Rafael Montero

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.

Alterations in primary and secondary metabolism in Vitis vinifera ‘Malvasía de Banyalbufar’ upon infection with Grapevine leafroll‐associated virus 3

Plant defense mechanisms against pathogens result in differential regulation of various processes of primary and secondary metabolism. Imaging techniques, such as fluorescence imaging and thermography, are very valuable tools providing spatial and temporal information about these processes. In this study, effects of Grapevine leafroll-associated virus 3 (GLRaV-3) on grapevine physiology were analyzed in pot-grown asymptomatic plants of the white cultivar Malvasía de Banyalbufar. The virus triggered changes in the activity of photosynthesis and secondary metabolism. There was a decrease in the photorespiratory intermediates glycine and serine in infected plants, possibly as a defense response against the infection. The content of malate, which plays an important role in plant metabolism, also decreased. These results correlate with the increased non-photochemical quenching found in infected plants. On the other hand, the concentration of flavonols (represented by myricetin, kaempferol and quercetin derivatives) and hydroxycinnamic acids (which include derivatives of caffeic acid) increased following infection by the virus. These compounds could be responsible for the increase in multicolor fluorescence F440 (blue fluorescence) and F520 (green fluorescence) on the leaves, and changes in the fluorescence parameters F440/F680, F440/F740, F520/F680, F520/F740 and F680/F740. The combined analysis of chlorophyll fluorescence kinetics and blue-green fluorescence emitted by phenolics could constitute disease signatures allowing the discrimination between GLRaV-3 infected and non-infected plants at very early stage of infection, prior to the development of symptoms.

Combined effect of virus infection and water stress on water flow and water economy in grapevines

Water limitation is one of the major threats affecting grapevine production. Thus, improving water-use efficiency (WUE) is crucial for a sustainable viticulture industry in Mediterranean regions. Under field conditions, water stress (WS) is often combined with viral infections as those are present in major grape-growing areas worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting grapevines. Indeed, the optimization of water use in a real context of virus infection is an important topic that needs to be understood. In this work, we have focused our attention on determining the interaction of biotic and abiotic stresses on WUE and hydraulic conductance (Kh ) parameters in two white grapevine cultivars (Malvasia de Banyalbufar and Giró Ros). Under well-watered (WW) conditions, virus infection provokes a strong reduction (P < 0.001) in Kpetiole in both cultivars; however, Kleaf was only reduced in Malvasia de Banyalbufar. Moreover, the presence of virus also reduced whole-plant hydraulic conductance (Khplant ) in 2013 and 2014 for Malvasia de Banyalbufar and in 2014 for Giró Ros. Thus, the effect of virus infection on water flow might explain the imposed stomatal limitation. Under WS conditions, the virus effect on Kplant was negligible, because of the bigger effect of WS than virus infection. Whole-plant WUE (WUEWP ) was not affected by the presence of virus neither under WW nor under WS conditions, indicating that plants may adjust their physiology to counteract the virus infection by maintaining a tight stomatal control and by sustaining a balanced carbon change.

Effects of grapevine leafroll associated virus 3 (GLRaV-3) on plant carbon balance in Vitis vinifera L. cv. Giró Ros

Phloem systemic viruses like Grapevine leafroll associated virus-3 (GLRaV-3) cause significant economic losses in many vineyards around the world. Previous studies have shown that this virus could limit carbon assimilation, without affecting aerial plant growth. However, most of the studies were focused only in the effects of the virus on photosynthesis, i.e. a reduction in the availability of fixed carbon, neglecting the importance carbon losses by respiration, not only in leaves, but in all plant tissues. In this study, we analysed the effects of GLRaV-3 on the plant carbon balance (PCB). The results showed an absence of virus effects on the total biomass increment despite the fact that there was a reduction in carbon assimilation. The discrepancy was found due to an adjustment of carbon losses by respiration caused by the presence of the virus, which compensated the lower carbon assimilation with the result that the total PCB was unaffected. The carbon balance estimated by measuring photosynthesis and respiration in different plant organs matched well with that measured as total biomass increments. In addition, upper leaves and roots were identified as the most sensitive parts of the plant to GLRaV-3 infection.

Effects of Grapevine leafroll-associated virus 3 on the physiology in asymptomatic plants of Vitis vinifera

Grapevine leafroll disease is one of the most important viral diseases of grapevine (Vitis vinifera) worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is the most predominant virus species causing this disease. Therefore, it is important to identify GLRaV-3 effects, especially in plants which do not systematically show visual symptoms. In this study, effects of GLRaV-3 on grapevine physiology were evaluated in asymptomatic plants of Malvasia de Banyalbufar and Cabernet Sauvignon cvs. Absolute virus quantification was performed in order to determine the level of infection of the treatment. The net carbon dioxide (CO2) assimilation (AN) and electron transport rate (Jflux) were the main parameters affected by the virus. The AN reduction in infected plants was attributed to restrictions in CO2 diffusion caused by anatomical leaf changes and a reduction of Rubisco activity. Those effects were more evident in Malvasia de Banyalbufar plants. The reduction of AN leads to a decrease in the total oxygen uptake rate by the activity of the cytochrome oxidase pathway, producing slight differences in plant growth. Therefore, even though no symptoms were expressed in the plants, the effects of the virus compromised the plant vital processes, showing the importance of early detection of the virus in order to fight against the infection.

Improving respiration measurements with gas exchange analyzers

Dark respiration measurements with open-flow gas exchange analyzers are often questioned for their low accuracy as their low values often reach the precision limit of the instrument. Respiration was measured in five species, two hypostomatous (Vitis Vinifera L. and Acanthus mollis) and three amphistomatous, one with similar amount of stomata in both sides (Eucalyptus citriodora) and two with different stomata density (Brassica oleracea and Vicia faba). CO2 differential (ΔCO2) increased two-fold with no change in apparent Rd, when the two leaves with higher stomatal density faced outside. These results showed a clear effect of the position of stomata on ΔCO2. Therefore, it can be concluded that leaf position is important to guarantee the improvement of respiration measurements increasing ΔCO2 without affecting the respiration results by leaf or mass units. This method will help to increase the accuracy of leaf respiration measurements using gas exchange analyzers.

Combined drought and virus infection trigger aspects of respiratory metabolism related to grapevine physiological responses

In the Mediterranean region, grapevines usually deal with drought during their summer growth season. Concurrently, grapevines are hosts to a large number of viruses from which grapevine leafroll associated virus-3 is one of the most widespread and provokes considerable economic losses in many vineyards. However, information concerning grapevine metabolic responses to the combination of drought and viral infection is scarce. Gas-chromatography coupled to mass-spectrometry based metabolite profiling was used in combination with growth analysis, viral loads and gas exchange data to perform an integrative study of the effects of individual and combined stress in two Majorcan grapevine varieties at two experimental years. Metabolic responses of both varieties to the combination of water stress and virus infection were specific and not predicted from the sum of single stress responses. Correlations between respiration, biomass and key metabolites highlight specific adjustments of respiratory and amino acid metabolism possibly underlying the maintenance of carbon balance and growth in grapevines under stress combination.