Trigiano R N, Rinehart T A, Dee M M, Wadl P A, Poplawski L, Ownley B H
Department of Entomology and Plant Pathology, University of Tennessee, Knoxville 37996-4560.
USDA-ARS, Thad Chcoran Southern Horticultural Laboratory, Poplarville, MS 39470.
Plant Dis. 2014 Jun;98(6):855. doi: 10.1094/PDIS-11-13-1181-PDN.
Ruth's golden aster (Pityopsis ruthii (Small) Small: Asteraceae) is an endangered, herbaceous perennial that occurs only at a few sites along the Hiwassee and Ocoee rivers in Polk County, Tennessee. This species is drought, heat, and submergence tolerant and has ornamental potential as a fall flowering landscape plant. In 2012, we vegetatively propagated various genotypes and established plantings in a landscape at Poplarville, Mississippi. In June and July of 2013, during periods of hot and humid weather, several well-established plants exhibited black or brown necrotic aerial blight symptoms including desiccation of stems and leaves. Blighted leaf samples were surface sterilized (10% commercial bleach, active ingredient 8.25% sodium hypochlorite, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). Rhizoctonia sp. was identified based on hyphal morphology and cultures were maintained on potato dextrose agar. Colonies were fast growing, consisting of light tan to brown mycelia and tufts of crystalline aerial hyphae. Within 10 days, brown exudates were present in cultures and there was no pigmented reverse to the agar. Hyphae were a mean of 5.2 μm wide (4.6 to 6.1 μm; n = 10) and each compartment contained three or more nuclei. Hyphae were constricted at septa with right angle branching and no clamp connections, which is typical for Rhizoctonia solani (1). Light- to medium-brown, oblong to irregularly shaped sclerotia measuring 1.2 mm long (0.7 to 2.1 mm) × 0.9 mm wide (0.5 to 1.2 mm; n = 20) were formed in cultures after 3 weeks of growth. Total genomic DNA was extracted from two different colonies grown in potato dextrose broth for 7 days, amplified with PCR using ITS1 and ITS4 primers for amplification of the 18S rDNA subunit (2), the products purified, and sequenced. A consensus sequence of 657 bp was deposited in GenBank (Accession Nos. KF843729 and KF843730) and was 96% identical to two R. solani Kühn ITS sequences in GenBank (HF678125 and HF678122). R. solani was grown on twice autoclaved oats for 2 weeks at 21°C and incorporated into Pro-Mix BX, low fertility soilless medium (Premier Horticulture, Rivière-du-Loup, Quebec, Canada) at 4% (w/w) to inoculate seven P. ruthii plants grown in 10 cm-diameter pots; seven additional plants were grown in the same medium amended with 4% (w/w) sterile oats. Plants were grown in a greenhouse and covered with a plastic dome to maintain high humidity. After 2 weeks, six of the seven inoculated plants exhibited the same aerial blight symptoms as did the original infected plants from the field; none of the control plants developed disease symptoms. Colony morphology and hyphal characteristics as well as the sequence for the ITS region of rDNA from the re-isolated fungus were identical to the original isolate. To our knowledge, this is the first report of R. solani infecting Ruth's golden aster. We are not aware of the disease occurring in wild populations of the plant, but may impact plants grown in the landscape or greenhouse. References: (1) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St Paul, MN, 1991. (2) 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, CA, 1990.
鲁思金鸡菊(Pityopsis ruthii (Small) Small:菊科)是一种濒危的多年生草本植物,仅生长在田纳西州波尔克县希瓦西和奥科伊河沿岸的少数地点。该物种耐旱、耐热且耐水淹,作为秋季开花的园林植物具有观赏潜力。2012年,我们对不同基因型进行了营养繁殖,并在密西西比州波普拉维尔的一处园林中进行了种植。2013年6月和7月,在炎热潮湿的天气期间,几株生长良好的植株出现了黑色或褐色坏死性叶枯病症状,包括茎和叶的干枯。将患病叶片样本进行表面消毒(10%商业漂白剂,有效成分8.25%次氯酸钠,1分钟),用无菌水冲洗,空气干燥,然后接种到添加了3.45毫克甲氰菊酯/升(敌杀死2.4乳油,瓦伦特化学公司,加利福尼亚州核桃溪)和10毫克/升利福平(西格玛奥德里奇公司,密苏里州圣路易斯)的2%水琼脂上。根据菌丝形态鉴定为立枯丝核菌,并将培养物保存在马铃薯葡萄糖琼脂上。菌落生长迅速,由浅棕褐色菌丝体和晶状气生菌丝束组成。10天内,培养物中出现褐色渗出物,琼脂背面无色素沉着。菌丝平均宽度为5.2微米(4.6至6.1微米;n = 10),每个隔室包含三个或更多细胞核。菌丝在隔膜处缢缩,呈直角分支,无锁状联合,这是茄丝核菌的典型特征(1)。生长3周后,在培养物中形成浅至中褐色、长椭圆形至不规则形状的菌核,长1.2毫米(0.7至2.1毫米)×宽0.9毫米(0.5至1.2毫米;n = 20)。从在马铃薯葡萄糖肉汤中培养7天的两个不同菌落中提取总基因组DNA,使用ITS1和ITS4引物通过PCR扩增18S rDNA亚基(2),对产物进行纯化并测序。一个657 bp的共有序列保存在GenBank中(登录号KF843729和KF843730),与GenBank中两个茄丝核菌库恩ITS序列(HF678125和HF678122)的相似度为96%。将茄丝核菌在两次高压灭菌的燕麦上于21°C培养2周,然后以4%(w/w)的比例掺入低肥力无土培养基Pro-Mix BX(魁北克省里维耶尔迪卢普的Premier Horticulture公司)中,接种到7株种植在10厘米直径花盆中的鲁思金鸡菊植株上;另外7株植株在添加了4%(w/w)无菌燕麦的相同培养基中生长。植株在温室中生长,并用塑料穹顶覆盖以保持高湿度。2周后,7株接种植株中有6株表现出与田间原始感染植株相同的叶枯病症状;对照植株均未出现病害症状。重新分离的真菌的菌落形态、菌丝特征以及rDNA ITS区域的序列与原始分离株相同。据我们所知,这是关于茄丝核菌感染鲁思金鸡菊的首次报道。我们不知道该病害在该植物的野生种群中是否发生,但可能会影响园林或温室中种植的植株。参考文献:(1)B. Sneh等人。立枯丝核菌物种的鉴定。美国植物病理学会,明尼苏达州圣保罗,1991年。(2)T. J. White等人。载于:《PCR协议:方法与应用指南》第315页。M. A. Innis等人编。学术出版社,加利福尼亚州圣地亚哥,1990年。