Development of Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of <i>Pyrenophora graminea</i> in Barley Seeds
Zhangwei Hu,
Liyifan Chen,
Chunmei Du,
Yaoxia Liu,
Jiahui Yan,
Qingyun Guo,
Qiang Yao
Affiliations
Zhangwei Hu
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
Liyifan Chen
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
Chunmei Du
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
Yaoxia Liu
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
Jiahui Yan
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
Qingyun Guo
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
Qiang Yao
Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest in Xining, Ministry of Agriculture and Rural Affairs, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
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.
