Bassoriello M M I, Jordan K S
Department of Plant Agriculture, University of Guelph, ON N1G 2W1, Canada.
Plant Dis. 2012 Nov;96(11):1698. doi: 10.1094/PDIS-06-12-0530-PDN.
The ectotrophic, root-infecting fungus Magnaporthe poae Landschoot & Jackson, the causal agent of summer patch disease in the U.S. (2), is implicated in the damage and loss of annual bluegrass (Poa annua L.) on golf course greens. This pathogenic fungus, one of the important root pathogens of turfgrass, attacks and colonizes susceptible turfgrass roots suffering from environmental or cultural stresses. Over 100 turf samples that exhibited symptoms (chlorotic circular or irregular patches of ≥15 cm in diameter with necrotic crowns and discolored roots) reminiscent of summer patch were collected from 77 southwestern Ontario golf courses from July to August of 2009 and 2010. Roots and crowns were often covered with dark, ectotrophic runner hyphae, lobed hyphopodia, and growth cessation structures, characteristic of M. poae. Sections of root tissue were surface sterilized in 0.6% sodium hypochlorite (NaOCl) for 5 min. Sterilized root tissue was plated on potato dextrose agar (PDA) containing 50 mg L streptomycin sulfate and incubated at 28°C for 7 to 10 days. A fungus with morphological characteristics (hyaline mycelium that appears gray or olive-brown when mature) similar to those of M. poae (1) was consistently isolated (≥100 isolates were obtained) and used to identify M. poae through molecular techniques and Koch's postulates. DNA was extracted from the fungal mycelium of the collected isolates using the PowerPlant DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA). The rDNA internal transcribed spacer (ITS) regions of the isolates (≥100 isolates) were amplified by PCR using universal fungal rDNA primers ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS 5 (5'- GGAAGTAAAAGTCGTAACAAGG-3') (3). The purified PCR products were sequenced (GenBank Accession No. JX134588 through JX134601) and a BLAST search exhibited seven isolates with 99% (MAG3, MAG6, MAG13, MAG16, and MAG17) and 100% (MAG1 and MAG14) similarity to M. poae in the GenBank database. Pathogenicity of four isolates (MAG1, MAG3, MAG6, and MAG14) was confirmed with Koch's postulates. Sixteen healthy P. annua core samples (four replicates of each treatment/isolate) collected from an Ontario golf course were inoculated with 25 mg M. poae-infested Kentucky bluegrass seed (Poa pratensis L.; 12.5 mg inoculum applied at the surface of the potting medium and 12.5 mg inoculum applied on the foliar surface) and were placed in a growth chamber with 12-h day/night cycles at 30/25°C and approximate relative humidity. After 2 to 3 weeks, inoculated plants exhibited chlorotic foliage and necrotic roots covered with dark ectotrophic runner hyphae and lobed hyphopodia. Infected root sections from each replication were surface sterilized and placed on PDA containing 50 mg L streptomycin sulfate. The fungal cultures exhibited morphological characteristics consistent with M. poae (1). To our knowledge, this is the first report of summer patch caused by M. poae in Canada. References: (1) B. B. Clarke and A. B. Gould, eds. Turfgrass Patch Diseases Caused by Ectotrophic Root-Infecting Fungi. The American Phytopathological Society, St. Paul, MN, 1993. (2) P. J. Landschoot and N. Jackson. Mycol. Res. 93:59, 1989. (3) T. J. White et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al. eds. Academic Press, San Diego, CA, 1990.
外生型、侵染根部的真菌匍柄霉叶斑病菌（Magnaporthe poae Landschoot & Jackson）是美国夏季斑病的病原菌（2），与高尔夫球场果岭上一年生早熟禾（Poa annua L.）的损害和损失有关。这种致病真菌是草坪草重要的根部病原菌之一，会侵袭并定殖于遭受环境或栽培胁迫的易感草坪草根部。2009年7月至8月以及2010年7月至8月期间，从安大略省西南部的77个高尔夫球场收集了100多个表现出类似夏季斑症状（直径≥15 cm的褪绿圆形或不规则斑块，伴有坏死的叶冠和变色的根部）的草坪样本。根部和叶冠上常常覆盖着深色的外生匍匐菌丝、叶状附着胞以及生长停止结构，这些都是匍柄霉叶斑病菌的特征。将根组织切片在0.6%次氯酸钠（NaOCl）中表面消毒5分钟。将消毒后的根组织接种到含有50 mg/L硫酸链霉素的马铃薯葡萄糖琼脂（PDA）上，在28°C下培养7至10天。持续分离出一种形态特征（成熟时呈灰色或橄榄褐色的透明菌丝体）与匍柄霉叶斑病菌相似的真菌（获得了≥100个分离株），并通过分子技术和柯赫氏法则来鉴定匍柄霉叶斑病菌。使用PowerPlant DNA提取试剂盒（MO BIO Laboratories, Inc., Carlsbad, CA）从收集到的分离株的真菌菌丝体中提取DNA。使用通用真菌rDNA引物ITS 4（5'-TCCTCCGCTTATTGATATGC-3'）和ITS 5（5'- GGAAGTAAAAGTCGTAACAAGG-3'）（3）通过PCR扩增分离株（≥100个分离株）的rDNA内部转录间隔区（ITS）。对纯化的PCR产物进行测序（GenBank登录号为JX134588至JX134601），BLAST搜索显示有7个分离株与GenBank数据库中的匍柄霉叶斑病菌相似度为99%（MAG3、MAG6、MAG13、MAG16和MAG17），2个分离株（MAG1和MAG14）相似度为100%。通过柯赫氏法则证实了4个分离株（MAG1、MAG3、MAG6和MAG14）的致病性。从安大略省一个高尔夫球场收集的16个健康一年生早熟禾核心样本（每个处理/分离株4个重复）接种25 mg受匍柄霉叶斑病菌侵染的草地早熟禾种子（Poa pratensis L.；12.5 mg接种物施于盆栽基质表面，12.5 mg接种物施于叶面），并置于生长室中，昼夜周期为12小时，温度为30/25°C，相对湿度近似。2至3周后，接种的植株出现叶片褪绿和根部坏死，覆盖着深色的外生匍匐菌丝和叶状附着胞。对每个重复的感染根段进行表面消毒，并置于含有50 mg/L硫酸链霉素的PDA上。真菌培养物表现出与匍柄霉叶斑病菌一致的形态特征（1）。据我们所知，这是加拿大关于匍柄霉叶斑病菌引起夏季斑病的首次报道。参考文献：（1）B. B. Clarke和A. B. Gould编著。由外生型侵染根部真菌引起的草坪草斑块病。美国植物病理学会，明尼苏达州圣保罗，1993年。（2）P. J. Landschoot和N. Jackson。真菌学研究93:59，1989年。（3）T. J. White等人。用于系统发育学的真菌核糖体RNA基因的扩增和直接测序。载于：PCR实验方案：方法与应用指南。M. A. Innis等人编著。学术出版社，加利福尼亚州圣地亚哥，1990年。
