Differentiation of Gaeumannomyces graminis from other turfgrass fungi by amplification with primers from ribosomal internal transcribed spacers

A region comprising the 5.8S RNA gene and internal transcribed spacers 1 and 2 of the take-all patch fungus, Gaeumannomyces graminis var. avenae, was cloned and sequenced using primers from the flanking 17S and 26S ribosomal RNA genes. The sequenced region had 99% similarity between the two G. graminis isolates, and from 70% to 80% similarity between these two isolates and several other species of fungi. From the sequence, oligonucleotide primers were selected which permitted specific amplification of DNA from G. graminis vars. avenae and graminis using the polymerase chain reaction (PCR). The assay could detect DNA of G. graminis strains obtained from a wide variety of hosts but did not amplify DNA from many other fungi including the important turfgrass root pathogens Magnaporthe poae and Leptosphaeria korrae. The primers also did not amplify DNA from G. graminis var. tritici, M. rhizophila or Phialophora graminicola. The PCR-based assay shows promise as a diagnostic tool for the take-all pathogen in turfgrass pathology. INTRODUCTION Take-all disease, caused by Gaeumannomyces graminis, is a widespread root and crown disease of many cereals and grasses. Three varieties of G. graminis have been differentiated based on pathogenicity and the morphology of the perithecia, ascospores and appressorium-like adhesive cells called hyphopodia (Walker, 1981). Variety avenae is the main causal agent of take-all of oats and bentgrasses; var. graminis attacks rice, St. Augustinegrass and bermudagrass; and var. tritici infects wheat and barley. The anamorph of G. graminis is Phialophora; however, the taxonomy of Phialophora remains unclear and the genus includes nonpathogenic species (Walker, 1981; Ward & Gray, 1992). For turfgrass pathology, G. graminis var. avenae is particularly important as a severe disease of bentgrass (Agrostis spp.) grown under intensive culture in cool temperate regions (Smiley et al., 1992). Accurate diagnosis of turfgrass diseases, such as take-all patch, is complicated by several factors such as nondistinctive symptoms and symptom variability. A ring-shaped pattern of dead grass, which is characteristic of take-all patch, is also commonly observed for several other patch diseases such as necrotic ring spot, summer patch, fusarium patch, pythium blight, rhizoctonia blight, and sclerotium blight (Couch, 1985). Another complicating factor in the identification of take-all patch is the presence of saprophytic Phialophora spp. in the turfgrass rhizosphere. The occurrence of dark runner hyphae on roots is commonly used for preliminary diagnosis of take-all patch, necrotic ring spot and summer patch diseases of turfgrass; however, saprophytic Phialophora spp. and other fungi will also produce dark runner hyphae (Smiley et al., 1992). For practical diagnosis, host specificity can be used to distinguish turfgrass ring-patch diseases (T. Hsiang, unpublished); however, this is not entirely reliable because G. graminis, Leptosphaeria korrae (causing necrotic ring spot), and Magnaporthe poae (causing summer patch) have all been isolated from species of Poa and Agrostis, which are the major cool-season turfgrasses grown under intensive culture (Smith et al., 1989). Because of the difficulties in diagnosing various take-all diseases, molecular techniques have been applied to the identification of G. graminis. A DNA hybridization probe and subsequently a polymerase chain reaction (PCR)-based assay were developed from a mitochondrial DNA fragment of G. graminis var. tritici (Henson, 1989; Schesser et al., 1991; Elliott et al., 1993; Henson et al., 1993). Although these assays were relatively specific to G. graminis, the hybridization probe showed homology to other Phialophora spp. as well as Neurospora crassa, and the PCR-based assay resulted in amplification of DNA from Phialophora, Magnaporthe and other species of Gaeumannomyces even when nested primers were used (Henson, 1989; Henson, 1992).