Olive mild mosaic virus transmission by Olpidium virulentus

The ability of Olpidium virulentus to vector Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) and Tobacco necrosis virus D (TNV-D) was evaluated. Transmission assays involved zoospore acquisition of each virus, inoculation onto cabbage plant roots followed by viral detection. Assays revealed that these viruses are transmitted in the absence of the fungus, but the transmission rates of OMMV are much higher when OMMV is incubated with O. virulentus zoospores prior to inoculation, while the transmission rates of each OLV-1 and TNV-D do not change when they are incubated with the fungus. Our data shows that O. virulentus is an efficient vector of OMMV, greatly enhancing virus transmissibility, but not that of OLV-1 nor TNV-D. Prior studies have shown that another fungus, O. brassicae, also enhances OMMV transmission. To our knowledge, this is the first report of a plant virus, OMMV, being efficiently transmitted by two different species of fungi

Co infection of OMMV and OLV-1 enhances symptoms and increases both viruses accumulation and viral derived siRNAs in plants

Previous extensive field surveys in olive orchards have revealed high levels of Olive mild mosaic virus (OMMV) and Olive latent virus 1 (OLV-1), frequently appearing in mixed infections. These viruses belong to genus Alphanecrovirus and their RNA dependent RNA polymerase (RdRp),as well as their p6 and p8 amino acid sequences share over 87% identity. Preliminary studies have shown that co infection of OMMV and OLV-1 is associated to an intensification of symptoms, as well to an increase in transmission efficiency, suggesting a synergistic effect. Single and double infections of OMMV and OLV-1 were obtained through mechanical inoculation of Nicotiana benthamiana plants and the second upper leaf from each inoculated plant was collected at different stages and used for quantitative PCR. In this study we found that the co infection of OMMV and OLV-1 causes an exacerbation of symptoms and increases the accumulation of both viruses in N. benthamiana plants. Highthroughput sequencing of siRNAs from both viruses in singly and co infected plants showed that OMMV and OLV-1 co infection increased the accumulation of siRNAs, mainly of 21 and 22 nt in length, with most non distinguishable between OMMV and OLV-1 siRNAs. Our findings suggest that siRNAs of both viruses have possible roles in the synergistic interaction between OLV-1 and OMMV in N. benthamiana plants. Whether a similar situation occurs in olive fields is not yet known and studies are being pursued

Fungal endophytic communities associated to the phyllosphere of grapevine cultivars under different types of management

Fungal endophytes present in different asymptomatic grapevine plants (Vitis vinifera L.) located in different vineyards within Alentejo, a highly important viticulture region in Portugal, were identified in this study. Sampled grapevine plants included the three most representative cultivars in the region, Syrah, Cabernet Sauvignon, and Aragonez, growing under two different modes of management, conventional and biological. Sixteen fungal taxa were identified through sequencing of the internal transcribed spacer region. Total number of endophytic fungi isolated showed significant differences both in management mode and in cultivars, with higher numbers in grapevines under conventional mode and from Syrah cultivar. The composition of fungal endophytic communities did not show significant differences among cultivars, but differences were observed between fungal communities isolated from grapevines under biological or conventional modes. The most fungal taxa isolated from grapevines cultivated under biological mode were Alternaria alternata, Cladosporium sp., and Nigrospora oryzae, and under conventional mode Botrytis cinerea, Epicoccum nigrum, and Epicoccum sp. These differences suggest that the different products used in grapevine production have impacts in fungal endophytic composition. Further investigation of the identified fungi with respect to their antagonistic characteristics and potential use in plant protection to ensure food safety is now in course

A degenerate pair of primers for simultaneous detection of four alphaand betanecroviruses

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5 and parCoat3 was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced

Specific amino acids of Olive mild mosaic virus coat protein are involved in transmission by Olpidium brassicae

Abstract: Transmission of Olive mild mosaic virus (OMMV) is facilitated by Olpidium brassicae (Wor.) Dang. An OMMV mutant (OMMVL11) containing two changes in the coat protein (CP), asparagine to tyrosine at position 189 and alanine to threonine at position 216, has been shown not to be Olpidium brassicae-transmissible owing to inefficient attachment of virions to zoospores. In this study, these amino acid changes were separately introduced into the OMMV genome through site-directed mutagenesis, and the asparagine-to-tyrosine change was shown to be largely responsible for the loss of transmission. Analysis of the structure of OMMV CP by comparative modelling approaches showed that this change is located in the interior of the virus particle and the alanine-to-threonine change is exposed on the surface. The asparagine-to-tyrosine change may indirectly affect attachment via changes in the conformation of viral CP subunits, altering the receptor binding site and thus preventing binding to the fungal zoospore

Development of a viral vector to control TSWV in tomato plants

Tomato spotted wilt virus (TSWV) is a virus that causes a disease of major concern in tomato plants all over the world. TSWV is responsible for extremely significant economic losses in this crop, which constitutes 72% of the value of fresh vegetables produced worldwide. So far, plant viral control strategies rely only on preventive sanitary measures or genetic resistance as antiviral products are not available for field use which turns essential the development of innovative and efficient means of control. Viruses are responsible for several important plant diseases, however, they have also been used in biotechnology with different purposes. Virus induced gene silencing (VIGS) allows specific silencing of foreign genes that can be inserted in an optimized virus vector and then inoculated in plants. When a sequence of a viral gene is introduced in a VIGS vector, the plant infected with this vector will be signalized to target that foreign viral RNA. This will give to the plant a significant advantage in its protection against a possible infection of that virus. The VIGS approach provides the generation of rapid phenotype, no need for plant transformation and can be used for plant protection purposes, at a relatively low cost. Several plant viruses have been used as VIGS vectors however, their large genomes, their difficult manipulation and the reduced number of hosts they infect restrain their use as vectors. The Alphanecrovirus Olive mild mosaic virus (OMMV) has characteristics that place it as a very promising vector tool. Its small genome makes it easy to manipulate, in addition, it causes only mild systemic symptoms in a wide range of crops, which will facilitate their manipulation into symptomless constructs and allow its application to a high number of plants. To our knowledge no studies have been done so far concerning this matter using necroviruses. This new virus-based vector for protection of tomato plants against TSWV through silencing, with no need of plant genome transformation, is being developed under the project TOMVIRPROTECT (ongoing, from October 2018 to October 2021) here presented, and where it is also intended to turn the vector available for the control of other important plant diseases

COOPEDU IV — Cooperação e Educação de Qualidade

O quarto Congresso Internacional de Cooperação e Educação-IV COOPEDU, organizado pelo Centro de Estudos Internacionais (CEI) do Instituto Universitário de Lisboa e pela Escola Superior de Educação e Ciências Sociais do Instituto Politécnico de Leiria decorreu nos dias 8 e 9 de novembro de 2018, subordinado à temática Cooperação e Educação de Qualidade. Este congresso insere-se numa linha de continuidade de intervenção por parte das duas instituições organizadoras e dos elementos coordenadores e este ano beneficiou do financiamento do Instituto Camões, obtido através de um procedimento concursal, que nos permitiu contar com a participação presencial de elementos dos Países Africanos de Língua Portuguesa, fortemente implicados nas problemáticas da Educação e da Formação. Contou também com a participação do Instituto Camões e da Fundação Calouste Gulbenkian, entidades que sistematizaram a sua intervenção nos domínios da cooperação na área da educação nos últimos anos. A opção pela temática da qualidade pareceu aos organizadores pertinente e actual. Com efeito os sistemas educativos dos países que constituem a Comunidade de países de língua portuguesa têm implementado várias reformas mas em vários domínios mantem-se a insatisfação de responsáveis políticos, pedagogos, técnicos sociais face aos resultados obtidos. Aliás o caminho de procura da Qualidade é interminável porque vai a par da aposta na exigência e na promoção da cidadania e responsabilidade social. As comunicações que agora se publicam estão organizadas em dois eixos: o das Políticas da Educação e Formação e o das dimensões em que se traduzem essas políticas. Neste último eixo encontramos fios condutores para agregarmos as comunicações apresentadas