Enrique Garzón-Jimeno’s research while affiliated with University of Leon and other places

Global warming boosted by climate change affects grape quality, with increasing total soluble solids (TSS) content and decreasing total acidity (TA). However, current wine preferences increasingly include moderate alcohol content, higher acidity and the preservation of primary aromas reminiscent of grapes. Therefore, we hypothesised that applying phytohormones or mineral nutrients to leaves or carrying out defoliation can improve grape must properties in the face of climate warming and in accordance with current oenological trends. The effects of these three viticultural strategies were assessed independently from one another during three growing seasons in a Vitis vinifera L. cv. Tempranillo vineyard in northern Spain. Specifically, three 1-naphtaleneacetic acid (NAA) treatments, two early defoliations (ED; moderate and severe) and two foliar fertilisations with magnesium (Mg) were applied. Treatment with NAA was the most encouraging strategy for decreasing must TSS while increasing TA: it had slight effects on TSS in general and also slight effects on TA when applied close to veraison. The effects of the Mg treatments and moderate ED had null to slightly adverse effects. Finally, severe ED was clearly counter-productive. This study contributes to understanding the effects of both auxin and early defoliation treatments on grape must TSS, acidity and even yeast assimilable nitrogen (YAN) at harvest time. The favourable effects of NAA application are shown to be consistent though slight. Therefore, according to these results, the application of auxins may be an adequate choice for balancing sugars with acidity in grape musts. However, the results also suggest that more research needs to be undertaken to better characterise the effects of auxin treatments on grape must properties at harvest. In particular, different types of auxins, rates, concentrations and number of applications should be tested in the quest for more marked effects.

In addition to aluminum and other heavy metal toxicities, acidic soils also feature nutrient deficits that are not easily overcome by merely adding the required amounts of mineral fertilizers. One of the most critically scarce nutrients in acidic soils is phosphorus, which reacts with aluminum and iron to form phosphates that keep soil phosphorus availability significantly low. Liming ameliorates acidic soils by increasing pH and decreasing aluminum contents; however, it also increases the amount of calcium, which can react with phosphorus to form low-solubility phosphates. In the present work, three liming materials, namely, dolomitic limestone, limestone and sugar foam, were applied on a Typic Palexerult cropped with rye. The effects of these materials on soil properties, including soil available phosphorus extracted with the Olsen and Bray-1 methods, rye phosphorus content in stems and stem and spike harvested biomasses were monitored for nine years. According to the Olsen extraction, the amount of soil available phosphorus generally decreased following liming, with limestone presenting the lowest values; however, the amount of soil available phosphorus increased according to the Bray-1 extraction, though only to a significant extent with the sugar foam from the third year onward. Regardless, the phosphorus content in rye and the relative biomass yield in both stems and spikes generally increased as a consequence of liming. Since crop uptake and growth are the ultimate tests of soil nutrient availability, the inconsistent stem phosphorus content results following the Olsen and Bray-1 extraction methods suggest a lowered efficiency of both extractants regarding crops in soils rich in both aluminum and calcium ions. This decrease can lead to important interpretation errors in the specific conditions of these limed acidic soils, so other methods should be applied and/or researched to better mimic the crop roots’ phosphorus extraction ability. Consequently, the effects of the liming of acidic soils on phosphorus availability and crop performance in the short and long term will be better understood.

Vineyard calcareous soils are usually low in organic matter, which makes them prone to physical, chemical, and biological degradation. Besides, these soils are also usually poor in various nutrients in plant-available form, e.g., iron. To make up for this lack of soil fertility, on the one hand, manures, and on the other, iron chelates are usually used. However, the soil application of these materials is not free from problems, and other amendments based on leonardites could be advantageously used as an alternative. Therefore, two organic amendments, one leonardite alone (1 Mg/ha), and the other leonardite (1 Mg/ha) plus ferrous sulphate heptahydrate (0.5 Mg/ha), were tested for three years in a commercial vineyard calcareous plot under Mediterranean climate. The effects of these amendments on soil fertility, plant nutrient contents, and berry quality were studied against a control of bare soil by means of a fully randomized trial with three repetitions per treatment. Soil organic matter (SOM) increased as a consequence of both leonardite treatments, but much more than expected on the basis of a simple mass transfer from the amendments. With the ferrous-sulphate-heptahydrate-supplemented leonardite, the increase in SOM was noticeably higher. This is explained on the basis of nutrient quantity and intensity-pH-related effects, which increased soil nutrient plant-availability and presumably enhanced vine root growth. In response to the higher plant availability of nutrients, the petiole nutrient concentrations were observed to increase under the leonardite treatments. However, only a trend to increase potassium in petioles and in grape must, linked to a decrease of grape must pH, was observed in harvest quality under the leonardite treatments. Leonardite and adequately supplemented leonardite seem to have potential for increasing SOM contents and nutrient plant-availability, thus improving the soil fertility of vineyard calcareous soils.

Aluminium phytotoxicity is considered the main limiting factor for crop productivity in agricultural acid soils. Liming is a common practice used to improve acidic soil properties, but an appropriate liming material is essential for both agricultural productivity and environmental sustainability. A long-term field experiment with two liming amendments (dolomitic limestone and limestone) was developed during 10 years to determine the changes in soil acidity and assess the effects on crop (rye) yields. Although the adverse effects of the soil acidity conditions were alleviated with both amendments tested, dolomitic limestone was the most effective in the short- and long-term period. In terms of the saturation of exchange complex, dolomitic limestone had a better efficiency, likely based on its rate of dissolution. No significant changes in soil organic matter and exchangeable potassium levels between the treatments tested were found. Both liming materials significantly increased the rye total biomass, but interestingly, significant correlations were showed between tissue levels of magnesium and biomass production, but not between the latter and calcium. The increases in rye biomass production compared with control soils at the end of the research were the following: dolomitic limestone, 47%, and limestone, 32%. A link between an increase in magnesium bioavailability and biomass production was found, as well as between magnesium rye content and total, spike and stem biomass. Hence, it could conceivably be hypothesized that since magnesium is crucial for the transport of assimilates from source leaves to sink organs, alleviating its deficiency leads to avoiding the reducing growth rate of sink organs. Although further investigations are needed to gain a better understanding of liming on the biological, chemical and physical soil properties in the long term, our research provides support for the conceptual premise that an appropriate selection of liming material is crucial for the productivity of acid soils.

Liming is a common practice used to improve acidic soil properties, as is essential for agricultural quality. A long-term field experiment with one lime rate (6000 kg/ha of carbonate calcium equivalent) and three calcium-based liming amendments (gypsum, limestone and sugar foam) was maintained on a Typic Palexerult for 10 years in order to determine changes in soil acidity and to assess the effects on crop (rye) yields. The soil acidity conditions decreased with all the amendments tested, but the sugar foam and limestone was more effective than gypsum over a long-term period. No significant changes in organic soil matter levels between the treatments tested were found. Interestingly, an increase in the leaching of organic soil matter was observed in limed soils. Lime application significantly increased the total rye biomass compared to the control soils during the whole experiment (2002–2011). Yield trends observed in spike and stem biomass were similar to those reported for total rye biomass. In this respect, at the end of the research, gypsum, limestone and sugar foam increased in relation to the total production of rye biomass by 16%, 32% and 38%, respectively, as compared to the control soils. Additionally, a significant and prolonged difference in calcium concentrations in rye stems between unlimed and limed subplots was observed. However, in spite of the results presented here, further investigations are needed to gain a better understanding of the long-term effects of liming on the chemical properties of soil.

The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents.

Correlation between mean values of DNA amounts quantified by qPCR from Streptomyces sp. VV/E1 and VV/R4 strains and cell numbers. (DOCX)

Limit of quantification (LOQ) from Streptomyces sp. VV/E1 and VV/R4 strains. Coefficient of variation [CV = 100 x (SD/mean value)] of the back-calculated amounts of genomic DNA from (A) Streptomyces sp. VV/E1 and (B) Streptomyces sp. VV/R4 by qPCR assays (5 replicates). Horizontal dashed lines correspond to CV = 35% and vertical dashed lines indicate the lowest quantity of DNA with a CV below 35% (obtained by interpolation). Grey symbols indicate the presence of negative (“non-detected”) replicates among samples. (PDF)

qPCR standard curves supplemented with grapevine genomic DNA. Standard curves obtained by SYBR Green qPCR using the primer sets that amplified the SCAR markers and generated with 10-fold serial dilutions of genomic DNA from (A) VV/E1 and (B) VV/R4 strains are shown in black lines. Grey lines correspond to the curves generated in the presence of grapevine genomic DNA. The final ratio of actinobacteria-to-grapevine DNA ranged from 1:1 to 1:100,000. (PDF)

Correlation between quantity of genomic DNA and number of cells. Standard curves obtained by plotting (A) the mean Ct values with respect to the logarithm of DNA copy number from purified and quantified PCR products. Standard curves obtained by plotting (B) the mean Ct values with respect to the logarithm of genomic DNA quantity (10-fold serial dilutions). (C) Correlation (logarithm scales) between genomic DNA quantity and the corresponding number of copies of each amplicon. Streptomyces sp. VV/E1 (black line) and Streptomyces sp. VV/R4 (grey line). (PDF)