Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody

Authors: DING, FANG - Huazhong Agricultural University; Duan, Ping; Paul, Cristina; BRLANSKY, RONALD - University Of Florida; Hartung, John

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/9/2015
Publication Date: 5/6/2015
Publication URL: http://handle.nal.usda.gov/10113/61521
Citation: Ding, F., Duan, Y., Paul, C., Brlansky, R., Hartung, J.S. 2015. Localization and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immuno assay with an anti-OmpA polyclonal antibody. PLoS One. 10(5):e0123939. doi:10.1371/journal.pone.0123939.

Interpretive Summary: ‘Candidatus Liberibacter asiaticus’ (CaLas), is the causal agent of citrus Huanglongbing (HLB). Antibodies provide rapid and inexpensive assays for plant pathogens, but until now, antibodies against CaLas have not been widely used in studies of this pathogen. We demonstrated and used a simple antibody based assay to detect CaLas in different citrus tissues and in periwinkle leaves. Our assay preserved the anatomical detail of the plant tissues that were sampled, something that was not possible with previous assays. CaLas was unevenly distributed in the tubes that the plant used to conduct sugar solutions and where the insect that transmits the pathogen feeds, throughout the roots, stems, leaves and fruit. Colonies of CaLas in these tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves, both with an uneven distribution pattern. Our data provide direct spatial and anatomical information for CaLas in planta, which has not been possible with previous methods. Our method is simple and scalable method may facilitate the future research on the interaction of CaLas and host plant. It is also faster cheaper and better for field diagnosis of HLB than current methods, and may be useful for diagnostic application.

Technical Abstract: ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the a-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.