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.

Keywords: DNA; endophyte; fungi; surface sterilization.

Figure 1

PCR of all treatments. Lanes 1 and 20, 100 bp molecular weight marker (Thermo Fisher Scientific); Lanes 2 to 5, Treatment A (physical); Lanes 6 to 9, Treatment B (chemical); Lanes 10 to 13, Control Y; Lanes 14 to 17, Control N; Lane 18, positive PCR control from pure yeast DNA; Lane 19, template-free control. The S. cerevisiae band is absent in the negative control samples and the treatments and positive controls show differing intensities of the band corresponding to the 530 bp pure S. cerevisiae band.

Figure 2

Contour map indicating band position and intensities from the DGGE gel data. This shows different regions (P, Q and R) with varying effects of surface and control treatments in the columns A, B, Y and N. The yeast ITS1 fragment is represented by two bands (S) in Region P. The key indicates the increasing intensity of the band with increasing darkness, corresponding to the square root of the percentage of maximum band intensity.

Figure 3

An NMDS plot of DGGE bands, showing the clustering of replicate samples from different treatments.

Figure 4

VPSEM micrographs of A-an uninoculated and untreated leaf surface (Negative Control-N) showing fungal hyphae (W), B-inoculated and untreated leaf surfaces (Positive Control-Y) showing abundant yeast cells (X), C-inoculated and physically abraded leaf surfaces (Treatment A) indicating a significant reduction in debris and S. cerevisiae cells, and D-inoculated and chemically surface sterilized leaf surfaces (Treatment B) indicating the presence of S. cerevisiae cells (y) and debris (z).