Zhang J, Zou Q, Li G Q, Jiang D H, Huang H C
The State Key Lab of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alberta, T1J 4B1, Canada. This study was supported by the National Science Foundation of China (Grant 30570079).
Plant Dis. 2008 Jul;92(7):1133. doi: 10.1094/PDIS-92-7-1133B.
During the spring of 2006, onion bulbs with gray mold symptoms on the surface were observed in a few supermarkets in Wuhan, China. Onions mummified as they decayed. Further surveys of five randomly selected batches of onion bulbs in one of the supermarkets indicated that the disease occurred in all batches and the disease incidence ranged from 6 to 50%. Eight diseased onion bulbs were collected arbitrarily and isolations were made using homemade potato dextrose agar (PDA). Single-spore cultures of the isolated Botrytis sp. were established and maintained on PDA plates at 20°C. The 10-day-old PDA cultures of all of these isolates were gray and covered with abundant beige, ovoid- or oblong-shaped conidia, which were budded from terminal ampullae formed on dichotomously branching conidiophores. Conidia from these isolates measured 7.6 to 10.4 μm long and 4.2 to 5.6 μm wide, with an average of 8.4 × 5.0 μm. No sclerotia were produced from any of these PDA cultures after incubation at 20°C for 30 days. Morphological characteristics of colonies and conidia of these isolates were similar to Botrytis aclada according to the description made by Yohalem et al. (3). Inoculation of healthy onion bulbs with one of the eight fungal strains, OnionBc-15, resulted in gray mold symptoms similar to those observed in the supermarkets. Microscopic examinations showed that the size and shape of conidia that formed on the surface of diseased bulbs of onion were identical to the size and shape of conidia of OnionBc-15, indicating that this isolate can cause onion bulb rot. The isolate OnionBc-15 was further characterized by molecular techniques. Genomic DNA was extracted from mycelia of this strain and used as a template for amplification of two previously reported DNA regions, the internal transcribed spacer (ITS) region of the ribosomal RNA genes and the L45-550 sequence (1), which can be used to distinguish B. aclada and two closely related species, B. allii and B. byssoidea (3). Universal primers ITS1 and ITS4 were used to amplify the ITS region (2). A 539-bp DNA sequence was generated, cloned, and sequenced (GenBank Accession No. EU093077). The sequence contained two SphI restriction sites and was 99% identical in nucleotides to that of B. aclada strain PRI006 (GenBank Accession No. AJ716295). It is different from B. allii and B. byssoidea, which have only one SphI restriction site for the ITS1/ITS4-amplified DNA sequence (2). The Botrytis-specific primers, BA2f and BA1r, were used to amplify the L45-550 sequence (2). A 413-bp DNA sequence was generated, cloned, and sequenced. The sequence did not contain any ApoI restriction sites. This is also similar to B. aclada, but different from B. allii and B. byssoidea, which contains one ApoI restriction site in the BA2f/BA1r-amplified DNA sequence (2,3). On the basis of morphological characteristics and the two molecular features, it is concluded that the isolate OnionBc-15 belongs to B. aclada. To our knowledge, this is the first report on the occurrence of B. aclada causing onion bulb rot in China. References: (1) K. Nielsen and D. S. Yohalem. Mycologia 93:264, 2001. (2) K. Nielsen et al. Plant Dis. 86:682, 2002. (3) D. S. Yohalem et al. Mycotaxon 85:175, 2003.
2006年春季，在中国武汉的几家超市中，发现了一些表面带有灰霉病症状的洋葱鳞茎。洋葱腐烂时会干瘪。对其中一家超市随机抽取的五批洋葱鳞茎进行进一步调查发现，所有批次均出现病害，发病率在6%至50%之间。任意收集了八个患病洋葱鳞茎，并用自制的马铃薯葡萄糖琼脂（PDA）进行分离培养。将分离得到的葡萄孢属真菌进行单孢培养，并在20°C的PDA平板上保存。所有这些分离菌株在PDA上培养10天后呈灰色，表面覆盖着大量米色、卵形或长方形的分生孢子，这些分生孢子从二叉状分枝分生孢子梗顶端的壶形结构上芽生而出。这些分离菌株的分生孢子长7.6至10.4μm，宽4.2至5.6μm，平均为8.4×5.0μm。在20°C下培养30天后，这些PDA培养物均未产生菌核。根据约哈勒姆等人（3）的描述，这些分离菌株的菌落和分生孢子的形态特征与葱葡萄孢相似。用八种真菌菌株之一的洋葱葡萄孢菌-15（OnionBc-15）接种健康洋葱鳞茎，导致出现与超市中观察到的类似的灰霉病症状。显微镜检查表明，患病洋葱鳞茎表面形成的分生孢子的大小和形状与洋葱葡萄孢菌-15的分生孢子相同，表明该分离菌株可引起洋葱鳞茎腐烂。通过分子技术对分离菌株洋葱葡萄孢菌-15进行了进一步鉴定。从该菌株的菌丝体中提取基因组DNA，并用作扩增两个先前报道的DNA区域的模板，即核糖体RNA基因的内部转录间隔区（ITS）和L45-550序列（1），这两个区域可用于区分葱葡萄孢与两个近缘种，葱腐葡萄孢和棉絮状葡萄孢（3）。使用通用引物ITS1和ITS4扩增ITS区域（2）。产生了一个539bp的DNA序列，进行克隆和测序（GenBank登录号：EU093077）。该序列包含两个SphI限制性酶切位点，核苷酸序列与葱葡萄孢菌株PRI006（GenBank登录号：AJ716295）的序列99%相同。它与葱腐葡萄孢和棉絮状葡萄孢不同，后两者对于ITS1/ITS4扩增的DNA序列只有一个SphI限制性酶切位点（2）。使用葡萄孢属特异性引物BA2f和BA1r扩增L45-550序列（2）。产生了一个413bp的DNA序列，进行克隆和测序。该序列不包含任何ApoI限制性酶切位点。这也与葱葡萄孢相似，但与葱腐葡萄孢和棉絮状葡萄孢不同（2,3），后两者在BA2f/BA1r扩增的DNA序列中包含一个ApoI限制性酶切位点。根据形态特征和这两个分子特征，得出结论：分离菌株洋葱葡萄孢菌-15属于葱葡萄孢。据我们所知，这是中国关于葱葡萄孢引起洋葱鳞茎腐烂的首次报道。参考文献：（1）K. 尼尔森和D. S. 约哈勒姆。《真菌学》93:264，2001年。（2）K. 尼尔森等人。《植物病害》86:682，2002年。（3）D. S. 约哈勒姆等人。《真菌分类学报》85:175，2003年。
