草莓根颈腐烂病的病原鉴定

【目的】明确草莓根颈腐烂病的病原，为草莓根颈腐烂病的防治及抗病育种提供依据。【方法】对取自湖北省武汉市的发病草莓植株分别进行发病组织培养、分离纯化。采用牙签根颈接种方法进行致病性测定；观察病原微生物在马铃薯葡萄糖琼脂培养基（potato dextrose agar，PDA）上的菌落形态；从所分离到的菌株中选择PDA培养基平板上形态不同的Zhd-3、Zhd-4-1和Zhd-5进行后续试验和分析。测定各菌株在18℃黑暗条件下的菌丝生长速度，然后用方差分析程序（ANOVA）对不同菌株间的差异显著性进行分析；采用光学显微镜和扫描电子显微镜对病原菌在草莓叶柄上产生的分生孢子形态进行分析。分别以Zhd-3、Zhd-4-1和Zhd-5的基因组DNA为模板对这3个菌株的部分肌动蛋白基因（Actin，ACT）、β-维管蛋白基因（β-Tubulin 2，TUB2）和钙调蛋白基因（Calmodulin, CAL）进行PCR扩增，并将PCR产物测序。将Zhd-3、Zhd-4-1和Zhd-5与胶孢炭疽复合种内全部23个种的代表性菌株一起进行系统进化分析。采用Mega4.1软件和邻接法进行基于病原菌ACT、TUB2和CAL序列的多基因位点系统进化分析。【结果】从武汉市郊发病草莓植株上共分离纯化到15个菌株，不同菌株在PDA培养基平板上的正面形态没有显著差异，但是背面形态却明显不同，根据各菌株的PDA平板背面形态将15个菌株分成3组，分别从每组中选择Zhd-3、Zhd-4-1和Zhd-5进行后续形态学鉴定和系统进化分析。3个菌株在采用牙签接种法接种草莓根颈时均能使草莓植株发病；使用这3个菌株接种草莓叶柄时均能在病斑处产生分生孢子，但分生孢子形态没有显著差异；20个分生孢子的平均大小为11 μm×3.8 μm；3个菌株在18℃黑暗条件下的菌丝生长速度分别为0.82、0.68和0.88 cm?d-1；这些生物学特征表明这些菌株属于胶孢炭疽复合种。通过基于ACT、TUB2和CAL的多基因系统进化分析结果表明菌株Zhd-3、Zhd-4-1和Zhd-5与已鉴定为Colletotrichum siamense的菌株ICMP12567、ICMP17795、ICMP18121、CBS113199和CBS112983归在1个分支，其自展支持率为84%，能够与胶孢炭疽复合种内的其他物种区分开。【结论】通过对草莓根颈腐烂病菌的生物学特性、柯赫氏法则证病过程及基于ACT、TUB2和CAL的多基因系统进化分析，明确了引起武汉地区草莓根颈腐烂病的病原为胶孢炭疽菌复合种内的C. siamense。

Identification of Pathogen Causing Crown and Root Rot on Strawberry

【Objective】The objective of this study is to identify the pathogen causing crown rot on strawberry in Wuhan, and to provide a basis for disease prevention and resistance breeding.【Method】Diseased samples from Wuhan, Hubei Province in central China were collected, pieces of infected tissue from the crowns were surface disinfested and plated on acidified potato dextrose agar (PDA). The growing edges of any fungal hyphae developing from the tissues were then transferred aseptically to another PDA plate. Isolates were characterized by colony morphology, conidial characteristics and mycelia growth rate under 18℃ in dark. One-way analyses of variance (ANOVA) were conducted to determine the significance of differences in growth rates of different isolates. According to the morphological characteristics, isolates Zhd-3, Zhd-4-1 and Zhd-5 were selected as representatives for next analysis. Pathogenicity test was carried out by inoculating conidia of the three isolates to crown of strawberry with toothpick, respectively. Conidia produced on petioles were observed by optical microscope and scanning electron microscopy. The length and width of 20 conidia of each isolate were measured. Partial actin (ACT), beta-tubulin (TUB2) and calmodulin (CAL) genes of Zhd-3, Zhd-4-1 and Zhd-5 were amplified and sequenced. The ACT, TUB2 and CAL sequences of Zhd-3, Zhd-4-1, Zhd-5, along with the 23 different representative isolates in C. gloeosporioides species complex were used for multigene loci analyses with Mega 4.1 and Neighbour-Joining method. 【Result】Fifteen isolates were morphologically identified as Colletotrichum spp.. Colony morphology of these isolates on the above of PDA plate appeared similar but significantly different on the back. The 15 isolates were divided into three groups based on the colony morphology, and isolates Zhd-3, Zhd-4-1 and Zhd-5 belonged to each group, respectively, were selected for follow-up tests and analysis. The pathogenicity tests showed that the symptoms identical to those observed in field were produced on all inoculated plants. Control plants remained symptomless. The same pathogen was reisolated from the diseased tissues. Biological tests showed that mycelial growth rates of Zhd-3, Zhd-4-1 and Zhd-5 were 0.82, 0.68 and 0.88 cm?d-1, respectively. The average size of 20 conidia was 11 μm × 3.8 μm. The biological characteristics indicated these isolates were C. gloeosporioides species complex. The result of multigene loci analysis based on ACT, TUB2 and CAL indicated that isolates Zhd-3, Zhd-4-1 and Zhd-5 were divided into one group with C. siamense strains ICMP12567, ICMP17795, ICMP18121, CBS113199 and CBS112983.【Conclusion】The results of biological characteristics and multigene loci analysis based on the sequences of TUB2, ACT and CAL are concluded that the crown rot disease of strawberry occurred in Wuhan is caused by C. siamense.