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Effects of Drying Temperature on Quality Parameters of Thai Fermented Fish Dip (Jaew Bong)

Fermented fish dip is a popular condiment in Thailand and the Lao People’s Democratic Republic. Thai fermented fish dip (TFFD) can be dried to increase its shelf life and ease of transportation. Dried TFFD can be rehydrated to return the powder to its original, paste-like form. Pre-cooked TFFD paste was dried at three different temperatures (40, 60, and 80 C). Total plate count, yeast and mould count, CIE colour values (L*, a*, and b*), non-enzymatic browning, and sensory scores of the resulting powders were determined. The CIE colour values and sensory scores were also analysed for rehydrated TFFD. Increasing the drying temperature did not affect the total plate count or yeast and mould count. When dried at 80 C , the L* value of TFFD powder was reduced, although the a* and b* values were unaffected compared with lower temperatures. All CIE colour values of rehydrated TFFD decreased as drying temperature increased. Drying temperature did not affect the sensory scores of dried TFFD powder. However, rehydrated samples that had been dried at 80 C had significantly lower sensory colour scores than those dried at 40 or 60 C . Overall preference rankings of dried and rehydrated TFFD dried at 40 and 60 C were better than for those dried at 80 C. Due to an undesirable colour change in the rehydrated product, 80 C was deemed to be an unsuitable temperature for drying TFFD paste. In conclusion, both 40 and 60 C are appropriate temperatures for drying the product.

Introduction
Fermented fish dip is a condiment that is popular in the north-eastern and central regions of Thailand. The product is also commonly consumed in Lao People's Democratic Republic (Lao PDR), a neighbouring country of Thailand. Fermented fish dip is known in the Thai language as Jaew Bong, Plara Bong, or Nam Prik Plara; and in the Lao PDR language as Jaew Pla Dak. Thai fermented fish dip (TFFD) is composed of fermented fish paste (Plara), dried chilli, and fresh herbs including galangal, lemon grass, red shallot, and garlic. Galangal and lemon grass are sliced and used fresh. Red shallot and garlic are pan-roasted, grilled, or baked and peeled prior to combination. The ingredients are combined and pounded into a paste (Thai Industrial Standards Institute, 2013), after which taste-and flavour-enhancing ingredients such as fermented fish sauce, tamarind paste, and sugar may be added (Duangsai, Srisataporn, Hauisan & Gawbor-Copyright ©2020 ISEKI-Food Association (IFA) 10.7455/ijfs/9.1.2020.a10 isut, 2019; Posri, 2008). TFFD paste is typically served with glutinous rice, fried or roasted meat, and fresh or steamed vegetables. The viscous paste is able to stick well to the rice, meat, or vegetables, which balances the taste and heat of TFFD (Duangsai et al., 2019;Posri, 2008;Teaupun, 2009). In the past, TFFD was prepared in the home and served uncooked. At the present time, TFFD is primarily manufactured in small-scale factories, and the product is fully cooked to eliminate food-borne pathogens such as Salmonella spp. and Staphylococcus aureus (Thai Industrial Standards Institute, 2013). The quality of TFFD varies depending on the ratio of ingredients and the processing methods used (Posri, 2008). High-quality TFFD contains wellblended ingredients and a desirable herbal flavour (Thai Industrial Standards Institute, 2013). Poor-quality TFFD may show visible separation of liquid from the paste, or have offensive musty or sour flavours associated with spoilage (Thai Industrial Standards Institute, 2013). Colour is a vital element in assessing the quality of uncooked TFFD, because it can be indicative of the freshness and level of heat in the product (Posri, 2008;Ratchatachaiyos, 2007). Cooked TFFD can be expected to be darker in colour than the raw product due to non-enzymatic browning reactions that occur during the cooking process (Posri, 2008). The pH of commercial TFFD ranges from 4.42 to 5.4 (Posri, 2008). The moisture content and water activity (a w ) of commercial TFFD are 48.61-64.40% and 0.82-0.86 respectively (Posri, 2008). Its medium/low acidity and intermediate moisture and a w levels make TFFD prone to spoilage, therefore shortening its shelf life (Duangsai et al., 2019;Hiraga, Stonsaovapak, Sittipod & Mahakarnchanakul, 2008;Posri, 2008). Additionally, the high moisture content of the product means that liquid leakage may occur during transportation, and the transportation weight may be high (Duangsai et al., 2019). Drying is a widely used method for preserving food. Reduction of the a w to < 0.66 prevents microbial spoilage and prolongs the shelf life of food products (Ulloa et al., 2015). In the case of TFFD, the removal of moisture also eliminates the transportation issues described previously. Dried TFFD may be ground or pulverized, to be used as an instant powder. Appropriate amounts of water can be added to rehydrate the powder, returning the dried TFFD to the original pastelike form. Utilization of the drying process is limited in foods containing heat-sensitive compounds (Ahmed, Shivhare & Ramaswamy, 2002). Chilli and herbs -key ingredients contributing to the freshness, heat, and flavour of TFFD -contain heat-sensitive colour pigments and volatile oils, respectively. Although the effects of the drying process on the quality of dried TFFD have not been reported, it is likely that these ingredients will be adversely affected by the heat of the process. The negative effects of dehydration on the colour of capsicums and chilli-based products have been found to be caused by non-enzymatic browning reactions and thermal degradation of colour pigments (Ahmed et al., 2002;Arslan & Ozcan, 2011;Kim, Lee, Park, Lee & Hwang, 2006;Topuz, Feng & Kushad, 2009;Vega-Galvez, Lemus-Mondaca, Bilbao-Sainz, Fito & Andres, 2008;Vega-Galvez et al., 2009). Using a proper drying temperature may minimize adverse effects and maintain the desired colour and flavour of dried TFFD. The aim of this research was to investigate the quality parameters of dried TFFD powder and rehydrated TFFD, after drying at different temperatures (40, 60, and 80 o C), in order to ascertain the appropriate temperature for drying TFFD.

Preparation of cooked TFFD
The TFFD was prepared in accordance with a protocol modified from Thathiwan (2017). Briefly, 6 g fermented tilapia paste, 480 g minced galangal, 360 g thinly sliced lemon grass, 900 g minced, peeled, and roasted shallot, 900 g minced, peeled, and roasted garlic, and 240 g tamarind paste were blended using a food processor (MCM 640660, Bosch, Bratislava, Slovakia). Then, 264 g chilli powder, 204 g sugar, and 72 g julienned kaffir lime leaves were handmixed into the blended herbs. Fermented tilapia paste was purchased from a local factory (Phetdam Foods Co. Ltd., Kalasin, Thailand) and IJFS April 2020 Volume 9 pages 251-263 kept at ambient temperature as recommended by the manufacturer. Other ingredients were purchased from a local supermarket (Tesco Lotus, Khon Kaen, Thailand). The resulting TFFD was divided into three equal portions, each of which was weighed and cooked as recommended by the Thai Industrial Standards Institute (2013). This involved heating on the stovetop until the internal temperature reached 70 o C and then simmering for 15 min (Official Methods of Analysis of the Association of Official Analytical Chemists, 1990). The cooked TFFD portions were packed into polypropylene plastic bags, kept in a refrigerator at 4 o C, and used for the experiment within 3 h.

Drying and rehydration of cooked TFFD
The three portions of cooked TFFD were spread on silicon baking mats to a thickness of 2 mm and dried in a hot-air oven at either 40, 60, or 80 o C until the a w reached 0.66 or lower (Ulloa et al., 2015). The a w assessment was conducted using an Aqua Lab Series 4TEV water activity meter (Aqua Lab, Pullman, WA, USA). Each sample was then aseptically removed from the mat with a sterilized spatula, weighed, and the microbial content analysed. The samples were pulverized prior to assessment of CIE colour values, nonenzymatic browning, and sensory acceptability. Dried TFFD powder samples were rehydrated with hot water. The rehydrated pastes were also analysed for CIE colour values and sensory acceptability. All experiments were repeated in triplicate using three lots of fermented fish.

Microbial content
Analysis of microbial content involved measurement of the total plate count (TPC) and yeast and mould count (YM

CIE colour values
The samples were analysed for CIE colour values (L*, a*, and b*) using a Konica Minolta CM-2600d spectrophotometer (Konica Minolta, Inc., Japan). A D65 artificial daylight bulb and 10 o standard angle observer were used to illuminate the samples. The L* value indicates lightness, while a* and b* are the red/green and yellow/blue coordinates respectively.

Non-enzymatic browning
Non-enzymatic browning was assessed using the method described by Dissaraphong, Benjakul, Visessanguan and Kishimura (2006). Briefly, 5 g of dried TFFD powder was combined with 50 mL of ethanol 50% (v/v), stirred continuously for 60 min, and filtered with Whatman No. 1 filter paper. The absorbance of the filtrate was determined at 420 nm using a Spectronic-15 spectrophotometer (Thermo Scientific, Thermo Fisher Scientific India Pvt. Ltd., Nasik, India).

Sensory score evaluation
Sensory acceptability (colour, odour, texture, flavour, and overall acceptability) was evaluated using a nine-point hedonic scale according to Meilgaard, Civille and Carr (1991) (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely). Prior to evaluation, samples were randomly assigned a three-digit number and presented to 45 panellists acquainted with TFFD. The panel was composed of 18 females and 17 males with ages between 20-50 years. All sensory evaluations were conducted in an air-conditioned room at 25 o C. A score of 5 was considered the limit of acceptability for all sensory parameters. Overall preference ranking according to Lu (2017) was also evaluated, with a ranking of '1' meaning most preferred. Any undesirable sensory characteristics noted by the panellists were recorded.

Analysis of rehydrated TFFD
Samples of dried TFFD powder were rehydrated with hot water (temperature > 90 o C ). The amount of water added to the powder was calculated according to equation (1). The samples were allowed to absorb the water for 30 min and then stirred continuously for 2 min. CIE colour values and sensory tests were then evaluated as described in section 2.3.
where WT reh is the amount of hot water used for rehydrating the sample (g), W uncook is the weight of uncooked TFFD (g), and W dried is the weight of dried TFFD (g).

Statistical analysis
The experiment was carried out using a randomized complete block design (RCBD). Data were analysed using SAS University Edition (SAS Institute Inc., Cary, NC) with a 95% confidence level. The calculated means were compared using the least significant difference (LSD) test.
Overall preference rankings were compared using Friedman's test as recommended by Meilgaard, Carr and Civille (2006).

Microbial content
The calculated TPC and YM values of dried TFFD samples are shown in Fig. 1. TPC and YM showed no significant difference (p > 0.05) between the three drying temperatures. The results indicated that increasing the drying temperature did not promote microbial destruction. It has been reported that some microorganisms are destroyed in the drying process. However, many microorganisms -such as bacterial endospores, yeasts, moulds, and several Gram-negative and Gram-positive bacteria -are resistant to dehydration (Jay, 2000). Our results indicated that the tested TFFD may have contained microorganisms that were able to withstand drying tem-peratures of ≤ 80 o C . Herbs and spices contain microorganisms indigenous to the soil and plants where they are grown, often including heat-resistant spore-forming bacteria which are able to survive the drying process (Farkas, 2001;Fellows, 2000). It is likely that the heat-resistant microorganisms in the TFFD originated from the herbal ingredients in the product. These results were in agreement with Orphanides, Goulas, Botsaris and Gekas (2017)

CIE colour values
The CIE colour values of dried TFFD are shown in Fig. 2. The results showed that increasing the drying temperature to 80 o C significantly reduced the L* value of dried TFFD, compared with 40 and 60 o C (p < 0.05) (Fig. 2 a). However, L* values showed no significant difference (p > 0.05) between 40 and 60 o C . The three temperatures had no effect on the a* and b* values (p > 0.05) (Fig. 2 b and c). The reduction of L* value may have resulted from increased non-enzymatic browning at higher temperatures. To our knowledge, the effect of drying temperature on the colour values of dried TFFD has not yet been reported. In both uncooked and cooked TFFD, non-enzymatic browning has been reported to be a cause of colour change (Posri, 2008). Changes in the colour values of dried jumbo squid and Atlantic salmon fillets are reported to be due to non-enzymatic browning, and are more prominent in samples exposed to high drying temperatures of 50 o C or more (Ortiz et al., 2013;Vega-Galvez et al., 2011). Additionally, the colour changes in dried salted cod (Gadus morhua) that occur at higher temperatures have been found to be due to contraction of  IJFS April 2020 Volume 9 pages 251-263 muscle myotomes caused by protein aggregation (Ozuna, Gomez Alvarez-Arenas, Riera, Carcel & Garcia-Perez, 2014). Similar contraction of the fish muscle in TFFD at increased drying temperatures may contribute to reduction of the L* value.
Colour changes in dried red pepper and chilli are reported to be caused by non-enzymatic browning and the thermal degradation of colour pigments (Ahmed et al., 2002;Arslan & Ozcan, 2011;Kim et al., 2006). These reports showed that high drying temperatures cause more brown pigments to form, contributing to discolouration of the dried products. Discolouration of dried chilli in TFFD may have occured at 80 o C , thus reducing the L* value of the dried product. From these studies, it can be hypothesized that non-enzymatic browning of the fish in TFFD contributed to the reduction in the L* value of TFFD dried at 80 o C . To prevent colour changes in TFFD caused by non-enzymatic browning of chilli, drying temperatures of 40 and 60 o C are more appropriate than 80 o C .

Non-enzymatic browning
The results of the assessment for non-enzymatic browning of dried TFFD samples are shown in Fig. 3. The lowest value (0.76) was detected in the sample exposed to 40 o C , while the highest value (1.97) was found in the sample exposed to 80 o C . The rate of browning increased significantly as the drying temperature increased (p < 0.05). Non-enzymatic browning involves the reaction of carbonyl compounds with amino groups. In muscle-based foods, the carbonyl compounds mainly originate from carbohydrates, in the form of glycogen, reducing sugars, and nucleotides. Amino groups are readily available from the muscle protein (Vega-Galvez et al., 2011). The rate of non-enzymatic browning is reported to be highly sensitive to heat, increasing 2-3-fold for each 10 o C rise (Gögüs, Fadıloglu & Soysal, 2009). It has been reported that non-enzymatic browning reactions reduce the colour quality of uncooked TFFD (Posri, 2008), but the effect of drying temperature on the non-enzymatic browning of dried TFFD has not, to our knowledge, been previously reported. Heat from the cook-ing process increased the rate of browning in TFFD, resulting in the cooked product displaying a darker colour (Posri, 2008). Drying temperatures of 50-90 o C have been shown to increase non-enzymatic browning reactions in both dried and rehydrated jumbo squid (Dosidicus gigas) (Vega-Galvez et al., 2011). Furthermore, Atlantic salmon (Salmo salar L.) fillets dried at 60 o C exhibit more colour change due to nonenzymatic browning compared with those dried at 40 or 50 o C (Ortiz et al., 2013).
The results led to the conclusion that fermented fish may influence the non-enzymatic browning of TFFD. This ingredient is composed of fish, salt, and carbohydrates such as rice bran or roasted rice, fermented for at least 6 months at ambient temperatures. During the fermentation process, proteolytic enzymes -naturally present in the guts and muscle of fish and those produced by microorganisms -break down the muscular protein into amino acids. Amylases and lipases produced by the microorganisms also cause chemical changes in the product, by releasing sugars from the rice bran or roasted rice (Krusong, 2004). Fermented fish in TFFD may therefore supply significant amounts of free amino acids and reducing sugars, which could contribute to excessive non-enzymatic browning of the product. This theory is supported by the fact that squid -which is also rich in free amino acids -is susceptible to excessive browning. This poses a significant quality problem in dried squid products, especially during the drying process and subsequent storage (Vega-Galvez et al., 2011).

Sensory score evaluation
The sensory acceptability scores of dried TFFD are shown in Fig. 4. Although a slight decrease in the scores was noted as the drying temperature increased, statistical analysis revealed that there was no significant difference in the sensory scores (p > 0.05). All samples were rated > 7 (like moderately), higher than the cut-off score of 5. Therefore, dried TFFD powder was considered acceptable.
Overall preference rankings of dried TFFD are illustrated in Fig. 5. There were no significant differences in the scores for TFFD dried at 40 and 60 o C (p > 0.05). The results show that the IJFS April 2020 Volume 9 pages 251-263  IJFS April 2020 Volume 9 pages 251-263 Figure 5: Overall preference ranking of dried Thai fermented fish dip as affected by drying temperature. Identical letters above the bars indicate rankings that are not significantly different at a confidence level of 95%.
average rankings of TFFD exposed to 40 and 60 o C were significantly better (close to 1) than of those dried at 80 o C (p < 0.05). Undesirable dark colour of TFFD exposed to 80 o C , recorded by panellists, may cause a worse rank (close to 3). It was clear that treatment at 40 and 60 o C gave better rankings compared to 80 o C treatment. Based on these results, drying temperatures of 40 and 60 o C were considered to be more appropriate for drying TFFD than 80 o C .

CIE colour values
The CIE colour values of rehydrated TFFD are presented in Fig. 6. The results show that the L*, a*, and b* values of rehydrated TFFD decreased significantly as drying temperature increased (p < 0.05). Non-enzymatic browning may play an important role in the colour changes of rehydrated TFFD, as previously described for dried TFFD. The results revealed that drying at 80 o C significantly reduced the redness (indicated by the a* value) of the rehydrated TFFD samples. Redness plays an important role in the colour quality of TFFD, because it reflects the freshness and level of heat of the product (Posri, 2008). Drying at a temperature of 80 o C is therefore not appropriate for TFFD, due to the obvious changes in CIE colour values, particularly the a* value, that occur at this temperature. Several studies have investigated the colour values of rehydrated food products, most commonly products that are intended to be consumed after rehydration (Ulloa et al., 2015;Vega-Galvez et al., 2008;Vega-Galvez et al., 2009;Vega-Galvez et al., 2011). The effects of drying temperature on the colour values of rehydrated fish products have been investigated by Vega-Galvez et al. rehydrated jumbo squid decreased (Vega-Galvez et al., 2011). Non-enzymatic browning was suggested to be the cause of the colour changes observed in the product. The results showed a relation between the L* values of dried TFFD and those of rehydrated TFFD. Similar decreasing trends in L* values of dried and rehydrated TFFD were observed as drying temperature decreased (Fig. 2 a and Fig.  6a). However, a* and b* values did not show any relation between dried and rehydrated samples. No reduction of a* and b* values was detected in dried samples ( Fig. 2b and c), but reductions were found in rehydrated samples (Fig. 6 b and  c). Addition of water to rehydrate TFFD may help the product gain a more homogenous structure, thus able to display more colour variation. Therefore, measurement of colour values in rehydrated TFFD along with dried TFFD is recommended.

Sensory score evaluation
The sensory acceptability scores recorded for rehydrated TFFD are shown in Fig. 7. Odour, texture, flavour, and overall acceptability scores were not significantly different between the drying temperatures (p > 0.05). However, the sensory colour score of the samples dried at 80 o C was significantly lower than those dried at 40 and 60 o C (p < 0.05; Fig. 7). Examination of panellists' records revealed that TFFD dried at 80 o C exhibited a darker colour, which was deemed to be less acceptable compared with those dried at 40 or 60 o C . Excessive brown pigments caused by non-enzymatic browning reactions during hightemperature drying may have contributed to a darker colour. The sensory colour scores correlated well with the CIE colour values of rehydrated TFFD (Fig. 6), in which the colour values decreased significantly when TFFD was exposed IJFS April 2020 Volume 9 pages 251-263 Figure 7: Sensory acceptability scores of rehydrated Thai fermented fish dip as affected by drying temperature. Identical letters above the bars within each attribute indicate scores that are not significantly different at a confidence level of 95%. to 80 o C drying temperature. Drying temperatures of 40 and 60 o C were not significantly different in term of sensory scores (p > 0.05; Fig. 7).
Overall preference rankings of rehydrated TFFD samples are presented in Fig. 8. The samples exposed to 40 and 60 o C had rankings of 1.56 and 1.41, respectively. These scores represented better ranks (close to 1) compared to drying at 80 o C which produced a score of 2.62. Panellists' records confirmed that 80 o C caused an unpleasant dark colour in rehydrated TFFD. The rankings of rehydrated TFFD (Fig. 8) showed a similar trend to those of dried TFFD (Fig. 5). Therefore, it is clear that 40 and 60 o C are more appropriate temperatures for drying TFFD than 80 o C .

Conclusions
The microbial content, CIE colour values, and sensory acceptability scores did not differ significantly between TFFD dried at 40 o C and that dried at 60 o C . Drying at 60 o C resulted in increased non-enzymatic browning compared with drying at 40 o C , but this did not have an adverse effect on sensory colour scores. Drying at a temperature of 80 o C caused significant changes in the CIE colour values and sensorial colour score, which were particularly evident when rehydrated product was examined. Overall preference rankings of dried and rehydrated TFFD confirmed that 40 and 60 o C gained better ranks than 80 o C . Therefore, both 40 and 60 o C are appropriate temperatures for drying TFFD, but at 80 o C there was an unacceptable amount of undesirable changes in the product and so that temperature should not be used for drying cooked TFFD. Based on this study, optimization of drying factors such as time, temperature, and thickness of TFFD for commercial preparation using a mathematic model of data with multivariate analysis should be further investigated. Although dried TFFD is intended to be used as an instant powder, which should be rehydrated prior to consumption, it was found that the powder needed a rehydrating period of 30 min, due to its poor water absorption. Rehydrating for less than 30 min caused solid particles to form at the bottom of the container and separate from the liquid portion. To solve this problem, the addition of food binding or thickening agents to dried TFFD powder should be further explored.


extfieldsofstudy: Medicine Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Preliminary evidences of the presence of extracellular DNA single stranded forms in soil

The relevance of extracellular DNA (eDNA) in the soil ecosystem is becoming more and more evident to the scientific community by the progressive discovery of functions accompanying to natural gene transformation. However, despite the increased number of published articles dedicated to eDNA in soil, so far only few are focused on its single stranded form (eDNAss). The present paper is the first to investigate the quantitative relevance of eDNAss in the total soil eDNA pool, discriminating between its linear (eDNAssl) and circular (eDNAssc) forms and the respective weakly (wa) and tightly (ta) adsorbed fractions. The results showed the prevalence of eDNAss and its linear form in both the total soil eDNA pool and its wa and ta fractions. Both of the eDNAss fractions (linear and circular) were characterized by small fragments.

Introduction
Extracellular DNA (eDNA) can represent up to 40% of the total soil DNA pool [1,2]. Despite the interest of the scientific community on eDNA in soil is quite recent, the studies have highlighted several additional ecological functions to genetic exchange through natural transformation [3]. In fact, in relation to the soil bacterial community, eDNA can act as a source of nutrients [4,5,6], as constituent of biofilms [7,8,9] and bio-crusts [10], as agent to influence the porosity of soil aggregates [11,12], and as signaling-or chemoattractant molecule [13,14,7]. For plants, the soil eDNA can act as source of nutrients [15], as constitutive component of roots extracellular traps defense against biotic [16] and abiotic [17] hazards, as hormone simulant [15,18] and allelopathic [18] molecules.
All these functions and peculiar traits make eDNA to one of the most interesting molecules in soil, capable to affect both the composition and activity of microbial and plant communities. However, despite the increasing evidences on multiple roles and functions of eDNA in soil, the discrimination between its single and double stranded fractions has been up to now neglected although they are characterized by different reactivity and adsorption capacity [19,20,21]. To the best of our knowledge, there are only few studies dealing with eDNAss with focus on its adsorption onto clays [19,21] and other minerals [22] under laboratory a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 conditions. Thus, the present study aimed to assess the most mobile fraction of soil eDNA, based on the sequential extraction method by Ascher et al. [1], that is capable to specifically evaluate and characterize the eDNA fractions that are weakly (adsorbed) and strongly bound onto soil colloids, thus discriminating between the DNA forms with high and low mobility in soil.
The experimental approaches tested were an indirect and a direct method, to improve the discriminatory assessment of the single stranded (ss) and double stranded (ds) DNA forms. The proposed indirect approach is an optimization of the method by Gardner and Gunsch [21] that originally discriminated between the extracellular DNA fractions, ds and ssDNA, via subtraction of the amount of dsDNA (determined by Qubit using the DNA intercalating fluorochrome PicoGreen) from the total DNA (determined by Nanodrop UV-Vis spectrophotometer). Our modifications are based on the assumption that the soil total eDNA pool represents a mix of ds and ss forms within a wide range of different molecular sizes (molecular weight). Compared to the indirect approach, our proposed direct approach provides more precise information about eDNAss by discriminating between circular and linear forms, that can be further characterized in detail via downstream analysis.
The present paper is a first attempt to investigate in-depth on eDNAss in soil with the objectives to i) quantitatively assess the eDNAss as well as to discriminate between its linear (eDNAssl) and circular (eDNAssc) forms; ii) avoid bias in the quantification of eDNAss; and iii) define the adsorption strength of the eDNAss linear and circular fractions.

Material and methods
The sampling site, named Cavalla, is located in Vallombrosa forest at Tuscany Appennine 50 km east of Florence, Italy with an extension of 13 km 2 at an altitude between 600-1300 m above sea level. Mean annual temperature and precipitation ranging from 8-12 ºC and 1000-25000 mm, respectively. The soil is classified as a fragic distrudept soil (Table 1) with Silver fir (Abies alba Mill.) and European beech (Fagus sylvatica L.) as dominant trees [23]. Samples were collected from public land and permits were not required as local people could have access to this site and could collect different forest products except forest animals. Further, soil sampling is permitted for research in which protected or threatened species or locations are not involved, such as in our case. Moreover, we confirm that no protected or threatened species or locations were involved in the study. The sampling area, Cavalla, cover an area of 1200 m 2 and is characterised by high soil, vegetable cover, slope and exposure homogeneity. The soil was sampled in May 2018 to a depth of 10 cm (includes horizon A1 and part of A2 horizon), air dried, sieved at 2 mm and stored at -20˚C prior to further analyses. Soil samples (one kg) were collected randomly from three different sampling points. The extraction of the weakly (wa) and tightly (ta) eDNA fractions from the three field replicates was done by sequential elution with deionized distilled water (ddH 2 O) and Na 2 HPO 4 (NaP) 0.12M, respectively [24]. With the intent to increase the yields of extractable eDNA from soil, the number and duration of washing cycles were scheduled on the base of our experience on Vallombrosa soil from previous studies. In detail, the duration of the elution washing cycles for the different eDNA fractions was extended from 30 min to 1 h (ddH 2 O) for the weekly bound (wa) fraction, and from 30 min to 2 h (NaPI) and 4 h for the first one and for each of the subsequent three washings (NaPII, NaPIII, NaPIV) for the tightly bound (ta) fractions. The number of elution washings was increased only for the extraction of eDNAta from one to three washings (NaPII, NaPIII, NaPIV). The total number of samples was 15 including replications (3 -eDNAwa and 12 -eDNAta). All of the eDNA fractions were finally purified as described by Ascher et al. [1] (Fig 1A). The detection of the eDNA ds and ss forms in the wa and ta fractions was performed by the indirect approach, whereas their additional discrimination into the linear and circular forms was obtained by the direct approach as illustrated in Fig 1B.

Indirect approach
The amount of soil eDNAds was quantified by Qubit1 2.0 fluorimeter in both wa and ta fractions. The quantification of eDNAss was performed by UV-Vis spectrophotometer (PicoDrop 260 nm), modifying the method of Gardner and Gunsch [21] by assuming that the total soil eDNA pool is a mix of differently sized ds and ss forms. Thus, its spectrophotometric quantification is biased by the utilized conversion factor (CF); CF 50 for DNAds and CF 33 for DNAss. To overcome this bias, the total amount of eDNA was thermally denatured into its single stranded form by PCR treatment at 94˚C for 20 minutes [25] and then spectrophotometrically determined by utilizing the DNAss specific CF 33 (according to the manufacturer's manual). To avoid the re-association process, the total eDNAss samples were stored on ice and frequently checked by Qubit1 2.0 fluorimeter and, if detected, converted in ss forms by the equation: The amount of eDNAss was then calculated by subtracting the amount of eDNAds (Qubit) from the total eDNA denatured into the ss form. The molecular weights of the eDNA wa and ta fractions were determined by agarose gel electrophoresis (1x Tris Acetate-EDTA buffer; 1:10,000 EtBr; 0.8% w/v; 100 V 60 min) in comparison to a DNA Mass Ladder Mix (Fermentas, 75 bp-20 Kb), without discriminating between its ss and ds forms (S1A Fig).

Direct approach
We tested also a direct approach to discriminate between the eDNAss linear (eDNAssl) and circular (eDNAssc) forms by Ca 2+ -Hydroxyapatite (HAP) chromatography [26] through elution of purified eDNA wa and ta samples (Fig 1B). The approach is based on the different interaction strengths of the eDNA molecules with Ca 2+ ions on the surface of HAP in relation to the number of involved phosphate groups that depends on its molecular size and conformation. Thus, the sequential washing of the Ca 2+ -HAP column with NaP buffer 0,12M and 0,18M is capable to elute the eDNAss circular and linear forms, respectively. The obtained eDNAss wa and ta circular (eDNAsswac, DNAsstac) and linear (eDNAsswal, eDNAsstal) forms were then quantified at 260 nm by PicoDrop spectrophotometer by utilizing the DNAss specific CF 33.

Statistical analysis
Two-way analysis of variance (ANOVA, extraction time x DNA form) was applied to check for any significant effect of extraction time, DNA ds and ss forms and their interaction on the variability of the eDNA yield. Further, a multiple pairwise comparison of means was done by Tukey's HSD (honestly significant difference) test at P <0.05 level of significance, to assess individual effects of each factor. Statistical data processing was done using Past 3.06 [27].

Results
The indirect approach is suggested to be capable of i) determining the soil eDNAds and eDNAss pool and ii) discriminating their wa (ddH 2 O) and ta (NaPI, NaPII, NaPIII, NaPIV) fractions (Fig 2). Furthermore, the total eDNAss desorption quantification by the original indirect approach [21] evidenced a significant overestimation of total eDNAss amount [21]. The observed overestimation is most probably due to the utilization of the CF 50 in the UV-Vis quantification that is only accurate for the DNAds ( Table 2) (see Materials and Methods).
The results of two factorial ANOVA analysis of the modified indirect approach have confirmed that the extraction steps (time) and DNA conformation significantly affected the yields of eDNA, both in its single stranded and double stranded form (Fig 2). Furthermore, the oneway ANOVA (pair wise comparison) results showed that the eDNAss concentration was significantly higher compared to eDNAds for both wa (ddH 2 O) and ta (NaPI, NaPII, NaPIII, NaPIV) fractions (Fig 2). The proposed direct approach has successfully detected the presence of eDNAss, linear and circular forms (Fig 3), highlighting the prevalence of eDNAssl in the eDNAsswa (ddH 2 O) and eDNAssta (NaPI, NaPII, NaPIII, NaPIV) fractions. The relevance of the linear form in the eDNAssta fraction is attributable to the NaPI washing with high extraction yield, whereas the subsequent three washings (NaPII, NaPIII, NaPIV) have shown the progressive increase in the quantitative relevance of the circular form (Fig 3). Regarding the detection of total eDNAsswa, it has to be pointed out that the significant lower detection of eDNAsswa was obtained by the direct approach compared to the indirect one. This discrepancy casts also doubt on the data obtained with respect to the linear and circular fractions of eDNAsswa.
The one-way ANOVA analysis of the results of the direct approach revealed significant differences only as a function of the desorbing agent (Fig 3). Furthermore, the qualitative characterization of the eDNA ds and ss forms by agarose gel electrophoresis evidenced a higher fragment size (molecular weight) for eDNAds with respect to eDNAss (S1A and S1B Fig). The agarose gel of eDNAss revealed its extremely small molecular size, as reflected by the presence of a smear in correspondence to the minimum fragment size of the utilized DNA Mass Ladder (S1B Fig).

Discussion
The results of our proposed comparative approach, although being preliminary, revealed quantitatively relevant amounts of eDNAss in the soil eDNA pool (Fig 2), thus, providing the very first evidence of an important ecological aspect related to the eDNA degradation in soil. In fact, the experimental evidences on the dominance of eDNAss in the total soil eDNA pool combined with its extremely small molecular size (S1B Fig) and the absence of the eDNAds forms in the eDNAwa fraction (Fig 2) indicated that eDNAss results from the final step of the degradation process of eDNA in soil. Furthermore, based on the detected prevalence of DNAss in the eDNAwa fraction, DNAss can be clearly defined as the dominant form of the mobile soil eDNA pool. These results were confirmed by both the direct and indirect approach, thus, further supporting our findings.
In addition, both the properties of the eDNAss molecules and those of the studied soil (Table 1) further provide a plausible explanation of the obtained results [28]. First, the detected dominance of Mg 2+ and Ca 2+ cations in the soil solution (Table 1) have limited the interaction of the phosphate groups (strong) of eDNAds and eDNAss with the -O(H), -Si, -Al, -Fe sites of the soil minerals [29,19], and stimulated their electrostatic interactions (weak) [30,31]. Furthermore, the small molecular size of eDNAss (S1B Fig) has favored its adsorption in high amounts but also limited the binding sites per molecule, thus, facilitating its high desorption as evidenced by the high amounts of both wa and ta fractions (Fig 2) [32]. Also the prevalent hydrophilic characteristics of the acid mull humus form present in the sampled soil [23] might have reduced the strength of their bonds with the adsorbed eDNAss by limiting the contribution of its hydrophobic N bases and thus, facilitating its desorption [33].  Table 2. Yields of single stranded DNA (μg/g dry soil) extracted by ddH 2 O and alkaline NaP buffer by sequential washings and measured by comparing the method by Gardner and Gunsch (2017) Fig 1] and our proposed methods. The statistically significant differences between the different extraction methods are shown in different letters. The high fragmentation observed for eDNAss was probably due to its biotic degradation as supported by the high microbial activity [34] and phosphoesterases activitiy [23] in the soil. Despite a pending in-depth discussion of our findings due to the lack of related research, an additional explanation of the degradative processes of eDNA might also come from the activity of earthworms burrowing, reported for the studied soil [23], through their gut's microbial community. It is also relevant to underline that the small molecular size of the adsorbed eDNAss fragments has drastically limited the degradative efficiency of microbes and extracellular enzymes [35,36], thus promoting their persistence and a potential accumulation in soil over time [37].
Regarding the eDNAssl and eDNAssc ta fractions, their similar desorption values (Fig 3) contradict, which might be due to the soil pH. In fact, the soil sub-acid pH (5.0) value, correspondent to the isoelectric point of DNAds [38], should have induced a positive charge on the N-base of the eDNAss molecule by protonation, thus differentiating the molecular reactivity of its linear and circular forms. Furthermore, the detected prevalence of eDNAssl in both the eDNAsswa and eDNAssta fractions (Fig 3) is of ecological relevance, considering the dominance of the circular form in nature [39]. Moreover, the higher yield of extracted total eDNAss by the direct approach in the last two NaP washing cycles (III-IV) suggests a higher protection rate of the eDNAss fractions.
Finally, part of the desorbed eDNAssta potentially represents detached fragments of partially degraded large double stranded molecules, previously strongly adsorbed onto soil components [35,36,40].
Importantly, the proposed direct and indirect approach highlighted two peculiarities: • The overestimation of the amounts of desorbed total eDNAss by the original indirect approach [21] with respect to our proposed modified version, that is ascribable to the selected CF for the DNA quantification by UV-Vis spectrometry (see Materials and Methods); thus, our modified method provides an optimization of the method by Gardner and Gunsch (2017) [21], yielding reliable quantitative results; • The lower total amount of eDNAsswa desorbed from soil detected by the direct approach with respect to the indirect approach can be ascribed to the elution of the smaller DNAss fragments by Ca-HAP chromatography and the high reactivity of the nucleotides at their molecular ends (NTE). In fact, the two free hydroxyl groups of the NETs' phosphate significantly increase the bonding strength on Ca-HAP for small DNA molecules.
With respect to our research hypotheses, the present research provided evidences on the quantitatively relevant presence of single stranded forms in the soil eDNA pool, discriminating also between its circular and linear fractions. This approach overcomes methodological restrictions, thus improving the potential of an in-depth assessment of the eDNA degradation processes in soil [41], basic for its ecological relevance in terms of integrity [24], persistence [37] and mobility [42]. In the light of these results, it is relevant to consider that some of the published studies on eDNA in soil [21,24,34,40] have elaborated data and, consequently, interpreted findings potentially biased also by the presence of relevant amounts of eDNAss. Finally, although the eDNAsswa seems to be affected by a bias, the proposed (optimized) indirect and direct approaches are suggested to provide a powerful tool for an in-depth assessment of the extracellular soil DNA pool, capable to also discriminate between the circular and linear forms of the eDNAss molecules, contributing thus to a more correct interpretation of the genetic information deriving from soil DNA based downstream analysis.


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Effect of integrated nutrient management (INM) on nutrient uptake, yield and quality of potato (Solanum tuberosum L.)

A field experiment was conducted at Instructional farm, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, West Bengal, India during rabi season of 2017-18 to evaluate the performance of potato under different integrated nutrient management (INM) options. The experiment was placed in randomised block design (RBD) with seven nutrient management options (T1-100% RDF, T2-100% RDF + yeast vinasse @ 2 t/ha, T3-75% RDF + yeast vinasse @ 2 t/ha, T4-50% RDF + yeast vinasse @ 2 t/ha, T5-100% RDF + vermicompost @ 2 t/ha, T6-75% RDF + vermicompost @ 2 t/ha, T7-50% RDF + vermicompost@ 2 t/ha) and replicated thrice. Results from the experiment expressed that application of 100% RDF along with supplementation of either vermicompost or yeast vinasse @ 2 t/ha ensured greater nutrient uptake such as N (144.6 kg/ha or 138.9 kg/ha respectively), P (18.3 kg/ha or 16.8 kg/ha respectively) and K (130.2 kg/ha or 134.8 kg/ha respectively), tuber yield (27.29 t/ha or 26.97 t/ha respectively), dry matter yield (6.75 t/ha or 6.66 t/ha respectively) and quality such as carbohydrate (74.7% or 74.9% respectively) and protein (1.23% or 1.22% respectively) of potato over sole application of 100% RDF through chemical sources. Thus, both these integrated nutrient management options can be recommended in place of sole chemical fertilizers to obtain improved potato performance.

Introduction
Potato (Solanum tuberosum L.) is the most important temperate food crop of the world. High nutrition (carbohydrate, protein, dietary fibre, vitamins, minerals, amino acids etc.), easy digestibility, bulk quantity of production etc. have made potato the most popular vegetable food crop over the years around the world. Potato has achieved the status of 'poor man's friend' due its constant assurance of feeding and supplying low cost energy and high nutrition to people irrespective of economic categories. Globally, India ranks second in terms of potato production after China, where potato is cultivated in the year 2018-19 on an average over 2.18 million ha with annual production of 52.58 million tonnes and productivity of 24.07 tonnes/ha (Horticulture Statistics Division, 2019) [14] . In India, potato cultivation is mostly concentrated in northern hills and plains, eastern hills, plateau region and southern hills. Among the states of India, Uttar Pradesh tops the chart in terms of potato area and production which is followed by West Bengal. Other major potato growing states are Bihar, Madhya Pradesh, Assam, Gujarat, Punjab, Jharkhand, Karnataka, Haryana, Chattisgarh etc. (Horticulture Statistics Division, 2019) [14] . Potato has different forms of use for table consumptions viz. vegetable ingredient in many cooked dishes, potato flour in making snacks, potato chips, slice or shredded potato, smashed potato, french fries etc. Besides, potato also has industrial value in production of dextrin, alcohol and starch (i.e. farina, used in laundries and textiles). In the present context of rapidly growing population and limited agricultural area with no scope of further area spreading, in order to avoid disparity between demand and supply, potato cultivation highly urges for more and more return. Proper quantity and timely supply of nutrients is the most important factor in realising high productivity of potato cultivation as potato is a heavy feeder and highly responsive to nutrient supply (Westermann, 2005;Pandit et al., 2018) [29,22] . So far, potato cultivation has mostly remained with the supply of nutrients (nitrogen, phosphorus, potassium etc.) through chemical fertilizers (Pandit et al., 2018) [22] . However, with the increment of the price of these chemical fertilizers as well as its issues of environmental hazards, potato cultivation is now required to be shifted to a promising alternative of inorganic nutrition, called integrated nutrient management (INM). INM involves supplies of nutrients from various sources (inorganic and organic) to achieve balanced nutrition covering benefits and curtailing limitations of both the sources. Specifically, it addresses the adverse effects of chemical fertilizers on soil health and crop qualities as well as incorporates the benefits of organic manures to improve soil fertility and crop performance (Chang et al., 1991;Ahmed et al., 1996;Brady, 1996;Chung et al., 2000;Keupper and Gegner, 2004) [7,6,8,18] . Previous studies (Raghav et al., 2009;Baishya et al., 2012, Pandit et al., 2018 [23,4,22] have already stated the importance of INM on quality and quantity of potato crop over sole application of chemical fertilizers. The present experiment has been framed for confirmation of previous works and also to investigate further the best INM option for potato cultivation.

Materials and Methods
The field experiment was carried out at Instructional farm, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, West Bengal, India during rabi (winter) season of 2017-18 comprising seven nutrient management options (Table 1), replicated thrice under randomised block design (RBD). The soil of the experimental site was sandy clay loam in texture with 61.24% sand, 14.20% silt and 24.56% clay. The soil had 1.51 Mg/m 3 bulk density, 1.2 cm/hour infiltration rate, 6.65 pH, 0.29 dS/m electrical conductivity. Initial soil organic carbon, available nitrogen, phosphorus and potassium were 5.8 g/kg, 159.2 kg/kg, 44.9 kg/kg and 101.8 kg/kg of soil respectively. Climatic condition during the experimental period has been shown in figure 1, 2 and 3. Potato variety 'Kufri Jyoti' was planted (approximately 40-50 g weight of each tuber) on flat bed on 19 th November, 2017 at 60cm x 25 cm spacing and harvested on 10 th March, 2018. Individual plot size was 5m x 5m. Earthing up was done twice at 30 and 60 days after planting (DAP). Late blight infestation was checked through application of Dithane M 45 as and when required. Agronomic and other plant protection measures were used as per standard recommendations.  Recommended dose of fertilizer (RDF) was applied through urea, S.S.P. and M.O.P. respectively for nitrogen (N), phosphorus (P2O5) and potassium (K2O). Entire quantities of P2O5 and K2O and half of N were applied at basal and rest half of N was applied at the time of first earthing up (30 DAP). Entire quantity of yeast vinasse and vermicompost were applied 15 and 7 days before planting respectively during land preparation. Yeast vinasse is a fine brown to dark brown powder obtained after evaporation and drying of semiliquid rejected part derived from reverse osmosis after anaerobic and aerobic microbial digestion of residual liquid part from bakery yeast fermentation (Fig 4). Chemical composition of yeast vinasse analysed is presented in table 2.  Observations from the experimental field included tuber yield and dry matter yield of potato. Nutrient uptake (nitrogen, phosphorus and potassium), carbohydrate and protein contents of potato were analysed in laboratory. Data collected from the experiment were statistically analysed through OP-STAT online portal using analysis of variance (ANOVA) method (Goulden, 1952 andCochran andCox, 1959) [12,9] and treatment means were compared at 5% level of significance using critical differences (CD) as stated by Gomez and Gomez (1984) [11] .

Results and Discussion Effect of INM on nutrient uptake of potato
Experimental results explored variability in nutrient uptake of potato under different nutrient management options (Table 3). Highest uptake of N (144.6 kg/ha) by potato was noticed when 100% RDF along with vermicompost @ 2 t/ha (T5) was applied which was closely followed by application of 100% RDF along with yeast vinasse @ 2 t/ha (T2) (138.9 kg/ha) and both remained statistically at par with each other. Application of 75% RDF along with vermicompost @ 2 t/ha (T6) (129.3 kg/ha) and application of 75% RDF along with yeast vinasse @ 2 t/ha (T3) (121.8 kg/ha) remained statistically at par with each other and with sole application 100% RDF (T1) (125.8 kg/ha) in terms of N (nitrogen) uptake by potato (Table 3 and  [13] and Yourtchi et al. (2013) [30] . Application of 100% RDF along with vermicompost @ 2 t/ha (T5) similarly recorded highest P (phosphorus) uptake by potato (18.3 kg/ha), followed by 100% RDF along with yeast vinasse @ 2 t/ha (T2) (16.8 kg/ha) and application of 75% RDF along with vermicompost @ 2 t/ha (T6) (15.9 kg/ha) and all the three INM options remained statistically similar to each other (Table 3 and Fig 5). Enhancement of P uptake of various crops by addition of vermicompost in INM was also reported by Sainz et al. (1998) [25] , Shivaputra et al. (2004) [27] , Mohanty et al. (2006) [21] , Zaller (2007) [31] and Yourtchi et al. (2013) [30] . Application of 75% RDF along with yeast vinasse @ 2 t/ha (T3) recorded statistically similar P uptake by potato (12.4 kg/ha) as recorded by sole application of 100% RDF (T1) (13.1 kg/ha). In case of K (potassium) uptake by potato, slightly different trend was observed, where application of 100% RDF along with yeast vinasse @ 2 t/ha (T2) recorded highest K uptake (134.8 kg/ha), followed by application of 100% RDF along with vermicompost @ 2 t/ha (T5) (130.2 kg/ha) and both remained statistically indifferent to each other following the previous trend again (Table 3 and Fig 5). No statistical difference was also noticed among application of 75% RDF along with yeast vinasse @ 2 t/ha (T3) (119.4 kg/ha), application of 75% RDF along with vermicompost @ 2 t/ha (T6) (116.6 kg/ha) and sole application of 100% RDF (T1) (122.3 kg/ha). Improvement of K uptake by potato by application of vermicompost along with chemical fertilizers has been confirmed by Yourtchi et al. (2013) [30] . Use of organic sources of nutrients such as vermicompost and yeast vinasse in INM options induced better root system of potato (Barman et al., 2018) [5] resulting greater uptake of nutrients than sole application of chemical fertilizers. Vermicompost is a rich source of nutrients and an excellent factor for improvement of soil bio-physical health which enhances root proliferation of crops and helps in higher availability and uptake of nutrients (Khan et al., 2017) [16] . Higher availability of nutrients for crops on application of vermicompost has been also reported earlier by Vasanthi and Kumaraswamy (1996) [28] . Enhanced soil microbial activity by application of vermicompost on improvement of uptake nutrients (Alam et al., 2007) [2] might be another reason for such results. Beside positive impacts of vermicompost in INM on nutrient uptake, results from the present study (Table 3) stated equal effectiveness of yeast vinasse as organic sources in INM. Specially, best uptake of K by potato through application of yeast vinasse along with chemical fertilizers might be due to high potassium content of yeast vinasse as mentioned in table 2. Increased nutrient uptake by application of yeast vinasse over sole application of chemical fertilizers was earlier reported by Komdorfer and Anderson (1993) in sugarcane, maize, wheat and pigeon pea. High concentration of K and moderate concentrations of P and N in yeast vinasse exerting positive influence on soil properties and nutrient uptake was also stated by Zhou et al. (2009) [32] , Rajagopal et al. (2014) [24] and Mahmoud et al. (2019) [20] .

Effect of INM on yield and quality of potato
Experimental results from table 4 further revealed that various integrated nutrient management options exerted significant effect on potato tuber and dry matter yields. Application of 100% RDF along with vermicompost @ 2 t/ha (T5) produced maximum potato tuber yield (27.29 t/ha) which was closely followed by application of 100% RDF along with yeast vinasse @ 2 t/ha (T2) (26.97 t/ha) and both those INM options (T5 and T2) remained statistically indifferent to each other. Sole application of chemical fertilizers (100% RDF i.e. T1) produced potato tuber yield of 25.61 t/ha and both application of 75% RDF along with vermicompost @ 2 t/ha (T6) (24.96 t/ha) and application of 75% RDF along with yeast vinasse @ 2 t/ha (T3) (24.68 t/ha) were statistically at par with 100% RDF (T1) and among each other (Table 4 and Fig 6), indicating the positive effects of vermicompost and yeast vinasse as organic sources under INM on potato tuber yield over sole application of chemical fertilizers. Similarly, maximum dry matter yield (6.75 t/ha) was achieved by application of 100% RDF along with vermicompost @ 2 t/ha (T5) which was statistically at par with application of 100% RDF along with yeast vinasse @ 2t/ha (T2) (6.66 t/ha). Applications of 75% RDF along with vermicompost @ 2 t/ha (T6) (5.63 t/ha) and 75% RDF along with yeast vinasse @ 2 t/ha (T3) (5.46 t/ha) remained statistically similar with application of chemical fertilizers alone (5.87 t/ha) and among each other in terms of potato dry matter yield (Table 4 and Fig  6). Application of vermicompost or yeast vinasse as a part of INM enhancing the tuber and dry matter yield of potato might be due to improved soil health (as they are rich various macro nutrients, micro nutrients, growth regulators etc.) resulting in better availability, uptakes of nutrients and its translocation inside the plant. Improved vegetative growth in response to application of vermicompost or yeast vinasse along with chemical fertilizers facilitating higher photosynthesis and translocation of assimilates in to reproductive part of the plant, might be another reason for recording such a higher yield of potato under INM as compared to sole application of chemical fertilization. analogous type of results confirming yield enhancement of potato through use of vermicompost was reported by Ghosh et al. (1999) [10] , Alam et al. (2007) [2] , Ansari (2008) [3] , Yourtchi et al. (2013) [30] , Pandit et al. (2018) [22] and many more. Similarly, yield enhancement through yeast vinasse corroborated the findings of Mahmoud et al. (2019) [20] in spinach and barley. Quality attributes of potato as shown in  Table 4). Application of 100% RDF along with vermicompost @ 2 t/ha (T5) helped potato tuber to contain highest protein content (1.23%) which was closely followed by application of 100% RDF along with yeast vinasse @ 2 t/ha (T2) (1.22%) and sole application of 100% RDF (T1) (1.21%) and all those three remained statistically same in terms of providing protein content of potato tuber. Nitrogen is the precursor of protein and thus, higher protein content under application of vermicompost or yeast vinasse along with chemical fertilizers might be due the better uptake of nitrogen as compared to application of sole chemical fertilizers. Joshi et al. (2014) reported improved quality of crops in terms of carbohydrate, protein and many others through application of vermicompost.

Conclusion
Finally, the study confirms the wise choice of incorporating integrated nutrient management (INM) in place of sole use of chemical fertilizers for potato cultivation. Supplemental application of either vermicompost or yeast vinasse @ 2 t/ha along with 100% RDF can be recommended to potato growers of new alluvial zone of West Bengal, India for achieving improved nutrient uptake, yield and quality enhancement of potato.


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A Water Footprint Review of Italian Wine: Drivers, Barriers, and Practices for Sustainable Stewardship

Wine constitutes the dominant Italian agricultural product with respect to both production quantity and economic value. Italy is the top wine producer worldwide in terms of volume and the second one below France in terms of national income. As the Italian agricultural production accounts for 85% of the national freshwater appropriation, the country’s agricultural sector strains freshwater resources, especially in the central and southern regions, which constitute important winemaking areas in terms of quantity and quality. To this end, we first perform a review of the existing research efforts on wine water footprint assessment to investigate the water dynamics of wine production in Italy compared to the rest of the world. The results indicate a prevalence of studies on the water footprint of Italian wine, emphasising the need for deeper research on the sector’s water efficiency. Then, we aim at exploring the major drivers, barriers, and good practises for systematic water stewardship in the Italian winemaking industry, considering the product and territorial characteristics. This research is anticipated to contribute towards providing insights for practitioners in the Italian wine sector to develop water-friendly corporate schemes for enhancing the added value of their products.

Introduction
The winemaking industry plays a critical role in the economy of the primary sector of the Southern European and Mediterranean regions [1]. Thus, there is an increased pressure towards minimising the environmental impacts of wine production [2] to improve the sustainability of the sector in terms of climate change and natural resources [3]. More specifically, consumers' environmental expectations further motivate winemakers to adopt green technological interventions for efficient water use during irrigation or wastewater reuse [4]. In addition, given that a considerable number of consumers, especially young ones [5], express willingness to pay a premium for a sustainable wine label [6], the production of water-friendly wine could be an ambitious strategy for increasing profitability through quality improvement [7].
The winemaking efficiency in terms of freshwater use can be expressed through the water footprint (WF) concept, which refers to the total volume of freshwater consumed and polluted at national,

Water Footprint of Wine: Literature Background
In this section, we perform a review of the global wine WF literature to identify the position of the Italian case studies in this research field. Within the extant literature, we have identified 20 articles in total that include the terms "water footprint" (or "water management" or "freshwater resources") and "wine" (or "winemaking") in the Scopus and Web of Science databases. Then, we present the major descriptive statistics of the review findings along with a brief discussion. Finally, the taxonomy of the Italian literature provides a detailed analysis of the papers under study in a structured manner.
The WF of a product is defined as the total volume of freshwater used directly or indirectly across its end-to-end supply chain [20]. Figure 1 illustrates the different stages of a wine supply chain, highlighting the viticulture and the vinification phases as the prevalent WF contributors. To quantify the WF of wine, several methodological approaches exist; the WF assessment manual that focusses on the volumetric measurement of water consumption and pollution [8] and the life cycle assessment (LCA) techniques (e.g., the ISO 14046 [21]), further including the impact of water scarcity (which varies spatially and temporally) on the WF indicator [14], constitute the most common ones. Although a comparison among the different WF assessment approaches is considered out of the scope of this research, a more detailed analysis of their unique charasterics is provided by Chenoweth et al. [22]. on the volumetric measurement of water consumption and pollution [8] and the life cycle assessment (LCA) techniques (e.g., the ISO 14046 [21]), further including the impact of water scarcity (which varies spatially and temporally) on the WF indicator [14], constitute the most common ones. Although a comparison among the different WF assessment approaches is considered out of the scope of this research, a more detailed analysis of their unique charasterics is provided by Chenoweth et al. [22].

Research Efforts Worldwide
Several case studies on wine WF assessment have been identified in the literature, indicating the increasing academic interest in evaluating the use of freshwater resources in the winemaking sector worldwide. Outside Europe, efforts have been made to quantify the WF of New Zealand's wines. First, a combination of an LCA-based approach and a hydrological water-balance technique was implemented to quantify the volume of water consumed and polluted during wine production in two New Zealand regions [23]. In a later study, the authors extended the WF research of New Zealand's wine by comparing the results obtained using diverse methodologies, further including the traditional WF assessment method, to investigate freshwater utilisation from different perspectives [24]. In North America, a US study assessed the greenhouse gas emissions, the energy use, and the freshwater use across the life cycle of wine produced in California, beginning from the cultivation of grapes up to their delivery at the winery gate, to provide a holistic evaluation of the wine's environmental impact [25]. Moreover, a preliminary research effort was made for assessing the grey WF associated with wastewater produced during the winemaking process in a Canadian winery and co-treated by municipal wastewater treatment plants [26]. In Latin America, a recent study quantified the consumptive blue and green WF of several varieties of grapes for wine production in five Argentinian regions, using different irrigation systems [27].
Within Europe, although the majority of research on wine WF assessment has been documented across southern countries, two publications refer to Northern Europe. In Romania, the WF of a bottled wine produced in a medium-sized winemaking plant was quantified in the stages of viticulture and vinification, further evaluating the socio-economic potential of winemaking and the related waterrelated schemes within the country [28]. In Hungary, a recent study developed a framework for WF assessment during grapes' cultivation and processing to optimise the consumption of both rainwater and freshwater consumed [29]. Moving to the south, several researchers evaluated the WF of Iberian wines. More specifically, an evaluation of both direct and indirect freshwater use was performed for a Portuguese white wine variety during the viticulture and the winemaking stages, further analysing the related environmental impacts of water use [30]. In addition, a more comprehensive analysis included the LCA of the carbon, water, and energy footprints, as well as the material intensity and solid and water wastes, of a bottle wine during the phases of grapes' cultivation, wine production, bottling and packaging in Portugal [31]. More recently, the water-related ISO 14046 was used to analyse the WF profile of a Spanish grape variety for vinification and to address the impacts due to water scarcity and degradation from a life cycle perspective [32]. Moreover, an indicator of water

Research Efforts Worldwide
Several case studies on wine WF assessment have been identified in the literature, indicating the increasing academic interest in evaluating the use of freshwater resources in the winemaking sector worldwide. Outside Europe, efforts have been made to quantify the WF of New Zealand's wines. First, a combination of an LCA-based approach and a hydrological water-balance technique was implemented to quantify the volume of water consumed and polluted during wine production in two New Zealand regions [23]. In a later study, the authors extended the WF research of New Zealand's wine by comparing the results obtained using diverse methodologies, further including the traditional WF assessment method, to investigate freshwater utilisation from different perspectives [24]. In North America, a US study assessed the greenhouse gas emissions, the energy use, and the freshwater use across the life cycle of wine produced in California, beginning from the cultivation of grapes up to their delivery at the winery gate, to provide a holistic evaluation of the wine's environmental impact [25]. Moreover, a preliminary research effort was made for assessing the grey WF associated with wastewater produced during the winemaking process in a Canadian winery and co-treated by municipal wastewater treatment plants [26]. In Latin America, a recent study quantified the consumptive blue and green WF of several varieties of grapes for wine production in five Argentinian regions, using different irrigation systems [27].
Within Europe, although the majority of research on wine WF assessment has been documented across southern countries, two publications refer to Northern Europe. In Romania, the WF of a bottled wine produced in a medium-sized winemaking plant was quantified in the stages of viticulture and vinification, further evaluating the socio-economic potential of winemaking and the related water-related schemes within the country [28]. In Hungary, a recent study developed a framework for WF assessment during grapes' cultivation and processing to optimise the consumption of both rainwater and freshwater consumed [29]. Moving to the south, several researchers evaluated the WF of Iberian wines. More specifically, an evaluation of both direct and indirect freshwater use was performed for a Portuguese white wine variety during the viticulture and the winemaking stages, further analysing the related environmental impacts of water use [30]. In addition, a more comprehensive analysis included the LCA of the carbon, water, and energy footprints, as well as the material intensity and solid and water wastes, of a bottle wine during the phases of grapes' cultivation, wine production, bottling and packaging in Portugal [31]. More recently, the water-related ISO 14046 was used to analyse the WF profile of a Spanish grape variety for vinification and to address the impacts due to water scarcity and degradation from a life cycle perspective [32]. Moreover, an indicator of water depletion, mainly due to irrigation during the viticulture stage, was evaluated in the context of a complete LCA of an aged red wine produced in Catalonia, Spain [33].

Italian Case Studies
An increased number of case studies on wine WF has been mapped across the Italian territory. Lamastra et al. [34] proposed a new WF quantification approach (Valutazione Impatto Viticoltura sull'Ambiente -V.I.V.A. tool) to improve the WF assessment manual technique [8], emphasising in detail the calculation of the grey WF of six different wine varieties of a Sicilian winery. Bonamente et al. [35] quantified the direct green, blue, and grey WFs of a typical red wine produced from a blend of grape varieties by a medium-sized winery in Umbria, based on the V.I.V.A. tool [34] and following the ISO 14046 principles [21]. In a later study, the authors performed a combined carbon and WF assessment in the life cycle of the Italian red wine using the same dataset and methodological approach [36]. In the same vein, Rinaldi et al. [37] performed a cradle-to-grave analysis for juxtaposing the carbon and WF indicators of a red and a white wine of an Umbrian producer, using the same system boundaries, functional unit, and input data, based on the relevant ISO guidelines [21]. In Umbria again, Bartocci et al. [38] calculated the carbon, ecological, and WF, along with several LCA-related environmental impacts, for two different varieties of grapes during cultivation, wine production, vinegar ageing, and bottling, following the ISO approach [21]. Recently, Borsato et al. [39] compared the WF outcomes of a volumetric (i.e., the V.I.V.A. tool [34]) and two LCA-based approaches (i.e., Available WAter REmaing -AWARE [40] and Water Scarcity Index [14]) during the production of a white wine variety in Northeast Italy to improve water management. Miglietta et al. [41] investigated the WF of two types of wines indicated with designation of origin whose vineyards are situated in Northern (Piedmont) and Southern (Sicily) Italy to compare the geographical impact of grapes' cultivation on freshwater consumption and pollution. More recently, Miglietta et al. [42] quantified the water efficiency (i.e., the ratio of total wine WF to total wine production) and the economic water productivity (i.e., the ratio of wine price to wine WF) of all Italian wines indicated with appellation of origin. In addition, Miglietta and Morrone [43] studied the virtual water flows and economic water productivity of wine trade between Italy and Balkan countries. The latter three research efforts were conducted based on the WF assessment manual estimates [8]. Figure 2 illustrates the distribution of the case studies on wine WF assessment by country. Notably, Italy dominates the wine WF research (i.e., nine out of 20 studies), confirming (i) the leading role of the Italian winemaking industry both within the country [16] and abroad [1], and (ii) the increased water scarcity concerns in the region [14], followed by New Zealand, Portugal, and Spain (i.e., two out of 20 studies each). Notably, there is an apparent absence of WF studies for French wines, despite the major economic impact of the country's winemaking sector worldwide [17], which is potentially due to lower water scarcity indices compared to Italy [14]. In addition, Figure 3 depicts the distribution of the studies by year of publication. In fact, the research on wine WF assessment has received a rather constant interest during the last 6.5 years (i.e., the first paper was identified in 2013), exhibiting an average of 2.9 studies per year worldwide and 1.2 studies annually in Italy.
Finally, Table 1 provides a taxonomy of the literature in the field of wine WF assessment in Italy. More specifically, the type of the study, the period in which the data were collected, the location of the study, the wine variety examined, the winemaking phase considered, the WF assessment method used, as well as the type and volume of the WF quantified, are documented to provide detailed information in the field of wine WF assessment in a supplementary manner. Notably, a comparative analysis of the studies could be challenging due to significant differences concerning the (i) methodological approaches implemented, (ii) databases utilised, (iii) assumptions articulated, and (iv) temporal or spatial characteristics considered. However, as the diverse WF assessment approaches exhibited vary with respect to the manner that they quantify water use [22], it is not infeasible to compare WF results derived from different methods, even though the calculations are performed using the same dataset [44]. In fact, Bonamente et al. [36] confirm this statement through providing different results compared to Bonamente et al. [35], although they use the same input data. Even by applying the same methodology, the different wine variety types [34], as well as the diverse climatic and geographical conditions of the Italian regions from North to South [41], influence the wine WF assessment findings. Nevertheless, in general, green water emerges as the typical source of water for wine production, even in semi-arid environments such as Central Italy.
Water 2020, 12, 369 5 of 16 climatic and geographical conditions of the Italian regions from North to South [41], influence the wine WF assessment findings. Nevertheless, in general, green water emerges as the typical source of water for wine production, even in semi-arid environments such as Central Italy.   climatic and geographical conditions of the Italian regions from North to South [41], influence the wine WF assessment findings. Nevertheless, in general, green water emerges as the typical source of water for wine production, even in semi-arid environments such as Central Italy.

Water Stewardship in the Italian Wine Industry: Drivers and Barriers
The food and beverage industry's contribution to global freshwater withdrawal is well documented in the extant scientific literature, while freshwater resources are dwindling at an alarming rate [18]. As satisfying the supply of food products requires a sufficient and consistent availability of freshwater resources, collaborative and harmonised interventions across supply chains are essential in order to ensure sustainable and efficient water use [45]. Considering that the Italian wines exhibit substantial WFs especially in water-stressed areas, we discuss the main drivers and barriers of water management in the wine sector based on the existing Italian research efforts. Table 2 summarises the identified drivers and barriers along with a taxonomy of the citing articles.

Drivers
From an environmental point of view, the elevated global water stress levels foster the adoption and application of water management policies and practices in the wine industry [19]. More specifically, wine quality is correlated to grapes' quality, thus motivating the wine industry to investigate irrigation practises [34]. In particular, a wine's identify is defined by grape maturation, aroma, and coloration [46], which are attributes that are amenable to the vine's geographical location and climate conditions that determine the chemical composition and sensory characteristics of grapes [47].
As freshwater appropriation is characterised by space-temporal dimensions, the adoption of advocated practises in the winemaking industry is eminent to mitigate water stress phenomena at both local and global freshwater bodies [33,35,43]. This need is even more pronounced in regions where the nexus of water scarcity, vineyards, production seasonality, and climatic conditions' severity aggravate water consumption. In this regard, targeted institutional and state-specific policies and directives (e.g., European Program of Sustainability, New Zealand Winegrowing Program, Italian initiative on Valutazione dell' Impatto in Vitivinicoltura sull' Ambiente) motivate circular economy and water-use minimisation in wine [39], while they further support investments in related infrastructure to protect water quality and quantity [41]. Furthermore, the plethora of research studies and corporate reports pertinent to water consumption across the wine supply chains operations allows benchmarking [36,37], thus further enabling the continuous improvement and proliferation of water management policies and practises among industry stakeholders.
From a socio-economic angle, water security has a prominent role in the United Nations Sustainable Development Goals, as it is recognised as a key determinant to the delivery of a viable ecosystem to future generations and a critical factor towards ensuring continuity to food manufacturing operations [48]. In particular, securing freshwater resources' sustainability allows winegrowers to improve economic water productivity of their wine supply chains (i.e., monetary value attained per cubic meter of water used), hence ensuring high-quality winery products at a low level of water use [42]. In addition, the implementation of water management policies and practises (e.g., water reuse) allows grape growers and wine manufacturers to reduce the resources' scarcity burden linked with their production [35,36]. To a greater extent, reducing the utilisation of freshwater as a production material results in operational cost savings for companies [43].
From a market perspective, consumers' awareness and attentiveness over the sustainability impact of wine products drive demand growth in the sector, especially in case water-related eco-certification is provided [39]. Indicatively, Rugani et al. [49] critically analysed LCA and carbon footprint-based studies on the wine-making industry and stressed that carbon footprint labelling in wines provides a market differentiation element that could influence consumers' purchasing behaviour. Moreover, wine produced with sustainable techniques has a greater export potential [42], while customers have a willingness to pay a premium price for environmentally friendly wine products [38]. Specifically, given that around 80% of wine sales occur in-store, clear communication of sustainably produced wine is deemed critical for increasing sales [50]. Therefore, as sustainable marketing has nowadays a dominant role in consumer purchasing behaviour and market sales, the communication of the water-related identity of wines could be an additional driver for approaching water-sensitive market segments. To some extent, consumers and policy-makers should also become aware of the virtual water flows, particularly blue water, embedded in international wine trade, considering that agri-food trade greatly influences water appropriation in a country [43]. To this end, national initiatives and businesses in the wine sector actively engage multispectral WF mitigation initiatives to reduce operating costs and communicate the water stewardship of their products to increase consumer value. In particular, better communication to consumers could be achieved via calculating a single-score indicator for labelling purposes [5,51].
In the case of the Italian wines, which are traded under the "controlled designation of origin" and "controlled and guaranteed designation of origin" labels, the adoption of water management policies can deliver a compelling marketing narrative linked to the territorial culture and history of each specific production wine site, promote the valorisation of local freshwater resources, and ultimately drive rural development [41]. Notably, on average, young wine consumers in Italy value water saving labelled wines and demonstrate a willingness to pay a premium price for such product offerings; determinats include wine consumption frequency, environmental-friendly attitude, label use, and label trust [5]. Therefore, as young consumers represent the most common market segment regarding wine consumption, policy-makers could act as a driving force for supporting the winemaking industry to adopt more environmental-friendly production methods (e.g., the Common Agricultural Policy of the EU [52]).

Barriers
Notwithstanding the pronounced need to apply water management policies and practises in the winemaking industry [46], dominant barriers hinder their adoption and maturity. The greatest peril in this process regards the poor alignment between water and agricultural policies [43], which is supported by the existence of different views among scientists regarding the system boundaries to apply water management policies (e.g., indirect WF from raw materials, transportation, end-of-life processes, etc.) [36,37,39]. Indicatively, Italian wines are associated with a lower WF exclusively due to the particular production specifications to guarantee designation of origin (i.e., irrigation and fertilisation are prohibited) [41]. To that effect, WF assessment methodologies generate different results even in the case that the same water management techniques are considered [35].
To a greater extent, established methodologies used for the ex ante evaluation of manufacturing operations' water impact (e.g., LCA) myopically leverage secondary data sources and neglect geographically related characteristics, such as diverse climatic conditions and applied production techniques [36,39]; thus, inconsistencies and discrepancies in the derived results are possible, but they have a detrimental effect on specific regions considering the localised supply of the embedded production inputs. At a more granular level, an evident gap in existing databases regarding indirect water consumption (e.g., green water) further raises evaluation challenges [36]. The lack of detailed data input further inhibits the contextualisation of the results in real-world operations [34], subsequently affecting the decision-making over the investments in related practises.
Furthermore, most water management-related studies focus on the academic merit of the applied methodological approaches in the pursuit of accuracy and precision of calculations. However, business stakeholders, who in principal operationalise water mitigation policies, cannot make inferences about the associated economic water productivities [42]. Moreover, the diverse alternative eco-labels for certifying the adoption of good practices for freshwater utilisation does not always provide businesses with an opportunity to differentiate from the competition [43].

Discussion
Food production and consumption are considered to have a rather detrimental impact on the environment [39,53]. Particularly, in the winemaking industry, sustainability is a key driver for competitiveness, market differentiation, and process innovation [4]. WF could become a meaningful indicator in sustainability initiatives for wines (as lower water consumption is also connected with a better wine quality and taste [41,42]); thus, the winemaking industry is exploring practises to improve the related environmental impact.
At a national level (e.g., Chile, Australia, New Zealand), frameworks to inform sustainability in the winemaking industry exist. Flores [3] reviewed the process-based winemaking frameworks in six countries and reported three categories where common water management practises are recognised: (i) soil management-protection of water resources from pollution, (ii) water management-registration of water use, selection of irrigation system, and control of water quality, and (iii) wastewater-monitoring of effluents and treatment of winery wastewater. Focussing on wastewater treatment, in countries such as France, Italy, and Spain, where the wine cellars are generally located close to urban areas, the use of advanced biological processes is crucial [54].
At a more granular level of operations and to cultivate grapes that result in high-quality wines, agricultural practices are required to enable the control of particular properties of grapes, such as the concentration of phenols, which determine the taste, color, and mouthfeel of wine. From a WF point of view, regulated deficit irrigation at the phenological stage is applied to increase the phenols' content [27]. Alongside the different quality of the cultivated grapes for winemaking, irrigation systems and practises are also dictated by the edaphoclimatic and related infrastructure conditions at each region. Indicatively, in the province of Mendoza, Argentina, 88% of the vineyards are irrigated through surface irrigation (i.e., gravity-based systems), whereas the remaining vine-growing area is irrigated through pressurised systems (e.g., dripping) to grow wine grapes of different qualities [27]. Moreover, the selection of good practices in water management for the winemaking industry could be influenced by the assessment methodology applied [39]. Overall, this selection depends on a range of decision-making constituents. More specifically, alternative irrigation options (e.g., drip, deficit) [55,56] and wastewater treatment techniques (i.e., aerobic, anaerobic, or their combination) [54] result in different levels of water savings. In addition, the implementation of digital technologies (e.g., sensors used during viticulture) [57,58] or holistic approaches for water-friendly activities across the whole wine supply chain [59] can further support an advanced and complete water stewardship plan. An indicative list of WF mitigation practices in the winemaking industry is tabulated in Table 3.

Conclusions
The Italian wine industry constitutes a global leader in terms of production quantity and quality. Within the country, the economic scale of winemaking renders the industry as the key production component of the Italian agrifood sector related to respective freshwater appropriation. To that end, the investigation of water use needs during viticulture and vinification is imperative to support water stewardship within the country's wine sector. According to the scientific literature, Italian wine dominates the research efforts with respect to WF assessment, thus validating the important role of economic and environmental sustainability in the national winemaking industry. To a greater extent, given the consumers' awareness and positive purchasing behaviour towards water-efficient wines, the sustainability profile of the Italian wines could further link to the designation of origin and receive international market's appreciation.
As research regarding water management in the winemaking industry is limited, this paper acts as an initial mapping that captures the major drivers and barriers of water stewardship considering the unique geographic and socio-economic characteristics of the Italian landscape. Our research findings indicate that the environmental, socio-economic, and market drivers outperform the existing, mainly technical and methodological, barriers. This proliferation of drivers and the identification of good practises in the industry motivate the development of operationalisable water stewardship frameworks for the Italian wine sector.

Practical Implications
Based on the literature evidence, it is critical that the practitioners of the Italian wine industry should act towards the direction of water management to support the preservation of freshwater resources and enhance the economic water productivity of their products. This research validates that water management policies and practices should be systematically applied in the Italian winemaking industry, from an end-to-end supply chain perspective, to enhance the sustainable brand image of the national production and foster its trade potential and market appreciation.
At an operational level, this research suggests that vine growers and winemaking practitioners should focus on water management interventions at three levels, including (i) soil management, (ii) freshwater management, and (iii) wastewater treatment [3]. In particular, we propose that the type of irrigation systems and practises to be applied should also consider the edaphoclimatic and related infrastructure conditions at each winemaking region to increase the efficiency of water resources appropriation [27]. Regarding wastewater treatment, aerobic processes (e.g., membrane bioreactors [60]) could offer an efficient and easy-to-use solution compared to anaerobic ones that constitute a more economic option [54].
To a greater extent, we propose the digitalisaiton of the wine supply chain, particularly at the farming echelon. In this regard, sensor technologies are reported to support the decision-making process concerning the water stewardship of agrifood commodities [57], which could also be pertinent to the case of the wine supply chain. The use of sensors in grapes' farming for monitor freshwater use and other related parameters (e.g., soil moisture) is an indicative digital intervention that relates to the quality of the wine production. The introduction of advanced technologies can assist in (i) mitigating methodological errors in water-use estimations, (ii) gathering field-level data, (iii) calculating water consumption in viticulture in a more accurate way, (iv) extrapolating information with regard to the WF of their supply chain, and (v) devising sound marketing strategies to engage with consumers [61].

Future Research
With regard to future research directions, both researchers and practitioners of the winemaking production field may focus on developing analytical and computer-based tools for multi-objective analysis and simulation to solve freshwater resource planning and operational problems. To that end, Aivazidou et al. [7] suggest a framework that guides the ex ante evaluation of applied water management policies through developing a pertinent simulation model that enbables the assessment of water utilisation on the supply chain financial performance. Notably, the modelling effort captures the concept of consumers' environmental sensitivity with regard to blue WF efficiency as a supply chain profitability factor. In addition, the economic evaluation of the green WF to the overall production value of wine, as inspired by the study of Grammatikopoulou et al. [9] for the case of cereals, is highly recommended considering that the majority of wine grapes across the Mediterranean are grown under rainfed conditions. To a greater extent, the economic water productivity could be combined with water scarcity indicators to account for the inter-annual variability of the green and blue WFs at a regional level to improve the management of grapes' production, supply, and wine trade in the winemaking sector [62].
To wrap up, based on the environmental, economic, and technical managerial insights obtained by the analysis of the major drivers, barriers, and good practises, it is crucial that industry stakeholders should systematically focus towards developing a concrete water management scheme in the Italian winemaking sector for fostering its sustainability.