Abstract
The identification of fungal endophytes often relies on culturing isolates from surface-sterilized plant tissue. However, molecular techniques have enabled the rapid detection and identification of targeted endophyte species, and next-generation sequencing technology provides an opportunity to obtain comprehensive information on endophytic communities, directly from plant tissue. In order to achieve accurate detection from internal tissues, surface microbes and associated deoxyribonucleic acid (DNA) must be eliminated, with particular consideration for the type of plant tissue and the efficacy of the surface sterilization procedure used. The methodology described later was developed specifically for detection of DNA from the entomopathogenic fungal endophyte Beauveria bassiana (Vuillemin) (Ascomycota: Hypocreales) in various tissues of Zea mays (L.). However, the protocol may be easily applied to other fungi and bacterial endophytes. Included is a stringent sodium hypochlorite-based surface sterilization protocol for plant material in preparation for polymerase chain reaction (PCR) to detect target DNA within plant tissue. Included are a treatment for dealing with surface DNA contamination and a novel procedure for assessing the efficacy of surface sterilization using PCR.
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References
Hyde KD, Soytong K (2008) The fungal endophyte dilemma. Fungal Divers 33:163–173
Wilson D (1995) Endophyte—the evolution of a term, and classification of its use and definition. Oikos 73(2):274–276
Eilenberg J, Hajek A, Lomer C (2001) Suggestions for unifying the terminology in biological control. BioControl 46(4):387–400
Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686
Schulz B, Krohn K, Meier K, Draeger S (2011) Isolation of endophytic fungi for the production of biologically active secondary metabolites. In: Pirttilä AM, Sorvari S (eds) Prospects and applications for plant-associated microbes. A laboratory manual, Part B: Fungi, 1st edn. Biobien Innovations, Germany
Hallmann J, Berg G, Schulz B (2006) Microbial root endophytes. In: Czeschlik D (ed) Soil biology. Springer, Berlin
Zhang SZ, Xia YX, Kim B, Keyhani NO (2011) Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development and pathogenesis in the entomopathogenic fungus, Beauveria bassiana. Mol Microbiol 80(3):811–826
Wagner BL, Lewis LC (2000) Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana. Appl Environ Microbiol 66(8):3468–3473
Nocker A, Cheung C-Y, Camper AK (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67(2):310–320
Nocker A, Sossa-Fernandez P, Burr MD, Camper AK (2007) Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 73(16):5111–5117
Brownbridge M, Reay SD, Nelson TL, Glare TR (2012) Persistence of Beauveria bassiana (Ascomycota: Hypocreales) as an endophyte following inoculation of radiata pine seed and seedlings. Biol Control 61(3):194–200
Nocker A, Sossa KE, Camper AK (2007) Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. J Microbiol Methods 70(2):252–260
Petrini O (1986) Taxonomy of endophytic fungi of aerial plant tissues. In: van den Huevel J, Fokkema NJ (eds) Microbiology of the phyllosphere. Cambridge University Press, Cambridge, UK
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McKinnon, A.C. (2016). Plant Tissue Preparation for the Detection of an Endophytic Fungus In Planta . In: Glare, T., Moran-Diez, M. (eds) Microbial-Based Biopesticides. Methods in Molecular Biology, vol 1477. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6367-6_13
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DOI: https://doi.org/10.1007/978-1-4939-6367-6_13
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6367-6
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