A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects

Abstract

Four pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay.

Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method

“…A total of 141 plant and insect samples and bacterial suspensions were quantified by real-time PCR ( de Chaves et al, 2023 ) and comparatively analyzed by the new real-time RPA developed in this work ( Supplementary Table S1 ). Particularly, 131 samples of nine different ‘ Ca .…”

“…The presence/absence and absolute quantification of ‘ Ca . Liberibacter spp.’ in all samples were determined using the real-time qPCR detection protocol described by de Chaves et al. (2023) from DNA purification of each sample.…”

“…For HLB-infected material, three replicates of 10-fold serially diluted (from 10 −1 to 10 −6 ) crude CaLas-infected extracts (samples 12,112.6 and 11,661.7) in healthy extracts were analyzed. Absolute quantification of CaLas was performed in each dilution by real-time PCR according to de Chaves et al. (2023) .…”

“…Furthermore, in order to evaluate the limit of detection (LOD) in infected samples, 76 positive samples infected with CaLas, CaLaf, and CaLam isolates were quantified by real-time qPCR ( de Chaves et al., 2023 ), and the absolute result was compared with the new real-time RPA protocol.…”

“…Within the molecular detection techniques, the polymerase chain reaction (PCR) is the gold standard reference technique for HLB diagnosis ( Valdés et al., 2016 ; Morán et al., 2020 ). In this regard, according to the latest HLB diagnostic protocols published by the European and Mediterranean Plant Protection Organization (EPPO) and the International Plant Protection Convention (IPPC), PCR protocols that amplify partial regions of several genes ( Jagoueix et al., 1996 ; Hocquellet et al., 1999 ; Teixeira et al., 2005 ; Li et al., 2006 ; Lin et al., 2010 ; Morgan et al., 2012 ; Zheng et al., 2016 ; de Chaves et al., 2023 ) are the most recommended for the detection of ‘ Ca . Liberibacter’ spp.…”