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Brief Report

Variability in Stomatal Adaptation to Drought among Grapevine Cultivars: Genotype-Dependent Responses

by
Luca Nerva
1,†,
Walter Chitarra
1,†,
Gianni Fila
2,
Lorenzo Lovat
1 and
Federica Gaiotti
1,*
1
Research Centre for Viticulture and Enology, Council for Agricultural Research and Economics (CREA-VE), Via XXVIII Aprile 26, 31015 Conegliano, Italy
2
Research Centre Agriculture and Environment, Council for Agricultural Research and Economics, Sericulture Laboratory, Via Leonardo Eulero 6a, 35143 Padova, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2023, 13(12), 2186; https://doi.org/10.3390/agriculture13122186
Submission received: 19 October 2023 / Revised: 14 November 2023 / Accepted: 21 November 2023 / Published: 22 November 2023
(This article belongs to the Special Issue Agricultural Crops Subjected to Drought and Salinity Stress)
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Abstract

:
Leaf stomata are the primary determinants of the plant water relations. Physiological adaptations of stomata in response to water stress have been extensively reported for grapevine. On the contrary, little is known about how the plasticity in stomatal anatomical features may affect their adaptability to drought conditions. In this study, we investigated, at the molecular and anatomical level, the effect of water stress on the stomatal anatomical features of four grapevine varieties extensively cultivated in the north of Italy. Potted plants of Garganega, Glera, Moscato giallo, and Merlot varieties were subjected to a 12–13 day period of water restriction during two consecutive seasons. Stomatal density and size were investigated in newly developed young leaves, 7 days after tip separation, following the occurrence of a water stress event. Furthermore, the gene expression of three key stomagenesis genes (VvEPFL9, VvEPF1, and VvEPF2) was analysed. The response of stomatal anatomical features to drought varied among the studied varieties. Moscato and Glera showed an increase in stomatal density and a decrease in stomatal size. On the contrary, Merlot displayed a reduction in stomatal number, while Garganega remained unchanged in terms of these values. Transcript levels of VvEPFL9 were overall in agreement with stomatal densities measured in the four varieties, showing an up-regulation when drought induced an increase in stomatal density or a down-regulation when the stomatal number decreased. The wide variability in stomatal response observed in the four varieties under study suggests that anatomical changes in stomatal characteristics are genotype dependent. These variations contribute to the intra-specific variability in grapevine’s response to water stress.

1. Introduction

Drought stress poses a major threat to grapevine production and quality worldwide and is predicted to increase in intensity as a consequence of the ongoing climate change [1]. In view of this, the identification of strategies that can counteract the effects of climate is crucial to maintain the economic sustainability of viticulture in the near future. This requires a comprehensive knowledge of the mechanisms underlying grapevine drought responses and the definition of phenotypic, physiological, or molecular traits that can be used to identify stress-tolerant genotypes.
Leaf stomata, the minute apertures found mainly in the epidermis of leaves, are major determinants of the plant water stress response. Rapid changes in leaf stomatal conductance and photosynthesis in response to water stress have been extensively reported for grapevine and several other crops [2,3,4]. The regulation of water consumption by stomatal control plays a key role in determining the genotype adaptability to limited water conditions and has been shown to vary greatly among grapevine varieties and clones [5,6,7]. Leaf morphoanatomy also takes part in determining the genotype adaptability to drought conditions. Among leaf features, stomatal density and size on the leaf epidermis are key anatomical drivers in determining the transpiration rate [8]. Plants can adjust stomatal development to optimise gas exchange in response to water stress [8]. These anatomical adaptations to drought have been shown to vary greatly by species, cultivar, and the level of stress. In response to water limitation, stomatal density was reported to increase in wheat [9], olive [10], apricot [11], and sugarcane [12], and it was often coupled with a reduction in stomatal size [10,11,13].
In grapevine, stomatal density and size have been reported to vary in response to several environmental factors, including temperature and CO2 levels [14], wind [15], and water availability [16,17,18]. Palliotti et al. [16] found that the Sangiovese variety exhibited a notable rise in stomatal count and a decrease in stomatal dimensions during periods of drought. Theodorou et al. [17] reported a similar behaviour for Grenache and Xinomavro varieties. However, in the same study, opposite results (reduced stomatal density and increased size) were observed in Agiorgitiko and Syrah. Overall, these studies concur that grapevine can regulate stomatal development on new leaves in response to water limitation. Variability in the response of stomatal density and size across cultivars suggests a variety-dependent adaptation response.
The molecular processes regulating stomatal development in response to environmental signals in plants, including grapevines, remain poorly understood, limiting our knowledge of how vines (and woody crops in general) may sense environmental constraints and change stomatal development to adapt to protracted water deficits [8,19,20] and references therein. In the model plant Arabidopsis, stomata development is under the control of a complex molecular network, which is regulated by a signalling pathway involving three key regulators, i.e., the epidermal patterning factors EPF1, EPF2, and EPFL9 (also known as STOMAGEN). EPF1 and EPF2 are negative regulators of stomatal density, whereas EPFL9 promotes stomata development [20,21,22]. The latter was recently functionally characterized in edited grapevine plants of the “Sugraone” variety, demonstrating its role in stomatal density determination [23]. Until now, no study has investigated the gene expression patterns of VvEPFL9 and other putative genes associated with stomagenesis in grapevine plants under varying water availability. Furthermore, it is unclear whether immature leaves possess the ability to detect water deficit both at the biochemical level (such as responses mediated by the abscisic acid—ABA hormone) and at the molecular level, by activating gene pathways in response to drought (such as ABA-responsive genes like dehydrin (DH), a well-recognized marker of drought in plants, including grapevines) [24,25,26]. These data could enhance our understanding of the molecular pathways involved in stomatal development under drought conditions.
Glera, Garganega, Moscato giallo, and Merlot are four varieties widely cultivated in the north of Italy [27]. In a recent study, we analysed the physiological responses to water stress of these cultivars and revealed differences in their water-use strategy [28]. For instance, a more typical near-isohydric behaviour was found for Moscato, and a near-anisohydric one for Garganega, Glera, and Merlot.
In this study, we examined the impact of water limitation on the stomatal characteristics of young leaves (7 days after tip separation) of Garganega, Glera, Merlot, and Moscato varieties, to determine whether the variation in stomatal density and size could serve as an adaptive mechanism enabling tolerance to extended periods of water stress conditions. Moreover, to gain deeper insights into stomatal adaptations to water deficit in the selected varieties, we investigated the gene expression patterns of three pivotal genes (VvEPFL9, VvEPF1, and VvEPF2) that regulate stomatal development. These genes are homologous to those previously characterized in Arabidopsis. In summary, the objective of this study is to enhance our comprehension of the morpho-anatomical mechanisms contributing to varietal differences in water stress tolerance. Additionally, the study aims to elucidate the molecular regulation of stomagenesis in newly developed grapevine leaves exposed to water limitation.

2. Materials and Methods

2.1. Plant Material and Experimental Design

In this study, the plant material and experimental design were consistent with those employed in our recent study, which focused on comparative ecophysiological responses among various grapevine cultivars [28]. Briefly, four-year-old V. vinifera L. potted plants of Garganega, Glera, Merlot, and Moscato giallo, grafted onto Kober 5BB rootstock, were selected for this experiment. Glera is a white variety grown in the Friuli and Veneto Regions and is used to produce the recognized Prosecco wine. Garganega is a white variety mainly grown in the provinces of Verona and Vicenza and is used to produce the Soave wines. Moscato giallo belongs to the Moscato family and is cultivated mostly in the Colli Euganei area to produce the Moscato Fior d’Arancio wine. All three of these varieties are characterised by good vigour and productivity. Merlot is a well-known international variety cultivated worldwide.
The vines were grown in open air at the experimental farm of the Research Centre for Viticulture and Enology (CREA-VE) in Susegana (45°51′ N–12°15′ E), Italy, in 80 L plastic pots filled with a sand–peat–clay mixture (50–35–15% in volume) and covered with plastic waterproof sheets. Vines were positioned in rows at a spacing of 1 m and pruned to one single cane that is 14–15 nodes in length. Shoot thinning was performed in spring to standardize the number of shoots to 14–15 for all plants. Ten vines per cultivar were randomly selected and initially maintained well watered at field capacity. At the beginning of the experiment (start of veraison, BBCH 81 stage) [29], the vines of each cultivar were divided into two groups of five plants and assigned to the following treatments: well-watered vines (WW) received 100% of the total daily water requirement and water-stressed vines (WS) received 30% of the total daily water requirement. Daily water consumption was monitored through continuously weighing two reference potted vines with Laumas Elettronica ISC scales connected to a D1 Flex log 1.9 datalogger (Tecnopenta, Teolo, PD, Italy).
Plant water status during the experiment was monitored by measuring the midday stem water potential (Ψstem) with a Scholander pressure chamber. Stem data recorded in the two years of study are available in Gaiotti et al. (2023) [28] and are summarised in Figure S1. Water restriction was applied for 12 days in 2017 and 13 days in 2018, until vines reached severe water stress, defined as Ψstem ≤ 1.3 MPa [30,31]. Thereafter, all vines were rehydrated by applying 100% of the total daily water usage. For the profiling of stomagenesis target genes by the means of qPCR, unfolded leaves were collected at the end of water stress imposition, immediately before the rewatering phase, from WS plants and WW ones used as controls. For stomatal density and size measurements, the experiment was concluded one week after the re-watering, when samples of young leaves were collected from WW and WS vines for the analysis of stomatal characteristics. All samples for stomatal density and gene expression analysis were collected from shoots bearing one single cluster.
Weather conditions were monitored using the local CREA-VE weather station coupled to Watch Dog 1400 datalogger (Spectrum Technologies, Bridgend, UK). Mean temperature during the trial ranged between 27 and 28 °C in the two study seasons. Mean air humidity and irradiance during the experiment were 60.7% and 22.6 MJ/m−2 day−1, respectively.

2.2. Stomatal Density in Developing Leaves

To explore the stomatal developmental changes induced by water limitation in the four varieties, stomatal density and length were measured in young leaves collected from WW and WS plants. Leaves developed from shoot tips that had experienced water stress during the period of water restriction were sampled one week after re-watering. From each variety, a young leaf with an at least 40 mm long main vein was collected from the tip of each vine’s main stem (n = 5, randomly selected leaves per treatment for each variety). The impression method was used to determine leaf stomatal density, expressed as the number of stomata per unit leaf area, and the stomata length, defined as the length in micrometres between the junctions of the guard cells at each end of the stoma. In detail, clear nail polish was applied to three different areas of the abaxial epidermis of each leaf avoiding the midvein and allowed to dry as previously reported [32]. According to Düring (1990) [2], stomata are uniformly distributed across the abaxial epidermis; thus, the selection of areas for stomatal counting was considered irrelevant. The nail polish was then removed using transparent tape and transferred onto a microscope glass slide. Images were captured for each film strip using an optical microscope system equipped with a built-in camera (microscope Leica DM750, Camera Leica ICC50HD, lens HIPLAN 20X/04, software Leica Application Suite ver. 4.4.0, Leica, Wetzlar, Germany). Stomatal density and length were counted on a standard area (655 × 491 µm) for each strip.

2.3. RNA Isolation and RT-qPCR Analysis of Target Genes Involved in Stomatal Development

In order to explore the effect of water stress on the expression of genes involved in stomatal development, during the second year of the experiment (2018), shoot tips containing unfolded leaves were collected from WW and WS plants on the final day of water stress imposition. For each treatment and cultivar, three shoot tips (independent biological replicates) from three independent plants were collected and promptly frozen at −80 °C for subsequent gene expression analysis. Total RNA was isolated from the unfolded lyophilized leaf samples using the SpectrumTM Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) following manufacturer’s instructions. RNA concentration of the extracted samples was quantified using the NanoDropTM (Thermo Fisher Scientific, Waltham, MA, USA). DNAse treatment and cDNA synthesis was performed as previously reported starting from 1 µg of total RNA [33]. The absence of genomic DNA contamination was checked before cDNA synthesis with qPCR using ubiquitin (VvUBI) specific primers of grapevine. RT-qPCR reactions were carried out in a final volume of 10 µL containing 5 µL of SYBR® Green Master Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 5 µM specific primers, and 1:10 of diluted cDNA. Reactions were run in the CFX 96 apparatus (Bio-Rad Laboratories, Inc.) using the following program: 10 min preincubation at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 30 s at 60 °C. Each amplification was followed by melting curve analysis (60–94 °C) with a heating rate of 0.5 °C every 15 s. All reactions were performed with at least two technical replicates for three biological replicates. Transcripts expression levels were normalized to the geometric mean of the ubiquitin (VvUBI) and actin (VvACT) transcripts and calculated with the 2−ΔCt method using CFX Maestro software v. 2.3 (Bio-Rad Laboratories, Inc.). Oligonucleotide sequences are listed in Table S1 [25,26,34].

2.4. Statistical Analysis

The significance of differences between WS and WW treatments for stomatal characteristics and gene expression was assessed using Student’s t-test. The tests were performed using the software Statistica 7.0 (StatSoft Inc., Tulsa, OK, USA).

3. Results

3.1. Stomatal Density and Size in Developing Leaves

Densities observed in the four varieties under WW conditions ranged between 130 and 230 stomata/mm2 in the two study years (Figure 1A–D). Changes induced by water limitation were different among the cultivars under study. Moscato and Glera responded similarly, with an increase in stomatal density and a decrease in stomatal size in WS plants relative to WW plants. Although the differences in Moscato stomatal density were significant only during the second season of the study, the results were consistent in both years, with a similar increase of 22–23% in WS vines compared to WW ones. Merlot is the variety that exhibited the lowest stomatal density (between 130 and 160 stomata/mm2 under WW conditions), and displayed the opposite response as WS plants showed a decrease in the stomatal number relative to WW plants, while the stomatal size remained unchanged. The stomatal characteristics of Garganega were not significantly affected by water restriction over the two years of the experiment. Figure S2 displays comparative images of the stomata of the four varieties under study in the WW and WS conditions.

3.2. Expression of Target Genes Involved in Water Stress Sensing and Stomagenesis in Developing Leaves

To better understand how newly forming leaves sense water constraints and modulate their stomatal development in the four varieties under study, the expression of a dehydrin (VvDH) and three key genes related to stomatal development were examined using RT-qPCR, i.e., VvEPFL9 that competes with VvEPF1 and VvEPF2 for receptor binding and thus promotes stomatal development. It is worth noting that developing leaves could perceive water stress, as evidenced by a significant VvDH up-regulation in all WS conditions (Figure 2A) across all varieties. Upon WS, VvEPFL9 transcripts levels increased in Moscato and Glera, although only in Glera this trend was statistically significant (Figure 2B). In contrast, transcript levels for this gene decreased in Merlot exposed to WS.
Under water stress, the negative regulator VvEPF1 responded differently in Merlot and Glera, being up-regulated in the former and down-regulated in the latter (Figure 2C). The transcript levels of this gene did not vary significantly in Moscato and Garganega. Under WS condition, the gene expression of VvEPF2 was suppressed in all four varieties (Figure 2D).

4. Discussion

Grapevine has demonstrated the ability to modulate leaf stomatal development, adjusting stomata density and size to better cope with prolonged water stress [16,17]. As no information regarding this adaptive strategy has been previously reported for Glera, Garganega, Moscato, or Merlot, we analysed their stomatal characteristics in young leaves (7 days after tip separation) developed under different water availability conditions.
The response of stomatal anatomical features to drought was not consistent among the studied varieties, with some exhibiting an increase, while others demonstrating a decrease or no changes in their values. This wide range of responses suggests that morpho-anatomical changes in stomatal characteristics are genotype-dependent and can contribute to the intra-specific variability in the response to water stress observed in grapevine. When the four cultivars were compared, Moscato and Glera responded similarly, with a rise in stomatal density and a decrease in stomatal size in newly formed leaves over the drought stress imposition. A comparable response to water stress has been extensively reported in various crops as a water deficit adaptation [11,13,35,36]. According to these studies, a higher stomatal density allows for the maintenance or improvement of CO2 external supply, whereas small stomata are reported to provide a quicker adjustment of aperture response [35,37]. Merlot WS, unlike Glera and Moscato, exhibited a decrease in stomatal density as previously found in Argiorgitiko and Shyrah varieties under water stress [17]. Despite the fact that this adjustment has been documented less frequently in response to water stress, studies on transgenic plants with reduced stomatal density revealed that this modification can improve Water Use Efficiency (WUE) and drought tolerance in several species, including grapevine [23,38,39,40].
Our results indicated that adjustments in stomatal anatomical features were not related to the varietal water use strategy. In fact, similar changes in density and size were observed for Glera that displays a near-anisohydric behaviour and Moscato that displays a near-isohydric behaviour. On the contrary, opposite adjustments in stomatal characteristics were observed in Merlot and Glera, despite the common anisohydric water use strategy of these varieties [28].
To better understand the stomatal adaptations to water deficit in the cultivars under study, we analysed the gene expression of a dehydrin (VvDH) and of three key genes (VvEPFL9, VvEPF1, and VvEPF2) controlling stomatal development, homologues to those characterized in Arabidopsis. As, in Arabidopsis, stomata formation is determined in the early stages of leaf development, when the epidermal cells of leaf primordia either differentiate into pavement cells or into meristemoid mother cell that initiate the stomata lineage [41,42], gene expression analysis was performed on unfolded leaves collected from the main shoot tips of WW and WS treatments. Apexes analogous to those collected for gene analysis were allowed to develop over the subsequent week. Stomatal density measurements were conducted on the young leaves developed from these structures. The dehydrin gene (VvDH) has been generally regarded as a drought perception marker, since its expression has been shown to be induced by water stress in grapevine [25,43]. VvDH expression analysis demonstrated that unfolded leaves could perceive water stress, as attested by the considerable up-regulation of this gene in all WS-exposed cultivars. Although VvEPFL9 has been shown to promote stomatal development [21,22,23], no prior studies have looked into its regulation in developing leaves in grapevines exposed to water restriction. The transcript levels of VvEPFL9 were overall in agreement with stomata densities on young leaves in the four varieties, showing an up-regulation when drought induced an increase in stomatal density (in WS Glera and WS Moscato) and a down-regulation when the stomatal number decreased (in WS Merlot).
In model plants, VvEPF1 and VvEPF2 genes have been shown to act as negative regulators of VvEPFL9, thus negatively regulating stomatal density [8,21]. The relation between the expression of these genes and stomatal density was not clear in all varieties. VvEPF1, as a negative regulator of stomagenesis, was expected to be down-regulated in Glera and Moscato varieties in which water stress induced an increase in stomatal density. Surprisingly, the comparison of the transcript levels of WS and WW plants revealed that only Glera exhibited a lower level of expression. Merlot was the only variety in which drought induced an up-regulation of VvEPF1, and this result supports, at the molecular level, the decrease in stomatal density observed in WS Merlot plants. The function of VvEPF2 is similar to that of VvEPF1, but EPF2 acts as a negative regulator of stomatal development slightly earlier than EPF1 [22]. Even though stomatal density responses to water stress varied among cultivars, the expression of the VvEPF2 gene was down-regulated in all four WS plants.
Overall, the expression analysis of VvEPFL9 showed a trend similar to that observed for stomatal density, also confirming the putative role of this gene in promoting stomagenesis in grapevine as recently demonstrated in the “Sugraone” grape variety [23]. Additionally, results indicate that VvEPF1 and VvEPF2 may act antagonistically to VvEPFL9. Nonetheless, the up- or down-regulation of a single gene among those mentioned above could not entirely account for the observed alterations in stomatal density. This suggests that stomatal development is probably controlled by the combined effects of all the analysed genes and potentially by other unidentified components that require further exploration.

5. Conclusions

The findings of this study, both at the anatomical and molecular levels, confirm that water stress can influence the stomatal development in young leaves (7 days after tip separation). This adaptive response potentially equips the plant for enhanced drought tolerance for future drought events. The wide variability in stomatal responses observed in the four varieties under study reinforces the idea that adjustments in stomatal anatomical features represent a genotype-dependent mechanism of adaptation to prolonged water stress rather than a strategy common to all grapevine cultivars.
This short report clearly illustrates the substantial impact of drought on stomagenesis. However, our data do not permit an assessment of the functional significance of the observed variations in stomatal traits. This limitation arises from the unsuitability of young leaves for physiological measurements, given their known lack of full photosynthetic activity. Future studies should address this aspect, planning long-term drought experiments to analyse if changes induced in stomata density and size in mature leaves imply improved grapevine physiological performances.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture13122186/s1, Table S1: List of the oligonucleotides used in the study; Figure S1: Midday stem water potential (Ψstem) measured in well-watered (A) and water stress treatments (B) of the four varieties under study during the experiment. Each point is the average of measurements taken on five individual plants over 2 seasons (2017–2018). This figure summarises the data published in extended form in Gaiotti et al. (2023), [28]; Figure S2: Comparative images of the stomata of the four varieties under study under well-watered (WW) and water stress (WS) conditions. Photomicrographies are taken from nail polish impressions of the abaxial leaf surface of young leaves (7 days after tip separations).

Author Contributions

Conceptualization, L.N., W.C. and F.G.; methodology, L.N., W.C. and F.G.; investigation, L.N., W.C. and F.G.; data curation, L.L., G.F., L.N., W.C. and F.G.; writing—original draft preparation, L.N., W.C. and F.G.; writing—review and editing, L.L. and G.F.; visualization, L.L. and G.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 13 02186 g001
Figure 1. Box plot for stomatal density and stomata length measured in young leaves (7 days after tip separation) of well-watered (WW) and water-stressed (WS) treatments of Garganega, Glera, Merlot, and Moscato varieties. Analysis were performed in 2017 (A,B) and 2018 (C,D) on leaf samples collected one week after rewatering. The lines that delimit the bottom and the top of the boxes are the 25th and 75th percentiles, respectively. The 10th and 90th percentiles are indicated by whiskers. Outlayers are indicated by dots. For each variety, asterisks indicate a significant difference between WS and WW treatments according to t-test analysis (p ≤ 0.05).
Figure 1. Box plot for stomatal density and stomata length measured in young leaves (7 days after tip separation) of well-watered (WW) and water-stressed (WS) treatments of Garganega, Glera, Merlot, and Moscato varieties. Analysis were performed in 2017 (A,B) and 2018 (C,D) on leaf samples collected one week after rewatering. The lines that delimit the bottom and the top of the boxes are the 25th and 75th percentiles, respectively. The 10th and 90th percentiles are indicated by whiskers. Outlayers are indicated by dots. For each variety, asterisks indicate a significant difference between WS and WW treatments according to t-test analysis (p ≤ 0.05).
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Figure 2. Expression changes of water-stress- and stomagenesis-related genes in unfolded leaves collected on the last day of water restriction in 2018 from WW (grey bars) and WS (black bars) treatments of the four varieties: (A) VvDH; (B) VvEPFL9; (C) VvEPF1; and (D) VvEPF2. For each cultivar. asterisks indicate significant differences between treatments as attested by student t-test (p ≤ 0.05).
Figure 2. Expression changes of water-stress- and stomagenesis-related genes in unfolded leaves collected on the last day of water restriction in 2018 from WW (grey bars) and WS (black bars) treatments of the four varieties: (A) VvDH; (B) VvEPFL9; (C) VvEPF1; and (D) VvEPF2. For each cultivar. asterisks indicate significant differences between treatments as attested by student t-test (p ≤ 0.05).
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MDPI and ACS Style

Nerva, L.; Chitarra, W.; Fila, G.; Lovat, L.; Gaiotti, F. Variability in Stomatal Adaptation to Drought among Grapevine Cultivars: Genotype-Dependent Responses. Agriculture 2023, 13, 2186. https://doi.org/10.3390/agriculture13122186

AMA Style

Nerva L, Chitarra W, Fila G, Lovat L, Gaiotti F. Variability in Stomatal Adaptation to Drought among Grapevine Cultivars: Genotype-Dependent Responses. Agriculture. 2023; 13(12):2186. https://doi.org/10.3390/agriculture13122186

Chicago/Turabian Style

Nerva, Luca, Walter Chitarra, Gianni Fila, Lorenzo Lovat, and Federica Gaiotti. 2023. "Variability in Stomatal Adaptation to Drought among Grapevine Cultivars: Genotype-Dependent Responses" Agriculture 13, no. 12: 2186. https://doi.org/10.3390/agriculture13122186

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