Mobile sulphur status in soils of organic and conventional vineyards of the Southern coast of Crimea

. The data on the content of mobile sulfur in soils of vineyards on the Southern coast of Crimea with organic and conventional land use systems as well as in fallow soils after long-term application of chemical pesticides are presented. As a result of the use of sulphur-containing pesticides allowed in organic farming and the activation of organic matter mineralization processes due to regional soil and climatic conditions, a significantly higher accumulation of mobile sulphur in the upper soil horizons of organic farms was observed. Higher levels of mobile sulphur in organic vineyard soils can adversely affect the organoleptic and physico-1


Introduction
Recent studies have confirmed the multifunctional role of sulphur in managing soil fertility and crop productivity.Sulphur is classified as a macronutrient which, along with nitrogen, phosphorus and potassium, affects plant physiological and biochemical processes [1].The scientific literature extensively discusses sulphur deficiency in agroecosystems [2,3].However, the potential consequences of sulphur excess are neglected.The extensive and prolonged use of sulphur-containing pesticides, particularly in orchards and vineyards, results in the accumulation of sulphur in the upper parts of the soil profile [4].This phenomenon can induce toxicity in plants and soil microorganisms, with adverse effects on biodiversity and human health [5,6].
Throughout history, sulphur has been used as a natural insecticide to control parasites, as a fungicide to protect plants, as a wine preservative, as a bactericide, and as an antioxidant.Since the mid-20th century, sulphur, in combination with copper compounds, has been used in plantation and horticultural agroecosystems as an inorganic contact fungicide and low hazard acaricide.Thus, various sulphur-based products are used to control powdery mildew (Uncinula necator (Schw.)Burr.), the most common grapevine disease worldwide.The toxicity of sulphur is attributed to its liposolubility.It enters the mycelium and spores of the fungus either in the form of vapour or by diffusion through lipid membranes.
Sulphur is preferred over alternative organic fungicides due to its low cost, availability, high efficacy and lower toxicity.Additionally, it has a lower risk of resistance development [7].For these reasons, Russia and other countries allow the use of sulphur and sulphur-based preparations, even in organic and biodynamic viticulture [8,9].Calcium polysulphide (lime sulphur) and elemental sulphur are permitted for treatment on organic farms in the Russian Federation, which are required to be used only if there is an immediate threat to the crop (GOST 33980-2016).In traditional farming under different trade names preparative forms of sulphur in the form of water-dispersible granules (Tiovit Jet, Cumulus DF, Vitashans), suspension concentrate (Sulphur 400, Flosul) are used.
Sulphur-based pesticides are also widely used in European countries.According to Eurostat (2022), inorganic fungicides containing copper and inorganic sulphur accounted for almost 57% of total fungicide and bactericide sales in European countries in 2020.It is also used as a component of some herbicides.According to the EU Pesticides Database, examples of authorized sulphur-containing pesticides are preparations with the following active ingredients: sulfosulphuron, sulfoxaflor, sulphuryl fluoride, calcium polysulfide.
Soil sulphur derived from pesticides and agrochemicals is actively involved in natural aqueous and biogeochemical fluxes and various transformation processes.As a result, the chemical form, nature and duration of deposition, mobility and bioavailability of sulphur in the soil are constantly changing [10].Up to 98% of the total element content is found in the soil in organic form as part of plant residues and humus and is represented by various complex organic compounds (e.g.sulphate esters).
Sulphur as part of organic compounds is not available for uptake by plant roots.The bioavailability of the element is ensured by the mineralization processes of its organic compounds to sulphate forms through microbial mediated processes [11].Therefore, the biomass and activity of soil microbial populations are key factors that regulate the reversible processes involved in the conversion of organic sulphur to inorganic sulphate, playing an essential role in the biogeochemical cycling of sulphur and determining its bioavailability [12,13].
The sulphur biogeochemical cycle is complex, affected by various biotic and abiotic factors.However, among all, the crucial factors in the context of agroecosystems are climate and agro-technological practices.In particular, the specific features of applied grape cultivation technologies, as well as regional climatic conditions can significantly change the balance of sulphur in vineyard soils.The mobility of sulfate sulphur, resulting from the oxidation of elemental sulphur when used as a pesticide, largely depends on the soil's water regime.Applied sulphur accumulates in the top layer of the vineyard soil when grapes are grown in arid conditions without irrigation until the rainy season starts.
Under conditions of high soil moisture, sulphates become saturated in the soil solution and have the potential to migrate.This can pose an ecological risk to surface and groundwater quality, especially where vineyards are located on sloping landforms [14].The ideal combination of temperature and humidity, together with certain agricultural techniques, in particular the use of organic fertilizers, increases the activity of soil microbes, which promote the mineralization process of organic matter and thus increase the content of mobile sulphur in the soil.
The limited number of studies investigating the impact of sulphur-containing pesticides on agrochemical and biological properties of soils in vineyards using different agrotechnologies has motivated the objective of this research paper.The aim of the study was to identify regional patterns of mobile sulphur accumulation in soils of vineyards on the Southern coast of Crimea, where organic and traditional land use systems are practiced.Additionally, the study included fallow soils used for long-term grape cultivation with the application of chemical plant protection system.
The study was carried out in the Sevastopol Region, Republic of Crimea.The soil of six organic wineries that were certified in accordance with the GOST 33980-2016 standard and five conventional wineries with chemical plant protection systems were investigated.Fallow land in former wineries that had previously used traditional agricultural techniques over a long period of time was also studied.Soils on all farms were identified as Haplic Kastanozems/Haplic Calcisols according to WRB.The locations of the farms where soil samples were collected are shown in Fig. 1.According to the Köppen classification, the Sevastopol wine growing area has subtropical Mediterranean climate conditions, characterized by moderately hot and dry summers and mild winters with frequent thaws, as shown in Fig. 2.
The daily average high (red line) and low (blue line) temperature, with 25th to 75th and 10th to 90th percentile bands.The thin dotted lines are the corresponding average perceived temperatures.During the autumn-winter period, precipitation is significantly higher than the annual average, while during the spring-summer period it is significantly lower than the average (Fig. 3).In this regard, the quantity of rainfall is a limiting environmental factor in arable farming, and without irrigation it is impossible to achieve sustainable yields.However, the climatic and soil conditions favour the development of wine-growing and winemaking.Soil samples were collected using a soil auger at depths of 0-10 cm and 10-20 cm.Soil sampling was carried out using the envelope method.Five samples of 0.5 kg each were taken from a 5 m x 5 m plot.The five individual samples were then mixed and one combined sample weighing 1.0 kg was analyzed.Soil preparations included milling, removing plant roots, stones and other inclusions.

The average rainfall (solid line) accumulated over the course of a sliding 31-day period centered on the day in question, with 25th to 75th and 10th to 90th percentile bands. The thin dotted line is the corresponding average snowfall.
Water pH was analyzed potentiometrically with a glass electrode using a pH meter.The organic matter content was measured by the photometric method using a strong oxidizing agent (K2Cr2O7) in the presence of H2SO4 with a spectrophotometer.Mobile sulphur content was determined using a 0.15 percent CaCl2 solution.The mobile phosphorus and potassium content in soils was determined by an ammonium carbonate extraction, according to the Machigin method (with pH = 9.0 and 1:20 soil/solution ratio).After extraction, potassium content was determined using flame emission photometry.Phosphorus was determined by a spectrophotometer after color development with ammonium molybdate and SnCl2.All analyses were performed in triplicate.
The determination of basal respiration (BR) was carried out in accordance with CEN EN ISO 16072-2011 Soil Quality Laboratory methods for the determination of microbial soil respiration (ISO 16072:2002).Soil was moistened with distillated water of 0.2 mL and incubated for 24 h at 22 ± 0.5°C.After incubation, the carbon dioxide content in 3 mL of the air probe extracted by a syringe was measured using a Chromatec-Crystal 5000.1 gas chromatograph.The BR rate was expressed in µg CO2 C g −1 soil per hour, with a five-fold repeatability.
Substrate-induced respiration (SIR) was determined in a similar manner, but the soil was moistened with an aqueous glucose solution (10 mg glucose g −1 soil) instead of distilled water and incubated for 3.5 h at 22 ± 0.5°C.The SIR rate was expressed in µL CO2 g −1 soil per hour, with a five-fold repeatability.
The data were statistically processed using the R language and Software Package STATISTICA.

Results
Table 1 shows pH, organic matter, mobile phosphorus and potassium content, mobile sulphur content, basal and substrate-induced respiration indicators in vineyard soils according to the land use system.The pH values for the aqueous extracts in the soils of the farms studied ranged from 7.18 to 8.38 pH units, indicating a moderately alkaline to neutral environment.
The organic matter content of the topsoil varied widely, from 1.48% to 4.34%, and showed a decreasing trend from fallow soils to the soils of the conventional farms and the organic farms.Organic matter generally declines with depth, although there are some exceptions.
The mobile phosphorus content varied significantly across the farms studied, ranging from 3.8 to 138.3 mg P2O5/kg of soil.These levels corresponded to soil categories I to VI, indicating very low to very high availability of phosphorus.The mobile potassium content of the vineyard soils studied was high, high and very high, ranging from 359 to 1109 mg K2O/kg soil in the topsoil layer (0-10 cm).
The mobile sulphur content in vineyard soils ranged from 2.1 to 39.2 mg/kg and correlated with the land use system applied on the farms, according to the Kraskell-Wallis criterion (Fig. 4).The average mobile sulphur content of organic vineyard soils was 18.3 mg/kg.In comparison, the corresponding values for conventional vineyard soils and fallow were 8.0 and 5.5 mg/kg, respectively.Based on the classification used by the Russian Agrochemical Service, the soils of conventional vineyards and fallow in general corresponded to the group with low mobile sulphur content (less than 6.0 mg/kg) or close to it.Organic farms had an average sulphur content of 21.7 and 14.8 mg/kg at a depth of 0-10 and 10-20 cm, respectively, which was 1.8 -3.4 and 2.9 -3.7 times higher than in conventional farms and on fallow land.To identify trends in the change of agrochemical indicators of soils depending on the content of mobile sulphur, the studied farms were divided into groups with very low (less than 5 mg/kg), low (5-10 mg/kg), medium (10-35 mg/kg) and high (more than 35 mg/kg) levels of mobile sulphur in horizons 0-10 (Table 2) and 10-20 cm (Table 3).This grouping used in some other countries was more appropriate for a wide range of mobile sulphur content in vineyard soils found in this study.The data from Table 2 show that the group with low and very low mobile sulphur in the top horizon included soils from fallow and conventional farms.The group with medium and high mobile sulphur included soils from all organic farms and only one conventional farm.
Furthermore, the grouping demonstrated the following trend: as the average mobile sulphur content in the top horizon increased from 3.1 to 38.5 mg/kg, the average pH values decreased from 8.1 to 7.3-7.6pH units, the organic matter content decreased from 3.8 to 2.7% and the mobile potassium content decreased from 721 to 467 mg/kg.
The mobile sulphur content was lower in the 10-20 cm horizon than in the upper horizon, as observed in other studies [15].The soils here are characterized by a predominance of low and very low levels of elements.None of the farms is classified as having high levels of mobile sulphur.The range of this indicator was not as wide as in the upper horizon, varying from 3.5 mg/kg in the low element group to 18.0 mg/kg in the medium element group (Table 3).The general trends that were observed for the upper layer remained valid to a depth of 10 -20 cm.Thus, with an increase in mobile sulphur content from very low to medium, the pH decreased from 8.0 to 7.7, and the organic matter content reduced from 3.3% to 2.3%.Additionally, the mobile phosphorus and potassium content also decreased from 33.2 to 16.7-22.4and from 678 to 364 mg/kg soil, respectively.It should be noted that the differences in the groups of indicators observed at the depth of 10-20 cm were not as contrasting as in the top soil horizon of the vineyards studied.
Statistical analysis showed a reliable inverse correlation between mobile sulphur content and organic matter content only in the 0-10 cm and 10-20 cm horizons of fallow soils, with correlation coefficients of -0.98 and -0.99, respectively (Fig. 5).
Table 1 shows that the average value of basal respiration in the soils of the organic farms was 2.7 times higher than that of the conventional farms and fallow land.This difference indicates a more favorable state of the soil microbiome in the soil of organic vineyards.This conclusion is supported by even more contrasting values of substrate-induced respiration.These values were 3.5 and 5.9 times higher in the top soil horizon of organic farms, compared to conventional farms and fallow land respectively.coefficients of 0.86 and 0.84 respectively.A significant inverse correlation was found between substrate-induced respiration and pH in the organic vineyard (Fig. 6) and fallow soils, with correlation coefficients of -0.87 and -0.99, respectively.
It is important to note that the soil respiration indices gradually increase with increasing mobile sulphur content, reaching their maximum values in the group of soils with medium sulphur content, ranging from 10 to 35 mg/kg.In the group with a high level of S in the 0-10 cm horizon there was a twofold reduction in both basal and substrate-induced respiration, which could be attributed to the suppression of the functional state of the soil microbiome.At a depth of 10-20 cm, the BR and SIR indices increased by a factor of 4 and 1.5 respectively in the low mobile sulphur group compared to the very low level, but there was almost no change in the indices when reaching the medium level.

Discussion
Vineyards provide a unique opportunity to study the processes of sulphur input, transformation and migration in the soil due to the scale of sulphur-containing pesticide use, the diversity of landscapes, and the soil-climatic and hydrological conditions of the growing regions.The interaction of natural, agrogenic, and anthropogenic factors significantly affects the status of sulphur in ampelocenoses.As local conditions can be highly variable, it is difficult to identify general patterns in soil sulphur behavior and accumulation in different areas.
In the context of the Southern coast of Crimea, the accumulation of mobile sulphur in the top layer of the soil of organic vineyards was 2-3 times higher than in the soil of conventional vineyards.The significant sulphur inputs are assumed to be a result of the approved use of fungicides, acaricides and repellents during organic production.Furthermore, due to climatic changes in the region, the emergence of resistant strains and new pathogens, and the use of susceptible varieties, the number of such treatments in vineyards on the Southern coast of Crimea is expected to increase in the coming years [16].In this regard, it is to be noted that the increased sulphur content in the soils of ampelocenoses enhances the availability and assimilation of nitrogen by plants and reduces the accumulation of phenolic compounds in grape berries, which determine the quality of the wine material and its belonging to a particular terroir.As a result, the organoleptic and physico-chemical characteristics of the wine products obtained are adversely affected [17].
Soil properties such as organic carbon content and soil pH have the greatest influence on the distribution of sulphur between soil layers and its availability to plants [18].Some authors have observed a direct correlation between soil organic matter and total sulphur content [9].Our study showed a significant inverse correlation between organic matter and mobile sulfur content only in fallow soils without agrogenic load.The application of agrochemicals, technological treatments of upper soil horizons of conventional farms, and the use of sulfur in the plant protection system of organic farms apparently eliminated possible correlations between the content of mobile sulfur and the content of organic matter, and therefore no reliable correlation was found between these indicators.
As soil microorganisms play a crucial role in transforming sulphur compounds between organic and inorganic forms, soil biological activity was assumed as an estimated integral indicator in this study.Its most informative component is microbial respiration: basal respiration is widely used to determine the current physiological status of soil microbiota [19,20], substrate-induced respiration can be considered as a model of the potential ability of microorganisms to mineralize organic matter [21].Our research identified high levels of soil microbial activity in relation to both BR and SIR, where these indicators were found to be 3.5 and 5.9 times higher, respectively, than those found in soils from conventional farms and fallow land.Therefore, it is expected that the mineralization of organic matter and the release of sulphate as a by-product of this process occurred more intensively in the soils of organic farms, especially considering the high temperature background in the conditions of the Crimean peninsula.This is also indicated by the lowest organic matter content in organic vineyard soils among the farms studied, averaging 2.6% compared to 3.4-3.5% in conventional vineyard and fallow soils.
The pH indicator (water extract) in both horizons of the soils studied decreased with increasing mobile sulphur content.Field and laboratory studies by other authors also confirmed a decrease in pH with elemental sulphur application [22,23].In alkaline soils, such as those used for vineyards on the Southern Crimean coast, this effect neutralizes soil alkalinity and activates heterotrophic microorganisms that mineralize organic matter.This illustrates the significant negative correlation found in our study between pH and substrateinduced respiration indices in soils from organic farm.Consequently, sulphur is assimilated into the microbial biomass and partly transferred to the soil in the form of sulphate.Soil acidification caused by use of sulphur-containing plant protection products in organic vineyards on Crimean south coast induces mineralization of organic matter and release of additional bioavailable sulphur, but quantitative characteristics of this process remain unknown and require further study.
The accumulation of sulphur in vineyard soil may have negative ecological consequences for the surrounding environment.Leaching of sulphate ions from soil is considered as one of the primary pathways for element losses in agroecosystems, but this process is highly dependent on soil and climatic conditions.The few studies carried out on ampelocenoses suggest that sulphur is effectively released from the root layer in vineyards, particularly in areas with adequate moisture and where irrigation is used.In particular, intense leaching of sulphate ions was observed in the warm and humid climate of the Apulia region in southeastern Italy, as well as in the irrigated vineyards of northern California [9,24].Under severe overwatering and anaerobic conditions, sulfur can also be lost from the soil as gas.
In the Sevastopol region of the Republic of Crimea, where soil moisture supply is frequently a limiting factor for crop yield, the losses of mobile sulphur resulting from leaching processes during the growing season are considered insignificant in the absence of irrigation systems.An imbalance between sulphur input and sulphur output towards the former item may lead to sulphur accumulation in the upper soil horizons during the vegetation of grape plants, which was found in the present study for soils of organic vineyards.
Our studies highlight the importance of investigating the impact of sulphur as a pesticide or agrochemical, particularly in organic farming systems, taking into account regional and local climatic characteristics.

Conclusions
A study on Haplic Kastanozems in vineyards on the South Coast of Crimea, cultivated by organic and conventional technologies, showed a significant accumulation of mobile sulphur in the soils of organic farms, with levels 1.8 -3.4 times higher than this indicator in the soils of conventional farms and fallow land, as a result of the permitted use of sulphur-containing pesticides in organic viticulture.
The increase in bioavailable sulphur content in organic farm soils was due to higher organic matter mineralization resulting from the increased activity of soil microbiome and favourable soil-climatic conditions.Thus, the index of substrate-induced respiration in the soils of organic farms was 3.5 and 5.9 times higher than in the soils of conventional farms and fallow land, respectively.
The neutralization of the alkaline reaction of the soil solution by the increased content of sulphate ions further promoted the mineralization of organic matter, as confirmed by the observed inverse correlation between pH and the index of substrate-induced respiration in the soils of organic farms.
Considering the low potential for sulphate leaching from the root layer due to the low rainfall during the growing season in the context of the Southern coast of Crimea, we can expect an increase in the sulphur content of wine products, which will negatively affect their quality characteristics and terroir identity.

Fig 1 .
Fig 1. Location of the studied wineries.

Fig. 4 .
Fig. 4. Mobile sulphur content (mg/kg soil) compared between different land-use types in the upper and lower horizons of the studied vineyard soils according to the Kruskal-Wallis criterion (p=0.07).

Fig. 6 .
Fig. 6.Correlation between pH (H2O) and substrate-induced respiration (µg CO2 g −1 soil per hour) in soils of organic vineyards at a depth of 10-20 cm (α = 0,05).Basal respiration indices showed a direct and reliable relationship with mobile phosphorus and potassium contents in organic vineyard soils at 10-20 cm, with correlation

Table 2 .
Distribution of mean values of agrochemical indicators and soil respiration by groups according to the mobile sulphur content (Smob) in the 0-10 cm horizon of the vineyard soils studied

Table 3 .
Distribution of mean values of agrochemical indicators and soil respiration by groups according to the mobile sulphur content (Smob) in the 10-20 cm horizon of the vineyard soils studied