Immune Tissue Print and Immune Capture-PCR for Diagnosis and Detection of Candidatus Liberibacter Asiaticus

Abstract

'Candidatus Liberibacter asiaticus' (CaLas), associated with citrus Huanglongbing (HLB), is a non culturable member of the α-proteobacteria. In this study serologically based methods for the detection of CaLas were developed. An anti-outer membrane protein A (OmpA) polyclonal antibody previously produced (in our laboratory) was highly effective for the detection of CaLas from citrus tissues in a simple tissue printing format. The antibody was also used to capture bacteria from periwinkle extracts. About 80% of all field 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 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 highlights 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'.

Figure 1. Detection of ‘Ca. Liberibacter asiaticus’ in different tissues of sweet orange using tissue prints.

(a–d) Sections of petiole, stem, seed and root; respectively. ‘Ca. Liberibacter asiaticus’ in sweet orange is on the left in each panel with healthy sweet orange (h) on the right. (a1–d1) Higher magnification of the portions of the images indicated by arrows in a–d shows the localization of the purple spots in the phloem cells.

Figure 2. Immune capture-PCR for the detection of ‘Ca. Liberibacter asiaticus’ isolate B432 (Florida).

M: 1 kb DNA Ladder, +: Purified CaLas DNA positive control; -: water only control. Lane 1, B432 extract and anti-OmpA Pab (1:100); Lane 2, Healthy control with anti-OmpA Pab (1:100); Lane 3, B432 extract and anti-OmpA Pab (1:200); Lane 4, Healthy control with anti- OmpA Pab (1:200); Lane 5, B432 extract and anti-OmpA Pab (1:250); Lane 6, Healthy control with anti- OmpA Pab (1:250); Lane 7, No anti-OmpA Pab was added.

Figure 3. Immune capture-PCR for the detection of ‘Ca. Liberibacter asiaticus’ in periwinkle.

M: 1 kb Marker.+: Purified CaLas DNA positive control. -: water only control. Crude extract of periwinkle captured in 0.2 ml PCR tubes pre-coated with anti-OmpA Pab at a dilution of 1:200 and sample volumes of 20 μl, 50 μl, 100 μl and 200 μl in lanes 1,2; 4,5; 7,8; and 10,11 respectively. Crude extract of periwinkle captured in 0.2 ml PCR tubes without pre-coating with anti-Omp A Pab and sample volumes of 20 μl, 50 μl, 100 μl and 200 μl in lanes 3, 6, 9 and 12, respectively. Anti-OmpA Pab was used at a 1:200 dilution throughout.

Figure 4. Immune capture-PCR for the detection of ‘Ca. Liberibacter asiaticus’ in periwinkle and citrus samples.

M: 1 kb Marker.+: Purified CaLas DNA positive control; −: water only control. Lanes 1, 3, 5 and 7: Periwinkle plants DF1, DF2, DF3 and DF4, respectively with anti-OmpA Pab (1:200 dilution); 2, 4, 6 and 8: Periwinkle DF1, DF2, DF3 and DF4 respectively, without anti-OmpA polyclonal antibody; 9. Sweet orange CaLas B232 (Thailand) with anti-OmpA Pab (1:200 dilution); 10. CaLas B232 (Thailand) without anti-Omp A Pab; 11. Sweet orange B430 (Japan) with anti-Omp A Pab (1:200 dilution); 12: B430 without anti-OmpA Pab; 13. Healthy sweet orange control.

Figure 5. Detection of ‘Ca. Liberibacter asiaticus’ by tissue printing of symptomatic and asymptomatic sweet orange leaves collected from Florida citrus groves.

Cq values are from qPCR assays performed on the same samples that were printed. Three leaves with blotchy mottle symptoms are on the left (a); and six leaves from the same tree but without symptoms are in the center (b); 3 leaves from a healthy tree on the right (c). Panel d contains higher magnification images of the tissue prints from the samples in the top row to illustrate the strict localization of the specific color reaction in phloem vessels.