A novel real-time PCR assay based on the TaqMan probe was developed for the detection of Gnomoniopsis castaneae, causal agent of brown rot of chestnut kernels, and responsible for leaf necrosis, shoot blight and bark canker. A part of the pathogen life cycle is endophytic, colonizing all tissues of chestnut and additional hosts, which is suspected to play a key role in its epidemiology. Thus, a molecular tool for sensitive detection and quantification of G. castaneae in symptomatic and asymptomatic host tissues is urgently required to better understand G. castaneae ecology, biology and epidemiology. Primers and a species-specific probe for G. castaneae were designed based on the sequence of the single-copy elongation factor 1 alpha (EF1α) gene. The amplification efficiency of target DNA was 105.3% and the limit of detection of the assay was calculated at approximately 40 fg of pure fungal DNA. The pathogen was consistently detected in artificial mixtures of plant and pathogen DNAs with the same Limit of Detection (LOD) as pure fungal DNA. In naturally infected samples, the assay rapidly revealed the presence of the pathogen in all symptomatic specimens, as well as in asymptomatic tissues. Notably, a significant relationship between the results of a metagenomic HTS analysis and the qPCR assay on DNAs extracted from bulk fruit was found. This molecular tool will be of substantial aid in detecting and quantifying G. castaneae, even in the endophytic state, and in different host tissues.

中文翻译：

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开发用于检测和定量板栗组织中 Gnomoniopsis castaneae 的 TaqMan qPCR 检测方法

开发了一种基于 TaqMan 探针的新型实时 PCR 检测方法，用于检测板栗褐腐病的病原体 Gnomoniopsis castaneae，并负责叶片坏死、茎枯病和树皮溃疡病。病原体生命周期的一部分是内生的，定植于栗子的所有组织和其他宿主，这被怀疑在其流行病学中起关键作用。因此，一种用于灵敏检测和定量G的分子工具。 castaneae在有症状和无症状的宿主组织迫切需要更好地理解ģ。 栗科生态学、生物学和流行病学。G 的引物和物种特异性探针。 栗科基于单拷贝延伸因子 1 α (EF1α) 基因的序列设计。目标 DNA 的扩增效率为 105.3%，检测限计算为大约 40 fg 的纯真菌 DNA。在植物和病原体 DNA 的人工混合物中始终检测到病原体，其检测限 (LOD) 与纯真菌 DNA 相同。在自然感染的样本中，该检测迅速揭示了所有有症状样本以及无症状组织中都存在病原体。值得注意的是，宏基因组 HTS 分析的结果与从散装水果中提取的 DNA 的 qPCR 分析结果之间存在显着关系。这种分子工具将大大有助于检测和量化G。 栗科，甚至在内生状态下，以及在不同的宿主组织中。