Olive Mild Mosaic Virus Coat Protein and P6 Are Suppressors of RNA Silencing, and Their Silencing Confers Resistance against OMMV.

RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. In this study, the genome of the Olive mild mosaic virus (OMMV) was screened for silencing suppressors. The full OMMV cDNA and 5 OMMV open reading frames (ORFs) were cloned into the Gateway binary vector pK7WG2, transformed into Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana 16C plants. CP and p6 showed suppressor activity, with CP showing significantly higher activity than p6, yet activity that was lower than the full OMMV, suggesting a complementary action of CP and p6. These viral suppressors were then used to induce OMMV resistance in plants based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants, which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a significant reduction in viral accumulation and symptom attenuation was observed as compared to those of the controls, as well as to when each construct was used alone, proving them to be effective against OMMV infection. This is the first time that a silencing suppressor was found in a necrovirus, and that two independent proteins act as silencing suppressors in a virus member of the Tombusviridae family.

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.

Influence of Temperature and Copper on Oxalobacteraceae in Soil Enrichments.

ß-Proteobacteria is one of the most abundant phylum in soils, including autotrophic and heterotrophic ammonium-consumers with relevance in N circulation in soils. The effects of high-temperature events and phytosanitary treatments, such as copper amendments, on soil bacterial communities relevant to N-cycling remain to be studied. As an example, South Portugal soils are seasonally exposed to high-temperature periods, the temperature at the upper soil layers can reach over 40 °C. Here, we evaluated the dynamics of mesophilic and thermophilic bacteria from a temperate soil, in particular of heterotrophic ß-Proteobacteria, regarding the ammonium equilibrium, as a function of temperature and copper treatment. Soil samples were collected from an olive orchard in southern Portugal. Selective enrichments were performed from samples under different conditions of temperature (30 and 50 °C) and copper supplementation (100 and 500 µM) in order to mime seasonal variations and phytosanitary treatments. Changes in the microbial communities under these conditions were examined by denaturing gradient gel electrophoresis, a molecular fingerprint technique. At moderate temperature--30 °C--either without or with copper addition, dominant members were identified as different strains belonging to genus Massilia, a genus of the Oxalobacteraceae (ß-Proteobacteria), while at 50 °C, members of the Brevibacillus genus, phylum Firmicutes were also represented. Ammonium production during bacterial growth at moderate and high temperatures was not affected by copper addition. Results indicate that both copper and temperature selected specific tolerant bacterial strains with consequences for N-cycling in copper-treated orchards.

Detection of sweet potato virus C, sweet potato virus 2 and sweet potato feathery mottle virus in Portugal.

Field sweet potato plants showing virus-like symptoms, as stunting, leaf distortion, mosaic and chlorosis, were collected in southwest Portugal and tested for the presence of four potyviruses, sweet potato virus C (SPVC), sweet potato virus 2 (SPV2), sweet potato feathery mottle virus (SPFMV), sweet potato virus G (SPVG), and the crinivirus sweet potato chlorotic stunt virus (SPCSV). DsRNA fractions were extracted from symptomatic leaves and used as templates in single and multiplex RT-PCR assays using previously described specific primers for each analyzed virus. The amplified reaction products for SPVC, SPV2 and SPFMV were of expected size, and direct sequencing of PCR products revealed that they correspond to the coat protein gene (CP) and showed 98%, 99% and 99% identity, respectively, to those viruses. Comparison of the CP genomic and amino acid sequences of the Portuguese viral isolates recovered here with those of ten other sequences of isolates obtained in different countries retrieved from the GenBank showed very few differences. The application of the RT-PCR assays revealed for the first time the presence of SPVC and SPFMV in the sweet potato crop in Portugal, the absence of SPVG and SPCSV in tested plants, as well as the occurrence of triple virus infections under field conditions.

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

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.