Abstract The causative agent of crayfish plague, Aphanomyces astaci (Saprolegniales, Oomycota), is one of the 100 world’s worst invasive alien species and represents a major threat to freshwater crayfish species worldwide. A better understanding of the biology and epidemiology of A. astaci relies on the application of efficient tools to detect the pathogen and assess its genetic diversity. In this study, we validated the specificity of two recently developed PCR-based approaches used to detect A. astaci groups. The first relies on the analysis of mitochondrial ribosomal rnnS (small) and rnnL (large) subunit sequences and the second, of sequences obtained by using genotype-specific primers designed from A. astaci whole genome sequencing. For this purpose, we tested the specificity against 76 selected isolates, including other oomycete species and the recently described species Aphanomyces fennicus, which, when used in nrITS-based specific tests for A. astaci, is known to result in a false positive. Under both approaches, we were able to efficiently and accurately identify A. astaci and its genetic groups in both pure cultures and clinical samples. We report that sequence analysis of the rnnS region alone is sufficient for the identification of A. astaci and a partial characterization of haplogroups. In contrast, the rnnL region alone is not sufficiently informative for A. astaci identification as other oomycete species present sequences identical to those of A. astaci.

中文翻译：

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Aphanomyces astaci mtDNA：深入了解病原体的分化及其与其他密切相关的卵菌的遗传多样性

摘要 小龙虾瘟疫的病原体 Aphanomyces astaci (Saprolegniales, Oomycota) 是世界上 100 种最严重的外来入侵物种之一，对全世界淡水小龙虾物种构成重大威胁。对 A. astaci 生物学和流行病学的更好理解依赖于有效工具的应用来检测病原体并评估其遗传多样性。在这项研究中，我们验证了两种最近开发的用于检测 A. astaci 组的基于 PCR 的方法的特异性。第一个依赖于线粒体核糖体 rnnS（小）和 rnnL（大）亚基序列的分析，第二个依赖于使用从 A. astaci 全基因组测序设计的基因型特异性引物获得的序列。为此，我们测试了对 76 个选定分离株的特异性，包括其他卵菌物种和最近描述的物种 Aphanomyces fennicus，当在基于 nrITS 的 A. astaci 特定测试中使用时，已知会导致假阳性。在这两种方法下，我们都能够在纯培养物和临床样本中高效准确地识别 A. astaci 及其遗传组。我们报告说，仅对 rnnS 区域进行序列分析就足以鉴定 A. astaci 和单倍群的部分特征。相比之下，单独的 rnnL 区域对于 A. astaci 鉴定的信息不足，因为其他卵菌物种存在与 A. astaci 相同的序列。我们能够在纯培养物和临床样本中高效准确地识别 A. astaci 及其遗传群。我们报告说，仅对 rnnS 区域进行序列分析就足以鉴定 A. astaci 和单倍群的部分特征。相比之下，单独的 rnnL 区域对于 A. astaci 鉴定的信息不足，因为其他卵菌物种存在与 A. astaci 相同的序列。我们能够在纯培养物和临床样本中高效准确地识别 A. astaci 及其遗传群。我们报告说，仅对 rnnS 区域进行序列分析就足以鉴定 A. astaci 和单倍群的部分特征。相比之下，单独的 rnnL 区域对于 A. astaci 鉴定的信息不足，因为其他卵菌物种存在与 A. astaci 相同的序列。