Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.
The Asian citrus psyllid, Diaphorina citri Kuwayama, one of the known vectors for citrus greening disease or Huanglongbing (HLB) pathogens, has been present in Texas for over a decade, but the detection of the disease is recent. HLB has been confirmed in only two adjacent commercial citrus groves of grapefruit and sweet orange. A study was conducted to compare the population of Candidatus Liberibacter asiaticus (CLas) cells in different plant parts including peduncle, columella, leaves, seeds, young shoots, flower buds, flowers, and bark of 6-year-old known infected grapefruit and sweet orange trees. The bacterial population was estimated using a previously described grand universal regression equation Y = 13.82 – 0.2866X, where Y is the log of the target copy number and X is the Ct (threshold cycle) of the assay. Except for bark tissue, there was no significant difference in the concentration of CLas cells in other plant parts between the two cultivars. Within the cultivar, the bacterial concentration also varied with the plant part, with peduncle, columella, midrib having significantly higher titer of CLas compared with other plant parts. The obtained results here are in agreement with previous studies conducted on Florida samples, but the consistently lowest bacterial titer recorded in young shoots, leaf blade, and especially leaf margins relative to the midrib has never been previously reported.