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.

A highly diverse fungal community associated with leaves of the mangrove plant Acanthus ilicifolius var. xiamenensis revealed by isolation and metabarcoding analyses

“…The leaves were surface-sterilized before isolation and therefore the diversity of fungi recovered from isolation represented the endophytic fungal diversity. On the other hand, the diversity obtained from the metabarcoding analysis represented predominantly endophytic fungi and might represent partial diversity of the epiphytic fungi as surface sterilization of leaves by sodium hypochlorite and ethanol does not completely eliminate all fungal DNA on the surface of the leaves (Burgdorf et al, 2014). This might have resulted in the differences in fungal richness (Margalef species richness, total richness) between the two methods, i.e., generally higher in the metabarcoding analysis (winter: 7.50 (Margalef), 96 species, summer: 5.29, 70) than in the isolation study (winter: 6.24, 30, summer: 5.44, 26) although the Shannon–Wiener diversity index of the two samples was comparable.…”

Fungal endophytes as priority colonizers initiating wood decomposition

“…Both sets of these stem rounds (i.e. autoclaved and not) were surface sterilized prior to addition to microcosms by swabbing with 70% ethanol, in line with recommendations of Burgdorf et al (2014) for surface sterilizing large samples.…”

Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach

“…After treating the leaves with 70 % (w/v) ethanol for microbial decontamination and 5 % (w/v) sodium hypochlorite for DNA decontamination together with several times washing with sterile water, no growth of microorganisms and no PCR products of the D1/D2 domain from the washed leaves were detected, indicating adequate sterilization and DNA removal of the leaf surface, respectively. The inoculation of Saccharomyces cerevisiae as a control organism on the leaf surface of wheat was proposed for the evaluation of the surface sterilization efficiency in culture-independent fungal endophyte studies (Burdorf et al 2014); however, a known species used as control organism must not be an endophyte of the plant and must produce amplified products that are different in size from the native endophytes. Therefore, we used epiphytic microorganisms as control organisms to elucidate the reduction in the epiphytic DNA and the comparison of the PCR products with and without sterilization revealed that the sterilization process could significantly reduce the contamination of epiphytic yeast DNA and sufficiently prevent the amplification of the D1/D2 domain of the epiphytic yeast DNA.…”