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Differentiating ryegrass, turfgrass and wheat T. indica TM, and other similar species, relying on morphological characteristics, rely mainly on molecular biology methods. A brief review of molecular biology in the identification of ryegrass turfgrass was made.
In 2000, Frederick Reid of the National Agricultural Service Center of the U.S. Department of Agriculture designed five pairs of specific new primers and amplifications for wheat T. indica according to the mitochondrial DNA of T. indica and T. horrida. Three new pairs of specific primers were used to establish the new PCR detection method for wheat T. indica. At the same time, 212 bp amplicon was established as the target of fluorescent 5 nuclease. TaqMan real-time fluorescence PCR was used to detect the discrimination between wheat smut and ryegrass.
In 2000, McDonald of the Center for Phytosanitary Pests of the Canadian Ministry of Agriculture, Agri-Food and Agriculture used the repeat sequence PCR technique to amplify T. indica and T. indica by using Tilletia's DNA primers (BOX, ERIC and REP). Genetic fingerprints of walkeri, T.controversa, T.laevis, T.tritici, T.goloskokovii, T.barclayana, and T.fusca, using computer-analyzed sequences, based on the similarity of between 35% and 40% of species differences The subspecies are divided into 3 groups, the first group including T. indica and T. walkeri; the second group including T. fusca, T. controversa, T. laevis, T. tritic, and T. goloskokovii; the third group includes T.barclayana.
In 2001, Levy Laurene, the United States Department of Agriculture's System Botany and Mycology Laboratory, used the PCR-RFLP method to distinguish between T. indica and T. walkeri. The ITS region of the ribosomal DNA repeat units of T. indica, T. walkeri, T. horrida, and other S. obliquus strains was amplified and sequenced. A unique restriction site for the identification of T. walkeri was identified in the ITS1 region of T.walkeri. A developmental tree analysis based on ITS sequences shows that there is a close relationship between T.indica and T.walkeri.
In 2001, Cheng Yinghui of South China Agricultural University and Zhang Guiming of Shenzhen Entry-Exit Inspection and Quarantine Bureau applied the PCR method to wheat Tilletia indica and its related species or related species, including Ryegrass truncatum, Pennisetum sp. A total of 14 strains of ten Trichoderma spp., such as Oryza sativa, were tested and examined. According to the sequence of mitochondrial DNA, specific primers for amplifying wheat T. indica and amplified primers for T. striolata were designed, and the amplified Tb was designed based on the ribosomal transcribed region (ITS) DNA fragment. The primers of the fungus genus fungi can be used to distinguish the wheat indica sclerotia from the ryegrass smut black and other similar species or related species by PCR.
In 2003, Shanghai Entry-Exit Inspection and Quarantine Bureau Yi Jianping designed Tilletia indica and Tilletia walkeri common primers H4/H7 and H11/H12 in combination with T.indica specificity. Primers Tin3/Tin4 and T.walkeri specific primers Tin11/Tin4 were used to establish nested-PCR detection methods for the detection of T. indica and T. walkeri teliospores. The sensitivity of the test was up to 1 teliospores, and the detection time was shortened to 8 h.
In 2004, the Jiangsu Entry-Exit Inspection and Quarantine Bureau Wu Cuiping and others found a teliosia similar to Tilletia indica from the seeds of imported ryegrass. Specific primers Tin3/Tin4 and T11 walker specific primers Tin11/Tin4 and common primers H4/H7 and H11/H12 were selected for the samples from wheat T. indica. The teliospore was subjected to nested PCR amplification and the samples were detected to contain T. walkeri. The detection sensitivity was 3 teliospores and the detection time was about 6 h. Compared with the conventional detection method, the working efficiency is increased at least 6 times.
In 2005, Yi Jianping from Shanghai Entry-Exit Inspection and Quarantine Bureau and Liu Suping from South China Agricultural University designed two pairs of universal primers and two specificities based on the ITS sequences of Tilletia indica and T. walkeri ribosomes of wheat. A real-time fluorescence PCR detection method was established for the probes of Tilletia indica and T. walkeri. The detection sensitivity was 1 teliospores, and the whole detection process was shortened to 1 day.
In 2006, Shenzhen Entry-Exit Inspection and Quarantine Bureau Zhang Guiming and Shanghai Entry-Exit Inspection and Quarantine Bureau Yi Jianping took 9 strains of wheat Indica sclerotia and 5 strains of ryegrass smut smut and their similar species or related species: Rice Prussophila, Pennisetum japonicum, Grifola versicolor, Setaria virescens, Smmat sphaerotheca, Falconia viridis, Triticum reticulata, and Wheat dwarf smut Nine strains of 22 strains were selected as subjects. TaqMan MGB real-time fluorescent PCR primers and probes for the detection of T. indica and T. communis were designed to optimize the reaction conditions. A real-time fluorescence single-plex PCR and real-time fluorescence dual PCR detection method was developed for the detection of wheat indica smut and ryegrass smut (strains and spores), respectively. The real-time fluorescence dual PCR detection method was implemented in the same PCR. Only 5 μL of reaction system was used in the tube and PCR was performed to specifically detect wheat T. indica or ryegrass smut.
Molecular Biology Detection Techniques of Rye Black Grass
T. walkeri (TW) is a morphological and molecular biological feature of the ryegrass, T. walkeri, T. barclayana, T. inolens, and T. eragrostidis. Fungi that are very similar to the smut fungus mainly damage the ryegrass and ryegrass. So far. The bacteria are distributed in the United States, Australia, Canada, New Zealand, Denmark, the Netherlands, Japan, Spain, and other countries. In recent years, a large number of other grass seeds such as ryegrass have been imported from abroad for pasture, urban afforestation or golf course construction. Therefore, TW is also There is a risk of passing into our country.