Background

Phytophthora pluvialis was first described in 2013 and is the causal agent of red needle cast (RNC) in Pinus radiata as well as infection in Douglas fir (Pseudotsuga menziesii). A species-specific PCR is necessary for detection of this pathogen and diagnosis of RNC.

Objective

To design and validate a species-specific molecular assay for P. pluvialis using isolates from infected pine needles.

Methods

Species-specific PCR primers were generated from the ras-related GTP-binding protein 1 gene (ypt1) gene sequence, concentrating on DNA regions unique to P. pluvialis, and real-time and quantitative polymerase chain reaction (qPCR) were used to detect P. pluvialis from both artificially inoculated and naturally infected samples.

Results

The species-specific PCR assay was generated following P. pluvialis DNA sequence analysis. In vitro tests of the specificity of the probe-based, quantitative, polymerase chain reaction (qPCR) assay showed that no amplification was observed with other Phytophthora species including other closely-related clade 3 species, or with fungal species associated with pine or with pine DNA. The limit of detection of the qPCR assay was 2 pg/μl. When the qPCR assay was used to detect P. pluvialis in artificially-inoculated and naturally infected P. radiata needles, a PCR product was detected in all inoculated samples; the mean concentration ranges of P. pluvialis DNA in the inoculated and naturally infected samples tested were 5.9–124.5 pg/μl and 8.1–340.2 pg/μl, respectively. The assays described herein were used with serological diagnostic strips, providing the ability to identify to species level.

Conclusions

The assay described herein detects P. pluvialis with high specificity and sensitivity from a range of DNA samples, including those extracted from infected plant material and serological diagnostic strips. The ability to detect and identify P. pluvialis, from infected tissues directly, provides value and practicality to diagnostics, biosecurity and research.

中文翻译：

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辐射松红针管型病原体雨生疫霉的分子检测

背景

雨生疫霉于 2013 年首次被描述，是辐射松红针管型 (RNC)以及花旗松( Pseudotsuga menziesii ) 感染的病原体。物种特异性 PCR 是检测这种病原体和诊断 RNC 所必需的。

客观的

使用从受感染的松针中分离出来的雨生杨梅的物种特异性分子分析方法进行设计和验证。

方法

物种特异性 PCR 引物由 ras 相关的 GTP 结合蛋白 1 基因 (y pt1 ) 基因序列产生，集中在P. pluvialis特有的 DNA 区域，并使用实时和定量聚合酶链反应 (qPCR)从人工接种和自然感染的样本中检测P. pluvialis 。

结果

在P. pluvialis DNA 序列分析之后产生了物种特异性 PCR 测定。对基于探针的定量聚合酶链反应 (qPCR) 测定的特异性的体外测试表明，在其他疫霉属物种（包括其他密切相关的进化枝 3 物种）或与松树或与松树相关的真菌物种中未观察到扩增脱氧核糖核酸。qPCR 测定的检测限为 2 pg/μl。当使用 qPCR 法检测人工接种和自然感染的P. radiata针中的P. pluvialis时，在所有接种样品中均检测到 PCR 产物；P. pluvialis的平均浓度范围测试的接种和自然感染样本中的 DNA 分别为 5.9-124.5 pg/μl 和 8.1-340.2 pg/μl。本文所述的测定与血清学诊断条一起使用，提供了鉴定到物种水平的能力。

结论

本文所述的测定从一系列 DNA 样品中检测出具有高特异性和敏感性的雨生杨梅，包括从受感染的植物材料和血清学诊断条中提取的样品。直接从受感染的组织中检测和识别雨生杨梅的能力为诊断、生物安全和研究提供了价值和实用性。