Holistic understanding of the response of grapevines to foliar application of seaweed extracts.

Viticulture is highly dependent on phytochemicals to maintain good vineyard health. However, to reduce their accumulation in the environment, green regulations are driving the development of eco-friendly strategies. In this respect, seaweeds have proven to be one of the marine resources with the highest potential as plant protective agents, representing an environmentally-friendly alternative approach for sustainable wine production. The current work follows an interdisciplinary framework to evaluate the capacity of Ulva ohnoi and Rugulopteryx okamurae seaweeds to induce defense mechanisms in grapevine plants. To our knowledge, this is the first study to evaluate Rugulopteryx okamurae as a biostimulator . This macroalgae is relevant since it is an invasive species on the Atlantic and Mediterranean coast causing incalculable economic and environmental burdens. Four extracts (UL1, UL2, RU1 and RU2 developed from Ulva and Rugulopteryx, respectively) were foliar applied to Tempranillo plants cultivated under greenhouse conditions. UL1 and RU2 stood out for their capacity to induce defense genes, such as a PR10, PAL, STS48 and GST1, mainly 24 hours after the first application. The increased expression level of these genes agreed with i) an increase in trans-piceid and trans-resveratrol content, mainly in the RU2 treated leaves, and, ii) an increase in jasmonic acid and decrease in salicylic acid. Moreover, an induction of the activity of the antioxidant enzymes was observed at the end of the experiment, with an increase in superoxide dismutase and catalase in the RU2-treated leaves in particular. Interestingly, while foliar fungal diversity was not influenced by the treatments, alga extract amendment modified fungal composition, RU2 application enriching the content of various groups known for their biocontrol activity. Overall, the results evidenced the capacity of Rugulopteryx okamurae for grapevine biostimulation, inducing the activation of several secondary metabolite pathways and promoting the abundance of beneficial microbiota involved in grapevine protection. While further studies are needed to unravel the bioactive compound(s) involved, including conducting field experiments etc., the current findings are the first steps towards the inclusion of Rugulopteryx okamurae in a circular scheme that would reduce its accumulation on the coast and benefit the viticulture sector at the same time.

Genetic Characterization by SSR Markers of a Comprehensive Wine Grape Collection Conserved at Rancho de la Merced (Andalusia, Spain).

The IFAPA research center "Rancho de la Merced" (Jerez, Spain) hosts one of the oldest and most diverse grapevine germplasm repositories in Europe, and is aimed at providing feasible solutions to deal with any agronomic trait by exploring its genetic variability and by means of association and Deoxyribonucleic Acid (DNA) editing studies. In this work, we focused on a wine and dual-use grapevine subcollection that consists of 930 accessions. Genetic analysis allowed to identify 521 unique genotypes. After comparing them with several databases, matches were found for 476 genetic profiles while the remaining 45 have not been previously described. Combination with clustering analysis suggested a total pool of 481 Vitis vinifera accessions that included some table cultivars. Several synonymies, homonymies and mislabeling have also been detected. Structure analysis allowed identifying six clusters according to eco-geographic cultivation areas and one additional group including non-vinifera accessions. Diversity analysis pointed out that Spanish Mediterranean varieties are genetically closer to oriental genotypes than to European varieties typical of oceanic and continental climates. The origin of Spanish varieties is discussed in depth considering our data and previous studies. Analysis of molecular variance partition confirmed a well-structured germplasm, although differentiation among groups had a much lower effect on genetic variability than differences within groups, which are strongly related to a very high heterozygosity. A core collection that covers all allele richness is proposed. It is constituted of about 13% of total accessions, and each cluster inferred by structure analysis is represented.

Two novel partitiviruses that accumulate differentially in Rosellinia necatrix and Entoleuca sp. infecting avocado.

Rosellinia necatrix is responsible for the white rot root disease of avocado in Southern Spain. Entoleuca sp. is a fungus isolated from roots of these same trees, but it is not pathogenic in avocado. Here, we describe two new species of partitiviruses detected in isolates of the avocado sympatric fungi Entoleuca sp. and R. necatrix, termed Entoleuca partitivirus 1 (EnPV1), genus Alphapartitivirus, and Entoleuca partitivirus 2 (EnPV2), genus Betapartitivirus. For both R. necatrix and Entoleuca sp., the dsRNA of the RdRp genomic segment of EnPV1 accumulates at a higher rate than the CP dsRNA, except for a set of Entoleuca sp. isolates where titers of the CP dsRNA are 35-50 times higher than those of the RdRp dsRNA and between 250-380 times higher than the CP dsRNA titers found in the rest of Entoleuca sp. and R. necatrix isolates. For EnPV2, the accumulation rates of the RdRp dsRNA in Entoleuca sp., is in most of the cases, higher than the CP dsRNA. In contrast, in R. necatrix isolates, EnPV2 dsRNA2 generally accumulates at a higher rate. Genetic analysis of the partitiviruses revealed that there is no apparent variation in the nucleotide sequences among the strains. RNA silencing of the partitiviruses appears to be limited in Entoleuca sp., as shown by small RNA sequencing. Finally, the investigation of the presence of these partitiviruses in a fungal collection revealed that they have no role in the pathogenicity of R. necatrix in avocado or in the avirulence of Entoleuca sp. in this host.

Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

TransFlow: a modular framework for assembling and assessing accurate de novo transcriptomes in non-model organisms.

BACKGROUND: The advances in high-throughput sequencing technologies are allowing more and more de novo assembling of transcriptomes from many new organisms. Some degree of automation and evaluation is required to warrant reproducibility, repetitivity and the selection of the best possible transcriptome. Workflows and pipelines are becoming an absolute requirement for such a purpose, but the issue of assembling evaluation for de novo transcriptomes in organisms lacking a sequenced genome remains unsolved. An automated, reproducible and flexible framework called TransFlow to accomplish this task is described. RESULTS: TransFlow with its five independent modules was designed to build different workflows depending on the nature of the original reads. This architecture enables different combinations of Illumina and Roche/454 sequencing data, and can be extended to other sequencing platforms. Its capabilities are illustrated with the selection of reliable plant reference transcriptomes and the assembling six transcriptomes (three case studies for grapevine leaves, olive tree pollen, and chestnut stem, and other three for haustorium, epiphytic structures and their combination for the phytopathogenic fungus Podosphaera xanthii). Arabidopsis and poplar transcriptomes revealed to be the best references. A common result regarding de novo assemblies is that Illumina paired-end reads of 100 nt in length assembled with OASES can provide reliable transcriptomes, while the contribution of longer reads is noticeable only when they complement a set of short, single-reads. CONCLUSIONS: TransFlow can handle up to 181 different assembling strategies. Evaluation based on principal component analyses allows its self-adaptation to different sets of reads to provide a suitable transcriptome for each combination of reads and assemblers. As a result, each case study has its own behaviour, prioritises evaluation parameters, and gives an objective and automated way for detecting the best transcriptome within a pool of them. Sequencing data type and quantity (preferably several hundred millions of 2×100 nt or longer), assemblers (OASES for Illumina, MIRA4 and EULER-SR reconciled with CAP3 for Roche/454) and strategy (preferably scaffolding with OASES, and probably merging with Roche/454 when available) arise as the most impacting factors.

A Novel Hypovirus Species From Xylariaceae Fungi Infecting Avocado.

The white rot root disease caused by Rosellinia necatrix is a major concern for avocado cultivation in Spain. Healthy escapes of avocado trees surrounded by diseased trees prompted us to hypothesize the presence of hypovirulent R. necatrix due to mycovirus infections. Recently, we reported the presence of another fungal species, Entoleuca sp., belonging to the Xylariaceae, that was also found in healthy avocado trees and frequently co-infecting the same roots than R. necatrix. We investigated the presence of mycoviruses that might explain the hypovirulence. For that, we performed deep sequencing of dsRNAs from two isolates of Entoleuca sp. that revealed the simultaneous infection of several mycoviruses, not described previously. In this work, we report a new member of the Hypoviridae, tentatively named Entoleuca hypovirus 1 (EnHV1). The complete genome sequence was obtained for two EnHV1 strains, which lengths resulted to be 14,958 and 14,984 nt, respectively, excluding the poly(A) tails. The genome shows two ORFs separated by a 32-nt inter-ORF, and both 5'- and 3'-UTRs longer than any other hypovirus reported to date. The analysis of virus-derived siRNA populations obtained from Entoleuca sp. demonstrated antiviral silencing activity in this fungus. We screened a collection of Entoleuca sp. and R. necatrix isolates and found that EnHV1 was present in both fungal species. A genetic population analysis of EnHV1 strains revealed the presence of two main clades, each of them including members from both Entoleuca sp. and R. necatrix, which suggests intra- and interspecific virus transmission in the field. Several attempts failed to cure Entoleuca sp. from EnHV1. However, all Entoleuca sp. isolates collected from avocado, whether harboring the virus or not, showed hypovirulence. Conversely, all R. necatrix isolates were pathogenic to that crop, regardless of being infected by EnHV1.

Differences of Three Ampeloviruses' Multiplication in Plant May Explain Their Incidences in Vineyards.

Grapevine leafroll ampeloviruses have been recently grouped into two major clades, one for Grapevine leafroll associated virus (GLRaV) 1 and 3 and another one grouping GLRaV-4 and its variants. In order to understand biological factors mediating differential ampelovirus incidences in vineyards, quantitative real-time polymerase chain reactions were performed to assess virus populations in three grapevine varieties in which different infection status were detected: GLRaV-3 + GLRaV-4, GLRaV-3 + GLRaV-4 strain 5, and GLRaV-4 alone. Specific primers based on the RNA-dependent RNA polymerase (RdRp) domains of GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 were used. Absolute and relative quantitations of the three viruses were achieved by normalization of data to the concentration of the endogenous gene actin. In spring, the populations of GLRaV-4 and GLRaV-4 strain 5 were 1.7 × 104 to 5.0 × 105 genomic RNA copies/mg of petiole tissue whereas, for GLRaV-3, values were significantly higher, ranging from 5.6 × 105 and 1.0 × 107 copies mg-1. In autumn, GLRaV-4 and GLRaV-4 strain 5 populations increased significantly, displaying values for genome copies between 4.1 × 105 and 6.3 × 106 copies mg-1, whereas GLRaV-3 populations displayed a less pronounced boost but were still significantly higher, ranging from 4.1 × 106 to 1.6 × 107 copies mg-1. To investigate whether additional viruses may interfere in the quantifications the small RNA populations, vines were analyzed by Ion Torrent high-throughput sequencing. It allowed the identification of additional viruses and viroids, including Grapevine virus A, Hop stunt viroid, Grapevine yellow speckle viroid 1, and Australian grapevine viroid. The significance of these findings is discussed.