Molecular Identification of Armillaria gallica from the Niobrara Valley Preserve in Nebraska

Armillaria isolates were collected from a unique forest ecosystem in the Niobrara Valley Preserve in Nebraska, USA, which comprises a glacial and early postglacial refugium in the central plains of North America. The isolates were collected from diverse forest trees representing a unique mixture of forest types. Combined methods of rDNA sequencing and flow cytometric measurements of nuclear DNA content determined that all Armillaria isolates collected from the site were A. gallica. Introduction Armillaria species are diverse in pathogenicity, host specificity, and environmental requirements. Some Armillaria spp. are aggressive pathogens, while other Armillaria spp. are predominately saprophytic, and may help sustain forest productivity via nutrient cycling. Thus, Armillaria species identification is a critical component for monitoring forest health and understanding forest ecosystem functions. Current classification of Armillaria spp. is based in part on morphology and in vitro compatibility of isolates. In recent decades, molecular genetic methods were developed to augment identification of Armillaria species (Kim et al. 2006), and the nuclear DNA content of North American Armillaria spp. has been determined using laser flow cytometry (Kim et al. 2000). The Niobrara Valley Preserve is a glacial and early postglacial refugium in the central plains of North America where six major ecosystems converge. The middle Niobrara Valley contains a unique mixture of three forest types (northern boreal, western coniferous, and eastern deciduous) (Kaul et al. 1988). This forest refugium offers a unique opportunity to study biological diversity of this ecosystem that has harboured enriched flora and fauna since early postglacial to glacial times (ca. 9000–12 000 years B.P.). However, little is known about the fungal community associated with tree species in this ecologically unique area, and Armillaria species have not been previously reported from this ecosystem. Because Armillaria fungi can be major drivers of forest ecosystem processes, our objectives were to (i) determine if Armillaria spp. are present on diverse tree species within the Niobrara Valley Preserve forest refugium and (ii) identify any Armillaria spp. that were collected in this forest refugium. Materials and Methods On the Niobrara Valley Preserve in Nebraska, USA (Latitude 42 55¢16¢¢N, Longitude 100 26¢21¢¢ W, elevation 791 m), 10 trees were randomly selected within the three forest types present (Table 1). On each tree, major lateral roots were excavated and inspected for external rhizomorphs or internal mycelial fans of Armillaria spp. Samples of Armillaria spp. were collected along with host tree data, which included species and general health status. Armillaria spp. collections were established in culture following the protocol of Hanna et al. (2007). The protocol of Kim et al. (2006) was used for PCR amplification of the intergenic spacer 1 (IGS-1) region of rDNA. The PCR products of IGS-1 region were sequenced with an ABI 3700 DNA sequencer at the Davis Sequencing Facility (Davis, CA, USA), and the IGS-1 sequences of the Armillaria isolates have been deposited in GenBank (Table 1). Methods of Kim et al. (2000) were used to determine nuclear DNA content. Nuclei were isolated from mycelia and stained with propidium iodide (PI). Fluorescence of the PI-stained nuclei was analysed at the University of Nebraska-Flow Cytometry Core Research Facilities using a FACScan Flow Cytometer J Phytopathol 159:69–71 (2011) doi: 10.1111/j.1439-0434.2010.01718.x Published 2010. This article is a US Government work and is in the public domain in the USA