A Phage/prophage-Based Pcr System for Sensitive and Specific Detection of “candidatus Liberibacter Asiaticus and Spiroplasma
Crop Diseases, Pests and Genetics
2013 Annual Report
1a.Objectives (from AD-416):
The proposed research project is to develop PCR detection systems that are highly sensitive and specific in detection of “Ca. L. asiaticus” and S. citri.
1b.Approach (from AD-416):
1. Identification and characterization of phages/prophages in “Ca. L. asiaticus” and S. citri through data-mining from available DNA sequences.
2. Selection and evaluation of candidate phage/prophage genes for their capacity in pathogen detection.
3. Testing the phage/prophage-based detection systems using clinical field insect and plant DNA samples.
This research is in support of Objective 1.B (Genome sequence analyses of plant pathogens) of the in-house project. A sequence-based polymerase chain reaction (PCR) protocol was developed for sensitive detection of Spiroplasma citri, the pathogen causing citrus stubborn disease in California. Based on recent information that the citrus stubborn pathogen harbors multiple copies of prophage genes, two PCR primer sets were developed. The primer sets were tested against 18 pure bacterial cultures and 250 field samples collected in two citrus orchards in California from 2007 to 2011. The prophage PCR primer sets were 1,000 times more sensitive than conventional PCR primer sets designed from single copy genes. The higher detection sensitivity of the PCR assay based on multicopy prophage genes will improve stubborn disease management, as early pathogen detection will allow early removal of infected trees.