Science and Technology, Plant Protection and Quarantine, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Beltsville, MD, U.S.A.
Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.
Phytopathology. 2020 Jun;110(6):1174-1179. doi: 10.1094/PHYTO-12-18-0453-R. Epub 2020 Jan 10.
pv. (syn. pv. ) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as pv. in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of pv. -like strains of pv. in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of pv. strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 pv. isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the pv. isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of pv. isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the pv. isolates that cause more severe symptoms on corn.
pv. （同义词：pv. ）最初被鉴定为南非玉米细菌性叶斑病的病原体。该血清型可引起甘蔗和玉米发病，但该血清型的某些菌株在玉米上的致病严重度更高。这些菌株的亚群在 20 世纪 90 年代初被重新分类为 pv. ，并被发现与甘蔗分离株相比具有略微不同的生化和遗传特性。自 2010 年以来，美国和阿根廷都出现了 pv. 样菌株。我们对美国分离株进行了全基因组测序以确认其身份。通过比较基因组学，我们随后开发了用于检测该特定 pv. 菌株亚群的 TaqMan qPCR 和环介导等温扩增（LAMP）检测方法。qPCR 4909 检测方法针对 27 种黄单胞菌（多样性代表）、32 种玉米叶片的 DNA 提取物（经证实为细菌阳性或阴性）、来自美国和阿根廷的 41 种玉米 pv. 分离株以及 31 种与玉米、甘蔗或高粱相关的其他细菌进行了测试。在所有情况下，该检测方法均显示出对引起玉米更严重疾病的 pv. 分离株具有特异性。然后，我们使用 LAMP 166 检测方法对 27 种黄单胞菌和 32 种玉米叶片 DNA 样本进行了测试，我们发现该检测方法也对该 pv. 分离株具有特异性。我们还开发了一种在 DNA 提取前使用吖啶橙进行活/死细胞区分的方案，用于分析使用这些检测方法的种子冲洗物。这两种检测方法可用于诊断医生和研究人员，以特异性鉴定在玉米上引起更严重症状的 pv. 分离株。
