Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR

Abstract

‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.

An effective DNA extraction method from Cajanus cajan seeds suitable for PCR analysis

“…This method is characterized by its speed, low cost and suitability for routine use in the processing of hundreds of samples simultaneously, such as population analyzes for breeding selection. The elimination of the purification step compensates for the use of small biological samples, resulting in less waste and allowing successful PCR amplification reactions to run smoothly (Warner et al, 2001;Quintana et al, 2022).…”

Interaction between the flagellum of Candidatus Liberibacter asiaticus and the vitellogenin-like protein of Diaphorina citri significantly influences CLas titer

“…Two μL of the supernatant obtained from method A was aspirated as the PCR template with primers OI1 and OI2 ( Supplementary Table 1 ). Method B extracted DNA from D. citri according to the CTAB method, and the specific steps were performed according to Quintana et al (2022) . A two-step assessment of C Las titer in a single D. citri was addressed: (1) the presence of C Las was detected by ordinary PCR; and (2) the C Las titer in infected D. citri was quantified by qPCR.…”

Plant Pathogenic Microorganisms: State-of-the-Art Research in Spain

“…The availability of sensitive and accurate pathogen detection methods is crucial for plant pathologists and disease management. In this Special Issue, the paper by Quintana et al [ 1 ] focused on this topic. The authors assessed different DNA extraction methods for the detection of the uncultured bacterium ‘ Candidatus Liberibacter solanacearum’ (CaLsol) by qPCR in the psyllid vector.…”