s11274-022-03356-8
Basu S, Bose C, Ojha N et al (2015) Evolution of bacterial and fungal growth media. Bioinformation 11(4):182–184. https://doi.org/10.6026/97320630011182
Carroll G (1995) Forest endophytes: pattern and process. Can J Bot 73(S1):1316–1324. https://doi.org/10.1139/b95-393
Chi MH, Park SY, Lee YH (2009) A quick and safe method for fungal DNA extraction. Plant Pathol J 25(1):108–111. https://doi.org/10.5423/PPJ.2009.25.1.108
Cubero OF, Crespo ANA, Fatehi J et al (1999) DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi. Plant Syst Evol 216(3):243–249. https://doi.org/10.1007/BF01084401
Dairawan M, Shetty PJ (2020) The evolution of DNA extraction methods. Am J Biomed Sci Res 8(1):39–46. https://doi.org/10.34297/AJBSR.2020.08.001234
Faggi E, Pini G, Campisi E (2005) Use of magnetic beads to extract fungal DNA. Mycoses 48(1):3–7. https://doi.org/10.1111/j.1439-0507.2004.01030.x
Hatamzadeh S, Rahnama K, Nasrollahnejad S et al (2020) Isolation and identification of L-asparaginase-producing endophytic fungi from the Asteraceae family plant species of Iran. PeerJ 8(e8309. https://doi.org/10.7717/peerj.8309
Hatamzadeh S, Rahnama K, White JF et al (2022) Investigation of some endophytic fungi from five medicinal plants with growth promoting ability on maize (Zea mays L.). Journal of Applied Microbiology 134(1):lxac015. https://doi.org/10.1093/jambio/lxac015
Liu D, Coloe S, Baird R et al (2000) Rapid mini-preparation of fungal DNA for PCR. J Clin Microbiol 38(1):471–471
Lu H, Wei T, Lou H et al (2021) A critical review on communication mechanism within plant-endophytic fungi interactions to cope with biotic and abiotic stresses. J Fungi 7(9):719. https://doi.org/10.3390/jof7090719
Mahuku GS (2004) A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA. Plant Mol Biol Rep 22(1):71–81. https://doi.org/10.1007/BF02773351
Manganyi MA, Njie C (2020) Untapped potentials of endophytic fungi: A review of novel bioactive compounds with biological applications. Microorganisms 8(12):1934. https://doi.org/10.3390/microorganisms8121934
Natale M, Maresca B, Abrescia P et al (2011) Image analysis workflow for 2-D electrophoresis gels based on ImageJ. Proteome Insights 4(PRI. S7971. https://doi.org/10.4137/PRI.S7971
Pamphile JA, Santos Ribeiro MAd, Polonio JC (2017) Secondary metabolites of endophyte fungi: techniques and biotechnological approaches. In: Springer, Cham, pp 185–206. https://doi.org/10.1007/978-3-319-55804-2_9
Rana KL, Kour D, Sheikh I et al (2019) Biodiversity of endophytic fungi from diverse niches and their biotechnological applications. Adv.Endophytic Fungal Res. 105–144. https://doi.org/10.1007/978-3-030-03589-1_6
Rao RG, Ravichandran A, Dhali A et al (2018) A rapid microwave method for isolation of genomic DNA and identification of white rot fungi. bioRxiv 307066. https://doi.org/10.1101/307066
Saitoh K-i, Togashi K, Arie T et al (2006) A simple method for a mini-preparation of fungal DNA. J Gen Plant Pathol 72(6):348–350. https://doi.org/10.1007/s10327-006-0300-1
Samarrai TH, Schmid J (2000) A simple method for extraction of fungal genomic DNA. Lett Appl Microbiol 30(1):53–56. https://doi.org/10.1046/j.1472-765x.2000.00664.x
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675. https://doi.org/10.1038/nmeth.2089
Schulz B, Römmert A-K, Dammann U et al (1999) The endophyte-host interaction: a balanced antagonism? Mycol Res 103(10):1275–1283. https://doi.org/10.1017/S0953756299008540
Sudhakar T, Dash S, Rao R et al (2013) Do endophytic fungi possess pathway genes for plant secondary metabolites. Curr Sci 104(2):178–182
Sun X, Guo L-D (2012) Endophytic fungal diversity: review of traditional and molecular techniques. Mycology 3(1):65–76
Voigt K, James TY, Kirk PM et al (2021) Early-diverging fungal phyla: taxonomy, species concept, ecology, distribution, anthropogenic impact, and novel phylogenetic proposals. Fungal Divers. 109(59–98. https://doi.org/10.1007/s13225-021-00480-y
Yan L, Zhu J, Zhao X et al (2019) Beneficial effects of endophytic fungi colonization on plants. Appl Microbiol Biotechnol 103(8):3327–3340. https://doi.org/10.1007/s00253-019-09713-2
Zhang YJ, Zhang S, Liu XZ et al (2010) A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains. Lett Appl Microbiol 51(1):114–118. https://doi.org/10.1111/j.1472-765X.2010.02867.x
Acknowledgements
The authors are grateful to professor Hassan Vatandoost (Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences) and professor James Francis White (Department of Plant Biology, Rutgers University) for their valuable scientific advices during this research project.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Performing all tests, visualization, investigation and methodology were performed by Sareh Hatamzadeh. Data analysis and writing the original draft were performed by Nima Akbari Oghaz. Conceptualization, supervision, validation, review and editing original draft were performed by Kamran Rahnama and Fatemeh Noori. All participating authors read, commented and approved on previous versions of the manuscript.
Corresponding author
Ethics declarations
Consent for publication
All authors have read and approved the final manuscript.
Competing interests
Authors do not have any competing interests.
Financial interests
The authors declare they have no financial interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Hatamzadeh, S., Akbari Oghaz, N., Rahnama, K. et al. Combination of a hierarchical and thermal shock process with a specific aqueous buffer: a safe, rapid and reliable DNA extraction method for plant endophytic fungi. Biologia 79, 597–604 (2024). https://doi.org/10.1007/s11756-023-01579-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11756-023-01579-0