Downy mildew is a major disease of boysenberries in New Zealand, caused by Peronospora sparsa. Most boysenberry plant material, including tissue culture propagated plants are systemically infected and this pathogen also presents as a latent infection. The current nested PCR method to detect latent infection of P. sparsa in asymptomatic boysenberry plants is time consuming as it employs two separate PCR, with potential contamination producing false positive and/or false negative results. To overcome these issues a one step nested PCR method was developed. The method was optimised for primer concentrations, PCR cycle number and DNA concentration. DNA was extracted using a CTAB method. The one step nested PCR method could detect latent infection of P. sparsa in both dormant and active plant growth at 0.4 pg genomic DNA. The most reliable detection was achieved from crown or root tissues. For surety of the infection status, replicate plant tissues should be assessed by PCR as inconsistency between the one step nested PCR and fluorescence microscopy indicated that P. sparsa colonisation is discontinuous through the plant. This method can be recommended for screening P. sparsa latent infection in boysenberry mother plants and daughter plants in nurseries, due to high sensitivity, improved throughput, low cost, and low contamination risk.

霜霉病是新西兰波森莓的主要病害，由稀疏霜霉病菌引起。大多数波森莓植物材料，包括组织培养繁殖的植物，都被系统感染，这种病原体也表现为潜伏感染。当前用于检测无症状波森莓植物中P. sparsa潜伏感染的巢式 PCR 方法非常耗时，因为它采用了两个独立的 PCR，潜在的污染会产生假阳性和/或假阴性结果。为了克服这些问题，开发了一种单步嵌套 PCR 方法。该方法针对引物浓度、PCR 循环数和 DNA 浓度进行了优化。使用 CTAB 方法提取 DNA。一步巢式PCR方法可检测P. sparsa的潜伏感染在 0.4 pg 基因组 DNA 的休眠和活跃植物生长中。最可靠的检测是从冠或根组织中实现的。为确保感染状态，应通过 PCR 评估复制植物组织，因为一步嵌套式 PCR 和荧光显微镜之间的不一致表明P. sparsa定植在整个植物中是不连续的。由于灵敏度高、通量提高、成本低和污染风险低，该方法可推荐用于筛选苗圃中波森莓母株和子株中的P. sparsa潜伏感染。