Development of Grapevine Leafroll Associated Virus 3 symptoms within a month on rooted Cabernet Sauvignon cuttings

Symptoms of GLRaV-3 are visible in the form of leaves reddening which initially appears on smaller leaves surface. As the infection spreads, the entire leaf turns red, while only the leaf veins remain green. The leaves start to downroll. The symptoms first affect the older leaves but as the infection spreads, they become visible throughout the whole plant.

Back to Lab

After summer vacation, we are in the lab again! Together with our colleagues, we perform morphological and physiological analyzes on healthy and GLRaV-3 infected plants. We analyze plant growth, membrane permeability, leaf water and nutrient content, enzymes and stress hormone, and much more. This is also the time when changes typical for GLRaV-3 infection can be clearly determined in plants.

Spring and summer project activities

To verify the success of virus transmission by grafting, we used ELISA  (Enzyme-linked immunosorbent assay) method for the detection of GLRaV-3 virus in the Microbiological laboratory. It is a serological method that enables the detection of viral antigens in a sample if the virus titar is satisfactory. Although we have adopted the method, the preferred time of virus detection in the plant is winter, and the sample type phloem tissue, so we will repeat this testing in the winter period in the dormant phase of grapevine plants.

In the meantime, we inoculated a small number of healthy indicator plants with selected GLRaV-3 isolates from the field. The type of grafting technique was “green grafting”. After only two days, the buds from the inserted plants began to grow, what guarantees virus transmission from the donor plant to the indicator host.

In the second half of July, we began to measure the photosynthesis in the experimental plants. Hopefully, infected plants will show differences in photosynthetic activity compared to control uninfected plants. We also hope for differences in photosynthesis activity between different GLRaV-3 isolates as well as between different host plants infected with the same isolate. Significant…?! It is to be seen.. 🙂

Grapevine inoculation with GLRaV-3 isolates

At the beginning of Mart our variety of grapevine plants were inoculated with different GLRaV-3 isiolates. Plants were infected with only one of GLRaV-3 variant (monophyletic isolates), and with combination of genetic variants (polyphyletic isolates). In some minor extend plant were also infected with mixt viral isolates composed of GLRaV-3 and other viruses that are the most abundant in our grapevine plants. Those are field isolates. In total we inoculated around 300 plants. Some part of the plants remained uninoculated and will serve as control plants. This experiment was designed to examine the biological characteristics of the genetic variants of GLRaV-3, their combinations and field isolates and to elucidate the behavior of GLRaV-3 virus and its variants in terms of replication in host plants and host responses to virus presence.

The year 2019 is going toward its end, so as the first year of the GRAPE-PATH3 project. It has been a great year! Our team is now wealthier for one new team member, Mate a fresh PhD student. Our lab is now equipped with modern UV-DOC system, ELISA reader and system for vertical electrophoresis.  We grow plants in newly improved vegetative chamber. We have results on viral distribution and GLRaV-3 population structure among autochthonous grapevine cultivars. And plenty more.. Above all, we had a great time working together!

Have a great New Year and plenty of great new results!

GRAPE-PATH3 Team

Lecture at Sabatina

PhD Katarina Hančević participated on November 8th as a speaker at the traditional 29th international meeting of winemakers and winegrowers Sabatina 2019, organized by the Association of the Dalmation Cooperatives, held in Seget Donji near Trogir.

Presentation entitled: “Pathogenicity of grapevine viruses in indigenous Dalmatian cultivars” was part of a scientific and professional conference organized by the Faculty of Agriculture. Part of the presentation was dedicated to the presentation of the CSF project GRAPE-PATH3.

Dear colleague from the Institute and our associate on the project, PhD Ana Mucalo was honoured with the “Zlatan Plenković” award by the “Zlatan Otok” from Sveta Nedjelja for a young wine expert.  Although this well-deserved award is not related to the project,  the entire GRAPE-PATH3 team congratulates Ana on this award!

Preparation of viral inoculum for grafting indicator plants

The grapevine plants from the collection vineyard in which we proved the presence of singe GLRaV-3 virus infection were rooted and transplanted in pots for the purpose of biological indexing. Since we did not find all genetic variants of GLRaV-3 as a single infection, we have obtained individual GLRaV-3 genetic variants which we will use to infect indicator plants. The cuttings containing monophyletic variants of GLRaV-3 were gained by the courtesy of dr. Rachelle Bester from the Stellenbosch University (Department of Genetics), South Africa. These cuttings are properly stored until spring next year when they will be used as a source of virus inoculum for grafting of indicator plants.

Symptoms observation of a viral infection in the vineyard

In parallel with laboratory analyzes of GLRaV-3 genetic diversity that we had detected in autochthonous varieties, since spring we have been monitoring the occurrence and intensity of symptoms of all viral diseases on grapevine plants in collection vineyard. The first symptoms we noticed were the yellowing and leaf malformations associated with viruses from the infectious degeneration group. With the beginning of the summer, symptoms of leaf redness between leaf veins began to develop in grapevines infected with viruses from the leafroll associated virus group. Over time, these leaves became redder and twisted. Because of this symptom of leaf downrolling, leafroll viruses justifiably bear the name given.

Short training of PhD Mate Čarija

Mate Čarija was in the short scientific training from 17.06.-30.06. 2019 at the Institute for Sustainable Plant Protection in Bari, Italy. The host was prof. dr. sc. Pasquale Saldarelli, also a collaborator on the project.

While we in Croatia were celebrating two holidays during that period, Mate was in the Laboratory of prof. Saldarelli learning and adopting the Real-Time PCR method, advanced analytical detection and quantification method for grapevine viruses. In the second week of his stay in Bari, Mate was hosted at the Agritest Laboratory to perform ELISA on  the Croatian autochthonous grapevine samples.

Bravo Mate for great learning and results and we owe you two days off the work :-)!

Internal methodological workshop

We attended an internal methodological workshop at the Faculty of Science  in Zagreb. At the workshop, we were theoretically and practically introduced with the method of  SSCP (Single-Strand Conformation Polymorphism)  used to determine genetic variants present in the sample. The method was used to determine the genetic structure of GLRaV.-3 virus in grapevine plants.  The workshop has lasted for 5 days and was led by PhD Silvija Černi, associate on the project.

The program of the workshop was adapted on an everyday basis in accordance with the protocol for the implementation of the SSCP method and results obtained.