|DING, FANG - Huazhong Agricultural University
|BRLANSKY, RONALD - University Of Florida
Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/2017
Publication Date: 4/18/2017
Citation: Ding, F., Paul, C., Brlansky, R.H., Hartung, J.S. 2017. Immune tissue print and immune capture-PCR for diagnosis and detection of ‘Candidatus Liberibacter asiaticus’. Scientific Reports. 7:46467. doi:10.1038/srep46467.
Interpretive Summary: Citrus greening or huanglongbing disease has caused tremendous damage to the citrus industry of Florida, reducing production to only 30% of previous levels. The disease is caused by infection of citrus trees by a bacterial pathogen that has not been cultured in the laboratory, is difficult to detect and impossible to cure. We describe the development and optimization of two new methods to detect the pathogen in infected citrus trees. Both methods are based on a novel antibody that we have developed that binds to the surface of the pathogen. The first method produces images of plant tissues on a special paper that and can be used to see the bacterium in infected plants with microscopic detail. The second method uses the antibody to purify the bacterium from infected plants and is combined with molecular methods to detect the DNA of the bacterium with high sensitivity and specificity. Both methods are improvements over current methods for detection of the pathogen. Our methods will be useful to researchers who study huanglongbing disease of citrus and to regulatory officials who seek to prevent its further spread in the United States.
Technical Abstract: ‘Candidatus Liberibacter asiaticus’ (CaLas), associated with citrus Huanglongbing (HLB), is a non culturable member of the a-proteobacteria. Because CaLas has not been cultured in vitro, there has been little work done on serologically based methods for its detection. In the present study, a simple, in situ immune tissue print method for the detection of CaLas was optimized and methods for immune capture followed by PCR were developed. The results showed that an anti OmpA polyclonal antibody was highly effective for the detection of CaLas from citrus petioles, stems, seeds and roots in a simple tissue printing format. The antibody was also used to capture bacteria from both citrus and periwinkle extracts. When field samples of known CaLas-infected citrus were tested, about 80% of all samples analyzed tested positive with both immune tissue printing and qPCR; whereas 95% were positive with at least one of these two methods. When asymptomatic citrus tissues were tested, the tissue printing method gave a higher rate of detection (83%) than the qPCR method (64%). This result is consistent with a lower concentration of CaLas DNA, but a higher proportion of viable cells, in the asymptomatic tissues. The immune tissue printing method also preserves the detail of the spatial distribution of ‘Ca. Liberibacter asiaticus’ in diseased citrus tissues. Both the immune capture PCR and immune tissue printing methods offer the advantages of low cost, high throughput, ease of scaling for multiple samples and simplicity over current PCR-based methods for the detection of ‘Ca. Liberibacter asiaticus’.