Lee H B, Hong J P, Kim S B
Division of Applied Bioscience and Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea.
Department of Microbiology, Chungnam National University, Daejeon 305-764, Republic of Korea. This study was in part supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
Plant Dis. 2010 Nov;94(11):1372. doi: 10.1094/PDIS-05-10-0374.
In September 2009, leaf blights were observed on rice (Oryza sativa L., variety Dongjin 1 and Hopyeong) in paddy fields located in Gwangyang and Naju, Jeonnam Province, Korea. Lesions appeared first as water-soaked stripes or light brown-to-slightly reddish spots on the upper blades of the leaves, ultimately causing leaf blight and stalk rot. Ten strains of bacteria were isolated from the blighted leaf samples and four isolates (EML-ORY1, -ORY2, -ORY3, and -ORY4) suspected to be Pantoea spp. were selected on the basis of colony types and sampling sites. The isolates readily grew at 27 to 32°C but growth was significantly lower at 35°C. Using the API 20E system, EML-ORY1, 2, and 3 showed the same reaction patterns and gave 15 positive reactions whereas EML-ORY4 gave 11 positive reactions, but results were negative for arginine dihydrolase, citrate utilization, sorbitol fermentation, and rhamnose fermentation. All strains were considerably different from Pantoea agglomerans ATCC27155, which produced nine positive reactions. The strains were identified based on the 16S rRNA gene sequence analyses. A neighbor-joining tree was generated for the four isolates using PHYLIP with the following known bacterial strains: P. agglomerans DSM3493; P. vagans LMG24199; P. eucalypti LMG24197; P. ananatis ATCC19321; and Kluyvera georgiana ATCC51603. The four isolates from rice formed a monophyletic cluster and were most closely related to P. agglomerans DSM3493 (GenBank AJ2334231) with an average 16S rRNA sequence similarity of 99.0%. GenBank Accession numbers for the four isolates are: EML-ORY1, HM854282; -ORY2, HM854283; -ORY3, HM854284; and -ORY4, HM854285. On the basis of molecular phylogenetic analyses and API 20E test, we determined that the causal pathogen might be a subspecies of P. agglomerans. Pathogenicity tests were performed on 2-week-old rice seedlings (variety Hopyeong) in duplicate with bacterial suspensions containing 1.5 × 10 CFU/ml with 0.001% Tween 20. Of the isolates, EML-ORY3 demonstrated the strongest pathogenicity to rice seedlings when evaluated by five scoring systems on the basis of symptom development and severity levels. Disease symptoms appeared 3 days after artificial inoculation. Symptoms on the inoculated leaves were similar to those of natural infection and included water-soaked stems with a light brown color, blighted leaves, and stalk rot, with no symptoms found on water-treated controls. P. agglomerans, formerly called Enterobacter agglomerans (or Erwinia herbicola), is a group of gram-negative bacteria that belong to the family Enterobacteriaceae (3). Pantoea spp. are known to cause different diseases on a broad range of host plants including gypsophila, cotton, pineapple, maize, barley, onion, melons, and eucalyptus and also have been implicated as opportunistic pathogens in humans (1,2). P. agglomerans has been widely found in nature on leaves, fruits, and the seeds of many crops and is a known endophyte (1,2). To our knowledge, this is the first report of rice leaf blight caused by a putative subspecies of P. agglomerans in Korea. The importance of this pathogen to rice production in Korea is unknown. References: (1) Y. Feng et al. J. Appl. Microbiol. 100:938, 2006. (2) S. Manulis and I. Barash. Mol. Plant Pathol. 4:307, 2003. (3) M. P. Starr. The genus Erwinia. Page 1260 in: The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria. Springer-Verlag, NewYork, 1981.
2009年9月,在韩国全罗南道光阳和罗州的稻田中,观察到水稻(Oryza sativa L.,品种东津1号和宝平)出现叶枯病。病斑最初表现为叶片上部的水渍状条纹或浅褐色至微红色斑点,最终导致叶片枯萎和茎腐病。从病叶样本中分离出10株细菌,根据菌落类型和采样地点,选择了4株疑似泛菌属(Pantoea spp.)的分离株(EML-ORY1、-ORY2、-ORY3和-ORY4)。这些分离株在27至32°C下易于生长,但在35°C下生长显著降低。使用API 20E系统,EML-ORY1、2和显示相同的反应模式,有15个阳性反应,而EML-ORY4有11个阳性反应,但精氨酸双水解酶、柠檬酸盐利用、山梨醇发酵和鼠李糖发酵的结果为阴性。所有菌株与成团泛菌(Pantoea agglomerans)ATCC27155有很大不同,后者产生9个阳性反应。基于16S rRNA基因序列分析对菌株进行鉴定。使用PHYLIP为这4株分离株与以下已知细菌菌株生成了邻接树:成团泛菌DSM3493;漫游泛菌(P. vagans)LMG24199;桉木泛菌(P. eucalypti)LMG24197;菠萝泛菌(P. ananatis)ATCC19321;以及乔治克鲁维菌(Kluyvera georgiana)ATCC51603。从水稻中分离出的这4株菌株形成一个单系簇,与成团泛菌DSM3493(GenBank AJ2334231)关系最密切,16S rRNA序列平均相似性为99.0%。这4株分离株的GenBank登录号分别为:EML-ORY1,HM854282;-ORY2,HM854283;-ORY3,HM854284;以及-ORY4,HM854285。基于分子系统发育分析和API 20E测试,我们确定致病病原体可能是成团泛菌的一个亚种。对2周龄的水稻幼苗(宝平品种)进行致病性测试,一式两份,使用含有1.5×10 CFU/ml且添加0.001%吐温20的细菌悬液。在根据症状发展和严重程度水平的5种评分系统评估时,分离株EML-ORY3对水稻幼苗表现出最强的致病性。人工接种3天后出现病害症状。接种叶片上的症状与自然感染相似,包括浅褐色水渍状茎、枯萎叶片和茎腐病,水处理对照未出现症状。成团泛菌,以前称为聚团肠杆菌(Enterobacter agglomerans)(或草生欧文氏菌(Erwinia herbicola))),是一组革兰氏阴性细菌,属于肠杆菌科(3)。已知泛菌属在包括满天星、棉花、菠萝、玉米、大麦、洋葱、甜瓜和桉树在内的多种寄主植物上引起不同病害,并且在人类中也被认为是机会致病菌(1,2)。成团泛菌在自然界中广泛存在于许多作物的叶片、果实和种子上,是一种已知的内生菌(1,2)。据我们所知,这是韩国首次报道由成团泛菌的一个假定亚种引起的水稻叶枯病。该病原体对韩国水稻生产的重要性尚不清楚。参考文献:(1)Y. Feng等人,《应用微生物学杂志》100:938,2006年。(2)S. Manulis和I. Barash,《分子植物病理学》4:307,2003年。(3)M. P. Starr,欧文氏菌属。载于《原核生物:细菌栖息地、分离和鉴定手册》第1260页。施普林格出版社,纽约,1981年。