Genome-based PCR primers for specific and sensitive detection and quantification of Xylella fastidiosa

被引:126
作者
Francis, Marta
Lin, Hong
Cabrera-La Rosa, Juan
Doddapaneni, Harshavardhan
Civerolo, Edwin L.
机构
[1] USDA, ARS, San Joaquin Valley Agr Sci Ctr, Parlier, CA 93648 USA
[2] Univ Calif Davis, Dept Plant Pathol, Davis, CA 95616 USA
[3] Univ Calif Riverside, Dept Entomol, Riverside, CA 92521 USA
[4] Univ Calif Davis, Dept Viticulture & Enol, Davis, CA 95616 USA
关键词
almond leaf scorch; citrus variegated chlorosis; conventional PCR; oleander leaf scorch; Pierce's disease of grapes; tquantitative PCR;
D O I
10.1007/s10658-006-9009-4
中图分类号
S3 [农学(农艺学)];
学科分类号
0901 ;
摘要
Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan (TM)) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce's disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10(5) cells per reaction in water and grape extracts and 10-10(5)cells in insect DNA. Regression curves were similar, with correlation coefficients of r(2) > 0.97. In quantitative PCR, C-t values ranged between 20 and 36 cycles for 5-10(5) bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.
引用
收藏
页码:203 / 213
页数:11
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