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

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Jason Griffin / Feeding preference experiments with the pine sawyer beetle (Monochamus carolinensis Olivier) were conducted using eleven taxa of Pinaceae. One newly emerged adult beetle (≤ 24 hours) was placed into each feeding arena (n = 124) containing three or four shoots of current season's growth from different tree species (one shoot per species) for choice experiments. Beetles were allowed to feed for 48 (2011) or 72 (2012-2014) hours, at which point shoots were removed and data collected on feeding occurrence and percent feeding area. Augmented design analyses of feeding occurrence and percent feeding area of the eleven taxa did not indicate significant evidence for feeding preferences of the pine sawyer beetle on most taxa except for a higher preference for both scots (Pinus sylvestris L.) and eastern white (P. strobus L.) pines compared to deodar cedar [Cedrus deodara (Roxb. ex D. Don) G. Don]. The feeding preference experiments suggest that pine sawyer beetle may feed on a wide-range of Pinaceae taxa. Virtual plant walk maps were developed using a web-application for two semesters of an ornamental plant identification course (n = 87). The maps allowed students to revisit plants and information covered in lecture and laboratory sections at their own convenience, using either a computer or mobile device. Survey results indicated 63% of the students used the virtual plant walk maps as a study resource. Students mainly used the maps to study the most current plant list. Most students who used the virtual maps did so to review the plants online. When asked to rate usefulness of the maps on a scale from slightly useful (1) to very useful (3), 43% of students indicated that the virtual maps study tool was very useful, 25% indicated the maps were useful, and 8% indicated that the maps were slightly useful. A significant dependence between student use frequency and student usefulness ratings of virtual plant walk maps was observed. As students’ use of the virtual maps increased, they perceived the maps to be more useful to their studies in preparing for identification quizzes. No differences between plant identification quiz scores were observed between students who used and did not use the virtual plant walk maps or between learning styles. Our survey indicated students used the virtual plant walk maps as a resource and perceived the maps as a useful tool in preparation for identification quizzes.

>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.

The longhorn beetle Monochamus galloprovincialis (Oliver, 1795)) is a secondary wood-boring pest of pine trees. It is also an important vector of the pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer, 1934)), a quarantine organism that has caused large damage to pine forests in Asia and Portugal. Therefore, it is desirable to monitore the vector and its associated organisms. The aim of this work was to gain insight into the nematodes transmitted by Monochamus galloprovincialis and to evaluate their biological interactions based on literary sources. The trapping of the longhorn beetle was counducted at ten locations in the Czech Republic by teflon coated pheromone traps in 2015, 2016 and 2017. The extraction of nematodes was done by macerating the captured beetles in distilled water. Determination of nematodes was done on the basic of morphological and molecular features. In total, 1704 individuals of Monochamus galloprovincialis were analyzed, 12,6 % of them carried nematodes. Following taxa of nematodes were found: Aphelenchoides sp., Aphelenchoides stammeri, Bursaphelenchus mucronatus, Contortylenchus sp., Deladenus sp., Diplogasteroides sp., Ektaphelenchoides sp., Parasitaphelenchus sp., Parasitorhabditis sp., and larvae of the order Tylenchida.

Le nématode du pin Bursaphelenchus xylophilus, originaire des Etats-Unis, est responsable de dépérissements massifs dans les forêts de pin des pays où il a été introduit. Il utilise comme vecteur un insecte du genre Monochamus, notamment M. galloprovincialis en Europe. L’objectif de cette thèse est de fournir des informations concernant les capacités de dispersion de ce coléoptère afin de mieux prédire le risque d'invasion. A l'aide d'expériences en manège de vol, nous avons montré que les adultes émergents, encore immatures, sont déjà en mesure de maintenir des vols soutenus. Ils peuvent parcourir une distance d'environ 500m par jour et les capacités de vol augmentent avec la prise de poids pendant la phase de maturation sexuelle. Il n'existe pas de compromis d'allocation de l'énergie entre fonctions de dispersion et de reproduction pendant cette période et nous proposons de qualifier ces insectes de "income breeder" et "capital disperser". A l'état mature M. galloprovincialis est capable de voler un total de 16 km, avec des vols individuels d'environ1 km; ces performances augmentent avec l'âge et le poids des insectes. Le maintien des capacités de vol tout au long de la vie adulte de M. galloprovincialis conduit à proposer que les distances potentiellement parcourues dépendent surtout de la longévité de l'insecte. Dans une expérience d'élevage en conditions contrôlées nous avons montré que la vie adulte dure au moins 4 mois, augmentant quand les fluctuations thermiques sont atténuées. Ces résultats ont des implications pour la gestion de B. xylophilus en Europe puisqu'ils démontrent que la méthode d'éradication par coupe rase sur un rayon de 500m est probablement inopérante. / The pinewood nematode (PWN) Bursaphelenchus xylophilus, native to the USA, is responsible of the pine wilt disease, causing large economic losses in the pine forests of all countries where it has been introduced. It uses as vector an insect of the genus Monochamus, e.g. M.galloprovincialis in Europe The objective of this thesis is to provide information about the dispersalabilities of, this beetle in order to better predict the spread rate of the PWN. Using flight mill experiments, we showed that immature adult beetles were able to fly since their emergence. The meanflight performance was ca. 500m per day, increasing with weight gain during the maturation period.There was no trade-offs in energy allocation between reproduction and dispersion. We therefore suggest that M. galloprovincialis is both "income breeder" and "capital disperser". Mature beetles were able to fly a total of 16km on average during their lifetime, with individual flights of ca. 1km. Flight performances increased with age and body weight, being equal for males and females. Because flight capacity remained constant across time, we suggested that the total distance potentially covered by beetles was mainly driven by their longevity. In a rearing experiment under controlled conditions we estimated the adult lifespan at 4 months. It was reduced under temperature regime with higheryearly thermal fluctuations. These results have important implications for the management of the PWN since they demonstrate that precautionary clear-cuts of 500m radius are likely to be ineffective in eradicating the disease.

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.