Evaluation of DNA Amplification Methods for Improved Detection of “Candidatus Liberibacter Species” Associated with Citrus Huanglongbing

Abstract

Citrus huanglongbing (HLB), also known as citrus greening or citrus yellow shoot, is considered the most serious disease of citrus worldwide. The disease has Asian, African, and American forms caused by “Candidatus Liberibacter asiaticus”, “Ca. L. africanus”, and “Ca. L. americanus”, respectively, which can be spread efficiently by the psyllid vectors Diaphorina citri and Trioza erytreae and through contaminated plant materials. Infected citrus groves are usually destroyed or become unproductive in 5 to 8 years. The presumed low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to detect consistently. In this study, we compared and validated four conventional polymerase chain reaction (PCR)-based protocols, one loop-mediated isothermal amplification (LAMP) protocol, and three TaqMan real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays reported are improved compared with the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests for the presence of “Ca. Liberibacter spp.” in symptomatic samples. There were no differences in assay specificity among the standard format PCR-based methods evaluated. The TaqMan real-time PCR was 10- to 100-fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of “Ca. Liberibacter spp.” prior to the appearance of disease symptoms. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a “Ca. Liberibacter spp.”-free nursery system.

In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

“…1b). Similar Ct values (from 16 to 17) and a CLas concentration (10 7 CN g −1 of tissue) were observed by Li et al (2007 and and Tatineni et al (2008) in HLB symptomatic samples that had been naturally or artificially inoculated. Therefore, our estimate of the CLas population in symptomatic leaves of sweet orange plants inoculated by grafting seems consistent with published values.…”

“…This factor compensates for the use of 0.25 g in the extraction, and 25% of the extract obtained. Although recently Kim and Wang (2009) determined three sets of ribosomal genes in CLas our data were not corrected against the bacterial concentration obtained for the already published papers (Li et al 2006(Li et al , 2007(Li et al , 2008Manjunath et al 2008;Tatineni et al 2008), which assumed one copy of 16S rRNA gene in the CLas genome.…”

Technological Advances in Huanglongbing (HLB) or Citrus Greening Disease Management

“…The low concentration and irregular distribution of the pathogen in host plants, along with the inhibitors of PCR present in citrus extracts made the detection of the pathogen difficult. Although conventional PCR and qPCR are accepted techniques for the confirmation of trees symptomatic for HLB in Brazil and the United States, qPCR is considered much more sensitive and robust than conventional PCR and the technique has been validated with DNA extracts from different species of citrus and different tissues from diverse geographic regions (Li et al 2006(Li et al , 2007.…”

Detection of ‘Candidatus Liberibacter Asiaticus’ the Causal Organism of Huanglongbing, in Mandarin Group of Citrus

“…For successful management of HLB, accurate detection of pathogen is very important but, the detection of the pathogen is very difficult, because of its low titer and uneven distribution in its citrus hosts (Bove, 2006;Li et al, 2007). For HLB diagnosis, different parameters have been used including monitoring of natural vector of HLB in citrus groves, observation of HLB symptoms on citrus leaves and fruits, biochemical tests for the assessment of presence of HLB pathogen, microscopic identification of HLB pathogen and molecular detection of HLB pathogen.…”