Rapid detection of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing (Greening) disease using PCR.

摘要

A study was conducted to detect the presence of Liberibacter (greening disease; Huanglongbing, HLB) by PCR in samples of citrus (Citrus sinensis) plants from various places in Maharashtra, India, in 2003. Buds were collected from symptomatic trees. Shoots were side grafted to sweet orange indicator plants. DNA was extracted from leaf midribs and bark of the indicator plants. Primers OI1 and O12c defined from the 16S rDNA sequence of Candidatus Liberibacter asiaticus (strain Poona, India) were designed. Infected leaves were small, upright and frequently had various types of chlorotic mottling and Zn deficiency-like symptoms. Typical symptoms (severe vein yellowing and leaf mottle on the emerging shoots) on the inoculated indicator plants developed within 4-6 months after grafting. PCR produced an amplified fragment of expected size (1160 bp), which was observed in indicator plant samples infected by various isolates of Candidatus Liberibacter asiaticus from various places in Maharashtra. No amplification was obtained from water or DNA extracted from healthy citrus. Compared to the more conventional biological indexing, this PCR method permitted much more rapid detection of HLB bacterium. The protocol consists of three major steps: (i) extraction of template DNA from citrus tissue; (ii) PCR amplification and (iii) analysis of PCR products. The entire procedure can be completed within a 6-h time period: 2 h for DNA extraction, 3 h for PCR, and 1 h for analysis of PCR products by agarose gel electrophoresis. The PCR-based assay was also able to overcome difficulties caused by the low concentration and uneven distribution of HLB bacterium in citrus hosts.