THE DETECTION AND CHARACTERIZATION OF SOME VIRUSES INFECTING BLACKBERRY AND CHERRY IN SOUTH CAROLINA

Three separate virus research projects were conducted. Blackberry yellow vein disease (BYVD), a disorder caused by virus complexes, has become a major threat to blackberry production in the United States, especially in the southeastern part of the country where blackberries are grown for the fresh market. More than 30 viruses have been found to be associated with the disease. Most of these induce no symptoms when infecting the plant alone. However, when more than a single virus is present in the host visible symptoms are displayed. The incidence of 6 different viruses (Blackberry yellow vein-associated virus, Blackberry virus Y, Blackberry chlorotic ringspot virus, Blackberry virus E, Blackberry virus Ω, and Tobacco ringspot virus) that have been commonly found in BYVD-infected plants was studied using sentinel plants dispersed in plantings of blackberry in the field. Experiments were completed at the two largest commercial blackberry farms in South Carolina using more than 1200 sentinel plants over the course of three years. The sentinel plants were tested for the presence of the 6 viruses before they were exposed in the field and were again tested for the presence of the 6 viruses after the plants had been recovered from the field and allowed to overwinter in the greenhouse. Both Blackberry virus E, and Blackberry virus Ω were found infecting blackberry in South Carolina for the first time. A potential new ilarvirus was identified in blackberry and veronica. Partial sequence information for the 3 genomic molecules has been obtained. The virus shows closest homology to the members of subgroup 1 of the genus Ilarvirus, but is unique. This subgroup includes BCRV, one of the viruses previously associated with the BYVD complex. Symptoms typical of virus infection were observed in the suckers/watersprouts growing from the ‘Mazzard’ rootstock of a flowering cherry tree growing at Musser Farm, Clemson University in 2011. However, the scion of the tree, Prunus serrulata cv. Shirofugen, displayed no symptoms. Double-stranded RNA was isolated from the symptomatic tissues of the rootstock and used to provide templates for cDNA cloning and for nucleotide sequencing. Sequence data showed the virus to be most closely related to Cherry rusty mottle-associated virus

The molecular characterization of the virus and virus-line agents present in Ta Tao 5 germplasm of Prunus persica

Tesis para obtener el grado de Doctor of Philosophy Plant and Environmental Sciences, de Clemson University, diciembre 2007Peach production in the southeastern United States is limited by late spring freezes. Ta Tao 5 germplasm, used either as an interstem or by chip bud inoculation, has been shown to delay bloom and avoid the effects of these late freezes. The growth modification is graft transmissible and the germplasm has been found to be infected with ACLSV, APruV-3, and PLMVd. Using a combination of PCR, cloning, and sequencing techniques, a molecular characterization of the three graft-transmissible agents present in Ta Tao 5 has been completed. The complete nucleotide sequence of the genome of the isolate of ACLSV (ACLSV-Ta Tao 5) was determined. The genomic organization was typical of other isolates of ACLSV, but the sequence showed only 73% nucleotide identity to the Batalon1 isolate of ACLSV. This distant relationship with characterized isolates of ACLSV explains why primers recommended for PCR reactions used to identify the virus failed to detect the isolate from Ta Tao 5 reliably. This is the first complete genomic sequence of an isolate of ACLSV from peach. The 3' terminal third of the complete sequence of APruV-3 isolated from Ta Tao 5 was obtained. Four ORFs and one long 819 nt NCR region were identified. The ORFs encoded for the TGB proteins and the CP, respectively. The aa sequence of the CP showed 94% identity with the corresponding published sequence of APruV- 3. The genome of the isolate of PLMVd present in Ta Tao 5 was 337 nt in length and showed no obvious insertions or variations. The sequence showed more than 96% sequence identity with PLMVd isolates found in other parts of the world. Reliable and sensitive techniques for the detection of the agents infecting Ta Tao 5 are described in this study. One Step PCR was used to detect all three agents, and PLMVd also was detected readily by dot blot hybridization. The further studies necessary to determine the relationship between these three agents found in Ta Tao 5 and the bloom delay phenomenon now can be completed.EEA JunínFil: Marini, Diana Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Junín; Argentin

THE MOLECULAR CHARACTERIZATION OF THE VIRUS AND VIRUS-LIKE AGENTS PRESENT IN TA TAO 5 GERMPLASM OF \u3cem\u3ePRUNUS PERSICA\u3c/em\u3e

Peach production in the southeastern United States is limited by late spring freezes. Ta Tao 5 germplasm, used either as an interstem or by chip bud inoculation, has been shown to delay bloom and avoid the effects of these late freezes. The growth modification is graft transmissible and the germplasm has been found to be infected with ACLSV, APruV-3, and PLMVd. Using a combination of PCR, cloning, and sequencing techniques, a molecular characterization of the three graft-transmissible agents present in Ta Tao 5 has been completed. The complete nucleotide sequence of the genome of the isolate of ACLSV (ACLSV-Ta Tao 5) was determined. The genomic organization was typical of other isolates of ACLSV, but the sequence showed only 73% nucleotide identity to the Batalon1 isolate of ACLSV. This distant relationship with characterized isolates of ACLSV explains why primers recommended for PCR reactions used to identify the virus failed to detect the isolate from Ta Tao 5 reliably. This is the first complete genomic sequence of an isolate of ACLSV from peach. The 3\u27 terminal third of the complete sequence of APruV-3 isolated from Ta Tao 5 was obtained. Four ORFs and one long 819 nt NCR region were identified. The ORFs encoded for the TGB proteins and the CP, respectively. The aa sequence of the CP showed 94% identity with the corresponding published sequence of APruV- 3. The genome of the isolate of PLMVd present in Ta Tao 5 was 337 nt in length and showed no obvious insertions or variations. The sequence showed more than 96% sequence identity with PLMVd isolates found in other parts of the world. Reliable and sensitive techniques for the detection of the agents infecting Ta Tao 5 are described in this study. One Step PCR was used to detect all three agents, and PLMVd also was detected readily by dot blot hybridization. The further studies necessary to determine the relationship between these three agents found in Ta Tao 5 and the bloom delay phenomenon now can be completed

Small RNA NGS revealed the presence of cherry virus A and little cherry virus 1 on apricots in Hungary

Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation. © 2018 by the authors. Licensee MDPI, Basel, Switzerland

Plant viruses and viroids in Japan

An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms (Shotokuvirae, Orthornavirae, Pararnavirae) across 2 realms (Monodnaviria and Riboviria). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy

Molecular identification and characterization of viral pathogens infecting sweet cherry

Stone fruits are a valuable crop grown worldwide, however pathogens such as viruses threaten fruit production by reducing tree health and fruit yield. In an orchard within the Niagara region of Ontario, symptoms typical of viral infection such as chlorosis and leaf deformation were seen on sweet cherry (Prunus avium L.) trees. Next generation sequencing was performed on symptomatic and asymptomatic leaves and four viruses were identified. On the tree displaying the most severe symptoms, Prune dwarf virus (PDV), was the only virus detected. A survey conducted during this work showed 42% of cherry trees on a single orchard plot are infected by PDV. The first infectious clone of PDV was developed for molecular characterization of this virus. Introduction of the infectious clone into cherry revealed PDV caused dwarfing symptoms but did not induce the foliar symptoms found on orchard grown trees. A mass spectrometry (MS)-based label-free quantitative proteomic analysis was performed to identify host proteins affected by PDV infection. The results show in PDV infected cherry many defense related proteins are upregulated, and many photosynthesis-related proteins are downregulated. In the model plant cucumber (Cucumis sativus L.) infected by PDV, significant accumulation changes of proteins related to translation and photosynthesis were identified using proteomics, suggesting a possible role of these proteins in the viral infection cycle of PDV. Two proteins identified through proteomic analysis of cucumber were further studied. These proteins are predicted to be important in the infection cycle of PDV as both co-localized with the viral coat protein (CP) of PDV when visualized using confocal microscopy. Finally, to further understand the intra-host spread of PDV, the movement protein (MP) of PDV was characterized. In plant cells, MP expressed alone formed tubules, a typical structure for virus movement. Additionally, domains of MP crucial for tubule formation and subcellular localization were identified. Taken together, this work advances knowledge in the molecular biology of PDV and host impact caused by PDV infection. In the long run, these findings will assist the development of novel strategies against PDV for the sustainable production of cherry and related Prunus fruits

Metagenomic analysis of the begomovirus diversity in tomatoes in Central Brazil and impact of the Ty-1 tolerance gene on viral evolutionary dynamics

Tese (doutorado)—Univesidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, 2020.O tomateiro (Solanum lycopersicum L.) é uma das principais hortaliças cultivadas no Brasil. Até o início década de 1990, a ocorrência de doenças causadas por espécies de Begomovirus (família Geminiviridae) era esporádica no país. Entretanto, a partir deste período, um complexo extremamente diverso de espécies de begomovírus emergiu no cultivo do tomateiro, coincidindo com a ampla dispersão geográfica do vetor Bemisia tabaci MEAM 1 (Middle East-Asian Minor 1= biótipo B). A maioria dos begomovírus apresenta genoma bipartido e apresentam níveis variados de eficiência de transmissão pelo vetor. A utilização mais intensa de híbridos resistentes/tolerantes (principalmente com o gene Ty–1) é um potentical fator no processo de evolução deste grupo de vírus no Brasil. A metagenômica aliada ao Next-Generation Sequencing – NGS é uma das ferramentas mais eficientes para analisar, em larga escala, a diversidade de populações virais em diferentes condições ambientais. Neste contexto, o objetivo geral do presente trabalho foi conduzir estudos de metagenômica sobre a diversidade de begomovírus em tomateiro no Brasil. Os objetivos específicos foram: (a) conduzir estudos comparativos da diversidade de begomovírus infectando tomateiros com e sem o gene Ty–1 e (b) catalogar as espécies virais predominantes e/ou novas espécies de Begomovirus ocorrendo em tomateiros com e sem o gene Ty–1. Para isto, 107 amostras foram coletadas em campos de produção em Goiás (n=56), Distrito Federal (n=27) e Minas Gerais (n=24) entre os anos de 2002 e 2016. O DNA total das amostras foi extraído e submetido a PCR usando primers para detecção de begomovírus e também com primers para região genômica ligada ao gene Ty–1. Posteriormente as amostras foram submetidas a um enriquecimento via RCA (Rolling Circle Amplification) e divididas em dois pools: tomateiros sem o gene Ty–1 (n=64) e com o gene Ty–1 (n=43). Os dois pools foram sequenciados em uma plataforma Illumina HiSeq2500. As sequências obtidas foram montadas no programa CLC Genomics Workbench 11.0 e analisadas no Geneious 10.1. As sequências então foram comparadas com sequências virais presentes no GenBank utilizando o algoritmo BLASTn. Pares de primers específicos foram desenhados visando recuperar o genoma completo e confirmar a presença dos vírus em amostras individuais dentro de cada pool. Os resultados destas análises estão descritos no capítulo 2. Foi observada uma maior diversidade de espécies virais (n=14) no pool de amostras sem o gene Ty–1 em comparação com aquelas obtidas de plantas com gene Ty–1 (n=6). Observou-se uma aparente filtragem entre as espécies detectadas nos dois pools. Foi também observada uma grande frequência de infecções mistas nas amostras, tendo casos da ocorrência simultânea de até cinco espécies em uma única amostra. Três potenciais novas espécies foram detectadas, duas em amostras sem o gene Ty– 1 (MG-378 e GO-169) e uma em amostras contendo o gene Ty–1 (DF-640). Além disso, uma espécie do gênero Gemycircularvirus e um novo Alfasatélite foram detectados. Tomato golden vein virus (TGVV) foi uma das espécies amplamente detectadas nessas análises. Estudos conduzidos no capítulo 3, mostraram que TGVV e Tomato yellow vein streak virus (ToYVSV) estão intimamente relacionados como indicado por análises empregando Sequence Demarcation Tool (SDT) e alinhamento MUSCLE. Dois grupos bem definidos foram identificados, consistentes com os critérios atuais para demarcação de espécies de Begomovirus, sendo também identificado um conjunto distinto características genômicas, biológicas e ecológicas específicas para cada espécie viral. Uma reavaliação dos isolados de TGVV e ToYVSV disponíveis no GenBank mostrou que uma grande fração está erroneamente classificada ao nível de espécie. A espécie Bean golden mosaic virus (BGMV) foi detectada em associação com tomateiro nas análises conduzidas no capítulo 1. No capítulo 4 a diversidade de 161 isolados classificados como BGMV foi catalogada comparando suas sequências completas com o DNA–A e DNA–B do isolado de referência. Análises filogenéticas e com SDT indicaram que os isolados descritos coletivamente como BGMV compreendem, de fato, duas espécies distintas: uma que engloba isolados de BGMV de Phaseolus vulgaris e de uma ampla gama de hospedeiros (incluindo o tomateiro) e uma espécie estreitamente relacionada (com identidade variando de 89 a 91% em comparação com o isolado de referência de BGMV) principalmente associada ao feijão-lima (P. lunatus). O capítulo 5 descreve as características moleculares de um Gemycircularvirus (2.189 nucleotídeos) identificado em associação com o tomateiro no Brasil Central. As análises mostraram que a espécie identificada compartilhou 99% de identidade com um vírus provisoriamente denominado como Plant-associated genomovirus 12 de Larrea tridentata. O capítulo 6 descreve duas novas espécies de Begomovirus que foram identificadas em amostras de Minas Gerais e Goiás. Os genomas virais completos foram clonados, sequenciados via Sanger e provisoriamente denominados Tomato golden net virus – ToGNV (2.649 nucleotídeos) e Tomato yellow net virus – ToYNV (2.636 nucleotídeos). Ambos os vírus exibiram a organização do DNA–A com características típicas das espécies de begomovírus do Novo Mundo. No entanto, nenhum componente cognato do DNA–B foi encontrado, indicando que ToGNV e ToYNV provavelmente compreendem um grupo peculiar de begomovírus neotropicais monopartidos.Conselho Nacional de Desenvolvimento Científico e Tecnológico e Fundação de Apoio a Pesquisa do Distrito Federal (FAP/DF).Tomato (Solanum lycopersicum L.) is one of the main vegetable crops cultivated in Brazil. Until the early 1990s, the occurrence of diseases caused by Begomovirus species (family Geminiviridae) was sporadic in the country. However, from this period on, an extremely diverse complex of begomoviruses emerged in tomato fields, coinciding with the wide geographical dispersion of the Bemisia tabaci MEAM 1 (Middle East-Asian Minor 1 = biotype B). Most begomoviruses have a bipartite genome and have varying levels of transmission efficiency by the vector. The more intense use of resistant/tolerant hybrids (mainly with the Ty–1 gene) is a potentical factor in the evolution process of this group of viruses in Brazil. Metagenomics combined with Next-Generation Sequencing (NGS) is one of the most efficient tools for large scale analysis of the diversity of viral populations in different environmental conditions. In this context, the general objective of the present work was to conduct metagenomics studies on the diversity of begomoviruses in tomatoes in Brazil. The specific objectives were: (a) to carry out comparative studies of the begomovirus diversity infecting tomato plants with and without the Ty– 1 gene and (b) to catalog the predominant viral species and/or new begomoviruses occurring in tomato plants with and without the Ty–1 gene. For this, 107 samples were collected in production fields in Goiás (n = 56), Distrito Federal (n = 27) and Minas Gerais (n = 24) between the years 2002 and 2016. The total DNA of the samples was extracted and submitted to PCR using primers to detect begomovirus and also with primers for the genomic region linked to the Ty–1 gene. Subsequently, the samples were subjected to enrichment via RCA (Rolling Circle Amplification) and divided into two pools: tomatoes without the Ty–1 gene (n = 64) and with the Ty–1 gene (n = 43). The two pools were sequenced on an Illumina HiSeq 2500 platform. The obtained sequences were assembled using the CLC Genomics Workbench 11.0 program and analyzed in Geneious 10.1. The sequences were then compared to viral sequences present on GenBank using the BLASTn algorithm. Specific primer pairs were designed to recover the complete genome and confirm the presence of viruses in individual samples within each pool. The results of these analyzes are described in Chapter 2. A greater diversity of viral species (n = 14) was observed in the sample pool without the Ty – 1 gene compared to those obtained from plants with the Ty – 1 gene (n = 6). It was observed an apparent filtering effect among viral species detected in the two pools. A high frequency of mixed infections was also observed in the samples, with cases of the simultaneous occurrence of up to five species in a single sample. Three potential new species were detected, two in samples without the Ty–1 gene (MG-378 and GO-169) and one in samples containing the Ty–1 gene (DF-640). In addition, a species of the genus Gemycircularvirus and a new alpha-satellite were detected. Tomato golden vein virus (TGVV) was one of the species widely detected in these analyzes. Studies conducted in Chapter 3 have shown that TGVV and Tomato yellow vein streak virus (ToYVSV) are closely related as indicated by analyzes using Sequence Demarcation Tool (SDT) and MUSCLE alignment. Two well-defined clusters were identified, consistent with the current criteria for demarcation of Begomovirus species. In addition, a distinct set of genomic, biological and ecological characteristics specific to each viral species was identified. A reassessment of the TGVV and ToYVSV isolates available on GenBank showed that a large fraction of them is erroneously classified at the species level. Bean golden mosaic virus (BGMV) was also detected in association with tomato in the analyzes carried out in Chapter 1. In Chapter 4 the diversity of 161 isolates classified as BGMV was cataloged by comparing their complete sequences with the DNA–A and DNA–B components of reference isolate. Phylogenetic and SDT analyzes indicated that the isolates collectively described as BGMV actually comprise two distinct species: one that encompasses isolates of BGMV from Phaseolus vulgaris and from a wide range of hosts (including tomato) and a closely related species (with identity ranging from 89 to 91% compared to the reference BGMV isolate), which were mainly associated with lima beans (P. lunatus). Chapter 5 describes the molecular characterization of a Gemycircularvirus (2,189 nucleotides) identified in association with tomato in Central Brazil. The analyzes showed that the gemycircularvirus shared 99% of identity with a virus tentatively named as Plant-associated genomovirus 12 of Larrea tridentata. Chapter 6 describes two new Begomovirus species that were identified in samples from Minas Gerais and Goiás states. The complete viral genomes were cloned, sequenced via Sanger and tentatively named as Tomato golden net virus – ToGNV (2,649 nucleotides) and Tomato yellow net virus – ToYNV (2,636 nucleotides). Both viruses exhibited DNA–A organization with typical features of the New World begomovirus species. However, no cognate components of DNA–B were found, indicating that ToGNV and ToYNV might comprise a peculiar group of monopartite neotropical begomoviruses

Entwicklung, Optimierung und Erprobung von Nachweisverfahren von Viren an Apfel und Untersuchungen zur Ätiologie der Gummiholzkrankheit (ARW)

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Mycoviruses

A virus (from the Latin word ‘v?rus’ meaning ‘venom’ or ‘poison’) is a microorganism invisible to the naked eye. Viruses can multiply exclusively by entering a cell and using the cell’s resources to create copies of themselves. As the origin of their name suggests, viruses are generally considered dangerous, harmful and often deadly. Some of the most well-studied and widely known viruses, such as HIV and influenza, infect humans. However, viruses can also infect animals, plants and microorganisms, including fungi. Many fungi are medically, ecologically and economically significant, for example, causing diseases to humans, plants and insects or being used in industry to produce bread, cheese, beer and wine. Viruses that infect fungi are called mycoviruses (from the Greek work ‘myco’, meaning ‘fungus’). Mycoviruses do not cause harm to or kill the infected fungus; in contrast, they are ‘friendly’ viruses and we can utilize them to control the growth, pathogenicity and toxin production of fungi. This book describes a range of different mycoviruses and their geographical distribution, transmission and evolution, together with their effects on the fungal hosts and how these are brought about