T. walkeri (TW) is a fungal species that shares morphological and molecular characteristics with several ryegrass-infecting smut fungi, including T. barclayana, T. inolens, and T. eragrostidis. These fungi are closely related to the smut fungus and primarily affect ryegrass and other grasses. The distribution of these fungi spans across multiple countries, such as the United States, Australia, Canada, New Zealand, Denmark, the Netherlands, Japan, and Spain. In recent years, an increasing number of grass seeds, including ryegrass, have been imported for purposes like pasture development, urban greening, and golf course construction. As a result, there is a growing risk of introducing T. walkeri into our country.
Distinguishing between ryegrass smut, turfgrass smut, and wheat smut (T. indica) and similar species relies heavily on both morphological traits and molecular biology techniques. Recent advances in molecular methods have significantly improved the accuracy and efficiency of identification. A review of these methods highlights the importance of DNA-based approaches in differentiating these closely related species.
In 2000, Frederick Reid from the U.S. Department of Agriculture's National Agricultural Service Center designed five new specific primers targeting mitochondrial DNA sequences of T. indica and T. horrida. Using these, three pairs of primers were developed to create a PCR detection method for wheat smut. A 212 bp amplicon was used as a target for fluorescent 5' nuclease assays, enabling real-time TaqMan PCR to distinguish between wheat smut and ryegrass smut effectively.
In the same year, McDonald from Canada’s Ministry of Agriculture, Agri-Food and Rural Development used repeat sequence PCR with primers such as BOX, ERIC, and REP to generate genetic fingerprints of various Tilletia species, including T. walkeri, T. controversa, T. laevis, and others. Based on sequence similarity, the species were grouped into three clusters, with T. indica and T. walkeri falling into the first group.
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 based on ITS sequences revealed a close evolutionary relationship between T. indica and T. walkeri.
Also in 2001, Cheng Yinghui and Zhang Guiming tested 14 strains of various Trichoderma and other fungi using PCR. They designed specific primers targeting mitochondrial DNA and ITS regions to distinguish wheat T. indica from ryegrass smut and related species. This method proved effective in identifying smut fungi in imported seed samples.
In 2003, Yi Jianping from the Shanghai Entry-Exit Inspection and Quarantine Bureau developed nested PCR methods using common and specific primers for T. indica and T. walkeri. This technique achieved a sensitivity of one teliospore and reduced detection time to 8 hours, improving efficiency significantly.
In 2004, Wu Cuiping and colleagues from Jiangsu Inspection and Quarantine Bureau detected T. walkeri in imported ryegrass seeds using nested PCR. The method had a sensitivity of three teliospores and reduced detection time to six hours, offering a sixfold increase in efficiency compared to traditional methods.
By 2005, Yi Jianping and Liu Suping from Shanghai and South China Agricultural University developed a real-time fluorescence PCR method using universal and specific primers targeting ITS sequences. This allowed for rapid detection of T. indica and T. walkeri with a sensitivity of one teliospore and a total detection time of one day.
In 2006, Zhang Guiming and Yi Jianping optimized TaqMan MGB real-time PCR for detecting T. indica and T. communis. They developed single- and dual-plex methods that could detect both fungi simultaneously in a 5 µL reaction volume, enhancing detection speed and accuracy.
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