Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

Abstract

'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

Fig 1. Localization and distribution of CaLas in sweet orange leaf midribs with blotchy mottling and zinc nutrient deficiency symptoms.

Rabbit polyclonal anti-OmpA antibody (1:5000) and goat anti-rabbit secondary antibody (1:50,000) conjugated with alkaline phosphatase were used. A1-A5: CaLas in individual phloem sieve cells of leaf midribs with symptoms of zinc nutrient deficiency. B1-B4: CaLas in both phloem sieve cells of petioles and secondary veins of leaves with blotchy mottle symptoms. C1-E4: Uneven distribution of CaLas in phloem cells in serial sections of leaf midribs from leaves with blotchy mottle symptoms. E5: Healthy sweet orange. The arrow in panel E4 indicates CaLas infected individual phloem cells densely stained by the antibody reaction and the arrow in panel E5 indicates the lack of color localized in the phloem cells of the healthy control specimen.

Fig 2. Localization and distribution of CaLas in a sweet orange leaf with yellowing symptoms.

Samples were taken from the abscission zone (A) to the proximal portion of the midrib (N). A-D: Lower titer of CaLas in the sections of petioles close to the abscission zone between the leaf and the stem. E-H: higher titer of CaLas in petiole between the abscission zones. I-K: Lower titer of CaLas in the sections of petioles close to abscission zone between the leaf blade and the petiole. M-N: higher titer of CaLas in the proximal portion of the midrib. O: healthy sweet orange control, sampled at the midpoint between the abscission zones. The arrow in panel N indicates CaLas infected individual phloem cells densely stained by the antibody reaction and the arrow in panel O indicates the lack of color localized in the phloem cells of the healthy control specimen.

Fig 3. Irregular distribution of CaLas in phloem sieve cells.

A-D: Localized infection of phloem cells by CaLas and E-K: generalized infection of phloem cells by CaLas. L: healthy sweet orange control, sampled at the midpoint between the abscission zones.

Fig 4. Localization and distribution of CaLas in sweet orange stem sections.

A-D: CaLas in stem with symptomatic leaves. E-K: CaLas in stem with asymptomatic leaves. L: healthy sweet orange stem. A’, F’, H’ and L’ are higher magnifications of A, F, H and L.

Fig 5. Localization and distribution of CaLas in sweet orange receptacle and peduncle.

A-D: Calas in the receptacle; E-K: CaLas in the peduncle. L: healthy sweet orange control. N: Branch, and M: peduncle for orientation.

Fig 6. Localization and distribution of CaLas in sweet orange seed.

A-G: CaLas infected and symptomatic ‘Valencia’ seeds collected from symptomatic fruit in Florida. H-K: Sweet orange seed from Florida (asymptomatic commercial fruit from supermarket). L: Healthy sweet orange seed.

Fig 7. Localization and distribution of CaLas in citrus roots.

A-H: CaLas in roots collected from HLB-symptomatic ‘Valencia’ orange tree 1. I-K: CaLas in roots collected from HLB-symptomatic ‘Valencia’ orange 2. L: Healthy sweet orange. M, N: Roots of ‘Valencia’ orange trees 1 and 2.

Fig 8. Localization and distribution of CaLas in whole leaves of periwinkle.

A-G: DTBIA of leaves from CaLas-infected periwinkle plants. H: Healthy periwinkle.

Fig 9. Visual symptoms in periwinkle leaves used for the DTBIA in Fig 8 with the Cq values for relative CaLas concentrations estimated by qPCR.