Modern agriculture is dependent on both global supply chains and crop monocultures. These features aid the evolution and spread of novel plant pathogens. Limited genetic diversity in commercial crop lines can result in widespread susceptibility to emerging pathogens. Pathogen resistance may be developed through conventional breeding approaches, or a number of transgenic strategies. This thesis focuses on the characterisation of an emerging maize disease, Maize lethal necrosis (MLN), and engineering resistant maize lines using an artificial microRNA (amiRNA) approach. MLN is a synergistic viral disease caused by the interaction of Maize chlorotic mottle virus (MCMV) with any maize-infecting member of the potyviridae. I used next-generation RNA sequencing to characterise the MLN outbreak in East Africa, discovering that local and Chinese strains of the potyvirus Sugarcane mosaic virus (SCMV) typically coinfect with MCMV. A first global MCMV phylogeny was constructed using these samples combined with new Sanger sequencing of samples in Ecuador and Hawaii. The phylogeny supported previous hypotheses of a link between the Chinese and African outbreaks, and suggested a novel link between the Hawaiian and Ecuadorian outbreaks. The SCMV sequences generated demonstrated strong evidence of extensive recombination, in line with previous reports on SCMV and potyviruses. These data also produced first reports of a number of RNA viruses in East Africa, and five novel viral-like sequences, with their presence confirmed by RT-PCR. RNA silencing is an important component of the plant immune response to viral infection. amiRNAs can be used to generate specific and effective viral resistance through Watson- Crick base pairing between the amiRNA and the (RNA) viral genome. Previous amiRNA approaches have targeted invariable genomic regions using consensus sequences. However, the high mutation rate of RNA viruses means single cells contain a variety of mutant genomes, collectively called a quasispecies. To deter the evolution of resistance breaking I devised a novel strategy to include intra-sample variation from NGS data in amiRNA design, and constructs, each containing five of these amiRNAs, were transformed into tropical maize lines. RNA silencing may be hampered by the expression of viral suppressors of silencing (VSRs). Local VSR assays demonstrated that there are no local VSRs in the MCMV genome, while systemic VSR assays showed a possible systemic VSR role for the unique P32 protein, and an interesting link between photoperiod and systemic silencing more generally.

Sweet potato is one of the 15 most important food crops worldwide. At least 30 different virus species, belonging to different taxonomic groups affect sweet potato. Little is known about the viruses present in sweet potato crops in Central America, which is the primary origin of sweet potato. The objective of this study was to design and implement primers for use in polymerase chain reaction (PCR) and Reverse transcription-PCR (RT-PCR) to identify and survey the diversity of plant viruses infecting sweet potato in Honduras and Guatemala. Primers were designed and used to amplify, clone, and sequence a taxonomically informative fragment of the coat protein (CP) gene for whitefly-transmitted geminiviruses (herein, sweepoviruses) and potyviruses, and of the heat shock protein 70 (HSP70) for the Crinivirus, Sweet potato chlorotic stunt virus (SPCSV). The partial genome sequences were used for identification based on phylogenetic relationships with reference sequences available in the GenBank database. All three of the plant virus groups identified in this study were found to occur either in single or in multiple infections. Results of the sequence analyses indicated that the genomic regions amplified in this study were capable of discriminating among potyvirus species, and strains of SPCSV. With respect to potyvirus, all isolates were identified as Sweet potato feathery mottle virus (SPFMV) species, except for two, which grouped phylogenetically with Sweet potato virus G (SPVG) and Sweet potato virus C (SPVC). All sweepoviruses detected in sweet potato plants belonged to a single phylogenetically, well-supported group that contains all other previously described geminiviruses (sweepoviruses) associated with sweet potato or closely related host species. These results demonstrate that the primers designed for amplification of plant virus species commonly recognized to infect sweet potato, effectively detected the viruses singly and in mixtures from symptomatic plants, and that the resultant fragment, when subjected to cloning and DNA sequenced, was phylogenetically informative at the species and/or strain levels, depending on the virus group.

[ES] En este trabajo, se detectó por primera vez el virus de la clorosis amarilla de las cucurbitáceas (CCYV, cucurbit chlorotic yellows virus) infectando pepino y calabacín en Argelia. El CCYV (género Crinivirus, familia Closteroviridae) forma parte de un complejo de virus transmitidos por la mosca blanca Bemisia tabaci, que causan enfermedades de amarillamiento en las cucurbitáceas. Se obtuvo la secuencia completa del gen que codifica la proteína de la cápside (CP) y secuencias parciales de los genes RdRp y Hsp70 de un aislado argelino de CCYV y se compararon con las secuencias presentes en las bases de datos. ToLCNDV (género Begomovirus, familia Geminiviridae) también se detectó por primera vez infectando plantas de cucurbitáceas en Argelia. ToLCNDV es un begomovirus bipartito que causa importantes epidemias en cultivos económicamente valiosos de las familias Solanaceae y Cucurbitaceae. Se obtuvo la secuencia genómica completa de un aislado de ToLCNDV de pepino de Argelia (ToLCNDV-Biskra). La alineación de las secuencias de nucleótidos de los segmentos de ADN-A y ADN-B reveló identidades del 98,7% y 97,6%, respectivamente, con los segmentos de los aislados de la cuenca del Mediterráneo, mientras que la identidad con los aislados de Asia fue de aproximadamente del 90% y 81%. Los aislados presentes en la cuenca mediterránea son monofiléticos y forman un único grupo, mientras que los aislados de la cepa asiática presentan una mayor variabilidad genética y forman varios grupos. Todos los aislados de Argelia mostraron una variación de nucleótidos muy baja. Solo se observó una duplicación de 17 nucleótidos en el ADNB de algunos aislados que daría lugar a una proteína de movimiento 53 aminoácidos más larga, aunque la funcionalidad de esta supuesta proteína es desconocida. A pesar de esa duplicación, no se detectaron eventos de recombinación entre los aislados secuenciados. La baja variación genética podría dificultar la detección de recombinantes. / [CA] En aquest treball, es va detectar per primera vegada el virus de la clorosi groga de les cucurbitàcies (CCYV, cucurbit chlorotic yellows virus) infectant cogombres i carabassetes a Algèria. El CCYV (gènere Crinivirus, família Closteroviridae) forma part d'un complex de virus transmesos per mosca blanca que causen malalties d'esgrogueïment en les cucurbitàcies. Es va realitzar la determinació de la seqüència completa de la proteïna de la càpsida (CP) i seqüències parcials dels gens RdRp i Hsp70 d'un aïllat algerià de CCYV i es van comparar amb les seqüències publicades en les bases de dades. ToLCNDV (gènere Begomovirus, família Geminiviridae) també va ser detectat per primera vegada infectant plantes de cucurbitàcies a Algèria. ToLCNDV és un begomovirus bipartit que causa importants epidèmies en cultius econòmicament valuosos de les famílies Solanaceae i Cucurbitaceae. Es va obtindre la seqüència genòmica completa d'un aïllat de ToLCNDV de cogombre d'Algèria (ToLCNDV-Biskra). La alineació de les seqüències de nucleòtids del segments d'ADN-A i ADN-B van mostrar identitats del 98.7% i 97.6%, respectivament, amb els segments corresponents dels aïllats de la conca del Mediterrani, mentre que la identitat amb els aïllats d'Àsia va ser d'aproximadament el 90% i 81%. Els aïllats presents a la conca del Mediterrani són monofilètics i formen un únic grup, mentre que els aïllats de la soca asiàtica presenten una major variabilitat genètica i s'agrupen en diversos grups. Tots els aïllats algerians van mostrar una variació de nucleòtids molt baixa. Només es va observar una duplicació de 17 nucleòtids en d'ADNB d'alguns aïllats que lonaria lloc a una proteïna de moviment 53 aminoàcids més llarga, encara que la funcionalitat d¿aquesta suposada proteïnaés desconeguda. A més, malgrat la duplicació, no es van detectar esdeveniments de recombinació entre els aïllats seqüenciats. La baixa variació genètica podria dificultar la detecció de recombinants. / [EN] In this work, cucurbit chlorotic yellows virus (CCYV) was first detected infecting cucumber and zucchini in Algeria. CCYV (genus Crinivirus, family Closteroviridae) is part of a complex of whitefly-transmitted viruses that cause yellowing disease in cucurbits. Determination of the complete CP, and partial RdRp and Hsp70 sequences of an Algerian CCYV isolate was conducted to unveil the evolutionary relationships with the published isolates in databases. The phylogenetic analysis showed that the Algerian isolate clustered into group I together with the majority of the reported CCYV isolates. ToLCNDV (genus Begomovirus, family Geminiviridae) was also detected for the first time infecting cucurbit plants in Algeria. ToLCNDV is a bipartite begomovirus that causes major epidemics in economically valuable crops. epidemics in economically valuable crops of the Solanaceae and Cucurbitaceae families. Cucurbitaceae. The complete genome sequence of a ToLCNDV isolate from Algerian cucumber (ToLCNDV from Algeria (ToLCNDV-Biskra) was obtained. Alignment of the nucleotide sequences of the A-DNA and B-DNA segments revealed the identity of the DNA-A and DNA-B segments revealed identities of 98.7% and 97.6%, respectively, with the segments of the isolates from the Algerian segments of the Mediterranean basin isolates, while the identity with the Asian isolates was approximately 90% and 81%, respectively. was approximately 90% and 81%. The isolates present in the Mediterranean Basin are monophyletic and form a single monophyletic and form a single cluster, whereas isolates from the Asian strain have a higher genetic variability and form several clusters. genetic variability and form several clusters. All isolates from Algeria showed very low nucleotide variation. very low nucleotide variation. Only a duplication of 17 nucleotides was observed in the B-DNA of some isolates which would result in a longer 53 amino acid movement protein, although the functionality of this putative protein is unknown. Despite this duplication, no recombination events recombination events were detected among the sequenced isolates. The low genetic variation could hinder the detection of recombinants. / This work was supported by the following grants: Programa de Ayudas de Investigación y Desarrollo (PAID-11-21) of the Universitat Politècnica de València; PID2021-125787OR-C33 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; and PROMETEO project for excellence groups 2021/072 Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana). / Kheireddine, A. (2023). First report of Cucurbit Chlorotic Yellows Virus (CCYV) and Tomato Leaf Curl New Delhi Virus (ToLCNDV) in Algeria and Lack of Evidence for Seed Transmission of ToLCNDV in Melon and Pumpkin [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196840

Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen. / Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.