Characterization of Pacific golden chanterelle (Cantharellus formosus) genet size using co-dominant microsatellite markers.

We characterized five co-dominant microsatellite markers and used them to study Pacific golden chanterelle (Cantharellus formosus) genet size and its relation to forest age and disturbance. Fruit-bodies were mapped in and collected from nine replicate study plots in old-growth, recently thinned, and unthinned 40-60-year-old second-growth stands dominated by Douglas fir (Pseudotsuga menziesii). Information from microsatellite loci, combined with random fragment length polymorphism analysis of the nuclear DNA internal transcribed spacer indicates that putative 'C. formosus' fruit-body collections may include a cryptic chanterelle species. Small genets were characterized for both genetic types with mean maximum widths of 3.2 +/- 3.6 m for C. formosus and 1.5 +/- 1.7 m for the alternative genetic group. Variance in genet size was high and some multilocus genotypes were observed on multiple plots separated by 0.3 km or more, indicating that genets were not fully resolved by the loci described here. There was no evidence that genet size differed across the three disturbance treatments.

Regional specialization of Sarcodes sanguinea (Ericaceae) on a single fungal symbiont from the Rhizopogon ellenae (Rhizopogonaceae) species complex.

We have sampled the mycorrhizal roots of 76 snow plants (Sarcodes sanguinea, Monotropoideae, Ericaceae) in two areas of the Sierra Nevada of California that are ∼180 km apart. To identify the fungal symbionts associated with these plants, we first analyzed restriction fragment length polymorphisms (RFLPs) of the internal transcribed spacer region (ITS) of the fungal nuclear ribosomal repeat. Fungal ITS-RFLPs were successfully produced from 57 of the 76 plants sampled, and all symbionts shared the same DNA fragment pattern. The morphology of S. sanguinea mycorrhizae was consistent with that expected from a Rhizopogon species in section Amylopogon. To confirm and refine this identification, a total of six fungal ITS sequences were determined from S. sanguinea mycorrhizae. These sequences were analyzed together with eight existing and eight newly determined ITS sequences from Rhizopogon section Amylopogon. The newly determined sequences include an ITS sequence from the fungal symbiont of pine drops (Pterospora andromedea, Monotropoideae, Ericaceae), a plant that was previously reported to be exclusively associated with the Rhizopogon subcaerulescens group. When these sequences were analyzed together, the Sarcodes symbionts grouped tightly with several collections of R. ellenae including the holotype, one collection of R. idahoensis, and one collection of R. semireticulatus. A different lineage comprised collections of R. subgelatinosus, R. subcaerulescens, another collection of R. semireticulatus, and the Pterospora symbiont. We conclude that S. sanguinea associates exclusively with a single species in the R. ellenae species complex throughout our sampling range. These results indicate a much higher level of specificity in S. sanguinea than was previously reported and confirm the emerging pattern that nonphotosynthetic, monotropoid plants generally associate very specifically with a narrow range of ectomycorrhizal fungi.

High root concentration and uneven ectomycorrhizal diversity near Sarcodes sanguinea (Ericaceae): a cheater that stimulates its victims?

Sarcodes sanguinea is a nonphotosynthetic mycoheterotrophic plant that obtains all of its fixed carbon from neighboring trees through a shared ectomycorrhizal fungus. We studied the spatial structuring of this tripartite symbiosis in a forest where Sarcodes is abundant, and its only fungal and photosynthetic plant associates are Rhizopogon ellenae and Abies magnifica, respectively. We found disproportionately high concentrations of Abies roots adjacent to Sarcodes roots compared to the surrounding soil. Rhizopogon ellenae colonizes the vast majority of those Abies roots (86-98%), and its abundance tends to decrease with increasing distance from Sarcodes plants. At 500 cm from Sarcodes plants we did not detect R. ellenae, and the ectomycorrhizal community instead was dominated by members of the Russulaceae and Thelephoraceae, which are commonly dominant in other California pinaceous forests. The highly clumped distribution of Abies-R. ellenae ectomycorrhizas indicates that Sarcodes plants either establish within pre-existing clumps, or they stimulate clump formation. Several lines of evidence favor the latter interpretation, suggesting an unexpected mutualistic aspect to the symbiosis. However, the mechanism involved remains unknown.

Use of atp6 in fungal phylogenetics: an example from the boletales.

Complete nucleotide sequences have been determined for atp6 from Suillus luteus and cox3 from Suillus sinuspaulianus (Boletales, Hymenomycetes, Basidiomycota), which code for ATPase subunit 6 and cytochrome oxidase subunit 3, respectively. These sequences were used to design PCR primers for the amplification of partial atp6 and cox3 sequences from other members of the Boletales and outgroup taxa. In atp6 and cox3 from Russula rosacea, one of the outgroup taxa, we observed a number of in-frame TGA(trp) codons, which imply a Neurospora crassa-type mitochondrial code in R. rosacea and possibly in basidiomycetes in general. Interestingly, however, most basidiomycetes other than R. rosacea appear to strongly prefer the TGG(trp) codon, which is unusual, given the strong A + T bias in fungal mitochondrial genomes. Pairwise comparisons were performed between atp6 sequences from increasingly divergent fungal lineages, and results show that all three codon positions become saturated in substitutions after an estimated divergence time of approx 300 Ma. This means that atp6 is likely to provide phylogenetic resolution within fungal classes but not at higher taxonomic levels. Also, because of the strong A + T bias in fungal mitochondrial genomes, A/T transversions were found to be more common than any other type of substitution, resulting in transversions being about two to three times more common in most pairwise sequence comparisons. Finally, atp6 sequences were used to infer phylogenetic relationships between 27 taxa from the Boletales and 4 outgroup taxa. Analyses were performed (i) on nucleotide sequence data using parsimony (successive approximation) as well as maximum likelihood methods and (ii) on deduced amino acid sequences using distance methods based on empirical substitution probabilities. Results from the various analyses are largely concordant with each other as well as with prior analyses of partial mitochondrial large-subunit rDNA (mtLSU rDNA). Analysis of the combined atp6 and mtLSU rDNA sequences results in increased bootstrap support for several key branches. Relationships that have been resolved for the first time in the current analysis are discussed.

Catabolism of isonicotinate by Mycobacterium sp. INA1: extended description of the pathway and purification of the molybdoenzyme isonicotinate dehydrogenase.

Catabolism of isonicotinate by Mycobacterium sp. INA1 has been shown to proceed via 2-hydroxyisonicotinate, 2,6-dihydroxyisonicotinate (citrazinate), citrazyl-CoA and 2,6-dioxopiperidine-4-carboxyl-CoA. An extended pathway involving propane-1,2,3-tricarboxylate as a further intermediate is presented in this paper. Propane-1,2,3-tricarboxylate was oxidized stepwise to 2-oxoglutarate involving an oxidase, aconitase and isocitrate dehydrogenase. Isonicotinate dehydrogenase catalyses the first step of isonicotinate metabolism in Mycobacterium sp. INA1. The enzyme was purified to apparent homogeneity by a three-step procedure. Enrichment was accompanied by partial loss in specific activity. The native enzyme had a molecular mass of either 125 kDa or 250 kDa, when estimated by native gradient PAGE or gel filtration, respectively. SDS-gel electrophoresis revealed three types of subunits with molecular masses of approximately 83, 31 and 19 kDa. N-Terminal amino acid sequences of all three subunits have been determined. Molybdenum, iron, acid-labile sulphur and FAD were present at molar ratios of 1, 4, 4, 1 per protomer (125 kDa). The molybdenum-complexing cofactor was shown to be molybdopterin cytosine dinucleotide. Besides isonicotinate, only quinoline-4-carboxylate was found to be oxidized at appreciable rates.

Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria.

Eighteen different aerobic bacteria were isolated which utilized quinoline as sole source of carbon, nitrogen, and energy. Attempts were unsuccessful at isolating anaerobic quinoline-degrading bacteria. The optimal concentration of quinoline for growth was in the range of 2.5 to 5 mM. Some organisms excreted 2-hydroxyquinoline as the first intermediate. Hydroxylation of quinoline was catalyzed by a dehydrogenase which was induced in the presence of quinoline or 2-hydroxyquinoline. Quinoline dehydrogenase activity was dependent on the availability of molybdate in the growth medium. Growth on quinoline was inhibited by tungstate, an antagonist of molybdate. Partially purified quinoline dehydrogenase from Pseudomonas putida Chin IK indicated the presence of flavin, iron-sulfur centers, and molybdenum-binding pterin. Mr of quinoline dehydrogenase was about 300 kDa in all isolates investigated.