Robert C. Ullrich

Biological species of Armillaria mellea in North America.

SUMMARYArmillaria mellea consists of at least ten reproductively isolated groups, the equivalent of “biological species.” Each biological species possesses bifactorial heterothallism with compatibi...

A method for extracting high-molecular-weight deoxyribonucleic acid from fungi.

Abstract A new method for isolating high-molecular-weight DNA from mycelium of the basidiomycete Schizophyllum commune is reported. Lyophilized mycelium is broken with mortar and pestle, and DNA extracted during gentle shaking in buffer containing 2% sodium dodecyl sulfate and toluene. DNA with double-strand length in excess of 80 kilobase pairs and singlestrand length of 36 kilobases can be prepared routinely in milligram quantities. This DNA is of high purity and suitable for reassociations and recombinant DNA studies. The procedure has also been used to prepare DNA from another filamentous basidiomycete, Heterobasidion annosum , and from yeast. With slight modification the procedure is attractive for isolating recombinant plasmids from yeast.

Sex and diploidy in Armillaria mellea.

The life cycle and sexuality of Armillaria mellea are poorly understood. The mating behavior is atypical, and laboratory fruiting is rare. Genetic studies reported herein are based upon monosporous progeny from 27 distinct fruiting bodies and reveal A. mellea as a bifactorial heterothallic fungus. Both loci determining sexuality are multiallelic. The simplest, internally consistent explanation of wild-type mating interactions, morphogenesis, mycelial aspect, and cytology, coupled with examination of the mating process with genetically marked, auxotrophic strains, is that the naturally occurring persistent vegetative stage is diploid. This would be the first wild-type Basidiomycete shown to possess a life cycle emphasizing an extended diploid phase. The occurrence and distribution of reproductively isolated “biological species” are also reported.

Relationships between European and North American biological species ofArmillaria mellea

Abstract Pairings of monosporous isolates from five European and ten North American biological species of Armillaria reveal that certain species from Europe are interfertile with certain species from North American. All other pairings between species are intersterile. These pairings of isolates derived from different continents reveal three interactions not observed in earlier studies with isolates exclusively from one continent or the other. One, pairings from species demonstrated to be interfertile have a reduced frequency of compatibility, i.e., some determinant, in addition to mating type, affects the compatibility of specific pairings. Two, the pairing of isolates from intersterile species occasionally results in an unexplained reduction in growth in one or both members of the pairing. Three, in a single case, members of a species from one continent are compatible with members of two different intersterile species from the other continent. In fact, individual strains from one European species are compatible with members of two rigorously intersterile North American species.

Transformation of the basidiomycete, Schizophyllum commune

Protoplasts of a Schizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp+ with plasmid DNA containing the Schizophyllum TRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp+ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.

Sexuality, distribution, and dispersal of Heterobasidion annosum in pine plantations of Vermont.

Mating experiments with monosporous isolates of H. annosum from red pine plantations in Vermont demonstrate that the fungus is heterothallic and unifactorial. Clamp connections are present in tissue isolates from basidiocarps and in mycelia formed from compatible matings. The distribution of clamp connections within these mycelia is irregular and the features of this character are described in detail. The incompatibility factor is multiallelic and 40 alleles were identified in our collections from 53 basidiocarps. These alleles were used as naturally-occurring genetic markers to examine the distribution of the fungus within plantations. The distributional data are used to contribute information regarding dispersal. The genetic evidence suggests that centers of infection within a plantation are not due to the extensive vegetative spread of single clones, but that multiple inoculations are common in the development of infection centers. Nevertheless, the vegetative growth of mycelium does occur across the root systems of adjacent trees.

Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

SummaryAn Aα mating-type allele (Aα4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the Aα1 allele isolated from the walk was used as a probe to recover the Aα1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an Aα allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that Aα encodes a diffusible product. Restriction mapping shows that Aα1 and Aα4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of Aα1 or Aα4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other Aα alleles. Aα1 and Aα4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.

Strain specific differences in ribosomal DNA from the fungus Schizophyllum commune

SummaryCsCl-bisbenzimide gradients were used to purify ribosomal DNA (rDNA) from Schizophyllum commune total DNA. Southern hybridizations demonstrate that this DNA codes for rRNA. Restriction mapping of the rDNA from four strains revealed strain variation with repeat lengths of 9.2–9.6 kbp. Specific differences in the length of the rDNA repeat in different strains are due to insertions of 0.2 or 0.4 kbp of DNA at a single site. Different strains also show restriction site polymorphisms. Our analysis demonstrates the caution that must be exercised when interpreting restriction data from genomes containing restriction polymorphisms. Restriction digests with MspI and HpaII indicate that the rDNA contains 5-methylcytosine and that the unit repeats are not methylated identically, but rather differentially. This is the first report of methylated rDNA in fungi.

Transformation ofSchizophyllum commune: An analysis of parameters for improving transformation frequencies

Abstract The procedure used for transforming protoplasts prepared from germinated basidiospores of Schizophyllum commune has been improved to yield 10 3 transformants per microgram of DNA when using 10 7 protoplasts. Important to the protocol are keeping the protoplasts on ice and creating an osmotic shock when mixing DNA and protoplasts. Protoplasts may be stored at −70°C; this eliminates the need to prepare fresh protoplasts for each experiment. Titrations of CaCl 2 , polyethylene glycol, DNA, and protoplasts were tested individually to maximize the effect of each. Transformation does not occur without CaCl 2 or polyethylene glycol. The optimal concentration for CaCl 2 is 50 m M and that for polyethylene glycol ( M r = 3350) is 25% (w/v). At optimal polyethylene glycol and CaCl 2 concentrations, the number of transformants obtained and the calculated transformation frequencies (transformants per microgram of DNA) are dependent on protoplast and DNA concentrations.

Isolation and characterization of mitochondrial DNA from the basidiomyceteSchizophyllum commune

Abstract Mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations from a homokaryotic strain of the woodrotting basidiomycete Schizophyllum commune . An AT-rich satellite DNA was also isolated from both homokaryotic and dikaryotic strains using CsCl-bisbenzimide gradients. The satellite DNA and the DNA from DNase I-treated preparations of mitochondria have identical buoyant densities and hybridize to the same restriction fragments in Southern hybridizations. It is concluded that this satellite DNA is mitochondrial DNA. Restriction analyses of the mitochondrial DNA of strain 4–40 with five endonucleases show the mitochondrial genome to be about 49.85 kbp. Mitochondrial DNA from the 4–39 × 4–40 dikaryon has identical restriction fragments. The satellite, which we have shown to be mitochondrial DNA, represents 3–4% of the total cellular DNA and averages 22–29 copies per haploid cell under our growth conditions. Mitochondrial DNA prepared from three other strains of Schizophyllum revealed restriction enzyme polymorphisms and ranged in size from 50.3 to 52.2 kbp.