Quantification of ‘Candidatus Liberibacter Asiaticus' in Fruit Tissues of Citrus Aurantifolia Christ. (Swingle), Poster Board #357

In order to begin the study of in planta distribution of Candidatus Liberibacter asiaticus (Las) in Mexican lime [Citrus aurantifolia Christ. (Swingle)], quantitative real-time PCR analyses were carried out in total DNA purified from different fruit tissues [seed, peduncle, flavedo (exocarp), albedo (mesocarp) and endocarp (juice vesicles plus lamella)] of six PCR (A2/J5)-positive huanglongbing-infected trees from Colima, México. The qPCR protocol was developed in a LightCycler^® 1.5 thermal cycler using a Taqman probe labeled with 6-FAM and BHQ-1 as reporter and quencher dyes, respectively, Las-specific primers previously published, and reaction conditions of 10 µL (final volume), 100 ng of total DNA, 50 cycles and ramp rates of 3 °C/s. For quantification of Las, a standard curve (error: 0.0209, efficiency: 1.868) constructed by serial dilutions (3.08 x 10³ to 3.08 x 10⁷ copies/µL, four replicates per dilution) of Las-16S rRNA gene cloned in pCR^®2.1-TOPO^® (analyzed by sequencing), was used to calculate the samples’ Ct values and bacterial titers. Results indicate that seeds, followed by fruit peduncle and flavedo contain the higher bacterial titers, which ranged from 9.16 x 10⁴ (Ct 37.75) to 3.60 x 10⁶ (Ct 33.50) bacterial cells/g of fruit tissue. This experiment supports previous ideas that Las distributes unevenly throughout the plant and specifically in fruit tissues, and reinforces current research in Mexico to understand huanglongbing pathology in this economically important crop. Fund FORDECyT-CONACyT agreement No. 139259.