La necrosis bacteriana de la vid, causada por la bacteria de cuarentena Xylophilus ampelinus, es una enfermedad de difícil control químico y que ha causado en España importantes pérdidas económicas. Actualmente, es endémica en distintas zonas de Aragón y Galicia y se han identificado focos aislados en La Rioja. Ante la carencia de métodos sensibles y específicos de detección de esta bacteria que se puedan utilizar para el análisis rutinario, se ha puesto a punto un método de extracción y un protocolo de detección serológica de X. ampelinus. El método de extracción se basa en el lavado interno a presión de los sarmientos a analizar. El protocolo de detección se basa en la técnica ELISA-DASI, utilizando anticuerpos monoclonales específicos. Ambos hacen posible la detección de esta bacteria de cuarentena en material vegetal durante cualquier época del año. También se ha puesto a punto la detección mediante inmunoimpresión en membrana de nitrocelulosa, que puede ser utilizada para detección rápida o para confirmación de sintomatologías dudosas. Se ha realizado una prospección en las denominaciones de origen Campo de Borja y Somontano, utilizando el lavado interno de los sarmientos y la técnica ELISA-DASI. Se detectó la bacteria en el 4'7% y 0'9% de los sarmientos analizados, siendo la primera vez que mediante dicha técnica se detecta la enfermedad en sarmientos asintomáticos y en la zona de Somontano. En la prospección se observó una distribución irregular de X. ampelinus en las cepas analizadas. La distribución de la bacteria en las plantas de una parcela naturalmente infectada fue al azar. Se realizó un estudio de sensibilidad a la necrosis bacteriana en plantas de 19 variedades españolas injertadas en los patrones Rupestris de Lot, R-110 y 41-B. Se observó que el primer patrón confería mayor sensibilidad a las variedades injertadas y que todas las variedades ensayadas se mostraron sensibles a X. ampelinus. Entre ellas, las variedades Merseguera, Palomino, Airén, Bobal, Juan Ibáñez y Granegro se comportaron como muy sensibles y Mazuela, Tinto basto y Garnacha peluda mostraron baja sensibilidad. / Cambra Alvarez, M. (1997). La necrosis bacteriana de la vid, causada por xylophilus ampelinus. Detección serológica, distribución en Aragón y sensibilidad varietal [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/10952

>Magister Scientiae - MSc / Xylophilus ampelinus, the causal agent of bacterial blight and canker of grapevines, has long been a threat to the table grape industry in the Western Cape, leading to severe economic losses due to the reduced productivity and shortened lifespan of infected grapevines. Very little is known about the genetic makeup of the organism, especially with regard to the factors that contribute to its pathogenicity. Generally, bacterial pathogens directly inject the effector proteins into host cells via Type III secretion system (T3SS). In the attempts to identify and characterize the T3 secreted effectors, different reporter plasmid systems have been used to study the secretion and translocation mechanisms the effectors employ during pathogenicity. The aim of the study was to generate a T3 reporter plasmid system for X. ampelinus that will allow the identification and classification of potential pathogenicity factors as members of the Type III secretion class of effectors. First, the avrBs1 family genes avrBs1 and avrA were identified and characterized. The two avirulence genes induced HR on Nicotiana tabacum leaves. Due to the relatedness of the X. ampelinus avr sequences to those of xanthomonads, and the fact that Xanthomonas avrBs1 has been successfully used in a number T3 effector studies, it was decided to construct an X. ampelinus T3 effector reporter vector based on the avrBs1 gene. The minimal segment of the X. ampelinus AvrBs1 protein C-terminus, sufficient for recognition inside host cells and also responsible for HR-induction was identified and characterized using Agrobacterium-mediated transient expression. The AvrBs157-413 HR-inducing domain was cloned in-frame with the 3x FLAG epitope, into a broad-host range vector. To test the reporter vector, the full length avrBs1 sequences of X. ampelinus and Xanthomonas campestris pv. campestris were cloned ahead of the 3x FLAG epitope and the constructs were transferred into XaΔavrBs1 knockout mutant to test for protein secretion. Furthermore, the reporter construct was tested for Type III protein translocation on Bs1 resistant pepper cultivar STAR 6657. Optimization of protein secretion and translocation assays is however required for the improved results. This might include the application of an alternative protein tag to identify candidate X. ampelinus T3SS effectors.

Thesis (MSc (Genetics))--University of Stellenbosch, 2011. / Includes bibliography / ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development. / AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.