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Barley leaf stripe, caused by Pyrenophora graminea, is an essential systemic seed-borne disease in barley worldwide. Barley is a major cereal crop in the Qinghai–Tibet Plateau, and barley production has been threatened by leaf stripe in this region, particularly in organic farming regions. Detecting the pathogen in infected barley seeds is crucial for managing barley leaf stripe. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the pathogen based on primers designed based on the sequence of the pig 14 gene (GenBank: AJ277800) of P. graminea. The optimal concentrations of MgSO4, dNTPs, and enzymes in the LAMP reaction system were established as 10.0 mM, 1.0 mM, and 8 U in a 25 μL reaction volume, respectively. The established LAMP methods for detecting P. graminea were optimally performed at 63 °C for 70 min with high reliability. The minimum detection limit was 1 × 10−2 ng·μL−1 in the 25 μL reaction system. The specificity of LAMP for P. graminea was validated with eight fungal species. All DNA extracts from P. graminea-infected barley seeds with incubation, intact, and smashed treatments were applied in LAMP and confirmed to enable the detection of the pathogen. The LAMP assay in this study could facilitate the detection of P. graminea in barley seeds onsite, provide information for seed health certificates, and help decide on seed treatment in leaf stripe management. [ABSTRACT FROM AUTHOR]

[Objective] To provide a reference for detection of barley seeds and effective prevention and control of barley stripe, this study established a detection system for barley stripe based on loop-mediated isothermal amplification (LAMP) technology. [Method] In this study, five sets of specific primers were de-signed based on the ITS-rDNA sequences of P. graminea, and one set was screened to optimize reaction temperature based on six gradients (60, 61, 62, 63, 64 and 65 °C). Then, the LAMP system for efficient detection of the pathogen was established. Its specificity was tested based on LAMP amplification of DNA of genes from P. graminea, Ustilago hordei, Ustilago nuda, Dactylobotrys graminicola, Alternaria alternata and Fusarium graminearum. The sensitivity was analyzed by testing genes at levels of 310, 31, 3. 1, 0.31, 0.031 and 0.003 1 pg/µL. At last, it was used for testing and verifying seeds of five barley varieties. [Result] The established LAMP methods for detecting P. graminea performed optimally at 63 °C for 70 min with specific primers of set 2. Under the optimal reaction conditions, this method had good specificity for P. graminea with minimum detection limit of 0.31 pg/µL. The P. graminea-carrying rates of Dulihuang, Chaiqing 1, 1141, Beiqing 9 and Kunlun 15 seeds were 68.18%, 72.73%, 100%, 77.27% and 68.18%, respectively. [Conclusion] The established LAMP method can be used to detect the P. graminea in barley seeds within 70 min at 63 µC. [ABSTRACT FROM AUTHOR]