Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm³ volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates.

中文翻译：

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哈里斯单核打孔或激光显微切割收集的苹果再植病感染根中的油桃科分子鉴定

苹果再植病（ARD）对全球苹果植物的生长和产量产生负面影响。属于油桃科的真菌通常是从生长在补种土壤中的根中分离出来的，因此被提出作为该病复合体的一种生物原因。对受ARD影响的根的显微分析显示与真菌感染部位相关的特征性症状。在这里，这种组织部位的两种提取方法被应用于直接识别未知的真菌，该真菌在患病的根皮层细胞中形成典型的菜花状结构。冲压约0.5 mm ^3的小组织样本Harris Uni-Core的容积测量是一种快速简便的方法来收获有症状的物质。其次，建立了针对苹果根的激光显微解剖（LMD）协议。该技术允许从薄的冷冻切片中提取确定的细胞或组织级分。组织收获后，通过两个基因片段的PCR扩增和Sanger测序鉴定真菌。对于哈里斯（Harris）样品，将Chelex用于DNA稳定化，而将LMD样品直接提交PCR。在哈里斯（Harris）样品中，主要鉴定到的是油桃属植物（Dactylonectria torresensis），鲁棒伊兰氏菌（Ilyonectriarobusta）和皱皮芸香（Rugonectria rugulosa）。除了这些，在LMD样品中Cylindrocladiella sp.。和检出了欧洲鸢尾。因此，细胞内CF结构在受ARD影响的皮层细胞中含有不同的油桃科植物。这些结果大大有助于ARD的病因。两种方案都提供了通过分子工具从选定的有症状小根部中鉴定真菌的可能性，从而避免了分离和随后的单一真菌分离物的纯合菌培养。