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An Unsaturated/Saturated Coupled Hydrogeological Model for the Llamara Salt Flat, Chile, to Investigate Prosopis Tamarugo Survival

: The Propopis tamarugo Phil, also known as Tamarugo, is an endemic and protected tree that survives in the Atacama Desert — a hyper arid and highly saline environment. The Tamarugo is threatened because of groundwater overexploitation, and its preservation depends on the soil moisture in the vadose zone, as many of the tree roots do not reach the current water table levels. To improve the estimation of soil moisture available for the Tamarugo trees, we applied a hydrogeological model that couples the unsaturated and saturated zones. The model was used to represent different groundwater exploitation and recharge scenarios between February 2006 and September 2030 to predict simultaneously groundwater levels and soil moisture. The model results show that even at locations where water table depletion is relatively small (~1 – 1.5 m), soil moisture can drastically decrease (0.25 – 0.30 m 3 /m 3 ). Therefore, Tamarugo survival can be better addressed, as the applied model provides a management tool to estimate response of Tamarugo trees to changing soil moisture. To further improve the model and its use to assess Tamarugo survival, more field data, such as soil hydrodynamic properties and soil moisture, should be collected. Additionally, relationships between the state of the Tamarugo trees and soil moisture should be further constructed. In this way, the developed model will be able to predict future conditions associated to the Tamarugo’s health state.

Introduction
Arid and semi-arid regions cover approximately 32.4% of the world's land area and are inhabited by more than 2500 million people [1]. In these regions, groundwater-dependent environments endangered by natural or anthropogenic causes are common [2][3][4][5]. The Prosopis tamarugo Phil, also known as Tamarugo tree, is found in groundwater-dependent ecosystems, and are adapted to extreme arid environments [3]. It is one of the few species that lives in the Atacama Desert, Chile, in a zone comprised by many salt flats that is known as Pampa del Tamarugal [6][7][8].
The Tamarugo tree is one of the 44 Prosopis species in the world [9]. This legume tree is an endemic and protected flora that survives in hyper arid and highly saline environments [10]. It lives in zones where the water table is relatively shallow (4-20 m depth) [3], and near areas where groundwater is extracted for mining purposes [10,11]. The preservation of the Tamarugo tree depends on the soil moisture in the vadose zone, because in many of these trees, the roots do not reach the water table levels [12,13]. The Tamarugo root system is composed by a root mat at less than a meter depth from the soil and by long pivoting roots that reach the water table [10]. To protect the forests of Tamarugo, the Chilean government imposes environmental monitoring plans where different parameters must be periodically measured to ensure the sustainability of these systems. For instance, in the Resolution N° 890/2010 of the Chilean Environmental Assessment System, the moisture profile in the Llamara salt flat basin near the Tamarugo trees is among the six biophysical parameters that must be monitored in the following decades. Different studies have quantified the environmental risk to which the Pampa del Tamarugal is exposed due to the exploitation of aquifers and how this affects the survival of the Tamarugo tree [3,10,14,15].
In 2012, in the Pampa del Tamarugal and its surroundings, a total water withdrawal equivalent to four times the estimated natural recharge of the aquifer was registered [3]. This aquifer overexploitation constitutes a real threat to the survival of the Tamarugo and the local flora. Hydrogeological models that simulate the groundwater flow in the Pampa del Tamarugal have been developed primarily for environmental assessment studies [11,14,15], and recent studies have updated the conceptual hydrogeological model of the Pampa del Tamarugal aquifer [6,7]. Furthermore, it has been demonstrated that anthropogenic pressures are the dominant factor of the affecting water table fluctuations, which has resulted in an overuse of the aquifer [8]. A relationship between the water table depth and the vital state of the trees has been proposed by [15], in which the water table depth below the trees in the Llamara salt flat was obtained from monitoring wells, and the state of the Tamarugo trees was estimated using high-resolution satellite images. Based on a statistical analysis, it has been determined that water table depths of more than 13.3 m increase the probability of finding an ill-conditioned Tamarugo tree [15]. Satellite indices, such as the green canopy fraction (GCF) [16] and the normalized difference vegetation index (NDVI) [17], have been used for analyzing the health of the Tamarugo trees. The GCF is the ratio between the volumes of green canopy and total canopy and corresponds to an indirect measurement of the photosynthetic activity of the trees [16], whereas the NDVI represents the fraction of photosynthetically active radiation absorbed by the vegetation [17]. Chávez et al. [18] proposed a relationship between water table depth and the vital state of the Tamarugo tree in which the GCF is integrated along with the NDVI, the 18 O isotope enrichment in foliar tissue and the twig growth to determine a range of tree damage as a function of water table depth. In this way, they estimated that for a water table depth of 6 m, the Tamarugo tree begins to suffer damage that increases until the water table depth reaches 20 m, where the tree dies [13]. Decuyper et al. [3] studied two Tamarugo plantations that were exposed for 25 years to a decrease in the water table level in the Pampa del Tamarugal. Plantation A had an approximate decline of 3 m in 25 years (high descent) in the water table level, whereas plantation B experienced a decrease of 0.4 m in the same 25 years (low decline). The effect on trees of this groundwater depletion was quantified using the GCF. Their results showed that plantations A and B had 60% and 47% of the trees in poor condition, respectively. Despite the large difference in water table depletion observed between the plantations, the percentage of trees in poor condition did not differ substantially. This suggests that depth to groundwater may not be the best proxy for assessing soil water status for Tamarugo trees. Therefore, in order to correctly assess the amount of water that the Tamarugo trees will have due to a decrease in the groundwater level, it is essential to further simulate the evolution of the volumetric water content (or the suction) in the unsaturated zone, which is what supplies water to the roots of these trees [13,19,20].
The aim of this work was to improve the estimation of the volumetric water content available for the Tamarugo trees in an arid basin through the development of a hydrogeological model that couples the unsaturated and saturated zones. The model combines a three-dimensional MODFLOW model to simulate the saturated flow with a one-dimensional model developed in HYDRUS to represent the unsaturated flow. This hydrogeological model represents the response of the aquifer to an intensive exploitation or to fluctuations in groundwater recharge and, at the same time, it predicts the evolution of the volumetric water content in the vadose zone. Thus, with a better understanding of the behavior of the entire system in which the Tamarugo trees subsist, more informed decisions could be made for its sustainable exploitation.

Study Zone
The study was conducted in the Llamara basin, in the southern section of the Pampa del Tamarugal, located in the Atacama Desert, northern Chile ( Figure 1). According to the Köepen climatic classification, the study site is a subtropical marine desert [21] with almost absolute absence of precipitation, low relative humidity and a large daily thermal amplitude. For the warmest and coolest months, the minimum absolute temperatures are of −5 and −12 °C, the maximum absolute temperatures are of 35 and 36 °C, and the mean temperatures are of 32 and 0 °C, respectively [13]. Annual evaporation in the Llamara basin is of ~2.680 mm [15].
The delineation of the basin was kept from that presented in an environmental impact study carried out at the study site [15]. The Tamarugo forest is located at the north of the study area, as shown in Figure 1. The location of the four monitoring sites used in the calibration of the unsaturated model is also presented in Figure 1. These sites correspond to the soil that is beneath the canopy of the T1, T2, T3, and T4 Tamarugo trees [15]. The location of the pumping wells in the study area is also shown in Figure 1.

Description of the Unsaturated/Saturated Coupled Hydrogeological Model
MODFLOW-2000 [22] was used to represent water flow under saturated conditions. MODFLOW is a widely used numerical model that represents groundwater flow [23,24]. MODFLOW is a three-dimensional model that uses finite-difference equations to solve mass conservation in confined and unconfined aquifers [22,25]: where where  [L 3 L −3 ] is the volumetric water content; h [L] is the soil water pressure head; S [T-−1 ] is a volumetric sink/source terms that is typically used to represent root water uptake [26]; K(h) [L T −1 ] is the unsaturated hydraulic conductivity curve; and all the other parameters were previously defined.
Using the Richards' [27] equation to represent water movement in the vadose zone allows incorporating processes such as precipitation, infiltration and evapotranspiration, among others [26,28]. To solve Equation (2), it is required to know the soil hydrodynamic characteristics. In this work, we used the water retention curve, h() (Equation (3)), proposed by van Genuchten [29], and the K(h) (Equation (4)) described by Mualem [30]: where s and r [L 3 L −3 ] are the saturated and residual volumetric water contents, respectively; is the inverse of the bubbling pressure; l [-] is the pore-connectivity parameter, assumed to be 0.5 [30]; The HYDRUS package [26,28] was used to couple MODFLOW as a variable-flow boundary condition imposed at the vadose zone of each MODFLOW cell, at each time-step. As these models are fully coupled, the HYDRUS package provides MODFLOW with recharge fluxes into the aquifer, whereas MODFLOW provides HYDRUS with the position of the water table. Note that there are other MODFLOW packages that have been developed to couple the flow in the vadose and saturated zones, such as the Unsaturated Zone Flow (UZF1) [31] and the Variably Saturated Flow (VSF) [32] packages, but the HYDRUS package was selected as it is highly applicable to represent the water flow through the vadose zone in arid and semiarid areas, with moderate computational efforts [28].

Model Implementation in the Llamara Salt Flat Basin
The flow in the saturated zone was represented using a previously developed MODFLOW numerical model freely available for the Llamara salt flat basin [15]. This numerical model was approved by the National Water Authority; hence, it is the model that should be used by stakeholders interested in groundwater characterization in the area. For this reason, the conceptual model to represent the flow in the saturated zone was based on the information included in the approved numerical model ( Figure 2) and on publicly available information [15]. A horizontal spatial discretization of 250 × 150 cells (236 m × 236 m) and a vertical distribution of two strata of variable thicknesses were defined. The hydrogeological properties of the model, such as saturated hydraulic conductivities and storage coefficients, were also obtained from the MODFLOW model. Nonetheless, as described below, the model was completely reviewed, and the hydraulic conductivities were recalibrated to improve its initial calibration. Groundwater recharge was estimated using a hydrological model based on the soil moisture accounting (SMA) algorithm [33]. This hydrological model was carried out for an environmental impact study of the study area [15]. The hydrological model represents precipitation, runoff, actual evapotranspiration, infiltration and deep percolation using the hydrological response units of the basin. The historical hydrological data to estimate model recharge were available between January 1978 and January 2006 [15]. The groundwater recharge comes mainly from precipitation that falls into the neighboring basins of Guatacondo, Maní, Sipuca, Mal Paso and Sama ( Figure 2). This recharge enters the study zone as a lateral groundwater input and corresponds to a total of 465 L/s (estimated average recharge based on the historical data). Since there is almost absolute absence of precipitation in the study area, there is no recharge from the top of the model domain. The groundwater discharge of the aquifer is comprised by 2 L/s of transpiration from the Tamarugo forest; 325 L/s of bare soil evaporation, which is calculated using the potential evaporation rate (4.6 mm/day on average over the historical time period); a spring discharge of 128 L/s in the Amarga ravine, and an estimated discharge of 10 L/s towards the Loa river; and an extinction depth that varies between 1.5 and 6.0 m depending on the soil texture, as estimated by [15]. The extinction depth corresponds to the depth below land surface below which groundwater evapotranspiration does not occur. When the water table is above the extinction depth, groundwater evapotranspiration is represented in an exponential way, ranging between zero (at the extinction depth) and the potential evapotranspiration rate (at the soil surface). When the water table is deeper than the extinction depth, the groundwater evapotranspiration flux is set to zero.
The MODFLOW model was built to simulate 300 stress periods. The first stress period corresponded to a steady state simulation using the historical groundwater recharge. The groundwater levels obtained in the first stress period were used as initial conditions for the following stress periods, in which a transient simulation between February 2006 and September 2030 was performed. In absence of available hydrological data between October 2012 and September 2030, the historical groundwater recharge was used for this time period, although different simulations were performed to account for this issue (this is described below).
The model calibration was performed in two stages. The first stage consisted in the recalibration the saturated zone model developed by [15], which was performed for the time- In the recalibration of the saturated zone model, the saturated hydraulic conductivity distribution of the study zone was adjusted by comparing simulated and observed groundwater levels at 75 observation wells located in the aquifer (Figure 1). An isotropic aquifer was assumed in the horizontal directions, whereas the hydraulic conductivity in the vertical direction was assumed to be 10% of the horizontal hydraulic conductivity, i.e., Kz = 0.1 Kx = Ky [34]. The calibration process was performed using the PEST software [35]. PEST is a public domain code that uses an uncertainty analysis for parameter estimation [35]. Note that the recalibration only modified saturated hydraulic conductivities as it is considered that the specific storage distribution estimated by [15] is representative of the aquifer, as the obtained values were consistent among the different pumping tests performed throughout the basin. PEST develops a calibration process that minimizes the sum of the squared errors between simulated and observed groundwater levels, i.e., the objective function (Equation (5)), as a function of the model response to a value of the hydraulic conductivity: where i is the difference between the observed and simulated value of a variable at position i in the model domain, X(pi) is the function that represents the model (i.e., the simulated hydraulic head), pi is the parameter that will be calibrated at position i (i.e., the saturated hydraulic conductivity), and Oi is the observation performed at position i (i.e., the observed hydraulic head). The initial hydraulic conductivity distribution was that reported by [15] and it was improved using the pilot points approach [36]. In this approach, instead of creating a homogeneous zone and finding its hydraulic conductivity using inverse modeling, the value of the parameter is interpolated from the pilot points (locations where the parameter values are known). Then, an inverse model is used to adjust the hydraulic conductivity at the pilot points, and the surface defining the variation of hydraulic conductivity throughout the domain is warped until the objective function (Equation (5)) is minimized. Note that PEST interpolates the hydraulic conductivity distribution using the Kriging method [35]. In this way, the regions of the model with more groundwater level observations had a continuous distribution of the hydraulic conductivity. The calibration process in the unsaturated/saturated coupled model was performed manually and aimed to replicate the observed volumetric water content profile beneath the canopy of the T1, T2, T3 and T4 Tamarugo trees (Figure 1). The calibration consisted in adjusting the s, r, , and n parameters of the van Genuchten [29] water retention model, and assuming m = 1 -1/n [29]. The observed volumetric water contents were measured between May and July 2006, as part of the environmental monitoring plan. These data are public and can be found in the Chilean Environmental Assessment Service (https://bit.ly/31fOeFe). Additionally, the soil hydrodynamic characteristics were validated using volumetric water contents measured between November 2007 and January 2008. Unfortunately, the data available for calibration and validation of the unsaturated model were collected for a very brief period (6 months). For simplicity, we considered a uniform distribution of the water retention parameters throughout the model domain. Additionally, at each cell of the model, the vertical hydraulic conductivity, Kz, obtained from the calibration of the saturated zone model, was used as the saturated hydraulic conductivity in the vadose zone, i.e., Ks = Kz. Therefore, the relative hydraulic conductivity, K(h)/Ks, was determining by using the calibrated water retention curve parameters and Equation (4).

Impact of Groundwater Withdrawal and Recharge Rates on Water Table Levels and Soil Moisture Beneath the Tamarugo Trees
To investigate the impact of groundwater withdrawal on the survival of the Tamarugo trees, the coupled model was used to simulate the water flow in both the unsaturated and the saturated zones of the study site between February 2006 and September 2030. Specifically, the model was used to assess the impact of groundwater extraction from five pumping wells, as well as of groundwater recharge, on the water table level and the volumetric water content profile in the Tamarugo forest.
Four simulations were performed with the aim of observing the aquifer response to different water scarcity scenarios. Simulation 1 considers the impact of the five pumping wells located in the study site and their corresponding groundwater withdrawal rates, according to the water rights granted by the Chilean water authority. Simulation 2 considers that the groundwater withdrawal rates increase in 25% with respect to the water rights granted. Simulation 3 uses the groundwater withdrawal rates of simulation 1, but with a 20% reduction in the groundwater recharge that enters the model domain, i.e., a total of 372 L/s, between October 2012 and September 2030. Finally, simulation 4 also uses the groundwater withdrawal rates of simulation 1, but the groundwater recharge imposed in the model for the period October 2012 and September 2030 was that estimated using the last year with reliable data for the calculation of groundwater recharge, i.e., the monthly groundwater recharge time series estimated between October 2011 and September 2012 was repeated until September 2030. Simulations 3 and 4 provide possible future climate scenarios. The groundwater withdrawal rates and the groundwater recharge used in each simulation are presented in Table 1.

Model Calibration and Validation
To assess model calibration and validation, among many calibration metrics, we selected the root mean squared error (RMSE) [37]. For the saturated zone model, the RMSE between the simulated and observed groundwater levels of the initial distribution of the hydraulic conductivity in the study site was of 19.9 m, and the hydraulic conductivity was represented in approximately 40 zones that had uniform hydraulic conductivity [15]. These zones were based on the geological characteristics of the study site [15]. After the calibration of the saturated zone model, the RMSE between simulated and observed groundwater levels was of 15.2 m (24% reduction). The final hydraulic conductivity distribution of the study site is shown in Figure 3 and the drawdown at selected monitoring wells for the validation period is presented in Figure 4. The worse representation of the water table was observed at MW1 and MW4 (Figure 1), which are located north of the selected Tamarugo trees. In general, in all the other locations in the aquifer, the water table levels were represented fairly well. Moreover, the simulated flows at the Amarga ravine springs and at the discharge towards the Loa River were of 128.6 and 10.6 L/s, respectively, which are very similar to those observed (see Figure  2).  The results of the unsaturated/saturated model calibration are shown in Table 2 and Figure 5. Table 2 presents the calibrated parameters of the van Genuchten [29] water retention curve, whereas the simulated and observed volumetric water contents beneath the canopy of the Tamarugo trees are shown in Figure 5. The simulated and observed moisture profiles agree well with root-mean squared errors (RMSEs) ranging between 0.007 and 0.026 m 3 /m 3 , with an average value of 0.014 m 3 /m 3 for the 12 volumetric water content profiles used in the calibration process. The break in the soil moisture profile of the T3 tree at a depth of ~0.2-0.5 m is due to root water uptake from the zone where the highest root density was defined. Note also that the variations in soil moisture at the Tamarugo trees are small. As the period of available data to calibrate/validate the unsaturated conditions of the model was short, caution must be taken when interpreting the transient behavior of the flow in the vadose zone. Table 2. Calibrated water retention curve parameters of the van Genuchten model [29]. The simulated and observed volumetric water contents beneath the canopy of the Tamarugo trees agree well for the validation period ( Figure 6). Therefore, the calibrated hydrodynamic properties found in the calibration process are adequate to represent the soil processes. The RMSE in the validation period ranges between 0.006 and 0.023 m 3 /m 3 , with an average value of 0.011 m 3 /m 3 for the 12 volumetric water content profiles used in the validation.

Water table depth evolution
The evolution of the water level depth at each pumping well for all the simulations is presented in Figure 7. All the simulations show a water table depletion that ranges between 2 and 10 m at the different pumping wells. In general, towards the end of the simulations, the water table levels stabilize, except for those occurring at the 3X-14A pumping well. In addition, when the groundwater withdrawal rates are increased in 25% (simulation 2), the water depletion is larger than that observed for the other simulations. Figure 8 shows the temporal evolution of the simulated water table depth at the T1, T2, T3 and T4 Tamarugo trees. In general, no significant differences of the water table levels are observed between the simulations. When the groundwater withdrawal rates are increased (simulation 2), the water levels in the Tamarugo forest are slightly lower than those simulated for the current water rights granted (simulation 1). Note also that the trees T3 and T4 exhibit a recovery on the water table level that occurs because the average groundwater recharge is larger than that estimated at October, 2006. However, this behavior is not seen at trees T1 and T2 because they are located much closer to the pumping wells of the study site (Figure 1).   Table 3 shows the simulated drawdown at PW1 and at the T1, T2, T3 and T4 Tamarugo trees for simulation 1 (baseline), and the distance of each Tamarugo tree to PW1. On the one hand, after 6 months of pumping, only the T1 and T2 trees are within the radius of influence of the pumping wells.
On the other hand, the T3 and T4 trees are exposed to a water table level recovery for the years 2010 and 2030. As explained before, this recovery is due to the groundwater recharge considered in the simulations. Using the HYDRUS package, the soil moisture evolution in the model domain was simulated. A comparison between the volumetric water content profiles at the T1, T2, T3 and T4 Tamarugo trees for the beginning (2006) and for the end (2030) of simulation 1 is presented in Figure 8 (baseline). The T3 and T4 trees, which are further away from PW1 (Figure 1), show a larger volumetric water content at depths deeper than 0.5 m, compared to the T1 and T2 Tamarugo trees (that are closer to PW1), which exhibit a similar volumetric water content (~0.25 m 3 /m 3 ) at depths of 5 m. At trees T1 and T2, the difference in volumetric water contents between the beginning and the end of the baseline simulation does not exceed 0.05 m 3 /m 3 up to 4 m deep. However, at a depth of 6 m in the T1 Tamarugo tree, the difference between the volumetric water content at the beginning and at the end of the baseline simulation is more important (~0.3 m 3 /m 3 ). Similarly, at a 6 m depth in the T2 Tamarugo tree, at the beginning of the simulation, the soil is fully saturated (0.45 m 3 /m 3 ), whereas at the end of the simulation, the volumetric water content reached ~0.20 m 3 /m 3 .

Discussion
The Tamarugo trees are subject to multiple abiotic and biotic stress factors that affect plant productivity. When a stress factor surpasses the physiological performance threshold, plant survival is threatened [10]. Groundwater overexploitation is one factor that can damage the Tamarugos trees, not only because as the water table drops, the roots of the Tamarugo cannot reach the saturated zone, but also because less water is available in the unsaturated zone for root water uptake [13]. Water stress can result in biomass reduction for an individual tree, but also in a reduction of the tree population. Hence, the ecosystem dynamics can be permanently modified [10]. For this reason, it is important to monitor the impacts of groundwater extraction on different variables that can be used to assess the survival of the Tamarugo tree, as well as to develop predictive tools for water resource management that incorporate ecological constrains.
Nowadays, there are mining projects in the study area that have water rights granted to extract groundwater for decades. Consequently, there are environmental monitoring plans that aim for Tamarugo survival. The state of the Tamarugo trees is being monitored using relationships that combine the GFC, NDVI, 18 O enrichment in the foliar tissue, twig growth with the water table depth. Soil moisture is also being monitored beneath the canopy of the trees. Unfortunately, continuous measurement of soil moisture using time-domain reflectometry or other methods is extremely complex due to the soil's high salinity [38] so there are only discrete observations of this key variable. The gravimetric method, which is highly destructive and time consuming, has been used to get insights of volumetric water content levels near the Tamarugo trees. The latter reinforces the need for tools, such as the coupled unsaturated/saturated model presented in this work, to predict variables that are directly associated to water stress, i.e., the soil's volumetric water content.
Our results show that an increase in groundwater withdrawal rates results in additional drawdown of water table levels near the selected Tamarugo trees. However, changes in future groundwater recharge do not have a notorious influence on modeled groundwater levels near the pumping wells or beneath the selected Tamarugo trees (Figures 7 and 8), i.e., the water table evolution in this area of the model is similar in the four simulations investigated in this work. This behavior is explained by the large distance between the eastern recharge boundary and the pumping wells and trees, as well as because the simulated groundwater extraction rates are generally smaller than the groundwater recharge. Consequently, changes in groundwater recharge and in withdrawal rates are mainly balanced by changes in bare soil evaporation and groundwater storage. However, when groundwater extraction rates are larger than groundwater recharge, water table levels will drop, and groundwater storage will be depleted [34]. In simulation 4 (recharge maintained), which has a groundwater recharge that is smaller than the overall pumping rates, the groundwater levels modeled were similar to those simulated in the other scenarios ( Figure 8). Nonetheless, near the model boundaries groundwater storage and water table levels were slowly decreasing, which is consistent with the field evidence that the flow at the Amarga ravine springs has been decreasing over time [15]. Therefore, caution must be taken to ensure that the groundwater rights that have been granted do not surpass groundwater recharge. This issue is of extremely importance in the study area because during the last 30 years, there has been a sustained increase in groundwater withdrawal rates in the Pampa del Tamarugal aquifer [3,8,14], and it is likely that climate change will reduce even more the groundwater recharge.
Even when the water table levels decreased up to ~10 m at PW1 for the different simulations, water table levels at the selected Tamarugo trees did not reach the depth that, according to [15], damages the trees (i.e., 13.3 m). However, according to [13], the T1 and T2 could be damaged because the water table levels are below 6 m. This depth corresponds to the first physiological threshold of the damage scale reported in [13], in which the Tamarugo tree keeps its water status by decreasing water demand through partial defoliation and decreased twig growth. Given the differences between these methods to assess Tamarugo survival, which only use the water table depth to infer tree  damage, other key variables, such as volumetric water content, must be included in this analysis.
The evolution of the soil's volumetric water content was achieved by incorporating the HYDRUS package in the MODFLOW model. Our results show that the T3 and T4 sites show a rapid increase with depth in the volumetric water content at shallow depths. The moisture content increase deeper into the soil is more gradual, reaching saturation values at depths of 3-4 m (Figure 9). The difference between the initial (2006) and final (2030) soil moisture profile for these sites does not surpass 0.05 and 0.02 m 3 /m 3 , for T4 and T3, respectively. At T2, a water table drop of 1.0 m results in a moisture reduction of ~0.25 m 3 /m 3 at ~5.5 m depth between 2006 and 2030. Therefore, even if the water table depletion is relatively small, soil moisture can drastically decrease. These results help explaining what was observed by Decuyper et al. [3], in which small water table drops greatly reduced the water available for the Tamarugo tree at a state where half of a Tamarugo forest exhibits damages.
As described previously, the applied model was based on a previously developed MODFLOW model available for the Llamara salt flat basin, and on public information [15]. This model was approved by the national water authority. For this reason, we did not change boundary conditions or groundwater recharge or discharge; however, as the water table evolution near the pumping wells and trees was similar in all the simulations, the groundwater recharge rates should be further explored to ensure that there are no flaws in their estimation. Our focus, instead, was to improve the hydraulic conductivity distribution and the couple the unsaturated model to provide a management tool to estimate the response of the Tamarugo tree to soil moisture fluctuations. Therefore, the applied model has limitations that can be overcome so there are many opportunities to improve it. First, to avoid structural errors of the model, a new calibration can be implemented using lateral head differences instead of absolute heads [39,40]. It has been recognized that as a result of uncertainties and simplifications in the model, some parameters most likely will have values that compensate for structural defects in the model and that are biased by incorrect model boundaries [39]. Secondly, a new type of observational data can be used to improve the calibration. For instance, the method proposed by Schilling et al. [41], which reduces uncertainty in model parametrization, uses tree ring growth data and transpiration to calibrate a hydrogeological model that represents interactions between surface water, vadose zone, groundwater, and vegetation. In our case, the Schilling et al. [41] method could be modified to use tree distribution as another target in the model validation.
Future research should focus on improving the current unsaturated/saturated model and its input parameters. The following are some future research directions that we foresee: (1) update of hydrological, hydrogeological and ecological data to improve the model prediction capability; (2) perform a new calibration that ensures minimization of structural errors of the model by using lateral head differences [39,40]; (3) incorporate a new type of experimental data for improved calibration. For instance, methods such as that proposed by Schilling et al. [41] could be used to reduce uncertainty in model parametrization. In our case, this method can be modified to use tree distribution for model validation; (4) perform an uncertainty analysis to better understand the groundwater dynamics in the study site, which is a remote location with scarce data; (5) collect samples to improve the spatial distribution of the soil hydrodynamic properties, as currently our model assumes that these properties are constant; (6) maintain a monitoring program that can be used for both the Tamarugo survival and the improvement of the coupled hydrogeological model; (6) investigate relationships between the state of Tamarugo trees and soil moisture; and (7) integrate the different stakeholders so an integrated water resource management that incorporates ecological constrains can be achieved.


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

text:
Influence of Seed Priming with Chemicals, Micronutrients and Bio-Inoculants on Growth and Yield Attributes in Foxtail Millet (Setaria italica L.)

Foxtail millet is commonly known as Italian millet, German millet, Siberian millet, and foxtail bristle grass. Foxtail millet grows 2–5 feet tall and can be cultivated in drier and cooler regions when compared with other millets. It is currently grown in China, India, Europe, particularly in Portugal, Turkey, Hungary, France and Spain, in Asia, primarily in Korea, Pakistan, Myanmar, Bhutan, Nepal, and South Africa. It is used for feeding birds and as livestock feed in the developed countries and for food in some parts of Asia. It International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 10 (2019) Journal homepage: http://www.ijcmas.com

is suitable for inclusion in multiple or intercropping systems because of its short duration nature.
Seed enhancement is a term used in industry and in scientific literature to describe beneficial techniques performed on seeds, after harvesting but prior to sowing for improving a crop's harvested yield and quantity. Rapid germination and emergence is an important determinant of successful plant establishment (Heydecker et al., 1975) and seed priming has presented promising results, for many crop seeds (Bradford, 1986).
Seed priming is a controlled hydration process that involves exposing seeds to low water potentials that restrict germination (radicle protrusion), but permits pre germinative physiological and biochemical changes to occur (Heydecker and Coolbear, 1977;Bradford, 1986;Khan, 1992). Upon rehydration, primed seeds may exhibit faster rate of germination, more uniform emergence, greater tolerance to environmental stresses, and reduced dormancy in many species (Khan, 1992). The present investigation was carried out to know the different seed priming effect on growth and grain yield of foxtail millet.

Results and Discussion
Foxtail millet seeds primed with T 12 (Seed priming with Azospirillum sp. @ 20% + Pseudomonas fluorescens @ 20% + Phosphobacter @ 20%+ ZnSO 4 0.1% + Boron 0.1%) recorded highest plant height (52.25cm, 133.70cm and 134.12cm), number of tillers (5.18, 5.37 and 5.37 respectively)at 30, 60 DAS and at harvest respectively. Days to 50% flowering and days to maturity recorded lower number of days (52 and 92 days respectively) in T 12 . While, control (T 1 ) recorded lowest plant height (35.70cm, 109.30cm and 116.80cm) and number of tillers (3.54, 3.85 and 3.8) at 30,60DAS and at harvest respectively. Days to 50% flowering and days to maturity recorded highest number of days (55 and 96 DAS respectively) in control (T 1 ) ( Table 1). Improvement in growth parameters might be due to combined effect of bio fertilizers and micronutrients. The enhanced plant height may also be due to the improved and faster plant emergence in bio-primed seeds which might have created nitrogen fixation by the plant, phosphorous solubilization and also cooperative competition among the plants for light and resulted in taller plants. In addition increased the nutrient availability, Azospirillum and PSB also effects the plant growth through production of growth harmone like IAA, GA 3 , Cytokinin (Sattar and Gaur, 1987).  Janardan Yadav et al., (2010) in chickpea concluded that the increase in plant height might be due to the early emergence of the bioprimed seeds which makes the plant to compete well with the weeds and higher rate of accumulation of dry matter due to atmospheric nitrogen fixation and phosphorous solubilization. Raj et al., (2004) and Abdullahi et al., (2014) reported significant difference for number of tillers per plant in pearl millet, Gangwar and Sinha (2014) in rice. Increased leaf production due to seed bio-priming might be due to Pseudomonas fluorescence which contains physiologically active substances viz., growth regulators and nutrients that promote profuse number of tillers per plant as noticed by Sharifi et al., (2011) in maize.
Significant difference is found for all the yield parameters due to seed priming treatment. The increase in the panicle length may be due to the synthesis of amino acid and chlorophyll and better carbohydrates transformation which resulted in better growth and length of panicle which ultimately produced more number of seed per panicle resulting in increasing panicle weight there by increasing the yield. Similar results were also reported by Niranjan raj et al., (2004) in finger millet and Prasad et al., (2009) in wheat. Increase in yield was also contributed by Zn which attributed to the enhanced synthesis of carbohydrates and their transport to the site of grain production crop (Pedda-Babu et al., 2007). Similar results were also reported by various researchers due to Zinc seed priming on seed yield of wheat (Nazir et al., 2000;Harris et al., 2005;Aboutalebian et al., 2012).
The influence of bio-inoculants on the grain yield might be through their effect on actively growing regions in such a way that they encourage nitrogen fixation, phosphorous solubilization and mobilize the nutrients absorbed elsewhere towards the shoot resulting in better vegetative growth and subsequently increased yield as noticed by Nezarat and Gholami (2009) in maize. Azospirillum sp. and phosphobacter have improved photosynthesis by increasing water and nutrients absorption leading to produce more assimilate and improves translocation of metabolites from sources to sink which in turn increased the test weight in seeds (Mirzaei et al., 2010).


text:
Antifungal activity of essential oils on mycelial growth of Fusarium oxysporum and Bortytis cinerea

In vitro study of the effect of different volumes of twelve essential oils on the mycelial growth of economically significant phytopathogenic fungi ( Fusarium oxysporum and Botrytis cinerea ) and it was compared to the effect of a fungicide. The antifungal activity of essential oils is decreased with the duration of incubation and it differs depending on the type of phytopathogenic fungus and the applied volume. The most effective antifungal effect on both tested fungi was in the essential oil of thyme, with lowest values of IC 50 while the weakest effect was in essential oils of eucalyptus and lemon, with the highest values of IC 50 . Certain essential oils, when applied in certain volumes, had the same or even better effect on the inhibition of the growth of mycelium when compared to the tested fungicides.

INTRODUCTION
Plants pathogens cause deceases of plants in the field and warehouses and decrease the yield and quality of crop which eventually leads to great economic losses. The causes of plant deceases on the global level still cause losses of between 10 and 16 percent (Chakraborty and Newton, 2011). According to the opinion of the international community, Botrytis cinerea Pers is in the first place, with Fusarium oxysporum Schl. in the fifth place of economically most significant plants pathogens (Dean et al., 2012). The ability to adjust to various agroclimatic conditions allows the species of Fusarium to spread all over the world (Ćosić et al., 2004). B. cinerea adjusts its enzymes, depending on the specificity of the host and has the ability to adjust to the host it is infecting (Fillinger and Elad, 2016). The most dangerous fungal products are mycotoxins, secondary metabolites, which have a toxic effect on the organisms of people and animals. The exposure of population to mycotoxins is a growing problem in African countries due to everyday consumption of the infected corn (Degraeve et al., 2016).
The causes of plant deceases are still most effectively fought by the use of synthetic fungicides. However, the excessive, frequent and inappropriate use of fungicides has caused many problems in their control, especially due to the development of resistant pathogen populations (da Cruz Cabral et al., 2013), through the residue accumulation in food above the permitted limit (Ogah et al., 2016), environmental pollution and negative effects on human health. All of these have led to an increased pressure of the public to decrease the use of synthetic fungicides (Bhagwat and Datar, 2014) and to conduct many studies in the search for the new and alternative agrochemicals (Combrinck et al., 2011).
Essential oils (EO) are secondary plant metabolites which often possess antimicrobial features and, as such, have an important role in the defence of the plants (Hyldgaard et al., 2012). Due to their antifungal, antivirus and insecticidal characteristics, they are a desirable source of alternative methods in the protection of plants (Bassolé and Juliani, 2012). EO can also have a significant role in prolonging the food shelf-life and overcoming losses caused by storing agricultural products (Farzaneh et al., 2015;Prakash et al., 2015). They mostly consist of monoterpenes and sesquiterpenes and their oxygenated derivatives, such as alcohol, ketones, acids, phenols, ethers, esters, etc. (Fornari et al., 2012). They may contain more than 60 different ingredients, two or three of which are present in high concentration while others are present only in traces (Bakkali et al., 2008). Their antimicrobial effect depends on all chemical components (Queiroga et al., 2007) and the interactions among these components may lead to antagonistic, additive or synergistic effects (Bassolé and Juliani, 2012).
As the biological activity of essential oils depends on the type of plants and their chemical composition, applied concentrations, that is, volumes, environmental conditions, agro-technical practice (Ćosić et al., 2014), manner of application (Suhr and Nielsen,2003) and other factors, there are studies being conducted on antifungal effect of essential oils (Palfi et al., 2018;Li et al., 2017;Pedrotti et al., 2017;Sharma et al., 2017;Nosrati et al., 2011;Ćosić et al., 2010;Huang et al., 2010) in order to find the most effective essential oils or their combinations.
The purpose of this research was to test the effect of twelve commercial essential oils in the in vitro conditions on the growth of mycelium Fusarium oxysporum and Botrytis cinerea and compare them with the effects of commercial fungicides but also IC 50 for every essential oil.

Essential oils
Twelve commercial essential oils were used in the study Essential oils were produced in Pranarôm International Ltd. (Belgium) except the essential oil of anise which was produced in Kemig Ltd., Sesvete-Soblinec, Croatia.
The study was conducted with eight volumes of essential oils: 3,5,7,9,15,30,50 and 70 µL/10 mL potato dextrose agar (PDA). PDA substrates, produced by Merck KGaA, (Darmstadt, Germany), were used in the experiment, as well as the auxiliary agent Polysorbatum 80 (Tween 80) produced in Kemig Ltd., Donja Zelina, Croatia. The concentration of Tween 80 was 0.05%. Distilled water was used in the control variable of the experiment instead of essential oils.

Fungicides
The effect of the fungicide Prosaro 250 EC (prothioconazole + tebuconazole) was tested for the phytopathogenic fungus Fusarium oxysporum, and for Bortytis cinerea, the effect of the fungicide Switch 62.5 WG (cyprodinil + fludioxonil) was tested. The amounts of fungicides were determined according to the manufacturer's instructions for each used fungicide. The Prosaro 250 EC fungicide was used in four quantities (75 mL/20 L H 2 O, 75 mL/40 L H 2 O, 100 mL/20 L H 2 O, 100 mL/40 L H 2 O) while Switch 62,5 WG fungicide was used in two quantities (60 g/100 L H 2 O, 100 g/100 L H 2 O). Instead of water, the same amount of prepared PDA substrate was used.

Phytopathogenic fungi
Single spore isolates of F. oxysporum i B. cinerea were obtained from the culture collection of Faculty of Agriculture in Osijek, Osijek, Croatia. Procedure 10 mL of the PDA substrate was poured into each Petri dish (diameter 90 mm), and auxiliary agent Tween 80 was added as well as the previously determined volumes of essential oils. Discs of mycelium of eight-day old phytopathegenic fungi (diameter 4 mm) were placed in the middle of the Petri dish. The experiment was done for each volume of essential oils, fungicides and for the control variable in four replicates. After the inoculation, the Petri dishes were placed into an incubator, at the temperature of 20 °C and under the light regime of 12 h of light/12 h of dark.
The fourth and the eighth day since the beginning of the incubations, the diameter of the growth of mycelium of each pathogenic fungus was measured and expressed in mm.

Statistical data interpretation
Data analysis and graphs were made using the GraphPad Prism Version 7 program (GraphPad Prism, https://www. graphpad.com/scientific-software/prism/). The data are expressed as an arithmetic mean, standard deviation and 95 percent confidence interval for the IC 50 parameter. IC 50 was calculated as a concentration of the tested compound which decreases the mycelial growth by half between the base and maximum response by using the subroutine in the GraphPad Prism program.
Other data are expressed as mean and standard deviation. P value is the result of comparison with control using Dunnett's multiple comparison test, which prevents false positive results (Type I error). P values less or equal 0.05 were considered statistically significant.

The effect of the used fungicide doses
The ratio between the mycelial growth diameters for the phytopathogenic fungi Fusarium oxysporum and Botrytis cinerea was compared and expressed in millimetres, in variants of fungicide and control variable without the use of fungicide was compared.
The results, shown in Table 1, show that the fungicide Prosaro 250 EC in all used doses, completely (100%) counteracts the growth of F. oxysporum fungus on the fourth and eighth day after the mycelial inoculation. In the control variable the mycelial growth of 68 ± 0.0 mm four days after the inoculation was measured, while on the eighth day the diameter of the mycelial growth was 90 ± 0.0 mm. Fungicide Switch 62.5 WG completely inhibited the growth of B. cinerea fungus on the fourth day, while on the eighth day after the inoculation, the mycelial growth was 4 ± 3.1 mm, that is, 23 ± 2.9 mm, depending on the used dosage. In the control variable, the maximum mycelial growth was measured at 90 ± 0.0 mm already on the fourth day after the inoculation.

Ratio between the used volumes of essential oils and the diameter of the mycelial growth of Fusarium oxysporum and Botrytis cinerea
On the fourth day after the inoculation of fungus F. oxysporum, the weakest effect was determined in the  volume of 3 µL/10 mL PDA, while, as expected, the weakest mycelial growth was determined while using 70 µL/10 mL PDA ( Table 2).
The complete inhibition of the mycelial growth was seen in the essential oil of thyme, in the volume of 7 µL/10 mL PDA, which is followed by the essential oil of clove (9 µL/10 mL PDA), fennel (15 µL/10 mL PDA) and peppermint and anise (30 µL/10 mL PDA). The essential oils of tea and lavender completely inhibited the mycelial growth in the volume of 50 µL/10 mL PDA, and the essential oils of basil and sage in the volume of 70 µL/10 mL PDA. Essential oils of rosemary, lemon and eucalyptus did not completely inhibit the mycelial growth even at the highest used volume.
The results show that on the fourth day the essential oils of thyme, clove, fennel, peppermint, anise, tea, true lavender, sage and basil, used in certain volumes, had the same effect as did the fungicide Prosaro 250 EC in the control variable, which completely inhibited the mycelial growth. On the eighth day, just as on the fourth, the measurement results of mycelial growth of F. oxysporum showed a decrease in the diameter of the mycelium with the increase of essential oil volume relative to the control variable (Table 3).
Essential oils of thyme, clove, peppermint, tea, sage and basil completely inhibited the mycelial growth in the same volume as on the fourth day, while essential oils of anise, fennel, and true lavender completely inhibited the mycelial growth of F. oxysporum in a greater volume. Essential oils, applied in certain volumes, had the same effect as did the fungicide Prosaro 250 EC in the control variable on the eighth day, which completely inhibited the mycelial growth. Essential oils of rosemary and lemon did not completely inhibit the mycelial growth even in the larges used volume, while the essential oil of eucalyptus did not inhibit the mycelial growth at all.
Measuring the mycelium diameter four days after the inoculation of fungus B. cinerea, it was determined that the weakest effect was found with essential oils in the lowest doses, whereas the best effect was when the oils were applied in the volume of 70 µL/10 mL PDA ( Table 4). The complete inhibition of the mycelial growth was seen in the essential oil of thyme in the volume of 9 µL/10 mL PDA, fennel and peppermint at 50 µL/10 mL PDA and tea, basil rosemary and sage at 70 µL/10 mL PDA (Table 4). Those oils did not have the same effect on the mycelial growth in relation to the control as well as the fungicide Switch 62.5 WG.
On the eighth day after the inoculation of fungus B. cinerea, the complete inhibition was caused by the essential oil of thyme in the volume of 15 µL/10 mL PDA, peppermint Table 3: The relationship between the applied volumes of essential oils (μL/10 mL PDA) and diameter (mm) of Fusarium oxysporum mycelium in which total mycelium inhibition was observed -eight days Essential oils Volumes (μL/10 mL PDA) and P value (    in the volume of 50 µL/10 mL PDA and fennel and basil in the volume of 70 µL/10 mL PDA ( Table 5). The oils, both used in those volumes, when compared to the effect of the fungicide Switch 62.5 WG had a better effect on the decrease of the mycelial growth of fungus B. cinerea. The essential oils of clove and sage, even though they did not completely inhibit the mycelial growth, had a better effect relative to the control than the fungicide. The essential oils of eucalyptus and lemon did not inhibit the mycelial growth at all, even at highest applied volume.

Estimated values of IC 50 parameters for essential oils according to effect on mycelial growth of Fusarium oxysporum and Botrytis cinerea
Based on the size of diameter of mycelium for every essential oil in all applied volumes, values of IC 50 parameters (95 % CI) for F. oxysporum and B. cinerea.
On the eighth day after the inoculation with fungus F. oxysporum the lowest value IC 50 ( 95% CI) was noted in, as was on the fourth day, in the essential oil of thyme 0.85 (0.54 -1.21) and clove 1.71 (1.29 -2.18). They are followed by the essential oils of sage 9. 76 (8.27 -11.52), anise 15.71 (11.57 -21.39) and fennel 15.84 (11.83 -21.27). The essential oil of eucalyptus did not inhibit the mycelial growth (Figure 1).
The values of IC 50 parameters (95% CI), for the fourth and the eighth day of the experiment, show that in all tested essential oils, the values are larger for B. cinerea than for F. oxysporum. On the fourth day after inoculation of B. cinerea, the lowest IC 50 (95% CI) was in essential oil of thyme 2.63 (1.77 -3.72), clove 2.21 (1.79 -2.67), anise 9.41 (7.48 -11.82), peppermint 13.42 (9.82 -18.37) and fennel 13.82 (10.28 -18.59). Essential oils of lemon and eucalyptus did not inhibit the mycelial growth ( Figure 2).

DISCUSSION
Although it was noted that many efforts have been taken and many studies conducted with the aim of achieving a more efficient protection of agricultural crops and adjusting the amount of pesticides used to the real need with a less negative effect on the environment (Villalobos and Fereres, 2016), the possibility of not using plant protection products has never been completely addressed, including the negative effect the products have on the health of people and the pollution of the environment. Using natural plant products, including the essential oils, is a promising alternative aimed at saving the nature, the environment and the human health.
In order to determine the mycelial growth of fungi Fusarium oxysporum and Botrytis cinerea, as well as the effect of fungicide on pathogens, the relationships between the applied doses of fungicide with control was tested. The results showed a complete growth inhibition of fungus Fusarium oxysporum on the fourth and eighth day after the change since changing the Prosaro 250 EC fungicide in all applied doses.
Fungicide Switch 62.5 WG worked in accordance with all the producer's specifications after the fourth day of testing. However, the effect on the Botrytis cinerea fungus in in vitro coditions kept decreasing and on the eighth day it did not stop it as effectively as we expected.
The results of measuring mycelial growth of the tested pathogens showed a decrease in the growth, depending on the applied volume of essential oils. The achieved results were in the accordance with El-Mohamedy et al. (2013) tests which tested inhibitory activity of some alternatives to fungicides on the growth of eight soil phytopathogenic fungi and determined that the mycelial growth decreases significantly with the increase of applied essential oil concentration. The minimal mycelial growth was determined at the highest concentration, and the complete inhibition was determined at 1.5% concentration of all tested essential oils. Li et al. (2017) determined that the effect of the tea essential oil on the mycelial growth of B. cinerea and Penicillium expansum depends on the amount of essential oil. At 1.00 mL/L, the essential oil of tea inhibited the growth of B. cinerea 77.16% and P. expansum 27.77%. The inhibition of the mycelial growth is increased with the increase of the volume of essential oil. With the volume of 1.5 mL of oil/L, the inhibition of B. cinerea was 99.02%, while for P. expansum the amount of 4.0 mL/L resulted in 98.62% inhibition of mycelial growth. The mycelial growth was completely inhibited with 2.0 and 6.5 mL oil/L for B. cinerea odnosno P. expansum. The achieved results of our research is also in accordance with results of many other scientists who tested the antifungal activity of essential oils on phytopathogenic fungi and who claim that their activity depended on the applied volume, that is, concentration (Palfi et al., 2018;Duduk at al., 2015;Adabayo et al., 2013;Hung et al., 2013;Lu et al., 2013;Moghaddam et al., 2013;Nguyen et al., 2017;Viuda-Martos et al., 2008). However, antifungal properties of essential oils depend on the properties of plants they are extracted from (Piyo et al., 2009) and their chemical composition (Ćosić et al., 2014).
The positive effect of essential oil of thyme in this research is in a accordance with the research of Alam et al. (2014) who claim that the essential of Thymus capitatus L. in the volume of 2 µg/mL completely inhibited the growth of Fusarium oxysporum, Alternaria solani, Aspergillus niger, Penicillium sp1 and Penicillium sp2. Marandi et al. (2011) determined that Thymus kotschyanus had the strongest antifungal activity in in vitro conditions and completely inhibited the fungal growth in volumes above 200 µL/L. Furthermore, Elshafie et al. (2015) tested potential fungistatic or fungicidal activity of essential oil of thyme (Thymus vulgaris) and verbena (Verbena officinalis) in in vivo conditions in various concentrations against Monilinia laxa, Monilinia fructigena and Monilinia fructicola. They determined that the greatest applied volumes of the essential oil of verbena (1000 ppm) and thyme (500 ppm) considerably reduced the diameter of brown lesions while smaller volumes of the essential of verbena (500 ppm) and thyme (250 ppm) obtained low effect. A good antifungal activity of essential oils of thyme and anise on the increase of mycelial growth of twelve phytopathogenic fungi was also determined by Ćosić et al. (2010).
In our research, the essential oils of lemon and eucalyptus did not inhibit the mycelial growth of B. cinerea at all. Moreover, those oils did not completely inhibit the mycelial growth of F. oxysporum even at highest applied volumes. Our results are not in accordance with the research of Viuda-Martos at al. (2008) who claim that the essential oil of citrus show a certain antifungal activity, and the research of Lee at al. (2007) who claim that the eucalyptus oil (Eucalyptus citriodora) has a strong antifungal effect on the mycelial growth of fungus B. cinerea and that the inhibition grows with the increase of the applied volume of the essential oil.
In our research, the essential oils of thyme, clove, fennel, peppermint, anise, tea, true lavender, sage and basil were applied in volumes which completely inhibited the mycelial growth of F. oxysporum, and had the same effect relative to the control as the fungicide Prosaro 250 EC. The complete inhibition of mycelial growth of B. cinerea at the end of the experiment caused by essential oils of thyme, fennel, peppermint and basil. Those oils had a better effect on the mycelial growth relative to control than the fungicide Switch 62.5 WG. The essential oils of clove and sage, although they did not inhibit the mycelial growth, had a better effect relative to control than a fungicide. Therefore, after comparing the effect of essential oils with the effect of chemical fungicides, it can be concluded that in certain volumes essential oils could have the same or better inhibitory effect on the mycelial growth than fungicides, which is in accordance with the results of Moghtader et al. (2011) andSitara et al. (2008).
The decreased antifungal activity of essential oils in this paper, which was determined in the last measurement, is in accordance with results of Nosrati at al. (2011), who state that the inhibitory effect depends on the volume of essential oils, but also the duration of inhibition. The author states that the samples were treated with 1 µL of essential oil of mint showed a slow decrease on the inhibition of mycelial growth of Fusarium oxysporum f. sp. radicis-cucumerinum throughout the incubation period. Similar results were stated by Sumalan et al. (2013) and Adabayo et al. (2013).
IC 50 (95% CI) parameter values were calculated based on the diameter of mycelium for each essential oil in all applied volumes for Fusarium oxysporum and Botrytis cinerea.
On the fourth day of the experiment, it was determined that the lowest IC 50 (95% CI) for Fusarium oxysporum was in the essential oils of thyme, clove, fennel and anise, and the highest values of the essential oils of eucalyptus and rosemary. On the eight day, the lowest IC 50 (95 % CI) was in the essential oils of thyme, clove, sage, anise and fennel. The essential oil of lemon had the highest value of IC 50 (95% CI), while the essential oil of eucalyptus did not inhibit the growth of F. oxysporum. On the fourth day after the inoculation of mycelium of Botrytis cinerea the lowest IC 50 was in the essential oils of thyme, clove, anise, peppermint and fennel. On the eight day, the lowest IC 50 was in the essential oils of thyme, clove, peppermint and fennel, while the essential oils of lemon and eucalyptus did not inhibit the mycelial growth at all, not even in the highest applied volumes. Also, an increase of IC 50 was noted on the eighth day in relation to the fourth day in both tested phytopathogenic fungi. Bi at al. (2012) stated that among the fourteen tested commercial essential oils, the essential oils Origanum syriacum, Cymbopogon martini and Thymus vulgaris had the lowest values of EC 50 (< 0.15 µg/mL). Perez-Sanchez et al. (2007) determined by testing antifungal properties of essential oils in six Thymus zygis Loefl. ex L. on phytopathogenic fungi Phytium irregulare, Rhizoctonia solani, Colletotrichum acutatum, Fusarium oxysporum i Sclerotinia sclerotiorum that the EC 50 is in the range between 86 ppm and 577 ppm. Good antifungal activity of essential oil Thymus vulgaris on B. cinerea in the amount of 500 μL/mL was also determined by Gebel and Magurno (2014). Huang et al. (2010) states that during the testing of antifungal activity of essential oil of star anise (Illicium verum) and its main component trans-anethole, the IC 50 for all isolates of F. oxysporum was between 0.14 and 0,20 mg/mL for trans-anethole, and for the oil it was 0.16, to 0.25 mg/mL.
The results of our research have showed that the essential oil of eucalyptus has the highest IC 50 which is in accordance with the results of Sharma et al. (2017) that state that the eucalyptus oil shows inhibitory activity at relatively high concentrations and that IC 50 for the essential oil of eucalyptus was 207.86 ppm. On the other hand, Derwich et al. (2013) state that the essential oil Eucalyptus globulus has a good antifungal effect on the fungus Penicillium citrinum and that the minimum inhibitory concentration was 3.07 and 96.14 μL/mL. Fonseca et al. (2015) state that the essential oils Mentha piperita and Rosmarinus officinalis had MIC 50 0.44 mg/mL, and MIC 90 3.5 mg/mL for Pythium insidiosum. Moghaddam et al. (2013) determined that the essential oil M. piperita in in vitro conditions and in the volume of 800 ppm and 1600 ppm completely inhibited the mycelial growth of Dreschlera spicifera, and Fusarium oxysporum f.sp. ciceris in the concentration of ppm, while Sharma et al. (2017) state the IC 50 values for essential oil of mint from 60.05 ppm. Authors state that the essential oils of mint show inhibitory activity at relatively higher concentrations.
Comparing the estimated values of IC 50 (95% CI) parameters of essential oils according to their effect on F. oxysporum and B. cinerea through the entire duration of the experiment, it is visible that with all the tested essential oils, the IC 50 (95% CI) values are higher for B. cinerea than for F. oxysporum. The results of this research are in accordance with research done by authors who claim that essential oils have different effects, depending on the type of phytopathogenic fungus (Karimi et al., 2016;Türkölmez and Soylu, 2014;Moghaddam et al., 2013;Amini at al., 2012;Bahraminejad et al., 2011;Ćosić at al., 2010) and the incubation time (Al-Reza et al., 2010). Li at al. (2017) state that in in vitro conditions, fungus B. cinerea is more sensitive to essential oil Melaleuca alternifolia than Penicillium expansum. Also, a higher sensitivity of B. cinerea was also determined in in vivo conditions with artificial infection. Various antifungal activity to certain fungi was also noted by Angioni et al. (2006) who tested the activity of essential oil Lavandula stoechas L. ssp. stoechas and its components. They determined a strong antifungal activity of the essential oil on pathogenic fungi Rhizoctonia solani and Fusarium oxysporum, and less effective on Aspergillus flavus.
The results of the research with the same essential oils, plant types and pathogen microorganisms can be very different (Hyldgaard et al., 2012) and it is necessary to conduct continuous research, especially in combination with other antimicrobial compounds which enable synergistic effect (Adeyinka and Richard, 2015) or innovative methods of application.

CONCLUSIONS
Based on the conducted in vitro research of antifungal activity of twelve essential oils on the mycelial growth of Fusarium oxysporum and Botrytis cinerea in different volumes and achieved results, we can conclude that some essential oils had the same negative effect on the mycelial growth relative to the control as fungicide Prosaro 250 EC and Switch 62.5 WG. The antifungal effect of essential oils is decreased with the duration of the incubation, while with the increase of applied volumes of essential oils, the mycelial growth of F. oxysporum and B. cinerea is slowed down. The best antifungal effect on the tested phytopathogenic fungi was the essential oil of thyme. That essential oil completely inhibited the mycelial growth of both phytopathogenic fungi in the smallest applied volume on the eighth day of inoculation.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Study to Detect Seed Borne Mycoflora Associated with Chickpea (Cicer aeritinum L.) Seeds

In Indian Agriculture pulses play an important role not only from economical point of view but also due to their nutritional value. Among all the pulses chickpea is the most important one being rich sources of protein (21%), carbohydrates (61.57%) and vitamin C. Chickpea producing states in India are Madhya Pradesh, Uttar Pradesh, Rajasthan, Maharashtra and Andhra Pradesh. Madhya Pradesh contributes 40.96 percent of the total production of chickpea in the country. In Madhya Pradesh it ranks 1 st in both area (3.59 m. ha.) and production (4.6 m. tones) and the average productivity is estimated 1280 kg./ha. International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 11 (2019) Journal homepage: http://www.ijcmas.com

Introduction
In Indian Agriculture pulses play an important role not only from economical point of view but also due to their nutritional value. Among all the pulses chickpea is the most important one being rich sources of protein (21%), carbohydrates (61.57%) and vitamin C.
Agri. Statistics at a glance, . The number of diseases recorded on the chickpea has been increased and those which were formally of little importance have now become more significant.
There are more than 50 pathogens known to attack chickpea of which about 40 are of economic important. Many factors is involved in reducing chickpea seed quality but a combination of susceptibility to pathogenic fungi, environmental condition that favour disease development and field deterioration of seed. Moreover chickpea seeds are rich in protein contain and therefore easily infected by several field and storage fungi which not only affect seed quality but also emergence in field.
Detected several fungal species belonging to many genera from cultivars of chickpea seedborne fungi of chickpea have been also reported by various workers from time to time (Rathod et al., 2012 andNarayan et al., 2013).
Hence the present study on detection of seedborne myco-flora of chickpea was conducted to know the seed mycoflora associated with the chickpea seeds from different locations of Madhya Pradesh.

Blotter paper method
Standard blotter method was used for the detection of seed borne fungi of chickpea. The 100 seeds of each variety were sown on three layers of pre-soaked moist blotter paper having 9 cm diameter. In each plate 5 seed were arranged, 4 seeds in the outer ring and one in the center of petri plates.

Agar plate method
Agar plate method is preferred mostly in plant pathological studies as it provides nutrients rich substrate for development of mycelial growth and sporulation of pathogen on seed, particularly for slow growing fungi, PDA medium was autoclaved at 1.04 kg/cm2 for 15 minute and warm medium approximately 15 ml per plate was poured. On solidification 100 seeds were plated, each plate containing 5 seeds.

Deep freeze method
This method allows better growth of certain fungi as the imbibed seeds on moist blotters are killed by deep freezing and the enclosed nutrients in seed are utilized by fungi. One hundred seeds were plated as in blotter method and incubated for 24hr at room temperature (27 ± 2°C) then the plated seeds were deep frozen at -20°C for 24 hr.

2, 4 -D Method
2,4-D, is a herbicide retards seed germination and seedlings growth due to which the seeds are not displaced and the examination of fungi becomes easy, the blotter were soaked in 0.1 per cent 2,4-D suspension and then placed in plates. Two hundred seeds were incubated for seven days as in blotter method. The plated seeds were then incubated at 27± 20°C, under alternate cycle of 12 hr light and 12hr darkness for seven days by using two 40W white fluorescent tubes. After seven days of incubation, seeds were examined under stereoscopic microscope by using a magnification of 6X to 50X.
Research microscope was also used to confirm the identification of fungi based on morphological characters given in standard mycological books.

Rolled paper towel method
Randomly selected 100 seeds were placed on two layers of moist germination papers, which will be placed on a polythene paper, at ten seeds per row. These seeds will be were covered with another moist germination paper and rolled carefully to avoid any excess pressure on seeds. The rolled towel papers will be kept in slanting position along the wall of laboratory tables and incubated at 26+ 2°Cfor 7 days. The first count of germination was taken on 4 th day and final count of 10 th day. Morphologically normal seedlings were examined with stereo binocular microscope.

Seedling symptoms test
This method is developed by Khare, Mathur and Neergaard in 1977. 10 ml water agar is taken in each 16 mm diameter rimless test tube, sterilized and solidified with slight slant. One seed is sown in each test tube.
The tubes are placed for incubation at 20°C under artificial day light tubes, 12/12 h cycle. To retain moisture they are covered individually or in groups by a plastic sheet or aluminium foil which is removed when the seedlings have reached the cover. Seedlings are examined after 14 days for the typical symptoms of disease in the coleoptiles. The symptoms can be easily studied being visible on roots as well as on green parts. Table 1 showed that the result of comparative performance of various seed health detection methods employed in present study. The results revealed that, all the six seed health detection methods attempted were found to be efficient in detecting various myco-flora associated with the seeds of thirty chickpea varieties. However, higher percentages of the major eight seedborne fungi were evidenced by the seed health detection methods standard blotter paper method was found best method (110.0) for testing of seed associated fungi followed by Agar plate method (103.2 %), Deep freeze method (90.8 %), Paper towel method (80.1 %), Seedling symptoms test (75.3 %) and 2, 4 -D blotter method. These findings are inconformity with findings of Khan et al., (1988), Arya et al., (2004 and Warude et al., (2016). Among all the method tested Aspergillus niger was observed maximum (30.6 %) in standard blotter paper followed by Agar plate (26.9 %), Deep freeze (24.9 %) and paper towel (24.5) was found predominant in all the thirty varieties of chickpea. Aspergillus flavus was observed maximum in standard blotter (23.3 %) followed by agar plate (20.8 %) while minimum was recorded in 2,4-D blotter (11.7%). In all eight mycoflora associated with chickpea seeds Rhizopus spp. was noticed maximum in standard blotter (22.4 %) while minimum was recorded in 2,4-D blotter (10.0 %) followed by paper towel (14.4%), seedling symptoms (16.8%). In agar plate detection method Fusarium oxysporum was observed maximum 20.0% followed by standard blotter 19.6% and Deep freeze 19.5%. Minimum association of Rhizoctonia bataticola was recorded in seedling symptoms method while maximum was observed in agar plate. In standard blotter Alternaria alternata (5.1%) was observed maximum. Curvularia lunata recorded ranged from 1.3 to 1.7 % in 2,4-D blotter and standard blotter, respectively. In agar plate method Penicillium spp. was observed maximum (8.2 %) followed by 5.5 % in standard blotter method. In present study, among six seed health detection methods attempted. The most efficient were Standard blotter paper and Agar plate methods.

Results and Discussion
These methods of seed health detection were also reported as most efficient and reliable for detection of various mycoflora associated with the seeds of soybean, sesame, sunflower, niger, chickpea, pigeonpea etc, by several earlier workers (Mandhare et al., 2009). Sontakke and Hedawoo (2014) and Patil et al., (2018).
It was concluded from the present study, amongst the six method used for testing of thirty chickpea varieties, the Standard Blotter Paper Method was found best method (110.0%) for testing of seed associated mycoflora followed by Agar Plate Method


extfieldsofstudy: Medicine Chemistry

text:
Volatilome in Milk for Grana Padano and Parmigiano Reggiano Cheeses: A First Survey

Milk characteristics in terms of volatile compounds can influence the subsequent product characteristics and can give indications about metabolism. These features can strongly depend on feeding and management. In this perspective, the screening of milk samples intended for Parmigiano-Reggiano and Grana Padano located in Northern Italy was performed, focusing on a panel of volatile molecules. The work was carried out on a total number of 25 bovine milk samples for the production of Parmigiano-Reggiano and Grana Padano. Milk samples were collected from May to September and analyzed for volatile molecules using headspace gas chromatography mass spectrometry. A range of several volatile molecule classes was considered (aldehydes, ketones, alcohols, carboxylic acids, esters, aromatic hydrocarbons, solforates). Results showed a significant influence of the month and destination of milk due to the time period and subsequent use in cheesemaking (Parmigiano-Reggiano and Grana Padano). Significant differences between months were observed for all volatiles. These preliminary results indicate differences between the two types of milk due to the period and destination. The study of volatile molecules in milk will give important information about the physiology of milk and the evolution of dairy products. These features must be extended and confirmed by the sensory analysis of milk and derived products, leading to a more complete characterization of milk biology and derived products.

Introduction
Ruminant milk has different physical and chemical characteristics involving differences in sensory properties [1]. Flavor is probably the most relevant attribute in milk preference, and the milk flavor is the result of a balance of volatile compounds present in a broad concentration spectrum in raw milk.
There is a complex interaction between animal metabolism, the environment, and the involved molecules implied in the definition of flavor and aroma [2]; milk aroma is characteristic for each specie, and is strictly related to the volatilome footprint [3]. The environmental changes and the treatment imposed to milk production can deeply modify the volatile compounds' profile with the possible amelioration of flavor or the appearance of off-flavors.
While it is well-known that small ruminant milk differs from cow milk, discrepancies can be appreciated also between bovine breeds. Flavor compounds derive from fodder (direct passage), rumen Milk samples were collected every month of the year 2017 in the first teen days of the spring-summer months (May-September), during the morning milking. Samples were refrigerated (4-5 • C) soon after collection and carried to the laboratory for the analysis.
A panel of 54 volatile molecules was determined by headspace analysis as described by Panseri et al. [8]; the molecules analyzed were: 1.
The volatile compounds of milk were analyzed according to Panseri et al. [8]. The technique applied was as follows:

HS-SPME Extraction
All milk samples were prepared by weighing exactly 10 mL of milk sample in a 20 mL glass vial, fitted with a cap equipped with silicon/polytetrafluoretylene (PTFE) septa (Supelco, Bellefonte, PA, USA), and by adding 1 mL of the internal standard solution (IS) in water (4-methyl-2-pentanone 1 µg/mL). At the end of the sample equilibration period (1 nh), a conditioned (1.5 h at 280 • C) 1 µL carboxentm/polydimethylsiloxane (CAR/PDMS) StableFlextm fiber (Supelco; Bellefonte, PA, USA) was exposed to the headspace of the sample for extraction (3 h) by a CombiPAL system injector autosampler (CTC analytics, Zwingen, Switzerland). A temperature of 4 • C was selected as the extraction temperature in order to prevent possible matrix alterations (oxidation of some compounds, particularly aldehydes). To keep a constant temperature during analysis, the vials were maintained on a heater plate (CTC Analytics, Zwingen, Switzerland).

Gas Chromatography-Mass Spectrometry Analysis of Volatile Organic Compounds (VOCs)
HS-SPME analysis was performed using a Trace GC Ultra (Thermo-Fisher Scientific; Waltham, MA, USA) Gas Chromatograph coupled to a quadrupole Mass Spectrometer Trace DSQ (Thermo-Fisher Scientific; Waltham, MA, USA) and equipped with an Rtx-Wax column (30 m; 0.25 mm i.d.; 0.25 µm film thickness, Restek, USA). The oven temperature program was: From 35 • C, hold 8 min, to 60 • C at 4 • C/min, then from 60 • C to 160 • C at a rate of 6 • C/min, and finally from 160 • C to 200 • C at a rate of 20 • C/min. Carry over and peaks originating from the fiber were regularly assessed by running blank samples. After each analysis, fibers were immediately thermally desorbed in the GC injector for 5 min at 250 • C to prevent contamination. The injections were performed in splitless mode (5 min). The carrier gas was helium at a constant flow of 1 mL min -1 . The transfer line to the mass spectrometer was maintained at 230 • C, and the ion source temperature was set at 250 • C. The mass spectra were obtained by using a mass selective detector with the electronic impact at 70 eV, a multiplier voltage of 1456 V, and by collecting the data at rate of 1 scan s −1 over the m/z range of 30-350. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic compounds analyzed under the same conditions when available, or by comparing the Kovats retention indices with the literature data. The identification of MS fragmentation patterns was performed either by comparison with those of pure compounds or using the National Institute of Standards and Technology (NIST) MS spectral database. The quantitative evaluation was performed using the internal standard procedure, assuming a response factor of one. Thus, the quantitative results (µg/g) of each volatile compound were computed by relating the peak intensity of the volatile compounds to the intensity of the internal standard added to the sample in a known amount.
Data are shown as mean ± standard deviation. The effects of the cheese destination and month of collection were analyzed by the two-way analysis of variance (ANOVA), considering destination and season as fixed factors. Due to the nature of the data, ANOVA was performed on natural logarithmic data, calculated as: where y' is the new value of the variable, y is the original value. The term, y + 1, was introduced in order to avoid errors in the logarithm calculation due to zero values. In order to evaluate possible multivariate differences between the destination and month groups, a multiple factor analysis (MFA), considered as an evolution of principal component analysis, was applied. In this way, multivariate relations between two groups of qualitative variables (month and milk destination) and a group of response variables (volatile classes) were calculated.
In order to better refine the characteristics of the volatile composition of milks, the MFA procedure was extended-for Padano and Reggiano groups-to all volatiles, putting in evidence the correlations of all variables with the first two dimensions, and the correlations of groups with the abovementioned dimensions. Single volatile milk contents were processed by a two-way ANOVA on log-values as described. Month and milk destination were set as fixed factors, and the statistical significance was set at p < 0.05.
The univariate descriptive statistics and the ANOVA were performed with the software, JMP ver. 7.0, while the MFA analysis with the software, R ver. 3.5.2. for Windows platform.

Results
The mean values, the standard deviations, and the effects calculated by ANOVA are reported in Table 2. ANOVA results underline that the period, at least in the spring-summer period, has a deep influence on the volatile levels, since each volatile class differs significantly during that time. In particular, aldehydes, acids, and aromatic hydrocarbons increased in concentration in late spring-early summer, while ketones, alcohols, esters, and solforate molecules reached their zenith in August (Table 2).
When milk destination was considered as a factor, statistical evidence is seen for aldehydes, alcohols, and acids, more abundant in the Parmigiano-Reggiano destined milk, while aromatic hydrocarbons are more present in the Grana Padano destined milk (Table 2). Although not statistically significant, a slight difference in the solforates mean values is evident for Grana Padano milk ( Table 2).
The MFA analysis put in evidence the interesting multivariate relation between volatile classes and factors; the first two dimensions explain 39.88% of the total variability ( Figure 1). Grana Padano and Parmigiano-Reggiano milk samples ( Figure 1a) separate themselves well in the multivariate space, as well as the months of milk collection. In particular, the June-July months form a doublet in the multivariate plane, and the spring-summer months (May-June) form another group of months. An evident singleton is represented by August (Figure 1a). Figure 1b highlights the relation between the quantitative variables (volatile classes): Alcohols, esters, and ketones correlate positively in the first two component planes, while aldehydes and aromatic hydrocarbons, negatively related each other, appear uncorrelated with respect to the abovementioned volatiles. Acids and solforates lay in an intermediate field. From the paired examination of Figure 1a,b, it seems that "mild" months, intended as climatically favorable, are characterized by milks slightly richer in aromatic hydrocarbons and solforates, while August milk, as reported also in the univariate statistics, is abundant in solforates and compounds, such as alcohols, ketones, and acids, and poor in aldehydes.  Figures 1a and 1b, it seems that "mild" months, intended as climatically favorable, are characterized by milks slightly richer in aromatic hydrocarbons and solforates, while August milk, as reported also in the univariate statistics, is abundant in solforates and compounds, such as alcohols, ketones, and acids, and poor in aldehydes. The univariate distinction between the two different destination milks is repeated also with MFA: Grana Padano samples tend to be richer in solforates and aromatic hydrocarbons with respect to Parmigiano-Reggiano milk, characterized mainly by an abundance of aldehydes. The two milk groups are quite well separated by the zero abscissa of the two-dimensional component plan.
The MFA data processing put in evidence again the sharp separation between the milk groups. Table 3 reports the results for the first two dimensions calculated by MFA, the only dimensions in which the separation between the milks is statistically significant.
By examining dimension 1, Parmigiano milk correlates well with the presence of several esters, alcohols, and ketones, whereas Padano milk appears inversely related with these components ( Table  3). The negative correlation between Padano milk and dimension 1 evidences positive correlations with this type of milk and the presence of p-xylene (negatively correlated with dimension 1) ( Table  3).
Dimension 2 relates positively with Padano milk, except for methanol (Table 3). Also, in this case, several branched esters and alcohols can be appreciated and, notably, dimethylsulfide, directly related to Padano milk group.
The effects of the month of production and destination on the single volatile levels are reported in Table 4. It is clear that the month of production can heavily influence the volatile levels, while the types of milk differ only in four molecules (2-methylbutanal, 2,3-butanedione, 2-propanol, p-xylene). So, multivariate analysis seems to uncover relations between molecules in a more complete way than univariate statistics. The univariate distinction between the two different destination milks is repeated also with MFA: Grana Padano samples tend to be richer in solforates and aromatic hydrocarbons with respect to Parmigiano-Reggiano milk, characterized mainly by an abundance of aldehydes. The two milk groups are quite well separated by the zero abscissa of the two-dimensional component plan.
The MFA data processing put in evidence again the sharp separation between the milk groups. Table 3 reports the results for the first two dimensions calculated by MFA, the only dimensions in which the separation between the milks is statistically significant.
By examining dimension 1, Parmigiano milk correlates well with the presence of several esters, alcohols, and ketones, whereas Padano milk appears inversely related with these components ( Table 3). The negative correlation between Padano milk and dimension 1 evidences positive correlations with this type of milk and the presence of p-xylene (negatively correlated with dimension 1) ( Table 3).
Dimension 2 relates positively with Padano milk, except for methanol (Table 3). Also, in this case, several branched esters and alcohols can be appreciated and, notably, dimethylsulfide, directly related to Padano milk group.
The effects of the month of production and destination on the single volatile levels are reported in Table 4. It is clear that the month of production can heavily influence the volatile levels, while the types of milk differ only in four molecules (2-methylbutanal, 2,3-butanedione, 2-propanol, p-xylene). So, multivariate analysis seems to uncover relations between molecules in a more complete way than univariate statistics.

Discussion
This survey on the volatile characteristics of milk destined for the production of two broadly diffused cheeses evidences differences between products before transformation. From our results, the year period can heavily influence the abundance or lack of substances, according to [9]. The presence of certain classes of volatiles is the result of a complex interaction between the physiological status of forages/grazing, the environment, and the metabolism of the cow. Therefore, it might be very difficult to separate these sources to allow forecasting of, e.g., the characteristics of a given subject metabolism or the influence of a given pasture, but the selection of a group of volatile molecules in milk could characterize several aspects of ruminant physiology. The differences in the two groups of milk samples destined for the production of Grana Padano and Parmigiano-Reggiano could primarily be due to the different production methods (e.g., forbidden use of silages in Parmigiano-Reggiano production, used instead in Grana Padano), although the significant differences in volatiles could be due to the broad dispersion of data, as evidenced by the ample standard deviations, and ANOVA analysis was performed on log-transformed data. The month of milking seems then to influence in a more marked fashion the volatile composition with respect to the milk destination groups, maybe due to variations in hay and fresh forage seasonality or metabolic reactions to higher temperatures. The multivariate analysis (MFA) on single volatile molecules, on the opposite side, showed a quite different pattern, leading to evidence of a net distinction between the two types of milk seen in a multidimensional space. Some milk components (more or less volatile) can have a great impact on the transformed product following ripening and the action of bacterial microflora, even if these modifications in cheese or milk are often missed by untrained consumers that cannot easily perceive these differences [10,11].
Parmigiano-Reggiano milk contains higher concentrations of aldehydes, alcohols, and acids than milk destined for Grana Padano production. Aldehydes as a pentanal can contribute to the appearance of grassy aromas, mainly when feed derives from pastures with respect to concentrates [12]. An inverse trend is generally reported for alcohols, ethanol in particular, which are mainly concentrated in silage-fed animals. Our results seem to follow an opposite direction, since Parmigiano-Reggiano milk was found to be richer in alcohols, but was milked from cows in which silage feeding of cows is forbidden. This feature could suggest a non-alimentary source of alcohols. Organic acids could derive, at least in part, directly from feed. It is reported [13] that higher levels of carboxylic acids are found in animals fed with hay with respect to pasture, probably because of a concentration effect. However, [14] presents a review with contrasting results about this aspect.
The picture of volatile compounds for milk before cheesemaking could add precious information about the physiological condition of animals, as well as help to make forecasts on the evolution of transformed products, as a predictor of cheese quality and characterization.