A crush on small fungi: An efficient and quick method for obtainingDNAfrom minute ascomycetes

Abstract

We have developed a reliable technique for extracting DNA from single microscopic fungal thalli, including efficient cell disruption and transfer of cell content for subsequent polymerase chain reaction (PCR). The technique was primarily developed for members of the ascomycete order Laboulbeniales, which are minute fungi with tough cell walls that are exceedingly difficult to disrupt with standard extraction techniques. Our method makes routine amplification of DNA from single thalli possible, even from small species or poorly developed individuals. DNA release is accomplished in an entirely mechanical manner using an arbor press fitted with custom‐made components. This approach has eliminated additional treatment such as laborious freeze‐thaw cycles, enzymes, or lysing agents. The overall PCR success rate of 89% is comparable to or better than alternative protocols that make use of substantially larger amounts of fungal tissue. From 97% of the successful PCRs a total of 156 sequences from four gene regions were produced. Being able to restrict DNA extractions to a single thallus is critical to all genetic studies requiring data at the level of individual, e.g. population genetics. As all researchers working with minute uncultivable organisms in many respects face the same problems (effective handling of the material, small quantities of DNA etc.), the methodology described here has a potential to be widely applicable. Necessary custom‐made components can be manufactured at fairly low cost by any precision‐tool workshop using our detail drawings.

Laboulbeniales (Ascomycota) of the Boston Harbor Islands II (and Other Localities): Species Parasitizing Carabidae, and the Laboulbenia flagellata Species Complex

“…25, Special Issue 9 Table 1. Overview of Laboulbeniales sequences generated and/or used in this study, with indication of DNA extraction protocol (REPLI = REPLI-g Single Cell Kit, ISOLATE II = ISOLATE II Plant DNA Kit, Sundberg = methods of Sundberg et al (2018) , QIAamp = QIAamp DNA Micro Kit, and Extract-N-Amp = Extract-N-Amp Plant PCR Kit) and numbers of thalli used per extraction (juv = juvenile, sub = subadult, ad = adult), host species, growth position on the host, and country. All isolates of which sequences were used are listed, with GenBank accession numbers for ITS and nrLSU rDNA.…”

“…Although commonly accepted in mycology (Hibbett et al 2011(Hibbett et al , 2016Taylor et al 2000), applying sequence-based taxonomy and phylogenetic species recognition to Laboulbeniales was long hindered by technical issues (Haelewaters et al 2015b, Sundberg et al 2018, Weir and Blackwell 2001. That DNA characters can provide answers to the issues of morphological variability and host specificity in Laboulbeniales was confirmed by Goldmann and Weir (2012).…”

Position specificity in the genus Coreomyces (Laboulbeniomycetes, Ascomycota)

“…Thalli were detached from 76 corixid individuals and crushed according to the protocol by Sundberg et al (2018). We used from one (or when poorly developed) up to six thalli from each position.…”

“…Our sampling included corixid populations in southern Sweden and Denmark, and we obtained DNA sequence data from several markers and numerous Coremomyces individuals from different host positions, sexes and species. This approach was made possible by recent advances in acquiring DNA sequence data from Laboulbeniales (Haelewaters et al 2015, Sundberg et al 2018.…”

Draft Genome Sequence of the Globally Distributed Cockroach-Infecting Fungus Herpomyces periplanetae Strain D. Haelew. 1187d

“…Species of both Herpomycetales and Laboulbeniales are unique among related fungi in that they do not form hyphae but instead produce multicellular units of hundreds to thousands of cells, referred to as "thalli," through coordinated cell divisions from a single ascospore (3,(6)(7)(8). To date, these fungi have not been grown apart from their host; DNA needs to be isolated from individual thalli removed from the host (9)(10)(11).…”