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MelonMixVir Report Summary

Project ID: 657527
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - MelonMixVir (Mixed viral infections in melon: disease, vector transmission, RNA silencing suppression and plant defense)

Reporting period: 2015-06-16 to 2017-06-15

Summary of the context and overall objectives of the project

As all living beings, plants are subjected to the attack by different pathogens (bacteria, fungi and viruses) that lead to huge looses in the agriculture worldwide.
Viruses are one of the most damaging biotic stresses affecting crops. In nature, frequently single viral infections (SVI) cause mild or no symptoms, facilitating the spread of viruses by the dispersion of symptomless infected plants. In contrast, in mixed viral infections (MVI), probably the most common, the plant is infected by two or more viruses at the same time. It can lead to synergistic effects, with the resultant disease showing stronger symptoms than with SVI. A successful control of such viral diseases requires epidemiological information regarding their spread (about 80% of all plant viruses are transmitted by insect vectors). Furthermore, MVI might modify virus dissemination parameters by altering the virus-vector-host interactions.
In few cases the importance of MVI is well known, for example in the sweet potato virus disease (SPVD), but in many other cases the scarce knowledge available makes difficult to evaluate the risk represented by MVI. Cucurbits like melon are good examples of this situation: despite the frequent occurrence of MVI (as proved by recent field surveys), only a few in-depth studies have been initiated in this crop until the moment.
A deeper knowledge of the mechanisms involved in the MVI would help to minimize their negative effects on several crops worldwide, increasing the productivity and avoiding looses, leading to direct economic benefits for the society.
MelonMixVir dealt with the plant health challenge posed by MVI in a crop of extreme importance in Europe: melon. The main purpose was to provide scientific-based advice to minimize the damage caused by MVI in melon, with the expected outcome of improving the production and quality of this crop, and accordingly of rising the economic benefits to Europe. We proposed a systematic analysis of pathosystems combining hosts, viruses and vectors to explore the effects caused by their interactions. MelonMixVir general objectives were the following: 1) Pathology of mixed infections: determine the severity and extend of a melon mixed infection, and seek pro-active measures favouring virus control and potential solutions to minimize their damages; 2) Entomology and vectors transmission: analysis of virus transmission by insect vectors (aphids/whiteflies) during a MVI to provide advice and practical approaches to control virus dissemination; 3) Molecular biology and resistance: determine the role of the viral defence system acting during the MVI in melon that could modulate the resistance response of host plants.
We concluded that in the case of a MVI in melon plants by WMV (Watermelon mosaic virus) and CYSDV (Cucurbit yellow stunting disorder virus) there is probably a synergism where WMV benefits of the presence of CYSDV (at least in transmission). The relative amount of each virus is altered by the presence of the other, indicating that somehow they interact inside the plant.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The idea of the project was to understand MVI in melon and explore modes to interfere with essential steps to reduce damages. To attain the objectives, we analysed a MVI using susceptible melon plants infected with CYSDV and WMV. The virus defence mechanism inside the plant is the inhibition of the plant RNA silencing by the activity of proteins that block/decrease this defence mechanism. These viral proteins, the RNA silencing suppressors (RSS), play an essential role during a plant infection.
First of all, we developed a protocol to infect plants using the natural vectors of CYSDV and WMV, whiteflies and aphids, respectively, and to obtain MVI. We then followed the infection symptoms comparing the single and mixed infected plants and we took samples each 12 days until 60 days of infection to compare the amount of virus. Our observations indicated that the MVI plants were more severely affected than the SVI. However, after 60 days the MVI plants presented a recovery in the disease symptoms compared to the WMV infected plants (see picture). Virus quantification revealed that amount of CYSDV was always higher in mixed infected plants. On the other hand, WMV behaved in the opposite manner (less WMV in mixed infected plants).
Next we studied virus transmission to know if it is altered when the insects acquire the viruses in single or mixed infected plants. To this aim, we let the insects acquire either in SVI or in MVI plants (whiteflies and aphids to transmit CYSDV and WMV) and transmit the virus to healthy melon seedlings (primary transmission). We also did secondary transmission where the receptor plants were pre-infected with one virus (aphids transmitted WMV to CYSDV infected plants and vice-versa), to see if the presence of one virus inhibited the arrival of the second one. In both primary and secondary transmissions there was no difference in CYSDV transmission rate when the whiteflies acquired in single or mixed infected plants. However, to our surprise, in the case of WMV transmission we observed a higher transmission rate when aphids acquired in mixed infected plants. This result was unexpected because the virus quantification showed that the amount of WMV decreases in MVI plants compared to SVI ones. That is: in MVI plants there is less amount of WMV but there is a higher rate of virus transmission when this is the source plant. Complementary experiments are being performed currently by group members to better elucidate these results.
Finally, we investigated if there was any interaction/relation between the RSS from both viruses during the MVI. We first confirmed the RSS activity of the described proteins for each virus and then performed subcellular localization experiments that points to a colocalization of some of these proteins inside the plant cell.
To fulfil the most important goal of a scientific research, the results obtained we disseminated both to the scientific community and to the general public. They were presented in 5 scientific meetings (national and international) and regularly in the internal seminars in the host institute. Concerning the general public, several approaches were made to bring closer the science to the society and up to 6 dissemination activities were carried out.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

To our best knowledge this is the first work addressing the MVI in melon under a systematic and controlled way. We expect that these results will help decipher the MVI not only in melon but in other crops. Tools developed to manage the insect’s population will certainly be very useful for future works addressing similar issues. Also transmission results can have great value to control virus dissemination in the field (as the control of the whiteflies population to try to minimize the presence of CYSDV and thus the presence of mixed infected plants).
Results obtained here are the basis to produce melon plants with improved traits in virus resistance. This will have a direct positive impact in European economy through the improvement of the industry competitiveness, and will benefit the society by allowing production, and thus consumption, of better-quality fruits. Moreover, potential uses and commercial value of the tools generated can be extended to other crops, expanding the impact of MelonMixVir results beyond the limits of a single crop and geographical area.

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