Xiao F, Zhang J Z, Tu Y L
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.
Agricultural Bureau, Yueqing City, Zhejiang Province 325600, China.
Plant Dis. 2012 Sep;96(9):1377. doi: 10.1094/PDIS-03-12-0304-PDN.
White Dendrobium (Dendrobium candidum Wall. ex Lindl.) is a traditional Chinese medicinal herb that is used raw or processed for health care products in China (4). It is propagated by tissue-cultured seedlings largely because of its dramatic decrease in the wild from heavy market demand. In July 2011, a wilt disease was observed on 1-year-old seedlings in a cultivated field in Yueqing City, Zhejiang Province, China. Initial symptoms were one to several chlorotic leaves in a cluster of plants. As the disease progressed, leaves wilted before dropping. Vascular discoloration was not observed in infected stems. Individual plants wilted, bent, and rotted starting from the base and eventually all plants in a cluster wilted. Three strains isolated from stem tissues collected at different sites were grown on potato dextrose agar (PDA) and carnation leaf agar (CLA) under a 12-h alternating cycle of light and dark at a temperature of 25°C. Macroconidia were sparse, three-septate, slightly curved, and ranged from (21.1) 23.9 to 32.7 (36.9) × (2.8) 3.3 to 4.2 (4.8) μm. Microconidia were abundant, unicellular, oval to reniform, and ranged from (5.3) 6.0 to 8.2 (9.4) × (2.2) 2.5 to 3.3 (3.6) μm on PDA. Abundant chlamydospores formed after 3 weeks on CLA. Chlamydospores were single and sometimes in pairs, terminal and intercalary, rough walled, and measured (6.7) 7.6 to 10.2 (11.9) × (5.9) 6.8 to 8.8 (10.4) μm. Strains were identified as Fusarium oxysporum on the basis of morphological features (2). PCR amplification of genomic DNA using the primers ITS6 and ITS4 as well as ef1 and ef2 (translation elongation factor 1-α gene) generated sequences of approximately 500 bp and 670 bp, respectively. ITS sequences were submitted to the NCBI database (Accession Nos. JQ809654, JQ809655, and JQ809656), and were identical to the sequence for F. oxysporum, Accession No. HQ379654 (3). Three sequences of a portion of TEF-1α (Accession Nos. JQ809657, JQ809658, and JQ809659) had 99% similarity with an isolate of F. oxysporum isolate from chickpea (Accession No. HQ731057) (1), thus confirming the identity of the pathogen. The roots of five to seven transplanted seedlings of a cluster grown for 2 months in pasteurized potting media (primarily chipped pine bark) were inoculated with 15 ml of a 10 conidia ml suspension of each isolate. There were 10 replicates for each isolate treatment. Sterile water was used as a negative control. Plants were grown in the greenhouse at 28 ± 2°C. Lateral roots turned brown 15 days after inoculation. Leaves started to turn yellow 1 month after inoculation and the plants began to wilt 2 months after inoculation. Noninoculated controls did not develop disease symptoms. The pathogenic isolates were reisolated from all diseased plants. Isolates have been deposited at the Biotechnology Institute, Zhejiang University (F_01, F_02 and F_03). To the best of our knowledge, this is first record of Fusarium wilt of D. candidum in China. References: (1) D. Jiménez-Fernández et al. Plant Dis. 95:860, 2011. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) N. Rajmohan et al. Plant Pathol. 131:631, 2011. (4) J. Z. Zhang et al. Plant Pathol. 57:370, 2008.
铁皮石斛（Dendrobium candidum Wall. ex Lindl.）是一种传统的中药材，在中国可直接使用或加工制成保健品（4）。由于市场需求巨大导致其野生资源急剧减少，目前主要通过组培育苗进行繁殖。2011年7月，在中国浙江省乐清市的一块种植地里，1年生的组培育苗出现了枯萎病症状。最初症状是一丛植株中有一两片至几片褪绿叶片。随着病情发展，叶片在掉落前枯萎。受感染的茎中未观察到维管束变色。单株植株从基部开始枯萎、弯曲并腐烂，最终一丛中的所有植株都会枯萎。从不同地点采集的茎组织中分离出的三个菌株，在25°C、光照和黑暗交替12小时的条件下，在马铃薯葡萄糖琼脂（PDA）和香石竹叶琼脂（CLA）上培养。大分生孢子稀少，有3个隔膜，略弯曲，大小为（21.1）23.9至32.7（36.9）×（2.8）3.3至4.2（4.8）μm。在PDA上，小分生孢子丰富，单细胞，椭圆形至肾形，大小为（5.3）6.0至8.2（9.4）×（2.2）2.5至3.3（3.6）μm。在CLA上培养3周后形成大量厚垣孢子。厚垣孢子单个存在，有时成对，顶生和间生，壁粗糙，大小为（6.7）7.6至10.2（11.9）×（5.9）6.8至8.8（10.4）μm。根据形态特征，这些菌株被鉴定为尖孢镰刀菌（2）。使用引物ITS6和ITS4以及ef1和ef2（翻译延伸因子1-α基因）对基因组DNA进行PCR扩增，分别产生了约500 bp和670 bp的序列。ITS序列提交到NCBI数据库（登录号JQ809654、JQ809655和JQ809656），与尖孢镰刀菌序列（登录号HQ379654）相同（3）。TEF-1α部分的三个序列（登录号JQ809657、JQ809658和JQ809659）与鹰嘴豆分离的尖孢镰刀菌分离株（登录号HQ731057）有99%的相似性（1），从而确认了病原菌的身份。将15 ml浓度为每毫升10个分生孢子的各分离株悬浮液接种到在经过巴氏消毒的盆栽基质（主要是碎松树皮）中生长2个月的一丛5至7株移栽组培育苗的根部。每个分离株处理有10个重复。用无菌水作为阴性对照。植株在28±2°C的温室中生长。接种15天后侧根变为褐色。接种1个月后叶片开始变黄，接种2个月后植株开始枯萎。未接种的对照未出现病害症状。从所有患病植株中重新分离出致病分离株。这些分离株已保藏于浙江大学生物技术研究所（F_01、F_02和F_03）。据我们所知，这是中国铁皮石斛镰刀菌枯萎病的首次报道。参考文献：（1）D. Jiménez-Fernández等人，《植物病害》95:860，2011年。（2）J. F. Leslie和B. A. Summerell，《镰刀菌实验室手册》，Blackwell Publishing，艾姆斯，爱荷华州，2006年。（3）N. Rajmohan等人，《植物病理学》131:631，2011年。（4）J. Z. Zhang等人，《植物病理学》57:370，2008年。
