In vivo endophytic, rhizospheric and epiphytic colonization of vitis vinifera by the plant-growth promoting and antifungal strain pseudomonas protegens MP12

An evaluation was conducted of the colonization of Pseudomonas protegens MP12, a plantgrowth promoting and antagonistic strain, inoculated in vine plants during a standard process of grapevine nursery propagation. Three in vivo inoculation protocols (endophytic, rhizospheric, and epiphytic) were implemented and monitored by means of both culture-dependent and independent techniques. Endophytic treatment resulted in the colonization of the bacterium inside the vine cuttings, which spread to young leaves during the forcing period. Microscopy analysis performed on transformed dsRed-tagged P. protegens MP12 cells confirmed the bacterium’s ability to penetrate the inner part of the roots. However, endophytic MP12 strain was no longer detected once the plant materials had been placed in the vine nursery field. The bacterium also displayed an ability to colonize the rhizosphere and, when the plants were uprooted at the end of the vegetative season, its persistence was confirmed. Epiphytic inoculation, performed by foliar spraying of cell suspension, was effective in controlling artificially-induced Botrytis cinerea infection in detached leaves. The success of rhizospheric and leaf colonization in vine plants suggests potential for the future exploitation of P. protegens MP12 as biofertilizer and biopesticide. Further investigation is required into the stability of the bacterium’s colonization of vine plants under real-world conditions in vineyards

THE STUDY OF THREE GRAPEVINE CLONES TO UNCOVER THE GENETIC TRAITS RESPONSIBLE FOR THE LOW SUSCEPTIBILITY TO FLAVESCENCE DORéE

Flavescence dorée (FD) is one of the most destructive grapevine yellows diseases and a quarantine pest in the European Community. It is caused by phytoplasmas, which are transmitted in vineyard by the leafhopper Scaphoideus titanus. Inter and intraspecific differences in susceptibility to FD have already been observed among grapevine varieties and clones of the same variety. Grapevine varieties and clones completely resistant to FD have not been uncovered yet, however these differences suggest the presence of genetic traits in grapevine related to high or low susceptibility to FD. Cultivated grapevines are clonally propagated and the genome of each cultivar is preserved, except for the accumulation of mutations over time that can generate distinguishable clones with several notable phenotypes. The differences between the genome of clones of the same variety are less than those between different varieties; thus, in order to highlight the genetic features responsible for the different phenotypes, the study of the genomes of clones with different susceptibility to FD can be a valid technique to achieve the goal. The aim of this work is to find out the genetic traits responsible for the different susceptibility to FD among three Chardonnay clones, analyzing the diversity in their genomes and transcriptomic profiles. The clones were sequenced by accurate whole genome techniques, Hi-Fi reads sequencing on PacBio platform coupled with Illumina, and then the genomes were de novo assembled. Moreover, two of them were experimentally infected in field by means of the insect vector and the transcriptomic profiles in the early stage of FD infection were analyzed. In particular, the clones were compared in absence of the disease and the vector, in presence of the healthy vector and with the FD infective vector. The results obtained from the preliminary genome comparison showed higher similarity between the three clones than compared with the reference genome from variety Pinot noir, while the analysis to identify the genomic differences among clones are still in progress. The transcriptomic profiles showed interesting differences in some pathways expressed in presence of healthy vectors, while the clones shared a similar expression profile before being in contact with the vectors as well as after the challenge with the FD infective vectors. These findings might suggest the involvement of antibiosis mechanisms in the partial resistance of the specific Chardonnay clone to FD. The results obtained, and those that will be achieved in future, are useful for new breeding programs and clonal selection. Grapevine plants more resistant to FD will permit to decrease the insecticides used to control the disease in vineyards. Moreover, the knowledge of the molecular and metabolic mechanisms involved in scarcely susceptible clones could pave the way for the development, for example, of biostimulants capable to induce such defenses even in the most susceptible plants

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

SEAWINES: Use of macroalgae as biostimulants against fungal diseases in grapevines

The outlook for climate change foresees major impacts on vineyards worldwide, shifting pathogens distribution and dynamics demanding more intense plant protection measures in certain regions, increasing viticulture's dependence on phytochemicals and pesticides. However, the European Commission is applying restrictions on their use, encouraging the development of more sustainable strategies efficient for disease control. Seaweeds represent an ecological alternative for a more sustainable production. Previous studies have shown that algae extracts contain compounds capable of reducing the abundance of plant fungal pathogens. Despite it, little is known about the molecular mechanism underlying this response. SEAWINES project is evaluating the efficacy of the foliar application of Ulva ohnoi and Rugulopteryx okamurae extracts to control powdery and downy mildew, in addition to testing their effect on grape and wine quality. To our knowledge, this is the first study evaluating R. okamurae biostimulant capacity and fungicidal effect in viticulture. This macroalgae is relevant since it is an invasive species in our coasts, causing incalculable economic and environmental burdens. We aim to 1- Reduce the usage of chemicals in grapevines; 2- Reduce fungal diseases in viticulture; 3- Valorize polysaccharides from seaweeds; 4- Increase the added-value to wines (ecological and quality); and 5- Provide an alternative use to seaweed biomass, contributing to bio-circular economy and reducing its accumulation in our coasts

Two New Putative Plant Viruses from Wood Metagenomics Analysis of an Esca Diseased Vineyard

The concept of plant as a holobiont is now spreading among the scientific community and the importance to study plant-associated microorganisms is becoming more and more necessary. Along with bacteria and fungi, also viruses can play important roles during the holobiont-environment interactions. In grapevine, viruses are studied mainly as pathological agents, and many species (more than 80) are known to be able to replicate inside its tissues. In this study two new viral species associated with grape wood tissues are presented, one belongs to the Potyviridae family and one to the Bunyavirales order. Due to the ability of potyviruses to enhance heterologous virus replication, it will be important to assess the presence of such a virus in the grapevine population to understand its ecological role. Furthermore, the association of the cogu-like virus with esca symptomatic samples opens new questions and the necessity of a more detailed characterization of this virus

Genetic variability and pathological properties of Grapevine Leafroll-associated Virus 2 isolates

The genetic variability among Grapevine leafroll-associated virus 2 (GLRaV-2) isolates was investigated in several grapevine accessions from various geographic origins in three genomic fragments, which encode the 70 kDa heat-shock protein homologue, the coat protein and the ∼60 kDa protein. The majority of the isolates were identical or only slightly different from one another and formed a monophyletic group. Several other variants were found which differed greatly from the main cluster and were mostly present in autochthonous or specific vine cultivars. Phylogenetic analyses on the CP gene sequences, including the isolates analyzed in this work and some others from related literature, allowed five clades to be identified (PN, H4, RG, BD and PV20). Biological trials for graft incompatibility and leafroll symptoms revealed that GLRaV-2 divergent variants had different pathological properties, mainly according to their phylogenetic grouping. The BD isolate seemed to be a mild variant of the virus, because it was unable to induce graft incompatibility and it rarely caused leafroll symptoms. The RG isolate appeared to be a more virulent variant, given the strong decrease in rooted grafted grapevines obtained in nursery, in particular with the use of certain rootstocks; however, it does not induce leafroll symptoms. The variants belonging to the PN group, which were the most widespread, caused both graft incompatibility and leafroll symptoms, according to the viral variant. Graft incompatibility also proved to depend strongly on the rootstock used

Evolution of the new grapevine disease of Pinot gris and of Grapevine Pinot gris virus (GPGV)

An emergent grapevine disease, discovered about 10 year ago, has been associated to a new virus, called Grapevine Pinot gris virus (GPGV). The aim of the present work was to survey the presence of both the disease and the virus in Veneto (North Eastern Italy), and the presence of the virus in Europe in the last 15 years, using molecular techniques. The visual survey in Veneto showed that the disease is present in about 70% of the investigated vineyards, with a general low occurrence of symptomatic plants. However, the increase of the number of symptomatic grapevines in 8 much damaged vineyards was around 25% per year. The virus occurs in more than 80% of the plants. Finally, our data suggest that GPGV was limited to some Eastern European countries at least before 2005, and that it spread more widely throughout Europe after 2010

Evolution of the new grapevine disease of Pinot gris and of Grapevine Pinot gris virus (GPGV)

An emergent grapevine disease, discovered about 10 year ago, has been associated to a new virus, called Grapevine Pinot gris virus (GPGV). The aim of the present work was to survey the presence of both the disease and the virus in Veneto (North Eastern Italy), and the presence of the virus in Europe in the last 15 years, using molecular techniques. The visual survey in Veneto showed that the disease is present in about 70% of the investigated vineyards, with a general low occurrence of symptomatic plants. However, the increase of the number of symptomatic grapevines in 8 much damaged vineyards was around 25% per year. The virus occurs in more than 80% of the plants. Finally, our data suggest that GPGV was limited to some Eastern European countries at least before 2005, and that it spread more widely throughout Europe after 2010