Improved isolation of nucleic acids from basidiomycete fungi.

Although current interest in basidiomycete fungi is focused mainly on taxonomy and exploitation of selected species in environmental biotechnology, isolation and characterization of their nucleic acids is becoming still more and more important for many purposes. Differences in cell wall composition and the mode of growth of some basidiomycetes are the major limitations for utilization of common techniques for nucleic acids preparation. So far, methods developed for nucleic acid isolation are usually either time-consuming and difficult (3,11,12) or vary in efficiency with different groups of fungi (5,7,8,13). Some of the common methods mentioned above are limited in use on the laboratory-grown mycelium as a source, and none of them can cope with the rigidity of fruit bodies of some fungal species. Mini-prep methods for fungal DNA isolation described until now are often either time-consuming, or require the use of potentially hazardous reagents (e.g., chloroform, phenol or 2-mercaptoethanol) or expensive purification kits (2,4). Our method described here is fast, low in cost, not laborious and works well with many various fungal species and with both fungal in vitro cultures and fruit bodies collected in the natural environment. We used this method mainly for analyses of and in a search for new fungal extrachromosomal inheritable elements like plasmids and mycoviruses and also when the processing of multiple fungal samples is necessary. Taking into account the advantages mentioned above and the quality of isolated nucleic acids, we propose it as a method of choice (i) in wide surveys, especially when simultaneous isolation of both fungal DNA and RNA is necessary (e.g., searching for plasmids and mycoviruses), (ii) for analyses of nucleic acids from huge number of fungal samples, (iii) for taxonomical and biotechnological studies in the field, (iv) for nucleic acid isolation from fungi, which are difficult to prepare and grow in a pure culture (e.g., mycorrhizal basidiomycetes), and (v) for isolation of nucleic acids from rigid and hard to disintegrate fruit bodies, like those of some wood-rotting fungal species. This method is based on the spontaneous autolysis of fungal mycelium. Autolysis in fungi, including basidiomycetes, have been known for several decades. It either occurs as a normal final stage of a life-cycle of a particular species, or it can be induced by some chemical substances (1,10). The phenomenon of autolysis was mostly studied in yeast, and a method for microisolation of yeast nucleic acids has been developed (6). However, autolysis proceeds also in most (if not all) basidiomycetes. Our procedure (Table 1) yields sufficient amount of nucleic acids from both laboratory cultures and fruit bodies. Other features of this protocol are as follows: (i) it is possible to induce autolysis in a wide range of fungal genera; no further addition of lytic enzymes or mechanical treatment of the mycelium is necessary; (ii) small amount of mycelia (5–10 mg fresh weight) is sufficient for successful isolation; (iii) high number of samples can be processed in parallel; (iv) the procedure usually works equally well with mycelium both from laboratory culture and from naturally occurring fruit bodies; (v) the method is simple, inexpensive and not laborious; and (vi) both DNA and RNA are isolated at the same time. The major advantage is that the use of autolysis for disintegration of mycelium can also be used in case of rigid fruit bodies (especially wood-rotting basidiomycetes), non-susceptible to lytic enzymes and proof against mechanical disruption. Here, we demonstrate the use of the method for isolation of nucleic acids from both laboratory-cultured mycelia and fruit bodies of wood-rotting fungi and also fruit bodies of some edible or poisonous mycorrhizal, saprophytic and parasitic basidiomycetes (Figure 1). The method was used for the nucleic acid isolation of 29 strains of wood-rotBenchmarks