Testing for Candidatus Liberibacter asiaticus in leaf and root tissue of citrus and orange jasmine

Huanglongbing (HLB), a bacterial disease associated with CandidatusLiberibacter spp., is one of the major threats faced by the citrus industry worldwide. Ca. Liberibacter spp. are phloem limited, Gram-negative, alpha-proteo bacteria. These bacterial pathogens are naturally transmitted bypsyllid vectors, Diaphorina citri Kuwayama and Triozaerytreae (Del Guercio). In this study, a compilation of data obtained from the Texas A&M University--Kingsville's Citrus Center Diagnostics Laboratory was statistically analyzed from 2010 to 2012 to better visualize the distribution of threshold cycle (CT) values obtained from HLB suspicious samples. With the present threat on the rise, the preferred host for D. citri Kuwayama and HLB pathogen reservoir, Murraya paniculata (Orange Jasmine) plants were studied. An average of 40 M. paniculata residential plants were collected at different times of the year between 2011 and 2012 and were subjected toa multi-loci polymerase chain reaction using six distinct sets of primers targeting different genomeregions of the Ca. Liberibacterasiaticus (Las) strain. The results showed that the tested primer sets did not detect the presence of Lasin any of the M. paniculata samples. The presence of phytoplasmas was also verified using 40 randomly selected samples of previously tested M. paniculata plants. These samples were assayed through Conventional Polymerase chain reaction (cPCR) using the phytoplasma---specific primer set fU5/rU3 nested with primer set P1/P7 yielding no positive results for phytoplasmas in M. paniculata samples. The obtained results show that MOBio's PowerSoil RTM DNA extraction kit is more effective in removing root inhibitors yielding useful DNA for the qPCR assay. The results show that there was no significant difference (p>0.05) among CT values for root samples collected from different quadrants (N, E, S and W) at different distances from the tree trunk. There was also no significant difference (p>0.05) found among the CT values for root samples from previously confirmed Las-infected trees and Las infected tree stumps. There was, however, a significant difference (p<0.05) among different root sections of varying thickness with the thinnest (feeder roots) having lower CT values than the thicker roots. There was also a significant difference (p<0.05) found among the root samples collected from visually---determined HLB symptomatic and asymptomatic mature 'Valencia' sweet orange trees with symptomatic trees having an average bacterial titer of 3.32x10 8 cells/g and 2.6x108 cells/g for asymptomatic root samples. Moreover, a significant difference (p<0.05) was also found in collected samples from the same tree with a 6.72 CT difference among corresponding leaf and root samples.