Polizzi G, Aiello D, Vitale A, Lahoz E, Nicoletti R, Hyakumachi M
Dipartimento di Scienze e Tecnologie Fitosanitarie, University of Catania, Italy.
CRA, Unità di Ricerca per le Colture Alternative al Tabacco, Scafati, Salerno, Italy.
Plant Dis. 2009 Apr;93(4):433. doi: 10.1094/PDIS-93-4-0433B.
Laurustinus (Viburnum tinus L.), native to the Mediterranean Region, is an evergreen shrub belonging to the Caprifoliaceae that is commonly cultured as an ornamental shrub or small tree. During the summer and autumn of 2007 and 2008, a widespread yellowing, partial foliar necrosis, or death of the whole plant was observed on 3- to 4-year-old potted plants of V. tinus in a commercial nursery in eastern Sicily (Italy). More than 20% of the plants showed disease symptoms. Infected roots, crowns, and stems turned dark brown, leaves gradually became necrotic, and infected plants were often killed. Diseased tissues were disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissue of Laurustinus. Fungal colonies were initially white, then turned light brown or brown with age, and formed irregularly shaped, light brown sclerotia after 10 days. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined with a microscope at ×400. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (4). Pairings were made with tester strains of binucleate Rhizoctonia AG-A through AG-S, except AG-J and AG-M. Anastomosis was observed only with tester isolates of AG-G. The rDNA-ITS of two isolates of BNR (DISTEF-Vt 31 and DISTEF-Vt 32) was sequenced (GenBank Accession Nos. AB478783 and AB478784, respectively) (3). The sequence from these two isolates exhibited 100% homology with BNR AG-G (GenBank Accession No. AY927334). Pathogenicity tests were conducted on potted, healthy, 6-month-old laurustinus. Twenty plants were inoculated by placing 1-cm plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1-cm PDA plugs as control. Plants were kept at 25°C and 95% relative humidity with a 12-h fluorescent light/dark regimen. Stem, crown, and root rot symptoms, identical to ones observed in nursery, appeared 20 days after inoculation, and all the inoculated plants showed symptoms within 1 month. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. R. solani was previously reported on Viburnum sp. in the United States (2). To our knowledge, this is the first report of binucleate Rhizoctonia causing disease on V. tinus. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. F. Farr et al. Page 1252 in: Fungi on Plants and Plant Products in the United States. The American Phytopathological Society. St. Paul, MN, 1989. (3) M. Hyakumachi et al. Phytopathology 95:784, 2005. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.
地中海荚蒾（Viburnum tinus L.）原产于地中海地区，是忍冬科的一种常绿灌木，通常作为观赏灌木或小树进行栽培。在2007年和2008年的夏秋季节，意大利西西里岛东部一家商业苗圃中，3至4年生的盆栽地中海荚蒾出现了普遍的黄化、部分叶片坏死或整株死亡现象。超过20%的植株表现出病害症状。受感染的根、根茎和茎变成深褐色，叶片逐渐坏死，受感染的植株常死亡。将患病组织在1%次氯酸钠中消毒1分钟，用无菌水冲洗，接种到添加了100毫克/升硫酸链霉素的马铃薯葡萄糖琼脂（PDA）平板上，然后在25°C下培养。从地中海荚蒾的患病组织中一直分离出一种双核丝核菌（BNR）。真菌菌落最初为白色，随后随着时间变为浅褐色或褐色，10天后形成不规则形状的浅褐色菌核。用1%番红O和3%氢氧化钾溶液染色（1），在400倍显微镜下观察，确定菌丝细胞为双核。通过在培养皿中的2%水琼脂上配对分离株来确定融合群（4）。除AG-J和AG-M外，与双核丝核菌AG-A至AG-S的测试菌株进行配对。仅与AG-G的测试分离株观察到融合现象。对两个BNR分离株（DISTEF-Vt 31和DISTEF-Vt 32）的rDNA-ITS进行了测序（GenBank登录号分别为AB478783和AB478784）（3）。这两个分离株的序列与BNR AG-G（GenBank登录号AY927334）具有100%的同源性。对6个月大的健康盆栽地中海荚蒾进行致病性测试。通过在茎基部附近放置来自5天龄菌丝体培养物的1厘米PDA菌块对接种20株植株。用同样数量的1厘米PDA菌块处理相同数量的植株作为对照。将植株置于25°C、相对湿度95%、12小时荧光光照/黑暗周期的环境中。接种20天后出现与苗圃中观察到的相同的茎、根茎和根腐症状，所有接种植株在1个月内均出现症状。对照植株保持健康。从有症状的组织中重新分离出双核丝核菌，完成了柯赫氏法则验证。此前在美国报道过茄丝核菌在荚蒾属植物上的情况（2）。据我们所知，这是关于双核丝核菌引起地中海荚蒾病害的首次报道。参考文献：（1）R. J. Bandoni. Mycologia 71:873, 1979.（2）D. F. Farr等人。载于《美国植物和植物产品上的真菌》第1252页。美国植物病理学会。明尼苏达州圣保罗，1989年。（3）M. Hyakumachi等人。Phytopathology 95:784, 2005.（4）C. C. Tu和J. W. Kimbrough. Mycologia 65:941, 1973.
