Identification protocol for sixArmillariaspecies from northeastern North America

Abstract

DNA sequences (~3 kb long) extending from the intergenic spacer 1 (IGS1) region to the 18S gene were obtained for isolates of Armillaria ostoyae , Armillaria calvescens , Armillaria gallica , and Armillaria sinapina . Additional investigation of 16 A. ostoyae, 11 Armillaria gemina , 21 A. calvescens, 18 A. gallica, and 15 A. sinapina isolates produced 117 sequences spanning the 3′ end of the IGS1 through the 5S gene and into the 5′ end of the IGS2 region. Additional sequences spanning the 3′ IGS2 to 5′ 18S gene region were obtained for two A. ostoyae, three A. gemina, two A. calvescens, two A. gallica, and three A. sinapina isolates. This is the first report of complete IGS2 sequences from Armillaria spp. A species identification protocol involving species-specific primers and restriction fragment length polymorphism analysis was devised based on species-specific polymorphisms. The protocol successfully identified all 16 A. ostoyae, 11 A. gemina, three of three Armillaria mellea , 18 A. gallica, 14 of 15 A. sinapina (11/12 diploid and 3/3 haploid), and 14 of 21 A. calvescens (13/15 diploid and 1/6 haploid) isolates included in this study. To the best of our knowledge, this success rate has not been matched by other methods.