Montero-Astúa M, Saborío-R G, Chacón-Díaz C, Villalobos W, Rodríguez C M, Moreira L, Rivera C
Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica (UCR), CP 11501-2060, San José, Costa Rica.
CIBCM-UCR and Facultad de Microbiología, UCR, San José, Costa Rica.
Plant Dis. 2008 Aug;92(8):1249. doi: 10.1094/PDIS-92-8-1249A.
Oleander (Nerium oleander L.) shrubs presenting mottling, leaf tip and margin scorch, short internodes, defoliation, and branch dieback were observed at different localities in the Central Valley in Costa Rica. Severity of the symptoms ranged widely, and most plants showed both diseased and healthy branches. In severe cases, entire sections of the plant were defoliated. Symptoms resembled those described for oleander leaf scorch (OLS) caused by the bacterium Xylella fastidiosa in the United States (3). This bacterium has been reported in coffee and citrus plants in Costa Rica. Sixty plants from five different places were sampled and tested using ELISA (Agdia Inc., Elkhart, IN) against X. fastidiosa. Thirty-five plants showed absorbance mean value of duplicate wells greater than the mean of control wells plus three times the standard deviation, and therefore were considered positive. Thirty-three of the sixty samples were processed for an immunofluorescence assay modified from Carbajal et al. (1) with antibody to X. fastidiosa (Agdia Inc.). Thirteen samples showed fluorescent rod-shaped bacilli with morphology similar to those observed from a pure culture of X. fastidiosa obtained from coffee. Ten of these thirteen samples were positive by ELISA. DNA extracts (2) from three of the oleander plants with high ELISA absorbance values were tested by nested PCR with primer pair 272-1/272-2 followed by the pair 272-1 int/272-2 int (4). Two of the samples were positive for the bacterium and one of the PCR products was cloned and sequenced in both directions (GenBank Accession No. EU009615). The negative (PCR mix) and positive (pure culture of X. fastidiosa isolated from grapevine) controls for nested-PCR were indeed negative and positive, respectively. The BLAST program was used to compare the sequence to the nucleotide collection (nr/nt) and Microbe Assembled Genomes databases in GenBank. All matches corresponded to X. fastidiosa sequences. The sequence showed 97% similarity with strains Found-4 (coffee strain from Brazil) and Found-5 (citrus strain from Brazil) and 96% similarity with strain Ann-1 from oleander in California. On the basis of serological, microscopic, and molecular detection of X. fastidiosa from oleander exhibiting symptoms of OLS similar to those reported in the literature, this pathogen likely is causing the symptoms we observed in Costa Rica. References: (1) D. Carbajal et al. Curr. Microbiol. 49:372, 2004. (2) M. J. Green et al. Plant Dis. 83:482, 1999. (3) Q. Huang et al. Plant Dis. 88:1049, 2004. (4) M. R. Pooler and J. S. Hartung. Curr. Microbiol. 31:377, 1995.
在哥斯达黎加中央山谷的不同地点，观察到夹竹桃（Nerium oleander L.）灌木出现斑驳、叶尖和叶缘焦枯、节间短、落叶以及枝条枯死的情况。症状的严重程度差异很大，大多数植株都有患病和健康的枝条。在严重的情况下，植株的整个部分都会落叶。这些症状与美国报道的由木质部难养菌（Xylella fastidiosa）引起的夹竹桃叶焦枯病（OLS）症状相似。这种细菌在哥斯达黎加的咖啡和柑橘植株中已有报道。从五个不同地点采集了60株夹竹桃植株样本，使用酶联免疫吸附测定法（ELISA，美国印第安纳州埃尔克哈特市的Agdia公司产品）检测是否感染木质部难养菌。35株植株重复孔的吸光度平均值大于对照孔平均值加上三倍标准差，因此被视为阳性。对60个样本中的33个进行了免疫荧光测定，该方法是根据卡瓦哈尔等人（1）的方法改进而来，使用了针对木质部难养菌的抗体（Agdia公司产品）。13个样本显示出荧光杆状杆菌，其形态与从咖啡中获得的木质部难养菌纯培养物中观察到的相似。这13个样本中有10个通过ELISA检测呈阳性。对3株ELISA吸光度值高的夹竹桃植株的DNA提取物（2）进行巢式PCR检测，引物对为272 - 1/272 - 2，随后使用引物对272 - 1 int/272 - 2 int（4）。其中2个样本检测到该细菌呈阳性，其中一个PCR产物进行了双向克隆和测序（GenBank登录号为EU009615）。巢式PCR的阴性对照（PCR混合液）和阳性对照（从葡萄中分离的木质部难养菌纯培养物）分别确实为阴性和阳性。使用BLAST程序将该序列与GenBank中的核苷酸数据库（nr/nt）和微生物组装基因组数据库进行比较。所有匹配结果均与木质部难养菌序列相对应。该序列与巴西咖啡菌株Found - 4和巴西柑橘菌株Found - 5的相似性为97%，与加利福尼亚夹竹桃菌株Ann - 1的相似性为96%。基于对表现出与文献报道相似的OLS症状的夹竹桃中木质部难养菌的血清学、显微镜和分子检测，这种病原体很可能是导致我们在哥斯达黎加观察到的症状的原因。参考文献：（1）D. 卡瓦哈尔等人，《当前微生物学》49:372，2004年。（2）M. J. 格林等人，《植物病害》83:482，1999年。（3）Q. 黄等人，《植物病害》88:1049，2004年。（4）M. R. 普勒和J. S. 哈通，《当前微生物学》31:377，1995年。
