Identification of Armillaria nabsnona in Gastrodia Tubers

Abstract

Armillaria species (Basidiomycota, Agaricales, Tricholomataceae) are well known to be the cause of root rot and death to woody plants in boreal, temperate and tropical regions worldwide.1) Generally this species has a broad variety of hosts, presenting pathogenic or symbiotic properties depending on the available substrate. 1,2)Armillaria has been identified as an associate in certain achlorophyllous taxa of Orchidaceae including Gastrodia elata BL. 3,4) and Galeola septentrionalis REICHB. 5) Although many reports refer to Armillaria mellea as the main fungus symbiont of G. elata, 3,6) 4)In eastern traditional medicine, the tubers of this orchid have been used for the treatment of many kinds of diseases including headache, rheumatism, seizure, dizziness and circulation problems. 7,8) G. elata is considered as a difficult species to cultivate due to the need for different kinds of fungi colonization to achieve its full development.6) To increase the knowledge of the cultivation process and understand its relationship with the Armillaria, it is important to first identify which Armillaria species is involved in the association.In past decades, the identification of Armillaria spp. was based on mating tests and morphological characteristics. 2,9,10) However, those methods are very limited due to the great number of biological species and similar morphological characteristics among them.11,12) Furthermore, mating pairings require fresh haploid cultures that sometimes are not available and a long period is required to obtain results. 13,14) Methods based on molecular techniques are nowadays routinely used and provided a new perspective for phylogenetic and taxonomic studies.12,15-17) PCR-RFLP analysis profiles of intergenic spacer 1 (IGS-1) have been successfully applied to the Armillaria species identification. 12,13,18,19) Sequencing of the internal transcribed spacer (ITS) and intergenic spacer 1 (IGS-1) regions 14,20,21) has also been used for identification. The ITS and IGS sequences available in the GenBank and the development of efficient software have provided a valuable tool for differentiation as well as for the detection of polymorphism and heterogeneity among species. The ITS and IGS-1 of the ribosomal DNA are known to be highly polymorphic enabling the accurate species differentiation of the Armillaria.11) In some cases, however, ITS1 region sequences were proven to be nearly uniform among the Armillaria of northern hemisphere species. 11)The aim of this study was to apply molecular biological tools to identify which Armillaria species are involved in the association with G. elata. The ITS and IGS-1 regions of rDNA were amplified and sequenced for further comparison with other Armillaria species. PCR-RFLP of the IGS-1 region was performed with three different endonucleases. Morphological characteristics and compatibility tests were also used in the analysis. MATERIALS AND METHODS Fungal Isolation and CultivationFungal samples were harvested in July 2005, found in different root parts of G. elata, which ...

Secrets of the subterranean pathosystem of Armillaria

“…The orchid populations are endangered in China and Japan, and cultivation of their corms, which are important in eastern traditional medicine, is hindered by a poor understanding of the relationship between Armillaria and the orchid. The interaction is poorly characterized with respect to Armillaria , aside from species' identity (Cha and Igarashi, 1995; Sekizaki et al ., 2008), but has been better characterized with respect to the orchid over the past 20 years. In fact, the first attempts to examine the molecular basis of an Armillaria –host interaction were based on the orchid.…”

Phylogenetic relationships among biological species of Armillaria from China

“…Phylogenetic methods utilising DNA sequence data have been widely employed to elucidate the identity of field isolates of Armillaria (Coetzee et al 2003a(Coetzee et al , b, 2005bKeča et al 2006;Sekizaki et al 2008;Kikuchi and Yamaji 2010;Elías-Román et al 2013) and to resolve the phylogenetic relationships of Armillaria species from various parts of the world (Maphosa et al 2006;Coetzee et al 2011). For phylogenetic inference, the internally transcribed spacer regions (ITS) and intergenic spacer region one (IGS-1) have been useful in studies focused on the relationships of taxa from Africa (Coetzee et al 2005a), South America (Pildain et al 2009), Australasia (Coetzee et al 2001), North America (Anderson and Stasovski 1992), Europe (Chillali et al 1998) and Asia (Terashima et al 1998;Coetzee et al 2000).…”

Populations of Armillaria species in pine plantations in west-central Poland

“…the internal transcribed spacer (ITS) and intergenic spacer (IGS) (Anderson and Stasovski 1992;Harrington and Wingfield 1995;Banik et al 1996;Terashima et al 1998a,b;Pérez-Sierra et al 1999;Fukuda et al 2003;Lochman et al 2004a;Matsushita and Suzuki 2005;Schnabel et al 2005;Keča et al 2006;Sekizaki et al 2008). More recently the translation elongation factor-1 alpha (EF-1 alpha) gene, which encodes an essential part of the protein translation machinery, was shown to have high phylogenetic utility and has been used in studies on fungi.…”