Location: Subtropical Plant Pathology Research
Title: Nested PCR is essential for the detection of extremely low titer of Candidatus Liberibacter asiaticus from citrus and its vector psyllid Diaphorina citri Authors
Submitted to: American Phytopathology Society
Publication Type: Abstract Only
Publication Acceptance Date: March 13, 2008
Publication Date: N/A
Technical Abstract: Citrus huanglongbing (HLB), transmitted by the psyllids Diaphorina citri and Trioza erytreae, is one of the most devastating diseases of citrus worldwide. The disease is associated with three different species of Candidatus Liberibacter: Ca. L. asiaticus (Las), Ca. L. americanus and Ca. L. africanus. Currently detection and diagnosis of HLB rely on the typical symptoms of HLB such as leaf blotchy mottle and PCR confirmation using various sets of Las-specific primers. Due to the complex nature of HLB, conventional PCR and real-time PCR were not able to detect the bacterium in certain phenotypes of the disease or from psyllids that carried a low titer of the bacterium. Three sets of primers for nested PCR were developed which targeted three different genetic loci, 16S rDNA, ß-operon and the outer membrane protein gene of the bacterium. With any one or a combination of these nested PCR Las-specific primer sets, the detection rate of Las bacterium in psyllids collected from HLB infected citrus groves was 20-30% higher than the detection rate by real-time PCR. Using nested PCR coupled with improved sampling and bacterial DNA isolation methods, we also increased detection of the Las bacterium from citrus exhibiting atypical HLB symptoms. These results demonstrated that nested PCR is essential in the detection of Las bacterium when the pathogen is present at a very low level, and it is extremely important in screening for HLB-free germplasm in citrus.