First Report of White Root Rot of Japanese Serissa Caused by Rosellinia necatrix in Taiwan

Japanese serissa (Serissa japonica (Thunb.) Thunb.) is a very popular ornamental in Taiwan. During the summer of 2005, serissa plants in a central Taiwan nursery had decaying roots, leaf yellowing, and were wilting. Wilted plants had white fan-like mycelium under the bark. The disease caused 70% loss of seedlings at the nursery. Associated synnemata were rigid, erect, dark, setaceous, 0.8 to 2.1 mm long, 90 to 200 µm wide, and tapering to enlarged whitish gray heads composed of geniculate conidiophores and conidia. Conidia were 3.1 to 5.8 × 2.6 to 3.4 µm, unicellular, hyaline, and subglobal with a truncated base. Root rots were washed, disinfested for 1 min in 0.5% NaOCl, cut into 3 mm3 pieces, plated on Merck (Darmstadt, Germany) potato dextrose agar (PDA) amended with 100 ppm of ampicillin (Sigma, St. Louis, MO), and incubated at 24°C in the dark until hyphae emerged. Single hyphal tips were transferred to PDA, and two isolates were established as pure cultures. Mycelia were cut, stained with 1% cotton blue in lactophenol, and pear-shaped hyphal swellings adjacent to the septa were observed. According to these hyphal, synnematal, and conidial characteristics, the fungus was identified as Dematophora necatrix Hartig, the anamorph of Rosellinia necatrix Prill. Inoculum for pathogenicity tests were produced on oat-wheat medium composed of 20 ml of oat grain and 20 ml of wheat grain, mixed, and soaked in water for 3 h. The grains were placed in 200- ml flasks, autoclaved at 121°C for 30 min, inoculated with two isolates of D. necatrix separately, and grown for 14 days. Six 3-month-old Japanese serissa seedlings were grown in pots. The grain inoculum was added to unsterilized field soil and the plants were transplanted into this mix. Control plants were transplanted into a similar mix without the inoculum. Two replicates were used for a total of 24 inoculated plants and 24 control plants. All plants were kept in a growth chamber at 25 to 35°C with 20 min of irrigation per day, 12 h of irradiation, and relative humidity at more than 95%. Inoculated plants developed root rots after 1 month, and after 4 months, all plants were dead, while control plants remained healthy. D. necatrix was reisolated, hyphal characteristics confirmed, and synnemata were observed on collars of dead plants. The teleomorph was not formed by our cultures, and the identification of Rosellinia necatrix was confirmed by molecular studies. The nuclear ribosomal internal transcribed spacer (ITS) amplified with two primers, ITS1 and ITS4, from our representative isolate demonstrated 99.63, 99.81, and 99.27% similarity to two R. necatrix isolates from Japan and one R. necatrix isolate from Italy, respectively. This disease has been reported on many species of plants (1), but to our knowledge, this is the first report of white root rot of Japanese serissa seedlings caused by R. necatrix in Taiwan.六月雪（Japanese serissa，学名Serissa japonica (Thunb.) Thunb.）是台湾地区极具人气的观赏植物。2005年夏季，台湾中部某苗圃内的六月雪植株出现根系腐烂、叶片黄化及萎蔫症状，萎蔫植株的树皮下方可见白色扇状菌丝体（mycelium）。该病害导致该苗圃的幼苗损失率达70%。伴随病害产生的分生孢子座（synnemata）质地坚硬、直立、色深且呈刚毛状，大小为0.8~2.1 mm长、90~200 µm宽，顶端渐狭为膨大的灰白至浅灰色头部，该头部由膝状分生孢子梗（geniculate conidiophores）与分生孢子（conidia）构成。分生孢子大小为3.1~5.8 × 2.6~3.4 µm，单细胞、透明，呈近球形且基部平截。将发病根系清洗后，于0.5%次氯酸钠（NaOCl）溶液中消毒1 min，切成3 mm³的小块，接种于添加100 ppm氨苄青霉素（ampicillin，Sigma，美国密苏里州圣路易斯市）的默克（Merck，德国达姆施塔特）马铃薯葡萄糖琼脂（potato dextrose agar, PDA）培养基上，于24℃黑暗条件下培养至菌丝长出。挑取单菌丝尖端转接至PDA培养基，获得2个纯培养菌株。切取菌丝体，用1%棉蓝乳酸酚溶液染色后，可观察到隔膜附近存在梨形菌丝膨大体。结合上述菌丝、分生孢子座及分生孢子的形态特征，该真菌被鉴定为毁囊壳霉（Dematophora necatrix Hartig），即褐座坚壳菌（Rosellinia necatrix Prill.）的无性型。致病性测试所用接种物制备于燕麦-小麦培养基：取20 mL燕麦粒与20 mL小麦粒混合，于水中浸泡3 h，分装至200 mL三角瓶中，于121℃高压灭菌30 min，分别接种2株D. necatrix菌株，培养14 d。选取6株3月龄的六月雪幼苗进行盆栽试验。将谷物接种物添加至未灭菌的大田土壤中混匀，将植株移栽至该混合基质中；对照组植株移栽至不含接种物的同款基质中。试验设置2次重复，总计接种组植株24株、对照组植株24株。所有植株置于生长箱中培养，培养条件为：温度25~35℃，每日灌溉20 min，光照时长12 h，相对湿度不低于95%。接种组植株于1个月后出现根系腐烂症状，4个月后全部植株死亡，而对照组植株始终保持健康状态。再次从死亡植株中分离得到D. necatrix，其菌丝特征得到验证，且在死株的根颈部位观察到分生孢子座。本研究获得的菌株未产生有性型，通过分子生物学研究确认了褐座坚壳菌（Rosellinia necatrix）的鉴定结果：以引物ITS1和ITS4扩增代表性菌株的核核糖体内部转录间隔区（nuclear ribosomal internal transcribed spacer, ITS），其序列与2株日本来源的R. necatrix菌株、1株意大利来源的R. necatrix菌株的相似性分别为99.63%、99.81%和99.27%。目前已有诸多植物被报道可发生该病害(1)，但据我们所知，这是台湾地区首例由褐座坚壳菌（Rosellinia necatrix）引起的六月雪幼苗白根腐病。