Hu M J, Chen Y, Chen S N, Liu X L, Yin L F, Luo C X
Department of Plant Pathology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
Department of Plant Pathology, College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China.
Plant Dis. 2011 Feb;95(2):225. doi: 10.1094/PDIS-11-10-0779.
In 2009 and 2010, peaches (Prunus persica) with brown rot symptoms were collected from Zhejiang Plant Protection State Research Farm and a commercial orchard in Fujian Province in southeastern China. Affected fruit showed brown decay with zones of sporulation. Single-spore isolates from the diseased fruit were cultured on potato dextrose agar. After incubation at 25°C in the dark for 5 days, colonies were gray with concentric rings of sporulation. Mean mycelial growth of isolates MZ09-2a from Zhejiang Province and 0907-a from Fujian Province was 4.46 ± 0.58 and 7.05 ± 0.81 cm after 4 and 7 days of incubation, respectively. Lemon-shaped conidia were formed in branched, monilioid chains and mean size was 14.6 (9.6 to 21.6) × 10.3 (7.2 to 13.2) μm. Mean conidial germination was 97 ± 1% with one straight germ tube per conidium. These characteristics were consistent with descriptions of Monilinia fructicola (G. Wint.) Honey (3). Morphology-based species identification was confirmed by sequencing and analysis of ribosomal internal transcribed spacer (ITS) sequences. A 496-bp fragment including ITS 1 and 2 and the gene encoding the 5.8S small subunit of the ribosomal RNA from isolates MZ09-2a and 0907-a was amplified using the universal primer pair ITS1/ITS4 (4) and sequenced. Nucleotide sequences of both isolates were identical. Blast searches of the ITS sequences in GenBank showed the highest similarity (100%) with sequences of M. fructicola isolates from China (FJ515894), Italy (FJ411109), and Spain (EF207423). The isolates were also identified as M. fructicola using the Monilinia spp. PCR detection protocol based on sequence-characterized amplification region marker DNA sequences (2). Pathogenicity was confirmed by inoculating surface-sterilized, mature cv. Zhonghua 2 peaches with mycelial plugs of representative isolates. Fruit was stabbed at two points with a 5-mm-diameter sterile cork borer, mycelial plugs (5 mm in diameter) were removed from the periphery of a 4-day-old colony of each isolate and placed upside down into each wound; control fruit received water agar. Inoculated fruit developed typical brown rot symptoms with sporulating fungi while control fruit remained healthy after 3 days of incubation at 22°C in a moist chamber. Pathogens were reisolated from the inoculated fruit and confirmed to be M. fructicola on the basis of morphological characteristics. To our knowledge, this is the first report of M. fructicola in Zhejiang and Fujian provinces. Both provinces are located more than 1,000 km south of Beijing, Hebei, and Shandong provinces, where M. fructicola had been reported previously (1). References: (1) J. Y. Fan et al. Acta Phytophylacica Sin. (in Chinese) 34:289, 2007. (2) I. Gell et al. J. Appl. Microbiol. 103:2629, 2007. (3) G. C. M. van Leeuwen and H. A. van Kesteren. Can. J. Bot. 76:2041, 1998. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, 1990.
2009年和2010年,从浙江省植物保护国营试验场以及中国东南部福建省的一个商业果园采集了表现出褐腐病症状的桃子(Prunus persica)。受感染的果实呈现出带有产孢区域的褐色腐烂。从患病果实上分离得到的单孢菌株在马铃薯葡萄糖琼脂培养基上进行培养。在25°C黑暗条件下培养5天后,菌落呈灰色,带有同心环状的产孢结构。来自浙江省的分离株MZ09 - 2a和来自福建省的0907 - a在培养4天和7天后,平均菌丝生长分别为4.46 ± 0.58厘米和7.05 ± 0.81厘米。柠檬形分生孢子形成于分枝的、念珠状的链中,平均大小为14.6(9.6至21.6)× 10.3(7.2至13.2)微米。平均分生孢子萌发率为97 ± 1%,每个分生孢子有一根直的芽管。这些特征与桃褐腐病菌(Monilinia fructicola (G. Wint.) Honey)的描述一致(3)。基于核糖体内部转录间隔区(ITS)序列的测序和分析证实了基于形态学的物种鉴定。使用通用引物对ITS1/ITS4(4)扩增了分离株MZ09 - 2a和0907 - a的一个包含ITS 1和2以及编码核糖体RNA 5.8S小亚基的基因的496碱基对片段,并进行了测序。两个分离株的核苷酸序列相同。在GenBank中对ITS序列进行Blast搜索显示,与来自中国(FJ515894)、意大利(FJ411109)和西班牙(EF207423)的桃褐腐病菌分离株的序列相似度最高(100%)。还使用基于序列特征扩增区域标记DNA序列的褐腐病菌属PCR检测方案(2)将这些分离株鉴定为桃褐腐病菌。通过用代表性分离株的菌丝块接种表面消毒的成熟中华2号桃子来确认致病性。用直径5毫米的无菌钻孔器在果实上刺两个点,从每个分离株4天龄菌落的边缘取下直径5毫米的菌丝块,将其倒置放入每个伤口中;对照果实接种水琼脂。接种后的果实在22°C潮湿环境中培养3天后出现典型的褐腐病症状并带有产孢真菌,而对照果实保持健康。从接种的果实中重新分离出病原菌,并根据形态学特征确认为桃褐腐病菌。据我们所知,这是桃褐腐病菌在浙江和福建两省的首次报道。这两个省份位于北京、河北和山东省以南1000多公里处,此前已报道过桃褐腐病菌在这些省份存在(1)。参考文献:(1)范洁瑜等,《植物保护学报》(中文)34:289,2007。(2)I. Gell等,《应用微生物学杂志》103:2629,2007。(3)G. C. M. van Leeuwen和H. A. van Kesteren,《加拿大植物学杂志》76:2041,1998。(4)T. J. White等,见《PCR协议:方法与应用指南》。M. A. Innis等编,学术出版社,圣地亚哥,1990年,第315页。