PCR-Based Test for Differentiating Varieties of <i>Gaeumannomyces graminis</i>, The Take-All Pathogens
Take-all is the most devastating root disease of wheat worldwide. The causal agent is Gaeumannomyces graminis (Sacc.) Arx & Olivier. Based on morphological characteristics and host ranges, three varieties of G. graminis have been recognized. G. graminis var. tritici Walker (Ggt) is the major causal agent of take-all of wheat and barley and the most economically important take-all pathogen. G. graminis var. avenae (Turner) Dennis (Gga) attack oats and causes take-all patch of turf grasses while G. graminis var. graminis (Ggg) is pathogenic on turf grasses but is non-pathogenic on wheat. Conventional diagnosis of take-all pathogens is based on field symptoms such as blackened roots, stunted growth, and white-heads and morphological characteristics such as hyphopodia type, size of perithecia, asci, and ascospores. These procedures are time-consuming, laborious, and often inconclusive. The objective of this study was to develop a rapid, simple, and specific method for differentiation of G. graminis varieties using PCR and molecular-based technology. Exploitation of genes associated with pathogenicity of G. graminis as markers for the test was proposed. Metabolic activities of G. graminis associated with pathogenesis were investigated, namely, the abilities to produce avenacinase and to oxidize manganese. Avenacinase, an avenacin detoxifying enzyme, was associated with Gga pathogenicity for oats but this enzyme is not important in Ggt pathogenicity for wheat. Manganese oxidation was also correlated with Ggt virulence. In this study, avenacinase-like genes were discovered in Ggt and Ggg and manganese oxidation was confirmed for Ggt, Gga, and Ggg. All isolates of Ggt except isolate ATCC 28230 were manganese oxidizers. Ggg and Gga isolates could oxidize manganese but their precipitation patterns were not as intense or closely correlated with mycelial growth as for Ggt. Pathogenicity assays on oats for Ggt, Gga, and Ggg isolates confirmed that Ggt isolates could not cause disease on oats aside from occasional slight root damage. Root weight was reduced for oat seedlings inoculated with Gga isolates. Comparison of partial sequences of avenacinase-like genes from Ggt and Ggg showed strong homology to that of Gga (94.8% identity to Ggt and 94.6% identity to Ggg). However, the Ggt gene was more closely related to that of Ggg (99.2% identity) than to Gga. DNA restriction endonuclease polymorphisms of the genes supported DNA sequencing information and revealed that there were variations within the genes among Ggt, Gga, and Ggg. Variety-specific electrophoretic patterns were obtained when the genes were digested with HaeIII. Ggt, Gga, and Ggg upstream (5') variety-specific primers and a downstream (3') universal primer were designed from the avenacinase and avenacinase-like DNA sequences. PCR amplification with Ggt-, Gga-, and Ggg-specific primers generated fragments of 870, 617, and 1,086 bp, respectively. Each 5'-specific primer showed high specificity for its own DNA template in mixed populations of DNA templates. The optimized PCR procedure was sensitive to DNA template concentration as low as 100 pg. Genomic DNA of sixteen Ggt isolates, seven Gga isolates, and five Ggg isolates were tested. Although all Ggt isolates were originally isolated from wheat, seven isolates produced Ggg-specific fragments. This result corresponded well with HaeIII DNA polymorphisms, pathogenicity assay, and manganese oxidizing ability. All but one Gga isolates produced the variety-specific fragment. Ggt- and Gga- specific products were generated from Gga isolate RB-W. Although Ggg-specific fragments were produced from all Ggg isolates, non-specific products were also observed from isolates that were not from wheat origin suggesting some genetic variations due to host ranges. Additionally, no non-specific amplification was obtained from any closely related fungi such as Gaeumannomyces cylindrosporus or Phialophora spp. The test developed in this study is the first test capable of identification of Ggt, Gga, and Ggg in a single PCR tube with a basic PCR protocol. The test is rapid and specific. Interpretation of results is simple and conclusive based on differences in size of each variety-specific fragment.