title: A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies.
abstract: Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.
authors.affiliation: Department of Microbiology University of KwaZulu-Natal Pietermaritzburg South Africa Department of Microbiology, University of KwaZulu-Natal, Pietermaritzburg, South Africa.
authors.forename: R J
authors.initials: RJ
authors.lastname: Burgdorf
authors.affiliation: Department of Plant Pathology University of KwaZulu-Natal Pietermaritzburg South Africa Department of Plant Pathology, University of KwaZulu-Natal, Pietermaritzburg, South Africa.
authors.forename: M D
authors.initials: MD
authors.lastname: Laing
authors.affiliation: Agricultural Research Council c/o School of Life Sciences University of KwaZulu-Natal Pietermaritzburg South Africa Agricultural Research Council, c/o School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, South Africa.
authors.forename: C D
authors.initials: CD
authors.lastname: Morris
authors.affiliation: Department of Microbiology University of KwaZulu-Natal Pietermaritzburg South Africa Department of Microbiology, University of KwaZulu-Natal, Pietermaritzburg, South Africa.
authors.forename: S F
authors.initials: SF
authors.lastname: Jamal-Ally
mesh_terms: D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics / Q000302:isolation & purification; D058645:Denaturing Gradient Gel Electrophoresis; D060026:Endophytes / Q000302:isolation & purification*; D008828:Microbiological Techniques / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D018515:Plant Leaves / Q000382:microbiology / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D013242:Sterilization / Q000379:methods*; D013499:Surface Properties; D014908:Triticum / Q000382:microbiology* / Q000648:ultrastructure
chemical_list: D004271:DNA, Fungal; D018744:DNA, Plant; D021903:DNA, Ribosomal Spacer
keywords: DNA; endophyte; fungi; surface sterilization