甘薯丛枝病植原体TaqMan探针qPCR检测方法的建立与应用OA

Phytoplasma-induced sweet potato infection is a significant disease that impacts sweet potato(Ipomoea batatas)production in China,particularly in southeastern coastal regions.This study developed specific primers and TaqMan probes based on the secY sequence of the sweet potato witches'-broom phytoplasma transporter protein gene for sensitive and rapid detection of the sweet potato witches'-broom phytoplasma and its presence in propagation media.A qPCR detection method was established using TaqMan,and was applied to test collected sweet potato samples,insect vectors,and various sweet potato tissues infected with phytoplasma.The results demonstrated that this TaqMan qPCR detection method could detect sweet potato witches'-broom phytoplasma sensitively,rapidly,and accurately,with a detection limit of 1.71×10¹ copies/μL.It could also specifically distinguish between Melia azedarach witches'-broom phytoplasma(16SrⅠ-B),Areca catechu yellowing phytoplasma(16SrⅠ-B),Catharanthus roseus periwinkle little leaf phytoplasma(16SrⅠ-A),the causal agent of sweet potato bacterial wilt(Ralstonia solanacearum),and black rot pathogen of sweet potato(Dickeya dadantii).This method was used to quantitatively detect the pathogen content in different tissues of sweet potatoes infected with phytoplasma,revealing a pathogen content range of 3.98×10⁴～6.03×105 copies/μL,and identifying the sweet potato stems and the stem base as having the highest phytoplasma content.The insect vectors in the field were also quantitatively tested using this detection method,confirming Orosius orientalis as the transmission medium of sweet potato witches'-broom phytoplasma.In conclusion,the qPCR detection method developed in this study demonstrated good sensitivity,specificity,and repeatability.It not only enables rapid detection of sweet potato witches'-broom phytoplasma,but also provides a reference for grading the disease severity of sweet potato witches'-broom disease according to pathogen quantity.