T. walkeri is a fungal species closely related to several ryegrass smuts, including T. barclayana, T. inolens, and T. eragrostidis. These fungi, which resemble the smut fungus, primarily affect ryegrass and other grasses. The bacteria are found in various countries such as the United States, Australia, Canada, New Zealand, Denmark, the Netherlands, Japan, and Spain. With the increasing importation of grass seeds for pastures, urban greening, and golf courses, there is a growing risk of T. walkeri entering our country.
Distinguishing between ryegrass, turfgrass, and wheat smut species like T. indica requires both morphological analysis and molecular techniques. In recent years, molecular biology has become a key tool in identifying these similar species. A brief review of molecular methods used in the identification of ryegrass smuts highlights their importance in accurate detection and differentiation.
In 2000, Frederick Reid from the U.S. Department of Agriculture’s National Agricultural Service Center designed five new specific primers targeting mitochondrial DNA of T. indica and T. horrida. These primers were used to develop a PCR method for detecting wheat T. indica, with a 212 bp amplicon serving as a target for fluorescent 5’ nuclease assays. This method enabled real-time TaqMan PCR to distinguish between wheat smut and ryegrass smut effectively.
In the same year, McDonald from Canada's Ministry of Agriculture used repeat sequence PCR (BOX, ERIC, and REP) to amplify DNA from various Tilletia species. Genetic fingerprints were analyzed, and based on sequence similarity, species were grouped into three categories. T. indica and T. walkeri fell into the first group, while others formed separate clusters.
In 2001, Laurene Levy from the USDA Systematic Botany and Mycology Laboratory applied PCR-RFLP to differentiate T. indica and T. walkeri. By amplifying and sequencing the ITS region of ribosomal DNA, a unique restriction site was identified in the ITS1 region of T. walkeri. Phylogenetic analysis showed a close relationship between T. indica and T. walkeri.
Also in 2001, Cheng Yinghui and Zhang Guiming used PCR to test 14 strains of wheat Tilletia and related species. Specific primers were designed based on mitochondrial DNA and ITS regions, allowing for the differentiation of wheat T. indica from ryegrass smut and similar species.
In 2003, Yi Jianping from Shanghai Entry-Exit Inspection and Quarantine Bureau developed nested PCR methods using common and specific primers for T. indica and T. walkeri. The method achieved a sensitivity of one teliospore and reduced detection time to 8 hours.
In 2004, Wu Cuiping from Jiangsu Entry-Exit Inspection and Quarantine Bureau detected T. walkeri in imported ryegrass seeds. Using nested PCR, they confirmed the presence of T. walkeri with a sensitivity of three teliospores and a detection time of about six hours, significantly improving efficiency.
In 2005, Yi Jianping and Liu Suping designed universal and specific primers based on ITS sequences for real-time fluorescence PCR detection of T. indica and T. walkeri. The method could detect as few as one teliospore within a day.
In 2006, Zhang Guiming and Yi Jianping optimized a real-time PCR method using TaqMan MGB probes for detecting T. indica and T. walkeri. They developed both single-plex and dual-plex PCR methods, enabling efficient and accurate detection of these smut species in a single reaction.
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