Identification of Armillaria isolates from Bhutan based on DNA sequence comparisons

Armillaria root rot is a serious disease in fir and mixed conifer forests of Bhutan, Eastern Himalayas. The species causing this disease have, however, never been identified. The aim of this study was to identify field isolates collected at four localities in Bhutan. Identification was based on RFLP analysis of the IGS-1 region, comparisons of ITS and IGS-1 sequence data with those available on GenBank, cladistic analyses and sexual compatibility studies. Isolates were found to reside in two distinct RFLP groups. RFLP group 1 isolates from Pinus wallichiana at Yusipang had RFLP profiles and IGS-1 sequences similar to those of Armillaria mellea ssp. nipponica. Although ITS sequence data are not available for A. mellea ssp. nipponica, sequences from this DNA region were most similar to the closely related A. mellea from Asia. The RFLP profile and IGS-1 sequences for RFLP group 2 isolates from Abies densa at Changaphug, Tsuga dumosa at Chimithanka as well as Picea spinulosa and T. dumosa in the Phobjikha valley were similar to those published for Armillaria borealis, Armillaria cepistipes, Armillaria gemina and Armillaria ostoyae. Distance analysis based on IGS-1 and ITS sequence data indicated that these isolates are closely related to A. cepistipes, Armillaria gallica and Armillaria sinapina. The isolates were, however, sexually incompatible with tester strains of A. cepistipes, A. gallica and A. sinapina. Although closely related to these species, they appear to represent a distinct taxon that will be referred to as Bhutanese phylogenetic species I (BPS I) until basidiocarps are found and the species can be described.