A Case Study about the Use of Precision Agriculture Technology Applied to a Zn Biofortiﬁcation Workﬂow for Grapevine Vitis vinifera cv Moscatel †

: As the human population is growing worldwide, the food demand is sharply increasing. Following this assumption, strategies to enhance the food production are being explored, namely, smart farming, for monitoring crops during the production cycle. In this study, a vineyard of Vitis vinifera cv. Moscatel located in Palmela (N 38 ◦ 35 (cid:48) 47.113 (cid:48)(cid:48) O 8 ◦ 40 (cid:48) 46.651) was submitted to a Zn biofortiﬁcation workﬂow, through foliar application of zinc oxide (ZnO) or zinc sulfate (ZnSO 4 ) (at a concentration of 60% and 90%—900 g · ha − 1 and 1350 g · ha − 1 , respectively). The ﬁeld morphology and vigor of the vineyard was performed through Unmanned Aerial Vehicles (UAVs) images (assessed with altimetric measurement sensors), synchronized by GPS. Drainage capacity and slopes showed one-third of the ﬁeld with reduced surface drainage and a maximum variation of 0.80 m between the extremes (almost ﬂat), respectively. The NDVI (Normalized Difference Vegetation Index) values reﬂected a greater vigor in treated grapes with treatment SZn90 showing a higher value. These data were interpolated with mineral content, monitored with atomic absorption analysis (showing a 1.3-fold increase for the biofortiﬁcation index). It was concluded that the used technologies furnishes speciﬁc target information in real time about the crops production.


Introduction
By 2050, due to the increase in the world population, to avoid hunger, food production must significantly increase [1].In addition, to ensure safety, food also must have high quality, specifically at a prophylactic level, providing the necessary nutrients, since it is expected that their deficiency might affect the health of more than two billion people worldwide [2,3].In this context, some alternatives are being suggested, namely, agronomic biofortification to increase target nutrients in edible plant tissues.This alternative can be accomplished through soil and foliar application, and this latter application seems to allow plants to assimilate micronutrients with high efficiency, as it does not depend upon root-to-shoot translocation [4,5].Beyond the main aims of agronomic biofortification, some evidence showed that yield and nutritional quality increases with this practice [6].
Zinc deficiency continues to affect around three billion people worldwide, leading to the appearance of neurological disorders, autoimmune, degenerative diseases related to age, Wilson's disease, cardiovascular problems, and diabetes mellitus, among other conditions [7].
To address this increase in food demand, other factors also may be considered, such as climate change, the limited availability of arable lands, as well as the growing necessity for freshwater, making it indispensable to resort to new technologies such as Unmanned Aerial Vehicle (UAVs).This technology can carry different types of cameras, including multispectral cameras that allow users to obtain vegetation indices translated by Normalized Difference Vegetation Index (NDVI), providing us with information about biomass levels and stress conditions such as crop diseases, water stress, pest infestations, nutrient deficiencies and other factors that affect crop productivity [8].Regarding other advantages of UAVs, acquirement of field data its carried out more easily, and in a fast and cost-effective way [9].Following this assumption, the present study used multispectral images from UAVs to monitor Zn biofortified vineyards, once this fruit plays a predominant role in the development of the world and, according to the Food and Agriculture Organization (FAO), covers 75.866 square kilometers worldwide, additionally helping in some health problems [10].

Field Morphology and Vigor of the Vine
Flight planning and execution was performed to obtain images with a high resolution RGB (20 Mp) and Parrot Sequoia Plus installed multispectral cameras in an unmanned aerial vehicle (UAV), model DJI Phantom 4 Pro +.The multispectral camera had four band sensors: Green (550 BP 40), Red (660 BP 40), Red Edge (735 BP 10) and Near Infrared (790 BP 40).After acquisition of images, an orthophotomap was processed and, using the altimetry data, the digital model of the terrain (MDT) was obtained, as well as the surface drainage model (using the ARCGIS and Agisoft Photoscan software), creating the vegetation index maps that reflect the vigor of the plants (NDVI) [11].

Quantification of Zn in Grapes
At harvest, grapes were cut, dried (until constant weight, at 60 • C) and subjected to an acid digestion procedure with a mixture of HNO 3 -HCL (4:1), according to [12].Then, the samples were filtrated, and Zn contents were measured using an atomic absorption spectrophotometer model Perkin Elmer AAnalyst 200, fitted with a deuterium background corrector, and the AA WinLab software program.

Statistical Analyses
Data were statistically analyzed using a One-Way ANOVA (p ≤ 0.05) to access differences, followed by a Tukey's test for mean comparison (95% confidence level).

Results
The slopes of the experimental field were determined, being found a moderate surface drainage prevailing, with 63.86% of infiltration capacity (Table 1).Zn contents in treated grapes showed, relatively to the control, 1.2-1.3-foldincreases in the higher concentrations of treatments, OZn90 and SZn90, respectively (Table 2).After the fourth treatment, Moscatel-treated grapes revealed a positive response, showing higher NDVI values than the control, with treatments by SZn displaying the highest foliage densities (Table 3; Figure 1).After the fourth treatment, Moscatel-treated grapes revealed a positive response, showing higher NDVI values than the control, with treatments by SZn displaying the highest foliage densities (Table 3; Figure 1).

Discussion
Climate changes are a concern among winemakers, as grapes are one of the fruit crops most sensitive to severe drought conditions and water shortage [13].Water shortage is considered one of the most common causes of stress conditions in grapes, leading to yield and quality losses.Under these conditions, decreases in relative water content (RWC), leaf dry matter, chlorophyll (Chl) content, net photosynthetic rate (PN), ribulose-1,5-bisphosphate carboxylase (RuBPC) and nitrate reductase (NR) activities of Riesling grapevines can develop [14].
The Portuguese climate is becoming dryer, and it is indispensable to develop adaptation strategies to face water scarcity.In this framework, a smart irrigation, optimizing grape composition and providing a balanced solution between environment and plant requirements can become a relevant option.Indeed, with higher dryness, and without irrigation, yield reductions were already found in Portugal (i.e., Alentejo, Lisboa, Minho and Terras-da-Beira) [15].Moreover, an efficient irrigation showed a diminishing volume of water applied to crops fields by 30-70% and an increase in crop yields by 20-90% [16].According to our data, the experimental field with Moscatel grapes showed a moderate capacity of surface drainage in 63.86% of the area (Table 1), which determines a moderate infiltration capacity, contributing to the groundwater recharge (i.e., water available for plant growth) [17].Additionally, as this field is being irrigated, the potential hydric stress derived by rain scarcity, observed in recent decades, is being mitigated.
In Turkey, fertilization with Zn was carried out by leaves spraying, which determined increases in productivity of about 25% in cereals, with the concurrent augmentation of Zn contents in the edible parts of the plants [18].Our study also showed an increase in Zn content in grapes sprayed with ZnO and ZnSO 4 (Table 2).In addition, following [19,20], Zn biofortification through foliar application, also affected yield parameters.Indeed, through vegetation indices, namely NDVI, is possible to access health conditions, providing information of photosynthetic capacity, which can be correlated with plant vigor and vegetation abundance, health and growth [21,22].This index has values normalized between +1 and −1, with higher values indicating a denser vegetation [22].In fact, our data showed higher values for NDVI in the vines fertilized with ZnSO 4 and ZnO, relative to the control, but treatments with ZnSO 4 triggered a higher vigor (Table 3).

Conclusions
Through images obtained with cameras attached to UAVs, it is possible to obtain information about morphology of the field and potential limiting conditions for vines development.Using the Moscatel field as a test system, important characteristics-namely, moderate infiltration capacity and the use of irrigation, enabling vines to have more resistance to hydric stress-were optimized.The obtained images further provided information regarding the crops' state, detecting a positive response to Zn fertilization with an increase in the Zn content and vigor of vines subjected to ZnO and ZnSO 4 .

Table 1 .
Slope characterization before foliar application of Lau Novo field.

Table 3 .
Average vigor ± S.E.(n = 3) in fruits of Vitis vinifera L. variety Moscatel after the 4th application.Letter indicates the absence of significant differences among treatments (p ≤ 0.05).