The complex etiology of cranberry fruit rot (CFR) (Oudemans et al., 1998) has made it difficult to precisely identify the fungi involved in CFR and their relative importance in North America. To remedy this situation, a multiplex PCR approach targeting the 12 most commonly reported fungi in CFR was recently developed (Conti et al., 2019). However, in surveys conducted in Eastern Canada, the molecular tool revealed the presence of an unknown fungus in more than 30% of the collected samples. Analyses were thus undertaken to identify this species. From 117 rotten fruit collected at harvest in 2017, 34 samples of the unknown fungus, all morphologically similar, were isolated but not detected using the molecular tool. Their ITS ribosomal regions were sequenced using universal primers (Vilgalys and Hester, 1990; White et al., 1990) and searched against the GenBank database using the Blastn tool (Altschul et al., 1990). The top match was obtained with Godronia cassandrae (accession number: MH855281 (Vu et al., 2019), 98-100% of identity and an E-value of 0.0), even though some isolates had minor nucleotide differences, as presented in the tree. Sequences were deposited in GenBank as accession numbers MT599989 to MT600022. Since G. cassandrae had been reported, albeit rarely, on cranberry in Michigan (Olatinwo et al., 2003), it was supposed to amplify with the molecular tool developed from the strain DAOM C216021 (AAFC, Ottawa, ON) identified in 1993 on Vaccinium angustifolium as G. cassandrae. Analysis of the sequences used to build the specific primers from this strain confirmed the DAOM strain as being Neocucurbitaria juglandicola, which was never diagnosed in our cranberry samples. To confirm this revised diagnosis, a multi-sequence alignment (MSA) was performed on the ITS regions of the isolates from rotten cranberries and sequences available for the genus Godronia in the NCBI nucleotide database (NCBI txid269064). This MSA allowed us to find discriminant regions between Godronia spp. A pair of PCR primers specific to G. cassandrae found on cranberry fruit was then designed (the forward and reverse sequences are AAT CAG TGG CGG TGC CTG TC and TAC CGC TTC ACT CGC CGT TAC, respectively), generating 196 bp amplicons, with an annealing temperature of 65°C. The diagnosis of 7,835 fruit sampled at three time points (harvest, after three and after six weeks of storage) in 2018, from four cranberry farms located in Québec (CA) and Nova Scotia (CA), detected G. cassandrae in 2350 samples (30%). To assess the pathogenicity of four specimens from 2017, Koch's postulates were completed on two healthy fruit per isolate. The fruit were wounded with a sterilized pick and individually inoculated; two fruit were used as control. Based on our observations, the fungi isolated from cranberry fruit displayed a pale lemon yellow mycelium and black pycnidia. Conidia are hyaline, cylindrical and divided by a single septum. These morphological characters are similar to the ones described in the literature for G. cassandrae (Polashock et al., 2017). Rot symptoms appear as a discoloration from the firm, red and healthy cranberry fruit to a yellowish-orange softer fruit. Molecular characterization of the re-isolated fungus confirmed the presence of G. cassandrae. We report Godronia cassandrae for the first time as a major cause of CFR in Eastern Canada. Its prevalence in cranberry fields of Québec and Nova Scotia suggests that it supplants Physalospora as the main fungus involved in CFR in Eastern Canada.

中文翻译：

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首次报道 Godronia cassandrae 作为加拿大东部主要蔓越莓果腐病病原体。


蔓越莓果腐病 (CFR) 的复杂病因学（Oudemans 等人，1998）使得精确识别与 CFR 相关的真菌及其在北美的相对重要性变得困难。为了纠正这种情况，最近开发了一种针对 CFR 中 12 种最常见真菌的多重 PCR 方法（Conti 等人，2019）。然而，在加拿大东部进行的调查中，分子工具显示超过 30% 的收集样本中存在未知真菌。因此进行了分析以确定该物种。从 2017 年收获时收集的 117 个腐烂水果中，分离出 34 个形态相似的未知真菌样本，但未使用分子工具检测到。使用通用引物（Vilgalys 和 Hester，1990；White 等，1990）对它们的 ITS 核糖体区域进行测序，并使用 Blastn 工具（Altschul 等，1990）在 GenBank 数据库中进行搜索。最匹配的是 Godronia cassandrae（登录号：MH855281（Vu 等人，2019），同一性为 98-100%，E 值为 0.0），尽管一些分离株具有微小的核苷酸差异，如表中所示。树。序列以登录号 MT599989 至 MT600022 保存在 GenBank 中。由于 G. cassandrae 曾在密歇根州的蔓越莓上被报道过（尽管很少）（Olatinwo 等人，2003 年），因此应该使用 1993 年鉴定的菌株 DAOM C216021（AAFC，渥太华，安大略省）开发的分子工具对其进行扩增。狭叶越橘（Vaccinium angustifolium），即 G. cassandrae。用于构建该菌株特异性引物的序列分析证实，DAOM 菌株为 Neocucurbitaria juglandicola，而我们的蔓越莓样本中从未诊断出该菌株。 为了确认这一修订后的诊断，对来自腐烂蔓越莓的分离物的 ITS 区域和 NCBI 核苷酸数据库 (NCBI txid269064) 中可用于 Godronia 属的序列进行了多序列比对 (MSA)。该 MSA 使我们能够找到 Godronia spp 之间的区分区域。然后设计了一对针对蔓越莓果实上发现的 G. cassandrae 特异的 PCR 引物（正向和反向序列分别为 AAT CAG TGG CGG TGC CTG TC 和 TAC CGC TTC ACT CGC CGT TAC），生成 196 bp 的扩增子，退火温度65℃。 2018 年，对来自魁北克省 (CA) 和新斯科舍省 (CA) 四个蔓越莓农场的 7,835 个水果样本（采收、三周后和六周储存后）进行诊断，在 2350 个样本中检测到 G. cassandrae（ 30%）。为了评估 2017 年四个样本的致病性，对每个分离株的两个健康水果完成了科赫假设。用消毒过的镐将果实划伤并单独接种；使用两种水果作为对照。根据我们的观察，从蔓越莓果实中分离出的真菌显示出淡柠檬黄色的菌丝体和黑色的分生孢子器。分生孢子是透明的、圆柱形的并且被单个隔膜分开。这些形态特征与文献中描述的 G. cassandrae 相似（Polashock 等，2017）。腐烂症状表现为从坚硬、红色、健康的蔓越莓果实变色为黄橙色较软的果实。重新分离的真菌的分子表征证实了 G. cassandrae 的存在。我们首次将 Godronia cassandrae 报告为加拿大东部 CFR 的主要原因。 它在魁北克省和新斯科舍省的蔓越莓田中盛行，表明它取代轮纹孢菌成为加拿大东部 CFR 的主要真菌。