Improvements in the detection of plant pathogenic bacteria of the genus 'candidatus liberibacter' and evaluation of the transmission of 'ca. L. Solanacearum' to citrus plants
- Autores: María Quintana González de Chaves
- Directores de la Tesis: Felipe Siverio (dir. tes.), Estrella Marina Hernandez Suarez (codir. tes.)
- Lectura: En la Universidad de La Laguna ( España ) en 2021
- Idioma: español
- Tribunal Calificador de la Tesis: Cristina Giménez Mariño (presid.), Collin Jeffries (secret.), Maria del Carmen Asensio Sanchez Manzanera (voc.)
- Programa de doctorado: Programa de Doctorado en Biodiversidad y Conservación por la Universidad de La Laguna
- Materias:
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- Resumen
The uncultured bacterial genus 'Candidatus Liberibacter' is transmitted by psyllids (Hemiptera: Psyllidae) and associated with several plant diseases. ‘Ca. L. asiaticus' (CaLas), 'Ca. L. americanus' (CaLam) and 'Ca. L. africanus' (CaLaf) are associated with Huanglongbing (HLB) of citrus, and transmitted by Diaphorina citri and Trioza erytreae. ‘Ca. L. solanacearum' (CaLsol) is associated with several diseases in the solanaceous and apiaceous crops, as zebra chip in potato and is transmitted by Bactericera cockerelli, T. apicalis and B. trigonica.
Normally, the DNA extraction requires specimens destruction, preventing subsequent identification. Many laboratories prioritise the detection of the pathogen rather than the identification of their vectors, as some psyllid species require a specialist for its morphological identification. Therefore, it is necessary to find a method that allows the psyllid identification after DNA extraction. For this purpose, four specimen preparations (grinding, whole, cut-off head and punctured abdomen) and seven DNA extraction methods (CTAB, TRIsure™, DNeasy®, HotSHOT, Chelex, squashed on membrane and PBS) were compared. Psyllid grinding and HotSHOT were respectively the preparation and the method which obtained the the best results by qPCR, although the other non-destructive preparations and methods also allowed its detection. CaLsol could be detected after a long-term storage, without lack of sensitivity by using DNeasy®. In addition, cross-contamination was evaluated between CaLsol-infected and non-infected B. trigonica specimens in ethanol, obtaining that CaLsol-free psyllids were positive in qPCR analysis after incubation in ethanol with CaLsol-positive specimens in the same vials.
So far it has not been possible to cultivate CaLspp bacteria, so their detection is only possible by PCR. Because some of the current protocols have produced cross-reactions with non-infected tissues, a new protocol for qPCR on the rpoB gene with TaqManTM probe has been designed, which allows the detection of the plant pathogenic bacteria of the genus CaLspp in plant and insect tissues, with and without DNA extraction. The new qPCR protocol detected CaLaf, CaLam, CaLas and CaLsol in infected tissues, and showed no cross-reactions with the non-target samples tested. Comparative analyses with previous protocols showed that this new protocol has high specificity and sensitivity. In addition, the new method detected CaLsol and CaLas directly from crude plant and insect extracts.
The coexistence of citrus crops, affected by T. erytreae, with carrot crops, with high prevalence of CaLsol, could imply a risk of CaLsol transmission to citrus. Therefore, the transmission of CaLsol from carrot plants to citrus plants by cuscuta, grafting (budding) and T. erytreae was evaluated. CaLsol was transmitted to citrus by dodder, but infection was not systemically established. In assays by T. erytreae, it was able to acquire CaLsol from infected carrots and transmit it to healthy carrots immediately after 7 and 14 days (inoculation access period), although CaLsol was not detected after one-month post-inoculation. Trioza erytreae was unable to transmit CaLsol to citrus plants. The feeding behaviour of T. erytreae was studied by electrical penetration graphs (EPG) and its oviposition on carrots and citrus, reaching the phloem only on citrus and being unable to complete its life cycle on carrots. In summary, T. erytreae could acquire and transmit the bacterium from carrot to carrot with a low efficiency, but unable to transmit CaLsol from carrot to citrus.
In conclusion, this Doctoral Thesis has tried to improve the detection of CaLspp bacteria, and increase knowledge on the transmission of CaLsol. A characterization of various methods of DNA extraction and preparations of specimens that can be applied to the detection of CaLspp bacteria in their respective vectors has been done, and can be used as a guide for the choice of the most suitable method according to the purpose of the research. A qPCR protocol has been designed to detect the four pathogenic species of CaLspp in a single step, without non-specific amplification in non-infected tissues, for used in both rapid screening tests and epidemiological studies of CaLspp bacteria associated with disease. Finally, it has been concluded that transmission of CaLsol to citrus by T. erytreae is unlikely.
